Introduction

Environmental contamination by toxic heavy metals and organometals has become a major global concern as they pose a serious threat to the biota along with humans (Nehru and Kaushal 1992; Hernandez et al. 1998; Nies 1999; Cerebasi and Yetis 2001; Hartwing et al. 2002; Dubey and Roy 2003; Dubey et al. 2006). Heavy metal and organometallic pollutants, viz. Pb, Hg, Cd, tributyltin, triphenyltin and tetraethyl lead contaminate the environment from various anthropogenic sources such as industrial wastes, effluents, shipyard wastes, automobile emissions, mining drainage and agricultural wastes. Toxic heavy metals, viz. Hg, Cd and Pb are persistent in the environment which ultimately results in bioaccumulation, causing DNA damage, oxidative damage to proteins and lipids as they bind to essential proteins, lipids and metabolic enzymes (Nies 1999; Asmub et al. 2000; Hartwing et al. 2002). Therefore, these toxic metals are included as hazardous wastes in the list of priority pollutants by Environmental Protection Agency of USA (Cameron 1992).

Bioremediation is environmental friendly, economically viable and highly efficient technique as compared to physicochemical methods to clean up environmental sites polluted with potentially hazardous heavy metals (Gadd and White 1993). Bacteria need to develop different mechanisms to survive in extreme environmental conditions such as heavy metal stress. General resistant mechanisms to counteract heavy metals and organometals include energy-dependent efflux, enzymatic detoxification, bioaccumulation, biosorption, precipitation and sequestration (Gadd 1990; Nies et al.1995; Pain and Cooney 1998; Levinson and Mahler 1998; Nies 1999; Roane 1999; Canovas et al. 2003; Dubey and Roy 2003; Dubey et al. 2006; Cruz et al. 2007; Martinez et al. 2007; Taghavi et al. 2009; Naik and Dubey 2011).

Bacteria show resistance to multiple heavy metals since bacterial genomes possess multiple open reading frames involved in conferring resistance to wide range of metals (Canovas et al. 2003). Expression of bacterial stress induced proteins in response to stress stimuli, viz. heavy metals and organometalic toxicants and their role in resistance is well-known (Dubey et al. 2006; Sharma et al. 2006; Ramachandran and Dubey 2009). Bacterial isolates have been reported to exhibit alteration in cell morphology in response to environmental stresses, viz. toxic heavy metals, temperature and organic compounds (Shi and Xia 2003; Neumann et al. 2005; Chakravarty and Banerjee 2008; Naik and Dubey 2011). Modification in cell envelopes gives adaptive advantage to microbes to survive under extreme environmental conditions (Neumann et al. 2005). Bacterial strains resistant to Tributyltin were also found resistant to multiple heavy metals, viz. Hg, Cu, Zn, Cd and Pb (Dubey and Roy 2003; Pain and Cooney 1998; Cruz et al. 2007).

The present communication focuses on screening and identification of highly lead- and TBTC-resistant estuarine bacterial isolate and its possible resistance mechanisms, viz. biosorption, synthesis of lead-induced proteins and alteration in cell morphology along with resistance to mercury, cadmium, copper and zinc and multiple antibiotics.

Materials and methods

Isolation of lead-resistant bacterial strain

Lead-resistant bacterial strain was isolated from surface water sample of Mandovi estuary Goa, India. Water sample was serially diluted and plated on Zobell marine agar (Zobell 1941) amended with 0.5 mM Pb(NO3)2. Bacterial colonies appeared on the ZMA plate were further streaked on mineral salt medium (MSM) with varying concentration of lead nitrate (0.2–1.6 mM). Slight modification in the composition of MSM was done by replacing inorganic phosphate with 0.4 mM β- glycerol phosphate to avoid lead precipitation. Discrete bacterial colony growing at highest lead level was selected for identification and further biological characterization.

Identification of the bacterial strain

Identification of selected highly lead-resistant bacterial strain was done on the basis of morphological and biochemical characteristics according to Bergey’s Manual of Systematic Bacteriology (Krieg and Holt 1984) and this strain was designated as KS -1. This strain was also confirmed by FAME analysis (Sherlock version 6.0B)

Growth behaviour of the bacterial strain under lead and TBTC stress

Lead and TBTC resistance of bacterial strain KS-1 was determined by its growth in mineral salt medium supplemented with 0.4 % glucose and different concentrations of lead and TBTC separately at 28 ± 2 °C and pH 7.5 with constant shaking at 150 rpm. The minimum inhibitory concentration (MIC) was determined as the lowest concentration of lead or TBTC at which no visible growth of test bacterium was observed. Growth behaviour of the bacterial isolate under stress of lead and TBTC separately was studied by recording absorbance at 600 nm at definite time intervals using UV–Vis spectrophotometer (Shimadzu, Model –UV 2450, Japan). Tolerance of the test bacterium to other heavy metals, viz. CuSO4, ZnSO4, HgCl2 and CdSO4 was also determined in MSM (HiMedia, India).

