Novel silver nanoparticle-antibiotic combinations as promising antibacterial and anti-biofilm candidates against multiple-antibiotic resistant ESKAPE microorganisms.

HYPOTHESIS: The emergence of Multiple Antibiotic Resistance (MAR) in ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) pathogens is a global challenge to public health. The inherent antimicrobial nature of silver nanoparticles (AgNPs) makes them promising antimicrobial candidates against antibiotic-resistant pathogens. This study explores the combination of AgNPs with antibiotics (SACs) to create new antimicrobial agents effective against MAR ESKAPE microorganisms. METHODS: AgNPs were synthesized using Streptococcus pneumoniae ATCC 49619 and characterized for structure and surface properties. The SACs were tested against ESKAPE microorganisms using growth kinetics and time-kill curve methods. The effect of SACs on bacterial biofilms and the disruption of cell membranes was determined. The in-vitro cytotoxicity effect of the AgNPs was also studied. FINDINGS: The synthesized AgNPs (spherical, 7.37±4.55 nm diameter) were antimicrobial against MAR ESKAPE microorganisms. The SACs showed synergy with multiple conventional antibiotics, reducing their antibacterial concentrations up to 32-fold. Growth kinetics and time-kill studies confirmed the growth retardation effect and bactericidal activity of SACs. Mechanistic studies suggested that these biofilm-eradicating SACs probably resulted in the loss of bacterial cell membrane integrity, leading to leakage of the cytoplasmic content. The AgNPs were highly cytotoxic against skin melanoma cells but non-cytotoxic to normal Vero cells.

MSMEG_0311 is a conserved essential polar protein involved in mycobacterium cell wall metabolism.

Cell wall synthesis and cell division are two closely linked pathways in a bacterial cell which distinctly influence the growth and survival of a bacterium. This requires an appreciable coordination between the two processes, more so, in case of mycobacteria with an intricate multi-layered cell wall structure. In this study, we investigated a conserved gene cluster using CRISPR-Cas12 based gene silencing technology to show that knockdown of most of the genes in this cluster leads to growth defects. Investigating conserved genes is important as they likely perform vital cellular functions and the functional insights on such genes can be extended to other mycobacterial species. We characterised one of the genes in the locus, MSMEG_0311. The repression of this gene not only imparts severe growth defect but also changes colony morphology. We demonstrate that the protein preferentially localises to the polar region and investigate its influence on the polar growth of the bacillus. A combination of permeability and drug susceptibility assay strongly suggests a cell wall associated function of this gene which is also corroborated by transcriptomic analysis of the knockdown where a number of cell wall associated genes, particularly iniA and sigF regulon get altered. Considering the gene is highly conserved across mycobacterial species and appears to be essential for growth, it may serve as a potential drug target.

Effective gene silencing using type I-E CRISPR system in the multiploid, radiation-resistant bacterium Deinococcus radiodurans.

The extremely radiation-resistant bacterium, Deinococcus radiodurans, is a microbe of importance, both, for studying stress tolerance mechanisms and as a chassis for industrial biotechnology. However, the molecular tools available for use in this organism continue to be limiting, with its multiploid genome presenting an additional challenge. In view of this, the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas tools provide a large repertoire of applications for gene manipulation. We show the utility of the type I-E Cascade system for knocking down gene expression in this organism. A single-vector system was designed for the expression of the Cascade components as well as the crRNA. The type I-E Cascade system was better tolerated than the type II-A dCas9 system in D. radiodurans. An assayable acid phosphatase gene, phoN integrated into the genome of this organism could be knocked down to 10% of its activity using the Cascade system. Cascade-based knockdown of ssb, a gene important for radiation resistance resulted in poor recovery post-irradiation. Targeting the Radiation and Desiccation Response Motif (RDRM), upstream of the ssb, prevented de-repression of its expression upon radiation exposure. In addition to this, multi-locus targeting was demonstrated on the deinococcal genome, by knocking down both phoN and ssb expression simultaneously. The programmable CRISPR interference tool developed in this study will facilitate the study of essential genes, hypothetical genes, and cis-elements involved in radiation response as well as enable metabolic engineering in this organism. Further, the tool can be extended for implementing high-throughput approaches in such studies. IMPORTANCE Deinococcus radiodurans is a microbe that exhibits a very high degree of radiation resistance. In addition, it is also identified as an organism of industrial importance. We report the development of a gene-knockdown system in this organism by engineering a type I-E clustered regularly interspaced short palindromic repeat (CRISPR)-Cascade system. We used this system to silence an assayable acid phosphatase gene, phoN to 10% of its activity. The study further shows the application of the Cascade system to target an essential gene ssb, that caused poor recovery from radiation. We demonstrate the utility of CRISPR-Cascade to study the role of a regulatory cis-element in radiation response as well as for multi-gene silencing. This easy-to-implement CRISPR interference system would provide an effective tool for better understanding of complex phenomena such as radiation response in D. radiodurans and may also enhance the potential of this microbe for industrial application.

