Reverchon Sylvie, Rouanet Carine, Expert Dominique, Nasser William
Unité de Microbiologie et Génétique CNRS-INSA-UCB UMR 5122, INSA, Bâtiment Louis Pasteur, 11 Avenue Jean Capelle, 69621 Villeurbanne Cedex, France.
J Bacteriol. 2002 Feb;184(3):654-65. doi: 10.1128/JB.184.3.654-665.2002.
In the plant-pathogenic bacterium Erwinia chrysanthemi production of pectate lyases, the main virulence determinant, is modulated by a complex network involving several regulatory proteins. One of these regulators, PecS, also controls the synthesis of a blue pigment identified as indigoidine. Since production of this pigment is cryptic in the wild-type strain, E. chrysanthemi ind mutants deficient in indigoidine synthesis were isolated by screening a library of Tn5-B21 insertions in a pecS mutant. These ind mutations were localized close to the regulatory pecS-pecM locus, immediately downstream of pecM. Sequence analysis of this DNA region revealed three open reading frames, indA, indB, and indC, involved in indigoidine biosynthesis. No specific function could be assigned to IndA. In contrast, IndB displays similarity to various phosphatases involved in antibiotic synthesis and IndC reveals significant homology with many nonribosomal peptide synthetases (NRPS). The IndC product contains an adenylation domain showing the signature sequence DAWCFGLI for glutamine recognition and an oxidation domain similar to that found in various thiazole-forming NRPS. These data suggest that glutamine is the precursor of indigoidine. We assume that indigoidine results from the condensation of two glutamine molecules that have been previously cyclized by intramolecular amide bond formation and then dehydrogenated. Expression of ind genes is strongly derepressed in the pecS background, indicating that PecS is the main regulator of this secondary metabolite synthesis. DNA band shift assays support a model whereby the PecS protein represses indA and indC expression by binding to indA and indC promoter regions. The regulatory link, via pecS, between indigoidine and virulence factor production led us to explore a potential role of indigoidine in E. chrysanthemi pathogenicity. Mutants impaired in indigoidine production were unable to cause systemic invasion of potted Saintpaulia ionantha. Moreover, indigoidine production conferred an increased resistance to oxidative stress, indicating that indigoidine may protect the bacteria against the reactive oxygen species generated during the plant defense response.
在植物致病细菌菊欧文氏菌中，主要毒力决定因素果胶酸裂解酶的产生受一个涉及多种调节蛋白的复杂网络调控。这些调节因子之一PecS，还控制一种被鉴定为靛玉红的蓝色色素的合成。由于该色素在野生型菌株中产量很低，通过筛选pecS突变体中的Tn5 - B21插入文库，分离出了缺乏靛玉红合成能力的菊欧文氏菌ind突变体。这些ind突变位于调控性pecS - pecM基因座附近，紧挨着pecM的下游。对该DNA区域的序列分析揭示了参与靛玉红生物合成的三个开放阅读框，即indA、indB和indC。IndA没有明确的特定功能。相比之下，IndB与参与抗生素合成的多种磷酸酶具有相似性，而IndC与许多非核糖体肽合成酶（NRPS）具有显著同源性。IndC产物包含一个具有识别谷氨酰胺的特征序列DAWCFGLI的腺苷化结构域和一个与各种形成噻唑的NRPS中发现的氧化结构域相似的氧化结构域。这些数据表明谷氨酰胺是靛玉红的前体。我们推测靛玉红是由两个谷氨酰胺分子缩合而成，这两个谷氨酰胺分子先前通过分子内酰胺键形成而环化，然后脱氢。ind基因在pecS背景下的表达强烈去抑制，表明PecS是这种次生代谢物合成的主要调节因子。DNA条带迁移分析支持这样一个模型，即PecS蛋白通过结合indA和indC启动子区域来抑制它们的表达。通过pecS，靛玉红与毒力因子产生之间的调控联系促使我们探究靛玉红在菊欧文氏菌致病性中的潜在作用。靛玉红产生受损的突变体无法引起盆栽非洲紫罗兰的系统侵染。此外，靛玉红的产生赋予了对氧化应激的增强抗性，表明靛玉红可能保护细菌免受植物防御反应过程中产生的活性氧的伤害。
