- Volume 152, Issue 9, 2006

Rho-dependent transcription terminators participate in sophisticated genetic regulatory mechanisms, in both bacteria and phages; they occur in regulatory regions preceding the coding sequences of genes and within coding sequences, as well as at the end of transcriptional units, to prevent readthrough transcription. Most Rho-dependent terminators have been found in enteric bacteria, but they also occur in Gram-positive bacteria and may be widespread among bacteria. Rho-dependent termination requires both cis-acting elements, on the mRNA, and trans-acting factors. The only cis-acting element common to Rho-dependent terminators is richness in rC residues. Additional sequence elements have been observed at different Rho termination sites. These ‘auxiliary elements' may assist in the termination process; they differ among terminators, their occurrence possibly depending on the function and sequence context of the terminator. Specific nucleotides required for termination have also been identified at Rho sites. Rho is the main factor required for termination; it is a ring-shaped hexameric protein with ATPase and helicase activities. NusG, NusA and NusB are additional factors participating in the termination process. Rho-dependent termination occurs by binding of Rho to ribosome-free mRNA, C-rich sites being good candidates for binding. Rho's ATPase is activated by Rho–mRNA binding, and provides the energy for Rho translocation along the mRNA; translocation requires sliding of the message into the central hole of the hexamer. When a polymerase pause site is encountered, the actual termination occurs, and the transcript is released by Rho's helicase activity. Many aspects of this process are still being studied. The isolation of mutants suppressing termination, site-directed mutagenesis of cis-acting elements in Rho-dependent termination, and biochemistry, are and will be contributing to unravelling the still undefined aspects of the Rho termination machinery. Analysis of the more sophisticated regulatory mechanisms relying on Rho-dependent termination may be crucial in identifying new essential elements for termination.

Conditional mutants of pbpB with an IPTG-inducible promoter were used to compare the effects of interrupted transcription of this gene in a meticillin-sensitive (MSSA) and a meticillin-resistant (MRSA) strain of Staphylococcus aureus. After 3 h growth following the removal of IPTG, multiplication of the MSSA strain stopped abruptly, cells began to lyse, and membrane preparations showed greatly decreased quantities of penicillin-binding protein (PBP) 2. In contrast, the MRSA strain continued to grow for at least 20 h in the IPTG-free medium, but with gradually increasing doubling times, which eventually reached 180 min. The peptidoglycan produced during this period of extremely slow growth showed only minor alterations, but cells with abnormal morphology accumulated in the culture, the abundance of mecA transcript gradually declined, and the cellular amounts of PBP2A were significantly decreased. Adding back the IPTG inducer caused rapid resumption in the transcription of pbpB, followed by an increase in the transcription of mecA. No changes were detected in the transcription of pbpA, C and D, the determinant of 16S rRNA or the housekeeping gene pta. Promoter fusion experiments suggested that the transcription of the resistance gene mecA may respond to some regulatory signal generated in the bacteria during changes in the transcription of pbpB.

Chlamydia trachomatis L2 was used to infect BGMK cells at an m.o.i. of 1.0, and the developmental cycle was followed by transmission electron microscopy and quantitative PCR (QPCR) for both chromosomal and plasmid DNA. Samples were taken at sequential 6 h time points. Subsequent analysis by QPCR showed that there was an initial slow replication period (0–18 h), followed by a rapid phase (18–36 h) coinciding with exponential division when the DNA doubling time was 4.6 h. Chromosomal DNA was amplified 100–200-fold corresponding to 7–8 generations for the complete developmental cycle. Penicillin (10 and 100 units ml−1) was added to cultures at 20 h post-infection (p.i.). This blocked binary fission and also prevented reticulate body (RB) to elementary body transition. However, exposure to penicillin did not prevent chromosomal or plasmid DNA replication. After a short lag period, following the addition of penicillin, chlamydial chromosomal DNA replication resumed at the same rate as in control C. trachomatis-infected cells. C. trachomatis-infected host cells exposed to penicillin did not lyse, but instead harboured large, aberrant RBs in massive inclusions that completely filled the cell cytoplasm. In these RBs, the DNA continued to replicate well beyond the end of the normal developmental cycle. At 60 h p.i. each aberrant RB contained a minimum of 16 chromosomal copies.

