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Mutational analysis of the Rhodobacter Capsulates ORF162B gene Harmer, Andrea Leigh 1998

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MUTATIONAL ANALYSIS OF THE RHODOBACTER  CAPSULATUS  ORF162B  GENE  by ANDREA LEIGH HARMER B.Sc,  The University of British Columbia,  1994  A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE F A C U L T Y OF GRADUATE STUDIES (Department of Microbiology  and Immunology)  We accept this thesis as to the  required  conforming  standard  T H E UNIVERSITY OF BRITISH COLUMBIA April  1998  © Andrea Leigh Harmer,  1998  In  presenting  degree  this  at the  thesis  in  partial  fulfilment  of  University of  British  Columbia,  I agree  freely available for reference and study. copying  of  department  this or  thesis by  publication of this  for  his thesis  or  scholarly her  Department The University of British Columbia Vancouver, Canada  (2/88)  requirements  purposes  may  representatives.  be  It  is  for  an  advanced  that the Library shall  I further agree  for financial gain shall not  permission.  DE-6  the  that  permission  granted  for extensive  by the head  understood  make it  that  be allowed without  of  my  copying  or  my written  ABSTRACT  There are several open reading frames (orfs) of unknown function in the photosynthesis  gene cluster of Rhodobacter  that  including  these  orfs,  photosynthesis. orfl62b  orfl  62 b,  When R. capsulatus  capsulatus.  have  a  It is possible  function  in  bacterial  R N A was probed for the presence of an  transcript, a 4 kb polycistronic mRNA was detected. Strains were  then created which possessed either a polar or non-polar deletion mutation of orfl62b.  These mutant strains were evaluated on the basis of their light  harvesting protein  (LH) and reaction center  band  Mutants were orfl62b  intensities  complemented  a  polar  growth  in  rate  mutation  their  ability  with orfl62b  strain, decreases  photosynthetic When  and  (RC) complex to  levels,  grow  was  restored inserted  downstream orfs cotranscribed with 162b  photosynthetically.  in trans. In a non-polar mutant  L H and R C complex  were  chromatophore  by into  levels  trans orfl  as  well  as  complementation. 62b  such  that  any  would no longer be expressed, a  reduction in L H and R C complex levels as well as photosynthetic growth rate was observed. As with the non-polar mutation, the L H and R C complex levels were increased upon trans the  wild  orfl62b orfl62b  type  generation  time  complementation with orfl62b, was  not  restored.  It  is  whereas  concluded  that  and at least one orf found downstream of, and cotranscribed with, are genes that encode  photosynthesis.  A hypothesis  proteins which are involved in bacterial  that  orfl62b  interaction of complexes is proposed.  functions in the assembly or  TABLE OF CONTENTS  ABSTRACT  ii  T A B L E OF CONTENTS  iii  LIST OF T A B L E S  vi  LIST OF FIGURES  vii  ABBREVIATIONS  x  ACKNOWLEDGMENT  xii  Chapter 1. INTRODUCTION. 1 . 1 Photosynthesis in Rhodobacter  1 capsulatus  1  1 . 2 The photosynthetic apparatus of R. capsulatus  2  1 . 3 The photosynthesis gene cluster  6  1 . 4 Thesis objectives  10  Chapter 2. MATERIALS AND METHODS.  15  2 . 1 Bacterial strains  15  2 . 2 In-vitro  15  recombinant D N A techniques  2 . 3 Growth conditions  16  2 . 4 Spectral analysis of light harvesting and reaction center complexes  17  2 . 5 Bacterial conjugation  18  2 . 6 Gene transfer agent transduction  19  2 . 7 Construction of a translationally in-frame deletion of orfl62b  20  2 . 8 Construction of the suicide plasmid pAH2  23  2 . 9 Ultraviolet light mutagenesis of R.  capsulatus  recombinants 2.10  Construction of a deletion mutation in pAH4 by disruption  of orj162b 2.11 PflM 2.12  26  with a kanamycin resistance  cartridge  Construction of a polar mutation in the orfl62b  26 BsaB  I to  I deletion site  27  Construction of the complementation plasmid pAH8  27  2.13  Southern blots  32  2.14  Northern blots  36  2 . 1 5 Chromatophore isolation  38  2.16  39  Gel electrophoresis of chromatophore proteins  Chapter 3 . R E S U L T S 3 . 1 Construction of R. capsulatus orfl62b 3.1.1  41 mutants  Construction of a non-polar mutation in  41 orfl62b 41  3.1.2  Construction of a polar mutation in orfl62b  3 . 2 Analyses of deletion mutants MWK1 and SBK1  52 52  3.2.1  Southern blot analysis of chromosomal D N A  52  3.2.2  Northern blot analysis of R. capsulatus R N A  55  3.2.3  Photosynthetic growth studies  58  3.2.4  Absorption spectroscopy analysis of L H and RC  complex  levels  62  3.2.5  SDS-PAGE analysis of chromatophore proteins  3 . 3 Analyses of deletion mutants MWSpec and SBSpec  66 69  3.3.1  Southern blot analysis of chromosomal D N A  69  3.3.2  Photosynthetic growth studies  69  3.3.3  Absorption spectroscopy analysis of L H and R C  complex levels  72  3.3.4  72  3 . 4 Trans  SDS-PAGE analysis of chromatophore proteins complementation of orfl62b  mutants  73  3.4.1  Photosynthetic growth studies  73  3.4.2  Absorption spectra  74  Chapter 4 . D I S C U S S I O N  80  Chapter 5 . C O N C L U S I O N S  89  Chapter 6 . R E F E R E N C E S  90  vi  LIST OF TABLES  Table I.  Doubling time of photosynthetically grown R. cells during log-phase  Table II.  61  Doubling time of photosynthetically grown R. cells during log-phase  Table III.  growth  growth  Doubling time of orfl62^-complemented mutants during log-phase low light intensity  photosynthetic  capsulatus  capsulatus 72  R.  capsulatus  growth under 74  Vll  LIST OF FIGURES  Figure  1.  The photosynthetic apparatus of R. capsulatus  Figure  2.  Genes located in the R. capsulatus  3  photosynthesis gene  cluster  Figure 3 .  7  An alignment of the R. rubrum orfl3087 sequence with that of R. capsulatus orfl62b identity  Figure 4 .  shows 25%  sequences  11  Hydropathy plots of the putative gene products of R. capsulatus  Figure 5 .  between the  amino acid  orfl62b  and R. rubrum 13087  12  Outline of the construction of p A H l which contains a translationally in-frame deletion of orfl62b  Figure  6.  21  Construction of the mobilizable plasmid pAH2 which contains a translationally in-frame deletion of  orfl62b 24  Figure 7 .  Construction of pAH4 which contains a non-polar mutation in the deletion site of orfl62b  Figure  8.  28  Construction of pAH3 which contains a polar mutation in the deletion site of orfl62b  30  Vlll  Figure  9.  Construction of the orfl62b  complementation  plasmid  pAH8....  Figure 10.  33  A n example of how homologous  recombination between  strain DW21 and pAH2 would integrate the plasmid into the host chromosome and confer kanamycin resistance to this recombinant organism  Figure 11.  42  Southern blot hybridization analysis  of chromosomal  D N A isolated from strain DW21K1  Figure 12.  44  Homologous recombination between the duplicated orfs located downstream of the Omega cartridge in the P G C of strain DW21K1  Figure 13.  46  Fusion proteins created by the insertion of the K I X X cartridge into orfl62b  at the BsaB  I to PflM  1 deletion  site  Figure 14.  Southern blot analysis of BamU DNA  Figure 15.  49  from orfl62b  I digested chromosomal  mutants  Northern blot analysis of R. capsulatus  53  R N A using an  orf 162b probe  Figure 16.  Photosynthetic  56  growth of R. capsulatus  under variable light intensities  strains grown 59  IX  Figure 17.  Absorption spectra of R. capsulatus  LHII" strains grown  under low aeration conditions  Figure 18 . Absorption spectra of R. capsulatus under low  Figure 19.  64  LHII+ strains grown  aeration conditions  65  S D S - P A G E analysis of chromatophore proteins from R. capsulatus  isolated  SB 1003, MW442 and related strains  grown under low aeration conditions  Figure 2 0.  Photosynthetic  67  growth of R. capsulatus  strains grown  under variable light intensities  Figure 21.  Absorption spectra of LHII" strains grown under low aeration  Figure 2 2.  conditions  Absorption spectra of LHII" strains aeration  Figure 2 3.  70  75  grown under low  conditions  76  Absorption spectra of LHII+ strains grown under low aeration  conditions  78  Figure 2 4. Absorption spectra of LHII+ strains grown under low aeration  conditions  79  ABBREVIATIONS  a a Ap  amino acid ampicillin  r  resistance  ATP  adenosine  5'-triphosphate  ATPase  adenosine  5'-triphosphatase  Bchl  bacteriochlorophyll  Ble  bleomycin  r  resistance  bp  base pair  BSA  bovine  CFU  colony forming unit  Cyt  cytochrome  DNA  deoxyribonucleic  dUTP  2'-deoxy nucleoside  EDTA  ethylenediaminetetra-acetic  Gly  glycine  G m  r  serum albumin  gentamycin  uridine-5'-triph acid  resistance  GTA  gene transfer  ICM  intracytoplasmic  k b  kilobase  KDa  kilodalton  Km  kanamycin  1  acid  agent membrane  resistance  KU  klett  unit  LH  light  harvesting  MOPS  3-(N-morpholino)propanesulfonate  o rf  open reading frame  PAGE  polyacrylamide  PNS  purple  p si  pounds per square inch  RC  reaction  RNA  ribonucleic acid  SDS  sodium dodecyl  Spr  spectinomycin  TBE  tris borate E D T A  TE  tris E D T A  UV  ultraviolet  gel  electrophoresis  non-sulfur  center  sulfate resistance  ACKNOWLEDGMENT  I would like to thank my supervisor, Dr. J. T. Beatty for his support and  guidance throughout the course of my research. His patience, non-  judgmental  manner, enthusiasm  and unwavering dedication  to research  make him an outstanding graduate supervisor. I would also like to thank the members of the Beatty laboratory past and present including Xiao-yi Chen, Farahad Dastoor, Andrew Lang, Heidi LeBlanc, Tim Lilburn, Romina Reyes, A l i Tehrani, Danny Wong, Conan Young and Vladimir Yurkov for their assistance and suggestions. Finally, I would like to thank my committee members, Dr. W. Mohn and Dr. J. Smit, for their helpful advice during my research.  1  1  1.1  Photosynthesis  The inability  in  INTRODUCTION  Rhodobacter  capsulatus  purple non-sulfur (PNS) bacteria, designated as such due to the of  most  considerably  in  Rhodobacter  species  their  to  oxidize  metabolic  capsulatus  elemental  and growth  sulfur  to  capabilities  sulfate,  (Imhoff  vary 1995).  is a PNS bacterium capable of aerobic respiration  as well as anaerobic photosynthetic growth, and so the effects of mutations that  prevent  organism coupled  photosynthetic  by  aerobic  with  availability  the  of  growth  cultivation  simplicity  considerable  be  readily  evaluated  of  mutant  strains.  These  the  PNS  bacterial  of  amounts  can  of  R.  in  this  properties,  photosystem,  capsulatus  genomic  the DNA  sequences, and the wealth of information about the structure and function of  the  bacterial photosynthetic  apparatus, make  R.  capsulatus  an ideal  system to study the function and regulation of genes involved in bacterial photosynthesis  (Bauer  Photosynthesis energy carbon  1995).  is widely  thought of as the process  is used in plants, algae dioxide  definition  has  for  phosphodiester  carbohydrate  been  which light energy  and photosynthetic  set is  bonds  forth,  synthesis.  defining  bacteria to  However,  photosynthesis  transformed into chemical energy, within A T P molecules.  by which light  a as  more  assimilate general  a process  by  in the form of  In PNS bacteria, the A T P  formed in this process may be the sole source of energy during anaerobic photoheterotropic  growth,  in which  organic  compounds  are  oxidized  to  generate  reducing power  [NAD(P)H]  and biosynthetic  intermediates  (Gest  1993).  Photosynthetic energy conversion in R.  capsulatus  takes place in the  intracytoplasmic membranes (ICM). These invaginations of the cytoplasmic membrane generate  spherical as well  the  pigment-protein  and  Golecki  complexes  of  1995). Under low  as tube-like structures that contain  the  photosynthetic  concentrations  of  apparatus  oxygen,  (Drews  photosynthetic  pigments are synthesized which associate with the reaction center (RC) and light harvesting (LH) complex proteins in the I C M (Fig. 1) (Drews 1986). The  process  of  photosynthetic  energy  conversion  begins  with  the  absorption of a photon by a L H complex, followed by the transfer of this absorbed sequence  energy of  to  a reaction center  electron  transfers.  