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  • 1
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 27 March 2012, Vol.109(13), pp.E757-64
    Description: SgrS RNA is a model for the large class of Hfq-associated small RNAs that act to posttranscriptionally regulate bacterial mRNAs. The function of SgrS is well-characterized in nonpathogenic Escherichia coli, where it was originally shown to counteract glucose-phosphate stress by acting as a repressor of the ptsG mRNA, which encodes the major glucose transporter. We have discovered additional SgrS targets in Salmonella Typhimurium, a pathogen related to E. coli that recently acquired one-quarter of all genes by horizontal gene transfer. We show that the conserved short seed region of SgrS that recognizes ptsG was recruited to target the Salmonella-specific sopD mRNA of a secreted virulence protein. The SgrS-sopD interaction is exceptionally selective; we find that sopD2 mRNA, whose gene arose from sopD duplication during Salmonella evolution, is deaf to SgrS because of a nonproductive G-U pair in the potential SgrS-sopD2 RNA duplex vs. G-C in SgrS-sopD. In other words, SgrS discriminates the two virulence factor mRNAs at the level of a single hydrogen bond. Our study suggests that bacterial pathogens use their large suites of conserved Hfq-associated regulators to integrate horizontally acquired genes into existing posttranscriptional networks, just as conserved transcription factors are recruited to tame foreign genes at the DNA level. The results graphically illustrate the importance of the seed regions of bacterial small RNAs to select new targets with high fidelity and suggest that target predictions must consider all or none decisions by individual seed nucleotides.
    Keywords: Phylogeny ; Base Pairing -- Genetics ; Gene Transfer, Horizontal -- Genetics ; RNA, Bacterial -- Genetics ; Salmonella -- Genetics
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 2
    Language: English
    In: Molecular microbiology, 2010, Vol.77(3), pp.771-786
    Description: The transcriptional regulator CsgD of Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major regulator of biofilm formation required for the expression of csgBA, which encodes curli fimbriae, and adrA, coding for a diguanylate cyclase. CsgD is a response regulator with an N-terminal receiver domain with a conserved aspartate (D59) as a putative target site for phosphorylation and a C-terminal LuxR-like helix-turn-helix DNA binding motif, but the mechanisms of target gene activation remained unclear. To study the DNA-binding properties of CsgD we used electrophoretic mobility shift assays and DNase I footprint analysis to show that unphosphorylated CsgD-His₆ binds specifically to the csgBA and adrA promoter regions. In vitro transcription analysis revealed that CsgD-His₆ is crucial for the expression of csgBA and adrA. CsgD-His₆ is phosphorylated by acetyl phosphate in vitro, which decreases its DNA-binding properties. The functional impact of D59 in vivo was demonstrated as S. Typhimurium strains expressing modified CsgD protein (D59E and D59N) were dramatically reduced in biofilm formation due to decreased protein stability and DNA-binding properties in the case of D59E. In summary, our findings suggest that the response regulator CsgD functions in its unphosphorylated form under the conditions of biofilm formation investigated in this study. ; Includes references ; p. 771-786.
    Keywords: Salmonella -- Analysis ; Phosphates -- Analysis ; Aspartate -- Analysis ; Nucleases -- Analysis;
    ISSN: 0950-382X
    E-ISSN: 13652958
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  • 3
    In: Molecular Microbiology, September 2011, Vol.81(5), pp.1144-1165
    Description: GcvB is one of the most highly conserved Hfq‐associated small RNAs in Gram‐negative bacteria and was previously reported to repress several ABC transporters for amino acids. To determine the full extent of GcvB‐mediated regulation in , we combined a genome‐wide experimental approach with biocomputational target prediction. Comparative pulse expression of wild‐type versus mutant sRNA variants revealed that GcvB governs a large post‐transcriptional regulon, impacting ∼1% of all genes via its conserved G/U‐rich domain R1. Complementary predictions of C/A‐rich binding sites in mRNAs and reporter fusion experiments increased the number of validated GcvB targets to more than 20, and doubled the number of regulated amino acid transporters. Unlike the previously described targeting via the single R1 domain, GcvB represses the glycine transporter CycA by exceptionally redundant base‐pairing. This novel ability of GcvB is focused upon the one target that could feedback‐regulate the glycine‐responsive synthesis of GcvB. Several newly discovered mRNA targets involved in amino acid metabolism, including the global regulator Lrp, question the previous assumption that GcvB simply acts to limit unnecessary amino acid uptake. Rather, GcvB rewires primary transcriptional control circuits and seems to act as a distinct regulatory node in amino acid metabolism.
