RNA-sequencing based analysis of Listeria monocytogenes 10403S::ΔBCHL Prha-sigH and ΔBCHL Prha
ABSTRACT: In this study, RNA-seq was used to compare the transcriptomes of Listeria monocytogenes 10403S::ΔBCHL Prha-sigH and ΔBCHL Prha. RNA-seq was performed on ΔBCHL Prha-sigH and ΔBCHL Prha RNA samples representing three independent biological replicates at log phase in Brain Heart Infusion (BHI) broth under rhamnose induction. Indexed and purified cDNA libraries (6 libraries including 3 replicates for 2 strains) were loaded together onto an independent flow cell without any other samples; sequencing was carried out by running Hiseq 2500 (single-end, 150-bp per read). Reads alignment was carried out using the Burrows-Wheeler Aligner (BWA). Differential expression of genes in different strains was statistically assessed using the BaySeq method. To identify sigH-dependent promoters, a new method of moving sliding windows of 50 nt along the whole genome was used to compare the normalized RNA-seq coverage (NRC) between the two strains. Using the standard whole gene differential expression analysis, significant upregulation of 5 genes in 4 operons was found in the sigH overexpressing strain. While with the sliding windiow analysis, 2 additional σH-dependent promoters were identified. Our results show that three σH-dependent transcritption units that encode competence proteins, including the comEABC , comGABCDEFG and coiA. Transcriptome profiles of L. monocytogenes 10403S::ΔBCHL Prha-sigH and ΔBCHL Prha were generated by deep sequencing, in triplicate, using Illumina Hiseq 2500.
Project description:The stationary phase stress response transcriptome of the human bacterial pathogen Listeria monocytogenes was defined using RNA sequencing (RNA-Seq) with the Illumina Genome Analyzer. Specifically, bacterial transcriptomes were compared between stationary phase cells of L. monocytogenes 10403S and an otherwise isogenic DsigB mutant, which does not express the alternative sigma factor σB, a major regulator of genes contributing to stress response. Keywords: Transcriptome and differential expression analyses a laboratory strain, 10403S and its otherwise isogenic mutant lacking sigB were analyzed. Two replicates of each strain were analyzed for a total of 4 runs
Project description:Among the three major genetic lineages of L. monocytogenes (i.e. LI, LII, and LIII), LI and LII are predominantly associated with foodborne listeriosis outbreaks, whereas LIII is rarely implicated in human infections. In a previous study, we identified a Crp/Fnr family transcription factor lmo0753 that was highly specific to outbreak-associated LI and LII but absent from LIII. Lmo0753 shares two conserved functional domains including a DNA-binding domain with the well-characterized master virulence regulator PrfA in L. monocytogenes. In this study, we constructed a lmo0753 deletion and complementation mutants of the fully sequenced L. monocytogenes LII strain EGDe. We found that deletion of lmo0753 led to the loss of L-rhamnose utilization in EGDe. Transcriptomic comparison of the EGDe lmo0753 deletion mutant and the wild type incubated in phenol-red medium containing L-rhamnose as the sole carbon source revealed 126 (4.5%) and 546 (19.5%) out of 2,798 genes in the EGDe genome that were up- and down-regulated for more than 2-fold, respectively. Genes involved in biotin biosynthesis, general stress response and rhamnose metabolism were shown to be differentially regulated by Lmo0753. Findings from this study may partially explain why LIII of L. monocytogenes is underrepresented in the environment and rarely associated with human listeriosis outbreaks due to the inability of rhamnose utilization. We report the transcriptomic profile of L. monocytogenes Δlmo0753 LII strain (EGDe) in broth media with L-rhamnose as the sole carbon source. Examination of deletion of Lmo0753 on L-rhamnose utilization in L. monocytogenes. Two biological replicates per WT and Δlmo0753.
Project description:To characterize regulons of alternative sigma factor SigH, SigL, and SigC in Listeria monocytogenes, in-frame mutant strains were created in the 10403S background. Regulons controlled by these 3 alternative sigma factors were characterized by whole-genome microarrays. The L. monocytogenes 10403S wild type and sigma factor null mutation strains were grown at 37 °C to stationary phase (defined in this study as growth to OD600 = 1.0, followed by incubation for an additional 3 h) prior to RNA isolation. Transcriptional profiles of 10403S wild type were compared to those of null mutation strain. In addition to stationary phase condition, SigC regulon was further characterized using heat stress (cultures grown to log phase at OD600 = 0.4, 37 °C and then exposed to heat at 55 °C for 10 min) and a condition with IPTG-inducible expression of sigC (sigC gene is placed under Pspac promoter using pLIV2 vector in wild type 10403S background). Under these conditions, expression profiles were compared between (i) wild type and sigC null mutant for heat stress and (ii) IPTG-inducible sigC strain and sigC null mutant, respectively. Using adjusted P < 0.05 and ≥ 1.5 fold change as cutoff values, microarray analyses identified 169 SigH-dependent, 51 SigL-dependent, and 3 SigC-dependent genes. Keywords: Listeria monocytogenes, alternative sigma factor, SigH, SigL, SigC Overall design: Independent RNA isolations were performed for cultures grown to stationary phase (OD600 = 1 + additional incubation for 3 hours) at 37°C. In addition to RNA from stationary phase cultures, for SigC regulon characterization, cultures were also grown to log phase (OD600 = 0.4) then exposed to heat at 55 °C for 10 min) and grown in BHI with 0.5 mM IPTG to log phase prior to RNA isolations. Three biological replicates were used in competitive whole-genome microarray experiments. The LIMMA package from the R/BioConductor software project was used for data pre-processing and differential expression analyses.
