The SXT/R391 Family of Integrative Conjugative Elements Is Composed of Two Exclusion Groups

Eex sequences from diverse ICEs segregate into two groups.

We sequenced eex genes from 19 SXT/R391 family ICEs to explore the diversity of exclusion proteins found in this group of conjugative elements. These ICEs were obtained from five different genera of gram-negative organisms isolated from either clinical or environmental specimens from diverse locations (Table 1). PCR primers for the amplification of these eex genes were complementary to the sequences that flank eexS in the ICE SXT^MO10. We also obtained the sequences of eex genes from the SXT/R391-related ICEs pMERPH (17) and ICESpuPO1 (19) from the NCBI database. Unexpectedly, the predicted Eex proteins segregated unambiguously into two groups (Fig. 1) and were almost identical (>93%) to either EexS or EexR. Like the residues that distinguish EexS and EexR, the majority of amino acids that distinguished EexS-like proteins from EexR-like proteins were found within the carboxyl-terminal 56 amino acids. Since this region determines the exclusion specificities of EexS and EexR, and since the corresponding regions of the newly sequenced genes appear to encode proteins essentially identical to either EexS or EexR, it appears that the SXT/R391 family of ICEs is divided into two exclusion groups: the S and R exclusion groups.

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FIG. 1.

ClustalW alignment of the predicted amino acid sequences of Eex proteins from SXT/R391 family ICEs. (A) Map of the traG and eex regions depicting the regions encoding exclusion specificity (yellow) and the primers used in this study. (B) The Eex sequences of the S and R groups are presented. Amino acids that are conserved in all sequences are shown with dashed lines; amino acids that distinguish EexS and related proteins from EexR and related proteins are shown in blue and red, respectively; amino acids that are divergent in some members of the EexS or EexR groups are shown in boldface black.

S exclusion group.

The S group is composed of eight ICEs that were derived from bacteria isolated in distinct locations, ranging from an environmental isolate from Sri Lanka to a fish pathogen from Spain (Table 1). There are six different EexS-like sequences in our ICE collection. EexS was found in three V. cholerae O139 clinical isolates, those carrying ICEs SXT^MO10, ICEVchBan7, and ICEVchInd4, from the Indian subcontinent (Table 1). However, these ICEs do not carry the same antibiotic resistance genes, indicating that eexS is found in ICEs that are not identical (Table 1). On the other hand, ICEVchSL1, an ICE derived from an environmental V. cholerae O139 isolate, carries eexS5, demonstrating that not all V. cholerae O139 strains contain eexS. Overall, the predicted differences between the six EexS-related sequences are slight, and most of them are located within the amino termini and thus are not expected to influence exclusion specificity (Fig. 1).

R exclusion group.

The R group is composed of 13 ICEs derived from four continents (Table 1). Overall, this group consists of only five distinct Eex sequences, as nine of the ICEs appear to encode the same protein, EexR1 (Table 1). The ICEs encoding EexR1 were all derived from V. cholerae O1 clinical isolates from India or Bangladesh (Table 1). Although our sample size was fairly small, it is interesting that the exclusion proteins encoded by SXT/R391 family ICEs in V. cholerae O1 and V. cholerae O139 belong to the R and S exclusion groups, respectively. These findings suggest that more than one SXT/R391 family ICE may have been acquired by pathogenic V. cholerae strains relatively recently, as was also suggested by Hochhut et al. (12) based on differences between the antibiotic resistance genes in ICEs from V. cholerae O1 and O139 clinical isolates. As that among the EexS-related sequences, the variation among the five R exclusion group protein sequences is minor (Fig. 1). The predicted carboxyl-terminal 3 amino acids of two of the five EexR group sequences, EexR and EexR4, differ from the amino acids found at the C termini of all the other Eex sequences (Fig. 1). R391 and pMERPH, the ICEs that contain eexR and eexR4, respectively, both contain an insertion of genes conferring resistance to mercury immediately downstream of the eexR and eexR4 genes (4, 17). It is possible that the acquisition of mercury resistance genes at this locus was accompanied by the alteration of the 3′ end of eexR in both R391 and pMERPH.

For each ICE, the corresponding TraG and Eex proteins belong to the same exclusion group.

