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					  19th Annual Pow-Wow

Loyola university chicago
Stritch school of medicine
       Maywood, il

    March 16-17, 2007




                             1
                    2007 Pow-Wow Schedule


    FRIDAY, MARCH 16 th

    8:00-8:30         Breakfast

    8:40-8:45         Welcome (Karen Visick)
    8:45-9:00         Introductory remarks (Ned Ruby)

    9:00-10:00        Doug Bartlett
                      Environmental adaptation in the Vibrionaceae: examples
                      from the deep blue sea and the human gut

    10:00-10:30       Break

    10:30-11:00       Josh Troll ................................................................... Page 6
                      Characterization of the squid peptidoglycan recognition
                      proteins 1, 2 and 3

    11:00-11:30       Anne Dunn ................................................................. Page 8
                      Toward understanding the role of twin arginine translocation
                      (Tat) system-targeted proteins in the Vibrio fischeri-
                      Euprymna scolopes symbiosis

    11:30-12:00       Michael Wollenberg ................................................ Page 10
                      'Endless Forms Most Beautiful and Most Wonderful':
                      Subspecies microdiversity of V. fischeri populations found
                      in the adult E. scolopes light organ

    12:00-1:30        Lunch

    1:30-4:00         Poster Session I

    4:00-4:30         Ricardo Gue rrero.................................................... Page 12
                      Luminescent symbionts from Thailand squids of the genera
                      Uroteuthis, Loliolus, and Euprymna (Mollusca:
                      Cephalopoda)

    4:30-5:00         Emily Yip ................................................................. Page 14
                      The role of the symbiosis polysaccharide (syp) cluster in
                      biofilm formation and polysaccharide production

    5:00-5:30         General discussion

    5:30              Dinner (Loyola)



2
3
FRIDAY, March 16th – POSTER SESSION I



Melissa Altura ......................................................................................................... Page 16
Regulation of Nitric Oxide and NFκB in the Host during the Establishment of the
Euprymna-Vibrio Symbiosis

Don Sanjiv Ariyakumar ......................................................................................... Page 17
The roles of UDP- glucose-6-dehydrogenase and mshA in colonization of the squid-
luminescent bacterial symbiosis

Kati Geszvain........................................................................................................... Page 18
RscS: the structure of a symbiosis regulator

Jessica (Sook Yuin) Ho ........................................................................................... Page 19
Two Structural Analogs of 3-oxohexanoyl Homoserine Lactone can Super-Agonize or
Antagonize Quorum Sensing in Vibrio fischeri

Noreen Lyell............................................................................................................. Page 20
Identification of unknown players in Vibrio fischeri lux regulation: the isolation
and characterization of transposon mutants of ES1114 with differing luminescence

Mark Mandel........................................................................................................... Page 21
Comparative genomic analysis of Vibrio fischeri strains colonizing Hawaiian squid
and Japanese fish

Sarah Studer ............................................................................................................ Page 22
AinS quorum sensing regulates the Vibrio fischeri acetate switch

Barb Wimpee........................................................................................................... Page 23
Differential Gene Expression in the 2-Hour Apo-Symbiotic and Symbiotic Squid,
Euprymna scolopes




4
                    2007 Pow-Wow Schedule


SATURDAY, March 17th

8:00-9:00         Breakfast

9:00-9:30         Andre w Wier ........................................................... Page 24
                  Diel changes in global gene expression of host and
                  symbiont in the squid- vibrio association

9:30-10:00        Alicia Ballok............................................................. Page 26
                  A Brighter Future for GacA Mutants: Small RNAs Mediate
                  the Regulation of Symbiosis Traits in Vibrio fischeri

10:00-10:30       Break

10:30-11:00       Will Soto................................................................... Page 28
                  Microbial Experimental Evolution in the Bobtail Squid-
                  Vibrio Model System

11:00-11:30       Maria Castillo .......................................................... Page 30
                  Identification and Molecular Characterization of
                  Complement Components in the Squid Euprymna scolopes

11:30-12:00       General Discussion

12:00-1:30        Lunch

1:30-4:00         Poster Session II

4:00-4:30         Beth Hussa ............................................................... Page 32
                  Two Component Signal Transduction Systems and the
                  Vibrio-Squid Symbiosis

4:30-5:00         Amber Pollack ......................................................... Page 34
                  Chitin and Competence: Investigating the mechanism of
                  DNA uptake in Vibrio fischeri

5:00              Concluding remarks (Karen and Margaret)


7:00              Dinner (Karen‟s)




                                                                                                    5
SATURDAY, March 17th – POSTER SESSION II



Christine Ande rson ................................................................................................. Page 36
Exploring the roles of Vibrio fischeri response regulators in symbiosis with Euprymna
scolopes

Caitlin Brennan ....................................................................................................... Page 37
Asynchronous swimmers: A high- throughput motility screen of a transposon mutant
collection of Vibrio fischeri

Wendy Castle ........................................................................................................... Page 38
Detecting Selection in the Bacterial Symbiont, Vibrio fischeri

Cindy Darnell .......................................................................................................... Page 39
Complex control of syp transcription and Syp-dependent phenotypes

Kasia Skrzpczynka.................................................................................................. Page 40
Light-induced behavioral and genomic responses in a beneficial bacterial association

Deyan (Lily) Tong ................................................................................................... Page 41
Extraocular expression of phototransduction proteins in the light organ of the squid
Euprymna scolopes

Yanling Wang .......................................................................................................... Page 42
NO Detoxification Systems in Vibrio fischeri

Daniel Zamborsky................................................................................................... Page 43
The population genetic structure of a sympatric Squid-Vibrio symbiosis in the
Mediterranean Sea




6
CHARACTERIZATION OF THE SQUID PEPTIDOGLYCAN RECOGNITION
PROTEINS 1, 2 AND 3

Joshua Troll1 , Nicholas Pacquette2 , Michael Apicella3 , William Goldman4 , Neal
Silverman2 and Margaret McFall-Ngai1
1
  University of Wisconsin School of Medicine and Public Health, Department of Medical
Microbiology and Immunology, Madison, WI
2
  Department of Medicine, University of Massachusetts Medical School, Worchester, MA
3
  Department of Microbiology, University of Iowa, Iowa City, IA
4
  Department of Molecular Medicine, Washington University School of Medicine, St.
Louis, MO

        Studies of the Euprymna scolopes/Vibrio fischeri symbiosis have shown that
selective harvesting of V. fischeri from the environment and subsequent colonization of
the squid light organ are orchestrated through signaling events involving components of
the partners‟ cell surfaces. Recent studies of this association have shown that following
colonization, monomeric V. fischeri peptidoglycan (PGN) and lipopolysaccharide
synergistically induce developmental processes, including apoptosis, in the host squid
light organ. We aim to understand the mechanisms by which the squid distinguishes and
responds to these diverse signaling molecules to promote the establishment of a spec ific
symbiosis. We have identified four putative E. scolopes receptors orthologous to the
peptidoglycan recognition proteins (PGRPs) from insects and mammals, where they have
been demonstrated to be receptors for PGN, acting as either membrane proteins or
circulating blood/hemolymph factors. Immunocytochemistry, visualized by confocal
microscopy, revealed an unexpected nuclear localization for PGRP1 in the light organ
epithelial cells (LOECs). In symbiotic animals, a subpopulation of the LOECs excluded
or lost the nuclear PGRP1 signal. PGRP1 negative LOECs appeared with similar
frequency and morphology to apoptotic cells. Cells positive for TUNEL staining (an
indicator of late stage apoptosis) always lacked PGRP1 in their nuclei; whereas some
PGRP1-negative nuclei were also TUNEL negative. These data suggest that loss of
PGRP1 from the nucleus occurs prior to late stage apoptosis. Our current hypothesis is
that PGRP1 acts as apoptotic „checkpoint‟, preventing the entry into late stage apoptosis
until the correct PGN signals are received. If this hypothesis is correct, the nuclear
protein EsPGRP1 would represent a novel type of PGRP receptor for PGN ligands.
        In contrast to PGRP1, PGRP2 and PGRP3 were present in vesicle-like structures
lining the apical side of the light organ epithelia. Consistent with this vesicular
localization, both PGRP2 and PGRP3 were secreted into the deep crypts of symbiotic
animals at 24 and 48 h post colonization and PGRP2 was found in the mucus lining the
light organ surface of 72 h aposymbiotic animals. PGRP2 was purified by anti-FLAG tag
affinity chromatography and found to have anti- microbial activity against a Gram-
positive bacterium (Micrococcus luteus) but not against two Gram-negative bacteria
(Escherichia coli and V. fischeri). HPLC analysis demonstrated that the PGRP2 protein
is capable of degrading PGRP2, most likely through amidase activity. These data suggest
that PGRP2 and PGRP3 may be important in establishing and maintaining the specificity
of the symbiosis, by inhibiting Gram-positive bacteria from aggregating in the mucus or
establishing an infection in the deep crypts.



