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Supersize me: Cronobacter sakazakii phage GAP32
Abstract and Figures
Cronobacter sakazakii is a Gram-negative pathogen found in milk-based formulae that causes infant meningitis. Bacteriophages have been proposed to control bacterial pathogens; however, comprehensive knowledge about a phage is required to ensure its safety before clinical application. We have characterized C. sakazakii phage vB_CsaM_GAP32 (GAP32), which possesses the second largest sequenced phage genome (358,663 bp). A total of 571 genes including 545 protein coding sequences and 26 tRNAs were identified, thus more genes than in the smallest bacterium, Mycoplasma genitalium G37. BLASTP and HHpred searches, together with proteomic analyses reveal that only 23.9% of the putative proteins have defined functions. Some of the unique features of this phage include: a chromosome condensation protein, two copies of the large subunit terminase, a predicted signal-arrest-release lysin; and an RpoD-like protein, which is possibly involved in the switch from immediate early to delayed early transcription. Its closest relatives are all extremely large myoviruses, namely coliphage PBECO4 and Klebsiella phage vB_KleM-RaK2, with whom it shares approximately 44% homologous proteins. Since the homologs are not evenly distributed, we propose that these three phages belong to a new subfamily.
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... The C-terminus contains the enzymatic depolymerase domain that defines capsule serotype and LPS-O-antigen specificity. The Cterminus may also include additional chaperones or carbohydrate-binding domains [42][43][44][45][46][47]. Based on recent studies [40,41,60], the structural model of HBTSs is postulated to resemble a multilevel branch bifurcation, similar to a branched tree (Figure 2). ...
... For example, phage ϕKp24 has 14 putative depolymerases, but is classified as a four-level HBTS phage, based on the length of the Nterminal structural domains [38]. Recent research has focused on understanding the function of HBTSs in different phages, as well as their potential applications in biotechnology and medicine [42,43]. ...
... So far, 29 jumbo phages with genome sizes of more than 300 kbp have been characterized and fully sequenced. Some of them were found recently to form a new phylogenetic clade, termed "Rak2-like phages" (Abbasifar et al., 2014), while some of the other jumbo phages are distantly related to the Rak2like jumbo phages (Attai et al., 2018;Yoshikawa et al., 2018). Two of the jumbo phages, Agrobacterium virus Atuph07 (Attai et al., 2018) and Xanthomonas phage XacN1 (Yoshikawa et al., 2018), infect plant pathogenic bacteria including Agrobacterium tumefaciens, the causal agent of crown gall disease of many economically important crops, and Xanthomonas citri, a causal agent of devastating Asian citrus canker disease. ...
... The phage, however, did not infect tested Xanthomonas campestris and Pseudomonas syringae strains, indicating its specificity to the three Ralstonia species (Table 1). RsoM2USA has a lytic infection cycle, which was determined to be 360 min, with a latent period of 270 min, the longest latent period determined so far for jumbo phages (Monson et al., 2011;Abbasifar et al., 2014;Yoshikawa et al., 2018), followed by a 90-min rise period with a burst size of 32 ± 3 particles per infected cell (Figure 2A). A similar burst size of approximately 30 was found for jumbo phage XacN1, although its latent period was determined to be 90 min and growth cycle completed within 240 min (Yoshikawa et al., 2018). ...
A jumbo phage infecting Ralstonia solanacearum species complex strains, designated RsoM2USA, was isolated from soil of a tomato field in Florida, United States, and belongs to the family Myoviridae. The phage has a long latent period of 270 min and completed its infection cycle in 360 min with a burst size of approximately 32 particles per cell. With a genome size of 343,806 bp, phage RsoM2USA is the largest Ralstonia-infecting phage sequenced and reported to date. Out of the 486 ORFs annotated for RsoM2USA, only 80 could be assigned putative functions in replication, transcription, translation including 44 tRNAs, and structure with the main structural proteins experimentally confirmed. Phylogenetic analyses placed RsoM2USA in the same clade as Xanthomonas phage XacN1, prompting a proposal of a new genus for the two jumbo phages. Jumbo phage RsoM2USA is a lytic phage and has a wide host range, infecting each of the three newly established Ralstonia species: R. solanacearum, R. pseudosolanacearum, and R. syzygii, and significantly reduced the virulence of its susceptible R. solanacearum strain RUN302 in tomato plants, suggesting that this jumbo phage has the potential to be developed into an effective control against diseases caused by R. solanacearum species complex strains.
