Transposable Phage Research Articles

Exploiting CRISPR/Cas system in controlling antibiotic resistance of bacteria

The emergence and spread of antimicrobial resistance is a threat to human health, therefore there is a need to explore new strategies to combat antibiotic-resistant bacteria. Antimicrobial resistance genes are mainly transmitted through horizontal gene transfer (HGT) mediated by transposable elements, phages, and plasmids, which leads to the emergence of multidrug-resistant bacteria. Clustered Regularly interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated proteins (Cas) protect archaebacteria and eubacteria from potentially harmful foreign DNA such as plasmids, transposable elements, and phages. Several studies have introduced the CRISPR/Cas system as an innovative and effective strategy to trigger antibiotic resistance and virulent genes in bacteria. Therefore, this study aimed to introduce the CRISPR-Cas system in controlling antibiotic resistance of bacteria. We hope exploiting CRISPR/Cas could provide a bright future for the treatment of disastrous infections with resistant and virulent organisms.

Comparative genomic analysis of clinical Enterococcus faecalis distinguishes strains isolated from the bladder

BackgroundEnterococcus faecalis is the most commonly isolated enterococcal species in clinical infection. This bacterium is notorious for its ability to share genetic content within and outside of its species. With this increased proficiency for horizontal gene transfer, tremendous genomic diversity within this species has been identified. Many researchers have hypothesized E. faecalis exhibits niche adaptation to establish infections or colonize various parts of the human body. Here, we hypothesize that E. faecalis strains isolated from the human bladder will carry unique genomic content compared to clinical strains isolated from other sources.ResultsThis analysis includes comparison of 111 E. faecalis genomes isolated from bladder, urogenital, blood, and fecal samples. Phylogenomic comparison shows no association between isolation source and lineage; however, accessory genome comparison differentiates blood and bladder genomes. Further gene enrichment analysis identifies gene functions, virulence factors, antibiotic resistance genes, and plasmid-associated genes that are enriched or rare in bladder genomes compared to urogenital, blood, and fecal genomes. Using these findings as training data and 682 publicly available genomes as test data, machine learning classifiers successfully distinguished between bladder and non-bladder strains with high accuracy. Genes identified as important for this differentiation were often related to transposable elements and phage, including 3 prophage species found almost exclusively in bladder and urogenital genomes.ConclusionsE. faecalis strains isolated from the bladder contain unique genomic content when compared to strains isolated from other body sites. This genomic diversity is most likely due to horizontal gene transfer, as evidenced by lack of phylogenomic clustering and enrichment of transposable elements and prophages. Investigation into how these enriched genes influence host-microbe interactions may elucidate gene functions required for successful bladder colonization and disease establishment.

Unexplored diversity and ecological functions of transposable phages.

Phages are prevalent in diverse environments and play major ecological roles attributed to their tremendous diversity and abundance. Among these viruses, transposable phages (TBPs) are exceptional in terms of their unique lifestyle, especially their replicative transposition. Although several TBPs have been isolated and the life cycle of the representative phage Mu has been extensively studied, the diversity distribution and ecological functions of TBPs on the global scale remain unknown. Here, by mining TBPs from enormous microbial genomes and viromes, we established a TBP genome dataset (TBPGD), that expands the number of accessible TBP genomes 384-fold. TBPs are prevalent in diverse biomes and show great genetic diversity. Based on taxonomic evaluations, we propose the categorization of TBPs into four viral groups, including 11 candidate subfamilies. TBPs infect multiple bacterial phyla, and seem to infect a wider range of hosts than non-TBPs. Diverse auxiliary metabolic genes (AMGs) are identified in the TBP genomes, and genes related to glycoside hydrolases and pyrimidine deoxyribonucleotide biosynthesis are highly enriched. Finally, the influences of TBPs on their hosts are experimentally examined by using the marine bacterium Shewanella psychrophila WP2 and its infecting transposable phage SP2. Collectively, our findings greatly expand the genetic diversity of TBPs, and comprehensively reveal their potential influences in various ecosystems.

