pH and a mixed carbon-substrate spectrum influence FocA- and FocB-dependent, formate-driven H2 production in Escherichia coli.

Escherichia coli encodes two formate channels, FocA and FocB, that either export formate or import it for further disproportionation by the formate hydrogenlyase (FHL) complex to H2 and CO2. We show that FocA/B appear to change their substrate-translocation direction depending on pH and electron donor. When cells were grown on glucose and glycerol at pH 7.5, formate accumulated in focB or focA-focB mutants when glucose or formate was used as electron donor because H2 production increased ∼2- and ∼1.5-fold, respectively. Moreover, addition of external formate to the growth medium increased H2 production in a focA-focB mutant. This indicates that in the wild type, formate is preferentially exported at pH 7.5 and that another FocA/B-independent uptake system exists. At pH 6.5 and 5.5, the formate channel mutants showed reduced H2 production, suggesting that formate is usually imported by them to produce H2 at acidic pH. Addition of formate to the growth medium increased H2 production at these pHs. Notably, glycerol failed to act as an effective electron donor for formate production. Taken together, our results suggest that regulation of formate translocation direction by FocA/FocB channels is important for maintaining internal pH and proton motive force by modulating H2 production.

Evidence that the Rhizobium regulatory protein RirA binds to cis-acting iron-responsive operators (IROs) at promoters of some Fe-regulated genes.

Mutations in rirA of Rhizobium have been shown to deregulate expression of several genes that are normally repressed by iron. A conserved sequence, the iron-responsive operator (IRO), was identified near promoters of vbsC (involved in the synthesis of the siderophore vicibactin), rpoI (specifies an ECF sigma factor needed for vicibactin synthesis) and the two fhuA genes (encoding vicibactin receptor). Removal of these IRO sequences abolished Fe-responsive repression. Most of these genes were constitutively expressed in the heterologous host, Paracoccus denitrificans, but introduction of the cloned rirA gene repressed expression of these Rhizobium genes in this heterologous host if the corresponding IRO sequences were also intact. These observations are the first to examine the mechanisms of RirA, which has no sequence similarity to well-known iron-responsive regulators such as Fur or DtxR. They provide strong circumstantial evidence that RirA is a transcriptional regulator that binds to cis-acting regulatory sequences near the promoters of at least some of the genes whose expression it controls in response to Fe availability.

The Fur-like protein Mur of Rhizobium leguminosarum is a Mn(2+)-responsive transcriptional regulator.

In wild-type Rhizobium leguminosarum, the sitABCD operon specifies a Mn(2+) transporter whose expression is severely reduced in cells grown in the presence of this metal. Mutations in the R. leguminosarum gene, mur (manganese uptake regulator), whose product resembles the Fur transcriptional regulator, cause high-level expression of sitABCD in the presence of Mn(2+). In gel-shift mobility assays, purified R. leguminosarum Mur protein bound to at least two regions near the sitABCD promoter region, although this DNA has no conventional consensus Fur-binding sequences (fur boxes). Thus, in contrast to gamma-proteobacteria, where Fur binds Fe(2+), the R. leguminosarum Fur homologue, Mur, act as a Mn(2)-responsive transcriptional regulator.

Characterization of the expression and activity of the periplasmic nitrate reductase of Paracoccus pantotrophus in chemostat cultures.

The periplasmic nitrate reductase (Nap) from Paracoccus pantotrophus has a role in cellular redox balancing. Previously, transcription from the nap promoter in P. pantotrophus was shown to be responsive to the oxidation state of the carbon substrate. During batch culture, expression was higher during growth on reduced substrates such as butyrate compared to more oxidized substrates such as succinate. In the present study the effect of growth rate on nap expression in succinate-, acetate- and butyrate-limited chemostat cultures was investigated. In all three cases transcription from the nap promoter and Nap enzyme activity showed a strong correlation. At the fastest growth rates tested for the three substrates nap expression and Nap activity were highest when growth occurred on the most reduced substrate (butyrate > acetate > succinate). However, in all three cases a bell-shaped pattern of expression was observed as a function of growth rate, with the highest levels of nap expression and Nap activity being observed at intermediate growth rates. This effect was most pronounced on succinate, where an approximately fivefold variation was observed, and at intermediate dilution rates nap expression and Nap activity were comparable on all three carbon substrates. Analysis of mRNA prepared from the succinate-grown cultures revealed that different transcription initiation start sites for the nap operon were utilized as the growth rate changed. This study establishes a new regulatory feature of nap expression in P. pantotrophus that occurs at the level of transcription in response to growth rate in carbon-limited cultures.

