Characterization andin vivoregulon determination of an ECF sigma factor and its cognate anti-sigma factor inNostoc punctiforme

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Based on primary sequence comparisons and genomic context, Npun_F4153 (SigG)/Npun_F4154 (SapG) of the cyanobacterium Nostoc punctiforme were hypothesized to encode an ECF sigma factor/anti-sigma factor pair. Transcription of sigG increased in heterocysts and akinetes, and after EDTA treatment. Interaction between SigG and the predicted cytoplasmic domain of SapG was observed in vitro. A SigG-GFP translational fusion protein localized to the periphery of vegetative cells in vivo, but lost this association following heat stress. A sigG mutant was unable to survive envelope damage caused by heat or EDTA, but was able to form functional heterocysts. Akinetes in the mutant strain appeared normal, but these cultures were less resistant to lysozyme and cold treatments than those of the wild-type strain. The SigG in vivo regulon was determined before and during akinete differentiation using DNA microarray analysis, and found to include multiple genes with putative association to the cell envelope. Mapped promoters common to both arrays enabled identification of a SigG promoter-binding motif that was supported in vivo by reporter studies, and in vitro by run-off transcription experiments. These findings support SigG/SapG as a sigma/anti-sigma pair involved in repair of envelope damage resulting from exogenous sources or cellular differentiation.

In the heterocyst and akinete forming cyanobacterium Nostoc punctiforme, SigG is shown to be an ECF sigma factor expressed at low levels in vegetative cells that increases in akinetes (A), heterocysts (H), and cells adjacent to heterocysts. Although essential for stress survival of vegetative cells, SigG is not essential to the function of differentiated cells, indicating SigG is likely activated in response to envelope stress caused by differentiation. The SigG regulon includes many genes encoding proteins related to the bacterial envelope, and promoter mapping revealed a binding motif that was supported in vivo by reporters and in vitro by transcription assays. A SigG-GFP chimeric protein localized to the cell membrane and was released following envelope stress, likely due to interaction with a trans-membrane anti-sigma factor (SapG) that SigG was shown to bind in vitro.

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