Structural basis for topological regulation of Tn3 resolvase

Site-specific DNA recombinases play a variety of biological roles, often related to the dissemination of antibiotic resistance, and are also useful synthetic biology tools. The simplest site-specific recombination systems will recombine any two cognate sites regardless of context. Other systems have evolved elaborate mechanisms, often sensing DNA topology, to ensure that only one of multiple possible recombination products is produced. The closely-related resolvases from the Tn3 and γδ transposons have historically served as paradigms for the regulation of recombinase activity by DNA topology. However, despite many proposals, models of the multi-subunit protein-DNA complex (termed the synaptosome) that enforces this regulation have been unsatisfying due to a lack of experimental constraints and incomplete concordance with experimental data. Here we present new structural and biochemical data that lead to a new, detailed model of the Tn3 synaptosome, and discuss how it harnesses DNA topology to regulate the enzymatic activity of the recombinase.

Use of orthogonal serine integrases to multiplex plasmid conjugation and integration from E. coli into Streptomyces

Some major producers of useful bioactive natural products belong to the genus Streptomyces or related actinobacteria. Genetic engineering of these bacteria and the pathways that synthesize their valuable products often relies on serine integrases. To further improve the flexibility and efficiency of genome engineering via serine integrases, we explored whether multiple integrating vectors encoding orthogonally active serine integrases can be introduced simultaneously into Streptomyces recipients via conjugal transfer and integration. Pairwise combinations of Escherichia coli donors containing vectors encoding orthogonal serine integrases were used in each conjugation. Using donors containing plasmids (of various sizes) encoding either the φBT1 or the φC31 integration systems, we observed reproducible simultaneous plasmid integration into Streptomyces coelicolor and Streptomyces lividans at moderate frequencies after conjugation. This work demonstrated how site-specific recombination based on orthogonal serine integrases can save researchers time in genome engineering experiments in Streptomyces .

Multiple regulatory mechanisms control the production of CmrRST, an atypical signal transduction system in Clostridioides difficile

Clostridioides difficile, an intestinal pathogen and leading cause of nosocomial infection, exhibits extensive phenotypic heterogeneity through phase variation by site-specific recombination. The signal transduction system CmrRST, which encodes two response regulators (CmrR and CmrT) and a sensor kinase (CmrS), impacts C. difficile cell and colony morphology, surface and swimming motility, biofilm formation, and virulence in an animal model. CmrRST is subject to phase variation through site-specific recombination and reversible inversion of the ‘cmr switch’, and expression of cmrRST is also regulated by c-di-GMP through a riboswitch. The goal of this study was to determine how the cmr switch and c-di-GMP work together to regulate cmrRST expression. We generated “phase locked” strains by mutating key residues in the right inverted repeat flanking the cmr switch. Phenotypic characterization of these phase locked cmr-ON and -OFF strains demonstrates that they cannot switch between rough and smooth colony morphologies, respectively, or other CmrRST-associated phenotypes. Manipulation of c-di-GMP levels in these mutants showed that c-di-GMP promotes cmrRST expression and associated phenotypes independent of cmr switch orientation. We identified multiple promoters controlling cmrRST transcription, including one within the ON orientation of cmr switch and another that is positively autoregulated by CmrR. Overall, this work reveals a complex regulatory network that governs cmrRST expression and a unique intersection of phase variation and c-di-GMP signaling. These findings suggest that multiple environmental signals impact the production of this signaling transduction system.

Marker-free genome engineering in Amycolatopsis using the pSAM2 site-specific recombination system

