Some Background on Research in the Sandrin Lab
Life likely began in metal-laden waters. Today, life continues to rely
upon metals to help carry out countless essential processes. Approximately
1/3 of all enzymes with known structures utilize metals (i.e., cofactors).
These metal-mediated reactions include redox reactions involved in carbon
and nitrogen cycling and fixation as well as free radical detoxification.
Life would not be possible without metals, because most of the oxygen
we breathe and many of the nutrients we consume result from a metal-mediated
set of reactions. Undoubtedly, if microbial life exists elsewhere in
the universe, it has also become reliant on this inorganic side of biochemistry.
Ironically, metal chemistry represents a double-edged sword. Many metals,
such as iron, zinc and molybdenum, are necessary for the normal functioning
of many enzymes, but at high concentrations, metals become toxic. Metal
toxicity, while poorly understood, is thought to result primarily from
the tight, non-specific binding of metals to enzymes. Some metals, such
as mercury, bind so tightly to proteins that they are not used as cofactors.
The scientific community is only beginning to understand how life balances
its requirements for metals with their potential danger. Research on
metal effects on life processes is inherently interdisciplinary and
requires a synergistic synthesis and fusion of microbiology, biochemistry,
geochemistry and inorganic chemistry. As poignantly illustrated in a
recent cover story in Science (May 9, 2003), results of research in
this area are incredibly relevant to the needs of society as metals
dramatically affect the intertwined realms of human health and the environment.
Effects of metals on microbial physiology and ecology unify nearly
all of the research projects active in my lab. Microorganisms utilize
remarkable and largely unexplored mechanisms to survive in these ubiquitous,
toxic environments. Previous research in my lab has also focused on
how metals affect processes such as pollutant biodegradation (the process
by which bacteria can be used to clean up the environment). My research
group has also focused on characterizing how metals affect the bacteria
that dwell in sediments (i.e., muddy bottoms) of a nearby, lead-polluted
lake (Rush Lake). A few years ago, I realized that some of the most
relevant, yet bewildering questions regarding metal effects on microorganisms
might not be easily answered without comprehensively considering metal
effects on microbial physiology and ecology. Proteomics and functional
genomics represent a holistic, interdisciplinary set of tools well-suited
to do just this. Utilizing a host of proteomics and functional genomics-based
approaches, I have been leading my group of student researchers in delving
more deeply into effects of metals on microbial physiology and ecology.
Below, I have highlighted a research projects that REU student participants
can explore during their time at UW Oshkosh.
How Do Bacteria Cope with Toxic Metals?
The scientific community is only beginning to understand and appreciate
impacts of metals on bacterial physiology. The fact that the literature
reports inhibitory concentrations of one metal, zinc, that vary by six
orders of magnitude underscores the current embryonic state of knowledge
in this field. A more lucid understanding of bacterial responses to
metals will benefit society directly by providing a foundation from
which to develop strategies to minimize deleterious impacts of metals
in polluted environments. For example, additional knowledge should lead
to the development of: 1) strategies to optimize biodegradation in polluted
environments and 2) more accurate models to predict the extent of metal
effects on bacterial physiology and ecosystem health.
Literature reports of inhibitory concentrations of metals vary so dramatically,
in part, because microbiologists commonly assume that metals exist in
single forms, typically the ionic forms (e.g., Cd2+). This is in spite
of the fact that metals often exist in several different forms (species)
that vary in their toxicity. Speciation (i.e., the process of forming
additional metal species) of metals occurs even in microbiological media
and is strongly dependent on the composition and pH of the medium. The
free, ionic metal species is largely held to be the most toxic form,
yet the abilities of other species to affect bacterial physiology has
not been investigated. Species besides the free, ionic forms may be
more toxic and may elicit as yet uncharacterized responses in bacteria.
Suggesting that the free, ionic metal species may not be the only players
in determining bacterial responses to metals, my students and I have
recently observed a sediment bacterium (Comamonas testosteroni)
to be inhibited more by cadmium in one microbiological medium (Pipes-buffered
minimal salts medium) than in another medium (Tris-buffered minimal
salts medium), even though concentrations of free, ionic cadmium in
both media were comparable. Growth rates observed in both media not
containing cadmium were also similar, suggesting that medium components
other than cadmium do not explain the observed effect. Next, we will
be characterizing this phenomenon by determining effects of these two
cadmium-containing media (and the speciation events that occur in each
medium) on bacterial physiology at the level of the proteome. We are
also developing chemically defined media in which we can better predict
and measure metal speciation. We will then use these media to explore
effects of different metal species on global gene and protein expression
using DNA microarrays and proteomics-based approaches.
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CSI Meets Proteomics: Molecular source-tracking of microorganisms
My research group's interest in microbial ecology is not limited to
effects of metals on microbial sediment communities. Recently, we have
begun utilizing molecular approaches to gain insight into sources of
microbial contamination present in recreational waters and on beaches.
Microbial contamination of recreational waters is a problem that Wisconsin
has not escaped. My research group is currently using repetitive element
PCR to obtain DNA fingerprints of E. coli obtained from Wisconsin beaches.
These PCR-based DNA fingerprinting methods can be useful in microbial
source-tracking, but they are rather labor-intensive and may lack reproducibility.
Alternative methods of microbial fingerprinting are warranted. One promising
approach involves obtaining mass spectra of intact or fractionated cells
using MALDI-TOF MS. Such fingerprinting offers advantages of greater
sensitivity and much higher sample throughput. In collaboration with
a member of our Computer Science department (Dr. Wing Huen), my lab
group is developing protocols and software tools to allow rapid acquisition
and comparison of mass spectra-based microbial fingerprints. Applications
of such rapid microbial fingerprinting technologies are not limited
to microbial source-tracking in recreational waters. Agencies interested
in biodefense issues should express keen interest in this technology
as a possible method of rapid detection and identification of potential
bioterror agents.
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