Fourier Transform Infrared (FTIR) Spectroscopic Study of Biofilms Formed by the Rhizobacterium Azospirillum baldaniorum Sp245: Aspects of Methodology and Matrix Composition.

Biofilms represent the main mode of existence of bacteria and play very significant roles in many industrial, medical and agricultural fields. Analysis of biofilms is a challenging task owing to their sophisticated composition, heterogeneity and variability. In this study, biofilms formed by the rhizobacterium Azospirillum baldaniorum (strain Sp245), isolated biofilm matrix and its macrocomponents have for the first time been studied in detail, using Fourier transform infrared (FTIR) spectroscopy, with a special emphasis on the methodology. The accompanying novel data of comparative chemical analyses of the biofilm matrix, its fractions and lipopolysaccharide isolated from the outer membrane of the cells of this strain, as well as their electrophoretic analyses (SDS-PAGE) have been found to be in good agreement with the FTIR spectroscopic results.

Selenite Reduction by Proteus sp. YS02: New Insights Revealed by Comparative Transcriptomics and Antibacterial Effectiveness of the Biogenic Se0 Nanoparticles.

Biotransformation of selenite by microorganisms is an effective detoxification (in cases of dissimilatory reduction, e.g., to Se0) and assimilation process (when Se is assimilated by cells). However, the current knowledge of the molecular mechanism of selenite reduction remains limited. In this study, a selenite-resistant bacterium was isolated and identified as Proteus sp. YS02. Strain YS02 reduced 93.2% of 5.0 mM selenite to selenium nanoparticles (SeNPs) within 24 h, and the produced SeNPs were spherical and localized intracellularly or extracellularly, with an average dimension of 140 ± 43 nm. The morphology and composition of the isolated and purified SeNPs were characterized using dynamic light scattering (DLS), scanning electron microscopy (SEM) with energy-dispersive X-ray (EDX) spectrometry, and Fourier transform infrared (FTIR) spectroscopy. FTIR spectroscopy indicated the presence of proteins, polysaccharides, and lipids on the surface of the isolated SeNPs. Furthermore, the SeNPs showed excellent antimicrobial activity against several Gram-positive and Gram-negative pathogenic bacteria. Comparative transcriptome analysis was performed to elucidate the selenite reduction mechanism and biosynthesis of SeNPs. It is revealed that 197 genes were significantly upregulated, and 276 genes were significantly downregulated under selenite treatment. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that genes associated with ABC transporters, sulfur metabolism, pentose phosphate pathway (PPP), and pyruvate dehydrogenase were significantly enhanced, indicating selenite is reduced by sulfite reductase with PPP and pyruvate dehydrogenase supplying reducing equivalents and energy. This work suggests numerous genes are involved in the response to selenite stress, providing new insights into the molecular mechanisms of selenite bioreduction with the formation of SeNPs.

Fourier Transform Infrared (FTIR) Spectroscopic Analyses of Microbiological Samples and Biogenic Selenium Nanoparticles of Microbial Origin: Sample Preparation Effects.

To demonstrate the importance of sample preparation used in Fourier transform infrared (FTIR) spectroscopy of microbiological materials, bacterial biomass samples with and without grinding and after different drying periods (1.5-23 h at 45 °C), as well as biogenic selenium nanoparticles (SeNPs; without washing and after one to three washing steps) were comparatively studied by transmission FTIR spectroscopy. For preparing bacterial biomass samples, Azospirillum brasilense Sp7 and A. baldaniorum Sp245 (earlier known as A. brasilense Sp245) were used. The SeNPs were obtained using A. brasilense Sp7 incubated with selenite. Grinding of the biomass samples was shown to result in slight downshifting of the bands related to cellular poly-3-hydroxybutyrate (PHB) present in the samples in small amounts (under ~10%), reflecting its partial crystallisation. Drying for 23 h was shown to give more reproducible FTIR spectra of bacterial samples. SeNPs were shown to contain capping layers of proteins, polysaccharides and lipids. The as-prepared SeNPs contained significant amounts of carboxylated components in their bioorganic capping, which appeared to be weakly bound and were largely removed after washing. Spectroscopic characteristics and changes induced by various sample preparation steps are discussed with regard to optimising sample treatment procedures for FTIR spectroscopic analyses of microbiological specimens.

