Insertion sequence excision is enhanced by a protein that catalyzes branch migration and promotes microhomology-mediated end joining.

Insertion sequence (IS)-excision enhancer (IEE) promotes the excision of ISs in the genome of enterohemorrhagic Escherichia coli O157. Because IEE-dependent IS excision occurs in the presence of transposase, the process of IS transposition may be involved in IS excision; however, little is understood about the molecular mechanisms of IS excision. Our in vitro analysis revealed that IEE exhibits DNA-dependent ATPase activity, which is activated by branched DNA. IEE also catalyzes the branch migration of fork-structured DNA. These results suggest that IEE remodels branched structures of the IS transposition intermediate. Sequence analysis of recombination sites in IS-excision products suggested that microhomologous sequences near the ends of the IS are involved in IS excision. IEE promoted microhomology-mediated end joining (MMEJ), in which base pairing between 6-nucleotides complementary ends of two 3'-protruding DNAs and subsequent elongation of the paired DNA strand occurred. IS-excision frequencies were significantly decreased in cells producing IEE mutants that had lost either branch migration or MMEJ activity, which suggests that these activities of IEE are required for IS excision. Based on our results, we propose a model for IS excision triggered by IEE and transposase.

The insertion sequence excision enhancer: A PrimPol-based primer invasion system for immobilizing transposon-transmitted antibiotic resistance genes.

Evolutionary studies often identify genes that have been exchanged between different organisms and the phrase Lateral or Horizontal Gene Transfer is often used in this context. However, they rarely provide any mechanistic information concerning how these gene transfers might have occurred. With the astonishing increase in the number of sequences in public databases over the past two or three decades, identical antibiotic resistance genes have been identified in many different sequence contexts. One explanation for this would be that genes are initially transmitted by transposons which have subsequently decayed and can no longer be detected. Here, we provide an overview of a protein, IEE (Insertion Sequence Excision Enhancer) observed to facilitate high-frequency excision of IS629 from clinically important Escherichia coli O157:H7 and subsequently shown to affect a large class of bacterial insertion sequences which all transpose using the copy-out-paste-in transposition mechanism. Excision depends on both IEE and transposase indicating association with the transposition process itself. We review genetic and biochemical data and propose that IEE immobilizes genes carried by compound transposons by removing the flanking insertion sequence (IS) copies. The biochemical activities of IEE as a primase with the capacity to recognize DNA microhomologies and the observation that its effect appears restricted to IS families which use copy-out-paste-in transposition, suggests IS deletion occurs by abortive transposition involving strand switching (primer invasion) during the copy-out step. This reinforces the proposal made for understanding the widespread phenomenon loss of ISApl1 flanking mcr-1 in the compound transposon Tn6330 which we illustrate with a detailed model. This model also provides a convincing way to explain the high levels of IEE-induced precise IS excision.

Incidence of carbapenem-resistant Escherichia coli ST2437 of clinical origin harbouring blaOXA-144 gene: a report from India.

AIMS: Carbapenem-resistant Escherichia coli has been categorized as a pathogen of critical priority by the World Health Organization as it is highly infectious with high mortality and morbidity rates and widespread transmission potential. Carbapenem resistance is primarily mediated by carbapenemase-encoding genes and, additionally, through intrinsic factors. In India, over the years, carbapenemase-encoding genes have been reported from diverse clinically significant pathogens. The present study identifies E. coli of clinical origin that harbours blaOXA-144. METHODS AND RESULTS: The study isolate was obtained from a tertiary referral hospital in northeast India. Carbapenemase production was investigated through culture on chromogenic agar and Rapidec Carba NP test as per manufacturer's instructions. Susceptibility of the isolate was performed by the Kirby-Bauer disc diffusion method and agar dilution method following CLSI guidelines. PCR targeting carbapenemase-encoding genes was performed, followed by transformation and conjugation experiments. Whole-genome sequencing of the isolate was done through the Illumina sequencing platform and the data were analysed using the Centre for Genomic Epidemiology database. BJD_EC180 is 6 919 180 bp in length and consists of six rRNA operons, 111 tRNA, and 6849 predicted protein-coding sequences. BJD_EC180 belonged to ST2437 and harboured the carbapenemase-encoding gene blaOXA-144 with ISAba1 upstream, along with multiple antibiotic resistance genes conferring clinical resistance towards beta-lactams, aminoglycosides, amphenicols, sulphonamides, tetracyclines, trimethoprim, and rifampin. CONCLUSIONS: Carbapenem-resistant E. coli harbouring blaOXA-144 associated with insertion sequence pose a serious health threat as their mobilization into carbapenem non-susceptible strains that will contribute to the resistance burden and therefore, needs urgent monitoring.

