Background/objectives: Pseudomonas aeruginosa can cause community-acquired infections affecting various body sites. The present retrospective study investigated the genetic diversity of 173 isolates (166 clinical, 7 environmental) of P. aeruginosa collected from clinical pathology laboratories in Abidjan, Côte d’Ivoire (2001–2011). Methods: Multiple-Locus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) using 13 loci was applied to all isolates and compared to published MLVA data. The antibiotics status of the isolates was compiled when available and compared to published profiles. Results: Among 95 isolates analyzed for their antibiotics status, 14 displayed concerning resistance profiles: five multidrug-resistant (MDR) and nine extensively drug-resistant (XDR). MLVA typing revealed a high genetic diversity (>130 genotypes), with many genotypes represented by a single strain. Notably, thirteen clusters (≥4 related isolates) were observed. Some clusters displayed close genetic relatedness to isolates from France, Korea, and well-studied strains (ST560, LES and PA14). Comparative analysis suggested the presence of international high-risk MDR clones (CC233, CC111) in Côte d’Ivoire. Importantly, MLVA clustering revealed a close relationship of CC235-MDR strains with a locally identified cluster (group 9). Conclusions: These findings support MLVA as a reliable and cost-effective tool for low-resource settings, allowing the selection of relevant strains for future whole genome sequence analyses. This approach can improve outbreak investigations and public health interventions aimed at curbing MDR P. aeruginosa transmission within hospitals and at the national level.

ABSTRACT We report the discovery of a satellite-helper phage system with a novel type of dependence on a tail donor. The Acinetobacter baumannii satellite podovirus Aci01-2-Phanie (short name Phanie) uses a phage phi29-like DNA replication and packaging mode. Its linear 11,885 bp dsDNA genome bears 171 bp inverted terminal repeats (ITR). Phanie is related to phage DU-PP-III from Pectobacterium and to members of the Astrithrvirus from Salmonella enterica . Together, they form a new clade of phages with 27% to 30% identity over the whole genome. Detailed 3D protein structure prediction and mass spectrometry analyses demonstrate that Phanie encodes its capsid structural genes and genes necessary to form a short tail. However, our study reveals that Phanie virions are non-infectious unless they associate with the contractile tail of an unrelated phage, Aci01-1, to produce chimeric myoviruses. Following the coinfection of Phanie with myovirus Aci01-1, hybrid viral particles composed of Phanie capsids and Aci01-1 contractile tails are assembled together with Phanie and Aci01-1 particles. IMPORTANCE There are few reported cases of satellite-helper phage interactions but many more may be yet undiscovered. Here we describe a new mode of satellite phage dependence on a helper phage. Phanie, like phage phi29, replicates its linear dsDNA by a protein primed-mechanism and protects it inside podovirus-like particles. However, these particles are defective, requiring the acquisition of the tail from a myovirus helper for production of infectious virions. The formation of chimeras between a phi29-like podovirus and a helper contractile tail reveals an unexpected association between very different bacterial viruses.

The Acinetobacter baumannii bacteriophage Aci01-1, which belongs to the genus Saclayvirus of the order Caudoviricetes, has an icosahedral head and a contractile rigid tail. We report that Aci01-1 has, attached to the tail conical tip, a remarkable 146-nm-long flexible fiber with seven beads and a terminal knot. Its putative gene coding for a 241.36-kDa tail fiber protein is homologous to genes in Aci01-1-related and unrelated phages. Analysis of its 3D structure using AlphaFold provides a structural model for the fiber observed by electron microscopy. We also identified a putative receptor of the phage on the bacterial capsule that is hypothesized to interact with the Aci01-1 long fiber.