Poly-3-hydroxybutyrate synthesis by different Azospirillum brasilense strains under varying nitrogen deficiency: A comparative in-situ FTIR spectroscopic analysis.

Monitoring of poly-3-hydroxybutyrate accumulation and changes in its relative contents in biomass of the plant-growth-promoting bacteria Azospirillum brasilense (strains Sp7, Cd and Sp245) was performed during aerobic cultivation for 1 to 8 days at various initial concentrations of bound nitrogen (0.1 to 0.5 g∙L-1 NH4Cl) in the culture medium using in-situ transmission FTIR spectroscopy. A methodology has been proposed based on calculating band areas in FTIR spectra (instead of band intensities commonly used earlier) for determining relative contents of PHB in dry bacterial biomass, using the ester ν(C=O) band as a PHB marker (in the region 1750-1720 cm-1) and amide II band of cellular proteins (at ca. 1540 cm-1). Differences in PHB accumulation levels and their changes in the course of cultivation under various trophic stress for the three strains are discussed in relation to their different ecological niches which they occupy in the rhizosphere.

Developments in the study and applications of bacterial transformations of selenium species.

Microbial bio-transformations of the essential trace element selenium are now recognized to occur among a wide variety of microorganisms. These transformations are used to convert this element into its assimilated form of selenocysteine, which is at the active center of a number of key enzymes, and to produce selenium nanoparticles, quantum dots, metal selenides, and methylated selenium species that are indispensable for biotechnological and bioremediation applications. The focus of this review is to present the state-of-the-art of all aspects of the investigations into the bacterial transformations of selenium species, and to consider the characterization and biotechnological uses of these transformations and their products.

Selenite reduction by the rhizobacterium Azospirillum brasilense, synthesis of extracellular selenium nanoparticles and their characterisation.

Microbial reduction of selenium oxyanions has attracted attention in recent years. In this study, an original and simple method for the synthesis of extracellular selenium nanoparticles (Se NPs) of relatively uniform size has been developed using strains Sp7 and Sp245 of the ubiquitous plant-growth promoting rhizobacterium Azospirillum brasilense, both capable of selenite (SeO32-) reduction. In addition, a reliable purification protocol for the recovery of the Se NPs has been perfected, which could be applied with minor modifications to cultures of other microbial species. Importantly, it was found that, by changing the conditions of bacterial reduction of selenite, extracellularly localised Se NPs can be obtained using bacteria which would otherwise produce intracellular Se NPs. In particular, bacterial cultures grown up to the end of the logarithmic growth phase, washed free of culture medium and then incubated with selenite, were used to obtain extracellular Se NPs. Their sizes depended on the initial selenite concentration (∼25-80 nm in diameter at 50-10 mM selenite, respectively). The Se NPs obtained were characterised by transmission electron microscopy (TEM), dynamic light scattering, as well as Raman and UV-vis spectroscopies. Their zeta potential was found to be negative (ca. minus 21-24 mV). Bacterial selenite reduction was also studied in the presence of the efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP). In this case, TEM indicated the formation only of intracellular selenium crystallites. The data show that the formation of extracellular Se NPs requires normal bacterial metabolic activity, while CCCP evidently blocks the membrane export of Se0 nuclei.

Diffuse reflectance infrared Fourier transform (DRIFT) and Mössbauer spectroscopic study of Azospirillum brasilense Sp7: Evidence for intracellular iron(II) oxidation in bacterial biomass upon lyophilisation.

Microbial cells are well known to be capable of remaining viable when desiccated, and a variety of beneficial microorganisms can thus be preserved for storage. For the ubiquitous widely studied soil bacterium Azospirillum brasilense (wild-type strain Sp7), which has a significant agrobiotechnological potential owing to its plant-growth-promoting capabilities perspective for its use in biofertilisers, Fourier transform infrared (FTIR) spectroscopy (in the diffuse reflectance mode, DRIFT) was used to control the state of biomass, together with 57Fe transmission Mössbauer spectroscopy to monitor intracellular iron speciation in live rapidly frozen cell suspension and in the lyophilised biomass (both measured at T = 80 K). It has been shown for the first time that a relatively large part of ferrous iron in live cells (22% of the whole cellular iron pool, represented by two high-spin Fe(II) forms, in the 18-h culture grown on 57Fe(III) complex with nitrilotriacetic acid as the sole source of iron) gets largely oxidised upon lyophilisation. The remaining part of iron(II) in the resulting dry biomass was found to be ca. 3% only. The major part of ferric iron in the dry biomass was shown to be comprised of ferritin-like ferric species (giving a typical magnetically split sextet at T = 5 K), while the iron(III) formed from cellular iron(II) by oxidation in air in the course of drying remained in a paramagnetic state even at T = 5 K. The possibility of intracellular iron(II) oxidation to iron(III) upon desiccation may be a specific natural strategy to avoid cell damage caused by Fenton-type reactions in dormant (frozen, dried) cells. The results obtained may have important implications related to iron speciation and redox transformations in dried bacterial preparations intended for long-term storage.

