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ORGANIZATION AND REGULATION OF THE
RHODOBACTER CAPSULATUS CO2 FIXATION GENES
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of the Ohio State University
By
George Carl Paoli, B.S., M.S.
*****
The Ohio State University 1997
Dissertation Committee Approved by: F.R. Tabita, Adviser
W.R. Strohl
K.E. Kendrick viser
C.J. Daniels Department of Microbiology UMI Number: 9721152
UMI Microform 9721152 Copyright 1997, by UMI Company. All rights reserved.
This microform edition is protected against unauthorized copying under Title 17, United States Code.
UMI 300 North Zeeb Road Ann Arbor, MI 48103 ABSTRACT
Rhodobacter capsulatus is a nonsulfur purple photosynthetic bacterium that fixes
CO2 primarily via the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway. R. capsulatus is one of only a few bacterial species known to synthesize two forms of the key CBB cycle enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase
(RubisCO). The organization of several genes encoding CBB cycle enzymes {ebb genes) in R. capsulatus was determined. The R. capsulatus form I and form II RubisCO genes, cbbLcbbS and cbbM, respectively, were functionally expressed in Escherichia colt and
Rhodobacter sphaeroides. Several of the ebb genes were sequenced. Phylogenetic analysis revealed that the R. capsulatus form I RubisCO genes and the genes immediately upstream and downstream of cbbLcbbS were acquired by R. capsulatus as a result of a horizontal gene transfer. A combination of ebb gene disruption strains and ebb promoter fusion constructs was employed to examine aspects of ebb gene regulation in
R. capsulatus.
Chemoautotrophic growth of mutant strains of R. capsulatus, and a spontaneous mutant of Rhodobacter sphaeroides, that synthesize only form I or form II RubisCO was examined. The results of these experiments suggest that these strains might be useful in providing a system for biological selection for RubisCO with desirable biochemical properties.
Ill Dedicated to Renee, Cassie, and Courtney
IV ACKNOWLEDGEMENTS
I wish to thank my adviser. Dr. F. Robert Tabita for his support and patience, as well as the training I received while working on these studies. Thanks also to Dr. Janet L.
Gibson for her advice and input. For his contributions to this work, I thank Dr. Jessup
Shively. I wish to express my gratitude to Drs. Charles E. Daniels, Kathleen E. Kendrick, and William R. Strohl for their time and input, both as advisory members of my dissertation committee and as teachers. Thanks to past and present members of Dr.
Tabita's laboratory, too many to list, with whom working has been a pleasure. To my parents. Lido and Mary Ann Paoli, my thanks for continued support and encouragement, and to my wife Renee, for her sacrifice and support. VTTA
December 6, 1963 ...... Bom - Hancock, Michigan
1986 ...... B.S., Michigan Technological University, Houghton, Michigan
1988...... M.S., Michigan Technological University, Houghton, Michigan
1988 - Present...... Graduate Teaching and Research Associate, The Ohio State University, Columbus, Ohio
PUBLICATIONS
Tabita, F.R., J.L. Gibson, D.L. Falcone, X. Wang, L.-A. Li, B.A. Read, K.C. Terlesky, and G.C. Paoli. 1991. Current studies on the molecular biology and biochemistry of CO? fixation in phototrophic bacteria, p.469-479. In J.C. Murrell and D.P. Kelly (ed.). Microbial Growth on C| Compounds. Intercept Ltd, Andover, U.K.
Paoli, G.C., N. Strom-Morgan, J.M. Shively, and F.R. Tabita. 1995. Expression of the cbbLcbbS and cbbM genes and distinct organization of the ebb Calvin cycle structural genes of Rhodobacter capsulatus. Arch. Microbiol. 164:396-405.
Gibson, J.L., Y. Qian, G.C. Paoli, J.M. Dubbs, H. Xu. H.V. Modak, K.M. Horken, T.M. Wahlund, G.M.F. Watson, and F.R. Tabita. 1996. Molecular control and biochemistry of CO? fixation in photosynthetic bacteria. In, M.E. Lidstrom and F.R. Tabita (ed.). Microbial Growth on Ci Compounds. Kluwer Academic Publishers, Dordrecht, The Netherlands.
FIELDS OF STUDY
Major Field: Microbiology
vi TABLE OF CONTENTS
ABSTRACT...... ii
DEDICATION...... iv
ACKNOWLEDGEMENTS ...... v
VITA ...... vi
LIST OF TA BLES...... ix
LIST OF FIGURES...... xi
INTRODUCTION...... 1
CHAPTERS
1. Expression of the cbbLcbbS and cbbM Genes and Distinct Organization of theebb Calvin Cycle Structural Genes of Rhodobacter capsulatus ...... 4
Introduction ...... 4 Materials and M ethods ...... 7 Results...... 13 Discussion ...... 27 Acknowledgments ...... 30
2. Organization and Nucleotide Sequence of the Rhodobacter capsulatus cbbLcbbS and Neighboring Genes. Phylogenetic Analysis Suggests That These Genes Were Acquired by a Horizontal Gene Transfer ...... 31
Introduction ...... 31 Materials and M ethods ...... 33 Results...... 39 Discussion ...... 60 Acknowledgments ...... 63
vii 3. Studies of the Regulation of the ebb Structural Genes of Rhodobacter capsulatus ...... 64
Introduction ...... 64 Materials and M ethods ...... 68 Results...... 80 Discussion ...... 130 Acknowledgments ...... 134
4. Aerobic ChemoUthoautotrophic Growth of Rhodobacter capsulatus strain SB 1003 and a Spontaneous Mutant of Rhodobacter sphaeroides strain HR.... 135
Introduction ...... 135 Materials and M ethods ...... 138 Results...... 141 Discussion ...... 159 Acknowledgments ...... 162
5. SUM M ARY...... 163
REFERENCES...... 169
V lll UST OF TABLES
Table Page
1.1. Plasmids used in Chapter 1 ...... 8
1.2. RubisCO activity in R. sphaeroides strain 16 complemented withR. capsulatus RubisCO clones ...... 21
1.3. Expression of R. capsulatus cbbLcbbS and cbbM genes in E .co li ...... 24
2.1. Plasmids used in Chapter 2 ...... 34
3.1. Plasmids used in Chapter 3 ...... 69
3.2. Bacterial strains used in Chapter 3 ...... 73
3.3. Growth Rates, RubisCO activities, and PRK activities in R. capsulatus wild-type, cbbL and cbbM' strains ...... ^
3.4. Growth Rates, RubisCO activities, and PRK activities in R. capsulatus wild-type and cbbL'cbbM' strains ......
3.5. Growth rates and RubisCO activities in R. capsulatus RubisCO-minus strain SBI-II complemented with R. capsulatus RubisCO clones ...... 114
3.6. Growth rates, RubisCO activities, and PRK activities in photoheterotrophically grown R. capsulatus wild-type, cbbF, and complemented strains ...... 120
3.7. Growth rates, RubisCO activities, and PRK activities in R. capsulatus wild-type, cbbR\i, and complemented strains. 124
4.1. Bacterial strains used in Chapter 4 ...... 140
IX Table Page
4.2. Comparison of growth characteristics of the GAG strain withR. sphaeroides strain HR and R. capsulatus strain SB 1003...... 144
4J. Growth rates and RubisGO activities in R. sphaeroides strains HR and HR-GAG and R. capsulatus strain SB 1003.. 146
4.4. RubisGO Protein Levels in R. sphaeroides strains HR and HR-GAG...... 148
4.5. Chemoautotrophic growth of R. sphaeroides and R. capsulatus strain s ...... 150
4.6. Ability of R. sphaeroides HR-derivatives to acquire the G AG-Phenotype ...... 151
4.7. RubisGO activity in R. sphaeroides strains grown chemo- autotrophically at various GO 2 concentrations ...... 154
4.8. RubisGO activity in R. capsulatus strains grown chemoauto- trophically at various GO; concentrations ...... LIST OF FIGURES
Figure Page
1.1. Restriction m ^ s and gene organization of the two R. capsulatus ebb gene clusters ...... 15
1.2. Southern hybridization analysis of R. capsulatus ebb gene clones ...... 18
1.3. Photoheterotrophic growth of R. sphaeroides strain 16 containing the R. capsulatus cbbM gene in plasmids pRFSFE-I and pRPSFH-H ...... 22
1.4. Western immunoblots of R. capsulatus RubisCO synthesized in E. coll and R. capsulatus ...... 26
2.1. Organization of the R. capsulatus cbbi gene cluster 36
2.2. Nucleotide and deduced amino acid sequence of the 42 R. capsulatus cbb\ gene cluster ......
23. CbbQ amino acid sequence alignment ...... 50
2.4. RubisCO large subunit phylogenetic tree ...... 53
2.5. Multiple sequence alignment of CbbR conserved regions. . . 56
2.6. CbbR phylogenetic tree ...... 58
3.1. Organization of the cbb\i operon region of R. capsulatus and sequence of the 5'-end of the cbba operon and upstream region ...... 82
3.2. Alignment of the deduced amino acid sequence of the partial 5'-0RF with theAgrobacterium tumefaciens and human PGM * 1+ phosphoglucomutase ...... 90
XI Figure Page
33. Amino acid sequence alignment of the R. capsulatus QOR with the QOR fromE. coli and P. aeruginosa ...... 92
3.4. Phosphoribulokinase amino acid sequence alignment ...... 96
3.5. Alignment of the R. capsulatus cbbTa partial deduced amino acid sequence with R. capsulatus TktA and R. sphaeroides CbbTa...... 98
3.6. Insertional mutagenesis of the R. capsulatus cbbL and cbbRi genes ...... 101
3.7. Insertional mutagenesis of R. capsulatus cbbn genes 103
3.8. Western blot analysis of R. capsulatus RubisCO-minus strains ...... 107
3.9. Growth phenotype of the R. capsulatus RubisCO-deletion strains ...... 109
3.10. Southern blot analysis of R. capsulatus cbbP at low stringency ...... 116
3.11. Growth phenotype of the R. capsulatus PRK-minus strain.. 119
3.12. Growth phenotype of the R capsulatus CbbRn-strain . • • • • 123
3.13. R. capsulatus ebb promoter fusion activity ...... 127
4.1. Growth of R. sphaeroides strains HR and HR-CAC under aerobic chemoautotrophic conditions ...... 143
5.1. Organization of the ebb genes in R. capsulatus and R. sphaeroides ...... 165
XU INTRODUCTION
The importance of the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway as a life sustaining process cannot be overstated. As the major mechanism for biological CO 2 fixation, this pathway is critical in global carbon cycling and is at the base of the global food chain. Although a number of pathways are known to enable autotrophic growth of organisms (Fuchs, 1989; Strauss and Fuchs, 1993), the predominance the CBB cycle in biological CO 2 fixation stems from its broad phylogenetic distribution. The CBB pathway is the means by which all plants, algae, and cyanobacteria, and most photosynthetic and chemoautotrophic bacteria acquire carbon from CO 2 .
The enzyme that catalyzes the CO2 fixation reaction in the CBB cycle, ribulose
1,5-bisphosphate carboxylase/oxygenase (RubisCO), is unique to this pathway. RubisCO exists in two biochemically and genetically distinct forms. The form I enzyme is a hexadecameric enzyme (LgSg) composed of eight large (Mr ~ 50,000) and eight small
(Mr ~ 10,000 - 15,000) subunits. The form II enzyme is by far the less common form and consists of large subunits only (L 2x)- Although form I and form II RubisCO large subunits share only about 25 % overall amino acid sequence identity, they both perform the same reactions at a highly conserved active site. As its name indicates, RubisCO catalyzes both the carboxylation and oxygenation of ribulose 1,5-bisphosphate (RuBP). Not only does the oxygenase activity reduce the efficiency of carboxylation by competing for RuBP at the active site, but the subsequent metabolism of the oxygenase products leads to the net loss of carbon from the cell. Thus the oxygenase reaction reduces net CO 2 fixation by RubisCO. This fact, combined with the fact that CO2 fixation is the rate-limiting step in photosynthetic carbon assimilation, has led to great interest in RubisCO biochemistry. It has long been hoped that an increase in the carboxylase activity or a decrease in the oxygenase activity would result in increased plant productivity. The ability of RubisCO to discriminate between the two gaseous substrates varies widely in nature, suggesting that genetic manipulation of
RubisCO could very well lead to an improved enzyme.
Phosphoribulokinase (PRK), which generates the RubisCO substrate ribulose 1,5- bisphosphate (RuBP) from ribulose 5-phosphate (Ru-5-P) and ATP, is the only other enzyme unique to the CBB pathway. The other reactions in the cycle serve to reduce the
CO2 fixation product so that it can be assimilated into the cell, and to regenerate Ru-5-P so that the cycle can continue.
In some species of autotrophic bacteria several of the genes encoding CBB cycle enzymes {ebb genes) are arranged in large opérons (Gibson and Tabita, 1996). The arrangement of these genes in large transcriptional units allows the coordinate control of their expression. Among photosynthetic bacteria, the ebb genes have been most extensively studied in the nonsulfur purple photosynthetic bacterium Rhodobaeter sphaeroides (Gibson, 1995; Gibson and Tabita, 1996; Tabita, 1988, 1995). R. sphaeroides was the first organism shown to possess both form I and form II RubisCO
(Gibson and Tabita, 1977a, 1977b, 1985), and the organization and regulation of the ebb
genes in this organism have been studied thoroughly (Falcone and Tabita, 1991; Gibson
et al., 1990, 1991; Gibson and Tabita, 1986, 1987, 1988, 1993; Jouanneau and Tabita,
1986; Quivey and Tabita, 1984).
Rhodobacter capsulatus is a nonsulfur purple photosynthetic bacterium that is closely related to R. sphaeroides and, like R. sphaeroides, possesses both form I and form
n RubisCO (Gibson and Tabita, 1977c; Shively et al, 1984; 1986). Earlier reports
indicated that the form I RubisCO of R. capsulatus was immunologically distinct from the R. sphaeroides form I RubisCO (Gibson and Tabita, 1977c) and that the levels of form I and form II RubisCO in R. sphaeroides (Jouanneau and Tabita, 1986) and
R. capsulatus (Shively et a i, 1984) differed under similar growth conditions.
The studies presented in this dissertation address the differences between
R.. sphaeroides and R. capsulatus form I RubisCO. During the course of these studies the organization of the R. capsulatus ebb genes was determined. The nucleotide sequence of several of these genes was also determined. Aspects of ebb gene regulation in
R. capsulatus were examined using a combination of ebb gene disruption strains and ebb promoter fusions. Mutant strains of R. sphaeroides and R. capsulatus that were isolated or constructed may provide a useful system for the biological selection of RubisCO with increased CO 2-O2 substrate specificity. CHAPTER 1
Expression of the cbbLcbbS and cbbM Genes and Distinct Organization of the ebb Calvin
Cycle Structural Genes of Rhodobacter capsulatus
INTRODUCTION
The Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway is the means by which all plants, algae, and cyanobacteria, and most photosynthetic and chemoautotrophic bacteria acquire carbon from CO 2 for subsequent cell metabolism and growth (Tabita, 1988). The enzyme that catalyzes the actual CO, fixation reaction is ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). Together with phosphoribulokinase (PRK), these two enzymes are unique to the CBB cycle and allow other enzymes common to intermediary metabolism to function in a biosynthetic capacity so that CO2 may be reduced and subsequently assimilated (Tabita, 1994; 1995).
There are two distinct forms of RubisCO, form I and form II. Form I is a hexadecameric enzyme (LgSg) composed of eight large subunits of Mr - 55,000 and eight small subunits of Mr - 15,000. The form II RubisCO (LJ, initially found in species of nonsulfur purple bacteria, is both biochemically and genetically distinct from the form I enzyme and is composed of only large subunits, which show little homology to form I large subunits. Form I RubisCO is found in virtually all prokaryotic and eukaryotic autotrophs, however an increasing number of photosynthetic and chemoautotrophic bacteria have been shown to possess either form I, form II, or both forms of RubisCO (Tabita, 1995). Studies with specific cbbLcbbS and cbbM deletion strains have further shown that form I and form II RubisCO may fulfill different physiological functions
(Wang et al, 1993), reflective of their differentially regulated expression in Rhodobacter sphaeroides (Gibson, 1995; Gibson and Tabita, 1996).
hi R. sphaeroides the genes for form I (cbbLicbbS{) and form H (cbbMu) RubisCO are associated with other CBB cycle structural genes in separate unlinked (ebb) opérons
(Gibson, 1995), each of which is localized on one of the two major genetic elements of this organism (Suwanto and Kaplan, 1989). The cbbLicbbSi genes are at the distal end of the cbbi operon, associated with other genes encoding enzymes of the pathway (Gibson et ai, 1991). Likewise, thecbbMn gene is at the distal end of the cbbu operon (Gibson et al. 1990; Chen et al, 1991). The cbbR gene, which is upstream of and divergently transcribed from the cbbi operon, encodes a LysR-type regulatory protein that positively regulates the expression of both the cbb\ and cbba opérons (Gibson and Tabita, 1993).
Transcriptional regulation of the ebb genes by CbbR has been demonstrated in other autotrophic bacteria as well (Windhovel and Bowien, 1991; Viale et al, 1991; Kusano and Sugawara, 1993; van den Bergh et al, 1993). In Rhodospirillum rubrum, which has only a single RubisCO (form H), the cbbM gene is also clustered with other ebb genes.
However, in this organism, cbbR is directly upstream of cbbM and is transcribed in the same direction, while the other ebb genes are upstream of and divergently transcribed from cbbR and cbbM (Falcone and Tabita, 1993).
In many respects, R. capsulatus presents an interesting system to study CO? fixation control. This organism metabolizes a wider array of organic and inorganic compounds (Hansen and Van Gemerden, 1972; Weaver et al, 1975; Madigan and Gest,
1979; Madigan, 1988) and it grows somewhat faster than R sphaeroides, its close relative, under conditions where CO 2 fixation is of special physiological importance. In addition, several years ago it was shown that R capsulatus synthesizes both form I and
5 form n RubisCO (Gibson and Tabita, 1977c; Shively et al., 1984), however the form I enzyme is surprisingly immunologically distinct from the corresponding R. sphaeroides enzyme. Preliminary studies indicated that the form I and form II RubisCO genes might be unlinked in R. capsulatus (Shively et ai, 1986), yet this organism, unlike
R. sphaeroides, contains only a single large chromosome which has been extensively mapped (Fonstein et al., 1995). The current study was undertaken to isolate the cbbLcbbS and cbbM genes of R. capsulatus and, as a prelude to further studies of regulation, determine the organization of genes encoding CBB cycle enzymes of this organism. I have found that the ebb genes are organized quite differently in R. capsulatus, suggestive of distinct regulation. In addition, both the cbbLcbbS and cbbM genes of R. capsulatus were functionally, expressed in Escherichia coli and in a RubisCO deletion strain of
R. sphaeroides. MATERIALS AND METHODS
Bacterial strains and plasmids. A genomic library of Rhodobacter capsulatus
SB 1003 (Yen and Marrs, 1976) was obtained from Pablo Scolnik. The library was
constructed (Scolnik and Haselkom, 1984) by cloning a /7/ndin partial digest of genomic
DNA into cosmid pDPTSCm (Taylor, 1984) maintained in Escherichia coli HBlOl
(Boyer and Roulland-Dussoix, 1969). E. coli JM107 (Yanisch-Perron et a i, 1985) was
the host strain for the propagation of newly constructed plasmids. Rhodobacter
sphaeroides strain 16, containing deletions in the cbbLcbbS and cbbM coding sequences
(Falcone and Tabita, 1991), was used as a host for the expression of R. capsulatus
RubisCO genes. Plasmid vectors and constructions used in this study are listed in Table
1 . 1 .
Media and growth conditions. E. coli was grown in LB broth (Ausubel et ai,
1987) at 37°C. Aerobic cultures of R. sphaeroides were grown in PYE medium (Weaver and Tabita, 1983) at 30°C. Photosynthetic cultures of R. sphaeroides and R. capsulatus were grown under anoxic conditions at 30°C in Ormerod's medium (Ormerod et ai,
1961) supplemented with 1 pg/ml thiamine, 1 pg/ml nicotinic acid, and 0.1 pg/ml biotin under photoautotrophic (1.5% CO 2/H2) and photoheterotrophic (malate) conditions as previously described (Falcone and Tabita, 1991).
DNA manipulations. Routine DNA manipulations, including plasmid preparation, restriction endonuclease digestion, agarose gel electrophoresis, fragment ligation, and bacterial transformation were performed by standard methods (Ausubel et ai, 1987). R. capsulatus chromosomal DNA was prepared as described by Grimberg et al. 1989. Plasmids were conjugated into R. capsulatus by tri-parental matings on filter pads as previously described (Weaver and Tabita, 1983) using the helper plasmid pRK2013 (Figurski and Helinski, 1979). Plasmid Relevant characteristics Source or reference pK18, pK19 Km , pUC derivatives Pridmore, 1987 pUC1318 Ap^, pUC derivative with modified Kay and multiple cloning site McPherson, 1987 pRK415 Tc**, broad-host-range cloning vector, lacZa Keen era/., 1988 pRPS-1 Tc**, broad host range expression vector containing R. rubrum cbbM Falcone and promoter and c66R gene in pRK404 Tabita, 1991 pDPTSCm Cm*^ and T c \ pBR322-based cosmid vector Taylor, 1984 pRCFI Cm*^, pDPTSCm containing an approximately 31 kb Hindm insert hybridizing to a cbbL probe This work pRCFll Cm^, pDPTCm containing an approximately 39 kb Hindis, insert hybridizing to a cbbM probe This work pRKFIP pRK415 containing the 9 kb Pstl fragment of pRCFI containing the R. capsulatus cbbLcbbS genes This work pRFSH-I pRPS-1 containing the R. capsulatus cbbLcbbS genes on a 4.7 kb BamHI fragment cloned from pRKFIP This work pR Psn-n Same as pRPSFI-I with the 4.7 kbBamIS fragment in the opposite orientation This work pK18H pK18 containing the R. capsulatus cbbLcbbS genes on a 4.7 kb ___ BamlS-Pstl fragment from pRKFIP This work pK19FI pK19 containing the R. capsulatus cbbLcbbS genes on a 4.7 kb ___ BamiS-Pstl fragment from pRKFIP This work
Table 1.1. Plasmids used in this study (continued) Table 1.1. (continued)
Plasmid Relevant characteristics Source or reference pKlSFDEH pK18 containing the R. capsulatus cbbR, cbbF, cbbP, cbbT, cbbG, and ebb A' genes on an 8.4 kb EcoRI-ffrndDI fragment from pRCFIl This work pK18FnS4.4 pK18 containing a 4.4 kb SaR fragment from pK18MlHH This work pKI8FÏÏS4 pK18 containing a 4 kb SaR fragment frompK18FllHH This work pK18FnS2-I pK18 containing the R. capsulatus cbbM gene on a 2 kb SaR fragment from pRCFn This work pK18FnS2-n Same as pKI8FIIS-I with the 2 kbSaR fragment in the opposite orientation This work pUC1318::FnS pUC1318 containing the R. capsulatus cbbM gene on a 2 kb SaR fragment from pK18FnS2-I This work pRKFII-I pRK415 containing the R. capsulatus cbbM gene on a 2 kb BamYR fragment cloned from pUC1318::FIIS This work pRKFE-n Same as pRKF'li-I with the 2 kbBamYR fragment in the opposite orientation This work pRPSFn-I pRPS-1 containing the R. capsulatus cbbM gene on a 2 kb BamYR fragment cloned from pUC1318::FIIS This work pRPSFn-n Same as pRPSFII-I with the 2 kbBamYR fragment in the opposite orientation This work pRR116 pBR325 containing the R. rubrum Somerville and cbbM gene on a 6.6 kb EcoRI fragment Somerville, 1984 pANP1155 pBR322 containing the Synechococcus sp. strain PCC 6301 rbcL gene on a 2.3 kb Shinozaki et al, Pstl Aagment 1983
(continued) Table 1.1. (continued)
Plasmid Relevant characteristics Source or reference p6B pUC8 containing a 650 bp BamYH-EcoRL fragment encoding part of the Gibson et al., R. sphaeroides cbbL\ gene 1991 pJG29 pUC8 containing a 4.2 kb Smal fragment encoding R. sphaeroides cbbAi, cbbL\, and Gibson and cbbS\ genes Tabita, 1986 pRQ52 pUC8 containing a 3.0 kb EcoRI fragment Quivey and encoding the R. sphaeroides cbbMa gene Tabita, 1984
PÜC12EH pUC19 containing a 1.8 kb EcoRI-HindDI fragment encoding the R. sphaeroides Gibson and cbbR and cbbFî genes Tabita, 1993 pJG8 pUC8 containing a 3.7 kb Pstl fragment encoding the R. sphaeroides cbbFù, Gibson and cbbPa, cbbTu, and cbbGn genes Tabita, 1987 p42 pUC8 containing a 1.8 kb BamHl fragment encoding the R. sphaeroides cbbTn gene Chen era/., 1991 p5410A5/?u3 pUC8 containing a 1.3 kb BamHl-Smal fragment encoding the R. sphaeroides cbbTn and cbbGn genes; constructed by removal of a 0.6 kb Smal fragment Gibson and from p5410 Tabita, 1988 p3D pK18 containing a an 800 bp fragment derived from a set of nested deletions Falcone and and encoding the R. rubrum cbbE gene Tabita, 1993
10 Southern blotting and hybridization. The conditions for the preparation and
hybridization of colony blots for screening the R. capsulants genomic library have been
described previously (Shively et at., 1986) as were procedures for Southern blotting,
hybridization, and preparation of probes used for whole cosmid mapping. The
R. sphaeroides cbbF, cbbP, cbbG, and cbbA gene probes used for cosmid mapping were
those of Stoner and Shively (1993).
Additional Southern hybridization experiments were performed using Gene-
Screen Plus membranes (NEN, DuPont, Boston, Mass., USA). Hybridizations were
conducted according to the protocols provided by the manufacturer using formamide
stringent conditions. Probes were labeled with a-[^‘P]-dCTP (NEN, DuPont) by the
random prime labeling method (Feinberg and Vogelstein, 1983) using a kit purchased
from United States Biochemical Corporation (Cleveland, Ohio, USA). Gene-specific
probes were prepared from plasmids listed in Table 1.1 as follows: R. sphaeroides cbbL,
650 bp Hindül-EcoRÎ fragment from p6B; R. capsulants cbbL, 530 bp HindSL-Smal
fragment from pK18FISB; rbcL, 1.5 kg EcoRl-PSIl fragment from pANPl 155; cbbR, 350
bp PvuII fragment for the 5'-probe or a 950 bp HindQl-Xhol fragment for the full length-
probe, both from pl2EH; cbbF, 600 bp AflSi-EcdRl fragment from pl2EH; cbbP, 970 bp
BgHL-Smal fragment from pJG8; cbbT, 1.8 kb Smal-BamHl fragment from p42; cbbG,
600 bp SaB-EcoBl fragment from p5410A5ma; cbbA, 6(X) bp Pstl fragment from pJG29;
cbbM, 1 kb Pstl fragment from pRQ52 or two SaB fragments of 260 and 320 bp from
pRRl 16; and cbbE, 520 bp Sall-ffindin fragment from p3D.
Induction of R. capsulatus cbbLcbbS and cbbM expression in E. coli. The
R. capsulatus cbbLcbbS and cbbM RubisCO genes were cloned into pK18 or pK19 to
allow IPTG-inducible expression in E. coll. Overnight cultures were diluted 1/100 in LB
medium and grown at 37°C. When the culture reached an Asoo of 0.4 to 0.6, IPTG was
added to the culture to a final concentration of 1 mM. Samples of the culture were taken
11 prior to the addition of IPTG and every 4 h over a 24 h period for the quantitation of
RubisCO activity.
Preparation of extracts, RubisCO assay, and hnmunological analysis. Culture samples (20-30 ml) were washed twice in 100 mM Tris-HCl (pH 8.0) and 1 mM EDTA and frozen at -70°C. Thawed pellets were resuspended in 1 ml TEM (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 5 mM P-mercaptoethanol) and disrupted by sonication at 0°C.
Cell debris was removed by centrifugation for 10 min in a microcentrifuge at 4°C. The resultant crude extract was used for enzyme assays. RubisCO activity, assayed as RuBP- dependent ^^COz fixation into acid-stable material, and protein concentrations were measured by standard procedures as described earlier (Gibson et al, 1991).
For Western immunoblot analysis the recombinant RubisCOs were partially purified. Magnesium/heat soluble fractions (Gibson et al, 1991) were prepared from crude extracts and RubisCO was resolved by sucrose density gradient centrifugation using a 39 ml linear sucrose gradient (0.2 M to 0.9 M) in TEMMB buffer [20 mM Tris-HCl
(pH 7,5), 1 mM EDTA, 10 mM P-mercaptoethanol, 10 mM magnesium acetate, and 20 mM bicarbonate]. The gradients were centrifuged for 20 h at 50,000 rpm in a Beckman
VC53 rotor (200,000 x g). Proteins were resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970). After SDS-PAGE the proteins were transferred to nitrocellulose membranes (Immobilon-NC, Millipore,
Bedford, Mass., USA) (Towbin et al., 1979) using a Bio-Rad Transblot semi-dry cell.
Antibody binding was assessed using an alkaline-phosphatase-conjugated anti-rabbit antibody (Bio-Rad, Hercules, Calif., USA) as described (Blake et al., 1984) except that
0.05% Tween 20 was used in all buffers.
12 RESULTS
Isolation of RubisCO clones from the R. capsulatus library. Both
Synechococcus sp, strain PCC 6301 (form I) and R. rubrum (form II) RubisCO gene probes hybridize to R. capsulatus chromosomal DNA (Shively et ai, 1986). To facilitate the cloning of the RubisCO genes, these same RubisCO gene probes (Shively et a i, 1986;
Table 1.1.) were used to screen a genomic cosmid library of R. capsulatus SB 1003.
