Molecular Microbiology

Gotfredsen, Marie; Gerdes, Kenn

doi: 10.1046/j.1365-2958.1998.00993.xpmid: 9767574

Toxin–antitoxin systems are defined as a group of plasmid‐ and chromosome‐encoded loci that specify a cell toxin and a protein antitoxin. Plasmid‐encoded toxin–antitoxin systems stabilize their replicons by killing plasmid‐free cells. Here, we show that the relBE genes of Escherichia coli K‐12 have all the basic features previously connected with toxin–antitoxin systems: (i) relE encodes a cytotoxin lethal or inhibitory to host cells; (ii) relB encodes an antitoxin that prevents the lethal action of the relE‐encoded toxin; (iii) the relBE genes stabilize a mini‐R1 test plasmid; and (iv) the RelB antitoxin autoregulates the relBEF operon at the level of transcription. Using database searching, we found relBE homologues on the chromosomes of E. coli K‐12, Haemophilus influenzae and Vibrio choleraeA fifth relBE homologue was identified on the enterotoxin encoding E. coli plasmid P307. Indirect evidence suggests that the toxicity of RelE may be related to the inhibition of protein synthesis. Based on these observations, we propose a model that explains the delayed relaxed phenotype associated with mutations in relB.

Sawers, Gary; Watson, Gregory

doi: 10.1046/j.1365-2958.1998.00941.xpmid: 9767563

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.

Dougherty, Brian A.; Hill, Colin; Weidman, Janice F.; Richardson, Delwood R.; Venter, J. Craig; Ross, R. Paul

doi: 10.1046/j.1365-2958.1998.00988.xpmid: 9767571

The complete sequence of pMRC01, a large conjugative plasmid from Lactococcus lactis ssp. lactis DPC3147, has been determined. Using a shotgun sequencing approach, the 60 232 bp plasmid sequence was obtained by the assembly of 1056 underlying sequences (sevenfold average redundancy). Sixty‐four open reading frames (ORFs) were identified. Analysis of the gene organization of pMRC01 suggests that the plasmid can be divided into three functional domains, with each approximately 20 kb region separated by insertion sequence (IS) elements. The three regions are (i) the conjugative transfer region, including a 16‐gene Tra (transfer) operon; (ii) the bacteriocin production region, including an operon responsible for the synthesis of the novel bacteriocin lacticin 3147; and (iii) the phage resistance and plasmid replication region of the plasmid. The complete sequence of pMRC01 provides important information about these industrially relevant phenotypes and gives insight into the structure, function and evolution of large Gram‐positive conjugative plasmids in general. The completely sequenced pMRC01 plasmid should also provide a useful framework for the design of novel plasmids to be incorporated into starter strain improvement programmes for the dairy industry.

Sacerdot, Christine; Caillet, Joel; Graffe, Monique; Eyermann, Flore; Ehresmann, Bernard; Ehresmann, Chantal; Springer, Mathias; Romby, Pascale

doi: 10.1046/j.1365-2958.1998.00995.xpmid: 9767575

The expression of the gene encoding Escherichia coli threonyl‐tRNA synthetase (ThrRS) is negatively autoregulated at the translational level. ThrRS binds to its own mRNA leader, which consists of four structural and functional domains: the Shine–Dalgarno (SD) sequence and the initiation codon region (domain 1); two upstream hairpins (domains 2 and 4) connected by a single‐stranded region (domain 3). Using a combination of in vivo and in vitro approaches, we show here that the ribosome binds to thrS mRNA at two non‐contiguous sites: region −12 to +16 comprising the SD sequence and the AUG codon and, unexpectedly, an upstream single‐stranded sequence in domain 3. These two regions are brought into close proximity by a 38‐nucleotide‐long hairpin structure (domain 2). This domain, although adjacent to the 5′ edge of the SD sequence, does not inhibit ribosome binding as long as the single‐stranded region of domain 3 is present. A stretch of unpaired nucleotides in domain 3, but not a specific sequence, is required for efficient translation. As the repressor and the ribosome bind to interspersed domains, the competition between ThrRS and ribosome for thrS mRNA binding can be explained by steric hindrance.

