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Studies of the cell surface of caulobacter crescentus Walker, Stephen George 1994

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STUDIES OF THE CELL SURFACE OF CAULOBACTER CRESCENTUS By STEPHEN GEORGE WALKER B. Sc. Hons., The University of Western Ontario, 1984 M. Sc., The University of Guelph, 1987  A THESIS SUBMITI’ED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES  (Department of Microbiology and Immunology)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA  September 1994 © Stephen George Walker, 1994  thesis In presenting this in of partial fulfillment the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department of  IV\J-(RO iSIoCOy  The University of British Columbia Vancouver, Canada Date  Abstract  Caulobacter produces  a  crescentus is a Gram-negative eubacteria which  surface  layer (S-layer).  S-layers  are paracrystalline  assemblies of protein that cover the outer surface of some eubacteria and archaebacteria cells.  The method by which the protein subunits  composing the S-layer of C.  crescentus, RsaA, interact to form the  array and attach to the cell was examined in this thesis. The  S-layer  was  extracted  from  the  cell  surface  of  C  crescentus NA1000 by treating cells with a pH 2 solution or a solution containing 10 mM ethylene glycol-bis(f-aminoethylether) N,N,N,N’-tetraacetic  acid  (EGTA).  The  examined by sodium dodecyl sulfate (SDS)  extracted -  extract  was  polyacrylamide gel  electrophoresis (PAGE) and found to consist of nearly pure RsaA.  The  isolated S-layer was amorphous in structure but could reassemble  in vitro into a crystalline array in the presence of calcium ions. Two mutants of C.  crescentus NA1000, JS1001 and JS1002,  selected for the ability to grow in the absence of calcium had the additional phenotype of being unable to attach the S-layer to the cell surface although they produced a wild-type RsaA. shed  the  S-layer  into  the  surrounding  medium  These mutants during  growth.  Methods were developed to identify, isolate and purify the cell surface molecules of the wild-type and S-layer attachment-defective strains.  It was determined that the mutant strains did not produce a  smooth lipopolysaccharide (LPS) although they produced the wild-  type rough LPS and extracellular polysaccharide.  The smooth LPS  (termed the S-layer associated oligosaccharide or SAO) was very homogeneous in length as determined by SDS-PAGE and silver staining.  RsaA negative strains that could form and attach an S-layer  on the cell surface if RsaA was expressed on a plasmid vector were also  shown  mutants  to  produce  examined  did  SAO.  All  not produced  S-layer  attachment-defective  SAO.  Two  cosmids  were  identified that partially restored the production of SÃO in the mutant strain JS1001  however restoration of S-layer attachment did not  occur.  III  TABLE OF CONTENTS  Page  Abstract  ii  Table of Contents  iv  List of Figures  ix  List of Tables  xiv  List  xv  of Abbreviations  Acknowledgments  xvii  Dedication  xviii  1.  2.  1  Introduction 1 .1  Bacterial surface arrays  1.2  The S-layer of Caulobacter crescentus  Materials  and  2 11  16  Methods  2.1  Chemicals  16  2.2  Bacterial strains  16  2.3  Growth media  16  2.4  Isolation of calcium-independent mutants  19  2.5  Growth studies  19  2.6  Colourimetric assays  20  2.7  Isolation and purification of cell surface molecules  20  2.7.1  20  LPS isolation iv  2.7.2  EPS isolation  22  2.7.3  RsaA isolation  23  2.7.4  SAO isolation and purification  24  Antisera production  2.8  Production of antiserum to low pH  2.8.1  2.8.2  extracted RsaA  25  Antisera to SAO  26  2.9  In vitro crystallization of S-layer  26  2. 10  Electrophoretic methods  27  2.10.1  SDS-PAGE  27  2.10.2  Western blotting  28  2.10.3  Sample preparation  28 29  2. 11  Silver staining  2. 12  Cell preparation for thin-section electron microscopy  30  2. 13  Negative stain electron microscopy  31  2.14  Transposon mutagenesis of NA1000  32  2. 15  Complementation of mutant strains using  2. 16  3.  25  cosmids  32  Carbohydrate and lipid chemical analysis  33  34  Results 3. 1  Analysis of S-layer attachment by Western 34  blotting 3.2  Isolation and purification of EPS V  38  3.2.1  Assessment of EPS cell association  38  3.2.2  Chemical characterization of EPS  39  Isolation and purification of LPS  3.3  41  3.3.1  Electrophoretic analysis of LPS  41  3.3.2  Isolation LPS, monitored by lipid analysis  43  3.3.3  Colourimetric analysis of LPS  45  3.3.4  Detailed chemical analysis of LPS  45  3.4  Identification of SÃO  47  3.5  Isolation and purification of SÃO  52  3.5.1  Extraction of cell surface molecules  52  3.5.2  Examination of cell extracts  52  3.5.3  Examination of extracted cells  55  3.5.4  Purification of SÃO  59  Purification of S-layer protein  61  3.6  3.6.1  Extraction of the S-layer of C. crescentus 61  NA1000 3.6.2  3.6.3  In vitro recrystallization of NA1000 S-layer  65  Anti-RsaA sera  65  Comparison of S-layers among Caulobacter  3.7  68  isolates S-layer extraction  68  3.7.2  Western blot analysis of extracted proteins  73  3.7.3  Polysaccharide analysis  79  3.8  3.7.1  Metal ion requirements for C. crescentus growth  vi  and S-layer assembly 3.8.1  Influence of calcium on cell growth  86  3.8.2  Influence of metal ions on growth rate  86  3.8.3  Influence of metal ions on S-layer crystallization  3.8.4  3.8.5  90  + or Sr 2 + concentration on 2 Influence of Ca S-layer crystallization  95  The localization of non-crystallized S-layer  95  protein Genetic studies of the calcium-independent  3.9  100  phenotype 3.9.1  Production and screening of a Tn5 library  3.9.2  Complementation of mutants with a cosmid  107  Discussion The C. crescentus surface polysaccharides  4.1  100  104  library  4.  85  107  4.1.1  C. crescentus “rough” LPS  1 07  4.1 .2  C. crescentus SAO  111  4.1.3  C. crescentus EPS  118  4.2  RsaA extraction and in vitro recrystallization  4.3  Distribution of RsaA- and SAO-like molecules in environmental Caulobacter isolates  4.4  Ionic requirements for C. crescentus NA1000  vii  121  123  growth and expression/crystallization of RsaA  4.5  1 28  Genetic studies of the calcium-independent / S-layer  4.6  attachment-defective phenotype  137  Conclusions The relationship between calcium-  4.6.1  independence and loss of SAO  1 37  2 in the The role of Ca 2 or Sr  4.6.2  4.6.3 4.7  1 35  crystallization of S-layer  140  The role of SAO in S-layer attachment  140 141  Summary  5.  References  142  6.  Appendix  169  I  6.1  Isolation of Calcium-independent mutants  1 69  6.2  Production of anti-SAO sera  169  6.3  Colony immunoblot for identification of S-layer 170  mutants  7.  Appendix  171  II  Figure 1  172  Figure 2  174  Figure 3  176  Table I  177  Table II  178  Table III  1 79 VIII  LIST OF FIGURES Page  Figure  1.  Detection of RsaA in Caulobacter crescentus strains by Western Blot analysis.  2.  Fractionation of the EPS of CB2A and NA1000 on 40  Sephacryl S-400. 3.  Analysis of the LPS of CB2A and NA1000 by SDS-PAGE and silver stained.  4.  35  42  Analysis of the LPS of various Caulobacter crescentus strains by SDS-PAGE and silver 44  stained.  5.  Gas chromatographic analysis of the fatty acid methyl esters from the LPS of NA 1000.  6.  46  Analysis of the LPS of NA1000 containing contaminating SAO by SDS-PAGE and silver stained or examined by Western blotting using 48  a-SAO sera.  7.  Analysis of various Caulobacter crescentus strains by SDS-PAGE. LPS and SAO.  (B)  (A)  Silver stained to detect  Examined by Western blotting 50  using a-SAO sera. 8.  Analysis of proteinase K treated NA 1000 NaC1IEDTA extract by SDS-PAGE and silver  ix  stained or examined by Western blotting using x-SAO sera. 9.  53  Analysis of NA1000 NaC1JEDTA extract by SDS-PAGE and coomassie-blue stained or examined by Western blotting using x-RsaA sera. 54  10.  Gas chromatographic analysis of the aiditol acetates from NA1000 EPS isolated by the method of Darveau and Hancock (1983) (A) or NaC1/EDTA extraction (B).  11.  56  Transmission electron micrograph of thin sectioned NA1000 cell (A) and NA1000 cell extracted with NaC1/EDTA (B).  12.  57  Analysis of the carbohydrates purified from JS1003 NaC1/EDTA extracts by SDS-PAGE and silver staining.  13.  60  Analysis of low pH extract from NA 1000 by SDS-PAGE and stained by Coomassie blue or examined by Western blotting using x-RsaA sera. 6 2  14.  Analysis of urea-solubilized protein from JS1001 cultures by SDS-PAGE and stained by 64  Coomassie blue. 15.  Negative-stain transmission electron micrograph of the in vitro recrystallization product of RsaA.  16.  Analysis of low pH extract of NA 1000 and whole cell lysate of NA1000 by Western blotting x  66  using unabsorbed cc-RsaA sera. 17A.  67  Analysis of low pH extracts from Caulobacter strains by SDS-PAGE and Coomassie blue staining. 69  17B.  Analysis of EGTA extracts from Caulobacter strains by SDS-PAGE and Coomassie blue staining. 7 1  18.  Analysis of low pH (A) or EGTA (B) extracts from Caulobacter strains by Western blotting using 74  x-RsaA sera. 19.  Analysis of proteinase K-treated whole cell lysates of Caulobacter strains by SDS-PAGE and 80  silver staining. 20.  Analysis of proteinase K-treated whole cell lysates of Caulobacter strains by Western blotting 83  using c-SAO sera. 21.  The influence of calcium concentration on the  growth of Caulobacter crescentus NA1000 and JS1001 cultured in Higg 10 medium. M  22.  87  The influence of calcium concentration on the  generation time of Caulobacter crescentus NA1000 cultured in Higg 10 medium. M 23.  88  The influence of the metal ion supplement on the  generation time of Caulobacter crescentus 10 medium. M NA 1000 cultured in Higg 24.  The influence of the metal ion supplement on the  generation time of Caulobacter crescentus  xi  89  0 medium. Higg 1 JS1001 cultured in M 25.  91  The influence of the metal ion supplement on the  lag phase of Caulobacter crescentus NA1000 0 medium. Higg 1 cultured in M 26.  92  The influence of the metal ion supplement on the  lag phase of Caulobacter crescentus JS1001 0 medium. Higg 1 cultured in M 27.  93  Negative-stain transmission electron micrograph of strontium mediated crystallization of RsaA  in a Caulobacter crescentus JS1001 colony. 28.  94  Analysis of whole cell lysates of Caulobacter 0 Higg 1 crescentus JS1001 cells, cultured in M liquid medium supplemented with a chloride metal salt, by Western blotting using x-RsaA sera.  29.  98  Analysis of whole cell lysates of Caulobacter 0 Higg 1 crescentus JS1001 cells, cultured on M medium plates supplemented with a chloride metal salt, by Western blotting using x-RsaA sera.  30.  99  Analysis of proteinase K-treated NaC1JEDTA  extracts of Caulobacter crescentus NA1000 Tn5 mutants by SDS-PAGE and silver staining. 3 1.  102  Analysis of protein ase K-treated cell lysates  of Caulobacter crescentus strains by Western blotting using x-SAO sera. 32.  Analysis of proteinase K-treated cell lysates  xii  1 05  of Caulobacter crescentus strains by Western blotting using o-SAO sera. 33.  1 06  A representation of the cell surface of Caulobacter crescentus NA1000.  XIII  1 08  LIST OF TABLES  Page  Table  I.  Bacterial strains.  17  II.  Relevant characteristics of Caulobacter strains.  77  III  The influence of the template and cation concentration on the crystallization of RsaA.  xiv  96  List of Abbreviations  C terminal  carboxy terminal  cm  centimeter  cFU  colony forming unit  DDW  distilled deionized water  DNA  deoxyribonucleic acid  DNase  deoxyribonuclease  EDTA  ethylendiaminetetra-acetic  EGTA  1 ,2-Di(2-aminoethoxy)ethane-NNN’N’ -tetra-acetic acid  EPS  extracellular polysaccharide  FWC  freshwater Caulobacter  F1EPES  N-2-hydroxyethylpiperazine-N’ -2-ethane  ct  gas  g  gravity  h  hour  kDa  kilodalton  kV  kilovolts  KDO  2-keto-3-deoxyoctonate  LPS  lipopolysaccharide  L  litre  MCS  marine Caulobacter strain  M  molar  mg  milligram  mm  minute  chromatography  xv  acid  sulfonic  acid  ml  milliliter  MS  mass  spectrometry  microgram microlitre $.Lm  micrometer  N  normal  nm  nanometer  NMR  nuclear magnetic resonance  N terminal  amino terminal  ODyj  optical density at 600 nm ohms  PAGE  polyacrylamide gel electrophoresis  PCII  phenol-chloroform-hexane  PYE  peptone yeast extract  PBS  phosphate-buffered  RNA  ribonucleic acid  RNase  ribonuclease  SÃO  S-layer associated oligosaccharide  S-layer  surface layer  SDS  sodium dodecyl sulfate  SEC  steric exclusion chromatography  TEM  transmission electron microscopy  ‘]lC  thin-layer  Tris  Tris(hydroxymethyl)methylamine  saline  chromatography  xvi  Acknowledgments I would like to thank my supervisor Dr. John Smit for his guidance and encouragement during my studies.  I would also like to  thank Dr. R. E. W. Hancock, Dr. R. S. Molday and Dr. G. B. Spiegelman for serving on my advisory committee.  A special thanks goes to Dr.  Spiegelman for reading and early draft of this thesis. I  would  Karunaratne  like for  to thank Dr. analyzing  the  work.  xvii  Neil  Ravenscroft and Dr Nedra  carbohydrates  purified  during  my  Dedication This thesis is dedicated to my parents Dr. and Mrs. G. R. Walker. I would like to thank them for their love, support and guidance throughout my life.  Thanks Mom and Dad.  xviii  1.  Introduction  Within cells enzymes synthesize biomolecules by the formation The biomolecules that are  of covalent bonds between substrates.  formed, be they proteins, nucleic acids, lipids or polysaccharides, The components  must then be organized into functional structures. of  the  functional,  or  supramolecular,  interact  structure  mainly  through the formation of weak (hydrogen, ionic, hydrophobic) bonds. Supramolecular structures can be composed of identical biomolecules such as the protein actin in the case of an actin filament or can be composed of a variety of different biomolecules as in a bacterial ribosome.  The supramolecular structure can be constructed either or “self-assembly”  of “instructed morphogenesis”  by the process  (Cohen 1977; Sitte 1981). structures  Supramolecular morphogenesis  require  the  action  fashioned of  by  components  instructed that  are  not  Thus the final components of the  retained in the final structure.  structure do not contain all the information required for assembly. These  template/scaffold 1990).  components  additional or  a  proteolytic  may  take  cleavage  the event  form  of  a  (Kellenberger  Supramolecular structures built through self-assembly do not  require any additional components to achieve the final functional form.  All the information required for assembly is contained in the  biomolecules that make up the structure.  The only requirements are  a sufficient concentration of the subunits and suitable environmental 1  conditions  1981).  (Sitte  supramolecular  structure  appropriate  under  Thus that  the  forms  conditions,  isolated  components  through  spontaneously  of  self-assembly reassemble  a  can,  into  the  examples  of  final structure in vitro. Bacterial  surface  layers  supramolecular structures This thesis reports on  (S-layers)  built through  the  are  self-assembly process.  studies of the mechanisms by which the  protein subunits that form the S-layer of Caulobacter  crescentus  interact to produce a crystalline array and the method by which the S-layer associates with the bacterial cell surface.  The introduction  serves as a brief review on bacterial S-layers and the S-layer of C. crescentus.  For more in depth information on S-layers the reader is  referred to published reviews by Baumeister et a!. (1988), Koval (1988), Messner and Sleytr (1992), Sleytr and Messner (1983, 1988), and Smit (1987).  1. 1  Bacterial  surface  S-layers  are  two  arrays dimensional  crystalline  arrays  of  proteinaceous subunits forming surface layers on prokaryotic cells (Sleytr et al. 1988). protein  or  The subunits are usually composed of a single  glycoprotein  species  which  self-assembles  characteristic lattice (Sleytr and Messner 1983).  to  form  a  The subunits vary  in molecular weight, between species and strains, from 30  -  220 kDa.  S-layers are common components of prokaryotic cell design, being found on over 200 species of eubacteria and archaebacteria, however 2  in comparison to other wall components little is known about these This lack of knowledge is due,  structures (Messner and Sleytr 1992).  for the most part, to the absence of S-layers on the enteric bacteria which are the most thoroughly studied of all prokaryotic groups. focused on  structural  studies to determine how bacteria maintain these layers.  S-layers  Historically, research  S-layers has  on  are identified in transmission electron microscopy studies by virtue of their characteristic periodic morphology (Smit 1987).  The detailed  structure of the S-layer subunits are not readily discernible however due to the limited resolution in these images (Hovmöller et a!. To  1988a).  overcome  this,  computerized  image  processing  techniques are used to obtain an unbiased averaged image in the form of a two dimensional density map (Amos et al. 1982).  By  combining two dimensional information from a tilt series of the same specimen, three dimensional reconstructions with 1.3 nm resolution have been produced (Baumeister and Engelhardt 1987; Chalcroft et Such analytical techniques have determined that S-layer  al. 1986).  proteins, within the crystal lattice, consist of a large core domain and a smaller connecting domain.  This asymmetrical conformation allows  these subunits to arrange themselves into a variety of patterns or morphological units the most common containing 2, 4 or 6 monomers. , (p ) Crystallization of these building blocks results in hexagonal 6 tetragonal (p4.) or linear (p2) lattice types (Saxton and Baumeister 1986). species  The and  resulting strains  patterns  of the  same 3  are  very  species  heterogeneous  between  with respect to lattice  symmetry and the centre-to-centre spacing of the unit cell.  Three  and two dimensional reconstructions indicate that the S-layers are The surface facing  also asymmetric with respect to the two surfaces.  the external environment is smooth in character while the surface proximal to the cell is generally rough (Sleytr and Messner 1988).  It  has been assumed that these layers maintain a standard pore size under all growth conditions, however the S-layer of Aeromonas salmonicida (Garduflo and Kay 1992; Stewart et al. 1986) and some thermophilic Bacillus Sp. (Sleytr and Sara 1986) have been shown to undergo  structural  which  transformations  apparently  alter  the  porosity. Figure 1 in appendix II illustrates some of the features of 5layers using the S-layer of C. crescentus as an example.  Two S-layer  monomers are shown in Fig. 1A (appendix II) illustrating the two domains.  Six S-layer monomers assemble to produce a hexagonal  “unit cell” or “morphological unit” (Fig. 1B; appendix II).  Unit cells  then interact to form the final array structure (Fig. 1C; appendix II). Kinetic  studies  have  determined  that  crystallization  of  S-layer  monomers into the final array proceeds by a two step mechanism as illustrated in Fig. 1 (appendix II) and the first step occurs at a faster rate than the second (Sleytr and Messner 1983). The majority of S-layer producing bacteria have a single layer, although  double  S-layers  have  been  found  on  Aquaspirillum  metamorphum (Beveridge and Murray 1975), A. “Ordal” (Beveridge and  Murray  1976c), A. serpens MW5 (Kist and Murray 1984), A.  4  sinuosum (Smith and Murray 1990), Lampropedia  oceanus (Remsen et al. 1970) and  and Murray 1990), Nitrocystis  Bacillus  hyalina (Austin  brevis 47 (Tsuboi et al. 1982), and three layers have been  observed on Waisby’s “square bacterium” (Stoeckenius 1981).  The  double layer of A. “Ordal” (Beveridge and Murray 1976c) contains an  of hexagonal  layer  outer  tetragonal symmetry.  an  and  symmetry  layer  inner  of  The two S-layers of A. serpens MW5 are both  hexagonal in symmetry but are antigenically unrelated (Koval et a!.  brevis 47  1988) while the proteins of the two S-layers of Bacillus  differ in molecular weight they are produced as a cotranscriptional unit.  An S-layer is usually composed of a single protein.  However,  the very complex S-layers of L. hyalina (Austin and Murray 1990),  Flexibacter Chiamydia  polymorphus  and  trachomatis (Chang et al. 1982) are composed of two or  more polypeptides.  The pathogen Campylobacter fetus produces an  S-layer that undergoes produce  1983)  Lewin  and  (Ridgeway  S-layer  an  proteins  antigenic that  A  shift.  vary  by  strain can  single  molecular  weight  and  antigenic character (Dubreuil et al. 1990; Wang et a!. 1990). Biochemical studies of S-layers have demonstrated that general similarities exist between the protein subunits produced by diverse species.  Most subunits are held together, and to the underlying cell  surface,  by non-covalent (hydrophobic,  ionic,  hydrogen  or polar)  bonds and are similar with respect to amino acid composition (Koval and Murray 1984a; Messner and Sleytr 1992; Sleytr and Messner 1983).  They generally contain a large proportion of acidic and  hydrophobic amino acids and little or no sulphur-containing amino 5  These proteins contain a high proportion of random coil, 20  acids.  35 percent beta sheet and very little alpha helix (< 2  -  14 percent)  -  However, not all S-layer  (Koval 1988; Sleytr and Messner 1988).  The very thermophilic  proteins conform to these generalizations.  and sheathed archaebacterial (Konig and Stetter 1986), Chiamydiace .Sp. (Newhall and Jones 1983), and the inner tetragonal layer of A. (Smith  sinuosum covalently  bonded  and and  Murray are  have  1990)  resistant  highly  covalently attached carbohydrates.  archaebacterial  and have and  denaturation  to  are by  Glycoprotein containing subunits  were first identified in Halobacterium 1976)  that  Subunits can also be modified with  physical or chemical methods.  Strominger  subunits  since  salinarium  located in a number of  been  species.  eubacterial  (Mescher and  bacterial  These  glycoproteins contain substantial differences compared to those in eukaryotes  with respect to both  glycan chains  the  and  linkages  (Messner and Sleytr 1988; Messner and Sleytr 1991). Although similarities exist between S-layer proteins produced by unrelated species, analysis of the S-layer genes has identified little to no sequence homology (Gilchrist et al. 1992; Messner and Sleytr 1992).  1992; Messner and Sleytr  Genetic (Gilchrist et al.  1992),  ultrastructural  (Hovmöller  (Sleytr  and  1983)  Messner  et  al.  and  1988b)  comparisons  have  led  to  biochemical a  general  consensus among researchers that S-layers are of a non-conserved nature  and  evolution.  have  arisen  independently  in  species  by  convergent  An S-layer may evolve in a given species to fulfill a 6  specific function(s); However, the protein subunits of all species must be capable of three major tasks: secretion, self-assembly or bonding with adjacent subunits, and attachment to the underlying cell surface It is assumed that the similarities between S-layer  (Smit 1987).  subunits with respect to general amino acid composition, method of subunit bonding and gross morphology are due to the proteins all having to fulfill the above tasks. The mechanisms by which S-layers are secreted have not been studied in great detail in comparison to the mechanisms involved in assembly and attachment to the cell surface.  fetus (Blaser and Gotschlich  layer genes all but 4; Campylobacter 1990), Caulobacter  Of the 18 sequenced S  crescentus (Gilchrist et al. 1992), Rickettsia  prowazekii (Carl et al. 1990), and R. rickettsii (Gilmore et al. 1989), a  cleaved  N-terminal  membrane  proteins  are  contain  signal  believed  to  sequence. be  periplasm to the outer membrane through 1979)  Many  transported  outer  from  the  adhesion zones (Bayer  However, Belland and Trust (1985) have indicated that the S salmonicida is transported from the  layer monomer of Aeromonas  cytoplasm to the distal side of the outer membrane by a mechanism which includes a step where the protein is free in the periplasm.  