Microoxic Conditions Research Articles

The Sinorhizobium meliloti RNA chaperone Hfq influences central carbon metabolism and the symbiotic interaction with alfalfa

BackgroundThe bacterial Hfq protein is able to interact with diverse RNA molecules, including regulatory small non-coding RNAs (sRNAs), and thus it is recognized as a global post-transcriptional regulator of gene expression. Loss of Hfq has an extensive impact in bacterial physiology which in several animal pathogens influences virulence. Sinorhizobium meliloti is a model soil bacterium known for its ability to establish a beneficial nitrogen-fixing intracellular symbiosis with alfalfa. Despite the predicted general involvement of Hfq in the establishment of successful bacteria-eukaryote interactions, its function in S. meliloti has remained unexplored.ResultsTwo independent S. meliloti mutants, 2011-3.4 and 1021Δhfq, were obtained by disruption and deletion of the hfq gene in the wild-type strains 2011 and 1021, respectively, both exhibiting similar growth defects as free-living bacteria. Transcriptomic profiling of 1021Δhfq revealed a general down-regulation of genes of sugar transporters and some enzymes of the central carbon metabolism, whereas transcripts specifying the uptake and metabolism of nitrogen sources (mainly amino acids) were more abundant than in the wild-type strain. Proteomic analysis of the 2011-3.4 mutant independently confirmed these observations. Symbiotic tests showed that lack of Hfq led to a delayed nodulation, severely compromised bacterial competitiveness on alfalfa roots and impaired normal plant growth. Furthermore, a large proportion of nodules (55%-64%) elicited by the 1021Δhfq mutant were non-fixing, with scarce content in bacteroids and signs of premature senescence of endosymbiotic bacteria. RT-PCR experiments on RNA from bacteria grown under aerobic and microoxic conditions revealed that Hfq contributes to regulation of nifA and fixK1/K2, the genes controlling nitrogen fixation, although the Hfq-mediated regulation of fixK is only aerobiosis dependent. Finally, we found that some of the recently identified S. meliloti sRNAs co-inmunoprecipitate with a FLAG-epitope tagged Hfq protein.ConclusionsOur results support that the S. meliloti RNA chaperone Hfq contributes to the control of central metabolic pathways in free-living bacteria and influences rhizospheric competence, survival of the microsymbiont within the nodule cells and nitrogen fixation during the symbiotic interaction with its legume host alfalfa. The identified S. meliloti Hfq-binding sRNAs are predicted to participate in the Hfq regulatory network.

Expression and Mutational Analysis of the glnB Genomic Region in the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120

In the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, the glnB gene is expressed at considerable levels both in the presence and in the absence of combined nitrogen, although induction, influenced by NtcA, takes place upon combined-nitrogen deprivation likely localized to vegetative cells. In spite of extensive efforts, a derivative of PCC 7120 lacking a functional glnB gene could be obtained only with constructs that lead to overexpression of a downstream open reading frames (ORF), particularly all2318. Strain CSP10 [glnB all2318(Con)] exhibited growth rates similar to those of the wild type when it was using nitrate or ammonium, but its diazotrophic growth was impaired. However, it differentiated heterocysts with a time course and distribution pattern similar to those of the wild type, although with no cyanophycin-containing polar granules, and exhibited impaired nitrogenase activity under oxic conditions, but not under microoxic conditions. In the mutant, NtcA-dependent induction of the hetC and nifH genes was unaltered, but induction of the urtA gene and urea transport activity were increased. Active uptake of nitrite was also increased and insensitive to the ammonium-promoted inhibition observed for the wild type. Thus, regulation of the nitrite transport activity requires the glnB gene product. In the presence of a wild-type glnB gene, neither inactivation nor overexpression of all2318 produced an apparent phenotype. Thus, in an otherwise wild-type background, the glnB gene appears to be essential for growth of strain PCC 7120. For growth with combined nitrogen but not for diazotrophic growth, the requirement for glnB can be overridden by increasing the expression of all2318 (and/or ORFs downstream of it).

