RESUMO
The Bacillus subtilis strain A1/3 shows exceptionally diverse antibiotic capacities compared to other B. subtilis strains. To analyze this phenomenon, mutants for the putative pantotheinyltransferase gene (pptS), and for several genes involved in non-ribosomal peptide synthesis and polyketide synthesis were constructed and characterized, using bioassays with blood cells, bacterial and fungal cells, and mass spectrometry. Among at least nine distinct bioactive compounds, five antibiotics and one siderophore activity were identified. The anti-fungal and hemolytic activities of strain A1/3 could be eliminated by mutation of the fen and srf genes essential for the synthesis of fengycins and surfactins. Both pptS- and dhb -type mutants were defective in iron uptake, indicating an inability to produce a 2,3-dihydroxybenzoate-type iron siderophore. Transposon mutants in the malonyl CoA transacylase gene resulted in the loss of hemolytic and anti-fungal activities due to the inhibition of bacillomycin L synthesis, and this led to the discovery of bmyLD-LA-LB* genes. In mutants bearing disruption mutations in polyketide (pksM- and/or pksR -like) genes, the biosynthesis of bacillaene and difficidins, respectively, was inactivated and was accompanied by the loss of discrete antibacterial activities. The formation of biofilms (pellicles) was shown to require the production of surfactins, but no other lipopeptides, indicating that surfactins serve specific developmental functions.
Assuntos
Antibacterianos/biossíntese , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Biofilmes , Ferro/metabolismo , Biossíntese de Peptídeos Independentes de Ácido Nucleico/genética , Proteína de Transporte de Acila S-Maloniltransferase , Aciltransferases/genética , Sequência de Aminoácidos , Bacillus subtilis/metabolismo , Bioensaio , Primers do DNA , Elementos de DNA Transponíveis/genética , Componentes do Gene , Lipopeptídeos , Lipoproteínas , Espectrometria de Massas , Dados de Sequência Molecular , Mutagênese , Mutação/genética , Peptídeos Cíclicos , Plasmídeos/genética , Policetídeo Sintases/genética , Especificidade da Espécie , Transferases (Outros Grupos de Fosfato Substituídos)/genéticaRESUMO
Two amylolytic active protein fractions (named alpha-amylase 1 and alpha-amylase 2) were isolated from the bacterium Thermoactinomyces vulgaris strain 94-2A. alpha-Amylase 1 had a molecular mass of 51.6 kDa, whereas alpha-amylase 2 consists of two fragments which have molecular masses of 17.0 and 34.6 kDa, respectively. These two fragments are products from a proteolytic cleavage of alpha-amylase 1 at amino acid position 303 (tryptophan) by a serine protease (thermitase) which is also produced by T. vulgaris. The purified alpha-amylase 1 and 2 follow the Michaelis-Menten kinetics in the presence of starch as substrate with Km values of 1.37 +/- 0.07 and 1.29 +/- 0.18 mg/mL, respectively. In effect they differ in their stability characteristics. The amino acid sequence of alpha-amylase from T. vulgaris derived from DNA sequence (1) was compared with those of other alpha-amylases. It reveals high homologies to alpha-amylases from other microorganisms (e.g. B. polymyxa, A. oryzae, S. occidentalis and S. fibuligera). A three-dimensional structure model for alpha-amylase 1 on the basis of the 3 A X-ray structure of Taka-amylase was constructed.
Assuntos
Micromonosporaceae/enzimologia , Estrutura Secundária de Proteína , alfa-Amilases/química , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Sequência Conservada , Cristalografia por Raios X , Brometo de Cianogênio , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Homologia de Sequência de Aminoácidos , Serina Endopeptidases , Tripsina , alfa-Amilases/biossíntese , alfa-Amilases/isolamento & purificaçãoRESUMO
We isolated the gene amyE(TV1) from Thermoactinomyces vulgaris 94-2A encoding a nonglucogenic alpha-amylase (AmyTV1). A chromosomal DNA fragment of 2,247 bp contained an open reading frame of 483 codons, which was expressed in Escherichia coli and Bacillus subtilis. The deduced amino acid sequence of the AmyTV1 protein was confirmed by sequencing of several peptides derived from the enzyme isolated from a T. vulgaris 94-2A culture. The amino acid sequence was aligned with several known alpha-amylase sequences. We found 83% homology with the 48-kDa alpha-amylase part of the Bacillus polymyxa beta-alpha-amylase polyprotein and 50% homology with Taka amylase A of Aspergillus oryzae but only 45% homology with another T. vulgaris amylase (neopullulanase, TVA II) recently cloned from strain R-47. The putative promoter region was characterized with primer extension and deletion experiments and by expression studies with B. subtilis. Multiple promoter sites (P3, P2, and P1) were found; P1 alone drives about 1/10 of the AmyTV1 expression directed by the native tandem configuration P3P2P1. The expression levels in B. subtilis could be enhanced by fusion of the amyE(TV1) coding region to the promoter of the Bacillus amyloliquefaciens alpha-amylase gene.
