Table 2.
Plasmids used in this study.
| Designation | Descriptiona | Source |
|---|---|---|
| PLASMIDS FOR penA DELETION AND OVEREXPRESSION | ||
| pUCP20 | Apr; Broad host-range cloning vector | West et al. (1994) |
| pEXKm5 | Kmr; Allelic-exchange plasmid | López et al. (2009) |
| pTNS3 | Apr; Tn7 insertion helper plasmid | Choi et al. (2008) |
| pUC18T-mini-Tn7T-Km-LAC | Apr Kmr; Tn7 cassette vector with Ptac and lacIq for regulated expression of cloned genes | This study |
| pPS2370 | Apr; pUCP20 with a 1,308 bp penA fragment (amplified with primers 1687 + 1712) inserted into SmaI site | This study |
| pPS2549 | Apr; pPS2370 with 291 bp NsiI-PmlI fragment deleted from penA | This study |
| pPS2550 | Kmr; 1,339 bp PvuII fragment from pPS2549 was inserted into the SmaI site of pEXKm5 | This study |
| pPS2605 | Apr Kmr; pCR2.1 with 1,230 bp penA fragment (amplified with primers 2003 + 2005) | This study |
| pPS2608 | Apr Kmr; pCR2.1 with the 1,261 bp penA fragment from pPS2605 (amplified with primers 2003 + 2004 to modify the 5′ region) | This study |
| pPS2627 | Apr Kmr; pUC18T-mini-Tn7T-Km-LAC with the 1,266 bp penA fragment from pPS2608 | This study |
| PLASMIDS FOR penA PUTATIVE REGULATORY GENE DELETION AND OVEREXPRESSION | ||
| pPS2609 | Apr Kmr; 1,043 bp SOEing PCR product using primer sets 2014 + 2015 and 2016 + 2017 was ligated into pCR2.1 | This study |
| pPS2610 | Apr Kmr; 1,300 bp SOEing PCR product using primer sets 2010 + 2011 and 2012 + 2013 was ligated into pCR2.1 | This study |
| pPS2611 | Apr Kmr; 1,239 bp SOEing PCR product using primer sets 2006 + 2007 and 2008 + 2009 was ligated into pCR2.1 | This study |
| pPS2614 | Kmr; blunt ended 1,085 bp BstXI fragment from pPS2609 ligated into SmaI site of pEXKm5 | This study |
| pPS2615 | Kmr; 1,316 bp EcoRI fragment from pPS2610 ligated into EcoRI site of pEXKm5 | This study |
| pPS2616 | Kmr; 1,255 bp EcoRI fragment from pPS2611 ligated into EcoRI site of pEXKm5 | This study |
| pUC18-mini-Tn7T-Gm-Gateway | Apr Gmr; mini-Tn7T-Gm with GATEWAY cassette. GenBank accession number AY737004 | Choi et al. (2005) |
| pPS2735 | Apr Kmr; pUC18T-mini-Tn7T-Km-FRT with Ps12 | This study |
| pPS2737 | Apr Kmr; Gateway-ready Tn7 cassette vector with s12 promoter toward insert (KpnI-AfeI fragment containing 1,824 bp Gateway cassette from pPS1612 ligated into pPS2735 between KpnI-StuI) | This study |
| pPS2745 | Kmr; Nested PCR with primers 2015 + 2016, then 2155 + 2156 for 1,051 bp fragment, cloned into pENTR-SD-D-TOPO | This study |
| pPS2746 | Kmr; Nested PCR with primers 2013 + 2010, then 2157 + 2158 for 1,056 bp fragment, cloned into pENTR-SD-D-TOPO | This study |
| pPS2747 | Kmr; Nested PCR with primers 2009 + 2006, then 2159 + 2160 for 1,456 bp fragment, cloned into pENTR-SD-D-TOPO | This study |
| pPS2748 | Apr Kmr; Gateway LR recombination reaction with pPS2737 + pPS2745 | This study |
| pPS2749 | Apr Kmr; Gateway LR recombination reaction with pPS2737 + pPS2746 | This study |
| pPS2750 | Apr Kmr; Gateway LR recombination reaction with pPS2737 + pPS2747 | This study |
| PLASMIDS FOR ENGINEERING OF TAT-SIGNAL SEQUENCE MUTATIONS | ||
| pPS2674 | Apr Kmr; 740 bp of the 5′ region of penA gene amplified with primers 2010 + 2011 and cloned into pCR2.1 | This study |
| pPS2613 | Apr Kmr; Mutagenic primer 2022 substituted AAG for CGC at bases 19-21 of penA in pPS2674 to provide a R7K substitution | This study |
| pPS2618 | Kmr; 736 bp EcoRI fragment from pPS2676 ligated into EcoRI of pEXKm5 | This study |
| pPS2676 | Apr Kmr; Mutagenic primer 2076 substituted GC for CG at bases 22-23 of penA in pPS2674 to provide a R8A substitution | This study |
| pPS2678 | Kmr; 736 bp EcoRI fragment from pPS2676 ligated into EcoRI of pEXKm5 | This study |
| PLASMIDS FOR ENGINEERING OF penA POINT MUTATIONS | ||
| pPS2675 | Apr Kmr; Mutagenic primer 2075 mutated G to A at base 224 of penA to provide a C69Y substitution in pPS2674 sequence | This study |
| pPS2677 | Kmr; 736 bp EcoRI fragment from pPS2675 ligated into EcoRI of pEXKm5 | This study |
| pPS2712 | Kmr; 1,094 bp NruI-HincII fragment (entire penA) from pPS2370 ligated into the SmaI site of pEXKm5 | This study |
| pPS2721 | Kmr; Mutagenic primer 2136 mutated C to T at base 517 of penA to provide a P167S substitution using pPS2712 (pEXKm5-based) | This study |
| pPS2722 | Kmr; Mutagenic primer 2137 C to T at base 233 of penA to provide a S72F mutation using pPS2712 (pEXKm5-based) | This study |
| PLASMIDS FOR tatABC DELETION | ||
| pPS2612 | Apr; 1,038 bp Soeing PCR product using primer sets 2018 + 2019 and 2020 + 2021 ligated into pGem-T Easy (Promega, Madison, WI, USA) | This study |
| pPS2617 | Kmr; 1,058 bp EcoRI fragment from pPS2612 ligated into EcoRI of pEXKm5 | This study |
aAp, ampicillin; Km, kanamycin; Gm, gentamicin; Ps12, B. thailandensis ribosomal s12 gene promoter.