Article Figures & Data
Figures
- FIG. 1.
(A) Schematic representation of the trdX-lrp-ftsK region on the C. rodentium chromosome. Short arrows indicate the position of annealing of synthetic oligonucleotides. (B) β-Galactosidase assay performed with the E. coli strains CV975 (ilvIH::lacZ), CV1008 (ilvIH::lacZ lrp::Tn10), and AC13 (CV1008 carrying plasmid pAC12), indicated as lrp+, lrp−, and lrp−/pAC12, respectively. Cells were grown in minimal medium (white bars) and leucine-supplemented minimal medium (gray bars).
- FIG. 2.
β-Glucuronidase assay performed on C. rodentium strains AC49 (ATCC 51459, wild type carrying plasmid pAC47), AC52 (EM2, lrp null carrying plasmid pAC47), AC62 (ATCC 51459, wild type carrying plasmid pAC61), indicated as wild type/pAC47(gusA), lrp−/pAC47 (gusA), and wild type/pAC61 (gusA lrp), respectively. Cells were grown in minimal medium (white bars) and leucine-supplemented minimal medium (gray bars). The activity value obtained for strain AC49 grown in the absence of leucine was considered 100% activity. All values are the average of at least three independent experiments.
- FIG. 3.
Schematic representation of the fim region on the C. rodentium chromosome. Arrows indicate the transcription orientation; short arrows indicate the position of annealing of synthetic oligonucleotides. Enlarged is the fimS element in the ON orientation. Three Lrp boxes and two inverted repeats (IRL and IRR) are also indicated. The three Lrp boxes are indicated in the order 2-1-3 in homology to the E. coli model (19).
- FIG. 4.
Real-time PCR experiment performed to monitor fimAICDFGH expression in various growth conditions. Cells of the wild-type ATCC 51459 strain were grown in minimal medium (white bars), rich medium in aerated conditions (gray bars), and rich medium in static conditions (black bars) and collected during the exponential growth phase, at entry into stationary growth phase, or after 3 h of stationary growth phase. Total RNA was extracted, and cDNA was synthesized and used in the reactions with an ABI PRISM 7500 sequence detection system (PE Applied Biosystems). The fluorescence signal due to SYBR Green intercalation was monitored to quantify the double-stranded DNA product formed in each PCR cycle. The ΔΔCt method was used to calculate the relative amount of specific RNA present in each sample, and the transcriptional induction was estimated by comparison to values relative to the wild-type strain grown in minimal medium at exponential phase.
- FIG. 5.
Yeast agglutination experiments. We mixed 10 μl of 3% (wt/vol) Saccharomyces cerevisiae yeast cells on a glass slide with the same volume of three bacterial cultures of Citrobacter rodentium EM2 (A), wild-type (wt) (B), and EM3 (C) strains at an optical density of 595 nm.
Tables
- TABLE 1.
