In-Depth Specificity Profiling of Endopeptidases Using Dedicated Mix-and-Split Synthetic Peptide Libraries and Mass Spectrometry.

Proteases comprise the class of enzymes that catalyzes the hydrolysis of peptide bonds, thereby playing a pivotal role in many aspects of life. The amino acids surrounding the scissile bond determine the susceptibility toward protease-mediated hydrolysis. A detailed understanding of the cleavage specificity of a protease can lead to the identification of its endogenous substrates, while it is also essential for the design of inhibitors. Although many methods for protease activity and specificity profiling exist, none of these combine the advantages of combinatorial synthetic libraries, i.e., high diversity, equimolar concentration, custom design regarding peptide length, and randomization, with the sensitivity and detection power of mass spectrometry. Here, we developed such a method and applied it to study a group of bacterial metalloproteases that have the unique specificity to cleave between two prolines, i.e., Pro-Pro endopeptidases (PPEPs). We not only confirmed the prime-side specificity of PPEP-1 and PPEP-2, but also revealed some new unexpected peptide substrates. Moreover, we have characterized a new PPEP (PPEP-3) that has a prime-side specificity that is very different from that of the other two PPEPs. Importantly, the approach that we present in this study is generic and can be extended to investigate the specificity of other proteases.

Immobilized Regenerable Active Chlorine within a Zirconium-Based MOF Textile Composite to Eliminate Biological and Chemical Threats.

The most recent global health crisis caused by the SARS-CoV-2 outbreak and the alarming use of chemical warfare agents highlight the necessity to produce efficient protective clothing and masks against biohazard and chemical threats. However, the development of a multifunctional protective textile is still behind to supply adequate protection for the public. To tackle this challenge, we designed multifunctional and regenerable N-chlorine based biocidal and detoxifying textiles using a robust zirconium metal-organic framework (MOF), UiO-66-NH2, as a chlorine carrier which can be easily coated on textile fibers. A chlorine bleaching converted the amine groups located on the MOF linker to active N-chlorine structures. The fibrous composite exhibited rapid biocidal activity against both Gram-negative bacteria (E. coli) and Gram-positive bacteria (S. aureus) with up to a 7 log reduction within 5 min for each strain as well as a 5 log reduction of SARS-CoV-2 within 15 min. Moreover, the active chlorine loaded MOF/fiber composite selectively and rapidly degraded sulfur mustard and its chemical simulant 2-chloroethyl ethyl sulfide (CEES) with half-lives less than 3 minutes. The versatile MOF-based fibrous composite designed here has the potential to serve as protective cloth against both biological and chemical threats.

Untargeted accurate identification of highly pathogenic bacteria directly from blood culture flasks.

To improve the preparedness against exposure to highly pathogenic bacteria and to anticipate the wide variety of bacteria that can cause bloodstream infections (BSIs), a safe, unbiased and highly accurate identification method was developed. Our liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method can identify highly pathogenic bacteria, their near-neighbors and bacteria that are common causes of BSIs directly from positive blood culture flasks. The developed Peptide-Based Microbe Detection Engine (http://proteome2pathogen.com) relies on a two-step workflow: a genus-level search followed by a species-level search. This strategy enables the rapid identification of microorganisms based on the analyzed proteome. This method was successfully used to identify strains of Bacillus anthracis, Brucella abortus, Brucella melitensis, Brucella suis, Burkholderia pseudomallei, Burkholderia mallei, Francisella tularensis, Yersinia pestis and closely related species from simulated blood culture flasks. This newly developed LC-MS/MS method is a safe and rapid method for accurately identifying bacteria directly from positive blood culture flasks.

Discovery of a new Pro-Pro endopeptidase, PPEP-2, provides mechanistic insights into the differences in substrate specificity within the PPEP family.

