The prophages of Citrobacter rodentium represent a conserved family of horizontally-acquired mobile genetic elements associated with enteric evolution towards pathogenicity

Prophage mediated horizontal gene transfer (HGT) plays a key role in the evolution of bacteria, enabling access to new environmental niches, including pathogenicity. Citrobacter rodentium is a host-adapted intestinal mouse pathogen and important model organism for attaching and effacing (A/E) pathogens including the clinically significant enterohaemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli. Despite containing ten prophage genomic regions, including an active temperate phage, ΦNP, little was known regarding the nature of C. rodentium prophages in the bacterium’s evolution towards pathogenicity. In this study, our characterization of ΦNP led to the discovery of a second, fully functional temperate phage, named ΦSM. We identify the bacterial host-receptor for both phages as lipopolysaccharide (LPS). ΦNP and ΦSM are likely important mediators of HGT in C. rodentium. Bioinformatic analysis of the ten prophage regions reveals cargo genes encoding known virulence factors, including several Type III secretion system (T3SS) effectors. C. rodentium prophages are conserved across a wide range of pathogenic enteric bacteria, including EPEC and EHEC as well as pathogenic strains of Salmonella enterica, Shigella boydii, and Klebsiella pneumoniae. Phylogenetic analysis of core enteric backbone genes compared against prophage evolutionary models suggests that these prophages represent an important, conserved family of horizontally acquired enteric-associated pathogenicity determinants. In addition to highlighting the transformative role of bacteriophage mediated HGT in C. rodentium’s evolution towards pathogenicity, these data suggest that the examination of conserved families of prophages in other pathogenic bacteria and disease outbreaks might provide deeper evolutionary and pathological insights otherwise obscured by more classical analysis.BBSRC and China Scholarship Council and the Cambridge Commonwealth, European, and International Trust

Type I-F CRISPR-Cas resistance against virulent phages results in abortive infection and provides population-level immunity

This is the final version. Available from the publisher via the DOI in this record.Type I CRISPR-Cas systems are abundant and widespread adaptive immune systems in bacteria and can greatly enhance bacterial survival in the face of phage infection. Upon phage infection, some CRISPR-Cas immune responses result in bacterial dormancy or slowed growth, which suggests the outcomes for infected cells may vary between systems. Here we demonstrate that type I CRISPR immunity of Pectobacterium atrosepticum leads to suppression of two unrelated virulent phages, ɸTE and ɸM1. Immunity results in an abortive infection response, where infected cells do not survive, but viral propagation is severely decreased, resulting in population protection due to the reduced phage epidemic. Our findings challenge the view of CRISPR-Cas as a system that protects the individual cell and supports growing evidence of abortive infection by some types of CRISPR-Cas systems

The LacI–family transcription factor, RbsR, is a pleiotropic regulator of motility, virulence, siderophore and antibiotic production, gas vesicle morphogenesis and flotation in Serratia

Gas vesicles (GVs) are proteinaceous, gas-filled organelles used by some bacteria to enable upward movement into favorable air/liquid interfaces in aquatic environments. Serratia sp. ATCC39006 (S39006) was the first enterobacterium discovered to produce GVs naturally. The regulation of GV assembly in this host is complex and part of a wider regulatory network affecting various phenotypes, including antibiotic biosynthesis. To identify new regulators of GVs, a comprehensive mutant library containing 71,000 insertion mutants was generated by random transposon mutagenesis and 311 putative GV-defective mutants identified. Three of these mutants were found to have a transposon inserted in a LacI family transcription regulator gene (rbsR) of the putative ribose operon. Each of these rbsR mutants was GV-defective; no GVs were visible by phase contrast microscopy (PCM) or transmission electron microscopy (TEM). GV deficiency was caused by the reduction of gvpA1 and gvrA transcription (the first genes of the two contiguous operons in the GV gene locus). Our results also showed that a mutation in rbsR was highly pleiotropic; the production of two secondary metabolites (carbapenem and prodigiosin antibiotics) was abolished. Interestingly, the intrinsic resistance to the carbapenem antibiotic was not affected by the rbsR mutation. In addition, the production of a siderophore, cellulase and plant virulence was reduced in the mutant, whereas it exhibited increased swimming and swarming motility. The RbsR protein was predicted to bind to regions upstream of at least18 genes in S39006 including rbsD (the first gene of the ribose operon) and gvrA. Electrophoretic mobility shift assays (EMSA) confirmed that RbsR bound to DNA sequences upstream of rbsD, but not gvrA. The results of this study indicate that RbsR is a global regulator that affects the modulation of GV biogenesis, but also with complex pleiotropic physiological impacts in S39006.CL was sponsored under the Academic Staff Training Scheme from the Malaysian Ministry of Education. Work in the Salmond lab is funded by the Biotechnology and Biological Sciences Research Council (BBSRC)—UK

