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Directed mutagenesis and gene fusion analysis of the Rhodobacter capsulatus puc operon LeBlanc, Heidi 1994

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DIRECTED MUTAGENESIS AND GENE FUSION ANALYSIS OF THE RHODOBACTER CAPS 1JLATUS PUC OPERON  Heidi LeBlanc  B.Sc. (Honours, Co-op), University of Waterloo, 1988  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in THE FACULTY OF GRADUATE STUDIES (DEPARTMENT OF MICROBIOLOGY AND IMMUNOLOGY)  Signature(s) removed to protect privacy  THE UNIVERSITY OF BRITISH COLUMBIA JUNE 1995 © Heidi LeBlanc, 1995  Signature(s) removed to protect privacy  __  In presenting this thesis in  partial fulfilment of the requirements for an advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by  his  or  her  representatives,  It  is  understood  that  copying  or  publication of this thesis for financial gain shall not be allowed without my written permission.  Signature(s) removed to protect privacy  (Signature)  Department of 1  De91O  The University of British Columbia Vancouver, Canada Date  DE-6 (2/88)  /,  11  ABSTRACT The puc operon of Rhodobacter capsulatus encodes the genes for the pigment binding peptides of the light harvesting (LH) II complex, pucB and pucA, followed by three open reading frames named pucC, pucD and pucE. RNA blot analysis and promoter mapping experiments using translational  fusions of lac’Z to the distal gene, pucE, showed that all five genes are primarily transcribed from a promoter upstream of pucB. The primary transcript from this promoter is of low abundance and is processed to give a 1.0 kb transcript containing pucDt sequences. A minor promoter was detected between the 3’ end of pucC and the middle of pucD; a 0.5 kb RNA molecule detected by hybridisation probes to the pucDE region could be the transcription product of this minor promoter. The most abundant transcript detected was the previously characterised 0.55 kb pucBA mRNA. Strain ALHII, which lacked the LHII complex, was created by replacement of the five chromosomal puc genes with the spectinomycin cartridge. This strain was complemented by plasmids carrying deletions of the pucCDE genes. The pucC gene was required for the presence of the LHII complex, and deletion of the pucE gene, which encodes the subunit of the LHII complex, led to a reduced level of the complex. Deletion of the pucD gene had no discernable effect on LHII complex levels. Photosynthetic growth of strain ALHII was similar to that of wild type cells at light levels greater than 30 p.E•m-2.s-1, but strains containing deletions of pucC or pucE were significantly impaired for photosynthetic growth at all light levels tested. In the case of pucC-deleted cells, chromosomal suppressor mutations frequently arose that restored the LHII complex and/or photosynthetic growth ability. A model for the trans-membrane topology of the PucC protein was derived by theoretical analyses and tested using the genetic system of alkaline phosphatase fusions. The model predicts 12 transmembrane domains, with both the N and C termini on the cytoplasmic side of the membrane. The fusions were also used for a deletion analysis of pucC. None of the truncated alleles allowed the cells to synthesize the LHII complex, but several had unexpected pleiotropic effects on LHI and RC complex levels.  111  TABLE OF CONTENTS  LIST OF TABLES LIST OF FIGURES  ii v vi  ABBREVIATIONS  viii  ABSTRACT  ACKNOWLEDGEMENTS  INTRODUCTION MATERIALS AND METHODS  ix  1 16  a.  Bacterial strains and growth conditions  16  b. c.  DNA manipulations Plasmids  18  d.  Conjugations  19  e.  Spectral analysis  22  £  Construction of R. capsulatus strain iLHII Southern blot analysis  22 25 25  g. h.  Plasmid deletions of pucC, pucD and pucE  19  31  j.  Fluorescence measurements Construction and screening of pucC::phoA fusions  k.  Alkaline phosphatase assays  36  1. m.  Hydropathy analysis Construction of plasmids for expression of pucC and C-terminal  36  deletions of PucC  37 38  o.  RNA isolation, blot analysis and probe construction pucE’-lac’Z fusion construction  p.  fl-galactosidase assays  i.  n.  RESULTS Chapter 1. Phenotypic properties of puc operon mutants Chromosomal deletion of the puc operon a. Effects of pucCDE deletions on LHII complex absorption spectra b.  34  40 41 42 42 42 45  c.  Photosynthetic growth characteristics of puc gene deletions  49  d.  Fluorescence analysis of z\LHII(ptE)  52  iv e. f.  Effects of light intensity on LH complex levels Isolation of strains with secondary mutations that suppressed  54 58  g. h.  pucC deletion Phenotypic characterisation of suppressors of pucC deletion Conclusions  Chapter 2. PucC structure and function Hydropathy and positive inside rule analysis of PucC a. Construction and analysis of pucC’:.pho’A fusions b. c. d. e. f.  59 65 69 69 75  Sequence differences between wild type strains SB1003 and 3Th4 Deletion analysis of pucC Role of PucC in LHI assembly  82  Conclusions  95  Chapter 3: Analysis of puc operon transcription a. puc operon promoter mapping by deletion analysis of  86 89  101  pucE’::lac’Z gene fusions.  101  b. c.  Is there translational coupling between pucD and pucE? RNA blot analysis of the puc operon  d.  Conclusions  103 104 111  DISCUSSION a. Transcriptional organisation of the puc operon b. Deletion of chromosomal pucBACDE genes in strain ALHII c. d. e. f. g.  115 115 122  The phenotypes of pucD and pucE mutants The phenotypes of pucC and pucCDE deletion mutants Deletion analysis of PucC using pucC’:.pho’A fusions and effects  123  on LHI complex levels The structure of the PucC protein Concluding remarks  128 132  REFERENCES  125  134 137  V  LIST OF TABLES Table 1.  Bacterial strains  17  Table 2.  Plasmids  20  Table 3.  Summary of translationally in-frame fusions between PucC and  Table 4.  PhoA Amino acid differences between the predicted protein sequences of PucC from strains SBIOO3 and 3Th4  78 85  vi  LIST OF FIGURES Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Figure 8.  The photosynthetic apparatus of R. capsulatus. The photosynthesis gene cluster of R. capsulatus. The puc promoter region of R. sphaeroides. The puf operon of R. capsulatus. The puc operon of R. capsulatus, ca. 1989. Construction of plasmids pHLBMpuc::2 and pUCiCDE. Construction of plasmid pUCAC. Construction of plasmid pUCzD(IF). Construction of plasmid pUCAE.  Figure 9. Figure 10. Construction of pucC’::pho’A fusions. Figure 11. Absorption spectra of strains SBIOO3 and ALHII. Figure 12. Southern blot analysis of strains SB1003 and ALHII. Figure 13. The Pst I EcoR I plasmid inserts used to complement ALHII. -  Figure 14. Absorption spectra of pucCDE deletion strains. Figure 15. Photosynthetic growth of pucCDE deletion strains. Figure 16. Photosynthetic growth of R. capsulatus strains at different light  2 7 9 II 13 23 27 29 32 35 43 44 46 47 50  intensities. Figure 17. Fluorescence emission of R. capsulatus strains. Figure 18. Spectra of R. capsulatus strains grown photosynthetically at  53 55  different light intensities. Figure 19. Spectra of secondary suppressor strains. Figure 20. Spectra of ALHII-1 (pzC) grown photosynthetically at different light intensities. Figure 21. Hydropathy predictions for PucC. Figure 22. Possible arrangements of trans-membrane helices in the  56  C-terminus of PucC. Figure 23. A model of the topology of the PucC protein. Figure 24. Complementation of z\LHII(pAC) with truncated pucC alleles. Figure 25. Complementation of F1696 mutant strains with pucC. Figure 26. Absorption spectra of strains containing pucC’::pho’A fusions. Figure 27. Strains containing pucC’::pho’A fusions grown  81  photosynthetically. Figure 28. j3-galactosidase activities in cells containing pucE’::lac’Z fusions.  60 63 70  83 87 91 93 96 102  vii Figure 29. RNA blot analysis of wild type puc operon transcripts. Figure 30. RNA blot analysis of puc deletion strains. Figure 31. Transcriptional organisation of the puc operon Figure 32. Superoperonal organisation of the photosynthetic gene cluster  105 107 116 119  viii ABBREVIATIONS  aa  amino acid  Ap  ampicillin  bch  R. capsulatus bacteriochiorophyll biosynthesis gene  BSA bovine serum albumin cfu  crt  colony forming units R. capsulatus carotenoid biosynthesis gene  FNR fumarate nitrate regulator 1CM intracytoplasmic membrane IHF integration host factor plasmid incompatibility group Inc LH  kanamycin light harvesting  OR orf  operator region open reading frame  PS PSA R  photosynthetic photosynthetic apparatus  RC  reaction centre  Km  resistance/resistant  tetracycline Tc URS upstream regulatory site XGa1 5-bromo-4-chloro-3-indolyl-f3-D-galactopyranoside 5-bromo-4-chloro-3-indolylphosphate-p-toluidine salt XP denotes a novel joint (prime) denotes a truncated gene at the indicated 5’ or 3’ side  ix ACKNOWLEDGEMENTS I would like to thank my supervisor, Tom Beatty, for six years of advice and support, and for assembling a wonderful group of people to work with. Past members of the lab, Cheryl Wellington and Joanna Zilsel, were very supportive when I first started my work, and current members have been a valuable source of advice as I continued it. These people, Tim Lilburn, Farahad Dastoor, Conan Young and Andrew Lang, have also been unfailingly patient with me as I wrote my thesis and monopolised our computer. Two other former lab members, Danny Wong and Diane Driver, were invariably helpful. My committee members, Bob Hancock, Brett Finlay and Doug Kilburn, have been constructive with their criticisms, for which I thank them. Beverly Green was very generous with her help and advice about integral membrane proteins. Other faculty members, especially George Spiegelman and Rosie Redfield, have been valuable sources of advice on a wide range of subjects, and I will always be grateful to a former supervisor, July Evans, for serving as an exemplary role model. I also thank two former students in the lab, Grace Wong and Christine Chiang, for help in construction of some plasmids, and with enzyme assays. Finally, thank you, John, for moral support and patience over the past six years.  1  INTRODUCTION  The purple non-sulphur photosynthetic bacterium Rhodobacter capsulatus has been used extensively as a model organism to study genetic regulation of bacterial photosynthesis [reviewed in 70 and 2]. It is metabolically versatile, and under growth conditions of high aeration derives energy for growth by respiration of oxygen. When oxygen partial pressure is reduced the photosynthetic apparatus (PSA) is gratuitously induced, making it possible to study interactions of the components of the PSA in the absence of a requirement for function.  Induction of the PSA is accompanied by synthesis of a specialised membrane system known as the intracytoplasmic membrane (1CM) which forms by invagination of the cytoplasmic membrane. The 1CM is thus continuous with cytoplasmic membrane but is biochemically distinct, primarily containing components of the PSA that are absent from the cytoplasmic membrane in both induced and uninduced cells [55].  The integral membrane protein components of the PSA include the reaction centre complex (RC), and the two surrounding light harvesting complexes, light harvesting I (LHI; sometimes designated B870) and light harvesting II (LHII; B800-850) [for reviews see 33 and 18]. The LH complexes absorb light energy (Fig. 1) and transfer it to the RC where it is converted to chemical energy when a quinone associated with the RC is reduced with a concomitant uptake of two protons from the cytosol. The quinol then diffuses  2  Figure 1. The photosynthetic apparatus of R. capsulatus. Light energy (wavy  arrow) is absorbed by the bacteriochiorophyll molecules (shown as four-ringed structures) of the light harvesting antenna complexes, LHI and LHII, and transferred to the reaction centre (RC) “special pair” of bacteriochlorophylls. Reduction of a quinone (hexagon) results in uptake of protons from the 1 complex, cytoplasm. The quinol is then reoxidised at the cytochrome (cyt) b/c releasing protons to the periplasm and creating a proton gradient. The electron . The proton 2 cycle is completed by re-reduction of the RC special pair by cyt c gradient drives synthesis of cellular ATP by diffusion of protons through the ATPase complex. Electron and proton transfer is indicated by bold arrows, and the subunits of the pigment-binding complexes are indicated by bold letters. (Figure adapted from 83).  H  +  H +  XYN  (  cyt C 2  ATPase  CYTOPLASM  ATP  ( ADP +  Pi  cyt b/c 1 H  RC  innflnir (°1n1rn  PERIPLASM  LHI  ‘  LHII  ui  4 through the membrane to the cytochrome b/c 1 complex where it is oxidised, releasing protons to the periplasm. A proton gradient is thus produced and its potential energy is used to drive synthesis of ATP by passage of the protons through the ATPase complex. Meanwhile, electrons from the cytochrome b/c 1 complex are transferred back to the RC by a cytochrome c. Electron transfer is thus cyclic and oxygen is not generated in the process [611.  Our understanding of the mechanism of electron transfer in the RC has been greatly helped by the solution of the three dimensional structure of the bacterial reaction centre by X-ray crystallography [reviewed in 681. The RC consists of three peptides, the L, M and H subunits, which are encoded by the pufL, pufM and puhA genes. The H subunit crosses the membrane only once and does not bind photosynthetic pigments. The L and M polypeptides each cross the membrane five times in a symmetrical arrangement. These subunits bind four bacteriochiorophyll molecules and two bacteriopheophytins, and the pigments are also arranged around a two-fold axis of symmetry. Despite this structural symmetry, electron transfer occurs only through the chromophores associated with the L subunit [73].  Both of the LH complexes have two small, pigment-binding peptide components, the a and /3 subunits. Similarities in sequence and arrangement of the genes for these peptides (pucBA for LHII and pufBA for LHI) suggest that LHII complexes arose from a duplication of the genes for an ancestral LHI complex [901. Although LHII complexes are relatively easy to purify, they have proven recalcitrant to crystallisation [141. To the great excitement of the bacterial photosynthesis community the crystal structure of the LHII complex from  5  Rhodopseudomonas acidophila has finally been published [50], and a low resolution projection for the LHT complex of Rhodospirillum rubrum has also recently been reported [31]. Both structures show a$ dimers arranged in rings. In the case of the LHI complex, 16 subunits form a ring with a central hole large enough to accomodate the RC complex. Nine LHII complex cxf3 dimers form two concentric rings with the a peptides forming the inner cylinder and the /3 peptides arranged around the outside. No ordered molecules were detected within this ring.  The pigment-binding peptides of the LHII complex each have a single membrane-spanning a helix, perpendicular to the plane of the membrane [50]. The domains flanking the central a helix are amphipathic helices associated with the surface of the membrane, and the N-termini are located on the cytoplasmic side of the membrane. Three bacteriochlorophyll molecules are associated with each a$ dimer, suspended between the transmembrane cx helices, and the local protein environment shifts the absorbance maximum of the pigments. The two located close to the periplasmic face of the cytoplasmic membrane are complexed to conserved histidine residues and absorb light maximally at 850 nm (B850). The third, coordinated to the carbonyl of the N-terminal formylmethionine of the /3 subunit near the cytoplasmic side of the membrane, has an absorbance peak at 800 nm (B800). The B800 molecules are oriented parallel to the plane of the membrane, and are coordinated with the B850 molecules by interactions of their phytol tails and by a carotenoid molecule which spans the membrane. The 18 B850 molecules are perpendicular to the membrane plane, stacked in an overlapping ring which allows rapid delocalisation of the excited state. The B870 molecules of the LHI complex are probably arranged in a similar ring,  6 coordinated to conserved histidines that would place the B870 ring at the same position within the membrane as the B850 ring. The RC complex special pair are also located at this level within the membrane, so the spacing and orientation of the chromophores favours efficient energy transfer within and among the complexes of the PSA.  Synthesis of the PSA in R. capsulatus occurs only in conditions of low aeration, and the amount and composition of the system are further controlled by levels of illumination. As light intensity decreases the size of the lightcollecting surface is increased by increasing the amount of the PSA and the amount of LHII complex relative to the RC and LHI complexes.  The arrangement of the genes known to be required for photosynthesis is remarkably conserved amongst several species of purple photosynthetic bacteria. The operons encoding enzymes for the biosynthesis of the two photosynthetic pigments, bacteriochiorophyll (the bch genes) and carotenoids (the crt genes), are clustered; for example, in R. capsulatus, within a 50 kb segment of the chromosome (Fig. 2). They are flanked by the puh and puf operons which encode the structural genes for the RC and LHI complexes. The LHII complex is not found in all purple photosynthetic bacteria, by contrast, and those organisms that have LHII complexes have radically different genomic locations for the puc operon (which encodes the LHII structural genes). In R. capsulatus, for example, the puc operon has been mapped to the far side of the chromosome from the  puh operon [23] whereas in the closely related species Rhodobacter sphaeroides it has been found to lie 18 kb downstream of puh [76]. Other bacteria, notably  Rhodopseudomonas palustris, have several different forms of LHII complex  orf469  orf428  pufQ B A L MX  Figure 2. The photosynthesis gene cluster of R. capsulatus. Genes for structural peptides of the RC and LHI complexes are indicated by hatched boxes; shaded boxes indicate genes encoding bacteriochiorophyll biosynthetic enzymes; carotenoid biosynthesis genes are dotted; cross-hatching indicates genes of unknown function. The names of genes discussed in the text are indicated.  puhAFl696  8 and up to five copies of the puc operon [771.  Several mechanisms control the levels of the PSA components. Transcription of the bch and crt operons under aerobic conditions is between 62% and 131% of the maximally induced anaerobic level [91]. This seems, at least in part, to be negatively regulated since aerobic transcription of some of these operons increases 2 fold in an orf469 mutant background [591, and R. sphaeroides mutants in an orf469 homologue, ppsR, overproduce pigments [561. Additionally, pigment biosynthesis is inhibited by oxygen at the level of enzyme activity [49] and a non-enzymatic factor, the pufQ gene product, is required post-transcriptionally for bacteriochiorophyll synthesis [4].  Transcription of the puf operon, which encodes the pigment-binding peptides of the LHI and RC complexes, is strongly induced by low oxygen conditions [2]. This operon is not regulated by orf469 [591; rather a twocomponent regulatory system (RegA/RegB) induces transcription of the operon under anaerobic conditions [72, 54]. A second two component system, HvrA/HvrB, weakly induces the operon under conditions of decreased illumination [12].  Transcription of the puc operon is similarly induced by the RegA/RegB two component system in response to decreased oxygen levels [54]. Additionally, as with the crt and bch genes, orf469 appears to repress transcription in aerobic conditions [59]. An additional gene has been identified which is required for induction of both the puc and puf operons anaerobically; this gene has no homology to known genes or R. capsulatus sequences [58]. The puc operon is  9  Promoter  Activator  URS -629  -117 -106  -84  -66  R2 -52  -35  -27  -10  0  117  02 PS  -  Figure 3. The puc promoter region of R. sphaeroides. Sequence elements upstream of the transcription start site (0) are represented by boxes and labels above the figure; their position relative to the transcription start is indicated  below. The relative amount of 13-galactosidase activity from a iacZ’::puc’B fusion (B::Z) to the promoter under aerobic (02) or photosynthetic (PS) growth conditions is indicated by the thickness of the black arrows. The activities of similar fusions to mutated promoters are indicated by the thickness of the bars positioned under the regions affected. Black bars represent deletions; grey bars point mutations. URS: upstream regulatory site; FNR: homology to the fumarate nitrate regulator binding site; IHF: homology to the E. coil integration host factor binding site (the overlap in these two sequences is indicated by the hatched box); OR1 and 0R2: inverted repeat sequences. (Figure adapted from 30).  10 not transcriptionally regulated in response to decreased illumination; instead the amount of LHII complex increases by a post-transcriptional mechanism in dim light  [931. The promoter region of the puc operon in Rhodobacter sphaeroides has  been analysed and several transcriptional trans-acting factors and cis regulatory sequences have been identified [reviewed in 30]. Figure 3 shows the cis-acting sequences: the upstream regulatory site (URS) is required for aerobic repression of transcription; an activator required for transcription has been identified by deletion in the region 70 nucleotides upstream of the transcriptional start site; and point mutations in one of two regions of dyad symmetry (OR1) reduce aerobic repression of transcription. Sequences 100 nudeotides upstream of the transcriptional start site show similarity with the recognition sites for the E. coli integration host factor (IHF) and fumarate nitrate regulator (FNR). Il-IF from E. coli has been reported to bind to the puc promoter region [44].  