RecA is a highly conserved ∼38 kDa protein that plays a critical role in homologous recombination and also acts to stimulate LexA self-cleavage.⁴³ Structurally, monomeric RecA consists of three domains with a central core RecA fold that is flanked by smaller regulatory domains.⁴⁴ These monomers can form large nucleoprotein filaments on ssDNA (Figure 4A), which can extend across thousands of base pairs via cooperative oligomerization mediated by the core RecA fold.⁴⁵ Filamentous RecA has a deep helical groove that envelopes, stretches, and unwinds the bound DNA, preparing it for homology searching and subsequent DNA strand exchange. The core RecA fold binds ATP at the monomer–monomer interface (Figure 4A).⁴⁴ While only binding of ATP is required for filament formation and simple DNA strand exchange reactions, RecA also catalyzes ATP hydrolysis, which is important for filament depolymerization as well as some specific types of recombination activities.⁴³

Filamentous RecA acts as a co-protease to stimulate self-cleavage of LexA (discussed below), as well as other related members of the LexA/signal peptidase superfamily, such as phage λ repressor and UmuD in E. coli. In the case of phage repressor, cleavage stimulates the prophage to enter the lytic cycle.⁴⁶ Interestingly, RecA serves two roles in association with UmuD: it stimulates self-cleavage of UmuD to UmuD′ and is also, itself, an essential component of the associated Pol V mutasome.⁴⁷ The LexA binding site on the RecA filament has not been fully elucidated, but current models suggest that LexA may span adjacent RecA monomers across the deep helical groove.⁴⁸ ATP binding, but not hydrolysis, is required for the co-protease activity.

As ΔrecA strains are hypersensitive to antibiotics and less prone to acquired resistance,^35,36 RecA has been proposed as a novel target for slowing the evolution of antibiotic resistance. The feasibility of targeting RecA is further supported by the existence of biological protein modulators of RecA, including RecX and DinI, which both can antagonize SOS induction.⁴⁹ In an effort to discover small molecule RecA inhibitors, Singleton and colleagues have designed several high-throughput screens largely focused on E. coli RecA ATPase activity and identified potential inhibitors.⁵⁰⁻⁵³ While antimicrobial activity against E. coli has not yet been described, one lead RecA probe, suramin, was characterized against Mtb, where the inhibitor was suggested to potentiate the activity of the fluoroquinolone ciprofloxacin.⁵⁴ Although studies aimed at inhibiting RecA are promising, specificity is one important consideration that needs to be explored. In mammals, RecA has up to seven important homologues (Rad51 family).⁵⁵ In this context, rational approaches using nucleotide analogues to target RecA’s ATP binding site have been examined to a limited extent.⁵⁶ These offer a potential starting point for applying strategies that have yielded analogous protein kinase inhibitors that are ATP competitive and selective.⁵⁷

The ∼22 kDa LexA molecule consists of two domains separated by a short flexible linker and exists as a homodimer in solution. The N-terminal domain (NTD) contains specific DNA binding activity, and the C-terminal domain (CTD) contains protease activity (Figure 4B).^58,59 Dimeric LexA binds to SOS box DNA through a winged helix–turn–helix motif in the NTD with dimerization mediated by the CTDs.⁵⁹ The CTD contains a protease active site, with a serine-lysine catalytic dyad. Self-cleavage occurs at a protein loop in the same monomer, located near the linker between the two domains.⁵⁸ Crystal structures of well-characterized LexA mutants show that this cleavage loop can exist in two distinct states. In the “non-cleavable” state, the loop is far removed from the active site. In the “cleavable” state, it undergoes a large ∼20 Å conformational change, positioning the scissile peptide bond adjacent to the active site serine.⁵⁸ Interestingly, in the “cleavable” state, LexA binds its peptide substrate in a sharp β-turn, rather than the extended β-sheet peptide conformation common to canonical proteases.^58,60

Given the promising genetic studies on bacteria with noncleavable LexA discussed above, small molecule inhibition of LexA’s protease domain has been proposed.^15,34 To this end, while LexA’s distinct active site architecture offers potential advantages, it also poses two major challenges. First, as the substrate is tethered in cis, any competitive inhibitor will have to overcome the high local substrate concentration of the internal cleavage loop. Indeed, LexA shows inhibition only under large excesses of nonspecific protease inhibitors, such as diisopropyl fluorophosphates.⁶¹ Second, given self-cleavage, classical high-throughput protease assays, such as using fluorophore quencher-containing peptides in trans, cannot be readily translated to LexA. Despite these challenges, rational or screening-based approaches to the discovery of LexA inhibitors are well-justified.

