GRINS: Genetic elements that recode assembly-line polyketide synthases and accelerate their diversification.

Assembly-line polyketide synthases (PKSs) are large and complex enzymatic machineries with a multimodular architecture, typically encoded in bacterial genomes by biosynthetic gene clusters. Their modularity has led to an astounding diversity of biosynthesized molecules, many with medical relevance. Thus, understanding the mechanisms that drive PKS evolution is fundamental for both functional prediction of natural PKSs as well as for the engineering of novel PKSs. Here, we describe a repetitive genetic element in assembly-line PKS genes which appears to play a role in accelerating the diversification of closely related biosynthetic clusters. We named this element GRINS: genetic repeats of intense nucleotide skews. GRINS appear to recode PKS protein regions with a biased nucleotide composition and to promote gene conversion. GRINS are present in a large number of assembly-line PKS gene clusters and are particularly widespread in the actinobacterial genus Streptomyces While the molecular mechanisms associated with GRINS appearance, dissemination, and maintenance are unknown, the presence of GRINS in a broad range of bacterial phyla and gene families indicates that these genetic elements could play a fundamental role in protein evolution.

Evolution and Diversity of Assembly-Line Polyketide Synthases.

Assembly-line polyketide synthases (PKSs) are among the most complex protein machineries known in nature, responsible for the biosynthesis of numerous compounds used in the clinic. Their present-day diversity is the result of an evolutionary path that has involved the emergence of a multimodular architecture and further diversification of assembly-line PKSs. In this review, we provide an overview of previous studies that investigated PKS evolution and propose a model that challenges the currently prevailing view that gene duplication has played a major role in the emergence of multimodularity. We also analyze the ensemble of orphan PKS clusters sequenced so far to evaluate how large the entire diversity of assembly-line PKS clusters and their chemical products could be. Finally, we examine the existing techniques to access the natural PKS diversity in natural and heterologous hosts and describe approaches to further expand this diversity through engineering.

Structural heterogeneity of attC integron recombination sites revealed by optical tweezers.

A predominant tool for adaptation in Gram-negative bacteria is the functional genetic platform called integron. Integrons capture and rearrange promoterless gene cassettes in a unique recombination process involving the recognition of folded single-stranded DNA hairpins-so-called attC sites-with a strong preference for the attC bottom strand. While structural elements have been identified to promote this preference, their mechanistic action remains incomplete. Here, we used high-resolution single-molecule optical tweezers (OT) to characterize secondary structures formed by the attC bottom (${{att}}{{{C}}_{{\rm{bs}}}}$) and top (${{att}}{{{C}}_{{\rm{ts}}}}$) strands of the paradigmatic attCaadA7 site. We found for both sequences two structures-a straight, canonical hairpin and a kinked hairpin. Remarkably, the recombination-preferred ${{att}}{{{C}}_{{\rm{bs}}}}$ predominantly formed the straight hairpin, while the ${{att}}{{{C}}_{{\rm{ts}}}}$ preferentially adopted the kinked structure, which exposes only one complete recombinase binding box. By a mutational analysis, we identified three bases in the unpaired central spacer, which could invert the preferred conformations and increase the recombination frequency of the ${{att}}{{{C}}_{{\rm{ts}}}}$in vivo. A bioinformatics screen revealed structural bias toward a straight, canonical hairpin conformation in the bottom strand of many antibiotic resistance cassettes attC sites. Thus, we anticipate that structural fine tuning could be a mechanism in many biologically active DNA hairpins.

Dynamic stepwise opening of integron attC DNA hairpins by SSB prevents toxicity and ensures functionality.

