Relative Strengths of Promoters Provided by Common Mobile Genetic Elements Associated with Resistance Gene Expression in Gram-Negative Bacteria

Abstract

Comparison of green fluorescent protein expression from outward-facing promoters (P_OUT) of ISAba1, ISEcp1, and ISAba125 revealed approximate equivalence in strength, intermediate between P_CS (strong) and P_CW_TGN-10 (weak) class 1 integron promoter variants, >30-fold stronger than P_OUT of ISCR1, and >5 times stronger than Ptac. Consistent with its usual role, P_CW_TGN-10 produces more mRNA from a “downstream” gfp gene transcriptionally linked to a “usual” P_CW_TGN-10-associated gene cassette than does P_OUT of ISAba1.

TEXT

Antibiotic resistance genes in Gram-negative bacteria are generally associated with mobile genetic elements (MGE) such as transposons, integrons, and insertion sequences (IS) (1). These MGE typically provide the promoter responsible for resistance gene expression, and as each different resistance gene is generally associated with one particular MGE (1), the strength of the MGE promoter is expected to be a major determinant of the level of antibiotic resistance.

Complete outward-facing promoters (P_OUT) have been identified in many IS, including ISAba1 (2), ISEcp1 (3), and ISCR1 (4), which are associated with a variety of important resistance genes (1). Some IS include only a partial promoter; for example, ISAba125 provides only a −35 element in the right terminal inverted repeat, a new promoter being generated when this IS inserts itself at the correct distance from a −10 element (5). Gene cassettes captured by class 1 integrons are expressed from a common promoter (Pc), sometimes in combination with a secondary promoter (P2) (6, 7). The relative strengths of several Pc variants have been described previously (6–12), but a direct systematic comparison with P_OUT of various IS has not been made.

We therefore measured the relative expression of green fluorescent protein (GFP) from P_OUT promoters of some important IS (ISAba1, ISAba125, ISEcp1, and ISCR1) and two Pc variants from class 1 integrons (without an active P2), PcS (“strong”) and PcW_TGN-10 (a variant of the “weak” version of Pc with an extended −10 motif that increases the PcW efficiency [9]), in both Escherichia coli and Klebsiella pneumoniae. Each promoter was amplified from a suitable isolate (see Table S1 in the supplemental material) with primers containing specific restriction sites (see Table S2), the bla_NDM-1 −10 region being included in the case of ISAba125, and cloned into the unique BamHI site of low-copy-number promoter detection vector pANT3 (13) to direct the expression of gfpmut3 (gfp here) (Table 1; see Fig. S1 in the supplemental material). Sequencing confirmed a single copy of each promoter in the correct orientation.

TABLE 1.

Plasmids used in this study

Plasmid	Characteristic(s)	Reference
pANT3	Low-copy-number, broad-host-range cloning vector; Kanr, gfpmut3 with no promoter	13
pANT5	Low-copy-number, broad-host-range cloning vector; Kanr, gfpmut3 gene under control of tac promotera	13
pJIBE401	Naturally occurring conjugative IncL/M plasmid from Kp1239 containing blaIMP-4 qacG aacA4 catB3 in a class 1 integron	14
pJIJP001	pANT3 with 210-bp fragment containing POUT from ISAba1 inserted into BamHI site upstream of gfpmut3	This work
pJIJP002	pANT3 with 238-bp fragment containing POUT from ISEcp1 inserted into BamHI site upstream of gfpmut3	This work
pJIJP003	pANT3 with 251-bp fragment containing POUT from ISCR1 inserted into BamHI site upstream of gfpmut3	This work
pJIJP004	pANT3 with 245-bp fragment containing class 1 integron PCWTGN-10 promoter inserted into BamHI site upstream of gfpmut3	This work
pJIJP005	pANT3 with 245-bp fragment containing class 1 integron PCS promoter inserted into BamHI site upstream of gfpmut3	This work
pJISS125	pANT3 with 120-bp fragment containing −35 from ISAba125 and −10 from blaNDM inserted into BamHI site upstream of gfpmut3	This work
pJIMK007	pANT3 with 1,040-bp PCWTGN-10-blaIMP-4 region from pJIBE401 inserted into BamHI and XbaI sites	This work
pJIMK51	pJIJP001 with 808-bp fragment containing blaIMP-4 inserted into XbaI site between gfpmut3 and POUT from ISAba1	This work
pJIMK52	pJIJP003 with 808-bp fragment containing blaIMP-4 inserted into XbaI site between gfpmut3 and POUT from ISCR1	This work

^aSequencing of the trp-lac (tac) fusion promoter region of pANT5 (with primer U-GFP-R1; see Table S2 in the supplemental material) revealed a “standard” tac promoter sequence, corresponding to nucleotides 20 to 92 of the 96-bp HindIII/BamHI fragment previously used by Lévesque et al. (6) as a comparator for Pc.

