Rhodobacter Capsulatus Mutants Research Articles

Kinetic analysis of photosynthetic growth and photohydrogen production of two strains of Rhodobacter Capsulatus

Two mutants of Rhodobacter Capsulatus (JP91 and IR3), a photosynthetic purple non-sulfur bacterium, were grown in a batch photobioreactor under illumination with 30 mmol l −1 dl-lactate and 5 mmol l −1 l-glutamate as carbon and nitrogen source, respectively. Bacterial growth was measured by monitoring the increase in absorbance at 660 nm. The photosynthetic growth processes under different cultivated temperatures are well fitted by a specific logistic model to analyze the kinetics of photosynthetic growth of two strains, thus the apparent growth rates ( k) of these photosynthetic bacteria, the variations of cell dry weight (CDW) as well as their relationship with temperature are obtained. In present work, k is (0.1465 ± 0.0146), (0.2266 ± 0.0207) and (0.3963 ± 0.0257) h −1 for JP91 and (0.1117 ± 0.0122), (0.1218 ± 0.0133) and (0.2223 ± 0.0152) h −1 for IR3 at 26, 30 and 34 °C, respectively. And the difference between CDW max and CDW 0 is (0.8997 ± 0.0097), (0.8585 ± 0.0093) and (0.9241 ± 0.0099) g l −1 for JP91 and (0.8167 ± 0.0089), (0.7878 ± 0.0086) and (0.8358 ± 0.0091) g l −1 for IR3 at 26, 30 and 34 °C, respectively. Also real-time monitoring of hydrogen production rates is acquired by recording the flow rates of photohydrogen for these two strains under different temperatures. The effects of temperature on the bacteria growth, hydrogen production capability and substrate conversion efficiency are discussed based on these results. The most preferment temperature, 30 °C, showed good substrate conversion efficiency of 52.7 and 68.2% for JP91 and IR3, respectively.

Positive and Negative Selection of Mutant Forms of Prokaryotic (Cyanobacterial) Ribulose-1,5-bisphosphate Carboxylase/Oxygenase

A system for biological selection of randomly mutagenized ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) genes from the cyanobacterium Synechococcus PCC6301 was designed in which a Rubisco deletion mutant of the photosynthetic bacterium Rhodobacter capsulatus served as a host. Trans-complementation with the Synechococcus PCC6301 rbcLS genes enabled anaerobic photoautotrophic growth of the R. capsulatus deletion strain with 5% CO 2, but not with 1.5% CO 2 in the atmosphere, and this strain could not grow under aerobic chemoautotrophic conditions. Phenotypic differences between the R. capsulatus host strain complemented with the wild-type rbcLS genes and transconjugates carrying mutated genes were used to identify mutants that were able to complement to photoautotrophic growth with 1.5% CO 2. These “positive” mutant proteins were unaffected for any measured kinetic properties, with a single exception. A mutant with a valine substitution at phenylalanine 342 had an increased affinity for ribulose-1,5-bisphosphate. Mutants with changes in the affinity for CO 2 were isolated through negative selection, in which mutants incapable of complementing R. capsulatus to photoautotrophic growth with 5% CO 2 were identified. Mutations at aspartate 103 resulted in enzymes that were greatly affected for different kinetic parameters, including an increased K m for CO 2. This study demonstrated that random mutagenesis and bioselection procedures could be used to identify mutations that influence important properties of bacterial Rubisco; these residues would not have been identified by other methods.

Detergent effects on primary charge separation in wild-type and mutant Rhodobacter capsulatus reaction centers

The primary electron-transfer processes in reaction centers (RCs) from wild-type and several mutants of Rhodobacter capsulatus have been investigated as a function of the detergent used to extract the RC protein from the membrane. Wild-type and L(M212)H mutant RCs that have been isolated using the detergent Deriphat 160-C both display somewhat slower initial charge separation (longer P* lifetimes) than the same RCs isolated using the detergent LDAO. For the F(L181)Y/Y(M208)F/L(M212)H triple mutant, the differences in the initial charge separation events for Deriphat-versus LDAO-isolated RCs are more significant. In all cases, use of Deriphat 160-C to extract the protein from the membrane yields RCs in which the QY band of P is retained at its native position near 865 nm, whereas LDAO extraction yields RCs that have the P band near 850 nm. Origins of the differences in both the ground state spectrum and the photochemistry, including possible RC–lipid associations, are considered.

Complex I and its involvement in redox homeostasis and carbon and nitrogen metabolism in Rhodobacter capsulatus.

