A Putative O-Linked β- N-Acetylglucosamine Transferase Is Essential for Hormogonium Development and Motility in the Filamentous Cyanobacterium Nostoc punctiforme

Abstract

Most species of filamentous cyanobacteria are capable of gliding motility, likely via a conserved type IV pilus-like system that may also secrete a motility-associated polysaccharide. In a subset of these organisms, motility is achieved only after the transient differentiation of hormogonia, which are specialized filaments that enter a nongrowth state dedicated to motility. Despite the fundamental importance of hormogonia to the life cycles of many filamentous cyanobacteria, the molecular regulation of hormogonium development is largely undefined. To systematically identify genes essential for hormogonium development and motility in the model heterocyst-forming filamentous cyanobacterium Nostoc punctiforme, a forward genetic screen was employed. The first gene identified using this screen, designated ogtA, encodes a putative O-linked β-N-acetylglucosamine transferase (OGT). The deletion of ogtA abolished motility, while ectopic expression of ogtA induced hormogonium development even under hormogonium-repressing conditions. Transcription of ogtA is rapidly upregulated (1 h) following hormogonium induction, and an OgtA-GFPuv fusion protein localized to the cytoplasm. In developing hormogonia, accumulation of PilA but not HmpD is dependent on ogtA Reverse transcription-quantitative PCR (RT-qPCR) analysis indicated equivalent levels of pilA transcript in the wild-type and ΔogtA mutant strains, while a reporter construct consisting of the intergenic region in the 5' direction of pilA fused to gfp produced lower levels of fluorescence in the ΔogtA mutant strain than in the wild type. The production of hormogonium polysaccharide in the ΔogtA mutant strain is reduced compared to that in the wild type but comparable to that in a pilA deletion strain. Collectively, these results imply that O-GlcNAc protein modification regulates the accumulation of PilA via a posttranscriptional mechanism in developing hormogonia.IMPORTANCE Filamentous cyanobacteria are among the most developmentally complex prokaryotes. Species such as Nostoc punctiforme develop an array of cell types, including nitrogen-fixing heterocysts, spore-like akinetes, and motile hormogonia, that function in dispersal as well as the establishment of nitrogen-fixing symbioses with plants and fungi. These symbioses are major contributors to global nitrogen fixation. Despite the fundamental importance of hormogonia to the life cycle of filamentous cyanobacteria and the establishment of symbioses, the molecular regulation of hormogonium development is largely undefined. We employed a genetic screen to identify genes essential for hormogonium development and motility in Nostoc punctiforme The first gene identified using this screen encodes a eukaryotic-like O-linked β-N-acetylglucosamine transferase that is required for accumulation of PilA in hormogonia.

Keywords: Nostoc punctiforme; O-linked β-N-acetylglucosamine transferase; gliding motility; hormogonia; type IV pili.