Tn10 insertional mutations of Bacillus subtilis that block the biosynthesis of bacilysin

Transposon mutagenesis was employed to isolate the gene(s) related with the biosynthesis of dipeptide antibiotic in Bacillus subtilis PY79 (a prototrophic derivative of the standard 168 strain). The blocked mutants were phenotypically selected from the transposon library by bioassay and the complete loss of biosynthetic ability was verified through ESI-mass spectrometry analysis. Four different bacilysin nonproducer mutants (Bac(-)::Tn10(ori-spc)) were isolated from the transposon library. The genes involved in bacilysin biosynthesis were identified as thyA (thymidilate synthetase), ybgG (unknown; similar to homocysteine methyl transferase) and oppA (oligopeptide permease), respectively. The other blocked gene was yvgW (unknown; similar to heavy metal-transporting ATPase); however, backcross studies did not verify its involvement in bacilysin biosynthesis. This gene, on the other hand, appeared to be necessary for efficient sporulation and transformation. Opp involvement was significant as it suggested that bacilysin biosynthesis is under or a component of the quorum sensing pathway which has been shown to be responsible for the establishment of sporulation, competence development and onset of surfactin biosynthesis. For verification, it was necessary to check the involvement of peptide pheromones (PhrA or PhrC) internalized by the Opp system and response regulator ComA as the essential components of this global control. phrA, phrC and comA deleted mutants of PY79 were thus constructed and the latter two genes were shown to be essential for bacilysin biosynthesis.     

Nikkomycin-resistant mutants ofMucor rouxii: physiological and biochemical properties

We isolated three nikkomycin-resistant mutants of the dimorphic fungusM. rouxii which were physiologically characterized regarding their response to yeast-phase inducing conditions and their sensitivity to bacilysin. Mutant strains G21 and G23, showed a qualitatively normal, though delayed, dimorphic transition and partial cross-resistance to bacilysin. Mutant strain G27 showed an altered dimorphism, producing a high proportion (50%) of hyphal cells, and a wild-type sensitivity to bacilysin. Cell-free extracts from this mutant exhibited an activity of both basal and protease-activated chitin synthetase which was overexpressed as compared with the parental strain and mutants G21 and G23. Results are discussed in terms of the different genetic background of the mutants.Abbreviations NTG N-methyl-N-nitro-N-nitrosoguanidine - UDP-GlcNAc uridine 5-diphospho-N-acetylglucosamine - GlcNAc N-acetylglucosamine     

Bacilysin production byBacillus subtilis: Effects of bacilysin,pH and temperature

The dipeptide antibiotic bacilysin, when added externally to the early exponential-phase cultures, markedly limited its own synthesis. It was shown in cell-free extracts that the feedback effect does not involve the inhibition of bacilysin synthetase, the enzyme catalyzing bacilysin formation. We also studied pH and temperature dependence of bacilysin production. Production was highest at about pH 6.8 and at 25 °C.     

Regulation of biosynthesis of bacilysin byBacillus subtilis

Summary Production of the dipeptide antibiotic bacilysin byBacillus subtilis 168 was growth associated and showed no evidence of repression by glucose or sucrose. Carbohydrates other than glucose and sucrose yielded lower specific titers of bacilysin. Bacilysin production in three such carbon sources (maltose, xylose, ribose) was delayed until growth slowed down. Ammonium salts were poor for bacilysin production when used as the sole nitrogen source. When added to the standard medium containing glutamate, they suppressed antibiotic production. Aspartate was slightly better than glutamate for antibiotic production as sole nitrogen source. No other nitrogen source tested, including inorganic, organic or complex, approached the activity of glutamate or aspartate. When added to glutamate, casamino acids, phenylalanine and alanine (a substrate of bacilysin synthetase) suppressed bacilysin production while stimulating growth. Phosphate provided for optimum growth and production at 7.5 mM and both processes were inhibited at higher concentrations. Ferric citrate stimulated growth and inhibited bacilysin production, the effects being due to both the iron and the citrate components. Elimination of ferric citrate stimulated production as did increasing the concentration of Mn to its optimum concentration of 6.6×10^–4M.     