A transposon mutant of Rhodobacter capsulatus, strain Mal7, that was incapable of photoautotrophic and chemoautotrophic growth and could not grow photoheterotrophically in the absence of an exogenous electron acceptor was isolated. The phenotype of strain Mal7 suggested that the mutation was in some gene(s) not previously shown to be involved in CO(2) fixation control. The site of transposition in strain Mal7 was identified and shown to be in the gene nuoF, which encodes one of the 14 subunits for NADH ubiquinone-oxidoreductase, or complex I. To confirm the role of complex I and nuoF for CO(2)-dependent growth, a site-directed nuoF mutant was constructed (strain SBC1) in wild-type strain SB1003. The complex I-deficient strains Mal7 and SBC1 exhibited identical phenotypes, and the pattern of CO(2) fixation control through the Calvin-Benson-Bassham pathway was the same for both strains. It addition, it was shown that electron transport through complex I led to differential control of the two major cbb operons of this organism. Complex I was further shown to be linked to the control of nitrogen metabolism during anaerobic photosynthetic growth of R. capsulatus.

Investigation of bacteriochlorophyll biosynthesis genes in Blastochloris viridis by functional complementation of mutants of Rhodobacter capsulatus

Blastochloris (formerly Rhodopseudomonas) viridis produces bacteriochlorophyll (BChl) b which is distinguished from BChl a by the possession of an ethylidene group at C8 instead of an ethyl. This chemical structure enables to BChl b to absorb longer wave length light (~1100 nm) than BChl a. To investigate genes involved in the BChl b biosynthesis, we have constructed genomic DNA libraries from B. viridis and introduced them to mutants of the nonsulfur purple bacterium Rhodobacter capsulatus which is deficient in BChl a biosynthesis. Total ten kinds of R. capsulatus mutants, three Mg-chelatase (BchD, H, I) mutants, three protochlorophyllide reductase (BchB, L, N) mutants, Mg-protoporphyrin IX methyltransferase (BchM) mutant, Mg-protoporphyrin IX monomethylester oxidative cyclase (BchE) mutant, bacteriochlorophyll synthase (Bch G) mutant, and 2-vinyl-bacteriochlorophyllide hydratase (BchF) mutant, were functionally complemented by B. viridis library DNA. The complemented R. capsulatus strains were demonstrated to be capable of synthesizing BChl a under photoautotrophic growth conditions.

Reversible inactivation of nitrogenase in Rhodobacter capsulatus strain W107I deleted in the draTG gene region

Upon addition of ammonium or removal of light, nitrogenase was reversibly inactivated in a draTG gene deletion mutant of Rhodobacter capsulatus. Reversible inactivation by exposure of the mutant to darkness followed the same kinetics as in control strains containing the draTG gene region. Reactivation of nitrogenase by light was significantly faster than removal of ADP-ribose from component 2 of nitrogenase. The results show that, in R. capsulatus, reversible inactivation of nitrogenase is independent of reversible ADP-ribosylation by the DRAT/DRAG enzyme system encoded by the draTG gene region.

Reversible inactivation of nitrogenase inRhodobacter capsulatusstrain W107I deleted in thedraTGgene region

Upon addition of ammonium or removal of light, nitrogenase was reversibly inactivated in a draTG gene deletion mutant of Rhodobacter capsulatus. Reversible inactivation by exposure of the mutant to darkness followed the same kinetics as in control strains containing the draTG gene region. Reactivation of nitrogenase by light was significantly faster than removal of ADP-ribose from component 2 of nitrogenase. The results show that, in R. capsulatus, reversible inactivation of nitrogenase is independent of reversible ADP-ribosylation by the DRAT/DRAG enzyme system encoded by the draTG gene region.

Isolation and characterization of a two-subunit cytochrome b-c1 subcomplex from Rhodobacter capsulatus and reconstitution of its ubihydroquinone oxidation (Qo) site with purified Fe-S protein subunit.

