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.

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.

A radical-chemical route to acetyl-CoA: the anaerobically induced pyruvate formate-lyase system of Escherichia coli.

Anaerobically growing Escherichia coli cells contain the enzyme pyruvate formate-lyase which catalyses the non-oxidative cleavage of pyruvate to acetyl-CoA and formate. The enzyme is subject to interconversion between inactive and active forms. The active form contains an oxygen-sensitive organic free radical located on the polypeptide chain which is essential for catalysis. It affords a novel homolytic C-C bond cleavage of the pyruvate substrate. The radical is generated by an iron-dependent converter enzyme which requires reduced flavodoxin and adenosyl methionine as co-substrates and pyruvate as a positive allosteric effector. A second converter enzyme, also iron-dependent, accomplishes the removal of the radical. This post-translational interconversion cycle controls the activity state of pyruvate formate-lyase in the anaerobic cell. Anaerobic conditions also regulate pyruvate formate-lyase at the level of gene expression. Multiple promoters are responsible for effecting a twelve to fifteen fold induction and they are coordinately controlled in response to the oxygen and metabolic status of the cell by sequences which are located far upstream of the pfl coding region. The transcription factor Fnr has been identified as being responsible for part of the anaerobic control of pfl expression, probably through direct interaction with the upstream sequences. In contrast, the expression of the gene encoding the first iron-dependent converter enzyme is unaffected by anaerobiosis and is independent of the Fnr protein.

Signal transduction and bacterial conjugation: characterization of the role of ArcA in regulating conjugative transfer of the resistance plasmid R1.

The role of the two-component response regulator ArcA protein in the transfer of the conjugative resistance plasmid R1 was investigated using a variety of in vivo and in vitro assays. The frequency of conjugal DNA transfer of plasmid R1-16, a derepressed variant of R1, was reduced by four orders of magnitude in an Escherichia coli host with a mutation in the arcA gene. Measurements of mRNAs transcribed from key plasmid transfer genes revealed that the abundance of each of the mRNA species investigated was reduced significantly in an arcA background. Gene fusion studies with the R1 PY promoter, the major promoter of the transfer operon, and a lacZ reporter gene, indicated that arcA is required for maximal expression from this promoter. However, a stimulating effect of arcA could only be detected when the plasmid-specified positive regulator of the transfer genes, traJ, was present. Electrophoretic mobility shift assays were used to demonstrate specific binding of purified ArcA protein and a purified and phosphorylated oligohistidine-tagged ArcA (His6-ArcA) to a DNA fragment containing the PY promoter region. The binding of phosphorylated His6-ArcA to the PY promoter was further characterized by DNase I footprinting. The observed protection pattern was characteristic for ArcA acting as a transcriptional activator.

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.

Pyruvate formate-lyase is not essential for nitrate respiration by Escherichia coli.

Defined deletion mutants of Escherichia coli defective for the synthesis of pyruvate formate-lyase (PFL) or pyruvate dehydrogenase (PDH) were analysed in regards their growth in batch culture and their enzyme levels under fermentative and nitrate respiratory conditions. A pfl mutant proved not to be completely auxotrophic for acetate when grown anaerobically in glucose minimal medium. In contrast, a pfl aceEF double mutant exhibited an absolute requirement for acetate, indicating that PDH is the source of acetyl-CoA in the pfl mutant. Growth of both pfl and aceEF single mutants under nitrate respiratory conditions was essentially indistinguishable from the wild-type. Thus, either PFL or PDH can be used to catabolize pyruvate in nitrate-respiring cells. The activities of PFL and PDH measured after growth with nitrate are commensurate with this proposal.

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.

The tdcE gene in Escherichia coli strain W3110 is separated from the rest of the tdc operon by insertion of IS5 elements.

Unlike other Escherichia coli K-12 strains, W3110 contains multiple copies of the insertion sequence IS5. Some of these IS5 elements have been involved in tandem duplication of a portion of the chromosome which includes, amonst others, the tdcABC-DEFG operon genes. The nucleotide sequence and insertion site of one of these elements, IS5P, was determined. It was shown that IS5P has inserted within the coding sequence of the tdcA gene and is flanked, not by the remaining portion of the tdcA gene, but by the extreme 3' end of the tdcD gene. In other E. coli K-12 strains the tdcD gene and three other genes, tdcE, tdcF and tdcG, all form part of the tdc operon. Our results demonstrate that during the duplication event the tdcABCgenes have been amplified and separated from the remaining genes tdcE, tdcF and tdcG of the operon, which are each present in single copy.

The hydrogenases and formate dehydrogenases of Escherichia coli.

