Delivery of functional Cas:DNA nucleoprotein complexes into recipient bacteria through a type IV secretion system.

CRISPR-associated (Cas) endonucleases and their derivatives are widespread tools for the targeted genetic modification of both prokaryotic and eukaryotic genomes. A critical step of all CRISPR-Cas technologies is the delivery of the Cas endonuclease to the target cell. Here, we investigate the possibility of using bacterial conjugation to translocate Cas proteins into recipient bacteria.

A virally encoded tRNA neutralizes the PARIS antiviral defence system.

Viruses compete with each other for limited cellular resources, and some deliver defence mechanisms that protect the host from competing genetic parasites. The phage antirestriction induced system (PARIS) is a defence system, often encoded in viral genomes, that is composed of a 55 kDa ABC ATPase (AriA) and a 35 kDa TOPRIM nuclease (AriB). However, the mechanism by which AriA and AriB function in phage defence is unknown.

An antiplasmid system drives antibiotic resistance gene integration in carbapenemase-producing Escherichia coli lineages.

Plasmids carrying antibiotic resistance genes (ARG) are the main mechanism of resistance dissemination in Enterobacterales. However, the fitness-resistance trade-off may result in their elimination. Chromosomal integration of ARGs preserves resistance advantage while relieving the selective pressure for keeping costly plasmids.

Computational design of novel Cas9 PAM-interacting domains using evolution-based modelling and structural quality assessment.

We present here an approach to protein design that combines (i) scarce functional information such as experimental data (ii) evolutionary information learned from a natural sequence variants and (iii) physics-grounded modeling. Using a Restricted Boltzmann Machine (RBM), we learn a sequence model of a protein family. We use semi-supervision to leverage available functional information during the RBM training.

Phages and their satellites encode hotspots of antiviral systems.

Bacteria carry diverse genetic systems to defend against viral infection, some of which are found within prophages where they inhibit competing viruses. Phage satellites pose additional pressures on phages by hijacking key viral elements to their own benefit. Here, we show that E.

Generating functional protein variants with variational autoencoders.

The vast expansion of protein sequence databases provides an opportunity for new protein design approaches which seek to learn the sequence-function relationship directly from natural sequence variation. Deep generative models trained on protein sequence data have been shown to learn biologically meaningful representations helpful for a variety of downstream tasks, but their potential for direct use in the design of novel proteins remains largely unexplored. Here we show that variational autoencoders trained on a dataset of almost 70000 luciferase-like oxidoreductases can be used to generate novel, functional variants of the luxA bacterial luciferase.

Gene silencing with CRISPRi in bacteria and optimization of dCas9 expression levels.

The catalytic null mutant of the Cas9 endonuclease from the bacterial CRISPR immune system, known as dCas9, can be guided by a small RNA to bind DNA sequences of interest and block gene transcription in a strategy known as CRISPRi. This powerful gene silencing method has already been used in a large number of species and in high throughput screens. Here we provide detailed design rules, methods and novel vectors to perform CRISPRi experiments in S.

Genome-wide CRISPR-dCas9 screens in E. coli identify essential genes and phage host factors.

High-throughput genetic screens are powerful methods to identify genes linked to a given phenotype. The catalytic null mutant of the Cas9 RNA-guided nuclease (dCas9) can be conveniently used to silence genes of interest in a method also known as CRISPRi. Here, we report a genome-wide CRISPR-dCas9 screen using a starting pool of ~ 92,000 sgRNAs which target random positions in the chromosome of E.

PhageTerm: a tool for fast and accurate determination of phage termini and packaging mechanism using next-generation sequencing data.

The worrying rise of antibiotic resistance in pathogenic bacteria is leading to a renewed interest in bacteriophages as a treatment option. Novel sequencing technologies enable description of an increasing number of phage genomes, a critical piece of information to understand their life cycle, phage-host interactions, and evolution. In this work, we demonstrate how it is possible to recover more information from sequencing data than just the phage genome.

Competition between VanU(G) repressor and VanR(G) activator leads to rheostatic control of vanG vancomycin resistance operon expression.