Fur is not the global regulator of iron uptake genes in Rhizobium leguminosarum.

Rhizobium leguminosarum fur mutants were unaffected in Fe-dependent regulation of several operons that specify different Fe uptake systems, yet cloned R. leguminosarum fur partially corrected an Escherichia coli fur mutant and R. leguminosarum Fur protein bound to canonical fur boxes. The lack of a phenotype in fur mutants is not due to functional redundancy with Irr, another member of the Fur superfamily found in the rhizobia, since irr fur double mutants are also unaffected in Fe-responsive regulation of several operons involved in Fe uptake. Neither Irr nor Fur is needed for symbiotic N(2) fixation on peas. As in Bradyrhizobium japonicum, irr mutants accumulated protoporphyrin IX. R. leguminosarum irr is not regulated by Fur and its Irr protein lacks the motif needed for haem-dependent post-translational modification that occurs in B. japonicum Irr. The similarities and differences in the Fur superfamily in the rhizobia and other Gram-negative bacteria are discussed.

Hierarchy of carbon source selection in Paracoccus pantotrophus: strict correlation between reduction state of the carbon substrate and aerobic expression of the nap operon.

Paracoccus pantotrophus can express a periplasmic nitrate reductase (Nap) during aerobic growth. A proposed role for this enzyme is the dissipation of excess redox energy during oxidative metabolism of reduced carbon substrates. To investigate the regulation of nap expression, a transcriptional fusion between the nap promoter region of P. pantotrophus and the lacZ gene was constructed. When this fusion was used, analyses showed that transcription from the nap promoter increases as the average reduction state of the carbon atoms increases. Thus, beta-galactosidase activities increase as the carbon source changes in the order succinate-acetate-butyrate. This result was obtained regardless of which of the three carbon sources was used for culture of the inoculum. If two carbon sources were presented together, the beta-galactosidase activity was always the same as it was when the least-reduced carbon source was added alone. This suggests that the regulation is dependent upon metabolism of the more-reduced carbon sources rather than just their presence in the medium. Analysis of culture medium by (1)H nuclear magnetic resonance showed that for aerobic growth P. pantotrophus strictly selected its carbon source in the order succinate-acetate-butyrate. This was reflected by diauxic growth kinetics on medium containing mixed carbon substrates. The regulatory mechanism underpinning such a selection is unknown but is likely to be related to the mechanism which controls the transcription of the nap operon.

The vbs genes that direct synthesis of the siderophore vicibactin in Rhizobium leguminosarum: their expression in other genera requires ECF sigma factor RpoI.

A cluster of eight genes, vbsGSO, vbsADL, vbsC and vbsP, are involved in the synthesis of vicibactin, a cyclic, trihydroxamate siderophore made by the symbiotic bacterium Rhizobium leguminosarum. None of these vbs genes was required for symbiotic N2 fixation on peas or Vicia. Transcription of vbsC, vbsGSO and vbsADL (but not vbsP) was enhanced by growth in low levels of Fe. Transcription of vbsGSO and vbsADL, but not vbsP or vbsC, required the closely linked gene rpoI, which encodes an ECF sigma factor of RNA polymerase. Transfer of the cloned vbs genes, plus rpoI, to Rhodobacter, Paracoccus and Sinorhizobium conferred the ability to make vicibactin on these other genera. We present a biochemical genetic model of vicibactin synthesis, which accommodates the phenotypes of different vbs mutants and the homologies of the vbs gene products. In this model, VbsS, which is similar to many non-ribosomal peptide synthetase multienzymes, has a central role. It is proposed that VbsS activates L-N5-hydroxyornithine via covalent attachment as an acyl thioester to a peptidyl carrier protein domain. Subsequent VbsA-catalysed acylation of the hydroxyornithine, followed by VbsL-mediated epimerization and acetylation catalysed by VbsC, yields the vicibactin subunit, which is then trimerized and cyclized by the thioesterase domain of VbsS to give the completed siderophore.

dpp genes of Rhizobium leguminosarum specify uptake of delta-aminolevulinic acid.

