Article Figures & Data
Figures
- Fig 1
(A) Comparison of genomic organizations of rarA (KPN_02968) locus in Klebsiella pneumoniae strain MGH 78578 to those of other Enterobacteriaceae harboring the locus. (B) Predicted phylogenetic tree of MarA-type protein homologues in Klebsiella pneumoniae determined using PhyML 3.0 (14). The amino acid sequences for MarA (KPN_01624), Rob (KPN_04851), RarA (KPN_02968), RamA (KPN_00556), SoxS (KPN_04462), and KPN_01709 were used in Muscle alignment software to generate the phylogenetic tree. All amino acid sequences were obtained from the genome sequence of MGH 78578 (NC_009648). Of note, different branch lengths denote relative amino acid identities.
- Fig 2
Nucleotide changes within the intergenic region between KPN_02968 (rarA) and KPN_02969 (oqxA). Changes observed within the intergenic region between rarA and oqxA in the different clinical strains tested are shown. The numbering scheme is based on the first nucleotide before the ATG of rarA as position 1. The transcription start site of rarA, determined by 5′ RACE analysis, is labeled TSS. The Shine-Dalgarno sequence is underlined; putative −10 and −35 promoter regions determined through Softberry analysis are shown boxed and labeled accordingly.
- Fig 3
Fold change in expression levels of rarA and oqxA among clinical isolates compared to K. pneumoniae Ecl8. All QPCR experiments were performed as outlined in Materials and Methods. For strains Ecl8Mdr1 [represented by EcL8(R)], Kp342, TS152, TS165, TS202, GC9, GC12, GC19, and GC21, fold change values were generated after normalizing to 16S levels for each strain and then using oqxA and rarA levels derived from the sensitive K. pneumoniae Ecl8 strain. To assess whether rarA could activate the expression of the efflux pump oqxAB, transcription of oqxA was assessed in Ecl8/pACrarA-1 [EcL8(P)] and Ecl8 ΔrarA/pACrarA-2 [EcL8(Δ)]. In this case, the fold change was normalized as mentioned before but calibrated against either Ecl8/pACYC177 or Ecl8ΔrarA/pACYC184.
Tables
- Table 1
List of strains used in experiments
Strain Relevant genotype and phenotypea Reference or source E. coli strains AG100 E. coli wt strain 11 AG100/pACrarA-1 AG100 + pACrarA-1 (wt rarA cloned into pACYC177 (BamHI, ScaI), Kanr This work AG100/pACYC177 AG100 + pACYC177 (Kanr, Ampr) This work AG100/pACrarA-2 AG100 + pACrarA-2 (wt rarA cloned into pACYC184 [BamHI, HindIII]), Cmr This work AG100/pACYC184 AG100 + pACYC184 (Cmr, Tetr) This work AG100A acrAB efflux pump-deleted strain 28 AG100A/pACrarA-2 AG100A + pACrarA-2 (wt rarA cloned into pACYC184 [BamHI, HindIII]), Cmr