Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Studies on inter-species expression of photosynthesis genes in Rhodobacter capsulatus Zilsel, Joanna 1990

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1991_A6_7 Z54.pdf [ 5.88MB ]
Metadata
JSON: 831-1.0098553.json
JSON-LD: 831-1.0098553-ld.json
RDF/XML (Pretty): 831-1.0098553-rdf.xml
RDF/JSON: 831-1.0098553-rdf.json
Turtle: 831-1.0098553-turtle.txt
N-Triples: 831-1.0098553-rdf-ntriples.txt
Original Record: 831-1.0098553-source.json
Full Text
831-1.0098553-fulltext.txt
Citation
831-1.0098553.ris

Full Text

STUDIES ON INTER-SPECIES EXPRESSION OF PHOTOSYNTHESIS GENES IN RHODOBACTER  CAPSULATUS  by Joanna Zilsel B. Sc., University of British Columbia, 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT O F THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Microbiology) We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA September 1990 © Joanna Zilsel, 1990  In  presenting  degree at the  this  thesis  in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  scholarly purposes may be her  representatives.  permission.  of  Microbiology  The University of British Columbia Vancouver, Canada  D a t e  DE-6 (2/88)  September 2 5 t h , . 1990  for  an advanced  Library shall make  it  agree that permission for extensive  It  publication of this thesis for financial gain shall not  Department  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  is  granted  by the  understood  that  head of copying  my or  be allowed without my written  ii  ABSTRACT The  primary  amino  acid  sequences  photosynthetic reaction center peptide purple  non-sulfur bacteria,  Rhodobacter  including  sphaeroides,  previously  shown  to  of  the  highly  M,  Rhodopseudomonas  non-sulfur  shown that conserved.  all  bacteria,  capsulatus  homologous, and  Rps.  viridis  recognized structural  Experiments  were  H  viridis, have been  detailed  crystallographic analyses of reaction centers from two purple  and  subunits from a number of  and Rhodobacter  be  L,  and R.  and  species of  sphaeroides  functional  undertaken  X-ray  to  have  features  determine  are  whether  genes encoding reaction center and light harvesting peptide subunits from one species could be functionally expressed in other species. Plasmid-borne copies of R binding-peptide  genes  photosynthetically deficient  in  sphaeroides  all  sphaeroides  were  independently  incompetent  known  and Rps.  R.  pigment  introduced  capsulatus  pigment-binding  viridis  into  a  mutant host strain,  peptide  genes.  The  R.  puf operon, which encodes the L and M subunits of the  reaction center as well as both peptide subunits of light harvesting complex I, was shown to be capable of complementing the mutant R. capsulatus  host.  Hybrid  sphaeroides-encotied  reaction  centers,  comprised  L and M subunits and an  R.  LHI  complexes.  photosynthetic light  growth,  conditions  relative  to  and  cells  but their  These their  hybrid slower  higher  containing  growth  fluorescence  native  impairment in energy transduction.  cells  sphaeroides-  were rates  capable under  emission  complexes,  viridis center,  puhA into  Introduction  The Rps. viridis puf operon  host  cells  already  H subunit of the  containing  the  Rps.  low an was  capsulatus  of a plasmid-borne copy of the  gene, which encodes the  of  levels  indicated  found to be incapable of functional expression in the R. mutant host.  R.  capsulatus-  encoded H subunit, were formed in addition to the R. encoded  of  Rps.  reaction  viridis  puf  iii  operon, such that all structural peptides of the Rps.  viridis  reaction  center were present, still did not permit stable assembly of viridis  photosynthetic complexes.  that the  barrier  transcription.  RNA blot analysis demonstrated  to functional expression was  not at the  Differences between Rps. viridis and R.  level of sphaeroides  that may account for their differing abilities to complement the capsulatus  Rps.  mutant host strain are discussed.  R.  iv  TABLE OF CONTENTS Abstract  ii  Table of Contents  iv  List of Tables  vi  List of Figures  vii  Abbreviations and symbols  x  Acknowledgements  xi  INTRODUCTION  1  MATERIALS AND METHODS  15  1. Growth and manipulation of bacterial strains  15  2. Plasmid constructions  17  3. RNA isolation  28  4. RNA blotting and probing  30  5. Fluorescence detection  32  6. Spectrophotometric analyses  32  7. Bete-galactosidase assays  33  RESULTS 1. Rhodobacter  34 sphaeroides  study  a. Absorption spectroscopy  34 34  b. S D S - P A G E analysis of chromatophore membrane proteins....  36  V  c. Fluorescence emission  36  d. Photosynthetic growth rates  39  2. Rhodopseudomonas  viridis study  a. Oxygen and ammonia effects on  39 nifHDK  promoter activity in expression vector pNIF215  39  b. Absorption spectroscopy  43  (i) Rps. viridis put operon expression in U43(pJZ1) (ii) Rps.  43  viridis puhA gene expression in  U43(pJZ1)+(pJZ6)  48  c. RNA blot analysis.  52  (i) Rps. viridis put operon expression in U43  52  (ii) Rps. viridis puhA gene expression in U43  55  d. R. capsulatus  LHI complex levels in cells with  and without reaction center complexes as determined by absorption spectroscopy  DISCUSSION 1. R. sphaeroides study 2. Rps. viridis study  56  60 61 64  CONCLUSIONS  78  REFERENCES  81  APPENDIX  94  vi  List of Tables  Table  I:  Percent amino acid identities  in alignments  of reaction center and light harvesting subunits from ft capsulatus,  ft  sphaeroides  and Rps. viridis  13  Table  II:  Plasmids and bacterial strains used  Table  III:  Befa-galactosidase ft  capsulatus  29  specific activities  in  strains U43(pJZ4) and U43(pJZ5)  grown in ammonia-free  or ammonia-supplemented  media under high O 2 . low O 2 and anaerobic conditions Table  IV:'  43  Putative Shine-Dalgarno sites in and Rps. viridis pufoperon  ft  capsulatus  and puhA genes  68  vii  List of Figures Figure  1:  Schematic diagram of Rps.  viridis  reaction center  and o/Ci complex, showing cofactor arrangements and electron transfer times between  primary  reactants' Figure  2:  2  Schematic diagram of the photosynthetic apparatus of R. capsulatus  and relevant genes and  corresponding Rps. viridis genes  9  Expression vectors pJAJ9 and pNIF215  19  Figure 4:  Plasmids pCT1 and pTB999  20  Figure  5:  Construction of plasmids pJZ1 and pJZ2  21  Figure  6:  Construction of plasmids pJZ4 and pJZ5  24  Figure  7:  Construction of plasmid pJZ6  26  Figure  8:  Absorption spectra of intact cells of  Figure  3:  R. capsulatus  strains U43, U43(pJAJ9),  U43(pCT1),  and U43(pTB999) Figure  9:  SDS-polyacrylamide  35 gel  chromatophore vesicles from R.  electrophoresis  of  capsulatus  strains U43, U43(pCT1) and U43(pTB999)  37  viii  Figure  10:  Relative fluorescence emission levels of U43(pTB999) and U43(pCT1)  Figure  11:  Relative  growth  38  rates  of  ft  capsulatus  B10, and strain U43 containing plasmids (pJAJ9), U43(pTB999) and U43(pCT1) Figure  12:  40  Absorption spectra of membranes prepared  from  YPS-grown  cells  of  R.  capsulatus  strains U43, U43(pJAJ9), and U43(pJZ1), with second derivatives inset Figure  13:  Absorption pared  from  46  spectra  of  membranes  pre-  RCWDMSO/pyruvate/fructose-grown  cells of R. capsulatus  strains U43, U43(pJAJ9),  U43(pJZ1) and U43(pJZ1+pJZ6) Figure  14:  49  A . Agarose/formaldehyde gel of RNA from photosynthetically grown Rps.  viridis and low 0 2  grown (+/-  U43 strains containing  NH4)  ft  capsulatus  various plasmids, and B. autoradiogram of an [a- P]-labelled 32  Rps. viridis put DNA-probed blot  of this gel Figure  15:  53  Autoradiogram of an [a- P]-labelled 32  puhA  Rps.  viridis  DNA-probed blot of the gel described in  Figure 14 Figure  16:  Absorption spectra of intact cells of strains U43(pRC77) and U43(pTB999)  57 ft  capsulatus 59  ix  Figure  17:  Molecular structures of pigment and cofactor molecules of the reaction centers of and Rps. viridis  ft  capsulatus 70  X  ABBREVIATIONS AND SYMBOLS Bchl  bacteriochlorophyll  bp  base pair  BSA  bovine serum albumin  DNA  deoxyribonucleic acid  DMSO  dimethyl  EDTA  ethylenediaminetetra-acetic  kan  kanamycin  kb  kilobase  LH  light  mRNA  messenger RNA  ORF  open reading frame  PAGE  polyacrylamide  PSI I  photosystem II of higher plants  pufA,  pufB  sulfoxide acid  harvesting  gel  electrophoresis  structural genes of LHI a and p peptides  pufL, pufM  structural genes of the RC L and M subunits  pufQ, pufX  genes of unknown function, required for photosynthetic growth in R. capsulatus sphaeroides  puhA  structural gene of the RC H subunit  RC  reaction  RCV*  ammonia-free  RNA  ribonucleic acid  rRNA  ribosomal RNA  SDS  sodium dodecyl sulfate  SSC  standard sodium citrate  TBE  tris borate  Tc  tetracycline  UV  ultraviolet  center R C V medium  EDTA  and R.  xi  Acknowledgements  I  thank  Tom  Beatty,  for  his  patience, encouragement, and enthusiastic guidance throughout  the  course of my research.  thesis  supervisor  A rare atmosphere of good will, co-operation  and excitement permeates his lab. with Beatty  It has been a privilege to work  lab members Cheryl Wellington, Tim Lilburn, Farahad  Dastoor and Heidi LeBlanc. been  extraordinaire,  considerable, and their  Their contributions to this thesis have friendships wonderful.  I especially  thank Tim Lilburn, who did the protein work in the R. study.  I am grateful  to Dan Walker  approach used in the appendix.  for help with the  sphaeroides statistical  To my parents, who have helped me in  so many ways over the years, I express heart-felt gratitude.  This  thesis is dedicated to my partner, Andre Sobolewski, who put his own career "on hold" for two years, allowing me to return to school while he took primary responsibility for the care of our son, Daniel. Without  his constant support and encouragement, this thesis would  not have been possible.  1  INTRODUCTION  Photosynthesis is a fundamental biological process by which light is  converted  capable  to chemical  of  membrane  performing  this  pigment-peptide  reaction center (RC). membrane  energy.  The minimal  energy  conversion  complex  termed  structural is  the  an  unit  integral  photosynthetic  Within the R C , charge separation across the  occurs upon absorption of light.  The process can be  simply summarized as follows (see below and Fig. 1):  absorption of  light energy by pigment molecules located near the periplasmic side of the R C causes an electron discrete  intermediates,  cytoplasmic side. is  returned  electron  to  flow  membrane.  to be transferred,  to an acceptor  molecule  through located  several near the  After a complex series of reactions, the electron the  is  photo-oxidized  coupled  to  initial  proton  donor.  This  translocation  cyclical  across  the  The net effect is the production of a trans-membrane pH  and electrical potential, which can be used to drive A T P synthesis (23,34,52,70).  For many years, researchers have sought to elucidate the precise molecular mechanisms by which energy conversion in the R C and associated light harvesting  (LH) complexes occurs.  One group of  photosynthetic organisms, the purple non-sulfur bacteria, have extraordinarily fundamental apparatus  useful as experimental problems  is relatively  of  systems for the study of such  photosynthesis.  simple,  been  although  Their clearly  photosynthetic structurally  and  functionally related to photosystem II of higher plants (7,55,89). addition,  they  are facultative  phototrophs  whose  photosynthesis  genes can be fully induced during non-photosynthetic growth. essential  photosynthesis genes  can be mutated  In  and the  studied in living cells expressing the genes encoding the  Thus, effects  2  Figure reaction  1.  A center  simplified and  schematic  cytochrome  diagram b/c\  of the  complex.  Rps.  viridis  Electron  flow  between cofactors in the reaction center is shown by arrows, with transfer times indicated.  Dashed arrows outside the reaction center  show movement of protons, electrons and cofactors.  Abbreviations:  Cyt, cytochrome subunit of the reaction center; L, M and H, positions of the b/c\  respective reaction center subunits; Cyt. b/c-\,  cytochrome  complex; Cyt c 2 , cytochrome c 2 ; P, special pair; A, accessory  bacteriochlorophyll;  BP, bacteriopheophytin;  QB, secondary quinone; Q B H protonated  2 j  Q A , primary  quinone;  ubiquinol (doubly-reduced and doubly  secondary quinone);  detailed explanation see the text.  Fe, non-haem  iron.  For a  more  PERIPLASM  MEMBRANE  CYTOPLASM  LIGHT  4  photosynthetic photosynthetic  apparatus.  Major  energy transduction  in  largely  bacteria.  Three species in particular - Rhodobacter  extensively The  experiments  sphaeroides,  understanding  have occurred in the  decades, Rhodobacter  through  advances involving  purple  and Rhodopseudomonas  past  two  non-sulfur capsulatus,  viridis  have been  characterized.  first  sphaeroides  purified  photosynthetic  R C s were  obtained  by Reed and Clayton in 1968 (66).  from  R.  Within two years,  isolated R C s from a number of other purple non-sulfur bacteria were available  (40,81).  The peptides and associated cofactor molecules  were characterized. comprised  of  In most cases the complexes were found to be  three  peptide  subunits,  designated  L  (light),  M  (medium), and H (heavy) based on their apparent molecular weights according to S D S - P A G E .  The RC from Rps.  viridis  was found to  contain a fourth peptide subunit, a cytochrome, and in some species (e.g.  Rhodopseudomonas  gelatinosa)  the R C s were reported to  contain only L and M subunits (40). The  RC-associated cofactors  molecules  of  bacteriochlorophyll  bacteriopheophytin ubiquinone  and  exceptional,  were  generally  a (Bchl a),  found two  be  4  molecules  of  a, one carotenoid molecule, two  one  non-haem  containing  iron.  Rps.  bacteriochlorophyll  viridis  been determined that the Rps.  viridis  molecules of again  b (Bchl b)  a, and one menaquinone rather than two ubiquinones.  to  proved  rather  than  It has since  RC in fact contains a loosely  bound ubiquinone (as well as the more tightly bound menaquinone). Much information about RC function was obtained through various biophysical and biochemical studies on intact complexes.  By the  middle  primary  electron  of  the  1970s  donor  is  it a  had  been  Bchl  established  dimer  that  the  (31,48,59,60),  that  a  5  bacteriopheophytin that the  two  acceptors  molecule  is transiently  reduced (31,75,84)  quinones act  in series as  primary  (33,48,61).  It  was  recognized,  and  and secondary  however,  that  a  full  understanding of the mechanism of energy transduction within the R C would require a detailed knowledge of the spatial arrangement of all of the components. In  a  landmark  achievement  in  1982,  Harmut  Michel  and co-  workers succeeded in producing large, well ordered photochemically active crystals of R C s from Rps. good quality crystals of R. (3).  viridis  sphaeroides  (24,57).  Within two years,  RCs had also been obtained  Recently, high resolution X-ray analysis of these crystals (to  2.3 and 2.8 A for Rps. permitted  detailed  constructed.  viridis and R.  three  sphaeroides  dimensional  maps  of  respectively) these  has  R C s to  be  Thus the precise configuration of all of the peptide and  cofactor components of RCs from two species is now known. In  conjunction  with  X-ray  crystallography,  knowledge  of  the  primary amino acid sequences of the peptide subunits was required for an accurate determination the  RCs.  determine  The the  poor amino  of the three dimensional structures of  solubility acid  of  R C s had  sequences  by  made  it difficult  traditional  to  methods.  However in 1983, a year after the crystals were obtained from  Rps.  viridis,  from  the  first  molecular  cloning of photosynthesis genes  any organism was reported for the species Rhodobacter (80).  Subsequently,  successfully  applied  to  molecular other  genetic  techniques  species, including  (89,90), and (to a lesser extent) Rps. viridis  capsulatus  R.  (54,56,87).  were  sphaeroides The genes  encoding the peptide subunits of the RCs from all three species were sequenced, and knowledge  of the deduced amino acid sequences  assisted in construction of the three dimensional map.  6  In  addition  approaches  to  have  sequence  yielded  function and regulation (73).  The  complex,  insights  into  biogenesis of the  known genes in R. capsulatus,  genetic  questions of  of photosynthesis genes  photosynthetic  co-ordinate  cytochrome b/c-j  molecular  fundamental  of a wide variety  requiring the  C2, the  information,  regulation  apparatus  is highly  of approximately  30  including those encoding cytochrome  complex  components, the  R C and  LH  structural peptides, and the enzymes involved in Bchl and carotenoid biosynthesis.  Induction  of the  response  reduced  oxygen  to  components Regulation  additionally has  transcriptionally  been  photosynthetic apparatus occurs in concentration  regulated  found to  (1,11,97),  by  occur  and  (18),  light  with  intensity  transcriptionally  post-translationally  some  (38,97).  (44),  post-  (25,26,87,88).  The synthesis of the pigment and peptide components is coupled, such that free Bchl does not accumulate in mutant strains deficient in  pigment-binding  proteins,  and  conversely, strains  deficient  Bchl biosynthesis do not accumulate pigment-binding peptides  in  (45).  The first step of photosynthesis, the absorption of incident light, generally  does not take place in the  category  of  harvesting  pigment-peptide (LH)  RC itself,  complexes  complexes.  Light  termed  energy  or  inductive  resonance transfer  antenna  absorbed  molecules in these complexes migrates to the exciton  but in a second by  or  light  pigment  R C by delocalized  (70).  