A multicopy suppressor screening approach as a means to identify antibiotic resistance determinant candidates in Yersinia pestis.

BACKGROUND: Yersinia pestis is the causative agent of plague and a potential agent of bioterrorism and biowarfare. The plague biothreat and the emergence of multidrug-resistant plague underscore the need to increase our understanding of the intrinsic potential of Y. pestis for developing antimicrobial resistance and to anticipate the mechanisms of resistance that may emerge in Y. pestis. Identification of Y. pestis genes that, when overexpressed, are capable of reducing antibiotic susceptibility is a useful strategy to expose genes that this pathogen may rely upon to evolve antibiotic resistance via a vertical modality. In this study, we explored the use of a multicopy suppressor, Escherichia coli host-based screening approach as a means to expose antibiotic resistance determinant candidates in Y. pestis.
RESULTS: We constructed a multicopy plasmid-based, Y. pestis genome-wide expression library of nearly 16,000 clones in E. coli and screened the library for suppressors of the antimicrobial activity of ofloxacin, a fluoroquinolone antibiotic. The screen permitted the identification of a transcriptional regulator-encoding gene (robAYp) that increased the MIC99 of ofloxacin by 23-fold when overexpressed from a multicopy plasmid in Y. pestis. Additionally, we found that robAYp overexpression in Y. pestis conferred low-level resistance to many other antibiotics and increased organic solvent tolerance. Overexpression of robAYp also upregulated the expression of several efflux pumps in Y. pestis.
CONCLUSION: Our study provides proof of principle for the use of multicopy suppressor screening based on the tractable and easy-to-manipulate E. coli host as a means to identify antibiotic resistance determinant candidates of Y. pestis.

Background

Yersinia pestis (Yp) is one of the most virulent known bacteria [1] and a potential agent of bioterrorism and biowarfare [2,3] included in the Category A of biological agents for public health preparedness against bioterrorism [4]. Yp is the etiologic agent of plague, a disease responsible for millions of human deaths during the history of civilization [5,6]. Cases are reported every year in many parts of the world [7] and the increasing number of worldwide cases has placed plague in the category of re-emerging diseases [8].

Patients with plague need prompt antibiotic treatment or else death may be unavoidable. The aminoglycosides streptomycin (STR) and gentamicin (GEN) are the preferred antibiotics for treatment, but a number of other drugs are also effective [9,10]. Tetracyclines [such as doxycycline (DOX)], chloramphenicol (CHL), or selected sulfonamides are the recommended antibiotics for prophylactic therapy in the event of exposure or high risk of exposure to Yp [2,9,10]. Fluoroquinolones have also been suggested for treatment and prophylaxis and are noted as a chemotherapeutic alternative against strains resistant to the first line anti-plague drugs [2,10].

The threat of bioterrorism-generated plague outbreaks with engineered (multi)drug-resistant Yp strains [2,3] and the documented outbreak of multidrug-resistant plague [11] underscore the need to develop alternative chemotherapeutic solutions to this disease. In line with this view, we are exploring the development of anti-infectives that target the high-affinity iron acquisition system of Yp [12-14] and may offer novel therapeutic possibilities [15]. The plague biothreat also underscores the need to increase our understanding of the intrinsic potential of Yp for developing antimicrobial resistance and to anticipate the mechanisms of resistance that may emerge in Yp clinical isolates in the future. With this consideration in mind, we explored herein the use of a multicopy suppressor screening approach as a means to expose antibiotic resistance determinant candidates in Yp. Multicopy suppressor screening has been useful to study potential drug targets or mechanisms of antibiotic resistance in other species [16]. We constructed a multicopy plasmid-based, Yp genome-wide expression library of nearly 15,000 clones in E. coli (Ec), a tractable and easy-to-manipulate surrogate bacterial host, and screened the library for suppressors of the antimicrobial activity of the fluoroquinolone antibiotic ofloxacin (OFX). Noteworthy, fluoroquinolones have been suggested by the Working Group on Civilian Biodefense as alternative drugs in the event of the use of aerosolized Yp as a bioweapon against a civilian population [2]. The screen permitted the identification of a gene that reduced the susceptibility of Yp to fluoroquinolones and other antibiotic classes when overexpressed from a multicopy plasmid. Our study provides proof of principle for the utilization of multicopy suppressor screening using an Ec host as a means to identify antibiotic resistance determinant candidates in Yp.

Results and Discussion

A multicopy suppressor screen led to the isolation of a Y. pestis genomic fragment involved in ofloxacin resistance

We constructed a plasmid-based expression library of the Yp genome comprised of 15,648 Ec clones and screened the library for strains with reduced OFX susceptibility. A strain (Ec pGEM-OFXr1) selected in the screen exhibiting reduced susceptibility that was confirmed to be plasmid-mediated and transferable to Yp was chosen for further characterization (Figure 1). The plasmid (pGEM-OFXr1) carried by this strain was isolated and the restriction digestion pattern and sequence of its genomic insert were examined. This analysis revealed a 4,158-bp fragment (Yp KIM chromosome coordinates 4,137,482 to 4,141,639) (Figure 2). The 5' and 3' ends of the fragment included the 5' end of y3722 (creA) and the 3' end of y3727 (slt), respectively. The products of creA and slt are annotated as a conserved hypothetical protein and a putative soluble lytic murein transglycosylase, respectively, in the Yp genome database. The center of the fragment encompassed four genes: y3723 (robA, herein referred to as robAYp); y3724 (gpmB); y3725; and y3726 (trpR). The products of gpmB and trpR are annotated as a putative phosphoglyceromutase and a putative regulator of tryptophan metabolism genes, respectively. The product of y3725 is annotated as a conserved hypothetical protein. Our in silico search for conserved domains (via CD-Search; please see Availability & requirements for more details) revealed the presence of an NTPase (PRK05074) domain in this protein. The NTPase domain is characteristic of proteins with pyrophosphatase activity [17,18]. This suggested that y3725 may be involved in nucleoside triphosphate metabolism. Lastly, the predicted product of robAYp (RobAYp) is annotated as an orthologue of Ec RobA (RobAEc), a transcriptional regulator of unclear physiological function and member of the AraC/XylS family [19]. Importantly, overexpression of robAEc and Enterobacter cloacae robA confers low-level resistance in Ec and E. cloacae, respectively, to a number of unrelated antibiotics [20-22]. Thus, the analysis of the insert in pGEM-OFXr1 suggested that robAYp is responsible for the reduced OFX susceptibility observed in Ec pGEM-OFXr1 and Yp pGEM-OFXr1 (Figure 1). These results validate the utility of our library and suppressor screen approach as a means to identify antibiotic resistance determinant candidates in Yp.

