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Bacteriophages (phages) are critical players in the dynamics and function of microbial communities and drive processes as diverse as global biogeochemical cycles and human health. Phages tend to be predators finely tuned to attack specific hosts, even down to the strain level, which in turn defend themselves using an array of mechanisms. However, to date, efforts to rapidly and comprehensively identify bacterial host factors important in phage infection and resistance have yet to be fully realized. Here, we globally map the host genetic determinants involved in resistance to 14 phylogenetically diverse double-stranded DNA phages using two model Escherichia coli strains (K-12 and BL21) with known sequence divergence to demonstrate strain-specific differences. Using genome-wide loss-of-function and gain-of-function genetic technologies, we are able to confirm previously described phage receptors as well as uncover a number of previously unknown host factors that confer resistance to one or more of these phages. We uncover differences in resistance factors that strongly align with the susceptibility of K-12 and BL21 to specific phage. We also identify both phage-specific mechanisms, such as the unexpected role of cyclic-di-GMP in host sensitivity to phage N4, and more generic defenses, such as the overproduction of colanic acid capsular polysaccharide that defends against a wide array of phages. Our results indicate that host responses to phages can occur via diverse cellular mechanisms. Our systematic and high-throughput genetic workflow to characterize phage-host interaction determinants can be extended to diverse bacteria to generate datasets that allow predictive models of how phage-mediated selection will shape bacterial phenotype and evolution. The results of this study and future efforts to map the phage resistance landscape will lead to new insights into the coevolution of hosts and their phage, which can ultimately be used to design better phage therapeutic treatments and tools for precision microbiome engineering.

Bacteriophages (phages) are critical players in the dynamics and function of microbial communities and drive processes as diverse as global biogeochemical cycles and human health. Phages tend to be predators finely tuned to attack specific hosts, even down to the strain level, which in turn defend themselves using an array of mechanisms. However, to date, efforts to rapidly and comprehensively identify bacterial host factors important in phage infection and resistance have yet to be fully realized. Here, we globally map the host genetic determinants involved in resistance to 14 phylogenetically diverse double-stranded DNA phages using two model Escherichia coli strains (K-12 and BL21) with known sequence divergence to demonstrate strain-specific differences. Using genome-wide loss-of-function and gain-of-function genetic technologies, we are able to confirm previously described phage receptors as well as uncover a number of previously unknown host factors that confer resistance to one or more of these phages. We uncover differences in resistance factors that strongly align with the susceptibility of K-12 and BL21 to specific phage. We also identify both phage specific mechanisms, such as the unexpected role of cyclic-di-GMP in host sensitivity to phage N4, and more generic defenses, such as the overproduction of colanic acid capsular polysaccharide that defends against a wide array of phages. Our results indicate that host responses to phages can occur via diverse cellular mechanisms. Our systematic and high-throughput genetic workflow to characterize phage-host interaction determinants can be extended to diverse bacteria to generate datasets that allow predictive models of how phage-mediated selection will shape bacterial phenotype and evolution. The results of this study and future efforts to map the phage resistance landscape will lead to new insights into the coevolution of hosts and their phage, which can ultimately be used to design better phage therapeutic treatments and tools for precision microbiome engineering.

Here we present vB_BanS-Tsamsa, a novel temperate phage isolated from Bacillus anthracis, the agent responsible for anthrax infections in wildlife, livestock and humans. Tsamsa phage is a giant siphovirus (order Caudovirales), featuring a long, flexible and non-contractile tail of 440 nm (not including baseplate structure) and an isometric head of 82 nm in diameter. We induced Tsamsa phage in samples from two different carcass sites in Etosha National Park, Namibia. The Tsamsa phage genome is the largest sequenced Bacillus siphovirus, containing 168,876 bp and 272 ORFs. The genome features an integrase/recombinase enzyme, indicative of a temperate lifestyle. Among bacterial strains tested, the phage infected only certain members of the Bacillus cereus sensu lato group (B. anthracis, B. cereus and B. thuringiensis) and exhibited moderate specificity for B. anthracis. Tsamsa lysed seven out of 25 B. cereus strains, two out of five B. thuringiensis strains and six out of seven B. anthracis strains tested. It did not lyse B. anthracis PAK-1, an atypical strain that is also resistant to both gamma phage and cherry phage. The Tsamsa endolysin features a broader lytic spectrum than the phage host range, indicating possible use of the enzyme in Bacillus biocontrol.

