Genetic and Transcriptional Analyses of the Flagellar Gene Cluster in Actinoplanes missouriensis

Abstract

ABSTRACT 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(N 15–17 )GCCGAA. This result suggests that a sigma factor is responsible for the transcription of all flagellar genes and that the flagellar structure assembles simultaneously. IMPORTANCE The 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 .