ISAnalytics enables longitudinal and high-throughput clonal tracking studies in hematopoietic stem cell gene therapy applications.

Longitudinal clonal tracking studies based on high-throughput sequencing technologies supported safety and long-term efficacy and unraveled hematopoietic reconstitution in many gene therapy applications with unprecedented resolution. However, monitoring patients over a decade-long follow-up entails a constant increase of large data volume with the emergence of critical computational challenges, unfortunately not addressed by currently available tools. Here we present ISAnalytics, a new R package for comprehensive and high-throughput clonal tracking studies using vector integration sites as markers of cellular identity. Once identified the clones externally from ISAnalytics and imported in the package, a wide range of implemented functionalities are available to users for assessing the safety and long-term efficacy of the treatment, here described in a clinical trial use case for Hurler disease, and for supporting hematopoietic stem cell biology in vivo with longitudinal analysis of clones over time, proliferation and differentiation. ISAnalytics is conceived to be metadata-driven, enabling users to focus on biological questions and hypotheses rather than on computational aspects. ISAnalytics can be fully integrated within laboratory workflows and standard procedures. Moreover, ISAnalytics is designed with efficient and scalable data structures, benchmarked with previous methods, and grants reproducibility and full analytical control through interactive web-reports and a module with Shiny interface. The implemented functionalities are flexible for all viral vector-based clonal tracking applications as well as genetic barcoding or cancer immunotherapies.

Intrathymic AAV delivery results in therapeutic site-specific integration at TCR loci in mice.

Adeno-associated virus (AAV) vectors have been successfully exploited in gene therapy applications for the treatment of several genetic disorders. AAV is considered an episomal vector, but it has been shown to integrate within the host cell genome after the generation of double-strand DNA breaks or nicks. Although AAV integration raises some safety concerns, it can also provide therapeutic benefit; the direct intrathymic injection of an AAV harboring a therapeutic transgene results in integration in T-cell progenitors and long-term T-cell immunity. To assess the mechanisms of AAV integration, we retrieved and analyzed hundreds of AAV integration sites from lymph node-derived mature T cells and compared these with liver and brain tissue from treated mice. Notably, we found that although AAV integrations in the liver and brain were distributed across the entire mouse genome, >90% of the integrations in T cells were clustered within the T-cell receptor α, ß, and γ genes. More precisely, the insertion mapped to DNA breaks created by the enzymatic activity of recombination activating genes (RAGs) during variable, diversity, and joining recombination. Our data indicate that RAG activity during T-cell receptor maturation induces a site-specific integration of AAV genomes and opens new therapeutic avenues for achieving long-term AAV-mediated gene transfer in dividing cells.

TNAP limits TGF-ß-dependent cardiac and skeletal muscle fibrosis by inactivating the SMAD2/3 transcription factors.

Fibrosis is associated with almost all forms of chronic cardiac and skeletal muscle diseases. The accumulation of extracellular matrix impairs the contractility of muscle cells contributing to organ failure. Transforming growth factor ß (TGF-ß) plays a pivotal role in fibrosis, activating pro-fibrotic gene programmes via phosphorylation of SMAD2/3 transcription factors. However, the mechanisms that control de-phosphorylation of SMAD2 and SMAD3 (SMAD2/3) have remained poorly characterized. Here, we show that tissue non-specific alkaline phosphatase (TNAP, also known as ALPL) is highly upregulated in hypertrophic hearts and in dystrophic skeletal muscles, and that the abrogation of TGF-ß signalling in TNAP-positive cells reduces vascular and interstitial fibrosis. We show that TNAP colocalizes and interacts with SMAD2. The TNAP inhibitor MLS-0038949 increases SMAD2/3 phosphorylation, while TNAP overexpression reduces SMAD2/3 phosphorylation and the expression of downstream fibrotic genes. Overall our data demonstrate that TNAP negatively regulates TGF-ß signalling and likely represents a mechanism to limit fibrosis.

Transgelin-expressing myofibroblasts orchestrate ventral midline closure through TGFß signalling.

