Distribution of EmrE over 29 strains of E. coli
Analysis of the genome of 29 E. coli strains available from the National Center for Biotechnology Information (NCBI) database showed that 18 strains contained a single copy of the emrE gene, three strains contained two copies, and eight strains lacked the gene (Table 1). Analysis of the genomic context divided the 24 emrE genes into two groups. A group of 10 genes showed a well-defined context. At one side, they were flanked by flagellar region IIIB, containing genes involved in flagellum assembly, and at the other side by a putative kinase inhibitor gene and a regulatory gene (Fig. 1; FLA). The other 14 emrE genes were flanked downstream by a gene encoding a phage recombinase and upstream by a gene encoding the REN protein. Differences in the upstream region of the REN gene discriminated four different subgroups (Fig. 1; REN_1 to REN_4). The variable upstream regions contained many genes encoding integrases (REN_1, REN_2, and REN_3), replication proteins (REN_1 and REN_4), exonucleases (REN_2), and IS elements (REN_2) that may be involved in the insertion/deinsertion of mobile elements, suggesting that the emrE gene, together with the two flanking genes, was inserted in these sites at some point in evolution. The two emrE genes found in the genome of strain EC4115 are both of the REN type, whereas strains 55989 and SE11 contain both an FLA type and a REN type.
Table 1. Distribution of EmrE proteins over E. coli strains. EmrE (GI): GI number NCBI in protein database. Insertion site: see text and Fig. 1
|Strain||EmrE (GI)||Insertion site||Number of residues|
A phylogenetic tree of the emrE genes found in the different E. coli strains was constructed on the basis of the alignment of the corresponding parts of the DNA (see below). The alignment contained no gaps, and revealed pairwise nucleotide sequence identities of between 90% and 99%, indicating that the tree represents the very recent divergence of the genes in the different strains. The tree revealed two well-separated clusters represented by the FLA and REN types of gene (Fig. 2). With the exception of the gene in strain 55989 (GI: 218694205), the different genomic contexts of the REN types were found as different branches on the tree as well, indicating that the divergence of the genes took place after insertion into the genome. No correlation was found between the phylogenetic tree of the genes encoding EmrE proteins and the whole-genome phylogenetic tree of E. coli strains computed with feature frequency profiles . The strains containing the FLA-type EmrE did not group together on one branch of the strain tree, and, similarly, the strains containing the REN type were scattered over the tree. This suggests that the genes encoding the different types of EmrE were spread by horizontal gene transfer (HGT).
Figure 2. Phylogenetic tree of the emrE genes from different E. coli strains. The tree was constructed on the basis of a multiple sequence alignment of the corresponding 330-nucleotide fragments of the genes. Sequences encoding for EMRE165 were truncated at the 5′-end, resulting in nucleotides with the same length as those encoding EMRE110. This avoided a bias in the tree because of the different lengths of the genes encoding EMRE165. The insertion sites FLA and REN_1–4 are defined in Fig. 1. EmrE proteins are indicated by the strain in which they were identified.
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The well-studied EmrE from strain K-12 consists of the usual 110 residues found for most of the members of the SMR family. The K-12 protein is of the REN type, and all EmrE proteins of this type from the other strains were annotated in the databases as proteins of the same size. Surprisingly, FLA-type EmrE proteins were annotated as proteins containing 165 or 170 residues. The additional base pairs resulting in the larger proteins were found at the 5′-end of the gene. Inspection of the nucleotide sequence revealed that all FLA-type EmrE proteins had the same upstream sequence, which differed from that of the REN-type EmrE proteins. Annotation differences (165 versus 170) were mainly the consequence of two closely spaced initiation codons. It follows that the genes in the FLA and REN insertion sites encode a long and a short version of the EmrE protein, EMRE165 and EMRE110, respectively.
Sequence analysis of EMRE110 and EMRE165
The difference between the nucleotide sequences encoding EMRE165 and EMRE110 is in the 5′-end of the genes (Fig. 3A). From a position that is 76 nucleotides upstream of the start codon of the short version, the two sequences overlap and are highly identical, i.e. 91% in the 536 and K-12 strains. The corresponding parts contain identical ribosomal binding sites, and ATG start and UAA stop codons. Upstream of the corresponding parts, the sequences are unrelated. EMRE165-encoding DNA contains a GTG start codon that is in frame with the ATG start codon of the short version, whereas this is not the case in EMRE110-encoding DNA. It follows that the messenger produced from EMRE165-encoding DNA is likely to produce a mixture of EMRE165 and EMRE110.
