Microbial Research Commons Including Viruses

Prof. A.S. KolaskarBioinformatics Center

University of PunePune, India

Introduction

• Increasing research in life sciences and biotechnology in Indian Universities and national research institutions

• Increased need for microbial and genetic resources

• Establishment of microbial and other biological culture collections in universities and research institutions

Culture Collections In India

• Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh – World Intellectual Property Organization (WIPO, recognized as International Depository Authority)

• National Collection of Industrial Microorganisms (NCIM), Pune – Cultures are deposited for patenting

• Virus cultures at National Institute of Virology (NIV)• National Facility for Animal Tissue and Cell Cuture, Pune

Culture Collections In India

• Anaerobic Bacterial Resource Center (ABRC), Hyderabad

• National Collection of Dairy Cultures, Karnal

• National Fungal Culture Collection of India, Pune

• University of Mumbai Food and Fermentation Technology Division

21 Culture Collections from India registered with WDCM

Thailand Network of Culture Collections

• Biotech Culture Collection (BCC) – 3430

• Department of Medical Sciences Thailand (DMST) – 442

• Department of Agriculture (DOA) – 1163

• Thailand Institute of Scientific and Technological Research – 515

Issues• Limited characterization• Very few cultures characterized at DNA finger printing level• Data not fully computerized and information not available on the

web• Duplication of cultures in the repository• Material Transfer Agreement similar to that in ATCC is followed by

most repositories• No systems in place to detect or prevent misuse of MTA• Redistribution of cultures at informal level• Very few scientists conversant with taxonomic classification even at

the national culture collections• Issues related to Biosafety and National security are not given due

importance

PUMP-E: Salient Features

• Dynamic Representation of pathways• Dynamically building the organism-specific pathways

from genomic data• Development of Software for

– Automated data updating (Perl scripts) – Reformatting and organization of relevant

information from different databases– Drawing pathways diagrams – Comparison of pathways– Visualization of ligands, enzymes– Prediction of enzyme-substrate interactions

• URL- http://202.41.70.51/mpe/

Approaches

Data acquisition & Integration Dynamic Visualization of Metabolic

Pathways Query Interface Molecular Visualization Structure Prediction of Proteins Simulation of 3D Structures of Enzymes

and Metabolites

PUMP-E

Database

Enzyme

Reaction

Compound

Pathway

Organism Gene

User-friendly Query interface

Search by keywords

Dynamic generation of queried pathway

Molecular viewer

Homology models

Source Databases for Data Acquisition 

• Sequence databases: TIGR, NCBI, EBI• Metabolite databases: KEGG• Metabolic pathway database: KEGG• 3D Structure database: PDB • Enzyme Database: KEGG, EXPASY,

IUBMB, BRENDA• Kinetics Data: NIST• Organism List : GOLD

Motifs, patterns & signatures : PROSITE

• Web-based query interface • Supports complex advanced queries • Developed using ASP, HTML and java • Tested by various testing tools such as

Winrunner, Test Director etc.

PUMP-E Front End and Query System

PUMP-E : Front End and Query System

PUMP-E

Organism name Phylum Genome Size (Mbp)

Total number of pathways

Agrobacterium tumefaciens

Bacillus anthracis

Bacillus subtilis

Caulobacter crescentus

Chlamydia trachomatis

Escherichia coli

Francisella tularensis

Haemophilus influenzae

Helicobacter pylori

Mycoplasma pneumoniae

Mycobacterium tuberculosis CDC1551

Mycobacterium tuberculosis H37Rv

Shigella flexneri

Treponema pallidum

Vibrio cholerae

Proteobacteria

Firmicutes

Firmicutes Proteobacteria

Chlamydiae

Proteobacteria

Proteobacteria

Proteobacteria

Proteobacteria

Firmicutes

Actinobacteria

Actinobacteria

Proteobacteria

Spirochaetes

Proteobacteria

5.673462

5.22729

4.21463

4.01695

1.04252

4.63968

1.89282

1.83014

1.66787

0.816394

4.40384

4.41153

4.6072

1.13801

4.03346

207

254

145

176

61

198

184

127

123

48

186

184

179

56

207

Total number of pathways in bacteria under study as per BioCyc 9.1

Hamming Distance Calculations

• Identical Pathways (0): – Start and end products are identical;

intermediate steps are same.