Ventral body wall (VBW) defects are among the most common congenital malformations, yet their embryonic origin and underlying molecular mechanisms remain poorly characterised. Transforming growth factor beta (TGFß) signalling is essential for VBW closure, but the responding cells are not known. Here, we identify in mouse a population of migratory myofibroblasts at the leading edge of the closing VBW that express the actin-binding protein transgelin (TAGLN) and TGFß receptor (TGFßR). These cells respond to a temporally regulated TGFß2 gradient originating from the epithelium of the primary body wall. Targeted elimination of TGFßR2 in TAGLN+ cells impairs midline closure and prevents the correct subsequent patterning of the musculature and skeletal components. Remarkably, deletion of Tgfbr2 in myogenic or chondrogenic progenitor cells does not manifest in midline defects. Our results indicate a pivotal significance of VBW myofibroblasts in orchestrating ventral midline closure by mediating the response to the TGFß gradient. Altogether, our data enable us to distinguish highly regulated epithelial-mesenchymal signalling and successive cellular migration events in VBW closure that explain early morphological changes underlying the development of congenital VBW defects.

Distinct Cellular Mechanisms Underlie Smooth Muscle Turnover in Vascular Development and Repair.

RATIONALE: Vascular smooth muscle turnover has important implications for blood vessel repair and for the development of cardiovascular diseases, yet lack of specific transgenic animal models has prevented it's in vivo analysis. OBJECTIVE: The objective of this study was to characterize the dynamics and mechanisms of vascular smooth muscle turnover from the earliest stages of embryonic development to arterial repair in the adult. METHODS AND RESULTS: We show that CD146 is transiently expressed in vascular smooth muscle development. By using CRISPR-Cas9 genome editing and in vitro smooth muscle differentiation assay, we demonstrate that CD146 regulates the balance between proliferation and differentiation. We developed a triple-transgenic mouse model to map the fate of NG2+CD146+ immature smooth muscle cells. A series of pulse-chase experiments revealed that the origin of aortic vascular smooth muscle cells can be traced back to progenitor cells that reside in the wall of the dorsal aorta of the embryo at E10.5. A distinct population of CD146+ smooth muscle progenitor cells emerges during embryonic development and is maintained postnatally at arterial branch sites. To characterize the contribution of different cell types to arterial repair, we used 2 injury models. In limited wire-induced injury response, existing smooth muscle cells are the primary contributors to neointima formation. In contrast, microanastomosis leads to early smooth muscle death and subsequent colonization of the vascular wall by proliferative adventitial cells that contribute to the repair. CONCLUSIONS: Extensive proliferation of immature smooth muscle cells in the primitive embryonic dorsal aorta establishes the long-lived lineages of smooth muscle cells that make up the wall of the adult aorta. A discrete population of smooth muscle cells forms in the embryo and is postnatally sustained at arterial branch sites. In response to arterial injuries, existing smooth muscle cells give rise to neointima, but on extensive damage, they are replaced by adventitial cells.

Interplay of CodY and ScoC in the Regulation of Major Extracellular Protease Genes of Bacillus subtilis.

UNLABELLED: AprE and NprE are two major extracellular proteases in Bacillus subtilis whose expression is directly regulated by several pleiotropic transcriptional factors, including AbrB, DegU, ScoC, and SinR. In cells growing in a rich, complex medium, the aprE and nprE genes are strongly expressed only during the post-exponential growth phase; mutations in genes encoding the known regulators affect the level of post-exponential-phase gene expression but do not permit high-level expression during the exponential growth phase. Using DNA-binding assays and expression and mutational analyses, we have shown that the genes for both exoproteases are also under strong, direct, negative control by the global transcriptional regulator CodY. However, because CodY also represses scoC, little or no derepression of aprE and nprE was seen in a codY null mutant due to overexpression of scoC. Thus, CodY is also an indirect positive regulator of these genes by limiting the synthesis of a second repressor. In addition, in cells growing under conditions that activate CodY, a scoC null mutation had little effect on aprE or nprE expression; full effects of scoC or codY null mutations could be seen only in the absence of the other regulator. However, even the codY scoC double mutant did not show high levels of aprE and nprE gene expression during exponential growth phase in a rich, complex medium. Only a third mutation, in abrB, allowed such expression. Thus, three repressors can contribute to reducing exoprotease gene expression during growth in the presence of excess nutrients. IMPORTANCE: The major Bacillus subtilis exoproteases, AprE and NprE, are important metabolic enzymes whose genes are subject to complex regulation by multiple transcription factors. We show here that expression of the aprE and nprE genes is also controlled, both directly and indirectly, by CodY, a global transcriptional regulator that responds to the intracellular pools of amino acids. Direct CodY-mediated repression explains a long-standing puzzle, that is, why exoproteases are not produced when cells are growing exponentially in a medium containing abundant quantities of proteins or their degradation products. Indirect regulation of aprE and nprE through CodY-mediated repression of the scoC gene, encoding another pleiotropic repressor, serves to maintain a significant level of repression of exoprotease genes when CodY loses activity.

