pBaSysBioII: an integrative plasmid generating gfp transcriptional fusions for high-throughput analysis of gene expression in Bacillus subtilis

Botella, Eric; Fogg, Mark J.; Jules, Matthieu; Piersma, Sjouke; Doherty, Geoff; Hansen, Annette G.; Denham, Emma L.; Chat, Ludovic Le; Veiga, Patrick; Bailey, Kirra; Lewis, Peter J.; Dijl, Jan Maarten van; Aymerich, Stéphane; Wilkinson, Anthony J.; Devine, Kevin M.

doi:10.1099/mic.0.035758-0

Cited by 55 publications

(71 citation statements)

References 23 publications

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“…Strain LSB434 was created by amplifying a 400-bp DNA fragment containing the promoter region of S1052 using the primer pair PpipA fwd/PpipA rev and cloning it into plasmid pGPFamy (28) using the ligation-independent cloning (LIC) procedure described by Botella et al (29). The resulting pLIS065 plasmid was linearized using PstI and transformed into B. subtilis 168.…”

Section: Methodsmentioning

confidence: 99%

Genome-Wide Analysis of Phosphorylated PhoP Binding to Chromosomal DNA Reveals Several Novel Features of the PhoPR-Mediated Phosphate Limitation Response in Bacillus subtilis

et al. 2015

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The PhoPR two-component signal transduction system controls one of three responses activated by Bacillus subtilis to adapt to phosphate-limiting conditions (PHO response). The response involves the production of enzymes and transporters that scavenge for phosphate in the environment and assimilate it into the cell. However, in B. subtilis and some other Firmicutes bacteria, cell wall metabolism is also part of the PHO response due to the high phosphate content of the teichoic acids attached either to peptidoglycan (wall teichoic acid) or to the cytoplasmic membrane (lipoteichoic acid). Prompted by our observation that the phosphorylated WalR (WalRϳP) response regulator binds to more chromosomal loci than are revealed by transcriptome analysis, we established the PhoPϳP bindome in phosphate-limited cells. Here, we show that PhoPϳP binds to the chromosome at 25 loci: 12 are within the promoters of previously identified PhoPR regulon genes, while 13 are newly identified. We extend the role of PhoPR in cell wall metabolism showing that PhoPϳP binds to the promoters of four cell wall-associated operons (ggaAB, yqgS, wapA, and dacA), although none show PhoPR-dependent expression under the conditions of this study. We also show that positive autoregulation of phoPR expression and full induction of the PHO response upon phosphate limitation require PhoPϳP binding to the 3= end of the phoPR operon. IMPORTANCEThe PhoPR two-component system controls one of three responses mounted by B. subtilis to adapt to phosphate limitation (PHO response). Here, establishment of the phosphorylated PhoP (PhoPϳP) bindome enhances our understanding of the PHO response in two important ways. First, PhoPR plays a more extensive role in adaptation to phosphate-limiting conditions than was deduced from transcriptome analyses. Among 13 newly identified binding sites, 4 are cell wall associated (ggaAB, yqgS, wapA, and dacA), revealing that PhoPR has an extended involvement in cell wall metabolism. Second, amplification of the PHO response must occur by a novel mechanism since positive autoregulation of phoPR expression requires PhoPϳP binding to the 3= end of the operon. The PhoPR two-component signal transduction system (TCS) controls one of three responses activated by Bacillus subtilis to adapt to phosphate limiting conditions (PHO response). PhoPR is well characterized, with orthologous TCS being widely distributed among Gram-negative (e.g., PhoBR in Escherichia coli) and Gram-positive (e.g., PhoPR in B. subtilis and Staphylococcus aureus) bacteria. Although the PHO response is activated upon phosphate depletion in both E. coli and B. subtilis, the PhoR kinase senses a completely different signal in each bacterium. In E. coli the signal emanates from phosphate transport mediated by the PstSCAB 2 phosphate transporter and the PhoU chaperone-like protein (1). It is proposed that formation of a Pst-SCAB 2 /PhoU/PhoR complex under conditions of phosphate excess (Ͼ4 M) inhibits PhoR autokinase activity, thereby keeping the PHO response of...

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Section: Methodsmentioning

confidence: 99%

Genome-Wide Analysis of Phosphorylated PhoP Binding to Chromosomal DNA Reveals Several Novel Features of the PhoPR-Mediated Phosphate Limitation Response in Bacillus subtilis

et al. 2015

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“…Primers designed to specifically amplify each promoter region had ligation-independent cloning (LIC) tails (59-CCGCGGGC-TTTCCCAGC-39 forward primer and 59-GTTCCTCCTTCCCACC-39 reverse primer) added for high throughput cloning. Promoter fragments were generated from genomic B. subtilis 168 DNA by PCR using the primers listed in Supplementary Table S3 and were cloned into pBaSysBioII as described by Botella et al (2010). The resulting plasmids (see Supplementary Table S4) were sequenced using primer GFP1 and then transformed into BSB168 and AH024 (DphoPR) recipient strains.…”

