Discovery of phage determinants that confer sensitivity to bacterial immune systems.

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.

Bacterial Retrons Function In Anti-Phage Defense.

Retrons are bacterial genetic elements comprised of a reverse transcriptase (RT) and a non-coding RNA (ncRNA). The RT uses the ncRNA as template, generating a chimeric RNA/DNA molecule in which the RNA and DNA components are covalently linked. Although retrons were discovered three decades ago, their function remained unknown. We report that retrons function as anti-phage defense systems. The defensive unit is composed of three components: the RT, the ncRNA, and an effector protein. We examined multiple retron systems and show that they confer defense against a broad range of phages via abortive infection. Focusing on retron Ec48, we show evidence that it "guards" RecBCD, a complex with central anti-phage functions in bacteria. Inhibition of RecBCD by phage proteins activates the retron, leading to abortive infection and cell death. Thus, the Ec48 retron forms a second line of defense that is triggered if the first lines of defense have collapsed.

The recombination initiation functions DprA and RecFOR suppress microindel mutations in Acinetobacter baylyi ADP1.

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.

chi sequences switch the RecBCD helicase-nuclease complex from degradative to replicative modes during the completion of DNA replication.

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.

Thymine-starvation-induced chromosomal fragmentation is not required for thymineless death in Escherichia coli.

Thymine or thymidine starvation induces robust chromosomal fragmentation in Escherichia coli thyA deoCABD mutants and is proposed to be the cause of thymineless death (TLD). However, fragmentation kinetics challenges the idea that fragmentation causes TLD, by peaking before the onset of TLD and disappearing by the time TLD accelerates. Quantity and kinetics of fragmentation also stay unchanged in hyper-TLD-exhibiting recBCD mutant, making its faster and deeper TLD independent of fragmentation as well. Elimination of fragmentation without affecting cellular metabolism did not abolish TLD in the thyA mutant, but reduced early TLD in the thyA recBCD mutant, suggesting replication-dependent, but undetectable by pulsed-field gel, double-strand breaks contributed to TLD. Chromosomal fragmentation, but not TLD, was eliminated in both the thyA and thyA recBCD mutants harboring deoCABD operon. The expression of a single gene, deoA, encoding thymidine phosphorylase, was sufficient to abolish fragmentation, suggesting thymidine-to-thymine interconversion during T-starvation being a key factor. Overall, this study reveals that chromosomal fragmentation, a direct consequence of T-starvation, is either dispensable or redundant for the overall TLD pathology, including hyper-TLD in the recBCD mutant. Replication forks, unlike chromosomal fragmentation, may provide a minor contribution to TLD, but only in the repair-deficient thyA deoCABD recBCD mutant.

UV-induced DNA damage disrupts the coordination between replication initiation, elongation and completion.

Replication initiation, elongation and completion are tightly coordinated to ensure that all sequences replicate precisely once each generation. UV-induced DNA damage disrupts replication and delays elongation, which may compromise this coordination leading to genome instability and cell death. Here, we profiled the Escherichia coli genome as it recovers from UV irradiation to determine how these replicational processes respond. We show that oriC initiations continue to occur, leading to copy number enrichments in this region. At late times, the combination of new oriC initiations and delayed elongating forks converging in the terminus appear to stress or impair the completion reaction, leading to a transient over-replication in this region of the chromosome. In mutants impaired for restoring elongation, including recA, recF and uvrA, the genome degrades or remains static, suggesting that cell death occurs early after replication is disrupted, leaving partially duplicated genomes. In mutants impaired for completing replication, including recBC, sbcCD xonA and recG, the recovery of elongation and initiation leads to a bottleneck, where the nonterminus region of the genome is amplified and accumulates, indicating that a delayed cell death occurs in these mutants, likely resulting from mis-segregation of unbalanced or unresolved chromosomes when cells divide.

Phage Mu Gam protein promotes NHEJ in concert with Escherichia coli ligase.

