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Heterologous acyl-homoserine lactone synthase expression in Pseudomonas putida

ABSTRACT: Many bacteria harbor an incomplete quorum sensing system, wherein they possess LuxR homologues without the quorum sensing acyl-homoserine lactone (AHL) synthase, which is encoded by a luxI homolog. An artificial AHL-producing plasmid was constructed using a cviI gene encoding for C6-AHL (HHL) synthase from Chromobacterium violaceum and was introduced successfully into both wild-type and the ppoR (a luxR homolog) mutant. Our data provides evidence to suggest that the PpoR-HHL complex, but neither PpoR nor HHL alone, could attenuate growth, antibiotic resistance, and biofilm formation ability. In contrast, swimming motility, siderophore production, and indole degradation were enhanced by PpoR-HHL. The addition of exogenous indole increased biofilm formation and reduce swimming motility. Interestingly, indole proved ineffective in the presence of PpoR-HHL, thereby suggesting that the PpoR-HHL complex masks the effects of indole. Our data was supported by transcriptome analyses showing that the presence of the plasmid-encoded AHL synthase altered the expression of many genes on the chromosome in strain KT2440. Our results showed that heterologous luxI expression occurring via horizontal gene transfer can regulate a broad range of specific target genes, resulting in alterations of the phenotype and physiology of host cells.

ORGANISM(S): Pseudomonas putida KT2440 Pseudomonas putida

PROVIDER: GSE23632 | GEO | 2010/08/22

SECONDARY ACCESSION(S): PRJNA130935

REPOSITORIES: GEO

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Project description:The Escherichia coli quorum-sensing regulator, SdiA, belongs to the LuxR family of transcriptional regulators and is responsible for detecting signals from other bacteria. Previously we found that SdiA is necessary for E. coli to control its biofilm formation with indole just as SdiA is necessary for E. coli to alter its biofilm formation in the presence of N-acylhomoserine lactones (AHLs). Here we engineered SdiA by random mutagenesis to further control biofilm formation in the presence of indole and AHLs. After screening of 477?? mutants with indole and two AHLs (N-butyryl-DL-homoserine lactone, and N-(3-oxooctanoyl)-L-homoserine lactone, C6HSL), five SdiA variants were obtained that altered biofilm with and without signals of indole and AHLs. Two truncation variants (1E11 and 14C3) lacking the C-terminal DNA-binding domain of SdiA showed the reduction of biofilm formation by 5-fold and 10-fold in LB and LB glu, respectively. DNA microarrays show that the evolved SdiA (1E11) compared to wild-type SdiA influences indole synthesis genes, AI-2 uptake genes, acid-resistance genes, motility related genes, cold-shock protein genes, and several regulatory protein genes. Corroborating DNA microarrays, SdiA variants produced various amounts of indole which led to different survivals in low pH and influenced swimming motility and final cell density. Also, an AHL sensitive variant (2D10) 2-fold increased biofilm formation in the presence of C6HSL, while another variant (6B12) lowered biofilm formation in the presence of C6HSL. Hence, the results clearly showed that mutation of SdiA itself directly controls biofilm formation and SdiA variants could be further recognized by the inter-species signal AHLs. This is the first random protein engineering to control biofilm formation. Experiment Overall Design: For the microarray experiments, 10 g glass wool (Corning Glass Works, Corning, N.Y.) were used to form biofilms in 250 mL LB ( 1 mM IPTG + Cm30) in 1 L Erlenmeyer shake flasks which were inoculated with overnight cultures diluted that were 1:100. The cells were shaken at 250 rpm and 30C for 12 hours to form biofilms on the glass wool, and RNA was isolated from the the biofilm cells.

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Heterologous acyl-homoserine lactone synthase expression in Pseudomonas putida

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