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Volume 162,
Issue 3,
2016
Volume 162, Issue 3, 2016
- Review
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Transcription termination factor Rho: a hub linking diverse physiological processes in bacteria
More LessFactor-dependent termination of transcription in bacteria relies on the activity of a specific RNA helicase, the termination factor Rho. Rho is nearly ubiquitous in bacteria, but the extent to which its physiological functions are conserved throughout the different phyla remains unknown. Most of our current knowledge concerning the mechanism of Rho's activity and its physiological roles comes from the model micro-organism Escherichia coli, where Rho is essential and involved in the control of several important biological processes. However, the rather comprehensive knowledge about the general mechanisms of action and activities of Rho based on the E. coli paradigm cannot be directly extrapolated to other bacteria. Recent studies performed in different species favour the view that Rho-dependent termination plays a significant role even in bacteria where Rho is not essential. Here, we summarize the current state of the ever-increasing knowledge about the various aspects of the physiological functions of Rho, such as limitation of deleterious foreign DNA expression, control of gene expression, suppression of pervasive transcription, prevention of R-loops and maintenance of chromosome integrity, focusing on similarities and differences of the activities of Rho in various bacterial species.
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- Cell Biology
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Ribosome dimerization is essential for the efficient regrowth of Bacillus subtilis
Ribosome dimers are a translationally inactive form of ribosomes found in Escherichia coli and many other bacterial cells. In this study, we found that the 70S ribosomes of Bacillus subtilis dimerized during the early stationary phase and these dimers remained in the cytoplasm until regrowth was initiated. Ribosome dimerization during the stationary phase required the hpf gene, which encodes a homologue of the E. coli hibernation-promoting factor (Hpf). The expression of hpf was induced at an early stationary phase and its expression was observed throughout the rest of the experimental period, including the entire 6 h of the stationary phase. Ribosome dimerization followed the induction of hpf in WT cells, but the dimerization was impaired in cells harbouring a deletion in the hpf gene. Although the absence of ribosome dimerization in these Hpf-deficient cells did not affect their viability in the stationary phase, their ability to regrow from the stationary phase decreased. Thus, following the transfer of stationary-phase cells to fresh LB medium, Δhpf mutant cells grew slower than WT cells. This observed lag in growth of Δhpf cells was probably due to a delay in restoring their translational activity. During regrowth, the abundance of ribosome dimers in WT cells decreased with a concomitant increase in the abundance of 70S ribosomes and growth rate. These results suggest that the ribosome dimers, by providing 70S ribosomes to the cells, play an important role in facilitating rapid and efficient regrowth of cells under nutrient-rich conditions.
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- Environmental Biology
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Natural variation in methane emission of sheep fed on a lucerne pellet diet is unrelated to rumen ciliate community type
Only limited information is available on the roles of different rumen ciliate community types, first described by Eadie in 1962, in enteric methane (CH4) formation by their ruminant hosts. If the different types were differentially associated with CH4 formation, then ciliate community typing could be used to identify naturally high and low CH4-emitting animals. Here we measured the CH4 yields [g CH4 (kg feed dry matter intake, DMI)− 1] of 118 sheep fed a standard pelleted lucerne diet at two different times, at least 2 weeks apart. There were significant differences (P < 2.2 × 10− 16, Wilcoxon rank sum test) in the CH4 yields ( ± sd) from sheep selected as high [16.7 ± 1.5 g CH4 (kg DMI)− 1] and low emitters [13.3 ± 1.5 g CH4 (kg DMI)− 1]. A rumen sample was collected after each of the two measurements, and ciliate composition was analysed using barcoded 454 Titanium pyrosequencing of 18S rRNA genes. The genera found, in order of mean relative abundance, were Epidinium, Entodinium, Dasytricha, Eudiplodinium, Polyplastron, Isotricha and Anoplodinium–Diplodinium, none of which was significantly correlated with the CH4 emissions ranking associated with the rumen sample. Ciliate communities naturally assembled into four types (A, AB, B and O), characterized by the presence and absence of key genera. There was no difference in CH4 yield between sheep that harboured different ciliate community types, suggesting that these did not underlie the natural variation in CH4 yields. Further research is needed to unravel the nature of interactions between ciliate protozoa and other rumen micro-organisms, which may ultimately lead to contrasting CH4 emission phenotypes.
