Jeffrey G. Lawrence is an Assistant Professor in the Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pa.

Article Links:

• Interview: Tracing Genomic Flux

Links to Other ASM Pages:

• Table of Contents
• ASM News Issues

This model helps in understanding transfers of gene clusters whose constituent genes contribute to a single ecological function

Jeffrey G. Lawrence

The dissemination of antibiotic resistance genes among bacterial genomes has attracted the interest of microbiologists and epidemiologists alike. From a medical standpoint, disease-causing organisms resistant to multiple antibiotics appear with alarming frequency. For example, Streptococcus pneumoniae infections, a major health problem in developing countries, were once treated routinely with penicillin. Now, however, the majority of these cases in some regions involve penicillin-resistant strains that often are also resistant to tetracycline, chloramphenicol, cotrimoxazole, and erythromycin.

From a genetic perspective, the localization of antibiotic resistance genes on mobile genetic elements allows for their frequent and facile transfer among bacterial strains and species. This organization is not surprising, since antibiotic resistance genes can provide astonishing benefits to their host cells in certain environments and the mobile elements bearing them would be expected to proliferate. Indeed, if we consider them as selfish elements, antibiotic resistance genes found on mobile genetic elements are stronger competitors in the invasion of naive bacterial strains than are chromosome-bound antibiotic resistance genes with their less-obvious means for dissemination.

However, one facet of the distribution and organization of antibiotic resistance genes is not so easily explained. Some plasmids and transposons contain clusters of genes that provide resistance to different antibiotics. The most striking examples of such clusters are borne on integrons, families of mobile genetic elements that contain exchangeable antibiotic resistance gene cassettes. Moreover, these natural elements came into existence well before the modern era of antibiotic production and use. Thus, although the frequency of antibiotic resistance elements has increased among bacterial lineages since the advent of medicinal antibiotics, resistance genes have conferred upon bacteria a tolerance of antimicrobial substances in natural environments since well before the 20th century. For instance, integrons are found in Vibrio cholerae strains that were isolated prior to 1900.

Although several models have been proposed to explain why genes contributing to a single metabolic function--for instance, histidine biosynthesis--are often found in clusters, different antibiotic resistance genes found in clusters presumably do not contribute to a single metabolic function. Rather, each gene (or set of such genes) confers resistance to a class of antibiotic independent from the functions of the other genes. In this context, the Selfish Operon Model provides a framework for understanding the origin and persistence of this class of gene clusters, whose constituent genes contribute to a single ecological function.

Many Bacterial Genes Are Clustered

A notable feature of prokaryotic genomes is the clustering of genes whose products contribute to a single function or phenotype. Striking examples of such gene organization were noted during the infancy of bacterial genetics in the 1950s, as the genes for lactose utilization (lac), galactose utilization (gal), histidine biosynthesis (his), and tryptophan biosynthesis (trp), among others, were found to be adjacent to one another in the genomes of enteric bacteria such as Escherichia coli and Salmonella enterica serovar Typhimurium.

Four hypotheses have been proposed to explain the organization of these and other prokaryotic genes into such clusters:

• The Natal Model proposes that some genes within clusters arose there by gene duplication and divergence. This model for how clusters may arise can readily explain the clustering of mammalian globin genes, or yeast hexose transport genes, but it fails to explain the assembly of prokaryotic genes whose products belong to distinct protein families, such as in an operon comprising a kinase, a dehydrogenase, and an epimerase.
  
• The Coregulation Model arose from the observation that cotranscription of genes allows coordination of gene expression and is advantageous to the cell; this observation led to the tacit assumption that cotranscription provided selection for the origin of gene clusters. Although such an advantage could provide for the selective maintenance of preexisting gene clusters, it is more difficult to conceive how such benefits--derived only after the cluster is formed and absent from any intermediate forms--could lead to the formation of clusters from previously dispersed genes.
  
• According to the Fisher Model, alleles in diploid systems may be in linkage disequilibrium if their products interact. Alleles at one locus may encode products which perform best with the products of alleles at the second locus. If so, recombination between these coadapted alleles would result in less-fit offspring, and the genes would appear to be physically linked. Several workers extended this idea to propose that bacterial genes were physically clustered so that recombination would not disrupt coadapted alleles. However, this model requires frequent recombination to select for gene clustering. Moreover, it is difficult to show that alleles of enzymes that do not physically interact are coadapted to work with isozymes encoded by other loci.
  
