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...

The structural and functional organization of the fengycin synthetase system from B. subtilis b213 has been characterized in detail and correlated with the corresponding pps and fen genes in B. subtilis strains 168, A1/3 and F29-3. Biosynthesis of the peptide part of fengycin involves five multifunctional modular proteins that assemble the lipopeptide chain using a nonribosomal, multiple carrier thiotemplate mechanism.

The adaptation and application of the Escherichia coli T7 RNA polymerase system for regulated and promoter-specific gene expression in Bacillus subtilis is reported. The expression cassette used in Bacillus subtilis was tightly regulated and T7 RnA polymerase (T7 RNAP)appeared 30 minutes after induction. The efficiency of T7 promoter-specific gene expression in B.subtilis was studied using one secretory and two cytosolic proteins of heterologous origin. The accumulation of E. coli beta-galactosidase, as well as a 1,4-beta-glucosidase from Thermoanaerobacter brockii in B. subtilis after T7 RNAP induction was strongly enhanced by rifampicin inhibition of host RNAP activity. The alpha-amylase of Thermactinomyces vulgaris, a secretory protein, was found to accumulate in the culture supernatant up to levels of about 70 mg/l 10-20 h after T7 RNAP induction, but was also deposited in cellular fractions. The addition of rifampicin inhibited chi-amylase secretion, but unexpectedly, after a short period, also prevented its further (intra)cellular accumulation.

A series of 33 single and mosaic hybrid alpha-amylases was constructed from the genes amyBA or amyLI, encoding the alpha-amylases from Bacillus amyloliquefaciens (AmyBA) and Bacillus licheniformis (AmyLI). The hybrid proteins, consisting of the entire alpha-amylase sequence with a variable portion of AmyBA or AmyLI origin, were characterized in order to find enzymes with new properties (thermostability, temperature and pH optima, and substrate specificity), and to localize the amino acid sequence regions responsible for the changes. The thermostability of the AmyBA/AmyLI (AL-type) hybrid proteins correlated with the position and the length of the hybrid sequence. The hybrid enzymes fell into six groups retaining, in comparison to AmyBA, a certain value of the extra-thermostability of AmyLI or becoming more thermolabile than AmyBA. Four regions are proposed to contain thermostability determinants (TSDs). They map between amino acid residues 34-76, 112-142, 174-179 and 263-276 of the respective hybrid enzymes, indicating the dominance of the N-terminal half of AmyLI for these hybrid enzymes' resistance against irreversible inactivation. Two (TSD3 and TSD4) coincide with regions I and II that had already been suggested to stabilise AmyLI [Suzuki, Y., Ito, N., Yuuki, T., Yamagata, H. & Udake, S. (1989) J. Biol. Chem. 264, 18,933-18,938]. The temperature dependence of activity of the AL-type hybrid alpha-amylases was compared at pH 6.4 and pH 7.6 and the hybrid enzymes of one thermostability group were found to have similar temperature responses. A hybrid region between residues 34-76 is demonstrated to correlate with the alpha-amylases' substrate specificity, i.e. either hydrolysis or accumulation of maltohexaose. This region was therefore named the G6G5 region. The exchange of internal sequences between residues 17-201 of AmyBA by the AmyLI counterpart in ALA-type mosaic hybrid alpha-amylases, with one exception (ALA99-429), unexpectedly destabilized the respective ALA-type hybrids. Two of these hybrid alpha-amylases (ALA17-151 and ALA76-151) were less thermostable than AmyBA, while others (ALA112-151, ALA112-201) were enzymically inactive. These data support specific roles of the predicted A1-B domain portion between residues 17-201 of those Bacillus alpha-amylases probably for correct folding and enzymic activity.