Connecting quorum sensing, c-di-GMP, pel polysaccharide, and biofilm formation in Pseudomonas aeruginosa through tyrosine phosphatase TpbA (PA3885).
Connecting quorum sensing, c-di-GMP, pel polysaccharide, and biofilm formation in Pseudomonas aeruginosa through tyrosine phosphatase TpbA (PA3885).
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DOI：
10.1371/journal.ppat.1000483
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发表时间：
2009-06
期刊：
影响因子：
6.7
通讯作者：
Wood TK
中科院分区：
文献类型：
--
作者：
Ueda A;Wood TK
对于条件致病菌铜绿假单胞菌，发现基于高丝氨酸内酯的群体感应影响生物膜的形成。在这里，我们通过筛选5850个转座子突变体的铜绿假单胞菌PA 14改变生物膜形成来识别群体感应控制生物膜形成的机制。该筛选鉴定了PA 3885突变体，其具有比野生型菌株多147倍的生物膜。PA 3885的损失减少游泳，废除群集，并增加附件，虽然这并不影响生产的鼠李糖脂。PA 3885突变体也具有一个菌落表型，形成明显的表膜，有更多的聚集，并有28倍以上的胞外多糖生产。PA 3885在反式中的表达减少了生物膜形成并消除了聚集。全转录组分析显示，PA 3885的缺失激活了pel基因座的表达，pel基因座是编码细胞外基质多糖合成的操纵子。遗传筛选鉴定出PelABDEG和PA 1120蛋白（其含有GGDEF基序）的缺失抑制了PA 3885突变体的表型，表明PA 3885蛋白的功能是作为磷酸酶调节3，5-环二鸟苷酸（c-di-GMP）浓度，因为c-di-GMP通过激活PelD增强生物膜形成，并且c-di-GMP抑制群集。PA 3885蛋白的缺失增加了细胞c-di-GMP浓度;因此，PA 3885蛋白是c-di-GMP产生的负调节剂。纯化的PA 3885蛋白具有针对磷酸酪氨酸肽的磷酸酶活性，并转移到周质。Las-mediated quorum sensing正向调节PA 3885基因的表达。这些结果表明，PA 3885蛋白响应阿勒信号并可能使PA 1120去磷酸化，这导致c-di-GMP产生减少。这抑制了基质胞外多糖的形成，从而导致生物膜形成减少;因此，我们提供了一种通过pel基因座控制生物膜形成的群体感应机制，并建议PA 3885应命名为TpbA，用于与生物膜形成相关的酪氨酸磷酸酶，PA 1120应命名为TpbB。大多数细菌生活在生物膜中，生物膜是通过多糖附着在表面的复杂微生物群落;这些生物膜是大多数人类细菌疾病的原因。病原体铜绿假单胞菌是生物膜形成的最佳研究。目前，人们认识到细胞通讯或群体感应对生物膜形成很重要，但这些外部信号如何转化为内部信号来调节导致生物膜形成的基因网络还不清楚。在这里，通过研究5850个细菌菌株，其中每一个都缺乏一个单一的蛋白质，我们确定了一种新的酶铜绿假单胞菌，酪氨酸磷酸酶（TpbA），连接胞外群体感应信号的多糖生产和生物膜形成。我们发现，TpbA是受控制的群体感应信号，它是在周质，它控制的水平的细胞内第二信使3，5-环二鸟苷酸（c-di-GMP）。通过控制c-di-GMP浓度，TpbA用于调节生物膜形成、表面上的快速细胞运动、集落形态、细胞聚集和多糖产生。本研究的重要性在于，它表明第二信使c-di-GMP可能受酪氨酸磷酸化的调节，因此，它为控制细菌的社会行为提供了一个新的靶点。
With the opportunistic pathogen Pseudomonas aeruginosa, quorum sensing based on homoserine lactones was found to influence biofilm formation. Here we discern a mechanism by which quorum sensing controls biofilm formation by screening 5850 transposon mutants of P. aeruginosa PA14 for altered biofilm formation. This screen identified the PA3885 mutant, which had 147-fold more biofilm than the wild-type strain. Loss of PA3885 decreased swimming, abolished swarming, and increased attachment, although this did not affect production of rhamnolipids. The PA3885 mutant also had a wrinkly colony phenotype, formed pronounced pellicles, had substantially more aggregation, and had 28-fold more exopolysaccharide production. Expression of PA3885 in trans reduced biofilm formation and abolished aggregation. Whole transcriptome analysis showed that loss of PA3885 activated expression of the pel locus, an operon that encodes for the synthesis of extracellular matrix polysaccharide. Genetic screening identified that loss of PelABDEG and the PA1120 protein (which contains a GGDEF-motif) suppressed the phenotypes of the PA3885 mutant, suggesting that the function of the PA3885 protein is to regulate 3,5-cyclic