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.

A lantibiotic gene cluster was identified inLantibiotics are amphiphilic peptide antibiotics of bacterial origin and are nearly exclusively produced by gram-positive bacteria. They contain unusual constituents like nonproteinogenic didehydroamino acids and lanthionines (49; for reviews, see references 16, 30, 47, and 51). Out of the about 26 known lantibiotics, the nisins (A and Z) of Lactococcus lactis cheese starter organisms (6, 15) are the best-studied members which are also of commercial value (5,14,21,31,33,41). Subtilin was the first lantibiotic isolated from Bacillus subtilis ATCC 6633 (22; for review, see reference 16). A variant of subtilin (subtilin B) was found to have reduced antibiotic activity due to posttranslational succinylation of the amino group of the N-terminal tryptophan residue (7). Sublancin from B. subtilis 168 is quite different and contains a single lanthionine linkage and two disulfide bridges (43). A relative small lantibiotic, mersacidin of Bacillus sp., shows unusual properties with respect to bridging, amphiphilic character, and C-terminal modification (30). Lantibiotics are ribosomally synthesized as precursor peptides consisting of an N-terminal leader and the propeptide sequence. The latter becomes posttranslationally modified by dehydration and thioether formation (49). The biochemistry of these modifications is still unknown but is associated in one group of lantibiotics with proteins LanB and LanC (24,35) and in a second group with LanM (16, 46; for review, see reference 47). A multimeric enzyme complex consisting of LanBTC was demonstrated for subtilin and nisin to be membrane associated and to catalyze modification and transport (32, 50).The B. subtilis strain A1/3 attracted our attention due to a broad spectrum of inhibitory activities against fungi and phytoviruses (28), as well as against diverse bacteria. Notable among these is the causative agent of tomato bacterial canker, Clavibacter michiganensis (20). In this paper we report the discovery of a lantibiotic gene cluster of B. subtilis A1/3, which shows conserved character to subtilin genes but encodes two distinct lantibiotic peptides, ericin S and ericin A. Both ericins were isolated from culture supernatants of B. subtilis A1/3, studied by high-performance liquid chromatography (HPLC), peptidase digestion, and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The complete ericin gene cluster has been sequenced. Mutant studies indicated that both peptides are processed by the LanB homologue EriB. MATERIALS AND METHODSStrains, plasmids, and growth conditions. The original B. subtilis A1/3 (20, 28) contained at least two plasmids. A derivative GB709 of strain A1/3 was cured from plasmid DNA by repeated protoplasting and protoplast regeneration (8) and was used throughout these studies synonymously to A1/3, as it exhibited no detectable phenotypic differences from the parental strain. For antibiotic activity tests the following were used: B. subtilis strains DSM 402 (Spizi...

We isolated the gene amyE(TV1) from Thermoactinomyces vulgaris 94-2A encoding a nonglucogenic a-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 Bacilus 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 oa-amylase sequences. We found 83% homology with the 48-kDa a-amylase part of the Bacilus polymyxa Pj-a-amylase polyprotein and 50%o homology with Taka amylase A of AspergiUlus 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 Bacilus amyloliquefaciens a-amylase gene. A number of thermostable enzymes have been isolated from Thermoactinomyces vulganis strains. Among these were thermitase (4, 13, 14, 28, 29), a protease from the subtilisin family (2, 5, 36), and a variety of oa-amylases (pullulanases). The a-amylases of strains R47 and 42, like fungal glucoamylases (1, 15, 49, 54), hydrolyze starch and pullulan (1, 54). The a-amylase of T. vulgaris 94-2A, however, utilizes only starch and glycogen as substrates, not pullulan. a-Amylase 1 of T. vulganis 94-2A (AmyTV1) is a protein of 53 kDa and was previously shown to exhibit striking homology to Taka amylase A of Aspergillus oryzae for a short N-terminal sequence (22, 60, 67). Smaller peptides of 33 and 18 kDa have been shown to be products of limited AmyTV1 proteolysis (21). The AmyTV1 amylase is unusual because of its temperature optimum at 62.5°C in a low pH range (4.8 to 6), its relatively short half-life of about 5 min at 70°C, and the production of maltose and maltotriose in the hydrolysate, which lacks glucose (47). In order to overcome problems of poor growth and product yield in T. vulgaris 94-2A, we cloned the gene encoding the AmyTV1 amylase and performed expression studies with Bacillus subtilis. The transcription start site(s) of the gene in B. subtilis was studied after deletion of the 5'-flanking region and after its replacement with the promoter of the B. amyloliquefaciens cx-amylase gene. MATERIALS AND METHODS Bacterial strains, plasmids, and phages. The T. vulgaris strain used was originally isolated and described by Klingenberg et al. (29, 47). The strain 94-2A was selected for higher enzyme production (29). The EMBL3 lambda phage (12) was used for preparation of a T. vulgaris DNA bank in Escherichia

The use of Bacillus amyloliquefaciens for enzyme production and its exceptional high protein export capacity initiated this study where the presence and function of multiple type I signal peptidase isoforms was investigated. In addition to type I signal peptidases SipS(ba) [Meijer, W.J.J., de Jong, A., Bea, G., Wisman, A., Tjalsma, H., Venema, G., Bron, S. & van Dijl, J.M. (1995) Mol. Microbiol.17, 621–631] and SipT(ba) [Hoang, V. & Hofemeister, J. (1995) Biochim. Biophys. Acta1269, 64–68] which were previously identified, here we present evidence for two other Sip‐like genes in B. amyloliquefaciens. Same map positions as well as sequence motifs verified that these genes encode homologues of Bacillus subtilis SipV and SipW. SipU‐encoding DNA was not found in B. amyloliquefaciens. SipW‐encoding DNA was also found for other Bacillus strains representing different phylogenetic groups, but not for Bacillus stearothermophilus and Thermoactinomyces vulgaris. The absence of these genes, however, could have been overlooked due to sequence diversity. Sequence alignments of 23 known Sip‐like proteins from Bacillus origin indicated further branching of the P‐group signal peptidases into clusters represented by B. subtilis SipV, SipS‐SipT‐SipU and B. anthracis Sip3‐Sip5 proteins, respectively. Each B. amyloliquefaciens sip(ba) gene was expressed in an Escherichia coli LepBts mutant and tested for genetic complementation of the temperature sensitive (TS) phenotype as well as pre‐OmpA processing. Although SipS(ba) as well as SipT(ba) efficiently restored processing of pre‐OmpA in E. coli, only SipS(ba) supported growth at TS conditions, indicating functional diversity. Changed properties of the sip(ba) gene disruption mutants, including cell autolysis, motility, sporulation, and nuclease activities, seemed to correlate with specificities and/or localization of B. amyloliquefaciens SipS, SipT and SipV isoforms.

Two amylolytic active protein fractions (named α‐amylase 1 and α‐amylase 2) were isolated from the bacterium Thermoactinomyces vulgaris strain 94‐2A. α‐Amylase 1 had a molecular mass of 51.6 kDa, whereas α‐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 a‐amylase 1 at amino acid position 303 (tryptophan) by a serine protease (thermitase) which is also produced by T. vulgaris. The purified α‐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 α‐amylase from T. vulgaris derived from DNA sequence (1) was compared with those of other α‐amylases. It reveals high homologies to α‐amylases from other microorganisms (e.g. B. polymyxa, A. oryzae, S. occidentalis and S.fibuligera). A three‐dimensional structure model for α‐amylase 1 on the basis of the 3 Å X‐ray structure of Taka‐amylase was constructed.