Factors controlling transcription of the R. sphaeroides puc operon in trans have been reported by several groups. Lee and Kaplan [42] identified oxyA and  oxyB genetically as mutants unable to repress puc transcription aerobically [42]. oxyB maps to the same region of the PS gene cluster as ppsR [56]; the characterisation of ppsR and its R. capsulatus homologue, orf469, is consistent with ppsR and oxyB being the same gene. A suppressor of oxyB mutants was identified and named prrA; by sequence comparison it is the R. sphaeroides homologue of the R. capsulatus regA gene [21]. Finally, a 26 kDa protein which binds the R. sphaeroides puc promoter region 120 nucleotides upstream of the transcriptional start site has been identified biochemically [51].  11  <0.5 mm  8 mm  4 4  33 mm  Figure 4. The puf operon of R. capsulatus. The puf genes are represented by boxes; genes for pigment-binding proteins are shaded. The promoter is indicated as a bent arrow and the positions of predicted stem loop structures within puf mRNA are shown as lollipops. The major mRNA molecules are represented as arrows under the sequences encoded; the thickness of the arrow reflects the  abundance of the message in induced cells. Vertical arrows show processing events and the measured half-life (t 1 /2) of each mRNA is indicated in minutes.  12 The relative amounts of LHI and RC complexes are constant and are determined post-transcriptionally by differential degradation of their respective segments of the polycistronic puf mRNA (Fig. 4) [reviewed in  351.  The region  encoding the RC L and M subunits contains endonucleolytic processing sites which lead to rapid degradation of this message [36]. A stem ioop structure between the pufA and pufL genes protects the LHI mRNA segment from exonucleolytic decay, leading to a 12:1 ratio of pufBA: pufLM messages [13].  The final level of control of the amounts of the complexes of the PSA is post-translational. The RC and LH pigment binding peptides are stable only if they are assembled into complexes. The LHI /3 peptide, for example, is synthesized and inserted in the 1CM in the absence of the LHI a peptide, but is then rapidly degraded [64]. In the complementary experiment the a peptide is not detectable in the absence of the /3 peptide. Furthermore, in mutants unable to synthesize bacteriochiorophyll none of the pigment complex peptides accumulate in cell membranes [15]. Coloured carotenoids do not seem to be required for synthesis of RC and LHI complexes, but mutants in carotenoid synthesis have variable amounts of LHII complex [17]. In R. sphaeroides it has been shown that if the carotenoid biosynthetic pathway is blocked before the colourless intermediates have been converted to coloured carotenoid derivatives the LHII complex is not formed and the LHII peptides are unstable [39].  The two pigment binding peptides of the LHII complex are encoded by the pucBA genes [901. Two dicistronic RNA species of approximately 550 nt in length encoding these genes have been characterized [93] and when first identified the puc operon was thought to consist of only these two genes. The  13  BA  Figure 5. The puc operon of R. capsulatus,  C  Ca.  D  E  1989. The structural genes for  the a and 13 subunits of the LHII complex, pucA and pucB, had been identified (shaded boxes) [90] and the ends of an abundant mRNA (arrow) had been mapped to regions corresponding to promoter activity (bent arrow) and sequences resembling a rho-independant transcriptional terminator (lollipop) [93]. Three new open reading frames (open boxes) followed by a second putative transcriptional terminator had been identified downstream of pucBA [791.  14 LHII mutant strain NK3 was then discovered to have a transposon inserted  downstream of the pucBA genes, and three new open reading frames were discovered in this region by DNA sequencing [791. The new open reading frames were named the pucC, pucD and pucE genes since a transposon disruption of the pucC gene led to loss of the LHII complex, and the predicted protein sequence of the pucE gene matched the partial amino acid sequence of one of the two 14 kD peptides, named the y subunit, which co-purified with the LHII complex [791. The organisation of these genes is given in Figure 5.  A pucC homologue has been discovered downstream of pucBA in R. sphaeroides, but further sequencing did not show any evidence of sequences homologous to pucDE [25]. In Rubrivivax gelatinosus a pucC homologue has also been found downstream of pucBA, but interestingly, in the opposite orientation [Adrian Simmons, personal communication]. Again, pUCDE homologues have not been found.  Because the transposon insertion mutation of the pucC gene in strain NK3 [79, 781 could be polar on the pucD and pucE genes and interfere with their expression, it was not known which of the pucC, pucD and pucE genes were required for formation of the LHII complex. Furthermore, although the pucC and pucE genes were reported to be required for normal levels of the LHII complex [78], the effects of puc gene deletions on growth had not been reported. It was also not known if all of the pucCDE genes were transcribed from the pucBA promoter region, or whether there was a promoter located between the pucC and pucDE regions, although a complementation analysis indicated that  the pucC gene could not be expressed if the pucBA promoter region was not  15 present [79].  For these reasons I chose to investigate the requirements of the pucC, pucD and pucE genes, individually, for cell growth as well as for formation of the LHII complex. I created a mutant strain in which the chromosomal pucBACDE genes were replaced by an omega cartridge [60], and complemented this deletion with plasmids carrying various combinations of puc operon genes. I also used pucE ‘::lac ‘Z fusions to evaluate the location of the promoter of this gene fusion,  and in wild type cells detected RNA species long enough to encode the entire pucBACDE region that hybridized to probes specific for the pucBA, pucC and pucDE regions.  Finally, I derived a model for the structure of the PucC protein by theoretical and genetic analyses, and used truncated PucC alleles in a variety of genetic backgrounds to assess the function of the pucC gene product. Only the wild type PucC protein allowed synthesis of LI-ill complexes, but different pucC alleles had unexpected effects on the levels of LHI complex in induced cells.  16 MATERIALS AND METHODS  a. Bacterial strains and growth conditions  The Eschericia coli strains used in this thesis have been previously described. Subcloning was done in the E. coli strains JM83 [881, an hsdR derivative of C600 [7], RB404 [11] and SM1O [74]. Strains SM1O [74] and HB1O1(pRK2OI3) [161 were used to transfer plasmids by conjugation to R. capsulatus. Fusions of puc’C to pho’A were expressed in the phoA deletion strain CC1I8 [48]. E. coli strains were grown at 30°C or 37°C in Luria broth [461 supplemented with the appropriate antibiotics at the following concentrations: ampicillin, 200 g/mL, kanamycin sulphate, 20-40 jig/mL, tetracyclineHCl, 10 j.tg/mL and spectinomycin sulphate, 100 .tg/mL.  The relevant genotypes and phenotypes of the Rhodobacter capsulatus strains used in this thesis are summarized in Table 1. The strain SB1003 [75] was used for RNA analysis, /3-galactosidase specific activity determinations in the promoter localization experiments and as the parent strain for construction of a  pucBACDE deletion strain (see below). This puc operon deletion strain, R. capsulatus ALHII, was used for analyses of the effects of deletion of segments of the puc operon on LHII complex formation, and in RNA blot analyses. The chromosomal second site mutants ALHII-1, zLHTI-2, ALHII-3, ALHII-4 and ALHII 5 arose spontaneously during photosynthetic growth experiments (see Results). ANco and AStu are derivatives of strain ALHII containing an additional deletion of the C-terminal 348 and 340 amino acids, respectively, of the gene F1696 [Conan Young, personal communication], They were used in studies to determine the  17 Table 1. R. capsulatus strains. Strain  Genotype  Phenotype  Reference  SB1003  rif-lO  Wild type for PSA  ARC6  \puf::KmR  RC, LHI, LHII  75 13  ALHII  1puc::2  LHII  41  ALHII-1  Xpuc::2; unidentified  See text  41  See text  41  See text  This work  See text  This work  See text  This work  second site mutation XLHII-2  lxpuc::2; unidentified  second site mutation zLHII-3  tpuc::2; unidentified  second site mutation zLHII-4  lipuc::2; unidentified  second site mutation z\LHII-5  ipuc::2; unidentified  second site mutation zNco  zpuc::c24F1696::Km’  LHI, LHII  C. Young  AStu  1 zpuc::Q4F1696::Km  LHr, LHII  C. Young  18 effect of pucC expression on LHI complex levels. Strain ARC6(pA4) [11 lacks the RC and LHI complex, but expresses the LHII complex because pufQ is present on plasmid pA4. Light absorbed by LHII is reemitted as fluorescence. This strain was used as a positive control in measuring fluorescence emission of puc gene deletion strains.  R. capsulatus strains were grown in RCV medium [51, supplemented with an appropriate antibiotic for plasmid maintenance if appropriate, at 34°C. Tetracycline-HC1 was used at a concentration of 0.5 ig/mL, spectinomycin sulphate at 10 .tg/mL and kanamycin sulphate at 5-10 iig/mL. High aeration growth conditions were defined as cultures grown in Erlenmeyer flasks filled to 8% of their nominal volumes and shaken at 300 rpm in an orbital shaker. Low aeration growth conditions were obtained by filling flasks to 80% of their nominal capacities and shaking at 150 rpm. Stationary phase low oxygen cultures were used as inocula for photosynthetic cultures in screw cap tubes filled to capacity, and incubated with illumination provided by tungsten filament 2 incandescent lamps with light intensities ranging from 5 to 300 pEm  .4.  Light  intensity was measured with a Li-Cor photometer equipped with a LIO19OSB quantum sensor (Li-Cor, Lincoln Nebraska). Growth was monitored by measuring turbidity with a Klett-Summerson photometer (filter #66).  b. DNA manipulations  Standard methods of DNA purification and manipulation were used throughout this thesis [66]. Double stranded plasmid DNA for screening and  19 sequencing pucC’::pho’A fusions was prepared according to the Promega Applications and Protocols Guide. Briefly, the supernatant liquid from potassium acetate-precipitated alkaline cell lysates was treated with RNase A then extracted with phenol/chloroform and chloroform.  c. Plasmids  Most of the plasmids used in this thesis are summarized in Table 2. Plasmids containing fusions of puc’C to phoA’ are listed separately in Table 3 (see Results). The characteristics or construction of the plasmids are described in greater detail below.  d. Conjugations  Mobilizable plasmids were transferred to R. capsulatus by conjugation in biparental (when the donor strain was SM1O) or triparental matings (using the helper strain HB1OI(pRK2O13)). Approximately equal numbers of E. coli donor/helper and R. capsulatus recipient cells were concentrated by centrifugation and spotted as a slurry on an RCV plate without antibiotic and incubated overnight at 30°C. Cells were then streaked sequentially on RCV (minimal) and YPS (rich) [82] media containing the selective antibiotic until isolated R. capsulatus colonies were obtained.  20  Table 2. Plasmids.  Plasmid  Description  Reference  pUCI3  £. coil cloning vector  88  pSUP2O2  Mobilizable suicide vector for Gram negative bacteria  74  pXCA6OI  iac’Z promoter fusion vector; TcR; IncP  I  pRK4I5  Broad host range vector; TcR; IncP  32  pJAJ9  R. capsuiatus expression vector; contains the puf promoter and  29  pufQ; Tc’; IncP pPUFP::C01E1 R. capsuiatus expression vector; contains the puf promoter and  J.T. Beatty  pufQ; Ap’, KmR; IncQ; ColE I pHLB1  pUCI3 containing a 4.5 kb Pst I-EcoR I puc operon fragment  41  pHLB2  Suicide plasmid for construction of strain tLHII  This work  pRK415::1I  Vector for ALHII control strains  41  pBACDE  4.5 kb Pst 1-EcoR I fragment bearing the puc operon in pRK4J5::2  41  pACDE  Pst 1-EcoR I fragment bearing the pucCDE deletion in pRK415::  41  pz\C  Pst I-EcoR I fragment bearing the pucC deletion in pRK4I5::2  41  ptD  Pst I-EcoR I fragment bearing the pucD deletion in pRK415::  41  pzE  Pst I-EcoR I fragment bearing the pucE deletion in pRK4I5::2  41  pM  pXCA6OI containing the puf promoter and the pufQ gene  I  pPEZ  pXCA6OI containing the pucE’::iac’Z fusion with 5’ end at Pst I  41  pPEZ-OOF  pPEZ with a frameshift mutation in pucD  This work  pCEZ  pXCA6OI containing the pucE’::iac’Z fusion with 5’ end at Cia I  41  pBEZ  pXCA6OI containing the pucE’::iac’Z fusion with 5’ end at BsaB I  41  pBEZ-OOF  pBEZ with a frameshift mutation in pucD  This work  pHEZ  pXCA6OI containing the pucE’::iac’Z fusion with 5’ end at Hinc II  41  pUC::pucC(+) pUC13 containing the pucC gene cloned in the orientation of  This work  transcription from the lac promoter pUC::pucC(-)  pUCI3 containing the pucC gene cloned in the orientation  This work  opposite to transcription from the lac promoter pUC19::phoA Contains the truncated phoA sequence and JR from TnphoA  8  pPQ::C  pucC expressed from pPUFP::C01EI  This work  pPQ::E4-33  pucC, truncated at aa 439, expressed from pPUFP::ColEl  This work  21 pPQ::E4-36  pucC, truncated at aa 412, expressed from pPUFP::C0IEI  This work  pPQ::E4-38  pucC, truncated at aa 387, expressed from pPUFP::C01EI  This work  pPQ::S8-1O  pucC::phoA fusion at aa 63, expressed from pPUFP::C01EI  This work  pPQ::E8-5  pucC::phoA fusion at aa 291, expressed from pPUFP::C01EI  This work  pPQ::E8-25  pucC::phoA fusion at aa 329, expressed from pPUFP::C01EI  This work  pPQ::E4-25  pucC::phoA fusion at aa 422, expressed from pPUFP::C0IEI  This work  pC  pucC expressed from pJAJ9  This work  22 e. Spectral analysis  Absorption spectra of intact cells (1.8 x 1O 9 cells suspended in 1 mL of 22.5% BSA in RCV medium) were measured with a Hitachi U-2000 spectrophotometer, and data were collected with the Spectra Caic software package (Galactic Industries Corporation). All spectra are normalised to an absorbance of 0.2 at 650 nm to correct for cell numbers.  f. Construction of R. capsulatus strain ALHII  The 4.5 kb Pst 1-EcoR I fragment bearing the pucBACDE genes from pRPSLHII [901 was first subcloned into pUCI3 [881 for ease of subsequent manipulations, yielding pHLBI. After digestion of pHLB1 with Cia I and partial digestion with Bcl I (Fig. 6), the 4.7 kb vector fragment containing the sequences 3’ of the Bc! I site at position 3030 of the published sequence  [791 and 5’ of the  Cia I site in the puc operon (including the first 24 codons of the pucB gene) was purified and treated with the Klenow fragment of DNA polymerase I to generate blunt ends. This fragment was recircularized, inserting a BamH I linker (5’-CCGGATCCGG-3’) at the ligation site by linker tailing [40]. The omega fragment [601 was then inserted into this BamH I site. The Pst I- EcoR I fragment of the resultant pHLB1ipuc::2 was inserted into the suicide vector pSUP2O2 [74] and introduced into SB1003 by conjugation. SpectinomycinR colonies were selected. Lighter coloured colonies were screened for loss of LHII by spectral analysis. Replacement of the puc operon resulting from a double cross-over event was confirmed by Southern blot analysis.  23  Figure 6. Construction of plasmids pHLBThpuc::2 and pUCACDE. See Materials and Methods, sections (f) and (h) for details. The positions of sites for restriction enzymes used are indicated; pUCI3 sequences are represented by medium, shaded boxes; wide boxes represent coding sequences (pucC, pucD, pucE or the omega fragment); wide shaded boxes show pucB and pucA; and thin lines designate R. capsulatus sequences flanking the puc operon.  Cia I  24  Bc! I  Bc! I  Bcl I  Bc! I  BclI partial digest  I Cia I Cia I  4.7 kb fragment  Cia I  Bc! I  Kienow BamH I linker tailing BamHI BamH I 2 fragment  Bcl I  I  /  Pst I  Pst I  EcoR  EcoR  BclI  BamHI  Bcl I  25 g. Southern blot analysis  Chromosomal DNA from SB1003 and tLHII (5 j.tg) was separately doubly digested with EcoR I and BamH I, run on a 1% agarose gel and transferred to nitrocellulose paper. A non-radioactive DNA labelling kit (Boehringer Mannheim) was used to prepare digoxigenin-labelled probes from gel-purified DNA fragments. The blots were treated according to the kit’s specifications until the development stage when a fluorescent dye substrate for alkaline phosphatase (Gibco-BRL) was used to develop the blots, after which they were exposed to X ray film at room temperature for varying lengths of time before development.  h. Plasmid deletions of pucC, pucD and pucE  The following deletions were made in the plasmid pHLB1 (see above) and transferred to the broad host range plasmid pRK4IS [32] as Pst 1-EcoR I fragments (Fig. 13, Results). The omega fragment was inserted at the Hind III site upstream of the Pst I site to reduce transcriptional read through from plasmid promoters [84]. The positions of restriction enzyme sites given below refer to the numbering in the published DNA sequence [79].  Plasmid pACDE was created by deleting the pucCDE genes from the Bc! I site at position 1463 to the Bc! I site at 3030 (Fig. 6). The 4.7 kb Cia Ito Bc! I fragment generated in making strain ALHII (see above) was ligated to a 0.95 kb Cia I to Bc! I fragment containing the C-terminal sequences of pucB, pucA and the first 750 bp of pucC.  26  The deletion of the pucC gene in plasmid pAC extended from the Bcl I site at position 1463 to the BsaB I site at 2217 (Fig. 7). The Pst I-BsaB I fragment from pHLBI containing the sequences downstream of pucC was ligated to the Pst I-Bc! I (filled) fragment bearing the pucBA genes and the first 750 bp of pucC. Fusion of the filled-in Bcl I site to the blunt BsaB I end created a translational stop codon.  In order to facilitate the deletion of pucD, a 0.75 kb Sma I-BsaB I fragment from pHLB1 was first subcloned into pUCI3 (Fig. 8), making the Bcl I site (position 2348) and the BssH I site (position 2598) unique in the resultant plasmid pUC::BS. The plasmid was cut at these sites and the ends were then filled in using the Klenow fragment of DNA polymerase I. Subsequent religation created a translationally in-frame deletion of pucD (pUC::BSzD). This construction was returned to the puc operon sequences by replacing the wild type Bsm I to Eco47 III fragment with the corresponding fragment from pUC::BSAD to generate pUCAD(IF). The deletion was confirmed by DNA sequencing (data not shown).  The pucE deletion extends from the Eco47 III site at 2761 to the Bc! I site at 3030 (Fig. 9). To ensure that translation beginning at the pucE start codon did not continue into the presumed transcription stop signal downstream of the deletion, an in-frame translational stop codon was created by adding a BamH I linker (5’-CGCGGATCCGCG-3’) at the Eco47 III site of pHLBI. This plasmid was then cut with BamH I and EcoR I, and ligated to the EcoR I-BamH I fragment from pHLBlApuc::Q (described above) to restore the sequences downstream of  27  Figure 7. Construction of plasmid pUCAC. See Materials and Methods, section (h) for details. The positions of sites for restriction enzymes used are indicated; B, Bcl I; 0, filled in site; z, site of deletion of pucC sequences; pUCI3 sequences are represented by medium, shaded boxes; wide boxes represent pucC, pucD, and pucE; wide shaded boxes show pucB and pucA; and thin lines designate R. capsulatus sequences flanking the puc operon.  28  Pst I Bcl I B BsaB I  Bcl I Kienow  BsaB I PstJ  Pst I  Pst I  Pst I  BcI I 0  BsaB I  Ligate  Pst I  A  EcoR I  29  Figure 8. Construction of plasmid pUCAD(IF). See Materials and Methods, section (h) for details. The positions of sites for restriction enzymes used are indicated, but only 1 of 6 Sma I sites is shown for clarity; 0, site destroyed by ligation; , site of deletion of pucD sequences; pUCI3 sequences are represented by medium, shaded boxes; wide boxes represent pucC, pucD, and pucE; wide shaded boxes show pucB and pucA; and thin lines designate R. capsulatus sequences flanking the puc operon.  30  0  pUC13  BclI BssH II Sma I  Sma I Ligate  Sma I BsaB I  Bc! I BssH I  Small fragment  Kienow Religate  Ill  I  Bsm I Eco47 III  Large fragment  Bsm I Eco47 III Pst I  4  EcoR I  Small fragment  31  the stop codon of pucE. The deletion of the pucE gene was confirmed by DNA sequencing (data not shown).  All experiments that utilized strain ALT-HI as a control in experiments using the plasmids described above were done with this strain containing the vector pRK4l5 carrying the omega fragment (pRK4l5::2).  To reconstitute the wild type phenotype to strain ALT-ill, the 4.5 kb Pst I EcoR I fragment that contained the pucBACDE genes was subcloned into pRK4l5::2 to yield plasmid pBACDE.  i Fluorescence measurements  Infrared fluorescence was measured using a method based on that of Youvan et al. [891. Stationary phase cells grown in low oxygen conditions were resuspended at a concentration of 6 x 1O cells/mL in RCV medium. The cell suspensions were then serially diluted 2 fold in a microtiter plate with flatbottomed wells. The plate placed over a 1 cm thick 1 M cupric sulphate solution illuminated from below by fluorescent lamps and photographed using Kodak H1E135 high speed infrared film with and without an additional Wratten 87C infrared filter (Eastman Kodak). Several exposures ranging up to 2 minutes were taken; an approximately 20 second exposure at f-stop 5.6 was used for the prints shown in Fig. 17B.  32  Figure 9. Construction of plasmid pUCz\E. See Materials and Methods, section (h) for details. The positions of sites for restriction enzymes used are indicated; B, Bcl I sites shown for reference; pUCI3 sequences are represented by medium, shaded boxes; wide boxes represent pucC, pucD, and pucE; the 2 fragment is shown as a very wide box; wide shaded boxes show pucB and pucA; and thin lines designate R. capsulatus sequences flanking the p1ic operon.  33 BamH I  Pst I  EcoR I  B  B  Eco47 Ill  EcoR I  Eco47 Ill, BamH I linker tailing  BamH I EcoR I  BamH I EcoR I 4  EcoR I I  BamHI EcoR I  Ligate  EcoRI  B  BamHI  B BamH I I I  34  j.  