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2003
Fall MEETING of the MICHIGAN ASM
POSTER PRESENTERS AND
ABSTRACTS |
Abstracts are posted
in the order they are received. Winners of "Best Posters", as judged by members of the
MI-ASM Board at the meeting, are indicated below.
SPECIAL AWARD
ANNOUNCEMENT
A.J. Matthews
of CMU is the very first recipient of our newly established PHILLIP AND VERA
GERHARDT STUDENT TRAVEL AWARD. Congratulations, A.
J.
COLONIZATION OF BEACH SAND BY BIOFILMS CONTAINING
ENTERIC PATHOGENS
A. J. MATTHEWS* and E.W. ALM
Central Michigan University Mount Pleasant, MI (adriane88@hotmail.com)
Fecal contamination of public recreational waters is a growing problem
around the world. Bacterial pathogens associated with fecal waste are
responsible for many intestinal ailments that threaten the health of
susceptible individuals like young children and seniors. Although not
mandatory, some local groups voluntarily monitor water from bathing beaches
for the abundance of fecal indicators (e.g., Escherichia coli and
enterococci), which are easier to detect than specific human pathogens.
Beach sand is not monitored, but may be a region of the beach that harbors
viable populations of enteric bacteria. The purpose of this study was to
test for the presence of enteric bacteria in biofilms developed in a natural
beach environment. Glass slides were buried on a private beach on Lake
Huron and retrieved every two weeks from June to September 2003. Confocal
laser scanning microscopy (CLSM) was used to observe the viability of
bacteria in biofilms that formed on the glass slides. The presence of a
specific human pathogen was determined by molecular DNA analysis and
fluorescent antibodies specific to E .coli 0157. E. coli 0157 was
observed in sand biofilms using CLSM. The presence of viable bacteria and a
human pathogen in sand biofilms suggests that enteric bacteria are
persisting and possibly even reproducing in the sand environment where they
may pose a health threat to beach users.
Toxicity and Effects of Ionic Liquids Upon Three Groundwater Microbial
Communities
K.M. Docherty* and C.F. Kulpa
University of Notre Dame, Dept. Biological Sciences
Ionic liquids (ILs) are novel organic salts with a wide liquid range and have
enormous potential for green industrial use. Their chemical properties, such
as miscibility with water and toxicity can be altered by varying the anions and
cation substituents. Before their potential release into the environment, it
is crucial to determine their toxicity to aquatic ecosystems, particularly
microbial communities. This study examines the toxicity of 5 ILs using a
Microtox Acute Toxicity test. Toxicity among these ILs depends solely upon the
identity of the cation; the anion does not effect toxicity. Three ionic liquids
were then added in concentrations of 10 ppm, 100 ppm and 1000 ppm to water
samples collected from 2 groundwater wells and 1 agricultural stream to
determine the effect on viable bacterial counts and microbial community changes
using denaturing gradient gel electrophoresis. Only the highest concentration,
1000 ppm, yielded a significant decrease in viable counts compared to the
control, though changes in microbial community banding pattern are seen in some
sites at 100 ppm and 1000 ppm. The stream site microbial communities appear to
be more resistant to change than the 2 well sites. This may indicate that
certain bacterial groups may be more resistant to high concentrations of ILs,
and possibly capable of degradation, but that their addition to the water
column could change the functional microbial community.