小麦赤霉病原菌拮抗菌Bacillus amyloliquefaciens 7M1产抗菌素的研究

【目的】从小麦根际土壤分离鉴定一株赤霉病拮抗菌,对该菌产的抗菌素进行生物学性质研究、种类鉴定和抑菌实验。【方法】利用牛津杯法和光照培养箱实验对其抑菌活性进行测定,通过16S r RNA基因序列分析对目标菌株的种属进行初步鉴定,根据抗菌素相关基因进行PCR扩增和测序,利用在线软件Pro Param tool和TMHMM对编码蛋白进行生物信息学分析。【结果】7M1菌体和抗菌素对禾谷镰刀菌的抑菌圈直径分别为16.33±0.13 mm和15.43±0.21 mm,16S r RNA基因序列分析结果显示其为芽孢杆菌,并与解淀粉芽孢杆菌具有较近的亲缘关系,菌株7M1抗菌素对小麦赤霉病的温室防治效果为76.41%,而且热稳定性好,可被蛋白酶K、胰蛋白酶、胃蛋白酶降解,在p H 5.0-10.0有较好的抑菌活性,但是紫外线辐射会降低其抑菌活性。菌株7M1含有bac AB、itu C、bam D 3种基因,通过与Gen Bank中相关的抗菌素基因进行比对,发现其编码的氨基酸序列与Gen Bank库中的芽孢杆菌溶素、伊枯草菌素和杆菌抗霉素D等抗菌素的相似性达到99%。bac AB编码蛋白和itu C编码蛋白是稳定蛋白,bam D编码蛋白是不稳定蛋白,此外,3种基因的编码产物不具有明显的跨膜结构。【结论】从该菌发酵液提取的抗菌素有很好的抗禾谷镰刀菌活性而且性质稳定,因而在小麦赤霉病的生物防治中具有潜在的应用价值。     

ScoC Regulates Bacilysin Production at the Transcription Level in Bacillus subtilis

Bacillus subtilis mutants with high expression of the bacilysin operon ywfBCDEFG were isolated. Comparative genome sequencing analysis revealed that all of these mutants have a mutation in the scoC gene. The disruption of scoC by genetic engineering also resulted in increased expression of ywfBCDEFG. Primer extension and gel mobility shift analyses showed that the ScoC protein binds directly to the promoter region of ywfBCDEFG. Our results indicate that the transition state regulator ScoC, together with CodY and AbrB, negatively regulates bacilysin production in B. subtilis.Gram-positive model bacterium Bacillus subtilis produces the dipeptide antibiotic bacilysin, which consists of an l-alanine and an unusual amino acid, l-anticapsin (15). We previously reported that a polycistronic operon, ywfBCDEFG, and a monocistronic gene, ywfH, are required for bacilysin production (7). The gene products of ywfB and ywfG are thought to participate in the l-anticapsin biosynthesis pathway, while the ywfE gene product has been assigned as an amino acid ligase involved in alanine-anticapsin ligation (14). The protein encoded by the ywfF gene is necessary for self-protection against bacilysin (13). Thus, the ywfBCDEFG operon has an obligate role in bacilysin production.We previously showed that a certain rifampin (rifampicin) resistance mutation can activate the B. subtilis dormant secondary metabolism, neotrehalosadiamine (3,3′-diamino-3,3′-dideoxy-α,β-trehalose) synthesis (8). Subsequently, we attempted to activate bacilysin production in the same way. Unexpectedly, we found that the expression of the bacilysin operon ywfBCDEFG was induced by a mechanism independent of the rifampin resistance mutation. Although the expression of the bacilysin operon ywfBCDEFG was previously reported to be negatively regulated by transition state regulators CodY (7) and AbrB (11), the mechanism we found was apparently different from these known mechanisms. Here, we report a novel regulatory mechanism involved in bacilysin production.     