IS6 family insertion sequences promote optrA dissemination between plasmids varying in transfer abilities.

Plasmids are the primary vectors for intercellular transfer of the oxazolidinone and phenicol cross-resistance gene optrA, while insertion sequences (ISs) are mobile genetic elements that can mobilize plasmid-borne optrA intracellularly. However, little is known about how the IS-mediated intracellular mobility facilitates the dissemination of the optrA gene between plasmid categories that vary in transfer abilities, including non-mobilizable, mobilizable, and conjugative plasmids. Here, we performed a holistic genomic study of 52 optrA-carrying plasmids obtained from searches guided by the Comprehensive Antibiotic Resistance Database. Among the 132 ISs identified within 10 kbp from the optrA gene in the plasmids, IS6 family genes were the most prevalent (86/132). Homologous gene arrays containing IS6 family genes were shared between different plasmids, especially between mobilizable and conjugative plasmids. All these indicated the central role of IS6 family genes in disseminating plasmid-borne optrA. Thirty-three of the 52 plasmids were harbored by Enterococcus faecalis found mainly in humans and animals. By Nanopore sequencing and inverse PCR, the potential of the enterococcal optrA to be transmitted from a mobilizable plasmid to a conjugative plasmid mediated by IS6 family genes was further confirmed in Enterococcus faecalis strains recovered from the effluents of anaerobic digestion systems for treating chicken manure. Our findings highlight the increased intercellular transfer abilities and dissemination risk of plasmid-borne optrA gene caused by IS-mediated intracellular mobility, and underscore the importance of routinely monitoring the dynamic genetic contexts of clinically important antibiotic resistance genes to effectively control this critical public health threat. KEY POINTS: • IS6 was prevalent in optrA-plasmids varying in intercellular transfer abilities. • Enterococcal optrA-plasmids were widespread among human, animal, and the environment. • IS6 elevated the dissemination risk of enterococcal optrA-plasmids.

Molecular characterization and antibiotic resistance of clinical Bacteroides and related genera from a tertiary care center in Türkiye.

OBJECTIVES: This study was conducted to measure the prevalence of antibiotic resistance, and corresponding resistance genes among Bacteroides and related genera in a tertiary hospital. METHODS: We examined 138 clinical strains of Bacteroides, Phocaeicola and Parabacteroides species isolated between July 2018 and June 2022. Antibiotic susceptibility tests were conducted using agar dilution. The bft gene and antibiotic resistance genes were targeted by real-time PCR. RESULTS: Resistance rates of all strains against ampicillin, cefoxitin, piperacillin-tazobactam, meropenem, imipenem, clindamycin, metronidazole, and tigecycline were 97.8 %, 28.3 %, 11.6 %, 7.9 %, 5.1 %, 47.8 %, 0 % and 4.3 %, respectively. Non-fragilis Bacteroidales spp. (NFB) exhibited lower susceptibility rates compared to B. fragilis for cefoxitin, clindamycin, and piperacillin-tazobactam. The prevalence of meropenem resistance was higher in B. fragilis (15.5 %) than in NFB (0 %). Among all strains, the rates of cepA, cfxA, cfiA, ermF, ermG, ermB, nim, linA, mefA, msrSA, tetQ, tetX, tetX1 and bft genes were 42.8 %, 44.9 %, 8.7 %, 44.2 %, 10.9 %, 2.2 %, 0.7 %, 29.0 %, 17.4 %, 7.2 %, 76.1 %, 8.0 %, 37.7 % and 16.7 %, respectively. In five B. fragilis strains, insertion sequences [IS1187(n = 3), ISBf6(n = 1), IS612B(n = 1)] were detected in the upstream region of cfiA. NimE with ISBf6 on plasmid pBFM29b was detected in one B. fragilis strain, intermediate to metronidazole (MIC = 16 µg/mL). ErmF was the most abundant gene responsible for clindamycin resistance. TetQ and tetX1 genes exhibited a higher frequency in strains that were not susceptible to tigecycline (MIC ≥8 µg/ml). CONCLUSIONS: Monitoring the resistance trends of Bacteroides and related genera is crucial given the observed resistance to all classes of antibiotics and the presence of various resistance mechanisms.

