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Volume 160,
Issue 10,
2014
Volume 160, Issue 10, 2014

- Review
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Biogenesis and multifaceted roles of outer membrane vesicles from Gram-negative bacteria
More LessOuter membrane vesicles (OMVs) released from Gram-negative bacteria consist of lipids, proteins, lipopolysaccharides and other molecules. OMVs are associated with several biological functions such as horizontal gene transfer, intracellular and intercellular communication, transfer of contents to host cells, and eliciting an immune response in host cells. Although hypotheses have been made concerning the mechanism of biogenesis of these vesicles, research on OMV formation is far from complete. The roles of outer membrane components, bacterial quorum sensing molecules and some specific proteins in OMV biogenesis have been studied. This review discusses the different models that have been proposed for OMV biogenesis, along with details of the biological functions of OMVs and the likely scope of future research.
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- Cell and Molecular Biology of Microbes
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Flippase (FLP) recombinase-mediated marker recycling in the dermatophyte Arthroderma vanbreuseghemii
More LessBiological processes can be elucidated by investigating complex networks of relevant factors and genes. However, this is not possible in species for which dominant selectable markers for genetic studies are unavailable. To overcome the limitation in selectable markers for the dermatophyte Arthroderma vanbreuseghemii (anamorph: Trichophyton mentagrophytes), we adapted the flippase (FLP) recombinase-recombination target (FRT) site-specific recombination system from the yeast Saccharomyces cerevisiae as a selectable marker recycling system for this fungus. Taking into account practical applicability, we designed FLP/FRT modules carrying two FRT sequences as well as the flp gene adapted to the pathogenic yeast Candida albicans (caflp) or a synthetic codon-optimized flp (avflp) gene with neomycin resistance (nptII) cassette for one-step marker excision. Both flp genes were under control of the Trichophyton rubrum copper-repressible promoter (PCTR4 ). Molecular analyses of resultant transformants showed that only the avflp-harbouring module was functional in A. vanbreuseghemii. Applying this system, we successfully produced the Ku80 recessive mutant strain devoid of any selectable markers. This strain was subsequently used as the recipient for sequential multiple disruptions of secreted metalloprotease (fungalysin) (MEP) or serine protease (SUB) genes, producing mutant strains with double MEP or triple SUB gene deletions. These results confirmed the feasibility of this system for broad-scale genetic manipulation of dermatophytes, advancing our understanding of functions and networks of individual genes in these fungi.
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Unravelling the genetic basis for competence development of auxotrophic Bacillus licheniformis 9945A strains
Bacterial natural genetic competence – well studied in Bacillus subtilis – enables cells to take up and integrate extracellularly supplied DNA into their own genome. However, little is known about competence development and its regulation in other members of the genus, although DNA uptake machineries are routinely encoded. Auxotrophic Bacillus licheniformis 9945A derivatives, obtained from repeated rounds of random mutagenesis, were long known to develop natural competence. Inspection of the colony morphology and extracellular enzyme secretion of two of these derivatives, M28 and M18, suggested that regulator genes are collaterally hit. M28 emerged as a 14 bp deletion mutant concomitantly displaying a shift in the reading frame of degS that encodes the sensor histidine kinase, which is part of the molecular switch that directs cells to genetic competence, the synthesis of extracellular enzymes or biofilm formation, while for M18, sequencing of the suspected gene revealed a 375 bp deletion in abrB, encoding the major transition state regulator. With respect to colony morphology, enzyme secretion and competence development, both of the mutations, when newly generated on the wild-type B. licheniformis 9945A genetic background, resulted in phenotypes resembling M28 and M18, respectively. All of the known naturally competent B. licheniformis representatives, hitherto thoroughly investigated in this regard, carry mutations in regulator genes, and hence genetic competence observed in domesticated strains supposedly results from deregulation.
