Fate, inducibility, and behavior of Latilactobacillus curvatus temperate phage TMW 1.591 P1 during sausage fermentation.

AIMS: Temperate phages insert their genome into the host's chromosome. As prophages, they remain latent in the genome until an induction event leads to lytic phage production. When this occurs in a starter culture that has been added to food fermentation, this can impair the fermentation success. This study aimed to analyze prophage inducibility in the Latilactobacillus curvatus TMW 1.591 strain during meat fermentation and investigate whether an induction signal before cryopreservation is maintained during storage and can lead to phage-induced lysis after culture activation. METHODS AND RESULTS: A prophage-free isogenic derivative of the model starter organism, L. curvatus TMW 1.591, was developed as a negative control (L. curvatus TMW 1.2406). Raw meat fermentation was performed with the wild-type (WT) and phage-cured strains. The WT strain produced high numbers of phages (5.2 ± 1.8 × 107 plaque-forming units g-1) in the meat batter. However, the prophage did not significantly affect the meat fermentation process. Induction experiments suggested an acidic environment as a potential trigger for prophage induction. Phage induction by ultraviolet light before strain cryopreservation remains functional for at least 10 weeks of storage. CONCLUSIONS: Intact prophages are active during meat fermentation. However, in this study, this has no measurable consequences for fermentation, suggesting a high resiliency of meat fermentation against phages. Inadequate handling of lysogenic starter strains, even before preservation, can lead to phage introduction into food fermentation and unintended host lysis.

Isolation and Characterization of the First Temperate Virus Infecting Psychrobacillus from Marine Sediments.

Viruses are far more abundant than cellular microorganisms in the marine ecosystem. However, very few viruses have so far been isolated from marine sediments, especially hydrothermal vent sediments, hindering the understanding of the biology and ecological functions of these tiny organisms. Here, we report the isolation and characterization of a temperate bacteriophage, named PVJ1, which infects Psychrobacillus from a hydrothermal vent field in Okinawa Trough. PVJ1 belongs to the Myoviridae family of the order Caudovirales. The tailed phage possesses a 53,187 bp linear dsDNA genome, with 84 ORFs encoding structural proteins, genome replication, host lysis, etc. in a modular pattern. The phage genome is integrated into the host chromosome near the 3'-end of deoD, a gene encoding purine nucleoside phosphorylase (PNP). The phage integration does not appear to disrupt the function of PNP. The phage DNA is packaged by the headful mechanism. Release of PVJ1 from the host cell was drastically enhanced by treatment with mitomycin C. Phages encoding an MCP sharing significant similarity (≥70% identical amino acids) with that of PVJ1 are widespread in diverse environments, including marine and freshwater sediments, soils, artificial ecosystems, and animal intestines, and primarily infect Firmicutes. These results are valuable to the understanding of the lifestyle and host interactions of bacterial viruses at the bottom of the ocean.

A novel thermophilic Aeribacillus bacteriophage AP45 isolated from the Valley of Geysers, Kamchatka: genome analysis suggests the existence of a new genus within the Siphoviridae family.

A novel thermophilic bacteriophage AP45 and its host strain Aeribacillus sp. CEMTC656 were isolated from the Valley of Geysers, Kamchatka Peninsula, Russia. Bacteriophage AP45 was identified as a member of the Siphoviridae family by electron microscopy. It showed high thermostability and had a slow cycle of reproduction. The AP45 genome had 51,606 base pairs (bp) and contained 71 open reading frames (ORFs), 40 of them encoding proteins of predicted function. Genes encoding DNA and RNA polymerases were not identified, indicating that AP45 used host polymerases. Based on the ORF65 encoding putative endolysin, the recombinant protein rAP45Lys was developed and its peptidoglycan-hydrolyzing activity was demonstrated. The AP45 genome exhibited limited identity to other phage sequences; the highest identity, 36%, was with the genome of the thermophilic Geobacillus myovirus D6E. The majority of putative proteins encoded by the AP45 genome had higher similarity to proteins from bacteria belonging to the Bacillaceae family, than to bacteriophages. In addition, more than half of the putative ORFs in the AP45 genome were highly similar to prophage sequences of A. pallidus strain 8m3, which was isolated in north-east China. The AP45 phage and revealed prophages might be members of a new genus belonging to the Siphoviridae family.

Characterization of a novel bacteriophage specific to Exiguobacterium indicum isolated from a plateau eutrophic lake.

Exiguobacterium is a versatile genus with potential in industry and agriculture. No bacteriophage that infects Exiguobacterium has been reported, despite its potential impacts on the utilization of Exiguobacterium. E. indicum EI9 was isolated from Dianchi Lake, a plateau eutrophic lake in China, which can significantly inhibit the growth of Microcystis aeruginosa. To isolate and characterize Exiguobacterium-infecting bacteriophage, a virulent bacteriophage, DCEIV-9 that specifically infects E. indicum EI9 was isolated from Dianchi lake water sample. DCEIV-9 produced tiny, round, and clear plaques with 0.5-1 mm in diameter. Electron microscopy showed that DCEIV-9 is a typical representative of the Siphoviridae, with an icosahedral head (56 nm in diameter) and a non-contractile tail (163 nm in length). Based on a one-step growth curve, latent period of 20 min and burst size of 51 PFU/infected cell were determined. DCEIV-9 was sensitive to temperature over 50 °C and prefers acid environment. DCEIV-9 was extremely sensitive to proteinase K, chloroform, ethanol, Triton X-100 but not sensitive to SDS. Restriction endonucleases analysis indicated that DCEIV-9 is a dsDNA virus. DCEIV-9 can only infect E. indicum, indicates that it has a narrow host range. DCEIV-9 is a potential new species.