Construction and screening of pucC’:.pho’A fusions  A 1.7 kb Nae I fragment bearing the pucC gene was subcloned into plasmid pUC13 in the orientation allowing transcription of pucC from the lac promoter. As shown in Figure 10, the resultant plasmid, pUC::pucC(+), was digested at unique restriction endonuclease sites in the middle (Stu I) or at the 3’ end (EcoR I) of pucC, then treated with the exonuclease Bat 31 to generate fragments with deletions of the 3’ end of pucC.  As recommended by Sambrook et at. [66], 30 minute trial reactions were carried out on aliquots of 1.1 jig linearized DNA using enzyme concentrations ranging from 0.32 units to 0.01 units Bat 31 (Boehringer Mannheim) per 10 jiL reaction. Enzyme concentrations of 0.02, 0.04 and 0.08 units per reaction digested 0.2-0.8 kb from each 3’ end, an appropriate range to cover the length of the pucC gene starting from either the EcoR I or Stu I sites, so these reactions were scaled up 2-fold. The exonucleolytic digestion reactions were stopped by the addition of EDTA to 50 mM and heating at 65°C for 5 minutes, then the products were cleaved with Hind III. The pucC digestion products were separated from vector fragments by gel electrophoresis and fragments greater than 0.8 kb were purified using the Qiaex (Qiagen Inc.) gel extraction kit. The smallest digestion products resulted in bands too diffuse to purify by this method, so they were electrophoresed onto DEAE cellulose paper and eluted in a high salt buffer (1M NaC1, 0.1 M Tris-HC1 (pH 7.5), 10 mM EDTA). Acidification prior to ethanol precipitation aided in recovery of the DNA. The purified fragments were shotgun cloned into pUC19::phoA [8] which had been digested with Hind III and Sma I. The ligation products were used to transform the phoA deletion strain  35  Stu I  EcoR I  Linearize with EcoR I or Stu I Digest with exonuclease Bal 31 Digest with Hind III Gel purify fragments  —c-f -0--f -0--f -0-1E Ligate into pUCI9::phoA cut with Hind III and Sma I Plac —  —  puC  I  phoA  I  —  —  Screen for blue/white colonies on XP  Figure 10. Construction of pucC’::pho’A fusions, See Materials and Methods, section (j) for details. The arrow head represents the lac promoter.  36 CC118 [481, and transformants were screened on plates containing the chromogenic substrate XP (5-bromo-4-chloro-3-indolylphosphate-p-toluidine salt) at 40  A second screening of clones was done by restriction enzyme  digestion and gel electrophoresis of plasmid DNA and selected clones were subsequently sequenced. Standard dideoxynucleotide sequencing was done using a primer (5’-AATCACGCAGAGCGGCAGTC-3’) complementary to phoA sequences near the site of fusion.  k. Alkaline phosphatase assays  Approximately 1.2 x io cells from logarithmically growing cultures were harvested and washed in IM Tris-HC1 buffer (pH 8). The cells were resuspended in 0.5 mL of the same buffer and made permeable with a drop each of 0.1% SDS and chloroform for 5 minutes at 37°C. Timed assays were started with the addition of 0.5 mL of the alkaline phosphatase substrate (p-nitrophenyl phosphate at 0.8 mg/mL in 1M Tris-HC1, pH 8). Once a perceptible yellow colour had developed due to release of p-nitrophenol, the reaction was stopped with HPO and the cells were removed by 2 K the addition of 0.1 mL of 1M 4 centrifugation. Activities are expressed as 1000xAA42O/Atime (minutes).  1. Hydropathy analysis  The hydropathy profiles predicted for the PucC protein by the programs available in PC/GENE were compared with additional analyses run using ENDIT  37 [28]. PC/GENE offers SOAP [34], which is based on the hydrophobicity scale of Kyte and Doolittle [38] and assesses the probability that 17 amino acid hydrophobic segments are integral or peripheral; the method of Eisenberg et al [20], which identifies 21 residue segments as membrane associated helices likely to be globular or transmembrane based on the average hydrophobicity of the segment; and the method of Rao and Argos [62] which identifies segments of greater than 16 amino acids having a hydrophobicity peak above 1.13. ENDIT permits the user to choose any of several algorithms and calculates hydropathy values over both a large (19) and a small (11) amino acid window. It also calculates probabilities of N and C caps [631, and comparison of all these data helps in determining probable ends for transmembrane segments identified as peaks in the hydropathy profile plotted using the larger window. ENDIT was run using the hydropathy scales of Kyte and Doolittle [38], Chothia [53], Michel [281 and Eisenberg [19]. The results of all the hydropathy analyses were compared but the Chothia results were analysed in most detail in arriving at the model for the topology of PucC presented in the second chapter of Results.  m. Construction of plasmids for expression of pucC and terminally deleted alleles of pucC  In order to complement the pucC mutation in ALHII(pAC) in trans the pucC gene was cloned into the R. capsulatus expression vector pPUFP::ColEl. This plasmid was chosen to be compatible with pAC both in terms of its origin of replication and its selectable markers. The pucC gene was obtained as an EcoR I -BamH I fragment from pUC::pucC(-) and inserted downstream of the pufQ  38 gene in pPUFP::Co1EI, yielding plasmid pPQ::C. Expression of pucC was therefore driven from the puf promoter in an oxygen-repressible fashion. The plasmid was transferred to iLHII(pzC) by conjugation and TcRKm’ recipients were selected.  Translationally out of frame fusions between puc’C and phoA’ (see above) were used as C-terminal deletions of pucC. In such fusions translation terminates 4 or 10 codons after the last pucC sequence, depending on the reading frame. Three such fusions, occurring at residues 387 (E4-38), 412 (E4-36) and 439 (E4-33) of PucC, were subcloned from the original pUC19::pucC’::pho’A fusion plasmids as Xba I-Pst I fragments and inserted into pPUFP::Co1EI cut with Xba I and Nsi I to allow transcription from the puf promoter. These plasmids, pPQ::E4-38, pPQ::E4-36 and pPQ::E4-33, were transferred to ALHII(pAC) by conjugation and TcRKmR recipients were selected.  To express pucC in the F1696 deletion strains z.Nco and AStu, in which F1696 has been disrupted by a kanamycin resistance cartridge [C. Young, personal communication], the puf promoter, pufQ and pucC were transferred from pPQ::C to pRK4l5 (which encodes TcR) as a Hind III-BamH I fragment to give pC. The plasmid was introduced into the ANco and AStu strains and TcR recipients were purified.  n. RNA isolation, blot analysis and probe construction  RNA was isolated from R. capsulatus by the hot phenol method as  39 previously described [801. Electrophoresis samples were ethanol precipitated and denatured in a buffer containing formaldehyde and ethidium bromide [651. Five pg of RNA per lane were run on a 1.4% agarose/formaldehyde gel beside 3 tg per lane of a 0.24-9.5 kb RNA ladder (BRL). After electrophoresis the gel was equilibrated in 0.5X TBE buffer, and photographed with UV illumination before electroblotting overnight at 30V in 0.5X TBE buffer onto a Biotrans nylon membrane (ICN). After blotting the membrane was dried at 80°C under vacuum and exposed to UV light for comparison with the gel photograph to evaluate the efficiency of transfer.  Blotted membranes were prehybridized in 5X SSC (1X SSC is 0.15 M NaC1, 0.015 M sodium citrate, pH 7.0), 1% SDS, 10 mM EDTA and 50% formamide containing 0.5 mg/mL denatured sheared salmon sperm DNA for 4-8 hours at 42°C before addition of the denatured labelled probe. The blots were hybridized with the probes for 18 hours at 42°C.  Hybridization probes were prepared using purified DNA fragments as P-dATP by the random primer method [221. 32 templates for labelling with cxUnincorporated nucleotides were removed using the Qiaex DNA purification procedure (Qiagen). The Qiaex eluate in TE buffer was denatured at 90°C for 10 minutes and used directly for hybridization. After hybridisation the membranes were washed twice for 10-15 minutes in 2X SSC, 1% SDS at room temperature, twice in the same solution at 50°C for 10-15 minutes and once for 5 minutes in 0.2X SSC, 1% SDS at 50°C. Blots were then exposed to Kodak X-Omat film in a cassette with an intensifying screen at -75°C for varying lengths of time before development.  40 If necessary, blots were stripped for reprobing by brief incubation with boiling 0.1% SDS then this treatment was repeated and the solution allowed to cool to room temperature.  o. pucE’::lac’Z fusion construction  A translationally in-frame fusion of the pucE’ to lac ‘Z genes was obtained by cutting pHLB1 (see section f.) at the BspH I site in the pucE gene and filling in the overhanging  5t  ends with the Kienow fragment of DNA  polymerase I, followed by digestion with Pst I. The resultant 3.5 kb Pst I blunt-  ended fragment was then inserted into pUC13 digested with Pst I and Xba I (filled in with the Kienow enzyme), next to the BamH I site of pUC13, creating a translationally in-frame fusion of the pUC13 lacZ allele to the fifth codon of pucE (pUC-PEZ). A Pst I linker (5’-CCTGCAGG-3’) was inserted at the Cia I site in the pucB gene (pUC-CEZ), at the BsaB I site at the 3’ end of the pucC gene (pUC-BEZ), or at the Hinc II site in the pucD gene (pUC-HEZ) by linker tailing [401. (Construction of pUC-HEZ deleted all the sequences between a Hinc II site in the 5’ unsequenced region and the middle of the pucD gene.) A stop codon was created at the 13th codon of pucD by first cufting pUC-BEZ with Pst I and recircularizing the plasmid to eliminate the sequences upstream of the BsaB I site in pucC. The resulting plasmid was then cut with Bcl I, the ends were filled in and the plasmid was again recircularized, changing the sequence from 5’-GTG ATC ACA-3’ to 5’-GTG ATC GAT CAC A-3’, thus causing a frame-shift mutation in pucD (pUC-BEZ-OOF). The same frameshift was transferred to pUC-PEZ by replacing the Pst I-Bsm I fragment of pUC-BEZ-OOF with the wild  41 type sequences upstream of the Bsm I site, creating pUC-PEZ-OOF [Grace Wong, personal communication]. The fragments shown in Figure 28 (see Results) were inserted into the promoter probe vector pXCA6OI [1] as Pst I-BamH I fragments. The resultant plasmids were transferred to SB1003 by conjugation and TcR recipients purified.  p. 13-galactosidase assays  In early experiments, cells were grown to a density of 90-100 Klett units (3.5-3.8 x 108 cfu/mL) under high or low aeration conditions, harvested by centrifugation, resuspended and sonicated in 13-galactosidase assay buffer [521 on ice. After centrifugation the cleared supernatant liquids were assayed for /3galactosidase activity in a continuous assay [52]. Protein concentrations were determined by a modified Lowry procedure [57]. In later experiments [Christine Chiang, personal communication], J3-galactosidase activity was measured in a timed assay where logarithmically-growing cells concentrated 20-fold in /3galactosidase assay buffer were made permeable with SDS and chloroform [47]. Because of this difference, activities are expressed as percentages of the activity of low oxygen grown SBIOO3(pPEZ) assayed in the same experiment. For sonicated , for cells 1 cells, this specific activity was 52.9 nmol.ig protein-I .minute permeabilised with SDS and chloroform the measured activity was 518 . All determinations were performed on 1 1 mL Klett unitmin 420 OD duplicate samples in at least 2 experiments.  42 RESULTS  1. Phenotvpic properties of puc operon mutants  a. Chromosomal deletion of the puc operon  Absorption spectra of wild type cells show two peaks in the near infra-red region, one centred at 800 nm and the other at 850 nm (Fig. 11, dotted trace). These peaks are primarily due to the absorbance of the bacteriochlorophyll molecules associated with the LHII complex and reflect the predominance of this complex in the intracytoplasmic membrane. A small amount of the 800 nm peak is contributed by absorption of the less abundant reaction centre. The 850 nm peak is asymmetrical, having a shoulder due to the absorption of the LHI complex at 870 nm.  The pigment binding proteins of the LHII complex are encoded by the pucB and pucA genes [901. Three additional open reading frames, reading in the same direction, were discovered downstream of pucBA and named pucC, pucD and pucE  [791. In order to characterize these putative genes and determine their role  in LHII complex synthesis I made a chromosomal deletion of all five puc genes [41].  The puc derivative of the wild type R. capsulatus strain SBIOO3, designated strain ALHII, was created by chromosomal replacement of the puc genes with the 2 fragment (see Materials and Methods and Fig. 12B).  43  0 r1  600  800  1000  Wavelength (nm)  Figure 11. Whole cell absorption spectra of wild-type SBIOO3 (dotted line), the puc deletion strain ALHII (heavy line) and ALHII complemented with the wild type puc operon on the plasmid pBACDE (solid line). Cells were grown to stationary phase in low oxygen conditions and spectra were normalized to the same A650 value.  44  1 kb  A  E  P  I  I  C  8  I BA  B  E I  P I  E  C  D  E  BK  BK  Ii  I  C  E  12 25.8 9.8 5.1  2.4 2.2  1.3 0.96  Figure 12. Southern blot analysis of DNA from wild-type strain SB1003 and puc operon deletion/insertion strain ALHII. A) Representation of the 5.5 kb EcoR I fragment that contains the puc operon. B) Representation of the EcoR I fragment with the puc genes replaced by the omega cartridge. Hatched boxes designate puc genes, the open box the omega cartridge, and heavy lines unsequenced regions. Gene designations are given below the boxes, restriction enzyme sites above. Abbreviations of restriction enzyme sites: B, Bcl I; BH, BamH I; C, Cia I; E, EcoR I; P, Pst I. C) Results of Southern blot analysis. DNA from SB1003 (lane 1) and ALFIII (lane2) was doubly digested with EcoR I and BamH I and probed with the 5.5 kb EcoR I fragment shown in (A). The sizes of molecular weight markers are shown in kb to the left of the blot.  45 Strain ALHII lacked the LHII complex, as evidenced by the loss of absorption at 800 and 850 nm (Fig. 11, heavy trace). Residual peaks at 800 and 870 nm are due to absorption of the RC and LHI complexes, respectively. The deletion of the puc genes was confirmed by Southern blot analysis (Fig. 12). Chromosomal DNA from SB1003 and tLHII digested with EcoR I and BamH I was run on an agarose gel and transferred to nitrocellulose. When probed with the 5.5 kb EcoR I fragment shown in Figure 12A only one band of approximately 5.5 kb was seen in the lane containing wild type DNA. The lane containing zLHII DNA had two bands of 2.3 and 0.9 kb in size, consistent with a double cross-over having replaced the puc genes with the 2 fragment. If the suicide plasmid had inserted by a single cross-over event 3 bands of 5.5, 0.8 and 1.3 kb (or 4.5, 0.8 and 2.3 kb depending on the site of recombination) would have been detected. Probing a similar blot with the 2 fragment confirmed its presence. at a single site in the ALHII chromosome (data not shown).  b. Effects of pucCDE deletions on LHII complex absorption spectra  A series of plasmids was made that carried versions of the puc operon with different deletions of the pucC, pucD and pucE genes (Fig. 13), and these were conjugated into the ALHII strain [41]. Spectral analysis of cells grown under conditions of low aeration showed that the LHII complex could be restored by complementation in trans with the plasmid pBACDE, which contained the entire puc operon (Fig. 13 and Fig. 11, solid trace).  Strain zXLHII(pzE) (Fig. 14C) showed about 64% of the level of LHII complex  46  P  C  I  I  B  BBBBSE  B  ER  II  I  I  II  D  C  pBACDE  H  E I  pACDE pAC pAD pAE  Figure 13. The Pst I EcoR I plasmid inserts used to complement the ALHII deletion. puc operon structural genes are indicated as boxes, with the gene designations given below the map of pBACDE. Deletions are shown by dashed lines. Restriction enzyme sites used in constructing the deletions are shown above the maps and are abbreviated as: B, Bcl I; BB, BsaB I; BS, BspH I; C, Cia I; E, Eco47 III; ER, EcoR I; P, Pst I. Heavy lines represent unsequenced regions [79] and potential mRNA stem-loop structures are marked with loops. *  47  Figure 14. Absorption spectra of intact cells grown under low aeration (see Materials and Methods). All spectra are normalised to the same A650, and the heights of the major peaks are indicated. A) ALHII(pBACDE); B) ALHII(ptD); C) zXLHII(pAE); D) LHII(pRK415::Q); E) tLHII(pz\CDE); F) ALHII(pAC).  C  1-  D  A  1,7  1000  400  E  B  800  Wavelength (nm)  600  0.44  1000  400  F  C  600  800  0.36  1.4  1000  49  absorption found with tLHll(pBACDE) (Fig. 14A), based on integration of the areas under their respective 800 nm peaks, whereas the spectrum of zLHII(ptD) showed no difference from the profile obtained with ALHII(pBACDE) (Fig. 14B). In contrast, strains ALHII(pAC) and z\LHII(pACDE) had undetectable levels of LHII absorption (Fig. 14F and 14E respectively) and an apparent reduction in the levels of LHI complex compared to strain ALHII(pRK415::2) (Fig. 14D).  c. Photosynthetic growth characteristics of puc gene deletions  Because the LHII complex is thought to increase the intracellular area of photon absorption and thus might aid photosynthetic growth at low light intensities [18], the puc gene deletion strains were tested for the ability to grow 1 (Fig. 15A). At this s 2 photosynthetically at the low light intensity of 30 tEm intensity of light, strain ALHII(pBACDE) was light-limited for growth since it had a doubling time of 10 hours, whereas at saturating light intensities the doubling time was typically about two hours (see below).  Surprisingly, strain ALHII(pRK4I5::i2) grew only slightly slower at 30 1 than ALHII(pBACDE), with a doubling time of 15 hours, despite Ems undetectable levels of the LHII complex (see Fig. lAD). The kinetics and extent of growth of ALHII(pt\D) were indistinguishable from LHII(pBACDE). Strain z\LHlI(pzE), which had reduced levels of LHII (see Fig. 14C), had a much slower doubling time (28 hours) than strain zLHII(pBACDE) and did not reach as great a density in stationary phase.  50  A  1000  C  100  C-)  300 Time (hours)  B  1000 U)  o zLHII(pBACDE) D zLHII(pRK41 5::c) /iLHII(pzCDE) VLH II (ptC) OLLH I I(pzD) •zLHII(pzE)  D 4-, 4-,  U)  100 U) U)  U) C,)  0  10  30  20  1000  C  50  40  60  70  80  Time (hours)  U)  4-4  C 44 4-,  U)  100 4-,  U) C 1)  U)  C-) 0  10  20  30  40  50  60  70  80  90  Time (days)  -s, 2 Figure 15. Photosynthetic growth of cells with illumination at A) 30 E.m s- The growth of tLHhI(pACDE) in (A) and 2 j.iE•m. -s-l and C) 5 1 2 B) 300 iE.m/XLHII(ptC) in (C) show the appearance of a secondary mutants. In experiments where none arose the growth curves of ALHII(pAC) and zLHII(pACDE) were identical (not shown).  51 The photosynthetic growth of strains zLHll(pACDE) and ALHII(pAC) was the most impaired of the strains tested, doubling in 40 hours (Fig. 15A). Occasionally in cultures grown at low light intensities, faster growing secondary mutants arose, distinguished by a marked increase in the slope of the growth curve and, in one culture of ALHII(ptC), by an unusual greenish colour (see section f). The appearance of secondary mutants was not observed in cultures of any of the other strains described here, or in zLHII(pAC) or ALHII(pzCDE) cultures grown aerobically or photosynthetically with a light intensity greater than 60 tEm . 1 s 2  Because the ALHII(pAC), ALHII(pACDE) and ALHII(pAE) strains grew more , these 1 s 2 slowly than ALHII(pRK4I5::Q) at the low light intensity of 30 Em 2s strains were grown photosynthetically at 300 Em 1 to determine if they were also impaired in photosynthetic growth at this high light intensity. Strains zLHII(pBACDE), ALFIII(pAD) and ALHII(pRK415::2) were found to grow equally well with a doubling time of about 2 hours (Fig. 15B). Strain zLHll(pAE) grew more slowly with a doubling time of  Ca.  7 hours, whereas strains ALHII(pAC)  and ALHII(pACDE) grew very poorly with about 10 hour doubling times. When these strains were grown aerobically with high or low oxygen conditions no differences in growth rates were observed (data not shown).  Under photosynthetic growth conditions at the lowest light levels tested (5 ) only strains ALHII(pBACDE) and z\LHII(pAD) grew well (Fig. 15C) 1 s 2 tEm The slight reduction in the growth rate of z\LHII(pAD), with an 85 hour doubling time, compared to the pseudo-wild type strain (65 hours) is the only phenotype yet observed for the pucD deletion strain. After an approximately 50 day lag,  52  strain zLHII(pL\E) began to grow with a doubling time of roughly 12 days. This late growth did not seem to be the result of secondary mutation: duplicate samples grew with exactly the same profile, and spectra of these cultures resembled usual zLHII(piE) profiles. Although it is not shown in Figure 15, the  remaining strains in the experiment were followed for up to 130 days. Strain zXLFffl(pRK415::2) grew very slowly with an estimated doubling time of 55 days. Strain ALHII(piCDE) never exhibited growth at this light level, and growth in ALHII(pAC) cultures proved to be due to suppression of the effects of the pucC deletion by a secondary mutation (see section f).  The photosynthetic growth characteristics of the wild type strain SB1003, and strains ALHII(pBACDE), ALHII(pRK415::2) and L\LHII(pAC) were compared in greater detail (Fig. 16). tLI-llI(pBACDE) grew at the same rate as the wild type ’. Significant differences in Em s strain at light levels ranging from 200 to 30 2 the growth rate of zLHII(pRK415::2) compared to the wild type strain appeared s’ and strain iLHII(pAC) 2 once the light intensity had fallen to 30 tEm consistently grew more slowly than any of the other strains tested, generating . 