Physical map of the genome of Rhodobacter capsulatus SB 1003.

A map of the chromosome of Rhodobacter capsulatus was constructed by overlapping the large restriction fragments generated by endonucleases AseI and XbaI. The analyses were done by hybridization of single fragments with the restriction fragments blotted from pulsed-field gels and by grouping cosmids of a genomic library of R. capsulatus into contigs, corresponding to the restriction fragments, and further overlapping of the contigs. A technical difficulty due to a repeated sequence made it necessary to use hybridization with cloned genes and prior knowledge of the genetic map in order to close the physical circle in a unique way. In all, 41 restriction sites were mapped on the 3.6-Mb circular genome and 22 genes were positioned at 26 loci of the map. Cosmid clones were grouped in about 80 subcontigs, forming two groups, one corresponding to the chromosome of R. capsulatus and the other corresponding to a 134-kb plasmid. cos site end labeling and partial digestion of cosmids were used to construct a high-resolution EcoRV map of the 134-kb plasmid. The same method can be extended to the entire chromosome. The cosmid clones derived in this work can be used as a hybridization panel for the physical mapping of new genes as soon as they are cloned.     

Phototrophic Fe(II) oxidation promotes organic carbon acquisition by Rhodobacter capsulatus SB1003

Anoxygenic phototrophic Fe(II) oxidation is usually considered to be a lithoautotrophic metabolism that contributes to primary production in Fe-based ecosystems. In this study, we employed Rhodobacter capsulatus SB1003 as a model organism to test the hypothesis that phototrophic Fe(II) oxidation can be coupled to organic carbon acquisition. R. capsulatus SB1003 oxidized Fe(II) under anoxic conditions in a light-dependent manner, but it failed to grow lithoautotrophically on soluble Fe(II). When the strain was provided with Fe(II)-citrate, however, growth was observed that was dependent upon microbially catalyzed Fe(II) oxidation, resulting in the formation of Fe(III)-citrate. Subsequent photochemical breakdown of Fe(III)-citrate yielded acetoacetic acid that supported growth in the light but not the dark. The deletion of genes (RRC00247 and RRC00248) that encode homologs of atoA and atoD, required for acetoacetic acid utilization, severely impaired the ability of R. capsulatus SB1003 to grow on Fe(II)-citrate. The growth yield achieved by R. capsulatus SB1003 in the presence of citrate cannot be explained by lithoautotrophic growth on Fe(II) enabled by indirect effects of the ligand [such as altering the thermodynamics of Fe(II) oxidation or preventing cell encrustation]. Together, these results demonstrate that R. capsulatus SB1003 grows photoheterotrophically on Fe(II)-citrate. Nitrilotriacetic acid also supported light-dependent growth on Fe(II), suggesting that Fe(II) oxidation may be a general mechanism whereby some Fe(II)-oxidizing bacteria mine otherwise inaccessible organic carbon sources.     

Phototrophic Fe(II) Oxidation Promotes Organic Carbon Acquisition by Rhodobacter capsulatus SB1003

Anoxygenic phototrophic Fe(II) oxidation is usually considered to be a lithoautotrophic metabolism that contributes to primary production in Fe-based ecosystems. In this study, we employed Rhodobacter capsulatus SB1003 as a model organism to test the hypothesis that phototrophic Fe(II) oxidation can be coupled to organic carbon acquisition. R. capsulatus SB1003 oxidized Fe(II) under anoxic conditions in a light-dependent manner, but it failed to grow lithoautotrophically on soluble Fe(II). When the strain was provided with Fe(II)-citrate, however, growth was observed that was dependent upon microbially catalyzed Fe(II) oxidation, resulting in the formation of Fe(III)-citrate. Subsequent photochemical breakdown of Fe(III)-citrate yielded acetoacetic acid that supported growth in the light but not the dark. The deletion of genes (RRC00247 and RRC00248) that encode homologs of atoA and atoD, required for acetoacetic acid utilization, severely impaired the ability of R. capsulatus SB1003 to grow on Fe(II)-citrate. The growth yield achieved by R. capsulatus SB1003 in the presence of citrate cannot be explained by lithoautotrophic growth on Fe(II) enabled by indirect effects of the ligand [such as altering the thermodynamics of Fe(II) oxidation or preventing cell encrustation]. Together, these results demonstrate that R. capsulatus SB1003 grows photoheterotrophically on Fe(II)-citrate. Nitrilotriacetic acid also supported light-dependent growth on Fe(II), suggesting that Fe(II) oxidation may be a general mechanism whereby some Fe(II)-oxidizing bacteria mine otherwise inaccessible organic carbon sources.     