All  photosynthetic  organisms contain LH complexes in vast molar excess over R C s . These serve not only to increase the number of pigment molecules available  for  photon  absorption  photosynthetic pigments are but  also to  increase the  (on  of  total  associated with LH complexes)  (81),  probability  average,  of capturing  wavelength not absorbed efficiently by the RC. molecules  associated with  LH  and  ~95%  RCs  a  photon  of  a  Although the pigment  may  be  identical,  the  wavelengths which they maximally absorb is strongly influenced by their  microenvironment  -  i.e.  by  pigment-pigment  and  pigment-  7  peptide interactions.  Thus, LH complexes function to broaden the  spectrum of absorbable light. there  Among purple  non-sulfur  bacteria,  are species with one, two or three different types  complexes  (81).  Each  species has a characteristic  of LH  absorption  spectrum, determined by the particular combination of LH and R C complexes present. Within the membrane, the LH and R C complexes are organized in a specific  fashion  with  respect  to each  other,  permitting  energy  transfer to the R C to occur with excellent efficiency (70,81). arrangement  in the photosynthetic  membrane  represented schematically in Figure 2.  of  ft  Their  capsulatus is  The L and M subunits of the  RC are each comprised of 5 trans-membrane a-helices (not shown), to  which  the Bchl  covalently bound.  a  and other  cofactor  molecules  are non-  Although the function of the H subunit is not yet  understood, it forms a cap on the cytoplasmic side of the membrane, which is anchored by a single membrane-spanning a-helix.  The R C  absorbs light maximally at 800 and 870 nm (apart from the Soret band 760 nm, shared with all Bchl a species). Each R C is surrounded by LH complexes termed LHI with a fixed stoichiometry of ~12:1 (29). and  carotenoid  designated  molecules  LHI complexes are comprised of Bchl a noncovalently  bound to two peptides,  a and p, which form single trans-membrane  a-helices.  LHI complexes absorb light maximally at approximately 875 nm. A second, more peripheral  LH complex termed LHII is comprised of  three peptides, designated a , p and y.  As in LHI complexes, the LHII a  and p peptides form single membrane-spanning a-helices to which pigment  molecules are non-covalently bound. The function of the y  peptide, which is largely hydrophilic, is not known.  LHII complexes  have two absorption maxima, at 800 and 850 nm. The number of LHII complexes in the membrane varies, being inversely proportional to  8  both light intensity and oxygen concentration. The  R C , LHI  components  and LHII  complexes  of the photosynthetic  absorption of light in R.  comprise  apparatus  capsulatus.  that  the  structural  participate  Below the diagram  in  of the  complexes in Figure 2, the chromosomal organization of the genes encoding these components is schematically  represented.  subunit of the R C is encoded by the puhA  gene  (92), which is  believed to form part of a larger transcriptional unit. open reading frame (ORF1696) immediately  The H  Recently, an  upstream of puhA  and  transcribed in the same direction, has been shown to be necessary for assembly of LHI complexes (9).  Approximately 40 kb away, and  transcribed in the opposite orientation, is an operon designated which is comprised of 6 genes (92). designated pufQ  and pufX  put,  The 5' and 3' most genes,  respectively, have both been shown to be  required for photosynthetic growth, but their exact functions are as yet unknown (1,32). subunits  The pufB  of the LHI complex  and A genes encode the p and a respectively,  encode the L and M subunits of the RC (92).  whereas  pufL  and M  The a , p\ and y subunits  of the LHII complex are encoded by genes B A and E of the puc operon, which is located over 100 kb away on the chromosome (93). The pucC  gene, which has high sequence identity with ORF1696, has  been show to be required for assembly of LHII complexes (83). The ca.  40 kb interval separating the puh and put operons  by  operons  that  encode  enzymes  involved  is occupied  in carotenoid and  bacteriochlorophyll biosynthesis (92).  Figure 2 depicts the structural photosynthetic  apparatus  of R.  and genetic organization capsulatus,  but the same  of the figure,  unchanged, would serve to describe the photosynthetic apparatus of R.  sphaeroides. The photosynthetic apparatus of Rps. viridis differs at the gross  9  Figure 2. Schematic diagram  showing the organization  of reaction  center and light harvesting I and II complexes in the photosynthetic membrane of R. capsulatus  and R. sphaeroides.  pigment-binding  operons  sphaeroides, subunits  peptide  of  The organization of  R.  capsulatus  and R.  with structural genes shaded to match the specific they  diagramatic  encode,  is depicted  representation  enclosed in a box.  of  the  schematically Rps.  viridis  below. puf  A  operon is  Abbreviations: R C , reaction center; LHI, light  harvesting I complex; LHII, light harvesting II complex; L, M and H, light,  medium  respectively;  and  heavy  subunits  a , p and y, the  subunits  of  the  of  the  reaction light  center  harvesting  complexes; puc, operon encoding the subunits of LHII; puf,  operon  encoding the L and M subunits of the reaction center and the a and p subunits of light harvesting I complex; puhA, subunit  of  transcription.  the  reaction  The dotted  center.  gene encoding the H  Arrows  show  direction  of  line connecting ORF1696  and the  puf  operon indicates ~40 kb of DNA occupied by operons encoding genes involved  in bacteriochlorophyll  and carotenoid  biosynthesis.  Note  that a y peptide has not been reported to co-purify with the a and p peptides of the R. sphaeorides  LHII complex.  10  PERIPLASM  LHII  LHI  CYTOPLASM  8  ORFC  A  Put  Q  put  B  B  ORFD  A  E  M  M  A  C  mmm Rps.  viridis  11  structural level from that of R. two principle ways. RC  contains  and R.  viridis  subunit  in  addition  well  as the  to  in  Rps.  L,  viridis  M and  contains only one LH complex, termed  which is comprised of three trans-membrane p and y (14).  sphaeroides  Firstly, as mentioned earlier, the  a cytochrome  Secondly, Rps.  capsulatus  H.  B1015,  peptides designated a ,  The organization of the existing structural subunits as genes  encoding them  in  Rps.  viridis,  however,  is  essentially the same as in the above two  species.  representation  is enclosed in a box in  Figure 2.  of the Rps.  The Rps.  viridis  viridis  pufoperon  pufB, A, L,  exactly to their counterparts in R. Notable  differences  are the  A schematic  and M g e n e s  capsulatus  correspond  and R.  absence of puf  sphaeroides.  genes Q and X ( 8 8 ) .  The Rps. viridis pufC gene encodes the R C cytochrome subunit.  The  location of the gene encoding the B1015 y subunit is not known  (88).  As in R.  Rps.  viridis  capsulatus  and R.  sphaeroides,  the H subunit of the  RG is encoded by a gene (also designated puhA)  an operon separate and distant from the puf  located in  operon (not shown in  Figure 2).  It is clear from the above that the fundamental the  photosynthetic  similar  in all three species.  similarity  between  sphaeroides shows  apparatus,  a  the  cofactors in the Rps.  RC  structures by X-ray  representation viridis  structurally  Perhaps even  as determined schematic  both  RC.  of  organization  of  and genetically,  is  more  striking  Rps.  viridis  crystallography.  of the  spatial  the  membrane,  and  The cofactors are  R.  Figure 1  arrangement  of  non-covalently  bound to the L and M subunits, such that two nearly branches are formed.  is the  symmetrical  The axis of symmetry runs perpendicular to  from the Bchl dimer (termed the  "special pair", or  "primary donor") near the periplasmic surface, to the non-haem iron molecule near the cytoplasmic surface. light  energy,  the  special pair  Upon excitation by absorbed  rapidly transfers  an electron to  the  12  bacteriopheophytin molecule on the L branch. whether  the  intervening  Bchl  molecule  is  It is as yet unclear involved  in  electron  transfer, thus it is termed the "voyeur" or "accessory" Bchl . bacteriopheophytin  is only transiently  The  reduced, rapidly passing the  electron to a tightly bound menaquinone molecule (Q ), termed the A  "primary acceptor", on the periplasmic side of the L branch.  (Note  that near the periplasm, helices of the L and M subunits "crossover", such that the residues,  while  the  Q Q  binding-site  A  is comprised of  M  subunit  binding site on the M branch, is in fact  B  comprised of L subunit residues.  [Not shown in Figure 2])  From Q  A  the electron is transferred past the non-haem iron to the ubiquinone (Q ), termed the "secondary acceptor", on the M branch. B  Q  B  The reduced  becomes protonated to Q H , and the photo-oxidized special pair B  is re-reduced by the cytochrome subunit.  After a second photo-  oxidation event, Q H is again reduced and subsequently protonated to B  form  a  hydroquinone, Q H . B  diffuses  toward  the  replaced at the Q After  B  Q H  2  B  periplasmic  2  dissociates from the  side  of  the  RC and  membrane,  being  site by a quinone from the bulk cytoplasmic pool.  a complex series of reactions involving a cytochrome  b/c\  complex, the electrons are returned to the cytochrome subunit (for subsequent donation to the oxidized special pair), and protons are translocated to the periplasm.  This  detailed  spectroscopic reactants.  structural  findings  The  information  regarding  apparently  the  largely  confirmed  identities  of  symmetrical configuration  the  earlier primary  of cofactors  was unexpected however, as it raises the question of why electrons flow selectively amino  along the  L branch.  acid sequences of the  in the  primary  L and M subunits clearly  exert a  profound influence on electron transfer. of the  cofactors to the  branches (23).  peptide  Differences  Thus, the detailed binding  backbone is different in the  Recently, genetically  engineered  R C s have  two been  13  artificially  symmetrized  (67),  and  structural  basis for  unidirectional  R C of R.  sphaeroides,  progress electron  in  understanding  flow  is  rapidly  the  being  made. In the  arrangement periplasmic firmly  and soluble  bound  fundamentally  of in  evidence the  analyzed (23).  electron  cytochrome  cytochrome  spectroscopic  peptide  path  same  the  in  all  flow  is  performing  Rps.  that  fundamental  viridis  the  identical,  the  function  (4,5).  There  cofactor  purple  cofactor with of  the  is good  arrangement  non-sulfur  bacteria  a  is  so  far  Furthermore, the primary amino acid sequences of R C  subunits  from  a  number  of  purple  non-sulfur  bacterial  species have been determined, and a high degree of similarity is evident.  Table  I shows the  amino acid residues of R. viridis  per cent identities  capsulatus,  R.  R C ( and LHI) complexes (89,96).  subunit, which forms the conserved, participate  while directly  the  H  sphaeroides  and  of  Rps.  It is noteworthy that the L  "active branch" of the subunit,  in alignments  which  in the charge separation  R C , is the  apparently  does  reaction, is the  most not least  conserved.  Table  I:  Percent  Identities from  alignments  of RC and LH  Subunits  R  capsulatus/R.  sphaeroides  R. capsulatus/Rps.  RC L:  78  59  RCM:  76  50  RCH:  64  38  LHI a:  78  37  LHI p:  76  32  viridis  14  It is perhaps appropriate to mention at this point the extent to which RCs from purple non-sulfur bacteria are similar to those of photosystem fundamental  II  (PSII)  in  chloroplasts  and  processes in both are entirely  cyanobacteria. analogous:  The  In PSII, a  photo-oxidized chlorophyll special pair donates an electron first to a  pheophytin, and then  to two  quinones acting  in series.  The  secondary quinone is exchangeable with the bulk quinone pool (7). The  cofactors  in  PSII  are  associated  with  peptide  subunits,  designated D1 and D2, which show weak but significant sequence similarities with the bacterial L and M subunits (55).  Furthermore,  R C s from purple non-sulfur bacteria and PSII are both sensitive to herbicides of the s-triazine its binding site.  type, which act by displacing Q  from  B  Mutations conferring herbicide resistance in purple  non-sulfur and PSII RCs and have been shown to change homologous residues  (16,62).  These  structural  strongly suggest that purple green  plants,  algae  and  and  functional  non-sulfur bacterial  cyanobacteria  similarities  R C s and PSII of  evolved  from  a common  ancestor. Given  these  components  of  striking the  similarities,  photosynthetic  the  possibility  apparatus  from  existed one  that  organism  might be capable of functional expression in other organisms.  As a  first step in addressing this possibility, I wished to test whether LH and RC genes from one species of purple non-sulfur bacteria could be functionally expressed in another species.  Specifically, I wished to  introduce  capsulatus  heterologous genes into  an  R.  mutant host  strain, deficient in all known pigment-binding peptide genes. hoped that characterization shed  of inter-species hybrid complexes might  light on structure-function  apparatus. possible,  It was  relationships in the  photosynthetic  Furthermore, if functional expression were shown to be it would  imply  that  R.  capsulatus,  which  is  relatively  15  easily  manipulated  molecular-biological  genetically, studies  of  might a wide  be  used  variety  as  of  a  host  for  photosynthesis  genes from genetically recalcitrant species. I report here the results of two independent sets of experiments. In the  first,  I demonstrate  sphaeroides puf-  put  that a plasmid-borne copy of the  R.  operon is capable of genetically complementing a  puc- R. capsulatus  sphaeroides-encotieti  host.  Hybrid  R C s , comprised of  L and M subunits, and an R.  R.  capsulatus-  encoded H subunit, assemble in vivo and are capable of supporting photosynthetic growth.  Cells containing hybrid R C s are compared to  cells containing native RCs with respect to absorption spectroscopy, peptide  subunit content,  growth  rates.  infra-red  fluorescence and  photosynthetic  In the second set of experiments, I show that the Rps. operon does not functionally complement the same R. mutant  host  strain.  Furthermore,  stable  heterologous  viridis  puf  capsulatus pigment-  peptide complexes do not form even when a plasmid-borne copy of the Rps.  viridis  puf operon.  puhA  gene is introduced along with the Rps.  viridis  The implications in each case are discussed.  The R C and LH genes from the vast majority of photosynthetic organisms are as yet uncharacterized.  In an attached appendix, I  briefly describe a technique which could be used to detect such uncharacterized genes in R. capsulatus.  Theoretical considerations  regarding construction of plasmid expression libraries and screening in R. capsulatus  are included.  16  MATERIALS AND METHODS  Growth  1.  and manipulation of bacterial strains.  The R. capsulatus  strains used in this study were:  strain B10 (50), puf'puc' (1).  strain U43 (74), and puf  wild type  strain A R C 6  Strain U43 was made by deletion of a 2,778 bp DNA fragment part of pufQ  spanning capsulatus  and all of pufBALMX  from the puc- R.  strain MW422 (74). The nature of the puc mutation in  this strain is unknown. All strains were routinely grown in YPS medium (86). Oxygenlimited (low O 2 ) cultures were grown in Erlenmeyer flasks filled to 8 0 % of their nominal capacity and shaken on a rotor and shaker at 150 RPM.  Under these conditions the expression of photosynthesis  genes is induced.  Cultures to be used for transfer to anaerobic  photoheterotrophic  growth  conditions  were  first  stationary phase under oxygen-limited conditions.  grown to  Aliquots were  diluted to 20 Klett units (about 6 X10 colony forming units/ml) in 7  completely filled 20 ml screw cap tubes. occurred  in aquaria  intensities.  illuminated  with  Photosynthetic growth  light  sources  of varying  Dark anaerobic cultures were grown in RCV medium (10)  supplemented  with  20 mM  (DMSO), and 5 % pyruvate.  fructose, 30 mM  dimethylsulphoxide  DMSO served as an electron acceptor for  anaerobic respiration (37). When induction of the R. nifHDK or  promoter was required, cultures were grown anaerobically  under  low O 2 conditions  supplemented with 7 mM For  capsulatus  in ammonia-free  RCV  (RCV*)  sodium-glutamate as a nitrogen source.  fluorescence measurements colonies of cells  were  9 o r  w n  17 aerobically  on  RCV  medium  supplemented  with  1.5%  agar.  Photosynthetic growth on plates was obtained in anaerobic jars in the aquarium.  Plasmid-containing strains were grown in media supplemented with antibiotics as follows: pJAJ9 and its derivatives, tetracycline (0.5 ng/ml);  pNIF215 and its derivatives, kanamycin (10 ng/ml);  pTB999, tetracycline (0.5 jig/ml); Antibiotics  were  omitted  from  p R C 7 7 , kanamycin (10 ng/ml).  photosynthetically  grown  cultures.  All cultures were grown at 34° C.  Rhodobacter  sphaeroides  strain  2.4.1.  was  photosynthetically in Y P S medium as described for R. with ca. 20 W / m  2  grown  capsulatus,  illumination provided by incandescent lamps.  Rhodopseudomonas  viridis  strain  D S M 133 was  grown  photosynthetically as above, in medium comprised of 50% R C V / 50% Y P S supplemented with 0.2 ug/ml para-amino benzoic acid. Escherichia  coli strains C600r-m+ (13) and HB101 (pRK2013)  (71) were used to deliver plasmids by triparental conjugation to R.  capsulatus,  (27)  and were cultured at 37° C in LB medium (49)  supplemented when appropriate with tetracycline or kanamycin (10 ug/ml). 2.  Plasmid  constructions.  Flow charts for the relevant plasmid constructions are given in Figures 8 - 1 1 , and all plasmids are listed in Table II. host  range  plasmids were  used throughout  this  Two broad  study  for  the  18  expression of heterologous cloned genes in  ft  capsulatus.  