1 s 2 secondary mutants at light intensities below 60 tEm  d. Fluorescence analysis of ALHII(pAE)  To evaluate whether the slow photosynthetic growth of ALHII(pAE) might be because the LHII complexes present in the strain functioned improperly, the cells were tested for emission of fluorescence [921. When light energy absorbed by the LHII complex is not efficiently transferred through the LHI complex to the  0  a)  ci  a)  C,,  4-’  >  a)  4-, 4-..  4-,  C’,  0  a)  ci  a)  c  U,  4-’  >  ‘I)  10  100  1000  10  50  50  0  100  100  150  a tLHII(pBACDE)  o SB1003  Time (hours)  Time (hours) D  a)  (-)  a)  ci  (I) C  >  4-,  a)  4-, 4-,  C :3  (IC  4  0  50  50  Time (hours)  100  Time (hours)  iLHII(pRK415::cI) V zLHIl(piC)  100  1000  1100  s’  a)  4-,  4-..  4-.. 4-’  0  1000 C,,  C  100  B  C :3  4-,  C,,  1000  150  200  100  . tEm s 2  Figure 16. Photosynthetic growth of wild type SBIOO3, zLHII(pBACDE), L\LHII(pRK415::2) and ALHII(pAC) at s. D) 30 2 -s. C) 60 Em 2 s. B) 100 iEm 2 different light intensities. (See key for symbols) A) 200 Em  A  01 03  54 RC it is reemitted as fluorescence. As shown in Figure 17, the control strain RC6(pA4) fluoresces strongly because it contains LHII complex but no LHI or RC. However the fluorescence detected from z\LHII(pzE) was not greater than that of ALHII(pBACDE) or ALHII(pAD).  e. Effects of light intensity on LH complex levels  LHII complex levels are known to increase by a post-transcriptional mechanism as illumination levels decrease  [931. This response can be seen in the  wild type strain SBIOO3 (Fig. 18A). The area under the LI-ill absorption peak at , 1 s 2 800 nm increased 2.5 fold as the light intensity dropped from 200 to 30 Em where the maximum amount of LHII complex was reached. A decrease in LHII . 1 s 2 complex levels was seen at the light intensity of 15 Em  The pseudo-wild type strain ALHII(pBACDE) (Fig. 18B) showed a similar pattern of increasing LHII complex levels as light intensity decrease from 200 to 30 Em , but the absolute amounts of the complex were approximately 60% 1 s 2 of those in SB1003. Both strains nonetheless grew at the same rates (see Fig. 16).  Although LHI levels have been reported to increase in response to decreasing illumination [27], in Figure 18C it can be seen from the absorption peak at 870 nm that there was little difference between the LHI levels when strain ALHII(pRK4I5::12) was grown at light intensities ranging from 15 to 200 s’. In strain ALHII(pzC), however, the LHI levels increased steadily as 2 iEm 1 the s 2 light intensity dropped from 200 to 60 tEm 1 (Fig. 18D). At 200 Em s 2  55  A 1 2 3 4 5  B -  —  1 -  —  2 3 4  •..• .••.  5  Figure 17. Fluorescence emission of R. capsulatus strains. Cells were serially diluted and photographed under visible light (A) or through an infra-red filter (B). Row 1: zRC6(pz4) (RC-, LHII); Row 2: zLHII(pRK415::2); Row 3 ALHII(pBACDE); Row 4: ALI-ffl(pAD); Row 5: zLFffl(pAE).  56  Figure 18. Spectra of intact cells grown photosynthetically at 200 .tE.m•s-’, 100 2 .s-l, 60 i.tE.m2 p.E.ms- The light intensity is 2 tE.m. •s- and 15 1 2 .iE.m•s-’, 30 1 2 indicated to the right of the major peak. All spectra were normalised to the same A650 A) SB1003, B) ALHII(pBACDE), C) ALHII(pRK415), D) ALHII(pAC).  57  30  A  A  B  60  II  60  ri  A  Il  \  30 15  2-  fj  100  200  200  0-  2-  C  D  30  60 15  30 100  1-  60  200  100 200  -j 5 .  0I  I  I  700  800  900  i  I  1000 700  Wavelength (nm)  I  800  900  1000  58 amount of LHI complex in this strain was much lower than in strain ALHII(pRK415::2), which might explain why the pucC deletion mutant grew ’, however, Em s more slowly (see Fig. 15). At the lower light intensity of 60 2 the amount of the LHI complex was 76% of that in strain LHII(pRK415::2) but this relative increase in LHI complex level in strain ALHII(pzC) was not accompanied by an improved photosynthetic growth rate (see Fig. 16). At light 1 the slow growth rate provided enough of a s 2 intensities of less than 60 Em disadvantage to the pucC mutant that cells containing second site suppressor mutations out-grew the primary mutant. This is reflected in the spectra for these cultures (Fig. 18D).  f. Isolation of strains with secondary mutations that suppressed pucC deletion  The impaired photosynthetic growth of strains ALHII(pACDE) and ALFffl(pzC) apparently provided an appreciable selective advantage for cells containing second site suppressor mutations. When these strains, especially ALHll(pzC), were grown photosynthetically at light levels lower than 60 ’ faster growing secondary mutants often arose, distinguished by a Em s 2 marked increase in the slope of the growth curve and, occasionally, by an unusual greenish colour.  When plates streaked from unusually fast-growing ALHII(pzCDE) or ALHII(pAC) photosynthetic cultures were incubated aerobically at least two colony types were seen: pink colonies similar to the original ALHII(pACDE) or ALFIII(pAC) strains, and darker red colonies. A total of five second site  59 suppressors of pucC deletion were isolated from photosynthetic cultures grown at low light intensities and named ALHII-1 (pt\C), ALHII-2(pACDE), ALHII-3(pAC), ALHII-4(pAC) and ALHII-5(pAC), for the order in which they were found. For all five suppressor mutants I determined that the site of the second mutation was not on the pAC (or pACDE) plasmid by isolating the plasmid and transferring it back to LHII. In all cases these back crosses had the phenotype of the primary mutant. The suppressor strains varied considerably from each other and are described in more detail below.  g. Phenotypic characterisation of suppressors of pucC deletion  Strain ALHII-1(pAC) regained only a moderate amount of LHII complex (Fig. 19B) compared to strain ALHII(pBACDE) (Fig. 19A), and as shown below the relative amounts of LHI and LHII varied considerably. The ability of this strain to grow photosynthetically was improved compared to the parental strain ALHII(pAC) but still lagged behind that of wild type cells at light intensities of less than 100 tEm 1 (data not shown). This retarded growth at low light levels s 2 made this strain unstable: for instance plates spread from cultures grown at 5 s’ showed a mix of colony types indicating that the strain had mutated at 2 tEm additional site(s). These triple mutants were not characterized further.  The occurence of tertiary mutants sometimes made it difficut to tell whether the strain had mutated or if its amount of LHII complex was repressed to an unusual degree at high light intensities. An experiment was designed to systematically test this notion by growing identical inocula photosynthetically at  60  Figure 19. Spectra of intact cells of secondary suppressor strains grown photosynthetically. For reference, the spectrum of the primary mutant, ALHII(pAC) or ALHII(pACDE), is shown with each strain (fine line) and the pseudo-wild type strain ALHII(pBACDE) is given in (A). The secondary mutants shown are: B) ALHII-1(pzC); C) ALHII-2(pACDE); D) zLHII-3(pz\C); E) zL1-llI-4(pAC); F) ALHII-5(pAC).  61 2-  B  0— 2-  1.5-  C  C  1.5*  0 I  I  700  800  900  I  I  1000  700  Wavelength (urn)  I  800  900  1000  62 . The relative amounts of the 1 s 2 light intensities ranging from 15 to 200 Em two LH complexes varied considerably in strain zLH1I-1(pAC) (Fig. 20). The shoulder at 870 nm was always very pronounced, indicating that the amount of LHI complex was high relative to the level of LHII complex. Strain zLHil-1 (ptXC) had much less LI-HI complex than the pseudo-wild type strain (compare Fig. 18B  and Fig. 20) but the levels of LHII increased to a greater extent in tLHII-1(pzC) as . The level of LI-il complex, 1 s 2 the light intensity decreased from 200 to 30 tEm indicated by the absorbance at 870 nm, can also be seen to have increased in strain ALI-ilI-1(pAC) as light intensity decreased. The absorbance spectrum of the cells , in which a tertiary mutation arose, resembled a wild type 4 s 2 grown at 15 Em spectrum in that the shoulder at 870 nm was much less pronounced than in the original mutant. The absorbance value at 870 nm of this mixed culture grown at 1 suggesting s 2 15 Em 1 was lower than for ALHII-1(pAC) grown at 30 Em s 2 either that ALHII-1(pAC) has higher than normal amounts of LHI complex or 1 has a reduced amount of the s 2 that the tertiary mutant that arose at 15 tEm LHI complex.  The suppressor strain z\LHII-2(pACDE) had spectral characteristics similar to those of wild type cells (Fig. 19C) and doubled at wild type rates when grown 1 (data not shown). When this strain was s 2 photosynthetically at 300 or 30 Em ) it actually had a shorter doubling 1 s 2 grown at very low light levels (5 tEm time than wild type cells (35 as opposed to 65 hours). This robust photosynthetic growth suggested it might be possible to clone the suppressor mutation from this strain by making a gene library which could then be used to complement LHII(pACDE) under low light photosynthetic growth conditions. Unfortunately, attempts to make such a library from ALHII—2(pzCDE) were  63  30 15  60 C 100  C  200 ie  700  800  900  1000  Wavelength (nm)  Figure 20. Whole cell spectra of ALHII-1(pzXC) cells grown photosynthetically at 200 jiE.m.s-I (blue), 100 jiE.m2 .s-l (green), 60 1 2 .s-1 2 .s- (pink), 30 jiE.m2 .tE.m(turquoise), 15 tE.m.s-l (black). All spectra were normalised to the same A650. 2  64  unsuccessful.  Suppressor strain ALHII-3(pAC) regained LHII complex levels in approximately wild type proportion to LHI levels, judging by the size of the LHI shoulder of absorption at 870 nm (Fig. 19D), although the peak heights were only about 50% of wild type. Unusually, this strain seemed to be unstable under aerobic conditions. Dark coloured colonies from the original mixed culture were difficult to propagate in aerobic cultures or on aerobically-incubated streak plates. A plate spread with cells from the original (mixed) culture which was incubated photosynthetically and then stored aerobically initially had both pale and dark colonies. The pale colonies continued to increase in size while the dark colonies did not. Light pink and green colonies then appeared at the periphery of some of these dark colonies. For this reason strain tLHII-3(pzC) was subsequently only propagated under photosynthetic conditions where it appeared stable.  Suppressor strain zXLHII-4(pzC) was isolated from a photosynthetic culture 2 of ALHII(pAC) grown at 30 tEm  When a sample from this culture was  spread on plates and incubated photosynthetically to isolate the secondary mutant several types of colonies were seen instead of two as expected. Some variation may have been due to differences in light levels on different regions of the plate, but there were clearly small, pale colonies resembling the primary mutant, small dark colonies which had regained LHII complex and large, pale colonies. Pure cultures of this last type had no LHII complex (Fig. 19E), but nonetheless grew well photosynthetically. This improved growth was apparently due to an elevated amount of RC and LHI complex. This is the strain that was retained as tLHII-4(pAC).  65  The secondary mutant strain z\LHII-5(pAC) was isolated from a sample grown at 15 tEm 2s 1 in the same experiment that generated ALHII-4(ptC), so both cultures were started from the same low oxygen inoculum. Only one secondary mutant was isolated from this sample, and its spectral characteristics resembled those of pure cultures started from the small dark colonies that were co-isolated with ALI-llI-4(pAC). The LHII complex levels in this strain varied 1 the cells had s 2 considerably. When grown photosynthetically at 15 tEm approximately wild type levels of LHII complex. At higher light intensities, however, the levels of LHII absorption at 850 nm are slightly lower than the LHI absorption at 870 nm (Fig. 19F). The pronounced shoulder on the 800 nm peak was associated with the culture supernatant; when cells were removed by centrifugation the culture medium appeared blue and a broad peak at 760 nm was seen. This soluble pigment is probably a degradation product of bacteriochlorophyll present in stationary phase cultures.  g. Conclusions  A chromosomal deletion of the puc operon was made that eliminated LHII complex absorption from induced cells. This deletion strain, ALHII, retained high levels of the LHI complex and was able to grow photosynthetically at rates approaching those of wild type cells at levels of illumination greater than 30 1 (Fig. 16). As light intensity fell below this value a significant difference s 2 tEm was apparent, and at the lowest light intensity tested these cells grew much more slowly than wild type. When complemented with a plasmid-borne copy of the  66 puc operon the strain had wild type photosynthetic growth characteristics (Fig. 16) but slightly reduced levels of LHII complex (Fig. 18).  Strain ALHII was complemented with plasmids carrying deletions of one or more of the pucC, pucD and pucE genes to determine their involvement in LI-ifi complex formation. The mutant strains were evaluated for the presence of LH complex absorption peaks in the near infrared and for their ability to grow photosynthetically.  Deletion of the pucD gene had very little effect on LHII complex formation (Fig. 13B) and photosynthetic growth (Fig. 15). The only difference that could be detected between strain ALI-llI(pAD) and strain ALHII(pBACDE) was a slight reduction in photosynthetic growth rate at the lowest level of illumination tested. The function of pucD thus remains unknown.  Deletion of pucE caused a reduction in levels of LHII complex (Fig. 13C) and impaired photosynthetic growth (Fig. 15). This strain grew more slowly under most light intensities than ALHII(pRK415::2) despite retaining some LHII complex. The exception to this was at 5 Em 1 where ALHII(pRK415:2) s 2 barely grew but ALHII(pAE) grew reasonably well after a long lag period. The reduction in photosynthetic capability at light intensities greater than 30 1 did not seem to result from inefficient transfer of light energy from s 2 tEm LHII to the rest of the photosynthetic unit because no increase in fluorescence was detected in ALHII(pAE) cells compared to the pseudo-wild type strain (Fig. 17).  67 Of the three genes deleted only pucC was absolutely required for LHII complex formation. Deletion of pucC alone or along with pucD and pucE abolished the LHII complex from induced cells (Fig. 13) and greatly diminished the cells’ ability to grow photosynthetically (Fig. 15). This was not a general physiological impairment, however, because the cells grew at normal rates under conditions of low aeration where the puc operon is induced but the cells derive their energy from respiration. The strains lacking the pucC gene had reduced levels of LHI complex compared to ALHII(pRK4I5::Q) (Fig. 13), which could account for their poor photosynthetic growth. The relative abilities of the two strains to grow photosynthetically did not parallel their respective levels of LHI 1 the levels of the s 2 complex, however. At the low light intensity of 60 Em complex were comparable in the two strains (Fig. 18) but this relative increase in the amount of the LHI complex in z\LHII(pAC) was not accompanied by a corresponding increase in photosynthetic growth rate (Fig. 16). In fact, at even lower light intensities the slow growth rate provided enough of a disadvantage that cells containing second site suppressor mutations frequently came to predominate in cultures.  Five such suppressor mutants were isolated in separate experiments in which they took over slowly growing cultures of ALHII(pAC) or z\LHII(pt\CDE). None of the suppressor mutations mapped to the plasmids containing the puc operon deletions. Three of these secondary mutant strains, ALHII-1 (pAC), ALHII 3(pAC) and ALHII-4(pAC) had radically different phenotypes, suggesting that multiple sites can be mutated to suppress the loss of pucC. Interestingly, two of these strains were unstable. zLHII-1 (pAC) only partially restored LHII complex levels (Fig. 19B) and generated tertiary mutants under conditions of low light photosynthetic growth. ALHII-3(pAC) had moderate levels of LHII complex (Fig.  68 19D) but was unstable when grown aerobically. Finally, strain tXLHII-4(pAC) grew well photosynthetically without restoring LHII complex, perhaps because it had elevated levels of the LHI and RC complexes (Fig. 19E). This result, along with the growth characteristics of ALHII(pRK4I5::Q), showed that the reduction in the photosynthetic growth rates of pucC mutants was not due solely to the loss of LHII complex.  69 2. PucC structure and function  a. Hydropathy and positive inside rule analysis of PucC  In order to gain some insights into the functional domains of the pucC gene product the structure of the PucC protein was studied. The predicted product of the pucC gene is extremely hydrophobic [79] and is almost certainly an integral membrane protein, but when the amino acid sequence was analysed by several hydropathy algorithms there was considerable disagreement over the assignment of transmembrane segments (Fig. 21). Green [281 examined the ability of several hydropathy programs to correctly predict the transmembrane x helices of the Rhodopseudomonas viridis reaction centre L and M subunits, which are known from X-ray crystallography data.  She concluded that although  no one program unambiguously identified all the membrane-spanning domains, the consensus of several programs gave fairly accurate results. Analyses using Green’s program ENDIT and several algorithms in the commercial package PCGENE led to a theoretical model for PucC and are described below.  Along with hydropathy predictions of membrane-spanning helices, the distribution of positively charged amino acids must also be considered according to the “Positive Inside Rule” of von Heijne [811. This principle states that hydrophilic loops containing clusters of basic amino acids are generally restricted to the cytoplasmic side of the cell membrane. Interestingly, only the absolute number of positively charged residues is relevant; there is no correlation seen in the distribution of acidic residues nor is net charge important. For this reason  70  A  ELZEZ ——ft  It—  -  —-  B —1---I••  S  •  •epe  .  .  S  •  ••  I  •S  If  S  ..  S  a  IS••  I  a  S  SW  IS  a  5S  •I  S  C  D  100  200  300  400  I  I  I  Amino Acid Residues  Figure 21. Transmembrane domains (boxes) predicted by various hydropathy  analyses. Shaded boxes represent weak predictions, or, in the case of the Eisenberg algorithm, membrane associated helices. Dots below each model indicate the position of an arginine or lysine residue. The software package PCGENE was used to predict integral membrane domains using the methods of A) Kyte and Doolittle [381 (SOAP), B) Eisenberg [201 (HELLXMEM), and C) Rao and Argus [62] (RAOARGUS). The Chothia analysis (D) was run using Green’s program ENDIT [281. The positions of amino acid residues are indicated at the bottom of the figure.  71 the position of lysine and arginine residues in PucC are shown as filled cirdes under each hydropathy prediction in Figure 21. Application of the positive inside rule indicated that the clusters of basic residues at both termini and around positions 1.00, 240 and 330 were likely to be in cytoplasmic domains.  PucC was first analysed using the SOAP program of PCGENE. This method uses the hydropathy scale of Kyte and Doolittle in which the amino acids are assigned a hydropathy index that reflects the free energy of transfer from the water to vapour phase, the probability of being buried in the interior of a protein and, in some cases, the authors’ personal bias [381. SOAP modifies Kyte and Doolittle’s original analysis by first identifying the most hydrophobic 17 amino acid segment in a protein sequence. The segment, and therefore the protein, is then classified as integral (I) rather than peripheral (P) if the average hydropathy across the 17 residue window is above a cutoff value that was arrived at by examination of a database of proteins known to be either peripheral or integral membrane proteins [34]. If an integral segment is identified it is saved and removed from consideration while the analysis continues on the remainder of the sequence until no more integral domains are found. Figure 21A shows the nine hydrophobic domains (boxes) predicted by SOAP. The open boxes represent hydrophobic domains with a predicted P:I ratio of less than 0.05, while the shaded boxes are hydrophobic domains less likely to be membrane-spanning segments according to this algorithm. This method seemed to overlook some transmembrane segments. There are two clusters of basic residues at the N terminus of the protein which should be separated by an even number of transmembrane sequences according to the positive inside rule. The intervening segment should therefore cross the membrane twice or not at all. The P:I  72 probabilities given for the first two boxes were 0.90 and 0.0034 respectively, however  —  the algorithm predicted one transmembrane helix in this region.  Similarly, odd numbers of predicted membrane-spanning segment separated all the other clusters of basic residues from each other. The suggestion that this algorithm overlooked transmembrane domains was strengthened when the results of other methods were examined.  The other three hydropathy scales used predicted eleven hydrophobic domains in common even though they are based on different principles.  The Eisenberg consensus scale, used to predict the average hydropathy over a sliding window of 21 residues, is a composite scale that averages four previous hydropathy scales [20]. It was intended to minimize artifacts built into other scales because of choice of data base or other non-random factors. Twelve hydrophobic domains were predicted in PucC using this scale (Fig. 21B). The PCGENE program HELIXMEM then classified these segments as globular (shaded boxes) or transmembrane (open boxes) according to their average hydrophobicity. The cutoff distinguishing the two classes is somewhat arbitrary, and all three domains classified as globular were sufficiently hydrophobic to be in the uncertain range. To satisfy the positive inside rule all three of these weakly hydrophobic segments must cross the membrane, or an adjacent strongly hydrophobic segment must not.  