Article Figures & Data
Figures
- FIG. 1.
Repetitive sequence in phnE of E. coli. The figure shows part of the phn gene cluster in E. coli and focuses on phnE and the location and sequences of a direct triple repeat in the K-12 strain and a direct double repeat in the B strain, where A corresponds to the octamer 5′-CGCTGGCG-3′ and B corresponds to the octamer 5′-TGCTGGCG-3′. The relative positions of pairs of primer used in this work to amplify two different segments of phnE of E. coli K-12 are shown under the gene organization. Amplicon 1 was used in the study in the AFLP analysis, and amplicon 2 was used for gene sequencing.
- FIG. 2.
AFLP analysis of phnE in E. coli. Primer pair 1 (Fig. 1) was used to amplify a small fragment containing the octameric repetitive element in phnE from populations of two strains of E. coli grown to log phase in LB. PCR products were analyzed as described in Materials and Methods. Shown are fragments detected as peaks in the AFLP analysis. Tracks 1 and 5, PCR fragments produced from cultures of K-12 strain MC4100 and B strain BL21(DE3), respectively; tracks 2 to 4, artificial mixtures of MC4100 and BL21(DE3), each grown to an optical density at 600 nm of 0.7 and mixed prior to extraction of template and amplification in the ratios 9:1, 6:4, and 2:8; track M, 200-bp marker peak from the 50-bp standard ladder set used to determine fragment lengths.
- FIG. 3.
AFLP analysis of phnE in cultures of E. coli K-12 MC4100 growing with different phosphonates. Cultures of K-12 MC4100 were grown in MNM with different phosphonates as sole P sources, which had been inoculated from Phn+ variants isolated and purified on MNM-agarose containing the respective phosphonate. PCR products were analyzed as described in Materials and Methods. Shown are fragments detected as peaks in the AFLP analysis. Tracks correspond to the phosphonates supporting growth as follows: 2, MePn, log phase; 3, MePn, stationary phase; 4, EPn, log phase; 5, AMPn, log phase; 6, 2-AEPn, log phase; 7, 3-APPn, log phase; M, 200-bp marker peak from the 50-bp standard ladder set used to determine fragment lengths.
- FIG. 4.
AFLP analysis of phnE in cultures of E. coli K-12 MC4100 growing with different phosphonates. Shown is AFLP analysis of phnE at the stationary phase of successive subcultures of a Phn+ variant of E. coli K-12 MC4100 selected and purified on MNM-agarose. The inoculum level used for each round of subculture was 0.03%. The tracks show the fragments produced after amplification performed at the end of each subculture in the following succession of media: 1, MNM with MePn as the sole added P source; 2 to 4, LB. Tracks M, 200-bp marker peak from the 50-bp standard ladder set used to determine fragment lengths.
- FIG. 5.
Variation in the sequences of phnE genes in various isolates of E. coli. (Top) Sequences determined in this work for that part of phnE containing the octameric repetitive element for the following strains and conditions: MC4100/Pi, MC4100 grown in MNM with Pi as the sole added P source; MC4100/MePn, a Phn+ variant isolated and purified on MNM-agarose with MePn as the sole P source; MC4100/MePn→LB, revertant obtained after successive subculture of a Phn+ variant in LB. Asterisks, positions of the direct repeat sequences. (Bottom) Sequences published for four strains of E. coli: K-12 and B (14), the enterohemorrhagic strain 0157:H7 (9, 16), and the uropathogenic strain CFT073 (22).
Tables
- TABLE 1.
E. coli strains used
Strain Genotype Source or reference AB1157 F−thi-1 hisG4 Δ(gpt-proA)62 argE3 thr-1 leuB6 kdgK51 rfbD1 ara-14 lacY-1 galK2 xyl-5 mtl-1 tsx-33 supE44 rpsL-31 Rac− λ− 1 BL21 (DE3) F−ompT hsdSB (rB mB) dcm gal λ(DE3) 19 CD4 Hfr metD88 proA3 Δ(lacI-Y)6 tsx-76 λ−relAl malA36(λr) metB1 7 DH5α φ80dlacZΔM15 recA1 endA1 gyrA96 thi-1 hsdR17 (rk− mk+) supE44 relA1 deoR Δ(lacZYA-argF)U169 KA796 ara thi Δpro lac 7 MC4100 araD139 Δ(argF-leu)169 LAM− e14−FlhD5301 fruA25 relA1 rpsL150 RbsR22 deoC1 4 NR9458 KA796 mutD5 zaf13::Tn10 8 NR9807 CD4 dnaQ49 8 STL1671 AB1157 sbcB15 Δ(slr-recA)304 3 STL2172 AB1157 mutS201::Tn5 Δ(slr-recA)304 13 STL2314 AB1157 dnaQ Δ(slr-recA)304 17 NWA3 Wild-type isolate from sewerage sludge North West Water UK NWA4 Wild-type isolate from sewerage sludge North West Water UK NWA5 Wild-type isolate from sewerage sludge North West Water UK NWD7 Wild-type isolate from sewerage sludge North West Water UK NW23341 Wild-type isolate from raw water North West Water UK NW211585 Wild-type isolate from raw water North West Water UK ST1 Wild-type isolate from water Severn Trent Water UK ST8 Wild-type isolate from water Severn Trent Water UK ST11 Wild-type isolate from water Severn Trent Water UK ST15 Wild-type isolate from water Severn Trent Water UK
