Assessing the impact of denitrifier-produced nitric oxide on other bacteria

A series of experiments was undertaken to learn more about the impact on other bacteria of nitric oxide (NO) produced during denitrification. The denitrifier Rhodobacter sphaeroides 2.4.3 was chosen as a denitrifier for these experiments. To learn more about NO production by this bacterium, NO levels during denitrification were measured by using differential mass spectrometry. This revealed that NO levels produced during nitrate respiration by this bacterium were in the low muM range. This concentration of NO is higher than that previously measured in denitrifiers, including Achromobacter cycloclastes and Paracoccus denitrificans. Therefore, both 2.4.3 and A. cycloclastes were used in this work to compare the effects of various NO levels on nondenitrifying bacteria. By use of bacterial overlays, it was found that the NO generated by A. cycloclastes and 2.4.3 cells during denitrification inhibited the growth of both Bacillus subtilis and R. sphaeroides 2.4.1 but that R. sphaeroides 2.4.3 caused larger zones of inhibition in the overlays than A. cycloclastes. Both R. sphaeroides 2.4.3 and A. cycloclastes induced the expression of the NO stress response gene hmp in B. subtilis. Taken together, these results indicate that there is variability in the NO concentrations produced by denitrifiers, but, irrespective of the NO levels produced, microbes in the surrounding environment were responsive to the NO produced during denitrification.     

Enzymatic removal of nitric oxide catalyzed by cytochrome c' in Rhodobacter capsulatus

Cytochrome c' from Rhodobacter capsulatus has been shown to confer resistance to nitric oxide (NO). In this study, we demonstrated that the amount of cytochrome c' synthesized for buffering of NO is insufficient to account for the resistance to NO but that the cytochrome-dependent resistance mechanism involves the catalytic breakdown of NO, under aerobic and anaerobic conditions. Even under aerobic conditions, the NO removal is independent of molecular oxygen, suggesting cytochrome c' is a NO reductase. Indeed, we have measured the product of NO breakdown to be nitrous oxide (N(2)O), thus showing that cytochrome c' is behaving as a NO reductase. The increased resistance to NO conferred by cytochrome c' is distinct from the NO reductase pathway that is involved in denitrification. Cytochrome c' is not required for denitrification, but it has a role in the removal of externally supplied NO. Cytochrome c' synthesis occurs aerobically and anaerobically but is partly repressed under denitrifying growth conditions when other NO removal systems are operative. The inhibition of respiratory oxidase activity of R. capsulatus by NO suggests that one role for cytochrome c' is to maintain oxidase activity when both NO and O(2) are present.     

Identification of two new denitrifying strains of Rhodobacter sphaeroides

Abstract Highly specific polyclonal and antibodies against either nitrate, nitrite or nitrous oxide reductases from a photosynthetic denitrifying bacterium Rhodobacter sphaeroides f. sp. denitrificans were used to show the presence of immunologically reactive proteins in strains that Pellerin and Gest had shown to grow in the dark with nitrate as a terminal acceptor [9]. Two strains of this bacterium, namely 81-3 and 2.4.3 synthesized the three denitrifying enzymes and were capable of denitrification. Strains 81-1 and 2.4.1 (neotype) both expressed nitrate reductase activities but nitrite reductase was not detected since these strains did not reduce nitrite. They also did not grow in the dark with nitrate as a terminal acceptor. Each of strains 81-1, 81-3, 2.4.1 and 2.4.3 contain four plasmids. R. sphaeroides f. sp. denitrificans , however, contains only one large 108 kb plasmid, which is distinctly different in size from those detected in the other strains. This indicates that the 108 kb plasmid is not necessarily specific for denitrification.     

Analysis of the role of the nnrR gene product in the response of Rhodobacter sphaeroides 2.4.1 to exogenous nitric oxide.

Rhodobacter sphaeroides 2.4.1, which is incapable of denitrification, has been found to carry nnrR, the nor operon, and nnrS, which are utilized for denitrification in R. sphaeroides 2.4.3. The gene encoding nitrite reductase was not found in 2.4.1. Expression of beta-galactosidase activity from a norB-lacZ fusion was activated when cells of 2.4.1 were incubated with NO-producing bacteria. This result indicates that the products of nnrR and the genes flanking it are utilized when 2.4.1 is growing in an environment where denitrification occurs.     

