Direct Repeats Research Articles

Thermotoga maritima oriC involves a DNA unwinding element with distinct modules and a DnaA-oligomerizing region with a novel directional binding mode

Initiation of chromosomal replication requires dynamic nucleoprotein complexes. In most eubacteria, the origin oriC contains multiple DnaA box sequences to which the ubiquitous DnaA initiators bind. In Escherichia coli oriC, DnaA boxes sustain construction of higher-order complexes via DnaA-DnaA interactions, promoting the unwinding of the DNA unwinding element (DUE) within oriC and concomitantly binding the single-stranded (ss) DUE to install replication machinery. Despite the significant sequence homologies among DnaA proteins, oriC sequences are highly diverse. The present study investigated the design of oriC (tma-oriC) from Thermotoga maritima, an evolutionarily ancient eubacterium. The minimal tma-oriC sequence includes a DUE and a flanking region containing five DnaA boxes recognized by the cognate DnaA (tmaDnaA). This DUE was comprised of two distinct functional modules, an unwinding module and a tmaDnaA-binding module. Three direct repeats of the trinucleotide TAG within DUE were essential for both unwinding and ssDUE binding by tmaDnaA complexes constructed on the DnaA boxes. Its surrounding AT-rich sequences stimulated only duplex unwinding. Moreover, head-to-tail oligomers of ATP-bound tmaDnaA were constructed within tma-oriC, irrespective of the directions of the DnaA boxes. This binding mode was considered to be induced by flexible swiveling of DnaA domains III and IV, which were responsible for DnaA-DnaA interactions and DnaA box binding, respectively. Phasing of specific tmaDnaA boxes in tma-oriC was also responsible for unwinding. These findings indicate that a ssDUE recruitment mechanism was responsible for unwinding and would enhance understanding of the fundamental molecular nature of the origin sequences present in evolutionarily divergent bacteria.

Structural basis of λCII-dependent transcription activation

The CII protein of bacteriophage λ activates transcription from the phage promoters PRE, PI, and PAQ by binding to two direct repeats that straddle the promoter -35 element. Although genetic, biochemical, and structural studies have elucidated many aspects of λCII-mediated transcription activation, no precise structure of the transcription machinery in the process is available. Here, we report a 3.1-Å cryo-electron microscopy (cryo-EM) structure of an intact λCII-dependent transcription activation complex (TAC-λCII), which comprises λCII, E.coli RNAP-σ70 holoenzyme, and the phage promoter PRE. The structure reveals the interactions between λCII and the direct repeats responsible for promoter specificity and the interactions between λCII and RNAP α subunit C-terminal domain responsible for transcription activation. We also determined a 3.4-Å cryo-EM structure of an RNAP-promoter open complex (RPo-PRE) from the same dataset. Structural comparison between TAC-λCII and RPo-PRE provides new insights into λCII-dependent transcription activation.

Mutation-site localized non-B DNA burden and survival heterogeneity in early-stage pancreatic cancer.

