# Intracellular formation of carbonates by bacteria as a new case of genetically controlled biomineralization

Karim Benzerara, Khan Monis Athar, Isabelle Callebaut, Elodie Duprat, Geoffroy Gaschignard, Christopher T. Lefèvre, Camille Mangin, Neha Mehta, Nicolas Menguy, Caroline Monteil, et al.

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## To cite this version:

Karim Benzerara, Khan Monis Athar, Isabelle Callebaut, Elodie Duprat, Geoffroy Gaschignard, et al. Intracellular formation of carbonates by bacteria as a new case of genetically controlled biomineralization. MTM2024 - 8th International Meeting on Magnetotactic Bacteria, Jun 2024, Bilbao, Spain. hal-04871270

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https://hal.science/hal-04871270v1

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# Intracellular formation of carbonates by bacteria as a new case of genetically controlled biomineralization

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Karim Benzerara, Khan M. Athar, Isabelle Callebaut, Elodie Duprat, Geoffroy Gaschignard, Christopher T. Lefevre, Camille Mangin, Neha Mehta, Nicolas Menguy, Caroline L. Monteil, Fériel Skouri-Panet, ANR Carbomagnet and the ANR Harley consortia

Institut de Minéralogie et de Physique des Milieux Condensés (IMPMC)
CNRS, Sorbonne Université & Muséum National d'Histoire Naturelle
Bacterial biomineralization is often considered as a non controlled mineralization process (except for MTB)

<figure><img src="image_4.png" /><figcaption>Modern stromatolite formed in Sardinia</figcaption></figure>

Debrie et al. (2022) *Chem Geol*

e.g., extracellular CaCO₃ precipitation by cyanobacteria

$$6Ca^{2+} + 12HCO_3^- \rightleftharpoons 6CaCO_3 + C_6H_{12}O_6 + 6O_2$$
Serendipitous discovery of a cyanobacterium forming intracellular amorphous CaCO₃ in a controlled manner

→ *Gloeomargarita lithophora*

<figure><img src="image_5.png" /></figure>
<figure><img src="image_6.png" /><figcaption>Now hundreds of cyanobacteria from diverse families known to form intracellular ACC</figcaption></figure>

PCC 6716, 6717, 6312

<figure><img src="image_7.png" /></figure>

Benzerara et al. (2014) PNAS

<figure><img src="image_8.png" /></figure>

Benzerara et al. (2022) GBE

Single cell cyanobacteria; Multicellular cyanobacteria; Marine cyanobacteria ...
<figure><img src="image_9.png" /></figure>

Widespread distribution of iACC-forming cyanobacteria in phylogenetic tree

Indications that intracellular biomineralization is ancestral in
Cyanobacteria
G. lithophora is the phylogenetically closest extant cyanobacterium to plastids

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

Ponce-Toledo et al (2017) *Current Biology*

Was the formation of intracellular CaCO₃ transferred to photosynthetic eukaryotes ?
Amorphous calcium carbonates (ACC)

TEM

<figure><img src="image_12.png" /></figure>

Li et al. (2016) Minerals

XAS at the Ca
K-edge

<figure><img src="image_13.png" /></figure>

Mehta et al. (2023) Chem Geol

FTIR

<figure><img src="image_14.png" /></figure>

Mehta et al. (2022) Spectrochimica Acta

They seem to stay intracellular and amorphous in a culture
## Apparent out-of-equilibrium process

### Formation of intracellular CaCO₃ in environments undersaturated with CaCO₃ phases

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

## Fate of intracellular ACC after cell death?

<figure><img src="image_18.png" /></figure>
Unusual element fractionation by, e.g., *Gloeomargarita lithophora*
= preferential uptake of high-Z alkaline earth metals

Cam et al. (2016) ES&T; Blondeau et al. (2018) Chem Geol.

