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.
Members of the genus Sphingomonas are important catalysts for removal of polycyclic aromatic hydrocarbons (PAHs) in soil, but their activity can be affected by various stress factors. This study examines the physiological and genome-wide transcription response of the phenanthrene-degrading Sphingomonas sp. strain LH128 in biofilms to solute stress (invoked by 450 mM NaCl solution), either as an acute (4-h) or a chronic (3-day) exposure. The degree of membrane fatty acid saturation was increased as a response to chronic stress. Oxygen consumption in the biofilms and phenanthrene mineralization activities of biofilm cells were, however, not significantly affected after imposing either acute or chronic stress. This finding was in agreement with the transcriptomic data, since genes involved in PAH degradation were not differentially expressed in stressed conditions compared to nonstressed conditions. The transcriptomic data suggest that LH128 adapts to NaCl stress by (i) increasing the expression of genes coping with osmolytic and ionic stress such as biosynthesis of compatible solutes and regulation of ion homeostasis, (ii) increasing the expression of genes involved in general stress response, (iii) changing the expression of general and specific regulatory functions, and (iv) decreasing the expression of protein synthesis such as proteins involved in motility. Differences in gene expression between cells under acute and chronic stress suggest that LH128 goes through changes in genome-wide expression to fully adapt to NaCl stress, without significantly changing phenanthrene degrading activity.
Although the study of phage infection has a long history and catalyzed much of our current understanding in bacterial genetics, molecular biology, evolution and ecology, it seems that microbiologists have only just begun to explore the intricacy of phage–host interactions. In a recent manuscript by Cenens et al. we found molecular and genetic support for pseudolysogenic development in the Salmonella Typhimurium–phage P22 model system. More specifically, we observed the existence of phage carrier cells harboring an episomal P22 element that segregated asymmetrically upon subsequent divisions. Moreover, a newly discovered P22 ORFan protein (Pid) able to derepress a metabolic operon of the host (dgo) proved to be specifically expressed in these phage carrier cells. In this addendum we expand on our view regarding pseudolysogeny and its effects on bacterial and phage biology.
Salmonella Typhimurium; phage P22; phage carrier state; phage–host interactions; pseudolysogeny
In contrast to the rapidly increasing knowledge on genome content and diversity of bacterial viruses, insights in intracellular phage development and its impact on bacterial physiology are very limited. We present a multifaceted study combining quantitative PCR (qPCR), microarray, RNA-seq, and two-dimensional gel electrophoresis (2D-GE), to obtain a global overview of alterations in DNA, RNA, and protein content in Pseudomonas aeruginosa PAO1 cells upon infection with the strictly lytic phage LUZ19. Viral genome replication occurs in the second half of the phage infection cycle and coincides with degradation of the bacterial genome. At the RNA level, there is a sharp increase in viral mRNAs from 23 to 60% of all transcripts after 5 and 15 min of infection, respectively. Although microarray analysis revealed a complex pattern of bacterial up- and downregulated genes, the accumulation of viral mRNA clearly coincides with a general breakdown of abundant bacterial transcripts. Two-dimensional gel electrophoretic analyses shows no bacterial protein degradation during phage infection, and seven stress-related bacterial proteins appear. Moreover, the two most abundantly expressed early and late-early phage proteins, LUZ19 gene product 13 (Gp13) and Gp21, completely inhibit P. aeruginosa growth when expressed from a single-copy plasmid. Since Gp13 encodes a predicted GNAT acetyltransferase, this observation points at a crucial but yet unexplored level of posttranslational viral control during infection.
Massive genome sequencing has led to important insights into the enormous genetic diversity of bacterial viruses (bacteriophages). However, for nearly all known phages, information on the impact of the phage infection on host physiology and intracellular phage development is scarce. This aspect of phage research should be revitalized, as phages evolved genes which can shut down or redirect bacterial processes in a very efficient way, which can be exploited towards antibacterial design. In this context, we initiated a study of the human opportunistic pathogen Pseudomonas aeruginosa under attack by one its most common predators, the Phikmvlikevirus. By analyzing various stages of infection at different levels, this study uncovers new features of phage infection, representing a cornerstone for future studies on members of this phage genus.
