ABSTRACT
The study was undertaken to determine the effects of HLA-B27 subtypes on susceptibility to ankylosing spondylitis (AS) in Taiwan Chinese, a polymerase chain reaction-restrictive fragment length polymorphism (PCR-RFLP) method was developed for subtyping of HLA-B27. In this series, there are 62 patients with AS who were tested HLA-B27 positive serologically and 738 normal persons over the age of 65. Among the 738 normal controls, 42 (5.7%) were HLA-B27 positive. There were six (14.3%) homozygous for B2704, 18 (42.9%) heterozygous for B2704, 2 (4.8%) double heterozygous for B2704 and B2705, one (2.3%) double heterozygous for B2704 and B2706, 2 (4.8%) homozygous for B2705, 11 (26.1%) heterozygous for B2705, and 2 (4.8%) heterozygous for B2706. In our patients with AS, 37 (59.7%) were homozygous for B2704 and 25 (40.3%) were heterozygous for B2704. The HLA-B27 carrier rate in Taiwan healthy old persons is estimated at 5.7%. Susceptibility to AS is determined by homozygosity for B2704. However, B2705 may be an indicator of protection against AS in Taiwan Chinese.
Subject(s)
Genetic Predisposition to Disease , HLA-B27 Antigen/genetics , Spondylitis, Ankylosing/genetics , Aged , Alleles , HLA-B27 Antigen/classification , Homozygote , Humans , TaiwanABSTRACT
A 37-kb DNA fragment containing five fengycin synthetase genes, including fenC, fenD, fenE, fenA, and fenB, was cloned and sequenced. Among these genes, fenC encodes a fengycin synthetase 2,560 amino acids long with an estimated molecular mass of 287 kDa. This protein contains two amino acid activation modules, FenC1 and FenC2, which activate L-glutamic acid and L-ornithine, respectively. Primer extension, using mRNA isolated from the log-phase cells, identified a transcription start site located 86 nucleotides upstream from the initiation codon of fenC, implying that a promoter is located upstream from the start site. Primer extension using total RNA isolated from stationary-phase cells also identified a transcription start site located 61 nucleotides upstream from the initiation codon of fenC. Gene fusion studies demonstrated that in nHA medium, the cells transcribe the fengycin synthetase genes at two different stages of cell growth. The promoter is active during the log phase, and the activity reaches the highest level during the late log phase. The activity decreases sharply but is maintained at a low level for approximately 24 h after cells enter the early stationary phase. The results of this investigation also suggest that the transcription of fenC is positively regulated during the late log phase. Results presented herein provide further insight into fengycin synthesis by B. subtilis F29-3.
Subject(s)
Bacillus subtilis/enzymology , Bacillus subtilis/genetics , Isoenzymes/genetics , Peptide Synthases/genetics , Adenine/metabolism , Base Sequence , Catalytic Domain , Cloning, Molecular , Cosmids , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Enzymologic , Isoenzymes/metabolism , Molecular Sequence Data , Mutagenesis, Insertional , Operon , Peptide Synthases/metabolism , Sequence Analysis, DNA , Substrate Specificity , Transcription, GeneticABSTRACT
A fengycin synthetase gene, fenB, has been cloned and sequenced. The protein (FenB) encoded by this gene has a predicted molecular mass of 143.6 kDa. This protein was overexpressed in Escherichia coli and was purified to near homogeneity by affinity chromatography. Experimental results indicated that the recombinant FenB has a substrate specificity toward isoleucine with an optimum temperature of 25 degrees C, an optimum pH of 4.5, a Km value of 922 microM, and a turnover number of 236 s(-1). FenB also consists of a thioesterase domain, suggesting that this protein may be involved in the activation of the last amino acid of fengycin.