CRISPR-Cas12a assisted specific detection of mpox virus.

Mpox virus, a member of genus Orthopoxvirus, causes rash and flu-like symptoms in humans. In the recent global outbreak, it was reported from several geographical areas that have not historically reported mpox. Point of care, sensitive and specific mpox diagnostic assays are critical in checking the spread of the disease. We have developed a clustered regularly interspaced short palindromic repeats associated Cas12a nuclease-based assay for detecting mpox virus. Mpox specific conserved sequences were identified in polA (E9L) gene which differ by a single nucleotide polymorphism (SNP) from all the viruses present in the genus Orthopoxvirus. This SNP was exploited in our assay to specifically distinguish mpox virus from other related orthopox viruses with a limit of detection of 1 copy/µl in 30 min. The assay exhibits a sensitive and specific detection of mpox virus which can prove to be of practical value for its surveillance in areas infected with multiple orthopox viruses, especially in hotspots of mpox virus infections.

Harnessing CRISRP-Cas9 as an anti-mycobacterial system.

Rapid emergence of drug resistance has posed new challenges to the treatment of mycobacterial infections. As the pace of development of new drugs is slow, alternate treatment approaches are required. Recently, CRISPR-Cas systems have emerged as potential antimicrobials. These sequence-specific nucleases introduce double strand cuts in the target DNA, which if left unrepaired, prove fatal to the host. For most bacteria, homologous recombination repair (HRR) is the only pathway for repair and survival. Mycobacteria is one of the few bacteria which possesses the non-homologous end joining (NHEJ) system in addition to HRR for double strand break repair. To assess the antimicrobial potential of CRISPR-system, Cas9-induced breaks were introduced in the genome of Mycobacterium smegmatis and the survival was studied. While the single strand breaks were efficiently repaired, the organism was unable to repair the double strand breaks efficiently. In a mixed population of antibiotic-resistant and sensitive mycobacterial cells, selectively targeting a factor that confers hygromycin resistance, turned the entire population sensitive to the drug. Further, we demonstrate that the sequence-specific targeting could also be used for curing plasmids from mycobacterium cells. Considering the growing interest in nucleic acid-based therapy to curtail infections and combat antimicrobial resistance, our data shows that CRISPR-systems hold promise for future use as an antimicrobial against drug-resistant mycobacterial infections.

Loss of 2-Cys-Prx affects cellular ultrastructure, disturbs redox poise and impairs photosynthesis in cyanobacteria.

In a striking similarity to plant chloroplasts, the cyanobacterium Anabaena displays very low catalase activity, but expresses several peroxiredoxins (Prxs), including the typical 2-Cys-Prx (annotated as Alr4641), that detoxify H2 O2 . Due to the presence of multiple Prxs, the precise contribution of Alr4641 to the oxidative stress response of Anabaena is not well-defined. To unambiguously assess its in vivo function, the Alr4641 protein was knocked down using the CRISPRi approach in Anabaena PCC 7120. The knockdown strain (An-KD4641), which showed over 85% decrease in the content of Alr4641, was viable, but grew slower than the control strain (An-dCas9). An-KD4641 showed elevated levels of reactive oxygen species and the expression of several redox-responsive genes was analogous to that of An-dCas9 subjected to oxidative stress. The knockdown strain displayed reduced filament size, altered thylakoid ultrastructure, a marked drop in the ratio of phycocyanin to chlorophyll a and decreased photosynthetic parameters compared to An-dCas9. In comparison to the control strain, exposure to H2 O2 had a more severe effect on the photosynthetic parameters or survival of An-KD4641. Thus, in the absence of adequate catalase activity, 2-Cys-Prx appears to be the principal Prx responsible for maintaining redox homoeostasis in diverse photosynthetic systems ranging from chloroplasts to cyanobacteria.