Abstract Background Vibrio cholerae O139 Bengal is the only serogroup other than O1 implicated in cholera epidemics. We describe the isolation and characterization of an O139 serogroup-specific phage, vB_VchP_VchO139-I (ϕVchO139-I) that has similar host range and virion morphology as phage vB_VchP_JA1 (ϕJA1) described previously. We aimed at a complete molecular characterization of both phages and elucidation of their genetic and structural differences and assessment of their genetic relatedness to the N4-like phage group. Methods Host-range analysis and plaque morphology screening were done for both ϕJA1 and ϕVchO139-I. Both phage genomes were sequenced by a 454 and Sanger hybrid approach. Genomes were annotated and protein homologies were determined by Blast and HHPred. Restriction profiles, PFGE patterns and data on the physical genome structure were acquired and phylogenetic analyses were performed. Results The host specificity of ϕJA1 has been attributed to the unique capsular O-antigen produced by O139 strains. Plaque morphologies of the two phages were different; ϕVchO139-I produced a larger halo around the plaques than ϕJA1. Restriction profiles of ϕJA1 and ϕVchO139-I genomes were also different. The genomes of ϕJA1 and ϕVchO139-I consisted of linear double-stranded DNA of 71,252 and 70,938 base pairs. The presence of direct terminal repeats of around 1974 base pairs was demonstrated. Whole genome comparison revealed single nucleotide polymorphisms, small insertions/deletions and differences in gene content. Both genomes had 79 predicted protein encoding sequences, of which only 59 were identical between the two closely related phages. They also encoded one tRNA-Arg gene, an intein within the large terminase gene, and four homing endonuclease genes. Whole genome phylogenetic analyses of ϕJA1 and ϕVchO139-I against other sequenced N4-like phages delineate three novel subgroups or clades within this phage family. Conclusions The closely related phages feature significant genetic differences, in spite of being morphologically identical. The phage morphology, genetic organization, genomic content and large terminase protein based phylogeny support the placement of these two phages in the Podoviridae family, more specifically within the N4-like phage group. The physical genome structure of ϕJA1 could be demonstrated experimentally. Our data pave the way for potential use of ϕJA1 and ϕVchO139-I in Vibrio cholerae typing and control.

Abstract Background Spontaneous Bacillus anthracis mutants resistant to infection by phage AP50c (AP50R) exhibit a mucoid colony phenotype and secrete an extracellular matrix. Methods Here we utilized a Roche/454-based whole genome sequencing approach to identify mutations that are candidates for conferring AP50c phage resistance, followed by genetic deletion and complementation studies to validate the whole genome sequence data and demonstrate that the implicated gene is necessary for AP50c phage infection. Results Using whole genome sequence data, we mapped the relevant mutations in six AP50R strains to csaB. Eleven additional spontaneous mutants, isolated in two different genetic backgrounds, were screened by PCR followed by Sanger sequencing of the csaB gene. In each spontaneous mutant, we found either a non-synonymous substitution, a nonsense mutation, or a frame-shift mutation caused by single nucleotide polymorphisms or a 5 base pair insertion in csaB. All together, 5 and 12 of the 17 spontaneous mutations are predicted to yield altered full length and truncated CsaB proteins respectively. As expected from these results, a targeted deletion or frame-shift mutations introduced into csaB in a different genetic background, in a strain not exposed to AP50c, resulted in a phage resistant phenotype. Also, substitution of a highly conserved histidine residue with an alanine residue (H270A) in CsaB resulted in phage resistance, suggesting that a functional CsaB is necessary for phage sensitivity. Conversely, introduction of the wild type allele of csaB in cis into the csaB deletion mutant by homologous recombination or supplying the wild type CsaB protein in trans from a plasmid restored phage sensitivity. The csaB mutants accumulated cell wall material and appeared to have a defective S-layer, whereas these phenotypes were reverted in the complemented strains. Conclusions Taken together, these data suggest an essential role for csaB in AP50c phage infection, most likely in phage adsorption. (The whole genome sequences generated from this study have been submitted to GenBank under SRA project ID: SRA023659.1 and sample IDs: AP50 R1: SRS113675.1, AP50 R2: SRS113676.1, AP50 R3: SRS113728.1, AP50 R4: SRS113733.1, AP50 R6: SRS113734.1, JB220 Parent: SRS150209.1, JB220 Mutant: SRS150211.1).

