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.
The Bacillus subtilis transcriptional regulator Fnr is an integral part of the regulatory cascade required for the adaptation of the bacterium to low oxygen tension. The B. subtilis Fnr regulon was defined via transcriptomic analysis in combination with bioinformatic-based binding site prediction. Four distinct groups of Fnr-dependent genes were observed. Group 1 genes (narKfnr, narGHJI, and arfM) are generally induced by Fnr under anaerobic conditions. All corresponding promoters contain an essential Fnr-binding site centered ؊41.5/؊40. The gram-positive model organism Bacillus subtilis adapts to an anaerobic environment by changing its metabolic activity (26). Under anaerobic conditions, B. subtilis performs a mixed-acid fermentation with lactate, acetate, and acetoin as the major products (7, 24). In the presence of nitrate, B. subtilis performs the respiratory process of ammonification (8,13,14,22).The regulatory network underlying anaerobic adaptation has been extensively studied during the last decade. A regulatory cascade describing the coordinated regulation of genes involved in anaerobiosis was established (7). One major regulatory switch in the adaptation to anaerobiosis is the two-component system ResDE (32,35). While the mechanism of signal perception by ResDE is still unknown, the downstream regulatory network is elucidated to a significant depth. Activated ResD binds to promoter regions of nasDE, encoding the nitrite reductase, the flavohemoglobin gene hmp, and the gene encoding the redox regulator Fnr (23, 25). Fnr in turn is responsible for the induction of the narGHJI operon and narK, encoding the respiratory nitrate reductase and a potential nitrite extrusion protein, respectively (6, 24). Mutation of fnr strongly affects anaerobic growth of B. subtilis on nitrate (6, 24). Furthermore, Fnr activates the expression of the arfM gene encoding an anaerobic respiration and fermentation modulator protein by direct interaction with the arfM promoter region (16). The promoter regions of all three Fnr-regulated genes carry the highly conserved potential B. subtilis Fnr-binding site (TGTGA-N 6 -TCACA) centered 41.5/40.5 bp from the transcriptional start point. Complementation experiments using an Escherichia coli crp mutant revealed that the DNA-binding domain of Fnr of B. subtilis is similar to that of Crp from E. coli, the well-studied cyclic AMP receptor protein (6). In B. subtilis, additional potential Fnr-binding sites were found in the promoter regions of a second potential nitrite transporter gene, ywcJ, as well as the fermentation operons ldh-lctP and alsSD (6,7,34). The latter operons encode lactate dehydrogenase, lactate permease, and acetolactate synthase and acetolactate decarboxylase, respectively. Transcription of alsSD and ldh-lctP was found to be anaerobically induced and repressed by the presence of nitrate (7). Nitrate repression was related to nitrate reductase activity (7).Global transcriptional profiling was used to analyze changes in the mRNA population after adaptation to anaerobic...
SummaryThe oxygen regulator Fnr is part of the regulatory cascade in Bacillus subtilis for the adaptation to anaerobic growth conditions. In vivo complementation experiments revealed the essential role of only three cysteine residues (C227, C230, C235) at the Cterminus of B. subtilis Fnr for the transcriptional activation of the nitrate reductase operon ( narGHJI ) and nitrite extrusion protein gene ( narK ) promoters. UV/ VIS, electron paramagnetic spin resonance (EPR) and Mössbauer spectroscopy experiments in combination with iron and sulphide content determinations using anaerobically purified recombinant B. subtilis Fnr identified the role of these three cysteine residues in the formation of one
Bacillus subtilis forms acetoin under anaerobic fermentative growth conditions and as a product of the aerobic carbon overflow metabolism. Acetoin formation from pyruvate requires ␣-acetolactate synthase and acetolactate decarboxylase, both encoded by the alsSD operon. The alsR gene, encoding the LysR-type transcriptional regulator AlsR, was found to be essential for the in vivo expression of alsSD in response to anaerobic acetate accumulation, the addition of acetate, low pH, and the aerobic stationary phase. The expressions of the alsSD operon and the alsR regulatory gene were independent of other regulators of the anaerobic regulatory network, including ResDE, Fnr, and ArfM. A negative autoregulation of alsR was observed. In vitro transcription from the alsSD promoter using purified B. subtilis RNA polymerase required AlsR. DNA binding studies with purified recombinant AlsR in combination with promoter mutagenesis experiments identified a 19-bp high-affinity palindromic binding site (TA AT-N 11 -ATTA) at positions ؊76 to ؊58 (regulatory binding site [RBS]) and a low-affinity site (AT-N 11 -AT) at positions ؊41 to ؊27 (activator binding site [ABS]) upstream of the transcriptional start site of alsSD. The RBS and ABS were found to be essential for in vivo alsSD transcription. AlsR binding to both sites induced the formation of higher-order, transcription-competent complexes. The AlsR protein carrying the S100A substitution at the potential coinducer binding site still bound to the RBS and ABS. However, AlsR(S100A) failed to form the higher-order complex and to initiate in vivo and in vitro transcription. A model for AlsR promoter binding and transcriptional activation was deduced.
During infection of the cystic fibrosis (CF) lung, Pseudomonas aeruginosa microcolonies are embedded in the anaerobic CF mucus. This anaerobic environment seems to contribute to the formation of more robust P. aeruginosa biofilms and to an increased antibiotic tolerance and therefore promotes persistent infection. This study characterizes the P. aeruginosa protein PA4352, which is important for survival under anaerobic energy stress conditions. PA4352 belongs to the universal stress protein (Usp) superfamily and harbors two Usp domains in tandem. In Escherichia coli, Usp-type stress proteins are involved in survival during aerobic growth arrest and under various other stresses. A P. aeruginosa PA4352 knockout mutant was tested for survival under several stress conditions. We found a decrease in viability of this mutant compared to the P. aeruginosa wild type during anaerobic energy starvation caused by the missing electron acceptors oxygen and nitrate. Consistent with this phenotype under anaerobic conditions, the PA4352 knockout mutant was also highly sensitive to carbonyl cyanide m-chlorophenylhydrazone, the chemical uncoupler of the electron transport chain. Primer extension experiments identified two promoters upstream of the PA4352 gene. One promoter is activated in response to oxygen limitation by the oxygensensing regulatory protein Anr. The center of a putative Anr binding site was identified 41.5 bp upstream of the transcriptional start site. The second promoter is active only in the stationary phase, however, independently of RpoS, RelA, or quorum sensing. This is the second P. aeruginosa Usp-type stress protein that we have identified as important for survival under anaerobic conditions, which resembles the environment during persistent infection.
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