Pro-Pro endopeptidases (PPEPs) belong to a recently discovered family of proteases capable of hydrolyzing a Pro-Pro bond. The first member from the bacterial pathogen Clostridium difficile (PPEP-1) cleaves two C. difficile cell-surface proteins involved in adhesion, one of which is encoded by the gene adjacent to the ppep-1 gene. However, related PPEPs may exist in other bacteria and may shed light on substrate specificity in this enzyme family. Here, we report on the homolog of PPEP-1 in Paenibacillus alvei, which we denoted PPEP-2. We found that PPEP-2 is a secreted metalloprotease, which likewise cleaved a cell-surface protein encoded by an adjacent gene. However, the cleavage motif of PPEP-2, PLP↓PVP, is distinct from that of PPEP-1 (VNP↓PVP). As a result, an optimal substrate peptide for PPEP-2 was not cleaved by PPEP-1 and vice versa. To gain insight into the specificity mechanism of PPEP-2, we determined its crystal structure at 1.75 Å resolution and further confirmed the structure in solution using small-angle X-ray scattering (SAXS). We show that a four-amino-acid loop, which is distinct in PPEP-1 and -2 (GGST in PPEP-1 and SERV in PPEP-2), plays a crucial role in substrate specificity. A PPEP-2 variant, in which the four loop residues had been swapped for those from PPEP-1, displayed a shift in substrate specificity toward PPEP-1 substrates. Our results provide detailed insights into the PPEP-2 structure and the structural determinants of substrate specificity in this new family of PPEP proteases.

Covalent attachment and Pro-Pro endopeptidase (PPEP-1)-mediated release of Clostridium difficile cell surface proteins involved in adhesion.

In the past decade, Clostridium difficile has emerged as an important gut pathogen. This anaerobic, Gram-positive bacterium is the main cause of infectious nosocomial diarrhea. Whereas much is known about the mechanism through which the C. difficile toxins cause diarrhea, relatively little is known about the dynamics of adhesion and motility, which is mediated by cell surface proteins. This review will discuss the recent advances in our understanding of the sortase-mediated covalent attachment of cell surface (adhesion) proteins to the peptidoglycan layer of C. difficile and their release through the action of a highly specific secreted metalloprotease (Pro-Pro endopeptidase 1, PPEP-1). Specific emphasis will be on a model in which PPEP-1 and its substrates control the switch from a sessile to motile phenotype in C. difficile, and how this is regulated by the cyclic dinucleotide c-di-GMP (3'-5' cyclic dimeric guanosine monophosphate).

A Novel Fic (Filamentation Induced by cAMP) Protein from Clostridium difficile Reveals an Inhibitory Motif-independent Adenylylation/AMPylation Mechanism.

Filamentation induced by cAMP (Fic) domain proteins have been shown to catalyze the transfer of the AMP moiety from ATP onto a protein target. This type of post-translational modification was recently shown to play a crucial role in pathogenicity mediated by two bacterial virulence factors. Herein we characterize a novel Fic domain protein that we identified from the human pathogen Clostridium difficile The crystal structure shows that the protein adopts a classical all-helical Fic fold, which belongs to class II of Fic domain proteins characterized by an intrinsic N-terminal autoinhibitory α-helix. A conserved glutamate residue in the inhibitory helix motif was previously shown in other Fic domain proteins to prevent proper binding of the ATP γ-phosphate. However, here we demonstrate that both ATP binding and autoadenylylation activity of the C. difficile Fic domain protein are independent of the inhibitory motif. In support of this, the crystal structure of a mutant of this Fic protein in complex with ATP reveals that the γ-phosphate adopts a conformation unique among Fic domains that seems to override the effect of the inhibitory helix. These results provide important structural insight into the adenylylation reaction mechanism catalyzed by Fic domains. Our findings reveal the presence of a class II Fic domain protein in the human pathogen C. difficile that is not regulated by autoinhibition and challenge the current dogma that all class I-III Fic domain proteins are inhibited by the inhibitory α-helix.