The detection of genome variants, including point mutations, indels and structural variants, is a fundamental and challenging computational problem. We address here the problem of variant detection between two deep-sequencing (DNA-seq) samples, such as two human samples from an individual patient, or two samples from distinct bacterial strains. The preferred strategy in such a case is to align each sample to a common reference genome, collect all variants and compare these variants between samples. Such mapping-based protocols have several limitations. DNA sequences with large indels, aggregated mutations and structural variants are hard to map to the reference. Furthermore, DNA sequences cannot be mapped reliably to genomic low complexity regions and repeats. We introduce 2-kupl, a k-mer based, mapping-free protocol to detect variants between two DNA-seq samples. On simulated and actual data, 2-kupl achieves higher accuracy than other mapping-free protocols. Applying 2-kupl to prostate cancer whole exome sequencing data, we identify a number of candidate variants in hard-to-map regions and propose potential novel recurrent variants in this disease. We developed a mapping-free protocol for variant calling between matched DNA-seq samples. Our protocol is suitable for variant detection in unmappable genome regions or in the absence of a reference genome.

Sixteen bacteriophages of Achromobacter xylosoxidans distributed into four genera have been isolated from sewage water in Abidjan, Côte d'Ivoire, using a single clinical strain, and their genomes have been sequenced. Three podoviruses belonged to the genus Phikmvvirus, and these represent the first A. xylosoxidans phages of this genus. Seven podoviruses, distributed into three groups, belonged to the genus Jwalphavirus. Among the siphoviruses, three revealed similarities to Pseudomonas phage 73 and members of the genus Septimatrevirus, and three were YuA-like phages. The virulence of these phages toward a panel of 10 genetically diverse strains was tested, with the phiKMV-like phages showing the broadest host range.

