Quorum-sensing control of antibiotic synthesis in Burkholderia thailandensis

Abstract

The genome of Burkholderia thailandensis codes for several LuxR-LuxI quorum-sensing systems. We used B. thailandensis quorum-sensing deletion mutants and recombinant Escherichia coli to determine the nature of the signals produced by one of the systems, BtaR2-BtaI2, and to show that this system controls genes required for the synthesis of an antibiotic. BtaI2 is an acyl-homoserine lactone (acyl-HSL) synthase that produces two hydroxylated acyl-HSLs, N-3-hydroxy-decanoyl-HSL (3OHC(10)-HSL) and N-3-hydroxy-octanoyl-HSL (3OHC(8)-HSL). The btaI2 gene is positively regulated by BtaR2 in response to either 3OHC(10)-HSL or 3OHC(8)-HSL. The btaR2-btaI2 genes are located within clusters of genes with annotations that suggest they are involved in the synthesis of polyketide or peptide antibiotics. Stationary-phase cultures of wild-type B. thailandensis, but not a btaR2 mutant or a strain deficient in acyl-HSL synthesis, produced an antibiotic effective against gram-positive bacteria. Two of the putative antibiotic synthesis gene clusters require BtaR2 and either 3OHC(10)-HSL or 3OHC(8)-HSL for activation. This represents another example where antibiotic synthesis is controlled by quorum sensing, and it has implications for the evolutionary divergence of B. thailandensis and its close relatives Burkholderia pseudomallei and Burkholderia mallei.

FIG. 1.

Transcriptional activation of genes in the btaI2 operon requires 3OHC₁₀-HSL or 3OHC₈-HSL and BtaR2. (A) Acyl-HSL dose responses of the btaI2 promoter in E. coli containing a BtaR2 expression vector (pJNR2) and a btaI2-lacZ fusion vector (pI2P50). The following acyl-HSLs were tested: 3OHC₁₀-HSL (▪), 3OHC₈-HSL (•), 3OHC6-HSL (□), C₁₂-HSL (▾), C₁₀-HSL (○), and C₈-HSL (▴). The open diamonds indicate the 3OHC₁₀-HSL response in the absence of BtaR2. The error bars represent the range of three independent experiments. β-Galactosidase activity is given as millions of relative light units. (B) Relative transcript levels of btaI2 and the downstream gene BTH_II1224 from wild-type B. thailandensis (gray bars) and the btaR2 mutant strain JBT108 (white bars). The error bars represent the range of two independent experiments assayed in triplicate.

FIG. 2.

Organization of the B. thailandensis btaR2-btaI2 genomic region. Shown is a map of the genes in the immediate vicinity of btaR2 and btaI2. The btaR2 and btaI2 genes are separated by three open reading frames encompassing about 3.3 kb of DNA. btaI2 resides in a predicted five-gene operon that contains open reading frames annotated to function in antibiotic synthesis. btaR2 is 3 kb upstream of a cluster containing putative antibiotic biosynthesis genes. The blue arrows labeled R2 and I2 represent btaR2 and btaI2, respectively. The red arrows are annotated as NRPS genes, green arrows indicate PKS genes, pink arrows are potential accessory antibiotic synthesis genes, orange arrows are putative transport genes, the brown arrow is a metallopeptidase, and gray indicates genes of unknown function. The black lines between coding regions represent intergenic DNA. The purple bars represent the btaR2-btaI2 genomic region and the flanking DNA, which is conserved in B. pseudomallei and mostly absent from the B. mallei chromosome. The genomic sequences were obtained from the publicly available genome sequences of B. thailandensis strain E264, B. pseudomallei strain K96243, and B. mallei strain ATCC 23344. The alignments were generated using the nucleotide BLAST algorithm (73). The B. thailandensis E264 genomic sequence was used as the reference sequence. A solid purple bar indicates congruence in nucleic acid sequence; the amino acid sequences within these regions share >90% identity. Vertical black bars represent nucleic acid sequence with dissimilarity, and gaps between purple bars are missing sequences. The arrows representing btaR2, btaI2, and their surrounding genes are drawn to scale.