where  The  it is  electron  utilized to initiate a  transfers  result  in  the  formation of a proton gradient which is used to drive A T P synthesis (Bauer et al,  1.2  1993).  The  LH function  photosynthetic  complexes to  increase  apparatus  contain the  light  photon  of  R.  capsulatus  absorbing  pigment  capturing area  of  molecules  the  that  photosynthetic  apparatus. The majority of light energy harvested by the R C is received from  these  complexes.  bacteriochlorophyll molecules  (Miroslav  Pigments  (Bchl)  and,  involved for  in  shorter  energy  transfer  wavelengths,  include  carotenoid  et al., 1996). The L H I complex of purple bacteria is  thought to contain about 32 Bchl molecules, based on a structure obtained  3  Figure 1. The photosynthetic apparatus of R.  capsulatus.  Light energy  (depicted as wavy lines) is absorbed by bacteriochlorophyll molecules (ringed structures) of the L H I and LHII complexes before being transferred to the R C . A charge separation in the R C initiates a series of electron transfers, which in turn generates a proton gradient utilized in A T P synthesis (Fig. taken from Wellington et al,  1991).  4  CYTOPLASM  5 with the Rhodospirillum molecules  can  be  rubrum  distinguished  L H I (Karrasch by  their  long  et al.,  1995).  wavelength  These  absorption  maximum, resulting from protein-pigment interactions within the complex, of 870 to 875 nm. Protein-free Bchl molecules  absorb light at about 770  nm, and it is through poorly understood interactions with amino acid side chains in proteins that the long wavelength shifted  (Zuber  (including R. B800-850  and  capsulatus)  complex.  Rhodopseudomonas possess  Cogdell  According  molecules  In  some  photosynthetic  bacteria  there is an additional antenna called the LHII or  acidophila  18 Bchl  1995).  absorbance maxima are red-  to  crystal  structure  analyses  in  strain 10050, each LHII is thought to  which absorb at 850  nm, and 9 Bchl which  absorb at 800 nm for a total of 27 Bchl molecules (McDermott et al., 1995).  Both LHI and LHII antennae are composed of two hydrophobic, low molecular  mass  proteins,  designated  a and (3, which are organized in  heterodimer pairs to form subunit structures. The light absorbing pigments are noncovalently attached to these dimers. The intact antenna are  oligomers  of  (Miroslav et al,  a (3 protein  subunits  and  their  associated  complexes pigments  1996).  Three protein subunits designated heavy (H), medium (M) and light (L) according to their SDS-PAGE mobilities make up the R C complex which spans the I C M . A special pair of Bchl molecules  within the R C captures  energy from the LHI, which seems to be arranged as a ring of 16 a (3 dimers that encircles  the R C . The L H I - R C  (Miroslav et al,  1996).  structure is called the core complex  The  LHII  antenna,  when  present,  is  arranged as  a collection  of  separate, excentric rings around the concentric L H I / R C core structure. The number  of  LHII  complexes  associated  with  each  core  complex  varies  depending on the growth conditions of the bacteria. Cells grown under low light conditions synthesize  more LHII structures relative to L H I / R C cores  (Garcia et al., 1987). Between 8 to 10 LHII rings, each composed of 9 a (3 pairs  and their associated  complex  such  neighboring together  that  LHII  they  make  structures  close  are believed contact  (Miroslav  to  with  et al.,  surround  the  the  L H I as  1996). The core  core  well  as  complex  with peripheral LHII is called the photosynthetic unit. In purple  bacteria, photosynthetic the  pigments,  membrane,  extended  are  regions  units, which are arrayed parallel to the plane of  densely  packed  of complexes  in  the  I C M and  appear  that are functionally connected  to  form  for light  energy transfer (Sundstrom and van Grondelle 1995).  1.3  The  photosynthesis  gene  cluster  Many of the genes that code for the polypetides pigment  biosynthesis  photosynthetic the R. (Fig.  (Alberti  overlapping transcription  and  structural  proteins  of  the  apparatus are grouped together in a 46 kilobase region of  capsulatus 2)  enzymes,  that comprise the  genome  termed the photosynthesis  et al.,  1995).  operons of  or  specific  This  "superoperons" photosynthesis  gene which  gene cluster (PGC)  cluster  is  organized  result  in  the  genes  from  more  into  concurrent than  one  promoter. This feature of the P G C is thought to allow for the efficient shift from aerobic respiratory to anaerobic .photosynthetic growth in response to  7  Figure 2. Genes located in the R. capsulatus Bacteriochlorophyll biosynthesis  photosynthesis  genes (bch)  diagonal lines, carotenoid biosynthesis  gene cluster.  are designated by  genes (crt) are shown as  cross-hatched boxes, L H and R C genes (puh and puf) are represented by lightly shaded boxes, and open reading frames of uncertain function (orfs) are shown as darkly shaded regions. The arrows represent transcripts of the puh A region that were reported in (Bauer et al., 1991).  o r f 6 4  7  pufM pufL pufA pufB pufQ bchl bchY  [ X X X X X I  kAAAAj  bchX bchC crtF crtE crtD crtC crtK crtB crtl crtA bchl bchD orf284 orfl 76 bchP orf428 bchG bchJ bchE orf469 bchW bchF bchB bchK bchH  Bam H  I  Bam H I  ft  bchl bchM FT 696 f puhA or f214 orfl 62b  orf55 orf274 orfl 62a  changes in environmental conditions (Beatty 1995). Located in the P G C are: the puf P  genes which encode the R C L and M polypeptides and the LHI a and  protein  functioning  subunits; in  the  crt  carotenoid  and bch  genes,  which  encode  enzymes  and bacteriochlorophyll biosynthesis;  and  the  puh A gene encoding the R C H subunit. Five open reading frames (orfs) are located  downstream  puh A.  It is possible  orfl62b,  orf55,  of  orf274  with puhA  (Wong  experiments  revealed  and  in  the  same  that at least some of these orfs, termed and orfl62a,  the  et al.,  1996),  only  the two  orfs  downstream  as  orf214,  are genes transcribed as a unit along although  puh A  molecules  which have 3' ends shortly after the puhA  Since  transcriptional orientation  transcript  shown  in Fig. 2,  gene (Bauer et al.,  of puhA  mapping both of  1991).  were first revealed  by D N A  sequence analysis, it was not clear if they were expressed (i.e., were genes) and, if so, had a function in photosynthetic and  complementation  containing frame  either  (non-polar)  that orf214,  experiments  growth. Recent gene disruption  carried out  on  transcriptionally polar disruptions deletion  mutations  within puhA  R.  capsulatus  strains  or translationally inand orf214  suggested  and at least one additional gene downstream of orf214,  were  required to obtain wild type levels of L H I and R C complexes. For example, when a polar antibiotic resistance directly  downstream  mutant was strain was  incapable  of  puhA  cartridge was placed in orf214, and upstream of orfl62b  of photosynthetic  complemented  the orf  (Fig. 2),  growth. When this  this  polar mutant  with a plasmid containing orf214  the organism  did not fully regain the ability to grow photosynthetically.  In contrast, a  strain containing  a translationally in-frame  photosynthetically  impaired but when  it was  deletion  of  orf214  complemented  was also  in trans with  orf214,  it was capable of photosynthetic growth at a rate similar to that of  the parental strain (Wong et al.,  1996). These data indicated that at least  one gene downstream of orf214  (and within the same transcription unit)  has an affect on the ability of R. capsulatus  In addition, Rhodospirillum similarity to R.  capsulatus  are located  at the  capsulatus  homologues.  acid  to  rubrum  photosynthesize.  contains two orfs with sequence  orf214 and orf!62b,  same  positions  relative  Although R.  sequence with only  and these R.  to the puhA  rubrum orfI3087  25% identity  to R.  rubrum  orfs  gene as the  encodes an amino  capsulatus  orfl  62b  in an  alignment (Fig. 3), hydropathy plots of the putative gene products of capsulatus show  orfl62b  the presence  and R.  rubrum orf13087  of a possible  R.  R.  are qualitatively similar and  transmembrane sequence near the amino  terminus of both proteins, followed by similar hydropathy profiles of less hydrophobic capsulatus  sequences  orf 162b  with an analogous orf 162b  (Fig.  and R.  4).  These  similarities  rubrum orf13087  encode  function. It is conceivable  and orf13087  serve as  suggest  homologous  that  R .  proteins  that the amino termini of  anchors which localize these polypeptides  to the intracytoplasmic membrane, while  the rest of the proteins, which  possess a cluster of highly conserved amino acids from Glyl29 to Glyl39 (R.  capsulatus  putative  1.4  sequence;  Fig. 3) provide a functional sequence to these  polypeptides.  Thesis  objectives  It would be  useful  to  learn more information about  the  possible  11  R.rubrum 13087.aa R.capsulatus 162b.aa  M SAG HRDPIFPRGL KIATLGLVLL TFGLIGFS-- RLTDVGHSTL MAQLPLSPAP QRPETKTPGK PEAELIPKPL LRAMIGIALL SLALTTYAVL  R.rubrum 13087.aa R.capsulatus 162b.aa  PAATAEGSAW ISFLPRENGD V--AWERD- --SAREIAIL AS-GDNNFAV 86 TGRPHEGVPA PGKWAEKLV VLKDIDARHA TVSDPEGNIL LDLPEGGFVD 100  R.rubrum 13 087.aa R.capsulatus 162b.aa  GMLRGLARQR ARIGVAATDP YELTRWTDGR LTMTDPATGH IIVANAFGSK 13 6 VMAAAVRRSR AVARITDNPP VRIVRYDNGR LAMEDPATGW STELYAFGAD 150  R.rubrum 13087.aa R.capsulatus 162b.aa  SAAQMNGFYD AAQAARDGR •-ERILDMK SKA -AF-  Figure 3.  A n alignment o f the R.  rubrum  with that of R.  capsulatus  the  Identical a m i n o  sequences.  42 50  155 162  orf 13087  orf 162b  amino acid  sequence  shows 2 5 % identity between  acid  residues  are  shaded.  12  Figure 4. Hydropathy plots of the putative gene products of R. orf 162b  and R. rubrum  capsulatus  13087. Hydrophobic sequences of  residues are given positive values on the hydropathy index shown in the y-axis. Amino acid positions are shown in the xaxis. The hydropathy index was calculated using a window of 11 amino acid residues. The certain and putative boundaries on the y-axis represent the probability of a transmembrane segment based on the Goldman, Engelman and Steitz (GES) algorithm included in the TopPred computer software program (Claros and von Heijne 1995).  R. rubrum orf!3087  function of the orfs located downstream of puhA  to see if they are genuine  genes, to see if they are required for normal photosynthetic growth, and if so,  to  use  understanding  this of  information the  process  to  design  of  bacterial  experiments  to  improve our  photosynthesis.  The research  described in this thesis is centered on the orfs of unknown function located downstream  of puhA  ascertain if orf 162b  on the P G C . The explicit aim of this thesis is to encodes a protein and, if so, to determine if this gene  product is involved in the process of photosynthesis in R.  capsulatus.  15  2  2.1  MATERIALS AND METHODS  Bacterial strains  The R. capsulatus  parental strains used in this thesis were  SB 1003  (Solioz and Marrs 1977), a derivative of the natural isolate BIO (Weaver et al.,  1975), and MW442 (Scolnik and Marrs 1987), an LHII" derivative of  SB 1003.  R. capsulatus  DE442, a gene transfer agent (GTA) overproducer  that produces phage-like gene transducing particles (Yen et al., 1979), was used  as  a G T A phage  strains Escherichia 1983)  were  donor in transductions.  The plasmid mobilizing  coli TEC5 (Taylor et al., 1983) and SI7-1 (Simon et al.,  used in conjugation experiments  with R.  capsulatus.  E.  coli  C600 r-m+ (Bibb and Cohen 1982) and DH5a (Life Technologies, GIBCO BRL) were  used  as  host  strains  for  the  construction  and  maintenance  of  plasmids.  2.2 In-vitro  recombinant  DNA techniques  Plasmid D N A was isolated from E. coli cultures by the alkaline lysis technique (Sambrook et al., 1989) and purified with the use of Qiagen D N A affinity  columns. D N A was  purified  from  agarose  gels by adsorption to  silica beads, using a QIAEX D N A Extraction Kit (QIAGEN Inc.).  