Ahmad_ proposal - Rochester Institute of Technology

COS Undergraduate Student Summer Research Proposal
Nurul Hawa Ahmad
Gene Tagging in Novosphingobium sp. : Mutation of a luxI homolog
1. Abstract
Bacterium Rr 2-17 isolated from grapevine crown gall tumor is a member of
Novosphingomonas genus and produces bacterial communication signals known as N-acylhomoserine lactone (AHL). This signal functions in a cellular process called quorum sensing
(QS) which acts in gene regulation. Bacterium Rr 2-17 will be subjected to genetic mutagenesis
and the resulting mutants will be screened for a disruption in AHL signal production. The goal of
this project is to produce a population of genetic mutants in newly identified Novosphingobium
sp. strain (Rr 2-17) and screen for mutants deficient in bacterial communication signal synthesis.
The identification of a genetic mutant in AHL signal production will enable the genetic
identification of the DNA sequence responsible for AHL signal synthesis in Novosphingobium
sp. Rr2-17.
2. Background and Significance
i) Grapevine crown gall disease. Infection of plants by
pathogenic strains of Agrobacterium vitis causes crown gall
tumor disease. This disease can cause economic losses in
vineyards [5]. A. vitis, a soil-borne bacterium that commonly
associated with crown gall disease in grapevine, causes a poor
xylem development of grapevine, which impairs water flow into
the young parts of the shoot above the gall [1]. The presence of
A. vitis on grapevine seldom causes tumor production unless the
vine is injured. The intermittent freezing and thawing weather
conditions can be the source of the entry of A. vitis on grape
tissues and cells [2].
Figure 1: Small, pimple-like
galls (arrows) of crown gall (A.
vitis) extending up the trunk of a
grapevine (Courtesy T.G. Burr).
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COS Undergraduate Student Summer Research Proposal
Nurul Hawa Ahmad
ii) Quorum sensing. Bacteria exist in populations and communicate with each other to
facilitate their adaptation to changing environmental conditions. Quorum sensing (QS), or the
control of gene expression in response to cell density, is used by bacteria to modulate both intraand inter- species cell-cell communication [2]. Bacteria produce and secrete chemical signals
called N-acyl-homoserine lactones (AHL) to their surroundings and these signaling molecules
can be sensed by cells which then allow the whole population to initiate a concerted action once
it reaches a critical threshold concentration. The understanding of bacterial cell-cell
communication is derived from the discovery of symbiotic relationship between bacterium
Vibrio fischeri that produce AHL signaling molecules and Euprymna scolopes (small squid lives
in the shallow sand flats associated with coral reefs in the Hawaian archipelago). E. scolopes has
bioluminescent appearances in the dark environments due to the maintenance of high density V.
fischeri populations in a specialized light organ. Thus, the bacterium protects the squid from
predators by eliminating a visible shadow created by moonlight and in return, the squid provides
V. fischeri with nutrients [2,6].
iii) Gram negative bacterial communication: the LuxI/LuxR language. Over 30
species of Gram negative bacteria employ the control of different cell-density-dependent
functions where these systems all have in common, the synthesis of AHL is dependent on a luxl
homologue (gene encoding AHL synthase) as well as a luxR homologue (gene encoding a
receptor and transcriptional activator) of target gene (Figure 2) [2].
Figure 2: The LuxI / LuxR quorum-sensing system. In Gram-negative quorum-sensing bacteria, LuxI (square) are
responsible for production of specific AHL signals (triangles). Upon reaching a critical concentration, its cognate
receptor protein, LuxR (circle), binds the AHL signal and together the LuxR-AHL complex activates transcription of
the target gene(s).
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COS Undergraduate Student Summer Research Proposal
Nurul Hawa Ahmad
3. Significance
Production of genetic mutants in grapevine tumor-associated Novosphingobuim sp. Rr 217 strain will enable the identification of the gene involved in AHL signal production. This will
facilitate the development of novel strategies to control the pathogenic bacterium, A. vitis, on
grapevine plants. Besides grapes, over 600 types of plants are known to be susceptible to crown
gall including apples, stone fruits and brambles, so broad disease control measures may be
developed from this proposed research [5].
4. Project Goals and Design
i) Production of Mutants. Approximately 4000 to 5000 mutants will be produced by
introducing into Novosphingobium sp. Rr 2-17 strain a genetic element called a transposon (Tn5
mutagenesis) using EpicentreTM Tn5 Kit [3]. To select for mutants, the resulting
Novosphingobium sp. Rr 2-17 cells will be recovered and grown in potato dextrose agar (PDA)
medium supplemented with selection agent, kanamycin (Km25). Each of the colonies observed
on the PDA+ kanamycin plates will be retested on PDA medium containing a higher
concentration of kanamycin (Km50) to ensure the mutants are true.
ii) Screening Mutants for a Deficiency in AHL Signal Production. After the selection
of colonies on PDA + kanamycin, each mutant will be individually screened for disruption of
AHL signal producing using a bacterial biosensor strain, Chromobacterium violaceum (CV026).
Tryptone yeast extract-potato dextrose (TYE-PD) agar medium (1:1) is used to undergo the
screening process because this media allow the growth for both bacteria, biosensor CV026 and
Novosphingobium Rr 2-17 mutants [4]. These cells are cultured for 48 hours in the 28˚C
incubator and purple pigments will be produced by biosensor CV026 in the presence of
exogenous AHL signals produced from mutant test colonies. Disruption of signal production in a
mutant will lead to a failure of color change in the biosensor CV026 and result in the absence of
purple pigment synthesis. E.coli, which do not produce a AHL signal and wild type
Novosphingobium sp. Rr 2-17, which produces an AHL signal, will be used as a control strains.
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COS Undergraduate Student Summer Research Proposal
Nurul Hawa Ahmad
References Cited
1. Aloni, Roni. 2005. Tumor-Induced Ethylene Controls Crown Gall Morphogenesis.
Darmstadt University of Technology, Germany. Plant Physiology 4th edition. Chapter
22:1
2. Bassier, Bonnie L. 2000. How bacteria talk to each other: regulation of gene expression
by quorum sensing. Department of Molecular Biology., Princeton University.
Microbiology 2: 582-587
3. Enzyme System for RNA and DNA Research. EZ-Tn5TM <R60Kyori/KAN-2> Tnp
TransposomeTM Kit. Retrieved on January 27,2009 from Epicentre Biotechnologies. Web
site: http://www.epibio.com/item.asp?ID=291
4. Gan, Han Ming, Buckley L., Szegedi E., Hudson A. O., Savka MA. 2009.
Identification of an rsh gene from a Novosphingobium sp. necessary for quorum sensing
signal accumulation. Journal of Bacteriology (accepted)
5. Hartman, John. 2007. Grape Crown Gall. University of Kentucky.
6. Whitehead, Neil A., Barnard, Anne M.L., Slater, Holly, Simpson, Natalie J.L,
Salmond, George P.C. 2001. Quorum-sensing in Gram Negative Bacteria. FEMS
Microbiology 25: 365-404
7. Williams, Paul. 2007. Quorum Sensing, communication and cross-kingdom signaling in
the bacterial world. Institute of Infection Immunity and Inflammation Centre for
Biomolecular Sciences, University of Nottingham. Microbiology. 153: 3923-393
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