Novel Mechanism for Surface Layer Shedding and Regenerating in Bacteria Exposed to Metal-Contaminated Conditions Archjana Chandramohan, Elodie Duprat, Laurent Remusat, Severine Zirah, Carine Lombard, Adrienne Kish To cite this version: Archjana Chandramohan, Elodie Duprat, Laurent Remusat, Severine Zirah, Carine Lombard, et al.. Novel Mechanism for Surface Layer Shedding and Regenerating in Bacteria Exposed to Metal- Contaminated Conditions. Frontiers in Microbiology, Frontiers Media, 2019, 9 (Janvier 2019), pp.3210. 10.3389/fmicb.2018.03210. hal-01996862 HAL Id: hal-01996862 https://hal.sorbonne-universite.fr/hal-01996862 Submitted on 28 Jan 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. fmicb-09-03210 January 14, 2019 Time: 17:59 # 1 ORIGINAL RESEARCH published: 15 January 2019 doi: 10.3389/fmicb.2018.03210 Novel Mechanism for Surface Layer Shedding and Regenerating in Bacteria Exposed to Metal-Contaminated Conditions Archjana Chandramohan1, Elodie Duprat2, Laurent Remusat2, Severine Zirah1, Carine Lombard1 and Adrienne Kish1* 1 Unité Molécules de Communication et Adaptation des Microorganismes (MCAM), Muséum National d’Histoire Naturelle, CNRS UMR 7245, Paris, France, 2 Institut de Minéralogie, Physique des Matériaux et de Cosmochimie (IMPMC), Sorbonne Université, Muséum National d’Histoire Naturelle, CNRS UMR 7590, IRD UMR 206, Paris, France Surface layers (S-layers) are components of the cell walls throughout the Bacteria and the Archaea that provide protection for microorganisms against diverse environmental stresses, including metal stress. We have previously characterized the process by which S-layers serve as a nucleation site for metal mineralization in an archaeon Edited by: for which the S-layer represents the only cell wall component. Here, we test the Stéphane Pesce, hypothesis originally proposed in cyanobacteria that a “shedding” mechanism exists for National Research Institute of Science and Technology for Environment replacing S-layers that have become mineral-encrusted, using Lysinibacillus sp. TchIII and Agriculture (IRSTEA), France 20n38, metallotolerant gram-positive bacterium, as a model organism. We characterize Reviewed by: for the first time a mechanism for resistance to metals through S-layer shedding Mariusz Cycon,´ and regeneration. S-layers nucleate the formation of Fe-mineral on the cell surface, Medical University of Silesia, Poland Sukhendu Mandal, depending on physiological state of the cells and metal exposure times, leading to the University of Calcutta, India encrustation of the S-layer and changes in the cell morphology as observed by scanning *Correspondence: electron microscopy. Using Nanoscale Secondary Ion Mass Spectrometry, we show Adrienne Kish [email protected] that mineral-encrusted S-layers are shed by the bacterial cells after a period of latency (2 days under the conditions tested) in a heterogeneous fashion likely reflecting natural Specialty section: variations in metal stress resistance. The emerging cells regenerate new S-layers as This article was submitted to Microbiotechnology, Ecotoxicology part of their cell wall structure. Given the wide diversity of S-layer bearing prokaryotes, and Bioremediation, S-layer shedding may represent an important mechanism for microbial survival in a section of the journal metal-contaminated environments. Frontiers in Microbiology Received: 13 September 2018 Keywords: S-layer, metal, biomineralization, metallotolerance, Lysinibacillus Accepted: 11 December 2018 Published: 15 January 2019 Citation: INTRODUCTION Chandramohan A, Duprat E, Remusat L, Zirah S, Lombard C and Environmental contamination by metals and radionuclides from activities such as mining and Kish A (2019) Novel Mechanism nuclear power generation pose a serious risk to human health. The sudden, accidental release of for Surface Layer Shedding and Regenerating in Bacteria high concentrations of iron from acid mine drainage from the Gold King Mine polluted the Animas Exposed to Metal-Contaminated River in 2015, mixing downstream with phosphates from agricultural runoff (Rodriguez-Freire Conditions. Front. Microbiol. 