were plasmid pJAJ9 and plasmid pNIF215 (Fig. 3). an  RK2 (71)  derivative  containing the  R.  Plasmid pJAJ9 is  capsulatus  promoter upstream of a multiple cloning site (43). is an RSF1010 derivative (72) operon  promoter  upstream  Wellington, unpublished).  containing the R. of  a  multiple  puf  capsulatus  cloning  puf  site  (Cheryl  such that  promoters.  carries a complete wild-type  copy of the  operon, and was constructed as follows.  pJW1 (90) contains the fragment.  nifHDK  Unless stated otherwise, all heterologous  their expression was driven by the puf or nif  sphaeroides  operon  Plasmid pNIF215  genes were inserted into these vectors in an orientation  Plasmid pCT1  These  ft  sphaeroides  R.  Plasmid  puf operon on a 4.5 kb Pst\  Digestion with Psfl released this fragment,  then gel purified and ligated into the unique Pst\  which  was  site of pJAJ9 (see  Fig. 4a). Plasmid pTB999 is a pRC11 (17) ft  capsulatus  derivative carrying the entire  puf operon (Fig. 4b).  Plasmid pJZ1, which carries a complete wild-type copy of the Rps.  viridis  Plasmid viridis  puf  pKVS1 puf  operon, was constructed as follows (see Fig. 5a). (gift of Joe Farchaus) which  operon,  Oligodeoxynucleotide (5'-CGAGCTCG-3'  )  was  linearized  linkers  containing  (synthesized  by  Dept., U.B.C.) were ligated to the Hpa\ resultant  plasmid  was  digested  fragment containing the Rps.  by  viridis  with  contains the  digestion an  Tom  Sst\  with  Rps. Hpa\.  recognition  Atkinson, Biochemistry  - generated blunt ends. Sst  site  I,  puf operon.  releasing  a 6.7  The kb  The fragment was  gel purified and ligated into the unique Sst\ site in pJAJ9. Plasmid pJZ2, which carries a complete wild-type copy of the  19  B  Figure 3. A, A representation of expression vector pJAJ9. The open arrow around pufQB' indicates the direction of transcription initiated at the R. capsulatus puf promoter. The R. capsulatus pufQ gene, as well as the first 20 codons of the pufB gene are present in this vector. Tc indicates the approximate position of the tetracyline resistance determinant, transcribed in the direction shown by the arrow. All unique restriction sites following the puf promoter are indicated. B, A representation of expression vector pNIF215. The open arrow around NIFHDK indicates the direction of transcription initiated at the nifHDK promoter. kan and sm designate kanamycin and streptomycin resistance determinants respectively, with arrows showing direction of transcription. Unique restriction sites following the nifHDK promoter are shown.  20  Figure 4. A. A representation of plasmid pTB999. The solid black line represents the enitreR capsulatus puf operon, transcribed in the direction shown by the arrow. Tc designates the tetractycline resistance determinant. B. A representation of plasmid pCT1. The solid black line represents 4.5 kb of R sphaeroides DNA including the complete puf operon and flanking sequences, inserted into the unique Pst\ site in pJAJ9 and transcribed in the direction shown by the arrow. A detailed description of the construction can be found in section 2 of Materials and Methods.  21  Figure 5.  Construction of A. plasmid pJZ1 and B. pJZ2.  represent  vector  DNA.  described in Figure 3B. puf  The open arrows  The thin lines  around pufQB'  are as  The thick black lines represent Rps.  viridis  operon DNA, transcribed in the direction indicated by the arrow  above.  The thick shaded lines represent Rps.  the puf  operon.  resistance direction  viridis  DNA flanking  Tc and amp designate tetracycline  and ampicillin  determinants of  transcription.  respectively, A  with  detailed  arrows  description  indicating of  both  constructions can be found in section 2 of Materials and Methods.  22  ligation  23  Rps.  viridis  viridis  puf operon plus an additional 2.1  kb of upstream  sequence, was constructed as follows (see Fig. 5b).  pKVS1  was  digested with H / n d l l l ,  containing the Rps. The fragment  viridis  releasing  an  8.8  Rps.  Plasmid  kb  fragment  puf operon plus the upstream sequence.  was gel purified and inserted into a unique  site in plasmid pJAJ9, which was generated  as follows.  pJAJ9 was linearized by digestion with S a m H I .  Hmdlll Plasmid  Overhanging ends  were filled in by DNA polymerase I (Klenow fragment) treatment to generate  blunt  ends.  oligodeoxynucleotide  The  linkers  termini  were  containing  a  ligated  H/ndlll  to  synthetic  recognition  (5'-CCAAGCTTGG-3') (P-L Biochemicals, Inc., Milwaukee, Wis.). generated  a  unique  site  H/ndlll  in pJAJ9,  site This  while duplicating  the  original (unique) B a m H I site. Plasmid additional  ca.  constructed contains  pJZ4 carries the  the  0.9  as E.  entire  kb upstream  follows coli  (see  lac  trp  E. coli operon  Fig. 6a).  operon  lac  sequence,  Plasmid  plus the  operon plus an and  pMC903,  upstream  trp  was which  operon  sequence, was digested with S a m H I and BglW, releasing a ca.  7.6  kb  trp  fragment  sequences.  containing  the above  This fragment  described lac operon and  was gel purified and inserted  into  the  Plasmid pJZ5 is identical to plasmid pJZ4 except that the  7.6  unique S a m H I site in pNIF215.  kb BamH\-Bgl\\  fragment  has  been  inserted  in  the  opposite  orientation with respect to the nif promoter (see Fig. 6b). Plasmid pJZ6 carries a complete wild type copy of the viridis  puhA  gene, and was constructed as follows  (see  Fig. 7).  Plasmid pDG4B ( gift of Joe Farchaus), which contains the viridis  puhA  gene, was digested with Xma\\\,  Rps. Rps.  and overhanging ends  were filled in by treatment with Klenow fragment.  The linearized  24  Figure 6.  Construction of A. plasmid pJZ4 and B. pJZ5.  represent vector DNA.  The thick lines represent E. coli lac operon  DNA, inserted into the unique BarnHI site in pNIF215. of transcription  initiated at the  the open arrow. the arrow above.  The thin lines  nifHDK  promoter  The direction  is indicated  by  The orientation of the insert (5' - 3') is shown by A detailed description of the construction can be  found in section 2 of Materials and Methods.  25  BomHI  26  Figure 7.  Construction of pJZ6.  Thick black lines indicate the Rps. shaded lines indicate designate  ampicillin,  Rps.  respectively,  transcription.  A detailed  viridis puhA gene DNA.  viridis flanking DNA.  kanamycin  determinants  Thin lines indicate vector DNA.  with  amp, kan and sm  and  streptomycin  arrows  indicating  description of the  found in section 2 of Materials and Methods.  Thick  resistance direction  construction  can  of be  27  28  resultant  plasmid was  viridis puhA  then  cut with  gene on a ca. 0.1  EcoRI,  releasing the  kb blunt - E c o R I  Rps.  fragment.  The  fragment was gel purified and inserted into plasmid pNIF215 which had  been  digested  with  Hin6\\\,  blunt-ended  by  filling  in with  Klenow fragment, and subsequently digested with EcoRI. Plasmid pRC77 is a pRC11 derivative carrying a complete but modified R. capsulatus insertion  of  a  puf operon (17). synthetic  The modification involves  oligodeoxynucleotide  GCCCACCGGCAGCTGCCGGTGGGC-3') naturally insert  occurring hairpin downstream  functions as a  (5'-  immediately  following  the  of the pufBA  genes.  The  strong transcriptional  terminator,  such that  there is little or no transcription of the downstream pufLMX  genes  in this construct. DNA  was purified from agarose gels by adsorption to glass  beads, using the commercially available "Gene Clean" kit (BioRad). Digestion of DNA with restriction endonuclease enzymes, agarose gel  electrophoresis,  other  recombinant  DNA  transformation  of  DNA procedures were performed  standard procedures  4.  ligations,  E. coli and according to  (49).  RNA isolation. RNA was harvested as described (85)  follows.  Rps.  viridis strain DSM 133 was grown  as described in section 1. U43(pJZ1),  U43(pJZ1+pJZ6)  under  O2  low  or  RCWDMSO/fructose  R.  capsulatus  strains U43,  conditions  supplemented supplemented  in  two  with  U43(pJAJ9),  with 7  different  10mM  mM  was also grown in Y P S medium.  added as required.  photosynthetically  and U43(pJZ1+pNIF215) were all grown  anaerobic  RCVVDMSO/fructose  U43(pJZ1+pJZ6)  from cultures grown as  media:  NH , and 4  sodium-glutamate. Antibiotics were  In all cases, cells were harvested at the mid to  late log phase of growth.  29  T A B L E II:  Plasmids and bacterial strains used  Plasmids  Description (source or reference)  pRK2013  Mobilizing plasmid  pJAJ9  Expression vector utilizing  (27) puf  promoter  (contains pufQ gene and 1st 20 codons of pufB) (43; pNIF215  Fig. 3a)  Expression  vector  utilizing  promoter(C. Wellington; pTB999  pRC11 XhoW  derivative segment  promoter.  Fig. 3b)  missing  5'  nifHDK  the  of the  Contains R.  puf  EcoR\operon  capsulatus  puf  operon (95; Fig. 4A) pCT1  pJAJ9  derivative  sphaeroides puf operon (95; pJZ1  pJAJ9  derivative with 6.7  fragment  R.  containing  containing  Fig. 4B) kb  Rps.  Hpa\-Sst\ viridis  puf  operon (this work; Fig. 9A) pJZ2  pJAJ9  derivative  fragment  with  containing  8.8 Rps.  kb  H/ndlll  viridis  puf  operon plus 2.1 kb of upstream sequence (this work; Fig. 9B) pJZ4  pNIF215 derivative BglW  fragment  with 7.6  kb  containing  E.  operon (this work; Fig. 10A)  BamH\coli  lac  30  T A B L E II  (continued)  Plasmids  Description (source or reference) pNIF215  pJZ5  derivative with E. coli lac operon  in opposite orientation (this work; Fig. 10) pNIF215  pJZ6  EcoRI  derivative with 0.1 fragment  containing  kb  XmaIII-  Rps. viridis  puhA gene (this work; Fig. 11) pRC11 derivative containing R.  pRC77  puf  operon  with  transcriptional  terminator following puf A  Bacterial  r  E. coli HB101  (17)  Description (source or reference)  strains  E. coli C600 -m+  capsulatus  Donor strain in conjugations (13) -Helper  plasmid host strain in triparental  conjugations  (27)  R. capsulatus B10  wild type  (50)  R. capsulatus U43  puf- puc- (74)  R. capsulatus A R C 6  puf-  R. sphaeroides 2.4.1.  wild type (gift of Joanne Williams)  Rps. viridis DSM 133  wild type (gift of Joe Farchaus)  (1)  31  5. RNA blotting and probing. Prior to loading on 1.4 % agarose/formaldehyde gels, a 34 u.g aliquot of Rps. plasmid  viridis  containing  RNA and 17 \ig  R.  capsulatus  aliquots of RNA from  strains were denatured  all  in the  presence of ethidium bromide (final cone. 0.04 |ig/ml) as described (68,69).  Each denatured sample was then divided into two equal  portions, and two gels were loaded identically - each with 17 u\g of Rps.  viridis RNA and 8.5 ng of R.  capsulatus  RNA.  After blotting,  one gel was used to probe for the presence of Rps. the  transcripts.  Gels were run for 3 hours at 100V, rinsed for 5 minutes and  determine  for  the  photographed  presence  through  the position of the  a  of  Rps.  U.V.  viridis  puf  transcripts, in dhteO  other  viridis  puhA  transilluminator  rRNA with respect to the  to  molecular  weight markers (see Fig. 14A). Transfer  to  nylon  membranes  (ICN  Bio-Trans)  was  accomplished by electroblotting at 30 V in 0.5X T B E buffer (49) about 16 hours at 4 ° C.  for  The membranes were then baked for 2 hours  at 8 0 ° C , U.V. cross-linked for 3 minutes and again photographed through a U.V. transilluminator  to determine  quality  and extent of  transfer. Membranes  were  prehybridized  with  heat-denatured  salmon  sperm DNA at a final concentration of 500 ug/ml ( 9 5 ° C, 10') in 5 X S S C (49), 1% SDS, 50% formamide and 10 mM EDTA for a minimum of two hours at 4 2 ° C.  Heat denatured DNA probes (a- P-labelled 32  the random oligonucleotide primer method [36]) the  prehybridization  mixture.  When  by  were then added to  probing for  Rps.  viridis  puf  transcripts the probe DNA used was identical to the DNA sequence inserted into plasmid pJAJ9 to create pJZ2 (see  Fig. 5B).  probing  probe  identical  for  Rps.  to the  viridis  puhA  transcripts,  DNA sequence inserted  the  into  used  When was  plasmid pNIF215 to  32  create pJZ6 (see Fig. 7). Hybridization occurred overnight at 4 2 ° C .  Membranes were  then washed twice for 10 minutes at room temperature in 2 X S S C + 0.1% S D S , twice for 10 minutes at 50° C in 2 X S S C + 0.1 % S D S and twice for 5 minutes at 55° C in 0.2 X S S C + 0.1 % S D S . They were exposed to X-ray films for 2 to 13 days at -80°  C with intensifier  screens. Fluorescence  6.  The (95).  R  detection.  infra-red fluorescence of cells was evaluated as described capsulatus  strain  negative control, and R positive  U43(pJAJ9) was routinely  capsulatus strain ARC6(pJAJ9) served as a  control.  Spectrophotometric  7.  used as a  Absorption  analyses.  s c a n s were  obtained  using  a  Hitachi  U2000  spectrophotometer.  All cells were grown under low O2 conditions to  induce  of  synthesis  the  photosynthetic  apparatus,  and  were  strains  U43,  harvested at the mid to late log phase of growth. In the R  sphaeroides  study,  R  capsulatus  U43(pJAJ9), U43(pCT1) and U43(pTB999) and were grown in Y P S medium.  Intact cells (ca. 1.8 X 10 9 cells suspended in 22.5% B S A  [77] in Y P S medium) were scanned. In the Rps. U43(pJAJ9),  viridis  study,  R  capsulatus  strains  U43,  U43(pJZ1), U43(pJZ1+pJZ6) and U43(pJZ1 +pNIF215),  were grown both in R C V * medium supplemented with 7mM^ sodiumglutamate/DMSO/fructose/pyruvate supplemented  with  Membranes were  10mM  and  in  RCV*  medium  NH4 + / D M S O / f r u c t o s e / p y r u v a t e .  prepared as follows.  Forty  ml  of cells were  33  harvested, washed by resuspension in 10 ml RCV medium, and finally resuspended  in  750  sonication with a  |il  of  RCV.  Membranes  Branson microtip  probe  (2  X  were  released  by  15"  treatment  at  setting "2" on a Sonifier cell disrupter 350 sonicator, Branson Sonic Power Company).  The samples were centrifuged for 60" to  cellular debris, and supernatant fluids were scanned. strains  U43,  U43(pJAJ9)  R.  pellet  capsulatus  and U43(pJZ1) were also grown  in Y P S  medium, and membranes, prepared as described above, were scanned (Fig.  12).  The supernatant  spectrophotometer  fluids were then  recovered from  cuvettes, diluted to 4 ml in R C V medium  centrifuged  for  30  minutes  supernatant  fluids and pellets  at  100,000  rpm.  The  (resuspended in 750  the and  resultant  uJ RCV) were  both scanned.  8.  Beta-galactosidase The  capsulatus  amount strains  of  assays.  (3-galactosidase U43(pJZ4) (58).  present  U43(pJZ5)  Specifically,  20  was -  30  in  R.  assayed  essentially  as  capsulatus  cells were pelleted and resuspended in 1 ml of Z-buffer  (58).  described  and  activity  ml  of  R.  The resuspended samples were transferred to Eppendorf tubes  and sonicated as above.  Various proportions of supernatant  fluid  (containing the p-galactosidase enzyme) and Z-buffer to give a total volume of 800 ul were added to a 1 ml cuvette. nitrophenyl-p-D-galactoside  (ONPG;  The substrate o-  200 jil of a 4 jig/jil solution in  Z-buffer) was then added and the sample mixed well by inversion. minutes.  repeated  Increase in absorbancy at 420 nm was followed for 1 - 2  34  RESULTS 1. Rhodobacter  a.  Sphaeroides  Absorption  Study.  spectroscopy  Figure 8 shows an overlay of typical absorption spectra of intact cells of R. capsulatus and U43(pCT1).  U43(pTB999), which contains a plasmid-borne copy  of the R. capsulatus 875 nm. complex  strains U43, U43(pJAJ9), U43(pTB999)  puf  operon, has absorption maxima at 800 and  These peaks result from RC (800 and 870 nm) and LHI (875 nm) absorbancy respectively.  U43(pCT1), which  contains plasmid pJAJ9 into which the R. sphaeroides has been similar  inserted, has an absorption  to that  of U43(pTB999),  puf  operon  profile qualitatively very  whereas  U43(pJAJ9), which  contains the expression vector alone, does not. Thus, the presence of the R. sphaeroides  puf  genes in R. capsulatus  U43 results in the  formation of stable heterologous LH and hybrid RC complexes with superficial capsulatus  spectroscopic  properties  very  similar  to native  R.  complexes.  In general, peak amplitudes in absorption spectra are roughly proportional to quantities of absorbing pigment-peptide complexes. Measurements  of peak to baseline ratios in these spectra showed  about 10 - 3 0 % greater levels of pigment-peptide complexes in U43(pTB999) cells relative to U43(pCT1).  There are a number of  possible  differences  reasons  for this,  including  accumulation, translation, protein  stability  in mRNA  or assembly  barriers  (see Rps. viridis and Discussion Sections).. Note that the absorption spectrum of U43(pJAJ9) shows a slight increase in absorbancy at approximately 800 nm not seen in U43  35  Figure U43 (R.  8.  Absorption spectra of intact cells of R.  containing various plasmids. capsulatus  operon);  c.  puf  capsulatus  strain  T r a c e s : a. cells containing  pTB999  operon); b. cells with pCT1  cells with  cells with no plasmid.  pJAJ9  (expression  (R.  vector  sphaeroides lacking; insert);  puf d.  36  alone.  This is believed to be associated with the R.  pufQgene  present on pJAJ9 (see the Rps.  viridis  capsulatus  and Discussion  sections). b.  SDS-PAGE  analysis  of  chromatophore  membrane  proteins Confirmation of the presence of all the peptide components of the LHI and RC complexes was obtained by S D S - P A G E , the results of which are shown in Figure 9.  The presence of protein bands with  mobilities predicted for the subunits of the R C and LHI complexes was evident in samples from cells of U43 containing either the capsulatus  or R. sphaeroides  puf genes, whereas those bands were  absent in samples of U43 host cells. sphaeroides  It is noteworthy that the  R C L and M subunits have slightly different  compared with the equivalent R. capsulatus b.  Fluorescence The  native  relative- efficiency  mobilities  peptides.  and  of light energy  transduction  hybrid] photosynthetic  be seen in Figure 10, U43(pCT1) cells containing R fluorescence capsulatus  hybrid  than  R C s , emit  cells  of  significantly  U43(pTB999)  pigment-peptide complexes).  