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1.  High incidence of multiple antibiotic resistant cells in cultures of in enterohemorrhagic Escherichia coli O157:H7 
The spontaneous incidence of chloramphenicol (Cam) resistant mutant bacteria is at least ten-fold higher in cultures of enterohemorrhagic E. coli O157:H7 strain EDL933 than in E. coli K-12. It is at least 100-fold higher in the dam (DNA adenine methyltransferase) derivative of EDL933, compared to the dam strain of E. coli K-12, thereby preventing the use of Cam resistance as a marker in gene replacement technology. Genome sequencing of Cam-resistant isolates of EDL933 and its dam derivatives showed that the marR (multiple antibiotic resistance) gene was mutated in every case but not in the Cam-sensitive parental strains. As expected from mutation in the marR gene, the Cam-resistant bacteria were also found to be resistant to tetracycline and nalidixic acid. The marR gene in strain EDL933 is annotated as a shorter open reading frame than that in E. coli K-12 but the longer marR+ open reading frame was more efficient at complementing the marR antibiotic-resistance phenotype of strain EDL933. Beta-lactamase-tolerant derivatives were present at frequencies 10–100 times greater in cultures of marR derivatives of strain EDL933 than the parent strain. Spontaneous mutation frequency to rifampicin, spectinomycin and streptomycin resistance was the same in E. coli O157:H7 and E. coli K-12 strains.
doi:10.1016/j.mrfmmm.2013.11.008
PMCID: PMC3913999  PMID: 24361397
Escherichia coli O157; multiple antibiotic resistance; mutation; genes; repressor protein; DNA
2.  DNA Methylation and Mutator Genes in Escherichia coli K-12 
Mutation research  2010;705(2):71-76.
Mutator strains of Escherichia coli have been used to define mechanisms that account for the high fidelity of chromosome duplication and chromosome stability. Mutant strains defective in post-replicative mismatch repair display a strong mutator phenotype consistent with a role for correction of mismatches arising from replication errors. Inactivation of the gene (dam) encoding DNA adenine methyltransferase results in a mutator phenotype consistent with a role for DNA methylation in strand discrimination during mismatch repair. This review gives a personal perspective on the discovery of dam mutants in E. coli and their relationship to mismatch repair and mutator phenotypes.
doi:10.1016/j.mrrev.2010.05.001
PMCID: PMC2932865  PMID: 20471491
Alkylating agents; DNA repair; Escherichia coli; Mutator; Methylation; Recombination
3.  Using stable MutS dimers and tetramers to quantitatively analyze DNA mismatch recognition and sliding clamp formation 
Nucleic Acids Research  2013;41(17):8166-8181.
The process of DNA mismatch repair is initiated when MutS recognizes mismatched DNA bases and starts the repair cascade. The Escherichia coli MutS protein exists in an equilibrium between dimers and tetramers, which has compromised biophysical analysis. To uncouple these states, we have generated stable dimers and tetramers, respectively. These proteins allowed kinetic analysis of DNA recognition and structural analysis of the full-length protein by X-ray crystallography and small angle X-ray scattering. Our structural data reveal that the tetramerization domains are flexible with respect to the body of the protein, resulting in mostly extended structures. Tetrameric MutS has a slow dissociation from DNA, which can be due to occasional bending over and binding DNA in its two binding sites. In contrast, the dimer dissociation is faster, primarily dependent on a combination of the type of mismatch and the flanking sequence. In the presence of ATP, we could distinguish two kinetic groups: DNA sequences where MutS forms sliding clamps and those where sliding clamps are not formed efficiently. Interestingly, this inability to undergo a conformational change rather than mismatch affinity is correlated with mismatch repair.
doi:10.1093/nar/gkt582
PMCID: PMC3783165  PMID: 23821665
4.  RecA-independent single-stranded DNA oligonucleotide-mediated mutagenesis 
F1000 biology reports  2010;2(1):56.
