Anderson Mark T, Mitchell Lindsay A, Mobley Harry L T
University of Michigan Medical School, Department of Microbiology and Immunology, Ann Arbor, Michigan, USA.
University of Michigan Medical School, Department of Microbiology and Immunology, Ann Arbor, Michigan, USA
J Bacteriol. 2017 Jul 25;199(16). doi: 10.1128/JB.00159-17. Print 2017 Aug 15.
causes health care-associated opportunistic infections that can be difficult to treat due to a high incidence of antibiotic resistance. One of the many secreted proteins of is the PhlA phospholipase enzyme. Genes involved in the production and secretion of PhlA were identified by screening a transposon insertion library for phospholipase-deficient mutants on phosphatidylcholine-containing medium. Mutations were identified in four genes (, , , and ) that are involved in the flagellum-dependent PhlA secretion pathway. An additional phospholipase-deficient isolate harbored a transposon insertion in the gene encoding a predicted serine -acetyltransferase required for cysteine biosynthesis. The requirement for extracellular phospholipase activity was confirmed using a fluorogenic phospholipase substrate. Phospholipase activity was restored to the mutant by the addition of exogenous l-cysteine or -acetylserine to the culture medium and by genetic complementation. Additionally, transcript levels were decreased 6-fold in bacteria lacking and were restored with added cysteine, indicating a role for cysteine-dependent transcriptional regulation of phospholipase activity. mutants also exhibited a defect in swarming motility that was correlated with reduced levels of and flagellar regulator gene transcription. Together, these findings suggest a model in which cysteine is required for the regulation of both extracellular phospholipase activity and surface motility in is known to secrete multiple extracellular enzymes, but PhlA is unusual in that this protein is thought to be exported by the flagellar transport apparatus. In this study, we demonstrate that both extracellular phospholipase activity and flagellar function are dependent on the cysteine biosynthesis pathway. Furthermore, a disruption of cysteine biosynthesis results in decreased and flagellar gene transcription, which can be restored by supplying bacteria with exogenous cysteine. These results identify a previously unrecognized role for CysE and cysteine in the secretion of phospholipase and in bacterial motility.
引发与医疗保健相关的机会性感染，由于抗生素耐药性高发，这些感染可能难以治疗。[细菌名称]众多分泌蛋白之一是PhlA磷脂酶。通过在含磷脂酰胆碱的培养基上筛选转座子插入文库以寻找磷脂酶缺陷型突变体，鉴定出了参与PhlA产生和分泌的基因。在参与鞭毛依赖性PhlA分泌途径的四个基因（[基因名称1]、[基因名称2]、[基因名称3]和[基因名称4]）中发现了突变。另一个磷脂酶缺陷型分离株在编码半胱氨酸生物合成所需的预测丝氨酸 - 乙酰转移酶的[基因名称]中存在转座子插入。使用荧光磷脂酶底物证实了[细菌名称]对细胞外磷脂酶活性的需求。通过向培养基中添加外源性L - 半胱氨酸或O - 乙酰丝氨酸以及基因互补，使磷脂酶活性恢复到[细菌名称]突变体中。此外，在缺乏[基因名称]的细菌中，[基因名称]转录水平降低了6倍，并通过添加半胱氨酸得以恢复，表明半胱氨酸依赖性转录调控对[细菌名称]磷脂酶活性有作用。[细菌名称]突变体在群体运动方面也表现出缺陷，这与[基因名称]和鞭毛调节基因转录水平降低相关。总之，这些发现提示了一个模型，即半胱氨酸是[细菌名称]中细胞外磷脂酶活性和表面运动调节所必需。[细菌名称]已知分泌多种细胞外酶，但PhlA不同寻常之处在于该蛋白被认为是通过鞭毛运输装置输出的。在本研究中，我们证明细胞外磷脂酶活性和鞭毛功能均依赖于半胱氨酸生物合成途径。此外，半胱氨酸生物合成的破坏导致[基因名称]和鞭毛基因转录减少，通过向细菌提供外源性半胱氨酸可恢复。这些结果确定了CysE和半胱氨酸在[细菌名称]磷脂酶分泌和细菌运动中以前未被认识到的作用。