Cell integrity in yeasts is ensured by a rigid cell wall whose synthesis is triggered by a MAP kinase-mediated signal-transduction cascade. Upstream regulatory components of this pathway in Saccharomyces cerevisiae involve a single protein kinase C, which is regulated by interaction with the small GTPase Rho1. Here, two genes were isolated which encode these proteins from Kluyveromyces lactis (KlPKC1 and KlRHO1). Sequencing showed ORFs which encode proteins of 1161 and 208 amino acids, respectively. The deduced proteins shared 59 and 85 % overall amino acid identities, respectively, with their homologues from S. cerevisiae. Null mutants in both genes were non-viable, as shown by tetrad analyses of the heterozygous diploid strains. Overexpression of the KlRHO1 gene under the control of the ScGAL1 promoter severely impaired growth in both S. cerevisiae and K. lactis. On the other hand, a similar construct with KlPKC1 did not show a pronounced phenotype. Two-hybrid analyses showed interaction between Rho1 and Pkc1 for the K. lactis proteins and their S. cerevisiae homologues. A green fluorescent protein (GFP) fusion to the C-terminal end of KlPkc1 located the protein to patches in the growing bud, and at certain stages of the division process also to the bud neck. N-terminal GFP fusions to KlRho1 localized mainly to the cell surface (presumably the cytoplasmic side of the plasma membrane) and to the vacuole, with some indications of traffic from the former to the latter. Thus, KlPkc1 and KlRho1 have been shown to serve vital functions in K. lactis, to interact in cell integrity signalling and to traffic between the plasma membrane and the vacuole.

The larvicidal activity of Bacillus thuringiensis subsp. israelensis against dipteran larvae is determined by four major polypeptides of the parasporal crystalline body produced during sporulation. Cyt1Aa shows the lowest toxicity when used alone but is the most synergistic with any of the other proteins. The sequence of the plasmid pBtoxis, which contains all the toxin genes in this subspecies, revealed a new cyt-like coding sequence named cyt1Ca. In addition to the Cyt-like region, the predicted Cyt1Ca contained an extra domain at the C terminus, which appeared to be a β-trefoil carbohydrate-binding motif, as found in several ricin-like toxins. The gene was PCR-amplified from pBtoxis and cloned in several vectors, allowing high-level expression in Escherichia coli. Cyt1Ca was purified by nickel-nitrilotriacetic acid affinity chromatography, characterized, and its biological activity was determined. Toxicity against larvae of Aedes aegypti of Cyt1Ca in recombinant E. coli cells was compared with that of Cyt1Aa and Cyt2Ba, and the ability of these proteins to enhance the activity of Cry4Aa was assessed. Although Cyt2Ba appeared able to interact with Cry4Aa, no activity for Cyt1Ca was observed, even when produced in truncated form. Furthermore, in contrast to Cyt1Aa, Cyt1Ca did not lyse sheep erythrocytes, and it was not bactericidal to the host cell.