Local Genomic Surveillance of Invasive Streptococcus pyogenes in Eastern North Carolina (ENC) in 2022-2023.

The recent increase in Group A Streptococcus (GAS) incidences in several countries across Europe and some areas of the Unites States (U.S.) has raised concerns. To understand GAS diversity and prevalence, we conducted a local genomic surveillance in Eastern North Carolina (ENC) in 2022-2023 with 95 isolates and compared its results to those of the existing national genomic surveillance in the U.S. in 2015-2021 with 13,064 isolates. We observed their epidemiological changes before and during the COVID-19 pandemic and detected a unique sub-lineage in ENC among the most common invasive GAS strain, ST28/emm1. We further discovered a multiple-copy insertion sequence, ISLgar5, in ST399/emm77 and its single-copy variants in some other GAS strains. We discovered ISLgar5 was linked to a Tn5801-like tetM-carrying integrative and conjugative element, and its copy number was associated with an ermT-carrying pRW35-like plasmid. The dynamic insertions of ISLgar5 may play a vital role in genome fitness and adaptation, driving GAS evolution relevant to antimicrobial resistance and potentially GAS virulence.

Co-production of metallo-ß-lactamase and OXA-type ß-lactamases in carbapenem-resistant Acinetobacter baumannii clinical isolates in North East India.

Carbapenem-resistant Acinetobacter baumannii poses a significant threat to public health globally, especially due to its ability to produce multiple carbapenemases, leading to treatment challenges. This study aimed to investigate the antibiotic resistance pattern of carbapenem-resistant A. baumannii isolates collected from different clinical settings in North East India, focusing on their genotypic and phenotypic resistance profiles. A total of 172 multidrug-resistant A. baumannii isolates were collected and subjected to antibiotic susceptibility test using the Kirby-Bauer disk diffusion method. Various phenotypic tests were performed to detect extended-spectrum ß-lactamase (ESBL), metallo-ß-lactamase (MBL), class C AmpC ß-lactamase (AmpC), and carbapenem hydrolyzing class D ß-lactamase (CHDL) production among the isolates. Overexpression of carbapenemase and cephalosporinase genes was detected among the isolates through both phenotypic and genotypic investigation. The antibiotic resistance profile of the isolates revealed that all were multidrug-resistant; 25% were extensively drug-resistant, 9.30% were pan-drug-resistant, whereas 91.27% were resistant to carbapenems. In the genotypic investigation, 80.81% of isolates were reported harbouring at least one metallo-ß-lactamase encoding gene, with blaNDM being the most prevalent at 70.34%, followed by blaIMP at 51.16% of isolates. Regarding class D carbapenemases, blaOXA-51 and blaOXA-23 genes were detected in all the tested isolates, while blaOXA-24, blaOXA-48, and blaOXA-58 were found in 15.11%, 6.97%, and 1.74% isolates respectively. Further analysis showed that 31.97% of isolates co-harboured ESBL, MBL, AmpC, and CHDL genes, while 31.39% of isolates co-harboured ESBL, MBL, and CHDL genes with or without ISAba1 leading to extensively drug-resistant or pan drug-resistant phenotypes. This study highlights the complex genetic profile and antimicrobial-resistant pattern of the isolates circulating in North East India, emphasizing the urgent need for effective infection control measures and the development of alternative treatment strategies to combat these challenging pathogens.

Insertion sequence contributes to the evolution and environmental adaptation of Acidithiobacillus.

BACKGROUND: The genus Acidithiobacillus has been widely concerned due to its superior survival and oxidation ability in acid mine drainage (AMD). However, the contribution of insertion sequence (IS) to their biological evolution and environmental adaptation is very limited. ISs are the simplest kinds of mobile genetic elements (MGEs), capable of interrupting genes, operons, or regulating the expression of genes through transposition activity. ISs could be classified into different families with their own members, possessing different copies. RESULTS: In this study, the distribution and evolution of ISs, as well as the functions of the genes around ISs in 36 Acidithiobacillus genomes, were analyzed. The results showed that 248 members belonging to 23 IS families with a total of 10,652 copies were identified within the target genomes. The IS families and copy numbers among each species were significantly different, indicating that the IS distribution of Acidithiobacillus were not even. A. ferrooxidans had 166 IS members, which may develop more gene transposition strategies compared with other Acidithiobacillus spp. What's more, A. thiooxidans harbored the most IS copies, suggesting that their ISs were the most active and more likely to transpose. The ISs clustered in the phylogenetic tree approximately according to the family, which were mostly different from the evolutionary trends of their host genomes. Thus, it was suggested that the recent activity of ISs of Acidithiobacillus was not only determined by their genetic characteristics, but related with the environmental pressure. In addition, many ISs especially Tn3 and IS110 families were inserted around the regions whose functions were As/Hg/Cu/Co/Zn/Cd translocation and sulfur oxidation, implying that ISs could improve the adaptive capacities of Acidithiobacillus to the extremely acidic environment by enhancing their resistance to heavy metals and utilization of sulfur. CONCLUSIONS: This study provided the genomic evidence for the contribution of IS to evolution and adaptation of Acidithiobacillus, opening novel sights into the genome plasticity of those acidophiles.

