Notes: When complexed with Escherichia coli RNA polymerase core enzyme, purified RpoI protein of Rhizobium leguminosarum initiated transcription in vitro from promoters of the vbsADL and vbsGSO operons, which are needed to synthesise the siderophore vicibactin. There is a single transcription initiation site for rpoI, regardless of whether the cells are grown in Fe-replete or Fe-depleted media, but levels of rpoI mRNA were reduced, though not abolished, in the presence of Fe. Unlike PvdS, a similar Pseudomonasσ factor needed to transcribe genes involved in pyoverdine synthesis, RpoI transcribes vbsADL and vbsGSO in the absence of the cognate siderophore. The RpoI σ factor is not required for transcription of rpoI.

Notes: 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 β-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 β-ketothiolase and acetoacetyl-CoA reductase activities in this mutant. The cydR mutant accumulated poly-β-hydroxybutyrate throughout the exponential growth phase, unlike the wild-type strain that only accumulated poly-β-hydroxybutyrate during stationary phase. The results demonstrate that CydR controls poly-β-hydroxybutyrate synthesis in A. vinelandii.

Notes: Enzymes that contain a glycyl radical are functional only in the absence of oxygen. When synthesised, these proteins are catalytically inactive and must undergo activation through the process of hydrogen atom abstraction from a C-terminally located glycyl residue in the polypeptide chain. This process is catalysed by an iron–sulfur-containing activating enzyme that utilises S-adenosylmethionine as substrate, and a one-electron donor, usually dihydroflavodoxin. The 4Fe–4S cluster in the activating enzyme is directly involved in redox catalysis, generating a transient 5′-deoxyadenosyl radical intermediate that is proposed to be the hydrogen atom abstracting species. DNA sequence analysis has revealed that the genes encoding the glycyl radical enzyme and its cognate activating protein are either co-transcribed or are adjacent on the chromosome. Recent genomic analyses have revealed that glycyl radical enzymes may be widespread amongst obligate and facultative anaerobic microbes.

Notes: 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.

Notes: Anaerobic expression of the focA pfl operon is dependent on the transcription factors ArcA and FNR and transcription is directed by multiple, anaerobically regulated promoters. A FNR-binding site is centred at −41.5 bp relative to the P6 promoter, inactivation of which severely impairs anaerobic expression of the complete operon. Mutations were introduced into this binding site to create a consensus recognition site for the cAMP-receptor protein, CRP (CC-site), and one that was recognised by both CRP and FNR (CF-site). Transcription directed by these mutant binding sites in vivo in different promoter constructs was analysed by primer extension and by constructing lacZ operon fusions. With a derivative including only the P6 promoter and the CF-binding site, transcription was shown to be independent of oxygen and was activated by CRP or FNR. In agreement with previous findings, FNR only activated transcription anaerobically. In a construct including the CC-binding site transcription was strong, CRP dependent and initiated at the identical site to the wild-type promoter. Transcription activation from the CC-site was exquisitely sensitive to low cAMP concentration. Surprisingly, in a crp mutant, anaerobically inducible, FNR-dependent transcription directed by the CC-site was detected, indicating that FNR can recognise a consensus CRP-binding site in vivo. A strain unable to synthesise CRP or FNR exhibited no transcription from the P6 promoter. Essentially the same results were observed in a series of constructs that also included the promoter P7 and its regulatory sequences. Evidence is also presented which demonstrates that CRP activates transcription from the natural FNR-binding site of the P6 promoter. In vitro DNA-binding studies showed that CRP specifically interacted with the FNR-binding site, protecting exactly the same sequence as that protected by the FNR protein. Interaction of CRP with the natural FNR-binding site was reduced greater than 50-fold compared to its interaction with the mutant CC-binding site. Although we could not demonstrate that FNR interacted with the CC-binding site in vitro, it did bind to the CF-site giving the same protection as observed with the wild-type FNR-binding site. FNR also activated transcription from the CF-site in vitro, giving further support to the idea that a single functional DNA half-site is sufficient to direct binding and transcription activation by a dimeric transcription factor.

