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Directed mutational analysis of the Rhodobacter capsulatus puha gene and downstream open reading frames Wong, Danny Ka-Ho 1994

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DIRECTED MUTATIONAL ANALYSIS OF THE RHODOBACTER CAPSULATUS PUHA GENE AND DOWNSTREAM OPEN READING FRAMES by DANNY KA-HO WONG B.Sc, The University of Manitoba, 1990 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Microbiology and Immunology) We accept this thesis as conforming to the required standard 422 THE UNIVERSITY OF BRITISH COLUMBIA May 1994 © Danny Ka-Ho Wong, 1994 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my writ ten permission. (Signature) VpO^wu /J& I&VWA t-Jena Department of Mhtbhtolafa ^ ^ f v ~ , W The University of British Columbia Vancouver, Canada DE-6 (2/88) 11 ABSTRACT Two mutant strains of Rhodobacter capsulatus which lacked the heavy (H) subunit of the reaction center (RC) were constructed. One strain, DW5, contains a translationally in-frame deletion of the puhA gene, which encodes the RC H subunit. The other strain, DW1, is a puhA polar mutation strain containing the same deletion of the puhA gene coupled with the insertion of a streptomydn/spectinomycin resistance omega (Ω) cartridge. Both strains were unable to grow under photosynthetic conditions (PS ). Absorption spectroscopy and SDS-PAGE analysis showed that these two strains have a reduction in the amount of the light-harvesting I (LHI) complex. In DW5, both photosynthetic growth and LHI complex level were restored by complementation in trans with plasmid pPUHA, which contains the puhA gene. However, when pPUHA was introduced into DW1, photosynthetic growth was only partially restored, and the LHI complex level was not restored to the wild type phenotype. When DW1 was doubly complemented with pPUHA and pORF214/162b (which contains orf214 and orfl62b, two open reading frames [orfs] downstream of puhA), photosynthetic growth was restored to the wild type level. However, LHI complex level was not restored in this doubly complemented strain. In a third mutant strain, DW2, orf214 was directly mutated with an insertion of the Ω. cartridge. DW2 also showed a PS" phenotype and a reduction in LHI complex level. Interestingly, SDS-PAGE analysis showed that the RC H subunit was missing in the intracytoplasmic membrane of DW2. Photosynthetic growth, but not LHI complex level, was restored when DW2 was complemented with pORFF214/162b. The roles of the RC H subunit and the gene products of the orfs downstream of puhA in photosynthesis are discussed. Ill TABLE OF CONTENTS Abstract ii Table of Contents iii List of Tables v List of Figures vi Abbreviations and Symbols ix Acknowledgement xi Introduction 1 Materials and Methods 12 1. Bacterial strains 12 2. Growth conditions 12 3. In vitro DNA techniques 17 4. Bacterial conjugation and GTA transduction 17 5. Construction of R. capsulatus strains DW1, DW2, DW5, DW6, and DW13 19 6. Complementation plasmid constructions 32 7. DNA and protein sequence analyses 38 8. Southern blots 38 9. Spectrophotometric analyses 40 10. Isolation of chromatophores 40 11. Protein concentration determinations 41 12. Gel electrophoresis of proteins 41 iv Results 42 1. Analyses of the translationally in-frame puhA deletion mutants DW5andDW13 42 A. Southern blot analysis 42 B. Growth studies 45 C. Absorption spectroscopy 45 D. SDS-PAGE analysis of ICM proteins 50 2. Phenotypic analysis of the puhA deletion/Q. insertion mutants D W l a n d D W 6 56 A. Growth studies 56 B. Absorption spectroscopy 59 C. SDS-PAGE analysis of ICM proteins 64 3. Phenotypic analysis of the orf214::Q insertion mutant DW2 66 A. Growth studies 66 B. Absorption spectroscopy 66 C SDS-PAGE analysis of ICM proteins 66 Discussion 72 Conclusions 83 References 84 V LIST OF TABLES Table I: Bacterial strains and plasmids used 13 VI LIST OF FIGURES Figure 1: Representation of genes and transcripts of the Rhodobacter capsulatus photosynthesis gene cluster 5 Figure 2: Operonal organization and restriction map of the R. capsulatus bchF-puh operon 7 Figure 3: Hydropathy plot of putative gene product of orf214 10 Figure 4: Genetic arrangement of the puhA and orf214 mutant strains 20 Figure 5: Outline of the construction of the plasmids used to make the translationally in-frame deletion of puhA 21 Figure 6: Representation of the wild type (SB1003) and the translationally in-frame deletion allele of puhA 23 Figure 7: Outline of the screening of the translationally in-frame deletion of puhA 25 Figure 8: Outline of the construction of the plasmids used to make the puhA deletion/ Q insertion 28 Figure 9: Outline of the construction of the plasmids used to make the Q. insertion into orf214 30 Figure 10: Outline of the construction of the plasmid pPUHA 33 Figure 11: Outline of the construction of the plasmid pORF214/162b 36 Figure 12: Southern hybridization analysis of Pst I cut chromosomal DNA of R. capsulatus SB1003 and DW5 43 Vll Figure 13: Photosynthetic growth of R. capsulatus DW5 and related strains 46 Figure 14: Photosynthetic growth of R. capsulatus DW13 and related strains 47 Figure 15: Absorption spectra of intact cells of R. capsulatus DW5 and related strains grown under low aeration 48 Figure 16: Absorption spectra of intact cells of R. capsulatus DW13 and related strains grown under low aeration 51 Figure 17: Absorption spectra of intact cells of R. capsulatus ALHII and DW13(pPUHA) grown under photosynthetic conditions 53 Figure 18: SDS-PAGE analysis of chromatophore proteins isolated from R. capsulatus DW5 and related strains grown under low aeration 55 Figure 19: Photosynthetic growth of R. capsulatus DW1 and related strains 57 Figure 20: Photosynthetic growth of R. capsulatus DW6 and related strains 58 Figure 21: Absorption spectra of intact cells of R. capsulatus DW1 and related strains grown under low aeration 60 Figure 22: Absorption spectra of intact cells of R. capsulatus DW6 and related strains grown under low aeration 62 Figure 23: SDS-PAGE analysis of chromatophore proteins isolated from R. capsulatus DW1 and related strains grown under low aeration 65 viii Figure 24: Photosynthetic growth of R. capsulatus DW2 and related strains 67 Figure 25: Absorption spectra of intact cells of R. capsulatus DW2 and related strains grown under low aeration 68 Figure 26: SDS-PAGE analysis of chromatophore proteins isolated from R. capsulatus, DW2 and related strains grown under low aeration 70 Figure 27: Summary of the effects of the deletion of puhA and possible functions of the downstream open reading frames 82 IX ABBREVIATIONS AND SYMBOLS Ap ATP ATPase Bchl bp BSA ca cfu Cm cyt b/ci complex dATP DNA EDTA GTA kDa km kb LH MOPS M r mRNA orf PAGE psi pucA, pucB pucC pucD pucE ampicillin adenosine 5'-triphosphate adenosine 5'-triphosphatase bacteriochlorophyll base pair bovine serum albumin approximately colony forming unit chlor ampheni col ubiquinol: cytochrome b/cj oxidoreductase complex 2'-deoxynucleoside adenosine 5'-triphosphate deoxyribonucleic acid ethylenediaminetetra-acetic acid gene transfer agent kilodaltons kanamycin kilobases light-harvesting 3-(N-morpholino)propanesulfonate relative mobility of proteins in a gel messenger RNA open reading frame poly aery lamide gel electrophoresis pounds per square inch structural genes of the R. capsulatus LHII a and f3 polypeptides a gene in the R. capsulatus puc operon required for normal LHII function a gene in the R. capsulatus puc operon of unknown function structural gene of R. capsulatus LHII y polypeptide X pufA, pufB structural genes of R. capsulatus LHI a and f3 polypeptides pufL, pufM structural genes of the R. capsulatus RC L and M subunits pufQ a gene in the R. capsulatus puf operon required for bacteriochlorophyll biosynthesis pufX a gene in the R. capsulatus puf operon required for electron transfer from the RC to the cyt b/cj complex puhA structural gene of the R. capsulatus RC H subunit PS photosynthesis RC reaction center RNA ribonucleic acid RPM revolutions per minute SDS sodium dodecyl sulfate SSPE saline sodium phosphate EDTA TBE tris borate EDTA Tc (or Tcs) tetracycline (or tetracycline sensitive) xi ACKNOWLEDGEMENT I would like to thank my thesis supervisor, Dr. J.T. Beatty, for his guidance throughout the course of my research. His diligence, kindness, enthusiasm, approachable manner, and tremendous patience make him an excellent supervisor. The Beatty lab is where I could find a cooperative atmosphere for research. It has been a pleasure to work with my present and former lab colleagues Heidi LeBlanc, Tim Lilburn, Farahad Dastoor, Conan Young, Dr. Nora Lem, and Dr. Jorg Overmann. I thank them for their help and friendship during these years. I also thank my thesis committee members, Dr. R.A.J. Warren and Dr. J.A. Benbasat, for their helpful suggestions during my research. I express my appreciation to my family, who has been very understanding and supportive. I thank my brothers and sisters in the Pacific Grace Chinese Church, for their support and prayers. Finally, I thank God for answering these prayers. 1 INTRODUCTION Rhodobacter capsulatus is a purple non-sulfur bacterium that is capable of growth by several mechanisms, including aerobic respiration and anaerobic photosynthesis. Unlike plants, non-photosynthetic mutants of R. capsulatus are viable, so the phenotypic effects of photosynthesis gene mutations can be readily evaluated. Virtually all of the R. capsulatus genes encoding the known components essential for photosynthesis have been cloned and sequenced, and several have been used for site-directed mutagenesis studies (Coleman and Youvan, 1990; Richter, et al., 1992). When the external oxygen concentration drops below a threshold level, R. capsulatus becomes photosynthetically competent by inducing the synthesis of extensive invaginations of the inner membrane. These invaginations constitute the intracytoplasmic membrane (ICM) system which, although physically continuous with the cytoplasmic membrane, is structurally different from the cytoplasmic membrane. The ICM contains the components necessary and specific for photosynthetic growth. These components include two light-harvesting (LH) antenna complexes, the reaction center (RC) complex, and the ubiquinohcytochrome b/cj oxidoreductase complex (the cyt b/cj complex). The two LH complexes function to increase the cross-sectional area for absorption of light energy. They transfer energy to the reaction center where electrons from bacteriochlorophyll a (Bchl a) are used to reduce a quinone molecule. The reduced quinone (quinol) is oxidized at the cyt b/cj complex, which generates a proton gradient and cycles electrons back to the RC (Prince, 1990). The proton gradient is utilized to generate ATP via coupling factor (ATPase). 