Figure 1

Reduction of ofloxacin susceptibility conferred by plasmids pGEM-OFXr1 and pGEM-Rob. E. coli (Ec) and Y. pestis (Yp) strains were streaked on solid media without or with ofloxacin: 0.35 μg/ml (the concentration used in the screen) for E. coli and 0.15 μg/ml for Y. pestis. Ampicillin (100 μg/ml) was also added to the media for plasmid-carrying strains.

Figure 2

Genetic map of the robAYp-containing region of the Y. pestis KIM chromosome and inserts of pGEM-OFXr1 and pGEM-RobYp.

Overexpression of robAYp affects susceptibility to multiple antibiotics

We investigated whether overexpression of robAYp alone would reduce OFX susceptibility in Ec and, more importantly, in Yp. To this end, we evaluated the antibiotic susceptibility of Ec pGEM-RobYp and Yp pGEM-RobYp. These test strains carried pGEM-RobYp, a plasmid constructed by inserting the fragment encompassing robAYp and its promoter region (identified by using the robAEc promoter as reference [23]) into the vector pGEM-4Z. The antibiotic susceptibilities of these test strains were compared to that of the corresponding Ec pGEM-4Z and Yp pGEM-4Z control strains. These control and test strains were isogenic, except for the lack of the plasmid-borne robAYp, and their growth in ampicillin (AMP)-containing liquid media was indistinguishable from that of their cognate test strains (not shown). A first examination of Ec pGEM-RobYp and Yp pGEM-RobYp indicated that these strains retained the reduced OFX susceptibility phenotype seen in Ec pGEM-OFXr1 and Yp pGEM-OFXr1 on solid media (Figure 1), thus indicating that robAYp alone was sufficient to reduce OFX susceptibility. In view of this, we conducted further OFX susceptibility testing in liquid media. In addition, we compared the susceptibility of the test and control strains to two other fluoroquinolones [ciprofloxacin (CIP) and levofloxacin (LVX)], a quinolone (NAL), and antibiotics of other classes, including two tetracyclines [tetracycline (TET) and DOX], four aminoglycosides [STR, GEN, kanamycin (KAN), and apramycin (APR)], and CHL.

The IC50 and MIC99 values determined for the aforementioned antibiotics are shown in Table 1. Comparison of the OFX IC50 and OFX MIC99 values of the test strains and their respective control strains revealed that overexpression of robAYp reduced OFX susceptibility in both Yp and Ec. In Yp, robAYp overexpression increased OFX IC50 and OFX MIC99 values by 5-fold and 23-fold, respectively. The IC50 and MIC99 values of CIP, LVX, and NAL also increased significantly (3- to 5-fold change range) in Yp pGEM-RobYp compared with Yp pGEM-4Z. The reduced OFX and LVX susceptibility of Yp pGEM-RobYp was also revealed by time-kill experiments described below. In Ec, robAYp overexpression produced an increase in the IC50 and MIC99 of the fluoroquinolone antibiotics (2- to 4-fold change range), but had no significant effect (<2-fold change) on NAL susceptibility.

Table 1

Effect of robAYp overexpression on antibiotic susceptibility

	IC50 (μg/ml)a			FCc	MIC99 (μg/ml)b			FC
Y. pestis	no plasmid	pGEM-4Z	pGEM-RobYp		no plasmid	pGEM-4Z	pGEM-RobYp
Ofloxacin	0.01	0.01	0.05	5	0.03	0.03	0.7	23
Ciprofloxacin	0.009	0.009	0.04	4	0.02	0.02	0.09	5
Levofloxacin	0.01	0.01	0.04	4	0.02	0.02	0.09	5
Nalidixic Acid	0.5	0.8	2	3	2	2	6	3
Chloramphenicol	0.3	0.4	0.9	2	1	1	3	3
Tetracycline	0.7	1	6	6	1	3	10	3
Doxycycline	0.4	0.5	1	2	0.6	1	3	3
Kanamycin	1	0.9	0.4	0.4	2	2	2	1
Apramycin	2	3	1	0.3	6	6	4	0.7
Streptomycin	1	2	0.8	0.4	3	3	3	1
Gentamicin	0.4	0.4	0.2	0.5	1	0.4	0.4	1
E. coli	no plasmid	pGEM-4Z	pGEM-RobYp		no plasmid	pGEM-4Z	pGEM-RobYp
Ofloxacin	0.03	0.04	0.09	2	0.08	0.2	0.4	2
Ciprofloxacin	0.01	0.009	0.04	4	0.05	0.08	0.2	3
Levofloxacin	0.04	0.04	0.1	3	0.2	0.2	0.4	2
Nalidixic Acid	27	26	28	1	83	100	100	1
Chloramphenicol	0.3	0.3	0.8	3	2	2	13	7
Tetracycline	0.8	0.6	2	3	3	3	10	3
Doxycycline	0.3	0.2	0.6	3	1	0.6	3	5
Kanamycin	2	1	2	2	10	6	6	1
Apramycin	3	3	4	1	21	17	21	1
Streptomycin	3	2	3	2	17	13	13	1
Gentamicin	0.9	0.6	2	3	6	5	8	2

a IC50 values were calculated from sigmoidal curves fitted to triplicate sets of dose-response data. b MIC99 values are means of triplicates. c Fold change (FC) values were calculated as the ratio of the IC50 or MIC99 of the pGEM-RobYp transformants to the IC50 or MIC99 of the pGEM-4Z transformants. IC50, MIC99, and FC values <1 and values >1 were rounded to one significant digit and to the nearest non-fractional number, respectively.