                                                                                        7
CHARACTERIZATION OF THE SQUID PEPTIDOGLYCAN RECOGNITION
PROTEINS 1, 2 AND 3

Joshua Troll1 , Nicholas Pacquette2 , Michael Apicella3 , William Goldman4 , Neal
Silverman2 and Margaret McFall-Ngai1
1
  University of Wisconsin School of Medicine and Public Health, Department of Medical
Microbiology and Immunology, Madison, WI
2
  Department of Medicine, University of Massachusetts Medical School, Worchester, MA
3
  Department of Microbiology, University of Iowa, Iowa City, IA
4
  Department of Molecular Medicine, Washington University School of Medicine, St.
Louis, MO




8
Toward understanding the role of twin arginine translocation (Tat) system-targeted
proteins in the Vibrio fischeri-Euprymna scolopes symbiosis

Anne K. Dunn and Eric V. Stabb
Department of Microbiology, University of Georgia, Athens, GA

Underlying the ability of bacteria to respond to and survive environmental change and
stress is the presence of multiple, branched respiratory pathways. The ability of bacteria
to utilize numerous compounds as terminal electron acceptors and the presence of
multiple pathways for individual acceptors suggests a fine-tuning of specific pathways to
particular physiological conditions, including survival in and outside of a host
environment. Our goal is to utilize the Vibrio fischeri-Euprymna scolopes model system
to characterize the role of aerobic and anaerobic respiratory pathways in bacterial
survival during host-associated and free- living lifestyles. The completion of the genome
sequence of V. fischeri revealed diverse anaerobic respiratory pathways, including those
utilizing nitrate, nitrite, and trimethylamine-N-oxide as terminal electron acceptors. In
other bacterial systems, these respiratory proteins are translocated across the cytoplasmic
membrane using the twin arginine translocation (Tat) system, a Sec-independent
pathway. The absence of the Tat system results in the mislocalization of the proteins and
anaerobic growth defects. In addition, symbiotic and pathogenic colonization can be
disrupted in bacterial strains lacking the Tat system. To determine the role of Tat-
targeted proteins in E. scolopes colonization, we generated a strain of V. fischeri lacking
the tatABC operon, which encodes the structural proteins for the Tat system. When
mixed with the wild type, the tatABC mutant is outcompeted during colonization of E.
scolopes. Bioinformatic analyses of the V. fischeri genome using TatP, a program that
predicts Tat-targeted proteins, revealed at least 11 potential targets, including anaerobic
respiratory proteins and other proteins of unknown function. We are interested in
knowing which of these potentially Tat-targeted proteins contribute to successful host
colonization. Our current approach involves generating mutant strains of V. fischeri
lacking individual anaerobic respiratory pathways and assaying colonization ability of
these mutants. In addition to the bioinformatics approach, we plan to use proteomics to
determine the identity of additional Tat-targeted proteins in V. fischeri.




                                                                                         9
Toward understanding the role of twin arginine translocation (Tat) system-targeted
proteins in the Vibrio fischeri-Euprymna scolopes symbiosis

Anne K. Dunn and Eric V. Stabb
Department of Microbiology, University of Georgia, Athens, GA




10
'Endless Forms Most Beautiful and Most Wonderful': Subspecies microdive rsity of
V. fischeri populations found in the adult E. scolopes light organ.

Michael Wollenberg and Edward G. Ruby
University of Wisconsin School of Medicine and Public Health, Department of Medical
Microbiology and Immunology, Madison, WI

Kyu-Ho, Michelle, and others have demonstrated the considerable richness and
abundance of V. fischeri found in association with E. scolopes on environmental scales
ranging from Kaneohe Bay to the greater Pacific Rim. In contrast, we have focused on
the 'inner space' scale of individual E. scolopes by posing a simple question:

Is there evidence that multiple strains of V. fischeri inhabit a single light organ lobe of an
adult E. scolopes?

Data from multiple phenotypic and genetic characters suggests one to three distinct
strains reside within each adult's light organ lobe(s); my talk will discuss evidence we
have accumulated for this assertion. Also, patterning of these strains at three levels of
scale (light organ lobe, individual squid, and squid population) and the beginnings of
preliminary work probing both ecological and evolutionary time-scales for information
on this observed variation will round out our survey of this tangled bank within E.
scolopes.




                                                                                           11
'Endless Forms Most Beautiful and Most Wonderful': Subspecies microdive rsity of
V. fischeri populations found in the adult E. scolopes light organ.

Michael Wollenberg and Edward G. Ruby
University of Wisconsin School of Medicine and Public Health, Department of Medical
Microbiology and Immunology, Madison, WI




12
Luminescent symbionts from Thailand s quids of the genera Uroteuthis, Loliolus,
and Euprymna (Mollusca: Cephalopoda)

R.C. Guerrero-Ferreira and M.K. Nishiguchi
Department of Biology, New Mexico State University Las Cruces, NM

Luminescent bacteria in the family Vibrionaceae (Bacteria: γ-Proteobacteria) are
commonly found in complex, bilobed light organs of sepiolid and loliginid squids.
Morphology of these organs in both families of squids is similar, but the species of
bacteria that inhabit loliginid hosts has yet to be verified. We utilized sequences of 16S
ribosomal RNA, luciferase -subunit (luxA) and the glyceraldehyde-3-phosphate
dehydrogenase (gapA) genes to determine phylogenetic relationships between 63 strains
of Vibrio bacteria, which included representatives from different e nvironments as well as
unidentified luminescent isolates from loliginid and sepiolid squids from Thailand. A
combined phylogenetic analysis was completed using all loci and biochemical data in the
form of carbon use profiles. Results demonstrated that certain symbiotic Thailand
isolates from a common geographical location were part of a clade containing bacterial
species phenotypically suitable to colonize light organs, but never before reported to
establish symbiosis with squid. Moreover, strains isolated from a particular squid host
were found phylogenetically related to more than one bacteria species in our phylogeny.
This research presents evidence of species of bacteria that have not been previously
described as symbiotic strains colonizing light organs of Indo-west Pacific squids, and
also supports the hypothesis of a non-specific association between sepiolid and loliginid
squids and marine luminescent bacteria.