Identification of an operon and its regulator required for autoaggregation in Tetragenococcus halophilus.

IMPORTANCE: Tetragenococcus halophilus is a halophilic lactic acid bacterium generally used as a starter culture in fermenting soy and fish sauces. Aggregating strains can be useful in fermenting and obtaining clear soy sauce because cell clumps are trapped by the filter cake when the soy sauce mash is pressed. However, the genetic mechanisms of aggregation in T. halophilus are unknown. In this study, we identified genes encoding aggregation factor and its regulator. These findings may provide a foundation for developing improved T. halophilus starter cultures for soy sauce fermentation, leading to more efficient and consistent clear soy sauce production.

Targeted Screening for Spontaneous Insertion Mutations in a Lactic Acid Bacterium, Tetragenococcus halophilus.

Studies on the microorganisms used in food production are of interest because microbial genotypes are reflected in food qualities such as taste, flavor, and yield. However, several microbes are nonmodel organisms, and their analysis is often limited by the lack of genetic tools. Tetragenococcus halophilus, a halophilic lactic acid bacterium used in soy sauce fermentation starter culture, is one such microorganism. The lack of DNA transformation techniques for T. halophilus makes gene complementation and disruption assays difficult. Here, we report that the endogenous insertion sequence ISTeha4, belonging to the IS4 family, is translocated at an extremely high frequency in T. halophilus and causes insertional mutations at various loci. We developed a method named targeting spontaneous insertional mutations in genomes (TIMING), which combines high-frequency insertional mutations and efficient PCR screening, enabling the isolation of gene mutants of interest from a library. The method provides a reverse genetics and strain improvement tool, does not require the introduction of exogenous DNA constructs, and enables the analysis of nonmodel microorganisms lacking DNA transformation techniques. Our results highlight the important role of insertion sequences as a source of spontaneous mutagenesis and genetic diversity in bacteria. IMPORTANCE Genetic and strain improvement tools to manipulate a gene of interest are required for the nontransformable lactic acid bacterium Tetragenococcus halophilus. Here, we demonstrate that an endogenous transposable element, ISTeha4, is transposed into the host genome at an extremely high frequency. A genotype-based and non-genetically engineered screening system was constructed to isolate knockout mutants using this transposable element. The method described enables a better understanding of the genotype-phenotype relationship and serves as a tool to develop food-grade-appropriate mutants of T. halophilus.

Identification of lthB, a Gene Encoding a Putative Glycosyltransferase Family 8 Protein Required for Leptothrix Sheath Formation.