A  linkage between S-layer and lipopolysaccharide (LPS) translocation from the cytoplasm to the outer membrane has been suggested for Acinetobacter  199A and Aeromonas  salmonicida (Thorne et al.  1976; Belland and Trust 1985). The non-covalent forces responsible for 7  subunit-subunit and  subunit-cell  surface  stability  in  S-layers  identifying  conditions  under  which  the  disintegrate  and then reassemble into  1981; Koval and Murray  1984a;  are  determined  crystalline  by will  arrays  a regular array (Beveridge  Smit  1987;  Sleytr and Messner  Within the same S-layer, the subunit-subunit and subunit-  1983).  cell surface bonds may be of a different nature although it is often difficult to differentiate between the two interactions (Smit 1987).  It  has been noted that when metal ions are required for reassembly an S-layer Ca 2 is the ion of choice. 1982) and Sporosarcina requirement  Only Bacillus brevis (Tsuboi et al.  ureae (Beveridge 1979) have a absolute  + while Aeromonas 2 Mg  for  salmonicida  appears  to  2 (Garduno et al. 1992b). 2 and Mg require both Ca The cell surface molecules  with which  S-layers interact to  maintain cell association have been identified in only a few bacterial species.  The S-layer of Deinococcus  cell via proteins  (Thompson et al.  radiodurans is anchored to the 1982) whereas Clostridium  difficile utilizes neutral cell surface polysaccharides (Masuda and Kawata 1981).  Other Gram-positive species may use anionic sites on  the peptidoglycan (Beveridge 1981; Hastie and Brinton 1979).  The  S-layers of Gram-negative eubacteria have been shown to interact with the outer membrane via protein(s) in Acinetobacter (Thorne et al.  1975), and perhaps Spirillum  putridiconchylium  (Beveridge and Murray 1976b), and LPS in Aeromonas (Belland and Trust 1985), A. and Carnpylobacter  199A  salmonicida  hydrophila (Dooley and Trust 1988)  fetus (Yang et a!. 1992). 8  Spirillum  serpens  For species  requires both LPS and lipid (Chester and Murray 1978). possessing  a  double  layer  upper  the  S-layer,  will  only  often  reassemble in the presence of the lower layer as illustrated with Aquaspirillum serpens MW5 (Kist and Murray 1983). Although much is known about the structure and biochemical composition of S-layers no one definitive function has been proposed Because S-layer producing bacteria are founded in  for these layers.  almost every environmental niche it is unlikely that all S-layers have It is assumed that S-layers have evolved to  an identical function. different  serve  functional  roles  due  to  particular  stresses that a species encounters in its habitat.  environmental  The function 5-  layers serve must be important to the survival of the cell when one S-layer monomers  considers the great energetic cost of the layer.  account for up to 10 percent of the total cellular protein and the energy  expenditure  is  even  (Sleytr and Messner 1988). that  many  greater  glycosylization  when  occurs  Considering this cost, it is not surprising strains  S-layer producing  lose  them  upon  laboratory  cultivation in the absence of environmental stress (Blaser et al. 1985; Buckmire  1971;  Beveridge,  1980).  Luckevich  and  Beveridge  1989;  Stewart  and  The observation of pore-like structures formed within S-layers indicates that these layers may act as a molecular sieve and restrict the diffusion of molecules larger than the exclusion limit of the pore. S-layers with exclusion limits less than that of a harmful molecule would thus protect the cell.  S-layers protect some bacterial strains  from lysozyme (Nermut and Murray 1967), various proteases (Sleytr 9  1976), predation (Buckmire 1971; Koval and Hynes 1991) and host virulence  factors  (Ishiguro  et  al.  1981;  Blaser  et  a!.  1988)  presumably by exclusion. The most obvious function for an S-layer is in the type three archaebacterial wall, consisting of a plasma membrane surrounded by an S-layer (Kandler and Konig 1985), where it determines the cell shape (Sleytr et al.  Shape determination and  1986a).  structural  integrity has also been attributed to the S-layer sheaths of some methanogenic archaebacteria (Patel et al. 1986). functions have been suggested for S-layers.  A number of other  Beveridge and Murray  (1976a) proposed that the electronegative character of most S-layers may serve to concentrate essential cations from a dilute environment or  protect  cells  by  immobilizing  toxic  ions  (Beveridge  1979).  Alternatively, S-layer interaction with soluble ions may also act to buffer the environment immediately surrounding the cell and inhibit large changes in pH  (Stewart and Beveridge 1980).  S-layers have  also been implicated as a means to promote bacterial adhesion to macrophages (Garduflo et al.  1992a; Trust et al.  1983), epidermal  cells (Baumeister and Hegerl 1986), porphyrin and immunoglobulin (Kay et al. 1988; Phipps and Kay 1988), fibronectin and laminin (Doig et al. 1992; Kay and Trust 1991), bacteriophage (Edwards and Smit 1991;  Howard  and  Tipper  and  1973)  between  bacteria  via  autoagglutination (Evenberg and Lugtenberg 1982). This brief review of bacterial S-layers illustrates that although these  structures  appear  “similar” 10  at  a  superficial  level  (two  dimensional arrays composed of acidic proteins lacking cysteine and of similar secondary structure) detailed ultrastructural, biochemical and  genetic  studies  have  that  revealed  these  layers  are  very  heterogeneous, sometimes even between strains of the same species, and  are  evolutionaraly  Therefore,  unrelated.  when  an  uncharacterized S-layer is studied it is difficult to predict how the protein subunits are secreted to the outer membrane, assembled into an array and attached to the cell surface.  1.2  The S-layer of C.  C.  crescentus  crescentus  is  Gram-negative  a  eubacterium  which  undergoes a sequence of morphological changes at specific polar membrane sites during its life cycle (for review, see Poindexter 1964 and 1981; Shapiro 1976). bacteriophage  receptors,  Swarmer cells express a single flagellum, pili  and  an  adhesive  substance  termed  holdfast all at one cell pole.  All polar features but the holdfast are  lost  at  and  a  stalk  develops,  differentiates into a stalked cell.  the  same  pole,  as  the  swarmer  The stalk, which is an outgrowth of  the cell envelope and contains no cytoplasmic  material, remains  Swarmers  are produced by  through  all  subsequent generations.  growth and division of the stalked cell with the swarmer cell polar surface appendages being produced at the pole distal to the stalk cell. With the exception of the pilus (Smit and Agabian 1982a) control of the production of the polar structures is linked to DNA replication.  A  single round of replication occurs during the life cycle and these 11  polar events are initiated at the midpoint by an unknown signal.  The  majority of research conducted with C. crescentus has focused on dissecting the developmental process at the genetic level (Dingwall et al.  1990; Shapiro 1993).  Throughout the entire life cycle of C.  crescentus the cell surface is completely covered with an S-layer  (Smit et al. 1981). The C. crescentus S-layer is of hexagonal symmetry (Smit et al. 1981).  Smit  et  al.  have  (1992)  produced  a  three-dimensional  reconstruction to a resolution of 2.0 nm (see Fig. 1; appendix II).  The  reconstruction shows that the morphological unit is formed by six protein subunits that are arranged on a p6 lattice.  The subunits  forming the array contain a heavy domain, that interacts to form a central hexagonal core, and a lighter domain that connects adjacent morphological units.  The centre to centre distance between the The interaction of the heavy domain  morphological units is 22 nm.  regions produces a central pore that has a diameter of 2.5  -  3.5 nm.  A similar size of gap is found in the space between the unit cells. These breeches in the array would allow the passage of globular proteins no larger than approximately 17 kDa (Smit et al. 1992). The gene encoding the protein that forms the S-layer (rsaA) has been cloned (Smit and Agabian 1984) and sequenced (Fisher et al. 1988; Gilchrist et al. 1992).  The predicted molecular weight of  RsaA is 98,132 and the predicted p1 is 3.46.  Apart from the removal  of the initial methionine no N-terminal or C-terminal processing of the protein occurs during secretion (Gilchrist et aT. 1992). 12  RsaA is a major cellular protein accounting for 5-7% of total Indirect immunocytochemical  protein synthesis (Smit et al. 1981). have  methods  been  used  examine  to  where  new  S-layer  incorporated into the crystalline lattice during cell growth.  is  Two  systems of S-layer incorporation were distinguished by Smit and (1982b).  Agabian  During  growth  newly  synthesized  S-layer is  incorporated at random locations on the cell body but only new 5layer was incorporated onto the growing stalk and the newly formed Because array components are synthesized uniformly during  pole.  the cell cycle (Agabian et al. 1979) and a cell possesses only one copy of rsaA (Smit and Agabian 1984), control of S-layer incorporation must work at the level of assembly rather than transcription. account  for  postulated  the  that  mechanisms  two on  the  cell  body  of  S-layer  S-layer  assembly,  arrived  via  it  To was  transient  adhesion zones between the inner and outer membrane, whereas at the newly formed pole and stalk more stable sites of membrane adhesion exist.  At present no information is available on how the 5-  layer protein is secreted or targeted to the cell surface. Calcium  has  been  implicated  in  the  assembly  of the  C  crescentus S-layer and/or in the attachment of the layer to the cell surface.  Gilchrist et a!. (1992) identified 4 or 5 putative calcium  binding motifs in the C-terminal region of the predicted amino acid sequence of rsaA.  Similar glycine-rich repeats have been identified  in hemolysins and proteases of other species which require calcium for biological activity.  Apart from the homology analysis there is no  direct demonstration that the S-layer binds calcium. 13  Poindexter  that  showed  (1982)  wild-type  C.  crescentus  has  a  growth  requirement for calcium and that mutants could be selected which no Analysis of the mutants revealed that they  longer require calcium.  could no longer attach RsaA to the cell surface and that in high density liquid cultures a macroscopic debris, presumed to consist of RsaA, was formed. Smit et a!. (1992; unpublished studies; see appendix I [method A])  isolated a number of calcium-independent mutants It  NA1000.  crescentus  was  demonstrated  these  that  from C. mutants  produced large sheets of non-cell-associated, but assembled S-layer, when cultured on calcium-containing agar.  Smit et a!. (1992) showed  that these sheets were composed of two S-layers that associate via the side of the array which is proximal to the cell surface in the The two layers were in such precise alignment  wild-type situation.  that three-dimensional image reconstruction was required to resolve that a double layer existed. were  grown  detected.  on  When the calcium-independent mutants plates  calcium-free  no  assembled  S-layer  was  This data indicated that the mutants were not defective in  S-layer assembly and that calcium was involved in the assembly process.  The  S-layer  gene  was  cloned  from  three  calcium  independent mutants and each was individually introduced into C. crescentus JS1003.  The chromosomal copy of rsaA of the parent  strain, NA1000, was deleted to produce JS1003 (Smit, unpublished). Negative-stain electron microscopy and indirect immunofluorescence microscopy (Smit et al. unpublished) showed that a wild-type S-layer 14  was produced on the cell surface of JS1003 when any of the S-layer genes from the calcium-independent mutants were expressed on a This implied that the mutation resulting in the S  plasmid vector.  layer attachment-defective phenotype was not located in rsaA but in some other gene whose product is involved in the attachment the array to the cell surface. The central goal of this thesis was to identify, isolate and characterize the cell surface molecule(s) of C.  crescentus which  interact with the RsaA in order to attach the S-layer to the cell.  The  cell  surface of calcium-independent / S-layer attachment-defective  C.  crescentus  strains  wild-type  the  and  strain,  NA1000,  were  examined in order to identify any differences between the wild-type An examination of the LPS of the Caulobacter  and mutant strains.  strains determined that the mutants did not produce a smooth LPS. The smooth LPS, termed the “S-layer associated oligosaccharide” (SAO),  was  competent  produced  strains  by  but not by  attachment-defective  wild-type  all  any  of the  /  S-layer  attachment  calcium-independent /  mutants.  Some of the data presented in this thesis has been previously published as Ravenscroft et al. 1991, Ravenscroft et al. 1992, Walker et a!. 1992 and Walker et al. 1994.  15  Materials  2.1  Chemicals Unless  Methods  and  2.  stated all chemicals  otherwise  Sigma (Sigma Chemical Company,  were purchased from  St. Louis,  MO) and were of  analytical grade.  2.2  strains  Bacterial  The Caulobacter strains used in this thesis are described in Table I.  All Caulobacter  strains  were  at  30°C  and  (Poindexter  1964)  was  grown  Escherichia coli B and DH5x were grown at 37°C.  2.3  Growth  media  Peptone-yeast extract (PYE)  medium  used for the growth of of all Caulobacter strains unless otherwise specified and contains per litre; 2 g peptone (Difco Laboratories, 2 Detroit, MI), 1 g yeast extract (BDH Inc., Darrnstadt, Ger.), 0.01% CaC1 4 (BDH). (BDH), and 0.02% MgSO routinely  for  harvested 0.6  Cells grown in PYE medium were  experiments  at  mid-logarithmic  growth  Solid media, of all types, contained 1.2%  600 phase (OD  =  agar (Difco).  For growth of freshwater Caulobacter isolates PYE  -  0.7).  liquid was supplemented with riboflavin at 2 ig/m1. igg medium (Smit et al. 1981) contains 5 mM imidazole M H 3  -  O (pH 6.8), 0.3% glucose, 0.3% L-glutamic 4 P 2 HC1 (pH 7.0), 2 mM KH 1, and 1% modified Hutner NH C acid (monosodium salt; pH 7.0), 0.05% 4 16  Bacterial strains  TABLE I.  strain  Bacterial  Description  Reference  genotype  or  of  source  C.  crescentus  CB2A  S.1ayer minus variant of wild-type  Smit and Agabian  CB2, Tpr, Rfr, Apr.  1984  Spontaneous  CB2NY66R  S-layer plus  J.  mutant  Poindexter*  of CB2, Tpr, Apr. CB2NY66Rmg1  Calcium-independent  mutant  J. Poindexter*  of  CB2NY66R, Tpr, Apr. NA1000  Variant of wild-type strain CB15,  Smit and Agabian  ATCC19O89, synchronous cultures  1984  readily prepared  from this strain,  Tpr, Apr.  JS1001  Calcium-independent of NA000.  3. Smit’  JS1002  Calcium-independent of NA1000.  351003  NA 1000 with rsaA interrupted with  I. Smit 3. Smit*  KSAC Kmr cassette. JS1001 with rsaA interrupted with  JS1004  J. Smit*  KSAC Kmr cassette. JS1002 with rsaA interrupted with  JS1005  J. Smit*  KSAC Kmr cassette.  Fresh  Water  42 FWC strains  isolates  Caulobacter  Isolated from aquatic and wastewater MacRae and Smit, 1991  sources.  Legend:  *  unpublished strain, Km’ resistant, Apr  =  =  kanamycin resistant, Tpr  Ampicillin resistant, Rf  17  =  =  trimethoprim  Rifampicin resistant  Higg or 3 Cells grown in M  mineral base (Cohen-Bazire et a!. 1957). M 10 Higg  were  routinely  600 logarithmic growth phase (0D  =  2.0  -  at  experiments  for  harvested  mid-  0 Higg 1 Higg and M 3 3.0). M  media were used for physiological studies to determine the cation requirements for Caulobacter growth and their influence on S-layer Higg medium is: 3 The final metal ion concentration in M  structure.  ; 9.1 p.M 4 ; 25.1 p.M FeSO 4 ; 38 p.M ZnSO 2 2.2 mM MgSO ; 454 p.M CaC1 4 ; and 0.15 p.M 7 O 4 B 2 ; 0.5 p.M Na 2 ) 3 ; 0.9 p.M Co(N0 4 ; 1.6 p.M CuSO 4 MnSO o 4 . M 6 ) 4 (NH 2 O 7 Higg medium except that the 3 0 medium is identical to M Higg 1 M 2 and 18 M2 distilled Hutner mineral base was prepared without CaCI -  deionized water, produced by a Barnsted “NANOpure” ultrapure  water system, was used. M 10 Higg  medium  All containers used to prepare or store  were washed  with  10  mM Na-EDTA  (Fisher  Scientific Co., Nepean, Ont.) (pH 8.0), to remove any trace calcium, then washed with 18 M2 distilled  -  deionized water.  Only new 16 x  150 mm S/P® diSPo® culture tubes (Baxter Healthcare Corporation, 0 medium. Higg 1 McGaw Park, IL) were used for growth of cells in M 10 medium plates was suspended in 50 mM M The agar used for Higg EDTA (pH 8.0) and allowed to settle.  The supernatant was decanted  and the procedure was repeated four times.  The agar was then  washed in, the same manner, twice with 18 M2 distilled water.  -  deionized  The slurry was then suction-filtered through a hardened  -  ashless Whatman (Whatman International Ltd., Maidstone, England) 541 filter.  The agar was then dried under negative pressure in a 18  dessicator oven at 85°C.  Plastic petri dishes (Fisher) were used for  0 medium. Higg 1 solid M L medium was used for the growth of E. coil (Miller, 1972) and contains per L; 10 g tryptone (Difco), 5 g yeast extract and 5 g NaC1. When  antibiotics  required  were  added  media  to  at  the  Ap(100) [sodium salt], Km(50)  following concentrations (in p.g/ml):  [sulfate salt] (ICN Biomedicals, Inc., Cleveland, OH), Sm(50 or 10 when pSUP2O21  [Simon et al. 1983] was used in NA1000) [sulfate salt],  Tc(10 or 4 when pLAF5 [Keen et al. 1988] was used in JS1004) [chloride  salt]  Inc.,  Biochemicals,  (P-L  Milwaukee,  Wis).  All  antibiotics were prepared and stored as recommended by Sambrook et al (1989).  2.4  of  Isolation  independent  calcium  mutants  The calcium independent mutant CB2NY66Rmg1 was isolated by Dr. J. Poindexter (unpublished). mutants  JS1001  (unpublished).  and  JS1002  The calcium independent mutant were  isolated  by  Dr.  John  Smit  See appendix I (method A) for the method by which  these mutants were selected.  2.5  Growth  studies  For studies on the ion requirements of NA1000 and JS1001 the following procedure was used. 600 to mid-log phase (0D  =  Higg medium 3 Cells were grown in M  2.0-3.0), harvested by centrifugation, and  0 by centrifugation and resuspension. washed four times in M Higg 1 19  0 (with or without a metal ion [chloride salt] Higg 1 Five ml of M supplement) was inoculated with 5 x 106 washed cells and incubated at 30°C on a tube roller (VWR Scientific Canada, Ltd., London, Ont) at 60  rpm.  Plate  crescentus  counts  NA1000  that  determined grown  in  igg M H 3  approximately 1 x iO CFU’s per ml. 600 between 0D  2.6  =  an  600 0D  =  medium  1.0 of C. contains  Growth rates were estimated  0.100 and 1.000.  Colourimetric  assays  An LKB Biochrom Ultraspec II UV/VIS spectrophotometer was used for all colourimetric assays.  Protein levels were determined by  the method of Markwell et al. (1978) using egg white lysozyme as a standard or using the Bio-Rad protein assay (Bio-Rad laboratories, Missassauga, Ontario), which is based on the method of Bradford (1976), using bovine gamma globulin as a standard.  3-deoxy-2-  octulosonic acid (KDO) was estimated by the method of Karkhanis et al.  (1978) using  authentic KDO (ammonium salt) as a standard.  Inorganic phosphate was determined by the method of Ames and HPO as a standard. 2 K Dubin (1960) using 4  Sugars were estimated by  the method of Dubois et al. (1956) using D-glucose as a standard. Uronic acids were estimated by the method of Dische (1947) using D glucuronic acid as a standard.  2.7 2.7.1  Isolation LPS  and  purification  isolation. C.  of cell  surface  molecules  crescentus strains CB2A and NA1000  were grown in PYE as 500 ml cultures, in 2 L Erlenmeyer flasks, on a 20  600 rotary shaker (200 rpm) and harvested during late log-phase (0D =  Cells were harvested with a Sorvall RC-5B centrifuge (Du  0.6-0.7).  Pont Instruments, Wilmington, Delaware) using a GSA rotor (Du Pont) (10,000  for  xg  10 mm) and washed once with 0.1  M HEPES  (Research Organics, Inc., Cleveland, Ohio) buffer (pH 7.2).  LPS was  isolated using a modification of the method of Darveau and Hancock After nuclease digestion of the disrupted cells, the cell lysate  (1983).  was made to contain 0.1 M EDTA, 2% SDS, and 10 mM Tris-HC1 (pH 8.0)  and was  at 37°C  then incubated  for  The extended  2 h.  incubation was required to completely dissociate the Caulobacter cell  The  membranes.  published  procedure  completion of the final ultracentrifugation step.  until  followed  was  The supernatant The LPS  from this ultracentrifugation step contained “crude” EPS.  pellet was resuspended in 10 mM Tris-HCI (pH 8.0) and washed five times  by  Beckman  xg for 2 h at 15°C), using a  ultracentrifugation (200,000 (Beckman  Instruments,  Inc.,  Palo  Alto,  L8-55  CA)  ultracentrifuge and a Type 6OTi rotor (Beckman), and resuspension. ’ LPS fraction. 1 The final pellet was considered to be the “crude The crude LPS was extracted following the Sonesson et al. (1989) modification of the Galanos et al. (1969) procedure.  The  freeze-dried crude LPS preparation was extracted three times with phenol:chloroform:hexane, 2:5:8, (PCH) (at 8 ml per g of original dry weight  of the cells)  supernatants  were  and  centrifuged  evaporated  using  at 2000 xg.  a rotary  The pooled  evaporator  and  the  phenol was removed by dialysis against distilled deionized water (4 21  x 2L).  The LPS was recovered by lyophilization and washed three  times with 10 ml of chloroform:methanol (2:1) and insoluble material The precipitate  was pelleted by centrifugation (200,000 xg for 2 h).  was dried under a stream of nitrogen gas, dissolved in distilled ’ LPS fraction. t deionized water then freeze-dried to yield the “pure  EPS  2.7.2  The “crude” EPS obtained during the LPS  isolation.  isolation procedure  above)  (see  was  1/10 the original volume,  DDW to  freeze  dried,  resuspended  in  treated with bovine pancreatic  RNase (25 jig/mi at 37°C for 24h), dialyzed against 4L DDW at 4°C for 24h  then  and  remove  ultracentrifuged  any remaining  resuspended  in  LPS.  M  0.1  at  200,000  The  EPS  pyridinium  xg (30 hr at 4°C) to  was  acetate  then  buffer  freeze (pH  dried,  7.0)  and  fractionated by steric-exciusion chromatography (SEC) on a Sephacryl S-400 (Pharmacia LKB Biotechnology, Uppsala, Sweden) column (60 x 2 cm) using 0.1 M pyridinium acetate buffer (pH 7.0) as eluent. Fractions  (2.5  were collected using  ml)  fraction collector.  a Pharmacia FRAC-100  Each fraction was analysed for carbohydrate to  determine peak locations. The  nature  of  the  cell  surface  EPS  was  investigated  by  assessing the degree to which the carbohydrate remained associated with the cells.  The yield of EPS from unwashed cells was compared  to yield from cells that were washed 5 times by centrifugation and resuspension in 0.1 M HEPES buffer (pH 7.2).  22  series  C.  RsaA isolation.  2.7.3 of  experiments  to  crescentus NA1000 was used in a  determine  an  effective  extracting the S-layer protein from the cell surface.  method  for  In a typical  600 experiment a 100 ml culture was grown in PYE to an 0D and harvested by centrifugation (10,000 xg for 10 mm).  0.6-0.7  =  The cell  pellet was washed twice in 1 volume of 10 mM HEPES buffer (pH 7.2) by suspension and centrifugation and then resuspended in 15 ml of the same buffer.  One ml of the cell suspension was placed into 1.7 Two  ml microfuge tubes, pelleted and the supernatant removed.  