Recombination of bacteriophage λ in recD mutants of Escherichia coli

RecBCD enzyme is centrally important in homologous recombination in Escherichia coli and is the source of ExoV activity. Null alleles of either the recB or the recC genes, which encode the B and C subunits, respectively, manifest no recombination and none of the nuclease functions characteristic of the holoenzyme. Loss of the D subunit, by a recD mutation, likewise results in loss of ExoV activity. However, mutants lacking the D subunit are competent for homologous recombination. We report that the distribution of exchanges along the chromosome of Red−Gam−phage λ is strikingly altered by recD null mutations in the host. When λ DNA replication is blocked, recombination in recD mutant strains is high near λ's right end. In contrast, recombination in isogenic recD+ strains is approximately uniform along λ unless the λ chromosome contains a χ sequence. Recombination in recD mutant strains is focused toward the site of action of a type II restriction enzyme acting in vivo on λ. The distribution of exchanges in isogenic recD+ strains is scarcely altered by the restriction enzyme (unless the phage contains an otherwise silent χ). The distribution of exchanges in recD mutants is strongly affected by λ DNA replication. The distribution of exchanges on λ growing in rec+ cells is not influenced by DNA replication. The exchange distribution along λ in recD mutant cells is independent of χ in a variety of conditions. Recombination in rec+ cells is χ influenced. Recombination in recD mutants depends on recC function, occurs in strains deleted for rac prophage, and is independent of recJ, which is known to be required for λ recombination via the RecF pathway. We entertain two models for recombination in recD mutants: (i) recombination in recD mutants may proceed via double-chain break–repair, as it does in λ's Red pathway and E. coli's RecE pathway; (ii) the RecBC enzyme, missing its D subunit, is equivalent to the wild-type, RecBCD, enzyme after that enzyme has been activated by a χ sequence.Key words: χ sequence, RecBCD pathway, Red pathway, RecBC‡ pathway.

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Homologous recombination-dependent initiation of DNA replication from DNA damage-inducible origins in Escherichia coli.

The EMBO Journal ◽

10.1002/j.1460-2075.1993.tb05998.x ◽

1993 ◽

Vol 12 (8) ◽

pp. 3287-3295 ◽

Cited By ~ 62

Author(s):

T. Asai ◽

S. Sommer ◽

A. Bailone ◽

T. Kogoma

Keyword(s):

Escherichia Coli ◽

Dna Damage ◽

Dna Replication ◽

Homologous Recombination ◽

Initiation Of Dna Replication

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Overproduction of Inactive Variants of the Murein Synthase PBP1B Causes Lysis in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.185.18.5342-5348.2003 ◽

2003 ◽

Vol 185 (18) ◽

pp. 5342-5348 ◽

Cited By ~ 34

Author(s):

Ute Meisel ◽

Joachim-Volker Höltje ◽

Waldemar Vollmer

Keyword(s):

Escherichia Coli ◽

Single Amino Acid ◽

Penicillin Binding Protein ◽

Amino Acid Exchange ◽

Lytic Transglycosylases ◽

Mutant Strains ◽

Ph Conditions ◽

Expression Plasmids ◽

Acid Exchange

ABSTRACT Penicillin-binding protein 1B (PBP1B) of Escherichia coli is a bifunctional murein synthase containing both a transpeptidase domain and a transglycosylase domain. The protein is present in three forms (α, β, and γ) which differ in the length of their N-terminal cytoplasmic region. Expression plasmids allowing the production of native PBP1B or of PBP1B variants with an inactive transpeptidase or transglycosylase domain or both were constructed. The inactive domains contained a single amino acid exchange in an essential active-site residue. Overproduction of the inactive PBP1B variants, but not of the active proteins, caused lysis of wild-type cells. The cells became tolerant to lysis by inactive PBP1B at a pH of 5.0, which is similar to the known tolerance for penicillin-induced lysis under acid pH conditions. Lysis was also reduced in mutant strains lacking several murein hydrolases. In particular, a strain devoid of activity of all known lytic transglycosylases was virtually tolerant, indicating that mainly the lytic transglycosylases are responsible for the observed lysis effect. A possible structural interaction between PBP1B and murein hydrolases in vivo by the formation of a multienzyme complex is discussed.