The CRISPR JournalVol. 3, No. 6 First CutFree AccessNew Insights into CRISPR ArraysChristine PourcelChristine PourcelAddress correspondence to: Christine Pourcel, PhD, , Institute for Integrative Biology of the Cell (I2BC), SSFA, Université Paris-Saclay, Bât 400, 91400 Orsay, France, E-mail Address: christine.pourcel@i2bc.paris-saclay.frUniversité Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France.Search for more papers by this authorPublished Online:18 Dec 2020https://doi.org/10.1089/crispr.2020.29111.cpoAboutSectionsView articleView PDFView PDF Plus ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleA new study in this issue from Eugene Koonin and his colleagues (Shmakov et al. page 535) investigates isolated CRISPR arrays and defines true “orphan” arrays that may be associated with yet unknown CRISPR-Cas systemsCRISPR-Cas systems have evolved into a very complex family, as reflected by the composition and functionality of the cas gene clusters and the diversity of CRISPR repeats. Despite a remarkably conserved structure, CRISPR arrays display heterogeneity in size, nature and length of their leader, and their capacity to expand, all characteristics that are poorly understood.Whereas the arrays in archaea and many extremophiles can store >100 spacers, in most species, CRISPR arrays are no longer than a few dozen spacers, which may be due to deletion by recombination between repeats. The leader, a 100–200 bp sequence flanking the array on its proximal side,1 is involved in transcription and in adaptation through binding of the Cas1–Cas2 complex onto the leader-repeat junction.2 It coevolves with repeats, the Cas1 protein, and the protospacer adjacent motif.3 Interestingly, in many genomes, a single cluster of cas genes may be associated with several arrays (up to 24 in Nocardiopsis alba ATCC BAA-2165 for a single Type IA cas gene cluster; https://crisprcas.i2bc.paris-saclay.fr/4), which possess the same or very similar repeat sequences and do not share any spacer. They are generally flanked by a leader with 70–90% similarity over >100 bp, and can be targets for spacer acquisition.Multiple arrays are found more frequently in genomes bearing a Type I and/or Type III system, as in most archaea and bacteria, but are rare in Type II systems. They can be scattered or grouped, often near the cas gene cluster. The multiplication of CRISPR arrays with their leader is probably not an accident. An analysis of 5,796 genomes bearing a single Type I or Type III cas gene cluster shows that the number of spacers increases with the number of CRISPR arrays (Fig. 1A). This may suggest that in cells where the size of the CRISPR arrays seems to be limited, the existence of multiple copies that can be transcribed efficiently and are available for insertion of spacers provides a selective advantage. This would prevent the loss of isolated CRISPR arrays, as observed for different gene families.5FIG. 1. Multiplication of CRISPR arrays. (A) Box plot representation of the correlation between the number of CRISPR arrays and the total number of spacers in bacteria possessing a single Type I or Type III CRISPR-Cas system. Half (50%) of the observations are inside the box, and the median is represented by a continuous orange line. Outliers are represented by dots outside the whiskers. (B) Proposed mechanisms to account for the creation of new CRISPR arrays by duplication or reverse transcription and insertion of a leader-repeat RNA. The black diamonds represent repeats. Colored boxes are spacers. The leader is shown with a gray bar marked L. The red arrows are transcription starts. The Cas1–Cas2 complex binds to the leader repeat junction and allows insertion of a new spacer and duplication of the repeat.Writing in this issue of The CRISPR Journal, Shmakov et al. present the results of their exploration of 13,116 complete genome sequences in search for isolated CRISPR arrays, that is, those with no nearby cas genes.6 Over the past 15 years, Eugene Koonin's group has been a leader in cataloguing Cas proteins, allowing the precise definition and classification of the CRISPR-Cas types and subtypes. In the latest work, they apply the minCED tool (https://github.com/ctSkennerton/minced) and a filtering pipeline to identify CRISPR arrays that are not adjacent to cas genes. In agreement with previous observations made with a more limited number of sequences, they find that 90% of arrays that have no cas gene in their vicinity possess a repeat shared with a cas-accompanied array in the same or different genomes and a leader. Interestingly, they identify 116 unique bona fide arrays distributed across 89 clusters, for which repeats show no similarity to known CRISPR, and are therefore “orphans” until the cas they complement is uncovered.To explain the existence of isolated arrays with similarity to cas-adjacent arrays, the authors analyzed their region of insertion, and found that transposable elements were three times more frequent than in other part of the genome. Therefore, they propose a plausible scenario: the loss of the cas gene cluster following transfer of a complete CRISPR-Cas system. Another hypothesis is de novo generation by off-target insertion of spacers into a repeat-like sequence as observed experimentally7 and dissemination by mobile genetic elements.These mechanisms may apply in some situations but can hardly explain the majority of isolated-CRISPR arrays. The systematic deletion of cas clusters seems improbable, and the creation of arrays following off-target insertion of spacers is incompatible with the presence of the conserved leader sequence in a majority of isolated arrays. I propose two additional mechanisms that could lead to production of copies of the leader and the first repeat of a cas-associated array and allow their insertion at different positions. In cases where arrays are clustered and separated only by their leader, such as in Fusobacterium pseudoperiodonticum KCOM 2555 (with 11 arrays within 9 kb), it might result from tandem duplication of the original array and exclusion of preexisting spacers by recombination between repeats (Fig. 1B). Excision of some arrays and nonhomologous recombination could account for scattered distribution of new copies.Another mechanism could involve retro-transcription of a mature RNA bearing the leader and the proximal repeat (Fig. 1C). RNA-seq analysis has shown that in addition to transcription initiated at the leader, antisense transcripts can be common due to the presence of promoters within repeats or in some spacers.8,9 Furthermore, in some arrays such as in Myxococcus xhantus, the leader is transcribed from the cas gene cluster readthrough.10 Ancillary proteins with reverse transcriptase activity, group II intron or RT-Cas1 fusion proteins in Type III systems, might be actors in the generation of new CRISPR arrays.11,12In the past few years, the term “orphan” has been used somewhat indiscriminately to define any array that is not physically associated with cas genes, whether the repeat was shared with a cas-associated array or not. They were also called “split arrays,”13 although the presence of the leader shows that the majority do not correspond to an array that has been fragmented. Shmakov et al. propose a list of arrays that do not seem to be associated with known cas genes and will be the subject of future investigations. It is important that each genome presumably holding orphan arrays be reexamined with caution such as the cited Ktedonobacterales bacterium SCAWS-G2 genome which does possess Type IIID and Type ID cas genes and 8 isolated arrays.Much work remains in order to understand the dynamics of the CRISPR-Cas systems and particularly the birth and multiplication of CRISPR arrays. The organization we see today is the result of millions of years of slow evolution, and we need to analyze more genomes to reconstruct these ancient events.References1. Alkhnbashi OS, Shah SA, Garrett RA, et al. Characterizing leader sequences of CRISPR loci. Bioinformatics 2016;32:i576–i585. DOI: 10.1093/bioinformatics/btw454. Crossref, Medline, Google Scholar2. Sasnauskas G, Siksnys V. CRISPR adaptation from a structural perspective. Curr Opin Struct Biol 2020;65:17–25. DOI: 10.1016/j.sbi.2020.05.015. Crossref, Medline, Google Scholar3. Shah SA, Garrett RA. CRISPR/Cas and Cmr modules, mobility and evolution of adaptive immune systems. Res Microbiol 2011;162:27–38. DOI: 10.1016/j.resmic.2010.09.001. Crossref, Medline, Google Scholar4. Pourcel C, Touchon M, Villeriot N, et al. CRISPRCasdb a successor of CRISPRdb containing CRISPR arrays and cas genes from complete genome sequences, and tools to download and query lists of repeats and spacers. Nucleic Acids Res 2020;48:D535–D544. DOI: 10.1093/nar/gkz915. Crossref, Medline, Google Scholar5. Reams AB, Neidle EL. Selection for gene clustering by tandem duplication. Annu Rev Microbiol 2004;58:119–142. DOI: 10.1146/annurev.micro.58.030603.123806. Crossref, Medline, Google Scholar6. Shmakov SA, Utkina I, Wolf YI, et al. CRISPR arrays away from cas genes. CRISPR J 2020. Google Scholar7. Nivala J, Shipman SL, Church GM. Spontaneous CRISPR loci generation in vivo by non-canonical spacer integration. Nat Microbiol 2018;3:310–318. DOI: 10.1038/s41564-017-0097-z. Crossref, Medline, Google Scholar8. Zoephel J, Randau L. RNA-Seq analyses reveal CRISPR RNA processing and regulation patterns. Biochem Soc Trans 2013;41:1459–1463. DOI: 10.1042/BST20130129. Crossref, Medline, Google Scholar9. Heidrich N, Dugar G, Vogel J, et al. Investigating CRISPR RNA biogenesis and function using RNA-seq. Methods Mol Biol 2015;1311:1–21. DOI: 10.1007/978-1-4939-2687-9_1. Crossref, Medline, Google Scholar10. Bernal-Bernal D, Abellon-Ruiz J, Iniesta AA, et al. Multifactorial control of the expression of a CRISPR-Cas system by an extracytoplasmic function sigma/anti-sigma pair and a global regulatory complex. Nucleic Acids Res 2018;46:6726–6745. DOI: 10.1093/nar/gky475. Crossref, Medline, Google Scholar11. Toro N, Martinez-Abarca F, Gonzalez-Delgado A. The reverse transcriptases associated with CRISPR-Cas systems. Sci Rep 2017;7:7089. DOI: 10.1038/s41598-017-07828-y. Crossref, Medline, Google Scholar12. Gonzalez-Delgado A, Mestre MR, Martinez-Abarca F, et al. Spacer acquisition from RNA mediated by a natural reverse transcriptase-Cas1 fusion protein associated with a type III-D CRISPR-Cas system in Vibrio vulnificus. Nucleic Acids Res 2019;47:10202–10211. DOI: 10.1093/nar/gkz746. Crossref, Medline, Google Scholar13. Crawley A, Henriksen JR, Barrangou R. CRISPRdisco: an automated pipeline for the discovery and analysis of CRISPR-Cas systems. CRISPR J 2018;1:171–181. DOI: 10.1089/crispr.2017.0022. Link, Google ScholarFiguresReferencesRelatedDetails Volume 3Issue 6Dec 2020 InformationCopyright 2020, Mary Ann Liebert, Inc., publishersTo cite this article:Christine Pourcel.The CRISPR Journal.Dec 2020.422-424.http://doi.org/10.1089/crispr.2020.29111.cpoPublished in Volume: 3 Issue 6: December 18, 2020