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The TrpA protein of Trichodesmium erythraeum IMS101 is a non-fibril-forming collagen and a component of the outer sheath
More LessCollagen molecules are structural in nature and primarily found in eukaryotic, multicellular organisms. Recently, a collagen-like protein, TrpA, was identified and characterized in the marine cyanobacterium Trichodesmium erythraeum IMS 101, and it was shown to be involved in maintaining the structural integrity of the trichomes. The TrpA protein contains one glycine interruption in the otherwise perfectly uninterrupted collagenous domain. In this study, we used phylogenetic analysis to determine that the TrpA protein sequence is most closely associated with non-fibril-forming collagen proteins. Structural modelling and circular dichroism data suggest that the glycine insertion decreases the stability of TrpA compared to uninterrupted collagen sequences. Additionally, scanning electron microscopy revealed that TrpA is expressed entirely on the surface of the trichomes, with no specific pattern of localization. These data indicate that the TrpA protein is part of the outer sheath of this organism. As such, this protein may function to promote adhesion between individual T. erythraeum trichomes, and between this organism and heterotrophic bacteria found in the same environment.
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Bacteriophage-derived CHAP domain protein, P128, kills Staphylococcus cells by cleaving interpeptide cross-bridge of peptidoglycan
Sudarson Sundarrajan, Junjappa Raghupatil, Aradhana Vipra, Nagalakshmi Narasimhaswamy, Sanjeev Saravanan, Chemira Appaiah, Nethravathi Poonacha, Srividya Desai, Sandhya Nair, Rajagopala Narayana Bhatt, Panchali Roy, Ravisha Chikkamadaiah, Murali Durgaiah, Bharathi Sriram, Sriram Padmanabhan and Umender SharmaP128 is an anti-staphylococcal protein consisting of the Staphylococcus aureus phage-K-derived tail-associated muralytic enzyme (TAME) catalytic domain (Lys16) fused with the cell-wall-binding SH3b domain of lysostaphin. In order to understand the mechanism of action and emergence of resistance to P128, we isolated mutants of Staphylococcus spp., including meticillin-resistant Staphylococcus aureus (MRSA), resistant to P128. In addition to P128, the mutants also showed resistance to Lys16, the catalytic domain of P128. The mutants showed loss of fitness as shown by reduced rate of growth in vitro. One of the mutants tested was found to show reduced virulence in animal models of S. aureus septicaemia suggesting loss of fitness in vivo as well. Analysis of the antibiotic sensitivity pattern showed that the mutants derived from MRSA strains had become sensitive to meticillin and other β-lactams. Interestingly, the mutant cells were resistant to the lytic action of phage K, although the phage was able to adsorb to these cells. Sequencing of the femA gene of three P128-resistant mutants showed either a truncation or deletion in femA, suggesting that improper cross-bridge formation in S. aureus could be causing resistance to P128. Using glutathione S-transferase (GST) fusion peptides as substrates it was found that both P128 and Lys16 were capable of cleaving a pentaglycine sequence, suggesting that P128 might be killing S. aureus by cleaving the pentaglycine cross-bridge of peptidoglycan. Moreover, peptides corresponding to the reported cross-bridge of Staphylococcus haemolyticus (GGSGG, AGSGG), which were not cleaved by lysostaphin, were cleaved efficiently by P128. This was also reflected in high sensitivity of S. haemolyticus to P128. This showed that in spite of sharing a common mechanism of action with lysostaphin, P128 has unique properties, which allow it to act on certain lysostaphin-resistant Staphylococcus strains.
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Production of acylated homoserine lactone by a novel marine strain of Proteus vulgaris and inhibition of its swarming by phytochemicals
More LessA marine strain of Proteus vulgaris capable of activating multiple acylated homoserine lactone (AHL)-based reporter cultures was isolated. The cognate signal molecule was characterized as octanoyl homoserine lactone (OHL) and its production was observed to be growth dependent, with maximum production (5.675 µg l−1) at 24 h growth. The strain exhibited swarming, but its motility was not affected upon addition of pure OHL or culture supernatant. Phytochemicals such as quercitin and berberine chloride inhibited OHL production and reduced swarming. FliA, the predominantly upregulated protein during swarming, was considered as a possible target for these inhibitors, and docking of the two most active and two least active inhibitors to this protein suggested preferential binding of the former set of compounds. Apart from adding new evidence to AHL production in Proteus vulgaris, active inhibitors shortlisted from this study could help in identifying lead compounds to act against this opportunistic pathogen of the respiratory and gastrointestinal tract.
