TABLE 1.
E. coli strains and plasmids used in this studya
| Strain or plasmid | Genotype or description |
|---|---|
| Strains | |
| MC4100 | Δ(argF-lac)U169 rpsL150 relA1 spoT1 araD139 flbB5301 deoC1 ptsF25 |
| JD17 | MC4100 ΔptsN::Kan |
| JD466 | MC4100 ΔycgO::Kan |
| JD509 | MC4100 ΔptsN ΔycgO::Kan |
| JD624 | MC4100 kup trkA |
| JD637 | JD624 ΔkdpA |
| JD656 | MC4100 ΔkdpA |
| JD657 | JD637 zha-203::Tn10 |
| JD658 | JD657 ΔptsN::Kan |
| JD660 | JD624 ΔptsN::Kan |
| JD662 | JD656 ΔptsN::Kan |
| JD693 | JD637 ΔycgO::Kan |
| JD694 | JD656 ΔycgO::Kan |
| JD696 | JD624 ΔycgO |
| JD710 | JD624 ΔycgO ΔptsN::Kan |
| JD714 | JD637 ΔycgO ΔptsN::Kan zha-203::Tn10 |
| JD723 | JD624 ycgOFL ΔKan |
| JD724 | JD723 ΔptsN::Kan |
| JD725 | JD656 ΔycgO ΔptsN::Kan |
| JD726 | MC4100 attλ::(Ptrc99A bla) |
| JD727 | MC4100 attλ::(PtrcycgO bla) |
| JD728 | JD624 attλ::(Ptrc99A bla) |
| JD729 | JD624 attλ::(PtrcycgO bla) |
| JD732 | JD624 attλ::(PtrcycgO bla::Kan) |
| JD734 | JD637 attλ::(Ptrc99A bla) |
| JD735 | JD637 attλ::(PtrcycgO bla) |
| GJ14949 | JD656 attλ::(Ptrc99A bla) |
| GJ14950 | JD656 attλ::(PtrcycgO bla) |
| Plasmids | |
| pHYD3025 | Derivative of plasmid pTrc99A, described in reference 41 |
| pHYD724 | pTrc99A in which kdpA′, which encodes N-terminal 135 amino acids of KdpA, is placed under expression control of Ptrc promoter, described in reference 43 |
| pHYD1852 | pHYD3025 bearing kup expressed from Ptrc promoter, present within EcoRI and HindIII sites |
| pHYD1858 | pTrc99A containing ycgO, present within NcoI and HindIII sites, and expressed from Ptrc promoter |
| pHYD5006 | pHYD3025 containing ptsN appended at its 3′ end with DNA sequence encoding hexahistidine, placed under expression control of Ptrc promoter, present within NdeI and HindIII sites |
| pHYD5007 | Derivative of pHYD5006 encoding PtsN bearing H73A amino acid substitution |
| pHYD5008 | Derivative of pHYD5006 encoding PtsN bearing H73D amino acid substitution |
| pHYD5009 | Derivative of pHYD5006 encoding PtsN bearing H73E amino acid substitution |
All strains listed are E. coli K-12 strains, and MC4100 was from our laboratory collection. The ΔptsN::Kan, ΔycgO::Kan, and ΔkdpA::Kan deletion insertion mutations were obtained from strains JW3171, JW5184, and JW0686, respectively, of the Keio collection (28) and introduced into appropriate strains by P1 transduction. Removal of the antibiotic cassette was performed as described previously (30), and such mutations are indicated in the table by “Δ.” trkA and kup represent the trkA405 and trkD1 alleles, respectively, which were obtained from strain TL1105A (13) and introduced into MC4100 by P1 transduction in multiple steps. zha-203::Tn10 was obtained from CAG12072 (33) and introduced into appropriate strains by P1 transduction. ycgOFL encodes YcgO bearing a 3× FLAG epitope attached to the C terminus of YcgO, and the attλ::(PtrcycgO bla), attλ::(Ptrc99A bla), and attλ::(PtrcycgO bla::Kan) chromosomal constructs were obtained by the plasmid-to-chromosome shuttling system (see the “Methods” section in the supplemental material for details). The ancestral plasmid pTrc99A is described in reference 34.