International Journal of Biometrics and Bioinformatics (IJBB), Volume (4): Issue (5)

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International Journal of
Biometrics and Bioinformatics
(IJBB)







Volume 4, Issue 5, 2010









Edited By
Computer Science Journals
www.cscjournals.org

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Editor in Chief Professor João Manuel R. S. Tavares

International
Journal
of
Biometrics
and
Bioinformatics (IJBB)
Book: 2010 Volume 4, Issue 5
Publishing Date: 20-12-2010
Proceedings
ISSN (Online): 1985-2347

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© IJBB Journal
Published in Malaysia

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Editorial Preface

This is the fifth issue of volume four of International Journal of Biometric and
Bioinformatics (IJBB). The Journal is published bi-monthly, with papers being
peer reviewed to high international standards. The International Journal of
Biometric and Bioinformatics are not limited to a specific aspect of Biology
but it is devoted to the publication of high quality papers on all division of Bio
in general. IJBB intends to disseminate knowledge in the various disciplines
of the Biometric field from theoretical, practical and analytical research to
physical implications and theoretical or quantitative discussion intended for
academic and industrial progress. In order to position IJBB as one of the
good journal on Bio-sciences, a group of highly valuable scholars are serving
on the editorial board. The International Editorial Board ensures that
significant developments in Biometrics from around the world are reflected in
the Journal. Some important topics covers by journal are Bio-grid, biomedical
image processing (fusion), Computational structural biology, Molecular
sequence analysis, Genetic algorithms etc.

The coverage of the journal includes all new theoretical and experimental
findings in the fields of Biometrics which enhance the knowledge of scientist,
industrials, researchers and all those persons who are coupled with
Bioscience field. IJBB objective is to publish articles that are not only
technically proficient but also contains information and ideas of fresh interest
for International readership. IJBB aims to handle submissions courteously
and promptly. IJBB objectives are to promote and extend the use of all
methods in the principal disciplines of Bioscience.


IJBB editors understand that how much it is important for authors and
researchers to have their work published with a minimum delay after
submission of their papers. They also strongly believe that the direct
communication between the editors and authors are important for the
welfare, quality and wellbeing of the Journal and its readers. Therefore, all
activities from paper submission to paper publication are controlled through
electronic systems that include electronic submission, editorial panel and
review system that ensures rapid decision with least delays in the publication
processes.

To build its international reputation, we are disseminating the publication
information through Google Books, Google Scholar, Directory of Open Access
Journals (DOAJ), Open J Gate, ScientificCommons, Docstoc and many more.
Our International Editors are working on establishing ISI listing and a good
impact factor for IJBB. We would like to remind you that the success of our
journal depends directly on the number of quality articles submitted for
review. Accordingly, we would like to request your participation by
submitting quality manuscripts for review and encouraging your colleagues to
submit quality manuscripts for review. One of the great benefits we can

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provide to our prospective authors is the mentoring nature of our review
process. IJBB provides authors with high quality, helpful reviews that are
shaped to assist authors in improving their manuscripts.


Editorial Board Members
International Journal of Biometrics and Bioinformatics (IJBB)

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Editorial Board

Editor-in-Chief (EiC)

Professor. João Manuel R. S. Tavares
University of Porto (Portugal)

Associate Editors (AEiCs)

Assistant Professor. Yongjie Jessica Zhang
Mellon University (United States of America)
Professor. Jimmy Thomas Efird
University of North Carolina (United States of America)
Professor. H. Fai Poon
Sigma-Aldrich Inc (United States of America)
Professor. Fadiel Ahmed
Tennessee State University (United States of America)
Mr. Somnath Tagore (AEiC - Marketing)
Dr. D.Y. Patil University (India)
Professor. Yu Xue
Huazhong University of Science and Technology (China)
Professor. Calvin Yu-Chian Chen
China Medical university (Taiwan)
Associate Professor. Chang-Tsun Li
University of Warwick (United Kingdom)

Editorial Board Members (EBMs)