Typing Pseudomonas aeruginosa Isolates with Ultrahigh Resolution MALDI-FTICR Mass Spectrometry.

The introduction of standardized matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platforms in the medical microbiological practice has revolutionized the way microbial species identification is performed on a daily basis. To a large extent, this is due to the ease of operation. Acquired spectra are compared to profiles obtained from cultured colonies present in a reference spectra database. It is fast and reliable, and costs are low compared to previous diagnostic approaches. However, the low resolution and dynamic range of the MALDI-TOF profiles have shown limited applicability for the discrimination of different bacterial strains, as achieved with typing based on genetic markers. This is pivotal in cases where certain strains are associated with, e.g., virulence or antibiotic resistance. Ultrahigh resolution MALDI-FTICR MS allows the measurement of small proteins at isotopic resolution and can be used to analyze complex mixtures with increased dynamic range and higher precision than MALDI-TOF MS, while still generating results in a similar time frame. Here, we propose to use ultrahigh resolution 15T MALDI-Fourier transform ion cyclotron resonance (FTICR) MS to discriminate clinically relevant bacterial strains after species identification performed by MALDI-TOF MS. We used a collection of well characterized Pseudomonas aeruginosa strains, featuring distinct antibiotic resistance profiles, and isolates obtained during hospital outbreaks. Following cluster analysis based on amplification fragment length polymorphism (AFLP), these strains were grouped into three different clusters. The same clusters were obtained using protein profiles generated by MALDI-FTICR MS. Subsequent intact protein analysis by electrospray ionization (ESI)-collision-induced dissociation (CID)-FTICR MS was applied to identify protein isoforms that contribute to the separation of the different clusters, illustrating the additional advantage of this analytical platform.

A novel secreted metalloprotease (CD2830) from Clostridium difficile cleaves specific proline sequences in LPXTG cell surface proteins.

Bacterial secreted proteins constitute a biologically important subset of proteins involved in key processes related to infection such as adhesion, colonization, and dissemination. Bacterial extracellular proteases, in particular, have attracted considerable attention, as they have been shown to be indispensable for bacterial virulence. Here, we analyzed the extracellular subproteome of Clostridium difficile and identified a hypothetical protein, CD2830, as a novel secreted metalloprotease. Following the identification of a CD2830 cleavage site in human HSP90ß, a series of synthetic peptide substrates was used to identify the favorable CD2830 cleavage motif. This motif was characterized by a high prevalence of proline residues. Intriguingly, CD2830 has a preference for cleaving Pro-Pro bonds, unique among all hitherto described proteases. Strikingly, within the C. difficile proteome two putative adhesion molecules, CD2831 and CD3246, were identified that contain multiple CD2830 cleavage sites (13 in total). We subsequently found that CD2830 efficiently cleaves CD2831 between two prolines at all predicted cleavage sites. Moreover, native CD2830, secreted by live cells, cleaves endogenous CD2831 and CD3246. These findings highlight CD2830 as a highly specific endoproteinase with a preference for proline residues surrounding the scissile bond. Moreover, the efficient cleavage of two putative surface adhesion proteins points to a possible role of CD2830 in the regulation of C. difficile adhesion.

Exploring genetic variation in the tomato (Solanum section Lycopersicon) clade by whole-genome sequencing.