2 The names of the two LH antenna complexes, B870 and B800-850, refer to their major Bchl a absorption peaks in the near-infrared spectrum (Feick and Drews, 1978). The B870 complex, also called the light-harvesting I (LHI) complex, is made up of two pigment-binding polypeptides: the a subunit and the P subunit which have apparent M r 's of 12 and 7 kilodaltons (kDa) respectively, according to SDS-PAGE analysis (Peters and Drews, 1983). This complex occurs in a relatively fixed stoichiometry to the RC with about ten to twenty B870 LH complexes per RC (Drews, 1985). The B800-850 complex, also called the l ight-harvesting II (LHII) complex, consists of three polypeptides: the a, P, and y subunits. According to SDS-PAGE analysis, the apparent Mr 's of these subunits are 10, 8, and 14 kDa respectively (Feick and Drews, 1978; Shiozawa, et al, 1980). The number of B800-850 complexes in the ICM relative to the RC is highly variable and inversely proportional to light intensity and pC>2 (Schumacher and Drews, 1979; Oelze, 1983; Drews, 1986). The pigments are bound non-covalently to the a and P subunits of both LHI and LHII complexes, whereas the y subunit of the B800-850 complex does not bind pigment molecules (Feick and Drews, 1978). The RC complexes from two closely related bacteria, Rhodobacter sphaeroides and Rhodopseudomonas viridis, have been crystallized, and their 3-dimensional structures are very similar (Deisenhofer, et al., 1985; Feher, et al, 1989). The RC subunit primary structures of R. sphaeroides and Rps. viridis are highly similar to the R. capsulatus RC subunit sequences (Williams, et al, 1986). The RC consists of a Bchl a dimer (called the special pair), two "voyeur" Bchl a molecules (named because they are not thought to take part directly in electron transfer), two bacteriopheophytins, and a pair of quinones (associated with an iron atom). These cofactors are bound non-3 covalently and held in a precise orientation to one another by two polypeptides, the light (L) and medium (M) subunits. A third polypeptide, the heavy (H) subunit, which has a single transmembrane a-helix and a large cytoplasmic domain, holds the L and M subunits together to form a RC complex. In a photosynthetic ICM, the L, M, and H subunits are found in the RC in a 1:1:1 ratio and, according to SDS-PAGE and crystallography analyses the M r 's of these subunits are 21, 24, and 28 kDa respectively (Nieth, et al., 1975; Feher and Okamura, 1978). The L and M subunits are directly involved in the energy transfer within the RC. The role of the RC H subunit, which does not bind pigments or other cofactors, is less clear. A previous study found, with a preparation from JR. sphaeroides, a minimal "functional" RC which contained the L and M subunits, but not the H subunit. This purified minimal RC had in vitro spectral characteristics similar to those of the native RC and performed primary photochemistry (reduction of the first of the two quinones) in the absence of H (Feher and Okamura, 1978). However, the second quinone was not stably bound and the ability of such a two-component RC to donate reduced quinone to the cytochrome bjci complex in vivo is unknown. Nevertheless, it was reported that the RC complex of the photosynthetic bacterium Rhodocyclus gelatinosus has only the two L and M subunits (Clayton and Clayton, 1978). Despite these results, the H subunit has been suggested to play an important role in photosynthesis. On the basis of the 3-dimensional crystal structure of RCs and the primary amino acid sequences of the H subunits, it has been proposed that some amino acid residues in the H subunit might act as proton carriers between the cytoplasm and the Q B site (Deisenhofer, et al, 1985; Allen, et al., 1988). Other studies reported that the H subunit seemed to 4 be required for the assembly and/or maintenance of a functional RC and the correct alignment and stabilization of the RC L and M subunits in R. sphaeroides (Chory, et al, 1984; Varga and Kaplan, 1993). Therefore, when I began my thesis research the function of the RC H subunit in photosynthesis in purple, non-sulfur bacteria was unclear. Many of the essential pigment biosynthetic and pigment-protein complex structural genes involved in photosynthesis are located in a 46 kb region of the R. capsulatus genome, termed the photosynthesis gene cluster (Fig. 1) (Taylor, et al., 1983; Zsebo and Hearst, 1984). The DNA sequence of the entire region shown in Fig. 1 has been obtained by J. Hearst's laboratory (deposited in the EMBL library, accession #Z11165). The puf operon encodes the structural genes for LHI and the RC L and M subunits, a gene necessary for bacteriochlorophyll biosynthesis (pufQ), and the pufX gene, which is involved in quinone transfer to the cyt b/ci complex (Youvan, et al, 1984; Belasco, et al, 1985; Bauer, et al., 1988; Lilburn, et al., 1992). The puc operon, which is not located within the photosynthesis gene cluster, encodes the LHII structural polypeptides as well as gene products essential for wild type levels of LHII complex (Youvan and Ismail, 1985; Tichy, et al., 1989; LeBlanc and Beatty, 1993). The puhA gene, which is located in the photosynthesis gene cluster 39 kb away from the puf operon and transcribed in an opposite direction (see Fig. 1), encodes the RC H subunit (Taylor, et al, 1983; Zsebo and Hearst, 1984). Like the puf operon, the puhA gene has overlapping transcripts, which (for the puhA gene) originate from the bchFBKHLM-F1696 operon (herein referred to as bchF operon; Fig. 2A) (Bauer, et al., 1991; Wellington, et al, 1991; Wellington, et al, 1992). A large 11 kb transcript that encodes puhA is a product of read-through transcription from the bchF 5 Figure 1. Representation of genes and transcripts of the Rhodobacter capsulatus photosynthesis gene cluster. Bacteriochlorophyll biosynthesis genes (bch) are designated by gray shading, carotenoid biosynthesis genes (crt) are shown as cross-hatched boxes, light-harvesting and reaction center genes (puf and puh) are represented by diagonal lines, and open reading frames of uncertain function are shown by spots. Proposed transcripts are designated by arrows, with possible read-through extensions shown as dotted lines (after Beatty, in press). orf641 X M L A B pufQ Z Y X bchC F crtE crtD C K B crtl crtA bchl D orf284 orfl76 bchP orf428 G J bchE orf469 bchW bchF B K H L M ^niA F1696 °f62b *«* 55 274 162a KZZZ, ^ fflffi l l l Hi h » * M t 1 mrmfmrnm 7 Figure 2. A. Operonal organization of the bchF operon in R. capsulatus. Designations of genes and open reading frames are the same as in Fig. 1. The three proposed puhA messages are shown as horizontal arrows (Bauer, et al., 1991). These include a large 11 kb transcript originated from the bchF promoter, a 1.1 kb puhA transcript derived from mRNA processing of the 11 kb transcript, and a highly expressed 0.9 kb transcript initiated from the puhA promoter located with F1696. Possible read-through extensions (this thesis) are shown as dotted lines. B. Restriction map of the puhA region in the R. capsulatus photosynthesis gene cluster. Designations of genes and open reading frames are the same as in Fig. 1. The restriction sites shown here are sites used in this thesis for subcloning. 8 0.5 kb mRNA processing -a i mrncmMimm-bchF bchB bchK bchH bchL bchM puhA F1696 orf214 orfl62b orf55 orf274 orfl62a B 0.5 kb Pstl Mlu I Msc I Sal I Sty I BamH I EcoR I X z. WEEEmMMMZ^^MMMm bchM F1696 puhA orf214 orfl62b orf55 9 promoter, which encodes several bacteriochlorophyll biosynthesis genes. A second 1.1 kb puhA mRNA is derived from the 11 kb bchF transcript by mRNA processing and a third, highly expressed 0.95 kb transcript is initiated from a promoter located within the gene immediately upstream of the puhA structural gene, namely F1696. The data reported by Bauer et al. are consistent with the view that all puhA transcripts terminate at an inverted repeat sequence located about 12 bp 3' (downstream) of the stop codon of the puhA gene (Bauer, et al, 1991). The dashed extensions of transcripts, shown in Fig. 2A, are based upon results presented in this thesis. This thesis describes the use of a directed mutagenesis approach with the puhA gene to elucidate the role of the RC H subunit in photosynthesis. This study also investigates the possibility of the presence of genes downstream of puhA in the photosynthesis gene cluster and their effects on photosynthesis. Open reading frames (orfs) downstream of the puhA exist, according to sequencing data provided by J. Hearst's laboratory (EMBL library accession # Z11165). These open reading frames are designated orf214, orfl62b, orf55, orf274, and orfl62a (Fig. 2A). The functions of these gene products possibly encoded by the downstream orfs are not known. The putative gene product of orf214 is a 23.7 kDa protein which, shown by hydropathy plot, is hydrophilic at both ends and hydrophobic at the central sequence (Fig. 3). This hydropathy plot indicates a relatively hydrophobic protein, perhaps even an integral membrane protein. The possible importance of puhA and orf214 in photosynthesis was assessed by directed mutagenesis. The approach that I took was to mutate puhA and orf214 separately, followed by characterization of the mutant strains. Two kinds of mutations were used: omega (Q.) interposon insertions (Prentki and Krisch, 10 -58 " 1 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 128 168 288 p i i i i n n r 48 88 Figure 3. Hydropathy plot of putative gene product of orf214, using the method of Kyte and Doolittle (Kyte and Doolittle, 1982). The hydropathy index is shown on the y-axis (more positive values indicate more hydrophobic segments), whereas the amino acid residues are numbered on the x-axis. The hydrophobic domains are above the dotted line and the hydrophilic domains are below it. The hydropathy index is computed using an interval of 15 amino acids. 11 1984) and a translationally in-frame deletion of puhA. Omega interposon insertion has a polar effect on transcription of downstream genes if they are co-transcribed with the gene being mutated, whereas an in-frame deletion should not. After mutagenesis, the mutant strains were characterized to determine if they grew photosynthetically and had normal levels of pigment-protein complexes. The mutant strains were complemented in trans with plasmids expressing puhA, orf214 and orfl62b to see if photosynthetic growth or other phenotypic changes were restored. All mutations were made in the photosynthetically wild type strain SB1003, and some mutations were also made in strains that lacked LHII complex (encoded by the puc genes) to simplify spectral analyses. (The LHII complex partially obscures the less abundant light-harvesting complex I [LHI] and reaction center absorption spectra.) 