Search

Salmonellosis is one of the most frequently reported zoonotic foodborne diseases worldwide, and poultry is the most important reservoir of Salmonella enterica serovar Enteritidis. The use of lytic bacteriophages (phages) to reduce foodborne pathogens has emerged as a promising biocontrol intervention for Salmonella spp. Here, we describe and evaluate the newly isolated Salmonella phage STGO-35-1, including: (i) genomic and phenotypic characterization, (ii) an analysis of the reduction of Salmonella in chicken meat, and (iii) genome plasticity testing. Phage STGO-35-1 represents an unclassified siphovirus, with a length of 47,483 bp, a G + C content of 46.5%, a headful strategy of packaging, and a virulent lifestyle. Phage STGO-35-1 reduced S. Enteritidis counts in chicken meat by 2.5 orders of magnitude at 4 °C. We identified two receptor-binding proteins with affinity to LPS, and their encoding genes showed plasticity during an exposure assay. Phenotypic, proteomic, and genomic characteristics of STGO-35-1, as well as the Salmonella reduction in chicken meat, support the potential use of STGO-35-1 as a targeted biocontrol agent against S. Enteritidis in chicken meat. Additionally, computational analysis and a short exposure time assay allowed us to predict the plasticity of genes encoding putative receptor-binding proteins. ispartof: MICROORGANISMS vol:10 issue:3 ispartof: location:Switzerland status: published

Xanthomonas campestris pv. campestris (Xcc) is a vascular pathogen that invades the xylem of Brassica crops. Current chemical and antibiotics-based control measures for this bacterium are unsustainable and inefficient. After establishing a representative collection of Xcc strains, we isolated and characterized bacteriophages from two clades of phages to assess their potential in phage-based biocontrol. The most promising phages, FoX2 and FoX6, specifically recognize (lipo) polysaccharides, associated with the wxc gene cluster, on the surface of the bacterial cell wall. Next, we determined and optimized the applicability of FoX2 and FoX6 in an array of complementary bioassays, ranging from seed decontamination to irrigation- and spray-based applications. Here, an irrigation-based application showed promising results. In a final proof-of-concept, a CaCl2-formulated phage cocktail was shown to control the outbreak of Xcc in the open field. This comprehensive approach illustrates the potential of phage biocontrol of black rot disease in Brassica and serves as a reference for the broader implementation of phage biocontrol in integrated pest management strategies. The authors thank A. Volckaert for her input, Dr. L. van der Heijden (Bejo Zaden B.V.), Ms. K. Sereti (Rijk Zwaan Zaadteelt en zaadhandel B.V.) for providing seed material and Prof. L. Cruz for providing us with a wellcharacterized Xcc collection. They further thank Miss Y. Busschots for her technical assistance. This research was supported by the ‘Vlaams Agentschap Innoveren en Ondernemen’ (VLAIO) agriculture programme (LA) grant IWT.150914 and by the European Union’s Horizon H2020 Research and Innovation Programme (grant agreement N. 773567). DH holds a predoctoral scholarship from the ‘Fonds voor Wetenschappelijk Onderzoek Vlaanderen’ (FWO) strategic basic research grant 1S02520N.

Phages are generally considered species- or even strain-specific, yet polyvalent phages are able to infect bacteria from different genera. Here, we characterize the novel polyvalent phage S144, a member of the Loughboroughvirus genus. By screening 211 Enterobacteriaceae strains, we found that phage S144 forms plaques on specific serovars of Salmonella enterica subsp. enterica and on Cronobacter sakazakii. Analysis of phage resistant mutants suggests that the O-antigen of lipopolysaccharide is the phage receptor in both bacterial genera. The S144 genome consists of 53,628 bp and encodes 80 open reading frames (ORFs), but no tRNA genes. In total, 32 ORFs coding for structural proteins were confirmed by ESI-MS/MS analysis, whereas 45 gene products were functionally annotated within DNA metabolism, packaging, nucleotide biosynthesis and phage morphogenesis. Transmission electron microscopy showed that phage S144 is a myovirus, with a prolate head and short tail fibers. The putative S144 tail fiber structure is, overall, similar to the tail fiber of phage Mu and the C-terminus shows amino acid similarity to tail fibers of otherwise unrelated phages infecting Cronobacter. Since all phages in the Loughboroughvirus genus encode tail fibers similar to S144, we suggest that phages in this genus infect Cronobacter sakazakii and are polyvalent Funding: This research was funded by Danish AgriFish Agency of Ministry of Environment and Food, grant number 34009-14-0873. Acknowledgments: We thank Mark van Raaij for his advice (CNB–CSIC, Madrid, Spain) and John E. Olsen (Department of Veterinary and Animal Sciences, University of Copenhagen, Copenhagen, Denmark), Anne Elsser-Gravesen (ISI Food Protection, Århus, Denmark) and Yinghua Xiao (Arla Foods, Århus, Denmark) for providing bacterial strains used in the determination of phage host range. Angela Back (Max Rubner-Institut) is acknowledged for her technical assistance with the electron microscopy

Providencia stuartii is emerging as a significant drug-resistant nosocomial pathogen, which encourages the search for alternative therapies. Here, we have isolated Providencia stuartii phage Stuart, a novel podovirus infecting multidrug-resistant hospital isolates of this bacterium. Phage Stuart is a proposed member of a new Autographivirinae subfamily genus, with a 41,218-bp genome, direct 345-bp repeats at virion DNA ends, and limited sequence similarity of proteins to proteins in databases. Twelve out of the 52 predicted Stuart proteins are virion components. We found one to be a tailspike with depolymerase activity. The tailspike could form a highly thermostable oligomeric β-structure migrating close to the expected trimer in a nondenaturing gel. It appeared to be essential for the infection of three out of four P. stuartii hosts infected by phage Stuart. Moreover, it degraded the exopolysaccharide of relevant phage Stuart hosts, making the bacteria susceptible to serum killing. Prolonged exposure of a sensitive host to the tailspike did not cause the emergence of bacteria resistant to the phage or to serum killing, opposite to the prolonged exposure to the phage. This indicates that phage tail-associated depolymerases are attractive antivirulence agents that could complement the immune system in the fight with P. stuartii.IMPORTANCE The pace at which multidrug-resistant strains emerge has been alarming. P. stuartii is an infrequent but relevant drug-resistant nosocomial pathogen causing local to systemic life-threatening infections. We propose an alternative approach to fight this bacterium based on the properties of phage tailspikes with depolymerase activity that degrade the surface bacterial polymers, making the bacteria susceptible to the immune system. Unlike antibiotics, phage tailspikes have narrow and specific substrate spectra, and by acting as antivirulent but not bactericidal agents they do not cause the selection of resistant bacteria., This study was supported by the Portuguese Foundation for Science and Technol ogy (FCT) under the scope of the strategic funding of UID/BIO/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte. R.L. and H.H. are supported by the GOA grant from KU Leuven “Phage Biosystems.” G.P. acknowledges the FCT grant SFRH/BD/117365/2016., info:eu-repo/semantics/publishedVersion

