Comment on “the IS6 family, a clinically important group of insertion sequences including IS26” by Varani and co-authors

The insertion sequence IS26 has long been known to play a major role in the recruitment of antibiotic resistance genes into the mobile resistance gene pool of Gram-negative bacteria and IS26 also plays a major role in their subsequent broad dissemination. Related IS, IS431/257 and IS1216 are important in the same roles in Gram positive bacteria. However, until recently the properties of IS26 movement that could potentially explain this ability had not been explored. A much needed insight has come from our recent demonstration that IS26 uses a novel targeted mechanism that is conservative. The targeted conservative mechanism is much more efficient than the known replicative mechanism, which is now more accurately called copy-in. A recent review “The IS6 family, a clinically important group of insertion sequences including IS26” by Varani, He, Siguier, Ross and Chandler published in Mobile DNA has substantially misrepresented the recent studies on the targeted conservative mechanism and at the same time incorrectly implied that any mechanism established for IS26 can be assumed to apply to a range of IS that are at best very distantly related. A few of the most important issues are examined in this comment. Readers are advised to consult the original literature to check facts before drawing firm conclusions.

Scarless excision of an insertion sequence restores capsule production and virulence in Acinetobacter baumannii

We identify a new mechanism mediating capsule production and virulence in the WHO and CDC priority ESKAPE pathogen Acinetobacter baumannii. Non-capsulated and avirulent bacteria can revert into a capsulated and virulent state upon scarless excision of an ISAba13 insertion sequence under stress conditions. Reversion events fully restore capsule production and in vivo virulence. This increases our knowledge about A. baumannii genome dynamics, and the regulation of capsule production, virulence and resistance.

Evaluation of the occurrence of genetic determinants of multi-drug resistance among Acinetobacter baumannii strains

Introduction: The aim of the study was the analysis of occurrence of genetic determinants of multi-drug resistance and the assessment of genetic relationship among Acinetobacter baumannii strains. Methods: Multiplex-PCR method was performed in order to: (1) confirm the phenotypic identification and (2) detect the presence of CHDL oxacillinases in the group of thirty A.baumannii strains. Further PCR studies included the analysis of the occurrence of genetic determinants associated with efflux pump, insertion sequence and biofilm formation. The relationship between bacterial strains was assayed using 6 primers in RAPD-PCR method. Results: Detection of the blaOXA-51-like gene confirmed that the strains belong to the A. baumannii species. In the multiplex-PCR, the presence of the blaOXA-23-like and blaOXA-40-like genes was detected in 3 (10%) and 27 (90%) isolates, respectively. Moreover, some strains showed the coexistence of the blaOXA-51-like and blaOXA-23-like genes (10%, n=3) or blaOXA-51-like and blaOXA-40-like (90%, n=27). In the group of analysed strains the presence of the efflux pump gene (adeA) and the insertion sequence ISAba1 were demonstrated in all tested isolates. Biofilm-related genes (abaI, csuE) were found in 100% and 97% (n=29) tested strains adequately. The RAPD-PCR studies revealed the presence of 10 unrelated genotypes. Conclusions: The obtained results suggest that the phenomenon of multi-drug resistance in the studied A. baumannii strains could be attributed to the occurrence of CHDL oxacillinases, AdeABC efflux pump, insertion sequence ISAba1 and the biofilm formation.

Occurrence of Pseudomonas spp. in Raw Vegetables: Molecular and Phenotypical Analysis of Their Antimicrobial Resistance and Virulence-Related Traits

Pseudomonas is characterized by its great capacity to colonize different ecological niches, but also by its antimicrobial resistance and pathogenicity, causing human, animal, or plant diseases. Raw and undercooked food is a potential carrier of foodborne disease. The aim of this study was to determine the occurrence of Pseudomonas spp. among raw vegetables, analysing their antimicrobial resistance, virulence, and molecular typing. A total of 163 Pseudomonas spp. isolates (12 different species) were recovered from 77 of the 145 analysed samples (53.1%) and were classified into 139 different pulsed-field gel electrophoresis patterns. Low antimicrobial resistance levels, but one multidrug-resistant isolate, were found. Among the 37 recovered P. aeruginosa strains, 28 sequence-types and nine serotypes were detected. Eleven OprD patterns and an insertion sequence (ISPa1635) truncating the oprD gene of one imipenem-resistant strain were found. Ten virulotypes were observed, including four exoU-positive and thirty-one exoS-positive strains. The lasR gene was absent in three ST155 strains and was truncated by different insertion sequences (ISPre2, IS1411, and ISPst7) in other three strains. High biofilm, motility, pigment, elastase, and rhamnolipid production were detected. Our study demonstrated a low occurrence of P. aeruginosa (18%) and low antimicrobial resistance, but a high number of virulence-related traits in these P. aeruginosa strains, highlighting their pathological importance.

Predicting efficiency of writing short sequences into the genome using prime editing

Any short sequence can be precisely written into a selected genomic target using prime editing. This ability facilitates protein tagging, correction of pathogenic deletions, and many other exciting applications. However, it remains unclear what types of sequences prime editors can efficiently insert, and how to choose optimal reagents for a desired outcome. To characterize features that influence insertion efficiency, we designed a library of 2,666 sequences up to 69 nt in length and measured the frequency of their insertion into four genomic sites in three human cell lines, using different prime editor systems. We discover that insertion sequence length, nucleotide composition and secondary structure all affect insertion rates, and that mismatch repair proficiency is a strong determinant for the shortest insertions. Combining the sequence and repair features into a machine learning model, we can predict insertion frequency for new sequences with R = 0.69. The tools we provide allow users to choose optimal constructs for DNA insertion using prime editing.

