scholarly journals Role of DNA Ligase in the Illegitimate Recombination That Generates λbio-Transducing Phages in Escherichia coli

Genetics ◽  
2001 ◽  
Vol 158 (1) ◽  
pp. 29-39
Author(s):  
Masaaki Onda ◽  
Junko Yamaguchi ◽  
Katsuhiro Hanada ◽  
Yasuo Asami ◽  
Hideo Ikeda
Keyword(s):  
Escherichia Coli ◽  
Uv Irradiation ◽  
Sequence Homology ◽  
Illegitimate Recombination ◽  
Dna Ligase ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Recombinant Phage ◽  
Dna Break

Abstract We studied the role of DNA ligase in illegitimate recombination in Escherichia coli. A temperature-sensitive mutation in the lig gene reduced the frequency with which λbio-transducing phages were generated to 10-14% of that of wild type under UV irradiation. Reintroduction of the lig gene into this mutant restored the frequency of recombinant phage generation to that of wild type. Furthermore, overexpression of DNA ligase enhanced illegitimate recombination by 10-fold with or without UV irradiation. In addition, when DNA ligase was present in only limited amounts, UV-induced or spontaneous illegitimate recombination occurred exclusively at hotspot sites that have relatively long sequences of homology (9 or 13 bp). However, when DNA ligase was overexpressed, most of the illegitimate recombination took place at non-hotspot sites having only short sequences of homology (<4 bp). Thus, the level of ligase activity affects the frequency of illegitimate recombination, the length of sequence homology at the recombination sites, and the preference for recombination at hotspots, at least after UV irradiation. These observations support our hypothesis that the illegitimate recombination that generates λbio-transducing phages is mediated by the DNA break-and-join mechanism.

scholarly journals Roles of RecJ, RecO, and RecR in RecET-Mediated Illegitimate Recombination in Escherichia coli

2002 ◽  
Vol 184 (17) ◽  
pp. 4715-4721 ◽  
Author(s):  
Kouya Shiraishi ◽  
Katsuhiro Hanada ◽  
Yoichiro Iwakura ◽  
Hideo Ikeda
Keyword(s):  
Escherichia Coli ◽  
Uv Irradiation ◽  
The Other ◽  
Illegitimate Recombination ◽  
Wild Type ◽  
Prophage Induction ◽  
Other Hand ◽  
In The Wild ◽  
Recombination Pathway

ABSTRACT We analyzed effects of overexpression of RecE and RecT on illegitimate recombination during prophage induction in Escherichia coli and found that frequencies of spontaneous and UV-induced illegitimate recombination are enhanced by coexpression of RecE and RecT in the wild type, but the enhanced recombination was reduced by recJ, recO, or recR mutation. The results indicated that RecET-mediated illegitimate recombination depends on the functions of RecJ, RecO, and RecR, suggesting that the RecE and RecJ exonucleases play different roles in this recombination pathway and that the RecO and RecR proteins also play important roles in the recombination. On the other hand, the frequency of the RecET-mediated illegitimate recombination was enhanced by a recQ mutation, implying that the RecQ protein plays a role in suppression of RecET-mediated illegitimate recombination. It was also found that RecET-mediated illegitimate recombination is independent of the RecA function with UV irradiation, but it is enhanced by the recA mutation without UV irradiation. Based on these results, we propose a model for the roles of RecJOR on RecET-mediated illegitimate recombination.

scholarly journals Escherichia coli MutM Suppresses Illegitimate Recombination Induced by Oxidative Stress

Genetics ◽  
1999 ◽  
Vol 151 (2) ◽  
pp. 439-446 ◽  
Author(s):  
Masaaki Onda ◽  
Katsuhiro Hanada ◽  
Hirokazu Kawachi ◽  
Hideo Ikeda
Keyword(s):  
Oxidative Stress ◽  
Escherichia Coli ◽  
Hydrogen Peroxide ◽  
Dna Damage ◽  
Double Strand Breaks ◽  
Illegitimate Recombination ◽  
Recombination Analysis ◽  
Wild Type ◽  
Strand Breaks ◽  
In The Wild