Approximately 1200 colonies from this library were screened. Three and five positive clones were obtained, using the form I {Synechococcus) and form II {R. rubrum) probes, respectively. After preliminary mapping, it was apparent that cbbLcbbS and cbbM were not closely linked. Therefore, one clone of each type was selected for further study.
Mapping cosmids pRCFi and pRCFU. Cosmids containing cbbLcbbS and cbbM genes, pRCFI and pRCFH, respectively, were subjected to restriction endonuclease digestion with HindSn., EcoRL SaE, BamHl, and Xhol. Single and double enzyme digests were used for general restriction mapping in combination with Southern blotting and hybridization analysis. Southern blots were probed with junction fragments obtained from restriction enzyme digests of the cosmids, as well as R. sphaeroides ebb genes, and cosmid maps were generated (Figs. 1.1 A and B). R. sphaeroides cbbR, cbbF, cbbP, cbbT, cbbG, cbbA, and R. rubrum cbbE gene probes did not hybridize to pRCFI but, with the exception of cbbE, did hybridize to pRCFH DNA. These putative CBB cycle genes mapped very near the cbbM gene of R. sphaeroides (Fig. 1.1 B). In all cases, hybridizing bands derived from restriction enzyme digests of R. capsulatus chromosomal DNA comigrated with bands present in DNA isolated from the cosmid clones (data not shown).
Organization and subcloning of the R. capsulatus CBB cycle genes. DNA fragments from cosmid clones pRCFI and pRCFH that hybridized to the specific ebb probes were subcloned in order to more carefully map the genes. In addition, subclones
13 Fig. 1.1. Restriction maps and gene organization of the two R. capsulatus ebb gene clusters found on cosmid clones pRCFT (A) and pRCFH (B). BamHl, £coRI, HindM,
SaB, and Xhol restriction sites for both cosmids were mapped by hybridization analysis.
Additional restriction mapping, including Smal sites, was carried out using subclones of the R. capsulatus ebb containing regions from cosmids pRCFI and pRCFH. The subcloned regions have been enlarged for clarity. Locations of structural genes are indicated by boxes and gene designations are shown below the boxes. The direction of transcription is indicated with arrows. (C), Subclones and sizes of restriction fragments used in Southern blotting and hybridization analysis (presented in Fig. 1.2). The subclones are aligned with the genetic map in (B) above. Restriction sites: B, BamHl', E,
EcoRI; H, ffmdDI; P, Pstl; S, SaB; Sm, Smal; X, Xhol.
14 I kb
HX H E S X X H E B E S HBH I I I I I I______I I I I I I pRCFl
pRKFIP cbbLf cbbS, ►
I kb
H E X H B XB XSEXEHHS E X B H B pRCFIl
HS SmXB
cbbR„ cbbF„ cbbP„ cbbT„ cbbG„ cbbA„ cbbM„
HB B B _ u _ pKlSFIIEH f 1.8 kb ^ 2.3 kb BS I kb
pK18FIIS4.4 2.0 kb B 1,8 kb
pK18FIIS4 s 2.0 kb S I______I Figure 1.1. pKI8FlIS2 of cbbLcbbS and cbbM were obtained to design expression vectors to synthesize
recombinant RubisCO in E. coli as well as complement the R. sphaeroides RubisCO
deletion strain (Falcone and Tabita, 1991). A 9 kb Pstl fragment from pRCFI hybridized to an R. sphaeroides cbbL gene probe and was cloned in pRK41S to yield plasmid pRKFIP. Additional hybridization and sequence analysis of subclones revealed that the predicted start of the R. capsulatus cbbL gene was near the HindBl site within thisPstl fragment. Hybridization of the R. sphaeroides cbbL probe with RamHI-PjrI-digested pRKFIP revealed a single reactive band of 4.7 kb (Fig. 1.2 A, B).
The cbbu gene cluster of R. capsulatus was subcloned (from pRCFH) as two separate DNA fragments, an 8.4 kb MndlH-fcoRI fragment (pKlBFUEH) and a 2 kb SaE fragment (pK18FHS2-I) (Fig. 1.1 C). The overlap between these two subclones was confirmed by Southern hybridization experiments using the cbbA\ probe (Fig. 1.2 J). A detailed restriction map of the subcloned region is shown (Fig. 1.1 B). In order to distinguish between the two 1.8 kb SaE-BamlE fragments of pKlBFHEH, it was necessary to further subclone the two larger SaE fragments derived from pKlBFHEH, generating plasmids pK18FHS4.4 and pK18FHS4 (Fig. 1.1 C). Restriction endonuclease digestion of pK18FHS4.4 and pK18FHS4 with SaE and BamHl and digestion of pK18FHS2-I with SaE (Figs. 1.1 C and 1.2 C), followed by hybridization with the
R. sphaeroides heterologous probes (Figs. 1.2 D-K), established the order of the cbbu genes of R. capsulatus (Figs. 1.1 B,C). Partial DNA sequencing of the cbbP, cbbT, cbbA, and cbbM genes (data not shown) was used to determine the location of the Calvin cycle genes within the cbbu cluster more accurately and revealed that these genes are all transcribed in the same direction (Fig. 1.1 B). Furthermore, these genes were highly homologous to their counterparts from R. sphaeroides, with partial deduced amino acid sequences exhibiting 79-92% identity. Hybridization with R. sphaeroides "full length" and 5'-cbbR probes (Figs. 1.2 CJD) showed that the R. capsulatus cbbRu gene was
16 Fig. 1.2. Southern hybridization analysis of R. capsulatus ebb gene clones. (A), ethidium bromide stained agarose gel; lane 1, pRKFIP digested with flamHI and Psri; lane 2, A,
DNA digested with H/ndin. (B), hybridization results of gel in (A) probed with cbbL.
(C), ethidium bromide stained agarose gel; lane 1, pK18FUS4.4 digested with fiawiHI and
SaW, lane 2, pK18FIIS4, digested with BamHL and SaW, lane 3, pK18FIIS2-I digested with Sail; and lane 4, A, DNA digested with HindHl. (D-K), hybridization results of lanes
1-3 of gel from (C) probed with R. sphaeroides cbbR "full-length" probe (D); cbbR "N- terminal" probe (E); probe (F); probe (G); c66T probe (H); probe (I); cbbA probe (J); cbbM probe (K). Probes were derived from plasmids listed in Table
1.1. Numbers refer to sizes of fragments in kilobases and "v" refers to vector hybridization.
17 Figure 1.2, transcribed divergently from the rest of the ebbjj cluster (Fig. 1.1 B); sequencing the ends of the BamHl fragment derived from pK18FIS4.4 revealed homology to cbbP and cbbRa and also indicated that the genes were divergently transcribed. The partial deduced sequence indicated that the R. capsulatus CbbR protein exhibited 38% identity to
R. sphaeroides. ThecbbA and 5'-cbbR probes also hybridized to vector DNA (Fig. 1.2 E,
J) probably because of contamination of the gene probe with a small amount of vector
DNA. No hybridization to a 2.7 kb fragment was observed when cosmid pRCFH was digested with BamHl and SaB and probed with either the cbbA or 5'-cbbR probe (data not shown).
Complementation of R. sphaeroides strain 16 by R. capsulatus cbbLcbbS and cbbM clones. R. sphaeroides strain 16 is a RubisCO deletion strain {cbbLcbbS'cbbM^ that was originally constructed for the expression of heterologous RubisCO genes and the selection of mutant forms of this enzyme (Falcone and Tabita, 1991). This strain lacks the ability to grow photoautotrophically and can grow photoheterotrophically only if an electron acceptor other than 00% is supplied, such as DMSO. To determine if the homologous R. capsulatus cbbLcbbS and cbbM sequences encoded functional form I and form n RubisCO enzymes, each of these putative genes was cloned into the broad host range vector pRK415 (Keen et al., 1988) and the broad host range R. sphaeroides
RubisCO expression vector pRPS-1 (Falcone and Tabita, 1991).
To construct the R. capsulatus form 1 RubisCO expression clone, the 4.7 kb
BamHl-Pstl fragment was removed from plasmid pRKFlP and cloned into pRPS-1 in both orientations, yielding plasmids pRPSFI-I and pRPSFI-U. Neither pRK415 or pRPS-1 has a unique SaB site, so the 2 kb SaB fragment encoding the R. capsulatus cbbM gene was first cloned into pUC1318. The 2.0 kb SaB fragment was removed from pUC1318::FnS as a BamHl fragment and cloned into both pRK415 and pRPS-1 in both orientations. The resulting plasmids were pRKFH-I, pRKFH-H, pRPSFH-I, and
19 pRPSFE-n. All of these plasmids were introduced via conjugation into R. sphaeroides strain 16. Tetracycline-resistant clones of each recombinant strain were tested for the ability to complement R. sphaeroides strain 16 to photoheterotrophic growth using CO 2 as the electron acceptor. Only plasmids pRPSFI-I, pRKFE-I, and pRPSFH-I (Fig. 1.3) enabled R. sphaeroides strain 16 to grow under these conditions, yet all of the plasmid- bearing strains grew when DMSO was used as the electron acceptor. Thus, complementation by the cbbLcbbS or cbbM genes was orientation specific. Only strain
16 (pRPSFI-I) was able to grow photoautotrophically.
The doubling times and RubisCO activity of the complemented strains were determined (Table 1.2.). Photoheterotrophic and photoautotrophic growth rates were similar to those previously observed for R. sphaeroides strain 16 complemented by a variety of heterologous RubisCO genes (Falcone and Tabita, 1991). The increase in
RubisCO activity observed in photoautotrophically-grown (1.5% 002/98.5% H 2) over photoheterotrophically-grown R. sphaeroides strain 16 (pRPSFI-I) is a regulatory feature of wild-type R. sphaeroides retained by the R. sphaeroides strain 16 (pRPS-1) expression system (Falcone and Tabita, 1991). Thus, the high level of RubisCO activity in photoautotrophically-grown R. sphaeroides strain 16 (pRPSFI-I) was not surprising. A relatively high level of R. capsulatus form II RubisCO activity was observed in R. sphaeroides strain 16 (pRKFH-I). This expression does not appear to be due to promoter activity within the 2 kb Sail fragment because expression of R. capsulatus cbbM in pRK415 was orientation-dependent. Although the RubisCO activity of photoheterotrophically grown R. sphaeroides strain 16 (pRPSFH-I) approached that observed for photoautotrophically growing wild-type R. sphaeroides, this strain was unable to grow photoautotrophically.
A correlation between RubisCO activity and complementation was made by measuring the RubisCO levels in cultures grown photoheterotrophically in the presence
20 Plasmid Growth Doubling RubisCO Construct Mode^ Time Activity^ (hours)
pRPSn-I MAL 12 261 AUT 40 756
pRKFH-I MAL 11 112
pRPSFH-I MAL 10 315
^ MAL - photoheterotrophic growth on malate; AUT - photoautotrophic growth
^ expressed in nmoles CO 2 fixed/min/mg All values represent averages from duplicate experiments.
Table 1.2. RubisCO activity in R. sphaeroides strain 16 complemented with R. capsulatus RubisCO clones
21 10
0.1
0.01 0 50 100 150 200 Time (h)
Fig. 1.3. Photoheterotrophic growth of R. sphaeroides strain 16 containing the
R. capsulatus cbbM gene in plasmids pRPSFH-I ( # ) and pRPSFH-H ( O ).
22 and absence of DMSO. For the complemented strains, RubisCO activity levels were not significantly different when cultures were grown with and without DMSO, however, strains that contained plasmids that did not complement showed no RubisCO activity when grown under permissive conditions (using DMSO as electron acceptor; results not shown).
Expression of R. capsulatus cbbLcbbS and cbbM genes in E. coli. The complementation data strongly suggested that the isolated R. capsulatus fragments encoded functional form I and form II RubisCO. In order to further establish this, as well as to develop a system for future site-directed mutagenesis studies of the R. capsulatus genes, hybridizing and complementing DNA was inserted into E. coli expression vectors under the control of the lac promoter by using plasmids pK18 or pK19. Although some bacterial RubisCO genes are not expressed well in E. coli (Meijer et al., 1991), the
R. capsulatus cbbLcbbS and cbbM genes were both expressed (Table 1.3.). In all cases, expression was orientation-dependent and significantly increased by lac promoter induction. Antisera to the form I RubisCO from R. sphaeroides failed to cross react on
Western immunoblots with the recombinant form I R. capsulatus protein, and only slight cross reaction was observed with antibodies to the Xanthobacter enzyme (results not shown). The immunoblot studies with the R. sphaeroides form I antibodies are in agreement with earlier studies using R. capsulatus extracts (Gibson and Tabita, 1977).
Most interestingly, however, antibodies to the Synechococcus 6301 RubisCO strongly reacted to the recombinant R. capsulatus form I enzyme (Fig. 1.4), suggesting that the R. capsulatus protein might be more similar to the cyanobacterial RubisCO. The form II
R. capsulatus enzyme strongly reacted with antibodies to its counterpart from
R. sphaeroides.
23 RubisCO Activity^ (nmol/min/mg) Plasmid Constructs -IPTG +DPTG
pKI9 <0.01 <0.01
p K isn 3 66
pK19H 1 5
pK18FnS2-I <0.01 63
pK18FnS2-n <0.01 2
^ All numbers are the averages of duplicate experiments. Samples were removed every 4 h after the addition of IPTG; the highest level of RubisCO for E. coli (pKlSFI) was obtained after 20 h induction, while the maximum activity for E. coli (pK18FIIS2-I) was obtained after 12 h induction.
Table 1.3. Expression of R. capsulatus cbbLcbbS and cbbM genes in E. coli
24 Fig. 1.4. Western immunoblots of R. capsulatus RubisCO synthesized in E. coli and
R. capsulatus. Western blots were prepared from SDS-PAGE gels: lane 1, purified
Synechococcus sp. strain PCC 6301 RubisCO; lane 2, purified R. sphaeroides form H
RubisCO; lane 3, partially purified recombinant R. capsulatus form I RubisCO; lane 4, partially purified recombinant R. capsulatus form II RubisCO; lane 5, magnesium/heat soluble extract of photoheterotrophically grown R. capsulatus. Immunoblots were incubated with antibody raised against (A) Synechococcus strain PCC 6301 RubisCO and
(B) R. sphaeroides form II RubisCO.
25 A
B
1 2 3 4 5
Figure 1.4.
26 DISCUSSION
I have cloned the R. capsulatus cbbLcbbS and cbbM genes, which encode form I and form E RubisCO, respectively, and expressed these genes in E. coli and in
R. sphaeroides. In addition, we determined the organization of several other structural genes of the CBB cycle in R. capsulatus. Most interestingly, and unlike the situation in the related organism R. sphaeroides, the cbbLcbbS genes of R. capsulatus were not clustered with other CBB cycle genes (Fig. 1.1 A). However, the cbbM gene was clustered with other ebb genes (Fig. 1.1 B), much like the form E CBB cycle operon
{cbbxd of R. sphaeroides (Gibson et ai, 1990). Unlike R. sphaeroides, however, a divergently transcribed cbbR gene was found directly upstream of the first gene of the form E gene cluster of R. capsulatus. In R. sphaeroides, a divergently transcribed cbbR gene is directly upstream of the form I (cbb\) operon and its gene product activates transcription of both the cbbi and cbbu opérons (Gibson and Tabita, 1993). By analogy to
R. sphaeroides (i.e., the order and tight spacing of the genes, and the direction of transcription), it seems likely that the form E CBB cycle gene cluster of R. capsulatus is transcribed as a single operon. However, this remains to be established, as does the function of cbbR.
Despite the presence of a common transcriptional activator, independent regulation of the two forms of RubisCO has been observed in R. sphaeroides (Jouanneau and Tabita, 1986) and R. capsulatus (Shively et al., 1984). For R. sphaeroides, the 11- fold increase in RubisCO activity observed when cultures were shifted from photoheterotrophic growth on malate to photoautotrophic conditions (1.5% C02/98.5%
Hz) was found to be due to a four-fold increase in the form E RubisCO protein level and a
17-fold increase in the form I RubisCO protein level. In contrast, the R. capsulatus form I
27 RubisCO was completely repressed when cells were grown photoheterotrophically on
malate and under photoautotrophic conditions at high levels of CO 2 (5-10% CO2). Form
I RubisCO of R. capsulatus was present only when cultures were grown
photoheterotrophically on butyrate, at limiting CO 2 concentrations, whereas form H
RubisCO was observed under all growth conditions (Shively et ai, 1984). Although
immunological quantitation of form I and form II RubisCO synthesis in R. capsulatus is
lacking, since antibodies to these enzymes are not yet available, the above findings
suggest very distinct regulatory schemes in these two organisms. This perhaps reflects
the different arrangement of the ebb genes in R. sphaeroides and R. capsulatus. For
example, other than RubisCO, PRK is the only enzyme unique to the CBB cycle, and all
of the available evidence suggests that R. capsulatus possesses only a single form of
PRK. Purification of PRK from R. capsulatus has revealed only a single form of the
enzyme (Tabita, 1980). In addition, the results of Southern hybridization studies with R.
capsulatus chromosomal DNA performed here, using a R. sphaeroides cbbP probe, did
not indicate the presence of a second cbbP gene. If additional cbbP genes are present,
they must differ substantially from existing nonsulfur purple and chemoautotrophic
bacterial cbbP genes (Gibson, 1995). Likewise, if the R. capsulatus cbbn gene cluster is
indeed transcribed as an operon, the expression of this operon would be obligatory under
any growth condition in which the CBB cycle is required since there is no other discernible cbbP gene. Thus, the cbbM gene, encoding form II RubisCO, would be expressed under all growth conditions that require the Calvin cycle. By the same token,
the cbbLcbbS genes, encoding form I RubisCO, could be regulated independently of the other ebb genes. This scenario would be consistent with the observations described previously (Shively et ai, 1984) and discussed above.
R. capsulatus is the third species of the familyRhodospirillaceae from which the
RubisCO gene(s) have been cloned. In each case, the RubisCO genes have been
28 functionally expressed in E. coli (Quivey and Tabita, 1984; Somerville and Somerville,
1984; Muller et al., 1985; Gibson and Tabita, 1986), and the recombinant enzymes have all proven to possess unique properties that reflect important structure-function relationships of this key enzyme (Tabita, 1995). Surprisingly, recombinant form I
RubisCO of R. capsulatus reacted poorly, if at all, with antibodies directed against the R. sphaeroides and X. flavus enzymes, while showing substantial reactivity towards anti-
Synechococcus form I RubisCO. These results suggest that the R. capsulatus form I
RubisCO may be more similar to the cyanobacterial enzyme than the enzyme from the closely related organism, R. sphaeroides. In addition, these results, combined with the unusual elution behavior of the R. capsulatus form I protein from anion-exchange columns (Gibson and Tabita, 1977), suggest that the E. coli cbbLcbbS expression vector prepared here might prove to be extremely useful for comparative structure-function studies. Indeed, detailed enzymological studies of this protein might provide a rationale for the ability of R. capsulatus, unlike R. sphaeroides and Rs. rubrum, to fix CO2 and grow under aerobic lithoautotrophic conditions (Madigan and Gest, 1979).
The R. capsulatus RubisCO genes were also expressed in the R. sphaeroides
RubisCO-minus strain, strain 16 (Table 1.2.). Both the cbbLcbbS and the cbbM genes of
R. capsulatus complemented R. sphaeroides strain 16 to photoheterotrophic growth when cloned in expression vector pRPS-1 in the proper orientation, but only the cbbLcbbS genes in vector pRPS-1 complemented this strain to photoautotrophic growth. By contrast, R. rubrum cbbM, the Synechococcus form I RubisCO genes, and the X. flavus and B. japonicum cbbLcbbS genes complemented R. sphaeroides strain 16 to photoautotrophic growth when expressed using the promoter of plasmid pRPS-1 or the endogenous promoters associated with the respective RubisCO genes (Falcone and
Tabita, 1991; 1993). Thus, the inability of theR. capsulatus cbbM to complement
R. sphaeroides strain 16 to photoautotrophic growth in vector pRPS-1 is unique and not
29 easily explained. The R. capsulatus cbbM gene also able complemented R. sphaeroides strain 16 to photoheterotrophic, but not photoautotrophic, growth when cloned in broad host-range vector pRK415. However, complementation in this case was probably not due to the presence of an endogenous internal cbbM promoter on the cloned fragment since complementation was orientation-dependent. Transcription from a plasmid-based promoter or the generation of a promoter as an artifact of cloning may be the cause of the observed RubisCO synthesis.
Finally, it is intriguing that the cbbLcbbS genes of R. capsulatus did not complement R. sphaeroides strain 16 when cloned in the broad-host range plasmid pRK415. In addition, no RubisCO activity was expressed when plasmid pRKFIP was introduced into strain 16. Analysis of the DNA sequence upstream of the R. capsulatus cbbLcbbS genes revealed the presence of multiple copies of the T-Nn-A motif which is common to LysR (CbbR)-activated promoters (Goethals et al., 1992), suggesting that the cbbLcbbS genes of R. capsulatus may be activated by CbbR. A similar case was found in
R. rubrum, where its cbbM gene was not expressed in strain 16 unless its cognate cbbR was present (Falcone and Tabita, 1991). Thus, one potential explanation for the lack of
R. capsulatus cbbLcbbS expression using plasmid pRKFIP may be the absence of a cbbR gene on this clone and the inability of R. sphaeroides CbbR to activate transcription from theR. capsulatus cbbLcbbS promoter.
ACKNOWLEDGEMENTS
The author would like to thank Dr. Jessup Shively for providing the R. capsulatus cosmid clones and Dr. Wim Meijer for providing antibodies raised against the
Xanthobacter flavus RubisCO.
30 CHAPTER 2
Organization and Nucleotide Sequence of the Rhodobacter capsulatus
cbbLcbbS and Neighboring Genes. Phylogenetic Analysis Suggests That
These Genes Were Acquired by a Horizontal Gene Transfer.
INTRODUCTION
Rhodobacter capsulatus, a nonsulfur purple photosynthetic bacterium, assimilates
CO2 via the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway. The
CBB enzyme responsible for fixing CO 2 is ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). RubisCO exists in two distinct forms. The form I
RubisCO is a large hexadecameric enzyme (LgSg) composed of eight large subunits (Mr
-55,000) and eight small subunits (Mr - 15,000), which are encoded by the cbbL and cbbS genes, respectively. The form II RubisCO is composed of large subunits only (L%), which are biochemically and genetically distinct from the form I large subunits, and are encoded by the cbbM gene (Gibson, 1995). The nonsulfur purple photosynthetic bacterium Rhodobacter sphaeroides was the first organism shown to synthesize both forms of RubisCO (Gibson and Tabita, 1977a). The organization and regulation of the genes encoding CBB enzymes (ebb genes) have been intensely studied in R. sphaeroides and other autotrophic bacteria (Gibson and Tabita, 1996).
31 The organization of the ebb genes in the closely related organism, R. capsulatus, was determined recently (Paoli et al, 1995). TheR. capsulatus form II RubisCO gene, cbbM, has been sequenced (Larimer et ai, 1995) and encodes a protein very similar to the form H RubisCO from R. sphaeroides. In earlier studies the R. capsulatus form I
RubisCO behaved quite differently from the R. sphaeroides form I enzyme in its elution behavior from anion exchange columns and antibody cross-reactivity (Gibson and Tabita,
1977c). Recently, recombinant R. capsulatus form I RubisCO was shown to cross-react with antibody directed against cyanobacterial RubisCO, despite the lack of cross-reaction to anti-R. sphaeroides form I RubisCO (Paoli et al, 1995). In hopes of better understanding its unusual properties, the current study was undertaken to determine the primary structure of the R. capsulatus form I RubisCO. During the process of sequencing the R. capsulatus cbbL and cbbS genes, other ebb genes were found upstream and downstream of the R. capsulatus cbbLcbbS genes. A phylogenetic analysis of the unusual
R. capsulatus form I RubisCO large subunit amino acid sequence indicated that the genes encoding the R. capsulatus RubisCO were acquired by a horizontal gene transfer.
32 MATERIALS AND METHODS
DNA sequencing and analysis. The sequence of the R. capsulatus cbbLSQ genes from cosmid pRCFI was determined at the Clemson University Core Nucleotide
Sequencing Facility. Automated sequencing was performed with an ABI 377a DNA sequencer using ABI Taq DyeDeoxy Cycle sequencing reagents and a Perkin-EImer
Cetus thermal cycler as described by the manufacturer (Perkin Elmer, Foster City, Calif.).
Plasmids pKlSFIP, pKlSFISB, pKlSFISEH, pK18FI6E, and pKlSFTTEH (Table 2.1.) were used, and, as necessary, oligonucleotide primer walking was used to generate complete double stranded sequence. Synthetic oligonucleotides were purchased from
Integrated DNA Technologies (Coralville, Iowa). Because of the unusual nature of the
R. capsulatus cbbLS sequence, the identity of the cbbL gene was confirmed from partial sequencing of clones derived from 2 additional sources (pEULA4 and pK181H2E7; Fig.
2.1, Table 2.1.). The nucleotide sequences of the R. capsulatus cbbR\ and cbbL from plasmids pEULA4 and PK181H2E7 were determined using an ABI Prism 310 Genetic
Analyzer. A Dye Terminator Cycle Sequencing Kit and a Perkin Elmer Cetus thermal cycler were used as described by the manufacturer (Perkin Elmer, Foster City, Calif.).
The M13/pUC forward 23-base sequencing primer
(5'-CCCAGTCACGACGTTGTAAAACG-3'), M13 reverse (-48) primer
(5'-AGCGGATAACAATTTCACACAGGA-3 ), and sequence-specific synthetic primers were used to complete the double stranded sequence. Sequence analysis was carried out using the University of Wisconsin Genetics Computing Group (GCG) Software, the
33 Plasmid Relevant characteristics Source or reference pK18, pK19 Km*^, pUC derivatives Pridmore, 1987 pRKFIP pRK415 containing the 9 kb Pstl fragment of pRCFI containing the R. capsulatus This work cbbLcbbS genes pKlSnSB pKI8 containing part of the R. capsulatus cbbL gene on a 1.8 kb BamlSr SaR fragment cloned from pRKFIP This work pKlSnSEH pK18 containing part of the R. capsulatus cbbL gene on a 0.5 kb TfmdlH-EcoRI fragment cloned from pK18FISB This work pK18FI6E pK18 containing part of the R. capsulatus cbbL gene on a 0.6 kb EcdRl fragment cloned from pKlSFISB This work pK18F7EH pK18 containing part of the R. capsulatus cbbL gene on a 0.7 kb EcoRI-£fi/idIH fragment cloned from pK18FISB This work pEULA4 pK19 containing part of the R. capsulatus cbbL gene and upstream sequence on a 4 kb EcoRl fragment This work pULHSm531 pK19 containing part of the R. capsulatus cbbL and upstream sequence on a 0.5 kb HirvSR-Smal fragment cloned form pEULA4 This work pULHSm320 pK19 containing part of the R. capsulatus cbbRi sequence on a 0.36 kb HindlR-Smal fragment cloned form pEULA4 This work plH2 A cbbL hybridizing clone derived from an R. capsulatus SB 1003 overlapping Fonstein, cosmid library etal, 1993 pK181H2E7 pK18 containing part of the R. capsulatus cbbL gene on a 0.7 kb EcoRI fragment cloned form plH2 This work
Table 2.1. Plasmids used in this study
34 Fig. 2.1. Organization of the R. capsulatus cbbi gene cluster. (A) Genetic and physical map of the R. capsulatus cbbi gene cluster. Arrows below the genes indicate size and direction of potential transcripts. Restriction enzymes sites: E, EcoRl', H, HindHl; P,
S, SaE', Sm, Smal; and X, Xhol. (B) Clones from which the map was generated.
The thicker lines represent cosmid DNA that was subcloned along with the R. capsulatus chromosomal DNA in the original library clones. Only 100 bp of R. capsulatus chromosomal DNA is present upstream of the cbbL gene in pRKFIP. pEULA4 is a 4 kb
EcoRI fragment cloned directly from R. capsulatus chromosomal DNA. pKlH2E7 contains a 700 bp EcoRI fragment subcloned from cosmid plH2 (Fonstein et ai, 1993).
As indicated, the insert in plasmid plH2E7 is in part cosmid DNA, because cosmid plH2 did not contain the entire cbbL gene. The Sau3A (S3) site at the end of the R. capsulatus chromosomal DNA insert in plH2 is shown.