The fox operon from Rhodobacter strain SW2 promotes phototrophic Fe(II) oxidation in Rhodobacter capsulatus SB1003

Anoxygenic photosynthesis based on Fe(II) is thought to be one of the most ancient forms of metabolism and is hypothesized to represent a transition step in the evolution of oxygenic photosynthesis. However, little is known about the molecular basis of this process because, until recently (Y. Jiao and D. K. Newman, J. Bacteriol. 189:1765-1773, 2007), most phototrophic Fe(II)-oxidizing bacteria have been genetically intractable. In this study, we circumvented this problem by taking a heterologous-complementation approach to identify a three-gene operon (the foxEYZ operon) from Rhodobacter sp. strain SW2 that confers enhanced light-dependent Fe(II) oxidation activity when expressed in its genetically tractable relative Rhodobacter capsulatus SB1003. The first gene in this operon, foxE, encodes a c-type cytochrome with no significant similarity to other known proteins. Expression of foxE alone confers significant light-dependent Fe(II) oxidation activity on SB1003, but maximal activity is achieved when foxE is expressed with the two downstream genes foxY and foxZ. In SW2, the foxE and foxY genes are cotranscribed in the presence of Fe(II) and/or hydrogen, with foxZ being transcribed only in the presence of Fe(II). Sequence analysis predicts that foxY encodes a protein containing the redox cofactor pyrroloquinoline quinone and that foxZ encodes a protein with a transport function. Future biochemical studies will permit the localization and function of the Fox proteins in SW2 to be determined.     

ORF90, a Gene Required for Photoreactivation in Rhodobacter capsulatus SB1003 Encodes a Cyclobutane Pyrimidine Dimer Photolyase

Based on deduced amino-acid sequence similarities to class-I photolyases, the open reading frame ORF90 was identified from the genome sequence of Rhodobacter capsulatus SB1003. Photoreactivation activity is not detectable in an ORF90 deletion mutant of R. capsulatus SB1003. The phenotype of R. capsulatus wild-type cells was restored by plasmid borne ORF90 of R. capsulatus DeltaORF90. Furthermore, we detected an ORF90-related CPD-specific photoreactivation activity in R. capsulatus cell extracts. The results show that the gene product of ORF90 is involved in photoreactivation and encodes a class-I cyclobutane pyrimidine dimer photolyase.     

Effects of light intensity distribution on growth of Rhodobacter capsulatus

For cultivation of photosynthetic cells under defined light intensity distributions, the repeated batch culture, in which a part of culture broth containing grown cells was repeatedly replaced at predetermined time intervals with a fresh medium to keep the cell concentration constant at an initial value, was employed. By use of this method the effects of the light intensity distribution on the growth characteristics of Rhodobacter capsulatus were studied. Unexpected decreases in the specific growth rate were observed in culture of R. capsulatus at high cell concentrations and a long light path length. Big differences in the light intensities of lightly and darkly illuminated portions in photobioreactors, which reflects the light intensity distribution, seemed to cause this phenomenon, which must be taken into consideration for stable growth of photosynthetic cells.     

Cytochrome c2-independent respiratory growth of Rhodobacter capsulatus.

To assess the role of cytochrome c2 as a respiratory electron carrier, we obtained a double mutant of Rhodobacter capsulatus defective in cytochrome c2 and in the quinol oxidase260. This mutant was able to grow chemoheterotrophically, indicating that an electron pathway independent of cytochrome c2 was functional between the ubiquinol:cytochrome c2 oxidoreductase and the cytochrome oxidase410.     

