TABLE 2.
Strains and plasmids used in this study
| Strain or plasmid | Genotype or descriptiona | Reference(s) or source |
|---|---|---|
| Strains | ||
| E. coli | ||
| DH5α MCR | Strain used for general cloning | Life Technologies (Grand Island, NY) |
| S17-1 λpir | Strain used for conjugation | 49 |
| HB101 | Strain used with pRK2013 for triparental conjugation | 50, 51 |
| F. johnsoniae | ||
| ATCC 17061T (UW101) | Wild type | 6, 32, 43 |
| ATCC 17061T (FJ1) | Wild type | 6, 32 |
| UW102-9 | gldA mutant encoding GldAG40R | 27 |
| UW102-41 | gldI-null mutant | 32 |
| UW102-55 | gldJ-null mutant | 28 |
| UW102-80 | gldJ-null mutant | 28 |
| UW102-81 | gldJ mutant encoding truncated GldJ of 542 amino acids | 28 |
| UW102-168 | gldA mutant encoding GldAR137C | 34 |
| CJ101-288 | gldA insertion mutant; (Emr) | 27 |
| CJ282 | gldD Tn4351 mutant; (Emr) | 31 |
| CJ569 | gldB Tn4351 mutant; (Emr) | 30 |
| CJ787 | gldF Tn4351 mutant; (Emr) | 29 |
| CJ1043 | gldH Tn4351 mutant; (Emr) | 33 |
| CJ1808 | chiA insertion mutant; (Emr) | 16 |
| CJ1827 | rpsL2; wild-type strain derived from ATCC 17061T (UW101) and used in construction of deletion mutants described below; (Smr) | 44 |
| CJ1922 | ΔsprB rpsL2 (Smr) | 44 |
| CJ2083 | remA::myc-tag-1 rpsL2 (Smr) | 11 |
| CJ2090 | ΔgldNO rpsL2 (Smr) | 11 |
| CJ2122 | ΔgldK rpsL2 (Smr) | 10 |
| CJ2157 | ΔgldL rpsL2 (Smr) | 10 |
| CJ2262 | ΔgldM rpsL2 (Smr) | 10 |
| CJ2302 | ΔsprA rpsL2 (Smr) | 10 |
| CJ2360 | ΔgldJ remA::myc-tag-1 rpsL2 (Smr) | This study |
| CJ2386 | gldJ548 rpsL2; gldJ deletion mutation that results in production of GldJ protein truncated after the 548th amino acid; (Smr) | This study |
| CJ2443 | gldJ553 rpsL2; gldJ deletion mutation that results in production of GldJ protein truncated after the 553rd amino acid; (Smr) | This study |
| FJ149 | sprE HimarEm2 mutant; (Emr) | 9 |
| KDF001 | sprT insertion mutant; (Emr) | 15 |
| Plasmids | ||
| pCP11 | E. coli-F. johnsoniae shuttle plasmid; Apr (Emr) | 45 |
| pCP23 | E. coli-F. johnsoniae shuttle plasmid; Apr (Tcr) | 27 |
| pCP29 | E. coli-F. johnsoniae shuttle plasmid; Apr (Cfr Emr) | 52 |
| pSA21 | pCP23 carrying gldA; Apr (Tcr) | 27 |
| pDH223 | pCP11 carrying gldB; Apr (Emr) | 30 |
| pMM209 | pCP23 carrying gldD; Apr (Tcr) | 31 |
| pMK314 | pCP29 carrying gldF and gldG; Apr (Cfr Emr) | 29 |
| pMM293 | pCP23 carrying gldH; Apr (Tcr) | 33 |
| pMM291 | pCP11 carrying gldI; Apr (Emr) | 32 |
| pMM313 | pCP11 carrying gldJ; Apr (Emr) | 28 |
| pTB99 | pCP23 carrying gldK; Apr (Tcr) | 6 |
| pRR51 | rpsl-containing suicide vector used for constructing deletion mutants; Apr (Emr) | 44 |
| pMM317 | Fragment encoding the first 337 amino acids of GldJ inserted in pCP11; Apr (Emr) | 28 |
| pMM318 | Fragment encoding the first 547 amino acids of GldJ inserted in pCP11; Apr (Emr) | 28 |
| pAB31 | Fragment encoding the first 542 amino acids of GldJ inserted in pCP23; Apr (Tcr) | This study |
| pJJ07 | Construct used to delete gldJ; 2.3-kbp upstream and 3.0-kbp downstream of gldJ amplified and ligated into pRR51; Apr (Emr) | This study |
| pJJ09 | 1.9-kbp region upstream of base 1644 of gldJ (numbered from the “A” of gldJ start codon) amplified with primers 1502 and 1503 and ligated into BamHI- and SalI-digested pRR51; Apr (Emr) | This study |
| pJJ10 | Construct used to generate truncated gldJ encoding the first 548 amino acids of GldJ (GldJ548); 1.9-kbp region downstream of gldJ amplified with primers 1504 and 1505 and ligated into SalI- and SphI-digested pJJ09; Apr (Emr) | This study |
| pJJ11 | Construct used to generate truncated gldJ encoding the first 553 amino acids of GldJ (GldJ553); 1.9-kbp region upstream of base 1659 of gldJ (numbered from the “A” of gldJ start codon) amplified with primers 1502 and 1555 and ligated into BamHI- and SalI-digested pJJ10; Apr (Emr) | This study |
a
Antibiotic resistance phenotypes are as follows: Apr, ampicillin resistance; Cfr, cefoxitin resistance; Emr, erythromycin resistance; Smr, streptomycin resistance; Tcr, tetracycline resistance. The antibiotic resistance phenotypes given in parentheses are those expressed in F. johnsoniae but not in E. coli. The antibiotic resistance phenotypes without parentheses are those expressed in E. coli but not in F. johnsoniae.