Fourier transform infrared spectroscopic characterisation of heavy metal-induced metabolic changes in the plant-associated soil bacterium Azospirillum brasilense Sp7

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Abstract

Structural and compositional features of whole cells of the plant-growth-promoting rhizobacterium Azospirillum brasilense Sp7 under standard and heavy metal-stressed conditions are analysed using Fourier transform infrared (FTIR) spectroscopy and compared with the FT-Raman spectroscopic data obtained previously [J. Mol. Struct. 563–564 (2001) 199]. The structural spectroscopic information is considered together with inductively coupled plasma-mass spectrometric (ICP-MS) analytical data on the content of the heavy metal cations (Co2+, Cu2+ and Zn2+) in the bacterial cells. As a bacterial response to heavy metal stress, all the three metals, being taken up by bacterial cells from the culture medium (0.2 mM) in significant amounts (ca. 0.12, 0.48 and 4.2 mg per gram of dry biomass for Co, Cu and Zn, respectively), are shown to induce essential metabolic changes in the bacterium revealed in the spectra, including the accumulation of polyester compounds in bacterial cells and their enhanced hydration affecting certain IR vibrational modes of functional groups involved.

Introduction

Over the past decade, vibrational spectroscopy based on the rapidly expanding use of modern Fourier-transform (FT) IR and Raman spectrometers has been shown to be a valuable and informative tool for the structural characterisation of diverse biological objects, starting from functional groups of biopolymers and supramolecular assemblies up to intact cells [1], [2], [3], [4], [5], [6], [7], [8], [9]. Some of our previous research was focused on structural spectroscopic and analytical studies on whole cells and cell constituents of the plant-associated soil bacterium Azospirillum brasilense[10], [11], [12], [13], [14] which, among other Azospirillum species, attracts the world-wide attention of researchers owing to its plant growth-promoting effects (for reviews see [15], [16], [17], [18], [19], [20]). In line with our data previously obtained using FT-Raman spectroscopy [13], in the present work we analyse heavy metal-induced metabolic changes in A. brasilense (wild-type strain Sp7) reflected by certain FTIR spectroscopic features of its whole cells. The structural spectroscopic data are complemented by inductively coupled plasma-mass spectrometric (ICP-MS) analyses of the cell samples of the bacterium, grown both in a standard medium and in the presence of a certain metal cation (Co2+, Cu2+ or Zn2+).

Section snippets

Preparation of bacterial cultures

Azospirillum brasilense (wild-type strain Sp7; the collection of IBPPM RAS, Saratov, Russia) was cultivated in a standard synthetic phosphate- and malate-containing medium as reported elsewhere [10], [11], [12], [13], with 0.5 g/l NH4Cl as a bound nitrogen source (pH 6.9), under aeration by stirring on a rotary shaker. Along with the standard medium (control), the bacterium was similarly cultured also in the same media to which CoCl2, CuSO4 or ZnSO4 had been added up to 2.0×10−4 M. Bacterial

Results and discussion

Binding of heavy metals by the cell surface in Gram-negative bacteria, to which azospirilla belong, is mediated primarily by capsular polysaccharide (PS) and lipopolysaccharide (LPS) materials [21], [22], [23]. The latter are characteristic for A. brasilense and, along with its outer-membrane proteins, are believed to be involved in contact interactions with host plant roots [17], [24], [25] and other surfaces [24], [26], as well as in bacterial cell aggregation [17], [27], [28].

Our previous

Conclusions

The results obtained, based on FTIR spectra of whole bacterial cells, in line with the assumptions made on the basis of previously reported FT-Raman data [13], have revealed essential metabolic changes induced by heavy metal cations (Co2+, Cu2+ or Zn2+) taken up by the bacterium from the medium. It is proposed that, along with a possible increase in PL synthesis under metal stress which was assumed earlier for another A. brasilense strain (Sp245) [12] and reported for another soil bacterium [35]

Acknowledgements

The authors are grateful to Drs M. Ristić (Zagreb, Croatia), M. Colina (Sheffield, UK; permanent address: Maracaibo, Venezuela) and L.A. Bespalova (Saratov, Russia) for their skilful assistance in experimental work. This study was supported in part by the EC (INTAS, Brussels, Belgium; Project 96-1015), NATO (Collaborative Linkage Grant LST.CLG.977664) and the Russian Academy of Sciences' Commission for the Work with Young Scientists (Grant No. 205 under the 6th Competition-Expertise of research

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