Abstract DNA damage by oxidative stress is one of the causes of mutagenesis. However, whether or not DNA damage induces illegitimate recombination has not been determined. To study the effect of oxidative stress on illegitimate recombination, we examined the frequency of λbio transducing phage in the presence of hydrogen peroxide and found that this reagent enhances illegitimate recombination. To clarify the types of illegitimate recombination, we examined the effect of mutations in mutM and related genes on the process. The frequency of λbio transducing phage was 5- to 12-fold higher in the mutM mutant than in the wild type, while the frequency in the mutY and mutT mutants was comparable to that of the wild type. Because 7,8-dihydro-8-oxoguanine (8-oxoG) and formamido pyrimidine (Fapy) lesions can be removed from DNA by MutM protein, these lesions are thought to induce illegitimate recombination. Analysis of recombination junctions showed that the recombination at Hotspot I accounts for 22 or 4% of total λbio transducing phages in the wild type or in the mutM mutant, respectively. The preferential increase of recombination at nonhotspot sites with hydrogen peroxide in the mutM mutant was discussed on the basis of a new model, in which 8-oxoG and/or Fapy residues may introduce double-strand breaks into DNA.

scholarly journals Role of SpoVA Proteins in Release of Dipicolinic Acid during Germination of Bacillus subtilis Spores Triggered by Dodecylamine or Lysozyme

2006 ◽  
Vol 189 (5) ◽  
pp. 1565-1572 ◽  
Author(s):  
Venkata Ramana Vepachedu ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Small Molecules ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Ph Optimum ◽  
Inner Membrane ◽  
Temperature Sensitive Mutants

ABSTRACT The release of dipicolinic acid (DPA) during the germination of Bacillus subtilis spores by the cationic surfactant dodecylamine exhibited a pH optimum of ∼9 and a temperature optimum of 60°C. DPA release during dodecylamine germination of B. subtilis spores with fourfold-elevated levels of the SpoVA proteins that have been suggested to be involved in the release of DPA during nutrient germination was about fourfold faster than DPA release during dodecylamine germination of wild-type spores and was inhibited by HgCl2. Spores carrying temperature-sensitive mutants in the spoVA operon were also temperature sensitive in DPA release during dodecylamine germination as well as in lysozyme germination of decoated spores. In addition to DPA, dodecylamine triggered the release of amounts of Ca2+ almost equivalent to those of DPA, and at least one other abundant spore small molecule, glutamic acid, was released in parallel with Ca2+ and DPA. These data indicate that (i) dodecylamine triggers spore germination by opening a channel in the inner membrane for Ca2+-DPA and other small molecules, (ii) this channel is composed at least in part of proteins, and (iii) SpoVA proteins are involved in the release of Ca2+-DPA and other small molecules during spore germination, perhaps by being a part of a channel in the spore's inner membrane.

scholarly journals Lethal Action of Quinolones against a Temperature-Sensitive dnaB Replication Mutant of Escherichia coli

2006 ◽  
Vol 50 (1) ◽  
pp. 362-364 ◽  
Author(s):  
Xilin Zhao ◽  
Muhammad Malik ◽  
Nymph Chan ◽  
Alex Drlica-Wagner ◽  
Jian-Ying Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Dna Replication ◽  
Nalidixic Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Lethal Action ◽  
Inhibition Of Protein Synthesis

ABSTRACT Inhibition of DNA replication in an Escherichia coli dnaB-22 mutant failed to block quinolone-mediated lethality. Inhibition of protein synthesis by chloramphenicol inhibited nalidixic acid lethality and, to a lesser extent, ciprofloxacin lethality in both dnaB-22 and wild-type cells. Thus, major features of quinolone-mediated lethality do not depend on ongoing replication.

Involvement of the cgtA gene function in stimulation of DNA repair in Escherichia coli and Vibrio harveyi

Microbiology ◽  
2003 ◽  
Vol 149 (7) ◽  
pp. 1763-1770 ◽  
Author(s):  
Ryszard Zielke ◽  
Aleksandra Sikora ◽  
Rafał Dutkiewicz ◽  
Grzegorz Wegrzyn ◽  
Agata Czyż
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Dna Repair ◽  
Gene Product ◽  
Uv Irradiation ◽  
Vibrio Harveyi ◽  
Bacterial Species ◽  
Wild Type ◽  
E Coli ◽  
Stimulation Of

