Elucidating the Mechanism of pH-Dependent Ni Tolerance in Burkholderia cepacia PR1₃₀₁

J. D. Van Nostrand¹, A. G. Sowder², J. M. Arthur³, P. M. Bertsch^1,2, and P. J. Morris^1,4

¹ Marine Biomedicine and Environmental Sciences Center, Medical University of South Carolina, Charleston, SC

² Savannah River Ecology Laboratory, The University of Georgia, Aiken, SC

³ Department of Medicine, Medical University of South Carolina, Charleston, SC

⁴ NOAA/National Ocean Service, Charleston, SC

The U.S. Department of Energy's Savannah River Site was a nuclear materials processing facility that discharged metallurgical process wastes that led to extensive co-contamination of groundwater, stream sediments, and soil with chlorinated solvents and heavy metals (predominately Ni and U).  Nickel concentrations, ranging from 2 to 5300 ppm, are present in the soils and sediments, and this divalent cation appears to be providing a greater selection pressure on the microbial community than U, with more Ni (~100 ppm) found in the porewater (where it would be accessible to the microorganisms) than U (~10 ppm).  In our initial studies, we examined Ni toxicity in Burkholderia cepacia PR1, a constitutive TCE degrader, which grows well at pH 5, 6, and 7.  Burkholderia cepacia PR1₃₀₁ (PR1) grew in 17.04, 3.41, and 0.85 mM Ni at pH 5, 6, and 7, respectively, displaying Ni tolerance at pH 5 but Ni sensitivity at pH 7.  This suggests that either Ni speciation is altered by pH or that Ni tolerance mechanisms are more efficient during growth at lower pH.  Ni speciation, calculated using the MinteqA2 thermodynamic speciation model, predicts Ni to be the same at pH 5-7 with 3.4 mM Ni, although at this concentration, PR1 grew at levels comparable to the absence of Ni at pH 5 while no growth was observed at pH 7.  Therefore, mechanisms of Ni tolerance/resistance were examined in PR1.  Known Ni efflux genes (i.e., cnr, ncc, and nre) were not detected using PCR primers designed using GenBank sequences.  While these genes were not detected, further analysis of PR1 is underway using a DNA microarray containing >2000 genes involved in metal resistance.  Sequestration of Ni by PR1 was examined after incubation with 3.4 mM Ni; PR1 sorbed 1.5, 1.1, and 3.9 mg Ni g^-1 dry weight (0.9, 0.7, and 2.4% of the total Ni present) at pH 5, 6 and 7, respectively.  The amount of Ni sorbed was considered too low to explain the differences in toxicity observed.  Size exclusion chromatography coupled to inductively coupled plasma mass spectrometry was used to determine if Ni-binding substances were released from PR1.  Only low molecular weight (< 1000 Da) substances were detected associated with Ni, suggestive of medium components (lactate or nitrilotriacetic acid).  To determine if the presence of stress proteins were involved, changes in protein expression were examined at pH 5 and 7 after exposure with (3.4 mM) and without Ni.  One protein was identified by peptide mass fingerprinting and database searches within SWISS-PROT using the Mascot search engine as GroEL, a stress protein, which assists in protein folding of new or damaged proteins.  GroEL expression was highest at pH 5 after 3 h exposure to Ni.  These results suggest that the increased expression of GroEL may directly or indirectly assist PR1 in tolerating higher concentrations of Ni at low pH.