12 MATERIALS AND METHODS 1. Bacterial strains The strains used in this thesis are listed in Table I. R. capsulatus SB1003, which is a phage-free, rifampicin resistant derivative of the natural isolate strain BIO, was used as a wild type parental strain (Weaver, et al., 1975; Solioz and Marrs, 1977). R. capsulatus MW442 and ALHII were used as LHII" background strains in spectral analysis. R. capsulatus MW442 (Scolnik, et ah, 1980) is a LHII' derivative of BIO. R. capsulatus ALHII (LeBlanc and Beatty, 1993) is a LHII" derivative of SB1003 in which the pucBACDE genes have been replaced by the Q cartridge (Prentki and Krisch, 1984). The R. capsulatus GTA (Gene Transfer Agent; a generalized transducing phage) over-producer strain DE442 was used as a GTA phage donor in GTA-aided transduction (Yen, et ah, 1979). £. coli strains C600 r"m+ (Bibb and Cohen, 1982), JM83 (Yanisch-Perron, et al, 1985) and JM101 (Yanisch-Perron, et ai, 1985) were host strains used for maintenance of plasmids. E. coli SM10 (Simon, et ai, 1983) and TEC5 (Taylor, et al, 1983) were used as mobilizing strains for inter-generic conjugation with R. capsulatus. When necessary, E. coli HB101(pRK2013) was used as a mobilization helper strain for tri-parental conjugation (Ditta, et al, 1985). 2. Growth conditions All R. capsulatus strains were routinely grown in either RCV (a minimal m a l a t e / N H 4 + medium) or YPS medium (which contains yeast extract and peptone) at 34°C (Weaver, et ai, 1975; Beatty and Gest, 1981). A modified RCV medium, which has 7 mM glutamate rather than NH4+ as the 13 Table I. Bacterial strains and plasmids used Strains / Plasmids Genotype /Pescription Reference /Source A. Ecoli C600r-m+ F- el4-(mcrA-) hsdR thr-1 leuB6 thi-1 lacYl supE44 fhuA21 mcrB TEC5 C600(pDPT51) plasmid mobilizing strain JM101 F traD36 lacl1A(lacZ)M15 proA+B+/supE thi A(lac-proAB) JM83 F" ara A(lac-proAB) rpsL (Strr) [08O dlacA(lacZ)M15] SM10 Plasmid mobilizing strain HB101 F" A(gpt-proA)62 leu supE44 aral4 galK2 lacYl A(mcrC-mrr) rpsL20 (Strr) xyl-5 mtl-1 recA13 Host strain for pRK2013 helper plasmid used in tri-parental matings B. R. capsulatus (Bibb and Cohen, 1982) (Taylor, et al, 1983) (Yanisch-Perron, et al, 1985) (Yanisch-Perron, et al, 1985) (Simon, et al, 1983) (Schmidhauser and Helinski, 1985) SB1003 DE442 MW442 ALHII DW1 DW2 rif-10 crtD GTA over-producer L H i r derivative of BIO Apucy.Q. derivative of SB1003 ApuhAr.Q. derivative of SB1003 orf214::Q. derivative of SB1003 (Solioz and Marrs, 1977) (Yen, et al, 1979) (Scolnik, et al, 1980) (LeBlanc and Beatty, 1993) This thesis This thesis 14 DW5 DW6 DW13 IFApuhA derivative of SB1003 ApuhAr.Q derivative of MW442 IFApuhA derivative of ALHII This thesis This thesis This thesis C Plasmids pNIF215 Expression vector with the (Zilsel, 1990) R. capsulatus nifHDK operon promoter; RSF1010 derivative pRK2013 pRK415 pRK415* pRPSE2 pSUP202 pUC13 pUC13::EcoF pUCpuhAl pIFApuhAl pIFApuhA2 pIFApuhA3 Mobilizing plasmid; Km r Broad host range vector; Tc r; pRK404 derivative with the pUC19 multiple cloning site; lacZa BamH. I filled-in derivative of pRK415 EcoF fragment cloned in pDPT44 Suicide plasmid; pBR325 derivative; Mob+; Apf; Cm r ; Tc r Ap r ; lacZa EcoF fragment cloned in pUC13 pUC13 with 1.1 kb Mlu I-BamH I puhA insert pUC13 with 0.4 kb Mlu I-BamH I IFApuhA insert pIFApuhAl with 1.3 kb Pst l-Mlu I sequence upstream of puhA pIFApuhA2 with 1.1 kb BamH I-EcoR I (Ditta, et ah, 1985) (Keen, et ai, 1988) This thesis (Taylor, et ah, 1983) (Simon, et ai, 1983) (Messing, 1983) This thesis This thesis This thesis This thesis This thesis sequence downstream of puhA 15 pSUP-IFApuhA pSUP202 with 2.8 kb Pstl-EcoR I This thesis IFApuhA insert pApuhA::Ql pUC13 with 2.4 kb Mlu 1-BamH I This thesis ApuhA::Q insert pApuhA::Q2 pApuhA::Ql with 1.3 kb Pst l-Mlu I This thesis sequence upstream of puhA pApuhA::Q3 pApuhA::Q2 with 1.1 kb Sfu I-EcoR I This thesis sequence downstream of puhA pRKpuhAl pRK415* with 5.1 kb Sst I puhA insert This thesis pRK214Q pRKpuhAl with the 2.0 kb Q cartridge This thesis insert into the BamH I site inside orf214 pPUHA pRK415 derivative; expression vector This thesis for puhA pORF214/162b pNIF215 derivative; expression vector This thesis for orf214 and orfl62b 16 nitrogen source, was used for the expression of the nifHDK promoter on plasmid pNIF215 derivatives (Pollock, et a\., 1988; Zilsel, 1990). All cultures used in growth experiments were inoculated to a turbidity of about 20 Klett units (ca. 8 x 107 cfu/ml) and growth was followed by measuring the turbidity of the culture using a Klett-Summerson Photometer with a red (No. 66) filter. Oxygen-limited cultures were grown in long neck metal-capped Erlenmeyer flasks filled to 80% of their nominal volumes and shaken at 150 RPM. Photosynthetic cultures were inoculated from oxygen-limited cultures in stationary phase. Photosynthetically grown cultures were grown in screw-cap tubes filled to capacity and incubated in a glass-sided water bath illuminated with several Sylvania lumiline incandescent lamps, depending on the light intensity needed. Light intensity was measured with a Li-Cor photometer equipped with an LI-190SB quantum sensor (Li-Cor, Lincoln, NE). Plate cultures were grown on media supplemented with agar at 15 g/L. Photosynthetically grown plate cultures were grown in BBL GasPak anaerobic jars (Becton Dickinson and Co., Cockeysville, MD) at 34°C. Unless specified, all E. coli strains were grown in LB medium (Sambrook, et al., 1989). Media were supplemented with antibiotics at the following concentrations: for R. capsulatus, spectinomycin: 10 ug /ml , tetracycline: 0.5 u g / m l , kanamycin: 10 u g / m l ; for E. coli, ampicill in: 200 u g / m l , spectinomycin: 50 ug /ml , tetracycline: 10 ug /ml , kanamycin: 50 ug /ml , streptomycin: 20 ug/ml , trimethoprim: 40 ug/ml. 17 3. In vitro DNA techniques The plasmids used and constructed in this thesis are listed in Table I. Plasmid DNA was routinely isolated from E. coli cultures by the alkaline lysis method (Sambrook, et al, 1989). For large-scale purification, E. coli was grown in either LB or Terrific Broth for plasmid amplification (Sambrook, et al, 1989) and cells were lysed and plasmids were isolated using the QIAGEN DNA-affinity column procedures (QIAGEN Inc., Chatsworth, CA). General in vitro DNA techniques such as restriction endonuclease digestion, DNA ligation, agarose gel electrophoresis, transformation of E. coli and other recombinant DNA procedures were performed as described (Sambrook, et al, 1989). DNA was purified from agarose gel by adsorption to glass or silicagel particles, using the commercially available GeneClean II (Bio/Can Scientific, Mississauga, Canada) or QIAEX (QIAGEN Inc., Chatsworth, CA) procedures. Electro-transformation of E. coli was performed using the Gene Pulser apparatus , and cells were grown, harvested, and electro-transformed according to the manufacturer's instruction manual (Bio-Rad Laboratories, Richmond, CA). 4. Bacterial conjugation and GTA transduction Conjugation of plasmid DNA into R. capsulatus strains was accomplished using E. coli SM10 (Simon, et al, 1983) or TEC5 (Taylor, et al, 1983) as plasmid donor strains. When other E. coli strains were used as plasmid donors, E. coli HB101(pRK2013) (Ditta, et al, 1985) was used as a mobilization helper strain. Equal volumes of overnight stationary phase cultures of donor and recipient cells (and helper cells when necessary) were mixed, pelleted (30 seconds, 15,000 x g in an Eppendorf benchtop 18 microcentrifuge), and resuspended in an equal volume of RCV medium. A 10 |il portion of the suspension was spotted onto a RCV plate. After the spot was dried, the plate was incubated at 30°C overnight to allow for conjugation. R. capsulatus exconjugants were purified from £. coli cells by subsequent streaking onto RCV plates with an appropriate antibiotic(s), and purity was checked by streaking onto RCV plates supplemented with 0.1 % yeast extract. The R. capsulatus GTA over-producer strain DE442 (Yen, et al., 1979) was used as the donor in GTA transductions. Plasmids were transferred from E. coli strains to R. capsulatus DE442 by conjugation. The exconjugant R. capsulatus strains were grown to stationary phase in YPS medium under photosynthetic conditions. GTAs were isolated by filtration of this culture through a 0.45 |im syringe filter (Nalge Company, Rochester, NY), collected in a polycarbonate plastic vial, and then used for the transduction into an appropriate recipient. The recipient R. capsulatus cells, which were grown aerobically to early stationary phase, were harvested by centrifugation and resuspended in an equal volume of filter sterilized G buffer. (G buffer consists of 10 mM Tris-HCl pH 7.8, 1.0 mM MgCl2, 1.0 mM CaCl2, 1.0 mM NaCl, 500 | ig /ml BSA.) Equal volumes (0.1 ml) of GTAs and resuspended recipient cells were mixed with 4 volumes of G buffer, and the mixture was incubated at 35°C for an hour to allow GTA phage adsorption to recipient cells. The mixture was then transferred to a test tube containing 3 ml of YPS medium, and incubated aerobically at 30°C overnight. Portions of this culture were spread on YPS plates with an appropriate antibiotic for the selection of the transductants. 19 5. Construction of R. capsulatus strains DW1, DW2, DW5, DW6, and DW13 The chromosomal arrangements of genes and orfs in the puhA and orf214 mutant strains are summarized in Fig. 4. The puhA gene is located in the EcoF fragment of pRPS404 (Marrs, 1981), which contains the 46 kb photosynthesis gene cluster, and the EcoF fragment has been cloned in pDPT44 to make pRPSE2 (Taylor, et al, 1983). For easy manipulation, the EcoF fragment was subcloned into pUC13 to make pUC13::EcoF. A Mlu I (ends made blunt with the large fragment of DNA polymerase I, Klenow fragment) to BamH I 1.1 kb puhA fragment was obtained from plasmid pUC13::EcoF and inserted into plasmid pUCpuhAl (Fig. 5). Plasmid pUCpuhAl was then digested with Msc I and Sty I (ends made blunt with the Klenow fragment) such that a 701 bp fragment (which accounts for the central 92% of the coding sequence) was deleted from the puhA gene, and a 10 bp Hind III linker was inserted such that the deletion did not cause a translational frame shift, as confirmed by DNA sequencing (see Fig. 6). This deletion leaves only 33 bp of the puhA coding sequence upstream and 29 bp downstream of the deletion site. To this plasmid containing the Mlu I to BamH. I translationally in-frame deleted puhA fragment (IFApuhA), designated as pIFApuhAl, the upstream Pst I to Mlu I flanking sequence and downstream BamH I to EcoR I flanking sequences were added, making the plasmid pIFApuhA3. Plasmid pIFApuhA3 was then partially digested with Pst I, followed by digestion with EcoR I. The 2.7 kb Pst I to EcoR I fragment containing the IFApuhA gene was then ligated into the suicide plasmid pSUP202 (Simon, et al., 1983). The resulting construct, named pSUP-IFApuhA, was mobilized into the wild type R. capsulatus strain SB1003 by conjugation. Exconjugants were selected by tetracycline resistance, one of the 20 SB 1003 Mlul Mscl Sty I BamHl dzzzzzz^-kmm}-Lc puhA orf214 DW5 orDW13 Hind HI Mscl \-dzH. [Sty I] Mlul \/BamHl ' 1 • i • i •, i • i •, i " i IFApuhA orf214 DW1 orDW6 Mlu I [Msc I] Leb= "IT ApuhA::Q Sty I BamHl orf214 DW2 Mlul Mscl Sty I BamHl ^-^zzzzzhA puhA JL BamHl orf214::Q. Figure 4. Genetic arrangement of the puhA and orf 214 mutant strains. Only the puhA and orf214 loci are shown. Restriction sites used for making deletion or insertion are shown above the genes. Filled-in sites which were not regenerated after ligation are shown in square brackets. 