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Transcriptional regulation of the l-lactate permease gene lutP by the LutR repressor of Bacillus subtilis RO-NN-1
More LessThe Bacillus subtilis lutABC operon encodes three iron–sulfur-containing proteins required for l-lactate utilization and involved in biofilm formation. The transcriptional regulator LutR of the GntR family negatively controls lutABC expression. The lutP gene, which is situated immediately upstream of lutR, encodes an l-lactate permease. Here, we show that lutP expression can be strongly induced by l-lactate and is subject to partial catabolite repression by glucose. Disruption of the lutR gene led to a strong derepression of lutP and no further induction by l-lactate, suggesting that the LutR repressor can also negatively control lutP expression. Electrophoretic mobility shift assay revealed a LutR-binding site located downstream of the promoter of lutA or lutP and containing a consensus inverted repeat sequence 5′-TCATC-N1-GATGA-3′. Reporter gene analysis showed that deletion of each LutR-binding site caused a strong derepression of lutA or lutP. These results indicated that these two LutR-binding sites can function as operators in vivo. Moreover, deletion analysis identified a DNA segment upstream of the lutP promoter to be important for lutP expression. In contrast to the truncated LutR of laboratory strains 168 and PY79, the full-length LutR of the undomesticated strain RO-NN-1, and probably many other B. subtilis strains, can directly and negatively regulate lutP transcription. The absence or presence of the N-terminal 21 aa of the full-length LutR, which encompass a small part of the predicted winged helix–turn–helix DNA-binding motif, may probably alter the DNA-binding specificity or affinity of LutR.
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The RcsCDB regulatory system plays a crucial role in the protection of Salmonella enterica serovar Typhimurium against oxidative stress
More LessDps, the most abundant protein during the stationary growth phase, in Salmonella enterica is required for resistance to reactive oxygen species produced by the host during infection. It has been reported that in Salmonella dps expression is controlled by RpoS and Fur proteins. However, the regulation and function of Dps remain to be resolved. In the present work we demonstrate that activation of the complex RcsCDB regulatory system increases dps expression during exponential growth of Salmonella. In addition, we show that such dps upregulation produces high levels of H2O2 resistance. This phenotype allows the bacteria to avoid reactive oxygen species killing at early stages of growth, thus protecting its genetic material.
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The archaic chaperone–usher pathways may depend on donor strand exchange for intersubunit interactions
More LessSubunit–subunit interactions of the classical and alternate chaperone–usher (CU) systems have been shown to proceed through a donor strand exchange (DSE) mechanism. However, it is not known whether DSE is required for intersubunit interactions in the archaic CU system. We have previously shown that the Myxococcus xanthus Mcu system, a member of the archaic CU family that functions in spore coat formation, is likely to use the principle of donor strand complementation to medicate chaperone–subunit interactions analogous to the classical CU pathway. Here we describe the results of studies on Mcu subunit–subunit interactions. We constructed a series of N-terminal-deleted, single amino acid-mutated and donor strand-complemented Mcu subunits, and characterized their abilities to participate in subunit–subunit interactions. It appears that certain residues in both the N and C termini of McuA, a subunit of the Mcu system, play a critical role in intersubunit interactions and these interactions may involve the general principle of DSE of the classical and alternate CU systems. In addition, the specificity of the M. xanthus CU system for Mcu subunits over other spore coat proteins is demonstrated.