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- Genomics and Systems Biology
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Comparison of Lactobacillus crispatus isolates from Lactobacillus-dominated vaginal microbiomes with isolates from microbiomes containing bacterial vaginosis-associated bacteria
Vaginal lactobacilli can inhibit colonization by and growth of other bacteria, thereby preventing development of bacterial vaginosis (BV). Amongst the lactobacilli, Lactobacillus crispatus appears to be particularly effective at inhibiting growth of BV-associated bacteria. Nonetheless, some women who are colonized with this species can still develop clinical BV. Therefore, we sought to determine whether strains of L. crispatus that colonize women with lactobacilli-dominated vaginal microbiomes are distinct from strains that colonize women who develop BV. The genomes of L. crispatus isolates from four women with lactobacilli-dominated vaginal microbiomes ( < 1 % 16S rRNA reads above threshold from genera other than Lactobacillus) and four women with microbiomes containing BV-associated bacteria (>12 % 16S rRNA reads from bacterial taxa associated with BV) were sequenced and compared. Lactic acid production by the different strains was quantified. Phage induction in the strains was also analysed. There was considerable genetic diversity between strains, and several genes were exclusive to either the strains from Lactobacillus-dominated microbiomes or those containing BV-associated bacteria. Overall, strains from microbiomes dominated by lactobacilli did not differ from strains from microbiomes containing BV-associated bacteria with respect to lactic acid production. All of the strains contained multiple phage, but there was no clear distinction between the presence or absence of BV-associated bacteria with respect to phage-induced lysis. Genes found to be exclusive to the Lactobacillus-dominated versus BV-associated bacteria-containing microbiomes could play a role in the maintenance of vaginal health and the development of BV, respectively.
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- Host-microbe Interaction
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Salivaricin E and abundant dextranase activity may contribute to the anti-cariogenic potential of the probiotic candidate Streptococcus salivarius JH
More LessDental caries is an infectious disease that is continuing to increase in prevalence, reducing the quality of life for millions worldwide as well as causing considerable expense, with an estimated US$108 billion spent on dental care in the USA each year. Oral probiotics are now being investigated to determine whether they could play a role in the prevention and treatment of this disease. Streptococcus salivarius strain JH is a potential probiotic candidate that produces multiple proteinaceous antimicrobials (bacteriocins), the inhibitory spectrum of which includes Streptococcus mutans, one of the principal causative agents of dental caries. The genome of strain JH has previously been shown to contain the biosynthetic loci for the bacteriocins salivaricin A3, streptin and streptococcin SA-FF22. Here we show that strain JH also produces salivaricin E, a 32 aa lantibiotic with a mass of 3565.9 Da, which is responsible for the inhibition of S. mutans growth. In addition, strain JH was shown to produce dextranase, an enzyme that hydrolyses (1 → 6)-α-d-glucosidic linkages, at levels higher than any other S. salivarius tested. In vitro testing showed that partial hydrolysis of the exopolymeric substances of S. mutans, using strain JH dextranase, improved the anti-S. mutans inhibitory activity of the lytic bacteriocin, zoocin A. The multiple bacteriocin and dextranase activities of strain JH support its candidature for development as an oral probiotic.
