In vivo studies on the interaction of RecBCD enzyme and lambda Gam protein

Maršić, Nataša; Roje, Sanja; Stojiljković, Igor; Salaj-Šmic, Erika; Trgovčević, Željko

doi:10.1128/jb.175.15.4738-4743.1993

Cited by 41 publications

(30 citation statements)

References 42 publications

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“…M9 medium contained 0.5 g of NaCl, 1 g of NH 4 Cl, 3 g of KH 2 PO 4 , 7.5 g of Na 2 HPO 4 ⅐ 2H 2 O, 4 g of glucose, 120 mg of MgSO 4 , 10 mg of CaCl 2 , and water added to create a volume of 1,000 ml. For minimal selective plates, M9 medium was supplemented with appropriate amino acids, 1 mg of thiamine, and 16 g of agar (33). All experiments were done with exponentially growing cells at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Effects ofrecJ,recQ, andrecFORMutations on Recombination in Nuclease-DeficientrecB recDDouble Mutants ofEscherichia coli

2005

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The two main recombination pathways in Escherichia coli (RecBCD and RecF) have different recombination machineries that act independently in the initiation of recombination. Three essential enzymatic activities are required for early recombinational processing of double-stranded DNA ends and breaks: a helicase, a 533 exonuclease, and loading of RecA protein onto single-stranded DNA tails. The RecBCD enzyme performs all of these activities, whereas the recombination machinery of the RecF pathway consists of RecQ (helicase), RecJ (533 exonuclease), and RecFOR (RecA-single-stranded DNA filament formation). The recombination pathway operating in recB (nuclease-deficient) mutants is a hybrid because it includes elements of both the RecBCD and RecF recombination machineries. In this study, genetic analysis of recombination in a recB (nucleasedeficient) recD double mutant was performed. We show that conjugational recombination and DNA repair after UV and gamma irradiation in this mutant are highly dependent on recJ, partially dependent on recFOR, and independent of recQ. These results suggest that the recombination pathway operating in a nuclease-deficient recB recD double mutant is also a hybrid. We propose that the helicase and RecA loading activities belong to the RecBCD recombination machinery, while the RecJ-mediated 533 exonuclease is an element of the RecF recombination machinery.

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Section: Methodsmentioning

confidence: 99%

Effects ofrecJ,recQ, andrecFORMutations on Recombination in Nuclease-DeficientrecB recDDouble Mutants ofEscherichia coli

2005

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“…A gene 2 mutant of T4 plates equally poorly on nonlysogens and lambda lysogens, indicating no expression of the lambda gam gene from lambda prophage (62). In contrast to lambda lysogens, Lula/phi80 lysogens allow T4 2 mutants to plate (Fig.…”

Section: Mutagenesis Of the Nonessential Genesmentioning

confidence: 99%

Genome of Enterobacteriophage Lula/phi80 and Insights into Its Ability To Spread in the Laboratory Environment