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Sro7 and Sro77, the yeast homologues of the Drosophila lethal giant larvae (Lgl), regulate cell proliferation via the Rho1–Tor1 pathway
More LessSaccharomyces cerevisiae Sro7 and Sro77 are homologues of the Drosophila tumour suppressor lethal giant larvae (Lgl), which regulates cell polarity in Drosophila epithelial cells. Here, we showed that double mutation of SRO7/SRO77 was defective in colony growth. The colony of the SRO7/SRO77 double deletion was much smaller than the WT and appeared to be round with a smooth surface, compared with the WT. Analysis using transmission electron microscopy revealed multiple defects of the colony cells, including multiple budding, multiple nuclei, cell lysis and dead cells, suggesting that the double deletion caused defects in cell polarity and cell wall integrity (CWI). Overexpression of RHO1, one of the central regulators of cell polarity and CWI, fully recovered the sro7Δ/sro77Δ phenotype. We further demonstrated that sro7Δ/sro77Δ caused a decrease of the GTP-bound, active Rho1, which in turn caused an upregulation of TOR1. Deletion of TOR1 in sro7Δ/sro77Δ (sro7Δ/sro77Δ/tor1Δ) recovered the cell growth and colony morphology, similar to WT. Our results suggested that the tumour suppressor homologue SRO7/SRO77 regulated cell proliferation and yeast colony development via the Rho1–Tor1 pathway.
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Global regulator Anr represses PlcH phospholipase activity in Pseudomonas aeruginosa when oxygen is limiting
More LessHaemolytic phospholipase C (PlcH) is a potent virulence and colonization factor that is expressed at high levels by Pseudomonas aeruginosa within the mammalian host. The phosphorylcholine liberated from phosphatidylcholine and sphingomyelin by PlcH is further catabolized into molecules that both support growth and further induce plcH expression. We have shown previously that the catabolism of PlcH-released choline leads to increased activity of Anr, a global transcriptional regulator that promotes biofilm formation and virulence. Here, we demonstrated the presence of a negative feedback loop in which Anr repressed plcH transcription and we proposed that this regulation allowed for PlcH levels to be maintained in a way that promotes productive host–pathogen interactions. Evidence for Anr-mediated regulation of PlcH came from data showing that growth at low oxygen (1 %) repressed PlcH abundance and plcH transcription in the WT, and that plcH transcription was enhanced in an Δanr mutant. The plcH promoter featured an Anr consensus sequence that was conserved across all P. aeruginosa genomes and mutation of conserved nucleotides within the Anr consensus sequence increased plcH expression under hypoxic conditions. The Anr-regulated transcription factor Dnr was not required for this effect. The loss of Anr was not sufficient to completely derepress plcH transcription as GbdR, a positive regulator of plcH, was required for expression. Overexpression of Anr was sufficient to repress plcH transcription even at 21 % oxygen. Anr repressed plcH expression and phospholipase C activity in a cell culture model for P. aeruginosa–epithelial cell interactions.
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- Environmental and Evolutionary Microbiology
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Characterization of uncultured giant rod-shaped magnetotactic Gammaproteobacteria from a freshwater pond in Kanazawa, Japan
More LessMagnetotactic bacteria (MTB) are widespread aquatic bacteria, and are a phylogenetically, physiologically and morphologically heterogeneous group, but they all have the ability to orientate and move along the geomagnetic field using intracellular magnetic organelles called magnetosomes. Isolation and cultivation of novel MTB are necessary for a comprehensive understanding of magnetosome formation and function in divergent MTB. In this study, we enriched a giant rod-shaped magnetotactic bacterium (strain GRS-1) from a freshwater pond in Kanazawa, Japan. Cells of strain GRS-1 were unusually large (~13×~8 µm). They swam in a helical trajectory towards the south pole of a bar magnet by means of a polar bundle of flagella. Another striking feature of GRS-1 was the presence of two distinct intracellular biomineralized structures: large electron-dense granules composed of calcium and long chains of magnetosomes that surround the large calcium granules. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that this strain belongs to the Gammaproteobacteria and represents a new genus of MTB.