Figure 3. Comparison of the long and short versions of EmrE. (A) Alignment of base sequences containing the genes encoding EMRE165 from strain 536 and EMRE110 from strain K-12. The alignment is annotated with start and stop codons. (B) Dual topology of EMRE110 (top) and topology model for EMRE165 before (bottom, left) and after (bottom, right) cleavage of the signal sequence. The signal sequence was predicted by the signalp server with a probability of 0.967 (HMM mode). (C) Amino acid sequence of the EMRE165 preprotein. Double-headed arrows indicate the positions of predicted TMSs. The putative signal cleavage site is indicated by an arrow, the first residue of EMRE110 by a triangle, and positively charged residues by dots.
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The widely used membrane topology predictor tmhmm 2.0  predicts the presence of four transmembrane segments (TMSs) in EMRE110, which is in agreement with experimental data [25-27]. The protein is predicted to have no preference for one of the two orientations in the membrane, because of the lack of a positive charge bias over the loops at both sides of the membrane (positive-inside rule) . The prediction is in line with the dual topology character of EMRE110, which was convincingly demonstrated experimentally (Fig. 3B) [10, 11, 13, 28]. In the EMRE165 sequence, tmhmm predicts an additional TMS positioned at the N-terminus. The putative TMS would be connected to the four TMS bundle by a loop of 34 residues. The signal peptide predictor signalp [29, 30] predicted the N-terminal TMS to be part of a signal sequence with maximal cleavage site probability between positions 23 and 24. The presence of the signal sequence suggested that the protein would be inserted in the membrane in one specific orientation and, following cleavage of the leader sequence by leader peptidase, would leave the matured protein in the NoutCout orientation (Fig. 3B).
Membrane topology of EMRE110 and EMRE165
The ORFs encoding EMRE110 and EMRE165 of strains K-12 and 536, respectively, were cloned in pLIC vectors , yielding pLIC_EMRE110 and pLIC_EMRE165, respectively. The pLIC vectors produce the proteins with a His6-tag at the N-terminus and AP (pLIC1) or GFP (pLIC2) fused to the C-terminus. The two reporters AP and GFP allow for the determination of the cellular location of the C-termini of EMRE110 and EMRE165. GFP is properly folded and fluorescent only when targeted to the cytoplasm, whereas AP is enzymatically active only when exported to the periplasm. High GFP fluorescence and low AP activity indicate that the C-terminus is located in the cytoplasm, whereas high AP activity and low GFP fluorescence indicate a periplasmic C-terminus localization. Significant activity of both reporters indicates dual topology. It is important to stress here that expression of the EMRE165-encoding gene from pLIC plasmids is likely to produce reporter fusions of both EMRE165 and EMRE110, only the former of which contains the N-terminal His6-tag. To produce only EMRE165, the Met at position 56 (start of EMRE110) was mutated to Ala, yielding the vectors pLIC1_EMRE165(M56A) and pLIC2_EMRE165(M56A). The same set of plasmids was constructed by using the pBADcLIC vectors that produce the fusion proteins with a His10-tag at the C-termini of the reporter proteins. In this case, both long and short versions produced from the EMRE165-encoding gene contain the His-tag (see Table 2 for an overview of the constructs).