• Similar Pathways (1):– Start and end products are identical;

intermediate steps are different

• Pathways are absent (2):– Start or end products are not same

Metabolic pathway path profile

Columns represents ‘n’ number of pathways and rows represent 15 bacteria under study. Each column corresponds to a particular type of pathway. 2 denote pathway follows same path, 1 denotes pathway follows different path while 0 denotes absence of pathway. This represents a part of the organism specific metabolic pathway path profile.

Metabolic pathway path profile based tree

Comparison of Pathways from Genus Bacillus with E.Coli

Bacillus anthracis Bacillus cereus 10987

Bacillus subtilis Bacillus cereus Zk

Bacillus anthracis Sterne Bacillus halodurans C-125

Bacillus anthracis strain A2012 Bacillus licheniformis ATCC 14580

Bacillus cereus ATCC14579 Bacillus anthracis Ames Ancestor

198 Pathways of E.Coli are compared with pathways data from Biocyc for each of these organisms

Pathways absent in Genus Bacillus; Present in E.Coli

• Electron transport (aerobic and anaerobic)• Phenyl ethyl amine degradation• L-lyxose degradation• Pyridoxal 5’-phosphate salvage pathway• Super pathway of pyridoxal 5’-phosphate

biosynthesis and salvage• D-allose degradation• Fructose lysine degradation • Taurine degradation

Effect of pathways absent in genus Bacillus

• Because of absence of L-lyxose degradation pathways in genus bacillus, it cannot utilize L-lyxose sugar as source of energy

• D-Allose cannot be utilized as a sole carbon source by bacteria of genus bacillus as D-allose degradation pathway is absent

• Under sulfate starvation conditions, bacteria from genus bacillus cannot utilizes taurine as a sulfur source owing to absence of Taurine degradation pathway.

• Bacillus cannot grow on fructoselysine or psicoselysine as the sole carbon source because of absence of Fructose lysine degradation.

Pathways present in Genus Bacillus; Absent in E.Coli

• 2 Nitro propane degradation• Denitrification pathway• Folate transformations• Formaldehyde assimilation• Methanogenesis from acetate• Octane oxidation• Spermine biosynthesis• Xylulose monophosphate cycle

Effect of pathways absent in E.coli

• Xylulose monophosphate cycle and Methanogenesis from acetate are characteristic pathways of methanogenic bacteria and E.coli is not a methanogenic bacteria. Hence these pathways are absent in E.coli

• E.coli cannot reduce nitrate to dinitrogen because of absence of Denitrification pathway

• Formaldehyde produced from the oxidation of methane and methanol by methanotrophic bacteria is assimilated by Formaldehyde assimilation pathway. This pathway is absent in E.coli as it is not methanogenic

Issues

• Taxonomic classification as per NCBI and thus errors can creep in

• No standard system to represent metabolic pathways

• Errors in annotation at gene level translate into errors in metabolic pathways

• Usefulness of metabolic pathways for characterization of microbes is not exploited

Animal Virus Information System

Signature peptide sequences for animal virus families Family Genus Protein Peptide

Togaviridae 

Alphavirus Structural polyprotein AYEHXXV/TXPN

Filoviridae Filovirus Nucleocapsid protein PQLSAIALGVATAHGSTLAGVNVGEQYQQLREAA

Iridoviridae LymphocystivirusIridovirus

Capsid protein TSXFIDXATIEKXXYGGSRXGDYXL

Papovaviridae Papillomavirus L1 protein CKYPDF/YGHPLF/YNKV/L

Polyomavirus Coat protein VP1 PDPXXNENGVGPLCKQVEEVR

Coat protein VP2 WXLPLXLGLYG

Arenaviridae Arenavirus Surface glycoprotein MLXKEYXXRQXXTPPTHXHIXGXXCPXPHRLXLXGRSC

Flaviviridae Flavivirus Non structural protein 1 CWYXMEIRP

Envelope glycoprotein DRGWGNXCGXFGKG

Adenoviridae   Hexon protein FKPYSGTAGVLAGQPNYCFPL ,NPFNHHRN

Species specific peptides Family – Flaviviridae Protein –

Envelope glycoproteins  

Virus Peptide

Unique upto -number of mismatches

St. Louis encephalitis virus VNPFISTGGAN 3EGRPAT 0

Murray valley encephalitis virus VTANPYVASSTA 3

Japanese encephalitis virus LDVRMINIEA[S/V]Q 3West Nile virus TTKATGWIIQK 3Kunjin virus STKATGRTILKE 3Langat virus DGAEAWNEAGR 3

