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Berlin Brandenburg

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  • 1
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 25 August 2015, Vol.112(34), pp.E4772-81
    Description: Horizontal gene transfer via plasmid conjugation is a major driving force in microbial evolution but constitutes a complex process that requires synchronization with the physiological state of the host bacteria. Although several host transcription factors are known to regulate plasmid-borne transfer genes, RNA-based regulatory circuits for host-plasmid communication remain unknown. We describe a posttranscriptional mechanism whereby the Hfq-dependent small RNA, RprA, inhibits transfer of pSLT, the virulence plasmid of Salmonella enterica. RprA employs two separate seed-pairing domains to activate the mRNAs of both the sigma-factor σ(S) and the RicI protein, a previously uncharacterized membrane protein here shown to inhibit conjugation. Transcription of ricI requires σ(S) and, together, RprA and σ(S) orchestrate a coherent feedforward loop with AND-gate logic to tightly control the activation of RicI synthesis. RicI interacts with the conjugation apparatus protein TraV and limits plasmid transfer under membrane-damaging conditions. To our knowledge, this study reports the first small RNA-controlled feedforward loop relying on posttranscriptional activation of two independent targets and an unexpected role of the conserved RprA small RNA in controlling extrachromosomal DNA transfer.
    Keywords: Hfq ; Rpra ; Feedforward Control ; Plasmid Conjugation ; Srna ; Chromosomes, Bacterial ; DNA, Bacterial -- Genetics ; RNA, Bacterial -- Genetics ; Salmonella -- Genetics
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 2
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 07 January 2014, Vol.111(1), pp.355-60
    Description: Antibiotic-resistant isolates of Salmonella enterica were selected on plates containing lethal concentrations of rifampicin, kanamycin, and nalidixic acid. The stability of the resistance phenotype was scored after nonselective growth. Rifampicin-resistant (Rif(r)) isolates were stable, suggesting that they had arisen by mutation. Mutations in the rpoB gene were detected indeed in Rif(r) mutants. In contrast, a fraction of kanamycin-resistant (Km(r)) and nalidixic acid-resistant (Nal(r)) isolates showed reduced resistance after nonselective growth, suggesting that mechanisms other than mutation had contributed to bacterial survival upon lethal selection. Single-cell analysis revealed heterogeneity in expression of the porin gene ompC, and subpopulation separation provided evidence that reduced ompC expression confers adaptive resistance to kanamycin. In the case of Nal(r) isolates, mutations in the gyrA gene were present in most nalidixic acid-resistant isolates. However, the efflux pump inhibitor Phe-Arg-β-naphtylamide (PAβN) reduced the level of resistance in Nal(r) mutants, indicating that active efflux contributes to the overall level of nalidixic acid resistance. Heterogeneous efflux pump activity was detected in single cells and colonies, and a correlation between high efflux and increased resistance to nalidixic acid was found. These observations suggest that fluctuations in the expression and the activity of critical functions of the bacterial cell, alone or combined with mutations, can contribute to adaptive resistance to antibiotics.
    Keywords: Anti-Bacterial Agents -- Pharmacology ; Drug Resistance, Bacterial -- Genetics ; Kanamycin -- Pharmacology ; Nalidixic Acid -- Pharmacology ; Rifampin -- Pharmacology ; Salmonella Enterica -- Genetics
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 3
    Language: English
    In: Future Microbiology, Feb, 2011, Vol.6(2), p.133(3)
    Description: Salmonella is a human pathogen that causes various types of infections, ranging from mild gastroenteritis to life-threatening typhoid fever. For decades, research on Salmonella has attracted not just clinical microbiologists and epidemiologists but also geneticists and molecular biologists who use Salmonella as a model organism. Hence, meetings dealing with Salmonella can be truly interdisciplinary. Such was the case in the ESCMID-FEMS Conference held in Villars-sur-Ollon (Switzerland) in October 2010. The meeting fostered interactive views of Salmonella biology and prompted the discussion of strategies to prevent and combat Salmonella infections.