Escherichia coli has the capacity to synthesise three distinct formate dehydrogenase isoenzymes and three hydrogenase isoenzymes. All six are multisubunit, membrane-associated proteins that are functional in the anaerobic metabolism of the organism. One of the formate dehydrogenase isoenzymes is also synthesised in aerobic cells. Two of the formate dehydrogenase enzymes and two hydrogenases have a respiratory function while the formate dehydrogenase and hydrogenase associated with the formate hydrogenlyase pathway are not involved in energy conservation. The three formate dehydrogenases are molybdo-selenoproteins while the three hydrogenases are nickel enzymes; all six enzymes have an abundance of iron-sulfur clusters. These metal requirements alone invoke the necessity for a profusion of ancillary enzymes which are involved in the preparation and incorporation of these cofactors. The characterisation of a large number of pleiotropic mutants unable to synthesise either functionally active formate dehydrogenases or hydrogenases has led to the identification of a number of these enzymes. However, it is apparent that there are many more accessory proteins involved in the biosynthesis of these isoenzymes than originally anticipated. The biochemical function of the vast majority of these enzymes is not understood. Nevertheless, through the construction and study of defined mutants, together with sequence comparisons with homologous proteins from other organisms, it has been possible at least to categorise them with regard to a general requirement for the biosynthesis of all three isoenzymes or whether they have a specific function in the assembly of a particular enzyme. The identification of the structural genes encoding the formate dehydrogenase and hydrogenase isoenzymes has enabled a detailed dissection of how their expression is coordinated to the metabolic requirement for their products. Slowly, a picture is emerging of the extremely complex and involved path of events leading to the regulated synthesis, processing and assembly of catalytically active formate dehydrogenase and hydrogenase isoenzymes. This article aims to review the current state of knowledge regarding the biochemistry, genetics, molecular biology and physiology of these enzymes.

Specific transcriptional requirements for positive regulation of the anaerobically inducible pfl operon by ArcA and FNR.

Expression of the pfl operon of Escherichia coli is induced 12- to 15-fold by anaerobiosis and transcription is mediated by seven co-ordinately regulated promoters. The 5' non-translated regulatory region of the operon is approximately 450bp in length and contains two of the seven promoters, termed promoter 6 and promoter 7. Site-directed mutagenesis was used to aid the identification of DNA sequences important in directing transcription from the two promoters and to examine the effects such mutations had on the regulation of anaerobic pfl operon expression. Introduction of chromosomal mutations either in the FNR-binding site or -10 region of promoter 6 blocked transcription from this promoter, as determined by primer extension. Similarly, mutation of the -10 region or the putative FNR half-site located at -50 relative to the transcription start site of promoter 7 severely reduced transcription from that promoter. Prevention of transcription from promoter 6 by the -10 box mutation had no influence on promoter 7 transcription. Surprisingly, however, alteration of the FNR-binding site at promoter 6 did reduce transcription from promoter 7. Thus, a cis mutation located 280 bp downstream on the DNA had a profound effect on promoter 7 transcription. This effect would be commensurate with this mutation disrupting an important interaction between proteins bound at promoter 7 with those bound at promoter 6. Primer extension demonstrated that the promoter 7 mutations had no apparent influence on promoter 6 transcription. By using pfl-lacZ gene fusions it could be shown that the FNR-binding site and -10 region mutations at promoter 6 abolished FNR-dependent anaerobic regulation of pfl operon expression. The equivalent mutations at promoter 7 caused a 25% reduction in anaerobic expression. The residual anaerobic expression in such constructs was FNR-, but no longer ArcA-dependent. A construct in which the -10 region of both promoters 6 and 7 was mutated showed no anaerobic induction of pfl operon expression. This indicates that transcription from both promoters is required for maximal anaerobic regulation by ArcA and FNR.

The anaerobic degradation of L-serine and L-threonine in enterobacteria: networks of pathways and regulatory signals.

The mechanisms controlling the biosynthesis and degradation of l-serine and l-threonine are remarkably complex. Their metabolism forms a network of pathways linking several amino acids, central primary metabolites such as pyruvate, oxaloacetate and 3-phosphoglycerate, and C1 metabolism. Studies on the degradation of these amino acids in Escherichia coli have revealed the involvement of fascinating enzymes that utilise quite diverse catalytic mechanisms. Moreover, it is emerging that both environmental and metabolic signals have a major impact in controlling enzyme synthesis This is exemplified by the anaerobically regulated tdc operon, which encodes a metabolic pathway for the degradation of serine and threonine. Studies on this pathway are beginning to provide insights into how an organism adapts its genetic makeup to meet the physiological demands of the cell.

