Gene Clusters Related To Biofilm Formation In Lysobacter Enzymogenes OH11

Lysobacter enzymogenes OH11 is a gram-negative bacterium isolated and purified from pepper rhizosphere soil.It is a new type of biocontrol bacteria that can secrete a variety of extracellular hydrolases and produce abundant secondary metabolites.It has a good control effect on a variety of pathogenic fungi,bacteria,nematodes,etc.But there are few research reports on its biofilm formation.In the early stage,we analyzed the gene cluster for the synthesis of secondary metabolites of Lysobacter enzymogenes OH11 by anti SMASH,and predicted that the gene Cluster16 might be related to the formation of biofilm.The orf5 gene in Cluster16 encoded non-ribosomal peptide synthase（NRPS）,which was the structural gene of the expression product of this gene cluster.Based on this,the orf5 gene deletion mutant strains and overexpressed engineered strains of Lysobacter enzymogenes OH11 were constructed.The differences in biofilm formation and secondary metabolites between strains,as well as the antistress effect of pyrrole-2-carboxylic acid（PCA）which was a biosynthetic substrate of NRPS were investigated in the paper.Cluster16-orf5 gene deletion mutant strains were constructed by plasmid-mediated homologous recombination technique.In addition,the strong promoter P_HSAF（promoter of HSAF gene cluster）was inserted in front of orf5 gene to construct overexpressed engineered strains.Four engineered strains were constructed:（1）OH11-N4T1,which was the mutant strain constructed by knocking out orf5 gene in wild-type strain OH11-WT;（2）OH11-B5,which was the overexpressed strain constructed by inserting the strong promoter P_HSAF before orf5 gene in wild-type strain OH11-WT;（3）HW-N2S1,which was the mutant strain constructed by knocking out orf5 gene in strain HW-WT（constructed by knocking out the genes of compounds HSAF and WAP in strain OH11-WT）;（4）HW-C1a,which was the overexpressed strain constructed by inserting the strong promoter P_HSAFbefore orf5 gene in strain HW-WT.The transcription level of the orf5 gene in the overexpression engineered strains were detected by real-time PCR.The result showed that the transcription of the orf5 biosynthetic gene in the engineered strain OH11-B5/HW-C1a was 3-4 times higher than in the wild-type OH11-WT/HW-WT.Based on the successful construction of mutant and overexpressed engineered strains,the biofilm formation capacity of the constructed 4 engineered strains and the wild strains OH11-WT and HW-WT was measured.The OD value of 33%glacial acetic acid solution dissolved in crystal violet was measured under the wavelength of 590 nm by UV spectrophotometer after the biofilm was stained with crystal violet to compare the difference of biofilm volume formed by these 6 strains.The results showed that the biofilm formation of overexpressed strains OH11-B5 and HW-C1a was the most significant among6 strains,which was 2-3 times of the biofilm amount of wild-type strain OH11-WT and strain HW-WT on average.This initially indicated that Cluster16 in Lysobacter enzymogenes OH11 was involved in the formation of biofilm.By culturing in a static way,adding different chemical inducers and culturing in different medium,the differences of secondary metabolites between strains were compared.No significant differences in secondary metabolites have been found between strains by thin-layer chromatography and high-performance liquid analysis.On the web of anti SMASH,it was predicted that the product of secondary metabolite synthesis gene Cluster16 in Lysobacter enzymogenes OH11 was a pyrrole-2-carboxylic acid derivative,and the precursor of the compound was pyrrole-2-carboxylic acid.Under adverse condition,bacteria would produce physiological reactions such as biofilm formation.Then the influence of PCA which was a precursor of metabolites related to biofilm formation on the growth of Lysobacter enzymogenes OH11 wild-type and mutant strains under the stress of heavy metals Cd Cl₂ and Cu SO₄ was explored.In the heavy metal Cd Cl₂ stress experiment,when the concentration of 0.0017 mg/m L,0.0138 mg/m L,0.0276mg/m L and 0.0552 mg/m L,PCA could alleviate the inhibition effect of 0.25m M Cd Cl₂ on the growth of OH11-WT and OH11-N4T1 strains cultured for 96 h.The concentration of0.0552 mg/m L PCA had the strongest protective effect among the Cd Cl₂ stress experiments.In the heavy metal Cu SO₄ stress experiment,when the concentration of 0.0017 mg/m L,0.0138 mg/m L and 0.11 mg/m L,PCA could alleviate the inhibition effect of 0.8m M Cu SO₄on the growth of OH11-WT and OH11-N4T1 strains cultured for 72 h.The concentration of 0.11 mg/m L PCA had the strongest protective effect among the Cu SO₄ stress experiments.At concentrations of 0.0017 mg/m L and 0.0138 mg/m L,PCA could alleviate the inhibitory effect of 0.8m M Cu SO₄ on the growth of strains OH11-WT and OH11-N4T1cultured for 96 h.The results showed that the appropriate concentration of PCA could alleviate the inhibitory effect of heavy metals Cd Cl₂ and Cu SO₄ on the growth of the strains.In this paper,the study showed that the Cluster16 for the synthesis of secondary metabolites in Lysobacter enzymogenes OH11 was related to the formation of biofilm.PCA,the biosynthetic substrate of this gene cluster,had the effect of anti-heavy metals Cd Cl₂ and Cu SO₄ at a certain concentration.But the secondary metabolites of its gene cluster need to be further studied.This paper lays the foundation for the study on the growth regulation of Lysobacter enzymogenes OH11.