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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�([email protected])


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 M
I

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.�

winner.gifDevelopment�of�a�Continuous�Culture�Model�to�Assess�Production�of�a
������������������������������������ Bacteriocin-like�Inhibitor�by�Enterococcus�faecium�62-6:�Significance�to
������������������������������������ Bacterial�Vaginosis�


KELSEY�JOHNSONAND�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.�

winner.gifMolecular 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.

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Last updated: August 15, 2017