A large proportion of non-coding sequences in prokaryotes are transcribed, playing an important role in the cell metabolism and defense against exogenous elements. This is the case of small RNAs and of clustered regularly interspaced short palindromic repeats “CRISPR” arrays. The CRISPR-Cas system is a defense mechanism that protects bacterial and archaeal genomes against invasions by mobile genetic elements such as viruses and plasmids. The CRISPR array, made of repeats separated by unique sequences called spacers, is transcribed but the nature of the promoter and of the transcription regulation is not well known. We describe the Transcription Orientation Pipeline (TOP) which makes use of transcriptome sequence reads to recover those corresponding to a selected sequence, and determine the direction of the transcription. CRISPR repeat sequences extracted from CRISPRCasdb were used to test the performances of the program. Statistical tests show that CRISPR elements can be reliably oriented with as little as 100 mapped reads. TOP was applied to all the available RNA-Seq Illumina sequencing archives from species possessing a CRISPR array, allowing comparisons with programs dedicated to the orientation of CRISPR repeats. In addition TOP was used to analyze small non-coding RNAs in Staphylococcus aureus , demonstrating that it is a valuable and convenient tool to investigate the transcription orientation of any sequence of interest. Availability and implementation TOPs is implemented in Python and is freely available via the I2BC github repository at https://github.com/i2bc/TOP .

