Origin and Direction of Replication of Bacteriophage 186 DNA

Chattoraj, Dhruba K.; Inman, Ross B.

doi:10.1073/pnas.70.6.1768

Cited by 18 publications

(6 citation statements)

References 10 publications

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“…Interestingly, in a BLAST 2 alignment with the P2 A gene, the rep gene of K139 was found to be 90% identical to a small region of the A gene (30 bp), which corresponds to the mapped origin of replication in P2. The same region of K139 rep is also 94% identical to a small sequence of the A gene of phage 186 (data not shown), which is also in good accordance with the location of the 186 ori estimated by electron microscopy studies (10). The conservation of this small region suggests the same location for the ori sites within these phages and could possibly reflect a common binding specificity for the related replication proteins.…”

Section: Resultssupporting

confidence: 71%

Vibrio choleraePhage K139: Complete Genome Sequence and Comparative Genomics of Related Phages

et al. 2002

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In this report, we characterize the complete genome sequence of the temperate phage K139, which morphologically belongs to the Myoviridae phage family (P2 and 186). The prophage genome consists of 33,106 bp, and the overall GC content is 48.9%. Forty-four open reading frames were identified. Homology analysis and motif search were used to assign possible functions for the genes, revealing a close relationship to P2-like phages. By Southern blot screening of a Vibrio cholerae strain collection, two highly K139-related phage sequences were detected in non-O1, non-O139 strains. Combinatorial PCR analysis revealed almost identical genome organizations. One region of variable gene content was identified and sequenced. Additionally, the tail fiber genes were analyzed, leading to the identification of putative host-specific sequence variations. Furthermore, a K139-encoded Dam methyltransferase was characterized.At present, 183 different tailed and 10 filamentous Vibrio phages have been described. On the basis of the morphotypes, the tailed phages were grouped into seven basic forms belonging to the families of tailed phages (Myoviridae, Siphoviridae, and Podoviridae) and the filamentous phages were typed to the Inoviridae family (1). Due to the importance of the filamentous phage CTX for the virulence of Vibrio cholerae, sequencing efforts have been focused mainly on this group of phages (CTX [56], fsl [26], and fs-2 [27]). To our knowledge, K139 is the first tailed vibriophage for which information for the entire sequence is available. K139 was originally isolated from the V. cholerae serogroup O139 (48), which emerged for the first time in 1992 as the causative agent of cholera epidemics (1a). Subsequently, we found that the phage can also be recovered very frequently from various V. cholerae strains of serogroup O1 biotype El Tor. The observation that nonlysogenic O139 strains could not be infected with K139 was confirmed by the identification of the O1 antigen as the primary phage receptor (41). Analysis of the lysogeny-lysis switch genes already indicated a relationship to P2-like phages (40), which belong morphologically to the Myoviridae family. Members of this phage group (Escherichia coli phages P2 and 186 [14], Pseudomonas aeruginosa phage CTX [39], and Haemophilus influenzae phages HP1 [15] and HP2 [direct submission, GenBank accession no. NC_003315]) typically contain approximately 31-to 36-kb double-stranded DNA with single-stranded cohesive ends, but several subgroups, defined by the presence of differently derived genes, exist. For example, HP1 and HP2 contain tail genes different from those of the P2/186/CTX group, whereas CTX differs from all P2-like phages in its content of the early and delayed early genes. The evolution of such mosaic-like phage genomes may result from horizontal exchange of whole functional units (modules) (7) or of smaller units (single genes or gene fragments) acquired from a common gene pool shared by all double-stranded DNA tailed bacteriophages (24).Here we completely sequence...

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Section: Resultssupporting

confidence: 71%

Vibrio choleraePhage K139: Complete Genome Sequence and Comparative Genomics of Related Phages

et al. 2002

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“…Because the available evidence indicates that the chromosomes of OX, P2, and 186 replicate unidirectionally (2,10,25), we investigated whether the E. coli rep chromosome replicates unidirectionally as would be the case if one of the growing forks in the bidirectionally replicating chromosome required the rep function. We found that although the rep chromosome is, in fact, replicated in a bidirectional manner from an origin in approximately the same position as in rep+, there are some interesting alterations in the structure of the replicating chromosome.…”

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confidence: 99%

TherepMutation III. Altered Structure of the ReplicatingEscherichia coliChromosome

