Role of surface biomolecules in the heavy metals sequestrant capacity of lactic acid bacteria. A Vibrational Spectroscopy study

E. GERBINO; P. MOBILI; E.E. TYMCZYSZYN; C. ARAUJO-ANDRADE; C. FRAUSTO-REYES; RUI FAUSTO; A. GÓMEZ-ZAVAGLIA

Lisboa

Simposio; International Symposium on Metal Complexes (ISMEC) 2012; 2012

INTRODUCTION: Toxic metals are not degradable and tend to accumulate in the exposedorganisms causing serious health effects. The use of inactivated microbial biomass asadsorbents (biosorption) has become an efficient tool to remove such metals from differentsubstrates (i.e. water, food, etc.) [1,2]. For this purpose, several species of microorganisms,including lactic acid bacteria, have been successfully employed. However, the molecularbases involved in this process have not been properly explored. For this reason, the aim of thiswork was to evaluate the bacteria/heavy metal interaction from a chemical point of view.MATERIALS AND METHODS: Lactobacillus kefir strains CIDCA 8348 and JCM 5818were cultured in MRS broth [3] for 48 h at 30 oC (stationary phase). One millilitre of culturesin the stationary phase was harvested and washed twice with ultra pure water (Milli-Q plus;Millipore Cop., USA). The pellets were resuspended into 1 ml milli Q water containing Pb+2[from Pb(NO3)2], Cd+2 [from Cd(NO3)2] or Ni+2 [from Ni(NO3)2.6H2O] ranging from 0 to 0.9mM. The suspensions were further incubated for 1 h at 30oC (pH 5.5) and then centrifuged for4 min at 6600 g. The pellets were kept to register the Raman spectra.Raman Spectroscopy: The Raman spectra of both bacteria containing the adsorbedheavy metals and nontreated bacteria (controls) were measured by placing them onto analuminium substrate and then under a Leica microscope integrated to the Raman system(Renishaw 1000B). The wavelength of excitation was 830 nm and the power irradiation overthe samples, 45 mW. Each spectrum was registered with an exposure of 30 s, twoaccumulations, and collected in the 1800-200 cm-1 region (spectral resolution: 2 cm-1).S-layer proteins preparation: Bacterial cells harvested in the stationary phase weremixed with 5 M LiCl and the S-layer proteins were extracted and purified as describedpreviously [4]. S-layer proteins were suspended in 1 ml milli Q water containing 0.3 mMPb(NO3)2, Cd(NO3)2 or Ni(NO3)2.6H2O. The suspensions were incubated for 24 h at 30 ºCand then, centrifuged for 4 min at 6600 g. The pellets were used for the FTIR determinations.FTIR spectroscopy: 30 ml of the S-layer pellets were put on a CaF2 window and furtherdried for 15 min at 45 ºC to get a transparent film, used for FTIR experiments. FTIR spectrawere recorded in the 4000-500 cm-1 range in transmission mode. The IR spectra were obtained co-adding 128 scans at room temperature (spectral resolution: 4 cm-1).Deconvolution of amide I band and assignment of protein secondary structures was carriedout as previously reported [4].RESULTS: The Raman spectra of both nontreated L. kefir strains (controls) werecompared with those obtained after the treatment with each heavy metal. The main differencesobserved between controls and bacteria/metal samples clearly denoted the sites where metalsare attached. Modifications of both nCOO- s (1445 cm-1) and nCOO- as (1655 cm-1) bandsindicated that bacteria⁄metal interaction occurs through carboxylate groups. The band at 1260cm-1, related with phosphate groups disappeared after the treatment with metals and in theregion 1000-850 cm-1, ascribed to polysaccharides, further changes were observed.The outmost structures in L. kefir strains are the S-layer proteins. They aremacromolecular paracrystalline arrays that completely cover the bacterial cell surface [5,6].They are attached to the underlying cell wall by non-covalent bonds and usually may bedissociated and solubilized into protein monomers by chaotropic agents (i.e.: LiCl).Considering this background, the S-layers of the two strains under study were purifiedand the S-layer/heavy metal interaction was analyzed using FTIR spectroscopy. Themetal/protein interaction occurred mainly through the COO- groups of the side chains of Aspand Glut residues, with some contribution of the NH groups of the peptide backbone. Thefrequency separation between the nCOO- as and nCOO- s vibrations in the spectra of the Slayersin presence of the metal ions was found to be ca. 190 cm-1 for S-layer CIDCA 8348 andca. 170 cm-1 for JCM 5818, denoting an unidentate coordination in both cases [7].The secondary structure of the proteins was also altered after the interaction with heavymetals: a general trend to increase the amount of b-sheet structures and to reduce the amountof a-helices was observed. These changes allow the proteins to adjust their structure to thepresence of the metal ions at minimum energy expense.CONCLUSION: This analysis allowed obtaining a deeper insight on the molecularinteractions involved when heavy metals are attached to bacterial surfaces.