Chromium resistance of bacteria from contaminated soil

CHROMIUM RESISTANCE OF BACTERIA FROM CONTAMINATED SOIL

Cindy H. Nakatsu* (Department of Agronomy, Purdue University, W. Lafayette, IN 47907, USA, cnakatsu@purdue.edu), Jennifer Steger, David Petros and Allan Konopka (Dept. Biol. Sci., Purdue University, W. Lafayette, IN 47907, USA).

ABSTRACT

To improve our understanding of the ecology of heavy metal resistance in bacteria, chromium resistant strains were isolated from contaminated soils. Soil was collected from an old tannery site contaminated with Cr. The minimum inhibitory concentrations of metals for growth were used to determine metal tolerance of bacteria isolated from these soils. Chromium resistant bacteria could grow on concentrations as high as 50 mM CrO₄^-2. One of these strains, Cr15, was studied in greater detail. Metal sensitive mutants of this strain were obtained by growth in the absence of Cr. The Cr resistance genes were determined to be on a mobile element by transferring the trait from the resistant to sensitive strain using conjugation experiments. Molecular genetic analysis revealed the presence of plasmids in both strains, however there was a deletion in the plasmid of the sensitive strain. Nucleotide sequence analysis of the deleted region showed presence of chromium resistance genes with amino acid similarity to ChrA and ChrB.

INTRODUCTION

Despite the fact that heavy metals are acutely toxic to most microbes, there are metal-resistant bacteria. The toxic effects of heavy metals immediately upon introduction to environmental samples have been documented for a broad array of microbial processes. Long term exposure to metals imposes a selection pressure that favors the proliferation of microbes that are tolerant/resistant to this stress. This has been investigated by assaying habitats exposed to anthropogenic or natural metal contamination over an extended period of time (Hutchinson and Symington, 1997), or by experimentally adding heavy metals to samples, and assaying changes over periods up to a few years (Diaz-Ravina and Baath, 1996).

Biochemical studies have shown that a broad spectrum of microbes can reduce soluble, toxic hexavalent chromium to insoluble, less toxic Cr⁺³ (Wang and Shen, 1995). Soil humic acids can also reduce hexavalent chromium. Most studies have been conducted with Gram negative bacteria, although more recent work has found Gram positive isolates resistant to as much as 5 mM Cr(VI) (Basu et al., 1997). Reduction occurs both under aerobic and anaerobic conditions. The aerobic chromate reductase activities probably involve soluble proteins, whereas anaerobic reduction occurs with a membrane preparation.

Genetic analysis of chromate resistant bacteria, Pseudomonas aeruginosa (Cervantes et al., 1990) and Alcaligenes eutrophus (Nies et al., 1990), has shown that reduced accumulation of CrO₄^-2 is plasmid-mediated. A hydrophobic membrane protein, ChrA, catalyzes the active efflux of chromate from the cytoplasm, driven by the membrane potential (Alvarez et al., 1999). Chromate or dichromate induces expression of the chrA gene. The mechanisms for tolerance or resistance of other bacteria to Cr are unclear but another possible mechanism is binding of Cr⁺³ to an extracellular polysaccharide. The overall objective of this project is to gain insight into the physiology and genetics of chromium resistance by bacteria isolated from Cr contaminated sites. This paper describes the isolation of Cr resistant bacteria from a Cr contaminated tannery site. The genetic traits associated with Cr resistance in one particular Gram positive strain Cr15 are also described.

METHODS

Sampling Site. Soil samples were collected from an old tannery site in upper Michigan contaminated with chromium (Cr). This soil contained extremely high total Cr of 263 g/kg soil.

Cr resistant strain isolation and Minimum Inhibitory Concentration (MIC). Soil slurries were made and serially diluted then plated onto PYT80 plates (80 mg/L of peptone, tryptone, and yeast extract) with 0.0, 0.1-, 1.0-, and 10.0-mM K₂CrO₄. The proportion of Cr resistant/tolerance microbes were by enumerating by comparing number of culturable bacteria on PYT 80 agar without Cr or with 1 mM or 10 mM K₂CrO₄. For the MIC experiment 20 bacteria were selected based on variant morphology and concentration of chromium used in viable plate counts. Cultures were grown overnight in PYT80 + 10 mM MES (pH 6.5) media then 50 mL of culture was inoculated into tubes of PYT80 plus K₂CrO₄ concentrations of 0, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, and 20.0 mM up to a final volume of 5 mL. MIC were also determined for the same strains using 0.5, 10, 25, 50, 100, and 200 mM of Pb(NO₃)₂, CuSO₄, CdSO₄, or NiCl₂. The tubes were left at room temperature for two weeks. The minimum inhibitory concentration was determined by examining the cell pellet at the bottom of the tubes. When the cell pellet was significantly reduced in size as compared to the tube with no metal addition the isolate was unable to grow at that concentration of metal. The MIC was then recorded as the concentration of metal just prior to the concentration that inhibited growth.

Chromium sensitive mutants and gene transfer. Strain Cr15 was tested for loss of Cr-resistance phenotype after growth overnight in nutritionally rich non-selective media. Cultures were spread onto nutrient agar plates then individual colonies were replica-plated on media with and without Cr addition. A streptomycin-resistant derivative was created for one of the mutants that had lost the Cr phenotype and it was used as a recipient in mating experiments. Patch mating experiments were carried out between the metal sensitive recipient and metal resistant donor. Transconjugants were selected on media containing Cr and streptomycin.