Effect of aerobic growth conditions on the soluble cytochrome content of the purple phototrophic bacterium Rhodobacter sphaeroides: induction of cytochrome c554

When grown anaerobically in the light, Rhodobacter sphaeroides contains appreciable quantities of cytochromes c2 and c', but smaller amounts of other soluble cytochromes such as cytochrome c551.5, cytochrome c554, and an oxygen-binding heme protein. When R. sphaeroides is mass cultured aerobically in the dark to stationary phase, the content of cytochrome c2 does not change appreciably, whereas cytochrome c554 is approximately 8-fold more abundant, cytochrome c' is at least 10-fold less abundant, and cytochrome c551.5 is fivefold lower than in the phototrophically grown cells. These observations confirm previous literature reports that in this organism a cytochrome c553 (or c554 in our experience) is more abundant when cells are grown aerobically. Furthermore, the aerobic cytochrome c554 is positively identified with the previously characterized minor cytochrome c554 component of anaerobic photosynthetic cells. Preliminary sequence results show that cytochrome c554 is a member of the cytochrome c' structural family, but differs from normal cytochromes c' in having a methionine sixth ligand to the heme. The levels of electron carrier proteins of low redox potential had previously been reported to be less in aerobic than in photoheterotrophic cells and we have verified that observation for the specific examples of cytochromes c' and c551.5. The oxygen binding heme protein, SHP, is not induced by aerobic growth.     

Use of a green fluorescent protein-based reporter fusion for detection of nitric oxide produced by denitrifiers

To determine if green fluorescent protein could be used as a reporter for detecting nitric oxide production, gfp was fused to nnrS from Rhodobacter sphaeroides 2.4.3. nnrS was chosen because its expression requires nitric oxide. The presence of the fusion in R. sphaeroides 2.4.3 resulted in a significant increase in fluorescent intensity of the cells, but only when nitrite reductase was active. Cells lacking nitrite reductase activity and consequently the ability to generate nitric oxide were only weakly fluorescent when grown under denitrification-inducing conditions. One of the R. sphaeroides strains unable to generate nitric oxide endogenously was used as a reporter to detect exogenously produced nitric oxide. Incubation of this strain with sodium nitroprusside, a nitric oxide generator, significantly increased its fluorescence intensity. Mixing of known denitrifiers with the reporter strain also led to significant increases in fluorescence intensity, although the level varied depending on the denitrifier used. The reporter was tested on unknown isolates capable of growing anaerobically in the presence of nitrate, and one of these was able to induce expression of the fusion. Analysis of the 16S rRNA gene sequence of this isolate placed it within the Thauera aromatica subgroup, which is known to contain denitrifiers. These experiments demonstrate that this green fluorescent protein-based assay provides a useful method for assessing the ability of bacteria to produce nitric oxide.     

Assessing the Impact of Denitrifier-Produced Nitric Oxide on Other Bacteria

A series of experiments was undertaken to learn more about the impact on other bacteria of nitric oxide (NO) produced during denitrification. The denitrifier Rhodobacter sphaeroides 2.4.3 was chosen as a denitrifier for these experiments. To learn more about NO production by this bacterium, NO levels during denitrification were measured by using differential mass spectrometry. This revealed that NO levels produced during nitrate respiration by this bacterium were in the low μM range. This concentration of NO is higher than that previously measured in denitrifiers, including Achromobacter cycloclastes and Paracoccus denitrificans. Therefore, both 2.4.3 and A. cycloclastes were used in this work to compare the effects of various NO levels on nondenitrifying bacteria. By use of bacterial overlays, it was found that the NO generated by A. cycloclastes and 2.4.3 cells during denitrification inhibited the growth of both Bacillus subtilis and R. sphaeroides 2.4.1 but that R. sphaeroides 2.4.3 caused larger zones of inhibition in the overlays than A. cycloclastes. Both R. sphaeroides 2.4.3 and A. cycloclastes induced the expression of the NO stress response gene hmp in B. subtilis. Taken together, these results indicate that there is variability in the NO concentrations produced by denitrifiers, but, irrespective of the NO levels produced, microbes in the surrounding environment were responsive to the NO produced during denitrification.     

