Genome sequences characterizing five mutations in RNA polymerase and major capsid of phages ?A318 and ?As51 of Vibrio alginolyticus with different burst efficiencies

Background

The burst size of a phage is important prior to phage therapy and probiotic usage. The efficiency for a phage to burst its host bacterium can result from molecular domino effects of the phage gene expressions which dominate to control host machinery after infection. We found two Podoviridae phages, ϕA318 and ϕAs51, burst a common host V. alginolyticus with different efficiencies of 72 and 10 PFU/bacterium, respectively. Presumably, the genome sequences can be compared to explain their differences in burst sizes.

Results

Among genes in 42.5 kb genomes with a GC content of 43.5%, 16 out of 47 open-reading frames (ORFs) were annotated to known functions, including RNA polymerase (RNAP) and phage structure proteins. 11 strong phage promoters and three terminators were found. The consensus sequence for the new vibriophage promoters is AATAAAGTTGCCCTATA, where the AGTTG bases of −8 through −12 are important for the vibriophage specificity, especially a consensus T at −9 position eliminating RNAP of K1E, T7 and SP6 phages to transcribe the genes. ϕA318 and ϕAs51 RNAP shared their own specific promoters. In comparing ϕAs51 with ϕA318 genomes, only two nucleotides were deleted in the RNAP gene and three mutating nucleotides were found in the major capsid genes.

Conclusion

Subtle analyses on the residue alterations uncovered the effects of five nucleotide mutations on the functions of the RNAP and capsid proteins, which account for the host-bursting efficiency. The deletion of two nucleotides in RNAP gene truncates the primary translation due to early stop codon, while a second translational peptide starting from GTG just at deletion point can remediate the polymerase activity. Out of three nucleotide mutations in major capsid gene, H53N mutation weakens the subunit assembly between capsomeres for the phage head; E313K reduces the fold binding between β-sheet and Spine Helix inside the peptide.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-505) contains supplementary material, which is available to authorized users.     

Thirteen Virulent and Temperate Bacteriophages of Lactobacillus bulgaricus and Lactobacillus lactis Belong to a Single DNA Homology Group

Thirteen virulent phages and two temperate phages of two closely related bacterial species (Lactobacillus lactis and L. bulgaricus) were compared for their protein composition, their antigenic properties, their restriction endonuclease patterns, and their DNA homology. The immunoblotting studies and the DNA-DNA hybridizations showed that the phages could be differentiated into two groups. One group contained 2 temperate phages of L. bulgaricus and 11 virulent phages of L. lactis. Inside each group, at least two common proteins of identical sizes could be detected for each phage. These proteins were able to cross-react in immunoblotting experiments with an antiserum raised against one phage of the same group. Temperate phage DNAs showed partial homology with DNAs from some virulent phages. These homologies seem to be located on the region coding for the structural proteins since recombinant plasmids coding for one of the major phage proteins of one phage were able to hybridize with the DNAs from phages of the same group. These results suggest that temperate and virulent phages may be related to one another.     

Compensatory evolution in response to a novel RNA polymerase: orthologous replacement of a central network gene

A bacteriophage genome was forced to evolve a new system of regulation by replacing its RNA polymerase (RNAP) gene, a central component of the phage developmental pathway, with that of a relative. The experiment used the obligate lytic phage T7 and the RNAP gene of phage T3. T7 RNAP uses 17 phage promoters, which are responsible for all middle and late gene expression, DNA replication, and progeny maturation, but the enzyme has known physical contacts with only 2 other phage proteins. T3 RNAP was supplied in trans by the bacterial host to a T7 genome lacking its own RNAP gene and the phage population was continually propagated on naive bacteria throughout the adaptation. Evolution of the T3 RNAP gene was thereby prevented, and selection was for the evolution of regulatory signals throughout the phage genome. T3 RNAP transcribes from T7 promoters only at low levels, but a single mutation in the promoter confers high expression, providing a ready mechanism for reevolution of gene expression in this system. When selected for rapid growth, fitness of the engineered phage evolved from a low of 5 doublings/h to 33 doublings/h, close to the expected maximum of 37 doublings/h. However, the experiment was terminated before it could be determined accurately that fitness had reached an obvious plateau, and it is not known whether further adaptation could have resulted in complete recovery of fitness. More than 30 mutations were observed in the evolved genome, but changes were found in only 9 of the 16 promoters, and several coding changes occurred in genes with no known contacts with the RNAP. Surprisingly, the T7 genome adapted to T3 RNAP also maintained high fitness when using T7 RNAP, suggesting that the extreme incompatibility of T7 elements with T3 RNAP is not an invariant property of divergence in these expression systems.     

