ESI LC-MS and MS/MS characterization of antifungal cyclic lipopeptides produced by Bacillus subtilis XF-1

Bacillus subtilis XF-1 (CGMCC No. 2357), a patent strain with good effects for controlling the clubroot of crucifer and many pathogenic fungi, was predicted to produce cyclic lipopeptide (CLP) antibiotics based on its genomic analysis. In this study, the CLPs were purified and determined with the following protocol: the supernatant of XF-1 cultivating mixture was firstly precipitated, then the precipitants were extracted with methanol and further separated by Sephadex LH-20 chromatography to test its antifungal activities. Fungi-inhibiting fractions were further characterized with LC/ESI-MS and LC/ESI-MS/MS. The results show that four molecular ion peaks [M+H]? (m/z 1,464, 1,478, 1,492 and 1,506) from fungi suppression fraction were identified as fengycin A with fatty acid of C??-C??, fengycin B (C??-C??), fengycin C (C??-C??), fengycin D (C??-C??) and fengycin S (C??-C??). Fengycin C (C?? and C??), fengycin D (C??, C?? and C??) and fengycin S (C??, C?? and C??) were reported for the first time. The diversity of the fengycins that exist in this strain will help the elucidation of their biocontrol mechanisms.     

Regulation of polyglutamic acid synthesis by glutamate in Bacillus licheniformis and Bacillus subtilis

The synthesis of polyglutamic acid (PGA) was repressed by exogenous glutamate in strains of Bacillus licheniformis but not in strains of Bacillus subtilis, indicating a clear difference in the regulation of synthesis of capsular slime in these two species. Although extracellular gamma-glutamyltranspeptidase (GGT) activity was always present in PGA-producing cultures of B. licheniformis under various growth conditions, there was no correlation between the quantity of PGA and enzyme activity. Moreover, the synthesis of PGA in the absence of detectable GGT activity in B. subtilis S317 indicated that this enzyme was not involved in PGA biosynthesis in this bacterium. Glutamate repression of PGA biosynthesis may offer a simple means of preventing unwanted slime production in industrial fermentations using B. licheniformis.     

Comparison of the inactivation of Bacillus subtilis spores and MS2 bacteriophage by MIOX, ClorTec and hypochlorite

AIMS: To compare the disinfection ability of two widely used electrolytic generation systems (ClorTec and MIOX) and the conventional chlorine disinfectant (sodium hypochlorite) using three strains of Bacillus subtilis spores and MS2 bacteriophage. METHODS AND RESULTS: Three B. subtilis aerobic spore strains (ATCC1A1, 35021 and 35946) and the bacteriophage MS2 (ATCC 15597-B1) were propagated and sporulated. Four indicator organisms were exposed to four disinfectant treatments for comparing the effectiveness of inactivation: hypochlorite, ClorTec, MIOX and MIOX-anode. The results indicated that the two electrolytic generation systems were as effective as the conventional chlorination for the inactivation of micro-organisms used. Some data points showed the variation using anova analysis, in which the inactivation of MIOX and ClorTec was higher than that of hypochlorite. CONCLUSIONS: The ClorTec and MIOX systems are quite similar to hypochlorite in the inactivation-effectiveness for aerobic spores and bacteriophage in drinking water. SIGNIFICANCE AND IMPACT OF THE STUDY: Laboratory-scale investigation proved that gaseous chlorine could be replaced by either ClorTec or MIOX systems for the drinking water treatment utilities, which still could maintain the same disinfection efficiency.     

Carbamyl phosphate synthesis in Bacillus subtilis

In vitro and "in situ" assays have been developed to test the carbamyl phosphate synthetase (CPSase) activity of a series of pyrimidine-requiring mutants of Bacillus subtilis. The enzyme has been shown to be highly unstable, and was successfully extracted only in the presence of 10% glycerol and 1 mM dithiothreitol (Cleland's reagent). It loses activity rapidly when sonicated or when treated with lysozyme. Genetic studies, using mutants, indicate that B. subtilis may possess two CPSases. This possibility and its physiological consequences were probed enzymatically. CPSase activity has been shown to undergo inhibition by both uridine triphosphate and dihydroorotate; activation has been demonstrated in response to phosphoribosyl pyrophosphate (PRPP) and (to a lesser extent) ornithine.     

