E-Book 2nd Congress

  • Detection of the pathoadaptibility gene in Escherichia coli facilitating genetic adaptation
  • Elham Talebi,1 Keihan Kookli,2 Rabee Movagharnia,3 Alireza Yahyaee,4 Danial Jafari,5,*
    1. Department of Genetics, Faculty of Advanced Science and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran
    2. International Campus, Iran University of Medical Sciences, Tehran, Iran
    3. Department of Genetics and Biotechnology, School of Biological Sciences, Varamin-Pishva Branch, Islamic Azad University, Varamin, Iran
    4. Qazvin university of medical science
    5. MD, Medicine faculty of Islamic Azad University of Shahrood, Iran


  • Introduction: Escherichia coli is considered as a facultative anaerobe (that creates ATP through aerobic respiration when oxygen is accessible, but it can switch to anaerobic respiration or fermentation when oxygen is absent). The cells of this bacterium have characteristically rod form, (2.0 μm long and 0.25–1.0 μm diameter), having a cell volume of 0.6–0.7 μm3. Escherichia coli is Gram-negative since its cell wall contains, in addition to the cytoplasmic membrane, a thin layer of peptidoglycan and an outer membrane. Strains that own flagella are motile. Escherichia coli own the capability to laterally transfer DNA through bacterial transformation, transduction, and conjugation, which allows the foreign DNA to spread over the bacterial population. The transduction process is mediated via bacteriophages. Escherichia coli strain IHE3034 is a neonatal meningitis-associated K1 pathovariant. The K1 capsule is very similar to the capsule formed by the Group B Neisseria meningitidis strains. The ycgG (pdeG) gene is found among most of the genomes of E. coli strains. It codes for an EAL phosphodiesterase that contains a membrane-binding domain. In the genome of IHE3034, the ycgG gene is evolved to a new allelic variant, i.e. ycgG2 (pdeG2) that only encodes the EAL domain of the protein. Escherichia coli K1 IHE3034 is a neonatal meningitis isolate that belongs to the group of extra-intestinal pathogenic strains. The focus of this study is to delete ycgG to comprehend the role of modified ycgG in E. coli IHE3034 in terms of biofilm formation, motility, and pathogenicity.
  • Methods: PCR-amplification of antibiotic resistance maker carried on a plasmid for one-step inactivation of chromosomal genes using PCR products. Recombination between H1 & H2 on the PCR fragment and H1 & H2 on the chromosome was done. PCR amplification for deletion fragment using pKD3 as a template was carried out. To Electro-competent cell preparation, the IHE3034 strain carrying pSIM6 plasmid was grown at 30˚C, overnight. For activation of the red system in pSIM6 plasmid, bacterial culture with OD = 0.5 was incubated at 42˚C for 30 min. Centrifugation was done at 2700 rmp. Pellet was washed with 20% glycerol 3x. To check if transductants are MG1655ΔycgG::aph, we used blue-white screening Xgal-IPTG plate by knowing JW5174 chromosomal markers: Δ(araD-araB)567, ΔlacZ4787(::rmB-3), λ-, ΔycgG757::aph, rph-1, Δ(rhaDrhaB)568, hsdR514. Two mutant candidates were selected on a Kanamycin-containing plate and verified by sequencing.
  • Results: YcgG2 regulated the IHE3034 virulence features. The ycgG2 deletion mutant displayed lower expression of the S-fimbriae, consistent with the immunoblotting against SfaA and the SEM of the cells, in comparison with the wild type. The agglutination test confirmed the agglutination of the K1-encapsulated mutant cells. The detection of YcgG2 expression was accomplished in a medium with high osmolarity, which also linked YcgG2 to adaptation to osmotic stress. IHE3034ΔycgG2 (NMEC) is less fimbriated as compared to IHE3034. The ycgG2 mutant cells make more K1 capsules shown by anti-Grp. B meningococcal agglutination assay. Protein expression levels of YcgG2 and its effector factors. NMEC produces more K1 capsules indicated by agglutination assay.
  • Conclusion: we suggest phenotypic assays (e.g. acid stress), a comparison of growth rate between wild-type and mutants at 37, 42, and 16˚C, and checking for YcgG protein levels in wild-type and mutants in different conditions for future plans.
  • Keywords: Escherichia coli, Neonatal Meningitis, Pathoadaptibility, C-di-GMP, Genetic Adaptation