Surveillance and Processing of Foreign DNA by the Escherichia coli CRISPR-Cas System.

CRISPR-Cas adaptive immune systems protect bacteria and archaea against foreign genetic elements. In Escherichia coli, Cascade (CRISPR-associated complex for antiviral defense) is an RNA-guided surveillance complex that binds foreign DNA and recruits Cas3, a trans-acting nuclease helicase for target degradation. Here, we use single-molecule imaging to visualize Cascade and Cas3 binding to foreign DNA targets. Our analysis reveals two distinct pathways dictated by the presence or absence of a protospacer-adjacent motif (PAM). Binding to a protospacer flanked by a PAM recruits a nuclease-active Cas3 for degradation of short single-stranded regions of target DNA, whereas PAM mutations elicit an alternative pathway that recruits a nuclease-inactive Cas3 through a mechanism that is dependent on the Cas1 and Cas2 proteins. These findings explain how target recognition by Cascade can elicit distinct outcomes and support a model for acquisition of new spacer sequences through a mechanism involving processive, ATP-dependent Cas3 translocation along foreign DNA.

DNA sequence alignment by microhomology sampling during homologous recombination.

Homologous recombination (HR) mediates the exchange of genetic information between sister or homologous chromatids. During HR, members of the RecA/Rad51 family of recombinases must somehow search through vast quantities of DNA sequence to align and pair single-strand DNA (ssDNA) with a homologous double-strand DNA (dsDNA) template. Here, we use single-molecule imaging to visualize Rad51 as it aligns and pairs homologous DNA sequences in real time. We show that Rad51 uses a length-based recognition mechanism while interrogating dsDNA, enabling robust kinetic selection of 8-nucleotide (nt) tracts of microhomology, which kinetically confines the search to sites with a high probability of being a homologous target. Successful pairing with a ninth nucleotide coincides with an additional reduction in binding free energy, and subsequent strand exchange occurs in precise 3-nt steps, reflecting the base triplet organization of the presynaptic complex. These findings provide crucial new insights into the physical and evolutionary underpinnings of DNA recombination.

Protein dynamics of human RPA and RAD51 on ssDNA during assembly and disassembly of the RAD51 filament.

Homologous recombination (HR) is a crucial pathway for double-stranded DNA break (DSB) repair. During the early stages of HR, the newly generated DSB ends are processed to yield long single-stranded DNA (ssDNA) overhangs, which are quickly bound by replication protein A (RPA). RPA is then replaced by the DNA recombinase Rad51, which forms extended helical filaments on the ssDNA. The resulting nucleoprotein filament, known as the presynaptic complex, is responsible for pairing the ssDNA with homologous double-stranded DNA (dsDNA), which serves as the template to guide DSB repair. Here, we use single-molecule imaging to visualize the interplay between human RPA (hRPA) and human RAD51 during presynaptic complex assembly and disassembly. We demonstrate that ssDNA-bound hRPA can undergo facilitated exchange, enabling hRPA to undergo rapid exchange between free and ssDNA-bound states only when free hRPA is present in solution. Our results also indicate that the presence of free hRPA inhibits RAD51 filament nucleation, but has a lesser impact upon filament elongation. This finding suggests that hRPA exerts important regulatory influence over RAD51 and may in turn affect the properties of the assembled RAD51 filament. These experiments provide an important basis for further investigations into the regulation of human presynaptic complex assembly.

Abundance and diversity of methicillin-resistant bacteria from bathroom surfaces at workplaces using CHROMagar media, 16S, and dnaJ gene sequence typing.