21 Figure 5. Outline of the construction of the plasmids used to make the translationally in-frame deletion of puhA. Plasmid sequences are shown as thin lines. Genes and origin of replication in the plasmids are shown as open arrows. R. capsulatus sequences (DNA inserts) are shown as shaded boxes and the puhA gene is shown as a shaded arrow. The restriction sites are abbreviated as follows: E, EcoR I; B, BamH I; S, Sal I; St, Sty I; Ms, Msc I; P, Pst I; M, Mlu I; H, Hind III. Filled-in sites which were not regenerated after ligation are shown in square brackets. 22 Tc 1. Sal I digest; Klenow fill-in 2. BamH I digest 3. ligate with 1.1 kb puhA fragment 1. Sty I digest; Klenow fill-in 2. Msc I digest 3. ligate with Hind III linker M l.Pst I digest [S] 2. Mlu I digest P 3. ligate with 1.3 kb upstream flanking sequence 1. EcoR I digest 2. BamH I digest 3. ligate with 1.1 kb downstream flanking sequence [St] l.Pst I partial digest M" 2. EcoR I digest 3. isolate 2.8 kb IFApuhA insert ligation Mob\ pSUP202 8.1 kb l.Pst I digest 2. EcoR I digest 3. isolate 7.0 kb vector Ap Cam. SB 1003 DW5 Pstl MlulMscl Styl BamHl EcoRl Pstl MVGVNFFGDFDLAKLGKGVKPYAL Figure 6. Representation of wild type (SB1003) and the translationally in-frame deletion allele of the puhA gene, and flanking regions. Restriction endonuclease sites used in subcloning experiments are given at the top. The puhA gene is designated as hatched box, and the two flanking open reading frames are shown by spots. The underlined amino acid residues (single letter code) indicate the amino acids present in both the wild type and the deletion allele of the puhA gene. 24 antibiotic-resistance markers coded for by the plasmid vector. Since this suicide plasmid is unable to replicate in R. capsulatus, the most likely way for the host to acquire tetracycline resistance is by a single homologous recombination between either the upstream or the downstream R. capsulatus sequences on the plasmid with the homologous sequence on the host chromosome. Colonies of recombinants were then subjected to replica plating, after growth in the absence of tetracycline, to obtain a secondary recombinant by screening for the loss of tetracycline resistance and the loss of photosynthetic growth. This screening was done to select for a recombinant which had undergone a second homologous recombination, such that the in-frame deleted copy of puhA replaced the adjacent wild type allele (see Fig. 7). I assumed that this strain would not be capable of photosynthetic growth since the puhA locus was originally defined as a site that, when mutated, resulted in the loss of photosynthetic growth (Youvan, et al., 1983). Of the approximately 2000 colonies screened, about 3% were tetracycline sensitive Tcs. Among these Tcs colonies, two were found to be incapable of growth under photosynthetic conditions (PS"). The genetic arrangement of one of these PS", Tcs isolates was confirmed by Southern hybridization (see Results section l.A). This IFApuhA strain was named DW5 (Fig. 4). With further GTA-aided transduction of the Apucr.Q. allele from ALHII into DW5, the LHII" puhA' strain was made and named DW13. DW1 and DW6 are R. capsulatus strains with the central part of the puhA gene coding sequence deleted and replaced by the 2 kb streptomycin/spectinomycin resistance omega (Q) interposon (Fig. 4). This disrupted puhA allele is herein referred to as ApuhAv.Q. As in the construction of DW5 (see above), plasmid pUCpuhAl, which contains the 25 Figure 7. Outline of the screening of the recombinants which incorporated an translationally in-frame deleted copy of the puhA gene into the chromosome. A. Plasmid pSUP-IFApuhA was conjugated into the wild type strain SB1003. B. Tc r transductants were selected. These were found to be ones with the plasmid sequence incorporated into the chromosome through a single homologous recombination. Plasmid pSUP202 sequence, which has been incorporated into the chromosome is shown as a dotted line in the middle. C. A single homologous recombinant was grown in the absence of Tc, allowing a second homologous recombination. Two possible outcomes could occur: i) the single cross-over strain reverts to wild type genotype, giving Tcs, puhA+; or ii) a second cross-over occurs such that the IFApuhA allele replaces the wild type puhA allele, giving Tcs, puhA'. The restriction sites are abbreviated as follows: E, EcoR I; P, Pst I; St, Sty I; Ms, Msc I; H, Hind III. Filled-in sites which were not regenerated after ligation are shown in square brackets. WZ2i^Si—' First homologous recombination Tcr, PS+(puhA+) F1696 puhA orf214 A H Ms I [St] i) Second homologous recombination Revert to wild-type Tcs , PS+(puhA+) M Tc1 #--C y~/s •k wzm IFApuhA orf214 F1696 puhA o ii) Second homologous recombination Tcs , PS" (puhA-) IT H Ms [St] D-H F1696 IFApuhA orf214 27 intact puhA gene, was used (Fig. 8). The same 701 bp Msc I and Sty I fragment was deleted from the puhA gene and replaced by the Q interposon, which contains both transcriptional and translational stop signals at both ends, thereby causing a polar effect on the expression of downstream genes that might be co-transcribed with puhA. Again, only 33 bp of the puhA coding sequence upstream and 29 bp downstream of the Q interposon remained (Fig 4). The fl-inserted/deleted puhA gene, together with its flanking sequences, was present in plasmid pApuhA::Q3 as a 4.8 kb Pst I to EcoR I fragment (Fig. 8). Plasmid pApuhA::Q3 was subsequently transferred to the R. capsulatus GTA over-producer strain DE442, using the mobilizing E. coli strain TEC5. Through GTA-aided transduction into the wild type strain SB1003, followed by selection for spectinomycin resistance, a ApuhAr.Q. strain was obtained and named DW1. Southern blot was performed to confirm the correct genotype (data not shown). The same procedures were performed in the LHII" strain, MW442, making the mutant strain DW6. R. capsulatus strain DW2 was constructed by disruption of the open reading frame downstream of puhA, orf214 (Fig. 4). The disruption was obtained by insertion of the Q. interposon at the BamH I site located 36 bp downstream of the start codon of this orf, in plasmid pRKpuhAl containing the cloned orf214 (Fig. 9). (The BamH I site in the plasmid sequence of pRK415, from which pRKpuhAl was derived, was previously deleted by filling-in of the BamH I digested termini, followed by re-ligation of these ends.) The resultant plasmid, designated pRK214Q, was transferred from the mobilizing E. coli strain SM10 to the R. capsulatus GTA over-producer strain DE442 by conjugation. GTA from this strain was used to infect the wild type R. capsulatus strain SB1003. Recipients which were transduced by 28 Figure 8. Outline of the construction of the plasmids used to make the puhA dele t ion/Q insertion. Plasmid sequences are shown as thin lines. Genes and origin of replication in the plasmids are shown as open arrows. R. capsulatus sequences (DNA inserts) are shown as shaded boxes, the puhA gene is shown as a shaded arrow, and the Q. cartridge is shown in black. The restriction sites are abbreviated as follows: E, EcoR I; B, BamH I; S, Sal I; St, Sty I; Ms, Msc I; P, Pst I; M, Mlu I; Sf, Sfu I. Filled-in sites which were not regenerated after ligation are shown in square brackets. 29 P I. Sail digest; Klenow fill-in 2. BamH I digest 3. ligate with 1.1 kb puhA fragment 1. 5ry I digest; Klenow fill-in E/BSf 2. Msc I digest ^ St 3. ligate with 2.0 kb Sma\ Q. fragment l.Pst I digest 2. M/H I digest 3. ligate with 1.3 kb upstream flanking sequence 1. EcoR I digest 2. Sfu I digest 3. ligate with 1.1 kb downstream flanking | f sequence St 30 Figure 9. Outline of the construction of the plasmids used to make the Q insertion into orf214. Plasmid sequences are shown as thin lines. Genes and origin of replication in the plasmids are shown as open arrows. R. capsulatus sequences (DNA inserts) are shown as shaded boxes, the puhA gene and orf214 axe shown as shaded arrows, and the Q cartridge is shown in black. The restriction sites are abbreviated as follows: E, EcoR I; Ss, Sst I; B, BamH I; H, Hind III. Filled-in sites which were not regenerated after ligation are shown in square brackets. 31 1. BamH I digest 2. Klenow fill-in 3. Re-ligation 1. Sst I digest 2. isolate 5.1 kb insert Sst I digest 1. BamH I digest 2. ligate with 2 kb E BamH IQ. fragment Ss / E 32 the Q-inserted copy of orf214 would have incorporated this mutated copy into their chromosome by homologous recombination, resulting in the loss of the corresponding wild type orf214 allele and the gain of spectinomycin resistance. These transductants were selected by spectinomycin resistance, yielding the mutant strain DW2 (Fig. 4), and the genotype was confirmed by Southern blot (data not shown). 6. Complementation plasmid constructions Since puhA has one of its two endogenous promoters located inside F1696 and at about 0.25 kb upstream of the puhA coding sequence (Fig. 2A; Bauer, et ah, 1991), an expression vector was made using this promoter for the expression of the puhA gene. The puhA gene with this promoter was subcloned from pUC13::EcoF into the mobilizable broad host-range vector pRK415 as a 2.4 kb Pst I to BamH I fragment (Fig. 10). This fragment contains the entire F1696 and puhA coding sequences, but the only promoter that is known to function in R. capsulatus is the puhA promoter located within F1696 (Johnson, et al., 1986; Bauer, et al., 1988). The resulting plasmid, pPUHA, was then transferred by conjugation to appropriate R. capsulatus hosts for the expression of puhA (which would produce the RC H subunit). The location of the promoter for orf214 is not known, although orf214 transcription seems likely to originate from upstream of puhA (see Discussion section). Therefore, an expression vector pNIF215, with the R. capsulatus nifHDK promoter, was used for the expression of orf214. A 1.5 kb Sal I-EcoR I fragment containing orf214 and orf!62b (the open reading frame downstream of orf214) was subcloned from plasmid pUC13::EcoF into 33 Figure 10. Outline of the construction of the plasmid pPUHA. Plasmid sequences are shown as thin lines. Genes and origin of replication in the plasmids are shown as open arrows. R. capsulatus sequences (DNA inserts) are shown as shaded boxes, and F1696, puhA, orf214, and orfl62b are shown as shaded arrows. The puhA promoter is shown as arrows above the plasmids. The restriction sites are abbreviated as follows: E, EcoR I; B, BamH I; P, Pst I; H, Hind III. 34 l.Pst I digest 2. BamH I digest 1. Pst I digest 2. BamH I digest 3. isolate 2.4 kb puhA insert ligation 35 pNIF215 (Zilsel, 1990) to construct the orf214 expression vector pORF214/162b (Fig. 11). The plasmids pPUHA and pORF214/162b are members of different incompatibility groups and can stably co-exist in the same cell. 36 Figure 11. Outline of the construction of the plasmid pORF214/162b. Plasmid sequences are shown as thin lines. Genes and origin of replication in the plasmids are shown as open arrows. R. capsulatus sequences (inserts) are shown as shaded boxes, and F1696, puhA, orf214, and orfl62b are shown as shaded arrows. The puhA promoter and nifHDK promoter are shown as arrows above the plasmids. The restriction sites are abbreviated as follows: S, Sal I; E, EcoR I. 37 nifHDK promoter 1. EcoR I digest 2. Sal I digest 3. isolate 9.1 kb vector 1. EcoR I digest 2. 