Novel genomic islands and a new vanD-subtype in the first sporadic VanD-type vancomycin resistant enterococci in Norway

Background Vancomycin-resistant enterococci (VRE) represent several types of transferable vancomycin resistance gene clusters. The vanD type, associated with moderate to high level vancomycin resistance, has only sporadically been described in clinical isolates. The aim of this study was to perform a genetic characterization of the first VanD-type VRE strains detected in Norway. Methods The VanD-type VRE-strains (n = 6) from two patient cases were examined by antimicrobial susceptibility testing and whole genome sequencing (WGS) to uncover Van-phenotype, strain phylogeny, the vanD gene clusters, and their genetic surroundings. The putative transferability of vanD was examined by circularization PCR and filter mating. Results The VanD-type Enterococcus faecium (n = 4) and Enterococcus casseliflavus (n = 2) strains recovered from two cases (A and B), expressed moderate to high level vancomycin resistance (MIC 64—>256 mg/L) and various levels of teicoplanin susceptibility (MIC 2—>256 mg/L). WGS analyses revealed phylogenetically different E. faecium strains (A1, A2, and A3 of case A and B1 from case B) as well as vanD gene clusters located on different novel genomic islands (GIs). The E. casseliflavus strains (B2 and B3 of case B) were not clonally related, but harbored nearly identical novel GIs. The vanD cluster of case B strains represents a novel vanD-subtype. All the vanD-GIs were integrated at the same chromosomal site and contained genes consistent with a Clostridiales origin. Circular forms of the vanD-GIs were detected in all strains except B1. Transfer of vanD to an E. faecium recipient was unsuccessful. Conclusions We describe the first VanD-type E. casseliflavus strains, a novel vanD-subtype, and three novel vanD-GIs with a genetic content consistent with a Clostridiales order origin. Despite temporal occurrence, case A and B E. faecium strains were phylogenetically diverse and harbored different vanD subtypes and vanD-GIs.

Truly epigenetic: A centromere finds a “neo” home

Murillo-Pineda and colleagues (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202007210) use CRISPR-Cas9–based genetic engineering in human cells to induce a new functional centromere at a naive chromosomal site. Long-read DNA sequencing at the neocentromere provides firm evidence that centromere establishment is a truly epigenetic event.

Polymorphic centromere locations in the pathogenic yeast Candida parapsilosis

ABSTRACTCentromeres pose an evolutionary paradox: strongly conserved in function, but rapidly changing in sequence and structure. However, in the absence of damage, centromere locations are usually conserved within a species. We report here that isolates of the pathogenic yeast species Candida parapsilosis exhibit within-species polymorphism for the location of centromeres on two of its eight chromosomes. Its old centromeres have an inverted-repeat (IR) structure, whereas its new centromeres have no obvious structural features, but are located within 30 kb of the old site. Centromeres can therefore move naturally from one chromosomal site to another, apparently spontaneously and in the absence of any significant changes in DNA sequence. Our observations are consistent with a model where all centromeres are genetically determined, such as by the presence of short or long IRs, or the ability to form cruciforms. We also find that centromeres have been hotspots for genomic rearrangements in the C. parapsilosis clade.

TFIIIC localizes budding yeast ETC sites to the nuclear periphery

Chromatin function requires specific three-dimensional architectures of chromosomes. We investigated whether Saccharomyces cerevisiae extra TFIIIC (ETC) sites, which bind the TFIIIC transcription factor but do not recruit RNA polymerase III, show specific intranuclear positioning. We show that six of the eight known S. cerevisiae ETC sites localize predominantly at the nuclear periphery, and that ETC sites retain their tethering function when moved to a new chromosomal location. Several lines of evidence indicate that TFIIIC is central to the ETC peripheral localization mechanism. Mutating or deleting the TFIIIC-binding consensus ablated ETC -site peripheral positioning, and inducing degradation of the TFIIIC subunit Tfc3 led to rapid release of an ETC site from the nuclear periphery. We find, moreover, that anchoring one TFIIIC subunit at an ectopic chromosomal site causes recruitment of others and drives peripheral tethering. Localization of ETC sites at the nuclear periphery also requires Mps3, a Sad1-UNC-84–domain protein that spans the inner nuclear membrane. Surprisingly, we find that the chromatin barrier and insulator functions of an ETC site do not depend on correct peripheral localization. In summary, TFIIIC and Mps3 together direct the intranuclear positioning of a new class of S. cerevisiae genomic loci positioned at the nuclear periphery.

