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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 THERHODOBACTER CAPS1JLATUS PUC OPERONHeidi LeBlancB.Sc. (Honours, Co-op), University of Waterloo, 1988A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIES(DEPARTMENT OF MICROBIOLOGYAND IMMUNOLOGY)THE UNIVERSITY OF BRITISH COLUMBIAJUNE 1995© Heidi LeBlanc, 1995Signature(s) removed to protect privacySignature(s) removed to protect privacyIn presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives, It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)__________________________Department of 1 De91O /,The University of British ColumbiaVancouver, CanadaDateDE-6 (2/88)Signature(s) removed to protect privacy11ABSTRACTThe puc operon of Rhodobacter capsulatus encodes the genes for the pigmentbinding peptides of the light harvesting (LH) II complex, pucB and pucA, followedby three open reading frames named pucC, pucD and pucE.RNA blot analysis and promoter mapping experiments using translationalfusions of lac’Z to the distal gene, pucE, showed that all five genes are primarilytranscribed from a promoter upstream of pucB. The primary transcript from thispromoter is of low abundance and is processed to give a 1.0 kb transcript containingpucDt sequences. A minor promoter was detected between the 3’ end of pucC andthe middle of pucD; a 0.5 kb RNA molecule detected by hybridisation probes to thepucDE region could be the transcription product of this minor promoter. The mostabundant transcript detected was the previously characterised 0.55 kb pucBAmRNA.Strain ALHII, which lacked the LHII complex, was created by replacement of thefive chromosomal puc genes with the spectinomycin cartridge. This strain wascomplemented by plasmids carrying deletions of the pucCDE genes. The pucCgene was required for the presence of the LHII complex, and deletion of the pucEgene, which encodes the subunit of the LHII complex, led to a reduced level of thecomplex. Deletion of the pucD gene had no discernable effect on LHII complexlevels. Photosynthetic growth of strain ALHII was similar to that of wild type cells atlight levels greater than 30 p.E•m-2.s-1, but strains containing deletions of pucC orpucE were significantly impaired for photosynthetic growth at all light levels tested.In the case of pucC-deleted cells, chromosomal suppressor mutations frequentlyarose that restored the LHII complex and/or photosynthetic growth ability.A model for the trans-membrane topology of the PucC protein was derived bytheoretical analyses and tested using the genetic system of alkaline phosphatasefusions. The model predicts 12 transmembrane domains, with both the N and Ctermini on the cytoplasmic side of the membrane. The fusions were also used for adeletion analysis of pucC. None of the truncated alleles allowed the cells tosynthesize the LHII complex, but several had unexpected pleiotropic effects on LHIand RC complex levels.111TABLE OF CONTENTSABSTRACT iiLIST OF TABLES vLIST OF FIGURES viABBREVIATIONS viiiACKNOWLEDGEMENTS ixINTRODUCTION 1MATERIALS AND METHODS 16a. Bacterial strains and growth conditions 16b. DNA manipulations 18c. Plasmids 19d. Conjugations 19e. Spectral analysis 22£ Construction of R. capsulatus strain iLHII 22g. Southern blot analysis 25h. Plasmid deletions of pucC, pucD and pucE 25i. Fluorescence measurements 31j. Construction and screening of pucC::phoA fusions 34k. Alkaline phosphatase assays 361. Hydropathy analysis 36m. Construction of plasmids for expression of pucC and C-terminaldeletions of PucC 37n. RNA isolation, blot analysis and probe construction 38o. pucE’-lac’Z fusion construction 40p. fl-galactosidase assays 41RESULTS 42Chapter 1. Phenotypic properties of puc operon mutants 42a. Chromosomal deletion of the puc operon 42b. Effects of pucCDE deletions on LHII complex absorption spectra 45c. Photosynthetic growth characteristics of puc gene deletions 49d. Fluorescence analysis of z\LHII(ptE) 52ive. Effects of light intensity on LH complex levels 54f. Isolation of strains with secondary mutations that suppressedpucC deletion 58g. Phenotypic characterisation of suppressors of pucC deletion 59h. Conclusions 65Chapter 2. PucC structure and function 69a. Hydropathy and positive inside rule analysis of PucC 69b. Construction and analysis of pucC’:.pho’A fusions 75c. Sequence differences between wild type strains SB1003 and 3Th4 82d. Deletion analysis of pucC 86e. Role of PucC in LHI assembly 89f. Conclusions 95Chapter 3: Analysis of puc operon transcription 101a. puc operon promoter mapping by deletion analysis ofpucE’::lac’Z gene fusions. 101b. Is there translational coupling between pucD and pucE? 103c. RNA blot analysis of the puc operon 104d. Conclusions 111DISCUSSION 115a. Transcriptional organisation of the puc operon 115b. Deletion of chromosomal pucBACDE genes in strain ALHII 122c. The phenotypes of pucD and pucE mutants 123d. The phenotypes of pucC and pucCDE deletion mutants 125e. Deletion analysis of PucC using pucC’:.pho’A fusions and effectson LHI complex levels 128f. The structure of the PucC protein 132g. Concluding remarks 134REFERENCES 137VLIST OF TABLESTable 1. Bacterial strains 17Table 2. Plasmids 20Table 3. Summary of translationally in-frame fusions between PucC andPhoA 78Table 4. Amino acid differences between the predicted protein sequencesof PucC from strains SBIOO3 and 3Th4 85viLIST OF FIGURESFigure 1. The photosynthetic apparatus of R. capsulatus. 2Figure 2. The photosynthesis gene cluster of R. capsulatus. 7Figure 3. The puc promoter region of R. sphaeroides. 9Figure 4. The puf operon of R. capsulatus. IIFigure 5. The puc operon of R. capsulatus, ca. 1989. 13Figure 6. Construction of plasmids pHLBMpuc::2 and pUCiCDE. 23Figure 7. Construction of plasmid pUCAC. 27Figure 8. Construction of plasmid pUCzD(IF). 29Figure 9. Construction of plasmid pUCAE. 32Figure 10. Construction of pucC’::pho’A fusions. 35Figure 11. Absorption spectra of strains SBIOO3 and ALHII. 43Figure 12. Southern blot analysis of strains SB1003 and ALHII. 44Figure 13. The Pst I - EcoR I plasmid inserts used to complement ALHII. 46Figure 14. Absorption spectra of pucCDE deletion strains. 47Figure 15. Photosynthetic growth of pucCDE deletion strains. 50Figure 16. Photosynthetic growth of R. capsulatus strains at different lightintensities. 53Figure 17. Fluorescence emission of R. capsulatus strains. 55Figure 18. Spectra of R. capsulatus strains grown photosynthetically atdifferent light intensities. 56Figure 19. Spectra of secondary suppressor strains. 60Figure 20. Spectra of ALHII-1 (pzC) grown photosynthetically at differentlight intensities. 63Figure 21. Hydropathy predictions for PucC. 70Figure 22. Possible arrangements of trans-membrane helices in theC-terminus of PucC. 81Figure 23. A model of the topology of the PucC protein. 83Figure 24. Complementation of z\LHII(pAC) with truncated pucC alleles. 87Figure 25. Complementation of F1696 mutant strains with pucC. 91Figure 26. Absorption spectra of strains containing pucC’::pho’A fusions. 93Figure 27. Strains containing pucC’::pho’A fusions grownphotosynthetically. 96Figure 28. j3-galactosidase activities in cells containing pucE’::lac’Z fusions. 102viiFigure 29. RNA blot analysis of wild type puc operon transcripts. 105Figure 30. RNA blot analysis of puc deletion strains. 107Figure 31. Transcriptional organisation of the puc operon 116Figure 32. Superoperonal organisation of the photosynthetic gene cluster 119viiiABBREVIATIONSaa amino acidAp ampicillinbch R. capsulatus bacteriochiorophyll biosynthesis geneBSA bovine serum albumincfu colony forming unitscrt R. capsulatus carotenoid biosynthesis geneFNR fumarate nitrate regulator1CM intracytoplasmic membraneIHF integration host factorInc plasmid incompatibility groupKm kanamycinLH light harvestingOR operator regionorf open reading framePS photosyntheticPSA photosynthetic apparatusR resistance/resistantRC reaction centreTc tetracyclineURS upstream regulatory siteXGa1 5-bromo-4-chloro-3-indolyl-f3-D-galactopyranosideXP 5-bromo-4-chloro-3-indolylphosphate-p-toluidine saltdenotes a novel joint(prime) denotes a truncated gene at the indicated 5’ or 3’ sideixACKNOWLEDGEMENTSI would like to thank my supervisor, Tom Beatty, for six years of advice andsupport, and for assembling a wonderful group of people to work with. Pastmembers of the lab, Cheryl Wellington and Joanna Zilsel, were very supportivewhen I first started my work, and current members have been a valuable sourceof advice as I continued it. These people, Tim Lilburn, Farahad Dastoor, ConanYoung and Andrew Lang, have also been unfailingly patient with me as I wrotemy 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, havebeen constructive with their criticisms, for which I thank them. Beverly Greenwas very generous with her help and advice about integral membrane proteins.Other faculty members, especially George Spiegelman and Rosie Redfield, havebeen valuable sources of advice on a wide range of subjects, and I will always begrateful to a former supervisor, July Evans, for serving as an exemplary rolemodel.I also thank two former students in the lab, Grace Wong and ChristineChiang, for help in construction of some plasmids, and with enzyme assays.Finally, thank you, John, for moral support and patience over the past sixyears.1INTRODUCTIONThe purple non-sulphur photosynthetic bacterium Rhodobacter capsulatushas been used extensively as a model organism to study genetic regulation ofbacterial photosynthesis [reviewed in 70 and 2]. It is metabolically versatile, andunder growth conditions of high aeration derives energy for growth byrespiration of oxygen. When oxygen partial pressure is reduced thephotosynthetic apparatus (PSA) is gratuitously induced, making it possible tostudy interactions of the components of the PSA in the absence of a requirementfor function.Induction of the PSA is accompanied by synthesis of a specialised membranesystem known as the intracytoplasmic membrane (1CM) which forms byinvagination of the cytoplasmic membrane. The 1CM is thus continuous withcytoplasmic membrane but is biochemically distinct, primarily containingcomponents of the PSA that are absent from the cytoplasmic membrane in bothinduced and uninduced cells [55].The integral membrane protein components of the PSA include thereaction centre complex (RC), and the two surrounding light harvestingcomplexes, light harvesting I (LHI; sometimes designated B870) and lightharvesting II (LHII; B800-850) [for reviews see 33 and 18]. The LH complexesabsorb light energy (Fig. 1) and transfer it to the RC where it is converted tochemical energy when a quinone associated with the RC is reduced with aconcomitant uptake of two protons from the cytosol. The quinol then diffuses2Figure 1. The photosynthetic apparatus of R. capsulatus. Light energy (wavyarrow) is absorbed by the bacteriochiorophyll molecules (shown as four-ringedstructures) of the light harvesting antenna complexes, LHI and LHII, andtransferred to the reaction centre (RC) “special pair” of bacteriochlorophylls.Reduction of a quinone (hexagon) results in uptake of protons from thecytoplasm. The quinol is then reoxidised at the cytochrome (cyt) b/c1 complex,releasing protons to the periplasm and creating a proton gradient. The electroncycle is completed by re-reduction of the RC special pair by cyt c2. The protongradient drives synthesis of cellular ATP by diffusion of protons through theATPase complex. Electron and proton transfer is indicated by bold arrows, andthe subunits of the pigment-binding complexes are indicated by bold letters.(Figure adapted from 83).PERIPLASMcytC2H+H+(XYNinnflnir (°1n1rn‘__ui(cytb/c1LHILHIIATPATPaseADP+PiRCHCYTOPLASM4through the membrane to the cytochrome b/c1 complex where it is oxidised,releasing protons to the periplasm. A proton gradient is thus produced and itspotential energy is used to drive synthesis of ATP by passage of the protonsthrough the ATPase complex. Meanwhile, electrons from the cytochrome b/c1complex are transferred back to the RC by a cytochrome c. Electron transfer isthus cyclic and oxygen is not generated in the process [611.Our understanding of the mechanism of electron transfer in the RC hasbeen greatly helped by the solution of the three dimensional structure of thebacterial reaction centre by X-ray crystallography [reviewed in 681. The RCconsists of three peptides, the L, M and H subunits, which are encoded by thepufL, pufM and puhA genes. The H subunit crosses the membrane only onceand does not bind photosynthetic pigments. The L and M polypeptides eachcross the membrane five times in a symmetrical arrangement. These subunitsbind four bacteriochiorophyll molecules and two bacteriopheophytins, and thepigments are also arranged around a two-fold axis of symmetry. Despite thisstructural symmetry, electron transfer occurs only through the chromophoresassociated with the L subunit [73].Both of the LH complexes have two small, pigment-binding peptidecomponents, the a and /3 subunits. Similarities in sequence and arrangementof the genes for these peptides (pucBA for LHII and pufBA for LHI) suggest thatLHII complexes arose from a duplication of the genes for an ancestral LHIcomplex [901. Although LHII complexes are relatively easy to purify, they haveproven recalcitrant to crystallisation [141. To the great excitement of the bacterialphotosynthesis community the crystal structure of the LHII complex from5Rhodopseudomonas acidophila has finally been published [50], and a lowresolution projection for the LHT complex of Rhodospirillum rubrum has alsorecently 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 largeenough to accomodate the RC complex. Nine LHII complex cxf3 dimers formtwo concentric rings with the a peptides forming the inner cylinder and the /3peptides arranged around the outside. No ordered molecules were detectedwithin this ring.The pigment-binding peptides of the LHII complex each have a singlemembrane-spanning a helix, perpendicular to the plane of the membrane [50].The domains flanking the central a helix are amphipathic helices associated withthe surface of the membrane, and the N-termini are located on the cytoplasmicside of the membrane. Three bacteriochlorophyll molecules are associated witheach a$ dimer, suspended between the transmembrane cx helices, and the localprotein environment shifts the absorbance maximum of the pigments. The twolocated close to the periplasmic face of the cytoplasmic membrane are complexedto conserved histidine residues and absorb light maximally at 850 nm (B850).The third, coordinated to the carbonyl of the N-terminal formylmethionine ofthe /3 subunit near the cytoplasmic side of the membrane, has an absorbancepeak at 800 nm (B800). The B800 molecules are oriented parallel to the plane ofthe membrane, and are coordinated with the B850 molecules by interactions oftheir phytol tails and by a carotenoid molecule which spans the membrane. The18 B850 molecules are perpendicular to the membrane plane, stacked in anoverlapping ring which allows rapid delocalisation of the excited state. The B870molecules of the LHI complex are probably arranged in a similar ring,6coordinated to conserved histidines that would place the B870 ring at the sameposition within the membrane as the B850 ring. The RC complex special pair arealso located at this level within the membrane, so the spacing and orientation ofthe chromophores favours efficient energy transfer within and among thecomplexes of the PSA.Synthesis of the PSA in R. capsulatus occurs only in conditions of lowaeration, and the amount and composition of the system are further controlledby levels of illumination. As light intensity decreases the size of the light-collecting surface is increased by increasing the amount of the PSA and theamount of LHII complex relative to the RC and LHI complexes.The arrangement of the genes known to be required for photosynthesis isremarkably conserved amongst several species of purple photosynthetic bacteria.The operons encoding enzymes for the biosynthesis of the two photosyntheticpigments, bacteriochiorophyll (the bch genes) and carotenoids (the crt genes),are clustered; for example, in R. capsulatus, within a 50 kb segment of thechromosome (Fig. 2). They are flanked by the puh and puf operons whichencode the structural genes for the RC and LHI complexes. The LHII complex isnot found in all purple photosynthetic bacteria, by contrast, and those organismsthat have LHII complexes have radically different genomic locations for the pucoperon (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 thepuh operon [23] whereas in the closely related species Rhodobacter sphaeroidesit has been found to lie 18 kb downstream of puh [76]. Other bacteria, notablyRhodopseudomonas palustris, have several different forms of LHII complexFigure2.ThephotosynthesisgeneclusterofR.capsulatus.GenesforstructuralpeptidesoftheRCandLHIcomplexesareindicatedbyhatchedboxes;shadedboxesindicategenesencodingbacteriochiorophyll biosyntheticenzymes;carotenoidbiosynthesisgenesaredotted;cross-hatchingindicatesgenesofunknownfunction.Thenamesofgenesdiscussedinthetextareindicated.puhAFl696orf469orf428pufQBALMX8and 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 between62% and 131% of the maximally induced anaerobic level [91]. This seems, at leastin part, to be negatively regulated since aerobic transcription of some of theseoperons 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 ofenzyme activity [49] and a non-enzymatic factor, the pufQ gene product, isrequired post-transcriptionally for bacteriochiorophyll synthesis [4].Transcription of the puf operon, which encodes the pigment-bindingpeptides of the LHI and RC complexes, is strongly induced by low oxygenconditions [2]. This operon is not regulated by orf469 [591; rather a two-component regulatory system (RegA/RegB) induces transcription of the operonunder anaerobic conditions [72, 54]. A second two component system,HvrA/HvrB, weakly induces the operon under conditions of decreasedillumination [12].Transcription of the puc operon is similarly induced by the RegA/RegBtwo component system in response to decreased oxygen levels [54]. Additionally,as with the crt and bch genes, orf469 appears to repress transcription in aerobicconditions [59]. An additional gene has been identified which is required forinduction of both the puc and puf operons anaerobically; this gene has nohomology to known genes or R. capsulatus sequences [58]. The puc operon is9PromoterURS Activator R2-629 -117 -106 -84 -66 -52 -35 -27 -10 0 11702PS -Figure 3. The puc promoter region of R. sphaeroides. Sequence elementsupstream of the transcription start site (0) are represented by boxes and labelsabove the figure; their position relative to the transcription start is indicatedbelow. The relative amount of 13-galactosidase activity from a iacZ’::puc’Bfusion (B::Z) to the promoter under aerobic (02) or photosynthetic (PS) growthconditions is indicated by the thickness of the black arrows. The activities ofsimilar fusions to mutated promoters are indicated by the thickness of the barspositioned under the regions affected. Black bars represent deletions; grey barspoint mutations. URS: upstream regulatory site; FNR: homology to thefumarate nitrate regulator binding site; IHF: homology to the E. coil integrationhost factor binding site (the overlap in these two sequences is indicated by thehatched box); OR1 and 0R2: inverted repeat sequences. (Figure adapted from 30).10not transcriptionally regulated in response to decreased illumination; instead theamount of LHII complex increases by a post-transcriptional mechanism in dimlight [931.The promoter region of the puc operon in Rhodobacter sphaeroides hasbeen analysed and several transcriptional trans-acting factors and cis regulatorysequences have been identified [reviewed in 30]. Figure 3 shows the cis-actingsequences: the upstream regulatory site (URS) is required for aerobic repressionof transcription; an activator required for transcription has been identified bydeletion in the region 70 nucleotides upstream of