Molecular Biology of Bacteriophage Mu

Howe, M M; Bade, Ernesto G.

doi:10.1126/science.1103291

Cited by 185 publications

(96 citation statements)

References 72 publications

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“…Gel permeation chromatography of chlAl extract was carried out by using a Superose 12 column to estimate the size of the active material in Mono Q fractions 4 and 18. The elution of the activities from the column was monitored by using the nit-i assay with added material from the complementary Mono Q fraction.…”

Section: Resultsmentioning

confidence: 99%

Two proteins encoded at the chlA locus constitute the converting factor of Escherichia coli chlA1

Pitterle

Rajagopalan

1989

J Bacteriol

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Molybdopterin (MPT) is not produced by the Escherichia coli mutants chlA1, chlM, or chlN or by the Neurospora crassa mutant nit-1. Extracts of E. coli chlA1 contain an activity, the converting factor, which is functionally defined by its ability to convert a low-molecular-weight precursor present in crude extracts of N. crassa nit-1 into molybdopterin in vitro. In this study, it has been shown that the converting factor consists of two associative proteins (10 and 25 kilodaltons [kDa]) which can be separated by using either anion-exchange or gel filtration chromatography. Neither protein is able to complement extracts of nit-1 by itself. Analysis of chlA Mu insertion mutants has shown that the two proteins are distinct gene products encoded at the chlA locus. Twelve chlA Mu insertion strains which lacked converting factor activity were deficient in one or both of the proteins. Converting factor activity could be generated by mixing extracts from strains having the 25-kDa protein with those having the 10-kDa protein but not those lacking both proteins. Finally, it was shown that the chlM mutant lacks the 10-kDa protein while the chlN mutant, which contains both the 10- and 25-kDa proteins, lacks a function required to activate the 10-kDa protein.

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Section: Resultsmentioning

confidence: 99%

Two proteins encoded at the chlA locus constitute the converting factor of Escherichia coli chlA1

Pitterle

Rajagopalan

1989

J Bacteriol

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“…Incidentally, spontaneously induced coliphage h allowed one to estimate a virus-host ratio of 0.005 (Birge 1994). Phage Mu lysogens release phages spontaneously at a low level virus-host ratio of -0.0001 but are not induced by treatment with UV or other DNA-damaging agents (Howe & Bade 1975, Ljungquest & Bukhari 1977, Howe 1987. Although the values observed in this study were biased due to filtration, marine strains have shown considerably high rates of spontaneous VLP induction.…”

Section: Bacterial Growth and Vlp Productionmentioning

confidence: 99%

Generalized gene transfer by virus-like particles from marine bacteria

Chiura¹

1997

Aquat. Microb. Ecol.

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Spontaneous VLP (virus-like particle) production and VLP-mediated gene transfer into Escherichia coli AB1157 as recipient was demonstrated. Five marine isolates (Alc 096, Alc 233, Alc 252, Agrobacterium kieliense and Flavobacterium sp. 11604) were investigated for their potential to produce VLP as well as for the gene transfer capability of these VLPs to the E. col1 recipient. These strains are classified as ubiquinone-10-possessing marine bacteria (QIOMB) in the 16s-rRNA Superfamily IV VLPs were obtained from 100 h cultured broth of all strains examined. VLP-host ratio after 100 h growth culture was: Alc 233, 1.54; Alc 252, 1.26; Alc 096, 1.06; Flavobacterjum sp. 11604, 0.69; and A. kieliense, 0.06. These ratios were smaller than those found in the marine environment. However, the spontaneously produced VLP number can be considered as high because the reported numbers are relatively low from coliphage h (0.005) and phage Mu (-0.0001). VLP-mediated gene transfer was examined using an auxotrophic mutant of E. coli (AB1157) with 4 amino acid deficiencies (leu, pro, his, arg)as recipient at multiplicity of infection (MOI) of 0.1. Through this treatment, VLPs showed lethal effect on the recipient. The survival rate of control was: Alc 096, 7%; Alc 252, 8 % ; A. kieliense, 17%; Flavobacterium sp. 11604,31%; and Alc 233,40%. At the same time, all the purified VLPs derived from these 5 strains successfully transferred genes to rescue genetlc defects of the recipi.ent. Overall average efficiency of VLP-mediated gene transfer at MO1 of 0.1 was estimated to be between 2.62 X' 10-3 and 3.58 X 10-'per VLP particle. Loci of employed genetic markers were dispersed on the E. coli chromosome with mutual distance of 121, 1154, 1397 and 364 kb between them. Since VLPs from different sources showed similar gene transfer efficiency in respect to the genet~c marker rescued, it is suggested that VLPs from QlOMB transferred genes as generalized transduction. These results indicate that the VLPs produced by certain marine bacteria may be an important element for both non-specific generalzed horizontal gene transfer towards a broad range of bacterial hosts and population control in the marine environment.

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“…Insertions may be precise or may result in deletion of host sequences at the site of integration (5,6). 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).…”

mentioning

confidence: 99%

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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Molecular Biology of Bacteriophage Mu

Cited by 185 publications

References 72 publications

Two proteins encoded at the chlA locus constitute the converting factor of Escherichia coli chlA1

Two proteins encoded at the chlA locus constitute the converting factor of Escherichia coli chlA1

Generalized gene transfer by virus-like particles from marine bacteria

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

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