Insights into the Origin of Clostridium botulinum Strains: Evolution of Distinct Restriction Endonuclease Sites in rrs (16S rRNA gene)

Abstract

Diversity analysis of Clostridium botulinum strains is complicated by high microheterogeneity caused by the presence of 9-22 copies of rrs (16S rRNA gene). The need is to mine genetic markers to identify very closely related strains. Multiple alignments of the nucleotide sequences of the 212 rrs of 13 C. botulinum strains revealed intra- and inter-genomic heterogeneity. Low intragenomic heterogeneity in rrs was evident in strains 230613, Alaska E43, Okra, Eklund 17B, Langeland, 657, Kyoto, BKT015925, and Loch Maree. The most heterogenous rrs sequences were those of C. botulinum strains ATCC 19397, Hall, H04402065, and ATCC 3502. In silico restriction mapping of these rrs sequences was observable with 137 type II Restriction endonucleases (REs). Nucleotide changes (NC) at these RE sites resulted in appearance of distinct and additional sites, and loss in certain others. De novo appearances of RE sites due to NC were recorded at different positions in rrs gene. A nucleotide transition A>G in rrs of C. botulinum Loch Maree and 657 resulted in the generation of 4 and 10 distinct RE sites, respectively. Transitions A>G, G>A, and T>C led to the loss of RE sites. A perusal of the entire NC and in silico RE mapping of rrs of C. botulinum strains provided insights into their evolution. Segregation of strains on the basis of RE digestion patterns of rrs was validated by the cladistic analysis involving six house keeping genes: dnaN, gyrB, metG, prfA, pyrG, and Rho.

Potential Emergence of Multi-quorum Sensing Inhibitor Resistant (MQSIR) Bacteria

“…Clostridium has a broad range of biotechnological applications, but can cause illness and deadly diseases like pneumonia, bacteremia, botulism, myonecrosis, and tetanus [21][22][23]. Clostridium are noted to contain either single or multiple QSS, such as: (1) LuxS, (2) Agr, (3) Agr2 that are regulated by peptide signals e.g., AIP ( Table 1) [24][25][26][27][28][29].…”

A Genome-Wide Profiling Strategy as an Aid for Searching Unique Identification Biomarkers for Streptococcus

“…Among the various genes based identification methods, the most widely employed has been the usage of rrs gene [12][13][14][15][16]. It has proved instrumental in bacterial identification; however, the major difficulty encountered is in the cases where the organism has multiple copies of rrs within the genome [13,14,[18][19][20].…”

“…Although, recA is one of those genes which are used widely for identification of Streptococcus [27], however, our analysis revealed that it is not among the best candidates, which can be exploited for Table 5 continued detection of Streptococcus infections especially in the cases of S. mutans (2 strains), S. pneumoniae (20 strains), S. pyogenes (3/7 strains), and S. suis (5/9 strains) (Table 5a, b). In the similar manner, analysis of gyrB gene, commonly used as biomarker for identification in general [13], was also not very effective as the unique RE patterns could not be deduced in the following cases: S. pneumoniae (18/20 strains), S. pyogenes (4/7 strains), and S. suis (6/9 strains) (Table 6a, b).…”

“…: S. pneumoniae, S. pseudopneumoniae, S. mitis, and S. oralis show more than 99 % similarity leading to unsuccessful or misleading results [10]. Recent studies have elucidated certain latent but unique characteristics in rrs: (1) 30-50 nucleotides (nts) long signatures, and (2) restriction endonuclease (RE) digestion patterns [12][13][14][15][16]. These features enable easy identification of organisms up to the species level.…”

“…However, rrs gene does not prove helpful in the following scenarios: (1) in phylogenetically very closely related strains, which differ in less than 1 % nts along their length, and (2) in strains with multiple copies of this gene in each genome [17][18][19][20]. An obvious solution to circumvent this problem is to use other HKGs [13,21]. Streptococcus poses a big challenge as most pathogenic strains are genetically very close to each other and have multiple copies of rrs per genome.…”