35 1 kb Sm Sm EX Sm Sm P H EXES Sm L anfA cbbRt cbbLt cbbS^ cbbQ^ w Os H pRKFIP
pEULA4 E
pKlH2E7
Figure 2.1. EGCG Extension Programs (The Sanger Centre, Hinxton,
England) and the Mac Vector Sequence Analysis Software (Eitemational Biotechnology,
Inc., New Haven, Conn.). Phylogenetic analysis of R. capsulatus RubisCO and CbbR protein sequences. Multiple sequence alignments were performed using Clustal W (Thompson et ai, 1994) and trees were generated by neighbor-joining and maximum parsimony methods using Phylip 3.5c (Felsenstein, 1993) and by maximum-likelihood using Puzzle
2.5.1 (Strimmer and von Haeseler, 1996). Accession numbers [Swiss Protein Database or GenBank (gb)] of sequences used for phylogenetic analysis of form I RubisCO large subunit proteins (CbbL or RbcL) are as follows: Anabaena sp. PCC7120, P00879;
Chlamydomonas reinhardtii, P00877; Chromatium vinosum I, P22849; Chromatium vinosum 2, P22S59; Cryptomonas <^,P14951; Cyanophora paradoxa, P24312; Cuscuta reflexa, P30401; Cylindrotheca sp. Nl, P24673; Euglena gracilis, P00878;
Hydrogenovibrio marinus 1, D43621 (gb); Hydrogenovibrio marinus 2, D43622 (gb);
Ipomoea purpurea, P28260; Mollugo verticillata, P25832; Nicotiana tabacum, P00876;
Nitrobacter vulgaris, L22885 (gb); Odontella sinensis, P49520; Olisthodiscus luteus,
P 14959; Porphyra purpurea, P50255; Prochlorococcus marinus, D21833 (gb);
Prochloron sp., D21834 (gb); Prochlorothrix hollandica, P27586; Pseudomonas hydrogenothermophila, D30764 (gb); Rhodobacter capsulatus, GSDB:S:S 1079287;
Rhodobacter sphaeroides, P27997; Ralstonia eutropha ATCC17707 (chromosomal),
M 17744 (gb); Ralstonia eutropha ATCC17699 (chromosomal), P09657; Ralstonia eutropha ATCC 17699 (plasmid), U20585 (gb); Spinacia oleracea, P00875
Synechococcus sp. PCC6301, P00880; Synechococcus sp. PCC7002, D 13971 (gb)
Synechococcus sp. WH7803, U46156 (gb); Thiobacillus denitrificans, L42920 (gb)
Thiobacillus ferrooxidans, P28895; vent worm (Alvinoconcha) symbiont (potential
Thiobacillus sp.), P24672; Triticum aestivum, PI 1383; Xanthobacterflavus, P23011.
37 Accession numbers of sequences used for phylogenetic analysis of form II
RubisCO large subunit proteins (CbbM or RbcL) are as follows: Gonyaulux polyedra,
LL41063 (gb); Hydrogenovibrio marinus, D28135 (gb); Rhodobacter capsulatus,
P50922; Rhodobacter sphaeroides, (j^y, Rhodospirillum rubrum,Ÿ{W1\%
Symbiodinium sp., U43532 (gb); Thiobacillus denitrificans, L37437 (gb).
Accession numbers of sequences used for phylogenetic analysis of the CbbR proteins are as follows: Chromatium vinosum (CbbR), P25544; Cyanophora paradoxa
(plastid) (YCF30/RbcR) P48271; Odontella sinensis (plastid) (YCF30/RbcR), P49518;
Porphyra purpurea (plastid) (YCF30/RbcR), P51205; Ralstonia eutropha (CbbR),
P42722; Rhodobacter capsulatus (AmpR), P14145; Rhodobacter capsulatus (CbbRi),
U87283 (gb); Rhodobacter capsulatus (CbbRn), U87282 (gb); Rhodobacter sphaeroides
(CbbR), P52690; Rhodospirillum rubrum (CbbR), P52595; Synechocystis sp. PCC6803
(RbcRl), CyanoBase ORF I.D.=SLIXX)30; Synechocystis sp. PCC6803 (RbcR2),
CyanoBase ORF I.D.=SLL1594; Thiobacillus ferrooxidans (CbbR), Q06610;
Xanthobacter flavus (CbbR), P25545.
Nucleotide sequence accession numbers. The National Center for Genome
Research genome sequence database accession number for the R. capsulatus cbbLSQ genes is GSDB:S: 1079287. The GenBank nucleotide sequence accession number for
R. capsulatus cbbR\ is U87283.
38 RESULTS
Organization of the R. capsulatus cbbLcbbS genes and neighboring regions.
Earlier studies using R. sphaeroides gene-specific probes and a c661-hybridizing
R. capsulatus library clone indicated that the R. capsulatus cbbLcbbS genes, encoding form I RubisCO, were not associated with any other ebb genes (Paoli et al., 1995). DNA sequence analysis of subclones derived from the c66ZLc6M-containing cosmid pRCFI revealed that the 31 kb chromosomal DNA insert in this library clone contained only 100 bp of R. capsulatus DNA upstream of the cbbL gene. A 4 kb EcoRI fragment containing part of the R. capsulatus cbbL gene and upstream DNA was cloned from the
R. capsulatus SB 1003 chromosome into pK19 (pEULA4, Fig. 2.1) in order to determine if other ebb genes were present upstream of the form I RubisCO genes. The organization of the cbbLcbbS and the neighboring region is presented in Fig. 2.1. Nucleotide sequencing revealed an open reading frame (ORF) with a deduced amino acid sequence similar to the CbbR class of LysR-type transcriptional activators upstream and divergently transcribed from cbbL, in addition, the previously identified anfA gene
(Kutsche et al, 1996) was found to be about 2 kb downstream of cbbR\. The subscript I indicates that cbbR\ is divergently transcribed from the form I RubisCO genes. A second cbbR gene, cbbR\i, is present upstream and divergently transcribed from a group of ebb genes associated with the form II RubisCO gene of R. capsulatus (Paoli et ai, 1995).
Additional nucleotide sequencing indicated that no other known ebb genes were present in the region between cbbRi and an/A (J.L. Gibson, unpublished). Sequencing
39 downstream of cbbS revealed an ORF with a predicted amino acid sequence having
significant similarity to the cbbQ gene product ft’om Pseudomonas hydrogenothermophila
(Yokoyama gr a/., 1995).
Nucleotide sequence of cbbRi, cbbL, cbbS, and cbbQ. The nucleotide sequence
for both strands of a 3993 nucleotide region including the form I RubisCO genes and
approximately I kb upstream and downstream of cbbLcbbS was determined (Fig. 2.2).
An 897 nucleotide ORF, 110 nucleotides upstream of cbbL and divergently
transcribed from cbbL, extends from an ATG start codon at position 948 to a TGA stop codon at position 52 (Fig. 2.2). The open reading frame is preceded by a potential
ribosome binding site, GGA, 11 bp upstream of the putative start site. The coding region
is 298 amino acids long with a deduced molecular weight of 31,766. A database search
for proteins with amino acid homology revealed that the product of this ORF was most similar to members of the CbbR family of LysR-type transcriptional activators. A cbbR is divergently transcribed from ebb genes in a number of autotrophic bacteria (Gibson and
Tabita, 1996), and in several cases CbbR has been shown to activate transcription of ebb genes (Gibson and Tabita, 1993; Kusano and Sugawara, 1993; van den Bergh et ai,
1993; Windhovel and Bowien, 1990; Viale et al., 1991). Recently, genome sequencing has revealed ORFs with similarity to CbbR in the cyanobacterium Synechocystis sp.
PCC6803, in the plastids of the non-green algae Odontella sinensis and Porphyra purpurea, and in the plastids of the glaucophyte Cyanophora paradoxa. Interestingly, cbbR\ shares 51.0 % deduced amino acid sequence identity with CbbR from Thiobacillus
40 Fig. 2.2. Nucleotide and deduced amino acid sequence of the R. capsulatus cbbi gene cluster. Predicted amino acid sequences for the cbbL, cbbS, and cbbQ genes are given below the nucleotide sequence. The predicted amino acid sequence of the cbbRi gene, which is transcribed in the opposite direction and from the DNA strand complementary to the one shown, is given above the nucleotide sequence. The names of the genes and the direction of transcription are shown near the start of each ORF. Putative ribosome binding sites are underlined and stop codons are represented by asterisks. The T-Nn-A sequences are indicated by “<+—/—+>”, where the + is above the T or A and the “< >” indicate the short inverted repeats. The longer inverted repeats downstream of cbbS that may form hairpins and function in transcriptional termination are indicated by arrows below the sequence.
41 * s p GCTTGGGGCTAGCATGAAACCGGTGTTTTCGCAAGCGGGCCAGGCGGCGCGTCAGCTCGG 6 0
aASOAAZiEALFARAAVSLHK GCCCGCGCTGCCCGCCGCCAGATGCGCCAGAAAGGCTCTGGCCGCCACCGACAGATGCTT 120
GRPYAVNWVRIiLPFGQVDLV GCCGCGCGGATAAGCGACGTTCCAGACCCGCAGCAGCGGAAAGCCCTGCACATCCAGCAC 180
ALRGTBAELTITDQSIiIAIiG CGCAAGCCGCCCGGTCTCGGCCTCCAGCGTGATCGTGTCCTGCGACAGGATCGCCAGGCC 240
LGAAVAAKIAENSGLEMRVQ CAGCCCTGCCGCCACCGCCGCCTTGATCGCCTCGTTCGAGCCCAGCTCCATCCGCACCTG 300
PRIGNEAFFKEAAARTGSGP CGGCCGGATGCCGTTCTCGGCAAAGAACTTTTCCGCCGCAGCCCGGGTGCCGGACCCGGG 360
ERIiIFPAEAIiAAFPVATVGA CTCGCGCAGAATGAAGGGCGCCTCGGCCAGCGCGGCAAAGGGCACCGCGGTCACGCCCGC 420
LPHGPPALVLIANRAIPRAV CAGCGGATGGCCCGGCGGCGCCAGCACGAGGATCGCATTGCGCGCAATCGGGCGGGCCAC 480
VDLOEPPQGLICIiDDENAAL CACATCCAGCCCCTCGGGCGGCTGGCCCAGAATGCACAGGTCATCCTCGTTCGCGGCCAG 540
RAIVTQRNTVTZiAVEZGPNE ACGGGCGATCACCGTTTGCCGGTTGGTGACGGTCAGCGCCACTTCGATCCCGGGGTTTTC 600
TCFAGLLRPLFYQATSVIAL GGTGCAAAAGGCGCCCAGAAGCCGCGGCAGGAAATATTGCGCGGTGGAGACGATGGCCAG 660
RLRGRRIiGQLDAIiQHELREI CCGCAGCCGCCCTCGCCGCAGCCCCTGCAGATCGGCCAGCTGCATCTCCAGCCGCTCGAT 720
GELTERATALVAQGAETLFL CCCCTCCAGCGTCTCGCGCGCGGTGGCCAGCACCGCCTGCCCCGCCTCGGTCAGGAAAAG 780
RRGIRDLLVAGLADELQKVQ CCGCTTGCCGATCCGGTCAAGCAGCACGGCGCCGAGTGCGTCTTCGAGCTGCTTGACCTG 840
TFVAPQTZ.HLEEAARTYSAH GGTGAAGACCGCGGGCTGGGTCAGGTGCAATTCCTCGGCGGCGCGGGTGTAGCTGGCGTG 900
< - c b b R i RAVAAFVQLQRLTCRM CCGCGCCACCGCGGCAAAGACCTGCAACTGGCGAAGCGTGCAACGCATGATAAGCTTTTC 960
S/D « + + » ^ + ^ — — — — — — — — — ^ + ^ — — — — — — — — — CCTATTTCATCACTAAAAACTATTCAGTTTTGTAAAGTCCCGCAAGGCCGCATAGTCCCC 1020
Figure 2.2 (continued)
42 F i g . 2.2. (continued)
GCAAGCCCCGCCAAACGGGCCAACCAACGAGGGAACCATGGCCGCCAAGACCTATGACGC 1080 S /D HAAKTTDA cbbL ->
CGGTGTCAAAGACTACCGCTCGATCTATTGGGAACCGCAATATCAGGTGAAGGACAGCGA 1140 OVKDTRSIYWBPQYQVKDSD
CATTCTGGCCGTCTTCAAGGTGGTGCCGCAGCCGGGCGTCAGCCGCGAGGAAGCCGCCGC 1200 ILAVFKVVPQPOVSRBEAAA
CGCCGTCGCCGCCGAAAGCTCCACCGCGACCTGGACGACGGTCTGGACCGACCTTCTGAC 1260 AVAAESSTATWTTVWTDLIiT
CGATCTCGATTACTACAAGGGCCGCGCCTATGCGATCGAGGATGTCCCCGGCAGCGACGA 1320 DLDYYKORAYAIEDVPOSDE
GGCCTTTTATGCCTTCATCGCCTATCCGATGGACCTGTTCGAGGAAGGCTCGGTCGTCAA 1380 AFYAFIAYPHDIiFEEGSVVN
CGTCTTCACCTCGCTCGTCGGAAACGTCTTTGGTTTCAAGGCGGTGCGGGCGCTGCGGCT 1440 VFTSI.VONVFGFKAVRALRL
GGAAGACGTGCGGTTTCCGCTCTGGTTCGTGATGACCTGCCCCGGGGCGCCGCACGGGAT 1500 EDVRFPLWFVMTCPOAPHGM
GAAGGTCGAGCGCGACCTTCTGGACAAATACGGCCGCCCGCTGCTGGGCTGCACGATCAA 1560 KVBRDLLDKYGRPLLGCTIK
GCCGAAACTGGGTCTTGCGGCCAAGAACTACGGCCGGGCGGTCTATGAATGCCTGCGCGG 1620 PKLGIiAARNYGRAVYECLRG
CGGGCTGGATTTCACCAAGGATGACGAGAACGTCAACAGCCAGCCTTTCCTGCGCTGGCG 1680 GIiDFTKDDENVNSQPFliRWR
CGACCGCTTCCTGTTCTGTCAGGAGGCGATCCAGAAGGCCGAGGCCGAAACCGGCGAGCG 1740 DRFZ.FCQEAIQKAEAETGER
CAAGGGCCATTACATGAACGTCACCGCCGGGACGATGGAGGAGATCTACGAACGCGCGGA 1800 KGHYHNVTAaTHEEIYERAE
ATTCGCCAAGGAAATCGGCACGCCGATCATCATGTCGGATTATCTGACCGTCGGCTGGGC 1860 FAKEIGTPIZHSDYIiTVGWA
(continued) 43 F i g . 2.2. (continued)
GGCGCATACGAGCCTGTCGCGCTGGTGCCGCAAGAACGGGATGCTGCTGCATGTCCACCG 1920 AHTSLSRWCRKHGMX.LHVRR
CGCCATGCATGCGGTGATGGACCGCAACCCCAATCACGGCATCAACTTCCGGGTGCTGGC 1980 AMHAVMDRNPHBGINFRVZiA
GAAGATCCTGCGGCTGATGGGGGGCGATCACCTGCATTCGGGCACCGTCGTCGGCAAGCT 2040 KILRLMGGDBI.HSGTVVGRI.
CGAGGGCGACCGCGAGGCGACGATCGGCTGGATCAACCTTCTGCGCGATCGCTTCATCAA 2100 EGDREATIGWINZiLRDRFIK
GGCCGACCGGTCGCGGGGGATCTTTTTCGATCAGGACTGGGGGCCGCAGCCGGGGCTGTT 2160 ADRSRGIFFDQDWGPQPGI.F
CCCGGTGGCTTCGGGCGGCATCCATGTCTGGCACATGCCGGCGCTGGTCTCGATCTTCGG 2220 PVASGGIBVWBHPALVSIFG
CAATGACTCGGTGCTGCAATTCGGCGGCGGCACGCTGGGCCACCCCTGGGGCAATGCCGC 2280 NDSVZiQFGGGTLGBPWGNAA
GGGCGCCTGTGCGAACCGGGTGGCGCTGGAGGCCTGCGTGCAGGCCCGCAACGAGGGCCG 2340 GACANRVALEACVQARNEGR
CCATCTGGAAAAGGAAGGCAAGGAGATCCTGACCAAGGCCGCGCAATCGAGCCCCGAGCT 2400 BIiEREGREILTKAAQSSPEL
GCGCATGGCGATGGAGACCTGGAAAGAGATCAAGTTCGAATTCGACACCGTCGACAAGCT 2460 RMAHETWKEIKFEFDTVDRI.
CGACGTGCAGCACCGCTGAGACCGGCCCGAAAGGACATCAGATGAGCACCGTTCAGGACT 2520 D V Q B R * S/D M S T V Q D ebbs ->
ACCCCTCCCGCCTCTCGCGCCCGGAAAGCCGCAAGATGGGCACCTTTTCCTATCTGCCGC 2580 YPSRL8RPE8RKMGTFSYLP
CGATGGGCGAGGCCGAGATCCGTCGTCAGGTCGAGTGGATCGTGAAGAACGGCTGGAACC 2640 PHGEAEIRRQVEWIVKNGWN
CGGGGATCGAACATACCGAACCCGACTTCGCCGCGCAGATCTATTGGTACATGTGGAAAC 2700 PGIEBTEPDFAAQIYW7HWK
(continued) 44 F i g . 2.2. (continued)
TGCCGATGTTCGGCGAAACCGATGTCGATGCGATCCTGGCCGAGCTGAAAGCCTGTCACG 2760 LPHFOBTDVDAILAELKACH
AAGCGAACCCCGGCCATCATGTCCGGCTGATCGGCTATGACAATTTCACCCAAAGCCAGG 2820 EANPGHHVRLIGYDNFTQSQ
GCGCCAACATGATCGTCTATCGCGGCACCGCCGTCTGAGCCGACACCCTGCCCGCCGCCC 2880 GANMIVYRGTAV* *'
+< . . . +<— CTCCCCTCCTGGCGGCGGTTCACGGCCCGCCGCGACCTTCCTTGCGGCGGGCCCCTTTCC 2940 4------► 4------>+ ..... CCATCCACGAGGCTGTCATGACCGATCCCACCCAGCCTTTCCGCATTCCGGCCGAGCCCT 3000 S/D HTDPTQPFRIPAEP cbbQ ->
GGTATCGCCCGGTCGCCGATGAAATCGCGCTGTTCGAGGCCGCCCATGCGGCGCGGATGC 3060 WYRPVADEIALFEAAHAARH
CGGTGATGCTCAAGGGCCCGACCGGCTGCGGCAAGACCCGTTTCGTCGAGCACATGGCCT 3120 PVHLKGPTGCGKTRFVEHMA
GGCGGCTGGGCAAGCCGCTCGTCACCGTCGCCTGCAACGAGGACATGACCGCCTCGGATC 3180 WRIiGKPIiVTVACNEDMTASD
TGGTCGGGCGCTTCCTGCTGGATGCCACGGGCACGCGCTGGCAGGACGGGCCCTTGACCT 3240 LVGRFLIiDATGTRWQDGPIiT
TTGCCGCGCGGCATGGCGCGATTTGCTATCTGGACGAAGTGGTCGAGGCGCGGCAGGACA 3300 FAARHGAICYLDEVVEARQD
CGACCATCGCCATCCACCCGCTGACCGACAATCGCCGCGTGCTGCCGCTTGAAAAGAAAG 3360 TTIAXHPLTDNRRVLPLEKK
GCGAATTGCTGCGCGCCCACCCGGATTTCCAGCTCGTGATCAGCTATAACCCCGGCTATC 3420 GELLRAHPDFQLVI SYNPGY
AGGCGCTGATGAAGGATCTCAAGCAATCGACGAAGCAACGCTTCGGCGCGCTCGATTTCA 3480 QALMRDLKQSTKQRFGALDF
CCTGGCCCGAGCATGGCGTCGAGGTCGAGATCGTCGCCCATGAAACCGGCATCGACCCGG 3540 TWPEHGVEVEIVAHETGIDP
CCTTGGCGCAAAAGCTGGTGGCGATCGCGGAACGGGCGCGCAACCTCAAGGGGCACGGCC 3600 ALAQKLVAIAERARNZ.KGHG (continued) 45 F i g . 2.2. (continued)
TTGACGAGGGGATTTCGACGCGGATGCTGGTGCATGCGGGCGGGCTGATCGCCCAGGGCG 3660 LDEGISTRMLVHAGGLIAQQ
TGGCGCCGCTGGCCGCCTGCCGGATGGCGCTGGTGCGCCCGATCACCGACGATCCCGACA 3720 VAPLAACRMAXiVRPITDDPO
TGCGCGACGCGCTTGACGCCGCCGTCACCACCTATTTCTGAGGCCCGCCATGTCCGCCCC 3780 MRDAZ.DAAVTTYF*
GCTTGCCCCCGAGCCCCTGCCCGATGCGGCGCAGGACGCCCTGACGCGCCTCGCCCCCGA 3840
GGCGGCGCGGGCGCTTTCGGCGCAGGGCCGCGCGGTCTGGCACGAGGGCGCGGCGGCGCT 3900 - '■ —' ► 4 -----
TTCCCGCAGCGGCAAGGGCGCCGAGGCGGTGCAGGCCTGGGCCGAGGCGGCCCTGCCCGT 3960
CGCCCGCGATCTGGGGGAAGACGTTCTGCCGGA 3993
46 ferrooxidans, but only 42.7 % identity with R. capsulatus CbbRn, and 37.2 % identity
withR. sphaeroides CbbR.
R. capsulatus cbbL starts at position 1058 and is preceded by a ribosome binding
site 5 nucleotides upstream of the ATG start codon (Fig. 2.2). A T-Nn-A motif was
identified as a conunon feature of the binding site for LysR-type transcriptional activators
(Goethals et al., 1992). This motif usually has a 3-bp inverted repeat at each end and is
often repeated 2 or 3 times upstream of the regulated gene or operon. Three such T-Nn-
A motifs are present in the cbbRrcbbL intergenic region (Fig. 2.2). The cbbL gene is
1422 nucleotides in length and encodes a protein of 473 amino acids with a predicted
molecular weight of 53,036. The highest sequence identity (86.0 %) was found with the
CbbL of Pseudomonas hydrogenothermophila. Significantly less sequence identity
(57.8 %) was noted when the R. capsulatus CbbL was compared to the R. sphaeroides
RubisCO large subunit. This is particularly interesting in light of the earlier reports that
the R. capsulatus form I RubisCO was inununologically distinct from that of
R. sphaeroides (Gibson and Tabita, 1977c; Paoli et ai, 1995).
ThecbbL sequence was obtained from subclones of pRCFI (Fig. 2.1, Table 2.1), a
cosmid clone from a R. capsulatus SB 1003 chromosomal DNA library (Scolnik and
Haselkom, 1984). Because of the unusual sequence of the cbbL gene, the sequence was
confirmed by sequencing of clones from two additional sources. Portions of the cbbL
gene present on the R. capsulatus SB 1003 chromosomal DNA fragment in plasmid
pEULA4 (Fig. 2.1, Table 2.1) and a subclone from a cosmid clone obtained from an
R. capsulatus SB 1003 overlapping cosmid library (Fonstein et al, 1993) were sequenced.
47 The sequence derived from these clones was identical to the sequence derived from the
pRCFI subclones.
An ORF encoding the R. capsulatus form I RubisCO small subunit starts at an
ATG codon 22 nucleotides downstream of the cbbL stop codon (Fig. 2.2). A potential
ribosome binding site is present 6 nucleotides upstream of the cbbS start codon. The
R. capsulatus CbbS is predicted to be 118 amino acids in length with a molecular weight
of 13,548. The highest sequence identity, 67.8 %, was to the Thiobacillus denitrificans
CbbS. The P. hydrogenothermophila CbbS was 60.7 % identical and the R. sphaeroides shared only 33.0 % identity. Two inverted repeat sequences that might function in transcriptional termination are present downstream of cbbS (Fig. 2.2).
Downstream of cbbS is an ORF predicted to encode a protein of 267 amino acids with a molecular weight of 29,429. The putative ATG codon at position 2958 is 100 nucleotides downstream of the cbbS stop codon and preceded by a plausible ribosome binding site (Fig. 2.2). The deduced amino acid sequence of this ORF is 72.0 % identical to CbbQ of P. hydrogenothermophila and 52.3 % identical to P. aeruginosa NirQ.
Alignment of the predicted amino acid sequence of the R. capsulatus CbbQ with theP. hydrogenothermophila CbbQ and P. aeruginosa NirQ (Fig. 2.3) revealed several regions of sequence identity, including a putative ATP-binding domain. Two T-Nn-A LysR binding motifs are present within the cbbS-cbbQ intergenic region (Fig. 2.2).
Phylogenetic analysis of the R. capsulatus form I RubisCO large subunit
The unusual properties of R. capsulatus form I RubisCO, including the unexpected
48 Fig. 23. CbbQ amino acid sequence alignment. Amino acid alignment of the presumptive CbbQ of R. capsulatus (Reap) compared to CbbQ of P. hydrogenothermophila (Phyd) and the P. aeruginosa NirQ (Paer). The putative p-loop nucleotide binding motif [(G/A)X 4GK(S/T)] (Walker et al, 1982) is indicated by the dark bar over the alignment.
49 Reap MTDPTQPFRIPAEPWYRPVADEIALFEAAHAARMPVMLKGPTGCGKTRFVEHMAWRLGKPLVTVACNEDMTASDL 7 5 Phyd -MDLRNQYLVRSEPYYHAVGDEIERFEAAYANRIPMMLKGPTGCGKSRFVEYMAWKLGKPLITVACNEDMTAADL 7 4 Paer ------MRDATPFYEATGHEIEVFERAWRHGLPVLLKGPTGCGKTRFVQYMARRLELPLYSVACHDDLGAADL 6 7 * * * * ★★ ★ * * * * * * * * * * * * * * * * * * * * * * * * * • ••• • • • ••••• Rc ap VGRFLLDATGTRWQDGPLTFAARHGAICYLDEWEARQDTTIAIHPLTDNRRVLPLEKKGELLRAHPDFQLVISY 150 Phyd VGRFLLDKEGTRWQDGPLTTAARIGAICYLDEWEARQDTTWIHPLTDHRRILPLDKKGEWEAHPDFQIVISY 149 Paer LGRHLIGADGTWWQDGPLTRAVREGGICYLDEWEARQDTTVAIHPLADDRRELYLERTGETLQAPPSFMLWSY 142 * * * ** ******* * * * *************** * * * * * * * * * * * * * * * * * % Reap NPGYQALMKDLKQSTKQRFGALDFTWPEHGVEVEIVAHETGIDPALAQKLVAIAERARNLKGHGLDEGISTRMLV 225 Phyd NPGYQSAMKDLKTSTKQRFAAMDFDYPAPEVESEIVAHESGVDAATAKKLVEVAIRSRHLKGHGLDEGISTRLLV 224 Paer NPGYQNLLKGLKPSTRQRFVALRFDYPAAQQEARILVGESGCAETLAQRLVQLGQALRRLEQHDLEEVASTRLLI 217 ***** * * * * * * * * * * * * * * * * * * * * * * * * * * * • • • •••••• • , , Reap HAGGLIAQGVAPLAACRMALVRPITDDPDMRDALDAAVTTYF- 267 Phyd YAGSLITKGIAPLIACEMALICPITDDPDLRYALRAAAQTLFA 267 Paer FAARLIGDGMDPREACRVALAEPLSDDPATVAALMDIVDLHVA 260 * * * * * * * * * * * * * * *
Figure 2.3. immunological properties, similarity of its sequence to that of P. hydrogenothermophila
CbbL, and its relative dissimilarity to R. sphaeroides CbbL, prompted a phylogenetic analysis of the R. capsulatus CbbL. Phylogenetic analysis of a broad range of RubisCO large subunits was conducted using the neighbor-joining method (Fig. 2.4). The tree is modified from the one presented by Watson and Tabita (1997) by the exclusion of the
Symbiodinium sp. RbcG and the addition of the R. capsulatus, Porphyra purpurea, and
Odontella sinensis form I RubisCO large subunit sequences. Both form I and form II
RubisCO were included in the analysis and the form I RubisCO “red-like” and “green like” classification of Delwiche and Palmer (1996) is indicated. Despite the close phylogenetic relationship between R. capsulatus and R. sphaeroides on the basis of 16S rRNA sequences (Woese et al, 1984) and the phylogenetic relatedness of the R capsulatus and R. sphaeroides form H RubisCO large subunits (Fig. 2.4), the RubisCO phylogenetic tree indicates that the form I RubisCO large subunits from these two organisms are not closely related. R. capsulatus CbbL is within a “green-like” radiation containing species of o/p/y purple bacteria and cyanobacterial species. The R. capsulatus sequence shares a branch with the sequence fromP. hydrogenothermophila, the most similar protein on the basis of comparisons of deduced amino acid sequences. In contrast, R. sphaeroides CbbL is within the “red-like” radiation. Bootstrap values are indicated and strongly support the tree topology. Trees with similar topology were generated by maximum parsimony and maximum likelihood analysis.
Phylogenetic analysis of the ChhR class of LysR-type transcriptional activators. Like the R. capsulatus form I RubisCO large subunit, the CbbRj sequence
51 Fig. 2.4. RubisCO large subunit phylogenetic tree. Amino acid sequence alignment was performed using Clustal W. The 412 amino acid alignment corresponds to the sequence of the R. capsulatus CbbL from W59 to T454. The tree was generated by neighbor- joining (Kimura distances) using the Protdist and Neighbor programs of Phylip 3.5.
Bootstrap values were calculated from 1000 replicates of the Seqboot, Protdist, Neighbor, and Consense programs of Phylip 3.5., and are indicated at the nodes as percentages.
This tree is unrooted. (*, this branch did not occur in the majority rule consensus tree)
52 "Green-like" R. capsulatus Form I R hydrogatothennophUa H .m ariniu 1
T.ferwoxuians PCC7120 N. vulgaris iynechococctts sp JCC7002 Vent Symbiont 'ynechococcus sp.PCC630I T. daiitrifieans R hollandica C vtnoium 1 Rrochlomn sp. H, marinus C paradoxa ' reinhardtii Synechococcus C. vinosum 2 1. T. aesttvum Cryptomonas 2. C reflexa 3 .1, purpurea _ Green Red& Porphyra purpurea Brown . 4. M tabacum Plastids Plasdds Cyiindrotheca sp. strain N l 5. M. verticillata Odontella sinensis 6. S. oleracea Olisthodiscus luteus
Vühoxidiziog bacterium O.I substitutions "Red-like" X .flavus per site R. eutropha ATCC17707 (cfarom.) R. eutropha ATCC 17699 (plasmid) R. eutropha ATCC 17699 (chrom.)
