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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 tr

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 tr

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 tr

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 tr

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.

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.

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 li

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 li

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.

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.

Tyrosine phosphatase (PTP)-like proteins exist in many bacteria and are segregated into two major groups: low molecular weight and conventional. The latter group also has activity as phosphoinositide phosphatases. These two kinds of PTP are suggested to be involved in many aspects of bacterial physiology including stress response, DNA binding proteins, virulence, and capsule/cell wall production. By annotation, Listeria monocytogenes possesses two potential low molecular weight and two conventional PTPs. Using L. monocytogenes wild-type (WT) strain 10403S, we have created an in-frame deletion mutant lacking all four PTPs, as well as four additional complemented strains harboring each of the PTPs. No major physiological differences were observed between the WT and the mutant lacking all four PTPs. However, the deletion mutant strain was resistant to Listeria phages A511 and P35 and sensitive to other Listeria phages. This was attributed to reduced attachment to the cell wall. The mutant lacking all PTPs was found to lack N-acetylglucosamine in its wall teichoic acid. Phage sensitivity and attachment was rescued in a complemented strain harboring a low molecular weight PTP (LMRG1707)