Human mismatch-repair protein MutL homologue 1 (MLH1) interacts with Escherichia coli MutL and MutS in vivo and in vitro: a simple genetic system to assay MLH1 function

A simple genetic system has been developed to test the effect of over-expression of wild-type or mutated human MutL homologue 1 (hMLH1) proteins on methyl-directed mismatch repair (MMR) in Escherichia coli. The system relies on detection of Lac+ revertants using MMR-proficient or MMR-deficient E. coli strains carrying a lac +1 frameshift mutation expressing hMLH1 proteins. We report that expression of wild-type hMLH1 protein causes an approx. 19-fold increase in mutation rates. The mutator phenotype was due to the ability of hMLH1 protein to interact with bacterial MutL and MutS proteins, thereby interfering with the formation of complexes between MMR proteins and mismatched DNA. Conversely, expression of proteins encoded by alleles deriving from hereditary-non-polyposis-colon-cancer (HNPCC) families decreases mutation rates, depending on the specific amino acid substitutions. These effects parallel the MutL-and MutS-binding and ATP-binding/hydrolysis activities of the mutated proteins.

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Antagonism of Ultraviolet-Light Mutagenesis by the Methyl-Directed Mismatch-Repair System of Escherichia coli

Genetics ◽

10.1093/genetics/154.2.503 ◽

2000 ◽

Vol 154 (2) ◽

pp. 503-512 ◽

Cited By ~ 1

Author(s):

Hongbo Liu ◽

Stephen R Hewitt ◽

John B Hays

Keyword(s):

Escherichia Coli ◽

Mismatch Repair ◽

Excision Repair ◽

Spontaneous Mutation ◽

Repair System ◽

Uv Mutagenesis ◽

Repair Protein ◽

Mismatch Repair System

Abstract Previous studies have demonstrated that the Escherichia coli MutHLS mismatch-repair system can process UV-irradiated DNA in vivo and that the human MSH2·MSH6 mismatch-repair protein binds more strongly in vitro to photoproduct/base mismatches than to “matched” photoproducts in DNA. We tested the hypothesis that mismatch repair directed against incorrect bases opposite photoproducts might reduce UV mutagenesis, using two alleles at E. coli lacZ codon 461, which revert, respectively, via CCC → CTC and CTT → CTC transitions. F′ lacZ targets were mated from mut+ donors into mutH, mutL, or mutS recipients, once cells were at substantial densities, to minimize spontaneous mutation prior to irradiation. In umu+ mut+ recipients, a range of UV fluences induced lac+ revertant frequencies of 4–25 × 10−8; these frequencies were consistently 2-fold higher in mutH, mutL, or mutS recipients. Since this effect on mutation frequency was unaltered by an Mfd− defect, it appears not to involve transcription-coupled excision repair. In mut+ umuC122::Tn5 bacteria, UV mutagenesis (at 60 J/m2) was very low, but mutH or mutL or mutS mutations increased reversion of both lacZ alleles roughly 25-fold, to 5–10 × 10−8. Thus, at UV doses too low to induce SOS functions, such as Umu2′D, most incorrect bases opposite occasional photoproducts may be removed by mismatch repair, whereas in heavily irradiated (SOS-induced) cells, mismatch repair may only correct some photoproduct/base mismatches, so UV mutagenesis remains substantial.

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The Escherichia coli Mismatch Repair Protein MutL Recruits the Vsr and MutH Endonucleases in Response to DNA Damage

Journal of Bacteriology ◽

10.1128/jb.00066-09 ◽

2009 ◽

Vol 191 (12) ◽

pp. 4041-4043 ◽

Cited By ~ 11

Author(s):

Yaroslava Y. Polosina ◽

Justin Mui ◽

Photini Pitsikas ◽

Claire G. Cupples

Keyword(s):

Escherichia Coli ◽

Dna Damage ◽

Mismatch Repair ◽

Repair Protein ◽

Mismatch Repair Protein

ABSTRACT The activities of the Vsr and MutH endonucleases of Escherichia coli are stimulated by MutL. The interaction of MutL with each enzyme is enhanced in vivo by 2-aminopurine treatment and by inactivation of the mutY gene. We hypothesize that MutL recruits the endonucleases to sites of DNA damage.