FTIR and Raman spectroscopic studies of selenium nanoparticles synthesised by the bacterium Azospirillum thiophilum.

Vibrational (Fourier transform infrared (FTIR) and Raman) spectroscopic techniques can provide unique molecular-level information on the structural and compositional characteristics of complicated biological objects. Thus, their applications in microbiology and related fields are steadily increasing. In this communication, biogenic selenium nanoparticles (Se NPs) were obtained via selenite (SeO32-) reduction by the bacterium Azospirillum thiophilum (strain VKM B-2513) for the first time, using an original methodology for obtaining extracellular NPs. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) showed the Se NPs to have average diameters within 160-250nm; their zeta potential was measured to be minus 18.5mV. Transmission FTIR spectra of the Se NPs separated from bacterial cells showed typical proteinacious, polysaccharide and lipid-related bands, in line with TEM data showing a thin layer covering the Se NPs surface. Raman spectra of dried Se NPs layer in the low-frequency region (under 500cm-1 down to 150cm-1) showed a single very strong band with a maximum at 250cm-1 which, in line with its increased width (ca. 30cm-1 at half intensity), can be attributed to amorphous elementary Se. Thus, a combination of FTIR and Raman spectroscopic approaches is highly informative in non-destructive analysis of structural and compositional properties of biogenic Se NPs.

Proteins in microbial synthesis of selenium nanoparticles.

Biogenic formation of nano-sized particles composed of various materials (in particular, selenium) by live microorganisms is widespread in nature. This phenomenon has been increasingly attracting the attention of researchers over the last decade not only owing to a range of diverse applications of such nanoparticles (NPs) in nanobiotechnology, but also because of the specificity of methodologies and mechanisms of NPs formation related to "green synthesis". In this mini-review, recent data are discussed on the multifaceted role of proteins in the processes of microbial reduction of selenium oxyanions and the formation of Se NPs. Besides the involvement of proteins in reducing selenites and selenates, their participation in the microbially driven growth and stabilisation of Se NPs is analysed, which results in the formation of unique nanostructured materials differing from those obtained chemically. This mini-review is thus focussed on proteins involved in microbial synthesis of Se NPs and on instrumental analysis of these processes and their products (biogenic nanostructured selenium particles functionalised by a surface-capping layer of various biomacromolecules).

Sample treatment in Mössbauer spectroscopy for protein-related analyses: Nondestructive possibilities to look inside metal-containing biosystems.

In this review, the unique possibilities are considered of the 57Fe transmission (TMS) and 57Co emission (EMS) variants of Mössbauer (nuclear γ-resonance) spectroscopy as nondestructive techniques with minimal sample preparation/treatment and a significant analytical potential, with a focus on the analysis of cation-binding sites in metalloproteins. The techniques are shown to provide unique structural and quantitative information on the coordination microenvironment, the chemical state and transformations of the Mössbauer nuclides in sophisticated metal-containing proteins, including those within complicated supramolecular structures, and in microbial cells or tissues. Recent representative examples of analyses of Fe-containing proteins by 57Fe TMS are briefly discussed, along with the newly emerging data on using 57Co EMS for probing the structural organisation of 57Co-doped cation-binding sites in sophisticated biocomplexes including metalloenzymes. Finally, some rare or exotic applications of Mössbauer spectroscopy (including the synchrotron-based methodology) in protein-related studies are outlined.

Cobalt(II) complexation with small biomolecules as studied by 57Co emission Mössbauer spectroscopy.