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.

Fnr activates transcription from the P6 promoter of the pfl operon in vitro.

Expression of the Escherichia coli focA-pfl operon is induced by anaerobiosis and transcription is controlled by seven promoters. Anaerobic induction is mediated by the ArcA and Fnr transcription factors. Fnr was purified and its specific interaction with the pfl promoter-regulatory region was characterized. A single binding site could be identified by DNase I footprinting, which was centred at -40.5 bp relative to the start site of promoter 6 (P6) transcription. Activation of P6 transcription in vitro was completely dependent on the Fnr protein. Fnr-dependent transcription could be prevented by mutating the Fnr-binding site, indicating that Fnr must bind to its recognition sequence to be able to activate transcription. An Fnr-independent promoter, which overlaps the P6 promoter, was also identified and characterized in vivo and in vitro. Transcription from this promoter (termed P6A) initiated 10 bp upstream of the P6 start site and assures that low-level synthesis of the FocA protein occurs under aerobic growth conditions.

Isolation and characterization of hypophosphite--resistant mutants of Escherichia coli: identification of the FocA protein, encoded by the pfl operon, as a putative formate transporter.

Hypophosphite was used as a toxic analogue to identify genes whose products have a putative function in the transport of formate. Two Tn10-derived insertion mutants were identified that exhibited increased resistance to high concentrations of hypophosphite in the culture medium. The transposon was located in the identical position in the focA (formate channel; previously termed orf) gene of the pfl operon in both mutants. A defined chromosomal focA nonsense mutant, which showed minimal polarity effects on pfl gene expression, had the same phenotype as the insertion mutants. Results obtained using a hycA-lacZ fusion to monitor changes in the intracellular formate concentration in a focA mutant indicated that the level of formate inside the cell was elevated compared with the wild type. Moreover, it could be shown that there was a corresponding reduction of approximately 50% in the amount of formate excreted by a focA mutant into the culture medium. Taken together, these results indicate that formate accumulates in anaerobic cells which do not have a functional focA gene product and that one function of FocA may be to export formate from the cell. A further significant result was that hypophosphite could substitute for formate in activating hycA gene expression. This hypophosphite-dependent activation of hycA gene expression was reduced 10-fold in a focA null mutant, suggesting that hypophosphite must first enter the cell before it can act as a signal to activate hycA expression. By analogy, these data suggest that focA may also be functional in the import of formate into anaerobic Escherichia coli cells. Site-specific mutagenesis identified the translation initiation codon of focA as a GUG. Therefore, the FocA polypeptide has a molecular weight of 30,958. FocA shows significant similarity at both the primary and secondary structural levels with the NirC protein of E. coli and the FdhC protein of Methanobacterium formicicum. All three proteins are predicted to be integral membrane proteins. A detailed in vivo TnphoA mutagenesis study predicted that FocA has six membrane-spanning segments.

Nitrate repression of the Escherichia coli pfl operon is mediated by the dual sensors NarQ and NarX and the dual regulators NarL and NarP.

The pfl operon is expressed at high levels anaerobically. Growth of Escherichia coli in the presence of nitrate or nitrite led to a 45% decrease in expression when cells were cultivated in rich medium. Nitrate repression, however, was significantly enhanced (sevenfold) when the cells were cultured in minimal medium. Regulation of pfl expression by nitrate was dependent on the NarL, NarP, NarQ, and NarX proteins but independent of FNR, ArcA, and integration host factor, which are additional regulators of pfl expression. Strains unable to synthesize any one of the NarL, NarP, NarQ, or NarX proteins, but retaining the capacity to synthesize the remaining three, exhibited essentially normal nitrate regulation. In contrast, narL narP and narX narQ double null mutants were devoid of nitrate regulation when cultured in rich medium but they retained some nitrate repression (1.3-fold) when grown in minimal medium. By using lacZ fusions, it was possible to localize the DNA sequences required to mediate nitrate repression to the pfl promoter-regulatory region. DNase I footprinting studies identified five potential binding sites for the wild-type NarL protein in the pfl promoter-regulatory region. Specific footprints were obtained only when NarL was phosphorylated with acetyl phosphate before the binding reaction was performed. Each of the protected regions contained at least one heptamer sequence which has been deduced from mutagenesis studies to be essential for NarL binding (K. Tyson, A. Bell, J. Cole, and S. Busby, Mol. Microbiol. 7:151-157, 1993).

Purification of ArcA and analysis of its specific interaction with the pfl promoter-regulatory region.

