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Overexpression of pknE blocks heterocyst development in Anabaena sp. strain PCC 7120.

ABSTRACT: The upstream intergenic regions for each of four genes encoding Ser/Thr kinases, all2334, pknE (alr3732), all4668, and all4838, were fused to a gfpmut2 reporter gene to determine their expression during heterocyst development in the cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120. P(pknE)-gfp was upregulated after nitrogen step-down and showed strong expression in differentiating cells. Developmental regulation of pknE required a 118-bp upstream region and was abolished in a hetR mutant. A pknE mutant strain had shorter filaments with slightly higher heterocyst frequency than did the wild type. Overexpression of pknE from its native promoter inhibited heterocyst development in the wild type and in four mutant backgrounds that overproduce heterocysts. Overexpression of pknE from the copper-inducible petE promoter did not completely inhibit heterocyst development but caused a 24-h delay in heterocyst differentiation and cell bleaching 4 to 5 days after nitrogen step-down. Strains overexpressing pknE and containing P(hetR)-gfp or P(patS)-gfp reporters failed to show developmental regulation of the reporters and had undetectable levels of HetR protein. Genetic epistasis experiments suggest that overexpression of pknE blocks HetR activity or downstream regulation.

SUBMITTER: Saha SK

PROVIDER: S-EPMC3133150 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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Overexpression of pknE blocks heterocyst development in Anabaena sp. strain PCC 7120.

Saha Sushanta K SK Golden James W JW

Journal of bacteriology 20110318 10

The upstream intergenic regions for each of four genes encoding Ser/Thr kinases, all2334, pknE (alr3732), all4668, and all4838, were fused to a gfpmut2 reporter gene to determine their expression during heterocyst development in the cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120. P(pknE)-gfp was upregulated after nitrogen step-down and showed strong expression in differentiating cells. Developmental regulation of pknE required a 118-bp upstream region and was abolished in a hetR mutant. A ...[more]

PMID: 21421755

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Project description:Cyanobacteria are monophyletic organisms that perform oxygenic photosynthesis. While they exhibit great diversity, they have a common set of genes. However, the essentiality of them for viability has hampered the elucidation of their functions. One example of these genes is cyabrB1 (also known as calA in Anabaena sp. strain PCC 7120), encoding a transcriptional regulator. In the present study, we investigated the function of calA/cyabrB1 in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 through CRISPR interference, a method that we recently utilized for the photosynthetic production of a useful chemical in this strain. Conditional knockdown of calA/cyabrB1 in the presence of nitrate resulted in the formation of heterocysts. Two genes, hetP and hepA, which are required for heterocyst formation, were upregulated by calA/cyabrB1 knockdown in the presence of combined nitrogen sources. These genes are known to be induced by HetR, a master regulator of heterocyst formation. hetR was not induced by calA/cyabrB1 knockdown. hetP and hepA were repressed by direct binding of CalA/cyAbrB1 to their promoter regions in a HetR-independent manner. In addition, the overexpression of calA/cyabrB1 abolished heterocyst formation upon nitrogen depletion. Also, knockout of calB/cyabrB2 (a paralogue gene of calA/cyabrB1), in addition to knockdown of calA/cyabrB1, enhanced heterocyst formation in the presence of nitrate, suggesting functional redundancy of cyAbrB proteins. We propose that a balance between amounts of HetR and CalA/cyAbrB1 is a key factor influencing heterocyst differentiation during nitrogen stepdown. We concluded that cyAbrB proteins are essential safety devices that inhibit heterocyst differentiation.IMPORTANCE Spore formation in Bacillus subtilis and Streptomyces has been extensively studied as models of prokaryotic nonterminal cell differentiation. In these organisms, many cells/hyphae differentiate simultaneously, which is governed by a network in which one regulator stands at the top. Differentiation of heterocysts in Anabaena sp. strain PCC 7120 is unique because it is terminal, and only 5 to 10% of vegetative cells differentiate into heterocysts. In this study, we identified CalA/cyAbrB1 as a repressor of two genes that are essential for heterocyst formation independently of HetR, a master activator for heterocyst differentiation. This finding is reasonable for unique cell differentiation of Anabaena because CalA/cyAbrB1 could suppress heterocyst differentiation tightly in vegetative cells, while only cells in which HetR is overexpressed could differentiate into heterocysts.

Project description:UnlabelledTo stabilize cellular integrity in the face of environmental perturbations, most bacteria, including cyanobacteria, synthesize and maintain a strong, flexible, three-dimensional peptidoglycan lattice. Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium capable of differentiating morphologically distinct nitrogen-fixing heterocyst cells in a periodic pattern. While heterocyst development has been shown to require proper peptidoglycan remodeling, the role of peptidoglycan synthesis has remained unclear. Here we report the identification of two peptidoglycan synthesis genes, murC (alr5065) and murB (alr5066), as required for heterocyst development. The murC and murB genes are predicted to encode a UDP-N-acetylmuramate:L-alanine ligase and a UDP-N-acetylenolpyruvoylglucosamine reductase, respectively, and we confirm enzymatic function through complementation of Escherichia coli strains deficient for these enzymes. Cells depleted of either murC or murB expression failed to differentiate heterocysts under normally inducing conditions and displayed decreased filament integrity. To identify the stage(s) of development affected by murC or murB depletion, the spatial distribution of expression of the patterning marker gene, patS, was examined. Whereas murB depletion did not affect the pattern of patS expression, murC depletion led to aberrant expression of patS in all cells of the filament. Finally, expression of gfp controlled by the region of DNA immediately upstream of murC was enriched in differentiating cells and was repressed by the transcription factor NtcA. Collectively, the data in this work provide evidence for a direct link between peptidoglycan synthesis and the maintenance of a biological pattern in a multicellular organism.ImportanceMulticellular organisms that differentiate specialized cells must regulate morphological changes such that both cellular integrity and the dissemination of developmental signals are preserved. Here we show that the multicellular bacterium Anabaena, which differentiates a periodic pattern of specialized heterocyst cells, requires peptidoglycan synthesis by the murine ligase genes murC (alr5065) and murB (alr5066) for maintenance of patterned gene expression, filament integrity, and overall development. This work highlights the significant influence that intracellular structure and intercellular connections can have on the execution of a developmental program.

Dataset Information

Overexpression of pknE blocks heterocyst development in Anabaena sp. strain PCC 7120.

Publications

Overexpression of pknE blocks heterocyst development in Anabaena sp. strain PCC 7120.

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