Kingdom Bacteria Phylum Proteobacteria Biology Essay

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Vibrio.cholerae M66-J belongs to the family Vibrionaceae . It is a facultative motile and gram negative organism. It is free living, non sporulating and mostly found in fresh waters. It is responsible for causing cholera to humans and it is mostly found in fresh water. It is found to be closely related with enteric bacteria and some features with Pseudomonas species but is distinguished from Pseudomonas species by being both fermentative and oxidative in metabolic profiles.

While coming to the genome of V.cholerae it contains about 3938905 base pairs of genes in the entire chromosome. And they constitute about 3812 genes of which 3693 are protein coding genes and rest of them are RNA coding genes about 119. All these genes form into several clusters and perform varied functions.

So in order to know the features of this organism in detail one of the strain is taken and studied by using several bioinformatics tools that are available online. Similarly phylogenetic analysis of the organism is carried out with the help of ClustalW and found out the organism is closely related to Pseudomonas species with most of the respects.

Introduction:

Kingdom: Bacteria

Phylum: Proteobacteria

Class: Gamma proteobacteria

Order: Vibrionales

Genus: Vibrio

Species: V. cholera

Vibrio genus mainly consists of gram negative straight or curved rods motility is mainly due to flagellum. They are capable of both respiratory and fermentative metabolism and most of them are oxidase positive. They are mostly related to enteric bacteria but some share their futures with pseudomonas. But they are distinguished from pseudomonas by being fermentative as well as oxidative mechanisms in their metabolisms.

Vibrio cholera is the major agent that is responsible for causing cholera in humans as well as in most of the animals. They require simple growth factors for their growth and development they can grow in synthetic as well as in simple environment where glucose is sole carbon source for their growth and energy. They are mostly marine organisms so they require 2-3% of Nacl for their growth which are the major habitats of sea water. They live both in fresh water as well as in marine water environments and some are bioluminescent and some exhibit mutualism with other marine organisms. They show nutritional versatility because they have a capacity to grow on more than 150 different organic compounds which forms their carbon source and energy.

Two species of vibrios are mainly responsible for causing cholera in humans

V. Cholera: Non-invasive and mainly affects small intestine by the release of enterotoxins.

V.parahaemolyticus- invasive organism which mainly affects colon.

As our present study is about v.cholera which is mainly responsible for causing " Cholera" a diarrheal infection. The main process by which the transmission takes place in not yet known but is believed to be through food or water .In this diseased condition they produce an enterotoxin which mainly affects the mucosal epithelium and results in causing diarrhea.

METHODS:

In order to carry out the draft analysis of the genome V.cholerae M66-J several online tools are employed. In order to know about the background information about the organism ENTERZ database is used. It is a integrated text-based search and retrieval system used at NCBI for the several major databases such as PubMed, Nucleotide and Protein Sequences etc. (http://www.ncbi.nlm.nih.gov/sites/gquery?itool=toolbar)

In order to found out the genome statistics of the organism we go to 'Joint genome Institute-IMG' it constitutes integrated microbial genomes which serves as a community resource for comparative analysis and annotation of publicly available genomes from three domains of life and integrated them in a unique context. The IMG user interface allows users to navigate microbial date along the three dimensions (that is genes, genomes and functions) which forms the basis for comparative analysis of any organism.(http://img.jgi.doe.gov/cgi-bin/pub/main.cgi)

In order to know more about the organism restriction digestion and PFGE is carried out with the help of online server 'In silico simulation of molecular biology experiments' . This site is mainly useful for carrying theoretical experiments such as PCR analysis, restriction digestion and PFGE , PCR-RFLP etc. It collects the updated databases from NCBI and can able to perform all the experiments by computer basis. So, the results obtained forms the prerequisite for practical experiments.( http://insilico.ehu.es/ )

In order to found out the relationship between the Vibrio species and Pseudomonas species comparative genomic analysis is carried out by online curative server ClustalW. It is a general purpose multiple sequence alignment program for DNA and proteins. It is helpful to draw the evolutionary relationships of the organisms which are represented by means of Phylograms and cladograms. ( http://www.ebi.ac.uk/Tools/clustalw2/index.html )

In order to found investigate biochemical pathways, uptake mechanisms, secretion systems etc. We go for KEGG (Kyoto Encyclopedia of Genes and Genomes). It is a database which helps in computational prediction of higher-level complexity of cellular processes and organism behaviors using genomic and molecular information. (http://www.genome.jp/kegg/)