PhREEPred: Phage Resistance Emergence Prediction Web Tool to Foresee Encapsulated Bacterial Escape from Phage Cocktail Treatment

Phages, as well as phage-derived proteins, especially lysins and depolymerases, are intensively studied to become prospective alternatives or supportive antibacterials used alone or in combination. In the common phage therapy approach, the unwanted emergence of phage-resistant variants from the treated bacterial population can be postponed or reduced by the utilization of an effective phage cocktail. In this work, we present a publicly available web tool PhREEPred (Phage Resistance Emergence Prediction) (https://phartner.shinyapps.io/PhREEPred/), which will allow an informed choice of the composition of phage cocktails by predicting the outcome of phage cocktail or phage/depolymerase combination treatments against encapsulated bacterial pathogens given a mutating population that escapes single phage treatment. PhREEPred simulates solutions of our mathematical model calibrated and tested on the experimental Klebsiella pneumoniae setup and Klebsiella-specific lytic phages: K63 type-specific phage KP34 equipped with a capsule-degrading enzyme (KP34p57), capsule-independent myoviruses KP15 and KP27, and recombinant capsule depolymerase KP34p57. The model can calculate the phage-resistance emergence depending on the bacterial growth rate and initial density, the multiplicity of infection, phage latent period, its infectiveness and the cocktail composition, as well as initial depolymerase concentration and activity rate. This model reproduced the experimental results and showed that (i) the phage cocktail of parallelly infecting phages is less effective than the one composed of sequentially infecting phages; (ii) depolymerase can delay or prevent bacterial resistance by unveiling an alternative receptor for initially inactive phages. In our opinion, this customer-friendly web tool will allow for the primary design of the phage cocktail and phage-depolymerase combination effectiveness against encapsulated pathogens.     

Impact of Phages on Two-Species Bacterial Communities

A long history of experimental work has shown that addition of bacteriophages to a monoculture of bacteria leads to only a temporary depression of bacterial levels. Resistant bacteria usually become abundant, despite reduced growth rates relative to those of phage-sensitive bacteria. This restoration of high bacterial density occurs even if the phages evolve to overcome bacterial resistance. We believe that the generality of this result may be limited to monocultures, in which the resistant bacteria do not face competition from bacterial species unaffected by the phage. As a simple case, we investigated the impact of phages attacking one species in a two-species culture of bacteria. In the absence of phages, Escherichia coli B and Salmonella enterica serovar Typhimurium were stably maintained during daily serial passage in glucose minimal medium (M9). When either of two E. coli-specific phages (T7 or T5) was added to the mixed culture, E. coli became extinct or was maintained at densities that were orders of magnitude lower than those before phage introduction, even though the E. coli densities with phage reached high levels when Salmonella was absent. In contrast, the addition of a phage that attacked only Salmonella (SP6) led to transient decreases in the bacterial number whether E. coli was absent or present. These results suggest that phages can sometimes, although not always, provide long-term suppression of target bacteria.     

Structural and Functional Studies of gpX of Escherichia coli Phage P2 Reveal a Widespread Role for LysM Domains in the Baseplates of Contractile-Tailed Phages