Enterococcus faecalis BM4518 is resistant to vancomycin by synthesis of peptidoglycan precursors ending in D-alanyl-D-serine. In the chromosomal vanG locus, transcription of the resistance genes from the PYG resistance promoter is inducible and, upstream from these genes, there is an unusual three-component regulatory system encoded by the vanURS(G) operon from the P(UG) regulatory promoter. In contrast to the other van operons in enterococci, the vanG operon possesses the additional vanU(G) gene which encodes a transcriptional regulator whose role remains unknown.

D-Ala-d-Ser VanN-type transferable vancomycin resistance in Enterococcus faecium.

Enterococcus faecium UCN71, isolated from a blood culture, was resistant to low levels of vancomycin (MIC, 16 μg/ml) but susceptible to teicoplanin (MIC, 0.5 μg/ml). No amplification was observed with primers specific for the previously described glycopeptide resistance ligase genes, but a PCR product corresponding to a gene called vanN was obtained using degenerate primers and was sequenced.

Inducible expression eliminates the fitness cost of vancomycin resistance in enterococci.

Inducible vancomycin resistance in enterococci is due to a sophisticated mechanism that combines synthesis of cell wall peptidoglycan precursors with low affinity for glycopeptides and elimination of the normal target precursors. Although this dual mechanism, which involves seven genes organized in two operons, is predicted to have a high fitness cost, resistant enterococci have disseminated worldwide. We have evaluated the biological cost of VanB-type resistance due to acquisition of conjugative transposon Tn1549 in Enterococcus faecium and Enterococcus faecalis.

VanB-type Enterococcus faecium clinical isolate successively inducibly resistant to, dependent on, and constitutively resistant to vancomycin.

Three Enterococcus faecium strains isolated successively from the same patient, vancomycin-resistant strain BM4659, vancomycin-dependent strain BM4660, and vancomycin-revertant strain BM4661, were indistinguishable by pulsed-field gel electrophoresis and harbored plasmid pIP846, which confers VanB-type resistance. The vancomycin dependence of strain BM4660 was due to mutation P(175)L, which suppressed the activity of the host Ddl D-Ala:D-Ala ligase. Reversion to resistance in strain BM4661 was due to a G-to-C transversion in the transcription terminator of the vanRS(B) operon that lowered the free energy of pairing from -13.

New combinations of mutations in VanD-Type vancomycin-resistant Enterococcus faecium, Enterococcus faecalis, and Enterococcus avium strains.

We studied the clinical isolates Enterococcus faecium NEF1, resistant to high levels of vancomycin (MIC, 512 microg/ml) and teicoplanin (MIC, 64 microg/ml); Enterococcus faecium BM4653 and BM4656 and Enterococcus avium BM4655, resistant to moderate levels of vancomycin (MIC, 32 microg/ml) and to low levels of teicoplanin (MIC, 4 microg/ml); and Enterococcus faecalis BM4654, moderately resistant to vancomycin (MIC, 16 microg/ml) but susceptible to teicoplanin (MIC, 0.5 microg/ml). The strains were distinct, were constitutively resistant via the synthesis of peptidoglycan precursors ending in D-alanyl-D-lactate, and harbored a chromosomal vanD gene cluster that was not transferable.

Modes and modulations of antibiotic resistance gene expression.

Since antibiotic resistance usually affords a gain of function, there is an associated biological cost resulting in a loss of fitness of the bacterial host. Considering that antibiotic resistance is most often only transiently advantageous to bacteria, an efficient and elegant way for them to escape the lethal action of drugs is the alteration of resistance gene expression. It appears that expression of bacterial resistance to antibiotics is frequently regulated, which indicates that modulation of gene expression probably reflects a good compromise between energy saving and adjustment to a rapidly evolving environment.

Binding sites of VanRB and sigma70 RNA polymerase in the vanB vancomycin resistance operon of Enterococcus faecium BM4524.

The vanB operon of Enterococcus faecium BM4524 which confers inducible resistance to vancomycin is composed of the vanR(B)S(B) gene encoding a two-component regulatory system and the vanY(B)WH(B)BX(B) resistance genes that are transcribed from promoters P(RB) and P(YB) respectively. In this study, primer extension revealed transcription start sites at 13 and 48 bp upstream from the start codon of vanR(B) and vanY(B), respectively, that allowed identification of -10 and -35 promoter motifs. The VanR(B) protein was overproduced in Escherichia coli, purified and phosphorylated (VanR(B)-P) non-enzymically with acetylphosphate.