The RAOARGUS algorithm of PCGENE predicted 11 transmembrane helices in PucC if the first 53 residue hydrophobic segment was considered to consist of two domains (Fig. 21C). This method is based on the probability of a  73 given amino acid being found in a membrane-soluble helix versus elsewhere in a membrane-associated protein [62]. The authors derived this “buried helix parameter” by first predicting transmembrane helices for a group of known integral membrane proteins using complicated and diverse criteria. The buried helix parameter for each amino acid was then calculated simply as the ratio of the frequency of its occurrence within these predicted transmembrane domains to its frequency at any position within the integral membrane proteins in their database. Candidate integral membrane proteins are analysed by plotting the buried helix parameter for each residue and smoothing the curve with a slidingaverage window of 7. Transmembrane helices are identified as peaks meeting cut-off values for height and width which were assigned somewhat arbitrarily by analysis of a limited number of sequences for which transmembrane domains had been determined by other means. Although the derivation of the method is somewhat tortuous, all the transmembrane helices predicted for PucC by the RAOARGUS program were also predicted by the Eisenberg and Chothia algorithms (Fig. 21B, C, and D), and except at the C-terminus were consistent with the positive inside rule. The C-terminal region is considered in more detail below.  The final program used to analyse the PucC sequence was ENDIT, written by Beverley Green [28]. ENDIT permits analysis using several hydropathy scales for sliding windows of two sizes, and additionally calculates the probability of omega loops, alpha and beta turns and N- and C-terminal caps. Several hydropathy scales were used to analyse PucC, including the Eisenberg consensus and the Kyte-Doolittle scales described above. The predictions of the Chothia free energy scale were analysed in the greatest detail and combined with turn and cap  74 predictions to generate the model shown in Figure 21D.  The Chothia free energy scale is based on an analysis of the distribution of the 20 amino acids in crystal structures of soluble globular proteins [53]. The frequency of a particular amino acid occuring on the surface of the protein was compared to the frequency of its volume being less than 5% accessible to exterior water, the authors’ definition of an interior location. The ratios of these frequencies were then used to calculate implied free energies of transfer from the interior to the surface for each amino acid. Amino acids defined as hydrophobic by chemical criteria generally had a large free energy values while charged amino acids had negative free energies for such a transfer. This scale was used to predict hydrophobic segments in PucC. A large window (19 aa) was used to identify hydrophobic segments long enough to be membrane-spanning helices. An 11 amino acid window plot, along with the N- and C-terminal cap predictions, helped to determine the outer boundaries of predicted transmembrane domains.  Other secondary structure predictions from ENDIT were used to help define the ends of membrane-spanning helices. Cap predictions were based on relative frequencies of particular amino acids at residues adjacent to alpha helices in the structures of soluble proteins [63], and as such might be of questionable value when considering membrane associated helices. For the PucC protein strong predictions of N-caps were less frequent than C-cap predictions, and never occurred within hydrophobic segments. This was not that surprising since asparagine and proline residues are strongly favoured in calculating N-cap probabilities and are not commonly found within hydrophobic cc-helical segments. However while Richardson and Richardson found that these residues were much more common at the N-terminal end of soluble helices than at the  75 C-terminal end, in my analysis strong N-cap predictions coincided more often with the C-terminal end of predicted hydrophobic helices. The N-terminal boundaries were more difficult to predict. They sometimes coincided with a strong prediction of a C-cap but these were so common along the length of the protein sequence that this could have been fortuitous. In practice, the region close to the boundaries of a hydrophobic segment was examined and the residue corresponding to the strongest prediction of a cap or alpha turn was chosen as the outer boundary.  The results of this analysis are shown in Figure 21D, where open boxes represent the more hydrophobic predicted transmembrane domains and shaded boxes the less hydrophobic ones. Twelve potential membrane-spanning helices were predicted and, as with the Eisenberg prediction, all of the weakly hydrophobic segments are required to cross the membrane in order to satisfy the positive inside rule.  In sum, comparison of the four hydropathy analyses along with the restrictions of the positive inside rule indicated that the first 8 hydrophobic domains predicted by both the Eisenberg and Chothia scales, most of which are in agreement with the other two predictions (see Fig. 21), were strong candidates for transmembrane helices. In the C-terminal 100 amino acids of the protein, however, it could only be determined that there were likely to be either 2 or 4 membrane-spanning helices from this theoretical analysis.  76 b. Construction and analysis of pucC’:.pho’A fusions  The theoretical model of PucC topology was tested using the genetic system of phoA fusions [48]. The phoA gene encodes alkaline phosphatase which requires the formation of disulfide bonds for activity. These bonds cannot form in the cell cytoplasm, a characteristic which has allowed phoA to be used as a reporter gene in topological analyses of integral membrane proteins [47]. Typically, several translationally in-frame fusions are made between N-terminal segments of the gene of interest and a truncated phoA which lacks translational start and leader peptide sequences. When the alkaline phosphatase activities of such fusion proteins are assayed high activity generally indicates that the fusion site occurred in a periplasmic domain.  A collection of fragments having the same 5’ but different 3’ ends was generated by exonucleolytic digestion from the 3’ end of pucC (see Materials and Methods). The fragments were then shotgun cloned into the plasmid pUC19::phoA, which has phoA (without its translational start and leader peptide sequences) inserted in the multiple cloning site of plasmid pUCI9 [8]. The phoA deletion strain CC1 18 was transformed with the ligation products and used to screen for alkaline phosphatase activity on agar containing the chromogenic substrate XP.  Initially, only blue colonies were screened by restriction digestion analysis, followed by sequencing across the fusion joint. Essentially all the blue colonies screened contained translationally in-frame fusions of pucC to phoA. The exceptions to this were colonies which stained extremely dark blue and  77 contained plasmids with no detectable insert. In the undigested plasmid the  phoA gene is not in the same reading frame as the lacZ a fragment and colonies appear white on )(P. If the reading frame is altered to allow translation of alkaline phosphatase from the lacZ a start codon blue colonies result (data not shown). I cannot explain this observation since translation through the multiple cloning site is not expected to generate an effective leader peptide.  It was found that the fusion sites were not dispersed over the entire gene but instead clustered at approximately 200 base pair intervals, so white colonies were then screened. Since both low activity and translationally out-of-frame fusions would be expected to give white colonies, only plasmids which had inserts of sizes not found amongst the blue colonies were sequenced. Five out of 34 white colonies whose plasmids were sequenced contained fusions which were translationally in-frame. In all, 44 different translationally in-frame fusions of  pucC to phoA were generated. These results are summarized in Table 3.  In order to avoid ambiguous results associated with, for example, fusions half-way through a transmembrane segment, the fusions most likely to reside in regions predicted in the foregoing theoretical analysis to be hydrophilic were chosen for alkaline phosphatase assays. All the fusions in the C-terminal region of PucC were also analysed to resolve the ambiguities of the theoretical analyses. As shown in Table 3, there was generally an approximately 20-fold difference between high activity fusions and low activity fusions which is typical of the relative levels of activity for periplasmic vs. cytoplasmic fusions [67]. This difference was too great to be likely to be due to differences in the rate of synthesis of the different fusion proteins, especially when the size and consistency of the data set were taken into account. Synthesis rates of fusion  78 Table 3. Summary of translationally in-frame fusions between puc’C and phoA’. Residues which differ from the sequence published by Tichy et a!.  [791 are underlined. Alkaline phosphatase  activities are given in relative units and are the average of at least 3 different experiments.  Fusion  1 (last intact codon) Sequence 2  Colony  Alk Phos  3 Colour  Activity  S8-7  ACC GTC GGG ATG ACC TTG AC  (48)  B  S8-12  GTC GGG ATG ACC TTG ACC CT  (49)  B  S8-1O  GTG ATG ATC GTC GAG ITG GC  (63)  B  S8-6  GAG ITG GCG GTT CCG GCC TC  (67)  B  S8-8  CTC GTC TC GTG ATG CTG GC  (74)  B  S8-3  TC GTG ATG CTG GCG ATG CC  (76)  B  S8-11  ATG CTG GCG ATG CCG ATG CT  (78)  B  S4-20  ACG CTG ATC GGC TTN AAG TC  (90)  W  4.7  S4-15  CCC TGG ATC TGG AAG GGA AC  (109)  W  4.5  S2—9  CCC TTC GCA CTT CTG GTG CT  (126)  B  S2-7  TTC GCA CTT CTG GTG CTG TC  (127)  B  S4-7  TCG GGQ TTC GG GAA TCC GT  (133)  B  S4-9  TTC GG GAA TCC GTG GAT GC  (135)  B  S4-6  GG GAA TCC GTG GAT GCG CC  (136)  B  151  S8-5  CTG GCA ACC GAC CTC GTC GC  (171)  B  30  S2-2  TAT GTG ATG CTG CTC TTC GG  (189)  B  S2-5  CTG CTC TTC GGC ATG GTG AT  (192)  B  S2—8  ATG GTG ATC AGC GCG CTC  (196)  B  S2-10  AGC GCG CTC C TAC GGG GC  (199)  B  104  S2-6  CTG GCG GAC TAC ACG NNC GN  (207)  B  108  E8-29  TTC AG GAG GCC TTC GGC CT  (256)  w  3.7  E8-26  GGC CGT CCG GGG ATG CTG GC  (265)  W  E8-3  GCG CTG CTG ACC GTG ATC GC  (271)  B  E8-1  ATC GCG CTG GGA ACG TTC GG  (276)  B  E8-2  GCG CTG GG ACG TTC GGC TC  (277)  B  E8-5  GAA CCC TAI GG GGI CAG GC  (291)  B  101  E8-25  AAC GGG GCA AGG CCG ATG CQ  (329)  W  4.2  E4-9  GGG TGC ACT GAT CGG GTT CC  (339)  B  137  116  E8-6  CGG GTT CCC GGG TTT GTC GC (343)  B  E4-5  TTT GTC GCC ATC ATI ATG TC  (347)  B  E4-31  GTC GCC ATC AT ATG TCC TC  (348)  B  E8-9  AGC CA G GGT ATC TGG IT  (357)  B  148  (359)  B  118  E4-6  G GGT ATC TGG ITG TTC CT  79 E4-2  TGG TTG TTC CT GIG GGC AC  (362)  B  E8-7  TTC CT GIG GGC ACC TTI GC  (364)  B  E4-27  NNA TGG GGG GCN GTG CAG GC  (401)  B  15  E4-12  GGG GCN GTG CAG GCG ACG GC  (403)  B  35  E4-30  GCC GG TG GG GTC GCG CT  (410)  B  51  E4-25  GAC GGT TTG GTT GCC CTG CC  (422)  B  86  E4-22  TTG GTT GCC CTG CCG GGG AC  (424)  B  77  E4-26  ACT TTC GGG TCC GGT GTG GQ  (431)  B  59  E4-1O  GGG TCC GGT GTG GQG GGG CC  (432)  B  49  E4-29  GTG GG GGG CCT TAT AAT AC  (435)  B  33  E4-43  ACG GTG TTC GCC ATC GAG GC  (441)  B  64  1. N represents a termination in more than one lane of the sequencing gel. 2. The sequence distal to the fusion joint is: C GGG GTA CCT GAC TCT. where the underlined sequences represent the first two codons of phoA. 3. B: blue; W: white colony on XP.  80 proteins can differ, but significantly different rates of synthesis are normally associated with toxicity of the fusion protein [671. No evidence of toxicity was seen with any of the fusions in this study. The only apparently anomalous point in the N-terminal part of the protein, the value of 30 for fusion S8-5, was assigned to the cytoplasmic side of the membrane for three reasons. First, in many experiments of this type it has been found that fusions in cytoplasmic loops that are not preceded by a basic residue seem to be exported to the periplasm at a low rate [10], possibly because a positive charge is needed to anchor the hydrophilic domain in the cytoplasm [10]. Second, the two regions flanking the hydrophilic region that contains fusion S8-5 are predicted to be the most highly hydrophobic regions of the protein, and therefore could reasonably be expected to cross the membrane. Thirdly, the high values obtained with fusions in the hydrophilic segments located on either side of this fusion indicated periplasmic locations; thus it seemed more likely that the alkaline phosphatase activity of this fusion was an abnormally high activity cytoplasmic fusion rather than a low activity periplasmic fusion, and this fusion site was assigned to the cytoplasmic side of the membrane.  The theoretical analyses allowed three possible assignments of transmembrane helices in the C-terminal part of the PucC protein. Figure 22 shows the potential membrane-spanning domains (boxes) and indicates whether the fusions, indicated below (Fig. 22D), would be predicted to have high (thick lines) or low (thin lines) alkaline phosphatase activity. The first model (Fig. 22A) has only the two hydrophobic regions, centred at amino acids 370 and 440, which were picked by all four algorithms. This possibility, and the second alternative that the four helices predicted by Eisenberg exist (Fig. 22B), were ruled out by the  81  A  II  B  IF—  r  C  F—  III  IHI  64 4.2  0  I E8-25  118 148  33 77 49 86 59  III  111111  35 15  II E8-9 E4-6  51  E4-26 E4-25 E4-27 E4-22 E4-1O E4-12 E4-29 E4-30 E4-43  -f  +  +  +  +  330  360  390  420  450  Figure 22. Possible arrangements of transmembrane helices (boxes) in the Cterminal 150 amino acids of PucC. Thin lines represent predicted cytoplasmic domains and consequently low Alkaline phosphatase (AP) activity. Thick lines represent predicted periplasmic domains which would be expected to have high AP activity. The positions of amino acid residues are indicated below the figure. A) The pair of membrane spanning helices predicted by all 4 hydropathy algorithms (see Fig. 21). B) The Eisenberg prediction. C) The Chothia prediction. D) The positions of pucC::phoA fusions in this region. The fusion designation is indicated below the line, while the relative units of AP activity are shown above.  82 high activities seen in fusions E8-9 and E4-6, the low activity of fusion E4-27 and the indirect evidence of the absence of fusions found between residues 364 and  401, despite extensive screening of blue colonies. Additional evidence against the Eisenberg prediction is the fairly high activity (51 units) seen in fusion E4-30, which this model predicted to be a cytoplasmic domain.  The four transmembrane domains predicted by the Chothia model (Fig. 22C), however, are consistent with all the data. Fusions P8-9 and P4-6 had high alkaline phosphatase activity, consistent with their predicted periplasmic location. The AP activities of fusions P4-27 to E4-25 increased steadily, as has been seen for fusions within “outgoing” transmembrane helices [471. The remaining fusions did not help to distinguish between the models, but were not inconsistent with any of them.  The results of the alkaline phosphatase fusions, taken together with the hydropathy predictions and the distribution of positively charged residues, led to the model proposed in Figure 23 for the membrane topology of the PucC protein. It has 12 membrane spanning helices, with the N- and C-termini in the cytoplasm. Cytoplasmic loops tend to be longer than periplasmic ones, and most of the protein is embedded in the membrane.  c. Sequence differences between wild type strains SB1003 and 3Th4  When Tichy et al.  [791 reported the extended sequence of the puc operon  for R. capsulatus strain 37b4 they found only a few differences between the  Figure 23. A model of the topology of the PucC protein using the single letter amino acid code. The periplasmic side of the membrane is above, the cytoplasmic side below the membrane. Positively charged amino acids are circled. The positions of fusions important in determining topology are indicated; their activities are listed in Table 3. Residues predicted for the SBIOO3 sequence which differ from the published PucC sequence from strain 37b4 are shown in lower case letters.  E8- S  84 previously published sequence of pUCBA from strain SB1003 [901 and the part of their sequence that overlapped it [79]. One of these discrepancies, an extra pair of C’s at position 833 in Tichy et al’s numbering, predicts that DNA from strain SB1003 but not 37b4 will cut with the restriction enzyme Bsm I. I found that this enzyme will not digest puc DNA from SB1003 at this site (unpublished data), so the SBIOO3 sequence probably agrees with that of 37b4 in this location. In sequencing the pucC’::pho’A fusion joints, however, a number of differences were noted between the sequence of pucC published for strain 37b4 and the sequence of the strain used here, SBI 003. The differences are marked in Table 3 as underlined bases, and have only been included if they were consistently found in at least two independent fusions. Very few of the sequence differences result in amino acid changes; those that do are listed in Table 4 and shown as lower case letters in Figure 23. Most of the changes are conservative and do not cause the hydropathy predictions to change when the revised PucC sequence is analysed. The predicted aspartic acid to glycine change at position 354, however, increases the hydropathy of this region enough that the transmembrane helix predicted by RAOARGUS (see above) at positions 355-382 is extended to 341-382 and HELIXMEM predicts an additional membrane associated helix at residues 331-351. These predictions are in better agreement with the model derived from  phoA analysis of SB1003 PucC topology than the original hydropathy analyses conducted on the 37b4 sequence. The only other nonconservative amino acid change predicted, lysine to glutamine at residue 252, does not affect the topology predictions as it occurs in a hydrophilic segment containing five additional basic residues.  85  Table 4. Amino acid differences between the predicted protein sequences of PucC from strains SBI.003 and 37b4  Residue  37b4  SBIOO3  197  valine  threonine  252  lysine  glutamine  332 354  serine aspartate  glycine glycine  361  alanine  valine  407  leucine valine  valine alanine  431  86  d. Deletion analysis of pucC  C-terminal deletions of PucC were created to determine whether truncated PucC retained function (see Materials and Methods). ALHII(pL\C) was complemented in trans with a compatible plasmid carrying the full-length  pucC gene (pPQ::C) or pucC alleles truncated near the C-terminus. Expression of pucC was driven by the oxygen-regulated puf promoter [1].  Expression of the full-length pucC gene in trans to z\LHII(pAC) restored LHII complex, although not to wild type levels (Fig. 24A, inset). The proportion of LHII to LHI complex, judged by the lack of obvious shoulder at 870 nm, were similar to that of ALHII(pBACDE) when the cells were grown under low aeration, but when ALHII(pt\C; pPQ::C) was grown photosynthetically at 100 1 the spectrum showed an unusually high proportion of LHI complex s 2 Em (Fig. 24B, inset).  None of the deleted pucC alleles, which removed one (pPQ::E4-33 and pPQ::E4-36) or two (pPQ::E4-38) of the last transmembrane domains, restored LHII complex to strain zLHII(pt\C) (Fig. 24A). In photosynthetic growth experiments the ALHII(pAC) strains complemented with the deletions did not grow faster than the vector control (Fig. 24C), although the amount of LHI complex in these strains increased compared to ALHII(pAC; pPUFP) (Fig. 24C).  87  Figure 24. Complementation of ALHII(pAC) with truncated pucC alleles. Spectra of whole cells grown under conditions of low aeration (A) or tEm (B), and the photosynthetic growth curve of 1 s photosynthetically at 100 2 these cells (C). The insets show strains z\LHII(pBACDE) (fine line) and iXLHII(pAC; pPQ::C) (heavy trace). (a) LHII(pzC; pPQ::C); (b) ALHII(pAC; pPUFP); (c) ALHll(ptC; pPQ::E4-38); (d) ALHII(pAC; pPQ::E4-36); (e) ALHII(ptC; pPQ::E4-33).  88  A  B  1.52-  2-I  a  /\  I  1-  C  d,e  a  1 0 C  b b d  C  .5-  0700 1000  -  I  I  I  800  900  1000  I  800  700  900  1000  Wavelength (nm)  C  cd  pPQ::C  z  • pPUFP .  ioo  x  -  pPQ::E4-33  z pPQ::E4-36  o  U  10  I  —  0  10  20  I  30  I  I  40 50 Time (hours)  I  I  I  60  70  80  pPQ::E4-38  89 e. Role of PucC in LHI assembly  Several observations suggested that deletion of pucC affected levels of the LHI complex as well as eliminating the LHII complex. First, the pucC deletion strain zLHII(pAC) generally had lower levels of LHI complex than ALI-llI(pRK415::2) (see Fig. 14). The pzC plasmid carries a version of pucC truncated at residue 193, midway through the fifth transmembrane domain, along with the pucBA and pUCDE genes whereas pRK415:2 has no puc sequences. The presence of the truncated pucC and/or the additional puc sequences thus leads to a lower level of expression of LHI complex than the complete absence of the puc operon. Second, when strain zXLHII(pzC) contained some plasmids that expressed pucC’::pho’A fusions in trans it was found to have different levels of LHI than ALHII(pAC) alone (see below). Different pucC alleles therefore affected the amounts of LHI complex seen.  Two questions thus arose: Does PucC participate in LHI complex assembly? Do truncated versions of PucC interfere with LHI assembly?  