This work AG100A/pACYC184 AG100A + pACYC184 (Cmr, Tetr) This work MG1655 E. coli K-12 laboratory isolate In-house strain collection MG1655/pACrarA-1 MG1655 + pACrarA-1 (wt rarA cloned into pACYC177 [BamHI, ScaI]), Kanr This work MG1655/pACYC177 MG1655 + pACYC177 (Kanr, Ampr) This work MG1655 ΔmarA marA-deleted strain S. B. Levy, L. McMurry MG1655 ΔmarA/pACrarA-1 MG1655 ΔmarA + pACrarA-1 (wt rarA cloned into pACYC177 [BamHI, ScaI]), Kanr This work MG1655 ΔmarA/pACYC177 MG1655 ΔmarA + pACYC177 (Kanr, Ampr) This work AG100 ΔsoxS Δrob ΔmarA soxS-, rob-, marA-deleted strain S. B. Levy, L. McMurry AG100 ΔsoxS Δrob ΔmarA/pACrarA-2 AG100 ΔsoxS Δrob ΔmarA + pACrarA-2 (wt rarA cloned into pACYC184 [BamHI, HindIII]), Cmr This work AG100 ΔsoxS Δrob ΔmarA/pACYC184 AG100 ΔsoxS Δrob ΔmarA + pACYC184 (Cmr, Tetr) This work K. pneumoniae strains MGH 78578 ATCC 700721 19 Ecl8 K. pneumoniae wt strain 8 Ecl8/pACrarA-1 Ecl8 + pACrarA-1 (wt rarA cloned into pACYC177 [BamHI, ScaI]), Kanr This work Ecl8/pACYC177 Ecl8 + pACYC177 (Kanr, Ampr) This work Ecl8Mdr1 Spontaneous MDR mutant of Ecl8 10 Ecl8 ΔramA ramA-deleted strain derived from Ecl8 27 Ecl8 ΔramA/pACrarA-1 Ecl8 ΔramA + pACrarA-1 (wt rarA cloned into pACYC177 [BamHI, ScaI]), Kanr This work Ecl8 ΔramA/pACYC177 Ecl8 ΔramA + pACYC177 (Kanr, Ampr) This work Kp342 K. pneumoniae isolated from maize 9 Ecl8 ΔacrAB acrAB-deleted strain derived from Ecl8, Kanr This work Ecl8 ΔacrAB/pACrarA-2 Ecl8 ΔacrAB + pACrarA-2 (wt rarA cloned into pACYC184 [BamHI, HindIII]), Cmr This work Ecl8 ΔacrAB/pACYC184 Ecl8 ΔacrAB + pACYC184 (Cmr, Tetr) This work Ecl8 ΔrarA rarA (KPN_02968)-deleted strain derived from Ecl8, Kanr This work Ecl8 ΔrarA/pACrarA-2 Ecl8 ΔrarA + pACrarA-2 (wt rarA cloned into pACYC184 [BamHI, HindIII]), Cmr This work Ecl8 ΔrarA/pACYC184 Ecl8 ΔrarA + pACYC184 (Cmr, Tetr) This work TS152, TS165 K. pneumoniae clinical isolates (Turkey) 27 TS180, TS202 K. pneumoniae clinical isolates (Chile) 27 GC9, GC12, GC19, GC21 K. pneumoniae clinical isolate (Germany) This work GC9, GC12, GC19, GC21/pACoqxR GC isolates + pACoqxR (wt oqxR cloned into pACYC177 [BamHI, ScaI]), Kanr This work GC9, GC12, GC19, GC21/pACYC177 GC isolates + pACYC177 (Kanr, Ampr) This work ↵a wt, wild type; Kan, kanamycin; Amp, ampicillin; Cm, chloramphenicol; Tet, tetracycline.
- Table 2
List of primers used in experiments
