Glycerol Metabolism in Bacillus subtilis: Gene-Enzyme Relationships

Bacillus subtilis mutants unable to catabolize glycerol (Glp mutants) were isolated and mapped. The location of the mutations on the chromosome was determined by a density transfer technique and confirmed by PBS1 transduction and transformation. The different mutations were ordered relative to each other. Mutations rendering the cells glycerol auxotrophic were also mapped and found not to be linked to the Glp mutations.     

Gene-Enzyme Relationships in Neurospora Invertase

A spontaneous, single-gene mutation responsible for a total lack of invertase activity in Neurospora crassa is described. The mutation is believed to lie in the structural gene for invertase, since an immunologically cross-reacting protein is made by the mutant strain. In addition, there was no evidence for a defect in regulation of invertase activity or synthesis by the following criteria. (i) The invertaseless condition was recessive in heterokaryons; (ii) no invertase inhibitor was found in mutant extracts by mixing experiments; and (iii) none of the several sugars able to induce activity in wild-type strains was able to induce activity in the mutant strain. It was also discovered that most of the wild-type enzyme (55 to 75%) cannot be washed free from the rapidly sedimenting cell debris. This finding provided additional support for the hypothesis that Neurospora invertase is located within or about the cell wall.     

Pathway for Ubiquinone Biosynthesis in Escherichia coli K-12: Gene-Enzyme Relationships and Intermediates

Seven ubiquinone-deficient mutants of Escherichia coli, each of which accumulates two phenolic precursors of ubiquinone, have been characterized, and the accumulated compounds have been identified. The mutants accumulate small quantities of 2-octaprenyl-6-methoxyphenol, which was isolated and characterized by nuclear magnetic resonance and mass spectrometry, and relatively large amounts of 2-octaprenylphenol, a compound previously identified from E. coli. They also accumulate small quantities of a compound identified as 2-(hydroxyoctaprenyl)phenol although the relevance of this compound to the biosynthesis of ubiquinone is not clear. The results of genetic analysis suggest that each of the mutants carries a mutation in a gene (designated ubiH) which is located at about min 56 on the E. coli chromosome and is co-transducible with the serA and lysB genes. Based on information obtained from this and previous studies with ubiquinone-deficient mutants, a pathway is proposed for ubiquinone biosynthesis in E. coli, and a summary of the known gene-enzyme relationships is given.     

Dominance Relationships in Mixedly Infected Bacillus subtilis

The progeny released from Bacillus subtilis cells mixedly infected with bacteriophages beta22, SP82, and SP02(c1) have been studied at varying multiplicities of infection and orders of addition and with different host strains of the bacterium. In B. subtilis 168, SP02(c1) was subordinate to both SP82 and beta22 and did not yield significant numbers of progeny even when added 5 min before the superior phage. Dominance in mixed infections of beta22 and SP82 was host-dependent. In B. subtilis 168, SP82 was dominant and greatly reduced the yield of beta22 if added simultaneously or before the subordinate partner. However, in the same mixed infection in B. subtilis SB11, beta22 was the dominant phage and totally suppressed the production of SP82 even when added 5 min after the latter.     

Biosynthesis of wall polymers in Bacillus subtilis.

Preparations of membrane plus wall derived from Bacillus subtilis W23 were used to study the in vitro synthesis of peptidoglycan and teichoic acid and their linkage to the preexisting cell wall. The teichoic acid synthesis showed an ordered requirement for the incorporation of N-acetylglucosamine from uridine 5'-diphosphate (UDP)-N-acetylglucosamine followed by addition of glycerol phosphate from cytidine 5'-diphosphate (CDP)-glycerol and finally by addition of ribitol phosphate from CDP-ribitol. UDP-N-acetylglucosamine was not only required for the synthesis of the teichoic acid, but N-acetylglucosamine residues formed an integral part of the linkage unit attaching polyribitol phosphate to the cell wall. Synthesis of the teichoic acid was exquisitely sensitive to the antibiotic tunicamycin, and this was shown to be due to the inhibition of incorporation of N-acetylglucosamine units from UDP-N-acetylglucosamine.     