Interactive regulation by the Bacillus subtilis global regulators CodY and ScoC.

CodY and ScoC are Bacillus subtilis transcriptional regulators that control the expression of dozens of genes and operons. Using scoC-lacZ fusions and DNA-binding experiments, we show here that scoC is directly repressed by CodY. This effect creates multiple forms of cascade regulation. For instance, expression of the dtpT gene, which is directly and negatively controlled by ScoC and encodes a putative oligopeptide permease, was activated indirectly by CodY due to CodY-mediated repression of scoC. The opp operon, which encodes an oligopeptide permease that is essential for sporulation and genetic competence development, proved to be a direct target of repression by both ScoC and CodY but was not significantly affected in codY or scoC single mutants. The combined actions of CodY and ScoC maintain opp repression when either one of the regulators loses activity but limit the level of repression to that provided by one of the regulators acting alone. Under conditions of nitrogen limitation, repression by ScoC of dtpT and opp was partly prevented by TnrA. Thus, the functioning of ScoC is determined by other transcription factors via modulation of its expression or DNA binding.

CodY regulates expression of the Bacillus subtilis extracellular proteases Vpr and Mpr.

UNLABELLED: CodY is a global transcriptional regulator in low-G+C Gram-positive bacteria that is responsive to GTP and branched-chain amino acids. By interacting with its two cofactors, it is able to sense the nutritional and energetic status of the cell and respond by regulating expression of adaptive genetic programs. In Bacillus subtilis, more than 200 genes, including those for peptide transporters, intracellular proteolytic enzymes, and amino acid degradative pathways, are controlled by CodY. In this study, we demonstrated that expression of two extracellular proteases, Vpr and Mpr, is negatively controlled by CodY. By gel mobility shift and DNase I footprinting assays, we showed that CodY binds to the regulatory regions of both genes, in the vicinity of their transcription start points. The mpr gene is also characterized by the presence of a second, higher-affinity CodY-binding site located at the beginning of its coding sequence. Using strains carrying vpr- or mpr-lacZ transcriptional fusions in which CodY-binding sites were mutated, we demonstrated that repression of both protease genes is due to the direct effect by CodY and that the mpr internal site is required for regulation. The vpr promoter is a rare example of a sigma H-dependent promoter that is regulated by CodY. In a codY null mutant, Vpr became one of the more abundant proteins of the B. subtilis exoproteome. IMPORTANCE: CodY is a global transcriptional regulator of metabolism and virulence in low-G+C Gram-positive bacteria. In B. subtilis, more than 200 genes, including those for peptide transporters, intracellular proteolytic enzymes, and amino acid degradative pathways, are controlled by CodY. However, no role for B. subtilis CodY in regulating expression of extracellular proteases has been established to date. In this work, we demonstrate that by binding to the regulatory regions of the corresponding genes, B. subtilis CodY negatively controls expression of Vpr and Mpr, two extracellular proteases. Thus, in B. subtilis, CodY can now be seen to regulate the entire protein utilization pathway.

Assessment of immobilized PGA orientation via the LC-MS analysis of tryptic digests of the wild type and its 3K-PGA mutant assists in the rational design of a high-performance biocatalyst.