Table 2. Vectors used in this study to express EMRE110, EMRE165, and EMRE165(M56A). All vectors are pBAD24-based, and use the arabinose promoter for induction of expression. N-terminus: tag fused at the N-terminus of EmrE. C-terminus: tag/reporter fused at the C-terminus of EmrE
|pLIC1||His6-tag||AP||  |
|pLIC2||His6-tag||GFP||  |
|pBADcLIC–GFP||–||GFP/His10-tag||  |
|pBADhis||His6-tag||–||  |
|pBADBAD||His6-tag/BAD||–||  |
The normalized activities (see 'Experimental procedures') of the reporter proteins GFP and AP fused to EMRE110 produced from both pLIC_EMRE110 and pBADcLIC_EMRE110 were similar (Fig. 4A,B), indicating that the short version inserted into the membrane was distributed more or less equally over the two orientations (dual topology), as documented many times before [10, 11, 13]. The distribution shifted significantly to the orientation with the C-terminus in the periplasm with the EMRE165-encoding gene in plasmids pLIC_EMRE165 and pBADcLIC_EMRE165 (Fig. 4A,B). Importantly, the signals obtained from the reporters are likely to be the sum of the contributions of both long and short EmrE versions. Apparently, the long version contributes significantly to the fraction of molecules with the C-terminus in the periplasm. Within the limits of experimental error, the result was independent of the position of the His-tag at the N-terminus or C-terminus of the fusion proteins (Fig. 4A,B). Also, the levels of expression deduced from the reporter activities were of the same order of magnitude for the different constructs. In contrast, the level of expression dropped significantly for both pLIC_EMRE165(M56A) and pBADcLIC_EMRE165(M56A), when only the long version was produced. Relative to EMRE110, the distribution of the version with the His-tag at the N-terminus was shifted to the orientation with the C-terminus in the periplasm, but a significant fraction had the opposite orientation (Fig. 4A). With the His-tag at the C-terminus, the orientation of EMRE165(M56A) with the C-terminus in the periplasm was dominant (Fig. 4B). Optimization of the expression level of the latter construct by using a range of inducer concentrations resulted in a two-fold increase in expression level. Importantly, the orientation of the protein in the membrane was unaffected (Fig. 4B).
Figure 4. Membrane orientation of EmrE variants. Normalized AP (nAP) activity is plotted against GFP fluorescence of cells producing EMRE110 (gray square), EMRE165 (white square) and EMRE165(M56A) (black square) with (A) an N-terminal His6-tag and a C-terminal reporter, and (B) a C-terminal reporter and His10-tag. EMRE165(M56A) was induced with 0.04% (black triangle) and 0.1% (white triangle) arabinose.
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The EmrE–GFP fusion proteins produced from the pBADcLIC–GFP and pLIC2 vectors were purified from isolated membranes by Ni2+–nitrilotriacetic acid affinity chromatography. The yield of the proteins was too low to be visualized by protein staining after SDS/PAGE or western blotting with antibodies raised against GFP or the His-tag. Rather, the proteins were visualized by in-gel GFP fluorescence (Fig. 5). The EMRE110 fusion protein expressed from the pBADcLIC–GFP vector and carrying the His10-tag at the C-terminus bound strongly to the resin, and was only observed in the elution fractions. The band represents the membrane-bound protein molecules that originally had the C-terminus in the cytoplasm, where GFP matures to its fluorescent state. The same EMRE110–GFP fusion was observed with membranes isolated from cells containing EMRE165-encoding DNA from which both EMRE110 and EMRE165 are produced. In contrast, no fluorescent EMRE165–GFP fusion protein was observed, suggesting that the GFP moiety was exported to the periplasm during biosynthesis. In agreement with this, no fluorescent band was observed when EMRE165 was produced alone [EMRE165(M56A)] (Fig. 5A). Apparently, within the detection limit, all EMRE165–GFP fusion proteins insert with the C-terminus in the periplasm, which is in agreement with the orientation assay above (Fig. 4B). The majority of the EMRE110–GFP fusion protein carrying the His6-tag at the N-terminus produced from the pLIC2 vector was found in the elution fraction, but binding to the resin was clearly weaker, leaving significant fractions in the flowthrough and wash steps (Fig. 5B). As expected, EMRE110 produced from EMRE165-encoding DNA showed up in the flowthrough because it was translated from an internal ORF without a His-tag. No clear band for EMRE165 was observed. In contrast to the protein carrying the C-terminal His-tag, the EMRE165(M56A) fusion protein with the N-terminal His-tag was clearly observed, indicating that some of the molecules were inserted with their C-termini in the cytoplasm, which, again, was in line with the orientation assay above (Fig. 4A). Possibly, the N-terminal His-tag interferes with proper insertion of the protein in the membrane, which is also supported by the breakdown products observed in the flowthrough and elution fractions.
Figure 5. In-gel fluorescence of GFP fusion proteins isolated from membranes prepared from cells harboring (A) pBADcLIC–GFP vectors encoding the indicated EmrE variants containing a C-terminal His10-tag, and (B) pLIC2 vectors encoding the same EmrE variants containing an N-terminal His6-tag (Table 2). The EmrE variants were purified by Ni2+–nitrilotriacetic acid affinity chromatography, and samples from the flowthrough (FT), wash (W1 and W2) and elution (E1 and E2) steps were analyzed by SDS/PAGE followed by fluorescence imaging of the gel, as detailed in 'Experimental procedures'. ●, EMRE110–GFP, ○, EMRE165–GFP, □, free GFP.
