E. coli plasmids and phages.
The most relevant plasmids, phages, and strains used in this study are listed in Table and depicted in Fig. .
E. coli strains, plasmids, and phages used in this study
FIG. 1. Genetic constructs. Shown are the E. coli mre (A) and mrd (B) loci, chromosomal deletion-replacements, and inserts present on plasmids and phages. Portions of the chromosome that were replaced with an aph cassette, or an frt scar sequence remaining after (more ...)
Plasmids pBR322 (11
), pZAQ (88
), pJF118EH (33
), pMEL1 (85
), pMLB1113 (24
), pCX19 (87
), pCP20 (16
), pDB346 (66
), pDR107 (67
), pDR120 (37
), pKD13 (23
), pCH151 (8
), pCH157 (51
), and pTB59 (6
) were described previously.
Unless indicated otherwise, MG1655 chromosomal DNA was used as a template in amplification reactions. Sites of interest (e.g., relevant restriction sites) are underlined in primer sequences.
To construct pCH221 (Plac::gfpmut2-T7-mrdB), mrdB (rodA) was amplified using primers 5′-TATAGAATTCATATGACGGATAATCCGAATAAAAAAACATTCTGG-3′ and 5′-CATTGTCGACTTACACGCTTTTCGACAACATTTTCC-3′. The product was treated with EcoRI and SalI, and the 1,127-bp fragment was used to replace the 13-bp EcoRI-SalI fragment of pDR107c, yielding pCH218 (PT7::gfpmut2-T7-mrdB). The 1,970-bp BglII-HindIII fragment of pCH218 was next used to replace the 20-bp BamHI-HindIII fragment of pMLB1113, resulting in pCH221.
For pCH222 [Plac::mrdB-gfpmut2], mrdB was amplified using primers 5′-TATAGAATTCATATGACGGATAATCCGAATAAAAAAACATTCTGG-3′ and 5′-GCACCTCGAGCACGCTTTTCGACAACATTTTCC-3′. The product was treated with NdeI and XhoI, and the 1,119-bp fragment was used to replace the 77-bp NdeI-XhoI fragment of pET21a, yielding pCH219 (PT7::mrdB-His6). The 1,152-bp XbaI-XhoI fragment of pCH219 was used to replace the 1,025-bp XbaI-XhoI fragment of pCH151 (Plac::zipA-gfpmut2), resulting in pCH222.
Construction of plasmid pCH235 (Plac::mreD-LE) involved several steps. The annealed product of oligonucleotides 5′-TCGAGTAAGTCGACACGGTACCA-3′ (sense) and 5′-AGCTTGGTACCGTGTCGACTTAC-3′ (antisense) was used to replace the 122-bp XhoI-HindIII fragment of pCH157 (Plac::gfpmut2-T7-minD minE-His6). This resulted in pCH181 (Plac::gfpmut2-T7-minD minE-LE), in which the His6 tag sequence in pCH157 was replaced with an XhoI site, encoding the dipeptide LE, followed by the TAA stop codon. The mreD gene was amplified using primers 5′-TATAGAATTCATATGGCGAGCTATCGTAGCCAGGGACGCTG-3′ and 5′-CGTTCTCGAGTTGCACTGCAAACTGCTGACGGAC-3′ and digested with EcoRI and XhoI. The 494-bp fragment was used to replace the 34-bp EcoRI-XhoI fragment of pDR107c, resulting in pCH217 (PT7::gfpmut2-T7-mreD-His6). Circularization of the 5,837-bp NdeI fragment of pCH217 yielded pCH223 (PT7::mreD-His6). pCH235 was finally obtained by replacing the 1,859-bp XbaI-XhoI fragment of pCH181 with the 512-bp XbaI-XhoI mreD fragment of pCH223.
Plasmid pCH244 (Plac::mreB mreC mreD yhdE) was obtained after several steps. The 3,359-bp ApoI fragment of pMEL1 was inserted in the EcoRI site of pDR107a, yielding pDB364 [PT7::gfpmut2-T7-mreB(5-347) mreC mreD yhdE]. The 4,223-bp BglII-HindIII fragment of pDB364 was next used to replace the 20-bp BamHI-HindIII fragment of pMLB1113, resulting in pDB366 [Plac::gfpmut2-T7-mreB(5-347) mreC mreD yhdE]. The mreB gene was amplified using primers 5′-CGACTCTAGACAGCTTTCAGGATTATCCCTTAGTATG-3′ and 5′-GCAAAAGCTTACTCTTCGCTGAACAGGTCGCC-3′. The product was treated with XbaI and HindIII, and the 1,072-bp fragment ligated to the 7,639-bp XbaI-HindIII fragment of pCH151 (Plac::zipA-gfpmut2), generating pCH214 (Plac::mreB). Finally, replacement of the 534-bp KpnI-HindIII fragment of pCH214 with the 2,879-bp KpnI-HindIII fragment of pDB366 resulted in pCH244.