In both Yp and Ec, robAYp overexpression also correlated with an increase in the IC50 and MIC99 of the two tetracyclines tested and CHL (2- to 7-fold change range). No substantial impact (<2-fold change) on the MIC99 values of four aminoglycosides tested was detected in Yp upon overexpression of robAYp. Interestingly, however, the IC50 values of these aminoglycosides were reproducibly and consistently lower (2- to 3-fold reduction range) in Yp pGEM-RobYp compared with Yp pGEM-4Z. These results indicated that robAYp overexpression increased the susceptibility of Yp to aminoglycosides. The hypersensitivity of Yp pGEM-RobYp to aminoglycosides was also observed in time-kill experiments described below. Aminoglycoside hypersensitivity was not observed in Ec pGEM-RobYp. On the contrary, the strain had a modest decrease in the susceptibility to GEN, STR, and KAN (2- to 3-fold change range) relative to Ec pGEM-4Z.

Overall, the phenotypic comparison of the antibiotic susceptibility of pGEM-RobYp-bearing strains and pGEM-4Z-bearing strains clearly demonstrates that robAYp overexpression affects antibiotic susceptibility in both Yp and Ec, yet in a noticeably species-specific manner. As discussed below, the effects on antibiotic susceptibility induced by robAYp overexpression are likely due to an upregulation of efflux pumps. Thus, the species-specific differences in antibiotic susceptibility are probably produced by species-specific differences in efflux pump upregulation.

Overexpression of robAYp in Y. pestis reduces killing by fluoroquinolones but enhances killing by aminoglycosides

The comparative analysis of IC50 and MIC99 values described above indicated that Yp pGEM-RobYp has reduced fluoroquinolone susceptibility and increased aminoglycoside susceptibility compared with Yp pGEM-4Z. To further probe these phenotypes, we examined the killing kinetics of these two strains when exposed to OFX, CIP, STR, and GEN (Figure 3). The profiles of the time-kill curves for OFX and CIP demonstrated that the Yp pGEM-4Z control was more rapidly killed by the fluoroquinolones than Yp pGEM-RobYp. Conversely, the profiles of the time-kill curves for STR and GEN revealed that Yp pGEM-RobYp was more rapidly killed by the aminoglycosides than the Yp pGEM-4Z control. Both strains had comparable growth in the absence of fluoroquinolone or aminoglycoside antibiotics during the time frame of the time-kill assays. The contrasting effects of robAYp overexpression on fluoroquinolone- and aminoglycoside-mediated killing are consistent with the results of the comparative analysis of IC50 and MIC99 values (Table 1). The observed aminoglycoside hypersensitivity is somewhat unexpected and contrasts with both the reduced susceptibility observed for all other antibiotics tested and the increased tolerance to organic solvents described below.

Figure 3

Effect of . Yp pGEM-RobYp (overexpressing robAYp) and Yp pGEM-4Z (vector control) were treated with the indicated antibiotics at 5 × MIC99. The means of triplicate treated cultures were plotted and standard error bars are shown.

Overexpression of robAYp increases tolerance to organic solvents

We investigated whether robAYp overexpression affected the susceptibility of Yp and Ec to O2.--generating compounds (paraquat, menadione, and plumbagin), heavy metals (zinc, cobalt, and copper), and organic solvents (n-pentane, n-hexane, cyclohexane, p-xylene, and diphenyl ether). No effect on the susceptibility to O2.--generating compounds, cobalt, and copper was observed (not shown). Conversely, overexpression of robAYp drastically increased organic solvent tolerance in both Yp and Ec (Figure 4) and reduced the susceptibility of Yp to zinc (not shown). Overexpression of robAYp increased the tolerance of Yp to n-hexane and cyclohexane and the tolerance of Ec to cyclohexane and n-pentane. All the Yp and Ec strains were resistant to diphenyl ether and sensitive to p-xylene. Ec was also resistant to n-hexane, a result that is in agreement with previous reports [24]. These findings parallel the reduction of organic solvent susceptibility induced by robAEc overexpression in Ec [21].

Figure 4

Effect of . Cultures of Y. pestis (Yp) and E. coli (Ec) strains carrying the plasmids indicated were spotted on solid medium. The surface of the medium was overlaid with the organic solvent and growth was recorded after incubation. H, n-hexane; CH, cyclohexane; P, n-pentane; X, p-xylene; DE, diphenyl ether; -, no solvent control.

Overexpression of robAYp in Y. pestis induces changes in efflux pump gene expression

In Ec, the RobAEc-induced multidrug resistance and solvent tolerance phenotypes have been shown to be largely dependent on the upregulation of the multidrug efflux pump AcrAB, which belongs to the resistance-nodulation-cell division (RND) superfamily [25,26]. With this precedent in mind, we compared the expression of genes belonging to 34 drug efflux pumps between Yp pGEM-RobYp and Yp pGEM-4Z using quantitative real-time PCR (qRT-PCR). These pumps were identified using a variety of bioinformatic approaches (see Methods) to compile an extensive list that included most, if not all, putative drug efflux pump systems encoded in the genome of Yp. It is worth mentioning as a reference that there are 37 drug efflux pumps annotated in the Ec genome [27,28]. Our expression analysis detected transcripts for 33 of the 34 genes investigated and revealed that four efflux pumps were significantly upregulated (≥ 5-fold change) in Yp pGEM-RobYp compared with Yp pGEM-4Z (Table 2). Interestingly, two of these upregulated pumps (y3392-y3393 and y1050-y1049) are Ec AcrAB homologs. The other two upregulated pumps (y2173 and y0010) belong to the major facilitator superfamily (MFS). The transcript level of hasF (y3516), encoding the ortholog of Ec TolC, which is the outer membrane protein channel that partners with Ec AcrAB and other RND and MFS pumps [29,30], was drastically upregulated as well (5.8-fold change; not shown).