hundred tl of one of the following agents was used to resuspend the cells: 10 mM EDTA in 10 mM HEPES buffer (pH 7.5); 10 mM EGTA in 10 mM HEPES buffer (pH 7.5); 100 mM HEPES (pH 2, 4, 6, 7.5, 8 or 10); 200 mM glycine-HC1 buffer (ICN) (pH 2, 3 or 4); 100 mM TRIS buffer (ICN) (pH 7.2); 0.5% 8-mercaptoethanol (Bio-Rad) in 10 mM HEPES (pH 7.5); 1 M urea (BDH); 1 M guanidine-HC1 (BRL); 10 mM NaCI (Fisher);  ; and 100 mM HEPES (pH 7.5) with 2 10 mM CaC1  incubation at 65°C. temperature  (unless  The samples were incubated for 15 mm otherwise  pelleted by centrifugation.  stated)  and  then  the  at room  cells  were  Ten il of supernatant was analyzed by  SDS-PAGE and the proteins in the gel were visualized by Coomassie blue staining (See below). Subsequently, as a standard method for isolation of S-layer 600 protein, 5 ml cultures of Caulobacter strains were grown to 0D 0.6 and the cells harvested by centrifugation.  =  The cells were washed  twice by centrifugation and resuspension with 5 ml of 10 mM HEPES 23  (pH 7.2) and then the washed pellet was suspended in 200 j.tl of 100 mM HEPES (pH 2) or 200 il of 10 mM EGTA in 10 mM HEPES (pH  7.5).  The cell suspension was incubated for 10 mm  at 20°C, pelleted  by centrifugation and the supernatant retained for examination by SDS-PAGE.  The acid samples were immediately adjusted to pH 7  with 5 N NaOH (BDH). JS1001 and JS1002 produce a macroscopic particulate “debris” in high density cultures.  A debris sample (approximately 100 mg  wet weight) was collected with a pasteur pipet and suspended in 10 mM HEPES  The material was pelleted in a  buffer (pH 7.2).  microcentrifuge (5 sec, 14,000 rpm) and the supernatant discarded. The pellet was suspended in the same buffer and washed a total of three times.  The washed pellet was suspended 400 ml of 8 M urea  (pH 8.5) at room temperature for 8  h.  Insoluble material was  removed by centrifugation for 10 mill in an Eppendorf centrifuge and the supernatant was dialyzed against 10 mM HEPES (pH 7.2) at 4°C.  2.7.4  SÃO  purification.  isolation and  All procedures were carried  purified from JS1003 cultured in PYE. out at room  temperature  (20  -  22°C).  suspension and centrifugation (10,000 HEPES (pH 7.2).  SAO was isolated and  xg;  Cells  were  washed  by  10 mm) with 20 mM  The washed cell pellets were suspended in 0.77 M  NaC1 / 0.12 M EDTA (pH 7.2) [at 25 mi/lO g (wet weight) of cells (Kabir 1986)], stirred for 5 mill and then pelleted by centrifugation. The supernatant was saved and the pellet was extracted a second 24  The combined supernatants were ultracentrifuged at 225,000  time. xg.  PO 4 H 2 Na The pellet was resuspended in PBS (consisting of 1.23 g ,  O and 8.5 g NaC1 per L) by sonication and dialyzed 4 P 2 0.18 g NaH The extracts, containing approximately 5 mg  against PBS at 4°C.  protein and 450 pg KDO per ml, were mixed with an equal volume of then cooled  SDS-PAGE sample buffer and heated at 100°C for 10 mm to  room  Proteinase  temperature.  concentration of 0.5  K  was  added  to  a  mg/mi and the sample was placed at 60°C  The sample was then heated at 100°C for 10 mm  overnight.  final  and  fractionated by preparative SDS-PAGE (12 x 14 cm separating gel cast using 1.5 mm spacers).  The region of the gel containing SAO was  excised, using prestained molecular weight markers (Gibco BRL Life Technologies, Inc.) as a guide to estimate its location, placed in a dialysis membrane [12  -  14 Kda cutoff. (Spectrum)] containing SDS The SAO was  PAGE running buffer and electroeluted at 100 mA.  concentrated and washed extensively with water using an Centricon 30 microconcentrator (Amicon Canada Ltd., Oakville, Ont.).  2.8  Antisera  2.8.1  production  Production of antiserum to low pH  extracted  RsaA.  S-layer protein was extracted from NA1000 using 100 mM HEPES (pH 2) as described above. immunized quantity  with  A New Zealand white female rabbit was  this preparation,  after combining  of Freund’s incomplete adjuvant,  by  with  an equal  an initial injection  containing 1 mg protein and subsequent booster injections at days 25  Sera with the highest titer was  21, 28 and 35 with 0.3 mg protein.  collected on days 55 and 62 and was processed by standard methods to  according  Heide  and  (1978).  Schwick  Serum  activity  was  determined by the Ouchterlony double diffusion assay (Ouchterlony The antisera (c.c-RsaA) was  1949) and Western immunoblot analysis. adsorbed  against  whole  cells  JS1003,  of  and  against  Western  The pre-immune sera gave no  immunoblots of JS1003 cell lysate.  activity against RsaA by Ouchterlony or Western immunoblot assays. Unless otherwise noted the antibody was used at a concentration of 1:50,000 for use in Western blot experiments.  2.8.2  Smit  Antisera to SAO.  and  Merker  (unpublished)  produced a sera that completely labeled the cell surface of S-layer negative C. strains.  crescentus strains while not labeling S-layer producing  The epitopes on the cell surface that the antibody recognized  were unknown.  See appendix I (method B) for details on antigen  preparation.  2.9  In  vitro  crystallization  of  S-layer  Low pH and EGTA extracted NA1000 S-layer samples and 5layer isolated by urea extraction of the macroscopic debris from from JS1001 cultures were dialyzed [Spectra/Por cellulose dialysis tubing with a molecular weight cutoff of 12  -  14 KDa (Spectrum Medical  Industries, Inc., Los Angeles, CA)] overnight against 10 mM HEPES (pH 7.5 at 4°C).  Portions of the dialyzed samples were 26  examined by  and  SDS-PAGE (TEM).  transmission  negative-stain  electron  microscopy  The remainder was dialyzed overnight at 4°C against 10 mM  , 1 mM, 5 2 , 1 mM SrC1 2 HEPES (pH 7.5) containing one of: 1 mM MgC1 Each sample was then examined by negative-  . 2 mM or 10 mM CaC1 stain TEM.  methods  2.10 Electrophoretic 2.10.1  Samples were analysed by SDS-PAGE using  SD S-PAGE.  the buffer system of Laemmli (1970).  Polyacrylamide (Bio-Rad)  -  bisacrylamide (Bio-Rad) stock solutions contained 29.2 g and 0.8 g, respectively, per 100 ml.  Sample buffer consisted of 40% 0.5M Tris  HC1 (pH 6.8), 40% glycerol, 4% SDS, 4% 3 mercaptoethanol and 0.005% Samples loads were normalized by  bromophenol blue (Bio-Rad).  When C. crescentus S-layer was to be  assaying for protein or KDO. analysed  by  SDS-PAGE,  samples  were  not  heated  prior  to  electrophoresis because little or no S-layer protein will enter the gel if heated to Protein  100°C in  molecular  sample buffer (Smit and  weights  were  estimated  molecular weight protein standards. Western  blotting,  prestained  protein  using  Agabian  Bio-Rad  low  If gels were to be used for molecular  weight  standards  (BRL Life Technologies, Burlington, Ontario) were employed. were stained with: A)  1984).  Gels  0.1% Coomassie brilliant blue R-250 (Bio-Rad)  in fixative (40% methanol [Fisher], 10% glacial acetic acid [Fisher]) then destained in fixative or B) below. 27  one of the silver stains outlined  2.10.2  were  carbohydrates  Following SDS-PAGE proteins or  Blotting.  Western  transferred  to  nitrocellulose  membranes  (Schleicher and Scheull, Inc., Keene, N.H.) by the method of Burnette After blotting, membranes were processed as described by  (1981).  All primary antibody was used at a  Smit and Agabian (1984).  Goat x  dilution of 1:50,000 for cc-RsaA and 1:20,000 for x-SAO.  rabbit antibody coupled to horseradish peroxidase (Antibodies Inc., Davis, CA) secondary antibody was used at a dilution of 1:2000. blots  were  developed  using  4-chloro-l-naphthol  as  described  The by  Smit and Agabian (1982b).  2.10.3  600 Five ml of PYE grown cells (0D  Sample preparation.  600 Higg/M grown cells (0D 3 M Higg 0.6-0.7) or 1 ml of 10  =  =  2.0-3.0)  were pelleted and washed with 10 mM HEPES (pH 7.2) by suspension The pellet was suspended in 250 jil of 10 mM  and centrifugation.  Tris-HC1 / 1 mM EDTA (pH 7.2), frozen at -20°C and then thawed at room  temperature.  concentration.  A  sample was removed  to estimate protein  To the remainder of the sample 1 pA of proteinase  free bovine pancreatic DNase (0.5 mg/ml), 20 j.tl of lysozyme (10 2 were added and the sample mg/ml) (ICN), and 3 p.1 of 1 M MgC1 lysate was incubated at room temperature for 15 mm.  If lysate was  to be used for detection of LPS, cell lysate containing 1 p.g of KDO was suspended in 20 p.1 of SDS-PAGE sample buffer, heated at 100°C for 10  mm,  cooled  to room  temperature, 28  made  to  0.5  mg/mi with  Proteinase K and incubated at 60°C for 1 h.  This method is referred  to as the modification of the sample preparation method of Hitchcock and Brown (1983) for the qualitative analysis of Caulobacter LPS. Cells were prepared for analysis of LPS by the method of Hitchcock and Brown (1983) as follows. culture  600 (0D  =  The cells from 5 ml of  0.6-0.7) were pelleted and washed with 10 mM  HEPES (pH 7.2) by suspension and centrifugation then resuspended to a concentration of 200 Klett units (blue filter, Klett-Summerson colourimeter) in the same buffer. pelleted  and  the pellet  One and one half ml was then  was resuspended  in  50  pl of a sample  dissociation solution containing 2% SDS, 1 M Tris-HC1 (pH 6.8), 4% mercaptoethanol, 10% glycerol, and 0.005% bromophenol blue.  13  The  sample was heated at 100°C for 10 mm, cooled to room temperature and 10 p.1 of the above sample dissociation solution containing 2.5 mg/ml of proteinase K was added.  The sample was then incubated at  60°C for 1 h.  2. 1 1 Silver  staining  Following electrophoresis gels were stained using the Bio-Rad silver stain kit (Merril et al. 1981), a modification of the BioRadTM silver stain kit (Cava et al. 1989), the method of Tsai and Frasch (1982) or a modification of the method of Tsai and Frasch. The Bio-Rad silver stain kit was used as directed by the manufacturer.  In the modification of the Bio-Rad silver stain kit  the oxidizer solution was replaced with 0.7% sodium metaperiodate (BDH) dissolved in 0.65% isopropanol and 0.26% glacial acetic acid. 29  Briefly, the method of Tsai and Frasch (1982) involves an overnight fixation of the gel in 40% ethanol-5% acetic acid followed by a 5 mm oxidation with 0.7% periodic acid in 40% ethanol-5% acetic acid. After washing with water the gel is stained for  10 mm  ammonium-silver reagent then washed extensively with water.  in an The  gel was developed at 25°C with a citric acid-formaldehyde solution. This development temperature inhibits the visualization of proteins (Hitchcock and Brown 1983).  The Tsai and Frasch (1982) procedure  was modified in that the gels were fixed for only 1 h with two changes of the fixation solution, the periodic acid oxidation step was extended from 5 to 15 mm, and the staining step was extended to 20 mm.  2.12 Cell  preparation  for  thin-section  electron  microscopy  Treated and control cells were pelleted in a microcentrifuge tube and resuspended in Burdett’s buffer (Burdett and Murray 1974)  [5% acrolein, 0.25% glutaraldehyde (J. B. E. M. Services Inc., Point Claire  -  Durval, PQ) in 50 mM cacodylate (Electron Microscopy  Sciences, Fort Washington, PA) buffer (pH 7.4)] and incubated at room temperature for 1 h then at 4°C overnight.  Cells were then  pelleted, washed twice with 50 mM cacodylate buffer (pH 7.4) by centrifugation and resuspension, and the washed pellet was then resuspended in 0.8% tannic acid (Mallinckrodt, Inc., Paris, KY) (in 50 mM cacodylate buffer [pH 7.4]) and incubated at room temperature for 30 mm.  The cells were again washed twice in 50 mM cacodylate 30  buffer (pH 7.4) and enrobed in 2% nobel agar (in 50 mM cacodylate 4 [J. B. E. M.] and 0.5 Blocks were post fixed (1% 0s0  buffer [pH 7.41).  mg/ml ruthenium red in 50 mM cacodylate buffer [pH 7.4]) for lh at 4°C, washed three times with 50 mM cacodylate buffer (pH 7.4), twice with water and en bloc stained in saturated aqueous uranyl Blocks were then washed twice with water  acetate (Fisher) for lh.  and dehydrated in an ethanol series, infiltrated with Spurr’s (3. B .E. M.) resin/ethanol [(1:1) for 30 mm], Spurr’s resin [100% for 30 mm two  times]  and  finally  embedded  Specimens  acetate and  resin  and  heat  Thin sections were cut then stained  polymerized at 65°C for 24h. with uranyl  Spurr’s  in  Reynolds’  lead citrate (Reynolds  were viewed in a Siemens  lOlA  1963).  electron microscope  operating at 80 kV.  2.13 Negative  stain  electron  microscopy  To examine colonies for the presence of S-layer, a colony was suspended in 10 p.1 of water containing 1 p.1 of bacitracin (1 mg/ml) and a carbon-stabilized, parlodion (3. B. E. M.)-coated 400 mesh copper grid was floated on top of the drop for a few mm.  To examine  samples for in vitro reassembly of S-layer a grid was placed on a droplet containing RsaA.  Grids were then lifted and excess liquid  removed by wicking with filter paper.  After drying, the sample was ammonium  molybdate  (Mallinckrodt) (pH 7.5) or 2% methylamine tungstate.  Specimens  negatively-stained  using  2%  aqueous  were examined in a Siemens lOlA transmission electron microscope 31  operated at 60 kV.  2.14 Transposon A  Tn5  mutagenesis  library  was  of  NA1000  constructed  by  the  electroporation  of  pSUP2O21 (Simon et al. 1983) into electrocompetent NA1000 cells. Transposition events were selected by plating on PYE supplemented with Km and Sm.  pSUP2O21 was isolated from E. coli S17-1 by a  mini-alkaline plasmid preparation procedure (Sambrook et al. 1989). Electrocompetent NA1000 were prepared as described by Gilchrist and Smit (1991).  A BioRadTM Gene Pulser, Pulse Controller, and  cuvettes with 0.2-cm interelectrodal gaps was used as described by the manufacturer.  The Gene Pulser was set at 2.5 kV and 25 .tF.  Pulse Controller was set at 400 2.  Twenty-thousand  The  Km/Sm  resistant colonies were pooled to form the library.  2.15 Complementation  an  NA1000  cosmid  of  JS1004  for  SÃO  production  with  library  A NA1000 cosmid library, using pLAF5 (Keen et al. 1988) as a vector, was supplied by Dr. L. Shapiro (Stanford University) and was electroporated into electrocompetent JS1004.  Nine hundred and fifty  Tcr colonies were inoculated into 96 well microtiter plates containing PYE-Tc using sterile tooth picks and grown for 40 h at 30°C.  Two j.tl  of each culture, including JS1003 as a positive control and JS1004 as a negative control, was placed on a nitrocellulose sheet and allowed to dry for lh.  Sterile DMSO was added to each microtiter well to a  32  final concentration of 5% and the plates were frozen at -70°C.  The  dry nitrocellulose was processed in the same manner as a Western One of the 950  blot using cz-SAO sera as the primary antibody.  The cosmid  electroporants reacted positively in the antibody screen.  DNA was obtained from this clone by an alkaline lysis  method  (Sambrook et a!. 1989) and the cosmid DNA was electroporated into The cosmid DNA  E. coli DH5x (BRL Laboratories, Gaithersburg, MD).  was isolated by an alkaline lysis method, digested with B amHI and electrophoresed on a 0.7 % agarose (Bio Rad) gel using a Tris-acetate The DNA fragments  EDTA buffer system (Sambrook et al. 1989).  running lower than the top band (pLAF5 plus some Caulobacter DNA) were isolated using a GENECLEAN Il® Kit (Bio 101 Inc., La Jolla, CA). a!.  P labeled by nick translation (Rigby et The isolated DNA was 32 1977)  and  used  to  screen  a cosmid  library  by  colony-blot  hybridization to identify overlapping cosmids (Maniatis et al. 1983).  2.16 Carbohydrate Detailed carbohydrates  and  chemical and  lipid analysis  lipids  Ravenscroft et al. (1991;  were 1992).  chemical on  the  carried  analysis isolated out  as  and  purified  described  by  The rough LPS and EPS was  analysed by Dr. N. Ravenscroft and the SAO was analysed by Dr. D. N. Karunaratne.  33  3  Results  3.1  Analysis  Western  the  S-layer  attachment  phenotype  by  blotting  Smit  coworkers  and  negative-stain mutants of C. plates,  of  electron  (manuscript  microscopy  submitted)  that  showed  by  calcium-independent  crescentus, when grown on calcium-containing PYE  produced  sheets  of  assembled  S-layer  that  were  not  associated with the bacterial cells.  The S-layer gene from the  calcium-independent mutant JS1001  was cloned and expressed in  the spontaneous S-layer minus C.  crescentus CB2A and a wild-type,  cell-associated, S-layer resulted.  Thus although CB2A is S-layer  negative it is S-layer “attachment competent”.  When the cloned S  layer gene from the wild-type strain NA1000 was introduced into the calcium-independent,  S-layer negative,  associated S-layer sheets were produced.  strain JS1004 non-cell  These electron microscopy  studies determined that the defect in calcium-independent mutants, resulting in the inability to attach the S-layer to the cell surface, was not due to a defect in the S-layer protein but in some other cellular locus.  Thus  calcium-independent  “attachment-defective”  phenotype  mutants as  exhibited  well  as  an  the  S-layer calcium  independent phenotype for which the mutants were selected. Western blotting experiments were conducted to corroborate the electron microscopy observations of Smit and coworkers.  S-layer  attachment-defective mutants could be distinguished from wild-type 34  Western blot analysis of whole cell lysates reacted with FIG. 1. unadsorbed anti-RsaA antisera. “L” indicates cells were grown in a liquid culture and washed prior to analysis. “P” indicates cells were grown on plates and were not washed prior to analysis. Lanes: 1, prestained molecular mass markers; 2, NA1000 (L); 3, JS1003 (P); 4, JS1003/pKT23O-A19 (L); 5, JS1003/pKT23O-Ca5 (L); 6, JS1002 (L); 7, JSlOOl (P); 8, JS1004 (P); 9, JS1002 (P); 10, JS1005 (P); 11, NA1000 (L); 12, JS1003/pKT23O-CB2AD (L); 13, JS1005/pKT23OA19 (L); 14, JS1005/pKT23O-A19 (P); 15, prestained molecular mass 16, CB2A (P); 17, CB2A/pKT23O-A19 (L); 18, markers; CB2A/pKT23O-CalO 19, CB2A/pKT23O-Ca5 (L); 20, (L); CB2A/pKT23O-CB2AD (L); 21, JS1004/pKT23O-CalO (P); 22, JS1004/pKT23O-CalO (L); 23, prestained molecular mass markers; 24, CB2NY66R (L); 25, CB2NY66Rmg1 (L); 26, CB2NY66Rmg1 (P). Only the region of the gel containing S-layer is shown. The prestained molecular mass markers are 97.4 and 200 kDa. Lanes were loaded with samples containing 5 tg of protein as estimated by the method of Markwell et al. (1978). Samples were fractionated on a resolving gel containing 10% acrylamide prior to blotting onto nitrocellulose. Unless otherwise noted this and all subsequent polyacrylamide gels utilized a stacking gel containing 4 % acrylamide. The discontinuous buffer and sample dissociation method of Laemmli (1970) was used in this and all subsequent polyacrylamide gels as described in the Materials and Methods.  35  11 12  13  21  14  22  15 16  17 18  23 24 25  36  26  19  20  strains  on  the  electrophoresis.  basis  of  how  the  cells  were  prepared  for  S-layer protein could be detected in colonies of  calcium-independent  mutants  only  when  the  mutant  cells  were  scraped directly from plate cultures (Fig. 1; lanes 7, 9, 14, 21 and 26) whereas  washed  liquid-grown  calcium-independent  cells  almost negative for RsaA (Fig 1; lanes 6, 13, 22 and 25).  were  Western  blots of plate-grown strains in which the chromosomal S-layer gene had  been  deleted  which  or  were  spontaneous  S-layer  negative  mutants were RsaA negative (Fig. 1; lanes 3 and 16).  Washed liquid  medium-grown  negative  but  S-layer blot when  any  cells  of  strains  that  were  attachment-competent produced a positive cloned  rsaA  gene,  including  those  S-layer  from  attachment-defective  strains, was expressed in the cell on a plasmid vector (Fig. 1; lanes 4, 5, 12, 17, 18,19 and 20).  When rsaA, cloned from an attachment-  competent  expressed  strain,  was  in  an  attachment-defective  background the attachment-defective phenotype persisted in that S layer protein was detected only when the cells were harvested from plate cultures (Fig 1; lane 14).  If the same strain is cultured in liquid  medium RsaA is not detected (Fig. corroborated coworkers  the  electron  (manuscript  1;  microscopy  submitted)  lane  13).  observations  which  These results of  demonstrate  Smit  and  that  the  defect in “calcium-independent” mutants, resulting in the inability to attach the S-layer to the cell surface, was not due to a defect in the S-layer protein but in some other cellular locus.  37  3.2  Isolation  and  JS1001  and  purification  of EPS  from  CB2A,  NA1000  An EPS was identified on the surface of wild-type and calciumindependent type  strains  Caulobacter strains. was  isolated  and  The EPS from mutant and wildcharacterized  to  determine  if an  alteration in the EPS was responsible for the S-layer attachmentdefective phenotype. Fractionation, by steric-exciusion chromatography, of the crude EPS isolated from NA 1000 and CB2A yielded similar profiles for both strains (Fig. 2).  JS1001, a calcium-independent mutant of NA1000,  produced the same fractionation profile as NA 1000 Carbohydrate contained  a  analysis  showed  that  heteropolysaccharide  contained only ribose.  the  whereas  (not shown).  void  volume  peak  (A)  the  second  peak  (C)  Additionally, the second peak (C) contained  significant amounts of phosphate and had a maximum absorbance at 260 nm so the peak was attributed to undegraded RNA.  The CB2A  crude EPS also contained an additional minor peak (B) between the heteropolysaccharide (A) and the RNA (C) peaks.  On basis of  exclusion limits reported for Sephacryl S-400 the appearance of the EPS peaks in the void volume indicates a minimum molecular mass of 1-2 million daltons.  3.2.1  Assessing  the  degree  of  EPS  cell  association.  Bacterial cell surface carbohydrates are qualitatively categorized as true capsules I BPS or as slime layers based on their ability to  38  maintain cell association.  True capsules maintain cell association  during growth or when the cells are subjected to mild sheer forces whereas slime layers are easily detached from the cell (Boulnois and Roberts  1990). In one trial of the EPS isolation procedure NA1000,  JS1001 and CB2A cells were washed five times by centrifugation with 0.1 M HEPES buffer (pH 7.2) before extracting the EPS to determine if the EPS could be readily washed from the cells.  The  yield of EPS from this experiment was not significantly different than that of cells which had been washed only once or not at all (not shown).  The culture supernatants from 500 ml batch cultures of  CB2A, JS1001 and NA1000 were freeze-dried, dialyzed against water, freeze-dried  again  chromatography.  and  analyzed  The analysis  for  carbohydrate  by  gas  did not produce a sugar profile  similar to that produced by purified EPS  (see below).  therefore concluded that the EPS produced by the 3  It was  strains was  significantly adherent to the cell.  3.2.2  Chemical  NA1000  and  characterization  JS1001.  of  EPS  from  The isolated EPS was found to be in a  purified state suitable for detailed structural analysis (see II, Fig. 2.). wild-type  CB2A,  appendix  Dr. Neil Ravenscroft concluded that the EPS isolated from  C.  crescentus  NA 1000  mutant JS1001 were identical.  and  the  calcium-independent  The EPS produced by C.  CB2A differed from that of NA1000.  39  crescentus  0.3  A C  o a’  02  .  o  0.1-  C  a’  .  C 0  z  B  0.0  •  0  FIG. 2.  5  Blue dextran  •  10  traction  20  No.  Fractionation of EPS of CB2A  Sephacryl S-400.  0.0 J’25 Glucose  I  15  ()  and NA 1000 (+) on  Carbohydrate monitored by the phenol-sulfuric  acid assay as described in Materials and Methods (optical density at 490 nm).  Peak A contains CB2A and NA1000 EPS fraction.  contains a minor polysaccharide found only in CB2A. RNA.  Each fraction consisted of 2.5 ml.  40  Peak B  Peak C contains  3.3  Isolation  and  JS1001  and  purification  of LPS  from  CB2A,  NA1000  The LPS from mutant and wild-type Caulobacter strains was characterized and isolated to determine if an alteration in the LPS was responsible for the S-layer attachment-defective phenotype. 3.3.1  Electrophoretic  analysis  of  LPS.  SDS-PAGE of the  purified LPS and subsequent staining, by the method of Tsai and Frasch (1982) or using the Bio-Rad silver stain kit, revealed that the  C. crescentus strains produced a rough LPS (Fig. 3).  