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Chi-Dependent Intramolecular Recombination in Escherichia coli

Genetics ◽

10.1093/genetics/148.2.545 ◽

1998 ◽

Vol 148 (2) ◽

pp. 545-557

Author(s):

Rachel Friedman-Ohana ◽

Iris Karunker ◽

Amikam Cohen

Keyword(s):

Escherichia Coli ◽

Homologous Recombination ◽

Experimental System ◽

Cis Acting ◽

Enzymes Activity ◽

Intramolecular Recombination ◽

Insight Into

Abstract Homologous recombination in Escherichia coli is enhanced by a cis-acting octamer sequence named Chi (5′-GCTGGTGG-3′) that interacts with RecBCD. To gain insight into the mechanism of Chi-enhanced recombination, we recruited an experimental system that permits physical monitoring of intramolecular recombination by linear substrates released by in vivo restriction from infecting chimera phage. Recombination of the released substrates depended on recA, recBCD and cis-acting Chi octamers. Recombination proficiency was lowered by a xonA mutation and by mutations that inactivated the RuvABC and RecG resolution enzymes. Activity of Chi sites was influenced by their locations and by the number of Chi octamers at each site. A single Chi site stimulated recombination, but a combination of Chi sites on the two homologs was synergistic. These data suggest a role for Chi at both ends of the linear substrate. Chi was lost in all recombinational exchanges stimulated by a single Chi site. Exchanges in substrates with Chi sites on both homologs occurred in the interval between the sites as well as in the flanking interval. These observations suggest that the generation of circular products by intramolecular recombination involves Chi-dependent processing of one end by RecBCD and pairing of the processed end with its duplex homolog.

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An Efficient Site-Directed Mutagenesis Method In Vivo in Escherichia Coli

Applied Mechanics and Materials ◽

10.4028/www.scientific.net/amm.195-196.407 ◽

2012 ◽

Vol 195-196 ◽

pp. 407-411

Author(s):

Mu Qing Qiu

Keyword(s):

Escherichia Coli ◽

Homologous Recombination ◽

Recombinant Plasmid ◽

Genomic Dna ◽

Gene Replacement ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis

In order to develop an efficient site-directed mutagenesis method in vivo, the tests were tested by the following methods. The methods that the fragment knockouted ompR gene was constructed through overlapping PCR, digested by Notand Sal, ligated to plasmid pKOV were applied. The recombination plasmid was transformed into Escherichia coli WMC-001 strain, integrated into the genomic DNA through two step homologous recombination. The Escherichia coli WMC-001/ompR-mutant was obtained due to gene replacement. The fragment of the mutant ompR gene was amplified through overlapping PCR, cloned into pKOV vector. The recombinant plasmid was introduced into Escherichia coli WMC-001/ompR-mutant. The Escherichia coli WMC-001/ompR mutant was also obtained due to gene replacement. Results: The site-directed mutagenesis has been successfully constructed in the ompR gene by sequencing. Conclusion: The method is effective for construction of gene site-directed mutagenesis in vivo.

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RecBCD enzyme overproduction impairs DNA repair and homologous recombination in Escherichia coli

Research in Microbiology ◽

10.1016/j.resmic.2004.10.005 ◽

2005 ◽

Vol 156 (3) ◽

pp. 304-311 ◽

Cited By ~ 10

Author(s):

Damir Đermić ◽

Edyta Halupecki ◽

Davor Zahradka ◽

Mirjana Petranović

Keyword(s):

Escherichia Coli ◽

Dna Repair ◽

Homologous Recombination ◽

Enzyme Overproduction ◽

Recbcd Enzyme

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Mechanical Link between Cohesion Establishment and DNA Replication: Ctf7p/Eco1p, a Cohesion Establishment Factor, Associates with Three Different Replication Factor C Complexes