Ralstonia pickettii, an environmental bacterium that may also be responsible for human infections, produces two unrelated, inducible and chromosomally encoded oxacillinases, OXA-22 and OXA-60. In order to study the molecular basis of the induction process of these oxacillinase genes, the induction kinetics, the promoter/operator regions necessary for expression and induction, and the role of several ORFs located upstream and downstream of the bla OXA genes were investigated. The β-lactamase production reached a maximal level after 1 h induction, returned to its basal level within the following 3 h and was then again inducible. Using 5′RACE experiments, the promoter sequences of both oxacillinases were determined. These sequences showed weak promoter activities, which could, however, be increased approximately 200-fold by mutating the −35 promoter sequence. Deletion of the sequences located upstream of the promoter regions did not modify the basal β-lactamase expression in R. pickettii, but resulted in the lack of induction. A minimum of 240 and 270 bp upstream of the transcription initiation sites was required for inducible expression of the bla OXA-22 and bla OXA-60 genes, respectively. Analysis of the genetic environment of both bla OXA genes revealed several ORFs that were inactivated by homologous recombination. Disruption of ORF-RP3, located 190 bp upstream of bla OXA-60 and divergently transcribed, abolished induction of both β-lactamases. ORF-RP3, which encoded a polypeptide of 532 aa with an estimated molecular mass of 58.7 kDa, displayed no obvious sequence homology with known regulatory proteins. Trans-complementation of ORF-RP3 restored the basal and inducible expression of both oxacillinase genes, indicating that the induction of both enzymes was related to the presence of ORF-RP3. In addition to the loss of induction, inactivation of the ORF-RP3 in R. pickettii resulted in a complex pleiotropic phenotype, with increased lag phase and reduced survival after heat exposure, suggesting that ORF-RP3 might be a global regulator involved in unrelated regulatory pathways.

Mycobacterium tuberculosis under stress stores triacylglycerol (TG). There are 15 genes in M. tuberculosis that belong to a novel family of TG synthase genes (tgs), but it is not known which of them is responsible for this accumulation of TG. In this paper, it is reported that M. tuberculosis H37Rv accumulated TG under acidic, static or hypoxic growth conditions, or upon treatment with NO, whereas TG accumulation was drastically reduced in the tgs1 (Rv3130c) disrupted mutant. Complementation with tgs1 restored this TG accumulation. C26 was a major fatty acid in this TG, indicating that the TGS1 gene product uses C26 fatty acid, which is known to be produced by the mycobacterial fatty acid synthase. TGS1 expressed in Escherichia coli preferred C26 : 0-CoA for TG synthesis. If TG storage is needed for the long-term survival of M. tuberculosis under dormant conditions, the tgs1 product could be a suitable target for antilatency drugs.

Adenylyltransferase, GlnE, has a predicted role in controlling the enzymic activity of glutamine synthetase, the key enzyme in ammonia assimilation. It was previously demonstrated that glnE is an essential gene in Mycobacterium tuberculosis. glnE is located downstream of glnA2, one of four glutamine synthetases. The expression of GlnE under various conditions was determined. Although a co-transcript of glnA2 and glnE was detectable, the major transcript was monocistronic. A transcriptional start site immediately upstream of glnE was identified and it was shown by site-directed mutagenesis that the predicted −10 region is a functional promoter. It was demonstrated that in a Mycobacterium smegmatis background M. tuberculosis PglnE was up-regulated in ammonia- or glutamine-containing media.

Aeromonas salmonicida subsp. salmonicida contains a functional type III secretion system that is responsible for the secretion of the ADP-ribosylating toxin AexT. In this study, the authors identified AopP as a second effector protein secreted by this system. The aopP gene was detected in both typical and atypical A. salmonicida isolates and was found to be encoded on a small plasmid of approximately 6.4 kb. Sequence analysis indicates that AopP is a member of the YopJ family of effector proteins, a group of proteins that interfere with mitogen-activated protein kinase (MAPK) and/or nuclear factor kappa B (NF-κB) signalling pathways. AopP inhibits the NF-κB pathway downstream of IκB kinase (IKK) activation, while a catalytically inactivated mutant, AopPC177A, does not possess this inhibitory effect. Unlike other effectors of the YopJ family, such as YopJ and VopA, AopP does not inhibit the MAPK signalling pathway.