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The results suggest that the long version of EmrE inserts into the membrane in a single orientation, with the C-terminus in the periplasm, which would be in line with the presence of a signal sequence at the N-terminus of the protein (Fig. 3B).
Maturation of EMRE165
The genes encoding EMRE110 and the EMRE165(M56A) were cloned in the pBADhis vector (Table 2) to study the fate of the putative signal sequence present in EMRE165. The pBADhis vector produces the inserts with an N-terminal His6-tag. Following isolation of cytoplasmic membranes from the cells harboring the plasmids, and purification by Ni2+–nitrilotriacetic acid affinity chromatography from the solubilized membranes, the expression levels were too low to be detected by staining of the gel after SDS/PAGE (not shown). To enhance the sensitivity of the detection, the biotin acceptor domain (BAD) of the oxaloacetate decarboxylase of Klebsiella pneumoniae was inserted in between the His6-tag and the EmrE protein (pBADBAD vectors; Table 2). BAD is biotinylated in vivo, and can be detected with high sensitivity by western blotting with streptactin. BAD fused to the short version of EmrE produced from pBADBAD_EMRE110 was readily detected in the membrane fraction as a protein with an apparent molecular mass of ∼20–22 kDa, which is in line with the masses of EMRE110 and the 10-kDa BAD (Fig. 6). In contrast, no BAD was detected in the membrane fraction from cells expressing EMRE165(M56A). Rather, the cytoplasmic fraction of the cells contained a low amount of biotinylated protein with an apparent molecular mass that was slightly more than expected for BAD itself. The results demonstrate that the N-terminal BAD was efficiently removed from the membrane-bound EmrE part.
Figure 6. Processing of EMRE165: western blotting of membrane fractions of EMRE110 (lane 1), EMRE165(M56A) (lane 2), and the cytoplasmic fraction of EMRE165(M56A) (lane 3). The proteins were expressed with BAD fused at the N-terminus. Membranes and cytoplasm of cells producing the EmrE variants were separated by centrifugation, after which proteins were separated by SDS/PAGE and, following blotting, biotinylated proteins were detected with streptactin.
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Activity of EMRE110 and EMRE165
EmrE makes the cells resistant to toxic compounds. EtBr is a known substrate of EmrE of strain K-12 (EMRE110). The ability of the EmrE variants to confer resistance to E. coli SF100 was assayed by spotting 10-fold serial dilutions on LB plates containing 500 μg·mL−1 EtBr (Fig. 7). The host E. coli SF100 by itself or harboring the pBADhis vector grew well on the plates in the absence of EtBr, whereas growth was completely inhibited in its presence. Plasmid pBADhis_EMRE165 producing both EMRE110 and EMRE165 conferred significant resistance to the cells. Plasmid pBADhis_EMRE165(M56A) producing only EMRE165 did not confer resistance, suggesting that EMRE110 was responsible for the resistance in the former case. However, surprisingly, plasmid pBADhis_EMRE110 producing only EMRE110 did not confer resistance. Control experiments showed that cells harboring the three plasmids all showed the same growth on plates without EtBr.
Figure 7. Activity of EmrE variants. Ten-fold serial dilutions of SF100 cells expressing EMRE165, EMRE165(M56A) and EMRE110 with (top) and without (bottom) an N-terminal His6-tag were spotted on LB plates supplemented with the indicated concentrations of EtBr.
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The EmrE proteins produced from the pBADhis vectors carry an N-terminal His6-tag. The three genes were recloned in pBAD24 vectors that produce the proteins without any tags. The untagged EMRE110 made the SF100 cells resistant to EtBr (Fig. 7), demonstrating that the N-terminal His6-tag inhibited the activity of the protein. The matured EMRE165 did not confer resistance, whereas the mixture of the two versions did confer resistance, as was observed with the His-tagged versions. The activity of the latter is explained by the fact that EMRE110 produced from pBADhis_EMRE165 does not carry a His6-tag.
The pattern of resistance was the same for the EmrE versions produced from the pLIC1 and pLIC2 vectors when plated on LB plates containing 500 μg·mL−1 EtBr (not shown). Plasmids pLIC1_EMRE165 and pLIC2_EMRE165 conferred resistance to EtBr, demonstrating, in addition, that EMRE110 with the reporters fused at the C-terminus are active proteins. Most importantly, matured EMRE165 inserted into the membrane in one orientation did not confer resistance against EtBr to the cells.