It seemed unlikely that PucC normally contributes to LHI complex formation because strain ALHII, deleted for the entire puc operon, appears to have high levels of the LHI complex. However it was conceivable that PucC has a small role that might be revealed in strains containing a second mutation that caused low levels of LHI complex. Therefore a pucC expression plasmid was introduced into strain ALT-ill and two derivatives which have deletions of the gene F1696. F1696 shows strong homology (47% identity) to PucC, and F1696 mutants are known to have decreased levels of LHI complex [87]. In an otherwise wild type background LHII complex levels are not affected by deletion  90 of F1696 [871. To facilitate spectral monitoring of LHI complex amounts, deletions of F1696 were introduced into the chromosome of strain ALHII [C. Young, personal communication]. These deletion strains, z\Nco and AStu, thus lack the entire puc operon and due to the F1696 disruption have only 20% of the amount of LHI complex seen in z\LHII.  LHII, zStu and tNco were complemented with plasmid pC. The only puc gene on this plasmid is pucC, and it has been shown to complement the LHII strain MW442 [7[] thought to have a point mutation in pucC (unpublished results). Transcription of pucC is driven by the puf promoter carried on a fragment which includes the pufQ gene. pufQ has been implicated in bacteriochlorophyll biosynthesis [4], so two control plasmids were also introduced into the three chromosomal backgrounds: the pRK4l5 vector and pJAJ9, which carries the same pufQ fragment as pC [29].  In Figure 25 it can be seen that neither pucC nor pufQ expression led to increased levels of LHI complex in the F1696 backgrounds tested. Small differences were seen, but were not consistent between experiments, or from strain to strain. Wild type PucC therefore does not seem to participate directly in LHI complex formation, even in the F1696 mutants AStu and ANco.  Several different pucC deletion alleles had been cloned into a vector compatible with pAC such that they would be transcribed from the puf promoter. Three of these alleles were derived from translationally out-of-frame  pucC’::pho’A fusions and had been shown to be incapable of restoring LHII  91  A  B  C  D  E  F  G  II  I  1-  0 .0  .4  700  800  900  100WOO  800  900  100WOO  800  900  1000  Wavelength (nm) Figure 25. Complementation of F1696 mutant strains with pucC. Whole cell . A) ALHII(pRK4I5) 1 s 2 spectra of strains grown photosynthetically at 120 Em E) ANco(pJAJ9) F) ANco(pC) D) ANco(pRK415) B) ALHII(pJAJ9) C) zLHII(pC) G) AStu(pRK415) H) AStu(pJAJ9) I) AStu(pC)  92 complexes to strain z\LHII(pAC) when expressed in trans (see section d). Another four alleles were translationally in-frame pucC’::pho’A fusions. Although none of these fusions restored LHII complex to ALHII(pAC) cells, I noticed that one of them, S8-1O, caused the cells to be unusually pale. Spectra of low oxygen-grown zLHII(pAC; pPQ::58-1O) had dramatically reduced LHI and RC absorption peaks (Fig. 26A). Comparison of all four of these in-frame  pucC’::pho’A fusions in trans to pzC showed that LHI absorption correlated roughly to the length of the PucC sequences remaining in the fusion. It thus seemed that these truncated pucC alleles interfered with LHI assembly and that shorter peptides had more effect than longer ones. When these fusions were conjugated into strain zLHII in the absence of pzXC, however, all the strains had amounts of LHI complex similar to the vector control (Fig. 26B). Thus expression of truncated PucC per se does not affect LHI complex formation; instead the combination of the pAC plasmid and these pucC’::pho’A fusions reduces the amount of LHI complex in cells.  Because these strains had different amounts of LHI complex they provided a direct test of whether the decreased photosynthetic growth rate of tLHII(pzC) compared to ALHII(pRK415::12) was due to relatively low levels of LHI complex. I argued above, on the basis of photosynthetic growth of tXLHII(p\C) at different light intensities, that the strain’s growth rate does not correlate to its amount of LHI complex (see Results, Ch. 1, section e). When z\LHII(pAC) was complemented with the translationally in-frame pucC’::pho’A fusions and grown photosynthetically at 100 Em 1 the strains showed a gradient of LHI s 2 complex levels (Fig. 27A) similar to that seen with cells from low oxygen cultures (see Fig. 26A). Their photosynthetic growth rates did not correlate to their  93  Figure 26. Whole cell spectra of low oxygen grown strains containing translationally in-frame fusions of pucC’ to pho’A. Fusions pPQ::S8-1O (c), pPQ::E8-5 (d), pPQ::E8-25 (e), pPQ::E4-25 (f), and plasmids pPQ::C (a) and pPUFP (b) were expressed in ALHII(pAC) (A) and ALHII (B).  400  A  600  a  1000  700  Wavelength (nm)  b, f  B  800  f  900  d  b  e  C  95 relative levels of complex, however (Fig. 27B). Strains tXLHII(pAC; pPUFP), ALHII(pAC; pPQ::E8-25) and ALHII(pz\C; pPQ::E4-25) grew at similar rates and the only strain which might have had a slightly faster growth rate, strain ALHII(pAC; pPQ::E8-5), had approximately 50% of the LHI complex levels seen in LHII(piC; pPUFP). The exception to this lack of correlation of photosythetic growth rate to amount of LHI complex was ALHII(pzC; pPQ::S8-10) which had only 12% of the amount of LHI and RC complexes seen in zLHII(pAC; pPUFP) under low oxygen growth conditions (see section d). This strain did not grow at all photosynthetically, presumably due to its low RC content.  f. Conclusions  Theoretical models for the topology of PucC were derived from four hydropathy analyses combined with the restrictions of von Heijne’s positive inside rule (Fig. 21). Three of the four hydropathy algorithms predicted 11 similar transmembrane segments. The fourth hydropathy scale, that of Kyte and Doolittle, was less useful. It predicted only 9 of the 12 membrane-spanning segments that were consistant with the phoA fusion analysis.  The C-terminal region of PucC was difficult to analyse, being largely hydrophobic without strongly hydrophilic intervening segments. Three possible arrangements of 2 or 4 transmembrane helices could not be resolved without experimentation.  The consensus of these analyses predicted 10 or, more probably, 12  96  Figure 27. Strain ALHII(pAC) containing translationally in-frame fusions of  pucC’ to pho’A grown photosynthetically at 100 2 tEm A) Whole cell . 1 s spectra: pPQ::E8-5 (d), pPQ::E8-25 (e), pPQ::E4-25 (f), pPQ::C (a) and pPUFP (b) B) Photosynthetic growth (see legend for symbols).  97  1.5-  A  a  e  1b 0 d .5-  0400  600  800  1000  Wavelength (nm) B  cr  100  LJ  pPQ::C  •  pPUFP  0 pPQ::E8-5 L  pPQ::E8-25  •  pPQ::E4-25 pPQ::S8-1O  10  I  0  10  I  I  20  30  I  I  40 50 Time (hours)  I  I  60  70  80  98 transmembrane domains, with both the N- and C-termini located in the cytoplasm. The theoretical models were tested experimentally using the genetic system of translationally in-frame phoA fusions.  Alkaline phosphatase, the product of the phoA gene, can be used as a reporter gene in topology analyses because it has greater activity when it is transported past the cytoplasmic membrane than if it remains within the cell. Forty-four random, translationally in-frame fusions were made between the 5’ end of pucC and a phoA allele whose N-terminus and signal peptide had been deleted. Alkaline phosphatase activities of some of the fusions were assayed and the resulting pattern of high or low activity (Table 3) was compared to the theoretical models (Fig. 21). High activity correlated well with fusion joints located in regions predicted to be periplasmic. Predicted cytoplasmic loops were sites of fusions having approximately 20-fold lower alkaline phosphatase activity. The only anomalous fusion occured between the fourth and fifth predicted transmembrane domains, and had an intermediate activity. This region was assigned to the cytoplasm for two reasons. First, the theoretical analysis predicted that the two flanking segments were transmembrane domains as they were strongly hydrophobic, and the hydrophilic regions flanking them both contained high activity alkaline phosphatase fusions. It was thus reasonable that they might cross the membrane and that the intervening segment would be in the cytoplasm. The second justification for this arrangement is that the anomalous fusion occurs in a region that is not preceded by any basic residues. In the many phoA analyses that have been carried out it has been noted that cytoplasmic fusions that are not anchored by positively charged amino acids are exported to the periplasm at a low rate and thus have  99 unusually high activities compared to other cytoplasmic fusions.  The arrangement of transmembrane domains in the C-terminal region of PucC was the most ambiguous part of the theoretical analysis. Testing of  pucC’::pho’A fusions in this region made it possible to distinguish which of three theoretically plausible models was most likely to be correct (Fig. 22). The models predicted different patterns of high and low alkaline phosphatase activity, only one of which was consistent with the observed data.  On the basis of the theoretical analysis and the information provided by activities of pucC’::pho’A fusions a model for PucC topology was proposed in which there are 12 membrane-spanning domains and both the N- and C-termini are located in the cytoplasm (Fig. 23). In the course of characterising the fusion sites, DNA sequence differences, some of which result in amino acid changes (Table 4), were noted between the strain used here and that reported in the literature (Table 3). The theoretical and experimental data were in even better agreement when the theoretical analysis was repeated for this slightly different amino acid sequence.  Expression of pucC in trans to ALHII(pAC) restored the LHII complex although to less than the wild type level (Fig. 24). Three pucC deletion mutants, missing 1 or 2 of the C-terminal transmembrane helices, did not restore detectable amounts of the LI-ill complex to this strain. The C-terminal region of PucC is thus required either directly for function, or for proper folding or stability (and thus indirectly for function). Although these deletion mutants were unable to restore the LHII complex to ALHII(pAC), they did increase the amount of the  100 LHI complex seen when the cells were grown photosynthetically (Fig. 24B).  A second set of pucC’ alleles, translationally in-frame pucC’::pho’A fusions, also influenced LHI complex levels in this strain (Fig. 26). One such fusion, containing only the N-terminal PucC transmembrane domain, decreased RC and LHI complex levels to 12% of strain tLHII(pzC; pPUFP). These fusions did not produce this effect in the absence of pAC, however, nor did expression of  pucC alone increase LHI complex levels in ALHII or in F1696 mutant derivatives which have an 80% reduction in LHI complex levels (Fig. 25). It was therefore concluded that PucC does not directly participate in LHI complex formation and that truncated pucC alleles do not interfere with LHI complex assembly in the absence of the other puc genes carried on pzC.  101 Chapter 3: Analysis of puc operon transcription  a. puc operon promoter mapping by deletion analysis of pucE’::lac’Z gene fusions.  Although previous work had shown that the pucC gene is probably transcribed from the pucBA promoter [79; 78], it was not clear where transcription of the pucDE genes originates. Therefore, to test for the existence and positions of other possible puc operon promoters, translationally in-frame fusions of the pucE gene were made to a E. coli lac ‘Z allele (Fig. 28) in the promoter-probe plasmid pXCA6O1 [1]. /3-galactosidase activities were measured in cell extracts from R. capsulatus strain SBIOO3 containing the different pucE ‘::lac ‘Z fusions, grown under either high or low aeration (see Materials and Methods). Figure 28 shows that approximately 90% of transcription of the pucE gene derived from transcription initiated upstream of pucB. The remaining 10% of J3-galactosidase activity was lost when the region between the 3’ end of the pucC gene and the 5’ end of the pucD gene was deleted.  Comparison of /3-galactosidase specific activities between SB1003(pPEZ) cells grown under high and low aeration shows that expression of this pucE’::lac’Z fusion was approximately two-fold higher in cultures that were oxygen-starved. This is in good agreement with the results obtained using a pucB ‘::lac ‘Z fusion in cells grown under the same conditions [24].  ‘I  pPEZ -OOF pCEZ  -  D.ZEJ  pBEZ-OOF 0.49 (.02)  8.3 (1.o)*  8.5 (.62)  6.7 (.51)  83 (21)*  100 (8.5)  0.16 (.07)  4.2 (.36)  6.4 (.26)  57 (5.)  High 02  Figure 28. Specific activities of fl-galactosidase in cell extracts from wild type R. capsulatus strain SB1003 containing plasmids with a pucE ‘::lac ‘Z fusion preceded by different amounts of upstream sequences, as shown. Cells were grown under conditions of low (Low 02) or high (High 02) aeration. Activities are expressed as percentages of the activity of SB1003(pPEZ) measured in the same experiment. Values are the averages of 3 assays and standard deviations are given within brackets. *These values were measured using permeabilised cells (see Materials and Methods). The inverted triangle represents insertion of 4 nucleotides that cause a frameshift in the pucD reading frame, boxes represent puc genes, designated above pPEZ and the predicted RNA stem loop structure is indicated with a lollipop.  pHEZ  ‘‘  I I  U-’  I  pBEZ  I  D E’  II  —  )-‘  C  ii  0  I  i.U  —  i—I  .-----  pPEZ -  T  0 BA  Low 02  Relative activity  C  103 b. Is there translational coupling between pucD and pucE?  /3-galactosidase activity decreased approximately 10 fold when the region between the 3’ end of pucC and the 5’ end of pucD was deleted (pBEZ vs. pHEZ in Fig. 28). Normally this decrease in activity would be interpreted as evidence for minor promoter activity in the deleted fragment. In this particular case, however, an alternative explanation was possible. The sequences of pucD and pucE overlap by four nucleotides so the pucE start codon (ATG) is buried in the pucD stop codon (TGA)  [791. In other systems with similar overlapping open  reading frames it has been shown that translational coupling exists between the two genes, such that translation of the first gene promotes translation of the second one [reviewed in 261. The reduced ,i3-galactosidase activity of pHEZ could therefore have been due to decreased translation of the fusion protein in the absence of pucD translation, with transcription originating from vector sequences. Alternatively, the decrease in /3-galactosidase activity in pHEZ relative to pBEZ could have been due to reduced transcription after deletion of a minor promoter located between pucC and pucD.  To test whether translational coupling between pucD and pucE contributed significantly to expression of the pucE ‘::lac ‘Z fusion a frameshift mutation was introduced at the 5’ end of pucD which resulted in early termination of translation (see Materials and Methods). The mutation was transferred into pBEZ (pBEZ-OOF) and pPEZ (pPEZ-OOF), and /3-galactosidase activities were measured (Fig. 28). In both constructs /3-galactosidase activity decreased modestly (by 17% in pPEZ-OOF, not statistically significant) compared to the equivalent unmutated plasmid, but pBEZ-OOF activity was not decreased  104  to the level seen in pHEZ.  c. RNA blot analysis of the puc operon  Because pucE ‘::lac ‘Z fusions showed that 90% of transcription of the pucE gene originates upstream of the pucB gene, RNA blot analyses were performed [41] to determine whether RNA species long enough to encode all five of the puc genes could be detected in addition to the previously characterized  Ca,  550 nt pucBA messages  [931. When RNA from the wild type strain SB1003 was  probed with a 2.0 kb fragment extending from the Cia I site in pucB to the BspH I site in pucE (probe 1 in Fig. 29A), a 0.5 kb message was detected along with two other less abundant species (Fig. 29B, lane 1). These two weaker signals corresponded to sizes of 2.4 kb and approximately 1.0 kb. (The faint bands of approximately 1.5 kb visible in lanes 1, 3 and 4 are artifacts caused by rRNA bands above and below this position that interfere with hybridization.) By scanning the autoradiogram on a densitometer, the relative amounts of the 0.5 kb: 1.0 kb : 2.4 kb species were estimated as 35 : 3: 1.  The 2.4 kb species described above was large enough to encode all five puc genes. In order to evaluate whether it might, and to identify the sequences giving rise to the smaller species, further RNA blot analyses were carried out using probes specific for smaller segments of the puc region (Fig. 29A). A probe specific for the pucBA genes detected only the largest (2.4 kb) and smallest (0.5 kb) bands (Fig. 29B, lane 3). A probe specific for the pucDE region detected bands comigrating with the 1.0 and 2.4 kb species, as well as a previously undetected  105  A  1kb  ER  2  3  4  B 1  2  3  4  5  9.5... 7.5— 4.4—  I-  2.4— 1.35—  0.24—  IL  Figure 29. RNA blot analysis of puc operon transcripts. A) DNA probes used. Hatched boxes represent puc genes, potential mRNA stem ioop structures are marked with loops. P: Pst I; ER: EcoR I. B) Autoradiograms of hybridized blots of RNA isolated from SB1003 (lanes 1, 3-5) and ALT-ill (lane 2) cells grown under low aeration (see Materials and Methods). Lanes I and 2 were probed with the 2.2 kb Cia I BspH I fragment (probe 1), which contains the pucBACDE genes; lane 3 with a 435 nt Ban II fragment (pucBA, probe 2); lane 4 with the 1.1 kb Ban II fragment (pucC, probe 3); and lane 5 with the 635 nt Bc! I fragment -  (pucDE, probe 4). The approximate positions of RNA size markers (kb) are indicated to the left of the blots.  106 message that appeared to be about 0.7 kb (Fig. 29B, lane 5). When a probe containing only the pucC region was used, a signal comigrating with the 1.0 kb band detected by the pUCDE probe was detected, along with a fainter 2.4 kb signal (Fig. 29B, lane 4). A smear extending down from the 1.0 kb band ended approximately where the 0.5 kb signal was found with the pucBA-containing probes.  When RNA from the L\LHII strain was probed as above no bands were detected (Fig. 29B, lane 2), indicating that none of the signals resulted from crosshybridization to non-puc operon derived messages, as was reported with R. sphaeroides  [431.  The mRNA species of the pucCDE deletion strains described in Results, Chapter 1 were examined in a second RNA blot experiment (Fig. 30). Equal amounts of RNA from ALHII complemented with pRK415::2 (lane 1), pBACDE (lane 2), pACDE (lane 3), pAC (lane 4), pAD (lane 5) and pAE (lane 6) were analysed. The same membrane was hybridised sequentially with the probes shown in Figure 30A. As above, in no case was any signal detected in the lane containing RNA from ALHII(pRK415::12).  Hybridisation of the pseudo-wild type strain (lane 2) gave results similar to those described for SB1003. Because the same membrane was probed each time, it was possible to compare the migration of the species detected with each probe directly. It became apparent that the smaller (“0.7 kb”; see above) message detected with the probe to the pucDE region was not bigger than the 0.5 kb pucBA message.  107 e  A  b  pEACDE  c  II  1kb d  ____  piCDE  i  I  --  p\C  Th  pAE  11111  --  B  ___  D  C  E  123456123456123456123456  2.4  q  *4  *  1.4  1’•.  I  .24  Figure 30. RNA blot of puc deletion strains. Equal amounts of RNA harvested from cells grown under low oxygen conditions were run in each lane and transferred to nylon membrane electrophoretically. The same blot was repeatedly probed then stripped for reprobing as described in Materials and Methods. A) Diagrammatic representation of the DNA fragments used to make probes (b-e) and the puc gene deletion plasmids of the strains tested (plasmid pRK4l5:2, not shown, had no puc sequences). B-E) Autoradiograms of RNA blots of strains ALHII(pRK4I5) (lane 1), ALHII(pBACDE) (lane 2), zXLFIII(pACDE) (lane 3), ALHII(pAC) (lane 4), ALHII(pAD) (lane 5), and ALHII(pAE) (lane 6). The probe used in (B) was the 435 nt Ban II fragment (b). Panel (C) was probed with the 0.95 kb Xmn I-ApaL I fragment (c). A 586 nt Xmn I fragment (d) was used as the probe in panel (D) and panel (E) was probed with the 2.3 kb Cia I to Eco47 III fragment (e). Positions of RNA size markers (kb) are shown to the left of the blots. Symbols indicate RNA species discussed in the text.  108 The probe to the pucBA region (probe b) was expected to hybridise to the 0.5 kb pucBA mRNA (white circle) and full-length puc transcripts (star) from each construct (Panel B). Transcription of the puc sequences present on pzCDE would yield a 1 kb RNA, pAC primary transcripts would be approximately 1.8 kb and ptD and pAE transcripts would each be approximately 2.2 kb. A signal migrating to greater than 0.5 kb was detected only in the pseudo-wild type strain (lane 2), however. The intensity of the 0.5 kb signal was the same in all five strains that contained puc sequences.  The only discrete band detected by the probe carrying pucC sequences (probe c, panel C) was the wild type primary transcript in lane 2 (*). Weak smeary signals beginning at approximately 1.5 kb were present in all the lanes except lane 1 and, as in Figure 28, rRNA running at approximately 1.5 and 1.2 kb interfered with hybridisation, creating the appearance of faint bands at approximately 1.4 kb. Within the smear in lane 2 there was a region of more intense hybridisation at approximately 1.0 kb (black dot) which coincided with the 1.0 kb band detected by the pucDE probe (see below). This RNA might therefore overlap slightly with the 3’ end of probe c to give a weak signal. In the first RNA blot experiment described above the 3’ end of the fragment used to probe for pucC transcripts (probe 3, Fig. 29) was located 280 bp from the 3’ end of the pucC open reading frame and a relatively strong signal was detected running at 1.0 kb. The 3’ end of probe c is located 100 bp further upstream. The weakness of the signal seen using this probe suggested that the 5’ end of this 1.0 kb message is approximately 400 bp upstream from the 3’ end of the pucC gene. If this were the case, corresponding bands of hybridisation would be expected at 0.7 kb in the lanes containing RNA from ALHII(pAD) and ALHII(pz\E), both of which contain 250 bp  109 deletions in regions shown to hybridise with this message. In fact, very weak signals were seen in this position in lanes 5 and 6, but not in the ALHU(pAC) lane (Fig. 30C, lane 4). All of these weak signals aligned perfectly with the strong bands detected by probe d when the original autoradiograms were overlaid.  