A variety of bacterial pathogenicity determinants, including the type VI secretion system and the virulence cassettes from Photorhabdus and Serratia, share an evolutionary origin with contractile-tailed myophages. The well-characterized Escherichia coli phage P2 provides an excellent system for studies related to these systems, as its protein composition appears to represent the “minimal” myophage tail. In this study, we used nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of gpX, a 68-residue tail baseplate protein. Although the sequence and structure of gpX are similar to those of LysM domains, which are a large family associated with peptidoglycan binding, we did not detect a peptidoglycan-binding activity for gpX. However, bioinformatic analysis revealed that half of all myophages, including all that possess phage T4-like baseplates, encode a tail protein with a LysM-like domain, emphasizing a widespread role for this domain in baseplate function. While phage P2 gpX comprises only a single LysM domain, many myophages display LysM domain fusions with other tail proteins, such as the DNA circulation protein found in Mu-like phages and gp53 of T4-like phages. Electron microscopy of P2 phage particles with an incorporated gpX-maltose binding protein fusion revealed that gpX is located at the top of the baseplate, near the junction of the baseplate and tail tube. gpW, the orthologue of phage T4 gp25, was also found to localize to this region. A general colocalization of LysM-like domains and gpW homologues in diverse phages is supported by our bioinformatic analysis.     

Ambivalent bacteriophages of different species active on Escherichia coli K12 and Salmonella sp. strains

A study was made of several bacteriophages (including phages U2 and LB related to T-even phages of Escherichia coli) that grow both on E. coli K12 and on some Salmonella strains. Such phages were termed ambivalent. T-even ambivalent phages (U2 and LB) are rare and have a limited number of hosts among Salmonella strains. U2 and LB are similar to canonical E. coli-specific T-even phages in morphological type and size of the phage particle and in reaction with specific anti-T4 serum. Phages U2 and LB have identical sets of structural proteins, some of which are similar in size to structural proteins of phages T2 and T4. DNA restriction patterns of phages U2 and LB differ from each other and from those of T2 and T4. Still, DNAs of all four phages have considerable homology. Unexpectedly, phages U2 and LB grown on Salmonella bongori were unstable during centrifugation in a CsCl gradient. Ambivalent bacteriophages were found in species other than T-even phages and were similar in morphotype to lambdoid and other E. coli phages. One of the ambivalent phages was highly similar to well-known Felix01, which is specific for Salmonella. Ambivalent phages can be used to develop a new set for phage typing in Salmonella. An obvious advantage is that ambivalent phages can be reproduced in the E. coli K12 laboratory strain, which does not produce active temperature phages. Consequently, the resulting typing phage preparation is devoid of an admixture of temperate phages, which are common in Salmonella. The presence of temperate phages in phage-typing preparations may cause false-positive results in identifying specific Salmonella strains isolated from the environment or salmonellosis patients. Ambivalent phages are potentially useful for phage therapy and prevention of salmonellosis in humans and animals.     

Complete Genomic Sequence of Bacteriophage φEcoM-GJ1, a Novel Phage That Has Myovirus Morphology and a Podovirus-Like RNA Polymerase

The complete genome of EcoM-GJ1, a lytic phage that attacks porcine enterotoxigenic Escherichia coli of serotype O149:H10:F4, was sequenced and analyzed. The morphology of the phage and the identity of the structural proteins were also determined. The genome consisted of 52,975 bp with a G+C content of 44% and was terminally redundant and circularly permuted. Seventy-five potential open reading frames (ORFs) were identified and annotated, but only 29 possessed homologs. The proteins of five ORFs showed homology with proteins of phages of the family Myoviridae, nine with proteins of phages of the family Podoviridae, and six with proteins of phages of the family Siphoviridae. ORF 1 encoded a T7-like single-subunit RNA polymerase and was preceded by a putative E. coli σ⁷⁰-like promoter. Nine putative phage promoters were detected throughout the genome. The genome included a tRNA gene of 95 bp that had a putative 18-bp intron. The phage morphology was typical of phages of the family Myoviridae, with an icosahedral head, a neck, and a long contractile tail with tail fibers. The analysis shows that EcoM-GJ1 is unique, having the morphology of the Myoviridae, a gene for RNA polymerase, which is characteristic of phages of the T7 group of the Podoviridae, and several genes that encode proteins with homology to proteins of phages of the family Siphoviridae.     

