The smallest ssDNA phage infecting a marine bacterium

In the marine environment, only a few lytic single-stranded DNA (ssDNA) phages have been isolated and characterized, despite the fact that diverse ssDNA bacteriophages have been discovered via metagenomic studies. In this study, we isolated and characterized a new ssDNA phage, vB_RpoMi-Mini, which infects a marine bacterium Ruegeria pomeroyi DSS-3. With a genome size of 4248 bp and only four putative open reading frames (ORF), vB_RpoMi-Mini becomes the smallest ssDNA phage among the known ssDNA phage isolates and represents the DNA bacteriophage with the least number of ORFs. Genome-wide analysis reveals that bacteriophage Mini is distantly related to the known ssDNA phages and belongs to an unclassified ssDNA phage within the Microviridae family. The presence of peptidase in vB_RpoMi-Mini genome further implies that horizontal gene transfer could be an important driving force in the evolution of ssDNA phages. Bacteriophage Mini seems to have lost the spike protein commonly seen in ssDNA phages, suggesting that ssDNA phage can be more diverse than previously thought. Metagenomic analysis indicates that Mini-like phages are widely distributed in the environments. The discovery of vB_RpoMi-Mini expands our understanding of ssDNA phages in nature, and also indicates our dearth of knowledge regarding of ssDNA phages.     

The Effect of Mutation and Selection on Codon Adaptation in Escherichia coli Bacteriophage

Studying phage codon adaptation is important not only for understanding the process of translation elongation, but also for reengineering phages for medical and industrial purposes. To evaluate the effect of mutation and selection on phage codon usage, we developed an index to measure selection imposed by host translation machinery, based on the difference in codon usage between all host genes and highly expressed host genes. We developed linear and nonlinear models to estimate the C→T mutation bias in different phage lineages and to evaluate the relative effect of mutation and host selection on phage codon usage. C→T-biased mutations occur more frequently in single-stranded DNA (ssDNA) phages than in double-stranded DNA (dsDNA) phages and affect not only synonymous codon usage, but also nonsynonymous substitutions at second codon positions, especially in ssDNA phages. The host translation machinery affects codon adaptation in both dsDNA and ssDNA phages, with a stronger effect on dsDNA phages than on ssDNA phages. Strand asymmetry with the associated local variation in mutation bias can significantly interfere with codon adaptation in both dsDNA and ssDNA phages.     

Diversity and distribution of single-stranded DNA phages in the North Atlantic Ocean

Knowledge of marine phages is highly biased toward double-stranded DNA (dsDNA) phages; however, recent metagenomic surveys have also identified single-stranded DNA (ssDNA) phages in the oceans. Here, we describe two complete ssDNA phage genomes that were reconstructed from a viral metagenome from 80 m depth at the Bermuda Atlantic Time-series Study (BATS) site in the northwestern Sargasso Sea and examine their spatial and temporal distributions. Both genomes (SARssφ1 and SARssφ2) exhibited similarity to known phages of the Microviridae family in terms of size, GC content, genome organization and protein sequence. PCR amplification of the replication initiation protein (Rep) gene revealed narrow and distinct depth distributions for the newly described ssDNA phages within the upper 200 m of the water column at the BATS site. Comparison of Rep gene sequences obtained from the BATS site over time revealed changes in the diversity of ssDNA phages over monthly time scales, although some nearly identical sequences were recovered from samples collected 4 years apart. Examination of ssDNA phage diversity along transects through the North Atlantic Ocean revealed a positive correlation between genetic distance and geographic distance between sampling sites. Together, the data suggest fundamental differences between the distribution of these ssDNA phages and the distribution of known marine dsDNA phages, possibly because of differences in host range, host distribution, virion stability, or viral evolution mechanisms and rates. Future work needs to elucidate the host ranges for oceanic ssDNA phages and determine their ecological roles in the marine ecosystem.     

Experimental evolution of RNA versus DNA viruses

Based on their extremely high mutation rates, RNA viruses have been traditionally considered as the fastest evolving entities in nature. However, recent work has revealed that, despite their greater replication fidelity, single-stranded (ss) DNA viruses can evolve fast in a similar way. To further investigate this issue, we have compared the rates of adaptation and molecular evolution of ssRNA and ssDNA viruses under highly controlled laboratory conditions using the bacteriophages ΦX174, G4, f1, Qβ, SP, and MS2 as model systems. Our results indicate that ssRNA phages evolve faster than ssDNA phages under strong selective pressure, and that their extremely high mutation rates appear to be optimal for this kind of scenario. However, their performance becomes similar to that of ssDNA phages over the longer term or when the population is moderately well-adapted. Interestingly, the roughly 100-fold difference between the mutation rates of ssRNA and ssDNA phages yields less than a fivefold difference in adaptation and nucleotide substitution rates. The results are therefore consistent with the observation that, despite their lower mutation rates, ssDNA viruses can sometimes match the evolvability of RNA viruses.     