4166 Background: Pancreatic cancer is a highly aggressive disease with a poor prognosis, and one in which most measure of genomic instability (e.g., tumor mutation burden (TMB), fraction of genome altered (FGA)) have thus far, not proven informative in differentiating survival. Non-B DNA are alternative DNA forms that deviate from the canonical B-DNA structure with potential to increase susceptibility to mutations, leading to the development of cancer. The role of non-B in pancreatic cancer has not been fully explored. Herein, we investigate the relationship between gene mutation sites co-localized with non-B DNA motifs in terms of survival and B-DNA features. Methods: Using TCGA data, we derived a genome-wide mutation signature on 104 early-stage pancreatic patients. We introduced a metric, mutation-localized non-B burden (MLNB) by the number of non-b motifs containing mutation sites, normalized to the number of mutations and motif library size. We applied MLNB to patient-specific mutation signatures to derive an MLNB burden for non-B DNA structure motifs: G-quadruplexes (G4), Z-DNA, inverted repeats (IR), mirror repeats (MR), direct repeats (DR), and short tandem repeats (STR). We performed a cluster analysis on MLNB and compared groups based on overall survival (OS) in months (mos) using a log-rank test. Comparisons of B-DNA-derived molecular features among clusters were performed using a Kruskal-Wallis and Fisher Exact test. Results: Among the 104 early-stage pancreatic patients with a mutation signature, MLNB clustering resulted in six patient clusters that differentiated by non-B DNA structure, with DR burden the longest OS cluster (n=23, median OS=30mos), significantly (p < 0.05) differing as compared to IR (n=23, median OS=15 mos), STR (n=20, median OS=16mos), MR with lack of IR (n=14, median OS=13mos) and MR with IR (n=16, median OS=8mos). A mix of Z-DNA and G4 burden defined a cluster with shorter OS (n=22, median OS=14mos) as compared to DR, though not significant. Patients with the longest OS, MLNB-DR burden cluster had mutation signatures enriched in MAPK and Notch signaling pathways, as compared to the other clusters enriched with double-stranded break and mismatch repair (IR), hedgehog and WNT signaling (STR), and interleukin-4 signaling (MR) pathways. Among all clusters, CDKN2A deletion was most prevalent except for MLNB-DR burden cluster. No Significant differences were found between the MLNB clusters with age, race gender, KRAS and TP53 mutations, FGA, TMB, tumor purity, Bailey and Moffit subtypes. Conclusions: Our results point towards the potential use of MLNB as an emerging marker of site-structure genomic instability. Considering that triplex DNA is emerging as a marker genomic instability, and that we distinguish between two MR groups based on IR burden, our results may provide insights into various instability mechanisms associated with poor survival in pancreatic cancer.

ICESpsuAH0906, a novel optrA-carrying element conferring resistance to phenicols and oxazolidinones from Streptococcus parasuis, is transferable to Streptococcus suis

Streptococcus parasuis is a potential opportunistic zoonotic pathogen which is a close relative to Streptococcus suis, which exhibit extensive genetic exchange. The occurrence and dissemination of oxazolidinone resistance poses a severe threat to public health. However, such knowledge about the optrA gene in S. parasuis is limited. Herein, we characterized an optrA-positive multi-resistant S. parasuis isolate AH0906, in which the capsular polysaccharide locus exhibited a hybrid structure of S. suis serotype 11 and S. parasuis serotype 26. The optrA and erm(B) genes were co-located on a novel ICE of the ICESsuYZDH1 family, designated ICESpsuAH0906. IS1216E-optrA-carrying translocatable unit could be formed when excised from ICESpsuAH0906. ICESpsuAH0906 was found to be transferable from isolate AH0906 to Streptococcus suis P1/7RF at a relative high frequency of ∼ 10-5. Nonconservative integrations of ICESpsuAH0906 into the primary site SSU0877 and secondary site SSU1797 with 2-/4-nt imperfect direct repeats in recipient P1/7RF were observed. Upon transfer, the transconjugant displayed elevated MICs of the corresponding antimicrobial agents and performed a weak fitness cost when compared with the recipient strain. To our knowledge, it is the first description of the transfer of optrA in S. prarasuis and the first report of interspecies transfer of ICE with triplet serine integrases (of the ICESsuYZDH1 family). Considering the high transmission frequency of the ICEs and the extensive genetic exchange potential of S. parasuis with other streptococci, attention should be paid to the dissemination of the optrA gene from S. parasuis to clinically more important bacterial pathogens.

Global Effects of the Developmental Regulator BldB in Streptomyces venezuelae.