Methylotrophy in Freshwater Beggiatoa alba Strains

Two freshwater strains of the gammaproteobacterium Beggiatoa alba, B18LD and OH75-2a, are able to use methanol as a sole carbon and energy source under microoxic conditions. Genes encoding a methanol dehydrogenase large-subunit homolog and four enzymes of the tetrahydromethanopterin-dependent C(1) oxidation pathway were identified in B18LD. No evidence of methanotrophy was detected.

Haloalkaliphilic diazotrophs in soda solonchak soils

Nitrogen fixation (NF) potential was measured in more than 40 samples of soda solonchak soils with the pH of water extract between 9.5 and 11.0 collected in several locations of Central Asia and in Egypt, using the acetylene reduction method. NF was detected in most of the samples. Maximal rates were observed under microaerophilic-anaerobic conditions with glucose as a substrate. In most cases, the NF negatively correlated with salt content and alkalinity. Five enrichments at pH 10 under micro-oxic conditions with glucose resulted in stable haloalkaliphilic mixed cultures, with diazotrophic component(s) active up to 2.0-3.0 M total Na(+). The cultures were dominated by Gram-positive spore-forming bacteria. Molecular cloning of nifH genes demonstrated the presence of two phylogenetic lineages of diazotrophs in the enrichments affiliated with the low-GC Gram-positive bacteria (in rRNA groups 1 and 6 of bacilli and in Clostridiales). Isolation of pure cultures of haloalkaliphilic diazotrophs from micro-oxic enrichments yielded nine strains, comprising two phylogenetic lineages. Most of the isolates (eight) were affiliated with the aerotolerant fermentative haloalkaliphilic bacterium Amphibacillus tropicus and a single strain clustered with the obligately anaerobic haloalkaliphile Bacillus arseniciselenatis. Diazotrophy has never been recognized previously in these groups of Gram-positive bacteria. Overall, the results demonstrated the existence, in soda solonchak soils, of a novel group of free-living fermentative diazotrophic bacteria active at extremely haloalkaline conditions.

Thiohalospira halophila gen. nov., sp. nov. and Thiohalospira alkaliphila sp. nov., novel obligately chemolithoautotrophic, halophilic, sulfur-oxidizing gammaproteobacteria from hypersaline habitats

A previously unknown ecotype of obligately chemolithoautotrophic, sulfur-oxidizing bacteria was discovered in sediments of various inland hypersaline lakes and a solar saltern. The salt requirements for these bacteria were similar to those of haloarchaea, representing the first example of extreme halophiles occurring among the chemolithoautotrophs. They were enriched and isolated at 4 M NaCl under aerobic conditions with thiosulfate or tetrathionate as the electron donor or under micro-oxic conditions with sulfide. In total, 20 strains were obtained from hypersaline inland lakes in central Asia, central Russia and Crimea and a sea saltern of the Adriatic Sea. The isolates were thin, motile spirilla, some of which possessed a yellow, membrane-bound pigment. They were obligately aerobic, chemolithoautotrophic, sulfur-oxidizing bacteria that used thiosulfate, sulfide, sulfur and tetrathionate as electron donors. The characteristic feature of the group was the production of large amounts of tetrathionate as an intermediate during the oxidation of thiosulfate to sulfate. All but one of the strains grew within the pH range 6.5-8.2 (optimally at pH 7.3-7.8) and at NaCl concentrations from 2.0 to 5 M (optimally at 3.0 M). A single strain, designated ALgr 6sp(T), obtained (by enrichment) from the hypersaline alkaline lakes of the Wadi Natrun valley, was found to be moderately halophilic and facultatively alkaliphilic (capable of growth at pH 10). The predominant cellular fatty acids were quite unusual, with 10-methyl C(16 : 0) and C(16 : 0) predominating. Cells grown at 4 M NaCl accumulated extremely high concentrations of glycine betaine as a compatible solute. The 20 neutrophilic isolates contained three genospecies (on the basis of DNA-DNA relatedness data) but could not be discriminated phenotypically. On the basis of the phenotypic and genotypic analyses, the isolates constitute a novel genus and species, for which the name Thiohalospira halophila gen. nov., sp. nov. is proposed. The type strain of Thiohalospira halophila is HL 3(T) (=DSM 15071(T)=UNIQEM U219(T)). The haloalkaliphilic strain ALgr 6sp(T) represents a second species of the new genus, for which the name Thiohalospira alkaliphila sp. nov. is proposed. The type strain of Thiohalospira alkaliphila is ALgr 6sp(T) (=DSM 17116(T)=UNIQEM U372(T)).