General  DNA  manipulations  such  as  restriction  digestion, D N A dephosphorylation and ligation, agarose  gel  endonuclease electrophoresis  and  calcium  (Sambrook  chloride  transformation  performed  as  described  et al., 1989). New restriction enzyme sites were inserted by the  linker tailing technique (Lathe et al.,  A  were  Gene  Pulser  apparatus  1984).  was  used  as  per  the  manufacturer's  instructions to electro-transform E. coli cells (BIO-RAD Laboratories). D N A sequencing was performed by the Nucleic Acid Protein Service Unit at the University of British Columbia using an ABI automated D N A sequencer.  2.3  Growth  R.  conditions  capsulatus  strains were grown at 30 to 3 4 ° C in either R C V , a  minimal medium containing malate as the sole carbon source, or YPS, a rich medium Strains  containing grown  yeast  extract  on plates were  Photosynthetically  grown  anaerobic jars (Becton,  plate  and  grown  peptone on media  cultures  were  (Weaver  et al.,  containing grown  15  1975).  g/1  agar.  in  B B L GasPak  Dickinson and Co.). E. coli strains  were grown in  Luria-Bertani (LB) medium (Sambrook et al., 1989).  Antibiotics, the following 10 pg/ml, E.coli  when present  concentrations:  streptomycin  10  in R.  kanamycin sulfate  kanamycin sulfate  gentamycin sulfate 10 p g / m l .  cultures, were added to 10 p.g/ml,  pg/ml and gentamycin  cultures, higher concentrations  200 pg/ml,  capsulatus  50  of antibiotics  (ig/ml,  sulfate  spectinomycin 2 p,g/ml.  For  were used: ampicillin  spectinomycin  50  pg/ml  and  In grown  preparation for photosynthetic under oxygen-limited  growth, R.  conditions  in  capsulatus  cells were  R C V medium in Erlenmeyer  flasks filled to 80% of their nominal capacity. These cultures were shaken at 150 R P M in a gyrorotary water bath shaker at 3 4 ° C for 2-3 days until the cultures reached a density of approximately 300 Klett units ( K U ; 1 K U is about 3.3 x 10 pigmented.  For  CFU/ml).  6  anaerobic  These  low-oxygen  photosynthetic  cultures  were  liquid  cultures  growth,  inoculated to a turbidity of approximately 30 K U from the cultures.  These  capacity  with  filled  cultures liquid  were placed in screw-cap  growth  aquarium. The water  incandescent  lamps  bath was  at various  with a L I - C O R photometer (LI-COR  medium  and incubated  Light  were  oxygen-limited  Pyrex tubes filled at  to  3 4 ° C in a water  illuminated with  intensities.  highly  tungsten  intensity  was  filament measured  equipped with the LI-190SB quantum sensor  Inc.). Water temperature and circulation in the water bath were  maintained using a Haake D3-V circulator (Haake Mess-Technik GmbH Co.). Growth rate was followed by measuring the culture turbidity using a KlettSummerson Photometer with a red (No. 66) filter.  2.4  Spectral  analysis  of  light  harvesting  and  reaction  center  complexes  R. capsulatus  cultures were grown under oxygen-limited conditions to  early stationary phase (150 KU). About 1.3 ml (6.5 x 10  8  CFU) from each  culture was harvested and the cells were resuspended in a solution of 1 ml of  22.5% bovine serum albumin (BSA) in R C V medium. The presence  of  B S A reduces light scattering by cells within the sample and thus results in  a better measure of absolute absorption. Each sample was scanned for light absorption over a range of wavelengths (350 U-2000 and  spectrophotometer.  to 1000 nm) using a Hitachi  Data were collected using SpectraCalc software  spectra were analyzed with the Grams 386 software  Industries  Corporation).  Pigments  Therefore,  in  compare  order  to  do  not  spectra,  absorb scans  package (Galactic  light were  of  650  nm.  normalized  by  multiplication of spectra by a factor to yield a light scattering A650 of 0.2.  2.5  Bacterial  conjugation  Conjugation  of  plasmid  D N A into  R.  capsulatus  cells (with  the  exception of strain DE442) was usually accomplished using E. coli SI7-1 as the  plasmid  capsulatus ratio  donor  strain.  Overnight  cultures  of  donor  cells  and  R .  recipient cells grown to stationary phase were mixed in a 1:2  by  volume,  microcentrifuge  pelleted  (1  minute,  and resuspended  in  an  15,000 equal  g)  in  volume  a  of  bench  top  antibiotic-free  R C V medium. A 10 pi volume of the mixture was deposited onto a dry R C V agar  plate.  overnight  After  at 3 0 ° C  the  liquid  had  absorbed,  allowing conjugation  the  plates  to take place.  were Negative  incubated controls  consisted of equal concentrations of recipient cells in the absence of donor cells.  Cells from each spot were streaked onto R C V minimal medium agar plates containing the appropriate antibiotic in order to purify R. transconjugants. After 3-5 days of growth at 3 0 ° C , isolated  capsulatus  colonies  were  restreaked onto YPS medium containing antibiotics. Although the growth of  auxotrophic E. coli donor cells is not possible on R C V medium, the YPS plate which permits growth of the donor cells, ensures that the exconjugant not been contaminated with E.  coli.  2.6  transduction  Gene  The  transfer  agent  G T A over-producer, R. capsulatus  has  DE442, was used as the donor  strain in G T A transductions. Recombinant pUC plasmids of interest from E. coli  hosts were first transformed into the plasmid donor strain, E. coli TEC5.  This strain possesses a mobilizable vector (pDPT51) that contains a region of  homology  recombination  to  the  al.,  Mobilizable  plasmid  1983). and  Thus  a  of the  DE442.  recombinant  et  plasmid is thought to allow for both the transfer and maintenance in  the  (Taylor  TEC5  plasmid  between  plasmids  the  recombinant  event  pUC  recombinant  plasmids  transferred to DE442 by conjugation were selected for by the acquisition of antibiotic  resistance on R C V agar plates. The exconjugants  photosynthetically  in rich  YPS medium to  stationary  were grown  phase in order to  induce G T A production. The DE442 culture was filtered through a 0.45 p,m Gelman Supor membrane filter in order to remove the bacterial cells and obtain the GTA-containing filtrate.  Recipient R. capsulatus to  stationary  phase  and  cells were grown aerobically in YPS medium 0.1  microcentrifugation and resuspended  ml  of  in 0.1  culture  was  pelleted  ml of filter-sterilized  by  G buffer  (10 mM Tris-HCl, pH 7.8, 1.0 mM CaCl2, 1.0 m M NaCl, 500 p.g/ml BSA). A volume of 0.2 ml of G T A filtrate was mixed with 0.4 ml of G T A buffer and  20 0.1 ml of resuspended recipient cells and incubated for 60 minutes at 3 5 ° C . During this period, G T A particles adhere to the recipient cells. After phage adsorption, 0.9 ml of YPS broth were added to the culture and the recipient cells  were  incubated  aerobically  recombination and expression  at  30°C  for 4 hours to allow  of the antibiotic resistance  genetic  gene. Portions of  this culture were spread on YPS plates, with the appropriate antibiotic, for the  selection  of  transductants.  Negative  controls  consisted  of  the G T A  suspension without recipient cells and recipient cells in the absence of the GTA-containing  filtrate.  2.7  Construction  orfl  62b  of  a  Digestion of R. capsulatus PGC  into  a  series  of  translationally  in-frame  chromosomal D N A with BamH  fragments  termed  Bam  deletion  of  I cleaves the  fragments,  labelled  alphabetically on the basis of decreasing size (Taylor et al., 1983). A 2051 bp section of the R.  capsulatus  P G C , generated via BamH  called the BamK fragment contains orf214, orfl62b, and  has been cloned into the BamH  orf55,  I digestion and orf274  I site of pUC13 (Messing  (Fig. 2) 1983)  to  create pUC13::BamK (W. Collins, personal communication).  In  order to make  a translationally  pUC13::BamK was cleaved with PflM located  within orfl62b).  region of orfl62b  in-frame deletion  I and BsaB  in  orfl62b,  I (restriction enzyme sites  This double digest removed 63% of the coding  (Fig. 5). Treatment of the PflM  I to BsaB  I fragment with  T4 D N A polymerase removed the 3' protruding terminus created by PflM  I  2 1  Figure 5. Outline of the construction o f p A H l  which contains a  translationally in-frame deletion of R. capsulatus  orfl62b.  R. capsulatus orfs are shown as unshaded arrows. The lacZ ( a allele) and kanamycin resistance genes are shown as lightly shaded arrows. D a r k l y shaded replication.  arrows represent the o r i g i n o f  Hind III  23 digestion. When the blunt ends were joined together in a ligation reaction, an in-frame deletion of orfl62b frame across  the deletion  on pUC13::BamK was created. The reading  site was  confirmed by D N A sequence  analysis  (data not shown), and the plasmid was designated pUC13::BamKAl62b.  Digestion of pUC13::BamKAl62b with Xba I and Mlu I removed 81 bp including  one  photosynthesis polymerase  of  the  two  BamH  I sites flanking the R.  capsulatus  genes of the BamK fragment. The Klenow fragment of D N A  I was  restriction enzyme  used  to  fill  in  3'  recessed  digest. A kanamycin resistance  was cloned into the former BamH  termini  created  by  the  (Km ) cartridge, K I X X , r  I site. The K I X X cartridge was removed  from pUC4::KIXX as a 1.3 kb Sma I fragment containing the K m gene as r  well as the first (Barany  1985).  153 bp of the 380 bp bleomycin resistance Thus,  a Km  deletion of orfl62b  r  marker was  and the entire R.  removed as a Hind  III to BamH  associated  capsulatus  with  (Ble ) r  the  gene  in-frame  D N A segment could be  I fragment from this plasmid,  designated  p A H l (Fig. 5).  2.8  Construction  The R. flanking  of  capsulatus  orf214  the  suicide  BamK  plasmid  fragment  containing  was removed from p A H l  6).  plasmid  The suicide pSUP203  genes, a 7.5 kb BamH  plasmid p R R l  (Simon  et al.,  lll/BamH  was  1983)  the  as a 3 kb Hind  fragment and cloned into p R R l at the Hind (Fig.  pAH2  KIXX  cartridge  III to BamH  I  I site to create pAH2  derived from the  mobilizable  but in addition to the pSUP203  I to Bgl II fragment containing the lacZ  gene was  24  Figure 6. Construction of the mobilizable plasmid pAH2 which contains a translationally in-frame deletion of orf 162b. R. are shown as unshaded arrows. The lacZ  capsulatus  orfs  and K m genes are r  shown as lightly shaded arrows. Darkly shaded arrows represent the origin of replication.  25  Hind III  Hind III  26 cloned into the BamH (R.  Reyes, personal communication). The new plasmid, p A H 2 ,  and,  is mobilizable  although it may be transferred by conjugation from an E.  an R.  capsulatus  expressed  into the R.  in R.  Ultraviolet  R. about  10  of  light  capsulatus 9  capsulatus  capsulatus,  screen for the presence  2.9  coli  host to  recipient, it will not be replicated in the latter cell  unless it integrates be  I site of pSUP203 to generate a 16.1 kb plasmid  chromosome.  allowing  blue/red  The  lacZ  screening  type  gene can  of  cells  to  pAH2.  mutagenesis  of  cells were grown  R.  capsulatus  aerobically  at 3 4 ° C  recombinants  to a density  of  cells per ml. A 5 ml volume of this culture was placed into each  of 5 sterile petri dishes and exposed  to either no U V light (dish #1) or U V  light for 30, 45, 60, or 75 seconds (dish 2, 3, 4, and 5 respectively).  Besides  the negative control, only dish #2  greater  than 0.5%. survival light  2.10  exposure,  the  of  capsulatus  acquiring other  culture exhibiting  an 8%  cultures exhibiting a low mutations  survival rate  during the U V was  chosen  for  experimentation.  Construction  of orf 162b  In KIXX  survival) had a survival rate  A s it was feared that the R.  rate ran a high risk  further  (8%  with  a second  cartridge  of a  a  deletion  kanamycin  approach to create was  inserted  into  mutation resistance  in  pAH4  by  disruption  cartridge  a non-polar mutation in orfl62b, the  deleted  orf 162b  sequence.  the This  27 cartridge rarely has a polar effect when it is inserted into a gene which is cotranscibed with genes located downstream of the cartridge insertion site (Bollivar et al,  1994). A 10 bp dephosphorylated linker ( 5 ' - A G C G G C C G C T -  3') containing a Not  I restriction enzyme site was inserted into the (BsaB  to PflM  site of pUC13::BamKA 162b.  