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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 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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