Summary auto-generated
This study characterizes the genome structure of bacteriophage B40-8, which infects Bacteroides fragilis, a common anaerobic bacterium found in human intestines and a frequent cause of clinical infections. B40-8 was chosen as a model phage because it represents the most common morphotype of B. fragilis phages in natural environments and can detect human-origin fecal contamination. The phage belongs to the Siphoviridae family and contains a 51.7 kb double-stranded DNA genome with low GC content (38.9 mol%). The DNA has permuted ends with 7.3% terminal redundancy rather than cohesive ends. A detailed restriction map was constructed using six endonucleases. Three major structural proteins were identified and located: MP1 and MP3 are head proteins, while MP2 is a tail protein, as confirmed by immunogold electron microscopy. A genomic library was created and a clone containing the MP2 gene was sequenced. The work provides essential molecular characterization of this phage, laying groundwork for developing B. fragilis-based genetic vectors for molecular studies and understanding viral contamination markers in wastewater.
Key findings
- Phage B40-8 has a 51.7 kb double-stranded DNA genome with permuted ends and 7.3% terminal redundancy, lacking cohesive ends characteristic of lambda-like phages
- The genome shows 38.9 mol% GC content and is resistant to digestion by many restriction endonucleases, indicating modified or unusual base composition
- Three major structural proteins identified: MP1 and MP3 are head components while MP2 is a tail component, located on the genomic map using degenerate probes and Southern blotting
- A complete restriction map was constructed for six endonucleases, identifying packaging initiation sites and major protein gene locations for molecular studies
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Abstract
Very few data are available on the molecular biology of Bacteroides fragilis bacteriophages, which have been considered in several studies as indicators of faecal contamination. Phage B40-8, initially isolated from an urban sewage sample using a strain of B. fragilis (HSP40) isolated from a clinical specimen, was chosen in this study as a prototype for morphological and molecular studies. Like most of the phages infective for B. fragilis, B40-8 belongs to the Siphoviridae family. Its genome has been found to be a double-stranded DNA molecule, of approximately 51.7 kb, containing a rather low percentage (38.9 mol%) of G+C. The ends of the molecule appeared not to be cohesive but permuted, with a terminal redundancy of 7.3%. A genomic map was constructed. Three major proteins (MP) out of 15 peptides in the SDS-PAGE profile were selected for N-terminal sequencing. From these data, degenerate probes were designed to locate the ORFs in the genomic map. Immunodetection by electron microscopy revealed that MP1 and MP3 were structural proteins of the phage head and that MP2 was a constituent of the tail. A genomic library of the phage was prepared, and a clone including the MP2 ORF was identified and sequenced.