Cytochrome c' from Paracoccus denitrificans: spectroscopic studies consistent with a role for the protein in nitric oxide metabolism

Cytochrome c' was purified from the denitrifying bacterium Paracoccus denitrificans and the interaction of the protein with nitric oxide was examined spectroscopically. Two distinct types of haem-nitrosyl electronic absorption spectrum were observed, which were dependent upon [NO]. When cytochrome c' was saturated with NO, alpha and beta bands were centred at 562 nm and 530 nm, whereas with sub-saturating concentrations of NO the alpha and beta bands were red-shifted to 578 nm and 542 nm respectively. Further spectroscopic analysis showed that purified cytochrome c', added to suspensions of P. denitrificans, is able to complex with the NO which is formed as a freely diffusible intermediate of denitrification. In the presence of added NO-3 or NO-2, 40-60% of Fe(II)-cytochrome c' forms a 6-coordinate haem-nitrosyl complex. In the absence of nitrogen oxyanions or NO whole denitrifying cells are able to remove the NO from a Fe(II)-cytochrome c'-NO complex. These findings support the hypothesis that the physiological function of this enigmatic cytochrome involves the reversible binding of nitric oxide.     

Nitrite and Nitrous Oxide Reductase Regulation by Nitrogen Oxides in Rhodobacter sphaeroides f. sp. denitrificans IL106

We have cloned the nap locus encoding the periplasmic nitrate reductase in Rhodobacter sphaeroides f. sp. denitrificans IL106. A mutant with this enzyme deleted is unable to grow under denitrifying conditions. Biochemical analysis of this mutant shows that in contrast to the wild-type strain, the level of synthesis of the nitrite and N(2)O reductases is not increased by the addition of nitrate. Growth under denitrifying conditions and induction of N oxide reductase synthesis are both restored by the presence of a plasmid containing the genes encoding the nitrate reductase. This demonstrates that R. sphaeroides f. sp. denitrificans IL106 does not possess an efficient membrane-bound nitrate reductase and that nitrate is not the direct inducer for the nitrite and N(2)O reductases in this species. In contrast, we show that nitrite induces the synthesis of the nitrate reductase.     

Regulation of a cytochrome c2 isoform in wild-type and cytochrome c2 mutant strains of Rhodobacter sphaeroides.

In Rhodobacter sphaeroides, mutations that suppress the photosynthetic deficiency (spd mutations) of strains lacking cytochrome c2 (cyt c2) cause accumulation of a periplasmic cyt c2 isoform that has been designated isocytochrome c2 (isocyt c2). In this study, a new method for purification of both cyt c2 and isocyt c2 is described that uses periplasmic fluid as a starting material. In addition, antiserum to isocyt c2 has been used to demonstrate that all suppressor mutants contain an isocyt c2 of approximately 15 kDa. Western blot analysis indicates that isocyt c2 was present at lower levels in both wild-type and cyt c2 mutants than in spd-containing mutants. Although isocyt c2 is detectable under all growth conditions in wild-type cells, the highest level of isocyt c2 is present under aerobic conditions. Our results demonstrate that spd mutations increase the steady state level of isocyt c2 under photosynthetic conditions. Although the physiological function of isocyt c2 in wild-type cells is not known, we show that a nitrate-regulated protein in Rhodobacter sphaeroides f. sp. denitrificans also reacts with the isocyt c2 antiserum.     

Physiological roles for two periplasmic nitrate reductases in Rhodobacter sphaeroides 2.4.3 (ATCC 17025)

The metabolically versatile purple bacterium Rhodobacter sphaeroides 2.4.3 is a denitrifier whose genome contains two periplasmic nitrate reductase-encoding gene clusters. This work demonstrates nonredundant physiological roles for these two enzymes. One cluster is expressed aerobically and repressed under low oxygen while the second is maximally expressed under low oxygen. Insertional inactivation of the aerobically expressed nitrate reductase eliminated aerobic nitrate reduction, but cells of this strain could still respire nitrate anaerobically. In contrast, when the anaerobic nitrate reductase was absent, aerobic nitrate reduction was detectable, but anaerobic nitrate reduction was impaired. The aerobic nitrate reductase was expressed but not utilized in liquid culture but was utilized during growth on solid medium. Growth on a variety of carbon sources, with the exception of malate, the most oxidized substrate used, resulted in nitrite production on solid medium. This is consistent with a role for the aerobic nitrate reductase in redox homeostasis. These results show that one of the nitrate reductases is specific for respiration and denitrification while the other likely plays a role in redox homeostasis during aerobic growth.     

