sbcB15 and ΔsbcB Mutations Activate Two Types of RecF Recombination Pathways in Escherichia coli

ABSTRACT Escherichia coli cells with mutations in recBC genes are defective for the main RecBCD pathway of recombination and have severe reductions in conjugational and transductional recombination, as well as in recombinational repair of double-stranded DNA breaks. This phenotype can be corrected by suppressor mutations in sbcB and sbcC(D) genes, which activate an alternative RecF pathway of recombination. It was previously suggested that sbcB15 and ΔsbcB mutations, both of which inactivate exonuclease I, are equally efficient in suppressing the recBC phenotype. In the present work we reexamined the effects of sbcB15 and ΔsbcB mutations on DNA repair after UV and γ irradiation, on conjugational recombination, and on the viability of recBC (sbcC) cells. We found that the sbcB15 mutation is a stronger recBC suppressor than ΔsbcB, suggesting that some unspecified activity of the mutant SbcB15 protein may be favorable for recombination in the RecF pathway. We also showed that the xonA2 mutation, a member of another class of ExoI mutations, had the same effect on recombination as ΔsbcB, suggesting that it is an sbcB null mutation. In addition, we demonstrated that recombination in a recBC sbcB15 sbcC mutant is less affected by recF and recQ mutations than recombination in recBC ΔsbcB sbcC and recBC xonA2 sbcC strains is, indicating that SbcB15 alleviates the requirement for the RecFOR complex and RecQ helicase in recombination processes. Our results suggest that two types of sbcB-sensitive RecF pathways can be distinguished in E. coli, one that is activated by the sbcB15 mutation and one that is activated by sbcB null mutations. Possible roles of SbcB15 in recombination reactions in the RecF pathway are discussed.

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Effects of recJ, recQ, and recFOR Mutations on Recombination in Nuclease-Deficient recB recD Double Mutants of Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.187.4.1350-1356.2005 ◽

2005 ◽

Vol 187 (4) ◽

pp. 1350-1356 ◽

Cited By ~ 28

Author(s):

Ivana Ivančić-Baće ◽

Erika Salaj-Šmic ◽

Krunoslav Brčić-Kostić

Keyword(s):

Escherichia Coli ◽

Double Mutant ◽

Reca Protein ◽

Recq Helicase ◽

Single Stranded Dna ◽

Double Stranded Dna ◽

Recf Pathway ◽

Recombination Pathway ◽

Recbcd Enzyme ◽

Dna Ends

ABSTRACT 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 5′→3′ 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 (5′→3′ 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 (nuclease-deficient) 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 5′→3′ exonuclease is an element of the RecF recombination machinery.

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Escherichia coli and colorectal cancer: Unfolding the enigmatic relationship

Current Pharmaceutical Biotechnology ◽

10.2174/1389201022666210910094827 ◽

2021 ◽

Vol 22 ◽

Author(s):

Rogayeh Nouri ◽

Alka Hasani ◽

Kourosh Masnadi Shirazi ◽

Mohammad Reza Aliand ◽

Bita Sepehri ◽

...

Keyword(s):

Colorectal Cancer ◽

Escherichia Coli ◽

Mammalian Cells ◽

Chromosomal Rearrangements ◽

Current Evidence ◽

Dna Breaks ◽

Colon Cells ◽

E Coli ◽

Dna Mismatch ◽

Double Strand Dna Breaks

: Colorectal cancer (CRC) is one of the deadliest cancers in the world. Specific strains of intestinal Escherichia coli (E. coli) may influence the initiation and development of CRC by exploiting virulence factors and inflammatory pathways. Mucosa-associated E. coli strains are more prevalent in CRC biopsies in comparison to healthy controls. Moreover, these strains can survive and replicate within macrophages and induce a pro-inflammatory response. Chronic exposure to inflammatory mediators can lead to increased cell proliferation and cancer. Production of colobactin toxin by the majority of mucosa-associated E. coli isolated from CRC patients is another notable finding. Colibactin-producing E. coli strains, in particular, induce double-strand DNA breaks, stop the cell cycle, involve in chromosomal rearrangements of mammalian cells and are implicated in carcinogenic effects in animal models. Moreover, some enteropathogenic E. coli (EPEC) strains are able to survive and replicate in colon cells as chronic intracellular pathogens and may promote susceptibility to CRC by downregulation of DNA Mismatch Repair (MMR) proteins. In this review, we discuss current evidence and focus on the mechanisms by which E. coli can influence the development of CRC.

