- Volume 153, Issue 5, 2007

MbtH-like proteins are a family of small proteins encoded by genes found in many, but not all, non-ribosomal peptide synthetase-encoding gene clusters that direct the biosynthesis of peptide antibiotics and siderophores. Studies published to date have not elucidated the function of MbtH-like proteins, nor have they clarified whether they are required for metabolite biosynthesis. Here it is shown that only one of two genes (cdaX or cchK) encoding MbtH-like proteins in Streptomyces coelicolor is required for biosynthesis of the peptide siderophore coelichelin and the calcium-dependent peptide antibiotic (CDA). The cdaX and cchK genes can functionally complement each other in trans, suggesting that CdaX and CchK can cross-talk with the coelichelin and CDA biosynthetic pathways, respectively. Transcriptional analyses of wild-type S. coelicolor and a double cchK/cdaX replacement mutant indicate that CchK and CdaX may not be involved in transcriptional regulation of coelichelin and CDA biosynthetic gene clusters.

In the biosynthetic gene cluster of the aminocoumarin antibiotic clorobiocin, the small ORF cloY encodes a 71 aa protein which shows significant sequence similarity to mbtH from the mycobactin biosynthetic gene cluster of Mycobacterium tuberculosis. mbtH-like genes are frequently found in the biosynthetic gene clusters of peptide antibiotics and siderophores, but their function has remained enigmatic. In a recent publication it has been suggested that these genes may have no function for secondary metabolite biosynthesis. An in-frame deletion of cloY in the clorobiocin cluster has now been carried out. When the modified cluster was expressed in the heterologous host Streptomyces coelicolor M512, clorobiocin was still formed. However, when the two further mbtH-like genes from elsewhere in the host genome were inactivated as well, clorobiocin formation was reduced dramatically. Complementation with cloY or with any of three other mbtH-like genes restored clorobiocin formation. This is the first report proving the requirement of an mbtH-like gene for secondary metabolite formation, and the first proof that different mbtH-like genes can functionally replace each other. Feeding of an mbtH-defective triple mutant strain with an intact 3-amino-4,7-dihydroxy-coumarin moiety restored antibiotic production, showing that cloY is specifically required for the formation of this moiety of the clorobiocin molecule.

Several accessory proteins are required for the maturation of two nickel-containing enzymes in the gastric pathogen Helicobacter pylori. These two enzymes are hydrogenase and urease. Among the accessory/maturation proteins, the nickel-binding HypA protein has been previously shown to be required for the full activity of both the hydrogenase and the urease enzymes, while another nickel-binding protein, UreE, is known to be solely involved in the urease maturation process. In this study, UreE was shown to be required under all nickel levels for full activation of the apourease. By use of cross-linking studies, an interaction between purified HypA and UreE proteins was identified, leading to the formation of a 34 kDa heterodimer complex. The cross-linked adduct was detected by immunoblotting with either anti-HypA or anti-UreE antiserum. By using a two-plasmid system in Escherichia coli, the highest urease activity was achieved under low nickel conditions only when the UreE protein was expressed along with the wild-type HypA protein, but not with its nickel-binding-deficient variant HypA H2A. Addition of only 1 μM NiCl2 into minimal medium abolished the need for HypA to activate the urease. Although various attempts to show direct nickel transfer from HypA to UreE failed, these results suggest that interactions between the nickel-binding accessory proteins HypA and UreE are required to allow nickel transfer from HypA eventually to the apourease in H. pylori.

The Escherichia coli FNR protein is an oxygen-responsive global transcription factor, and OxyR is a key regulator of the peroxide stress response. Here both FNR and OxyR are shown to regulate expression of the E. coli yhjA gene. The yhjA gene encodes a predicted cytochrome c peroxidase, a bacterial haem-containing protein involved in the peroxide stress response through its ability to convert hydrogen peroxide to water. It is shown that the yhjA gene of E. coli possesses a class II FNR site and an OxyR site upstream of the yhjA transcript start. Expression of yhjA was found to be dependent on this unusual combination of FNR and OxyR under conditions of oxygen starvation. Phenotypic analysis of the yhjA mutant revealed increased sensitivity to exogenous hydrogen peroxide and organic peroxides during growth under anaerobic conditions, consistent with the observed regulation and predicted function of the yhjA gene product.

