To throw some light on transcriptional regulation of nblA in response to nutrient starvation, we performed Northern and primer extension analyses in wild type, NblR- and NtcA- strains from Synechococcus sp. PCC 7942 and gel retardation assays with purified NblR and NtcA. Results show that (i) after nitrogen depletion, nblA transcript accumulation was impaired in NtcA- cells; (ii) nblA expression was still responsive to nitrogen in the absence of NblR; (iii) purified NtcA and NblR bind to the nblA promoter region; and (iv) the NtcA-mediated increase in nblA transcripts is not via NblR, since nblR transcript levels were not affected by ntcA inactivation or by the nutritional conditions tested. As a whole, these results demonstrate that NtcA directly activates nblA transcription under nitrogen starvation conditions.
Primer extension and sequence analysis also indicate that nblA transcription can initiate at several promoters. The most active one, PnblA-2, is indeed directly regulated by NtcA under nitrogen starvation conditions and constitutes a novel type of NtcA activated promoter, with putative NtcA binding sites centered at -68 and -100.5 from the transcription star point.
Our results indicate that nblA promoter region is a complex regulatory region and that both NtcA and NblR greatly stimulate transcription from PnbL4-2 in response to nitrogen starvation and, in the case of NblR, also in response to other signals. Lack of viability of the NblR'-NtcA" double mutant and other observations made in the course of this work anticipate additional regulatory connections between the NblR and NtcA régulons.
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