Transposable Prophages in Leptospira: An Ancient, Now Diverse, Group Predominant in Causative Agents of Weil's Disease.

The virome associated with the corkscrew shaped bacterium Leptospira, responsible for Weil’s disease, is scarcely known, and genetic tools available for these bacteria remain limited. To reduce these two issues, potential transposable prophages were searched in Leptospiraceae genomes. The 236 predicted transposable prophages were particularly abundant in the most pathogenic leptospiral clade, being potentially involved in the acquisition of virulent traits. According to genomic similarities and phylogenies, these prophages are distantly related to known transposable phages and are organized into six groups, one of them encompassing prophages with unusual TA-TA ends. Interestingly, structural and transposition proteins reconstruct different relationships between groups, suggesting ancestral recombinations. Based on the baseplate phylogeny, two large clades emerge, with specific gene-contents and high sequence divergence reflecting their ancient origin. Despite their high divergence, the size and overall genomic organization of all prophages are very conserved, a testimony to the highly constrained nature of their genomes. Finally, similarities between these prophages and the three known non-transposable phages infecting L. biflexa, suggest gene transfer between different Caudovirales inside their leptospiral host, and the possibility to use some of the transposable prophages in that model strain.

Characterization and Genomic Analysis of ɸSHP3, a New Transposable Bacteriophage Infecting Stenotrophomonas maltophilia.

This study describes a novel transposable bacteriophage, ɸSHP3, continuously released by Stenotrophomonas maltophilia strain c31. Morphological observation and genomic analysis revealed that ɸSHP3 is a siphovirus with a 37,611-bp genome that encodes 51 putative proteins. Genomic comparisons indicated that ɸSHP3 is a B3-like transposable phage. Its genome configuration is similar to that of Pseudomonas phage B3, except for the DNA modification module. Similar to B3-like phages, the putative transposase B of ɸSHP3 is a homolog of the type two secretion component ExeA, which is proposed to serve as a potential virulence factor. Moreover, most proteins of ɸSHP3 have homologs in transposable phages, but only ɸSHP3 carries an RdgC-like protein encoded by gene 3, which exhibits exonuclease activity in vitro Two genes and their promoters coding for ɸSHP3 regulatory proteins were identified and appear to control the lytic-lysogenic switch. One of the proteins represses one promoter activity and confers immunity to ɸSHP3 superinfection in vivo The short regulatory region, in addition to the canonical bacterial promoter sequences, displays one LexA and two CpxR recognition sequences. This suggests that LexA and the CpxR/CpxA two-component system might be involved in the control of the ɸSHP3 genetic switch.IMPORTANCES. maltophilia is an emerging global pathogenic bacterium that displays genetic diversity in both environmental and clinical strains. Transposable phages have long been known to improve the genetic diversity of bacterial strains by transposition. More than a dozen phages of S. maltophilia have been characterized. However, no transposable phage infecting S. maltophilia has been reported to date. Characterization of the first transposable phage, ɸSHP3, from S. maltophilia will contribute to our understanding of host-phage interactions and genetic diversity, especially the interchange of genetic materials among S. maltophilia.

A novel vieuvirus from multidrug-resistant Acinetobacter baumannii.

Bacteriophages are considered the most abundant biological entities on earth, and they are able to modulate the populations of their bacterial hosts. Although the potential of bacteriophages has been accepted as an alternative strategy to combat multidrug-resistant pathogenic bacteria, there still exists a considerable knowledge gap regarding their genetic diversity, which hinders their use as antimicrobial agents. In this study, we undertook a genomic and phylogenetic characterization of the phage Ab11510-phi, which was isolated from a multidrug-resistant Acinetobacter baumannii strain (Ab11510). We found that Ab11510-phi has a narrow host range and belongs to a small group of transposable phages of the genus Vieuvirus that have only been reported to infect Acinetobacter bacteria. Finally, we showed that Ab11510-phi (as well as other vieuvirus phages) has a high level of mosaicism. On a broader level, we demonstrate that comparative genomics and phylogenetic analysis are necessary tools for the proper characterization of phage diversity.

