Inactivation of Lactobacillus Virulent Bacteriophage by Thermal and Chemical Treatments

ABSTRACT The uses of thermal and chemical treatments were evaluated with respect to the inactivation of the Lactobacillus virulent bacteriophage P2. Thermal treatments consisted of heating the phage at 63, 72, and 90°C in three broth media: de Man Rogosa Sharpe broth, reconstituted skim milk, and Tris magnesium gelatin buffer. Chemical treatments evaluated were ethanol, isopropanol, sodium hypochlorite, and peracetic acid at various concentrations. Phage P2 was completely inactivated in 20 and 5 min at 72 and 90°C, respectively. Reconstituted skim milk and de Man Rogosa Sharpe broth provided optimum and minimum heat protection, respectively. Only sodium hypochlorite at 400 and 800 ppm completely inactivated the phage in 50 and 30 min, respectively. Treatment with 100% ethanol and isopropanol resulted in only a ca. 5.1-log reduction. Peracetic acid at the highest concentration used (0.45%) resulted in only a 1.40-log reduction of the phage within 60 min. These results provide additional data for establishing effective methods of controlling phage contamination in dairy and laboratory environments.

Structural insight into DNA binding and oligomerization of the multifunctional Cox protein of bacteriophage P2

The Cox protein from bacteriophage P2 is a small multifunctional DNA-binding protein. It is involved in site-specific recombination leading to P2 prophage excision and functions as a transcriptional repressor of the P2 Pc promoter. Furthermore, it transcriptionally activates the unrelated, defective prophage P4 that depends on phage P2 late gene products for lytic growth. In this article, we have investigated the structural determinants to understand how P2 Cox performs these different functions. We have solved the structure of P2 Cox to 2.4 Å resolution. Interestingly, P2 Cox crystallized in a continuous oligomeric spiral with its DNA-binding helix and wing positioned outwards. The extended C-terminal part of P2 Cox is largely responsible for the oligomerization in the structure. The spacing between the repeating DNA-binding elements along the helical P2 Cox filament is consistent with DNA binding along the filament. Functional analyses of alanine mutants in P2 Cox argue for the importance of key residues for protein function. We here present the first structure from the Cox protein family and, together with previous biochemical observations, propose that P2 Cox achieves its various functions by specific binding of DNA while wrapping the DNA around its helical oligomer.

The Comparative Analysis of Two Homologous Genomic Islands Associated with the Noncoding Sequence in Enterobacter cloacae

The genomic islands (GIs) are usually the products of horizontal gene transfer (HGT) that is evolution pattern in prokaryote. Two homologous GIs (WSU1GINoand KU01GINo) containing the homologous integrase of Bacteriophage P2 were determined inEnterobacter cloacaethrough flanking sequence alignment of the homologous integrase. The homologous GIs were integrated into the noncoding sequence. Their common flanking sequence is 5-AAGGCTCCCTCAGGAGC-3, and their integrases share 97% similarity. About two-thirds of the nucleotide sequences between WSU1GINoand KU01GINoare highly similar. The different regions between WSU1GINoand KU01GINomainly include hypothetical gene, Phage-related tail gene, capsid gene, and baseplate assembly gene. In conclusion, the tandem arrangement of WSU1GINoand KU01GINowill be artificially constructed because of their similar structural characteristics, and phage coat protein assembly will also be analyzed.

Bacteriophage-Encoding Cytolethal Distending Toxin Type V Gene Induced from Nonclinical Escherichia coli Isolates

ABSTRACTCytolethal distending toxin (Cdt) is produced by a variety of pathogenic bacteria, including pathogenic serotypes of Shiga toxin-producingEscherichia coli(STEC). The Cdt family comprises five variants (Cdt-I to Cdt-V) encoded by three genes located within the chromosome or plasmids or, in the case of Cdt-I, within bacteriophages. In this study, we evaluated the occurrence of thecdtgene in a collection of 140 environmental STEC isolates.cdtwas detected in 12.1% of strains, of which five strains carried inducible bacteriophages containing the Cdt-V variant. Two Cdt-V phages of theSiphoviridaemorphology lysogenizedShigella sonnei, generating two lysogens: a single Cdt phage lysogen and a double lysogen, containing a Cdt phage and an Stx phage, both from the wild-type strain. The rates of induction of Cdt phages were evaluated by quantitative PCR, and spontaneous induction of Cdt-V phage was observed, whereas induction of Stx phage in the double lysogen was mitomycin C dependent. The Cdt distending effect was observed in HeLa cells inoculated with the supernatant of the Cdt-V phage lysogen. A ClaI fragment containing thecdt-Vgene of one phage was cloned, and sequencing confirmed the presence of Cdt-V, as well as a fragment downstream from thecdthomolog togpA, encoding a replication protein of bacteriophage P2. Evaluation of Cdt-V phages in nonclinical water samples showed densities of 102to 109gene copies in 100 ml, suggesting the high prevalence of Cdt phages in nonclinical environments.