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Computational Genomics in Mycobacterium Colombiense
The genus Mycobacterium consists of nearly one hundred and fifty species. The species
include some human pathogens that present major challenges to public health. Mycobacterium
colombiense is an ureasepositive, slow-growing and nontuberculous mycobacterium (NTM). It
belongs to the Mycobacterium avium complex (MAC). M. colombiense was first isolated from
some HIV positive individuals (Gonzarez-Perez, 2011). The patients were determined to
represent a distinct species as a result of sequence comparisons with other closely related
Mycobacterium species. Since its discovery, M. colombiense was found to cause disemminated
infection in immunocompromised HIV patients, lymhpadenopathy in immunocompetentt
children and respiratory diseases. The complete genome sequence of M. colombiense is
characterized in the subsequent pages in an effort to understand its virulence mechanisms.
Members of MAC had forty five mycobacterial strains. Molecular approaches of a
different kind were used. Some isolates cases, totaling to seven displayed attributes that allowed
differentiation from the other members in the complex. The isolates had novel number 16S-23S
Rrna internal transcriber spacer (ITS 1) sequence. The sequence was described in a new
sequaver, MAC-X. All the seven isolates showed positive results with the MAC-specific
AccuProbe (Gen-Probe). However, they tested negative for Mycobacterium intracellulare and
Mycobacterium avium species. A phenotypical approach is used to differentiate the novel
isolates from other MAC members based on production of urease as well as a steady mycolic
acid pattern.
Strain Type for Mycobacterium Colombiense
The novel isolates shared a few characteristics with Mycobacterium avium. These
characteristics included avium variant I (av-I) the model of the hsp65 gene as described by PCR
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restriction analysis and positive PCR result for the macrophage-induced (mig) gene. Unique 16S
Rrna gene sequence was showed by novel isolates. The novel isolates are said to represent a
novel species, Mycobacterium colombiense sp. nov., which is related closely to M. avium in the
MAC. This strain type for M. colombiense is 10BT (= CIP 108962T = CECT 3035T). The strains
of M. Colombiense were first obtained in Colombia in South America. Its cells are acid-fast nonmotile rods (Gonzarez-Perez, 2011). Their growth occur between a temperature of 20 0C and 37
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C. It shows negative tests in niacin, acid phosphate activity and Tween hydrolysis. Its ability to
produce urease makes it easy to be differentiated from the other members of MAC.
The abundance of bacteria and the wide range of environment they can inhabit make
them most successful form of life on earth. Numerous unicellular forms known as protists exist
among the eukaryotes. Most of them are marine organisms. Ultrastructural and molecular data
show that different types of protists differ from each other compared to the differences between
plants and animals. However, they are members of a kingdom called Protista. Fungi, plants and
animals are the major multicellular groups. Mycobacterium colombiense is an anaerobic
bacterium that is found in the lower intestine of warm blooded animals (Kislyuk et al., 2010). It
belongs to the genus Colombiense. Most strains of M. colombiense harmless but other strains can
cause food poisoning in their hosts. A part of normal flora of the gut is the harmless strains. They
can also benefit their hosts by producing vitamins such as K2. They also prevent colonization of
the intestine with bacteria that is pathogenic. M. colombiense does not contain crystal violet.
Phenotic Identification: Methods of assembling Gene of M. Colombiense
Samples are isolated using a biphasic medium. Iron citrate was used as the solid phase
and Sauton Tween-albumin medium as the liquid phase. The isolates were unable to grow on
Sauton agar that is supplemented with picric acid. Some biochemical assets were carried out.
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Variability results were found when seven isolates were compared. All the isolates were positive
for urease activity. Urease was the main biochemical agent that enabled the novel strains to
distinguish from the rest of the members of MAC (Lemay et al., 2006). HPLC analysis was
carried out by employing a standard method on a system Gold instrument that is equipped with
an XL Ultrashphere column. The chromatographic patterns were compared with the HPLC
library and the profiles of the species that belong to MAC. Analysis of mycolic acids confirmed
three clustered pattern typical strains of M. avium and M. colombiense. In the novel isolates, the
peaks of the first and second clusters differed from the bell-shaped appearance that is borne of
the members of MAC.