The Gam protein of transposable phage Mu is an ortholog of eukaryotic and bacterial Ku proteins, which carry out nonhomologous DNA end joining (NHEJ) with the help of dedicated ATP-dependent ligases. Many bacteria carry Gam homologs associated with either complete or defective Mu-like prophages, but the role of Gam in the life cycle of Mu or in bacteria is unknown. Here, we show that MuGam is part of a two-component bacterial NHEJ DNA repair system. Ensemble and single-molecule experiments reveal that MuGam binds to DNA ends, slows the progress of RecBCD exonuclease, promotes binding of NAD+-dependent Escherichia coli ligase A, and stimulates ligation. In vivo, Gam equally promotes both precise and imprecise joining of restriction enzyme-digested linear plasmid DNA, as well as of a double-strand break (DSB) at an engineered I-SceI site in the chromosome. Cell survival after the induced DSB is specific to the stationary phase. In long-term growth competition experiments, particularly upon treatment with a clastogen, the presence of gam in a Mu lysogen confers a distinct fitness advantage. We also show that the role of Gam in the life of phage Mu is related not to transposition but to protection of genomic Mu copies from RecBCD when viral DNA packaging begins. Taken together, our data show that MuGam provides bacteria with an NHEJ system and suggest that the resulting fitness advantage is a reason that bacteria continue to retain the gam gene in the absence of an intact prophage.

How Acts of Infidelity Promote DNA Break Repair: Collision and Collusion Between DNA Repair and Transcription.

Transcription is a fundamental cellular process and the first step in gene regulation. Although RNA polymerase (RNAP) is highly processive, in growing cells the progression of transcription can be hindered by obstacles on the DNA template, such as damaged DNA. The authors recent findings highlight a trade-off between transcription fidelity and DNA break repair. While a lot of work has focused on the interaction between transcription and nucleotide excision repair, less is known about how transcription influences the repair of DNA breaks. The authors suggest that when the cell experiences stress from DNA breaks, the control of RNAP processivity affects the balance between preserving transcription integrity and DNA repair. Here, how the conflict between transcription and DNA double-strand break (DSB) repair threatens the integrity of both RNA and DNA are discussed. In reviewing this field, the authors speculate on cellular paradigms where this equilibrium is well sustained, and instances where the maintenance of transcription fidelity is favored over genome stability.

Sequential eviction of crowded nucleoprotein complexes by the exonuclease RecBCD molecular motor.

In physiological settings, all nucleic acids motor proteins must travel along substrates that are crowded with other proteins. However, the physical basis for how motor proteins behave in these highly crowded environments remains unknown. Here, we use real-time single-molecule imaging to determine how the ATP-dependent translocase RecBCD travels along DNA occupied by tandem arrays of high-affinity DNA binding proteins. We show that RecBCD forces each protein into its nearest adjacent neighbor, causing rapid disruption of the protein-nucleic acid interaction. This mechanism is not the same way that RecBCD disrupts isolated nucleoprotein complexes on otherwise naked DNA. Instead, molecular crowding itself completely alters the mechanism by which RecBCD removes tightly bound protein obstacles from DNA.

How Chi Sequence Modifies RecBCD Single-Stranded DNA Translocase Activity.

E. coli RecBCD initiates homologous repair as well as degrades foreign DNA. Recognition of chi sequence (5'-GCTGGTGG-3') switches RecBCD from a destructive, nucleolytic mode into a repair-active one that promotes RecA-mediated recombination. RecBCD includes a 3'-to-5' single-stranded DNA (ssDNA) translocase in RecB subunit, a 5'-to-3' translocase in RecD, and a secondary translocase activity associated with RecBC. To understand how chi specifically affects each translocase activity, we directly visualized individual RecBCD translocating along DNA substrates containing a ssDNA gap of different polarities, with or without chi. Disappearance of RecBCD from the ssDNA signals the loss of the ssDNA translocase activity. For substrates containing a ssDNA gap that RecBCD encounters in the 3'-to-5' polarity (3'-to-5' ssDNA), wild-type RecBCD disappears from the DNA substrates with similarly high percentage, either with chi or without. This suggests that (1) the 3'-to-5' translocase in RecB is unaffected by chi and (2) it is low in processivity. With substrates containing a ssDNA gap that RecBCD encounters in the 5'-to-3' polarity (5'-to-3' ssDNA), we found that the leaving percentage increases significantly with chi, implying inactivation of the 5'-to-3' translocase of RecD upon chi recognition. Surprisingly, the RecD defective mutant RecBCDK177Q showed only ≈50 % leaving on 5'-to-3' ssDNA, directly revealing the presence of RecBC secondary translocase and its activity is unaffected by chi. Multiple ssDNA translocases within the RecBCD complex both before and after chi ensures processive unwinding of DNA substrates required for efficient recombination events.

