Identification of a putative cell wall-hydrolyzing amidase involved in sporangiospore maturation in Actinoplanes missouriensis

Abstract

ABSTRACT Actinoplanes missouriensis is a filamentous bacterium that differentiates into terminal sporangia, each containing a few hundred spores. Previously, we reported that a cell wall-hydrolyzing N -acetylglucosaminidase, GsmA, is required for the maturation process of sporangiospores in A. missouriensis ; sporangia of the gsmA null mutant (Δ gsmA ) strain released chains of 2–20 spores under sporangium dehiscence-inducing conditions. In this study, we identified and characterized a putative cell wall hydrolase (AsmA) that is also involved in sporangiospore maturation. AsmA was predicted to have a signal peptide for the general secretion pathway and an N -acetylmuramoyl- l -alanine amidase domain. The transcript level of asmA increased during the early stages of sporangium formation. The asmA null mutant (Δ asmA ) strain showed phenotypes similar to those of the wild-type strain, but sporangia of the Δ gsmA Δ asmA double mutant released longer spore chains than those from the Δ gsmA sporangia. Furthermore, a weak interaction between AsmA and GsmA was detected in a bacterial two-hybrid assay using Escherichia coli as the host. Based on these results, we propose that AsmA is an enzyme that hydrolyzes peptidoglycan at septum-forming sites to separate adjacent spores during sporangiospore maturation in cooperation with GsmA in A. missouriensis . IMPORTANCE Actinoplanes missouriensis produces sporangiospores as dormant cells. The spores inside the sporangia are assumed to be formed from prespores generated by the compartmentalization of intrasporangium hyphae via septation. Previously, we identified GsmA as a cell wall hydrolase responsible for the separation of adjacent spores inside sporangia. However, we predicted that an additional cell wall hydrolase(s) is inevitably involved in the maturation process of sporangiospores because the sporangia of the gsmA null mutant strain released not only tandemly connected spore chains (2–20 spores) but also single spores. In this study, we successfully identified a putative cell wall hydrolase (AsmA) that is involved in sporangiospore maturation in A. missouriensis .