ComGA‐RelA interaction and persistence in the Bacillus subtilis K‐state

The bistably expressed K‐state of Bacillus subtilis is characterized by two distinct features; transformability and arrested growth when K‐state cells are exposed to fresh medium. The arrest is manifested by a failure to assemble replisomes and by decreased rates of cell growth and rRNA synthesis. These phenotypes are all partially explained by the presence of the AAA⁺ protein ComGA, which is also required for the binding of transforming DNA to the cell surface and for the assembly of the transformation pilus that mediates DNA transport. We have discovered that ComGA interacts with RelA and that the ComGA‐dependent inhibition of rRNA synthesis is largely bypassed in strains that cannot synthesize the alarmone (p)ppGpp. We propose that the interaction of ComGA with RelA prevents the hydrolysis of (p)ppGpp in K‐state cells, which are thus trapped in a non‐growing state until ComGA is degraded. We show that some K‐state cells exhibit tolerance to antibiotics, a form of type 1 persistence, and we propose that the bistable expression of both transformability and the growth arrest are bet‐hedging adaptations that improve fitness in the face of varying environments, such as those presumably encountered by B. subtilis in the soil.     

Expression level of a chimeric kinase governs entry into sporulation in Bacillus subtilis

Upon starvation, Bacillus subtilis cells switch from growth to sporulation. It is believed that the N-terminal sensor domain of the cytoplasmic histidine kinase KinA is responsible for detection of the sporulation-specific signal(s) that appears to be produced only under starvation conditions. Following the sensing of the signal, KinA triggers autophosphorylation of the catalytic histidine residue in the C-terminal domain to transmit the phosphate moiety, via phosphorelay, to the master regulator for sporulation, Spo0A. However, there is no direct evidence to support the function of the sensor domain, because the specific signal(s) has never been found. To investigate the role of the N-terminal sensor domain, we replaced the endogenous three-PAS repeat in the N-terminal domain of KinA with a two-PAS repeat derived from Escherichia coli and examined the function of the resulting chimeric protein. Despite the introduction of a foreign domain, we found that the resulting chimeric protein, in a concentration-dependent manner, triggered sporulation by activating Spo0A through phosphorelay, irrespective of nutrient availability. Further, by using chemical cross-linking, we showed that the chimeric protein exists predominantly as a tetramer, mediated by the N-terminal domain, as was found for KinA. These results suggest that tetramer formation mediated by the N-terminal domain, regardless of the origin of the protein, is important and sufficient for the kinase activity catalyzed by the C-terminal domain. Taken together with our previous observations, we propose that the primary role of the N-terminal domain of KinA is to form a functional tetramer, but not for sensing an unknown signal.     

DegU-phosphate activates expression of the anti-sigma factor FlgM in Bacillus subtilis

The bacterial flagellum is a complex molecular machine that is assembled by more than 30 proteins and is rotated to propel cells either through liquids or over solid surfaces. Flagellar gene expression is extensively regulated to co-ordinate flagellar assembly in both space and time. In Bacillus subtilis, the proteins of unknown function, SwrA and SwrB, and the alternative sigma factor σ(D) are required to activate expression of the flagellar filament protein, flagellin. Here we determine that in the absence of SwrA and SwrB, the phosphorylated form of the response regulator DegU inhibits σ(D) -dependent gene expression indirectly by binding to the P(flgM) promoter region and activating expression of the anti-sigma factor FlgM. We further demonstrate that DegU-P-dependent activation of FlgM is essential to inhibit flagellin expression when flagellar basal body assembly is disrupted. Regulation of FlgM is poorly understood outside of Salmonella, and differential control of FlgM expression may be a common means of coupling flagellin expression to flagellar assembly.     

Surface-associated flagellum formation and swarming differentiation in Bacillus subtilis are controlled by the ifm locus

Knowledge of the highly regulated processes governing the production of flagella in Bacillus subtilis is the result of several observations obtained from growing this microorganism in liquid cultures. No information is available regarding the regulation of flagellar formation in B. subtilis in response to contact with a solid surface. One of the best-characterized responses of flagellated eubacteria to surfaces is swarming motility, a coordinate cell differentiation process that allows collective movement of bacteria over solid substrates. This study describes the swarming ability of a B. subtilis hypermotile mutant harboring a mutation in the ifm locus that has long been known to affect the degree of flagellation and motility in liquid media. On solid media, the mutant produces elongated and hyperflagellated cells displaying a 10-fold increase in extracellular flagellin. In contrast to the mutant, the parental strain, as well as other laboratory strains carrying a wild-type ifm locus, fails to activate a swarm response. Furthermore, it stops to produce flagella when transferred from liquid to solid medium. Evidence is provided that the absence of flagella is due to the lack of flagellin gene expression. However, restoration of flagellin synthesis in cells overexpressing sigma(D) or carrying a deletion of flgM does not recover the ability to assemble flagella. Thus, the ifm gene plays a determinantal role in the ability of B. subtilis to contact with solid surfaces.     

