Characterization and the host specificity of Pet-CM3-4, a new phage infecting Cronobacter and Enterobacter strains.

Bacteria belonging to Cronobacter and Enterobacter genera are opportunistic pathogens responsible for infections in immunocompromised patients including neonates. Phage therapy offers a safe method for pathogen elimination, however, phages must be well characterized before application. In the present study we isolated four closely related bacteriophages from the subfamily Tevenvirinae infecting Cronobacter and Enterobacter strains. Bacteriophage Pet-CM3-4 which was isolated on C. malonaticus strain possessed broader host specificity than other three phages with primary Enterobacter hosts. Based on genome sequences all these phages have been assigned to the genus Karamvirus. We also studied factors influencing the host specificity of Pet-CM3-4 phage and its host range mutant Pet-CM3-1 and observed that a lysine to glutamine substitution in the long tail fiber adhesin was the reason of the Pet-CM3-1 reduced host specificity. By characterization of phage-resistant mutants from transposon library of C. malonaticus KMB-72 strain we identified that LPS is the receptor of both phages. C. malonaticus O:3 antigen is the receptor of Pet-CM3-1 phage and the Pet-CM3-4 phage binds to structures of the LPS core region. Obtained results will contribute to our understanding of biology and evolution of Tevenvirinae phages.

Characterization of Dev-CD-23823 and Dev-CT57, new Autographivirinae bacteriophages infecting Cronobacter spp.

Cronobacter spp. are opportunistic pathogenic bacteria responsible for severe infections in neonates. Powdered infant formula has been confirmed to be the source of infection in some cases. Bacteriophages offer a safe means for eliminating this pathogen. In the present study, we characterized two closely related Cronobacter-specific bacteriophages of the proposed genus "GAP227virus". The phages Dev-CD-23823 and Dev-CT57 possessed broad host specificity, as they infected 88% and 80% of the Cronobacter strains tested. Genome sequence comparisons of phages Dev-CD-23823 and Dev-CT57 showed different levels of similarity to the prototype GAP227 phage. The Dev-CT57 phage was highly similar, whereas the Dev-CD-23823 phage showed only 75% sequence identity. A phylogenic tree based on the RNA polymerase (RNAP) gene from selected representatives of the subfamily Autographivirinae confirmed the grouping of Dev-CD-23823, Dev-CT57 and GAP227 in one cluster together with phages PP2, Phi80-18 and PhiR8-01. A common conserved motif was also detected in the RNAP promoters of these phages. The functional activity of these RNAP promoters was confirmed experimentally using a promoter probe vector, and a phage-specific signal was observed; however, some cross-specificity of Dev-CD-23823 and Dev-CT57 promoters was also detected. These results will contribute to our understanding of the biology and evolution of Autographivirinae phages.

Characterization of newly identified DnaA and DnaB proteins from Acetobacter.

Although chromosomal replication is an essential feature of the bacterial life cycle, the replication mechanism and involved molecular players have never been properly characterized in the Acetobacter genera. Thanks to whole-genome sequencing, the unknown replication proteins from Acetobacter pasteurianus and Acetobacter orleanensis, DnaA-like and DnaB-like, could be identified. Despite the low nucleotide or amino acid similarity to the respective orthologs from Escherichia coli, their involvement during replication regulation was corroborated by artificial microRNA. In the Acetobacter genome, a novel replication origin, oriAo, was detected with three 9-nucleotide-long DnaA boxes to which DnaA-like proteins bind actively. Bacterial two-hybrid systems and co-immunoprecipitation confirmed the homologous and heterologous interactions between DnaA-like and DnaB-like proteins with their E. coli orthologs. This communication is due to the conserved tryptophan at position 6 for E. coli or 25 for Acetobacter that unables DnaA-like proteins to form oligomeric protein structures after its substitution. Altogether, these results provide novel insights into the genome replication mechanism in Acetobacter.

The Rep20 replication initiator from the pAG20 plasmid of Acetobacter aceti.

In the previously isolated pAG20 plasmid from the Acetobacter aceti CCM3610 strain, the Rep20 protein was characterized as a main replication initiator. The pAG20 plasmid origin was localized in the vicinity of the rep20 gene and contained two 21-nucleotide-long iteron sequences, two 13-nucleotide-long direct repeats, and a DnaA-binding site. Electrophoretic mobility shift assay and nonradioactive fragment analysis confirmed that the Rep20 protein interacted with two direct repeats (5'-TCCAAATTTGGAT'-3') and their requirement during plasmid replication was verified by mutagenesis. Although the association could not be validated of the DnaA protein of from the host cells of Escherichia coli with the plasmid-encoded replication initiator that usually occurs during replication initiation, Rep20 was able to form dimeric structures by which it could bind the sequence of the rep20 gene and autoregulate its own expression. Targeted mutagenesis of the Rep20 protein revealed the importance of the third α-helix and 6³Lys, specifically during DNA binding. The second, closely adjacent ß-sheet also took part in this process in which 5²Asn played a significant role.