Prophages and Past Prophage-Host Interactions Revealed by CRISPR Spacer Content in a Fish Pathogen.

The role of prophages in the evolution, diversification, or virulence of the fish pathogen Flavobacterium columnare has not been studied thus far. Here, we describe a functional spontaneously inducing prophage fF4 from the F. columnare type strain ATCC 23463, which is not detectable with commonly used prophage search methods. We show that this prophage type has a global distribution and is present in strains isolated from Finland, Thailand, Japan, and North America. The virions of fF4 are myoviruses with contractile tails and infect only bacterial strains originating from Northern Finland. The fF4 resembles transposable phages by similar genome organization and several gene orthologs. Additional bioinformatic analyses reveal several species in the phylum Bacteroidetes that host a similar type of putative prophage, including bacteria that are important animal and human pathogens. Furthermore, a survey of F. columnare Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) spacers indicate a shared evolutionary history between F. columnare strains and the fF4 phage, and another putative prophage in the F. columnare strain ATCC 49512, named p49512. First, CRISPR spacer content from the two CRISPR loci (types II-C and VI-B) of the fF4 lysogen F. columnare ATCC 23463 revealed a phage terminase protein-matching spacer in the VI-B locus. This spacer is also present in two Chinese F. columnare strains. Second, CRISPR analysis revealed four F. columnare strains that contain unique spacers targeting different regions of the putative prophage p49512 in the F. columnare strain ATCC 49512, despite the geographical distance or genomovar of the different strains. This suggests a common ancestry for the F. columnare prophages and different host strains.

Role of enterotoxigenic Escherichia coli prophage in spreading antibiotic resistance in a porcine-derived environment.

Enterotoxigenic Escherichia coli (ETEC) cause acute secretory diarrhoea in pigs, posing a great economic loss to the swine industry. This study analysed the prevalence and genetic characteristics of prophages from 132 ETEC isolates from symptomatic pigs to determine their potential for spreading antibiotic resistance. A total of 1105 potential prophages were identified, and the distribution of the genome size showed three 'overlapping' trends. Similarity matrix comparison showed that prophages correlated with the ETEC lineage distribution, and further identification of these prophages corroborated the lineage specificity. In total, 1206 antibiotic resistance genes (ARGs) of 52 different categories were identified in 132 ETEC strains; among these, 2.65% (32/1206) of ARGs were found to be carried by prophages. Analysis of flanking sequences showed that almost all the ARGs could be grouped into two types: 'blaTEM-1B ' and 'classic class 1 integron (IntI1)'. They co-occurred with a strictly conserved recombinase and transposon Tn3 family but with a difference: the 'blaTEM-1B type' prophages exhibited a classic Tn2 transposon structure with 100% sequence identity, whereas the 'IntI1 type' co-occurred with the TnAs2 transposon with only 84% sequence identity. These results imply that ARGs might be pervasive in natural bacterial populations through transmission by transposable bacteriophages.

Transposable temperate phages promote the evolution of divergent social strategies in Pseudomonas aeruginosa populations

Transposable temperate phages randomly insert into bacterial genomes, providing increased supply and altered spectra of mutations available to selection, thus opening alternative evolutionary trajectories. Transposable phages accelerate bacterial adaptation to new environments, but their effect on adaptation to the social environment is unclear. Using experimental evolution of Pseudomonas aeruginosa in iron-limited and iron-rich environments, where the cost of producing cooperative iron-chelating siderophores is high and low, respectively, we show that transposable phages promote divergence into extreme siderophore production phenotypes. Iron-limited populations with transposable phages evolved siderophore over-producing clones alongside siderophore non-producing cheats. Low siderophore production was associated with parallel mutations in pvdgenes, encoding pyoverdine biosynthesis, and pqs genes, encoding quinolone signaling, while high siderophore production was associated with parallel mutations in phenazine-associated gene clusters. Notably, some of these parallel mutations were caused by phage insertional inactivation. These data suggest that transposable phages, which are widespread in microbial communities, can mediate the evolutionary divergence of social strategies.