Regioselective synthesis of kojic acid esters by Bacillus subtilis protease

The lipophilicity of kojic acid [5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one] was improved by esterifying kojic acid with either divinyl adipate, vinyl hexanoate, vinyl octanoate or vinyl decanoate using protease from Bacillus subtilis for 7 d. ¹H-NMR and ¹³C-NMR showed that the primary hydroxyl group at the C-7 position of kojic acid was regioselectively esterified to afford 7-O-vinyl adipoyl kojic acid, 7-O-hexanoyl kojic acid, 7-O-octanoyl kojic acid and 7-O-decanoyl kojic acid (13–27% yield). The kojic acid esters had radical scavenging activities, inhibited tyrosinase activity and was biodegradable.     

The program of protein synthesis during sporulation in Bacillus subtilis

The program of protein synthesis was examined during sporulation in Bacillus subtilis as an index of the control of gene expression. At various stages of growth and spore formation, cells of B. subtilis were pulse-labeled with ³⁵S-methionine. Protein was extracted from the radioactively labeled bacteria and then subjected to high resolution one-dimensional and two-dimensional slab gel electrophoresis. We report that sporulating cells restricted or “turned off” the synthesis of certain polypeptides characteristic of the vegetative phase of growth. In certain cases, this “turn off” was prevented in a mutant (SpoOa-5NA) blocked at the first stage of spore formation. Sporulating bacteria also elaborated new polypeptide species that could not be detected in vegetatively growing cells or in cells of the asporogenous mutant SpoOa-5NA in sporulation medium. The synthesis of these sporulation-specific proteins was “turned on” in a temporally defined sequence throughout the period of spore formation. Spore coat protein, for example, was first synthesized at 4 hr after the onset of sporulation, the time at which refractile prespores appeared. Certain sporulation-specific polypeptides including the coat protein were among the most actively produced polypeptides in sporulating cells.     

Thymidylate synthesis and aminopterin resistance in Bacillus subtilis

Wilson, Melba Carr (Brown University, Providence, R.I.), James L. Farmer, and Frank Rothman. Thymidylate synthesis and aminopterin resistance in Bacillus subtilis. J. Bacteriol. 92:186-196. 1966.-The thymine-requirement of Bacillus subtilis 168 thy results from mutation in two unlinked genes (i.e., genetic loci) designated thyA and thyB. The thyB gene is located between the met and ile markers. Both thyA(+)thyB and thyA thyB(+) strains are phenotypically thy(+). ThyA(+)thyB strains resemble the wild type in their sensitivity to aminopterin, poor incorporation of exogenous thymine into deoxyribonucleic acid (DNA), and high level of thymidylate synthetase activity in crude extracts. ThyA thyB(+) strains are resistant to aminopterin in the presence of thymine, incorporate exogenous thymine into DNA, and have no detectable thymidylate synthetase activity. Experiments designed to elucidate the role of the thyB gene indicate that it specifies an alternate pathway of thymidylate synthesis, similar to thymidylate synthetase but requiring a cofactor other than tetrahydrofolate. The mechanism of selection of thymine-requiring mutants by aminopterin is revealed by these results.     

Ribosomal ribonucleic acid synthesis in Bacillus subtilis

The mode of biosynthesis of the 16S and 23S ribosomal ribonucleic acids (rRNA) was studied in Bacillus subtilis 168thy(-). Three criteria were used to define the characteristics of the rRNA species: (i) the time required at 37 degrees C to synthesize 16S and 23S rRNA chains de novo in growing cultures; (ii) the degree of reactivity of the 3'-terminal groups of the rRNA molecules with periodate and [carbonyl-(14)C]isonicotinic acid hydrazide; and (iii) the reactivity of the 5'-terminal regions of the rRNA molecules with the bacterial exonuclease purified by Riley (1969). The 16S and 23S chains of B. subtilis were synthesized at rates of 22+/-2 and 21+/-2 nucleotides added/s. The periodate-[(14)C]isonicotinic acid hydrazide and the exonuclease techniques for estimating apparent chain lengths of RNA indicated that the chain length of the 23S rRNA was 1.8 times that of the 16S fraction. The apparent chain lengths of each rRNA species were: 16S rRNA, 1650+/-50 nucleotide residues; 23S rRNA, 3050+/-90 nucleotide residues. It appears that, the 16S and 23S rRNA molecules in B. subtilis are synthesized in the expected manner, by simple polymerization of the final products on independent cistrons.     