Phylogenetic and complementation analysis of a single-stranded DNA binding protein family from lactococcal phages indicates a non-bacterial origin

Background

The single-stranded-nucleic acid binding (SSB) protein superfamily includes proteins encoded by different organisms from Bacteria and their phages to Eukaryotes. SSB proteins share common structural characteristics and have been suggested to descend from an ancestor polypeptide. However, as other proteins involved in DNA replication, bacterial SSB proteins are clearly different from those found in Archaea and Eukaryotes. It was proposed that the corresponding genes in the phage genomes were transferred from the bacterial hosts. Recently new SSB proteins encoded by the virulent lactococcal bacteriophages (Orf14_bIL67-like proteins) have been identified and characterized structurally and biochemically.

Methodology/Principal Findings

This study focused on the determination of phylogenetic relationships between Orf14_bIL67-like proteins and other SSBs. We have performed a large scale phylogenetic analysis and pairwise sequence comparisons of SSB proteins from different phyla. The results show that, in remarkable contrast to other phage SSBs, the Orf14_bIL67–like proteins form a distinct, self-contained and well supported phylogenetic group connected to the archaeal SSBs. Functional studies demonstrated that, despite the structural and amino acid sequence differences from bacterial SSBs, Orf14_bIL67 protein complements the conditional lethal ssb-1 mutation of Escherichia coli.

Conclusions/Significance

Here we identified for the first time a group of phages encoded SSBs which are clearly distinct from their bacterial counterparts. All methods supported the recognition of these phage proteins as a new family within the SSB superfamily. Our findings suggest that unlike other phages, the virulent lactococcal phages carry ssb genes that were not acquired from their hosts, but transferred from an archaeal genome. This represents a unique example of a horizontal gene transfer between Archaea and bacterial phages.     

Comparison of new giant bacteriophages OBP and Lu11 of soil pseudomonads with bacteriophages of the ϕKZ-supergroup of Pseudomonas aeruginosa

Study of two recently isolated giant bacteriophages Lu11 and OBP that are active on Pseudomonas putida var. Manila and Pseudomonas fluorescens, respectively, demonstrated their similarity in morphotype, genome size, and size of phage particles, with giant bacteriophages of Pseudomonas aeruginosa assigned to the supergroup of ?KZ-like phages of the family Myoviridae. This supergroup was designated in this manner according to the best studied phage ?KZ that belongs to the species of this group widely distributed in nature. Comparison of major polypeptide sizes of mature particles suggests similarity of certain proteins in the phages examined. In OBP particles visualized with an electron microscope, an “inner body” was detected, which points to specific DNA package intrinsic to phages of ?KZ group. In the meantime, phages Lu11 and OBP do not exhibit resemblance among themselves or with any of earlier described ?KZ-like phages in respect to detectable DNA homology. Note that phage Lu11 of P. putida var. Manila exhibits very slight homology with phage Lin68 of the family of P. aeruginosa ?KZ-like phages detected only in blot hybridization. This suggests the possible involvement of these phages in interspecies recombination (“gene shuffling”) between phages of various bacterial species. Results of partial sequencing of phage genomes confirmed the phylogenetic relatedness of phage OBP to phages of the ?KZ supergroup, whereas phage Lu11 most probably belongs to a novel species that is not a member of supergroup ?KZ composition. The results of the study are discussed in terms of the evolution of these phages.     

Immune electron microscopy in the study of biological matter

A brief review of literature data and our investigations on the antibodies used for specific labeling in electron microscopy is presented. Considered are the problems connected with structure and function of separate components of bacterial viruses revealed by means of specific antibodies. The results of fine differentiation of antigenic components in the case of phages of the colidysentery group allowed to elucidate the functional role of the adsorption apparatus in the course of phage interaction with the bacterial cell. The topology of structural proteins (gene-products 35, 36, 37) of the tail's long strands for phages T4, DDVI+h and DDVIh is determined. Antigenic properties of proteins that are found in the composition of two forms of Bacillus mycoides are demonstrated immune-electronmicroscopically. On the basis of this finding, a conclusion was made that one of these phages acted as precursor, the other--as satellite during the simultaneous development of these phages in the bacterial cell. It was also established that temperate and virulent phages are related antigenically, which proves that lysogenic bacteria can be one of the phage sources on the environmental conditions. Visualization of non-ribosomal genes of procaryots that code for structural proteins of a defective phage proves the efficiency of the immune-electronmicroscopic method for investigating of biological objects.     