Table 2

Effect of robAYp overexpression on the transcript levels of efflux pump genes in Y. pestis

Gene namea	University of Wisconsinb	TIGR	SANGER	Pump family or protein functionc	FCd
(floR)	y2173	NT02YP2579	YPO2148	MFS	12.7 ± 2.5
(yieO)	y0010	NT02YP0010	YPO0009	MFS	8.6 ± 1.1
(acrA*-acrB)	y3392*-y3393	NT02YP4041	YPO1000	RND	6.9 ± 0.4
acrA*-acrB	y1050*-y1049	NT02YP1227	YPO3132	RND	5.0 ± 0.4
(acrA*-acrB)	y3760*-y3759	NT02YP4463	YPO0420	RND	2.7 ± 0.1
-	y4041	NT02YP4789	YPO4020	DMT	1.7 ± 0.9
yegM*-yegN-yegO-yegB	y1386*-y1385-y1384-y1383	NT02YP1646	YPO2847	RND	1.4 ± 0.5
(ybjY*-ybjZ)	y2814*-y2813	NT02YP3366	YPO1364	ABC	1.4 ± 0.4
(acrA*-acrB)	y0702*-y0703	NT02YP0804	YPO3483	RND	1.3 ± 0.3
emrA*-emrB	y0922*-y0921	NT02YP1066	YPO3267	MFS	1.3 ± 0.4
(macA*-macB)	y1481*-y1480	NT02YP1756	YPO2999	RND	1.3 ± 0.5
ygeD	y3180	NT02YP3786	YPO0792	MFS	1.3 ± 0.2
fieF	y0060	NT02YP0067	YPO0077	CDF	1.2 ± 0.1
(abgT)	y3402	NT02YP4052	YPO1008	IT	1.1 ± 0.2
acrD	y1439	NT02YP1705	YPO3043	RND	1.1 ± 0.1
sugE	y0613	NT02YP0702	YPO0355	SMR	1.1 ± 0.4
(ynfM)	y2108	NT02YP2497	YPO2266	MFS	1.1 ± 0.4
bcr	y2916	NT02YP3488	YPO1267	MFS	1.1 ± 0.3
aaeA*-aaeB	y0178*-y0177	NT02YP0192	YPO3685	ArAE	1.1 ± 0.0
(yjcR*-Q)	y3558*-y3559	NT02YP4231	YPO0619	RND	1.0 ± 0.2
yajR	y1017	NT02YP1187	YPO3169	MFS	1.0 ± 0.1
arsB	y0844	NT02YP0968	YPO3347	IT	1.0 ± 0.4
corC	y1191	NT02YP1397	YPO2617	HCC	0.9 ± 0.1
mdtJ*-mdtI	y2242*-y2241	NT02YP2670	YPO2068	SMR	0.8 ± 0.2
rosA	y1087	NT02YP1268	YPO3093	MFS	0.8 ± 0.1
ydhC	y1948	NT02YP2306	YPO2389	MFS	0.8 ± 0.2
	y3186	NT02YP3792	YPO0798	MFS	0.7 ± 0.1
(ydhE)	y1945	NT02YP2302	YPO2392	MATE	0.7 ± 0.1
(ybeQ)	y1874	NT02YP2220	YPO1712	MFS	0.7 ± 0.0
mdlA*-mdlB	y1039*-y1040	NT02YP1213	YPO3145	ABC	0.7 ± 0.0
ydeF	y2653	NT02YP3168	YPO1515	MFS	0.5 ± 0.1
emrE, gacE	y2000	NT02YP2368	YPO2333	SMR	0.5 ± 0.0
mdfA, cmr	y4067	NT02YP4824	YPO4048	MFS	0.2 ± 0.0
(ydjV)	y2272	NT02YP2703	YPO2040	MFS	nd

a Gene name as annotated for Y. pestis strain KIM and/or CO92 or gene names (in parentheses) given herein based on the name of their E. coli homologs. Multiple names for the same gene are separated by commas. Genes of multi-component pumps are separated by dashes. The star (*) marks genes from multi-component pump gene clusters whose transcripts were analyzed by qRT-PCR. b Gene designations in the University of Wisconsin, TIGR, and SANGER Y. pestis genome databases. The Wisconsin column shows designations for all the genes in each predicted multi-component pump. TIGR and SANGER columns show only genes targeted in qRT-PCR. c Pump families assigned based on homology to known (super)family members from other organisms. MFS, major facilitator superfamily; SMR, small multidrug resistance family; ABC, ATP-binding cassette superfamily; RND, resistance-nodulation-cell division superfamily; MATE, multidrug and toxic compound extrusion family; DMT, drug/metabolite transporter superfamily; CDF, cation diffusion facilitator family; ArAE, aromatic acid exporter family; IT, Ion transporter superfamily; HCC, HlyC/CorC family. d Fold change values (FC) are means of triplicates ± standard errors and are presented in decreasing order. For polycistronic transcripts, qRT-PCR was conducted with primers targeting the first pump component-encoding gene of the operon. These genes are marked with a star. nd, transcript not detected in any of the three Y. pestis strains examined (wild-type, Yp pGEM-4Z, and Yp pGEM-RobYp).

Inspection of the promoter regions upstream of the upregulated genes in Yp pGEM-RobYp revealed the presence of a putative RobAEc binding site in each of these regions (Figure 5). These results suggest that RobAYp may act as a positive regulator for the y0010, y1050-y1049, y2173 and y3392-y3393 systems. This possible regulatory scenario is consistent with the upregulation in the expression levels of these pumps induced by robAYp overexpression in Yp.