We discovered a novel interaction between phage P22 and its host Salmonella Typhimurium LT2 that is characterized by a phage mediated and targeted derepression of the host dgo operon. Upon further investigation, this interaction was found to be instigated by an ORFan gene (designated pid for phage P22 encoded instigator of dgo expression) located on a previously unannotated moron locus in the late region of the P22 genome, and encoding an 86 amino acid protein of 9.3 kDa. Surprisingly, the Pid/dgo interaction was not observed during strict lytic or lysogenic proliferation of P22, and expression of pid was instead found to arise in cells that upon infection stably maintained an unintegrated phage chromosome that segregated asymmetrically upon subsequent cell divisions. Interestingly, among the emerging siblings, the feature of pid expression remained tightly linked to the cell inheriting this phage carrier state and became quenched in the other. As such, this study is the first to reveal molecular and genetic markers authenticating pseudolysogenic development, thereby exposing a novel mechanism, timing, and populational distribution in the realm of phage–host interactions.
Viruses of bacteria, also referred to as (bacterio)phages, are the most abundant biological entity on earth and have a tremendous impact on the ecology of their hosts. It has traditionally been recognized that upon infection by a temperate phage the host cell is forced either to produce and release new virions during lytic development or to replicate and segregate the phage chromosome together with its own genetic material during lysogenic development. These developmental paths are orchestrated by a dedicated set of phage–host interactions that are able to sense and redirect host cell physiology. In addition to this classical bifurcation of temperate phage development, many studies on phage biology in natural ecosystems hypothesize the existence and significance of stable phage carrier cells that are not engaged in either lytic or lysogenic proliferation. Using Salmonella Typhimurium and phage P22 as a model system, we provide substantial evidence authenticating the existence of the phage carrier state and demonstrate that this state (i) is asymmetrically inherited among carrier cell siblings and (ii) enables the execution of a novel phage–host interaction that is not encountered during lytic or lysogenic proliferation.
Serratia plymuthica strain RVH1, initially isolated from an industrial food processing environment, displays potent antimicrobial activity towards a broad spectrum of Gram-positive and Gram-negative bacterial pathogens. Isolation and subsequent structure determination of bioactive molecules led to the identification of two polyamino antibiotics with the same molecular structure as zeamine and zeamine II as well as a third, closely related analogue, designated zeamine I. The gene cluster encoding the biosynthesis of the zeamine antibiotics was cloned and sequenced and shown to encode FAS, PKS as well as NRPS related enzymes in addition to putative tailoring and export enzymes. Interestingly, several genes show strong homology to the pfa cluster of genes involved in the biosynthesis of long chain polyunsaturated fatty acids in marine bacteria. We postulate that a mixed FAS/PKS and a hybrid NRPS/PKS assembly line each synthesize parts of the backbone that are linked together post-assembly in the case of zeamine and zeamine I. This interaction reflects a unique interplay between secondary lipid and secondary metabolite biosynthesis. Most likely, the zeamine antibiotics are produced as prodrugs that undergo activation in which a nonribosomal peptide sequence is cleaved off.
In 2011, a novel strain of O104:H4 Escherichia coli caused a serious outbreak of foodborne hemolytic uremic syndrome and bloody diarrhea in Germany. Antibiotics were of questionable use and 54 deaths occurred. Candidate therapeutic bacteriophages that efficiently lyse the E. coli O104:H4 outbreak strain could be selected rather easily from a phage bank or isolated from the environment. It is argued that phage therapy should be more considered as a potential armament against the growing threat of (resistant) bacterial infections.
The total synthesis is reported of the peptaibol Septocylindrin B which is related to the well documented channel forming peptaibol antibiotic Alamethicin. Several analogues were synthesized with a modified C-terminus, to investigate the SAR of the terminal residue Phaol. All these peptides were tested for their membrane perturbation properties by fluorescent dye leakage assay and for their antibacterial activity.
The 283,757-bp double-stranded DNA genome of Pseudomonas fluorescens phage OBP shares a general genomic organization with Pseudomonas aeruginosa phage EL. Comparison of this genomic organization, assembled in syntenic genomic blocks interspersed with hyperplastic regions of the ϕKZ-related phages, supports the proposed division in the “EL-like viruses,” and the “phiKZ-like viruses” within a larger subfamily. Identification of putative early transcription promoters scattered throughout the hyperplastic regions explains several features of the ϕKZ-related genome organization (existence of genomic islands) and evolution (multi-inversion in hyperplastic regions). When hidden Markov modeling was used, typical conserved core genes could be identified, including the portal protein, the injection needle, and two polypeptides with respective similarity to the 3′-5′ exonuclease domain and the polymerase domain of the T4 DNA polymerase. While the N-terminal domains of the tail fiber module and peptidoglycan-degrading proteins are conserved, the observation of C-terminal catalytic domains typical for the different genera supports the further subdivision of the ϕKZ-related phages.