To date, Certrevirus is one of two genera of bacteriophage (phage), with phages infecting Pectobacterium atrosepticum, an economically important phytopathogen that causes potato blackleg and soft rot disease. This study provides a detailed description of Pectobacterium phage CB7 (vB_PatM_CB7), which specifically infects P. atrosepticum. Host range, morphology, latent period, burst size and stability at different conditions of temperature and pH were examined. Analysis of its genome (142.8 kbp) shows that the phage forms a new species of Certrevirus, sharing sequence similarity with other members, highlighting conservation within the genus. Conserved elements include a putative early promoter like that of the Escherichia coli sigma70 promoter, which was found to be shared with other genus members. A number of dissimilarities were observed, relating to DNA methylation and nucleotide metabolism. Some members do not have homologues of a cytosine methylase and anaerobic nucleotide reductase subunits NrdD and NrdG, respectively. Furthermore, the genome of CB7 contains one of the largest numbers of homing endonucleases described in a single phage genome in the literature to date, with a total of 23 belonging to the HNH and LAGLIDADG families. Analysis by RT-PCR of the HNH homing endonuclease residing within introns of genes for the large terminase, DNA polymerase, ribonucleotide reductase subunits NrdA and NrdB show that they are splicing competent. Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) was also performed on the virion of CB7, allowing the identification of 26 structural proteins&mdash, 20 of which were found to be shared with the type phages of the genera of Vequintavirus and Seunavirus. The results of this study provide greater insights into the phages of the Certrevirus genus as well as the subfamily Vequintavirinae.

Volume 93, no. 4, e01163-18, 2019, https://doi.org/10.1128/JVI.01163-18. Page 14, Acknowledgments, line 4: (POCI-01-0145-FEDER-016678) should read (POCI-01-0145-FEDER-016643)., info:eu-repo/semantics/publishedVersion

The bacterial DNA gyrase complex (GyrA/GyrB) plays a crucial role during DNA replication and serves as a target for multiple antibiotics, including the fluoroquinolones. Despite it being a valuable antibiotics target, resistance emergence by pathogens including Pseudomonas aeruginosa are proving problematic. Here, we describe Igy, a peptide inhibitor of gyrase, encoded by Pseudomonas bacteriophage LUZ24 and other members of the Bruynoghevirus genus. Igy (5.6 kDa) inhibits in vitro gyrase activity and interacts with the P. aeruginosa GyrB subunit, possibly by DNA mimicry, as indicated by a de novo model of the peptide and mutagenesis. In vivo, overproduction of Igy blocks DNA replication and leads to cell death also in fluoroquinolone-resistant bacterial isolates. These data highlight the potential of discovering phage-inspired leads for antibiotics development, supported by co-evolution, as Igy may serve as a scaffold for small molecule mimicry to target the DNA gyrase complex, without cross-resistance to existing molecules. "Fonds Wetenschappelijk Onderzoek'' (FWO) [G.0323.09]; KULeuven project GOA "Phage Biosystems.''

Pseudomonas virus vB_PaeM_PA5oct is proposed as a model jumbo bacteriophage to investigate phage-bacteria interactions and is a candidate for phage therapy applications. Combining hybrid sequencing, RNA-Seq and mass spectrometry allowed us to accurately annotate its 286,783 bp genome with 461 coding regions including four non-coding RNAs (ncRNAs) and 93 virion-associated proteins. PA5oct relies on the host RNA polymerase for the infection cycle and RNA-Seq revealed a gradual take-over of the total cell transcriptome from 21% in early infection to 93% in late infection. PA5oct is not organized into strictly contiguous regions of temporal transcription, but some genomic regions transcribed in early, middle and late phases of infection can be discriminated. Interestingly, we observe regions showing limited transcription activity throughout the infection cycle. We show that PA5oct upregulates specific bacterial operons during infection including operons pncA-pncB1-nadE involved in NAD biosynthesis, psl for exopolysaccharide biosynthesis and nap for periplasmic nitrate reductase production. We also observe a downregulation of T4P gene products suggesting mechanisms of superinfection exclusion. We used the proteome of PA5oct to position our isolate amongst other phages using a gene-sharing network. This integrative omics study illustrates the molecular diversity of jumbo viruses and raises new questions towards cellular regulation and phage-encoded hijacking mechanisms. We are grateful to Steve Abedon, Harald Brüssow and the reviewers for their critical comments and helpful suggestions on the manuscript. This study was supported by research grant no. 2012/04/M/NZ6/00335 of the National Science Centre, Poland. KDW was co-financed by the European Union as part of the European Social Fund and a postdoctoral fellowship at KU Leuven, Belgium. CL is supported by an SB PhD fellowship from FWO Vlaanderen (1S64718N). RL, YB and ZDK are members of the ‘PhageBiotics’ research community, supported by the FWO Vlaanderen. ZDK was a members of the EU COST BM1003 Action in Microbial cell surface determinants of virulence (http://www.cost.eu/COST_Actions/bmbs/BM1003). JPN is supported by a grant from the Hercules Foundation (project R-3986).

Acinetobacter baumannii is an important pathogen causative of healthcare-associated infections and is able to rapidly develop resistance to all known antibiotics including colistin. As an alternative therapeutic agent, we have isolated a novel myovirus (vB_AbM_B9) which specifically infects and makes lysis from without in strains of the K45 and K30 capsule type, respectively. Phage B9 has a genome of 93,641 bp and encodes 167 predicted proteins, of which 29 were identified by mass spectrometry. This phage holds a capsule depolymerase (B9gp69) able to digest extracted exopolysaccharides of both K30 and K45 strains and that remains active in a wide range of pH values (5 to 9), ionic strengths (0 to 500 mM), and temperatures (20 to 80\textdegreeC). B9gp69 demonstrated to be non-toxic in a cell line model of the human lung, and to make the K45 strain fully susceptible to serum killing in vitro. Contrary to the phage, no resistance development was observed by bacteria targeted with the B9gp69. Therefore, capsular depolymerases may represent attractive antimicrobial agents against A. baumannii infections.IMPORTANCE Currently, phage therapy has revived interest for controlling hard-to-treat bacterial infections. Acinetobacter baumannii is an emerging Gram-negative pathogen able to cause a variety of nosocomial infections. Additionally, this species is becoming more resistant to several classes of antibiotics. Herein, we describe the isolation of a novel lytic myophage B9 and its recombinant depolymerase. While the phage can be a promising alternative antibacterial agent, its success in the market will ultimately depend on new regulatory frameworks and general public acceptance. We therefore characterised the phage-encoded depolymerase which is a natural enzyme that can be more easily managed and used. To our knowledge, the therapeutic potential of phage depolymerase against A. baumannii is still unknown. We show for the first time that K45 capsule type is an important virulence factor of A. baumannii and that capsule removal via the recombinant depolymerase activity helps the host immune system to combat the bacterial infection., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145-FEDER-006684) and the Projects PTDC/BBB-BSS/ 6471/2014 (POCI-01-0145-FEDER-016678) and PTDC/CVT-CVT/29628/2017 (POCI-01- 0145-FEDER-029628). This work was also supported by the BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte. H.O. and A.R.C. acknowledge FCT for grants SFRH/BPD/111653/2015 and SFRH/BPD/ 94648/2013, respectively. Support from Hercules Foundation project R-3986 for J.-P.N. is also acknowledged. The work of R.L. and M.B. was supported by a GOA grant from KU Leuven. J.-P.N. acknowledges the support of Hercules Foundation project R-3986., info:eu-repo/semantics/publishedVersion