We explored genetic variation by sequencing a selection of 84 tomato accessions and related wild species representative of the Lycopersicon, Arcanum, Eriopersicon and Neolycopersicon groups, which has yielded a huge amount of precious data on sequence diversity in the tomato clade. Three new reference genomes were reconstructed to support our comparative genome analyses. Comparative sequence alignment revealed group-, species- and accession-specific polymorphisms, explaining characteristic fruit traits and growth habits in the various cultivars. Using gene models from the annotated Heinz 1706 reference genome, we observed differences in the ratio between non-synonymous and synonymous SNPs (dN/dS) in fruit diversification and plant growth genes compared to a random set of genes, indicating positive selection and differences in selection pressure between crop accessions and wild species. In wild species, the number of single-nucleotide polymorphisms (SNPs) exceeds 10 million, i.e. 20-fold higher than found in most of the crop accessions, indicating dramatic genetic erosion of crop and heirloom tomatoes. In addition, the highest levels of heterozygosity were found for allogamous self-incompatible wild species, while facultative and autogamous self-compatible species display a lower heterozygosity level. Using whole-genome SNP information for maximum-likelihood analysis, we achieved complete tree resolution, whereas maximum-likelihood trees based on SNPs from ten fruit and growth genes show incomplete resolution for the crop accessions, partly due to the effect of heterozygous SNPs. Finally, results suggest that phylogenetic relationships are correlated with habitat, indicating the occurrence of geographical races within these groups, which is of practical importance for Solanum genome evolution studies.

Clostridium difficile TcdC protein binds four-stranded G-quadruplex structures.

Clostridium difficile infections are increasing worldwide due to emergence of virulent strains. Infections can result in diarrhea and potentially fatal pseudomembranous colitis. The main virulence factors of C. difficile are clostridial toxins TcdA and TcdB. Transcription of the toxins is positively regulated by the sigma factor TcdR. Negative regulation is believed to occur through TcdC, a proposed anti-sigma factor. Here, we describe the biochemical properties of TcdC to understand the mechanism of TcdC action. Bioinformatic analysis of the TcdC protein sequence predicted the presence of a hydrophobic stretch [amino acids (aa) 30-50], a potential dimerization domain (aa 90-130) and a C-terminal oligonucleotide-binding fold. Gel filtration chromatography of two truncated recombinant TcdC proteins (TcdCΔ1-89 and TcdCΔ1-130) showed that the domain between aa 90 and 130 is involved in dimerization. Binding of recombinant TcdC to single-stranded DNA was studied using a single-stranded Systematic Evolution of Ligands by Exponential enrichment approach. This involved specific binding of single-stranded DNA sequences from a pool of random oligonucleotides, as monitored by electrophoretic-mobility shift assay. Analysis of the oligonucleotides bound showed that the oligonucleotide-binding fold domain of TcdC can bind specifically to DNA folded into G-quadruplex structures containing repetitive guanine nucleotides forming a four-stranded structure. In summary, we provide evidence for DNA binding of TcdC, which suggests an alternative function for this proposed anti-sigma factor.

A functional isopenicillin N synthase in an animal genome.

Horizontal transfer of genes is widespread among prokaryotes, but is less common between microorganisms and animals. Here, we present evidence for the presence of a gene encoding functional isopenicillin N synthase, an enzyme in the ß-lactam antibiotics biosynthesis pathway, in the genome of the soil-living collembolan species, Folsomia candida (FcIPNS). At present, this gene is only known from bacteria and fungi, as is the capacity to produce ß-lactam antibiotics. The FcIPNS gene was located on two genomic contigs, was physically linked to a predicted insect ATP-binding cassette transporter gene, and contained three introns each flanked by eukaryotic splicing recognition sites (GT/AG). Homology searches revealed no similarity between these introns and the FcIPNS regions of bacteria or fungi. All amino acids conserved across bacteria and fungi were also conserved in F. candida. Recombinant FcIPNS was able to convert its substrate amino δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine into isopenicillin N, providing strong evidence that FcIPNS is functional. Phylogenetic analysis clustered FcIPNS outside the bacterial IPNS clade, and also outside the fungal IPNS clade, suggesting an ancient gene transfer followed by divergence in the F. candida genome. In conclusion, the data suggest that the soil-living collembolan F. candida has assimilated the capacity for antibacterial activity by horizontal gene transfer, which may be an important adaptive trait in the microbe-dominated soil ecosystem.

Capillary-electrophoresis mass spectrometry for the detection of carbapenemases in (multi-)drug-resistant Gram-negative bacteria.

