Integration Of Genetic Elements Research Articles

Genetic characterization of non-O1/non-O139 <i>Vibrio cholerae</i> mobilome: a strategy for understanding and discriminating emerging environmental bacterial strains

Acute diarrhea and cholera (AWD/C) result in more than 21000 to 143000 global mortality annually and are associated with Vibrio cholerae. The pathogen has shown increasing evolutionary/emerging dynamics linked with mobilome or ubiquitous nature of mobile integrative genetic and conjugative elements (MIGCE), however, such dynamics are rarely reported amongst somatic-antigen non-agglutinating Type-1/-139 V. cholerae (SA-NAG-T-1/139Vc). The study reports the genetic detection of mobilome-associated indices in SA-NAG-T-1/139Vc as a potential strategy for differentiating/discriminating emerging environmental bacteria. Presumptive V. cholerae isolates were retrieved from five water sources, while strains were characterized/serogrouped and confirmed using simplex and comparative-genomic-multiplex Polymerase Chain Reaction (PCR). Genomic island (GI-12det, GI-14det, GI-15det); Phages (TLC-phagedet, Kappa-phagedet) and ICEs of the SXT/R391 family genes (SXT/R391-ICEs integrase, SXT-Hotspot-IV, ICEVchInd5Hotspot-IV, ICEVchMoz10Hotspot-IV) were detected. Other rare ICE members such as the ICEVcBan8att gene and Vibrio Seventh Pandemic island detection (VSP-II Integrase, Prototypical VSP-II) were also detected. Results revealed that the 8.22% (61/742) SA-NAG-T-1/139Vc serogroup observed harbors the Vibrio Seventh Pandemic island integrase (34/61; 55.7%) and other rare genetic traits including; attB/attP (29/61; 47.5%, 14/61; 23%), integrative genetic elements (4/61; 6.56%), phage types (TLC-phagedet: 2/61; 3.28% and Kappa-phagedet: 7/61; 11.48%) as well as the integrase genes (INT1, Sul1, Sul2) (29/61: 47.5%; 21/61: 34.4%; 25/61: 41%). Such genetic detection of mobilome determinants/MIGCE suggests potential discriminatory tendencies amongst SA-NAG-T-1/139Vcwhich may be applied in mobilome typing of evolving/emerging environmental bacteria. The need to encourage the application of such mobilome typing indices and continuous study of these strains is suggestive of interest in controlling future potential emerging environmental strains.

On distinguishing between canonical tRNA genes and tRNA gene fragments in prokaryotes

ABSTRACT Automated genome annotation is essential for extracting biological information from sequence data. The identification and annotation of tRNA genes is frequently performed by the software package tRNAscan-SE, the output of which is listed for selected genomes in the Genomic tRNA database (GtRNAdb). Here, we highlight a pervasive error in prokaryotic tRNA gene sets on GtRNAdb: the mis-categorization of partial, non-canonical tRNA genes as standard, canonical tRNA genes. Firstly, we demonstrate the issue using the tRNA gene sets of 20 organisms from the archaeal taxon Thermococcaceae. According to GtRNAdb, these organisms collectively deviate from the expected set of tRNA genes in 15 instances, including the listing of eleven putative canonical tRNA genes. However, after detailed manual annotation, only one of these eleven remains; the others are either partial, non-canonical tRNA genes resulting from the integration of genetic elements or CRISPR-Cas activity (seven instances), or attributable to ambiguities in input sequences (three instances). Secondly, we show that similar examples of the mis-categorization of predicted tRNA sequences occur throughout the prokaryotic sections of GtRNAdb. While both canonical and non-canonical prokaryotic tRNA gene sequences identified by tRNAscan-SE are biologically interesting, the challenge of reliably distinguishing between them remains. We recommend employing a combination of (i) screening input sequences for the genetic elements typically associated with non-canonical tRNA genes, and ambiguities, (ii) activating the tRNAscan-SE automated pseudogene detection function, and (iii) scrutinizing predicted tRNA genes with low isotype scores. These measures greatly reduce manual annotation efforts, and lead to improved prokaryotic tRNA gene set predictions.

New candidates for regulated gene integrity revealed through precise mapping of integrative genetic elements.

Integrative genetic elements (IGEs) are mobile multigene DNA units that integrate into and excise from host bacterial genomes. Each IGE usually targets a specific site within a conserved host gene, integrating in a manner that preserves target gene function. However, a small number of bacterial genes are known to be inactivated upon IGE integration and reactivated upon excision, regulating phenotypes of virulence, mutation rate, and terminal differentiation in multicellular bacteria. The list of regulated gene integrity (RGI) cases has been slow-growing because IGEs have been challenging to precisely and comprehensively locate in genomes. We present software (TIGER) that maps IGEs with unprecedented precision and without attB site bias. TIGER uses a comparative genomic, ping-pong BLAST approach, based on the principle that the IGE integration module (i.e. its int-attP region) is cohesive. The resultant IGEs from 2168 genomes, along with integrase phylogenetic analysis and gene inactivation tests, revealed 19 new cases of genes whose integrity is regulated by IGEs (including dut, eccCa1, gntT, hrpB, merA, ompN, prkA, tqsA, traG, yifB, yfaT and ynfE), as well as recovering previously known cases (in sigK, spsM, comK, mlrA and hlb genes). It also recovered known clades of site-promiscuous integrases and identified possible new ones.