To obtain pCH268 (Plac::gfpmut2-T7-zapA), zapA was amplified using primers 5′-GAAGGATCCATGTCTGCACAACCCGTC-3′ and 5′-CGAGTCGACTCATTCAAAGTTTTGGTTAG-3′. The product was treated with BamHI and SalI, and the 336-bp fragment was used to replace the 1,164-bp BamHI-SalI fragment of pDR120 (Plac::gfpmut2-T7-ftsZ).
For plasmid pFB112 (tet sdiA), the 1,312-bp EcoRI-PstI fragment of pCX19 was ligated to the 3,615-bp EcoRI-PstI fragment of pBR322.
For pFB118 (Plac::mreB), the 2,696-bp ClaI fragment of pCH244 was deleted.
To obtain pFB120 (Plac::mreC-LE), mreC was amplified using primers 5′-CTAGTCTAGAATACGAGAATACGCATAACTT-3′ and 5′-CGTTCTCGAGTTGCCCTCCCGGCGCACGCGCAGGC-3′. The product was treated with XbaI and XhoI, and the 1,128-bp fragment was used to replace the 512-bp XbaI-XhoI fragment of pCH235.
For pFB121 (Plac::mreC mreD-LE), an mreCD fragment was amplified using primers 5′-CTAGTCTAGAATACGAGAATACGCATAACTT-3′ and 5′-CGTTCTCGAGTTGCACTGCAAACTGCTGACGGAC-3′. The product was treated with XbaI and XhoI, and the 1,616-bp fragment was used to replace the 512-bp XbaI-XhoI fragment of pCH235.
Plasmid pFB124 [cI857(Ts) PλR::mreC, mreD-LE] was obtained by replacing the 1,196-bp XbaI-SalI fragment of pDB346 [cI857(Ts) PλR::ftsZ] with the 1,625-bp XbaI-SalI fragment of pFB121.
In turn, pFB128 [cI857(Ts) PλR::mreD-LE] was created by replacing the 1,625-bp XbaI-SalI fragment of pFB124 with the 521-bp XbaI-SalI fragment of pCH235.
Plasmid pFB142 (Plac::mreB, mreC-LE) was created in two steps. The 1,271-bp XbaI-XhoI fragment of pCH217 was used to replace the 1,859-bp XbaI-XhoI fragment of pCH181, yielding pCH233 (Plac::gfpmut2-T7-mreD-LE). An mreBC fragment was amplified using primers 5′-CGACTCTAGACAGCTTTCAGGATTATCCCTTAGTATG-3′ and 5′-CGTTCTCGAGTTGCCCTCCCGGCGCACGCGCAGGC-3′. The product was treated with XbaI and XhoI, and the 2,240-bp fragment was used to replace the 1,271-bp XbaI-XhoI fragment of pCH233.
For pFB149 (Plac::mreB mreC mreD-LE), the 1,033-bp BamHI-SalI fragment of pCH244 was replaced with the 359-bp BamHI-SalI fragment of pFB124.
To create pFB174 (PBAD::mreB mreC mreD-LE), the 1,451-bp XbaI-HindIII fragment of pLL116 (a pBAD33 derivative that will be described elsewhere) was replaced with the 2,743-bp XbaI-HindIII fragment of pFB149.
For pFB185 (Plac::mrdB), the 508-bp NsiI-HindIII fragment of pCH221 was used to replace the 1,252-bp NsiI-HindIII fragment of pCH222.
To construct pFB190 (Plac::mrdA), pTB59 (Plac::mrdAB) was used as a template to amplify mrdA (pbpA) with primers 5′-CTCTGAATTCCCGTGAGTGATAAGGGAGCTTTGAGTAG-3′ and 5′-GCCAAGCTTGGTCGACTTAATGGTCCTCCGCTGCGGC-3′. The product was treated with EcoRI and HindIII, and the 1,954-bp fragment was used to replace the 3,084-bp EcoRI-HindIII fragment of pTB59.
