Project description:In prokaryotes and eukaryotes, cell-cell communication and recognition of self are critical to coordinate multicellular functions. While kin and kind discrimination are increasingly appreciated to shape naturally occurring microbe populations, the underlying mechanisms that govern these interbacterial interactions are insufficiently understood. Here we identify a mechanism of interbacterial signal transduction that is mediated by contact-dependent growth inhibition (CDI) system proteins. CDI systems have been characterized by their ability to deliver a polymorphic protein toxin into the cytoplasm of a neighboring bacterium, resulting in growth inhibition or death unless the recipient bacterium produces a corresponding immunity protein. Using the model organism Burkholderia thailandensis, we show that delivery of a catalytically active CDI system toxin to immune (self) bacteria results in gene expression and phenotypic changes within the recipient cells. Termed contact-dependent signaling (CDS), this response promotes biofilm formation and other community-associated behaviors. Examination of wild-type Burkholderia thailandensis and two mutant strains, each in triplicate (9 samples total). mutant BtEKA contains two amino acid substitutions (E3064A and K3066A) within the coding sequence of gene Bth_I2723. In mutant PS12-WT, the native promoter of gene Bth_I2723 has been replaced with the strong constitutive promoter of the E264 rpsL gene, PS12.
Project description:Gene silencing by nucleoid-associated protein H-NS is sensitive to neighbouring transcription. This effect does not require neighbouring transcription to elongate into the silenced region. Here we measured how activating transcription of a tetRA cassette positioned upstream of either hilD or leuO promoters of Salmonella affected the activity of these promoters. The tetRA cassette included a strong Rho-independent transcription terminator at the 3' end of tetA. Transcription of the cassette was activated with anhydrotetracycline (AHTc). The activities of hilD and leuO promoters were measured upon performing template-switching reverse transcription follwed by semi-quantitative PCR and Illumina high-throughput sequencing.
Project description:Several members of the Bacillus cereus group of bacteria carry lysogenic phages of the family Tectiviridae. The Bacilus cereus reference strain (ATCC 14579) harbor a linear plasmid (pBClin15) that is highly similar to the genomes of the Tectiviruses. Production of active phages has however never been reported for pBClin15 and the role of pBClin15 in B. cereus physiology has been enigmatic. We have for the first time demonstrated an effect of pBClin15 on the physiology of B. cereus ATCC 14579 whereby the wild type is more sensitive to DNA damaging antibiotics compared to a pBClin15 cured strain. The microarray experiments in this study were designed to highlight transcriptional differences between the B. cereus ATCC 14579 wild type and a plasmid cured variant that potentially could explain the impact of pBClin15 on the B. cereus physiology. Microarray experiments were carried out under non-stressful conditions (growth in logarithmic phase in LB medium) under which there were no phenotypic difference between the wild type and pBClin15 cured strain as well as under stressful conditions under which there were phenotypic differences between the two strain (growth in LB medium supplemented with 0.5M-5g norfloxacin per ml).
Project description:Drugs targeting DNA and RNA in mammalian cells or viruses can also affect bacteria present in the host and thereby induce the bacterial SOS system. This has the potential to increase mutagenesis and the development of antimicrobial resistance (AMR). Here we have examined nucleoside analogues (NAs) commonly used in anti-viral and anti-cancer therapies for potential effects on mutagenesis in Escherichia coli using the Rifampicin mutagenicity assay. To further explore the mode of action of the NAs, we appliedanalyzed metabolome and proteome of E.coli deletion mutants., and metabolome and proteome analyses. Five out of the thirteen NAs examined, including three nucleoside reverse transcriptase inhibitors (NRTIs) and two anti-cancer drugs, increased the mutation frequency in E. coli more than 25-fold at doses that were within reported plasma concentration range (Pl.CR), but that did not affect bacterial growth. We show that the SOS response is induced and that the increase in mutation frequency is mediated by the TLS polymerase Pol V. Quantitative mass spectrometry based metabolite profiling did not reveal large changes in nucleoside phosphate or other central carbon metabolite pools, which suggests that the SOS induction is an effect of increased replicative stress. Our results suggest that NAs/NRTIs can contribute to the development of AMR.
Project description:SecDF is a highly conserved accessory protein of the Sec-translocase located in the cytoplasmic membrane. The deletion mutant (delta Bc4405) of Bacillus cereus ATCC14579 shows multiple phenotypic changes, including aberrent cell division starting at transitionary phase. To understand the underlying processes genotypic profiling was carried out at 3h and 4h after inoculation. The morphology of the mutant seems to be more severe if glucose is added to the LB medium, thus all cultures contained 1% glucose.