SDS-PAGE analysis of whole-cell proteins of bacterial strain exposed to lead

Whole-cell protein profile of the bacterial isolate exposed to 1.4 mM lead nitrate was determined by whole-cell protein extraction and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis (Laemmli 1970; Sambrook et al. 1989). Cells grown without lead nitrate served as control. Protein gels were stained with 0.25 % Coomassie Brilliant Blue-250 for 2 h and destained overnight with destaining solution (methanol/acetic acid/distilled water, 40:10:50).

Morphological characterization of the bacterial strain

Alteration in cell morphology and lead biosorption of the bacterial isolate grown under the stress of TBTC and Pb(NO3)2, respectively, was studied separately using scanning electron microscope coupled with energy dispersive X-ray spectroscopy (SEM-EDX) ( Naik and Dubey 2011). Cultures (both control and stressed) in the exponential growth phase were harvested at 8,000 rpm for 10 min and washed with 0.1 M phosphate buffer saline (PBS). Cells were fixed with (2.5 % v/v) glutaraldehyde on glass cover slip in the same buffer and kept overnight at room temperature. Cells were washed with PBS prior to dehydration using different concentrations of ethanol, i.e. 10, 20, 40, 50, 70, 80, 90, and 95 % and absolute ethanol for 10 min each. The Scanning electron microscope (model-JEOL JSM-6360 LV) was used to observe alteration in cell morphology and lead adsorption on the cell surface was recorded by EDX microanalysis.

Antibiotic susceptibility of bacterial strain

Susceptibility of the bacterial strain to common antibiotics was tested by Kirby–Bauer disc diffusion method (Bauer et al. 1966), using Mueller-Hinton agar and antibiotic discs (HiMedia, India).

Results

Identification of the bacterial strain

The bacterial strain KS-1 isolated from water sample of Mandovi estuary Goa, India was gram negative, motile, rod shaped and oxidative. This isolate also showed presence of various enzymes, viz. oxidase, catalase, nitrate reductase, and β-galactosidase and could utilise citrate. The strain was negative for indole, methyl red, Voges–Proskauer, ornithine utilisation and urease. Based on these biochemical characteristics and following Bergey’s Manual of Systematic Bacteriology (Krieg and Holt 1984) and fatty acid methyl ester analysis, this bacterial strain KS-1 was identified as Aeromonas caviae.

Growth behaviour of the bacterial strain under TBTC and lead stress

A. caviae strain KS -1 showed tolerance to lead nitrate and TBTC upto 1.4 and 1.0 mM with their MIC values 1.6 and 1.2 mM, respectively (Figs. 1 and 2). Cross-tolerance to other heavy metals was also noticed as MIC values were 1.2 mM, 30 μM, 0.4 mM and 0.9 mM for ZnSO4, HgCl2, CdSO4 and CuSO4, respectively.

Fig. 1
figure 1

Growth behaviour of A. caviae strain KS-1 in MSM amended with different concentrations of lead nitrate

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Fig. 2
figure 2

Growth behaviour of A. caviae strain KS-1 in MSM amended with different concentration of TBTC

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Antibiotic susceptibility of the bacterial strain

Besides heavy metal resistance A. caviae strain KS-1 clearly exhibited resistance to various common antibiotics, viz. amikacin (10 μg/ml), ciprofloxacin (100 μg/ml), kanamycin (30 μg/ml), streptomycin (10 μg/ml), cephalothin (15 μg/ml), sulphatriad (200 μg/ml) and colistin methane sulphonate (25 μg/ml).

Morphological characterization of the bacterial strain

Alteration in cell morphology as increase in cell size along with a unique morphological pattern in the form of long inter-connected chains of cells was observed in the presence of 1.0 mM TBTC which was clearly revealed by SEM whereas no significant change in cell morphology was noticed when cells were grown in MSM with 1.4 mM lead nitrate (Fig. 3). SEM-EDX analysis of cells exposed to 1.4 mM lead nitrate interestingly revealed 8 % surface biosorption of lead as compared to other major and minor elements present on the cell surface (Fig. 4).