Genetic Studies of Leucine Biosynthesis in Bacillus subtilis

The mutations in a series of leucine auxotrophs isolated after treatment with nitrosoguanidine, ultraviolet light, and ICR-191 have been mapped between ilvC and pheA on the Bacillus subtilis chromosome. A fine structure map of the region was constructed by transformation. Analysis of several strains by assaying levels of their leucine bioysnthetic enzymes has shown that the region encodes three enzymes. The order of the genes with respect to the biosynthetic steps catalyzed by the gene products is 1–3–2.     

Pyrimidine Synthesis in Neurospora crassa: Gene-Enzyme Relationships

A new series of pyrimidine-requiring mutants of Neurospora has been isolated and all enzymes involved in pyrimidine biosynthesis are represented by at least one mutant. Among these mutants is included a single isolate for a new locus, pyr-6. This mutant is deficient in dihydroorotase (DHOase) and represents the only enzymatic step in orotate synthesis for which no mutant previously had been found. This mutant, which mapped genetically on the right arm of linkage group V, is unlinked to any of the other pyrimidine mutants. The DHOase-deficient mutant is also characterized by an unexpected growth behavior. The pyr-1 locus has been specifically associated with a lack of dihydroorotate dehydrogenase (DHOdehase). Mutants isolated in this series for other pyrimidine loci have been related to previously isolated mutants by allelism, recombination, and accumulation studies.     

Antibacterial Agents that Inhibit Histidine Protein Kinase YycG of Bacillus subtilis

We demonstrated in vitro that YycG-YycF of Bacillus subtillis constitutes a two-component system and shows a specificity of the sensor protein for the cognate phosphorylation partner. Based on inhibition of such an autophosphorylation of YycG, we searched imidazole and zerumbone derivatives to identify the antibacterial agents such as NH125, NH126, NH127, and NH0891.     

Biosynthesis of Bacillus licheniformis penicillinase in Escherichia coli and in Bacillus subtilis.

Modified prepenicillinase was accumulated in both Escherichia coli and Bacillus subtilis treated with globomycin. Although the inhibitions of processings of prepenicillinase and prolipoprotein by globomycin in E. coli are qualitatively similar, they differ in the degree of inhibition at given concentrations of globomycin. The processing of prepenicillinase proceeds much more rapidly in E. coli than in B. subtilis.     

Biosynthesis and membrane binding of succinate dehydrogenase in Bacillus subtilis

Antibodies specific for the Mr 65,000 (flavoprotein) and the Mr 28,000 subunits of the succinic dehydrogenase (SDH) of Bacillus subtilis were obtained. By using these antibodies it was shown that both subunits accumulated in the cytoplasm during 5-aminolevulinic acid starvation of a 5-aminolevulinic acid auxotroph. In the cytoplasm the subunits were not associated since they precipitated essentially independently of each other with subunit-specific antibody. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis the cytoplasmic subunits migrated identically with the corresponding subunits from the purified membrane-bound SDH complex. Cytoplasmic subunits were pulse-labeled with L-[35S]methionine during 5-aminolevulinic acid starvation. The labeled subunits bound to the membrane when heme synthesis was resumed and also when protein synthesis was blocked by chloramphenicol before readdition of 5-aminolevulinic acid. The experiments thus demonstrated a precursor relationship between cytoplasmic subunits and the subunits of the membrane-bound SDH complex. All SDH-negative mutants isolated so far carry mutations in the citF locus. None of the mutants was found to have either the Mr 65,000 or the Mr 28,000 SDH subunits in the membrane. Four citF mutants, however, contained both subunits in the cytoplasm. Three of these mutants lacked spectrally detectable cytochrome b558. The respective mutations mapped at one end of the citF locus. These results strongly support our previous suggestion that cytochrome b558 is (part of) a membrane binding site for SDH in B. subtilis.     

