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The entomopathogen Bacillus thuringiensis produces dense biofilms under various conditions. Here, we report that the transition phase regulators Spo0A, AbrB and SinR control biofilm formation and swimming motility in B. thuringiensis, just as they control biofilm formation and swarming motility in the closely related saprophyte species B. subtilis. However, microarray analysis indicated that in B. thuringiensis, in contrast to B. subtilis, SinR does not control an eps operon involved in exopolysaccharides production, but regulates genes involved in the biosynthesis of the lipopeptide kurstakin. This lipopeptide is required for biofilm formation and was previously shown to be important for survival in the host cadaver (necrotrophism). Microarray analysis also revealed that the SinR regulon contains genes coding for the Hbl enterotoxin. Transcriptional fusion assays, Western blots and hemolysis assays confirmed that SinR controls Hbl expression, together with PlcR, the main virulence regulator in B. thuringiensis. We show that Hbl is expressed in a sustained way in a small subpopulation of the biofilm, whereas almost all the planktonic population transiently expresses Hbl. The gene coding for SinI, an antagonist of SinR, is expressed in the same biofilm subpopulation as hbl, suggesting that hbl transcription heterogeneity is SinI-dependent. B. thuringiensis and B. cereus are enteric bacteria which possibly form biofilms lining the host intestinal epithelium. Toxins produced in biofilms could therefore be delivered directly to the target tissue.

One of the key ways in which microbes are thought to regulate their metabolism is by modulating the availability of enzymes through transcriptional regulation. However, the limited success of efforts to manipulate metabolic fluxes by rewiring the transcriptional network has cast doubt on the idea that transcript abundance controls metabolic fluxes. In this study, we investigate control of metabolic flux in the model bacterium Bacillus subtilis by quantifying fluxes, transcripts, and metabolites in eight metabolic states enforced by different environmental conditions. We find that most enzymes whose flux switches between on and off states, such as those involved in substrate uptake, exhibit large corresponding transcriptional changes. However, for the majority of enzymes in central metabolism, enzyme concentrations were insufficient to explain the observed fluxes--only for a number of reactions in the tricarboxylic acid cycle were enzyme changes approximately proportional to flux changes. Surprisingly, substrate changes revealed by metabolomics were also insufficient to explain observed fluxes, leaving a large role for allosteric regulation and enzyme modification in the control of metabolic fluxes.

Bacteria adapt to environmental stimuli by adjusting their transcriptomes in a complex manner, the full potential of which has yet to be established for any individual bacterial species. Here, we report the transcriptomes of Bacillus subtilis exposed to a wide range of environmental and nutritional conditions that the organism might encounter in nature. We comprehensively mapped transcription units (TUs) and grouped 2935 promoters into regulons controlled by various RNA polymerase sigma factors, accounting for ~66% of the observed variance in transcriptional activity. This global classification of promoters and detailed description of TUs revealed that a large proportion of the detected antisense RNAs arose from potentially spurious transcription initiation by alternative sigma factors and from imperfect control of transcription termination.

Assessing gene expression noise in order to obtain mechanistic insights requires accurate quantification of gene expression on many individual cells over a large dynamic range. We used a unique method based on 2-photon fluorescence fluctuation microscopy to measure directly, at the single cell level and with single-molecule sensitivity, the absolute concentration of fluorescent proteins produced from the two Bacillus subtilis promoters that control the switch between glycolysis and gluconeogenesis. We quantified cell-to-cell variations in GFP concentrations in reporter strains grown on glucose or malate, including very weakly transcribed genes under strong catabolite repression. Results revealed strong transcriptional bursting, particularly for the glycolytic promoter. Noise pattern parameters of the two antagonistic promoters controlling the nutrient switch were differentially affected on glycolytic and gluconeogenic carbon sources, discriminating between the different mechanisms that control their activity. Our stochastic model for the transcription events reproduced the observed noise patterns and identified the critical parameters responsible for the differences in expression profiles of the promoters. The model also resolved apparent contradictions between in vitro operator affinity and in vivo repressor activity at these promoters. Finally, our results demonstrate that negative feedback is not noise-reducing in the case of strong transcriptional bursting.

Quantification of promoter activity or protein expression in gene regulatory networks is generally achieved via measurement of fluorescent protein (FP) intensity, which is related to the true FP concentration by an unknown scaling factor, thereby limiting analysis and interpretation. Here, using approaches originally developed for eukaryotic cells, we show that two-photon (2p) fluorescence fluctuation microscopy, specifically scanning number and brightness (sN&B) analysis, can be applied to determine the absolute concentrations of diffusing FPs in live bacterial cells. First, we demonstrate the validity of the approach, despite the small size of the bacteria, using the central pixels and spatial averaging. We established the lower detection limit at or below 75 nM (~3 molecules of FP/vol(ex)) and the upper detection limit at approximately 10 μM, which can be extended using intensity measurements. We found that the uncertainty inherent in our measurements (<5%) was smaller than the high cell-cell variations observed for stochastic leakage from FP fusions of the lac promoter in the repressed state or the 10 to 25% variation observed on induction. This demonstrates that a reliable and absolute measure of transcriptional noise can be made using our approach, which should make it particularly appropriate for the investigation of stochasticity in gene expression networks., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The transcriptional regulator CcpN of Bacillus subtilis has been recently characterized as a repressor of two gluconeogenic genes, gapB and pckA, and of a small noncoding regulatory RNA, sr1, involved in arginine catabolism. Deletion of ccpN impairs growth on glucose and strongly alters the distribution of intracellular fluxes, rerouting the main glucose catabolism from glycolysis to the pentose phosphate (PP) pathway. Using transcriptome analysis, we show that during growth on glucose, gapB and pckA are the only protein-coding genes directly repressed by CcpN. By quantifying intracellular fluxes in deletion mutants, we demonstrate that derepression of pckA under glycolytic condition causes the growth defect observed in the ccpN mutant due to extensive futile cycling through the pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and pyruvate kinase. Beyond ATP dissipation via this cycle, PckA activity causes a drain on tricarboxylic acid cycle intermediates, which we show to be the main reason for the reduced growth of a ccpN mutant. The high flux through the PP pathway in the ccpN mutant is modulated by the flux through the alternative glyceraldehyde-3-phosphate dehydrogenases, GapA and GapB. Strongly increased concentrations of intermediates in upper glycolysis indicate that GapB overexpression causes a metabolic jamming of this pathway and, consequently, increases the relative flux through the PP pathway. In contrast, derepression of sr1, the third known target of CcpN, plays only a marginal role in ccpN mutant phenotypes.