Peptidoglycan synthesis in L-phase variants of Bacillus licheniformis and Bacillus subtilis.

Stable L-phase variants isolated from Bacillus licheniformis and Bacillus subtilis, when grown in osmotically stabilized media, do not synthesize peptidoglycan but have been found to accumulate the nucleotide precursors of this polymer. The enzymes involved in the synthesis of these precursors and the later membrane-bound stages of peptidoglycan synthesis have been investigated, and the L-phase variants have been shown to contain lesions, which provide a rational explanation for the absence of peptidoglycan and for the nature of the precursor accumulated. The majority of the L-phase variants contained a single enzymic defect, but two strains were isolated with double lesions. Five out of seven strains examined accumulated uridine 5'-diphosphate (UDP)-MurAc-L-ala-D-glu and were unable to synthesize diaminopimelic acid as a consequence of a defect in aspartate-beta-semialdehyde dehydrogenase activity. Two strains were deficient in UDP-MurAc: L-alanine ligase and accumulated UDP-MurAc. One strain accumulated the complete nucleotide precursor UDP-MurAc-L-ala-D-glu-mA2pm-D-ala-D-ala and was deficient in phospho-N-acetylmuramyl pentapeptide translocase. A second strain also had this lesion, together with defective aspartate-beta-semialdehyde dehydrogenase activity. The other enzymes of peptidoglycan synthesis were present in the L-phase variants, with activities similar to those found in the parent bacilli grown under identical conditions. Membrane preparations from certain of the L-phase variants were also capable of synthesizing the secondary polymers poly(glycerol phosphate) teichoic acid and teichuronic acid and also a polymer of N-acetylglucosamine.     

Bacteriophage SPO1-induced macromolecular synthesis in minicells of Bacillus subtilis.

SPO1 bacteriophage injects its DNA into minicells produced by Bacillus subtilis CU403 divIVB1. The injected DNA is partially degraded to small trichloracetic acid-precipitable material and trichloroacetic acid-soluble material. The injected DNA is not replicated; however, it serves as a template for RNA and protein synthesis. The RNA produced specifically hybridizes to SPO1 DNA, and the amount of RNA hybridized can be reduced by competition with RNA isolated at all stages of the phage cycle from infected nucleate cells of the B. subtilis CU403 divIVB1. An unrelated phage, SPP1, also induces phage-specific RNA in infected minicells. Translation occurs in SPO1-infected minicells resulting in at least eight proteins which have been separated by gel electrophoresis, and two of these proteins have mobilities similar to proteins found only in infected B. subtilis CU403 divIVB1 nucleate cells. A large proportion of the polypeptide material synthesized in infected minicells is very small and heterogeneous in size.     

DNA synthesis in vivo in Bacillus subtilis

DNA polymerase III is the enzyme responsible for deoxynucleotide addition to nascent DNA fragments in Bacillus subtilis protoplasts. Nascent single-stranded fragments separated from bulk DNA by hydroxyapatite chromatography cannot self-anneal. Partial inhibition of DNA polymerase III by 6-(hydroxyphenylazo)-uracil, a specific inhibitor, slows the rate of nascent fragment synthesis but has no effect on final size. Neither DNA polymerase I nor II can elongate nascent fragments in protoplasts when DNA polymerase III is completely inhibited.     

The production of antifungal volatiles by Bacillus subtilis

A strain of Bacillus subtilis which produces an antibiotic metabolite was also found to produce a volatile compound(s) which was antifungal to Rhizoctonia solani and Pythium ultimum.
Growth of the fungi was severely impaired in the presence of the volatiles and physiological abnormalities of the hyphae were observed, including hyphal distortion and vacuolation. A range of media were tested for volatile production and potato dextrose agar (PDA) was found to be the most active. Temperature had a considerable effect on antifungal volatile activity with the greatest inhibition occurring at 30°C. Addition of iron (III) chloride to Sabouraud's glucose agar (SGA) also enhanced the antifungal effect. The volatiles were found to be water soluble and remained active when trapped in SGA.