Notes: In the N2-fixing bacterium Rhizobium leguminosarum, mutations in a homologue of tonB (tonBRl) block the import of vicibactin and haem as iron sources in free-living bacteria. TonBRl mutants were normal for growth with ferric dicitrate and slightly reduced for growth with haemoglobin as sole iron sources. The deduced TonBRl product is larger than that of (for example) Escherichia coli, on account of an extended N-terminal domain. Transcription of tonBRl was enhanced in low-Fe growth conditions; this was not controlled by Fur, nor RpoI, an Fe-regulated extracytoplasmic σ factor. Upstream of tonBRl 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 σ factor was identified, but this gene, rpoZ, did not affect the transcription of tonBRl or hmuPSTUV. Mutations in tonBRl, hmu genes and rpoZ did not affect symbiotic N2 fixation in peas.

Notes: 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.

Notes: 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.

Notes: Pyruvate formate-lyase (PFL) catalyses the non-oxidative dissimilation of pyruvate to formate and acetyl-CoA using a radical–chemical mechanism. The enzyme is enzymically interconverted between inactive and active forms, the active form contains an organic free radical located on a glycyl residue in the C-terminal portion of the polypeptide chain. Introduction of the radical into PFL only occurs anaerobically, and the activating enzyme responsible is an iron–sulphur protein that uses S-adenosyl methionine as cofactor and reduced flavodoxin as reductant. As the radical form of PFL is inactivated by molecular oxygen it is safeguarded during the transition to aerobiosis by conversion back to the radical-free, oxygen-stable form. This reaction is catalysed by the anaerobically induced multimeric enzyme alcohol dehydrogenase. The genes encoding PFL and its activating enzyme are adjacent on the chromosome but form discrete transcriptional units. This genetic organization is highly conserved in many, but not all, organisms that have PFL. Recent studies have shown that proteins exhibiting significant similarity to PFL and its activating enzyme are relatively widespread in facultative and obligate anaerobic eubacteria, as well as archaea. The physiological function of many of these PFL-like enzymes remains to be established. It is becoming increasingly apparent that glycyl radical enzymes are more prevalent than previously surmised. They represent a class of enzymes with unusual biochemistry and probably predate the appearance of molecular oxygen.

Notes: An immunological analysis of an Escherichia coli strain unable to synthesize the main pyruvate formate-lyase enzyme Pfl revealed the existence of a weak, cross-reacting 85 kDa polypeptide that exhibited the characteristic oxygen-dependent fragmentation typical of a glycyl radical enzyme. Polypeptide fragmentation of this cross-reacting species was shown to be dependent on Pfl activase. Cloning and sequence analysis of the gene encoding this protein revealed that it coded for a new enzyme, termed TdcE, which has 82% identity with Pfl. On the basis of RNA analyses, the tdcE gene was shown to be part of a large operon that included the tdcABC genes, encoding an anaerobic threonine dehydratase, tdcD, coding for a propionate kinase, tdcF, the function of which is unknown, and the tdcG gene, which encodes a L-serine dehydratase. Expression of the tdcABCDEFG operon was strongly catabolite repressed. Enzyme studies showed that TdcE has both pyruvate formate-lyase and 2-ketobutyrate formate-lyase activity, whereas the TdcD protein is a new propionate/acetate kinase. By monitoring culture supernatants from various mutants using 1H nuclear magnetic resonance (NMR), we followed the anaerobic conversion of L-threonine to propionate. These studies confirmed that 2-ketobutyrate, the product of threonine deamination, is converted in vivo by TdcE to propionyl-CoA. These studies also revealed that Pfl and an as yet unidentified thiamine pyrophosphate-dependent enzyme(s) can perform this reaction. Double null mutants deficient in phosphotransacetylase (Pta) and acetate kinase (AckA) or AckA and TdcD were unable to metabolize threonine to propionate, indicating that propionyl-CoA and propionyl-phosphate are intermediates in the pathway and that ATP is generated during the conversion of propionyl-P to propionate by AckA or TdcD.