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Genome Sequence of a T4-like Phage, Escherichia Phage vB_EcoM-Sa45lw, Infecting Shiga Toxin-Producing Escherichia coli Strains

Microbiology Resource Announcements ◽

10.1128/mra.00804-19 ◽

2019 ◽

Vol 8 (32) ◽

Author(s):

Yen-Te Liao ◽

Yujie Zhang ◽

Alexandra Salvador ◽

Vivian C. H. Wu

Keyword(s):

Escherichia Coli ◽

Surface Water ◽

Genome Size ◽

Shiga Toxin ◽

Open Reading Frames ◽

Content Type ◽

E Coli ◽

Double Stranded Dna ◽

A Genome ◽

Reading Frames

Escherichia phage vB_EcoM-Sa45lw, a new member of the T4-like phages, was isolated from surface water in a produce-growing area. The phage, containing double-stranded DNA with a genome size of 167,353 bp and 282 predicted open reading frames (ORFs), is able to infect generic Escherichia coli and Shiga toxin-producing E. coli O45 and O157 strains.

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Histone-like Nucleoid-Structuring Protein (H-NS) Paralogue StpA Activates the Type I-E CRISPR-Cas System against Natural Transformation in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.00731-20 ◽

2020 ◽

Vol 86 (14) ◽

Author(s):

Dongchang Sun ◽

Xudan Mao ◽

Mingyue Fei ◽

Ziyan Chen ◽

Tingheng Zhu ◽

...

Keyword(s):

Escherichia Coli ◽

Binding Site ◽

Natural Transformation ◽

Inducible Promoter ◽

Regulation Mechanism ◽

Type I ◽

Content Type ◽

E Coli ◽

Double Stranded Dna ◽

Dna Binding Site

ABSTRACT Working mechanisms of CRISPR-Cas systems have been intensively studied. However, far less is known about how they are regulated. The histone-like nucleoid-structuring protein H-NS binds the promoter of cas genes (Pcas) and suppresses the type I-E CRISPR-Cas system in Escherichia coli. Although the H-NS paralogue StpA also binds Pcas, its role in regulating the CRISPR-Cas system remains unidentified. Our previous work established that E. coli is able to take up double-stranded DNA during natural transformation. Here, we investigated the function of StpA in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. We first documented that although the activated type I-E CRISPR-Cas system, due to hns deletion, interfered with CRISPR-Cas-targeted plasmid transfer, stpA inactivation restored the level of natural transformation. Second, we showed that inactivating stpA reduced the transcriptional activity of Pcas. Third, by comparing transcriptional activities of the intact Pcas and the Pcas with a disrupted H-NS binding site in the hns and hns stpA null deletion mutants, we demonstrated that StpA activated transcription of cas genes by binding to the same site as H-NS in Pcas. Fourth, by expressing StpA with an arabinose-inducible promoter, we confirmed that StpA expressed at a low level stimulated the activity of Pcas. Finally, by quantifying the level of mature CRISPR RNA (crRNA), we demonstrated that StpA was able to promote the amount of crRNA. Taken together, our work establishes that StpA serves as a transcriptional activator in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. IMPORTANCE StpA is normally considered a molecular backup of the nucleoid-structuring protein H-NS, which was reported as a transcriptional repressor of the type I-E CRISPR-Cas system in Escherichia coli. However, the role of StpA in regulating the type I-E CRISPR-Cas system remains elusive. Our previous work uncovered a new route for double-stranded DNA (dsDNA) entry during natural transformation of E. coli. In this study, we show that StpA plays a role opposite to that of its paralogue H-NS in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. Our work not only expands our knowledge on CRISPR-Cas-mediated adaptive immunity against extracellular nucleic acids but also sheds new light on understanding the complex regulation mechanism of the CRISPR-Cas system. Moreover, the finding that paralogues StpA and H-NS share a DNA binding site but play opposite roles in transcriptional regulation indicates that higher-order compaction of bacterial chromatin by histone-like proteins could switch prokaryotic transcriptional modes.