... The MCP, TerL protein and portal vertex protein are major conserved proteins in phage genomes and are thus used as phylogenetic markers to organize the phage families through single gene analysis (Smith et al., 2013;Prevelige and Cortines, 2018). Analysis of phylogenetic trees based on these three genes revealed that EJP2 is grouped with Rak2-like phage family (Šimoliūnas et al., 2013), which includes E. coli phage PBECO4 (Kim et al., 2013), E. coli phage 121Q (accession number: NC_025447), Cronobacter phage vB_CsaM_ GAP32 (Abbasifar et al., 2014), Enterobacteria phage vB_PcaM_CBB (Buttimer et al., 2017), and Salmonella phage Munch (accession number: MK268344.1). EJP2 appeared to be evolutionally closest to Salmonella phage Munch for three proteins ( Figure 3B). ...
The emergence of antimicrobial resistance (AMR) Escherichia coli has noticeably increased in recent years worldwide and causes serious public health concerns. As alternatives to antibiotics, bacteriophages are regarded as promising antimicrobial agents. In this study, we isolated and characterized a novel jumbo phage EJP2 that specifically targets AMR E. coli strains. EJP2 belonged to the Myoviridae family with an icosahedral head (120.9 ± 2.9 nm) and a non-contractile tail (111.1 ± 0.6 nm), and contained 349,185 bp double-stranded DNA genome with 540 putative ORFs, suggesting that EJP2 could be classified as jumbo phage. The functions of genes identified in EJP2 genome were mainly related to nucleotide metabolism, DNA replication, and recombination. Comparative genomic analysis revealed that EJP2 was categorized in the group of Rak2-related virus and presented low sequence similarity at the nucleotide and amino acid level compared to other E. coli jumbo phages. EJP2 had a broad host spectrum against AMR E. coli as well as pathogenic E. coli and recognized LPS as a receptor for infection. Moreover, EJP2 treatment could remove over 80% of AMR E. coli biofilms on 96-well polystyrene, and exhibit synergistic antimicrobial activity with cefotaxime against AMR E. coli . These results suggest that jumbo phage EJP2 could be used as a potential biocontrol agent to combat the AMR issue in food processing and clinical environments.
... 27.15% of 7-11 and 32.03% of phiEco32 ORFs are homologous to each other [1]. The genome of phage GAP52 [20] encodes 79 proteins homologous to 7-11 gene products, which corresponds to~52% of 7-11 ORFs and~69% of GAP52 ORFs (Supplementary Table S2). Despite the relatively high similarity of many proteins, the genomes of GAP52 and 7-11 share only 10% similarity at the nucleotide sequence level. ...
Salmonella enterica serovar Newport bacteriophage 7-11 shares 41 homologous ORFs with Escherichia coli phage phiEco32, and both phages encode a protein similar to bacterial RNA polymerase promoter specificity σ subunit. Here, we investigated the temporal pattern of 7-11 gene expression during infection and compared it to the previously determined transcription strategy of phiEco32. Using primer extension and in vitro transcription assays, we identified eight promoters recognized by host RNA polymerase holoenzyme containing 7-11 σ subunit SaPh711_gp47. These promoters are characterized by a bipartite consensus, GTAAtg-(16)-aCTA, and are located upstream of late phage genes. While dissimilar from single-element middle and late promoters of phiEco32 recognized by holoenzymes formed by the phi32_gp36 σ factor, the 7-11 late promoters are located at genome positions similar to those of phiEco32 middle and late promoters. Two early 7-11 promoters are recognized by the RNA polymerase holoenzyme containing the host primary σ70 factor. Unlike the case of phiEco32, no shut-off of σ70-dependent transcription is observed during 7-11 infection and there are no middle promoters. These differences can be explained by the fact that phage 7-11 does not encode a homologue of phi32_gp79, an inhibitor of host and early phage transcription and an activator of transcription by the phi32_gp36-holoenzyme.
... Comparison of phiEco32 and 7-11 genomes is schematically presented in Fig. 1. 27.15% of 7-11 and 32.03% of phiEco32 ORFs are homologous to each other [1]. The genome of phage GAP52 [20] encodes 79 proteins homologous to 7-11 gene products, which corresponds to ~52% of 7-11 ORFs and ~69% of GAP52 ORFs (Supplementary Table 2). Despite the relatively high similarity of many proteins, the genomes of GAP52 and 7-11 share only 10% of similarity at the level of DNA. ...
Salmonella enterica serovar Newport bacteriophage 7-11 shares 41 homologous ORFs with Escherichia coli phage phiEco32 and both phages encode a protein similar to bacterial RNA polymerase promoter specificity subunit. Here, we investigated the temporal pattern of 7-11 gene expression during the infection and compared it to the previously determined transcription strategy of phiEco32. Using primer extension and in vitro transcription assays we identified eight promoters recognized by host RNA polymerase holoenzyme containing 7-11 subunit SaPh711_gp47. These promoters are characterized by a bipartite consensus GTAAtg-(16)-aCTA and are located upstream of late phage genes. While dissimilar from single-element middle and late promoters of phiEco32 recognized by holoenzyme formed by the phi32_gp36 factor, the 7-11 late promoters are located at genome positions similar to those of phiEco32 middle and late promoters. Two early 7-11 promoters are recognized by RNA polymerase holoenzyme containing host primary σ70 factor. Unlike the case of phiEco32, no shut off of σ70-dependent transcription is observed during 7-11 infection and there are no middle promoters. These differences can be explained by the fact that phage 7-11 does not encode a homologue of phi32_gp79, an inhibitor of host and early phage transcription and an activator of transcription by the phi32_gp36-holoenzyme.