Three different Bacillus bacteriophages designated TP21 are known from the literature. We have determined the sequence and structure of the TP21-L genome, and compared it to the other phages. The genome is 37.5 kb in size, possesses fixed invariable genome ends and features the typical modular organization of a temperate siphovirus. TP21-L is neither identical to TP21 isolated by Thorne (TP21-T), as shown by a PCR-based approach nor to TP21 isolated by He et al. (TP21-H), as estimated from phage dimensions. For reasons of clarity, we suggest renaming the different TP21 isolates.

The genomes of six Listeria bacteriophages were sequenced and analyzed. Phages A006, A500, B025, P35, and P40 are members of the Siphoviridae and contain double-stranded DNA genomes of between 35.6 kb and 42.7 kb. Phage B054 is a unique myovirus and features a 48.2-kb genome. Phage B025 features 3' overlapping single-stranded genome ends, whereas the other viruses contain collections of terminally redundant, circularly permuted DNA molecules. Phages P35 and P40 have a broad host range and lack lysogeny functions, correlating with their virulent lifestyle. Phages A500, A006, and B025 integrate into bacterial tRNA genes, whereas B054 targets the 3' end of translation elongation factor gene tsf. This is the first reported case of phage integration into such an evolutionarily conserved genetic element. Peptide fingerprinting of viral proteins revealed that both A118 and A500 utilize +1 and -1 programmed translational frameshifting for generating major capsid and tail shaft proteins with C termini of different lengths. In both cases, the unusual +1 frameshift at the 3' ends of the tsh coding sequences is induced by overlapping proline codons and cis-acting shifty stops. Although Listeria phage genomes feature a conserved organization, they also show extensive mosaicism within the genome building blocks. Of particular interest is B025, which harbors a collection of modules and sequences with relatedness not only to other Listeria phages but also to viruses infecting other members of the Firmicutes. In conclusion, our results yield insights into the composition and diversity of Listeria phages and provide new information on their function, genome adaptation, and evolution. doi: 10.1128/JB.01041-09

Only little information on a particular class of myoviruses, the SPO1-like bacteriophages infecting lowG+C-content, gram-positive host bacteria (Firmicutes), is available. We present the genome analysis and molecular characterization of the large, virulent, broad-host-range Listeria phage A511. A511 contains a unit (informational) genome of 134,494 bp, encompassing 190 putative open reading frames (ORFs) and 16 tRNA genes, organized in a modular fashion common among the Caudovirales. Electron microscopy, enzymatic fragmentation analyses, and sequencing revealed that the A511 DNA molecule contains linear terminal repeats of a total of 3,125 bp, encompassing nine small putative ORFs. This particular genome structure explains why A511 is unable to perform general transduction. A511 features significant sequence homologies to Listeria phage P100 and other morphologically related phages infecting Firmicutes such as Staphylococcus phage K and Lactobacillus phage LP65. Equivalent but more-extensive terminal repeats also exist in phages P100 (~6 kb) and K (~20 kb). High-resolution electron microscopy revealed, for the first time, the presence of long tail fibers organized in a sixfold symmetry in these viruses. Mass spectrometry-based peptide fingerprinting permitted assignment of individual proteins to A511 structural components. On the basis of the data available for A511 and relatives, we propose that SPO1-like myoviruses are characterized by (i) their infection of gram-positive, low-G+C-content bacteria; (ii) a wide host range within the host bacterial genus and a strictly virulent lifestyle; (iii) similar morphology, sequence relatedness, and collinearity of the phage genome organization; and (iv) large double-stranded DNA genomes featuring nonpermuted terminal repeats of various sizes.