in  complexes  evaluated! by comparison! of their intensities of fluorescence. and  R.  emission!  R. capsulatus;  complexes  R.  the was  As cart  sphaeroides  greater  (containing  Wild type R.  levels native  LHI of R.  sphaeroides  strain. 2.4.1 emits marginally more fluorescence than, does wild, type R.  capsulatus-  strain  B10/  (data  not  shown),  however  the  significantly enhanced; fluorescence of U43(pCT1) is suggestive of a  :  dysfunction  in- energy transduction specific to cells containing; the>  hybrid R C .  In general* cells that contain functionally impaired' L H or.  RC  complexes  are  unable  to  electrochemical energy as efficiently  convert  absorbed  as wild-type  some of the absorbed light as fluorescence. In R.  light  cells, and capsulatus  to emit  37  1  2  3  29 18.4 B875  6.2-  Figure 9. vesicles.  S D S - p o l y a c r y l a m i d e gel electrophoresis of Lanes:  chromatophores chromatophores  1.  from from  chromatophores U43  from  containing  U43(pTB999).  The  chromatophore  strain  plasmid bands  i  U43;  2,  pCT1;  3,  corresponding  to  reaction center subunits H, M and L are indicated on the right, as are the  LHI  complex  bands  (designated  B875).  The  molecular m a s s markers (in kDa) are shown on the left.  positions  of  38  Figure 10.  Relative fluorescence of colonies of R. capsulatus  A. cells of strain  U43 containing  plasmids  cells.  pTB999 viewed  with  visible light; B. U43(pTB999) viewed through the infra-red filter; C. U43(pCT1) viewed with visible light; D. U43(pCT1) viewed the infra-red  filter.  through  39  U43(pCT1),  efficient  transfer  of  light  energy  from  the  R.  sphaeroides  LHI complexes to the hybrid R C may in some way be  impeded, and/or creation of a stable charge separation by the hybrid RC may be impaired (see Discussion). c.  Photosynthetic  Growth  Rates  The data given above indicated. that hybrid photosynthetic R C s , comprised of R. sphaeroides L and M subunits and an R. H subunit, were capable of stable assembly in R. along with R.  sphaeroides-encoded  capsulatus  capsulatus  LHI complexes.  U43  I wished to  determine whether this assemblage was functional enough to permit photosynthetic growth. kinetics  of  intensities.  Figure 11 shows the photosynthetic  U43(pCT1) and  U43(pTB999) grown  growth  at various  light  At 20 W / m (considered a moderate light intensity), no 2  significant difference observed,  while  photosynthetic  in the growth  U43(pJAJ9)  growth  was  (Fig. 11 A).  rates of the two strains was shown  to  W h e n the  be  incapable  light intensity  of was  dropped to 4 W / m (considered a low light; intensity),; U43(pCT1) 2  grew somewhat  more slowly than  U43(pTB999) and, due to  the  absence of LHII complexes in, U43(pCT1) and U43(pTB999), they both grew  more  (Fig.11B).  slowly  than  At  W/m ,  2  wild-type 2  R.  U4'3(pCT1'>  capsulatus; was  unable  strain  B10  to  grow  photosynthetically, whereas U43(pTB999) began to grow after a lag of 163 hours (Fig. 11C).  At 1 W / m , 2  neither  U43(pTB999)  nor  U43(pCT1) were capable of photosynthetic growth (Fig.. 11 D). 2'.;. Rhodooseudomonas a.  Determination  regulation plasmid  of  R.  viridis of  Study.  oxygen  capsulatus  and nifHDK  ammonia promoter  effects activity  pNIF:215  The R. capsulatus nifHDK operon promoter was introduced  on in  40  Figure 11.  Comparison of photosynthetic growth of R.  capsulatus  strains containing either the hybrid or native reaction center. W/m2; B. 4 W/m?; C. 2 W/m.2; D. 11 W/m.2.  A. 20  Klett  Units  Klett  Units  Klett  O O  M O O  Units  42  into plasmid pJRD215 by Cheryl Wellington (as described inMaterials and Methods) to create an expression vector (pNIF215) capable of co-replicating along with the pre-existing vector pJAJ9,  which  utilizes the  R.  fr  capsulatus  expression of cloned genes (see Fig. 3B). transcription from the presence  of  nifHDK  ammonia,  but  obtained in ammonia-free  (64).  promoter  for  It has been shown (64) that  that  high  levels  of  transcription  are  medium supplemented with (e.g.) glutamic  promoter  Furthermore, transcription from the is independent  of the  nitrogen  R.  source  Both the puf and nif promoters are known to be repressed by  oxygen. genes  puf  puf  expression  promoter is totally repressed in the  acid as a nitrogen source. capsulatus  capsulatus  Thus, it should be possible to obtain transcription of cloned from  both  expression  vectors  by  growing  cells  either  anaerobically or under reduced aeration in ammonia-free medium.  In order to determine  the optimal  conditions for expression of  heterologous genes introduced into pNIF215, I first created a fusion to the nifHDK grown  under  a  promoter (pJZ4).  variety  of  R. capsulatus U43(pJZ4)  conditions  (various  combinations  lac was of  high/low/anaerobic oxygen and presence/absence of ammonia),  and  nifHDK  was  promoter  determined  by  activity  monitoring  under  each  of  these  p-galactosidase  conditions  activities.  Parallel  experiments were performed on U43 cells containing plasmid pJZ5, a pNIF215  derivative  containing  the  lac  operon  inserted  in  the  opposite orientation to pJZ4, such that expression of the lac genes is not driven  by the  nifHDK  expression derived from the lac  promoter.  In  this way,  any  lac  DNA fragment could be accounted  for. Table III promoter  shows that, as expected, the highest levels of  activity  were  obtained  under  conditions (1,854 nm ONPG/min/mg).  anaerobic,  However  nifHDK  ammonia-free  significant  activity  43  (598  nm ONPG/min/mg) was also obtained under low O2,  free conditions.  U43 cells containing the pNIF215 derivative pJZ6,  which contains the Rps. RCV*  viridis puhA gene, were initially grown in  supplemented  with  glutamate/DMSO/fructose/pyruvate conditions.  RNA  expression  blots  showed  of  strong  considerably  U43(pJZ1+pJZ6) transcription  photosynthetic  faster  anaerobic  sodium-  under both low O2  anaerobic conditions (data not shown). is  ammonia-  and  probed both  for low  puhA and  O2  Because growth under low  more  growth,  under  and anaerobic  reproducible  U43(pJZ1+pJZ6)  than was  O2  non-  routinely  grown under low O2 conditions.  b. Absorption spectroscopy (i) Rps. viridis puf operon expression in U43(pJZ"n Absorption  spectra  of  intact  cells  indistinguishable from those of U43(pJAJ9) increase  resolution  membranes  of  any  small  prepared from U43,  peaks  of  U43(pJZ1)  (data not shown). that  might  be  shows  a  typical  To  present,  U43(pJAJ9), and U43(pJZ1) cells  grown under low O2 conditions in Y P S medium were scanned. 12A  were  membrane  absorption  spectrum  of  Figure U43.  Although whole cell scans of U43 showed no absorption peaks ( Fig. 5), membrane scans showed peaks at -754, 803 and, at the limits of detection,  864  determined  nm.  The  precise locations of these  peaks were  in 2nd order derivative scans (shown as insets in Fig.  12), in which valleys correspond to peaks in the original scan, and vice  versa).  The origin of these peaks is not known.  peak is probably due to free bacteriopheophytin a product of Bchl a);  The 754 nm (a  break-down  the 803 nm and 864 nm peaks may be associated  with some as yet uncharacterized pigment-peptide complex(es)  such  as  (see  the  putative  Discussion).  assembly  peptide  encoded  by  ORF1696  Alternatively, the observed peaks may result from very  low level "leaky" LHII expression in U43.  As stated in  44  Table  III:  Specific  cultures  grown  conditions supplemented  in  activities  under  high  either  of O2,  p-galactosidase low  ammonia-free  O2  or or  in  anaerobic ammonia-  media  AMMONIA-FREE: HIGH 0 U43(pJZ4)  1  U43(pJZ5)  2  197  LOW 0 2  2  3  22  ANAEROBIC  591  1,854  36  48  AMMONIA-SUPPLEMENTED : 4  LOW 0 2  ANAEROBIC  U43(pJZ4)  22  161  U43(pJZ5)  22  12  1  pJZ4 is the nifHDK-lac  fusion in the correct orientation for  lacZ  expression 2  3  pJZ5 is the nifHDK-lac  fusion in the incorrect orientation  Specific activities are expressed as nmoles O N P G cleaved per minute per mg protein  4  Final ammonia concentration = 10mM  45  Materials and Methods, the nature of the mutation in U43 resulting in the LHII" phenotype has not been determined  (74).  A typical absorption spectrum of U43(pJAJ9) shown in Figure 12B.  cell membranes is  The amplitude of all absorption peaks was  increased compared to U43 cells, the 864 nm peak was consistently blue-shifted to 855 nm, and a small peak at ~688 nm appeared. increased  absorbancy in U43(pJAJ9),  which was  The  sufficient to  be  detectable in whole cell scans as a slight rise at ~800 nm (see Fig. 8d), is believed to be associated with the pufQ pJAJ9.  gene present on  The pufQ gene is known to be required for Bchl biosynthesis  (see below and Discussion). In addition to pufQ,  plasmid pJAJ9 contains a truncated  pufB  gene, encoding the first 20 amino acids of the LHI p peptide (see Fig. 4a).  It is unlikely that the peaks observed in U43(pJAJ9)  resulted  from Bchl associated with a peptide encoded by the 5' end of  pufB  fused to the downstream lac sequence on pJAJ9 because histidine residue 21,  part of the  highly conserved Ala-X-X-X-His sequence  believed to be involved in Bchla binding (96),  is not encoded on  pJAJ9 (see Discussion). A  representative  U43(pJZ1), puf  operon  absorption spectrum of cell  membranes  which contains plasmid pJAJ9 into which the R. has  been  inserted,  is shown  in  Figure  12C.  from viridis The  amplitudes of both the 754 nm and 685 nm peaks were consistently reduced in U43(pJZ1)  relative to U43(pJAJ9).  The reduction in 754  nm absorbancy suggests that Bchl a might be bound to peptides more tightly in U43(pJZ1) cells, thus reducing the amount of the free Bchl a that the  break-down  product,  bacteriopheophytin  peaks observed in U43(pJZ1)  do not  formation of Rps. viridis LHI or R C complexes.  a. reflect  It is evident the  However, the  stable  46  Figure  12.  YPS-grown plasmids.  Absorption spectra of cell membranes prepared cells  of  A. U43;  lacking insert);  R.  capsulatus  B. U43  strain  U43,  containing pJAJ9  G. U43 containing pJZ1 (Rps.  with  or  from  without  (expression vector viridis puf  operon).  Second derivatives are inset, with valleys in the second derivative scans corresponding to peaks in the first.  47  Wavelength (nm)  48  absorption  profile  distinguishable  ofU43(pJZ1)  from  that  of  was  consistently,  U43(pJAJ9),  though  subtly,  suggesting that  Rps.  viridis pu/-encoded peptides interacted in some way with Bchl a. (ii) Rps. viridis puhA gene expression in Cell  membranes  U43(pJZ1+pJZ6)  prepared  from  glutamate/DMSO/fructose/pyruvate-grown U43(pJAJ9), U43(pJZ1) and U43(pJZ1+pJZ6) shows  that  U43(pJZ1)  the  absorption  and amplitude  of  were scanned.  of  U43,  observed  Figure 13  U43(pJAJ9)  and  from those  The media-dependency of peak location  by  other  workers  U43(pJZ1+pJZ6)  in order to induce the nifHDK viridis  U43,  in these strains is not understood, although it has  communication). Rps.  cells  grown in this medium appear quite different  grown in Y P S (see Fig. 12). been  profiles  RCWsodium  puhA  gene.  (Doug  Youvan,  personal  was grown on supplemented RCV* promoter driving expression of  The other  the  strains were grown on this  medium in order to allow valid comparisons of absorption profiles to be made. The absorption spectra of U43(pJZ1) found to  be essentially  13C and D).  and U43(pJZ1+pJZ6)  indistinguishable from each other. (Figure  Thus, although genes encoding all of the  peptides of the Rps.  were  structural  viridis RC (i.e. the L, M, H and cytochrome  subunits) as well as the a and (3 subunits of the LH complex were present  in  U43(pJZ1+pJZ6),  encoded complexes occurred.  no stable  assembly of  Rps.  viridis-  A number of possible explanations for  this lack of functional expression were considered, some of which were  readily  amenable  to  experimental  verification.  Several  experiments, described below, were thus undertaken.  To  test  whether  stable  assembly  of  Rps.  viridis-encoded  pigment-binding peptides might occur in an R. capsulatus host  49  Figure  13.  Absorption spectra of cell membranes  prepared  RCV*/sodium-glutamate/DMSO/pyruvate/fructose-grown ft  capsulatus  strain U43 containing various plasmids.  B. U43(pJAJ9); C. U43(JZ1); D. U43(pJZ1+pJZ6).  cells  from of  A. U43 alone;  50  Wavelength (nm)  51  strain synthesizing LHII complexes, plasmids pJZ1 and pJZ6 were introduced into the puf puc absorption  R. capsulatus  +  spectroscopy  indicated  that  strain A R C 6 .  However,  heterologous  pigment-  peptide complexes were not formed in this strain (data not shown). At the time of this study, the DNA region upstream of the viridis  pufB  gene had not been characterized.  The Rps. viridis  Rps. puf  operon was known to be comprised of five genes, as follows: B A L M C. The R. capsulatus  puf operon is comprised of six genes in the  following order: Q B A L M X. pufX  The exact functions of the pufQ  gene products in R. capsulatus  as discussed above, the pufQ  are as yet unknown.  and  However,  gene product has been shown to be  required for Bchl a biosynthesis and thus, indirectly, for pigmentpeptide complex formation. be  a  "carrier  protein",  There is evidence to suggest that it may necessary  at  each  step  of  the  Bchl  biosynthetic pathway, as well as for delivery of mature Bchl a to the pigment-binding peptides of the R C and LH complexes (1,8). The possibility existed that an Rps. viridis might  be present  upstream  of the pufB  pufQ  gene  equivalent  gene on the Rps.  chromosome, and that its presence in R. capsulatus  viridis  U43(pJZ1+pJZ6)  might be required for delivery of Bchl a to the Rps. viridis- en c o d e d pigment-binding  peptides.  Hence,  plasmid pJZ2 was constructed  (see Fig. 5B). As described in Materials and Methods, pJZ2 is a pJAJ9 derivative  carrying  the  entire  Rps.  additional 2.1 kb of upstream DNA. along with pJZ6 into R. absorption  spectra  ARC6(pJZ2+pJZ6)  of  viridis  operon  plus an  Plasmid pJZ2 was conjugated  capsulatus intact  puf  cells  strains U43 and A R C 6 , of  U43(pJZ2+pJZ6)  but and  showed that inclusion of this upstream region had  no discernible effect- on absorption profiles (data not shown). Recently, the sequence the Rps. viridis pufB and A genes plus an  52  additional reported  958  base pairs of the upstream  (88).  capsulatus  DNA region has been  No open reading frame corresponding to the  pufQ  R.  gene was found, although an O R F (designated O R F  R), with the same transcriptional orientation as the puf  operon was  found.  O R F R shows a high sequence similarity to the R.  bchA  gene,  which  encodes  a  bacteriochlorophyll  capsulatus  biosynthetic  enzyme, and which lies immediately upstream of the R.  capsulatus  puf operon (92).  c. RNA blot analysis. (i) Rps. viridis  ouf operon expression in U43  Because absorption spectroscopy demonstrated lack of stable Rps.  Wr/d/s-encoded  determine  complex  if in fact the Rps.  formation,  viridis  puf  it  was  essential  operon and puhA  to  genes  were being transcribed from their respective expression vectors in the R. capsulatus  U43 host.  RNA from Rps.  viridis  DSM 133 and  from U43 strains containing various combinations of plasmids pJZ1, pJZ6, pJAJ9 and pNIF215 was therefore size fractionated on agarose gels, electro-blotted onto nylon membranes and hybridized with [oc32  P]-labelled  probes specific for either the  Rps.  viridis  puf or the  Rps. viridis puh operons (see Materials and Methods). The size of ribosomal RNA transcripts differs in Rps. R.  capsulatus.  whereas  Rps.  viridis  and  Both species contain two major rRNA classes, but viridis  contains the  standard  16s  and  23s  rRNA  classes found in most prokaryotes, very little 23s rRNA is observed in preparations  of R.  capsulatus  RNA.  addition to a 16s rRNA) is observed. R.  capsulatus  Instead, a 14s rRNA (in  It is believed that cleavage of  23s rRNA gives rise to a 16s rRNA (that co-migrates  with the standard 16s rRNA) and the 14s rRNA (39,44).  The location  of the rRNA bands was determined by photographing the RNA gel prior to blotting.  Figure 14A lane 6 shows rRNA bands at ~2.4 and  53  Figure  14.  A. An ethidium bromide-stained agarose/formaldehyde  gel of RNA from photosynthetically grown Rps. 6) and low 02-grown plasmids (8.5 with (+)  R.  capsulatus  viridis  (17 \ig,  strain U43 containing various  ng, all other lanes) grown either in Y P S or in RCV*  or without (-)  ammonia; and B. Autoradiogram of a blot of  the above gel, probed with [oc- P]-labelled 32  Rps.  viridis  puf  Lane 1, U43(pJAJ9) (+); lane 2, pJAJ9 (-); lane 3, pJZ1 (+); pJZ1  (-);  lane  lane 5, pJZ1 (YPS-grown); lane 6,  Rps.  viridis;  DNA. lane 4,  lane  7,  pJZ1+pJZ6 (+); lane 8, pJZ1+pJZ6 (-); lane 9, pJZ1+pJZ6 (YPS-grown); lane 10, pJZ1+pNIF215 (+); lane 11, pJZ1+pNIF215 (-).  The numbers  on the right refer to the approximate size in kilobases of A. rRNA bands and B. hybridizing transcripts.  54  1 2 3 4 5 6 7 8 91011  2.4 1.4 1.1  1 2 3 4  5 6 7 8 9  10 11  55  1.4  kb in Rps.  respectively) capsulatus other  viridis  in  (corresponding  contrast  to  bands  of  to  23s  ~1.4  and  and  1.1  16s  rRNAs  kb  in  R.  (corresponding to 16s and 14s rRNAs respectively, all  lanes).  interpretation  This of the  information  was  essential  autoradiograms, where the  for  subsequent  presence of  large  quantities of rRNA was found to block hybridization to co-migrating messages. The autoradiogram  of a [a- P]-labelled  Rps.  32  probed blot of this gel is shown in Figure 14b.  viridis puf  DNA-  Lanes 1 and 2 show  that the R. viridis puf probe did not hybridize to mRNA present in U43  (pJAJ9) (host strain containing the expression vector without  insert). 