TABLE 1.
Strains and plasmids used in this work
| Strain or plasmid | recA | uvrD | Other relevant genotype | Source or derivation |
|---|---|---|---|---|
| Strains | ||||
| CAG18491 | + | + | metE3079::Tn10 | 44 |
| GJ1989 | + | + | lexA3 malB::Tn9 | 39 |
| JC13509a | + | + | + | Lab stock |
| JC19108 | + | + | lexA71::Tn5 malE::Tn10 | Lab stock |
| JC19328 | 306 | + | + | Lab stock |
| JJC145 | + | + | PO66-HfrPK191 srlC::Tn10 | 4 |
| JJC2457 | + | cath | + | 16 |
| MV1138 | + | + | srlC300::Tn10 recAo281 | M. Volkert |
| SK9041 | + | 294 | + | 53 |
| SS775 | + | + | lexA3 malE::Tn10-9 | GJ1989→JC13509e |
| SS996 | + | + | del(attB)::psulA-gfp | 31 |
| SS1054 | + | + | metE3079::Tn10 | CAG18491→JC13509d |
| SS1465 | + | + | gal76::Tn10 del(attB)::psulA-gfp | Lab stock |
| SS2533 | + | cat | del(attB)::psulA-gfp | JJC2457→SS996e |
| SS3085 | 4136, 4155b | + | + | 37 |
| SS3368 | 4136, 4155b | cat | + | 6 |
| SS4414 | + | + | lexA3 malE::Tn10-9 | SS775/pKD46 |
| SS4629 | 730 | + | del(attB)::psulA-gfp | Lab stock |
| SS4952 | 100c | + | + | 3 |
| SS4976 | 4142 | + | recAo1403 del(attB)::psulA-gfp | Lab stock |
| SS5112 | 730 | + | metE3079::Tn10 del(attB)::psulA-gfp | CAG18491→SS4629d |
| SS5129 | + | + | del(uvrA)100::kan | 3 |
| SS5141 | + | 303, 705 | lexA3 malE::Tn10-9 | This study |
| SS5145 | + | 701, 706 | lexA3 malE::Tn10-9 | This study |
| SS5191 | + | 701, 706 | + | SS5145→JC13509f |
| SS5402 | + | 701, 706 | + | SS5191/pRC35 |
| SS5428 | + | 701 | + | This study |
| SS5450 | + | 701 | + | SS5428→SS1054g |
| SS5482 | + | 303, 705 | + | SS5141→JC13509f |
| SS5489 | + | 303, 705 | + | SS5482/pRC40 |
| SS5496 | + | 303 | + | This study |
| SS5704 | + | 303 | + | SS5496→SS1054g |
| SS5711 | 4136, 4155b | 303 | + | SS3085→SS5704f |
| SS5737 | + | 701 | gal76::Tn10 del(attB)::psulA-gfp | SS1465→SS5450d |
| SS5756 | 306 | 303 | + | JC19328→SS5704d |
| SS5764 | + | 303 | del(uvrA)100::kan | SS5129→SS5704f |
| SS5766 | + | + | del(uvrA)100::kan | SS5129→JC13509f |
| SS5769 | 306 | + | del(uvrA)100::kan | SS5756→ SS5766d |
| SS5801 | 730 | 303 | del(attB)::psulA-gfp | SS5496→SS5112g |
| SS5802 | + | + | metE3079::Tn10 del(attB)::psulA-gfp | CAG18491→SS996d |
| SS5803 | + | 303 | del(attB)::psulA-gfp | SS5496→SS5802g |
| SS5807 | 4142 | + | metE3079::Tn10 recAo1403 del(attB)::psulA-gfp | CAG18491→SS4976d |
| SS5808 | 4142 | cat | recAo1403 del(attB)::psulA-gfp | JJC2457→SS5807g |
| SS5809 | 4142 | 303 | recAo1403 del(attB)::psulA-gfp | SS5496→SS5807g |
| SS5907 | 100c | + | + | SS4952→JC13509f |
| SS5912 | 100c | + | + | SS5907/pLH29 |
| SS5915 | 200c | + | + | SS5912/FLP |
| SS5959 | + | + | lexA71::Tn5 | JC19108→JC13509f |
| SS5960 | + | 303 | lexA71::Tn5 | JC19108→SS5704f |
| SS5962 | + | + | srlC300::Tn10 recAo281 | MV1138→JC13509d |
| SS5963 | + | 303 | srlC300::Tn10 recAo281 | MV1138→SS5704d |
| SS5985 | + | 303, 705 | + | SS5482/pRC49 |
| SS5986 | + | 303, 701 | + | This study |
| SS5987 | + | 303, 701 | + | SS5986→SS1054g |
| SS5988 | + | 303, 701 | del(attB)::psulA-gfp | SS5986→SS5802g |
| SS5989 | 730 | 303, 701 | del(attB)::psulA-gfp | SS5986→SS5112g |
| SS5990 | 730 | 701 | del(attB)::psulA-gfp | SS5450→SS5112g |
| SS5993 | + | 294 | + | SK9041→JC13509f |
| Plasmids | ||||
| pGZK20 | 53 | |||
| pGZK31 | 53 | |||
| pKD46 | 14 | |||
| pKO3 | 27 | |||
| pLH29 | 22 | |||
| pRC19 | This study | |||
| pRC32 | This study | |||
| pRC33 | This study | |||
| pRC35 | This study | |||
| pRC40 | This study | |||
| pRC49 | This study | |||
| pSJS1488 | 37 |
JC13509 has the following genotype: sulB103 lacMS286 φ80dIIlacBK1 argE3 his-4 thi-1 xyl-5 mtl-1 rpsL31 tsx. The lacMS286 φ80dIIlacBK1 genes code for two partial nonoverlapping deletions of the lac operon (23, 54).
The full genotype is recAo1403 ygaD1::kan recA4155,4136::gfp-901 (37). This is abbreviated to recA4155,4136 in the table. recAo1403 is an operator mutation that increases the basal or non-SOS-induced level of transcription twofold (49). ygaD is the open reading frame in front of the recA gene. It has no known function. gfp-901 refers to mut-2 (9) with the additional “monomeric” mutation A206T (52). recA4155 is a mutant allele of recA with an arginine-to-alanine change at codon 28. It does not make storage structures in vivo (37). recA4136 refers to the specific fusion of recA to gfp.
The 100 allele (from the Keio collection [3]) essentially replaces the gene in question with a kan gene between two FRT sites. The 200 allele is a result of removing the kan gene with FLP recombinase, induced by isopropyl-β-d-thiogalactopyranoside (IPTG) and expressed from the chloramphenicol resistance plasmid pLH29 (2). The strain was then grown in the presence of IPTG, and Kans Cams strains were screened.
Select for tetracycline resistance. Screen by PCR if necessary.
Select for chloramphenicol resistance. Screen by PCR if necessary.
Select for kanamycin resistance. Screen by PCR if necessary.
Select for methionine auxotrophy. Screen by PCR if necessary.
We have sequenced the insertion-deletion mutation originally called del(uvrD-yigB)::cam in reference 16. We find that the mutation is actually a deletion-insertion mutation of uvrD and yigE. The endpoints of the deletion are GAAGAAGACGAAG in uvrD and CAGGCAAAATCATA in yigE. The last sequence in the uvrD gene is GAAGAAGACGAAG and the first sequence in the yigE gene is CAGGCAAAATCATA (all sequences between those two are deleted). The cat gene from pACYC184 (inclusive of the sequences starting with CCAAGCTCGAATT and ending with AAGTTGGAACCT) are in place of the uvrD and yigE sequences.