I) deletion  linearized  with  Not  I and treated  with  the  This  plasmid was  Klenow fragment  then  of D N A  polymerase I to fill in the 5' overhangs created by Not I digestion. K I X X cartridge, as a 1.3 kb Sma  I  The  I fragment, was ligated into the filled in  Not I site (Fig. 7). The resulting plasmid was termed pAH4.  2.11  Construction of a polar mutation in the  In order to create a polar mutation in orfl62b expression  of  orfl62b  as  well  as  downstream  transcribed from the same promoter as orfl62b, was  62b  that  would prevent  orfs  that might  I deletion site  PflM  the  BsaB  inserted  into  the  deletion  cartridge confers  spectinomycin  in  regions  the  flanking  of  removed  from  The Omega  and streptomycin resistance and contains,  the  fragment,  pHP45Q as a 2 kb Sma  transcription  and  translation  1984). The Omega cartridge was I fragment. The resulting plasmid,  shown in Fig. 8, was named pAH3.  Construction of the  be  the Omega (Q.) cartridge  site of pUC13::BamKA 162b.  termination signals (Prentki and Krisch  2.12  I to  orfl  complementation  plasmid pAH8  28  Figure  7. Construction of pAH4 which contains a non-polar mutation in a deletion site located within orfl62b. shown as unshaded arrows. The lacZ  R.  capsulatus  orfs are  (a allele) and K m genes r  are shown as lightly shaded arrows. Darkly shaded arrows represent the origin of replication.  SamH I  BamH I  Figure 8. Construction of pAH3 which contains a polar mutation in the deletion site of orf 162b. R. capsulatus  orfs are shown as  unshaded arrows. The Omega cartridge and the lacZ (a) gene are shown as lightly shaded arrows.  3) Ligate the Omega cartridge into the orfl 62b deletion site  An expression vector was created so that orf 162b from a plasmid in R.  capsulatus orfl62b  could be expressed  mutants. Plasmid  p\JCl3::orfl62b  (W. Collins, personal communication) contains orf 162b as a 797 bp Cla I to EcoR  I fragment, including a small region of the orf214  directly upstream of orf 162b. site located yielding  An EcoR  within the orf214  plasmid  I linker was inserted into the Pst I  sequence,  into  the  linker tailing  method, orfl62b  I fragment and reduced the size  fragment by 49 bp to 212 bp.  The EcoRl ligated  by  p U C 1 3 : : o r / 7 6 2 b l (Fig. 9). This allowed the  sequence to be removed as a 748 bp EcoR of the orf214  sequence found  fragment containing orf 162b the  mobilizable  communication) at an EcoR  plasmid  from pUCl3::orfl62b2  pRR5C  (R.  Reyes,  was personal  I site located downstream of a R. capsulatus  puf  promoter (Fig. 9). The plasmid pRR5C is a derivative of the broad host range expression vector pPUFPl being maintained in R. capabilities,  pRR5C  (Bollivar et al., 1994) which is capable of  capsulatus  confers  conjugation of this construct,  cells. In addition to its broad host range  gentamycin  resistance  termed pAH8,  (Gm ), thus  into R.  r  capsulatus  following orf  162b  mutants, the presence of the plasmid was selected for on the basis of G m . r  2.13  Southern  A 100 ml R.  blots  capsulatus  culture was  oxygen conditions to early log phase (150  aerobically  grown under low  K U ) , pelleted by centrifugation  and resuspended in 2 ml of 25% sucrose solution in 50 m M Tris-HCl (pH 8). Added to this solution was 5 mg of lysozyme, 0.5 ml of 0.5 M E D T A and 2  33  Figure 9. Construction of the orfl62b  complementation  plasmid,  pAH8. The G m and lacZ (a) genes are shown as lightly shaded r  arrows. The puf promoter of pRR5C and pAH8 is shown as an arrow located upstream of EcoR represented  I. The origin of replication is  as a darkly shaded arrow.  34  1) Remove 7 4 8 bp orfl 62b seqence as an EcoR I fragment 2) Ligate orfl 62b into the EcoR I site of pRR5C  1) EcoR I digest  pAH8 12.3 kb  ml of Triton-X lysis solution (50 mM Tris-HCl [pH 8], 10 mM E D T A , 2% Triton-X-100  [octyl  phenoxy  polyethoxyethanol]).  This  suspension  was  mixed and incubated at 5 0 ° C for 10 minutes to allow cell lysis. One gram of CsCl  per  ml  transferred solution  of  to  (10  lysate  5.5  ml  mg/ml).  was  added,  ultracentrifuge  and portions tubes  of  the  containing  The tubes were centrifuged  solution  200  were  p i of EtBr  at 297,805 x g for  16  hours at 1 5 ° C . The highly visible chromosomal D N A band was removed and extracted remove  with the  isopropanol  equilibrated  ethidium bromide from the  with  CsCl-saturated  chromosomal  water  D N A . The  to  cesium  chloride was removed by dialysis in l.Ox T E buffer (Sambrook et al., 1989).  Ten p.g  of  chromosomal  D N A were  digested  with  BamH  I and  electrophoresed in a 1% agarose mini-gel in 0.5x T B E buffer (89 mM Trisbase, 89 mM boric acid, 2 mM E D T A ) at room temperature. The D N A was denatured by soaking the gel in 500 ml of a 0.2 M NaOH and 0.5 M NaCl solution for 30 minutes at room temperature. To neutralize the gel, it was washed 2-3 times in 500 ml 5x T B E for 10 minutes at room temperature. The ionic composition of the gel was reduced by soaking it in 500 ml 0.5x T B E for 20 minutes.  A positively  charged nylon membrane (Boehringer Mannheim GmbH)  which had been cut to the size of the mini-gel was equilibrated in 0.5x T B E for 10 minutes at room temperature before being placed on the gel. D N A was transferred to the membrane by electro-blotting for  16 hours, followed  at 30 V in 0.5x T B E  by 80 V for 2 hours in a B I O - R A D  Electrophoretic Transfer Cell  Trans-Blot  (BIO-RAD Laboratories). The membrane  was  rinsed in T B E and air dried. The transferred D N A was fixed to the nylon  36 membrane under vacuum in an 8 0 ° C oven for 2 hours.  The  probe  was  digoxigenin-dUTP (Boehringer detection  by  (DIG-dUTP)  were  provided with  random primed  using  Mannheim GmbH). procedures  instructions  created  a D N A labeling  with  and detection  kit  The prehybridization, hybridization and  carried  the  D N A labelling  out  detection  as  per  the  manufacturer's  kit. Membranes were  incubated  with colour detection solution for 8 hours.  2.14  Northern  blots  Total R N A was isolated from R. oxygen-limited  conditions  or  capsulatus  cells grown under either  photosynthetically  (anaerobically)  density of 150 K U . A volume of 25 ml of R. capsulatus  to  a  culture was added to  a 50 ml centrifuge tube packed with ice and centrifuged for 10 minutes at 12,100 x g, and the cells were resuspended in 125 pi of 0.3 M sucrose, M  sodium acetate (pH 4.5).  To lyse the cells and denature  0.01  the cellular  proteins, 125 pi of a 0.01 M sodium acetate (pH 4.5) and 2% SDS solution were added. The sample was immediately vortexed and heated to 6 5 ° C for 90 seconds and 250 pi of 6 5 ° C unbuffered, dH20-equilibriated phenol were added. The sample was vortexed, cooled in an ethanol-dry ice bath for 15 seconds,  centrifuged  extractions aqueous  were  and  repeated  the on  aqueous the  aqueous  layer layer  was 2-4  retained. times,  Phenol  until  the  phase appeared clear. The R N A was precipitated by the addition  of 30 pi of 3 M sodium acetate and 900 pi of 95% ethanol. The R N A was dissolved and stored at on ice in 180 p i of R N A storage buffer (20 m M  sodium phosphate, 1 mM E D T A [pH 6.6]).  Residual D N A was removed from the R N A preparation by addition of 30 units of DNase I in incubation buffer (100  mM sodium acetate, 5 m M  MgSC»4,  for  pH  temperature.  5.0)  After  was resuspended RNA  followed  by  incubation  phenol extraction  and ethanol  30  minutes  at  room  precipitation, the R N A  in 40 pi R N A storage buffer and stored at - 8 0 ° C .  concentration  was  determined  through  absorbance  values  Total of  the  sample at 260 nm. One absorbance unit was taken to equal 40 pg R N A .  Twenty pg of total R N A were mixed with 2 volumes of sample mix (500  p i formamide,  MOPS,  120 p i formaldehyde  50 m M anhydrous sodium acetate,  heated  to 6 8 ° C for  volume  10 minutes  of loading buffer  ethidium bromide was 1.2%,  and lOx MOPS  (5  10 m M E D T A ,  to remove  ml glycerol,  buffer  any secondary 4 ml dH20,  added to each sample  before  [0.2  pH 6.5])  M and  structure. One 1 ml 10 mg/ml  being loaded onto a  2 M formaldehyde gel and electrophoresed in lx MOPS buffer at 30  V for 16 hours.  After electrophoresis, at room temperature  the gel was soaked in 0.05  for 20  minutes  to fragment  N sodium hydroxide  high molecular weight  R N A molecules for improved transfer. The gel was then rinsed in sterile dH20  and equilibrated 4 times in 0.5x  electro-blotted  onto a Biotrans positively  T B E for charged  10 minutes.  R N A was  nylon membrane (ICN  Biomedicals Inc.) in a B I O - R A D Trans-Blot Electrophoretic Transfer Cell. The  transfer  process  was  carried out  followed by 80 V for 2 hours.  in 0.5x  T B E at  30  V  overnight,  38 The  nylon membrane  was  prehybridization buffer (12.5  prehybridized for  3 hours  in  10  ml of  ml formamide [50%], 5 ml 5x P buffer  [1%  BSA,  1% ficol, 250 mM Tris-HCl pH 7.5, 0.5% sodium pyrophosphate, 5%  SDS,  1% polyvinylpyrrolidone], 5 ml of 50% dextran sulfate,  1.45  g of  sodium chloride and denatured salmon sperm D N A to a concentration 0.1 mg/ml) at 4 2 ° C . The probe was synthesized from the 424 bp BsaB EcoR  I orf 162b  capsulatus  sequence removed from the BamK  of  the  I to R.  PGC (Fig. 2). The Q I A E X agarose gel purified 424 bp fragment  was used to create a Primer  fragment  of  3 2  Labelling kit  manufacturer's  P  radio-labelled probe using  (Amersham  instructions.  International) The  in  a Rediprime Random accordance  prehybridization  with  buffer  the was  supplemented  with approximately 50 ng of alkali denatured probe and the  hybridization  reaction  was  allowed  to  proceed  overnight  at  42°C.  The  hybridization of the D N A probe to its complementary mRNA sequence was visualized -80°C  by  on X - O M A T A R X-ray  intensifying  2.15  autoradiography.  The  nylon  membrane  film (Eastman Kodak  was  incubated  at  Company) with an  screen for 23-72 hours.  Chromatophore  isolation  Chromatophores were isolated from 100 ml cultures of R.  capsulatus  cells grown in R C V medium under low oxygen conditions to a density of 300  K U . These cells were disrupted by  (15,000  psi)  (chromatophores)  to  release  and  passage through a French press,  fragment  the  which contain the photosynthetic  debris was removed from the suspension  ICM  into  vesicles  apparatus. The cellular  by centrifugation  at 25,800 x g  for  8 minutes, and the supernatant was centrifuged at 412,000 x g for 14  minutes  to  pellet  the  chromatophores.  This  crude  chromatophore  preparation was resuspended in 1 ml of chromatophore buffer (20 m M 3[N-morpholino]propanesulfonate (Jackson et al,  7.2])  sucrose  1986)  [MOPS],  100 m M KC1, 1 m M M g C l  and loaded onto  2  [pH  a 3-layered (20%-40%-60%)  step gradient in 50 m M Tris (pH 8) for further purification. The  sucrose gradient was centrifuged at 68,600 x g for 17 hours at 4 ° C . The purified  chromatophores  were  collected  from  the  20%-40%  sucrose  interface, diluted 1:1 in 50 m M Tris-HCl (pH 8), centrifuged at 450,000 x g for  15 minutes  to pellet  the chromatophores, and resuspended  in 500  pi  chromatophore buffer. A typical yield was 2 to 4 mg of total membrane protein.  The  protein concentrations  determined  using  a  modified  of  the  Lowry  chromatophore preparations were assay  (Peterson  1983).  Protein  absorbance values were measured at 660 nm with B S A as the standard.  2.16  Gel  electrophoresis  Chromatophore  of  proteins  chromatophore  were  separated  by  proteins  electrophoresis  on  a  sodium dodecyl sulfate (SDS) polyacrylamide gel by the Schagger and von Jagow  method  capsulatus  (Schagger  SB 1003  and  Jagow  1987).  to  visualize  it  appeared  chromatophores contained a larger percentage  harvesting complex proteins than R. capsulatus order  As  and compare  levels  of  that  R .  