Bands of similar  mobility were detected in purified LPS preparations (Fig 3, lanes 3 6)  -  No high molecular weight morphological heterogeneous forms,  typical of smooth LPS, were noted.  LPS profiles of these strains  prepared by the method of Hitchcock and Brown (1983) also showed no high molecular weight bands (Fig. 3, lanes 1 and 2) indicating that the isolation method did not select against recovery of smooth LPS species.  The heterogeneity of the LPS bands is typical of the  microheterogeneity found in “rough” LPS species when examined by SDS-PAGE or TLC (Nowotny 1984).  The samples prepared by the  Hitchcock and Brown method (Fig. 3; lanes 1 and 2) produced bands that were broader in the horizontal plane and more condensed in the vertical plane than the bands produced by the purified samples. This is most likely a result of the Hitchcock and Brown samples containing  bulk cellular components,  such as undigested protein,  peptidoglycan and nucleic acids which slightly alter the mobility of the LPS through the polyacrylamide gel. The electrophoretic profile of a number of C. crescentus  41  1  FIG. 3.  2  3  4  5  6  SDS-PAGE of LPS prepared by the methods of Hitchcock and  Brown (1983) [lanes 3 to 61.  [lanes 1 and 2]  and Darveau and Hancock (1983)  Gels were stained by the method of Tsai and Frasch  (1982) [lanes 1 to 41 or by using the Bio-Rad silver stain kit [lanes 5 and 6].  Odd-numbered lanes contained LPS from CB2A; even  numbered lanes contained LPS from NA1000.  All lanes were loaded  with samples containing 1 p.g of KDO as estimated by the method of Karknanis et al. (1978).  The resolving gel contained 14% acrylamide.  42  strains,  including  the  calcium-independent  strains  JS1001  and  JS1002, were examined by the method of Hitchcock and Brown (1983) (Fig. 4). gel.  The LPS from all strains ran at the dye front of the  Purified LPS from JS1001  and JS1002 produced the same  banding pattern (gel not shown).  3.3.2  Isolation  of LPS,  monitored  by  lipid  analysis.  The  cold ethanol extraction procedure of Darveau and Hancock (1983) yielded the crude LPS fraction.  Analysis of the fatty acids of the  crude LPS showed the presence of saturated and mono-unsaturated 16- and 18-carbon fatty acids together with 3-OH-dodecanoic acid indicating contamination by phospholipids (Fig. 19821).  5A;  [Lelts et al.  Extraction of the “crude” LPS by PCH and chloroform-  methanol yielded a “pure” LPS product that is largely free of these C16 and C18 fatty acids (Fig. 5B). contaminates  from  the  PCH  and  Lipid analysis of the soluble chloroform-methanol  extraction  steps revealed negligible amounts of 3-OH-dodecanoic acid indicating that little LPS was lost during these purification steps (see below). The material extracted from the final “pure” LPS was also examined by SDS-PAGE and stained with the Bio Rad’ silver stain kit and Coomassie blue.  The gels showed that residual protein but not  carbohydrate was extracted.  Coomassie blue stained gels showed  that a pronase-resistant protein of approximately 31 kDa was the major protein contaminant in the “crude” LPS (data not shown).  The  final freeze dried “purified” LPS was approximately 40% (by weight) 43  1 FIG. 4.  234  SDS—PAGB of LPS prepared by the method of Hitchcock and  Brown (1983) and stained by the method of Tsai and Frasch (1982). Lanes: 1, CB2A; 2, CB2NY66R; 3, NA1000; 4, JS1003; 5, JS1002; 6, JS1005; 7, JS1001; 8, JS1004; 9, CB2NY66Rmg1.  All lanes were  loaded with samples containing 0.5 .tg of KDO as estimated by the method of Karknanis et a!. (1978). acrylamide.  44  The resolving gel contained 12.5%  lighter that the “crude” LPS  indicating  that  the two  procedures removed substantial amounts of impurities.  extraction The PCH  extraction resulted in a 10% decrease in the dry weight of the “crude” LPS whereas the chloroform-methanol wash decreased the weight by 30%.  3.3.3  Colourimetric  analysis.  for protein, phosphate and KDO.  The “pure” LPS was analyzed No protein was detected and  phosphate and KDO were found to account for 0.5% and 12% of the LPS dry weight, respectively.  The molar ratio of phosphate to KDO,  determined colourimetrically, was approximately 1:3.  When the LPS  was analysed for KDO under more severe hydrolysis conditions, than that recommended by Karkhanis et al. (1978), no increase in the amount of KDO per dry weight LPS was noted indicating that the KDO was likely not phosphorylated (Caroff et al. 1987). The total amount of thiobarbiturate-positive material (KDO) contained in freeze dried cells of CB2A, NA1000 and E. coli B was also examined.  For these studies identical weights of the three cell  types were simultaneously analyzed along with KDO standards.  The  Caulobacter strains were found to contain less KDO per dry weight than E. coli B; the ratio being E. coli B:CB2A:NA1000  3.3.4  Detailed  chemical  analysis.  =  1:0.83:0.68.  The purified LPS was  found suitable for detailed chemical analysis.  Dr. Neil Ravenscroft  concluded  from  that  LPS  isolated  and  purified  wild-type  crescentus strains CB2A and NA1000 (Ravenscroft et al. 1992) and 45  C.  A  HLL B  S  FIG. 5.  A GC trace of fatty acid methyl esters from the LPS of  NA1000  prepared  and  Materials and Methods. extractions. sample  chromatographed  as  described  in  the  (A) Crude LPS preparation prior to organic  (B) Pure LPS resulting from extraction of the crude with  phenol/chloroform/hexane  followed  chloroform/methanol as described in the Materials and Methods. methyl ester of octadocanoic acid as an internal standard.  by S  =  The crude  and purified LPS was prepared for GC and the major peak remaining in  the  purified  LPS  was  identified  as  determined by GC-MS) by Dr. Neil Ravenscroft. 46  3-OH-dodeconate  (as  the calcium-independent mutant JS1001  (Ravenscroft, unpublished)  were structurally and chemically identical (see appendix II, Table I and II).  3.4  Identification  of  an  S-layer  associated  oligosaccharide  During one experiment to isolate LPS from NA1000, by the Darveau  and  Hancock  procedure  (1993),  an  additional  polysaccharide-containing molecule that migrated more slowly than LPS on SDS-PAGE was detected (Fig. 6).  This molecule (which was  subsequently referred to as the S-layer associated oligosaccharide or SÃO) did not stain with the Tsai and Frasch silver stain procedure for LPS but was reliably stained using the more general Bio-Rad silver stain kit (Fig. 6; lane 1).  [Note:  It will be determined that the SAO is  a smooth LPS species.]  When this LPS preparation was examined by  Western  using  blot  analysis,  anti-SÃO  sera  (see  Materials  and  Methods), the SÃO band was specifically labeled (Fig. 6; lane 2).  No  reactivity was seen with the rough LPS.  The various steps in the  Darveau and Hancock procedure were monitored by SDS-PAGE with silver-staining and immunoblotting to determine where the SÃO was lost.  It was found that the SÃO and significant amounts of rough LPS  remained  in  the  supernatant  following  the  cold  2 ethanol-Mg  +  precipitation procedure (data not shown). The rapid LPS analysis method of Hitchcock and Brown did not reveal the SÃO along with the rough LPS  (Fig.  3 and 4).  modification of both the Hitchcock and Brown sample preparation 47  A  FIG. 6.  Electrophoretic analysis of purified NA1000 LPS containing  contaminating SAO.  Lane 1: SDS-PAGE of LPS stained using the Bio  Rad silver stain kit.  Lane 2: Western blot of LPS, fractionated by  SDS-PAGE, reacted with cc-SAO sera and visualized as described in the Materials and Methods.  LPS was prepared by the method of  Darveau and Hancock (1983) and fractionated using a resolving gel containing 13% acrylamide.  Both lanes were loaded with samples  containing 0.5 jig of KDO as estimated by the method of Karknanis et a!. (1978).  The arrow indicates the region of the gel containing SAO.  48  procedure and the Tsai and Frasch staining process was developed in order to visualize  SAO  in polyacrylamide gels.  This  modified  procedure was used to examine a number of Caulobacter strains (Fig.  7A).  Wild-type  S-layer  producing  strains,  NA1000  and  CB2NY66R, and strains that produce a wild-type S-layer when rsaA is  expressed  on  a plasmid  additional polysaccharide comparable  band  was  (CB2A  (Fig. not  7A;  and JS1003), lanes  detected  in  1, the  4,  contained  this  and 6).  A  5,  S-layer  attachment-  defective strains JS1001 and JS1002 (Fig. 7A; lanes 2 and 3). other  calcium-independent strains  isolated  by  Smit  Eight  (unpublished)  were also examined by these methods and none contained the SAO band (data not shown). revealed competent  the  C.  SÃO  in  The modified procedure of Tsai and Frasch whole  cell  lysates  of  S-layer attachment-  crescentus strains (Fig. 7A, lanes 1, 4  -  6) although  increased sample loadings were required to that which is normally used to detect LPS (note the overloading of the rough LPS in Fig. 7A). Tsai and Frasch (1982) recommends loading 1 to 5 .tg of LPS per lane to obtain a satisfactory LPS profile.  Western blotting of these  preparations, using anti-SÃO sera, showed that the SÃO of all strains producing the polysaccharide cross reacted with the antibody which was raised against CB2A cell membranes (Fig. 7B).  JSlOOl and  JS1002 showed only a faint immunoreactive band in the SÃO region. These experiments were repeated using cells directly removed from plates and identical results were obtained (data not shown). confirmed that the SÃO was not sloughed off the cell surface of  49  This  FIG. 7. A)  SDS-PAGE of whole cell lysates treated with proteinase K  as described in the Materials and Methods, using a modification of the method of Hitchcock and Brown (1983), and fractionated using a resolving gel containing 13% acrylamide.  The gel was stained using a  modification of the method of Tsai and Frasch (1982) as described in the Materials and Methods.  B)  Western blot of samples shown in  “A” reacted with c-SAO sera and visualized as described in Materials and Methods.  Lanes in “A” and “B  “:  1, NA1000; 2, JS1001; 3,  JS1002; 4, JS1003; 5, CB2A; 6, CB2NY66R.  All lanes in “A” and “B”  were loaded with samples containing 0.75 j.tg KDO as estimated by the method of Karknanis et a!. (1978). indicates the region containing SAO.  50  The arrow in “A” and “B”  It)  calcium-independent  mutants.  Isolation and purification of SÃO  3.5 3.5.1  Extraction  of cell  surface  killed by the concentration of Na saline (PBS) (Poindexter 1964).  molecules. C. found  in  crescentus is  phosphate-buffered  When cells incubated in PBS were  examined by negative-stain electron microscopy small vesicles were observed to be extracted from the cell surface.  The isolated vesicles  were shown to contain SAO when examined by Western blotting using anti-SÃO sera and the amount of material extracted from the cells increased if the PBS was supplemented with 10 mM EDTA (data not  shown).  Multiple  extractions  were  required  to  completely  remove all of this material from the cells and resulted in an extract that contained a low concentration of SÃO.  When 0.77 M NaC1 / 0.12  M EDTA (pH 7.2) [25 ml/10 g (wet weight)] was used, in place of PBS / 10 mM EDTA, 95% of the cellular KDO was solubilized with two extractions.  3.5.2  Therefore this method was used to isolate the SÃO.  Examination  ultracentrifugation  and  of  extract.  The extract was subjected to  the  pelleted  material  from  NÃ1000  was  shown to contain a protein/KDO at a ratio of 19.2 while pellets from JS1003 extract had ratio of 10.7.  Proteinase K treatment of the  pelleted extract followed by SDS-PÃGE with silver-staining using the Bio Rad kit and Western blotting using anti-SÃO sera revealed that the pelleted extract contained rough LPS and SAO (Fig. 8). 52  Coomassie  97,400 4  66,200  45,000  -4  31,000  21,500 -4  1  FIG. 8.  2  14,400  3  Electrophoretic analysis of proteinase K-treated NaCl/EDTA  extract of NA1000.  Lanes: 1, Western blot, of the polyacrylamide gel  shown in lane 2, reacted with x-SAO sera and visualized as described in the Materials and Methods. Rad’ silver stain kit. 1 and 2  2, SDS-PAGE stained using the Bio  3, Molecular mass standards in daltons.  were loaded with  samples containing 0.5  estimated by the method of Karknanis et al. (1978).  pg KDO  SAO is the  band that runs adjacent to the 45,000 molecular mass marker.  53  as  Samples were  fractionated on a resolving gel containing 12% acrylamide.  rough LPS runs at the dye front.  Lanes  The  —  4  97,400  4  66,200  4  45,000  4  31,000  4  21,500 14,400  1  FIG. 9.  2  3  Electrophoretic analysis of NaC1/EDTA extract of NA1000.  Lanes: 1, Western blot, of the polyacrylamide gel shown in lane 2, reacted  with  cc -RsaA  Materials and Methods. brilliant blue R-250. 1  and 2 were  sera 2,  and  visualized  SDS-PAGE  as  stained  described using  in  Coomassie  3, Molecular mass standards in daltons.  loaded  with  samples  containing  3  the  and  Lanes 10  t  g,  respectively, as estimated by the method of Markwell et al. (1978). Samples  were  fractionated  on  acrylamide. 54  a  resolving  gel  containing  12%  blue staining of proteinase K treated extracts detected the minor band running above the SAO in lane 2 of Fig. 8 (data not shown). This band most likely represented the 66 kDa protein found in the non-proteinase  K  treated  extracts  (see  below).  The  proteins  contained in the pelleted extracts were examined by SDS-PAGE with Coomassie blue staining and Western blotting using anti-RsaA sera (Fig. 9).  Many bands were detected with the major protein bands  being approximately 20 kDa, 66 kDa and 105 kDa (RsaA).  The 105  kDa protein reacted with -RsaA sera confirming that it was the S layer protein. The supernatant following ultracentrifugation of the NA1000 extract was found to contain a high concentration of carbohydrate. Chemical analysis of this purified carbohydrate indicated that it was the EPS (Ravenscroft et al. unpublished).  Figure 10 shows that the  BPS was not contaminated with RNA as was the case when the EPS was isolated by the method of Darveau and Hancock (1983) before the SEC chromatographic step.  3.5.3 cells  Examination were  prepared  of extracted for  and  microscopy (Fig. 1 1A and B).  cells.  examined  by  Extracted and control thin-section  electron  The extracted cells remained intact, no  breeches in the peptidoglycan layer were noted,  and the bilayer  appearance of the outer and cytoplasmic membrane was maintained. Two major ultrastructural changes were noted.  The electron-dense  material between the peptidoglycan and the outer membrane was extracted from treated cells (Fig. 1 1A and B; arrow in inset) and  55  A  s R  F  B  FIG. 10. using:  GC trace of alditol acetates from EPS of NA1000 isolated A.  The method of Darveau and Hancock (1983).  NaC1/EDTA extraction.  R  =  ribose; F  =  fucose; S  =  B.  inositol standard.  The same amount of inositol standard was included in both samples. The EPS was prepared for GC by Dr. Neil Ravenscroft.  56  FIG. 11.  Thin-section TEM micrograph of: A.  NaC1/EDTA extracted NA1000 cell.  Control NA1000 cell. B.  Note the even distribution of  chromosomal material and ribosomes in control cell (A) and the rearrangement of cytoplasmic constituents into two distinct regions containing ribosomes or chromosomal material (B).  Arrow in (B)  shows  ribosome-free  region  containing  chromosomal  material.  Arrow  in  figures  illustrates  electron-dense  material  between membrane  the  inset  peptidoglycan  layer  and  inner  leaflet  of  (A) which is absent in extracted cells (B).  the  outer  All of 100  longitudinally sectioned control and extracted cells examined showed the features demonstrated in this figure. taken by Mr. S. H. Smith.  57  Bar  =  0.5 .im.  Micrograph  A.  T  B.  58  there  was  redistribution  a  the  of  nuclear  material  within  the  cytoplasm of these cells (Fig. 11B; arrow in main figure).  This  redistribution has been proposed by Whitfield and Murray (1956) to be  due  to  of  loss  permeability  barrier  the to  plasma  membrane  monovalent  losing  cations.  A  its total  selective of  100  longitudinally sectioned control and extracted cells were examined and  all  of the  extracted cells  contained  the  two  ultrastructural  changes.  3.5.4  Purification of the SAO.  Proteinase K treated extracts  from JS1003 were subjected to SDS-PAGE and the region of the gel containing SAO was removed. acrylamide  by  The SAO was extracted from the  electroelution  AmiconTM filtration.  then  concentrated  and  washed  by  A portion of the SAO was subjected to SDS-PAGE  to determine its purity.  Figure  12  shows  contamination by protein and rough LPS.  that it was free of  It was noted that although  both the SAO and rough LPS were stained by the Bio-Rad silverstain kit this method was approximately five-fold less sensitive for the detection of rough Caulobacter LPS in comparison to the method of Tsai and Frasch (1982) (Gel not shown). Colourimetric  analysis  indicated  that  the  contained, per mg dry wt; 25 j.ig KDO, 454 pg  purified  SAO  phenol-sulphuric  positive carbohydrate, 1 pg phosphate and 4.7 .tg of uronic acid.  On a  molar basis the KDO:Pi ratio was 3:1; the same ratio as that found in the rough LPS.  Detailed chemical analysis of the SAO, preformed by  Dr. D. N. Karunaratne, indicated the only major lipid present was the 59  -  3  2  FIG.  12.  extracts  SDS-PAGE of carbohydrates, purified from NaC1/EDTA from  JS1003,  using  a  resolving  gel  containing  acrylamide and stained using the Bio-Rad silver stain kit.  Lanes:  12% 1,  purified rough LPS; 2, protein molecular mass standard; 3, purified SÃO.  Lanes 1 and 3 were loaded with samples containing 1.0 pg and  0.1 .tg of KDO, respectively, as estimated by the method of Karknanis et al. (1978).  Molecular mass standards are, from top, 66.2, 45, 31,  21.5, and 14.4 kDa.  Note:  The 31 kDa marker is poorly stained.  60  esterified  fatty  acid  3 -OH-dodecanoate.  Carbohydrate  analysis  indicated three major sugars and a number of minor sugars (see appendix II, Table III).  The minor sugars were mannose, glucose,  galactose and heptose which have been detected in the rough LPS. The major sugars were tentatively identified as 4,6-dideoxy-4-amino hexose, 3,6-dideoxy-3-amino hexose and glycerol.  3. 6  Purification of C.  crescentus  S-layer protein  It was of interest to determine a rapid and effective method to extract and isolate a relatively pure preparation of RsaA from whole cells.  With  purified  RsaA  it  will  enable  both  the  in  vitro  crystallization experiments and the production of polyclonal antisera against RsaA.  3.6.1  Extraction of the S-layer of C.  crescentus  NA1000.  Of the agents tested, 100 mM HEPES at pH 2 was the most effective at extracting RsaA with the least contamination from other proteins. By Coomassie blue staining the preparations appeared to contain nearly pure RsaA in this single step purification procedure (Fig. 13). The smearing of RsaA above the major band in the immunoblot shown in Figure 13 is commonly seen in acrylamide gels stained with Coomassie blue if sufficient RsaA protein is loaded on the lane.  This  is assumed to be a consequence of RsaA aggregating or polymerizing before or during electrophoresis (Smit and Agabian 1984).  RsaA also  stained poorly in polyacrylamide gels by Coomassie blue staining methods presumably due to the low content of basic amino acids in 61  200,000 97,400 -4-  68,000  43,000 29,000 1  FIG. 13.  Electrophoretic analysis of proteins extracted from whole  cells of NA1000 using 0.1 M HEPES (pH  =  2.0).  Lanes:  1, SDS-PAGE  using a resolving gel containing 12% acrylamide and stained with Coomassie brilliant blue R; reacted  with  x -RsaA  Materials and Methods. and 2  sera  2, Western blot of gel shown in lane 1 and  visualized  as  described  3, Molecular mass markers in kDa.  were loaded with samples containing  estimated by the BioRadTM protein assay.  62  3  in  the  Lanes 1  jig of protein as  the protein (Wilson 1983).  Lysine, histidine and arginine account for  only 2.3% of the total of amino acids in RsaA (Gilchrist et al. 1992). Occasionally, a minor amount of protein migrating with a faster electrophoretic mobility than RsaA was noted in low pH extracts. EGTA  treatment  preparations  also  efficiently  removed  were more contaminated  Other methods were less effective:  with  RsaA  although  other protein  the  species.  HEPES at pH 4 extracted RsaA as  well as a number of other proteins while HEPES at pH 6, 7.5, 8, and 10 did not extract RsaA.  Glycine-HC1 at pH 2 yielded RsaA as a  prominent protein but significant amounts of lower molecular weight proteins were also present. showed  further  increases  Glycine-HC1 treatment at pH 3 and 4 of  other  proteins.  Similarly,  65°C  treatment produced a prominent RsaA protein band but many lower molecular weight proteins were also present.  Guanidine-HC1, urea,  Tris (pH 7.2), 8-mercaptoethanol and EDTA all extracted numerous proteins without RsaA predominating.  NaCI and CaC1 2 treatments did  not yield significant amounts of protein. The macroscopic precipitate formed in high density cultures of JS1001 was extracted with 8 M urea for 8 hrs and particulate matter was then removed by centrifugation.  The urea was removed by  dialysis and the solubilized protein was examined by SDS-PAGE and Coomassie blue staining (Fig. 14).  Western blotting using anti-RsaA  sera confirmed that the major protein species was RsaA (not shown).  63  97,400  66,200  -  45,000  -  -  31,000  a 1  FIG. 14.  2  SDS-PAGE using a resolving gel containing 10% acrylamide  and stained with Coomassie brilliant blue R. mass standards.  Lanes: 1,  Molecular  2, Protein solubilized by urea from macroscopic  precipitates produced in high density cultures of JS1001.  Lane 2 was  loaded with a sample containing 5 jig of protein as estimated by the Bio-Rad protein assay.  64  3.6.2 layer.  In  vitro  crystallization  of  the  isolated  NA1000  S  After dialysis overnight against 100 mM HEPES buffer (pH  7.5), protein samples extracted by the EGTA or low pH methods had no visible turbidity and TEM negative-stain analysis showed only amorphous structures (not shown).  Dialysis of the sample against 1  mM MgC1 , or 1 mM SrC1 2 2 did not promote crystallization of the S layer protein. sample  After overnight dialysis against  became  turbid  and  TEM  showed  that  1  mM CaC1 2 the the  protein  had  crystallized into a regularly structured array of hexagonal symmetry (Fig. 15) with center-to-center spacing comparable to the native S layer.  Higher concentrations of CaCl 2 also promoted turbid solutions  but ordered S-layer regions were much more difficult to detect by TEM.  RsaA solubilized by urea from the macroscopic precipitates  formed in cultures of JS1001 did not recrystallize.  3.63  Anti-RsaA  sera.  The anti-RsaA sera was used at a  dilution of 1:100,000 for a Western blot of whole cell lysates of NA1000 (Fig. 16).  The blot indicated that the low pH extracted RsaA  used as an antigen was not pure but contained a number of other proteins.  Preadsorption of the antisera with cell lysates of JS1003  effectively removed antibody activity to proteins other than RsaA. The urea solubilized protein from JS1001 reacted with the anti-RsaA sera  by  Western  blotting  indicating  that  although  recrystallize into a regular array it was in fact RsaA.  it  failed  The adsorbed  RsaA antisera bound to NA1000 cells and did not bind to JS1003 or 65  to  FIG. 15.  vitro  Negative-stain transmission electron micrograph of the In  recrystallization of RsaA.  extraction from NA1000 cells.  RsaA was purified by low-pH Recrystallization was mediated by  dialysis of monomeric RsaA against 10 mM HEPES buffer (pH containing 1 mM CaC1 2 at 4°C for 18 hrs. Bar micrograph was taken by Mr. S. H. Smith. 66  =  0.1 $.im.  =  7.5)  This electron  1  FIG. 16.  2  Western blot reacted with unadsorbed  x-RsaA sera and  visualized as described in the Materials and Methods.  