0.68  kb  However hybridization to transcripts of ~4.2, 3.5, 0.82 occurred  in  all  U43  strains  containing  plasmid  and pJZ1  (expression vector into which the Rps. viridis puf operon had been inserted)  (Fig.  14b,  lanes  3-5  and  7-11).  Interestingly,  transcript size pattern is similar to that observed in Rps. itself, where transcripts of ~3.5 and ~0.68 6).  this viridis  kb occur (Fig. 14b lane  Recent RNA blot analysis and end-mapping experiments (88) have  revealed four classes of puf was  operon transcripts  hypothesized that a major transcript of 3.6  in Rps. yiridis.  kb encoding the  entire puf operon is processed to give rise to a more abundant kb transcript encoding only the pufBA 3.7 and 0.76 puf promoter.  genes.  It  0.62  Two minor mRNAs of  kb were postulated to arise from a second,  upstream  Processing of puf operon transcripts seems to occur  in several species of purple non-sulfur bacteria, and was originally reported in R  capsulatus  (11)  where major transcripts of 2.7  and  0.5 kb are found (see Discussion). The 3.5  and 0.68  kb transcripts in U43(pJZ1) almost  correspond to their counterparts in Rps.  viridis.  certainly  The -4.2 and 0.82  kb transcripts seen in U43(pJZ1) may be fusion mRNAs, derived from  56  ft capsulatus pufQB'v.Rps. viridis pufBALMC sequences. (ii) Rps. viridis puhA gene expression in U43 Unlike the Rps. did hybridize weakly  viridis puf probe, the Rps. viridis puhA probe to transcripts present in the  (Fig. 15, lanes 1-5, 7 and 9-11). mutated  ft  at both the puf and puc  the puhA between  gene. Rps.  U43  capsulatus strain  host strain  U43,  although  loci, is wild type with respect to  Figure 15 indicates that the low level of homology viridis and  ft  capsulatus  puhA genes (38%  at the  amino acid level) was sufficient for weak hybridization, even under the stringent conditions employed. However U43(pJZ1+pJZ6) cells grown under conditions that induce the  ft  viridis puhA gene (low O2, - N H 4 ;  Fig. 15 lane 8) contained  transcripts that hybridized much more strongly to the puhA  probe than  (low O 2 , puhA  +NH4;  either  this strain  in Y P S is evidently  transcription of the puhA (lanes 8 and 9) and Rps.  viridis  A transcript of ~1.0 kb is faintly  in YPS-grown U43(pJZ1+pJZ6) (lane 9).  concentration  viridis  non-inducing conditions  lane 7) or than U43 strains lacking the Rps.  gene (lanes 1-5, 10 and 11).  detectable  under  Rps.  low enough  gene in pJZ6.  to  The  ammonia  allow  low-level  In both U43(pJZ1+pJZ6)  viridis (lane 6) the major transcript size  was ~1.0 kb.  d. Absorption spectroscopy of U43(pRC77) and The above data show that both the Rps. puhA  genes  were  transcribed  in  ft  U43(pTB999).  viridis puf operon and capsulatus  strain  U43(pJZ1+pJZ6) when grown under the appropriate conditions. absorbance spectra showed that stable pigment-peptide did not assemble in this strain. peaks were absent,  Yet  complexes  Because both LH and R C complex  I wished to investigate  the  possibility that, in  general, LH and RC complexes may exhibit some form of inter-  Figure 15. probed  Autoradiogram of a blot of the gel shown in Figure 14A,  with  [a-  3 2  P]-labelled  designations are as for Figure 14B.  Rps.  viridis  puhA  DNA.  All  58  dependence with regard to assembly and/or photosynthetic membranes of R  stable  modified  in Materials R  terminator  and  capsulatus  of the pufLMX  inserted terminator,  genes, which  genes.  capsulatus  (11).  a  transcriptional No detectable  lie downstream  occurs in this construct  plasmid pRC77 into R  U43. As  p R C 7 7 contains  operon, with a strong  inserted downstream of the pufBA  transcription  capsulatus  Methods, plasmid  puf  into  capsulatus.  I therefore conjugated plasmid pRC77 into R described  insertion  of  the  Introduction of  U43 therefore provided a means to  determine whether stable assembly of LHI complexes would occur in the absence of R C s , and if so at what levels compared to strains synthesizing both R C and LHI complexes.  In R  capsulatus,  R C s have  two near infra-red absorbance maxima, at 800 and 870 nm.  The LHI  complex absorbs maximally at 875 nm. Typical absorption spectra of intact cells of U43(pRC77), which contains the modified puf operon, and L)43(pTB999), which contains a wild-type Although present  R  the  capsulatus  puf  operon, are shown  875 nm peak characteristic  of  in Figure  LHI complexes  in both strains, measurements of peak to baseline  showed a - 5 0 % reduction U43(pTB999).  16\ was  ratios  in amplitude, in U43(pRC77) relative  In order to accurately quantitate  differences  to  in LHI  complex levels between the two strains however, the R C absorbance in U43(pTB999) must be taken into consideration. peak is "buried" under the 875 hm peak. per R C in the photosynthetic  The 870 hm R C .  There are - 1 2 LH si complexes  membrane of R  capsulatus  (11), so  that a maximum of 10% of the 875 nm peak in U43(ptB999) could be attributed to RC absorbance.  If the peak amplitude at 875 nm in  U43(pTB999) were reduced by 10%, it would still be 30% higher than in U43(pRC77).  Thus, stable assembly of LHI complexes in the  photosynthetic membrane of U43(pRC77) was reduced by at least  r  400  :  !  r  600  T  800  1-  1000  Wavelength (nm)  Figure 16.  Absorption spectra of intact cells of R.  capsulatus  strain U43 containing A, plasmid pRC77 and B, plasmid! pTB999.  60  30%  relative to U43(pTB999).  complex  levels  in  Reductions of 50 to 70% in LHI  R C - strains of both  fr  and  capsulatus  R.  have recently been reported (32,46).  sphaeroides  DISCUSSION I have described two independent sets of experiments involving inter-species expression of pigment-binding peptide genes from two purple  non-sulfur  bacterial  species,  viridis,  in cells of a third species, R.  R.  and  sphaeroides  capsulatus.  Rps.  Genes encoding  the peptide subunits of the R C and LH complexes from all three species had been cloned, and were readily available for manipulation. The  first set of experiments  sphaeroides  puf  involved  introduction  of the  R.  operon, which encodes the L and M subunits of the  RC as well as both peptide subunits of the LHI complex, into an R mutant  capsulatus  host  deficient  in  all  known  structural  components of the photosynthetic apparatus, except the H subunit of the R C and the: cytochrome b/C\ complex. hybrid R C s , comprised! of R and an R  with the  sphaeroides-encoded  L and M! subunits  H subunit, assemble  capsulatus-encoded  ft  The results showed that  LHI complex.  sphaeroides-encoded  in vivo a l o n g  C e l l s containing  these hybrid complexes are capable of photosynthetic growth, but: are less efficient at photosynthetic energy transduction thani either of the wild type parental strains. In a subsequent set of experiments,: the? Rps. was  introduced! into; the R  capsulatus  occurred , nor did! Rps. Introduction of the Rps.  viridis  -  R  viridis puhA  puf. operon  mutant host strain.  case no stable assembly of Rps. viridis-encoded 1  viridis  capsulatus  In this;  LHI! complexes hybrid R C s form'.  gene, which: encodes the H  subunit of the R C , along, with the Rps. viridis puf operon still did  61  not result in stable R C complex formation, although the possibility existed that native Rps. viridis  R C s could have assembled, because  genes encoding all of the Rps. the R. capsulatus  viridis  R C peptides were present in  host.  When this project was initially undertaken, a wide spectrum of results was theoretically  possible.  The results actually  fall at the extreme ends of this spectrum.  obtained  Among the possibilities  were, for example, the stable assembly of nonfunctional hybrid R C s along with LHI complexes (functional or not) or assembly of only LHI complexes (again, functional or not). sphaeroides  and Rps.  viridis  The results of both the  studies  provide  insights  R. into  requirements for functional expression of the structural components of the photosynthetic apparatus.  They point to a distinction between  requirements for complex a s s e m b l y in contrast to requirements for assembled complex function. sphaeroides impaired. Rps,  study,  Both requirements were met in the R.  although the  hybrids RCs. were  functionally  Because the requirement for assembly was not met in the  viridis  study, it can not be determined whether assembled  complexes would or would not have been functional-. 1  The impairment of the R. sphaeroides results  from  interactions  subunits with the R.  between the  - R. capsulatus R.  capsulatus H subunit  sphaeroides  hybrid: RC L and Mi  The absence of stable)  heterologous complexes in the Rps. viridis study points to hitherto unknown assembly requirements. discussed individually  1.  Rhodobacter  The two sets of experiments are  below.  sphaeroides  study.  U43(pCT1) cells containing the hybrid R C were shown to  62  suffer  some  degree  of  impairment  transduction by two  methods:  grew  than  more  containing  slowly native  R.  1)  in  energy  under low light conditions they  U43(pTB999)  capsulatus  photosynthetic  cells  (their  counterparts  complexes), and 2)  they  emitted  significantly more fluorescence than cells containing either  native  R. capsulatus or native R. sphaeroides complexes. In principle, there are two stages at which the impairment could occur.  First, efficient  energy transfer from LH to  RC complexes  requires that the two types of complexes be correctly oriented with respect to each other in the membrane.  If the conformation of the  hybrid R C caused a perturbation in the normal arrangement of the surrounding LHI complexes, transfer of excitation energy might be impaired. the  Second, within the RC efficient electron transfer requires  highly  components.  precise  spatial  arrangement  of  all  participating  Excitation of the special pair within the  hybrid R C  might not produce a stable charge separated state as efficiently as in a native R C .  The phenotype observed with the hybrid R C may  result from impairment at either or both stages. Preliminary Youvan's special  characterization  laboratory pair  incomplete.  purified  hybrid  RCs  by  indicated that photobleaching of the  absorbancy  separation - i.e.  of  band  loss of the  Complete  (which  is  indicative  electron from the  bleaching was  obtained  Doug  850  of  nm  charge  special pair) was upon addition  of  exogenous quinone, suggesting a problem in either QA or QB binding (or both).  In order to determine if the Q  A  site was affected, the rate  of the "back-reaction" (electron flow in the opposite direction,, back to the special pair) was measured.  Figure extremely  1 shows that excitation  of the  special pair  results in  rapid electron transfer to the bacteriopheophytin (~3  X  63  10  seconds at room temperature ).  -12  from the bacteriopheophytin to Q transfer time between X 10"  ~1  seconds.  4  seconds  Q  to Q , A  and Q  A  Thus,  if  an  electron  and then is impeded in reaching Q , the backB  seconds.  An electron impeded in  electron  flow  in  (personal  the  hybrid  between the bacteriopheophytin and Q . A  the Q  1  is successfully  re-reduces the special pair in less than 0.1  that  The and 1  10"  8  latter rate was observed by Youvan etal. indicating  seconds.  10  is considerably slower, requiring  B  reaction takes between 1 and 0.1 reaching Q  takes ~2 X 10'  A  The back-reaction rates are 10" ,  respectively.  transferred  A  The subsequent transfer step,  seconds.  The  communication),  RC  was  disrupted  Thus, the conformation of  binding pocket, which is formed predominantly by residues of  A  the R.  sphaeroides-encoded  the presence of the  R.  M subunit, is apparently perturbed by capsulatus-encoded  H subunit.  known whether the effect is localised, or whether  It is not  long-range non-  specific conformational changes have occurred.  The  role  understood.  of  the  H subunit  in  RC  function  is  not  yet  fully  It has been postulated to act as a nucleus around which  the L and M subunits aggregate (76), and cross linking experiments have indicated that the H peptide may interact with LHI complexes Interestingly, the conformation of the Q  (30).  to be influenced by the  H subunit (20),  B  site has been shown  but whether  this site is  abberant in the hybrid reaction center has not been established. It has recently been shown that Q " is protonated prior to the B  second  photo-oxidation  cycle,  protonated at a second site. Q H B  _  after  which  it  is  reduced  and  Protons are believed to reach Q ~  and  from the cytoplasm via two different pathways.  B  The pathways  have not yet been traced back toward the cytoplasm, but they may well proceed through the H subunit (see Fig. 1).  64  The primary amino acid sequences of the R. sphaeroides  capsulatus and R.  H subunit peptides are 64% identical after introduction  of gaps to maximize alignment (89). This means that the 254 amino acid R.  capsulatus  sphaeroides substitute  H subunit at 101 ft  capsulatus  representations differing  H subunit differs from the 260  of  the  residues  conformation,  residues.  amino acid  R.  It would be interesting to  H subunit residues in computer graphics R.  sphaeroides  R C to see if any of the  might  potentially  alter  especially  around  the  the  quinone  hybrid  binding  Hypotheses based on such an analysis could be tested  RC sites.  by site-  directed mutagenesis.  2. Rhodopseudomonas  viridis  study.  RNA blot analyses showed that both the puf puhA  genes were transcribed when  operon and  U43(pJZ1+pJZ6) was  grown  under inducing conditions.  Because Shine-Dalgarno sequences are  highly  prokaryotes,  unlikely  conserved (see  among  below).  Therefore, the  translational  lack of stable  blocks  are  LH and  RC  complex formation in U43(pJZ1+pJZ6),  in which the genes encoding  all structural components of the Rps  viridis reaction center as well  as the a and p  subunits  of  the  suggested that some fundamental  B1015  complex  requirement(s)  were  present,  for assembly of  these complexes was not met in the R. capsulatus host strain. In wild type R.  capsulatus cells, puf operon mRNA is processed  to give rise to two major classes of transcripts, 2.7 and 0.5 kb in length (11). genes.  The 2.7 kb message encodes the pufB, A, L, M and X  The smaller transcript, which is nine times more abundant,  encodes pufB and A genes only. transcripts  accounts  for  the  The relative abundance of ~ 12:1  stoichiometric  between LHI and R C complexes in the membrane.  pufBA  relationship  Recently, it has  been shown that Rps. viridis puf operon mRNA is processed in an  65  analogous manner, giving rise to two major transcripts of 3.5 0.6  and  kb, with the smaller transcript being more abundant (88).  In  both species the comparatively stable 5' segment of the transcript ends in a large stem-loop structure, which has been shown in capsulatus  R.  to be necessary (but not sufficient) for stabilization of  the upstream mRNA ( ). Figure  14  capsulatus  shows that  Rps.  viridis puf  operon  strains U43(pJZ1) and U43(pJZ1+pJZ6) viridis genes transcribed in the R.  but it appears that the puf  operon  transcripts  in  R.  was processed in  a manner similar to that in both parental strains. were the Rps.  mRNA  Thus, not only capsulatus  were  host,  appropriately  processed as well. Table  IV  shows  putative  viridis  and R.  capsulatus  viridis  sequences are  Shine-Dalgarno  puf and puhA  compatible  with  sites  on  both  Rps.  transcripts.  The  Rps.  R.  capsulatus  ribosome  binding since they are very similar to the R.  capsulatus  sequences  and complementary to the 3' terminus of the R (92).  Thus,  it  is  unlikely  transcripts in U43(pJZ1+pJZ6)  that translation  capsulatus 16S rRNA of  would not occur.  verify the presence of the Rps.  viridis peptides  the  Rps.  viridis  I did not attempt to in  U43(pJZ1+pJZ6)  because studies in R  capsulatus have shown that in the absence of  stable pigment-peptide  complex formation, the peptide subunits are  rapidly degraded, and thus not detectable on S D S - P A G E (16).  Joe  Farchaus' attempts to detect the Rps. viridis M subunit in U43(pJZ1) with Western (personal  blots probed with anti-M  antibody were unsuccessful  communication).  Membranes prepared from R  capsulatus strain U43 grown in Y P S  medium show absorption peaks at ~754, detection, 864 nm.  803  and, at the limits of  As mentioned in the Results Section, the origin  66  of these peaks is not known, although they may be associated with some as yet uncharacterized pigment-peptide the  putative  assembly peptide  contains four histidine residues.  complex(es)  such as  encoded by ORF1696.  ORF1696  One is located four residues from  an alanine (Ala-X-X-X-His) and two are located four residues from a glycine  (Gly-X-X-X-His).  The Ala-X-X-X-His sequence has been  shown to be highly conserved in LH complex peptides of purple nonsulfur bacteria, viridis,  including R.  capsulatus,  R.  sphaeroides  and Rps.  and is believed to be involved in Bchl binding (15).  postulated  that  the histidine  residue  provides  a  ligand  It is  for the  central Mg2+ ion of Bchl, while the alanine side chain may be in van der Waals contact with the Bchl ring (15). site-directed  mutagenesis  that  It has been shown by  Ala can be functionally  replaced  with Gly (16). In order to determine sequences  occur  whether  frequently  Ala-X-X-X-His or Gly-X-X-X-His  in integral  membrane  proteins  not  involved in Bchl a binding, the amino acid sequences of the PufX protein, the amino terminal protein  membrane-spanning helices of the R C H  and the LHII y peptide, as well as the cytochrome  complex cytochrome b protein of R. capsulatus  were analyzed.  b/c\ No  Ala-X-X-X-His or Gly-X-X-X-His sequences were found in the PufX, H or y peptides. cytochrome Thus, binding  However, 2 of 11 histidine residues in R.  capsulatus  b protein occur 3 amino acids away from an alanine.  