Comparative (Meta)genomic Analysis and Ecological Profiling of Human Gut-Specific Bacteriophage φB124-14

Bacteriophage associated with the human gut microbiome are likely to have an important impact on community structure and function, and provide a wealth of biotechnological opportunities. Despite this, knowledge of the ecology and composition of bacteriophage in the gut bacterial community remains poor, with few well characterized gut-associated phage genomes currently available. Here we describe the identification and in-depth (meta)genomic, proteomic, and ecological analysis of a human gut-specific bacteriophage (designated φB124-14). In doing so we illuminate a fraction of the biological dark matter extant in this ecosystem and its surrounding eco-genomic landscape, identifying a novel and uncharted bacteriophage gene-space in this community. φB124-14 infects only a subset of closely related gut-associated Bacteroides fragilis strains, and the circular genome encodes functions previously found to be rare in viral genomes and human gut viral metagenome sequences, including those which potentially confer advantages upon phage and/or host bacteria. Comparative genomic analyses revealed φB124-14 is most closely related to φB40-8, the only other publically available Bacteroides sp. phage genome, whilst comparative metagenomic analysis of both phage failed to identify any homologous sequences in 136 non-human gut metagenomic datasets searched, supporting the human gut-specific nature of this phage. Moreover, a potential geographic variation in the carriage of these and related phage was revealed by analysis of their distribution and prevalence within 151 human gut microbiomes and viromes from Europe, America and Japan. Finally, ecological profiling of φB124-14 and φB40-8, using both gene-centric alignment-driven phylogenetic analyses, as well as alignment-free gene-independent approaches was undertaken. This not only verified the human gut-specific nature of both phage, but also indicated that these phage populate a distinct and unexplored ecological landscape within the human gut microbiome

Attachment sites for bacteriophage P2 on the Escherichia coli chromosome: DNA sequences, localization on the physical map, and detection of a P2-like remnant in E. coli K-12 derivatives.

Integration of bacteriophage P2 into the Escherichia coli genome involves recombination between two attachment sites, attP and attB, one on the phage and one on the host genome, respectively. At least 10 different attB sites have been identified over the years. In E. coli C, one site, called locI, is preferred, being occupied before any of the others. In E. coli K-12, no such preference is seen (reviewed in L. E. Bertani and E. W. Six, p. 73-143, in R. Calendar, ed., The Bacteriophages, vol. 2, 1988). The DNA sequence of locI has been determined, and it shows a core sequence of 27 nucleotides identical to attP (A. Yu, L. E. Bertani, and E. Haggård-Ljungquist, Gene 80:1-12, 1989). By inverse polymerase chain reactions, the prophage-host junctions of DNA extracted from P2 lysogenic strains have been amplified, cloned, and sequenced. By combining the attL and attR sequences, the attB sequences of locations II, III, and H have been deduced. The core sequence of location II had 20 matches to the 27-nucleotide core sequence of attP; the sequences of locations III and H had 17 matches. Thus, the P2 integrase accepts at least up to 37% mismatches within the core sequence. The E. coli K-12 strains examined all contain a 639-nucleotide-long cryptic remnant of P2 at a site with a sequence similar to that of locI but that may have a different map position. The P2 remnant consists of the C-terminal part of gene D, all of gene ogr, and attR. Locations II, III, and H have been located on Kohara's physical map to positions 3670, 1570 to 1575, and 2085, respectively

Characterization of the binding sites of two proteins involved in the bacteriophage P2 site-specific recombination system.

Integration of the bacteriophage P2 genome into the Escherichia coli host chromosome occurs by site-specific recombination between the phage attP and E. coli attB sites. The phage-encoded 38-kDa protein, integrase, is known to be necessary for both phage integration as well as excision. In order to begin the molecular characterization of this recombination event, we have cloned the int gene and overproduced and partially purified the Int protein and an N-terminal truncated form of Int. Both the wild-type Int protein and the integration host factor (IHF) of E. coli were required to mediate integrative recombination in vitro between a supercoiled attP plasmid and a linear attB substrate. Footprint experiments revealed one Int-protected region on both of the attP arms, each containing direct repeats of the consensus sequence TGTGGACA. The common core sequences at attP and attB were also protected by Int from nuclease digestion, and these contained a different consensus sequence, AA T/A T/A C/A T/G CCC, arranged as inverted repeats at each core. A single IHF-protected site was located on the P (left) arm, placed between the core- and P arm-binding site for Int. Cooperative binding by Int and IHF to the attP region was demonstrated with band-shift assays and footprinting studies. Our data support the existence of two DNA-binding domains on Int, having unrelated sequence specificities. We propose that P2 Int, IHF, attP, and attB assemble in a higher-order complex, or intasome, prior to site-specific integrative recombination analogous to that formed during lambda integration

Studies of bacteriophage P2 DNA replication: localization of the cleavage site of the A protein.

Bacteriophage P2 replicates via a modified rolling circle-type of mechanism, where the P2 A protein acts as an initiator of the replication by inducing a single-stranded cut at the origin of replication (ori). The exact location of the cut induced by the A protein in vivo is determined in this report by: (i) restriction analysis; (ii) DNA sequence analysis; and (iii) primer extensions. It is located 89.2% from the left end of the P2 genome, which is within the coding part of the A gene, in a region devoid of secondary structures. The A gene has been cloned into an expression vector, and the A protein has been purified. The purified A protein does not bind to double-stranded ori containing DNA, but it cleaves single-stranded ori containing DNA, which indicates that a special DNA structure and/or protein is required to make the ori accessible for the A protein

DNA sequences of the tail fiber genes of bacteriophage P2: evidence for horizontal transfer of tail fiber genes among unrelated bacteriophages.

We have determined the DNA sequence of the bacteriophage P2 tail genes G and H, which code for polypeptides of 175 and 669 residues, respectively. Gene H probably codes for the distal part of the P2 tail fiber, since the deduced sequence of its product contains regions similar to tail fiber proteins from phages Mu, P1, lambda, K3, and T2. The similarities of the carboxy-terminal portions of the P2, Mu, ann P1 tail fiber proteins may explain the observation that these phages in general have the same host range. The P2 H gene product is similar to the products of both lambda open reading frame (ORF) 401 (stf, side tail fiber) and its downstream ORF, ORF 314. If 1 bp is inserted near the end of ORF 401, this reading frame becomes fused with ORF 314, creating an ORF that may represent the complete stf gene that encodes a 774-amino-acid-long side tail fiber protein. Thus, a frameshift mutation seems to be present in the common laboratory strain of lambda. Gene G of P2 probably codes for a protein required for assembly of the tail fibers of the virion. The entire G gene product is very similar to the products of genes U and U' of phage Mu; a region of these proteins is also found in the tail fiber assembly proteins of phages TuIa, TuIb, T4, and lambda. The similarities in the tail fiber genes of phages of different families provide evidence that illegitimate recombination occurs at previously unappreciated levels and that phages are taking advantage of the gene pool available to them to alter their host ranges under selective pressures