    Keywords: Salmonella -- Distribution ; Salmonella -- Research ; Salmonella -- Health Aspects ; Microbial Drug Resistance -- Health Aspects ; Microbial Drug Resistance -- Research
    ISSN: 1746-0913
    E-ISSN: 17460921
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  • 4
    In: Molecular Microbiology, March 2014, Vol.91(6), pp.1057-1069
    Description: is a quiescent ‐type regulator belonging to the ‐ regulon. Activation of transcription represses expression of pathogenicity island 1 (‐1) in serovar yphimurium and inhibits invasion of epithelial cells. Loss of suppresses ‐mediated downregulation of ‐1. Activation of transcription reduces the level of protein, and loss of restores the wild type level. Hence, ‐mediated downregulation of ‐1 may involve inhibition of activity by , a view consistent with the fact that is a inhibitor. analyses using β‐galactosidase fusions indicate that activates transcription. analyses by slot blotting, electrophoretic mobility shift analysis and footprinting show that binds the promoter region. Although residual ‐1 repression by is observed in the absence of , the ‐‐ ‘pathway’ appears to be the major mechanism. Because both and ‐1 are repressed by ‐, activation of transcription may provide a backup mechanism for ‐1 repression under conditions that impair ‐‐mediated silencing.
    Keywords: Salmonella -- Analysis;
    ISSN: 0950-382X
    E-ISSN: 1365-2958
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  • 5
    Language: English
    In: Genetics, March 2010, Vol.184(3), pp.637-49
    Description: DNA adenine methylase (Dam(-)) mutants of Salmonella enterica are attenuated in the mouse model and present multiple virulence-related defects. Impaired interaction of Salmonella Dam(-) mutants with the intestinal epithelium has been tentatively correlated with reduced secretion of pathogenicity island 1 (SPI-1) effectors. In this study, we show that S. enterica Dam(-) mutants contain lowered levels of the SPI-1 transcriptional regulators HilA, HilC, HilD, and InvF. Epistasis analysis indicates that Dam-dependent regulation of SPI-1 requires HilD, while HilA, HilC, and InvF are dispensable. A transcriptional hilDlac fusion is expressed at similar levels in Dam(+) and Dam(-) hosts. However, lower levels of hilD mRNA are found in a Dam(-) background, thus providing unsuspected evidence that Dam methylation might exert post-transcriptional regulation of hilD expression. This hypothesis is supported by the following lines of evidence: (i) lowered levels of hilD mRNA are found in Salmonella Dam(-) mutants when hilD is transcribed from a heterologous promoter; (ii) increased hilD mRNA turnover is observed in Dam(-) mutants; (iii) lack of the Hfq RNA chaperone enhances hilD mRNA instability in Dam(-) mutants; and (iv) lack of the RNA degradosome components polynucleotide phosphorylase and ribonuclease E suppresses hilD mRNA instability in a Dam(-) background. Our report of Dam-dependent control of hilD mRNA stability suggests that DNA adenine methylation plays hitherto unknown roles in post-transcriptional control of gene expression.
