The evolution of MarR family transcription factors as counter-silencers in regulatory networks

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Highlights

  • MarR family transcription factors (MFTFs) are ancient regulatory proteins, predating the divergence of Archaea and Bacteria.

  • Gene duplication has accommodated the adaptation of MFTFs to multiple regulatory functions.

  • Allosteric inhibition by small aromatic molecules confers MFTF responsiveness to environmental and physiological stimuli.

  • Variation in DNA-binding domains and promoters contributes to MFTF specificity.

Gene duplication facilitates the evolution of biological complexity, as one copy of a gene retains its original function while a duplicate copy can acquire mutations that would otherwise diminish fitness. Duplication has played a particularly important role in the evolution of regulatory networks by permitting novel regulatory interactions and responses to stimuli. The diverse MarR family of transcription factors (MFTFs) illustrate this concept, ranging from highly specific repressors of single operons to pleiotropic global regulators controlling hundreds of genes. MFTFs are often genetically and functionally linked to antimicrobial efflux systems. However, the SlyA MFTF lineage in the Enterobacteriaceae plays little or no role in regulating efflux but rather functions as transcriptional counter-silencers, which alleviate xenogeneic silencing of horizontally acquired genes and facilitate bacterial evolution by horizontal gene transfer. This review will explore recent advances in our understanding of MFTF traits that have contributed to their functional evolution.

Introduction

In his book Evolution by Gene Duplication [1], Susumu Ohno argued that gene duplication is a driving force in evolution, suggesting that ‘natural selection (has) merely modified, while redundancy created.’ He posited that gene duplications and the redundancy they produce provide the evolutionary space necessary for functional diversification and innovation. Duplication allows the exploration of otherwise ‘forbidden’ mutations, generating novel functions unique from those of the ancestral gene, ultimately resulting in greater organismal complexity. In the absence of gene duplication, mutations are limited to those that do not disrupt essential gene function, constraining potential evolutionary trajectories. Although more recent evolutionary studies suggest that horizontal gene transfer has a greater impact on bacterial evolution than classical intragenomic duplication [2, 3, 4], Ohno’s ideas are relevant here as well, as the expansion of a gene family by extensive lateral transfer also provides valuable evolutionary space. These processes are exemplified by the evolution of gene regulatory networks. Transcription is typically regulated by transcription factors (TFs), which bind near gene promoters to modulate their transcription by RNA polymerase (RNAP). Although one genome may encode hundreds of unique TFs, these belong to as few as 10 unique TF families [5,6]. The TFs contained within each family are the products of gene duplication, and the maintenance of these duplicates implies a fitness advantage. This can occur through neofunctionalization, wherein a duplicate acquires a novel function not present in the original gene, or subfunctionalization, wherein the function of an original gene is divided between two or more copies via mutational divergence [7]. TF gene duplication allows both in cis variation, resulting from changes in the promoter driving expression of a TF, which affects the binding and activity of upstream TFs and RNAP, or in trans variation, resulting from changes in the coding sequence of the TF, which alters interactions with cognate targets or other interaction partners. The net result of these duplication events and the resulting variation are increasingly complex regulatory networks that are able to respond to a variety of environmental and physiological stimuli.

The MarR (Multiple antibiotic resistance Regulator) family of TFs (MFTFs) exemplifies these processes. MFTFs are ancient, predating the divergence of Archaea and Bacteria [8] and presently comprising one of the most common TF families in bacteria. Although the average bacterial genome encodes seven unique MFTFs [9], the number can vary widely: Bacillus subtilis and Streptomyces coelicolor encode at least 20 each (depending on the strain), whereas Salmonella enterica serovar Typhimurium encodes seven, and the related enteric species Yersinia pseudotuberculosis encodes only three. Even endosymbiotic species, which have undergone substantial genome loss, encode multiple MFTFs, including Sodalis glossinidius, which encodes five [10••,11]. Despite their significant presence in bacteria, MFTFs exhibit limited sequence conservation between lineages, typically with less than 30% identity. This variability may reflect the inherent versatility of the MFTF backbone that allows them to interact with a variety of targets and respond to a variety of physiological and environmental signals. The ubiquity of MFTFs, particularly in the reduced genomes of endosymbiotic species, suggests that they serve an underappreciated role as central regulators of bacterial gene expression.