Quantitative genomic analysis of RecA protein binding during DNA double-strand break repair reveals RecBCD action in vivo.

Understanding molecular mechanisms in the context of living cells requires the development of new methods of in vivo biochemical analysis to complement established in vitro biochemistry. A critically important molecular mechanism is genetic recombination, required for the beneficial reassortment of genetic information and for DNA double-strand break repair (DSBR). Central to recombination is the RecA (Rad51) protein that assembles into a spiral filament on DNA and mediates genetic exchange. Here we have developed a method that combines chromatin immunoprecipitation with next-generation sequencing (ChIP-Seq) and mathematical modeling to quantify RecA protein binding during the active repair of a single DSB in the chromosome of Escherichia coli. We have used quantitative genomic analysis to infer the key in vivo molecular parameters governing RecA loading by the helicase/nuclease RecBCD at recombination hot-spots, known as Chi. Our genomic analysis has also revealed that DSBR at the lacZ locus causes a second RecBCD-mediated DSBR event to occur in the terminus region of the chromosome, over 1 Mb away.

Short DNA containing χ sites enhances DNA stability and gene expression in E. coli cell-free transcription-translation systems.

Escherichia coli cell-free transcription-translation (TXTL) systems offer versatile platforms for advanced biomanufacturing and for prototyping synthetic biological parts and devices. Production and testing could be accelerated with the use of linear DNA, which can be rapidly and cheaply synthesized. However, linear DNA is efficiently degraded in TXTL preparations from E. coli. Here, we show that double-stranded DNA encoding χ sites-eight base-pair sequences preferentially bound by the RecBCD recombination machinery-stabilizes linear DNA and greatly enhances the TXTL-based expression and activity of a fluorescent reporter gene, simple regulatory cascades, and T7 bacteriophage particles. The χ-site DNA and the DNA-binding λ protein Gam yielded similar enhancements, and DNA with as few as four χ sites was sufficient to ensure robust gene expression in TXTL. Given the affordability and scalability of producing the short χ-site DNA, this generalized strategy is expected to advance the broad use of TXTL systems across its many applications. Biotechnol. Bioeng. 2017;114: 2137-2141. © 2017 Wiley Periodicals, Inc.

Completion of DNA replication in Escherichia coli.

The mechanism by which cells recognize and complete replicated regions at their precise doubling point must be remarkably efficient, occurring thousands of times per cell division along the chromosomes of humans. However, this process remains poorly understood. Here we show that, in Escherichia coli, the completion of replication involves an enzymatic system that effectively counts pairs and limits cellular replication to its doubling point by allowing converging replication forks to transiently continue through the doubling point before the excess, over-replicated regions are incised, resected, and joined. Completion requires RecBCD and involves several proteins associated with repairing double-strand breaks including, ExoI, SbcDC, and RecG. However, unlike double-strand break repair, completion occurs independently of homologous recombination and RecA. In some bacterial viruses, the completion mechanism is specifically targeted for inactivation to allow over-replication to occur during lytic replication. The results suggest that a primary cause of genomic instabilities in many double-strand-break-repair mutants arises from an impaired ability to complete replication, independent from DNA damage.

Single-strand gap repair involves both RecF and RecBCD pathways.

Homologous recombination repairs discontinuities in DNA including single-strand gaps (SSGs) and double-strand breaks (DSBs). This commentary describes how the RecBCD and RecF pathways might be exchangeable for the repair of their respective DSB and SSG canonical substrates. In particular, I will discuss how the RecBCD pathway could engage in the repair of an SSG even when the latter is not associated with a DSB.

Inhibition of RecBCD in Klebsiella pneumoniae by Gam and its effect on the efficiency of gene replacement.