the transcriptional start site;and point mutations in one of two regions of dyad symmetry (OR1) reduceaerobic repression of transcription. Sequences 100 nudeotides upstream of thetranscriptional start site show similarity with the recognition sites for the E. coliintegration 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 transhave been reported by several groups. Lee and Kaplan [42] identified oxyA andoxyB genetically as mutants unable to repress puc transcription aerobically [42].oxyB maps to the same region of the PS gene cluster as ppsR [56]; thecharacterisation of ppsR and its R. capsulatus homologue, orf469, is consistentwith ppsR and oxyB being the same gene. A suppressor of oxyB mutants wasidentified and named prrA; by sequence comparison it is the R. sphaeroideshomologue of the R. capsulatus regA gene [21]. Finally, a 26 kDa protein whichbinds the R. sphaeroides puc promoter region 120 nucleotides upstream of thetranscriptional start site has been identified biochemically [51].11<0.5 mm8 mm33 mm4Figure 4. The puf operon of R. capsulatus. The puf genes are represented byboxes; genes for pigment-binding proteins are shaded. The promoter is indicatedas a bent arrow and the positions of predicted stem loop structures within pufmRNA are shown as lollipops. The major mRNA molecules are represented asarrows under the sequences encoded; the thickness of the arrow reflects theabundance of the message in induced cells. Vertical arrows show processingevents and the measured half-life (t1 /2) of each mRNA is indicated in minutes.412The relative amounts of LHI and RC complexes are constant and aredetermined post-transcriptionally by differential degradation of their respectivesegments of the polycistronic puf mRNA (Fig. 4) [reviewed in 351. The regionencoding the RC L and M subunits contains endonucleolytic processing siteswhich lead to rapid degradation of this message [36]. A stem ioop structurebetween the pufA and pufL genes protects the LHI mRNA segment fromexonucleolytic 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 ispost-translational. The RC and LH pigment binding peptides are stable only ifthey are assembled into complexes. The LHI /3 peptide, for example, issynthesized and inserted in the 1CM in the absence of the LHI a peptide, but isthen rapidly degraded [64]. In the complementary experiment the a peptide isnot detectable in the absence of the /3 peptide. Furthermore, in mutants unableto synthesize bacteriochiorophyll none of the pigment complex peptidesaccumulate in cell membranes [15]. Coloured carotenoids do not seem to berequired for synthesis of RC and LHI complexes, but mutants in carotenoidsynthesis have variable amounts of LHII complex [17]. In R. sphaeroides it hasbeen shown that if the carotenoid biosynthetic pathway is blocked before thecolourless intermediates have been converted to coloured carotenoid derivativesthe LHII complex is not formed and the LHII peptides are unstable [39].The two pigment binding peptides of the LHII complex are encoded by thepucBA genes [901. Two dicistronic RNA species of approximately 550 nt inlength encoding these genes have been characterized [93] and when firstidentified the puc operon was thought to consist of only these two genes. The13____HIBA C D EFigure 5. The puc operon of R. capsulatus, Ca. 1989. The structural genes forthe a and 13 subunits of the LHII complex, pucA and pucB, had beenidentified (shaded boxes) [90] and the ends of an abundant mRNA (arrow) hadbeen mapped to regions corresponding to promoter activity (bent arrow) andsequences resembling a rho-independant transcriptional terminator (lollipop)[93]. Three new open reading frames (open boxes) followed by a second putativetranscriptional terminator had been identified downstream of pucBA [791.14LHII mutant strain NK3 was then discovered to have a transposon inserteddownstream of the pucBA genes, and three new open reading frames werediscovered in this region by DNA sequencing [791. The new open reading frameswere named the pucC, pucD and pucE genes since a transposon disruption ofthe pucC gene led to loss of the LHII complex, and the predicted proteinsequence of the pucE gene matched the partial amino acid sequence of one ofthe two 14 kD peptides, named the y subunit, which co-purified with the LHIIcomplex [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 sequenceshomologous to pucDE [25]. In Rubrivivax gelatinosus a pucC homologue hasalso been found downstream of pucBA, but interestingly, in the oppositeorientation [Adrian Simmons, personal communication]. Again, pUCDEhomologues 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 theirexpression, it was not known which of the pucC, pucD and pucE genes wererequired for formation of the LHII complex. Furthermore, although the pucCand pucE genes were reported to be required for normal levels of the LHIIcomplex [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 thepucBA promoter region, or whether there was a promoter located between thepucC and pucDE regions, although a complementation analysis indicated thatthe pucC gene could not be expressed if the pucBA promoter region was not15present [79].For these reasons I chose to investigate the requirements of the pucC, pucDand pucE genes, individually, for cell growth as well as for formation of the LHIIcomplex. I created a mutant strain in which the chromosomal pucBACDE geneswere replaced by an omega cartridge [60], and complemented this deletion withplasmids carrying various combinations of puc operon genes. I also usedpucE ‘::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 entirepucBACDE region that hybridized to probes specific for the pucBA, pucC andpucDE regions.Finally, I derived a model for the structure of the PucC protein by theoreticaland genetic analyses, and used truncated PucC alleles in a variety of geneticbackgrounds to assess the function of the pucC gene product. Only the wild typePucC protein allowed synthesis of LI-ill complexes, but different pucC alleles hadunexpected effects on the levels of LHI complex in induced cells.16MATERIALS AND METHODSa. Bacterial strains and growth conditionsThe Eschericia coli strains used in this thesis have been previouslydescribed. Subcloning was done in the E. coli strains JM83 [881, an hsdRderivative of C600 [7], RB404 [11] and SM1O [74]. Strains SM1O [74] andHB1O1(pRK2OI3) [161 were used to transfer plasmids by conjugation to R.capsulatus. Fusions of puc’C to pho’A were expressed in the phoA deletionstrain CC1I8 [48]. E. coli strains were grown at 30°C or 37°C in Luria broth [461supplemented with the appropriate antibiotics at the following concentrations:ampicillin, 200 g/mL, kanamycin sulphate, 20-40 jig/mL, tetracyclineHCl, 10j.tg/mL and spectinomycin sulphate, 100 .tg/mL.The relevant genotypes and phenotypes of the Rhodobacter capsulatusstrains used in this thesis are summarized in Table 1. The strain SB1003 [75] wasused for RNA analysis, /3-galactosidase specific activity determinations in thepromoter localization experiments and as the parent strain for construction of apucBACDE deletion strain (see below). This puc operon deletion strain, R.capsulatus ALHII, was used for analyses of the effects of deletion of segments ofthe puc operon on LHII complex formation, and in RNA blot analyses. Thechromosomal second site mutants ALHII-1, zLHTI-2, ALHII-3, ALHII-4 and ALHII5 arose spontaneously during photosynthetic growth experiments (see Results).ANco and AStu are derivatives of strain ALHII containing an additional deletionof the C-terminal 348 and 340 amino acids, respectively, of the gene F1696 [ConanYoung, personal communication], They were used in studies to determine theTable 1. R. capsulatus strains.Strain Genotype Phenotype ReferenceSB1003 rif-lO Wild type for PSA 75ARC6 \puf::KmR RC, LHI, LHII 13ALHII 1puc::2 LHII 41ALHII-1 Xpuc::2; unidentified See text 41second site mutationXLHII-2 lxpuc::2; unidentified See text 41second site mutationzLHII-3 tpuc::2; unidentified See text This worksecond site mutationzLHII-4 lipuc::2; unidentified See text This worksecond site mutationz\LHII-5 ipuc::2; unidentified See text This worksecond site mutationzNco zpuc::c24F1696::Km’ LHI, LHII C. YoungAStu zpuc::Q4F1696::Km1 LHr, LHII C. Young1718effect of pucC expression on LHI complex levels. Strain ARC6(pA4) [11 lacks theRC and LHI complex, but expresses the LHII complex because pufQ is present onplasmid pA4. Light absorbed by LHII is reemitted as fluorescence. This strain wasused as a positive control in measuring fluorescence emission of puc genedeletion strains.R. capsulatus strains were grown in RCV medium [51, supplemented withan appropriate antibiotic for plasmid maintenance if appropriate, at 34°C.Tetracycline-HC1 was used at a concentration of 0.5 ig/mL, spectinomycinsulphate at 10 .tg/mL and kanamycin sulphate at 5-10 iig/mL. High aerationgrowth conditions were defined as cultures grown in Erlenmeyer flasks filled to8% of their nominal volumes and shaken at 300 rpm in an orbital shaker. Lowaeration growth conditions were obtained by filling flasks to 80% of theirnominal capacities and shaking at 150 rpm. Stationary phase low oxygencultures were used as inocula for photosynthetic cultures in screw cap tubes filledto capacity, and incubated with illumination provided by tungsten filamentincandescent lamps with light intensities ranging from 5 to 300 pEm2 .4. Lightintensity was measured with a Li-Cor photometer equipped with a LIO19OSBquantum sensor (Li-Cor, Lincoln Nebraska). Growth was monitored bymeasuring turbidity with a Klett-Summerson photometer (filter #66).b. DNA manipulationsStandard methods of DNA purification and manipulation were usedthroughout this thesis [66]. Double stranded plasmid DNA for screening and19sequencing pucC’::pho’A fusions was prepared according to the PromegaApplications and Protocols Guide. Briefly, the supernatant liquid frompotassium acetate-precipitated alkaline cell lysates was treated with RNase Athen extracted with phenol/chloroform and chloroform.c. PlasmidsMost 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 ingreater detail below.d. ConjugationsMobilizable plasmids were transferred to R. capsulatus by conjugation inbiparental (when the donor strain was SM1O) or triparental matings (using thehelper strain HB1OI(pRK2O13)). Approximately equal numbers of E. colidonor/helper and R. capsulatus recipient cells were concentrated bycentrifugation and spotted as a slurry on an RCV plate without antibiotic andincubated overnight at 30°C. Cells were then streaked sequentially on RCV(minimal) and YPS (rich) [82] media containing the selective antibiotic untilisolated R. capsulatus colonies were obtained.20Table 2. Plasmids.Plasmid Description ReferencepUCI3 £. coil cloning vector 88pSUP2O2 Mobilizable suicide vector for Gram negative bacteria 74pXCA6OI iac’Z promoter fusion vector; TcR; IncP IpRK4I5 Broad host range vector; TcR; IncP 32pJAJ9 R. capsuiatus expression vector; contains the puf promoter and 29pufQ; Tc’; IncPpPUFP::C01E1 R. capsuiatus expression vector; contains the puf promoter and J.T. BeattypufQ; Ap’, KmR; IncQ; ColE IpHLB1 pUCI3 containing a 4.5 kb Pst I-EcoR I puc operon fragment 41pHLB2 Suicide plasmid for construction of strain tLHII This workpRK415::1I Vector for ALHII control strains 41pBACDE 4.5 kb Pst 1-EcoR I fragment bearing the puc operon in pRK4J5::2 41pACDE Pst 1-EcoR I fragment bearing the pucCDE deletion in pRK415:: 41pz\C Pst I-EcoR I fragment bearing the pucC deletion in pRK4I5::2 41ptD Pst I-EcoR I fragment bearing the pucD deletion in pRK415:: 41pzE Pst I-EcoR I fragment bearing the pucE deletion in pRK4I5::2 41pM pXCA6OI containing the puf promoter and the pufQ gene IpPEZ pXCA6OI containing the pucE’::iac’Z fusion with 5’ end at Pst I 41pPEZ-OOF pPEZ with a frameshift mutation in pucD This workpCEZ pXCA6OI containing the pucE’::iac’Z fusion with 5’ end at Cia I 41pBEZ pXCA6OI containing the pucE’::iac’Z fusion with 5’ end at BsaB I 41pBEZ-OOF pBEZ with a frameshift mutation in pucD This workpHEZ pXCA6OI containing the pucE’::iac’Z fusion with 5’ end at Hinc II 41pUC::pucC(+) pUC13 containing the pucC gene cloned in the orientation of This worktranscription from the lac promoterpUC::pucC(-) pUCI3 containing the pucC gene cloned in the orientation This workopposite to transcription from the lac promoterpUC19::phoA Contains the truncated phoA sequence and JR from TnphoA 8pPQ::C pucC expressed from pPUFP::C01EI This workpPQ::E4-33 pucC, truncated at aa 439, expressed from pPUFP::ColEl This work21pPQ::E4-36 pucC, truncated at aa 412, expressed from pPUFP::C0IEI This workpPQ::E4-38 pucC, truncated at aa 387, expressed from pPUFP::C01EI This workpPQ::S8-1O pucC::phoA fusion at aa 63, expressed from pPUFP::C01EI This workpPQ::E8-5 pucC::phoA fusion at aa 291, expressed from pPUFP::C01EI This workpPQ::E8-25 pucC::phoA fusion at aa 329, expressed from pPUFP::C01EI This workpPQ::E4-25 pucC::phoA fusion at aa 422, expressed from pPUFP::C0IEI This workpC pucC expressed from pJAJ9 This work22e. Spectral analysisAbsorption spectra of intact cells (1.8 x 1O9 cells suspended in 1 mL of22.5% BSA in RCV medium) were measured with a Hitachi U-2000spectrophotometer, and data were collected with the Spectra Caic softwarepackage (Galactic Industries Corporation). All spectra are normalised to anabsorbance of 0.2 at 650 nm to correct for cell numbers.f. Construction of R. capsulatus strain ALHIIThe 4.5 kb Pst 1-EcoR I fragment bearing the pucBACDE genes frompRPSLHII [901 was first subcloned into pUCI3 [881 for ease of subsequentmanipulations, yielding pHLBI. After digestion of pHLB1 with Cia I and partialdigestion with Bcl I (Fig. 6), the 4.7 kb vector fragment containing the sequences3’ of the Bc! I site at position 3030 of the published sequence [791 and 5’ of theCia 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 togenerate blunt ends. This fragment was recircularized, inserting a BamH Ilinker (5’-CCGGATCCGG-3’) at the ligation site by linker tailing [40]. The omegafragment [601 was then inserted into this BamH I site. The Pst I- EcoR Ifragment of the resultant pHLB1ipuc::2 was inserted into the suicide vectorpSUP2O2 [74] and introduced into SB1003 by conjugation. SpectinomycinRcolonies were selected. Lighter coloured colonies were screened for loss of LHIIby spectral analysis. Replacement of the puc operon resulting from a doublecross-over event was confirmed by Southern blot analysis.23Figure 6. Construction of plasmids pHLBThpuc::2 and pUCACDE. SeeMaterials and Methods, sections (f) and (h) for details. The positions of sites forrestriction enzymes used are indicated; pUCI3 sequences are represented bymedium, shaded boxes; wide boxes represent coding sequences (pucC, pucD,pucE or the omega fragment); wide shaded boxes show pucB and pucA; andthin lines designate R. capsulatus sequences flanking the puc operon.Cia I 24BclIpartial I Cia IdigestBc! IBc! IBcl IBc! ICia I4.7 kb fragmentBcl I IKienowBamH I linkertailingBamHI /BamH I 2fragmentCia IBc! IPst IPst IEcoR EcoR Bcl IBclI BamHI25g. Southern blot analysisChromosomal DNA from SB1003 and tLHII (5 j.tg) was separately doublydigested with EcoR I and BamH I, run on a 1% agarose gel and transferred tonitrocellulose paper. A non-radioactive DNA labelling kit (BoehringerMannheim) was used to prepare digoxigenin-labelled probes from gel-purifiedDNA fragments. The blots were treated according to the kit’s specifications untilthe development stage when a fluorescent dye substrate for alkaline phosphatase(Gibco-BRL) was used to develop the blots, after which they were exposed to Xray film at room temperature for varying lengths of time before development.h. Plasmid deletions of pucC, pucD and pucEThe following deletions were made in the plasmid pHLB1 (see above) andtransferred to the broad host range plasmid pRK4IS [32] as Pst 1-EcoR Ifragments (Fig. 13, Results). The omega fragment was inserted at the Hind IIIsite upstream of the Pst I site to reduce transcriptional read through fromplasmid promoters [84]. The positions of restriction enzyme sites given belowrefer to the numbering in the published DNA sequence [79].Plasmid pACDE was created by deleting the pucCDE genes from the Bc! Isite at position 1463 to the Bc! I site at 3030 (Fig. 6). The 4.7 kb Cia Ito Bc! Ifragment generated in making strain ALHII (see above) was ligated to a 0.95 kbCia I to Bc! I fragment containing the C-terminal sequences of pucB, pucAand the first 750 bp of pucC.26The deletion of the pucC gene in plasmid pAC extended from the Bcl Isite at position 1463 to the BsaB I site at 2217 (Fig. 7). The Pst I-BsaB I fragmentfrom pHLBI containing the sequences downstream of pucC was ligated to thePst I-Bc! I (filled) fragment bearing the pucBA genes and the first 750 bp ofpucC. Fusion of the filled-in Bcl I site to the blunt BsaB I end created atranslational stop codon.In order to facilitate the deletion of pucD, a 0.75 kb Sma I-BsaB Ifragment from pHLB1 was first subcloned into pUCI3 (Fig. 8), making the Bcl Isite (position 2348) and the BssH I site (position 2598) unique in the resultantplasmid pUC::BS. The plasmid was cut at these sites and the ends were thenfilled in using the Klenow fragment of DNA polymerase I. Subsequentreligation created a translationally in-frame deletion of pucD (pUC::BSzD). Thisconstruction was returned to the puc operon sequences by replacing the wildtype Bsm I to Eco47 III fragment with the corresponding fragment frompUC::BSAD to generate pUCAD(IF). The deletion was confirmed by DNAsequencing (data not shown).The pucE deletion extends from the Eco47 III site at 2761 to the Bc! I siteat 3030 (Fig. 9). To ensure that translation beginning at the pucE start codon didnot continue into the presumed transcription stop signal downstream of thedeletion, an in-frame translational stop codon was created by adding a BamH Ilinker (5’-CGCGGATCCGCG-3’) at the Eco47 III site of pHLBI. This plasmid wasthen cut with BamH I and EcoR I, and ligated to the EcoR I-BamH I fragmentfrom pHLBlApuc::Q (described above) to restore the sequences downstream of27Figure 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 sequencesare represented by medium, shaded boxes; wide boxes represent pucC, pucD, andpucE; wide shaded boxes show pucB and pucA; and thin lines designate R.capsulatus sequences flanking the puc operon.28Pst IBsaB IPst IBcI I 0LigateAPst IBcl IBBsaB IBcl IKienowBsaB IPstJPst IPst IEcoR I29Figure 8. Construction of plasmid pUCAD(IF). See Materials and Methods,section (h) for details. The positions of sites for restriction enzymes used areindicated, but only 1 of 6 Sma I sites is shown for clarity; 0, site destroyed byligation; , site of deletion of pucD sequences; pUCI3 sequences are representedby medium, shaded boxes; wide boxes represent pucC, pucD, and pucE; wideshaded boxes show pucB and pucA; and thin lines designate R. capsulatussequences flanking the puc operon.30pUC130 BclIBssH IISma ISma ISma IBsaB ILigateSmallfragmentBc! IBssH IIKienowReligateIllSmallfragmentBsm IEco47 IIILargefragmentPst IBsm IEco47 III4EcoR I31the stop codon of pucE. The deletion of the pucE gene was confirmed by DNAsequencing (data not shown).All experiments that utilized strain ALT-HI as a control in experimentsusing the plasmids described above were done with this strain containing thevector pRK4l5 carrying the omega fragment (pRK4l5::2).To reconstitute the wild type phenotype to strain ALT-ill, the 4.5 kb Pst IEcoR I fragment that contained the pucBACDE genes was subcloned intopRK4l5::2 to yield plasmid pBACDE.i Fluorescence measurementsInfrared fluorescence was measured using a method based on that ofYouvan et al. [891. Stationary phase cells grown in low oxygen conditions wereresuspended at a concentration of 6 x 1O cells/mL in RCV medium. The cellsuspensions were then serially diluted 2 fold in a microtiter plate with flat-bottomed wells. The plate placed over a 1 cm thick 1 M cupric sulphate solutionilluminated from below by fluorescent lamps and photographed using KodakH1E135 high speed infrared film with and without an additional