The Bacillus subtilis phage phi 29 protein p16.7, involved in phi 29 DNA replication, is a membrane-localized single-stranded DNA-binding protein.

The functional role of the phi 29-encoded integral membrane protein p16.7 in phage DNA replication was studied using a soluble variant, p16.7A, lacking the N-terminal membrane-spanning domain. Because of the protein-primed mechanism of DNA replication, the bacteriophage phi 29 replication intermediates contain long stretches of single-stranded DNA (ssDNA). Protein p16.7A was found to be an ssDNA-binding protein. In addition, by direct and functional analysis we show that protein p16.7A binds to the stretches of ssDNA of the phi 29 DNA replication intermediates. Properties of protein p16.7A were compared with those of the phi 29-encoded single-stranded DNA-binding protein p5. The results obtained show that both proteins have different, non-overlapping functions. The likely role of p16.7 in attaching phi 29 DNA replication intermediates to the membrane of the infected cell is discussed. Homologues of gene 16.7 are present in phi 29-related phages, suggesting that the proposed role of p16.7 is conserved in this family of phages.     

Single-stranded DNA intermediates in IS91 rolling-circle transposition

IS91 displays a number of characteristics unique among insertion sequence (IS) elements, suggesting that it transposes by a novel mechanism called rolling-circle (RC) transposition. We reported previously that IS91 transposase (TnpA) amino acid sequence shares a series of five conserved signatures with A proteins of RC replicating phages, including a pair of invariant tyrosines that catalyse two successive transesterification reactions during replication initiation and termination. To analyse their role in IS91 transposition, we constructed a series of TnpA derivatives in which the invariant Tyr-249 and/or Tyr-253 were mutated to either phenylalanine or serine. Mutation of either tyrosine resulted in complete loss of transposition activity in vivo. This result was taken as a first new line of evidence that TnpA is a functional analogue of phiX174 phage A protein. Secondly, RC replication plasmids and phages accumulate single-stranded DNA (ssDNA) intermediates as a result of uncoupled leading and lagging DNA strand synthesis. Using a plasmid carrying an IS91-derived IRLkan-IRR transposable cassette, in which the left (IRL)- and right (IRR)-terminal sequences of IS91 flank a kanamycin resistance gene (kan), we demonstrated the in vivo formation of two new DNA species after induction of transposase expression. The first was a circular ssDNA that contained the transposable cassette covalently joined at its exact termini, whereas the second was a double-stranded circle of the same element. When this experiment was repeated using the mutant transposases described above, the ssDNA and dsDNA intermediates could not be observed, indicating that the integrity of both Y249 and Y253 was essential for their appearance. The presence of ssDNA intermediate products is the first biochemical evidence for a RC mechanism of IS91 transposition.     

The DNA binding mechanism of a SSB protein from Lactococcus lactis siphophage p2

Virulent lactococcal phages of the Siphoviridae family are responsible for the industrial milk fermentation failures worldwide. Lactococcus lactis, a Gram-positive bacterium widely used for the manufacture of fermented dairy products, is subjected to infections by virulent phages, predominantly those of the 936 group, including phage p2. Among the proteins coded by lactococcal phage genomes, of special interest are those expressed early, which are crucial to efficiently carry out the phage lytic cycle. We previously identified and solved the 3D structure of lactococcal phage p2 ORF34, a single stranded DNA binding protein (SSBp2). Here we investigated the molecular basis of ORF34 binding mechanism to DNA. DNA docking on SSBp2 and Molecular Dynamics simulations of the resulting complex identified R15 as a crucial residue for ssDNA binding. Electrophoretic Mobility Shift Assays (EMSA) and Atomic Force Microscopy (AFM) imaging revealed the inability of the Arg15Ala mutant to bind ssDNA, as compared to the native protein. Since R15 is highly conserved among lactococcal SSBs, we propose that its role in the SSBp2/DNA complex stabilization might be extended to all the members of this protein family.     

