Ends of bacteriophage Mu DNA

Bukhari, Ahmad I.; Froshauer, S; Botchan, Michael R.

doi:10.1038/264580a0

Cited by 63 publications

(18 citation statements)

References 17 publications

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“…For phage Mu, this results in ~100 bp of host DNA at one end of the phage genome and ~2 kb at the other end 98 , whereas for a Mu-like phage in Rhodobacter capsulatus, RcapMu, the particles contain ~30 bp of host DNA at one end and ~3 kb at the other 99 . The presence of such transposing phages in viral samples used for metagenomics would result in cellular genes being detected, as is common 86 .…”

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Gene transfer agents: phage-like elements of genetic exchange

2012

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Horizontal gene transfer is important in the evolution of bacterial and archaeal genomes. An interesting genetic exchange process is carried out by diverse phage-like gene transfer agents (GTAs) that are found in a wide range of prokaryotes. Although GTAs resemble phages, they lack the hallmark capabilities that define typical phages, and they package random pieces of the producing cell's genome. In this Review, we discuss the defining characteristics of the GTAs that have been identified to date, along with potential functions for these agents and the possible evolutionary forces that act on the genes involved in their production.The prevalence of horizontal gene transfer (HGT), in which DNA is exchanged between closely or distantly related lineages, has altered the way we think about evolution 1,2 , having profound effects on our views of the evolutionary relationships among living organisms. It has caused a shift from a bifurcating 'tree of life' view to a 'net of life' or 'web of life' view [3][4][5] , in which "highways of gene sharing" (REF. 6) represent major connections, with genes (or DNA segments) viewed as "public goods, available for all organisms to integrate into their genomes" (REF. 7). HGT occurs within and between all three domains of life, and it has been estimated that 0.05-80% of genes in bacterial and archaeal genomes have been affected by HGT, depending on the analytical method used and the genome analysed [8][9][10] . Although HGT is widespread, it is not random: patterns of preferential gene exchange among specific groups of organisms that share ancestry or habitat have been * Present address. aslang@mun.ca; olgazh@dartmouth.edu; j.beatty@mail.ubc.ca Competing interests statementThe authors declare no competing financial interests. 6,9,11,12 . Such patterns are probably a consequence of existing barriers to HGT, which have been reviewed in detail elsewhere 13,14 . FURTHER INFORMATIONAlthough we have known about some HGT mechanisms for decades, novel processes that expand the existing repertoire of gene exchange methods are continually being identified. Transformation was the first mechanism of gene exchange to be discovered 15 , wherein DNA is taken up directly from the environment (reviewed elsewhere 16,17 ). In transduction, DNA transfer is mediated by viral particles (BOX 1). In conjugation, which was discovered in the 1940s 18 , DNA is transferred from a donor to a recipient cell during cell-to-cell contact; conjugation is used in the transfer of plasmids, transposons, integrons, and integrative and conjugative elements (ICEs) [19][20][21] . There are other modes of gene exchange that do not fit well into any of these three canonical mechanisms, including temporary cell fusion followed by chromosomal recombination and plasmid exchange 22 , intercellular connection through nanotubes 23 , and the release of membrane vesicles containing chromosomal, plasmid and phage DNA that can then merge with nearby cells 24 . In addition, a novel mechanism of gene transfer that has feature...

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Gene transfer agents: phage-like elements of genetic exchange

2012

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“…Because the DNA of Mu prophage is colinear with that of vegetative Mu, it appears that specific sites near or at the ends of the Mu genome mediate integration (7,8). In a lytic cycle of infection, multiple insertion events occur that are believed to generate the heterogeneity in the host DNA that is covalently attached to both ends of Mu in mature phage particles (3,9,10). Mu can also promote the integration of plasmids and direct the transposition of chromosomal markers (11)(12)(13).…”

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“…DNA synthesis in five Hfr strains of Escherichia coli K-12 (3,9,10). Mu can also promote the integration of plasmids and direct the transposition of chromosomal markers (11-13).…”

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Integration of bacteriophage Mu at host chromosomal replication forks during lytic development.

Fitts¹,

Taylor²

1980

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

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The target site for bacteriophage Mu integration in a lytic cycle of infection was investigated. DNA synthesis in five Hfr strains of Escherichia coli K-12 (3,9,10). Mu can also promote the integration of plasmids and direct the transposition of chromosomal markers (11-13). As is the case with some insertion elements and transposons, Mu prophages in stable lysogens are flanked by a direct repeat of five base pairs of host DNA (14,15). No form of Mu that is free of host DNA has yet been observed.One possible explanation for the ability of this phage to integrate randomly utilizes host chromosomal replication forks as target sites for Mu insertion. In a population of cells replicating asynchronously, the chromosomal replication forks must be distributed over all parts of the bacterial genome; integration of Mu at host replication forks would therefore result in an apparent lack of site specificity for insertion. Integration at host replication forks has been associated with Mu insertion events leading to the formation of stable lysogens (16,17).In this study we asked if the integration events that occur in a lytic cycle of infection involve host chromosomal replication forks. We constructed a system consisting of five Escherichia coli K-12 Fig. 1. Nonsuppressing (Su-) derivatives of the Hfr stocks were isolated by using the procedure of Templin et al. (22). The his-4 allele from strain AB1157 (23) was introduced into each Hfr strain by phage P1 transduction. Strain AT3757 was constructed by phage P1 cotransduction of the pdxAl::Mu -and ara + alleles from AT796 into a spontaneous arabinose-negative mutant of strain AT3753.Bacteriophages. Mucts62SamlO04 was obtained by heat induction of strain MH1671. Mucts62AamlO93 and Mucts62Bam7234 were obtained by heat induction of strains MH219 and MH2808, respectively. A phage strains JC1929 (XN7N53cI26) and JC1930 (h80iXN7N53cI26), used for the construction of Su-strains, were the gift of A. J. Clark.Media. Bacteria were cultured in L broth or in M9 minimal medium (24). Dilutions and washings were done in 0.85% NaCI, except where otherwise noted. Agar plates contained 1% agar (Difco) in L broth plus 5 mM MgSO4 and nalidixic acid (Sigma) at 30 ,ug/ml; agar overlays contained 0.3% agar in L broth plus nalidixic acid at 15 ,ug/ml. Amino Acid Starvation and Nalidixic Acid Treatment.Overnight cultures of Hfr strains in M9 minimal medium supplemented with histidine at 40 ug/ml were diluted 1:100 in 40 ml of the same medium and grown at 37°C for 2 hr with vigorous shaking. After this period of incubation, the cells were collected by filtration (Amicon, 0.45 ,m), washed, resuspended in 40 ml of M9 without histidine, and incubated at 37°C for 200 min to allow existing rounds of replication to terminate (25)(26)(27). We have previously determined by thymidine incorporation assays that DNA synthesis ceases in these his-4 strains after 180 min of starvation (data not shown). Starved cells were collected by filtration and resuspended in 10 ml of L broth Abbreviations: cfu, ...

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“…DNA was transferred from agarose gels to nitrocellulose paper by using the method of Southern (22). Hybridization with nick-translated Mu or pSC101 was performed as described (23) with the following modifications. Treatment with Denhardt's medium prior to hybridization was elimated.…”

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