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Single-molecule visualization of RecQ helicase reveals DNA melting, nucleation, and assembly are required for processive DNA unwinding

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1518028112 ◽

2015 ◽

Vol 112 (50) ◽

pp. E6852-E6861 ◽

Cited By ~ 23

Author(s):

Behzad Rad ◽

Anthony L. Forget ◽

Ronald J. Baskin ◽

Stephen C. Kowalczykowski

Keyword(s):

Single Molecule ◽

Fluorescent Sensor ◽

Holliday Junctions ◽

Dna Unwinding ◽

Recq Helicase ◽

Dna Breaks ◽

Dna Helicases ◽

Double Stranded Dna ◽

Functional Forms ◽

And Migration

DNA helicases are motor proteins that unwind double-stranded DNA (dsDNA) to reveal single-stranded DNA (ssDNA) needed for many biological processes. The RecQ helicase is involved in repairing damage caused by DNA breaks and stalled replication forks via homologous recombination. Here, the helicase activity of RecQ was visualized on single molecules of DNA using a fluorescent sensor that directly detects ssDNA. By monitoring the formation and progression of individual unwinding forks, we observed that both the frequency of initiation and the rate of unwinding are highly dependent on RecQ concentration. We establish that unwinding forks can initiate internally by melting dsDNA and can proceed in both directions at up to 40–60 bp/s. The findings suggest that initiation requires a RecQ dimer, and that continued processive unwinding of several kilobases involves multiple monomers at the DNA unwinding fork. We propose a distinctive model wherein RecQ melts dsDNA internally to initiate unwinding and subsequently assembles at the fork into a distribution of multimeric species, each encompassing a broad distribution of rates, to unwind DNA. These studies define the species that promote resection of DNA, proofreading of homologous pairing, and migration of Holliday junctions, and they suggest that various functional forms of RecQ can be assembled that unwind at rates tailored to the diverse biological functions of RecQ helicase.

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A Role for Single-Stranded Exonucleases in the Use of DNA as a Nutrient

Journal of Bacteriology ◽

10.1128/jb.01678-08 ◽

2009 ◽

Vol 191 (11) ◽

pp. 3712-3716 ◽

Cited By ~ 13

Author(s):

Vyacheslav Palchevskiy ◽

Steven E. Finkel

Keyword(s):

Escherichia Coli ◽

Bacterial Cells ◽

Wild Type ◽

Nutrient Source ◽

Term Survival ◽

Natural Competence ◽

E Coli ◽

Double Stranded Dna ◽

Better Than

ABSTRACT Nutritional competence is the ability of bacterial cells to utilize exogenous double-stranded DNA molecules as a nutrient source. We previously identified several genes in Escherichia coli that are important for this process and proposed a model, based on models of natural competence and transformation in bacteria, where it is assumed that single-stranded DNA (ssDNA) is degraded following entry into the cytoplasm. Since E. coli has several exonucleases, we determined whether they play a role in the long-term survival and the catabolism of DNA as a nutrient. We show here that mutants lacking either ExoI, ExoVII, ExoX, or RecJ are viable during all phases of the bacterial life cycle yet cannot compete with wild-type cells during long-term stationary-phase incubation. We also show that nuclease mutants, alone or in combination, are defective in DNA catabolism, with the exception of the ExoX− single mutant. The ExoX− mutant consumes double-stranded DNA better than wild-type cells, possibly implying the presence of two pathways in E. coli for the processing of ssDNA as it enters the cytoplasm.

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Double-stranded gap repair of DNA by gene conversion in Escherichia coli.

Genetics ◽

10.1093/genetics/119.4.751 ◽

1988 ◽

Vol 119 (4) ◽

pp. 751-757

Author(s):

I Kobayashi ◽

N Takahashi

Keyword(s):

Escherichia Coli ◽

Gene Conversion ◽

Genetic Selection ◽

Coli Strain ◽

Repair Mechanism ◽

E Coli ◽

Double Stranded Dna ◽

Inverted Orientation ◽

Plasmid Recombination ◽

Flanking Sequences

Abstract We demonstrated repair of a double-stranded DNA gap through gene conversion by a homologous DNA sequence in Escherichia coli. We made a double-stranded gap in one of the two regions of homology in an inverted orientation on a plasmid DNA molecule and introduced it into an E. coli strain which has the RecE system of recombination (genotype; sbcA23 recB21 recC22). We detected repair products by genetic selection. The repair products were those expected by the double-strand-gap repair model. Gene conversion was frequently accompanied by crossing over of the flanking sequences as in eukaryotes. This double-strand gap repair mechanism can explain plasmid recombination in the absence of an artificial double-stranded break reported in a companion study by Yamamoto et al.

