Genetic evidence that multiple proteases are involved in modulation of heat-induced activation of the sigma factor SigI in Bacillus subtilis.

The Bacillus subtilis sigI-rsgI operon encodes the heat-inducible sigma factor SigI and its cognate anti-sigma factor RsgI. The heat-activated SigI positively regulates expression of sigI itself and genes involved in cell wall homeostasis and heat resistance. It remains unknown which protease(s) may contribute to degradation of RsgI and heat-induced activation of SigI. In this study, we found that transcription of sigI from its σI-dependent promoter under heat stress was downregulated in a strain lacking the heat-inducible sigma factor SigB. Deletion of protease-relevant clpP, clpC or rasP severely impaired sigI expression during heat stress, whereas deletion of clpE partially impaired sigI expression. Complementation of mutations with corresponding intact genes restored sigI expression. In a null mutant of rsgI, SigI was activated and sigI expression was strongly upregulated during normal growth and under heat stress. In this rsgI mutant, further inactivation of rasP or clpE did not affect sigI expression, whereas further inactivation of clpP or clpC severely or partially impaired sigI expression. Spx negatively influenced sigI expression during heat stress. Possible implications are discussed. Given that clpC, clpP and spx are directly regulated by SigB, SigB appears to control sigI expression under heat stress via ClpC, ClpP and Spx.

A membrane transporter required for 3-hydroxybutyrate uptake during the early sporulation stage in Bacillus subtilis.

Exogenous 3-hydroxybutyrate can be utilized by a variety of soil bacteria as a carbon and energy source. However, the membrane transporter responsible for 3-hydroxybutyrate uptake remains unidentified. The Bacillus subtilis strain 168 gene yxjC (herein renamed hbuT) encodes a putative gluconate transporter GntT-type membrane transporter with a previously unknown function. hbuT is organized within the same operon with genes that are used for metabolism of 3-hydroxybutyrate. Here we report that a null mutation of hbuT reduced uptake of 3-hydroxybutyrate by B. subtilis cells grown in nutrient sporulation medium. The SigE-controlled HbuT transporter apparently plays a major role in the uptake of 3-hydroxybutyrate. Uptake of 3-hydroxybutyrate by the HbuT transporter occurred in a specific manner at the early sporulation stage. SigE-controlled hbuT expression and 3-hydroxybutyrate uptake were also subject to CcpA-mediated glucose repression. hbuT expression was not induced by exogenous 3-hydroxybutyrate and B. subtilis cells could not utilize 3-hydroxybutyrate as a sole carbon source for growth. HbuT homologs are present in a wide variety of Gram-positive Bacillus species, some Gram-negative Acinetobacter species and a small group of other bacteria. This is the first tentative identification of a membrane transporter responsible for the uptake of 3-hydroxybutyrate in bacteria.

Crystallization and preliminary crystallographic analysis of poly(3-hydroxybutyrate) depolymerase from Bacillus thuringiensis.

Poly[(R)-3-hydroxybutyrate] (PHB) is a microbial biopolymer that has been commercialized as biodegradable plastics. The key enzyme for the degradation is PHB depolymerase (PhaZ). A new intracellular PhaZ from Bacillus thuringiensis (BtPhaZ) has been screened for potential applications in polymer biodegradation. Recombinant BtPhaZ was crystallized using 25% polyethylene glycol 3350, 0.2 M ammonium acetate, 0.1 M bis-tris pH 6.5 at 288 K. The crystals belonged to space group P1, with unit-cell parameters a = 42.97, b = 83.23, c = 85.50 Å, α = 73.45, ß = 82.83, γ = 83.49°. An X-ray diffraction data set was collected to 1.42 Šresolution with an Rmerge of 6.4%. Unexpectedly, a molecular-replacement solution was obtained using the crystal structure of Streptomyces lividans chloroperoxidase as a template, which shares 24% sequence identity to BtPhaZ. This is the first crystal structure of an intracellular poly(3-hydroxybutyrate) depolymerase.

Transcriptional regulation of the l-lactate permease gene lutP by the LutR repressor of Bacillus subtilis RO-NN-1.

