CHAPTER 3 Effect of heavy metals on E. coli

CHAPTER 3 Effect of heavy metals on E. coli

3.1 CHAPTER INTRODUCTION Heavy metals are toxic and harmful to organisms. A number of bacteria develop processes which withstand the effects of these pollutants. Microbes near environments polluted with metals are generally metal tolerant (Duxbury and Bicknell, 1983). Waste dump sites in villages have been reported to contain heavy metal-tolerant bacteria (Liu et al., 2009; Nwachukwu et al., 2010). An accumulation of heavy metals in different bacterial strains in soil could be used in the process of remediation of soils contaminated by heavy-metal-pollutants. Although some heavy metals are essential trace elements, most of them, at high concentrations are toxic to all branches of life, including microbes, by forming complex compounds within the cell. Because heavy metals are increasingly found in microbial habitats due to natural and industrial processes, microbes have evolved several mechanisms to tolerate their presence (by efflux, complexation, or reduction of metal ions) or to use them as terminal electron acceptors in anaerobic respiration (Nies, 1999). Thus far, tolerance mechanisms for metals such as copper, zinc, arsenic, chromium, cadmium, and nickel have been identified and described in detail. Most mechanisms studied involve the efflux of metal ions outside the cell, and genes for this general type of mechanism have been found on both chromosomes and plasmids. The intake and subsequent efflux of heavy metal ions by microbes usually includes a redox reaction involving the metal (Spain, 2003). Some bacteria can even use them for energy and growth. Bacteria that are resistant to and grow on metals also play an important role in the biogeochemical cycling of those metal ions. This is an important implication of microbial heavy metal tolerance because the oxidation state of a heavy metal relates to the solubility and toxicity of the metal itself. When looking at the microbial communities of metal-contaminated environments, it has been found that among the bacteria present, there is more potential for unique forms of respiration. Also, since the oxidation state of a metal ion may determine its solubility, the microbes that are able to oxidize or reduce heavy metals are used to remediate metal-contaminated sites (Wuana and Okieimen, 2011). Tolerance to heavy metals in the environment may contribute to the maintenance of antibiotic resistance genes by increasing the selective pressure of the environment. Correlation exists between metal tolerance and antibiotic resistance in

bacteria because the resistance genes to both (antibiotics and heavy metals) may be located closely on the same plasmid in bacteria and are thus more likely to be transferred together in the environment. Because of the prevalence of antibioticresistant pathogenic bacteria, infectious diseases are becoming more difficult and more expensive to treat. Thus, we need to not only be more careful of the drastic overuse of antibiotics in our society, but also be aware of other xenobiotics, such as heavy metals, that we release into the environment. Minimal inhibitory concentrations (MIC) refer to the smallest concentration necessary to inhibit growth. Thus, lower MIC values indicate more toxic metals and higher MICs lesser toxicity. A P-type ATPase is defined as an ATPase that forms a phosphorylated intermediate while catalyzing a reaction (Nies and Silver, 1995). RND refers to a family of proteins that are involved in the transport of heavy metals (Nies, 1999). The purpose of this chapter is to study MIC of Cu ++, Cr +++, Hg ++, Fe ++ and Zn ++. Other relevant details are given in introduction and they refer to MIC and toxicity effects. Microorganisms have evolved several mechanisms that respond to the toxic effects of heavy metals. In the presence of toxic concentrations of metals, several resistance mechanisms are activated- the production of peptides of family of metalbinding proteins such as metallothioneins, the regulation of intracellular concentration of metals, with the expression of protein transporters of ligand-metal complexes from the cytoplasm to the inside of vacuoles, and efflux of metal ions by ion channels present in the cell wall. In bacteria, these tolerance mechanisms are often encoded by plasmids, which facilitate their dispersion from cell to cell (Valls and Lorenzo, 2002). Metallothioneins (MTs) are a group of low molecular mass, cysteine-rich proteins with a variety of functions including involvement in metal homeostasis, free radical scavenging, protection against heavy metal damage and metabolic regulation. The over-expression of MTs is drawing attention as novel and promising markers of tumor diseases. Besides, the levels of MTs in microorganisms correlate with heavy metal pollution of an environment and thus serve as bio-environmental marker. Detection and quantification of MTs is challenging due to high cysteine content and relatively low molecular mass (Egli et al., 2003; Chaabouni et al., 2012; Ryvolova et al., 2011). Gel electrophoresis is a routine method used in biochemical labs for the identification of bio analytes such as MTs. Due to its structural and chemical properties, low