In response to iron starvation, Pseudomonas aeruginosa produces the siderophore pyochelin. When secreted to the extracellular environment, pyochelin chelates iron and transports it to the bacterial cytoplasm via its specific outer-membrane receptor FptA and the inner-membrane permease FptX. Exogenously added pyochelin also acts as a signal which induces the expression of the pyochelin biosynthesis and uptake genes by activating PchR, a cytoplasmic regulatory protein of the AraC/XylS family. The importance of ferripyochelin uptake genes in this regulation was evaluated. The fptA and fptX genes were shown to be part of the fptABCX ferripyochelin transport operon, which is conserved in Burkholderia sp. and Rhodospirillum rubrum. The fptB and fptC genes were found to be dispensable for utilization of pyochelin as an iron source, for signalling and for pyochelin production. By contrast, mutations in fptA and fptX not only interfered with pyochelin utilization, but also affected signalling and diminished siderophore production. It is concluded from this that pyochelin-mediated signalling operates to a large extent via the ferripyochelin transport system.

Centrifugation of cells of Schizosaccharomyces pombe in liquid medium prompted a marked activation of Sty1 and Pmk1, which are the effector mitogen-activated protein kinases (MAPKs) of the stress-activated protein kinase pathway and the cell-integrity pathway, respectively. Transduction of the centrifugation signals showed a sensitivity threshold above which the response was dependent on time and temperature. Centrifugation-induced phosphorylation of Sty1 and Pmk1 required the presence of the main functional components of the respective signalling cascades, i.e. Wak1 or Win1 plus Wis1, and Mkh1 plus Pek1. The transcription factor Atf1 also became phosphorylated in a Sty1-dependent way upon centrifugation. Hypergravity was an important factor in the activation of Sty1 induced by centrifugation, whilst activation of Pmk1 was mostly due to gravity-associated shear forces. Centrifugation did not increase cell survival against other stresses. Rather, the increased gravitational forces produced a delay in the cell cycle, probably related to alterations in the actin-polarization pattern. Phosphorylation of the MAPK Sty1 was needed for the depolarization of actin patches induced by the centrifugation stress.

The pathogenesis of Staphylococcus aureus infections is dependent upon expression of various virulence factors, which are under the control of multiple regulatory systems, including two-component regulatory systems and transcriptional regulators such as the SarA family of proteins. As a part of a continuing effort to understand the regulatory mechanisms that involve the SarA protein family, the regulation and physical characterization of rot transcription is described here. The rot gene, a member of the sarA family of genes, was previously characterized and has been shown to regulate a large number of genes. The rot locus is composed of multiple overlapping transcripts as determined by primer extension and was proposed to encode an open reading frame of 133 residues. Transcription of rot was significantly increased in the sarA mutant. Gel shift and transcriptional studies revealed that SarA could bind to the rot promoter region, probably acting as a repressor for rot transcription. The data indicate that the expression of rot transcription is significantly repressed only by SarA among the sarA family of mutants tested at the post-exponential phase of growth.