An operon with homology to the dppABCDF genes required to transport dipeptides in bacteria was identified in the N2-fixing symbiont, Rhizobium leguminosarum. As in other bacteria, dpp mutants were severely affected in the import of delta-aminolevulinic acid (ALA), a heme precursor. ALA uptake was antagonized by adding dipeptides, indicating that these two classes of molecule share the same transporter. Mutations in dppABCDF did not affect symbiotic N2 fixation on peas, suggesting that the ALA needed for heme synthesis is not supplied by the plant or that another uptake system functions in the bacteroids. The dppABCDF operon of R. leguminosarum resembles that in other bacteria, with a gap between dppA and dppB containing inverted repeats that may stabilize mRNA and may explain why transcription of dppA alone was higher than that of dppBCDF. The dppABCDF promoter was mapped and is most likely recognized by sigma70.

The Rhizobium leguminosarum tonB gene is required for the uptake of siderophore and haem as sources of iron.

In the N2-fixing bacterium Rhizobium leguminosarum, mutations in a homologue of tonB (tonB(Rl)) block the import of vicibactin and haem as iron sources in free-living bacteria. TonB(Rl) mutants were normal for growth with ferric dicitrate and slightly reduced for growth with haemoglobin as sole iron sources. The deduced TonB(Rl) product is larger than that of (for example) Escherichia coli, on account of an extended N-terminal domain. Transcription of tonB(Rl) was enhanced in low-Fe growth conditions; this was not controlled by Fur, nor RpoI, an Fe-regulated extracytoplasmic sigma factor. Upstream of tonB(Rl) and transcribed divergently is an operon, hmuPSTUV, whose products are homologous to ABC transporters involved in haem uptake in pathogenic bacteria. Expression of hmuPSTUV was enhanced in low-Fe conditions, and hmu mutants show slightly diminished growth on haem as sole Fe source, suggesting that there is more than one system for the uptake of this molecule. hmuPSTUV expression appears to be from three closely linked promoters. Downstream of hmuPSTUV, a gene that may encode an extracytoplasmic sigma factor was identified, but this gene, rpoZ, did not affect the transcription of tonB(Rl) or hmuPSTUV. Mutations in tonB(Rl), hmu genes and rpoZ did not affect symbiotic N(2) fixation in peas.

X-ray structure of a blue copper nitrite reductase at high pH and in copper-free form at 1.9 A resolution.

Copper-containing nitrite reductases possess a trimeric structure where the catalytic Cu site, located at the monomer-monomer interface, resembles the catalytic sites of a number of Zn enzymes. Nitrite reductase from Alcaligenes xylosoxidans has optimum activity at pH 5.2 which decreases to a negligible level at pH 8. The structure of this nitrite reductase has previously been determined at pH 4.6. It has now been crystallized under new conditions at pH 8.5. Its crystallographic structure provides a structural explanation for the greatly reduced activity of the enzyme at high pH. Characterization of overexpressed protein in solution by EXAFS suggested that the protein lacked Cu in the catalytic type 2 Cu site and that the site was most probably occupied by Zn. Using the anomalous signals from Cu and Zn, the crystal structure revealed that the expressed protein was devoid of Cu in the catalytic site and that only a trace amount (<10%) of Zn was present at this site in the crystal. Despite the close structural similarity of the catalytic site to a number of Zn enzymes, these data suggest that Zn, if it binds at the catalytic copper site, binds weakly in nitrite reductase.

A novel mechanism controls anaerobic and catabolite regulation of the Escherichia coli tdc operon.