Erwinia amylovora is a Gram-negative plant pathogen that causes fire blight. This disease affects most members of the Rosaceae family including apple and pear. Here, an infection model is introduced to study proteomic changes in a highly virulent E. amylovora strain upon interaction with its host as compared to a lower virulent strain. For this purpose separate shoots of apple rootstocks were wound-infected and when infection became systemic, bacterial cells were isolated and processed for analysis in a proteomics platform combining 2-D fluorescence difference gel electrophoresis and mass spectrometry. Comparing the proteome of the isolates, significant abundance changes were observed in proteins involved in sorbitol metabolism, amylovoran production as well as in protection against plant defense mechanisms. Furthermore several proteins associated with virulence were more abundant in the higher virulent strain. Changes at the proteome level showed good accordance at the transcript level, as was verified by RT-qPCR. In conclusion, this infection model may be a valuable tool to unravel the complexity of plant-pathogen interactions and to gain insight in the molecular mechanisms associated with virulence of E. amylovora, paving the way for the development of plant-protective interventions against this detrimental disease. Significance During this research a first time investigation was performed on the proteome of E. amylovora, grown inside a susceptible host plant. This bacterium is the causal agent of fire blight, which can affect most members of the Rosaceae family including apple and pear. To do so, an artificial infection model on shoots of apple rootstocks was optimized and employed. When infection was systemic, bacterial cells were extracted from the plant tissue followed by extraction of the proteins from the bacteria. Further processing of the proteins was done by using a 2-D fluorescence difference gel electrophoresis analysis followed by mass spectrometry. By the use of two strains differing in their virulent ability, we were able to draw conclusions concerning virulence and behavior of different strains inside the host. This research provides a model to investigate plant-pathogen interactions and more importantly, we identified possible new targets for the development of novel control methods against this devastating disease.

Agrobacterium tumefaciens is a plant pathogen that causes crown gall disease, leading to the damage of agriculturally-important crops. As part of an effort to discover new phages that can potentially be used as biocontrol agents to prevent crown gall disease, we isolated and characterized phage Atu_ph07 from Sawyer Creek in Springfield, MO, using the virulent Agrobacterium tumefaciens strain C58 as a host. After surveying its host range, we found that Atu_ph07 exclusively infects Agrobacterium tumefaciens. Timelapse microscopy of A. tumefaciens cells subjected to infection at a multiplicity of infection (MOI) of 10 with Atu_ph07 reveals that lysis occurs within 3 h. Transmission electron microscopy (TEM) of virions shows that Atu_ph07 has a typical Myoviridae morphology with an icosahedral head, long tail, and tail fibers. The sequenced genome of Atu_ph07 is 490 kbp, defining it as a jumbo phage. The Atu_ph07 genome contains 714 open reading frames (ORFs), including 390 ORFs with no discernable homologs in other lineages (ORFans), 214 predicted conserved hypothetical proteins with no assigned function, and 110 predicted proteins with a functional annotation based on similarity to conserved proteins. The proteins with predicted functional annotations share sequence similarity with proteins from bacteriophages and bacteria. The functionally annotated genes are predicted to encode DNA replication proteins, structural proteins, lysis proteins, proteins involved in nucleotide metabolism, and tRNAs. Characterization of the gene products reveals that Atu_ph07 encodes homologs of 16 T4 core proteins and is closely related to Rak2-like phages. Using ESI-MS/MS, the majority of predicted structural proteins could be experimentally confirmed and 112 additional virion-associated proteins were identified. The genomic characterization of Atu_ph07 suggests that this phage is lytic and the dynamics of Atu_ph07 interaction with its host indicate that this phage may be suitable for inclusion in a phage cocktail to be used as a biocontrol agent. This research is supported by startup funds, a research council grant (URC 14-051), and a research board grant (3786-2) from the University of Missouri to PJBB. This work was supported in part by the Excellence in Electron Microscopy Award provided by the University of Missouri Electron Microscopy Core and the Office of Research. HA has been supported by the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) under award number T32GM008396 and the U.S. Department of Education Graduate Assistance in Areas of National Need (GAANN) Fellowship. KP was supported by the IMSD EXPRESS Program via grant number R25GM056901 from the NIGMS, a component of the NIH. RL and MB are supported by a GOA grant from KU Leuven and J-PN was supported by Hercules Foundation project R-3986.

Fire blight, caused by the enterobacterium Erwinia amylovora, is a destructive disease, which can affect most members of the Rosaceae family. Since no significant genomic differences have been found by others to explain differences in virulence, we used here a gel-based proteomic approach to elucidate mechanisms and key players that allow the pathogen to survive, grow and multiply inside its host. Therefore, two strains with proven difference in virulence were grown under controlled conditions in vitro as well as in planta (infected apple rootstocks). Proteomic analysis including 2DE and mass spectrometry revealed that proteins involved in transcription regulation were more abundant in the in planta condition for both strains. In addition, genes involved in RNA processing were upregulated in planta for the highly virulent strain PFB5. Moreover, the upregulation of structural components of the F0F1-ATP synthase are major findings, giving important information on the infection strategy of this devastating pathogen. Overall, this research provides the first proteomic profile of E. amylovora during infection of apple rootstocks and insights into the response of the pathogen in interaction with its host.