The presence of a two-subunit cytochrome (cyt) b-c1 subcomplex in chromatophore membranes of Rhodobacter capsulatus mutants lacking the Rieske iron-sulfur (Fe-S) protein has been described previously [Davidson, E., Ohnishi, T., Tokito, M., and Daldal, F. (1992) Biochemistry 31, 3351-3358]. Here, this subcomplex was purified to homogeneity in large quantities, and its properties were characterized. As expected, it contained stoichiometric amounts of cyt b and cyt c1 subunits forming a stable entity devoid of the Fe-S protein subunit. The spectral and thermodynamic properties of its heme groups were largely similar to those of a wild-type bc1 complex, except that those of its cyt bL heme were modified as revealed by EPR spectroscopy. Dark potentiometric titrations indicated that the redox midpoint potential (Em7) values of cytochromes bH, bL, and c1 were very similar to those of a wild-type bc1 complex. The purified b-c1 subcomplex had a nonfunctional ubihydroquinone (UQH2) oxidation (Qo) site, but it contained an intact ubiquinone (UQ) reductase (Qi) site as judged by its ability to bind the Qi inhibitor antimycin A, and by the presence of antimycin A sensitive Qi semiquinone. Interestingly, its Qo site could be readily reconstituted by addition of purified Fe-S protein subunit. Reactivated complex exhibited myxothiazol, stigmatellin, and antimycin A sensitive cyt c reductase activity and an EPR gx signal comparable to that observed with a bc1 complex when the Qo site is partially occupied with UQ/UQH2. However, a mutant derivative of the Fe-S protein subunit lacking its first 43 amino acid residues was unable to reactivate the purified b-c1 subcomplex although it could bind to its Qo site in the presence of stigmatellin. These findings demonstrated for the first time that the amino-terminal membrane-anchoring domain of the Fe-S protein subunit is necessary for UQH2 oxidation even though its carboxyl-terminal domain is sufficient to provide wild-type-like interactions with stigmatellin at the Qo site of the bc1 complex.

Hydrogen bonding and circular dichroism of bacteriochlorophylls in the Rhodobacter capsulatus light-harvesting 2 complex altered by combinatorial mutagenesis.

We have investigated the spectroscopic properties of two classes of light-harvesting 2 (LH2, B800-850) mutants of Rhodobacter capsulatus obtained by combinatorial mutagenesis to the C-terminal half of the beta-apoprotein: a pseudoLH2 (pLH2) class, in which the 800-nm absorption was normal but the 850-nm peak was blue-shifted by up to 14 nm, and the other a pseudoLH1 (pLH1) class, which lacked the 800-nm absorption band and showed 850-nm absorption red-shifts of up to 30 nm. In several of the pLH1 antennae, carotenoid depletion contributed to the phenotype, while in the pLH2 complexes there was some carotenoid enrichment. A number of mutants from each class have also been characterized by low-temperature absorption and fluorescence spectroscopy, resonance Raman spectroscopy, and circular dichroism. In all of the mutants investigated, the B850 bacteriochlorophyll a binding site remained intact, conserving both the hydrogen bonding environment of the chromophores and their conformation and liganding. In contrast, the intensity of the CD spectra of pLH1 complexes was considerably reduced, relative to that of wild-type or pLH2 complexes, consistent with alterations in the interactions between pigments and in their relative orientation. Elevated fluorescence polarization over the red wing of the B850 band in the pLH2 complexes indicated a reduction of exciton mobility within the ring of BChl molecules. Possible structural alterations governing the spectral properties of the different mutants are discussed.

Xanthine dehydrogenase from the phototrophic purple bacterium Rhodobacter capsulatus is more similar to its eukaryotic counterparts than to prokaryotic molybdenum enzymes.

Fourteen Rhodobacter capsulatus mutants unable to grow with xanthine as sole nitrogen source were isolated by random Tn5 mutagenesis. Five of these Tn5 insertions were mapped within two adjacent chromosomal EcoRI fragments hybridizing to oligonucleotides synthesized according to conserved amino acid sequences of eukaryotic xanthine dehydrogenases. DNA sequence analysis of this region revealed two open reading frames, designated xdhA and xdhB, encoding xanthine dehydrogenase. The deduced amino acid sequence of XDHA contains binding sites for two [2Fe-2S] clusters and FAD, whereas XDHB is predicted to contain the molybdopterin cofactor. In contrast to R. capsulatus, these three cofactor binding sites reside within a single polypeptide chain in eukaryotic xanthine dehydrogenases. The amino acid sequence of xanthine dehydrogenase from R. capsulatus showed a higher degree of similarity to eukaryotic xanthine dehydrogenases than to the xanthine dehydrogenase-related aldehyde oxidoreductase from Desulphovibrio gigas. The expression of an xdhA-lacZ fusion was induced when hypoxanthine or xanthine was added as sole nitrogen source. Mutations in nifR1 (ntrC) and nifR4 (rpoN, encoding sigma54) had no influence on xdh gene expression. A putative activator sensing the availability of substrate seems to respond to xanthine but not to hypoxanthine. The transcriptional start site of xdhA was mapped by primer extension analysis. Comparison with known promoter elements revealed no significant homology. Xanthine dehydrogenase from R. capsulatus was purified to homogeneity. The enzyme consists of two subunits with molecular masses of 85 kDa and 50 kDa respectively. N-terminal amino acid sequencing of both subunits confirmed the predicted start codons. The molecular mass of the native enzyme was determined to be 275 kDa, indicating an alpha2beta2-subunit structure. Analysis of the molybdenum cofactor of xanthine dehydrogenase from R. capsulatus revealed that it contains the molybdopterin cofactor and not a molybdopterin dinucleotide derivative.

