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Cis-Acting sequences required for light-responsive expression of the psbDII gene in Synechococcus sp. strain PCC 7942.

ABSTRACT: We analyzed the sequences required for promoter activity and high-light responsiveness of the psbDII gene in the cyanobacterium Synechococcus sp. strain PCC 7942 by using transcriptional fusions to a lacZ reporter gene. The basal promoter drives high constitutive expression, although no canonical -35 element is evident. The smallest fragment that showed clear light-responsive expression extends from -38 to +160, which includes 52 bp of the psbDII open reading frame. Sequences downstream from the promoter, within the untranslated leader region from +11 to +24, were required for high-light induction.

SUBMITTER: Anandan S

PROVIDER: S-EPMC179622 | biostudies-other | 1997 Nov

REPOSITORIES: biostudies-other

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cis-Acting sequences required for light-responsive expression of the psbDII gene in Synechococcus sp. strain PCC 7942.

Anandan S S Golden S S SS

Journal of bacteriology 19971101 21

We analyzed the sequences required for promoter activity and high-light responsiveness of the psbDII gene in the cyanobacterium Synechococcus sp. strain PCC 7942 by using transcriptional fusions to a lacZ reporter gene. The basal promoter drives high constitutive expression, although no canonical -35 element is evident. The smallest fragment that showed clear light-responsive expression extends from -38 to +160, which includes 52 bp of the psbDII open reading frame. Sequences downstream from the ...[more]

PMID: 9352943

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Project description:Cyanobacteria are excellent autotrophic photosynthetic chassis employed in synthetic biology, and previous studies have suggested that they have alkaline tolerance but low acid tolerance, significantly limiting their productivity as photosynthetic chassis and necessitating investigations into the acid stress resistance mechanism. In this study, differentially expressed genes were obtained by RNA sequencing-based comparative transcriptomic analysis under long-term acidic stress conditions and acidic shock treatment, in the model cyanobacterium Synechococcus elongatus PCC 7942. A pathway enrichment analysis revealed the upregulated and downregulated pathways during long-term acidic and shock stress treatment. The subsequent single gene knockout and phenotype analysis showed that under acidic stress conditions, the strains with chlL, chlN, pex, synpcc7942_2038, synpcc7942_1890, or synpcc7942_2547 knocked out grew worse than the wild type, suggesting their involvement in acid tolerance. This finding was further confirmed by introducing the corresponding genes back into the knockout mutant individually. Moreover, individual overexpression of the chlL and chlN genes in the wild type successfully improved the tolerance of S. elongatus PCC 7942 to acidic stress. This work successfully identified six genes involved in acidic stress responses, and overexpressing chIL or chIN individually successfully improved acid tolerance in S. elongatus PCC 7942, providing valuable information to better understand the acid resistance mechanism in S. elongatus PCC 7942 and novel insights into the robustness and tolerance engineering of cyanobacterial chassis. KEY POINTS: • DEGs were identified by RNA-seq based transcriptomics analysis in response to acidic stress in S. elongatus PCC 7942. • Six genes were identified to be involved in acid tolerance in S. elongatus PCC 7942. • Overexpression of chIL or chIN individually successfully improved the acid tolerance of S. elongatus PCC 7942.

Project description:The heat shock protein CIpB (HSP100) is a member of the diverse group of Clp polypeptides that function as molecular chaperones and/or regulators of energy-dependent proteolysis. A single-copy gene coding for a ClpB homolog was cloned and sequenced from the unicellular cyanobacterium Synechococcus sp. strain PCC 7942. The predicted polypeptide sequence was most similar to sequences of cytosolic ClpB from bacteria and higher plants (i.e., 70 to 75%). Inactivation of clpB in Synechococcus sp. strain PCC 7942 resulted in no significant differences from the wild-type phenotype under optimal growth conditions. In the wild type, two forms of ClpB were induced during temperature shifts from 37 to 47.5 or 50 degrees C, one of 92 kDa, which matched the predicted size, and another smaller protein of 78 kDa. Both proteins were absent in the delta clpB strain. The level of induction of the two ClpB forms in the wild type increased with increasingly higher temperatures, while the level of the constitutive ClpC protein remained unchanged. In the delta clpB strain, however, the ClpC content almost doubled during the heating period, presumably to compensate for the loss of ClpB activity. Photosynthetic measurements at 47.5 and 50 degrees C showed that the null mutant was no more susceptible to thermal inactivation than the wild type. Using photosynthesis as a metabolic indicator, an assay was developed for Synechococcus spp. to determine the importance of ClpB for acquired thermotolerance. Complete inactivation of photosynthetic oxygen evolution occurred in both the wild type and the delta clpB strain when they were shifted from 37 directly to 55 degrees C for 10 min. By preexposing the cells at 50 degrees C for 1.5 h, however, a significant level of photosynthesis was retained in the wild type but not in the mutant after the treatment at 55 degrees C for 10 min. Cell survival determinations confirmed that the loss of ClpB synthesis caused a fivefold reduction in the ability of Synechococcus cells to develop thermotolerance. These results clearly show that induction of ClpB at high temperatures is vital for sustained thermotolerance in Synechococcus spp., the first such example for either a photosynthetic or a prokaryotic organism.

Dataset Information

Cis-Acting sequences required for light-responsive expression of the psbDII gene in Synechococcus sp. strain PCC 7942.

Publications

cis-Acting sequences required for light-responsive expression of the psbDII gene in Synechococcus sp. strain PCC 7942.

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