The mutant penicillin G acylase (PGA) 3K-PGA contains three additional Lys residues on the surface opposite the active site. This protein was designed to selectively drive its immobilization on aldehyde supports. We describe here a modified bottom-up proteomic method to assess the orientation of the immobilized wild-type and mutant proteins to verify our hypothesis of a driven immobilization induced by the mutations introduced. Tryptic digestion of the immobilized enzymes followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of released peptides was performed. This protocol generated peptides from the most accessible surface areas of the immobilized protein, thus not directly bound to the solid support, providing direct evidence of the areas involved in the linkage to the solid matrix. The results obtained suggest that 72 % of the wild-type PGA is immobilized on aldehyde agarose mainly through the Lys residues on the same side of the active site, whereas 3K-PGA reacted with the same support preferentially through the additional Lys residues introduced by mutation on the opposite side. This demonstrates that the active site of the 3K-PGA faces mostly (63 %) toward the reaction medium, resulting in significantly improved accessibility to the substrates. This finding is supported by the catalytic properties of the immobilized biocatalysts. The two immobilized preparations were tested in the synthesis of mandelyl-7-aminocephalosporanic acid (mandelyl-7-ACA) by N-acylation of the ß-lactam nucleus (7-aminocephalosporanic acid) with mandelic acid methyl ester: upon immobilization, the synthetic properties of wild-type PGA strongly decreased, whereas those of 3K-PGA were unaffected. Furthermore, the activity of 3K-PGA was not influenced by the physicochemical nature of the support used for immobilization (glyoxyl agarose or aldehyde Sepabeads) unlike that of wild-type PGA, whose active site is close to the matrix. The results obtained from the analytical characterization correlate well with those obtained by investigation of the synthetic properties of the immobilized enzymes both in the synthesis of mandelyl-7-ACA and in the preparative synthesis of cefazolin. This work highlights the effect exerted by site-directed mutagenesis on the orientation of PGA upon immobilization on solid matrices and suggests how protein engineering tools can be exploited in a synergistic fashion to rationally develop efficient biocatalysts.

DOF-binding sites additively contribute to guard cell-specificity of AtMYB60 promoter.

BACKGROUND: We previously demonstrated that the Arabidopsis thaliana AtMYB60 protein is an R2R3MYB transcription factor required for stomatal opening. AtMYB60 is specifically expressed in guard cells and down-regulated at the transcriptional levels by the phytohormone ABA. RESULTS: To investigate the molecular mechanisms governing AtMYB60 expression, its promoter was dissected through deletion and mutagenesis analyses. By studying different versions of AtMYB60 promoter::GUS reporter fusions in transgenic plants we were able to demonstrate a modular organization for the AtMYB60 promoter. Particularly we defined: a minimal promoter sufficient to confer guard cell-specific activity to the reporter gene; the distinct roles of different DOF-binding sites organised in a cluster in the minimal promoter in determining guard cell-specific expression; the promoter regions responsible for the enhancement of activity in guard cells; a promoter region responsible for the negative transcriptional regulation by ABA. Moreover from the analysis of single and multiple mutants we could rule out the involvement of a group of DOF proteins, known as CDFs, already characterised for their involvement in flowering time, in the regulation of AtMYB60 expression. CONCLUSIONS: These findings shed light on the regulation of gene expression in guard cells and provide new promoter modules as useful tools for manipulating gene expression in guard cells, both for physiological studies and future biotechnological applications.

Identification of a Novel Brevibacillus laterosporus Strain With Insecticidal Activity Against Aedes albopictus Larvae.

Bacterial species able to produce proteins that are toxic against insects have been discovered at the beginning of the last century. However, up to date only two of them have been used as pesticides in mosquito control strategies targeting larval breeding sites: Bacillus thuringensis var. israelensis and Lysinibacillus sphaericus. Aiming to expand the arsenal of biopesticides, bacterial cultures from 44 soil samples were assayed for their ability to kill larvae of Aedes albopictus. A method to select, grow and test the larvicidal capability of spore-forming bacteria from each soil sample was developed. This allowed identifying 13 soil samples containing strains capable of killing Ae. albopictus larvae. Among the active isolates, one strain with high toxicity was identified as Brevibacillus laterosporus by 16S rRNA gene sequencing and by morphological characterization using transmission electron microscopy. The new isolate showed a larvicidal activity significantly higher than the B. laterosporus LMG 15441 reference strain. Its genome was phylogenomically characterized and compared to the available Brevibacillus genomes. Thus, the new isolate can be considered as a candidate adjuvant to biopesticides formulations that would help preventing the insurgence of resistance.

MUTYH mutations associated with familial adenomatous polyposis: functional characterization by a mammalian cell-based assay.