diguanylic acid (c-di-GMP) concentrations as a phosphatase since c-di-GMP enhances biofilm formation by activating PelD, and c-di-GMP inhibits swarming. Loss of PA3885 protein increased cellular c-di-GMP concentrations; hence, PA3885 protein is a negative regulator of c-di-GMP production. Purified PA3885 protein has phosphatase activity against phosphotyrosine peptides and is translocated to the periplasm. Las-mediated quorum sensing positively regulates expression of the PA3885 gene. These results show that the PA3885 protein responds to AHL signals and likely dephosphorylates PA1120, which leads to reduced c-di-GMP production. This inhibits matrix exopolysaccharide formation, which leads to reduced biofilm formation; hence, we provide a mechanism for quorum sensing control of biofilm formation through the pel locus and suggest PA3885 should be named TpbA for tyrosine phosphatase related to biofilm formation and PA1120 should be TpbB. Most bacteria live in biofilms, which are complex communities of microorganisms attached to a surface via polysaccharides; these biofilms are responsible for most human bacterial diseases. The pathogen Pseudomonas aeruginosa is best-studied for biofilm formation. Currently, it is recognized that cell communication or quorum sensing is important for biofilm formation, but how these external signals are converted into internal signals to regulate the networks of genes that result in biofilm formation is not well understood. Here, by studying 5850 bacterial strains, each of which lacks a single protein, we identify a new enzyme of P. aeruginosa, a tyrosine phosphatase (TpbA), that links extracellular quorum sensing signals to polysaccharide production and biofilm formation. We find that TpbA is subject to control by quorum sensing signals, that it is in the periplasm, and that it controls the level of the intracellular secondary messenger 3,5-cyclic diguanylic acid (c-di-GMP). By controlling c-di-GMP concentrations, TpbA serves to regulate biofilm formation, rapid cell movement on the surface, colony morphology, cell aggregation, and polysaccharide production. The importance of our study is that it shows the secondary messenger c-di-GMP may be regulated by tyrosine phosphorylation; hence, it provides a new target for controlling bacterial social behavior.
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影响因子：
3.2
作者：
Barrios, AFG;Zuo, RJ;Wood, TK
通讯作者：
Wood, TK
DOI：
10.1073/pnas.77.12.7347
发表时间：
1980-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
DITTA, G;STANFIELD, S;HELINSKI, DR
通讯作者：
HELINSKI, DR
影响因子：
3.2
作者：
Kuchma, Sherry L.;Brothers, Kimberly M.;O'Toole, George A.
通讯作者：
O'Toole, George A.
DOI：
10.1073/pnas.0511090103
发表时间：
2006-02-21
影响因子：
11.1
作者：
Kulesekara, H;Lee, V;Lory, S
通讯作者：
Lory, S
DOI：
10.1073/pnas.76.4.1648
发表时间：
1979-01-01
影响因子：
11.1
作者：
FIGURSKI, DH;HELINSKI, DR
通讯作者：
HELINSKI, DR