The bacterium Leptothrix cholodnii generates cell chains encased in sheaths that are composed of woven nanofibrils. The nanofibrils are mainly composed of glycoconjugate repeats, and several glycosyltransferases (GTs) are required for its biosynthesis. However, only one GT (LthA) has been identified to date. In this study, we screened spontaneous variants of L. cholodnii SP6 to find those that form smooth colonies, which is one of the characteristics of sheathless variants. Genomic DNA sequencing of an isolated variant revealed an insertion in the locus Lcho_0972, which encodes a putative GT family 8 protein. We thus designated this protein LthB and characterized it using deletion mutants and antibodies. LthB localized adjacent to the cell envelope. ΔlthB cell chains were nanofibril free and thus sheathless, indicating that LthB is involved in nanofibril biosynthesis. Unlike the ΔlthA mutant and the wild-type strain, which often generate planktonic cells, most ΔlthB organisms presented as long cell chains under static conditions, resulting in deficient pellicle formation, which requires motile planktonic cells. These results imply that sheaths are not required for elongation of cell chains. Finally, calcium depletion, which induces cell chain breakage due to sheath loss, abrogated the expression of LthA, but not LthB, suggesting that these GTs cooperatively participate in glycoconjugate biosynthesis under different signaling controls. IMPORTANCE In recent years, the regulation of cell chain elongation of filamentous bacteria via extracellular signals has attracted attention as a potential strategy to prevent clogging of water distribution systems and filamentous bulking of activated sludge in industrial settings. However, a fundamental understanding of the ecology of filamentous bacteria remains elusive. Since sheath formation is associated with cell chain elongation in most of these bacteria, the molecular mechanisms underlying nanofibril sheath formation, including the intracellular signaling cascade in response to extracellular stimuli, must be elucidated. Here, we isolated a sheathless variant of L. cholodnii SP6 and thus identified a novel glycosyltransferase, LthB. Although mutants with deletions of lthA, encoding another GT, and lthB were both defective for nanofibril formation, they exhibited different phenotypes of cell chain elongation and pellicle formation. Moreover, LthA expression, but not LthB expression, was influenced by extracellular calcium, which is known to affect nanofibril formation, indicating the functional diversities of LthA and LthB. Such molecular insights are critical for a better understanding of ecology of filamentous bacteria, which, in turn, can be used to improve strategies to control filamentous bacteria in industrial facilities.

The extensively antibiotic resistant ST111 Acinetobacter baumannii isolate RBH2 carries an extensive mobile element complement of plasmids, transposons and insertion sequences.

The complete genome of RBH2, a sporadic, carbapenem resistant ST111 Acinetobacter baumannii isolate from Brisbane, Australia was determined and analysed. RBH2 is extensively resistant and the chromosome includes two transposons carrying antibiotic resistance genes, AbaR4 (oxa23 in Tn2006) and Tn7::Tn2006 (dfrA1, sat2, aadA1, oxa23). The chromosome also includes two copies of Tn6175, a transposon carrying putative copper resistance genes, and 1-17 copies of six different insertion sequences. RBH2 has six plasmids ranging in size from 6 kb - 141 kb, four carrying antibiotic resistance genes. Plasmids pRBH2-1 (aadB) and pRBH2-2 (aphA6 in TnaphA6) were found to be essentially identical to known plasmids pRAY*-v1 and pS21-1, respectively. The largest plasmids, pRBH2-5 (oxa23 in AbaR4) and pRBH2-6 (oxa23 in AbaR4::ISAba11 and sul2, tet(B), strA and strB in Tn6172) have known transfer-proficient relatives. pRBH2-5, an RP-T1 (RepAci6) plasmid, also carries a different putative copper resistance transposon related to Tn6177 found in pS21-2. The backbone of pRBH2-5 is related to those of previously described RepAci6 plasmids pAb-G7-2 and pA85-3 but has some distinctive features. Three different RepAci6 backbone types were distinguished, Type 1 (pAb-G7-2), Type 2 (pA85-3) and Type 3 (pRBH2-5 and pS21-2). pRBH2-6 is closely related to pAB3 and their backbones differ by only 5 SNPs. Plasmids pRBH2-3 and pRBH2-4 do not carry antibiotic resistance genes. pRBH2-3 does not include an identifiable rep gene and is a novel plasmid type. pRBH2-4 is of the R3-T3 type and includes segments of the larger pABTJ2 that heads this group. Other ST111 genomes carry different plasmids.

A subclass of the IS1202 family of bacterial insertion sequences targets XerCD recombination sites.

We describe here a new family of IS which are related to IS1202, originally isolated from Streptococcus pneumoniae in the mid-1990s and previously tagged as an emerging IS family in the ISfinder database. Members of this family have impacted some important properties of their hosts. We describe here another potentially important property of certain family members: specific targeting of xrs recombination sites. The family could be divided into three subgroups based on their transposase sequences and the length on the target repeats (DR) they generate on insertion: subgroup IS1202 (24-29 bp); ISTde1 (15-18 bp); and ISAba32 (5-6 bp). Members of the ISAba32 subgroup were repeatedly found abutting Xer recombinase recombination sites (xrs), separated by an intervening copy of a DR. These xrs sites, present in multiple copies in a number of Acinetobacter plasmids flanking antibiotic resistance genes, were proposed to form a new type of mobile genetic element using the chromosomally-encoded XerCD recombinase for mobility. Transposase alignments identified subgroup-specific indels which may be responsible for the differences in the transposition properties of the three subgroups (i.e. DR length and target specificity). We propose that this collection of IS be classed as a new insertion sequence family: the IS1202 family composed of three subgroups, only one of which specifically targets plasmid-borne xrs. We discuss the implications of xrs targeting for gene mobility.

