Table_2_GvmR – A Novel LysR-Type Transcriptional Regulator Involved in Virulence and Primary and Secondary Metabolism of Burkholderia pseudomallei.XLSX

Burkholderia pseudomallei is a soil-dwelling bacterium able to survive not only under adverse environmental conditions, but also within various hosts which can lead to the disease melioidosis. The capability of B. pseudomallei to adapt to environmental changes is facilitated by the large number of regulatory proteins encoded by its genome. Among them are more than 60 uncharacterized LysR-type transcriptional regulators (LTTRs). Here we analyzed a B. pseudomallei mutant harboring a transposon in the gene BPSL0117 annotated as a LTTR, which we named gvmR (globally acting virulence and metabolism regulator). The gvmR mutant displayed a growth defect in minimal medium and macrophages in comparison with the wild type. Moreover, disruption of gvmR rendered B. pseudomallei avirulent in mice indicating a critical role of GvmR in infection. These defects of the mutant were rescued by ectopic expression of gvmR. To identify genes whose expression is modulated by GvmR, global transcriptome analysis of the B. pseudomallei wild type and gvmR mutant was performed using whole genome tiling microarrays. Transcript levels of 190 genes were upregulated and 141 genes were downregulated in the gvmR mutant relative to the wild type. Among the most downregulated genes in the gvmR mutant were important virulence factor genes (T3SS3, T6SS1, and T6SS2), which could explain the virulence defect of the gvmR mutant. In addition, expression of genes related to amino acid synthesis, glyoxylate shunt, iron-sulfur cluster assembly, and syrbactin metabolism (secondary metabolite) was decreased in the mutant. On the other hand, inactivation of GvmR increased expression of genes involved in pyruvate metabolism, ATP synthesis, malleobactin, and porin genes. Quantitative real-time PCR verified the differential expression of 27 selected genes. In summary, our data show that GvmR acts as an activating and repressing global regulator that is required to coordinate expression of a diverse set of metabolic and virulence genes essential for the survival in the animal host and under nutrient limitation.

类鼻疽伯克霍尔德菌（Burkholderia pseudomallei）是一类土壤栖息细菌，不仅可在不利环境条件下存活，还能侵染多种宿主并引发类鼻疽病。该菌基因组编码大量调控蛋白，使其具备适应环境变化的能力，其中包含60余种尚未被表征的LysR型转录调控因子（LTTRs）。本研究针对一株在BPSL0117基因中插入转座子的类鼻疽伯克霍尔德菌突变株展开分析，该基因被注释为LysR型转录调控因子，我们将其命名为gvmR（全局作用型毒力与代谢调控因子，globally acting virulence and metabolism regulator）。与野生型菌株相比，该gvmR突变株在基础培养基与巨噬细胞中均表现出生长缺陷。此外，gvmR基因的插入失活使类鼻疽伯克霍尔德菌在小鼠体内丧失致病力，表明GvmR在感染过程中发挥关键作用。该突变株的上述缺陷可通过gvmR的异位表达得以修复。为鉴定受GvmR调控表达的基因，本研究利用全基因组平铺微阵列（whole genome tiling microarrays）对类鼻疽伯克霍尔德菌野生型与gvmR突变株开展全局转录组分析。结果显示，相较于野生型菌株，gvmR突变株中有190个基因表达上调，141个基因表达下调。其中下调幅度最大的基因包括三类关键致病因子基因：三型分泌系统3（T3SS3）、六型分泌系统1（T6SS1）与六型分泌系统2（T6SS2），这可解释gvmR突变株的致病力缺陷。除此之外，突变株中与氨基酸合成、乙醛酸分流、铁硫簇组装及syrbactin次级代谢相关的基因表达水平均出现下降。而GvmR的失活则上调了丙酮酸代谢、ATP合成、类鼻疽杆菌素（malleobactin）及孔蛋白编码基因的表达。实时定量PCR（quantitative real-time PCR）验证了27个筛选基因的差异表达情况。综上，本研究数据表明，GvmR是兼具激活与抑制功能的全局调控因子，可协调调控一系列代谢与毒力相关基因的表达，而这些基因对于细菌在动物宿主内的存活以及营养限制条件下的生存至关重要。