Fig. 3
figure 3

Scanning electron micrographs of cells of A. caviae strain KS-1 grown in MSM without TBTC and lead nitrate (control, a), with 1.4 mM lead nitrate (b) and with 1 mM TBTC (c and d)

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Fig. 4
figure 4

EDX analysis (in Fig. 3, arrow is pointing to the area considered for EDX analysis), control cells (no lead exposed, a), cells exposed to lead nitrate (1.4 mM, b)

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SDS-PAGE analysis of whole-cell proteins of the bacterial strain under lead stress

SDS-PAGE analysis of whole-cell proteins of A. caviae strain KS -1 in the presence of 1.4 mM lead nitrate clearly revealed lead-induced specific induction and upregulation of 15.7, 16.9 and 32.4 kDa proteins and repression and downregulation of a single 18.6 kDa protein (Fig. 5).

Fig. 5
figure 5

SDS-PAGE protein profile of cells grown without and with lead (i.e. 1.4 mM). Lane 1 and 3: cells grown with lead; lane 2: cells grown without lead; lane 4: protein molecular weight marker

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Discussion

Heavy metals and organometals, viz. lead, cadmium, mercury, tributyltin, triphenyltin and tetraethyl lead exert their toxic effects on microorganisms through various mechanisms. Even at micromolar levels, they inhibit growth of majority of bacteria whereas few natural microorganisms resist high levels of these toxicants. These resistant microorganisms have acquired a variety of mechanisms to overcome the stress of heavy metals and organometals which include ATP- mediated efflux, precipitation, intracellular sequestration, surface metal biosorption and alteration in cell morphology (Gadd 1990; Nies and Silver 1995; Levinson and Mahler 1998; Roane 1999; Nies 1999; Dubey and Roy 2003; Dubey et al. 2006; Cruz et al. 2007; Taghavi et al. 2009; Maldonado et al. 2010; Naik and Dubey 2011, 2012). Change in cell morphology of the organism has been explained as one of the protective mechanisms for the bacterial cell exposed to a stressful environment (Nepple et al. 1999; Shi and Xia 2003; Neumann et al. 2005; Chakravarty and Banerjee 2008; Naik and Dubey 2011). Bacterial exopolysaccharides play important role in sequestration of heavy metals and thus protect bacterial strains from their toxic effects (Pal and Paul 2008) and thus these bacterial strains can be employed to bioremediate heavy metal-contaminated sites.

In the presence of 1.4 mM lead nitrate, A. caviae strain KS-1 did not show any significant alteration in cell morphology, but exhibited a unique morphological pattern in the form of long inter-connected chains of cells which was clearly revealed by SEM, when exposed to 1 mM TBTC. Under TBTC stress, the cells have divided but did not separate as daughter cells resulting in the formation of enlarged, elongated chains of cells resulting in reduction of total surface area of cells with respect to its volume. This relative reduction of the cell surface to volume ratio serves as an effective mechanism for the cells to overcome the toxic effects of TBTC by reducing the exposed cell surface in relation to the cell volume. This is the first report on alteration in bacterial cell morphology in response to TBTC stress and serves as a novel resistance mechanism. Under the stress of lead nitrate, A. caviae strain KS-1, instead of changing its cell morphology, biosorbed 8 % of lead on the cell surface as evident from SEM-EDX analysis. This significant surface biosorption of lead may be due to entrapment of lead in bacterial exopolysaccharide which prevents entry of toxic lead inside cell and thus protects bacterial cell from its detrimental effects. Interestingly, it also showed induction/upregulation of three lead- induced proteins with molecular mass 15.7, 16.9 and 32.4 kDa, respectively, which clearly demonstrated their possible role in lead resistance.

We conclude that A. caviae strain KS-1 under the stress of TBTC protects itself by forming long chain of cells which reduces the surface to volume ratio and results in reducing the exposed cell surface for TBTC. But when cells are exposed to lead, it is adsorbed significantly (i.e. 8 % lead) on the cell surface itself and no significant alteration in cell morphology was observed which clearly indicates that A. caviae strain KS-1 has sequestered lead outside the cell and thus responds differently for two different stress conditions. Therefore, this lead- and TBTC-resistant A. caviae strain KS-1 may serve as a potential candidate for lead bioremediation in lead-contaminated environmental sites and also as a specific natural biosensor for TBTC biomonitoring since it showed significant alteration in cell morphology as long chains of cells when exposed to TBTC.