Dr. Wichian Sittiprapaporn
Mahasarakham University (Thailand)
Assistant Professor. M. Emre Celebi
Louisiana State University (United States of America)
Dr. Ganesan Pugalenthi
Genome Institute of Singapore (Singapore)
Dr. Vijayaraj Nagarajan
National Institutes of Health (United States of America)
Dr. Paola Lecca
University of Trento (Italy)
Associate Professor. Renato Natal Jorge
University of Porto (Portugal)
Assistant Professor. Daniela Iacoviello
Sapienza University of Rome (Italy)
Professor. Christos E. Constantinou
Stanford University School of Medicine (United States of America)
Professor. Fiorella SGALLARI
University of Bologna (Italy)
Professor. George Perry
University of Texas at San Antonio (United States of America)
Assistant Professor. Giuseppe Placidi
Università dell'Aquila (Italy)
Assistant Professor. Sae Hwang
University of Illinois (United States of America)
Assistant Professor. M. Emre Celebi
Louisiana State University (United States of America)

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Table of Content




Volume 4, Issue 5, December 2010



Pages
161-175
Structure and function predictions of hypothetical proteins in Vibrio

Phages

Swapnil G. Sanmukh, Waman Narayan Paunikar, Tarun Kanti

Ghosh, Tapan Chakrabarti
176-193
Multiple Features Based Two-stage Hybrid Classifier Ensembles for
Subcellular Phenotype Images Classification


Bailing Zhang, Tuan D. Pham









International Journal of Biometrics and Bioinformatics (IJBB), Volume (4): Issue (5)

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Swapnil G. Sanmukh, Waman N. Paunikar, Tarun K. Ghosh & Tapan Chakrabarti
Structure and Function Predictions of Hypothetical Proteins in
Vibrio Phages


Swapnil G. Sanmukh


swamukh1985in@rediffmail.com
Applied Aquatic Ecosystem Division,
National Environmental Engineering Research Institute,
Nehru Marg, Nagpur-440020, India

Waman N. Paunikar




wn_paunikar@neeri.res.in
Applied Aquatic Ecosystem Division,
National Environmental Engineering Research Institute,
Nehru Marg, Nagpur-440020, India

Tarun K. Ghosh
tk_ghosh@neeri.res.in
Applied Aquatic Ecosystem Division,
National Environmental Engineering Research Institute,
Nehru Marg, Nagpur-440020, India

Tapan Chakrabarti t_chakrabarti@neeri.res.in
National Environmental Engineering Research Institute,
Nehru Marg, Nagpur-440020, India

Abstract

The Vibriophages are the potential agents for the transfer of the virulence factor
to their host through lateral gene transfer. The complete genome sequencing of
various known vibriophages has been done which deciphered the presence of
various gene sequences for hypothetical proteins whose function is not yet
understood. We analyzed complete genome of 21such Vibriophages for
hypothetical proteins from which 13 phages were sorted for our studies. Our
attempt is to predict the structure and function of these hypothetical proteins by
the application of computational methods and Bioinformatics. The probable
function prediction of the hypothetical protein was done by using Bioinformatics
web tools like CDD-BLAST, INTERPROSCAN, PFAM and COGs by searching
sequence databases for the presence of orthologous enzymatic conserved
domains in the hypothetical sequences. While tertiary structures were
constructed using PS2 Server (Protein Structure Prediction server). These study
revealed presences of enzymatic functional domain in 92 uncharacterized
proteins; their roles are yet to be discovered in Vibriophages. These deciphered
enzymatic data for hypothetical proteins can be used for the understanding of
functional, structural, evolutionary and metabolic development of Vibriophages
and its life cycle along with their role in host evolution and pathogenicity.

Keywords: Bioinformatics Web Tools, Conserved Domains, Protein Structure Prediction, Uncharacterized
Proteins, Life Cycle and Pathogenicity.