Lactococcus lactis strains, being intensely used in the dairy industry, are particularly vulnerable to members of the so-called 936 group of phages. Sanitization and disinfection using purpose-made biocidal solutions is a critical step in controlling phage contamination in such dairy processing plants. The susceptibility of 36 936 group phages to biocidal treatments was examined using 14 biocides and commercially available sanitizers. The targets of a number of these biocides were investigated by means of electron microscopic and proteomic analyses. The results from this study highlight significant variations in phage resistance to biocides among 936 phages. Furthermore, rather than possessing resistance to specific biocides or biocide types, biocide-resistant phages tend to possess a broad tolerance to multiple classes of antimicrobial compounds. DVS the recipient of a Science Foundation Ireland (SFI) Investigator award (Ref. No. 13/IA/1953). SH is the recipient of an Irish Research Council Enterprise Partnership Scheme postgraduate scholarship. JMa is in receipt of a Technology Innovation Development Award (TIDA) (Ref. No. 14/TIDA/2287) and Starting Investigator Research Grant (SIRG) (Ref. No. 15/SIRG/3430) funded by SFI. We acknowledge the financial support from the Hercules Foundation in the framework of the project R-3986 LCMS@UHasselt: Linear Trap Quadrupool-Orbitrap mass spectrometer.

Enterobacteria phage vB_PcaM_CBB is a jumbo phage belonging to the family Myoviridae. It possesses highly atypical whisker-like structures along the length of its contractile tail. It has a broad host range with the capability of infecting species of the genera Erwinia, Pectobacterium, and Cronobacter. With a genome of 355,922 bp, excluding a predicted terminal repeat of 22,456 bp, phage CBB is the third largest phage sequenced to date. Its genome was predicted to encode 554 ORFs with 33 tRNAs. Based on prediction and proteome analysis of the virions, 29% of its predicted ORFs could be functionally assigned. Protein comparison shows that CBB shares between 3338% of its proteins with Cronobacter phage GAP32, coliphages PBECO4 and 121Q as well as Klebsiella phage vB_KleM_Rak2. This work presents a detailed and comparative analysis of vB_PcaM_CBB of a highly atypical jumbo myoviridae phage, contributing to a better understanding of phage diversity and biology., Funding was provided by Cork Institute of Technology as a PhD fellowship to CB.

Providencia rettgeri is emerging as a new opportunistic pathogen with high antibiotic resistance. The need to find alternative methods to control antibiotic-resistant bacteria and the recent advances in phage therapy motivate the search for new phages able to infect Providencia spp. This study describes the isolation and characterization of an obligatory lytic phage, vB_PreS_PR1 (PR1), with therapeutic potential against drug-resistant P. rettgeri. PR1 is a siphovirus. Its virion DNA size (118,537 bp), transcriptional organization, terminal repeats (10,461 bp), and nicks in the 3-to-5 strand are similar to those of phage T5. However, sequence similarities of PR1 to phages of the T5virus genus at the DNA and protein levels are limited, suggesting that it belongs to a new species within the Siphoviridae family. PR1 exhibits the ability to kill P. rettgeri antibiotic-resistant strains, is highly specific to the species, and did not present known genomic markers indicating a temperate lifestyle. The lack of homologies between its proteins and proteins of the only other sequenced Providencia prophage, Redjac, suggests that these two phages evolved separately and may target different host proteins., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145- FEDER-006684) and the Project PTDC/BBB-BSS/6471/2014 (POCI-01-0145-FEDER-016678). The Azeredo and Lavigne labs are members of the Phagebiotics research community, supported by the FWO Vlaanderen. HO isolated, sequenced, annotated and analysed the two genomes and drafted the manuscript. HO acknowledges the FCT grant SFRH/BPD/111653/2015. GP helped with the isolation and revised the manuscript. ML and JG helped with the phage annotation, analysis of the genome ends and nick sites, and revised the manuscript. AK helped with the phage taxonomy analysis. JN, HH and RL performed, analysed and wrote the proteomic related work. JA devised the study, assisted with experimental design and edited the manuscript., info:eu-repo/semantics/publishedVersion

Many bacteria are able to assume a transient cell wall-deficient (or L-form) state under favourable osmotic conditions. Cell wall stress such as expo- sure to β-lactam antibiotics can enforce the transition to and maintenance of this state. L-forms actively proliferate and can return to the walled state upon removal of the inducing agent. We have adopted Escherichia coli as a model system for the controlled transition to and reversion from the L-form state, and have studied these dynamics with genetics, cell biology and 'omics' technologies. As such, a transposon mutagenesis screen underscored the requirement for the Rcs phosphorelay and colanic acid synthesis, while proteomics show only little differences between rods and L-forms. In contrast, metabolome comparison reveals the high abundance of lysophospholipids and phospholipids with unsaturated or cyclopropanized fatty acids in E. coli L-forms. This increase of membrane lipids associated with increased membrane fluidity may facilitate proliferation through bud formation. Visualization of the residual peptidoglycan with a fluorescently labelled peptidoglycan binding protein indicates de novo cell wall synthesis and a role for septal peptidoglycan synthesis during bud constriction. The DD-carboxypeptidases PBP5 and PBP6 are threefold and fourfold upregulated in L-forms, indicating a specific role for regulation of crosslinking during L-form proliferation.

Addressing the functionality of predicted genes remains an enormous challenge in the postgenomic era. A prime example of genes lacking functional assignments are the poorly conserved, early expressed genes of lytic bacteriophages, whose products are involved in the subversion of the host metabolism. In this study, we focused on the composition of important macromolecular complexes of Pseudomonas aeruginosa involved in transcription, DNA replication, fatty acid biosynthesis, RNA regulation, energy metabolism, and cell division during infection with members of seven distinct clades of lytic phages. Using affinity purifications of these host protein complexes coupled to mass spectrometric analyses, 37 host complex-associated phage proteins could be identified. Importantly, eight of these show an inhibitory effect on bacterial growth upon episomal expression, suggesting that these phage proteins are potentially involved in hijacking the host complexes. Using complementary protein-protein interaction assays, we further mapped the inhibitory interaction of gp12 of phage 14-1 to the α subunit of the RNA polymerase. Together, our data demonstrate the powerful use of interactomics to unravel the biological role of hypothetical phage proteins, which constitute an enormous untapped source of novel antibacterial proteins. (Data are available via ProteomeXchange with identifier PXD001199.).

In a recent publication, we reported a unique interaction between a protein encoded by the giant myovirus phiKZ and the Pseudomonas aeruginosa RNA degradosome. Crystallography, site-directed mutagenesis and interactomics approaches revealed this ‘degradosome interacting protein’ or Dip, to adopt an ‘open-claw’ dimeric structure that presents acidic patches on its outer surface which hijack two conserved RNA binding sites on the scaffold domain of the RNase E component of the RNA degradosome. This interaction prevents substrate RNAs from being bound and degraded by the RNA degradosome during the virus infection cycle. In this commentary, we provide a perspective into the biological role of Dip, its structural analysis and its mysterious evolutionary origin, and we suggest some therapeutic and biotechnological applications of this distinctive viral protein. AV was doctoral fellow supported by the ‘Fonds voor Wetenschappelijk Onderzoek’ (FWO, Belgium). SH, KB and BFL are supported by the Wellcome Trust. This research was further supported by Grant G.0599.11 from the FWO, the SBO-project 100042 of the IWT (‘Agentschap voor Innovatie door Wetenschap en Technologie in Vlaanderen), the KULeuven GOA “Phage Biosystems”, the JPN project R-3986 of the Herculesstichting and the grants CREA/09/017 and IDO/10/012 from the KU Leuven Research Fund.