CgtA is a member of the Obg/Gtp1 subfamily of small GTP-binding proteins. CgtA homologues have been found in various prokaryotic and eukaryotic organisms, ranging from bacteria to humans. Nevertheless, despite the fact that cgtA is an essential gene in most bacterial species, its function in the regulation of cellular processes is largely unknown. Here it has been demonstrated that in two bacterial species, Escherichia coli and Vibrio harveyi, the cgtA gene product enhances survival of cells after UV irradiation. Expression of the cgtA gene was found to be enhanced after UV irradiation of both E. coli and V. harveyi. Moderate overexpression of cgtA resulted in higher UV resistance of E. coli wild-type and dnaQ strains, but not in uvrA, uvrB, umuC and recA mutant hosts. Overexpression of the E. coli recA gene in the V. harveyi cgtA mutant, which is very sensitive to UV light, restored the level of survival of UV-irradiated cells to the levels observed for wild-type bacteria. Moreover, the basal level of the RecA protein was lower in a temperature-sensitive cgtA mutant of E. coli than in the cgtA + strain, and contrary to wild-type bacteria, no significant increase in recA gene expression was observed after UV irradiation of this cgtA mutant. Finally, stimulation of uvrB gene transcription under these conditions was impaired in the V. harveyi cgtA mutant. All these results strongly suggest that the cgtA gene product is involved in DNA repair processes, most probably by stimulation of recA gene expression and resultant activation of RecA-dependent DNA repair pathways.

scholarly journals Fimbria-Encoding GeneyadCHas a Pleiotropic Effect on Several Biological Characteristics and Plays a Role in Avian Pathogenic Escherichia coli Pathogenicity

2015 ◽  
Vol 84 (1) ◽  
pp. 187-193 ◽  
Author(s):  
Renu Verma ◽  
Thaís Cabrera Galvão Rojas ◽  
Renato Pariz Maluta ◽  
Janaína Luisa Leite ◽  
Livia Pilatti Mendes da Silva ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Soft Agar ◽  
Pleiotropic Effect ◽  
Biological Characteristics ◽  
Wild Type ◽  
Content Type ◽  
Pathogenic Escherichia Coli

The extraintestinal pathogen termed avian pathogenicEscherichia coli(APEC) is known to cause colibacillosis in chickens. The molecular basis of APEC pathogenesis is not fully elucidated yet. In this work, we deleted a component of the Yad gene cluster (yadC) in order to understand the role of Yad in the pathogenicity of the APEC strain SCI-07.In vitro, the transcription level ofyadCwas upregulated at 41°C and downregulated at 22°C. TheyadCexpressionin vivowas more pronounced in lungs than in spleen, suggesting a role in the early steps of the infection. Chicks infected with the wild-type and mutant strains presented, respectively, 80% and 50% mortality rates. The ΔyadCstrain presented a slightly decreased ability to adhere to HeLa cells with or without thed-mannose analog compared with the wild type. Real-time PCR (RT-PCR) assays showed thatfimHwas downregulated (P< 0.05) andcsgAandecpAwere slightly upregulated in the mutant strain, showing thatyadCmodulates expression of other fimbriae. Bacterial internalization studies showed that the ΔyadCstrain had a lower number of intracellular bacteria recovered from Hep-2 cells and HD11 cells than the wild-type strain (P< 0.05). Motility assays in soft agar demonstrated that the ΔyadCstrain was less motile than the wild type (P< 0.01). Curiously, flagellum-associated genes were not dramatically downregulated in the ΔyadCstrain. Taken together, the results show that the fimbrial adhesin Yad contributes to the pathogenicity and modulates different biological characteristics of the APEC strain SCI-07.

scholarly journals Transposon Insertion in the purL Gene Induces Biofilm Depletion in Escherichia coli ATCC 25922

Pathogens ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 774
Author(s):  
Virginio Cepas ◽  
Victoria Ballén ◽  
Yaiza Gabasa ◽  
Miriam Ramírez ◽  
Yuly López ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
De Novo ◽  
Wild Type ◽  
Planktonic Cells ◽  
E Coli ◽  
Qrt Pcr ◽  
Transposon Insertion ◽  
De Novo Purine Biosynthesis

