Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Thu, 25 Oct 2018 17:30:21 Microbiology zyxwvutsrqp (1999), 145, 1661-1670 zyxwvutsrq Printed in Great Britain zy Genomic structure of phage B40-8 of Bacteroides fragilis Montserrat Puig and Rosina Girones Author for correspondence: zyxwvutsr Montserrat Puig. Tel: +34 93 4021491. Fax: +34 93 4110592. e-mail : mpuig@porthos.bio.ub.es Department of Microbiology, Faculty of Biology, University of Barcelona, Avgda Diagonal, 645-08028 Barcelona, Spain Very few data are available on the molecular biology of Bacteroides fragiks bacteriophages, which have been considered in several studies as indicators of faecal contamination. Phage 640-8, initially isolated from an urban sewage sample using a strain of B. fagilis (HSP4O) 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. fkagi/is, 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 zy (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. lmmunodetection by electron microscopy revealed that MPl 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. Keywords : zyxwvu Bacteroides zyxwvuts frugilis, phage B40-8, genomics I NTRObUCTlO N Bacteroides is one of the most abundant anaerobic bacterial genera in the gut of warm-blooded animals (107-1010 cells per g faeces) (Eggerth & Gagnon, 1933). Bacteroides fragilis is the anaerobic species most fre- quently isolated from clinical samples (Holland et al., 1977), particularly from infected human soft tissues and anaerobic bacteraemias (Holdeman et al., 1976; Polk & Kasper, 1977). Previous studies have attempted to understand the physiology and pathogenicity of B. fragilis, and some genes have been cloned and expressed in order to identify virulence factors (Russo et al., 1990; Ono et al., 1994), regulatory proteins (Rasmussen & Kovacs, 1993) or products related to the antibiotic resistance of bacteria (Rashtchian & Booth, 1981 ; Smith, 1985; Rasmussen et al., 1990). In these studies phage A-based vectors were used, and results were not always successful due to incompatibility of their ex- pression regulation systems. One possible way to deal Abbreviations: AFM, atomic force microscopy; CAP, calf alkaline phosphatase; MP, major protein; TEM, transmission electron microscopy. The CenBanWEMBUDDB1 accession number for the sequence reported in this paper is AF074719. with this may be the use of B. fragilis phage genetic elements in vector construction, which will require detailed characterization of individual phages. At pres- ent, very few data related to Bacteroides phage mol- ecular biology are available. There are more than 100 descriptions of Bacteroides phages in the literature but information on genome length, mol '/o G + C, hybridization patterns (Kory & Booth, 1986) and restriction profiles (Kai zy et al., 1985) is available for only seven phage isolates of B. fragilis. The group of phages that infect B. fragilis strain HSP40 have been proposed as a potential model for the study of viral contamination of faecal origin (Tartera & Jofre, 1987; IAWPRC Study Group on Health Related Water Microbiology, 1991 ; Havelaar, 1993). Furthermore, strain HSP40 selectively detects phages of human origin while it is resistant to infection by most of the phages present in animal faeces (Tartera & Jofre, 1987; Pig et al., 1997). Thus, the presence of B. fragilis HSP40 phages is informative of the origin of faecal contamination. The present study is focused on the molecular character- ization of phage B40-8 of zyx B. fragilis HSP40. B40-8 was selected as phage prototype for this study based on the following: (i) it is a Siphouiridae family member, like 0002-3087 0 1999 SGM 1661