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Differential expression of metallothioneins in response to heavy metals and their involvement in metal tolerance in the symbiotic basidiomycete Laccaria bicolor
More LessCysteine-rich peptides such as metallothioneins (MTs) are involved in metal homeostasis and detoxification in many eukaryotes. We report the characterization and expression of two MT genes, LbMT1 and LbMT2 from the ectomycorrhizal fungus Laccaria bicolor under metal stress conditions. LbMT1 and LbMT2 differ with respect to the length of the encoded peptides (58 versus 37 aa, respectively) and also by their expression patterns in response to metals. The expression levels of both LbMT1 and LbMT2 increased as a function of increased external Cu concentration, the expression levels for LbMT2 were always significantly higher compared with those of LbMT1. Only LbMT1, but not LbMT2, responded to Cd supply in the range of 25–100 µM while Zn did not affect the transcription of either LbMT1 or LbMT2. Both genes also responded to oxidative stress, but to a lesser extent compared to their responses to either Cu or Cd stress. Heterologous complementation assays in metal-sensitive yeast mutants indicated that both LbMT1 and LbMT2 encode peptides capable of conferring higher tolerance to both Cu and Cd. The present study identified LbMTs as potential determinants of the response of this mycorrhizal fungus to Cu and Cd stress.
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- Genes and Genomes
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Hierarchical management of carbon sources is regulated similarly by the CbrA/B systems in Pseudomonas aeruginosa and Pseudomonas putida
The CbrA/B system in pseudomonads is involved in the utilization of carbon sources and carbon catabolite repression (CCR) through the activation of the small RNAs crcZ in Pseudomonas aeruginosa, and crcZ and crcY in Pseudomonas putida. Interestingly, previous works reported that the CbrA/B system activity in P. aeruginosa PAO1 and P. putida KT2442 responded differently to the presence of different carbon sources, thus raising the question of the exact nature of the signal(s) detected by CbrA. Here, we demonstrated that the CbrA/B/CrcZ(Y) signal transduction pathway is similarly activated in the two Pseudomonas species. We show that the CbrA sensor kinase is fully interchangeable between the two species and, moreover, responds similarly to the presence of different carbon sources. In addition, a metabolomics analysis supported the hypothesis that CCR responds to the internal energy status of the cell, as the internal carbon/nitrogen ratio seems to determine CCR and non-CCR conditions. The strong difference found in the 2-oxoglutarate/glutamine ratio between CCR and non-CCR conditions points to the close relationship between carbon and nitrogen availability, or the relationship between the CbrA/B and NtrB/C systems, suggesting that both regulatory systems sense the same sort or interrelated signal.
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Genetic structure and biological properties of the first ancient multiresistance plasmid pKLH80 isolated from a permafrost bacterium
More LessA novel multidrug-resistance plasmid, pKLH80, previously isolated from Psychrobacter maritimus MR29-12 found in ancient permafrost, was completely sequenced and analysed. In our previous studies, we focused on the pKLH80 plasmid region containing streptomycin and tetracycline resistance genes, and their mobilization with an upstream-located ISPpy1 insertion sequence (IS) element. Here, we present the complete sequence of pKLH80 and analysis of its backbone genetic structure, including previously unknown features of the plasmid’s accessory region, notably a novel variant of the β-lactamase gene bla RTG-6. Plasmid pKLH80 was found to be a circular 14 835 bp molecule that has an overall G+C content of 40.3 mol% and encodes 20 putative ORFs. There are two distinctive functional modules within the plasmid backbone sequence: (i) the replication module consisting of repB and the oriV region; and (ii) the mobilization module consisting of mobA, mobC and oriT. All of the aforementioned genes share sequence identities with corresponding genes of different species of Psychrobacter. The plasmid accessory region contains antibiotic resistance genes and IS elements (ISPsma1 of the IS982 family, and ISPpy1 and ISAba14 of the IS3 family) found in environmental and clinical bacterial strains of different taxa. We revealed that the sequences flanking bla RTG-6 and closely related genes from clinical bacteria are nearly identical. This fact suggests that bla RTG-6 from the environmental strain of Psychrobacter is a progenitor of bla RTG genes of clinical bacteria. We also showed that pKLH80 can replicate in different strains of Acinetobacter and Psychrobacter genera. The roles of IS elements in the horizontal transfer of antibiotic resistance genes are examined and discussed.