In the emission (57Co) variant of Mössbauer spectroscopy (EMS), the 57Co radionuclide (with a half-life of 9months) is used that undergoes a nuclear decay 57Co→57Fe via electron capture followed by the emission of a γ-quantum, the energy of which is modified by the chemical state and the close coordination environment of the parent 57Co atom. While EMS has been used largely in materials science and nuclear chemistry, its high sensitivity can also be of great advantage in revealing fine structural features and for speciation analysis of biological complexes, whenever the 57Co2+ cation can be used directly as the coordinating metal or as a substitute for native cobalt or other metal ions. As such EMS applications are yet rare, in order to reliably interpret emission spectra of sophisticated 57Co2+-doped biosystems, model EMS studies of simple cobalt biocomplexes are necessary. In this work, EMS spectroscopic data are analysed and discussed for 57Co2+ complexes with a range of small biomolecules of different structures, including 4-n-hexylresorcinol, homoserine lactone and a few amino acids (spectra measured in rapidly frozen dilute aqueous solutions or in the dried state at T=80K). The EMS data obtained are discussed with regard to the available literature data related to the coordination modes of the biocomplexes under study.

Evidence for ferritin as dominant iron-bearing species in the rhizobacterium Azospirillum brasilense Sp7 provided by low-temperature/in-field Mössbauer spectroscopy.

For the ubiquitous diazotrophic rhizobacterium Azospirillum brasilense, which has been attracting the attention of researchers worldwide for the last 35 years owing to its significant agrobiotechnological and phytostimulating potential, the data on iron acquisition and its chemical speciation in cells are scarce. In this work, for the first time for azospirilla, low-temperature (at 80 K, 5 K, as well as at 2 K without and with an external magnetic field of 5 T) transmission Mössbauer spectroscopic studies were performed for lyophilised biomass of A. brasilense (wild-type strain Sp7 grown with (57)Fe(III) nitrilotriacetate complex as the sole source of iron) to enable quantitative chemical speciation analysis of the intracellular iron. In the Mössbauer spectrum at 80 K, a broadened quadrupole doublet of high-spin iron(III) was observed with a few percent of a high-spin iron(II) contribution. In the spectrum measured at 5 K, a dominant magnetically split component appeared with the parameters typical of ferritin species from other bacteria, together with a quadrupole doublet of a superparamagnetic iron(III) component and a similarly small contribution from the high-spin iron(II) component. The Mössbauer spectra recorded at 2 K (with or without a 5 T external field) confirmed the assignment of ferritin species. About 20% of total Fe in the dry cells of A. brasilense strain Sp7 were present in iron(III) forms superparamagnetic at both 5 and 2 K, i.e. either different from ferritin cores or as ferritin components with very small particle sizes.

Reduction of selenite by Azospirillum brasilense with the formation of selenium nanoparticles.

The ability to reduce selenite (SeO(3)(2-)) ions with the formation of selenium nanoparticles was demonstrated in Azospirillum brasilense for the first time. The influence of selenite ions on the growth of A. brasilense Sp7 and Sp245, two widely studied wild-type strains, was investigated. Growth of cultures on both liquid and solid (2 % agar) media in the presence of SeO(3)(2-) was found to be accompanied by the appearance of the typical red colouration. By means of transmission electron microscopy (TEM), electron energy loss spectroscopy (EELS) and X-ray fluorescence analysis (XFA), intracellular accumulation of elementary selenium in the form of nanoparticles (50 to 400 nm in diameter) was demonstrated for both strains. The proposed mechanism of selenite-to-selenium (0) reduction could involve SeO(3)(2-) in the denitrification process, which has been well studied in azospirilla, rather than a selenite detoxification strategy. The results obtained point to the possibility of using Azospirillum strains as endophytic or rhizospheric bacteria to assist phytoremediation of, and cereal cultivation on, selenium-contaminated soils. The ability of A. brasilense to synthesise selenium nanoparticles may be of interest to nanobiotechnology for "green synthesis" of bioavailable amorphous red selenium nanostructures.

Gold(III) reduction by the rhizobacterium Azospirillum brasilense with the formation of gold nanoparticles.