Identification of Two Homologous Genes, chlAI and chlAII, That Are Differentially Involved in Isocyclic Ring Formation of Chlorophyll a in the Cyanobacterium Synechocystis sp. PCC 6803

The isocyclic ring (E-ring) is a common structural feature of chlorophylls. The E-ring is formed by two structurally unrelated Mg-protoporphyrin IX monomethylester (MPE) cyclase systems, oxygen-dependent (AcsF), and oxygen-independent (BchE) systems, which involve incorporation of an oxygen atom from molecular oxygen and water into the C-13(1) position of MPE, respectively. Which system operates in cyanobacteria that can thrive in a variety of anaerobic environments remains an open question. The cyanobacterium Synechocystis sp. PCC 6803 has two acsF-like genes, sll1214 (chlA(I)) and sll1874 (chlA(II)), and three bchE-like genes, slr0905, sll1242, and slr0309. Five mutants lacking one of these genes were isolated. The DeltachlA(I) mutant failed to grow under aerobic conditions with anomalous accumulation of a pigment with fluorescence emission peak at 595 nm, which was identified 3,8-divinyl MPE by high-performance liquid chromatography-mass spectrometry analysis. The growth defect of DeltachlA(I) was restored by the cultivation under oxygen-limited (micro-oxic) conditions. MPE accumulation was also detected in DeltachlA(II) grown under microoxic conditions, but not in any of the bchE mutants. The phenotype was consistent with the expression pattern of two chlA genes: chlA(II) was induced under micro-oxic conditions in contrast to the constitutive expression of chlA(I). These findings suggested that ChlA(I) is the sole MPE cyclase system under aerobic conditions and that the induced ChlA(II) operates together with ChlA(I) under micro-oxic conditions. In addition, the accumulation of 3,8-divinyl MPE in the DeltachlA mutants suggested that the reduction of 8-vinyl group occurs after the formation of E-ring in Synechocystis sp. PCC 6803.

New Target Genes Controlled by the Bradyrhizobium japonicum Two-Component Regulatory System RegSR

RegSR-like proteins, members of the family of two-component regulatory systems, are present in a large number of proteobacteria in which they globally control gene expression mostly in a redox-responsive manner. The controlled target genes feature an enormous functional diversity. In Bradyrhizobium japonicum, the facultative root nodule symbiont of soybean, RegSR activate the transcription of the nitrogen fixation regulatory gene nifA, thus forming a RegSR-NifA cascade which is part of a complex regulatory network for gene regulation in response to changing oxygen concentrations. Whole-genome transcription profiling was performed here in order to assess the full regulatory scope of RegSR. The comparative analysis of wild-type and delta regR cells grown under oxic and microoxic conditions revealed that expression of almost 250 genes is dependent on RegR, a result that underscores the important contribution of RegR to oxygen- or redox-regulated gene expression in B. japonicum. Furthermore, transcription profiling of delta regR bacteroids compared with wild-type bacteroids revealed expression changes for about 1,200 genes in young and mature bacteroids. Incidentally, many of these were found to be induced in symbiosis when wild-type bacteroids were compared with free-living, culture-grown wild-type cells, and they appeared to encode diverse functions possibly related to symbiosis and nitrogen fixation. We demonstrated direct RegR-mediated control at promoter regions of several selected target genes by means of DNA binding experiments and in vitro transcription assays, which revealed six novel direct RegR target promoters.