In Archaea and Bacteria, the arrays called CRISPRs for 'clustered regularly interspaced short palin-dromic repeats' and the CRISPR associated genes or cas provide adaptive immunity against viruses, plas-mids and transposable elements. Short sequences called spacers, corresponding to fragments of invading DNA, are stored in-between repeated sequences. The CRISPR-Cas systems target sequences homologous to spacers leading to their degradation. To facilitate investigations of CRISPRs, we developed 12 years ago a website holding the CRISPRdb. We now propose CRISPRCasdb, a completely new version giving access to both CRISPRs and cas genes. We used CRISPRCasFinder, a program that identifies CRISPR arrays and cas genes and determine the system's type and subtype, to process public whole genome assemblies. Strains are displayed either in an alphabetic list or in taxonomic order. The database is part of the CRISPR-Cas(++) website which also offers the possibility to analyse submitted sequences and to download programs. A BLAST search against lists of repeats and spacers extracted from the database is proposed. To date, 16 990 complete prokaryote genomes (16 650 bacteria from 2973 species and 340 archaea from 300 species) are included. CRISPR-Cas systems were found in 36% of Bacteria and 75% of Archaea strains.

Pseudomonas aeruginosa is responsible for long-term infections and is particularly resistant to treatments when hiding inside the extracellular matrix or biofilms. Phage therapy might represent an alternative to antibiotic treatment, but up to 10% of clinical strains appear to resist multiple phages. We investigated the characteristics of P. aeruginosa clinical strains naturally resistant to phages and compared them to highly susceptible strains. The phage-resistant strains were defective in lipopolysaccharide (LPS) biosynthesis, were nonmotile and displayed an important degree of autolysis, releasing phages and pyocins. Complete genome sequencing of three resistant strains showed the existence of a large accessory genome made of multiple insertion elements, genomic islands, pyocins and prophages, including two phages performing lateral transduction. Mutations were found in genes responsible for the synthesis of LPS and/or type IV pilus, the major receptors for most phages. CRISPR-Cas systems appeared to be absent or inactive in phage-resistant strains, confirming that they do not play a role in the resistance to lytic phages but control the insertion of exogenous sequences. We show that, despite their apparent weakness, the multiphage-resistant strains described in this study displayed selective advantages through the possession of various functions, including weapons to eliminate other strains of the same or closely related species.

Five bacteriophages of Acinetobacter baumannii were isolated from sewage water in Abidjan, Côte d'Ivoire. Phages Aci01-1, Aci02-2, and Aci05 belong to an unclassified genus of the Myoviridae family, with double-stranded DNA (dsDNA) genomes, whereas Aci07 and Aci08 belong to the Fri1virus genus of the Podoviridae family of phages.

We report the first two cases of tuberculous coinfection with Mycobacterium tuberculosis and M. canettii. Both patients were young Djiboutian females with pulmonary tuberculosis (TB). One had a miliary pattern with concomitant human immunodeficiency virus infection. Both recovered completely with a standard four-drug anti-tuberculosis treatment regimen. Due to the different natural reservoirs and routes of infection of these two strains, our study supports the common belief that multiple strains of infection in TB are related to superinfection rather than concomitant infection.