Molecular and Cellular Biology ◽

10.1128/mcb.23.8.2999-3007.2003 ◽

2003 ◽

Vol 23 (8) ◽

pp. 2999-3007 ◽

Cited By ~ 73

Author(s):

Margaret A. Kenna ◽

Robert V. Skibbens

Keyword(s):

Dna Replication ◽

Replication Factor C ◽

Biologically Relevant ◽

Replication Factor ◽

Chromatid Cohesion ◽

Physical Linkage ◽

Factor C ◽

Redundant Functions ◽

Mutant Cells

ABSTRACT CTF7/ECO1 is an essential yeast gene required for the establishment of sister chromatid cohesion. The findings that CTF7/ECO1, POL30 (PCNA), and CHL12/CTF18 (a replication factor C [RFC] homolog) genetically interact provided the first evidence that the processes of cohesion establishment and DNA replication are intimately coupled—a link now confirmed by other studies. To date, however, it is unknown how Ctf7p/Eco1p function is coupled to DNA replication or whether Ctf7p/Eco1p physically associates with any components of the DNA replication machinery. Here, we report that Ctf7p/Eco1p associates with proteins that perform partially redundant functions in DNA replication. Chl12p/Ctf18p combines with Rfc2p to Rfc5p to form one of three independent RFC complexes. By chromatographic methods, Ctf7p/Eco1p was found to associate with Chl12/Ctf18p and with Rfc2p, Rfc3p, Rfc4p, and Rfc5p. The association between Ctf7p/Eco1p and this RFC complex is biologically relevant in that (i) Ctf7p/Eco1p cosediments with Chl12p/Ctf18p in vivo and (ii) rfc5-1 mutant cells exhibit precocious sister separation. Previous studies revealed that Rfc1p or Rad24p associates with Rfc2p to Rfc5p to form two other RFC complexes independent of Ctf18p-RFC complexes. These Rfc1p-RFC and Rad24p-RFC complexes function in DNA replication or repair and DNA damage checkpoint pathways. Importantly, Ctf7p/Eco1p also associates with Rfc1p and Rad24p, suggesting that these RFC complexes also play critical roles in cohesion establishment. The associations between Ctf7p/Eco1p and RFC subunits provide novel evidence regarding the physical linkage between cohesion establishment and DNA replication. Furthermore, the association of Ctf7p/Eco1p with each of three RFC complexes supplies new insights into the functional redundancy of RFC complexes in cohesion establishment.

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Increased Pho Regulon Activation Correlates with Decreased Virulence of an Avian Pathogenic Escherichia coli O78 Strain

Infection and Immunity ◽

10.1128/iai.00452-10 ◽

2010 ◽

Vol 78 (12) ◽

pp. 5324-5331 ◽

Cited By ~ 24

Author(s):

Nicolas Bertrand ◽

Sébastien Houle ◽

Guillaume LeBihan ◽

Édith Poirier ◽

Charles M. Dozois ◽

...

Keyword(s):

Escherichia Coli ◽

Regulatory System ◽

Rabbit Serum ◽

Infection Model ◽

Pho Regulon ◽

Wild Type ◽

Mutant Strains ◽

Pst System

ABSTRACT Avian pathogenic Escherichia coli (APEC) strains are associated with respiratory infections, septicemia, cellulitis, peritonitis, and other conditions, since colibacillosis manifests in many ways. The Pho regulon is jointly controlled by the two-component regulatory system PhoBR and by the phosphate-specific transport (Pst) system. To determine the specific roles of the PhoBR regulon and the Pst system in the pathogenesis of the APEC O78 strain χ7122, different phoBR and pst mutant strains were tested in vivo in chickens and in vitro for virulence traits. Mutations resulting in constitutive activation of the Pho regulon rendered strains more sensitive than the wild type to hydrogen peroxide and to the bactericidal effects of rabbit serum. In addition, production of type 1 fimbriae was also impaired in these strains. Using a chicken competitive infection model, all PhoB constitutive mutants were outcompeted by the wild-type parent, including strains containing a functional Pst system. Cumulative inactivation of the Pst system and the PhoB regulator resulted in a restoration of virulence. In addition, loss of the PhoB regulator alone did not affect virulence in the chicken infection model. Interestingly, the level of attenuation of the mutant strains correlated directly with the level of activation of the Pho regulon. Overall, results indicate that activation of the Pho regulon rather than phosphate transport by the Pst system plays a major role in the attenuation of the APEC O78 strain χ7122.