Biologically functional DNA hairpins are found in archaea, prokaryotes and eukaryotes, playing essential roles in various DNA transactions. However, during DNA replication, hairpin formation can stall the polymerase and is therefore prevented by the single-stranded DNA binding protein (SSB). Here, we address the question how hairpins maintain their functional secondary structure despite SSB's presence. As a model hairpin, we used the recombinogenic form of the attC site, essential for capturing antibiotic-resistance genes in the integrons of bacteria. We found that attC hairpins have a conserved high GC-content near their apical loop that creates a dynamic equilibrium between attC fully opened by SSB and a partially structured attC-6-SSB complex. This complex is recognized by the recombinase IntI, which extrudes the hairpin upon binding while displacing SSB. We anticipate that this intriguing regulation mechanism using a base pair distribution to balance hairpin structure formation and genetic stability is key to the dissemination of antibiotic resistance genes among bacteria and might be conserved among other functional hairpins.

Recoding of synonymous genes to expand evolutionary landscapes requires control of secondary structure affecting translation.

Synthetic DNA design needs to harness the many information layers embedded in a DNA string. We previously developed the Evolutionary Landscape Painter (ELP), an algorithm that exploits the degeneracy of the code to increase protein evolvability. Here, we have used ELP to recode the integron integrase gene (intI1) in two alternative alleles. Although synonymous, both alleles yielded less IntI1 protein and were less active in recombination assays than intI1. We spliced the three alleles and mapped the activity decrease to the beginning of alternative sequences. Mfold predicted the presence of more stable secondary structures in the alternative genes. Using synonymous mutations, we decreased their stability and recovered full activity. Following a design-build-test approach, we have now updated ELP to consider such structures and provide streamlined alternative sequences. Our results support the possibility of modulating gene activity through the ad hoc design of 5' secondary structures in synthetic genes.

Efficiency of integron cassette insertion in correct orientation is ensured by the interplay of the three unpaired features of attC recombination sites.

The integron is a bacterial recombination system that allows acquisition, stockpiling and expression of cassettes carrying protein-coding sequences, and is responsible for the emergence and rise of multiresistance in Gram-negative bacteria. The functionality of this system depends on the insertion of promoterless cassettes in correct orientation, allowing their expression from the promoter located upstream of the cassette array. Correct orientation is ensured by strand selectivity of integron integrases for the bottom strand of cassette recombination sites (attC), recombined in form of folded single-stranded hairpins. Here, we investigated the basis of such strand selectivity by comparing recombination of wild-type and mutated attC sites with different lengths, sequences and structures. We show that all three unpaired structural features that distinguish the bottom and top strands contribute to strand selectivity. The localization of Extra-Helical Bases (EHBs) directly favors integrase binding to the bottom strand. The Unpaired Central Spacer (UCS) and the Variable Terminal Structure (VTS) influence strand selectivity indirectly, probably through the stabilization of the bottom strand and the resulting synapse due to the nucleotide skew between the two strands. These results underscore the importance of the single-stranded nature of the attC site that allows such tight control over integron cassette orientation.

Primary and promiscuous functions coexist during evolutionary innovation through whole protein domain acquisitions.

Molecular examples of evolutionary innovation are scarce and generally involve point mutations. Innovation can occur through larger rearrangements, but here experimental data is extremely limited. Integron integrases innovated from double-strand- toward single-strand-DNA recombination through the acquisition of the I2 α-helix. To investigate how this transition was possible, we have evolved integrase IntI1 to what should correspond to an early innovation state by selecting for its ancestral activity. Using synonymous alleles to enlarge sequence space exploration, we have retrieved 13 mutations affecting both I2 and the multimerization domains of IntI1. We circumvented epistasis constraints among them using a combinatorial library that revealed their individual and collective fitness effects. We obtained up to 104-fold increases in ancestral activity with various asymmetrical trade-offs in single-strand-DNA recombination. We show that high levels of primary and promiscuous functions could have initially coexisted following I2 acquisition, paving the way for a gradual evolution toward innovation.

Integron Identification in Bacterial Genomes and Cassette Recombination Assays.