T. denitrificans H, ffuirinus
Canyaulax polyedra Symbiodinium rbcA Rs. rubrum R, capsulatu^xca R. sphaeroides Form n
Figure 2.4.
53 showed a significantly lower similarity to R. sphaeroides CbbR than to CbbR from more distantly related organisms. Thus, the possibility that R. capsulatus CbbRi has a similar phylogeny to the R. capsulatus form I RubisCO was investigated.
A phylogenetic analysis of 50 LysR-type transcriptional activators revealed that the CbbR-class of LysR-type proteins grouped together (not shown). The analysis of the
LysR proteins was carried out using the N-terminal region of the protein which includes the functionally conserved helix-tum-helix DNA binding domain. The region of the amino acid sequences used for tree building was a 145 residue sequence alignment generated by Clustal W and is represented by the gray bar in Fig. 2.5. Examination of the LysR multiple sequence alignment revealed no absolutely conserved residues and only four residues that were represented by conservative substitutions (not shown).
A multiple sequence alignment of the CbbR deduced amino acid sequences revealed additional sequence similarity within this class of LysR-type transcriptional activator. Most of the sequence similarity is limited to the helix-tum-helix motif and a second relatively conserved region of 36 residues corresponding to a previously recognized C-terminal domain (Schell, 1993) (Fig. 2.5). The C-terminal domain is thought to be involved in ligand binding or multimerization required for DNA binding
(Schell, 1993).
Fig. 2.6 shows the results of a neighbor-joining analysis of the CbbR proteins.
The tree is based on the Clustal W alignment of the N-terminal region represented by the gray bar in Fig. 2.5. Trees with identical topology were generated from an alignment of the complete CbbR sequences. Maximum parsimony and maximum likelihood analyses
54 Fig. 2.5. Multiple sequence alignment of CbbR conserved regions. The large dark bar represents the CbbR polypeptide. The gray bar indicates the region of the CbbR deduced amino acid multiple sequence alignment used for phylogenetic analysis. The aligned amino acid sequences of the two most conserved regions are shown. The sequences are labeled as follows: 6803 1, Synechocystis sp. 6803 RbcRl; 6803 2, Synechocystis sp.
6803 RbcR2; Rent, Ralstonia eutropha CbbR; Cpar, Cyanophora paradoxa
YCF30/RbcR; Cvin, Chromatium vinosum CbbR; Osin, Odontella sinensis
YCF30/RbcR; Ppur, Porphyra purpurea YCF30/RbcR; Reap I, Rhodobacter capsulatus
CbbRi; Reap H, Rhodobacter capsulatus CbbRn; Rhodospirillum rubrum CbbR; Rsph,
Rhodobacter capsulatus CbbR; Tfer, Thiobacillus ferrooxidans CbbR; Xfla,
Xanthobacter flavus CbbR.
55 Helix-Turn-helix 6803 1 SFTKAAEELFLTQPTVSQQMKQ 6803 2 SFTRAAEELYITQPTVSSQIKQ Reut SFVRAAEELHLTQPAVSMQVKQ Cpar SFKKAAESLYMTQPAISLQIQT Cvin SYTRAAEELHLSQPAVSMQVRQ Osin SFTRAAEVLFVSQPSLSKQIKT Ppur SFKKAANSLYVSQPAISLQIQN Reap I SYTRAAEELHLTQPAVFTQVKQ Reap II SLTAAGQALGLTTPAIHTQIKG Rrub SFSNAARELGLTQPAVSLQIKQ Rsph SLTGGATRLGLTPPAIHSQIRN Tfer SFARAAEELHLTPPALSIQVRQ X fla SYAKAAQDMGLSPPAVTAQMKA
% N-
6 8 0 3 1 MEISSNEAIKQAVYGGLGISILSLYSLALEGINGPL 6803 2 LELGSNEAIKQAIAGGMGISVLSQHTLVSEGARSEL R eut ITLGSNETIKQAVMAGMGISLLSLHTLGLELRTGEI Cpar MELNSIESIKNAVQSGLGAAFVSVSAXAKELELGIL C vin MQMTRNEAVKQAVRSGLGLSWSLHTIELELETRRL O sin MQLNSIEAIKTAVSLGLGAAFVSSSAIEKEIELKTI Ppur MELNSIEAIKNAVQSGLGAAFVSVSAIAKELELGIV Reap I MELGSNEAIKAAVAAGLGLAILSQDTITLEAETGRL Reap II MEMESNETIKQAVMAGLGIAFLSLHTVTEELGSGRL Rrub lEMSSNETIKQAVMAGMGISLLSRNTMSLELSVGRL Rsph lEMDSNETIKQSVIAGLGLAFLSLHWMDELRFGQL T f e r MEFGSNEAVKQSVAGGLGITVLSASTIRAELASGKL X f l a MEIGSNETIKQSVMAGLGLAFISAHTVAAEVADGRL
Conserved Region Figure 2.5. Fig. 2.6. CbbR phylogenetic tree. A 145 amino acid multiple sequence alignment
(corresponding to the region of the CbbR polypeptide indicated by the gray bar in Fig.
2.5., amino acids Q7 to G 148 of the R. capsulatus CbbRi) was generated by Clustal W.
The CbbR tree and bootstrap values were produced by the methods detailed in the legend to Fig. 2.4. The tree was rooted using R. capsulatus AmpR.
57 R. capsulatus I
51 T. ferrooxidans
70 Synechocystis sp. PCC6803 1 100 - Synechocystis sp. PCC6803 2 72 R. eutropha 69 R. rubrum
49 X. flavus 94 88 . R. capsulatus II
91 R. sphaeroides
O. sinensis Red 100 C. paradoxa Plastid 93 Encoded P. purpurea 0.1 substitutions per site
Figure 2.6.
58 generated trees of the same topology. The tree clearly shows the phylogenetic relationship between the R. capsulatus CbbRi and the T. ferrooxidans CbbR in addition to the more distant branching of the R. sphaeroides CbbR, which is itself closely related to
R. capsulatus CbbRn (Fig 2.6). The tree is in good agreement with the RubisCO phylogenetic tree (Fig. 2.4) in that the R. capsulatus CbbRi is on a branch with the CbbR from the “green-like” RubisCO-containing Thiobacillus ferrooxidans and the cyanobacterium Synechocystis sp. PCC6803.
It must be pointed out that the two CbbR homologues from Synechocystis sp.
PCC6803 and those encoded by the red plastid genomes are not transcribed divergently from ebb genes, and their designation as CbbR/RbcR proteins is based solely on the similarity of their deduced amino acid sequences to CbbR. The deep branching of the
Synechocystis RbcRs (Fig. 2.6) suggests that these proteins may well be CbbR homologues, despite the fact that neither is divergently transcribed from ebb genes. In contrast, the red plastid-encoded putative RbcRs form a separate branch in the CbbR tree.
As a matter of fact, the red plastid RbcR sequences grouped within the CbbR-family branch of the neighbor-joining and maximum likelihood LysR trees, but fell outside the
CbbR-family branch in a maximum parsimony LysR tree (not shown). Thus, the phylogenetic association of the red plastid RbcR proteins with the other CbbRs is tentative.
59 DISCUSSION
The nucleotide sequence of the genes encoding the R. capsulatus form I RubisCO was determined. Nucleotide sequencing upstream and downstream of the R. capsulatus cbbLcbbS revealed additional ebb genes (Figs. 2.1; 2.2).
The cbbR\ gene, upstream and divergently transcribed from cbbL, is the second cbbR found in R. capsulatus. The other cbbR, now designated cbbR^a, is upstream and divergently transcribed from the R. capsulatus cbba operon (Paoli et ai, 1995).
Thiobacillus denitrificans also has two cbbR genes (Lorbach and Shively, 1995). In
T. denitrificans cbbR\ and cbbRu are directly upstream and divergently transcribed from thecbbLcbbS and cbbM genes, respectively.
A single CbbR regulates the expression of the two ebb opérons in R. sphaeroides
(Gibson and Tabita, 1993) and the chromosomal and plasmid ebb opérons of Alcaligenes eutrophus (now called Ralstonia eutropha) (Windhovel and Bowien, 1991). In
Xanthobacter flavus, CbbR is required for the regulated expression of the ebb operon
(van den Bergh et al., 1993) and at least one other operon (Meijer et ai, 1996).
The presence of two CbbRs in R. capsulatus could allow independent regulation of the ebb opérons. If the CbbRs bind different inducer molecules, they could allow the expression of the two ebb opérons to be regulated by different environmental or metabolic signals. Thus, it will be interesting to determine the role of each CbbR in ebb gene expression in R. capsulatus.
60 A gene encoding an apparent CbbQ was found downstream of cbbS in
R. capsulatus. Likewise, the cbbQ gene is immediately downstream from cbbS in both
P. hydrogenothermophila and C. vinosum (Yokomada et al., 1995). CbbQ shares considerable amino acid homology with the NirQ protein of Pseudomonas spp.
(Yokomada et al. 1995; Fig. 2.3). The nirQ genes of Pseudomonas aeruginosa (Arai et ai, 1994) and Pseudomonas stutzeri (Jungst and Zumft, 1992) are within a cluster of genes encoding proteins required for nitrite respiration. A strain of Pseudomonas stutzeri in which nirQ had been disrupted lacked nitrite reductase or nitric oxide reductase activity despite the fact that the proteins were still synthesized, suggesting that
NirQ may be involved in post-translational assembly or modification of the nir gene products (Jungst and Zumft, 1992). A function has not yet been assigned to CbbQ, but preliminary investigations indicate that it may be involved in the post-translational activation of RubisCO (Igarashi and Kodama, 1996). The association of cbbQ and cbbLcbbS genes in R. capsulatus is very interesting. Because of the ease of constructing genetic null strains in this organism, it may be feasible to probe the function of CbbQ in greater depth than has been attempted thus far.
The assertion that theR. capsulatus form I RubisCO was acquired by horizontal gene transfer is not the first suggestion that such events have taken place. The incongruence of the RubisCO phylogenetic tree with that derived from the 16S rRNA phylogenetic tree necessitates at least 3 (now 4) horizontal transfers (Delwiche and
Palmer, 1996; Watson and Tabita, 1997). However, the evidence that R. capsulatus form
61 I RubisCO was acquired by such a mechanism is far more compelling than for any other presumed RubisCO gene transfer event
R. sphaeroides and R. capsulatus have been grouped in the same genus based on classical morphological and chemotaxonomic properties (Imhoff et a/., 1984). These two species are closely related on the basis of phylogenetic analysis of their 16S rRNA
(Woese et ai, 1984), 5S rRNA (Boulygina et ai, 1994), 23S rRNA (Ludwig et al, 1995) cytochrome c (Dickerson, 1980; Woese et ai, 1980), and reaction center L and M proteins (Blankenship, 1992). Even the amino acid sequences of the form II RubisCO of
R. capsulatus and R. sphaeroides are more than 94 % identical and show a close phylogenetic relatedness (Fig. 2.4). Nevertheless, the form I RubisCO proteins from these two organisms are very distantly related (Fig. 2.4).
The phylogenetic analysis of the R. capsulatus form I RubisCO is further supported by its unusual immunological properties. The enzyme cross-reacts poorly with antibody raised against R. sphaeroides form I RubisCO (Gibson and Tabita, 1977c; Paoli et ai, 1995) but cross-reacts well with antibody raised against the Synechococcus sp.
6301 RubisCO (Paoli et al, 1995). In addition, several years ago, Gibson, Whitman, and
Tabita (unpublished) examined cell extracts from several species of non-sulfur purple photosynthetic bacteria (Rhodocyclus tenuis, Rhodospirillum molischianum,
Rhodopseudomonas palustris, Rhodomicrobium vaniellii, Rhodobacter sphaeroides, and
Rhodobacter capsulatus). They found that antibody raised against the R. sphaeroides form I RubisCO cross-reacted with extracts from every species except R. capsulatus.
Thus, R. capsulatus may be unique among the non-sulfur purple photosynthetic bacteria
62 in having a “green-like” form I RubisCO. On the strength of this evidence, it would seem that theR. capsulatus form I RubisCO was acquired by a horizontal gene transfer from a “green-like” RubisCO-containing bacterial species.
Although the evidence is not as compelling, phylogenetic analysis of the CbbR deduced amino acid sequences (Fig. 2.6) suggests that the R. capsulatus CbbR; also may have been acquired from an organism containing a “green-like” RubisCO. In addition,
R. capsulatus RubisCO is most closely related to that of P. hydrogenothermophila (Fig.
2.4), which also has acbbQ gene directly downstream of its cbbS (Yokoyama et ai,
1995). The only other species known to have a cbbQ gene, also directly downstream of cbbS, is C. vinosum (Yokoyama et al., 1995). The C. vinosum RubisCO falls within the same “green-like” radiation as R. capsulatus and P. hydrogenothermophila (Fig 2.4.).
Thus, it seems likely that thecbbR\, cbbLcbbS, and cbbQ genes of R. capsulatus were acquired by a single gene transfer event from some as yet unidentified bacterial species whose RubisCO falls within the bacterial/cyanobacterial “green-like” RubisCO radiation
(Fig. 2.4.).
ACKNOWLEDGEMENTS
The author would like to thank Dr. Jessup Shively for coordinating the sequencing of the
R. capsulatus cbbLSQ, Dr. Gregory Watson for his considerable help with the phylogenetic analysis, and Drs. Mikle Fonstein and Robert Haselkom for providing the cbbL hybridizing cosmid plH2 from their R. capsulatus SB 1003 overlapping cosmid library.
63 CHAPTER 3
Studies of the Regulation of the ebb Structural Genes of Rhodobacter capsulatus
INTRODUCTION
Rhodobacter capsulatus is a purple nonsulfur photosynthetic bacterium that displays exceptional metabolic versatility (Hansen and Van Gemerden 1972; Weaver et ai, 1975; Madigan and Gest, 1979; Madigan, 1988). During photo- and chemoautotrophic growth, CO 2 , which is fixed by the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway, is the sole source of cellular carbon. When cells are grown photoheterotrophically CO 2 fixation is not obligatory, but functions primarily to help maintain cellular redox balance. Under these conditions, alternative electron acceptors such as DMSG can function in place of the CBB pathway (Richardson et ai,
1988; Falcone and Tabita, 1991; Wang et ai, 1993).
Phosphoribulokinase (PRK) and ribulose 1,5-bisphosphate carboxylase/oxygenase
(RubisCO) catalyze the only reactions unique to the CBB pathway, the ATP-dependent synthesis of ribulose 1,5-bisphosphate (RuBP) from ribulose 5-phosphate and the condensation of CO 2 to RuBP, respectively. The other enzymes in the CBB pathway carry out the phosphorylation and reduction of the primary CO 2 fixation product.
64 3-phosphoglycerate, and the regeneration of ribulose 5-phosphate. The net result is the synthesis of one molecule of triose phosphate for every three molecules of CO 2 fixed.
RubisCO exists in two distinct forms. Form I RubisCO is a complex enzyme composed of eight large and eight small subunits (LgSg). Form II RubisCO is composed of only large subunits (LO that are biochemically and genetically distinct from the form I
RubisCO large subunits (Tabita, 1995). R. capsulatus and the closely related organism
Rhodobacter sphaeroides synthesize both forms of RubisCO (Gibson and Tabita, 1977a;
1977c; 1985; Shively et al., 1984; Paoli et al, 1995). Studies conducted with
Rhodobacter sphaeroides indicate that, although either form of the enzyme can support autotrophic growth (Falcone and Tabita, 1991; this study. Chapter 4), form I and form II
RubisCO may fulfill different physiological functions in this organism (Wang et ai,
1993).
The organization and regulation of the genes encoding enzymes of the CBB pathway {ebb genes) have been extensively studied in R. sphaeroides. These genes are present in two separate opérons in R. sphaeroides. The form I operon contains the cbbF\, cbbPi, cbbA\, and cbbL\cbbS\ genes, encoding fructose 1,6/sedoheptulose 1,7- bisphosphatase (FBPase), phosphoribulokinase (PRK), fructose 1,6-bisphosphate aldolase
(FBP aldolase), and form I RubisCO, respectively. The form H operon contains the cbbF\i, cbbPa, cbbTn, cbbGa, cbbAn, and cbbMn genes, encoding FBPase, PRK, transketolase (TKL), glyceraldehyde 3-phosphate dehydrogenase, FBP aldolase, and form n RubisCO, respectively. A gene encoding a regulatory protein, CbbR, is present upstream and divergently transcribed from the R. sphaeroides cbb\ operon. CbbR is a
65 member of the LysR family of transcriptional activators and positively affects transcription of both the cbb\ and cbbu opérons in R. sphaeroides (Gibson and Tabita,
1993).
The organization of the ebb genes of R. capsulatus has recently been determined
(Paoli et ai, 1995). TheR. capsulatus form II RubisCO gene is associated with several other ebb genes that are organized in a manner identical to that of the R. sphaeroides ebbu operon (Paoli et ai, 1995, Larimer et ai, 1995) (Fig. 3.1 A). The arrangement and spacing of these genes suggests that the R. capsulatus cbbFPTGAM genes constitute an operon, as in R. sphaeroides. When thecbbM gene was sequenced by Larimer et al.
(1995), a cbbE gene, encoding pentose-5-phosphate 3-epimerase, was found downstream of cbbM. ThecbbE gene is probably not cotranscribed with the other cbbn genes (Larimer et al, 1995). Unlike the situation in R. sphaeroides, a gene encoding CbbR was found upstream and divergently transcribed from the cbbu operon of R. capsulatus.
No ebb genes encoding enzymes of the CBB pathway were associated with the
R. capsulatus cbbLcbbS genes. A cbbQ gene was found downstream of the R. capsulatus form I RubisCO genes. CbbQ is a protein of unknown function that is also found downstream of the cbbLcbbS genes of Pseudomonas hydrogenothermophila and
C. vinosum (Yokoyama et al, 1995). A second cbbR gene was found upstream and divergently transcribed from the R. capsulatus cbbLcbbS genes.
Transcriptional activation by CbbR is apparently a common mechanism of ebb gene regulation. Genes encoding CbbR transcriptional activators are immediately upstream and divergently transcribed from ebb opérons in several autotrophic bacteria
66 including Chromatium vinosum (Viale et al.. 1991), Ralstonia eutropha (formerly
Alcaligenes eutrophus) (Windhovel and Bowien, 1991), Xanthobacter fiavus (Meijer et
ai, 1991), Rhodobacter sphaeroides (Gibson and Tabita, 1993), Rhodospirillum rubrum
(Falcone and Tabita, 1993), Thiobacillus ferrooxidans (Kusano and Sugawara, 1993),
Thiobacillus denitrificans (Lorbach and Shively, 1995), and Rhodobacter capsulatus
(Paoli et ai, 1995). CbbR binds to the promoter of the ebb operon from which it is
divergently transcribed (Kusano and Sugawara, 1993: van den Bergh et al., 1993; Kusian
and Bowien, 1995) and a physiological role for CbbR has been established (Windhovel
and Bowien, 1991; Gibson and Tabita, 1993; van den Bergh etal., 1993).
The differences in the R. capsulatus and R. sphaeroides ebb gene organization, particularly the presence of two CbbRs in R. capsulatus, imply differences in ebb gene regulation in these two organisms. Notably, the R. capsulatus form I RubisCO is not expressed when cells are grown photoheterotrophically on malate (Shively et al, 1984;
Paoli et al., 1995; Fig. 1.5, lane 5) In addition, the R. capsulatus form I RubisCO is immunologically distinct from the R. sphaeroides form I enzyme (Gibson and Tabita,
1977c; Paoli et al., 1995) and appears to have been acquired by horizontal gene transfer
(This work. Chapter 2).
In this study, the nucleotide sequence of the R. capsulatus cbbRn, cifcFn, cbbP\i, and about 1.5 kb downstream of cbbRu was determined and R. capsulatus ebb gene regulation was examined using a combination of ebb gene disruption strains and ebb promoter fusions.
67 MATERIALS AND METHODS
Bacterial strains, plasmids, media, and growth conditions. Plasmids used or
constructed are listed in Table 3.1 and bacterial strains are listed in Table 3.2.
E. coli cultures were grown aerobically on LB medium (Ausubel et al., 1987) at
37°C. Aerobic cultures of R. sphaeroides were grown in PYE medium (Weaver and
Tabita, 1983) at 30°C. Photosynthetic cultures of R. sphaeroides and R. capsulatus were
grown anaerobically in front of a bank of incandescent lights at 30-33°C in Ormerod's
medium (Ormerod et a l, 1961) supplemented with 1 pg/ml thiamine, 1 pg/ml nicotinic
acid, and 0.1 pg/ml biotin. Photoautotrophic cultures were bubbled continuously with
1.5% CO2/H2 . Photoheterotrophic cultures contained 0.4 % DL-malate and were grown in filled screw cap tubes or continuously bubbled with argon. Chemoautotrophic growth was carried out in BEL GasPak jars (Becton Dickson Microbiology Systems,
Cockeysville, Maryland) under an atmosphere of C02/H2/air generated by an H 2/CO2 generating system without the palladium catalyst. Antibiotic concentrations used for
R. capsulatus strains were as follows: rifampicin (Rif), 100 pg/ml; kanamycin (Km), 5 jig/ml; spectinomycin (Sp), 10 pg/ml; tetracycline (Tc), 2 |ig/ml for plasmid maintenance, or 0.1 pg/ml for screening during gene disruption experiments. For E. coli, antibiotic concentrations were: Km, 30 pg/tnl; Sp, 50 |ig/ml; Tc, 12.5 pg/ml and; trimethoprim
(Tp), 200 pg/ml. Dimethylsulfoxide (DMSO) was used at 30 mM.
DNA manipulations. Routine DNA manipulations, including plasmid preparation, restriction endonuclease digestion, agarose gel electrophoresis, fragment ligation and bacterial transformation were performed by standard methods (Ausubel et al., 1987). R. capsulatus chromosomal DNA was prepared as described by Grimberg et al., 1989. For gene disruption experiments plasmid pJ5603 derivatives were conjugated into R. capsulatus SB 1003 using E. coli SI7-1 Xpir (Penfold and Pemberton,
68 Plasmid Relevant characteristics Source or reference
pK18, pK19 Km^, pUC derivatives Pridmore, 1987 pTZI8R Ap*^, pUC derivative Mead era/., 1986 pUC1318 Ap*^, pUC derivative with modified Kay and multiple cloning site McPherson, 1987 pUC1813 Ap^, pUC derivative with modified Kay and multiple cloning site McPherson, 1987 pRL648 Km*^, Ap^ pUC derivative with the Tn5 Elhai and Wolk, Km*^ gene cassette 1988 pRK415 Tc^, broad-host-range cloning vector, Keen et al, lacZa 1988 pRPS-l Tc**, broad host range expression vector containing R. rubrum cbbM Falcone and promoter and gene in pRK404 Tabita, 1991 pRPSKm Km** derivative of pRPS-1 Falcone and Tabita, unpub. pJP5603 Km**, mobilizable suicide vector Penfold and Pemberton, 1992 pTC5603 Tc** derivative of pJP5603 This work pHP450 Ap**,Sp**, containing O cassette encoding Prentki and spectinomycin resistance Krisch, 1984 pVKlOl Tc**,Km**, broad host-range vector Knauf and Nester, 1982 pUC1318K Ap**,Km**, pUC1318 with a 1.5 kb HirumSaK Meijer and Tabita, fragment from Tn5 encoding Km** 1992 pUC1813K Ap**,Km**, pUC1813 with a 1.5 kb Sati fragment from pUC1318K encoding the Tn5 Km** This work
Table 3.1. Plasmids used in this study (continued)
69 Table 3.1. (continued)
Plasmid Relevant characteristics Source or reference pXBAôOl Tc^, broad-host-range lacZ Adams et al, translational fusion vector 1989 pKlSFISN pKl8 containing the 3.2 kb SaK- Ncol fragment from pEULA4 such that the cbbL This work start codon is translationally fused to lacZa. pXLB pXBA601 containing the 3.2 kb BamYQ. fragment from pKIBFTSN, c b ^ translational fusion to lacZ This work pXLBP pXBAôOl containing the 367 Pstl-Bam¥l fragment from pKlBFISN, cbbi translational fusion to lacZ This work pKlBFnSN pK18 containing the 2.44 kb SaB-Ncol fragment from pK18FIIS4.4 such that cbbFu This work start codon is translationally fused to lacZxi pXFB pXBA601 containing 722 bp BamYQ. fragment from pKlBFnSN, cbbu translational fusion to lacZ This work pKlBFIffiH pK18 containing the R. capsulatus cbbR\i, cbbF, cbbP, cbbT, cbbG, and cbbA' genes on an 8.4 kb EcoBl-HindSR This work fragment from pRCMl pRKFHEH pRK415 containing the EcoRI-if/ndlH fragment from pKIBFDEH This work pRKFIP pRK415 with a 9 kbPstl fragment containing the R. capsulatus cbbLcbbS genes Paoli eta/., 1995 pUC1813::FIB pUC1813 containing the R. capsulatus cbbLcbbS genes on a 4.7 kb BomHI This work fragment from pRKFIP pUCl813::FK2 pUC1318::FTB with a EcoRI A and a Sp^ cartridge inserted This work pJP::FK2 pJP5603 containing the BamHl insert of pUC1813::FK2 This work pK18FnS2-I pK18 containing the R. capsulatus cbbM gene on a 2 kb Sail fragment Paoli era/., 1995 (continued)
70 Table 3.1. (continued)
Plasmid Relevant characteristics Source or reference p u c i s i s r a pUC1318 containing the R. capsulatus cbbM gene cloned on a 2 kb SaU. fragment This work pUC1318::FUKm pUC1318Fn with theHindJE A and a Km^ cartridge inserted This work pTC::FUKm pTC5603 with theSaU fragment of pUC1318::FUKm This work pK18FIIB2.3 pK18 with a 2.3 kbBaniHl fragment containing theR. capsulatus cbbR\i, cbbFu, and cbbPn Paoli era/., 1995 pK18FIIBSm pK18 containing part of the R. capsulatus cbbP on a 543 bp BamHl-Smal fragment This work pK18CBBPO pK18FilBSm with the Sp^ cartridge cloned into the SaB site of cbbP This work pJP::CBBPn pJP5603 containing the cbbPv.Q disruption of pK18CBBPQ cloned as This work a /findHI-EcoRI fragment pK18FnS4.4 pK18 with a 4.4 kbSaB fragment containing the 5’-end of the R. capsulatus cbbu cluster Paoli era/., 1995 pTZ::FE3.7 pTZ18R containing the R. capsulatus cbbR\i, and cbbFn genes on a 3.7 kb SaB-Smal This work fragment from pK18FilFH pTZr.CBBRKm pTZ::FII3.7 with a Km^ gene cartridge cloned into the BamHI site This work of cbbRjx pTCTZ:: pTZ::CBBRKm cloned into vector This work CBBRKm pTC5603 by linearizing with Xbal pJN940A theR. sphaeroides cbbPi gene cloned Novak and as an Aval fragment into pK18 Tabita, unpub. pUC1813::RsPi pUC1813 containing the R. sphaeroides cbbP\ gene cloned as a 1 kb Aval fragment This work from plasmid pJN940A (continued)
71 Table 3.1. (continued)
Plasmid Relevant characteristics Source or reference pRPS::Rs?iA pRPSKm containing the R. sphaeroides cbbP\ gene cloned from pUC1813::Rs?i as an This work Xbal fragment pRPS::Rs?iB pRsPiA containing the Xbal insert in the opposite orientation This work pJG106 pVK102 containing the R. sphaeroides cbbn Gibson and operon within a 26 kb insert Tabita, 1987 pJG336 pVK102 containing the R. sphaeroides cbbRi gene and the cbb\ and cbbXYZ opérons within Gibson and a 24 kb insert Tabita, 1987 pUC1813S4.4 pUC1813 containing the R. capsulatus cbbRn, cbbF, and cbbP' genes on a This work 4.4 kb SaU fragment pVK:;CBBRn the 4.4 kb SaK fragment containing the R. capsulatus cbbR[y cbbF, and cbbP' This work genes cloned into the Xhol site of pVKlOl pEULA4 4 kb EcoRl containing the R. capsulatus cbbL, cbbRi, anfA', and uncharacterized This work sequence between cbbRi and anfA' pULHSm the 361 bp HindHL-Smal fragment encoding the 5'-part of the This work R. capsulatus cbbR\ gene pULHSmMQ pULHSm with the 2 kb spectinomycin resistance Q fragment from pHP45A This work pTCHSmA pULHSm Q cloned into vector pTC5603 by linearizing with Kpnl This work
72 R. capsulatus Relevant characteristics Source or Strain reference
SB 1003 Rif*^ derivative of strain BIO Yen and Marrs, 1976
SBF c661::Sp^ derivative of SB 1003 This work
SBir cbbMv.Yixt^ derivative of SB 1003 This work
SBI-n cbbL::S^^, cbbMv.Y^ derivative of SB 1003 This work
SBF cbbPv.S^ derivative of SB 1003 This work
SBRn cbbR\^..Kx^^ derivative of SB 1003 This work
Table 3.2. Bacterial strains used in this study
73 1992). For complementation of mutant strains, plasmids were conjugated into
R. capsulatus by tri-parental matings on filter pads as previously described (Weaver and
Tabita, 1983) using the helper plasmid pRK2013 (Figurski and Helinski, 1979).
Southern blotting and hybridization. Southern transfer experiments were
performed using Gene-Screen Plus membranes (MEN, DuPont, Boston, Mass., USA) or
Hybond N+ (Amersham, Arlington Heights, Illinois, USA). Hybridizations were
conducted according to the protocols provided by the MEN, DuPont using formamide
under stringent conditions, except where noted otherwise. Probes were labeled with a-
[^■P]-dCTP (NEN, DuPont) by the random prime labeling method (Feinberg and
Vogelstein 1983) using a kit purchased from United States Biochemical Corporation
(Cleveland, Ohio, USA).