The Bacillus subtilis lutABC operon encodes three iron-sulfur-containing proteins required for l-lactate utilization and involved in biofilm formation. The transcriptional regulator LutR of the GntR family negatively controls lutABC expression. The lutP gene, which is situated immediately upstream of lutR, encodes an l-lactate permease. Here, we show that lutP expression can be strongly induced by l-lactate and is subject to partial catabolite repression by glucose. Disruption of the lutR gene led to a strong derepression of lutP and no further induction by l-lactate, suggesting that the LutR repressor can also negatively control lutP expression. Electrophoretic mobility shift assay revealed a LutR-binding site located downstream of the promoter of lutA or lutP and containing a consensus inverted repeat sequence 5'-TCATC-N1-GATGA-3'. Reporter gene analysis showed that deletion of each LutR-binding site caused a strong derepression of lutA or lutP. These results indicated that these two LutR-binding sites can function as operators in vivo. Moreover, deletion analysis identified a DNA segment upstream of the lutP promoter to be important for lutP expression. In contrast to the truncated LutR of laboratory strains 168 and PY79, the full-length LutR of the undomesticated strain RO-NN-1, and probably many other B. subtilis strains, can directly and negatively regulate lutP transcription. The absence or presence of the N-terminal 21 aa of the full-length LutR, which encompass a small part of the predicted winged helix-turn-helix DNA-binding motif, may probably alter the DNA-binding specificity or affinity of LutR.

Two enzymes, TilS and HprT, can form a complex to function as a transcriptional activator for the cell division protease gene ftsH in Bacillus subtilis.

The FtsH protein is an ATP-dependent cytoplasmic membrane protease involved in the control of membrane protein quality, cell division and heat shock response in Bacillus subtilis and many other bacteria. TilS, the tRNA(Ile2) lysidine synthetase, is a tRNA-binding protein that can modify pre-tRNA(Ile2). HprT, the hypoxanthine-guanine phosphoribosyltransferase, is implicated in purine salvage. Both tilS and hprT are essential for cell viability of B. subtilis. In this report, by co-purification experiments and gel filtration analyses, we show that there is complex formation between co-expressed TilS and HprT. Electrophoretic mobility shift assays and in vitro transcription analyses demonstrated that the TilS/HprT complex functions as a specific DNA-binding protein that can stimulate ftsH transcription in vitro. Two regions located upstream of the ftsH promoter have been identified as the TilS/HprT-binding sites and shown to be required for TilS/HprT-dependent ftsH transcription in vitro and in vivo. Results from gel supershift assays support the notion that the TilS/HprT complex likely employs its distinct segments for interaction with these two distinct TilS/HprT-binding sites, respectively. In conclusion, we present the first evidence that bi-functional TilS and HprT can form a complex to function as a transcriptional activator to stimulate ftsH transcription.

The heat-inducible essential response regulator WalR positively regulates transcription of sigI, mreBH and lytE in Bacillus subtilis under heat stress.

The actin homolog MreBH governs cell morphogenesis of Bacillus subtilis through localization of the cell wall hydrolase LytE. The alternative sigma factor SigI of B. subtilis coordinately regulates transcription of mreBH and lytE. Transcription of sigI, mreBH and lytE is heat-inducible. The essential response regulator WalR (YycF) plays a key role in coordinating cell wall metabolism with cell proliferation. We now demonstrate that mreBH is a new member of the WalR regulon. We also found that WalR can positively and directly regulate sigI transcription under heat stress through a binding site located upstream of the σ(I) promoter of sigI. In addition, we found that a WalR binding site located upstream of the SigI binding site in the regulatory region of lytE is important for lytE expression under heat stress. Moreover, we found that walR is a new member of the heat shock stimulon of B. subtilis. WalR appears to coordinately and positively regulate transcription of sigI, mreBH and lytE under heat stress. Finally, our work shows for the first time that WalR can stimulate activities of σ(I) promoters under heat stress.

Involvement of OpcR, a GbsR-type transcriptional regulator, in negative regulation of two evolutionarily closely related choline uptake genes in Bacillus subtilis.