Integrons are genetic elements involved in bacterial adaptation to the environment. Sedentary chromosomal integrons (SCIs) can stockpile and rearrange a myriad of different functions encoded in gene cassettes. Through their association with transposable elements and conjugative plasmids, some SCIs have acquired mobility and are now termed Mobile Integrons (MIs). MIs have reached the hospitals and are involved in the rise and spread of antibiotic resistance genes through horizontal gene transfer among numerous bacterial species. Here we aimed at describing methods for the detection of integrons in sequenced bacterial genomes as well as for the experimental characterization of the activity of their different components: the integrase and the recombination sites.

Structure-specific DNA recombination sites: Design, validation, and machine learning-based refinement.

Recombination systems are widely used as bioengineering tools, but their sites have to be highly similar to a consensus sequence or to each other. To develop a recombination system free of these constraints, we turned toward attC sites from the bacterial integron system: single-stranded DNA hairpins specifically recombined by the integrase. Here, we present an algorithm that generates synthetic attC sites with conserved structural features and minimal sequence-level constraints. We demonstrate that all generated sites are functional, their recombination efficiency can reach 60%, and they can be embedded into protein coding sequences. To improve recombination of less efficient sites, we applied large-scale mutagenesis and library enrichment coupled to next-generation sequencing and machine learning. Our results validated the efficiency of this approach and allowed us to refine synthetic attC design principles. They can be embedded into virtually any sequence and constitute a unique example of a structure-specific DNA recombination system.

Engineering of Chimeric Polyketide Synthases Using SYNZIP Docking Domains.

Engineering of assembly line polyketide synthases (PKSs) to produce novel bioactive compounds has been a goal for over 20 years. The apparent modularity of PKSs has inspired many engineering attempts in which entire modules or single domains were exchanged. In recent years, it has become evident that certain domain-domain interactions are evolutionarily optimized and, if disrupted, cause a decrease of the overall turnover rate of the chimeric PKS. In this study, we compared different types of chimeric PKSs in order to define the least invasive interface and to expand the toolbox for PKS engineering. We generated bimodular chimeric PKSs in which entire modules were exchanged, while either retaining a covalent linker between heterologous modules or introducing a noncovalent docking domain, or SYNZIP domain, mediated interface. These chimeric systems exhibited non-native domain-domain interactions during intermodular polyketide chain translocation. They were compared to otherwise equivalent bimodular PKSs in which a noncovalent interface was introduced between the condensing and processing parts of a module, resulting in non-native domain interactions during the extender unit acylation and polyketide chain elongation steps of their catalytic cycles. We show that the natural PKS docking domains can be efficiently substituted with SYNZIP domains and that the newly introduced noncovalent interface between the condensing and processing parts of a module can be harnessed for PKS engineering. Additionally, we established SYNZIP domains as a new tool for engineering PKSs by efficiently bridging non-native interfaces without perturbing PKS activity.

Differences in Integron Cassette Excision Dynamics Shape a Trade-Off between Evolvability and Genetic Capacitance.