An improved, simple and field-deployable CRISPR-Cas12a assay for the detection of SARS-CoV-2.

AIMS: The RT-PCR is the most popular confirmatory test for SARS-CoV-2. It is sensitive, but high instrumentation cost makes it difficult for use outside routine clinical setup. This has necessitated the development of alternative methods such as CRISPR-based DETECTR method which uses lateral flow technology. Although accurate and sensitive, this method is limited by complex steps and recurrent cost of high-quality lateral flow strips. The main goal of this study was to improve the Cas12a-based SARS-CoV-2 DETECTR method and develop a portable and field-deployable system to reduce the recurring consumable cost. METHODS AND RESULTS: Specific regions of N and E genes from SARS-CoV-2 virus and human RNase P (internal control) were reverse transcribed (RT) and amplified by loop-mediated isothermal amplification (LAMP). The amplified products were detected by a Cas12a-based trans-cleavage reaction that generated a fluorescent signal which could be easily visualized by naked eye. Detection of internal control, RNase P gene was improved and optimized by redesigning RT-LAMP primers. A number of steps were reduced by combining the reagents related to the detection of Cas12a trans-cleavage reaction into a single ready-to-use mix. A portable, cost-effective battery-operated instrument, CRISPR-CUBE was developed to run the assay and visualize the outcome. The method and instrument were validated using both contrived and patient samples. CONCLUSIONS: The simplified CRISPR-based SARS-CoV-2 detection and instrument developed in this study, along with improved design for internal control detection allows for easier, more definitive viral detection requiring only reagents, consumables and the battery operable CRISPR-CUBE. SIGNIFICANCE AND IMPACT OF STUDY: Significant improvement in Cas12 method, coupled with simple visualization of end point makes the method and instrument deployable at the point-of-care (POC) for SARS-CoV-2 detection, without any recurrent cost for the lateral flow strips which is used in other POC methods.

CLEVER assay: A visual and rapid RNA extraction-free detection of SARS-CoV-2 based on CRISPR-Cas integrated RT-LAMP technology.

AIM: The current scenario of COVID-19 pandemic has presented an almost insurmountable challenge even for the most sophisticated hospitals equipped with modern biomedical technology. There is an urgency to develop simple, fast and highly accurate methods for the rapid identification and isolation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected patients. To address the ongoing challenge, the present study offers a CLEVER assay (CRISPR-Cas integrated RT-LAMP Easy, Visual and Extraction-free RNA) which will allow RNA extraction-free method to visually diagnose COVID-19. RNA extraction is a major hurdle in preventing rapid and large-scale screening of samples particularly in low-resource regions because of the logistics and costs involved. METHOD AND RESULT: Herein, the visual SARS-CoV-2 detection method consists of RNA extraction-free method directly utilizing the patient's nasopharyngeal and oropharyngeal samples for reverse transcription loop-mediated isothermal amplification (RT-LAMP). Additionally, the assay also utilizes the integration of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas12-based system using different guide RNAs of N, E and an internal control POP7 (human RNase P) genes along with visual detection via lateral flow readout-based dip sticks with unaided eye (~100 min). Overall, the clinical sensitivity and specificity of the CLEVER assay were 89.6% and 100%, respectively. CONCLUSION: Together, our CLEVER assay offers a point-of-care tool with no equipment dependency and minimum technical expertise requirement for COVID-19 diagnosis. SIGNIFICANCE AND IMPACT OF THE STUDY: To address the challenges associated with COVID-19 diagnosis, we need a faster, direct and more versatile detection method for an efficient epidemiological management of the COVID-19 outbreak. The present study involves developing a method for detection of SARS-CoV-2 in human body without RNA isolation step that can visually be detected with unaided eye. Taken together, our assay offers to overcome one major defect of the prior art, that is, RNA extraction step, which could limit the deployment of the previous assays in a testing site having limited lab infrastructure.

Type I-E CRISPR-Cas System as a Defense System in Saccharomyces cerevisiae.