This study was conducted to propose a new strategy for preventing Pectobacterium carotovorum-mediated potato soft rot through the development of a carboxymethyl cellulose (CMC)-based antibacterial coating incorporated with green tea extract (GTE). GTE/CMC films resulted in increased water vapor permeability due to the incorporation of polar groups in GTE. In the antibacterial test against P. carotovorum, the MBC value of GTE was 2 mg/mL. The time-kill assay demonstrated that GTE/CMC (2 × MBC) completely eradicated bacteria within 0.5 h (~6.4 log CFU/mL reduction). The potential of GTE/CMC to prevent potato soft rot was evaluated by monitoring the potato appearance, maceration area, and texture properties. The GTE/CMC-coated potatoes exhibited significantly reduced maceration area and remained firm for 3 days. Moreover, there was no change in the antimicrobial efficacy for 8 weeks. The developed GTE/CMC could be used as a biological coating system for postharvest storage and soft rot prevention.
The use of antibiotics had reached a plateau due to antibiotic resistance, overuse, and residue. Bacteriophages have recently attracted considerable attention as alternative biocontrol agents. Here, we provide an up-to-date overview of phage applications in the food industry. We reviewed recently reported phages against ten typical foodborne pathogens, studies of competitive phage-encoded endolysins, and the primary outcomes of phage encapsulation in food packaging and pathogen detection. Furthermore, we identified existing barriers that still need to be addressed and proposed potential solutions to overcome these obstacles in the future.
Contamination of infant formula with Cronobacter sakazakii (C. sakazakii) can cause fatal infections in neonates. Phages have emerged as promising antibacterial agents for food safety, but their effectiveness may be limited by thermal processing. In this study, we isolated 27 C. sakazakii phages from environmental water samples and selected LPCS28 due to its broad lysis spectrum. The titer of LPCS28 will not be significantly affected by heating at a temperature of 60 °C for one hour. In both reconstituted powdered infant formula (RPIF) and liquid milk, the pre-added LPCS28, after the thermal processing at 63 °C for 30 min, significantly inhibited the post-contaminated C. sakazakii (103 CFU/mL) and eventually reduced the number of C. sakazakii to below the limit of detection (<10 CFU/mL) within 9 h at 37 °C and significantly delayed the increase of bacterial concentration in the samples at 23 °C. The phylogenetic analysis revealed that LPCS28 belonged to a new genus, we proposed as Nanhuvirus, under the family Straboviridae. These findings suggest that phage LPCS28 is a promising biological control agent for pathogenic C. sakazakii in the dairy industry.
Cronobacter species have adapted to survive harsh conditions, particularly in the food manufacture environment, and can cause life-threatening infections in susceptible hosts. These opportunistic pathogens employ a multitude of mechanisms to aid their virulence throughout three key stages: environmental persistence, infection strategy, and systemic persistence in the human host. Environmental persistence is aided by the formation of biofilms, development of subpopulations, and high tolerance to environmental stressors. Successful infection in the human host involves several mechanisms such as protein secretion, motility, quorum sensing, colonization, and translocation. Survival inside the host is achieved via competitive acquisition and utilization of minerals and metabolites respectively, coupled with host immune system evasion and antimicrobial resistance (AMR) mechanisms. Across the globe, Cronobacter sakazakii is associated with often fatal systemic infections in populations including neonates, infants, the elderly and the immunocompromised. By providing insight into the mechanisms of virulence utilized by this pathogen across these three stages, this review identifies current gaps in the literature. Further research into these virulence mechanisms is required to inform novel mitigation measures to improve global food safety with regards to this food-borne pathogen.
