Genetic and Transcriptional Analyses of the Flagellar Gene Cluster in Actinoplanes missouriensis
Genetic and Transcriptional Analyses of the Flagellar Gene Cluster in Actinoplanes missouriensis
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DOI：
10.1128/jb.00306-16
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发表时间：
2016-08-01
影响因子：
3.2
通讯作者：
Ohnishi, Yasuo
中科院分区：
文献类型：
--
作者：
Jang, Moon-Sun;Mouri, Yoshihiro;Ohnishi, Yasuo
密苏里放线菌是一种革兰氏阳性的土壤细菌，是一种罕见的放线菌。丝状细胞产生孢子囊，孢子囊中含有数百个鞭毛孢子，这些孢子可以在短时间内快速游动，直到找到发芽的生态位。这些游动的细胞被称为游动孢子，这种独特的时间鞭笞的机制尚未阐明。本文报道了细菌基因组中所有鞭毛基因及其对鞭毛形态发生的预期功能和贡献。我们发现了一个由33个基因组成的大型鞭毛基因簇，这些基因编码了组装革兰氏阳性细菌功能鞭毛所需的大部分蛋白质。一个值得注意的例外是fliQ基因的位置，它从集群中分离出来。我们检测了四个基因参与鞭毛生物合成的基因破坏，fliQ, fliC， fliK和lytA。此外，我们使用在产孢子囊琼脂培养基上培养1、3、6和40天的a . missouriensis制备的RNA样本对鞭毛基因进行转录分析。我们证明了鞭毛基因的转录在孢子囊形成的同时被激活。利用cDNA 5′端快速扩增（RACE）技术对鞭毛基因的11个转录起始点进行了分析，发现了高度保守的启动子序列CTCA(N15-17) GCCGAA。这一结果表明，一个sigma因子负责所有鞭毛基因的转录，并且鞭毛结构同时组装。从形态发生、生存策略和进化的角度来看，游动孢子的生物学是非常有趣的。在这里，我们分析了A. missouriensis的鞭毛基因，它产生孢子囊，每个孢子囊含有数百个鞭毛孢子。游动孢子从孢子囊中释放出来，在萌发前游动很短的时间。我们发现了一个大鞭毛基因簇和一个孤鞭毛基因（fliQ）。这些发现表明游动孢子鞭毛成分是典型的革兰氏阳性细菌。然而，转录分析显示，在孢子囊形成过程中，所有鞭毛基因都是同时转录的，这与其他细菌（如沙门氏菌和大肠杆菌）中鞭毛基因有序、受调控的表达模式不同。这些结果提示了一种新的鞭毛形成调控机制。
Actinoplanes missouriensis, a Gram-positive and soil-inhabiting bacterium, is a member of the rare actinomycetes. The filamentous cells produce sporangia, which contain hundreds of flagellated spores that can swim rapidly for a short period of time until they find niches for germination. These swimming cells are called zoospores, and the mechanism of this unique temporal flagellation has not been elucidated. Here, we report all of the flagellar genes in the bacterial genome and their expected function and contribution for flagellar morphogenesis. We identified a large flagellar gene cluster composed of 33 genes that encode the majority of proteins essential for assembling the functional flagella of Gram-positive bacteria. One noted exception to the cluster was the location of the fliQ gene, which was separated from the cluster. We examined the involvement of four genes in flagellar biosynthesis by gene disruption, fliQ, fliC, fliK, and lytA. Furthermore, we performed a transcriptional analysis of the flagellar genes using RNA samples prepared from A. missouriensis grown on a sporangium-producing agar medium for 1, 3, 6, and 40 days. We demonstrated that the transcription of the flagellar genes was activated in conjunction with sporangium formation. Eleven transcriptional start points of the flagellar genes were determined using the rapid amplification of cDNA 5' ends (RACE) procedure, which revealed the highly conserved promoter sequence CTCA(N15-17) GCCGAA. This result suggests that a sigma factor is responsible for the transcription of all flagellar genes and that the flagellar structure assembles simultaneously.IMPORTANCEThe biology of a zoospore is very interesting from the viewpoint of morphogenesis, survival strategy, and evolution. Here, we analyzed flagellar genes in A. missouriensis, which produces sporangia containing hundreds of flagellated spores each. Zoospores released from the sporangia swim for a short time before germination occurs. We identified a large flagellar gene cluster and an orphan flagellar gene (fliQ). These findings indicate that the zoospore flagellar components are typical of Gram-positive bacteria. However, the transcriptional analysis revealed that all flagellar genes are transcribed simultaneously during sporangium formation, a pattern differing from the orderly, regulated expression of flagellar genes in other bacteria, such as Salmonella and Escherichia coli. These results suggest a novel regulatory mechanism for flagellar formation in A. missouriensis.