Figure 5

Potential RobA binding sites in the promoter regions of genes upregulated in . The consensus shown is the 20-bp asymmetric marbox consensus sequence determined by Martin et al., 1999 [37]. R = A or G, Y = C or T, W = A or T, and N = A, T, G, C. Column C is the number of bp's in agreement with the 20-bp consensus sequence. The location of each RobA binding site with respect to the first codon of its cognate gene is indicated by the numbers flanking the putative binding site.

It is likely that the multidrug resistance and solvent tolerance phenotypes induced by robAYp overexpression are due, at least in part, to increases in compound extrusion by one or more of the upregulated pumps mentioned above. This idea is supported by the reported observation that the RobAEc-induced multidrug resistance and solvent tolerance in Ec is largely dependent on the AcrAB-TolC efflux pump system [25,26].

Conclusion

The identification of Yp genes that, when overexpressed, are capable of reducing antibiotic susceptibility is a useful strategy to expose genes that this pathogen may rely upon to evolve resistance via a vertical modality. In this study, we explored the use of a multicopy suppressor, Ec host-based screening approach as a means to identify antibiotic resistance determinant candidates in Yp. To seek proof of principle for this approach, we constructed a multicopy plasmid-based, Yp genome-wide expression library of nearly 16,000 clones in Ec and screened this library for suppressors of the antimicrobial activity of the fluoroquinolone antibiotic OFX. The screen permitted the identification of a gene that, when overexpressed, reduces the susceptibility of Yp not only to OFX, but also to other (fluoro)quinolones, tetracyclines and CHL. This gene (robAYp) encodes a putative transcriptional regulator, and our results clearly demonstrate that its overexpression in Yp and Ec confers low-level resistance to multiple antibiotics. Overexpression of robAYp also increases organic solvent tolerance in both Yp and Ec and reduces the susceptibility of Yp to zinc.

The molecular mechanism by which overexpression of robAYp leads to a reduction in the susceptibility to antibiotics and other compounds remains to be determined. Our results indicate that overexpression of robAYp induces a drastic upregulation in the transcript levels of four of the 34 predicted efflux pump gene systems and of hasF (tolC) in Yp. Increased expression of chromosomally encoded efflux pumps is a known cause of multidrug resistance in many bacteria [30]. Thus, it is likely that the reduction in the susceptibility to antibiotics and other compounds induced by robAYp overexpression is due, at least in part, to multidrug efflux pump-mediated increases in compound extrusion.

Overall, our findings provide proof of principle for the utilization of an Ec host-based suppressor screen to identify antibiotic resistance determinant candidates in Yp. This methodology will be useful in the identification of genetic determinants involved in target-dependent and target-independent resistance to antimicrobials with known and unknown mechanisms of action. Identification of such genetic determinants will provide first insights to guide further studies to obtain mechanistic information on novel modes of antimicrobial activity and antimicrobial resistance.

Methods

Construction of genomic library

Unless otherwise indicated, all molecular biology and microbiological manipulations were conducted using standard procedures [31] and reagents acquired from New England Biolabs or Sigma-Aldrich. Genomic DNA from the avirulent Yp strain KIM6+ [32] was used for the library. This strain lacks the Lcr virulence plasmid [5] and is excluded from the Select Agent Program (please see Availability & requirements for more details). Genomic DNA was prepared using AquaPure™ Genomic DNA Isolation Kit (Bio-Rad Laboratories) and partially digested with BsrFI, which, on average, cleaves the genome of Yp every ~760 bp. Independent partial digestions were resolved by agarose gel electrophoresis and the fragment populations in the 4,000-bp to 8,000-bp range were purified using QIAquick Gel Extraction Kit (Qiagen). The fragments were ligated to the multicopy plasmid vector pGEM-4Z (Promega) linearized with XmaI and dephosphorylated with calf intestine alkaline phosphatase. Genes inserted into pGEM-4Z can be transcribed from the gpt-lac hybrid promoter located at the 5'-end of the cloning site and, potentially, from their native promoters. Ligations were transformed into Ec DH5α (Invitrogen) and transformants were selected in Luria-Bertani (LB) agar plates containing AMP (100 μg/ml) and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (40 μg/ml) for blue/white colony screening [31]. White colonies were streaked onto the same medium to verify their white phenotype and 15,648 confirmed white clones were independently grown in AMP-containing LB broth in 96-well plates. After plate incubation for culture growth to early stationary phase, aliquots from each of the 96 cultures of each plate were pooled, cells from each pool were harvested, and plasmids from each pool were purified using QIAprep Spin Miniprep Kit (Qiagen). The cultures in the plates were supplemented with glycerol (25%) and this master library was stored at -70°C. Agarose gel electrophoresis analysis confirmed plasmid population heterogeneity and restriction digestion analysis of plasmids from several clones verified insert diversity (not shown). The library provides a theoretical ~760-fold genome coverage. Our in silico BsrFI restriction analysis of the Yp genome revealed the presence of four BsrFI fragments of ≥ 8,000 bp. These fragments, which add up to 39,118 bp, are unlikely to be represented in the library.