of light  MW442 chromatophores, in other  chromatophore  proteins  between the 2 species, more SB 1003 proteins had to be loaded on the gel.  40 It was empirically determined that when a total of 80 pg of chromatophore proteins  from  R.  capsulatus  chromatophore proteins  from  MW442-derived strains R.  capsulatus  and  SB1003-derived  110 strains  p g of were  loaded per lane on the gel, most of the bands were similar in intensity. Each gel was run for 16 hours at a constant current of 18 mA and stained overnight in a 0.25% solution of Coomassie brilliant blue G in 10% methanol and 40% acetic acid.  3  3.1  Construction  of  R.  RESULTS  capsulatus  mutants  orf 162b  3.1.1 Construction of a non-polar mutation in orf 162b  The mobilizable plasmid, pAH2, frame deletion of orf 162b (Aorfl62b)  which contains  a translationally in-  within the BamK fragment as well as  a Km " cartridge inserted upstream of orf214, 1  flanking the BamK orfs (Fig.  6), was used to attempt to create a non-polar mutation in strains SB 1003 and MW442. Purified pAH2 D N A was electroporated into E. coli SI7-1. The recipient  E. coli cells were mated with R. capsulatus DW21, a strain which  contains the Omega cartridge in the center of the otherwise intact orf 162b sequence  on  spectinomycin  the  chromosome.  resistance  concurrent loss of Sp  r  Since  the  Omega  (Sp ), the loss of orf 162b r  cartridge  confers  can be detected by the  in this strain. Because pAH2 is a suicide plasmid,  recipient DW21 cells would only acquire K m , in addition to Sp , if pAH2 r  r  integrated into the R. capsulatus chromosome (Fig. 10).  Three recipients, DW21K1, DW21K2 and DW21K3, were selected on the  basis of  their ability to  Southern blot analysis of BamK  grow  on a medium containing kanamycin.  I digested chromosomal D N A using a BamK  probe demonstrated that only one of these recipients, DW21K1, pAH2 integrated in the orf 162b  contained  region (Fig. 11). Lane 1, a positive control,  contained the 2 kb BamK fragment. Lane 2, contained chromosomal D N A from DW21K1. The 19 kb band shows that pAH2 had integrated into the  42  Figure 10. A n example of how homologous  recombination between  strain DW21 and pAH2 would incorporate this plasmid into the host chromosome and confer K m to the recombinant organism. r  An alternative recombination in the orfl62b also possible.  to orf274  region is  Omega  orf214 orfl 62b  or f214  orf274  orf214  <*  f55  Aorfl 62b  orfS5 orfl 62b  orf274  Omega orfl 62b  orf55 orfl 62b  or f 274  44  kb  1  2  19.0  4.0  2.0  Figure 1 1 . Southern blot hybridization analysis of chromosomal D N A isolated from strain DW21K1. Lane 1 contains the BamK fragment. A strong hybridization signal at 2 kb represents  the  BamK fragment used in the synthesis of the probe. Lane 2 contains BamH  I digested DW21K1 chromosomal D N A . The 19  kb band represents pAH2 that has been integrated into the DW21 chromosome upstream of orfl62b. represents  the Omega-disrupted BamK  The 4 kb signal fragment.  chromosome orfl62b  of DW21 through a single crossover  (Fig.  10),  BamK fragment. DNA  while  the  occurring upstream  4 kb band represents  the  of  Omega-disrupted  A 4 kb hybridization signal was detected from DW21K2  (data not shown),  corresponding to the  BamK::Omega fragment  of  DW21. The absence of a second band indicated that pAH2 did not integrate into  this  region  spontaneously was  the  chromosome,  and  so  perhaps  Km  r  arose  in this strain. Similarly, when DW21K3 chromosomal D N A  probed with  shown).  of  BamK,  only  one  2 kb signal  was  detected  (data  not  As the size of the wild type BamK fragment is 2 kb, this strain  possesses neither pAH2 nor the Omega fragment, indicating once again that Km  arose through means other than the K I X X cartridge.  r  In strain DW21K1, downstream  of  orf 162b  a second internal recombination event, occurring on  the  chromosome  and within  region of the P G C , would lead to the excision  the  duplicated  of the original orf  162b  containing the Omega cartridge as well as the surrounding duplicated orfs and non-homologous  plasmid D N A (Fig. 12). This event would result in the  replacement  original chromosomal  of  the  Omega-disrupted  orf 162b  with  the deletion construct from pAH2 and the concurrent loss of both Sp and r  Km . r  However,  after  approximately  10,000  DW21K1  colonies  were  screened for the loss of K m and Sp , no such colonies were found. r  Since  ultraviolet  (UV)  r  light  exposure  stimulates  RecA-dependent  recombination in some species of bacteria, the R. capsulatus was  exposed  recombination  to  U V light  event which  chromosome (see  for  30  would  seconds remove  in  the  an  DW21K1 strain  attempt  suicide  to  induce  plasmid from  Materials and Methods). This culture was grown under  a the  46  Figure  12. Homologous recombination between the duplicated orfs located downstream of the Omega cartridge in the P G C of strain DW21K1 would remove-the plasmid D N A as well as the K I X X and Omega cartridges from the chromosome, while the Aorfl62b  sequence would remain.  48 non-selective  conditions,  which time  in rich  the culture was  YPS medium, for  serially  several  transferred. Samples  days during  from the  third  sub-culture were plated onto R C V agar plates and screened for kanamycin and  spectinomycin  sensitivity.  Although 10 plates of  100  colonies  each  were tested, no such colonies were found.  Since described  it  seemed  second  extraordinarily  recombination,  an  380 bp B l e in  r  to  alternative  create a non-polar mutation in orfl62b. cartridge which contains  difficult  obtain  the  approach was  taken  to  It has been shown that the K I X X  a K m gene followed by the first 153 bp of the r  gene rarely has a polar effect when used in gene disruptions  which the transcriptional polarity of the K m gene is the same as the r  disrupted  gene  Aorfl62b,  (Bollivar et al.,  1994). Therefore, a K I X X  disruption of  provided by pAH4, was used in gene replacement  experiments.  On pAH4, the K I X X cartridge was inserted into the orfl62b  deletion site  (Fig. 7). If orfl 62b ::KIXX were transcribed and translated in R. two  above  fusion  possess 38  proteins  would  amino acids  be  produced.  One  of  these  proteins  from the N-terminus of orfl62b  acids from the beginning of the K I X X  capsulatus, would  and 14 amino  cartridge (Fig. 13A). The second  protein would have 51 amino acids from the N-terminus of the B l e  r  gene  followed by 24 amino acids, translated out of frame, from the 3' region of orfl62b  (Fig. 13B).  Plasmid pAH4  was  transformed into  the  mobilizing strain E.  coli  T E C 5 . Plasmids such as pAH4 which are derived from p U C plasmids are able to fuse with the mobilizable plasmid in E. coli  TEC5,  pDPT51,  by  homologous recombination, and thus are transferred at a low frequency to  49  Figure  13. Fusion proteins created by the insertion of the K I X X cartridge into orf 162b at the BsaB  I to PflM  I deletion site. (A) The  upstream fusion protein is composed of 38 amino acids (aa) from the N-terminus of orf 162b  (DNA codons underlined, aa  residues in bold type) and 14 aa from the beginning of the K I X X cartridge. (B) The downstream fusion protein possesses 51 aa from the N-terminus of the Ble  r  gene (DNA codons  underlined, aa residues in bold type) and 24 aa translated out-of-frame from the 3' region of orf 162b. the fusion sites.  Arrows indicate  A  1 2 4 3 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 a t g gca caq c t t c c g c t t t e c c c t gee ccq cag cgc c c c gag acg aaa acc c c c ggc aa M A L P L P A S P R P E T K K T P G Q Q  i 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 c c c gag. gee gag c t g a t e ccg aaa ccg c t g t t g egg g c g a t g a t e gag egg ccg gga ag M P E A E L I P K P L L R A I E R P G S  41 42 43 44 45 46 47 48 49 50 51 52 t t c t e g aga t t c t e a t g t t t g aca get t a t c a t cga t a a * R F L T A F S S c Y H R  B 4 1 2 3 5 7 6 8 9 10 11 12 13 14 15 16 17 18 19 20 a t g acc qac caa g c g acq c c c aac c t g cca t e a cga qat t t c qat t e c acc gec gee t t M T D A T P N L P S R D F D A A S T F Q  21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 t a t aaa agg t t g ggc t t c ggg a t e g t t t t c cag gac gec ggc a t g a t e e t c caq eg Y E R L G F G I V F R D A G M I L R Q W  tm  1r  41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ggg g a t e t c a t g c t g gag t t c t t c qcc cac ccc ggc cgc t c t ggt cca ccg aac t t t qc L M L E F G D F A H P G R P P S G N F T  61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 c c t t t g gcg ccg aca gca agg ccg c c t t e g age g t a t t c t e g aca t g a * A P T P L A P P V S S F T S R  R. capsulatus  cells.  The E. coli  TEC5  (pAH4) cells were conjugated with R.  capsulatus  DE442, a G T A overproducer strain. G T A particles resemble a generalized transducing phage which randomly packages host cell D N A in 4 kb linear fragments  (Yen  capsulatus  et al.,  1979). These particles are routinely used for  strain construction by  capsulatus  DE442  plasmid,  exconjugants  as the pDPT51  segment  plasmid to replicate in an R.  transduction. Kanamycin were  assumed  to  resistant  contain  the  host  while  the  pAH4  R.  pAH4  of the recombinant plasmid allows  capsulatus  R.  the  segment  confers K m . r  When R. capsulatus  DE442 cells  are grown photosynthetically, G T A  phage particles are produced which package 4 kb host cell D N A fragments which, in this case, would include segments of the TEC5::pAH4 plasmid. The GTA  particles  were  used  to  transduce  the  SB 1003, and the LHII" strain, R. capsulatus the homologous regions flanking orfl62b the  KlXX-disrupted  Aorfl62b  LHII+ strain, R.  capsulatus  MW442. A double crossover of on the bacterial chromosome and  gene (from pAH4) supplied by some G T A  particles, would result in the replacement of the wild type gene with the mutated version, thus conferring K m , due to the K I X X cartridge located in r  the orfl62b  Four selected  deletion site.  independent  K m orf!62b r  KlXX-disrupted  for study and called SB1003Kanl  MW442Kanl  (MWK1)  and MW442Kan2  (SBK1), (MWK2).  mutants are presented in sections 3.2 through 3.4.  mutants  were  SB1003Kan2 (SBK2), M y studies  of  these  3.1.2 Construction of a polar mutation in  orfl62b  In order to construct a polar mutant of orfl62b the  non-polar KlXX-disrupted  mutants,  pAH3  at the same site as in  was  transformed into  the  mobilizing strain, E. coli TEC5. Plasmid pAH3 (Fig. 8) contains an Omegadisrupted orfl62b TEC5  deletion construct (Aorfl62b::Omega).  (pAH3) and R.  capsulatus  DE442 conjugation,  After an E. the  G T A particles  produced by DE442 were used to transduce the Aorfl62b::Omega into R.  capsulatus  contains  both  strains  SB 1003  transcriptional and  and MW442. translational  coli  segment  As the Omega fragment stop  signals  within  sequences flanking the streptomycin and spectinomycin resistance  the  genes, a  polar effect on the expression of downstream genes that are cotranscribed with orfl62b  would be seen when the Omega cartridge is inserted into the  chromosome.  If  orfl62b be  a  double  crossover  replaced  sequence with the Aor/76~2£>::Omega  generated.  Two Sp  r  polar mutants  the  intact  fragment, a Sp  were chosen  SB 1003 and MW442 cultures for further study (see  chromosomal r  strain would  from the  transduced  section 3.3)  and called  SB1003Spec (SBSpec) and MW442Spec (MWSpec).  3.2  Analysis  3.2.1  The mutants  of  deletion  mutants  M W K 1 and S B K 1  Southern blot analyses of chromosomal D N A  chromosomal organization of the presumably non-polar was  confirmed by  Southern blot  analysis  of BamH.  