To help inform rational inhibitor discovery efforts, we have performed extensive mutagenesis of LexA to elucidate the substrate specificity determinants.⁶² These experiments suggested that several residues within the cleavage loop make essential recognition contacts, while other specificity determinants are likely involved in facilitating LexA’s conformational change. Interestingly, stabilization of the β-turn within the cleavage loop accelerates self-cleavage, suggesting that small cyclic peptides may be tractable rational inhibitor starting points.⁶² Alternatively, allosteric inhibitors that prevent LexA’s conformational change or small molecules that could disrupt the LexA–RecA interface are viable strategies for LexA inhibition; however, our understanding of the biochemical mechanisms involved is incomplete. Despite the available structural snapshots, the basis for LexA’s conformational dynamics has not been elucidated and, in particular, the LexA–RecA interface remains poorly characterized despite dedicated efforts.^63,64 Further studies are required to understand these essential elements of RecA-induced LexA catalysis to help drive the discovery of potential SOS inhibitors.

Foremost among the effectors in SOS mutagenesis are DNA polymerases (Pol II, IV, and V in E. coli).³³ These polymerases catalyze translesion synthesis (TLS) by replacing the replicative Pol III, which stalls when encountering a damaged DNA template.⁶⁵ The ability of these polymerases to catalyze TLS, however, is associated with an increased frequency of mutation because of the lack of 3′–5′ exonuclease proofreading activity, weak processivity, and low fidelity.^66,67

Several of the critical enzymes involved in TLS, including Pol IV and Pol V in E. coli, are dissimilar enough from replicative polymerases that their identity as DNA polymerases came long only after the discovery of their role in mutagenesis.^24,68 Crystal structures of several of these “Y-family” DNA polymerases have yielded insight into their function and fidelity (Figure 4C). Despite a low level of sequence identity, the error-prone polymerases share the palm, finger, and thumb domains characteristic of their high-fidelity relatives (Figure 4C);⁶⁹⁻⁷¹ however, a detailed comparison shows structural differences that likely account for their lower fidelity.^24,72,73 The finger and thumb domains of the error-prone polymerases are in general shorter and appended with an additional domain known as the little finger domain, which has specialized function in Y-family polymerases. Further, O-helices, which typically play a role in proper Watson–Crick base pairing, are absent from the finger domains. Overall, these modifications result in a more flexible and open active site that may facilitate TLS over DNA damage due to bulky adducts or strand cross-links. Notably, elegant studies using FRET or time-resolved crystallography have demonstrated that, despite the appearance of a more static open active site, enzyme dynamics are critical to lesion bypass and catalysis.^74,75

The importance of these error-prone polymerases in generating mutation and resistance, combined with their relaxed fidelity, suggests the opportunity to inhibit these enzymes with small molecules. While to the best of our knowledge no specific inhibitors of bacterial Y-family polymerases have been discovered, there is a rich precedent for use of specific nucleotide drugs to combat viral infection or cancer. The structural features of Y-family polymerases could potentially be exploited to achieve the required specificity needed for a polymerase inhibitor. For example, the more open active site and lack of requirement for canonical Watson–Crick base pairing may permit incorporation of bulky chain-terminating nucleotides that would be discriminated against by high-fidelity polymerases. Because genotoxic antibiotics induce the expression of these error-prone polymerases, Y-family polymerase inhibitors would be predicted to synergize with SOS-inducing antibiotics.

HGT is mediated by natural competency and transformation, transduction, and conjugation, with the latter mechanisms being more common in clinical isolates.⁸⁷ Whereas plasmid DNA can be maintained outside the chromosome, the life cycle of other types of mobile DNA relies on recombination with the host chromosome (Figure 5). These mobile DNA elements vary greatly in size and complexity, but all encode a specialized recombination enzyme. These enzymes catalyze DNA breaking–joining reactions at the element termini, thus allowing for mobilization of the DNA element from its chromosomal site to distant sites. The simplest mobile element, the insertion sequence (IS), encompasses only a transposase enzyme and a pair of short inverted repeat sequences that flank the transposase gene and function as recognition sites where the DNA breaking–joining reactions occur. Transposons are more complex, with a set of inverted repeats that capture additional genes between them. Finally, bacteriophage elements may be more complex yet, encoding all the proteins necessary to conduct the infectious life cycle. Thus, unlike a simple IS, a transposon or phage is capable of mobilizing accessory genes that aid in its own dissemination, often by accumulating and evolving genes that endow its host with an increased rate of survival or pathogenicity such as virulence factors and antibiotic resistance determinants.⁸⁸

Integrons make up a related class of DNA elements that function as gene assembly platforms to direct the expression of exogenous genes.⁸⁹ As with transposons, integrons may similarly be enriched with antibiotic resistance and virulence genes. Whereas transposons “hop” from location to location, occasionally capturing functionality along the way, integrons are stationary elements that collect exogenous genes and insert them into their DNA locus. Remarkably, once a set of genes is a part of the integron, the integrase enzyme can catalyze the rearrangement of those genes with respect to the promoter, thus changing which genes are expressed. In this way, integrons can serve as a cache of antibiotic-resistant genes to be deployed for future use if needed.

