Analysis of surfactin synthetase subunits in srfA mutants of Bacillus subtilis OKB105

Vollenbroich, Dirk; Mehta, Neena; Zuber, Peter; Vater, Joachim; Kamp, Roza Maria

doi:10.1128/jb.176.2.395-400.1994

Cited by 44 publications

(39 citation statements)

References 17 publications

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“…This domain organization implied that the nonribosomal peptide generated by this enzymology would contain an N-terminal lipid. This hypothesis was based on precedent whereby an N-terminal C domain recognizes a fatty acyl-coenzyme A or fatty acyl-acyl carrier protein derivative from primary metabolism and condenses the first amino acid tethered to the NRPS with the thioesterified fatty acid to generate a lipidated nonribosomal peptide (21,38,63).…”

Section: Resultsmentioning

confidence: 99%

Identification of a Biosynthetic Gene Cluster and the Six Associated Lipopeptides Involved in Swarming Motility ofPseudomonas syringaepv. tomato DC3000

Berti

Greve²,

Christensen³

et al. 2007

J Bacteriol

119

127

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Pseudomonas species are known to be prolific producers of secondary metabolites that are synthesized wholly or in part by nonribosomal peptide synthetases. In an effort to identify additional nonribosomal peptides produced by these bacteria, a bioinformatics approach was used to "mine" the genome of Pseudomonas syringae pv. tomato DC3000 for the metabolic potential to biosynthesize previously unknown nonribosomal peptides. Herein we describe the identification of a nonribosomal peptide biosynthetic gene cluster that codes for proteins involved in the production of six structurally related linear lipopeptides. Structures for each of these lipopeptides were proposed based on amino acid analysis and mass spectrometry analyses. Mutations in this cluster resulted in the loss of swarming motility of P. syringae pv. tomato DC3000 on medium containing a low percentage of agar. This phenotype is consistent with the loss of the ability to produce a lipopeptide that functions as a biosurfactant. This work gives additional evidence that mining the genomes of microorganisms followed by metabolite and phenotypic analyses leads to the identification of previously unknown secondary metabolites.Nonribosomal peptide synthetase (NRPS) enzymology is involved in the biosynthesis of many natural products with diverse biological activities, such as antifungal, antibacterial, anticancer, immunosuppressant, and metal chelation (reviewed in references 18 and 55). While the chemical structures of the natural products biosynthesized by NRPSs are diverse and explain their wide-ranging biological activities, the core enzymology used to synthesize these molecules is conserved. This conservation comes from NRPSs consisting of a set of repeating core protein domains grouped into enzymatic modules, with each module typically controlling the incorporation of one amino acid precursor into the nonribosomal peptide. The structural diversity of the NRPS-synthesized natural products comes from variations in the number and order of the modules, alterations in the substrate incorporated by the modules, and the potential addition of catalytic domains into modules that result in modifications to the growing peptide chain.The core domains that are repeated for each NRPS module are the adenylation (A) domain, peptidyl carrier protein (PCP) domain, and condensation (C) domain. The A domains are commonly referred to as the "gatekeepers" of a module, because they recognize the substrate that is incorporated into the growing peptide chain. In a significant breakthrough in understanding NRPS enzymology, an amino acid substrate specificity code was identified that enables one to deduce the amino acid that is likely recognized by an A domain, given the protein sequence of the A domain (8,9,35,60). This offers the ability to predict which module incorporates each amino acid of a nonribosomal peptide of known structure, but it also provides a means for proposing what amino acid is recognized by an NRPS module of unknown function. Once it recognizes the amino acid su...

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Section: Resultsmentioning

confidence: 99%

Identification of a Biosynthetic Gene Cluster and the Six Associated Lipopeptides Involved in Swarming Motility ofPseudomonas syringaepv. tomato DC3000

Berti

Greve²,

Christensen³

et al. 2007

J Bacteriol

119

127

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“…ZB307A is a prototrophic derivative of JH642, which is lysogenic for SPf8c2del2:: Tn917::pSK10A6 (18). Strain LAB848 bears the AsrfA:: pNAC14 mutation, which is a 19-kb deletion of srfAA and srfAB DNA (15).…”

Section: Materials and Methods Bacterial Strains And Plasmids Eschermentioning

confidence: 99%

Identification of comS, a gene of the srfA operon that regulates the establishment of genetic competence in Bacillus subtilis.

D’Souza

Nakano

Zuber

1994

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

145

117

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Genetic competence (the ability to internalize exogenous DNA) in Bacillus subtffis is dependent on a regulatory pathway that activates the expression of a battery of competence-specific genes. The srfA operon, encoding the subunits of surfactin synthetase, which catalyzes the nonribosomal synthesis of the peptide antibiotic sirfactin, also functions in the competence regulatory pathway. The DNA encoding only one of the seven amino acid-activating doma of surfactin synthetase, the valine-activating domain (srfABI), is necessary for competence. Deletion analysis revealed that a 569-bp fragment of srfABl, fused to the srfA promoter, complements a srfA deletion mutation (AsrfA) with respect to competence. This fragment contains an open reading frame consisting of 46 amino acids (of46), which is out of frame with srfABl. A frameshift mutation in srfAB upstream of orf46 has no effect on competence but a frameshift and nonsense mutation in odf46 resulted in failure to complement the AsrfA mutation. These results indicate that orf46 encodes the srfAassociated competence regulatory factor. Computer-aided analysis of the putative orf46 product (ComS) shows simily to the homeodomain of the POU domain class of eukaryotic transcriptional regulators.