University campus communities consist of dynamic and diverse human populations originated from different regions of the country or the world. Their national/global movement to and from campus may contribute to the spread and buildup of methicillin-resistant (MR) bacteria, including MR Staphylococci (MRS) on high-touch surfaces, sinks, and toilets. However, studies on MR bacteria contamination of surfaces, sinks, and toilets are scarce in workplaces outside of healthcare settings. Hence, little is known whether university communities contaminate campus bathrooms by MR bacteria. This study evaluated the abundance, identity, and phylogenetics of MR bacteria grown on CHROMagar MRSA media from bathrooms at workplaces. We collected 21 sink and 21 toilet swab samples from 10 buildings on campus and cultured them on CHROMagar MRSA media, extracted DNA from MR bacteria colonies, sequenced PCR products of 16S and dnaJ primers, determined the sequence identities by BLAST search, and constructed a phylogenetic tree. Of 42 samples, 57.1% (24/42) harbored MR bacteria. MR bacteria were more prevalent on the sink (61.9%) than in the toilet (52.2%) and in male bathrooms (54.2%) than in female bathrooms (41.7%). The colony count on the bathroom surfaces of 42 samples varied in that 42.9% (18/42), 33.3, 14.3, and 9.5% of samples harbored 0, 100, and > 1000 MR bacteria colonies, respectively. Of MR bacteria sequenced, BLAST search and phylogenetic analysis showed that Staphylococcus accounted for 60% of the MR bacteria and the rest were non-Staphylococci. Of Staphylococcus carrying MR (n = 15), 53.3% were S. hemolyticus followed by S. lugdunensis (26.7%), S. epidermidis (8%), and a newly discovered S. borealis in 2020 (4%). Of non-Staphylococci MR bacteria, 20% accounted for Sphingomonas koreensis. Campus bathrooms serve as a reservoir for diverse bacteria carrying MR, which pose a direct risk of infection and a potential source of horizontal gene transfer. To reduce the health risk posed by MR bacteria in high traffic areas such as bathrooms additional environmental monitoring and improved decontamination practices are needed.

Genome sequence of bacteriophage Janeemi isolated on Arthrobacter globiformis from soil collected in New York.

Janeemi is a bacteriophage that infects Arthrobacter globiformis B-2880, which was isolated from soil collected in New York City. The genome has a length of 43,877 bp and contains 69 predicted genes. Based on gene content similarity to phages in the actinobacteriophage database, Janeemi is assigned to phage cluster AZ1.

Single-stranded DNA curtains for real-time single-molecule visualization of protein-nucleic acid interactions.

Single-molecule imaging of biological macromolecules has dramatically impacted our understanding of many types of biochemical reactions. To facilitate these studies, we have established new strategies for anchoring and organizing DNA molecules on the surfaces of microfluidic sample chambers that are otherwise coated with fluid lipid bilayers. This previous work was reliant upon the use of double-stranded DNA, precluding access to information on biological processes involving single-stranded nucleic acid substrates. Here, we present procedures for aligning and visualizing single-stranded DNA molecules along the leading edges of nanofabricated barriers to lipid diffusion, in both "single-tethered" and "double-tethered" experimental formats. This new single-molecule approach provides long-awaited access to critical biological reactions involving single-stranded DNA binding proteins.

Requirements for catalysis in the Cre recombinase active site.

Members of the tyrosine recombinase (YR) family of site-specific recombinases catalyze DNA rearrangements using phosphoryl transfer chemistry that is identical to that used by the type IB topoisomerases (TopIBs). To better understand the requirements for YR catalysis and the relationship between the YRs and the TopIBs, we have analyzed the in vivo and in vitro recombination activities of all substitutions of the seven active site residues in Cre recombinase. We have also determined the structure of a vanadate transition state mimic for the Cre-loxP reaction that facilitates interpretation of mutant activities and allows for a comparison with similar structures from the related topoisomerases. We find that active site residues shared by the TopIBs are most sensitive to substitution. Only two, the tyrosine nucleophile and a conserved lysine residue that activates the 5'-hydroxyl leaving group, are strictly required to achieve >5% of wild-type activity. The two conserved arginine residues each tolerate one substitution that results in modest recombination activity and the remaining three active site positions can be substituted with several alternative amino acids while retaining a significant amount of activity. The results are discussed in the context of YR and TopIB structural models and data from related YR systems.

Complete Genome Sequences of Arthrobacter Phages Eraser, Kaylissa, and Phives.

Bacteriophages Phives, Kaylissa, and Eraser are siphoviruses infecting Arthrobacter globiformis B-2880 that were isolated in fall 2019 in Long Island, New York, from soil samples collected in Old Westbury, New York. All three bacteriophages are assigned to phage cluster AZ based on gene content similarity. While many aspects of the genomes are similar across the three phages, the endolysin genes for the phages are different and are located in different locations within the genomes.

The Relationship between Bacterial Load and Initial Run Time of a Surgical Helmet.

