Exploring Mechanisms of Resistance to Divalent Transition Metals in Gram-Positive Soil MicroorganismsEPA Grant Number: F5D20733
Title: Exploring Mechanisms of Resistance to Divalent Transition Metals in Gram-Positive Soil Microorganisms
Investigators: Neely, Benjamin A.
Institution: Medical University of South Carolina
EPA Project Officer: Boddie, Georgette
Project Period: August 1, 2005 through August 1, 2008
Project Amount: $111,172
RFA: STAR Graduate Fellowships (2005) RFA Text | Recipients Lists
Research Category: Academic Fellowships
The project will focus on the identification of the nickel (Ni) resistance mechanism in two Gram-positive microorganisms, Streptomyces aureofaciens and Kitasataspora cystarginea, isolated from sediments containing high concentrations of Ni. In addition, the project will focus on the influence of pH on the identified mechanism of Ni resistance. While most metal toxicity data in the literature are generated at circumneutral pH, many natural and anthropogenically-disturbed systems are at low pH. Our laboratory has observed that S. aureofaciens and K. cystarginea exhibited increased toxicity to Zn, Ni, and Co at pH 7 versus pH 6. This pH effect is inconsistent with the free ion activity and biotic ligand models that predict reduced toxicity of divalent first row transition metals with increasing pH. Chemical speciation modeling indicates that changes in complexation do not explain this pH effect. This same pH effect on metal toxicity has been observed in other Gram-positive and Gram-negative microorganisms and may be common in microbial populations at contaminated sites. Therefore, the project will also focus on how biodegradation of organic contaminants is influenced by pH when there is metal co-contamination. In a mixed waste contaminated site, metal toxicity can limit organic pollutant degradation by inhibiting the growth or altering the metabolism of key degrading microorganisms. It is critical to establish how pH affects co-contaminant metal resistance in microorganisms and how this can influence biodegradation.
To examine the influence of pH on the mechanism of Ni resistance in two Gram-positive Ni-resistant microorganisms and how pH effects influence biodegradation in mixed waste scenarios.
In order to examine the mechanism of metal resistance, we will initially perform DNA microarray analysis and screen for approximately 2000 metal resistance genes, including 70 genes specific to Ni transport and/or resistance. This will be conducted collaboratively with the laboratory of Dr. J. Zhou (Oak Ridge National Laboratory). The microarray results will help direct studies to identify the mechanism of Ni resistance in the two Gram-positive microorganisms. Additional studies will examine microbe-metal interactions. For example, size exclusion chromatography and field flow fractionation coupled with inductively coupled plasma mass spectrometry (ICP-MS) will be performed to determine whether any small molecular weight compounds, possibly microbially produced, are associated with Ni in solution. Mass spectrometry analysis will determine if the complexation agent is an extracellular polysaccharide or a different small molecular weight complexation agent. Additionally, atomic force microscopy could be used to investigate changes in the microorganism’s surface chemistry (e.g., surface Ni sequestration, formation of an impermeable layer). Transmission electron microscopy (TEM) coupled with energy dispersive X-ray (EDX) analysis could be used to observe the location and speciation of Ni if accumulated in the microorganism. Influx and efflux could also be studied by using 63Ni to evaluate cellular uptake or removal.
Once a Ni resistance mechanism is identified and characterized, the project will address the pH-dependent Ni toxicity. Speciation calculations and speciation data generated from coupled separations-ICP-MS techniques will be essential to develop a clear mechanistic understanding of increased Ni toxicity at pH 7. The data might demonstrate how pH limits the organism’s ability to sense Ni, possibly due to competitive binding with other complexing agents. These data might also demonstrate a potential biotic change with pH that modifies the number of Ni binding sites on the membrane. Additionally, we are interested in detecting metalloproteins that may function in Ni sensing, transport or resistance. Using 2D gel electrophoresis, differential expression can be evaluated and protein identification conducted by either peptide sequencing or MALDI-TOF. Also, by using native gel electrophoresis techniques coupled with laser ablation-ICP-MS, screening can be performed directly on gels for detection of proteins that have Ni bound in their native state. This would allow for identification of proteins that may function in activation of a resistance mechanism, Ni sequestration or Ni transport.
The proposed project will investigate how two microorganisms that were isolated from sediments contaminated with the metal nickel (Ni) are able to tolerate higher concentrations of Ni at low pH values. Initially, we will screen the microorganisms for genes of known Ni resistance mechanisms. Based on these findings, molecular and chemistry techniques will be used to identify and evaluate the specific mechanism of resistance. Next, the specific effect of pH on the resistance mechanism will be studied. Lastly, the project will focus on how pH influences biodegradation of organic contaminants when there is metal co-contamination.
The first expected result would be the identification of a mechanism of Ni resistance in two Gram-positive microorganisms isolated from Ni-contaminated riparian sediments. Secondly, the specific influence of pH on this mechanism will be determined. Thirdly, how these pH effects influence biodegradation in mixed waste scenarios where divalent metals are present will be examined. These results will aid in the development of more effective bioremediation techniques that can be applied to mixed waste sites.