The tdc operon is subject to CRP-controlled catabolite repression. Expression of the operon is also induced anaerobically, although this regulation does not rely on direct control by either FNR or ArcA. Recently, the anaerobic expression of the tdc operon was found to be fortuitously induced in the presence of glucose by a heterologous gene isolated from the Gram-positive anaerobe Clostridium butyricum. The gene, termed tcbC, encoded a histone-like protein of 14.5 kDa. Using tdc-lacZ fusions, it was shown that TcbC did not activate tdc expression by functionally replacing any of the operon regulators. In vitro transcription analyses with RNA polymerase and CRP revealed that faithful CRP-dependent transcription initiation occurred only on supercoiled templates. No specific, CRP-dependent transcription initiation was observed on relaxed or linear DNA templates. Surprisingly, purified His-tagged TcbC activated transcription from a relaxed, circular template, but not from supercoiled or linear templates. Examination of the CRP binding site of the tdc promoter revealed that it was located 43.5 bp upstream of the transcription initiation site. Repositioning of the CRP site at -41.5 bp abolished activation by the TcbC protein and allowed CRP-dependent transcription to occur on linear, relaxed and supercoiled templates. TcbC bound DNA non-specifically; however, in topoisomerase I relaxation assays, it was demonstrated that TcbC imposed torsional constraints on negatively supercoiled DNA, which influenced the ability of the enzyme to relax the topoisomers. Taken together, these results strongly suggest that TcbC activates transcription of tdc by altering the local topological status of the tdc promoter and that, in the wild-type tdc promoter, the CRP binding site is misaligned to allow transcription to occur only under optimal conditions. Indeed, in vivo transcription analyses revealed that repositioning of the CRP binding site to -41.5 bp resulted in high-level, CRP-dependent transcription, even under catabolite-repressing conditions, and that transcription was no longer influenced by TcbC. Remarkably, however, anaerobic regulation of the mutant promoter was retained. This indicates that the other tdc regulators, TdcA and TdcR, govern anaerobic transcription activation by CRP.

Constitutive expression of Escherichia coli tat genes indicates an important role for the twin-arginine translocase during aerobic and anaerobic growth.

The transcription start sites for the tatABCD and tatE loci, encoding components of the Tat (twin-arginine translocase) protein export pathway, have been identified. Expression studies indicate that the tatABCD and tatE transcription units are expressed constitutively. Translational fusion experiments suggest that TatA is synthesized at a much higher level than the other Tat proteins.

Biosynthesis of poly-beta-hydroxybutyrate (PHB) is controlled by CydR (Fnr) in the obligate aerobe Azotobacter vinelandii.

CydR is an Fnr-like protein in the obligatory aerobic nitrogen-fixing bacterium Azotobacter vinelandii. The cydR mutant overproduces the cytochrome bd terminal oxidase. Using two-dimensional polyacrylamide gel electrophoresis, we showed that beta-ketothiolase and acetoacetyl-CoA reductase were also overexpressed in the cydR mutant. Fumarase C and a coenzyme A transferase, possibly succinyl-SCoA transferase, were decreased in this mutant. Enzyme assays confirmed the elevated beta-ketothiolase and acetoacetyl-CoA reductase activities in this mutant. The cydR mutant accumulated poly-beta-hydroxybutyrate throughout the exponential growth phase, unlike the wild-type strain that only accumulated poly-beta-hydroxybutyrate during stationary phase. The results demonstrate that CydR controls poly-beta-hydroxybutyrate synthesis in A. vinelandii.

Catalytic and spectroscopic analysis of blue copper-containing nitrite reductase mutants altered in the environment of the type 2 copper centre: implications for substrate interaction.

The blue dissimilatory nitrite reductase (NiR) from Alcaligenes xylosoxidans is a trimer containing two types of Cu centre, three type 1 electron transfer centres and three type 2 centres. The latter have been implicated in the binding and reduction of nitrite. The Cu ion of the type 2 centre of the oxidized enzyme is ligated by three His residues, and additionally has a co-ordinated water molecule that is also hydrogen-bonded to the carboxyl of Asp(92) [Dodd, Van Beeumen, Eady and Hasnain (1998), J. Mol. Biol. 282, 369-382]. Two mutations of this residue have been made, one to a glutamic acid residue and a second to an asparagine residue; the effects of both mutations on the spectroscopic and catalytic properties of the enzyme have been analysed. EPR spectroscopy revealed that both mutants retained intact type 1 Cu centres with g( parallel)=2.12 (A( parallel)=0 mT) and g( perpendicular)=2.30 (A( perpendicular)=6.4 mT), which was consistent with their blue colour, but differed in their activities and in the spectroscopic properties of the type 2 centres. The D92E mutant had an altered geometry of its type 2 centre such that nitrite was no longer capable of binding to elicit changes in the EPR parameters of this centre. Accordingly, this mutation resulted in a form of NiR that had very low enzyme activity with the artificial electron donors reduced Methyl Viologen and sodium dithionite. As isolated, the EPR spectrum of the Asp(92)-->Asn (D92N) mutant showed no characteristic type 2 hyperfine lines. However, oxidation with iridium hexachloride partly restored a type 2 EPR signal, suggesting that type 2 copper is present in the enzyme but in a reduced, EPR-silent form. Like the Asp(92)-->Glu mutant, D92N had very low enzyme activities with either Methyl Viologen or dithionite. Remarkably, when the physiological electron donor reduced azurin I was used, both mutant proteins exhibited restoration of enzyme activity. The degree of restoration differed for the two mutants, with the D92N derivative exhibiting approx. 60% of the activity seen for the wild-type NiR. These findings suggest that on formation of an electron transfer complex with azurin, a conformational change in NiR occurs that returns the catalytic Cu centre to a functionally active state capable of binding and reducing nitrite.