Virulence of Mycobacterium tuberculosis

Abstract Different strains of Candida albicans show varied sensitivities to the peptide analogues bacilysin, polyoxins and nikkomycins. From a sensitive strain, B2630, spontaneous mutants were selected for resistance to each analogue; certain mutants showed cross-resistance to other analogues and associated defects in peptide transport. A bacilysin-resistant mutant was cross resistant to the other analogues and to m -fluorophenylalanyl-Ala (FPA) but retained sensitivity to m -fluorophenylalanyl-Ala—Ala (FPAA). It showed defective dipeptide transport but normal oligopeptide transport. A revertant, selected for its ability to utilize Ala-Ala as sole nitrogen source, regained wild-type dipeptide transport activity and analogue sensitivity. Thus, C. albicans has distinguishable mechanisms for dipeptide and oligopeptide transport which can be exploited for uptake of peptide-drug adducts.     

Some properties of glutathione biosynthesis-deficient mutants of Escherichia coli B

Mutants of Escherichia coli B that contain essentially no detectable glutathione were isolated. These mutants had a very low activity of gamma-glutamylcysteine synthetase or glutathione synthetase. No significant differences in growth in minimal medium were observed between the mutants and the parental strain. The mutants lacking gamma-glutamylcysteine synthetase activity were more susceptible to toxic compounds than either the parental strain or a glutathione synthetase-deficient strain. The mutants lacking gamma-glutamylcysteine synthetase activity were also susceptible to oxygen.     

The structure of bacilysin and other products of Bacillus subtillis

1. Mass spectra of the trimethylsilyl derivative and the methyl ester of the N-trifluoroacetyl derivative of bacilysin indicated that the antibiotic had a molecular weight of 270. Several peaks in the spectrum of the methyl ester were consistent with the presence of an N-terminal alanine residue in the molecule. 2. The proton-magnetic-resonance spectrum of bacilysin confirmed that the antibiotic contained an epoxide group and the spin-spin splitting of the protons of the epoxide group indicated that the side chain of the epoxycyclohexanone ring was attached at C-4 and was alphabeta to the keto group. 3. The formation of an alphabeta-unsaturated ketone on reduction of bacilysin with chromous chloride also showed that the epoxide was alphabeta to the keto group. 4. The optical-rotatory-dispersion curve of bacilysin showed a positive Cotton effect. On the assumption that the reversed Octant rule for alphabeta-epoxyketones was applicable this revealed the absolute stereochemistry and enabled a definitive structure to be assigned to the molecule. 5. Similar measurements showed that substance AA1, isolated from culture supernatants, was the C-terminal amino acid of bacilysin. 6. Hydrolysis of substance P2 with leucine aminopeptidase and the mass spectrum of the methyl ester of its N-trifluoroacetyl derivative showed that this substance was l-analyl-l-alanine. 7. These results are discussed in relation to the biogenesis of bacilysin.     

Molecular insights into the antifungal mechanism of bacilysin

Bacilysin is one of the simplest antimicrobial peptides and has drawn great attention for its excellent performance against Candida albicans. In this study, the antifungal mechanism of bacilysin was investigated. The target enzyme glucosamine-6-phosphate synthase (GFA) was expressed heterologously in Escherichia coli and its inhibition by bacilysin and derivatives was studied. It was concluded that bacilysin could be hydrolyzed by a proteinase of C. albicans, and that the released product, anticapsin, then inhibited the aminotransferase activity of GFA. This result was verified by molecular simulation, and the interaction mode of anticapsin with GFA was detailed, which provides data for the development of novel antifungal drugs. Transport of bacilysin into fungal cells was also simulated and it was shown that bacilysin is more readily transported into cells than anticapsin. Thus, our findings support a mechanism whereby bacilysin is transported into fungal pathogens, hydrolyzed to anticapsin, which then inhibits GFA.     