Lanes: 1,  Whole cell lysate of NA1000; 2, Low-pH extracted protein from NA1000.  Lanes 1 and 2 were loaded with samples containing 10 and  1 .tg of protein, respectively, as estimated using the Bio-Rad protein assay.  The samples were fractionated by SDS-PAGE using a resolving  gel containing 12% acrylamide.  67  CB2A cells  in indirect immunoflurescent microscopy experiments  (Smith and Smit, unpublished).  3.7  Comparison  of  S-layers  among  freshwater  Ca ul oh a c te rs It  was  environmental  of  interest  to  determine  if  the  cell  surface  of  Caulobacter isolates had the same general character  as the laboratory strains.  It will be shown that most environmental  Caulobacter isolates produce an S-layer, rough LPS and SÃO. 3.7.1  S-layer  extraction.  The HEPES (pH 2.0) extraction  method was applied to all of the freshwater Caulobacter  (FWC)  strains and, in general, proved to be a useful technique to specifically extract the S-layer proteins. molecular  weight  band,  That is, only a single major high  characteristic  of  S-layer  proteins  laboratory strains of Caulobacter, was seen by SDS-PAGE.  from  The SDS  PAGE profiles of low pH extracts yielding an S-layer like band are shown in Figure 17A.  The SDS-PAGE profiles of low pFT extracts from  FWC3O, -38, -40, and -43 did not show a prominent S-layer like band and are shown in Figure 17A (lanes 2, 8, 30 and 31) for comparison. Strains from which an S-layer band could not be extracted using the HEPES (pH 2.0) method were extracted using HEPES buffer (pH 7.5) containing  10 mM EGTA.  This treatment extracted a prominent  protein in almost every case (Fig. 17B).  FWC5, -14 and -21 (Fig. 17B;  lanes 17, 15 and 14 respectively) did not produce an extract showing a prominent S-layer like band using HEPES buffer (pH 7.5) containing 68  SDSPAGE of low pH extracted proteins from Caulobacter  FIG. 17A. strains.  Lanes: 1, molecular mass markers; 2, FWC38; 3, FWC28; 4,  FWC33; 5, FWC35; 6, FWC31; 7, FWC1; 8, FWC43; 9, NA1000; 10, FWC15;  11, molecular mass markers; 12, FWC2; 13, FWC44; 14,  FWC19;  15, FWC17; 16, FWC2O; 17, NA1000; 18, FWC37; 19,  NA1000; 20, molecular mass markers; 21, FWC16; 22, FWC26; 23, FWC18; 24, FWC22; 25, FWC25; 26, FWC46; 27, FWC11; 28, FWC23; 29, NA1000; 30, FWC4O; 31, FWC3O; 32, FWC39; 33, FWC27; 34, FWC24.  The gels were stained with Coomassie Brilliant blue and the  resolving gel contained 10% acrylamide. protein  as  estimated  by  the  Bio-Rad  Samples contained 3 jig of protein  assay  exception of FWC26, -30, -38, -40, -43, and -46 (see text). mass markers are: 200, 97.4, 68, 43, and 29 kDa.  69  with  the  Molecular  A  —————  -  8  18  19 20 21 22 23  9  24 25 26  10  11  27 28  12  13  29 30  14  15  16 17  31 32 33 34 F  70  FIG. 17B. strains.  SDS-PAGE of EGTA-extracted proteins from Caulobacter  Lanes: 1, molecular mass markers; 2, NA1000; 3, FWC32; 4,  FWC12; 5, FWC42; 6, FWC7; 7, FWC6; 8, FWC9; 9, FWC29; 10, FWC41; 11, FWC45; 12, molecular mass markers; 13, NA1000; 14, FWC21; 15, FWC14; 16, FWC4; 17, FWC5; 18, FWC8; 19, FWC34.  The gels  were stained with Coomassie Brilliant blue and the resolving gel contained 10% acrylamide.  Samples contained 3 tg of protein as  estimated by the Bio-Rad protein assay with the exception of FWC5, -14, -21, and -41 (see text).  Molecular mass markers are: 200, 97.4,  68, 43, and 29 kDa.  71  :“‘  *  w  10 mM EGTA or the low pH method.  As well, FWC -30, -38, -40 and  -43 did not produce an extract containing a prominent S-layer like band by using HEPES buffer (pH 7.5) containing 10 mM EGTA (not shown) or the low pH method (see above). Extracts containing 3 tg of protein were examined by SDS-PAGE in Figures 17A and 17B with the exception of FWC5, -14, -21, -26, 30, -38, -40, -41, -43 and -46.  -  Extraction of these strains by both  methods resulted in extracts containing very low concentrations of protein.  Therefore 3 pg of protein could not be loaded on the SDS  PAGE for these strains so 15 j.tl of extract from the method yielding the highest protein concentration was used.  3.7.2 protein  Western  blot  analysis  of  extracted  samples used for the Coomassie blue  proteins.  stained  The  gels were  analyzed by Western blotting using the anti-RsaA serum (Fig. 18). All of the broad S-layer-like bands in Figure 17 gave a positive reaction with the exception of FWC23 (see Fig 17A; lane 28 and Fig.l8; lane 28). S-layer bands. sometime  The antiserum was quite specific for the suspected Western blots of EGTA-extracted samples, which  contained  non-S-layer  proteins,  only  detected  one  prominent high molecular weight band. FWC5, -14, -21, -30, -38, -40, and -43 did not produce an 5layer (MacRae and Smit 1991) and did not show a high molecular weight band by Coomassie blue staining (See Fig. 17A; lanes 2, 8, 30, and 31 for FWC38, -43, -40, and -30 respectively and Fig 17B; lanes 73  FIG.  18.  Western  blot  analysis  of  proteins  extracted  from  Caulobacter strains using the low pH (A) or EGTA (B) method.  Blots  were reacted with cL-RsaA serum. contained 10% acrylamide.  The SDS-PAGE resolving gel  (A) Lanes: 1, molecular mass markers; 2,  FWC38; 3, FWC28; 4, FWC33; 5, FWC35; 6, FWC31; 7, FWC1; 8, FWC43;  9, NA1000; 10, FWC15; 11, molecular mass markers; 12,  FWC2; 13, FWC44; 14, FWC19; 15, FWC17; 16, FWC2O; 17, NA1000; 18, FWC37; 19, NA1000; 20, molecular mass markers; 21, FWC16; 22, FWC26; 23, FWC18; 24, FWC22; 25, FWC25; 26, FWC46; 27, FWC11; 28, FWC23; 29, NA1000; 30, FWC40; 31, FWC3O; 32, FWC39; 33, FWC27; 34, FWC24.  (B)  Lanes: 1, molecular mass markers; 2,  FWC32; 3, FWC12; 4, FWC42; 5, FWC7; 6, FWC6; 7, FWC9; 8, FWC29; 9, FWC41; 10, FWC45; 11, FWC21; 12, FWC14; 13, FWC4; 14, FWC5;  15, FWC8; 16, FWC34; 17, molecular mass markers.  Samples  contained 3 .tg of protein as estimated by the Bio-Rad protein assay with the exception of FWC5, -14, -21, -26, -30, -38, -40, -41, -43, and -46 (see text). kDa.  Note:  Molecular mass markers are: 200, 97.4, 68, 43, and 29 The samples in this figure are not in the same order as  samples in Figure 17A and 17B.  74  N  C,-)  (‘.J  0  N  U,  C,)  C”  to  14,  15 and 17 for FWC21, -14, and -5) or positive reaction by  Western blotting (See Fig. 18A; lanes 2, 8, 30 and 31 for FWC28, -43, -40, -30 respectively and Fig 18B; lanes 11, 12 and 14 for FWC21, 14, and -5).  -  The Coomassie blue stained gel of extracts from both  FWC14 and 5 showed a thin high molecular weight band (Fig 17B; lanes 15 and 17) but the bands did not react to anti-RsaA sera in immunoblot experiments (Fig 18B; lanes 12 and 14).  FWC23 was the  only strain without an S-layer, as determined by TEM, to show a prominent band by Coomassie blue staining (Fig. 17A; lane 28) but it did not label by Western blot analysis (Fig. 18A; lane 28).  FWC26,  -  41, and -46 have an S-layer and produced an S-layer band by the criteria of Coomassie blue staining (Fig. 17A; lanes 22 and 26 for FWC26 and -46 respectively and Fig. 17B; lane 10 for FWC41) and Western blot analysis (Fig. 18A; lanes 22 and 26 for FWC26 and -46 respectively and Fig. 18B; lane 9 for FWC41) although the bands were only weakly visible especially for the Western blots.  Extraction  of the S-layer protein from FWC26, -41 and -46 by both methods was considered poor in that the concentration of solubilized protein was much less that obtained from other strains (see above).  The  remaining 32 strains yielded significant amounts of protein with at least one of the extraction procedures and gave positive results by both Western immunoblots and Coomassie blue staining. The molecular weights of the S-layer proteins from the FWC strains were estimated from their mobility in SDS-PAGE relative to protein standards (Table II).  The S-layer proteins were quite  76  TABLE II. Relevant characteristics of Caulobacter strains Strain  S -layer Anti-RsaA protein b a response size (kDa)  S-layer-producing strains FWC1 FWC2 FWC4 FWC6 FWC7 FWC8 FWC9 FWC11 FWC12 FWC15 FWC16 FWC17 FWC18 FWC19 FWC2O FWC22 FWC24 FWC25 FWC26 FWC27 FWC28 FWC29 FWC31 FWC32 FWC33 FWC34 FWC35 FWC37 FWC39 FWC41 FWC42 FWC44 FWC45 FWC46  100 130 e 130 180 175 120 135 110 130 110 150 105 130 110 110 105 145 105 140 145 105 125 105 135 110 110 100 150 195 135 180 105 140 110  Strains without S layers wcs FWC14 FWC21 FWC23 FWC3O FWC38 FWC4O FWC43  NF NF NF 155 NF NF NP NF  Polysaccharides C detected  ÷ ÷  1 1 0 3 3 1 3 1 3  +  +  0  +  + + +  3 2 1 2 0 1 3 1 3 1 1 0 1 3 1 2 1 3 4 3 2 2 3 2  +  +  + +  +  +  + + +  -  + + + + +  ÷ + + +  + + +  + +  + +  + + +  + +  + + + + +  + + + + + + +  + + +  + +  + +  + +  + + +  + +  -  -  -  -  -  -  -  77  +  +  -  AntiSAO response d  +  -  Multiple bands Multiple bands + -  Multiple bands -  +  0 0 0 0 0 0 0 0  Legend for Table II.  a Molecular masses were estimated on the basis of SDS-PAGE. b Reaction with low pH or EGTA-extracted protein by Western  immunoblot analysis using anti-RsaA serum. C  Results of SDS-PAGE of proteinase K-treated samples stained  for polysaccharides by a modification of the procedure of Tsai and Frasch (1982). stained  bands  +,  single high-molecular-weight band.  running  slower  than  the rough  -,  no silver-  lipopolysaccharide  band. d  Reaction by Western immunoblot analysis with anti-SAO  serum to proteinase K-treated samples.  The response is rated on a  scale from 0 to 4, as follows: 0, no reaction; 1, slight reaction showing a doublet in the SAO region; 2, reaction showing the doublet and some smearing above the top doublet band;  3, definite smearing  reaction reminiscent of the silver-stained image of the SÃO; reaction equal in intensity to that obtained with the C.  4,  crescentus  NA1000 SÃO. e  S-layer presence was not confirmed by negative-stain TEM  although the protein was reactive with anti-Rsaà antibody.  f NF, not found.  78  heterogeneous in molecular weight ranging from 100 to 190 kDa. Proteins greater than 100 kDa are difficult to size by their SDS-PAGE mobility and so the molecular weights reported are only estimates, but are useful for comparative purposes.  3.7.3  Polysaccharide  analysis.  The FWC strains were also  examined for the electrophoretic mobility of their LPS using a rapid purification and staining procedure that was modified to visualize both SAO and rough LPS.  All strains produced a low molecular  weight polysaccharide-containing molecule, presumably rough LPS, which migrated at the dye front in the gel system used for these experiments (Fig. 19).  All strains producing an antibody reactive S  layer protein (Fig. 18, Table II) were found to also produce a single slower-migrating carbohydrate band with the exception (See Fig. 19; lane 20).  of FWC4  This SAO-like species varied in apparent  molecular weight from 60 to 95 kDa (such values are only for comparative purposes reflecting the approximate molecular weight of a protein migrating at that position).  Twenty two strains also  showed a stained band running with the electrophoretic mobility of a 43 kDa protein (see arrow in lane 1 of Fig. 19).  Lane 2 showed a  moderately stained band while lane 3 contained a strongly stained band.  A Coomassie blue stained gel of the proteinase K-treated  samples  also  detected  this  band  indicating  the  presence  proteinase K-resistant protein (data not shown). The S-layer negative strains showed a variety of high 79  of  a  FIG. 19.  SDS-PAGE of proteinase K-treated whole cell lysates of  Caulobacter strains stained using a modification of the method of Tsai and Frasch (1982).  Lanes: 1, NA1000; 2, FWC9; 3, FWC18; 4,  FWC41; 5, FWC45; 6, FWC24; 7, FWC27; 8, FWC16; 9, FWC25; 10, FWC2O; 11, FWC19; 12, FWC17; 13, FWC15; 14, FWC11; 15, FWC1; 16, FWC23; 17, NA1000; 18, FWC39; 19, FWC2; 20, FWC4; 21, FWC5; 22, FWC38; 23, FWC43; 24, FWC14; 25, FWC4O; 26, FWC29; 27, FWC8; 28, FWC46; 29, FWC44; 30, FWC35; 31, FWC33; 32, FWC28; 33, FWC21; 34, FWC3O; 35, FWC7; 36, FWC37; 37, FWC12; 38, NA1000; 39, FWC22; 40, FWC34; 41, MCS6; 42, FWC32; 43, FWC26; 44, NA1000; 45, FWC31; 46, FWC6; 47, FWC42.  Each lane was  loaded with sample containing 0.75 j.tg of KDO as estimated by the method of Karknanis et al. (1978). acrylamide.  The resolving gel contained 13%  The small arrow in lane 1 indicates the running position  of a proteinase-K resistant protein that was present in some samples. SAO runs as a poorly stained band above the small arrow in lane 1.  80  1  co  molecular  weight  carbohydrate  banding  patterns,  including  no  additional bands (FWC5, -30 and -40; see Fig. 19, lanes 21, 34, and 25  respectively), multiple bands reminiscent of the  smooth LPS  “ladder” seen with enteric bacteria (Peterson and McGroarty 1985) (FWC14, -21, -38; see Fig 19, lanes 24, 33, and 22 respectively), or a single band (FWC23, -43 and MCS6; see Fig. 19, lanes 16, 23 and 41 respectively). Western blot analysis, using anti-SÃO serum, of the proteinase K-treated samples (Fig. 20) showed no reaction with the probable rough LPS species of any of the strains.  When a reaction was seen in  S-layer producing strains it coincided with the SÃO-like bands.  No  immunoreactive bands were observed in Western blots, using anti SAO sera, for S-layer minus strains (Table II, Fig. 20; lanes 9, 21, 11, 12, 10, 22, 6). The majority of the “SÃO-like” carbohydrates, identified by silver-staining (Fig. 19) reacted with the anti-SÃO serum in Western blotting experiments (Fig. 20).  However, there was a variation in the  degree of immuno-reactivity between strains.  A qualitative analysis  of the degree to which the SÃO-like molecules reacted with anti-SÃO sera is presented in Table II.  The laboratory strain NÃ1000 stained  darkly and produced a major band that was smeared, reminiscent of the silver-stained image of the SÃO, and a minor band running slightly faster than the major band (See Fig. 20; lanes 4, 17, 24, 28, 37, and 47).  The band produced by FWC39 was as intense as that  produced by NA 1000 but no minor band was noted (Anti-SÃO response  =  4 in Table II; Fig. 20; lane 25).  Eight strains produced a  reaction that showed a definite smearing reaction, but was less 82  FIG. 20.  Western blot analysis of proteinase-K treated whole cell  lysates of Caulobacter strains.  Blots were reacted with c-SAO sera.  Lanes: 1, molecular mass markers; 2, FWC32; 3, FWC26; 4, NA1000;  5, FWC31; 6, FWC43; 7, FWC6; 8, FWC42; 9, FWC5; 10, FWC38; 11, FWC2I;  12, FWC3O; 13, molecular mass markers; 14, FWC7; 15,  FWC37; 16, FWC12; 17, NA1000; 18, FWC22; 19, FWC34; 20, MCS6; 21, FWC14; 22, FWC4O; 23, molecular mass markers; 24, NA1000; 25, FWC39; 26, FWC2; 27, FWC4; 28, NA1000; 29, molecular mass markers; 30, FWC9; 31, FWC18; 32, FWC41; 33, FWC45; 34, FWC24;  35, FWC27; 36, FWC16; 37, NA1000; 38, molecular mass markers; 39, FWC28; 40, FWC33; 41, FWC35; 42, FWC44; 43, FWC46; 44, FWC8; 45, FWC29; 46, molecular mass markers; 47, NA1000; 48, FWC23; 49, FWC1; 50, FWC11; 51, FWC15; 52, FWC17; 53, FWC19; 54, FWC2O;  55, FWC25.  Each lane  was loaded with  sample  containing 0.75 j.tg of KDO as estimated by the method of Karknanis et al. (1978).  Samples were fractionated by SDS-PAGE using resolving  gel that contained 13% acrylamide.  Note:  The samples in this figure  are not in the same order as samples in Figure 19.  83  I  U)  (N  U-)  In  cD  U)  C.-)  (N  0 0-) C.) C.)  C.)  intense than that seen for NA 1000, and produced a minor band below the smearing band (Anti-SÃO response 20; lane 3 [FWC26J as an example).  =  3 in Table II; see Fig.  Three strains, FWC6, 7 and 9  produced the definite smearing band but did not contain the lower minor band (Anti-SÃO response [FWC6] as an example).  3 in Table II; see Fig. 20; lane 7  =  Four strains showed some minor smearing  above a single band (Anti-SAO response lane 42  =  2 in Table II; see Fig. 20;  [FWC44] as an example) and two strains showed minor  smearing above a doublet band (Anti-SÃO response see Fig. 20; lane 53 [FWC19] as an example).  =  2 in Table II;  Eight strains produced a  doublet band with no smearing above them (Anti-SAO response  =  1  in Table II; see Fig. 20; lane 50 [FWC11J as an example) while four strains  showed  response  =  only  a  single  immunoreactive  band  (Anti-SAO  1 in Table II; see Fig. 20; lane 26 [FWC2J as an example).  Three strains producing a silver-stain positive band did not produce any immunoreactive bands (Anti-SÃO response FWC15,  -20,  -29).  20  Figure  illustrates  0 in Table II;  =  that  the  “SÃO-like”  carbohydrate produced by freshwater Caulobacters differed from strain  to  strain  with  respect  to  electrophoretic  mobility  and  growth  and  immunological reactivity to the anti-SÃO sera.  3.8  Metal  S-layer  ion  requirements  for  C.  crescentus  assembly  It has been shown that wild-type C. requires  calcium  for growth.  The 85  crescentus  experiments  NA1000  described below  examine this calcium requirement in more detail. 3.8.1  Influence  JS1001.  of  calcium  growth  on  of  NA1000  and  Higg grown NA1000 and JS1001 cells were 3 Washed M  inoculated  into  Higg 1 M 0  medium  supplemented  with  various  concentrations of calcium and the optical density was measured after 48  h of incubation (Fig. 21).  Calcium concentration had little  influence on the growth of the calcium-independent mutant JS1001 whereas  calcium concentrations  of less  decreased growth yield of NA1000.  than 75  p.M resulted in  Figure 22 illustrates that calcium  became growth rate limiting for NA1000 below 250 p.M. JS1003,  which was NA1000 with rsaA  When a Kmr  interrupted with  cassette, was used in place of NA1000, the same growth patterns were observed.  The washed NA1000 cells used to inoculate M Higg 1 0  medium containing no metal ion supplement did not lyse or die. After 48 hrs of incubation, phase contrast microscopy showed the cells were elongated, tapered consisting of a swarmer and a stalked cell frozen in mid-cell division.  Addition of calcium resulted in  growth following a brief lag period.  3.8.2  Influence  NA1000  and  of  metal  ions  on  the  growth  rate  of  10 medium, which contains 2.2 mM M JS1001. Higg  Mg + 2 , was supplemented with various cations to a final concentration of 500 p.M and the growth rate of NA1000 was determined by the method outlined in the Materials and Methods  section (Fig. 23).  NA1000 did not grow in M Higg 1 0 medium or M Higg 1 0 medium 86  5.  3.  I  I  NA1000  iM Calcium  FIG. 21.  The influence of calcium on growth of NA1000 and JS1001.  5 x 106 washed mid-logarithmic cells were inoculated into 5 mis of M 10 Higg medium containing 0 to 500 iiM calcium.  Cultures were  incubated at 30°C and the optical density at 600 nm was measured after 48 hrs.  Duplicate tubes were used for all concentrations of  calcium and the experiment was repeated 3 times.  The final optical  densities for each calcium concentration varied by less than 5% between  experiments.  87  —  0 -..  ‘-  p.M  FIG. 22. crescentus  Calcium  Influence of calcium on the generation time of Caulobacter NA1000.  5 x 106  washed mid-logarithmic  cells  were  inoculated into 5 mis of M Higg 1 0 medium containing 65.2 to 1000 M calcium.  Cultures were incubated at 30°C and the optical density at  600 nm was followed during growth.  The mean generation time was  determined for cultures between 0D 600  =  0.100 to 1.000.  Duplicate  tubes were used for all concentrations of calcium and the experiment was repeated 3 times.  The bar indicates the standard deviation.  88  4.0 3.0 2.0 1.0 0.0 C  C  0  0  0 OøNZ<OLIC .) i.  —  0  +  Z  500 .tM cation added  FIG. 23.  Influence of the metal ion supplement on the generation  time of Caulobacter medium.  crescentus NA1000 cultured in M Higg 1 0  5 x 106 washed mid-logarithmic cells were inoculated into  10 medium supplemented to 500 iM with a chloride M 5 mis of Higg metal salt.  Cultures were incubated at 30°C and the optical density  at 600 nm was followed during growth. was  determined  for  cultures  between  The mean generation time 600 0D  =  0.100 to 1.000.  Duplicate tubes were used for all metal salts and the experiment was repeated 3 times.  No growth was noted in unsupplemented medium  or medium supplemented with a monovalent cation. potassium or lithium. 2.2  mM  magnesium  M+  =  sodium,  Unsupplemented M Higg 1 0 medium contained chloride.  deviation.  89  The  bar  indicates  the  standard  supplemented  with  the  mono-valent  potassium (chloride salts).  cations  lithium,  sodium  Supplementation of M Higg 1 0  or  medium  with one of 8 divalent or 2 trivalent cations allowed NA1000 to grow, although  the resulting  generation  times  were  observed in medium supplemented with calcium.  greater  than those  Figure 24 indicates  that the growth of JS1001 was not greatly decreased by growth in the presence of any of the cations tested.  The cation used to  supplement M Higg 1 0 medium also had a pronounced influence on the lag time for NA1000 (Fig. 25) that was not noted in JS1001 cultures (Fig. 26).  The lag phase was defined as the number of hours for the  culture to reach an 0D 600 of 0.100.  Increasing the concentration of  magnesium in Higg 10 medium to 3.0 mM did permit limited growth M of NA1000, although culture lysis occurred as the 0D 600 approached 1.5 (data not shown).  3.8.3  Influence  of  metal  ions  on  S-layer  crystallization.  TEM was used to determine if crystallized S-layer was formed on the cell surface of NA1000 or in non-cell associated sheets in cultures of JS1001.  Assembled S-layer was observed in cultures of NA1000 or  JS1001 only when Higg 10 medium was supplemented with calcium M or strontium.  Figure 27 illustrates the non-cell associated sheets of  S-layer produced by JSlOOl cultured in the presence of strontium. Identical  sheets  were  produced  when  JS1001  Higg 1 M 0 medium supplemented with calcium.  was  cultured  on  TEM of NA1000 cells  demonstrated the presence of crystallized S-layer on the cell surface when grown on M Higg 1 0 medium supplemented with calcium or 90  3.0  2.0  1.0  0.0  500 p.M cation added  FIG. 24.  Influence of the metal ion supplement on the generation  time of Caulobacter crescentus JS1001 in M Higg 1 0 medium.  5 x 106  washed mid-logarithmic cells were inoculated into 5 mls of M Higg 1 0 medium  supplemented  to  500  p.M with a  chloride metal  salt.  Cultures were incubated at 30°C arid the optical density at 600 nm was  followed  during  growth.  The  determined for cultures between 0D 600  mean =  generation  0.100 to 1.000.  time  was  Duplicate  tubes were used for all metal salts and the experiment was repeated 3 times.  M+  =  sodium, potassium or lithium.  Unsupplemented  M 10 Higg medium contained 2.2 mM magnesium chloride. indicates the standard deviation. 91  The bar  .  I  —  .—  +  z 500 pM cation added  FIG. 25.  Influence of the metal ion supplement on the lag phase of  Caulobacter  crescentus NA1000 in M Higg 1 0 medium.  5 x 106  washed mid-logarithmic cells were inoculated into 5 mis of M Higg 1 0 medium  supplemented  to  500  M with a chloride metal salt.  Cultures were incubated at 30°C and the optical density at 600 nm was followed during growth.  Lag phase was defined as the number  of hours required for a culture to reach an 0D 600  =  0.100.  Duplicate  tubes were used for all metal salts and the experiment was repeated 3  times.  No  growth was noted in unsupplemented medium or  medium supplemented with a monovalent cation. potassium or lithium. 2.2  mM  magnesium  M+  =  sodium,  Unsupplemented M Higg 1 0 medium contained chloride.  deviation.  92  The  bar  indicates  the  standard  30  20  10  0 0  +  500 pM cation added  FIG. 26.  Influence of the metal ion supplement on the lag phase of  Caulobacter crescentus JS1001 in M Higg 1 0 medium.  5 x 106 washed  mid-logarithmic cells were inoculated into 5 mis of M Higg 1 0 medium supplemented to 500 tM with a chloride metal salt.  Cultures were  incubated at 30°C and the optical density at 600 nm was followed during growth.  Lag phase was defined as the number of hours  required for a culture to reach an 0D 600  =  0.100.  Duplicate tubes  were used for all metal salts and the experiment was repeated 3 times.  