In order to mine the K. pneumoniae genomes for other uncharacterized AraC-type transcriptional regulators, we used the amino acid sequence for the prototype regulator MarA (KPN_01624) from K. pneumoniae MGH 78578. We focused on the top five hits from the BLAST analyses, in descending order of identity, which were Rob (53%), RamA (46%), KPN_02968 (called rarA [46%]), SoxS (42%), and another putative AraC-type regulatory protein, KPN_01709 (34%). We decided to focus on rarA in further work for two reasons: first, because of its higher level of identity to MarA, and second, because of its predicted size of 121 amino acids being closer to that of the subset of AraC regulators such as RamA, SoxS, and MarA rather than the considerably larger KPN_01709 (326 amino acids). As expected, the phylogenetic tree generated using MUSCLE for multiple alignments of the five AraC regulators () shows that KPN_02968 is closely related to the MarA, SoxS, and RamA proteins; however, KPN_01709 appears to be an outlier, as shown by the branch lengths denoting relative sequence similarities.
Antimicrobial susceptibility testing.
Given the identity of RarA with the other AraC-type proteins such as MarA and RamA, we hypothesized that RarA might possess similar functional properties in conferring low-level multidrug resistance. In order to address this, we cloned the open reading frame encoding rarA with its putative promoter region into pACYC177 or pACYC184 and determined the multidrug resistance phenotype conferred by this regulator in both wild-type E. coli and K. pneumoniae strains as well as in strains harboring deletions in various loci such as marA, ramA, and the efflux operon acrAB (). Overexpression of rarA in the ΔmarA strain (E. coli K-12 MG1655 ΔmarA) led to increases in MICs as follows: a 2-fold increase in the tigecycline MIC, 4-fold increases in the ciprofloxacin, norfloxacin, and tetracycline MICs, and an 8-fold increase in the chloramphenicol MIC (). Similarly, in the AG100 ΔsoxS Δrob ΔmarA strain, the increased expression of pACrarA-2 resulted in increases in the olaquindox and ciprofloxacin MICs (2-fold) and the tigecycline and norfloxacin MICs (8-fold) relative to the MICs seen with the vector-only control (). However, when the pACrarA-2 construct was overexpressed in AG100A, there were no differences in the susceptibility profiles in comparison to the vector-only control, implying that the multidrug-resistant phenotype is dependent on the presence of a functional acrAB efflux pump. Regardless of the absence of marA, soxS, or rob, either singly or in combination, overexpression of rarA resulted in a low-level multidrug resistance phenotype in E. coli ().
Susceptibility profiles of E. coli strains transformed with pACrarA and vector-only controla
Correspondingly, in K. pneumoniae Ecl8 ΔramA, the absence of ramA did not affect the multidrug-resistant phenotype when rarA was overexpressed (). The increases in MIC ranged from 2-fold for tetracycline and norfloxacin to 4-fold for olaquindox, ciprofloxacin, and tigecycline and 8-fold for chloramphenicol. The deletion of the rarA open reading frame (Ecl8 ΔrarA) resulted in a 2- to 8-fold reduction in MIC (). As expected, the complementation of the rarA regulator in trans resulted in MIC levels higher than that observed for the parental strains. Similar to the experiments in E. coli, overexpression of rarA in K. pneumoniae Ecl8 ΔacrAB did not result in an MDR phenotype ().
Susceptibility profiles of K. pneumoniae strains transformed with pACrarA and vector-only controla
Gene expression levels of rarA in clinical multidrug-resistant isolates of K. pneumoniae.
In order to establish a role for rarA in clinical resistance, we determined by quantitative real-time RT-PCR whether rarA was upregulated in clinical isolates of K. pneumoniae obtained from various geographical locations. In our survey, we included 17 multidrug-resistant strains collected from Chile, Turkey, and Germany, where our results show that of the 17 strains tested, 7 overexpressed rarA-specific transcripts compared to the sensitive K. pneumoniae strain Ecl8 (see ), including two Turkish isolates, TS152 (6.62-fold) and TS165 (3.77-fold), as well as one from Chile (TS202; 7.37-fold). Most of the isolates from Germany also showed overexpression of the rarA regulator (GC9, 9.6-fold; GC12, 7.91-fold; GC19, 8.24-fold; GC21, 8.09-fold). For those strains that overexpressed rarA, we were also able to demonstrate that the levels of either marA or ramA were also elevated (4- to 5-fold) among all isolates showing expression (data not shown). Furthermore, analyses of rarA and oqxA levels in the constitutive rarA expresser, K. pneumoniae strain Ecl8/pACrarA-1 (), demonstrated the increased transcription of both genes (see ).
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, (more ...)
In all the strains where rarA overexpression was noted, we sought to determine the molecular basis of upregulation by initially focusing on identifying changes within the promoter and associated regions of the rarA regulator by (i) determining the transcriptional start site of rarA, (ii) mapping the changes within the intergenic region between rarA and the oqxAB operon relative to the transcription-relevant sequences (), and (iii) determining the sequence of the rarA regulator itself.
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 (more ...) DNA sequence analyses of the intergenic region and ORF of the rarA regulator. (i) Intergenic region.
