Commonalities and differences in regulation of N-acyl homoserine lactone quorum sensing in the beneficial plant-associated burkholderia species cluster

Abstract

The genus Burkholderia includes over 60 species isolated from a wide range of environmental niches and can be tentatively divided into two major species clusters. The first cluster includes pathogens such as Burkholderia glumae, B. pseudomallei, and B. mallei and 17 well-studied species of the Burkholderia cepacia complex. The other recently established cluster comprises at least 29 nonpathogenic species, which in most cases have been found to be associated with plants. It was previously established that Burkholderia kururiensis, a member of the latter cluster, possesses an N-acyl homoserine lactone (AHL) quorum-sensing (QS) system designated "BraI/R," which is found in all species of the plant-associated cluster. In the present study, two other BraI/R-like systems were characterized in B. xenovorans and B. unamae and were designated the BraI/R(XEN) and BraI/R(UNA) systems, respectively. Several phenotypes were analyzed, and it was determined that exopolysaccharide was positively regulated by the BraIR-like system in the species B. kururiensis, B. unamae, and B. xenovorans, highlighting commonality in targets. However, the three BraIR-like systems also revealed differences in targets since biofilm formation and plant colonization were differentially regulated. In addition, a second AHL QS system designated XenI2/R2 and an unpaired LuxR solo protein designated BxeR solo were also identified and characterized in B. xenovorans LB400(T). The two AHL QS systems of B. xenovorans are not transcriptionally regulating each other, whereas BxeR solo negatively regulated xenI2. The XenI2/R2 and BxeR solo proteins are not widespread in the Burkholderia species cluster. In conclusion, the present study represents an extensive analysis of AHL QS in the Burkholderia plant-associated cluster demonstrating both commonalities and differences, probably reflecting environmental adaptations of the various species.

FIG. 1.

(A) Genetic maps of the B. xenovorans LB400^T (braI_XEN and braR_XEN), B. kururiensis M130 (braI_KUR and braR_KUR), and B. unamae MTl-641^T (braI_UNA and braR_UNA) QS systems. An alignment of putative lux boxes is shown in several members of the Burkholderia cluster; numbers indicate the positions upstream where the lux box is centered with the respect to the putative ATG start codon. (B) Map of the xenI2 and xenR2 system of B. xenovorans LB400^T.

FIG. 2.

TLC analysis of the AHLs produced by the Burkholderia and mutant derivatives. (A and B) B. unamae profiles. (C and D) B. xenovorans LB400 ^T profiles. AHL extraction was performed as described in Materials and Methods, and TLCs were performed in 70% methanol for 6 h; for each strain, the equivalent of 2.5 × 10¹⁰ cells was loaded. In panels A and C, A. tumefaciens (pNTL4) was used to detect the AHL signals, and in panels B and D, E. coli pSB1075 was used. Synthetic AHL compounds were used as a reference. OC6, 3-oxo-C₆-HSL; OC8, 3-oxo-C₈-HSL; OC10, 3-oxo-C₁₀-HSL; OC12, 3-oxo-C₁₂-HSL; OC14, 3-oxo-C₁₄-HSL; OHC6, 3-OH-C₆-HSL; OHC8, 3-OH-C₈-HSL; C10, C₁₀-HSL, C12; C₁₂-HSL.

FIG. 3.

Determination of the biologically active AHL for BraR_XEN, XenR2, BraR_UNA, and BraR_KUR AHL sensor/regulators. (A) Determination of the cognate AHL for the BraI/R_XEN system of B. xenovorans. Bars correspond to β-galactosidase activities determined for E. coli harboring pQEXENR1 and pMPXENI1. (B) Determination of the cognate AHL for the BraI/R_UNA system of B. unamae. Bars correspond to β-galactosidase activities determined for E. coli harboring pQEUNAR and pMPUNAI combination. (C) Determination of the cognate AHL for the XenI2/R2 system of B. xenovorans. Bars correspond to β-galactosidase activities determined for E. coli harboring pQEXENR2 and pMPX2I. (D) Determination of the cognate AHL for the BraI/R_KUR system of B. kururiensis. Bars correspond to β-galactosidase activities determined for E. coli harboring pQEBRAR and PBRAI (73). Transcriptional fusions were harbored independently in E. coli expressing either BraR_XEN or XenR2 proteins; various exogenous AHLs (1 μM) were provided as indicated, and the β-galactosidase activities were determined. The results are mean values ± the standard deviations of three independent biological replicates. EA, ethyl acetate.

FIG. 4.

braI_XEN, xenI2, and bxeR promoter activities in wild-type and QS mutant strains of B. xenovorans LB400^T. Bacterial cultures were started with an initial inoculum of 5 × 10⁶ CFU in 20 ml of KB-Tc medium, and the β-galactosidase activities were measured over 12 h of growth. All experiments were performed in triplicate, and means values with standard deviations are indicated in the graph. ANOVA in combination with Dunnett's post-test and were performed with Prism 4.0 software (GraphPad). A P value of <0.05 was considered significant (*).

FIG. 5.

EPS Production of B. kururiensis M130 (A and B), B. unamae MTl-641^T (C and D), and B. xenovorans LB400^T wild-type and QS mutants (E). Single colonies were streaked in YEM agar plates. Chemical complementation was achieved by adding 1 μM AHL to the growth medium. Bar graphs show EPS quantification for B. kururiensis and B. unamae by using the boiling phenol method (described in Materials and Methods). Experiments were performed in triplicate, and means ± the standard deviations are plotted. ANOVA in combination with Dunnett's post-test was performed using Prism 4.0 software (GraphPad). A P value of <0.05 was considered significant.

FIG. 6.

Biofilm production in B. unamae (A), B. kururiensis (B), and B. xenovorans (C) wild-type and QS mutants after 72 h of incubation. “+AHLs” refers to complementation by adding 1 μM 3-oxo-C₁₄-HSL. Experiments were performed in triplicate, and means ± the standard error of the mean are plotted. ANOVA in combination with Dunnett's post-test were performed using Prism 4.0 software (GraphPad). A P value of <0.05 was considered significant compared to the wild type (*).

FIG. 7.

Phenol degradation profile in B. unamae wild type and QS mutants. A growth profile was obtained with 10³ CFU as starter the inoculum. As a control, phenol was replaced by mannitol as the carbon source, and all strains exhibited the same behavior.

FIG. 8.

Rice colonization assays performed with B. kururiensis M130 wild type and impaired QS mutants M130BRAI and M130BRAR. Plantlets were surface sterilized and inoculated as described in Materials and Methods. Bacterial colonization was measured by grinding and plating surface-sterilized plants after 12 days, and CFU/g levels are plotted. A P value of <0.05 was considered significant compared to the wild type (*). (A) Endophytic root colonization levels. (B) Endophytic aerial colonization. (C) Enhanced root development promoted by B. kururiensis M130 wild-type inoculation compared to QS mutants and noninoculated plants.