In Streptomyces, the Bld (Bald) regulators control formation of the reproductive aerial hyphae. The functions of some of these regulators have been well characterized, but BldB has remained enigmatic. In addition to the bldB gene itself, Streptomyces venezuelae has 10 paralogs of bldB that sit next to paralogs of whiJ and abaA. Transcriptome sequencing (RNA-seq) revealed that loss of BldB function causes the dramatic transcriptional upregulation of the abaA paralogs and a novel inhibitor of sporulation, iosA, and that cooverexpression of just two of these genes, iosA and abaA6, was sufficient to recapitulate the bldB mutant phenotype. Further RNA-seq analysis showed that the transcription factor WhiJ9 is required for the activation of iosA seen in the bldB mutant, and biochemical studies showed that WhiJ9 mediates the activation of iosA expression by binding to direct repeats in the iosA-whiJ9 intergenic region. BldB and BldB9 hetero-oligomerize, providing a potential link between BldB and the iosA-whiJ9-bldB9 locus. This work greatly expands our overall understanding of the global effects of the BldB developmental regulator. IMPORTANCE To reproduce and disperse, the filamentous bacterium Streptomyces develops specialized reproductive structures called aerial hyphae. The formation of these structures is controlled by the bld (bald) genes, many of which encode transcription factors whose functions have been characterized. An exception is BldB, a protein whose biochemical function is unknown. In this study, we gain insight into the global effects of BldB function by examining the genome-wide transcriptional effects of deleting bldB. We identify a small set of genes that are dramatically upregulated in the absence of BldB. We show that their overexpression causes the bldB phenotype and characterize a transcription factor that mediates the upregulation of one of these target genes. Our results provide new insight into how BldB influences Streptomyces development.

Analysis of CRISPR/Cas Genetic Structure, Spacer Content and Molecular Epidemiology in Brazilian Acinetobacter baumannii Clinical Isolates.

CRISPR/Cas is a molecular mechanism to prevent predatory viruses from invading bacteria via the insertion of small viral sequences (spacers) in its repetitive locus. The nature of spacer incorporation and the viral origins of spacers provide an overview of the genetic evolution of bacteria, their natural viral predators, and the mechanisms that prokaryotes may use to protect themselves, or to acquire mobile genetic elements such as plasmids. Here, we report on the CRISPR/Cas genetic structure, its spacer content, and strain epidemiology through MLST and CRISPR typing in Acinetobacter baumannii, an opportunistic pathogen intimately related to hospital infections and antimicrobial resistance. Results show distinct genetic characteristics, such as polymorphisms specific to ancestor direct repeats, a well-defined degenerate repeat, and a conserved leader sequence, as well as showing most spacers as targeting bacteriophages, and several self-targeting spacers, directed at prophages. There was a particular relationship between CRISPR/Cas and CC113 in the study of Brazilian isolates, and CRISPR-related typing techniques are interesting for subtyping strains with the same MLST profile. We want to emphasize the significance of descriptive genetic research on CRISPR loci, and we argue that spacer or CRISPR typing are helpful for small-scale investigations, preferably in conjunction with other molecular typing techniques such as MLST.

Low prevalence of mobilized resistance genes blaNDM, mcr-1, and tet(X4) in Escherichia coli from a hospital in China.

The emergence and spread of carbapenemase genes, colistin resistance genes mcr-1, and tigecycline resistance gene tet(X) represent a significant threat to clinical therapy and public health. In this study, we investigated the presence of carbapenemase genes, mcr-1, and tet(X) in 298 Escherichia coli strains obtained from a teaching hospital in China. In total, eight (2.68%), six (2.01%), and one (0.34%) E. coli isolates carried blaNDM, mcr-1, and tet(X4), respectively. The blaNDM gene was located on IncX3 (n = 4), F2:A-:B- (n = 3), and F2:A1:B1 (n = 1) plasmids, with high similarity to multiple plasmids belonging to the same incompatibility type from Enterobacteriaceae. Six MCR-producing strains contained mcr-1-carrying IncI2 plasmids, organized similarly to other mcr-1-bearing IncI2 plasmids from animals in China. The blaCTX-M-55/64/132/199 gene located within a typical transposition unit (ISEcp1-blaCTX-M-orf477Δ) was inserted near dnaJ to generate 5-bp direct repeats in four mcr-1-positive plasmids. The tet(X) and another four resistance genes [aadA2, tet(A), floR, and Δlnu(F)] were co-located on an IncX1 plasmid, highly similar to other tet(X4)-carrying IncX1 plasmids from Escherichia and Klebsiella of animal or food origin, except that the conjugative transfer region of IncX1 plasmids was absent in our plasmid. Although a low prevalence of blaNDM, mcr-1, and tet(X) was observed in E. coli from patients in this study, their dissemination associated with some successful pandemic plasmids is of great concern. The continued surveillance of these crucial resistance genes in patients should be strengthened.