ArcA is one of several transcription factors required for optimal anaerobic induction of the pyruvate formatelyase (pfl) operon. To aid the study at the molecular level of the interaction of ArcA with the pfl promoter-regulatory region we developed a procedure for the isolation of ArcA. The purification of ArcA involved chromatography in heparin agarose, hydroxylapatite and Mono-Q matrices and delivered a protein that was > 95% pure. Gel retardation assays demonstrated that ArcA bound specifically to the pfl regulatory region. Three distinct ArcA-DNA complexes could be resolved depending on the ArcA concentration used. This finding suggested that either multiple ArcA-binding sites are present in the regulatory region or that ArcA can oligomerize at one or more sites. The DNA-binding activity of ArcA could be increased as estimated 10-fold by prior incubation of the protein with carbamoyl phosphate, suggesting that phosphorylation activates DNA binding or oligomerisation. DNase I footprint analyses identified four sites that were protected by ArcA from cleavage. Two of these sites spanned the transcription start site and -10 regions of promoters 6 and 7, while a third site partially overlapped the characterized binding site of integration host factor (IHF). ArcA exhibited the highest affinity for a stretch of DNA located between the IHF site and the transcription start site of promoter 7. These results are congruent with the hypothesis that a higher-order nucleoprotein complex comprising several proteins, including ArcA, is required to activate transcription from the multiple promoters of the pfl operon.

Promoter 7 of the Escherichia coli pfl operon is a major determinant in the anaerobic regulation of expression by ArcA.

The anaerobically inducible pfl operon of Escherichia coli has a regulatory sequence comprising 494 bp, which includes two anaerobically regulated promoters, termed P6 and P7. In this study, we show that in its normal context the activity of P7 is constrained and that one important function of the promoter is to mediate controlled ArcA-dependent regulation of the operon.

Anaerobic induction of pyruvate formate-lyase gene expression is mediated by the ArcA and FNR proteins.

The pyruvate formate-lyase (pfl) gene of Escherichia coli is transcribed from seven promoters which are coordinately induced 12- to 15-fold by anaerobiosis. The FNR protein plays a major role in the anaerobic control of this system. A mutation in the fnr gene, however, only reduces anaerobic induction fivefold, indicating that FNR is not the only factor involved in the anaerobic activation process (Sawers and Böck, J. Bacteriol. 171:2485-2498, 1989). The residual anaerobic induction could be shown to be imparted by the transcriptional regulator ArcA; an arcA fnr double mutant was incapable of inducing pfl transcription anaerobically. A mutant strain unable to synthesize the membrane-associated histidine kinase (ArcB) that has been proposed to activate ArcA showed the same phenotype as an arcA mutant strain, indicating that a functional ArcB protein is also required for wild-type, anaerobic pfl transcriptional activation. Nuclease S1 analysis revealed that an arcA mutation abolished anaerobic transcription from promoter 7 and reduced expression from promoter 6 but did not affect transcription from promoters 1 to 5. On the other hand, an fnr mutation prevented anaerobic expression from promoters 6 and 7 and reduced transcription from promoters 1 to 5. These data indicate that both ArcA and FNR are essential for anaerobic activation of promoter 7 transcription, which suggests functional interaction between these proteins.

Anaerobic regulation of pyruvate formate-lyase from Escherichia coli K-12.

The anaerobic regulation of the gene encoding pyruvate formate-lyase from Escherichia coli was investigated. Expression of a pfl'-'lacZ protein fusion demonstrated that the gene is subject to a 12-fold anaerobic induction which can be stimulated a further 2-fold by the addition of pyruvate to the growth medium. Construction of a strain deleted for pfl verified that either pyruvate or a metabolite of glycolysis functions as an inducer of pfl gene expression. Complete anaerobic induction required the presence of a functional fnr gene product. However, the dependence was not absolute since a two- to threefold anaerobic induction could still be observed in an fnr mutant. These results could be confirmed immunologically by analyzing the levels of pyruvate formate-lyase protein present in cells grown under various conditions. It was also shown that pfl'-'lacZ expression was partially repressed by nitrate and that this repression was mediated by the narL gene product.

Alternative regulation principles for the production of recombinant proteins in Escherichia coli.

Established expression vectors exploiting regulated promoters such as the lac or tac promoters have economic and technical limitations when used for the industrial production of recombinant proteins. Consequently, alternative expression systems are being developed that can be more readily manipulated while maintaining high yields of protein. Several suitable expression vectors have been described for use in Escherichia coli that are based on promoters the activity of which is under metabolic control. This article discusses the advantages and disadvantages of a cross-section of these expression systems, how they compare with established systems and how they can be applied to the industrial-scale production of recombinant proteins.