DOI: 10.1128/IAI.70.7.3404-3412.2002
Article Figures & Data
Figures
- FIG. 1.
(a) RT-PCR was performed to detect the presence of cat mRNA in ivi strains i673, i675, i681, i682, i683, i684, i686, and i689 (lanes 1 to 8, respectively); RNA was isolated from infected mice livers. Lane 9, RNA isolated from the liver of a mouse challenged with the wild-type E. coli strain i484 carrying pKK232-8; lane 10, RNA from the liver of an uninfected mouse; lane M, molecular size marker (numbers indicate base pairs). (b) RT-PCR was performed with RNA from in vitro-grown E. coli strain i484 containing pKK232-8 (lane 1), RNA from in vitro-grown ivi clones (lane 2), and RNA from the livers of mice challenged with ivi clones i673, i681, and i689 (lanes 3, 5, and 7, respectively). In addition, the RNAs from the three ivi clones were treated with RNase prior to RT-PCR (lanes 4, 6, and 8). Lane M, molecular size marker.
Tables
- TABLE 1.
Bacterial strains and plasmids
Strain or plasmid Relevant characteristic(s) Reference or source E. coli strains i484 O25:H autoaglutinating; human isolate This study i659 Derivative of i484; Smr This study i665 E. coli strain HB101 containing pKK232-8 This study i668 E. coli i659 containing pKK232-8 This study i673 In vivo-induced clone This study i675 In vivo-induced clone This study i681 In vivo-induced clone This study i682 In vivo-induced clone This study i683 In vivo-induced clone This study i684 In vivo-induced clone This study i686 In vivo-induced clone This study i689 In vivo-induced clone This study HB101 Δ(gpt-proA)62 leuB6 thi-1 lacY1 recA rpsL20 (Strr) ara-14 galK2 xyl-5 mtl-1 supE44 Δ(mcrBC-hsdRMS-mrr) 3 SM10 λpir thi-1 thr-1 leuB6 supE44 tonA21 lacY1 rec::RP4-2-Tc::Mu Kmr 28 Plasmids pKK232-8 Ampr Cms; ColE1 origin 4 pGP704 Ampr; R6K origin 28 - TABLE 2.
Primers used for site-directed mutagenesis
Clone Gene or ORF Mutagenic primer Δivi814 bglG GCCGCAAAGCGGTAGATCTGGCAAGTTATGACGGAG Wild type bglG GCCGCAAAGCGGTAGAGGGCAAGTTATGACGGAG Δivi814 o761 GGATCAAGTTATCTAGAAAAAGGCGTGTTTTCAAGCCAGCAATAACGAAATAATTGCCG Wild type o761 GGATCAAGTTATCTGAAAAAGGCGTGTTTCTCGCCAGCAATAACGAAATAATTGCCG Δivi815 ycjF GCAAAAGCAATATTCAGCAAGGTACCAGCTTAAACAGGC Wild type ycjF GCAAAAGCAATATTCAGCAATACCAGCTTAAACAGGC Δivi816 clpB CCCGGATAAATTCACGATCGCGTTTAATAAAAGAATGGATCC Wild type clpB CCCGGATAAATTCACGTTCGCGAATAAAAGAATGGATCC Δivi817 —a GCCGTAGCGATTTAAAGATGCAATAACGGCG Wild type — GCCGTAGCGATTTAGATGCAATAACGGCG Δivi818 ORF V CATCACCAGGATGGCTTTAAAGCAATAAGAGTTGGCC Wild type ORF V CATCACCAGGATGGCTTTGAGCAATAAGAGTTGGCC Δivi819 aroA CAGTCTTATCAGTCCCCGGGTAGCTATTTGG Wild type aroA CAGTCTTATCAGTCTCCGGGTACTTATTTTGG ↵ a —, no similarity to sequences in GenBank.
- TABLE 3.