The soil bacterial isolate Variovorax sp. strain SRS16 mineralizes the phenylurea herbicide linuron. The proposed pathway initiates with hydrolysis of linuron to 3,4-dichloroaniline (DCA) and N,O-dimethylhydroxylamine, followed by conversion of DCA to Krebs cycle intermediates. Differential proteomic analysis showed a linuron-dependent upregulation of several enzymes that fit into this pathway, including an amidase (LibA), a multicomponent chloroaniline dioxygenase, and enzymes associated with a modified chlorocatechol ortho-cleavage pathway. Purified LibA is a monomeric linuron hydrolase of ∼55 kDa with a Km and a Vmax for linuron of 5.8 μM and 0.16 nmol min−1, respectively. This novel member of the amidase signature family is unrelated to phenylurea-hydrolyzing enzymes from Gram-positive bacteria and lacks activity toward other tested phenylurea herbicides. Orthologues of libA are present in all other tested linuron-degrading Variovorax strains with the exception of Variovorax strains WDL1 and PBS-H4, suggesting divergent evolution of the linuron catabolic pathway in different Variovorax strains. The organization of the linuron degradation genes identified in the draft SRS16 genome sequence indicates that gene patchwork assembly is at the origin of the pathway. Transcription analysis suggests that a catabolic intermediate, rather than linuron itself, acts as effector in activation of the pathway. Our study provides the first report on the genetic organization of a bacterial pathway for complete mineralization of a phenylurea herbicide and the first report on a linuron hydrolase in Gram-negative bacteria.
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 spots were visualized, and 181 protein spots were 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 protein may be detected in two or more 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.
Mass spectrometry (MS); proteomics; two-dimensional gel electrophoresis (2-DE)
The flagellotropic phage 7-7-1 infects motile cells of Agrobacterium sp H13-3 by attaching to and traveling along the rotating flagellar filament to the secondary receptor at the base, where it injects its DNA into the host cell. Here we describe the complete genomic sequence of 69,391 base pairs of this unusual bacteriophage.
The sequence of the 7-7-1 genome was determined by pyro(454)sequencing to a coverage of 378-fold. It was annotated using MyRAST and a variety of internet resources. The structural proteome was analyzed by SDS-PAGE coupled electrospray ionization-tandem mass spectrometry (MS/MS).
Sequence annotation and a structural proteome analysis revealed 127 open reading frames, 84 of which are unique. In six cases 7-7-1 proteins showed sequence similarity to proteins from the virulent Burkholderia myovirus BcepB1A. Unique features of the 7-7-1 genome are the physical separation of the genes encoding the small (orf100) and large (orf112) subunits of the DNA packaging complex and the apparent lack of a holin-lysin cassette. Proteomic analysis revealed the presence of 24 structural proteins, five of which were identified as baseplate (orf7), putative tail fibre (orf102), portal (orf113), major capsid (orf115) and tail sheath (orf126) proteins. In the latter case, the N-terminus was removed during capsid maturation, probably by a putative prohead protease (orf114).
Agrobacterium; Phage evolution; Phage ecology; Genome; Proteome; Complex flagellum; Bioinformatics; Posttranslational modification
Peptidoglycan lytic enzymes (endolysins) induce bacterial host cell lysis in the late phase of the lytic bacteriophage replication cycle. Endolysins OBPgp279 (from Pseudomonas fluorescens phage OBP), PVP-SE1gp146 (Salmonella enterica serovar Enteritidis phage PVP-SE1) and 201ϕ2-1gp229 (Pseudomonas chlororaphis phage 201ϕ2-1) all possess a modular structure with an N-terminal cell wall binding domain and a C-terminal catalytic domain, a unique property for endolysins with a Gram-negative background. All three modular endolysins showed strong muralytic activity on the peptidoglycan of a broad range of Gram-negative bacteria, partly due to the presence of the cell wall binding domain. In the case of PVP-SE1gp146, this domain shows a binding affinity for Salmonella peptidoglycan that falls within the range of typical cell adhesion molecules (Kaff = 1.26×106 M−1). Remarkably, PVP-SE1gp146 turns out to be thermoresistant up to temperatures of 90°C, making it a potential candidate as antibacterial component in hurdle technology for food preservation. OBPgp279, on the other hand, is suggested to intrinsically destabilize the outer membrane of Pseudomonas species, thereby gaining access to their peptidoglycan and exerts an antibacterial activity of 1 logarithmic unit reduction. Addition of 0.5 mM EDTA significantly increases the antibacterial activity of the three modular endolysins up to 2–3 logarithmic units reduction. This research work offers perspectives towards elucidation of the structural differences explaining the unique biochemical and antibacterial properties of OBPgp279, PVP-SE1gp146 and 201ϕ2-1gp229. Furthermore, these endolysins extensively enlarge the pool of potential antibacterial compounds used against multi-drug resistant Gram-negative bacterial infections.