Lane

Denhardt

1974

J Bacteriol

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The rep gene function of Escherichia coli is essential for the replication of P2 and 4X174 double-stranded deoxyribonucleic acid (DNA). Compared with isogenic rep+ strains, rep mutants show the following characteristics: larger cell size, more DNA per cell, and a slightly lower DNA/mass ratio. The replicating rep chromosomes show a steeper gradient of marker frequencies and contain more replicating forks per chromosome. The nucleoid body of rep mutants sediments faster and contains more DNA. We deduce that the rep function is required for the "normal" replication of the E. coli chromosome and that in its absence the E. coli chromosome replicates in an altered manner, perhaps involving slower-moving replicating forks. Denhardt et al. (13) originally isolated rep mutants of Escherichia coli as strains unable to support growth of bacteriophage 4X174. The

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“…for 10 min and then banded in a Cs gradient (12 When the h strand of Hy dis was used, the structures were also as predicted in Fig. 2; their distribution is shown in Table 1 with the exception of the ambiguous heteroduplexes).…”

Section: Methodsmentioning

confidence: 97%

“…point (Fig. 3A) and were thought to result from branch migration of the growing point (12,14,15). The combined length of the two strands was used in the plot of branch lengths (Fig.…”

Section: Methodsmentioning

confidence: 99%

Strand-specific break near the origin of bacteriophage P2 DNA replication.

Chattoraj¹

1978

Proc. Natl. Acad. Sci. U.S.A.

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Membrane-associated P2 DNA isolated early after infection under conditions that block replication (amB in phage and rep in Escherichia coli C) was analyzed by electron microscopy. Most DNA was in the form of relaxed circles (40%) and circles with short single-stranded tails (60%). When this DNA was hybridized with separate strands of linear P2 Hy dis DNA (which provides suitable reference points along the heteroduplex molecules), an interruption was located near the previously mapped origin of P2 DNA replication in one specific strand. The same strand was sometimes extended in the direction consistent with the unidirectional mode of P2 DNA replication. Similar conclusions were reached when the intracellular DNA was anal zed after partial denaturation. These results are consistent with the rolling circle mode of DNA replication. Bacteriophage P2 provided the first unambiguous evidence for unidirectional replication from a unique origin (1) in support of the rolling circle model of DNA replication (2). A prerequisite for unidirectional replication in the model is a strand-specific break at the origin. Indeed, when P2 replication was blocked, molecules with a strand-specific break accumulated probably at the cell membrane (3). The break was in the strand proper for the direction of normal P2 replication (1). Also, P2 cis-acting gene A, which is essential for P2 DNA replication, was involved in the formation of the break (3) and in early attachment of P2 to the membrane (4). In the present study, I show that the break occurs near the origin of normal P2 replication. Under the same replication-blocked conditions, a small amount of synthesis sometimes was observed. Both the origin and the direction of the synthesis were as observed when free replication was allowed (1). Thus, these molecules appear to be early intermediates of P2 DNA replication. MATERIALS AND METHODSPhage Strains. P2 vir79 amB116 oldl 7 (referred to here as P2 vir79) and a large-plaque derivative of P2 Hyl dis virl4 (referred to as P2 Hy dis) were used. Mutation vir79 is a small deletion and imparts insensitivity to immunity (6, 7) (Fig. 1). Mutation amB116, when not suppressed, produces a block in the synthesis of the phage DNA (8). Mutation oldl7 (7) is probably irrelevant in the present context. It was expected that P2 Hy dis would show similar nonhomology regions in respect to P2 DNA as the previously studied P2 Hyl dis (large) (9) (Fig. 1), and that its h and I strands would correspond to the h and 1 strands of P2. Both these assumptions were borne out in the present experiments. High-titer stocks of both phages were kindly supplied by G. Bertani.Strand Separation. Strands of P2 Hy dis phage DNA (30,gg) were separated with 30,gg of poly(U, G) (lot 2, Miles Laboratory) as described (10) There is a short region of homology beyond the right end of Hy disC substitution which is exaggerated both in this and Fig. 2 for clarity. The consequence of such a homology is discussed in Fig. 2. Assignment of h and I strands (drawn in thick and th...

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Origin and Direction of Replication of Bacteriophage 186 DNA

Cited by 18 publications

References 10 publications

Vibrio choleraePhage K139: Complete Genome Sequence and Comparative Genomics of Related Phages

Vibrio choleraePhage K139: Complete Genome Sequence and Comparative Genomics of Related Phages

TherepMutation III. Altered Structure of the ReplicatingEscherichia coliChromosome

Strand-specific break near the origin of bacteriophage P2 DNA replication.

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