Wratten 87Cinfrared filter (Eastman Kodak). Several exposures ranging up to 2 minutes weretaken; an approximately 20 second exposure at f-stop 5.6 was used for the printsshown in Fig. 17B.32Figure 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 isshown as a very wide box; wide shaded boxes show pucB and pucA; and thinlines designate R. capsulatus sequences flanking the p1ic operon.33BamHILigateBamHIEcoR I B BamH II I IBamH IPst IEcoR I B BEco47 Ill EcoR IEco47 Ill,BamH I linkertailingBamH I4EcoR IBamH IEcoR IEcoR IEcoRI B34j. Construction and screening of pucC’:.pho’A fusionsA 1.7 kb Nae I fragment bearing the pucC gene was subcloned intoplasmid pUC13 in the orientation allowing transcription of pucC from the lacpromoter. As shown in Figure 10, the resultant plasmid, pUC::pucC(+), wasdigested 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 generatefragments with deletions of the 3’ end of pucC.As recommended by Sambrook et at. [66], 30 minute trial reactions werecarried out on aliquots of 1.1 jig linearized DNA using enzyme concentrationsranging from 0.32 units to 0.01 units Bat 31 (Boehringer Mannheim) per 10 jiLreaction. Enzyme concentrations of 0.02, 0.04 and 0.08 units per reaction digested0.2-0.8 kb from each 3’ end, an appropriate range to cover the length of the pucCgene starting from either the EcoR I or Stu I sites, so these reactions were scaledup 2-fold. The exonucleolytic digestion reactions were stopped by the addition ofEDTA to 50 mM and heating at 65°C for 5 minutes, then the products werecleaved with Hind III. The pucC digestion products were separated from vectorfragments by gel electrophoresis and fragments greater than 0.8 kb were purifiedusing the Qiaex (Qiagen Inc.) gel extraction kit. The smallest digestion productsresulted in bands too diffuse to purify by this method, so they wereelectrophoresed onto DEAE cellulose paper and eluted in a high salt buffer (1MNaC1, 0.1 M Tris-HC1 (pH 7.5), 10 mM EDTA). Acidification prior to ethanolprecipitation aided in recovery of the DNA. The purified fragments wereshotgun cloned into pUC19::phoA [8] which had been digested with Hind III andSma I. The ligation products were used to transform the phoA deletion strain35Linearize with EcoR I or Stu IDigest with exonuclease Bal 31Digest with Hind IIIGel purify fragmentsLigate into pUCI9::phoAcut with Hind III and Sma IPlac— — puC I phoA I — —Screen for blue/white colonies on XPFigure 10. Construction of pucC’::pho’A fusions, See Materials and Methods,section (j) for details. The arrow head represents the lac promoter.Stu IEcoR I—c-f-0--f-0--f-0-1E36CC118 [481, and transformants were screened on plates containing thechromogenic substrate XP (5-bromo-4-chloro-3-indolylphosphate-p-toluidinesalt) at 40 A second screening of clones was done by restriction enzymedigestion and gel electrophoresis of plasmid DNA and selected clones weresubsequently sequenced. Standard dideoxynucleotide sequencing was done usinga primer (5’-AATCACGCAGAGCGGCAGTC-3’) complementary to phoAsequences near the site of fusion.k. Alkaline phosphatase assaysApproximately 1.2 x io cells from logarithmically growing cultures wereharvested and washed in IM Tris-HC1 buffer (pH 8). The cells were resuspendedin 0.5 mL of the same buffer and made permeable with a drop each of 0.1% SDSand chloroform for 5 minutes at 37°C. Timed assays were started with theaddition of 0.5 mL of the alkaline phosphatase substrate (p-nitrophenylphosphate at 0.8 mg/mL in 1M Tris-HC1, pH 8). Once a perceptible yellow colourhad developed due to release of p-nitrophenol, the reaction was stopped withthe addition of 0.1 mL of 1MK2HPO4and the cells were removed bycentrifugation. Activities are expressed as 1000xAA42O/Atime (minutes).1. Hydropathy analysisThe hydropathy profiles predicted for the PucC protein by the programsavailable in PC/GENE were compared with additional analyses run using ENDIT37[28]. PC/GENE offers SOAP [34], which is based on the hydrophobicity scale ofKyte and Doolittle [38] and assesses the probability that 17 amino acidhydrophobic segments are integral or peripheral; the method of Eisenberg et al[20], which identifies 21 residue segments as membrane associated helices likelyto be globular or transmembrane based on the average hydrophobicity of thesegment; and the method of Rao and Argos [62] which identifies segments ofgreater than 16 amino acids having a hydrophobicity peak above 1.13. ENDITpermits the user to choose any of several algorithms and calculates hydropathyvalues over both a large (19) and a small (11) amino acid window. It alsocalculates probabilities of N and C caps [631, and comparison of all these datahelps in determining probable ends for transmembrane segments identified aspeaks in the hydropathy profile plotted using the larger window. ENDIT wasrun using the hydropathy scales of Kyte and Doolittle [38], Chothia [53], Michel[281 and Eisenberg [19]. The results of all the hydropathy analyses were comparedbut the Chothia results were analysed in most detail in arriving at the model forthe topology of PucC presented in the second chapter of Results.m. Construction of plasmids for expression of pucC and terminally deletedalleles of pucCIn order to complement the pucC mutation in ALHII(pAC) in trans thepucC 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 ofreplication and its selectable markers. The pucC gene was obtained as an EcoR I-BamH I fragment from pUC::pucC(-) and inserted downstream of the pufQ38gene in pPUFP::Co1EI, yielding plasmid pPQ::C. Expression of pucC wastherefore driven from the puf promoter in an oxygen-repressible fashion. Theplasmid was transferred to iLHII(pzC) by conjugation and TcRKm’ recipientswere selected.Translationally out of frame fusions between puc’C and phoA’ (seeabove) were used as C-terminal deletions of pucC. In such fusions translationterminates 4 or 10 codons after the last pucC sequence, depending on the readingframe. 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 fusionplasmids as Xba I-Pst I fragments and inserted into pPUFP::Co1EI cut with XbaI 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) byconjugation and TcRKmR recipients were selected.To express pucC in the F1696 deletion strains z.Nco and AStu, in whichF1696 has been disrupted by a kanamycin resistance cartridge [C. Young, personalcommunication], the puf promoter, pufQ and pucC were transferred frompPQ::C to pRK4l5 (which encodes TcR) as a Hind III-BamH I fragment to givepC. The plasmid was introduced into the ANco and AStu strains and TcRrecipients were purified.n. RNA isolation, blot analysis and probe constructionRNA was isolated from R. capsulatus by the hot phenol method as39previously described [801. Electrophoresis samples were ethanol precipitated anddenatured in a buffer containing formaldehyde and ethidium bromide [651. Fivepg of RNA per lane were run on a 1.4% agarose/formaldehyde gel beside 3 tg perlane of a 0.24-9.5 kb RNA ladder (BRL). After electrophoresis the gel wasequilibrated in 0.5X TBE buffer, and photographed with UV illumination beforeelectroblotting overnight at 30V in 0.5X TBE buffer onto a Biotrans nylonmembrane (ICN). After blotting the membrane was dried at 80°C under vacuumand exposed to UV light for comparison with the gel photograph to evaluate theefficiency 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% formamidecontaining 0.5 mg/mL denatured sheared salmon sperm DNA for 4-8 hours at42°C before addition of the denatured labelled probe. The blots were hybridizedwith the probes for 18 hours at 42°C.Hybridization probes were prepared using purified DNA fragments astemplates for labelling with cx-32P-dATP by the random primer method [221.Unincorporated nucleotides were removed using the Qiaex DNA purificationprocedure (Qiagen). The Qiaex eluate in TE buffer was denatured at 90°C for 10minutes and used directly for hybridization. After hybridisation the membraneswere 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 in0.2X SSC, 1% SDS at 50°C. Blots were then exposed to Kodak X-Omat film in acassette with an intensifying screen at -75°C for varying lengths of time beforedevelopment.40If necessary, blots were stripped for reprobing by brief incubation withboiling 0.1% SDS then this treatment was repeated and the solution allowed tocool to room temperature.o. pucE’::lac’Z fusion constructionA translationally in-frame fusion of the pucE’ to lac ‘Z genes wasobtained by cutting pHLB1 (see section f.) at the BspH I site in the pucE geneand filling in the overhanging 5t ends with the Kienow fragment of DNApolymerase 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 atranslationally in-frame fusion of the pUC13 lacZ allele to the fifth codon ofpucE (pUC-PEZ). A Pst I linker (5’-CCTGCAGG-3’) was inserted at the Cia Isite in the pucB gene (pUC-CEZ), at the BsaB I site at the 3’ end of the pucCgene (pUC-BEZ), or at the Hinc II site in the pucD gene (pUC-HEZ) by linkertailing [401. (Construction of pUC-HEZ deleted all the sequences between a HincII site in the 5’ unsequenced region and the middle of the pucD gene.) A stopcodon was created at the 13th codon of pucD by first cufting pUC-BEZ with Pst Iand recircularizing the plasmid to eliminate the sequences upstream of the BsaBI site in pucC. The resulting plasmid was then cut with Bcl I, the ends werefilled in and the plasmid was again recircularized, changing the sequence from5’-GTG ATC ACA-3’ to 5’-GTG ATC GAT CAC A-3’, thus causing a frame-shiftmutation in pucD (pUC-BEZ-OOF). The same frameshift was transferred topUC-PEZ by replacing the Pst I-Bsm I fragment of pUC-BEZ-OOF with the wild41type sequences upstream of the Bsm I site, creating pUC-PEZ-OOF [Grace Wong,personal communication]. The fragments shown in Figure 28 (see Results) wereinserted into the promoter probe vector pXCA6OI [1] as Pst I-BamH I fragments.The resultant plasmids were transferred to SB1003 by conjugation and TcRrecipients purified.p. 13-galactosidase assaysIn 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 bycentrifugation, resuspended and sonicated in 13-galactosidase assay buffer [521 onice. After centrifugation the cleared supernatant liquids were assayed for /3-galactosidase activity in a continuous assay [52]. Protein concentrations weredetermined by a modified Lowry procedure [57]. In later experiments [ChristineChiang, personal communication], J3-galactosidase activity was measured in atimed assay where logarithmically-growing cells concentrated 20-fold in /3-galactosidase assay buffer were made permeable with SDS and chloroform [47].Because of this difference, activities are expressed as percentages of the activity oflow oxygen grown SBIOO3(pPEZ) assayed in the same experiment. For sonicatedcells, this specific activity was 52.9 nmol.ig protein-I .minute1,for cellspermeabilised with SDS and chloroform the measured activity was 518OD420min1mL Klett unit-1. All determinations were performed onduplicate samples in at least 2 experiments.42RESULTS1. Phenotvpic properties of puc operon mutantsa. Chromosomal deletion of the puc operonAbsorption spectra of wild type cells show two peaks in the near infra-redregion, 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 bacteriochlorophyllmolecules associated with the LHII complex and reflect the predominance of thiscomplex in the intracytoplasmic membrane. A small amount of the 800 nmpeak is contributed by absorption of the less abundant reaction centre. The 850nm peak is asymmetrical, having a shoulder due to the absorption of the LHIcomplex at 870 nm.The pigment binding proteins of the LHII complex are encoded by the pucBand pucA genes [901. Three additional open reading frames, reading in the samedirection, were discovered downstream of pucBA and named pucC, pucD andpucE [791. In order to characterize these putative genes and determine their rolein 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 pucgenes with the 2 fragment (see Materials and Methods and Fig. 12B).430r11000Figure 11. Whole cell absorption spectra of wild-type SBIOO3 (dotted line), thepuc deletion strain ALHII (heavy line) and ALHII complemented with the wildtype puc operon on the plasmid pBACDE (solid line). Cells were grown tostationary phase in low oxygen conditions and spectra were normalized to thesame A650 value.600 800Wavelength (nm)441 kbA E P C 8 EI I IBA C D EB E P BK BK EI I Ii IC 1225. 12. Southern blot analysis of DNA from wild-type strain SB1003 and pucoperon deletion/insertion strain ALHII. A) Representation of the 5.5 kb EcoR Ifragment that contains the puc operon. B) Representation of the EcoR Ifragment with the puc genes replaced by the omega cartridge. Hatched boxesdesignate puc genes, the open box the omega cartridge, and heavy linesunsequenced regions. Gene designations are given below the boxes, restrictionenzyme 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. DNAfrom SB1003 (lane 1) and ALFIII (lane2) was doubly digested with EcoR I andBamH I and probed with the 5.5 kb EcoR I fragment shown in (A). The sizes ofmolecular weight markers are shown in kb to the left of the blot.45Strain ALHII lacked the LHII complex, as evidenced by the loss of absorptionat 800 and 850 nm (Fig. 11, heavy trace). Residual peaks at 800 and 870 nm aredue to absorption of the RC and LHI complexes, respectively. The deletion of thepuc genes was confirmed by Southern blot analysis (Fig. 12). ChromosomalDNA from SB1003 and tLHII digested with EcoR I and BamH I was run on anagarose gel and transferred to nitrocellulose. When probed with the 5.5 kb EcoRI fragment shown in Figure 12A only one band of approximately 5.5 kb was seenin the lane containing wild type DNA. The lane containing zLHII DNA had twobands of 2.3 and 0.9 kb in size, consistent with a double cross-over havingreplaced the puc genes with the 2 fragment. If the suicide plasmid had insertedby a single cross-over event 3 bands of 5.5, 0.8 and 1.3 kb (or 4.5, 0.8 and 2.3 kbdepending on the site of recombination) would have been detected. Probing asimilar blot with the 2 fragment confirmed its presence. at a single site in theALHII chromosome (data not shown).b. Effects of pucCDE deletions on LHII complex absorption spectraA series of plasmids was made that carried versions of the puc operon withdifferent deletions of the pucC, pucD and pucE genes (Fig. 13), and these wereconjugated into the ALHII strain [41]. Spectral analysis of cells grown underconditions of low aeration showed that the LHII complex could be restored bycomplementation in trans with the plasmid pBACDE, which contained theentire puc operon (Fig. 13 and Fig. 11, solid trace).Strain zXLHII(pzE) (Fig. 14C) showed about 64% of the level of LHII complex46P C B BBBBSE B ERI I II II I IC D EpBACDE___.‘H IpACDEpACpAD_________pAE____ ____Figure 13. The Pst I * EcoR I plasmid inserts used to complement the ALHIIdeletion. puc operon structural genes are indicated as boxes, with the genedesignations given below the map of pBACDE. Deletions are shown by dashedlines. Restriction enzyme sites used in constructing the deletions are shownabove the maps and are abbreviated as: B, Bcl I; BB, BsaB I; BS, BspH I; C, CiaI; 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.47Figure 14. Absorption spectra of intact cells grown under low aeration (seeMaterials and Methods). All spectra are normalised to the same A650, and theheights 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).A1,7BCC1.4D1-EF0.440.361000400600800Wavelength(nm)1000400600800100049absorption found with tLHll(pBACDE) (Fig. 14A), based on integration of theareas 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 ofLHII absorption (Fig. 14F and 14E respectively) and an apparent reduction in thelevels of LHI complex compared to strain ALHII(pRK415::2) (Fig. 14D).c. Photosynthetic growth characteristics of puc gene deletionsBecause the LHII complex is thought to increase the intracellular area ofphoton absorption and thus might aid photosynthetic growth at low lightintensities [18], the puc gene deletion strains were tested for the ability to growphotosynthetically at the low light intensity of 30 tEm2s1 (Fig. 15A). At thisintensity of light, strain ALHII(pBACDE) was light-limited for growth since it hada doubling time of 10 hours, whereas at saturating light intensities the doublingtime was typically about two hours (see below).Surprisingly, strain ALHII(pRK4I5::i2) grew only slightly slower at 30Ems1 than ALHII(pBACDE), with a doubling time of 15 hours, despiteundetectable levels of the LHII complex (see Fig. lAD). The kinetics and extent ofgrowth of ALHII(pt\D) were indistinguishable from LHII(pBACDE). Strainz\LHlI(pzE), which had reduced levels of LHII (see Fig. 14C), had a much slowerdoubling time (28 hours) than strain zLHII(pBACDE) and did not reach as great adensity in stationary phase.50U)D4-,4-,U)U)U)U)C,)U)4-4C444-,U)4-,U)C1)U)C-)Figure 15. Photosynthetic growth of cells with illumination at A) 30 E.m2-s,B) 300 iE.m-2-s-l and C) 5 j.iE•m-2s-1The growth of tLHhI(pACDE) in (A) and/XLHII(ptC) in (C) show the appearance of a secondary mutants. In experimentswhere none arose the growth curves of ALHII(pAC) and zLHII(pACDE) wereidentical (not shown).1000C100C-)ABCTime (hours)3001000100o zLHII(pBACDE)D zLHII(pRK41 5::c)/iLHII(pzCDE)VLH II (ptC)OLLH I I(pzD)•zLHII(pzE)0 10 20 30 40 50 60Time (hours)100010070 800 10 20 30 40 50 60 70 80 90Time (days)51The photosynthetic growth of strains zLHll(pACDE) and ALHII(pAC) wasthe most impaired of the strains tested, doubling in 40 hours (Fig. 15A).Occasionally in cultures grown at low light intensities, faster growing secondarymutants arose, distinguished by a marked increase in the slope of the growthcurve and, in one culture of ALHII(ptC), by an unusual greenish colour (seesection f). The appearance of secondary mutants was not observed in cultures ofany of the other strains described here, or in zLHII(pAC) or ALHII(pzCDE)cultures grown aerobically or photosynthetically with a light intensity greaterthan 60 tEm2s1.Because the ALHII(pAC), ALHII(pACDE) and ALHII(pAE) strains grew moreslowly than ALHII(pRK4I5::Q) at the low light intensity of 30 Em2s1,thesestrains were grown photosynthetically at 300 Em2s1 to determine if they werealso impaired in photosynthetic growth at this high light intensity. StrainszLHII(pBACDE), ALFIII(pAD) and ALHII(pRK415::2) were found to grow equallywell with a doubling time of about 2 hours (Fig. 15B). Strain zLHll(pAE) grewmore 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. Whenthese strains were grown aerobically with high or low oxygen conditions nodifferences in growth rates were observed (data not shown).Under photosynthetic growth conditions at the lowest light levels tested (5tEm2s1)only strains ALHII(pBACDE) and z\LHII(pAD) grew well (Fig. 15C)The slight reduction in the growth rate of z\LHII(pAD), with an 85 hour doublingtime, compared to the pseudo-wild type strain (65 hours) is the only phenotypeyet observed for the pucD deletion strain. After an approximately 50 day lag,52strain zLHII(pL\E) began to grow with a doubling time of roughly 12 days. Thislate growth did not seem to be the result of secondary mutation: duplicatesamples grew with exactly the same profile, and spectra of these culturesresembled usual zLHII(piE) profiles. Although it is not shown in Figure 15, theremaining strains in the experiment were followed for up to 130 days. StrainzXLFffl(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 inALHII(pAC) cultures proved to be due to suppression of the effects of the pucCdeletion 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 comparedin greater detail (Fig. 16). tLI-llI(pBACDE) grew at the same rate as the wild typestrain at light levels ranging from 200 to 30 Em2s’. Significant differences inthe growth rate of zLHII(pRK415::2) compared to the wild type strain appearedonce the light intensity had fallen to 30 tEm2s’ and strain iLHII(pAC)consistently grew more slowly than any of the other strains tested, generatingsecondary mutants at light intensities below 60 tEm2s1.d. Fluorescence analysis of ALHII(pAE)To evaluate whether the slow photosynthetic growth of ALHII(pAE) mightbe because the LHII complexes present in the strain functioned improperly, thecells were tested for emission of fluorescence [921. When light energy absorbed bythe LHII complex is not efficiently transferred through the LHI complex to the1000C’, 4-,4-,4-.. a)100> 4-’ C,, a) ci a) 0AB1000C,,4-, C :3 4-..4-’‘I)100> 4-’ U, c a) ci a) 01000C,,4-.. C 4-,a)s’ 110010050Time(hours)10050Time(hours)100D1000(IC 4 C :3 4-, 4-, a)100> 4-, (I) C a) ci a) (-)10050100150Time(hours)050oSB1003atLHII(pBACDE)100Time(hours)150200iLHII(pRK415::cI)VzLHIl(piC)Figure16.PhotosyntheticgrowthofwildtypeSBIOO3,zLHII(pBACDE),L\LHII(pRK415::2)andALHII(pAC)atdifferentlightintensities.