The membrane-binding bacterial toxin long direct repeat D inhibits protein translation

Bacterial plasmids and chromosomes widely contain toxin-antitoxin (TA) loci, which are implicated in stress response, growth regulation and even tolerance to antibiotics and environmental stress. Type I TA systems consist of a stable toxin-expressing mRNA, which is counteracted by an unstable RNA antitoxin. The Long Direct Repeat (LDR-) D locus, a type I TA system of Escherichia Coli (E. coli) K12, encodes a 35 amino acid toxic peptide, LdrD. Despite being characterized as a bacterial toxin, causing rapid killing and nucleoid condensation, little was known about its function and its mechanism of toxicity. Here, we show that LdrD specifically interacts with ribosomes which potentially blocks translation. Indeed, in vitro translation of LdrD-coding mRNA greatly reduces translation efficiency. The structure of LdrD in a hydrophobic environment, similar to the one found in the interior of ribosomes was determined by NMR spectroscopy in 100% trifluoroethanol solution. A single compact α-helix was found which would fit nicely into the ribosomal exit tunnel. Therefore, we conclude that rather than destroying bacterial membranes, LdrD exerts its toxic activity by inhibiting protein synthesis through binding to the ribosomes.

Secondary structure of the human mitochondrial genome affects formation of deletions

BackgroundAging in postmitotic tissues is associated with clonal expansion of somatic mitochondrial deletions, the origin of which is not well understood. Such deletions are often flanked by direct nucleotide repeats, but this alone does not fully explain their distribution. Here, we hypothesized that the close proximity of direct repeats on single-stranded mitochondrial DNA (mtDNA) might play a role in the formation of deletions.ResultsBy analyzing human mtDNA deletions in the major arc of mtDNA, which is single-stranded during replication and is characterized by a high number of deletions, we found a non-uniform distribution with a “hot spot” where one deletion breakpoint occurred within the region of 6–9 kb and another within 13–16 kb of the mtDNA. This distribution was not explained by the presence of direct repeats, suggesting that other factors, such as the spatial proximity of these two regions, can be the cause. In silico analyses revealed that the single-stranded major arc may be organized as a large-scale hairpin-like loop with a center close to 11 kb and contacting regions between 6–9 kb and 13–16 kb, which would explain the high deletion activity in this contact zone. The direct repeats located within the contact zone, such as the well-known common repeat with a first arm at 8470–8482 bp (base pair) and a second arm at 13,447–13,459 bp, are three times more likely to cause deletions compared to direct repeats located outside of the contact zone. A comparison of age- and disease-associated deletions demonstrated that the contact zone plays a crucial role in explaining the age-associated deletions, emphasizing its importance in the rate of healthy aging.ConclusionsOverall, we provide topological insights into the mechanism of age-associated deletion formation in human mtDNA, which could be used to predict somatic deletion burden and maximum lifespan in different human haplogroups and mammalian species.

Identification and characterization of extrachromosomal circular DNA in the silk gland of Bombyx mori.