DNA Isolation. Plasmid DNA from Cr15 was isolated using the Qiagen large plasmid prep kit then digested with BamHI. The fragments were ligated into pGEM3Z(+) vector (Promega), and transformed into competent E. coli (Sambrook et al., 1989). Plasmids were isolated using an alkaline lysis method (Sambrook et al., 1989). Presence of inserts was confirmed by digestion with BamHI and separation in agarose gels. Nucleotide sequences were determined using the Thermo Sequenase cycle sequencing kit and ALFexpress automated sequencer (Amersham Pharmacia Biotech). Using the BLASTx and BLASTn algorithms (Altschul et al., 1997), sequences were compared to those in the Genbank database (NCIB) to identify potential gene products.

RESULTS

Table 1 lists the MIC for 15 of the 20 Cr resistant isolates. Values were not obtained for five of the twenty strains isolated because they were actinomycetes and they were difficult to grow in broth culture.

Table 1. Minimum Inhibitory Concentration (MIC) of Cr, Pb, Cu, Cd and Ni to bacteria isolated from chromium contaminated soils from an old tannery site in Michigan. (na means not available).

Strain Name	K₂CrO₄ (mM)	Pb(NO₃)₂ (mM)	CuSO₄ (mM)	CdSO₄ (mM)	NiCl₂ (mM)
Cr2	0.1	50.0	50.0	10.0	25.0
Cr3	10.0	50.0	100.0	10.0	25.0
Cr4	0.1	50.0	100.0	25.0	25.0
Cr7	5.0	50.0	100.0	10.0	25.0
Cr8	0.2	50.0	50.0	10.0	10.0
Cr9	2.0	0.0	100.0	25.0	50.0
Cr10	>20.0	50.0	100.0	50.0	50.0
Cr11	0.0	0.0	25.0	25.0	na
Cr12	1.0	50.0	100.0	10.0	50.0
Cr13	>20.0	50.0	100.0	10.0	50.0
Cr14	0.2	50.0	100.0	25.0	25.0
Cr15	>20.0	50.0	100.0	10.0	50.0
Cr18	10.0	0.0	25.0	10.0	100.0
Cr19	1.0	25.0	50.0	25.0	50.0
Cr20	2.0	0.0	50.0	10.0	100.0

After growth of strain Cr15 in non-selective media, 1% of the tested colonies had lost Cr-resistance. After the streptomycin-resistant, Cr-sensitive derivative of Cr15 was mated to the wild type strain transconjugants arose at a frequency of 1.7%.

The wild type Cr15 strain had a plasmid of approximately 60 kb, whereas the Cr-sensitive derivative contained a plasmid of lower molecular weight. Digestion of these plasmids with the restriction enzyme BamHI revealed 2 bands in the mutant strains and 10 bands in the wild type. The eight restriction fragments found only in the wild type comprise about 20 kb of the plasmid. Genetic characterization of this deleted region has commenced. Preliminary nucleotide sequence analysis of the deleted fragments has revealed regions with amino acid sequence similarity to ChrA (chromate efflux pump), ChrB (function unknown but require for Cr tolerance) and a transposase.

DISCUSSION

Input of heavy metals imposes a selective pressure that may favor the growth and activity of resistant/tolerant microbes. We investigated the microbial resistant/tolerance in Cr-contaminated soils by enumerating culturable bacteria on PYT 80 agar without Cr or with 1 mM or 10 mM K₂CrO₄. The population size of culturable microbes in control soil (without the addition of Cr) was in the order of magnitude of 10⁶, while the population sizes were a ten-fold and a hundred-fold lower in 1 mM and 10 mM K₂CrO₄treatments, respectively. The MICs of 14 of the isolates ranged from 0.1 to >20 mM K₂CrO₄. Most of them were also resistant to elevated concentrations of lead, copper, nickel, and cadmium. There was no obvious pattern between the presence and level of resistance to Cr and the other metals. Multiple metal resistance has been found in other bacteria and the resistance mechanisms and genes involved are typically not common (Nies and Silver, 1989; Trajanovska et al., 1997).

At this time, we do not know the mechanism by which Cr15 is chromium resistant. The trait, however, is not stable. The phenotype is readily lost when cells are grown in the absence of selection. Results from other research have shown that this can occur when a trait is encoded on a transposable element (Wyndham et al., 1994). Isolation of plasmid DNA from the wildtype and mutant strain of Cr15 suggests that Cr resistance may be transposon encoded. The plasmid in the sensitive strain is missing about 20-kb of sequence. Analysis of this deleted region showed the presence of putative chrA, chrB genes and a transposase. Mating experiments show that the phenotype can be transferred from the resistant to sensitive strain of Cr15. Altogether the evidence suggests that horizontal gene exchange can contribute to the dissemination of this trait.

The development of a metal-resistant population in a contaminated soil can result from: (i) vertical gene transfer (reproduction), (ii) horizontal gene transfer (including transposons and broad host range plasmids), and (iii) selection pressures on spontaneous mutants (due to the presence of metals). Transposable elements carrying mercury resistance genes have been linked to the distribution of this trait in nature (Bogdanova et al., 1998). The presence of Cr resistance genes on a putative transposon suggests that horizontal gene transfer may be an important factor for the development of metal resistant populations in contaminated sites. Future research will include hybridization experiments to determine the distribution of the same Cr resistant genes in other isolates from the same site and other Cr contaminated sites.

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