Author: Vipin Chandra Kalia
Introduction
Clostridium species are well known for their biotechnological applications: (i) solvents, (ii) biofuels, and (iii) therapeutic and cosmetics, etc. They are among the most dreaded by human beings. Clostridium strains are known to produce deadly toxins responsible for diseases like botulism, tetanus, gas gangrene, pseudomembranous colitis, etc. Cheese industry is one of those, which live in constant fear of getting infected by Clostridium strains. It is important, urgent and imperative to identify the bacteria responsible for causing disease, spoiling food, infecting wounds, before a treatment can be prescribed and implemented.
Diversity of Clostridium
The genus encompasses around 110 species with a G+C content ranging from 24 mol% in Clostridium perfringens at one extreme to 58 mol% in Clostridium barkeri at the other end of the spectrum. Biochemical methods for identifying Clostridium have been holding for quite some time. Things changed dramatically with the discovery of Molecular Biology tools, which revolutionized gene and genome sequencing.
Gene level identification
Bacterial identification has been employing the highly conserved gene: rrs. Individual researchers have been submitting partial and completely sequenced rrs genes to Ribosomal Data Base (RDP database - http://rdp.cme.msu.edu/). The number of rrs gene sequences submitted to RDP is increasing rapidly: RDP Release 11.4 - 3,224,600 seq. The taxonomical classification of Clostridium up to species level using rrs has been running into trouble due to: (i) Very high similarity between two sequences, and (ii) Presence of multiple copies per genome. Consequently, mostClostridium strains have been identified only up to genus level. High heterogeneity in the phylogenetic distribution of strains on the basis ofrrs has been recorded for: C. botulinum, C. perfringens, C. butyricum, C. acetobutylicum, C. beijerinckii, C. novyi, C. kluyveri, etc.
In case rrs based analysis is not proving helpful, scientist generally resort to other genes: (i) Heat shock proteins: hsp65 andhsp70, (ii) ATPase-ß-subunit, (iii) RNA polymerases, (iv) recombinase (recA). In addition, rpoB is used for identifying Mycobacterium, gyrB for Shewanella, Mycobacterium, Pseudomonas, and Acinetobacter, (v) gyrA for defining Bacillus subtilis and related taxa. At times up to 8 genes are needed to be sequenced, if there is no consensus gene specific to a strain.
Genomic tools to decipher novel features
• Single gene
• Whole genome
Single gene analysis
A novel approach has been used to find out the hidden features of rrs gene sequences of different isolates of Clostridium.
- Phylogenetic framework
- Restriction Endonuclease digestion pattern
- Unique signatures
Genome wide analysis
In case of the presence of multiple copies of rrs / genome, one needs to completely sequence all the copies of this gene. However, this exercise does not resolve the problem, because some of the rrs copies from different Clostridium species show 100% similarity. There is thus the risk of mislabeling of strains and overestimation of genetic diversity. Here, whole genome sequence comparisons can help to detect novel marker genes. The strategy employed to find species specific gene features was: (i) to find out genes, common to almost all the genomes and (ii) identify unique RE digestion patterns.
Combinations of genes and their unique RE digestion patterns: recN, dnaJ, secA, mutS, grpE -AluI, BfaI, Tru9I, can help in rapid identification of at least 9 Clostridium species.
Significance
- Hidden features of the rrs gene can be used for bacterial identification.
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About Author / Additional Info:
Researchers in Microbial Biotechnology and Genomics at CSIR-IGIB, Delhi.