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

Infection and Immunity ◽

10.1128/iai.01138-15 ◽

2015 ◽

Vol 84 (1) ◽

pp. 187-193 ◽

Cited By ~ 12

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.

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Regulatory Role of PlaR (YiaJ) for Plant Utilization in Escherichia coli K-12

Scientific Reports ◽

10.1038/s41598-019-56886-x ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Tomohiro Shimada ◽

Yui Yokoyama ◽

Takumi Anzai ◽

Kaneyoshi Yamamoto ◽

Akira Ishihama

Keyword(s):

Escherichia Coli ◽

Genetic System ◽

Regulatory Role ◽

E Coli ◽

Warm Blooded Animal ◽

Plant Utilization ◽

K 12

AbstractOutside a warm-blooded animal host, the enterobacterium Escherichia coli K-12 is also able to grow and survive in stressful nature. The major organic substance in nature is plant, but the genetic system of E. coli how to utilize plant-derived materials as nutrients is poorly understood. Here we describe the set of regulatory targets for uncharacterized IclR-family transcription factor YiaJ on the E. coli genome, using gSELEX screening system. Among a total of 18 high-affinity binding targets of YiaJ, the major regulatory target was identified to be the yiaLMNOPQRS operon for utilization of ascorbate from fruits and galacturonate from plant pectin. The targets of YiaJ also include the genes involved in the utilization for other plant-derived materials as nutrients such as fructose, sorbitol, glycerol and fructoselysine. Detailed in vitro and in vivo analyses suggest that L-ascorbate and α-D-galacturonate are the effector ligands for regulation of YiaJ function. These findings altogether indicate that YiaJ plays a major regulatory role in expression of a set of the genes for the utilization of plant-derived materials as nutrients for survival. PlaR was also suggested to play protecting roles of E. coli under stressful environments in nature, including the formation of biofilm. We then propose renaming YiaJ to PlaR (regulator of plant utilization).

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Mutational Analysis of Residues in PriA and PriC Affecting Their Ability To Interact with SSB in Escherichia coli K-12

Journal of Bacteriology ◽

10.1128/jb.00404-20 ◽

2020 ◽

Vol 202 (23) ◽

Author(s):

Anastasiia N. Klimova ◽

Steven J. Sandler

Keyword(s):

Escherichia Coli ◽

Mutational Analysis ◽

Wild Type ◽

Content Type ◽

Growth Defects ◽

C Terminus ◽

K 12 ◽

And Function

ABSTRACT Escherichia coli PriA and PriC recognize abandoned replication forks and direct reloading of the DnaB replicative helicase onto the lagging-strand template coated with single-stranded DNA-binding protein (SSB). Both PriA and PriC have been shown by biochemical and structural studies to physically interact with the C terminus of SSB. In vitro, these interactions trigger remodeling of the SSB on ssDNA. priA341(R697A) and priC351(R155A) negated the SSB remodeling reaction in vitro. Plasmid-carried priC351(R155A) did not complement priC303::kan, and priA341(R697A) has not yet been tested for complementation. Here, we further studied the SSB-binding pockets of PriA and PriC by placing priA341(R697A), priA344(R697E), priA345(Q701E), and priC351(R155A) on the chromosome and characterizing the mutant strains. All three priA mutants behaved like the wild type. In a ΔpriB strain, the mutations caused modest increases in SOS expression, cell size, and defects in nucleoid partitioning (Par−). Overproduction of SSB partially suppressed these phenotypes for priA341(R697A) and priA344(R697E). The priC351(R155A) mutant behaved as expected: there was no phenotype in a single mutant, and there were severe growth defects when this mutation was combined with ΔpriB. Analysis of the priBC mutant revealed two populations of cells: those with wild-type phenotypes and those that were extremely filamentous and Par− and had high SOS expression. We conclude that in vivo, priC351(R155A) identified an essential residue and function for PriC, that PriA R697 and Q701 are important only in the absence of PriB, and that this region of the protein may have a complicated relationship with SSB. IMPORTANCE Escherichia coli PriA and PriC recruit the replication machinery to a collapsed replication fork after it is repaired and needs to be restarted. In vitro studies suggest that the C terminus of SSB interacts with certain residues in PriA and PriC to recruit those proteins to the repaired fork, where they help remodel it for restart. Here, we placed those mutations on the chromosome and tested the effect of mutating these residues in vivo. The priC mutation completely abolished function. The priA mutations had no effect by themselves. They did, however, display modest phenotypes in a priB-null strain. These phenotypes were partially suppressed by SSB overproduction. These studies give us further insight into the reactions needed for replication restart.

