Phage Repressor Research Articles

Stability and gene strand bias of lambda prophages and chromosome organization in Escherichia coli.

Temperate phage-mediated horizontal gene transfer is a potent driver of genetic diversity in the evolution of bacteria. Most lambdoid prophages in Escherichia coli are integrated into the chromosome with the same orientation with respect to the direction of chromosomal replication, and their location on the chromosome is far from homogeneous. To better understand these features, we studied the interplay between lysogenic and lytic states of phage lambda in both native and inverted integration orientations at the wild-type integration site as well as at other sites on the bacterial chromosome. Measurements of free phage released by spontaneous induction showed that the stability of lysogenic states is affected by location and orientation along the chromosome, with stronger effects near the origin of replication. Competition experiments and range expansions between lysogenic strains with opposite orientations and insertion loci indicated that there are no major differences in growth. Moreover, measurements of the level of transcriptional bursts of the cI gene coding for the lambda phage repressor using single-molecule fluorescence in situ hybridization resulted in similar levels of transcription for both orientations and prophage location. We postulate that the preference for a given orientation and location is a result of a balance between the maintenance of lysogeny and the ability to lyse.IMPORTANCEThe integration of genetic material of temperate bacterial viruses (phages) into the chromosomes of bacteria is a potent evolutionary force, allowing bacteria to acquire in one stroke new traits and restructure the information in their chromosomes. Puzzlingly, this genetic material is preferentially integrated in a particular orientation and at non-random sites on the bacterial chromosome. The work described here reveals that the interplay between the maintenance of the stability of the integrated phage, its ability to excise, and its localization along the chromosome plays a key role in setting chromosomal organization in Escherichia coli.

Insight into the Mechanism of Lysogeny Control of phiCDKH01 Bacteriophage Infecting Clinical Isolate of Clostridioides difficile.

Clostridioides difficile is a causative agent of antibiotic-associated diarrhea as well as pseudomembranous colitis. So far, all known bacteriophages infecting these bacteria are temperate, which means that instead of prompt lysis of host cells, they can integrate into the host genome or replicate episomally. While C. difficile phages are capable of spontaneous induction and entering the lytic pathway, very little is known about the regulation of their maintenance in the state of lysogeny. In this study, we investigated the properties of a putative major repressor of the recently characterized C. difficile phiCDKH01 bacteriophage. A candidate protein belongs to the XRE family and controls the transcription of genes encoding putative phage antirepressors, known to be involved in the regulation of lytic development. Hence, the putative major phage repressor is likely to be responsible for maintenance of the lysogeny.

CI:Mor interactions in the lysogeny switches of Lactococcus lactis TP901-1 and Staphylococcus aureus φ13 bacteriophages.

Aim: To structurally characterize in detail the interactions between the phage repressor (CI) and the antirepressor (Mor) in the lysis-lysogeny switches of two Gram-positive bacteriophages, the lactococcal TP901-1 and staphylococcal φ13. Methods: We use crystallographic structure determination, computational structural modeling, and analysis, as well as biochemical methods, to elucidate similarities and differences in the CI:Mor interactions for the two genetic switches. Results: By comparing a newly determined and other available crystal structures for the N-terminal domain of CI (CI-NTD), we show that the CI interface involved in Mor binding undergoes structural changes upon binding in TP901-1. Most importantly, we show experimentally for the first time the direct interaction between CI and Mor for φ13, and model computationally the interaction interface. The computational modeling supports similar side chain rearrangements in TP901-1 and φ13. Conclusion: This study ascertains experimentally that, like in the TP901-1 lysogeny switch, staphylococcal φ13 CI and Mor interact with each other. The structural basis of the interaction of φ13 CI and Mor was computationally modeled and is similar to the interaction demonstrated experimentally between TP901-1 CI-NTD and Mor, likely involving similar rearrangement of residue side chains during the formation of the complex. The study identifies one CI residue, Glu69, which unusually interacts primarily through its aliphatic chain with an aromatic residue on Mor after changing its conformation compared to the un-complexed structure. This and other residues at the interface are suggested for investigation in future studies.

Arbitrium communication controls phage lysogeny through non-lethal modulation of a host toxin-antitoxin defence system.