    Keywords: Glycine -- Physiological Aspects ; Genetic Research -- Physiological Aspects ; Genomics -- Physiological Aspects ; Messenger Rna -- Physiological Aspects;
    ISSN: 0950-382X
    E-ISSN: 1365-2958
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  • 4
    Language: English
    In: Applied and Environmental Microbiology, 2010, Vol. 76(19), p.6514
    Description: The food-borne pathogen Escherichia coli O157:H7 is commonly exposed to organic acid in processed and preserved foods, allowing adaptation and the development of tolerance to pH levels otherwise lethal. Since little is known about the molecular basis of adaptation of E. coli to organic acids, we studied K-12 MG1655 and O157:H7 Sakai during exposure to acetic, lactic, and hydrochloric acid at pH 5.5. This is the first analysis of the pH-dependent transcriptomic response of stationary-phase E. coli. Thirty-four genes and three intergenic regions were upregulated by both strains during exposure to all acids. This universal acid response included genes involved in oxidative, envelope, and cold stress resistance and iron and manganese uptake, as well as 10 genes of unknown function. Acidulant- and strain-specific responses were also revealed. The acidulant-specific response reflects differences in the modes of microbial inactivation, even between weak organic acids. The two strains exhibited similar responses to lactic and hydrochloric acid, while the response to acetic acid was distinct. Acidulant-dependent differences between the strains involved induction of genes involved in the heat shock response, osmoregulation, inorganic ion and nucleotide transport and metabolism, translation, and energy production. E. coli O157:H7-specific acid-inducible genes were identified, suggesting that the enterohemorrhagic E. coli strain possesses additional molecular mechanisms contributing to acid resistance that are absent in K-12. While E. coli K-12 was most resistant to lactic and hydrochloric acid, O157:H7 may have a greater ability to survive in more complex acidic environments, such as those encountered in the host and during food processing. [PUBLICATION ]
    Keywords: Microbiology ; E Coli ; Cell Culture ; Organic Chemicals ; Resistance to Control;
    ISSN: 0099-2240
    ISSN: 00992240
    E-ISSN: 10985336
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  • 5
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 2018, Vol. 115(11), pp. E2614-E2623
    Description: Salmonella enterica serovar Typhimurium ST313 is a relatively newly emerged sequence type that is causing a devastating epidemic of bloodstream infections across sub-Saharan Africa. Analysis of hundreds of Salmonella genomes has revealed that ST313 is closely related to the ST19 group of S. Typhimurium that cause gastroenteritis across the world. The core genomes of ST313 and ST19 vary by only similar to 1,000 SNPs. We hypothesized that the phenotypic differences that distinguish African Salmonella from ST19 are caused by certain SNPs that directly modulate the transcription of virulence genes. Here we identified 3,597 transcriptional start sites of the ST313 strain D23580, and searched for a gene-expression signature linked to pathogenesis of Salmonella. We identified a SNP in the promoter of the pgtE gene that caused high expression of the PgtE virulence factor in African S. Typhimurium, increased the degradation of the factor B component of human complement, contributed to serum resistance, and modulated virulence in the chicken infection model. We propose that high levels of PgtE expression by African S. Typhimurium ST313 promote bacterial survival and dissemination during human infection. Our finding of a functional role for an extragenic SNP shows that approaches used to deduce the evolution of virulence in bacterial pathogens should include a focus on noncoding regions of the genome.
    Keywords: Salmonella ; Noncoding Genome ; Transcriptomics ; Evolution Of Virulence ; Host Adaptation ; Medical And Health Sciences ; Clinical Medicine ; Infectious Medicine ; Medicin Och Hälsovetenskap ; Klinisk Medicin ; Infektionsmedicin
    ISSN: 0027-8424
    E-ISSN: 10916490
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  • 6
    Language: English
    In: Applied and environmental microbiology, October 2015, Vol.81(19), pp.6800-11
    Description: Common salt (NaCl) is frequently used by the food industry to add flavor and to act as a humectant in order to reduce the water content of a food product. The improved health awareness of consumers is leading to a demand for food products with reduced salt content; thus, manufacturers require alternative water activity-reducing agents which elicit the same general effects as NaCl. Two examples include KCl and glycerol. These agents lower the water activity of a food matrix and also contribute to limit the growth of the microbiota, including foodborne pathogens. Little is currently known about how foodborne pathogens respond to these water activity-lowering agents. Here we examined the response of Salmonella enterica serovar Typhimurium 4/74 to NaCl, KCl, and glycerol at three time points, using a constant water activity level, compared with the response of a control inoculum. All conditions induced the upregulation of gluconate metabolic genes after 6 h of exposure. Bacteria exposed to NaCl and KCl demonstrated the upregulation of the osmoprotective transporter mechanisms encoded by the proP, proU, and osmU (STM1491 to STM1494) genes. Glycerol exposure elicited the downregulation of these osmoadaptive mechanisms but stimulated an increase in lipopolysaccharide and membrane protein-associated genes after 1 h. The most extensive changes in gene expression occurred following exposure to KCl. Because many of these genes were of unknown function, further characterization may identify KCl-specific adaptive processes that are not stimulated by NaCl. This study shows that the response of S. Typhimurium to different humectants does not simply reflect reduced water activity and likely involves systems that are linked to specific humectants.
    Keywords: Food Industry ; Hygroscopic Agents -- Pharmacology ; Salmonella Typhimurium -- Drug Effects
    ISSN: 00992240
    E-ISSN: 1098-5336
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  • 7
    Language: English
    In: Journal of bacteriology, 01 July 2017, Vol.199(13)
    Description: Deep sequencing has revolutionized our understanding of the bacterial RNA world and has facilitated the identification of 280 small RNAs (sRNAs) in Despite the suspicions that sRNAs may play important roles in pathogenesis, the functions of most sRNAs remain unknown. To advance our understanding of RNA biology in virulence, we searched for sRNAs required for bacterial invasion into nonphagocytic cells. After screening 75 sRNAs, we discovered that the ablation of InvS caused a significant decrease of invasion into epithelial cells. A proteomic analysis showed that InvS modulated the levels of several type III secreted proteins. The level of PrgH, a type III secretion apparatus protein, was significantly lower in the absence of InvS, consistent with the known roles of PrgH in effector secretion and bacterial invasion. We discovered that InvS modulates expression and hence flagellar gene expression and motility. We propose that InvS coordinates the increase of PrgH and decrease in FimZ that promote efficient invasion into nonphagocytic cells. Salmonellosis continues to be the most common foodborne infection reported by the CDC in the United States. Central to pathogenesis is the ability to invade nonphagocytic cells and to replicate inside host cells. Invasion genes are known to be regulated by protein transcriptional networks, but little is known about the role played by small RNAs (sRNAs) in this process. We have identified a novel sRNA, InvS, that is involved in invasion. Our result will likely provide an opportunity to better understand the fundamental question of how regulates invasion gene expression and may inform strategies for therapeutic intervention.
    Keywords: Salmonella ; Gene Regulation ; Host Cell Invasion ; Noncoding RNA ; Bacterial Proteins -- Metabolism ; Epithelial Cells -- Microbiology ; Salmonella Typhimurium -- Physiology
    ISSN: 00219193
    E-ISSN: 1098-5530
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  • 8
    Language: English
    In: Journal of bacteriology, February 2012, Vol.194(3), pp.686-701
    Description: Lag phase represents the earliest and most poorly understood stage of the bacterial growth cycle. We developed a reproducible experimental system and conducted functional genomic and physiological analyses of a 2-h lag phase in Salmonella enterica serovar Typhimurium. Adaptation began within 4 min of inoculation into fresh LB medium with the transient expression of genes involved in phosphate uptake. The main lag-phase transcriptional program initiated at 20 min with the upregulation of 945 genes encoding processes such as transcription, translation, iron-sulfur protein assembly, nucleotide metabolism, LPS biosynthesis, and aerobic respiration. ChIP-chip revealed that RNA polymerase was not "poised" upstream of the bacterial genes that are rapidly induced at the beginning of lag phase, suggesting a mechanism that involves de novo partitioning of RNA polymerase to transcribe 522 bacterial genes within 4 min of leaving stationary phase. We used inductively coupled plasma mass spectrometry (ICP-MS) to discover that iron, calcium, and manganese are accumulated by S. Typhimurium during lag phase, while levels of cobalt, nickel, and sodium showed distinct growth-phase-specific patterns. The high concentration of iron during lag phase was associated with transient sensitivity to oxidative stress. The study of lag phase promises to identify the physiological and regulatory processes responsible for adaptation to new environments.
    Keywords: Gene Expression Regulation, Bacterial ; Metals -- Metabolism ; Salmonella Typhimurium -- Metabolism
    ISSN: 00219193
    E-ISSN: 1098-5530
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  • 9
    In: Molecular Microbiology, June 2010, Vol.76(5), pp.1250-1265
    Description: The conjugative IncHI1 plasmid pSfR27 from 2a strain 2457T encodes the Sfh protein, a paralogue of the global transcriptional repressor H‐NS. Sfh allows pSfR27 to be transmitted to new bacterial hosts with minimal impact on host fitness, providing a ‘stealth’ function whose molecular mechanism has yet to be determined. The impact of the Sfh protein on the serovar Typhimurium transcriptome was assessed and binding sites for Sfh in the Typhimurium genome were identified by chromatin immunoprecipitation. Sfh did not bind uniquely to any sites. Instead, it bound to a subset of the larger H‐NS regulatory network. Analysis of Sfh binding in the absence of H‐NS revealed a greatly expanded population of Sfh binding sites that included the majority of H‐NS target genes. Furthermore, the presence of plasmid pSfR27 caused a decrease in H‐NS interactions with the Typhimurium chromosome, suggesting that the A + T‐rich DNA of this large plasmid acts to titrate H‐NS, removing it from chromosomal locations. It is proposed that Sfh acts as a molecular backup for H‐NS and that it provides its ‘stealth’ function by replacing H‐NS on the chromosome, thus minimizing disturbances to the H‐NS‐DNA binding pattern in cells that acquire pSfR27.
    Keywords: Fighter Planes -- Analysis ; Chromatin -- Analysis ; Preventive Medicine -- Analysis ; Salmonella Typhimurium -- Analysis ; Genomics -- Analysis;
    ISSN: 0950-382X
    E-ISSN: 1365-2958
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  • 10
    Language: English
    In: Current Opinion in Microbiology, February 2015, Vol.23, pp.ix-x
    Keywords: Biology
    ISSN: 1369-5274
    E-ISSN: 1879-0364
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