Reduction of Hexavalent Chromium and Detection of Chromate Reductase (ChrR) in Stenotrophomonas maltophilia

Abstract

An Gram negative strain of S. maltophilia, indigenous to environments contaminated by Cr(VI) and identified by biochemical methods and 16S rRNA gene analysis, reduced chromate by 100%, 98-99% and 92% at concentrations in the 10-70, 80-300, and 500 mg/L range, respectively at pH 7 and temperature 37 °C. Increasing concentrations of Cr(VI) in the medium lowered the growth rate but could not be directly correlated with the amount of Cr(VI) reduced. The strain also exhibited multiple resistance to antibiotics and tolerance and resistance to various heavy metals (Ni, Zn and Cu), with the exception of Hg. Hexavalent chromium reduction was mainly associated with the soluble fraction of the cell evaluated with crude cell-free extracts. A protein of molecular weight around 25 kDa was detected on SDS-PAGE gel depending on the concentration of hexavalent chromium in the medium (0, 100 and 500 mg/L). In silico analysis in this contribution, revealed the presence of the chromate reductase gene ChrR in S. maltophilia, evidenced through a fragment of around 468 bp obtained experimentally. High Cr(VI) concentration resistance and high Cr(VI) reducing ability of the strain make it a suitable candidate for bioremediation.

Keywords: ChrR; Cr(VI); Stenotrophomonas maltophilia; chromate reductase; heavy metal.

Figure 1

Growth of strain NA2. The cells were cultured on Luria–Bertani broth supplemented with 100, 200, 500, 1000, 1500, 5000, 6250, 7500, 8750 and 10,000 mg/L Cr(VI), respectively. The optical density was measured after incubation for 24 h at 37 °C.

Figure 2

(A) Phylogenetic analysis resulting from the multiple alignment of 16S rRNA gene sequence of NA2 with those of other especies of the family Xanthomonadaceae found in the EzBioClould database; (B) Neighbor-joining phylogenetic analysis resulting from the multiple alignment of 16S rRNA gene sequence of NA2 with those of other bacterial strains of the genus Stenotrophomonas found in the GenBank and JGI database. The accession numbers of the strains are given in brackets. The scale bar corresponds to 0.05 substitution per nucleotide position. Experimental evidence (+).

Figure 3

Effect of different metals on the growth of bacterial isolation.

Figure 4

Reduction of Cr(VI) by Stenotrophomonas maltophilia (strain NA2). The cells were cultured on Luria–Bertani broth supplemented of 0, 10, 15, 25, 50, 60, 70, 80, 100, 300 and 500 mg/L Cr(VI). The Cr(VI) reduction activity was measured after incubation for 0, 6, 12, 18, 24, 48, 72, 96 and 120 h at 37 °C.

Figure 5

SDS-PAGE gel prepared from cytosolic protein Stenotrophomonas maltophilia (Strain NA2): Lane 1. Native strain exposed to 0 mg/L of chromium VI. Lane 2. Native strain exposed to 500 mg/L of chromium VI. Lane 3. Native strain exposed to 100 mg/L of chromium VI. Lane 4. Protein molecular weight marker.

Figure 6

Chromate reductase in this study. Lane 1. 100 bp DNA Ladder-Thermo Fisher. Lanes 2–3. Gene positive ChR Patra et al. Lane 4. Negative Control. Lanes 5–6. Gene positive ChrR1 (Designed this study). Lane 7. Reactive Control. Lane 8. 100 bp DNA Ladder-Thermo Fisher.

Figure 7

Possible mechanisms of interaction of Stenotrophomonas maltophilia and chromate. A. Chromate enters the bacterial cell through sulfate transporter. B. Resistance: Plasmid DNA encoded efflux systems. * This system in some bacteria is ChrA transporter. C. Intracellular reduction of Cr(VI) to Cr(III) involves soluble chromate reductase ChrR which requires NAD(P)H as an electron donor. D. Tolerance: To combat the ROS generated oxidative stress, protective metabolic enzymes superoxide dismutase, catalase and glutathione are secreted. E. Damages: Cr(VI) and Cr(III) negatively affects DNA, RNA and proteins. F. SOS system: DNA repair system. Modified from [8,43,66].