Loss of Y in regulatory T lymphocytes in the tumor micro-environment of primary colorectal cancers and liver metastases.

Male sex is a risk factor for colorectal cancer (CRC) with higher illness burden and earlier onset. Thus, we hypothesized that loss of chromosome Y (LOY) in the tumor micro-environment (TME) might be involved in oncogenesis. Previous studies show that LOY in circulating leukocytes of aging men was associated with shorter survival and non-hematological cancer, as well as higher LOY in CD4 + T-lymphocytes in men with prostate cancer vs. controls. However, nothing is known about LOY in leukocytes infiltrating TME and we address this aspect here. We studied frequency and functional effects of LOY in blood, TME and non-tumorous tissue. Regulatory T-lymphocytes (Tregs) in TME had the highest frequency of LOY (22%) in comparison to CD4 + T-lymphocytes and cytotoxic CD8 + T-lymphocytes. LOY score using scRNA-seq was also linked to higher expression of PDCD1, TIGIT and IKZF2 in Tregs. PDCD1 and TIGIT encode immune checkpoint receptors involved in the regulation of Tregs function. Our study sets the direction for further functional research regarding a probable role of LOY in intensifying features related to the suppressive phenotype of Tregs in TME and consequently a possible influence on immunotherapy response in CRC patients.

Genomic and Metabolic Characterization of Plant Growth-Promoting Rhizobacteria Isolated from Nodules of Clovers Grown in Non-Farmed Soil.

The rhizosphere microbiota, which includes plant growth-promoting rhizobacteria (PGPR), is essential for nutrient acquisition, protection against pathogens, and abiotic stress tolerance in plants. However, agricultural practices affect the composition and functions of microbiota, reducing their beneficial effects on plant growth and health. Among PGPR, rhizobia form mutually beneficial symbiosis with legumes. In this study, we characterized 16 clover nodule isolates from non-farmed soil to explore their plant growth-promoting (PGP) potential, hypothesizing that these bacteria may possess unique, unaltered PGP traits, compared to those affected by common agricultural practices. Biolog profiling revealed their versatile metabolic capabilities, enabling them to utilize a wide range of carbon and energy sources. All isolates were effective phosphate solubilizers, and individual strains exhibited 1-aminocyclopropane-1-carboxylate deaminase and metal ion chelation activities. Metabolically active strains showed improved performance in symbiotic interactions with plants. Comparative genomics revealed that the genomes of five nodule isolates contained a significantly enriched fraction of unique genes associated with quorum sensing and aromatic compound degradation. As the potential of PGPR in agriculture grows, we emphasize the importance of the molecular and metabolic characterization of PGP traits as a fundamental step towards their subsequent application in the field as an alternative to chemical fertilizers and supplements.

Seasonality and intensity of airborne Boletus-type spores in relation to land use and weather pattern.

Forests are a natural source of airborne bolete spores. The timing of sporulation and its intensity as well as the dispersal of airborne spores and in consequence their concentrations depend in particular on the type of land use determining the availability of matter on which they develop and on meteorological factors. The aim of this study was to perform a spatial and temporal analysis of the occurrence of Boletus-type spores in the warm temperate climate of the Northern Hemisphere. An assumption was made that the spore concentrations depend on the type of land cover and weather conditions. The volumetric method was applied to investigate differences in spore concentrations and using spore traps installed at different heights and at locations with different land cover types. Boletus-type spores occurred in the air at high concentrations in late summer and in the autumn. The season start dates and maximum concentrations did not differ significantly between sites and seasons, but the season intensity varied. Higher spore concentrations were usually found in the region with a larger proportion of green areas, including forests. An analysis of the diurnal cycles showed that within 24 h spore concentration reached high levels twice, which was especially noticeable in ground level monitoring. Air temperature and air humidity were the main weather factors affecting the occurrence of airborne spores. This research indicates that when studying the effects of different factors on the concentration of airborne basidiospores, many environmental elements should be analyzed, including the characteristics of habitats in which basidiomycetes grow. Climate, weather, geobotany, and land use type should be taken into account in analysis and interpretation of aeromycological phenomena.