M+  =  sodium, potassium or lithium.  Unsupplemented M Higg 1 0  medium contained 2.2 mM magnesium chloride. the standard deviation.  93  The bar indicates  FIG. 27.  Negative-stain TEM micrograph of strontium mediated  crystallization of RsaA formed in a Caulobacter colony.  crescentus JS1001  A double S-layer sheet, as determined by optical diffraction,  that is not associated with the bacterial cells is formed. micrograph was taken by Dr. J. Smit.  94  Bar  0.1 jiM.  This electron  strontium (not shown). medium  TEM of NA1000 cells grown on M Higg 1 0  supplemented with ions other than strontium or calcium  showed that large amounts of material, resembling cell membranes, sloughed off the cells and that no crystallized S-layer could be found.  3.8.4  Influence  S-layer  crystallization.  of calcium  or  strontium  concentration  NA1000 and JS1001  on  were grown on  Higg plates that were supplemented with 5, 3, 1 and 0.5 mM M 10 calcium or strontium.  Colonies were examined by TEM for the  presence of crystallized S-layer. crystallization  in  the  Table III demonstrates that S-layer  presence  concentration dependent.  of  calcium  or  Higher concentrations  strontium of  was  strontium or  calcium were required for non-cell associated S-layer sheets to form in cultures of JS1001 than were required to allow detection of S layer assembled on the cell surface of NA1000. that  higher  concentrations  observation  of  strontium  of crystallized S-layer in  It was also noted were  both NA1000  required  for  and JS1001  cultures than that required for calcium mediated crystallization of S layer.  3.8.5  Localization  of  non-crystallized  S-layer  protein.  Washed NA1000 cells were used to inoculate M Higg 1 0 liquid medium supplemented logarithmic  to  phase  500 the  j.tM with a cation.  After growth to mid  cells  by centrifugation  were pelleted  washed twice with 20 mM FIEPES buffer (pH 7.2). 95  and  Whole cell lysates  TABLE III.  The influence of template and cation concentration on the  crystallization of RsaA.  Metal iona  Crystallized Slayerb NA1000  JS1001  Calcium e  C 05  1.0  +  +  3.0  +  +  5.0  +  +  1.0  +  -  3.0  +  +  5.0  +  +  Strontium  0.5  a  Chloride salt.  b  Forming an array on the cell surface of NA1000 and non cell associated sheets in JS1001.  C  Concentration in mM.  d  Crystallized S-layer observed by negative-stain TEM.  e  Crystallized S-layer not observed by negative-stain TEM.  96  of the washed cells were analysed by Western blotting using anti RsaA sera.  Figure 28 shows that S-layer protein was detected only in  cultures grown in the presence of calcium or strontium. Washed  NA1000  or  JS1001  cells  were  used  to  inoculate  Higg plates supplemented to 1 mM with a metal cation M 10  Following  growth the cells were scraped from the plate and suspended in 10 mM Tris  -  1 mM EDTA buffer.  The cell lysates were analysed by  Western blotting using anti-RsaA sera.  This procedure was used to  identify any RsaA within the cells, attached to the cell surface or RsaA that was translocated to the cell surface but not attached to the cell.  The method used to grow and prepare cells for analysis in Fig.  28 would wash unattached S-layer from the cell surface and thus not detect  the  protein.  S-layer  was  detected  only  when  unwashed  JS1001 or NA1000 cells were cultured in the presence of calcium or strontium (Fig. 29). determine unadsorbed  if  any  Unadsorbed sera was used as a control to  of the  sera (see Fig.  non-RsaA proteins  recognized  16) were repressed during  Higg medium supplemented with various cations. M 10  by  the  growth in  Figure 28 and  29 illustrate that only RsaA was inhibited by growth on cations other than calcium and strontium.  97  200,000  97,400  68,000  43,000 1  FIG. 28.  2  3  4  5  Western blot reacted with unadsorbed x-RsaA sera of whole  cell lysates of washed Caulobacter crescentus NA1000 cells grown in liquid M Higg 1 0 medium supplemented to 500 j.tM with a chloride metal salt.  Lanes: 1, Molecular mass markers in daltons; 2, calcium;  3, strontium; 4, manganese; 5, nickel.  Lanes 2 to 5 were loaded with  10 jig of protein as estimated by the method of Markwell et al. (1978).  Samples were fractionated by SDS-PAGE using a resolving  gel containing 10% acrylamide.  Unsupplemented M Higg 1 0  contained 2.2 mM magnesium chloride.  98  medium  1  FIG.  29.  2  Western  3  blot  4  5  of whole  Caulobacter crescentus cells.  cell  6  lysates  7  from  9  unwashed  Cells were grown on M Higg 1 0 medium  plates supplemented to 1 mM with a chloride metal salt. was reacted with unadsorbed NA1000.  8  x-RsaA sera.  Lanes 6, 7, 8, and 9  =  JS1001.  The blot  Lanes 1, 3, 4, and 5 Lanes: 1, calcium; 2,  molecular mass markers; 3, nickel; 4, manganese; 5, zinc; 6, no ions; 7, calcium; 8, manganese; 9, zinc.  All lanes but 2 were loaded with  10 .tg of protein as estimated by the method of Markwell et al. (1978).  Samples were fractionated by SDS-PAGE using a resolving  gel containing 10% acrylamide.  Unsupplemented M Higg 1 0  contained 2.2 mM magnesium chloride.  99  medium  3.9  Genetic  investigation  the  of  Calcium-independence  phenotype An attempt was made to characterize the calcium-independent, S-layer  attachment-defective phenotype  at the  genetic  level.  A  transposon library was constructed in attempt to isolate the gene responsible for the phenotype.  A cosmid library of wild-type C.  crescentus NA1000 was used in an attempt to complement the calcium-independent phenotype in the mutant strain JS1001. 3.9.1  Production  screening  and  The suicide vector pSUP2O21 library of C.  crescentus  transposon  a  library.  was used to produce a transposon  NA1000  transposon-insertion mutants.  of  containing  20,000  independent  The library was screened on calcium-  free M Higg plates for the identification of calcium-independent 10 mutants.  Although the medium supported the growth of JS1001 and  inhibited the growth of NA1000,  no calcium-independent mutants  could be isolated from the transposon library. S-layer attachment-defective Tn5 mutants were isolated from the library by use of a colony immunoblot screen (Awram and Smit, unpublished;  see  appendix  I  [method  C]).  These  attachment—  defective mutants were analysed by SDS-PAGE and silver-staining to determine the LPS banding pattern (Fig. 30). that  these  mutants  all  produce  altered  Figure 30 demonstrates LPS  banding  patterns.  However, as detailed below, none of these mutants were calcium independent. When SÃO is stained using the modification of the Bio-Rad 100  silver-stain method it is visualized as a golden-yellow band while the rough LPS stains black.  The arrow points to the running position of  the SÃO band in lane 1 of Figure 30A and 30B. could be grouped into 6 clusters patterns.  The Tn5 mutants  on the basis of LPS  banding  Cluster 1 consisted of one mutant, Fl, that produced a band  with the same electrophoretic mobility as SAO although the band was stained black (Fig. 30A; lane 3).  The diamond  ()  beside lane 1 of  Figure 30A and 30B denotes the running position of LPS species that have less electrophoretic mobility than the rough LPS in the Tn5 mutant strains.  Cluster 2 consisted of 5 mutants that produced a  band that was golden-yellow in colour like the SÃO band although it ran with a much greater electrophoretic mobility (Fig. 30A; lanes 4, 6 and 7 for F2, 4 and 5 respectively and Fig. 30B; lanes 3 and 10 for F12 and 19 respectively).  Cluster 3 consisted of 2 mutants that  produced a doublet band that were golden-yellow in colour running at the same electrophoretic mobility as the cluster 2 mutants (Fig. 30Ã; lane 13 for Fil and Fig. 30B; lane 12 for F21).  Cluster 4  consisted of one mutant that produced a doublet that stained black (Fig. 30A; lane 9 for F7).  Cluster 5 consisted of 7 Tn5 mutants which  produced less rough LPS and a small amount of a golden-yellow high molecular weight LPS species (Fig. 30A; lane 11 for F9 and Fig. 30B; lanes 4, 5, 6, 7, 8 and 9 for F13, 14, 15, 16, 17 and 18 respectively). Cluster 6 consisted of 5 mutants that produced a dark band at the running position of the diamond (Fig. 30A; lane 5, 8, 10 and 12 for F3, F6, F8 and FlO respectively and Fig. 30B; lane 11 and 13 for F20 101  FIG. 30.  SDS-PAGE of proteinase K treated NaC1/EDTA extracts of  Caulobacter  crescentus  Tn5  strains.  defective Tn5 mutants are designated “Fl  The -  S-layer attachment  F22”.  See appendix I  (method C) for details on the method used to isolate the mutants. The gel was stained using the modification of the Bio-Rad silverstain kit.  (A) Lanes: 1, NA1000; 2, JS1001; 3, Fl; 4, F2; 5, F3; 6, F4;  7, F5; 8, F6; 9, F7; 10, F8; 11, F9; 12, FlO; 13, Fil.  (B)  Lanes: 1,  NA1000; 2, JS1001; 3, F12; 4, F13; 5, F14; 6, Fl5; 7, Fl6; 8, F17; 9, F18; 10, F19; 11, F20; 12, F21; 13, F22.  The arrows designate the  running position of wild-type SAO while the diamonds designate the running positions of LPS species produced by the Tn5 mutants that run with a Samples  greater electrophoretic mobility than the rough LPS.  containing 0.5  pg KDO, as estimated by the method of  Karknanis et al. (1978), were loaded into each lane and fractionated with a resolving gel containing 13% acrylamide.  102  A  0  1  2  345  6789  10111213  B  0  1  2  3  4  5  6 103  78  910111213  and F22 respectively).  An S-layer attachment-defective Tn5 mutant  from each cluster was tested for the ability to grow in the absence of calcium.  None of the four mutants examined were capable of growth  Higg 1 in M 0 liquid medium indicating that although they produced altered SÃO they were not “calcium-independent” mutants.  3.9.2  Complementation  cosmid  library.  introduced  into  of  with  JS1004  an  NA1000  A cosmid library derived from NA1000 was the  calcium-independent  JS1004 by electroporation.  S-layer-negative  One of 680 cosmid containing clones  reacted with anti-SÃO sera in a dot blot immunological screen. cosmid was designated “D”. JS1001  containing  produced,  it was  cosmid at less  strain  This  A Western blot using anti-SAO sera of D  indicated that  although  than wild-type levels  (Fig.  SAO 31).  was The  Caulobacter DNA contained in cosmid D was isolated and used as a probe to identify 28 overlapping cosmids.  Restriction digests of  these cosmids indicated that 18 had unique banding patterns and these 18 were introduced into JS1001 separately by electroporation. The electroporants were screened by Western blot analysis using anti-SÃO sera and two were shown to produce SAO, although at levels less than wild-type (Fig. 32). D12 and D13.  These cosmids were designated  Washed cultures of JS1001 containing cosmid D12 or  D13 were analysed by Western blotting using anti-RsaA sera.  The  Western blots indicated that these cells were unable to attach most of the S-layer protein to the cell surface (not shown).  104  97,400 68,000 43,000 29,000 1  2  3  4  5  FIG. 31.  Western blot reacted with cc-SÃO sera of proteinase K treated whole cell lysates. Cells were grown in PYE liquid media. Lanes: 1, molecular mass markers in daltons; 2, NA1000; 3, JS1001; 4, JS1004 containing cosmid D; 5, JS10O1 containing cosmid D. Samples containing 1 pg KDO as estimated by the method of Karknanis et al. (1978) were loaded into each lane and fractionated by SDS-PAGE using a resolving gel containing 13% acrylamide.  105  1  FIG. 32.  2  3  4  Western blot reacted with cc-SAO sera of proteinase K  treated whole cell lysates.  Cells were grown in PYE liquid media.  Lanes: 1, NA1000; 2, JS1001 3, JS1001 containing cosmid D12; 4, JS1001 containing cosmid D13.  Samples containing 1 jig KDO as  estimated by the method of Karknanis et al. (1978) were loaded into each lane  and fractionated by SDS-PAGE using  containing 13% acrylamide.  106  a resolving  gel  4  Discussion  Figure 33 is a model diagram of the C.  crescentus cell surface  based, in part, on the information contained in this thesis.  The figure  illustrates the presence of three major polysaccharide species one of which, the SÃO or smooth LPS, plays a role in the attachment of the S-layer to the cell. in  Figure 33 also proposes that calcium is involved  the formation of the  membrane  assembly.  S-layer and may  The  following  is  also have a  a role in  discussion  of  the  experimental evidence which forms the basis of the model.  4. 1  C.  crescentus  The C.  cell  surface  polysaccharides  crescentus cell surface was examined and found to  produce three types of polysaccharides: an EPS, a “rough” LPS, and a “smooth” LPS termed the SÃO.  4.1.1  C.  crescentus  “rough” LPS.  The presence of LPS in C.  crescentus strains was initially revealed by SDS-PAGE analysis of washed whole cells treated with proteinase K (Fig. 3 and 4). The high electrophoretic mobility of the band sensitive to the Tsai and Frasch stain and the absence of bands of higher molecular weight indicated that the LPS species was “rough” in nature (Hitchcock et al. 1986). The LPS from all strains yielded similar electrophoretic profiles (Fig. 4). The LPS of CB2A and NA1000 was isolated and purified using a  107  phospholipid  Ca  S-layer protein  2+  [::1  FIG. 33.  porin protein  *  /l\ protein  EPS  Ii\  R LPS  SAD  -  Representation of the Caulobacter  surface.  108  negative charge  hydrophobic interaction  crescentus NA1000 cell  modification of the method of Darveau and Hancock (1983). purified NA1000 and CB2A LPS was analyzed by SDS-PAGE.  The  Half of  the gel was stained using the method of Tsai and Frasch (1982) while the other half was stained using the Bio-Rad silver-stain kit (Fig. 3). Both methods produced similar patterns of staining.  The bands  resulting from samples prepared by the method of Hitchcock and Brown  (1983)  (Fig.  3,  lanes  1  and  2)  had  a  slightly  slower  electrophoretic mobility and were wider in the horizontal plane than that of the purified LPS. containing  This may result from these samples  bulk cellular components,  such  as  undigested protein,  peptidoglycan and nucleic acids, which alters the mobility of the LPS though the gel.  Figure 3 also demonstrates that the purified LPS did  not contain contaminants.  The Bio-Rad silver-stain method detects  protein, carbohydrate, nucleic acid and contaminating  metal salts  (Bio-Rad 1987) whereas the staining procedure of Tsai and Frasch (1982) is very sensitive for detection of LPS but is insensitive for the detection of protein and nucleic acids (Hitchcock and Brown 1983). The Bio-Rad procedure allowed the visualization of contaminating protein and nucleic acid which were contained in the “crude” LPS preparation. Detailed chemical analysis of the purified LPS from CB2A, NA1000  and  JS1001  (Ravenscroft et al. 1992).  was  conducted  by  Dr.  N.  Ravenscroft  The purified LPS was shown to consist of  two definable regions: (i) an oligosaccharide region, consisting of an inner core of three residues of 2-keto-3-deoxyoctonate, two residues of x-L-glycero-D -mannoheptose, 109  and  one  x-D-glycero-D  mannoheptose and an outer core region containing one residue each of x-D-mannose,  x-D-galactose, and x-D-glucose, with the glucose  likely phosphorylated and (ii) a region equivalent to the lipid A of archetype LPS, consisting primarily of the esterified fatty acid 3-OHdodecanoate (see Table I, appendix II). The lipid A-like region was resistant to conclusive analysis. The major or only fatty acid component, 3-OH-dodecanoic acid, is less commonly found in LPS than 3-OH-tetradecanoic acid, but has been found, for example, in the Lipid A from Pseudomonas (Bhat et al. 1990).  aeruginosa  Mild acid hydrolysis readily cleaved the LPS into  Lipid A and core oligosaccharide fractions.  Yet despite extensive  efforts, no amino or diamino sugars, typical of the ’t backbone” region of other Lipid A moieties (Mayer et al. 1988; Rietschel et al. 1990) were detected during multiple approaches for amino sugar analysis (Ravenscroft et al. 1992). There is no clear explanation for the high stability of the Lipid A and thus its resistance to hydrolysis into assayable sugars. notable,  however,  that  Caulobacters are members of the  It is x-2  subdivision of the alpha proteobacteria, as defined by 16S rRNA sequence analysis (Stackebrandt et al. 1988; Stahl et al. 1992; Woese 1987), a group that contains members producing “unusual” Lipid A structures.  The  amino  sugar  2,3-diamino-2,3-dideoxy-D glucose -  (DAG) has been identified in the Lipid A backbone structures of some species in this phylogenetic group (Weckesser and Mayer 1988).  It  may be that a variation of the DAG-type Lipid A is present in C. 110  crescentus.  High-voltage paper  electrophoresis  has  proven  to  separate and detect such unusual Lipid A sugars in other species (Mayer et al. 1988) and may be appropriate in future studies with  the Caulobacter Lipid A. It has been reported that C.  crescentus  whole  membranes  contain from two-thirds to ten-fold less KDO than that reported for membranes of rough mutants of Salmonella type S.  typhimurium and wild-  typhimurium, respectively (Agabian and Unger 1978).  This  study showed that the total amount of KDO in whole cells of NA 1000 and CB2A is less than that found in E. coli B, but only to the extent of 20 to 30%.  This indicates that KDO could be used as an outer  membrane  marker  fractions.  The published methods  during  procedures  to  separate  for membrane  membrane  separation  and  isolation in C. crescentus do not account for the missing KDO in the “outer membrane” fractions (Agabian and Unger 1978; Clancy and Newton 1982; Koyasu et al. 1980).  Two of the protocols (Clancy and  Newton 1982; Koyasu et al. 1980) used PBS in the procedure.  It has  been since shown that PBS extracts LPS from the envelope of C. crescentus (Edwards and Smit 1991; Walker and Smit, unpublished observation)  and  the  absence  of  KDO  in  these  membrane  preparations might be explainable on that basis.  4.1.2  C. crescentus SAO.  SAO was initially identified as a  contaminant in a purified LPS sample (Fig. 6).  This proteinase  resistant molecule was detected in whole cell lysates of wild-type S 111  layer producing  strains  or  strains  that  are  attachment-competent  using a modification of the method of Tsai and Frasch (Fig. 7).  A  similar band was not detected in calcium-independent strains which are unable to attach the S-layer to the cell surface indicating that it may play a role in S-layer attachment.  Originally it was unclear if  this band represented a species of LPS with a homogeneous length 0antigen, as seen in Aeromonas  species (Belland and Trust 1985;  Dooley and Trust 1988), or if it was a unique carbohydrate species. The SAO was not detected by the SDS-PAGE and silver-staining methods of Hitchcock and Brown (1983) or Tsai and Frasch (1982) which are used widely to identify and qualitatively characterize LPS from many bacterial species.  Also, the SAO did not precipitate with  the rough LPS during the cold ethanol-MgC 2 1 step of the Darveau and Hancock (1983) procedure but remained in the supernatant along with a significant amount of rough LPS. purification  procedure  for  SAO  was  Thus an isolation and determined  in  order  to  chemically characterize the molecule. The cell surface extraction procedure using 0.77 M NaC1 / 0.12 M EDTA (pH 7.2) was effective at solublizing the cell surface components  without  releasing  large  amounts  of  cytoplasmic  constituents, although the redistribution of cytoplasmic material is indicative of the plasma membrane losing its selective permeability barrier toward ions (Whitfield and Murray 1956).  This extraction  method provided a convenient and rapid method to obtain rough LPS, SAO and EPS from liquid cultures ranging in volumes of 1.5 ml 112  to 60 liters.  For detailed chemical analysis, SAO was isolated from  JS1003 instead of NA1000 due to the deletion of rsaA in this strain which resulted in a lower protein concentration in the NaC1/EDTA extract.  The SAO was then separated from the rough LPS by SDS  PAGE and isolated from the polyacrylamide gel by electroelution. Detailed chemical analysis of the purified SAO was preformed by Dr. D. N. Karunaratne (See appendix II, Table III). to be composed of lipid and polysaccharide.  The SAO was shown  The major fatty acid was  identified as 3-OH-dodecanoate which is the same fatty acid as that found in the rough LPS.  Minor amounts of the same sugars detected  in the rough LPS were identified as well as large amounts of 4,6dideoxy-4-amino  hexose,  3 ,6-dideoxy-3 -amino hexose and glycerol  all in equal proportions.  Proton NMR studies on the purified SÃO  have determined that the amino group of both dideoxyamino hexoses are acetylated (W. R. Abraham, unpublished; see Table III, appendix II).  Given this data and the fact that colorimetric assays indicated  that SÃO and rough LPS have the same molar ratio of KDO:phosphate it is clear that SÃO is a species of smooth LPS with homogeneouslength 0-antigen. Dideoxyamino sugars  are regarded as  “rare”  and “unusual”  although they have been identified as component sugars in a number of bacterial species LPS (for a list of species and references see Ashwell  and  Hickman  1971;  Jann  Lindberg 1983; LUderitz et al.  and  Jann  1977;  Kenne  1968; and Wilkinson 1977).  and 4,6-  dideoxy-4-amino hexose or 3 ,6-dideoxy-3 -amino hexose containing 0-antigens often have altered solubility characteristics than that of 113  most LPS.  When some, but not all, species possessing these sugars  are subjected to the hot phenol/water LPS isolation procedure of Westphal et al. (1952) the 0-antigen is found in the phenol phase whereas the rough LPS is found in the aqueous phase.  The rough  and smooth LPS of most bacteria is partitioned into the aqueous phase.  It has been suggested that the phenol solubility of the  dideoxyamino hexose containing LPS is due to the increased number of non-polar groups (terminal methyl and N-acetyl residues) in these sugars (Hickman and Ashwell 1966).  Whatever the chemical basis  contributing to the phenol solubility of such an LPS it is clear that these 0-antigens possesses a hydrophobic character not found in most LPS species.  It is tempting to speculate that differences in  hydrophobic character between the C.  crescentus rough and smooth  LPS accounted for their separation during the Darveau and Hancock (1983) procedure. The inability to stain all components of LPS after SDS-PAGE using the standard methods of Hitchcock and Brown (1983) and Tsai and Frasch (1982) is relatively uncommon but is not unique to C.  crescentus. Cytophaga  johnsonae  (Godchaux  et  al.  1990),  Campylobacter jejuni (Preston and Penner 1987), C. coli (Mandatori and Penner 1989), E. coli 026 (Karch et al. 1984), Coxiella (Hackstadt et al. 1985), and Neisseria  burneti  gonorrhoeae (Mandrell et al.  1986) also have LPS species which do not stain using the standard LPS silver stain and immunological or alternative staining procedures must be used to visualize these molecules. 114  The Bio-Rad silver-stain  procedure detected both the rough LPS and the SÃO (Fig. 8) although its sensitivity towards the rough LPS was approximately five-fold less than that obtained using the method of Tsai and Frasch (1982). Therefore, different staining methods are required depending what species of Caulobacter LPS are being examined. The mechanism by which macromolecules are stained during various  silver  staining  protocols is  unknown  (Deh  Goldman and Merril 1982; Kropinski et a!. 1986).  et al.  1985;  Silver stains are  based on methods using either ammoniacal silver solutions (Oakley et al. 1980) or silver nitrate (Merrill et a!. 1981).  The method of Tsai  and Frasch (1982) uses ammoniacal silver whereas the Bio-Rad silver stain kit uses silver nitrate.  A modification of the Bio-Rad silver  stain (Cava et al.  1989) which substitutes  dichromate  oxidation  in  the  step  polyacrylamide gels (Fig. 30).  