Multicopy suppressor screening

The library was replicated using a 96-pin inoculator (Clonemaster™; Immusine Laboratories, Inc.) to inoculate 96-well plates loaded with fresh culture medium (150 μl/well). After plate incubation for culture growth (9 h, 37°C, 200 rpm), the 15,648 cultures were pooled and the cells of the pool were harvested. The pooled cells were resuspended in fresh medium (1/10 × pool's volume) containing AMP (100 μg/ml) and glycerol (25%), and the suspension was aliquoted (1 ml library stock aliquots) and stored at -70°C. Multicopy suppressor gene-containing clones were screened for by plating a 1/100 dilution of a library stock aliquot on LB agar plates containing AMP (100 μg/ml) and OFX at the MIC (0.35 μg/ml). The OFX MIC was determined by plating Ec carrying pGEM-4Z (Ec pGEM-4Z) on LB agar plates containing AMP (100 μg/ml) and OFX at increasing concentrations and defined as the concentration for which no colonies were obseved after plate incubation (37°C, 48 h). Clones identified in the screen were streaked on plates containing AMP (100 μg/ml) and OFX (0.35 μg/ml) to confirm their resistant phenotype. The plasmid from each confirmed clone was isolated and transformed into Ec and Yp. Each transformant was streaked on AMP and OFX-containing plates [LB agar for Ec and tryptose blood agar base (TBA; Difco Laboratories) for Yp] to ascertain whether the resistance was plasmid mediated. The insert of each plasmid conferring resistance was sequenced using M13 forward and reverse universal primers (Invitrogen). The sequences obtained were used as queries in sequence similarity searches against the Yp KIM genome using BLAST (please see Availability & requirements for more details) to determine the genome fragment carried by the plasmid.

Construction of pGEM-RobYp and transformation of Y. pestis

The fragment encompassing Yp robA (herein referred to as robAYp) and its promoter region was PCR-amplified from plasmid pGEM-OFXr1 (see results) with primers Robfor1 and Robrev1 (Table 3). The PCR product (1030 bp) was cloned into pCR2.1-TOPO (TOPO TA Cloning Kit, Invitrogen) and the fidelity of the insert was verified by DNA sequencing. The insert was recovered from the pCR2.1-TOPO clone as an EcoRI fragment and sub-cloned into the EcoRI site of pGEM-4Z using Ec DH5α as host. A clone with robAYp in the same orientation as the lacZ gene of pGEM-4Z was designated pGEM-RobYp. pGEM-RobYp and pGEM-4Z were introduced into avirulent Yp by electroporation as reported earlier [33] to create strains Yp pGEM-RobYp and Yp pGEM-4Z, respectively. Yp strains were grown in heart infusion broth (HIB; Difco Laboratories) and on TBA plates without or with antibiotics as appropriate.

Table 3

Oligonucleotides used in this study

Namea	Sequence
Robfor1	5'-TCTAGACGCTTTTTAACACACTGTACCAGT-3'
Robrev1	5'-GAATTCATTTAGATATGCCAGCACTTGATGA-3'
16sRNAF	5'-ATGACCAGCCACACTGGAACTGA-3'
16sRNAR	5'-TGACTTAACAAACCGCCTGCGT-3'
y3392F	5'-AGCGGCACCTTGGTCAATATTGT-3'
y3392R	5'-CAATTTGGTTATCCACCGATTCA-3'
y1050F	5'-GCTTATGACAGTGCAAAAGGTGA-3'
y1050R	5'-GATTAATGCGTGCAGACTCCAGT-3'
y0702F	5'-TATACCCAAGTGCGGGCACCCAT-3'
y0702R	5'-CATTCGCTACTGTGTCATTGCCT-3'
y3402F	5'-TCGATGCCACTGAATAGCGATCT-3'
y3402R	5'-ATCTGGTGAACGCAATAACGAGT-3'
y1439F	5'-CAGCCATCAAGAGGCTGCCCCAA-3'
y1439R	5'-ACCAAAGGCATCGACGCTGCCGA-3'
y1087F	5'-GGTGCTATCAGCGTATCTCACCT-3'
y1087R	5'-CCATACCGATGGGTAATGAGTAT-3'
y0060F	5'-GCAACCTGCTGATGAAGAACATA-3'
y0060R	5'-CACGAATCGCCTGACTGTGTGTT-3'
y0844F	5'-CGTTGCTAAACCGACTGGGTGAA-3'
y0844R	5'-TTGCGACAAAACATGCAGCCACA-3'
y0922F	5'-CGGCAGTGTGGTCAGTGTTCATT-3'
y0922R	5'-CACCCGACGCTTCAGGTCATTTT-3'
y1191F	5'-TCCTTAACCAGCTCTTCCACGGT-3'
y1191R	5'-CTTTATCTTCGCTGATCACCGGA-3'
y1386F	5'-GTGGTCAGGACACTAGCCATGGT-3'
y1386R	5'-CCTGCGTCAGTTGGACTTCGTAA-3'
y1481F	5'-AAAGCACAGAAGCAGGTGACTGT-3'
y1481R	5'-TTGTTGCTGACGCTGGTAGGTGA-3'
y1874F	5'-TTACCGACTATCGCACGTGACCT-3'
y1874R	5'-TTGTAGCACACGGGCAAACGTCA-3'
y1945F	5'-CCTGATGATGAGATGGTACGTAA-3'
y1945R	5'-TGAACTGAAGTTGAGGGCAATCT-3'
y2242F	5'-TTTAGTGACACTGATTGGTGGGA-3'
y2242R	5'-CATGATGCTCACCTGACTCAACA-3'
y3180F	5'-GGTAATGATGGTGGCTAATGGTT-3'
y3180R	5'-CAACCGAGCCAAGTAAGATCGCA-3'
y3558F	5'-GCCATTGATCCTGTTATCGGCTA-3'
y3558R	5'-ATAGGGAACAGATGAATGCCACA-3'
y4041F	5'-TTACGACCACAACGGTAGATGAA-3'
y4041R	5'-CATTGGTGCGGCAAGGTTCATAT-3'
y4067F	5'-GATATGATCCAGCCAGGTATGCT-3'
y4067R	5'-GCACCGATAAAGCACAAGCCAAT-3'
y0010F	5'-TCGCCGAAAGCCTTAACCGTTCT-3'
y0010R	5'-CCGAGAACGCACTAAGAAAGCCA-3'
y0178F	5'-TTTACCGCAGACGTGGTCGCTAT-3'
y0178R	5'-CCTACTGGACTCCCGTTGCTTCT-3'
y0613F	5'-GGGCTATTGGCCTGAAGTATTCT-3'
y0613R	5'-AAGCTTAGTATCCGCGCCAGACT-3'
y1017F	5'-AAATGACTCCGCTAGAGCTTCGA-3'
y1017R	5'-GGTTTACGACCGATACGATCAGA-3'
y1039F	5'-GCCGTGAATGGCACCGTTATGTA-3'
y1039R	5'-AGTTTGCCGACTCAGCTGACGAT-3'
y1948F	5'-CTGCCTGTAGTGCTGGCTTCTTT-3'
y1948R	5'-AGTACAGGGCAATCATGCTGCCA-3'
y2000F	5'-TGGCGATTATTGCCGAAGTGGTT-3'
y2000R	5'-AAAGGTGGCAGCGATTGAGACCA-3'
y2108F	5'-GGTTGTATATCAGCGGTAGTTCT-3'
y2108R	5'-TTGCACGAAAGTGTTTAGACGCT-3'
y2173F	5'-ACAAGGTGTCTCGGTATGCTGCA-3'
y2173R	5'-ATAATGCCAGGAACCAGAACGCT-3'
y2272F	5'-TTGGTATCGCAAGCTCGAAGCTT-3'
y2272R	5'-TCGCATTAGCATCCCGGTGACAA-3'
y2653F	5'-ATGACCGTCAATGCGACCATCGT-3'
y2653R	5'-AATGGCCATTGCCAGCATCCATA-3'
y2814F	5'-TCTGGACCAGGCAGTAACCGATT-3'
y2814R	5'-TACTCATATCGGCCAGGGTCAGA-3'
y2916F	5'-CCTTGGGTTGTTGTCGATGCTGA-3'
y2916R	5'-ACATGCCATACCTGCAAGCGCAA-3'
y3186F	5'-GTCAGTTGGACGTTACTGCTAAT-3'
y3186R	5'-CTTTCTTGCCATAAGCGACGACA-3'
y3760F	5'-TCTGGATATTCGCCGTGCAGAGA-3'
y3760R	5'-CGTGGTAAACAGACGCTCTGGAA-3'
y3516F	5'-TGCAACGACTAACCTGTATCAGT-3'
y3516R	5'-TTTGGCGAGTAGTATTCTCTGGT-3'