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Neisseria cinerea isolates can adhere to human epithelial cells by type IV pilus-independent mechanisms
In pathogenic Neisseria species the type IV pili (Tfp) are of primary importance in host–pathogen interactions. Tfp mediate initial bacterial attachment to cell surfaces and formation of microcolonies via pilus–pilus interactions. Based on genome analysis, many non-pathogenic Neisseria species are predicted to express Tfp, but aside from studies on Neisseria elongata, relatively little is known about the formation and function of pili in these organisms. Here, we have analysed pilin expression and the role of Tfp in Neisseria cinerea. This non-pathogenic species shares a close taxonomic relationship to the pathogen Neisseria meningitidis and also colonizes the human oropharyngeal cavity. Through analysis of non-pathogenic Neisseria genomes we identified two genes with homology to pilE, which encodes the major pilin of N. meningitidis. We show which of the two genes is required for Tfp expression in N. cinerea and that Tfp in this species are required for DNA competence, similar to other Neisseria. However, in contrast to the meningococcus, deletion of the pilin gene did not impact the association of N. cinerea to human epithelial cells, demonstrating that N. cinerea isolates can adhere to human epithelial cells by Tfp-independent mechanisms.
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Helicobacter pullorum induces nitric oxide release in murine macrophages that promotes phagocytosis and killing
More LessHelicobacter pullorum is an avian enterohepatic species that, more recently, has also been found as a naturally acquired infection in mice and rats, and isolated from patients with gastrointestinal and hepatobiliary diseases. In this work, the interaction between H. pullorum and murine macrophages was examined. Firstly, the impact of nitric oxide, which is an antimicrobial produced by mammalian macrophages, on H. pullorum 6350-92 viability and morphology was studied by colony-forming assays and light microscopy, respectively. Exposure to nitric oxide lowered H. pullorum viability, in a growth-phase-dependent manner, and decreased the mean cell size. However, the number of coccoid forms remained low, contrasting with what has been observed for other Helicobacter species. Confocal microscopy showed that H. pullorum is internalized by murine macrophages, triggering nitric oxide production that promotes phagocytosis and killing of the pathogen. Interaction between H. pullorum and macrophages stimulated secretion of pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-6 and MIP-2. These results show that H. pullorum is able to infect mammalian murine cells triggering an inflammatory response.
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Inner-membrane protein MorC is involved in fimbriae production and biofilm formation in Aggregatibacter actinomycetemcomitans
More LessFimbrial subunit synthesis, secretion and assembly on the surface of the periodontal pathogen Aggregatibacter actinomycetemcomitans are essential for biofilm formation. A recent quantitative proteomics study employing an afimbriated strain and a developed mutant isogenic for the inner-membrane protein morphogenesis protein C (MorC) revealed that the abundance of the proteins of the fimbrial secretion apparatus in the membrane is dependent on MorC. To investigate further the relationship between MorC and fimbriation, we identified and complemented the defect in fimbriae production in the afimbriated laboratory strain. The transformed strain expressing a plasmid containing genes encoding the WT fimbrial subunit and the prepilin peptidase displayed all of the hallmarks of a fimbriated bacterium including the distinct star-like colony morphology, robust biofilm formation, biofilm architecture composed of discrete microcolonies and the presence of fimbriae. When the identical plasmid was transformed into a morC mutant strain, the bacterium did not display any of the phenotypes of fimbriated strains. Extension of these studies to a naturally fimbriated clinical strain showed that the resulting morC mutant maintained the characteristic colony morphology of fimbriated strains. There was, however, a reduction in the secretion of fimbrial subunits, and fewer fimbriae were observed on the surface of the mutant strain. Furthermore, the morC mutant of the fimbriated strain displayed a significantly altered biofilm microcolony architecture, while maintaining a similar biofilm mass to the parent strain. These results suggest that MorC influences fimbrial secretion and microcolony formation in A. actinomycetemcomitans.