Glycerophosphodiester phosphodiesterases (GDPDs; EC 3.1.4.46) typically hydrolyze glycerophosphodiesters to sn-glycerol 3-phosphate (Gro3P) and their corresponding alcohol during patho/physiological processes in bacteria and eukaryotes. GDPD(-like) domains were identified in the structural particle of bacterial viruses (bacteriophages) specifically infecting Gram-positive bacteria. The GDPD of phage 17 (Ld17; GDPD(Ld17)), representative of the group b Lactobacillus delbrueckii subsp. bulgaricus (Ldb)-infecting bacteriophages, was shown to hydrolyze, besides the simple glycerophosphodiester, two complex surface-associated carbohydrates of the Ldb17 cell envelope: the Gro3P decoration of the major surface polysaccharide D-galactan and the oligo(glycerol phosphate) backbone of the partially glycosylated cell wall teichoic acid, a minor Ldb17 cell envelope component. Degradation of cell wall teichoic acid occurs according to an exolytic mechanism, and Gro3P substitution is presumed to be inhibitory for GDPD(Ld17) activity. The presence of the GDPD(Ld17) homotrimer in the viral baseplate structure involved in phage-host interaction together with the dependence of native GDPD activity, adsorption, and efficiency of plating of Ca2+ ions supports a role for GDPD(Ld17) activity during phage adsorption and/or phage genome injection. In contrast to GDPD(Ld17), we could not identify any enzymatic activity for the GDPD-like domain in the neck passage structure of phage 340, a 936-type Lactococcus lactis subsp. lactis bacteriophage. EMPOWER postdoctoral fellowship of the Irish Research Council; [08/IN.1/B1909]; [13/IA/1953]

In all domains of life, the catalysed degradation of RNA facilitates rapid adaptation to changing environmental conditions, while destruction of foreign RNA is an important mechanism to prevent host infection. We have identified a virus-encoded protein termed gp37/Dip, which directly binds and inhibits the RNA degradation machinery of its bacterial host. Encoded by giant phage фKZ, this protein associates with two RNA binding sites of the RNase E component of the Pseudomonas aeruginosa RNA degradosome, occluding them from substrates and resulting in effective inhibition of RNA degradation and processing. The 2.2 A˚ crystal structure reveals that this novel homo-dimeric protein has no identifiable structural homologues. Our biochemical data indicate that acidic patches on the convex outer surface bind RNase E. Through the activity of Dip, фKZ has evolved a unique mechanism to down regulate a key metabolic process of its host to allow accumulation of viral RNA in infected cells. We would like to thank Erik Royackers (Hasselt University, Belgium) for the technical support, the staff at Diamond Light source (Harwell, UK) for use of facilities and Boris Go¨ rke (University of Vienna, Austria) for providing the plasmids for the bacterial two-hybrid analyses. We acknowledge the PRIDE team for the deposition of our data to the ProteomeXchange Consortium. AV is doctoral fellow supported by the ‘Fonds voor Wetenschappelijk Onderzoek’ (FWO, Belgium). SH, KB and BFL are supported by the Wellcome Trust. This research was further supported by Grant G.0599.11 from the FWO, the SBO-project 100042 of the IWT (‘Agentschap voor Innovatie door Wetenschap en Technologie in Vlaanderen), the JPN project R-3986 of the Herculesstichting and the grants CREA/09/017 and IDO/10/012 from the KU Leuven Research Fund.

Bacteriophage therapy is currently resurging as a potential complement/alternative to antibiotic treatment. However, preclinical evaluation lacks streamlined approaches. We here focus on preclinical approaches which have been implemented to assess bacteriophage efficacy against Pseudomonas biofilms and infections. Laser interferometry and profilometry were applied to measure biofilm matrix permeability and surface geometry changes, respectively. These biophysical approaches were combined with an advanced Airway Surface Liquid infection model, which mimics in vitro the normal and CF lung environments, and an in vivo Galleria larvae model. These assays have been implemented to analyze KTN4 (279,593 bp dsDNA genome), a type-IV pili dependent, giant phage resembling phiKZ. Upon contact, KTN4 immediately disrupts the P. aeruginosa PAO1 biofilm and reduces pyocyanin and siderophore production. The gentamicin exclusion assay on NuLi-1 and CuFi-1 cell lines revealed the decrease of extracellular bacterial load between 4 and 7 logs and successfully prevents wild-type Pseudomonas internalization into CF epithelial cells. These properties and the significant rescue of Galleria larvae indicate that giant KTN4 phage is a suitable candidate for in vivo phage therapy evaluation for lung infection applications. This study was supported by research grant no. 2012/04/M/NZ6/00335 of the National Science Centre, Poland. Katarzyna Danis-Wlodarczyk was co-financed by the European Union as part of the European Social Fund. The authors thank Sylwia Nowak (UWR) for excellent technical assistance. We acknowledge Nikos Ekizoglou from Zeta Instruments Co., San Jose, USA, for providing the ZETA-20 profiler used in biofilm surface geometry assay. RL is a member of the "PhageBiotics" research community, supported by the FWO Vlaanderen. Zuzanna Drulis-Kawa and Brian Harvey are members of the EU COST Action in Microbial cell surface determinants of virulence (http://www.cost.eu/COST_Actions/bmbs/BM1003).

A total proteome map of the Pseudomonas aeruginosa PAO1 proteome is presented, generated by a combination of two-dimensional gel electrophoresis and protein identification by mass spectrometry. In total, 1128 spotswere visualized, and 181 protein spotswere characterized, corresponding to 159 different protein entries. In particular, protein chaperones and enzymes important in energy conversion and amino acid biosynthesis were identified. Spot analysis always resulted in the identification of a single protein, suggesting sufficient spot resolution, although the same proteinmay be detected in two ormore neighboring spots, possibly indicating posttranslational modifications. Comparison to the theoretical proteome revealed an underrepresentation of membrane proteins, though the identified proteins cover all predicted subcellular localizations and all functional classes. These data provide a basis for subsequent comparative studies of the biology and metabolism of P. aeruginosa, aimed at unraveling global regulatory networks. ispartof: MicrobiologyOpen vol:1 issue:2 pages:169-181 ispartof: location:England status: published

Summary We examined the genetic diversity of lytic Pseudomonas aeruginosa bacteriophage PB1 and four closely related phages (LBL3, LMA2, 14-1 and SN) isolated throughout Europe. They all encapsulate linear, non-permuted genomes between 64 427 and 66 530 bp within a solid, acid-resistant isometric capsid (diameter: 74 nm) and carry non-flexible, contractile tails of approximately 140 nm. The genomes are organized into at least seven transcriptional blocks, alternating on both strands, and encode between 88 (LBL3) and 95 (LMA2) proteins. Their virion particles are composed of at least 22 different proteins, which were identified using mass spectrometry. Post-translational modifications were suggested for two proteins, and a frameshift hotspot was identified within ORF42, encoding a structural protein. Despite large temporal and spatial separations between phage isolations, very high sequence similarity and limited horizontal gene transfer were found between the individual viruses. These PB1-like viruses constitute a new genus of environmentally very widespread phages within the Myoviridae.