Background: Periprosthetic joint infection (PJI) is a complication of arthroplasty surgery with significant morbidity and mortality. Surgical helmets are a possible source of infection. Pre-existing dust and microorganisms on its surface may be blown into the surgical field by the helmet ventilation system. Methods: Twenty surgical helmets at our institution were assessed through microscopy and polymerase chain reaction testing. Helmets were arranged with agar plates under the front and rear outflow vents. Helmets ran while plates were exchanged at different time points. Bacterial growth was assessed via colony counts and correlated with fan operating time. Gram staining and 16S sequencing were performed to identify bacterial species. Results: The primary microbiological contaminate identified was Burkholderia. There was an inverse relationship between colony formation and fan operating time. The highest number of colonies was found within the first minute of fan operating time. There was a significant decrease in the number of colonies formed from the zero-minute to the three (27 vs 5; P = <.01), four (27 vs 3; P = <.01), and five-minute (27 vs 4; P = <.01) time points for the front outflow plates. A significant difference was also observed between the one-minute and four-minute time points (P = .046). Conclusion: We observed an inverse relationship between bacterial spread helmet fan operation time, which may correlate with dispersion of pre-existing contaminates. To decrease contamination risk, we recommend that helmets are run for at least 3 min prior to entering the operating room.

Bacteriophage therapy for bone and joint infections.

Antibiotic resistance represents a threat to human health. It has been suggested that by 2050, antibiotic-resistant infections could cause ten million deaths each year. In orthopaedics, many patients undergoing surgery suffer from complications resulting from implant-associated infection. In these circumstances secondary surgery is usually required and chronic and/or relapsing disease may ensue. The development of effective treatments for antibiotic-resistant infections is needed. Recent evidence shows that bacteriophage (phages; viruses that infect bacteria) therapy may represent a viable and successful solution. In this review, a brief description of bone and joint infection and the nature of bacteriophages is presented, as well as a summary of our current knowledge on the use of bacteriophages in the treatment of bacterial infections. We present contemporary published in vitro and in vivo data as well as data from clinical trials, as they relate to bone and joint infections. We discuss the potential use of bacteriophage therapy in orthopaedic infections. This area of research is beginning to reveal successful results, but mostly in nonorthopaedic fields. We believe that bacteriophage therapy has potential therapeutic value for implant-associated infections in orthopaedics. Cite this article: Bone Joint J 2021;103-B(2):234-244.

The Many Applications of Engineered Bacteriophages-An Overview.

Since their independent discovery by Frederick Twort in 1915 and Felix d'Herelle in 1917, bacteriophages have captured the attention of scientists for more than a century. They are the most abundant organisms on the planet, often outnumbering their bacterial hosts by tenfold in a given environment, and they constitute a vast reservoir of unexplored genetic information. The increased prevalence of antibiotic resistant pathogens has renewed interest in the use of naturally obtained phages to combat bacterial infections, aka phage therapy. The development of tools to modify phages, genetically or chemically, combined with their structural flexibility, cargo capacity, ease of propagation, and overall safety in humans has opened the door to a myriad of applications. This review article will introduce readers to many of the varied and ingenious ways in which researchers are modifying phages to move them well beyond their innate ability to target and kill bacteria.

Dissociation of Rad51 Presynaptic Complexes and Heteroduplex DNA Joints by Tandem Assemblies of Srs2.

Srs2 is a superfamily 1 (SF1) helicase and antirecombinase that is required for genome integrity. However, the mechanisms that regulate Srs2 remain poorly understood. Here, we visualize Srs2 as it acts upon single-stranded DNA (ssDNA) bound by the Rad51 recombinase. We demonstrate that Srs2 is a processive translocase capable of stripping thousands of Rad51 molecules from ssDNA at a rate of ∼50 monomers/s. We show that Srs2 is recruited to RPA clusters embedded between Rad51 filaments and that multimeric arrays of Srs2 assemble during translocation on ssDNA through a mechanism involving iterative Srs2 loading events at sites cleared of Rad51. We also demonstrate that Srs2 acts on heteroduplex DNA joints through two alternative pathways, both of which result in rapid disruption of the heteroduplex intermediate. On the basis of these findings, we present a model describing the recruitment and regulation of Srs2 as it acts upon homologous recombination intermediates.

Visualizing protein movement on DNA at the single-molecule level using DNA curtains.

A fundamental feature of many nucleic-acid binding proteins is their ability to move along DNA either by diffusion-based mechanisms or by ATP-hydrolysis driven translocation. For example, most site-specific DNA-binding proteins must diffuse to some extent along DNA to either find their target sites, or to otherwise fulfill their biological roles. Similarly, nucleic-acid translocases such as helicases and polymerases must move along DNA to fulfill their functions. In both instances, the proteins must also be capable of moving in crowded environments while navigating through DNA-bound obstacles. These types of behaviors can be challenging to analyze by bulk biochemical methods because of the transient nature of the interactions, and/or heterogeneity of the reaction intermediates. The advent of single-molecule methodologies has overcome some of these problems, and has led to many new insights into the mechanisms that contribute to protein motion along DNA. We have developed DNA curtains as a tool to facilitate single molecule observations of protein-nucleic acid interactions, and we have applied these new research tools to systems involving both diffusive-based motion as well as ATP directed translocation. Here we highlight these studies by first discussing how diffusion contributes to target searches by proteins involved in post-replicative mismatch repair. We then discuss DNA curtain assays of two different DNA translocases, RecBCD and FtsK, which participate in homologous DNA recombination and site-specific DNA recombination, respectively.