5a/1 digest 3. isolate 1.5 kb insert ligation nifHDK promoter 38 7. DNA and protein sequence analyses The 46 kb R. capsulatus photosynthesis gene cluster has been sequenced by J. Hearst's laboratory (EMBL library, accession # Z11165). The DNA sequence of the IFApuhA allele was obtained by use of the T7Sequencing Kit (Pharmacia, Baie d'Urfe, Canada), and plasmid pIFApuhAl as template, in accordance with the manufacturer's instructions. The DNA sequence data and the protein sequence data predicted from the DNA sequence data were analyzed with the PC/GENE (Intelligenetics Inc., Switzerland) or DNA Strider (Commissariat a l'Energie Atomique, France) software package. 8. Southern blots Chromosomal DNA was purified from R. capsulatus cells by a Triton X-100 lysis procedure. Cell pellets from 150 ml of stationary phase cultures were resuspended in 4 ml of a 10% sucrose solution dissolved in Tris-HCl (pH 8.0). To these resuspended cells lysozyme was added to a concentration of 0.9 mg/ml, followed by incubation on ice for 10 minutes, addition of 0.4 ml of 0.5 M EDTA and a second 10 minutes incubation on ice. The cells were lysed by addition of 3.2 ml of Triton X-100 lysis buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 2% Triton X-100 [octyl phenoxy polyethoxyethanol]), and the lysate was incubated at 65°C for 10 minutes. CsCl (1 g/ml of crude lysate) was dissolved in the lysate after which it was dispensed into 13.5 ml ultracentrifuge tubes, followed by topping off with 250 |il of an ethidium bromide (10 mg/ml) stock solution. The DNA was recovered after standard CsCl density ultracentrifugation procedures, extracted with isopropanol (previously equilibrated with a saturated solution of CsCl in water), and 39 dialyzed against TE buffer (Sambrook, et al., 1989). Five ug of chromosomal DNA were digested with Pst I and electrophoresed in a 1% agarose gel in 0.5 X TBE buffer (0.089 M Tris-base, 0.089 M boric acid, 0.002 M EDTA) at room temperature. The DNA was denatured by soaking the gel twice in 20 gel volumes of 1.5 M NaCl, 0.5 M NaOH for 20 minutes at room temperature. The gel was neutralized by soaking twice in 20 gel volumes of 1.5 M NaCl , 1.0 M Tris (pH 7.5) for 20 minutes at room temperature. The neutralization step was repeated if the pH of the gel was > 7.8 (pH was checked by laying pH paper on the gel). The gel was then equilibrated in two changes of 0.5 X TBE buffer for 20 minutes each to reduce the ionic strength of the gel. DNA was transferred to ICN Bio-Trans nylon membranes (wetted in 0.5 X TBE buffer) (ICN Biomedicals Canada Ltd., Mississauga, Canada) by electroblotting at 30 V in 0.5 X TBE buffer for about 18 hours, followed by 80 V for 2 hours, in a BIO-RAD Trans-Blot Electrophoretic Transfer Cell (Bio-Rad Laboratories, Richmond, CA). The membrane was then soaked in 5 X SSPE (20 X SSPE: 3.6 M NaCl, 200 mM N a H 2 P 0 4 , 20 mM EDTA, pH 7.4) for 5 minutes at room temperature and air-dried for 30 minutes. The DNA was fixed to the membrane by drying in a vacuum oven at 80°C for 2 hours. Membranes (about 14 X 7 cm2) were prehybridized for about 4 hours at 42°C in 20 ml of 5 X SSPE, 0.3% SDS, 50% formamide, and 100 ug /ml denatured sheared salmon sperm DNA. The prehybridization buffer was then supplemented with approximately 50 ng of alkali denatured probe, which had been biotin-labelled by the random oligonucleotide primer method using biotin-14-dATP and 400 ng of template DNA (Bethesda Research Laboratories, Gaithersburg, MD). 40 Hybridization occurred overnight at 42°C, after which the membrane was washed twice for 5 minutes in 2 ml/cm2 of a solution of 5 X SSPE, 0.5% SDS at 65°C. The membrane was further washed once for 40 minutes in 2 ml /cm 2 of 0.1 X SSPE, 1% SDS at 65°C, followed by one wash in the same volume of 2 X SSPE for 5 minutes at room temperature. The non-radioactive Photogene Nucleic Acid Detection System (Bethesda Research Laboratories, Gaithersburg, MD) was used. The suggested protocol was followed except that the durations of the blocking solution and the final wash incubations were increased from one hour to two hours. Films were exposed for periods of time from 30 seconds to 5 minutes at room temperature. 9. Spectrophotometric analyses Absorption spectra of oxygen-limited and photosynthetically grown intact cells (about 1.8 x 109 cells suspended in 22.5% BSA in RCV medium) were obtained using a Hitachi U2000 spectrophotometer, and data were collected with the Spectra Calc software package (Galactic Industries Corporation, Salem, NH). 10. Isolation of chromatophores Chromatophores (ICM vesicles containing the photosynthetic apparatus) were prepared from cells grown under reduced aeration and disrupted by passage through a French pressure cell with a pressure of approximately 15,000 psi. The chromatophores were purified by centrifugation (25,800 x g, 8 minutes) of the French pressed cells to pellet intact cells and large debris, followed by centrifugation of this supernatant fluid (412,000 x g, 14 minutes) to pellet chromatophores. The pellet was 41 resuspended in 50 mM Tris (pH 8.0) and the chromatophores were further purified using a 3-layered (20-40-60 %) sucrose step gradient in 50 mM Tris (pH 8.0) as the buffer. After centrifugation at 100,000 x g for 7 hours, purified chromatophores were then collected from the 20%-40% interphase. The chromatophores were then diluted in 50 mM Tris (pH 8.0) and pelleted by centrifugation at 171,000 x g for 2 hours and resuspended in chromatophore buffer (20 mM 3-[N-morpholino]propanesulfonate [MOPS], 100 mM KC1, 1 mM MgCl2 [pH 7.2]) (Jackson, et al, 1986), to a concentration of about 10-20 mg protein/ml. Typically, 100 ml of cell culture (@~ 300 Klett units) were used to prepare about 0.5 ml of purified chromatophores. 11. Protein concentration determinations Chromatophore protein concentration was measured using a modified Lowry method (Peterson, 1983). 12. Gel electrophoresis of proteins A tricine-SDS polyacrylamide gel system was used for electrophoresis of purified chromatophore proteins (Schagger and von Jagow, 1987). The following polyacrylamide content for each gel layer were used : stacking gel, 4%; spacer gel, 10%; separating gel, 16.5%; with the buffers as suggested (Schagger and von Jagow, 1987). One hundred |ig of the purified chromatophore proteins were loaded in each lane. The gel was stained with Coomassie blue after electrophoresis. 42 RESULTS 1. Analyses of the translationally in-frame puhA deletion mutants DW5 and DW13 A. Southern blot analysis Chromosomal DNAs prepared from wild type SB1003 and DW5 were digested with Pst I and used for Southern blot analysis (Fig. 12). A 1.3 kb Pst I-Mlu I restriction endonuclease fragment containing SB1003 DNA sequences upstream of puhA was used to construct the biotin-labelled probe (see Materials and Methods). A strong hybridization signal at 1.3 kb was seen in the control lane with the non-labelled 1.3 kb Pst l-Mlu I DNA fragment as the hybridization target (Fig. 12, lane 1). When a Pst I digest of DNA from SB1003 was subjected to hybridization, a 2.7 kb signal was detected (Fig. 12, lane 2). This 2.7 kb band corresponds to the length of the Pst I chromosomal DNA fragment containing the wild type puhA gene and flanking sequences. A 2.0 kb hybridization band was observed in the Pst I digest of DW5 DNA, showing that the DNA sequence in the puhA region of DW5 is 0.7 kb shorter than that of the wild type SB1003 (Fig. 12, lane 3). This corresponds to the 0.7 kb deletion of wild type puhA coding sequence, giving rise to a signal of 2.0 kb instead of the wild type 2.7 kb (see Materials and Methods). As a control in lane 4 of Fig. 12 was a 2.0 kb Pst I restriction fragment, obtained from Pst I digestion of pIFApuhA3 (see Fig. 5), the plasmid used to generate the translationally in-frame deletion of puhA during the process of constructing DW5. A hybridization signal of 2.0 kb was seen with this Pst I fragment. This further confirmed that the 2.0 kb hybridization signal seen with Pst I-digested DW5 chromosomal DNA (Fig. 12, lane 3) was due to the integration 43 Figure 12. Southern hybridization analysis of Pst I-cut chromosomal DNA prepared from R. capsulatus strains SB1003 and DW5. A. Restriction maps of SB1003 and DW5, showing the restriction sites used in the construction of the in-frame puhA deletion and the probe. The corresponding sizes of the Pst I fragments in the two strains are shown. B. Southern blot result, using a 1.3 kb Pst I-Mlu I fragment as the probe (Panel A, thickened line above the map). Lane 1, 1.3 kb Pst I-Mlu I DNA probe fragment; lane 2, SB1003; lane 3, DW5; lane 4, 2.0 kb Pst I IFApuhA DNA fragment. 1.3 kb Probe SB 1003 DW5 Pst I Mlu I Msc I Sty I Pst I F1696 puhA orf214 2.7kb ///mi III Msc I [Sfy I] />wl Mlu\\/ Pst I M;:/:/>W</W^ F1696 orf214 -< • 2.0kb B kb - 23.1 - 9.4 - 6.7 - 4.4 2.3 2.0 - 0.56 45 of the in-frame deleted copy of puhA from the plasmid pSUP-IFApuhA (see Fig. 7) into the chromosome to replace the wild type copy of puhA. B. Growth studies All the strains (both wild type and mutants) discussed in this thesis grew at similar rates under either high O2 or low O2 conditions in the dark (data not shown). This means that neither deletion of puhA nor insertion of the Q cartridge into orf214 affected growth under aerobic, dark (respiratory) conditions. However, under photosynthetic conditions (anaerobic and a light intensity of 100-150 |iE m"2 s"1), their growth properties were obviously different. The photosynthetic growth properties of the puhA in-frame deletion mutants DW5 and DW13 are shown in Figs. 13 and 14. Both DW5 and DW13 were incapable of photosynthetic growth, consistent with the screen used to isolate DW5. When the plasmid pRK415 was present in these strains, they were still incapable of photosynthetic growth. However, when pPUHA, the plasmid that expressed the puhA gene, was present, photosynthetic growth was restored, with exponential growth rates comparable to the parental strains. This means that the PS growth phenotype of the puhA in-frame deletion was due to loss of production of the protein product of the puhA gene, which could be complemented in trans. C. Absorption spectroscopy Both DW5 and DW13 showed a reduction in the amount of the LHI complex, as evidenced by the reduction in the far-red shoulder of the LHII complex 855 nm peak for DW5 (Fig. 15), or the reduction of the LHI complex 46 1000 e c Q 3 u 20 30 Time (hours) 50 Figure 13. Photosynthetic growth of R. capsulatus DW5 and related strains. — • - , SB1003; — a - , DW5; — & - , DW5(pRK415); — O - , DW5(pPUHA). 