Short-Patch Double Illegitimate Recombination (SPDIR) has been recently identified as a rare mutation mechanism. During SPDIR, ectopic DNA single-strands anneal with genomic DNA at microhomologies and get integrated during DNA replication, presumably acting as primers for Okazaki fragments. The resulting microindel mutations are highly variable in size and sequence. In the soil bacterium Acinetobacter baylyi, SPDIR is tightly controlled by genome maintenance functions including RecA. It is thought that RecA scavenges DNA single-strands and renders them unable to anneal. To further elucidate the role of RecA in this process, we investigate the roles of the upstream functions DprA, RecFOR, and RecBCD, all of which load DNA single-strands with RecA. Here we show that all three functions suppress SPDIR mutations in the wildtype to levels below the detection limit. While SPDIR mutations are slightly elevated in the absence of DprA, they are strongly increased in the absence of both DprA and RecA. This SPDIR-avoiding function of DprA is not related to its role in natural transformation. These results suggest a function for DprA in combination with RecA to avoid potentially harmful microindel mutations, and offer an explanation for the ubiquity of dprA in the genomes of naturally non-transformable bacteria.

Cell-free protein synthesis (CFPS) is a method utilized for producing proteins without the limits of cell viability. The plug-and-play utility of CFPS is a key advantage over traditional plasmid-based expression systems and is foundational to the potential of this biotechnology. A key limitation of CFPS is the varying stability of DNA types, limiting the effectiveness of cell-free protein synthesis reactions. Researchers generally rely on plasmid DNA for its ability to support robust protein expression in vitro. However, the overhead required to clone, propagate, and purify plasmids reduces the potential of CFPS for rapid prototyping. While linear templates overcome the limits of plasmid DNA preparation, linear expression templates (LETs) were under-utilized due to their rapid degradation in extract based CFPS systems, limiting protein synthesis. To reach the potential of CFPS using LETs, researchers have made notable progress toward protection and stabilization of linear templates throughout the reaction. The current advancements range from modular solutions, such as supplementing nuclease inhibitors and genome engineering to produce strains lacking nuclease activity. Effective application of LET protection techniques improves expression yields of target proteins to match that of plasmid-based expression. The outcome of LET utilization in CFPS is rapid design-build-test-learn cycles to support synthetic biology applications. This review describes the various protection mechanisms for linear expression templates, methodological insights for implementation, and proposals for continued efforts that may further advance the field.

Over the past few years, numerous anti-phage defense systems have been discovered in bacteria. Although the mechanism of defense for some of these systems is understood, a major unanswered question is how these systems sense phage infection. To systematically address this question, we isolated 177 phage mutants that escape 15 different defense systems. In many cases, these escaper phages were mutated in the gene sensed by the defense system, enabling us to map the phage determinants that confer sensitivity to bacterial immunity. Our data identify specificity determinants of diverse retron systems and reveal phage-encoded triggers for multiple abortive infection systems. We find general themes in phage sensing and demonstrate that mechanistically diverse systems have converged to sense either the core replication machinery of the phage, phage structural components, or host takeover mechanisms. Combining our data with previous findings, we formulate key principles on how bacterial immune systems sense phage invaders.

Homologous recombination repairs potentially lethal DNA lesions such as double-strand DNA breaks (DSBs) and single-strand DNA gaps (SSGs). In Escherichia coli, DSB repair is initiated by the RecBCD enzyme that resects double-strand DNA ends and loads RecA recombinase to the emerging single-strand (ss) DNA tails. SSG repair is mediated by the RecFOR protein complex that loads RecA onto the ssDNA segment of gaped duplex. In both repair pathways, RecA catalyses reactions of homologous DNA pairing and strand exchange, while RuvABC complex and RecG helicase process recombination intermediates. In this work, we have characterised cytological changes in various recombination mutants of E. coli after three different DNA-damaging treatments: (i) expression of I-SceI endonuclease, (ii) γ-irradiation, and (iii) UV-irradiation. All three treatments caused severe chromosome segregation defects and DNA-less cell formation in the ruvABC, recG, and ruvABC recG mutants. After I-SceI expression and γ-irradiation, this phenotype was efficiently suppressed by the recB mutation, indicating that cytological defects result mostly from incomplete DSB repair. In UV-irradiated cells, the recB mutation abolished cytological defects of recG mutants and also partially suppressed the cytological defects of ruvABC recG mutants. However, neither recB nor recO mutation alone could suppress the cytological defects of UV-irradiated ruvABC mutants. The suppression was achieved only by simultaneous inactivation of the recB and recO genes. Cell survival and microscopic analysis suggest that chromosome segregation defects in UV-irradiated ruvABC mutants largely result from defective processing of stalled replication forks. The results of this study show that chromosome morphology is a valuable marker in genetic analyses of recombinational repair in E. coli.