DNA sequencing and analysis. Nucleotide sequences were determined using an
ABI Prism 310 Genetic Analyzer. A Perkin Elmer Cetus thermal cycler and Dye
Terminator Cycle Sequencing Kit were used as described by the manufacturer (Perkin
Elmer, Foster City, Calif.). The M13/pUC forward 23-base sequencing primer
(5'-CCCAGTCACGACGTTGTAAAACG-3’), M13 reverse (-48) primer
(5'-AGCGGATAACAATTTCACACAGGA-3'), and sequence specific synthetic primers were used to complete the double stranded sequence. Sequence analysis was carried out using the University of Wisconsin Genetics Computing Group (GCG) Software, the
EGCG Extension Programs (The Sanger Centre, Hinxton, England) and the MacVector
Sequence Analysis Software (International Biotechnology, Inc., New Haven, Conn.).
Preparation of cell extracts, enzyme assays. Culture samples (20-30 ml) were generally taken in late log phase (A^eo = 0.9-1.2) and washed twice in cold buffer [100 mM Tris-HCl (pH 8.0) and 1 mM EDTA (pH 8.0)] before freezing at -70 °C. Thawed pellets were resuspended in 1 ml TEM buffer [50 mM Tris-HCl (pH 7.5), 1 mM EDTA
74 (pH 7.5), and 5 mM P-mercaptoethanol] and disrupted by sonication in an ice bath. Cell debris was removed by centifugation for 10 min in a microcentrifuge at 4“C.
RubisCO activity was measured as RuBP-dependent *‘*CÜ 2 fixation into acid- stable 3-phosphoglycerate (Gibson et ai, 1991). PRK was assayed as previously described (Tabita, 1980) except that ribulose-5-phosphate was not added directly but generated from ribose-5-phosphate by the addition of 5 U of phosphoriboisomerase
(Sigma Chemical, St. Louis, Missouri).
P-galactosidase was measured by continuous assays in Z-buffer [SO mM sodium phosphate (pH 7.0), 10 mM KCl, 1 mM MgS 0 4 , and 50 mM P-mercaptoethanol] (Miller,
1972) containing 0.8 mg/ml o-nitrophenol P-D-galactopyranoside (ONPG). The production of o-nitrophenol from ONPG was measured by monitoring the increase in absorbance at 405 nm. P-galactosidase activities were calculated using the extinction coefficient of o-nitrophenol of 3.1 X 10^ cm^ / mmole (Wallenfels, 1962).
W estern immiinoblot analysis. Proteins were resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli ,1970). After
SDS-PAGE the proteins were transferred to PVDF-membranes (Immobilon-P, Millipore,
Bedford, Mass., USA) (Towbin et al., 1979) using a Bio-Rad Transblot semi-dry cell
(Bio-Rad, Hercules, Calif., USA). Blots were prewashed with Tris-buffered saline (TBS)
[100 mM Tris-HCl (pH 7.5) and 15 mM NaCl] containing 0.1 % Tween 20 (TTBS) before blocking with 2 % casein in TTBS for 3 or more h. Blots were incubated 1-2 h with the primary antibody, washed with TBS, developed using the Vistra ECF fluorescent detection system (Amersham Corporation, Buckinghamshire, England) and visualized with the Molecular Dynamics Storm 840 Imaging System (Molecular Dynamics,
Sunnyvale, Calif.).
75 Construction of mutant strains.
cbbU (strain SBI*). A 4.7 kb BamHL fragment containing the R. capsulatus cbbLcbbS genes was cloned from pRKFIP into pUC1813. The resulting plasmid,
pUC1813::FIB, lacked any EcoRI sites in the multiple cloning region so that the 639 bp
£coRI fragment within cbbL could be removed and replaced by the Sp resistance (Sp^)
gene from pHP45£2. The 6.5 kb BomHI fragment containing the disrupted gene was
moved from pUC1813::FIQ to pJP5603, resulting in plasmid pJP::FK2. Plasmid
pJP::FIQ was mobilized into R. capsulatus SB 1003 from E. coli S17-1 A,pir. Six of the
three hundred Sp^ exconjugants screened were sensitive to Km. Southern blot analysis
revealed that five of the six Sp^-Km sensitive (Km^) colonies resulted from double
recombination. One of these strains was used for subsequent experiments.
cbbbf (SBII ). The 2 kb SaH. fragment encoding the R. capsulatus cbbM gene
was cloned from plasmid pKI8FIIS2-I into plasmid pUC1318. The resulting construct,
pUC1318Fn, lacked HindIR sites within its multiple cloning region. In order to generate
a Km^ cassette with flanking Hi/idni sites, a 1.4 kb SaE fragment encoding the Tn5 Km^
gene was cloned from pUC1318K into plasmid pUC1813, generating pUC1813K. The
650 bp Hi/idin fragment within the cbbM gene in vector pUC1318FII was removed and
replaced by a TfmdlH fragment containing the Tn5 Km^ gene from plasmid pUC1813K.
The resulting cbbAf-deletion fragment was cloned as an Xbal fragment into plasmid pTC5603 yielding pTCr.FllKm. E. coli SI7-1 Xpir was used to mobilize pTC::FUKm
into R. capsulatus SB1(X)3. Three hundred Km^ clones were screened for sensitivity to
Tc. All of the exconjugants were sensitive to 2 pg/ml Tc, but only five clones were
sensitive to 0.1 pg/ml Tc. Due to the very low resistance to Tc, the three hundred clones
were examined for the loss of the pTC5603 by colony hybridization. The five Tc^ clones
did not hybridize to the pTC5603 probe, but the 295 Tc^ clones did hybridize to the
probe. Three of the five Tc^ clones were screened by Southern blot hybridization analysis
76 of chromosomal DNA and found to be the result of double recombination. One of these recombinants was used for subsequent experiments.
cbbL'cbbAT (SBI-II). In order to construct a strain lacking the genes for both forms of RubisCO, the cbbM deletion plasmid pTC.rMlKm was mobilized into R. capsulatus cbbL' strain SBF. Two hundred Km^ colonies were screened and 2 were
Tc^. Both of these Tc^ clones had lost the pTC5603 vector as determined by colony hybridization using pTC5603 as a probe. Southern blot analysis using cbbM and cbbL probes revealed that these strains were the result of double recombination, leaving behind a deletion within the chromosomal copy of cbbM, and that the cbbL gene deletion was still present.
cbbF (SBF). A 543 bp Smal-BamUÎ fragment encoding part of the R. capsulatus cbbP gene was cloned from pK18FUB2.3 into pK18, resulting in plasmid pK18::BSm. The cbbP gene was disrupted by cloning the Sp^ gene from pHP45D as a Pstl fragment into the unique Xhol site within thecbbP gene fragment in plasmid pK18::BSm, yielding plasmid pK18CBBPQ. The resulting disrupted gene fragment was cloned as a BamHl- Smal fragment into pJP5603 yielding plasmid pJP::CBBPQ. E. co/i S17-1 A.pir was used to mobilize plasmid pJP::CBBPQ into R. capsulatus SB 1003. Two hundred fifty Sp*^ exconjugants were screened and seven were sensitive to Km. One of these strains was screened by Southern blot hybridization analysis of the chromosomal DNA and found to be the result of a double recombination. This strain, SBP", was characterized further.
c^W?n*(SBRn’)- The 3.7 kb Sall-Smal fragment containing the R. capsulatus cbbRvi and cbbF genes was cloned from pK18FIIS4.4 into plasmid pTZ18R generating pTZ::FH3.7. Removal of the SaB-Smal fragment from the multiple cloning region of pTZ18R during the construction of pTZ::FII3.7 deleted the BamHl site. This allowed the disruption of the cbbRn gene in pTZ::FII3.7 by insertion of a BamHl fragment containing the Tn5 BCm*^ gene from plasmid pRL648 into the unique BamHl site within cbbR\[. The
77 resulting construct, pTZ::CBBRKm was linearized with Xbal and ligated to Xbal digested pTC5603 resulting in plasmid pTZTC::CBBRKm. This plasmid was mobilized into
R. capsulatus SB 1003 from E.coli S17-1 Xpir. Three hundred Km^ colonies were screened and 299 were sensitive to Tc. Hybridization of colony blots from the 300 Km*^ clones using a probe derived from the Tc^ region of pTC5603 (EcoRl to PvuII fragment of pBR322) revealed that only the single Tc^ clone contained the Tc^ gene.
Chromosomal DNA was prepared from three of the Tc^ and the Tc*^ clone. The Tc*^ clone was the result of a single recombination of pTZTC::CBBRKm into the SB 1003 chromosome and each of the three Tc^ clones was the result of double recombination.
One of the Km^-Tc^ double recombinants, strain SBRn’, was used in subsequent experiments.
cbbRi (SBRf). The 361 bp HinàSl-Smal fragment encoding the 5 -portion of the cbbR\ gene was cloned from pEULA4 into pK18, resulting in clone pULHSm. The Pstl site within the multiple cloning region of pK18 was removed in the construction of pULHSm, so that the pHP45Q Sp^ gene was cloned as a fragment into the unique
Pstl site within the cbbR\ gene fragment in pULHSm. The resulting construct, pULHSmA, was linearized with Kpnl and ligated to Kpnl-cut pTC5603 yielding pTCHSmQ. pTCHSmA was mobilized into R. capsulatus SB 1003 using E. coli S17-1.
One thousand two hundred fifty Sp*^ exconjugants were screened for sensitivity to Tc.
Only 4 Sp’^-Tc^ colonies were found, and none of these was the result of double recombination.
Construction of ebb translational fusions. The translational fusion vector pXBA601 (Adams et ai, 1989) was used for construction of the ebb promoter fusions to lacZ. pXBA601 requires that the fusion end of the promoter fragment be ligated to the unique BamHl site within this vector. For the construction of the cbbi translational fusion, the ends of a 3.2 kb SaE-Ncol fragment from pEULA4 were filled using the
78 Klenow fragment of DNA polymerase L The blunt ended fragment was cloned into the Srml site of pK18, yielding plasmid pKlSFISN. This resulted in an in-frame lacZ translational fusion to the cbbL ATG initiation codon that is within the Ncol recognition site. After screening for the proper orientation, the fusion was confirmed by sequencing.
A Pstl-BaniHl fragment and a BamHl fragment were cloned from pKlSFISN into pXBA601, resulting in constructs with 367 bp and 3.2 kb upstream of the cbbL initiation codon translationally fused to lacZ, pXLBP and pXLB, respectively. Inserts were detected by colony blot hybridization and the orientation of the insert in pXLB was determined by restriction enzyme digestion. The cbbn fusion was constructed by first filling the ends of the 2.44 kb SaB-Ncol fi-agment from plasmid pKFnS4.4 using the Klenow fragment of DNA polymerase I and ligating it to Smal cut pK18, yielding pK18FUSN. After screening for the orientation of the insert, the fusion was confirmed by nucleotide sequencing. This resulted in an in-frame LacZ translational fusion to the cbbF ATG initiation codon that is within the Ncol recognition site. A 722 bp BamHl fragment was subcloned from pK18FIISN into pXBA601. The presence of an insert was determined by colony blot hybridization and the orientation of the insert was determined by nucleotide sequencing. The resulting construct, pXFB, contained 722 bp upstream of cbbfn translationally fused to LacZ at the CbbFn start codon. Nucleotide sequence accession number. The nucleotide sequence accession number for the sequence presented in Fig. 3.1 is U87282.
79 RESULTS
Nucleotide sequence analysis and amino acid sequence comparisons. In a
previous study Southern hybridization analysis showed that several R. capsulatus ebb
genes are organized in a manner analogous to the cbbu operon of R. sphaeroides (Paoli et ai, 1995). Although the original convention for use of the subscript I or II was meant to
indicate the association of the ebb opérons of R. sphaeroides with chromosome I or chromosome H of this organism (Tabita et ai, 1992), thecbb\ and cbbu designations have also been used to refer to the association of these genes with form I RubisCO genes, cbbLS, or the form II RubisCO gene, cbbM. In accordance with the latter convention the
R. capsulatus genes associated with cbbM will be indicated by a subscript II (Fig. 3.1 A).
The DNA upstream of the presumptive R. capsulatus cbbu operon contains at least two regions of interest with respect to ebb gene regulation: the cbbRu gene, encoding a putative ebb transcriptional activator, and the cbbRxrcbbF intergenic region, containing the presumptive cbbu operon promoter. In order to better understand the regulation of the ebb genes in R. capsulatus and as a prelude to further studies, the nucleotide sequence of both strands of a 4537 bp region, from the SaU site 1.4 kb downstream of cbbRu to the 5'- end of the cbbTu, was determined (Fig. 3.1 A,B). In addition to the cbbRu, cbbFu, cbbPu genes, and part of the cbbTu gene previously identified in this region (Paoli et ai, 1995), database searches revealed one full-length open reading frame (ORF) and one partial
ORF downstream of cbbRu.
80 Fig. 3.1. Organization of the cbbxi operon region of R. capsulatus and sequence of the 5'-
end of the cbb^ operon and upstream region. (A) Map of R. capsulatus cbbu operon and
upstream region. Arrows indicate direction and size of potential transcripts. “
represent potential transcriptional terminator hairpin structures and “9 “ represents a
hairpin preceded by a RNase E recognition sequence. Restriction sites: B, BamHl, E,
EcoRI, H, Hmdni; S, SaU; Sm, Smal, and X, Xhol. The dark bar denotes the region that
was sequenced and is presented below. (B) Nucleotide sequence of the 5'-part of the cbbu operon and upstream region. The predicted amino acid sequence of pgm (partial), cbbFu, cbbPu, and cbbTu (partial) is given below the nucleotide sequence. For the qor
and cbbRu genes, which are transcribed in the opposite direction and from the DNA strand complementary to the one shown, the predicted amino acid sequence appears above the nucleotide sequence. The ORFs are identified above the start of each gene.
Putative ribosome binding sites are underlined and stop codons appear as asterisks. The
T-Nii-A potential CbbR binding sites are denoted as follows: "+" above the T or A and
"< >" above the short inverted repeats. Arrows underneath the sequence indicate inverted repeats which could form hairpin structures. The shaded sequence and inverted repeat between the cbbPu and cbbTu represent a potential RNase E cleavage site (Ehretsmann et al, 1992).
81 Ikb S SmXBiB
pgm qor cbbR^ cbbF„ cbbPa cbbT„ cbbG„ cbbA„ cbbMg cbbE
B
--—pgm -> ..... GTCGACGGCTCGATTTCCGCACATCAGGGCTTTCGCATCCTGTTCGAAGGTGGCTCGCGC 6 0 VD0SISAHQ0FRZLFE08SR
GCGGTCTTGCGTCTGTCCGGAACCGGCACCGAAGGCGCAACGCTGCGGGTCTATCTGGAG 120 AVIiRLSOTGTEaATLRVYLE
CGGTATGTCGCCGGACCGGAGGGCCTGACAGAAGATCCTCAGCACGCTTTGGCGCCGATC 180 RYVAGPEGLTEDPQBALAPI
ATCGCGGCGACAGAGGATCTTGTCGGGATCAAGGCCCGAACGGGACGGAAAGGCCCGGAT 240 lAATEDLVGIKARTGRKGPO
GTGATCACCTGACAATCAAAGGCGCCACTTCGAAGAGGTGGCGCCTTTGGTCTCACACAG 300 VIT* ------► 4------* P T GAAATCCTATAATCAGTATTATGTAAAATGCGGGTGCGATCCAGGACCCCTCACGGGGTC 360 ------+ 4------LITCGTTKRSELATHAAAVD AAAATCGTGCAGCCGGTGGTCTTTCGGCTTTCAAGCGCCGTATGGGCCGCGGCGACATCT 420
KLDYRQGIEVRITGKIiIIiDF TTCAGGTCATAGCGCTGACCGATCTCGACCCGGATCGTTCCCTTCAGGATCAGATCGAAC 480
MEASAKRLWGPQATBHFLTP ATTTCGGCGCTCGCCTTGCGCAGCCAGCCCGGTTGCGCCGTGTGATGAAACAGCGTCGGC 540
RTZ.RLSGRSIiDTIRFDI.APG CGTGTCAGACGCAAAGAGCCGCGGGAAAGATCGGTGATCCGAAAATCAAGCGCCGGTCCC 600
SSQGFSVLTGFTELVEVSKK GAGCTTTGCCCGAAACTGACCAGCGTTCCGAAAGTCTCCAGCACTTCGACCGATTTCTTC 660
Figure 3.1. (continued)
82 Fig. 3.1. (continued)
VTVAOVS DTVAKVGKOETLR ACCGTCACCGCGCCAACGGAATCGTAGACCGCCTTCACGCCCTTGCCCTCGGTCAGCCGC 720
MTEAVFDRTKTDIVAD7GHA ATCGTCTCGGCGACGAAATCTCTGGTCTTGTAATCGATCACCGCATCATAGCCATGCGCC 780
LALACKEATGATGLARVGKH AGCGCCAGCGCGCATTTCTCGGCCGTGCCCGCCGTGCCAAGCGCCCGCACGCCCTTGTGT 840
KLWQGALZiGVGGAAAHVLVC TTCAGCCATTGCCCGGCCAGCAGCCCGACACCGCCCGCCGCCGCATGCACCAGAACGCAG 900
DGAAAP7SHHILYBVTLGKL TCCCCCGCGGCGGCCGGATAGGAATGATGGATCAGGTAATGCACGGTCAGCCCCTTGAGC 960
MVAAAIEDSIGEPIPVLSEA ATCACCGCCGCCGCGATCTCGTCGCTGATCCCCTCCGGGATCGGCACCAGATCCTCGGCC 1020
AIVRHSAYAGNRVTYAVRQG GCAATCACCCGGTGGCTCGCATAGGCGCCATTGCGCACCGTATAGGCGACGCGCTGTCCC 1080
VPIiDVGPGVAEIVGAAEGGT ACCGGCAGATCGACGCCGGGACCGACCGCTTCGATCACCCCCGCAGCCTCGCCCCCGGTT 114 0
ILDAPVGWPYIiGTRFYVDIY ATCAGATCCGCAGGCACGCCCCAGGGATAAAGCCCGGTGCGAAAATAGACATCGATATAG 1 2 0 0
NIiGIATQRLLVEGPGPQPVD TTCAGCCCGATCGCGGTCTGCCGCAACAGCACTTCGCCCGGACCCGGCTGCGGCACATCG 1 260
RELLRFNEVGGPATVVMAYS CGCTCCAGCAGACGAAAATTCTCCACCCCGCCGGGAGCGGTCACCACCATCGCATAGCTC 1 320 < qor TNiiA TNiiA M +<-«<-»>->+ < SGRLALIAEWLREAVPRPVE CTGCCCCGAAGCGCCAGGATCGCTTCCCACAGCCGTTCGGCCACCGGCCTTGGCACCTCG 150 0 DERRVMFWNRVIPLGPASLE TCCTCGCGCCGCACCATGAACCAGTTGCGCACGATCGGCAGCCCCGGCGCCGAAAGCTCC 1 560 VLRGSGLEETVTBLSLFAIG ACCAGCCGCCCCGAGCCCAGCTCCTCGGTCACCGTATGCAGGCTGAGAAAGGCGATGCCC 1 620 (continued) 83 Fig. 3.1. (continued) LGAMVAQKITBNSBMEMTEF AGCCCCGCCATGACCGCCTGCTTGATCGTCTCGTTGCTCTCCATCTCCATCGTCTCGAAA 1680 ARGDAVRDIiWRBMLIRTOSO GCGCGTCCGTCCGCCACCCGGTCCAGCCAGCGCTCCATCAGGATCCGCGTCCCCGAGCCC 1740 PBRTLFVBDLLADASVGQGL GGTTCGCGCGTCAGGAACACCTCGTCCAGCAGCGCATCCGCACTGACCCCCTGCCCCAGC 1800 I.RBGPPVLI.THFHPGLPBAL AGCCGATGCCCCGGCGGCACGAGCAGCGTATGCGGATGCGGCCCCAGCGGCTCGGCCAGA 1860 VPPRRPPRGMIAIBFAGRBL ACCGGCGGGCGCCGCGGCGGCCGCCCCATGATCGCAATTTCAAAGGCGCCGCGTTCCAGC 1920 GBITTBRNGVRZ.AVBIDPHS CCCTCGATCGTGGTCTCGCGGTTGCCCACCCGCAGCGCCACCTCGATATCCGGGTGCGAA 1980 LSZiAKVLRPAFYKATSVVGL AGACTCAGGGCCTTGACCAGCCTTGGCGCGAAATATTTCGCCGTCGAGACCACGCCCAGC 2040 TVRGTRGRSZiAALDAGAQ SL GTCACCCGGCCGGTTTTCCCGCGTGATAGCGCTGCCAGATCGGCCCCGGCCTGCGACAAA 2100 ADBIRKAAALMAHGADTI.DS GCGTCTTCGATCCGCTTCGCCGCCGCCAGCATCGCATGACCGGCATCGGTCAGGTCCGAC 2160 gagdaarqllrvqvadbi . gr CCCGCGCCA t CGGCGGCCCGCTGCAAAAGACGCACTTGAACTGCGTCCTCCAAACCCTTG 2220 IQTHIAPTTLGLAQGAATLS ATTTGCGTATGAATTGCTGGCGTCGTCAGGCCAAGCGCCTGTCCCGCCGCAGTCAGCGAG 2280 GWKAVARLARLQRMTIGD LR CCCCATTTTGCAACCGCCCTCAGCGCGCGCAGTTGCTTCATCGTGATCCCGTCCAGCCGG 2340 < - c b b R x z TNiiA TNnA V M < + -< ------> -+ > <+------+> ACCATATTAAATTTTCCTGAAAGCGAGTTGCCGAATTTCAAATTTGCATGAAACCGCTTT 2400 S/D TNiiA cbbFjx .+< >+ . . . . CGGCGCAACGTCCGGCCAACCGAATGAGGAGGAGGCCATGGCGATCGAGCTTGAAGGTCT 2460 S/D MAZBLBGL CGGGCTTAGCCCGGAACTGGCGGATGTGATGACCCGGCTGGCGCGTGTGGGGGCGGATCT 2520 GLSPBLADVHTRLARVGADL (continued) 84 Fig. 3.1. (continued) GGCCCGCACCATTGCCCGCAACGGGGTGGAAACGGACCTGGCGGCGGGCGTCGGCACCAA 2 5 8 0 ARTIARHGVETDLAAGVaTM TGCCGGGGGCGATGGTCAAAAGGCGCTCGACGTGATGGCGGACGACGCCTTCCGCGAGGC 2 6 4 0 AGGDGQKALDVHADDAFREA GCTGACGGGCACCGCCGTCGCTTATTATGCTTCCGAAGAACAGGACGAAGTGGTGACGCT 2 7 0 0 LTGTAVAYYASEEQDEVVTL GGGCAAAGGTACGCTCGCCTTGGCCATCGATCCGCTCGACGGCTCCTCGAACATCGATGT 2 7 6 0 GKGTLALAIDPLDGSSNIDV GAACGTGTCGATCGGCACGATCTTCTCGATCTTCCCGGCGACCGACGATCCGAACACCAG 2 8 2 0 NVSIGTIFSIFPATDDFMTS CTTCCTGCGCAAGGGCTCGGAACAGATCGCGGGCGGCTATATCATCTACGGGCCACAATG 2 8 8 0 FLRKGSEQIAGG7IIYGPQC CGCGCTGGTGTGCAGCTTCGGGCGCGGCGTGCATCACTGGGTGCTTGATCTCGACAGCCG 2 9 4 0 ALVCSFGRGVHHWVI.DI1 DSR CAGCTTCAAGCGCCTGCCCGACATCAAGGCATTGCCGCAAGACACGTCGGAATATGCGAT 3 0 0 0 SFKRLPDIKALPQDTSEYAI CAACGCCTCGAACTACCGGCATTGGCCCTCGCCGATCCGCGCCTTCATCGACGATCTGGT 3 0 6 0 NASNYREWPSPIRAFIDDLV GGCGGGCGCCGAGGGGCCGCGCGGGCGCAACTTCAACATGCGCTGGATCGCCTCGCTCGT 3 1 2 0 AGAEGPRGRNFNMRWIASLV TGCCGAAACCCACCGCATCCTGATGCGCGGCGGCGTGTTCCTCTATCCGGGCGACGAACG 3 1 8 0 AETHRILMRGGVFLYPGOER CAAGGGCTATGCCCGCGGCCGTCTGCGCCATGTCTATGAATGTGCGCCGATCGCCTTTCT 3 2 4 0 KGYARGRLRHVYECAPIAFL GATCACGCAAGTGGGCGGCGGCGCCACCGACGGCTGCGAGGACATCCTCTCCGCCCTGCC 3 3 0 0 XTQVGGGATDGCEDIIiSALP CGACAAGCTGCATGCGCGCACGCCCTTCGTTTTCGGCTGCGCGGCCAAGGTGGCCCGCGT 3 3 6 0 DKLHARTPFVFGCAAKVARV (continued) 85 Fig. 3.1. ( continued) CACCGCCTATCACGACCTGCCCGGGCAGGAGACCTCGGCACCCTTCAACACCCGCGGTCT 3420 TATHDLPGQSTSAPFNTRGL S /D . cbbPzx->- .... TTTCCGGAGCTGATCCCATGTCGAAAAAATACCCCATCATCTCTGTCGTCGGTTCCTCGG 3480 FRS* MSKKYPIISVVGSS GGGCTGGCACCTCGACCGTCAAGGCGACCTTCGACCAGATCTTCCGCCGCGAGGGGGTGA 3540 GAGTSTVKATFDQIFRREGV AAGCCGTCTCGATCGAGGGCGACGCCTTCCACCGGTTCAACCGCGCCGACATGAAGGCGG 3600 KAVSIEGDAFHRFNRADMKA AACTGGAACGCCGCTATGCCGCGGGCGATGCCACCTTCTCGCATTTCTCCTACGAAGCGÀ 3660 ELERRYAAGDATFSHFSYEA ATGCGCTCGAGGATCTGGAACGCGTCTTCCGCGAATATGGCGAAACCGGCAAGGGCCGCA 3720 NALEDLERVFREYGETGKGR CCCGGCGTTATGTCCACGACGCCAATGAATCGGCGAAATACGGGGTCGAGCCGGGCCATT 3780 TRRYVEDANESAKYGVEPGH TCACCGACTGGGCGCCGTTCGAGGAAGATACCGACCTGCTCTTCTACGAGGGCCTGCATG 3840 FTDWAPFEEDTDLLFYEGLH GCTGCGTGACGAATGATCAGGTCAACATCGCTGCCCATGCCGACCTGAAAATCGGTGTCG 3900 GCVTNDQVNIAAHADLKIGV TGCCGGTGATCAACCTTGAATGGATCCAGAAAATCCACCGTGACCGGGCGCAGCGCGGCT 3960 VPVINLEWIQKIHRDRAQRG ACACGACCGAGGCCGTCACCGACGTGATCCTGCGTCGCATGCATGCCTATGTGCACTGCA 4020 YTTEAVTDVII.RRMHAYVHC TCGTGCCGCAATTCTCGCAGACCGACATCAACTTCCAGCGCGTGCCGGTTGTCGACACGT 4080 IVPQFSQTDINFQRVPVVDT CGAACCCGTTCATCACCCGCTGGATCCCGACGCCGGACGAAAGCCTGATCGTGATCCGCT 4140 SNPFITRWIPTPDESIiIVIR TCCGCAACCCGCGCGGCATCGATTTCCCCTATCTGACCTCGATGATCCATGGGTCGTGGA 4200 FRNPRGIDFPYLTSHIHGSW (continued) 86 Fig. 3.1. ( continued) TGAGCCGGGCAAACTCGATCGTCATTCCGGGCAACAAGCAGGATCTGGCGATGCAGCTGA 4 2 6 0 MSRANSIVIPGHKQDLAHQI, TCCTGACGCCGCTGATCGAGCGTCTGGTGCGCGAAGGCCGTCGCGCGCGGGCCTGAACCG 4 3 2 0 ILTPLIERLVREGRRARA* GAAqjmiAAGACATTGGCGAGGCCCGGCCCACCGGGCCCGCCGCTCGGACGAAAGGC 4 3 8 0 ------► 4------chbTjx .-> . TCATCAGGGAGGAGGAGCCATGAAGGATCTCGATATGGCGCAGGAAACGCGGATGGCCAA 4 4 4 0 S /D MRDLDHAQETRHAN TGCCATCCGGGCGCTGGCGATGGATGCGGTGGAACAGGCGAAATCCGGGCATCCCGGCAT 4 5 0 0 AXRALAMDAVEQAKSGHPOM GCCGATGGGCATGGCCGATGTCGCCACCGTTCTTTTC 4537 PMGHADVATVLF 87 The 252 nucleotides at the 5'-end of the region sequenced encode an 83 amino acid partial ORF (Fig. 3.1 A and B) that is 56.6 % identical (74.7 % similar) to the C-terminal portion of Agrobacterium tumtfaciens phosphoglucomutase and 49.4 % identical (69.9 % similar) to the Human PGM *14- isoform of phosphoglucomutase (Fig. 3.2). Phosphoglucomutase, which is not a CEE pathway enzyme, catalyzes the interconversion of glucose-6-phosphate and glucose-1-phosphate for the biosynthesis of polysaccharides. The pgm gene had not been previously identified in R. capsulatus. An ORF encoding a 322 amino acid gene product with a predicted molecular weight of 34,288 was found 81 nucleotides downstream from cbbRa (Fig. 3.1 E). This ORF, which starts at an ATG codon at nucleotide 1322 and ends at a TGA stop codon at nucleotide 351, is preceded by an excellent ribosome binding site, AAGGA, 7 nucleotides upstream of the start codon (Fig 3.1 E). The presence of two T-Nn-A sequences between cbbRa^nd this ORF makes it possible that its expression is CbbR-activated (Fig. 3.1 E). Nevertheless, no potential transcriptional terminator was found downstream of cbbRu and these genes could also be cotranscribed. The deduced amino acid sequence of this ORF shared 47.7 and 43.3 % identity to the NAD(P)H quinone oxidoreductase (QOR) from Pseudomonas aeruginosa and E. coll, respectively. QOR from E. colt has been crystallized (Thom et al., 1995) and every residue involved in substrate binding or in the catalytic site is conserved in the R. capsulatus enzyme (Fig. 3.3). The AXXGXXG sequence (Fig. 3.3) is an unusual nucleotide binding fingerprint motif found only among the QORs (Thom et ai, 1995). QOR is a soluble enzyme that catalyzes the transfer of 88 Fig. 3.2. Alignment of the deduced amino acid sequence of the partial 5 -ORF with that of Agrobacterium tumefaciens (Uttaro and Ugalde, 1994) and human PGM *1+ phosphoglucomutase (Whitehouse et al, 1992) amino acid sequences. Invariant amino acids are indicated by asterisks and conservative changes by dots. In each case the threonine residue is the C-terminal residue. 89 R. capsulatus VD6SZSAHQGFRILFEG6SRAVLRLS6T6TEGATLRVYLERYVAGPEGLTEDPQHA1APZ (?) A. tumefaciens VDQSVSKNQGZRZLFEGGSRZVLRLSGTGTAGATLRLYVERYEPDAARHGZETQSALADL (516) Human VDGSZSRNQGLRLZFTDGSRZVFRLSGTGSAGATZRLYZDSYEKDVAKZNQDPQVNLAPL (539) JR. capsulatus ZSVADTZAGZKAHTJADSEPTVZT (?) A. tumefaciens ZAATEDLVGZKARTGRKGPDVZT (542) Human ZSZALKVSQLQERTGRTAPTVZT (562) * * * * Figure 3.2. Fig. 3.3. Amino acid sequence alignment of the R. capsulatus QOR with the QOR from E. coli (Lilley et ai, 1993) and P. aeruginosa (GenBank Accession # X85015). Black boxes represent invariant residues and shaded boxes are conservative substitutions. The AXXGXXG is an unusual nucleotide binding motif (Thom et al., 1995) found in this protein. Additional residues in contact with the bound NADPH ( • ) and those that are within the catalytic site (Q ) are indicated. 91 R. capsulatus 1 YXgVVTQP FRL^^DVPQ^QI Y R G L Y P E. coU 1 y Q r SGLYP P. aeruginosa 1 YRMQB Y RSGLYP R. capsulatus 60 VRN- E. coli 61 P. aeruginosa 61 AXXGXXG R. capsulatus 118 E. coli 119 P. aeruginosa 119 s R. capsulatus 178 E. coli 179 P. aeruginosa 179 R. capsulatus 238 FHHTAQIi E. coli 239 P. aeruginosa 239 R. capsulatus 296 E. coli 299 P. aeruginosa 298 Figure 3.3. electrons from reduced pyridine nucleotides, with preference for NADPH, to membrane bound quinones. Its structure differs significantly firom the NADH quinone oxidoreductase involved in the respiratory chain. The function of NAD(P)H quinone oxidoreductase in bacteria is not known. The R. capsulatus CbbRn ORF extends 942 nucleotides from an ATG codon at position 2345 to a TGA codon at position 1404 (Fig 3.1 B). The cbbRxi gene is preceded by a plausible ribosome binding site 9 bp upstream of the ATG start codon. CbbRn is 313 amino acids long with a predicted molecular weight of 33,697. The CbbR proteins comprise a family of LysR-type transcriptional activators that are involved in the regulation of ebb genes, from which they are usually divergently transcribed (Gibson and Tabita, 1996). A second cbbR gene, cbbRi, was found upstream and divergently transcribed from the R. capsulatus cbbLcbbS genes (this work. Chapter 2). The R. capsulatus CbbRn is most similar to the R. sphaeroides CbbR (55.2 % identity) and less similar to the R. capsulatus CbbR; (42.5 % identity). A phylogenetic analysis of the CbbR proteins (Fig. 2.6) confirmed that the R. capsulatus CbbRn is more closely related to the R. sphaeroides CbbR than to the R. capsulatus CbbRi. It will be interesting to determine if the phylogenetic relationships have a functional significance with respect to ebb gene regulation in R. capsulatus. Nucleotides 2438 to 3433 comprise an ORF that could encode a protein with a predicted molecular weight of 35,269 and amino acid sequence similarity to a number of bacterial fructose 1,6-bisphosphatases (FBPase). The R. capsulatus FBPase II is most similar to the FBPases from R. sphaeroides, sharing 84.0 % identity with R. sphaeroides 93 FBPase II and 66.8 % identity with to R. sphaeroides FBPase L The next most similar FBPase was that of Xanthobacter flavus (47.4 % identity). A ribosome binding site was found 5 nucleotides upstream of the cbbFn ATG start codon. It has been suggested, by analogy to the R. sphaeroides cbbn operon, that the R. capsulatus cbbF\i promoter may function for the entire cbbFPTGAM (chèn) operon (Paoli et al., 1995). Thus, it is interesting that three T-Nn-A, potential CbbR binding sites, are found upstream of cbbF^i. TheR. capsulatus cbbPn gene, encoding a putative phosphoribulokinase (PRK), is 879 nucleotides long, starting at an ATG codon at nucleotide 3438, only 4 nucleotides downstream of the cbbFn stop codon, and terminating at a TGA codon at position 4316. A potential ribosome binding site is present 8 nucleotides upstream of the cbbPn start codon and is within the cbbFu coding region (Fig. 3.1 B); thus, cbbFn and cbbP]i may be translationally coupled and are almost certainly cotranscribed. This arrangement is similar to that of R. sphaeroides cbbF\- cbbP\ (Gibson et ai, 1991) and the cbbFvr cbbPu (Gibson et ai, 1990). The predicted molecular weight, 33,244, is very similar to the subunit molecular weight determined for the purified R. capsulatus PRK (Tabita, 1980). The R. capsulatus PRK H is highly homologous to R. sphaeroides PRK I (86.2 % identity) and PRK H (87.0 identity) of R. sphaeroides. The next most similar PRK is that of X. flavus (68.3 % identity). The homology between the R. capsulatus PRK and the R. sphaeroides PRK I and PRKII is obvious when the sequences are aligned (Fig. 3.4) The domains involved in binding ATP (Kreiger and Miziorko, 1986; Kreiger et ai, 1987) and pyridine nucleotide(Gibson et al., 1990; Charlier et al, 1994) are indicated 94 Fig. 3.4. Phosphoribulokinase amino acid sequence alignment. The R. capsulatus PRK amino acid sequence is aligned with the amino acid sequence of R. sphaeroides PRK I and PRK H. Conservative substitutions appear as gray boxes and nonconserved changes are shown in black boxes. The putative ATP binding domain is indicated by asterisks and the pyridine nucleotide binding site is indicated by the dark bar. Residues implicated in the R. sphaeroides form I enzyme in sugar phosphate binding (#) and catalysis (^) are noted. 