A denitrifying strain of Rhodobacter capsulatus

Abstract Repeated subculturing of Rhodobacter capsulatus strain BK5 under phototrophic conditions on a medium containing butyrate and nitrate led to the appearance of cultures that, unlike the original, produced gas. Isolation of a pure culture of the gas-forming organism revealed that it was a derivative of R. capsulatus BK5 that by virtue of expressing a nitrite reductase can catalyse the complete sequence of the denitrification reactions. The nitrite reductase is of the type that contains copper rather than haem.     

Non-reciprocal regulation of Rhodobacter capsulatus and Rhodobacter sphaeroides recA genes expression

Abstract The Rhodobacter capsulatus recA gene has been isolated and sequenced. Its deduced amino acid sequence showed the closest identity with the Rhodobacter sphaeroides RecA protein (91% identity). However, the promoter regions of both R. capsulatus and R. sphaeroides recA genes are only 64% similar. An Escherichia coli -like LexA binding site was not present in the upstream region of the R. capsulatus recA gene. Nevertheless, the R. capsulatus recA gene is inducible by DNA damage in both hetero- and phototrophically growing conditions. The R. capsulatus recA gene is poorly induced when inserted into the chromosome of R. sphaeroides , indicating that the recA gene of both bacteria possess different control sequences despite their phylogenetically close relationship.     

A structured model for Thiobacillus ferrooxidans growth on ferrous iron

A structured model for Thiobacillus ferrooxidans growth dependence on ferrous and ferric iron, arsenic, oxygen, carbon dioxide, pH, and temperature is presented. A new kinetic mechanism for ferrous oxidation by T. ferrooxidans is introduced. Data from several earlier experimental studies of T. ferroaxidans growth are used for model development. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 310-319, 1997.     

Aerobic chemolithoautotrophic growth and RubisCO function in Rhodobacter capsulatus and a spontaneous gain of function mutant of Rhodobacter sphaeroides

Photosynthetic prokaryotes that assimilate CO₂ under anoxic conditions may also grow chemolithoautotrophically with O₂ as the electron acceptor. Among the nonsulfur purple bacteria, two species (Rhodobacter capsulatus and Rhodopseudomonas acidophilus), exhibit aerobic chemolithoautotrophic growth with hydrogen as the electron donor. Although wild-type strains of Rhodobacter sphaeroides grow poorly, if at all, with hydrogen plus oxygen in the dark, we report here the isolation of a spontaneous mutant (strain HR-CAC) of Rba. sphaeroides strain HR that is fully capable of this mode of growth. Rba. sphaeroides and Rba. capsulatus fix CO₂ via the reductive pentose phosphate pathway and synthesize two forms of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). RubisCO levels in the aerobic-chemolithoautotrophic-positive strain of Rba. sphaeroides were similar to those in wild-type strains of Rba. sphaeroides and Rba. capsulatus during photoheterotrophic and photolithoautotrophic growth. Moreover, RubisCO levels of Rba. sphaeroides strain HR-CAC approximated levels obtained in Rba. capsulatus when the organisms were grown as aerobic chemolithoautotrophs. Either form I or form II RubisCO was able to support aerobic chemolithoautotrophic growth of Rba. capsulatus strain SB 1003 and Rba. sphaeroides strain HR-CAC at a variety of CO₂ concentrations, although form II RubisCO began to lose the capacity to support aerobic CO₂ fixation at high O₂ to CO₂ ratios. The latter property and other facets of the physiology of this system suggest that Rba. sphaeroides and Rba. capsulatus strains may be effectively employed for the biological selection of RubisCO molecules of altered substrate specificity. Received: 8 August 1997 / Accepted: 26 December 1997     

Intracytoplasmic membrane vesiculation in light-harvesting mutants of Rhodobacter sphaeroides and Rhodobacter capsulatus

Abstract In Chlamydomonas reinhardtii there are three glutamate dehydrogenase isozymes which can use both NADH and NADPH as cofactors and respond differently to different nitrogen sources and several stress conditions. From data of induction of isozymes in different metabolic situations, we propose a possible physiological role for each of them in algal carbon and nitrogen metabolism.     

