Induction of autolysis in Bacillus subtilis by ochratoxin A.

Ochratoxin A (OTA) added during the exponential growth phase at a concentration higher than 12 microgram/ml caused autolysis of Bacillus subtilis. Optical density of cultures decreased, and at higher concentrations the cultures became sterile. Optimum OTA-induced lysis was about pH 5. At concentrations below 10 microgram/ml, protein synthesis was inhibited more strongly than RNA synthesis. Cell wall synthesis was also strongly inhibited. A fraction extracted from the lysates had the property of a lysis inhibitor. The relevance of this fraction in respect to autolysis is discussed.     

Genetic evidence for interaction of sigma A with two promoters in Bacillus subtilis.

The specificity of promoter binding by RNA polymerase is governed by the sigma subunit. Recent studies, in which single-amino-acid substitutions in sigma factors have been found to suppress the effects of specific base pair substitutions in promoters, support the model that these sigma factors make sequence-specific contacts with nucleotides at the -10 and -35 regions of promoters. We found that single-amino-acid substitutions in the putative -35 region and -10 region recognition domains of sigma A specifically suppressed the effects of mutations in the -35 and -10 regions, respectively, of two promoters that are expressed in exponentially growing Bacillus subtilis. These mutations change the specificity of sigma A, the primary sigma factor in growing B. subtilis, and demonstrate that this sigma factor interacts with promoters in a manner similar to that of its homolog in Escherichia coli, sigma 70. These mutant derivatives of sigma A also provide a tool that may be useful for determining whether sigma A uses specific promoters in vivo.     

Existence of two alternative pathways for fructose and sorbitol metabolism in Bacillus subtilis Marburg.

Strains of Bacillus subtilis mutated for fructose phosphotransferase system (fruA), fructose-1-phosphate kinase (fruB), fructokinase (frucC) have been tested for their catabolism of sorbitol and fructose. It is shown that the previously known pathways of sorbitol and fructose degradation in B. subtilis, e.g.: (see article) may metabolize intracellular fructose produced either by sorbitol oxidation or by fructose-1-phosphate dephosphorylation. The intracellular fructore degradation via fructose-1-phosphate kinase has been found to require the fructose phosphotransferase system which ensures a vectorial transport of fructose.     

Physical interactions between tricarboxylic acid cycle enzymes in Bacillus subtilis: evidence for a metabolon

The majority of all proteins of a living cell is active in complexes rather than in an isolated way. These protein-protein interactions are of high relevance for many biological functions. In addition to many well established protein complexes an increasing number of protein-protein interactions, which form rather transient complexes has recently been discovered. The formation of such complexes seems to be a common feature especially for metabolic pathways. In the Gram-positive model organism Bacillus subtilis, we identified a protein complex of three citric acid cycle enzymes. This complex consists of the citrate synthase, the isocitrate dehydrogenase, and the malate dehydrogenase. Moreover, fumarase and aconitase interact with malate dehydrogenase and with each other. These five enzymes catalyze sequential reaction of the TCA cycle. Thus, this interaction might be important for a direct transfer of intermediates of the TCA cycle and thus for elevated metabolic fluxes via substrate channeling. In addition, we discovered a link between the TCA cycle and gluconeogenesis through a flexible interaction of two proteins: the association between the malate dehydrogenase and phosphoenolpyruvate carboxykinase is directly controlled by the metabolic flux. The phosphoenolpyruvate carboxykinase links the TCA cycle with gluconeogenesis and is essential for B. subtilis growing on gluconeogenic carbon sources. Only under gluconeogenic growth conditions an interaction of these two proteins is detectable and disappears under glycolytic growth conditions.     

Presence of a third sucrose hydrolyzing enzyme in Bacillus subtilis: constitutive levanase synthesis by mutants of Bacillus subtilis Marburg 168.

A beta-D-fructofuranosidase -- called levanase -- capable of the hydrolysis of sucrose, inulin and levans has been identified in Bacillus subtilis Marburg. This enzyme can not be detected in strain 168. However, sacL mutations -- mapped on the chromosome of strain 168 between the pheA and aroD reference markers -- lead to constitutive levanase synthesis. This synthesis is repressed by carbon sources such as glucose, glycerol or sucrose.     