Description of a cryptic thermophilic (pro)phage, CBP1 from Caldibacillus debilis strain GB1.

This study characterizes a cryptic (pro)phage-related sequence within the Caldibacillus debilis GB1 genome, designated CBP1.CBP1 is a Siphoviridae-like genome highly related to GBVS1 from Geobacillus sp. 6k51. The CBP1genome is a 37,315 bp region containing 69 putative ORFs with a GC content of 42% flanked on both sides by host DNA integrated into the main bacterial chromosome (contig 16). Bioinformatic analyses identified cassettes of genes within the CBP1 genome that were similar in function, yet distinct in sequence, from genes previously identified in GBVS1. All of CBP1 genes had less than 60% amino acid sequence identity with GBVS1by tBLASTx, with the exception of the TMP repeat gene. CBP1 possessed all the necessary genes to undergo a temperate/lytic phage life cycle, including excision, replication, structural genes, DNA packaging, and cell lyses. Proteomic analysis of CBP1 revealed the expression of 5 proteins. One of the expressed proteins was a transcriptional regulator protein homologous to the bacteriophage λ repressor protein (cI) expressed in high amounts from the CBP1 region, consistent with a lysogenic phage in a repressed state. The CBP1 protein expression profile during host growth provides unique insight into thermophilic Siphoviridae-like phages in the repressed state within their host cells.

Genomic and proteomic characterization of a thermophilic Geobacillus bacteriophage GBSV1.

Phages are present wherever life is found, and play roles in many biogeochemical and ecological processes. The thermophilic bacteriophages, however, have not been well studied. In this study, phage GBSV1 was obtained from a thermophilic bacterium Geobacillus sp. 6k51 isolated from a hot spring. GBSV1 contains a double-stranded linear DNA of 34,683bp, which encodes 54 putative open reading frames (ORFs). Thirty three of these 54 ORFs exhibit sequence similarities to genes from 7 species of Geobacillus or Bacillus bacteria, as well as of bacteriophages infecting these bacteria. Twenty-two ORFs have been functionally annotated based on both their sequence similarities to known genes and predicted Pfam protein domains. Five structural proteins of the purified GBSV1 virion have been identified by proteomic analyses. Surprisingly, 7 of the GBSV1 ORFs share sequence similarities with genes from bacteria relevant to human diseases. This is the first report that genes of human disease-inducing bacteria are found in a thermophilic phage. It is suggested that thermophilic phages may be the potential evolutionary link between thermophiles and human pathogens. The characterization of GBSV1 may possibly lead to new insights into virus-host interactions and to a better understanding of gene transfers and evolution of life on earth in general.

Deep-sea thermophilic Geobacillus bacteriophage GVE2 transcriptional profile and proteomic characterization of virions.

Thermophilic bacteria and viruses represent novel sources of genetic materials and enzymes with great potential for use in industry and biotechnology. In this study, GVE2, a virulent tailed Siphoviridae bacteriophage infecting deep-sea thermophilic Geobacillus sp. E263, was characterized. The bacteriophage contained a 40,863-bp linear double-stranded genomic deoxyribonucleic acid (DNA) with 62 presumptive open reading frames (ORFs). A viral DNA microarray was developed to monitor the viral gene transcription program. Microarray analysis indicated that 74.2% of the presumptive ORFs were expressed. The structural proteins of purified GVE2 virions were identified by mass spectrometric analysis. The purified virions contained six protein bands. Of the newly retrieved proteins, VP371 was further characterized. The immuno-electron microscopy indicated that the VP371 protein was a component of the viral capsid. Transcriptional analyses and proteomic characterization of GVE2 would be helpful to understand the complex host-virus interaction during virus infection.

Two novel bacteriophages of thermophilic bacteria isolated from deep-sea hydrothermal fields.

Bacteriophages of thermophiles are of great interest due to their important roles in many biogeochemical and ecological processes. However, no virion has been isolated from deep-sea thermophilic bacteria to date. In this investigation, two lytic bacteriophages (termed Bacillus virus W1 and Geobacillus virus E1) of thermophilic bacteria were purified from deep-sea hydrothermal fields in the Pacific for the first time. Bacillus virus W1 (BVW1) obtained from Bacillus sp. w13, had a long tail (300nm in length and 15 nm in width) and a hexagonal head (70 nm in diameter). Another virus, Geobacillus virus E1 (GVE1) from Geobacillus sp. E26323, was a typical Siphoviridae phage with a hexagonal head (130 nm in diameter) and a tail (180 nm in length and 30 nm in width). The two phages contained double-stranded genomic DNAs. The genomic DNA sizes of BVW1 and GVE1 were estimated to be about 18 and 41 kb, respectively. Based on SDS-PAGE of purified virions, six major proteins were revealed for each of the two phages. The findings in our study will be very helpful to realize the effect of virus on thermophiles as well as the communities in deep-sea hydrothermal fields.