GENOMIC
INSIGHTS INTO LOW TEMPERATURE GROWTH OF PSYCHROBACTER FROM ANCIENT
PERMAFROST
Monica
Ponder1,2, Hector Ayala-del-Rio1,2, Peter
Bergholz1,2, Patrick Chain3, Genevieve
DiBartolo3, Loren Hauser5, Miriam Land5, Frank
Larimer5, Paul Richardson4, Michael Thomashow 1,2
and James Tiedje1,2
1NASA Astrobiology
Institute’s Center for Genomic and Evolutionary Studies on Microbial Life at Low
Temperatures, Michigan State University, East Lansing , MI
2Center for Microbial
Ecology, Michigan State University, East Lansing , MI
3Lawrence
Livermore National Laboratory
4Joint Genome
Institute
5Genome Analysis and Systems
Modeling Life Sciences Division, Oak Ridge National
Laboratory
The basis for psychroactivity in bacteria is
poorly understood. To gain knowledge of microbial adaptation to low
temperature, the genomes of two Siberian permafrost bacteria were
sequenced. Exiguobacterium 255-15, a member of the
Firmicutes, and Psychrobacter 273-4, a gamma-
Proteobacterium, are both psychroactive and display marked physiological
changes under low temperature versus mesophilic growth. Genes known to be
differentially expressed in mesophiles and some psychrotrophs in response to low
temperatures are also present in both genomes in addition to stress responsive
hypothetical genes. In order to determine if there is evolutionary
evidence of cold adaptation, we performed a phylogenetic analysis of
isocitrate lyase (ICL), a thermolabile enzyme involved in central
metabolism. Maximum likelihood analysis of the amino acid sequences
revealed that Psychrobacter’s ICL clusters with ICLs of microbes that can
grow at low temperature but not with those of mesophiles. This group of
sequences possesses characteristic insertions that have been hypothesized as a
means of increasing the flexibility of the enzyme, a previously described
adaptation to low temperature. Microarrays consisting of 70-mer
oligonucleotides to 1,993 of the 2,056 predicted genes of were constructed.
Preliminary differentenial gene expression analyses at 4°C and 24°C
reveal 2-fold or greater upregulation for at the cold temperature for at
least 10 transport associated genes and 9 metabolic genes including a
potential amino acid metabolism operon, which has been implicated in compatible
solute production in other bacteria. Gene expression of
ribosomal proteins, regulatory proteins, metabolic genes and transporters not
upregulated at cold temperatures were upregulated at 24°C. The different
expression patterns of the transport genes support physiological evidence that
different carbon sources are utilized at the two temperatures.
Approximately 28% of the predicted ORFS in the two genomes are more than
80% similar in amino acid sequence. Of these 17 have been demonstrated to
be upregulated in Psychrobacter at 4°C and 16 have been demonstrated to
be upregulated at 24°C.
THE UV RESPONSES IN SHEWANELLA ONEIDENSIS MR-1
Xiaoyun Qiu
Center for Microbial Ecology,
Michigan State University, East Lansing MI
Shewanella oneidensis MR-1, a gamma proteobacterium, is capable of reducing
a variety of compounds including U and Cr. However, this bacterium showed
high sensitivity to UV radiation: a 20% survival rate with a dosage of 4.2
J/m 2 of UVC. We investigated the DNA repair and damage tolerance mechanisms
in MR-1 when it is exposed to UVR: UVC (254 nm), UVB (290-320 nm) and UVA
(320-400 nm). Gene expression profiles were compared using a cDNA array
containing 95% of MR-1 open reading frames. Briefly, there were about 3, 4.6
and 7.3 % of genes were up-regulated in response to UVC, UVB and UVA
respectively. Although the SOS response was observed in all three
treatments, the induction was most robust in response to UVC. The genes
involved in protecting cells from oxidative stresses/damages were
up-regulated in both UVB and UVA treatment. We also observed an increased
expression level of several genes that are involved in replication of
prophage MuSo1 and lambdaSo in both UVC and UVB irradiated cells.
Unexpectedly, we did not observe any inductions in gene expression of
nucleotide excision repair components (e.g. uvrA, uvrB and uvrD) in either
treatment. The contribution of photoreactivation and mutagenic repair to
cell survival were also evaluated. This study will enhance our understanding
on the survival of MR-1 in its natural habitats as well as improve our
management when applying it to clean up contaminated field.