although  Ala-X-X-X-His sequences are not obligatory  sites, they  seem  to occur  more  frequently  in  Bchl  pigment-  binding peptides, where they have been shown to be necessary for Bchl binding (15).  ORF1696 clearly has the potential to bind Bchl,  and could account for the low level absorbancy observed in U43. Introduction  of expression  vector  pJAJ9  into  U43 cells was  shown to result in significantly increased absorbancy (see Fig. 8d  67  for whole  cell scans  and  Fig. 12B for  increase in peak amplitude  membrane  scans).  This  is believed to be associated with the  pufQ gene on pJAJ9, which has been shown to be required for Bchl biosynthesis.  Klug  et  al.  (46)  studied  the  effects  of  pufQ  expression in RCV-grown U43, and reported no far-red absorbancy specific  for  pigment-peptide  complexes in  R.  capsulatus.  Peak  amplitudes in RCV-grown cells (Fig. 13B) are considerably reduced relative to YPS-grown cells (Fig 12B). Absorption  scans  U43(pJZ1),  which  viridis  operon  puf  distinguishable that the Rps.  of  membranes  contains  from  has  prepared  plasmid pJAJ9  been  equivalent  into  introduced, scans  of  from  YPS-grown  which  were  the  Rps.  consistently  U43(pJAJ9), suggesting  viridis encoded pigment-binding peptides interacted in  some way with Bchl a viridis-encoded  (Fig.  13C),  although  clearly stable  Rps.  complexes did not form.  The above data suggest that the barrier to functional expression of the Rps.  viridis LH and R C complexes in R.  the level of assembly.  capsulatus U43 is at  It is important to note that neither  complexes nor RCs are assembled.  B1015  The simplest explanation is that  some fundamental "assembly requirement(s)", common to both types of complexes is not met in R.  capsulatus strain U43.  However it is  also possible that each complex has unique requirements, neither of which are met.  Lastly, it may be that only one of the complexes has  an assembly requirement that is not met, but that formation of the two  complexes  is  inter-dependent,  such  that  absence  prohibits stable assembly of the other. The following structural features of the Rps.  viridis  of  one  68  Table R.  IV:  capsulatus  Comparison of and Rps.  putative  viridis  R. capsulatus  Shine-Dalgarno  puf and puhA  genes  Rps. viridis  pufQ  GGAAGG N A T G  pufB  GGAGG N ATG  GAGGG N ATG  puf A  A G G A G N ATG  GGAGG N ATG  pufL  GGAG N ATG  GGAGG N ATG  pufM  AGGAGG N ATG  G G A G N ATG  pufX  A G G A G N ATG  1 2  5  9  8  5  8  8  1 5  8  pufC puhA  7  G G A G N ATG 7  AGGAGG N ATG 6  GGAGG N ATG 5  sites  in  69  photosynthetic apparatus, which differ from both R. R  sphaeroides,  problems. primary  photosynthetic  is Bchl a,  acceptor  (Q ) A  ubiquinone whereas R  whereas the Rps.  pigment  whereas in R  Rps.  capsulatus and R  Rps.  in  in  Rps.  capsulatus  and R  sphaeroides  and R. The  sphaeroides  viridis utilizes menaquinone;  is  the R C s of  are comprised of three subunits,  viridis R C has an additional cytochrome subunit; capsulatus and R  comprised of vesicular invaginations  whereas  R  viridis utilizes Bchl b\  capsulatus  the photosynthetic membranes of R are  and  are the most obvious potential sources of assembly  The  sphaeroides  capsulatus  viridis  the  in the  photosynthetic  inner  sphaeroides membrane,  membranes  are  lammellar. In addition, it has recently been determined that several R p s . viridis  RC  and  modified (87,88).  LH  peptide  subunits  are  post-translationally  In principle, any of the above factors could be  responsible for or contribute to the lack of functional expression of Rps.  viridis  photosynthetic  complexes  in  R  capsulatus.  I will  attempt to assess each individually, beginning with a discussion of factors which could affect formation of both LH and R C complexes. a. Rps. viridis LH and RC peptides may be unable to bind Bchl a. Figure 17 shows the  molecular structures of Bchl a and b.  They  differ only at ring II, where Bchl b carries an exocyclic double bond on carbon 4.  All available evidence suggests that substituents on  ring II do not participate in binding to peptides of the RC and LH complexes.  In the R C , the central M g  2 +  ions of the special pair are  liganded to histidine residues on the L and M subunits.  Hydrogen  bonding to amino acid side chains on the L and M subunits involves the acetyl groups at carbon 2 on ring I, the keto carbonyl group at carbon 9 on ring V and possibly the ester carbonyl group at carbon 10 on ring V (53).  The M g  2 +  ions of the accessory Bchls are also  liganded to histidine residues, with apparently no hydrogen bonds to  C  D  Figure 17. Molecular structures of A. bacteriochlorophyll a ; B. bacteriochlorophyll b, with an arrow pointing to sole structural difference between the two molecules, the exocyclic double bond on carbon 4 of ring II. Functional groups on rings I, IV and V have been implicated in hydrogen bond formation to light harvesting and reaction center subunits; C. ubiquinone; D. menaquinone.  71  the surrounding amino acid residues (53).  The bacteriopheophytins  appear to be hydrogen bonded to residues on the L and M subunits via the carbonyl groups on ring V (53). A number of recent in vitro studies by Paul Loach and coworkers provide strong experimental evidence that functional groups on Bchl rings I and V are responsible for hydrogen bonding to the highly conserved Ala-X-X-X-His sequence occurring in both a and p subunits of LHI complexes in Rps  viridis, R.  capsulatus  as well as many other species (15). tested  for their ability to bind the  Rhodospirillum the  rubrum,  functional  Furthermore, obtaining  groups R.  sphaeroides  Various Bchl analogues were LHI  a  and p  peptides  from  and it was determined that alterations to on  rings  Parkes-Loach  stable  and R.  I  et al.  rubrum  LHI  and  V  prevent  (42,63), recently a/Rps.  viridis  binding  (47).  succeeded in LHI  p hybrid  complexes associated with Bchl a, and Loach has obtained both rubrum  LHI-Bchl  b  complexes, as well as  Rps.  peptide-Bchl a complexes (personal communication). the  bchl  a-b  difference  remains  barrier to stable assembly of Rps.  a viridis  formal  viridis  R.  LHI p  Thus, although  possibility  photosynthetic  for  the  complexes  in R.  capsulatus,  all available evidence suggests that it is unlikely.  b.  The pufQ  gene  biosynthesis in R. as  a  "carrier  has been  capsulatus,  protein"  shown to  be  required  for Bchl  and its product is postulated to act  involved  in delivery  mature  Bchl a to the  pigment-binding peptides of the RC and LH complexes (1,8). latter  step  involves protein-protein  interaction  Q/L and M ), then the R. capsulatus-eucoded be  able  to deliver  Bchl a to the Rps.  peptides for steric reasons.  The Rps.  encode a Q gene equivalent (88).  (e.g.  If this  Q / a and p or  Q gene product may not viridis  pigment-binding  viridis puf  operon does not  However, since the Bchl a a n d  Bchl b biosynthetic pathways are likely to be very similar, an  Rps.  72  viridis  Q gene equivalent  chromosome. in R.  may well be located elsewhere on the  It is possible that assembly of Rps. viridis complexes  capsulatus  U43 would be possible if this putative Rps.  viridis  pufQ gene were present. If either  of the  above two  possibilities is in fact correct, then  the assembly of both LHI and R C complexes would be prohibited.  I  will now consider possible barriers to assembly of the R C only. c. As mentioned earlier, the Rps. bound menaquinone in the Q R.  sphaeroides  structures). RCs  A  viridis R C contains a tightly  site, whereas both R. capsulatus and  RCs contain ubiquinone (see Fig. 17 for molecular  Although it is theoretically  require  experimental  menaquinone  for  stable  possible that Rps. assembly, there  viridis  is strong  evidence to suggest that this is not the case.  An  extensive and systematic study of the effect of replacing the native ubiquinone in purified reaction centers from R. variety  other quinones was undertaken  quinones  used  reconstitute  the  (including A  reactions in the isolated R C .  with a  by Gunner et al. (41).  menaquinone)  Q -dependent  sphaeroides  were  flash-activated  shown electron  to  fully  transfer  That is, not only did the R C s remain  intact, but they were functional with unnatural quinones in the site.  All  Q  A  Although the reciprocal experiment has not been carried out  with Rps.  viridis  reaction  centers,  Gunner's work  suggests that  ubiquinone and menaquinone would be functionally interchangeable at the Q  A  site of the Rps. viridis RC.  d. The fourth subunit of the Rps.  viridis  R C , the  cytochrome  subunit, may well be necessary for its stable assembly. the cytochrome is encoded by the pufC pJZ1,  it  is  post-translationally  significant for assembly.  modified  Although  gene present on plasmid in  ways  that  may  be  73  (i) The DNA sequence of the cytochrome subunit contains a typical  bacterial  signal peptide  of 20  present in the mature subunit (87). contain such a sequence.  amino  acids which  are  firmly  not  It is the only R C subunit to  It is also the only RC subunit which does  not have an intramembraneous peptide region. subunits  is  integrated  into  the  Both the L and M  membrane  with  5  trans-  membrane cc-helices each, and the H subunit is anchored by a single membrane-spanning  helix.  In  1987  Weyer  et al.  (87)  determined  that the cytochrome subunit is firmly anchored to the membrane by the post-translational  addition of two fatty acids, covalently bound  to the amino terminus of the protein via S-glycerocysteine. post-translational  modification  is undoubtedly  highly  there is no reason to believe that it would occur in R. The stability of the Rps.  viridis  This  specific, and capsulatus.  RC may require anchoring of the  cytochrome subunit in the membrane. (ii) are  Four haems, two high potential and two low potential,  post-translationally  inserted  into the  cytochrome.  haem lyases are known to be highly specific. translational  In  general,  This step in the post-  modification of the cytochrome would thus presumably  not occur in R. capsulatus. the Desulfovibrio  vulgaris  An attempt to express the gene encoding cytochrome c , which also contains four 3  covalently bound haems, in R. capsulatus  was  unsuccessful  (65).  The lack of functional expression was postulated to be due to the absence  of a specific haem  insertion  system in  R.  capsulatus.  Absence of the haem groups from the Rps. viridis cytochrome would clearly  impair  or destroy the  function  of the  R C , but whether  it  would interfere with its assembly is not clear. e.  It  is possible that  stable  assembly of  Rps.  viridis  RCs  requires assembly of B1015 complexes. I will turn now to possible explanations for absence of assembled B1015 complexes.  There are several important considerations.  74  f. Purified B1015  complexes from  subunits, a , p and y, in a 1:1:1 are  membrane-spanning  carotenoid molecules.  Rps.  viridis contain 3 peptide  stoichiometry.  peptides  known  The a and p subunits to  bind  Bchl  b  and  They are encoded by the B and A genes of  the puf operon, which are present in U43(pJZ1). y subunit gene is unknown.  The location of the  Unless it happens to be located within  the unsequenced 2.1 kb region upstream of the puf operon in plasmid pJZ2, then it is not present in any of the constructs used in these experiments. The function of the y subunit has not been determined. It has an unusually high proportion of aromatic residues relative to the a and p peptides, and has been postulated to be involved in the formation  of  regular  photosynthetic  arrays  membrane  of  of  Rps.  LH  complexes  viridis (14).  within  the  The possibility that  it is required for assembly and/or stabilization of B1015 complexes in R.  capsulatus  U43 cannot be ruled out, although Paul Loach has  shown that it is not required for in vitro formation of Rps.  viridis  LHI complexes (personal communication).  g. The recently sequenced Rps. viridis puf B and A genes have been shown to have carboxy terminal extensions of 13 and 10 amino acids for the a and p subunits respectively, which are not present in the polypeptides isolated from the photosynthetic membranes The carboxy terminal required  for  proteolytic  their  sphaeroides  extensions of the precursor proteins may be  correct  degradation  chromatophore  (88).  insertion  of  the  vesicles from  into  a and p Rps.  the  membrane.  peptides  viridis, R.  in  capsulatus  In situ inside-out and  R.  resulted in splitting off of parts of the amino terminal  domains (78,79,96).  Thus, although the LHI genes of the latter two  species do not encode carboxy terminal  extensions, the a and p  peptides are oriented in all three species such that the N terminus protrudes into the cytoplasm.  75  The mechanism by which the extensions are cleaved in viridis is not known.  Since R  not processed in this way,  capsulatus the  Rps.  LHI subunit peptides are  requisite  enzyme(s)  present, and stable assembly of unprocessed Rps.  may  not  be  viridis a and p  peptides may not be possible. h. There is strong evidence that an as yet uncharacterized open reading frame(s) around the puhA LHI complexes in both R  gene is required for assembly of  sphaeroides and R  capsulatus.  It has been  shown (76) that deletion of a -675 bp DNA fragment extending from 140 bp upstream of the start site of the R to bp 535 within the puhA  sphaeroides puhA  gene  gene resulted not only in the loss of  photosynthetic competence (due to absence of the H subunit of the RC), but also in loss of LHI complexes. with the puhA  gene alone  Complementation in trans  restored photosynthetic competence but  not LHI complexes, whereas both LHI and RCs were restored to the puhA  deletion mutant when complemented with the puhA gene plus  flanking sequences.  RNA blot analysis of the puhA  compared to wild type R  sphaeroides  strain 2.4.1  deletion strain  showed that puf  operon transcript levels are identical in the two strains. loss of LHI complexes in this puhA with loss of pufB A  transcripts.  deletion strain is not associated Bauer  recently determined that insertion of a kan located immediately  Thus, the  upstream of the puhA  results in an LHI* phenotype (9, in press).  and  co-workers  have  cartridge into ORF1696,  r  gene in R  capsulatus,  It is interesting to note  that the amino acid sequence of the putative ORF1696 peptide shows a high degree of similarity to the pucC gene product, which has been shown to be required for LHII complex assembly (83).  These data strongly suggest that expression of a gene or genes flanking  puhA  in both R  capsulatus  and R  necessary for assembly of LHI peptide subunits.  sphaeroides  is  It is possible that  76  assembly  of  requirement,  ftps,  viridis  and that the  cannot substitute for the  B1015  R.  complexes  capsulatus  Rps.  has  ORF1696  viridis equivalent.  barrier to stable assembly of B1015 complexes in  a  similar  gene  product  This may be a ft  capsulatus.  i. It is possible that formation of B1015 complexes requires the stable assembly of RCs. My data from the U43(pRC77) study (see Fig. 16 and the results section), as well as work from other laboratories (32,46), show that LHI levels decrease by 30 - 70% in the absence of RCs.  Although detectable  levels of LHI complexes are present in  these native studies, it is possible that such inter-dependence might be more pronounced in a heterologous system.  Furthermore, all of  the above mentioned studies were done in puhA+ strains.  No studies  on LHI assembly have been undertaken in a purely puhA- background, although cross linking studies in ft capsulatus may interact with LHI complexes (28). that in  ft  sphaeroides  have indicated that H  There is evidence to suggest  the H subunit is inserted into the membrane  first, and acts as a "nucleus" around which the L and M subunits and LHI  complexes assemble (76).  If the  Rps.  viridis  H subunit is  incapable of insertion into the ft capsulatus membrane, e.g. because of membrane structure differences or because of "competition" with the  ft  capsulatus  H subunit present in U43, then the Rps.  viridis  LHI complexes may be unable to assemble.  In  summary,  numerous  factors  may  be  responsible  contribute to lack of functional expression of Rps. photosynthetic above,  the  complexes  in  post-translational  ft  capsulatus.  or  viridis-encoded  Of those  modifications to the  for  discussed  R C cytochrome  and B1015 peptide subunits, the absence of the B1015 y subunit, and the  possible requirement  for "assembly peptides" seem to be the  most likely obstacles to stable complex formation.  77  CONCLUSIONS  The biogenesis of the photosynthetic apparatus sulfur  bacteria  transcriptional years,  is  highly  and  complex,  involving  post translational  post-translational  transcriptional,  regulation.  modifications  In  significant for  R C and LHI peptides have been discovered in Rps. and very recently an ORF(s) adjacent to the puhA capsulatus  and R. sphaeroides  sphaeroides-encoded  capsulatus  U43,  the  post-  past  3  assembly of viridis  (87,88),  gene in both  R.  has been shown to be necessary for  formation of LHI complexes (9,76). R.  in purple non-  My experiments have shown that  RC and LHI  whereas  Rps.  complexes assemble in  viridis-encoded  RC  R.  and  LHI  complexes do not. In retrospect, it is perhaps not surprising that the R. and Rps.  viridis  experimental  sphaeroides  results fell at extreme ends of the  wide range of possible outcomes.  Based on comparisons of primary  amino acid sequence identities of LHI peptide subunits, Zuber proposed the  following  those  non-sulfur  purple  sphaeroides viridis.  - R. capsulatus  R.  sphaeroides  of similarity (78% while R.  relative  bacterial  species  relationships  - Rps. gelatinosa  and R.  capsulatus  and Rps.  among  characterized: - R. rubrum  R. -  Rps.  show the highest degree  and 76% for the a and (3 peptides  sphaeroides  respectively).  