molecular weight, heavy metal content and high thiol groups, the protocols commonly used for protein electrophoresis may bring poor results. Treatment of MTs with denaturing SDS gel electrophoresis will result in the loss of metal. Gel electrophoresis may be challenging as acrylamide concentration of approximately 15-17.5% has to be used. Tricine-SDS-PAGE is commonly used to separate proteins in the mass range 1-100kDa. Tricine (N-2-Hydroxy 1-1-bis (hydroxyl methyl) ethyl glycine), used as the trailing ion, allows a resolution of small proteins at lower acrylamide concentrations than in glycine SDS-PAGE systems. It is the preferred electrophoretic system for the resolution of proteins smaller than 30kDa (Schagger, 2006).

3.2 MATERIALS AND METHODS Chemicals Heavy metals stock ZnSO 4.7H 2 O, CuSO 4.5H 2 O, HgCl 2, K 2 Cr 2 O 7, FeSO 4.7H 2 O were from Merck, India. HNO 3 and H 2 O 2 were also from Merck. Nutrient agar and Davis minimal Agar was from Sigma Chemical Co., St. Louis, MO, USA. All other chemicals used are as in chapter 2. 3.2.1Determination of MIC Five heavy metals ZnSO 4.7H 2 O, CuSO 4.5H 2 O, HgCl 2, K 2 Cr 2 O 7, FeSO 4.7H 2 O, were selected and their stocks were prepared as shown in Table 3.1. E. coli was revived and a single colony was inoculated in 5 ml of Luria Bertani broth and incubated at 37º C overnight (Step 1). Diluted inoculums (1:10) from Step 1 (200 µ l) were plated on LB media containing increasing concentrations of the heavy metals. MIC was noted when the isolates failed to grow on plates. MIC is defined as the lowest concentration at which no CFU (Colony forming unit) was observed after 24 h incubation at 37º C. Based on the threshold ranges shown in Table 3.2, further concentrations were explored.

Table 3.1 Preparation of heavy metal stocks Heavy Molecular Stock Preparation Metals weight concentration ZnSO 4.7H 2 O 287.56 20 mm 0.115 g dissolved to make up the volume to 20 ml using autoclaved distilled water. CuSO 4.5H 2 O 249.7 20 mm 0.099 g dissolved to make up the volume to 20 ml using autoclaved distilled water. HgCl 2 271.5 2 mm 0.011 g dissolved to make up the volume to 20 ml using autoclaved distilled water. K 2 Cr 2 O 7 294.185 100 mm 0.588 g dissolved to make up the volume to 20 ml using autoclaved distilled water. FeSO 4.7H 2 O 278.01 100 mm 0.556 g dissolved to make up the volume to 20 ml using autoclaved distilled water. Stocks were autoclaved and stored at RT.

3.2.2 Experiment to determine the percentage of absorption Nutrient broth (100 ml) at normal ph and temperature (30º C) was amended with 50 ppm (50 mg/l) Zn, Cr and Fe separately and each of the flasks was inoculated with 1 cm 3 of E. coli cells and incubated for 72 h at 30º C. Later, the cells were harvested at 100 rpm by centrifugation and the supernatant and biomass separated. Both were digested with 67% HNO 3 and 30% H 2 O 2 V/V and the metal concentration determined by atomic absorption spectroscopy (Appendix 3). Simultaneously a control is prepared without any metal. The percentage of absorption was calculated using the formula 3.2.3 Dead cell study Luria broth (150 ml) was inoculated with 1% of inoculum and incubated overnight at 37º C on an orbital shaker. Cells were harvested at 6000 rpm by centrifugation. Supernatant and biomass were separated. Biomass was harvested and washed thrice with distilled water. The pellet was dried and milled into fine particle of size 1 mm. Dried biomass (0.025 g) was added to Erlenmeyer flasks containing 25 ml of 50ppm each of Zn, Fe, Cr, Hg and Cu. The flasks were shaken at 120 rpm for 90 min at 30º C and the biomass removed. The supernatant was acid-digested and the concentration of heavy metals was measured by atomic absorption spectroscopy (Appendix 3). 3.2.4 To check MIC of FeSO4.7H 2 O on selected E. coli strain To 10 ml of Davis minimal agar plate 0.4 ml of FeSO 4.7H 2 O was added to the final concentration of 4 mm and 0.3 ml for 3 mm concentration. The strain selected from the IMViC test results was plated as 100 µl per plate containing 20, 10, 6, 5, 4 and 3mM concentrations of FeSO 4.7H 2 O. The MIC chosen was between 10 and 20 mm. Davis minimal broth (10 ml) containing 10 and 20 mm FeSO 4.7H 2 O was inoculated with the strain. Plain Davis minimal broth was also inoculated with the strain as control. Broths were incubated at 37º C in shaker overnight.