Thiohalomonas denitrificans gen. nov., sp. nov. and Thiohalomonas nitratireducens sp. nov., novel obligately chemolithoautotrophic, moderately halophilic, thiodenitrifying Gammaproteobacteria from hypersaline habitats

A novel group of moderately halophilic, obligately chemolithoautotrophic, sulfur-oxidizing Gammaproteobacteria was found in sediments of various inland hypersaline lakes and a solar saltern. These bacteria were enriched and isolated with thiosulfate as electron donor and nitrate as electron acceptor at 2 M NaCl. Ten isolates (HLD strains) were long non-motile rods. They grew anaerobically as complete denitrifiers, and aerobically under micro-oxic conditions. Sulfate was the final product of thiosulfate and sulfide oxidation, and nitrite and N(2)O were intermediates of nitrate reduction to N(2). The HLD strains grew optimally at pH 7.3-7.8, and at NaCl concentrations of 1.5-2.0 M. On the basis of phenotypic and genetic analysis, the moderately halophilic, thiodenitrifying isolates are proposed to be assigned to a new genus and species, Thiohalomonas denitrificans gen. nov., sp. nov. The type strain is HLD 2(T) (=DSM 15841(T)=UNIQEM U222(T) ). A single strain, HRhD 3sp(T), with vibrio-shaped cells, was obtained from a co-culture capable of complete denitrification of nitrate in the presence of either thiocyanate or thiosulfate as electron donor. It grew anaerobically with thiosulfate, reducing nitrate to nitrite, or under micro-oxic conditions at 1.0-2.5 M NaCl with an optimum at 1.0 M. Strain HRhD 3sp(T) was genetically related to the HLD strains at the level of a separate species and is described as Thiohalomonas nitratireducens sp. nov. The type strain is HRhD 3sp(T) (=DSM 16925(T)=UNIQEM U248(T)).

Rhodobium pfennigii sp. nov., a phototrophic purple non-sulfur bacterium with unusual bacteriochlorophyll a antennae, isolated from a brackish microbial mat on Rangiroa atoll, French Polynesia

A novel budding purple non-sulfur bacterium (strain AR2102(T)) was isolated in pure culture from a microbial mat that had developed in brackish-water ponds on the coral rim of the atoll of Rangiroa (Tuamotu Islands, French Polynesia). Single cells of this strain were rod-shaped and motile by means of polar flagella and divided by budding. Their intracellular photosynthetic membranes were of the lamellar type. Bacteriochlorophyll a and carotenoids of the normal spirilloxanthin series, with spirilloxanthin as the main carotenoid, were present as photosynthetic pigments. Bacteriochlorophyll a absorption in the infrared portion of the light spectrum exhibited an unusual in vivo absorption peak at 909 nm. The strain grew optimally under photoheterotrophic conditions, but could grow photolithotrophically on thiosulfate or chemo-organotrophically under micro-oxic conditions. Optimal growth occurred in the presence of 1-2 % NaCl. Comparative sequence analysis of the 16S rRNA gene placed strain AR2102(T) within the class Alphaproteobacteria, in a cluster with Rhodobium species. Representatives of this cluster form a closely related group of slightly to moderately halotolerant to halophilic, rod-shaped, purple non-sulfur bacteria that divide by budding. The new isolate exhibited some differences in physiology (no utilization of alcohols or carbohydrates) and genetic characteristics (low relatedness in DNA-DNA hybridization) as well as in its relation to light (differences in absorption wavelengths) from previously described Rhodobium species. Consequently, we propose that strain AR2102(T) (=DSM 17143(T)=ATCC BAA-1145(T)) should be considered as the type strain of a novel species within the genus Rhodobium, Rhodobium pfennigii sp. nov.

Denitrification in a binary culture and thiocyanate metabolism in Thiohalophilus thiocyanoxidans gen. nov. sp. nov. – a moderately halophilic chemolithoautotrophic sulfur-oxidizing Gammaproteobacterium from hypersaline lakes