phylogenetic  (96)  respectively)  viridis show the least (39% and 28%  It is interesting to note that R.  Zuber's comparisons is most closely related to  rubrum, Rps.  which by viridis,  has  recently been shown to also have carboxy terminal extensions of 13 and 9 amino acids on the LHI a and (3 peptides respectively (12). rubrum  and Rps.  viridis  bacterial  species in which  are such  the  only  two  purple  post-translational  R.  non-sulfur  modification  of  78  the LHI peptides has been reported. Further peptides  studies on inter-species expression of may  understanding function. be  be  very  instructive,  from  assembly requirements  pigment-binding  the  standpoint  as well as  hybrid complex  Several experiments immediately come to mind.  very  interesting  to  plasmid pJZ6 into an  introduce R.  the  Rps.  capsulatus  H-  viridis  mutant  of  It would  puhA host.  gene on Assembly  constraints in such a host should be minimal, as the LHI, LHII and RC L and M subunits would be R.  capsulatus-encoded.  opportunity to study the function of an Rps.  Thus,  viridis-R.  an  capsulatus  hybrid RC might be possible. When an R.  capsulatus  puf-puc-puhA-  multiple  deletion strain  becomes available (work is in progress) it might be interesting to introduce  plasmid-borne copies of both the  operon (pCT1) and the R. sphaeroides studies with U43(pCT1) assembly of native R capsulatus-encoded R  sphaeroides  puhA  R.  sphaeroides  puf  gene into this host.  Our  indicate that there would be no barrier to sphaeroides  complexes.  If, however, the  R.  ORF1696 gene product does not assemble the LHI complexes correctly, the cells would be expected  to emit more fluorescence than wild type  R  sphaeroides  cells.  Insights into the nature of the assembly process might be gained from such a study. It  would  be  extremely  peptide genes from R hosts.  interesting  gelatinosa  introduce  into various R  pigment-binding  capsulatus  mutant  On the basis of amino acid sequence identity comparisons it  seems that, except for R  sphaeroides,  most closely related to R  capsulatus  R  to  gelatinosa  complexes would  R. gelatinosa (96).  assemble  therefore greater than, for example, for Rps.  is the species  The likelihood that the in  R  viridis.  capsulatus  is  Furthermore,  79 the R C in R.  gelatinosa  subunits only  (40).  is reported to be comprised of L and M  Studies in both  R.  capsulatus  H- and  H+  background hosts might provide insight into the role of the H subunit in RC function. The  R C and  LH complex  peptides from  the  vast  majority of  photosynthetic organisms, both prokaryotic and eukaryotic, have not yet  been  emission  characterized. levels  from  The  technique  U43(pCT1)  cells  by which  was  fluorescence  evaluated  could  in  principle be applied to screen for the assembly of any heterologous pigment-binding peptides in U43, nothwithstanding those encountered with the Rps. viridis puf genes. appendix,  theoretical  considerations regarding  barriers such as In the attached  construction of  an  expression library and conjugation into R. capsulatus are discussed.  80  REFERENCES 1. Adams, C. W., M. E. Forrest, S. N. Cohen and J . T. Beatty.  1989.  Structural and functional analysis of transcriptional control of the Rhodobacter  capsulatus  puf operon. J . Bacteriol. 171: 473-482.  2. Agalidis, I., A. M. Nuijs and F. Reiss-Husson.  1987.  Characterization of an LM unit purified by affinity chromatography from Rhodobacter  sphaeroides  reaction centers and interactions  with the H subunit. Biochim. Biophys. Acta 890: 3. Allen, J . P. and G . Feher. center from Rhodopseudomonas  1984.  2422-250.  Crystallization of the reaction  sphaeroides:  Preliminary  characterization. Proc. Natl. Acad. Sci. USA 81:  4795-4799.  4. Allen, J . P., G . Feher, T. O. Yeates, H. Komiya, and D. C. Rees. 1987.  Structure of the reaction center from  sphaeroides  Rhodobacter  R-26: the cofactors. Proc. Natl. Acad. Sci. USA 84:  5730-5734. 5. Allen, J . P., G. Feher, T. O. Yeates, D. C. Rees, J . Deisenhofer, H. Michel and R. Huber. from Rhodopseudomonas viridis  1986.  Structural homology of reaction centers  sphaeroides  and  Rhodopseudomonas  as determined by X-ray diffraction. Proc. Natl. Acad. Sci.  USA. 83:  8589-8593.  6. Arkin, A. P., E. R. Goldman, S. J . Robles, C. A. Goddard, W. J . Coleman, M. M. Yang and D. C. Youvan. 1990. spectroscopy in molecular biology II. absorption spectra.  Applications of imaging  Colony screening based on  Bio/Technology 8: .746-749.  81  7. Arntzen C. J . and H. B. Pakrasi.  1986.  Photosystem II reaction  center: polypeptide subunits and functional cofactors. In L. A. Staehelin and C. J . Arntzen (eds.) Photosynthesis III  Photosynthetic  membranes and light harvesting systems. Springer-Verlag, Berlin. Heidelberg. 8. Bauer, C. E. and B. L Marrs. 1988  Rhodobacter capsulatus puf  operon encodes a regulatory protein (PufQ) for bacteriochlorophyll biosynthesis. Proc. Natl. Acad. Sci. USA 85:  7074-7078.  9. Bauer, C. E. personal communication 10. Beatty, J . T. and H. Gest. photosynthetic bacteria.  1981.  Generation of succinyl Co-A in  Arch. Microbiol. 129:  335-340.  11. Belasco, J . G., J . T. Beatty, C. W. Adams, A. Von Gabain and S. N. Cohen.  1985.  capsulatus  Differential expression of photosynthesis genes in R.  results from segmental differences in stability within  the polycistronic rxcA transcript.  Cell 40:  171-181.  12. Berard, J . G . Belanger, P. Corriveau, and G. Gingras.  1986.  Molecular cloning and sequence of the B880 holochrome gene from Rhodospirillum  rubrum.  J . Biol. Chem. 261:  13. Bibb, M.J. and S.N. Cohen. 1982. Streptomyces:  82-87.  Gene expression in  construction and application of promoter-probe  plasmid vectors in Streptomyces  lividans.  Mol. Gen. Genetics  187:  265-..277. 14. Brunisholz, R. A., F. Jay, F. Suter, and H. Zuber. light-harvesting  polypeptides of Rhodopseudomonas  Chem. Hoppe-Seyler. 366: 87-98.  1985.  The  viridis. Biol.  82  15. Brunisholz, R. A. and H. Zuber.  1988.  Primary structure  analyses of bacterial antenna polypeptides: - correlation of aromatic amino  acids with spectral properties - structural  similarities with  reaction center polypeptides. In (H. Scheer and S. Schneider, eds.), Photosynthetic light-harvesting systems.  Organization and Function.  Walter de Gruyter, New York. 16. Bylina, E. J . , S. J . Robles and D. C. Youvan. 1988. mutations  affecting  the  Directed  putative bacteriochlorophyll-binding sites  in the light harvesting I antenna of Rhodobacter  capsulatus.  Israel  J . of Chem. 28: 73-79. 17. Chen, C-Y. A., J . T. Beatty, S. N. Cohen and J . G . Belasco.  1988.  An intercistronic stem-loop structure functions as an mRNA decay terminator necessary but insufficient for puf mRNA stability. Cell 52:  609-619. 18. Clark, W. G . , E. Davidson and B. L. Marrs.  1984.  Variation of  levels of mRNA coding for antenna and reaction center polypeptides in Rhodopseudomonas concentration.  capsulata in response to changes in oxygen  J . Bacteriol. 157:  945-948.  19. Coleman, W. J . and D. C. Youvan. 1990.  Spectroscopic analysis  of genetically modified photosynthetic reaction centers.  Annu. Rev.  Biophy. Biophys. Chem. 19: 333-367. 20. Debus, R. J . , G . Feher, and M. Y. Okamura. 1985. reaction centers from Rhodopseudomonas  LM complex of  sphaeroides  R-26:  characterization and reconstitution with the H subunit. Biochemistry  24:  2488-2500.  21. Deisenhofer, J . and H. Michel.  1989.  The photosynthetic  83  reaction centre from the purple bacterium viridis.  EMBO Journal 8:  Rhodopseudomonas  2149-2169.  22. Deisenhofer, J . and H. Michel.  1989.  reaction center from Rhodopseudomonas  The photosynthetic  viridis. Biophys. J . 55:  Abstra. 1a. 23. Deisenhofer, J . R. Huber, and H. Michel.  1989.  The structure of  the photochemical reaction center of Rhodopseudomonas its implications for function.  viridis and  In G . D. Fasman (ed.), Prediction of  protein structure and the principles of protein conformation. Plenum Press, New York. 24. Deisenhofer, J;, O. Epp, K. Miki, R. Huber and H. Michel. Structure of the protein subunits in the photosynthetic centre of Rhodopseudomonas  1985.  reaction  viridis at 3A resolution. Nature  318:  618-624. 25. Dierstein, R. polypeptides  26. Dierstein, R. systems.  Biosynthesis of pigment-protein complex  in bacteriochlorophyll-less mutants  Rhodopseudomonas  toluene-treated  1983.  of  capsulata Y S . FEBS Lett. 160: 281-286. 1984.  Synthesis of pigment-binding protein in  Rhodopseudomonas  Eur. J . Biochem. 138:  capsulata and in cell-free  509-518.  27. Ditta, G., T. Schmidhauser, E. Yakobsdon, P. Lu, X.-W. Liang, D. R. Flnlay, D. Guiney and D. R. Helinski.  1985.  Plasmids related to the  broad host range vector pRK290 useful for gene cloning and for monitoring gene expression.  Plasmid 13: 149-153.  28. Drews, G. and J . Oelze.  1981.  Organization and differentiation  84  of membranes of prototrophic bacteria.  Adv. Microb. Physiol. 22: 1 -  92. 29. Drews, G . light-harvesting  1985.  Structure and functional organization of  complexes and photochemical reaction centers in  membranes of phototrophic bacteria. Microbiol. Rev. 49: 59-70. 30. Drews, G. J . Peters and R. Dierstein.  1983.  organization and biosynthesis of pigment-protein Rhodopseudomonas  Molecular complexes of  capsulata. Ann. Microbiol. (Inst. Pasteur)  134B,  151-158. 31. Fajer, J . , D. C. Brune, M. S. Davis, A. Forman and L. D. Spaulding. 1975.  Primary charge separation in bacterial photosynthesis:  oxidized chlorophyll and reduced pheophytin. Proc. Natl. Acad. Sci. USA 72: 4956. 32. Farchaus, J . W., H. Gruenberg and D. Oesterhelt. Complementation of a reaction center-deficient sphaeroides  1990.  Rhodobacter  pufLMX deletion strain in trans with pufBALM  does not  restore the photosynthesis-positive phenotype. J . Bacteriol.  172:  977-985. 33. Feher, G., A. J . Hoff, R. A. Isaacson and L. C. Ackerson.  1975.  Endor experiments on chlorophyll and bacteriochlorophyll in vitro and in the photosynthetic unit. Ann. NY Acad. Sci. 244: 34. Feher, G. J . P. Allen, M. Y. Okamura and D. C. Rees. Structure and function of bacterial photosynthetic Nature 339:  239-259. 1989.  reaction centres.  111-116.  35. Feher, G., M. Y. Okamura and J . D. McElroy. 1972.  Identification  85  of an electron acceptor in reaction centers of sphaeroides.  Biochim. Biophys. Acta 2 6 7 : 222-226.  36. Feinberg, A. P. and B. Volgelstein. radiolabeling  Rhodopseudomonas  1983.  A technique for  DNA restriction fragments to high specific activity.  Anal. Biochem. 1 3 2 : 6-13. 37. Ferguson, S. J . , J . B. Jackson and A. G . McEwan. Anaerobic respiration in the Rhodospirillaceae:  1987.  characterization of  pathways and evaluation of roles in redox balancing during photosynthesis.  FEMS Microbiol. Rev. 4 6 : 177-143.  38. Garcia, A., G . Venturoli, N. Gad'on, J . Fernandez-Velasco, B. A. Melandri and G . Drews.  1987.  The adaptation of the electron  transfer chain of Rhodopseudomonas intensities.  capsulata  Biochim. Biophys. Acta 890:  39. Gray, Ernest. 1978.  to different light  335-345.  Ribosomes and RNA metabolism.  Clayton and W. Sistrom (eds.), The photosynthetic bacteria.  In R. Plenum  Press, New York. 40. Gringas, G .  1978.  A comparitive review of photochemical  reaction center preparations from photosynthetic bacteria. Clayton and W. Sistrom (eds.), The photosynthetic bacteria.  In R. Plenum  Press, New York. 41. Gunner, M. R., B. S. Braun, J . M. Bruce and P. L. Dutton.  1985.  The characterization of the Q A binding site of the reaction center of Rhodopseudomonas  sphaeroides.  In M. E. Michel-Beyerle (ed.),  Antennas and reaction centers of photosynthetic bacteria.  Springer-  Verlag, Berlin Heidelberg. 42. Heller, B. A., P. S. Parkes-Loach, M. C. Chang and P. A. Loach.  86  1990.  Comparison of the structural subunit, B820, of core light-  harvesting complexes of photosynthetic bacteria. In M. Baltscheffsky (ed) Current Research in Photosynthesis Vol. II Academic Publishers, the  Kluwer  Netherlands.  43. Johnson, J . A., W. K. R. Wong, and J . T. Beatty. Expression of cellulase genes in Rhodobacter  capsulatus by use of  plasmid expression vectors. J . Bacteriol. 167: 44. Klug, G., N. Kaufmann and G . Drews.  1986.  604-610.  1985.  The expression of  genes encoding proteins of B800-850 antenna pigment complex and ribosomal RNA of Rhodopseudomonas  capsulata.  FEBS Lett.  177:  61-65. 45. Klug, G., R. Liebetanz, and G . Drews. bacteriochlorophyll  1986.  biosynthesis on formation  of  The influence of pigment-binding  proteins and assembly of pigment protein complexes in Rhodopseudomonas  capsulata.  Arch. Microbiol. 146:  46. Klug, G . and S. N. Cohen. localized Rhodobacter  1988.  capsulatus  Pleiotropic effects of  puf operon deletions on  production of light-absorbing pigment-protein Bacteriol. 170:  284-291.  complexes.  J.  5814-5821.  47. Loach, P. A., T. Michalski and P. S. Parkes-Loach. Probing the bacteriochlorophyll core light-harvesting  1990.  binding site requirements  of the  complex of photosynthetic bacteria using bchl  analogs. In M. Baltscheffsky (ed.), Current Research in Photosynthesis, Vol. II.  Kluwer Academic Publishers, the  Netherlands. 48. Loach, P. A. and R. L. Hall.  1972.  The question of the primary  87  electron acceptor in bacterial photosynthesis. Proc. Natl. Acad. Sci. U S A 6 9 : 786. 49. Maniatis, T., E. F. Fritsch and J . Sambrook.  1982.  Molecular  cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 50. Marrs.B.L. 1974.  Genetic recombination in R. capsulata.  Proc.  Natl. Acad. Sci. USA. 71: 971-973. 51. McElroy, J . D., G . Feher, D. C. Mauzerall. 1969. On the nature of the free radical formed during the primary process of bacterial photosynthesis. Biochim. Biophys. Acta 172: 52. Michel, H. and J . Deisenhofer.  1988.  180-183. Structure and function of  the photosynthetic reaction center from Rhodopseudomonas  viridis.  Pure and Appl. Chem. 7: 953-958. 53. Michel H., O. Epp and J . Deisenhofer. interactions  in the photosynthetic  Rhodopseudomonas  1986.  Pigment-protein  reaction centre from  viridis. E M B O Journal 5: 2445-2451.  54. Michel, H., K. A. Weyer, H. Gruenberg and F. Lottspeich. 1985. The 'heavy' subunit of the photosynthetic reaction centre from Rhodopseudomonas  viridis: isolation of the gene, nucleotide and  amino acid sequence. E M B O Journal. 4: 1667-1672. 55. Michel, H., and J . Deisenhofer. photosynthetic  1988.  Relevance of the  reaction center from purple bacteria to the  of photosystem II. Biochemistry 27:  structure  1-7.  56. Michel, H. K. A. Weyer, H. Gruenberg, I. Dunger, D. Oesterhelt  88  and F. Lottspeich.  1986.  The 'light' and 'medium' subunits of the  photosynthetic reaction centre from Rhodopseudomonas  viridis:  isolation of the genes, nucleotide and amino acid sequence. E M B O Journal 5:  1149-1158.  57. Michel, H. J . Deisenhofer. 19 membrane protein complex. Rhodopseudomonas 58. Miller, J . H.  Three dimensional crystals of a  The photosynthetic reaction centre from  viridis. Mol. Biol. 158: 1972.  567-572.  Experiments in Molecular Genetics.  Cold  Spring Harbor Laboratory, Cold Spring Harbor, New York. 59. Norris, J . R., R. A. Uphaus and H. L. Crespi.  1971.  Electron spin  resonance of chlorophyll and the origin of signal I in photosynthesis. Proc. Natl. Acad. Sci. USA 68: 625-628. 60. Norris, J . R., H. Scheer and J . J . Katz.  1975.  Models for  antenna and reaction center chlorophylls. Ann. NY Acad. Sci. 244: 260-280. 61. Okamura, M. Y., R. A. Isaacson and G. Feher. 1975.  Primary  acceptor in bacterial photosynthesis: obligatory role of ubiquinone in photoactive reaction centers of Rhodopseudomonas Proc. Natl. Acad. Sci. USA 72:  sphaeroides.  3491-3495.  62. Paddock, M. L , S. H. Rongey, E. C. Abresch, G. Feher and M. Y. Okamura.  1988.  Reaction centers from three herbicide-resistant  mutants of Rhodobacter sphaeroides 2.4.1: sequence analysis and preliminary  characterization.  Photosynthesis Research 17:  75-96.  63. Parkes-Loach, P. S., B. A. Heller, M. C. Chang, W. J . Bass, J . A. Chanatry and P. A. Loach.  1990.  Reconstitution of the core light-  harvesting complex of photosynthetic bacteria with selected  89  polypeptides. In M. Baltscheffsky (ed.) Current Research in Photosynthesis Vol. II. Kluwer Academic Publishers, the Netherlands. 64. Pollock, D., C. E. Bauer and P. A. Scolnik. of the Rhodobacter nitrogen source. on the nifHDK  capsulatus  1988.  Transcription  nifHDK operon is modulated by the  Construction of plasmid expression vectors based  promoter.  Gene 65: 269-275.  65. Pollock, W. B. R., P. J . Chemerika, M. E. Forrest, J . T. Beatty and G. Voordouw.  1989.  from Desulfovibrio  Expression of the gene encoding cytochrome C3 vulgaris (Hildenborough)  in Escherichia  coli:  export and processing of the apoprotein. J . of Gen. Microbiology  135:  2319-2328. 66. Reed. D. W. and R. K. Clayton.  1968.  center fraction from Rhodopseudomonas Biophys. Acta 30:  Isolation of a reaction  sphaeroides.  471-475.  67. Robles, J . Breton and D. C. Youvan. 1990. symmetrization  Biochim.  Partial  of the photosynthetic reaction center.  Science  248:  1402-1405. 68. Rosen, K. M. and L. Villa-Komaroff.  1990.  An alternative  method for the visualization of RNA in formaldehyde agarose gels. Focus 12:  23-24.  69. Rosen, K. M., E. D. Lamperti and L. Villa-Komaroff.  1990.  Optimizing the Northern blot procedure. BioTechniques 8: 398-403. 70. Sauer, K. Photosynthetic light reactions - physical aspects. 1986.  In L A. Staehelin and C . J . Arntzen (eds.), Photosynthesis III  Photosynthetic membranes and light harvesting systems. Springer-  90  Verlag, Berlin Heidelberg. 71. Schmidhauser, T. J . and D. K. Helinski.  1985.  Regions of broad  host range plasmid RK2 involved in replication and stable maintainance in nine species of gram-negative bacteria. Bacteriol. 