Temperate Bacillus phages often utilize arbitrium communication to control lysis/lysogeny decisions, but the mechanisms by which this control is exerted remains largely unknown. Here we find that the arbitrium system of Bacillus subtilis phage ϕ3T modulates the host-encoded MazEF toxin-antitoxin system to this aim. Upon infection, the MazF ribonuclease is activated by three phage genes. At low arbitrium signal concentrations, MazF is inactivated by two phage-encoded MazE homologues: the arbitrium-controlled AimX and the later-expressed YosL proteins. At high signal, MazF remains active, promoting lysogeny without harming the bacterial host. MazF cleavage sites are enriched on transcripts of phage lytic genes but absent from the phage repressor in ϕ3T and other Spβ-like phages. Combined with low activation levels of MazF during infections, this pattern explains the phage-specific effect. Our results show how a bacterial toxin-antitoxin system has been co-opted by a phage to control lysis/lysogeny decisions without compromising host viability.

CRISPRpi: Inducing and Curing Prophage Using the CRISPR Interference.

We present here a CRISPR-interference-based protocol to trigger prophage induction, even for non-inducible prophages. This method can also be used to cure the prophage from the bacterial host. The method is based on silencing of the phage's repressor transcription, thanks to CRISPR interference. Plasmid electroporation is used to bring the CRISPRi system into the bacteria, specifically on a plasmid carrying spacers targeting the prophage repressor. This method enables prophage induction and curation in a week or two with a high efficiency.

Differences in the Shiga Toxin (Stx) 2a Phage Regulatory Switch Region Influence Stx2 Localization and Virulence of Stx-Producing Escherichia coli in Mice.

Shiga toxin (Stx)-producing Escherichia coli (STEC) is a major cause of foodborne illness globally, and infection with serotype O157:H7 is associated with increased risk of hospitalization and death in the U.S. The Stxs are encoded on a temperate bacteriophage (stx-phage), and phage induction leads to Stx expression; subtype Stx2a in particular is associated with more severe disease. Our earlier studies showed significant levels of RecA-independent Stx2 production by STEC O157:H7 strain JH2010 (stx2astx2c), even though activated RecA is the canonical trigger for stx-phage induction. This study aimed to further compare and contrast RecA-independent toxin production in Stx2-producing clinical isolates. Deletion of recA in JH2010 resulted in higher in vitro supernatant cytotoxicity compared to that from JH2016ΔrecA, and the addition of the chelator ethylenediaminetetraacetic acid (EDTA) and various metal cations to the growth medium exacerbated the difference in cytotoxicity exhibited by the two deletion strains. Both the wild-type and ΔrecA deletion strains exhibited differential cytotoxicity in the feces of infected, streptomycin (Str)-treated mice. Comparison of the stx2a-phage predicted protein sequences from JH2010 and JH2016 revealed low amino acid identity of key phage regulatory proteins that are involved in RecA-mediated stx-phage induction. Additionally, other STEC isolates containing JH2010-like and JH2016-like stx2a-phage sequences led to similar Stx2 localization, as demonstrated by JH2010ΔrecA and JH2016ΔrecA, respectively. Deletion of the stx2a-phage regulatory region in the wild-type strains prevented the differential localization of Stx2 into the culture supernatant, a finding that suggests that the stx2a-phage regulatory region is involved in the differential ΔrecA phenotypes exhibited by the two strains. We hypothesize that the amino acid differences between the JH2010 and JH2016 phage repressor proteins (CIs) lead to structural differences that are responsible for differential interaction with RecA. Overall, we discovered that non-homologous stx2a-phage regulatory proteins differentially influence RecA-independent, and possibly RecA-dependent, Stx2 production. These findings emphasize the importance of studying non-homologous regulatory elements among stx2-phages and their influence on Stx2 production and virulence of STEC isolates.

Mutation-induced infections of phage-plasmids

Phage-plasmids are extra-chromosomal elements that act both as plasmids and as phages, whose eco-evolutionary dynamics remain poorly constrained. Here, we show that segregational drift and loss-of-function mutations play key roles in the infection dynamics of a cosmopolitan phage-plasmid, allowing it to create continuous productive infections in a population of marine Roseobacter. Recurrent loss-of-function mutations in the phage repressor that controls prophage induction leads to constitutively lytic phage-plasmids that spread rapidly throughout the population. The entire phage-plasmid genome is packaged into virions, which were horizontally transferred by re-infecting lysogenized cells, leading to an increase in phage-plasmid copy number and to heterozygosity in a phage repressor locus in re-infected cells. However, the uneven distribution of phage-plasmids after cell division (i.e., segregational drift) leads to the production of offspring carrying only the constitutively lytic phage-plasmid, thus restarting the lysis-reinfection-segregation life cycle. Mathematical models and experiments show that these dynamics lead to a continuous productive infection of the bacterial population, in which lytic and lysogenic phage-plasmids coexist. Furthermore, analyses of marine bacterial genome sequences indicate that the plasmid backbone here can carry different phages and disseminates trans-continentally. Our study highlights how the interplay between phage infection and plasmid genetics provides a unique eco-evolutionary strategy for phage-plasmids.