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- Physiology and Metabolism
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CphA2 is a novel type of cyanophycin synthetase in N2-fixing cyanobacteria
Most cyanobacteria use a single type of cyanophycin synthetase, CphA1, to synthesize the nitrogen-rich polymer cyanophycin. The genomes of many N2-fixing cyanobacteria contain an additional gene that encodes a second type of cyanophycin synthetase, CphA2. The potential function of this enzyme has been debated due to its reduced size and the lack of one of the two ATP-binding sites that are present in CphA1. Here, we analysed CphA2 from Anabaena variabilis ATCC 29413 and Cyanothece sp. PCC 7425. We found that CphA2 polymerized the dipeptide β-aspartyl-arginine to form cyanophycin. Thus, CphA2 represents a novel type of cyanophycin synthetase. A cphA2 disruption mutant of A. variabilis was generated. Growth of this mutant was impaired under high-light conditions and nitrogen deprivation, suggesting that CphA2 plays an important role in nitrogen metabolism under N2-fixing conditions. Electron micrographs revealed that the mutant had fewer cyanophycin granules, but no alteration in the distribution of granules in its cells was observed. Localization of CphA2 by immunogold electron microscopy demonstrated that the enzyme is attached to cyanophycin granules. Expression of CphA1 and CphA2 was examined in Anabaena WT and cphA mutant cells. Whilst the CphA1 level increased upon nitrogen deprivation, the CphA2 level remained nearly constant.
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GntR family regulator SCO6256 is involved in antibiotic production and conditionally regulates the transcription of myo-inositol catabolic genes in Streptomyces coelicolor A3(2)
More LessSCO6256 belongs to the GntR family and shows 74 % identity with SCO6974, which is the repressor of myo-inositol catabolism in Streptomyces coelicolor A3(2). Disruption of SCO6256 significantly enhanced the transcription of myo-inositol catabolic genes in R2YE medium. The purified recombinant SCO6256 directly bound to the upstream regions of SCO2727, SCO6978 and SCO6985, as well as its encoding gene. Footprinting assays demonstrated that SCO6256 bound to the same sites in the myo-inositol catabolic gene cluster as SCO6974. The expression of SCO6256 was repressed by SCO6974 in minimal medium with myo-inositol as the carbon source, but not in R2YE medium. Glutathione-S-transferase pull-down assays demonstrated that SCO6974 and SCO6256 interacted with each other; and both of the proteins controlled the transcription of myo-inositol catabolic genes in R2YE medium. These results indicated SCO6256 regulates the transcription of myo-inositol catabolic genes in coordination with SCO6974 in R2YE medium. In addition, SCO6256 negatively regulated the production of actinorhodin and calcium-dependent antibiotic via control of the transcription of actII-ORF4 and cdaR. SCO6256 bound to the upstream region of cdaR and the binding sequence was proved to be TTTCGGCACGCAGACAT, which was further confirmed through base substitution. Four putative targets (SCO2652, SCO4034, SCO4237 and SCO6377) of SCO6256 were found by screening the genome sequence of Strep. coelicolor A3(2) based on the conserved binding motif, and confirmed by transcriptional analysis and electrophoretic mobility shift assays. These results revealed that SCO6256 is involved in the regulation of myo-inositol catabolic gene transcription and antibiotic production in Strep. coelicolor A3(2).
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Sinorhizobium meliloti low molecular mass phosphotyrosine phosphatase SMc02309 modifies activity of the UDP-glucose pyrophosphorylase ExoN involved in succinoglycan biosynthesis
In Gram-negative bacteria, tyrosine phosphorylation has been shown to play a role in the control of exopolysaccharide (EPS) production. This study demonstrated that the chromosomal ORF SMc02309 from Sinorhizobium meliloti 2011 encodes a protein with significant sequence similarity to low molecular mass protein-tyrosine phosphatases (LMW-PTPs), such as the Escherichia coli Wzb. Unlike other well-characterized EPS biosynthesis gene clusters, which contain neighbouring LMW-PTPs and kinase, the S. meliloti succinoglycan (EPS I) gene cluster located on megaplasmid pSymB does not encode a phosphatase. Biochemical assays revealed that the SMc02309 protein hydrolyses p-nitrophenyl phosphate (p-NPP) with kinetic parameters similar to other bacterial LMW-PTPs. Furthermore, we show evidence that SMc02309 is not the LMW-PTP of the bacterial tyrosine-kinase (BY-kinase) ExoP. Nevertheless, ExoN, a UDP-glucose pyrophosphorylase involved in the first stages of EPS I biosynthesis, is phosphorylated at tyrosine residues and constitutes an endogenous substrate of the SMc02309 protein. Additionally, we show that the UDP-glucose pyrophosphorylase activity is modulated by SMc02309-mediated tyrosine dephosphorylation. Moreover, a mutation in the SMc02309 gene decreases EPS I production and delays nodulation on Medicago sativa roots.