The integration model of hepatitis B virus genome in hepatocellular carcinoma cells based on high-throughput long-read sequencing.

HBV integration and function has gradually been expanding. However, the exact mode of HBV integration remains unclear. In our research, the high-throughput long-read sequencing was combined with bioinformatics to study the complete mode of HBV integration in hepatocellular carcinoma (HCC) cells. The results demonstrated that: 1) The HBV insertion sequences of HBV integration events accounted for 49.5% of the total HBV sequences. 2) Short insertion segments with the length of 0-1 kbp accounted for 50% and the long insertion segments (>3 kbp) accounted for 25% of HBV insertion events. 3)There were different HBV insertion length in the breakpoints formed within different regions. 4) The occurrence of HBV integration events was accompanied by more frequent structural variations. 5)Furthermore, multiple HBV integration patterns were confirmed based on complete HBV insertion sequences. Our research not only clarified a variety of perfect HBV integration models but also determined multiple specific features of HBV integration.

The effects of identical nim gene-insertion sequence combinations on the expression of the nim genes and metronidazole resistance in Bacteroides fragilis strains.

In this study we examined whether the same nim gene-insertion sequence (IS) element combinations give rise to the same expression levels as they harbor shared IS element-borne promoters. From our quantitative analysis, we found that the expressions of the nimB and nimE genes with their cognate IS elements were similar, but the metronidazole resistance of these strains were more diverse.

The Mechanism of Tigecycline Resistance in Acinetobacter baumannii Revealed by Proteomic and Genomic Analysis.

The mechanism of tigecycline resistance in A. baumannii remains largely unclear. In this study, we selected a tigecycline-resistant and a tigecycline-susceptible strain from a tigecycline-susceptible and a resistant strain, respectively. Proteomic and genomic analyses were performed to elucidate the variations associated with tigecycline resistance. Our study showed proteins associated with efflux pump, biofilm formation, iron acquisition, stress response, and metabolic ability are upregulated in tigecycline resistant strains, and efflux pump should be the key mechanism for tigecycline resistance. By genomic analysis, we found several changes in the genome that can explain the increased level of efflux pump, including the loss of the global negative regulator hns in the plasmid and the disruption of the hns gene and acrR gene on the chromosome by the insertion of IS5. Collectively, we not only revealed the phenomenon that the efflux pump is mainly responsible for tigecycline resistance, but also highlighted the mechanism at the genomic level, which will help in understanding the resistance mechanism in detail and provide clues for the treatment of clinical multiple drug-resistant A. baumannii.

Efflux-Related Carbapenem Resistance in Acinetobacter baumannii Is Associated with Two-Component Regulatory Efflux Systems' Alteration and Insertion of ΔAbaR25-Type Island Fragment.

The efflux pumps, beside the class D carbapenem-hydrolysing enzymes (CHLDs), are being increasingly investigated as a mechanism of carbapenem resistance in Acinetobacter baumannii. This study investigates the contribution of efflux mechanism to carbapenem resistance in 61 acquired blaCHDL-genes-carrying A. baumannii clinical strains isolated in Warsaw, Poland. Studies were conducted using phenotypic (susceptibility testing to carbapenems ± efflux pump inhibitors (EPIs)) and molecular (determining expression levels of efflux operon with regulatory-gene and whole genome sequencing (WGS)) methods. EPIs reduced carbapenem resistance of 14/61 isolates. Upregulation (5-67-fold) of adeB was observed together with mutations in the sequences of AdeRS local and of BaeS global regulators in all 15 selected isolates. Long-read WGS of isolate no. AB96 revealed the presence of AbaR25 resistance island and its two disrupted elements: the first contained a duplicate ISAba1-blaOXA-23, and the second was located between adeR and adeA in the efflux operon. This insert was flanked by two copies of ISAba1, and one of them provides a strong promoter for adeABC, elevating the adeB expression levels. Our study for the first time reports the involvement of the insertion of the ΔAbaR25-type resistance island fragment with ISAba1 element upstream the efflux operon in the carbapenem resistance of A. baumannii.

Biosynthesis, Engineering, and Delivery of Selenoproteins.