Sequence analyses within the intergenic region for detection of polymorphisms highlighted changes within many of the rarA-overexpressing strains (). To clarify the possible significance of these intergenic changes, we performed 5′ RACE experiments using strain K. pneumoniae Ecl8Mdr1 to determine the transcription start site (TSS), which maps 58 bp upstream of the open reading frame of rarA (). Notably, none of the rarA-overexpressing clinical strains showed changes within regions, e.g., the rarA TSS, −10 or −35 hexamer, relevant to gene transcription (). In contrast, all of the rarA-overexpressing strains (TS152, TS165, Kp342, GC9, GC12, GC19, and GC21), with the exception of Ecl8MDR1 and TS202, harbored changes approximately 121 to 131 bp upstream of the rarA open reading frame (), where the most common change was a C insertion at 131 bp (). However, two of the rarA overexpressers (GC12 and GC21) showed identical changes (C8→T and T11→G) within the putative Shine-Dalgarno sequence (). Interestingly, no changes were found in multidrug-resistant strains Ecl8Mdr1 and TS202, but those strains still overexpressed rarA. Our findings suggest that the molecular basis for rarA upregulation may not be linked to the changes identified within the intergenic region.
(ii) rarA regulator.
Only 4 (GC19, Ecl8, Ecl8Mdr1, and Kp342) of the 10 strains sequenced (9 rarA
overexpressers and the sensitive Ecl8 strain) harbored changes within the rarA
regulator. Identical mutations in rarA
leading to a Glu96→Arg substitution were found in both nonexpresser K. pneumoniae
Ecl8 and rarA
overexpresser K. pneumoniae
Ecl8Mdr1. This change is located within the α-helix in the predicted HTH binding site (http://bioinf.cs.ucl.ac.uk/psipred/
); however, its presence in both strains implies that it is not a crucial residue. Several substitutions not present in other strains were identified in K. pneumoniae
Kp342: Ala31→Ser, Lys57→Gln, Ile63→Val, Val111→Ala, Ala112→Glu, and Thr114→Ala. This strain also harbors a mutation at position Arg117→STOP which leads to a premature stop codon (TGA). Of all the other clinical isolates that overexpress rarA
, only GC9 showed a unique change, Gln99→Lys, within the sequence between helices 6 and 7, proximal to the C-terminal end of the protein.
(KPN_02969 and KPN_02970) efflux operon lies downstream from the rarA
regulator where the oqxAB
pump has been associated with reduced susceptibility to olaquindox, ciprofloxacin, and chloramphenicol (15
). Interestingly, all clinical strains that showed upregulation of rarA
also demonstrated increased expression of KPN_02969 (oqxA
) (). In order to dissect the molecular basis for this upregulation, we determined the following: (i) whether there was an association between rarA
upregulation and (ii) whether the GntR-type regulator (OqxR_KPN_02971) encoded downstream of the oqxAB
operon would function as a repressor.
Our results show that plasmid-mediated overexpression of rarA resulted in increased levels of oqxA in K. pneumoniae Ecl8 and Ecl8ΔrarA (). Additionally, a reproducible (1.5-fold, mean of 4 experiments) increase in acrAB levels was also observed in the same strains. Therefore, we surmise that rarA may function as a positive regulator of oqxAB and acrAB levels.
Putative regulator OqxR. (i) Sequencing results.
We first sequenced all the rarA
- and oqxAB
-overexpressing strains and found that not all strains (e.g., TS202) that overexpressed oqxA
(KPN_02969) harbored changes within the oqxR
gene (). However, there were several recurring changes, namely, Gln11→Leu (GC12 and GC21), Asp95→Glu (GC12 and GC21), Val113→Ile (GC12, GC21, and and KP342) and Val130→Ala (TS152 and TS165). Based on alignments with other GntR family regulators, Gln11→Leu is located within the DNA-binding domain of the winged helix-turn-helix (WHTH) of OqxR. The other mutations were located within the predicted C-terminal effector binding and oligomerization domain (26
Sequence analysis of OqxR and rarA/oqxA expression levels of clinical K. pneumoniae isolatesa
(ii) oqxR overexpression effects on 02969 (oqxA) levels.
In order to confirm that the mutations noted within OqxR would be directly associated with increased expression of the oqxAB efflux pump, we performed complementation assays with wild-type oqxR on strains (Ecl8Mdr1, GC9, GC12, GC19, GC21, and Kp342) that overexpressed oqxAB. QPCR analyses showed that oqxA levels were lower in all of the complemented strains than in the vector-only control strains, while rarA levels remained unaffected (). Only two (Kp342 and Ecl8Mdr1) of the six strains were found to show reductions in olaquindox MICs (). The lack of reduction in olaquinodox MICs noted for the clinical strains (GC9, GC12, GC19, and GC21) may have been due to other mechanisms (). In all strains (Ecl8Mdr1, GC9, GC12, GC19, GC21, and Kp342) where we expressed the recombinant oqxR, we also ascertained that the levels of the AcrA protein (Western blot analyses for the AcrA protein; data not shown) remained identical to those of the vector-only controls. From our results, we surmise that a decrease in oqxAB transcription does result in the reduction of olaquindox MICs for some strains.
Susceptibility profiles of K. pneumoniae strains after complementation with pACoqxRa