Then we go for BLASTP by comparing the Vibrio species with Pseudomonas species by using 'Joint genome Institute-IMG' database. .(http://img.jgi.doe.gov/cgi-bin/pub/main.cgi)

Then we go for more deeper comparison by using WEBACT "|which

provides a database of sequence comparisons between all publicly available prokaryotic genome sequences, allowing the on-line visualisation of comparisons between up to five genomic sequences, using the Artemis Comparison Tool (ACT) developed by the Sanger Institute". ( http://www.webact.org/WebACT/home )

In order to more about the features of the organism we then go for Pub Med publications about the organism and found out several interesting thing about our organism. (http://www.ncbi.nlm.nih.gov/pubmed/ )

RESULTS AND DISCUSSION:

Our present study is regarding vibrio cholera M66-2 strain whose genome contains a total of 3938950(100%) base pairs of which 3514719(89.23%) are DNA coding for number of bases and 1875724(47.62%) are DNA G+C number of bases.

The total number of genes present on this organism are 3812 of which 3693 are protein coding genes and no pseudo genes found in this organism 119 are RNA genes of which 8 genes code for 16s rRNA. TRNA comprises of 97 genes. Protein coding genes with function predicted are about 2413 genes and without function prediction are 1280 genes and genes without function prediction and with similarity are about 1280 and there are no genes without similarity. Along with these genes there are several other genes which are mainly related with KEGG pathways, metacyclic pathways, genes coding for Swissprot protein products, genes coding for enzymes, genes connected with seed, proteins coding genes for COGs , protein coding genes for ortholog clusters, fused protein genes, genes coding for chromosomal cassettes, proteins coding genes for fusion components, signal peptides and transmenbrane proteins etc.

There are several other strains in Vibrio cholera which is also responsible for causing cholera. They are :

V.cholera MJ-1236.

V.cholera 01 biovar E1 Tor str N16961.

V.cholera 0395.

This MJ-1236 strain contains about 4236368 base pairs of which 3700593 are DNA coding number of bases and 2004640 are DNA G+C number of base pairs. They contain about 3894 protein coding genes of which 120 are RNA coding genes in which 7 genes code for 16s rRNA. They also contain all the genes that are responsible for various functions in M66-J strain.

V.cholera o1 biovar E1 Tor str N16961 contains about 4033429 base pairs of which 3564102 are DNA coding number of bases and 1915376 are DNA G+C number of base pairs. They contain about 3835 protein coding genes of which 163 are RNA coding genes in which 9 genes code for 16s rRNA. They also contain all the genes that are responsible for various functions in M66-J strain.

Similarly V.cholera 0395 contains about 4132319 bsae pairs of which 3683791 are DNA coding number of bases and 1964499 are DNA G+C number of base pairs. They contain about 3876 protein coding genes along with one pseudo gene of which 155 are RNA coding genes in which 8 genes code for 16s rRNA. They also contain all the genes that are responsible for various functions in M66-J strain.

By studying the above examples we noticed that lot of homology exists between various strains of V.cholera particularly while observing the 16s rRNA sequence of these strains.

M66-J strains contains about 7 genes which are coding for 16s rRNA these genes are located on the following chromosomes.

Gene object id: 643812214

Locus tag: VCM66_16S01

DNA coordinates: 53787..55321(+)

Gene location: Chromosome 1

Gene object id: 643812318

Locus tag: VCM66_16S02

DNA coordinates: 151030..152564 (+)

Gene location: Chromosome 1

Gene object id: 643812464

Locus tag: VCM66_16S03

DNA coordinates: 308873..310407 (+)

Gene location: Chromosome 1

Gene object id: 643812555

Locus tag: VCM66_16S04

DNA coordinates: 386478..388012 (+)

Gene location: Chromosome 1

Gene object id: 643812874

Locus tag: VCM66_16S05

DNA coordinates: 720224..721758 (+)

Gene location: Chromosome 1

6) Gene object id: 643814639

Locus tag: VCM66_16S06

DNA coordinates: 2616922..2618456 (-)

Gene location: Chromosome 1

7) Gene object id: 643814898

Locus tag: VCM66_16S07

DNA coordinates: 2868843..2870377 (-)

Gene location: Chromosome 1

In order to know more about V.cholera Pulse field gel electrophoresis is carried out along with restriction digestion with the help of online server. At first the genome of the organism is cut with EcoRII which cuts the sequence at CCWGG_ and generate about 1902 fragments which are further subjected to PFGE. We also noticed that when the genome is digested by AjnI ,Psp6I,PspGI similar kinds of bands are appeared. (We noticed that PFGE and restriction digestion is a useful tool for molecular typing of various gram positive and gram negative bacteria mainly during endemic conditions.)