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- Microbial Pathogenicity
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Strong mutator phenotype drives faster adaptation from growth on glucose to growth on acetate in Salmonella
The metabolic adaptation of strong mutator strains was studied to better understand the link between the strong mutator phenotype and virulence. Analysis of the growth curves of isogenic strains of Salmonella, which were previously grown in M63 glucose media, revealed that the exponential phase of growth was reached earlier in an M63 acetate medium with strong mutator strains (mutated in mutS or in mutL) than with normomutator strains (P<0.05). Complemented strains confirmed the direct role of the strong mutator phenotype in this faster metabolic adaptation to the assimilation of acetate. In a mixed cell population, proliferation of strong mutators over normomutators was observed when the carbon source was switched from glucose to acetate. These results add to the sparse body of knowledge about strong mutators and highlight the selective advantage conferred by the strong mutator phenotype to adapt to a switch of carbon source in the environment. This work may provide clinically useful information given that there is a high prevalence of strong mutators among pathogenic strains of Salmonella and that acetate is the principal short chain fatty acid of the human terminal ileum and colon where Salmonella infection is localized.
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Differences in vaginal microbiome in African American women versus women of European ancestry
Women of European ancestry are more likely to harbour a Lactobacillus-dominated microbiome, whereas African American women are more likely to exhibit a diverse microbial profile. African American women are also twice as likely to be diagnosed with bacterial vaginosis and are twice as likely to experience preterm birth. The objective of this study was to further characterize and contrast the vaginal microbial profiles in African American versus European ancestry women. Through the Vaginal Human Microbiome Project at Virginia Commonwealth University, 16S rRNA gene sequence analysis was used to compare the microbiomes of vaginal samples from 1268 African American women and 416 women of European ancestry. The results confirmed significant differences in the vaginal microbiomes of the two groups and identified several taxa relevant to these differences. Major community types were dominated by Gardnerella vaginalis and the uncultivated bacterial vaginosis-associated bacterium-1 (BVAB1) that were common among African Americans. Moreover, the prevalence of multiple bacterial taxa that are associated with microbial invasion of the amniotic cavity and preterm birth, including Mycoplasma, Gardnerella, Prevotella and Sneathia, differed between the two ethnic groups. We investigated the contributions of intrinsic and extrinsic factors, including pregnancy, body mass index, diet, smoking and alcohol use, number of sexual partners, and household income, to vaginal community composition. Ethnicity, pregnancy and alcohol use correlated significantly with the relative abundance of bacterial vaginosis-associated species. Trends between microbial profiles and smoking and number of sexual partners were observed; however, these associations were not statistically significant. These results support and extend previous findings that there are significant differences in the vaginal microbiome related to ethnicity and demonstrate that these differences are pronounced even in healthy women.