Codon Bias is a Major Factor Explaining Phage Evolution in Translationally Biased Hosts

The size and diversity of bacteriophage populations require methodologies to quantitatively study the landscape of phage differences. Statistical approaches are confronted with small genome sizes forbidding significant single-phage analysis, and comparative methods analyzing full phage genomes represent an alternative but they are of difficult interpretation due to lateral gene transfer, which creates a mosaic spectrum of related phage species. Based on a large-scale codon bias analysis of 116 DNA phages hosted by 11 translationally biased bacteria belonging to different phylogenetic families, we observe that phage genomes are almost always under codon selective pressure imposed by translationally biased hosts, and we propose a classification of phages with translationally biased hosts which is based on adaptation patterns. We introduce a computational method for comparing phages sharing homologous proteins, possibly accepted by different hosts. We observe that throughout phages, independently from the host, capsid genes appear to be the most affected by host translational bias. For coliphages, genes involved in virion morphogenesis, host interaction and ssDNA binding are also affected by adaptive pressure. Adaptation affects long and small phages in a significant way. We analyze in more detail the Microviridae phage space to illustrate the potentiality of the approach. The small number of directions in adaptation observed in phages grouped around ϕX174 is discussed in the light of functional bias. The adaptation analysis of the set of Microviridae phages defined around ϕMH2K shows that phage classification based on adaptation does not reflect bacterial phylogeny. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The putative single-stranded DNA-binding protein of the filamentous bacteriophage, Ifl. Amino acid sequence of the protein and structure of the gene.

The protein product corresponding to the gene located in the region of the coliphage Ifl genome shown to contain the code for the single-stranded DNA (ssDNA)-binding proteins of all filamentous phages so far studied has been isolated from infected bacterial cells and its amino acid sequence determined. The mature protein contains 95 amino acids (calculated molecular mass 10553 Da). Its sequence corresponds to that predicted from the DNA sequence but lacks the initiating methionine residue. Although there is little direct sequence homology between the phage Ifl protein and the ssDNA-binding proteins of the other filamentous phages that have been studied, computer-based comparisons of various physical and structural parameters showed that the phage Ifl protein contains a domain that is closely related to domains in the coliphage T4 gene 32 protein and the Pseudomonas phage Pfl ssDNA-binding protein and suggest that the Ifl protein does have a ssDNA-binding function although we were unable to show this directly.     

Prophage Tracer: precisely tracing prophages in prokaryotic genomes using overlapping split-read alignment

The life cycle of temperate phages includes a lysogenic cycle stage when the phage integrates into the host genome and becomes a prophage. However, the identification of prophages that are highly divergent from known phages remains challenging. In this study, by taking advantage of the lysis-lysogeny switch of temperate phages, we designed Prophage Tracer, a tool for recognizing active prophages in prokaryotic genomes using short-read sequencing data, independent of phage gene similarity searching. Prophage Tracer uses the criterion of overlapping split-read alignment to recognize discriminative reads that contain bacterial (attB) and phage (attP) att sites representing prophage excision signals. Performance testing showed that Prophage Tracer could predict known prophages with precise boundaries, as well as novel prophages. Two novel prophages, dsDNA and ssDNA, encoding highly divergent major capsid proteins, were identified in coral-associated bacteria. Prophage Tracer is a reliable data mining tool for the identification of novel temperate phages and mobile genetic elements. The code for the Prophage Tracer is publicly available at https://github.com/WangLab-SCSIO/Prophage_Tracer.     

Efficient point mutagenesis in mycobacteria using single-stranded DNA recombineering: characterization of antimycobacterial drug targets

Construction of genetically isogenic strains of mycobacteria is complicated by poor recombination rates and the lack of generalized transducing phages for Mycobacterium tuberculosis . We report here a powerful method for introducing single point mutations into mycobacterial genomes using oligonucleotide-derived single-stranded DNA recombineering and mycobacteriophage-encoded proteins. Phage Che9c gp61-mediated recombination is sufficiently efficient that single base changes can be introduced without requirement for direct selection, with isogenic mutant strains identified simply by PCR. Efficient recombination requires only short (50 nucleotide) oligonucleotides, but there is an unusually strong strand bias and an oligonucleotide targeting lagging strand DNA synthesis can recombine more than 10 000-fold efficiently than its complementary oligonucleotide. This ssDNA recombineering provides a simple assay for comparing the activities of related phage recombinases, and we find that both Escherichia coli RecET and phage λ Red recombination proteins function inefficiently in mycobacteria, illustrating the utility of developing recombineering in new bacterial systems using host-specific bacteriophage recombinases. ssDNA mycobacterial recombineering provides a simple approach to characterizing antimycobacterial drug targets, and we have constructed and characterized single point mutations that confer resistance to isoniazid, rifampicin, ofloxacin and streptomycin.     

Electrostatic potential distribution of the gene V protein from Ff phage facilitates cooperative DNA binding: a model of the GVP-ssDNA complex.