Effects of limited aeration and of the ArcAB system on intermediary pyruvate catabolism in Escherichia coli.

The capacity of Escherichia coli to adapt its catabolism to prevailing redox conditions resides mainly in three catabolic branch points involving (i) pyruvate formate-lyase (PFL) and the pyruvate dehydrogenase complex (PDHc), (ii) the exclusively fermentative enzymes and those of the Krebs cycle, and (iii) the alternative terminal cytochrome bd and cytochrome bo oxidases. A quantitative analysis of the relative catabolic fluxes through these pathways is presented for steady-state glucose-limited chemostat cultures with controlled oxygen availability ranging from full aerobiosis to complete anaerobiosis. Remarkably, PFL contributed significantly to the catabolic flux under microaerobic conditions and was found to be active simultaneously with PDHc and cytochrome bd oxidase-dependent respiration. The synthesis of PFL and cytochrome bd oxidase was found to be maximal in the lower microaerobic range but not in a delta ArcA mutant, and we conclude that the Arc system is more active with respect to regulation of these two positively regulated operons during microaerobiosis than during anaerobiosis.

Regulation of cytochrome bd expression in the obligate aerobe Azotobacter vinelandii by CydR (Fnr). Sensitivity to oxygen, reactive oxygen species, and nitric oxide.

Azotobacter vinelandii is an obligately aerobic bacterium in which aerotolerant nitrogen fixation requires cytochrome bd. Regulation of cytochrome bd expression is achieved by CydR (an Fnr homologue), which represses transcription of the oxidase genes cydAB. cydAB mRNA was mapped by primer extension; the transcriptional start site was determined, and putative -10 and -35 regions were deduced. Two "CydR boxes," one at the +1 site and one upstream of the -35 region, were identified. Transcriptionally inactive, purified CydR was converted, by adding NifS, cysteine, and Fe(2+), into an active form possessing acid-labile sulfide and spectra suggesting a [4Fe-4S](2+) cluster. Reconstituted CydR specifically bound both CydR boxes cooperatively, with higher affinity for the nearer consensus +1 site. Low concentrations of O(2) or NO ([O(2)]/[[CydR] or [NO]/[CydR] = 0.1-0. 6) elicited loss of the 420 nm absorbance attributed to the [4Fe-4S](2+) cluster, formation of a 315 nm species, and loss of ability to retard DNA migration. Retardation by reconstituted CydR was enhanced by superoxide dismutase and/or catalase, suggesting a role for reactive oxygen species in CydR inactivation. The role of CydR in regulating cydAB expression in the supposedly anoxic cytoplasm of A. vinelandii and similarities to cydAB regulation by Fnr in Escherichia coli are discussed.

Transcriptional regulation in response to oxygen and nitrate of the operons encoding the [NiFe] hydrogenases 1 and 2 of Escherichia coli.

