Massetolide A biosynthesis in Pseudomonas fluorescens

Abstract

Massetolide A is a cyclic lipopeptide (CLP) antibiotic produced by various Pseudomonas strains from diverse environments. Cloning, sequencing, site-directed mutagenesis, and complementation showed that massetolide A biosynthesis in P. fluorescens SS101 is governed by three nonribosomal peptide synthetase (NRPS) genes, designated massA, massB, and massC, spanning approximately 30 kb. Prediction of the nature and configuration of the amino acids by in silico analysis of adenylation and condensation domains of the NRPSs was consistent with the chemically determined structure of the peptide moiety of massetolide A. Structural analysis of massetolide A derivatives produced by SS101 indicated that most of the variations in the peptide moiety occur at amino acid positions 4 and 9. Regions flanking the mass genes contained several genes found in other Pseudomonas CLP biosynthesis clusters, which encode LuxR-type transcriptional regulators, ABC transporters, and an RND-like outer membrane protein. In contrast to most Pseudomonas CLP gene clusters known to date, the mass genes are not physically linked but are organized in two separate clusters, with massA disconnected from massB and massC. Quantitative real-time PCR analysis indicated that transcription of massC is strongly reduced when massB is mutated, suggesting that these two genes function in an operon, whereas transcription of massA is independent of massBC and vice versa. Massetolide A is produced in the early exponential growth phase, and biosynthesis appears not to be regulated by N-acylhomoserine lactone-based quorum sensing. Massetolide A production is essential in swarming motility of P. fluorescens SS101 and plays an important role in biofilm formation.

FIG. 1.

(A) Representation of the contig assembly of BAC clones 2F11, 3G1, 2H12, 7F11, 6F12, 8G2, and 7B4. The first contig (64 kb) harbors the massA gene, and the second contig (80 kb) contains the massB and massC genes. (B) Organization of the CLP gene cluster identified in P. fluorescens SS101 by sequence analysis of BAC clones 2H12 and 7B4. The three genes designated massA, massB, and massC are responsible for massetolide A biosynthesis. In the genome of strain SS101, massA is disconnected from massB and massC. Underneath the genes are the module and domain organization of MassA, MassB, and MassC. The domains are as follows: C, condensation; A, adenylation; T, thiolation; and TE, thioesterification. Underneath the domains are the amino acids that are incorporated into the CLP peptide moiety. The number associated with the amino acid refers to the position of the amino acid in the CLP peptide chain. Triangles represent the positions of the single transposon disruptions in the massABC genes obtained by random mutagenesis. The arrows indicate putative promoter sequences, and the closed circles represent putative terminator sequences. For each of the three mass genes, double arrows indicate the locations of the primers used for Q-PCR.

FIG. 2.

Phylogenetic analysis of amino acid sequences of 51 C domains identified in the known CLP biosynthesis clusters for massetolide A (mass), arthrofactin (arf), syringomycin (syr), and syringopeptin (syp). C domains predicted to have both condensation and epimerase activities are referred to as dual C/E domains and are indicated above the dotted line, whereas C domains predicted to function only in condensation (i.e., conventional C domains) are found below the dotted line. The C domains of the massetolide A biosynthesis cluster are boxed. Exceptions in this classification of C domains are indicated with an arrow followed by the upstream donor substrate. Dab, 2.4-diaminobutyric acid. C domains that are presumably involved in linking the fatty acid to the first amino acid of the CLP peptide moiety are the so-called C1 domains. The numbers at the nodes indicate the level of bootstrap support higher than 500, based on neighbor-joining analysis of 1,000 resampled data sets. The bar indicates the relative number of substitutions per site. The abbreviations in the tree indicate the gene and the C domain number, which refers to the module number in the CLP cluster.

FIG. 3.

(A) Alignment of the amino acid sequences of the 51 (C) domains identified in the known CLP biosynthesis clusters encoding the synthetases of massetolide A (mass), arthrofactin (arf), syringomycin (syr), and syringopeptin (syp). The C domains of the massetolide A biosynthesis cluster are boxed. The conserved motif (HHI/ LxxxxGD) for C domains with dual catalytic activity for condensation and epimerization is indicated by arrows. The abbreviations in the alignment indicate the gene and the C domain number, which refers to the module number in the CLP cluster. (B) Alignment of the amino acid sequences of 11 TE domains identified in known CLP biosynthesis clusters encoding the synthetases of massetolide A (mass), viscosin (visc), orfamide (orf), arthrofactin (arf), syringomycin (syr), and syringopeptin (syp). The TE domains of the massetolide A biosynthesis cluster are boxed. The conserved motif (GxSxG) for TE domains is indicated by arrows. Also indicated are Ser⁸⁰, Asp¹⁰⁷, and His²⁰⁷, which form a catalytic triad of the TE domain in the NPRS encoding surfactin biosynthesis. The abbreviations in the alignment indicate the gene and the TE domain number, referring to the first or second TE domain.

FIG. 4.

Identification of the flanking genes of massA, massB, and massC based on Blastx analysis. Indicated are the codes of the genes of other CLP-producing Pseudomonas strains present in the databases PseudoDB and Pseudomonas.com and the percentage of identical amino acids. Gene homologs shown in gray have an identity higher than 50% but are not located in close vicinity to CLP biosynthesis genes. SBW25, viscosin-producing P. fluorescens strain; Pf-5, orfamide-producing P. fluorescens strain; B728a, syringomycin- and syringopeptin-producing P. syringae pv. syringae strain; MIS36, arthrofactin-producing Pseudomonas sp. strain.

FIG. 5.

(A) HPLC profile of a crude surfactant extract of P. fluorescens SS101. The main peak (peak 9) represents massetolide A. The identities of the other eight peaks are given in panel B. (B) Peak numbers, retention times, names, and masses (m/z) of the pseudomolecular ions and amino acid sequence of the peptide moiety for each of the peaks in the chromatogram shown in panel A.

FIG. 6.

(A) Growth of SS101 and the ΔmassA, ΔmassB, and ΔmassC mutants at 25°C in liquid KB medium. Circles, SS101; triangles, ΔmassA; diamonds, ΔmassB; squares, ΔmassC. Closed symbols correspond to cell density, and open symbols correspond to the surface tension of the cell-free culture supernatant. Error bars represent standard deviations. OD600, optical density at 600 nm. (B) Transcript levels of massA, massB, and massC corrected for the transcript levels of the housekeeping gene rpoD [ΔC_T = C_{T(mass gene)} − C_T(rpoD)]. (C and D) Transcript levels of massA, massB, and massC in SS101 and in the ΔmassA, ΔmassB, and ΔmassC mutants after 12 h (C) and 16 h (D) of growth. For each time point, transcript levels are presented relative to the transcript level in wild-type SS101 (log RQ), with RQ = 2^{[ΔC_T(mutant) − ΔC_{T(wild type)}]}. For each time point, mean values for four biological replicates are given. Error bars represent the standard errors of the means.

FIG. 7.

Role of massetolide A in biofilm formation on an artificial surface by P. fluorescens SS101, its mutants, and the mutants complemented with massA or massBC. Wells of microtiter plates were filled with 200 μl of KB broth and inoculated with strain SS101 or its mutants at an initial density of 1 × 10⁸ cells/ml. After incubation for 24 h at 25 °C, cells were stained with crystal violet, wells were washed, and cells attached to the walls of the microtiter wells were quantified spectrophotometrically (optical density at 600 nm [OD600]). Asterisks indicate statistically significant (P < 0.05) differences relative to wild-type SS101. Error bars represent the standard errors of the means.