The crystal structure of the gene V protein (GVP) from the Ff filamentous phages (M13, fl, fd) has been solved for the wild-type and two mutant (Y41F and Y41H) proteins at high resolution. The Y41H mutant crystal structure revealed crystal packing interactions, which suggested a plausible scheme for constructing the polymeric protein shell of the GVP-single-stranded DNA (ssDNA) complex (Guan Y, et al., 1994, Biochemistry 33:7768-7778). The electrostatic potentials of the isolated and the cooperatively formed protein shell have been calculated using the program GRASP and they revealed a highly asymmetric pattern of the electrostatic charge distribution. The inner surface of the putative DNA-binding channel is positively charged, whereas the opposite outer surface is nearly neutral. The electrostatic calculation further demonstrated that the formation of the helical protein shell enhanced the asymmetry of the electrostatic distribution. A model of the GVP-ssDNA complex with the n = 4 DNA-binding mode could be built with only minor conformational perturbation to the GVP protein shell. The model is consistent with existing biochemical and biophysical data and provides clues to the properties of GVP, including the high cooperatively of the protein binding to ssDNA. The two antiparallel ssDNA strands form a helical ribbon with the sugar-phosphate backbones at the middle and the bases pointing away from each other. The bases are stacked and the Phe 73 residue is intercalated between two bases. The optimum binding to a tetranucleotide unit requires the participation of four GVP dimers, which may explain the cooperativity of the GVP binding to DNA.     

Resistance of Aerosolized Bacterial Viruses to Relative Humidity and Temperature

The use of aerosolized bacteriophages as surrogates for hazardous viruses might simplify and accelerate the discovery of links between viral components and their persistence in the airborne state under diverse environmental conditions. In this study, four structurally distinct lytic phages, MS2 (single-stranded RNA [ssRNA]), ϕ6 (double-stranded RNA [dsRNA]), ϕX174 (single-stranded DNA [ssDNA]), and PR772 (double-stranded DNA [dsDNA]), were nebulized into a rotating chamber and exposed to various levels of relative humidity (RH) and temperature as well as to germicidal UV radiation. The aerosolized viral particles were allowed to remain airborne for up to 14 h before being sampled for analysis by plaque assays and quantitative PCRs. Phages ϕ6 and MS2 were the most resistant at low levels of relative humidity, while ϕX174 was more resistant at 80% RH. Phage ϕ6 lost its infectivity immediately after exposure to 30°C and 80% RH. The infectivity of all tested phages rapidly declined as a function of the exposure time to UVC radiation, phage MS2 being the most resistant. Taken altogether, our data indicate that these aerosolized phages behave differently under various environmental conditions and highlight the necessity of carefully selecting viral simulants in bioaerosol studies.     

Differential functional behavior of viral phi29, Nf and GA-1 SSB proteins

DNA replication of 29 and related phages takes place via a strand displacement mechanism, a process that generates large amounts of single-stranded DNA (ssDNA). Consequently, phage-encoded ssDNA-binding proteins (SSBs) are essential proteins during phage 29-like DNA replication. In the present work we analyze the helix-destabilizing activity of the SSBs of 29 and the related phages Nf and GA-1, their ability to eliminate non-productive binding of 29 DNA polymerase to ssDNA and their stimulatory effect on replication by 29 DNA polymerase in primed M13 ssDNA replication, a situation that resembles type II replicative intermediates that occur during 29-like DNA replication. Significant differences have been appreciated in the functional behavior of the three SSBs. First, the GA-1 SSB is able to display helix-destabilizing activity and to stimulate dNTP incorporation by 29 DNA polymerase in the M13 DNA replication assay, even at SSB concentrations at which the 29 and Nf SSBs do not show any effect. On the other hand, the 29 SSB is the only one of the three SSBs able to increase the replication rate of 29 DNA polymerase in primed M13 ssDNA replication. From the fact that the 29 SSB, but not the Nf SSB, stimulates the replication rate of Nf DNA polymerase we conclude that the different behaviors of the SSBs on stimulation of the replication rate of 29 and Nf DNA polymerases is most likely due to formation of different nucleoprotein complexes of the SSBs with the ssDNA rather than to a specific interaction between the SSB and the corresponding DNA polymerase. A model that correlates the thermodynamic parameters that define SSB–ssDNA nucleoprotein complex formation with the functional stimulatory effect of the SSB on 29-like DNA replication has been proposed.     