Detection of the van alphabet and identification of enterococci and staphylococci at the species level by multiplex PCR.

A multiplex PCR assay was developed for detection of the six types of glycopeptide resistance characterized in enterococci and for identification of Enterococcus faecium, Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus epidermidis at the species level. Primers targeting the genes vanA, vanB, vanC, vanD, vanE, vanG, and ddl of E. faecium and E.

VanD-type vancomycin-resistant Enterococcus faecium and Enterococcus faecalis.

Enterococcus faecium clinical isolates A902 and BM4538, which were resistant to relatively high levels of vancomycin (128 and 64 microg/ml, respectively) and to low levels of teicoplanin (4 microg/ml), and Enterococcus faecalis clinical isolates BM4539 and BM4540, which were resistant to moderate levels of vancomycin (16 microg/ml) and susceptible to teicoplanin (0.25 microg/ml), were studied. They were constitutively resistant by synthesis of peptidoglycan precursors ending with d-alanyl-d-lactate and harbored a chromosomal vanD gene cluster which was not transferable by conjugation to other enterococci.

Expression of glycopeptide-resistance gene in response to vancomycin and teicoplanin in the cardiac vegetations of rabbits infected with VanB-type Enterococcus faecalis.

VanB-type resistance in enterococci corresponds to resistance to vancomycin but not to resistance to the related glycopeptide teicoplanin, because the vanB gene cluster is activated by the VanR(B)-VanS(B) 2-component regulatory system in response to vancomycin but not to teicoplanin. Mutations in the vanS(B) gene allow for constitutive or teicoplanin-inducible expression of the resistance genes. To analyze in vivo expression of the van genes in rabbits with experimental endocarditis, a VanB-type Enterococcus faecalis with a transcriptional fusion between the P(YB) promoter of resistance genes and the gfpmut1 gene for the green-fluorescent protein in the chromosome was constructed.

A six amino acid deletion, partially overlapping the VanSB G2 ATP-binding motif, leads to constitutive glycopeptide resistance in VanB-type Enterococcus faecium.

Enterococcus faecium clinical isolate BM4524, resistant to vancomycin and susceptible to teicoplanin, harboured a chromosomal vanB cluster, including the vanSB/vanRB two-component system regulatory genes. Enterococcus faecium strain BM4525, isolated two weeks later from the same patient, was resistant to high levels of both glycopeptides. The ddl gene of BM4525 had a 2 bp insertion leading to an impaired d-alanine:d-alanine ligase.

The vanG glycopeptide resistance operon from Enterococcus faecalis revisited.

Acquired VanG-type resistance to vancomycin (MIC = 16 micro g ml(-1)) but susceptibility to teicoplanin in Enterococcus faecalis BM4518 and WCH9 is due to the inducible synthesis of peptidoglycan precursors ending in d-alanine-d-serine. The vanG cluster, assigned to a chromosomal location, was composed of genes recruited from various van operons. The 3' end encoded VanG, a d-Ala:d-Ser ligase, VanXY(G), a putative bifunctional d,d-peptidase and VanT(G), a serine racemase: VanG and VanT(G) were implicated in the synthesis of d-Ala:d-Ser as in VanC- and VanE-type strains.

Influence of VanD type resistance on activities of glycopeptides in vitro and in experimental endocarditis due to Enterococcus faecium.

The consequences of VanD type glycopeptide resistance on the activity of vancomycin and teicoplanin were evaluated in vitro and in a rabbit model of aortic endocarditis with VanD type clinical isolate Enterococcus faecium BM4339 (MICs: vancomycin, 64 microg/ml; teicoplanin, 4 microg/ml) and its susceptible derivative BM4459 (MICs: vancomycin, 1 microg/ml; teicoplanin, 1 microg/ml). The two antibiotics were inactive against BM4339 in vivo, in terms both of reduction of bacterial counts and of prevention of emergence of glycopeptide-resistant subpopulations, despite using teicoplanin at concentrations greater than the MIC for VanD strains. This could be due to the high inoculum effect also observed in vitro with BM4339 and two other VanD type isolates against both antibiotics.