95 ******** _ A # # R. capsulatus 1 rFDQIFRREQVKAVSIEGDAFHRFNRADMKABXiBRRY R. sphaeroides I 1 PFDQIFRREOVKAV8IEOPAFHRFWRADMKAEI.lRRy R. sphaeroides H 1 ^QIFRREOVkIvSIEGDAFHRFNRADMKAEIiERRY R. capsulatus 61 ■ gi R. sphaeroides 1 61 AAGDATFSHFSyEANEZ.KEZiERVFREYGETGmRTRTYVHDDAEAARTGVAPGNFTDWQ3MG: R. sphaeroides II 61 i| g i R. capsulatus 121 FE^D B D 1.1. F YEGI.RGC vQndBVN I A fe AIADLKIGVVPVIHLEHIQRIHRDRAQRGYTTEAV R. sphaeroides I 121 BgDSaLLFYEGLHGQvVNgEVNIAggA: ! dx .k i g v v p v i n i .ehiqkihrdra Q r g y t t e a v s R. sphaeroides II 121 Î eBBsdi.i.fyeoi.hqcvvmpevmMb, EEa: a d l k | g v Q p v z n i .ewiqxihrdraqrgyttbav R. capsulatus 181 R. sphaeroides I 181 R. sphaeroides n 181 R. capsulatus 241 R. sphaeroides I 241 1 A -- R, sphaeroides II 2241 4 1 I dG p YI iTSMIQ g SWMSRANSIWPGNKQDLANQI iILTPLIE r | v R e ||PR A R A Figure 3.4. in Fig. 3.4. Residues implicated by site-directed mutagenesis of the R. sphaeroides form I PRK in sugar phosphate binding (Sandbaken et ai, 1992) and catalysis (Charlier et al.,. 1994) are also noted. The 46 N-terminal amino acids of the cbbT\i gene are encoded by the 138 nucleotides at the 3’-end of the sequenced region (Fig 3.1 AJB). Over this portion of the protein the R. capsulatus CbbTn is much more similar to the R. sphaeroides CbbTn (91.3% identity) than to the R. capsulatus TktA (69.6 % identical), a second transketolase in R. capsulatus (de Sury d’Aspremont et al, 1996) (Fig. 3.5). An inverted repeat (nucleotides 4344 through 4361) preceded by a sequence which matches a consensus RNase E cleavage site [(G/A)AUU(A/U)] (Ehretsmann et al, 1992) is present within the 83 nucleotide cbbPv-cbbTa intergenic region. (Fig. 3.1 B). The combination of a hairpin preceded by an RNase cleavage site has recently been shown to be sufficient for cleavage of the puf mRNA by an RNase E-like enzyme in R. capsulatus (Fritsch et a l, 1995). This potential RNase E cleavage site could function in cbb\i mRNA processing. Construction of R. capsulatus c66-minus strains. R. capsulatus strains with disruptions in cbbL, cbbM, cbbPu and cbbRu were constructed by mobilizing the appropriate pJP5603 derivative (see Materials and Methods) into strain SB 1003 from E. co/i S17-1 Xpir. Homologous recombination of the plasmid-bome disrupted gene into the wild-type copy in the chromosome is forced because pJP5603 will not replicate in R. capsulatus. 97 Rc CbbT (1) HKDUJM------AQETRMAIOaRAIAMDAVEQAKSGHPaHFMraiASVXTVU' (46) R8 CbbT (1 ) HKDIGA------AQETRMAHAIRAIJlHSXVEKAXSGHPaHPM(aiASVXTVI.F (46) Rc TktA (1 ) M-DZAALRAXTPDBWKIATAIR.VXJaDAVQAANSOaR(aiFMCafASVATVZiF (51) Fig. 3.5. Alignment of the R. capsulatus cbbTa partial deduced amino acid sequence with R. capsulatus TktA and /?. sphaeroides CbbTn- Invariant amino acids are indicated by asterisks and conservative changes by dots. 98 Recombinants were selected by aerobic growth on PYE plates supplemented with the antibiotic corresponding to the disrupting cassette. Rifampicin was added to the plates to select against the E. coli donor. Resistant clones were screened for sensitivity to the plasmid encoded antibiotic resistance marker to identify strains that may have undergone a second recombination event. Double recombinations were confirmed by Southern blotting and hybridization analysis of chromosomal DNA from the mutant and wild-type strains (Figs. 3.6B, 3.7 B,CJD). Strain SBF was constructed by incorporation of the cbbL::Sp^ deletion construct into the SB 1003 genome by a double recombination event. Due to the insertion of the 1.9 kb Sp^ cassette and deletion of the 639 bp EcoRL fragment of cbbL, a cbbL hybridizing restriction fragment from a cbbL::Sp^ double recombination strain would be expected to be about 1.3 kb larger than the corresponding fragment from the wild-type strain (Fig. 3.6 A). Chromosomal DNA fragments from the cbbL' strain SBF digested with eitherSail (Fig. 3.6 B, lane 2) or Pstl (Fig. 3.6 B, lane 4) were approximately 1.3 kb larger than the fragments from the wild-type strain (Fig. 3.6 B, lanes 1 and 3, respectively), confirming the double recombination in this strain. The c66M-hybridizing Sail fragment of the cbbM' strain SBIF was 700 bp larger than theSail fragment from SB 1003 (Fig. 3.7 D, lanes 1 and 2). This is due to the replacement of the 650 bp HindlH fragment of cbbM with the 1.4 kb Km*^ cassette (Fig. 3.7 A). Lanes 3 and 4 of Fig. 3.7 D show the loss of the 650 bp HindHl fragment and the presence of the wild-type fragments upstream (9.0 kb) and downstream (2.5 kb) of cbbM in strain SBIF. 99 Fig. 3.6. Insertional mutagenesis of the R. capsulatus cbbL and cbbR\ genes. (A) Organization of the cbbi genes and insertional inactivation of cbbL and cbbR\. The £coRI fragment within the cbbL gene was replaced by a spectinomycin resistance cassette. A spectinomycin resistance cassette was cloned into the Pstl of the cbbRi gene. Restriction sites: E, EcoRI, P, Pstl, S, SalL (B) Southern hybridization analysis of wild-type SB 1003 (lanes 1 and 3) and cbbL strain SBF (lanes 2 and 4) chromosomal DNA digested with Sail (lanes 1 and 2) and Pstl (lanes 3 and 4). The BamlH-Sall fragment from pKlSFIBS containing the R. capsulatus cbbL gene was used to synthesize the probe. Numbers in the margins refer to the sizes of the indicated fragments. 100 I kb ES UM anfA CbbR, cbbl^ \bbSi cbbQ R B ê 12 3 4 Figure 3.6. 101 Fig. 3.7. Insertional mutagenesis of R. capsulatus cbbu genes. (A) Organization of the cbbn operon region of R. capsulatus and insertional inactivation of cbbRu, cbbPn and cbbMa- ThecbbRa gene was disrupted by inserting a kanamycin resistance cassette at the BamHi site within cbbRa. A spectinomycin resistance cassette was cloned into the Xhol site within cbbP to disrupt this gene. The HindlR fragment within the cbbM gene was replaced by a kanamycin resistance cassette. (B) Southern hybridization analysis of wild- type SB 1003 (lane 1) and cbbRIT strain SBRIT (lane 2) chromosomal DNA digested with SaB. The 4.4 kbSaB fragment from pK18FIIS4.4 was used to synthesize the probe. (C) Southern hybridization analysis of wild-type SB 1003 (lanes 1 and 3) and cbbP' strain SBP (lanes 2 and 4) chromosomal DNA digested with HindIR (lanes 1 and 2) and BaniHl (lanes 3 and 4). The 543 bp BamHL fragment from pKlSFUBSm encoding part of the R. capsulatus cbbPa and cbbVa gene was used to synthesize the probe. (D) Southern hybridization analysis of wild-type SB 1(X)3 (lanes 1 and 3 ) and cbbM strain SBIT (lanes 2 and 4) chromosomal DNA digested with SaB (lanes 1 and 2) and HindIR (lanes 3 and 4). The 650 bp HindRl fragment from pK18FIIS2-I, containing part of the cbbM gene, was used to synthesize the probe. The sizes of the hybridizing fragments are indicated. 102 1 kb S X HSS m X B E H p g m qor cbhF, cbbT. cbbGn ebb An cqbMn\cbbE Km Km S 9.0 kb 9.0 kb 5.5 kb 5.2 kb 4^ 4.2 kb 3.5 kb 4.4 kb 2.7 kb ^ 2 .5 kb •4 2.3 kb 2.0 kb 0.65 kb 1 2 12 3 4 B Figure 3.7. A strain that was cbbL'cbbM', strain SBI H, was constructed by mating the cfcfeM-disruption construct pTC::FÜKm into the R. capsulatus cbbL' strain SBF. Kanamycin-resistant clones were screened for sensitivity to Tc and double recombination was confirmed by Southern hybridization analysis (data not shown). The construction of the cbbPu' double recombinant strain, SBP, was confirmed by Southern hybridization analysis of HindSl and BamKL digested chromosomal DNA (Fig. 3.7 C). The BamHL fragment from the mutant strain was 1.9 kb larger than the hybridizing fragment from the wild type strain (Fig 3.7 C, lanes 3 and 4) because of the insertion of the 1.9 kb Sp*^ cassette at the XhoL site within cbbP^ (Fig 3.7 A). HindSE sites are present on each end of the Sp^ cassette (Fig. 3.7 A) which results in the 9.0 kb cbbPu hybridizing Tfi/idlll fragment from the wild-type strain (Fig. 3.7 C, lane 1) to appear as 3.5 kb and 5.5 kb fragments when SBP' DNA is cut with TfmdHI (Fig. 3.7 C, lane 2). Insertion of the cbbRa:.Km^ construct into the SB 1003 genome was confirmed by digesting genomic DNA from the wild-type and mutant strains with Sail. The mutant copy of the cbbRa was about 0.8 kb larger than the wild-type copy (Fig. 3.7 D) due to the presence of the Tn5 Km*^ cassette (Fig. 3.7 A). Attempts to construct a cbbR{ strain were unsuccessful. Plasmid pTCHSmii containing the cbbRi.iSp^ construct in pTC5603 (Fig. 3.6 A; Method and Materials) was mobilized into R. capsulatus SB 1003. Of the 750 Sp^ colonies screened, 4 were Tc^. Despite the loss of the Tc^, when these clones were analyzed by Southern blotting and hybridization, they were found to be the result of a single recombination. A Sp’^-Tc** 104 clone, confirmed by Southern hybridization analysis to be the result of a single recombination, was grown aerobically in PYE liquid media supplemented with Sp and plated onto solid PYE supplemented with Sp to give isolated colonies. Five hundred of these Sp®^ clones were screened for Tc sensitivity, but no Tc^ clones were present. Characterization and complementation of RubisCO*mlnus strains. Southern blots established the disruption of the cbbL and cbbM genes in R. capsulatus strains SBF and SBIF, respectively (Figs. 3.6, 3.7), and western blot analyses confirmed the lack of RubisCO protein corresponding to each mutated gene (Figure 3.8). Either form of RubisCO supported photoheterotrophic, photoautotrophic, and chemoautotrophic growth (Fig. 3.9). Growth of these strains in liquid cultures revealed that the doubling times for the mutant strains were slightly longer than those of the wild-type strain (Table 3.3). Photoheterotrophic growth of strain SBIF is of particular interest. Form I RubisCO is not detectable in the wild-type strain grown photoheterotrophically on malate (Shively et al„ 1984; Paoli et a i, 1995; Fig. 1.4 A, lane 5), yet strain SBIF, which is unable to make form II RubisCO, is able to grow photoheterotrophically. Apparently the lack of form II RubisCO synthesis results in the compensatory synthesis of the form I RubisCO under photoheterotrophic conditions. This sort of compensation is analogous to that observed in R. sphaeroides. Analysis of R. sphaeroides cbbLcbbS' and cbbM' mutant strains revealed that, when the gene(s) encoding one form of RubisCO were disrupted by insertional mutagenesis, the level of RubisCO transcripts, protein, and activity associated with the remaining gene increased so that the observed level of activity 105 Fig. 3.8. Western blot analysis of R. capsulatus RubisCO-minus strains. Western blots were prepared from SDS-PAGE gels: lane 1, purified Synechococcus sp. strain PCC 6301 RubisCO; lane 2, crude extract from photoautotrophically grown R. capsulatus SB 1003; lane 3, crude extract from photoautotrophically grown R. capsulatus SEP; lane 4, crude extract from photoautotrophically grown R. capsulatus SBIT; lane 5, purified R. sphaeroides form II RubisCO. Blots were incubated with antibody raised against (A) Synechococcus strain PCC 6301 RubisCO and (B) R. sphaeroides form II RubisCO. 106 B Figure 3.8. 107 Fig. 3.9. Growth phenotype of the R. capsulatus RubisCO-deletion strains. The strains were grown (A) photoheterotrophically on malate, (B) photoheterotrophically on malate with DMSO (C), photoautotrophically, and (D) chemoautotrophically. WT, strain SB 1003; cbbL', strain SBF; cbbM', strain SBIF; cbbL'cbbM", strain SBI-II. 108 B cbbLT WT cbbM cbbL cbbM D Figure 3.9. Strain Growth “ Doubling RubisCO PRK Condition Time Activity Activity (hours) (nmol/min/mg) (nmol/min/mg) SB 1003 MAL 5.0 36.6 ± 6.4 40.4 ± 11.2 (wt) PA 12.5 642.0 ±25.7 343.8 ± 17.3 SBF MAL 6.5 51.3 ± 3.1 76.5 ± 15.7 {.CbbL) PA 15.5 362.8 ± 5.6 489.9 ±36.2 SBIF MAL 7.0 31.7± 1.3 193.0 ±25.3 {cbbM) PA 15.5 313.6 ± 18.4 506.3 ± 28.9 if w . T-----r: Table 3.3. Growth Rates, RubisCO activities, and PRK activities of R. capsulatus wild-type, cbbL\ and cbbM' strains 110 met or exceeded that present in the wild-type strain (Gibson et al., 1991). RubisCO and PRK activities of the R. capsulatus cbbL' and cbbAT strains were thus measured to more closely examine the observed compensation in RubisCO activity. The level of RubisCO activity in photoheterotrophically grown SBIT, which must be attributed to only form I RubisCO, is approximately the same as that observed in the photoheterotrophically grown wild-type strain (Table 3.3). The activity of form II RubisCO in photoheterotrophically grown SET exceeds that observed for the wild-type strain. No such compensation was observed for either strain grown photoautotrophically (Table 3.3). PRK activities in the RubisCO-minus strains exceeded those of strain SB 1003 under both photoheterotrophic and photoautotrophic conditions, with the SBIT strain displaying particularly higher PRK activities than the SET strain under photoheterotrophic growth conditions. A strain lacking both forms of RubisCO (cbbL'cbbMT) was unable to grow autotrophically or photoheterotrophically unless DMSO was supplied as an alternative electron acceptor (Fig. 3.9). Although the presence of DMSO did not support photoheterotrophic growth of strain SBI-E on solid media (Fig. 3.9), the strain was able to grow in liquid cultures containing DMSO with a generation time of 14.5 hours (Table 3.4). In the absence of DMSO as an alternative electron acceptor, strain SBI-E did not grow photoheterotrophically on malate in either standing or argon-bubbled broth cultures, nor did the strain grow photo- or chemoautotrophically in broth cultures. Strain SBI-E lacked any detectable RubisCO activity when grown photoheterotrophically on malate i n Strain Growth “ Doubling RubisCO PRK Condition Time Activity Activity (hours) (nmol/min/mg) (nmol/min/mg) SB 1003 MAL 6.5 88.9 ± 14.9 98.8 ± 6.2 MAL/DMSO 12.0 71.5 ± 10.4 74.4 ±10.8 __b SBI-n MAL __b __b {cbbUcbbNT) MAL/DMSO 14.5 0 25.7 ± 5.8 RC-PHC MAL 15.5 0 0 {cbbUcbbhT) MAL/DMSO 12.0 0 0 1 WA'r t- : -LJ DMSO ** ** Not determined, strain did not grow under these conditions Table 3.4. Growth Rates, RubisCO activities, and PRK activities in R. capsulatus wild- type and cbbUcbbM’ strains 112 with DMSO. This is not due to the presence of DMSO in the culture; despite the fact DMSO reduced the growth rate of the wild-type strain, RubisCO activity in the wild-type strain was not significantly reduced in the presence of DMSO (Table 3.4). Strain SBI-E expressed PRK activity, but at a reduced level compared to the wild-type strain (Table 3.4). Cosmids pJG336 and pJG106 (containing the R. sphaeroides cbbj and cbbn opérons, respectively) as well as the R. sphaeroides form I, R. sphaeroides form E, R. capsulatus form I, R. capsulatus form E, and Synechococcus strain PCC 6301 RubisCO genes cloned in pRPS-1 complemented strain SBI-E to photoheterotrophic and photo- and chemoautotrophic growth on solid media (results not shown). The RubisCO activity was determined for SBI-E complemented with the R. capsulatus form I and form E RubisCO genes cloned in the expression vector pRPS-1. The strains grew more slowly than wild-type strain SB 1003, particularly under photoautotrophic conditions (Table 3.5). The reduced growth rate cannot be attributed to the expression of only one form of RubisCO, because strains SBF and SBIT grew at rates comparable to the wild-type strain (Table 3.3). Furthermore, the level of RubisCO activity in the complemented strains was similar to that seen in the wild-type strain under both photoheterotrophic and photoautotrophic conditions. R. capsulatus has only one copy of cbbP. Previous studies indicated that R. capsulatus has only one cbbP gene, and that it is within the cbbu operon (Paoli et ai, 1995). Further evidence that this is indeed the case was provided by low stringency 113 Strain Growth “ Doubling RubisCO Condition Time Activity (hours) (nmol/min/mg) SBI-n MAL 9.5 34.5 ± 0.4 (pRPSFI-I) PA 49.5 269.4 ±13.9 SBI-n MAL 10.5 29.8 ± 4.9 (pRPSFn-D PA 40.0 492.8 ± 39.2 ' MAL - photoheterotrophically on malate; PA - photoautotrophically Table 3.5. Growth rates and RubisCO activities in R. capsulatus RubisCO- minus strain SBI-n complemented with R. capsulatus RubisCO clones 114 Fig. 3.10. Southern blot analysis of R. capsulatus cbbP at low stringency. R. capsulatus chromosomal DNA was digested to completion. Fragments were separated by agarose gel electrophoresis and transferred to Genescreen Plus membrane. The blot was hybridized for 18 h at low stringency, 37°C in a buffer containing 10% dextran sulfate, 6X SSC, 0.1% SDS, and 50% formamide. The BamHl-Smal fragment from pKlSFIEBSm containing the 5'-half of cbbPa and the 3'-end of cbbFn was used to synthesize the probe. The blot was washed twice (5 minutes each wash) with 6X SSC at room temperature prior to autoradiography. Lane 1, /fmdIII-£coRI; lane 2, BamHL-Sall; lane 3, SaB; lane 4, BamHl; lane 5, Smal; and lane 6, BaniHl-Smal. The sizes (kb) of the fragments resulting from A, DNA digestion with ffzndin are shown as a standard. 115 1 kb Sm B S E Sm - ► < pgm q o r cbbR a cbbF^y cbbP^ cbbT^ cbbG^ cbbA^ 12 3 4 5 6 B Figure 3.10. 116 Southern blot analysis of R. capsulatus genomic DNA using a probe derived from the R. capsulatus cbbPg gene (Fig. 3.10). hi each case, the size of the hybridizing fragment corresponded to the size expected for a chhPn-containing fragment. Although this does not disprove the presence of a second copy of cbbP in the R. capsulatus genome, the hybridization and wash stringency dictate that a second copy of cbbP would have to be less than 60 % identical to cbbPu- Characterization and complementation of the PRK-mlnus strain. The evidence presented above argues that, unlike R. sphaeroides, R. capsulatus has only a single copy of cbbP. Since cbbP encodes PRK, the only enzyme other than RubisCO that is unique to the CBB pathway, disruption of the R. capsulatus cbbPz gene should abolish the CBB pathway. AcbbPa strain was constructed. This strain, strain SBP', was unable to grow photoautotrophically, chemoautotrophically, or photoheterotrophically unless DMSO was supplied as an alternative electron acceptor (Fig. 3.11). hi addition, strain SBP lacked detectable PRK activity when grown photoheterotrophically on DMSO (Table 3.6). Since the PRK activity in strain SB 1003 is similar in photoheterotrophic cultures grown with or without DMSO, the absence of PRK activity in strain SBP is not due to the presence of DMSO in the media (Table 3.6). The RubisCO activity in strain SBP' grown photoheterotrophically with DMSO was much lower than in strain SB 1003. It was not determined if the RubisCO activity in SBP' was due to form I or form II RubisCO, but the likelihood that the cbbu genes are cotranscribed as a single operon necessitates that a disruption in cbbPjj would have a 117 Fig. 3.11. Growth phenotype of the R. capsulatus PRK-minus strain. Strains were grown (A) photoheterotrophically on malate, (B) photoheterotrophically on malate with DMSO, (C) photoautotrophically, and (D) chemoautotrophically. WT, strain SB 1003; cbbP', strain SBP'. 118 B é WT cbbPv VD X \ . C D Figure 3.11. Strain Growth “ Doubling RubisCO PRK Condition Time Activity Activity (hours) (nmol/min/mg) (nmol/min/mg) SB 1003 MAL 5.5 42.4 ± 12.0 112.9 ± 12.4 (wt) MAL/DMSO 12.5 32.4 ± 8.4 95.3 ± 14.2 SBFMAL ___b ___b ___b (cbbP-) MAL/DMSO 8.0 3.0 ± 1.1 0 SBP (pRPS::RsPiA) MAL 16.5 12.8+ 1.8 922.8 ± 202.4 SBP-PHC MAL 12.0 3.0 ± 2.5 0 (cbbF) MAL/DMSO 8.5 12.5 ± 6.7 0 MAL - photoheterotrophically on malate; MAL/DMSO - photoheterotrophically on malate with DMSO Not determined; strain did not grow under these conditions Table 3.6. Growth rates, RubisCO activities, and PRK activities in photoheterotro phically grown R. capsulatus wild-type, cbbP', and complemented strains 120 polar effect on the expression of the form II RubisCO gene. If the low level of RubisCO activity were due entirely to form I RubisCO synthesis, the lack of form II RubisCO synthesis would be consistent with the assertion that the R. capsulatus cbbvL genes form an operon. The R. sphaeroides cbbP\ gene in the expression vector pRPS-1 (pRPS::RsPi) complemented the R. capsulatus cbbF strain SBP' to photoheterotrophic growth without a requirement for DMSO. Complementation was dependent on the proper orientation of the inserted DNA fragment (not shown). Despite very high PRK activity in the complemented strain (Table 3.6), plasmid pRPSxRsP; did not complement strain SBP" to photoautotrophic growth. A 4-fold increase in RubisCO activity was also noted in the complemented strain (Table 3.6). Characterization and complementation of the CbhRn-minus strain. The regulation of ebb genes by a CbbR transcriptional activator is a common feature in autotrophic bacteria (Gibson and Tabita, 1996). In Alcaligenes eutrophus (now called Ralstonia eutropha) (Windhovel and Bowien, 1991), R. sphaeroides (Gibson and Tabita, 1993), and Xanthobacter flavus (van den Bergh et ai, 1993; Meijer et ai, 1996) the product of a single cbbR gene regulates transcription from at least two different promoters. Consequently, transcription from these promoters is coordinately activated. Only Thiobacillus denitrificans (Lorbach and Shively, 1995) and R. capsulatus (this work) have two potentially functional cbbR genes. The presence of two different CbbR proteins in R. capsulatus could allow independent regulation of the ebb opérons. 121 Fig. 3.12. Growth phenotype of the R. capsulatus CbbRn'-strain. Strains were grown (A) photoheterotrophically on malate, (B) photoheterotrophically on malate with DMSO, (C) photoautotrophically, and (D) chemoautotrophically. WT, strain SB 1003, cbbR\i, strain SBRn'. 122 B WT cbbR D Figure 3.12. Strain Growth® Doubling RubisCO PRK Condition Time Activity Activity (hours) (nmol/min/mg) (nmol/min/mg) SB 1003 MAL 5.5 42.4 ± 12.0 112.9 ± 12.4 (wt) PA 11.5 485.3 ± 65.7 451.1 ± 9.8 SBRn' MAL 10.5 10.1 ± 1.9 11.3 ± 1.3 (cbbRn) PA ___ b ___b ___b SBRn' MAL 7.5 11.8± 0.1 17.8 ± 3.4 (pVK::cbbRn) PA 47.0 49.1 ± 3.5 64.5 ± 6.9 S'i v v r " c-1 r : malate with DMSO Not determined, strain did not grow under these conditions Table 3.7. Growth rates, RubisCO activities, and PRK activities in R. capsulatus wild- type, cbbRù, and complemented strains 124 Attempts were made to establish a function for the two CbbR proteins in R. capsulatus. Unfortunately, construction of a cbbRi mutant was unsuccessful. To establish a physiological role for the function of CbbRn, a cbbR\i insertional mutant strain was constructed. This strain, SBRn, was unable to grow photo- or chemoautotrophically and grew photoheterotrophically on malate at a reduced rate (Fig. 3.12, Table 3.7). RubisCO and PRK activity in the cbbRn strain were reduced to 25 % and 10 % of the activity observed in wild-type strain SB 1003 (Table 3.7). The presence of PRK activity in strain SBRn indicates that transcription from the cbbn promoter occurs in the absence of CbbRn, albeit at an apparently reduced rate. Whether this transcription is due to “cross-talk” activation by CbbR; remains to be established. In R. sphaeroides, transcription from the cbbn promoter is not entirely dependent on CbbR (Gibson and Tabita, 1993). RubisCO activity in a R. sphaeroides cbbR' strain was approximately 30 % of that seen in the wild-type strain under photoheterotrophic growth conditions. Furthermore, all of this activity was due to the synthesis of form II RubisCO. Hence, transcriptional activity at cbbn in R. capsulatus SBRn does not necessarily imply activation by CbbRi. Strain SBRn' was complemented to photoautotrophic growth by the cbbRn gene in the low copy number vector pVKlOl. Despite the increased RubisCO and PRK activities in photoautotrophically grown SBRn (pVK::cbbRn), the enzyme activities in the complemented strain did not approach the levels seen in the wild-type strain (Table 3.7). The lack of complementation to wild-type enzyme activities might be due to the presence of the cbbn promoter, but not the cbbn genes, on the complementing plasmid 125 pVK::cbbRn- Binding of CbbRn to the cbbxi promoter on the plasmid would titrate the activator away from the chromosomal cbbu promoter without leading to productive transcription. Analysis of R. capsulatus ebb promoter fusions. The activity of both RubisCO and PRK in R. capsulatus is much higher under photoautotrophic conditions than under photoheterotrophic conditions (Table 3.3). The role of transcriptional activation of the ebb opérons in the observed increase in enzyme activities was examined using ebb promoter fusions. To construct R. capsulatus ebb promoter fusions, lacZ was translationally fused to the start codon of cbbL (jcbbi fusions, pXLB and pXLBP) and cbbFu {cbbu fusion, pXFB) (Fig. 3.13). The p-galactosidase activity measured in extracts from photoheterotrophically grown SB 1003 containing pXLB was nearly undetectable (Fig. 3.13). This result is consistent with the fact form I RubisCO was not detected in R. capsulatus grown photoheterotrophically on malate (Shively et al, 1984; Paoli et al., 1995; Fig. 1.4, lane 5). These previous studies showed that form II RubisCO is expressed under photoheterotrophic conditions. |3-galactosidase activities in photoheterotrophically grown SB 1003 containing pXFB indicate that transcription occurs from the cbbu promoter under these conditions (Fig. 3.13). Increased P-galactosidase activity was observed in photoautotrophically grown SB 1003 harboring either pXLB or pXFB, suggesting the transcription from cbb\ and cbbu is induced under photoautotrophic conditions. No P-galactosidase activity was detectable 126 P-Galactosidase Activity Fusion Fragment ( ' Strain SB 1003 Strain SBR» Sm Sm MAL PA MAL EX P Sm u n i u R L s Q pXLB <0.1 125.0 1.0 pXLBP 0 0 N.D. B SroXB X B X E E H H S wm M ké^md F P GAME pXFB 105.2 1013.8 6.4 Fig. 3.13. R. capsulatus cbb promoter fusion activity. The cbb promoter fragment that was fused to the lacZ gene in vector pXBA601 is represented by the arrow. The ebb, fusions pXLB and pXLBP were translationally fused to lacZ at the ATG start codon of cbbL and the cbb„ fusion pXFB was translationally fused to the ATG start codon of cbbF„. P-Galactosidase activity is expressed in nmol/min/mg. The growth conditions were: MAL, photoheterotrophically with malate as a carbon source; and PA, photoautotrophically. Strain SBR» did not grow photoautotrophically so the P-galactosidase activities was not determined. N.D., Not Determined. The values are the averages of several assays of extracts derived from two independent cultures. from the shorter cbb\ fusion construct pXLBP even under photoautotrophic conditions (Fig 3.13). The role of CbbRn as a transcriptional activator of c66n was established by measuring p-galactosidase activities in photoheterotrophically grown strain SBRn' harboring pXFB. The level of P-galactosidase activity expressed from the cbbvi fusion construct in the cbbR\i mutant strain was about 6 % of that observed in the wild-type strain (Fig. 3.13). Unfortunately, the lack of p-galactosidase activity from pXLB in SB 1003 under photoheterotrophic conditions and the inability of SBRn to grow under photoautotrophic conditions made it impossible to determine if CbbRn plays a role in activation of transcription from the cbb\ promoter. Nevertheless, the very low levels of P- galactosidase in photoheterotrophically grown SBRn- (pXFB) and the inability of SBRn’ to grow under photoautotrophic conditions suggest that CbbRi has little or no effect on transcription from cbbu- Photoheterotrophic growth of strains lacking the CBB pathway. It is well established that the CBB pathway allows CO2 to act as an electron sink under photoheterotrophic conditions (Richardson et al.. 1988; Falcone and Tabita, 1991; Wang et ai, 1993). A strain of R. sphaeroides that lacks the ability to synthesize RubisCO, strain 16, is unable to fix and reduce CO 2 ; thus, this strain is unable to grow autotrophically using H 2 as an electron donor, but can grow photoheterotrophically if an appropriate exogenous alternative electron acceptor, such as DMSO, is provided (Falcone and Tabita, 1991). Strain 16PHC is a spontaneous mutant, derived from strain 16, that lacks detectable RubisCO activity but is capable of photoheterotrophic growth in the 128 absence of DMSO (Wang et al., 1993). Strain I6PHC produced Ha gas and deregulated the expression of nitrogenase, showing considerable nitrogenase activity even when cells were grown in the presence of ammonium (Joshi and Tabita, 1996). Strain 16PHC is thus able to utilize protons as an alternative electron acceptor, catalyzing their reduction via nitrogenase. Spontaneous mutants of SBI-H and SBP', which have acquired the ability to grow photoheterotrophically in the absence of DMSO, have been obtained by prolonged incubation and serial transfer under selective conditions. The RubisCO-minus photoheterotrophically competent strain, RC-PHC, grows with a doubling time of about 15.5 hours in the absence of DMSO (Table 3.4). RC-PHC lacks RubisCO activity but, unlike the parent strain SBI-H, also lacks detectable PRK activity (Table 3.4). Strain SBP-PHC grows with a doubling time of 12 hours in the absence of DMSO, and, much like its parent strain SBP', lacks detectable PRK activity and shows only very low levels of RubisCO activity (Table 3.6). The physiological response that allows these CBB pathway-minus strains to grow under photoheterotrophic conditions is currently under investigation (Mary Tichi, this laboratory). Preliminary results indicate the strain SBP-PHC produces hydrogen and deregulates nitrogenase activity in a manner similar to R. sphaeroides 16PHC. The identity of the alternative electron acceptor used by RC-PHC has not yet been determined. 