Project description:Pervasive Transcription originates from spurious promoters present in both orientations within coding sequences. Spurious transcripts are typically untranslated and thus for the most part are terminated by transcription termination factor Rho. They can be studied by impairing Rho activity, which in this study was achieved by depleting cells for Rho co-factor NusG. To this end, strains in which the only chromosomal copy of the nusG gene is transcribed from a promoter that can be repressed by arabinose (ARA) were used. This study was aimed at determining how pervasive transcription affect H-NS mediated gene silencing in Salmonella Pathogenicity Island 1 (SPI-1). In particular, the analysis concentrated on the region surrounding the master regulator gene HilD. The activities of promoters in this region were assessed performing semiquantitative 5’RACE-Seq. RNA extracted from cells exposed to the indicated treatments was reverse-transcribed with gene-specific primers in the presence of a template-switching oligonucleotide (TSO). The latter carries three 3’-terminal riboguanosines that hybridize to the polycytosine overhang (typically 3 or 4 Cs) added by the RT enzyme when it reaches the 5’ end of the RNA.This allows the RT enzyme to switch template and polymerise the TSO. Subsequent PCR amplification, with a primer complementary to the TSO and another annealing inside the reverse transcribed region, followed by sequence analysis, allows identifying the 5’ end of the RNA as the sequence lying immediately adjacent to the poly-G track. Performing the PCR step semiquantitatively provides information as to relative RNA levels. Here, the technique was adapted to high throughput sequencing by using PCR primers carrying Illumina adapters at their 5’ ends. Three strains were used, all three carrying the ARA-repressible nusG allele, MA12996, MA14302 and MA14358. This last strain carries a deleted version of SPI-1 with the Anhydrotetracycline (AHTc)-inducible Ptet promoter positioned at edge of the H-NS patch covering hilD, which made it possible to study the effects of NusG depletion in the presence or absence of overlapping transcription.
Project description:Whole-genome transcriptional analysis has been performed to define the P. putida RoxS/RoxR regulon.<br>Transcriptome profiles of P. putida roxS cells harvested in stationary growth phase were compared with those of the wild-type strain.<br>
Project description:A long-term-survival (LTS) phase in Listeria monocytogenes was recently discovered. Cells in this phase are coccoid in shape, survive for at least 30 d without any decrease in viable cell numbers, and are very resistant to heat and high pressure. However, how cells of L. monocytogenes transition to this long-term-survival phase is little understood. Therefore, a whole-genome expression analysis was conducted to study the transcription profile of L. monocytogenes as it enters the LTS phase. Transcription profiles at log, stationary and death phases were analyzed since differential gene expressions at these phases may contribute to the eventual transition to the LTS phase. Specifically, cells of L. monocytogenes F2365 at log, stationary, death and LTS phases were obtained by incubating cultures in TSBYE at 35°C for 13 h, 17 h, 24 h and 168-336 h, respectively. Also, to study cells transitioning from the LTS phase back to the log phase, 1 ml of the LTS-phase culture at 336 h was reinoculated into 100 ml of fresh TSBYE with incubation at 35°C for 8 h. Total RNAs of all samples were extracted, reverse transcribed into cDNAs and then hybridized to the L. monocytogenes expression microarray (Roche NimbleGen). During the transition from log phase to stationary phase, differential changes in gene expression involved genes associated with cell envelope, cell division, stress response, energy metabolism, protein synthesis and material transport. During the transition from stationary to death phase, differential changes were observed in genes related to cell envelope, detoxification, pathogenesis, energy metabolism, protein synthesis and material transport. When cultures transitioned from death phase to 168-h LTS phase, significant downregulation of genes associated with amino acid and protein biosynthesis, as well as stress responses, were observed (P < 0.05), while multiple genes related to cell envelope, energy production and material transportation were significantly upregulated (P < 0.05). High similarity of transcription profiles (r = 0.93) within LTS phase was observed when comparing transcriptomes at 168 h and 336 h. RNA quality measurement revealed a high level of degradation of ribosomal RNA during the LTS phase. The transcription profile at 8-h (log-phase) after re-inoculation of LTS cells also resembled that at 13 h (r = 0.94). We hypothesize that the upregulation of some compatible solute transporters during the LTS phase may result in accumulation of these solutes, which may lower intracellular water activity and thus enhance resistance of L. monocytogenes to heat and high pressure. Dormancy may be induced at the LTS phase which is suggested by the downregulation of genes associated with transcription and translation. Once fresh nutrients are provided, LTS cells may quickly exit dormancy and become metabolically active as they transition to the log phase. Based on the annotated genome of L. monocytogenes F2365 (GenBank accession# NC_002973) (Donaldson et al., 2009), a gene expression microarray was designed to target 2821 protein-coding genes (including putative protein-coding genes). Each of the 2821 genes was targeted by 12 unique 60-mer oligonucleotide probes. Each unique probe was printed in duplicates on the microarray. The microarrays were in a format of 4 × 72 k (4 microarrays per slide, with each microarray containing 72,000 probes) and provided by Roche NimbleGen (Roche NimbleGen, WI).