• In contrast to the preceding three models, the Selfish Operon Model proposes that the organization of bacterial genes into clusters provides a fitness benefit to the constituent genes of a given cluster, enabling them to be transferred more readily between taxa. Horizontal transfer can drive the evolution of gene clusters because all genes providing for a single function need to be transferred in a single event to confer a selectable phenotype (Fig. 1). In this manner, genes may assemble into tight clusters by stepwise horizontal transfer and deletion of intervening DNA (which does not contribute to the selectable function). Cotranscription promotes facile gene transfer since a functional promoter can be provided at the site of insertion.

Intrinsic to the Selfish Operon Model is the notion that the cluster contains a set of genes that provide a selectable function only when cotransferred into a naive genome. Typical bacterial operons--whose gene products, for example, provide for the synthesis of an amino acid or for degradation of a carbon compound--encode products that conform to this model. But can this model explain the formation of clusters of genes whose products do not contribute to a single function?

Clusters of Antibiotic Resistance Genes

At first glance, the formation of clusters of antibiotic resistance genes does not appear to conform to any of these four models. For instance, because the genes could not arise by duplication and divergence--the encoded proteins belong to distinct families--the Natal Model certainly does not apply. Moreover, the genes are not cotranscribed, indicating that the Coregulation Model also does not apply. Finally, because recombination is not detrimental and is even facilitated by integron-encoded, site-specific recombinases, the Fisher Model also does not apply.

However, in considering the Selfish Operon Model it is possible, although not necessary, that the genes encoding the different antibiotic resistance cassettes contribute to a single selectable phenotype. That is, multiple antibiotic resistance genes may allow a host organism to persist in environments containing several antibiotics. Such environments form when several antibiotics are administered simultaneously to patients, which is currently a recommended clinical practice. However, this proposal is cumbersome from an evolutionary perspective. It would postulate that each integron with a different combination of antibiotic resistance genes would allow for growth in different specialized environments, each with a unique combination of antibiotics.

Alternatively, multiple antibiotic resistance genes may allow a host organism to tolerate exposure to different antibiotics at different times. In a clinical setting, this scenario corresponds to antibiotic cycling regimes. In both cases, the organization of antibiotic resistance genes into clusters increases the fitness of the constituent genes in competition for transfer among potential microbial hosts, which would benefit synergistically from the simultaneous acquisition of multiple antibiotic resistance genes. The increase of a gene's fitness by clustering with other genes is an essential feature of the Selfish Operon Model.

Consider an integron bearing an ampicillin resistance gene cassette (Ap^r). When introduced into a naive taxon, this integron will allow its host organism to exploit environments containing one class of antimicrobial agents. Consider another integron bearing a kanamycin resistance cassette (Kn^r). This integron will allow its host to exploit a different set of environments.

However, an integron bearing both genes would allow its host to exploit both environments. If a single organism is likely to benefit from exploiting both environments, then the two-gene operon would provide a stronger advantage than would either gene alone. Thus, the fitness of this two-gene integron will be higher than the fitness of either one-gene integron; consequently, the fitness of each individual antibiotic resistance gene increases when placed in the context of another antibiotic resistance gene. This synergism between antibiotic resistance genes reflects the ecological similarity of antibiotic resistance gene-encoded functions and serves to reinforce the mutual hitchhiking of such genes.

If so, one may ask why clusters of antibiotic resistance genes are not parasitized by genes that contribute little or no benefit to host cells. If the Apr gene could benefit from association with the Kn^r gene, all sorts of genes might also cluster with the Kn^r gene, each of them hitchhiking on the enormous selective benefit supplied by kanamycin resistance to assist their dissemination into naive hosts. While such hitchhiking may not be entirely avoidable, the resulting package is not a better competitor than the Kn^r gene alone because the hitchhiking genes provide no benefits. Moreover, hitchhiking genes decrease the fitness of the cluster since larger clusters are less likely to be transferred successfully among genomes (all modes of intercell transfer decrease in efficiency with increasing size). Therefore, we do not expect to find integrons with large numbers of useless genes hitchhiking their way to genomic stardom on the coattails of antibiotic resistance genes.

The lack of hitchhikers in integrons makes two strong statements regarding the benefits of antibiotic resistance genes to their hosts. First, the potential short-term selective benefit of an antibiotic resistance gene must be enormously high; this advantage is reflected in the high frequency of clustering among classes of antibiotic resistance that may be less frequent, but not unobserved, from other classes of genes. Second, there must be a large number of hosts that could benefit from antibiotic resistance genes; it is the transfer among hosts that selects for clusters of different antibiotic resistance genes.