YopO Localization and Lethality in Yeast
Laura Nejedlik
Department
of Biological Sciences, Western Michigan University
Yersinia species use a type III secretion system to deliver at least
six effector proteins (Yops H, O, M, E, T, P) into the target cell. We
have developed a new model system using Saccharomyces cerevisiae to
study Yersinia effectors. We obtained the Yop genes by PCR
amplification from pYV227, the virulence plasmid of Yersinia
enterocolitica. Each Yop gene was inserted into the yeast expression
vector using Gateway™ Cloning Technology. The effector genes are under
control of a GAL1 promoter, and a URA3 site also allows us to easily
select for our plasmid. We have used this system to overexpress YopO,
which is lethal to the yeast cell. YopO is a serine-threonine kinase
that causes disruption of actin filaments. YopO contains three
domains, an actin binding domain, a Rho binding domain and a kinase
region. Site-directed mutagenesis was used to create two mutant forms
of YopO. The first mutant K267A changes the Lysine at position 267 to
an Alanine, to disrupt the kinase activity. However this mutant is
still lethal to the yeast cell. The next mutant I543 replaces the
Isoleucine amino acid at position 543 with a stop codon. The
resulting prematurely truncated protein does not contain the Rho or
actin binding domain and is not lethal to the yeast cell. Yeast
containing YopO and K267A mutant cause a loss of actin cables by hour
2. Actin is still present at hour 8, however it is distributed
diffusely through out the cell. Yeast containing the I543 mutant are
similar to a yeast strain not expressing YopO. Using a V5 epitope we
have been able to show both YopO and the K267A mutant localize to the
cell periphery.
ZINC MEDIATED GENE
EXPRESSION IN PSEUDOMONAS FLUORESCENS MUTANTS
Jarrod Breeding,
Lindsay Berbiglia, and Dr. Silvia Rossbach, Dept. of Biological Sciences,
Western Michigan University
Metal cations are common in the environment
and are utilized by bacteria for metabolic purposes. However, when concentrations become too
high, even essential metals such as zinc and copper can become toxic. Bacteria had to develop ways to obtain
sufficient metal ions to grow while ridding themselves of excess ions before
damage can occur to the cell. We
are studying the response of Pseudomonas fluorescens to excess metal
ions. Our analysis uncovered a
two-component system that regulates the expression of a RND efflux system. In order to investigate how P.
fluorescens utilizes the efflux system to maintain metal homeostasis,
mutants were constructed with insertions in genes encoding a sensor kinase,
response regulator, cation/proton antiporter, and outer membrane porin. The mutants were exposed to various
metal concentrations and the internal metal concentrations were determined with
ICP-MS. Through this and other
analyses, the data gathered may provide clues as to how P.
fluorescens maintains metal homeostasis in metal polluted
environments.
HEAVY METAL-REGULATED
GENES IN SINORHIZOBIUM MELILOTI
Zarraz Lee, Rossbach S. and
Lynn J. C. Western Michigan University, Kalamazoo, MI
Sinorhizobium meliloti is a nitrogen-fixing bacterium that is commonly
associated with Medicago sativa in a nitrogen-fixing symbiotic
relationship. M. sativa, (alfalfa plants)are produced mainly for animal
feed in the United States. Because of alfalfa’s ability to improve soil
conditions, it would be interesting to make use of this plant for
phytoremediation of heavy metals. This study aims to identify genes of
Sinorzhobium meliloti that are regulated by heavy metals, specifically
cadmium and zinc. We approach this aim by analyzing the gene expression of S.
meliloti mutants. These mutants were created via random transposon
mutagenesis using a mini-transposon of Tn5 carrying the green fluorescent
protein (GFP) reporter gene. The anal! ysis of GFP expression on cadmium and
zinc exposed S. meliloti mutants showed that three mutants differentially
expressed the GFP gene, indicating that the mini-transposon has likely inserted
downstream of a metal-regulated promoter. DNA regions flanking the
mini-transposon of these mutants have been isolated and are currently
being analyzed. At the same time, these mutants are also tested for their
sensitivity towards other heavy metal ions, such as copper and nickel. The
sensitivity test identified one mutant that is sensitive to all four metals
tested in this study. Results from this study will help reveal the homeostatic
mechanism that S. meliloti uses to cope with heavy metals.