I  orfl62b digested  chromosomal D N A (Fig. 14). The 2 kb BamK fragment was used as a probe  53  Figure 14. Southern blot analysis of BamH from orfl62b  I digested chromosomal D N A  mutants. Lane 1 contains the BamK fragment  which generates a strong 2 kb signal. Lanes 2 and 6 contain D N A from the parental strains SB 1003 and MW442. Lanes 5 and 9 contain SBSpec and MWSpec D N A respectively. The 1.0 and 0.7 kb fragments flanking the Omega cartridge are detected by the probe. Lanes 3 (SBK1), 4 (SBK2), 7 (MWK1), and 8 (MWK2) contain D N A from the non-polar mutants. A l l show a strong hybridization signal at 3.0 kb representing the BamH  I fragment containing Aorfl62b  in the deletion site.  with the K m cartridge r  54  1 kb 3.0 2.0  1.0 0.7  2  3  4  5  6  7  8  9  to detect the BamK or other homologous  fragments  in the chromosomal  digests. A 2 kb hybridization signal in the control lane 1 corresponds to the BamH  I restriction fragment (BamK) obtained from pUC13::BamK. Although  the BamK sequence is 2 kb in length in the parental SB 1003 and MW442 strains,  shown in lanes 2 and 6,  SBK1/SBK2  possess a 0.3 kb deletion in orfl62b Km  r  and M W K 1 / M W K 2  should  coupled with the insertion of a 1.3 kb  cartridge in the deletion site, to generate a 3 kb BamH  I fragment. As  bands of 3 kb were seen with these strains, (Fig. 14, lanes 3, 4, 7, and 8), it was  concluded  from  this  experiment  that  the  gene  replacements  had  occurred as planned.  3.2.2 Northern blot analysis of R. capsulatus R N A  To therefore  determine  if  an expressed  carried out on SB 1003  orfl62b  R N A (Fig. 15).  transcription  read-through would  upstream of  puhA.  I agarose  R N A from strain DW1 was  expression)  possesses a polar mutation in puhA  EcoR  was also  not  could be determined. As DW1  (upstream of orfl62b), occur from any  in this strain  promoters  located  D N A probe was created from a 424 bp BsaB  I-  gel-purified D N A fragment removed from pUCT3::BamK.  This sequence extended from 101 nt into the orfl62b 32 nt into orf55  and is  so that the contribution of transcription read-  (to orfl62b  An orfl62b-specific  capsulatus  gene, a Northern blot hybridization experiment  included in this experiment through from puhA  is transcribed in R.  located downstream of orfl62b  coding sequence to  in the PGC (see Fig. 2).  56  Figure  15. Northern blot analysis of R.  capsulatus  R N A using an  orfl62b  probe. Lane 1 contains R N A harvested from SB 1003 cells grown photosynthetically, while lane 2 contains SB 1003 R N A purified from cells grown under oxygen-limited conditions. Lane 3 contains DW1 R N A harvested from cells grown under conditions of reduced aeration. A total of 20 pg of R N A was loaded per lane. The hybridization signal in lanes 1 and 2 indicates the presence of an unstable orfl62b  species of  approximately 4 kb. In lane 3, orfl 62b mRNA was not detected.  57  1 kb 4.0-  0.2  2 3  Strong hybridization signals which  contained  SB1003  appeared in lanes 1 and 2 of Fig. 15,  RNA  harvested  from  cells  grown  either  photosynthetically (lane 1) or under oxygen-limited conditions (lane 2). A n apparently unstable species of approximately 3.5  to 4 kb gave the largest  signal and a smear of smaller species extended down to the 0.2 kb range. Lane 3 contained DW1 R N A harvested from  cells grown under oxygen-  limited conditions (this strain is incapable of photosynthetic growth). When RNA  from strain DW1 was hybridized with the same orfl62b  or no mRNA was detected, indicating that orf 162b from the puhA  promoter and/or the bch  10 kb upstream of puhA  3.2.3  transcription originates  promoter located  approximately  on the P G C (Alberti et al., 1995).  Photosynthetic  Photosynthetic  probe, little  growth studies  growth experiments  conducted using several light intensities.  with  the  orf 162b  mutants  were  The growth curves presented in  this thesis utilized a high light intensity of 150 p E / m / s and a low light 2  intensity of 30 p E / m / s 2  separate  same  photosynthetic  observed  growth  the LHII Aorfl62b::KIXX -  doubling time  However,  of the results from three  experiments.  During intensity,  and were representative  as  the  a reduced rate  of  when photosynthetic  low light intensity (Fig. 16B).  LHII"  under  conditions  of  high  light  strain M W K 1 had approximately the parental  M W K 1 growth growth was  strain, MW442  (Fig.  compared to  MW442  16A).  measured under conditions  was of  59  Figure  16. Photosynthetic growth of R.  capsulatus  strains grown under  variable light intensities. (A) The growth rate of the LHII" mutant M W K 1 is compared to that of the parental strain, MW442, during high light and (B) low light growth (C) The growth rate of the LHII+ mutant SBK1 is compared to that of the wild type strain SB 1003 during high light and (D) low light growth.  Growth of Nonpolar Mutant in an LHIIBackground Under High Light Intensity  1000  Growth of Nonpolar Mutant in an LHII+ Background Under High Light Intensity  1000  Doubling High  Time  (g)  Light  Low  MW442  5.5  10.5  MWK1  5.3  19.1  SB1003  3.0  4.1  SBK1  8.2  16.5  Light  Table I. Doubling time of photosynthetically grown R. during log-phase  capsulatus  growth.  The photosynthetic growth rate of the LHII+ Aorfl62b::KIXX SBK1 low  strain  differed from that of the parental strain under conditions of high and light  intensity.  Under  both  conditions,  doubling time during photosynthetic 16D).  cells  strain SBK1 had a longer  growth than SB 1003  (Figs.  16C and  These data are summarized in Table I. It is interesting to note that,  although R.  capsulatus  photosynthetic  growth  approximately  350-400  cultures usually enter the stationary phase during when  the  K U , the  culture LHII+  reaches  a  cell  density  SBK1 strain entered  of  stationary  phase at a culture density of 100-150 K U during high light photosynthetic growth (Fig. 16C). After 40-60 hours, exponential growth resumed until a cell density of approximately 400 K U was reached. SBK1 colonies  isolated  from  did not  this  culture and regrown under photosynthetic  exhibit a transient early stationary phase, original  culture containing  a secondary  suggesting  conditions  that cells from  mutation overgrew  the  the  primary  mutant and allowed the SBK1 culture to eventually surpass a density of  150 K U during high light photosynthetic growth.  To  further  characterize  the  photosynthetic  growth  of  SBK1,  photosynthetic growth of individual cells was evaluated on solidified R C V medium by enumeration of the number and sizes of colonies  that formed.  When the SBK1 strain was compared to the wild type strain SB 1003 after 3 days of high light photosynthetic growth, the SBK1 colonies had a diameter of approximately 0.1 mm, whereas the wild type colonies measured 2 mm across.  After  discovered  an  that  photosynthetic frequency original mutants  of  additional 1%  growth,  the  When  isolated  days  of  photosynthetic  SBK1 colonies  which  a secondary  mutation. were  of  3  was  growth,  it  were  capable  of  interpreted to  indicate  a very  mutation that individual  suppressed  colonies  of  the  continued  effects  presumed  was  high  of  the  suppresser  and cultivated aerobically on YPS agar plates, a  change in pigmentation was observed for several of the colonies examined. While  colonies  of  strain  SBK1 are  normally  dark  red  when  grown  aerobically on agar plates, the double mutants varied in pigmentation from pale pink to brownish-red as well can  be  attributed  carotenoid carotenoid  molecules. and  to  the Since  as yellowish-green.  concentration different  of  operons  Cell  pigmentation  bacteriochlorophyll and within  bacteriochlorophyll biosynthesis  the  genes  P G C encode (Fig.  2),  the  variation in phenotypes of the suppresser mutants and the high frequency at which they arise suggests that numerous types of mutations at several loci give rise to the suppresser mutants (see Discussion).  3.2.4 Absorption spectroscopy analysis of L H and R C complex levels  63 As  described  earlier  (section  3.1.1),  non-polar orf 162b  deletion  mutants were created in both LHII+ and LHII' strains. The LHII+ strain, SB 1003  is the wild type strain whereas  that prevents grown  under  proteins LHI,  LHII  formation. Spectral analysis  oxygen-limited  involved  the LHII" strain has a mutation  conditions  in photosynthesis,  LHII and, in an LHII"  to  allows  of  R.  induce  capsulatus  the  quantitative  background, R C levels.  cells,  production  of  comparisons  of  The L H I complex  generates an absorbance peak at 870 nm whereas LHII yields two peaks at 800 and 850 nm, and one of the R C peaks is at approximately 800 nm. Thus, in a wild type strain the LHII 800 and 850 nm peaks dominate the spectrum, while  the less abundant R C 800  nm peak is  obscured by the  LHII peak at this wavelength. Similarly, in a wild type (LHII ) background +  the less abundant LHI 870 nm peak appears as a shoulder on the LHII 850 nm peak. However, in a LHII" background, in which the LHII 800 nm and 850 nm peaks are absent, the R C 800 nm and the LHI 870 nm peaks are visible. Since LHII absorbance can mask the L H I and R C absorbance peaks, its presence is undesirable when comparing in vivo LHI and R C levels in different  strains.  When  the  LHII" Aorfl62b::KIXX  strain M W K 1 was  compared to  MW442, M W K 1 clearly possessed lower L H I (870 nm) and R C (800 nm) levels (Fig. 17). Similarly, the LHII+ SBK1 mutant possessed less LHI in comparison to the wild type strain (evident from the decrease in the  870  nm shoulder of the LHII 850 nm peak; Fig. 18). Although it seemed that this strain also possessed less LHII, the apparent reductions in the LHII peaks could be due to reductions in R C and LHI, which contribute to these peaks (see  above).  64  1000  Wavelength (nm)  Figure 17. Whole cell absorption spectra of R.  capsulatus  LHII* strains  grown under low aeration conditions. The parental strain MW442 (blue) is compared with the mutant strains MWK1 (green) and MWSpec (red). Scans were normalized by multiplication of spectra by a factor to yield a light scattering A 6 5 0 value of  0.2.  700  800  900  1000  Wavelength (nm)  Figure 18. Whole cell absorption spectra of R.  capsulatus  LHII+ strains  grown under low aeration conditions. The parental strain SB 1003 (blue) is compared with the mutant strains SBK1 (green) and SBSpec (red). Scans were normalized by multiplication of spectra by a factor to yield a light scattering A  6 5  o of 0.2.  66 3.2.5 S D S - P A G E analysis of chromatophore proteins  In  order to evaluate  any changes  complex proteins in membranes of the R. analyses grown  were  in concentration of capsulatus  mutants,  carried out on chromatophore proteins purified  under conditions  of  low  aeration. As  it  R C or L H SDS-PAGE from  appeared that  cells  SB 1003  chromatophores contained a higher total protein count (perhaps due to the presence SB 1003  of  LHII)  than  protein was  MW442  chromatophores,  loaded per lane on the  chromatophore proteins  from  the  LHII"  gel.  a greater  +  of  A total of 80 pg of  MW442-derived  pg of chromatophore proteins from the L H I I  amount  strains  and  110  SB1003-derived strains were  loaded per lane. Relative amounts of L H and R C proteins were compared between  either  LHII" or LHII+  strains and not between the two species.  The R C and L H bands were identified on the basis of their electrophoretic mobilities.  As seen in Fig. 19, there were no major changes in R C H (28 kDa), R C M (24 kDa) and R C L (21 kDa) polypeptide levels in the LHII+ mutant SBK1 in lane 3 compared to the wild type SB 1003 strain in lane 2. Although the intensities of bands seemed to be slightly reduced in the mutant. Similarly, the levels of the three R C subunits in the LHII" strain M W K 1 , shown in lane 6, were about the same as in the parental strain, MW442 shown in lane 5. In regard to the L H subunit levels, there was a reduction in LHI a and LHI (3 polypeptides in the LHII" strain M W K 1 (lane MW442  (lane  5).  However, a reduction in L H I a  observed by this method in the L H I I  +  6) compared to  and L H I (3 was not  strain SBK1 (lane 3) when this strain  was compared to the wild type strain SB 1003.  67  Figure 19. S D S - P A G E analysis of chromatophore proteins isolated from R. capsulatus  SB 1003, MW442 and related strains grown under  low aeration conditions. A total of 80 pg of chromatophore proteins from the MW442-derived strains and 110 pg of chromatophore proteins from the SB1003-derived strains were loaded per lane. Lane 1, low molecular weight standards; lane 2, SB 1003; lane 3, SBK1; lane 4, SBSpec; lane 5, MW442; lane 6, M W K 1 ; lane 7, MWSpec.  