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In Vivo Titration of Mitomycin C Action by Four Escherichia coli Genomic Regions on Multicopy Plasmids

Journal of Bacteriology ◽

10.1128/jb.183.7.2259-2264.2001 ◽

2001 ◽

Vol 183 (7) ◽

pp. 2259-2264 ◽

Cited By ~ 26

Author(s):

Yan Wei ◽

Amy C. Vollmer ◽

Robert A. LaRossa

Keyword(s):

Escherichia Coli ◽

Mitomycin C ◽

Transcriptional Activation ◽

Efflux Pump ◽

Wild Type ◽

Potent Inducer ◽

E Coli ◽

Genomic Regions

ABSTRACT Mitomycin C (MMC), a DNA-damaging agent, is a potent inducer of the bacterial SOS response; surprisingly, it has not been used to select resistant mutants from wild-type Escherichia coli. MMC resistance is caused by the presence of any of four distinctE. coli genes (mdfA, gyrl, rob, andsdiA) on high-copy-number vectors. mdfAencodes a membrane efflux pump whose overexpression results in broad-spectrum chemical resistance. The gyrI (also called sbmC) gene product inhibits DNA gyrase activity in vitro, while the rob protein appears to function in transcriptional activation of efflux pumps. SdiA is a transcriptional activator of ftsQAZ genes involved in cell division.

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Cnu, a Novel oriC-Binding Protein of Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.187.20.6998-7008.2005 ◽

2005 ◽

Vol 187 (20) ◽

pp. 6998-7008 ◽

Cited By ~ 15

Author(s):

Myung Suk Kim ◽

Sung-Hun Bae ◽

Sang Hoon Yun ◽

Hee Jung Lee ◽

Sang Chun Ji ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acid Identity ◽

Replication Initiation ◽

In Vitro Assays ◽

Wild Type ◽

Genetic Method ◽

Dna Replication Initiation ◽

Pair Sequence

ABSTRACT We have found, using a newly developed genetic method, a protein (named Cnu, for oriC-binding nucleoid-associated) that binds to a specific 26-base-pair sequence (named cnb) in the origin of replication of Escherichia coli, oriC. Cnu is composed of 71 amino acids (8.4 kDa) and shows extensive amino acid identity to a group of proteins belonging to the Hha/YmoA family. Cnu was previously discovered as a protein that, like Hha, complexes with H-NS in vitro. Our in vivo and in vitro assays confirm the results and further suggest that the complex formation with H-NS is involved in Cnu/Hha binding to cnb. Unlike the hns mutants, elimination of either the cnu or hha gene did not disturb the growth rate, origin content, and synchrony of DNA replication initiation of the mutants compared to the wild-type cells. However, the cnu hha double mutant was moderately reduced in origin content. The Cnu/Hha complex with H-NS thus could play a role in optimal activity of oriC.