1000 0 10 20 30 40 50 Time (hours) Figure 14. Photosynthetic growth of R. capsulatus DW13 and related strains. —9-,AUm; - ^ ^ , D W 1 3 ; —a—, DW13(pRK415); — < ^ , DW13(pPUHA). 48 Figure 15. Absorption spectra of intact cells of R. capsulatus DW5 and related strains grown under low aeration. A, SB1003; B, DW5; C, DW5(pRK415); D, DW5(pPUHA). 49 400 — I 1 -600 800 Wavelength (nm) 1000 50 875 nm peak for the LHII" strain DW13 (Fig. 16). When these two in-frame puhA deletion strains were complemented with pPUHA, the LHI complex absorption level was increased. It is difficult to quantitatively evaluate the relative amounts of LHI complex in the presence of LHII complex (as in DW5; Fig. 15), but the LHI peak in DW13(pPUHA) (which lacks LHII complex) was only about 80% of that in the parental strain ALHII (Fig. 16). It was also noted that the RC absorption at 800 nm in strain DW13 was reduced; in strain DW13(pPUHA) the 800 nm peak was about 80% of the amount in the parental strain ALHII. This partial restoration of LHI and RC absorption may be due to a reduced level of expression of the plasmid copy of the puhA gene in DW13(pPUHA) compared to the expression of the chromosomal copy of puhA in ALHII. When DW13(pPUHA) was grown under photosynthetic conditions, both LHI and RC absorption was restored to wild type levels (Fig. 17). These results showed that complementation of the IFApuhA mutation with puhA in trans did restore wild type levels of LHI and RC complexes. D. SDS-PAGE analysis of ICM proteins To evaluate the presence or absence of the several protein subunits of each of the RC and LH complexes in the ICM, SDS-PAGE analyses were done on membrane vesicles (chromatophores) purified from cells by differential centrifugation and discontinuous sucrose density gradients. As shown in Fig. 18, the 28 kDa RC H subunit band was absent in the in-frame puhA deletion mutant DW5. Bands for the other two RC subunits, the M and L proteins (24 and 21 kDa respectively) were present at levels equivalent to the wild type SB1003 lane. Decreases in the intensities of the LHI a and (3 bands 51 Figure 16. Absorption spectra of intact cells of R. capsulatus DW13 and related strains grown under low aeration. Spectra are labelled in the order according to their relative amount of LHI absorption (875 nm). A, ALHII; B, DW13(pPUHA); C, DW13(pRK415); D, DW13. 52 1.5-e ee X5 u © en < 400 600 800 Wavelength (nm) 1000 53 Figure 17. Absorption spectra of intact cells of R. capsulatus ALHII and DW13(pPUHA) grown under photosynthetic conditions. Strains DW13 and DW13(pRK415) did not grow under photosynthetic conditions; therefore no absorption spectrum was collected. A, DW13(pPUHA); B, ALHII. 54 400 600 800 Wavelength (nm) 1000 55 kDa 1 2 3 4 43.7 28.9 .18.4 mm H M 14.7 5.5 2.9 * LHIa LHIIa LHIB LHIIp Figure 18. SDS-PAGE analysis of chromatophore proteins isolated from R. capsulatus DW5 and related strains grown under low aeration. Lane 1, low molecular weight standards; lane 2, SB1003; lane 3, DW5; lane 4, DW5(pPUHA). 56 were also observed, as was an decrease in the intensity of a band at ca. 26 kDa position in the DW5 lane and a doublet was seen. Increase in intensity of two other bands at ca. 30 and 11 kDa were also observed. The intensities of the RC H and the LHI a and (3 subunit bands, as well as those of the unidentified bands mentioned above, were restored to approximately wild type levels in the complementation strain DW5(pPUHA). 2. Phenotypic analysis of the puhA de le t ion /^ insertion mutants DWl and DW6 A. Growth studies The puhA deletion/Q. insertion strains DWl and DW6 were incapable of photosynthetic growth (Figs. 19 and 20). When these strains were complemented with pPUHA, they grew under photosynthetic conditions, although with rates much slower than the corresponding parental strains. These results are different from the results obtained with the puhA in-frame deletion mutants DW5 and DWl3, in which the complemented strains grew as well as the parental strains (see Results section l.B). The presence of the Q. cartridge could have a polar effect on the expression of downstream genes (such as orf214 and orfl62b) if they were part of a transcription unit with the puhA gene. Therefore, it was of interest to test for complementation of DWl and DW6 with plasmid pORF214/162b (which contains orf214 and orfl 62b downst ream of the R. capsulatus nifHDK promoter) . The plasmid pORF214/162b, when mobilized into DWl and DW6, did not restore photosynthetic growth. However, when DWl and DW6 were doubly 1000 0 20 40 60 Time (hours) 80 100 120 Figure 19. Photosynthetic growth of R. capsulatus DWl and related strains. — • - , SB1003; — D - , DWl; —O-, DWl(pPUHA); —+— , DWl(pORF214/162b); —o—, DWl(pPUHA)(pORF214/162b). 58 1000 r c -4-) £ 100 -t/2 e Q I u 10 0 50 100 150 Time (hours) 200 250 Figure 20. Photosynthetic growth of R. capsulatus DW6 and related strains. —•~,MW442; — Q - , DW6; — O - , DW6(pPUHA); —•—, DW6(pORF214/162b); —o—, DW6(pPUHA)(pORF214/162b). 59 complemented with both pPUHA and pORF214/162b, photosynthetic exponential growth rates were restored to the wild type level (Figs. 19 and 20). These results are interpreted to show that the Q, insertion into puhA had a polar effect on expression of either orf214 or orfl62b or both orf214 and orfl62b, and that or/224 or orfl62b (or both) are required for optimal photosynthetic growth. B. Absorption spectroscopy The LHI complex absorption levels of DW1 were reduced, as compared to that of SB1003 (Fig. 21). This reduction of LHI absorption can also be observed as the reduced ratio of 875 nm to 855 nm "valleys" (which point downward) in the corresponding 4th derivative of the spectrum. Using the LHII" strain MW442, the reduction in LHI absorption due to the ApuhAy.Q. mutation is easily seen as a reduction in the 875 nm peak (Fig. 22). Complementation of DW1 or DW6 with pPUHA alone did not restore LHI absorption, indicating that the Q. insertion into the deleted puhA gene had a polar effect on the expression of a downstream gene(s), which is required for normal LHI absorption level. To further investigate the effect of this possible downstream gene(s) on LHI absorption, DW1 and DW6 were simultaneously complemented with pPUHA and pORF214/162b, to express puhA, orf214 and orfl62b. These doubly complemented strains also did not fully restore LHI absorption, since the LHI absorption seemed to be the same as in the ApuhAy.Q. mutant strains DW1 and DW6 (Figs. 21 and 22). When DW6(pPUHA) and DW6(pPUHA)(pORF214/162b) were grown under photosynthetic conditions, LHI absorption was also not restored (data not shown). These results showed that, although puhA, orf214 and orfl62b were expressed, LHI absorption was not restored, implying that a gene(s) 60 Figure 21. Absorption spectra of intact cells of R. capsulatus DWl and related strains grown under low aeration. Their corresponding 4th derivatives are shown above the spectra. Panel A, SB1003; B, DWl; C, DWl(pPUHA); D, DWl(pPUHA)(pORF214/162b). 61 800 1000 400 600 Wavelength (nm) 1000 62 Figure 22. Absorption spectra of intact cells of R. capsulatus DW6 and related strains grown under low aeration. Spectra are labelled in the order according to their relative amount of LHI absorption (875 nm). A, MW442; B, DW6(pPUHA)(pORF214/162b); C, DW6(pPUHA); D, DW6. 63 H o 5 u o SB < .5H o-400 600 800 Wavelength (nm) 1000 64 downstream of orfl62b is required for normal LHI absorption. In the LHII" background (strain DW6), a reduction in the reaction center absorption at 800 nm in the ApuhAv.Q mutant was seen, which was only slightly increased in the complemented strains (Fig. 22). Although both LHI and RC absorption levels were low in strain DW6(pPUHA)(pORF214/162b), the doubly complemented strains were able to grow photosynthetically (Figs. 19 and 20). C. SDS-PAGE analysis of ICM proteins SDS-PAGE analysis showed that the RC H subunit band was absent in the DWl lane (Fig. 23, lane 2). This indicates that the RC H subunit was not in the ICM, evidently because the puhA gene was deleted. A great increase in the band at approximately 30 kDa and a decrease in the 26 kDa band were also observed. Interestingly, when DWl was complemented with pPUHA alone, only a small amount of the RC H polypeptide was seen. Although the increase in the intensity of the 30 kDa band was still present in DWl(pPUHA), the intensity of the 26 kDa band was only slightly increased. For the doubly complemented strain DWl(pPUHA)(pORF214/162b), the amount of H polypeptide was comparable to that of the SB1003 lane. The intensity of the 30 kDa band was reduced, whereas that of the 26 kDa band was increased to the wild type level. Interestingly, a slight increase in the intensity of the 11 kDa band was observed in the doubly complemented strain. The intensities of all of the LHI and LHII polypeptide bands were decreased in both the DWl and DWl(pPUHA) lanes. In the DWl(pPUHA)(pORF214/162b) lane, the amounts of LHII polypeptides were restored to the wild type levels, whereas the amounts of the LHI polypeptides, although increased, was slightly less than that of the SB1003 65 3 4 5 kDa L-43.7 28.9 -*» 18.4 14.7 LHIa LHHa LHI(3 LHnp 5.5 A 2.9 Figure 23. SDS-PAGE analysis of chromatophore proteins isolated from R. capsulatus DW1 and related strains grown under low aeration. Lane 1, SB1003; lane 2, D W 1 ; l a n e 3 , D W l ( p P U H A ) ; l a n e 4, DWl(pPUHA)(pORF214/162b); lane 5, low molecular weight standards. 66 lane. These results confirm the conclusion from the growth studies that the polar effect of the Q insertion into the puhA gene interferes with the expression of other genes located downstream of orf 162b, which are required for maximal levels of the RC H protein and the LHI complex. 3. Phenotypic analysis of the orf214::Q insertion mutant DW2 A. Growth studies DW2, the orf214::Q strain, was incapable of photosynthetic growth (Fig. 24). When this strain was complemented with the plasmid pORF214/162b / which contains both or[214 and or[162b (see Fig. 11), photosynthetic growth was restored. This result demonstrates that at least one of orf214 or orf!62b are required for optimal photosynthetic growth. B. Absorption spectroscopy The orf214::Q. strain DW2 also showed a reduction in the LHI absorption level, which was increased, but not restored to the wild type level by complementation with the pORF214/162b plasmid (Fig. 25). This result is cons i s t en t w i th the low level of LHI a b s o r p t i o n in the DWl(pPUHA)(pORF214/162b) doubly complemented strain (see Fig. 21), implying that other genes located downstream of orfl62b are required for accumulation of the LHI complex in membranes. C. SDS-PAGE analysis of ICM proteins Interestingly, SDS-PAGE gels of chromatophore proteins showed that the H polypeptide band was absent in the orf214' strain DW2, even though the puhA gene is intact in this strain (Fig. 26). The amounts of the LHI 67 0 10 20 30 40 50 Time (hours) Figure 24. Photosynthetic growth of R. capsulatus DW2 and related strains. — # - , SB1003; — O - , DW2; —A—, DW2(pNIF215); — O - , DW2(pORF214/162b). 