Table 1.
Bacterial strains, TraDIS libraries, plasmids and primers used in this study.
| Bacterial strains | ||
| Strain | Description/genotype (Genome accession no) | Source |
| K. pneumoniae ATCC 43816 | Hypervirulent, ST493, K-type 2, commonly used in mouse studies (CP009208.1) | Isolate: American Type Culture Collection; Genome: (Broberg et al. 2014) |
| K. pneumoniae RH201207 | Colistin-resistant UK gut isolate, ST258, K-type 106 | Isolate: (Jana et al. 2017); Genome: This study |
| Escherichia coli β2163 | F− RP4-2-Tc::Mu DdapA::(erm-pir) | (Demarre et al. 2005) |
| K. pneumoniae RH201207 | Tn5-rfaH | (Short et al. 2020) |
| K. pneumoniae RH201207 | Tn5-wza | (Short et al. 2020) |
| K. pneumoniae RH201207 | Tn5-wzxE | This study |
| K. pneumoniae RH201207 | ∆nfeR (KPNRH_00645) | This study |
| K. pneumoniae RH201207 | ∆nfeR (KPNRH_00645) complemented on chromosome | This study |
| K. pneumoniae RH201207 | ∆phoQ (KPNRH_03335) | This study |
| Transposon mutant libraries | ||
| Parent strain | Transposon (primers) | Source |
| K. pneumoniae RH201207 | Tn5, TetR (Tn5tetR-5Seq, Tn5tetR-5PCR) | (Jana et al. 2017) |
| K. pneumoniae ATCC 43816 | Tn5-derived from pDS1028 vector, CmR (FS107, FS108) | (Dorman et al. 2018) |
| Plasmids | ||
| Name | Description | Source |
| pKNG101-Tc | Allelic exchange vector, TcR | (Poulter et al. 2011) |
| pFLS27 | RH201207 nfeR knockout vector, pKNG101-Tc-derived, constructed with FS273-276 | This study |
| pFLS28 | RH201207 phoQ knockout vector, pKNG101-Tc-derived, constructed with FS277-280 | This study |
| pFLS36 | RH201207 nfeR complementation vector, pKNG101-Tc-derived, constructed with FS273-274 | This study |
| Primers | ||
| Name | Sequence 5’ – 3’ | Description |
| FS107 | GAGCTCGAATTCATCGATGATGGTTGAGATGTGTA | pDS1028 TraDIS 5’ Seq |
| FS108 | AATGATACGGCGACCACCGAGATCTACACCAGGAACACTTAACGGCTGACATGG | pDS1028 TraDIS 5’ PCR |
| Tn5tetR-5PCR | AATGATACGGCGACCACCGAGATCTACACACTGTGATAAACTACCGCATTAAAGCTTATCG | Tn5-Tet TraDIS 5’ PCR |
| Tn5tetR-5Seq | CGATGATAAGCTGTCAAACATTGATGGTTGAGATGTGTA | Tn5-Tet TraDIS 5’ Seq |
| FS273 | gctACTAGTTGCCCTACATTGAAGATGC | Mutant construction, SpeI site |
| FS274 | gctACTAGTCGTAAGCCAGCTCGTGA | Mutant construction, SpeI site |
| FS275 | GGTGGTGAAAAAAGAGGCGGTTACCTCCTGCTGTTTT | Mutant construction, overlap |
| FS276 | ACAGCAGGAGGTAACCGCCTCTTTTTTCACCACCGGC | Mutant construction, overlap |
| FS277 | gctACTAGTCGGGCAGTGCTGTTTCA | Mutant construction, SpeI site |
| FS278 | gctACTAGTGCGAACATCTCCCGGAT | Mutant construction, SpeI site |
| FS279 | GTGAAAAATCTCAACGACAGCGCAATTCGAACAGAT | Mutant construction, overlap |
| FS280 | TCGAATTGCGCTGTCGTTGAGATTTTTCACGGCG | Mutant construction, overlap |
| FS133 | TTAAACAGGCCGAATTCCAG | qRT-PCR recA |
| FS134 | CCGCTTTCTCAATCAGCTTC | qRT-PCR recA |
| FS135 | TCGCCGATGCGTCGTATAAA | qRT-PCR yqjH |
| FS136 | TGCTGATCGATGCTCTCGTC | qRT-PCR yqjH |