Synthesis of the [NiFe] hydrogenases 1 and 2 of Escherichia coli is induced in response to anaerobiosis and is repressed when nitrate is present in the growth medium. The hydrogenase 1 and hydrogenase 2 enzymes are encoded by the polycistronic hyaABCDEF and hybOABCDEFG operons, respectively. Primer extension analysis was used to determine the initiation site of transcription of both operons. This permitted the construction of single-copy lacZ operon fusions, which were used to examine the transcriptional regulation of the two operons. Expression of both was induced by anaerobiosis and repressed by nitrate, which is in complete accord with earlier biochemical studies. Anaerobic induction of the hyb operon was only partially dependent on the FNR protein and, surprisingly, was enhanced by an arcA mutation. This latter result indicated that ArcA suppresses anaerobic hyb expression and that a further factor, which remains to be identified, is involved in controlling anaerobic induction of operon expression. Nitrate repression of hyb expression was mediated by the NarL/NarX and NarP/NarQ two-component regulatory systems. Remarkably, a narP mutant lacked anaerobic induction of hyb expression, even in the absence of added nitrate. Anaerobic induction of hya expression was dependent on the ArcA and AppY regulators, which confirms earlier observations by other authors. Nitrate repression of the hya operon was mediated by both NarL and NarP. Taken together, these data indicate that although the hya and hyb operons share common regulators, there are important differences in the control of expression of the individual operons.

The aerobic/anaerobic interface.

Microorganisms have evolved intricate signal transduction mechanisms that respond both to dioxygen per se and to the consequences imparted by dioxygen on the metabolism of the cell. Escherichia coli provides examples of both types of signal sensing mechanisms, including FNR and the Arc system. The factors involved in these diverse sensory systems are proving to have a pervasive impact on controlling gene expression and cellular physiology. Similar signal transduction systems are prevalent in a diverse range of microorganisms.

The genetic basis of tetrathionate respiration in Salmonella typhimurium.

A range of bacteria are able to use tetrathionate as a terminal respiratory electron acceptor. Here we report the identification and characterization of the ttrRSBCA locus required for tetrathionate respiration in Salmonella typhimurium LT2a. The ttr genes are located within Salmonella pathogenicity island 2 at centisome 30.5. ttrA, ttrB and ttrC are the tetrathionate reductase structural genes. Sequence analysis suggests that TtrA contains a molybdopterin guanine dinucleotide cofactor and a [4Fe-4S] cluster, that TtrB binds four [4Fe-4S] clusters, and that TtrC is an integral membrane protein containing a quinol oxidation site. TtrA and TtrB are predicted to be anchored by TtrC to the periplasmic face of the cytoplasmic membrane implying a periplasmic site for tetrathionate reduction. It is inferred that the tetrathionate reductase, together with thiosulphate and polysulphide reductases, make up a previously unrecognized class of molybdopterin-dependent enzymes that carry out the reductive cleavage of sulphur-sulphur bonds. Cys-256 in TtrA is proposed to be the amino acid ligand to the molybdopterin cofactor. TtrS and TtrR are the sensor and response regulator components of a two-component regulatory system that is absolutely required for transcription of the ttrBCA operon. Expression of an active tetrathionate reduction system also requires the anoxia-responsive global transcriptional regulator Fnr. The ttrRSBCA gene cluster confers on Escherichia coli the ability to respire with tetrathionate as electron acceptor.

The blue copper-containing nitrite reductase from Alcaligenes xylosoxidans: cloning of the nirA gene and characterization of the recombinant enzyme.

The nirA gene encoding the blue dissimilatory nitrite reductase from Alcaligenes xylosoxidans has been cloned and sequenced. To our knowledge, this is the first report of the characterization of a gene encoding a blue copper-containing nitrite reductase. The deduced amino acid sequence exhibits a high degree of similarity to other copper-containing nitrite reductases from various bacterial sources. The full-length protein included a 24-amino-acid leader peptide. The nirA gene was overexpressed in Escherichia coli and was shown to be exported to the periplasm. Purification was achieved in a single step, and analysis of the recombinant Nir enzyme revealed that cleavage of the signal peptide occurred at a position identical to that for the native enzyme isolated from A. xylosoxidans. The recombinant Nir isolated directly was blue and trimeric and, on the basis of electron paramagnetic resonance spectroscopy and metal analysis, possessed only type 1 copper centers. This type 2-depleted enzyme preparation also had a low nitrite reductase enzyme activity. Incubation of the periplasmic fraction with copper sulfate prior to purification resulted in the isolation of an enzyme with a full complement of type 1 and type 2 copper centers and a high specific activity. The kinetic properties of the recombinant enzyme were indistinguishable from those of the native nitrite reductase isolated from A. xylosoxidans. This rapid isolation procedure will greatly facilitate genetic and biochemical characterization of both wild-type and mutant derivatives of this protein.