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Transforming DNA Uptake Gene Orthologs Do Not Mediate Spontaneous Plasmid Transformation in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.01130-08 ◽

2008 ◽

Vol 191 (3) ◽

pp. 713-719 ◽

Cited By ~ 20

Author(s):

Dongchang Sun ◽

Xuewu Zhang ◽

Lingyu Wang ◽

Marc Prudhomme ◽

Zhixiong Xie ◽

...

Keyword(s):

Escherichia Coli ◽

Carbon Source ◽

Outer Membrane ◽

Dna Uptake ◽

E Coli ◽

Plasmid Transformation ◽

Double Stranded Dna ◽

Outer Membrane Channel ◽

Gene Orthologs ◽

Stimulation Of

ABSTRACT Spontaneous plasmid transformation of Escherichia coli occurs on nutrient-containing agar plates. E. coli has also been reported to use double-stranded DNA (dsDNA) as a carbon source. The mechanism(s) of entry of exogenous dsDNA that allows plasmid establishment or the use of DNA as a nutrient remain(s) unknown. To further characterize plasmid transformation, we first documented the stimulation of transformation by agar and agarose. We provide evidence that stimulation is not due to agar contributing a supplement of Ca2+, Fe2+, Mg2+, Mn2+, or Zn2+. Second, we undertook to inactivate the E. coli orthologues of Haemophilus influenzae components of the transformation machine that allows the uptake of single-stranded DNA (ssDNA) from exogenous dsDNA. The putative outer membrane channel protein (HofQ), transformation pseudopilus component (PpdD), and transmembrane pore (YcaI) are not required for plasmid transformation. We conclude that plasmid DNA does not enter E. coli cells as ssDNA. The finding that purified plasmid monomers transform E. coli with single-hit kinetics supports this conclusion; it establishes that a unique monomer molecule is sufficient to give rise to a transformant, which is not consistent with the reconstitution of an intact replicon through annealing of partially overlapping complementary ssDNA, taken up from two independent monomers. We therefore propose that plasmid transformation involves internalization of intact dsDNA molecules. Our data together, with previous reports that HofQ is required for the use of dsDNA as a carbon source, suggest the existence of two routes for DNA entry, at least across the outer membrane of E. coli.

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Defective processing of methylated single-stranded DNA by E. coli alkB mutants

Genes & Development ◽

10.1101/gad.14.16.2097 ◽

2000 ◽

Vol 14 (16) ◽

pp. 2097-2105

Author(s):

Suneet Dinglay ◽

Sarah C. Trewick ◽

Tomas Lindahl ◽

Barbara Sedgwick

Keyword(s):

Dna Repair ◽

Replication Forks ◽

Human Homolog ◽

Γ Irradiation ◽

Single Stranded Dna ◽

E Coli ◽

Double Stranded Dna ◽

The Subject ◽

M13 Phage ◽

Methylating Agents

Escherichia coli alkB mutants are very sensitive to DNA methylating agents. Despite these mutants being the subject of many studies, no DNA repair or other function has been assigned to the AlkB protein or to its human homolog. Here, we report that reactivation of methylmethanesulfonate (MMS)-treated single-stranded DNA phages, M13, f1, and G4, was decreased dramatically in alkB mutants. No such decrease occurred when using methylated λ phage or M13 duplex DNA. These data show that alkB mutants have a marked defect in processing methylation damage in single-stranded DNA. Recombinant AlkB protein bound more efficiently to single- than double-stranded DNA. The single-strand damage processed by AlkB was primarily cytotoxic and not mutagenic and was induced by SN2 methylating agents, MMS, DMS, and MeI but not by SN1 agentN-methyl-N-nitrosourea or by γ irradiation. Strains lacking other DNA repair activities, alkA tag, xth nfo, uvrA, mutS, and umuC, were not defective in reactivation of methylated M13 phage and did not enhance the defect of an alkB mutant. ArecA mutation caused a small but additive defect. Thus, AlkB functions in a novel pathway independent of these activities. We propose that AlkB acts on alkylated single-stranded DNA in replication forks or at transcribed regions. Consistent with this theory, stationary phase alkB cells were less MMS sensitive than rapidly growing cells.

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