Operon formation by insertion sequence IS3 in Escherichia coli

Operons are a hallmark of the genomic and regulatory architecture of prokaryotes. However, the mechanism by which two genes placed far apart gradually come close and form operons remains to be elucidated. Here, we propose a new model of the origin of operons: Mobile genetic elements called insertion sequences can facilitate the formation of operons by consecutive insertion-deletion-excision reactions. This mechanism barely leaves traces of insertion sequences and is difficult to detect in evolution in nature. We performed, to the best of our knowledge, the first experimental demonstration of operon formation, as a proof of concept. The insertion sequence IS3 and the insertion sequence excision enhancer are genes found in a broad range of bacterial species. We introduced these genes into insertion sequence-less Escherichia coli and found that, supporting our hypothesis, the activity of the two genes altered the expression of genes surrounding IS3, closed a 2.7 kilobase pair gap between a pair of genes, and formed new operons. This study shows how insertion sequences can facilitate the rapid formation of operons through locally increasing the structural mutation rates and highlights how coevolution with mobile elements may shape the organization of prokaryotic genomes and gene regulation.

The expression of essential selenoproteins during zebrafish development requires SECIS binding protein 2-like

The dietary requirement for selenium is based on its incorporation into selenoproteins, which contain the amino acid selenocysteine (Sec). The Sec insertion sequence (SECIS) is an RNA structure found in the 3' UTR of all selenoprotein mRNAs, and it is required to convert in-frame UGA codons from termination to Sec-incorporating codons. There are two proteins that bind to SECIS elements, but only one, SECIS binding protein 2 (Sbp2), has been shown to be required for Sec incorporation. The Sbp2 paralogue, SECIS binding protein 2-like (Secisbp2l) is conserved in all vertebrates and shares many features with Sbp2, but its function is unknown. Here we set out to determine the relative roles of Sbp2 and Secisbp2l by introducing CRISPR mutations in both genes in zebrafish. By monitoring selenoprotein synthesis with 75Se labeling during embryogenesis, we found that sbp2-/- embryos still make a select subset of selenoproteins but secisbp2l-/- embryos retain the full complement. Abrogation of both genes completely prevents selenoprotein synthesis and juveniles die at 14 days post fertilization. Embryos lacking Sbp2 are sensitive to oxidative stress and express the stress marker Vtg1. We propose a model where Secisbp2l is required to promote essential selenoprotein synthesis during stress.

Prevalence of trimethoprim/sulfamethoxazole resistance genes among Stenotrophomonas maltophilia clinical isolates in Egypt

Stenotrophomonas maltophilia is an important multidrug resistant nosocomial pathogen. Trimethoprim/sulfamethoxazole (TMP/SMX) is considered the drug of choice for treatment of S. maltophilia infections, thus emerging resistance to TMP/SMX poses a serious threat. In the present study we aimed to investigate the frequency of TMP/SMX resistance genes (sul1, sul2, dfrA), and to evaluate their relatedness with integron 1 (int1), and insertion sequence common regions (ISCR) among 100 S. maltophilia from different clinical isolates in Egypt. Isolates were identified biochemically and confirmed by VITEK2. Detection of sul1, sul2, and dfrA genes, int1 and ISCR elements was performed by PCR. Among the 16 TMP/SMX resistant isolates, sul1 gene was detected in all of them, and it was associated with int1 gene presence in all resistant isolates. The sul2 gene was detected in 6 out of 16 resistant isolates (37.5%), and only 2 of the 16 resistant isolates (12.5%) harboured dfrA gene. ISCR was detected in 10 of the resistant isolates (62.5%) and in 4 of them it was associated with the presence of sul2 gene. Among the 84 TMP/SMX sensitive isolates, sul1 gene was detected in 15 (17.8%), int1 in 16 (19%) and ISCR in 6 (7.1%). None of the susceptible isolates had sul2 or dfrA genes. These findings point out an increasing frequency of TMP/SMX resistance genes among S. maltophilia clinical isolates in our region, so the adoption of prudent use of S. maltophilia antimicrobial agents and the establishment of a surveillance system are desperately needed.

Titin M-line insertion sequence 7 is required for proper cardiac function in mice

ABSTRACT Titin is a giant sarcomeric protein that is involved in a large number of functions, with a primary role in skeletal and cardiac sarcomere organization and stiffness. The titin gene (TTN) is subject to various alternative splicing events, but in the region that is present at the M-line, the only exon that can be spliced out is Mex5, which encodes for the insertion sequence 7 (is7). Interestingly, in the heart, the majority of titin isoforms are Mex5+, suggesting a cardiac role for is7. Here, we performed comprehensive functional, histological, transcriptomic, microscopic and molecular analyses of a mouse model lacking the Ttn Mex5 exon (ΔMex5), and revealed that the absence of the is7 is causative for dilated cardiomyopathy. ΔMex5 mice showed altered cardiac function accompanied by increased fibrosis and ultrastructural alterations. Abnormal expression of excitation–contraction coupling proteins was also observed. The results reported here confirm the importance of the C-terminal region of titin in cardiac function and are the first to suggest a possible relationship between the is7 and excitation–contraction coupling. Finally, these findings give important insights for the identification of new targets in the treatment of titinopathies.