Exopolysaccharide Biosynthesis in Rhizobium leguminosarum bv. trifolii Requires a Complementary Function of Two Homologous Glycosyltransferases PssG and PssI.

The Pss-I region of Rhizobium leguminosarum bv. trifolii TA1 comprises more than 20 genes coding for glycosyltransferases, modifying enzymes, and polymerization/export proteins, altogether determining the biosynthesis of symbiotically relevant exopolysaccharides. In this study, the role of homologous PssG and PssI glycosyltransferases in exopolysaccharide subunit synthesis were analyzed. It was shown that the glycosyltransferase-encoding genes of the Pss-I region were part of a single large transcriptional unit with potential downstream promoters activated in specific conditions. The ΔpssG and ΔpssI mutants produced significantly lower amounts of the exopolysaccharide, while the double deletion mutant ΔpssIΔpssG produced no exopolysaccharide. Complementation of double mutation with individual genes restored exopolysaccharide synthesis, but only to the level similar to that observed for the single ΔpssI or ΔpssG mutants, indicating that PssG and PssI serve complementary functions in the process. PssG and PssI interacted with each other in vivo and in vitro. Moreover, PssI displayed an expanded in vivo interaction network comprising other GTs involved in subunit assembly and polymerization/export proteins. PssG and PssI proteins were shown to interact with the inner membrane through amphipathic helices at their C-termini, and PssG also required other proteins involved in exopolysaccharide synthesis to localize in the membrane protein fraction.

A New Face of the Old Gene: Deletion of the PssA, Encoding Monotopic Inner Membrane Phosphoglycosyl Transferase in Rhizobium leguminosarum, Leads to Diverse Phenotypes That Could Be Attributable to Downstream Effects of the Lack of Exopolysaccharide.

The biosynthesis of subunits of rhizobial exopolysaccharides is dependent on glycosyltransferases, which are usually encoded by large gene clusters. PssA is a member of a large family of phosphoglycosyl transferases catalyzing the transfer of a phosphosugar moiety to polyprenol phosphate; thus, it can be considered as priming glycosyltransferase commencing synthesis of the EPS repeating units in Rhizobium leguminosarum. The comprehensive analysis of PssA protein features performed in this work confirmed its specificity for UDP-glucose and provided evidence that PssA is a monotopic inner membrane protein with a reentrant membrane helix rather than a transmembrane segment. The bacterial two-hybrid system screening revealed interactions of PssA with some GTs involved in the EPS octasaccharide synthesis. The distribution of differentially expressed genes in the transcriptome of the ΔpssA mutant into various functional categories indicated complexity of cell response to the deletion, which can mostly be attributed to the lack of exopolysaccharide and downstream effects caused by such deficiency. The block in the EPS biosynthesis at the pssA step, potentially leading to an increased pool of UDP-glucose, is likely to be filtered through to other pathways, and thus the absence of EPS may indirectly affect the expression of proteins involved in these pathways.

Personalized health risk assessment based on single-cell RNA sequencing analysis of a male with 45, X/48, XYYY karyotype.

Numeric sex chromosome abnormalities are commonly associated with an increased cancer risk. Here, we report a 14-year-old boy with a rare mosaic 45, X/48, XYYY karyotype presenting with subtle dysmorphic features and relative height deficiency, requiring growth hormone therapy. As only 12 postnatal cases have been described so far with very limited follow-up data, to assess the proband's long-term prognosis, including cancer risk, we performed high-throughput single-cell RNA sequencing (scRNA-seq) analysis. Although comprehensive cytogenetic analysis showed seemingly near perfect balance between 45, X and 48, XYYY cell populations, scRNA-seq revealed widespread differences in genotype distribution among immune cell fractions, specifically in monocytes, B- and T-cells. These results were confirmed at DNA level by digital-droplet PCR on flow-sorted immune cell types. Furthermore, deregulation of predominantly autosomal genes was observed, including TCL1A overexpression in 45, X B-lymphocytes and other known genes associated with hematological malignancies. Together with the standard hematological results, showing increased fractions of monocytes and CD4+/CD8+T lymphocytes ratio, long-term personalized hemato-oncological surveillance was recommended in the reported patient.