The expression of Beta, the single-stranded annealing protein (SSAP) of bacteriophage λ in Escherichia coli promotes high levels of oligonucleotide (oligo)-mediated mutagenesis and offers a quick way to create single or multiple base pair insertions, deletions, or substitutions in the bacterial chromosome. High rates of mutagenesis can be obtained by the use of mismatch repair (MMR)-resistant mismatches or MMR-deficient hosts, which allow for the isolation of unselected mutations. It has recently become clear that many bacteria can be mutagenized with oligos in the absence of any SSAP expression, albeit at a much lower frequency. Studies have shown that inactivation or inhibition of single-stranded DNA (ssDNA) exonucleases in vivo increases the rate of SSAP-independent oligo-mediated mutagenesis. These results suggest that λ Beta, in addition to its role in annealing the oligo to ssDNA regions of the replication fork, promotes high rates of oligo-mediated mutagenesis by protecting the oligo from destruction by host ssDNA exonucleases.
doi:10.3410/B2-56
PMCID: PMC2920528  PMID: 20711416
5.  The Alkaloid Compound Harmane Increases the Lifespan of Caenorhabditis elegans during Bacterial Infection, by Modulating the Nematode’s Innate Immune Response 
PLoS ONE  2013;8(3):e60519.
The nematode Caenorhabditis elegans has in recent years been proven to be a powerful in vivo model for testing antimicrobial compounds. We report here that the alkaloid compound Harmane (2-methyl-β-carboline) increases the lifespan of nematodes infected with a human pathogen, the Shiga toxin-producing Escherichia coli O157:H7 strain EDL933 and several other bacterial pathogens. This was shown to be unrelated to the weak antibiotic effect of Harmane. Using GFP-expressing E. coli EDL933, we showed that Harmane does not lower the colonization burden in the nematodes. We also found that the expression of the putative immune effector gene F35E12.5 was up-regulated in response to Harmane treatment. This indicates that Harmane stimulates the innate immune response of the nematode; thereby increasing its lifespan during bacterial infection. Expression of F35E12.5 is predominantly regulated through the p38 MAPK pathway; however, intriguingly the lifespan extension resulting from Harmane was higher in p38 MAPK-deficient nematodes. This indicates that Harmane has a complex effect on the innate immune system of C. elegans. Harmane could therefore be a useful tool in the further research into C. elegans immunity. Since the innate immunity of C. elegans has a high degree of evolutionary conservation, drugs such as Harmane could also be possible alternatives to classic antibiotics. The C. elegans model could prove to be useful for selection and development of such drugs.
doi:10.1371/journal.pone.0060519
PMCID: PMC3609739  PMID: 23544153
6.  High efficiency generalized transduction in Escherichia coli O157:H7 
F1000Research  2013;2:7.
Genetic manipulation in enterohemorrhagic E. coli O157:H7 is currently restricted to recombineering, a method that utilizes the recombination system of bacteriophage lambda, to introduce gene replacements and base changes inter alia into the genome. Bacteriophage 933W is a prophage in E. coli O157:H7 strain EDL933, which encodes the genes ( stx2AB) for the production of Shiga toxin which is the basis for the potentially fatal Hemolytic Uremic Syndrome in infected humans. We replaced the stx2AB genes with a kanamycin cassette using recombineering. After induction of the prophage by ultra-violet light, we found that bacteriophage lysates were capable of transducing to wildtype, point mutations in the lactose, arabinose and maltose genes. The lysates could also transduce tetracycline resistant cassettes. Bacteriophage 933W is also efficient at transducing markers in E. coli K-12. Co-transduction experiments indicated that the maximal amount of transferred DNA was likely the size of the bacteriophage genome, 61 kB. All tested transductants, in both E. coli K-12 and O157:H7, were kanamycin-sensitive indicating that the transducing particles contained host DNA.
doi:10.12688/f1000research.2-7.v1
PMCID: PMC3752737  PMID: 24358856
7.  Bleomycin Sensitivity in Escherichia coli is Medium-Dependent 
PLoS ONE  2012;7(3):e33256.
Bleomycin (BLM) is a glycopeptide antibiotic and anti-tumor agent that targets primarily the furanose rings of DNA and in the presence of ferrous ions produces oxidative damage and DNA strand breaks. Escherichia coli cells growing in broth medium and exposed to low concentrations of BLM contain double-strand breaks and require homologous recombination to survive. To a lesser extent, the cells also require the abasic (AP) endonucleases associated with base excision repair, presumably to repair oxidative damage. As expected, there is strong induction of the SOS system in treated cells. In contrast, E. coli cells growing in glucose or glycerol minimal medium are resistant to the lethal action of BLM and do not require either homologous recombination functions or AP-endonucleases for survival. DNA ligase activity, however, is needed for cells growing in minimal medium to resist the lethal effects of BLM. There is weak SOS induction in such treated cells.
doi:10.1371/journal.pone.0033256
PMCID: PMC3305319  PMID: 22438905
8.  Roles of DNA adenine methylation in host-pathogen interactions: mismatch repair, transcriptional regulation, and more 
FEMS microbiology reviews  2009;33(3):488-503.