CggR belongs to the SorC family of bacterial transcriptional regulators which control the expression of genes and operons involved in carbohydrate catabolism. CggR was first identified in Bacillus subtilis where it represses the gapA operon encoding the five enzymes that catalyze the central part of glycolysis. Here we present a structure/function study demonstrating that the C-terminal region of CggR regulates the DNA binding activity of this repressor in response to binding of a phosphorylated sugar. Molecular modeling of CggR revealed a winged-helix DNA-binding motif followed by a C-terminal domain presenting weak but significant homology with glucosamine-6-phosphate deaminases from the NagB family. In silico ligand screening suggested that the CggR C-terminal domain would bind preferentially bi-phosphorylated compounds, in agreement with previous studies that proposed fructuose-1,6-biphosphate (FBP) as the inducer metabolite. In vitro, FBP was the only sugar compound capable of interfering with CggR cooperative binding to DNA. FBP was also found to protect CggR against trypsin degradation at two arginine residues predicted to reside in a mobile loop forming the active site lid of the NagB enzymes. Replacement of residues predicted to interact with FBP led to mutant CggR with altered repressor activity in vivo but retaining their structural integrity and DNA binding activity in vitro. Interestingly, some of the mutant repressors responded with different specificity towards mono- and di-phospho-fructosides. Based on these results, we propose that the activity of the CggR-like repressors is controlled by a phospho-sugar binding (PSB) domain presenting structural and functional homology with NagB enzymes., ((c) 2008 Wiley-Liss, Inc.)

A transcriptome comparison of a wild-type Bacillus subtilis strain growing under glycolytic or gluconeogenic conditions was performed. In particular, it revealed that the ywkA gene, one of the four paralogues putatively encoding a malic enzyme, was more transcribed during gluconeogenesis. Using a lacZ reporter fusion to the ywkA promoter, it was shown that ywkA was specifically induced by external malate and not subject to glucose catabolite repression. Northern analysis confirmed this expression pattern and demonstrated that ywkA is cotranscribed with the downstream ywkB gene. The ywkA gene product was purified and biochemical studies demonstrated its malic enzyme activity, which was 10-fold higher with NAD than with NADP (kcat/Km 102 and 10 s(-1) mM(-1), respectively). However, physiological tests with single and multiple mutant strains affected in ywkA and/or in ywkA paralogues showed that ywkA does not contribute to efficient utilization of malate for growth. Transposon mutagenesis allowed the identification of the uncharacterized YufL/YufM two-component system as being responsible for the control of ywkA expression. Genetic analysis and in vitro studies with purified YufM protein showed that YufM binds just upstream of ywkA promoter and activates ywkA transcription in response to the presence of malate in the extracellular medium, transmitted by YufL. ywkA and yufL/yufM could thus be renamed maeA for malic enzyme and malK/malR for malate kinase sensor/malate response regulator, respectively.

The nucleotide sequence of 3 kb of Bacillus subtilis chromosomal DNA, including sacS, reveals that the regulatory locus for levansucrase synthesis consists of two genes, sacX and sacY. The sacX gene product is remarkably similar to sucrose-specific enzyme II of the B. subtilis phosphoenolpyruvate-dependent phosphotransferase system. The product of sacY is similar, both in amino acid sequence and most probably in its function, to BglG, an Escherichia coli transcriptional antitermination factor.

The target of the induction by sucrose of the levansucrase gene is a transcription terminator (sacRt) located upstream from the coding sequence, sacB. The two-gene locus sacX-sacY (formerly sacS) and the ptsI gene were previously shown to be involved in this induction. ptsI encodes enzyme I of the phosphoenolpyruvate-dependent phosphotransferase system. SacX is strongly homologous to sucrose-specific phosphotransferase system-dependent permeases. SacY is a positive regulator of sacB. Here we show that SacY is probably an antiterminator interacting directly with sacRt, since in Escherichia coli the presence of the sacY gene stimulates the expression of a reporter gene fused downstream from sacRt. Missense mutations affecting sacY were sequenced, and the sacB regulation was studied in isogenic strains carrying these mutations or in vitro-generated mutations affecting sacX, sacY, or ptsI. The phenotype of double mutants suggests a model in which SacX might be a sucrose sensor that would be phosphorylated by the phosphotransferase system and, in this state, could inhibit the SacY antiterminator. Exogenous sucrose, or a mutation inactivating the phosphotransferase system, would dephosphorylate SacX and allow antitermination at sacRt.