Loss of Y in leukocytes as a risk factor for critical COVID-19 in men.

BACKGROUND: The COVID-19 pandemic, which has a prominent social and economic impact worldwide, shows a largely unexplained male bias for the severity and mortality of the disease. Loss of chromosome Y (LOY) is a risk factor candidate in COVID-19 due to its prior association with many chronic age-related diseases, and its impact on immune gene transcription. METHODS: Publicly available scRNA-seq data of PBMC samples derived from male patients critically ill with COVID-19 were reanalyzed, and LOY status was added to the annotated cells. We further studied LOY in whole blood for 211 COVID-19 patients treated at intensive care units (ICU) from the first and second waves of the pandemic. Of these, 139 patients were subject to cell sorting for LOY analysis in granulocytes, low-density neutrophils (LDNs), monocytes, and PBMCs. RESULTS: Reanalysis of available scRNA-seq data revealed LDNs and monocytes as the cell types most affected by LOY. Subsequently, DNA analysis indicated that 46%, 32%, and 29% of critically ill patients showed LOY above 5% cut-off in LDNs, granulocytes, and monocytes, respectively. Hence, the myeloid lineage that is crucial for the development of severe COVID-19 phenotype is affected by LOY. Moreover, LOY correlated with increasing WHO score (median difference 1.59%, 95% HDI 0.46% to 2.71%, p=0.025), death during ICU treatment (median difference 1.46%, 95% HDI 0.47% to 2.43%, p=0.0036), and history of vessel disease (median difference 2.16%, 95% HDI 0.74% to 3.7%, p=0.004), among other variables. In 16 recovered patients, sampled during ICU stay and 93-143 days later, LOY decreased significantly in whole blood and PBMCs. Furthermore, the number of LDNs at the recovery stage decreased dramatically (median difference 76.4 per 10,000 cell sorting events, 95% HDI 55.5 to 104, p=6e-11). CONCLUSIONS: We present a link between LOY and an acute, life-threatening infectious disease. Furthermore, this study highlights LOY as the most prominent clonal mutation affecting the myeloid cell lineage during emergency myelopoiesis. The correlation between LOY level and COVID-19 severity might suggest that this mutation affects the functions of monocytes and neutrophils, which could have consequences for male innate immunity.

Comprehensive cancer-oriented biobanking resource of human samples for studies of post-zygotic genetic variation involved in cancer predisposition.

The progress in translational cancer research relies on access to well-characterized samples from a representative number of patients and controls. The rationale behind our biobanking are explorations of post-zygotic pathogenic gene variants, especially in non-tumoral tissue, which might predispose to cancers. The targeted diagnoses are carcinomas of the breast (via mastectomy or breast conserving surgery), colon and rectum, prostate, and urinary bladder (via cystectomy or transurethral resection), exocrine pancreatic carcinoma as well as metastases of colorectal cancer to the liver. The choice was based on the high incidence of these cancers and/or frequent fatal outcome. We also collect age-matched normal controls. Our still ongoing collection originates from five clinical centers and after nearly 2-year cooperation reached 1711 patients and controls, yielding a total of 23226 independent samples, with an average of 74 donors and 1010 samples collected per month. The predominant diagnosis is breast carcinoma, with 933 donors, followed by colorectal carcinoma (383 donors), prostate carcinoma (221 donors), bladder carcinoma (81 donors), exocrine pancreatic carcinoma (15 donors) and metachronous colorectal cancer metastases to liver (14 donors). Forty percent of the total sample count originates from macroscopically healthy cancer-neighboring tissue, while contribution from tumors is 12%, which adds to the uniqueness of our collection for cancer predisposition studies. Moreover, we developed two program packages, enabling registration of patients, clinical data and samples at the participating hospitals as well as the central system of sample/data management at coordinating center. The approach used by us may serve as a model for dispersed biobanking from multiple satellite hospitals. Our biobanking resource ought to stimulate research into genetic mechanisms underlying the development of common cancers. It will allow all available "-omics" approaches on DNA-, RNA-, protein- and tissue levels to be applied. The collected samples can be made available to other research groups.