164:  J.  446-455.  72. Scholz, P., V. Haring, B. Wittmann-Liebold, K. Ashman, M. Bagdasarian, and E. Scherzinger.  1989.  Complete nucleotide  sequence and gene organization of the broad-host-range plasmid RSF1010.  Gene 75: 271-288.  73. Scolnik, P. A. and B. L. Marrs.  1987.  Genetic research with  photosynthetic bacteria. Annu. Rev. Microbiol. 41: 703-726. 74. Scolnik, P. A., D. Zannoni and B. L .Marrs.  1980.  Spectral and  functional comparisons between the carotenoids of the two antenna complexes of Rhodopseudomonas 593:  230-240.  capsulata.  Biochim. Biophys. Acta  U43  75. Shuvalov, V. A. and V. V. Klimov.  1976.  The primary  photoreaction in the complex cytochrome P-890 P-760 (bacteriopheophytin  760)  of Chromatium  minutissimum  redox potentials. Biochim. Biophys. Acta 440:  at low  587.  76. Sockett, R. E., T. J . Donohue, A. R. Varga and S. Kaplan. Control of photosynthetic membrane assembly in sphaeroides 171:  Rhodobacter  mediated by puhA and flanking sequences. J . Bacteriol.  436-446.  77. Sojka, G. A., H. H. Freeze and H. Gest. 1970. estimation of bacteriochlorophyll in situ. 136:  1989.  5788-580.  Quantitative  Arch. Biochem. Biophys.  91  78. Stark, W. F. Jay  and K. Muehlethaler.  1986.  Localisation of  reaction centre and light harvesting complexes in the photosynthetic unit of Rhodopseudomonas  viridis.  Arch. Microbiol 146:  79. Tadros, M. H., D. Spormann and G . Drews. 1988.  130-133  The  localization of pigment-bionding polypeptides in membranes of Rhodopseudomonas viridis.  FEMS Microb. Lett. 55: 243-248.  80. Taylor, D. P., S. N. Cohen, W. G . Clark and B. L. Marrs.  1983.  Alignment of genetic and restriction maps of the photosynthesis region of the Rhodopseudomonas  capsulata chromosome by a  conjugation-mediated marker rescue technique. J . Bacteriol.  154:  580-590. 81. Thornber, J . P.  1986.  Biochemical characterization and  structure of pigment-proteins of photosynthetic organisms. Photosynthesis III  In  Photosynthetic membranes and light harvesting  systems. Springer-Verlag, Berlin Heidelberg. 83. Tichy, H.V., B. Oberle, H. Stiehle, E. Schiltz, and G. Drews. 1989.  Genes downstream from pucB and pucA are essential for  formation of the B800-850 complex of Rhodobacter Bacteriol. 171:  capsulatus.  J.  4914-4922.  84. Tiede, D. M., R. C. Prince, G. H. Reed and P. L Dutton. 1976. EPR properties of the electron carrier intermediate between the  reaction  center bacteriochlorophylls and the primary acceptor in Chromatium  vinosum. FEBS Lett. 65: 301  85. von Gabain, A., J . G. Belasco, J . L. Schottel, A. C. Y. Chang and S. N. Cohen.  1983.  Decay of mRNA in Escherichia coli: investigation of  92  the fate of specific segments of transcripts. Proc. Natl. Acad. Sci. USA  80: 653-657.  86. Weaver, P. F., J. D. Wall and H. Gest. 1975. Rhodopseudomonas  capsulata.  Characterization of  Arch. Microbiol. 105: 207-216.  87. Weyer, K. A., F. Lottspeich, H. Gruenberg, F. Lang, D. Oesterhelt and H. Michel. 1987.  Amino acid sequence of the cytochrome subunit  of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas  viridis. EMBO Journal 6: 2197-2202.  88. Wiessner, C. I, Dunger, and H. Michel. 1990. transcription of the genes encoding the B1015  Structure and  light-harvesting  complex |3 and a subunits and the photosynthetic reaction center L, M, and Cytochrome c subunits from Rhodopseudomonas Bacteriol. 172:  viridis. J.  2877-2887.  89. Williams, J. C, L. A. Steiner, and G. Feher. 1986. structure of the reaction center from sphaeroides.  Primary  Rhodopseudomonas  Proteins 1: 312-325.  90. Williams, J. C, L. A. Steiner, G. Feher, and M. I. Simon. 1984. Primary structure of the L subunit of the reaction center from Rhodopseudomonas  sphaeroides.  Proc. Natl. Acad. Sci. USA  81:  7307-7307. 91. Yang, M. M. and D. C. Youvan. 1988. Applications of imaging spectroscopy in molecular biology: I. screening photosynthetic bacteria. Bio/Technology  6:  939-942.  92 Youvan, D. C, E. J. Bylina, M. Alberti, H. Begusch and J. E. Hearst. 1984.  Nucleotide and deduced polypeptide sequences of the  93  photosynthetic reaction center, B870 antenna and flanking polypeptides from R. capsulata. Cell 37: 93. Youvan.D.C. and S. Ismail.  949-957.  1985.  Light harvesting II (B800-  850 complex) structural genes from Rhodopseudomonas  capsulata.  Proc. Natl. Acad. Sci. USA 82: 58-62. 94. Youvan, D. C , S. Ismail and E. J . Bylina. 1985.  Chromosomal  deletion and plasmid complementation of the photosynthetic reaction center and light-harvesting genes from capsulata.  Rhodopseudomonas  Gene 38: 19-30.  95. Zilsel, J . , T. G . Lilburn and J . T. Beatty.  1989.  Formation of  functional inter-species hybrid photosynthetic complexes in Rhodobacter  capsulatus.  96. Zuber, H.  FEBS Lett. 253:  247-252.  Comparative biochemistry of light-harvesting  systems. In Photosynthesis III  Photosynthetic membranes and light  harvesting systems. L. A. Staehelin and C . J . Arntzen (eds.), SpringerVerlag, Berlin Heidelberg. 97. Zucconi, A. P. and J . T. Beatty.  1988.  Post-transcriptional  regulation by light of the steady-state levels of mature B800-850 light-harvesting complexes in Rhodobacter 170:  877-882.  capsulatus.  J . Bacteriol.  94  APPENDIX R.  capsulatus  strain  emit very  low  pJAJ9  U43  cells that contain expression vector  levels of fluorescence.  If  pigment-binding  peptide genes inserted into pJAJ9 are functionally expressed in U43, fluorescence  emission  levels  increase  U43[pCT1]; see section 1 of Results). for  using  fluorescence  expression complex  libraries  genes  possible.  for  constructing  even  when  important  expression  to  screen  in  pJAJ9-derivative  heterologous  RC  and  photosynthetic growth  factors  libraries  (as  Thus, the possibility exists  expression of  in U43,  Several  emission  significantly  for  to  this  be  is not  considered  purpose  are  LH  when  discussed  below. Total genomic DNA from the organism of interest can be partially digested  with  fragments  a  ligated  restriction into  promoter on pJAJ9. transformed  into  endonuclease,  a compatible  and  the  site downstream  resultant  of the  puf  The collection of ligated plasmids would be  E. coli, after which it would be conjugated  into  U43. In  order  terminators interest  to  minimize  might  and  be  the  the  present  pJAJ9  possibility  between  promoter,  the  the  that  transcriptional  coding sequences of  fragment  sizes  should  correspond roughly to the predicted size of the gene(s) of interest. The optimal fragment size could vary considerably, depending on the source of the heterologous DNA. The  DNA  fragment  inserted into pJAJ9 correct  reading  containing  the  gene  of  interest  must  be  in the correct orientation, and possibly in the  frame.  Because prokaryotic  Shine-Dalgarno  sites  95  are  generally  heterologous frame  highly  conserved,  prokaryotic  genes  functional  may  not  require  expression  of  translationally  in-  insertion.  The  probability  (p)  that a given  recombinant vector will contain  a fragment with the gene(s) of interest is given by the expression:  (1)  p _  average  fragment  size  of  A s s u m i n g that the fragment equally  probable  interest  will  from the puf  be  p* =  (2)  NB:  if  then the  will  be  in the  F o r the  calculated  orientation,  kb)  kb)  inserted into the vector in probability  (p*)  two  that the g e n e of  correct orientation  for  transcription  i.e.:  average fragment size (in kb) size of genome (in kb) X 2  insertion  by 3.  the  in the  probability is 1/3  multiplied  can be  promoter is p/2,  (in  genome (in  orientations, present  size  only  although  correct reading the a b o v e , i.e. remainder  is also required,  the denominator must be  of this d i s c u s s i o n ,  considering  translationally  frame  the  probabilities  requirement  in-frame  for  insertions  correct  could  be  required for expression of eukaryotic (nuclear c D N A c o p i e s of) g e n e s  in R. capsulatus. It is n e c e s s a r y to determine transformed probability gene(s)  that  coli colonies) at  of interest  probability interest  E.  in  that the  a  least  one  how many recombinant vectors must  be  contains  obtained a  in the correct orientation. given  correct  recombinant  vector  orientation, then  the  to  fragment If p*  ensure a  (i.e. high  carrying  the  represents  the  contains the probability  (P)  gene  of  that at  96  least one copy of this vector will be present in a collection of N transformed colonies is:  P = 1 - (1-p*)  (3)  w  The derivation of this formula is perhaps most easily explained by analogy. comprised  For example; given a randomly mixed collection of coins of  one  million  each  of  pennies, nickles, dimes  and  quarters, how many coins must be picked to ensure a high probability that  at  least  orientation?  one  dime  will  be  picked  in  the  heads-up  (HU)  The biological counterpart would be that genomic DNA  from a culture comprised of 1 million cells is cut into 4 equal-sized fragments, designated "a" - "d".  If the 4 million resultant fragments  are present in a ligation mixture with linearized pJAJ9, how many recombinant  vectors  (transformed  E. coli colonies)  must  be  screened to have a high probability that at least one will contain fragment "a" in the correct orientation? The probability of picking a dime, 1/4, corresponds to "p" in (1). The probability that if a dime is picked it will be HU is 1/2, so that the probability of picking a HU dime (HUD) is 1/4 X 1/2 = 1/8. corresponds to "p*" in (2).  This  The probability of picking at least one  HUD in N tries, is equal to one minus the probability of picking up no. HUDs (derivation to follow).  Note that, because one is starting with  four million coins and making relatively few picks, one can assume for the process  sake of simplicity, that has  no  significant  removal  effect  on  of coins in the the  composition  picking of  the  collection. Thus, if one picks two coins, the probability that the first is a HUD is 1/8, and the probability that the second is a HUD is 1/8.  (1  97  million minus 2 ~= the total coin types.)  1 million, so that climes still comprise ~1/4  of  The probability that the first QL the second o_r  both are HUDs (i.e. at least one is a HUD) can be calculated as follows: The probability that the first is HUD and the second is not is equal to 1/8X7/8 = 7/64. The independent probability that the second is a HUD and the first is not is 7/8X1/8 = 7/64. The independent probability that both are HUDs is 1/8X1/8 = 1/64. The probability that the first or the second or both are HUDs is equal to the sum of their individual probabilities 7/64 + 7/64+1/64 = 15/64. The probability that neither are HUDs is 7/8 X 7/8 = (7/8) = 49/64. 2  Thus,  1 minus the probability that neither  are  HUDs (64/64 -  49/64 = 15/64) is equal to the probability that at least one is a HUD (15/64).  Stated generally, the  probability of picking at least one  (one or more) of an item is equal to one minus the probability of picking none.  98  Notice that the probability of n M picking a HUD on a try, 7/8, is equal to 1 minus the probability of picking a HUD (1 - 1/8), which in the general form = 1 - p*. coins were picked.  Furthermore, in the above example two  If three coins are picked, the probability of  getting at least one HUD =  1 - (7/8) ; in four picks it equals 1 3  (7/8) and in N picks it equals 1 - (7/8) . 4  N  Therefore,  the probability  tries is equal to 1 - {7/8)  N  P of picking at least one HUD in N = 1 - (l-p*)^.  The forgoing is the  derivation of the formula:  (3)  P = 1 - (1-p*)  w  After substitution (by analogy) of DNA fragments  (of a size such  that they comprise a fraction p* of the total genome), and numbers of E. coli colonies (A/), for the entities used above, the number of colonies required to have a high probability of having at least one of a  given  gene  in the correct orientation,  can be determined as  follows:  (4)  1-P = (1-p*)  w  and  In (1-P) = N In (1-p*)  (5) so that  (6)  N =  »n (1-P)  In  (1-p*)  In applying this formula, one must decide a) a suitable size range  99  of fragments to use, and b) the desired value of P.  The size range of  fragments will vary depending on the size of the genome of the organism that is tested. been  In the purple non-sulfur bacteria that have  characterized, pigment-binding  peptide  genes are  generally  organized into operons, which range in size from ~1 - ~4 kb.  To  generate an expression library from an as yet uncharacterized purple non-sulfur bacterial species, fragment sizes ranging from 0.5 - 5 kb would probably be reasonable.  The average fragment size would thus  be 2.75 kb, assuming equal distribution over the range given above. If the average bacterial genome is -4.0 X 10  3  kb, then p = 2.75/(4.0  X 103), and * = 2.75/(2 X 4.0 X 10 ) = 3.4 X 10-4. 3  p  If one desires a 99% probability of obtaining at least one copy of a given fragment  in the correct orientation  then, substituting into  the formula (6) above gives:  =  N  In (0.01) In (1 - 3.4 X  10-4)  = 13,544 colonies Thus,  using an average  colonies  transformed  ensure a 99% fragment  DNA fragment  with a  size of 2.75  recombinant  vector  are  kb,  13,544  required  to  probability that at least one colony will contain a  with the  gene of interest  in the  correct orientation  for  expression. There  are  two  important  points to  be  made.  Firstly,  13,544'  transformed colonies does not ensure a 99% probability of having a c o m p l e t e e x p r e s s i o n library notwithstanding  the  desired fragment  of the  organism.  This is true  fact that one can choose to screen for any  with this collection of transformed colonies in a  100  given  experiment,  and have  a 99% probability  that the chosen  fragment will be present in the correct orientation. If one wishes to create a complete expression library that can be frozen and used repeatedly for screening (or selecting) for any gene, then one requires a high probability that at least one of e a c h of all fragments is present in the correct orientation in the vector.  This  is given by the formula:  (7)  P  ( i 1  o f  a l l )  = ([1 - {1-p*>]".)<  1/p)  In the above example, 34,970 cells would be required to ensure a 99% probability of having at least one copy of each of the fragments in the correct orientation. This formula can be readily derived from (3) above, as follows: Using the analogy of the four coins again, one now wishes to have a high probability of obtaining at least one of each of a heads up penny (HUP), a heads-up nickel (HUN), a heads-up dime (HUD) and a heads-up quarter (HUQ).  The probability of picking at least one HUD in N tries  has already been shown to be  (8)  P(>1 HUD)= 1 " (7/8)"  The probability of picking at least one HUP is also 1 - (7/8)^ ( s e e [4]),  as is the probability of picking at least one HUN or of picking  one H U Q .  The probability of picking at least one of each, is the  product of their individual probabilities, namely  () 9  P  (>J  [HUP,HUN,HUD,HUQ]) = (I  1  Generally, 7/8 = 1 - p*, and 4 = 1/p. Thus,  " f ' }]^) 7  8  4  we have derived the  101  formula  (7)  P(>1 The  of all) =  second important  ([1-{1-P*}] ') A  point to be  1/P  made concerns creation of  expression libraries specifically for use in R a method for direct transformation of R  capsulatus.  capsulatus  Because  does not yet  exist, the transformation must be done into E. coli cells, which can then be used as donors for conjugation into R  capsulatus.  cultures of donor, helper and recipient cells are proportions,  spotted  onto  small sterile  mixed  millipore filters  Liquid in equal  placed on  RCV agar plates, and left to incubate at 3 7 ° C for > 6 hours to allow plasmid transfer to occur.  In order to recover all R  capsulatus  ex-  conjugant cells, the filters are resuspended by exhaustive vortexing in  selective  tetracycline),  medium  (RCV  followed  by  supplemented  overnight  with  incubation to  0.5  ng/ml  allow  plasmid-  containing cells (which are Tc resistant) to multiply.  The O . D .  this overnight  serial dilutions  culture  is determined,  and suitable  6 5 0  of  are made such that spread-plates of cultures grown on RCV/Tc will have ~ 200-500 colonies per plate.  These plates are then used for  fluorescence screening.  Therefore, one is again faced with a statistical question; namely "How many U43 ex-conjugants must be plated in order to ensure a 99%  probability that at  least one of each of the original  recombinant plasmid types is present?"  13,544  This is given by the formula  just derived in (8):  (7)  P(>1 of all) = 0  Note that in this case p = p*,  " [1-p]") ^  because  Thus, using the number N obtained above:  1  orientation  is  irrelevant.  102  0.99 = (1 - [ 1-1/13,544  3,544  In order to solve this for N, it is useful to express 0.99 as (1-.01). Thus, In (1-0.01) = 13,544 ln(1 - [ 13,543/13,544 ]") Since ln(1-x) ~= -x when x is very small, then -0.01 ~= 13,544 (-13.543/13,544)" and ln(0.01/13,544) = N  ln(  13,543/13,544  )  so N  =  ln(0.01/13.544) ln(13,543/13,544)  = 190,541 colonies. Thus, given the constraints assumed above, to ensure a 99% probability that at least one R. the  desired  recombinant  capsulatus  plasmid,  ex-conjugant  190,541  R.  contains  capsulatus  ex-  to allow  rapid  conjugant colonies must be screened. Recently,  the technology  has been  developed  screening of up to 500 colonies per plate, so that about four hundred plates would be required to screen ~200,00 cells (6). cells were required transcripts  from  If many more  (e.g. if a cDNA expression library  a eukaryotic  of nuclear  organism was to be screened), it  103  might  be  separate  possible to  use  fluorescence-activated  cells with enhanced fluorescence, which  spread on plates and screened as above.  cell  sorting  to  could then  be  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0098553/manifest

Comment

Related Items