a Primers used for qRT-PCR were named according to the University of Wisconsin gene designations for the Y. pestis KIM genome, except for 16sRNAF-R (used to amplify 16s rRNA).

MIC99 and IC50 determinations

Dose-response experiments were done in triplicate and using 96 well plate-based microdilution assays as reported [12,14]. Briefly, wells contained 200 μl of broth (LB for Ec, HIB for Yp) inoculated with 104 cfu/ml and supplemented with AMP (100 μg/ml) and a second antimicrobial compound at the concentration indicated below. Antimicrobial compounds were added from stock solutions in water, ethanol, or DMSO. Control cultures lacking the antimicrobial compounds contained water (2%), ethanol (1%), or DMSO (0.5%). After incubation (37°C, 200 rpm, 24 h for Yp and 16 h for Ec), growth was measured as optical density (A620) using a Spectra Max Plus spectrophotometer plate reader (Molecular Dynamics). IC50 values were calculated from sigmoidal curves fitted to triplicate sets of dose-response data using KaleidaGraph (Synergy Software). MIC99 values were calculated as the lowest concentration tested that inhibited growth by ≥ 99%. The range of concentrations tested were: OFX (Sigma), 2.5-0.001 μg/ml; KAN (Shelton Scientific), 50-0.024 μg/ml; CHL (Calbiochem), 10-0.005 μg/ml for Yp and 25-0.012 μg/ml for Ec; TET (Sigma), 40-0.020 μg/ml; APR (Sigma), 50-0.024 μg/ml; NAL (Sigma), 25-0.012 μg/ml for Yp and 400-0.195 μg/ml for Ec; STR (Sigma), 50-0.024 μg/ml for Yp and 100-0.049 μg/ml for Ec; GEN (EM Science), 25-0.012 μg/ml for Yp and 50-0.024 μg/ml for Ec; DOX (Sigma), 10-0.005 μg/ml; CIP (Fluka), 0.75-0.0004 μg/ml for Yp and 1.25-0.0006 μg/ml for Ec; LVX (Fluka), 0.75-0.0004 μg/ml for Yp and 1.25-0.0006 μg/ml for Ec; plumbagin (Sigma), 50-0.024 μg/ml for Yp and 200-0.098 μg/ml for Ec; menadione (Sigma), 50-0.024 μg/ml for Yp and 400-0.195 μg/ml for Ec; paraquat (Sigma), 200-0.098 μg/ml for Yp and 400-0.195 μg/ml for Ec; CoCl2 (Sigma), 1-0.0005 mg/ml; CuSO4 (Sigma), 2-0.001 mg/ml for Yp and 4-0.002 mg/ml for Ec; ZnCl2 (Sigma), 1-0.0005 mg/ml.

Organic solvent tolerance assay

The test for solvent tolerance was conducted essentially as reported previously [21]. Overnight cultures of Ec and Yp strains grown in LB broth and HIB, respectively, were inoculated (1%) into fresh media and allowed to grow to A620 = 0.4. Then, 5 μl of each culture were spotted on solid medium (LB agar for Ec, TBA for Yp) with 100 μg/ml AMP for transformants carrying pGEM plasmids or without antibiotic for other strains. The surface of the medium was then overlaid with the organic solvent (7 ml) to a thickness of ~3 mm. The plates were sealed and incubated for 24 h for Ec strains and 48 h for Yp strains before naked-eye examination for bacterial growth.