Production of hydrogen by a hydrogenase-deficient mutant of Rhodobacter capsulatus

The characteristics of Rhodobacter capsulatus ST410, a mutant of the wild strain B100 lacking hydrogenase activity, were investigated from the viewpoint of hydrogen production. When 30 mM dl-malate and 7 mM l-glutamate were used as carbon and nitrogen sources, respectively, in an argon atmosphere, a specific hydrogen evolution rate of 0.14 ml/h/mg-dry cells was obtained at 6600 lx and 33°C. The evolution rate strongly depended on the light intensity: the higher the light intensity, the larger the evolution rate became up to at least 6600 lx. R. capsulatus ST410 converted 60 mM malate to hydrogen at a yield of 68%, calculated as a percentage of the stoichiometric maximum for the complete conversion of the carbon source to H 2 and CO 2. On the other hand, when the wild strain was used under the same conditions, the yield was only 25%. R. capsulatus ST410 converted not only malate but also glucose and cellobiose to hydrogen with good yields (60% for 30 mM glucose and 66% for 7.5 mM cellobiose). Ethanolamine was found to be a good nitrogen source, which permitted a large amount of hydrogen to be evolved and also depressed the cell growth to low levels.

Association of LHIalpha(B870) polypeptide with phospholipids during insertion in the photosynthetic membrane of an LHII- mutant of Rhodobacter capsulatus.

Membranes from in vivo labeled cells of Rhodobacter capsulatus U43[pTX35] grown photosynthetically carried 60% of the [32P]-Pi in the "heavy" fraction (HM) after sucrose gradient sedimentation. Metal-chelating chromatography of either"heavy" or "light" (LM) membrane fractions rendered similar Bchl-protein complex profiles after octyl-glucoside treatment,including most of the radioactivity in the same corresponding elution fraction (F II). Similar labeling distribution of pigment-protein complexes was obtained for membranes of dark-grown cells induced by lowering oxygen tension. Fractions derived from HM showed highly labeled LHIalpha, whereas the same complex from LM was essentially [32P]-Pi-free, as revealed by SDS-PAGE followed by autoradiography. Phospholipid analysis showed a similar pattern for membranes isolated from cells photosynthetically or semiaerobically grown, being the most abundant: phosphatidylglycerol,phosphatidylethanolamine, cardiolipin, and phosphatidylcholine. Part of the phospholipids from HM comigrated with LHIalpha during SDS-PAGE and dissociated from the complexes only after solvent extraction and hydrophobic chromatography. However, a small amount remained always attached to LHIalpha,indicating an unusual strong interaction. These results suggest the existence of two operationally defined membrane regions carrying LHIalpha complexes differing in phosphorylation status and protein-phospholipid interaction.

Photosynthesis-independent regulation of bacteriochlorophyll synthesis by light intensity in Rhodobacter capsulatus

The amounts of bacteriochlorophyll a in a Rhodobacter capsulatus mutant strain which lacks the light-harvesting I and reaction center complexes were measured in cultures grown with different light intensities. Light intensity regulation of bacteriochlorophyll synthesis was found, manifested as differences in the amounts of a light harvesting complex II-like absorption peak in vivo, and the amount of organic solvent-extracted bacteriochlorophyll a, in comparisons between low and high light-grown cells. This light regulation was also evaluated by use of puc::lacZ fusions, which indicated that control is exerted independently of puc gene transcription or translation. We suggest that this light regulation might operate at the level of the enzymatic activity of bacteriochlorophyll biosynthetic enzymes.

