Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 8 Medical Drive, 117597 Singapore.
Life Science Institute, National University of Singapore, 28 Medical Drive, 117456 Singapore.
Microbiology (Reading). 2011 Apr;157(Pt 4):1176-1186. doi: 10.1099/mic.0.046540-0. Epub 2011 Jan 20.
The genome of Burkholderia pseudomallei encodes three acylhomoserine lactone (AHL) quorum sensing systems, each comprising an AHL synthase and a signal receptor/regulator. The BpsI-BpsR system produces N-octanoylhomoserine lactone (C8HL) and is positively auto-regulated by its AHL product. The products of the remaining two systems have not been identified. In this study, tandem MS was used to identify and quantify the AHL species produced by three clinical B. pseudomallei isolates - KHW, K96243 and H11 - three isogenic KHW mutants that each contain a null mutation in an AHL synthase gene, and recombinant Escherichia coli heterologously expressing each of the three B. pseudomallei AHL synthase genes. BpsI synthesized predominantly C8HL, which accounted for more than 95 % of the extracellular AHLs produced in stationary-phase KHW cultures. The major products of BpsI(2) and BpsI(3) were N-(3-hydroxy-octanoyl)homoserine lactone (OHC8HL) and N-(3-hydroxy-decanoyl)homoserine lactone, respectively, and their corresponding transcriptional regulators, BpsR(2) and BpsR(3), were capable of driving reporter gene expression in the presence of these cognate lactones. Formation of biofilm by B. pseudomallei KHW was severely impaired in mutants lacking either BpsI or BpsR but could be restored to near wild-type levels by exogenous C8HL. BpsI(2) was not required, and BpsI(3) was partially required for biofilm formation. Unlike the bpsI mutant, biofilm formation in the bpsI(3) mutant could not be restored to wild-type levels in the presence of OHC8HL, the product of BpsI(3). C8HL and OHC8HL had opposite effects on biofilm formation; exogenous C8HL enhanced biofilm formation in both the bpsI(3) mutant and wild-type KHW while exogenous OHC8HL suppressed the formation of biofilm in the same strains. We propose that exogenous OHC8HL antagonizes biofilm formation in B. pseudomallei, possibly by competing with endogenous C8HL for binding to BpsR.
伯克霍尔德氏菌基因组编码三个酰基高丝氨酸内酯（AHL）群体感应系统，每个系统都由 AHL 合酶和信号受体/调节剂组成。BpsI-BpsR 系统产生 N-辛酰基高丝氨酸内酯（C8HL），并被其 AHL 产物正向自动调节。其余两个系统的产物尚未确定。在这项研究中，串联质谱用于鉴定和定量三种临床分离的伯克霍尔德氏菌（KHW、K96243 和 H11）产生的 AHL 种类，这三种是具有相同遗传背景的 KHW 突变株，每个突变株都缺失一个 AHL 合酶基因，以及重组大肠杆菌异源表达三种伯克霍尔德氏菌 AHL 合酶基因中的每一种。BpsI 主要合成 C8HL，占静止期 KHW 培养物中外源 AHL 的 95%以上。BpsI(2)和 BpsI(3)的主要产物分别为 N-(3-羟基-辛酰基)高丝氨酸内酯（OHC8HL）和 N-(3-羟基-癸酰基)高丝氨酸内酯，它们各自的转录调节剂 BpsR(2)和 BpsR(3)能够在存在这些同源内酯的情况下驱动报告基因的表达。B. pseudomallei KHW 生物膜的形成在缺乏 BpsI 或 BpsR 的突变体中严重受损，但可以通过外源性 C8HL 恢复到接近野生型水平。BpsI(2)不是必需的，而 BpsI(3)对生物膜形成是部分必需的。与 bpsI 突变体不同，在 bpsI(3)突变体中，添加 OHC8HL（BpsI(3)的产物）不能将生物膜形成恢复到野生型水平。C8HL 和 OHC8HL 对生物膜形成有相反的影响；外源性 C8HL 增强了 bpsI(3)突变体和野生型 KHW 中的生物膜形成，而外源性 OHC8HL 抑制了相同菌株中生物膜的形成。我们提出，外源性 OHC8HL 可能通过与内源性 C8HL 竞争结合 BpsR 来拮抗 B. pseudomallei 生物膜的形成。