68 Figure 25. Absorption spectra of intact cells of R. capsulatus DW2 and related strains grown under low aeration. Their corresponding 4th derivatives are shown above the spectra. Panel A, SB1003; B, DW2; C, DW2(pORF214/162b). 69 B 400 600 800 Wavelength (nm) 1000 70 • « H M £3 SB 4 kDa 43.7 28.9 18.4 14.7 LHIcc LHIIoc LHIf3 LHIIp 5.5 2.9 Figure 26. SDS-PAGE analysis of chromatophore proteins isolated from R. capsulatus DW2 and related strains grown under low aeration. Lane 1, S B 1 0 0 3 ; l a n e 2, D W 2 ; l a n e 3, DW2(pORF214/162b); lane 4, low molecular weight standards. 71 polypeptides were also reduced. When DW2 was complemented with pORF214/162b, the amount of RC H and LHI subunit polypeptides was increased. However, the intensities of these bands in the DW2(pORF214/162b) lane were still less than those of the wild type SB1003 lane. These observations are consistent with the possibility that genes located downstream of orf214 and orfl62b are required to obtain normal amounts of the RC H subunit and LHI complex polypeptides in membranes of R. capsulatus, since the effects of the orf214::Q mutation are not completely complemented to wild type by pORF214/162b. Besides the RC H and LHI subunits bands, changes in intensities of other bands were also seen. An increase in intensities of bands at ca. 11, 9 and 8.6 kDa was observed in both the DW2 and DW2(pORF214/162b) lanes. However, the 30 kDa and 26 kDa bands, which demonstrated various changes in intensities in both the polar and non-polar puhA deletion mutant and complemented strains (Figs. 18 and 23), remain unchanged in intensities in both DW2 and DW2(pORF214/162b). It seems that the disruption of either puhA or orf214 has some intriguing effects on the composition of the ICM. 72 DISCUSSION I have reported here the construction of two puhA~ derivatives of R. capsulatus SB1003, designated strains DW5 and DW1. DW5 was constructed with a translationally in-frame deletion of the puhA gene, which codes for the H subunit of the RC complex in the ICM. Strain DW1 is a puhA deletion/Q insertion mutant in which the central 92% of the puhA coding sequence was deleted and replaced by an Q. cartridge (see Materials and Methods). The genotypes of these mutant strains were confirmed by Southern blot analysis. DW5 was photosynthetically incompetent, which I attribute to the lack of the RC H polypeptide. Photosynthetic growth was restored by complementation in trans with a wild type copy of the puhA gene on plasmid pPUHA. Although the H subunit of the RC does not bind Bchl and hence is not involved in capturing light energy directly, this finding shows that the H subunit itself plays an essential role in photosynthesis in vivo. Several possible functions of the RC H subunit have been suggested. One of the possible roles of the RC H subunit is that it is required for RC stability. A reduction in the amount of the M subunit polypeptide in the H-deficient R. sphaeroides mutant PUHA1 was attributed, at least in part, to a decrease in the stability of the RC M polypeptide (Varga and Kaplan, 1993). It was also reported that the R. sphaeroides mutant PUHA1 lacked the RC and LHI complexes (Sockett, et al, 1989). However, the R. sphaeroides strain PUHA1 was created by replacement of the puhA and the 5' flanking F1696 genes with an antibiotic resistance cassette (Sockett, et al., 1989). Subsequent experiments on R. capsulatus indicated that mutation of the F1696 gene 73 alone, which is located adjacent to and 5' of puhA in both species (see Fig. 2A) decreased the levels of LHI (Bauer, et al., 1991; Young and Beatty, unpublished results). Therefore, it is not clear what effect or combination of effects resulting from the deletion/insertion mutation in R. sphaeroides PUHA1 caused the absence of the LHI complex. This is because it is possible that, in addition to the direct effects of simultaneous replacement of parts of the F1696 and puhA genes with the antibiotic resistance cartridge, this cartridge could have interfered with transcription of genes 3' of puhA that are required for LHI complex formation (a polar effect). It is known that the puhA gene in R. capsulatus is transcribed as part of a large superoperon that includes Bchl biosynthesis genes and F1696 (Bauer, et al., 1991), and it was suggested that this transcriptional organization also exists in R. sphaeroides (Wellington, et al, 1992). It is significant that Sockett et al. (Sockett, et al, 1989) and Varga and Kaplan (Varga and Kaplan, 1993) were able to restore photosynthetic growth, but not the LHI complex, when strain PUHA1 was complemented with a plasmid containing the puhA gene (as well as ca. 450 bp of upstream and ca. 200 bp of downstream sequences). The possibility that sequences 3' of the puhA gene in R. capsulatus or R. sphaeroides might be transcribed and contribute to the photosynthetic phenotype had not been directly addressed until I began my thesis research. The absence of the H subunit in DW5 (IFApuhA) caused a decrease in LHI absorption, as shown by the increased slope of the far-red shoulder of the 855 nm peak (Fig. 15). This reduction of the LHI level was further confirmed using a LHII" derivative of DW5 to observe directly the decrease in LHI spectral absorption (Fig. 16). This decrease in LHI absorption in DW5 is correlated with a decrease in the levels of the LHI a and (3 polypeptides in the 74 ICM, as shown by SDS-PAGE analysis (Fig 18). However, it is not known whether this LHI level decrease is a result of an impairment at the level of puf operon transcription or translation, LHI complex a and (3 polypeptide stability, or their insertion into ICM. The previous study with R. sphaeroides cited above found that in strain PUHAl the amount of the pufBA mRNA transcript, which encodes the LHI a and P polypeptides, was comparable to that of the wild type R. sphaeroides 2.4.1 (Sockett, et al., 1989). If it is assumed that the decrease in LHI complex level in DW5 is also not due to a decrease in the amount of the pufBA transcript, then a post-transcriptional process must be affected by mutation of the puhA gene. When a wild type copy of the puhA gene on the plasmid pPUHA was introduced into DW5, wild type levels of LHI absorption and LHI a and (3 polypeptides were restored along with wild type photosynthetic growth (Figs 13, 15 and 18). This result is different from the complementation of the puhA disruption in R. sphaeroides described above (Sockett, et al., 1989). As noted above, it is impossible to assign the LHI" phenotype in the R. sphaeroides PUHAl strain to either deletion of the upstream F1696 gene or a polar effect of the Kmr cartridge used to replace the puhA gene on the expression of downstream genes. However, Varga and Kaplan attributed the LHI" phenotype of PUHAl to be due solely to the loss of the upstream F1696 gene (Varga and Kaplan, 1993). In contrast, my results show that the RC H subunit itself plays a role in the formation of a functional LHI complex. Since the deletion of puhA in DW5 is translationally in-frame (see Fig. 6), the genes flanking the puhA gene should not be affected. Therefore, the decrease in level of the LHI in DW5 should be a direct consequence of the absence of the RC H subunit. It is ironic that the RC H subunit has 75 previously been suggested to act as a nucleus of photosynthetic apparatus assembly (Sockett, et al, 1989) in spite of the paucity of direct experimental evidence. Another possibility is that the decrease in LHI level in DW5 is a consequence of the loss of the entire RC holocomplex due to the absence of the H subunit. However, it has been reported that both LHI and LHII were still present when only the RC structural polypeptides L and M were absent in R. sphaeroides (Jones, et al, 1992). Similar results were obtained with a R. capsulatus mutant (Zilsel, 1990). Therefore, it is likely that the RC H subunit, but not the L and M subunits or the complete RC, plays a special role in the maintenance of a functional LHI complex. The puhA de le t ion /Q insertion strain DW1 also displayed a PS" phenotype, which was expected since DW5, the translationally in-frame deletion of puhA, was also PS". However, when the puhA gene was introduced into this strain in trans, the resultant strain DWl(pPUHA), although photosynthetically competent, grew much more slowly than the wild type strain (Fig. 19). In contrast, when DW1 was doubly complemented with pPUHA and pORF214/162b, a wild type exponential photosynthetic growth rate was restored (Fig. 19). These results show the polar effect of the Q insertion, and the importance of orf214 and /o r orfl62b for photosynthetic growth. Spectral analysis on the puhA deletion/Q insertion strains revealed that both LHI and RC absorption levels were decreased (Figs. 21 and 22). In the doubly complemented strains, LHI and RC absorption levels were, although slightly increased, still very low. The amount of LHI subunits in the ICM in DWl(pPUHA)(pORF214/162b) was also less than that in the wild type SB1003 (Fig. 23). Despite these low levels of RC and LHI, these doubly complemented strains were able to grow photosynthetically at wild type 76 rates. It seems that, under reduced aeration, the wild type strain SB1003 is making an excess of RC and LHI complexes. Therefore, although the amount of both RC and LHI complexes are reduced in DWl(pPUHA)(pORF214/162b), there are still sufficient amount of RC and LHI complexes for photosynthetic growth at 100-150 |iE m"2 s"1. This excess level of RC and LHI complexes in the wild type cells may serve as a "reservoir" for photosynthetic apparatus and enhance the cells' ability to adapt to a shift from a high light to low light condition. Thus it is expected that these doubly complemented strains, although growing well under the light intensity used, may grow slower under a lower light condition. The open reading frame located immediately downstream of puhA is orf214 (Fig. 2A). The polar Q insertion mutant of orf214, DW2, is photosynthetically incompetent, indicating that either orf214 itself or genes further downstream of orf214 are essential for photosynthesis. This is consistent with the conclusions reached in the preceding paragraph. There are several open reading frames located downstream of orf214. According to the order determined by the DNA sequence (EMBL accession # Z11165), they are orfl62b, orf55, orf274, and orf!62a. Photosynthetic growth was restored to DW2 when pORF214/162b, in which both orf214 and orfl62b are transcribed, was present in DW2. This shows that either one or both of orf214 and orfl62b are essential for photosynthesis. However, in the complementation experiment of the puhA polar mutation strain, DWl(pPUHA) showed a poor PS growth, rather than a complete PS" phenotype (Fig. 19). If it is assumed that there is a low level of read-through transcription through the Q