Anaerobic enrichment culture with thiocyanate as electron donor and nitrate as electron acceptor at 2 M NaCl inoculated with a mixture of sediments from hypersaline lakes in Kulunda Steppe (Altai, Russia) resulted in a selection of a binary consortium of moderately halophilic, obligately chemolithoautotrophic sulfur-oxidizing bacteria (SOB) capable of complete denitrification of nitrate with thiosulfate as the electron donor. One consortium member, strain HRhD 3sp, was isolated into pure culture with nitrate and thiosulfate using a density gradient. This strain was responsible for the reduction of nitrate to nitrite in the consortium, while a second strain, HRhD 2, isolated under microoxic conditions with thiosulfate as substrate, was capable of anaerobic growth with nitrite and thiosulfate. Nitrite, either as substrate or as product, was already toxic at very low concentrations for both strains. As a result, optimal growth under anaerobic conditions could only be achieved within the consortium. On the basis of phylogenetic analysis, both organisms were identified as new lineages within the Gammaproteobacteria. As well as thiosulfate, strain HRhD 2 can also use thiocyanate as electron donor, representing a first halophilic SOB capable of growth with thiocyanate at 2-4 M NaCl. Product and enzymatic analysis identified the "carbonyl sulfide (COS) pathway" of primary thiocyanate degradation in this new species. On the basis of phenotypic and genetic analysis, strain HRhD 2 is proposed to be assigned to a new genus and species Thiohalophilus thiocyanoxidans.

Diversity of culturable halophilic sulfur-oxidizing bacteria in hypersaline habitats

Unexpectedly high culturable diversity of moderately and extremely halophilic obligately chemolithoautotrophic sulfur-oxidizing bacteria (SOB) was discovered in the sediments of various hypersaline habitats, including chloride-sulfate lakes in Mongolia, Russia and Ukraine, a sea saltern in Slovenia and a deep-sea salt brine from the Mediterranean. Six different groups of halophilic SOB, including four new genera, all belonging to the Gammaproteobacteria, were found. Two groups of moderately halophilic strictly aerobic SOB dominated at 2 M NaCl, including representatives of the genus Halothiobacillus (in fully aerobic conditions) and Thiomicrospira (in micro-oxic conditions). Under denitrifying conditions at 2 M NaCl, a group of moderately halophilic and facultatively anaerobic SOB was selected, capable of complete denitrification of nitrate. The group represents a new genus with closest relatives among as yet undescribed marine thiodenitrifying isolates. With thiocyanate as a substrate, an enrichment culture at 2 M NaCl yielded a pure culture of moderately halophilic SOB capable of aerobic growth with thiocyanate and thiosulfate at up to 4 M NaCl. Furthermore, this bacterium also grew anaerobically using nitrite as electron acceptor. It formed a new lineage distantly related to the genus Thiomicrospira. Enrichments at 4 M NaCl resulted in the domination of two different, previously unknown, groups of extremely halophilic SOB. Under oxic conditions, they were represented by strictly aerobic spiral-shaped bacteria, related to the Ectothiorhodospiraceae, while under denitrifying conditions a group of facultatively anaerobic nitrate-reducing bacteria with long rod-shaped cells was selected, distantly related to the genus Acidithiobacillus.

The ntrPR Operon of Sinorhizobium meliloti Is Organized and Functions as a Toxin-Antitoxin Module

The chromosomal ntrPR operon of Sinorhizobium meliloti encodes a protein pair that forms a toxin-antitoxin (TA) module, the first characterized functional TA system in Rhizobiaceae. Similarly to other bacterial TA systems, the toxin gene ntrR is preceded by and partially overlaps with the antitoxin gene ntrP. Based on protein homologies, the ntrPR operon belongs to the vapBC family of TA systems. The operon is negatively autoregulated by the NtrPNtrR complex. Promoter binding by NtrP is weak; stable complex formation also requires the presence of NtrR. The N-terminal part of NtrP is responsible for the interaction with promoter DNA, whereas the C-terminal part is required for protein-protein interactions. In the promoter region, a direct repeat sequence was identified as the binding site of the NtrPNtrR complex. NtrR expression resulted in the inhibition of cell growth and colony formation; this effect was counteracted by the presence of the antitoxin NtrP. These results and our earlier observations demonstrating a less effective downregulation of a wide range of symbiotic and metabolic functions in the ntrR mutant under microoxic conditions and an increased symbiotic efficiency with the host plant alfalfa suggest that the ntrPR module contributes to adjusting metabolic levels under symbiosis and other stressful conditions.

