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 motilityassociated 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 ␤-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 transcriptionquantitative 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 ␤-N-acetylglucosamine transferase that is required for accumulation of PilA in hormogonia.

Dynamic localization of HmpF regulates type IV pilus activity and directional motility in the filamentous cyanobacterium Nostoc punctiforme

“…Using a transposon mutagenic screen to identify genes essential for hormogonium development and motility (Khayatan et al ., ), two non‐motile isolates were identified, TNM634 and TNM1102, that harbored transposon insertions in an open reading frame, herein designated hmpF (locus tag Npun_R5959) (Fig. A).…”

“…Generation of transposon mutants and identification of transposon insertion sites was performed as previously described (Wolk et al ., ; Khayatan et al ., ). Gene deletions and allelic replacements were performed as previously described (Risser and Meeks, ) with N. punctiforme cultures supplemented with 5 mM sucralose to inhibit hormogonium development and enhance conjugation efficiency (Khayatan et al ., ). To construct the Δ hmpF strain, pDDR424 was introduced into wild‐type N. punctiforme , creating strain UOP 143.…”

“…Seven microliter of sample were used for immunoblot analysis following standard SDS‐PAGE (10% acrylamide) and immunoblot procedures. PilA, HmpD and RbcL, were simultaneously detected on a single membrane following electrophoretic transfer using a 1:10,000 dilution of each rabbit polyclonal antibody (Steinberg and Meeks, ; Risser et al ., ; Khayatan et al ., ) and a 1:20,000 dilution of an HRP‐conjugated anti‐rabbit secondary antibody (Chemicon). HmpF‐GFPuv was detected using a 1:2,000 dilution of the mouse monoclonal antibody GFP(B‐2) (Santa Cruz Biotechnology) and a 1:2,000 dilution of the HRP‐conjugated sheep anti‐mouse secondary antibody NXA931 (GE Healthcare Life Sciences).…”

A partner‐switching regulatory system controls hormogonium development in the filamentous cyanobacterium Nostoc punctiforme

“…For Npun_F4337, which encodes a protein with a C-terminal glycosyl transferase and N-terminal RsbVlike domain, only the first 369 nucleotides, encoding the RsbV-like domain, followed by the introduction of a stop codon, were cloned into pUT18c. Generation of transposon mutants and identification of transposon insertion sites was performed as previously described (Khayatan et al, 2017) using plasmid pRL1063a (Wolk et al, 1991). Gene deletions and allelic replacements were performed as previously described (Risser and Meeks, 2013) with N. punctiforme cultures supplemented with 4 mM sucralose to inhibit hormogonium development and enhance conjugation efficiency (Splitt and Risser, 2015;Khayatan et al, 2017).…”

“…Generation of transposon mutants and identification of transposon insertion sites was performed as previously described (Khayatan et al, 2017) using plasmid pRL1063a (Wolk et al, 1991). Gene deletions and allelic replacements were performed as previously described (Risser and Meeks, 2013) with N. punctiforme cultures supplemented with 4 mM sucralose to inhibit hormogonium development and enhance conjugation efficiency (Splitt and Risser, 2015;Khayatan et al, 2017). To construct the ΔhmpU, ΔhmpW and ΔhmpV strains, pDDR386, pDDR390 or pKR101, respectively, were introduced into wild-type N. punctiforme, creating strains UOP113, UOP119 and UOP150 respectively.…”