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Contributions of O Island 48 to Adherence of Enterohemorrhagic Escherichia coli O157:H7 to Epithelial Cells In Vitro and in Ligated Pig Ileal Loops

Applied and Environmental Microbiology ◽

10.1128/aem.00507-09 ◽

2009 ◽

Vol 75 (18) ◽

pp. 5779-5786 ◽

Cited By ~ 35

Author(s):

Xianhua Yin ◽

Roger Wheatcroft ◽

James R. Chambers ◽

Bianfang Liu ◽

Jing Zhu ◽

...

Keyword(s):

Escherichia Coli ◽

Epithelial Cells ◽

Cluster Formation ◽

Gene Clusters ◽

Enterohemorrhagic Escherichia Coli ◽

Wild Type ◽

The Difference ◽

Large Clusters

ABSTRACT O island 48 (OI-48) of Escherichia coli consists of three functional gene clusters that encode urease, tellurite resistance (Ter), and putative adhesins Iha and AIDA-1. The functions of these clusters in enterohemorrhagic E. coli (EHEC) O157:H7 infection are unknown. Deletion mutants for these three regions were constructed and evaluated for their ability to adhere to epithelial cells in vitro and in ligated pig ileal loops. Deletion of the Ter gene cluster reduced the ability of the organism to adhere to and form large clusters on IPEC-J2 and HEp-2 cells. Complementation of the mutation by introducing the wild-type ter genes restored adherence and large-cluster formation. Tests in ligated pig ileal loops showed a decrease in colonization by the Ter-negative mutant, but the difference was not significant compared to colonization by the wild type (26.4% ± 21.2% versus 40.1% ± 19.1%; P = 0.168). The OI-48 aidA gene deletion had no effect on adherence in vitro or in vivo. Deletion of the iha and ureC genes had no effect on adherence in vitro but significantly reduced the colonization of EHEC O157:H7 in the ligated pig intestine. These data suggest that Ter, Iha, and urease may contribute to EHEC O157:H7 pathogenesis by promoting adherence of the pathogen to the host intestinal epithelium.

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Characterization of Site-Specific Mutations in a Short-Chain-Length/Medium-Chain-Length Polyhydroxyalkanoate Synthase:In VivoandIn VitroStudies of Enzymatic Activity and Substrate Specificity

Applied and Environmental Microbiology ◽

10.1128/aem.00564-13 ◽

2013 ◽

Vol 79 (12) ◽

pp. 3813-3821 ◽

Cited By ~ 24

Author(s):

Jo-Ann Chuah ◽

Satoshi Tomizawa ◽

Miwa Yamada ◽

Takeharu Tsuge ◽

Yoshiharu Doi ◽

...

Keyword(s):

Chain Length ◽

Fold Increase ◽

Short Chain ◽

Pha Synthase ◽

Wild Type ◽

Short Chain Length ◽

Medium Chain ◽

Medium Chain Length

ABSTRACTSaturation point mutagenesis was carried out at position 479 in the polyhydroxyalkanoate (PHA) synthase fromChromobacteriumsp. strain USM2 (PhaCCs) with specificities for short-chain-length (SCL) [(R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate (3HV)] and medium-chain-length (MCL) [(R)-3-hydroxyhexanoate (3HHx)] monomers in an effort to enhance the specificity of the enzyme for 3HHx. A maximum 4-fold increase in 3HHx incorporation and a 1.6-fold increase in PHA biosynthesis, more than the wild-type synthase, was achieved using selected mutant synthases. These increases were subsequently correlated with improved synthase activity and increased preference of PhaCCsfor 3HHx monomers. We found that substitutions with uncharged residues were beneficial, as they resulted in enhanced PHA production and/or 3HHx incorporation. Further analysis led to postulations that the size and geometry of the substrate-binding pocket are determinants of PHA accumulation, 3HHx fraction, and chain length specificity.In vitroactivities for polymerization of 3HV and 3HHx monomers were consistent within vivosubstrate specificities. Ultimately, the preference shown by wild-type and mutant synthases for either SCL (C4and C5) or MCL (C6) substrates substantiates the fundamental classification of PHA synthases.

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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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