USA. 20 Wadsworth Center, New York State Department of Health, Albany, New York, USA. 21 Department of Medicine, University of Cambridge, Cambridge, UK. 22 Molecular and Experimental Mycobacteriology, Research Center Borstel, Borstel, Germany. 23 German Center for Infection Research, Partner site Hamburg-Lübeck-Borstel-Riems, Germany. 24 Department of Genetics, University of Cambridge, Cambridge, UK. 25

Bacteria and their bacteriophages coexist and coevolve for the benefit of both in a mutualistic association. Multiple mechanisms are used by bacteria to resist phages in a trade-off between survival and maintenance of fitness. In vitro studies allow inquiring into the fate of virus and host in different conditions aimed at mimicking natural environment. We analyse here the mutations emerging in a clinical Pseudomonas aeruginosa strain in response to infection by Ab09, a N4-like lytic podovirus and describe a variety of chromosomal deletions and mutations conferring resistance. Some deletions result from illegitimate recombination taking place during long-term maintenance of the phage genome. Phage variants with mutations in a tail fiber gene are selected during pseudolysogeny with the capacity to infect resistant cells and produce large plaques. These results highlight the complex host/phage association and suggest that phage Ab09 promotes bacterial chromosome rearrangements. Finally this study points to the possible role of two bacterial genes in Ab09 phage adhesion to the cell, rpsB encoding protein S2 of the 30S ribosomal subunit and ORF1587 encoding a Wzy-like membrane protein involved in LPS biosynthesis.

BACKGROUND:Mycobacterium canettii forms part of the Mycobacterium tuberculosis complex. Mycobacterium canettii infections are mainly described in the Horn of Africa. The permanent presence of French soldiers in Djibouti raises the question of the risk of being infected with M. canettii. Here, we describe M. canettii infections among French military and their families between 1998 and 2015.METHODS:This retrospective study relied on 3 sources of data: the reference center for mycobacteria in the Biology Department at Percy Military Hospital in Paris, the French Military Center for Epidemiology and Public Health, and the scientific literature. After an exhaustive census of the strains, we studied the epidemiological data on 20 cases among French soldiers and their families.RESULTS:Twenty cases of M. canettii infections are reported, including 5 unpublished cases. Adenitis predominates (n = 15), especially in the cervico facial area and among children; 1 case was observed 1 month after dental care in Djibouti. The pulmonary forms were less frequent (n = 6), and 3 atypical forms are described. All patients had stayed in Djibouti.CONCLUSIONS:Cases of M. canettii infection among the French military consisted mainly of adenitis; disseminated forms were possible with immunodeficiency. Their evolution under specific treatments was comparable to that of tuberculosis. The presumed origin of the infection seemed to be environmental, possibly a water reservoir, and not due to human-to-human contagion.

Certain Pseudomonas aeruginosa strains produce a homolog of colicin M, namely, PaeM, that specifically inhibits peptidoglycan biosynthesis of susceptible P. aeruginosa strains by hydrolyzing the lipid II intermediate precursor. Two variants of this pyocin were identified whose sequences mainly differed in the N-terminal protein moiety, i.e., the region involved in the binding to the FiuA outer membrane receptor and translocation into the periplasm. The antibacterial activity of these two variants, PaeM1 and PaeM2, was tested against various P. aeruginosa strains comprising reference strains PAO1 and PA14, PaeM-producing strains, and 60 clinical isolates. Seven of these strains, including PAO1, were susceptible to only one variant (2 to PaeM1 and 5 to PaeM2), and 11 were affected by both. The remaining strains, including PA14 and four PaeM1 producers, were resistant to both variants. The differences in the antibacterial spectra of the two PaeM homologs prompted us to investigate the molecular determinants allowing their internalization into P. aeruginosa cells, taking the PAO1 strain that is susceptible to PaeM2 but resistant to PaeM1 as the indicator strain. Heterologous expression of fiuA gene orthologs from different strains into PAO1, site-directed mutagenesis experiments, and construction of PaeM chimeric proteins provided evidence that the cell susceptibility and discrimination differences between the PaeM variants resulted from a polymorphism of both the pyocin and the outer membrane receptor FiuA. Moreover, we found that a third component, TonB1, a protein involved in iron transport in P. aeruginosa, working together with FiuA and the ExbB/ExbD complex, was directly implicated in this discrimination.IMPORTANCE Bacterial antibiotic resistance constitutes a threat to human health, imposing the need for identification of new targets and development of new strategies to fight multiresistant pathogens. Bacteriocins and other weapons that bacteria have themselves developed to kill competitors are therefore of great interest and a valuable source of inspiration for us. Attention was paid here to two variants of a colicin M homolog (PaeM) produced by certain strains of P. aeruginosa that inhibit the growth of their congeners by blocking cell wall peptidoglycan synthesis. Molecular determinants allowing recognition of these pyocins by the outer membrane receptor FiuA were identified, and a receptor polymorphism affecting the susceptibility of P. aeruginosa clinical strains was highlighted, providing new insights into the potential use of these pyocins as an alternative to antibiotics.

