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. 2014 Jun;355(2):170-6.
doi: 10.1111/1574-6968.12457. Epub 2014 Jun 3.

Involvement of both PKS and NRPS in antibacterial activity in Lysobacter enzymogenes OH11

Juan Zhang  1 , Liangcheng Du, Fengquan Liu, Feifei Xu, Baishi Hu, Vittorio Venturi, Guoliang Qian
Affiliations

Involvement of both PKS and NRPS in antibacterial activity in Lysobacter enzymogenes OH11

Juan Zhang et al. FEMS Microbiol Lett. 2014 Jun.

Abstract

Polyketides and nonribosomal peptides represent two large families of natural products (NPs) with diverse structures and important functions. They are synthesized by polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS), respectively. Lysobacter enzymogenes is emerging as a novel biocontrol agent against pathogens of crop plants and a new source of bioactive NPs, such as antibacterial antibiotic WAP-8294A2 and antifungal antibiotic HSAF. Genome survey of strain OH11, a Chinese L. enzymogenes isolate, detected four novel PKS, NRPS or hybrid gene clusters, designed as cluster A to D. We further individually mutated five genes (PKS or NRPS) located in these four gene clusters and showed that a PKS gene in cluster A and an NRPS gene in cluster D were involved in the antibacterial activity via a WAP-8294A2 dependent way. The data also showed that none of the five genes was associated with antifungal activity and the regulation of HSAF biosynthesis. Our results reveal the unusual regulatory role of these PKS and NRPS genes that were discovered from genome mining in L. enzymogenes.

Keywords: Lysobacter enzymogenes; WAP-8294A2; nonribosomal peptide synthetase; polyketide synthase.

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Figures

Figure 1
Figure 1. Four gene clusters containing PKS-NRPS in Lysobacter enzymogenes OH11
The PKS and NRPS genes were shown in red and blue respectively in each cluster. Of them, the genes highlighted with black circle were selected for gene deletion. The detailed information for each gene in these clusters (A to D) is provided in Table S4.
Figure 2
Figure 2. Antibacterial activity of the wild-type OH11 and the mutant strains
Mutation of 2856 and 3685 impaired the wild-type antibacterial activity against Clavibacter michiganensis, a Gram-positive bacterium causing potato ring rot. ΔWAP is a WAP-8294A2-deficient mutant (Zhang et al., 2011), which was used as a negative control.
Figure 3
Figure 3. Growth curve of the wild-type OH11 and mutant strains in 1/10 TSB broth
Mutation of 2856 and 3685 slightly impaired growth ability at logarithmic phase (approximately from 8 to 18 h), but not at stationary phase (approximately from 18 to 24 h).
Figure 4
Figure 4. Mutation of 2856 and 3685 impaired the production level of antibacterial antibiotic WAP-8294A2 in Lysobacter enzymogenes
A: HPLC determination of WAP-8294A2 production level. B: Antibacterial activity of WAP-8294A2 extracts (used for HPLC analysis) against Clavibacter michiganensis. C: Determination of transcriptional level of waps1 of the wild-type OH11 and the two mutants. Methanol, the solvent for dissolving WAP-8294A2, was used as a negative control in part A and B. The gene waps1 (annotated as 2168 in the genome of OH11) is a key gene responsible for WAP-8294A2 biosynthesis (Zhang et al., 2011). The arrow indicated the peak of WAP-8294A2. For each part, triple replicates for each treatment were used, and the experiment was performed three times. In part C, vertical bars represent standard errors. Different letters above bars indicate a significant difference in gene expression between the wild-type strain OH11 and the tested mutants (P<0.05; t-test).
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