(Bacterio)phage PVP-SE1, isolated from a German wastewater plant, presents a high potential value as a biocontrol agent and as a diagnostic tool, even compared to the well-studied typing phage Felix 01, due to its broad lytic spectrum against different Salmonella strains. Sequence analysis of its genome (145,964 bp) shows it to be terminally redundant and circularly permuted. Its G+C content, 45.6 mol%, is lower than that of its hosts (50 to 54 mol%). We found a total of 244 open reading frames (ORFs), representing 91.6% of the coding capacity of the genome. Approximately 46% of encoded proteins are unique to this phage, and 22.1% of the proteins could be functionally assigned. This myovirus encodes a large number of tRNAs (n=24), reflecting its lytic capacity and evolution through different hosts. Tandem mass spectrometric analysis using electron spray ionization revealed 25 structural proteins as part of the mature phage particle. The genome sequence was found to share homology with 140 proteins of the Escherichia coli bacteriophage rV5. Both phages are unrelated to any other known virus, which suggests that an “rV5-like virus” genus should be created within the Myoviridae to contain these two phages.
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 ViI. 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.
In 1921, Richard Bruynoghe and his student Joséph Maisin published on the first use of bacteriophages in a phage therapy context. At that time, Bruynoghe (a medical doctor) was affiliated as a professor at the KU Leuven (Belgium) for just over a decade, within the Bacteriological Institute which he founded and led. After a distinguished career (he was acting mayor of the city of Leuven-Belgium during the second World War), he received a special medical award in 1951 just before his retirement in 1952. In this perspective, he was asked to provide an overview of his research for a lay-audience within the local University magazine: Onze Alma Mater (Our alma mater). We, as current affiliates of the KU Leuven are honored to present some of his legacy, which to date has been largely overlooked in historical accounts.
bacteria; bacteriophages; historical overview; phage biology; phage therapy
Pantoea agglomerans is a common soil bacterium used in the biocontrol of fungi and bacteria but is also an opportunistic human pathogen. It has been described extensively in this context, but knowledge of bacteriophages infecting this species is limited. Bacteriophages LIMEzero and LIMElight of P. agglomerans are lytic phages, isolated from soil samples, belonging to the Podoviridae and are the first Pantoea phages of this family to be described. The double-stranded DNA (dsDNA) genomes (43,032 bp and 44,546 bp, respectively) encode 57 and 55 open reading frames (ORFs). Based on the presence of an RNA polymerase in their genomes and their overall genome architecture, these phages should be classified in the subfamily of the Autographivirinae, within the genus of the “phiKMV-like viruses.” Phylogenetic analysis of all the sequenced members of the Autographivirinae supports the classification of phages LIMElight and LIMEzero as members of the “phiKMV-like viruses” and corroborates the subdivision into the different genera. These data expand the knowledge of Pantoea phages and illustrate the wide host diversity of phages within the “phiKMV-like viruses.”
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.
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 ϕ15, a ‘T7-like virus’ with a virion-associated exopolysaccharide (EPS) depolymerase. Phage ϕ15 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 ϕ15-mediated biofilm degradation was observed with generally a maximum biofilm degradation 8 h after addition of the higher phage doses (104 and 106 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 ϕ15 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.
Although horizontal gene transfer plays a pivotal role in bacteriophage evolution, many lytic phage genomes are clearly shaped by vertical evolution. We investigated the influence of minor genomic deletions and insertions on various phage-related phenotypic and serological properties.
We collected ten different isolates of Pseudomonas aeruginosa bacteriophage ϕKMV. All sequenced genomes (42-43 kb, long direct terminal repeats) are nearly identical, which intuitively implied strongly similar infections cycles. However, their latent periods vary between 21 and 28 minutes and they are able to lyse between 5 and 58% of a collection of 107 clinical P. aeruginosa strains. We also noted that phages with identical tail structures displayed profound differences in host spectra. Moreover, point mutations in tail and spike proteins were sufficient to evade neutralization by two phage-specific antisera, isolated from rabbits.
Although all analyzed phages are 83-97% identical at the genome level, they display a surprisingly large variation in various phenotypic properties. The small overlap in host spectrum and their ability to readily escape immune defences against a nearly identical phage are promising elements for the application of these phages in phage therapy.