Photosynthesis-independent regulation of bacteriochlorophyll synthesis by light intensity inRhodobacter capsulatus

The amounts of bacteriochlorophyll a in a Rhodobacter capsulatus mutant strain which lacks the light-harvesting I and reaction center complexes were measured in cultures grown with different light intensities. Light intensity regulation of bacteriochlorophyll synthesis was found, manifested as differences in the amounts of a light harvesting complex II-like absorption peak in vivo, and the amount of organic solvent-extracted bacteriochlorophyll a, in comparisons between low and high light-grown cells. This light regulation was also evaluated by use of puc::lacZ fusions, which indicated that control is exerted independently of puc gene transcription or translation. We suggest that this light regulation might operate at the level of the enzymatic activity of bacteriochlorophyll biosynthetic enzymes.

Primary charge separation processes in reaction centers of an antenna-free mutant of Rhodobacter capsulatus

The primary charge separation processes of membrane-bound reaction centers from an antenna-free mutant of the purple bacterium Rhodobacter capsulatus have been investigated by picosecond time-resolved fluorescence and femtosecond stimulated emission measurements. Target analysis has been applied to decompose the time-resolved fluorescence emission spectrum into its components for open and closed (quinone-reduced) reaction centers. The reaction center environment, i.e. membrane vs. detergent, exerts a remarkable influence on the primary charge separation kinetics. For fully open reaction centers the main component of the primary electron transfer process from the excited special pair to bacteriopheophytin — measured both in fluorescence and in femtosecond stimulated emission — takes 5–6 ps, which is a factor of nearly two longer than in the corresponding detergent-isolated reaction centers (2.7 ps). For quinone-reduced RCs a very long charge separation time of approx. 10 ps is observed, concomitant with a more than five-fold increase in fluorescence yield. All of these parameters differ substantially from those of detergent-isolated RCs. Possible origins for the observed kinetics are discussed.

A glutamate/glutamine/aspartate/asparagine transport operon in Rhodobacter capsulatus.

A mutant of Rhodobacter capsulatus was identified in which an operon encoding a binding-protein-dependent transporter was interrupted by Tn5 transposition. Cloning and sequence analysis of the wild-type operon revealed a four-gene cluster with similarities to genes encoding periplasmic binding proteins (BztA), integral membrane proteins (BztB and BztC), and ATP-binding proteins (BztD). To assess the function of this putative binding-protein-dependent transport system, a mutant was constructed in which most of the bztABCD operon was deleted and replaced by an antibiotic-resistance marker. The deletion mutant grew more slowly than the wild type in NH4(+)-free medium supplemented by glutamate, glutamine, aspartate or asparagine; it was resistant to toxic analogues of Glu, Asp, and Asn at concentrations that inhibited growth of the wild type; and it was defective in the uptake of Glu, Gln, and Asp. A complementing plasmid containing the wildtype copy of bztABCD was able to rescue all the mutant phenotypes. Taken together, these results indicate that the proteins encoded by bztABCD are active in the uptake of Glu, Gln, Asp, and Asn. In addition, competition experiments, in which the ability of each of the four amino acids to compete for the transport of one another was examined, demonstrated that all four substrates share at least one component of this transport system.

Cloning and characterization of the chlorophyll biosynthesis gene chlM from Synechocystis PCC 6803 by complementation of a bacteriochlorophyll biosynthesis mutant of Rhodobacter capsulatus.

A bacteriochlorophyll a biosynthesis mutant of the purple photosynthetic bacterium Rhodobacter capsulatus was functionally complemented with a cosmid genomic library from Synechocystis sp. PCC 6803. The complemented R. capsulatus strain contains a defined mutation in the bchM gene that codes for Mg-protoporphyrin IX methyltransferase, the enzyme which converts Mg-protoporphyrin IX to Mg-protoporphyrin IX methylester using S-adenosyl-L-methionine as a cofactor. Since chlorophyll biosynthesis also requires the same methylation reaction, the Synechocystis genome should similarly code for a Mg-protoporphyrin IX methyltransferase. Sequence analysis of the complementing Synechocystis cosmid indicates that it contains an open reading frame exhibiting 29% sequence identity to BchM. In addition, expression of the Synechocystis gene in the R. capsulatus bchM mutant via the strong R. capsulatus puc promoter was shown to support nearly wild-type levels of bacteriochlorophyll a synthesis. To our knowledge, the Synechocystis sequence thus represents the first chlorophyll biosynthesis gene homolog of bchM. The complementing Synechocystis cosmid was also shown to code for a gene product that is a member of a highly conserved family of RNA binding proteins, the function of which in cyanobacteria remains undetermined.