As mentioned above that vibrio species are closely related with Psedomonas species so in order to prove that comparative genomic analysis is carried by using ClustalW which is online bioinformatics tool. It is a general purpose multiple sequence alignment program for DNA and protein sequences

It basically calculates the best match for the selected sequences, and also aligns them up so that the identities, similarities and differences can be seen. And Evolutionary relationships can be seen with the help of Cladograms or Phylograms.

So comparative genomic analysis is carried out by taking 16s rRNA sequences of one of Vibrio species and Psudomonas species because 16s rRNA sequence is more stable sequence during evolution that is it does not undergo rapid changes hence it is very useful for knowing evolutionary relationships of most of the organisms. Comparison is made between 16s rRNA of Vibrio. Cholera M66-2 and 16s r RNA of Pseudomonas putida F1 . The results show that both Pseudomonas and Vibrio species are closely related.

In order to determine the relationship between pseudomonas species and vibrio species more analysis is carried out by using BLASTP. The results obtained shows that about 12 hits for only one gene that is 'OmpA' gene. These results are different from one which we got by NCBI BLAST because here we are considering only one gene sequence for BLAST but in case of NCBI BLAST entire genome of the organism is considered. So here we are increasing the accuracy of our comparison with other organism. So the obtained results are more appropriate.

In order to know about the biochemical pathways of the organism We used online database known as KEGG (Kyoto Encyclopedia of genes and genomes) which stores large databases regarding several biochemical pathways of various organisms. From this KEGG database we can able to understand the pathways that leads to the cause of cholera in humans.

"Main virulence factor for causing cholera is Cholera toxin (CTX) . Once secreted, CTX B-chain (CTXB) binds to ganglioside GM1 on the surface of the host's cells. After binding takes place, the entire CTX complex is carried from plasma membrane (PM) to endoplasmic reticulum (ER). In the ER, the A-chain (CTXA) is recognized by protein disulfide isomerase (PDI), unfolded, and delivered to the membrane where the membrane-associated ER-oxidase, Ero1, oxidizes PDI to release the CTXA into the protein-conducting channel, Sec61. CTXA is then retro-translocated to the cytosol and induces water and electrolyte secretion by increasing cAMP levels via adenylate cyclase (AC) to exert toxicity". These organisms also secret several other toxins which causes deleterious effects to perilous and eukaryotic cells.

In order to know the functions of various genes in V.cholera M66-J we found out selected genes in the chromosome 1 of the V.cholera M66-J and we can able to found out the exact gene location as well as the function of that gene in the chromosome.

OmpA : It is between 22299338 nt and 2306337nt and it acts as a outer membrane protein.

Then we go for sequence comparison using WEBACT . we compared vibrio cholera (serovar O1 E1 Tor str N16961) with Pseudomonas putida ( strain KT 2440). We found out so many interesting things such as horizontal transfer of the genes between the two species at 247200 and 248000 of V. Cholera and 1457600 and 1459200 of P. putida. And we also found out that most of the genes are missing in V. Cholera. G+C content of the both strains shows several variations for example at 148800 nt the G+C content of V.cholera is very low. Similarly at 1367200 the G+C content of P.putida is very low. We can also able to found out the lengths of two genomes and we noticed that the length of Pseudomonas genome is more.

So, from the above obtained results we noticed that what are the general features of the organism and genome statistics of the organism and by doing comparative genomic analysis we can able to predict the evolutionary relationship of the organisms and also able to know the closely related species of that organism which are represented in the form of phylograms and cladograms. By using Insilico online database we can able to know the restriction digestion and PFGE bands of that organism. We can also able to analyse the genome sequence of the desired organism in depth. We can also able to predict the biochemical pathways carried out by that organism and also able to predict the genes which are responsible for carrying out particular biochemical pathway. Thereby we can able to know the function of a particular gene. BLAST results are useful to predict the relatedness of one species with the other species by means of number of hits. By comparing the sequences of the organisms in WEBACT we can able to predict which genes are horizontally transferred between two genomes and we can also notice the variations in G+C content of both the organisms we can also able to predict the missing genes between the two organisms and we can also able to notice the length of the genomes. With the help of PubMed search we can able to know more about the organism that is work carried by several scientists.

  

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