cartridge in puhA into orfl62b which gives the slow PS growth of DWl(pPUHA), then the Q insertion into orf214 in DW2 should also give a 77 slow (as opposed to no) PS growth. However, in DW2 no PS growth was obtained, which indicates that orf214 expression is essential for PS growth. Another possibility which accounts for the slow PS growth in DWl(pPUHA) and no PS growth in DW2 may be that there is a weak promoter downstream of the puhA deletion site in DW1 giving rise to a low level of orf214 transcription. This transcription itself, without the read-through transcription from the puhA promoter(s), could only result in a slow PS growth in DWl(pPUHA). In DW2, the disruption in orf214 expression by Q insertion resulted in no PS growth. Because of uncertainties about the levels of orf214 and orfl62b transcripts and the location of the promoter for these orfs in these strains, further complementation analysis with higher resolution is needed to be done to resolve the individual functions of orf214 and orf 162b. The way(s) in which orf214 and orf 162b affect photosynthesis is unclear. However, it was found that the RC H subunit was absent in SDS-PAGE of the ICM of DW2. Therefore, insertion of the Q. cartridge into orf214 had an effect on the amount of the RC H subunit in the ICM. This kind of gene arrangement, in which genes located downstream of pigment-protein complex structural genes in the same operon are essential to obtain optimal level of photosynthetic LH or RC complexes, is a feature of the puf and puc operons, which also encode structural polypeptides of the photosynthetic apparatus in R. capsulatus. Like the puh operon, the puf operon is part of a superoperon and is expressed from overlapping transcripts (Wellington, et al., 1992). Downstream of the puf operon-encoded LHI and RC structural genes is pufX. Deletion of pufX causes the cells to be photosynthetically incompetent in minimal medium (Lilburn, et al., 1992). Although the exact 78 function of the pufX gene product is unknown, it has been shown to be required for electron transfer from the RC to the cyt b/cj complex (Lilburn, et al, 1992). Downstream of pucB and pucA are three other genes, pucC, pucD and pucE. The gene pucC was shown to be necessary to obtain the LHII complex (LeBlanc and Beatty, 1993). This thesis shows that genes downstream of puhA are also important for photosynthesis and that their disruption had an effect on the levels of the RC and LHI complexes. When the expression of or/234 and/or orfl62b was disrupted, the amounts of RC H and LHI a and (3 polypeptides in the ICM were reduced, as were the amounts of the RC and LHI holocomplexes. This is analogous to the result of pucC deletion in the puc operon, in that pucC deletion affected the accumulation of the LHII complex encoded by the genes upstream of the deleted genes (LeBlanc and Beatty, 1993). I also discovered that the LHI complex 875 nm absorption level was decreased in DW2 (the orf214::Q strain; see Fig. 25), and the corresponding levels of the LHI polypeptides in the ICM were also decreased (Fig. 26). This decrease in LHI was not restored when pORF214/162b was present in DW2. Therefore , a l though both orf214 and orf 162b are expressed in DW2(pORF214/162b), other gene(s) downstream of orfl62b are needed to obtain the normal amount of the LHI complex. This finding is consistent with the results of a previous study in which it was reported that a relatively large cosmid containing puhA and its flanking sequences restored both photosynthetic competence and wild type LHI level in R. sphaeroides strain PUHA1 (Sockett, et al., 1989). Moreover, the LHI" phenotype in another R. sphaeroides mutant strain T i a was complemented by a 7.0 kb EcoR I 79 fragment containing puhA as well as flanking DNA sequences (Lee and Kaplan, 1992). In JR. capsulatus the open reading frames downstream of orfl62b are orf55, orf274 and orfl62a. This thesis shows that at least one of orf55, orf274, or orfl62a are essential for the wild type level of the LHI complex. Again, the exact functions of these genes are unclear. It is not known where the promoters for these genes are located. One possibility is that their transcription originates from the bchF operon promoter and/or the puhA promoter. A previous study showed that one of the puhA transcripts is a 11 kb read-through product from the upstream bchF operon (Yang and Bauer, 1990; Bauer, et al., 1991). However, since this thesis shows that expression of orf214 or a gene(s) beyond orf214 is dependent on read-through transcription from the puhA gene, it is possible that transcripts initiated at the bchF and/or puhA promoters extend into orf214 and beyond. Another possibility is that there is a promoter for orf214 located within the puhA gene, giving rise to transcripts encoding orf214 and gene(s) beyond. Detailed mRNA mapping is needed to better characterize transcription of these genes. The SDS-PAGE results of the puhA and orf214 mutants reveal an intriguing regulation on the components of the photosynthetic apparatus in the ICM. Even though one gene was mutated, there are changes in as many as 6-8 bands in the SDS-PAGE of the ICM proteins. Particularly, besides the changes in the RC H and LHI a and P subunit bands, changes in intensities in bands at approximate positions of 30, 26, 11, 9 and 8.6 kDa were also observed. It seems that there are an increase in the intensity of the 30 kDa band and a decrease in the intensity of the 26 kDa band in the puhA deletion strains DW1 and DW5, whereas the intensities of these bands were not 80 changed in the orf214::Q strain DW2. In DWl(pPUHA)(pORF214/162b) and DW5(pPUHA), the two complemented strains which demonstrated wild type PS growth, the intensities in the 30 and 26 kDa bands were restored to the wild type levels. This restoration was not seen in DWl(pPUHA), which showed a slow PS growth. It seems that the wild type amounts of the 30 kDa and the 26 kDa proteins are correlated with optimal photosynthetic growth. The intensities of the 11, 9 and 8.6 kDa bands were increased only in some of the mutant strains. As discussed above, the RC H subunit may have an effect on the correct assembly of the RC and LHI complexes. The SDS-PAGE results showed that the RC H subunit may also affect the insertion of some other proteins into the ICM or their stability. This dependency of the insertion or stability of one component in the ICM on other components was demonstrated between the LHI a and |3 subunits (Richter and Drews, 1991; Richter, et al, 1992). The insertion of the LHI a subunit into the ICM is dependent on its partner LHI [3 subunit, and the stability of the LHI |3 subunit in the ICM is dependent on the presence of the wild type LHI a subunit. In this thesis, a similar effect was exhibited as a decrease in band intensities perhaps resulting from defects in insertion or reductions in protein stability (for example, the 26 kDa protein). Otherwise it may be displayed as an increase in intensities resulting from dimer or aggregate formation (for example, the 30, 11, 9, and 8.6 kDa proteins). There is also a possibility that the mutation in either puhA or orf214 causes conformational changes in other proteins in the ICM. Another possibility may be that the mutations in some ways promote the insertion of other proteins into the ICM. It has been previously observed that some gene products other than the LH polypeptide subunits are involved in the formation of the functional LH complexes (Lee, et al, 1989; Sockett, et al, 1989; Tichy, et al, 1989; Lee and Kaplan, 1992; 81 LeBlanc and Beatty, 1993). Thus it is not surprising that RC complex formation may also involve some assembly factors, which may be encoded by the orfs downstream of puhA. Although the exact mechanism that causes the changes in the intensities of bands in the SDS-PAGE is unknown, this thesis demonstrates that there are some interesting interactions between different components in the ICM, and the disruptions of puhA and orf214 caused a variety of changes in the ICM composition. The effects of the mutations of puhA and downstream orfs are summarized in Fig. 27. The RC H subunit itself is essential for photosynthetic growth and normal LHI complex level. Deletion and complementation results shows that either one or both of the orf214 and orfl62b gene products are responsible optimal photosynthetic growth, whereas at least one of orf55,orf274 and orfl62a are responsible for normal LHI complex level. 82 0.5 kb mRNA processing - r > r * * ^ i' mmm®mM bchF bchB bchK bchH bchL bchM puhA orf55 F1696 /orf!14 orf274 orfl62b orfl62a H" LHlX PS" At least one of them At least one of them are responsible for are responsible for normal PS growth normal LHI level Figure 27. Summary of the effects of the deletion of puhA and the possible functions of the downstream open reading frames. The notations are the same as those in Fig. 2. 83 CONCLUSIONS I have reported a directed mutational analysis of the R. capsulatus puhA gene, which encodes the RC H subunit. Both of the puhA deletion mutant strains DW1 and DW5 were unable to grow under photosynthetic conditions, indicating that the RC H subunit is essential for photosynthesis. Spectral analyses and SDS-PAGE analyses of these strains also revealed that the RC H subunit is required to obtain normal levels of the LHI complex. The exact functions of different domains of the RC H subunits are still unclear. The precise in-frame puhA deletion mutant DW5 thus provides a good background for higher resolution site-directed mutagenesis studies of the RC H subunit, and the functions of individual domains and amino acids of the RC H subunit in RC and LHI complex formation can be evaluated. Complementation analysis showed that open reading frames downstream of puhA are also important in photosynthesis. Either one or both of the two orfs downstream of puhA, orf214 and orfl62b, are required for normal RC H subunit level and hence optimal photosynthetic growth. An open reading frame(s) downstream of orf 162b is required for normal LHI complex level. It seems that both the RC H subunit and LHI complex levels are affected by these orfs located downstream of puhA. However, it is not known how the transcription of these orfs are organized, and the location of the promoters of these genes are unclear. Detailed mRNA mapping and complementation studies are necessary to better characterize the transcription and the individual functions of these genes. 84 REFERENCES Allen, J. P., G. Feher, T. O. Yeates, H. Komiya and D. C. Rees (1988) Structure of the reaction center from Rhodobacter sphaeroides R-26: Protein-cofactor (quinones and Fe^+) interactions. Proc. Natl. Acad. Sci. USA 85:8487-8491. Bauer, C. E., J. J. Buggy, Z. Yang and B. L. Marrs (1991) The superoperonal organization of genes for pigment biosynthesis and reaction center proteins is a conserved feature in Rhodobacter capsulatus: analysis of overlapping bchB and puhA transcripts. Mol. Gen. 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