was  sodium periodate for  shown  to  stain  SÃO  in  SAO was not stained when periodic  acid was used to oxidize molecules prior to staining with ammoniacal silver in the method of Tsai and Frasch (1982) (Fig. 3 and 4).  This  indicates that the oxidized SÃO does not react with ammoniacal silver.  However, more detailed comparisons between the two LPS  staining methods will have to be carried out in order to determine the precise reason why SÃO does not stain by the method of Tsai and Frasch (1982). When  SAO  is  detected  by  the  Bio-Rad  method  or  the  modification of the Bio-Rad method it is stained a yellow-orange colour which is a common staining characteristic of 0-glycosidically 115  linked carbohydrate containing molecules (Deh et a!. 1985).  When  photographing such gels with black and white film the resulting image of the SÃO is much less intense in comparison to the rough LPS which stains dark black.  Therefore, the photographs in Figure 30 are  not accurate representations of the original polyacrylamide gel. Figures 8 and 12 show that the SAO was not resolved into discrete  bands  as  has  been  shown  for  other  0-antigen  homogeneous length (Chart et a!. 1984; Dooley et al. 1985).  of  Although  a variety of acrylamide concentrations and a number of gel protocols were used, heterogeneity in this region was not identified.  The  purified SÃO was subjected to laser desorption time of flight mass spectroscopy  analysis  to  identify  any  microheterogeneity in  region (A. Rudiger, unpublished data; See Fig.3, appendix II).  this The  analysis indicated that there is microheterogeneity present, but the average difference in mass between SAO molecules is approximately 176 daltons.  Therefore SDS-PAGE, under any conditions, would be  unable to detect this microheterogeneity. The cell surface defect responsible for the S-layer attachmentdefective  phenotype  of  calcium-independent  mutants  of  C.  crescentus appears to be the inability to produce an 0-antigen of uniform length (SÃO molecule).  This is reminiscent of the defect  thought to be responsible for the attachment-defective phenotype in  Aeromonas  salmonicida and A.  1985; Dooley and Trust 1988).  hydrophila (Belland and Trust  In mutants of both species inability  to attach the S-layer to the cell surface has been correlated with 116  defects in the LPS.  With A.  salmonicida strains the inability to  produce a homogeneous-length smooth LPS results in an attachmentdefective  phenotype  hydrophila  strains  (Belland also  and  Trust  produced  a  1985).  Wild-type  A.  homogeneous-length  0-  polysaccharide but mutants that generated only a core LPS could still maintain the S-layer.  Mutants of A.  hydrophila producing a deep-  rough LPS were, however, found to be S-layer attachment-defective (Dooley and Trust 1988).  Dooley and Trust (1988) have suggested  that a homogeneous-length 0-antigen may be required by all Gramnegative S-layer producing species. Campylobacter  fetus  has  smooth  However, the S-layer containing LPS  reminiscent of the LPS of enteric bacteria  of  heterogeneous-length  (Perez-Perez et a!. 1986)  although Yang et al. (1992) have implicated the LPS as the cell surface molecule to which the S-layer attaches. salmonicida and A.  The 0-antigen of A.  hydrophila extends past the S-layer into the  environment (Chart et al. 1984; Dooley et a!. 1988). Caulobacter  crescentus  (Smit,  unpublished  In contrast, the  observation)  and  Campylobacter fetus (Fogg et a!. 1990; McCoy et al. 1975) 0-antigens do not extend past the S-layer.  At present only these limited  number  producing  of  Gram-negative  S-layer  species  examined by SDS-PAGE to determine the LPS profile.  have  been  Therefore  broad generalizations cannot be made, however, it appears that S layers attach to the cell surface of Gram-negative bacteria via the LPS.  Like S-layers themselves, the mechanism of attachment to the  cell surface may prove to be a product of convergent evolution.  117  C. crescentus EPS.  4.1.3  CB2A, NA1000 and JS1001 produced  sufficient quantities of an EPS during growth in broth culture for it to be isolated in an aqueous phase as a by-product of the general purpose LPS isolation procedure of Darveau and Hancock (1983). inability  The  to  wash  the  EPS  off  the  surface  by  repeated  centrifugations and suspensions and the lack of significant amounts of polysaccharide located in the culture medium following growth indicated that the polymers were not a loosely associated “slime” layer but a true capsule or EPS layer (ørskov and ørskov 1990). Carbohydrate analysis of the purified EPS from NA1000, JS1001 and CB2A was conducted by Dr. N. Ravenscroft (Ravenscroft et al. 1991; see Fig. 2, appendix II).  These studies showed that NA1000 and  CB15A produce a unique neutral EPS. glucose,  The EPS of CB2A contained D  D-gulose and D-fructose in a ratio of 3:1:1 whereas the  NA1000 EPS contained D-galactose, D-glucose, D-mannose and D fucose  in  approximately equal  amounts.  analysis confirmed that the polymers  NMR  consist  and methylation  of repeating units,  NA1000 consisting of a tetrasaccharide and CB2A a pentasaccharide, containing both cz- and 13-linked sugar residues.  The repeating sugar  units indicated that the isolated polymers had the general features of a bacterial EPS or capsule.  The chemical differences in the EPS of  CB2A and NA1000 illustrate that they have evolved independently so  as  to  environment.  present  different  chemical  motifs  Like other bacterial species C.  produce many different EPS chemotypes. 118  to  the  external  crescentus strains may  The EPS isolated from the  calcium-independent  S -layer  attachment-defective  mutant  IS 1001  was chemically identical to that found in the parent strain NA1000. The classification of cell surface polysaccharides as capsules (EPS), slime layers or LPS are based both on chemical analysis of the polysaccharide material  as  (Costerton  well et  as the al.  biophysical  1981).  The  characteristics  firm  of the  attachment of the  Caulobacter EPS to the surface is a property shared with the LPS and it might be argued that the EPS is an LPS with a very long 0antigen.  LPS is often differentiated from EPS based on the criterion  that the LPS is pelleted by ultracentrifugation at 200,000 xg for 30 h in aqueous solution whereas the EPS remains in the supernatant (Whitfield  and  Valvano  1993).  Based  on  this  sedimentation  definition the carbohydrate described above is considered to be an EPS.  This term is also appropriate based on the chemical studies of  the LPS and SÃO that are discussed above.  The “rough” LPS and SÃO  had a completely different chemical composition from the EPS in both CB2A and NA1000 and no KDO, a constituent of all LPS, was detected in the EPS fraction (compare Fig. 2 with Table II and Table III, all in appendix II). It is still possible however, that the EPS fraction is technically a large species of “smooth” LPS due to the method by which it is attached to the cell surface. oligosaccharide  might  be  The long 4 or 5 sugar repeat structure anchored  to  the  outer  membrane  by  attachment to a single rough LPS molecule (consisting of lipid A and core oligosaccharide moieties).  Such an anchoring arrangement has 119  been suggested for the group I capsular polysaccharide antigens of Escherichia coli. (Jann and Jann 1990).  Anchoring of the EPS to the  surface might also be mediated by other lipids as has been shown for group II capsular polysaccharides of E. coil where the EPS is linked to the cell surface by phosphatidic acid (Jann and Jann 1990).  At this  point, the means of apparent surface adherence for the Caulobacter EPS is unresolved because of the difficulty in purifying sufficient quantities of the “anchor” portion of an EPS from the large excess of polymerized repeat unit after the two regions of the molecule are cleaved. Since the EPS remained on the cell following washing of the cells by centrifugation and resuspension it might be expected that this layer would be visible by thin section TEM methods.  However,  no indication of an EPS layer on cells prepared for thin section TEM by standard methods have been reported even when dyes commonly used  to  reveal  polysaccharides  (eg,  ruthenium  red)  were  incorporated into the procedures (Poindexter 1964, Ravenscroft et a! 1991; Smit et al. 1981).  Graham et a!. (1991), as part of a larger  study on the use of freeze-substitution methods, did not visualize an EPS  layer in  strain NA1000.  Yet the  same cryofixation/freeze  substitution technique has been used to successfully preserve and visualize the EPS layer on Leptothrix  discophora (Beveridge 1988)  and E. coil K30 (Whitfield et al. 1989).  However, the EPS of both  Cauiobacter strains contain only neutral monosaccharides and the cationic dyes and heavy metals used to stain surface polysaccharides 120  may not react with the neutral polymers and thus they remain. Other methods of capsular stabilization, such as pre-treatment with antibody  directed  against the EPS  or chemical  dehydration  and  Lowicryl embedding (Bayer 1990), may be required to visualize the layer by transmission electron microscopy. visualized an EPS-like structure on C. cryofixation/freeze technique.  substitution  Ravenscroft et al. (1991) crescentus CB2A using a  scanning  electron  In that procedure the sample is  microscopy  sputter coated with  heavy metals following cryofixation/freeze substitution.  Thus even  neutral molecules, such as the EPS, are rendered electron dense and therefore be visible when examined by electron microscopy. The composition  of the BPS  between CB2A and NA1000.  polymers  differed  sufficiently  In this context it is of interest to note  that CB2A no longer produces an S-layer (Smit et al. 1981) but does produce and correctly assemble the S-layer protein from NA1000 when  rsaA  is introduced into CB2A on a plasmid (Smit et al.  unpublished; See Fig. 1; lane 17).  Apparently S-layer assembly is not  affected by differing EPS molecules.  Since the EPS produced by the  S-layer attachment mutant JS1001 does not differ from its S-layer attachment  competent  parent  strain,  NA1000,  it  is  considered  unlikely that EPS plays a role in S-layer attachment to the cell surface.  4.2  RsaA extraction and in vitro  recrystallization  RsaA could be selectively extracted from whole cells of NA1000 121  using low pH.  Coomassie blue stained gels indicated that the extracts  contained almost exclusively RsaA (Fig. 13).  However, Western blots  of whole cell lysates of NA1000 probed with the unadsorbed anti RsaA sera indicated that the sample used as an antigen contained contaminating proteins (Fig.  16).  The presence of contaminating  non-RsaA in the low pH extracts indicated that further purification by gel exclusion chromatography or HPLC would be required if the protein was to be studied by high resolution methods.  However, the  purification of RsaA by the one step low pH extraction was sufficient for conducting the experiments discussed below. Low pH extraction has also been used for selective purification of  the  S-layer  of  other  bacteria  (Beveridge and Murray 1976a), S. Murray 1976b,c), Aeromonas  including  Sp irillum  “Ordal”  putridiconchylium (Beveridge and  hydrophila (Dooley and Trust 1988)  and Campylobacter fetus (McCoy et al. 1975).  For the Aeromonas  and Campylobacter species a low pH extraction procedure using a glycine-HC1 buffer was effective.  In contrast,  better results in  selectively removing RsaA were obtained using HEPES at low pH, recognizing that it is not a buffer in that range.  Perhaps with C.  crescentus, the protonated amino group of glycine at low pH also disrupts  other membrane-associated proteins.  The  reassembly  studies  with  the  purified  NA1000  protein  provided definitive data that only RsaA is responsible for the visible repeated structure. S-layer  Previously, Smit et al. (1981) had reported that  preparations  fragments  isolated  from by  NA1000  differential 122  (consisting  of  centrifugation)  shed  S-layer  contained  two  other proteins, the “74K” and “20K”, and membrane material. could  not  be  resolved  whether  the  additional  membrane-derived or were part of the Since  the  reassembly  experiments  visible  proteins  It were  S-layer structure.  reported  here  involved  preparations with very little contamination from the 74K and 20K proteins it seems clear that these additional proteins are not part of the S-layer structure.  The in vitro  reassembly  experiments  also  reinforce that calcium is specifically required for S-layer assembly; even the divalent strontium ion, which has a hydrated molecular diameter most similar to calcium and which has calcium in the in vitro  reassembly  of other  1976c), was unable to replace calcium.  substituted for  S-layers  (Beveridge  However, as discussed below,  strontium is capable of mediating in vivo RsaA assembly into an S layer.  4.3  Distribution  environmental  of  RsaA-  and  SAO-like  molecules  in  Ca ulobac ter isolates  NA1000, and its parent CB15 (Poindexter  1964), have been  maintained in pure culture as laboratory strains for almost thirty years.  Therefore it is of interest to determine if Caulobacters in  their natural environment possess equivalent cell surfaces as that found on strain NA1000 (see Fig. 33). the  cell  surface  of  many  It is generally accepted that  “domesticated”  bacteria  bare  little  resemblance to that of strains growing in their natural environment (Beveridge and Graham 1991, Costerton et al. 123  1981; 1987).  This  was  study  undertaken  determine  to  if  the  cell  surface  of  Caulobacters in nature have a similar S-layer and LPS composition as NA1000  and to determine the degree to which the  S-layers  produced by various environmental strains are conserved. Table II is a summary of the results of this study.  These  results indicate there is a similarity between the S-layers of the FWC isolates and that most of the isolates have a cell surface which resembles that of NA1000. several levels. specifically  The similarity was demonstrated at  The disruption methods used appeared in all cases to  disrupt  and  extract  the  S-layer  and  the  solubilized  protein was also, in all cases but one (FWC23), immunologically cross-reactive with anti-RsaA sera.  It is conceivable that as in strain  NA1000, calcium (or another divalent cation) is required for S-layer attachment or crystallization in the various freshwater isolates and the two extraction methods  used would disrupt calcium-mediated  ionic bonding (i.e., EGTA is a calcium-selective chelator and the protons of the low pH treatment would compete with calcium for anionic  sites).  In  addition,  oligosaccharide-containing  molecules  similar to SAO were present in all but one (FWC4) S-layer producing strain and in most cases the oligosaccharide had at least a degree of immunological reactivity with the anti-SÃO sera. then  that  there  is  not  only  a  degree  of  It can be argued  conservation  among  Caulobacter S-layer proteins but also a conservation of an SÃO-like molecule which may participate in surface attachment.  Conversely,  in the case of the atypical Caulobacters, when there is no S-layer, 124  there seems to be a different surface architecture as well.  However,  more detailed examination of the S-layer-like proteins and SAO-like polysaccharides would have to be conducted to initial  findings,  which  indicate  that  most  strengthen these  Caulobacters in the  environment have similar cell surface features as NA1000. FWC23 and FWC4 were exceptional strains in this study in that they could not be grouped with the typical or atypical Caulobacter strains.  A single prominent S-layer-like protein was extracted from  FWC23  and  an  staining  but  neither  SAO-like the  carbohydrate extracted  was  protein  detected or  the  by  silver-  carbohydrate  reacted in Western blots (See Fig. 17A; lane 28; Fig. 18; lane 28; Fig. 19; lane 16; Fig. 20; lane 48).  A regularly structured array was not  identified on FWC23 by negative-stain TEM.  EGTA extraction of  FWC4 yielded a high molecular weight S-layer like band, as well as a large number of lower molecular weight bands, that reacted with the anti-RsaA sera by Western blotting (see Fig. 17B; lane 16 and Fig 18; lane 13).  However, FWC4 lacked an SAO-like polysaccharide as  determined by silver-staining and Western blotting (see Fig. 19; lane 20 and Fig. 20; lane 27).  Negative-stain TEM has also failed to  visualize a regular structure on the surface of this strain. In a study by Stahl et al. (1992) involving 16s rRNA analysis of a number of these strains it was learned that the typical strains are a relatively closely-related subgroup of the freshwater Caulobacters, while examples  of the  atypical  strains  were  different from the  typical cluster and from each other (Stahl et al. 1992). 125  Nevertheless,  Caulobacters in the typical group were still measurably dissimilar. Since  the  group  of  S-layer  producing  Caulobacters  are  phylogenetically cohesive, yet clearly different from one another, it was difficult to predict a priori whether the S-layer proteins would be structurally similar.  Indeed, it might be expected that the S-layer  proteins of a collection of Caulobacter strains would show significant differences because they are not, for example, pathogenic strains with an S-layer attuned to parasite-host interactions, as with some other S-layer producing species (Dooley and Trust 1988; Dubreuil et al. 1990; Kay et a!. 1984; Murray et a!. 1988).  Thus, there might  seem to be little reason for genetic selection to favor a specific S layer structure, particularly at the level of immunological similarity. The anti-RsaA sera cross-reaction was specific in the Western blotting  experiments  of FWC’s, but the degree  of labeling was  relatively uniform between strains and significantly less than that obtained with RsaA.  It seems possible that there are conserved  regions in the S-layer proteins that are required for formation and surface attachment of the paracrystalline structure, while the rest of the protein is variable and may be dispensable.  RsaA is a member of  the group of smallest Caulobacter S-layer proteins (ca. 100 kDa) and therefore may be one that contains the minimal amount of essential assembly-attachment information.  This may mean, in some cases  (e.g., FWC39), that more than half of the protein serves some purpose other than essential structure information.  Dubreuil et al. (1990)  made a similar prediction of structurally nonessential regions in the  S-layer of Campylobacter fetus strains. 126  The  immunological  findings  are  also  reminiscent  of  gene  hybridization studies which analogously showed that the NA1000 S layer gene (rsaA) could be used to identify most Caulobacters isolated from the environment, since most produced S-layers, but only under reduced stringency conditions (MacRae and Smit 1991). It was hypothesized that conserved regions of the S-layer genes may be responsible for the hybridization noted.  Therefore, the data  presented in this thesis, that of MacRae and Smit (1991), and that of Stahl et al. (1992), indicates that the degree of S-layer structural conservation consequence  noted of  Caulobacter isolates may be a  between  common  mechanisms  of  self-assembly,  surface  attachment, and possibly export mechanisms conserved during the evolution of the various Caulobacter strains. Comparative studies between the S-layer proteins of related bacterial  strains  Aeromonas  have  been  conducted  in  other  species.  In  salmonicida there is a significant degree of structure  conservation among  the  S-layer protein  of strains  isolated from  diverse locals, as judged by N-terminal protein sequencing, Western immunoblot  and  immunofluorescence  ELISA analysis  analysis of  a  of larger  a  few  group  strains using  prepared against one of the S-layer proteins (Kay et al 1984). other hand, with A.  and  antibody On the  hydrophila, there were antigenic differences  among strains and no N-terminal amino acid sequence homology of the S-layer protein between two A. 1988).  hydrophila strains (Dooley et al.  In a similar analysis of Campylobacter 127  fetus there were  significant differences  in  the  S-layer proteins  of closely-related  strains and the suggestion that some form of antigenic variation was occurring (Dubreuil et al. 1990). strains  were  examined  by  In Aquaspirillum  peptide  mapping  and  serpens, two immunological  methods; there is apparently a degree of similarity between the S layer proteins (Koval et al. 1988).  Bacillus  stearothermophilus  A more general study of 39  strains,  focussing  primarily  on  molecular weight of the S-layer protein and appearance by electron microscopy, indicated remarkable variety not only in the presence or absence of S-layer but also the basic geometry of the paracrystalline structure and the size of the protein involved (Messner et al. 1984). A similar finding was made for several species of Desulfatomaculum nigrificans (Sleytr et al. 1986b).  Studies with strains of Bacillus  sphaericus also noted variation in presence or absence, molecular weight and antigenicity of the S-layer proteins (Lewis et al. 1987; Word et al. 1983).  A study of Bacillus  brevis strains, which often  produce a double S-layer, showed that the middle wall protein to be immunologically conserved between strains whereas the outer wall protein was not (Gruber et al. 1988).  Overall, these studies show that  the degree of S-layer conservation within related strains varies from species to species.  4.4  Ionic  and  expression  requirements  for  C.  crescentus NA1000  growth  I crystallization of RsaA  Wild-type C.  crescentus NA1000 does not grow in M Higg 1 0 128  medium and calcium titration experiments indicated that cultures became growth rate limited at concentrations less than 250 mM calcium (Fig. 22).  A number of other cations could substitute for  calcium and permit NA1000 to grow in the M Higg 1 0 medium.  All divalent cations, with the exception of magnesium, and  trivalent cations tested permitted growth. minimal  minimal  medium  supplemented  sodium, potassium or lithium.  with  Growth did not occur in the  mono-valent  cations  Cell growth in the presence of ions  other than calcium occurred with greater mean generation times and lag periods.  Furthermore, the growth rates with these other ions  varied from experiment to experiment resulting in greater standard deviations in comparison to calcium grown cells. electron  microscopy  supplemented  determined  Higg 1 M 0  medium  that  cells  blebbed  from large  Negative-stain non-calcium quantities  of  membranous material with the exception of cells grown in strontium supplemented calcium  Higg 1 M 0 medium.  This observation suggests that  and strontium act to maintain the integrity of the cell  membranes.  Metal ions are known to play an important role in  maintaining the cell membranes of other bacterial species (Beveridge 1981).  These growth studies indicated that ions other than calcium  or strontium could permit growth in M Higg 1 0 medium although such cells were less healthy. The growth characteristics of NA1000 in M Higg 1 0  medium  supplemented with 0.5 mM of one metal ion indicate that calcium and strontium are the preferred ions. 129  The ability of other ions to  substitute for the preferred ions indicates that the membrane is somewhat flexible with respect to the cations used for stabilization if membrane stabilization is the role of these cations in the physiology of NA1000.  Magnesium is clearly the poorest substitute for calcium  or strontium.  The M Higg 1 0 medium, containing 2.2 mM magnesium,  required an additional 800 mM magnesium to support any growth of NA1000.  Cells cultured in medium containing 3 mM magnesium had  the longest lag periods and greatest mean generations times.  This is  in contrast to the metal ion preference shown by Escherichia and Pseudomonas  coli  aeruginosa where magnesium is the preferred ion  for stabilization of the outer membrane (Coughlin et al. 1983; Ferris and Beveridge 1986; Nicas and Hancock 1983).  