3.2.5 To check MIC of K 2 Cr 2 O 7 on E. coli The MIC chosen was between 0.1 and 1 mm. LB broth (5 ml) containing 0.5, 0.25 and 0.1mM K 2 Cr 2 O 7 was inoculated with the strain culture. Plain LB broth was inoculated with the strain as positive control and LB broth containing 0.5 mm of K 2 Cr 2 O 7 as negative control. Broths were incubated at 37º C in shaker overnight. 3.6 To check MIC of ZnSO4.7H 2 O on E. coli The MIC chosen was between 1.25 and 2 mm. LB broth (5 ml) containing 1, 1.25 and 1.5 mm of ZnSO4.7H 2 O was inoculated with the strain culture. Plain LB broth was inoculated with the strain as positive control and LB broth containing 1.5 mm of ZnSO4.7H 2 O as negative control. Broths were incubated at 37º C in shaker overnight. 3.2.7 Cell lysis Control (10 ml) as well as broths containing heavy metal was pelleted at 10,000 rpm for 10 mins. The pellet was resuspended in 0.5 ml of 1XPBS and sonicated for 5 min. To the same vial, 100 µl of 10X Cell Lysis buffer was added and incubated for 15 min at 37º C. Supernatant was then separated by centrifugation at 10,000 rpm for 10 min and the pellet was discarded. The supernatant or the lysate was stored at -20º C till further use. 3.2.8 Estimation of Protein on Spectrophotometer The lysate was diluted 1:100 times using distilled water to check the absorbance at 280 nm, which indicated an approximation of protein concentration in lysate. All the lysates were loaded to get equal intensity bands on PAGE. Gel electrophoresis is done according to the procedure given in Appendix 2.

3.3 RESULTS AND DISCUSSION To survive under metal-stressed conditions, bacteria have evolved several types of mechanisms to tolerate the uptake of heavy metal ions. These mechanisms include the efflux of metal ions outside the cell, accumulation and complexation of the metal ions inside the cell, and reduction of the heavy metal to a less toxic state (Nies, 1999). Among the heavy metals tested, CuSO 4 was found to be the least toxic to the E. coli cells with an MIC of 4.75 mm (Fig. 3.2, Table 3.2). The percentage absorption of 50 ppm Cu by living E. coli cells was 96.77% (Table 3.3; Fig.3.7) and of dead cells 93% (Fig 3.8). Metallothioneins are induced by the presence of metals in the culture medium. In this case, the percentage of absorption of Cu by living and dead E. coli cells is almost the same. Probably, metallothioneins may not be induced except the Cu proteins already present in the cell. However, for Cu, no electrophoretic protein profiling was done which would have supported this conclusion. But some other mechanisms of metal absorption, accumulation and tolerance are possible in E. coli. Bacteria have evolved several types of mechanisms to resist toxicity due to high copper concentrations. Sixty-two percent were found to be copper resistant in copper corrosion distribution system. Of these resistant bacteria, 49% had cop or cop-like gene systems, including both compartmentalization and efflux systems (Cooksey, 1993). In E. coli, resistance to copper is based on an efflux mechanism by which copper is removed from the cell. The efflux proteins are expressed by plasmid-bound pco genes, which are in turn dependent on the expression of chromosomal cut genes (Cooksey, 1993). Two cut genes (cutc and cutf) were identified ( Gupta et al., 1995) and were shown to encode a copper-binding protein and an outer membrane. Most bacterial species in the environment have at least one of the aforementioned Cu management systems. MIC of E. coli to K 2 Cr 2 O 7 was found to be 1 mm (Fig. 3.4, Table 3.2) and the living cells when treated with 50 ppm Cr showed 95.5% absorption (Table 3.3; Fig. 3.7) and the dead cells showed 93% absorption of 50 ppm Cr (Fig. 3.8). SDS-PAGE results showed a prominent band of 27KDa which is speculated to be a metallothionein which is not seen in the control (Fig. 3.10). Chromium valance state