The osmoprotectant glycine betaine can be generated intracellularly from conversion of the exogenous precursor choline by enzymes encoded by the gbsAB operon in Bacillus subtilis. Uptake of choline from outside B. subtilis cells is mediated through two evolutionarily closely related ATP-binding cassette transporters, OpuB and OpuC. Expression of the opuB operon and of the opuC operon is known to be osmoinducible. Here, we show that choline exerts a suppressive effect on opuC expression during normal growth and under osmotic stress. In the absence of the choline-responsive repressor GbsR, opuB expression is also suppressed by choline. We also report that a gene (formerly yvbF, now designated opcR) located immediately upstream of the opuC operon negatively regulates transcription of the opuC operon and, in the absence of GbsR, also that of the opuB operon. An inverted repeat (TTGTAAA-N8-TTTACAA) that overlaps with the -35 hexamer of the promoters of both operons has been identified as the OpcR operator. OpcR belongs to the GbsR-type transcriptional regulators. Its orthologues with unknown function are present in some other Bacillus species. Moreover, deletion analyses revealed that a region located further upstream of the promoters of the opuB operon and the opuC operon is critical for expression of both operons during normal growth and under osmotic stress. Osmotic induction of these two operons appears not to be OpcR mediated. OpcR is not a choline-responsive repressor. The possible biological role of OpcR is discussed.

Reexamining transcriptional regulation of the Bacillus subtilis htpX gene and the ykrK gene, encoding a novel type of transcriptional regulator, and redefining the YkrK operator.

HtpX is an integral cytoplasmic membrane metalloprotease well conserved in numerous bacteria. A recent study showed that expression of the Bacillus subtilis htpX gene is under dual negative control by Rok and a novel type of transcriptional regulator, YkrK. Here we report that expression of the B. subtilis htpX gene is strongly heat inducible. Contrary to the previous prediction, ykrK expression has been found to be not subject to autoregulation. We have identified the htpX promoter and the authentic ykrK promoter, which is also distinct from the previously predicted one. We have redefined a conserved inverted repeat sequence to be the YkrK operator, which is somewhat different from the previously proposed one. We provide evidence that YkrK is not a substrate of HtpX and that heat induction of htpX is not YkrK mediated. We have also found that the absence of FtsH or HtpX alone did not impair B. subtilis cell viability on LB agar plates at high temperature, whereas the absence of both FtsH and HtpX caused a severe growth defect under heat stress. This finding supports the notion that FtsH and HtpX may have partially overlapping functions in heat resistance. Finally, we show that htpX expression is subject to transient negative control by sigB under heat stress in a Rok- and YkrK-independent manner. Triple negative control of htpX expression at high temperature by rok, sigB, and ykrK may help cells to prevent uncontrolled and detrimental oversynthesis of the HtpX protease.

Acid-induced type VI secretion system is regulated by ExoR-ChvG/ChvI signaling cascade in Agrobacterium tumefaciens.

The type VI secretion system (T6SS) is a widespread, versatile protein secretion system in pathogenic Proteobacteria. Several T6SSs are tightly regulated by various regulatory systems at multiple levels. However, the signals and/or regulatory mechanisms of many T6SSs remain unexplored. Here, we report on an acid-induced regulatory mechanism activating T6SS in Agrobacterium tumefaciens, a plant pathogenic bacterium causing crown gall disease in a wide range of plants. We monitored the secretion of the T6SS hallmark protein hemolysin-coregulated protein (Hcp) from A. tumefaciens and found that acidity is a T6SS-inducible signal. Expression analysis of the T6SS gene cluster comprising the imp and hcp operons revealed that imp expression and Hcp secretion are barely detected in A. tumefaciens grown in neutral minimal medium but are highly induced with acidic medium. Loss- and gain-of-function analysis revealed that the A. tumefaciens T6SS is positively regulated by a chvG/chvI two-component system and negatively regulated by exoR. Further epistasis analysis revealed that exoR functions upstream of the chvG sensor kinase in regulating T6SS. ChvG protein levels are greatly increased in the exoR deletion mutant and the periplasmic form of overexpressed ExoR is rapidly degraded under acidic conditions. Importantly, ExoR represses ChvG by direct physical interaction, but disruption of the physical interaction allows ChvG to activate T6SS. The phospho-mimic but not wild-type ChvI response regulator can bind to the T6SS promoter region in vitro and activate T6SS with growth in neutral minimal medium. We present the first evidence of T6SS activation by an ExoR-ChvG/ChvI cascade and propose that acidity triggers ExoR degradation, thereby derepressing ChvG/ChvI to activate T6SS in A. tumefaciens.

