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

Abstract

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...

The Structure of a Cyanobacterial Bicarbonate Transport Protein, CmpA

“…In cyanobacteria, Ca 2ϩ is an essential part of the reaction centers of photosynthetic proteins and may also be a signal for differentiation during heterocyst formation (28,29). In the case of CmpA, calcium may be used to either increase the binding affinity or selectivity of bicarbonate over the other anions such as nitrate.…”

Regulation of intracellular free calcium concentration during heterocyst differentiation by HetR and NtcA in Anabaena sp. PCC 7120

“…It has been demonstrated that mature heterocysts and proheterocysts have an increased [Ca 2ϩ ] i (10). To understand the mechanism for regulation of [Ca 2ϩ ] i during heterocyst differentiation, we studied the kinetics of [Ca 2ϩ ] i increase during heterocyst differentiation.…”

“…CcbP, a calcium-binding protein in heterocyst-forming cyanobacteria, plays an important role in the regulation of [Ca 2ϩ ] i and is absent in mature heterocysts. The expression of ccbP is also down-regulated in heterocysts (10). However, it is not known at present how Ca 2ϩ is released from CcbP and how the expression of ccbP is regulated.…”

“…Although the role of Ca 2ϩ in prokaryotic cellular activities is less clear (11)(12)(13), current evidence also shows that [Ca 2ϩ ] i is also tightly regulated in bacteria (14,15) and that Ca 2ϩ plays important roles in bacterial cell differentiation such as sporulation of Bacillus (16) and heterocyst formation of cyanobacteria (10). It was recently shown that [Ca 2ϩ ] i increases in differentiating cells after transfer from a nitrogen-replete condition to a nitrogen-deprived condition (10). CcbP, a calcium-binding protein in heterocyst-forming cyanobacteria, plays an important role in the regulation of [Ca 2ϩ ] i and is absent in mature heterocysts.…”

“…Another important signal in heterocyst differentiation is the intracellular concentration of free calcium, [Ca 2ϩ ] i (9,10). It is known that Ca 2ϩ ions play very important roles in cellular processes in eukaryotes and that eukaryotic [Ca 2ϩ ] i is tightly regulated and maintained in the nanomolar range.…”