Integrons ensure a rapid and "on demand" response to environmental stresses driving bacterial adaptation. They are able to capture, store, and reorder functional gene cassettes due to site-specific recombination catalyzed by their integrase. Integrons can be either sedentary and chromosomally located or mobile when they are associated with transposons and plasmids. They are respectively called sedentary chromosomal integrons (SCIs) and mobile integrons (MIs). MIs are key players in the dissemination of antibiotic resistance genes. Here, we used in silico and in vivo approaches to study cassette excision dynamics in MIs and SCIs. We show that the orientation of cassette arrays relative to replication influences attC site folding and cassette excision by placing the recombinogenic strands of attC sites on either the leading or lagging strand template. We also demonstrate that stability of attC sites and their propensity to form recombinogenic structures also regulate cassette excision. We observe that cassette excision dynamics driven by these factors differ between MIs and SCIs. Cassettes with high excision rates are more commonly found on MIs, which favors their dissemination relative to SCIs. This is especially true for SCIs carried in the Vibrio genus, where maintenance of large cassette arrays and vertical transmission are crucial to serve as a reservoir of adaptive functions. These results expand the repertoire of known processes regulating integron recombination that were previously established and demonstrate that, in terms of cassette dynamics, a subtle trade-off between evolvability and genetic capacitance has been established in bacteria.IMPORTANCE The integron system confers upon bacteria a rapid adaptation capability in changing environments. Specifically, integrons are involved in the continuous emergence of bacteria resistant to almost all antibiotic treatments. The international situation is critical, and in 2050, the annual number of deaths caused by multiresistant bacteria could reach 10 million, exceeding the incidence of deaths related to cancer. It is crucial to increase our understanding of antibiotic resistance dissemination and therefore integron recombination dynamics to find new approaches to cope with the worldwide problem of multiresistance. Here, we studied the dynamics of recombination and dissemination of gene encoding cassettes carried on integrons. By combining in silico and in vivo analyses, we show that cassette excision is highly regulated by replication and by the intrinsic properties of cassette recombination sites. We also demonstrated differences in the dynamics of cassette recombination between mobile and sedentary chromosomal integrons (MIs and SCIs). For MIs, a high cassette recombination rate is favored and timed to conditions when generating diversity (upon which selection can act) allows for a rapid response to environmental conditions and stresses. In contrast, for SCIs, cassette excisions are less frequent, limiting cassette loss and ensuring a large pool of cassettes. We therefore confirm a role of SCIs as reservoirs of adaptive functions and demonstrate that the remarkable adaptive success of integron recombination system is due to its intricate regulation.

Unmasking the ancestral activity of integron integrases reveals a smooth evolutionary transition during functional innovation.

Tyrosine (Y)-recombinases have evolved to deliver mechanistically different reactions on a variety of substrates, but these evolutionary transitions are poorly understood. Among them, integron integrases are hybrid systems recombining single- and double-stranded DNA partners. These reactions are asymmetric and need a replicative resolution pathway, an exception to the canonical second strand exchange model of Y-recombinases. Integron integrases possess a specific domain for this specialized pathway. Here we show that despite this, integrases are still capable of efficiently operating the ancestral second strand exchange in symmetrical reactions between double-stranded substrates. During these reactions, both strands are reactive and Holliday junction resolution can follow either pathway. A novel deep-sequencing approach allows mapping of the crossover point for the second strand exchange. The persistence of the ancestral activity in integrases illustrates their robustness and shows that innovation towards new recombination substrates and resolution pathways was a smooth evolutionary process.

The Integron: Adaptation On Demand.

The integron is a powerful system which, by capturing, stockpiling, and rearranging new functions carried by gene encoding cassettes, confers upon bacteria a rapid adaptation capability in changing environments. Chromosomally located integrons (CI) have been identified in a large number of environmental Gram-negative bacteria. Integron evolutionary history suggests that these sedentary CIs acquired mobility among bacterial species through their association with transposable elements and conjugative plasmids. As a result of massive antibiotic use, these so-called mobile integrons are now widespread in clinically relevant bacteria and are considered to be the principal agent in the emergence and rise of antibiotic multiresistance in Gram-negative bacteria. Cassette rearrangements are catalyzed by the integron integrase, a site-specific tyrosine recombinase. Central to these reactions is the single-stranded DNA nature of one of the recombination partners, the attC site. This makes the integron a unique recombination system. This review describes the current knowledge on this atypical recombination mechanism, its implications in the reactions involving the different types of sites, attC and attI, and focuses on the tight regulation exerted by the host on integron activity through the control of attC site folding. Furthermore, cassette and integrase expression are also highly controlled by host regulatory networks and the bacterial stress (SOS) response. These intimate connections to the host make the integron a genetically stable and efficient system, granting the bacteria a low cost, highly adaptive evolution potential "on demand".