                                                                                       13
Luminescent symbionts from Thailand s quids of the genera Uroteuthis, Loliolus,
and Euprymna (Mollusca: Cephalopoda)

R.C. Guerrero-Ferreira and M.K. Nishiguchi
Department of Biology, New Mexico State University Las Cruces, NM




14
The role of the symbiosis polysaccharide (syp) cluster in biofilm formation and
polysaccharide production

Emily S. Yip and Karen L. Visick
Dept. of Microbiology and Immunology, Loyola University Chicago, Maywood, IL

        Symbiotic association between Hawaiian squid Euprymna scolopes and marine
bacterium V. fischeri requires the symbiosis polysaccharide (syp) cluster. Previously, we
showed that under syp- inducing conditions, wild-type V. fischeri exhibited enhanced
biofilm behaviors, including the formation of wrinkled colonies and thick pellicles on
solid rich medium and minimal liquid medium, respectively. Therefore, we hypothesize
that syp modifies V. fischeri cell surface during symbiosis.
        To further understand the role of syp in biofilm formation, we analyzed cells by
confocal microscopy and found that both vector and syp-expressing wild-type cells were
able to attach to a glass cover slip. However, biofilms of syp-expressing cells were
substantially thicker, especially at the air/liquid interface and particularly at the later time
point (30 h). TEM analysis using ruthenium red, which is commonly used to recognize
acidic polysaccharides, revealed the presence of an extracellular matrix produced by syp-
expressing cells. We found both a layer of matrix at the colony surface, which was
consistent with previous SEM observations, and electron-dense materials that were
present between cells. We also examined the composition of this extracellular matrix by
lectin blot assays. Our data suggested that the syp-dependent polysaccharides were
composed of, in part, glucose and/or -linked mannose.
Currently, we are in the process of characterizing the role of each syp gene in biofilm
formation. We found that the majority of the syp genes are required for the formation of
wrinkled colonies and pellicles. To decipher the function of various syp genes in
polysaccharide production, we propose to extract surface polysaccharides from each syp
mutant and subject these extracts to a panel of lectins. Furthermore, we are examining
extracellular matrix production by these mutants using TEM. We expect that these
experiments will advance our understanding of the roles of the syp cluster in biofilm
formation and polysaccharide production.




                                                                                             15
The role of the symbiosis polysaccharide (syp) cluster in biofilm formation and
polysaccharide production

Emily S. Yip and Karen L. Visick
Dept. of Microbiology and Immunology, Loyola University Chicago, Maywood, IL




16
Regulation of Nitric Oxide and NFκB in the Host during the Establishme nt of the
Euprymna-Vibrio Symbiosis

Melissa Altura and Margaret McFall-Ngai
University of Wisconsin School of Medicine and Public Health, Department of Medical
Microbiology and Immunology, Madison, WI

During the establishment of its symbiotic relationship with the Gram- negative bacterium
Vibrio fischeri, the squid Euprymna scolopes undergoes a series of biochemical and
physiological changes. These changes include attenuation of reactive nitrogen species
and effacement and apoptosis of the symbiont-harvesting ciliated epithelium covering the
light organ. Two microbe-associated molecular patterns (MAMPs), the lipid A portion of
LPS and the tracheal cytotoxin (TCT) fragment of peptidoglycan, have been shown to
induce apoptosis of the light organ epithelium. I have shown that LPS and TCT can
induce a reduction in nitric oxide synthase (NOS) levels, which is the opposite of the
effect observed in pathogenic interactions. I will discuss my future plans to study nitric
oxide (NO) regulation and its effects, as well as the regulation of the NFκB protein, a
marker of late-stage apoptosis.




                                                                                       17
The roles of UDP-glucose-6-dehydrogenase and mshA in colonization of the squid-
luminescent bacterial symbiosis

ARIYAKUMAR, D,* and NISHIGUCHI, M.K.
Department of Biology, New Mexico State University, Las Cruces, NM

Mutualistic associations between the sepiolid squid Euprymna tasmanica (Mollusca:
Cephalopoda) and the luminescent bacterium Vibrio fischeri have been extensively
studied as a model to understand the interplay between hosts and various symbionts.
Juvenile squids lack symbiont bacteria, but obtain their Vibrio symbionts within a few
hours after hatching. Although this colonization event is specific, Vibrio bacteria must
overcome several obstacles to successfully attach and colonize the light organ. Here we
examine the effects of two possible symbiotic genes that are responsible for initial
attachment and biofilm formation in squid light organs. UDPDH (Uranyl diphospahte-6-
dehydrogenase) and mshA (mannose sensitive hemagglutinin) mutants were generated in
V. fischeri by single recombinant techniques, to determine whether each have an effect on
host colonization and specificity. Growth curves were generated to deter mine generation
times for mutant and wild types, along with bioluminescence measurements for light
emission. Biofilm assays were also completed to confirm differences in phenotypes of
UDPDH and mshA mutants. Colonization assays also confirmed differences in host
colonization within the wild-type and mutant strains. Results from this study reveal the
importance of UDPDH and mshA gene expression for successful light organ colonization
by V. fischeri, and also aids in the understanding of host specificity in a n environmentally
transmitted symbiosis.




18
RscS: the structure of a symbiosis regulator

Kati Geszvain1 , Sean Crosson2 and Karen L. Visick1
1
  Dept. of Microbiology and Immunology, Loyola University Chicago, Maywood, IL
2
  Dept. of Biochemistry and Molecular Biology and The Committee on Microbiology,
University of Chicago, Chicago, IL

        To initiate the symbiosis between itself and Euprymna scolopes, Vibrio fischeri
must sense the presence of the squid and respond appropriately. Bacteria commonly
employ two component regulatory systems to recognize and respond to their
environments. The sensor kinase component of one such regulatory system, RscS, is
required for initiation of the symbiosis through, at least in part, its role in the induction of
expression of the symbiosis polysaccharide, or syp, gene cluster. Multicopy expression of
a mutant allele (rscS1) increases syp expression even more so than the wild type allele.
The rscS1 allele contains two mutations that alter the RNA but not the protein sequence.
These are RBS1, a C to T transition in the putative ribosome binding site, and Leu25,
which changes a rare Leu codon to a more common codon. Preliminary results with an
anti-serum directed against a carboxy-terminal RscS peptide show that the rscS1
phenotype is due to increased levels of protein relative to that produced by multi-copy
expression of unaltered rscS. By assaying the ability of the individual mutations carried
on multicopy plasmids to induce syp cluster expression, I have shown that both RBS1
and Leu25 contribute to the rscS1 phenotype.
        A first step to identifying the signal detected by RscS will be to identify the
region(s) of the protein required for signal detection. In RscS, the sensor region of the
protein is made up of two predicted transmembrane helices, a large periplasmic loop and
a putative PAS domain located just inside the cytoplasm. PAS domains are signaling
modules that detect changes in light, oxygen or energy levels found in many sensor
proteins. Sequence alignment with PAS domains from other, well-characterized, proteins
predicts that the RscS PAS domain binds the redox sensitive flavin adenine dinucleotide
(FAD) cofactor. This suggests that RscS may be sensitive to the redox state of the cell.
To begin to determine the role of the PAS domain and its putative FAD cofactor in RscS
function, I have deleted the sequences coding for PAS from the rscS1 plasmid. The
resulting plasmid fails to induce syp cluster expression, providing the first evidence that
the predicted PAS domain is functional. Future experiments will focus on generating Ala
substitutions at residues predicted to interact with FAD and spectroscopic
characterization of the isolated, purified PAS domain to identify its bound cofactor.