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

Ba > Sr > Ca

<figure><img src="image_21.png" /></figure>

Cam et al. (2016) ES&T

→ **Formation of Sr and/or Ba-rich ACC despite inconsistent environmental chemical conditions**

= singularity of intracellular carbonates
Some of these bacteria may help remediate radioactive pollutions ($^{90}$Sr and $^{226}$Ra)

→ efficient trapping of heavy alkaline earth metals, incl. radioactive isotopes

nature

Subscribe

<figure><img src="image_22.png" /></figure>

A *Gloeomargarita lithophora* bacterium containing carbonate granules (white spheres). The microbe's ability to form such granules internally might explain how it draws radioactive isotopes from its surroundings. Credit: Karim Benzerara and Stefan Borensztajn/CNRS

ENVIRONMENTAL SCIENCES · 15 OCTOBER 2019

Nuclear waste? These microbes might help with the cleanup

Bacteria discovered in a Mexican lake accumulate two radioactive isotopes in their cells.

<figure><img src="image_23.png" /></figure>

NanoSIMS mapping of Ca, P, Ra

250 µM of Ca + 9.10⁻⁵ µM $^{90}$Sr (or Ra)

Mehta et al (2019) ES&T
Mehta et al (2022) ES&T Water
Some of these ACC-forming cyanobacteria form blooms (high cell densities)

→ cyanobacterium blooming in eutrophic aqueous environments

- major health/environmental issue

<figure><img src="image_24.png" /><figcaption>*Microcystis* bloom (Maimee River, Ohio, USA) @David J.Ruck</figcaption></figure>

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Optical microscopy
@EPIDOR

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→ Important Ca reservoir?
→ What is the biological role of ACC?

Gaetan et al. (2023) *Env. Microbiol*
# Comparative genomics of iACC⁻ vs iACC⁺ cyanobacteria

GBE

GENOME BIOLOGY AND EVOLUTION

Volume 14, Issue 2, March 2023

<figure><img src="image_29.png" /></figure>

ISBN 1759-0663

https://ncovsnc.org/ncovsnc

<figure><img src="image_30.png" /></figure>

Benzerara et al. (2022) GBE

⇒ One single "gene" shared by iACC⁺ and absent in iACC⁻

## iACC⁺ cyanobacteria

* Gloeomargarita_lithophora_Alchichica-D10
* Synechococcus_sp._PCC_6312
* Microcystis_aeruginosa_PCC_9443
* Fischerella_sp._NIES-4106

<figure><img src="image_31.png" /></figure>

GlyZip1

Alignment of aminoacids

★ No detectable full-length homologs with known function in databases

⇒ New family of genes (ccyA)/proteins (calcyanin)
Determining the structure of calcyanin

<figure><img src="image_32.png" /></figure>

AlphaFold2 (AI-based) prediction of the
calcyanin of *Cyanotheces* sp. PCC 7425

What is the role of these different modules (domains) in iACC formation?

What do we know about the CoBaHMA domain?
# What is CoBaHMA ?

Gaschignard et al. (2024) Proteins

<figure><img src="image_33.png" /></figure>

Blue: basic; red: acidic; cyan: histidine

CoBaHMA found in 2227 different bacterial proteins

>74 different types of protein modular organizations

## Membrane proteins involved in transport or lipid metabolism

<figure><img src="image_34.png" /><figcaption>P1B-ATPases</figcaption></figure>

<figure><img src="image_35.png" /><figcaption>ABC exporter</figcaption></figure>

<figure><img src="image_36.png" /><figcaption>Diacylglycerol kinase</figcaption></figure>

<figure><img src="image_37.png" /><figcaption>Unknown proteins with CoBaHMA+hairpins</figcaption></figure>

## Regulatory function, involving interactions with anionic lipids ?
Genomic neighboring of ccyA

→ *ccyA* in a megaplasmid in *Fischerella* sp NIES-4106.

<figure><img src="image_38.png" /></figure>

→ *ccyA* in the genome of *Microcystis aeruginosa* PCC 7806

<figure><img src="image_39.png" /></figure>

Some neighboring genes particularly interesting :

★ Ca²⁺/H⁺ exchanger (cax)

★ Domain of unknown function (DUF) encodes a CoBaHMA domain (no CoBaHMA in *Microcystis calcyanin*)
Day/night expression of the *ccyA* genes

<figure><img src="image_40.png" /></figure>

→ RNAseq experiments

Normalized expression of the *ccyA* gene

<figure><img src="image_41.png" /></figure>

Normalized expression of the *ccyA* gene

<figure><img src="image_42.png" /></figure>

DUF454

<figure><img src="image_43.png" /></figure>

Expression of *ccyA* varies over a day/night cycle with higher expression at the end of the night