Observations on the structure of bacilysin.

1. Elementary analysis and other properties of a highly purified preparation of bacilysin indicated that a possible molecular formula for the substance is C(12)H(18)N(2)O(5). The results of electrometric titration were consistent with the hypothesis that the substance was a peptide containing one free alpha-amino group and one free carboxyl group. 2. Hydrolysis of bacilysin with 6n-hydrochloric acid at 105 degrees yielded l-alanine and l-tyrosine, but the ultraviolet spectrum of the substance showed that no tyrosine residue was present in the molecule and a nuclear-magnetic-resonance spectrum indicated that olefinic and aromatic protons were absent. The dinitrophenyl (DNP) derivative of bacilysin yielded DNP-alanine on acid hydrolysis. 3. Bacilysin was hydrolysed by leucine aminopeptidase (EC 3.4.1.1) and by Pronase to give alanine and an uncharacterized amino acid. Its infrared spectrum was consistent with the presence of a peptide grouping in the molecule. 4. The optical rotatory dispersion of bacilysin and its reaction with thiosemicarbazide indicated that the substance contained an aldehyde or ketone group. Its behaviour on catalytic reduction and its reaction with sodium thiosulphate and with certain thiols suggested that an epoxide group was present. 5. A possible type of structure for bacilysin is considered in the light of its known properties.     

Regulation of nitrogen fixation. Nitrogenase-derepressed mutants of Klebsiella pneumoniae.

1. A new procedure is described for selecting nitrogenase-derepressed mutants based on the method of Brenchley et al. (Brenchley, J.E., Prival, M.J. and Magasanik, B. (1973) J. Biol. Chem. 248, 6122-6128) for isolating histidase-constitutive mutants of a non-N2-fixing bacterium. 2. Nitrogenase levels of the new mutants in the presence of NH4+ were as high as 100% of the nitrogenase activity detected in the absence of NH4+. 3. Biochemical characterization of these nitrogen fixation (nif) derepressed mutants reveals that they fall into three classes. Three mutants (strains SK-24, 28 and 29), requiring glutamate for growth, synthesize nitrogenase and glutamine synthetase constitutively (in the presence of NH4+). A second class of mutants (strains SK-27 and 37) requiring glutamine for growth produces derepressed levels of nitrogenase activity and synthesized catalytically inactive glutamine synthetase protein, as determined immunologically. A third class of glutamine-requiring, nitrogenase-derepressed mutants (strain SK-25 and 26) synthesizes neither a catalytically active glutamine synthetase enzyme nor an immunologically cross-reactive glutamine synthetase protein. 4. F-prime complementation analysis reveals that the mutant strains SK-25, 26, 27, 37 map in a segment of the Klebsiella chromosome corresponding to the region coding for glutamine synthetase. Since the mutant strains SK-27 and SK-37 produce inactive glutamine synthetase protein, it is concluded that these mutations map within the glutamine synthetase structural gene.     

Isolation and characterization of nitrogen-deregulated mutants of Streptomyces clavuligerus

Two screening methods for isolation of mutants of Streptomyces clavuligerus with altered control of nitrogen metabolism enzymes are described. Thirty-eight prototrophic mutants with simultaneous deregulation of urease and glutamine synthetase were isolated. Nine mutants were examined in more detail and they also showed deregulated formation of arginase and ornithine aminotransferase. Different patterns of altered control of all four enzymes were observed. Inactivation of glutamine synthetase after ammonium shock took place to different extents in these nine strains, and seven of them had a thermosensitive glutamine synthetase activity. It is concluded that a system of nitrogen control, in which glutamine synthetase has a key role, is present in S. clavuligerus. Cephalosporin production was depressed by ammonium in all the mutants, irrespective of the alterations in nitrogen control of primary metabolism.     