Subject(s)
Antifungal Agents/biosynthesis , Bacillus subtilis/genetics , Peptide Synthases/genetics , Amino Acid Sequence , Aminoacylation , Bacillus subtilis/enzymology , Chromatography, Affinity , Cloning, Molecular , Escherichia coli/genetics , Genes, Bacterial , Hydrogen-Ion Concentration , Isoleucine/metabolism , Kinetics , Lipopeptides , Lipoproteins/biosynthesis , Molecular Sequence Data , Molecular Weight , Peptide Synthases/chemistry , Peptide Synthases/isolation & purification , Peptide Synthases/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Substrate Specificity , TemperatureABSTRACT
A total of 20 Bacillus subtilis F29-3 mutants defective in fengycin biosynthesis was obtained by Tn917 mutagenesis. Cloning and mapping results showed that the transposon in these mutants was inserted in eleven different locations on the chromosome. We were able to use the chromosomal sequence adjacent to the transposon as a probe to screen for cosmid clones containing the fengycin biosynthesis genes. One of the clones obtained, pFC660, was 46 kb long. Eight transposon insertion sites were mapped within this plasmid. Among the eleven different mutants analyzed, four mutants had Tn917 inserted in regions which encoded peptide sequences similar to part of gramicidin S synthetase, surfactin synthetase, and tyrocidine synthetase. Our results suggest that fengycin is synthesized nonribosomally by the multienzyme thiotemplate mechanism.
Subject(s)
Antifungal Agents/biosynthesis , Bacillus subtilis/genetics , Bacterial Proteins , DNA Transposable Elements/genetics , Adenosine Triphosphatases/genetics , Amino Acid Isomerases/chemistry , Amino Acid Isomerases/genetics , Amino Acid Sequence , Bacillus subtilis/enzymology , Chromosome Mapping , Cloning, Molecular , Conserved Sequence/genetics , Lipopeptides , Lipoproteins/biosynthesis , Lipoproteins/genetics , Molecular Sequence Data , Multienzyme Complexes/chemistry , Multienzyme Complexes/genetics , Mutagenesis, Insertional , Peptide Synthases/chemistry , Peptide Synthases/genetics , Sequence Homology, Amino Acid , Transformation, GeneticABSTRACT
A Tn917 derivative was constructed for the purposes of mutagenesis and cloning of Bacillus subtilis genes. This transposon, Tn917ac1 (4.6 kb), consisted of terminal inverted repeats of Tn917, the res sequence, a ColE1 origin of replication (ori) and two drug-resistance genes. The plasmid carrying this transposon, named pD917, contained the erm-tnpR-tnpA gene cluster of Tn917 and a temperature-sensitive ori of pE194. For the purpose of mutagenesis, transposition of Tn917ac1 was induced by culturing strains harboring pD917 in a medium containing a low concentration of erythromycin. Cells with a Tn917ac1 insertion in the chromosome were selected on agar containing chloramphenicol after heat treatment to eliminate the plasmidic form of pD917. DNA fragments adjacent to Tn917ac1 could be cloned by restriction digestion of the chromosomal DNA and by transforming the self-ligated restriction fragments into Escherichia coli. Sequence analysis revealed that Tn917ac1 was integrated into the chromosome of B. subtilis by transposition in a recE strain and by transposition or integration of pD917 in a wild-type strain. Tn917ac1 has been demonstrated to be useful for mutating and cloning of the genes involved in the biosynthesis of fengycin in B. subtilis F29-3.
Subject(s)
Bacillus subtilis/genetics , Cloning, Molecular/methods , DNA Transposable Elements , Genes, Bacterial , Mutagenesis, Insertional/methods , Base Sequence , Blotting, Southern , DNA, Recombinant , Escherichia coli , Molecular Sequence Data , PlasmidsABSTRACT
An antifungal metabolite, bacereutin, was isolated from culture filtrate of Bacillus cereus CHU 130. The bacterium was isolated from soils collected in Changhwa County, Taiwan, and was grown in soybean meal-mannitol broth for production of the antibiotic metabolite. The antibiotic metabolite was isolated by adsorption column chromatography of Amberite XAD-2 and was purified by passing through the chromatographic columns packed with Dowex 50W-X8, Sephadex LH 20 and Biogel P-2. The antibiotic metabolite was soluble in water and 87% acetone, and was slightly soluble in methanol, but was not dissolved in n-propanol, n-butanol, acetone, benzene and ethyl acetate. The antibiotic metabolite was a heat-stable and ninhydrin-positive substance. The antibiotic activities of bacereutin were tested by means of the agar-diffusion plate method. The antibiotic metabolite inhibited the growth of Saccharomyces cerevisiae CHU 1, Paecilomyces variotii CHU 6, Rhizomucor miehei CHU 40 and Fusarium oxysportum CHU 98. Bacereutin was a ninhydrin-positive antifungal antibiotic.