List of E. coli genes identified to be in vivo induced
Class Gene or ORF Map position Product or function Amino acid metabolism dadX 26.688 Alanine racemase aroA 20.651 Chorismate biosynthesis argD 75.155 Acetylomithine aminotransferase metK 66.492 S-Adenosylmethionine synthetase RNA and protein biosynthesis, degradation, and modification me 24.582 mRNA turnover, maturation of 5S RNA rrsB 89.762 16S rRNA tufB 89.962 Elongation factor Tu fdhA 80.994 Selenocysteine synthesis msrA 95.687 Peptide methionine sulfoxide reductase hslV 88.794 Heat shock protein VU, proteasome-related peptidease subunit clpA 19.885 ATP-binding component of serine protease prpB 7.499 Phosphoprotein phosphatase DNA replication, repair, restriction, and modification dinD 82.242 DNA damage-inducible protein dinG 17.940 Probably ATP-dependent helicase parE 68.363 DNA topoisomerase IV subunit B uvrD 86.126 DNA-dependent ATPase I and helicase II uvrB 17.519 DNA repair; excision nuclease subunit B. dnaA 83.634 DNA biosynthesis, initiation of chromosome replication, can be transcription regulator recG 82.403 DNA helicase, resolution of Holliday junction, branch migration IS911 97.134 New member of the IS3 group of insertion sequences trs5 5.891 Transposon-related function Cell division ftsX 77.570 Cell division membrane protein Carbohydrate metabolism pgm 15.364 Phosphoglucomutase bgl 83.8 β-Glucoside metabolism malQ 76.427 Maltose degrading enzyme/amylomaltase Surface structure csgA 23.79 Fibronectin and Congo red binding curli pili of E. coli Peptidoglycan biosynthesis and metabolizing enzymes slt 99.764 Soluble lytic murein transglycosylase bacA 68.998 Bactracin resistance, possibly phosphorylates undecaprenol Transport system ugpB 77.354 sn-Glycerol 3-phosphate transport system, periplasmic binding protein fepE 13.310 Ferric enterobactin (enterochelin) transport iucA Aerobactin biosynthesis dcuA 94.047 Membrane transport of aspartase trkH 86.884 Potassium uptake. gltS 82.451 Sodium/glutamate symport carrier protein Outer membrane proteins nlpA 82.704 Lipoprotein-28 pldA 86.275 Outer membrane phospholipase A Anaerobic respiration narW 33.08 Appears to be essintial for nitrate reductase activity, probably by promoting the correct association of the alpha and beta subunits hybE 67.648 Energy metabolism, carbon: anaerobic respiration. Drug or analog sensitivity Macromolecule biosynthesis and degradation tehA 32.303 Tellurite resistance menG 88.731 Menaquinone biosynthesis protein menA 88.743 Menaquinone biosynthesis protein birA 89.9 Biotin-[acetyl-coenzyme A-carboxylase] ligase fadB 86.79 Fatty acid oxidation pathway tesB 10.207 Fatty acid and phosphatidic acid biosynthesis gloA 37.202 Methylglyoxal metabolism pntB 36.062 Beta subunit of pyridine nucleotide transhydrogenase appA 22.414 Phosphoanhydride phosphorylase mog 0.200 Required for efficient incorporation of molybdate into molybdoproteins udk 46.136 Uridine kinase Energy metabolism aceE 2.651 Pyruvate dehydrogenase glxB2 11.575 Glyoxylate-induced protein acn 28.752 Aconitate hydratase Cellular repressor of the SOS response dinR Cellular repressor of the SOS response in Bacillus subtilis Unclassified yejF 49.024 Putative ATP-binding component of a transport system yehU 47.658 Putative 2 component sensor protein hslU 88.765 Putative heat shock protein HslVU, ATPase-subunit, homologous to chaperones yihK 87.428 Putative GTP-binding factor b1284 28.919 Putative DEOR-type transcriptional regulator o169 9.57 Putative regulator of flagellar biosynthesis, transcription initiation factor? o808 35.70 Putative oxidoreductase f344 39.40 Putative DNA helicase II o196 95.651 Putative transport protein o275 8.32 Probable ABC transporter permease protein f278 21.41 Probable ABC transporter permease protein o251 41.83 Hypothetical ABC transporter o127 90.77 Putative arginine biosynthesis f216 23.749 Putative regulator hycF 61.265 Probable iron-sulfur protein of hydrogenase 3 b1806 40.7 Putative outer membrane protein yhjE 79.16 Putative transport protein f290 87.667 Putative aldose-1-epimerase Unknown function ygaF 60.095 Hypothetical protein pfs 3.846 Hypothetical protein yddE 33.912 Hypothetical protein yfhH 58.123 Hypothetical protein ycaJ 20.202 Hypothetical protein encoded in serS 5′ region f178 38.94 Hypothetical protein 375 87.043 Hypothetical RNA methyltransferase o425 87.551 Hypothetical protein ycjF 29.823 Hypothetical protein o423 78.000 Hypothetical protein b2434 54.96 Hypothetical protein b2362 53.3 Hypothetical protein b1839 41.4 Hypothetical protein o416 85.66 Hypothetical protein encoded in rffE-rffT intergenic region - TABLE 5.
Comparisons of mutant and wild-type growth in the mouse competition model of septicemia
Mutant Gene or ORF Function Competition index (CFU of wild type/ CFU of mutant) Δivi813 bglG β-Glucoside-metabolizing enzyme 2.1a Δivi814 o761 Unknown 1.8a Δivi815 ycjF Hypothetical protein 81.4a Δivi816 clpB ATP-dependent protease subunit 2.7 Δivi817 No significant similarity to sequences in GenBank 28.5a Δivi818 ORF V Similar to serine/threonine protein phosphatase 7.6a Δivi819 aroA Aromatic amino acid biosynthesis 2.7 ↵ a Significant (P > 0.05) based on Wilcoxon signed rank test.