Conversion of the coprogen transport protein FhuE and the ferrioxamine B transport protein FoxA into ferrichrome transport proteins

The FhuA protein of Escherichia coli K-12 transports ferrichrome and the structurally related antibiotic albomycin across the outer membrane and serves as a receptor for the phages T1, T5, and φ80 and for colicin M. In this paper, we show that chimeric proteins consisting of the central part of FhuA and the N- and C-terminal parts of FhuE (coprogen receptor) or the N- and/or C-terminal parts of FoxA (ferrioxamine B receptor), function as ferrichrome transport proteins. Although the hybrid proteins contained the previously identified gating loop of FhuA, which is the principal binding site of the phages T5, T1, and φ80, only the hybrid protein consisting of the N-terminal third of FoxA and the C-terminal two thirds of FhuA conferred weak phage sensitivity to cells. Apparently, the gating loop is essential, but not sufficient for wild-type levels of ferrichrome transport and for phage sensitivity. The properties of FhuA-FoxA hybrids suggest different regions of the two receptors for ferric siderophore uptake.     

A Study of 33 Bacteriophages of Rhizobium meliloti

A total of 33 Rhizobium meliloti bacteriophages were studied. Of those, 21 were isolated in northern France from field soil in which Medicago sativa L. was grown. The other 12 phages were obtained by UV light and mitomycin C induction from 46 R. meliloti strains. Rhizobiophages were characterized by their morphology, host range, serological properties, restriction endonuclease patterns, DNA-DNA homologies, and DNA molecular weights. Five morphotypes were observed showing tailed phages with icosahedral heads. The categories of morphotypes included the Myoviridae (11 phages), Siphoviridae (3 morphotypes and 20 phages), and Podoviridae (2 phages). Type NM1 phage (Siphoviridae) is highly unusual because of the presence of transverse bars on the phage tail. Soil phages had broad host ranges, whereas phages isolated from bacterial cultures showed more or less narrow host ranges. Restriction endonuclease patterns and DNA-DNA hybridization experiments showed that the five phage type genomes were unrelated. Molecular weights of phage type DNAs were estimated, and they corresponded to values expected for capsid sizes, except for phage NM8. Type M11S (Siphoviridae) did not correspond to any other described Rhizobium phages and represents a new species.     

Isolation and characterization of phage–host systems from the Baltic Sea ice

In search for sea ice bacteria and their phages from the Baltic Sea ice, two ice samples were collected from land-fast ice in a south-west Finland coastal site in February and March 2011. Bacteria were isolated from the melted sea ice samples and phages were screened from the same samples for 43 purified isolates. Plaque-producing phages were found for 15 bacterial isolates at 3 °C. Ten phage isolates were successfully plaque purified and eight of them were chosen for particle purification to analyze their morphology and structural proteins. Phage 1/32 infecting an isolate affiliated to phylum Bacteroidetes (Flavobacterium sp.) is a siphovirus and six phages infecting isolates affiliated to γ-Proteobacteria (Shewanella sp.) hosts were myoviruses. Cross titrations between the hosts showed that all studied phages are host specific. Phage solutions, host growth and phage infection were tested in different temperatures revealing phage temperature tolerance up to 45 °C, whereas phage infection was in most of the cases retarded above 15 °C. This study is the first to report isolation and cultivation of ice bacteria and cold-active phages from the Baltic Sea ice.     

Physical and genetical analysis of bacteriophage T4 generalized transduction

This report describes a comparison of the efficiency of transduction of genes in E. coli by the generalized transducing bacteriophages T4GT7 and P1CM. Both phages are capable of transducing many genetic markers in E. coli although the frequency of transduction for particular genes varies over a wide range. The frequency of transduction for most genes depends on which transducing phage is used as well as on the donor and recipient bacterial strains. Analysis of T4GT7 phage lysates by cesium chloride density gradient centrifugation shows that transducing phage particles contain primarily bacterial DNA and carry little, if any, phage DNA. In this regard transducing phages P1CM and T4GT7 are similar; both phages package either bacterial or phage DNA but not both DNAs into the same particle.