The lysis cassette of Pseudomonas aeruginosa phage ϕKMV encodes a holin, endolysin, Rz and Rz1 in the canonical order. It has a tight organization with a high degree of overlapping genes and is highly conserved (between 96 and 100% identity at the protein level) among several other members of the “phiKMV-like viruses.” The endolysin KMV45 exhibits characteristics as expected for a signal-arrest-release (SAR) endolysin, whereas the holin KMV44 is a typical pinholin. KMV45 is initially secreted as an inactive, membrane-anchored endolysin, which is subsequently released by membrane depolarization driven by the pinholin KMV44. The SAR domain of KMV45 is necessary for its full enzymatic activity, suggesting a refolding of the catalytic cleft upon release from the membrane. The physical proximity of the catalytic glutamic acid residue close to SAR domain suggests an alternative activation mechanism compared to the SAR endolysin of phages P1, ERA103 and 21. Expression of KMV44 leads to a quick cell lysis when paired with SAR endolysin KMV45, but not with the cytoplasmic phage λ endolysin, indicating the membrane depolarizing function of KMV44 rather than the large hole-making function characteristic of classical holins.
lysis cassette; endolysin; pinholin; ϕKMV; signal-arrest-release domain
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 74.9 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 Pseudomonasaeruginosa (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.
Bacteriophage; Genomics; Anaerobic infection; Pseudomonas aeruginosa; Phage N4; Mass spectrometry
The genomes of the two lytic mutant Staphylococcus aureus bacteriophages, vB_SauS-phiIPLA35 (phiIPLA35) and vB_SauS-phiIPLA88 (phiIPLA88), isolated from milk have been analyzed. Their genomes are 45,344 bp and 42,526 bp long, respectively, and contain 62 and 61 open reading frames (ORFS). Enzymatic analyses and sequencing revealed that the phiIPLA35 DNA molecule has 3′-protruding cohesive ends (cos) 10 bp long, whereas phiIPLA88 DNA is 4.5% terminally redundant and most likely is packaged by a headful mechanism. N-terminal amino acid sequencing, mass spectrometry, bioinformatic analyses, and functional analyses enabled the assignment of putative functions to 58 gene products, including DNA packaging proteins, morphogenetic proteins, lysis components, and proteins necessary for DNA recombination, modification, and replication. Point mutations in their lysogeny control-associated genes explain their strictly lytic behavior. Muralytic activity associated with other structural components has been detected in virions of both phages. Comparative analysis of phiIPLA35 and phiIPLA88 genome structures shows that they resemble those of φ12 and φ11, respectively, both representatives of large genomic groupings within the S. aureus-infecting phages.
Crystallization and X-ray data collection of the C-terminus of gp36 from bacteriophage ϕKMV (KMV36C) are reported.
The C-terminus of gp36 of bacteriophage ϕKMV (KMV36C) functions as a particle-associated muramidase, presumably as part of the injection needle of the ϕKMV genome during infection. Crystals of KMV36C were obtained by hanging-drop vapour diffusion and diffracted to a resolution of 1.6 Å. The crystals belong to the cubic space group P432, with unit-cell parameters a = b = c = 102.52 Å. KMV36C shows 30% sequence identity to T4 lysozyme (PDB code 1l56).
phage infection; endolysin; T4 lysozyme; ϕKMV
We advocate unifying classical and genomic classification of bacteriophages by integration of proteomic data and physicochemical parameters. Our previous application of this approach to the entirely sequenced members of the Podoviridae fully supported the current phage classification of the International Committee on Taxonomy of Viruses (ICTV). It appears that horizontal gene transfer generally does not totally obliterate evolutionary relationships between phages.
CoreGenes/CoreExtractor proteome comparison techniques applied to 102 Myoviridae suggest the establishment of three subfamilies (Peduovirinae, Teequatrovirinae, the Spounavirinae) and eight new independent genera (Bcep781, BcepMu, FelixO1, HAP1, Bzx1, PB1, phiCD119, and phiKZ-like viruses). The Peduovirinae subfamily, derived from the P2-related phages, is composed of two distinct genera: the "P2-like viruses", and the "HP1-like viruses". At present, the more complex Teequatrovirinae subfamily has two genera, the "T4-like" and "KVP40-like viruses". In the genus "T4-like viruses" proper, four groups sharing >70% proteins are distinguished: T4-type, 44RR-type, RB43-type, and RB49-type viruses. The Spounavirinae contain the "SPO1-"and "Twort-like viruses."
The hierarchical clustering of these groupings provide biologically significant subdivisions, which are consistent with our previous analysis of the Podoviridae.