CHARACTERIZATION OF BOVINE VIRAL DIARRHEA VIRUS ISOLATES FROM PREVIOUSLY
VACCINATED HERDS
Christopher Nowell LaRock
Medical Microbiology/Immunology
Lyman Briggs School,
Michigan State University, East Lansing MI
Bovine Viral Diarrhea Virus (BVDV) is one of the most significant causes of
disease in cattle worldwide, causing a multitude of clinical diseases. By
studying the genetic and serological characteristics of BVDV isolates from
beef and dairy herds that had been vaccinated against BVDV, we can expand
our pool of data on this disease and better tailor future vaccination
programs to challenge strains that might arise in the future. Briefly,
isolates from cattle meeting study criteria and internal controls were
subject to strain characterization by genomic sequencing and cross
neutralization techniques. Isolates were separated into two groups; one,
vaccinated in 2000, and the other, vaccinated in 2003 with a different
vaccine. In this period, average antibody titer increased significantly for
BVD of genotype 2, with a more slight increase for genotype 1. This
suggests that the second vaccine now contains a BVDV-II strain whereas the
program in 2000 may not have. No significant change occurred between
cytopathic and noncytopathic biotypes.
 Development of a Continuous Culture Model to Assess Production of a
Bacteriocin-like Inhibitor by Enterococcus faecium 62-6: Significance to
Bacterial Vaginosis
KELSEY JOHNSON AND VIVIEN PYBUS, PhD
Biology Department, Kalamazoo College, Kalamazoo MI 49006
Bacterial vaginosis (BV) is the most common vaginal tract infection
presenting in primary health care in the US. During BV, Lactobacillus
populations which are usually present in healthy women are replaced by a
consortium of organisms including Gardnerella vaginalis and anaerobes.
Currently, factors which initiate the shift in the ecology of the vaginal
tract resulting in BV are poorly understood. BV is associated with
sexual activity which provides the opportunity for the introduction of
new organisms into the vagina. Our laboratory is examining the
hypothesis that bacteria, producing a type of antibiotic known as a
bacteriocin, can inhibit the growth of lactobacilli. Should they be
introduced into the vagina they could pave the way for the establishment
of the BV-associated microflora. We have characterized a vaginal strain
of Enterococcus, Enterococcus faecium 62-6, that produces a
bacteriocin-like inhibitor antagonistic to the growth of vaginal
lactobacilli. During growth in batch culture we showed that inhibitor
production was concentration-dependent, requiring minimal concentrations
of strain 62-6 of ca. 7 log10 cfu/ml. Since growth in continuous culture
more accurately reflects in vivo conditions than growth in batch, the aim
of this study was to grow E. faecium 62-6 in a continuous culture model
to assess the significance of inhibitor production in vivo. Production
of the bacteriocin-like inhibitor was shown to be pH-dependent, being
detected at pH 5.4 but not at pH 5.1. It was also
concentration-dependent, requiring a minimum concentration of ca. 8 log10
cfu/ml at pH 5.4. Should enterococci be found in high concentration in
women with BV, inhibitor-producing strains may prevent the
reestablishment of lactobacilli and the restoration of the healthy
vaginal microflora.
Molecular Analysis of the Microbial Communities in Geologically
Distinct Bogs on Beaver Island
Mike Tjepkema, Christopher Blair, and Dr. Gregorgy Colores
Biology
Department, Central Michigan University, Mt Pleasant MI
Glaciers developed the bogs of Beaver Island approximately 11,000
years
before present (ybp) and 8,000 ybp, thus leaving a significant
age
difference among the respective bogs. This age difference was
the basis for
the comparisons between microbial communities in
these bogs. Sampling took
place on Egg and Fox Bog. Samples
were analyzed using techniques such as
PCR, DGGE, and gene
sequencing to amplify, separate, and analyze bacteria.
Initial results
indicate that some bacteria are common to these two bogs yet
others
appear to be distinct. Further purification and sequencing of bands
is
necessary to develop a more complete microbial characterization of
each bog.
If you have
questions about conference posters, email Dr. Rossbach
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