A CRISPRi-based investigation reveals that multiple promoter elements drive gene expression from the genome of mycobacteriophage D29.

A unique feature found in the genomes of mycobacteriophages such as L5 belonging to the A cluster is the presence of multiple dispersed repeated elements known as stoperators. The phage repressor binds these repeat elements, shutting off transcription globally and thereby promoting lysogeny. Interestingly, the sequence of these stoperators closely matches that of the consensus -35 region of prokaryotic promoters, leading us to propose that they may have a role to play in the initiation of transcription by serving as RNA polymerase binding sites. Mycobacteriophage D29 is closely related to phage L5, and their genome organizations are very similar. As in L5, there are multiple stoperators in the genome of D29. The positions occupied by the stoperators in the two genomes are almost identical. The significant difference between the two phages is that D29 lacks the gene encoding the equivalent of the L5 repressor. Since phage D29 does not produce a repressor, we considered it to be a suitable model for testing our hypothesis that the stoperators function as promoters in the absence of the repressor. To prove our point, we targeted CRISPR guide RNAs against six stoperators. In the case of five out of the six, we found a significant reduction in downstream gene expression and phage growth. Based on this observation and primer extension assays, we conclude that promoting gene expression is likely to be the primary function of stoperators.

Non-canonical Staphylococcus aureus pathogenicity island repression.

Mobile genetic elements control their life cycles by the expression of a master repressor, whose function must be disabled to allow the spread of these elements in nature. Here, we describe an unprecedented repression-derepression mechanism involved in the transfer of Staphylococcus aureus pathogenicity islands(SaPIs). Contrary to the classical phage and SaPI repressors, which are dimers, the SaPI1 repressor StlSaPI1 presents a unique tetrameric conformation never seen before. Importantly, not just one but two tetramers are required for SaPI1 repression, which increases the novelty of the system. To derepress SaPI1, the phage-encoded protein Sri binds to and induces a conformational change in the DNA binding domains of StlSaPI1, preventing the binding of the repressor to its cognate StlSaPI1 sites. Finally, our findings demonstrate that this system is not exclusive to SaPI1 but widespread in nature. Overall, our results characterize a novel repression-induction system involved in the transfer of MGE-encoded virulence factors in nature.

RNA polymerase efficiently transcribes through DNA-scaffolded, cooperative bacteriophage repressor complexes.

DNA can act as a scaffold for the cooperative binding of protein oligomers. For example, the phage 186 CI repressor forms a wheel of seven dimers wrapped in DNA with specific binding sites, while phage λ CI repressor dimers bind to two well-separated sets of operators, forming a DNA loop. Atomic force microscopy was used to measure transcription elongation by Escherichia coli RNA polymerase (RNAP) through these protein complexes. 186 CI, or λ CI, bound along unlooped DNA negligibly interfered with transcription by RNAP. Wrapped and looped topologies induced by these scaffolded, cooperatively bound repressor oligomers did not form significantly better roadblocks to transcription. Thus, despite binding with high affinity, these repressors are not effective roadblocks to transcription.

Tunable Temperature-Sensitive Transcriptional Activation Based on Lambda Repressor.

Temperature is a versatile input signal for the control of engineered cellular functions. Sharp induction of gene expression with heat has been established using bacteria- and phage-derived temperature-sensitive transcriptional repressors with tunable switching temperatures. However, few temperature-sensitive transcriptional activators have been reported that enable direct gene induction with cooling. Such activators would expand the application space for temperature control. In this technical note, we show that temperature-dependent versions of the Lambda phage repressor CI can serve as tunable cold-actuated transactivators. Natively, CI serves as both a repressor and activator of transcription. Previously, thermolabile mutants of CI, known as the TcI family, were used to repress the cognate promoters PR and PL. We hypothesized that TcI mutants can also serve as temperature-sensitive activators of transcription at CI’s natural PRM promoter, creating cold-inducible operons with a tunable response to temperature. Indeed, we demonstrate temperature-responsive activation by two variants of TcI with set points at 35.5 and 38.5 °C in E. coli. In addition, we show that TcI can serve as both an activator and a repressor of different genes in the same genetic circuit, leading to opposite thermal responses. Transcriptional activation by TcI expands the toolbox for control of cellular function using globally or locally applied thermal inputs.

