Involvement of three FliA-family sigma factors in the sporangium formation, spore dormancy and sporangium dehiscence in Actinoplanes missouriensis.

The rare actinomycete Actinoplanes missouriensis forms sporangia, which open up and release zoospores in response to water. Here, we report a genetic and functional analysis of four FliA-family sigma factors, FliA1, FliA2, FliA3 and FliA4. Transcription of fliA1, fliA2 and fliA3 was directly activated by the global transcriptional activator TcrA during sporangium formation and dehiscence, while fliA4 was almost always transcribed at low levels. Gene disruption analysis showed that (a) deletion of fliA2 reduced the zoospore swimming speed by half, (b) the fliA1-fliA2 double-deletion mutant formed abnormal sporangia in which mutant spores ectopically germinated and (c) deletion of fliA3 induced no phenotypic changes in the wild-type and mutant strains of fliA1 and/or fliA2. Comparative RNA-Seq analyses among the wild-type and gene deletion mutant strains showed probable targets of each FliA-family sigma factor, indicating that FliA1- and FliA2-dependent promoters are quite similar to each other, while the FliA3-dependent promoter is somewhat different. Gene complementation experiments also indicated that the FliA1 regulon overlaps with the FliA2 regulon. These results demonstrate that A. missouriensis has developed a complex transcriptional regulatory network involving multiple FliA-family sigma factors for the accomplishment of its characteristic reproduction process, including sporangium formation, spore dormancy and sporangium dehiscence.