Expression of neighboring genes is correlated with that of *ccyA*: transcription unit?
# Genetics: Expression of *ccyA* in a cyanobacterium not forming ACC

## *Synechococcus elongatus* PCC 7942, wild type and mutants expressing *ccyA*

<figure><img src="image_44.png" /></figure>

Benzerara et al. (2023) Journal of Electron Spectroscopy and Related Phenomena

### Mutants expressing *ccyA* have higher Ca contents

### They do not form ACC

### Need more than one gene to form intracellular ACC
Ongoing work

WT

pC-ccyA-6312

pC-ccyA-7425

pC-ccyA-Gloeo

pC-ccyA-Calci

5-5 5-4 5-3 5-2 5-1 0.1

<figure><img src="image_45.png" /></figure>

<figure><img src="image_46.png" /></figure>

→ Genetics and design of deletion mutants

→ iACC extraction and proteomics

...
# Are cyanobacteria the only ACC-forming bacteria? NO

## *Achromatium oxaliferum* (gammaproteobacterium)

<figure><img src="image_47.png" /><figcaption>Schewiakoff (1893)</figcaption></figure>

<figure><img src="image_48.png" /><figcaption>Benzerara et al. (2020) Geobiol.</figcaption></figure>

## Sulfoxidizing bacterium, massive amorphous carbonates
# The biomineralization of iACC in one gammaproteobacterium may be related to *Cyanobacteria = Methylococcus geothermalis*

<figure><img src="image_49.png" /></figure>

500 nm

## Calcyanin of a *Microcystis*

<figure><img src="image_50.png" /></figure>

<figure><img src="image_51.png" /></figure>

500 nm

## Calcyanin of a *Methylococcaceae*

<figure><img src="image_52.png" /></figure>

Benzerara et al., in prep

We find the *ccyA* gene in *Methylococcaceae*. Close to the one of *Microcystis* → Horizontal transfer?
# Cosmopolitism and diversity of MTB forming ACC

<figure><img src="image_53.png" /><figcaption>In Xingqinggong Lake, Xi'an City</figcaption></figure>

Alphaproteobacteria, Rhodospirillaceae (XQGS1),
Liu et al. (2021) AEM

<figure><img src="image_54.png" /><figcaption>Freshwater pond in Kanazawa, Japan</figcaption></figure>

Gammaproteobacteria, Candidatus order/family
CAIRSR01
Taoka et al. (2014) Microbiology
# Cosmopolitism and diversity of MTB forming ACC

In Lake Pavin, Lake Bois le Roi, etc...

## Gammaproteobacteria, Candidatus order/family CAIRSR01

<figure><img src="image_55.png" /></figure>

<figure><img src="image_56.png" /></figure>

<figure><img src="image_57.png" /></figure>

### Alphaproteobacteria, Azospirillaceae

<figure><img src="image_58.png" /></figure>

### Gammaproteobacteria, Chromatiaceae
ACC-forming MTB are abundant in Lake Pavin

Large SEM mosaics (5.9 Gpixels over ~4,3 cm²)

<figure><img src="image_59.png" /></figure>

<figure><img src="image_60.png" /></figure>

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ACC-forming MTB

<figure><img src="image_62.png" /></figure>

<figure><img src="image_63.png" /></figure>

<figure><img src="image_64.png" /></figure>
Tens to hundreds of cells per sample among millions (?) of particles. Need to detect them at different depths

<figure><img src="image_65.png" /></figure>

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Ongoing development of a method to detect all of them efficiently, count them and assess their diversity
Why have they been overlooked before?

<figure><img src="image_87.png" /></figure>

<figure><img src="image_88.png" /></figure>

<figure><img src="image_89.png" /></figure>

<figure><img src="image_90.png" /></figure>

Before cell
fixation
/glutaraldehyde)

<figure><img src="image_91.png" /></figure>

<figure><img src="image_92.png" /></figure>

<figure><img src="image_93.png" /></figure>

<figure><img src="image_94.png" /></figure>

After
cell
fixation

<figure><img src="image_95.png" /></figure>

<figure><img src="image_96.png" /></figure>

<figure><img src="image_97.png" /></figure>

<figure><img src="image_98.png" /></figure>

After
dehydration

Li et al. (2016) *Minerals*

→ ACC are lost upon classical sample preparation techniques
→ Different response to magnetic field for ACC-forming MTB (Monteil et al. 2020)
Link between biomineralization and primary metabolisms?