Nicas and Hancock  (1983) determined that growth medium containing 0.5 mM Mg 2 produced a wild-type outer membrane whereas medium containing Mg produced altered outer membrane. + 0.02 mM 2 The ability of various divalent and trivalent metal ions to substitute for calcium is somewhat surprising.  However, once a  metal ion is introduced into an aqueous environment such as a minimal medium, it is difficult to predict what chemical form the metal will adopt.  For example, metals can be in the free ion form,  adopt a form via the interaction with water molecules, or form a complex through the interactions with hydroxyl or carbonate species (Collins and Stotzky 1989).  Therefore, it is difficult to predict the  size and charge of the species of the metal ion that is active to permit cell growth. permitting  In order to unambiguously show that the metal ions growth  are  acting 130  to  stabilize  the  cell  membranes,  quantitative studies of the metal content of the inner and outer membrane need to be conducted.  Unfortunately, at present there is  no method available to separate the inner and outer membrane of C.  crescentus and all cell disruption methods used to isolate the cell membrane  fraction  from  the  cytoplasm  has  resulted  in  the  solublization of large amounts of LPS (Walker and Smit, unpublished data).  However, quantitation of the cations bound to purified LPS  and to whole cells grown in the presence of various metal ions may prove to be informative. Negative-stain electron microscopy indicated that crystallized S-layer could be found on the cell surface of NA1000 only when grown in M Higg 1 0  supplemented with calcium or  higher concentrations of strontium were required.  strontium, but  Higher strontium  concentrations were also required for the crystallization of S-layer sheets in cultures of JS1001.  The native template for the S-layer, the  cell surface of NA1000, could use a lower concentration of either divalent cation to mediate S-layer crystallization than the template provided by apposed S-layer subunits to produce the double sheets in JS1001 discussed  cultures. in  section  The in 4.2,  vitro  indicated  crystallization that  strontium would not substitute for calcium.  under  experiments,  those  conditions  The requirement for a  suitable template and the effect that the template has on the cation concentration required for crystallization is illustrated by Table III. The data indicates that if very high strontium concentrations had been used in vitro, crystallization of RsaA may have occurred. 131  The  effect of template quality on the concentration of cation required to mediate crystallization has been noted in other species.  Koval and  Murray (1984b) demonstrated that 10 mM calcium was required for S-layer to crystallize on naked envelopes of Aquaspirillum  serpens  whereas 0.5 mM calcium would mediate S-layer crystallization on the denuded cell surface. Unlike C.  crescentus NA1000,  other Gram-negative  S-layer  producing species will grow under sever calcium limitation. growth  Aquaspirillum  of  serpens  VHA,  putridiconchylium, and Azotobacter  vinelandii  containing  studied.  no  added  calcium  has  been  The  Sp irillum in  medium  Aeromonas  salmonicida has been studied in growth medium containing 0.5 M calcium.  Aquaspirillum  serpens VHA will grow but continuous  subculturing in such medium results in eventual cell lysis (Koval and Murray 1984b).  Spirillum  putridiconchylium grows, however, the  cells exuded membranous material into the medium (Beveridge and Murray 1976c).  For both Azotobacter  and Aeromonas  salmonicida (Garduno et a!. l992b), the cells grow  vinelandii (Doran et al. 1987)  but produce an S-layer with an altered conformation. The substitution of calcium with a number of cations allowed the growth of C.  crescentus in M Higg 1 0 medium, but these cells did  not produce an S-layer.  Thus, the cell is somewhat flexible with  respect to the ions that will allow growth but has a strict ionic requirement for S-layer production. for calcium  to mediate  Only strontium can substitute  S-layer crystallization. 132  The  ability for  strontium  to  substitute  for  calcium  in  for  vivo and in  vitro  crystallization of S-layer, in other Gram-negative species, has been noted for Spirillum  1976c), Azotobacter serpens  MW5  Lampropedia serpens  putridiconchylium  (Beveridge  and  Murray  vinelandii (Doran et al. 1978), Aquaspirillum  (Kist  and Murray,  1984),  the  outer  S-layer  of  hyalina (Austin and Murray 1990), and Spirillum  VHA  (Buckmire  and Murray  1970).  The  ability  for  strontium to substitute for calcium is not limited to the role these ions play in S-layer crystallization but has been noted in a number of physiological  processes  (Huh  attachment of Rhizobium  et  al.  1991).  For  example,  the  leguminosarum to the roots of leguminous  plants is mediated by a calcium salt bridge between a bacterial derived calcium binding protein, rhicadhesin, and the plant surface, and strontium can replace calcium in this interaction (Smit et al. 1991).  Calcium and strontium are very similar with respect to many  physicochemical properties, such as charge character and ionic radii, and this similarity is is often considered the reason that the two ions can substitute for one another in a number of biological processes (Fenton 1987; Huh et al. 1991; Martell 1961).  However, there are  only limited examples of strontium participating in the physiology of cells  in  their natural  environments  (Schultze-Lam  and Beveridge  1994). It was of interest to determine if the S-layer protein remained cell associated during growth on ions other than calcium or if it was released into the growth medium. 133  During growth without calcium  Aquaspirillum  serpens VHA secretes S-layer into the medium while  the S-layer of Azotobacter  vinelandii and Aeromonas  salmonicida  remains attached to the cell surface (Doran et al. 1987; Garduflo et al. 1992b; Koval and Murray 1984b).  The experiments using liquid  cultures indicated that unless cells were grown in the presence of calcium  or  strontium,  the  S-layer was not cell associated,  indicating that it was secreted into the medium (Fig. 28).  thus  However,  when NA1000 cells were scraped off plates and examined for S-layer protein, it was  detected only if the cells  presence of calcium or strontium. for cultures  of JS1001  were cultured in the  The same observation was made  even though the strain grows very well,  although with a slightly greater generation time, in unsupplemented Higg (Fig. 29). M 10 unknown,  At present the location of the S-layer protein is  but three possibilities  exist.  1.  The S-layer may be  degraded by a protease unless it is folded into a crystallized array. There are no reports in the literature to indicate that C.  crescentus  produces a secreted protease, although it is known that unfolded or improperly folded proteins are often more susceptible to proteases. 2. The S-layer may also be folded or aggregated in a form that will not enter a gel during SDS-PAGE.  It is known that RsaA, if boiled in  the presence of SDS, will not enter a gel (Smit et al. 1981) and that the  macroscopic  precipitate,  formed  in  calcium-containing  liquid  cultures of JS1001, composed of RsaA will not enter a gel unless first extracted  into  8  M  urea.  However,  during  growth  in  calcium/strontium minus liquid medium no macroscopic precipitate is formed in cultures of JS1001. 134  3. In the absence of calcium or  strontium the S-layer may be blocked at the level of transcription, translation or secretion.  It is known that calcium and magnesium act  to inhibit transcription of one of the major S-layer gene promoters of Bacillus brevis 47 (Adachi et a!. 1991).  C. crescentus also contains a  gene, flbF, which is very similar to a gene that is conserved in Yersinia species, lcrD, that has been implicated in calcium signal transduction (Piano et a!. 1991; Ramakrishnan et al. 1991; Sanders et al. 1992). to  So there is some indication that C. crescentus may be able  sense  environmental  calcium  levels.  Clearly,  further  experimentation into the fate of the S-layer protein during growth on ions other than strontium or calcium is required.  4.5  Investigations  independent  I  of  the  S-layer  genetic  basis  of  attachment-defective  the  calcium-  phenotype  The inability to isolate calcium-independent mutants from the transposon library indicates independent  that the locus defining the calcium-  I S-layer attachment-defective phenotype is  not  a  target for Tn5 integration or consists of more than one chromosomal site.  If the latter explanation is true then the calcium-independent  S-layer attachment-defective strains did not arise by a single step mutational event. mutants  were  The method by which the calcium-independent  selected  involved  an  “enrichment”  step  in  liquid  medium before plating and so it is possible that double mutants may have been selected (see appendix I; method A). The Tn5 library was screened by Mr. P. Awram using a colony 135  immunoblot  procedure  and  S-layer  attachment-defective  were identified (see appendix I; method C).  mutants  The cell surface of the  mutants were extracted with NaCJ[EDTA and the LPS was analyzed by SDS-PAGE and silver staining (Fig. 30).  All of the Tn5 S-layer  attachment-defective  to  banding patterns.  mutants  were  found  event  altered  LPS  However, none of the mutants tested were capable  of growth in unsupplemented M Higg 1 0 medium. mutational  have  resulting  in  the  This indicates that a  attachment-defective phenotype  does not also result in a calcium-independent phenotype.  Calculation  of the reversion rates from the spontaneous calcium-independent / attachment-defective phenotype to the wild-type phenotype  would  determine if the calcium-independent mutants resulted from a single or double mutational event. A cosmid library of NA1000 was electroporated into the S layer negative and calcium-independent strain JS1004 instead of the parent strain, JS1001, to allow better access of the anti-SAO sera to the cell surface during an immunoblot screen designed to detect renewed production of SAO.  Smit et a!. (unpublished) has shown that  the S-layer blocks access of antibody to the SAO.  Two cosmids, D12  and D13, were isolated that allowed production of SAO in JS1001 although at less than wild-type levels.  However, JS1001 containing  cosmid D12 or D13 were still unable to anchor the S-layer to the cell surface.  Perhaps  subcloning the Caulobacter  DNA to another  plasmid vector or deleting extraneous DNA will result in increased SAO production and S-layer attachment. 136  It has been indicated in  Aeromonas  salmonicida and Acinetobacter 199A that transport of  S-layer and LPS are coupled (Belland and Trust 1985; Thorne et al. If this is the case in C. crescentus complementation of the 0-  1976).  antigen in trans may not result in a functional attachment between the S-layer and the SÃO.  However, more studies with cosmid D12  and D13 must be undertaken before any conclusions can be made.  Conclusions  4.6 4.6.1  The  relationship  and loss of SÃO.  between  calcium-independence  Many of the experiments presented in this thesis  were designed to characterize the cell surface of the wild-type and the calcium-independent mutants of C. crescentus with the intention of discovering the defect in the mutants which rendered them S layer attachment-defective.  Figure 33 is a model of the wild-type  cell surface based, in part, on the information obtained in this study. The  wild-type  cell  produces  three  classes  of  polysaccharide  containing molecules: the “rough” LPS, the SÃO and an EPS.  The SÃO  molecule was found to be absent in all of the calcium-independent mutants  examined  whereas  unaltered in the mutants. calcium  apparently  the  rough  Thus for C.  required  LPS  and  the  EPS  were  crescentus growth without  a mutational event  that consistently  resulted in the loss of the SÃO molecule.  The attendant phenotype  was  and  that  production  of RsaA  continued  the  protein  could  crystallize into an S-layer, if calcium or strontium was available, but the S-layer did not attach to the cell surface. 137  Since no other  alterations of the surface were detected and it was demonstrated in these mutants that the attachment-defective phenotype could not be ascribed to a change in RsaA it was concluded that SAO was a necessary surface component for the attachment of the S-layer. On noting the absence of SAO in  all calcium-independent  mutants, it was hypothesized that SÃO had a net negative charge.  If  this was the case, then the simplest explanation for the necessity to delete SAO in order for the cell to be viable in the absence of calcium was that calcium binds to and neutralizes the charge on the SÃO molecule. between  Without calcium, it was envisioned that charge repulsion adjacent  SÃO  molecules  would  membrane and inhibit cell growth.  destabilize  the  outer  However, chemical analysis of  purified SÃO suggested that the 0-antigen is composed of three neutral molecules.  Therefore another hypothesis must be generated  to account for the relationship between a mutation that allows grow in the absence of calcium and the SAO-negative phenotype. The growth studies of NÃ1000 and JS1001 in M Higg 1 0 medium clearly  demonstrates  that  wild-type  C.  crescentus requires the  presence of calcium or strontium ions to grow normally and produce an this  S-layer, ionic  whereas calcium-independent mutants no longer have growth  requirement.  Examination  of  the  calcium  independent mutant cell surface revealed that SÃO was not present and this was suggested as the attachment-defective  phenotype.  structural basis for the S-layer The  Tn5  S-layer  attachment  defective mutants were all found to have an altered smooth LPS, thus strengthening the argument that the S-layer interacts with the 138  wild-type  smooth  LPS  to  remain  attached  the  to  cell  surface.  However, unlike JS1001 the Tn5 mutants did not share the additional phenotype  of being  calcium-independent.  The  Tn5  attachment-  defective mutants could not be isolated by plating the library on calcium-free medium and once isolated, by the immunoblot screen, they were unable to grow in calcium-free liquid medium. whole,  these results  hypothesis  that  the  can  best be  explained  spontaneous  by  Taken as a  entertaining  calcium-independent  the  mutants  JS1001  and JS1002 did not arise from a single point mutational  event.  The inability to fully complement the calcium-independent  mutant JS1004 with a cosmid also supports the notion of more than one  mutational  event rather  than  a  single  point  mutation  with  pleiotropic effects. If  the  spontaneous  calcium-independent  strains  are  double  mutants and the loss of SAO is not responsible for the calciumindependent phenotype some other alteration in the cell must have occurred.  It is  possible that an  alteration  composition of these mutants has taken place.  in the phospholipid  C.  crescentus has an  unusual phospholipid composition when compared to that of other eubacteria  in  that  it produces  no  phosphatidylethanolamine  (De  Siervo and Homola 1980; Contreras et al. 1978; Jones and Smith 1979).  Approximately 85% of the phospholipids of C.  crescentus  consist of the acidic species phosphatidylglycerol and cardiolipin. Johnson and Ely (1977) noted that the addition of calcium to PYE medium increased both the growth rate and yield of C. 139  crescentus.  Contreras et a!. (1978) suggested that this was a consequence of calcium binding to and neutralizing the high negative charge in the membranes produced by the acidic phospholipids. divalent  cations,  and  calcium  in  particular,  It is known that interact  with  and  influence the structure of acidic phospholipids (Cullis et al. 1983).  4.6.2  The  role  crystallization of the  of  calcium  S-layer.  or  strontium  in  the  Although a number of metal ions  were able to replace calcium to allow growth of NA1000 only strontium or calcium could mediate S-layer crystallization.  The  location in the S-layer protein where calcium or strontium acting to mediate  crystallization  illustrates  the  is  possible  unknown. locations  Figure where  1  in  metal  appendix  ion  II  I protein  interaction may occur.  Three potential calcium binding sites on RsaA  can be suggested:  Within the S-layer monomer thus altering its  1.  conformation to a form that will crystallize.  2.  Between the large  domains of the S-layer monomer allowing crystallization of the unit cell.  3.  Between the small domains of the S-layer monomer allowing  crystallization of the unit cells.  Site directed mutagenesis of the  predicted calcium binding motifs, identified by the rsaA  sequence  (Gilchrist et al. 1992), of RsaA may help determine the actual site(s) where calcium acts to allow crystallization.  4.6.3  The role of the SÃO in S-layer attachment.  Loss or  alteration of the SAO has been shown to result in the S-layer 140  attachment-defective phenotype.  Chemical studies of the purified  SAO indicate that the 0-antigen region if formed from sugars that may impart a hydrophobic character on the molecule.  It is tempting  to envision that the S-layer attaches to the cell surface by the interaction of hydrophobic regions on the S-layer protein and the If this is the case, the hydrophobic regions of the protein which  SAO.  interact with SAO could also interact between S-layer subunits to form the double S-layer sheets observed in mutant C. strains that do not produce SAO. that  the  two  S-layers  forming  crescentus  Smit et al. (1992) demonstrated the  non-cell  associated  sheets,  produced by calcium-independent mutants, interact via the surfaces of the S-layer that in the wild-type situation was proximal to the cell surface. layer  Definitive proof that the SAO molecule is responsible for S attachment  may  be  obtained  by  studies  of  the  S-layer  attachment-defective mutants produced by Tn5 mutagenesis.  4.7  Summary The information contained in this  thesis has enhanced our  understanding of the physiology and cell surface architecture of C. crescentus.  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Wilson, C. M. 1983. dyes and procedures.  Staining of proteins on gels: Comparisons of Meth. Enzym. 91:236-247.  Woese, C. R. 1987. Bacterial evolution. Microbiol. Rev. 51:221-271. Word, N. S., A. A. Yousten, and L. Howard. 1983. 167  Regularly  structured  and  non-regularly  structures  surface  layers  of  Bacillus sphaericus. FEMS Microbiol. Lett. 17:277-282. Yang, L., Z. Pci, S. Fujimoto, and M. 3. Blaser. 1992. of surface array proteins to Campylobacter Bacteriol. 174:1258-1267.  168  Reattachment  fetus cells. J.  6  Appendix  I  Appendix  I  outlines  unpublished  experimental  methods  conducted by other researchers that are pertinent to this thesis.  6.1  Isolation  of  Calcium-independent  mutants.  The following method was used for the isolation of calciumindependent mutants (Smit, unpublished).  C.  crescentus NA1000  was grown in PYE medium and then subcultured to M Higg 1 0 liquid medium. turbid  Four to six days of incubation were needed to develop growth  (whereas  with  calcium-sufficient  occurred with overnight incubation).  medium  growth  The cells were subcultured into  the same medium and incubated for two more days.  Cells were then  plated at appropriate dilutions onto M Higg 1 0 plate medium.  Colonies  that grew were examined for their S-layer characteristics and their ability to grow in the absence or presence of normal concentrations of calcium.  6.2  Production  of anti-SÃO  sera.  Antisera to the SAO was fortuitously raised during attempts to prepare antibody to the adhesive holdfast of strain CB2A (Merker and Smit, unpublished).  Colloidal gold particles (which bind to the  holdfast [Merker and Smit, 19881) were added to cultures of CB2A cells.  The cells were harvested by centrifugation, extensively treated  by sonic disruption to break the cells and treated with RNase and 169  DNase.  The preparation was then subjected to CsC1 density gradient  centrifugation (50% CsC1, wlv).  The colloidal gold particles (and  associated material) sedimented to the bottom of the gradient; these were collected and used for rabbit immunization in a similar fashion to the RsaA immunization.  Analysis of cells incubated with the sera  by indirect immunofluorescence microscopy showed that the sera had little activity to the holdfast material.  However, the cell surface  of S-layer minus but S-layer attachment competent strains CB2A and JS100I  were  completely  labeled  in  immunofluorescence  immunoelectron microscopy experiments using this sera.  and  When S  layer producing strains were examined by the same procedure no cell surface labeling was noted (Smit, unpublished).  63  Colony  immunoblot  attachment-defective  and  for  S-layer  identifying negative  S-layer  mutants.  Mr. Peter Awram developed a colony immunoblot screen using anti-RsaA sera that could differentiate between S-layer attachment defective, S-layer negative and wild-type NA1000 strains (Awram and Smit, unpublished).  While screening the NA1000 transposon  library for mutants that no longer produced RsaA on the cell surface 22 S-layer attachment-defective Tn5 mutants were also isolated.  170  7  Appendix  II  Appendix II lists the results of experiments conducted by other researchers  which  are  pertinent  to  this  thesis.  The  source  of  previously published data is listed in the tables or the figure legends. For  unpublished  data  the  researcher  acknowledged.  171  who  provided  the  data  is  FIG.  1.  Three  dimensional  Caulobacter crescentus NA1000.  reconstruction A.  of  S-layer monomer.  cell formed by crystallization of 6 S-layer monomers. formed by crystallization of unit cells.  the  C.  S-layer B.  The unit  The S-layer  Thin arrows in A, B and C  represent possible sites of calcium interaction with the protein. figure was adapted from Smit et al. (1992).  172  of  This  CA)  D  a  V  FIG.  2.  Proposed  structures  for the Caulobacter  crescentus  exopolysaccharides. (A) The two possible structures for the CB2A EPS repeating unit. (B) The structure of the NA1000 EPS repeating unit. Gic Man  =  D-glucose, Fuc  =  D-mannose.  =  D-fucose, Gui  =  D-gulose, Gal  =  Data from Ravenscroft et al. 1991.  174  D-galactose and  A.  —4’ 3)  -  Gic 4  -  (1  —  3)  -  Fuc  -  (1  —0’ 3)  -  1 Gic  Gic 4  -  (1 —0’  1 Gui  or  —  3)  -  Gic 4  -  (1 —  3)  -  1 Gic  Gb 4  -  (1  —  3)  -  Fuc  -  (1 —*  1 Gui  B.  —*4)-Fuc-(1--3)GIc(1.  4)-Man-(1-3  1 Gal  175  1. -  -  0.0065 0.0060  L()  0)  (.0  c  0)  r—  -  0.0-)0  o 0045 0.0040  .x1e D.0003 I  11000  .  FIG. 3.  200  -  260)0  Analysis of SAO by Laser desorption time of flight mass spectroscopy. The peaks are an average of 176 daltons apart. Unpublished data from A. Rüdiger (GBF-Gesellschaft für Biotechnologische Forschung).  176  iiz  TABLE I. Lipid analysis of Caulobacter crescentus rough LPStZ  Assignment  %  3-OH-C12:O  82  2-OH-C16:1  9  C16:O  5  C18:1  4  a Data from Raven scroft et al. 1992  177  Table II. Sugar composition of Caulobacter crescentus roug h LPS  Sugar  Residue per molecule  2-keto-3-deoxyoctonate  3  -L-glycero-D-mannoheptose  2  cx-D-glycero-D-mannoheptose  1  x-D-mannose  1  x-D-galactose  1  cDglucoseb  1  a Data from Ravenscro ft et al. 1992. b  phosphorylated  178  Table III. Sugar composition of SAO  Sugar  %  Glycerol  13.6  4,6-dideoxy-4-amino hexoseb  12.0  3,6-dideoxy-3-amino hexoseb  15.0  Mannose  1.7  Glucose  0.2  Galactose  0.2  D-Glycero-D-manno-heptose  0.2  L-Glycero-D-manno-heptose  0.5  a D. N. Karunaratne, unpublished (University of British Columbia). b  The amino is acetylated (W.  -R. Abraham, unpublished [GBF  Gesellschaft für B iotechnologische Forschung]).  179  

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