Symbiosis of a P2-family phage and deep-sea Shewanella putrefaciens.

Almost all bacterial genomes harbour prophages, yet it remains unknown why prophages integrate into tRNA-related genes. Approximately 1/3 of Shewanella isolates harbour a prophage at the tmRNA (ssrA) gene. Here, we discovered a P2-family prophage integrated at the 3'-end of ssrA in the deep-sea bacterium S. putrefaciens. We found that ~0.1% of host cells are lysed to release P2 constitutively during host growth. P2 phage production is induced by a prophage-encoded Rep protein and its excision is induced by the Cox protein. We also found that P2 genome excision leads to the disruption of wobble base pairing of SsrA due to site-specific recombination, thus disrupting the trans-translation function of SsrA. We further demonstrated that P2 excision greatly hinders growth in seawater medium and inhibits biofilm formation. Complementation with a functional SsrA in the P2-excised strain completely restores the growth defects in seawater medium and partially restores biofilm formation. Additionally, we found that products of the P2 genes also increase biofilm formation. Taken together, this study illustrates a symbiotic relationship between P2 and its marine host, thus providing multiple benefits for both sides when a phage is integrated but suffers from reduced fitness when the prophage is excised.

Isolation and characterization of virulent phages infecting Shewanella baltica and Shewanella putrefaciens, and their application for biopreservation of chilled channel catfish (Ictalurus punctatus).

The growth of Shewanella spp., mainly S. baltica and S. putrefaciens, is responsible for the spoilage of chilled fresh fish. Phages are an alternative tool to control bacterial growth. In this study, virulent phages infecting 4 S. baltica and 6 S. putrefaciens strains were isolated and characterized. Transmission electron microscopy revealed that 6 out of 10 phages (3 phages infecting S. baltica and 3 phages infecting S. putrefaciens) belonged to Myoviridae, while the other 4 phages (1 phage infecting S. baltica and 3 phages infecting S. putrefaciens) belonged to Siphoviridae. Phage SppYZU01 and SppYZU05 showed the broadest host range, being lytic towards all the 4 S. baltica strains and 5 out of the 6 S. putrefaciens strains, respectively. The genome sequence of SppYZU01 had no similarity with known genome sequences, while that of SppYZU05 was 88.5% similar to the genome of a virulent S. putrefaciens-infecting phage (Spp001). According to the host range and lytic activity, 3 phages, including SppYZU01, SppYZU05, and SppYZU06, were combined into a cocktail (designated as SPMIX3-156). SPMIX3-156 showed potential as an antimicrobial agent to control S. baltica and S. putrefaciens strain growth in catfish matrices. Bacterial growth in the catfish muscle juice inoculated with 104 colony-forming units (CFU)/mL of Shewanella strains was partially inhibited by 105 plaque-forming units (PFU)/mL of SPMIX3-156 at both 25 °C and 4 °C. The catfish fillets inoculated with Shewanella strains were used as a model to evaluate the biopreservative effects of SPMIX3-156. Total viable counts of fillet samples treated with 107 PFU/mL of SPMIX3-156 were reduced by 3.21 and 2.75 log units after 1 day at 25 °C and 10 day at 4 °C, respectively, compared to those of untreated samples. Fillet quality indices, including pH, total volatile basic nitrogen, and sensory value of the SPMIX3-156-treated samples, considerably improved compared to those of the control samples at both 4 °C and 25 °C. Our results suggest that SPMIX3-156 is a promising biological agent against S. baltica and S. putrefaciens, and may have a potential use in chilled fish fillet biopreservation.

Characteristics of Two Lysis-Related Proteins from a Shewanella putrefaciens Phage with High Lytic Activity and Wide Spectrum.

Although Shewanella putrefaciens is the specific spoilage organism in most seafood, only seven Shewanella phages have been sequenced and their endolysins have not been reported until now. In this study, we cloned and expressed two lysis-related proteins (Spp64 and Spp62) encoded by phage Spp001, the first sequenced S. putrefaciens phage. Both recombinant proteins showed strong lytic capability toward chilled S. putrefaciens Sp225 and presented a wider activity spectrum compared with bacteriophage Spp001. The enzymatic activity of crude Spp64, Spp62ΔTD, and Spp62ΔTD-GST can cause decreases of 0.691, 0.674, and 0.685, respectively, as tested through the turbidity reduction assay. Furthermore, purified enzyme Spp64 at concentrations of 537.5 and 4.20 µg/mL was enough to decrease the optical density of chilled S. putrefaciens by 0.881 and 0.492, respectively, within 15 min. The recombinant Spp64 has a peptidase catalytic domain and exhibits high temperature resistance. Moreover, Spp64 displayed superior enzymatic activity in a range of pH values that matches environmental conditions (pH between 5.0 and 10.0), which demonstrates that its application in seafood is feasible. The present work is to our knowledge the first report on lysis-related enzymes encoded in the Shewanella phage. Both proteins presented extraordinary potential to control S. putrefaciens; we hope that these proteins can be developed as novel antibacterial agents in further research.