The silk gland cells of silkworm are special cells which only replicate DNA in the nucleus without cell division throughout the larval stage. The extrachromosomal circular DNAs (eccDNAs) have not yet been reported in the silk gland of silkworms. Herein, we have explored the characterization of eccDNAs in the posterior silk gland of silkworms. A total of 35346 eccDNAs were identified with sizes ranging from 30 to 13569549bp. Motif analysis revealed that dual direct repeats are flanking the 5' and 3' breaking points of eccDNA. The sequences exceeding 1kb length in eccDNAs present palindromic sequence characteristics flanking the 5' and 3' breaking points of the eccDNA. These motifs might support possible models for eccDNA generation. Genomic annotation of the eccDNA population revealed that most eccDNAs (58.6%) were derived from intergenic regions, whereas full or partial genes were carried by 41.4% of eccDNAs. It was found that silk protein genes fib-H, fib-L, and P25, as well as the transcription factors SGF and sage, which play an important regulatory role in silk protein synthesis, could be carried by eccDNAs. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses showed that the genes carried by eccDNAs were mainly associated with the development and metabolism-related signaling pathways. Moreover, it was found that eccDNAfib-L could promote the transcription of fib-L gene. Overall, the results of the present study not only provide a novel perspective on the mechanism of silk gland development and silk protein synthesis but also complement previously reported genome-scale eccDNA data supporting that eccDNAs are common in eukaryotes.

A K-17 serotype specific Klebsiella phage JKP2 with biofilm reduction potential

Klebsiella pneumoniae is an opportunistic pathogen responsible for nearly one-third of all Gram-negative infections. Increasing antibiotic resistance has pushed scientists to look for alternative therapeutics. Bacteriophages have emerged as one of the promising alternatives. In the current study, the Klebsiella phage JKP2 was isolated from a sewage sample and characterized against the K-17 serotype of K. pneumoniae. It produced bulls-eye-shaped clear plaques and has a latent period of 45 min with a burst size of 70 pfu/cell. It remained stable at tested pH (5 to 10) and temperatures (37 to 60 °C). Its optimum temperature for long-term storage is 4 °C and -80 °C. The JKP2 showed its infectivity against the K. pneumoniae K-17 serotype only. It controlled planktonic cells of K. pneumoniae 12 h post-incubation. At MOI-1, it efficiently eliminated 98% of 24 and 96% of 48-hour-old biofilm and 86% and 82% of mature biofilm of day 3 and 4, respectively. The JKP2 has an icosahedral capsid of 54 ± 0.5 nm with a short, non-contractile tail, measuring 12 ± 0.2 nm. It possesses a double-stranded DNA genome of 43.2 kbp with 54.1% GC content and encodes 54 proteins, including 29 with known functions and 25 with unknown functions. JKP2 was classified as Drulisvirus within the Autographiviridae family. It uses a T7-like direct terminal repeat strategy for genome packaging. JKP2 can be applied safely for therapeutic purposes as it does not encode an integrase or repressor genes, antibiotic resistance genes, bacterial virulence factors, and mycotoxins.

The Dynamic Transcription Activator-Like Effector Family of Xanthomonas.

Transcription activator-like effectors (TALEs) are bacterial proteins that are injected into the eukaryotic nucleus to act as transcriptional factors and function as key virulence factors of the phytopathogen Xanthomonas. TALEs are translocated into plant host cells via the type III secretion system and induce the expression of host susceptibility (S) genes to facilitate disease. The unique modular DNA binding domains of TALEs comprise an array of nearly identical direct repeats that enable binding to DNA targets based on the recognition of a single nucleotide target per repeat. The very nature of TALE structure and function permits the proliferation of TALE genes and evolutionary adaptations in the host to counter TALE function, making the TALE-host interaction the most dynamic story in effector biology. The TALE genes appear to be a relatively young effector gene family, with a presence in all virulent members of some species and absent in others. Genome sequencing has revealed many TALE genes throughout the xanthomonads, and relatively few have been associated with a cognate S gene. Several species, including Xanthomonas oryzae pv. oryzae and X. citri pv. citri, have near absolute requirement for TALE gene function, while the genes appear to be just now entering the disease interactions with new fitness contributions to the pathogens of tomato and pepper among others. Deciphering the simple and effective DNA binding mechanism also has led to the development of DNA manipulation tools in fields of gene editing and transgenic research. In the three decades since their discovery, TALE research remains at the forefront of the study of bacterial evolution, plant-pathogen interactions, and synthetic biology. We also discuss critical questions that remain to be addressed regarding TALEs.

Bacteriolytic Potential of Enterococcus Phage iF6 Isolated from "Sextaphag®" Therapeutic Phage Cocktail and Properties of Its Endolysins, Gp82 and Gp84.