Using 894 phylogenetically diverse genomes of the Mycobacterium tuberculosis complex (MTBC), we simulated in silico the ability of the Hain Lifescience GenoType MTBC assay to differentiate the causative agents of tuberculosis. Here, we propose a revised interpretation of this assay to reflect its strengths (e.g., it can distinguish some strains of Mycobacterium canettii and variants of Mycobacterium bovis that are not intrinsically resistant to pyrazinamide) and limitations (e.g., Mycobacterium orygis cannot be differentiated from Mycobacterium africanum ).

From 1987 and during the following 20 years, a few research teams exploring bacteria and archea genome sequences uncover the prokaryotic adaptative immune system made of the CRISPR sequence and associated cas genes. First believed to be similar to the eukaryote RNA interference system, CRISPR-Cas turned out to be unique and of an amazing genetic complexity. The comparative studies of CRISPR arrays and of cas, and later of microbiotes metagenomes allowed to propose an evolution scenario for these systems. The results demonstrate the importance of a naturalistic approach, without a priori, for the understanding of living organisms.

Hepatitis B virus (HBV) belongs to a family of DNA viruses, the hepadnaviruses, that preferentially infect the liver and are responsible for disease states ranging from acute hepatitis to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Other members are the woodchuck hepatitis virus (WHV), the ground squirrel hepatitis virus (GSHV), and the duck hepatitis virus (DHBV). The viral replication mechanism was described in DHBV-infected ducks and then confirmed with the other viruses. A mouse is transgenic when it is able to transmit to its progeny new genetic material, the transgene, in a manner identical to the endogenous genes in its chromosomes. Methylation of the HBV core gene region in the hepatocellular carcinoma cell line PLC/PRF/5, which produces only HBsAg, indicating that core gene transcription might have been repressed during tumor progression. An approach toward understanding the tissue specificity of viral synthesis is the search for viral cis-acting sequences that would be activated by factors present in specific cells.

Bacteria and bacteriophages coexist and coevolve, bacteriophages being obligatory predators exerting an evolutionary pressure on their prey. Mechanisms in action vary depending on the bacterial genomic content and on the regulation of the bacteriophage cycle. To assess the multiplicity of bacterial genes involved in resistance as well as the changes in the bacteriophage interactions with the bacteria, it is necessary to isolate and investigate large numbers of independent resistant variants. Here we describe protocols that have been applied to the study of Pseudomonas aeruginosa and four of its virulent bacteriophages belonging to the Podoviridae and Myoviridae bacteriophage families. Mutations are identified using whole genome sequencing of resistant variants. Phenotypic analyses are performed to describe the changes conferred by the mutations.

CRISPR (clustered regularly interspaced short palindromic repeats) arrays and their associated (Cas) proteins confer bacteria and archaea adaptive immunity against exogenous mobile genetic elements, such as phages or plasmids. CRISPRCasFinder allows the identification of both CRISPR arrays and Cas proteins. The program includes: (i) an improved CRISPR array detection tool facilitating expert validation based on a rating system, (ii) prediction of CRISPR orientation and (iii) a Cas protein detection and typing tool updated to match the latest classification scheme of these systems. CRISPRCasFinder can either be used online or as a standalone tool compatible with Linux operating system. All third-party software packages employed by the program are freely available. CRISPRCasFinder is available at https://crisprcas.i2bc.paris-saclay.fr.