9:3210. et al., 2016). Metal contamination affected both water supplies from soluble metals, and sediments doi: 10.3389/fmicb.2018.03210 after the sedimentation of the majority of released metals. Sampling of soils contaminated by metals Frontiers in Microbiology| www.frontiersin.org 1 January 2019| Volume 9| Article 3210 fmicb-09-03210 January 14, 2019 Time: 17:59 # 2 Chandramohan et al. S-Layer Shedding and Regeneration and radionuclides near the former Chernobyl nuclear reactor for up to 5 days. Cells were filtered and observed by scanning site (Chapon et al., 2012) and in uranium mining waste piles in electron microscopy (SEM) as described below. Germany (Pollmann et al., 2006) have identified bacteria of the genre Lysinibacillus tolerant to these contaminants. Lysinibacillus Mineralization Recovery Time Course [formerly classified as part of the Bacillus genre (Ahmed et al., In order to test the hypothesis that mineral-encrusted S-layers 2007)] gram-positive bacteria, with a peptidoglycan cell wall are shed and regenerated, a time course of recovery was enclosed by a surface layer (“S-layer”) attached non-covalently followed after Fe-mineralization as follows: Lysinibacillus sp. to the lipopolysaccharides of the outer membrane (reviewed TchIII 20n38 cells were grown to mid-exponential growth phase ◦ in Sleytr et al., 2014). These S-layers have proven to be a key (OD600 nm = 0.3) in LB (30 C, 180 rpm). The culture medium mechanism for metallotolerance in Lysinibacillus as they have was then removed and the cells washed in MilliQ-H2O by gentle been shown to bind U, Pd(II), Cu, Pt(II), and Au(III) (Pollmann centrifugation (2600 × g, 15 min, room temperature). The cells et al., 2006). were then resuspended to an equivalent cell density in either S-Layers, however are not unique to Lysinibacillus. They are a mineralization solution (10 mM NaH2PO4, 10 mM FeSO4, common components of the cell envelopes of both bacteria pH = 4.5), or a nutrient-free buffered solution at the same pH ◦ and archaea. S-layers are formed by self-assembly of repeated (10 mM NaH2PO4, pH = 4.5), and agitated (30 C, 150 rpm) protein monomers into ordered structures (oblique, square, or for 16 h. The mineralization solution was then removed and the hexagonal) depending on the number of subunits composing the cells washed in MilliQ-H2O by gentle centrifugation (2600 × g, ordered structure. This self-assembly occurs even in the absence 15 min, room temperature), and replaced by growth medium (LB, of cells in vitro; a capacity has been exploited in biotechnology in or labeled growth medium). everything from the development of vaccine to nanomaterials to For nanoscale secondary ion mass spectrometry (NanoSIMS) filtration technologies (Sleytr et al., 2011). experiments, LB was replaced with a defined growth medium S-Layers form the interaction interface between prokaryotic approximating LB but containing either 14N or 15N (Celtone R , cells and their external environment, and are therefore in contact Cambridge Isotopes, United States). While specified for use with with metals and other ions present. Nucleation of mineralization bacteria for isotope analyses, this media was found to contain by S-layers was first noted in cyanobacteria by Schultze-Lam inhibitory concentrations of trace metals, which we were able et al.(1992). Cyanobacterial S-layers were demonstrated to to remove by precipitation by addition of an excess of buffered nucleate the formation of carbonates of calcium, magnesium, phosphate (10 mM) over 24 h with agitation at 150 rpm, followed and strontium (Schultze-Lam and Beveridge, 1994). Schultze- by filtration using a 0.22 mm filter. This metal-depleted medium Lam et al.(1992) proposed the hypothesis that mineral-encrusted containing 5 g/L Celtone R was completed with 5 g/L acetate as S-layers are shed from cyanobacteria as part of a protective a C-source, and basal salts (15 mM ammonium sulfate, 0.2 mM mechanism to ensure that essential cell activities are maintained MgSO4,17.6 mM KH2PO4, 32.7 mM NaH2PO4), as determined despite cell wall mineralization. This hypothesis was, however, by our preliminary optimized experiments with Lysinibacillus never fully tested. Since then, S-layer nucleation of mineralization sp. TchIII 20n38. In addition, cultures grown in the presence has been observed in a range of bacteria (Konhauser et al., 1994; of 14N rather than 15N rapidly ceased vegetative growth and Phoenix et al., 2000) and archaea (Kish et al., 2016). sporulated. Therefore instead of a standard labeling medium Here, we describe the shedding and regeneration of mineral- composed of both 14N and 15N, a 100% 15N-labeled medium, encrusted S-layers in the metallotolerant environmental isolate was used to follow the time course of recovery after Fe-exposure. Lysinibacillus sp. TchIII 20n38. An additional culture was resuspended in a 100% 14N medium and immediately sampled as a baseline control for N