For pFB194 [cI857(Ts) PλR::mrdB], the 1,155-bp XbaI-SalI fragment of pFB185 was used to replace the 1,625-bp XbaI-SalI fragment of pFB124.
Plasmid pTB182 (ftsQAZ) was obtained in several steps. The HindIII site within ftsA on pZAQ was removed by the QuikChange procedure (Stratagene), using the mutagenic primers 5′-CAGTTGCAGGAAAAGCTCCGCCAACAAGGGG-3′ and its reverse complement, resulting in a silent change (underlined) of FtsA codon 319 (Leu). The resulting plasmid (pTB178) was next mutagenized using primers 5′-TTATGAGGCCGACGATCTAGACGGCCTCAGGCGACAG-3′ and its reverse complement, creating an XbaI site in between ftsA and ftsZ. The 4,377-bp PstI-HindIII fragment of the resulting plasmid (pTB179) was then used to replace the 12-bp PstI-HindIII fragment of pGB2, yielding pTB182. The direction of ftsQAZ transcription from this plasmid is opposite that of the aadA gene.
For pTB188 (PλR::ftsZ), pDB346 [cI857(Ts) PλR::ftsZ] was used as a template in a PCR with 5′-CGTAGGATCCGCATGCGGGATAAATATCTAACACCGTGCGTG-3′ and 5′-GCTCAAGCTTGTCGACTTAATCAGCTTGCTTACGCAGGAATG-3′. The product was treated with BamHI and HindIII, and the 1,359-bp fragment was used to replace the 20-bp BamHI-HindIII fragment of pGB2, yielding pTB188. Note that this plasmid lacks a lambda repressor and that ftsZ is constitutively transcribed in the direction opposite that of aadA.
For pYT11 (Ptac::relA′), a portion of relA was amplified with primers 5′-CTTTTCTAGATTTCGGCAGGTCTGGTCCCTAAAGG-3′ and 5′-GGTCCTCGAGCTGGTAGGTGAACGGCACAATGCGCCC-3′. The product was treated with XbaI and XhoI, and the 1,401-bp fragment was used to replace an XbaI-XhoI fragment of pCH276, a plasmid whose construction will be detailed elsewhere. The 1,500-bp EcoRI-HindIII fragment of the resulting plasmid (pYT5) was next used to replace the 30-bp EcoRI-HindIII fragment of pJF118EH, yielding pYT11. The plasmid encodes the first 455 residues of RelA, followed by a glutamic acid residue and a stop codon.
Phages λCH221, λCH235, λCH268, λFB120, λFB185, λFB190, and λTB59 were obtained by crossing λNT5 with pCH221, pCH235, pCH268, pFB120, pFB185, pFB190, and pTB59, respectively, as described previously (24
E. coli strains. mre
knockout strains were constructed by λ red recombineering, using pKD13 as a template for amplification of an aph
cassette consisting of aph
flanked by FLP recombinase substrate sites (frt
) and appropriate mre
). Knockout alleles on linear fragments were recombined with the chromosome of strain DY329 carrying plasmid pCX16 [sdiA
]. With plating under standard conditions (LB-kanamycin [Kan] at 30°C), the number of recombinants recovered in the presence of pCX16 was, at least, 2 to 3 logs higher than in its absence.
We used the following primer sets (chromosomal sequences are underlined): for mreB<>aph, 5′-GACCTGGGTACTGCGAATACCCTCATTTATGTAAAAGGACAAGGCATCGTGTGTAGGCTGGAGCTGCTTC-3′ [primer mreB(KO)5′] and 5′-AGCCATCGGTTCTTCAATCAGGAAGACTTCACGGGCACCAGCGCCCTGCGATTCCGGGGATCCGTCGACC-3′ [mreB(KO)3′]; for mreC<>aph, 5′-ATCGGATGCAGGCAGGGGAAGTGTCTGTTTACCCTGCCTGGTCTGATACGATAAGTGTAGGCTGGAGCTGCTTC-3′ [mreC(KO)5′] and 5′-AGCGATCCCCGTTGCCGGTTCAGGTAACTTTGGCCCCATCGCGTCTGGCGAATTCCGGGGATCCGTCGACC-3′ [mreC(KO)3′]; for mreD<>aph, 5′-GTGGCGAGCTATCGTAGCCAGGGACGCTGGGTAATCTGGCTCTCTTTCCTCTAAGTGTAGGCTGGAGCTGCTTC-3′ [mreD(KO)5′] and 5′-TCAGCAAGAAAATCCACGGCCAGAGCACCCCATTGACTACACTACTCCAGAATTCCGGGGATCCGTCGACC-3′ [mreD(KO)3′]; for mreBCD<>aph, primers mreB(KO)5′ and mreD(KO)3′; for mreBC<>aph, primers mreB(KO)5′ and mreC(KO)3′; and for mreCD<>aph, primers mreC(KO)5′ and mreD(KO)3′.