Copper amine oxidases (CAOs) are found in almost every living kingdom. Although Saccharomyces cerevisiae is one of the few yeast species that lacks an endogenous CAO, heterologous gene expression of CAOs from other organisms produces a functional enzyme. To begin to characterize their function and mechanisms of copper acquisition, two putative cao + genes from Schizosaccharomyces pombe were expressed in S. cerevisiae. Expression of spao1 + resulted in the production of an active enzyme capable of catalysing the oxidative deamination of primary amines. On the other hand, expression of spao2 + failed to produce an active CAO. Using a functional spao1 +–GFP fusion allele, the SPAO1 protein was localized in the cytosol. Under copper-limiting conditions, yeast cells harbouring deletions of the MAC1, CTR1 and CTR3 genes were defective in amine oxidase activity. Likewise, atx1Δ null cells exhibited no CAO activity, while ccc2Δ mutant cells exhibited decreased levels of amine oxidase activity, and mutations in cox17Δ and ccs1Δ did not cause any defects in this activity. Copper-deprived S. cerevisiae cells expressing spao1 + required a functional atx1 + gene for growth on minimal medium containing ethylamine as the sole nitrogen source. Under these conditions, the inability of the atx1Δ cells to utilize ethylamine correlated with the lack of SPAO1 activity, in spite of the efficient expression of the protein. Cells carrying a disrupted ccc2Δ allele exhibited only weak growth on ethylamine medium containing a copper chelator. The results of these studies reveal that expression of the heterologous spao1 + gene in S. cerevisiae is required for its growth in medium containing ethylamine as the sole nitrogen source, and that expression of an active Schiz. pombe SPAO1 protein in S. cerevisiae depends on the acquisition of copper through the high-affinity copper transporters Ctr1 and Ctr3, and the copper chaperone Atx1.

The previous discovery of the Streptomyces lividans kcsA gene and its overexpression followed by the functional reconstitution of the purified gene product has resulted in new strategies to explore this channel protein in vitro. KcsA has evolved as a general model to investigate the structure/function relationship of ion channel proteins. Using specific antibodies raised against a domain of KcsA lacking membrane-spanning regions, KcsA has now been localized within numerous separated clusters between the outer face of the cytoplasm and the cell envelope in substrate hyphae of the S. lividans wild-type strain but not in a designed chromosomal disruption mutant ΔK, lacking a functional kcsA gene. Previous findings had revealed that caesium ions led to a block of KcsA channel activity within S. lividans protoplasts fused to giant vesicles. As caesium can be scored by electron energy loss spectroscopy better than potassium, this technique was applied to hyphae that had been briefly exposed to caesium instead of potassium ions. Caesium was found preferentially at the cell envelope. Compared to the ΔK mutant, the relative level of caesium was ≈30 % enhanced in the wild-type. This is attributed to the presence of KcsA channels. Additional visualization by electron spectroscopic imaging supported this conclusion. The data presented are believed to represent the first demonstration of in vivo monitoring of KcsA in its original host.

Mycobacterium bovis is the causative agent of bovine tuberculosis. Various genetic typing techniques have been used to trace the reservoirs of infection; however, they have limited success in population genetics and outbreak studies. Fourier-transform infrared spectroscopy (FT-IR) is a rapid phenotypic typing technique, which may be used to generate a metabolic fingerprinting and is increasingly used to characterize bacteria. When coupled with multivariate cluster analysis, this powerful combination has sufficient resolving power to discriminate bacteria down to subspecies level; however, to date this method has not been used in the differentiation of mycobacteria. Multiple isolates of the ten major spoligotypes in the UK, recovered from different geographical locations, were analysed using FT-IR. Hierarchical cluster analysis of the spectra showed that the isolates could be differentiated according to their spoligotypes. Six of the spoligotype FT-IR clusters were very homogeneous and all isolates were recovered together. However, the remaining four groups displayed a more heterogeneous phenotype, which may reflect greater variation than previously suspected within these groups. Included in the ten spoligotypes are the two most dominant isolates in the UK, designated types 9 and 17. Whilst type 17 showed a highly conserved phenotype as judged by FT-IR, type 9 showed a very heterogeneous metabolic profile and isolates were recovered throughout the dendrogram. This variation in type 9 is reflected in the high degree of diversity observed by variable number tandem repeats (VNTR) analysis, underlining the exquisite resolving power of FT-IR.