69  3.3  Analyses  3.3.1  The  of  deletion  mutants  MWSpec  and SBSpec  Southern blot analysis of chromosomal D N A  chromosomal organization of MWSpec and SBSpec was analyzed  by  Southern blotting using a BamK probe (Fig. 14). The presence of the 2  kb  Omega cartridge  generated  3 fragments  within  the  deletion  after BamH  site  in  orfl62b  should  have  I digestion: the 2 kb Omega cartridge  (which was not detected by the probe), and the 1 kb and 0.7 kb fragments, which  flank  the  Omega  cartridge  On the  chromosome  and  thus  were  detected by the BamK probe (Fig. 14, lanes 5 and 9). These results show that the  correct chromosomal organization  the goal to replace the wild type orfl62b  was  obtained,  consistent with  allele with the deletion construct  Aorfl 62b:: Omega.  3.3.2  The  Photosynthetic  results  of  growth studies  photosynthetic  growth  experiments  with  the  polar  mutants differed from those of the non-polar mutants. In the LHII" strain MWSpec,  regardless  of  whether  photosynthetic  growth  occurred under  conditions of high or low light intensity, the absence of orfl62b the possible  (as well as  decrease in transcription of downstream orfs) had a negative  effect on growth rate (Figs. 20A and B). Similarly, the LHII+ strain SBSpec exhibited  a reduced rate of photosynthetic  growth under both  although these cultures, at both light intensities, the  LHII+ non-polar orfl62b  conditions,  grew at a faster rate than  mutants and an early stationary phase  not observed (Figs. 20C and D). These data are summarized in Table II.  was  70  Figure  20. Photosynthetic growth of R.  capsulatus  strains grown under  variable light intensities. (A) The growth rate of the LHII" polar orf 162b  mutant, MWSpec, is compared to that of the  parental strain, MW442, during high light and (B) low light growth(C) The LHII+ polar orf 162b mutant, SBSpec, is compared to that of the wild type, SB 1003, during high light and(D) low light growth.  Growth of Polar Mutant in an LHII+ Background Under High Light Intensity  Growth of Polar Mutant in an LHIIBackground Under High Light Intensity  1000  g |  1000  1000  Growth of Polar Mutant in an LHIIBackground Under Low Light Intensity  D  1000  Growth of Polar Mutant in an LHII+ Background Under Low Light Intensity  72  Doubling Time (g) High Light  Low  MW442  5.5  10.5  MWSpec  8.1  25.1  SB1003  3.0  4.1  SBSpec  4.5  6.2  Table  light  II. Doubling time of photosynthetically grown R. capsulatus during log-phase  cells  growth.  3.3.3 Absorption spectroscopy analysis of L H and R C complex levels  When  the  LHII"  polar  mutant,  MWSpec,  was  compared  to  the  parental type strain MW442, the absorbance data indicated a reduction in LHI levels as well as R C levels (Fig. 17). In the LHII+ polar mutant, SBSpec, there was a small reduction in LHI evident from the decrease in the  870  nm shoulder of the LHII 850 nm peak (Fig. 18).  3.3.4 S D S - P A G E analysis of chromatophore proteins  Differences  in the concentrations  of  chromatophore proteins  in the  polar mutant strains were compared by S D S - P A G E analysis (Fig. 19). There was no obvious change in R C H , M and L polypeptides in the LHII+ mutant SBSpec (lane 4) compared to the wild type SB 1003 strain (lane 2) and the  73 LHII" mutant MWSpec in lane 7 compared to the parental strain, MW442 (lane  5).  Although the intensities  of these bands seemed to be  slightly  reduced in the SBSpec mutant. Furthermore, L H levels in the L H I I  +  strain  SBSpec (lane 4) were about the same (or, perhaps, slightly increased) as compared  to  the  parental strain SB 1003.  However,  there  was  a small  reduction in LHI a and LHI (3 band intensities in the LHII" MWSpec mutant shown in lane 7.  3.4 Trans  complementation  3.4.1  Photosynthetic  of  orfl62b  mutants  growth studies  An expression vector, pAH8, was created so that orf 162b expressed  in trans in R.  and non-polar orf 162b  capsulatus  complemented  mutants (Fig. 9). When polar  deletion mutations in LHII" and LHII+ strains were  complemented with pAH8, the  orf 162b  could be  the low light photosynthetic  mutants  (Table  III;  doubling times of  doubling  times  for  non-  complemented mutants are shown in brackets) decreased to approximately the value of the parental strains in the non-polar mutants but not in the polar mutants, MWSpec and SBSpec. (As the differences were  subtle  Therefore,  under high  complete  light  conditions,  this  data  was  in growth rates not  included.)  complementation did not occur in the MWSpec and  SBSpec strains. I conclude that this difference is due to the absence  of  expression of genes located downstream of orf 162b  in the P G C . The early  stationary  was  phase  complementation.  seen  previously  in  SBK1  eliminated  by  74  Doubling  Time Low  (g) light  (hr)  MW442  10.0  MWKl(pAH8)  12.0  (19.1)  MWSpec(pAH8)  19.1  (25.1)  SB1003  4.1  SBKl(pAH8)  4.2  (16.5)  SBSpec(pAH8)  5.2  (6.2)  Table III. Doubling time of c>r/7f52Z?-complemented during log-phase photosynthetic  R.  capsulatus  mutants  growth under low light intensity. Doubling  times for non-complemented mutants are shown in brackets.  3.4.2  Absorption spectra  When the non-polar deletion mutant M W K 1 was complemented with pAH8, spectral analysis showed that both L H I and R C levels increased to approximately the level of complexes (Fig.  21).  Similarly,  trans  found in the parental strain, MW442  complementation  in  the  polar  LHII"  strain  MWSpec(pAH8) increased LHI and R C levels (Fig. 22). However, there was a broad range of absorbance in the 770 complemented  strain MWSpec(pAH8)  to  800  nm range in the polar  that remains unexplained.  Wavelength (nm)  Figure 2 1 . Whole cell absorption spectra of LHII" strains grown under low aeration conditions. The parental strain MW442 (blue) is compared with strains M W K 1 (green) and M W K l ( p A H 8 )  (red).  Scans were normalized by multiplication of spectra by a factor to yield a light scattering A 6 5 0 of 0.2.  76  .8-1  Or\  700  1 800  1 900  1 1000  Wavelength (nm)  Figure 22.  Whole cell absorption spectra of LHII" strains grown under low aeration conditions. The parental strain MW442 (blue) is compared with strains MWSpec (green) and MWSpec(pAH8) (red). Scans were normalized by multiplication of spectra by a factor to yield a light scattering A 6 5 0 of 0.2.  77 When possibly  strain SBK1 was  R C complex  type strain (Fig. 23).  complemented  with pAH8,  L H I , LHII and  levels increased to levels associated Trans  complementation  in the  with the wild  polar LHII+  strain  SBSpec(pAH8) had no affect on spectral absorbance (Fig. 24). This result is not  surprising  since  the  original  replacement  of  orfl  62b  with  A or/7 (52/?:: Omega in SB1003 had a minimal affect on absorbance values in SBSpec. It should be noted that it is possible that the dominant LHII 800 and 850 nm abosrbance peaks are concealing small changes in LHI and R C complex levels in this strain.  1700  800  T  900  -1  1000  Wavelength (nm)  Figure 23. Whole cell absorption spectra of LHII+ strains grown under low aeration conditions. The parental strain SB 1003 (blue) is compared to strains SBK1 (green) and SBKl(pAH8) (red). Scans were normalized by multiplication of spectra by a factor to yield a light scattering A 6 5 0 of 0.2.  700  800  900  1000  Wavelength (nm)  Figure 24. Whole cell absorption spectra of LHII+ strains grown under low aeration conditions. The parental strain SB 1003 (blue) is compared to strains SBSpec (green) and SBSpec(pAH8) (red). Scans were normalized by multiplication of spectra by a factor to yield a light scattering A 6 5 0 of 0.2.  4  DISCUSSION  In order to determine if orfl62b from  the photosynthetically  is transcribed in R.  wild type  a radioactively labelled orfl62b  strain SB 1003  capsulatus,  was  RNA  hybridized with  D N A probe. This northern blot  analysis,  presented in Fig. 15, shows directly that a transcript containing  orfl62b  sequence limited  information is  present  (lane 2) as well  in  SB 1003  cells  grown  as anaerobic photosynthetic  under  conditions  oxygen(lane  1).  R N A from strain DW1 was also included in this experiment (lane 3). DW1 cells were grown under low oxygen conditions, as this strain is incapable of photosynthetic inserted  growth  because  into the puhA  the  gene (1.5  polar  Omega  cartridge  kb upstream of orfl62b;  has  been  see Fig. 2)  preventing synthesis of the R C H subunit. Since strain DW1 did not contain a transcript that hybridized to the orfl62b and  perhaps  downstream  genes  probe, the promoter of  cotranscribed  located upstream of the polar mutation in puhA.  with  this  orf,  orfl62b, must  be  In fact, based on the size  (approximately 4 kb), of the largest transcripts from SB 1003 R N A detected by the orfl62b  probe, it seems that the orfl62b  promoter located near the 5' end of the puhA  transcript arises from the gene (Bauer et al.,  However, the smear of transcripts detected in this northern blot that the orf 162b cellular RNases.  transcript It has been  is  unstable  shown  and  prone  to  1991). indicates  degradation  that very large photosynthesis  by  gene  messages are so unstable that the primary transcript is not detectable in a northern  blot,  although  segments  resulting  from  post  transcriptional  cleavages may still be detected (Wellington et al., 1991). Thus, it is possible that orfl62b  is also part of a larger transcript originating from the  bch  promoter located 10 kb upstream of puhA molecules  are  cleaved  into  smaller  on the P G C , and that these large segments  as  fast  as  they  are  orfl62b  to  synthesized.  Non-polar mutations were  created in the R.  determine if disruption of this growth were  and  also  between  synthesis generated  the  two  sequence had an effect  of  the  photosynthetic  for  two  reasons.  classes  of  capsulatus  mutations  on photosynthetic  apparatus. Polar  Firstly, would  mutations  a phenotypic  difference  indicate  the K m  that  r  cartridge, used to create the non-polar mutants, did not have a polar effect on the expression of orfs located downstream of orfl62b. photosynthetic  phenotype  Secondly, if the  of the polar mutants differed from  non-polar mutants this would indicate that orfl62b  was transcribed along  with at least one other gene situated downstream of orfl62b Moreover,  these  photosynthesis  genes  in R.  would  provide  capsulatus.  a  that of the  function  in  in the P G C .  the  process  of  To facilitate the interpretation of spectral  absorbance data, polar and non-polar orfl62b  mutations were created in a  LHII" as well as a LHII+ (wild type) background.  Southern  blot  analyses  of  chromosomal D N A from  the  orfl  62b  mutants confirmed that each strain contained a deletion in which 63% of the central coding region of orfl62b  had been removed and replaced with  a K I X X cartridge (conferring Km ) in the non-polar mutants, and an Omega r  cartridge (providing Sp ) in the case of the polar mutants. r  Photosynthetic growth rate comparisons of non-polar mutants to the parental  strain  showed  that  the  absence  of  orfl  62b  increases  the  generation time in both LHII" and LHII+ strains of R. Table  I).  suggesting  This  is  pronounced under conditions  of  capsulatus low  (Fig. 16;  light  intensity,  that there may be a deficiency in the mutants' ability to harvest  light energy during photosynthetic  growth. In addition, an unusual growth  pattern was observed when the SB1003-derived strain SBK1, thought to be more efficient  in regard to photosynthetic  MW442-derived  light harvesting than the LHII"  strains (due to the presence  of the LHII complex),  cultivated under high light conditions (Fig. 16C), whereas  was  no such effect  was seen for its LHII" counterpart, M W K 1 . It appears that, although the SBK1  strain  initially  grew  quickly  under  high  light  conditions,  the  generation time of these cells continuously increased until cell growth was barely perceptible. After 2-4 apparently  contained  photosynthetic  days of photosynthetic  secondary  growth, increased  mutations  incubation, cells that  which  allowed  in number sufficiently  to  normal  result  in an  increase in culture turbidity. However, the frequency at which these cells arose  ( I O ) is -2  too  mutational  event.  