Inactivation of Mg chelatase during transition from anaerobic to aerobic growth in Rhodobacter capsulatus

The facultative photosynthetic bacterium Rhodobacter capsulatus can adapt from an anaerobic photosynthetic mode of growth to aerobic heterotrophic metabolism. As this adaptation occurs, the cells must rapidly halt bacteriochlorophyll synthesis to prevent phototoxic tetrapyrroles from accumulating, while still allowing heme synthesis to continue. A likely control point is Mg chelatase, the enzyme that diverts protoporphyrin IX from heme biosynthesis toward the bacteriochlorophyll biosynthetic pathway by inserting Mg(2+) to form Mg-protoporphyrin IX. Mg chelatase is composed of three subunits that are encoded by the bchI, bchD, and bchH genes in R. capsulatus. We report that BchH is the rate-limiting component of Mg chelatase activity in cell extracts. BchH binds protoporphyrin IX, and BchH that has been expressed and purified from Escherichia coli is red in color due to the bound protoporphyrin IX. Recombinant BchH is rapidly inactivated by light in the presence of O(2), and the inactivation results in the formation of a covalent adduct between the protein and the bound protoporphyrin IX. When photosynthetically growing R. capsulatus cells are transferred to aerobic conditions, Mg chelatase is rapidly inactivated, and BchH is the component that is most rapidly inactivated in vivo when cells are exposed to aerobic conditions. The light- and O(2)-stimulated inactivation of BchH could account for the rapid inactivation of Mg chelatase in vivo and provide a mechanism for inhibiting the synthesis of bacteriochlorophyll during adaptation of photosynthetically grown cells to aerobic conditions while still allowing heme synthesis to occur for aerobic respiration.     

Cloning of the Rhodobacter capsulatus hemA gene.

Portions of the Rhodobacter capsulatus hemA gene have been cloned from a hemA::Tn5 insertion strain into the lambda bacteriophage derivative EMBL3. A cosmid containing the wild-type R. capsulatus hemA gene was isolated by complementation of the hemA::Tn5 mutant. The cosmid contains a 1.4-kilobase EcoRI fragment that spans the hemA::Tn5 insertion site. The entire hemA gene is contained in this fragment and the adjacent 0.6-kilobase EcoRI fragment.     

Cytochrome c2 mutants of Rhodobacter capsulatus.

Although structurally related to other members of the class I c-type cytochromes, the cytochromes c2 have little amino acid sequence homology to the eukaryotic cytochromes c. Moreover, the cytochromes c2 exhibit distinct properties such as redox potential and an isoelectric point. In an effort to understand the differences between the cytochromes c2 and the other class I c-type cytochromes, we have developed a genetic system to study Rhodobacter capsulatus cytochrome c2 by site-directed mutagenesis. We describe here overproduction of R. capsulatus wild-type cytochrome c2 in cytochrome c2-minus strains of R. capsulatus and Rhodobacter sphaeroides. We demonstrate that R. capsulatus wild-type cytochrome c2 can transcomplement for photosynthetic growth in R. sphaeroides. Further, we describe the generation, expression, and in vivo functionality properties of nine R. capsulatus site-directed mutants. We show that mutants K12D, K14E, K32E, K14E/K32E, P35A, W67Y, and Y75F are overproduced and functional in vivo. In contrast, mutants Y75C and Y75S are expressed at low levels and exhibit poor functionality in vivo. These findings establish an effective system for the production of R. capsulatus site-directed mutants and demonstrate that interspecies complementation can be used to detect defective cytochrome c2 mutants.     

Primary structure of porin from Rhodobacter capsulatus.

The primary structure of the integral membrane protein porin from the purple bacterium Rhodobacter capsulatus was determined. The protein was cleaved with trypsin, CNBr and Asp-N protease. The peptides were isolated, sequenced and aligned to a total length of 301 residues with an Mr of 31,536. The low isoelectric point of 3.9 is confirmed by the high excess of 34 Asp and 17 Glu (16.9%) over 10 Lys, 7 Arg and 2 His (6.3%). Overall sequence similarity to other porins is not evident when using sequence alignment programs. However, a partial relationship to Neisseria porins seems to exist. The established sequence has been used as the basis for a three-dimensional structure determination by X-ray diffraction at 0.18-nm resolution. The arrangement of the sequence in the 16-stranded beta-barrel of porin is given. Some sequence-structure correlations are discussed.