In a time in which the spread of multidrug resistant microorganisms is ever increasing, there is a need for fast and unequivocal identification of suspect organisms to supplement existing techniques in the clinical laboratory, especially in single bacterial colonies. Mass-spectrometry coupled with efficient peptide separation techniques offer great potential for identification of resistant-related proteins in complex microbiological samples in an unbiased manner. Here, we developed a capillary electrophoresis-electrospray ionization-tandem mass spectrometry CE-ESI-MS/MS bottom-up proteomics workflow for sensitive and specific peptide analysis with the emphasis on the identification of ß-lactamases (carbapenemases OXA-48 and KPC in particular) in bacterial species. For this purpose, tryptic peptides from whole cell lysates were analyzed by sheathless CE-ESI-MS/MS and proteins were identified after searching of the spectral data against bacterial protein databases. The CE-ESI-MS/MS workflow was first evaluated using a recombinant TEM-1 ß-lactamase, resulting in 68% of the amino acid sequence being covered by 20 different unique peptides. Subsequently, a resistant and susceptible Escherichia coli lab strain were analyzed and based on the observed ß-lactamase peptides, the two strains could easily be discriminated. Finally, the method was tested in an unbiased setup using a collection of in-house characterized OXA-48 (n = 17) and KPC (n = 10) clinical isolates. The developed CE-ESI-MS/MS method was able to identify the presence of OXA-48 and KPC in all of the carbapenemase positive samples, independent of species and degree of susceptibility. Four negative controls were tested and classified as negative by this method. Furthermore, a number of extended-spectrum beta-lactamases (ESBL) were identified in the same analyses, confirming the multiresistant character in 19 out of 27 clinical isolates. Importantly, the method performed equally well on protein lysates from single colonies. As such, it demonstrates CE-ESI-MS/MS as a potential next generation mass spectrometry platform within the clinical microbiology laboratory.

The human lactoferrin-derived peptide hLF1-11 exerts immunomodulatory effects by specific inhibition of myeloperoxidase activity.

Because of their ability to eliminate pathogens and to modulate various host immune responses, antimicrobial peptides are considered as candidate agents to fight infections by (antibiotic-resistant) pathogens. We recently reported that hLF1-11 (GRRRRSVQWCA), an antimicrobial peptide derived from the N terminus of human lactoferrin, displays diverse modulatory activities on monocytes, thereby enhancing their actions in innate immune responses. The aim of this study was to identify the cellular target of hLF1-11 that mediates these effects. Results revealed that hLF1-11 binds and subsequently penetrates human monocytes, after which it inhibits the enzymatic activities of myeloperoxidase (MPO). Moreover, a chemical inhibitor of MPO (aminobenzoic acid hydrazide) mimicked the effects of hLF1-11 on the inflammatory response by monocytes and on monocyte-macrophage differentiation. Computer-assisted molecular modeling predicted that hLF1-11 can bind to the edge of and within the crevice of the active site of MPO. Experiments with a set of hLF1-11 peptides with amino acid substitutions identified the stretch of arginines and the cysteine at position 10 as pivotal in these immunomodulatory properties of hLF1-11. We conclude that hLF1-11 may exert its modulatory effects on human monocytes by specific inhibition of MPO activity.

A TdT-driven amplification loop increases CRISPR-Cas12a DNA detection levels.