Mycobacteriophage D29 induced association of Mycobacterial RNA polymerase with ancillary factors leads to increased transcriptional activity.

Mycobacteriophage D29 infects species belonging to the genus Mycobacterium including the deadly pathogen Mycobacterium tuberculosis. D29 is a lytic phage, although, related to the lysogenic mycobacteriophage L5. This phage is unable to lysogenize in mycobacteria as it lacks the gene encoding the phage repressor. Infection by many mycobacteriophages cause various changes in the host that ultimately leads to inactivation of the latter. One of the host targets often modified in the process is RNA polymerase. During our investigations with phage D29 infected Mycobacterium smegmatis (Msm) we observed that the promoters from both phage, and to a lesser extent those of the host were found to be more active in cells that were exposed to D29, as compared to the unexposed. Further experiments indicate that the RNA polymerase purified from phage infected cells possessed higher affinity for promoters particularly those that were phage derived. Comparison of the purified RNA polymerase preparations from infected and uninfected cells showed that several ancillary transcription factors, Sigma factor F, Sigma factor H, CarD and RbpA are prominently associated with the RNA polymerase from infected cells. Based on our observations we conclude that the higher activity of RNA polymerase observed in D29 infected cells is due to its increased association with ancillary transcription factors.

Flexible linker modulates the binding affinity of the TP901-1 CI phage repressor to DNA.

Temperate bacteriophages can switch between two life cycles following infection of a host bacterium: the lytic or lysogenic life cycle. The choice between these is controlled by a bistable genetic switch. We investigated the genetic switch of the lactococcal temperate bacteriophage, TP901-1, which is controlled by two regulatory proteins, the Clear 1 (CI) repressor and modulator of repression (MOR) antirepressor. CI consists of a DNA-binding N-terminal domain and a C-terminal domain responsible for oligomerization, connected by a flexible interdomain linker. Full-length CI is hexameric, whereas the truncated version CI with 58 C-terminal residues truncated (CIΔ58), missing the second C-terminal subdomain, is dimeric, but binds with the same affinity as full-length CI to the OL operator site, responsible for lytic genes transcription repression. Three variants of CIΔ58 with shorter, longer, and PP substituted linkers were produced and confirmed by circular dichroism spectroscopy and nanodifferential scanning fluorimetry to be well folded. With small-angle X-ray scattering, we delineated the conformational space sampled by the variants and wild-type in solution and found that shortening and lengthening the linker decrease and increase this, respectively, as also substantiated by molecular dynamics and as intended. Isoelectric focusing electrophoresis confirmed that all variants are able to bind to the MOR antirepressor. However, using electrophoretic mobility shift assays, we showed that shortening and lengthening the linker lead to a 94 and 17 times decrease in affinity to OL , respectively. Thus, an appropriate linker length appears to be crucial for appropriate DNA-binding and subsequent TP901-1 genetic switch function.

Non-canonical LexA proteins regulate the SOS response in the Bacteroidetes.

Lesions to DNA compromise chromosome integrity, posing a direct threat to cell survival. The bacterial SOS response is a widespread transcriptional regulatory mechanism to address DNA damage. This response is coordinated by the LexA transcriptional repressor, which controls genes involved in DNA repair, mutagenesis and cell-cycle control. To date, the SOS response has been characterized in most major bacterial groups, with the notable exception of the Bacteroidetes. No LexA homologs had been identified in this large, diverse and ecologically important phylum, suggesting that it lacked an inducible mechanism to address DNA damage. Here, we report the identification of a novel family of transcriptional repressors in the Bacteroidetes that orchestrate a canonical response to DNA damage in this phylum. These proteins belong to the S24 peptidase family, but are structurally different from LexA. Their N-terminal domain is most closely related to CI-type bacteriophage repressors, suggesting that they may have originated from phage lytic phase repressors. Given their role as SOS regulators, however, we propose to designate them as non-canonical LexA proteins. The identification of a new class of repressors orchestrating the SOS response illuminates long-standing questions regarding the origin and plasticity of this transcriptional network.