Selenocysteine (Sec) was discovered as the 21st genetically encoded amino acid. In nature, site-directed incorporation of Sec into proteins requires specialized biosynthesis and recoding machinery that evolved distinctly in bacteria compared to archaea and eukaryotes. Many organisms, including higher plants and most fungi, lack the Sec-decoding trait. We review the discovery of Sec and its role in redox enzymes that are essential to human health and important targets in disease. We highlight recent genetic code expansion efforts to engineer site-directed incorporation of Sec in bacteria and yeast. We also review methods to produce selenoproteins with 21 or more amino acids and approaches to delivering recombinant selenoproteins to mammalian cells as new applications for selenoproteins in synthetic biology.

Characterization of the specific DNA-binding properties of Tnp26, the transposase of insertion sequence IS26.

The bacterial insertion sequence (IS) IS26 mobilizes and disseminates antibiotic resistance genes. It differs from bacterial IS that have been studied to date as it exclusively forms cointegrates via either a copy-in (replicative) or a recently discovered targeted conservative mode. To investigate how the Tnp26 transposase recognizes the 14-bp terminal inverted repeats (TIRs) that bound the IS, amino acids in two domains in the N-terminal (amino acids M1-P56) region were replaced. These changes substantially reduced cointegration in both modes. Tnp26 was purified as a maltose-binding fusion protein and shown to bind specifically to dsDNA fragments that included an IS26 TIR. However, Tnp26 with an R49A or a W50A substitution in helix 3 of a predicted trihelical helix-turn-helix domain (amino acids I13-R53) or an F4A or F9A substitution replacing the conserved amino acids in a unique disordered N-terminal domain (amino acids M1-D12) did not bind. The N-terminal M1-P56 fragment also bound to the TIR but only at substantially higher concentrations, indicating that other parts of Tnp26 enhance the binding affinity. The binding site was confined to the internal part of the TIR, and a G to T nucleotide substitution in the TGT at positions 6 to 8 of the TIR that is conserved in most IS26 family members abolished binding of both Tnp26 (M1-M234) and Tnp26 M1-P56 fragment. These findings indicate that the helix-turn-helix and disordered domains of Tnp26 play a role in Tnp26-TIR complex formation. Both domains are conserved in all members of the IS26 family.

From a large-scale genomic analysis of insertion sequences to insights into their regulatory roles in prokaryotes.

BACKGROUND: Insertion sequences (ISs) are mobile repeat sequences and most of them can copy themselves to new host genome locations, leading to genome plasticity and gene regulation in prokaryotes. In this study, we present functional and evolutionary relationships between IS and neighboring genes in a large-scale comparative genomic analysis. RESULTS: IS families were located in all prokaryotic phyla, with preferential occurrence of IS3, IS4, IS481, and IS5 families in Alpha-, Beta-, and Gammaproteobacteria, Actinobacteria and Firmicutes as well as in eukaryote host-associated organisms and autotrophic opportunistic pathogens. We defined the concept of the IS-Gene couple (IG), which allowed to highlight the functional and regulatory impacts of an IS on the closest gene. Genes involved in transcriptional regulation and transport activities were found overrepresented in IG. In particular, major facilitator superfamily (MFS) transporters, ATP-binding proteins and transposases raised as favorite neighboring gene functions of IS hotspots. Then, evolutionary conserved IS-Gene sets across taxonomic lineages enabled the classification of IS-gene couples into phylum, class-to-genus, and species syntenic IS-Gene couples. The IS5, IS21, IS4, IS607, IS91, ISL3 and IS200 families displayed two to four times more ISs in the phylum and/or class-to-genus syntenic IGs compared to other IS families. This indicates that those families were probably inserted earlier than others and then subjected to horizontal transfer, transposition and deletion events over time. In phylum syntenic IG category, Betaproteobacteria, Crenarchaeota, Calditrichae, Planctomycetes, Acidithiobacillia and Cyanobacteria phyla act as IS reservoirs for other phyla, and neighboring gene functions are mostly related to transcriptional regulators. Comparison of IS occurrences with predicted regulatory motifs led to ~ 26.5% of motif-containing ISs with 2 motifs per IS in average. These results, concomitantly with short IS-Gene distances, suggest that those ISs would interfere with the expression of neighboring genes and thus form strong candidates for an adaptive pairing. CONCLUSIONS: All together, our large-scale study provide new insights into the IS genetic context and strongly suggest their regulatory roles.