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“…From sequence homology, cores 2 and 4, SGTTGKPKG and TGD, are believed to be involved in ATP binding and hydrolysis (58), whereas the serine of core 6, LGGHSL, has been shown to be the site of thioester formation in the GrsB-Val and GrsB-Leu domains, the second and fourth domains in gramicidin S synthetase 2 (54). Furthermore, the serine of this core in the first to fourth domain of the surfactin-synthesizing enzymes is indispensable for the biosynthesis of surfactin and thioester formation (11,61,62). Core 6 is present only in the thioester-forming enzymes, not in the adenylate-forming enzymes.…”

mentioning

confidence: 99%

Analysis of core sequences in the D-Phe activating domain of the multifunctional peptide synthetase TycA by site-directed mutagenesis

1994

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The D-phenylalanine-activating enzyme tyrocidine synthetase I (TycA) from Bacilus brevis ATCC 8185 was overexpressed in Escherichia coli, purified to homogeneity, and assayed for ATP-PP. exchange and covalent binding of phenylalanine by the thiotemplate mechanism. Amino acid exchanges in four different cores of TycA created by site-directed mutagenesis revealed the amino acid residues involved in aminoacyladenylate formation and in covalent thioester formation. Mutations in the putative ATP-binding site SGTTGKPKG caused a decreased phenylalanine-dependent ATP-PP1 exchange activity to 10%v of the wild-type level for a Lys-186-to-Arg substitution and an almost complete loss of activity (<1%) for a Lys-186-to-Thr exchange. Under sporulation-inducing growth conditions, Bacillus brevis ATCC 8185 produces the peptide antibiotics tyrocidine and linear gramicidin (18). Both compounds are produced nonribosomally by large multifunctional enzymes via the protein thiotemplate mechanism (32). The enzymes involved in the biosynthesis of linear gramicidin have not been completely characterized (2, 23), but the biosynthesis of tyrocidine has been studied in detail (26,27,29,30,49). The cyclic decapeptide tyrocidine (D-Phe-Pro-Phe-D-Phe-Asn-Glu-Tyr-ValOrn-Leu-)c is synthesized by the aid of three multifunctional enzymes; tyrocidine synthetases 1 (TycA), 2 (TycB), and 3 (TycC) activate and polymerize the 10 substrate amino acids.TycA initiates peptide synthesis by activation of L-phenylalanine to the aminoacyladenylate by using ATP as the AMP donor. The activated phenylalanyladenylate is then covalently bound to TycA by a thioester linkage. After racemization, the D-isomer of phenylalanine is transferred from TycA to an activated proline residue on TycB. TycB activates proline and links it to two molecules of phenylalanine, while TycC, the third multienzyme, is responsible for the activation and condensation of the remaining six amino acids. Successive transpeptidation is achieved with the aid of the cofactor 4'-phosphopantetheine, which acts as an internal transport system for the growing peptide chain. To date, the presence of 4'-phosphopantetheine as a cofactor has been demonstrated for TycB and TycC (28, 30) but not for TycA. The cloning (34) and sequencing (64) of the tycA gene, however, revealed a putative 4'-phosphopantetheine-binding site similar to those found in grsB (58) and srfA (7,12 The tycA gene is located at the 5' end of the tyc operon (39), which is transcriptionally induced when cells enter the stationary phase of growth. Studies on the tyc promoter, which is located 330 bp upstream of the tycA initiation codon, revealed its dependence on the SpoOA-AbrB regulatory system (13,35,36,46). The nucleotide sequence of tycA codes for a protein of 1,087 residues with a calculated mass of 126 kDa.Sequence comparisons of TycA with other peptide synthetases and with a family of carboxy acid-activating enzymes revealed a high degree of similarity within a region of about 600 amino acids, which was designated the functional...

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Analysis of surfactin synthetase subunits in srfA mutants of Bacillus subtilis OKB105

Cited by 44 publications

References 17 publications

Identification of a Biosynthetic Gene Cluster and the Six Associated Lipopeptides Involved in Swarming Motility ofPseudomonas syringaepv. tomato DC3000

Identification of a Biosynthetic Gene Cluster and the Six Associated Lipopeptides Involved in Swarming Motility ofPseudomonas syringaepv. tomato DC3000

Identification of comS, a gene of the srfA operon that regulates the establishment of genetic competence in Bacillus subtilis.

Analysis of core sequences in the D-Phe activating domain of the multifunctional peptide synthetase TycA by site-directed mutagenesis

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