Gam protein is an inhibitor of the host RecBCD exonuclease, and this inhibition is essential to the proficiency of Red recombinase-mediated gene replacement. In Klebsiella pneumoniae, the efficiency of this gene replacement was lower than that in Escherichia coli, and the minimum length of homologous extensions required was longer. Thus, it was supposed that the inhibitory effect of Gam against RecBCD was weak in K. pneumoniae. To test this hypothesis, a Gam-deficient Red recombinase expression plasmid and a ΔrecB K. pneumoniae mutant were constructed. The Gam-deficient Red recombinase showed a reduced capacity for gene replacement compared with that of the complete Red recombinase. The efficiency of gene replacement in the ΔrecB mutant was 6-8 times higher than the wild-type strain, and the minimum length for the homologous extensions was reduced to 100 bp. These results indicate that Gam does inhibit the RecBCD exonuclease in K. pneumoniae, but that this inhibition is not stringent. Furthermore, mutation of recB presents a convenient and efficient method to enhance the Red recombinase assisted gene replacement in K. pneumoniae.

Chi hotspot Control of RecBCD Helicase-nuclease: Enzymatic Tests Support the Intramolecular Signal-transduction Model.

Repair of broken DNA is essential for life; the reactions involved can also promote genetic recombination to aid evolution. In Escherichia coli, RecBCD enzyme is required for the major pathway of these events. RecBCD is a complex ATP-dependent DNA helicase with nuclease activity controlled by Chi recombination hotspots (5'-GCTGGTGG-3'). During rapid DNA unwinding, when Chi is in a RecC tunnel, RecB nuclease nicks DNA at Chi. Here, we test our signal transduction model - upon binding Chi (step 1), RecC signals RecD helicase to stop unwinding (step 2); RecD then signals RecB (step 3) to nick at Chi (step 4) and to begin loading RecA DNA strand-exchange protein (step 5). We discovered that ATP-γ-S, like the small molecule RecBCD inhibitor NSAC1003, causes RecBCD to nick DNA, independent of Chi, at novel positions determined by the DNA substrate length. Two RecB ATPase-site mutants nick at novel positions determined by their RecB:RecD helicase rate ratios. In each case, we find that nicking at the novel position requires steps 3 and 4 but not step 1 or 2, as shown by mutants altered at the intersubunit contacts specific for each step; nicking also requires RecD helicase and RecB nuclease activities. Thus, altering the RecB ATPase site, by small molecules or mutation, sensitizes RecD to signal RecB to nick DNA (steps 4 and 3, respecitvely) without the signal from RecC or Chi (steps 1 and 2). These new, enzymatic results strongly support the signal transduction model and provide a paradigm for studying other complex enzymes.

CRISPR-Cas adaptation in Escherichia 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.

Established and Emerging Methods for Protecting Linear DNA in Cell-Free Expression Systems.

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.

A flexible RecC surface loop required for Chi hotspot control of RecBCD enzyme.

Escherichia coli RecBCD helicase-nuclease promotes vital homologous recombination-based repair of DNA double-strand breaks. The RecB nuclease domain (Nuc) is connected to the RecB helicase domain by a 19-amino-acid tether. When DNA binds to RecBCD, published evidence suggests that Nuc moves ∼50 Šfrom the exit of a RecC tunnel, from which the 3'-ended strand emerges during unwinding, to a distant position on RecC's surface. During subsequent ATP-dependent unwinding of DNA, Nuc nicks the 3'-ended strand near 5'-GCTGGTGG-3' (Chi recombination hotspot). Here, we test our model of Nuc swinging on the tether from the RecC tunnel exit to the RecC distant surface and back to the RecC tunnel exit to cut at Chi. We identify positions in a flexible surface loop on RecC and on RecB Nuc with complementary charges, mutation of which strongly reduces but does not eliminate Chi hotspot activity in cells. The recC loop mutation interacts with recB mutations hypothesized to be in the Chi-activated intramolecular signal transduction pathway; the double mutants, but not the single mutants, eliminate Chi hotspot activity. A RecC amino acid near the flexible loop is also essential for full Chi activity; its alteration likewise synergizes with a signal transduction mutation to eliminate Chi activity. We infer that altering the RecC surface loop reduces coordination among the subunits, which is critical for Chi hotspot activity. We discuss other RecBCD mutants with related properties.

Chromosome Segregation and Cell Division Defects in Escherichia coli Recombination Mutants Exposed to Different DNA-Damaging Treatments.

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.