MUTYH-associated polyposis (MAP) is a colorectal cancer syndrome, due to biallelic mutations of MUTYH. This Base Excision Repair gene encodes for a DNA glycosylase that specifically mitigates the high mutagenic potential of the 8-hydroxyguanine (8-oxodG) along the DNA. Aim of this study was to characterize the biological effects, in a mammalian cell background, of human MUTYH mutations identified in MAP patients (137insIW [c.411_416dupATGGAT; p.137insIleTrp]; R171W [c.511C>T; p.Arg171Trp]; E466del [c.1395_1397delGGA; p.Glu466del]; Y165C [c.494A>G; p.Tyr165Cys]; and G382D [c.1145G>A; p.Gly382Asp]). We set up a novel assay in which the human proteins were expressed in Mutyh(-/-) mouse defective cells. Several parameters, including accumulation of 8-oxodG in the genome and hypersensitivity to oxidative stress, were then used to evaluate the consequences of MUTYH expression. Human proteins were also obtained from Escherichia coli and their glycosylase activity was tested in vitro. The cell-based analysis demonstrated that all MUTYH variants we investigated were dysfunctional in Base Excision Repair. In vitro data complemented the in vivo observations, with the exception of the G382D mutant, which showed a glycosylase activity very similar to the wild-type protein. Our cell-based assay can provide useful information on the significance of MUTYH variants, improving molecular diagnosis and genetic counseling in families with mutations of uncertain pathogenicity.

Bacillus subtilis as a host for mosquitocidal toxins production.

Aedes albopictus transmits several arboviral infections. In the absence of vaccines, control of mosquito populations is the only strategy to prevent vector-borne diseases. As part of the search for novel, biological and environmentally friendly strategies for vector control, the isolation of new bacterial species with mosquitocidal activity represents a promising approach. However, new bacterial isolates may be difficult to grow and genetically manipulate. To overcome these limits, here we set up a system allowing the expression of mosquitocidal bacterial toxins in the well-known genetic background of Bacillus subtilis. As a proof of this concept, the ability of B. subtilis to express individual or combinations of toxins of Bacillus thuringiensis israelensis (Bti) was studied. Different expression systems in which toxin gene expression was driven by IPTG-inducible, auto-inducible or toxin gene-specific promoters were developed. The larvicidal activity of the resulting B. subtilis strains against second-instar Ae. albopictus larvae allowed studying the activity of individual toxins or the synergistic interaction among Cry and Cyt toxins. The expression systems here presented lay the foundation for a better improved system to be used in the future to characterize the larvicidal activity of toxin genes from new environmental isolates.

Gut Microbiota Analysis in Postoperative Lynch Syndrome Patients.

Lynch syndrome (LS) is a dominantly inherited condition with incomplete penetrance, characterized by high predisposition to colorectal cancer (CRC), endometrial and ovarian cancers, as well as to other tumors. LS is associated with constitutive DNA mismatch repair (MMR) gene defects, and carriers of the same pathogenic variants can show great phenotypic heterogeneity in terms of cancer spectrum. In the last years, human gut microbiota got a foothold among risk factors responsible for the onset and evolution of sporadic CRC, but its possible involvement in the modulation of LS patients' phenotype still needs to be investigated. In this pilot study, we performed 16S rRNA gene sequencing of bacterial DNA extracted from fecal samples of 10 postoperative LS female patients who had developed colonic lesions (L-CRC) or gynecological cancers (L-GC). Our preliminary data show no differences between microbial communities of L-CRC and L-GC patients, but they plant the seed of the possible existence of a fecal microbiota pattern associated with LS genetic background, with Faecalibacterium prausnitzii, Parabacteroides distasonis, Ruminococcus bromii, Bacteroides plebeius, Bacteroides fragilis and Bacteroides uniformis species being the most significantly over-represented in LS patients (comprising both L-CRC and L-GC groups) compared to healthy subjects.

Abrogation of TGF-beta signalling in TAGLN expressing cells recapitulates Pentalogy of Cantrell in the mouse.