Time-kill experiments

Yp pGEM-RobYp and Yp pGEM-4Z (control) were treated with STR, GEN, OFX, or CIP at 5 × MIC99. The MIC99 values were those determined using Yp pGEM-4Z in the dose-response experiments above (5 × MIC99 values: STR, 15.6 μg/ml; GEN, 5.2 μg/ml; OFX, 0.17 μg/ml; CIP, 0.12 μg/ml). For each antibiotic tested, three tubes with 10 ml of preheated (37°C) HIB containing AMP (100 μg/ml) were inoculated with 10 μl of an overnight culture of the corresponding Yp strain and incubated at 37°C with shaking at 200 rpm for 2 h. After incubation, a sample of each culture was taken and cfu/ml were determined by plating serial dilutions on TBA plates containing AMP (100 μg/ml) and enumerating colonies after plate incubation. Immediately after culture sampling, the test antibiotic was added (from stock solutions in water for GEN and STR or stock solutions in DMSO for OFX and CIP) and the cultures were returned to incubation (37°C, 200 rpm). Samples from these cultures were then taken at time points 0 (immediately after antibiotic addition), 30, 60, 90, 120, 180, 240, 300, 360, and 420 min for cfu/ml determination as above. Triplicate control cultures where water or DMSO was added in place of the antibiotic solution were included in the experiments and treated and analyzed in the same way as the antibiotic-treated cultures. The time-kill data were plotted using Kaleidagraph (Synergy software).

Isolation of total RNA and qRT-PCR

Yp and Ec were cultured in HIB and LB broth, respectively. AMP (100 μg/ml) was added to the medium for strains carrying pGEM-RobYp or pGEM-4Z. Cultures were incubated (37°C, 200 rpm) until they reached A620 of ~0.5 before RNA was isolated using the RiboPure-Bacteria Kit (Ambion) according to the manufacturer's instructions. RNA was isolated from triplicate cultures and treated with DNase I (Ambion) (4 units, 37°C, 30 min) in DNase I Buffer (Ambion). After the treatment, DNase I was inactivated by adding DNase Inactivation Reagent (Ambion) at 20% of the final volume of RNA treated. The inactivation was allowed to proceed at room temperature for 2 min. The RNA sample was then centrifuged at maximum speed in a microcentrifuge for 1 min to pellet the inactivation reagent. The RNA was then transferred to a new RNase-free microcentrifuge tube. cDNA was prepared from each RNA sample using TaqMan® Reverse Transcription Reagents Kit (Applied Biosystems) according to the manufacturer's instructions. Each cDNA sample was analyzed in triplicate by qRT-PCR using SYBR® Green Master Mix (Applied Biosystems) according to the manufacturer's instructions. cDNA was kept undiluted for qRT-PCR analysis of robA cDNA and diluted 1:5 for analysis of other cDNAs. qRT-PCR and target sequence relative quantification were carried out using a 384-multiwell platform with an ABI-PRISM 7900 HT Sequence Detection System (Applied Biosystems) as described previously [34,35]. The thermocycling program included 1 cycle of 95°C for 5 min followed by 40 cycles of 95°C for 30 sec, 55°C for 30 sec, and 72°C for 1 min. Relative quantification was conducted using the standard equation 2-ΔΔCT {i.e. 2- [(CT of target cDNA in sample1-CT of 16S rRNA cDNA in sample 1)-(CT of target cDNA in sample 2-CT of 16S rRNA cDNA in sample 2)]} [36]. The equation expresses n-fold difference of the target cDNA in the sample from the strain carrying pGEM-RobYp (sample 1) relative to the target cDNA in the sample from the strain carrying pGEM-4Z (sample 2, control) with normalization to an endogenous control (16S rRNA cDNA). The cycle threshold (CT) values utilized in the equation were the average of three independent cultures of the same strain, each analyzed in triplicate by qRT-PCR.

Identification of putative drug efflux pumps and RobAYp binding sites

Searches for efflux pumps were conducted in the sequenced genomes of Yp KIM and and Yp CO92 (please see Availability & requirements for more details). The search included the following strategies. First, the tables of functional classes in the genome websites were examined for annotated pumps. Second, the navigator function in the Artemis genome viewer software (please see Availability & requirements for more details) was used to search for the terms multidrug, efflux, transport, translocase, pump, and drug resistance as qualifiers in the annotated genome sequences. Third, the names of annotated Ec multidrug efflux pumps were used as search keywords using the navigator function in Artemis to find potential pumps not yet identified with the other strategies. Fourth, all putative pumps identified in Yp KIM were used as queries in BLASTP-based searches against the Yp CO92 genome and vice versa. Fifth, annotated Ec multidrug efflux pumps were used as queries in BLASTP-based searches against the Yp KIM and Yp CO92 genomes. Potential RobAEc binding sites were searched for using the navigator function in Artemis and the naked eye.

Abbreviations

AMP: ampicillin; APR: apramycin; CHL: chloramphenicol; CIP: ciprofloxacin; CT: cycle threshold; DOX: doxycycline; Ec: E. coli; GEN: gentamicin; HIB: heart infusion broth; KAN: kanamycin; LB: Luria-Bertani; LVX: levofloxacin; MDR: multidrug-resistant; MFS: major facilitator superfamily; NAL: nalidixic acid; OFX: ofloxacin; RND: resistance-nodulation-cell division; STR: streptomycin; TBA: tryptose blood agar base; TET: tetracycline; Yp: Y. pestis.

Availability & requirements

CD-Search:

Select Agent Program:

BLAST:

Yersinia pestis KIM Genome Page:

University of Wisconsin E. Coli Genome Project:

Wellcome Trust, Sanger Institute, Yersinia pestis data:

Artemis genome viewer software:

Authors' contributions

LENQ, KLS, and JAF conceived and designed the experiments. KLS, JAF, SMR, and FVF constructed the expression library. KLS and SMR screened the library. KLS carried out the gene expression analysis and bioinformatic-guided identification of efflux pump genes. KLS and RLM constructed all plasmids and strains and conducted the strain characterization experiments. All authors contributed to the preparation of the manuscript. LENQ and KLS wrote the final version of the manuscript. All authors read and approved the final version of the manuscript. LENQ directed and oversaw the project.