Different functions of HetR, a master regulator of heterocyst differentiation in
            <i>Anabaena</i>
            sp. PCC 7120, can be separated by mutation

Shi

Proc. Natl. Acad. Sci. U.S.A.

et al. 2005

Although it is known that calcium is a very important messenger involved in many eukaryotic cellular processes, much less is known about calcium's role in bacteria. CcbP, a Ca 2؉ -binding protein, was isolated from the heterocystous cyanobacterium Anabaena sp. PCC 7120, and the ccbP gene was cloned and inactivated. In the absence of combined nitrogen, inactivation of ccbP resulted in multiple contiguous heterocysts, whereas overexpression of ccbP suppressed heterocyst formation. Calmodulin, which is not present in Anabaena species, could also suppress heterocyst formation in both Anabaena sp. PCC 7120 and Anabaena variabilis. HetR induction upon nitrogen step-down was slow in the strain overexpressing ccbP. It has been demonstrated that [Ca 2ϩ ] i is tightly regulated in some bacteria (6), and there is evidence indicating that calcium is critical to some bacterial cellular processes such as sporulation of Bacillus (7), chemotaxis of Escherichia coli (8), and heterocyst differentiation of cyanobacteria (9). Heterocyst development may represent one of the earliest examples of pattern formation in evolution (10). Heterocysts are specialized cells for nitrogen fixation of some filamentous cyanobacteria (11)(12)(13)(14). When heterocystous cyanobacteria are subjected to nitrogen step-down, some vegetative cells differentiate and become heterocysts. Many genes are specifically involved in heterocyst differentiation. The genes that are required for initiation of cell differentiation include ntcA (15, 16) and hetR (17). HetR is a serine-type protease that plays an important role in heterocyst formation (18).Evidence obtained by manipulating extracellular calcium concentration and by using inhibitors of Ca 2ϩ -binding proteins suggested that Ca 2ϩ could be involved in heterocyst differentiation (9). Here we describe a Ca 2ϩ -binding protein, CcbP, from Anabaena sp. PCC 7120. Our study shows that free calcium accumulates in differentiating cells and mature heterocysts, correlated with a drop in the level of the Ca 2ϩ -binding protein.The free Ca 2ϩ concentration appears to be critical for the differentiation process. Materials and MethodsThe following protocols can be found in Supporting Text, which is published as supporting information on the PNAS web site: growth of the strains of Anabaena; isolation of the Ca 2ϩ -binding protein CcbP; isolation of the ccbP gene from total genomic DNA; expression of recombinant ccbP in Escherichia coli; construction of plasmids for inactivation of the ccbP gene, for copper-regulated expression of ccbP, for copper-regulated expression of rat calmodulin (CaM) gene, for ccbP promoter regulation of GFP expression, and for expression of obelin, a reporter for the level of free Ca 2ϩ .Assays for Ca 2؉ -Binding Proteins. 45 Ca 2ϩ overlay assay was performed as follows: Proteins were first separated by SDS͞PAGE and then transferred to a poly(vinylidene difluoride) membrane. It was then washed three times with buffer C (20 mM Tris⅐HCl, pH 7.2͞5 mM MgCl 2 ͞60 mM KCl͞10 mM imidazole). The memb...

Section: Discussionmentioning

confidence: 99%

CcbP, a calcium-binding protein from Anabaena sp. PCC 7120, provides evidence that calcium ions regulate heterocyst differentiation

Shi

Proc. Natl. Acad. Sci. U.S.A.

et al. 2005

“…More than 500 proteins are differentially expressed in heterocysts during cellular transformation from vegetative cells (162), showing that this complex process is under the control of many genes. Master regulators are HetR, a serine-type protease, and NtcA, a nitrogen control transcription factor in cyanobacteria (115,152,160,200). Expression of hetR is upregulated by nitrogen deprivation, and this upregulation depends on NtcA (62).…”

Section: Occurrence Of Nitrogenases In Heterocystsmentioning

confidence: 99%

Nitrogen Fixation and Hydrogen Metabolism in Cyanobacteria

Bothe

Schmitz²,

Yates

et al. 2010

Microbiol Mol Biol Rev

357

222

SUMMARY This review summarizes recent aspects of (di)nitrogen fixation and (di)hydrogen metabolism, with emphasis on cyanobacteria. These organisms possess several types of the enzyme complexes catalyzing N2 fixation and/or H2 formation or oxidation, namely, two Mo nitrogenases, a V nitrogenase, and two hydrogenases. The two cyanobacterial Ni hydrogenases are differentiated as either uptake or bidirectional hydrogenases. The different forms of both the nitrogenases and hydrogenases are encoded by different sets of genes, and their organization on the chromosome can vary from one cyanobacterium to another. Factors regulating the expression of these genes are emerging from recent studies. New ideas on the potential physiological and ecological roles of nitrogenases and hydrogenases are presented. There is a renewed interest in exploiting cyanobacteria in solar energy conversion programs to generate H2 as a source of combustible energy. To enhance the rates of H2 production, the emphasis perhaps needs not to be on more efficient hydrogenases and nitrogenases or on the transfer of foreign enzymes into cyanobacteria. A likely better strategy is to exploit the use of radiant solar energy by the photosynthetic electron transport system to enhance the rates of H2 formation and so improve the chances of utilizing cyanobacteria as a source for the generation of clean energy.

Proc. Natl. Acad. Sci. U.S.A.

“…Both patS and hetN are thought to produce diffusible inhibitors of heterocyst formation that act at the level of HetR, and both contain the RGSGR pentapeptide, which is capable of inhibiting heterocyst formation when exogenously added to a culture (7,8,14). Therefore, we tested the effect of the addition of RGSGR peptide on the distribution of HetR-GFP.…”

Section: Concentration Gradients Of Hetr Adjacent To Heterocystsmentioning

confidence: 99%

Genetic and cytological evidence that heterocyst patterning is regulated by inhibitor gradients that promote activator decay

Risser

Callahan

2009

111

The formation of a pattern of differentiated cells from a group of seemingly equivalent, undifferentiated cells is a central paradigm of developmental biology. Several species of filamentous cyanobacteria differentiate nitrogen-fixing heterocysts at regular intervals along unbranched filaments to form a periodic pattern of two distinct cell types. This patterning has been used to exemplify application of the activator-inhibitor model to periodic patterns in biology. The activator-inhibitor model proposes that activators and inhibitors of differentiation diffuse from source cells to form concentration gradients that in turn mediate patterning, but direct visualization of concentration gradients of activators and inhibitors has been difficult. Here we show that the periodic pattern of heterocysts produced by cyanobacteria relies on two inhibitors of heterocyst differentiation, PatS and HetN, in a manner consistent with the predictions of the activator-inhibitor model. Concentration gradients of the activator, HetR, were observed adjacent to heterocysts, the natural source of PatS and HetN, as well as adjacent to vegetative cells that were manipulated to overexpress a gene encoding either of the inhibitors. Gradients of HetR relied on posttranslational decay of HetR. Deletion of both patS and hetN genes prevented the formation of gradients of HetR, and a derivative of the inhibitors was shown to promote decay of HetR in a concentration-dependent manner. Our results provide strong support for application of the activator-inhibitor model to heterocyst patterning and, more generally, the formation of periodic patterns in biological systems.activator-inhibitor ͉ Anabaena ͉ development