                                                                                             19
Two Structural Analogs of 3-oxohexanoyl Homoserine Lactone can Super-Agonize or
Antagonize Quorum Sensing in Vibrio fischeri

Jessica Ho, Helen Blackwell and Ned Ruby
University of Wisconsin School of Medicine and Public Health, Department of Medical
Microbiology and Immunology, Madison, WI

The Vibrio fischeri luminescence genes are activated by the transcription factor LuxR in
a reversible association with a diffusible quorum signaling molecule, 3-oxo hexanoyl
homoserine lactone (OHHL). Synthetic structural analogs of OHHL therefore present the
possibility of displacing native OHHL in this association, thus altering the effectiveness
and outcome of quorum sensing in the cell population. The effects of such analogs can
range from agonism to antagonism. We recently identified two such analogs of OHHL: 3
nitrophenyl acetyl homoserine lactone (B17) and 4- iodophenyl acetyl homoserine lactone
(B13). B17 and B13 can, respectively, super-agonize and super-antagonize quorum
sensing in V.fischeri ; that is, they are able to either mimic or suppress the activity of
OHHL at a relatively lower molar concentration. We studied the impact of substituting
OHHL with either B17 or B13 on (i) inducing luminescence in V. fischeri and (ii) the
colonization competence of the OHHL deficient mutant luxI. We show that both B17 and
B13 appears to bind to LuxR to activate or repress V. fischeri luminescence, indicating
that they both interact effectively with the lux genes. We also showed that the addition of
B17 to luxI colonized animals was able to rescue not only luminescence, but also the
colonization persistence defect displayed by the luxI mutants. Because both B13 and B17
are structurally different from OHHL, the results from this analysis not only gives insight
into how OHHL might interact with LuxR, but also provides evidence that indicates that
the chemistry of quorum sensing molecules may also affect the host in a symbiont- host
relationship.




20
Identification of unknown players in Vibrio fischeri lux regulation: the isolation and
characterization of transposon mutants of ES1114 with differing luminescence

Noreen L. Lyell, Susan L. Vescovi, Erica M. Hall, Anne K. Dunn, Jeffrey L. Bose, and
Eric V. Stabb
Department of Microbiology, University of Georgia, Athens, GA

Vibrio fischeri isolates from the Euprymna scolopes light organ, such as strain ES114,
produce very low levels of bioluminescence in culture but induce luminescence ~1000-
fold when colonizing the host. Quorum sensing and cell-density dependent regulation
alone cannot completely explain these differences in luminescence, because ES114
colonies on plates do not achieve the same levels of luminescence as do cells colonizing
the light organ, although the cell density is equivalent. To better understand this
regulatory difference, and to identify previously unknown proteins affecting
luminescence, we performed random transposon mutagenesis using an improved miniTn5
system and assayed for mutants of ES114 with increased luminescence. Mutations
within 14 different loci with a range of „luminescence-up‟ phenotypes were isolated and
have been characterized in culture and in the squid. Two of the loci identified in our
screen, hns and lonA, were previously described as repressors of the V. fischeri lux genes
in E. coli. Similarly, insertions in arcA and arcB resulted in „luminescence-up‟
phenotypes, consistent with our studies of these regulators. Surprisingly we found that
ainS, encoding an autoinducer synthase, directs the repression of luminescence of ES114
on plates, although previous work showed it induces luminescence in broth cultures.
Other „luminescence-up‟ mutants of ES114 had insertions in acnB, pstA/C, topA, tfoX,
phoQ and specific tRNA genes. Further studies of these mutants will contribute to our
understanding of the „regulatory web‟ of luminescence.




                                                                                       21
Comparative genomic analysis of Vibrio fischeri strains colonizing Hawaiian s quid
and Japanese fish

Mark J. Mandel1 , Kati Geszvain2 , Karen L. Visick2 , Edward G. Ruby1 , and Eric V. Stabb3
1
  Dept. of Medical Microbiology and Immunology, University of Wisconsin, Madison, WI
2
  Dept. of Microbiology and Immunology, Loyola University Chicago, Maywood, IL
3
  Dept. of Microbiology, University of Georgia, Athens, GA

Genetic studies in Vibrio fischeri have identified gene products and physiological
processes required for colonization of animal tissue. In the best-studied system—
colonization of the Hawaiian bobtail squid Euprymna scolopes—a number of cellular
activities have been identified that are required to establish a successful symbiosis.
Genome sequencing of the V. fischeri strain ES114 has helped to advance these studies,
and here we expand our understanding of V. fischeri genomics by reporting the draft
sequence of strain MJ11, which was isolated from a distinct host, the Japanese pinecone
fish, Monocentris japonica.

The genome is similar in size (4.5 Mb) and G+C% (38%) to ES114. It includes two
circular chromosomes, plus a large (179 kb) plasmid. The MJ11 genome is predicted to
contain approximately 4061 ORFs. Reciprocal BLASTP and TBLASTN queries performed
between chromosomal genes identified orthologs and genes unique to each genome. 92%
of ES114 genes have an identifiable ortholog in MJ11, with those orthologous pairs
sharing a mean amino acid identity of 97%. Approximately 595 proteins are identical
between the two. Rapidly-evolved genes include those relevant for symbiotic functions
such as quorum sensing and luminescence (luxR, luxICDABEG, ainS), as well as genes
involved in LPS/EPS biogenesis. Especially well- conserved genes include those
regulating flagellar motility. In contrast, the plasmids in the two strains are largely
dissimilar and share no regions of synteny.

Among the ES114 genes absent from MJ11 is rscS (VFA0237). RscS is a hybrid sensor
kinase that activates production of the Syp capsular polysaccharide, and RscS and Syp
are each necessary for normal squid colonization in ES114. A preliminary population
analysis revealed that rscS is present in the genomes of V. fischeri isolates from squid in
Hawaii (E. scolopes) and Japan (E. morsei); it is absent from V. fischeri isolates of
Japanese fish and from V. fischeri and V. logei of Mediterranean squid (Sepiola spp.). On
the other hand, the syp locus is fully intact in MJ11, and largely intact in V.
parahaemolyticus, V. vulnificus, and Photobacterium profundum.

We therefore asked whether adding-back rscS to MJ11 is sufficient to induce Syp
polysaccharide. As in ES114, overexpression of rscS in MJ11 conferred a wrinkled-
colony phenotype, suggesting that the signal transduction pathway downstream of RscS
is conserved even in strains lacking the two-component regulator. We are currently
testing whether rscS is sufficient to enhance colonization in strain MJ11 and thus whether
acquisition of rscS is a mechanism by which Pacific V. fischeri may become competent to
associate with Euprymna spp. as hosts.




22
AinS quorum sensing regulates the Vibrio fischeri acetate switch

Sarah Studer and Edward G. Ruby
University of Wisconsin School of Medicine and Public Health, Department of Medical
Microbiology and Immunology, Madison, WI

Vibrio fischeri initiates a symbiosis with Euprymna scolopes during a species-specific
colonization of the host‟s light organ. The AinS quorum-sensing system is required for V.
fischeri to properly initiate and maintain this association. An ainS mutant was found to
have a growth yield defect in culture. Overnight cultures of ainS mutant cells grown in
rich media were found to accumulate high concentration of acetate. Wild-type V. fischeri
produced acetate during the early exponential phase of culture growth, but subsequent
growth was accompanied by consumption of this acetate. This pattern is consistent with a
metabolic shift called the acetate switch, in which increasing transcription of the Acetyl-
CoA synthetase gene (acs) leads to acetate uptake. In contrast, acetate secretion by the
ainS mutant continued throughout the growth of the culture until acidification became
lethal, suggesting that there was a defect in the switch. Transcriptional analysis using a
promoterless lacZ fused to acs confirmed that the wild-type cells induce acs transcription
late in growth and subsequently remove extra-cellular acetate, while the ainS mutant cells
do not induce acs transcription. This appears to be the first report of quorum sensing
control of the acetate switch.