We sequenced and analyzed the traG exclusion specificity regions in our set of 21 ICEs. As the predicted Eex amino acid sequences, the predicted amino acid sequences of the TraG exclusion regions segregated into two exclusion groups (Table 1). All of the ICEs that encoded an S exclusion group protein also encoded the TraG_S exclusion determinant residues P-G-E (Table 1). In contrast, all of the newly analyzed ICEs that encoded an R exclusion group protein encoded T-G-D at amino acids 606 to 608 of TraG. As the G residue at position 607 in the TraG exclusion determinant region is conserved between the two exclusion groups, this result suggests that only two TraG residues, those at positions 606 and 608, confer TraG exclusion specificity; the D residue at position 607 in TraG_R is unique and therefore unlikely to play a role in exclusion specificity. Thus, for all the ICEs examined, the corresponding Eex and TraG sequences were from the same exclusion group. This association likely reflects the functional interaction between these proteins that mediates exclusion.

Exclusion activity of EexS and EexR exclusion group proteins.

Since the EexS and EexR exclusion groups were defined by differences in their carboxyl termini, the part of the Eex protein that determines exclusion specificity, we hypothesized that S group proteins would exclude SXT transfer and not R391 transfer and that R group proteins would exclude R391 transfer and not SXT transfer. To test this hypothesis, we cloned eexS, eexS2, eexS3, eexR, eexR1, and eexR3 into an expression vector and then assessed whether the expression of these exclusion genes in a recipient cell excluded the transfer of either SXT or R391. We measured exclusion activity and specificity by calculating an exclusion index towards SXT (EI_S) or R391 (EI_R) transfer. This index is the ratio of an element's frequency of transfer to an ICE-free recipient to the frequency of transfer to the indicated recipient. We found that all Eex variants excluded ICE transfer as predicted. Thus, when expressed in a recipient, the three S exclusion group proteins excluded SXT transfer (EI_S ranged from 26 to 41) but did not exclude R391 transfer (EI_R ranged from 0.5 to 1) (Fig. 2). Similarly, R391 transfer was excluded by the three R exclusion group proteins (EI_R ranged from 18 to 174), but SXT transfer was excluded only to a small extent (EI_S ranged from 2 to 4) (Fig. 2). These results corroborate the previous report that the carboxyl termini of Eex proteins determine the specificities of the proteins. However, the exclusion potencies of the three EexR group proteins varied to a surprising extent, especially considering that the sequence of the highly active EexR1 protein (EI_R of 174) differs by only a single amino acid (at residue 43) from that of EexR3 (EI_R of 30). The basis for this variability requires further studies.

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FIG. 2.

Exclusion specificity and activity of SXT and R391 exclusion group proteins. In all cases, the recipient strain was a derivative of Escherichia coli CAG18439 (22) expressing the indicated Eex protein from pBAD-Topo. The donor strains were derivatives of MG1655 (13) harboring either SXT or R391. The exclusion index is the ratio of an element's frequency of transfer to an ICE-free recipient to the frequency of transfer to the indicated recipient. Each bar represents the mean of results from three experiments, with error bars indicating the standard deviations. Mating assays and calculations of transfer frequencies and exclusion indices were done as previously described (16).

Conclusions.

We report here the most comprehensive study to date of the diversity of exclusion determinants within a single family of conjugative elements. We analyzed the predicted amino acid sequences of 19 novel Eex and TraG proteins from the SXT/R391 family of ICEs derived from several bacterial species isolated from diverse parts of the world over the past four decades. All of the Eex proteins shared well-conserved amino termini. The distinguishing feature of these proteins was their carboxyl-terminal sequences, which determine exclusion specificity. All of the new Eex sequences contained carboxyl-terminal sequences that were nearly identical to that in either EexS or EexR. Remarkably, every ICE that encoded an S group Eex also encoded an S group TraG; this linkage was also observed in the ICEs of the R exclusion group. Thus, our sequence analyses strongly suggested that there are only two (S and R) exclusion groups in the SXT/R391 family of ICEs. Functional studies confirmed this prediction; the new EexS-like and EexR-like variants specifically excluded SXT and R391, respectively.

It is not clear why there are only two exclusion groups in the SXT/R391 family of ICEs. Considering that the traG and eex genes are linked, a potential scenario to explain the emergence of the S and R groups is a recombination event between the region encompassing exclusion specificities in both genes (Fig. 1) and a similar region from another conjugative element. Even though ICEs appear to have fairly plastic genomes (7, 8), their exclusion genes are conserved. Although entry exclusion in plasmids has been studied for many years (1, 10, 11, 21), there is relatively scant knowledge of the extent of the diversity of exclusion determinants within plasmid families. Further definition of the diversity of exclusion genes in plasmid families will provide useful tools for analyses of the bases of exclusion in plasmids and ultimately further our understanding of the conjugative process.

We are grateful for support from NIH grant AI42347, HHMI, and the Tufts-NEMC GRASP center.

We thank Linc Sonenshein, Brigid Davis, and Sarah McLeod for critically reading the manuscript.