The arbitrium system controls prophage induction

SummarySome Bacillus-infecting bacteriophages use a peptide-based communication system, termed arbitrium, to coordinate the lysis-lysogeny decision. In this system, the phage produces AimP peptide during the lytic cycle. Once internalized by the host cell, AimP binds to the transcription factor AimR, reducing aimX expression and promoting lysogeny. Although these systems are present in a variety of mobile genetic elements, their role in the phage life cycle has only been characterized in phage phi3T during phage infection. Here, using the B. subtilis SPβ prophage, we show that the arbitrium system is also required for normal prophage induction. Deletion of the aimP gene increased phage reproduction, although the aimR deletion significantly reduced the number of phage particles produced after prophage induction. Moreover, our results indicated that AimR is involved in a complex network of regulation and brought forward two new players in the SPβ lysis-lysogeny decision system, YopN and the phage repressor YopR. Importantly, these proteins are encoded in an operon, the function of which is conserved across all SPβ-like phages encoding the arbitrium system. Finally, we obtained mutant phages in the arbitrium system, which behaved almost identically to the wild-type (WT) phage, indicating that the arbitrium system is not essential in the laboratory but is likely beneficial for phage fitness in nature. In support of this, by possessing a functional arbitrium system, the SPβ phage can optimize production of infective particles while also preserving the number of cells that survive after prophage induction, a strategy that increases phage persistence in nature.

The Repressor C Protein, Pf4r, Controls Superinfection of Pseudomonas aeruginosa PAO1 by the Pf4 Filamentous Phage and Regulates Host Gene Expression.

It has been shown that the filamentous phage, Pf4, plays an important role in biofilm development, stress tolerance, genetic variant formation and virulence in Pseudomonas aeruginosa PAO1. These behaviours are linked to the appearance of superinfective phage variants. Here, we have investigated the molecular mechanism of superinfection as well as how the Pf4 phage can control host gene expression to modulate host behaviours. Pf4 exists as a prophage in PAO1 and encodes a homologue of the P2 phage repressor C and was recently named Pf4r. Through a combination of molecular techniques, ChIPseq and transcriptomic analyses, we show a critical site in repressor C (Pf4r) where a mutation in the site, 788799A>G (Ser4Pro), causes Pf4r to lose its function as the immunity factor against reinfection by Pf4. X-ray crystal structure analysis shows that Pf4r forms symmetric homo-dimers homologous to the E.coli bacteriophage P2 RepC protein. A mutation, Pf4r*, associated with the superinfective Pf4r variant, found at the dimer interface, suggests dimer formation may be disrupted, which derepresses phage replication. This is supported by multi-angle light scattering (MALS) analysis, where the Pf4r* protein only forms monomers. The loss of dimerisation also explains the loss of Pf4r’s immunity function. Phenotypic assays showed that Pf4r increased LasB activity and was also associated with a slight increase in the percentage of morphotypic variants. ChIPseq and transcriptomic analyses suggest that Pf4r also likely functions as a transcriptional regulator for other host genes. Collectively, these data suggest the mechanism by which filamentous phages play such an important role in P. aeruginosa biofilm development.

Proteomic Characterization of Bacteriophage Peptides from the Mastitis Producer Staphylococcus aureus by LC-ESI-MS/MS and the Bacteriophage Phylogenomic Analysis.

The present work describes LC-ESI-MS/MS MS (liquid chromatography-electrospray ionization-tandem mass spectrometry) analyses of tryptic digestion peptides from phages that infect mastitis-causing Staphylococcus aureus isolated from dairy products. A total of 1933 nonredundant peptides belonging to 1282 proteins were identified and analyzed. Among them, 79 staphylococcal peptides from phages were confirmed. These peptides belong to proteins such as phage repressors, structural phage proteins, uncharacterized phage proteins and complement inhibitors. Moreover, eighteen of the phage origin peptides found were specific to S. aureus strains. These diagnostic peptides could be useful for the identification and characterization of S. aureus strains that cause mastitis. Furthermore, a study of bacteriophage phylogeny and the relationship among the identified phage peptides and the bacteria they infect was also performed. The results show the specific peptides that are present in closely related phages and the existing links between bacteriophage phylogeny and the respective Staphylococcus spp. infected.