The number of infections caused by antibiotic-resistant strains of bacteria is growing by the year. The pathogenic bacterial species Enterococcus faecalis and Enterococcus faecium are among the high priority candidate targets for the development of new therapeutic antibacterial agents. One of the most promising antibacterial agents are bacteriophages. According to the WHO, two phage-based therapeutic cocktails and two medical drugs based on phage endolysins are currently undergoing clinical trials. In this paper, we describe the virulent bacteriophage iF6 and the properties of two of its endolysins. The chromosome of the iF6 phage is 156,592 bp long and contains two direct terminal repeats, each 2108 bp long. Phylogenetically, iF6 belongs to the Schiekvirus genus, whose representatives are described as phages with a high therapeutic potential. The phage demonstrated a high adsorption rate; about 90% of iF6 virions attached to the host cells within one minute after the phage was added. Two iF6 endolysins were able to lyse enterococci cultures in both logarithmic and stationary growth phases. Especially promising is the HU-Gp84 endolysin; it was active against 77% of enterococci strains tested and remained active even after 1 h incubation at 60 °C. Thus, iF6-like enterococci phages appear to be a promising platform for the selection and development of new candidates for phage therapy.

Excision of Integrated Human Herpesvirus 6A Genomes Using CRISPR/Cas9 Technology.

Human herpesviruses 6A and 6B are betaherpesviruses that can integrate their genomes into the telomeres of latently infected cells. Integration can also occur in germ cells, resulting in individuals who harbor the integrated virus in every cell of their body and can pass it on to their offspring. This condition is termed inherited chromosomally integrated HHV-6 (iciHHV-6) and affects about 1% of the human population. The integrated HHV-6A/B genome can reactivate in iciHHV-6 patients and in rare cases can also cause severe diseases including encephalitis and graft-versus-host disease. Until now, it has remained impossible to prevent virus reactivation or remove the integrated virus genome. Therefore, we developed a system that allows the removal of HHV-6A from the host telomeres using the CRISPR/Cas9 system. We used specific guide RNAs (gRNAs) targeting the direct repeat region at the ends of the viral genome to remove the virus from latently infected cells generated in vitro and iciHHV-6A patient cells. Fluorescence-activated cell sorting (FACS), quantitative PCR (qPCR), and fluorescence in situ hybridization (FISH) analyses revealed that the virus genome was efficiently excised and lost in most cells. Efficient excision was achieved with both constitutive and transient expression of Cas9. In addition, reverse transcription-qPCR (RT-qPCR) revealed that the virus genome did not reactivate upon excision. Taken together, our data show that our CRISPR/Cas9 approach allows efficient removal of the integrated virus genome from host telomeres. IMPORTANCE Human herpesvirus 6 (HHV-6) infects almost all humans and integrates into the telomeres of latently infected cells to persist in the host for life. In addition, HHV-6 can also integrate into the telomeres of germ cells, which results in about 80 million individuals worldwide who carry the virus in every cell of their body and can pass it on to their offspring. In this study, we develop the first system that allows excision of the integrated HHV-6 genome from host telomeres using CRISPR/Cas9 technology. Our data revealed that the integrated HHV-6 genome can be efficiently removed from the telomeres of latently infected cells and cells of patients harboring the virus in their germ line. Virus removal could be achieved with both stable and transient Cas9 expression, without inducing viral reactivation.

Revisiting mutagenesis at non-B DNA motifs in the human genome.

Non-B DNA structures formed by repetitive sequence motifs are known instigators of mutagenesis in experimental systems. Analyzing this phenomenon computationally in the human genome requires careful disentangling of intrinsic confounding factors, including overlapping and interrupted motifs and recurrent sequencing errors. Here, we show that accounting for these factors eliminates all signals of repeat-induced mutagenesis that extend beyond the motif boundary, and eliminates or dramatically shrinks the magnitude of mutagenesis within some motifs, contradicting previous reports. Mutagenesis not attributable to artifacts revealed several biological mechanisms. Polymerase slippage generates frequent indels within every variety of short tandem repeat motif, implicating slipped-strand structures. Interruption-correcting single nucleotide variants within short tandem repeats may originate from error-prone polymerases. Secondary-structure formation promotes single nucleotide variants within palindromic repeats and duplications within direct repeats. G-quadruplex motifs cause recurrent sequencing errors, whereas mutagenesis at Z-DNAs is conspicuously absent.

