Microbial excavation of solid carbonates powered by P-type ATPase-mediated transcellular Ca2+ transport

Abstract

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.

Fig. 1.

Strain BC008 and its ability to bore. (A) Photomicrograph of culture grown in liquid. (B) Close-up of a heavily infested calcite chip (infestation started from top). Single green filaments can be observed deep in the crystal matrix. Notice the reprecipitation of a yellow, light-scattering micrite layer close to the surface.

Fig. 2.

Evidence against separation between photosynthetic and boring activities in strain BC008. (A) growth rate (as doubling time, right axis) in liquid culture, and the intensity of infestation on blocky calcite chips attained at 5 wk of incubation (left axis), both as a function of the length of the illumination period when grown on a day/night cycle. (B) Cell-specific photosynthetic pigment content measured in situ during boring using fluorescence emission spectroscopy of confocal optical sections with excitation at 488 nm. Lines show average (n = 5) emission spectra of single cells for cells close to the boring front (proximal) compared with cells close to the surface of the solid (distal). Inset: Chlorophyll a-specific emission at 685 nm (error bars are 1 SD); differences are not significant (t test; P = 0.234). (C) Photosynthetic rates of boring BC008 cells retrieved from layers close to the surface of chips compared with those in the middle and deep zones and those from nonboring cultures (control). No significant differences were found (P > 0.33 in all t tests; n = 3).

Fig. 3.

Real-time free Ca²⁺ supersaturation around boreholes during boring of BC008 on calcite. LSCM Images are plane view from live illuminated cultures boring into calcite chips in custom-designed stage incubation chambers. (A) Three-dimensional reconstruction from single optical sections recorded parallel to the chip surface shown at an elevation angle of 30°. Green fluorescence denotes dissolved Ca²⁺ concentration reported by the externally supplied fluorophore. Red autofluorescence is from photosynthetic pigments (chlorophyll a and phycobilins) and tracks cyanobacterial filaments. (B) In an interpretation of A, thick lines denote that some filaments naturally stick out of the chip and thin lines denote filaments inside the chip. Note that Ca²⁺ inside a natural crack in the chip is much closer to saturation. (C) Vertical cross-section of the solid/liquid interface reconstructed from data in A (only the green channel is shown). (D) Temporal dynamics of Ca²⁺ concentration measured around boreholes after changes in stage illumination. Solid lines delimit 2 SD around the mean of n = 10 boreholes. Some source images are shown in the sequence of insets. Ca²⁺ decreased to saturation within 2 h of turning light (10 W m⁻²) off. Higher Ca²⁺ supersaturation was reached within 2 h of turning on illumination at 30 W m⁻².

Fig. 4.

Evidence for a boring mechanism based on transcellular Ca²⁺ transport. (A) LSCM calcium mapping of a heavily infested chip taken along the main boring direction shows supersaturation at the boundary layer (particularly in the heavily eroded surface) and within the boreholes close to the surface, and undersaturation around the deeper parts of the boreholes. (B) Quantitative data for Ca²⁺ concentration along the boreholes of single filaments measured in lightly bored chips (solid line is the average of 10 filaments; SDs shown as dashed lines); the dotted vertical line marks expected calcite-saturation equilibrium.

Fig. 5.

Effect of inhibitors on BC008 boring into calcite chips. All experiments measured the level of Ca²⁺ supersaturation at the chip's surface as a proxy for boring activity using Ca²⁺ imaging under LSCM as in Fig. 3D. Inhibitors were added at the time marked by the arrows in each panel. CCCP is a proton motive force inhibitor and oligomycin inhibits ATP synthase. Vanadate and lanthanum ions are typical inhibitors of P-type ATPases. Thapsigargin and tert-butylhydroquinone (t-BHQ) are known inhibitors of Ca²⁺-transporting P-type ATPases. Verapamil is a Ca²⁺ channel blocker.

Fig. 6.

Level of overexpression of two putative P-type Ca²⁺ ATPase genes detected in BC008, JGE01, and JGE04, with respect to the levels found in nonboring, free-living cultures, as measured by quantitative RT-PCR. Results are from three depths in a single chip, and with an analytical replication of three for each depth. Only overexpression levels that were statistically significant (P < 0.05; pair-wise fixed reallocation randomization test) are presented; an asterisk indicates overexpression that was not highly significant (P = 0.117). Lines on the bars indicate 95% confidence intervals for the mean ratio. Uncorrected PCR data for all samples and controls can be found in SI Text.

Fig. 7.

Working model for cyanobacterial excavation of calcium carbonates. Left: Concentrations and ranges of extracellular Ca²⁺ (measured; green intensity) and intracellular Ca²⁺ (inferred, red intensity) around the boring system, as well as the major direction of net Ca²⁺ movement. Right: Inferred nature and distribution of transporter components (legend at bottom), and the fate of Ca²⁺ and CO₃²⁻ after calcite dissolution.