A collection of 15 newly isolated (bacterio)phages infecting the opportunistic pathogen Pseudomonas aeruginosa was established to investigate their global diversity and potential in phage therapy. These phages were sampled in 14 different countries traversing four continents, from both natural environments and hospital sewage. They all display unique DNA and protein profiles and cluster morphologically into six groups within the three major families of the Caudovirales. Extensive host range studies on a library of 122 AFLP-genotyped clinical P. aeruginosa strains (of which 49 were newly isolated at the University Hospital of Leuven, Belgium) showed that the phages lysed 87% of the strains. Infection analysis of outer membrane mutants identified 10 phages as type IV pili-dependent. More detailed information about the evolutionary relatedness of the phages was gathered by de novo peptide sequencing of major virion proteins using tandem Matrix-Assisted Laser Desorption/Ionization Time of Flight technology. Applying this technique for the first time to viruses, seven groups of closely related phages were identified without the need of prior knowledge of genome content and/or electron microscopic imaging. This study demonstrates both the epidemic population structure of P. aeruginosa and the global spread of P. aeruginosa phage species, and points at the resistance of two clinically predominant, widespread P. aeruginosa strains against phage attack.

Introduction of the Agrobacterium ipt gene, coding for isopentenyl transferase,undercontrol of a tetracycline (Tc)-inducible promoter results in avery specificsystem in which cytokinin levels can be changed. Because Tc belongs to thegroup of antibiotics that affect 70S ribosomes, itis important to study the effectsof Tc on untransformed plants. Although 1 mg l

Until now, no data are available on the outer membrane (OM) proteome of Erwinia amylovora, a Gram-negative plant pathogen, causing fire blight in most of the members of the Rosaceae family. Since the OM forms the interface between the bacterial cell and its environment it is in direct contact with the host. Additionally, the type III secretion system, embedded in the OM, is a pathogenicity factor of E. amylovora. To assess the influence of the OM composition and the secretion behavior on virulence, a 2D-DIGE analysis and gene expression profiling were performed on a high and lower virulent strain, both in vitro and in planta. Proteome data showed an increase in flagellin for the lower virulent strain in vitro, whereas, in planta several interesting proteins were identified as being differently expressed between both the strains. Further, gene expression of nearly all type III secreted effectors was elevated for the higher virulent strain, both in vitro and in planta. As a first, we report that several characteristics of virulence can be assigned to the OM proteome. Moreover, we demonstrate that secreted proteins prove to be the important factors determining differences in virulence between the strains, otherwise regarded as homogeneous on a genome level. The authors would like to thank PC Fruit in Kerkom (Belgium) for the core facilities, and Greet Clerx, Inge Hermans, Hilde Schoofs, Robin Wozniak, and Erik Royackers for their technical support. This work was partially supported by the Agency of Innovation by Science and Technology (IWT-Flanders, Belgium) project no. 101513 and the Hercules Foundation project no. R-3986 "LC-MS@UHasselt: Linear TrapQuadrupool-Orbitrap mass spectrometer." M. Holtappels is indebted to the IWT for a predoctoral fellowship.

Ldl1 is a virulent phage infecting the dairy starter Lactobacillus delbrueckii subsp. lactis LdlS. Electron microscopy analysis revealed that this phage exhibits a large head and a long tail and bears little resemblance to other characterized phages infecting Lactobacillus delbrueckii . In vitro propagation of this phage revealed a latent period of 30 to 40 min and a burst size of 59.9 ± 1.9 phage particles. Comparative genomic and proteomic analyses showed remarkable similarity between the genome of Ldl1 and that of Lactobacillus plantarum phage ATCC 8014-B2. The genomic and proteomic characteristics of Ldl1 demonstrate that this phage does not belong to any of the four previously recognized L. delbrueckii phage groups, necessitating the creation of a new group, called group e, thus adding to the knowledge on the diversity of phages targeting strains of this industrially important lactic acid bacterial species.

Erwinia amylovora is a Gram-negative bacterium that causes the destructive disease fire blight affecting most members of the Rosaceae family, of which apple and pear are economically the most important hosts. E. amylovora has been considered as a homogeneous species in whole, although significant differences in virulence patterns have been observed. However, the underlying causes of the differences in virulence remain to be discovered. In a first-time comparative proteomic approach using E. amylovora, 2D differential in-gel electrophoresis (DIGE) was used to identify proteins that could explain the gradual difference in virulence between four different strains. Two important proteins were identified, FliC and CheY, both involved in flagella structure, motility and chemotaxis, which were more abundant in the least virulent strain. In the highly virulent strains the protein GalF, involved in amylovoran production, was more abundant, which was consistent with the higher expression of the gene and the higher amylovoran content in this strain in vitro. Together, these results confirm the involvement of amylovoran in virulence, but also imply an indirect role of flagellin in virulence as elicitor of plant defence. Biological significance This research provides new insights into our current understanding of the virulence of Erwinia amylovora. This plant-pathogen is considered a homogeneous species although different strains show differences in virulence. Despite the efforts made on the genomic level which resulted in the discovery of virulence factors, the reason for the different virulence patterns between strains has not yet been identified. In our lab we used a comparative proteomic approach, which has never been published before, to identify proteins involved in these differences between strains and hereby possibly involved in virulence. Our results provide interesting insights in virulence and present us with the opportunity to glance into the proteome of E. amylovora.

The structural proteome of ϕKMV, a lytic bacteriophage infecting Pseudomonas aeruginosa, was analysed using two approaches. In one approach, structural proteins of the phage were fractionated by SDS-PAGE for identification by liquid chromatography-mass spectrometry (LC-MS). In a second approach, a whole-phage shotgun analysis (WSA) was applied. WSA uses trypsin digestion of whole phage particles, followed by reversed-phase HPLC and gas-phase fractionation of the complex peptide mixture prior to MS. The results yield a comprehensive view of structure-related proteins in ϕKMV and suggest subtle structural differences from phage T7.