Qualitative analysis of the fluorophosphonate-based chemical probes using the serine hydrolases from mouse liver and poly-3-hydroxybutyrate depolymerase (PhaZ) from Bacillus thuringiensis.

The serine hydrolase family consists of more than 200 members and is one of the largest enzyme families in the human genome. Although up to 50 % of this family remains unannotated, there are increasing evidences that activities of certain serine hydrolases are associated with diseases like cancer neoplasia, invasiveness, etc. By now, several activity-based chemical probes have been developed and are applied to profile the global activity of serine hydrolases in diverse proteomes. In this study, two fluorophosphonate (FP)-based chemical probes were synthesized. Further examination of their abilities to label and pull down serine hydrolases was conducted. In addition, the poly-3-hydroxybutyrate depolymerase (PhaZ) from Bacillus thuringiensis was demonstrated as an appropriate standard serine hydrolase, which can be applied to measure the labeling ability and pull-down efficiency of FP-based probes. Furthermore, mass spectrometry (MS) was used to identify the serine residue that covalently bonded to the active probes. Finally, these FP-based probes were shown capable of establishing the serine hydrolase profiles in diverse mouse tissues; the serine hydrolases pulled down from mouse liver organ were further identified by MS. In summary, our study provides an adequate method to evaluate the reactivity of FP-based probes targeting serine hydrolases.

AdeR, a PucR-type transcription factor, activates expression of L-alanine dehydrogenase and is required for sporulation of Bacillus subtilis.

The Bacillus subtilis ald gene encodes L-alanine dehydrogenase, which catalyzes the NAD(+)-dependent deamination of L-alanine to pyruvate for the generation of energy and is required for normal sporulation. The transcription of ald is induced by alanine, but the mechanism underlying alanine induction remains unknown. Here we report that a gene (formerly yukF and now designated adeR) located upstream of ald is essential for the basal and alanine-inducible expression of ald. The disruption of the adeR gene caused a sporulation defect, whereas the complementation of an adeR mutation with an intact adeR gene restored the sporulation ability. adeR expression was not subject to autoregulation and alanine induction. Deletion and mutation analyses revealed that an inverted repeat, centered at position -74.5 relative to the transcriptional initiation site of ald, was required for ald expression and also likely served as a ρ-independent transcription terminator. Electrophoretic mobility shift assays showed that purified His-tagged AdeR was a specific DNA-binding protein and that this inverted repeat was required for AdeR binding. AdeR shows no significant amino acid sequence similarity to the known transcriptional activators of ald genes from other bacteria. AdeR appears to be a member of the PucR family of transcriptional regulators. Its orthologs of unknown function are present in some other Bacillus species. Collectively, these findings support the notion that AdeR is a transcriptional activator which mediates ald expression in response to alanine availability and is important for normal sporulation in B. subtilis.

PrcR, a PucR-type transcriptional activator, is essential for proline utilization and mediates proline-responsive expression of the proline utilization operon putBCP in Bacillus subtilis.

The soil bacterium Bacillus subtilis can utilize exogenous proline as a sole nitrogen or carbon source. The proline-inducible putBCP (formerly ycgMNO) operon encodes proteins responsible for proline uptake and two-step oxidation of proline to glutamate. We now report that a gene (formerly ycgP, now designated prcR) located downstream of the putBCP operon is essential for B. subtilis cells to utilize proline as a sole nitrogen or carbon source. Disruption of the prcR gene also abolished proline induction of putB transcription. prcR expression is not subject to autoregulation and proline induction. The PrcR protein shows no significant amino acid sequence similarity to the known transcriptional activators for proline utilization genes of other bacteria, but it does show partial amino acid sequence similarity to the transcriptional regulator PucR for the purine degradation genes of B. subtilis. PrcR orthologues of unknown function are present in some other Bacillus species. Primer-extension analysis suggests that both putB and prcR are transcribed by a σ(A)-dependent promoter. Deletion and mutation analysis revealed that an inverted repeat (5'-TTGTGG-N5-CCACAA-3') centred at position -76 relative to the transcriptional initiation site of putB is essential for putB expression. Electrophoretic mobility shift assays showed that the purified His-tagged PrcR was capable of binding specifically to this inverted repeat. Altogether, these results suggest that PrcR is a PucR-type transcriptional activator that mediates expression of the B. subtilis putBCP operon in response to proline availability.