ABSTRACTActinobacteria belonging to the genus Amycolatopsis are important for antibiotic production and other valuable biotechnological applications such as biodegradation or bioconversion. Despite their industrial importance, tools and methods for the genetic manipulation of Amycolatopsis are less developed than in other actinobacteria such as Streptomyces. Moreover, most of the existing methods do not support convenient marker-free genome engineering. Here, we report the use of the pSAM2 site-specific recombination system for the efficient deletion of marker genes or large DNA regions in Amycolatopsis. For this purpose, we constructed a shuttle vector, replicating in Escherichia coli and Amycolatopsis, expressing the Xis and Int proteins from the Streptomyces integrative and conjugative element pSAM2. These proteins are sufficient for site-specific recombination between the attachment sites attL and attR. We also constructed two plasmids, replicative in E. coli but not in Amycolatopsis, for the integration of the recombination sites attL and attR on each side of a region targeted for deletion. We exemplified the use of these tools in Amycolatopsis mediterranei DSM 40773 by obtaining with high efficiency (>95%) a marker-free deletion of one single gene in the rifamycin biosynthetic gene cluster or of the entire 90-kb cluster.IMPORTANCEThe genus Amycolatopsis is regarded as an important source of diverse specialized metabolites. Members of this genus are used in industry for the production of valuable antibiotics such as rifamycins or vancomycin. Amycolatopsis spp. also present a great interest for biotechnological applications such as biodegradation or bioconversion. Despite their importance, their genetic manipulation was somehow hampered by the lack of efficient tools. Here we report the successful use of the pSAM2 site-specific recombination system to construct unmarked deletion mutants, allowing marker recycling, or to create large deletions in A. mediterranei DSM 40773. The high efficiency of this site-specific recombination system and it possible application to other Amycolatopsis species open new opportunities for marker-free genome engineering in this genus.

A Guide to Cloning the Products of Polymerase Chain Reactions

This introduction outlines various methods to clone amplified DNAs and to facilitate the construction of complex multicomponent genetic units. Because of the ease with which the termini of amplified DNAs can be tailored by polymerase chain reaction (PCR), many of the methods outlined here use PCR not only to synthesize DNAs but also to link them together into purpose-designed constructs. The most recent refinements however have been the development of modular genetic units that can be harnessed to target DNAs not by PCR but by site-specific recombination enzymes.

Model-guided engineering of DNA sequences with predictable site-specific recombination rates

Site-specific recombination (SSR) is an important tool in genome editing and gene circuit design. However, its applications are limited by the inability to simply and predictably tune SSR reaction rates across orders of magnitude. Facile rate manipulation can in principle be achieved by modifying the nucleotide sequence of the DNA substrate of the recombinase, but the design principles for rationally doing so have not been elucidated. To enable predictable tuning of SSR reaction kinetics via DNA sequence, we developed an integrated experimental and computational method to parse individual nucleotide contributions to the overall reaction rate, which we used to analyze and engineer the DNA attachment sequence attP for the inversion reaction mediated by the serine recombinase Bxb1. A quantitative PCR method was developed to measure the Bxb1 reaction rate in vitro. Then, attP sequence libraries were designed, selected, and sequenced to inform a machine-learning model, which revealed that the Bxb1 reaction rate can be accurately represented assuming independent contributions of nucleotides at key positions. Next, we used the model to predict the performance of DNA site variants in reaction rate assays both in vitro and in Escherichia coli, with flipping rates ranging from 0.01- to 10-fold that of the wild-type attP sequence. Finally, we demonstrate that attP variants with predictable DNA recombination rates can be used in concert to achieve kinetic control in gene circuit design by coordinating the co-expression of two proteins in both their relative proportion and their total amount. Our high-throughput, data-driven method for rationally tuning SSR reaction rates through DNA sequence modification enhances our understanding of recombinase function and expands the synthetic biology toolbox.

Distribution of Prophages in the Oenococcus oeni Species

Oenococcus oeni is the most exploited lactic acid bacterium in the wine industry and drives the malolactic fermentation of wines. Although prophage-like sequences have been identified in the species, many are not characterized, and a global view of their integration and distribution amongst strains is currently lacking. In this work, we analyzed the complete genomes of 231 strains for the occurrence of prophages, and analyzed their size and positions of insertion. Our data show the limited variation in the number of prophages in O. oeni genomes, and that six sites of insertion within the bacterial genome are being used for site-specific recombination. Prophage diversity patterns varied significantly for different host lineages, and environmental niches. Overall, the findings highlight the pervasive presence of prophages in the O. oeni species, their role as a major source of within-species bacterial diversity and drivers of horizontal gene transfer. Our data also have implications for enhanced understanding of the prophage recombination events which occurred during evolution of O. oeni, as well as the potential of prophages in influencing the fitness of these bacteria in their distinct niches.