                                                                                        23
Differential Gene Expression in the 2-Hour Apo-Symbiotic and Symbiotic Squid,
Euprymna scolopes

Error! Bookmark not defined., Matt Rise, Margaret McFall-Ngai
University of Wisconsin School of Medicine and Public Health, Department of Medical
Microbiology and Immunology, Madison, WI

Within minutes of hatching, the light organ of Euprymna scolopes secretes mucous in
which bacterial symbionts aggregate before entering the organ. This study addressed
whether changes in host gene expression occur in specific response to aggregating V.
fischeri. mRNA from the light organs of juvenile aposymbiotic (exposed to Hawaiian
offshore seawater) and symbiotic squid (offshore seawater with 5000 V. fischeri cells/ml)
was isolated at 2 hours following hatching and compared to that of the light organ mRNA
from hatchlings. This experimental design allowed for the determination of genes
responsible for post-hatch development and determination of genes expressed in direct
response to the presence of V. fischeri. Genes were identified that are either up or down
regulated at least two- fold relative to hatchling. Comparison of aposymbiotic vs.
symbiotic gene expression reveal dysregulation of ~100 genes, principally involved in the
signal transduction. Primers to the specific genes of interest are now being used in real
time PCR to quantify the transcript from the original total RNA pool that was used in the
microarray.




24
Diel changes in global gene expression of host and symbiont in the squid-vibrio
association

Andrew M. Wier and Margaret McFall-Ngai
University of Wisconsin School of Medicine and Public Health, Department of Medical
Microbiology and Immunology, Madison, WI

The symbiosis between Hawaiian bobtail squid, Euprymna scolopes, and its light organ
symbiont, Vibrio fischeri, exhibits a profound diel rhythm. The symbionts are flushed
into the environment daily at dawn through pores on the light organ surface. The residual
5% of remaining bacteria re-colonize the light organ and the population is reestablished
after only nine hours into the daily cycle. The bacteria adhere to the crypt surfaces that
are lined with a simple columnar epithelium that undergoes distinct morphological
changes throughout the day. The bacterial symbionts themselves undergo a diel rhythm in
the light output per cell, in a cycle that reflects the changes in host tissues. The objective
of this study was to characterize the reciprocal dialogue between host and symbiont o ver
the diel cycle by characterizing global gene change in gene expression in both partners.

Methods: Adult squids were collected with dip nets in the shallow coastal waters off
leeward Oahu. The symbiont-containing, central-core epithelia were dissected from light
organs at four time points (400, 1000, 1600 and 2200 hours) and the total RNA was
extracted. Host samples were hybridized to a glass slide array, which had been generated
from a unique set of ~14,000 genes derived from libraries of juvenile light organs. V.
fischeri cDNA from the same time points was hybridized to Affymetrix chips. Array
results were analyzed with Genespring GX to reveal differential expressed genes between
successive time points.

Results: The data from these analyses demonstrate that this system is characterized by a
distinct molecular dialog between the squid host and its microbial symbionts over the
day-night cycle. Highest levels of host gene regulation occurred in the hours before
dawn, during effacement of the light-organ tissues and restructuring of squid epithelial
cells. This dysregulation in host genes was followed by a peak in changes in the
symbiont gene expression, which coincided with rapid re-growth and filling of the crypt
spaces. The regulation of some host and symbiont genes was consistent with these
observed phenotypes; however, the arrays revealed a variety of other changes that would
not have been predicted. Results of these squid-vibrio symbiosis studies constitute the
first in-depth molecular analyses of diel changes in gene expression in an animal-
microbial partnership.




                                                                                           25
Diel changes in global gene expression of host and symbiont in the squid-vibrio
association

Andrew M. Wier and Margaret McFall-Ngai
University of Wisconsin School of Medicine and Public Health, Department of Medical
Microbiology and Immunology, Madison, WI




26
A Brighter Future for GacA Mutants: Small RNAs Mediate the Regulation of
Symbiosis Traits in Vibrio fischeri

Alicia Ballock and Cheryl Whistler
Microbiology Department, University of New Hampshire, Durham, NH

The GacS/GacA two-component system regulates the expression of genes that mediate
host association in many gram negative bacteria. GacA mutants of Vibrio fischeri exhibit
defects in luminescence, motility, siderophore prod ution, LPS, growth on N-acetyl
glucosamine, and are severely impaired both in animal colonization and in triggering host
morphogenic responses. In other bacteria, GacA activates the transcription of small
untranslated RNAs (sRNAs) in the csrB family. The csrBs block the activity of CsrA, a
repressor of translation, that binds to specific transcripts and blocks access of RNA
polymerase. When present, the csrBs compete with the regulated transcripts for CsrA,
thus, antagonizing CsrA repression. It is probable that GacA activates the transcription of
two recently discovered csrB paralogs in V. fischeri, as they each contain GacA
concensus binding sites upstream of their promoters (P.R. Kulkarni, X. Cui, J.W.
Williams, A.M. Stevens, and R.V. Kulkarni, 2006, Nucleic Acids. Res., 34:3361-69).
However, whether the csrBs, or other GacA targets regulate the symbiotic phenotypes
still remains a mystery. Using gfp-promoter fusions to csrB2, we found GFP expression
was significantly diminished in the GacA mutant as compared to wild type. We next
elucidated the relationship between the CsrA antagonism by csrB and the phenotypes of
GacA. Multiple copies of either csrB gene in trans complemented the GacA mutant for
all in vitro phenotypes. In the future we will also look for complementation of squid
colonization phenotypes by these RNAs. Although we were unable to generate a csrA
deletion, two independent C-terminal spontaneous csrA mutations in GacA mutant
backgrounds suppressed all GacA defects in culture. These suppressors were also
restored in animal colonization. Therefore, all known phenotypes of the GacA mutant
likely result from CsrA repression of translation of target transcripts in the absence of the
two GacA regulated csrBs. To look at direct binding of CsrA to the lux transcript, we
will be generating a translational fusion of LuxI to LacZ. By generating mutations in the
putative binding site we should observe differences in -galactosidase expression.




                                                                                          27
A Brighter Future for GacA Mutants: Small RNAs Mediate the Regulation of
Symbiosis Traits in Vibrio fischeri

Alicia Ballock and Cheryl Whistler
Microbiology Department, University of New Hampshire, Durham, NH




28
Microbial Experimental Evolution in the Bobtail Squid-Vibrio Model System

SOTO, W. and M.K. NISHIGUCHI
Department of Biology, New Mexico State University, Las Cruces, NM

The mutualism between sepiolid squids (Cephalopoda: Sepiolidae) and their
bioluminescent symbionts (Genus Vibrio) has become an experimentally tractable system
for physiological, evolutionary, molecular, and ecological studies of symbiotic
relationships. Since the squid host and their symbionts can be grown and maintained
independently of each other in the laboratory, this association is feasible to empirical
manipulation, including studies examining differences between closely related host-
symbiont pairs. Previous research has shown that native strains of Vibrio fischeri will
out-compete non-native ones isolated from different host species when competitive
colonization experiments are conducted in the indigenous host. Since previous
experimental evolution studies with microorganisms have revealed that adaptation can be
observed relatively quickly, we designed a direct approach using experimental evolution
to identify symbiotic and ecological loci by evolving non-native V. fischeri through a
non-native Euprymna species for 500-750 generations. Since a “frozen fossil record” of
symbionts can be generated, the evolving non-native clone has been monitored every 50-
100 generations to determine if its competitive ability has improved relative to native
strains of V. fischeri. In addition, the relative competitive ability of the derived non-native
strain has been compared to the ancestral strain to determine if fitness has improved
through adaptation. This research addresses the potential of V. fischeri populations to
expand into novel host ranges, and whether non- native bacterial populations can displace
native ones in a particular geographical area.