Hierarchy in Gene Organization

The Selfish Operon Model proposes that clustering increases the fitness of the constituent genes if all genes are required to confer a selectable function; if one of the eight genes required for histidine biosynthesis is introduced into a naive cell, it cannot provide for histidine biosynthesis. Therefore, the cluster increases the probability of successful horizontal transfer of its constituent genes. The Selfish Operon Model is clearly extended when applied to antibiotic resistance genes by describing how particular clusters also can increase the fitness of the constituent genes by increasing their likelihood of successful horizontal transfer. The clustering of integron-borne antibiotic resistance genes is driven by mutual hitchhiking but is still a selfish property of the constituent genes.

With these notions in mind, we can further extend the Selfish Operon Model to consider a spectrum of organization among genes in microbial genomes. The most highly conserved gene clusters encode proteins that serve as subunits in a single enzyme (e.g., the Escherichia coli carAB genes); each of these genes has little chance of successful horizontal transfer without its partner. Operons represent promiscuous packages of genes that can confer complex selectable functions--such as the complete biosynthesis of histidine--and can be expressed at the site of insertion by a host promoter. Looser gene clusters can provide selectable functions in the form of operons--for instance, lactose degradation and transport by lacZYA--coupled with a tightly linked regulatory gene (lacI) whose cotransfer benefits, but is not essential for, the horizontal transfer of the operon of enzyme-encoding genes.

Figure 1

Such genetic synergism, reflected in broader genetic hierarchies, is not uncommon in bacterial genomes. Consider the selfish Salmonella pduR regulon, which was introduced into the Salmonella genome by horizontal transfer. The pdu operon enables B₁₂-dependent propanediol degradation and is found adjacent to the cob operon, which provides for coenzyme B₁₂ biosynthesis (Fig. 1). Moreover, two independently-transcribed cistrons--providing for propanediol transport (pduF) and for coregulation of the entire set of operons (pocR)--are found between them.

This organization represents a hierarchy of selfish gene clusters, where individual operons benefit from their proximity in the form of increased horizontal genetic transfer. The cob and pdu selfish operons allow cotransfer of genes required for these individual functions; however, their proximity can provide selective benefits to cells when introduced together into cells lacking de novo B₁₂ biosynthetic capacity. This hierarchical relationship confers benefits similar to those conferred by the juxtaposition of multiple antibiotic resistance genes; in both cases, groups of individual genetic entities (antibiotic genes or selfish operons) gain an advantage from mutual association if the phenotypes conferred by each member of the group provide a selective advantage to the host cell.

SUGGESTED READING

Baquero, F., and M. C. Negri. 1997. Selective compartments for resistant microorganisms in antibiotic gradients. Bioessays 19:731-736.

Baquero, F., M. C. Negri, M. I. Morosini, and J. Blazquez. 1998. Antibiotic-selective environments. Clin. Infect. Dis. 27:S5-S11.

Dandekar, T., B. Snel, M. Huynen, and P. Bork. 1998. Conservation of gene order: a fingerprint of proteins that physically interact. Trends Biochem. Sci. 23:324-328.

Davison, J. 1999. Genetic exchange between bacteria in the environment. Plasmid 42:73-91.

Hall, R. M., C. M. Collis, M. J. Kim, S. R. Partridge, G. D. Recchia, and H. W. Stokes. 1999. Mobile gene cassettes and integrons in evolution. Ann. N. Y. Acad. Sci. 870:68-80.

Jacob, F., D. Perrin, C. Sanchez, and J. Monod. 1960. L'operon: groupe de genes a expression coordonee par un operateur. C. R. Acad. Sci. 250:1727-1729.

Lawrence, J. G. 1999. Selfish operons: the evolutionary impact of gene clustering in prokaryotes and eukaryotes. Curr. Opin. Genet. Dev. 9:642-648.

Lawrence, J. G., and J. R. Roth. 1996. Selfish operons: horizontal transfer may drive the evolution of gene clusters. Genetics 143:1843-1860.

Liebert, C. A., R. M. Hall, and A. O. Summers. 1999. Transposon Tn21, flagship of the floating genome. Microbiol. Mol. Biol. Rev. 63:507-522.