Defense against viruses and other mobile genetic elements (MGEs) is important in many organisms. The CRISPR-Cas systems found in bacteria and archaea constitute adaptive immune systems that can acquire the ability to target previously unrecognized MGEs. No CRISPR-Cas system is found to occur naturally in eukaryotic cells, but here, we demonstrate interference by a type I-E CRISPR-Cas system from Escherichia coli introduced in Saccharomyces cerevisiae. The designed CRISPR arrays are expressed and processed properly in S. cerevisiae. Targeted plasmids display reduced transformation efficiency, indicative of DNA cleavage. IMPORTANCE Genetic inactivation of viruses and other MGEs is an important tool with application in both research and therapy. Gene editing using, e.g., Cas9-based systems, can be used to inactivate MGEs in eukaryotes by introducing specific mutations. However, type I-E systems processively degrade the target which allows for inactivation without detailed knowledge of gene function. A reconstituted CRISPR-Cas system in S. cerevisiae can also function as a basic research platform for testing the role of various factors in the interference process.

Isolation and characterization of a recombinant class C acid phosphatase from Sphingobium sp. RSMS strain.

Tributyl phosphate (TBP) is extensively used in nuclear industry and is a major environmental pollutant. The mechanism for TBP degradation is not identified in any TBP-degrading bacteria. Here, we report identification of an acid phosphatase from Sphingobium sp. RSMS (Aps) that exhibits high specific activity towards monobutyl phosphate (MBP) and could be a terminal component of the TBP degradation process. A genomic DNA library of the bacteria was screened using a histochemical method which yielded 35 phosphatase clones. Among these, the clone that showed the highest MBP degradation was studied further. DNA sequence analysis showed that the genomic insert encodes a protein (Aps) which belongs to class C acid phosphatase. The recombinant Aps was found to be a dimer and hydrolysed MBP with a Kcat 68.1 ± 5.46 s- 1 and Km 2.5 mM ± 0.50. The protein was found to be nonspecific for phosphatase activity and hydrolyzed disparate organophosphates.

The era of Cas12 and Cas13 CRISPR-based disease diagnosis.

Clustered regularly interspaced short palindromic repeats (CRISPR) and associated protein (Cas) systems, since their discovery, have found growing applications in cell imaging, transcription modulation, therapeutics and diagnostics. Discovery of Cas12 and Cas13 have brought a new dimension to the field of disease diagnosis. These endonucleases have been extensively used for diagnosis of viral diseases in humans and animals and to a lesser extent in plants. The exigency of SARS-CoV-2 pandemic has highlighted the potential of CRISPR-Cas systems and sparked the development of innovative point-of-care diagnostic technologies. Rapid adaptation of CRISPR-chemistry combined with sensitive read-outs for emerging pathogens make them ideal candidates for detection and management of diseases in future. CRISPR-based approaches have been recruited for the challenging task of cancer detection and prognosis. It stands to reason that the field of CRISPR-Cas-based diagnosis is likely to expand with Cas12 and Cas13 playing a pivotal role. Here we focus exclusively on Cas12- and Cas13-based molecular diagnosis in humans, animals and plants including the detection of SARS-coronavirus. The CRISPR-based diagnosis of plant and animal diseases have not found adequate mention in previous reviews. We discuss various advancements, the potential shortfalls and challenges in the widespread adaptation of this technology for disease diagnosis.

DNA repair pathways important for the survival of Escherichia coli to hydrogen peroxide mediated killing.

Escherichia coli exposed to 1-3 mM hydrogen peroxide undergo killing which is designated as the mode-one killing which is a result of oxidative DNA damage. Oxidative stress mediated DNA damage can be repaired by various DNA repair pathways like base excision repair, nucleotide excision repair and homologous recombination repair. In this study we have investigated the role of multiple DNA repair pathways in survival to oxidative killing and assessed their relative importance. Results show that both nucleotide excision repair pathway as well as the RecF pathway of recombination repair are important for repair of the DNA damage caused by exposure to hydrogen peroxide. The study also provides the evidence that RecG helicase which is known for the resolution of Holliday junction intermediates plays a critical role in the survival of mode-one killing by peroxide. There is a severe impact on the survival of repair mutants when parameters like aeration and growth medium are changed. Low aeration and growth in minimal medium provide significant protection from the mode-one killing suggesting that under natural conditions Escherichia coli cells are likely to be protected from the oxidative stress mediated DNA damage.

Whole-Genome Sequencing of Sphingobium sp. Strain RSMS, a Highly Efficient Tributyl Phosphate-Degrading Bacterium.