E. coli DH5α and K. pneumoniae ATCC 13883 containing a promoter construct or with pANT3 (promoterless gfp) or pANT5 (Ptac-gfp) were incubated overnight (16 h, stationary phase) at 37°C on nutrient agar with 50 μg/ml kanamycin and suspended in phosphate-buffered saline (PBS) adjusted to ∼1.5 × 10⁸ CFU/ml, as estimated with a nephelometer. Fluorescence was measured with a Victor 3 plate reader (PerkinElmer) with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Experiments were performed in triplicate on three separate occasions, and the mean and standard deviation were calculated (Fig. 1). Univariate analysis of variance (ANOVA) showed a statistically significant interaction (P < 0.001) between fluorescence intensities for promoters and species, indicating that results for each species should be considered separately. All further statistical analysis of multiple variables was performed by one-way ANOVA with Bonferroni's correction (SPSS v18; IBM Inc.). A P value of <0.05 was considered statistically significant.

FIG 1.

Relative GFP expression in E. coli DH5α (black) and K. pneumoniae ATCC 13883 (white) from control isolates (no plasmid) (A), promoter-gfp constructs (B), and promoter-bla_IMP-4-gfp constructs (C) in stationary phase. Fluorescence values are the means of nine data points; error bars indicate 1 standard deviation from the mean. The only significant (P < 0.05) differences in expression between species for the same promoter were for ISAba1 with or without bla_IMP-4 and ISEcp1. Differences in expression from different promoters were all significant (P < 0.05), except among ISAba1, ISEcp1, and ISAba125 in E. coli, between PcW_TGN-10 with and without bla_IMP-4 inserted in E. coli, and among ISCR1 (with or without bla_IMP-4), no plasmid, and no promoter. Abs, absorbance.

The relative GFP expression from the different promoters was PcS > ISEcp1 P_OUT ≈ ISAba125 P_OUT ≈ ISAba1 P_OUT > PcW_TGN-10 ≫ ISCR1 P_OUT (Fig. 1B) in both species, and in agreement with published data, the GFP fluorescence intensity from PcS was approximately 1.8-fold higher than that from PcW_TGN-10 (P < 0.001) (9) and ∼6.5-fold higher than that from the common tac expression promoter (6). PcS was 38-fold and PcW_TGN-10 was 21-fold stronger than ISCR1 P_OUT (P < 0.001) in both species. In E. coli, there was no significant difference among the relative strengths of ISAba1, ISAba125, and ISEcp1 P_OUT (P > 0.05). These three promoters were 32-fold stronger than ISCR1 P_OUT (P < 0.001) and 5.5-fold stronger than the tac promoter. GFP expression in exponential phase (measured with ∼1.5 × 10⁸ CFU/ml in PBS from cultures grown at 37°C for 3 h in LB with 50 μg/ml kanamycin after dilution from an overnight culture) was not significantly different (see Fig. S2 in the supplemental material) from that in stationary phase.

The cassette-borne bla_IMP-4 gene encodes a metallo-β-lactamase that is expressed from P_CW_TGN-10 in its native genetic context in pJIBE401 from K. pneumoniae Kp1239 (14) (GenBank accession no. AJ609296). To further understand the clinical relevance of promoter variation, bla_IMP-4 was inserted between selected promoters and gfp. In pJIBE401, the bla_IMP-4 gene is followed by 127 bp of the bla_IMP-4 attC site, which forms a stem-loop that may reduce the expression of downstream genes by impeding ribosome progression (15), and the transcriptionally coupled qacG gene cassette (14). P_CW_TGN-10 with the complete bla_IMP-4 gene, but not the downstream part of the attC site, was amplified from the native context in pJIBE401 with primers containing BamHI and XbaI sites (see Table S2 and Fig. S1 in the supplemental material) and cloned into these sites in pANT3 to generate pJIMK007 (Table 1). Primers including XbaI sites were used to amplify bla_IMP-4 with its native ribosome binding site (RBS) but none of the downstream attC site (see Table S2 and Fig. S1) for cloning into the XbaI site of pJIJP001 (ISAba1) and pJIJP003 (ISCR1) promoter constructs to generate pJIMK51 and pJIMK52, respectively.