What makes the bacteriophage λ Red system useful for genetic engineering: molecular mechanism and biological function

Recent studies have generated interest in the use of the homologous recombination system of bacteriophage lambda for genetic engineering. The system, called Red, consists primarily of three proteins: lambda exonuclease, which processively digests the 5'-ended strand of a dsDNA end; beta protein, which binds to ssDNA and promotes strand annealing; and gamma protein, which binds to the bacterial RecBCD enzyme and inhibits its activities. These proteins induce a 'hyper-rec' state in Escherichia coli and other bacteria, in which recombination events between DNA species with as little as 40 bp of shared sequence occur at high frequency. Red-mediated recombination in the hyper-rec bacterium proceeds via a number of different pathways, and with the involvement of different sets of bacterial proteins, depending in part on the nature of the recombining DNA species. The role of high-frequency double-strand break repair/recombination in the life cycle of the lambdoid phages is discussed.     

Functional and structural basis for a bacteriophage homolog of human RAD52

In eukaryotes, homologous recombination proteins such as RAD51 and RAD52 play crucial roles in DNA repair and genome stability. Human RAD52 is a member of a large single-strand annealing protein (SSAP) family [1] and stimulates Rad51-dependent recombination [2, 3]. In prokaryotes and phages, it has been difficult to establish the presence of RAD52 homologs with conserved sequences. Putative SSAPs were recently found in several phages that infect strains of Lactococcus lactis[4]. One of these SSAPs was identified as Sak and was found in the virulent L. lactis phage ul36, which belongs to the Siphoviridae family [4, 5]. In this study, we show that Sak is homologous to the N terminus of human RAD52. Purified Sak binds single-stranded DNA (ssDNA) preferentially over double-stranded DNA (dsDNA) and promotes the renaturation of long complementary ssDNAs. Sak also binds RecA and stimulates homologous recombination reactions. Mutations shown to modulate RAD52 DNA binding [6] affect Sak similarly. Remarkably, electron-microscopic reconstruction of Sak reveals an undecameric (11) subunit ring, similar to the crystal structure of the N-terminal fragment of human RAD52 [7, 8]. For the first time, we propose a viral homolog of RAD52 at the amino acid, phylogenic, functional, and structural levels.     

A New Microviridae Phage Isolated from a Failed Biotechnological Process Driven by Escherichia coli

Bacteriophages are present in every environment that supports bacterial growth, including manmade ecological niches. Virulent phages may even slow or, in more severe cases, interrupt bioprocesses driven by bacteria. Escherichia coli is one of the most widely used bacteria for large-scale bioprocesses; however, literature describing phage-host interactions in this industrial context is sparse. Here, we describe phage MED1 isolated from a failed industrial process. Phage MED1 (Microviridae family, with a single-stranded DNA [ssDNA] genome) is highly similar to the archetypal phage phiX174, sharing >95% identity between their genomic sequences. Whole-genome phylogenetic analysis of 52 microvirus genomes from public databases revealed three genotypes (alpha3, G4, and phiX174). Phage MED1 belongs to the phiX174 group. We analyzed the distribution of single nucleotide variants in MED1 and 18 other phiX174-like genomes and found that there are more missense mutations in genes G, B, and E than in the other genes of these genomes. Gene G encodes the spike protein, involved in host attachment. The evolution of this protein likely results from the selective pressure on phages to rapidly adapt to the molecular diversity found at the surface of their hosts.     

Isolation and Characterization of Bacteriophages from Inland Saline Aquaculture Environments to Control Vibrio parahaemolyticus Contamination in Shrimp

Among the various bacterial pathogens associated with the aquaculture environment, Vibrio parahaemolyticus the important one from shrimp and human health aspects. Though having been around for several decades, phage-based control of bacterial pathogens (phage therapy) has recently re-emerged as an attractive alternative due to the availability of modern phage characterization tools and the global emergence of antibiotic-resistant bacteria. In the present study, a total of 12 V. parahaemolyticus specific phages were isolated from 264 water samples collected from inland saline shrimp culture farms. During the host range analysis against standard/field isolates of V. parahaemolyticus and other bacterial species, lytic activity was observed against 2.3–45.5% of tested V. parahaemolyticus isolates. No lytic activity was observed against other bacterial species. For genomic characterization, high-quality phage nucleic acid with concentrations ranging from 7.66 to 220 ng/µl was isolated from 9 phages. After digestion treatments with DNase, RNase and S1 nuclease, the nature of phage nucleic acid was determined as ssDNA and dsDNA for 7 and 2 phages respectively. During transmission electron microscopy analysis of phage V5, it was found to have a filamentous shape making it a member of the family Inoviridae. During efficacy study of phage against V. parahaemolyticus in shrimp, 78.1% reduction in bacterial counts was observed within 1 h of phage application. These results indicate the potential of phage therapy for the control of V. parahaemoyticus in shrimp.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12088-021-00934-6.