Recent findings on CRISPR-Cas enzymes with collateral DNAse/RNAse activity have led to new and innovative methods for pathogen detection. However, many CRISPR-Cas assays necessitate DNA pre-amplification to boost sensitivity, restricting their utility for point-of-care applications. Achieving higher sensitivity without DNA pre-amplification presents a significant challenge. In this study, we introduce a Terminal deoxynucleotidyl Transferase (TdT)-based amplification loop, creating a positive feedback mechanism within the CRISPR-Cas12a pathogen detection system. Upon recognizing pathogenic target DNA, Cas12a triggers trans-cleavage of a FRET reporter and a specific enhancer molecule oligonucleotide, indicated by the acronym POISER (Partial Or Incomplete Sites for crRNA recognition). POISER comprises half of a CRISPR-RNA recognition site, which is subsequently elongated by TdT enzymatic activity. This process, involving pathogen recognition-induced Cas12a cleavage and TdT elongation, results in a novel single-stranded DNA target. This target can subsequently be recognized by a POISER-specific crRNA, activating more Cas12a enzymes. Our study demonstrates that these POISER-cycles enhance the signal strength in fluorescent-based CRISPR-Cas12a assays. Although further refinement is desirable, POISER holds promise as a valuable tool for the detection of pathogens in point-of-care testing, surveillance, and environmental monitoring.

EBV protein BNLF2a exploits host tail-anchored protein integration machinery to inhibit TAP.

EBV, the prototypic human γ(1)-herpesvirus, persists for life in infected individuals, despite the presence of vigorous antiviral immunity. CTLs play an important role in the protection against viral infections, which they detect through recognition of virus-encoded peptides presented in the context of HLA class I molecules at the cell surface. The viral peptides are generated in the cytosol and are transported into the endoplasmic reticulum (ER) by TAP. The EBV-encoded lytic-phase protein BNLF2a acts as a powerful inhibitor of TAP. Consequently, loading of antigenic peptides onto HLA class I molecules is hampered, and recognition of BNLF2a-expressing cells by cytotoxic T cells is avoided. In this study, we characterize BNLF2a as a tail-anchored (TA) protein and elucidate its mode of action. Its hydrophilic N-terminal domain is located in the cytosol, whereas its hydrophobic C-terminal domain is inserted into membranes posttranslationally. TAP has no role in membrane insertion of BNLF2a. Instead, Asna1 (also named TRC40), a cellular protein involved in posttranslational membrane insertion of TA proteins, is responsible for integration of BNLF2a into the ER membrane. Asna1 is thereby required for efficient BNLF2a-mediated HLA class I downregulation. To optimally accomplish immune evasion, BNLF2a is composed of two specialized domains: its C-terminal tail anchor ensures membrane integration and ER retention, whereas its cytosolic N terminus accomplishes inhibition of TAP function. These results illustrate how EBV exploits a cellular pathway for TA protein biogenesis to achieve immune evasion, and they highlight the exquisite adaptation of this virus to its host.

Expression of virulence and antimicrobial related proteins in Burkholderia mallei and Burkholderia pseudomallei.

BACKGROUND: Burkholderia mallei and Burkholderia pseudomallei are both potential biological threat agents. Melioidosis caused by B. pseudomallei is endemic in Southeast Asia and Northern Australia, while glanders caused by B. mallei infections are rare. Here we studied the proteomes of different B. mallei and B. pseudomallei isolates to determine species specific characteristics. METHODS: The expressed proteins of 5 B. mallei and 6 B. pseudomallei strains were characterized using liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). Subsequently, expression of potential resistance and virulence related characteristics were analyzed and compared. RESULTS: Proteome analysis can be used for the identification of B. mallei and B. pseudomallei. Both species were identified based on >60 discriminative peptides. Expression of proteins potentially involved in antimicrobial resistance, AmrAB-OprA, BpeAB-OprB, BpeEF-OprC, PenA as well as several other efflux pump related proteins and putative ß-lactamases was demonstrated. Despite, the fact that efflux pump BpeAB-OprB was expressed in all isolates, no clear correlation with an antimicrobial phenotype and the efflux-pump could be established. Also consistent with the phenotypes, no amino acid mutations in PenA known to result in ß-lactam resistance could be identified. In all studied isolates, the expression of virulence (related) factors Capsule-1 and T2SS was demonstrated. The expression of T6SS-1 was demonstrated in all 6 B. pseudomallei isolates and in 2 of the 5 B. mallei isolates. In all, except one B. pseudomallei isolate, poly-beta-1,6 N-acetyl-D-glucosamine export porin (Pga), important for biofilm formation, was detected, which were absent in the proteomes of B. mallei. Siderophores, iron binding proteins, malleobactin and malleilactone are possibly expressed in both species under standard laboratory growth conditions. Expression of multiple proteins from both the malleobactin and malleilactone polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) clusters was demonstrated in both species. All B. pseudomallei expressed at least seven of the nine proteins of the bactobolin synthase cluster (bactobolin, is a ribosome targeting antibiotic), while only in one B. mallei isolate expression of two proteins of this synthase cluster was identified. CONCLUSIONS: Analyzing the expressed proteomes revealed differences between B. mallei and B. pseudomallei but also between isolates from the same species. Proteome analysis can be used not only to identify B. mallei and B. pseudomallei but also to characterize the presence of important factors that putatively contribute to the pathogenesis of B. mallei and B. pseudomallei.