Sphingobium sp. strain RSMS was described earlier as an efficient degrader of tributyl phosphate, an organic pollutant. This report describes the generation and annotation of the genome sequence of Sphingobium sp. strain RSMS, which will facilitate future studies to identify genetic elements responsible for the degradation of tributyl phosphate.

Novel molecular aspects of the CRISPR backbone protein 'Cas7' from cyanobacteria.

The cyanobacterium Anabaena PCC 7120 shows the presence of Type I-D CRISPR system that can potentially confer adaptive immunity. The Cas7 protein (Alr1562), which forms the backbone of the type I-D surveillance complex, was characterized from Anabaena. Alr1562, showed the presence of the non-canonical RNA recognition motif and two intrinsically disordered regions (IDRs). When overexpressed in E. coli, the Alr1562 protein was soluble and could be purified by affinity chromatography, however, deletion of IDRs rendered Alr1562 completely insoluble. The purified Alr1562 was present in the dimeric or a RNA-associated higher oligomeric form, which appeared as spiral structures under electron microscope. With RNaseA and NaCl treatment, the higher oligomeric form converted to the lower oligomeric form, indicating that oligomerization occurred due to the association of Alr1562 with RNA. The secondary structure of both these forms was largely similar, resembling that of a partially folded protein. The dimeric Alr1562 was more prone to temperature-dependent aggregation than the higher oligomeric form. In vitro, the Alr1562 bound more specifically to a minimal CRISPR unit than to the non-specific RNA. Residues required for binding of Alr1562 to RNA, identified by protein modeling-based approaches, were mutated for functional validation. Interestingly, these mutant proteins, showing reduced ability to bind RNA were predominantly present in dimeric form. Alr1562 was detected with specific antiserum in Anabaena, suggesting that the type I-D system is expressed and may be functional in vivo. This is the first report that describes the characterization of a Cas protein from any photosynthetic organism.

Characterization of multiple antibiotic resistance of culturable microorganisms and metagenomic analysis of total microbial diversity of marine fish sold in retail shops in Mumbai, India.

Marine fish species were analyzed for culturable and total metagenomic microbial diversity, antibiotic resistance (AR) pattern, and horizontal gene transfer in culturable microorganisms. We observed a high AR microbial load of 3 to 4 log CFU g-1. Many fish pathogens like Providencia, Staphylococcus, Klebsiella pneumoniae, Enterobacter, Vagococcus, and Aeromonas veronii were isolated. Photobacterium and Vibrio were two major fish and human pathogens which were identified in the fish metagenome. Other pathogens that were identified were Shewanella, Acinetobacter, Psychrobacter, and Flavobacterium. Most of these pathogens were resistant to multiple antibiotics such as erythromycin, kanamycin, neomycin, streptomycin, penicillin, cefotaxime, bacitracin, rifampicin, trimethoprim, ciprofloxacin, and doxycycline with a high multiple antibiotic resistance index of 0.54-0.77. The fish microflora showed high prevalence of AR genes like bla TEM, Class I integron, tetA, aph(3')-IIIa, ermB, aadA, and sul1. Nineteen of 26 AR isolates harbored Class I integrons showing high co-resistance to trimethoprim, kanamycin, doxycycline, and cefotaxime. Mobile R-plasmids from 6 of the 12 AR pathogens were transferred to recipient E. coli after conjugation. The transconjugants harbored the same R-plasmid carrying bla CTX-M, dfr1, tetA, bla TEM, and cat genes. This study confirms that fish is a potential carrier of AR pathogens which can enter the human gut via food chain. To the best of our knowledge, this is the first study in the Indian subcontinent reporting a direct evidence of spread of AR pathogens to humans from specific marine fish consumption.

CRISPR-Cas-Mediated Gene Silencing Reveals RacR To Be a Negative Regulator of YdaS and YdaT Toxins in Escherichia coli K-12.

Bacterial genomes are rich in horizontally acquired prophages. racR is an essential gene located in the rac prophage that is resident in many Escherichia coli genomes. Employing a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas-based gene silencing approach, we show that RacR is a negative regulator of the divergently transcribed and adjacent ydaS-ydaT operon in Escherichia coli K-12. Overexpression of YdaS and YdaT due to RacR depletion leads to cell division defects and decrease in survival. We further show that both YdaS and YdaT can act independently as toxins and that RacR serves to counteract the toxicity by tightly downregulating the expression of these toxins. IMPORTANCEracR is an essential gene and one of the many poorly studied genes found on the rac prophage element that is present in many Escherichia coli genomes. Employing a CRISPR-based approach, we have silenced racR expression to various levels and elucidated its physiological consequences. We show that the downregulation of racR leads to upregulation of the adjacent ydaS-ydaT operon. Both YdaS and YdaT act as toxins by perturbing the cell division resulting in enhanced cell killing. This work establishes a physiological role for RacR, which is to keep the toxic effects of YdaS and YdaT in check and promote cell survival. We, thus, provide a rationale for the essentiality of racR in Escherichia coli K-12 strains.