Modulating the Cascade architecture of a minimal Type I-F CRISPR-Cas system.

Shewanella putrefaciens CN-32 contains a single Type I-Fv CRISPR-Cas system which confers adaptive immunity against bacteriophage infection. Three Cas proteins (Cas6f, Cas7fv, Cas5fv) and mature CRISPR RNAs were shown to be required for the assembly of an interference complex termed Cascade. The Cas protein-CRISPR RNA interaction sites within this complex were identified via mass spectrometry. Additional Cas proteins, commonly described as large and small subunits, that are present in all other investigated Cascade structures, were not detected. We introduced this minimal Type I system in Escherichia coli and show that it provides heterologous protection against lambda phage. The absence of a large subunit suggests that the length of the crRNA might not be fixed and recombinant Cascade complexes with drastically shortened and elongated crRNAs were engineered. Size-exclusion chromatography and small-angle X-ray scattering analyses revealed that the number of Cas7fv backbone subunits is adjusted in these shortened and extended Cascade variants. Larger Cascade complexes can still confer immunity against lambda phage infection in E. coli Minimized Type I CRISPR-Cas systems expand our understanding of the evolution of Cascade assembly and diversity. Their adjustable crRNA length opens the possibility for customizing target DNA specificity.

The novel Shewanella putrefaciens-infecting bacteriophage Spp001: genome sequence and lytic enzymes.

Shewanella putrefaciens has been identified as a specific spoilage organism commonly found in chilled fresh fish, which contributes to the spoilage of fish products. Limiting S. putrefaciens growth can extend the shelf-life of chilled fish. Endolysins, which are lytic enzymes produced by bacteriophages, have been considered an alternative to control bacterial growth, and have been useful in various applications, including food preservation. We report here, for the first time, the complete genome sequence of a novel phage Spp001, which lyses S. putrefaciens Sp225. The Spp001 genome comprises a 54,789-bp DNA molecule with 67 open reading frames and an average total G + C content of 49.42 %. In silico analysis revealed that the Spp001 open reading frames encode various putative functional proteins, including an endolysin (ORF 62); however, no sequence for genes encoding the holin polypeptides, which work in concert with endolysins, was identified. To examine further the lytic activity of Spp001, we analyzed the lytic enzyme-containing fraction from phages released at the end of the phage lytic cycle in S. putrefaciens, using diffusion and turbidimetric assays. The results show that the partially purified extract contained endolysin, as indicated by a high hydrolytic activity towards bacterial peptidoglycan decrease in the OD590 value by 0.160 in 15 min. The results will allow further investigation of the purification of natural Spp001 endolysin, the extension of Spp001 host range, and the applications of the phage-encoded enzymes.

Effects of bacteriophage on the quality and shelf life of Paralichthys olivaceus during chilled storage.

BACKGROUND: The microbiological spoilage of fishery foods is mainly due to specific spoilage organisms (SSOs), with Shewanella putrefaciens being the SSO of most chilled marine fish. Bacteriophages have shown excellent capability to control micro-organisms. The aim of this study was to determine a specific bacteriophage to prevent spoilage by reducing SSO (S. putrefaciens) levels in the marine fish Paralichthys olivaceus (olive flounder) under chilled storage. RESULTS: Chilled flounder fillets were inoculated with S. putrefaciens and treated with different concentrations of bacteriophage Spp001 ranging from 10(4) to 10(8) plaque-forming units (pfu) mL(-1) . Bacterial growth (including total viable count and SSO) of the bacteriophage-treated groups was significantly inhibited compared with that of the negative control group (P < 0.05). Sensory evaluation and biochemical parameters revealed that the bacteriophage could extend the shelf life of chilled flounder fillets (from <4 to 14 days) with good esthetic quality, even at low temperature (4 °C). Furthermore, bacteriophage concentrations of 10(6) and 10(8) pfu mL(-1) were more effective than the chemical preservative potassium sorbate (5 g L(-1) ). CONCLUSION: The bacteriophage Spp001 offered effective biocontrol of S. putrefaciens under chilled conditions, retaining the quality characteristics of spiked fish fillets, and thus could be a potential candidate for use in chilled fish fillet biopreservation.