Prokaryotes use the adaptive immunity mediated via the Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR associated (CRISPR-Cas) system for protection against invading elements such as phages and plasmids. The immunity is achieved by capturing small DNA fragments or spacers from foreign nucleic acids (protospacers) and integrating them into the host CRISPR locus. This step of CRISPR-Cas immunity called \'naïve CRISPR adaptation\' requires the conserved Cas1-Cas2 complex and is often supported by variable host proteins that assist in spacer processing and integration. Bacteria that have acquired new spacers become immune to the same invading elements when reinfected. CRISPR-Cas immunity can also be updated by integrating new spacers from the same invading elements, a process called \'primed adaptation\'. Only properly selected and integrated spacers are functional in the next steps of CRISPR immunity when their processed transcripts are used for RNA-guided target recognition and interference (target degradation). Capturing, trimming, and integrating new spacers in the correct orientation are universal steps of adaptation to all CRISPR-Cas systems, but some details are CRISPR-Cas type-specific and species-specific. In this review, we provide an overview of the mechanisms of CRISPR-Cas class 1 type I-E adaptation in Escherichia coli as a general model for adaptation processes (DNA capture and integration) that have been studied in detail. We focus on the role of host non-Cas proteins involved in adaptation, particularly on the role of homologous recombination.

Accurately completing DNA replication when two forks converge is essential to genomic stability. The RecBCD helicase-nuclease complex plays a central role in completion by promoting resection and joining of the excess DNA created when replisomes converge. chi sequences alter RecBCD activity and localize with crossover hotspots during sexual events in bacteria, yet their functional role during chromosome replication remains unknown. Here, we use two-dimensional agarose gel analysis to show that chi induces replication on substrates containing convergent forks. The induced replication is processive but uncoupled with respect to leading and lagging strand synthesis and can be suppressed by ter sites which limit replisome progression. Our observations demonstrate that convergent replisomes create a substrate that is processed by RecBCD and that chi, when encountered, switches RecBCD from a degradative to replicative function. We propose that chi serves to functionally differentiate DNA ends created during completion, which require degradation, from those created by chromosomal double-strand breaks, which require resynthesis.

Following infection of bacterial cells, bacteriophage modulate double-stranded DNA break repair pathways to protect themselves from host immunity systems and prioritise their own recombinases. Here, we present biochemical and structural analysis of two phage proteins, gp5.9 and Abc2, which target the DNA break resection complex RecBCD. These exemplify two contrasting mechanisms for control of DNA break repair in which the RecBCD complex is either inhibited or co-opted for the benefit of the invading phage. Gp5.9 completely inhibits RecBCD by preventing it from binding to DNA. The RecBCD-gp5.9 structure shows that gp5.9 acts by substrate mimicry, binding predominantly to the RecB arm domain and competing sterically for the DNA binding site. Gp5.9 adopts a parallel coiled-coil architecture that is unprecedented for a natural DNA mimic protein. In contrast, binding of Abc2 does not substantially affect the biochemical activities of isolated RecBCD. The RecBCD-Abc2 structure shows that Abc2 binds to the Chi-recognition domains of the RecC subunit in a position that might enable it to mediate the loading of phage recombinases onto its single-stranded DNA products.