Jennings, Michael P.; Virji, Mumtaz; Evans, Debbie; Foster, Virginia; Srikhanta, Yogitha N.; Steeghs, Liana; Van Der Ley, Peter; Moxon, E. Richard

doi: 10.1046/j.1365-2958.1998.00962.xpmid: 9767566

The pili of Neisseria meningitidis are a key virulence factor, being major adhesins of this capsulate organism that contribute to specificity for the human host. Recently it has been reported that meningococcal pili are post‐translationally modified by the addition of an O‐linked trisaccharide, Gal (β1–4) Gal (α1–3) 2,4‐diacetimido‐2,4,6‐trideoxyhexose. Using a set of random genomic sequences from N. meningitidis strain MC58, we have identified a novel gene homologous to a family of glycosyltransferases. A plasmid clone containing the gene was isolated from a genomic library of N. meningitidis strain MC58 and its nucleotide sequence determined. The clone contained a complete copy of the gene, here designated pglA (pilin glycosylation). Insertional mutations were constructed in pglA in a range of meningococcal strains with well‐defined lipopolysaccharide (LPS) or pilin‐linked glycan structures to determine whether pglA had a role in the biosynthesis of these molecules. There was no alteration in the phenotype of LPS from pglA mutant strains as judged by gel migration and the binding of monoclonal antibodies. In contrast, decreased gel migration of the pilin subunit molecules of pglA mutants was observed, which was similar to the migration of pilins of galE mutants of same strains, supporting the notion that pglA is a glycosyltransferase involved in the biosynthesis of the pilin‐linked trisaccharide structure. The pglA mutation, like the galE mutation reported previously, had no effect on pilus‐mediated adhesion to human epithelial or endothelial cells. Pilin from pglA mutants were unable to bind to monospecific antisera recognizing the Gal (β1–4) Gal structure, suggesting that PglA is a glycosyltransferase involved in the addition of galactose of the trisaccharide substituent of pilin.

Kao, R.; Shea, J. E.; Davies, J.; Holden, D. W.

doi: 10.1046/j.1365-2958.1998.00987.xpmid: 9767570

Fungal ribotoxins, such as mitogillin and the related Aspergillus toxins restrictocin and α‐sarcin, are highly specific ribonucleases, which inactivate the ribosome enzymatically by cleaving the eukaryotic 28S RNA of the large ribosomal subunit at a single phosphodiester bond. The site of cleavage occurs between G4325 and A4326, which are present in a 14‐base sequence (the α‐sarcin loop) conserved among the large subunit rRNAs of all living species. The amino acid residues involved in the cytotoxic activities of mitogillin were investigated by introducing point mutations using hydroxylamine into a recombinant Met‐mature mitogillin (mitogillin with a Met codon at the N‐terminus and no leader sequence) gene constructed from an Aspergillus fumigatus cDNA clone. These constructs were cloned into a yeast expression vector under the control of the GAL1 promoter and transformed into Saccharomyces cerevisiae. Upon induction of mitogillin expression, surviving transformants revealed that substitutions of certain amino acid residues on mitogillin abolished its cytotoxicity. Non‐toxic mutant genes were cloned into an Escherichia coli expression vector, the proteins overexpressed and purified to homogeneity and their activities examined by in vitro ribonucleolytic assays. These studies identified the His‐49Tyr, Glu‐95Lys, Arg‐120Lys and His‐136Tyr mutations to have a profound impact on the ribonucleolytic activities of mitogillin. We conclude that these residues are key components of the active site contributing to the catalytic activities of mitogillin.

Bernander, Rolf

doi: 10.1046/j.1365-2958.1998.00956.xpmid: 9767564

Sequence similarity data suggest that archaeal chromosome replication is eukaryotic in character. Putative nucleoid‐processing proteins display similarities to both eukaryotic and bacterial counterparts, whereas cell division may occur through a predominantly bacterial mechanism. Insights into the organization of the archaeal cell cycle are therefore of interest, not only for understanding archaeal biology, but also for investigating how components from the other two domains interact and work in concert within the same cell; in addition, archaea may have the potential to provide insights into eukaryotic initiation of chromosome replication.