The CRISPR-Cas immune system: biology, mechanisms and applications.

Viruses are a common threat to cellular life, not the least to bacteria and archaea who constitute the majority of life on Earth. Consequently, a variety of mechanisms to resist virus infection has evolved. A recent discovery is the adaptive immune system in prokaryotes, a type of system previously thought to be present only in vertebrates. The system, called CRISPR-Cas, provide sequence-specific adaptive immunity and fundamentally affect our understanding of virus-host interaction. CRISPR-based immunity acts by integrating short virus sequences in the cell's CRISPR locus, allowing the cell to remember, recognize and clear infections. There has been rapid advancement in our understanding of this immune system and its applications, but there are many aspects that await elucidation making the field an exciting area of research. This review provides an overview of the field and highlights unresolved issues.

Efficient programmable gene silencing by Cascade.

Methods that permit controlled changes in the expression of genes are important tools for biological and medical research, and for biotechnological applications. Conventional methods are directed at individually changing each gene, its regulatory elements or its mRNA's translation rate. We demonstrate that the CRISPR-associated DNA-binding Cascade complex can be used for efficient, long-lasting and programmable gene silencing. When Cascade is targeted to a promoter sequence the transcription of the downstream gene is inhibited, resulting in dramatically reduced expression. The specificity of Cascade binding is provided by the integral crRNA component, which is easily designed to target virtually any stretch of DNA. Cascade targeted to the ORF sequence of the gene can also silence expression, albeit at lower efficiency. The system can be used to silence plasmid and chromosome targets, simultaneously target several genes and is active in different bacterial species and strains. The findings described here are an addition to the expanding range of CRISPR-based technologies and may be adapted to additional organisms and cell systems.

Increased ultraviolet radiation sensitivity of Escherichia coli grown at low temperature.

The repair of DNA damage caused by ultraviolet radiation (UVR) is well understood in both lower and higher organisms. Genetic studies carried out at optimum temperature for growth, 37 °C in Escherichia coli, have revealed the major pathways of DNA repair. We show that E. coli cells grown at 20 °C are more sensitive to UVR than cells grown at 37 °C. The analysis of knockout mutants demonstrates that cells impaired in recombinational DNA repair pathways show increased UV sensitivity at 20 °C. Cells with mutations in the nucleotide excision repair (NER) pathway genes are highly sensitive to UVR when grown at 37 °C and retain that sensitivity when grown at 20 °C, whereas wild-type cells are not sensitive when grown at 37 °C but become more sensitive to UVR when grown at low temperatures. Our results taken along with reports from the literature suggest that the UVR sensitivity of E. coli cells at low temperature could be due to impaired NER function.

Involvement of pnp in survival of UV radiation in Escherichia coli K-12.

Polynucleotide phosphorylase (PNPase), a multifunctional protein, is a 3'→5' exoribonuclease or exoDNase in the presence of inorganic phosphate (P(i)), and extends a 3'-OH of RNA or ssDNA in the presence of ADP or dADP. In Escherichia coli, PNPase is known to protect against H(2)O(2)- and mitomycin C-induced damage. Recent reports show that Bacillus subtilis PNPase is required for repair of H(2)O(2)-induced double-strand breaks. Here we show that absence of PNPase makes E. coli cells sensitive to UV, indicating that PNPase has a role in survival of UV radiation damage. Analyses of various DNA repair pathways show that in the absence of nucleotide excision repair, survival of UV radiation depends critically on PNPase function. Consequently, uvrA pnp, uvrB pnp and uvrC pnp strains show hypersensitivity to UV radiation. Whereas the pnp mutation is non-epistatic to recJ, recQ and recG mutations with respect to the UV-sensitivity phenotype, it is epistatic to uvrD, recB and ruvA mutations, implicating it in the recombinational repair process.