International Journal of Biometrics and Bioinformatics, (IJBB), Volume (4): Issue (5)
161

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Swapnil G. Sanmukh, Waman N. Paunikar, Tarun K. Ghosh & Tapan Chakrabarti
1. INTRODUCTION
The etiologic agent of cholera, Vibrio cholerae is a gram negative bacterium which has been
reported to be infected by various specific filamentous phages (Campos, et al., 2003, Faruque, et
al., 2005, Waldor, et al., 1997, Ikema, et al., 1998, Jouravleva, et al., 1998, Kar, et al., 1996,
Honma, et al., 1996). CTXΦ phage has been the most studied due to its role in pathogenicity and
horizontal gene transfer (Davis, et al., 2003). The phage is potentially responsible for transducing
the cholera toxin genes into nonpathogenic environmental strains along with replicating directly
from the bacterial chromosome for producing infective phage particles (Davis, et al., 2003,
Waldor, et al., 2003). The VGJΦ is able to recombine with the CTXΦ genome to originate a
hybrid phage with the full potential for virulence conversion. The hybrid phage shows an
increased infectivity due to its specificity for the receptor mannose-sensitive hemagglutinin
(receptor mannose- sensitive hemagglutinin pilus), which is ubiquitous among environmental
strains (Campos, et al., 2003a, Campos, et al., 2003b). The vibriophages KVP40 differs from
many described vibriophages in having a broad host range and is reported to infect eight Vibrio
species, including Vibrio cholerae and Vibrio parahaemolyticus, the nonpathogenic species Vibrio
natriegens, and Photobacterium leiognathi (Matsuzaki, et al., 1992).

Vibriophages (family Vibrionaceae) contains the greatest number of reported phage-host systems
for the marine environment (Moebus 1987), with the genus Vibrio comprising most of the hosts
(Moebus & Nattkemper 1981). The phage VpVs phage infect only V. parahaemolyticus strains (
Koga et al., 1982; Kellogg et al., 1995), phage P4 (Baross et al., 1974) and KVP20 (Matsuzaki et
al., 1998) infect other Vibrio spp. (as the VpVs in this study), whereas phage V14 (Nakanishi et
al., 1966) and KVP40 (Matsuzaki et al., 1992) have been reported to infect other genera.
Vibriophage has also proved to be useful in studying the host chromosomes (Guidolin and
Manning, 1987).

Vibrio cholera-specific filamentous bacteriophages CTXf was first identified in 1996 (Waldor and
Mekalanos, 1996). Its genome includes the genes encoding cholera toxin, an AB 5- subunit type
toxin secreted by V. cholera during its growth in the small intestine which causes secretory
diarrhoea (Lencer and Tsai, 2003). The acquisition of CTXf is an important factor for V. cholera
virulence. Virulence factors are frequently encoded within mobile genetic elements such as
phages and plasmids (Davis and Waldor, 2002). The first reported filamentous phage horizontally
transmitting a virulence factor that results in lysogenic conversion of a host to become virulent
was CTXf (Waldor and Mekalanos, 1996; Ochman et al., 2000). Most of the characterized phages
that integrate into their respective host chromosomes also undergo a reverse reaction wherein
the phage genome excises from the chromosome (Azaro and Landy, 2002). However, excision of
the CTXf prophage from the V. cholera chromosome has never been observed (Davis and
Waldor, 2000). Instead, the chromosomally integrated CTXf prophage acts as a template for
synthesis of viral DNA (Davis and Waldor, 2000; Moyer et al., 2001).

The study of Vibriophages is limited to the expressed genetic characteristics which are observed
through experimental studies, but to get some insight of the Vibriophages and how its acquisition
imparts host to gain various new characteristics leading to virulence and evolution of both phage-
host systems, the study of phage genome is essential. The in-silico studies of hypothetical
proteins (Uncharacterized proteins) for identifying their structure and function is an attempt to
understand Vibriophages and their genomes with some possible implications.

Computational biology assists us to predict the functionality in the uncharacterized sequences
using the different strategies of comparative proteomics. The program’s ability of homology
searching using defined databases and by choosing standard parameters, the presence of the
enzymatic conserved domain/s in the sequences could be searched out and it may assist in the
categorizing protein into specific enzymatic family.

Bioinformatics web tools like CDD-BLAST,INTERPROSCAN, PFAM and COGs can search the
orthologous sequence in biological sequence databases for the target sequence, while assist in
classification of target sequence in particular family (Edward et al., 2000; Dilip and Alankar,
International Journal of Biometrics and Bioinformatics, (IJBB), Volume (4): Issue (5)
162

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Swapnil G. Sanmukh, Waman N. Paunikar, Tarun K. Ghosh & Tapan Chakrabarti
2009). This study will helps us to understand the probable functions of hypothetical proteins in
Vibriophages.