Current Escherichia coli antibiofilm treatments comprise a combination of antibiotics commonly used against planktonic cells, leading to treatment failure. A better understanding of the genes involved in biofilm formation could facilitate the development of efficient and specific new antibiofilm treatments. A total of 2578 E. coli mutants were generated by transposon insertion, of which 536 were analysed in this study. After sequencing, Tn263 mutant, classified as low biofilm-former (LF) compared to the wild-type (wt) strain (ATCC 25922), showed an interruption in the purL gene, involved in the de novo purine biosynthesis pathway. To elucidate the role of purL in biofilm formation, a knockout was generated showing reduced production of curli fibres, leading to an impaired biofilm formation. These conditions were restored by complementation of the strain or addition of exogenous inosine. Proteomic and transcriptional analyses were performed to characterise the differences caused by purL alterations. Thirteen proteins were altered compared to wt. The corresponding genes were analysed by qRT-PCR not only in the Tn263 and wt, but also in clinical strains with different biofilm activity. Overall, this study suggests that purL is essential for biofilm formation in E. coli and can be considered as a potential antibiofilm target.

scholarly journals The RcsCB His-Asp Phosphorelay System Is Essential To Overcome Chlorpromazine-Induced Stress in Escherichia coli

2002 ◽  
Vol 184 (10) ◽  
pp. 2850-2853 ◽  
Author(s):  
Annie Conter ◽  
Rachel Sturny ◽  
Claude Gutierrez ◽  
Kaymeuang Cam
Keyword(s):  
Escherichia Coli ◽  
Type Strain ◽  
Wild Type Strain ◽  
Cell Envelope ◽  
Wild Type ◽  
E Coli ◽  
Induced Stress ◽  
Mutant Strains ◽  
Molecular Nature

ABSTRACT The RcsCB His-Asp phosphorelay system regulates the expression of several genes of Escherichia coli, but the molecular nature of the inducing signal is still unknown. We show here that treatment of an exponentially growing culture of E. coli with the cationic amphipathic compound chlorpromazine (CPZ) stimulates expression of a set of genes positively regulated by the RcsCB system. This induction is abolished in rcsB or rcsC mutant strains. In addition, treatment with CPZ inhibits growth. The wild-type strain is able to recover from this inhibition and resume growth after a period of adaptation. In contrast, strains deficient in the RcsCB His-Asp phosphorelay system are hypersensitive to CPZ. These results suggest that cells must express specific RcsCB-regulated genes in order to cope with the CPZ-induced stress. This is the first report of the essential role of the RcsCB system in a stress situation. These results also strengthen the notion that alterations of the cell envelope induce a signal recognized by the RcsC sensor.

scholarly journals Role of Motility and the flhDC Operon in Escherichia coli MG1655 Colonization of the Mouse Intestine

2007 ◽  
Vol 75 (7) ◽  
pp. 3315-3324 ◽  
Author(s):  
Eric J. Gauger ◽  
Mary P. Leatham ◽  
Regino Mercado-Lubo ◽  
David C. Laux ◽  
Tyrrell Conway ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Regulatory Region ◽  
Flagellar Motor ◽  
Wild Type ◽  
E Coli ◽  
Mouse Intestine ◽  
Flagellum Biosynthesis ◽  
Colonizing Ability

ABSTRACT Previously, we reported that the mouse intestine selected mutants of Escherichia coli MG1655 that have improved colonizing ability (M. P. Leatham et al., Infect. Immun. 73:8039-8049, 2005). These mutants grew 10 to 20% faster than their parent in mouse cecal mucus in vitro and 15 to 30% faster on several sugars found in the mouse intestine. The mutants were nonmotile and had deletions of various lengths beginning immediately downstream of an IS1 element located within the regulatory region of the flhDC operon, which encodes the master regulator of flagellum biosynthesis, FlhD4C2. Here we show that during intestinal colonization by wild-type E. coli strain MG1655, 45 to 50% of the cells became nonmotile by day 3 after feeding of the strain to mice and between 80 and 90% of the cells were nonmotile by day 15 after feeding. Ten nonmotile mutants isolated from mice were sequenced, and all were found to have flhDC deletions of various lengths. Despite this strong selection, 10 to 20% of the E. coli MG1655 cells remained motile over a 15-day period, suggesting that there is an as-yet-undefined intestinal niche in which motility is an advantage. The deletions appear to be selected in the intestine for two reasons. First, genes unrelated to motility that are normally either directly or indirectly repressed by FlhD4C2 but can contribute to maximum colonizing ability are released from repression. Second, energy normally used to synthesize flagella and turn the flagellar motor is redirected to growth.

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