The Dam methylase of gamma-proteobacteria and the CcrM methylase of alpha-proteobacteria catalyze an identical reaction (methylation of adenosine moieties using S-adenosyl-methionine as methyl donor) at similar DNA targets (GATC and GANTC, respectively). Dam and CcrM are of independent evolutionary origin. Each may have evolved from an ancestral restriction-modification system that lost its restriction component, leaving an “orphan” methylase devoted solely to epigenetic genome modification. Formation of 6-methyladenine lowers the thermodynamic stability of DNA and changes DNA curvature. As a consequence, the methylation state of specific adenosine moieties can affect DNA-protein interactions. Well known examples include binding of the replication initiation complex to the methylated oriC, recognition of hemimethylated GATCs in newly replicated DNA by the MutHLS mismatch repair complex, and discrimination of methylation states in promoters and regulatory DNA motifs by RNA polymerase and transcription factors. In recent years, Dam and CcrM have been shown to play roles in host-pathogen interactions. These roles are diverse and only partially understood. Especially intriguing is the evidence that Dam methylation regulates virulence genes in E. coli, Salmonella, and Yersinia at the postranscriptional level.
doi:10.1111/j.1574-6976.2008.00159.x
PMCID: PMC2941194  PMID: 19175412
Dam; CcrM; Pathogenic bacteria; Transcription; GATC regulation
9.  RecA-independent single-stranded DNA oligonucleotide-mediated mutagenesis 
The expression of Beta, the single-stranded annealing protein (SSAP) of bacteriophage λ in Escherichia coli promotes high levels of oligonucleotide (oligo)-mediated mutagenesis and offers a quick way to create single or multiple base pair insertions, deletions, or substitutions in the bacterial chromosome. High rates of mutagenesis can be obtained by the use of mismatch repair (MMR)-resistant mismatches or MMR-deficient hosts, which allow for the isolation of unselected mutations. It has recently become clear that many bacteria can be mutagenized with oligos in the absence of any SSAP expression, albeit at a much lower frequency. Studies have shown that inactivation or inhibition of single-stranded DNA (ssDNA) exonucleases in vivo increases the rate of SSAP-independent oligo-mediated mutagenesis. These results suggest that λ Beta, in addition to its role in annealing the oligo to ssDNA regions of the replication fork, promotes high rates of oligo-mediated mutagenesis by protecting the oligo from destruction by host ssDNA exonucleases.
doi:10.3410/B2-56
PMCID: PMC2920528  PMID: 20711416
10.  DnaC Inactivation in Escherichia coli K-12 Induces the SOS Response and Expression of Nucleotide Biosynthesis Genes 
PLoS ONE  2008;3(8):e2984.
Background
Initiation of chromosome replication in E. coli requires the DnaA and DnaC proteins and conditionally-lethal dnaA and dnaC mutants are often used to synchronize cell populations.
Methodology/Principal Findings
DNA microarrays were used to measure mRNA steady-state levels in initiation-deficient dnaA46 and dnaC2 bacteria at permissive and non-permissive temperatures and their expression profiles were compared to MG1655 wildtype cells. For both mutants there was altered expression of genes involved in nucleotide biosynthesis at the non-permissive temperature. Transcription of the dnaA and dnaC genes was increased at the non-permissive temperature in the respective mutant strains indicating auto-regulation of both genes. Induction of the SOS regulon was observed in dnaC2 cells at 38°C and 42°C. Flow cytometric analysis revealed that dnaC2 mutant cells at non-permissive temperature had completed the early stages of chromosome replication initiation.
Conclusion/Significance
We suggest that in dnaC2 cells the SOS response is triggered by persistent open-complex formation at oriC and/or by arrested forks that require DnaC for replication restart.
doi:10.1371/journal.pone.0002984
PMCID: PMC2500167  PMID: 18714349
11.  YhdJ, a Nonessential CcrM-Like DNA Methyltransferase of Escherichia coli and Salmonella enterica▿  
Journal of Bacteriology  2007;189(11):4325-4327.
