�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 Ponder^1,2, Hector Ayala-del-Rio^1,2, Peter Bergholz^1,2, Patrick Chain³, Genevieve DiBartolo³, Loren Hauser⁵, Miriam Land⁵, Frank Larimer⁵, Paul Richardson⁴, Michael Thomashow ^1,2 and James Tiedje^1,2

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

�

�

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.�

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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.