Involvement of a putative acyltransferase gene in sporangium formation in Actinoplanes missouriensis.

The actinomycete Actinoplanes missouriensis forms branched substrate mycelia during vegetative growth and produces terminal sporangia, each of which contains a few hundred spherical flagellated spores, from the substrate mycelia through short sporangiophores. Based on the observation that remodeling of membrane lipid composition is involved in the morphological development of Streptomyces coelicolor A3(2), we hypothesized that remodeling of membrane lipid composition is also involved in sporangium formation in A. missouriensis. Because some acyltransferases are presumably involved in the remodeling of membrane lipid composition, we disrupted each of the 22 genes annotated as encoding putative acyltransferases in the A. missouriensis genome and evaluated their effects on sporangium formation. The atsA (AMIS_52390) null mutant (ΔatsA) strain formed irregular sporangia of various sizes. Transmission electron microscopy revealed that some ΔatsA sporangiospores did not mature properly. Phase-contrast microscopy revealed that sporangium dehiscence did not proceed properly in the abnormally small sporangia of the ΔatsA strain, whereas apparently normal sporangia opened to release the spores. Consistently, the number of spores released from ΔatsA sporangia was lower than that released from wild-type sporangia. These phenotypic changes were recovered by introducing atsA with its own promoter into the ΔatsA strain. These results demonstrate that AtsA is required for normal sporangium formation in A. missouriensis, although the involvement of AtsA in the remodeling of membrane lipid composition is unlikely because AtsA is an acyltransferase_3 (AT3) protein, which is an integral membrane protein that usually catalyzes the acetylation of cell surface structures.IMPORTANCEActinoplanes missouriensis goes through a life cycle involving complex morphological development, including mycelial growth, sporangium formation and dehiscence, swimming as zoospores, and germination to mycelial growth. In this study, we carried out a comprehensive gene disruption experiment of putative acyltransferase genes to search for acyltransferases involved in the morphological differentiation of A. missouriensis. We revealed that a stand-alone acyltransferase_3 domain-containing protein, named AtsA, is required for normal sporangium formation. Although the molecular mechanism of AtsA in sporangium formation, as well as the enzymatic activity of AtsA, remains to be elucidated, the identification of a putative acyltransferase involved in sporangium formation is significant in the study of morphological development of A. missouriensis. This finding will contribute to our understanding of a complex system for producing sporangia, a rare multicellular organism in bacteria.

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.

Extending the Salinilactone Family.

Five new members of the salinilactone family, salinilactones D-H, are reported. These bicyclic lactones are produced by Salinispora bacteria and display extended or shortened alkyl side chains relative to the recently reported salinilactones A-C. They were identified by GC/MS, gas chromatographic retention index, and comparison with synthetic samples. We further investigated the occurrence of salinilactones across six newly proposed Salinispora species to gain insight into how compound production varies among taxa. The growth-inhibiting effect of this compound family on multiple biological systems including non-Salinispora actinomycetes was analyzed. Additionally, we found strong evidence for significant cytotoxicity of the title compounds.