Isolation and molecular characterization of pMG160, a mobilizable cryptic plasmid from Rhodobacter blasticus

A 3.4-kb cryptic plasmid was obtained from a new isolate of Rhodobacter blasticus. This plasmid, designated pMG160, was mobilizable by the conjugative strain Escherichia coli S17.1 into Rhodobacter sphaeroides, Rhodobacter capsulatus, and Rhodopseudomonas palustris. It replicated in the latter strains but not in Rhodospirillum rubrum, Rhodocyclus gelatinosus, or Bradyrhizobium species. Plasmid pMG160 was stably maintained in R. sphaeroides for more than 100 generations in the absence of selection but showed segregational instability in R. palustris. Instability in R. palustris correlated with a decrease in plasmid copy number compared to the copy number in R. sphaeroides. The complete nucleotide sequence of plasmid pMG160 contained three open reading frames (ORFs). The deduced amino acid sequences encoded by ORF1 and ORF2 showed high degrees of homology to the MobS and MobL proteins that are involved in plasmid mobilization of certain plasmids. Based on homology with the Rep protein of several other plasmids, ORF3 encodes a putative rep gene initiator of plasmid replication. The functions of these sequences were demonstrated by deletion mapping, frameshift analysis, and analysis of point mutations. Two 6.1-kb pMG160-based E. coli-R. sphaeroides shuttle cloning vectors were constructed and designated pMG170 and pMG171. These two novel shuttle vectors were segregationally stable in R. sphaeroides growing under nonselective conditions.     

Features of Rhodobacter sphaeroides CcmFH

In this study, the in vivo function and properties of two cytochrome c maturation proteins, CcmF and CcmH from Rhodobacter sphaeroides, were analyzed. Strains lacking CcmH or both CcmF and CcmH are unable to grow under anaerobic conditions where c-type cytochromes are required, demonstrating their critical role in the assembly of these electron carriers. Consistent with this observation, strains lacking both CcmF and CcmH are deficient in c-type cytochromes when assayed under permissive growth conditions. In contrast, under permissive growth conditions, strains lacking only CcmH contain several soluble and membrane-bound c-type cytochromes, albeit at reduced levels, suggesting that this bacterium has a CcmH-independent route for their maturation. In addition, the function of CcmH that is needed to support anaerobic growth can be replaced by adding cysteine or cystine to growth media. The ability of exogenous thiol compounds to replace CcmH provides the first physiological evidence for a role of this protein in thiol chemistry during c-type cytochrome maturation. The properties of R. sphaeroides cells containing translational fusions between CcmF and CcmH and either Escherichia coli alkaline phosphatase or beta-galactosidase suggest that they are each integral cytoplasmic membrane proteins with their presumed catalytic domains facing the periplasm. Analysis of CcmH shows that it is synthesized as a higher-molecular-weight precursor protein with an N-terminal signal sequence.     

The significance of type II and PrxQ peroxiredoxins for antioxidative stress response in the purple bacterium Rhodobacter sphaeroides

Two peroxiredoxins, classified as Type II and PrxQ, were characterized in the purple non-sulfur photosynthetic bacterium Rhodobacter sphaeroides. Both recombinant proteins showed remarkable thioredoxin-dependent peroxidase activity with broad substrate specificity in vitro. Nevertheless, PrxQ of R. sphaeroides, unlike typical PrxQs studied to date, does not contain one of the two conserved catalytic Cys residues. We found that R. sphaeroides PrxQ and other PrxQ-like proteins from several organisms conserve a different second Cys residue, indicating that these proteins should be categorized into a novel PrxQ subfamily. Disruption of either the Type II or PrxQ gene in R. sphaeroides had a dramatic effect on cell viability when the cells were grown under aerobic light or oxidative stress conditions created by exogenous addition of reactive oxygen species to the medium. Growth rates of the mutants were significantly decreased compared with that of wild type under aerobic but not anaerobic conditions. These results indicate that the peroxiredoxins are crucial for antioxidative stress response in this bacterium. The gene disruptants also demonstrated reduced levels of photopigment synthesis, suggesting that the peroxiredoxins are directly or indirectly involved in regulated synthesis of the photosynthetic apparatus.     