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Anatomy of a twin DNA replication factory

Biochemical Society Transactions ◽

10.1042/bst20200640 ◽

2020 ◽

Vol 48 (6) ◽

pp. 2769-2778

Author(s):

Huilin Li ◽

Nina Y. Yao ◽

Michael E. O'Donnell

Keyword(s):

Escherichia Coli ◽

Saccharomyces Cerevisiae ◽

Bacillus Subtilis ◽

Dna Replication ◽

In Vivo Studies ◽

Replication Forks ◽

Structural Studies ◽

Replication Factory ◽

Higher Eukaryotes

The replication of DNA in chromosomes is initiated at sequences called origins at which two replisome machines are assembled at replication forks that move in opposite directions. Interestingly, in vivo studies observe that the two replication forks remain fastened together, often referred to as a replication factory. Replication factories containing two replisomes are well documented in cellular studies of bacteria (Escherichia coli and Bacillus subtilis) and the eukaryote, Saccharomyces cerevisiae. This basic twin replisome factory architecture may also be preserved in higher eukaryotes. Despite many years of documenting the existence of replication factories, the molecular details of how the two replisome machines are tethered together has been completely unknown in any organism. Recent structural studies shed new light on the architecture of a eukaryote replisome factory, which brings with it a new twist on how a replication factory may function.

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RecFOR Function Is Required for DNA Repair and Recombination in a RecA Loading-Deficient recB Mutant of Escherichia coli

Genetics ◽

10.1093/genetics/163.2.485 ◽

2003 ◽

Vol 163 (2) ◽

pp. 485-494 ◽

Cited By ~ 1

Author(s):

Ivana Ivančić-Baće ◽

Petra Peharec ◽

Sunčana Moslavac ◽

Nikolina Škrobot ◽

Erika Salaj-Šmic† ◽

...

Keyword(s):

Escherichia Coli ◽

Reca Protein ◽

Nuclease Activity ◽

Filament Formation ◽

Single Stranded Dna ◽

Catalytic Center ◽

In Vivo Assays ◽

Recbcd Enzyme ◽

Contradictory Data

Abstract The RecA loading activity of the RecBCD enzyme, together with its helicase and 5′ → 3′ exonuclease activities, is essential for recombination in Escherichia coli. One particular mutant in the nuclease catalytic center of RecB, i.e., recB1080, produces an enzyme that does not have nuclease activity and is unable to load RecA protein onto single-stranded DNA. There are, however, previously published contradictory data on the recombination proficiency of this mutant. In a recF– background the recB1080 mutant is recombination deficient, whereas in a recF+ genetic background it is recombination proficient. A possible explanation for these contrasting phenotypes may be that the RecFOR system promotes RecA-single-strand DNA filament formation and replaces the RecA loading defect of the RecB1080CD enzyme. We tested this hypothesis by using three in vivo assays. We compared the recombination proficiencies of recB1080, recO, recR, and recF single mutants and recB1080 recO, recB1080 recR, and recB1080 recF double mutants. We show that RecFOR functions rescue the repair and recombination deficiency of the recB1080 mutant and that RecA loading is independent of RecFOR in the recB1080 recD double mutant where this activity is provided by the RecB1080C(D–) enzyme. According to our results as well as previous data, three essential activities for the initiation of recombination in the recB1080 mutant are provided by different proteins, i.e., helicase activity by RecB1080CD, 5′ → 3′ exonuclease by RecJ- and RecA-single-stranded DNA filament formation by RecFOR.

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