A quantitative study of gene regulatory pathways in Bacillus subtilis for virulence and competence phenotype by quorum sensing

Quorum sensing (QS) is a process which allows a population of bacteria to coordinately regulate gene expression of their entire community. Bacillus subtilis is a soil organism which uses QS to alternate between competence for DNA uptake and sporulation. We propose a model to describe the components involved in QS and to analyze reaction species involved in the regulation of QS machinery. We targeted only those QS phenotypes for which the genetic organization and molecular characterization of the components are fully elucidated. We have analyzed simulations for concentration of different species involved in competence as well as sporulation pathways at diverse time period using quantitative methods. It was observed that there is possibility of achieving different measurement from reactions taken place between species by applying irreversible Michaelis–Menten kinetic law. We obtain variation in measurement on changing parameters such as concentrations ranging from 0.3 to 50 μM in stepwise manner by setting end time in the range of 0.1–100 ms. Additionally we observe covariance between different reaction species involved in QS by fluctuating their quantities in real-time simulations. Our model mimics correctly the phenotype for competence and virulence. We concluded that time factor play major role to determine rate kinetics of diverse reaction species as compared to their concentrations and support the hypothesis of getting genetic stability while colonies are in synchronization.     

Identification of two novel fibrinolytic enzymes from Bacillus subtilis QK02

Two fibrinolytic enzymes (QK-1 and QK-2) purified from the supernatant of Bacillus subtilis QK02 culture broth had molecular masses of 42,000 Da and 28,000 Da, respectively. The first 20 amino acids of the N-terminal sequence are AQSVPYGISQ IKAPALHSQG. The deduced protein sequence and its restriction enzyme map of the enzyme QK-2 are different from those of other proteases. The enzyme QK-2 digested not only fibrin but also a subtilisin substrate, and PMSF inhibited its fibrinolytic and amidolytic activities completely; while QK-1 hydrolyzed fibrin and a plasmin substrate, and PMSF as well as aprotinin inhibited its fibrinolytic activity. These results indicated QK-1 was a plasmin-like serine protease and QK-2 a subtilisin family serine protease. Therefore, these enzymes were designated subtilisin QK. The sequence of a DNA fragment encoding subtilisin QK contained an open reading frame of 1149 base pairs encoding 106 amino acids for signal peptide and 257 amino acids for subtilisin QK, which is highly similar with that of a fibrinolytic enzyme, subtilisin NAT (identities 96.8%). Asp32, His64 and Ser221 in the amino acid sequence deduced from the QK gene are identical to the active site of nattokinase (NK) produced by B. subtilis natto.     

Milk-clotting enzymes produced by culture of Bacillus subtilis natto

Factors affecting the production of milk-clotting enzyme (MCE) by Bacillus subtilis (natto) Takahashi, a ready available commercial natto starter, were studied. Remarkable milk-clotting activity (MCA), 685.7 SU/ml or 12,000 SU/g, was obtained when the bacteria were cultivated in the medium containing sucrose (50 g/L) and basal salts at pH 6, 37 °C with shaking at 175 rpm for 1 day. The MCA and MCA/PA ratio of the crude enzyme obtained are comparable with those of Pfizer microbial rennin and Mucor rennin. The crude enzyme showed excellent pH and thermal stability; it retained 96% of MCA after incubation for 40 min at 40 °C and retained more than 80% of its activity between pH 4 and pH 7 for more than 30 min at 30 °C. The MCE of B. subtilis (natto) Takahashi has potential as calf rennet substitutes.     

Further evidence for a partially folded intermediate in penicillinase secretion by Bacillus licheniformis.