Primer Sequence (5′–3′)a Cloning 02968FB CGGGATCCCACTATCGCGGCGATTGTA 02968RS AAAAGTACTTCATGCGGATCGCTGACG 02968HR CCCAAGCTTTCATGCGGATCGCTGACG 02968BF CGGGATCCTTGCACTTTATGTGCGGT OqxRbamHIF CGGGATCCTTTACTTCGCAGGCTAAC OqxRsca1R AGTACTTCATTTTCTGGTGACGAAAA Deletion Ci-02968 CCGTTCCAAGCGGCCGCAAGAGCGCATCTCGTCAGCGATCCGCATG Co-02968 AAAAAGTCGACTCAGTCCTGACCCGGCCCATG Ni-02968 CGCTCTTGCGGCCGCTTGGAACGGTCGCGGCCG GTTAAAAGCATC No-02968 AAAAACTGCAGGTATGGACTAATTTATCGGTGC 02968delchkF TCAGCCAGATGGCAACCG 02968delchkR ACCGCTTCAATGCGACCG Ci-acrB CGCTCTTGCGGCCGCTTGGAACGGGGCGCGCCTCTCCTGGTT Co-acrB AAAAACTCGAGCCACTTTTGCAAATCCGTAGAG Ni-acrA CGCTCTTGCGGCCGCTTGGAACGGGTGTCCAATTTCAAAATGTTC No-acrA AAAAACTGCAGGCGTAGCGTCGGG CAGAATTG AcrABdelchkF GAGCGAATGTGGCAATGTGC AcrABdelchkR CACGGTCATTTCACCGACG Sequencing 02968FB CGGGATCCCACTATCGCGGCGATTGTA 02968RS AAAAGTACTTCATGCGGATCGCTGACG 02698intF GACAGATCCTGAATGAT 02698intR TGAATGTTTCCCCAGGTT OqxRF GTCACCAGAAAATGATTAATGCGC OqxRR GCCTTTGCCCGTGAAATCAG Q-RTPCR Kp02968F TGGATCGACAACCATCTTGA Kp02968R AAGGACTGCTGGGAGTCAAA Kp_16SQ_F GTTACCCGCAGAAGAAGCAC Kp_16SQ_R CTACGCATTTCACCGCTACA 02969F AAGGTGCTGGTGAAGTCGAT 02969R GGAGACGAGGTTGGTATGGA Kp_ramAQ_F AGCCTGGGGCGCTATATT Kp_ramAQ_R GTGGTTCTCTTTGCGGTAGG Kp_marAF TCGAGGATAACCTGGAATCG Kp_marAR ACAAATGGGCTCATTGCTC oqxRqpcrF TAACGAAGCCTGCTCTGCTT oqxRqpcrR AATGGTTCCGCTAACTCGTG 5′ RACE GSP1 ATGTCTGCGCAACAG GSP2 GCTCGTCGGGCAACGGTGTC GSP3 CCAGACGGCTATCAAGATGGTTG ↵a Underlined sequences denote restriction enzyme cut sites.
- Table 3
Susceptibility profiles of E. coli strains transformed with pACrarA and vector-only controla
Strain MIC (μg/ml) CHL OQX TET TIG NOR CIP AG100 4 16 4 < 0.500 0.125 0.031 AG100/pACrarA-1 16 32 8 2 1 0.062 AG100/pACYC177 4 16 4 < 0.500 0.125 0.031 AG100/pACrarA-2 * 32 * 2 0.250 0.062 AG100/pACYC184 * 16 * < 0.5 0.125 0.031 AG100A * 8 * 0.250 0.008 0.004 AG100A/pACrarA-2 * 8 * 0.250 0.008 0.004 AG100A/pACYC184 * 8 * 0.250 0.008 0.004 AG100 ΔsoxS Δrob ΔmarA * 16 * 0.500 0.032 0.031 AG100 ΔsoxS Δrob ΔmarA/pACrarA-2 * 32 * 4 0.250 0.062 AG100 ΔsoxS Δrob ΔmarA/pACYC184 * 16 * 0.500 0.031 0.031 MG1655 4 16 4 4 0.500 0.062 MG1655/pACrarA-1 16 32 8 8 1 0.125 MG1655/pACYC177 4 16 4 4 0.500 0.062 MG1655 ΔmarA 4 8 4 4 0.250 0.031 MG1655 ΔmarA/pACrarA-1 32 16 16 8 1 0.125 MG1655 ΔmarA/pACYC177 4 8 4 4 0.250 0.031 ↵a A pACYC177 or pACYC184 backbone was used to clone the rarA open reading frame. Depending on the resistance cassettes already in situ, the appropriate recombinant constructs would be used. Asterisks denote that MIC testing for that particular antibiotic was not done due to presence of a conflicting antibiotic resistance cassette on the plasmid and/or construct. Entries in bold denote increased MICs over those seen with wild-type/parental strains. CHL, chloramphenicol, OQX, olaquindox, TET, tetracycline, TIG, tigecycline, NOR, norfloxacin, CIP, ciprofloxacin.