Characterization of Bacteriophage Peptides of Pathogenic Streptococcus by LC-ESI-MS/MS: Bacteriophage Phylogenomics and Their Relationship to Their Host.

The present work focuses on LC-ESI-MS/MS (liquid chromatography-electrospray ionization-tandem mass spectrometry) analysis of phage-origin tryptic digestion peptides from mastitis-causing Streptococcus spp. isolated from milk. A total of 2,546 non-redundant peptides belonging to 1,890 proteins were identified and analyzed. Among them, 65 phage-origin peptides were determined as specific Streptococcus spp. peptides. These peptides belong to proteins such as phage repressors, phage endopeptidases, structural phage proteins, and uncharacterized phage proteins. Studies involving bacteriophage phylogeny and the relationship between phages encoding the peptides determined and the bacteria they infect were also performed. The results show how specific peptides are present in closely related phages, and a link exists between bacteriophage phylogeny and the Streptococcus spp. they infect. Moreover, the phage peptide M∗ATNLGQAYVQIM∗PSAK is unique and specific for Streptococcus agalactiae. These results revealed that diagnostic peptides, among others, could be useful for the identification and characterization of mastitis-causing Streptococcus spp., particularly peptides that belong to specific functional proteins, such as phage-origin proteins, because of their specificity to bacterial hosts.

Prophage encoding toxin/antitoxin system PfiT/PfiA inhibits Pf4 production in Pseudomonas aeruginosa.

SummaryPf prophages are ssDNA filamentous prophages that are prevalent among various Pseudomonas aeruginosa strains. The genomes of Pf prophages contain not only core genes encoding functions involved in phage replication, structure and assembly but also accessory genes. By studying the accessory genes in the Pf4 prophage in P. aeruginosa PAO1, we provided experimental evidence to demonstrate that PA0729 and the upstream ORF Rorf0727 near the right attachment site of Pf4 form a type II toxin/antitoxin (TA) pair. Importantly, we found that the deletion of the toxin gene PA0729 greatly increased Pf4 phage production. We thus suggest the toxin PA0729 be named PfiT for Pf4 inhibition toxin and Rorf0727 be named PfiA for PfiT antitoxin. The PfiT toxin directly binds to PfiA and functions as a corepressor of PfiA for the TA operon. The PfiAT complex exhibited autoregulation by binding to a palindrome (5′‐AATTCN5 GTTAA‐3′) overlapping the ‐35 region of the TA operon. The deletion of pfiT disrupted TA autoregulation and activated pfiA expression. Additionally, the deletion of pfiT also activated the expression of the replication initiation factor gene PA0727. Moreover, the Pf4 phage released from the pfiT deletion mutant overcame the immunity provided by the phage repressor Pf4r. Therefore, this study reveals that the TA systems in Pf prophages can regulate phage production and phage immunity, providing new insights into the function of TAs in mobile genetic elements.

Analysis and the identification of DNA binding sites for the cI repressor of Bacteriophage ɛ34

Gene 46 of ɛ34 encodes a protein that is similar to other phage repressors. It has a C‐terminal protease domain which is about 80% identical to the repressors of phage λ, but its DNA binding N‐terminal portion is only about 18% identical to its closest known relatives in phages λ, and Lex A. The N‐terminal DNA‐binding domain is homologous to the HTH_XRE family of DNA‐binding domains. In another study we have generated homology models of this proteins and its interaction with DNA [1].The main goal of this project was to initiate a structure‐function study on the cI repressor protein of phage ɛ34. The cI repressor of ɛ34 protein with a 6X polyhistidine tag was first isolated and purified to homogeneity. We have identified regions for cI binding by using bioinformatics and gel‐mobility shift assay using PCR fragments encompassing the intergenic region of the cI and cro gene. Our binding sites compare well with the known intergenic regions of other phages such as λ, Φ11, 434. This project was done as a collaborative venture between two laboratories, Huntingdon College (Dr. Doba Jackson) and Alabama State University (laboratory of Dr. Robert Villafane). This project is in line with Dr. Robert Villafane's research into the Genomic analysis of bacteriophage ɛ34 of Salmonella enteric serovar Anatum.Support or Funding InformationSupport Support for this work was provided by Alabama State University and Huntingdon College.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.