(Seekeyforsymbols)A)200Em2s.B)100iEm2-s.C)60Em2s.D)30tEm2s.01 0354RC it is reemitted as fluorescence. As shown in Figure 17, the control strainRC6(pA4) fluoresces strongly because it contains LHII complex but no LHI orRC. However the fluorescence detected from z\LHII(pzE) was not greater thanthat of ALHII(pBACDE) or ALHII(pAD).e. Effects of light intensity on LH complex levelsLHII complex levels are known to increase by a post-transcriptionalmechanism as illumination levels decrease [931. This response can be seen in thewild type strain SBIOO3 (Fig. 18A). The area under the LI-ill absorption peak at800 nm increased 2.5 fold as the light intensity dropped from 200 to 30 Em2s1,where the maximum amount of LHII complex was reached. A decrease in LHIIcomplex levels was seen at the light intensity of 15 Em2s.The pseudo-wild type strain ALHII(pBACDE) (Fig. 18B) showed a similarpattern of increasing LHII complex levels as light intensity decrease from 200 to30 Em2s1,but the absolute amounts of the complex were approximately 60%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 todecreasing illumination [27], in Figure 18C it can be seen from the absorptionpeak at 870 nm that there was little difference between the LHI levels whenstrain ALHII(pRK4I5::12) was grown at light intensities ranging from 15 to 200iEm2s’. In strain ALHII(pzC), however, the LHI levels increased steadily aslight intensity dropped from 200 to 60 tEm2s1(Fig. 18D). At 200 Em2s1the55A12345B12345- —- —•..•.••.Figure 17. Fluorescence emission of R. capsulatus strains. Cells were seriallydiluted 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 3ALHII(pBACDE); Row 4: ALI-ffl(pAD); Row 5: zLFffl(pAE).56Figure 18. Spectra of intact cells grown photosynthetically at 200 .tE.m-2•s-’, 100p.E.m-2.s-l, 60 i.tE.m-2•s-’, 30 .iE.m-2•s-1and 15 tE.m-2s-. The light intensity isindicated to the right of the major peak. All spectra were normalised to the sameA650 A) SB1003, B) ALHII(pBACDE), C) ALHII(pRK415), D) ALHII(pAC).57A 30A B60II60AriIl \ 302- 15fj 200 1002000-2-C D306015301- 100200 60100200.5-j0-I I I i I I700 800 900 1000 700 800 900 1000Wavelength (nm)58amount of LHI complex in this strain was much lower than in strainALHII(pRK415::2), which might explain why the pucC deletion mutant grewmore slowly (see Fig. 15). At the lower light intensity of 60 Em2s’, however,the amount of the LHI complex was 76% of that in strain LHII(pRK415::2) butthis relative increase in LHI complex level in strain ALHII(pzC) was notaccompanied by an improved photosynthetic growth rate (see Fig. 16). At lightintensities of less than 60 Em2s1the slow growth rate provided enough of adisadvantage to the pucC mutant that cells containing second site suppressormutations out-grew the primary mutant. This is reflected in the spectra for thesecultures (Fig. 18D).f. Isolation of strains with secondary mutations that suppressed pucC deletionThe impaired photosynthetic growth of strains ALHII(pACDE) andALFffl(pzC) apparently provided an appreciable selective advantage for cellscontaining second site suppressor mutations. When these strains, especiallyALHll(pzC), were grown photosynthetically at light levels lower than 60Em2s’ faster growing secondary mutants often arose, distinguished by amarked increase in the slope of the growth curve and, occasionally, by anunusual greenish colour.When plates streaked from unusually fast-growing ALHII(pzCDE) orALHII(pAC) photosynthetic cultures were incubated aerobically at least twocolony types were seen: pink colonies similar to the original ALHII(pACDE) orALFIII(pAC) strains, and darker red colonies. A total of five second site59suppressors of pucC deletion were isolated from photosynthetic cultures grownat 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 allfive suppressor mutants I determined that the site of the second mutation wasnot on the pAC (or pACDE) plasmid by isolating the plasmid and transferring itback to LHII. In all cases these back crosses had the phenotype of the primarymutant. The suppressor strains varied considerably from each other and aredescribed in more detail below.g. Phenotypic characterisation of suppressors of pucC deletionStrain ALHII-1(pAC) regained only a moderate amount of LHII complex (Fig.19B) compared to strain ALHII(pBACDE) (Fig. 19A), and as shown below therelative amounts of LHI and LHII varied considerably. The ability of this strainto grow photosynthetically was improved compared to the parental strainALHII(pAC) but still lagged behind that of wild type cells at light intensities of lessthan 100 tEm2s1(data not shown). This retarded growth at low light levelsmade this strain unstable: for instance plates spread from cultures grown at 5tEm2s’ showed a mix of colony types indicating that the strain had mutated atadditional site(s). These triple mutants were not characterized further.The occurence of tertiary mutants sometimes made it difficut to tellwhether the strain had mutated or if its amount of LHII complex was repressedto an unusual degree at high light intensities. An experiment was designed tosystematically test this notion by growing identical inocula photosynthetically at60Figure 19. Spectra of intact cells of secondary suppressor strains grownphotosynthetically. For reference, the spectrum of the primary mutant,ALHII(pAC) or ALHII(pACDE), is shown with each strain (fine line) and thepseudo-wild type strain ALHII(pBACDE) is given in (A). The secondary mutantsshown are: B) ALHII-1(pzC); C) ALHII-2(pACDE); D) zLHII-3(pz\C);E) zL1-llI-4(pAC); F) ALHII-5(pAC).612-B0—2-1.5- CC1.5*0I I I I I700 800 900 1000 700 800 900 1000Wavelength (urn)62light intensities ranging from 15 to 200 Em2s1. The relative amounts of thetwo LH complexes varied considerably in strain zLH1I-1(pAC) (Fig. 20). Theshoulder at 870 nm was always very pronounced, indicating that the amount ofLHI 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. 18Band Fig. 20) but the levels of LHII increased to a greater extent in tLHII-1(pzC) asthe light intensity decreased from 200 to 30 tEm2s1. The level of LI-il complex,indicated by the absorbance at 870 nm, can also be seen to have increased in strainALI-ilI-1(pAC) as light intensity decreased. The absorbance spectrum of the cellsgrown at 15 Em2s4,in which a tertiary mutation arose, resembled a wild typespectrum in that the shoulder at 870 nm was much less pronounced than in theoriginal mutant. The absorbance value at 870 nm of this mixed culture grown at15 Em2swas lower than for ALHII-1(pAC) grown at 30 Em2s1suggestingeither that ALHII-1(pAC) has higher than normal amounts of LHI complex orthat the tertiary mutant that arose at 15 tEm2s1has a reduced amount of theLHI complex.The suppressor strain z\LHII-2(pACDE) had spectral characteristics similar tothose of wild type cells (Fig. 19C) and doubled at wild type rates when grownphotosynthetically at 300 or 30 Em2s1 (data not shown). When this strain wasgrown at very low light levels (5 tEm2s1)it actually had a shorter doublingtime than wild type cells (35 as opposed to 65 hours). This robust photosyntheticgrowth suggested it might be possible to clone the suppressor mutation from thisstrain by making a gene library which could then be used to complementLHII(pACDE) under low light photosynthetic growth conditions.Unfortunately, attempts to make such a library from ALHII—2(pzCDE) were63CCie1000Figure 20. Whole cell spectra of ALHII-1(pzXC) cells grown photosynthetically at200 jiE.m-2.s-I (blue), 100 jiE.m-2.s-l (green), 60 .tE.m-2s-1(pink), 30 jiE.m-2.s-1(turquoise), 15 tE.m-2.s-l (black). All spectra were normalised to the same A650.301560100200700 800 900Wavelength (nm)64unsuccessful.Suppressor strain ALHII-3(pAC) regained LHII complex levels inapproximately wild type proportion to LHI levels, judging by the size of the LHIshoulder of absorption at 870 nm (Fig. 19D), although the peak heights were onlyabout 50% of wild type. Unusually, this strain seemed to be unstable underaerobic conditions. Dark coloured colonies from the original mixed culture weredifficult to propagate in aerobic cultures or on aerobically-incubated streak plates.A plate spread with cells from the original (mixed) culture which was incubatedphotosynthetically and then stored aerobically initially had both pale and darkcolonies. The pale colonies continued to increase in size while the dark coloniesdid not. Light pink and green colonies then appeared at the periphery of some ofthese dark colonies. For this reason strain tLHII-3(pzC) was subsequently onlypropagated under photosynthetic conditions where it appeared stable.Suppressor strain zXLHII-4(pzC) was isolated from a photosynthetic cultureof ALHII(pAC) grown at 30 tEm2 When a sample from this culture wasspread on plates and incubated photosynthetically to isolate the secondarymutant several types of colonies were seen instead of two as expected. Somevariation may have been due to differences in light levels on different regions ofthe plate, but there were clearly small, pale colonies resembling the primarymutant, small dark colonies which had regained LHII complex and large, palecolonies. Pure cultures of this last type had no LHII complex (Fig. 19E), butnonetheless grew well photosynthetically. This improved growth wasapparently due to an elevated amount of RC and LHI complex. This is the strainthat was retained as tLHII-4(pAC).65The secondary mutant strain z\LHII-5(pAC) was isolated from a samplegrown at 15 tEm2s1 in the same experiment that generated ALHII-4(ptC), soboth cultures were started from the same low oxygen inoculum. Only onesecondary mutant was isolated from this sample, and its spectral characteristicsresembled those of pure cultures started from the small dark colonies that wereco-isolated with ALI-llI-4(pAC). The LHII complex levels in this strain variedconsiderably. When grown photosynthetically at 15 tEm2s1the cells hadapproximately wild type levels of LHII complex. At higher light intensities,however, the levels of LHII absorption at 850 nm are slightly lower than the LHIabsorption at 870 nm (Fig. 19F). The pronounced shoulder on the 800 nm peakwas associated with the culture supernatant; when cells were removed bycentrifugation the culture medium appeared blue and a broad peak at 760 nmwas seen. This soluble pigment is probably a degradation product ofbacteriochlorophyll present in stationary phase cultures.g. ConclusionsA chromosomal deletion of the puc operon was made that eliminated LHIIcomplex absorption from induced cells. This deletion strain, ALHII, retainedhigh levels of the LHI complex and was able to grow photosynthetically at ratesapproaching those of wild type cells at levels of illumination greater than 30tEm2s1(Fig. 16). As light intensity fell below this value a significant differencewas apparent, and at the lowest light intensity tested these cells grew much moreslowly than wild type. When complemented with a plasmid-borne copy of the66puc 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 ormore of the pucC, pucD and pucE genes to determine their involvement inLI-ifi complex formation. The mutant strains were evaluated for the presence ofLH complex absorption peaks in the near infrared and for their ability to growphotosynthetically.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 bedetected between strain ALI-llI(pAD) and strain ALHII(pBACDE) was a slightreduction in photosynthetic growth rate at the lowest level of illuminationtested. 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 slowlyunder most light intensities than ALHII(pRK415::2) despite retaining some LHIIcomplex. The exception to this was at 5 Em2s1where ALHII(pRK415:2)barely grew but ALHII(pAE) grew reasonably well after a long lag period. Thereduction in photosynthetic capability at light intensities greater than 30tEm2s1did not seem to result from inefficient transfer of light energy fromLHII to the rest of the photosynthetic unit because no increase in fluorescencewas detected in ALHII(pAE) cells compared to the pseudo-wild type strain (Fig.17).67Of the three genes deleted only pucC was absolutely required for LHIIcomplex formation. Deletion of pucC alone or along with pucD and pucEabolished the LHII complex from induced cells (Fig. 13) and greatly diminishedthe cells’ ability to grow photosynthetically (Fig. 15). This was not a generalphysiological impairment, however, because the cells grew at normal rates underconditions of low aeration where the puc operon is induced but the cells derivetheir energy from respiration. The strains lacking the pucC gene had reducedlevels of LHI complex compared to ALHII(pRK4I5::Q) (Fig. 13), which couldaccount for their poor photosynthetic growth. The relative abilities of the twostrains to grow photosynthetically did not parallel their respective levels of LHIcomplex, however. At the low light intensity of 60 Em2s1 the levels of thecomplex were comparable in the two strains (Fig. 18) but this relative increase inthe amount of the LHI complex in z\LHII(pAC) was not accompanied by acorresponding increase in photosynthetic growth rate (Fig. 16). In fact, at evenlower light intensities the slow growth rate provided enough of a disadvantagethat cells containing second site suppressor mutations frequently came topredominate in cultures.Five such suppressor mutants were isolated in separate experiments inwhich 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 pucoperon deletions. Three of these secondary mutant strains, ALHII-1 (pAC), ALHII3(pAC) and ALHII-4(pAC) had radically different phenotypes, suggesting thatmultiple sites can be mutated to suppress the loss of pucC. Interestingly, two ofthese strains were unstable. zLHII-1 (pAC) only partially restored LHII complexlevels (Fig. 19B) and generated tertiary mutants under conditions of low lightphotosynthetic growth. ALHII-3(pAC) had moderate levels of LHII complex (Fig.6819D) but was unstable when grown aerobically. Finally, strain tXLHII-4(pAC) grewwell photosynthetically without restoring LHII complex, perhaps because it hadelevated levels of the LHI and RC complexes (Fig. 19E). This result, along withthe growth characteristics of ALHII(pRK4I5::Q), showed that the reduction in thephotosynthetic growth rates of pucC mutants was not due solely to the loss ofLHII complex.692. PucC structure and functiona. Hydropathy and positive inside rule analysis of PucCIn order to gain some insights into the functional domains of the pucCgene product the structure of the PucC protein was studied. The predictedproduct of the pucC gene is extremely hydrophobic [79] and is almost certainlyan integral membrane protein, but when the amino acid sequence was analysedby several hydropathy algorithms there was considerable disagreement over theassignment of transmembrane segments (Fig. 21). Green [281 examined theability of several hydropathy programs to correctly predict the transmembrane xhelices of the Rhodopseudomonas viridis reaction centre L and M subunits,which are known from X-ray crystallography data. She concluded that althoughno one program unambiguously identified all the membrane-spanningdomains, the consensus of several programs gave fairly accurate results.Analyses using Green’s program ENDIT and several algorithms in thecommercial package PCGENE led to a theoretical model for PucC and aredescribed below.Along with hydropathy predictions of membrane-spanning helices, thedistribution of positively charged amino acids must also be considered accordingto the “Positive Inside Rule” of von Heijne [811. This principle states thathydrophilic loops containing clusters of basic amino acids are generally restrictedto the cytoplasmic side of the cell membrane. Interestingly, only the absolutenumber of positively charged residues is relevant; there is no correlation seen inthe distribution of acidic residues nor is net charge important. For this reason70AELZEZIt— ——ft —- -B—1----I•• S • •epe . . S • •• I •S If SC.. S a IS•• I a S SW IS a 5S •I SD100 200 300 400I I IAmino Acid ResiduesFigure 21. Transmembrane domains (boxes) predicted by various hydropathyanalyses. Shaded boxes represent weak predictions, or, in the case of theEisenberg algorithm, membrane associated helices. Dots below each modelindicate the position of an arginine or lysine residue. The software packagePCGENE was used to predict integral membrane domains using the methods ofA) Kyte and Doolittle [381 (SOAP), B) Eisenberg [201 (HELLXMEM), and C) Raoand Argus [62] (RAOARGUS). The Chothia analysis (D) was run using Green’sprogram ENDIT [281. The positions of amino acid residues are indicated at thebottom of the figure.71the position of lysine and arginine residues in PucC are shown as filled cirdesunder each hydropathy prediction in Figure 21. Application of the positiveinside rule indicated that the clusters of basic residues at both termini andaround positions 1.00, 240 and 330 were likely to be in cytoplasmic domains.PucC was first analysed using the SOAP program of PCGENE. This methoduses the hydropathy scale of Kyte and Doolittle in which the amino acids areassigned a hydropathy index that reflects the free energy of transfer from thewater to vapour phase, the probability of being buried in the interior of a proteinand, in some cases, the authors’ personal bias [381. SOAP modifies Kyte andDoolittle’s original analysis by first identifying the most hydrophobic 17 aminoacid segment in a protein sequence. The segment, and therefore the protein, isthen classified as integral (I) rather than peripheral (P) if the average hydropathyacross the 17 residue window is above a cutoff value that was arrived at byexamination of a database of proteins known to be either peripheral or integralmembrane proteins [34]. If an integral segment is identified it is saved andremoved from consideration while the analysis continues on the remainder ofthe sequence until no more integral domains are found. Figure 21A shows thenine hydrophobic domains (boxes) predicted by SOAP. The open boxes representhydrophobic domains with a predicted P:I ratio of less than 0.05, while theshaded boxes are hydrophobic domains less likely to be membrane-spanningsegments according to this algorithm. This method seemed to overlook sometransmembrane segments. There are two clusters of basic residues at the Nterminus of the protein which should be separated by an even number oftransmembrane sequences according to the positive inside rule. The interveningsegment should therefore cross the membrane twice or not at all. The P:I72probabilities 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 allthe other clusters of basic residues from each other. The suggestion that thisalgorithm overlooked transmembrane domains was strengthened when theresults of other methods were examined.The other three hydropathy scales used predicted eleven hydrophobicdomains in common even though they are based on different principles.The Eisenberg consensus scale, used to predict the average hydropathy overa sliding window of 21 residues, is a composite scale that averages four previoushydropathy scales [20]. It was intended to minimize artifacts built into otherscales because of choice of data base or other non-random factors. Twelvehydrophobic domains were predicted in PucC using this scale (Fig. 21B). ThePCGENE program HELIXMEM then classified these segments as globular (shadedboxes) or transmembrane (open boxes) according to their average hydrophobicity.The cutoff distinguishing the two classes is somewhat arbitrary, and all threedomains classified as globular were sufficiently hydrophobic to be in theuncertain range. To satisfy the positive inside rule all three of these weaklyhydrophobic segments must cross the membrane, or an adjacent stronglyhydrophobic segment must not.The RAOARGUS algorithm of PCGENE predicted 11 transmembranehelices in PucC if the first 53 residue hydrophobic segment was considered toconsist of two domains (Fig. 21C). This method is based on the probability of a73given amino acid being found in a membrane-soluble helix versus elsewhere ina membrane-associated protein [62]. The authors derived this “buried helixparameter” by first predicting transmembrane helices for a group of knownintegral membrane proteins using complicated and diverse criteria. The buriedhelix parameter for each amino acid was then calculated simply as the ratio ofthe frequency of its occurrence within these predicted transmembrane domainsto its frequency at any position within the integral membrane proteins in theirdatabase. Candidate integral membrane proteins are analysed by plotting theburied helix parameter for each residue and smoothing the curve with a sliding-average window of 7. Transmembrane helices are identified as peaks meetingcut-off values for height and width which were assigned somewhat arbitrarily byanalysis of a limited number of sequences for which transmembrane domainshad been determined by other means. Although the derivation of the method issomewhat tortuous, all the transmembrane helices predicted for PucC by theRAOARGUS program were also predicted by the Eisenberg and Chothiaalgorithms (Fig. 21B, C, and D), and except at the C-terminus were consistentwith the positive inside rule. The C-terminal region is considered in more detailbelow.The final program used to analyse the PucC sequence was ENDIT, written byBeverley Green [28]. ENDIT permits analysis using several hydropathy scales forsliding windows of two sizes, and additionally calculates the probability of omegaloops, alpha and beta turns and N- and C-terminal caps. Several hydropathyscales were used to analyse PucC, including the Eisenberg consensus and theKyte-Doolittle scales described above. The predictions of the Chothia free energyscale were analysed in the greatest detail and combined with turn and cap74predictions to generate the model shown in Figure 21D.The Chothia free energy scale is based on an analysis of the distribution ofthe 20 amino acids in crystal structures of soluble globular proteins [53]. Thefrequency of a particular amino acid occuring on the surface of the protein wascompared to the frequency of its volume being less than 5% accessible to exteriorwater, the authors’ definition of an interior location. The ratios of thesefrequencies were then used to calculate implied free energies of transfer from theinterior to the surface for each amino acid. Amino acids defined as hydrophobicby chemical criteria generally had a large free energy values while charged aminoacids had negative free energies for such a transfer. This scale was used to predicthydrophobic segments in PucC. A large window (19 aa) was used to identifyhydrophobic segments long enough to be membrane-spanning helices. An 11amino 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 definethe ends of membrane-spanning helices. Cap predictions were based on relativefrequencies of particular amino acids at residues adjacent to alpha helices in thestructures of soluble proteins [63], and as such might be of questionable valuewhen considering membrane associated helices. For the PucC protein strongpredictions of N-caps were less frequent than C-cap predictions, and neveroccurred within hydrophobic segments. This was not that surprising sinceasparagine and proline residues are strongly favoured in calculating N-capprobabilities and are not commonly found within hydrophobic cc-helicalsegments. However while Richardson and Richardson found that these residueswere much more common at the N-terminal end of soluble helices than at the75C-terminal end, in my analysis strong N-cap predictions coincided more oftenwith the C-terminal end of predicted hydrophobic helices. The N-terminalboundaries were more difficult to predict. They sometimes coincided with astrong prediction of a C-cap but these were so common along the length of theprotein sequence that this could have been fortuitous. In practice, the regionclose to the boundaries of a hydrophobic segment was examined and the residuecorresponding to the strongest prediction of a cap or alpha turn was chosen as theouter boundary.The results of this analysis are shown in Figure 21D, where open boxesrepresent the more hydrophobic predicted transmembrane domains and shadedboxes the less hydrophobic ones. Twelve potential membrane-spanning heliceswere predicted and, as with the Eisenberg prediction, all of the weaklyhydrophobic segments are required to cross the membrane in order to satisfy thepositive inside rule.In sum, comparison of the four hydropathy analyses along with therestrictions of the positive inside rule indicated that the first 8 hydrophobicdomains predicted by both the Eisenberg and Chothia scales, most of which are inagreement with the other two predictions (see Fig. 21), were strong candidates fortransmembrane 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 4membrane-spanning helices from this theoretical analysis.76b. Construction and analysis of pucC’:.pho’A fusionsThe theoretical model of PucC topology was tested using the genetic systemof phoA fusions [48]. The phoA gene encodes alkaline phosphatase whichrequires the formation of disulfide bonds for activity. These bonds cannot formin the cell cytoplasm, a characteristic which has allowed phoA to be used as areporter gene in topological analyses of integral membrane proteins [47].Typically, several translationally in-frame fusions are made between N-terminalsegments of the gene of interest and a truncated phoA which lacks translationalstart and leader peptide sequences. When the alkaline phosphatase activities ofsuch fusion proteins are assayed high activity generally indicates that the fusionsite occurred in a periplasmic domain.A collection of fragments having the same 5’ but different 3’ ends wasgenerated by exonucleolytic digestion from the 3’ end of pucC (see Materials andMethods). The fragments were then shotgun cloned into the plasmidpUC19::phoA, which has phoA (without its translational start and leaderpeptide sequences) inserted in the multiple cloning site of plasmid pUCI9 [8].The phoA deletion strain CC1 18 was transformed with the ligation products andused to screen for alkaline phosphatase activity on agar containing thechromogenic substrate XP.Initially, only blue colonies were screened by restriction digestion analysis,followed by sequencing across the fusion joint. Essentially all the blue coloniesscreened contained translationally in-frame fusions of pucC to phoA. Theexceptions to this were colonies which stained extremely dark blue and77contained plasmids with no detectable insert. In the undigested plasmid thephoA gene is not in the same reading frame as the lacZ a fragment andcolonies appear white on )(P. If the reading frame is altered to allow translationof alkaline phosphatase from the lacZ a start codon blue colonies result (datanot shown). I cannot explain this observation since translation through themultiple cloning site is not expected to generate an effective leader peptide.It was found that the fusion sites were not dispersed over the entire genebut instead clustered at approximately 200 base pair intervals, so white colonieswere then screened. Since both low activity and translationally out-of-framefusions would be expected to give white colonies, only plasmids which hadinserts of sizes not found amongst the blue colonies were sequenced. Five out of34 white colonies whose plasmids were sequenced contained fusions which weretranslationally in-frame. In all, 44 different translationally in-frame fusions ofpucC to phoA were generated. These results are summarized in Table 3.In order to avoid ambiguous results associated with, for example, fusionshalf-way through a transmembrane segment, the fusions most likely to reside inregions predicted in the foregoing theoretical analysis to be hydrophilic werechosen for alkaline phosphatase assays. All the fusions in the C-terminal regionof PucC were also analysed to resolve the ambiguities of the theoretical analyses.As shown in Table 3, there was generally an approximately 20-fold differencebetween high activity fusions and low activity fusions which is typical of therelative levels of activity for periplasmic vs. cytoplasmic fusions [67]. Thisdifference was too great to be likely to be due to differences in the rate ofsynthesis of the different fusion proteins, especially when the size andconsistency of the data set were taken into account. Synthesis rates of fusion78Table 3. Summary of translationally in-frame fusions between puc’C and phoA’. Residues whichdiffer from the sequence published by Tichy et a!. [791 are underlined. Alkaline phosphataseactivities are given in relative units and are the average of at least 3 different experiments.Fusion Sequence1(last intact codon)2 Colony Alk PhosColour3 ActivityS8-7 ACC GTC GGG ATG ACC TTG AC (48) BS8-12 GTC GGG ATG ACC TTG ACC CT (49) BS8-1O GTG ATG ATC GTC GAG ITG GC (63) B 137S8-6 GAG ITG GCG GTT CCG GCC TC (67) BS8-8 CTC GTC TC GTG ATG CTG GC (74) BS8-3 TC GTG ATG CTG GCG ATG CC (76) BS8-11 ATG CTG GCG ATG CCG ATG CT (78) BS4-20 ACG CTG ATC GGC TTN AAG TC (90) W 4.7S4-15 CCC TGG ATC TGG AAG GGA AC (109) W 4.5S2—9 CCC TTC GCA CTT CTG GTG CT (126) BS2-7 TTC GCA CTT CTG GTG CTG TC (127) BS4-7 TCG GGQ TTC GG GAA TCC GT (133) B 116S4-9 TTC GG GAA TCC GTG GAT GC (135) BS4-6 GG GAA TCC GTG GAT GCG CC (136) B 151S8-5 CTG GCA ACC GAC CTC GTC GC (171) B 30S2-2 TAT GTG ATG CTG CTC TTC GG (189) BS2-5 CTG CTC TTC GGC ATG GTG AT (192) BS2—8 ATG GTG ATC AGC GCG CTC (196) BS2-10 AGC GCG CTC C TAC GGG GC (199) B 104S2-6 CTG GCG GAC TAC ACG NNC GN (207) B 108E8-29 TTC AG GAG GCC TTC GGC CT (256) w 3.7E8-26 GGC CGT CCG GGG ATG CTG GC (265) WE8-3 GCG CTG CTG ACC GTG ATC GC (271) BE8-1 ATC GCG CTG GGA ACG TTC GG (276) BE8-2 GCG CTG GG ACG TTC GGC TC (277) BE8-5 GAA CCC TAI GG GGI CAG GC (291) B 101E8-25 AAC GGG GCA AGG CCG ATG CQ (329) W 4.2E4-9 GGG TGC ACT GAT CGG GTT CC (339) BE8-6 CGG GTT CCC GGG TTT GTC GC (343) BE4-5 TTT GTC GCC ATC ATI ATG TC (347) BE4-31 GTC GCC ATC AT ATG TCC TC (348) BE8-9 AGC CA G GGT ATC TGG IT (357) B 148E4-6 G GGT ATC TGG ITG TTC CT (359) B 11879E4-2 TGG TTG TTC CT GIG GGC AC (362) BE8-7 TTC CT GIG GGC ACC TTI GC (364) BE4-27 NNA TGG GGG GCN GTG CAG GC (401) B 15E4-12 GGG GCN GTG CAG GCG ACG GC (403) B 35E4-30 GCC GG TG GG GTC GCG CT (410) B 51E4-25 GAC GGT TTG GTT GCC CTG CC (422) B 86E4-22 TTG GTT GCC CTG CCG GGG AC (424) B 77E4-26 ACT TTC GGG TCC GGT GTG GQ (431) B 59E4-1O GGG TCC GGT GTG GQG GGG CC (432) B 49E4-29 GTG GG GGG CCT TAT AAT AC (435) B 33E4-43 ACG GTG TTC GCC ATC GAG GC (441) B 641. 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 underlinedsequences represent the first two codons of phoA.3. B: blue; W: white colony on XP.80proteins can differ, but significantly different rates of synthesis are normallyassociated with toxicity of the fusion protein [671. No evidence of toxicity wasseen with any of the fusions in this study. The only apparently anomalous pointin the N-terminal part of the protein, the value of 30 for fusion S8-5, wasassigned to the cytoplasmic side of the membrane for three reasons. First, inmany experiments of this type it has been found that fusions in cytoplasmicloops that are not preceded by a basic residue seem to be exported to theperiplasm at a low rate [10], possibly because a positive charge is needed to anchorthe hydrophilic domain in the cytoplasm [10]. Second, the two regions flankingthe hydrophilic region that contains fusion S8-5 are predicted to be the mosthighly hydrophobic regions of the protein, and therefore could reasonably beexpected to cross the membrane. Thirdly, the high values obtained with fusionsin the hydrophilic segments located on either side of this fusion indicatedperiplasmic locations; thus it seemed more likely that the alkaline phosphataseactivity of this fusion was an abnormally high activity cytoplasmic fusion ratherthan a low activity periplasmic fusion, and this fusion site was assigned to thecytoplasmic side of the membrane.The theoretical analyses allowed three possible assignments oftransmembrane helices in the C-terminal part of the PucC protein. Figure 22shows the potential membrane-spanning domains (boxes) and indicates whetherthe fusions, indicated below (Fig. 22D), would be predicted to have high (thicklines) 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, whichwere picked by all four algorithms. This possibility, and the second alternativethat the four helices predicted by Eisenberg exist (Fig. 22B), were ruled out by the81A II IF—B rC IHI III F—6451 33118 35 77 494.2 148 15 86 590 I II III 111111E8-25 E8-9 E4-27 E4-25 E4-26E4-6 E4-12 E4-22 E4-1OE4-30 E4-29E4-43Figure 22. Possible arrangements of transmembrane helices (boxes) in the C-terminal 150 amino acids of PucC. Thin lines represent predicted cytoplasmicdomains and consequently low Alkaline phosphatase (AP) activity. Thick linesrepresent predicted periplasmic domains which would be expected to have highAP activity. The positions of amino acid residues are indicated below the figure.A) The pair of membrane spanning helices predicted by all 4 hydropathyalgorithms (see Fig. 21). B) The Eisenberg prediction. C) The Chothiaprediction. D) The positions of pucC::phoA fusions in this region. The fusiondesignation is indicated below the line, while the relative units of AP activity areshown above.-f330+360+390+420+45082high activities seen in fusions E8-9 and E4-6, the low activity of fusion E4-27 andthe indirect evidence of the absence of fusions found between residues 364 and401, despite extensive screening of blue colonies. Additional evidence againstthe 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 highalkaline phosphatase activity, consistent with their predicted periplasmiclocation. The AP activities of fusions P4-27 to E4-25 increased steadily, as hasbeen seen for fusions within “outgoing” transmembrane helices [471. Theremaining fusions did not help to distinguish between the models, but were notinconsistent with any of them.The results of the alkaline phosphatase fusions, taken together with thehydropathy predictions and the distribution of positively charged residues, led tothe 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 thecytoplasm. Cytoplasmic loops tend to be longer than periplasmic ones, and mostof the protein is embedded in the membrane.c. Sequence differences between wild type strains SB1003 and 3Th4When Tichy et al. [791 reported the extended sequence of the puc operonfor R. capsulatus strain 37b4 they found only a few differences between theE8- SFigure23.AmodelofthetopologyofthePucCproteinusingthesingleletteraminoacidcode.Theperiplasmicsideofthemembraneisabove,thecytoplasmicsidebelowthemembrane.Positivelychargedaminoacidsarecircled.Thepositionsoffusionsimportantindeterminingtopologyareindicated;theiractivitiesarelistedinTable3.ResiduespredictedfortheSBIOO3sequencewhichdifferfromthepublishedPucCsequencefromstrain37b4areshowninlowercaseletters.84previously published sequence of pUCBA from strain SB1003 [901 and the part oftheir sequence that overlapped it [79]. One of these discrepancies, an extra pair ofC’s at position 833 in Tichy et al’s numbering, predicts that DNA from strainSB1003 but not 37b4 will cut with the restriction enzyme Bsm I. I found that thisenzyme will not digest puc DNA from SB1003 at this site (unpublished data), sothe SBIOO3 sequence probably agrees with that of 37b4 in this location. Insequencing the pucC’::pho’A fusion joints, however, a number of differenceswere noted between the sequence of pucC published for strain 37b4 and thesequence of the strain used here, SBI 003. The differences are marked in Table 3as underlined bases, and have only been included if they were consistently foundin at least two independent fusions. Very few of the sequence differences resultin amino acid changes; those that do are listed in Table 4 and shown as lowercase letters in Figure 23. Most of the changes are conservative and do not causethe hydropathy predictions to change when the revised PucC sequence isanalysed. The predicted aspartic acid to glycine change at position 354, however,increases the hydropathy of this region enough that the transmembrane helixpredicted by RAOARGUS (see above) at positions 355-382 is extended to 341-382and HELIXMEM predicts an additional membrane associated helix at residues331-351. These predictions are in better agreement with the model derived fromphoA analysis of SB1003 PucC topology than the original hydropathy analysesconducted on the 37b4 sequence. The only other nonconservative amino acidchange predicted, lysine to glutamine at residue 252, does not affect the topologypredictions as it occurs in a hydrophilic segment containing five additional basicresidues.85Table 4. Amino acid differences betweenthe predicted protein sequences of PucCfrom strains SBI.003 and 37b4Residue 37b4 SBIOO3197 valine threonine252 lysine glutamine332 serine glycine354 aspartate glycine361 alanine valine407 leucine valine431 valine alanine86d. Deletion analysis of pucCC-terminal deletions of PucC were created to determine whether truncatedPucC retained function (see Materials and Methods). ALHII(pL\C) wascomplemented in trans with a compatible plasmid carrying the full-lengthpucC gene (pPQ::C) or pucC alleles truncated near the C-terminus. Expressionof pucC was driven by the oxygen-regulated puf promoter [1].Expression of the full-length pucC gene in trans to z\LHII(pAC) restoredLHII complex, although not to wild type levels (Fig. 24A, inset). The proportionof LHII to LHI complex, judged by the lack of obvious shoulder at 870 nm, weresimilar to that of ALHII(pBACDE) when the cells were grown under lowaeration, but when ALHII(pt\C; pPQ::C) was grown photosynthetically at 100Em2s1the spectrum showed an unusually high proportion of LHI complex(Fig. 24B, inset).None of the deleted pucC alleles, which removed one (pPQ::E4-33 andpPQ::E4-36) or two (pPQ::E4-38) of the last transmembrane domains, restored LHIIcomplex to strain zLHII(pt\C) (Fig. 24A). In photosynthetic growth experimentsthe ALHII(pAC) strains complemented with the deletions did not grow fasterthan the vector control (Fig. 24C), although the amount of LHI complex in thesestrains increased compared to ALHII(pAC; pPUFP) (Fig. 24C).87Figure 24. Complementation of ALHII(pAC) with truncated pucC alleles.Spectra of whole cells grown under conditions of low aeration (A) orphotosynthetically at 100 tEm2s1 (B), and the photosynthetic growth curve ofthese cells (C). The insets show strains z\LHII(pBACDE) (fine line) andiXLHII(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).88A B1.5-2-2-II /\1 0aad,e1- CC bbdC.5-0- I I I I700 800 900 1000 700 800 900 10001000-Wavelength (nm)CcdpPQ::Cz• pPUFP. ioo-x pPQ::E4-33z pPQ::E4-36o pPQ::E4-38U10—I I I I I I I0 10 20 30 40 50 60 70 80Time (hours)89e. Role of PucC in LHI assemblySeveral observations suggested that deletion of pucC affected levels of theLHI complex as well as eliminating the LHII complex. First, the pucC deletionstrain zLHII(pAC) generally had lower levels of LHI complex thanALI-llI(pRK415::2) (see Fig. 14). The pzC plasmid carries a version of pucCtruncated at residue 193, midway through the fifth transmembrane domain,along with the pucBA and pUCDE genes whereas pRK415:2 has no pucsequences. The presence of the truncated pucC and/or the additional pucsequences thus leads to a lower level of expression of LHI complex than thecomplete absence of the puc operon. Second, when strain zXLHII(pzC) containedsome