Kallipolitis, Birgitte H.; Valentin‐Hansen, Poul

doi: 10.1046/j.1365-2958.1998.00999.xpmid: 9767576

In Escherichia coli, the rpoH gene encoding the essential heat‐shock regulator σ32, is expressed in a complex manner. Transcription occurs from four promoters (P1, P3, P4 and P5) and is modulated by several factors including (i) two σ factors (σ70 and σE); (ii) the global regulator CRP; and (iii) the DnaA protein. Here, a further dissection of the rpoH regulatory region has revealed that an additional transcription control exists that appears to link rpoH expression to nucleoside metabolism. The cAMP–CRP complex and the CytR anti‐activator bind co‐operatively to the promoter region forming a repression complex that overlaps the σE‐dependent P3 promoter and the σ70‐dependent P4 and P5 promoters. During steady‐state growth conditions with glycerol as the carbon and energy source, transcription from P3, P4 and P5 is reduced ≈threefold by CytR, whereas transcription from the upstream promoter, P1, appears to be unaffected. Furthermore, in strains that slightly overproduce CytR, transcription from P3, P4 and P5 is reduced even further (≈10‐fold), and repression can be fully neutralized by the addition of the inducer cytidine to the growth medium. In the induced state, P4 is the strongest promoter and, together with P3 and P5, it is responsible for most rpoH transcription (65–70%). At present, CytR has been shown to ‘fine tune’ transcription of two genes (rpoH and ppiA) that are connected with protein‐folding activities. These findings suggest that additional assistance in protein folding is required under conditions in which CytR is induced (i.e. in the presence of nucleosides).

Plumbridge, Jacqueline

doi: 10.1046/j.1365-2958.1998.00991.xpmid: 9767573

The gene for the glucose‐specific transporter of the phosphotransferase system, ptsG, is expressed from two promoters separated by 141 bp. The expression of the major, shorter transcript is very strongly dependent upon cAMP/CAP. However, unlike other CAP‐activated genes, the expression of ptsG is higher in glucose media than in glycerol, implying that ptsG is controlled by a glucose‐inducible regulator. A mutation in the mlc gene greatly enhances ptsG expression in a glycerol‐grown culture but has no effect on ptsG expression during growth on glucose. The mlc gene encodes a transcriptional regulator that has been shown to affect the expression of manXYZ and malT. ptsG mRNA levels are lower in the mlc strain grown on glucose than in the same strain grown on glycerol. This is presumably because of the greater catabolite repression in the glucose culture than in glycerol. The final level of expression of ptsG in a mlc+ strain in glucose is a compromise between specific induction by glucose and generalized catabolite repression. The result is that ptsG expression is very similar in glucose‐grown cultures of wild‐type and mlc strains. The Mlc protein binds to two sites centred at −6 and −175 upstream of the major ptsG transcript. CAP binds at −40.5 compared with this site, typical of class II CAP‐regulated promoters, and the binding of CAP and Mlc is co‐operative.

Wortinger, Mark A.; Quardokus, Ellen M.; Brun, Yves V.

doi: 10.1046/j.1365-2958.1998.00959.xpmid: 9767565

During exponential growth, each cell cycle of the α‐purple bacterium Caulobacter crescentus gives rise to two different cell types: a motile swarmer cell and a sessile stalked cell. When cultures of C. crescentus are grown for extended periods in complex (PYE) medium, cells undergo dramatic morphological changes and display increased resistance to stress. After cultures enter stationary phase, most cells are arrested at the predivisional stage. For the first 6–8 days after inoculation, the colony‐forming units (cfu) steadily decrease from 109 cfu ml−1 to a minimum of 3 × 107 cfu ml−1 after which cells gradually adopt an elongated helical morphology. For days 9–12, the cfu of the culture increase and stabilize around 2 × 108 cfu ml−1. The viable cells have an elongated helical morphology with no constrictions and an average length of 20 μm, which is 15–20 times longer than exponentially growing cells. The level of the cell division initiation protein FtsZ decreases during the first week in stationary phase and remains at a low constant level consistent with the lack of cell division. When resuspended in fresh medium, the elongated cells return to normal size and morphology within 12 h. Cells that have returned from stationary phase proceed through the same developmental changes when they are again grown for an extended period and have not acquired a heritable growth advantage in stationary phase (GASP) compared with overnight cultures. We conclude that the changes observed in prolonged cultures are the result of entry into a new developmental pathway and are not due to mutation.