The genes nepAB of a small multidrug resistance (SMR) pump were identified as part of the pAO1-encoded nicotine regulon responsible for nicotine catabolism in Arthrobacter nicotinovorans. When [14C]nicotine was added to the growth medium the bacteria exported the 14C-labelled end product of nicotine catabolism, methylamine. In the presence of the proton-motive force inhibitors 2,4-dinitrophenol (DNP), carbonyl cyanide m-chlorophenylhydrazone (CCCP) or the proton ionophore nigericin, export of methylamine was inhibited and radioactivity accumulated inside the bacteria. Efflux of [14C]nicotine-derived radioactivity from bacteria was also inhibited in a pmfR : cmx strain with downregulated nepAB expression. Because of low amine oxidase levels in the pmfR : cmx strain, γ-N-methylaminobutyrate, the methylamine precursor, accumulated. Complementation of this strain with the nepAB genes, carried on a plasmid, restored the efflux of nicotine breakdown products. Both NepA and NepB were required for full export activity, indicating that they form a two-component efflux pump. NepAB may function as a metabolic valve by exporting methylamine, the end product of nicotine catabolism, and, in conditions under which it accumulates, the intermediate γ-N-methylaminobutyrate.

Pseudomonas aeruginosa is an opportunistic pathogen, which secretes a wide variety of enzymes and toxins into the extracellular medium. Most exoproteins are exported by the type II secretion machinery, the Xcp system, which encompasses 12 different proteins. One of the core components of the Xcp system is the inner-membrane protein XcpS (GspF), homologues of which can be identified in type II secretion machineries as well as in type IV piliation systems. In this study, XcpS was shown to be stabilized by co-expression of the XcpR (GspE) and XcpY (GspL) components of the machinery, demonstrating an interaction between these three proteins. By replacing segments of P. aeruginosa XcpS with the corresponding parts of its Pseudomonas putida counterpart, XcpS domains were identified that are important for species-specific functioning and thus represent putative interaction domains. The cytoplasmic loop of XcpS was found to be involved in the stabilization by XcpR and XcpY.

Streptococcus uberis is commonly found in the environment and in association with various bovine body sites and is a major cause of bovine mastitis. Moreover, S. uberis is known to produce a variety of bacteriocin-like inhibitory substances, antimicrobial agents that generally inhibit closely related bacterial species. In this respect, S. uberis strain 42 has previously been shown to produce a novel nisin variant named nisin U. This paper reports that, in addition to nisin U, S. uberis strain 42 produces a second bacteriocin that induces the lysis of metabolically active, susceptible target bacteria and which has therefore been named uberolysin. Isolation of the native active antimicrobial agent revealed that uberolysin is a 7048 Da peptide that is refractory to sequence analysis by Edman degradation. Transposon mutagenesis was used to generate a uberolysin-negative mutant of S. uberis 42 and sequencing of DNA flanking the insertion site revealed, in addition to the structural gene (ublA), several open reading frames likely to be involved in post-translational modification, transport and producer self-protection (immunity), and possibly in regulation of the biosynthetic gene cluster. In addition, a pair of direct repeats that may be involved in bacteriocin acquisition were identified; indeed, ublA could be identified in 18 % of tested S. uberis strains. Enzymic hydrolysis of uberolysin was used to confirm that ublA does indeed encode the precursor of uberolysin, that an unusually short leader sequence of only six amino acids is cleaved during processing of the mature peptide and that uberolysin is post-translationally covalently modified to form a head-to-tail monocycle. Thus, uberolysin is a unique cyclic bacteriocin, belonging to the same family of bacteriocins as enterocin AS-48 and circularin A.