Characterization of Extensively Drug-Resistant Salmonella enterica Serovar Kentucky Sequence Type 198 Isolates from Chicken Meat Products in Xuancheng, China.

The purpose of this study was to characterize extensively drug-resistant Salmonella enterica serovar Kentucky sequence type 198 (ST198) isolates from chicken meat products. Ten S. Kentucky strains obtained from chicken meat products in Xuancheng, China, carried 12 to 17 resistance genes, such as blaCTX-M-55, rmtB, tet(A), floR, and fosA3, combined with mutations within gyrA (S83F and D87N) and parC (S80I), resulting in resistance to numerous antimicrobial agents, including the clinically important antibiotics cephalosporin, ciprofloxacin, tigecycline, and fosfomycin. These S. Kentucky isolates shared a close phylogenetic relationship (21 to 36 single-nucleotide polymorphisms [SNPs]) and showed close genetic relatedness to two human clinical isolates from China. Three S. Kentucky strains were subjected to whole-genome sequencing using Pacific Biosciences (PacBio) single-molecule real-time (SMRT) technology. All antimicrobial resistance genes were located on their chromosomes and clustered in one multiresistance region (MRR) and Salmonella genomic island (SGI) SGI1-K. The MRRs in three S. Kentucky strains were bounded by IS26 at both ends and were inserted downstream of the bcfABCDEFG cluster with 8-bp direct repeats. The MRRs were related to those of IncHI2 plasmids but differed by insertions, deletions, and rearrangements of multiple segments involving resistance genes and plasmid backbones. This finding suggests that the MRR fragment possibly originates from IncHI2 plasmids. Four SGI1-K variants with slight differences were identified in 10 S. Kentucky strains. Mobile elements, particularly IS26, play an essential role in forming distinct MRRs and SGI1-K structures. In conclusion, the emergence of extensively drug-resistant S. Kentucky ST198 strains containing numerous chromosomally located resistance genes is alarming and needs continued surveillance. IMPORTANCE Salmonella spp. are important foodborne pathogens, and multidrug-resistant (MDR) Salmonella strains have become a serious threat to clinical therapy. MDR S. Kentucky ST198 strains have been increasingly reported from various sources and have become a global risk. In this study, we described extensively drug-resistant S. Kentucky ST198 strains from chicken meat products from a city in China. Numerous resistance genes are clustered in the chromosomes of S. Kentucky ST198 strains, possibly acquired with the help of mobile elements. This would facilitate the spread of numerous resistance genes as intrinsic chromosomal genes within this global epidemic clone, with the potential to capture more resistance genes. The emergence and dissemination of extensively drug-resistant S. Kentucky ST198 pose a severe clinical and public health threat; therefore, continuous surveillance is warranted.

The Telomeric Repeats of HHV-6A Do Not Determine the Chromosome into Which the Virus Is Integrated

Human herpes virus 6A (HHV-6A) is able to integrate into the telomeric and subtelomeric regions of human chromosomes representing chromosomally integrated HHV-6A (ciHHV-6A). The integration starts from the right direct repeat (DRR) region. It has been shown experimentally that perfect telomeric repeats (pTMR) in the DRR region are required for the integration, while the absence of the imperfect telomeric repeats (impTMR) only slightly reduces the frequency of HHV-6 integration cases. The aim of this study was to determine whether telomeric repeats within DRR may define the chromosome into which the HHV-6A integrates. We analysed 66 HHV-6A genomes obtained from public databases. Insertion and deletion patterns of DRR regions were examined. We also compared TMR within the herpes virus DRR and human chromosome sequences retrieved from the Telomere-to-Telomere consortium. Our results show that telomeric repeats in DRR in circulating and ciHHV-6A have an affinity for all human chromosomes studied and thus do not define a chromosome for integration.