plasmids that expressed pucC’::pho’A fusions in trans it was found tohave different levels of LHI than ALHII(pAC) alone (see below). Different pucCalleles 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 complexformation because strain ALHII, deleted for the entire puc operon, appears tohave high levels of the LHI complex. However it was conceivable that PucC hasa small role that might be revealed in strains containing a second mutation thatcaused low levels of LHI complex. Therefore a pucC expression plasmid wasintroduced into strain ALT-ill and two derivatives which have deletions of thegene F1696. F1696 shows strong homology (47% identity) to PucC, and F1696mutants are known to have decreased levels of LHI complex [87]. In anotherwise wild type background LHII complex levels are not affected by deletion90of 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, thuslack the entire puc operon and due to the F1696 disruption have only 20% of theamount of LHI complex seen in z\LHII.LHII, zStu and tNco were complemented with plasmid pC. The only pucgene on this plasmid is pucC, and it has been shown to complement the LHIIstrain MW442 [7[] thought to have a point mutation in pucC (unpublishedresults). Transcription of pucC is driven by the puf promoter carried on afragment which includes the pufQ gene. pufQ has been implicated inbacteriochlorophyll biosynthesis [4], so two control plasmids were alsointroduced into the three chromosomal backgrounds: the pRK4l5 vector andpJAJ9, which carries the same pufQ fragment as pC [29].In Figure 25 it can be seen that neither pucC nor pufQ expression led toincreased levels of LHI complex in the F1696 backgrounds tested. Smalldifferences were seen, but were not consistent between experiments, or fromstrain to strain. Wild type PucC therefore does not seem to participate directly inLHI complex formation, even in the F1696 mutants AStu and ANco.Several different pucC deletion alleles had been cloned into a vectorcompatible with pAC such that they would be transcribed from the pufpromoter. Three of these alleles were derived from translationally out-of-framepucC’::pho’A fusions and had been shown to be incapable of restoring LHII910.0Wavelength (nm)Figure 25. Complementation of F1696 mutant strains with pucC. Whole cellspectra of strains grown photosynthetically at 120 Em1.A) ALHII(pRK4I5)B) ALHII(pJAJ9) C) zLHII(pC) D) ANco(pRK415) E) ANco(pJAJ9) F) ANco(pC)G) AStu(pRK415) H) AStu(pJAJ9) I) AStu(pC)A1-B CD E FG II I.4700 800 900 100WOO 800 900 100WOO 800 900 100092complexes 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, Inoticed that one of them, S8-1O, caused the cells to be unusually pale. Spectra oflow oxygen-grown zLHII(pAC; pPQ::58-1O) had dramatically reduced LHI and RCabsorption peaks (Fig. 26A). Comparison of all four of these in-framepucC’::pho’A fusions in trans to pzC showed that LHI absorption correlatedroughly to the length of the PucC sequences remaining in the fusion. It thusseemed that these truncated pucC alleles interfered with LHI assembly and thatshorter peptides had more effect than longer ones. When these fusions wereconjugated into strain zLHII in the absence of pzXC, however, all the strains hadamounts of LHI complex similar to the vector control (Fig. 26B). Thusexpression of truncated PucC per se does not affect LHI complex formation;instead the combination of the pAC plasmid and these pucC’::pho’A fusionsreduces the amount of LHI complex in cells.Because these strains had different amounts of LHI complex they provided adirect 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 differentlight intensities, that the strain’s growth rate does not correlate to its amount ofLHI complex (see Results, Ch. 1, section e). When z\LHII(pAC) wascomplemented with the translationally in-frame pucC’::pho’A fusions andgrown photosynthetically at 100 Em2s1the strains showed a gradient of LHIcomplex levels (Fig. 27A) similar to that seen with cells from low oxygen cultures(see Fig. 26A). Their photosynthetic growth rates did not correlate to their93Figure 26. Whole cell spectra of low oxygen grown strains containingtranslationally 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).AB1000700Wavelength(nm)f C e b dab,f40060080090095relative 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 theonly 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 toamount of LHI complex was ALHII(pzC; pPQ::S8-10) which had only 12% of theamount of LHI and RC complexes seen in zLHII(pAC; pPUFP) under low oxygengrowth conditions (see section d). This strain did not grow at allphotosynthetically, presumably due to its low RC content.f. ConclusionsTheoretical models for the topology of PucC were derived from fourhydropathy analyses combined with the restrictions of von Heijne’s positiveinside rule (Fig. 21). Three of the four hydropathy algorithms predicted 11similar transmembrane segments. The fourth hydropathy scale, that of Kyte andDoolittle, was less useful. It predicted only 9 of the 12 membrane-spanningsegments that were consistant with the phoA fusion analysis.The C-terminal region of PucC was difficult to analyse, being largelyhydrophobic without strongly hydrophilic intervening segments. Three possiblearrangements of 2 or 4 transmembrane helices could not be resolved withoutexperimentation.The consensus of these analyses predicted 10 or, more probably, 1296Figure 27. Strain ALHII(pAC) containing translationally in-frame fusions ofpucC’ to pho’A grown photosynthetically at 100 tEm2s1. A) Whole cellspectra: 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).97A1.5-a e1-b0d.5-0-400 600 800 1000Wavelength (nm)BcrLJ pPQ::C• pPUFP100 0 pPQ::E8-5L pPQ::E8-25• pPQ::E4-25pPQ::S8-1O10 I I I I I I I0 10 20 30 40 50 60 70 80Time (hours)98transmembrane domains, with both the N- and C-termini located in thecytoplasm. The theoretical models were tested experimentally using the geneticsystem of translationally in-frame phoA fusions.Alkaline phosphatase, the product of the phoA gene, can be used as areporter gene in topology analyses because it has greater activity when it istransported 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 beendeleted. Alkaline phosphatase activities of some of the fusions were assayed andthe resulting pattern of high or low activity (Table 3) was compared to thetheoretical models (Fig. 21). High activity correlated well with fusion jointslocated in regions predicted to be periplasmic. Predicted cytoplasmic loops weresites of fusions having approximately 20-fold lower alkaline phosphataseactivity. The only anomalous fusion occured between the fourth and fifthpredicted transmembrane domains, and had an intermediate activity. Thisregion was assigned to the cytoplasm for two reasons. First, the theoreticalanalysis predicted that the two flanking segments were transmembrane domainsas they were strongly hydrophobic, and the hydrophilic regions flanking themboth contained high activity alkaline phosphatase fusions. It was thusreasonable that they might cross the membrane and that the interveningsegment would be in the cytoplasm. The second justification for thisarrangement is that the anomalous fusion occurs in a region that is not precededby any basic residues. In the many phoA analyses that have been carried out ithas been noted that cytoplasmic fusions that are not anchored by positivelycharged amino acids are exported to the periplasm at a low rate and thus have99unusually high activities compared to other cytoplasmic fusions.The arrangement of transmembrane domains in the C-terminal region ofPucC was the most ambiguous part of the theoretical analysis. Testing ofpucC’::pho’A fusions in this region made it possible to distinguish which ofthree theoretically plausible models was most likely to be correct (Fig. 22). Themodels predicted different patterns of high and low alkaline phosphataseactivity, only one of which was consistent with the observed data.On the basis of the theoretical analysis and the information provided byactivities of pucC’::pho’A fusions a model for PucC topology was proposed inwhich there are 12 membrane-spanning domains and both the N- and C-terminiare located in the cytoplasm (Fig. 23). In the course of characterising the fusionsites, DNA sequence differences, some of which result in amino acid changes(Table 4), were noted between the strain used here and that reported in theliterature (Table 3). The theoretical and experimental data were in even betteragreement when the theoretical analysis was repeated for this slightly differentamino acid sequence.Expression of pucC in trans to ALHII(pAC) restored the LHII complexalthough 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 restoredetectable amounts of the LI-ill complex to this strain. The C-terminal region ofPucC is thus required either directly for function, or for proper folding or stability(and thus indirectly for function). Although these deletion mutants were unableto restore the LHII complex to ALHII(pAC), they did increase the amount of the100LHI complex seen when the cells were grown photosynthetically (Fig. 24B).A second set of pucC’ alleles, translationally in-frame pucC’::pho’Afusions, also influenced LHI complex levels in this strain (Fig. 26). One suchfusion, containing only the N-terminal PucC transmembrane domain, decreasedRC and LHI complex levels to 12% of strain tLHII(pzC; pPUFP). These fusionsdid not produce this effect in the absence of pAC, however, nor did expression ofpucC alone increase LHI complex levels in ALHII or in F1696 mutantderivatives which have an 80% reduction in LHI complex levels (Fig. 25). It wastherefore concluded that PucC does not directly participate in LHI complexformation and that truncated pucC alleles do not interfere with LHI complexassembly in the absence of the other puc genes carried on pzC.101Chapter 3: Analysis of puc operon transcriptiona. puc operon promoter mapping by deletion analysis of pucE’::lac’Z genefusions.Although previous work had shown that the pucC gene is probablytranscribed from the pucBA promoter [79; 78], it was not clear wheretranscription of the pucDE genes originates. Therefore, to test for the existenceand positions of other possible puc operon promoters, translationally in-framefusions of the pucE gene were made to a E. coli lac ‘Z allele (Fig. 28) in thepromoter-probe plasmid pXCA6O1 [1]. /3-galactosidase activities were measuredin cell extracts from R. capsulatus strain SBIOO3 containing the differentpucE ‘::lac ‘Z fusions, grown under either high or low aeration (see Materials andMethods). Figure 28 shows that approximately 90% of transcription of the pucEgene derived from transcription initiated upstream of pucB. The remaining10% of J3-galactosidase activity was lost when the region between the 3’ end ofthe 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 thispucE’::lac’Z fusion was approximately two-fold higher in cultures that wereoxygen-starved. This is in good agreement with the results obtained using apucB ‘::lac ‘Z fusion in cells grown under the same conditions [24].pBEZU-’‘‘8.5(.62)4.2(.36)D.ZEJ8.3(1.o)*pHEZ0.16(.07)Figure28.Specificactivitiesoffl-galactosidaseincellextractsfromwildtypeR.capsulatusstrainSB1003containingplasmidswithapucE‘::lac‘Zfusionprecededbydifferentamountsofupstreamsequences,asshown.Cellsweregrownunderconditionsoflow(Low02)orhigh(High02)aeration.ActivitiesareexpressedaspercentagesoftheactivityofSB1003(pPEZ)measuredinthesameexperiment.Valuesaretheaveragesof 3assaysandstandarddeviationsaregivenwithinbrackets.*Thesevaluesweremeasuredusingpermeabilisedcells(seeMaterialsandMethods).Theinvertedtrianglerepresentsinsertionof4nucleotidesthatcauseaframeshiftinthepucDreadingframe,boxesrepresentpucgenes,designatedabovepPEZandthepredictedRNAstemloopstructureisindicatedwithalollipop.BA0 TpPEZpPEZ-OOFpCEZC—I--0.-----‘Ii.Ui—IiiDE’—IIIIRelativeactivityLow02High02100(8.5)57(5.)83(21)*6.7(.51)6.4(.26)pBEZ-OOF)-‘II0.49(.02)C103b. Is there translational coupling between pucD and pucE?/3-galactosidase activity decreased approximately 10 fold when the regionbetween the 3’ end of pucC and the 5’ end of pucD was deleted (pBEZ vs. pHEZin Fig. 28). Normally this decrease in activity would be interpreted as evidencefor minor promoter activity in the deleted fragment. In this particular case,however, an alternative explanation was possible. The sequences of pucD andpucE overlap by four nucleotides so the pucE start codon (ATG) is buried in thepucD stop codon (TGA) [791. In other systems with similar overlapping openreading frames it has been shown that translational coupling exists between thetwo genes, such that translation of the first gene promotes translation of thesecond one [reviewed in 261. The reduced ,i3-galactosidase activity of pHEZ couldtherefore have been due to decreased translation of the fusion protein in theabsence of pucD translation, with transcription originating from vectorsequences. Alternatively, the decrease in /3-galactosidase activity in pHEZrelative to pBEZ could have been due to reduced transcription after deletion of aminor promoter located between pucC and pucD.To test whether translational coupling between pucD and pucEcontributed significantly to expression of the pucE ‘::lac ‘Z fusion a frameshiftmutation was introduced at the 5’ end of pucD which resulted in earlytermination of translation (see Materials and Methods). The mutation wastransferred into pBEZ (pBEZ-OOF) and pPEZ (pPEZ-OOF), and /3-galactosidaseactivities were measured (Fig. 28). In both constructs /3-galactosidase activitydecreased modestly (by 17% in pPEZ-OOF, not statistically significant) comparedto the equivalent unmutated plasmid, but pBEZ-OOF activity was not decreased104to the level seen in pHEZ.c. RNA blot analysis of the puc operonBecause pucE ‘::lac ‘Z fusions showed that 90% of transcription of the pucEgene originates upstream of the pucB gene, RNA blot analyses were performed[41] to determine whether RNA species long enough to encode all five of thepuc genes could be detected in addition to the Ca, 550 nt pucBA messagespreviously characterized [931. When RNA from the wild type strain SB1003 wasprobed with a 2.0 kb fragment extending from the Cia I site in pucB to theBspH I site in pucE (probe 1 in Fig. 29A), a 0.5 kb message was detected alongwith two other less abundant species (Fig. 29B, lane 1). These two weaker signalscorresponded to sizes of 2.4 kb and approximately 1.0 kb. (The faint bands ofapproximately 1.5 kb visible in lanes 1, 3 and 4 are artifacts caused by rRNA bandsabove and below this position that interfere with hybridization.) By scanning theautoradiogram on a densitometer, the relative amounts of the 0.5 kb: 1.0 kb : 2.4kb species were estimated as 35 : 3: 1.The 2.4 kb species described above was large enough to encode all five pucgenes. In order to evaluate whether it might, and to identify the sequencesgiving rise to the smaller species, further RNA blot analyses were carried outusing probes specific for smaller segments of the puc region (Fig. 29A). A probespecific for the pucBA genes detected only the largest (2.4 kb) and smallest (0.5kb) bands (Fig. 29B, lane 3). A probe specific for the pucDE region detected bandscomigrating with the 1.0 and 2.4 kb species, as well as a previously undetected105Figure 29. RNA blot analysis of puc operon transcripts. A) DNA probes used.Hatched boxes represent puc genes, potential mRNA stem ioop structures aremarked with loops. P: Pst I; ER: EcoR I. B) Autoradiograms of hybridizedblots of RNA isolated from SB1003 (lanes 1, 3-5) and ALT-ill (lane 2) cells grownunder low aeration (see Materials and Methods). Lanes I and 2 were probed withthe 2.2 kb Cia I - BspH I fragment (probe 1), which contains the pucBACDEgenes; lane 3 with a 435 nt Ban II fragment (pucBA, probe 2); lane 4 with the 1.1kb 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) areindicated to the left of the blots.A 1kbERB2 3 41 2 39.5...7.5—4.4—2.4—1.35—0.24—4 5I-IL106message that appeared to be about 0.7 kb (Fig. 29B, lane 5). When a probecontaining only the pucC region was used, a signal comigrating with the 1.0 kbband 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 endedapproximately where the 0.5 kb signal was found with the pucBA-containingprobes.When RNA from the L\LHII strain was probed as above no bands weredetected (Fig. 29B, lane 2), indicating that none of the signals resulted from cross-hybridization 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). Equalamounts 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) wereanalysed. The same membrane was hybridised sequentially with the probesshown in Figure 30A. As above, in no case was any signal detected in the lanecontaining RNA from ALHII(pRK415::12).Hybridisation of the pseudo-wild type strain (lane 2) gave results similar tothose described for SB1003. Because the same membrane was probed each time,it was possible to compare the migration of the species detected with each probedirectly. It became apparent that the smaller (“0.7 kb”; see above) messagedetected with the probe to the pucDE region was not bigger than the 0.5 kbpucBA message.107A e 1kbb c dpEACDE II____ --piCDE i Ip\C_ThpAE -- 11111 ___B C D E1234561234561234561234562.4 q *41.4.24Figure 30. RNA blot of puc deletion strains. Equal amounts of RNA harvestedfrom cells grown under low oxygen conditions were run in each lane andtransferred to nylon membrane electrophoretically. The same blot wasrepeatedly probed then stripped for reprobing as described in Materials andMethods. A) Diagrammatic representation of the DNA fragments used to makeprobes (b-e) and the puc gene deletion plasmids of the strains tested (plasmidpRK4l5:2, not shown, had no puc sequences). B-E) Autoradiograms of RNAblots 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). Theprobe used in (B) was the 435 nt Ban II fragment (b). Panel (C) was probed withthe 0.95 kb Xmn I-ApaL I fragment (c). A 586 nt Xmn I fragment (d) was usedas the probe in panel (D) and panel (E) was probed with the 2.3 kb Cia I to Eco47III fragment (e). Positions of RNA size markers (kb) are shown to the left of theblots. Symbols indicate RNA species discussed in the text.