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confidence: 99%

“…Growth and background-corrected fluorescence profiles of all cultures were aligned according to the T 0 , calculated as the time at which the culture stops growing exponentially using the Heat Map Generator web software. Promoter activities (P act ) were calculated by taking the derivative of the fluorescence divided by the OD 600 [(DGFP/DTime)/OD 600 ] at each time point and smoothed by averaging the activity values of three consecutive time points as described in detail by Botella et al (2010). At least three independent isolates of each fusion-containing strain (three biological replicates) were analysed and at least two P act determinations of each isolate were performed on two different Biotek microplate readers by different operatives under all conditions (at least two technical replicates).…”

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Cell envelope gene expression in phosphate-limited Bacillus subtilis cells

et al. 2011

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The high phosphate content of Bacillus subtilis cell walls dictates that cell wall metabolism is an important feature of the PhoPR-mediated phosphate limitation response. Here we report the expression profiles of cell-envelope-associated and PhoPR regulon genes, determined by live cell array and transcriptome analysis, in exponentially growing and phosphate-limited B. subtilis cells. Control by the WalRK two-component system confers a unique expression profile and high level of promoter activity on the genes of its regulon with yocH and cwlO expression differing both qualitatively and quantitatively from all other autolysin-encoding genes examined. The activity of the PhoPR two-component system is restricted to the phosphate-limited state, being rapidly induced in response to the cognate stimulus, and can be sustained for an extended phosphate limitation period. Constituent promoters of the PhoPR regulon show heterogeneous induction profiles and very high promoter activities. Phosphate-limited cells also show elevated expression of the actin-like protein MreBH and reduced expression of the WapA cell wall protein and WprA cell wall protease indicating that cell wall metabolism in this state is distinct from that of exponentially growing and stationary-phase cells. The PhoPR response is very rapidly deactivated upon removal of the phosphate limitation stimulus with concomitant increased expression of cell wall metabolic genes. Moreover expression of genes encoding enzymes involved in sulphur metabolism is significantly altered in the phosphate-limited state with distinct perturbations being observed in wild-type 168 and AH024 (DphoPR) cells.

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“…Green fluorescent protein (GFP) accumulation of the GFPmut3 stable variant was obtained as previously described for B. subtilis (5). Polynomial and exponential functions were used to fit the experimental data sets of GFP and biomass, respectively, and to deduce the rates of biomass and GFP production along the growth.…”

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Malate-Mediated Carbon Catabolite Repression in Bacillus subtilis Involves the HPrK/CcpA Pathway

et al. 2011

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Most organisms can choose their preferred carbon source from a mixture of nutrients. This process is called carbon catabolite repression. The Gram-positive bacterium Bacillus subtilis uses glucose as the preferred source of carbon and energy. Glucose-mediated catabolite repression is caused by binding of the CcpA transcription factor to the promoter regions of catabolic operons. CcpA binds DNA upon interaction with its cofactors HPr(Ser-P) and Crh(Ser-P). The formation of the cofactors is catalyzed by the metabolite-activated HPr kinase/phosphorylase. Recently, it has been shown that malate is a second preferred carbon source for B. subtilis that also causes catabolite repression. In this work, we addressed the mechanism by which malate causes catabolite repression. Genetic analyses revealed that malate-dependent catabolite repression requires CcpA and its cofactors. Moreover, we demonstrate that HPr(Ser-P) is present in malate-grown cells and that CcpA and HPr interact in vivo in the presence of glucose or malate but not in the absence of a repressing carbon source. The formation of the cofactor HPr(Ser-P) could be attributed to the concentrations of ATP and fructose 1,6-bisphosphate in cells growing with malate. Both metabolites are available at concentrations that are sufficient to stimulate HPr kinase activity. The adaptation of cells to environmental changes requires dynamic metabolic and regulatory adjustments. The repression strength of target promoters was similar to that observed in steady-state growth conditions, although it took somewhat longer to reach the second steady-state of expression when cells were shifted to malate.

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pBaSysBioII: an integrative plasmid generating gfp transcriptional fusions for high-throughput analysis of gene expression in Bacillus subtilis

Cited by 55 publications

References 23 publications

Genome-Wide Analysis of Phosphorylated PhoP Binding to Chromosomal DNA Reveals Several Novel Features of the PhoPR-Mediated Phosphate Limitation Response in Bacillus subtilis

Genome-Wide Analysis of Phosphorylated PhoP Binding to Chromosomal DNA Reveals Several Novel Features of the PhoPR-Mediated Phosphate Limitation Response in Bacillus subtilis

Cell envelope gene expression in phosphate-limited Bacillus subtilis cells

Malate-Mediated Carbon Catabolite Repression in Bacillus subtilis Involves the HPrK/CcpA Pathway

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