Identification and characterization of a novel class of extracellular poly(3-hydroxybutyrate) depolymerase from Bacillus sp. strain NRRL B-14911.

The catalytic, linker, and denatured poly(3-hydroxybutyrate) (dPHB)-binding domains of bacterial extracellular PHB depolymerases (PhaZs) are classified into several different types. We now report a novel class of extracellular PHB depolymerase from Bacillus sp. strain NRRL B-14911. Its catalytic domain belongs to type 1, whereas its putative linker region neither possesses the sequence features of the three known types of linker domains nor exhibits significant amino acid sequence similarity to them. Instead, this putative linker region can be divided into two distinct linker domains of novel types: LD1 and LD2. LD1 shows significant amino acid sequence similarity to certain regions of a large group of PHB depolymerase-unrelated proteins. LD2 and its homologs are present in a small group of PhaZs. The remaining C-terminal portion of this PhaZ can be further divided into two distinct domains: SBD1 and SBD2. Each domain showed strong binding to dPHB, and there is no significant sequence similarity between them. Each domain neither possesses the sequence features of the two known types of dPHB-binding domains nor shows significant amino acid sequence similarity to them. These unique features indicate the presence of two novel and distinct types of dPHB-binding domains. Homologs of these novel domains also are present in the extracellular PhaZ of Bacillus megaterium and the putative extracellular PhaZs of Bacillus pseudofirmus and Bacillus sp. strain SG-1. The Bacillus sp. NRRL B-14911 PhaZ appears to be a representative of a novel class of extracellular PHB depolymerases.

Genetic evidence for involvement of the alternative sigma factor SigI in controlling expression of the cell wall hydrolase gene lytE and contribution of LytE to heat survival of Bacillus subtilis.

The Bacillus subtilis cell wall hydrolase LytE is involved in cell wall turnover and cell separation during vegetative growth. lytE transcription is known to be driven by a YycF-activated SigA-dependent promoter. The cell wall regulator SigI is an alternative sigma factor that has been shown to be heat stress-inducible and to be essential for survival of B. subtilis at high temperature. However, none of the previously identified target genes of SigI contribute to heat resistance. We now demonstrate that lytE expression is heat-inducible and that heat induction of lytE expression is strongly dependent on SigI. We have also found that the lytE mutant shows the same growth defect at high temperature as the sigI mutant. Introducing an extra copy of lytE into the sigI mutant could rescue its growth defect. Our data strongly suggest that SigI-dependent lytE expression under heat stress is important for heat survival of B. subtilis.

The master transcription factor Spo0A is required for poly(3-hydroxybutyrate) (PHB) accumulation and expression of genes involved in PHB biosynthesis in Bacillus thuringiensis.

Bacillus thuringiensis is a gram-positive spore-forming bacterium that can accumulate poly(3-hydroxybutyrate) (PHB) as a carbon and energy storage substance in response to nutritional stress. The regulatory mechanism for PHB biosynthesis in B. thuringiensis and diverse Bacillus species is still poorly understood. We now report that disruption of the sigH gene or the gene encoding the master sporulation transcription factor Spo0A severely impaired PHB accumulation in B. thuringiensis. Complementation of the spo0A mutation with the spo0A gene restored PHB accumulation. We have found that the requirement of Spo0A for PHB accumulation is independent of the transition state regulator AbrB and of loss of sporulation ability. We also show that Spo0A is required for the expression of three genes involved in PHB biosynthesis. These findings have uncovered a new role of Spo0A in the regulation of stationary-phase-associated cellular events.

