All E. coli strains used in this study are derivatives of E. coli K-12 MG1655 and are listed in Table 2 . Plasmids were generally introduced into strains by TSS transformation (Chung and Miller, 1988 (link)). The ΔrybC∷kan, ΔgadX∷kan, ΔgadW∷kan and ΔgadXYW∷kan strains were generated by PCR amplification of the kan cassette of strain CRB316 (Ranquet and Gottesman, 2007 (link)) with the kan-for and kan-rev oligos listed in Table 3 followed by lambda Red recombinase-mediated gene replacement. Marked mutations were obtained and moved into the desired strain background using bacteriophage P1 transduction (Silhavy et al., 1984 ). Oligonucleotides used as probes and for polymerase chain reaction are described in Table 3 . For cloning procedures, PCR amplification was carried out using the Expand High Fidelity PCR system (Roche) and DH5a was used as the recipient strain.
Strain PM1004 was created by first amplifying the cat-sacB cassette from strain NC397 (Svenningsen et al., 2005 (link)) using primers PBAD-cat-For and lacZ-sacB-rev. The PCR product was then recombined in strain PM1002 using mini λ mediated recombination as previously described (Court et al., 2003 (link)). The resulting strain, PM1004, was lacI’∷kan-PBAD-cat-sacB-lacZ. Strain PM1205 was constructed by amplifying the PBAD-cat-sacB-lacZ cassette using oligonucleotides lacI-PBAD-for and lacZ-sacB-rev and strain PM1004 genomic DNA as a template. The resulting PCR DNA fragment was recombined as previously described (Court et al., 2003 (link)) in the chromosome of strain PM1203, giving rise to strain PM1205.
The rybC (-245)-lacZ and PBAD-(-89) dpiB-lacZ translational fusion were constructed as follows. A DNA fragment corresponding to nts -245 to +10 respective to the transcription start site of rybC was amplified using primers EcoRI-rybC(-245)-for and BamHI-rybC(-245)-rev. The PCR product was subsequently cloned in between the EcoRI and BamHI sites of the pRS415 plasmid (Simons et al., 1987 (link)), giving rise to pRSRybC. The PBAD-(-89) dpiB-lacZ translational fusion (strain PM1011) was obtained by first amplifying the PBAD promoter and a sequence corresponding to nts −89 to +10 respective to the ATG translation start codon of dpiB, using primers 5’PBAD and PBAD-dpiB(-89)-rev, and dpiB(-89)-for and SmaI-dpiB(-89)-rev, respectively. The two PCR fragments were then joined by an overlap extension PCR and the resulting PCR product was subsequently cloned in frame with lacZ between the EcoRI and SmaI sites of the pRS414 plasmid (Simons et al., 1987 (link)), resulting in pRSdpiB. The pRSRybC and pRSdpiB plasmids were then crossed with λRS468 bacteriophage and monolysogens were constructed in strain PM1001 as previously described (Simons et al., 1987 (link)), giving rise to strains PM1106 and PM1011, respectively.
The rybC (-101)-lacZ (PM1151) and rybC (-45)-lacZ (PM1152) lacZ fusions were constructed as follows. DNA fragments corresponding to nts -101 to +10 and to nts -45 to +10 respective to the transcription start site of rybC were amplified by PCR using oligonucleotides rybC(-101)-for or rybC(-45)-for and deeplac, and strain PM1106 as template. The PCR products were subsequently recombined in strain PM1004 as previously described (Court et al., 2003 (link)), resulting in strain PM1151 and PM1152, respectively.
The pRybC and pGadY plasmids were constructed by first PCR amplifying rybC and gadY from strain MG1655 using AatII-rybC-for and EcoRI-rybC-rev primers and AatII-gadY-for and EcoRI-gadY-rev primers, respectively. The PCR product was subsequently cloned into the pBR-plac vector digested with AatII and EcoRI. The C55G G56C G57C site directed mutants of pRybC was constructed using the Quickchange II site directed mutagenesis kit (Stratagene) following the manufacturers instructions, with pRybC as a template and primers QC-rybc-mut1-for and QC-rybc-mut1-rev.
Strain PM1004 was created by first amplifying the cat-sacB cassette from strain NC397 (Svenningsen et al., 2005 (link)) using primers PBAD-cat-For and lacZ-sacB-rev. The PCR product was then recombined in strain PM1002 using mini λ mediated recombination as previously described (Court et al., 2003 (link)). The resulting strain, PM1004, was lacI’∷kan-PBAD-cat-sacB-lacZ. Strain PM1205 was constructed by amplifying the PBAD-cat-sacB-lacZ cassette using oligonucleotides lacI-PBAD-for and lacZ-sacB-rev and strain PM1004 genomic DNA as a template. The resulting PCR DNA fragment was recombined as previously described (Court et al., 2003 (link)) in the chromosome of strain PM1203, giving rise to strain PM1205.
The rybC (-245)-lacZ and PBAD-(-89) dpiB-lacZ translational fusion were constructed as follows. A DNA fragment corresponding to nts -245 to +10 respective to the transcription start site of rybC was amplified using primers EcoRI-rybC(-245)-for and BamHI-rybC(-245)-rev. The PCR product was subsequently cloned in between the EcoRI and BamHI sites of the pRS415 plasmid (Simons et al., 1987 (link)), giving rise to pRSRybC. The PBAD-(-89) dpiB-lacZ translational fusion (strain PM1011) was obtained by first amplifying the PBAD promoter and a sequence corresponding to nts −89 to +10 respective to the ATG translation start codon of dpiB, using primers 5’PBAD and PBAD-dpiB(-89)-rev, and dpiB(-89)-for and SmaI-dpiB(-89)-rev, respectively. The two PCR fragments were then joined by an overlap extension PCR and the resulting PCR product was subsequently cloned in frame with lacZ between the EcoRI and SmaI sites of the pRS414 plasmid (Simons et al., 1987 (link)), resulting in pRSdpiB. The pRSRybC and pRSdpiB plasmids were then crossed with λRS468 bacteriophage and monolysogens were constructed in strain PM1001 as previously described (Simons et al., 1987 (link)), giving rise to strains PM1106 and PM1011, respectively.
The rybC (-101)-lacZ (PM1151) and rybC (-45)-lacZ (PM1152) lacZ fusions were constructed as follows. DNA fragments corresponding to nts -101 to +10 and to nts -45 to +10 respective to the transcription start site of rybC were amplified by PCR using oligonucleotides rybC(-101)-for or rybC(-45)-for and deeplac, and strain PM1106 as template. The PCR products were subsequently recombined in strain PM1004 as previously described (Court et al., 2003 (link)), resulting in strain PM1151 and PM1152, respectively.
The pRybC and pGadY plasmids were constructed by first PCR amplifying rybC and gadY from strain MG1655 using AatII-rybC-for and EcoRI-rybC-rev primers and AatII-gadY-for and EcoRI-gadY-rev primers, respectively. The PCR product was subsequently cloned into the pBR-plac vector digested with AatII and EcoRI. The C55G G56C G57C site directed mutants of pRybC was constructed using the Quickchange II site directed mutagenesis kit (Stratagene) following the manufacturers instructions, with pRybC as a template and primers QC-rybc-mut1-for and QC-rybc-mut1-rev.