                                                                                            29
Microbial Experimental Evolution in the Bobtail S quid-Vibrio Model System

SOTO, W. and M.K. NISHIGUCHI
Department of Biology, New Mexico State University, Las Cruces, NM




30
Identification and Molecular Characterization of Complement Components in the
Squid Euprymna scolopes

Maria G. Castillo and Margaret McFall-Ngai
University of Wisconsin School of Medicine and Public Health, Department of Medical
Microbiology and Immunology, Madison, WI


        The complement system consists of a group of serum proteins that play an
important role in immune processes such as cytolysis, opsonization, inflammation, and
linking the innate and adaptive immune systems. Orthologs of several vertebrate
complement components were recently identified in deuterostomes and ecdysozoans
including tunicates, sea urchin, and the horseshoe crab. The presence of complement
molecules in invertebrates suggests a more primitive origin of these immune components
than thought before. In this study we investigated the presence of orthologs of the
complement system in the EST library from E. scolopes light organ tissues. Preliminary
analysis revealed the presence of cDNA sequences with homology to several complement
components. Furthermore, microarray analysis from adult light organ tissues predicts
that the expression of some of the squid complement- like proteins changes during the diel
rhythm suggesting a possible role in the maintenance of the symbiosis with Vibrio
fischeri. One squid EST with sequence homology to the complement component C3,
considered to be the central element in the various complement enzymatic cascades, was
selected for further investigation. Following rapid amplification of cDNA ends (RACE)
methods have resulted in 520 amino acids (AA) corresponding to approximately 30% of
the expected full- size protein when compared to other known C3 molecules. The squid
C3-like sequence has an approximate 20% identical and 40% conserved AA residues
when compared to C3 from horseshoe crab and coral, its closest homologs. Future efforts
are focused on further characterizing the squid C3 as well as other complement
components both at the gene and protein level, in addition to investigating their possible
involvement in the onset and maintenance of the symbiotic partnership between the E.
scolopes and V. fisheri.




                                                                                       31
Identification and Molecular Characterization of Complement Components in the
Squid Euprymna scolopes

Maria G. Castillo and Margaret McFall-Ngai
University of Wisconsin School of Medicine and Public Health, Department of Medical
Microbiology and Immunology, Madison, WI




32
Two Component Signal Transduction Systems and the Vibrio-Squid Symbiosis

Elizabeth A. Hussa1 , Therese M. O‟Shea1 , Cynthia L. Darnell1 , Edward G. Ruby2 , and
Karen L. Visick1
1
  Dept. of Microbiology and Immunology, Loyola University Chicago, Maywood, IL
2
  Dept. of Medical Microbiology and Immunology, University of Wisconsin, Madison, WI

        Most bacteria utilize two-component signal transduction systems to sense and
respond to environmental stimuli, allowing rapid adaptation to changing conditions.
These signaling pathways often serve as an important mechanism of host-symbiont
communication in both pathogenic and mutualistic partnerships. The availability of the
Vibrio fischeri genome as well as the development of a rapid mutagenesis technique
allowed for a global mutagenesis study to investigate the contribution of bacterial two-
component systems to the squid-Vibrio symbiosis. In this work, we identified 40 putative
response regulators, which are responsible for the output of the two-component system,
encoded within the V. fischeri genome. Based on the type of DNA-binding effector
domain present, we classified 6 as NarL-type, 13 as OmpR-type, and 6 as NtrC-type; the
remaining 15 lacked a predicted DNA-binding domain. We subsequently mutated 35 of
these genes via a vector- integration approach, and analyzed the resulting mutants for
roles in bioluminescence, motility, and competitive colonization of squid. Through these
assays, we identified 3 novel regulators of V. fischeri luminescence and 7 regulators that
altered motility. Furthermore, we found 12 regulators with a previously undescribed
effect on competitive colonization of the host squid. Interestingly, 5 of the newly
characterized regulators each affected two or more of the phenotypes examined, strongly
suggesting inter-connectivity among systems. This work represents the first large-scale
mutagenesis of a class of genes in V. fischeri using a genomic approach, and emphasizes
the importance of two-component signal transduction in bacterial- host interactions.




                                                                                       33
Two Component Signal Transduction Systems and the Vibrio-Squid Symbiosis

Elizabeth A. Hussa1 , Therese M. O‟Shea1 , Cynthia L. Darnell1 , Edward G. Ruby2 , and
Karen L. Visick1
1
  Dept. of Microbiology and Immunology, Loyola University Chicago, Maywood, IL
2
  Dept. of Medical Microbiology and Immunology, University of Wisconsin, Madison, WI




34
Chitin and Competence: Investigating the mechanism of DNA uptake in Vibrio
fischeri

Amber G. Pollack*, Amy L. Schaefer, and Edward G. Ruby
University of Wisconsin School of Medicine and Public Health, Department of Medical
Microbiology and Immunology, Madison, WI

         Like other marine vibrios, Vibrio fischeri is capable of utilizing chitin, the most
abundant biopolymer in the marine environment, as a nutrient source. This characteristic
is useful not only to free- living V. fischeri: recent studies have revealed chitin is also
present in the squid light organ, and have indicated that it serves as both a nutrient and
chemoattractant during colonization by V. fischeri cells. In addition, transcriptional
results from V. fischeri microarray studies have suggested that genes potentially involved
in genetic competence are upregulated in cells grown in the presence of chitin
oligosaccharides in vivo, as well as within the light organ population. This evidence
suggested that V. fischeri cells exposed to chitin respond by upregulation of genetic
programs to mobilize extracellular DNA into the bacterium.
         To investigate DNA uptake in vitro, recipient strains were cultured in a minimal
medium supplemented with chitohexaose, a chitin break-down product, to induce
competence. The cells were then incubated with DNA from a V. fischeri strain carrying a
chloramphenicol-resistance cassette. Introduction of this marker by homologous
recombination was confirmed in antibiotic-resistant transformants by phenotypic analysis
and PCR. The phenomenon appears chitin- induced: cells grown to mid- log phase with
chitohexaose produced transformants, whereas cells provided with the chitin monomer,
N-acetylglucosamine (NAG), did not. The wild-type recipient strain could be transformed
with either chromosomal or plasmid DNA and, as in other Gram- negative bacteria, DNA
uptake by V. fischeri appears to involve type IV pili structure and function: a mutation of
either the pilA or pilT gene disrupted transformation.
         In vitro, the observed frequency of transformation ranged from 1E-09 to 1E-07, a
value that is lower than typically observed for optimized natural transformation.
Transformation may be limited either by the rate of DNA uptake, and/or by the rate of
recombination. To investigate the former, we are currently developing approaches to
directly measure uptake of DNA into the cell. Our work will continue to focus on
understanding the mechanism of DNA uptake. In addition, we will focus on identifying a
biological role for chitin- induced uptake of DNA within the light organ symbiont
population, perhaps one of facilitating genetic recombination amongst symbiont strains or
for utilizing DNA as a nutrient source.