FTCEDKK 0VGFSGTRP 0

Yellow fever virus MRVTKDTN[D/G][N/S]NL 3

Powassan virus KDNQDWNSVE 3Dengue type 1 virus GTVLVQV 0Dengue type 2 virus GTIVIRV 0Dengue type 3 virus TEATQL 0

GTILIKV 0Dengue type 4 virus TTAKEVA 0

GTTVVKV 0Tick borne encephalitis virus GFLTSVGKA 0Louping ill virus NPHWNNVER 0

VirGenComparative genomics & data

mining of viral genomes

Browse VirGen at

http://bioinfo.ernet.in/virgen/virgen.html

Salient Features of VirGen• Organizes genomic data in a structured fashion navigating from the

family to an isolate • Full genomes of viruses • Compilation of representative genome entries for every viral species

(Virus Taxonomy, 7th report of ICTV) • Complete annotation of every genomic entry  • Graphical representation of genome organization • Generation of alternative names of proteins • On-the-fly genome comparisons using BLAST2 • Multiple Sequence Alignment (MSA) of genomes, proteomes and

individual proteins • Whole genome phylogeny • Prediction of B-cell epitopes

VirGen Home

Menu to browse viral

families

Genome analysis &Comparative

genomics resourcesGuided tour

& Help

Navigation bar

Search using Keywords &

Motifs

Tabular display of genome annotation

Retrieve sequence

in FASTA format

Genome Sample Record in VirGen

‘Alternate names’ of proteins

Browsing the Module of Whole Genome Phylogenetic Trees

Most parsimonious tree of genus Flavivirus

Input data: Whole genome

Method: DNA parsimony

Bootstrapping: 1000

Most parsimonious tree of genus Flavivirus

Input data: Whole genome

Method: DNA parsimony

Bootstrapping: 1000

Case Study: Insertions in Pestivirus 1

891-1787 bp region remains unannotated using representative strain

BLAST with VirGen confirmed the non-viral origin of the insert

What is the origin of the

insert ???

BLAST with GenBank produced significant

match with Bos taurus J-domain protein

Issues

• ICTV classification and information available in published literature do not always match

• No standard method to describe viral isolates/strains • Electron micrograph and other image data are not

readily available making identification difficult and inaccurate

• Recombination occurs much faster in viruses than in bacteria/other microbes

• Host/vector information needs to be described in standard language

• Minimal availability of Immunological properties and therapeutic options in the databases

Suggestions• Devise measures to build confidence amongst underdeveloped and

developing nations that their resources will not be exploited• Networking and consortia among scientists, curators of culture

collections, policy makers from developed and developing countries• Material transfer agreements should be standardized by taking into

consideration national security and biosafety• Create awareness about open access and open educational

resources • Lobbying to policy makers to make publicly available the outcomes

of government funded research• Encouraging scientists to publish in open access journals • Organize training programs by international experts to improve

quality of culture collections and databases• Improve access to specialized culture collections

National Knowledge Commission

• The National Knowledge Commission (NKC) was constituted in 2005 as a high-level advisory body to the Prime Minister of India. The Commission has been given a mandate to guide policy and direct reforms, focusing on certain key areas such as education, science and technology, agriculture, industry, e-governance etc. Easy access to knowledge, creation and preservation of knowledge systems, dissemination of knowledge and better knowledge services are core concerns of the commission.

National Knowledge Commission

Access

Concepts

ApplicationsServices

Creation

NKC Working Model

• Identify focus areas/target groups• Consultations – formal and informal• Background research and analysis• Constitution of Working Groups• Internal deliberations of NKC• Finalization of recommendations• Submission to PM• Widespread dissemination• Implementation

Suggestions• Devise measures to build confidence amongst underdeveloped and

developing nations that their resources will not be exploited• Networking and consortia among scientists, curators of culture

collections, policy makers from developed and developing countries• Material transfer agreements should be standardized by taking into

consideration national security and biosafety• Create awareness about open access and open educational

resources • Lobbying to policy makers to make publicly available the outcomes

of government funded research• Encouraging scientists to publish in open access journals • Organize training programs by international experts to improve

quality of culture collections and databases• Improve access to specialized culture collections