FIG. 3.

Sensitivity of B. subtilis to a substance in B. thailandensis stationary-phase culture fluid. (A) Growth curve of the wild-type B. thailandensis strain E264. The arrowheads marked a to f indicate points where culture fluid was taken for the analysis shown in panel B. The open arrowheads indicate points in growth where antibiotic was produced. (B) Antibiotic sensitivity assays. Paper diffusion discs were saturated with fluid from a B. thailandensis E264 culture at the indicated points (a to f in panel A) and placed on lawns of B. subtilis. A zone of clearing around a diffusion disc indicates the region where B. subtilis growth was inhibited. (C) Antibiotic activity of B. thailandensis culture fluid against S. aureus COL, S. pyogenes MGAS5005, E. coli DH5α, and P. aeruginosa PAO1.

FIG. 4.

A B. thailandensis acyl-HSL synthesis mutant requires exogenous 3OHC₈-HSL or 3OHC₁₀-HSL for antibiotic production. Diffusion disc assays with fluid from a stationary-phase culture of the btaI1, btaI2, btaI3 triple mutant JBT112 grown without added signal or with 2 μM 3OHC₈-HSL or 3OHC₁₀-HSL, as indicated, are shown. Growth of B. subtilis is inhibited by culture fluid from 3OHC₁₀-HSL- or 3OHC₈-HSL-grown B. thailandensis JBT112, but not by JBT112 grown without an added acyl-HSL or in the presence of 2 μM C₈-HSL. The bottom panel shows a diffusion disc that had been soaked in sterile medium containing 2 μM 3OHC₁₀-HSL. This control shows that 3OHC₁₀-HSL itself is not an antimicrobial molecule.

FIG. 5.

B. thailandensis BtaR2 is required for antibiotic production. (A) A diffusion disc experiment showing antibiotic activity of fluid from a B. thailandensis E264 (wild-type) stationary-phase culture, a JBT108 (btaR2 mutant) stationary-phase culture, and JBT108 complemented with the BtaR2 expression plasmid pSCR2. The control is strain JBT108 carrying the empty vector pSCrhaB2. B. subtilis was used as the indicator strain. (B) Influence of B. thailandensis culture fluid (50% or 10% [vol/vol] as indicated) on growth of B. subtilis in broth assessed by colony counting. The white bars represent culture fluid from the parent B. thailandensis E264, the gray bars indicate fluid from the btaR2 mutant JBT108, and the hatched bar is a control B. subtilis culture with no added B. thailandensis culture fluid. The error bars indicate standard deviations.

FIG. 6.

Dependence of BTH_II1233, -1236, and -1239 transcription on the BtaI2-BtaR2 quorum-sensing system. (A) Acyl-HSL dose responses of the BTH_II1233-lacZ fusion on pQF1233 and btaR2 on pJNR2 in E. coli; 3OHC₁₀-HSL, ▪; 3OHC₈-HSL, •. The BTH_II1233-lacZ response to 3OHC₁₀-HSL in E. coli without a BtaR2 expression vector is also shown (▾). The error bars indicate the range of three independent experiments. β-Galactosidase activity is given as millions of relative light units. (B) Relative transcript levels of BTH_II1233 and the downstream genes BTH_II1236 and BTH_II1239 from wild-type B. thailandensis E264 (gray bars) and the btaR2 mutant strain JBT102 (white bars). The values represent the range of two independent experiments assayed in triplicate.

FIG. 7.

BTH_II1233 mutants do not produce antibiotic. The influence of B. thailandensis culture fluid (10% vol/vol) on growth of B. subtilis in broth was assessed by colony counting. The B. thailandensis wild-type strain E264 is indicated by the white bar. The BTH_II1233 kanamycin insertion mutant, BD909, and the in-frame deletion mutant, BD20, are indicated by the gray and hatched bars, respectively. For reference, a control culture with no added B. thailandensis culture fluid is shown (black bar). The error bars indicate standard deviations.