Characterization of a putative extracytoplasmic function (ECF) sigma factor, [sigma]G, and its cognate anti-sigma factor in Nostoc punctiforme

Akinetes are spore-like cells resistant to desiccation and cold that differentiate from vegetative cells of the filamentous cyanobacterium N. punctiforme in response to phosphate or potassium limitation and low light. Previous microarray data has identified a putative extracytoplasmic function (ECF) sigma factor, NpF4153 (SigG), and an adjacent downstream anti-sigma factor, NpF4154, as potential genes involved in akinete induction in N. punctiforme. GFP transcriptional reporter strains confirmed the array results, showing increased expression from the sigG promoter in akinetes, heterocysts, and following stresses imposed by heat or EDTA. The zwf model system of akinete induction was used to unveil a potential regulon for SigG by two separate microarray analyses comparing the zwf/sigG double mutant relative to a zwf strain under both vegetative growth conditions and during akinete formation. Genes showing decreased expression in the double mutant included outer membrane proteins, lipid biosynthesis proteins, proteases, as well as many hypothetical proteins. Transcriptional start sites of genes shown to be down-regulated due to deletion of sigG in both microarrays were determined by random amplification of cDNA ends, and a putative SigG consensus sequence was determined. To determine interaction between SigG and the N-terminal end of the NpF4154 anti-sigma factor, a GST pull down assay was performed. The N-terminal predicted cytoplasmic domain of the anti-sigma fused to GST was able to pull-down SigG-His6 from an E. coli crude lysate, indicating a specific interaction between SigG and F4154N-GST. A strain expressing a SigG-GFP protein fusion was shown by confocal laser microscopy to have GFP-fluorescence localized to the periphery of the cell under normal growth conditions. Such protein localization was lost following exposure to short periods of heat, indicating release of sigma factor from the cell membrane following stress. Survival studies comparing wild-type and ΔsigG mutant strains following exposure to heat or outer membrane disruption using EDTA showed reduced survival for the mutant strain. The mutant also exhibited a significant delay in heterocyst formation following nitrogen starvation relative to the wild-type strain. Together these data support the hypothesis that NpF4153-4154 encode proteins that act in a manner that is typical of an ECF sigma / anti-sigma factor pair that is important for normal expression of genes involved in cell differentiation and envelope biosynthesis/repair.