                                                                                         35
Chitin and Competence: Investigating the mechanism of DNA uptake in Vibrio
fischeri

Amber G. Pollack*, Amy L. Schaefer, and Edward G. Ruby
University of Wisconsin School of Medicine and Public Health, Department of Medical
Microbiology and Immunology, Madison, WI




36
Exploring the roles of Vibrio fischeri response regulators in symbiosis with
Euprymna scolopes

Christine M. Anderson and Karen L. Visick
Dept. of Microbiology and Immunology, Loyola University Chicago, Maywood, IL

To be a successful symbiont Vibrio fischeri must respond appropriately to its host
Euprymna scolopes and the environmental changes encountered in the transition from a
free living bacterium in sea water to a member of the densely packed symbiotic
community in the light organ of E. scolopes. One important way that bacteria sense and
respond to their environment is through two component signaling pathways consisting of
sensor kinases which activate response regulators via phosphorylation. In addition to the
previously known V. fischeri response regulators GacA, LuxO, ArcA, SypG and SypE,
35 other response regulators have been predicted from genome sequence analysis of V.
fischeri. Competition colonization experiments between response regulator mutants and
wild type V. fischeri revealed 7 response regulator mutants that were defective in
colonization of newly hatched E. scolopes but normal in motility and luminescence (see
E. A. Hussa et al. abstract). The goal of my work is to further understand the role of 6 of
these 7 response regulators in symbiosis. I will begin with single strain colonization
experiments and biofilm assays of response regulator mutants and V. fischeri strains
overexpressing the response regulator genes for each of the 6 response regulators. The
functions of the response regulators that play the biggest roles in colonization or control
the most striking phenotypes will be further investigated.




                                                                                        37
Asynchronous swimme rs: A high-throughput motility screen of a transposon
mutant collection of Vibrio fischeri

Caitlin Brennan, Mark Mandel, and Edward G. Ruby
University of Wisconsin School of Medicine and Public Health, Department of Medical
Microbiology and Immunology, Madison, WI

         Proper regulation of flagellar motility by Vibrio fischeri is essential for initiation
of the symbiosis with its host, Euprymna scolopes. Additionally, regulation of
flagellation appears to be important in the light organ when most V. fischeri cells are
aflagellate. However, much of what we know about the regulation of flagellation in V.
fischeri is from making comparisons to the pathways in the well-studied flagellar systems
of Vibrio cholerae and Vibrio parahaemolyticus. These comparisons are not necessarily
appropriate as there are noted differences in flagellar structure among these species: cells
of V. fischeri have a tuft of polar flagella in contrast to the single polar flagellum
commonly observed in cells of V. cholerae and V. parahaemolyticus. Work performed
by Debbie Millikan has exemplified these differences and demonstrated that the major
flagellin, FlaA, is not essential for motility in V. fischeri as it is in V. cholerae, but that
FlaA is required only for normal motility.
         Based on microarrary results comparing a flrA deletion mutant to a wild-type
strain, we identified two hypothetical proteins that are regulated by the master flagellar
activator FlrA, and that we have since shown to be necessary for normal motility. These
two open reading frames, VF1883 and VF1884, while present in V. parahaemolyticus,
were not identified in the saturating motility screen performed by the McCarter lab. This
suggests differences in the genetic basis of flagellar motility between V. fischeri and V.
parahaemolyticus, which highlights the importance to define the pathway that is
operating specifically in V. fischeri. We are interested in understanding the genes
involved in the regulation of flagellar motility in V. fischeri, which will help us
understand how flagellation is down-regulated in the light organ. We may also identify
novel genes involved in flagellar motility that account for the physiological differences
between the tufted V. fischeri and the monoflagellar V. cholerae and V.
parahaemolyticus.
         To find such novel regulators of flagellar motility, we are in the process of
generating a saturating transposon insertion mutant collection of V. fischeri ES114. In a
pilot screen of over 5700 mutants on soft agar, greater than 300 mutants have been
identified with altered motility and/or growth, ba sed on initial observations. This
collection also serves as a resource for identifying interrupted genes of interest using
basic PCR techniques.




38
r
Detecting Selection in the Bacterial Symbiont, Vibrio fischeri

W. L. Castle and M. K. Nishiguchi
Department of Biology, New Mexico State University, Las Cruces, NM

Mutualistic symbiosis between eukaryotes and their prokaryotic partners has been a
commonly studied theme to determine the effects of cospeciation, genetic drift, and the
various forms of selection that occur between stages of symbiosis during environmental
transfer. One approach to analyze the evolution of genes selected for symbiosis consists
of measuring the ratio of nonsynonymous vs. synonymous amino acid substitutions
within a gene to identify sites of directional selection. This nonsynonmous/synonymous
(Ka/Ks) rate ratio allows us to identify natural selection in action at the level of the
protein molecule. The goals of this project involve detecting modes of selection on V.
fischeri genes that have been identified as up or down regulated during symbiosis, as well
as those genes expressed in their free- living state (seawater). In order calculate Ka/Ks
ratios, two programs which use slightly different assumptions will be used: the codeml
program in the PAML package and the MEGA2 software package. This analysis will
afford us a view of which genes are under selective pressure in each of the very different
selective environments (squid light organ vs. seawater) that symbiotic V. fischeri must
contend with. We then hope to determine how these very different factors are
contributing to evolution on a molecular scale in this mutualisic symbiosis.




                                                                                       39
Complex control of syp transcription and Syp-dependent phenotypes

Elizabeth A. Hussa, Cynthia L. Darnell*, and Karen L. Visick
Dept. of Microbiology and Immunology, Loyola University Chicago, Maywood, IL
*Presenting author

Symbiotic colonization requires the symbiosis polysaccharide (syp) gene cluster. The
cluster encodes a number of putative polysaccharide synthesis genes, which produce a
biofilm that may enhance colonization by promoting adherence to the light organ.
Transcription of the syp genes depends on at least 2 two-component regulators: the sensor
kinase RscS (located distal to the syp genes) and the 54-dependent response regulator
SypG. In two-component regulatory systems, the sensor kinase is activated by an external
signal and autophospohorylates, and then passes the phosphate to a response regulator;
activated, the response regulator typically affects transcription of target genes (although
some response regulators impact protein function). The syp cluster contains two
additional two-component regulators, the sensor kinase SypF and the response regulator
SypE. To begin to understand transcriptional control of the syp cluster and control of
Syp-associated phenotypes, we overexpressed various regulators (RscS, SypG, SypE, and
SypF) in wild-type V. fischeri and in strains disrupted for putative syp regulators. In
general, multi- copy expression of RscS induced biofilm-associated phenotypes, including
wrinkled colonies, attachment to glass, and pellicles. These phenotypes depended on the
presence of wild-type copies of sypG and rpoN (encoding 54 ), and to a lesser extent,
sypE, but not sypF. Multi-copy expression of sypG exerted the greatest impact on syp
transcription (from the sypA and sypP promoters), and this effect depended on rpoN, but
not rscS, sypF or sypE. While expression of wild-type sypG did not induce wrinkled
colony formation, it did induce other biofilm-associated phenotypes such as attachment to
glass and pellicle formation. As with transcription, these phenotypes were rpoN-
dependent. Overexpression of sypF did not induce any syp-dependent phenotypes,
possibly due to lack of an activation signal in culture. Therefore, we isolated an altered
activity allele, sypF*, that both increased syp transcription (sypA) and induced wrinkled
colony formation. These phenotypes were entirely (syp transcription) or largely
(wrinkled colony formation) dependent on sypG and rpoN. Overexpression of sypF* also
enhanced adherence to a glass test tube in cultures grown either statically or with shaking
in minimal medium; surprisingly, this enhancement depended on sypE, but not sypG. The
sypF* allele carries two mutations (S247F and V439I). We then assessed the role of
SypE by examining the ability of multi-copy sypE to impact Syp-dependent phenotypes.
Surprisingly, multi-copy sypE caused a 2- fold increase in syp transcription, but only in
the absence of sypG. Finally, we are currently asking whether syp-dependent phenotypes
are impacted by disruptions in either rpoS or the rpoS- like gene, VFA1015. To date, our
data confirm the involvement of RscS and SypG, and reveal roles for SypE and SypF.
Taken together, these data demonstrate the complexity of syp transcription and in
particular, Syp-dependent phenotypes.