The Caulobacter crescentus DNA adenine methyltransferase CcrM and its homologs in the α-Proteobacteria are essential for viability. CcrM is 34% identical to the yhdJ gene products of Escherichia coli and Salmonella enterica. This study provides evidence that the E. coli yhdJ gene encodes a DNA adenine methyltransferase. In contrast to an earlier report, however, we show that yhdJ is not an essential gene in either E. coli or S. enterica.
doi:10.1128/JB.01854-06
PMCID: PMC1913422  PMID: 17400740
12.  SURVEY AND SUMMARY: A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes 
Nucleic Acids Research  2003;31(7):1805-1812.
A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identified and provides a system for naming the putative genes found by sequence analysis of microbial genomes.
PMCID: PMC152790  PMID: 12654995
13.  Nitric Oxide-Induced Homologous Recombination in Escherichia coli Is Promoted by DNA Glycosylases 
Journal of Bacteriology  2002;184(13):3501-3507.
Nitric oxide (NO.) is involved in neurotransmission, inflammation, and many other biological processes. Exposure of cells to NO. leads to DNA damage, including formation of deaminated and oxidized bases. Apurinic/apyrimidinic (AP) endonuclease-deficient cells are sensitive to NO. toxicity, which indicates that base excision repair (BER) intermediates are being generated. Here, we show that AP endonuclease-deficient cells can be protected from NO. toxicity by inactivation of the uracil (Ung) or formamidopyrimidine (Fpg) DNA glycosylases but not by inactivation of a 3-methyladenine (AlkA) DNA glycosylase. These results suggest that Ung and Fpg remove nontoxic NO.-induced base damage to create BER intermediates that are toxic if they are not processed by AP endonucleases. Our next goal was to learn how Ung and Fpg affect susceptibility to homologous recombination. The RecBCD complex is critical for repair of double-strand breaks via homologous recombination. When both Ung and Fpg were inactivated in recBCD cells, survival was significantly enhanced. We infer that both Ung and Fpg create substrates for recombinational repair, which is consistent with the observation that disrupting ung and fpg suppressed NO.-induced recombination. Taken together, a picture emerges in which the action of DNA glycosylases on NO.-induced base damage results in the accumulation of BER intermediates, which in turn can induce homologous recombination. These studies shed light on the underlying mechanism of NO.-induced homologous recombination.
doi:10.1128/JB.184.13.3501-3507.2002
PMCID: PMC135131  PMID: 12057944
14.  Recombinational Repair Is Critical for Survival of Escherichia coli Exposed to Nitric Oxide 
Journal of Bacteriology  2001;183(1):131-138.
Nitric oxide (NO⋅) is critical to numerous biological processes, including signal transduction and macrophage-mediated immunity. In this study, we have explored the biological effects of NO⋅-induced DNA damage on Escherichia coli. The relative importance of base excision repair, nucleotide excision repair (NER), and recombinational repair in preventing NO⋅-induced toxicity was determined. E. coli strains lacking either NER or DNA glycosylases (including those that repair alkylation damage [alkA tag strain], oxidative damage [fpg nei nth strain], and deaminated cytosine [ung strain]) showed essentially wild-type levels of NO⋅ resistance. However, apyrimidinic/apurinic (AP) endonuclease-deficient cells (xth nfo strain) were very sensitive to killing by NO⋅, which indicates that normal processing of abasic sites is critical for defense against NO⋅. In addition, recA mutant cells were exquisitely sensitive to NO⋅-induced killing. Both SOS-deficient (lexA3) and Holliday junction resolvase-deficient (ruvC) cells were very sensitive to NO⋅, indicating that both SOS and recombinational repair play important roles in defense against NO⋅. Furthermore, strains specifically lacking double-strand end repair (recBCD strains) were very sensitive to NO⋅, which suggests that NO⋅ exposure leads to the formation of double-strand ends. One consequence of these double-strand ends is that NO⋅ induces homologous recombination at a genetically engineered substrate. Taken together, it is now clear that, in addition to the known point mutagenic effects of NO⋅, it is also important to consider recombination events among the spectrum of genetic changes that NO⋅ can induce. Furthermore, the importance of recombinational repair for cellular survival of NO⋅ exposure reveals a potential susceptibility factor for invading microbes.
doi:10.1128/JB.183.1.131-138.2001
PMCID: PMC94858  PMID: 11114909

Results 1-14 (14)