The two major classes of specialized recombination enzymes are site-specific recombinases and transposases. Both conduct transesterification reactions of the DNA phosphodiester backbone without a requirement for high-energy cofactors such as ATP.^90,91 Site-specific recombinases contain an active site Tyr (or Ser) residue, which forms a covalent intermediate with the DNA backbone. This allows them to conduct a conservative two-step reaction that catalyzes strand exchange between two different pieces of double-stranded DNA (dsDNA), resulting in an intact, ligated dsDNA product. Bacterial transposases generally fall into the DDE/integrase superfamily of enzymes, containing protein folds remarkably similar to integrases encoded by retroviruses.⁹² The protein folds of these enzymes are topologically similar and bring at least three acidic residues (typically DDE) in the proximity of one another.⁹² The active site acidic residues bind divalent metal cations, required cofactors for catalysis, which promote transesterification reactions via a two-metal ion mechanism without any protein–DNA covalent intermediate.^93,94

Although there have been only a few studies identifying small molecule inhibitors of specialized bacterial recombination enzymes, the targeting of the retroviral integrase of HIV-1, a prominent integrase/DDE superfamily member, has been a significant clinical achievement and serves as a model for efforts to inhibit other specialized recombinases. The crystal structure of the Prototype Foamy Virus (PFV) retroviral integrase bound to donor DNA and different strand transfer inhibitors has been determined (Figure 5).⁹³ The structure supports a model in which the “diketo acid-like” pharmacophore of the inhibitors binds to the two active site divalent cations of the activated enzyme–donor DNA intasome complex. Inhibitor binding displaces the reactive 3′-OH of the donor DNA, thus deactivating the complex and inhibiting strand transfer. Diketo acid-like inhibitors have also been found for the Holliday junction (HJ) resolving enzyme encoded by poxviruses and for the Tn5 transposase,^95,96 suggesting that the motif may represent a general scheme for inhibiting members of this enzyme family. Small peptide inhibitors of site-specific Tyr recombinases that specifically bind to HJ DNA to interrupt enzymatic DNA transactions have also been identified.⁹⁷ One candidate peptide was able to block prophage excision by trapping such an intermediate, which also additionally resulted in antimicrobial activity, perhaps because of interference with DNA replication and repair.⁹⁸

For a DNA molecule to transfer between bacteria, it must cross lipid membranes, which is energetically disfavored. Large multiprotein molecular machines for natural competency, conjugation, and transduction allow for DNA shuttling and therefore represent potential targets for preventing the spread of resistance determinants (Figure 5). We refer our readers to recent reviews covering the molecular mechanisms and structural biology of each of these topics, noting that targeting of these processes with small molecules is virtually unexplored.⁹⁹⁻¹⁰¹ However, here we will highlight recent discoveries regarding the inhibition of a bacterial conjugation system with small molecules developed through rational design.

In bacterial conjugation, one strand (T-strand) of a dsDNA plasmid is transferred between organisms, ultimately resulting in the complete transfer of the genetic information encoded on the plasmid. The major components of conjugation are a relaxase enzyme, a type IV coupling protein (T4CP), and the membrane pore and pilus of a type IV secretion system (T4SS). The relaxase enzyme, often as part of a multiprotein “relaxasome”, recognizes the origin of transfer (oriT) sequence on the plasmid and nicks the T-strand to form a covalent 5′-phosphotyrosine linkage, thus creating a free DNA end to be transported through the membrane pore of the T4SS. After DNA transport and synthesis of the complementary strand, relaxase again nicks oriT, this time resulting in release and recircularization of the T-strand.

Recently, the Redinbo laboratory has discovered inhibitors for two types of relaxase enzymes using rational design based on structural insights. First, they determined the structure of an F-plasmid relaxase, whose catalytic cycle includes two simultaneous phosphotyrosine linkages to oriT. In the structure, they observed that a single divalent metal ion stabilized the formation of both phosphotyrosine linkages. On this basis, they reasoned that bisphosphonates could serve as functional mimics and potential inhibitors. After a directed screen against a small library of bisphosphonates, they found several that inhibited relaxase in vitro and inhibited conjugation in a relaxase and F-plasmid-dependent manner in cell-based assays.¹⁰² In a different study, the group also determined the crystal structure of the nicking enzyme of S. aureus (NES) in complex with oriT DNA (Figure 5). NES is present on clinically important conjugative plasmids known to result in vancomycin-resistant S. aureus. Unlike the F-plasmid relaxase, NES forms only one phosphotyrosine linkage. In this case, catalysis could be disrupted using a small polyamide designed to bind specifically to a five-nucleotide region of oriT that makes critical contacts with NES.¹⁰³

These examples, although preliminary, suggest specific targeting of the relaxasome is feasible and highlights the potential clinical utility of inhibitors of bacterial conjugation. Interestingly, in the F-plasmid case, inhibition of the relaxasome led to cell death.¹⁰² Because gene transfer mechanisms are highly activated during antibiotic stress, this finding raises the interesting possibility that inhibiting effector pathways in creative ways could not only curtail gene transfer but also lead to cell death by poisoning the cell with trapped intermediates.