Despite advances in modern technologies, the food industry is continuously challenged with the threat of microbial contamination. The overuse of antibiotics has further escalated this problem, resulting in the increasing emergence of antibiotic-resistant foodborne pathogens. Efforts to develop new methods for controlling microbial contamination in food and the food processing environment are extremely important. Accordingly, bacteriophages (phages) and their derivatives have emerged as novel, viable, and safe options for the prevention, treatment, and/or eradication of these contaminants in a range of foods and food processing environments. Whole phages, modified phages, and their derivatives are discussed in terms of current uses and future potential as antimicrobials in the traditional farm-to-fork context, encompassing areas such as primary production, postharvest processing, biosanitation, and biodetection. The review also presents some safety concerns to ensure safe and effective exploitation of bacteriophages in the future. Expected final online publication date for the Annual Review of Food Science and Technology Volume 5 is February 28, 2014. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
Phage ΦM12 is an important transducing phage of the nitrogen-fixing rhizobial bacterium Sinorhizobium meliloti. Here we report the genome, phylogenetic analysis, and proteome of ΦM12, the first report of the genome and proteome of a rhizobium-infecting T4-superfamily phage. The structural genes of ΦM12 are most similar to T4-superfamily phages of cyanobacteria. ΦM12 is the first reported T4-superfamily phage to lack genes encoding class I ribonucleotide reductase (RNR) and exonuclease dexA, and to possess a class II coenzyme B12-dependent RNR. ΦM12’s novel collection of genes establishes it as the founder of a new group of T4-superfamily phages, fusing features of cyanophages and phages of enteric bacteria.
We describe a program, tRNAscan-SE, which identifies 99-100% of transfer RNA genes in DNA sequence while giving less than one false positive per 15 gigabases. Two previously described tRNA detection programs are used as fast, first-pass prefilters to identify candidate tRNAs, which are then analyzed by a highly selective tRNA covariance model. This work represents a practical application of RNA covariance models, which are general, probabilistic secondary structure profiles based on stochastic context-free grammars. tRNAscan-SE searches at approximately 30 000 bp/s. Additional extensions to tRNAscan-SE detect unusual tRNA homologues such as selenocysteine tRNAs, tRNA-derived repetitive elements and tRNA pseudogenes.
Increased awareness of climate change has shifted the focus of water distribution system (WDS) optimization research from cost minimization only to the incorporation of energy or associated greenhouse gas (GHG) minimization. In this study, a sensitivity analysis is conducted to investigate the effect of electricity tariff and generation (emission factors) on the results of multiobjective WDS optimization accounting for both total economic cost (both capital and operating costs) and GHGs. A multiobjective genetic algorithm-based optimization approach is used to conduct the analysis. The results show that electricity tariff has a significant effect on the total economic cost of WDSs and the selection of optimal solutions. In contrast, the changes of emission factors in the future have a significant effect on the total GHGs from WDSs. However, it does not alter the final solutions on the Pareto-optimal front.
The use of lytic bacteriophages for the biocontrol of food-borne pathogens in food and in the food industry is gaining increasing acceptance. In this study, the effectiveness of a bacteriophage cocktail composed of three different lytic bacteriophages (UAB_Phi 20, UAB_Phi78, and UAB_Phi87) was determined in four different food matrices (pig skin, chicken breasts, fresh eggs, and packaged lettuce) experimentally contaminated with Salmonella enterica serovar Typhimurium and S. enterica serovar Enteritidis. A significant bacterial reduction (>4 and 2log/cm(2) for S. Typhimurium and S. Enteritidis, respectively; p≤0.005) was obtained in pig skin sprayed with the bacteriophage cocktail and then incubated at 33°C for 6h. Significant decreases in the concentration of S. Typhimurium and S. Enteritidis were also measured in chicken breasts dipped for 5min in a solution containing the bacteriophage cocktail and then refrigerated at 4°C for 7days (2.2 and 0.9log10 cfu/g, respectively; p≤0.0001) as well as in lettuce similarly treated for 60min at room temperature (3.9 and 2.2log10 cfu/g, respectively; p≤0.005). However, only a minor reduction of the bacterial concentration (0.9log10 cfu/cm(2) of S. Enteritidis and S. Typhimurium; p≤0.005) was achieved in fresh eggs sprayed with the bacteriophage cocktail and then incubated at 25°C for 2h. These results show the potential effectiveness of this bacteriophage cocktail as a biocontrol agent of Salmonella in several food matrices under conditions similar to those used in their production.
Escherichia coli O157:H7 is a human pathogen. We isolated a novel bacteriophage infecting this bacterium from a sewage water treatment facility. Phage PBECO4 belongs to the family Myoviridae, having an isometric head and a contractile tail. It has a linear double-stranded DNA genome of 348,113 base pairs in length with a GC content of 34.09 %. Whole-genome sequencing revealed that PBECO4 is distantly related to enterobacteria phage vB_KleM_RaK2, with 10 % similarity, and Cronobacter phage vB_CsaM_GAP32 with 6 % similarity. Five hundred fifty-one putative open reading frames (ORFs) and six tRNA genes were found. Eight ORFs are related to genes encoding structural proteins, nine to DNA packaging, two to DNA lysis activity, and 42 to replication and regulation. Four hundred ninety ORFs have not been functionally annotated.