MFTFs were first recognized when Escherichia coli mutants exhibiting heightened resistance to multiple antibiotics were observed to encode mutations in MarR, the prototypical MFTF [12]. Since that discovery, MFTFs have been found to play a role in a number of important biological processes, including antibiotic resistance, virulence [13], oxidative stress [14], central metabolism [9,15], and the catabolism of a variety of aromatic compounds [9]. Although MFTFs were originally regarded as classical repressors of transcription, often of very small regulons, more recent studies have demonstrated that some members of the family can also function as global regulators, both positively and negatively modulating gene expression [11,16,17]. This scenario is perhaps best exemplified by the SlyA MFTF lineage in Enterobacteriaceae, which has evolved to function as transcriptional counter-silencers [10••,16,18,19]. SlyA proteins alleviate xenogeneic silencing of horizontally -acquired genes by proteins such as H-NS [20,21], thereby playing a vital role in the regulatory integration of horizontally -acquired genes. This allows a bacterial cell to realize a potential fitness benefit from horizontally -acquired genes, which might be detrimental if expressed in an unregulated fashion. Notably, SlyA is strongly conserved, as it is present in most species in the Enterobacteriaceae, including endosymbionts such as S. glossinidius and Wigglesworthia glossinidia, in which it is under strong selective constraints [22]. The SlyA lineage is not unique in controlling large numbers of genes. ScoC, which belongs to a distinct MFTF lineage, positively and negatively regulates more than 560 genes in B. subtilis, which are involved in sporulation, transport, motility, and metabolism [23], although the mechanistic basis for its pleiotropic function is unknown. The existence of MFTFs functioning as both small regulon repressors and global counter-silencers provides a clear example of functional innovation and diversification as a consequence of gene duplication.

Section snippets

The MFTF DNA binding motif is highly variable

Regardless of their specific function, MFTFs are generally defined by four common features: (1) a single globular domain, containing both (2) a winged-helix-turn-helix (wHTH) DNA-binding motif [24,25] and (3) a ligand-binding site that allows allosteric inhibition by environmental or physiological signals [26], and (4) genetic linkage to a multi-drug efflux pump. The chimeric wHTH (Figure 1) domain consists of a classical prokaryotic helix-turn-helix motif, in which the recognition helix (α4)

MFTFs are allosterically inhibited by multiple stimuli

The mechanism of allosteric inhibition, which sensitizes MFTFs to physiological and environmental stimuli, is a point of conjecture for MarR. Early studies observed that multi-drug resistance could be induced by the addition of salicylate to E. coli cultures [43], which was later shown to result from the inhibition of MarR-mediated DNA binding and repression [44]. Because MFTFs are comprised of a singular globular domain, ligand binding can easily impact interaction with DNA. The binding of

Genetic linkage to transporters suggests a physiological function for MFTFs

The third common feature of MFTFs, genetic linkage to efflux pumps or transporters, may reflect the primordial function of this protein family. In S. Typhimurium, four out of seven MFTF genes are genetically linked to multi-drug efflux pump or transporter-encoding genes (Figure 3), including the pleiotropic counter-silencer SlyA, although this linkage has been lost in most other enteric species, including endosymbiotic species. This suggests that, although SlyA may be under selective

Variation in cis has contributed to pleiotropic function

As mentioned above, cis-level variation, which alters the expression of individual MFTFs, can also contribute to regulatory evolution and functional adaptation. This was recently demonstrated in an analysis of SlyA alleles from S. Typhimurium and E. coli. Although allelic exchange demonstrated that both alleles are capable of functioning as counter-silencers in S. Typhimurium [10••], SlyA does not play a significant role in the E. coli regulatory network [46,47,48], apparently because of low

Conclusions

Gene duplication appears to have facilitated the asymmetrical adaptation of MFTF lineages (Figure 2). Although recent studies have highlighted the prominence of horizontal gene transfer in bacterial evolution [2,4], these studies are typically limited to the last 100 million years. We do not dispute that horizontal gene transfer has played a role in MFTF expansion, as some lineages are suggested to have been acquired via horizontal gene transfer, such as HucR of Deinococcus radiodurans [64].

Conflict of interest statement

Nothing declared.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgement

The National Institutes of Health provided support to F.C.F. (Grant numbers: AI39557, AI44486, AI118962, and AI112640).

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