- Table 4
Susceptibility profiles of K. pneumoniae strains transformed with pACrarA and vector-only controla
Strain MIC (μg/ml) CHL OQX TET TIG NOR CIP Ecl8 4 16 4 2 0.250 0.031 Ecl8/pACrarA-1 8 32 8 4 0.500 0.062 Ecl8/pACYC177 4 16 4 2 0.250 0.031 Ecl8 ΔramA 0.5 8 1 1 0.250 0.016 Ecl8 ΔramA/pACrarA-1 4 32 2 4 0.500 0.062 Ecl8 ΔramA/pACYC177 0.5 8 1 1 0.250 0.016 Ecl8 ΔrarA * 8 * 0.500 0.031 0.016 Ecl8 ΔrarA/pACrarA-2 * 64 * 4 0.250 0.062 Ecl8 ΔrarA/pACYC184 * 8 * 0.500 0.031 0.016 Ecl8 ΔacrAB * 2 * 0.25 0.031 0.016 Ecl8 ΔacrAB/pACrarA-2 * 64 * 0.25 0.031 0.016 Ecl8 ΔacrAB/pACYC184 * 2 * 0.25 0.031 0.016 ↵a A pACYC177 or pACYC184 backbone was used to clone the rarA open reading frame. Depending on the resistance cassettes already in situ, the appropriate recombinant constructs would be used. Asterisks denote that MIC testing for that particular antibiotic was not done due to presence of a conflicting antibiotic resistance cassette on the plasmid and/or construct. Entries in bold denote increased MICs over those seen with wild-type/parental strains. CHL, chloramphenicol; OQX, olaquindox; TET, tetracycline; TIG, tigecycline; NOR, norfloxacin; CIP, ciprofloxacin.
- Table 5
Sequence analysis of OqxR and rarA/oqxA expression levels of clinical K. pneumoniae isolatesa
Strain OqxR change(s) Expression level of rarA calibrated against Ecl8 Con/Comp log2 expression level(s)b rarA oqxA 152 Val130→Ala 6.62 — 6.42/— 165 Arg25→His, Val130→Ala 3.77 — 4.76/— 202 Ala133→Thr 7.37 — 5.64/— GC9 Phe6→Ser 9.60 17.97/17.53 8.68/−1.4 GC12 Gln11→Leu, Asp95→Glu, V113→Ile 7.91 21.77/23.6 2.89/0.64 GC19 Frameshift Δ, aa 73-77 8.24 21.06/20.84 7.6/0.74 GC21 Gln11→Leu, Asp95→Glu, Val 113→Ile 8.09 23.34/21.75 3.72/0.01 Kp342 Asn38→Thr, Asp95→Glu, Val113→Ile, His159→Leu 6.21 21.76/22.03 1.77/−0.735 Ecl8Mdr1 Frameshift Δ, aa 88-94 10.46 17.88/17.44 9.06/0.28 ↵a Strains whose names are underlined were complemented with pACoqxR. Boldface font denotes amino acid changes present in several strains. Expression levels of rarA calibrated against sensitive K. pneumoniae strain Ecl8 are shown.
↵b Con, control data representing either the wild-type or vector-only strains (pACYC177); Comp, complementation data representing increases or decreases of rarA or oqxA expression levels in the different strains after complementation with pACOqxR. Negative values indicate reduction of oqxA levels below the levels noted in the vector-only calibrators. —, strains not complemented due to high levels of innate kanamycin resistance. aa, amino acids.
- Table 6
Susceptibility profiles of K. pneumoniae strains after complementation with pACoqxRa
Strain MICs (mg/liter) for strain transformed with pACoqxR/paCYC177 CHL OQX TET TIG NOR CIP GC9 64/64 768/768 16/16 4/4 8/8 8/8 GC12 24/24 768/768 16/16 8/8 8/8 12/12 GC19 256/256 768/768 32/32 4/4 4/4 1/1 GC21 24/24 768/768 16/16 8/8 8/8 12/12 Kp342 48/64 384/512 32/32 8/8 4/8 0.375/0.500 Ecl8Mdr1 32/32 384/512 4/4 1/1 8/8 0.500/0.500 ↵a MICs show values for strains complemented with pACoqxR/strains complemented with pACYC177. Numbers in bold indicate MIC reductions seen with the complemented strain versus the vector-only control. CHL, chloramphenicol, OQX, olaquindox, TET, tetracycline, TIG, tigecycline, NOR, norfloxacin, CIP, ciprofloxacin.