Escherichia coli strain 397c carries a temperature-sensitive mutation, rpoC397, that removes the last 50 amino acids of the RNA polymerase [beta]' subunit and is nonpermissive for plating of bacteriophage P2. P2 gor mutants productively infect 397c and define a new gene, lysC, encoded by a reading frame that extensively overlaps the P2 lysis accessory gene, lysB. The unusual location of lysC with respect to lysB is reminiscent of the Rz/Rz1 lysis gene pair of phage [lambda]. Indeed, coexpression of lysB and lysC complemented the growth defect of [lambda] Rz/Rz1 null mutants, indicating that the LysB/C pair is similar to Rz/Rz1 in both gene arrangement and function. Cells carrying the rpoC397 mutation exhibited an early onset of P2-induced lysis, which was suppressed by the gor mutation in lysC. We propose that changes in host gene expression resulting from the rpoC397 mutation result in changes in the composition of the bacterial cell wall, making the cell more susceptible to P2-mediated lysis and preventing accumulation of progeny phage sufficient for plaque formation.

We have carried out a mutational scan of the upstream region of the bacteriophage P2 FETUD late operon promoter, [P.sub.F], which spans an element of hyphenated dyad symmetry that is conserved among all six of the P2 and P4 late promoters. All mutants were assayed for activation by P4 Delta in vivo, by using a lacZ reporter plasmid, and a subset of mutants was assayed in vitro for Delta binding. The results confirm the critical role of the three complementary nucleotides in each half site of the upstream element for transcription factor binding and for activation of transcription. A trinucleotide DNA recognition site is consistent with a model in which these transcription factors bind via a zinc finger motif. The mutational scan also led to identification of the--35 region of the promoter. Introduction of a [[sigma].sup.70]--35 consensus sequence resulted in increased constitutive expression, which could be further stimulated by Delta. These results indicate that activator binding to the upstream region of P2 late promoters compensates in part for poor [[sigma].sup.70] contacts and helps to recruit RNA polymerase holoenzyme.

The site-specific shuttle integration vectors pPL1 and pPL2 use the listeriophage integrase and attachment site within the comK gene for chromosomal insertion. Data show that the vectors attach to Listeria monocytogenes forming stable, single-copy integrants at a frequency of approximately 10(sup)-4 per donor cell.

The sid gene promoter (Psid), which regulates expression of the late genes from satellite phage P4, is activated by a novel class of small DNA-binding proteins. In contrast to helper phage late promoters, Psid is activated better by helper phage activators than by satellite phage activators. Psid was observed to have an activator-binding site centered at site II. Transcription regulated by satellite phage activators was increased by a site II mutation in which it no longer binds activators.

The core RNA polymerase (RNAP) binding activity of the purified products of nine defective alleles of the rpoH gene, that encodes sigma-32 in Escherichia coli, was examined. The mutants were grouped into three classes depending on the calculated K(sub s)s from glycerol gradient sedimentation and in vitro transcription for the mutant sigma and core RNAP activity. The behavior of the class I mutant were similar to the wild type in the core RNAP binding, while the class II mutants exhibited reduced affinity for the core RNAP.

The ATPase and helicase activities of gene product alpha of phage 4 of Escherichia coli in a series of mutants that are modified in the NTP binding motif or at the termini of the protein were examined to define the helicase domain. Mutations of residues G506 and K507 abolish both NTPase and helicase activities and are defective in unwinding a forked helicase substrate, thus suggesting that the proposed P loop is essential for helicase activity. A series of truncations constructed at the 5' and 3' ends of the alpha gene showed that 142 residues of the C terminus are sufficient for the specific binding of ori and crr DNA. The C terminus was also found to be required for helicase activity and for specific DNA binding.

[[Sigma].sup.32], the product of the rpoH gene in Escherichia coli, provides promoter specificity by interacting with core RNAP. Amino acid sequence alignment of [[Sigma].sup.32] with other sigma factors in the [[Sigma].sup.70] family has revealed regions of sequence homology. We have investigated the function of the most highly conserved region, 2.2, using purified products of various rpoH alleles. Core RNAP binding analysis by glycerol gradient sedimentation has revealed reduced core RNAP affinity for one of the mutant [[Sigma].sup.32] proteins, Q80R. This reduced core interaction is exacerbated in the presence of [[Sigma].sup.70], which competes with [[Sigma].sup.32] for binding of core RNAP. When a different but more conserved amino acid was introduced at this position by site-directed mutagenesis (Q80N), this mutant sigma factor still displayed a significant reduction in its core RNAP affinity. Based on these results, we conclude that at least one specific amino acid in region 2.2 is involved in core RNAP interaction.