The extracellular matrix produced by Bacillus subtilis B-1, an environmental strain that forms robust floating biofilms, was purified, and determined to be composed predominantly of γ-polyglutamate (γ-PGA), with a molecular mass of over 1000 kDa. Both biofilm formation and γ-PGA production by B. subtilis B-1 increased with increasing Mn2+ or glycerol concentration. γ-PGA was produced in a growth-associated manner in standing culture, and floating biofilms were formed. However, γ-PGA was produced in a non-growth-associated manner in shaking culture conditions. When B. subtilis B-1 was grown in a microaerated culture system, floating biofilm formation and γ-PGA production were significantly retarded, suggesting that oxygen depletion is involved in the initial steps of floating biofilm formation in standing culture. Proteomic analysis of membrane proteins demonstrated that flagellin, oligopeptide permease and Vpr protease precursor were the major proteins produced by cells in a floating biofilm and a colony.

Nine mercury-resistance plasmids isolated from river epilithon were assessed for their ability to retrotransfer the non-conjugative IncQ plasmid, R300B, derivatives of which have commercial uses that may result in accidental or deliberate release into the environment. Retrotransfer frequencies ranging from 2.1×10−4 to 1.75×10−5 were obtained for five of the nine plasmids – the remaining plasmids showed low or undetectable retrotransfer ability. The majority of the retrotransfer-proficient plasmids could not be classified by the tests used. Classical incompatibility testing with RP4 identified pQKH6, pQKH54 and pQM719 as IncP-1. Hybridization to replicon probes confirmed this for pQKH6 and pQM719 and added pQKH33. PCR with primers designed to amplify trfA and korA regions of IncP-1 plasmids did not identify any other plasmids. Plasmids pQKH6 and pQM719 but not pQKH54 produced similar SphI restriction profiles to the IncP-1β subgroup. The complete nucleotide sequence of pQKH54 was determined, revealing it to have a complete IncP-1 backbone but belonging to a new distinct subgroup which was designated IncP-1γ. The results emphasize the ubiquity and diversity of IncP-1 plasmids in the environment but demonstrate that plasmids of as yet unknown groups are also able to retrotransfer IncQ plasmids efficiently.

In the microbial competition observed in enhanced biological phosphorus removal (EBPR) systems, an undesirable group of micro-organisms known as glycogen-accumulating organisms (GAOs) compete for carbon in the anaerobic period with the desired polyphosphate-accumulating organisms (PAOs). Some studies have suggested that a propionate carbon source provides PAOs with a competitive advantage over GAOs in EBPR systems; however, the metabolism of GAOs with this carbon source has not been previously investigated. In this study, GAOs were enriched in a laboratory-scale bioreactor with propionate as the sole carbon source, in an effort to better understand their biochemical processes. Based on comprehensive solid-, liquid- and gas-phase chemical analytical data from the bioreactor, a metabolic model was proposed for the metabolism of propionate by GAOs. The model adequately described the anaerobic stoichiometry observed through chemical analysis, and can be a valuable tool for further investigation of the competition between PAOs and GAOs, and for the optimization of the EBPR process. A group of Alphaproteobacteria dominated the biomass (96 % of Bacteria) from this bioreactor, while post-fluorescence in situ hybridization (FISH) chemical staining confirmed that these Alphaproteobacteria produced poly-β-hydroxyalkanoates (PHAs) anaerobically and utilized them aerobically, demonstrating that they were putative GAOs. Some of the Alphaproteobacteria were related to Defluvicoccus vanus (16 % of Bacteria), but the specific identity of many could not be determined by FISH. Further investigation into the identity of other GAOs is necessary.