Dichotomy in the evolution of pathogenicity island and bacteriophage encoded integrases from pathogenic Escherichia coli strains

The evolutionary histories of mobile and integrative genetic elements (MIGEs), key factors in the emergence of pathogenic bacteria, remain obscure due to their widespread distribution and diversity in gene content. Pathogenicity islands (PAIs) are large chromosomal regions that encode bacterial virulence factors present in pathogenic isolates and absent from non-pathogenic isolates. We examined the utility of PAI-encoded integrases as a marker to determine PAI phylogeny and evolutionary relationships to other MIGEs particularly bacteriophages. We examined 68 tyrosine recombinase (TR) integrases from 27 PAIs and 39 phages from five fully sequenced pathogenic Escherichia coli strains in the database. In general, all PAI regions identified were integrated adjacent to tRNA loci and had a percent GC content that differed from the host chromosome, which was not the case for most phages examined. Our phylogenetic analysis demonstrated that PAI-encoded integrases clustered within a distinct lineage, separate from phage-encoded integrases, which suggested a discrete evolutionary history. The tree branch lengths among PAI integrases were shorter than those among phage integrases suggesting that PAIs may be a more recent addition to E. coli. Further phylogenetic comparisons of these 68 integrases with 53 TR integrases from other characterized MIGEs also demonstrated that PAI-encode integrases form a distinct lineage, suggesting that these PAIs in E. coli are not remnants of other MIGEs. We identified recombination directionality factors/excisionases in the proximity of many of the E. coli PAI integrases, factors that were previously not shown to be present in E. coli PAIs. Overall this work is the first to demonstrate a dichotomy in the evolution of integrases encoded on PAIs and phages from pathogenic E. coli suggesting that PAIs are an evolutionary distinct genetic element.

Integrative and Sequence Characteristics of a Novel Genetic Element, ICE 6013 , in Staphylococcus aureus

A survey of chromosomal variation in the ST239 clonal group of methicillin-resistant Staphylococcus aureus (MRSA) revealed a novel genetic element, ICE6013. The element is 13,354 bp in length, excluding a 6,551-bp Tn552 insertion. ICE6013 is flanked by 3-bp direct repeats and is demarcated by 8-bp imperfect inverted repeats. The element was present in 6 of 15 genome-sequenced S. aureus strains, and it was detected using genetic markers in 19 of 44 diverse MRSA and methicillin-susceptible strains and in all 111 ST239 strains tested. Low integration site specificity was discerned. Multiple chromosomal copies and the presence of extrachromosomal circular forms of ICE6013 were detected in various strains. The circular forms included 3-bp coupling sequences, located between the 8-bp ends of the element, that corresponded to the 3-bp direct repeats flanking the chromosomal forms. ICE6013 is predicted to encode 15 open reading frames, including an IS30-like DDE transposase in place of a Tyr/Ser recombinase and homologs of gram-positive bacterial conjugation components. Further sequence analyses indicated that ICE6013 is more closely related to ICEBs1 from Bacillus subtilis than to the only other potential integrative conjugative element known from S. aureus, Tn5801. Evidence of recombination between ICE6013 elements is also presented. In summary, ICE6013 is the first member of a new family of active, integrative genetic elements that are widely dispersed within S. aureus strains.

Metabolic Diversity in Bacterial Degradation of Aromatic Compounds

Aromatic compounds pose a major threat to the environment, being mutagenic, carcinogenic, and recalcitrant. Microbes, however, have evolved the ability to utilize these highly reduced and recalcitrant compounds as a potential source of carbon and energy. Aerobic degradation of aromatics is initiated by oxidizing the aromatic ring, making them more susceptible to cleavage by ring-cleaving dioxygenases. A preponderance of aromatic degradation genes on plasmids, transposons, and integrative genetic elements (and their shuffling through horizontal gene transfer) have lead to the evolution of novel aromatic degradative pathways. This enables the microorganisms to utilize a multitude of aromatics via common routes of degradation leading to metabolic diversity. In this review, we emphasize the exquisiteness and relevance of bacterial degradation of aromatics, interlinked degradative pathways, genetic and metabolic regulation, carbon source preference, and biosurfactant production. We have also explored the avenue of metagenomics, which opens doors to a plethora of uncultured and uncharted microbial genetics and metabolism that can be used effectively for bioremediation.