Pentalogy of Cantrell (PC) is a rare multi-organ congenital anomaly that impedes ventral body wall closure and results in diaphragmatic hernia, intra- and pericardial defects. The underlying cellular and molecular changes that lead to these severe developmental defects have remained unknown largely due to the lack of representative animal models. Here we provide in depth characterization of a mouse model with conditional ablation of TGFßRII in Transgelin (Tagln) expressing cells. We show that Tagln is transiently expressed in a variety of cells that participate in the embryonic development and patterning of ventral structures. Genetic ablation of TGFßRII in these cells leads to ventral midline closure defect, diaphragmatic hernia, dilated cardiac outflow tract and aberrant cardiac septation, providing a reliable model to study the morphological changes leading to PC. We show that myogenisis in the diaphragm is independent of TGFß and the diaphragmatic hernia arises from fibroblast-specific migration defect. In the dorsal body wall Tagln expression is initiated after the closure process, revealing a remarkable difference between ventral and dorsal body walls development. Our study demonstrates the use of micro-CT scanning to obtain a 3-dimensional high-resolution overview of embryonic anomalies and provides the first mechanistic insight into the development of PC.

Shifts of Faecal Microbiota During Sporadic Colorectal Carcinogenesis.

Gut microbiota has been implicated in the etiopathogenesis of colorectal cancer. The development of colorectal cancer is a multistep process by which healthy epithelium slowly develops into preneoplastic lesions, which in turn progress into malignant carcinomas over time. In particular, sporadic colorectal cancers can arise from adenomas (about 85% of cases) or serrated polyps through the "adenoma-carcinoma" or the "serrated polyp-carcinoma" sequences, respectively. In this study, we performed 16 S rRNA gene sequencing of bacterial DNA extracted from faecal samples to compare the microbiota of healthy subjects and patients with different preneoplastic and neoplastic lesions. We identified putative microbial biomarkers associated with stage-specific progression of colorectal cancer. In particular, bacteria belonging to the Firmicutes and Actinobacteria phyla, as well as members of the Lachnospiraceae family, proved to be specific of the faecal microbiota of patients with preneoplastic lesions, including adenomas and hyperplastic polyps. On the other hand, two families of the Proteobacteria phylum, Alcaligeneaceae and Enterobacteriaceae, with Sutterella and Escherichia/Shigella being the most representative genera, appeared to be associated with malignancy. These findings, once confirmed on larger cohorts of patients, can represent an important step towards the development of more effective diagnostic strategies.

New active site oriented glyoxyl-agarose derivatives of Escherichia coli penicillin G acylase.

BACKGROUND: Immobilized Penicillin G Acylase (PGA) derivatives are biocatalysts that are industrially used for the hydrolysis of Penicillin G by fermentation and for the kinetically controlled synthesis of semi-synthetic beta-lactam antibiotics. One of the most used supports for immobilization is glyoxyl-activated agarose, which binds the protein by reacting through its superficial Lys residues. Since in E. coli PGA Lys are also present near the active site, an immobilization that occurs through these residues may negatively affect the performance of the biocatalyst due to the difficult diffusion of the substrate into the active site. A preferential orientation of the enzyme with the active site far from the support surface would be desirable to avoid this problem. RESULTS: Here we report how it is possible to induce a preferential orientation of the protein during the binding process on aldehyde activated supports. A superficial region of PGA, which is located on the opposite side of the active site, is enriched in its Lys content. The binding of the enzyme onto the support is consequently forced through the Lys rich region, thus leaving the active site fully accessible to the substrate. Different mutants with an increasing number of Lys have been designed and, when active, immobilized onto glyoxyl agarose. The synthetic performances of these new catalysts were compared with those of the immobilized wild-type (wt) PGA. Our results show that, while the synthetic performance of the wt PGA sensitively decreases after immobilization, the Lys enriched mutants have similar performances to the free enzyme even after immobilization. We also report the observations made with other mutants which were unable to undergo a successful maturation process for the production of active enzymes or which resulted toxic for the host cell. CONCLUSION: The desired orientation of immobilized PGA with the active site freely accessible can be obtained by increasing the density of Lys residues on a predetermined region of the enzyme. The newly designed biocatalysts display improved synthetic performances and are able to maintain a similar activity to the free enzymes. Finally, we found that the activity of the immobilized enzyme proportionally improves with the number of introduced Lys.

Functional analysis of MUTYH mutated proteins associated with familial adenomatous polyposis.