Bacteriophage P4 alpha mutants resistant to Cnr protein (alphacr) were isolated to determine the mechanisms that mediate the inhibition of P4 replication by Cnr. P4 mutants exhibiting alphacr mutations in the 60-amino-acid subdomain of the C-terminal 150-amino-acid-residue domain of gpalpha exhibited normal lytic growth. However, P4 propagation in the plasmid state was impaired due to the insensitivity of the protein to the negative control of Cnr which influences DNA binding affinity.

The hypothesis of a modular phage P4 alpha protein was supported by mutational analysis, with different modules performing the processes involved in bacteriophage P4 DNA replication. The replication system involves helicase, origin recognition and primase activity in physical association within one polypeptide. Three motifs were found within the alpha protein which shared elements with proteins performing similar functions.

The bacteriophage P4 recruits helper phages such as P2 to perform to grow and perform its role. The late genes of P2 are activated by a protein called Ogr but in the absence of Ogr, P4 delta activates those genes. Purification and characterization of the bacteriophage P4 delta protein using the MalE fusion system revealed two forms of delta, a fusion of MalE and delta and an unfused form. Nitrocellulose filtration and gel mobility shift assays showed that delta binds DNA while DNA footprinting studies showed that delta binds sequences located approximately -55 in the two late promoters of P4 and the four late promoters of its helper P2.

The Old protein of bacteriophage P2 intervenes in phage lambda growth and participates in recBC mutant Escherichia coli. It is fused to the maltose binding protein and exhibits nuclease function on single-stranded DNA and RNA, and exonuclease activity on double-stranded DNA. Double-stranded DNA is digested by the Old protein in the 5' to 3' direction, starting from the 5'-hydroxyl end. Nicked circular DNA exhibits the presence of active Old nuclease and is degraded by it.

Experiments conducted on open reading frames to identify and analyze the specific lysis functions of P2 in bacteriophages reveal four genes, gene X, gene Y, lysA and lysB. Both gene X and gene Y encode necessary functions while lysA and lysB do not encode essential functions. GeneY encodes a polypeptide similar to holin proteins while gene X encodes a tail protein. The gene product of lysA and lysB influences the timing of lysis in nonpermissive strains.

There are contractile tails comprising side tail fibers and icosahedral capsids in the satellite phage P4 and temperate coliphage P2. Capsid size is determined by the P4 sid gene product. There are 240 capsid protein subunits in P4, and 420 subunits in the P2 capsid. The premature termination of the transcription inhibits the P4 replication functions.

The genes coding for the two tail fiber proteins of the P2 bacteriophage were sequenced and compared with that of other virions. Some regions of the amino acid sequence of the tail proteins deduced from the genetic code of P2 exhibited remarkable similarity to that of other phages such as Mu, P1, Lambda, K3 and T2. This indicates an illegitimate recombination of genes among unrelated virions. Further, results indicate that phages take advantage of the available gene pool to modify their host ranges under stressful conditions.

The biochemical role of Escherichia coli mutant rpoC397 gene in the synthesis of ribonucleic acid (RNA) was analyzed in Escherichia coli 397c strains. The mutation of the rpoC gene has a shorter RNA polymerase beta' subunit compared to the wild type RNA polymerase betaprime subunit. Furthermore, the premature termination of genetic transcription contributed to the formation of the shorter betaprime subunit of RNA polymerase in the mutant strains.

The Serratia marcescens extracellular nuclease gene, nucA, is positively controlled by the product of the nucC gene. The upstream region needed for NucC-dependent nuclease expression was determined by utilizing fusions to the gene encoding chloramphenicol acetyltransferase (cat). This sequence has an element of hyphenated dyad symmetry as the binding site for the P2 Ogr family of activators. Members of this family of activator proteins were observed to bind to this site.