Roles for the Rhodobacter sphaeroides CcmA and CcmG Proteins

Rhodobacter sphaeroides cells containing an in-frame deletion within ccmA lack detectable soluble and membrane-bound c-type cytochromes and are unable to grow under conditions where these proteins are required. Only strains merodiploid for ccmABCDG were found after attempting to generate cells containing either a ccmG null mutation or a ccmA allele that should be polar on to expression of ccmBCDG, suggesting that CcmG has another important role in R. sphaeroides.     

Differential expression of the CO2 fixation operons of Rhodobacter sphaeroides by the Prr/Reg two-component system during chemoautotrophic growth

In Rhodobacter sphaeroides, the two cbb operons encoding duplicated Calvin-Benson Bassham (CBB) CO2 fixation reductive pentose phosphate cycle structural genes are differentially controlled. In attempts to define the molecular basis for the differential regulation, the effects of mutations in genes encoding a subunit of Cbb3 cytochrome oxidase, ccoP, and a global response regulator, prrA (regA), were characterized with respect to CO2 fixation (cbb) gene expression by using translational lac fusions to the R. sphaeroides cbb(I) and cbb(II) promoters. Inactivation of the ccoP gene resulted in derepression of both promoters during chemoheterotophic growth, where cbb expression is normally repressed; expression was also enhanced over normal levels during phototrophic growth. The prrA mutation effected reduced expression of cbb(I) and cbb(II) promoters during chemoheterotrophic growth, whereas intermediate levels of expression were observed in a double ccoP prrA mutant. PrrA and ccoP1 prrA strains cannot grow phototrophically, so it is impossible to examine cbb expression in these backgrounds under this growth mode. In this study, however, we found that PrrA mutants of R. sphaeroides were capable of chemoautotrophic growth, allowing, for the first time, an opportunity to directly examine the requirement of PrrA for cbb gene expression in vivo under growth conditions where the CBB cycle and CO2 fixation are required. Expression from the cbb(II) promoter was severely reduced in the PrrA mutants during chemoautotrophic growth, whereas cbb(I) expression was either unaffected or enhanced. Mutations in ccoQ had no effect on expression from either promoter. These observations suggest that the Prr signal transduction pathway is not always directly linked to Cbb3 cytochrome oxidase activity, at least with respect to cbb gene expression. In addition, lac fusions containing various lengths of the cbb(I) promoter demonstrated distinct sequences involved in positive regulation during photoautotrophic versus chemoautotrophic growth, suggesting that different regulatory proteins may be involved. In Rhodobacter capsulatus, ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) expression was not affected by cco mutations during photoheterotrophic growth, suggesting that differences exist in signal transduction pathways regulating cbb genes in the related organisms.     

Rhodobacter sphaeroides spd mutations allow cytochrome c2-independent photosynthetic growth.

In Rhodobacter sphaeroides, cytochrome c2 (cyt c2) is a periplasmic redox protein required for photosynthetic electron transfer. cyt c2-deficient mutants created by replacing the gene encoding the apoprotein for cyt c2 (cycA) with a kanamycin resistance cartridge are photosynthetically incompetent. Spontaneous mutations that suppress this photosynthesis deficiency (spd mutants) arise at a frequency of 1 to 10 in 10(7). We analyzed the cytochrome content of several spd mutants spectroscopically and by heme peroxidase assays. These suppressors lacked detectable cyt c2, but they contained a new soluble cytochrome which was designated isocytochrome c2 (isocyt c2) that was not detectable in either cycA+ or cycA mutant cells. When spd mutants were grown photosynthetically, isocyt c2 was present at approximately 20 to 40% of the level of cyt c2 found in photosynthetically grown wild type cells, and it was found in the periplasm with cytochromes c' and c554. These spd mutants also had several other pleiotropic phenotypes. Although photosynthetic growth rates of the spd mutants were comparable to those of wild-type strains at all light intensities tested, they contained elevated levels of B800-850 pigment-protein complexes. Several spd mutants contained detectable amounts of isocyt c2 under aerobic conditions. Finally, heme peroxidase assays indicated that, under anaerobic conditions, the spd mutants may contain another new cytochrome in addition to isocyt c2. These pleiotropic phenotypes, the frequency at which the spd mutants arise, and the fact that a frameshift mutagen is very effective in generating the spd phenotype suggest that some spd mutants contain a mutation in loci which regulate cytochrome synthesis.