Pseudomonas aeruginosa bacteriophage ϕKZ is the type representative of the giant phage genus, which is characterized by unusually large virions and genomes. By unraveling the transcriptional map of the ∼280-kb ϕKZ genome to single-nucleotide resolution, we combine 369 ϕKZ genes into 134 operons. Early transcription is initiated from highly conserved AT-rich promoters distributed across the ϕKZ genome and located on the same strand of the genome. Early transcription does not require phage or host protein synthesis. Transcription of middle and late genes is dependent on protein synthesis and mediated by poorly conserved middle and late promoters. Unique to ϕKZ is its ability to complete its infection in the absence of bacterial RNA polymerase (RNAP) enzyme activity. We propose that transcription of the ϕKZ genome is performed by the consecutive action of two ϕKZ-encoded, noncanonical multisubunit RNAPs, one of which is packed within the virion, another being the product of early genes. This unique, rifampin-resistant transcriptional machinery is conserved within the diverse giant phage genus. IMPORTANCE The data presented in this paper offer, for the first time, insight into the complex transcriptional scheme of giant bacteriophages. We show that Pseudomonas aeruginosa giant phage ϕKZ is able to infect and lyse its host cell and produce phage progeny in the absence of functional bacterial transcriptional machinery. This unique property can be attributed to two phage-encoded putative RNAP enzymes, which contain very distant homologues of bacterial β and β′-like RNAP subunits.

[Thoelen, M.; Noben, J. P.; Robben, J.; Valcke, R.] Hasselt Univ, Dept SBG BIOMED, B-3590 Diepenbeek, Belgium.

The renewed interest in controlling Staphylococcus aureus infections using their natural enemies, bacteriophages, has led to the isolation of a limited number of virulent phages so far. These phages are all members of the Twortlikevirus , displaying little variance. We present two novel closely related (95.9% DNA homology) lytic myoviruses, Romulus and Remus, with double-stranded DNA (dsDNA) genomes of 131,333 bp and 134,643 bp, respectively. Despite their relatedness to Staphylococcus phages K, G1, ISP, and Twort and Listeria phages A511 and P100, Romulus and Remus can be proposed as isolates of a new species within the Twortlikevirus genus. A distinguishing feature for these phage genomes is the unique distribution of group I introns compared to that in other staphylococcal myoviruses. In addition, a hedgehog/intein domain was found within their DNA polymerase genes, and an insertion sequence-encoded transposase exhibits splicing behavior and produces a functional portal protein. From a phage therapy application perspective, Romulus and Remus infected approximately 70% of the tested S. aureus isolates and displayed promising lytic activity against these isolates. Furthermore, both phages showed a rapid initial adsorption and demonstrated biofilm-degrading capacity in a proof-of-concept experiment.

Bacteriophage ZF40 is the only currently available, temperate Myoviridae phage infecting the potato pathogen Pectobacterium carotovorum subsp . carotovorum . Despite its unusual tail morphology, its major tail sheath and tube proteins remained uncharacterized after the initial genome annotation. Using ESI tandem mass-spectrometry, 24 structural proteins of the ZF40 virion were identified, with a sequence coverage ranging between 15,8 and 87,8%. The putative function of sixteen proteins could be elucidated based on secondary structure analysis and conservative domain searches. The experimental annotation of 35% of the encoded gene products within the structural region of the genome represents a complete view of the virion structure, which can serve as the basis for future structural analysis as a model phage.

We report the study of phage AF, the first member of the canonical lambdoid phage group infecting Pseudomonas putida . Its 42.6 kb genome is related to the “epsilon15-like viruses” and the “BPP-1-like viruses”, a clade of bacteriophages shaped by extensive horizontal gene transfer. The AF virions display exopolysaccharide (EPS)-degrading activity, which originates from the action of the C-terminal domain of the tail spike (Gp19). This protein shows high similarity to the tail spike of the T7-like P. putida -infecting phage φ15. These unrelated phages have an identical host spectrum and EPS degradation characteristics, designating the C-terminal part of Gp19 as sole determinant for these functions. While intact AF particles have biofilm-degrading properties, Gp19 and non-infectious AF particles do not, emphasizing the role of phage amplification in biofilm degradation.

The bacterium 'Dickeya solani', an aggressive biovar 3 variant of Dickeya dianthicola, causes rotting and blackleg in potato. To control this pathogen using bacteriophage therapy, we isolated and characterized two closely related and specific bacteriophages, vB_DsoM_LIMEstone1 and vB_DsoM_LIMEstone2. The LIMEstone phages have a T4-related genome organization and share DNA similarity with Salmonella phage Vil. Microbiological and molecular characterization of the phages deemed them suitable and promising for use in phage therapy. The phages reduced disease incidence and severity on potato tubers in laboratory assays. In addition, in a field trial of potato tubers, when infected with 'Dickeya solani', the experimental phage treatment resulted in a higher yield. These results form the basis for the development of a bacteriophage-based biocontrol of potato plants and tubers as an alternative for the use of antibiotics. EMA and the experimental work were funded through a PhD scholarship by the Katholieke Universiteit Leuven and the Institute for Agricultural and Fisheries Research Flanders (ILVO). PJC hold a post-doctoral fellowship of the Fonds voor Wetenschappelijk Onderzoek (FWO Flanders). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The increasing antibiotic resistance in bacterial populations requires alternatives for classical treatment of infectious diseases and therefore drives the renewed interest in phage therapy. Methicillin resistant Staphylococcus aureus (MRSA) is a major problem in health care settings and live-stock breeding across the world. This research aims at a thorough microbiological, genomic, and proteomic characterization of S. aureus phage ISP, required for therapeutic applications. Host range screening of a large batch of S. aureus isolates and subsequent fingerprint and DNA microarray analysis of the isolates revealed a substantial activity of ISP against 86% of the isolates, including relevant MRSA strains. From a phage therapy perspective, the infection parameters and the frequency of bacterial mutations conferring ISP resistance were determined. Further, ISP was proven to be stable in relevant in vivo conditions and subcutaneous as well as nasal and oral ISP administration to rabbits appeared to cause no adverse effects. ISP encodes 215 gene products on its 138,339 bp genome, 22 of which were confirmed as structural proteins using tandem electrospray ionization-mass spectrometry (ESI-MS/MS), and shares strong sequence homology with the 'Twort-like viruses'. No toxic or virulence-associated proteins were observed. The microbiological and molecular characterization of ISP supports its application in a phage cocktail for therapeutic purposes. PC holds a postdoctoral fellowship of the 'Fonds voor Wetenschappelijk Onderzoek-Vlaanderen' (FWO-Vlaanderen, Belgium). This study was performed by members of the research community 'PhageBiotics' and was funded by the Federal Public Service of Health, Food Chain Safety and Environment, Belgium (contract RT 08/6 PHAGE). DNA fingerprinting and hybridization experiments were supported by grant MED 19 of the Royal High Institute for Defence, Belgium. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Formation of a protected biofilm environment is recognized as one of the major causes of the increasing antibiotic resistance development and emphasizes the need to develop alternative antibacterial strategies, like phage therapy. This study investigates the in vitro degradation of single-species Pseudomonas putida biofilms, PpG1 and RD5PR2, by the novel phage phi 15, a 'T7-like virus' with a virion-associated exopolysaccharide (EPS) depolymerase. Phage phi 15 forms plaques surrounded by growing opaque halo zones, indicative for EPS degradation, on seven out of 53 P. putida strains. The absence of haloes on infection resistant strains suggests that the EPS probably act as a primary bacterial receptor for phage infection. Independent of bacterial strain or biofilm age, a time and dose dependent response of phi 15-mediated biofilm degradation was observed with generally a maximum biofilm degradation 8 h after addition of the higher phage doses (10(4) and 10(6) pfu) and resistance development after 24 h. Biofilm age, an in vivo very variable parameter, reduced markedly phage-mediated degradation of PpG1 biofilms, while degradation of RD5PR2 biofilms and phi 15 amplification were unaffected. Killing of the planktonic culture occurred in parallel with but was always more pronounced than biofilm degradation, accentuating the need for evaluating phages for therapeutic purposes in biofilm conditions. EPS degrading activity of recombinantly expressed viral tail spike was confirmed by capsule staining. These data suggests that the addition of high initial titers of specifically selected phages with a proper EPS depolymerase are crucial criteria in the development of phage therapy. This research was partially supported by the K.U. Leuven (STRT1/10/021TBA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study. The authors thank Prof. M. Vaneechoutte (Laboratory Bacteriology, Ghent University Hospital, Belgium), Prof. R. De Mot (Centre of Microbial and Plant Genetics, K.U.Leuven, Belgium) and Prof. D. Springael (Division Soil and Water Management, K.U.Leuven, Belgium) for providing bacteria for this study. The authors are also grateful to Dr. Hans-W. Ackermann (Laval University, Quebec, Canada) for expert electron microscopy and to Dr. Y. Briers and Y. Born for providing information on the capsule staining assay (Institute of Food Science and Nutrition, ETH Zurich, Switzerland). Anneleen Cornelissen holds a predoctoral fellowship of the 'Instituut voor de aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen' (I.W.T., Belgium). Pieter-Jan Ceyssens is a postdoctoral fellow of the 'Fonds voor Wetenschappelijk Onderzoek-Vlaanderen' (FWO-Vlaanderen, Belgium).