Synthetic oligonucleotides
Gene and Name Sequence (5′-3′)a Positions of annealingb lrp A GGGCCGGTCAGGTC −732/−719 B CGACACACGGACCTACG +427/+443 C TTAGCGCGTCTTAATAACC +477/+495 D AGCTGGGATCCGAGG +773/+787 E ACCAGAAGTGACGCATCC +210/+227 F GTAGGGAATTTACCGGC −505/−489 G GGTGAGAAAATGACGATTTG −373/−354 H GTGTTATCTGTGTGTCGC −252/−234 I GTCAGGCAGGAATAGGG −41/−25 L ATGGTAGATAGCAAGAAGCG +1/+20 N GCATGCCTTCTTGCTATCTACCAT +1/+18 O TGGATTGTAGGGAATTTACCGGC −512/−489 P CCGTTGCCTGACGGC −148/−133 Q GTGAGTAAACGTCGTTATCTTACC +1/+24 X GAAAATGACGATTTGACGCTGTTGGCAATGAATAACTGGgtgtaggctggagctgcttc +368/−329 Y TTGCTCTGTTTGACTTCTTCCATAACGACGTAGGTCCGcatatgaatatcctcctta +432/+469 Z CCGGTAAAGAAGTACAGGCTATGATGCTGGCCGCCCGGCATGGCgtgtaggctggagctgcttc +23/+66 W CGAATATTACGGTGGCCGAGATAATCCTGGATAAGCCGCGTcatatgaatatcctcctta +457/+497 c1 TTATACGCAAGGCGACAAGG (4) fim fimA-rt1 CGCTGACGCCACCTTCA +516/+532 fimA-rt2 GCCCTGAGTCACTCCCTGTCT +556/+576 fimB-rt1 CGGGCTACCGCTGGAGAT +396/+413 fimB-rt2 GTTTGCCAAAGCGAAACCA +441/+459 fimE-rt1 CGCCGCCTGAAAAATGG +187/+203 fimE-rt2 AACCGCTTCCCGCTCATC +232/+249 oNs AATAAAGAGGAAATATAAATCTGAACAAGTCA −419/−400 oNa AACCCCTCACAGGAAGCCAT −480/−449 sig70s TCCAGCGTAGAGTCCGAAATC +259/+279 sig70a TGCCCATTTCGCGCATAT +308/+325 A5 GCTGACGCCACCTTCAAAGT +517/+536 Cs CAGGAAATGACGGTG −12/+3 C4 GGTGATTGCCCGTGTTTTTTT +25/+45 Ds GCCGCCGGGAACCTATCGCG +219/+238 D1 CATTGAGGTAGATATCCACGCG +245/+267 D6 GGTGCAGCTCAGCGTCACCC +1583/+1602 Da GGCTGCCGCTCAGATAGA +1626/+1634 G5 TTGTCCTGAAGTTCGAGTGG −1/+21 G4 GATGAAATGGTTTCAATCCGG +318/+338 H3 CGACGTTCACTTGCGGTGTA +151/+170 - TABLE 2.
RT-PCR experimentsa
Oligonucleotide pairb Specific amplificationc B1-B2 + E1-E2 + B1-E2 − E1-C4 − A5-C4 + Cs-D1 + Ds-Da + D6-G5 + G4-H3 + a C. rodentium total RNA was used to produce cDNA as described in Materials and Methods. cDNA was then amplified with the oligonucleotide pairs here indicated.
↵ b> Oligonucleotides are described in Fig. 3 and Table 1.
↵ c For each oligonucleotide pair, a positive (chromosomal DNA as template) and two negative (RT and cDNA not added) control reactions were performed.
- TABLE 3.
Real-time PCR analysis of fim gene expression in wild-type and lrp null mutant strainsa
Gene(s) and medium Mean expression ± SD ATCC 51459 (wild type) EM2 (lrp) fimAICDFGH Min 1.00 ± 0.17 0.060 ± 0.014 Min + leu 2.20 ± 0.20 N.D. fimB Min 1.00 ± 0.20 0.90 ± 0.10 Min + leu 1.20 ± 0.10 N.D. fimE Min 1.00 ± 0.15 1.10 ± 0.26 Min + leu 1.60 ± 0.22 N.D. ↵ a Cells were grown in minimal medium (Min) or minimal medium plus leucine (Min + leu) and collected at the onset of stationary growth phase. Data are presented as arithmetic mean ± standard deviation. Data indicate relative levels of transcription compared to the wild-type value in minimal growth conditions for each individual transcriptional unit. N.D., not determined.
- TABLE 4.
Real-time PCR analysis of fimS orientation and expression of fimAICDFGHa
Strain Mean relative expression ± SD fimS-ON fimAICDFGH ATCC 51459 (wild type) 1.00 ± 0.25 1.0 ± 0.17 EM2 (lrp) 0.027 ± 0.016 0.06 ± 0.014 EM3 (fimE) 86.3 ± 14.3 50.3 ± 6.6 ↵ a Cells were grown in minimal medium and collected at the onset of stationary growth phase. Data are presented as arithmetic means ± standard deviations.