Pyruvate Fermentation by Oenococcus oeni and Leuconostoc mesenteroides and Role of Pyruvate Dehydrogenase in Anaerobic Fermentation

The heterofermentative lactic acid bacteria Oenococcus oeni and Leuconostoc mesenteroides are able to grow by fermentation of pyruvate as the carbon source (2 pyruvate --> 1 lactate + 1 acetate + 1 CO(2)). The growth yields amount to 4.0 and 5.3 g (dry weight)/mol of pyruvate, respectively, suggesting formation of 0.5 mol ATP/mol pyruvate. Pyruvate is oxidatively decarboxylated by pyruvate dehydrogenase to acetyl coenzyme A, which is then converted to acetate, yielding 1 mol of ATP. For NADH reoxidation, one further pyruvate molecule is reduced to lactate. The enzymes of the pathway were present after growth on pyruvate, and genome analysis showed the presence of the corresponding structural genes. The bacteria contain, in addition, pyruvate oxidase activity which is induced under microoxic conditions. Other homo- or heterofermentative lactic acid bacteria showed only low pyruvate fermentation activity.

Wide-range transcriptional modulating effect of ntrR under microaerobiosis in Sinorhizobium meliloti.

A mutation in the second gene in the ntrPR operon results in increased expression of nodulation (nod) and nitrogen fixation (nif) genes in Sinorhizobium meliloti. Since this pleiotropic effect is particularly pronounced in the presence of external combined nitrogen, a nitrogen regulatory function has been suggested for NtrR. To identify the complete set of protein-coding genes influenced by loss of ntrR function, microarray hybridizations were carried out to compare transcript levels in the wild type and mutant strains grown under aerobic and microaerobic conditions. Of the 6207 genes examined, representing the entire genome of S. meliloti, 7% exhibited altered expression: 4.5% of the genes are affected under oxic, 2.5% under microoxic conditions. 0.4% of all the genes are affected under both oxygen concentrations. A microoxic environment is required for the induction of genes related to symbiotic functions but results in the down-regulation of other (e.g. metabolic) functions. When the alterations in transcription levels at low oxygen concentration in the mutant strain were compared to those of the wild type, a modulating effect of the ntrR mutation was observed. For example, symbiotic nif/fix genes were induced in both strains, but the level of induction was higher in the ntrR mutant. In contrast, genes related to transcription/translation functions were down-regulated in both strains, and the effect was greater in the wild-type strain than in the ntrR mutant. A relatively wide range of functions was affected by this modulating influence, suggesting that ntrR is not a nitrogen regulatory gene. Since genes encoding various unrelated functions were affected, we propose that NtrR may either interfere with general regulatory mechanisms, such as phosphorylation/dephosphorylation, or may influence RNA stability.

F420H2 oxidase (FprA) from Methanobrevibacter arboriphilus, a coenzyme F420-dependent enzyme involved in O2 detoxification.

Cell suspensions of Methanobrevibacter arboriphilus catalyzed the reduction of O(2) with H(2) at a maximal specific rate of 0.4 U (micromol/min) per mg protein with an apparent K(m) for O(2) of 30 microM. The reaction was not inhibited by cyanide. The oxidase activity was traced back to a coenzyme F(420)-dependent enzyme that was purified to apparent homogeneity and that catalyzed the oxidation of 2 F(420)H(2) with 1 O(2) to 2 F(420) and 2 H(2)O. The apparent K(m) for F(420) was 30 microM and that for O(2) was 2 microM with a V(max) of 240 U/mg at 37 degrees C and pH 7.6, the pH optimum of the oxidase. The enzyme did not use NADH or NADPH as electron donor or H(2)O(2) as electron acceptor and was not inhibited by cyanide. The 45-kDa protein, whose gene was cloned and sequenced, contained 1 FMN per mol and harbored a binuclear iron center as indicated by the sequence motif H-X-E-X-D-X(62)-H-X(18)-D-X(60)-H. Sequence comparisons revealed that the F(420)H(2) oxidase from M. arboriphilus is phylogenetically closely related to FprA from Methanothermobacter marburgensis (71% sequence identity), a 45-kDa flavoprotein of hitherto unknown function, and to A-type flavoproteins from bacteria (30-40%), which all have dioxygen reductase activity. With heterologously produced FprA from M. marburgensis it is shown that this protein is also a highly efficient F(420)H(2) oxidase and that it contains 1 FMN and 2 iron atoms. The presence of F(420)H(2) oxidase in methanogenic archaea may explain why some methanogens, e.g., the Methanobrevibacter species in the termite hindgut, cannot only tolerate but thrive under microoxic conditions.