Changes in patterns of alkaline serine protease and bacilysin formation caused by common effectors of sporulation inBacillus subtilis 168

Bacilysin biosynthesis and alkaline serine protease production inBacillus subtilis 168 were monitored and compared in batch cultures when various effectors of sporulation were added at different stages of growth in a medium containing sucrose and glutamate. Depending on the time of addition, glucose affected sporulation and serine protease formation to the same extent, but had no effect on bacilysin production. Ammonium andl-alanine additions suppressed all three processes. Casamino acids severely interfered with bacilysin formation and sporulation, but not with protease formation. Decoyinine, a well-known inducer of sporulation, induced protease formation as well, but did not affect bacilysin biosynthesis. The extent of the observed effects depended largely on the time of metabolite additions. The results are discussed with reference to a possible coregulation of sporulation and the formation of bacilysin and alkaline serine protease inB. subtilis.     

Some properties of rat-liver glucose–adenosine triphosphate phosphotransferases

1. Bacilysin, a peptide which yields l-alanine and l-tyrosine on acid hydrolysis, was produced by a strain of Bacillus subtilis (A 14) in a chemically defined medium containing glucose, ammonium acetate or ammonium chloride, potassium phosphate and other inorganic salts, and ferric citrate. 2. Under the conditions used growth was diphasic. Bacilysin was formed during the second phase of slower growth, and there was little production during the stationary phase. Nevertheless, bacilysin production occurred when protein synthesis was inhibited by chloramphenicol. It thus appears that there is no obligatory coupling of protein synthesis and bacilysin synthesis. 3. When dl-[1-(14)C]alanine was added to a growing culture of B. subtilis, (14)C was incorporated into bacilysin, which contains an N-terminal alanine residue. 4. Under similar conditions virtually no (14)C was incorporated into bacilysin from dl-[2-(14)C]tyrosine, l-[U-(14)C]tyrosine or [1-(14)C]acetate, although these compounds were used by the cell for the biosynthesis of other substances. These results indicate that neither tyrosine nor acetate is a precursor of the fragment of bacilysin which yields tyrosine on hydrolysis with hot 6n-hydrochloric acid. 5. The tyrosine-yielding fragment of bacilysin was labelled with (14)C from [1,6-ring-(14)C(2)]shikimic acid. The biosynthesis of bacilysin thus appears to involve a diversion from the pathway leading to aromatic amino acids at the shikimic acid stage, or a subsequent one.     

Regulation of nitrogen fixation. Nitrogenase-derepressed mutants of Klebsiella pneumoniae

1. A new procedure is described for selecting nitrogenase-derepressed mutants based on the method of Brenchley et al. (Brenchley, J. E., Prival, M. J. and Magasanik, B. (1973) J. Biol. Chem. 248, 6122–6128) for isolating histidase-constitutive mutants of a non-N₂-fixing bacterium.2. Nitrogenase levels of the new mutants in the presence of NH₄⁺ were as high as 100% of the nitrogenase activity detected in the absence of NH₄⁺.3. Biochemical characterization of these nitrogen fixation (nif) derepressed mutants reveals that they fall into three classes. Three mutants (strains SK-24, 28 and 29), requiring glutamate for growth, synthesize nitrogenase and glutamine synthetase constitutively (in the presence of NH₄⁺). A second class of mutants (strains SK-27 and 37) requiring glutamine for growth produces derepressed levels of nitrogenase activity and synthesized catalytically inactive glutamine synthetase protein, as determined immunologically. A third class of glutamine-requiring, nitrogenase-derepressed mutants (strain SK-25 and 26) synthesizes neither a catalytically active glutamine synthetase enzyme nor an immunologically cross-reactive glutamine synthetase protein.4. F-prime complementation analysis reveals that the mutant strains SK-25, 26, 27, 37 map in a segment of the Klebsiella chromosome corresponding to the region coding for glutamine synthetase. Since the mutant strains SK-27 and SK-37 produce inactive glutamine synthetase protein, it is concluded that these mutations map within the glutamine synthetase structural gene.