ranges from -2 to +6. Hexavalent chromium is hundred fold more toxic than trivalent chromium and it is recognized as the most dangerous environmental pollutant as it can cause mutations, skin disorders and also lung cancer in humans (Ganguli and tripathi, 2002). Reduction of Cr (VI) to Cr (III) is a useful process for remediation of Cr (VI) pollution (Bhide et al., 1996). Bacterial Cr resistance is combined to plasmids (Viti et al., 2003). Transformation of Cr by chromium-resistant bacteria (CRB) is an eco- friendly option for detoxification of chromium (Pal et al., 2005). It is likely E. coli also exhibits Cr (VI) to Cr (III) as a detoxifying mechanism. In the present study it has not been experimentally proved in E. coli.

Heavy metal Table 3.2 Determination of MIC of heavy metals PC (mm) 1.0 2.0 5.0 1.25 1.5 1.75 2.0 MIC (mm) ZnSO 4. 7H 2 O +++ --- --- +++ +++ ++ --- 2.0 CuSO 4. 5H 2 O +++ ++ --- 3.0 +++ 3.5 +++ 4.0 +++ 4.5 --- 4.5 HgCl 2 0.01 +++ 0.05 ++ 0.1 --- 0.05 +++ 0.06 ++ 0.07 + 0.08 --- 0.08 K 2 Cr 2 O 7 0.5 ++ 0.75 ++ 1.0 --- 0.25 +++ 0.5 +++ 0.75 ++ 1.0 --- 1.0 FeSO 4.7H 2 O 3.0 +++ 4.0 ++ 5.0 --- 4.25 ++ 4.5 + 4.75 --- 5.0 --- 4.75 PC = Plated concentration; +++ lawn seen; ++ distinct colonies; + very few colonies; --- no growth

Figure 3.1 Control plate with no heavy metal shows a lawn of E. coli growing

Figure 3.2 Plates with increasing concentrations of heavy metal CuSO 4. Lawn seen till 3.5 mm concentration and the disappearance of colonies at 4.5 mm concentration of the heavy metal.

Figure3.3 Plates with increasing concentrations of heavy metal FeSO 4. Lawn seen in 4 mm concentration, distinct colonies seen at 4.5 mm concentration and colonies disappearing at 4.75 mm concentration of the heavy metal.

Figure 3. 4 Petri plates showing distinct colonies at 0.25, 0.5 and 0.75 mm concentrations of heavy metal K 2 Cr 2 O 7 and the disappearance of colonies at 1.0 mm concentration of the same heavy metal.

Figure 3.5 Petri plates with increasing concentrations of heavy metal HgCl 2. Lawn seen at 0.01 mm concentration, distinct colonies seen at 0.05 mm concentration and the disappearance of colonies at 0.07 and 1.0mM concentration.

Figure 3.6 Petri plates with increasing concentrations of heavy metal ZnSO 4. Lawn seen at 1.0 mm concentration and the disappearance of colonies at 1.7 and 2.0 mm concentrations.

Table 3.3 Percentage absorption of heavy metals by living E. coli cells Metal Initial Amount in Amount in % absorption concentration of Supernatant Pellet (ppm) broth (ppm) (ppm) after 72 h after 72 h incubation with incubation E. coli With E. coli Fe 48 3.8 43 92.083 Cr 50.2 2.245 47.24 95.527 Zn 49.2 1.45 47.3 97.052 Hg 50 7.105 43 85.79 Cu 50 1.612 47.04 96.77 As 48 46 0.652 4.1666 Au 5 1.80 3.2 64 The percentage was determined by atomic absorption spectroscopy

percentage absorption (ppm) 100 Percentage absorption of heavy metals by living E. coli 80 60 40 20 0 Fe Cr Zn Hg Cu heavy metals Figure 3.7 Cells were grown in LB medium with 50 ppm concentration of different heavy metals for 72 h and the percentage absorption was determined by atomic absorption spectroscopy.

percentage absorption (ppm) 100 Percentage absorption of heavy metals by E. coli dead cells 96 92 88 84 80 Fe Cr Zn Hg Cu heavy metals Figure 3.8 LB medium containing 50 ppm concentration of different heavy metals was inoculated with the dead cells of E. coli and was shaken at 120 rpm for 90 min before subjecting to atomic absorption spectroscopy.