For the soil nitrogen-fixing bacterium Azospirillum brasilense, the ability to reduce [AuCl4](-) and to form gold nanoparticles (GNPs) has been demonstrated, with the appearance of a mauve tint of the culture. To validate the shapes and chemical nature of nanoparticles, transmission electron microscopy (TEM) and X-ray fluorescence analysis were used. For the widely studied agriculturally important wild-type strains A. brasilense Sp7 and Sp245, GNPs formed after 10 days of incubation of cell biomass with 0.25 mM [AuCl4](-) were shown (using TEM) to be mainly of spherical form (5 to 20 nm in diameter), with rare occasional triangles. In the course of cultivation with [AuCl4](-), after 5 days, a mauve tint was already visible for cells of strain Sp245.5, after 6 days for Sp245 and after 10 days for Sp7. Thus, for the mutant strain Sp245.5 (which has significant differences in the structure and composition of cell-surface polysaccharides as compared with Sp245), a more rapid formation of GNPs was observed. Moreover, their TEM images (also obtained after 10 days) showed different shapes: nano-sized spheres, triangles, hexagons and rods, as well as larger round-shaped flower-like nanoparticles about 100 nm in size. Since by the time of GNP formation in our experiments the cells were found to be already not viable, this confirms the dominating role of cell surface structure and chemical composition in shaping the GNPs formed in the course of [AuCl4](-) reduction to Au(0). This finding may be useful for understanding the natural biogeochemical mechanisms of gold reduction and formation of GNPs involving microorganisms. The data obtained may also help in developing protocols for environmentally friendly synthesis of nanoparticles and possible use of bacterial cells with modified surface structure and composition for their fabrication.

Effects of Americium-241 and humic substances on Photobacterium phosphoreum: bioluminescence and diffuse reflectance FTIR spectroscopic studies.

The integral bioluminescence (BL) intensity of live Photobacterium phosphoreum cells (strain 1883 IBSO), sampled at the stationary growth stage (20 h), was monitored for further 300 h in the absence (control) and presence of (241)Am (an α-emitting radionuclide of a high specific activity) in the growth medium. The activity concentration of (241)Am was 2 kBq l(-1); [(241)Am]=6.5×10(-11) M. Parallel experiments were also performed with water-soluble humic substances (HS, 2.5 mg l(-1); containing over 70% potassium humate) added to the culture medium as a possible detoxifying agent. The BL spectra of all the bacterial samples were very similar (λ(max)=481±3 nm; FWHM=83±3 nm) showing that (241)Am (also with HS) influenced the bacterial BL system at stages prior to the formation of electronically excited states. The HS added per se virtually did not influence the integral BL intensity. In the presence of (241)Am, BL was initially activated but inhibited after 180 h, while the system (241)Am+HS showed an effective activation of BL up to 300 h which slowly decreased with time. Diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy, applied to dry cell biomass sampled at the stationary growth phase, was used to control possible metabolic responses of the bacteria to the α-radioactivity stress (observed earlier for other bacteria under other stresses). The DRIFT spectra were all very similar showing a low content of intracellular poly-3-hydroxybutyrate (at the level of a few percent of dry biomass) and no or negligible spectroscopic changes in the presence of (241)Am and/or HS. This assumes the α-radioactivity effect to be transmitted by live cells mainly to the bacterial BL enzyme system, with negligible structural or compositional changes in cellular macrocomponents at the stationary growth phase.

Emission ((57)Co) Mössbauer spectroscopy as a tool for probing speciation and metabolic transformations of cobalt(II) in bacterial cells.

The emission ((57)Co) variant of Mössbauer spectroscopy, rarely used in biology-related studies, was applied to study binding and possible transformations of (57)Co(II) traces in live and dead (hydrothermally treated) cells of the rhizobacterium Azospirillum brasilense (strain Sp7) at T=80 K in frozen aqueous suspensions and as their dried residues. The Mössbauer parameters calculated from the spectra were compared with the similarly obtained data reported earlier for another A. brasilense strain, Sp245 (which differs from strain Sp7 by the ecological niche occupied in the rhizosphere and was found earlier to exhibit different metabolic responses under similar environmental conditions). Similarly to strain Sp245, live cells of strain Sp7, rapidly frozen 2 min and 1 h after their contact with (57)Co(2+) (measured in frozen suspensions), showed marked differences in their Mössbauer parameters, reflecting metabolic transformations of (57)Co(2+) occurring within an hour. However, the parameters for strains Sp7 (this work) and Sp245 (reported earlier), obtained under similar conditions, were found to significantly differ, implying dissimilarity in their metabolic response to Co(2+). This is in line with their different metabolic responses to several heavy metals, including Co(2+), detected earlier using Fourier transform infrared spectroscopy.