Draft Genome Sequences of Enterobacter cloacae Strains CAPREx E7 and CAPREx E2-2

Enterobacter cloacae strains CAPREx E7 and CAPREx E2-2 were isolated from Ghanaian yams at a London market. The draft genome sequences indicate that the two strains are similar, with genomes of 5,042,838 and 5,039,930 bp and 56.19% and 55.05% G+C content, respectively. Both strains encoded three different β-lactamases, including one of the AmpC family.Work in the Salmond Lab is supported by the BBSRC. J.H. was supported by a Cambridge in Africa (CAPREx) fellowship and by the Cambridge-Africa Alborada fund

Environmental T4-Family Bacteriophages Evolve to Escape Abortive Infection via Multiple Routes in a Bacterial Host Employing “Altruistic Suicide” through Type III Toxin-Antitoxin Systems

Abortive infection is an anti-phage mechanism employed by a bacterium to initiate its own death upon phage infection. This reduces, or eliminates, production of viral progeny and protects clonal siblings in the bacterial population by an act akin to an “altruistic suicide.” Abortive infection can be mediated by a Type III toxin-antitoxin system called ToxIN$_\text{Pa}$ consisting of an endoribonuclease toxin and RNA antitoxin. ToxIN$_\text{Pa}$ is a heterohexameric quaternary complex in which pseudoknotted RNA inhibits the toxicity of the toxin until infection by certain phages causes destabilization of ToxIN$_\text{Pa}$, leading to bacteriostasis and, eventually, lethality. However, it is still unknown why only certain phages are able to activate ToxIN$_\text{Pa}$. To try to address this issue we first introduced ToxIN$_\text{Pa}$ into the Gram-negative enterobacterium, $\textit{Serratia}$ sp. ATCC 39006 (S 39006) and then isolated new environmental S 39006 phages that were scored for activation of ToxIN$_\text{Pa}$ and abortive infection capacity. We isolated three T4-like phages from a sewage treatment outflow point into the River Cam, each phage being isolated at least a year apart. These phages were susceptible to ToxIN$_\text{Pa}$-mediated abortive infection but produced spontaneous “escape” mutants that were insensitive to ToxIN$_\text{Pa}$. Analysis of these resistant mutants revealed three different routes of escaping ToxIN$_\text{Pa}$, namely by mutating $\textit{asiA}$ (the product of which is a phage transcriptional co-activator); by mutating a conserved, yet functionally unknown, $\textit{orf84}$; or by deleting a 6.5–10 kb region of the phage genome. Analysis of these evolved escape mutants may help uncover the nature of the corresponding phage product(s) involved in activation of ToxIN$_\text{Pa}$.This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC), UK. BC was funded by the Cambridge Commonwealth, European and International Trust. CA was funded by The Gates Cambridge Trust

Environmental Bacteriophages of the Emerging Enterobacterial Phytopathogen, Dickeya solani, Show Genomic Conservation and Capacity for Horizontal Gene Transfer between Their Bacterial Hosts

Dickeya solani is an economically important phytopathogen widespread in mainland Europe that can reduce potato crop yields by 25%. There are no effective environmentally-acceptable chemical systems available for diseases caused by Dickeya. Bacteriophages have been suggested for use in biocontrol of this pathogen in the field, and limited field trials have been conducted. To date only a small number of bacteriophages capable of infecting D. solani have been isolated and characterized, and so there is a need to expand the repertoire of phages that may have potential utility in phage therapy strategies. Here we describe 67 bacteriophages from environmental sources, the majority of which are members of the viral family Myoviridae. Full genomic sequencing of two isolates revealed a high degree of DNA identity with D. solani bacteriophages isolated in Europe in the past 5 years, suggesting a wide ecological distribution of this phage family. Transduction experiments showed that the majority of the new environmental bacteriophages are capable of facilitating efficient horizontal gene transfer. The possible risk of unintentional transfer of virulence or antibiotic resistance genes between hosts susceptible to transducing phages cautions against their environmental use for biocontrol, until specific phages are fully tested for transduction capabilities.This work was supported by the BBSRC, UK. AD was supported by a Cambridge Doctoral Training Partnership Award from the BBSRC, UK

Quorum Sensing Controls Adaptive Immunity through the Regulation of Multiple CRISPR-Cas Systems

Bacteria commonly exist in high cell density populations, making them prone to viral predation and horizontal gene transfer (HGT) through transformation and conjugation. To combat these invaders, bacteria possess an arsenal of defenses, such as CRISPR-Cas adaptive immunity. Many bacterial populations coordinate their behavior as cell density increases, using quorum sensing (QS) signaling. In this study, we demonstrate that QS regulation results in increased expression of the type I-E, I-F, and III-A CRISPR-Cas systems in $\textit{Serratia}$ cells in high-density populations. Strains unable to communicate via QS were less effective at defending against invaders targeted by any of the three CRISPR-Cas systems. Additionally, the acquisition of immunity by the type I-E and I-F systems was impaired in the absence of QS signaling. We propose that bacteria can use chemical communication to modulate the balance between community-level defense requirements in high cell density populations and host fitness costs of basal CRISPR-Cas activity.This work was supported by a Rutherford Discovery Fellowship (P.C.F.) from the Royal Society of New Zealand (RSNZ) and the Marsden Fund, RSNZ. A.G.P. was supported by a University of Otago Doctoral Scholarship. G.P.C.S. is funded by the Biotechnology and Biological Sciences Research Council, UK

Different genetic and morphological outcomes for phages targeted by single or multiple CRISPR-Cas spacers.

CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against genetic invaders, such as bacteriophages. The systems integrate short sequences from the phage genome into the bacterial CRISPR array. These 'spacers' provide sequence-specific immunity but drive natural selection of evolved phage mutants that escape the CRISPR-Cas defence. Spacer acquisition occurs by either naive or primed adaptation. Naive adaptation typically results in the incorporation of a single spacer. By contrast, priming is a positive feedback loop that often results in acquisition of multiple spacers, which occurs when a pre-existing spacer matches the invading phage. We predicted that single and multiple spacers, representative of naive and primed adaptation, respectively, would cause differing outcomes after phage infection. We investigated the response of two phages, ϕTE and ϕM1, to the Pectobacterium atrosepticum type I-F CRISPR-Cas system and observed that escape from single spacers typically occurred via point mutations. Alternatively, phages escaped multiple spacers through deletions, which can occur in genes encoding structural proteins. Cryo-EM analysis of the ϕTE structure revealed shortened tails in escape mutants with tape measure protein deletions. We conclude that CRISPR-Cas systems can drive phage genetic diversity, altering morphology and fitness, through selective pressures arising from naive and primed acquisition events. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.This work was supported by a Rutherford Discovery Fellow- ship from the Royal Society of New Zealand (RSNZ) (to P.C.F.), the Marsden Fund, RSNZ, the Bio-protection Research Centre (Tertiary Education Commission), a University of Otago Doctoral Scholarship (to B.N.J.W.), University of Otago Division of Health Sciences Career Development Post-doctoral Fellowship and a Veni grant (grant no. 016.Veni.171.047) from the The Netherlands Organization for Scienti- fic Research (to R.H.J.S.). G.P.C.S. was supported by the BBSRC, UK

Designing disordered materials using DNA-coated colloids of bacteriophage fd and gold.

DNA has emerged as an exciting binding agent for programmable colloidal self-assembly. Its popularity derives from its unique properties: it provides highly specific short-ranged interactions and at the same time it acts as a steric stabilizer against non-specific van der Waals and Coulomb interactions. Because complementary DNA strands are linked only via hydrogen bonds, DNA-mediated binding is thermally reversible: it provides an effective attraction that can be switched off by raising the temperature only by a few degrees. In this article we introduce a new binary system made of DNA-functionalized filamentous fd viruses of ∼880 nm length with an aspect ratio of ∼100, and 50 nm gold nanoparticles (gold NPs) coated with the complementary DNA strands. When quenching mixtures below the melt temperature Tm, at which the attraction is switched on, we observe aggregation. Conversely, above Tm the system melts into a homogenous particulate 'gas'. We present the aggregation behavior of three different gold NP to virus ratios and compare them to a gel made solely of gold NPs. In particular, we have investigated the aggregate structures as a function of cooling rate and determine how they evolve as function of time for given quench depths, employing fluorescence microscopy. Structural information was extracted in the form of an effective structure factor and chord length distributions. Rapid cooling rates lead to open aggregates, while slower controlled cooling rates closer to equilibrium DNA hybridization lead to more fine-stranded gels. Despite the different structures we find that for both cooling rates the quench into the two-phase region leads to initial spinodal decomposition, which becomes arrested. Surprisingly, although the fine-stranded gel is disordered, the overall structure and the corresponding length scale distributions in the system are remarkably reproducible. Such highly porous systems can be developed into new functional materials.This work would not have been possible without the financial support of various agencies: EE, CPG and ZR thank the Winton Program for the Physics of Sustainability. CPG and ZR acknowledge support from the EU ERC FP7 programme via an advanced fellowship for CPG. RU and SHN received support from the Engineering and Physical Sciences Research Council (EPSRC) for financial support. Work in the GPCS Lab is funded by the BBSRC, UK. DJ acknowledges the financial support from Udayan Care, NTCU and Schlumberger Foundation's FFTF program.This is the author accepted manuscript. The final version is available from the Royal Society of Chemistry via http://dx.doi.org/10.1039/C5FD00120

Predicting promoters in phage genomes using machine learning models

The renewed interest in phages as antibacterial agents has led to the exponentially growing number of sequenced phage genomes. Therefore, the development of novel bioinformatics methods to automate and facilitate phage genome annotation is of utmost importance. The most difficult step of phage genome annotation is the identification of promoters. As the existing methods for predicting promoters are not well suited for phages, we used machine learning models for locating promoters in phage genomes. Several models were created, using different algorithms and datasets, which consisted of known phage promoter and non-promoter sequences. All models showed good performance, but the ANN model provided better results for the smaller dataset (92% of accuracy, 89% of precision and 87% of recall) and the SVM model returned better results for the larger dataset (93% of accuracy, 91% of precision and 80% of recall). Both models were applied to the genome of Pseudomonas phage phiPsa17 and were able to identify both types of promoters, host and phage, found in phage genomes.This study was supported by the Portuguese Foundation for Science andTechnology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and theProject POCI-01-0145-FEDER-029628. This work was also supported by BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fundunder the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio