The sigE Gene Is Required for Normal Expression of Heterocyst-Specific Genes in Anabaena sp. Strain PCC 7120

Abstract

The filamentous cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120 produces specialized cells for nitrogen fixation called heterocysts. Previous work showed that the group 2 sigma factor sigE (alr4249; previously called sigF) is upregulated in differentiating heterocysts 16 h after nitrogen step-down. We now show that the sigE gene is required for normal heterocyst development and normal expression levels of several heterocyst-specific genes. Mobility shift assays showed that the transcription factor NtcA binds to sites in the upstream region of sigE and that this binding is enhanced by 2-oxoglutarate (2-OG). Deletions of the region containing the NtcA binding sites in P sigE -gfp reporter plasmids showed that the sites contribute to normal developmental regulation but are not essential for upregulation in heterocysts. Northern RNA blot analysis of nifH mRNA revealed delayed and reduced transcript levels during heterocyst differentiation in a sigE mutant background. Quantitative reverse transcription-PCR (qRT-PCR) analyses of the sigE mutant showed lower levels of transcripts for nifH, fdxH, and hglE2 but normal levels for hupL. We developed a P nifHD -gfp reporter construct that showed strong heterocyst-specific expression. Time-lapse microscopy of the P nifHD -gfp reporter in a sigE mutant background showed delayed development and undetectable green fluorescent protein (GFP) fluorescence. Overexpression of sigE caused accelerated heterocyst development, an increased heterocyst frequency, and premature expression of GFP fluorescence from the P nifHD -gfp reporter.

NrrA, a Nitrogen-regulated Response Regulator Protein, Controls Glycogen Catabolism in the Nitrogen-fixing Cyanobacterium Anabaena sp. Strain PCC 7120

“…5). Expression of sigE is shown to be up-regulated in differentiating cells at late stage of heterocyst differentiation using the sigE-gfp transcriptional reporter fusion (18), and SigE is involved in regulation of heterocyst-specific genes, such as nifH, fdxH, and hglE2 (19). In the present study, we showed that the transcription of sigE was up-regulated within 8 h after nitrogen deprivation, and NrrA regulated the sigE expression (Fig.…”

“…NrrA Directly Regulates the sigE Expression-The sigE transcript is shown to increase 12 h after nitrogen deprivation in Anabaena PCC 7120 (19). Changes in the transcript level of sigE were examined at earlier stages of nitrogen deprivation.…”

“…6). Interaction of NrrA with the region upstream of the sigE gene (PsigE), in which the promoter region necessary for the sigE expression was included (19), was determined by gel mobility shift assays (Fig. 7).…”

“…The sigE gene (alr4249; previously called sigF), encoding a group 2 factor of RNA polymerase (17), is up-regulated in heterocysts after nitrogen deprivation (18) and is required for normal expression of heterocyst-specific genes including nifH, fdxH, and hglE2 (19). NtcA binds to the upstream region of sigE, but it plays a partial role in the regulation of sigE expression (19). Another transcriptional regulator in the transcriptional regulation of sigE is suggested.…”

“…NrrA is required for the full induction of the hetR gene (11,14), encoding a master regulator of heterocyst differentiation (15,16). The sigE gene (alr4249; previously called sigF), encoding a group 2 factor of RNA polymerase (17), is up-regulated in heterocysts after nitrogen deprivation (18) and is required for normal expression of heterocyst-specific genes including nifH, fdxH, and hglE2 (19). NtcA binds to the upstream region of sigE, but it plays a partial role in the regulation of sigE expression (19).…”