A calcium signal is involved in heterocyst differentiation in the cyanobacterium Anabaena sp. PCC7120

Some cyanobacteria, known as euendoliths, excavate and grow into calcium carbonates, with their activity leading to significant marine and terrestrial carbonate erosion and to deleterious effects on coral reef and bivalve ecology. Despite their environmental relevance, the mechanisms by which they can bore have remained elusive and paradoxical, in that, as oxygenic phototrophs, cyanobacteria tend to alkalinize their surroundings, which will encourage carbonate precipitation, not dissolution. Therefore, cyanobacteria must rely on unique adaptations to bore. Studies with the filamentous euendolith, Mastigocoleus testarum, indicated that excavation requires both cellular energy and transcellular calcium transport, mediated by P-type ATPases, but the cellular basis for this phenomenon remains obscure. We present evidence that excavation in M. testarum involves two unique cellular adaptations. Long-range calcium transport is based on active pumping at multiple cells along boring filaments, orchestrated by the preferential localization of calcium ATPases at one cell pole, in a ring pattern, facing the cross-walls, and by repeating this placement and polarity, a pattern that breaks at branching and apical cells. In addition, M. testarum differentiates specialized cells we call calcicytes, that which accumulate calcium at concentrations more than 500-fold those found in other cyanobacteria, concomitantly and drastically lowering photosynthetic pigments and enduring severe cytoplasmatic alkalinization. Calcicytes occur commonly, but not exclusively, in apical parts of the filaments distal to the excavation front. We suggest that calcicytes allow for fast calcium flow at low, nontoxic concentrations through undifferentiated cells by providing buffering storage for excess calcium before final excretion to the outside medium.

supporting

confidence: 50%

Extreme cellular adaptations and cell differentiation required by a cyanobacterium for carbonate excavation

Guida

García-Pichel

2016

“…The detection of a transient increase of [Ca 2ϩ ] i during heterocyst differentiation (29) and the results obtained with manipulation of extracellular Ca 2ϩ concentrations (9) suggested that Ca 2ϩ might be a signal for the differentiation process. In this report, we describe a Ca 2ϩ -binding protein, CcbP, from Anabaena 7120 and suggest a role of CcbP in regulation of heterocyst differentiation.…”

Section: Discussionmentioning

confidence: 99%

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

Zhao

Shi

Zhao

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...

“…This may also explain how reprecipitation at the lagging end may cause microbial borers to act as cements agent in sedimentary structures when growth allows them to cross between mineral grains (27). Our model also opens biological questions as to how boring cyanobacteria might cope with altered intracellular Ca 2+ , which is otherwise kept low and tightly regulated (22). Because a lowering of ocean calcium carbonate saturation state driven by anthropogenic CO 2 emissions (28) will lower the bioenergetic cost of boring, a global increase in cyanobacterial bioerosion rates in shallow waters can be logically predicted, an outcome for which some experimental evidence is already available (29), likely affecting already impacted (30) coral reefs, as well as the bivalve fisheries industry (7).…”

Section: +mentioning

confidence: 97%

“…Intense extrusion of intracellular Ca 2+ by P-type ATPases strategically anchored on the plasma membrane of the distal cells, close to the borehole entrance, keep intracellular concentrations within the typical physiological micromolar (22) range and establish an intracellular diffusive mass transport down the concentration gradient that brings Ca 2+ along the trichome toward this region. Either through active transport with additional ATPases located at the cross walls, or through channels that allow facilitated diffusion of Ca 2+ and protons (the first variant is depicted in Fig.…”

Section: +mentioning

confidence: 99%

Microbial excavation of solid carbonates powered by P-type ATPase-mediated transcellular Ca ²⁺ transport

García-Pichel

Ramírez-Reinat

Gao

2010

104

106

Some microbes, among them a few species of cyanobacteria, are able to excavate carbonate minerals, from limestone to biogenic carbonates, including coral reefs, in a bioerosive activity that directly links biological and geological parts of the global carbon cycle. The physiological mechanisms that enable such endolithic cyanobacteria to bore, however, remain unknown. In fact, their boring constitutes a geochemical paradox, in that photoautotrophic metabolism will tend to precipitate carbonates, not dissolve them. We developed a stable microbe/mineral boring system based on a cyanobacterial isolate, strain BC008, with which to study the process of microbial excavation directly in the laboratory. Measurements of boring into calcite under different light regimes, and an analysis of photopigment content and photosynthetic rates along boring filaments, helped us reject mechanisms based on the spatial or temporal separation of alkali versus Acid-generating metabolism (i.e., photosynthesis and respiration). Instead, extracellular Ca 2+ imaging of boring cultures in vivo showed that BC008 was able to take up Ca 2+ at the excavation front, decreasing the local extracellular ion activity product of calcium carbonate enough to promote spontaneous dissolution there. Intracellular Ca 2+ was then transported away along the multicellular cyanobacterial trichomes and excreted at the distal borehole opening into the external medium. Inhibition assays and gene expression analyses indicate that the uptake and transport was driven by P-type Ca 2+ -ATPases. We believe such a chemically simple and biologically sophisticated mechanism for boring to be unparalleled among bacteria.bioerosion | calcium metabolism | endoliths | microbialites