As expected, probe d, derived from pucDE sequences, did not detect any complementary species in RNA isolated from strain ALHII(pACDE) (Fig. 30D, lane 3). RNA species of 2.4 kb (*), 0.5 kb (+) and a strong 1.0 kb band  (•) were  detected in the lane containing the pseudo-wild type RNA (lane 2). As described above, the 5’ end of this 1.0 kb RNA molecule probably contains sequences from the last 400 bp of pucC. Interestingly, a band of the same size was detected in lane 4, which contained RNA from ALHII(pAC). The size of this band did not seem to be affected by the deletion of the pucC sequences at the presumed 5’ end of this message. The results using pucC-specific probes had localised the 5’ end of this message (from wild type templates) quite specifically, and its 3’ end mapped to regions which should not have been affected by the deletion in pAC, so it seems likely that the 5’ end of the (still) 1.0 kb message was derived from sequences upstream of the deleted sequences which fortuitously resulted in a message whose size is similar to the wild type species. If this is the case, probe  C,  described above, would also be expected to detect this altered RNA. Reexamination of lane 4, panel C did not confirm or contradict this hypothesis. There was a weak signal which could be overlaid with the strong band in the same lane, panel D, but it was not convincingly stronger than the signal detected in the rest of the smear.  In RNA from both ALHII(pAD) and ALHII(pAE) (lanes 5 and 6) the only band  110 detected by probe d migrated at approximately 0.7 kb (open circle). This corresponds to the size of the wild type 1.0 kb molecule less the 0.25 kb deleted from each of these plasmids. The intensity of the band in lane 6 was somewhat lower than in lane 5, although the extent of sequences complementary to the probe was expected to be similar in both cases. This message may therefore be less abundant in strain zLHII(piE), presumably because the sequence removed or the sequence created by the deletion destabilised the molecule. The faint band of approximately 0.5 kb that was detected in lanes 2 and 4 was absent in RNA from zLHII(pAD) and ALHII(pt\E). This band did not appear when the blot was hybridised with the pucC-specific probe c, so it seems likely that this 0.5 kb message contains pucDE sequences.  A probe containing sequences from all five puc genes confirmed the results seen in the other RNA blots described (Fig. 30E). All the RNA species detected by region-specific probes were seen. The intensity of the approximately 0.5 kb band was less in strain ALHII(pAD) than in ALHTI(pzE). This signal would result from the combination of the signals of the pucBA 0.5 kb message (white circle) and the pucDE-specific 1.0 kb band which migrates at 0.7 kb (open circle) in these deletion strains. The 0.5 kb pucBA transcript was equally abundant in both strains (Fig. 30B) and the 0.7 kb pucDE band seemed more intense in the ALHII(pAD) lane than in ALHII(pAE) (Fig. 30D), so it initially seemed strange that the intensities of the signals in the 0.5 kb region were reversed when probed with probe e. The 3’ end of this probe extends only as far as the Eco47 III site 80 nt into pucE, however, so the entire sequence of this transcript from pAE would be homologous to probe e whereas a much smaller part of a transcript from the pUCDE region of pAD would overlap with the probe. Therefore, the signal for  111 the 0.7 kb RNA in ALHII(pzD) should be lower than in ALHII(pAE) using probe e.  d Conclusions  The promoter localisation studies using the pucE’::lac’Z fusions described here determined that the majority of pucE transcription originates from a promoter upstream of pucB. As shown in Figure 28, deletion of these sequences led to a ten-fold decrease in 13-galactosidase activity. No promoter activity was associated with the sequences between pucB and the 3’ end of pucC. However, when the interval containing the 3’ end of pucC and the 5’ end of pucD was deleted the relative level of f3-galactosidase activity was further reduced to less than I % of the maximum expression.  Two explanations for this additional decrease in reporter gene activity seemed possible. The first was that a minor promoter was located in the interval deleted and that its removal eliminated residual transcription of the pucE’::lac’Z fusion. The second possibility was suggested by the fact that the pucD and pucE open reading frames overlap by four nucleotides, a situation in which translational coupling could be important for efficient pucE translation. In this second case the 10% residual transcription would have to originate from vector sequences, and the observed drop in /3-galactosidase expression would be explained by decreased translation of the pucE’::lac’Z fusion message due to loss of pucD translation. The contribution of translational coupling to the level of expression of the pucE’::lac’Z fusion was tested by introducing a frameshift mutation within pucD that led to early termination of pucD translation. The  112 relative 13-galactosidase activities of pucE’::lac’Z fusions whose 5’ sequences either included the major promoter activity or extended only to the 3’ end of pucC were compared, with and without the frame-shift mutation. In the case of  pPEZ-OOF a modest but not statistically significant decrease in activity resulted from interruption of pucD translation. It is thus possible that translational coupling between pucD and pucE contributes a little to the level of pucE expression, but the decrease in /3-galactosidase activity in pHEZ is primarily due to deletion of a minor promoter located in the region between the 3’ end of pucC and the middle of pucD.  RNA blot analyses (Figs. 29 and 30) determined that there were several  RNA species encoding puc sequences. A 2.4 kb RNA molecule was the longest species detected in wild type and pseudo-wild type RNA preparations by probes specific to the pucBA, pucC and pucDE regions. I propose that this message extends from the major puc promoter region to the inverted repeat located just beyond the 3’ end of the pucE gene, and is thus the primary transcript of the puc operon. It was of relatively low abundance compared to the other signals  detected, which suggests that it is relatively unstable, and one of the other RNA species detected, the 1.0 kb message hybridising to pucDE sequences, can only be explained as a processing product of this primary transcript (see below). No analogous long transcripts were detected in any of the strains deleted for one or more of the pucCDE genes.  The most intense signal was generated by a message migrating at 0.5 kb and hybridising to pucBA-specific probes. This transcript is the previously characterised 0.55 kb pucBA mRNA [93]. Its relative abundance, judged by the  113 intensity of the signal, was the same in pseudo-wild type and pucCDE deleted strains. This is in contrast to a previous report based on dot blot analysis which claimed that interruption of pucC by a transposon caused a decrease in the level of pucBA mRNA [781.  Two signals were detected by probes carrying pucDE sequences. The less abundant of the two migrated to approximately 0.5 kb in extracts from pseudowild type cells, and could potentially represent a transcript originating from the minor promoter detected by pucE’::lac’Z fusion analysis. It is probably too small to encode both pucD and pucE, however, This transcript was unaffected by deletion of the 3’ pucC sequences but was not present in either the pucD or pucE deleted strains. It may therefore be unstable in these two strains, or it  might not be produced from a larger precursor if processing sequences in the pUCDE region were deleted, or it may primarily derive from sequences removed  in one of the deletions.  The final discrete signal seen in wild type strains was a message migrating to 1.0 kb and hybridising strongly to pucDE-specific probes. A band of this size was also detected by a probe whose 3’ end lay 280 bp upstream of the pucC stop codon (probe 3, Fig. 29), whereas a similar probe with a 3’ end 380 bp from the end of pucC hybridised very weakly at this position (probe c, Fig. 30). The 5’ end of  this 1.0 kb species thus seems to be delimited by these two probes, and appears to lie within the pucC coding region, approximately 400 bp upstream of the 3’ end of the gene. This position is well upstream of the region of the minor promoter activity detected by lacZ fusion analysis, so this molecule cannot be a product of this second promoter. Since no promoter activity was associated with the pucC  114 sequences, this 1.0 kb molecule is presumably a processing product of the 2.4 kb primary transcript. This signal was shifted to 0.7 kb in the strains deleted for pucD or pucE. This faster migration corresponds to the sizes of both deletions  and suggests that the 1.0 kb molecule encodes both genes. The size of the 1.0 kb message may be underestimated since its apparent size does not quite correspond to the distance between its 5’ end, determined by the pucC-specific probes, and the 3’ end of the pucE gene.  115  DISCUSSION  a. Transcriptional organisation of the puc operon  RNA blot analyses and promoter mapping by fusion of puc’E to lacZ’ led to the model shown in Figure 31 for the transcriptional organisation of the puc operon.  pucE’::lac’Z fusion analysis (Fig. 28) showed that the majority of pucE transcription originated from sequences upstream of pucB, and that this transcription showed the same regulation by oxygen as a similar pucB’::lac’Z fusion [93]. The same promoter (large bent arrow in Fig. 31) therefore seems to drive transcription of pUCBA and the genes distal to pucA. Consistent with this proposal, an RNA molecule long enough to encode all five puc genes was detected migrating at the same position by probes specific to each of the pucBA, pucC and pucDE regions (Figs. 29 and 30). This 2.4 kb transcript (fine black arrow in Fig. 31) was much less abundant than the 0.55 kb pucBA mRNA (thick grey arrow) that had previously been characterised [931 (Fig. 29B, lane 3).  There are two mechanisms that could result in an excess of the shorter message over the longer. They are not mutually exclusive and could both contribute to the steady-state levels of the two transcripts. The first is that the stem loop structure located downstream of pucA could act as an inefficient terminator of transcription, so that some transcription events produce a 0.55 kb molecule whereas others read through the stem ioop and produce the longer 2.4 kb transcript. Chen et a!. [13] measured rates of transcriptional termination  116  Figure 31. Transcriptional organisation of the puc operon. Coding sequences are shown as boxes with their gene designation below. Inverted repeats are indicated by stem-loops, and promoters are shown as bent arrows whose relative size indicates promoter strength. Putative primary transcripts are shown as black arrows, while messages likely to derive from processing of primary transcript(s) are shown in grey. The relative abundance of these RNA molecules is indicated by the thickness of the arrows.  117 when a hairpin structure based on the puc sequence from strain SBIOO3 [901, but modified at three residues to result in perfect dyad symmetry, was inserted within the puf operon. They found the amount of transcriptional readthrough depended on the sequence immediately downstream of the hairpin. The dyad symmetry element followed by ACCG caused very little termination, whereas when it was inserted in front of TTTT a very efficient transcriptional terminator was created. This finding is consistant with current models in E. coli that Rho independant termination of RNA synthesis occurs at CC-rich hairpins that are followed by a series of U residues [861. In the puc operon the imperfect inverted repeat is followed by ATTC, so it could act as a transcriptional attenuator. The reported sequence of the R. capsulatus puc operon from strain 37b4  [791 predicts  a stem 3 base pairs longer than that of strain SB1003, the wild type strain used in this thesis. However, the SBIOO3 sequence predicts a restriction enzyme site within this stem region which I have found does not exist (unpublished data), so the published sequence for SBIOO3 may be in error and the stem ioop may be more stable than previously thought. The relative abundance of the 1.0 kb RNA molecule that is a processing product of the 2.4 kb puc transcript (see below) shows that the degree of transcriptional readthrough at this site must be greater than the amount of the 2.4 kb message would seem to indicate. Site-directed mutagenesis of this inverted repeat in the pucE’::lac’Z fusion pPEZ could determine whether attenuation is occuring.  The second mechanism that could lead to an abundance of the 0.55 kb pucBA transcript over the 2.4 kb full-length puc transcript is differential stability of the two molecules. Like the 0.5 kb pufBA mRNA, the 0.55 kb pucBA transcript is very stable, having a half life of 20 minutes [93]. The stability of the  118 2.4 kb puc mRNA has not been measured in R. capsulatus, but in R. sphaeroides a 2.3 kb RNA molecule now known to encode pucBAC was found to have a half-life of less than 5 minutes [43]. My results indicate that processing of the R. capsulatus 2.4 kb puc transcript does occur (see below).  The stoichiometry of the R. capsulatus puf operon 0.5 and 2.7 kb messages has been shown to be due to differential stability of the messages [6], due in part to protection of the 3’ end of the 0.5 kb transcript from exonucleolytic degradation by a stem-loop structure [13]. As discussed above, a similar hairpin exists downstream of pucA, a modified version of which has been shown to protect pufBA transcripts from decay [13]. The instability of the 2.7 kb puf transcript has been shown to be caused by endonucleolytic cleavage at sites within pufLMX [reviewed in 35]. Unlike puc RNA (see below), there are no stable processing products from the 3’ end of the 2.7 kb puf transcript [35].  Minor promoter activity was detected in the region between the 3’ end of pucC and the middle of pucD. I have indicated this second promoter within pucD for the reasons described below, but it could be elsewhere in this region. No other promoters have been characterised within photosynthetic operons, but elaborate superoperons have been characterised at both ends of the photosynthetic gene cluster (Fig. 32) [83]. The overlap of the crtEF and bchCXYZ operons with the puf operon has been shown to reduce the lag phase of growth when uninduced cells are shifted to photosynthetic conditions [84], as has the overlap in the puhA region  [31. The physiological significance of the minor  puc promoter, if any, is unclear, although it may help in stoichiometric production of the LHII y subunit (PucE) and the a and /3 peptides.  H  bch K  BF  I  bchC X citE F  V  -  E.1  Z pufQ BA L MX  Figure 32. Superoperonal organisation of the photosynthetic gene cluster of R. capsulatus. Primary transcripts of the operons indicated are shown by solid arrows beginning at the promoter for the operons indicated. Genes for structural peptides of the RC and LI-il complexes are indicated by hatched boxes; shaded boxes indicate genes encoding bacteriochlorophyll biosynthetic enzymes; carotenoid biosynthesis genes are dotted; cross-hatching indicates genes of unknown function.  puhA F1696ML  120 In addition to the 2.4 kb puc transcript, two smaller RNA molecules were detected by probes to the pUCDE region in RNA blot analyses (Fig. 29B, lane 4 and Fig. 30D, lane 1). An inverted repeat downstream of pucE is followed by the sequence TTTTATTT, and as discussed above an inverted repeat followed by 4 T’s has been shown to efficiently terminate transcription in R. capsulatus [13]. It thus seems likely that puc transcription does not proceed beyond these sequences, and reasonable that messages ending in this region would have a stem-loop formed by these sequences at their 3’ ends. The 0.5 kb RNA molecule detected by pucDE probes (short black arrow, Fig. 31) would therefore have its 5’ end within the pucD gene, encode only pucE and could be the transcription product of the minor promoter described above. This molecule was not affected by deletion of pucC, but was absent from strains deleted for either pucD or pucE, which is consistent with the location I have suggested: deletion of pucD could remove the minor promoter, and the deletion of pucE would eliminate most of the sequences transcribed. If this is the cause of the lack of this molecule in ALHII(pAD), the minor promoter would be located between the Bcl I and Hinc II sites in pucD.  The larger of the two messages detected by pucDE probes was approximately 1.0 kb, and has also been reported by Tichy et at.  [791 (longer grey arrow in Fig.  31). The 5’ end of this molecule was mapped fairly accurately because it was detected by a probe with a 3’ end at the Ban II site 280 bp upstream of the 3’ end of pucC (Fig. 29B, lane 4), but only weakly detected by a similar probe with a 3’ end at the ApaL I site 380 bp from the 3’ end of the pucC gene (Fig. 30C, lane 2). The 5’ end of this 1.0 kb molecule therefore probably maps approximately 400 bp upstream of the 3’ end of the pucC gene. This is well upstream of the minor  121 promoter defined by the pucE’::lac’Z fusion deletion analysis, in a segment not associated with any detectable promoter activity. This transcript must therefore be a processing product of the primary transcript. Results of RNA blot analysis of the strains deleted for pucD and pucE showed that the mobility of this message increased by the size of each of these deletions, indicating that the 1.0 kb molecule encodes both pucD and pucE.  Given that the 5’ end of the 1.0 kb message maps within pucC, it appeared contradictory that an RNA species of the same mobility was detected in the strain containing the pucC deletion (Fig. 30D, lane 4), which should remove approximately 350 bp from the 1.0 kb molecule. The pucC coding sequences presumably contain endonuclease processing sites, however, some of which might have been removed or altered in making the deletion. It is therefore possible that the primary transcript from the pucC deletion strain is processed differently from the wild type puc transcript, and that it fortuitously produces a molecule of a similar size. It is clear that the primary transcript of the pzC deletion is rapidly degraded since no message of the appropriate size (1.8 kb) was detected with the pucBA probe (Fig. 30B, lane 4). In fact, full-length transcripts encoding the sequences remaining on plasmids pACDE, pAD and pAE were not detected in the strains harbouring these plasmids, either. I can only conclude that each of these deletions destabilised the primary transcript, either by removal of stability determinants, and/or by creation of endonuclease cleavage sites.  The only RNA molecule detected which could encode pucC was the low abundance 2.4 kb full-length puc transcript (Fig. 29B, lane 4). PucC is required for formation of the LHII complex (see below), and the fact that it is encoded on  122 such a low abundance transaipt suggests that its role is catalytic rather than as a structural component of the complex. Additionally, the pucD deletion strain has wild type levels of the LHII complex, but no detectable RNA molecule which could encode pucC (Fig. 30B, lane 5). This implies that very little pucC product is required for assembly of LHII complex.  Mutation of pucC has been reported to result in a reduced level of pucBA mRNA [781. This conclusion was based on dot blot analysis of a mutant (strain NK3) containing a transposon insertion in pucC, and was interpreted as evidence that pucC is required for high level transcription of puc mRNA. In Figure 30B it can be seen that all the strains tested (with the trivial exception of ALHII(pRK415::2)) had signals of similar intensity for the pucBA message. Thus I do not believe that loss of of pucC expression (or of pucD and pucE, which could also have been affected by the transposon in NK3) results in a decrease of pucBA mRNA levels. Rather, as discussed below, I think the PucC protein acts post-transcriptionally, probably in assembly of the LHII complex.  b. Deletion of chromosomal pucBACDE genes in strain ALHII.  I deleted almost the entire pucBACDE region, and demonstrated that all of the genes downstream of pucBA are transcribed primarily from a promoter upstream of pucB (see above). Previous studies of the expression of the pucCDE genes in R. capsulatus drew conclusions from studies of a mutant with a transposon inserted in the pucC gene (strain NK3), or a deletion of only the pucB, pucA and part of the pucC genes (strain U72) [78,  791. The interpretations  123 of these previous experiments were complicated by the likely polar effects of the transposon insertion and the possibility of a strong promoter located between the pucC and pucD genes that could allow expression of the pucD and pucE genes in the mutants NK3 or U72 [78]. Construction of the chromosomal puc deletion strain ALHII allowed me to unambiguously evaluate the phenotypes of pucC, pucD and pucE mutants by single gene deletions (see below).  Tichy et at. [78] reported that deletion of the entire puc region was lethal, whereas I found that deletion of the pucBACDE genes was not lethal or generally deleterious to cell growth. Photosynthetic growth of the deletion strain ALHII(pRK415::Q) was similar to that of wild type cells until the incident light level fell below 60 tEm 2 s (Fig. 16) and aerobic growth was not affected by the deletion (data not shown). Strain ALHII, lacking the LHII complex, has therefore been useful as a background strain for studies monitoring LHI and RC complex levels [C. Young, D. Wong, B. Collins, J.T. Beatty, personal communications] which would formerly have been conducted in relatively uncharacterised LHII mutants. As discussed below, some of these backgrounds may have unexpected pleiotropic effects on LHI complex expression.  