Molecular Biology is a set of tools that can be applied to topics ranging from anthropology to zoology. I have chosen to work mostly in the field of environmental molecular microbiology for a variety of reasons that are detailed below. During the 2008-2009 academic year, I am on sabbatical leave and will be a Visiting Scholar at Arizona State University working in the laboratory of Dr. Ferran Garcia-Pichel. There I will be pursuing a new research project on the molecular basis of desiccation resistance in desert crust organisms.
While the desert seems to be an inhospitable place, in reality it contains a multitude of diverse microorganisms. Many of these microorganisms live in the crust, the top several mm to cm of soil. In this environment, organisms survive high levels of UV irradiation with very little water. While Dr. Garcia-Pichel’s lab, which I will visit while on leave in 2008-09, is interested in the question, “Who is present in the desert crust?”, I am interested in the question, “How can they live in such an environment?”
Much of what is known about desiccation resistance comes from the Class Deinococci. Deinococcus radiodurans, the most famous member of the Class, was initially isolated from a can of spoiled meat that had been irradiated for sterilization. D. radiodurans has been shown to withstand 500,000 rads of radiation and still maintain the viability of some cells (Mattimore & Battista, 1996. J. Bacteriol. 178: 633-637). PFGE was used to analyze the effect of irradiation on the genome – the results showed the breakdown of the full length genome, approximately 3 Mbp in length, into small fragments estimated to be 50 kbp. Over time, the wild-type irradiated organisms were shown to rebuild a full-length functional, stable, genome from these fragments (Harris et al., 2004. PLoS Biol. 2: e304:1629-1639). Our overriding question is: does the mechanism that allows D. radiodurans to survive irradiation occur in organisms present in the desert crust?
A few bacterial species, of the more than 5000 identified, can infect animals and cause disease. My lab became interested in one of these organisms, Flavobacterium columnare, while I was at SUNY-Fredonia. F. columnare causes morbidity and mortality in both warmwater and coldwater fish; early work revealed a difference in the virulence of these organisms. Our initial studies on chondroitin AC lyase, an extracellular enzyme that breaks down chondroitin sulfate, showed a difference in chondroitin AC lyase activity between isolates from coldwater and warmwater fish. The molecular underpinnings of this difference are unknown. We have cloned cslA, the gene for chondroitin AC lyase, from six F. columnare isolates and have sequenced approximately 600 bp of the gene. In fall 2009, I will be looking for a student to help sequence and analyze the rest of this gene. Our long-term goal is to understand the molecular basis of the virulence differences.
Onondaga Lake, with its abundant brine deposits, was the site of a chlor-alkali facility in the early 1900’s, which produced chlorine gas and sodium hydroxide. This process used a floating mercury electrode as the cathode and as a solvent for sodium. Each electrolytic cell is estimated to have contained approximately four tons of mercury and there were dozens of electrolytic cells in operation (Chemistry in Context, 1994). Unfortunately, the chlor-alkali process resulted in the release of an estimated 76,000 kg of mercury into Onondaga Lake. Below is a brief description of the projects in which my research students have been engaged.
Molecular analysis of mercury-resistant microorganisms. Water and sediment samples were collected from several sites in Onondaga Lake. Mercury-resistant organisms were quantified through growth on plate count agar in the presence of 100 µM HgCl2. We detected an uneven distribution of mercury-resistant organisms in Onondaga Lake with the highest percentage in samples from Ley Creek.
The mer operon contains several accessory genes such as merB, encoding organomercurial lyase. The presence of MerB allows the organism to cleave the carbon-metal bond found in methyl- and dimethyl-mercury, producing mercuric ion, Hg2+, which is then reduced by MerA to Hg0. We analyzed the mercury-resistant population for the presence of MerB by growth in the presence of 25 µM phenylmercuric acetate and the presence of merB by PCR and Southern hybridization.
RFLP analysis of merA from mercury-resistant microorganisms. Using primers designed to conserved regions of merA, encoding mercuric reductase, we have found eight different NciI restriction fragment length polymorphism patterns in the mercury resistant microorganisms from Onondaga Lake. Six of these RFLP patterns were previously identified in fecal flora from primates with dental amalgams (Liebert et al., 1997, Appl. Environ. Microbiol. 63:1066-1076). The two unique merA RFLPs are being analyzed for sequence variations leading to the new NciI digest patterns.
Antibiotic resistance in mercury-resistant and susceptible microorganisms. An early observation in the studies of antibiotic resistance was that 65% of people tested carried multiple antibiotic-resistant bacteria (Levy et al., 1988, Antimicrob. Agents Chemother. 32:1801-1806). Studies show a genetic linkage between antibiotic resistance and heavy metal resistance, including mercury. Initial studies of our population of mercury-resistant organisms revealed several isolates with multiple antibiotic resistances. We have screened our mercury-resistant organisms for antibiotic resistance, the presence of a class 1 integron, and the capacity for transfer via conjugation.
Analysis of bacteria associated with aquatic macrophytes. Mercury is toxic to aquatic plants and animals, yet the macrophytes in Onondaga Lake are healthy and lack signs of mercury toxicity. Even after bringing macrophytes into the laboratory and exposing them to mercury through the sediments for up to six weeks, they remained healthy. This is in contrast with macrophytes from Pennsylvania’s Lake Nockamixon, which showed classic signs of metal toxicity. We undertook this study to investigate the mechanism by which macrophytes contend with mercury contamination.
I have contributed to several research projects initiated by faculty in Lafayette’s Department of Chemistry and in our Division of Engineering.
Solar Irradiation to Reduce Microbial Counts in Contaminated Water (with the Chemical Engineering Department). We used a fabricated solar disinfection unit to reduce the bacterial load in river water and partially processed water from two wastewater treatment plants. The unit reduced the bacterial load by more than 99.99% in highly contaminated water samples in less than 30 min in midday sunlight.
Stabilizing Sandy Soils With the Addition of Microbes (with the Civil & Environmental Engineering Department). Seismic loads result in an increase in the porewater pressure. In loose, sandy soils, this increase in pressure results in liquefaction of the soil. We are pursuing the use of bacteria to increase the strength of saturated sand, with the goal of possibly using this technique to reduce shifting of sandy sediments during earthquakes.
Chemical Reduction of Perchlorate by Microbes (with the Chemistry Department). Perchlorate has been identified as a significant water contaminant throughout the US. Some bacteria – predominantly in the Proteobacteria group – are capable of reducing perchlorate in the absence of oxygen. Our goal is to develop a combined process using (i) ion exchange to concentrate the perchlorate from groundwater, and (ii) perchlorate reducers to chemically reduce the perchlorate to chloride, an innocuous ion.
Many student-driven research projects lead to a presentation at a regional or national meeting or perhaps a publication in a scientific journal. These achievements are large feathers in their professional caps. Listed below are select recent abstracts and full publications by Lafayette students who have collaborated with me in research.
Abstracts with undergraduate research students (denoted by *):
My research students at Lafayette College and their current positions (when known). If your name is in this list and would like to update your information, click here).