The bla_IMP-4 and gfp mRNA levels were measured from promoter-bla_IMP-4-gfp constructs by quantitative reverse transcriptase PCR (qRT-PCR). One milliliter of exponential-phase culture (as described above, optical density at 600 nm [OD₆₀₀] of 0.4) was added to 2.0 ml of RNAprotect Bacteria Reagent (Qiagen), and RNA was extracted from cell pellets (RNeasy minikit, Qiagen) and treated with RNA-free TURBO DNase and then DNase Inactivation Reagent (Ambion), all according to the manufacturers' instructions. PCR with qRT-PCR primers (see Table S2 in the supplemental material) was performed to exclude contaminating DNA. RNA (1 μg, OD_260/280 ratio of 1.9 to 2.1) was converted to cDNA by RT-PCR (MultiScribe RT) and amplified (Power SYBR green PCR master mix [Applied Biosystems]; 95°C for 5 min and 40 cycles of 95°C for 5 s and 60°C for 10 s). mRNA levels, calculated by the ΔC_T method (16), were normalized to rpoB expression (see Table S2). Experiments were performed in triplicate, and mean values and standard deviations were calculated. Relative bla_IMP-4 and gfp mRNA expression from P_CW_TGN-10 and ISCR1 in both E. coli and K. pneumoniae (see Fig. S3 in the supplemental material) was consistent with GFP expression (Fig. 1C). bla_IMP-4 and gfp mRNA expression from ISAba1 (see Fig. S3) was also consistent with GFP fluorescence data but differed between the two species (Fig. 1C).

Interposition of bla_IMP-4 between gfp and the promoter had no effect on expression from P_CW_TGN-10 in either E. coli or K. pneumoniae, but GFP, gfp, and bla_IMP-4 mRNA expression from ISAba1 P_OUT was lower than that obtained with P_CW_TGN-10 after the interposition of bla_IMP-4 (Fig. 1; see Fig. S3 in the supplemental material), despite the presence of the same sequence between bla_IMP-4 and gfp (see Fig. S1). For all constructs with bla_IMP-4, the influence of promoter strength on imipenem MICs (determined by Etest; Liofilchem, Roseto degli Abruzzi, Italy) was less striking (Table 2) but was also consistent with GFP fluorescence data (Fig. 1C). The ISCR1|bla_IMP-4 construct did not significantly exceed the clinically defined MIC (17) beyond which therapeutic failure is predicted, even when expressed from this multicopy pANT plasmid. It is noteworthy that resistance genes found in association with ISCR1 typically have their own promoter (18).

TABLE 2.

Imipenem MICs for bla_IMP-4 with different promoters

Bacterial strain and plasmid	Promoter	MIC (μg/ml)
E. coli DH5α
None	None	0.125
pJIMK007	PCWTGN-10	3
pJIMK51	ISAba1	2
pJIMK52	ISCR1	0.75
K. pneumoniae ATCC 13883
None	None	0.38
pJIMK007	PCWTGN-10	4
pJIMK51	ISAba1	3
pJIMK52	ISCR1	1

Differences in replication and transcription machinery (19), codon preferences, and tRNA availability (20) may result in different levels of expression from identical promoters in different species. Expression of an Acinetobacter baumannii bla_ADC (ampC) gene (determined by qRT-PCR) was reported to be ∼2-fold higher from ISAba125 than from ISAba1 (21), and GFP expression from ISAba1 and ISEcp1 P_OUT was significantly lower in K. pneumoniae ATCC 13883 than in E. coli DH5α in our own experiments, although these differences were less striking than the differential effects of gene interposition. P_C promoters are found in a distinctive genetic context, usually with a tandem array of cassette-borne genes (four in pJIBE401 from clinical isolate Kp1239) that rely on them for expression, while IS promoters such as ISAba1 P_OUT are typically found upstream of a single gene that requires a shorter transcript for expression. Differential effects on polymerase processivity are known to result in different proportions of long transcripts from different promoters, despite very similar amounts of short transcripts from these same promoters (22–24). It is noteworthy that the relative expression of gfp mRNA was around twice that of bla_IMP-4 mRNA from all three promoter-bla_IMP-4-gfp constructs in both E. coli and K. pneumoniae (see Fig. S3 in the supplemental material), implicating additional processes such as RNase E-mediated degradation (25, 26) that may selectively modulate the expression of operon genes (27). The finding that P_CW_TGN-10 produces more bla_IMP-4 and gfp mRNA than ISAba1 (see Fig. S3) is consistent with minor MIC differences (Table 2), with a usual connection of P_CW_TGN-10 with bla_IMP-4 and similar gene cassettes, and with a usual role for P_CW_TGN-10 in the production of longer transcripts (gene cassette arrays) but contrasts with the apparently slightly greater potency of the ISAba1 promoter, as judged by GFP expression (Fig. 1).

Our findings are overall consistent with and extend previously published work. We show here that common MGE-associated promoters of resistance gene expression are generally stronger than familiar workhorses such as P_tac (28) with similar levels of expression of the proximate gene. Links between resistance genes and promoters in MGE are predictable and clearly important when considering the expression of a new resistance gene as it emerges in the mobile gene pool, but it is equally clear that the host strain, and especially the genetic context, should be carefully considered when making comparisons between promoters.