RecBCD helicase/nuclease supports replication fork progress via recombinational repair or linear DNA degradation, explaining recBC mutant synthetic lethality with replication elongation defects. Since replication initiation defects leave chromosomes without replication forks, these should be insensitive to the recBCD status. Surprisingly, we found that both Escherichia coli dnaA46(Ts) and dnaC2(Ts) initiation mutants at semi-permissive temperatures are also recBC-colethal. Interestingly, dnaA46 recBC lethality suppressors suggest underinitiation as the problem, while dnaC2 recBC suppressors signal overintiation. Using genetic and physical approaches, we studied the dnaA46 recBC synthetic lethality, for the possibility that RecBCD participates in replication initiation. Overproduced DnaA46 mutant protein interferes with growth of dnaA+ cells, while the residual viability of the dnaA46 recBC mutant depends on the auxiliary replicative helicase Rep, suggesting replication fork inhibition by the DnaA46 mutant protein. The dnaA46 mutant depends on linear DNA degradation by RecBCD, rather than on recombinational repair. At the same time, the dnaA46 defect also interacts with Holliday junction-moving defects, suggesting reversal of inhibited forks. However, in contrast to all known recBC-colethals, which fragment their chromosomes, the dnaA46 recBC mutant develops no chromosome fragmentation, indicating that its inhibited replication forks are stable. Physical measurements confirm replication inhibition in the dnaA46 mutant shifted to semi-permissive temperatures, both at the level of elongation and initiation, while RecBCD gradually restores elongation and then initiation. We propose that RecBCD-catalyzed resetting of inhibited replication forks allows replication to displace the \"sticky\" DnaA46(Ts) protein from the chromosomal DNA, mustering enough DnaA for new initiations.

DNA recombination repair systems are essential for organisms to maintain genomic stability. In recent years, we have improved our understanding of the mechanisms of RecBCD/AddAB family-mediated DNA double-strand break repair. In E. coli, it is RecBCD that plays a central role, and in Firmicute Bacillus subtilis it is the AddAB complex that functions. However, there are open questions about the mechanism of DNA repair in bacteria. For example, how bacteria containing crossover hotspot instigator (Chi) sites regulate the activity of proteins. In addition, we still do not know the exact process by which the RecB nuclease or AddA nuclease structural domains load RecA onto DNA. We also know little about the mechanism of DNA repair in the industrially important production bacterium Corynebacterium glutamicum (C. glutamicum). Therefore, exploring DNA repair mechanisms in bacteria may not only deepen our understanding of the DNA repair process in this species but also guide us in the targeted treatment of diseases associated with recombination defects, such as cancer. In this paper, we firstly review the classical proteins RecBCD and AddAB involved in DNA recombination repair, secondly focus on the novel helical nuclease AdnAB found in the genus Mycobacterium.

Lateral gene transfer (LGT) facilitates many processes in bacterial ecology and pathogenesis, especially regarding pathogen evolution and the spread of antibiotic resistance across species. The obligate intracellular chlamydiae, which cause a range of diseases in humans and animals, were historically thought to be highly deficient in this process. However, research over the past few decades has demonstrated that this was not the case. The first reports of homologous recombination in the Chlamydiaceae family were published in the early 1990s. Later, the advent of whole-genome sequencing uncovered clear evidence for LGT in the evolution of the Chlamydiaceae, although the acquisition of tetracycline resistance in Chlamydia (C.) suis is the only recent instance of interphylum LGT. In contrast, genome and in vitro studies have shown that intraspecies DNA exchange occurs frequently and can even cross species barriers between closely related chlamydiae, such as between C. trachomatis, C. muridarum, and C. suis. Additionally, whole-genome analysis led to the identification of various DNA repair and recombination systems in C. trachomatis, but the exact machinery of DNA uptake and homologous recombination in the chlamydiae has yet to be fully elucidated. Here, we reviewed the current state of knowledge concerning LGT in Chlamydia by focusing on the effect of homologous recombination on the chlamydial genome, the recombination machinery, and its potential as a genetic tool for Chlamydia.