Comparative genomics and pangenomics of vancomycin resistant and susceptible Enterococcus faecium from Irish hospitals across 20 years

Enterococcus faecium has emerged as an important nosocomial pathogen, which is increasingly difficult to treat due to the genetic acquisition of vancomycin resistance. Ireland exhibits a recalcitrant vancomycin resistant bloodstream infection rate compared to other developed countries. A set of 28 vancomycin resistant isolates was sequenced to construct a dataset alongside 61 other publicly available Irish genomes. This dataset was extensively analysed using in-silico methodologies and uncovered distinct evolutionary, coevolutionary, and clinically relevant population trends. These results suggest that a stable (in terms of genome size, GC%, and number of genes), yet genetically diverse population (in terms of gene content) of Enterococcus faecium persist in Ireland with acquired resistance arising via plasmid acquisition (vanA) or to a lesser extent, chromosomal recombination (vanB). Population analysis described five clusters with one cluster partitioned into four clades which transcend isolation dates. Pangenomic and recombination analyses revealed an open (whole genome and chromosomal specific) pangenome illustrating a rampant evolutionary pattern. Comparative resistomics and virulomics uncovered distinct chromosomal and mobilomal propensity for multidrug resistance, widespread chromosomal point-mutation mediated resistance, and chromosomal harboured arsenals of virulence factors. Comparative phagomics revealed a core prophagome of three prophages throughout the dataset. Interestingly, a potential difference in biofilm formation strategies was highlighted by coevolutionary analysis, suggesting differential biofilm genotypes between vanA and vanB isolates. These results highlight the evolutionary history of Irish Enterococcus faecium isolates and may provide an insight into underlying infection dynamics in a clinical setting.

Guide-directed DNA cleavage by a prokaryotic Argonaute protein induces chromosome recombination in Escherichia coli

Emerging evidence supports the argument that some prokaryotic argonautes (pAgos) serve as a defensive system against invasion of viruses and plasmids through guide DNAs (gDNAs) directed DNA cleavage. This DNA-guided DNA interference motivates research to induce genomic mutations via pAgo mediated cleavage. Here we demonstrate that CbAgo, a pAgo from Clostridium butyricum, is able to induce chromosomal recombination between direct repeat sequences via its gDNA-directed cleavage in Escherichia coli chromosome. We also show that CbAgo targeting can assist Lambda-Red recombineering in RecA-deficient strain. Our study reveals that cleavage by CbAgo in E. coli chromosome can be mutagenic and suggests its broader application in genetic manipulation.

DNA-RNA hybrids at DSBs interfere with repair by homologous recombination

DNA double strand breaks (DSBs) are the most harmful DNA lesions and their repair is crucial for cell viability and genome integrity. The readout of DSB repair may depend on whether DSBs occur at transcribed versus non-transcribed regions. Some studies have postulated that DNA-RNA hybrids form at DSBs to promote recombinational repair, but others have challenged this notion. To directly assess whether hybrids formed at DSBs promote or interfere with recombinational repair we have used plasmid and chromosomal-based systems for the analysis of DSB-induced recombination in Saccharomyces cerevisiae. We show that, as expected, DNA-RNA hybrid formation is stimulated at DSBs. In addition, mutations that promote DNA-RNA hybrid accumulation, such as hpr1∆ and rnh1∆ rnh201∆, cause high levels of plasmid loss when DNA breaks are induced at sites that are transcribed. Importantly, we show that high levels or unresolved DNA-RNA hybrids at the breaks interfere with their repair by homologous recombination. This interference is observed for both plasmid and chromosomal recombination and is independent of whether the DSB is generated by endonucleolytic cleavage or by DNA replication. These data support a model in which DNA-RNA hybrids form fortuitously at DNA breaks during transcription, and need to be removed to allow recombinational repair, rather than playing a positive role.

Human Spermatogenesis Tolerates Massive Size Reduction of the Pseudoautosomal Region

Mammalian male meiosis requires homologous recombination between the X and Y chromosomes. In humans, such recombination occurs exclusively in the short arm pseudoautosomal region (PAR1) of 2.699 Mb in size. Although it is known that complete deletion of PAR1 causes spermatogenic arrest, no studies have addressed to what extent male meiosis tolerates PAR1 size reduction. Here, we report two families in which PAR1 partial deletions were transmitted from fathers to their offspring. Cytogenetic analyses revealed that a ∼400-kb segment at the centromeric end of PAR1, which accounts for only 14.8% of normal PAR1 and 0.26% and 0.68% of the X and Y chromosomes, respectively, is sufficient to mediate sex chromosomal recombination during spermatogenesis. These results highlight the extreme recombinogenic activity of human PAR1. Our data, in conjunction with previous findings from animal studies, indicate that the minimal size requirement of mammalian PARs to maintain male fertility is fairly small.