Changes in motility of the rhizobacterium Azospirillum brasilense in the presence of plant lectins.

The plant-beneficial bacterium Azospirillum brasilense can swim in liquids and swarm or migrate with the formation of microcolonies in soft media. To get closer to understanding the influence of natural environments on A. brasilense motility, we studied the individual and social movement of the bacterium in the presence of various plant lectins. The lectins with specificity for N-acetyl-beta-d-glucosamine oligomers (wheat germ, Solanum tuberosum and Ulex europeus agglutinins) decreased A. brasilense swimming speed and induced the formation of branched-granular colonies instead of the swarming rings. These effects seemed to be a consequence of specific interactions between the agglutinins and the lectin-binding polymers present in the A. brasilense cell envelope. Concanavalin A (with an affinity for terminal alpha-d-mannosyl and alpha-d-glucosyl residues) and Phaseolus vulgaris phytohemagglutinin P (with unknown specificity) almost did not affect the motility of A. brasilense.

Instrumental analysis of bacterial cells using vibrational and emission Mössbauer spectroscopic techniques.

In biosciences and biotechnology, the expanding application of physicochemical approaches using modern instrumental techniques is an efficient strategy to obtain valuable and often unique information at the molecular level. In this work, we applied a combination of vibrational (Fourier transform infrared (FTIR), FT-Raman) spectroscopic techniques, useful in overall structural and compositional analysis of bacterial cells of the rhizobacterium Azospirillum brasilense, with 57Co emission Mössbauer spectroscopy (EMS) used for sensitive monitoring of metal binding and further transformations in live bacterial cells. The information obtained, together with ICP-MS analyses for metals taken up by the bacteria, is useful in analysing the impact of the environmental conditions (heavy metal stress) on the bacterial metabolism and some differences in the heavy metal stress-induced behaviour of non-endophytic (Sp7) and facultatively endophytic (Sp245) strains. The results show that, while both strains Sp7 and Sp245 take up noticeable and comparable amounts of heavy metals from the medium (0.12 and 0.13 mg Co, 0.48 and 0.44 mg Cu or 4.2 and 2.1 mg Zn per gram of dry biomass, respectively, at a metal concentration of 0.2 mM in the medium), their metabolic responses differ essentially. Whereas for strain Sp7 the FTIR measurements showed significant accumulation of polyhydroxyalkanoates as storage materials involved in stress endurance, strain Sp245 did not show any major changes in cellular composition. Nevertheless, EMS measurements showed rapid binding of cobalt(II) by live bacterial cells (chemically similar to metal binding by dead bacteria) and its further transformation in the live cells within an hour.

Effects of heavy metals on plant-associated rhizobacteria: comparison of endophytic and non-endophytic strains of Azospirillum brasilense.

The plant-associated nitrogen-fixing rhizobacterium Azospirillum brasilense attracts world-wide attention owing to its plant growth-promoting activities. Among hundreds of its strains known up to date, wild-type strain Sp245 has been proved to be capable of colonising both the plant-root interior and exterior (i.e. a facultative endophyte), whereas others are non-endophytes colonising the root surface only. Thus, the different ecological niches occupied by these strains in the rhizosphere suggest that their responses to environmental conditions might differ as well. In this study, responses of A. brasilense strains Sp245 and Sp7 to several heavy metals (Co2+, Cu2+, Zn2+), present in the medium at tolerable concentrations (up to 0.2 mmol/l) and taken up by the bacteria, were compared. Fourier transform infrared (FTIR) spectroscopy was used for controlling the compositional features of whole cells. The results obtained show that in strain Sp7 (non-endophyte) the heavy metals induced an enhanced accumulation of polyester compounds (poly-3-hydroxybutyrate; PHB). In contrast, the response of the endophytic strain Sp245 to heavy metal uptake was found to be much less pronounced. These dissimilarities in their behaviour may be caused by different adaptation abilities of these strains to stress conditions owing to their different ecological status. It was also found that adding 0.2 mmol/l Cu2+ or Cd2+ in the culture medium resulted in noticeably reducing the levels of indole-3-acetic acid (IAA, auxin) produced by both the strains of the bacterium. This can directly affect the efficiency of associative plant-bacterial symbioses involving A. brasilense in heavy-metal-contaminated soil.