    Keywords: Bacterial Proteins -- Metabolism ; DNA Methylation -- Physiology ; Genomic Islands -- Physiology ; RNA Stability -- Physiology ; RNA, Bacterial -- Metabolism ; Salmonella Enterica -- Metabolism ; Transcription Factors -- Metabolism
    ISSN: 00166731
    E-ISSN: 1943-2631
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  • 6
    Language: English
    In: PLoS ONE, 2012, Vol.7(1), p.e30499
    Description: Invasion of intestinal epithelial cells is a critical step in Salmonella infection and requires the expression of genes located in Salmonella pathogenicity island 1 (SPI-1). A key factor for SPI-1 expression is DNA adenine (Dam) methylation, which activates synthesis of the SPI-1 transcriptional activator HilD. Dam-dependent regulation of hilD is postranscriptional (and therefore indirect), indicating the involvement of unknown cell functions under Dam methylation control. A genetic screen has identified the std fimbrial operon as the missing link between Dam methylation and SPI-1. We show that all genes in the std operon are part of a single transcriptional unit, and describe three previously uncharacterized ORFs (renamed stdD , stdE , and stdF ). We present evidence that two such loci ( stdE and stdF ) are involved in Dam-dependent control of Salmonella SPI-1: in a Dam − background, deletion of stdE or stdF suppresses SPI-1 repression; in a Dam + background, constitutive expression of StdE and/or StdF represses SPI-1. Repression of SPI-1 by products of std operon explains the invasion defect of Salmonella Dam − mutants, which constitutively express the std operon. Dam-dependent repression of std in the ileum may be required to permit invasion, as indicated by two observations: constitutive expression of StdE and StdF reduces invasion of epithelial cells in vitro (1,000 fold) and attenuates Salmonella virulence in the mouse model (〉60 fold). In turn, crosstalk between std and SPI-1 may play a role in intestinal infections by preventing expression of SPI-1 in the caecum, an intestinal compartment in which the std operon is known to be expressed.
    Keywords: Research Article ; Biology ; Genetics And Genomics ; Microbiology
    E-ISSN: 1932-6203
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  • 7
    Language: English
    In: Genetics, July 2015, Vol.200(3), pp.807-19
    Description: Invasion of the intestinal epithelium is a critical step in Salmonella enterica infection and requires functions encoded in the gene cluster known as Salmonella Pathogenicity Island 1 (SPI-1). Expression of SPI-1 genes is repressed by L-arabinose, and not by other pentoses. Transport of L-arabinose is necessary to repress SPI-1; however, repression is independent of L-arabinose metabolism and of the L-arabinose-responsive regulator AraC. SPI-1 repression by L-arabinose is exerted at a single target, HilD, and the mechanism appears to be post-translational. As a consequence of SPI-1 repression, l-arabinose reduces translocation of SPI-1 effectors to epithelial cells and decreases Salmonella invasion in vitro. These observations reveal a hitherto unknown role of L-arabinose in gene expression control and raise the possibility that Salmonella may use L-arabinose as an environmental signal.
    Keywords: Hild ; Salmonella Invasion ; Salmonella Pathogenicity Island 1 ; L-Arabinose ; Gene Expression Regulation, Bacterial ; Genomic Islands ; Arabinose -- Metabolism ; Salmonella Enterica -- Genetics
    ISSN: 00166731
    E-ISSN: 1943-2631
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  • 8
    Language: English
    In: PLoS ONE, 2012, Vol.7(5), p.e36863
    Description: STM2209 and STM2208 are contiguous loci annotated as putative protein-coding genes in the chromosome of Salmonella enterica . Lack of homologs in related Enterobacteria and low G+C content suggest that S. enterica may have acquired STM2209-STM2208 by horizontal transfer. STM2209 and STM2208 are co-transcribed from a promoter upstream STM2209 , and their products are inner (cytoplasmic) membrane proteins. Analysis with the bacterial adenylate cyclase two-hybrid system suggests that STM2209 and STM2208 may interact. Expression of STM2209-STM2208 is subjected to phase variation in wild type Salmonella enterica serovar Typhimurium. Switching frequencies in LB medium are 6.1×10 −5 (OFF→ON) and 3.7×10 −2 (ON→OFF) per cell and generation. Lack of DNA adenine methylation locks STM2209-STM2208 in the ON state, and lack of the LysR-type factor OxyR locks STM2209-STM2208 in the OFF state. OxyR-dependent activation of STM2209-STM2208 expression is independent of the oxidation state of OxyR. Salmonella cultures locked in the ON state show alteration of O-antigen length in the lipopolysaccharide, reduced absorption of bacteriophage P22, impaired resistance to serum, and reduced proliferation in macrophages. Phenotypic heterogeneity generated by STM2209-STM2208 phase variation may thus provide defense against phages. In turn, formation of a subpopulation unable to proliferate in macrophages may restrain Salmonella spread in animal organs, potentially contributing to successful infection.