Mismatch repair systems might facilitate the chromosomal recombination induced by N-nitrosodimethylamine, but not by N-nitrosodiethylamine, in Drosophila

Mismatch repair (MMR) systems play important roles in maintaining the high fidelity of genomic DNA. It is well documented that a lack of MMR increases the mutation rate, including base exchanges and small insertion/deletion loops; however, it is unknown whether MMR deficiency affects the frequency of chromosomal recombination in somatic cells. To investigate the effects of MMR on chromosomal recombination, we used the Drosophila wing-spot test, which efficiently detects chromosomal recombination. We prepared MMR (MutS)-deficient flies (spel1(−/−)) using a fly line generated in this study. The spontaneous mutation rate as measured by the wing-spot test was slightly higher in MutS-deficient flies than in wild-type (spel1(+/−)) flies. Previously, we showed that N-nitrosodimethylamine (NDMA)-induced chromosomal recombination more frequently than N-nitrosodiethylamine (NDEA) in Drosophila. When the wing-spot test was performed using MMR-deficient flies, unexpectedly, the rate of NDMA-induced mutation was significantly lower in spel1(−/−) flies than in spel1(+/−) flies. In contrast, the rate of mutation induced by NDEA was higher in spel1(−/−) flies than in spel1(+/−) flies. These results suggest that in Drosophila, the MutS homologue protein recognises methylated DNA lesions more efficiently than ethylated ones, and that MMR might facilitate mutational chromosomal recombination due to DNA double-strand breaks via the futile cycle induced by MutS recognition of methylated lesions.

SPRTN protease and SUMOylation coordinate DNA-protein crosslink repair to prevent genome instability

DNA-protein crosslinks (DPCs) are a specific type of DNA lesions where proteins are covalently attached to DNA. Unrepaired DPCs lead to genomic instability, cancer, neurodegeneration and accelerated ageing. DPC proteolysis was recently discovered as a specialised pathway for DPC repair. The DNA-dependent SPRTN protease and 26S proteasome emerged as as two independent proteolytic systems for DPC repair. DPCs are also repaired by homologous recombination (HR), a canonical DNA repair pathway. While studying the role of ubiquitin and SUMO in DPC repair, we identified mutually exclusive signalling mechanisms associated with DPC repair pathway choice. DPC modification by SUMO-1 favours SPRTN proteolysis as the preferred pathway for DPC repair. DPC SUMOylation counteracts DPC ubiquitination, which promotes DNA breaks and the switch to HR. We propose that modification of DPCs by SUMO-1 promotes SPRTN proteolysis, which is essential for DPC removal to prevent DNA replication defects, chromosomal recombination and genomic instability.

Detection of Intron22 Mutations in Iraqi Female Carriers in Wasit City with Hemophilia A

The background:One of the prevalent main concerns in the medical world is the identification of Intron22 mutations in the Factor VIII gene carried by Iraqi patient in Wasit town, in Iraq suffering Hemophilia A (classical hemophilia) which is related to a X-chromosome recessive haemorrhage afflictions as the result of a flaw in the coagulation factor VIII (FVIII). It is essentially related with F8 mutations of Intron22 in version which forms the most typical kind of mutations of blood afflictions worldwide involving half the patients suffering from severe Hemophilia A that possesses mutations, in addition to Intron 1 inversion suffered by 5% of severe Hemophilia A patients.All of the inversion mutations are suffered mainly by males,and uncommonly by females due to the intra chromosomal recombination among the homologous areas, in inversion 1 or 22, with extragenic copy posited the telomeric to the Factor VIII gene. Unfortunately, there is an absence in Iraq on researches pertaining blood affliction gene identification in persons who carries the Intron22 mutations exception in the current research.Aims of study:The objectives of the research is to to analyze through the detection mechanisms, the existence of Intron 22 mutations in the Factor VIII gene of 10 Hemophilia A Iraqi carriers cohort families. The hypothesis and anticipated result is that there will be a minimal margin of hazardous possibility for the recurrence. The hereditary F8 mutation is unknown to be present on the maternal side of the patient sufferer due to the possibilty of germline mosaics that exists within the community.