Identification and characterization of a novel intracellular poly(3-hydroxybutyrate) depolymerase from Bacillus megaterium.

A gene that codes for a novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase, designated PhaZ1, has been identified in the genome of Bacillus megaterium. A native PHB (nPHB) granule-binding assay showed that purified soluble PhaZ1 had strong affinity for nPHB granules. Turbidimetric analyses revealed that PhaZ1 could rapidly degrade nPHB granules in vitro without the need for protease pretreatment of the granules to remove surface proteins. Notably, almost all the final hydrolytic products produced from the in vitro degradation of nPHB granules by PhaZ1 were 3-hydroxybutyric acid (3HB) monomers. Unexpectedly, PhaZ1 could also hydrolyze denatured semicrystalline PHB, with the generation of 3HB monomers. The disruption of the phaZ1 gene significantly affected intracellular PHB mobilization during the PHB-degrading stage in B. megaterium, as demonstrated by transmission electron microscopy and the measurement of the PHB content. These results indicate that PhaZ1 is functional in intracellular PHB mobilization in vivo. Some of these features, which are in striking contrast with those of other known nPHB granule-degrading PhaZs, may provide an advantage for B. megaterium PhaZ1 in fermentative production of the biotechnologically valuable chiral compound (R)-3HB.

Genetic evidence for the actin homolog gene mreBH and the bacitracin resistance gene bcrC as targets of the alternative sigma factor SigI of Bacillus subtilis.

The Bacillus subtilis sigI gene, which is a member of the class VI heat shock genes of the B. subtilis heat shock stimulon, encodes an alternative sigma factor whose regulon is poorly defined. In this study, by using a binary vector system, we showed that B. subtilis SigI could drive expression of a transcriptional fusion between the sigI regulatory region from Bacillus licheniformis, Bacillus sp. strain NRRL B-14911, B. subtilis, or Bacillus thuringiensis and the xylE reporter gene in B. subtilis. The transcriptional initiation sites of these fusions in B. subtilis were mapped by primer extension analyses. A putative consensus promoter sequence probably recognized by the B. subtilis SigI was thus deduced. Using a consensus sequence-based search procedure, we found putative sigmaI promoters preceding the actin homolog gene mreBH and the bacitracin resistance gene bcrC of B. subtilis. Overexpression of the B. subtilis sigI gene could specifically stimulate expression of both an mreBH promoter region-bgaB fusion and a bcrC promoter region-bgaB fusion. Expression of these two fusions at the amyE locus of the B. subtilis chromosome was heat inducible and SigI dependent as revealed by sigI gene disruption experiments. Primer extension analysis showed that the identified mreBH and bcrC transcriptional start sites were at appropriate distances from their sigmaI promoter elements. This further supports the notion that SigI can directly regulate mreBH and bcrC expression. Taken together, these results strongly suggest that mreBH and bcrC are new members of the SigI regulon.

Regulation of the kduID operon of Bacillus subtilis by the KdgR repressor and the ccpA gene: identification of two KdgR-binding sites within the kdgR-kduI intergenic region.

Transcription of the Bacillus subtilis kdgRKAT operon, which comprises genes involved in the late stage of galacturonate utilization, is known to be negatively regulated by the KdgR repressor. In this study, Northern analysis was carried out to demonstrate that the kdgR gene also negatively regulates the kduID operon, encoding ketodeoxyuronate isomerase and ketodeoxygluconate reductase. It has also been demonstrated that expression of the kduID operon can be induced by galacturonate and is subject to catabolite repression by glucose. The ccpA gene was found to be involved in this catabolite repression. Primer extension analysis identified a sigma(A)-like promoter sequence preceding kduI. Gel mobility shift assays and DNase I footprinting analyses indicated that KdgR is capable of binding specifically to two sites within the kdgR-kduI intergenic region in vitro. Reporter gene analysis revealed that these two KdgR-binding sites function in vivo. One site is centred 33.5 bp upstream of the translational start site of kdgR and can serve as an operator for controlling expression of the kdgRKAT operon. The other is centred 57.5 bp upstream of the translational start site of kduI and can serve as an operator for controlling expression of the kduID operon. Possible physiological significance of this regulation is discussed.