Blue light activates the sigmaB-dependent stress response of Bacillus subtilis via YtvA

Here we present evidence for a physiologically relevant light response mediated by the LOV domain-containing protein YtvA in the soil bacterium Bacillus subtilis. The loss and overproduction of YtvA abolish and enhance, respectively, the increase in sigma(B)-controlled ctc promoter activity at moderate light intensities. These effects were absent in the dark and in red light but present under blue-light illumination. Thus, activation of the general stress response in B. subtilis is modulated by blue light.     

Pre‐steady state kinetics of ATP hydrolysis by Na,K‐ATPase

Fast reaction kinetics of ATP hydrolysis by Na,K‐ATPase has been investigated by following absorption pattern of pH sensitive dye in stopped flow spectrophotometer. Distinct pre‐steady state phase signal could be recorded with an initial decrease in acidity followed by increase in acidity. Average half time for H⁺ absorption and peak alkalinity was, respectively, 30 ms and 60 ms. Under optimal Na⁺ (120 mM) and K⁺ (30 mM) concentrations, magnitude of both H⁺ absorption and H⁺ release are found to be about 1.0 H⁺/ATPase molecule. H⁺ absorption and release decreased with decrease in Na⁺ concentration, H⁺ release was more affected. Both H⁺ absorption and H⁺ release are found to be independent of K⁺ concentration in the pre‐steady state phase. No H⁺ absorption or release was observed following mixing of either ADP, Na⁺ or K⁺ alone with ATPase. Effect of delayed mixing of Na⁺ or K⁺ on two phases of pre‐steady state cycle indicates that ATP hydrolytic cycle starts without K⁺ ions if optimal Na⁺ is present. ATP hydrolytic cycle does not start in the absence of Na⁺ ions. Results obtained have been interpreted in terms of an extended kinetic scheme for Na,K‐ATPase. Copyright © 2009 John Wiley & Sons, Ltd.     

Carbonyl reduction by YmfI in Bacillus subtilis prevents accumulation of an inhibitory EF‐P modification state

Translation elongation factor P (EF‐P) in Bacillus subtilis is required for a form of surface migration called swarming motility. Furthermore, B. subtilis EF‐P is post‐translationally modified with a 5‐aminopentanol group but the pathway necessary for the synthesis and ligation of the modification is unknown. Here we determine that the protein YmfI catalyzes the reduction of EF‐P‐5 aminopentanone to EF‐P‐5 aminopentanol. In the absence of YmfI, accumulation of 5‐aminopentanonated EF‐P is inhibitory to swarming motility. Suppressor mutations that enhanced swarming in the absence of YmfI were found at two positions on EF‐P, including one that changed the conserved modification site (Lys 32) and abolished post‐translational modification. Thus, while modification of EF‐P is thought to be essential for EF‐P activity, here we show that in some cases it can be dispensable. YmfI is the first protein identified in the pathway leading to EF‐P modification in B. subtilis, and B. subtilis encodes the first EF‐P ortholog that retains function in the absence of modification.     

Mapping the twin‐arginine protein translocation network of Bacillus subtilis

Bacteria employ twin‐arginine translocation (Tat) pathways for the transport of folded proteins to extracytoplasmic destinations. In recent years, most studies on bacterial Tat pathways addressed the membrane‐bound TatA(B)C subunits of the Tat translocase, and the specific interactions between this translocase and its substrate proteins. In contrast, relatively few studies investigated possible coactors in the TatA(B)C‐dependent protein translocation process. The present studies were aimed at identifying interaction partners of the Tat pathway of Bacillus subtilis, which is a paradigm for studies on protein secretion by Gram‐positive bacteria. Specifically, 36 interaction partners of the TatA and TatC subunits were identified by rigorous application of the yeast two‐hybrid (Y2H) approach. Our Y2H analyses revealed that the three TatA isoforms of B. subtilis can form homo‐ and heterodimers. Subsequently, the secretion of the Tat substrates YwbN and PhoD was tested in mutant strains lacking genes for the TatAC interaction partners identified in our genome‐wide Y2H screens. Our results show that the cell wall‐bound protease WprA is important for YwbN secretion, and that the HemAT and CsbC proteins are required for PhoD secretion under phosphate starvation conditions. Taken together, our findings imply that the Bacillus Tat pathway is embedded in an intricate protein–protein interaction network.     