Streptococcus mutans strain K8 was shown to produce a newly identified type AII lantibiotic, mutacin K8. The mutacin K8-encoding muk locus consists of 13 ORFs, three of which (mukA1, A2 and A3) have close homology to scnA, the structural gene encoding the Streptococcus pyogenes lantibiotic SA-FF22, and another (mukA′) resembles scnA′, an ORF in the SA-FF22 locus that has no currently assigned function. Inactivation of the muk locus indicated that mutacin K8 is responsible for most of the inhibitory activity produced by strain K8 in deferred antagonism tests on Columbia blood agar base supplemented with 5 % human blood and 0.1 % CaCO3. By contrast, on tryptic soy agar plus 2 % yeast extract and 0.5 % CaCO3 most of the inhibitory activity of strain K8 appeared to be attributable either to mutacin IV or to some other inhibitory peptide(s) exported by the mutacin IV transporter nlmT. An inhibitory peptide purified from a derivative of strain K8 in which nlmT had been inactivated had a mass of 2734 Da and an N-terminal sequence identical to the predicted propeptide translation products of mukA1 and mukA3. The muk locus may be widely distributed in S. mutans, since 9 (35 %) of 26 strains tested contained at least part of the locus. In the genome sequence of strain UA159 the muk locus is incomplete, the sole residual components being the ORFs encoding the putative two-component regulatory system mukR (SMU.1815) and mukK (SMU.1814), followed by two transposases (SMU.1813 and SMU.1812) and then the ORFs mukF (SMU.1811), mukE (SMU.1810) and mukG (SMU.1809), thought to encode putative immunity peptides. Strains such as UA159 having incomplete loci did not produce detectable levels of mutacin K8.

The phytoene-related desaturases are the key enzymes in the carotenoid biosynthetic pathway. The gene encoding phytoene desaturase in the deinoxanthin synthesis pathway of Deinococcus radiodurans was identified and characterized. Two putative phytoene desaturase homologues (DR0861 and DR0810) were identified by analysis of conserved amino acid regions, and the former displayed the highest identity (68 %) with phytoene desaturase of the cyanobacterium Gloeobacter violaceus. DR0861 gene knockout and dinucleotide-binding motif deletion resulted in the arrest of lycopene synthesis and the accumulation of phytoene. The colourless DR0861 knockout mutant became more sensitive to acute ionizing radiation and oxygen stress. Complementation of the mutant with a heterologous or homologous gene restored its pigment and resistance. The desaturase activity of DR0861 (crtI) was further confirmed by the assay of enzyme activity in vitro and heterologous expression in Escherichia coli containing crtE and crtB genes (responsible for phytoene synthesis) from Erwinia uredovora. In addition, the amount of lycopene synthesis in E. coli resulting from the expression of crtI from D. radiodurans was determined, and this had significant dose-dependent effects on the survival rate of E. coli exposed to hydrogen peroxide and ionizing radiation.

SeqA protein, a main negative regulator of the replication initiation of the Escherichia coli chromosome, also has several other functions which are still poorly understood. It was demonstrated previously that in seqA mutants the copy number of another replicon, the λ plasmid, is decreased, and that the activity of the λ p R promoter (whose function is required for stimulation of oriλ) is lower than that in the wild-type host. Here, SeqA-mediated regulation of λ phage and plasmid replicons was investigated in more detail. No significant influence of SeqA on oriλ-dependent DNA replication in vitro was observed, indicating that a direct regulation of λ DNA replication by this protein is unlikely. On the other hand, density-shift experiments, in which the fate of labelled λ DNA was monitored after phage infection of host cells, strongly suggested the early appearance of σ replication intermediates and preferential rolling-circle replication of phage DNA in seqA mutants. The directionality of λ plasmid replication in such mutants was, however, only slightly affected. The stability of the heritable λ replication complex was decreased in the seqA mutant relative to the wild-type host, but a stable fraction of the λ O protein was easily detectable, indicating that such a heritable complex can function in the mutant. To investigate the influence of seqA gene function on heritable complex- and transcription-dependent λ DNA replication, the efficiency of λ plasmid replication in amino acid-starved relA seqA mutants was measured. Under these conditions, seqA dysfunction resulted in impairment of λ plasmid replication. These results indicate that unlike oriC, SeqA modulates λ DNA replication indirectly, most probably by influencing the stability of the λ replication complex and the transcriptional activation of oriλ.