*I1’•.108The probe to the pucBA region (probe b) was expected to hybridise to the0.5 kb pucBA mRNA (white circle) and full-length puc transcripts (star) fromeach construct (Panel B). Transcription of the puc sequences present on pzCDEwould yield a 1 kb RNA, pAC primary transcripts would be approximately 1.8 kband ptD and pAE transcripts would each be approximately 2.2 kb. A signalmigrating 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 fivestrains that contained puc sequences.The only discrete band detected by the probe carrying pucC sequences (probec, panel C) was the wild type primary transcript in lane 2 (*). Weak smearysignals beginning at approximately 1.5 kb were present in all the lanes except lane1 and, as in Figure 28, rRNA running at approximately 1.5 and 1.2 kb interferedwith 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 atapproximately 1.0 kb (black dot) which coincided with the 1.0 kb band detected bythe pucDE probe (see below). This RNA might therefore overlap slightly withthe 3’ end of probe c to give a weak signal. In the first RNA blot experimentdescribed 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 readingframe and a relatively strong signal was detected running at 1.0 kb. The 3’ end ofprobe c is located 100 bp further upstream. The weakness of the signal seenusing this probe suggested that the 5’ end of this 1.0 kb message is approximately400 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 lanescontaining RNA from ALHII(pAD) and ALHII(pz\E), both of which contain 250 bp109deletions in regions shown to hybridise with this message. In fact, very weaksignals 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 strongbands detected by probe d when the original autoradiograms were overlaid.As expected, probe d, derived from pucDE sequences, did not detect anycomplementary 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 (•) weredetected in the lane containing the pseudo-wild type RNA (lane 2). As describedabove, the 5’ end of this 1.0 kb RNA molecule probably contains sequences fromthe last 400 bp of pucC. Interestingly, a band of the same size was detected inlane 4, which contained RNA from ALHII(pAC). The size of this band did notseem to be affected by the deletion of the pucC sequences at the presumed 5’ endof this message. The results using pucC-specific probes had localised the 5’ endof this message (from wild type templates) quite specifically, and its 3’ endmapped 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 fromsequences upstream of the deleted sequences which fortuitously resulted in amessage 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 thesame lane, panel D, but it was not convincingly stronger than the signal detectedin the rest of the smear.In RNA from both ALHII(pAD) and ALHII(pAE) (lanes 5 and 6) the only band110detected by probe d migrated at approximately 0.7 kb (open circle). Thiscorresponds to the size of the wild type 1.0 kb molecule less the 0.25 kb deletedfrom each of these plasmids. The intensity of the band in lane 6 was somewhatlower than in lane 5, although the extent of sequences complementary to theprobe was expected to be similar in both cases. This message may therefore beless abundant in strain zLHII(piE), presumably because the sequence removedor the sequence created by the deletion destabilised the molecule. The faint bandof approximately 0.5 kb that was detected in lanes 2 and 4 was absent in RNAfrom zLHII(pAD) and ALHII(pt\E). This band did not appear when the blot washybridised with the pucC-specific probe c, so it seems likely that this 0.5 kbmessage contains pucDE sequences.A probe containing sequences from all five puc genes confirmed the resultsseen in the other RNA blots described (Fig. 30E). All the RNA species detected byregion-specific probes were seen. The intensity of the approximately 0.5 kb bandwas less in strain ALHII(pAD) than in ALHTI(pzE). This signal would result fromthe combination of the signals of the pucBA 0.5 kb message (white circle) andthe pucDE-specific 1.0 kb band which migrates at 0.7 kb (open circle) in thesedeletion strains. The 0.5 kb pucBA transcript was equally abundant in bothstrains (Fig. 30B) and the 0.7 kb pucDE band seemed more intense in theALHII(pAD) lane than in ALHII(pAE) (Fig. 30D), so it initially seemed strange thatthe intensities of the signals in the 0.5 kb region were reversed when probed withprobe e. The 3’ end of this probe extends only as far as the Eco47 III site 80 ntinto pucE, however, so the entire sequence of this transcript from pAE would behomologous to probe e whereas a much smaller part of a transcript from thepUCDE region of pAD would overlap with the probe. Therefore, the signal for111the 0.7 kb RNA in ALHII(pzD) should be lower than in ALHII(pAE) using probee.d ConclusionsThe promoter localisation studies using the pucE’::lac’Z fusions describedhere determined that the majority of pucE transcription originates from apromoter upstream of pucB. As shown in Figure 28, deletion of these sequencesled to a ten-fold decrease in 13-galactosidase activity. No promoter activity wasassociated 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 wasdeleted the relative level of f3-galactosidase activity was further reduced to lessthan I % of the maximum expression.Two explanations for this additional decrease in reporter gene activityseemed possible. The first was that a minor promoter was located in the intervaldeleted and that its removal eliminated residual transcription of the pucE’::lac’Zfusion. The second possibility was suggested by the fact that the pucD and pucEopen reading frames overlap by four nucleotides, a situation in whichtranslational coupling could be important for efficient pucE translation. In thissecond case the 10% residual transcription would have to originate from vectorsequences, and the observed drop in /3-galactosidase expression would beexplained by decreased translation of the pucE’::lac’Z fusion message due to lossof pucD translation. The contribution of translational coupling to the level ofexpression of the pucE’::lac’Z fusion was tested by introducing a frameshiftmutation within pucD that led to early termination of pucD translation. The112relative 13-galactosidase activities of pucE’::lac’Z fusions whose 5’ sequenceseither included the major promoter activity or extended only to the 3’ end ofpucC were compared, with and without the frame-shift mutation. In the case ofpPEZ-OOF a modest but not statistically significant decrease in activity resultedfrom interruption of pucD translation. It is thus possible that translationalcoupling between pucD and pucE contributes a little to the level of pucEexpression, but the decrease in /3-galactosidase activity in pHEZ is primarily dueto deletion of a minor promoter located in the region between the 3’ end ofpucC and the middle of pucD.RNA blot analyses (Figs. 29 and 30) determined that there were severalRNA species encoding puc sequences. A 2.4 kb RNA molecule was the longestspecies detected in wild type and pseudo-wild type RNA preparations by probesspecific to the pucBA, pucC and pucDE regions. I propose that this messageextends from the major puc promoter region to the inverted repeat located justbeyond the 3’ end of the pucE gene, and is thus the primary transcript of thepuc operon. It was of relatively low abundance compared to the other signalsdetected, which suggests that it is relatively unstable, and one of the other RNAspecies detected, the 1.0 kb message hybridising to pucDE sequences, can only beexplained as a processing product of this primary transcript (see below). Noanalogous long transcripts were detected in any of the strains deleted for one ormore of the pucCDE genes.The most intense signal was generated by a message migrating at 0.5 kb andhybridising to pucBA-specific probes. This transcript is the previouslycharacterised 0.55 kb pucBA mRNA [93]. Its relative abundance, judged by the113intensity of the signal, was the same in pseudo-wild type and pucCDE deletedstrains. This is in contrast to a previous report based on dot blot analysis whichclaimed that interruption of pucC by a transposon caused a decrease in the levelof pucBA mRNA [781.Two signals were detected by probes carrying pucDE sequences. The lessabundant of the two migrated to approximately 0.5 kb in extracts from pseudo-wild type cells, and could potentially represent a transcript originating from theminor promoter detected by pucE’::lac’Z fusion analysis. It is probably too smallto encode both pucD and pucE, however, This transcript was unaffected bydeletion of the 3’ pucC sequences but was not present in either the pucD orpucE deleted strains. It may therefore be unstable in these two strains, or itmight not be produced from a larger precursor if processing sequences in thepUCDE region were deleted, or it may primarily derive from sequences removedin one of the deletions.The final discrete signal seen in wild type strains was a message migrating to1.0 kb and hybridising strongly to pucDE-specific probes. A band of this size wasalso 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 ofpucC hybridised very weakly at this position (probe c, Fig. 30). The 5’ end ofthis 1.0 kb species thus seems to be delimited by these two probes, and appears tolie within the pucC coding region, approximately 400 bp upstream of the 3’ endof the gene. This position is well upstream of the region of the minor promoteractivity detected by lacZ fusion analysis, so this molecule cannot be a product ofthis second promoter. Since no promoter activity was associated with the pucC114sequences, this 1.0 kb molecule is presumably a processing product of the 2.4 kbprimary transcript. This signal was shifted to 0.7 kb in the strains deleted forpucD or pucE. This faster migration corresponds to the sizes of both deletionsand suggests that the 1.0 kb molecule encodes both genes. The size of the 1.0 kbmessage may be underestimated since its apparent size does not quite correspondto the distance between its 5’ end, determined by the pucC-specific probes, andthe 3’ end of the pucE gene.115DISCUSSIONa. Transcriptional organisation of the puc operonRNA blot analyses and promoter mapping by fusion of puc’E to lacZ’ ledto the model shown in Figure 31 for the transcriptional organisation of the pucoperon.pucE’::lac’Z fusion analysis (Fig. 28) showed that the majority of pucEtranscription originated from sequences upstream of pucB, and that thistranscription showed the same regulation by oxygen as a similar pucB’::lac’Zfusion [93]. The same promoter (large bent arrow in Fig. 31) therefore seems todrive transcription of pUCBA and the genes distal to pucA. Consistent with thisproposal, an RNA molecule long enough to encode all five puc genes wasdetected 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 blackarrow in Fig. 31) was much less abundant than the 0.55 kb pucBA mRNA (thickgrey arrow) that had previously been characterised [931 (Fig. 29B, lane 3).There are two mechanisms that could result in an excess of the shortermessage over the longer. They are not mutually exclusive and could bothcontribute to the steady-state levels of the two transcripts. The first is that thestem loop structure located downstream of pucA could act as an inefficientterminator of transcription, so that some transcription events produce a 0.55 kbmolecule whereas others read through the stem ioop and produce the longer 2.4kb transcript. Chen et a!. [13] measured rates of transcriptional termination116Figure 31. Transcriptional organisation of the puc operon. Coding sequencesare shown as boxes with their gene designation below. Inverted repeats areindicated by stem-loops, and promoters are shown as bent arrows whose relativesize indicates promoter strength. Putative primary transcripts are shown as blackarrows, while messages likely to derive from processing of primary transcript(s)are shown in grey. The relative abundance of these RNA molecules is indicatedby the thickness of the arrows.117when a hairpin structure based on the puc sequence from strain SBIOO3 [901, butmodified at three residues to result in perfect dyad symmetry, was insertedwithin the puf operon. They found the amount of transcriptional readthroughdepended on the sequence immediately downstream of the hairpin. The dyadsymmetry element followed by ACCG caused very little termination, whereaswhen it was inserted in front of TTTT a very efficient transcriptional terminatorwas created. This finding is consistant with current models in E. coli that Rhoindependant termination of RNA synthesis occurs at CC-rich hairpins that arefollowed by a series of U residues [861. In the puc operon the imperfect invertedrepeat is followed by ATTC, so it could act as a transcriptional attenuator. Thereported sequence of the R. capsulatus puc operon from strain 37b4 [791 predictsa stem 3 base pairs longer than that of strain SB1003, the wild type strain used inthis thesis. However, the SBIOO3 sequence predicts a restriction enzyme sitewithin this stem region which I have found does not exist (unpublished data), sothe published sequence for SBIOO3 may be in error and the stem ioop may bemore stable than previously thought. The relative abundance of the 1.0 kb RNAmolecule 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 greaterthan the amount of the 2.4 kb message would seem to indicate. Site-directedmutagenesis of this inverted repeat in the pucE’::lac’Z fusion pPEZ coulddetermine whether attenuation is occuring.The second mechanism that could lead to an abundance of the 0.55 kbpucBA transcript over the 2.4 kb full-length puc transcript is differentialstability of the two molecules. Like the 0.5 kb pufBA mRNA, the 0.55 kb pucBAtranscript is very stable, having a half life of 20 minutes [93]. The stability of the1182.4 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 foundto have a half-life of less than 5 minutes [43]. My results indicate that processingof 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 messageshas been shown to be due to differential stability of the messages [6], due in partto protection of the 3’ end of the 0.5 kb transcript from exonucleolytic degradationby a stem-loop structure [13]. As discussed above, a similar hairpin existsdownstream of pucA, a modified version of which has been shown to protectpufBA transcripts from decay [13]. The instability of the 2.7 kb puf transcripthas been shown to be caused by endonucleolytic cleavage at sites within pufLMX[reviewed in 35]. Unlike puc RNA (see below), there are no stable processingproducts from the 3’ end of the 2.7 kb puf transcript [35].Minor promoter activity was detected in the region between the 3’ end ofpucC and the middle of pucD. I have indicated this second promoter withinpucD for the reasons described below, but it could be elsewhere in this region.No other promoters have been characterised within photosynthetic operons, butelaborate superoperons have been characterised at both ends of thephotosynthetic gene cluster (Fig. 32) [83]. The overlap of the crtEF and bchCXYZoperons with the puf operon has been shown to reduce the lag phase of growthwhen uninduced cells are shifted to photosynthetic conditions [84], as has theoverlap in the puhA region [31. The physiological significance of the minorpuc promoter, if any, is unclear, although it may help in stoichiometricproduction of the LHII y subunit (PucE) and the a and /3 peptides.puhAbchbchCXVZIFigure32.SuperoperonalorganisationofthephotosyntheticgeneclusterofR.capsulatus.Primarytranscriptsoftheoperonsindicatedareshownbysolidarrowsbeginningatthepromoterfortheoperonsindicated.GenesforstructuralpeptidesoftheRCandLI-ilcomplexesareindicatedbyhatchedboxes;shadedboxesindicategenesencodingbacteriochlorophyllbiosyntheticenzymes;carotenoidbiosynthesisgenesaredotted;cross-hatchingF1696MLHKBFcitEFpufQBALMX E.1-indicatesgenesofunknownfunction.120In addition to the 2.4 kb puc transcript, two smaller RNA molecules weredetected by probes to the pUCDE region in RNA blot analyses (Fig. 29B, lane 4and Fig. 30D, lane 1). An inverted repeat downstream of pucE is followed by thesequence TTTTATTT, and as discussed above an inverted repeat followed by 4T’s has been shown to efficiently terminate transcription in R. capsulatus [13]. Itthus seems likely that puc transcription does not proceed beyond thesesequences, and reasonable that messages ending in this region would have astem-loop formed by these sequences at their 3’ ends. The 0.5 kb RNA moleculedetected 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 transcriptionproduct of the minor promoter described above. This molecule was not affectedby deletion of pucC, but was absent from strains deleted for either pucD orpucE, which is consistent with the location I have suggested: deletion of pucDcould remove the minor promoter, and the deletion of pucE would eliminatemost of the sequences transcribed. If this is the cause of the lack of this moleculein ALHII(pAD), the minor promoter would be located between the Bcl I andHinc II sites in pucD.The larger of the two messages detected by pucDE probes was approximately1.0 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 wasdetected by a probe with a 3’ end at the Ban II site 280 bp upstream of the 3’ endof 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 bpupstream of the 3’ end of the pucC gene. This is well upstream of the minor121promoter defined by the pucE’::lac’Z fusion deletion analysis, in a segment notassociated with any detectable promoter activity. This transcript must thereforebe a processing product of the primary transcript. Results of RNA blot analysis ofthe strains deleted for pucD and pucE showed that the mobility of this messageincreased by the size of each of these deletions, indicating that the 1.0 kbmolecule encodes both pucD and pucE.Given that the 5’ end of the 1.0 kb message maps within pucC, it appearedcontradictory that an RNA species of the same mobility was detected in the straincontaining the pucC deletion (Fig. 30D, lane 4), which should removeapproximately 350 bp from the 1.0 kb molecule. The pucC coding sequencespresumably contain endonuclease processing sites, however, some of whichmight have been removed or altered in making the deletion. It is thereforepossible that the primary transcript from the pucC deletion strain is processeddifferently from the wild type puc transcript, and that it fortuitously produces amolecule of a similar size. It is clear that the primary transcript of the pzCdeletion is rapidly degraded since no message of the appropriate size (1.8 kb) wasdetected with the pucBA probe (Fig. 30B, lane 4). In fact, full-length transcriptsencoding the sequences remaining on plasmids pACDE, pAD and pAE were notdetected in the strains harbouring these plasmids, either. I can only concludethat each of these deletions destabilised the primary transcript, either by removalof stability determinants, and/or by creation of endonuclease cleavage sites.The only RNA molecule detected which could encode pucC was the lowabundance 2.4 kb full-length puc transcript (Fig. 29B, lane 4). PucC is requiredfor formation of the LHII complex (see below), and the fact that it is encoded on122such a low abundance transaipt suggests that its role is catalytic rather than as astructural component of the complex. Additionally, the pucD deletion strainhas wild type levels of the LHII complex, but no detectable RNA molecule whichcould encode pucC (Fig. 30B, lane 5). This implies that very little pucC productis required for assembly of LHII complex.Mutation of pucC has been reported to result in a reduced level of pucBAmRNA [781. This conclusion was based on dot blot analysis of a mutant (strainNK3) containing a transposon insertion in pucC, and was interpreted asevidence that pucC is required for high level transcription of puc mRNA. InFigure 30B it can be seen that all the strains tested (with the trivial exception ofALHII(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 adecrease of pucBA mRNA levels. Rather, as discussed below, I think the PucCprotein 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 ofthe genes downstream of pucBA are transcribed primarily from a promoterupstream of pucB (see above). Previous studies of the expression of thepucCDE genes in R. capsulatus drew conclusions from studies of a mutant witha transposon inserted in the pucC gene (strain NK3), or a deletion of only thepucB, pucA and part of the pucC genes (strain U72) [78, 791. The interpretations123of these previous experiments were complicated by the likely polar effects of thetransposon insertion and the possibility of a strong promoter located between thepucC and pucD genes that could allow expression of the pucD and pucE genesin the mutants NK3 or U72 [78]. Construction of the chromosomal puc deletionstrain 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 orgenerally deleterious to cell growth. Photosynthetic growth of the deletion strainALHII(pRK415::Q) was similar to that of wild type cells until the incident lightlevel fell below 60 tEm2s (Fig. 16) and aerobic growth was not affected by thedeletion (data not shown). Strain ALHII, lacking the LHII complex, has thereforebeen useful as a background strain for studies monitoring LHI and RC complexlevels [C. Young, D. Wong, B. Collins, J.T. Beatty, personal communications]which would formerly have been conducted in relatively uncharacterised LHIImutants. As discussed below, some of these backgrounds may have unexpectedpleiotropic effects on LHI complex expression.c. The phenotypes of pucD and pucE mutantsThe deletion analysis described in this dissertation shows that two of thethree genes downstream of pucBA are required to obtain normal levels of theLHII complex. One of these genes, pucE, encodes the 14 kD y subunit that copurifies with the pigment binding proteins of the LHII complex [79], and itsdeletion resulted in decreased LHII complex levels. Strain ALHII(pAE)124synthesized 