Quorum-sensing system and stationary-phase sigma factor (rpoS) of the onion pathogen Burkholderia cepacia genomovar I type strain, ATCC 25416

Abstract

Bacterial strains belonging to Burkholderia cepacia can be human opportunistic pathogens, plant pathogens, and plant growth promoting and have remarkable catabolic activity. B. cepacia consists of several genomovars comprising what is now known as the B. cepacia complex. Here we report the quorum-sensing system of a genomovar I onion rot type strain ATCC 25416. Quorum sensing is a cell-density-dependent regulatory response which involves the production of N-acyl homoserine lactone (HSL) signal molecules. The cep locus has been inactivated in the chromosome, and it has been shown that CepI is responsible for the biosynthesis of an N-hexanoyl HSL (C(6)-HSL) and an N-octanoyl HSL (C(8)-HSL) and that the cep locus regulates protease production as well as onion pathogenicity via the expression of a secreted polygalacturonase. A cep-lacZ-based sensor plasmid has been constructed and used to demonstrate that CepR responded to C(6)-HSL with only 15% of the molar efficiency of C(8)-HSL, that a cepR knockout mutant synthesized 70% less HSLs, and that CepR responded best towards long-chain HSLs. In addition, we also report the cloning and characterization of the stationary-phase sigma factor gene rpoS of B. cepacia ATCC 25416. It was established that quorum sensing in B. cepacia has a negative effect on rpoS expression as determined by using an rpoS-lacZ transcriptional fusion; on the other hand, rpoS-null mutants displayed no difference in the accumulation of HSL signal molecules.

FIG. 1.

(a) Activation of bacterial sensor strain in cross-streak experiments. The sensor strain E. coli DH5α(pSCR1) was cross streaked on LB agar plates (containing ampicillin and X-Gal) with B. cepacia ATCC 25416 used as a tester strain. (b) HSL bioassay with E. coli DH5α(pSCR1). DH5α(pSCR1) was grown in the presence of variousconcentrations of either C₆-HSL (▪) or C₈-HSL (♦), and β-galactosidase activities were determined after 6 h. The values were determined with LB medium, the means of triplicate experiments are given, and the standard deviations are shown. (c) CepR-HSL response. E. coli DH5α(pSCR1) was grown for 6 h in the presence of a 100 nM concentration of either C₄-, C₆-, C₈-, C₁₀-, or C₁₂-HSL, and β-galactosidase activities were determined. The values were determined with LB medium, the means of triplicate experiments are given, and the standard deviations are shown. The values are expressed as percentages of the activity determined with C₈-HSL.

FIG. 2.

Role of quorum sensing in onion rot. One hundred microliters of a culture of the indicated strain with an OD₆₀₀ of 1 was inoculated on the right half of a sterile onion as described in the text. This picture was taken after 48 h of incubation at 30°C.

FIG.3.

(a) Map of the 5.5-kbp SmaI DNA fragment from B. cepacia ATCC 25416 isolated in this study. Shown are several enzyme restriction sites and the location of the rpoS gene within this fragment. Also shown is the position where a Km^r-containing BamHI fragment derived from pUC4K was cloned in the corresponding site of the rpoS gene to create pLCIKm. (b, c, and d) Effect of rpoS on stress responses of B. cepacia ATCC 25416. (b) Response to heat shock (50°C). Viability is expressed as a percentage of the number of CFU at time zero. (c) Response to osmotic shock (2 M NaCl). Viability is expressed as a percentage of the number of CFU at time zero. (d) Effect of rpoS mutation on oxidative stress. The sensitivity to H₂O₂ was measured on cells grown for 16 h in LB at 30°C. The zones of inhibition were measured in millimeters. Filled symbols, WT strain; open symbols, B. cepacia 25416-RPOS strain.

FIG. 4.

(a) Production of HSLs at different growth stages from B. cepacia ATCC 25416 (filled bars) and from B. cepacia ATCC 25416-RPOS (open bars). Values were determined as described in Materials and Methods. (b) rpoS promoter activity at different growth stages measured in B. cepacia ATCC 25416(pRPR2) (filled bars), B. cepacia 25416-I(pRPR2) (open bars), B. cepacia 25416-I(pRPR2) plus 100 nM C₈-HSL (shaded bars), and B. cepacia ATCC 25416(pMP190) (striped bars). Values were determined as described in Materials and Methods.