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- Physiology and Biochemistry
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Plant-derived compatible solutes proline betaine and betonicine confer enhanced osmotic and temperature stress tolerance to Bacillus subtilis
More Lessl-Proline is a widely used compatible solute and is employed by Bacillus subtilis, through both synthesis and uptake, as an osmostress protectant. Here, we assessed the stress-protective potential of the plant-derived l-proline derivatives N-methyl-l-proline, l-proline betaine (stachydrine), trans-4-l-hydroxproline and trans-4-hydroxy-l-proline betaine (betonicine) for cells challenged by high salinity or extremes in growth temperature. l-Proline betaine and betonicine conferred salt stress protection, but trans-4-l-hydroxyproline and N-methyl-l-proline was unable to do so. Except for l-proline, none of these compounds served as a nutrient for B. subtilis. l-Proline betaine was a considerably better osmostress protectant than betonicine, and its import strongly reduced the l-proline pool produced by B. subtilis under osmotic stress conditions, whereas a supply of betonicine affected the l-proline pool only modestly. Both compounds downregulated the transcription of the osmotically inducible opuA operon, albeit to different extents. Mutant studies revealed that l-proline betaine was taken up via the ATP-binding cassette transporters OpuA and OpuC, and the betaine-choline-carnitine-transporter-type carrier OpuD; betonicine was imported only through OpuA and OpuC. l-Proline betaine and betonicine also served as temperature stress protectants. A striking difference between these chemically closely related compounds was observed: l-proline betaine was an excellent cold stress protectant, but did not provide heat stress protection, whereas the reverse was true for betonicine. Both compounds were primarily imported in temperature-challenged cells via the high-capacity OpuA transporter. We developed an in silico model for the OpuAC–betonicine complex based on the crystal structure of the OpuAC solute receptor complexed with l-proline betaine.
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Lack of a surface layer in Tannerella forsythia mutants deficient in the type IX secretion system
Tannerella forsythia, a Gram-negative anaerobic bacterium, is an important pathogen in periodontal disease. This bacterium possesses genes encoding all known components of the type IX secretion system (T9SS). T. forsythia mutants deficient in genes orthologous to the T9SS-encoding genes porK, porT and sov were constructed. All porK, porT and sov single mutants lacked the surface layer (S-layer) and expressed less-glycosylated versions of the S-layer glycoproteins TfsA and TfsB. In addition, these mutants exhibited decreased haemagglutination and increased biofilm formation. Comparison of the proteins secreted by the porK and WT strains revealed that the secretion of several proteins containing C-terminal domain (CTD)-like sequences is dependent on the porK gene. These results indicate that the T9SS is functional in T. forsythia and contributes to the translocation of CTD proteins to the cell surface or into the extracellular milieu.
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Characterization of a dual-active enzyme, DcpA, involved in cyclic diguanosine monophosphate turnover in Mycobacterium smegmatis
More LessWe have reported previously that the long-term survival of Mycobacterium smegmatis is facilitated by a dual-active enzyme MSDGC-1 (renamed DcpA), which controls the cellular turnover of cyclic diguanosine monophosphate (c-di-GMP). Most mycobacterial species possess at least a single copy of a DcpA orthologue that is highly conserved in terms of sequence similarity and domain architecture. Here, we show that DcpA exists in monomeric and dimeric forms. The dimerization of DcpA is due to non-covalent interactions between two protomers that are arranged in a parallel orientation. The dimer shows both synthesis and hydrolysis activities, whereas the monomer shows only hydrolysis activity. In addition, we have shown that DcpA is associated with the cytoplasmic membrane and exhibits heterogeneous cellular localization with a predominance at the cell poles. Finally, we have also shown that DcpA is involved in the change in cell length and colony morphology of M. smegmatis. Taken together, our study provides additional evidence about the role of the bifunctional protein involved in c-di-GMP signalling in M. smegmatis.
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Volume 29 (1962)
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Volume 28 (1962)
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Volume 27 (1962)
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Volume 26 (1961)
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Volume 25 (1961)
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Volume 24 (1961)
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Volume 23 (1960)
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Volume 22 (1960)
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Volume 21 (1959)
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Volume 20 (1959)
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Volume 19 (1958)
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Volume 18 (1958)
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Volume 17 (1957)
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Volume 16 (1957)
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Volume 15 (1956)
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Volume 14 (1956)
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Volume 13 (1955)
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Volume 12 (1955)
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Volume 11 (1954)
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Volume 10 (1954)
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Volume 9 (1953)
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Volume 8 (1953)
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Volume 7 (1952)
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Volume 6 (1952)
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Volume 5 (1951)
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Volume 4 (1950)
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Volume 3 (1949)
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Volume 2 (1948)
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Volume 1 (1947)
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