Genomics of the ccoNOQP-encoded cbb3 oxidase complex in bacteria.

Many bacteria adapt to microoxic conditions by synthesizing a particular cytochrome c oxidase (cbb3) complex with a high affinity for O2, encoded by the ccoNOQP operon. A survey of genome databases indicates that ccoNOQP sequences are widespread in all sub-branches of Proteobacteria but otherwise are found only in bacteria of the CFB group ( Cytophaga, Flexibacter, Bacteroides). Our analysis of available genome sequences suggests four major strategies of regulating ccoNOQP expression in response to O2. The most widespread strategy involves direct regulation by the O2-responsive protein Fnr. The second strategy involves an O2-insensitive paralogue of Fnr, FixK, whose expression is regulated by the O2-responding FixLJ two-component system. A third strategy of mixed regulation operates in bacteria carrying both fnr and fixLJ-fixKgenes. Another, not yet identified, strategy is likely to operate in the epsilon-Proteobacteria Helicobacter pylori and Campylobacter jejuni which lack fnr and fixLJ-fixK genes. The FixLJ strategy appears specific for the alpha-subclass of Proteobacteria but is not restricted to rhizobia in which it was originally discovered.

A new purple sulfur bacterium isolated from a littoral microbial mat, Thiorhodococcus drewsii sp. nov.

A new strain of purple sulfur bacterium was isolated from a marine microbial mat sampled in Great Sippewissett Salt Marsh at the Atlantic coast (Woods Hole, Mass., USA). Single cells of strain AZ1 were coccus-shaped, highly motile by means of a single flagellum, and did not contain gas vesicles. Intracellular membranes were of the vesicular type. However, additional concentric membrane structures were present. The photosynthetic pigments were bacteriochlorophyll a and carotenoids of the normal spirilloxanthin series, with rhodopin as the dominant carotenoid. Hydrogen sulfide (up to 11 mM), sulfur, thiosulfate, and molecular hydrogen were used as electron donors during anaerobic phototrophic growth. During growth on sulfide, elemental sulfur globules were transiently stored inside the cells. Strain AZ1 is much more versatile than most other Chromatiaceae with respect to electron donor and organic substrates. In the presence of CO(2), it is capable of assimilating C(1)-C(5) fatty acids, alcohols, and intermediates of the tricarboxylic acid cycle. Strain AZ1 could also grow photoorganotrophically with acetate as the sole photosynthetic electron donor. Chemotrophic growth in the dark under microoxic conditions was not detected. Optimum growth occurred at pH 6.5-6.7, 30-35 degrees C, > or =50 micro mol quanta m(-2) s(-1), and 2.4-2.6% NaCl. The DNA base composition was 64.5 mol% G+C. Comparative sequence analysis of the 16S rRNA gene confirmed that the isolate is a member of the family Chromatiaceae. Sequence similarity to the most closely related species, Thiorhodococcus minor DSMZ 11518(T), was 97.8%; however, the value for DNA-DNA hybridization between both strains was only 20%. Because of the low genetic similarity and since strain AZ1 physiologically differs considerably from all other members of the Chromatiaceae, including Trc. minor, the new isolate is described as a new species of the genus Thiorhodococcus, Thiorhodococcus drewsii sp. nov.

Thioalkalispira microaerophila gen. nov., sp. nov., a novel lithoautotrophic, sulfur-oxidizing bacterium from a soda lake.