Giant bacteriophages phi KZ and EL of Pseudomonas aeruginosa contain 62 and 64 structural proteins, respectively, identified by ESI-MS/MS on total virion particle proteins. These identifications verify gene predictions and delineate the genomic regions dedicated to phage assembly and capsid formation (30 proteins were identified from a tailless phi KZ mutant). These data form the basis for future structural studies and provide insights into the relatedness of these large phages. The 4 KZ structural proteome strongly correlates to that of Pseudomonas chlororaphis bacteriophage 201 phi 2-1. Phage EL is more distantly related, shown by its 26 non-conserved structural proteins and the presence of genomic inversions.

Based on genotyping and host range, two newly isolated lytic bacteriophages, myovirus vB_AbaM_Acibel004 and podovirus vB_AbaP_Acibel007, active against Acinetobacter baumannii clinical strains, were selected from a new phage library for further characterization. The complete genomes of the two phages were analyzed. Both phages are characterized by broad host range and essential features of potential therapeutic phages, such as short latent period (27 and 21 min, respectively), high burst size (125 and 145, respectively), stability of activity in liquid culture and low frequency of occurrence of phage-resistant mutant bacterial cells. Genomic analysis showed that while Acibel004 represents a novel bacteriophage with resemblance to some unclassified Pseudomonas aeruginosa phages, Acibel007 belongs to the well-characterized genus of the Phikmvlikevirus. The newly isolated phages can serve as potential candidates for phage cocktails to control A. baumannii infections. This work was supported by the Royal Higher Institute for Defense of Belgium (grant MED 12). The authors would like to acknowledge the Research community “Phagebiotics” (WO.022.09) grant from the FWO Vlaanderen. The research of DV was funded by a Ph.D. grant of the Agency for Innovation by Science and Technology (IWT) Flanders. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

We present a detailed analysis of the genome architecture, structural proteome and infection-related properties of three Pseudomonas phages, designated LUZ7, LIT1 and PEV2 These podoviruses encapsulate 72 5 to 749 kb genomes and lyse their host after 25 min aerobic infection PEV2 can successfully infect under anaerobic conditions, but its latent period is tripled, the lysis proceeds far slower and the burst size decreases significantly While the overall genome structure of these phages resembles the well-studied coliphage N4, these Pseudomonas phages encode a cluster of tail genes which displays significant similarity to a Pseudomonas aeruginosa (cryptic) prophage region Using ESI-MS/MS, these tail proteins were shown to be part of the phage particle, as well as ten other proteins including a giant 370 kDa virion RNA polymerase These phages are the first described representatives of a novel kind of obligatory lytic P aeruginosa-infecting phages, belonging to the widespread "N4-like viruses" genus (C) 2010 Elsevier Inc All rights reserved We would also thank Dr Jane Burns at Children's Hospital, Seattle, for the gift of over 200 clinical pseudomonads. Pieter-Jan Ceyssens holds a postdoctoral scholarship (PDM) of the 'Bijzonder Onderzoeksfonds' at the K.U. Leuven

Cronobacter sakazakii is a Gram-negative pathogen found in milk-based formulae that causes infant meningitis. Bacteriophages have been proposed to control bacterial pathogens; however, comprehensive knowledge about a phage is required to ensure its safety before clinical application. We have characterized C. sakazakii phage vB_CsaM_GAP32 (GAP32), which possesses the second largest sequenced phage genome (358,663 bp). A total of 571 genes including 545 protein coding sequences and 26 tRNAs were identified, thus more genes than in the smallest bacterium, Mycoplasma genitalium G37. BLASTP and HHpred searches, together with proteomic analyses reveal that only 23.9% of the putative proteins have defined functions. Some of the unique features of this phage include: a chromosome condensation protein, two copies of the large subunit terminase, a predicted signal-arrest-release lysin; and an RpoD-like protein, which is possibly involved in the switch from immediate early to delayed early transcription. Its closest relatives are all extremely large myoviruses, namely coliphage PBECO4 and Klebsiella phage vB_KleM-RaK2, with whom it shares approximately 44% homologous proteins. Since the homologs are not evenly distributed, we propose that these three phages belong to a new subfamily. The authors thank Franco Pagotto, and Roger Stephan for providing bacterial strains: This work was financially supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) (NSERC IRCRJ 127376-07).