Project description:Bacteria are known to cope with environmental changes by using alternative sigma factors binding to RNA polymerase core enzyme. Sigma factor is one of the targets to modify transcription regulation in bacteria and to influence production capacities. In this study, the effect of overexpressing all annotated sigma factor genes on C. glutamicum WT was assayed using an IPTG inducible plasmid system and different IPTG concentrations. It was revealed that growth was severely decreased when sigD or sigH were overexpressed with IPTG concentrations higher than 50 μM. Overexpression of sigH led to an obvious phenotypic change, a yellow-colored supernatant. HPLC analysis revealed that riboflavin was excreted to the medium when sigH was overexpressed and DNA Microarray analysis confirmed increased expression of riboflavin biosynthesis genes. In addition, genes for enzymes of the pentose phosphate pathway and for enzymes dependent on FMN, FAD or NADPH as cofactor were upregulated when sigH was overexpressed. To test if sigH overexpression can be exploited for production of riboflavin-derived FMN or FAD, the endogenous gene for bifunctional riboflavin kinase/FMN adenyltransferase was co-expressed with sigH from a plasmid. Balanced expression of sigH and ribF improved accumulation of riboflavin (19.8 ± 0.3 μM) and allowed for its conversion to FMN (33.1 ± 1.8 μM) in the supernatant. While a proof-of-concept was reached, conversion was not complete and titers were not high. This study revealed that inducible and gradable overexpression of sigma factor genes is an interesting approach to switch gene expression profiles and to discover untapped potential of bacteria for chemical production. Endogenous sigma factor gene, sigH, was overexpressed in C. glutamicum ATCC13032 from IPTG inducible vector, pEKEx3. Two different concentration of IPTG (10 μM and 15 μM) was used for induction of SigH expression.
Project description:These studies were designed to examine the acute Listeria monocytogenes transcriptional response to mammalian (porcine) bile. Triplicate WT Listeria monocytogenes (strain 10403S) were grown to mid-log in BHI at 37 °C. Samples were divided, and either treated or not treated by addition of porcine bile (Sigma, to 1% final) for 30 minutes.
Project description:The extensively studied intracellular pathogen, L. monocytogenes, is an ideal model for identifying small-molecule agents for treating bacterial infections. By selecting specific biological targets in L. monocytogenes, which are common to Gram-positive pathogens, we could extrapolate drug discovery information derived from this well-studied bacterium. Attenuating the pathogen’s virulence and stress response attributes without killing it, eliminates selective pressure caused by disruption of essential gene functions (as done by current antibiotics) and reduces the likelihood of developing microbes that are impervious to the effects of antibiotics. To this end, we have assessed multiple libraries of small organic compounds to identify inhibitors of L. monocytogenes σB, the alternative sigma factor common to several clinically relevant Gram-positive pathogens, such as Staphylococcus aureus, Bacillus cereus, and Bacillus anthracis. The role of σB as a transcriptional regulator of stress response and virulence makes it an ideal, well conserved target for chemotherapeutic development. Independent RNA isolations were performed for each growth experiment (log phase cells exposed to BHI+0.3M NaCl and BHI+0.3M NaCl + CMPD). Four biological replicates were used in competitive whole-genome microarray experiments. For the hybridizations, RNA from the wildtype parent strain L. monocytogenes 10403S were hybridized to RNA from the wildtype parent strain 10403S treated with CMPD.
Project description:Listeria monocytogenes strain 10403S has been studied extensively for stress response activity toward multiple stressors (acid, osmotic, cold, high temperature, etc.) as well as multiple stress regulons (SigB, CtsR, HrcA, etc.). Here we aimed to determine the transcriptional response of Listeria monocytogenes in early log phase towards the strong oxidative stress imposed by ClO2. The elucidation of such a response allows for further a more completel understanding of the mechanism of inactivation by sanitizers, specifically ClO2. Independent RNA isolations were performed for strain 10403S with and without exposure to ClO2 from cells grown to early log phase. Four biological replicates were used in competitive whole-genome microarray experiments. For each set of hybridizations, RNA from a control sample of Listeria monocytogenes was hybridized with RNA from a culture of L. monocytogenes following exposure to ClO2. Dye swapping was performed for the four replicates to mitigate any concerns of dye bias.
Project description:Mycobacterium avium subspecies paratuberculosis (a.k.a. M. paratuberculosis) causes Johne’s disease, an enteric infection in cattle and other ruminants, greatly afflicting the dairy industry worldwide. Once inside the cell, M. paratuberculosis is known to survive harsh microenvironments, especially those inside activated macrophages. To improve our understanding of M. paratuberculosis pathogenesis, we examined the phagosome maturation associated with transcriptional responses of M. paratuberculosis during macrophage infection. Monitoring cellular markers, only live M. paratuberculosis bacilli were able to prevent phagosome maturation and reduce its acidification. On the transcriptional level, over 300 of M. paratuberculosis genes were significantly, differentially regulated in both naïve and IFN-γ-activated macrophages. These genes include the sigma factor H (sigH) that was shown to be important during persistent infection in M. tuberculosis. Interestingly, a sigH-knockout mutant showed increased sensitivity to a sustained level of thiol-specific oxidative stress. Large-scale RNA sequence analysis revealed that a large number of genes belong to the sigH regulon, especially following diamide stress. Genes involved in oxidative stress and virulence were among the induced genes in the sigH regulon with a putative consensus sequence for SigH binding was recognized in a subset of these genes (N=30), suggesting direct regulation with SigH. Finally, mice infections showed a significant attenuation of the ∆sigH mutant compared to its parental strain suggesting a role for sigH in M. paratuberculosis virulence. Such analysis could identify potential targets for further testing as vaccine candidates against Johne’s disease. Examination of role played by alternative sigma factor, SigH during M. paratuberculosis infection.