Integration of various plasmids into the Bacillus subtilis chromosome

Some features of integration of temperature-sensitive pE194, pGG10 and pGG20 plasmids into the Bacillus subtilis chromosome were studied. Several auxotrophic mutations were obtained using insertion of these plasmids into the chromosome. The sites of plasmids for illegitimate recombination were determined. It was shown that the integration into the Bac. subtilis chromosome is characteristic not only for the plasmid pE194 but is the property of Staphylococcus aureus plasmid pC194 and Escherichia coli pBR322 plasmid. The influence of different Bac. subtilis rec mutations on the frequency of integration was studied.     

Hydrolysis of polyethyleneterephthalate by p‐nitrobenzylesterase from Bacillus subtilis

From a screening on agar plates with bis(benzoyloxyethyl) terephthalate (3PET), a Bacillus subtilis p‐nitrobenzylesterase (BsEstB) was isolated and demonstrated to hydrolyze polyethyleneterephthalate (PET). PET‐hydrolase active strains produced clearing zones and led to the release of the 3PET hydrolysis products terephthalic acid (TA), benzoic acid (BA), 2‐hydroxyethyl benzoate (HEB), and mono‐(2‐hydroxyethyl) terephthalate (MHET) in 3PET supplemented liquid cultures. The 3PET‐hydrolase was isolated from non‐denaturating polyacrylamide gels using fluorescein diacetate (FDA) and identified as BsEstB by LC‐MS/MS analysis. BsEstB was expressed in Escherichia coli with C‐terminally fused StrepTag II for purification. The tagged enzyme had a molecular mass of 55.2 kDa and a specific activity of 77 U/mg on p‐nitrophenyl acetate and 108 U/mg on p‐nitrophenyl butyrate. BsEstB was most active at 40°C and pH 7.0 and stable for several days at pH 7.0 and 37°C while the half‐life times decreased to 3 days at 40°C and only 6 h at 45°C. From 3PET, BsEstB released TA, MHET, and BA, but neither bis(2‐hydroxyethyl) terephthalate (BHET) nor hydroxyethylbenzoate (HEB). The k_cat values decreased with increasing complexity of the substrate from 6 and 8 (s?1) for p‐nitrophenyl‐acetate (4NPA) and p‐nitrophenyl‐butyrate (4NPB), respectively, to 0.14 (s?1) for bis(2‐hydroxyethyl) terephthalate (BHET). The enzyme hydrolyzed PET films releasing TA and MHET with a concomitant decrease of the water‐contact angle (WCA) from 68.2° ± 1.7° to 62.6° ± 1.1° due to formation of novel hydroxyl and carboxyl groups. These data correlated with a fluorescence emission intensity increase seen for the enzyme treated sample after derivatization with 2‐(bromomethyl)naphthalene. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Unravelling the lipoyl‐relay of exogenous lipoate utilization in Bacillus subtilis

Lipoate is an essential cofactor for key enzymes of oxidative and one‐carbon metabolism. It is covalently attached to E2 subunits of dehydrogenase complexes and GcvH, the H subunit of the glycine cleavage system. Bacillus subtilis possess two protein lipoylation pathways: biosynthesis and scavenging. The former requires octanoylation of GcvH, insertion of sulfur atoms and amidotransfer of the lipoate to E2s, catalyzed by LipL. Lipoate scavenging is mediated by a lipoyl protein ligase (LplJ) that catalyzes a classical two‐step ATP‐dependent reaction. Although these pathways were thought to be redundant, a ?lipL mutant, in which the endogenous lipoylation pathway of E2 subunits is blocked, showed growth defects in minimal media even when supplemented with lipoate and despite the presence of a functional LplJ. In this study, we demonstrate that LipL is essential to modify E2 subunits of branched chain ketoacid and pyruvate dehydrogenases during lipoate scavenging. The crucial role of LipL during lipoate utilization relies on the strict substrate specificity of LplJ, determined by charge complementarity between the ligase and the lipoylable subunits. This new lipoyl‐relay required for lipoate scavenging highlights the relevance of the amidotransferase as a valid target for the design of new antimicrobial agents among Gram‐positive pathogens.     

Amplification of plasmid DNA inserted into the Bacillus subtilis chromosome

Amplification of plasmid pGG10 inserted into the Bacillus subtilis chromosome is described. The possibility of the 3.2 kb fragment of eucaryotic (wheat) DNA to be amplified within the bacterial genome is shown. The models explaining this phenomenon are discussed.     

Permeability of gentamicin into the inside of Bacillus subtilis spores

Abstract The penetration of gentamicin into the inside of Bacillus subtilis spores was examined by an immunoelectron microscopy method with colloidal gold-immunoglobulin G complex. The colloidal gold particles were located mainly in the coat regions of spores and were not observed in the cortex or core regions. This result suggests the existence of an outer membrane inside the coat region as the primary permeability barrier to gentamicin.