Involvement of two putative gene clusters for exopolysaccharide biosynthesis and modification in sporangium formation in Actinoplanes missouriensis

The terminal sporangium of the actinomycete Actinoplanes missouriensis contains a few hundred spores surrounded by a matrix substance, which is mainly composed of a polysaccharide synthesized by the imp gene cluster. However, the involvement of other extracellular polysaccharide (EPS) biosynthetic gene clusters in sporangium matrix formation remains to be examined. In this study, we characterized four putative EPS biosynthetic gene clusters by gene disruption, which were transcribed during sporangium formation and were thereby expected to be involved in the production or modification of EPSs inside sporangia. An in silico analysis proposed that three gene clusters, wps -1 ( AMIS_7280–AMIS_7380 ), wps -2 ( AMIS_56690–AMIS_56780 ), and wps -3 ( AMIS_58080–AMIS_58180 ), provide the Wzx/Wzy-dependent pathway, and that another gene cluster, gsf ( AMIS_70380–AMIS_70480 ), provides the GtrA-dependent pathway. Severely distorted sporangia were observed by scanning electron microscopy in the null mutant of the whole wps -1 cluster (Δ wps -1 strain). The null mutant of the whole gsf cluster (Δ gsf strain) formed sporangia smaller than the wild-type sporangia. Transmission electron microscopy revealed that the spores inside the Δ gsf sporangia were not fully matured. Spore release did not occur in the Δ wps -1 strain under appropriate aquatic conditions, whereas sporangia of the Δ gsf strain opened to release spores under the same conditions. Disruption of each gene in the wps -1 cluster indicated that all genes, except wpsD ( AMIS_7310 ), were indispensable for normal sporangium formation. We concluded that exopolysaccharide production and modification mediated by the wps -1 and gsf clusters, as well as the imp cluster, play crucial roles in sporangium formation in A. missouriensis . IMPORTANCE Actinoplanes missouriensis forms terminal sporangia, which consist of a sporangium envelope, spores, and a matrix substance. The sporangium matrix encapsulates the spores inside the sporangium. Recently, we reported that a major component of the sporangium matrix is a polysaccharide consisting of repeating oligosaccharides and that a gene cluster, named imp , is involved in the production of the polysaccharide. In the present study, through a gene disruption experiment, we revealed that two gene clusters, named wps -1 and gsf , are also required for normal sporangium formation in A. missouriensis , providing important insights into the components of the sporangium matrix. The wps -1 cluster appears to be responsible for producing sulfated polysaccharide(s). Sulfated polysaccharides are produced by animals and algae but are not common in bacteria, except cyanobacteria. Thus, although experimental verification is required, this study raises the possibility that the ability to produce sulfated polysaccharides is also present in phylogenetically distant actinomycetes.