Transposable temperate phages promote the evolution of divergent social strategies in Pseudomonas aeruginosa populations.

Transposable temperate phages randomly insert into bacterial genomes, providing increased supply and altered spectra of mutations available to selection, thus opening alternative evolutionary trajectories. Transposable phages accelerate bacterial adaptation to new environments, but their effect on adaptation to the social environment is unclear. Using experimental evolution of Pseudomonas aeruginosa in iron-limited and iron-rich environments, where the cost of producing cooperative iron-chelating siderophores is high and low, respectively, we show that transposable phages promote divergence into extreme siderophore production phenotypes. Iron-limited populations with transposable phages evolved siderophore overproducing clones alongside siderophore non-producing cheats. Low siderophore production was associated with parallel mutations in pvd genes, encoding pyoverdine biosynthesis, and pqs genes, encoding quinolone signalling, while high siderophore production was associated with parallel mutations in phenazine-associated gene clusters. Notably, some of these parallel mutations were caused by phage insertional inactivation. These data suggest that transposable phages, which are widespread in microbial communities, can mediate the evolutionary divergence of social strategies.

Complete Genome Sequence Analysis and Characterization of Selected Iron Regulation Genes of Pasteurella Multocida Serotype A Strain PMTB2.1.

Although more than 100 genome sequences of Pasteurella multocida are available, comprehensive and complete genome sequence analysis is limited. This study describes the analysis of complete genome sequence and pathogenomics of P. multocida strain PMTB2.1. The genome of PMTB2.1 has 2176 genes with more than 40 coding sequences associated with iron regulation and 140 virulence genes including the complete tad locus. The tad locus includes several previously uncharacterized genes such as flp2, rcpC and tadV genes. A transposable phage resembling to Mu phages was identified in P. multocida that has not been identified in any other serotype yet. The multi-locus sequence typing analysis assigned the PMTB2.1 genome sequence as type ST101, while the comparative genome analysis showed that PMTB2.1 is closely related to other P. multocida strains with the genomic distance of less than 0.13. The expression profiling of iron regulating-genes of PMTB2.1 was characterized under iron-limited environment. Results showed significant changes in the expression profiles of iron-regulating genes (p < 0.05) whereas the highest expression of fecE gene (281 fold) at 30 min suggests utilization of the outer-membrane proteins system in iron acquisition at an early stage of growth. This study showed the phylogenomic relatedness of P. multocida and improved annotation of important genes and functional characterization of iron-regulating genes of importance to the bacterial growth.

Phage Mu Gam protein promotes NHEJ in concert with Escherichia coli ligase

The Gam protein of transposable phage Mu is an ortholog of eukaryotic and bacterial Ku proteins, which carry out nonhomologous DNA end joining (NHEJ) with the help of dedicated ATP-dependent ligases. Many bacteria carry Gam homologs associated with either complete or defective Mu-like prophages, but the role of Gam in the life cycle of Mu or in bacteria is unknown. Here, we show that MuGam is part of a two-component bacterial NHEJ DNA repair system. Ensemble and single-molecule experiments reveal that MuGam binds to DNA ends, slows the progress of RecBCD exonuclease, promotes binding of NAD+-dependent Escherichia coli ligase A, and stimulates ligation. In vivo, Gam equally promotes both precise and imprecise joining of restriction enzyme-digested linear plasmid DNA, as well as of a double-strand break (DSB) at an engineered I-SceI site in the chromosome. Cell survival after the induced DSB is specific to the stationary phase. In long-term growth competition experiments, particularly upon treatment with a clastogen, the presence of gam in a Mu lysogen confers a distinct fitness advantage. We also show that the role of Gam in the life of phage Mu is related not to transposition but to protection of genomic Mu copies from RecBCD when viral DNA packaging begins. Taken together, our data show that MuGam provides bacteria with an NHEJ system and suggest that the resulting fitness advantage is a reason that bacteria continue to retain the gam gene in the absence of an intact prophage.