et al. 2012

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The novel temperate bacteriophage Lula, contaminating laboratory Escherichia coli strains, turned out to be the well-known lambdoid phage phi80. Our previous studies revealed that two characteristics of Lula/phi80 facilitate its spread in the laboratory environment: cryptic lysogen productivity and stealthy infectivity. To understand the genetics/genomics behind these traits, we sequenced and annotated the Lula/phi80 genome, encountering an E. coli-toxic gene revealed as a gap in the sequencing contig and analyzing a few genes in more detail. Lula/phi80's genome layout copies that of lambda, yet homology with other lambdoid phages is mostly limited to the capsid genes. Lula/phi80's DNA is resistant to cutting with several restriction enzymes, suggesting DNA modification, but deletion of the phage's damL gene, coding for DNA adenine methylase, did not make DNA cuttable. The damL mutation of Lula/phi80 also did not change the phage titer in lysogen cultures, whereas the host dam mutation did increase it almost 100-fold. Since the high phage titer in cultures of Lula/phi80 lysogens is apparently in response to endogenous DNA damage, we deleted the only Lula/phi80 SOS-controlled gene, dinL. We found that dinL mutant lysogens release fewer phage in response to endogenous DNA damage but are unchanged in their response to external DNA damage. The toxic gene of Lula/phi80, gamL, encodes an inhibitor of the host ATP-dependent exonucleases, RecBCD and SbcCD. Its own antidote, agt, apparently encoding a modifier protein, was found nearby. Interestingly, Lula/phi80 lysogens are recD and sbcCD phenocopies, so GamL and Agt are part of lysogenic conversion.T he laboratory setting is, by design, an inhospitable environment, as very few species are capable of overcoming the researcher-imposed barriers for growth, multiplication, and spread. The well-known examples of organisms successfully propagating in the laboratory environment are mycoplasmas (76) and HeLa cells (39), both of which cross-contaminate mammalian cell cultures and spread through the laboratories around the world with appalling ease (48). Part of their success is their rapid growth and hardiness, giving them the ability to outgrow and outlive any laboratory strain competitors. Until recently, however, no such creature was known for cultures of rapidly growing microbes, which, precisely because of their speed of growth, effectively deny such chances to a contaminating competitor. This has changed with a description of a temperate phage, Lula, that infects Escherichia coli, converts it into a lysogen, and efficiently spreads in the laboratory setting, stealthily contaminating laboratory strains without researchers' knowledge (75). Lula contamination is apparently widespread yet not broadly recognized, and, because of this combination, it can be a scourge in shared facilities that handle both contaminated cultures (lysogens) and noncontaminated cultures (nonlysogens), resulting in a frustratingly high frequency of lysis and growth inhibition of the latter "for no ap...

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“…Both plasmid-free and plasmid-containing strains were grown to the stationary phase in TP medium (31) and then used as indicator bacteria for platings of unmodified phages. All incubations were at 37ЊC.…”

Section: Methodsmentioning

confidence: 99%

Modulation of EcoKI restriction in vivo: role of the lambda Gam protein and plasmid metabolism

et al. 1997

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Two novel types of alleviation of DNA restriction by the EcoKI restriction endonuclease are described. The first type depends on the presence of the gam gene product (Gam protein) of bacteriophage . The efficiency of plating of unmodified phage is greatly increased when the restricting Escherichia coli K-12 host carries a gam ؉ plasmid. The effect is particularly striking in wild-type strains and, to a lesser extent, in the presence of sbcC and recA mutations. In all cases, Gam-dependent alleviation of restriction requires active recBCD genes of the host and recombination (red) genes of the infecting phage. The enhanced capacity of Gam-expressing cells to repair DNA strand breaks might account for this phenomenon. The second type is caused by the presence of a plasmid in a restricting host lacking RecBCD enzyme. Commonly used plasmids such as the cloning vector pACYC184 can produce such an effect in strains carrying recB single mutations or in recBC sbcBC strains. Plasmid-mediated restriction alleviation in recBC sbcBC strains is independent of the host RecF, RecJ, and RecA proteins and phage recombination functions. The presence of plasmids can also relieve restriction in recD strains. This effect depends, however, on the RecA function in the host. The molecular mechanism of the plasmid-mediated restriction alleviation remains unclear.

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In vivo studies on the interaction of RecBCD enzyme and lambda Gam protein

Cited by 41 publications

References 42 publications

Effects ofrecJ,recQ, andrecFORMutations on Recombination in Nuclease-DeficientrecB recDDouble Mutants ofEscherichia coli

Effects ofrecJ,recQ, andrecFORMutations on Recombination in Nuclease-DeficientrecB recDDouble Mutants ofEscherichia coli

Genome of Enterobacteriophage Lula/phi80 and Insights into Its Ability To Spread in the Laboratory Environment

Modulation of EcoKI restriction in vivo: role of the lambda Gam protein and plasmid metabolism

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