Characterisation of the complete mitochondrial genome of Taraxacum mongolicum revealed five repeat-mediated recombinations.

We reported the mitochondrial genome of Taraxacum mongolicum for the first time. Five pairs of repeats that can mediate recombination were validated, leading to multiple conformations of genome. Taraxacum mongolicum belongs to the Asteraceae family and has important pharmaceutical value. To explore the possible interaction between the organelle genomes, we assembled the complete mitochondrial genome (mitogenome) of T. mongolicum using Illumina and Oxford Nanopore sequencing data. This genome corresponded to a circular molecule 304,467bp long. It encodes 52 unique genes including 31 protein-coding, 3 ribosomal RNA (rRNA) and 18 transfer RNA (tRNA) genes. In addition to the single circular conformation, the existence of alternative conformations mediated by five repetitive sequences in the mitogenome was identified and validated. Recombination mediated by the inverted repeats resulted in two conformations. Conversely, recombination mediated by the two direct repeats broke one large circular molecule into two subgenomic circular molecules. Furthermore, we identified 12 homologous fragments by comparing the sequences of mitogenome and plastome, including eight complete tRNA genes. Lastly, we identified a total of 278 RNA-editing sites in protein-coding sequences based on RNA-seq data. Among them, cox1 and nad5 gene has the most sites (21), followed by the nad2 gene with 19 sites. We successfully validated 213 predicted RNA-editing sites using PCR amplification and Sanger sequencing. This project reported the first mitogenome of T. mongolicum and demonstrated its multiple conformations generated by repeat-mediated recombination. This genome could provide critical information for the molecular breeding of T. mongolicum, and also be used as a reference genome for other species of the genus Taraxacum.

Tandem repeats in giant archaeal Borg elements undergo rapid evolution and create new intrinsically disordered regions in proteins.

Borgs are huge, linear extrachromosomal elements associated with anaerobic methane-oxidizing archaea. Striking features of Borg genomes are pervasive tandem direct repeat (TR) regions. Here, we present six new Borg genomes and investigate the characteristics of TRs in all ten complete Borg genomes. We find that TR regions are rapidly evolving, recently formed, arise independently, and are virtually absent in host Methanoperedens genomes. Flanking partial repeats and A-enriched character constrain the TR formation mechanism. TRs can be in intergenic regions, where they might serve as regulatory RNAs, or in open reading frames (ORFs). TRs in ORFs are under very strong selective pressure, leading to perfect amino acid TRs (aaTRs) that are commonly intrinsically disordered regions. Proteins with aaTRs are often extracellular or membrane proteins, and functionally similar or homologous proteins often have aaTRs composed of the same amino acids. We propose that Borg aaTR-proteins functionally diversify Methanoperedens and all TRs are crucial for specific Borg-host associations and possibly cospeciation.

Deletions across the SARS-CoV-2 Genome: Molecular Mechanisms and Putative Functional Consequences of Deletions in Accessory Genes

The analysis of deletions may reveal evolutionary trends and provide new insight into the surprising variability and rapidly spreading capability that SARS-CoV-2 has shown since its emergence. To understand the factors governing genomic stability, it is important to define the molecular mechanisms of deletions in the viral genome. In this work, we performed a statistical analysis of deletions. Specifically, we analyzed correlations between deletions in the SARS-CoV-2 genome and repetitive elements and documented a significant association of deletions with runs of identical (poly-) nucleotides and direct repeats. Our analyses of deletions in the accessory genes of SARS-CoV-2 suggested that there may be a hypervariability in ORF7A and ORF8 that is not associated with repetitive elements. Such recurrent search in a "sequence space" of accessory genes (that might be driven by natural selection) did not yet cause increased viability of the SARS-CoV-2 variants. However, deletions in the accessory genes may ultimately produce new variants that are more successful compared to the viral strains with the conventional architecture of the SARS-CoV-2 accessory genes.