Transposition Behavior Revealed by High-Resolution Description of Pseudomonas Aeruginosa Saltovirus Integration Sites.

Transposable phages, also called saltoviruses, of which the Escherichia coli phage Mu is the reference, are temperate phages that multiply their genome through replicative transposition at multiple sites in their host chromosome. The viral genome is packaged together with host DNA at both ends. In the present work, genome sequencing of three Pseudomonas aeruginosa transposable phages, HW12, 2P1, and Ab30, incidentally gave us access to the location of thousands of replicative integration sites and revealed the existence of a variable number of hotspots. Taking advantage of deep sequencing, we then designed an experiment to study 13,000,000 transposon integration sites of bacteriophage Ab30. The investigation revealed the presence of 42 transposition hotspots adjacent to bacterial interspersed mosaic elements (BIME) accounting for 5% of all transposition sites. The rest of the sites appeared widely distributed with the exception of coldspots associated with low G-C content segments, including the putative O-antigen biosynthesis cluster. Surprisingly, 0.4% of the transposition events occurred in a copy of the phage genome itself, indicating that the previously described immunity against such events is slightly leaky. This observation allowed drawing an image of the phage chromosome supercoiling into four loops.

Extension of the transposable bacterial virus family: two genomic organisations among phages and prophages with a Tn552-related transposase

Mu-like transposable phages and prophages have been isolated from, or predicted, in a wide range of bacterial phyla. However, related B3-like transposable phages, which differ in their genome organisation and the DDE transposase and transposition activator they code for have thus far been restricted to a very limited set of hosts. Through sequence similarity searches, we have now expanded the number of predicted B3-like prophages and uncovered a third genomic organisation. These new genomes, although only prophages, further illustrate the previously reported mosaicism existing in the proposed “Saltoviridae” family of Caudovirales, and further support the proposal to move morphology criteria (contractile vs. flexible or short tail, i.e. Myo-vs. Sipho- or Podoviridae) from the family to the subfamily level in the taxonomic classification of the Caudovirales.

Transposable Bacteriophages as Genetic Tools.

Phage Mu is the paradigm of a growing family of bacteriophages that infect a wide range of bacterial species and replicate their genome by replicative transposition. This molecular process, which is used by other mobile genetic elements to move within genomes, involves the profound rearrangement of the host genome [chromosome(s) and plasmid(s)] and can be exploited for the genetic analysis of the host bacteria and the in vivo cloning of host genes. In this chapter we review Mu-derived constructs that optimize the phage as a series of genetic tools that could inspire the development of similarly efficient tools from other transposable phages for a large spectrum of bacteria.

Use of RP4::Mini-Mu for Gene Transfer.

Gene cloning is an invaluable technique in genetic analysis and exploitation of genetic properties of a broad range of bacteria. Numerous in vitro molecular cloning protocols have been devised but the efficiency of these techniques relies on the frequency with which the recombinant DNA can be introduced in the recipient strain. Here, we describe an in vivo gene transfer and cloning technique based on transposable bacteriophage Mu property to rearrange its host genome. This technique uses the broad host range plasmid RP4 carrying a transposable mini-MuA+ derivative and was successfully used as well in enteric as in environmental nonenteric bacteria.