Among the five heavy metals tested on E. coli, HgCl 2 is the most toxic with the lowest MIC of 0.08 mm (Fig. 3.5, Table 3.2). The percentage absorption by living E. coli cells was found to be 85.79 (Table 3.3; Fig 3.7) and the percentage absorption by the dead cells was 86% (Fig. 3.8). Lowest MIC and highest toxicity of Hg to E. coli was observed in the present study. It is interesting to note that E. coli has aquaporin Z-channels and Hg ion is a potent blocker of this channel, hence the highest toxicity was observed for Hg 2+ ion. Cells probably die due to the absence of osmoregulation and lack of water (Calamita et al., 1997). Many chemical forms of mercury are toxic to living organisms because of their affinity for cysteine residues in proteins (Osborn et al., 1997). Mercury inhibits RNA synthesis and protein synthesis retarding cell growth (Mortazavi et al., 2005). Some E. coli strains showing mercury resistance were identified. Mercury resistance was encoded by genes (Mer operon) located on plasmids and transposons (Schue et al., 2009; Moshafi et al., 2009). In majority of the strains, the role of R plasmids responsible for Hg resistance was also demonstrated (Nakahara et al., 1977). Two mercury detoxifying enzymes that convert toxic mercury to less toxic metallic mercury were identified (Foster, 1983; Russell, 1992). ZnSO 4 has medium toxicity with an MIC of 2 mm (Fig. 3.6, Table 3.2) and the percentage of absorption of 50 ppm Zn was 97.052% (Table 3.3; Fig. 3.7) and that of the dead cells was 93.5% (Fig. 3.8). No visible changes were observed in the protein profiles of E. coli treated with ZnSO 4 (Fig. 3.10); probably this doesn t induce metallothioneins. Zinc is essential trace element, not biologically active redox reactive and for this reason it is not used in respiration. It is important in forming complexes such as zinc fingers in DNA and is a component of cellular enzymes (Nies, 1999). Bacterial cells accumulate zinc by a fast, unspecified uptake mechanism and is normally found in higher concentrations (but is less toxic) than other heavy metals (Nies, 1999). Uptake of zinc ions is generally coupled to that of magnesium, and the two ions may be transported by similar mechanisms in bacteria (Nies and Silver, 1995).

Figure 3.9 SDS-PAGE Protein profiles with molecular weight marker protein. Lane 1is loaded with control, lanes 2 and 4 with 10 and 20 concentrations, respectively, of FeSO 4 and lane 3 with the marker.

Two general efflux mechanisms are responsible for bacterial resistance to zinc. One is a P-type ATPase efflux1 system that transports zinc ions across the cytoplasmic membrane by energy from ATP hydrolysis. A chromosomal gene, znta, was isolated from E. coli K-12 and was found to be responsible for the ATPase that transports zinc and other cations across cell membranes (Beard et al., 1997). MIC of FeSO 4.7H 2 O was found to be 4.75 mm (Fig. 3.3, Table 3.2). This heavy metal is least toxic to microorganisms. Living cells of E. coli showed 92.083% absorption at 50ppm concentration of the heavy metal (Table 3.3, Fig. 3.7) and the dead cells showed 93% absorption (Fig. 3.8). A protein band of 32 kda was observed in the lane treated with FeSO 4.7H 2 O (Fig. 3.9) which was not observed in the control lane. Metallothioneins are low-molecular weight cystein-rich proteins with an MW of less than 40 kda. The band observed could be that of metallothionein. E. coli, Gram negative bacteria form Fe 3+ siderophore and heme-transport system across outer membrane (Braun et al, 2002). The crystal structure of the three outer membrane transport proteins gives an idea of energy-coupled transport mechanisms. Dead cells take up metal ions through their channels alone, where as living cells have channels and when exposed to metals that enter them, metallothioneins are induced and are responsible for the additional uptake of metals by living cells as compared to dead cells.

Figure 3.10 SDS-PAGE Protein profiles with molecular weight marker protein. Lane 2 loaded with control, lane 3, 8 loaded with soluble protein extract from 0.5mM concentration of K 2 Cr 2 O 7 and lane 5, 9 loaded with soluble protein extract of 1mMZnSO 4