Dirk Vollenbroich

Structural and functional organization of the fengycin synthetase multienzyme system from Bacillus subtilis b213 and A1/3

BACKGROUND Bacillus subtilis strains produce a broad spectrum of lipopeptides that are potent biosurfactants and have specific antimicrobial and antiviral activities. The cyclic lipodecapeptide fengycin is one such compound. Although the fengycin biosynthetic genes in B. subtilis 168 (pps genes) and F29-3 (fen genes) have been well characterized, only limited information is available about the biochemical features of the fengycin synthetase multienzyme system. RESULTS Five multifunctional peptide synthetases (Fen1-5) that catalyze biosynthesis of the peptide portion of fengycin have been purified from crude extracts of the B. subtilis b213 and A1/3 strains. These enzymes activate all fengycin amino-acid components as aminoacyl adenylates or aminoacyl thioesters. Fen1, Fen2 and Fen3 are each approximately 286 kDa, Fen4 is approximately 400 kDa and Fen 5 is approximately 140kDa; each enzyme activates a different set of L-amino acids. A five-gene cluster (fen1-5) was detected in the B. subtilis A1/3 genome that shows high homology to the pps and fen genes in B. subtilis strains 168 and F29-3. Disruption of fen4 resulted in a loss of fengycin production. The fengycin synthetase enzymes isolated from B. subtilis b213 were assigned to the corresponding A1/3 fen genes by their amino-terminal sequences. CONCLUSIONS 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.

Analysis of a mutant amino acid-activating domain of surfactin synthetase bearing a serine-to-alanine substitution at the site of carboxylthioester formation

The reactive serine of the TGGHSL thioester binding motif of the first amino acid‐activating domain of surfactin synthetase was replaced by alanine using site‐directed mutagenesis. The multienzyme from cells of the resulting mutant lost its ability for thioester formation with l‐Glu and was therefore inactive in surfactin production. The thiolation reactions catalyzed by the other amino acid‐activating domains of surfactin synthetase were not affected by the mutation. The results show that l‐Glu is acativated at the first domain of surfactin synthetase, and give further evidence that a serine residue is essential for substrate amino acid activation at the reaction centers of peptide synthetases.

The modular organization of multifunctional peptide synthetases.

Gramicidin S synthetase 2 from B. brevis was affinity labeled at its valine thiolation center with the thiol reagent N-[3H]ethylmaleimide. From a tryptic digest of the enzyme–inhibitor complex a radioactive fragment was isolated in pure form by two reversed-phase HPLC steps. It was identified by liquid-phase N-terminal sequencing in combination with electrospray mass spectrometry (ESI-MS) as a hexadecapeptide containing the thiolation motif LGG(H/D)S(L/I). By ESI-MS it was demonstrated that a 4′-phosphopantetheine cofactor was attached to this fragment at its reactive serine. These results are consistent with the “Multiple Carrier Model” of nonribosomal peptide biosynthesis. Site-specific mutagenesis has been performed in thiolation, elongation, and epimerization motifs of some of the modules of surfactin synthetase from B. subtilis to clarify the function of prominent conserved amino acid residues in the intermediate steps of peptide biosynthesis. The modular structure of multifunctional peptide synthetases is discussed.

Application of surfactin for mycoplasma inactivation in virus stocks.

Dear Editor: A novel procedure for the purification of virus stocks from mycoplasma contaminations was established using the lipopeptide antibiotic and biosurfactant surfactin, composed of a 3-hydroxy-13-rnethylmyristic acid forming a lactone ring system with an anionic heptapeptide as antimycoplasma agent. We combined the mycoplasma inactivation procedure for cell cultures with the results of antiviral studies (Vollenbroich et al., submitted for publication). The enveloped viruses SHV-1 and BVDV and the nonenveloped viruses EMCV and PPV were investigated. Mycoplasma contamination of ceils and virus stocks were monitored by poly chain reaction (PCR) (7,11). For the inactivation of mycoplasmas in cell cultures, different more or less efficient techniques are available, but the most effective procedure published is the treatment with antibiotics (2,9,10). In virological research, one of the common sources for contaminations are virus stocks harvested from mycoplasma-contaminated cell cultures, collected in the field, or passaged through animals. Therefore~ in virology not only the cell cultures but also the virus stocks must be freed from mycoplasmas. Only a few techniques are published for the treatment of virus stocks (4,5,6). Source ofsurfactin. Surfactin was obtained from culture supematants of Bacillus subtilis OKB105 by acid precipitation, extraction with methanol, charcoal treatment, and geI filtration on Pharmacia LH20 as described previously (1). Otherwise, suffaetin can be purchased from Biomol (Hamburg, Germany) or Sigma Chemical Co. (Deisenhofen, Germany). In our experiments, we used an autoclaved suffaetin solution of 1 nel4 in phosphate-buffered saline (PBS). Virus, cells, and culture conditions. The following cell virus systems were used: nonenveloped viruses: murine eneephalomyoearditis virus (EMCV) / Hep2 cells (ATCC CCL 231), porcine parvovirus (PPV strain NADL) 1 ST cells {ATCC CRL 1746); enveloped viruses: bovine diarrhea virus (BVDV) / KL cells (embryonal calf lung), swine herpes virus type 1 (SHY-l, Pseudorabies) / ML cells (mink lung). In the last, both cell lines are propagated in our institute for a long time. Virus stocks were a gift from Behring Research Laboratories (Mannheim, Germany) and prepared by infecting subconfluent celt monolayers with a multiplicity of infection (m.o.i.) of 10 -3. The culture supernatants were harvested when a pronounced cytopathic effect (CPE) approximately 2 to 6 d postinfection was visible. Aliquots of the cell free supernatants were stored at 70 ° C. Cell lines were propagated as monolayers in Dulbeccos modified Eagles medium (DMEM; ICN Bioinedicals GmbH, Eschwege, Germany) supplemented with 8% heat-inactivated fetal calf serum (FCS; GIBCO, Great Britain) in 25 cm 2 tissue culture flasks (Greiner GmbH, Heidelberg, Germany). Virus titration. The virus titers were determined by a standard microtitration assay. Approximately 1 to 2 × 104 cells in 100 }.tl medium were plated into each well of a 96-wetl mierotiter plate (Nunc, Copenhagen, Denmark). The virus solution was serially diluted 1:5 or 1:10 in culture medium and 100 pl of each dilution were added to each of 8 wells of a 96-well microtiter plate. The microtiter plates were incubated at 37 ° C and evaluated microscopically for CPE. When a pronounced CPE was visible, the 50% tissue culture infectivity doses (TC1Dso) were calculated according to Reed and Muench (8). M)coplasma detection. Mycoplasma contamination was detected by the highly sensitive PCR technique with a mycoplasma groupspecific primer pair as outlined elsewhere (7,11). M)roplasma inactivation procedure with surfactin. 0.5 ml of the virus stocks were diluted in 4.5 ml cell culture medium without FCS supplemented with 80 ~M surfactin for nonenveloped and 20 btM for enveloped viruses. The reaction mixtures were incubated at room temperature by gentle shaking for 2 h. Thereafter, subconfluent cultures of mycoplasma-free cells were infected with the surfactintreated virus. The procedure was repeated with viruses harvested from these cultures to ensure that all mycoplasmas have been removed. Mycoplasma detection and virus titer determination of samples from the culture supernatant were performed when a pronounced CPE was visible. Mycoplasma-free virus stocks were subcultivated