Screening of the genome sequence of the Mycobacterium ulcerans strain Agy99 from Ghana with tandem repeats finder software revealed 34 novel non-degenerate tandem repeats containing loci suitable for variable number tandem repeats (VNTR) typing. All loci revealed polymorphism within M. ulcerans isolates of geographically diverse origins. The results confirm the evolutionary scenario suggested by multi-locus sequence typing in which a progenitor of all M. ulcerans lineages emerged from the environmental species Mycobacterium marinum and subsequently diverged into several geographical lineages. For further attempts to develop a VNTR-based genetic fingerprinting tool for M. ulcerans, it is suggested that the focus should rather be on M. marinum than on the African M. ulcerans Agy99 genome sequence as a starting point.

Approximately 200 serogroups of Vibrio cholerae exist, with only two, O1 and O139, responsible for epidemic and pandemic cholera. Strains from these serogroups have evolved from a common progenitor, with lateral gene transfer largely driving their emergence. These strains are so closely related that separation using single- or multi-locus phylogeny has proven difficult. V. cholerae strains contain a genetic system called the integron that is located in the chromosome and that can integrate and excise DNA elements called mobile gene cassettes (MGCs) by site-specific recombination. Large arrays of MGCs are found in V. cholerae strains. For instance, the O1 El Tor strain N16961 contains 179 MGCs. Since integron arrays are dynamic through recombination and excision of MGCs, it was hypothesized that the MGC composition in a given V. cholerae pandemic strain would be useful as a phylogenetic typing system. To address this, a PCR-based method was used to rapidly characterize the MGC composition of V. cholerae arrays. The results showed that the MGC composition of pandemic V. cholerae cassette arrays is relatively conserved, providing further evidence that these strains have evolved from a common progenitor. Comparison of MGC composition between the V. cholerae pandemic strains was also able to resolve the evolution of O139 from a subgroup of O1 El Tor. This level of differentiation of closely related V. cholerae isolates was more sensitive than conventional single-gene phylogeny or multi-locus sequence analysis. Using this method, novel MGCs from an O1 classical strain and an Argentinian O139 isolate were also identified, and a major deletion in the MGC array in all pandemic O139 strains and a subset of O1 El Tor strains was identified. Analysis of sequenced V. cholerae integron arrays showed that their evolution can proceed by rearrangements and deletions/insertions of large portions of MGCs in addition to the insertion or excision of single MGCs.

Pseudoalteromonas sp. SM9913 is a psychrotolerant bacterium isolated from deep-sea sediment. The structural characterization and ecological roles of the exopolysaccharide (EPS) secreted by this strain were studied in this work. The yield of the EPS increased as the culture temperature decreased in the range 30–10 °C, and it reached 5.25 g l−1 (dry weight) under optimal growth conditions (15 °C, 52 h). EPS fraction was purified and its structure was identified by the combination of NMR spectra, high-resolution mass spectrometry (HRMS) analysis and methylation analysis. The ratio of the sugar units, the acetyl group and the ethoxyl group was close to 4 : 5 : 1. The major sugar unit of the EPS was 6-linked glucose (61.8 %); other sugar units present included terminal arabinofuranosyl (11.0 %) and glucopyranosyl (11.2 %) residues and a small amount of other sugar derivatives. Its structure was different from EPSs reported for other marine bacteria. Besides the structural elucidation of the EPS, its ecological roles were studied. This EPS could enhance the stability of the cold-adapted protease MCP-01 secreted by the same strain through preventing its autolysis. It could bind many metal ions, including Fe2+, Zn2+, Cu2+, Co2+. It was also a very good flocculating agent and could conglomerate colloidal and suspended particles. These results indicated that the EPS secreted by strain SM9913 might help this strain enrich the proteinaceous particles and the trace metals in the deep-sea environment, stabilize the secreted cold-adapted proteases and avoid its diffusion. This is believed to be the first report on the structure of the EPS secreted by a deep-sea psychrotolerant bacterium and its ecological roles. According to these results and other studies, a schematic diagram of the lifestyle of the deep-sea psychrotolerant strain SM9913 is suggested.