枯草芽孢杆菌抗菌肽生物合成的研究进展

革兰氏阳性菌模式生物--枯草芽孢杆菌能分泌多种肽类及由肽类衍生的抗菌活性物质,按合成途径不同,可分为核糖体肽和非核糖体肽。其中,非核糖体肽分子量较小,一般为3000Da以下,其生物合成是通过多功能复合酶系--非核糖体肽链合成酶来完成的,多发生在菌体生长停止之后;而核糖体肽分子量较大,其合成多于菌体快速生长时期。非核糖体肽链合成酶和核糖体肽的合成及其调控均需基因参与,而这一系列基因就构成了各种抗菌肽生物合成的基因簇。对核糖体肽和非核糖体肽的生物合成及其相关调控机制进行了综述。     

Biosynthesis of nisin in the subtilin producer Bacillus subtilis ATCC6633

Summary Plasmids having the structural gene of nisin (nis A) combined with the subtilin or two hybrid subtilin-nisin leaders were integrated into the subtilin operon in the chromosome of a nisin-resistant and subtilin-producing strain of B. subtilis by single crossing over. Nisin was produced only when the leader consisted mainly of the nisin part. This indicates that nisin and its leader sequence might work as a couple that makes a recognizable conformation for the subtilin modification enzymes. Therefore recognition does not depend on the primary structure of the leader sequence itself.     

Histidinol Phosphate Phosphatase, Catalyzing the Penultimate Step of the Histidine Biosynthesis Pathway, Is Encoded by ytvP (hisJ) in Bacillus subtilis

The deduced product of the Bacillus subtilis ytvP gene is similar to that of ORF13, a gene of unknown function in the Lactococcus lactis histidine biosynthesis operon. A B. subtilis ytvP mutant was auxotrophic for histidine. The only enzyme of the histidine biosynthesis pathway that remained uncharacterized in B. subtilis was histidinol phosphate phosphatase (HolPase), catalyzing the penultimate step of this pathway. HolPase activity could not be detected in crude extracts of the ytvP mutant, while purified glutathione S-transferase-YtvP fusion protein exhibited strong HolPase activity. These observations demonstrated that HolPase is encoded by ytvP in B. subtilis and led us to rename this gene hisJ. Together with the HolPase of Saccharomyces cerevisiae and the presumed HolPases of L. lactis and Schizosaccharomyces pombe, HisJ constitutes a family of related enzymes that are not homologous to the HolPases of Escherichia coli, Salmonella typhimurium, and Haemophilus influenzae.     

Unusual Biosynthesis and Structure of Locillomycins from Bacillus subtilis 916

Three families of Bacillus cyclic lipopeptides—surfactins, iturins, and fengycins—have well-recognized potential uses in biotechnology and biopharmaceutical applications. This study outlines the isolation and characterization of locillomycins, a novel family of cyclic lipopeptides produced by Bacillus subtilis 916. Elucidation of the locillomycin structure revealed several molecular features not observed in other Bacillus lipopeptides, including a unique nonapeptide sequence and macrocyclization. Locillomycins are active against bacteria and viruses. Biochemical analysis and gene deletion studies have supported the assignment of a 38-kb gene cluster as the locillomycin biosynthetic gene cluster. Interestingly, this gene cluster encodes 4 proteins (LocA, LocB, LocC, and LocD) that form a hexamodular nonribosomal peptide synthetase to biosynthesize cyclic nonapeptides. Genome analysis and the chemical structures of the end products indicated that the biosynthetic pathway exhibits two distinct features: (i) a nonlinear hexamodular assembly line, with three modules in the middle utilized twice and the first and last two modules used only once and (ii) several domains that are skipped or optionally selected.