Aurora B dynamics at centromeres create a diffusion-based phosphorylation gradient

Aurora B kinase is essential for successful cell division and regulates spindle assembly and kinetochore–microtubule interactions. The kinase localizes to the inner centromere until anaphase, but many of its substrates have distinct localizations, for example on chromosome arms and at kinetochores. Furthermore, substrate phosphorylation depends on distance from the kinase. How the kinase reaches substrates at a distance and how spatial phosphorylation patterns are determined are unknown. In this paper, we show that a phosphorylation gradient is produced by Aurora B concentration and activation at centromeres and release and diffusion to reach substrates at a distance. Kinase concentration, either at centromeres or at another chromosomal site, is necessary for activity globally. By experimentally manipulating dynamic exchange at centromeres, we demonstrate that the kinase reaches its substrates by diffusion. We also directly observe, using a fluorescence resonance energy transfer–based biosensor, phosphorylation spreading from centromeres after kinase activation. We propose that Aurora B dynamics and diffusion from the inner centromere create spatial information to regulate cell division.

Evolution and Spread of a Multidrug-Resistant Proteus mirabilis Clone with Chromosomal AmpC-Type Cephalosporinases in Europe

ABSTRACTProteus mirabilisisolates obtained in 1999 to 2008 from three European countries were analyzed; all carried chromosomal AmpC-type cephalosporinaseblaCMYgenes from aCitrobacter freundiiorigin (blaCMY-2-like genes). Isolates from Poland harbored severalblaCMYgenes (blaCMY-4,blaCMY-12,blaCMY-14,blaCMY-15, andblaCMY-38and the new geneblaCMY-45), while isolates from Italy and Greece harboredblaCMY-16only. Earlier isolates withblaCMY-4orblaCMY-12, recovered in France from Greek and Algerian patients, were also studied. All isolates showed striking similarities. TheirblaCMYgenes resided within ISEcp1transposition modules, named Tn6093, characterized by a 110-bp distance between ISEcp1andblaCMY, and identical fragments of bothC. freundiiDNA and a ColE1-type plasmid backbone. Moreover, these modules were inserted into the same chromosomal site, within thepepQgene. Since ColE1 plasmids carrying ISEcp1with similarC. freundiiDNA fragments (Tn6114) had been identified earlier, it is likely that a similar molecule had mediated at some stage this DNA transfer betweenC. freundiiandP. mirabilis. In addition, isolates withblaCMY-12,blaCMY-15, andblaCMY-38genes harbored a secondblaCMYcopy within a shorter ISEcp1module (Tn6113), always inserted downstream of theppiDgene. Sequence analysis of all mobileblaCMY-2-like genes indicated that those integrated in theP. mirabilischromosome form a distinct cluster that may have evolved by the stepwise accumulation of mutations. All of these observations, coupled to strain typing data, suggest that theblaCMYgenes studied here may have originated from a single ISEcp1-mediated mobilization-transfer-integration process, followed by the spread and evolution of aP. mirabilisclone over time and a large geographic area.

Development of genetic tools for in vivo virulence analysis of Streptococcus sanguinis

Completion of the genome sequence of Streptococcus sanguinis SK36 necessitates tools for further characterization of this species. It is often desirable to insert antibiotic resistance markers and other exogenous genes into the chromosome; therefore, we sought to identify a chromosomal site for ectopic expression of foreign genes, and to verify that insertion into this site did not affect important cellular phenotypes. We designed three plasmid constructs for insertion of erm, aad9 or tetM resistance determinants into a genomic region encoding only a small (65 aa) hypothetical protein. To determine whether this insertion affected important cellular properties, SK36 and its erythromycin-resistant derivative, JFP36, were compared for: (i) growth in vitro, (ii) genetic competence, (iii) biofilm formation and (iv) virulence for endocarditis in the rabbit model of infective endocarditis (IE). The spectinomycin-resistant strain, JFP56, and tetracycline-resistant strain, JFP76, were also tested for virulence in vivo. Insertion of erm did not affect growth, competence or biofilm development of JFP36. Recovery of bacteria from heart valves of co-inoculated rabbits was similar to wild-type for JFP36, JFP56 and JFP76, indicating that IE virulence was not significantly affected. The capacity for mutant complementation in vivo was explored in an avirulent ssaB mutant background. Expression of ssaB from its predicted promoter in the target region restored IE virulence. Thus, the chromosomal site utilized is a good candidate for further manipulations of S. sanguinis. In addition, the resistant strains developed may be further applied as controls to facilitate screening for virulence factors in vivo.

Structural and temporal regulation of centromeric chromatinThis paper is one of a selection of papers published in this Special Issue, entitled 29th Annual International Asilomar Chromatin and Chromosomes Conference, and has undergone the Journal’s usual peer review process.

Normal inheritance of genetic material requires that chromosomes segregate faithfully during mitosis and meiosis. The kinetochore is a unique structure that attaches chromosomes to the microtubule spindle, monitors proper chromosome attachment to the spindle through the mitotic checkpoint, and couples spindle and motor protein forces to move chromosomes during prometaphase and anaphase. The centromere is a specialized chromosomal site that is the structural and functional foundation for kinetochore formation, and is characterized by a unique type of chromatin that needs to be reconstituted after each replication cycle. In this review, recent progress in understanding the structural nature of this chromatin and how it is specifically maintained through cell division are discussed.