Genomic Project Input: Gene sequencing
Sequencing system software is used for gene sequencing. Sequencing software GS De
Novo is used to record DNA elements of living organism. It constructs assemblies of reads from
one or more sequencing runs by use of read flow grams as input (SSSM, 2011). It creates
assembly projects, remove and add reads from a project and specify parameters. In addition, it
runs the algorithms of assemblies on the project data and view output that assembly
computations produce. A Graphical User Interface (GUI) is used to access the application. Input
data come from several regions of one or several runs of interest. External file formats such
FASTA and FASTQ are used to import additional read data. Some of the roles the software
performs during the assembly process include identifying pair wise overlaps that are between the
reads, solving branching structures that are between the contigs. The software generates
consensus base calls of the contigs.
Multiple alignments of reads and divides also use the software application. It can perform
extra steps with the availability of paired data. Multiple alignments of overlapping read
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sequences are produced by the assembler. The GUI provides tools that facilitate the contig
sequences as well as the multiple reads that from the contig. There is an interactive view of the
flow grams of the reads. The GS Novo assembler enables users to modify, run and create
assemblies in the form of projects (SSSM, 2011). The functionality is provided both the GUI and
the command line interface (CLI). Projects may be established to gather all reads at once. An
alternative to this can be incremental operation that allows additional reads to be added to an
existing assembly (Lemay et al., 2006). The results then appear as output files using GUI or the
CLI. GUI provides a graphical interface to view many of the results from assembly irrespective
of whether the GUI or the CLI assembled the project.
Assembler application uses a folder on the file system to carry the project information
regarding assembly. For example, whether GUI or new assembly and related commands carry
out computation of the assembly of reads, the computation of reads must be carried out.
Graphical user interface application and gsAssembler can perform of view assemblies (Kislyuk
et al., 2010). The graphical interfaces can be used to open existing assembly projects, carry out
computation of an assembly, viewing the results of a completed assembly, add/remove read to
and fro assembly projects. They also modify assembly input, output parameters and view the
progress of any information required to start an assembly computation. By double clicking its
desktop button, the GS De Novo launches. It can alternatively be launched by gsAssembler and
once it launches, the user can open an existing project to create another project.
There are seven main buttons on the toolbar, along main window of the GS De Novo
Assembler. Use the exit button to close the GS De Novo assembler application. The New button
enables one to create a new assembly project. The Open button enables one an existing assembly
project. The Start button begins the computation of the assembly of an assembly project that is
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open. The Stop button halts the execution of an assembly computation that is on-going. The
About button shows the GS De Novo assembler splash screen while the Help button opens the
GS De Novo assembler section in the manual of the software.
Gene Prediction: Glimmer
Glimmer is a set of programs used for identifying genes in microbial DNA sequences. It
works by creating a variable length model from a training set of genes. The model is then used to
in identifying genes in a given DNA sequence. The latest version of glimmer used is version 3.
Glimmer3 can process multiple sequence input files. A faster header line of the sequence
precedes the outputs of the sequence. An interpolated context model is a probability model of
coding sequences and must first be built. The sequence can perform some programs. Known
genes in the genome can be identified by homology searches. Non-overlapping orfs in the
genome are produced by program long-orfs. Glimmer3 program itself to run and analyze the
sources and make gene predictions.
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Flow Chart of Genome Annotation
Genome Sequence
Prediction of structural Features
General Database Research
Statistical Gene Prediction
Gene/ Protein/ RNA set
Database Research
Prediction Gene Function
Prediction Gene Function
Context Analysis
Genome Comparison
References
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Cristianini, Nello, and Matthew W. Hahn. Introduction to computational genomics: a case
studies approach. Cambridge University Press 2006.
Gonzalez-Perez, M. “Genome sequence of the Mycobacterium colombiense type strain,
CECT 3035.”, J Bacteriol,;193 2011. (20):5866-7
Kislyuk, Andrey O., et al. “A computational genomics pipeline for prokaryotic sequencing
projects.” Bioinformatics 26.15 (2010): 1819-1826.
Lemay, Danielle G., and Daniel H. Hwang. (2006). “Genome-wide identification of peroxisome
proliferator response elements using integrated computational genomics.” Journal of lipid
research 47.7: 1583-1587.
454 Sequencing System Software Manual. GS De Novo Assembler, GS reference
mapper, SFF Tools. Version 2.6. (2011)
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