    Keywords: Research Article ; Biology ; Medicine ; Genetics And Genomics ; Infectious Diseases ; Microbiology
    E-ISSN: 1932-6203
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  • 9
    Language: English
    In: Genetics, April 2010, Vol.184(4), pp.947-58
    Description: The virulence plasmid of Salmonella enterica (pSLT) is an F-like conjugative plasmid. High rates of pSLT transfer occur in the mammalian gut, a microaerobic environment. In this study, we describe genetic screens for host-encoded activators and repressors of the transfer operon (tra) of pSLT. We show that the transcription factor ArcA is an activator of conjugation, especially under microaerobiosis. In turn, succinate dehydrogenase (SdhABCD) is a repressor of mating in aerobiosis. ArcA binds upstream of the main tra promoter (p(traY)) and activates tra transcription, as previously described in F, R1, and R100. In the absence of ArcA, transfer of pSLT decreased 7-fold in aerobiosis and 〉100-fold in microaerobiosis. In aerobiosis, ArcA activates the traY promoter in an ArcB-independent manner, as described in other F-like plasmids. In microaerobiosis, however, the ArcB sensor is necessary for activation of p(traY). Lack of Sdh causes a 〉20-fold increase in pSLT transfer in aerobiosis, but has little effect under microaerobiosis. Sdh inhibits conjugal transfer by reducing traJ transcription, probably in an indirect manner. In turn, the sdhCDAB operon is repressed by the ArcAB system under microaerobiosis. Hence, the ArcAB two-component system of S. enterica stimulates pSLT transfer under microaerobiosis by two concerted actions: activation of the tra operon and repression of the sdhCDAB operon.
    Keywords: Aerobiosis ; Conjugation, Genetic ; Bacterial Proteins -- Metabolism ; Salmonella Enterica -- Genetics ; Succinate Dehydrogenase -- Metabolism ; Transcription Factors -- Metabolism
    ISSN: 00166731
    E-ISSN: 1943-2631
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  • 10
    Language: English
    In: Nucleic acids research, May 2014, Vol.42(9), pp.5894-906
    Description: Long 3' untranslated regions (3'UTRs) are common in eukaryotic mRNAs. In contrast, long 3'UTRs are rare in bacteria, and have not been characterized in detail. We describe a 3'UTR of 310 nucleotides in hilD mRNA, a transcript that encodes a transcriptional activator of Salmonella enterica pathogenicity island 1 (SPI-1). Deletion of the hilD 3'UTR increases the hilD mRNA level, suggesting that the hilD 3'UTR may play a role in hilD mRNA turnover. Cloning of the hilD 3'UTR downstream of the green fluorescent protein (gfp) gene decreases green fluorescent protein (GFP) activity in both Escherichia coli and S. enterica, indicating that the hilD 3'UTR can act as an independent module. S. enterica mutants lacking either ribonuclease E or polynucleotide phosphorylase contain similar amounts of hilD and hilD Δ3'UTR mRNAs, suggesting that the hilD 3'UTR is a target for hilD mRNA degradation by the degradosome. The hilD 3'UTR is also necessary for modulation of hilD and SPI-1 expression by the RNA chaperone Hfq. Overexpression of SPI-1 in the absence of the hilD 3'UTR retards Salmonella growth and causes uncontrolled invasion of epithelial cells. Based on these observations, we propose that the S. enterica hilD 3'UTR is a cis-acting element that contributes to cellular homeostasis by promoting hilD mRNA turnover.
    Keywords: Bacterial Proteins -- Genetics ; RNA, Bacterial -- Genetics ; RNA, Messenger -- Genetics ; Salmonella Typhimurium -- Genetics ; Transcription Factors -- Genetics
    ISSN: 03051048
    E-ISSN: 1362-4962
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