c. The phenotypes of pucD and pucE mutants  The deletion analysis described in this dissertation shows that two of the three genes downstream of pucBA are required to obtain normal levels of the LHII complex. One of these genes, pucE, encodes the 14 kD y subunit that co purifies with the pigment binding proteins of the LHII complex [79], and its deletion resulted in decreased LHII complex levels. Strain ALHII(pAE)  124 synthesized approximately 60% of the wild type levels of the LHII complex (Fig. 14F), and the absorption of the 800 nm peak seemed to be disproportionately low relative to that at 850 nm. The amount of the complex, and the relative sizes of the 800 and 850 nm peaks, varied with growth conditions and age of the culture. These results are consisent with those of Tichy et a!. [78] who showed that deletion of pucE decreases the in vitro stability of the LHII complex in cell-free chromatophore membrane preparations. The pucE gene is not absolutely required for wild type levels of LHII complex, however, since the secondary suppressor strain ALHII—2(pACDE) has high levels of the complex in the absence of pucE (Fig. 19C).  Despite retaining some LHII complex, strain ALHII(pAE) grew more slowly in photosynthetic conditions than LHII(pRK415::2), which had no LHII complex (Fig. 15). The decrease in photosynthetic growth rate did not appear to be due to inefficient transfer of light energy from the LHII complex to the rest of the PSA since no increase in fluorescence was detected for this strain compared to pseudo-wild type cells (Fig. 17). Nor was there any indication from protein gels that the pucE deletion strain had reduced levels of RC or LHI complex (data not shown). I therefore cannot explain this reduction in photosynthetic growth ability.  The pucD coding sequence overlaps that of pucE by four nucleotides, so pucD expression might be linked to that of pucE. Pairs of genes with overlapping coding sequences are often translationally coupled such that translation of the first results in efficient and stoichiometric translation of the second [26]. I found that a translationally in-frame deletion of the pucD gene  125 had no apparent effect on LHII complex levels detected by spectroscopy, and in this respect my results differ from those of Tichy et a!. [78]. The sequence removed in their deletion was not reported, however, so it was possible that their deletion was not translationally in-frame and, due to loss of translational coupling, had a polar effect on expression of pucE. I tested the contribution of translational coupling to expression of a pucE’::lac’Z fusion by introducing a frame-shift mutation which resulted in premature termination of PucD translation (Fig. 28). I found that /3-galactosidase levels decreased modestly, but not significantly given the high standard deviation in the assay. There may therefore be a small contribution of translational coupling to the levels of PucE expression, but it is certainly not required.  In addition to having no effect on LHII complex levels, deletion of pucD did not impair photosynthetic growth of strain zLHII(pAD) except at the very low 2 light level of 5 tEm  s*  This minor effect is the only phenotype yet detected  for this strain. The function of this gene, and the significance of the overlap between the pucD and pucE open reading frames, remains unknown.  d. The phenotypes of pucC and pucCDE deletion mutants  Deletion of pucC, alone or in combination with pucD and pucE, resulted in complete loss of the LHII complex, a reduction in the levels of RC and LHI complexes, and a severe decrease in the rate of photosynthetic growth at all light levels tested compared to strain ALHII(pRK415::2). As discussed below, the reduction in photosynthetic growth rate is probably not due solely to the lower  126 amount of LHI complex in these strains and I have already described results showing that the lack of LHII complex is not due to decreased pucBA transcription in strains ALHII(ptC) and ALHII(ptCDE) (see section a).  Strain z\LHII(pAC) contains a pucC allele truncated within the fifth transmembrane domain (see below for PucC structure). This strain has approximately 40% of the amount of LHI complex seen in strain ALHII(pRK4I5::2) when grown under low oxygen conditions (Fig. 14). Under photosynthetic conditions the amount of the LHI complex in ALHII(pRK415::2) changes very little with decreasing illumination (Fig. 18C) but strain zLHhI(pAC) experiences a 64% increase in the LHI absorption peak as the light intensity 2 1 to 60 jiEm 2 s decreases from 200 iEm  2 s ALHII(pAC) has At 60 Em  76% of the amount of LHI complex present in z\LHII(pRK4I5::Q) at the same light intensity. This relative increase in LHI complex levels is not accompanied by increased photosynthetic growth rates however, and at lower levels of illumination the decreased photosynthetic growth rates of pucC strains provided a selective advantage to secondary suppressor mutants (Fig. 16).  Most of these secondary mutants expressed LHII complexes, implying that at least one other gene is capable of providing the function of the missing pucC gene, and presumably its own normal function, when modified due to a mutation. The suppressor strain z\LHII-4(pAC) did not regain any LHII complex but grew faster than the parent strain, confirming that loss of LHII complex can be separated from the reduction in photosynthetic growth rate. I determined that the second site mutations are chromosomally located in all the suppressor strains I isolated, since transfer of the plasmids from these strains to strain ALHII gave the same phenotypes as plasmids pAC and pACDE (unpublished data). The  127  data presented here do not determine how many genes are represented in the collection of secondary mutants, but the diversity of phenotypes demonstrates that mutation at multiple sites can suppress the phenotypes of pucC or pucCDE deletion.  Two candidates as sites for a mutation that suppresses the effects of pucC gene deletion are F1696, a gene found upstream of the puhA gene, and 0RF428 (located in a bch operon; see Fig. 2). The amino acid sequence of 0RF428 has 27% identity and 69% similarity to PucC, but no mutant phenotype has yet been identified for this gene [9]. The predicted amino acid sequence of F1696, on the other hand, has 47% identity to that of PucC [3], and mutations in F1696 have been shown to decrease the amount of the LHI complex by up to 80% without affecting LI-ill or RC complex levels [3 and C. Young, unpublished results]. It therefore seems possible that the function of F1696 in formation of the LHI complex is analogous to that of pucC in LHII complex formation, and that mutation of F1696 can allow it to compensate for the deletion of pucC. This hypothesis could be tested by sequencing F1696 from the secondary supressor strains or by mutagenesis of F1696 and complementation of a pucC mutant.  F1696 mutants retain some LHI complex, so kinetic studies have been possible to determine whether F1696 maintains steady state levels of the LHI complex by facilitating assembly or by stabilising otherwise assembled LHI complexes. It was concluded that the primary role of F1696 is in assembly of the LHI complex [C. Young, J.T. Beatty, in preparation]. Similar experiments cannot be performed to investigate PucC’s role in determining LHII levels because all  pucC mutants examined so far have been completely devoid of LHII complex.  128 By analogy to Fl 696 it seems likely that PucC is required for assembly of LHII complexes rather than stabilisation of existing complexes. The low level of transcripts encoding pucC and the fact that the PucC protein has never been isolated with LHII complexes are consistent with a catalytic rather than structural role for PucC. It is hoped that comparison of detailed structural models for the two proteins will help in defining conserved elements required for similar function and differences important in the specificity of complex assembly.  F1696 mutation does not affect levels of LHII complex but strain zLHII(pAC) had decreased levels of the LHI and RC complexes, so it was formally possible that pucC is involved in LHI production and that residual levels of the LHI complex in F1696 strains is due to the presence of pucC. Strains containing deletions of both F1696 and the puc operon retained low levels of LHI complex which were not increased by expression of pucC (Fig. 25), and zLHII, which is wild type for F1696 but missing all the puc genes, has high amounts of the LI-il complex. Thus the PucC protein does not seem to participate in LHI formation and the decreased levels of the LHI and RC complexes in ALHII(pAC) must be due to an inhibitory effect of the presence of the other puc genes in the absence of pucC.  e. Deletion analysis of PucC using pucC’:qiho’A fusions and effects on LHI complex levels  Translationally out-of-frame pucC’::pho’A fusions were used to evaluate whether alleles of pucC, truncated at the C-terminus, retained any function.  129 None of the alleles tested, which lacked only one or two of the final transmembrane domains, conferred on ALHII(pzC) strains the ability to synthesize LHII complex when expressed in trans (Fig. 24). The C-terminus of PucC is thus required either for stability of the protein or for function in LHII complex formation.  The different pucC alleles examined had different phenotypes in terms of the amounts of RC and LHI complex present, however. The amounts of RC and LHI complex synthesized by these strains when grown photosynthetically was higher than that of the vector control strain, but this did not result in any increase in photosynthetic growth rate (Fig. 24).  Other pucC alleles, in effect truncated by translational fusion to phoA, also had unexpected effects on the levels of the LHI and RC complexes when expressed in LHII(pAC). The amount of LHI complex present in these cells was roughly correlated with the length of the pucC sequences remaining in the fusion, despite the presence of pAC (Fig. 26A). As above, with the exception of fusion pPQ::S8-1O which had almost no LHI or RC complex, the photosynthetic growth rates of these strains were similar regardless of the amount of LHI complex present (Fig. 27).  Thus truncated pucC alleles are not only unable to promote synthesis of the LHII complex, they can also lead to reductions in the amounts of the LHI and RC complexes, and to decreased rates of photosynthetic growth that are not due solely to the decrease in the components of the PSA. The effects on LHI complex level are only seen when the pitcB and pucA genes are also present, since the  130 truncated alleles have no effect when expressed in ALHII cells that do not harbour pIC (pucD and pucE are probably not important in these effects since strain ALHII(pACDE) has the same reduction in LHI complex level as ALHII(pAC)).  Reconciling these data is not a simple matter. Deletion of pucC alone is worse for the cells than deletion of the entire puc operon, leading to loss of LHII complex and to decreased amounts of RC and LHI complexes. Rates of photosynthetic growth are also impaired, but not simply because of these decreases in the components of the PSA. PucC does not participate in LHI formation, yet expression of truncated alleles leads to decreases in the amounts of the complex, an effect which is dependant on the presence of the pucBA genes. Finally, the reduction in LHI complex levels in ALHII(pAC) is relieved as light intensity decreases.  This final observation points to a possible reason for the decrease in LI-il complexes seen in strains containing pucB and pucA along with deletions in pucC. As light levels decrease, the transcription of the puc operon also decreases (in wild type cells the amount of LHII complex increases by a posttranscriptional mechanism  [931). It is possible that free LHII a and /3 peptides  can interfere with assembly of the LHI a and /3 peptides into complexes. One of the functions of PucC might be to shepherd the LHII pigment-binding peptides to prevent interactions that could interfere with LHI assembly. In the absence of pucB and pucA this function would be unnecessary, so strain ALHII has normal levels of LHI complex. The longer PucC peptides might retain more of this shepherding function while lacking the ability to promote LHII complex assembly, so the interference with assembly of the LHI complex is less with  131 longer alleles. In strain z\LHII(pAC), as light levels decrease the amount of LHII a and /3 peptides might decrease, so the interference with LHI assembly would also be reduced. The levels of LHI complex in ALHII did not change as light intensity varied, suggesting that the amount of LHI complex does not normally vary with changes in illumination.  This hypothesis is difficult to test, but one of its predictions is that increasing synthesis of the LHII cx and /3 peptides in a pucC mutant would decrease the amount of LHI complex in those cells. This could be tested by expressing pucB and pucA from the nif promoter and growing the cells on increasingly poor nitrogen sources to increase transcription of pucBA without affecting puf expression. This system has been used to evaluated the concentration dependance of bacteriochiorophyll synthesis on pufQ expression [4]. A second test would be to determine the point mutation in the LHIP mutant MW442. This strain is complemented by plasmid pC, which carries pucC, but its phenotype resembles that of ALHII rather than ALHII(pAC). My prediction would therefore be that the mutation in this strain is a missense mutation that causes an amino acid substitution that prevents LHII complex assembly, rather than a nonsense codon that results in a truncation of the PucC protein. This analysis might be complicated by sequence differences of the type seen between strains SB1003 and 37b4, so the mutation would have to be confirmed by duplication in a different background.  PucC is clearly required to obtain the LHII complex, much as F1696 is an assembly factor for the LHI complex. Unlike F1696, however, mutations in  pucC cause pleiotropic effects on the other complexes of the PSA. Putative RC  132 assembly factors have recently been identified that also have indirect effects on LI-il complex levels, possibly because the LHI complex is not stable in the absence of the RC [851.  f. The structure of the PucC protein  Theoretical models for the membrane topology of the PucC protein were constructed by comparison of hydropathy analyses and application of the positive inside rule. These models were tested by construction of over 40 translational fusions between pucC’ and pho’A to arrive at the model presented in Figure 23. It features 12 transmembrane domains and predicts that both the N- and C termini are located in the cytoplasm. In accordance with the positive inside rule none of the periplasmic domains have more than one basic residue, whereas most of the cytoplasmic loops have at least four. At the C-terminus the predicted hydrophilic domain is very short, but is anchored by three positively charged residues. The third cytoplasmic domain has only one lysine residue, and the only fusion in this region, S8-5, lacked this residue and had intermediate activity. The high hydrophobicity of neighbouring segments and precedents in the literature [67] argue that this intermediate activity is that of a cytoplasmic fusion that is inefficiently retained in the cytoplasm because it is not anchored by a basic residue.  Although the theoretical and experimental data sets are large and internally consistent, the model could be further tested by construction of different fusions which would provide positive rather than negative results for cytoplasmic  133 fusions. J3-galactosidase fusions have been used for such purposes [47], and replacement of pho’A with lac’Z in key fusions such as S8-5 would further support (or refute) the model. Screening blue colonies from new ligations between lac’Z and Ba! 31-digested pucC’ fragments on plates containing X-gal might also identify pucC’::lac’Z fusions in the first and last cytoplasmic domains. Each of the transmembrane domains would then be confirmed by positive data from each side of the membrane.  One limitation of analysis by these translational fusions is that truncation of the protein could lead to differences in stability of the remaining peptide or structural artifacts if important downstream structural determinants are removed. Biochemical analysis of PucC using monoclonal antibodies or chemical methods would not be subject to these caveats but is not currently possible.  In the course of sequencing the pucC’::pho’A fusions several differences were noted between the sequence of pucC in the strain used here, SBIOO3, and that published for strain 37b4 [79]. The number of differences in the DNA sequence is especially striking considering that the 600 bp sequence of the pucBA region reported from both these strains contains only 2 differences, one of which might be a sequencing error (see section a). All of the differences that resulted in amino acid changes are located in the second part of the protein, perhaps indicating that the first four transmembrane domains are more important for PucC function than the rest of the protein. Immediately upstream of the PucC start codon there were no differences noted. This is of interest because the preliminary results of an E. coli in vitro transcription/translation system reported that translation of PucC could not be detected until a stronger ribosome  134  binding site had been introduced  [371 whereas the levels of alkaline phosphatase  activity I detected in my study did not suggest that translation occured at low levels in E. coli.  PucC and the predicted protein product of the F1696 gene are strikingly similar: they share 47% identity and 64% similarity spread through the length of their sequences. Comparison of their structures will be of interest to see if membrane topology features are also conserved. The F1696 protein topology is currently being determined [C. Young, personal communication].  g. Concluding remarks  The results of this thesis extend our knowledge of the puc operon, and suggest several avenues for future investigations.  I have shown that the five genes of the puc operon are co-transcribed, and demonstrated the existence of a low-abundance pucBACDE transcript. My work indicates that this message is processed to generate an approximately 1.0 kb molecule that contains pucDE sequences. Extensive studies of processing events in the R. capsulatus puf operon mRNA have identified endonuclease recognition sites [35]; comparisons between the two operons might help establish general features of such processing sites. Primer extension studies would identify the 5’ end of the 1.0 kb molecule, and that of the 0.5 kb RNA species also detected by pUCDE probes, and might help to locate the minor promoter detected in my pucE’::lac’Z fusion analysis. Site-directed mutation of the inverted repeat  135 sequences downstream of pucA in a pucE’::lac’Z fusion would determine whether transcriptional attenuation occurs, a possible mechanism for generating the abundant 0.55 kb pucBA mRNA.  The most surprising result of the phenotypic characterisation of pucCDE deletion mutants was the pleiotropic effects of truncated pucC alleles on LHI and RC complex levels. Several experiments suggested that the reduction in photosynthetic growth rates was not due solely to the decreased amounts of the LHI and RC complexes, and that these reduced complex levels depended on the presence of the pUCBA genes. I have proposed that the PucC protein has the specific function of participating in assembly of the LHII complex and a less specific role in shepherding LHII a and /3 peptides, preventing them from interfering with LHI complex assembly.  Whether the LHII a and /3 peptides interfere with LHI complex assembly in the absence of full-length PucC protein could be tested by overexpression of pucBA, in the complete absence of pucC or with truncated alleles. Biochemical  reconstitution experiments might be able show whether LHI and LHII a and /3 peptides can interact; similar experiments have shown that LHI /3 peptides can interact, as can LHI a and /3 peptides from different species of photosynthetic bacteria [45].  The active site of PucC for assembly of the LHII complex might be identified by locating the mutation in the strain MW442 which renders it LHII without affecting LHI or RC complex levels. Further insight into the function of PucC could be obtained by cloning the suppressor mutations from strains such as  136 ALHll-2(pzCDE). Finally, mutational analyses of pucC can be planned on the basis of the topological model of the PucC protein, and should help in determining functional domains of the protein.  The final unexpected result of my studies was the observation that LHI complex levels in the strain ALHII did not vary in photosynthetic cultures grown at different light levels. Previous studies have reported that the number of photosynthetic units, including the LHI complex, increase as incident light levels decrease [69]. Mutation of the recently identified light-response regulator, hvrA, led to a 2-fold decrease in expression of a pufB’::lac’Z reporter gene under conditions of low illumination [121, suggesting that transcription of the puf operon is positively regulated by diminished light intensity, but the authors did not report whether in vivo amounts of LHI complex were affected. Mutation of hvrA in the strain ALHII would allow a direct evaluation of whether the 2-fold decrease in expression of the reporter gene is reflected by changes in the final amounts of LHI complex in response to low light intensity.  137 REFERENCES 1. Adams, C.W., M.E. Forrest, S.N. Cohen and J.T. 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