Oxygenic photosynthesis

$$6 \text{Ca}^{2+} + 12 \text{HCO}_3^{-} = 6 \text{CaCO}_3 + C_6H_{12}O_6 + 6\text{O}_2$$

Sulfoxidation

$$2\text{H}_2\text{S} + \text{O}_2 + \text{Ca}^{2+} + \text{HCO}_3^{-} = 2\text{S(O)} + 2\text{H}_2\text{O} + \text{CaCO}_3$$

Aerobic methane
oxidation/methylotrophy/carboxydotrophy

$$\text{CH}_4 + \text{O}_2 + \text{CaCO}_3 = \text{Ca}^{2+} + 2\text{HCO}_3^{-}$$

$$\text{CO} + 2\text{H}_2\text{O} + \text{CaCO}_3 = \text{Ca}^{2+} + \text{H}_2 + 2\text{HCO}_3^{-}$$

Some should favor ACC precipitation; other should favor dissolution

All seem to be able to fix CO₂
Shared origin among all bacteria?

→ No certainty as long as we do not know how exactly bacteria form ACC

→ If **primary metabolism** is part of the process, then it works differently for the different **ACC-forming bacteria**

→ If *ccyA* is involved in cyanobacterial biomineralization, it is not found in MTB (unless we missed a remote homolog) but found in *Methylococcus*

→ Lipid-bilayer compartment in MTB and *Achromatium*/ proteins in *Cyanobacteriota*

→ **Ca transport**: vacuolar-type pumps in *Achromatium* and MTB (?) vs Ca²⁺/H⁺ exchanger in cyanobacteria + other proteins

To be continued...
(Causal role) Function(s)?

Cosmidis and Benzerara (2022) Comptes rendus Géoscience

→ intracellular pH buffer/source of H⁺

→ Reservoir of inorganic C

→ Reservoir of Ca

→ Ballast

<figure><img src="image_99.png" /></figure>

$$Ca^{2+} + HCO_3^- = CaCO_3 + H^+$$

ACC very reactive

$$ACC \ density = 2.18 \ g/cm^3 (?)$$

Which parameter increased fitness so that this trait was eventually selected during evolution?
Conclusions

- Broad diversity of bacteria able to biomineralize intracellular amorphous carbonates

- This diversity is still most likely underestimated, including within MTB

- This may have some environmental impact

- Controlled mineralization but molecular mechanisms and function remain largely unknown

- It most likely involves more than one gene. Some of these genes encode proteins/domains annotated as hypothetical or of unknown function

- Evolutionary history?
Acknowledgements

<figure><img src="image_100.png" /></figure>

ANR Harley, ANR-19-CE44-0017-01

ANR Carbomagnet, ANR-21-CE01-0010-02

ERC Calcyan, AFP7/2007-2013 Grant 307110

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HARLEY

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SEM, TEM, Spectroscopy facilities + GEMME @IMPMC; HERMES beamline @SOLEIL

**IMPMC** : Apolline Bruley, Isabelle Callebaut, Geraldine Caumes, Manuela Dezi, Elodie Duprat, Imène Esteve, Juliette Gaëtan, Geoffroy Gaschignard, Neha Mehta, Nicolas Menguy, Maxime Millet, Feriel Skouri-Panet, Cynthia Travert

**BIAM (CEA Cadarache)**: Christopher Lefevre, Camille Mangin, Caroline Monteil, Daniel Chevrier, Catherine Berthomieu, Virginie Chapon

**IPGP**: Didier Jezequel, Eric Viollier

**I2BC (CEA Saclay)**: Corinne Cassier-Chauvat, Franck Chauvat, Monis Khan

**Institut Pasteur**: Muriel Gugger

**ESE (Univ Saclay)**: Purificacion Lopez-Garcia, David Moreira

**IEES**: Julie Leloup

**Key Laboratory of Earth and Planetary Physics, Beijing**: Jinhua Li