expression  of  recombination,  high to be  attributed solely  One possibility  genes,  whose  is  a random secondary  that under conditions  protein  are induced. Thus,  to  products  when  are  of  involved  SBK1 cultures  stress, in  ceased  the  genetic to  grow  photosynthetically, a genetic pathway may have been induced which led to a high rate of genetic recombination and therefore mutation.  The  growth kinetics of SBK1 resemble  depleted the medium of an essential  those of a culture that has  substance.  However, under low light  conditions the premature stationary phase did not occur (Fig. 16D), and so one would have to imagine a substance under high light but not for slow  that is required for rapid growth  growth under low  light. Furthermore,  83 high light cultures of the LHII" non-polar mutant, M W K 1 , did not exhibit a premature stationary phase (Fig. 16A). Therefore, one would have to also imagine a substance that is required by orfl62b but  mutants that contain LHII  is not required by equivalent mutants that lack LHII. Since the only  components of LHII that differ from L H I are the two protein subunits, the idea that the premature stationary phase  of high light SBK1 cultures is  caused by depletion of a substance seems unlikely.  Spectral analyses of L H and R C complex levels showed that the LHI and  R C levels dropped in the LHII" mutant M W K 1 in the  orfl62b. in  Similarly, in strain SBK1 the absence of orfl62b  absorption peaks,  presumably due  to  absence  of  led to decreases  decreases in the  L H I and R C  complexes (as in MWK1), although it is possible that there was a decrease in the amount of the LHII complex. These data suggest that orfl62b  may  act in vivo to facilitate the production of a functional photosynthetic unit. That is, orfl62b  alone or in conjunction with other gene products may aid  in the correct assembly  of the photosynthetic  unit, which contains a core  complex of LHI and R C in LHII" strains, or this core complex surrounded by LHII in L H I I possess LHII), and  +  the assembly  photosynthesis  orfl62b  strains. Perhaps in the  is  SB1003-derived strains  of the photosynthetic  affected  to  a greater  (which  unit is more complex  degree  in  the  absence  of  than in the MW442 (LHII") derived strains. That is, perhaps the  LHII complex somehow interferes with the assembly or proper function of the core complex, conceivably by competing for membrane sites or other factors that become rate-limiting for assembly, or for electron transport, in the absence of  orfl62b.  84 As photosynthetic units are present even in the absence of orf162b,  I  propose that assembly can occur in its absence, although the assembly of the constituent complexes occurs at a slower pace. This explains the more severe effects of orf 162b SBK1,  since  they  mutation on the SB1003-derived strains, such as  possess  more  complex  photosynthetic  units  that  presumably require a greater number of protein interactions for assembly. In the 2-3 days that R.  capsulatus  cells  are grown under oxygen-limited  conditions in preparation for photosynthetic photosynthetic  unit are synthesized  growth, the  and able  to  subunits  of  the  gradually assemble.  As  these units are not utilized in aerobic respiration, cell division and growth is  not  affected  by  their  concentration.  When  cultures  of  SBK1 are  transferred to anaerobic, high light conditions allowing for growth only by means of photosynthesis, this strain initially grows fairly quickly by means of  the  pre-assembled  quantity  complexes.  of photosynthetic  occurs more rapidly  cell  division, the  units per cell decreases because  cell division  than new  However, with each  units  are assembled.  As time  progresses,  each cell contains less and less of the photosynthetic complexes, until the level per cell drops to the point that a lack of chemical energy causes the cells to become dormant. This unusual growth pattern does not occur when SBK1 is grown photosynthetically under low light conditions because cells divide less frequently. With a doubling time of 16.5 hours, there is enough time between cell divisions for sufficient quantities of photosynthetic units to accumulate to allow for photosynthetic strain  M W K 1 , its  less  complex  enough in the absence of orf 162b low  growth. In the MW442-derived  photosynthetic  units  assemble  to allow photosynthesis  or high light conditions. In these LHII"  strains, the  quickly  to occur under doubling time  during photosynthetic growth was greater than in the parental strain only  85 under low light growth conditions.  The  hypothesis  described above  has  the  drawback that one would  think that growth of SBK1 would not completely cease upon depletion of the R C and LHI complexes by rapid cell division during high light growth, but that growth would simply slow to a rate that was governed by the rate of orfl  62b-independent  advantage  assembly.  that it could be  However,  this  tested in experiments  hypothesis  has  the  in which cells grown  under high aeration were shifted to low aeration and the rates of de  novo  assembly  rates  of  L H I and R C complexes  would be lower in orfl62b interesting  to  grow  were  determined. Assembly  mutants if my model is correct. It would also be  SBK1  cultures  under  photosynthetic  conditions until cells entered the premature stationary phase, such  cells for spectral analysis  proposed  hypothesis,  such  of photosynthetic  cells  would  be  complexes.  expected  to  high  light  and harvest Under  contain  the  lower  amounts of R C and LHI complexes than the cultures grown in conditions of reduced  aeration.  The  evaluation of chromatophore proteins in S D S - P A G E  showed a  small decrease in LHI a and (3 polypeptides in the LHII" mutants but not in the LHII+ mutants (Fig. 19). Moreover, there was no detectable decrease in R C polypeptides. The absence of a large decrease in polypeptide levels is consistent  with  the  proposed  function  of  orfl62b  in assembly  of a  functional photosynthetic unit but not in altering the expression of genes themselves.  That is, if the decreases in R C and L H I complex levels is not  due to a decrease in the amount of R C and LHI polypeptides, then a posttranslational process  such as complex  assembly  must be affected  by the  86 mutation of orf 162b.  The analyses of polar mutants revealed that these mutants possess a different least  phenotype  one  orf  light  the  located  photosynthesis, low  from  and is  non-polar mutants.  downstream  conditions,  20;  Table  photosynthetic  orf 162b  cotranscribed with orf 162b. the  LHII+  decreased rate of photosynthetic (Fig.  of  This  II).  As  cultures  the  was  and LHII"  indicates  has  that at  a function  in  Under both high and  polar mutants  exhibited a  growth compared to the parental strains generation  25.1  hours  time  of  whereas  MWSpec  the  low  light  M W K 1 generation  time was 19.1 hours, the orfs found downstream of orf 162b  probably play  a role in enhancing photosynthetic growth. However, strain SBSpec, with a generation  time  of  6.2  hours  under the  same  conditions  of  low  light  intensity, grew better photosynthetically than SBK1 at 16.5 hours, and so in orfs  this  instance  the  had a positive  absence  of  downstream  lack of effect  functional of  transcription read-through into downstream  on photosynthesis.  gene  orf 162b  products  slow  It is  encoded  conceivable by  the  the rate of photosynthetic  that the  orfs  found  growth to an  extent that allows the assembly of enough photosynthetic units to support photosynthetic mechanism, orf 162b  it  growth  after  seems  that  each one  cell or  division. Regardless  more  genes  located  of  the  exact  downstream  of  has a different effect on photosynthesis.  When the orf 162b  mutants were complemented in trans with pAH8,  the non-polar mutants increased in photosynthetic comparable  to the respective  growth rate to a value  parental strains whereas  did not (Table III). The absence  of complete  the polar mutants  complementation in strains  87 MWSpec(pAH8)  and  SBSpec(pAH8)  is  attributed  to  the  absence  expression of one or more genes located downstream of orfl62b, supports  the idea that orfl62b  transcription continues  of  and thus  through  and  is  that  in  required for expression of at least one additional gene.  Spectral  analysis  of  regard to photosynthetic  the  complemented  mutants  showed  complex levels, approximately wild type levels of  RC 800 nm and LHI 870 nm peaks were obtained in MWKl(pAH8)(Fig. 21). In MWSpec(pAH8), the L H I peak was restored to approximately the wild type level, but the RC peak was disrupted by a broad region of absorbance from about 760 nm to 800 nm (Fig. 22). A similar spectrum was obtained by  Conan Young  with  upstream of the puhA  a polar mutation in orfl696,  which  is  located  gene (Fig. 2). The basis of this unusual spectrum is  not yet known, although it seems to originate from bacteriochlorophyll that is abnormally associated  with proteins (Young 1997). In S B K l ( p A H 8 )  absorption peaks closely resemble the parental strain, SB 1003 contrast to MWSpec(pAH8), the SBSpec(pAH8) spectrum is  the  (Fig. 23). In superimposable  on the mutant SBSpec spectrum, both of which are deficient in the longwavelength LHI shoulder on the 850 nm LHII peak (Fig. 24).  All  of  the  above  data indicate  that  not  only  the  orfl62b  gene  product, but also some factor derived from transcription of 3' sequences is important  for  expression  of  photosynthetic these  genes  physiology.  have  complex  Alterations effects,  in  which  the are  level  of  manifested  differently depending on the presence or absence of the LHII complex. The importance of the work described in this  thesis is  not so much in the  provision of an exact, mechanistic explanation for how these gene products  88 function as it is in the discovery of these new genes, and the revelation of the  unsuspected  bacterial  system.  complexity  surrounding  photosynthesis  in  this  simple  89  5  Very about  the orfs  research, are  little  photosynthetic described are  found downstream  from  experiments  phenotypic  outlined  differences  and support  process  between  there  is  in  the  levels,  assembly  apparatus. orf 162b  is  interesting  a reasonable or  required  for  is necessary  to see what  in bacterial  of  others,  puhA.  that  are genes  presented  The  there  were  involved in  in this  thesis,  I  growth is inhibited. Since  has a direct  of  components  effect  of  on L H or R C  gene is involved  the  photosynthetic  at least one orf located  downstream of  to elucidate  future  analyses  affects the levels of functional L H and  optimal  gene  blot  and the parental  interpretation is that this  interaction  and downstream  genes function  mutants orfs  optimal  and possibly  revealed  the data  orf 162b  It is also probable that  experimentation orf 162b  that  and orf 162b  hence  upstream  thesis  these  and  orfs,  in such a way that photosynthetic  no indication  polypeptide  these  orf 162b  that  From  orf214  T h e northern  situated  the  the hypothesis  of photosynthesis.  complexes  that  in this  propose that the absence of orf 162b RC  1996).  promoter  that  R C levels  et al.,  demonstrate a  on the P G C when I began my  had suggested  L H I and  (Wong  thesis  than D N A sequence data was available of puhA  studies  optimal  growth  cotranscribed  strains,  recent  for  in this  additional  the  information other  although  required  CONCLUSIONS  photosynthetic the exact mode  products  experiments  photosynthesis.  i n R. will  growth. of action  capsulatus.  tell  Further  us about  of the  It will be how these  90  6  1.  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