Physiological and transcriptomic characterization of a fliA mutant of Pseudomonas putida KT2440.
Physiological and transcriptomic characterization of a fliA mutant of Pseudomonas putida KT2440.
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DOI：
10.1111/j.1758-2229.2009.00084.x
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发表时间：
2009-10
影响因子：
3.3
通讯作者：
J. Rodríguez-Herva;E. Duque;M. A. Molina-Henares;Gloria Navarro-Avilés;P. Dillewijn;J. Torre;A. Molina-Henares;A. S. D. L. Campa;A. S. D. L. Campa;F. Ran;A. Segura;V. Shingler;J. Ramos;J. Ramos
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文献类型：
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作者：
J. Rodríguez-Herva;E. Duque;M. A. Molina-Henares;Gloria Navarro-Avilés;P. Dillewijn;J. Torre;A. Molina-Henares;A. S. D. L. Campa;A. S. D. L. Campa;F. Ran;A. Segura;V. Shingler;J. Ramos;J. Ramos
恶臭假单胞菌KT2440编码23种不同的西格玛因子。该基因编码σ(28)，与其他参与鞭毛生物合成和趋化作用的基因在一起。逆转录聚合酶链式反应显示该簇由四个独立的转录单位组成：flhAF、fleNfliA、cheYZA和cheBmotAB。我们通过同源重组产生了一个非极性的fliA突变体，并测试了它的运动性、对生物和非生物表面的粘附性以及对各种胁迫条件的反应。该突变菌株不能移动，与玉米种子结合的能力降低，尽管其在植物根际的定殖能力没有受到影响。突变株与非生物表面的结合也受到影响，其形成生物膜的能力几乎下降了三倍。在fliA突变背景中，有25个基因的表达受到影响，其中2个基因表达上调，23个基因表达下调。除了一些运动性和趋化性基因外，fliA基因产物也是一些可能涉及氨基酸利用或胁迫反应的基因表达所必需的；然而，我们无法指定与这些基因连锁的特定表型，因为fliA突变体使用与亲本菌株相同的氨基酸范围，并且与野生型一样耐受热、抗生素、氯化钠、十二烷基硫酸钠、过氧化氢和苯甲酸盐施加的胁迫。根据FliA和基因组在计算机分析中识别的启动子的序列比对，我们认为恶臭假单胞菌σ(28)识别一个位于-34到-8之间的TCAAG-t-N12-GCCGATA共同序列，该序列优先与AT丰富的上游区域相关。
Pseudomonas putida KT2440 encodes 23 alternative sigma factors. The fliA gene, which encodes σ(28) , is in a cluster with other genes involved in flagella biosynthesis and chemotaxis. Reverse transcriptase-PCR revealed that this cluster is comprised of four independent transcriptional units: flhAF, fleNfliA, cheYZA and cheBmotAB. We generated a nonpolar fliA mutant by homologous recombination and tested its motility, adhesion to biotic and abiotic surfaces, and responses to various stress conditions. The mutant strain was nonmotile and exhibited decreased capacity to bind to corn seeds, although its ability to colonize the rhizosphere of plants was unaffected. The mutant was also affected in binding to abiotic surfaces and its ability to form biofilms decreased by almost threefold. In the fliA mutant background expression of 25 genes was affected: two genes were upregulated and 23 genes were downregulated. In addition to a number of motility and chemotaxis genes, the fliA gene product is also necessary for the expression of some genes potentially involved in amino acid utilization or stress responses; however, we were unable to assign specific phenotypes linked to these genes since the fliA mutant used the same range of amino acids as the parental strain, and was as tolerant as the wild type to stress imposed by heat, antibiotics, NaCl, sodium dodecyl sulfate, H2 O2 and benzoate. Based on the sequence alignment of promoters recognized by FliA and genome in silico analysis, we propose that P. putidaσ(28) recognizes a TCAAG-t-N12 -GCCGATA consensus sequence located between -34 and -8 and that this sequence is preferentially associated with an AT-rich upstream region.