Recombination yielded a set of six mre<>aph derivatives of DY329/pCX16, which all showed a spherical phenotype. The six strains were transformed with pCH244 (Plac::mreB mreC mreD yhdE), and transformants of each reverted to a rod shape in an IPTG (isopropyl-β-d-thiogalactopyranoside)-dependent manner. Phage P1 was grown on a transformant (containing both pCX16 and pCH244) of each strain in the presence of 250 μM IPTG, resulting in a high-titer transducing lysate for each mre<>aph allele. These lysates were then used to transduce Mre+ strains, PB103, or TB28, using various strategies to avoid selective pressure for the accumulation of undesired suppressor mutations. Generally, this was accomplished by the introduction of appropriate correcting or suppressing mre, sdiA, or ftsZ plasmids or phages into the Mre+ recipient before introduction of a chromosomal mre<>aph allele by transduction. For example, to obtain the MreBCD depletion strain FB30/pFB174 (mreBCD<>aph/cat araC PBAD::mreBCD), TB28 was transformed with pFB174 prior to transduction of mreBCD<>aph and transductants were recovered at 30°C on LB-Kan supplemented with chloramphenicol and 0.5% arabinose.
Similarly, derivatives of PB103 carrying chromosomal mre
alleles (Table ; also see Table S1 in the supplemental material) were obtained by introduction of pFB112 (tet sdiA
) prior to transduction with the corresponding mre
lysates. The resulting mre
/pFB112 strains were then transformed with pCP20 [bla cat repA
) and plated at 30°C on LB containing ampicillin (Amp) and tetracycline. Transformants were streaked on LB lacking Amp and incubated at 42°C to simultaneously induce production of Flp recombinase and block replication of pCP20. Kan- and Amp-sensitive clones were purified, resulting in the desired mre
/pFB112 strains. These strains were transformed with appropriate mre
plasmids and used for complementation analyses (see Table S1 in the supplemental material). The growth of some of these transformants at 37°C and in the presence of IPTG led to simultaneous correction of the rod phenotype and competitive loss of pFB112 (see Table S1 in the supplemental material), giving rise to depletion strains that lacked extra copies of sdiA
, such as the MreB depletion strain FB17/pFB118/pFB124 (mreBCD
For construction of mrd mutants, we used the following primer sets (chromosomal sequences are underlined): for mrdAB<>aph, 5′-CATCCTTATCACCGTGAGTGATAAGGGAGCTTTGAGTAGAAAACGCAGCGGGTGTAGGCTGGAGCTGCTTC-3′ [pbp2(KO)5′] and 5′-CGCCAGCCATGACGCGACCAAAGGTGGTTTGCGCTCTGGCGGCTATCCATTCCGGGGATCCGTCGACC-3′ [rodA(KO)3′]; and for mrdB<>aph, 5′-CGATCTGCCTGCGGAAAATCCAGCGGTTGCCGCAGCGGAGGACCATTAAGTGTAGGCTGGAGCTGCTTC-3′ [rodA(KO)5′ and rodA(KO)3′].
Recombination with the chromosome of DY329/pCX16 resulted in FB29/pCX16 (mrdAB<>aph/sdiA) and FB20/pCX16 (mrdB<>aph/sdiA), which propagated as spheres. These strains were transformed with pTB59 (Plac::mrdAB), which caused transformants to revert back to a rod shape in the presence of IPTG. P1 lysates were prepared on FB29/pCX16/pTB59 and FB20/pCX16/pTB59 transformants, and these were used to transduce mrdAB<>aph and mrdB<>aph into PB103 or TB28 derivatives carrying appropriate complementing plasmids and/or phages.
For the P1 transduction experiments whose results are shown in Tables and , we used the mre<>aph and mrd<>aph lysates described above, except for the mrdB<>aph and lacIZYA<>aph transducing lysates (Table ), which were prepared on strains FB22(λCH221) and TB12, respectively.
Suppression of Mre− and MrdB− lethality by multiple copies of sdiA
Recovery of mreBCD<>aph and mrdAB<>aph transductants on minimal mediuma