Temperate phages both mediate and drive adaptive evolution in pathogen biofilms

Temperate phages drive genomic diversification in bacterial pathogens. Phage-derived sequences are more common in pathogenic than nonpathogenic taxa and are associated with changes in pathogen virulence. High abundance and mobilization of temperate phages within hosts suggests that temperate phages could promote within-host evolution of bacterial pathogens. However, their role in pathogen evolution has not been experimentally tested. We experimentally evolved replicate populations of Pseudomonas aeruginosa with or without a community of three temperate phages active in cystic fibrosis (CF) lung infections, including the transposable phage, ɸ4, which is closely related to phage D3112. Populations grew as free-floating biofilms in artificial sputum medium, mimicking sputum of CF lungs where P. aeruginosa is an important pathogen and undergoes evolutionary adaptation and diversification during chronic infection. Although bacterial populations adapted to the biofilm environment in both treatments, population genomic analysis revealed that phages altered both the trajectory and mode of evolution. Populations evolving with phages exhibited a greater degree of parallel evolution and faster selective sweeps than populations without phages. Phage ɸ4 integrated randomly into the bacterial chromosome, but integrations into motility-associated genes and regulators of quorum sensing systems essential for virulence were selected in parallel, strongly suggesting that these insertional inactivation mutations were adaptive. Temperate phages, and in particular transposable phages, are therefore likely to facilitate adaptive evolution of bacterial pathogens within hosts.

Complete Genome Sequence of PM105, a New Pseudomonas aeruginosa B3-Like Transposable Phage.

The complete genome of the Pseudomonas aeruginosa bacteriophage PM105 is 39,593 bp long. The phage belongs to the B3 family of transposable Mu-like phages, as confirmed by the presence of bacterial DNA joined to the phage genome ends. PM105, together with other B3-like phages, form a newly arising species.

Repair of transposable phage Mu DNA insertions begins only when the E. coli replisome collides with the transpososome.

We report a new cellular interaction between the infecting transposable phage Mu and the host Escherichia coli replication machinery during repair of Mu insertions, which involves filling-in of short target gaps on either side of the insertion, concomitant with degradation of extraneous long flanking DNA (FD) linked to Mu. Using the FD as a marker to follow repair, we find that after transposition into the chromosome, the unrepaired Mu is indefinitely stable until the replication fork arrives at the insertion site, whereupon the FD is rapidly degraded. When the fork runs into a Mu target gap, a double strand end (DSE) will result; we demonstrate fork-dependent DSEs proximal to Mu. These findings suggest that Pol III stalled at the transpososome is exploited for co-ordinated repair of both target gaps flanking Mu without replicating the intervening 37 kb of Mu, disassembling the stable transpososome in the process. This work is relevant to all transposable elements, including retroviral elements like HIV-1, which share with Mu the common problem of repair of their flanking target gaps.

Identification and Molecular Characterisation of a Novel Mu-Like Bacteriophage, SfMu, of Shigella flexneri.

S. flexneri is the leading cause of bacillary dysentery in the developing countries. Several temperate phages originating from this host have been characterised. However, all S. flexneri phages known to date are lambdoid phages, which have the ability to confer the O-antigen modification of their host. In this study, we report the isolation and characterisation of a novel Mu-like phage from a serotype 4a strain of S. flexneri. The genome of phage SfMu is composed of 37,146 bp and is predicted to contain 55 open reading frames (orfs). Comparative genome analysis of phage SfMu with Mu and other Mu-like phages revealed that SfMu is closely related to phage Mu, sharing >90% identity with majority of its proteins. Moreover, investigation of phage SfMu receptor on the surface of the host cell revealed that the O-antigen of the host serves as the receptor for the adsorption of phage SfMu. This study also demonstrates pervasiveness of SfMu phage in S. flexneri, by identifying complete SfMu prophage strains of serotype X and Y, and remnants of SfMu in strains belonging to 4 other serotypes, thereby indicating that transposable phages in S. flexneri are not uncommon. The findings of this study contribute an advance in our current knowledge of S. flexneri phages and will also play a key role in understanding the evolution of S. flexneri.