Development and application of a 6.5 million feature Affymetrix Genechip® for massively parallel discovery of single position polymorphisms in lettuce (Lactuca spp.).

BACKGROUND: High-resolution genetic maps are needed in many crops to help characterize the genetic diversity that determines agriculturally important traits. Hybridization to microarrays to detect single feature polymorphisms is a powerful technique for marker discovery and genotyping because of its highly parallel nature. However, microarrays designed for gene expression analysis rarely provide sufficient gene coverage for optimal detection of nucleotide polymorphisms, which limits utility in species with low rates of polymorphism such as lettuce (Lactuca sativa). RESULTS: We developed a 6.5 million feature Affymetrix GeneChip® for efficient polymorphism discovery and genotyping, as well as for analysis of gene expression in lettuce. Probes on the microarray were designed from 26,809 unigenes from cultivated lettuce and an additional 8,819 unigenes from four related species (L. serriola, L. saligna, L. virosa and L. perennis). Where possible, probes were tiled with a 2 bp stagger, alternating on each DNA strand; providing an average of 187 probes covering approximately 600 bp for each of over 35,000 unigenes; resulting in up to 13 fold redundancy in coverage per nucleotide. We developed protocols for hybridization of genomic DNA to the GeneChip® and refined custom algorithms that utilized coverage from multiple, high quality probes to detect single position polymorphisms in 2 bp sliding windows across each unigene. This allowed us to detect greater than 18,000 polymorphisms between the parental lines of our core mapping population, as well as numerous polymorphisms between cultivated lettuce and wild species in the lettuce genepool. Using marker data from our diversity panel comprised of 52 accessions from the five species listed above, we were able to separate accessions by species using both phylogenetic and principal component analyses. Additionally, we estimated the diversity between different types of cultivated lettuce and distinguished morphological types. CONCLUSION: By hybridizing genomic DNA to a custom oligonucleotide array designed for maximum gene coverage, we were able to identify polymorphisms using two approaches for pair-wise comparisons, as well as a highly parallel method that compared all 52 genotypes simultaneously.

Analysis of a Clostridium difficile PCR ribotype 078 100 kilobase island reveals the presence of a novel transposon, Tn6164.