The MUTYH DNA glycosylase specifically removes adenine misincorporated by replicative polymerases opposite the oxidized purine 8-oxo-7,8-dihydroguanine (8-oxoG). A defective protein activity results in the accumulation of G>T transversions because of unrepaired 8-oxoG:A mismatches. In humans, MUTYH germline mutations are associated with a recessive form of familial adenomatous polyposis and colorectal cancer predisposition (MUTYH-associated polyposis, MAP). Here we studied the repair capacity of the MUTYH variants R171W, E466del, 137insIW, Y165C and G382D, identified in MAP patients. Following expression and purification of human proteins from a bacterial system, we investigated MUTYH incision capacity on an 8-oxoG:A substrate by standard glycosylase assays. For the first time, we employed the surface plasmon resonance (SPR) technology for real-time recording of the association/dissociation of wild-type and MUTYH variants from an 8-oxoG:A DNA substrate. When compared to the wild-type protein, R171W, E466del and Y165C variants showed a severe reduction in the binding affinity towards the substrate, while 137insIW and G382D mutants manifested only a slight decrease mainly due to a slower rate of association. This reduced binding was always associated with impairment of glycosylase activity, with adenine removal being totally abrogated in R171W, E466del and Y165C and only partially reduced in 137insIW and G382D. Our findings demonstrate that SPR analysis is suitable to identify defective enzymatic behaviour even when mutant proteins display minor alterations in substrate recognition.

Characterization of L-aspartate oxidase and quinolinate synthase from Bacillus subtilis.

NAD is an important cofactor and essential molecule in all living organisms. In many eubacteria, including several pathogens, the first two steps in the de novo synthesis of NAD are catalyzed by l-aspartate oxidase (NadB) and quinolinate synthase (NadA). Despite the important role played by these two enzymes in NAD metabolism, many of their biochemical and structural properties are still largely unknown. In the present study, we cloned, overexpressed and characterized NadA and NadB from Bacillus subtilis, one of the best studied bacteria and a model organism for low-GC Gram-positive bacteria. Our data demonstrated that NadA from B. subtilis possesses a [4Fe-4S]2+ cluster, and we also identified the cysteine residues involved in the cluster binding. The [4Fe-4S]2+ cluster is coordinated by three cysteine residues (Cys110, Cys230, and Cys320) that are conserved in all the NadA sequences reported so far, suggesting a new noncanonical binding motif that, on the basis of sequence alignment studies, may be common to other quinolinate synthases from different organisms. Moreover, for the first time, it was shown that the interaction between NadA and NadB is not species-specific between B. subtilis and Escherichia coli.

The Bacillus subtilis nrdEF genes, encoding a class Ib ribonucleotide reductase, are essential for aerobic and anaerobic growth.

Ribonucleotide reductases (RNRs) are essential for the biosynthesis of the deoxyribonucleoside triphosphates of DNA. Recently, it was proposed that externally supplied deoxyribonucleosides or DNA is required for the growth of Bacillus subtilis under strict anaerobic conditions (M. J. Folmsbee, M. J. McInerney, and D. P. Nagle, Appl. Environ. Microbiol. 70:5252-5257, 2004). Cultivation of B. subtilis on minimal medium in the presence of oxygen indicators in combination with oxygen electrode measurements and viable cell counting demonstrated that growth occurred under strict anaerobic conditions in the absence of externally supplied deoxyribonucleosides. The nrdEF genes encode the only obvious RNR in B. subtilis. A temperature-sensitive nrdE mutant failed to grow under aerobic and anaerobic conditions, indicating that this oxygen-dependent class I RNR has an essential role under both growth conditions. Aerobic growth and anaerobic growth of the nrdE mutant were rescued by addition of deoxynucleotides. The nrd locus consists of an nrdI-nrdE-nrdF-ymaB operon. The 5' end of the corresponding mRNA revealed transcriptional start sites 45 and 48 bp upstream of the translational start of nrdI. Anaerobic transcription of the operon was found to be dependent on the presence of intact genes for the ResDE two-component redox regulatory system. Two potential ResD binding sites were identified approximately 62 bp (site A) and 50 bp (site B) upstream of the transcriptional start sites by a bioinformatic approach. Only mutation of site B eliminated nrd expression. Aerobic transcription was ResDE independent but required additional promoter elements localized between 88 and 275 bp upstream of the transcriptional start.