An anaerobic enrichment medium (pH 10) with thiosulfate as electron donor and nitrate as electron acceptor was inoculated with sediment from soda lake Fazda (Wadi Natrun, Egypt); a novel strain, ALEN 1(T), was isolated from the subsequent enrichment culture. Cells of strain ALEN 1(T) had a spiral morphology (0.3-0.45 x 1-4 microm), were motile and had a single polar flagellum. Sphaeroplasts were formed by the cells and were rapidly lysed during prolonged aerobic incubation of cultures. Cells of strain ALEN 1(T) contained a membrane-associated yellow pigment. The metabolism of this novel organism was obligately chemolithoautotrophic, and thiosulfate or sulfide were utilized as electron donors. Washed cells of strain ALEN 1(T) oxidized thiosulfate, sulfide, polysulfide and elemental sulfur to sulfate. Best growth was observed when the strain was grown under micro-oxic conditions (1-2% O2 in gas phase), whereas growth was inhibited under fully oxic conditions. Nitrate was reduced to nitrite without growth of the novel organism, but other nitrogen oxides were not utilized as electron acceptors. Strain ALEN 1(T) was alkaliphilic and moderately halophilic. It grew between pH 8 and 10.4 (optimum around pH 10) with a salt concentration of between 0.3 and 1.5 M Na+ (optimum 0-5 M). The maximum growth rate (0.08 h(-1)) of the organism was achieved in a thiosulfate-limited micro-oxic continuous culture (pH 10). Phylogenetic analyses of the 16S rDNA sequences of strain ALEN 1(T) and its closest relatives demonstrated that this strain formed a deep branch within the gamma-Proteobacteria, with no obvious association to any described cluster of species/genera. On the basis of its unique physiological properties and distinct phylogenetic position, it is proposed that strain ALEN 1(T) (= DSM 14786(T) = UNICEM 212(T)) represents a novel genus within the gamma-Proteobacteria, for which the name Thioalkalispira is proposed. It is also proposed that the type species of this novel genus be named Thioalkalispira microaerophila.

Lithoautotrophic growth of the freshwater strain Beggiatoa D-402 and energy conservation in a homogeneous culture under microoxic conditions.

The freshwater filamentous bacterium Beggiatoa D-402 was shown to grow lithoautotrophically in a homogeneous culture under microoxic conditions only, the growth yield being the highest at 0.1 mg O(2) l(-1). High activities of the Calvin cycle key enzymes and of the dissimilatory path thiosulfate oxidation enzymes were found in the bacterial cells. The rate of CO(2) fixation above 112 nmol min(-1) (mg protein)(-1), an about 90% increase in the protein carbon at the expense of CO(2) carbon and an increase in the molar yield up to 12 mg dry weight (mmol oxidized thiosulfate)(-1) indicate the bacterial growth was autotrophic. Thiosulfate was oxidized by the strain almost completely into sulfate. The metabolically useful energy was conserved by oxidative phosphorylation that was coupled to oxidation of sulfur compounds. The bacterial membranes were found to contain CO-binding cytochromes b and two cytochromes c with M(r) 23 and 26 kDa, the terminal part of the respiratory chain containing presumably a cbb(3)-type oxidase. A cytochrome c with M(r) 12 kDa was detected in the soluble fraction.

Lithoautotrophic growth of the freshwater strain Beggiatoa D-402 and energy conservation in a homogeneous culture under microoxic conditions

The freshwater filamentous bacterium Beggiatoa D-402 was shown to grow lithoautotrophically in a homogeneous culture under microoxic conditions only, the growth yield being the highest at 0.1 mg O2 l−1. High activities of the Calvin cycle key enzymes and of the dissimilatory path thiosulfate oxidation enzymes were found in the bacterial cells. The rate of CO2 fixation above 112 nmol min−1 (mg protein)−1, an about 90% increase in the protein carbon at the expense of CO2 carbon and an increase in the molar yield up to 12 mg dry weight (mmol oxidized thiosulfate)−1 indicate the bacterial growth was autotrophic. Thiosulfate was oxidized by the strain almost completely into sulfate. The metabolically useful energy was conserved by oxidative phosphorylation that was coupled to oxidation of sulfur compounds. The bacterial membranes were found to contain CO-binding cytochromes b and two cytochromes c with Mr 23 and 26 kDa, the terminal part of the respiratory chain containing presumably a cbb3-type oxidase. A cytochrome c with Mr 12 kDa was detected in the soluble fraction.