40
r
Light-induced behavioral and genomic responses in a beneficial bacterial
association

Kasia Skrzypczynska and Edward G. Ruby
University of Wisconsin School of Medicine and Public Health, Department of Medical
Microbiology and Immunology, Madison, WI

It has been observed that non- luminous mutant strains of Vibrio fischeri have a
persistence defect when colonizing the light organ. The cause of this defect is unknown,
but it is hypothesized that the bacterial population may modulate the genes necessary for
proper colonization by using its own emitted light as a regulatory signal. To test this
assertion, the completely dark luxCDABEG deletion strain of V. fischeri and a wild-type
strain were grown under conditions that fully induce luminescence in the latter. RNA was
extracted from the cells, and subjected to microarray analysis. By comparing the
expression patterns of the two cell types, several genes that may be regulated by light
were identified. This list of candidate genes has been examined to eliminate any that
might simply reflect the detectably increased growth rate of the deletion mutant. Using
qRT PCR, I am confirming the microarray results for a representative subset of these
candidate genes. In addition, the question of light regulation of these genes is being
directly studied under conditions in which the luminescence defect in the luxCDABEG
mutant is complemented with external blue light.




                                                                                      41
Extraocular expression of phototransduction proteins in the light organ of the squid
Euprymna scolopes

Deyan (Lily) Tong and Margaret McFall-Ngai
University of Wisconsin School of Medicine and Public Health, Department of Medical
Microbiology and Immunology, Madison, WI

The sepiolid squid Euprymna scolopes harbors the marine luminous bacterium Vibrio
fischeri in a complex light organ in the center of its body cavity. Previous studies have
indicated that the bioluminescence of V. fischeri cells plays a critical role in both the
normal host-tissue development and symbiotic persistence. However, little is known
about the role of bacterial luminescence in communication between these partners during
the development and maintenance of the mutualistic state. With the striking similarity in
their anatomy and morphology to that of eyes, the light organ of E. scolopes may have
the ability to communicate with the symbiont V. fischeri by directly sensing the
luminescence. Analysis of a juvenile light organ EST database has provided evidence for
direct sensing of bacteria light. Eleven transcripts critical for the visual transduction
cascade and eye development have been identified in the photophores of E. scolopes,
including a visual pigment, signal transducers and other eye-specification proteins. Using
antibodies to a subset of these gene products, we also found visual-related protein
expression in the light organ of juvenile squid, as well as in the central core of adult
squid, the light organ epithelium that supports the culture of the bacterial symbiont. In
addition, our electroretinogram data showed both the eyes and the light organs of squid
have an electrical response to a light stimulus. Taken together, our data suggest that
extraocular phototransduction proteins of the light organ might function as the reciprocal
dialogue between host and symbiont in the squid-vibro association.




42
r
NO Detoxification Systems in Vibrio fischeri

Yanling Wang1 , Amy Schaefer1,2 , Edward G. Ruby1
1
  Department of Medical Microbiology and Immunology, University of Wisconsin-
Madison
2
  Department of Microbiology, University of Washington-Seattle

    Nitric oxide (NO) not only is an intermediary metabolite in the process of bacterial
denitrification, but also servers as a signaling and defense molecule of major biological
importance. Due to its high reactivity with DNA, proteins, low-molecular weight thiols
and reactive oxygen species, host- generated NO is recognized as a potent component of
innate immune system aimed against invading bacterial pathogens. Not surprisingly,
bacteria that live in association with animal tissues have evolved NO detoxifying systems
such as NO dioxygenase (Hmp) and flavorubredoxin NO reductase- flavorubredoxin-
NAD(+) reductase (NorVW). During the colonization process in the Euprymna scolopes-
Vibrio fischeri symbiosis, V. fischeri encounters high concentration of NO produced by
the epithelia of the superficial ciliated fields, ducts and crypt antechambers. NO is also
detectable in vesicles within the secreted mucus where the bacterial symbiont forms
aggregate. How does V. fischeri cope with the NO challenge during the establishment of
the symbiosis? We hypothesized that NO-detoxification systems are important for V.
fischeri to survive NO stress and to initiate successful colonization.
     Campbell mutants of both hmp and norV were constructed in V. fischeri. Under
aerobic condition, addition of 80µM DEA-NONOate (an NO-generator) to cells growing
in minimal medium severely affected the initiation of growth in hmp mutant, increasing
the lag phase by more than 2-fold, compared to the wild-type parent strain. In contrast,
this treatment had almost no effect on the growth of norV mutant. Similarly, in a kill
curve experiment, challenge with high concentration (800µM) of DEA-NONOate was
detrimental to both the wild-type and the two mutants, although the wild-type survived 4-
times better than both mutants, suggesting the important role of NO detoxification
mechanisms in surviving the NO stress in vitro.
     An in-frame deletion mutant of hmp was constructed. norV Campbell mutant and
hmp deletion mutant were competed against the wild-type in host-colonization assays .
Interestingly, hmp deletion mutant colonized the host squids almost well as the wild type
(RCI=0.96, n=20) while the wild-type out-competed norV Campbell mutant by
approximately 8-fold (RCI=0.12, n=13). The data may suggest microaerobic to anaerobic
conditions in the duct or crypt antechambers, where V. fischeri encounters high
concentration of host-generated NO at the initial stage of symbiosis.
     Currently, we are focusing on confirming the mutants‟ real phenotype in the
competition assay. And in- frame deletion mutant of norV will be made and tested for its
responses to NO challenge under anaerobic conditions. By studying the two
physiologically distinctive NO detoxification mechanisms in V. fischeri, we hope not
only to determine the relative contribution of each system to the initiation of the
symbiosis, but also to infer under what conditions (aerobic or anaerobic) NO stress
occurs in this beneficial association.




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The population genetic structure of a sympatric Squid-Vibrio symbiosis in the
Mediterranean Sea

DJ Zamborsky and MK Nishiguchi
Department of Biology, New Mexico State University, Las Cruces, NM

Squids from the genus Sepiola (Cephalopoda: Sepiolidae) living sympatrically in the
Mediterranean Sea form a mutualistic symbiosis with two species of the Vibrio genus: V.
fischeri and V. logei. Environmentally transmitted symbionts inhabit the light organs and
produce ventrally-directed luminescence used in anti-predatory behavior. The light organ
expulses symbiotic bacteria daily, and is subsequently re-colonized by the remaining
Vibrio bacteria in the light organ. This diurnal event has led to p hylogeographical
variation within the squid-Vibrio system in other regions of the world (Indo-west
Pacific). Although the symbiosis amongst sympatric Sepiola species and both species of
Vibrio in the Mediterranean has been well documented, the genetic structure of the host
squid and Vibrio symbiont has not been described. Preliminary results from the
sequencing of the 16S rRNA locus confirm the co-existence of both symbiont species (V.
logei and V. fischeri) in the light organs of Mediterranean Sepolid sq uid species.
Analysis of the glyceraldehyde phosphate dehydrogenase (gapA) sequence using the
Arlequin population genetic software program also reveals a number of different
haplotypes of both symbionts inhabiting light organs in the experimental populations
from Banyuls-sur- mer, France and Bari, Italy. Vibrio symbionts show a broad range of
haplotypes over a wide geographic area suggesting a strong influence of migratory
forces, both biotic and abiotic. Biotic factors such as host movement within the
Mediterranean as well as abiotic influences such as surface water movements may
contribute to the diversiform genetic architecture of the symbiont population observed in
this study.




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