Protoplasis of Bacillus licheniformis 749/C (a mutant constitutive for penicillinase production) continued to synthesize and release penicillinase in hypertonic growth medium in the presence of trypsin and chymotrypsin at 25 mug each per ml. When the protoplasts were stripped of about half of their membrane-bound penicillinase by pretreatment at pH 9.5 or with a higher level of trypsin, penicillinase activity no longer increased in the presence of the proteases. This effect was immediately eliminated after addition of soybean trypsin inhibitor. These proteases do not significantly inhibit general protein synthesis. Stripped protoplasts of strain 749/C and of uninduced strain 749 (unable to synthesize penicillinase) were incubated with 50 mug of chymotrypsin per ml, and the supernatent fluids were examined immunochemically for peptides derived from the penicillinase chain. Such fargments were found only with the protoplasts capable of synthesizing penicillinase (strain 749/C). The direct detection of the products of protease degradation of a susceptible form of penicillinase provides strong evidence that, in stripped protoplasts of B. licheniformis 749/C, penicillinase synthesis continues in the presence of trypsin or chymotrypsin and that, in these modified membranes, the protease is able to act on an early form of the enzyme that has not yet attained the protease-resistant conformation characteristic of the membrane-bound and exopenicillinases. This finding is discussed in terms of the current models of penicillinase secretion.     

Induction of sporulation in developmental mutants of Bacillus subtilis.

Summary Eight alleles of spineless-aristapedia (ss ^a ) were analysed for penetrance and expressivity at 18° C and 25° C. All alleles are recessive and none exhibits a maternal effect. Both ss ^a40a and ss ^aB are temperature sensitive with a higher penetrance at 18° C than 25° C. ss ^aUCI , ss ^ak , ss ^a , ss ^ax and ss ^a are viable alleles with variable penetrance and expressivity whereas ss ^aCam is a lethal allele. All mutants were tested as hemizygotes using the Df(3R) bxd ¹⁰⁰ deletion. The viable alleles showed a more extreme penetrance and expressivity when hemizygous, several becoming lethal near eclosion. Each of the eight alleles was examined in heterozygous combinations with each of the others. The lethal allele ss ^aCam was viable in combination with all other alleles. The temperature sensitivity (t.s.) of ss ^aB and ss ^a40a when heterozygous with the non-t.s. alleles was variable; some combinations were t.s. with respect to penetrance and others with respect to expressivity, whilst some heterozygotes completely lost their sensitivity to temperature.It seems, therefore, that various aspects of the spineless-aristapedia phenotype, such as the temperature sensitivity, can be separated from the expressivity and penetrance in certain allelic combinations. This suggests a very complex gene function which is, however, experimentally separable into its components.     

Independent genes for two threonyl-tRNA synthetases in Bacillus subtilis.

With the exception of Escherichia coli lysyl-tRNA synthetase, the genes coding for the different aminoacyl-tRNA synthetases in procaryotes are always unique. Here we report on the occurrence and cloning of two genes (thrSv and thrS2), both encoding functional threonyl-tRNA synthetase in Bacillus subtilis. The two proteins share only 51.5% identical residues, which makes them almost as distinct from each other as each is from E. coli threonyl-tRNA synthetase (42 and 47%). Both proteins complement an E. coli thrS mutant and effectively charge E. coli threonyl tRNA in vitro. Their genes have been mapped to 250 degrees (thrSv) and 344 degrees (thrS2) on the B. subtilis chromosome. The regulatory regions of both genes are quite complex and show structural similarities. During vegetative growth, only the thrSv gene is expressed.     

Induction of Bacillus subtilis sporulation by nucleosides: inosine appears to be sporogen.

Inosine completely reversed the selective inhibition of sporulation in Bacillus subtilis 168 caused bym-aminobenzeneboronic acid; guanosine and adenosine, but not xanthosine, partially reversed inhibition, whereas pyrimidine nucleosides were slightly effective. In addition, 0.005 to 0.025 mM inosine caused a four- to fivefold stimulation of sporulation of B. subtilis grown in minimal salts medium. Ultraviolet and infrared spectra and other physical and chemical properties of inosine were markedly similar to those of "sporogen," a previously described endogenous sporogenesis factor present in sporulating Bacillus species.