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 lowrelative to that at 850 nm. The amount of the complex, and the relative sizes ofthe 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 thatdeletion of pucE decreases the in vitro stability of the LHII complex in cell-freechromatophore membrane preparations. The pucE gene is not absolutelyrequired for wild type levels of LHII complex, however, since the secondarysuppressor strain ALHII—2(pACDE) has high levels of the complex in the absenceof pucE (Fig. 19C).Despite retaining some LHII complex, strain ALHII(pAE) grew more slowlyin photosynthetic conditions than LHII(pRK415::2), which had no LHIIcomplex (Fig. 15). The decrease in photosynthetic growth rate did not appear tobe due to inefficient transfer of light energy from the LHII complex to the rest ofthe PSA since no increase in fluorescence was detected for this strain comparedto pseudo-wild type cells (Fig. 17). Nor was there any indication from proteingels that the pucE deletion strain had reduced levels of RC or LHI complex (datanot shown). I therefore cannot explain this reduction in photosynthetic growthability.The pucD coding sequence overlaps that of pucE by four nucleotides, sopucD expression might be linked to that of pucE. Pairs of genes withoverlapping coding sequences are often translationally coupled such thattranslation of the first results in efficient and stoichiometric translation of thesecond [26]. I found that a translationally in-frame deletion of the pucD gene125had no apparent effect on LHII complex levels detected by spectroscopy, and inthis respect my results differ from those of Tichy et a!. [78]. The sequenceremoved in their deletion was not reported, however, so it was possible thattheir deletion was not translationally in-frame and, due to loss of translationalcoupling, had a polar effect on expression of pucE. I tested the contribution oftranslational coupling to expression of a pucE’::lac’Z fusion by introducing aframe-shift mutation which resulted in premature termination of PucDtranslation (Fig. 28). I found that /3-galactosidase levels decreased modestly, butnot significantly given the high standard deviation in the assay. There maytherefore be a small contribution of translational coupling to the levels of PucEexpression, but it is certainly not required.In addition to having no effect on LHII complex levels, deletion of pucDdid not impair photosynthetic growth of strain zLHII(pAD) except at the very lowlight level of 5 tEm2 s* This minor effect is the only phenotype yet detectedfor this strain. The function of this gene, and the significance of the overlapbetween the pucD and pucE open reading frames, remains unknown.d. The phenotypes of pucC and pucCDE deletion mutantsDeletion of pucC, alone or in combination with pucD and pucE, resultedin complete loss of the LHII complex, a reduction in the levels of RC and LHIcomplexes, and a severe decrease in the rate of photosynthetic growth at all lightlevels tested compared to strain ALHII(pRK415::2). As discussed below, thereduction in photosynthetic growth rate is probably not due solely to the lower126amount of LHI complex in these strains and I have already described resultsshowing that the lack of LHII complex is not due to decreased pucBAtranscription in strains ALHII(ptC) and ALHII(ptCDE) (see section a).Strain z\LHII(pAC) contains a pucC allele truncated within the fifthtransmembrane domain (see below for PucC structure). This strain hasapproximately 40% of the amount of LHI complex seen in strainALHII(pRK4I5::2) when grown under low oxygen conditions (Fig. 14). Underphotosynthetic 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 intensitydecreases from 200 iEm2 s1 to 60 jiEm2 At 60 Em2 s ALHII(pAC) has76% of the amount of LHI complex present in z\LHII(pRK4I5::Q) at the samelight intensity. This relative increase in LHI complex levels is not accompaniedby increased photosynthetic growth rates however, and at lower levels ofillumination the decreased photosynthetic growth rates of pucC strainsprovided a selective advantage to secondary suppressor mutants (Fig. 16).Most of these secondary mutants expressed LHII complexes, implying that atleast one other gene is capable of providing the function of the missing pucCgene, and presumably its own normal function, when modified due to amutation. The suppressor strain z\LHII-4(pAC) did not regain any LHII complexbut grew faster than the parent strain, confirming that loss of LHII complex canbe separated from the reduction in photosynthetic growth rate. I determined thatthe second site mutations are chromosomally located in all the suppressorstrains I isolated, since transfer of the plasmids from these strains to strain ALHIIgave the same phenotypes as plasmids pAC and pACDE (unpublished data). The127data presented here do not determine how many genes are represented in thecollection of secondary mutants, but the diversity of phenotypes demonstratesthat mutation at multiple sites can suppress the phenotypes of pucC or pucCDEdeletion.Two candidates as sites for a mutation that suppresses the effects of pucCgene 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 has27% identity and 69% similarity to PucC, but no mutant phenotype has yet beenidentified for this gene [9]. The predicted amino acid sequence of F1696, on theother hand, has 47% identity to that of PucC [3], and mutations in F1696 havebeen shown to decrease the amount of the LHI complex by up to 80% withoutaffecting LI-ill or RC complex levels [3 and C. Young, unpublished results]. Ittherefore seems possible that the function of F1696 in formation of the LHIcomplex is analogous to that of pucC in LHII complex formation, and thatmutation of F1696 can allow it to compensate for the deletion of pucC. Thishypothesis could be tested by sequencing F1696 from the secondary supressorstrains or by mutagenesis of F1696 and complementation of a pucC mutant.F1696 mutants retain some LHI complex, so kinetic studies have beenpossible to determine whether F1696 maintains steady state levels of the LHIcomplex by facilitating assembly or by stabilising otherwise assembled LHIcomplexes. It was concluded that the primary role of F1696 is in assembly of theLHI complex [C. Young, J.T. Beatty, in preparation]. Similar experiments cannotbe performed to investigate PucC’s role in determining LHII levels because allpucC mutants examined so far have been completely devoid of LHII complex.128By analogy to Fl 696 it seems likely that PucC is required for assembly of LHIIcomplexes rather than stabilisation of existing complexes. The low level oftranscripts encoding pucC and the fact that the PucC protein has never beenisolated with LHII complexes are consistent with a catalytic rather than structuralrole for PucC. It is hoped that comparison of detailed structural models for thetwo proteins will help in defining conserved elements required for similarfunction 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 possiblethat pucC is involved in LHI production and that residual levels of the LHIcomplex in F1696 strains is due to the presence of pucC. Strains containingdeletions of both F1696 and the puc operon retained low levels of LHI complexwhich were not increased by expression of pucC (Fig. 25), and zLHII, which iswild type for F1696 but missing all the puc genes, has high amounts of the LI-ilcomplex. Thus the PucC protein does not seem to participate in LHI formationand the decreased levels of the LHI and RC complexes in ALHII(pAC) must bedue to an inhibitory effect of the presence of the other puc genes in the absenceof pucC.e. Deletion analysis of PucC using pucC’:qiho’A fusions and effects on LHIcomplex levelsTranslationally out-of-frame pucC’::pho’A fusions were used to evaluatewhether alleles of pucC, truncated at the C-terminus, retained any function.129None of the alleles tested, which lacked only one or two of the finaltransmembrane domains, conferred on ALHII(pzC) strains the ability tosynthesize LHII complex when expressed in trans (Fig. 24). The C-terminus ofPucC is thus required either for stability of the protein or for function in LHIIcomplex formation.The different pucC alleles examined had different phenotypes in terms ofthe amounts of RC and LHI complex present, however. The amounts of RC andLHI complex synthesized by these strains when grown photosynthetically washigher than that of the vector control strain, but this did not result in anyincrease in photosynthetic growth rate (Fig. 24).Other pucC alleles, in effect truncated by translational fusion to phoA, alsohad unexpected effects on the levels of the LHI and RC complexes whenexpressed in LHII(pAC). The amount of LHI complex present in these cells wasroughly correlated with the length of the pucC sequences remaining in thefusion, despite the presence of pAC (Fig. 26A). As above, with the exception offusion pPQ::S8-1O which had almost no LHI or RC complex, the photosyntheticgrowth rates of these strains were similar regardless of the amount of LHIcomplex present (Fig. 27).Thus truncated pucC alleles are not only unable to promote synthesis ofthe LHII complex, they can also lead to reductions in the amounts of the LHI andRC complexes, and to decreased rates of photosynthetic growth that are not duesolely to the decrease in the components of the PSA. The effects on LHI complexlevel are only seen when the pitcB and pucA genes are also present, since the130truncated alleles have no effect when expressed in ALHII cells that do notharbour pIC (pucD and pucE are probably not important in these effects sincestrain ALHII(pACDE) has the same reduction in LHI complex level asALHII(pAC)).Reconciling these data is not a simple matter. Deletion of pucC alone isworse for the cells than deletion of the entire puc operon, leading to loss of LHIIcomplex and to decreased amounts of RC and LHI complexes. Rates ofphotosynthetic growth are also impaired, but not simply because of thesedecreases in the components of the PSA. PucC does not participate in LHIformation, yet expression of truncated alleles leads to decreases in the amountsof the complex, an effect which is dependant on the presence of the pucBAgenes. Finally, the reduction in LHI complex levels in ALHII(pAC) is relieved aslight intensity decreases.This final observation points to a possible reason for the decrease in LI-ilcomplexes seen in strains containing pucB and pucA along with deletions inpucC. As light levels decrease, the transcription of the puc operon alsodecreases (in wild type cells the amount of LHII complex increases by a post-transcriptional mechanism [931). It is possible that free LHII a and /3 peptidescan interfere with assembly of the LHI a and /3 peptides into complexes. One ofthe functions of PucC might be to shepherd the LHII pigment-binding peptides toprevent interactions that could interfere with LHI assembly. In the absence ofpucB and pucA this function would be unnecessary, so strain ALHII hasnormal levels of LHI complex. The longer PucC peptides might retain more ofthis shepherding function while lacking the ability to promote LHII complexassembly, so the interference with assembly of the LHI complex is less with131longer alleles. In strain z\LHII(pAC), as light levels decrease the amount of LHIIa and /3 peptides might decrease, so the interference with LHI assembly wouldalso be reduced. The levels of LHI complex in ALHII did not change as lightintensity varied, suggesting that the amount of LHI complex does not normallyvary with changes in illumination.This hypothesis is difficult to test, but one of its predictions is that increasingsynthesis of the LHII cx and /3 peptides in a pucC mutant would decrease theamount of LHI complex in those cells. This could be tested by expressing pucBand pucA from the nif promoter and growing the cells on increasingly poornitrogen sources to increase transcription of pucBA without affecting pufexpression. This system has been used to evaluated the concentrationdependance of bacteriochiorophyll synthesis on pufQ expression [4]. A secondtest would be to determine the point mutation in the LHIP mutant MW442.This strain is complemented by plasmid pC, which carries pucC, but itsphenotype resembles that of ALHII rather than ALHII(pAC). My predictionwould therefore be that the mutation in this strain is a missense mutation thatcauses an amino acid substitution that prevents LHII complex assembly, ratherthan a nonsense codon that results in a truncation of the PucC protein. Thisanalysis might be complicated by sequence differences of the type seen betweenstrains SB1003 and 37b4, so the mutation would have to be confirmed byduplication in a different background.PucC is clearly required to obtain the LHII complex, much as F1696 is anassembly factor for the LHI complex. Unlike F1696, however, mutations inpucC cause pleiotropic effects on the other complexes of the PSA. Putative RC132assembly factors have recently been identified that also have indirect effects onLI-il complex levels, possibly because the LHI complex is not stable in the absenceof the RC [851.f. The structure of the PucC proteinTheoretical models for the membrane topology of the PucC protein wereconstructed by comparison of hydropathy analyses and application of the positiveinside rule. These models were tested by construction of over 40 translationalfusions between pucC’ and pho’A to arrive at the model presented in Figure23. It features 12 transmembrane domains and predicts that both the N- and Ctermini are located in the cytoplasm. In accordance with the positive inside rulenone of the periplasmic domains have more than one basic residue, whereasmost of the cytoplasmic loops have at least four. At the C-terminus the predictedhydrophilic domain is very short, but is anchored by three positively chargedresidues. The third cytoplasmic domain has only one lysine residue, and theonly fusion in this region, S8-5, lacked this residue and had intermediate activity.The high hydrophobicity of neighbouring segments and precedents in theliterature [67] argue that this intermediate activity is that of a cytoplasmic fusionthat is inefficiently retained in the cytoplasm because it is not anchored by a basicresidue.Although the theoretical and experimental data sets are large and internallyconsistent, the model could be further tested by construction of different fusionswhich would provide positive rather than negative results for cytoplasmic133fusions. J3-galactosidase fusions have been used for such purposes [47], andreplacement of pho’A with lac’Z in key fusions such as S8-5 would furthersupport (or refute) the model. Screening blue colonies from new ligationsbetween lac’Z and Ba! 31-digested pucC’ fragments on plates containing X-galmight also identify pucC’::lac’Z fusions in the first and last cytoplasmic domains.Each of the transmembrane domains would then be confirmed by positive datafrom each side of the membrane.One limitation of analysis by these translational fusions is that truncation ofthe protein could lead to differences in stability of the remaining peptide orstructural artifacts if important downstream structural determinants areremoved. Biochemical analysis of PucC using monoclonal antibodies orchemical methods would not be subject to these caveats but is not currentlypossible.In the course of sequencing the pucC’::pho’A fusions several differenceswere noted between the sequence of pucC in the strain used here, SBIOO3, andthat published for strain 37b4 [79]. The number of differences in the DNAsequence is especially striking considering that the 600 bp sequence of the pucBAregion reported from both these strains contains only 2 differences, one of whichmight be a sequencing error (see section a). All of the differences that resulted inamino acid changes are located in the second part of the protein, perhapsindicating that the first four transmembrane domains are more important forPucC function than the rest of the protein. Immediately upstream of the PucCstart codon there were no differences noted. This is of interest because thepreliminary results of an E. coli in vitro transcription/translation systemreported that translation of PucC could not be detected until a stronger ribosome134binding site had been introduced [371 whereas the levels of alkaline phosphataseactivity I detected in my study did not suggest that translation occured at lowlevels in E. coli.PucC and the predicted protein product of the F1696 gene are strikinglysimilar: they share 47% identity and 64% similarity spread through the length oftheir sequences. Comparison of their structures will be of interest to see ifmembrane topology features are also conserved. The F1696 protein topology iscurrently being determined [C. Young, personal communication].g. Concluding remarksThe results of this thesis extend our knowledge of the puc operon, andsuggest several avenues for future investigations.I have shown that the five genes of the puc operon are co-transcribed, anddemonstrated the existence of a low-abundance pucBACDE transcript. My workindicates that this message is processed to generate an approximately 1.0 kbmolecule that contains pucDE sequences. Extensive studies of processing eventsin the R. capsulatus puf operon mRNA have identified endonucleaserecognition sites [35]; comparisons between the two operons might help establishgeneral features of such processing sites. Primer extension studies wouldidentify the 5’ end of the 1.0 kb molecule, and that of the 0.5 kb RNA species alsodetected by pUCDE probes, and might help to locate the minor promoter detectedin my pucE’::lac’Z fusion analysis. Site-directed mutation of the inverted repeat135sequences downstream of pucA in a pucE’::lac’Z fusion would determinewhether transcriptional attenuation occurs, a possible mechanism for generatingthe abundant 0.55 kb pucBA mRNA.The most surprising result of the phenotypic characterisation of pucCDEdeletion mutants was the pleiotropic effects of truncated pucC alleles on LHIand RC complex levels. Several experiments suggested that the reduction inphotosynthetic growth rates was not due solely to the decreased amounts of theLHI and RC complexes, and that these reduced complex levels depended on thepresence of the pUCBA genes. I have proposed that the PucC protein has thespecific function of participating in assembly of the LHII complex and a lessspecific role in shepherding LHII a and /3 peptides, preventing them frominterfering with LHI complex assembly.Whether the LHII a and /3 peptides interfere with LHI complex assemblyin the absence of full-length PucC protein could be tested by overexpression ofpucBA, in the complete absence of pucC or with truncated alleles. Biochemicalreconstitution experiments might be able show whether LHI and LHII a and /3peptides can interact; similar experiments have shown that LHI /3 peptides caninteract, as can LHI a and /3 peptides from different species of photosyntheticbacteria [45].The active site of PucC for assembly of the LHII complex might be identifiedby locating the mutation in the strain MW442 which renders it LHII withoutaffecting LHI or RC complex levels. Further insight into the function of PucCcould be obtained by cloning the suppressor mutations from strains such as136ALHll-2(pzCDE). Finally, mutational analyses of pucC can be planned on thebasis of the topological model of the PucC protein, and should help indetermining functional domains of the protein.The final unexpected result of my studies was the observation that LHIcomplex levels in the strain ALHII did not vary in photosynthetic cultures grownat different light levels. Previous studies have reported that the number ofphotosynthetic units, including the LHI complex, increase as incident light levelsdecrease [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 underconditions of low illumination [121, suggesting that transcription of the pufoperon is positively regulated by diminished light intensity, but the authors didnot report whether in vivo amounts of LHI complex were affected. 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