BACKGROUND: Clostridium difficile is the main cause of antibiotic associated diarrhea. In the past decade, the number of C. difficile patients has increased dramatically, coinciding with the emergence of two PCR ribotypes 027 and 078. PCR ribotype 078 is also frequently found during C. difficile outbreaks in pigfarms. Previously, the genome of the PCR ribotype 078 strain M120, a human isolate, was described to contain a unique insert of 100 kilobases. RESULTS: Analysis of this insert revealed over 90 open reading frames, encoding proteins originating from transposons, phages and plasmids. The insert was shown to be a transposon (Tn6164), as evidenced by the presence of an excised and circularised molecule, containing the ligated 5'and 3'ends of the insert. Transfer of the element could not be shown through filter-mating experiments. Whole genome sequencing of PCR ribotype 078 strain 31618, isolated from a diarrheic piglet, showed that Tn6164 was not present in this strain. To test the prevalence of Tn6164, a collection of 231 Clostridium difficile PCR ribotype 078 isolates from human (n = 173) and porcine (n = 58) origin was tested for the presence of this element by PCR. The transposon was present in 9 human, tetracycline resistant isolates, originating from various countries in Europe, and none of the pig strains. Nine other strains, also tetracycline resistant human isolates, contained half of the transposon, suggesting multiple insertion steps yielding the full Tn6164. Other PCR ribotypes (n = 66) were all negative for the presence of the transposon. Multi locus variable tandem repeat analysis revealed genetic relatedness among transposon containing isolates. Although the element contained several potential antibiotic resistance genes, it did not yield a readily distinguishable phenotype. CONCLUSIONS: Tn6164 is a newly described transposon, occurring sporadically in C. difficile PCR ribotype 078 strains. Although no transfer of the element could be shown, we hypothesize that the element could serve as a reservoir of antibiotic resistance genes for other bacteria. Further research is needed to investigate the transfer capabilities of the element and to substantiate the possible role of Tn6164 as a source of antibiotic resistance genes for other gut pathogens.

Combining CRISPR-Cas12a with terminal deoxynucleotidyl transferase dependent reporter elongation for pathogen detection using lateral flow test strips.

CRISPR-Cas (CC)-based detection technologies have some exceptional features, which hold the promise of developing into the next-generation diagnostic platforms. One of these features is the ability to trigger non-specific single-stranded DNA/RNA cleavage activity after specific target recognition and Cas enzyme activation. This cleavage activity can be visualized either by single-stranded DNA/RNA fluorescence resonance energy transfer quenching reporters or via lateral flow strips, which separate and detect the cleaved reporters. In a previous study, we reported coupling CC-cleavage activity with the enzyme terminal deoxynucleotidyl transferase (TdT) that elongates cleaved ssDNA reporter fragments with dTTP nucleotides. These elongated poly(thymine) tails then act as scaffolds for the formation of copper nanoparticles which generate a bright fluorescent signal upon UV excitation. In the current study, we visualize the poly(thymine) tails on lateral flow strips, using different combinations of biotinylated or fluorescein-labeled nucleotides, various reporters, and capture oligos. One particular approach, using a fluorescein reporter, reached a target sensitivity of <1 pM and was named Cas activity assay on a strip and was tested using Bacillus anthracis genomic DNA.

Genetic markers for Clostridium difficile lineages linked to hypervirulence.

Rapid identification of hypervirulent Clostridium difficile strains is essential for preventing their spread. Recent completion of several full-length C. difficile genomes provided an excellent opportunity to identify potentially unique genes that characterize hypervirulent strains. Based on sequence comparisons between C. difficile strains we describe two gene insertions into the genome of hypervirulent PCR ribotypes 078 and 027. Analysis of these regions, of 1.7 and 4.2 kb, respectively, revealed that they contain several interesting ORFs. The 078 region is inserted intergenically and introduces an enzyme that is involved in the biosynthesis of several antibiotics. The 027 insert disrupts the thymidylate synthetase (thyX) gene and replaces it with an equivalent, catalytically more efficient, thyA gene. Both gene insertions were used to develop ribotype-specific PCRs, which were validated by screening a large strain collection consisting of 68 different PCR ribotypes supplemented with diverse 078 and 027 strains derived from different geographical locations and individual outbreaks. The genetic markers were stably present in the hypervirulent PCR ribotypes 078 and 027, but were also found in several other PCR ribotypes. Comparative analysis of amplified fragment length polymorphisms, PCR ribotype banding patterns and toxin profiles showed that all PCR ribotypes sharing the same insert from phylogenetically coherent clusters. The identified loci are unique to these clusters, to which the hypervirulent ribotypes 078 and 027 belong. This provides valuable information on strains belonging to two distinct lineages within C. difficile that are highly related to hypervirulent strains.