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- Regulation
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Loss of σI affects heat-shock response and virulence gene expression in Bacillus anthracis
More LessThe pathogenesis of Bacillus anthracis depends on several virulence factors, including the anthrax toxin. Loss of the alternative sigma factor σI results in a coordinate decrease in expression of all three toxin subunits. Our observations suggest that loss of σI alters the activity of the master virulence regulator AtxA, but atxA transcription is unaffected by loss of σI. σI-containing RNA polymerase does not appear to directly transcribe either atxA or the toxin gene pagA. As in Bacillus subtilis, loss of σI in B. anthracis results in increased sensitivity to heat shock and transcription of sigI, encoding σI, is induced by elevated temperature. Encoded immediately downstream of and part of a bicistronic message with sigI is an anti-sigma factor, RsgI, which controls σI activity. Loss of RsgI has no direct effect on virulence gene expression. sigI appears to be expressed from both the σI and σA promoters, and transcription from the σA promoter is likely more significant to virulence regulation. We propose a model in which σI can be induced in response to heat shock, whilst, independently, σI is produced under non-heat-shock, toxin-inducing conditions to indirectly regulate virulence gene expression.
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MsaB activates capsule production at the transcription level in Staphylococcus aureus
More LessStaphylococcus aureus produces several virulence factors that allow it to cause a variety of infections. One of the major virulence factors is the capsule, which contributes to the survival of the pathogen within the host as a way to escape phagocytosis. The production of the capsular polysaccharide is encoded in a 16 gene operon, which is regulated in response to several environmental stimuli including nutrient availability. For instance, the capsule is produced in the late- and post-exponential growth phases, but not in the early- or mid-exponential growth phase. Several regulators are involved in capsule production, but the regulation of the cap operon is still poorly understood. In this study, we show that MsaB activates the cap operon by binding directly to a 10 bp repeat in the promoter region. We show that despite the fact that MsaB is expressed throughout four growth phases, it only activates capsule production in the late- and post-exponential growth phases. Furthermore, we find that MsaB does not bind to its target site in the early and mid-exponential growth phases. This correlates with decreased nutrient availability and capsule production. These data suggest either that MsaB binding ability changes in response to nutrients or that other cap operon regulators interfere with the binding of MsaB to its target site. This study increases our understanding of the regulation of capsule production and the mechanism of action of MsaB.
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Heat-shock-induced refolding entails rapid degradation of bsrG toxin mRNA by RNases Y and J1
More LessGene regulation accomplished by alternative folding of an mRNA is a widely used mechanism. Classical examples are the various transcriptional attenuation mechanisms that employ, for example, leader peptide translation, or binding of a modified protein, an uncharged tRNA or an antisense RNA to the 5′ untranslated region of an mRNA. With the discovery of transcriptional and translational riboswitches, it became clear that small metabolites or even metal ions can also alter RNA secondary structures and, hence, gene expression. In addition, biophysical factors like temperature can affect RNA folding, as exemplified by RNA thermometers. We have investigated in detail the type I toxin–antitoxin system bsrG/SR4 from Bacillus subtilis. The antitoxin SR4 is a cis-encoded regulatory RNA that neutralizes BsrG toxin action. SR4 prevents toxin expression by promoting degradation of the toxin mRNA and inhibiting its translation. In addition, upon temperature shock the amount of toxin mRNA decreases significantly. Here, we demonstrate that heat shock induces a refolding in the central region of the toxin mRNA that makes it more accessible to degradation by RNases Y and J1. Furthermore, we show that BsrG might play a role at the onset of stationary phase, when the antitoxin SR4 can no longer prevent toxin synthesis.
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