Characterization of an fdxN mutant of Rhodobacter capsulatus indicates that ferredoxin I serves as electron donor to nitrogenase

A mutant of Rhodobacter capsulatus, carrying an insertion into the fdxN gene encoding ferredoxin I (FdI), has been studied by biochemical analysis and genetic complementation experiments. When compared to the wild-type strain, the fdxN mutant exhibited altered nitrogen fixing ability and 20-fold lower levels of nitrogenase activity as assayed in vivo. When assayed in vitro with an artificial reductant, nitrogenase activity was only 3- to 4-fold lower than in the wild type. These results suggested that the FdI-deleted mutant had impaired electron transport to nitrogenase. Immunochemical assay of both nitrogenase components showed that the fdxN mutant contained about 4-fold less enzyme than wild-type cells. Results of pulse-chase labeling experiments using [ 35S]methionine indicated that nitrogenase was significantly less stable in the FdI-deleted mutant. When a copy of fdxN was introduced in the mutant in trans, the resulting strain appeared to be fully complemented with respect to both diazotrophic growth and nitrogenase activity. Depending on whether fdxN expression was driven by a nif promoter or a fructose-inducible promoter. FdI was synthesized either at wild-type level or in 10-fold lower amounts. The strain producing 10-fold less FdI did, however, display normal N 2-fixing ability. Analysis of cytosolic proteins by bidimensional electrophoresis revealed that the fdxN mutant produced a 14 kDa polypeptide in amounts about 3-fold greater than wild-type cells. This protein was identified by N-terminal microsequencing as a recently purified [2Fe-2S] ferredoxin, called FdV, which cannot reduce nitrogenase. It is concluded that FdI serves as the main electron donor to nitrogenase in R. capsulatus and that an ancillary electron carrier, distinct of FdV, is responsible for the residual nitrogenase activity observed in the FdI-deleted mutant.

Phenotypic characterisation and genetic complementation of dimethylsulfoxide respiratory mutants of Rhodobacter sphaeroides and Rhodobacter capsulatus

Two chlorate resistant mutants of Rhodobacter sphaeroides were isolated which were deficient in dimethylsulfoxide reductase activity. Immunoblotting experiments showed that the phenotype of these mutants and that of Rhodobacter capsulatus strain DK9, a mutant unable to reduce dimethylsulfoxide, was correlated with low or undetectable levels of the dimethylsulfoxide reductase apoprotein. All three mutants were complemented by a cosmid from a library of Rhodobacter sphaeroides genomic DNA. Further genetic complementation analysis revealed that functions required for restoration of dimethylsulfoxide reductase activity in the Rhodobacter sphaeroides mutants were encoded on an 9 kb EcoR1 DNA fragment derived from this cosmid. Expression of this 9 kb DNA fragment in Escherichia coli showed that it encoded the dimethylsulfoxide reductase structural gene of Rhodobacter sphaeroides.

Expression of the putA gene encoding proline dehydrogenase from Rhodobacter capsulatus is independent of NtrC regulation but requires an Lrp-like activator protein

Four Rhodobacter capsulatus mutants unable to grow with proline as the sole nitrogen source were isolated by random Tn5 mutagenesis. The Tn5 insertions were mapped within two adjacent chromosomal EcoRI fragments. DNA sequence analysis of this region revealed three open reading frames designated selD, putR, and putA. The putA gene codes for a protein of 1,127 amino acid residues which is homologous to PutA of Salmonella typhimurium and Escherichia coli. The central part of R. capsulatus PutA showed homology to proline dehydrogenase of Saccharomyces cerevisiae (Put1) and Drosophila melanogaster (SlgA). The C-terminal part of PutA exhibited homology to Put2 (pyrroline-5-carboxylate dehydrogenase) of S. cerevisiae and to aldehyde dehydrogenases from different organisms. Therefore, it seems likely that in R. capsulatus, as in enteric bacteria, both enzymatic steps for proline degradation are catalyzed by a single polypeptide (PutA). The deduced amino acid sequence of PutR (154 amino acid residues) showed homology to the small regulatory proteins Lrp, BkdR, and AsnC. The putR gene, which is divergently transcribed from putA, is essential for proline utilization and codes for an activator of putA expression. The expression of putA was induced by proline and was not affected by ammonia or other amino acids. In addition, putA expression was autoregulated by PutA itself. Mutations in glnB, nifR1 (ntrC), and NifR4 (ntrA encoding sigma 54) had no influence on put gene expression. The open reading frame located downstream of R. capsulatus putR exhibited strong homology to the E. coli selD gene, which is involved in selenium metabolism. R. capsulatus selD mutants exhibited a Put+ phenotype, demonstrating that selD is required neither for viability nor for proline utilization.