RPA antagonizes microhomology-mediated repair of DNA double-strand breaks.

Microhomology-mediated end joining (MMEJ) is a Ku- and ligase IV-independent mechanism for the repair of DNA double-strand breaks that contributes to chromosome rearrangements. Here we used a chromosomal end-joining assay to determine the genetic requirements for MMEJ in Saccharomyces cerevisiae. We found that end resection influences the ability to expose microhomologies; however, it is not rate limiting for MMEJ in wild-type cells. The frequency of MMEJ increased by up to 350-fold in rfa1 hypomorphic mutants, suggesting that replication protein A (RPA) bound to the single-stranded DNA (ssDNA) overhangs formed by resection prevents spontaneous annealing between microhomologies. In vitro, the mutant RPA complexes were unable to fully extend ssDNA and were compromised in their ability to prevent spontaneous annealing. We propose that the helix-destabilizing activity of RPA channels ssDNA intermediates from mutagenic MMEJ to error-free homologous recombination, thus preserving genome integrity.

Protein dynamics during presynaptic-complex assembly on individual single-stranded DNA molecules.

Homologous recombination is a conserved pathway for repairing double-stranded breaks, which are processed to yield single-stranded DNA overhangs that serve as platforms for presynaptic-complex assembly. Here we use single-molecule imaging to reveal the interplay between Saccharomyces cerevisiae RPA, Rad52 and Rad51 during presynaptic-complex assembly. We show that Rad52 binds RPA-ssDNA and suppresses RPA turnover, highlighting an unanticipated regulatory influence on protein dynamics. Rad51 binding extends the ssDNA, and Rad52-RPA clusters remain interspersed along the presynaptic complex. These clusters promote additional binding of RPA and Rad52. Our work illustrates the spatial and temporal progression of the association of RPA and Rad52 with the presynaptic complex and reveals a new RPA-Rad52-Rad51-ssDNA intermediate, with implications for how the activities of Rad52 and RPA are coordinated with Rad51 during the later stages of recombination.

Concentration-dependent exchange of replication protein A on single-stranded DNA revealed by single-molecule imaging.

Replication protein A (RPA) is a ubiquitous eukaryotic single-stranded DNA (ssDNA) binding protein necessary for all aspects of DNA metabolism involving an ssDNA intermediate, including DNA replication, repair, recombination, DNA damage response and checkpoint activation, and telomere maintenance. The role of RPA in most of these reactions is to protect the ssDNA until it can be delivered to downstream enzymes. Therefore a crucial feature of RPA is that it must bind very tightly to ssDNA, but must also be easily displaced from ssDNA to allow other proteins to gain access to the substrate. Here we use total internal reflection fluorescence microscopy and nanofabricated DNA curtains to visualize the behavior of Saccharomyces cerevisiae RPA on individual strands of ssDNA in real-time. Our results show that RPA remains bound to ssDNA for long periods of time when free protein is absent from solution. In contrast, RPA rapidly dissociates from ssDNA when free RPA is present in solution allowing rapid exchange between the free and bound states. In addition, the S. cerevisiae DNA recombinase Rad51 and E. coli single-stranded binding protein (SSB) also promote removal of RPA from ssDNA. These results reveal an unanticipated exchange between bound and free RPA suggesting a binding mechanism that can confer exceptionally slow off rates, yet also enables rapid displacement through a direct exchange mechanism that is reliant upon the presence of free ssDNA-binding proteins in solution. Our results indicate that RPA undergoes constant microscopic dissociation under all conditions, but this is only manifested as macroscopic dissociation (i.e. exchange) when free proteins are present in solution, and this effect is due to mass action. We propose that the dissociation of RPA from ssDNA involves a partially dissociated intermediate, which exposes a small section of ssDNA allowing other proteins to access to the DNA.

Sliding to the rescue of damaged DNA.

Single-molecule imaging experiments have shed new light on the methods used by the enzyme RecA to align single- and double-stranded DNA so that double-strand breaks can be repaired.