129 DISCUSSION The nucleotide sequence of the R. capsulatus cbbFn, cbbPn and part of the cbbTa genes provided further evidence that these genes are closely related to the cbbu operon genes of R. sphaeroides. This is particularly evident in the fact that the R. capsulatus cbbFu gene product is considerably more similar to the R. sphaeroides CbbFn (84 %) than to the R. sphaeroides CbbFi (67 %). Not only are the cbbu genes of R. capsulatus and R. sphaeroides organized in an identical manner, the intergenic regions share common features as well. Just as in R. sphaeroides the R. capsulatus cbbFu-cbbPu intergenic region is very short and the genes have overlapping translational sequences. The length of the cbbPu-cbbTu intergenic region is 80 nucleotides in R. sphaeroides and 83 nucleotides in R. capsulatus. Additional experiments are necessary to prove that the R. capsulatus cbbu genes are organized as an operon, but the available evidence suggests that these genes are arranged in a manner similar to those of R. sphaeroides and, like R. sphaeroides, most likely form an operon. Post-transcriptional processing of the R. sphaeroides ebb operon transcripts has been suggested (Gibson et al, 1991). The presence of a potential RNase E cleavage site within the R. capsulatus cbbPu-cbbTu intergenic region hints that post-transcriptional processing of the R. capsulatus cbbu message may occur. One significant difference between the R. capsulatus and R. sphaeroides cbbu opérons is the presence of a cbbR gene, cbbRu, divergently transcribed from the R. capsulatus cbbu operon. Phylogenetic analysis of the CbbR-class of the LysR-family of transcriptional activators revealed that the R. capsulatus CbbRn is most closely related to R. sphaeroides CbbR. This is especially interesting because the R. sphaeroides cbbR 130 gene is upstream and divergently transcribed from the cbbi operon. In R. capsulatus a second cbbR gene, cbbRi, is upstream of and divergently transcribed from the R. capsulatus cbb\ operon. In R. sphaeroides the single CbbR protein regulates and coordinates the expression of the cbb\ and cbbvi opérons (Gibson and Tabita, 1993). The presence of two CbbR proteins in R. capsulatus raises questions concerning the involvement of each form of CbbR in expression of the two ebb promoters. The possibility exists that CbbR; functions only to control cbb\ expression and that CbbRn activates transcription only for the c66n operon. This scenario is made more likely by the fact that thecbbR\, cbbLcbbS, and cbbQ genes were all apparently acquired by horizontal gene transfer (this work. Chapter 2). A physiological role for CbbRn of R. capsulatus was established. A strain (SBRn ) in which thecbbRn gene was disrupted was unable to grow autotrophically and grew at a reduced rate photoheterotrophically, showing reduced levels of both PRK and RubisCO activity. The P-galactosidase activity from the cbbn promoter fusion pXLFB in strain SERIF was only about 5 % of the activity of the wild-type strain under photoheterotrophic conditions, thus implicating CbbRn in the activation of transcription of the cbbn promoter. Although a cbbRi mutant could not be constructed, other experiments provide evidence that the CbbR proteins probably activate transcription from only the ebb operon from which they are divergently transcribed. The activation of transcription at the cbbn promoter by CbbRi is unlikely because of the reduced levels of P-galactosidase and PRK observed in strain SBRn. fr CbbRi were able to activate transcription at the cbbn promoter, the P-galactosidase and PRK activities in strain SERIF would not be significantly reduced. In addition, strain SERIF is unable to grow photoautotrophically; thus, CbbRi activation at the cbbn promoter does not appear to occur under photoautotrophic conditions either. The lack of form I RubisCO protein under 131 photoheterotrophic conditions suggests that CbbRn does not activate transcription from thecbbi promoter. This is further substantiated by the very low P-galactosidase activities from the cbbi promoter fusion construct pXLB under photoheterotrophic conditions. These observations indicate that “cross-talk” activation of the ebb opérons by the opposite CbbR protein does not occur. LysR-type transcriptional activators generally bind to the promoter they activate, even under non-inducing conditions. Thus, the binding of a low molecular weight co inducer molecule to the LysR-type protein is required, in most cases, to activate transcription (Schell, 1993). The identity of the inducer molecule(s) for CbbR proteins is not yet known. It will be interesting to determine if the different phylogenetic relationships of the R. capsulatus CbbR proteins can be correlated to the activation by unique co-inducer molecules and if this is related to the differential expression from the cbbi and cbbn promoters of R. capsulatus. The presence of two different CbbR proteins raises additional questions about DNA binding specificity. Since CbbR probably binds to the ebb promoter in the absence of the inducer molecule, binding must be specific for the proper promoter to prevent a repressive effect on the opposite promoter. For example, binding of CbbRn to the cbb\ promoter would not activate transcription, but could prevent the binding of CbbRi. Disruption of the form H RubisCO gene {cbbM\d revealed an interesting regulatory property of the R. capsulatus ebb opérons. Form I RubisCO protein is normally not detected in photoheterotrophically grown R. capsulatus (Shively et al., 1984; also see Fig. 1.4, lane 5). This is likely due to an apparent lack of transcription at the cbbi promoter under photoheterotrophic growth conditions, as shown by the lack of P-galactosidase activity in strain SB 1003 containing fusion plasmid pXLB. Nevertheless, when the form U RubisCO gene was disrupted, the resulting strain (SBIT) was able to grow photoheterotrophically concomitant with the apparent synthesis of form I RubisCO 132 in the mutant strain. Perhaps the lack of form II RubisCO causes the buildup of some CBB cycle intermediate or some other metabolite that acts as a co-inducer to activate CbbR-regulated transcription of the cbb\ operon. The product of the qor gene downstream of cbbRu may also serve to regulate ebb gene expression. This gene encodes a soluble NAD(P)H quinone oxidoreductase (QOR) that catalyzes the reversible transfer of electrons from reduced pyridine nucleotides, with a preference for NADPH, to membrane-bound quinones. Although this gene has been sequenced from both E. coli (Lilley et ai, 1993) and P. aeruginosa (GenBank Accession # X85015) and the E. coli protein has been crystallized (Thom et al., 1995), no physiological function for this enzyme has yet been determined in bacteria. On the basis of the reaction catalyzed by this enzyme, QOR could function to sense or maintain the redox state of the membrane quinone pool. Thus, as a redox sensor, QOR could be involved in the regulation of ebb gene expression. Although the co-inducer of CbbR has not yet been conclusively identified, there is limited evidence that the Xanthobacter flavus CbbR co-inducer may be NADPH (Wim Meijer, unpublished). Although the association of this gene with the R. capsulatus cbbu operon may be purely coincidental, the involvement of QOR in regulating ebb gene expression, or its involvement in regulating the CBB pathway enzymes, warrants further investigation. In summary, R. capsulatus ebb gene regulation is quite complex. Two different CbbR proteins are present that are potentially activated by the binding of unique co inducer molecules. Obviously, to allow efficient regulation, the CbbR proteins must bind specifically to their respective ebb promoter. The presence of a potential RNase E recognition site within the cbbn message suggests that it is post-transcriptionally processed. Further study of R. capsulatus ebb gene regulation will not only provide a better understanding of the control of C0% fixation but will also address more general 133 questions of gene regulation, such as the specificity of DNA-protein interactions and the significance of rnRNA processing in prokaryotes. ACKNOWLEDGEMENTS The author would like to thank Dr. J.T. Beatty for providing plasmid pXBAôOl, Wenona Stankiewicz for technical assistance, and Mary Tichi for providing preliminary physiological findings concerning the R. capsulatus PHC strains. 134 CHAPTER 4 Aerobic Chemollthoautotrophic Growth of Rhodobacter capsulatus strain SB 1003 and a Spontaneous Mutant of Rhodobacter sphaeroides strain HR INTRODUCTION Photosynthetic carbon metabolism and its regulation in purple bacteria have been the subject of intense study for several years (Gibson, 1995; Gibson and Tabita, 1996; Tabita, 1988; 1995). Phototrophic growth in these bacteria occurs only under anaerobic conditions, and CO 2 is fixed via the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway. Although not nearly as well studied, some species of photosynthetic purple bacteria are also able to grow chemoautotrophically under dark aerobic conditions. Several species of phototrophic purple sulfur bacteria (Chromatiaceae) can carry out aerobic chemoautotrophic metabolism using reduced sulfur compounds as a source of reductant (Kampf and Pfennig, 1980; 1986a; 1986b; Kondratieva et ai, 1976; Kondratieva, 1989). To date, only a few species of nonsulfur purple phototrophs iRhodospirillaceae) have been shown to grow under aerobic chemoautotrophic conditions. Rhodobacter capsulatus (Madigan and Gest, 1979; Siefert and Pfennig, 1979) and Rhodopseudomonas acidophilus (Siefert and Pfennig, 1979) grow under such conditions but only in the presence of molecular hydrogen as a source of reductant. Under these conditions the organisms grow in a completely inorganic medium in the presence of gaseous CO 2, O2, and H 2 , catalyzing the so called knallgas reaction ( 2 H2 + O2 ► H2O ; knall means explosion in German, and the term knallgas is used to refer 135 to the potentially explosive mixture of % and 0%). Growth under such conditions is equivalent to that of several species of nonphotosynthetic aerobic “hydrogen bacteria”. With the exception of a few species of thermophilic organisms of the genera Hydrogenobacter (Kawasumi et ai, 1984; Shibaet a i, 1985) and Aquifex (Beh et al., 1993), all bacteria that catalyze the Knallgas reaction fix CO2 via the CBB pathway (Schlegel, 1989). Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is the enzyme that catalyzes the CO2 fixation reaction of the CBB pathway, the carboxylation of ribulose 1,5-bisphosphate (RuBP). In the absence of oxygen, the net result of RubisCO catalysis and the CBB pathway is the generation of one molecule of hexose phosphate for every 6 carbons fixed. This is the case for phototrophic growth of purple photosynthetic bacteria, which occurs only under anaerobic conditions. As its name indicates, RubisCO also catalyzes the oxygenolysis of RuBP. The oxygenase reaction produces one molecule each of 3-phosphoglycerate and 2-phosphoglycolate. The subsequent metabolism of 2-phosphoglycolate results in the net loss of carbon from the cell. In plants the dissimilation of 2-phosphoglycolate proceeds via the photorespiratory pathway, and various estimates predict a loss of 0.5 carbons for each O2 fixed (Gutteridge, 1989). In bacteria the metabolism of phosphoglycolate is not thoroughly understood. In some cases phosphoglycolate is dephosphorylated and excreted directly. If all of the phosphoglycolate produced was metabolized in this manner, the oxygenase reaction would be truly costly in reducing net CO 2 fixation by RubisCO. The presence of glycolate oxidase activity in species of phototrophic (Codd and Smith, 1974; Codd and Turnbull, 1975) and chemoautotrophic bacteria (Codd et ai, 1976), and the increase in glycolate excretion when this enzyme is inhibited, indicates that some of the glycolate is further metabolized. The rates (v) at which RubisCO catalyzes the carboxylase and oxygenase reactions can be expressed by: V c/V q = T ([C02]/[02]), where T is the substrate specificity factor. X is defined by a ratio of the RubisCO carboxylase (c) and oxygenase (o) kinetic 136 parameters; X = VcKoA^oKc, where V is the and K is the Km for each gaseous substrate (Jordan and Ogren, 1981). Thus, the ratio of carboxylation to oxygenation (Vc/Vo) in a given atmosphere ([COJ/EOJ) is determined by X. RubisCO exists in two distinct forms. Form I RubisCO is a complex enzyme composed of eight large and eight small subunits (LgSg). Form II RubisCO is composed of only large subunits (L%) which are biochemically and genetically distinct from the form I RubisCO large subunits. Despite the fact that form I and form II RubisCO large subunits share only about 25 % amino acid sequence identity, both forms of the enzyme catalyze the carboxylation and oxygenolysis of RuBP at a highly conserved active site. One of the most notable differences between form I and form II RubisCO is in the degree to which they can discriminate between the two gaseous substrates. The specificity factor, X, of form II RubisCO is - 10. The X of form I RubisCO varies from 25 to 110 depending on the source of the enzyme (Tabita 1995). Rhodobacter sphaeroides and R. capsulatus, two species of photosynthetic nonsulfur purple bacteria, synthesize both form I and form U RubisCO (Gibson and Tabita, 1977a; 1977c; 1985; Shively er a/., 1984; Paoli et a/., 1995). These facultatively autotrophic organisms present a model system for examining the capacity of form I and form n RubisCO to support aerobic chemoautotrophic growth. Although R. capsulatus can grow under aerobic chemoautotrophic conditions, wild-type strains of R. sphaeroides apparently lack this ability (Madigan and Gest, 1979). We report here the isolation of a spontaneous mutant of R. sphaeroides that has acquired the ability to grow aerobically under chemolithoautotrophic conditions. The ability of form I or form H RubisCO to support aerobic chemoautotrophic growth was examined in R. capsulatus and R. sphaeroides strains synthesizing only one or the other form of RubisCO. The use of these strains for the biological selection of RubisCO with improved substrate specificity may be a direct consequence of these studies. 137 MATERIALS AND METHODS Bacterial strains and growth conditions. A list of bacterial strains used in this study is given in Table 4.1. A variety of wild-type R. sphaeroides and R. capsulatus strains were also used (Table 4.5) and were the generous gift of Dr. Michael T. Madigan (Southern Illinois University, Carbondale, Illinois). Aerobic cultures of R. sphaeroides and R. capsulatus were grown in PYE medium (Weaver and Tabita, 1983) at 30°C. Autotrophic cultures were grown in Ormerod's minimal medium (Ormerod et ai, 1961) supplemented with vitamins (1 pg/ml thiamine, 1 pg/ml nicotinic acid, and 0.1 pg/ml biotin). Photoautotrophic cultures were bubbled continuously with 1.5% CO 2 / 98.5% Hi and incubated in the light as previously described (Falcone and Tabita, 1991). Aerobic chemolithoautotrophic growth was carried out on solid media or in small liquid cultures (10 ml) at 30°C in BBL GasPak jars using a H 2/CO2 generating system without the palladium catalyst (Becton Dickson Microbiology Systems, Cockeysville, Maryland). Larger chemoautotrophic liquid cultures (300 ml) were grown with continuous bubbling (50 ml/min) with Hi/COi/Air. Gases were prepared by blending a mixture of H 2/CO2 (Liquid Carbonic Specialty Gases, Oak Brook, Illinois) with air (50:50) using a two tube rotometer (Matheson Gas Products, Montgomeryville, Penn.). For growth at different CO2 concentrations, different mixtures of H 2/CO2 were used. Under each condition O 2 was half the concentration found in air, 10.5 %. Photoheterotrophic cultures were grown in Ormerod’s medium plus vitamins supplemented with 0.4% malate or other carbon sources as specified, in filled screw cap tubes or by continuously bubbling with argon. Antibiotics were used where appropriate at the following concentrations: for R. sphaeroides, kanamycin, 30 pg/ml; trimethoprim, 200 |ig/ml; and streptomycin, 50 p.g/ml; for R. capsulatus, rifampicin, 100 p.g/ml; kanamycin, 5 |ig/ml; and 138 spectinomycin, 5 pg/ml. For comparison of the growth of R. sphaeroides CAC with that of R. capsulatus SB 1003 and R. sphaeroides HR, streptomycin was used at 50 p,g/ml and Penicillin G was used at 0.1 U/ml. Mutant strain isolation and construction. Chemolithoautotrophic competent (CAC) strains were selected by incubating cultures for 3-4 weeks in an Hz/COz/air atmosphere in a BBL GasPak jar using the H 2/CO2 generating system, without the palladium catalyst. The jar was opened weekly and a new H 2/CO2 generating envelope was used to refresh the atmosphere in the jar. E. coli SMIO (Simon et al., 1983) containing plasmid pSUP::E25A::Km (Falcone and Tabita, 1991) was used to construct the cbbLcbbS-déle.üoti strain CAC-FT from R. sphaeroides strain HR-CAC. The conjugal mating procedure and screening were described previously (Falcone and Tabita, 1991). Preparation of cell extracts and RubisCO Assays. Culture samples (20-30 ml) were washed twice in 100 mM Tris-HCl (pH 8.0) and 1 mM EDTA and frozen at -70°C. Thawed pellets were resuspended in 1 ml TEM (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 5 mM P-mercaptoethanol) and disrupted by sonication at 0°C. Cell debris was removed by centrifugation for 10 min in a microcentrifuge at 4°C. The resultant crude extract was used for enzyme assays. RubisCO activity was assayed as RuBP-dependent ‘‘‘CO2 fixation into acid-stable material (Gibson et al., 1991) and protein concentrations were measured by a modified Lowry procedure (Markwell et ai, 1978). Rocket inununoelectrophoresis. The amount of RubisCO protein in R. sphaeroides cell extracts was determined by rocket inununoelectrophoresis using antibodies specific for R. sphaeroides form I and form II RubisCO (Jouanneau and Tabita, 1986). 139 Strain Relevant characteristics Source or reference R. sohaeroides HR Sm*^ derivative of wild-type strain Weaver and ATCC 17023 Tabita, 1983 HR-CAC spontaneous mutant of HR able to This work grow chemolithoautotrophically HR-FT cbbLS'.'.Km^ derivative of HR Gibson et al., 1991 HR-Fir cbbM:: Tp^ derivative of HR Gibson et al., 1991 CAC-FT c66U::Tp^ derivative of HR-CAC This work Fir-CAC spontaneous mutant of strain HR-FIT able to grow chemolithoautotrophically This work 16 cô6L5::Km^, cbbM:: Tp*^ derivative of HR Falcone and Tabita, 1991 I6PHC spontaneous mutant of strain 16 able to Wang et al, grow photoheterotrophically 1993 1312 cbbR::Tp^ derivative of strain HR Gibson and Tabita, 1993 4X cbbX::Sp^ derivative of strain HR, cbbXYZ Gibson and Tabita, 1997 R. capsulatus SB 1003 Rif*^ derivative of wild-type strain B 10 Yen and Marrs, 1976 SBF cbbL:: Sp^ derivative of SB 1003 This work SBir cbbM:: Km*^ derivative of SB 1003 This work SBI-n cbbL:: Sp*^ ,cbbM:: Km^ derivative of SB 1003 This work Table 4.1. Bacterial strains used in this study 140 RESULTS Selection and characterization of R. sphaeroides strain HR-CAC. The initial observation concerning the ability of R. sphaeroides to acquire the capacity to grow chemoautotrophically came while examining the expression of lacZ from the Rhodospirillum rubrum RubisCO promoter in vector pRPS-1 (Falcone and Tabita, 1991). Expression from this promoter was examined in both R. sphaeroides and R. capsulatus under various growth conditions, including aerobic chemoautotrophic growth in GasPak jars. TheR. capsulatus strain grew within a week, at which time theR. sphaeroides strain showed little or no increase in turbidity. This is consistent with the observations of Madigan and Gest (1979), which indicated the ability of R. capsulatus to grow under aerobic chemoautotrophic conditions with R. sphaeroides growing very poorly, if at all, in this mode of growth. When theR. sphaeroides cultures were allowed to incubate for longer periods of time, the turbidity increased dramatically after 3 to 4 weeks. The experiment was repeated several times with the R. sphaeroides wild-type strain HR, and each time the culture showed little or no increase in turbidity for about three weeks, after which time the culture began to show a slow but steady increase to full density (O.D.%o > 1 -5) during the next week. It was not possible to monitor the growth turbidometrically because the cultures were grown in sealed jars. The lengthy time period required before the onset of chemoautotrophic growth suggested the selection of a spontaneous mutant. Nevertheless, the possibility still 141 existed that growth only occurred after an extensive physiological adaptation. This possibility was eliminated by growing the adapted culture under different physiological conditions prior to regrowing it chemoautotrophically. The culture was streaked on a PYE plate and, after it had grown up, a single colony was grown photoheterotrophically. This photoheterotrophic culture was used to inoculate minimal medium and incubated chemoautotrophically. After this regimen, the strain no longer showed the three week lag previously observed. The strain grew in approximately one week. Thus, the chemoautotrophic competent (CAC) phenotype of this strain, HR-CAC, appeared to be the result of a stable genetic change. The same observations were made when R. sphaeroides HR was grown aerobically chemoautotrophically by bubbling with a gas mixture of 5% CO? / 45% Hi / 50% air (Fig. 4.1) The culture density of the wild-type strain HR increased only slightly, then, after approximately 3 weeks, logarithmic growth ensued. Strain HR-CAC grew almost inunediately under these conditions. The fact that this CAC strain was derived directly from R. sphaeroides strain HR was confirmed by several means. Both R. sphaeroides and R. capsulatus can utilize malate as a carbon source, but only R. capsulatus can utilize propionate and only R. sphaeroides can utilize citrate (Weaver et ai, 1975). Strains of R. sphaeroides express a low level of resistance to penicillin G whereas R. capsulatus is sensitive (Weaver et ai, 1975). Furthermore, R. sphaeroides strain HR, the parent of the R. sphaeroides CAC strain, is resistant to streptomycin (Weaver et al., 1983). The CAC strain was 142 HR HR-CAC C Û g 2 U 0.1 0.01 0 510 15 20 25 3 0 3 5 Time (Days) Fig. 4.1. Growth of R. sphaeroides strains HR and HR-CAC under aerobic chemoautotrophic conditions. Strains HR (• ) and HR-CAC ( O ) were inoculated from photoautotrophically grown cultures into carbon-free Ormerod minimal medium. Cultures were incubated chemoautotrophically at 30°C in the dark with continuous bubbling at 50 ml/min using a gas mixture of 5% CO 2/ 45% H2 / 50% air. 143 Growth® Strain Malate** Propionate** Citrate** Sm' Pc** R. sphaeroides HR 4 4 4 - 4- HR-CAC 4-44- -I- R. capsulatus SB 1003 “( 4 4 + ) = very good; (-H -) = good; (4-) = poor, (-) = no growth **Photoheterotrophic growth in the presence of 0.4 % carbon source and 0.05% bicarbonate "^Photoheterotrophic growth on malate in the presence of 50 pg/ml streptomycin "*Photoheterotrophic growth on malate in the presence of 0.1 U/ml Penicillin G Table 4.2. Comparison of growth characteristics of the CAC strain with R. sphaeroides strain HR and R. capsulatus strain SB 1003 144 differentiated from R. capsulatus in all of these properties (Table 4.2). Since R. capsulatus form I RubisCO does not cross react with antibody raised against the R. sphaeroides form I RubisCO (Gibson and Tabita, 1977c; Paoli et al, 1995), the ability of anti-R. sphaeroides form I RubisCO antibody to cross-react with CAC extracts in Ouchterlony immunodiffusion experiments (not shown) and in rocket immunoelectrophoresis (Table 4.4) further differentiated the CAC strain from R. capsulatus. In addition, results of Southern hybridization analysis of CAC chromosomal DNA using R. sphaeroides cbbL and cbbM gene probes matched those observed for R. sphaeroides HR(data not shown). All of this evidence indicated that the CAC strain was derived from strain HR by a spontaneous mutation which resulted in the ability of strain HR-CAC to grow under aerobic chemoautotrophic conditions. Growth rate and RubisCO activity of R. sphaeroides HR-CAC cultured under different conditions. The growth rate and RubisCO activity of R. sphaeroides strain HR-CAC were determined under different growth conditions and compared to those of parent strain HR and R. capsulatus wild-type strain SB 1003 (Table 4.3). Comparisons were made to R. capsulatus because the wild-type strain of this species can grow chemoautotrophically (Madigan and Gest, 1979). Under photoheterotrophic conditions all three strains grew with a doubling time of 6.5 to 7 hours and had nearly the same RubisCO activity. When grown photoautotrophically, the R. sphaeroides strains were comparable in both doubling time and RubisCO activity (Table 4.3). Photoautotrophically grown R. capsulatus grew considerably faster than the 145 Doubling Time RubisCO Activity Strain (h) (nmoles/min/mg) Photoheterotrophic” HR 7.0 34 HR-CAC 6.5 40 SB 1003 6.5 34 Photoautotrophic^ HR 17 109 HR-CAC 14.5 123 SB 1003 10 186 Chemoautotrophic*^ HR-CAC 27 93 SB 1003 22 94 ” Growth in a malate-minimal salts medium bubbled with argon ** ** Growth in a minimal salts medium bubbled with 1.5% C02/98.5% Hz Growth in a minimal salts medium bubbled with 5% COz/45% Hz/50% Air Numbers represent averages of duplicate experiments Table 4.3. Growth rates and RubisCO activities in R. sphaeroides strains HR and HR-CAC and R. capsulatus strain SB 1003 146 R. sphaeroides strains, doubling every 10 hours. R. capsulatus strain SB 1003 grew faster than R. sphaeroides HR-CAC, but the RubisCO activities were similar (Table 4.3). In both R. sphaeroides and R. capsulatus, chemoautotrophic RubisCO activities were intermediate to the photoheterotrophic and photoautotrophic activities. RubisCO synthesis was not constitutive in R. sphaeroides strain HR-CAC, because, like strain HR, RubisCO activity was only barely detectable when cultures were grown under aerobic chemoheterotrophic conditions on minimal medium containing malate as carbon source (data not shown). The amount of form I and form H RubisCO protein in strain HR-CAC was quantitated by rocket immunoelectrophoresis. Similar experiments could not be done with R. capsulatus because antibodies against R. capsulatus RubisCO are not yet available. Considerably more form I and somewhat more form H RubisCO was synthesized in R. sphaeroides strain HR-CAC under both photoautotrophic and chemoautotrophic conditions compared to photoheterotrophic conditions (Table 4.4). These observations are consistent with previous experiments in which the amounts of form I and form II RubisCO was measured in strain HR grown photoheterotrophically and photoautotrophically (Jouarmeau and Tabita, 1986). Chemoautotrophic growth of other R. sphaeroides and R. capsulatus strains. To determine if chemoautotrophic competence is a species-specific trait, a number of R capsulatus and R. sphaeroides wild-type strains were tested for their ability to grow under aerobic chemoautotrophic conditions. The strains were streaked on solid medium and grown in GasPak jars. After three weeks the growth was evaluated. With the 147 Growth Condition RubisCO Level (% soluble protein)* Form I Formll Photoheterotrophic** 0.7 1.7 Photoautotrophic"* 4.1 4.2 Chemoautotrophic"* 6.6 2.6 “ Measured by rocket immunoelectrophoresis ** ** Growth in a malate-minimal salts medium bubbled with argon Growth in a minimal salts medium bubbled with 1.5% C02/98.5% Hz Growth in a minimal salts medium bubbled with 5% CÜ2/45% H2/50% Air Numbers represent averages of duplicate experiments Table 4.4. RubisCO Protein Levels in R. sphaeroides strains HR-CAC 148 exception of wild-type strain 2.4.7 and mutant HR-CAC, R. sphaeroides strains grew poorly, while most of the R. capsulatus strains grew well (Table 4.5). The ability of various R. sphaeroides HR mutant strains to acquire the CAC phenotype was determined . Each strain was incubated under selective conditions (liquid cultures in GasPak jars) to see if chemoautotrophic growth occurred within 4 weeks. Duplicate cultures of each strain were tested. For the strains where neither replicate grew, a third attempt was made to grow the strain under aerobic chemoautotrophic conditions. Four strains were unable to acquire the CAC-phenotype: strains 16, 16PHC, 1312, and HR-FT (Table 4.6). Strain 16 is an HR derivative that lacks both form I and form II RubisCO due to insertional inactivation of the respective genes. Strain 16PHC is a spontaneous mutant of strain 16 that has acquired the ability to grow photoheterotrophically (Wang et a/., 1993). This strain does not synthesize RubisCO but is able to grow photoheterotrophically in the absence of RubisCO by using protons as an alternative electron acceptor via reduction by nitrogenase (Joshi and Tabita, 1996). The inability of strains 16 and 16PHC to grow under the selective conditions is apparently due to the requirement for RubisCO and the CBB pathway for growth under aerobic chemoautotrophic conditions. Strain 1312 is a mutant strain of HR which is unable to grow photoautotrophically because of a disruption in the cbbR gene which encodes an activator of the ebb opérons (Gibson and Tabita, 1993). This strain grows photoheterotrophically but at a reduced rate due to decreased ebb gene expression (Gibson and Tabita, 1993). As is the case for photoautotrophic growth of strain 1312, the inability of this strain to acquire the CAC phenotype is probably due to its 149 R. sphaeroides G row th R. capsulatus G row th Strain Strain HR + /- BIO +++ HR-CAC ++ SB1003 +++ 2.4.1 + /- VW2 +++ 2.4.1 GA + /- Kbl + DSM158 + /- LB2 + Gelled + C3 +++ 21.7 + C4 +++ 2.4.7 ++ JH2 +++ SPlOl +++ “(+++) = very good; (++) = good; (+) = poor; (+/-) = very poor. Table 4.5. Chemoautotrophic growth of R. sphaeroides and R. capsulatus strains 150 R. sphaeroides Ability to Acquire CAC Strain® Phenotype HR HR-FT HR-FIT 16 16 (pJG336) 16 (pJG106) 16-PHC 1312 4X ® HR, wild-type; FT, form I RubisCO deletion; FIT, form H RubisCO deletion; 16, form I and form H RubisCO-minus; 16 (pJG106), strain 16 complemented with theR. sphaeroides cbb\ operon; 16 (pJG106), strain 16 complemented with the R. sphaeroides cbb^ operon; 16-PHC, spontaneous mutant of strain 16 able to grow photoheterotrophically; 1312, cbbR' deletion strain; 4X, cbbXYZ operon deletion Table 4.6. Ability of R. sphaeroides HR-derivatives to acquire the CAC-Phenotype 151 inability to express sufficient levels of RubisCO and other CBB pathway enzymes under autotrophic conditions. R. sphaeroides strain HR-FT is unable to synthesize form I RubisCO due to the disruption of the cbbLcbbS genes, but is still able to synthesize form n RubisCO, which supports photoheterotrophic and photoautotrophic growth (Gibson et aL, 1991). Despite the fact that this strain did not adapt under the selective conditions (Table 4.6), a form I RubisCO-minus strain constructed from HR-CAC (strain FT-CAC; data not shown) was able to grow under aerobic chemoautotrophic conditions. It is likely that, with additional attempts or an increased adaptation period, a CAC strain of HR-FT might be obtained. In addition to R. sphaeroides wild-type strain HR, strains HR-FIT, 4X, and strain 16 containing cosmids pJG336 or pJG106 were able to gain the ability to grow chemoautotrophically (Table 4.6). Strain HR-FIT is unable to synthesize form II RubisCO due to the disruption of the cbbM gene (Gibson et at., 1991), but can still synthesize form I RubisCO. The growth of strain 16 complemented with plasmid pJG336 (containing the R. sphaeroides form I ebb operon) or plasmid pJG106 (containing the R. sphaeroides form II ebb operon) under selective conditions is consistent with the requirement for the CBB pathway under aerobic chemoautotrophic conditions. Strain 4X was able to gain the CAC phenotype (Table 4.6) even though this strain lacks any detectable phosphoglycolate phosphatase activity (Gibson and Tabita, 1997). RubisCO activity of RubisCO-deletioii strains of R. sphaeroides and R. capsulatus. Strains of R. sphaeroides and R. capsulatus lacking either form I or form n RubisCO were tested for the ability to support chemoautotrophic growth at differing 152 COz concentrations. The oxygen concentration was constant at 10.5 %. As described above, the H. sphaeroides form II RubisCO-minus strain, RT-CAC, was a spontaneous CAC mutant of strain HR-FIT, and the R. sphaeroides form I RubisCO-minus strain, CAC-FT, was constructed by insertional inactivation of the form I RubisCO genes of HR- CAC. The R. capsulatus RubisCO deletion strains lacking form I or form II RubisCO, SET and SBIT, respectively, were constructed by insertional inactivation of the respective RubisCO gene in the wild-type strain SB 1003 (this work. Chapter 3). R. sphaeroides HR-CAC and RubisCO-minus derivatives of HR-CAC grew under aerobic chemoautotrophic conditions at each of the CO 2 concentrations tested, indicating that either form of RubisCO can support aerobic chemoautotrophic growth. All of the strains grew with a doubling time of 26 hours ± 15 % (results not shown). No decrease in growth rate was observed at the lower CO 2 concentrations. Since other studies have shown that photoautotrophic cultures of R. sphaeroides grown at 2.5 % CO 2 displayed only about two thirds of both RubisCO activity and RubisCO protein levels compared to cultures grown at 1.5 % CO2 (Jouanneau and Tabita, 1986), RubisCO activities were determined for each strain at different CO 2 concentrations under chemoautotrophic growth conditions. For strain HR-CAC the decrease in CO 2 concentration from 5.0 % to 2.5 % resulted in about twice the RubisCO activity (Table 4.7). No further increase in RubisCO activity was observed when HR- CAC was grown in an atmosphere containing 0.75 % CO 2 . 153 RubisCO Activity® Strain (% CO2) 5.0% 2.5% 0.75 % EÏR-CAC 93 185 183 CAC-FT 48 57 62 Fir-CAC 293 224 257 “nmol/min/mg Numbers are averages of duplicate experiments Table 4.7. RubisCO activity of R. sphaeroides strains grown chemoautotro phically at various 00% concentrations 154 When RubisCO activity of the R. sphaeroides form I or form H RubisCO-minus strains was measured under photoheterotrophic and photoautotrophic conditions it was found that dismption of one RubisCO gene resulted in elevated synthesis of the other form of RubisCO, such that RubisCO activity reached or exceeded the levels of wild-type cells (Gibson et al, 1991). The relatively high level of RubisCO activity in strain FIT-CAC compared to HR-CAC at each CO 2 concentration suggests that the synthesis of form I RubisCO increases in response to the absence of form II RubisCO under chemoautotrophic conditions (Table 4.7). It must be stressed that protein levels were not determined directly and this change in activity could also be the result of post- translational modification of RubisCO. No such compensation in form II activity by strain CAC-FT was observed, since the levels of form II activity were uniformly low at each CO2 concentration. As strain CAC-FT was generated fi-om a CAC derivative of strain HR and strain FIT-CAC was selected in an independent experiment, these strains are not necessarily isogenic. Thus, it is not clear whether the differences in compensation in RubisCO activity by these two strains are the result of the growth condition or reflect regulatory differences in the two strains. The level of RubisCO activity at various CO 2 concentrations was also determined for R. capsulatus wild-type and RubisCO-minus strains grown chemoautotrophically. R. capsulatus strains SB 1003 and SBIT grew at each of the CO 2 concentrations tested. The CO2 concentrations at which the R. capsulatus strains were grown (Table 4.8) are different from those at which theR. sphaeroides strains were grown (Table 4.7). Strain SET grew at both 5.0 % CO 2 and 1.5 % CO 2 . At a concentration of 0.25 % CO 2 155 RubisCO Activity “ Strain (% CO2) 5.0% 1.5% 0.25 % SB 1003 82 74 96 SBF 90 57 102” SBir 50 36 63 “nmol/min/mg ^This strain was able to grow under these conditions only after a significant lag and may represent a spontaneous mutant to chemoautotrophic competency. Although the activity presented was determined for the adapted strain, the culture that did not grow had the same RubisCO activity. Numbers are averages of duplicate experiments. Table 4.8. RubisCO activity in R. capsulatus strains grown chemoautotro phically at various CO? concentrations 156 one of the duplicate cultures of SBF failed to grow and the other culture began to grow after a 12 day lag (results not shown). Strains SB 1003 and SBIT showed no such lag, starting to grow after two days at 0.25 % CO;. All of the strains grew with comparable doubling times (26 hours ± 15 %) at each of the CO? concentrations tested, including the SBF culture that began to grow after a 12 day lag. Whether or not SBF is unable to grow at 0.25 % CO2 or if the SBF strain that grew after a long lag is a spontaneous mutant that has acquired the ability to grow under these conditions awaits confirmation. The only difference between strains SBIF and SBF is in the type of RubisCO each expresses. If the form I RubisCO-expressing strain (SBIT) is able to grow at 0.25 % CO2/IO. 5 % O2 but the form II RubisCO strain (SBF) is not, this difference should reside in the inherent substrate specificity of the RubisCO expressed by each strain. The RubisCO activities of R. sphaeroides HR-CAC and R. capsulatus SB 1003 are nearly the same at 5.0 % CO2 (Tables 4.7, 4.8). Unlike R. sphaeroides, the amount of RubisCO activity in R. capsulatus SB 1003 did not increase in response to lower CO 2 concentrations under aerobic chemoautotrophic conditions (Table 4.8). At each CO 2 concentration tested, the level of form I RubisCO activity in strain SBIF was about half of the form H RubisCO activity in strain SBF (Table 4.8). This is opposite to the response observed in the RubisCO-minus strains of R. sphaeroides where the form I expressing strain displayed higher activities than the form II expressing strain (Table 4.7). The differences in RubisCO activity between R. capsulatus and R. sphaeroides are most pronounced in the strains expressing only form I RubisCO, strains SBIF and Mi CAC, respectively. The differences in regulation of expression or activity of form I RubisCO in 157 R. capsulatus and R. sphaeroides may be the result of the different evolutionary origins of the form I RubisCO in these two organisms (this work. Chapter 2). In addition, the form I RubisCO from these two species may have different biochemical properties. Subsequent work in our laboratory (Kempton Horken, personal communication) has shown that the form I enzyme from R. capsulatus has an unusually poor affinity for CO 2, in keeping with its unique evolutionary lineage. 158 DISCUSSION In an earlier investigation R. capsulatus was shown to grow under aerobic chemoautotrophic conditions (Madigan and Gest, 1979). In the same study Rhodospirillum rubrum and Rhodobacter sphaeroides were found to be poorly capable or incapable of growth under these conditions. We report here the isolation of a spontaneous mutant of R. sphaeroides strain HR that has acquired the ability to grow under aerobic chemoautotrophic conditions. This is not the first demonstration of such a “gain of function” mutation in purple nonsulfur photosynthetic bacteria. Wild-type strains of R. capsulatus are unable to utilize glycerol as a carbon source, but Lucking et al. (1973) isolated a spontaneous mutant of R. capsulatus that could assimilate glycerol. The mutant, strain LI. showed an increase in the activities of both glycerokinase and glycerophosphate dehydrogenase. Although the specific mutation that allowed strain Li to utilize glycerol was not determined, the concomitant increase in the activity of two different enzymes suggests that the mutation was in a regulatory element affecting the expression or activity of these enzymes. Mutant strains of R. sphaeroides in which cytochrome C; has been disrupted are unable to grow photosynthetically. Spontaneous mutations occur that allow cytochrome cz-independent photosynthetic growth (Rott and Donohue, 1990). One such mutant strain was found to synthesize an alternative cytochrome, referred to as isocytochrome Ci, that was necessary and sufficient for cytochrome cz-independent photosynthetic growth (Rott et al., 1993). Detailed analysis of this mutant strain revealed a mutation within the promoter of the isocytochrome c-i gene. This mutation resulted in increased expression of this gene under the photosynthetic conditions in which the mutant strain was isolated (Tim Donohue, personal communication). Another example of a spontaneous “gain of function” 159 mutation occurred in the R. sphaeroides HR RubisCO-minus mutant, strain 16. Strain I6PHC is a spontaneous mutant, derived from strain 16, that is capable of photoheterotrophic growth in the absence of detectable RubisCO activity (Wang et al., 1993). Strain 16-PHC was found to produce H 2 gas and deregulate the expression of nitrogenase, showing considerable nitrogenase activity in the presence of ammonium (Joshi and Tabita, 1996). Strain 16PHC is thus able to utilize protons as an alternative electron acceptor, catalyzing their reduction via nitrogenase. The ability to adapt to such diverse physiological conditions underlie the considerable metabolic versatility of the purple nonsulfur photosynthetic bacteria. The chemoautotrophically competent mutant of R. sphaeroides , strain HR-CAC, possessed levels of RubisCO activity comparable to that of the wild-type strain under a variety of growth conditions. The ability to acquire the CAC phenotype was dependent upon RubisCO expression in the parent strain. Strains of R. capsulatus that have lost a functional CBB pathway, because of disruptions in either the genes encoding the two forms of RubisCO or the gene encoding phosphoribulokinase, lose the ability to grow chemoautotrophically. Thus, chemoautotrophic growth in purple nonsulfur bacteria is dependent on the CBB pathway for CO 2 fixation (see also. Chapter 3). Most of the wild- type R. capsulatus strains and at least one wild-type strain of R. sphaeroides grew chemoautotrophically, suggesting that the ability to grow under these conditions may have been recently lost in R. sphaeroides. The RubisCO substrate specificity factor, T, relates the ratio of the velocity of carboxylation and oxygenation to the ratio of CO 2 and O 2 concentration: V c ^ o = T([C02]/[02]). Since the oxygenase reaction causes the loss of carbon from the cell, Vc/Vo must exceed some critical value in order for RubisCO to catalyze net carbon assimilation. For example, in higher plants it has been estimated that 0.5 carbons are lost for each O2 fixed 160 (Gutteridge, 1989). Therefore, Vc/Vo must be greater than 0.5 in order for RubisCO to catalyze net carbon fixation and support autotrophic growth. Accordingly, the [COzj/EO?] ratio can be adjusted such that the Vc/Vo would be growth-limiting, hicubation of an organism at the growth-limiting condition would provide a biological selection for RubisCO with increased substrate specificity. The [COzl/COz] ratio at which growth limitation would occur would depend upon the T of the RubisCO expressed. A mutant strain of Synechocystis 6803 was constructed in which the natural RubisCO gene was replaced by the R. rubrum RubisCO gene (Pierce et ai, 1989). Growth of this “cyanorubrum” strain was dependent on increased atmospheric CO 2 due to the organism’s dependence on the form H RubisCO from R. rubrum. Cyanorubrum grew poorly at 0.5 % CO 2 and did not grow at 0.1 % CO 2 (in 21 % O 2). This was the first system developed to select mutant RubisCO with increased substrate specificity; however, inherent problems due to the physiology of the organism severely limited the flexibility of this system. The strains of R. capsulatus and R. sphaeroides that synthesize only form I or form n RubisCO differ only in the T of their respective RubisCO. A [COzj/COz] ratio at which the form I-synthesizing strain grows but the form H-synthesizing strain does not grow would be an ideal condition to select a form II RubisCO with increased substrate specificity. Analysis of such RubisCO mutants should allow a greater understanding of the structural basis for the discrimination between the gaseous substrates. Although confirmation of the results is necessary, preliminary experiments described here suggest that concentrations at or near 0.25 % CO 2 and 10.5 % O2 may be a proper gas ratio to effect such selection. In addition to the R. capsulatus strains expressing only one form of RubisCO, a strain has been constructed in which neither RubisCO is expressed. This strain, SBI-II, does not grow autotrophically, but can be complemented to both photoautotrophic and 161 chemoautotrophic growth by a variety of RubisCO genes cloned into the expression vector pRPS-1. RubisCO expression constructs in pRPS-1 could be randomly mutagenized and introduced into strain SBI-II to screen large numbers of mutant RubisCOs for increased substrate specificity. The metabolic versatility, genetic adaptability, and ease with which the nonsulfiir purple photosynthetic bacteria can be genetically manipulated make this combination of mutants strains and RubisCO clones an ideal system for the biological selection of RubisCO with increased substrate specificity. ACKNOWLEDGEMENTS The author would like to thank Dr. Michael Madigan for providing the R. capsulatus and R. sphaeroides wild-type strains and Mark Harding, whose work allowed the original observation of CAC growth in R. sphaeroides. 162 CHAPTERS SUMMARY »- Organization and Phylogeny of the R. capsulatus ebb genes. The organization of the R. capsulatus ebb genes (Fig. 5.1 A) was determined by a combination of Southern hybridization analysis and nucleotide sequencing. The arrangement of genes in the R. capsulatus presumptive c66n operon was identical to the cbbvi operon of R. sphaeroides (Fig. 5.1 A 3 ). Unlike the situation in the closely related organism, R. sphaeroides, a cbbR gene was found upstream and divergently transcribed from the R. capsulatus cbbn operon. This gene was designated cbbRn because of its association with the cbbn operon. In R. sphaeroides a set of genes with no homology to any other known genes is found upstream of the c66n operon (Fig. 5.1 B) The R. capsulatus cbbLcbbS and cbbM genes, encoding form I and form H RubisCO, respectively, were subcloned and functionally expressed in both E. coli and R. sphaeroides. Western immunoblot analysis of the recombinant R. capsulatus form I RubisCO expressed in E. coli confirmed that this RubisCO was distinct from the R. sphaeroides form I enzyme. Unlike the R. sphaeroides cbbLcbbS genes, the R. capsulatus form I RubisCO genes were not associated with any other genes encoding CBB pathway enzymes (Fig. 5.1 163 Fig. 5.1. Organization of the ebb genes in (A) R. capsulatus and (B) R. sphaeroides. The ebb genes are represented by single letter designations. Other genes are given full gene designations. The arrow under the genes represents the direction of transcription and the size of potential transcripts. The designation of transcripts for the R. capsulatus genes is tentative. Curved arrows indicate transcriptional activation at the specified ebb promoters by the designated CbbR. 164 anfA. R, L, S| Q I__ f pgm qor F. Tn Gp Ag Mg E B F, P, A, L, S, X Y 2 Gg Ag Mg orfW orfUlorfUl Figure 5.1 165 A3)- Nucleotide sequencing of the region upstream of the R. capsulatus form I RubisCO genes, cbbLcbbS, indicated that a cbbR gene was directly upstream and divergently transcribed from cbbL (Fig. 5.1 A). This gene was designated cbbR\ because of its association with the form I RubisCO genes. A cbbQ gene, encoding a protein of unknown function, was found just downstream of cbbS. Database searches revealed that the R. capsulatus cbbL deduced amino acid sequence was most similar to CbbL from Pseudomonas hydrogenothermophila, showing considerably less similarity to R. sphaeroides CbbL. This was particularly interesting because P. hydrogenothermophila is one of only two organisms (the other is Chromatium vinosum) in which a cbbQ gene is immediately downstream of cbbS. Subsequent phylogenetic analysis of R. capsulatus CbbL indicated that it is indeed closely related to the P. hydrogenothermophila RubisCO large subunit and very distantly related to R. sphaeroides CbbL. Phylogenetic analysis of the CbbR proteins revealed a similar relationship between CbbR; and CbbR of Thiobacillus ferrooxidans. Taken together, these results suggest that the R. capsulatus cbbR\, cbbLcbbS, and cbbQ were acquired by horizontal gene transfer from an organism related to P. hydrogenothermophila. Regulation of the R. capsulatus ebb genes. The regulation of the R. capsulatus ebb genes was studied by using a combination of gene disruption strains and ebb promoter fusions. Construction of cbbL' and cbbM~ strains showed that either form of RubisCO will support photoheterotrophic, photoautotrophic, and chemoautotrophic growth. Although the wild-type strain did not 166 express form I RubisCO under photoheterotrophic conditions, the cbbhT strain grew photoheterotrophically. Apparently the lack of form II RubisCO in this strain somehow signaled the organism to express the cbbLcbbS genes and synthesize form I RubisCO under photoheterotrophic conditions, hr addition, a strain was constructed in which both the cbbL and cbbM genes were disrupted. This strain was unable to grow autotrophically or photoheterotrophically in the absence of DMSO and lacked any detectable RubisCO activity. This RubisCO-minus strain could be complemented to autotrophic and photoheterotrophic growth by a number of RubisCO gene constructs. Complementation by either theR. capsulatus cbbLcbbS or cbbM genes in the expression vector pRPS-1 resulted in levels of RubisCO activity comparable to the wild-type strain under both photoheterotrophic and photoautotrophic growth conditions. Finally, southern blot analysis at low stringency indicated that, unlike R. sphaeroides, only one cbbP gene is present in R. capsulatus. The fact that acbbPa strain was unable to grow autotrophically or photoheterotrophically in the absence of DMSO and lacked any detectable PRK activity further confirmed the presence of only one cbbP gene in R. capsulatus. A strain in which the cbbRxL gene was disrupted was unable to grow autotrophically and grew photoheterotrophically at a reduced rate. This strain demonstrated reduced RubisCO and PRK activities compared to the wild-type strain under photoheterotrophic conditions. The cbbRxi strain was complemented to autotrophic growth when the cbbRn gene was cloned on a low copy number vector, but RubisCO and PRK activities did not return to wild-type levels. Plasmids containing the cbb\ promoter and cbb\i promoter translationally fused to P-galactosidase were constructed and 167 introduced into the wild-type and cbbRù strains. P-galactosidase activity in these strains showed that the cbb\ promoter was not active under photoheterotrophic conditions, however the cbbu promoter was active under photoheterotrophic conditions. Both the cbb\ and cbbu promoters were induced under photoautotrophic conditions. CbbRn activated transcription from the cbbn promoter; the p-galactosidase activity from the cbbn promoter construct in the cbbRn strain was only 5 % of the wild-type activity. Chemoautotrophic growth and biological selection for improved RuhisCO. A spontaneous mutant of R. sphaeroides strain HR was selected that had gained the ability to grow under aerobic chemoautotrophic conditions. Characterization of this chemoautotrophic competent strain (HR-CAC) indicated that RubisCO activity was regulated normally under aerobic chemoheterotrophic, photoheterotrophic and photoautotrophic conditions. Characterization of a number of wild-type strains of R. sphaeroides and R. capsulatus showed that the ability to grow chemoautotrophically is unusual in R. sphaeroides but quite common in R. capsulatus. In addition, CAC strains of R. sphaeroides that expressed only form I or form II RubisCO were selected or constructed. These strains, and the form I- or form Il-expressing strains of R. capsulatus, were grown under aerobic chemoautotrophic conditions at differing CO 2 concentrations. 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