Several online automated servers are available which can predict the three dimensional
structures for protein sequences by using the strategy of aligning target sequences with
orthologous sequences by virtue of sequence homology and based on that, constructs the
3Dstructure for target protein using best scored template of orthologous family member. Here, we
have predicted 3-D structure using Protein Structure Prediction Server (PS2 server) (Dilip and
Alankar, 2009; Zafer et al., 2006; Chih-Chieh et al., 2006).

2. MATERIALS AND METHODS

2.1 Sequence Retrieval
The Complete protein sequences for 21 different Vibrio phages were downloaded from the
Database of KEGG (http://www.genome.jp/kegg/). The phages under study includes Vibrio phage
kappa (Ehara, et. al., unpublished), Vibrio phage VP93,Vibrio phage VEJphi (Campos, 2010),
Vibrio phage N4 (Das, et. al., Unpublished),Vibrio phage fs1 (Honma, et. al., 1997),Vibrio phage
K139 (Kapfhammer, et. al., 2002),Vibrio phage KVP40 (Miller, et. al., 2003),Vibrio phage fs2
(Ikema, et. al., 1992),Vibrio phage VfO3K6,Vibrio phage VfO4K68,Vibrio phage Vf33,Vibrio phage
Vf12,Vibrio phage VSK (Basu, Unpublished), Vibrio phage VpV262 (Hardies, et. al., 2003),Vibrio
phage VHML, Vibrio phage VGJphi (Campos, et. al., Unpublished), Vibrio phage VP2 (Wang,
Unpublished),Vibrio phage VP5,Vibrio phage VP882,Vibrio phage KSF-1phi (Faruque, et. al.,
2005) and Vibrio phage VP4.

2.2 Functional Annotations
Hypothetical proteins were screened for the presence of enzymatic conserved domains using
sequence similarity search with close orthologous family members available in various protein
databases using the
web-tools. Four
bioinformatics web
tools like CDD-BLAST
(http://www.ncbi.nlm.nih.gov/BLAST/) (Altschul et al., 1997; Schaffer et al., 2001; Aron et al.,
2006), INTERPROSCAN (http://www.abi.ac.uk/interpro) (Zdobnov and Rolf, 2001), Pfam
(http://www.pfam.sanger.ac.uk/) (Alex et al., 2004) and COGs (http://www.ncbi.nih, gov/cog)
(Roman et al., 2000) were used, which shows the ability to search the defined conserved
domains in the sequences and assist in the classification of proteins in appropriate family.

2.3 Functional Categorization
Hypothetical proteins analyzed by the function prediction web tools such as CDD-BLAST,
INTERPROSCAN, PFAM and COGs have shown the variable results when searched for the
conserved domains in hypothetical sequences.

2.4 Protein Structure Prediction
Several online protein structure prediction servers are available. Out of that, online PS2 (PS
Squared) Protein Structure Prediction Server was used (http://www.ps2.life.nctu.edu.tw/) ( Chih-
Chieh et al., 2006; Altschul et al.,1997; Schaffer et al., 2001; Cédric et al., 2000; Wendy et
al.,2000), which accepts the protein (query) sequences in FASTA format and uses the strategies
of Pair-wise and multiple alignment by combining powers of the programs PSI-BLAST, IMPALA
and T-COFFEE in both target – template selection and target–template alignment and resultant
target proteins 3D structures were constructed using structural positioning information of atomic
coordinates for known template in PDB format using best scored alignment data. Where the
selection of template was based on the same conserved domain detected in the functional
annotations and which must be available in the structure alignment for modeling purpose.

3. RESULTS AND DISCUSSION
The in silico structure and function of the Vibriophages was worked out for 21 phages. Out of 21
Vibriophages, conserved domain prediction in hypothetical proteins was possible in 13 phages.
The hypothetical proteins were screened for the presence of enzymatic conserved domains using
International Journal of Biometrics and Bioinformatics, (IJBB), Volume (4): Issue (5)
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