Characteristics of the Surface Topography and Tribological Properties of Reinforced Aluminum Matrix Composite.

Due to their excellent synergistic properties, Aluminum Matrix Composites (AMC) have achieved a high degree of prominence in different industries. In addition to strength, the wear resistance of materials is also an important criterion for numerous applications. The wear resistance depends on the surface topography as well as the working conditions of the interacting parts. Therefore, extensive experiments are being conducted to improve the suitability of engineering materials (including AMC) for different applications. This paper presents research on manufactured aluminum metal matrix composites reinforced with 10 wt.% of Al2SiO5 (aluminum sillimanite). The manufactured and prepared samples were subjected to surface topography measurements and to tribological studies both with and without lubricant using a block-on-ring tester. Based on the results, analyses of the surface topography (i.e., surface roughness parameters, Abbott-Firestone curve, and surface defects) as well as of the tribological characteristics (i.a. friction coefficient, linear wear, and wear intensity) were performed. Differences in the surface topography of the manufactured elements were shown. The surface topography had a significant impact on tribological characteristics of the sliding joints in the tests where lubrication was and was not used. Better tribological characteristics were obtained for the surfaces characterized by greater roughness (determined on the basis of both the profile and surface texture parameters). In the case of tribological tests with lubrication, the friction coefficient as well as the wear intensity was significantly lower compared to tribological tests without lubrication. However, lower values of the friction coefficient and wear intensity were still recorded for the surfaces that were characterized by greater roughness. The obtained results showed that it is important to analyze the surface topography because surface characteristics influence tribological properties.

Bioaerosols on the atmospheric super highway: An example of long distance transport of Alternaria spores from the Pannonian Plain to Poland.

Alternaria spores are pathogenic to agricultural crops, and the longest and the most severe sporulation seasons are predominantly recorded in rural areas, e.g. the Pannonian Plain (PP) in South-Central Europe. In Poland (Central Europe), airborne Alternaria spore concentrations peak between July and August. In this study, we test the hypothesis that the PP is the source of Alternaria spores recorded in Poland after the main sporulation season (September-October). Airborne Alternaria spores (2005-2019) were collected using volumetric Hirst spore traps located in 38 locations along the potential pathways of air masses, i.e. from Serbia, Romania and Hungary, through the Czech Republic, Slovakia and Ukraine, to Northern Poland. Three potential episodes of Long Distance Transport (LDT) were selected and characterized in detail, including the analysis of Alternaria spore data, back trajectory analysis, dispersal modelling, and description of local weather and mesoscale synoptic conditions. During selected episodes, increases in Alternaria spore concentrations in Poznan were recorded at unusual times that deviated from the typical diurnal pattern, i.e. at night or during morning hours. Alternaria spore concentrations on the PP were very high (>1000 spores/m3) at that time. The presence of non-local Ambrosia pollen, common to the PP, were also observed in the air. Air mass trajectory analysis and dispersal modelling showed that the northwest part of the PP, north of the Transdanubian Mountains, was the potential source area of Alternaria spores. Our results show that Alternaria spores are transported over long distances from the PP to Poland. These spores may markedly increase local exposure to Alternaria spores in the receptor area and pose a risk to both human and plant health. Alternaria spores followed the same atmospheric route as previously described LDT ragweed pollen, revealing the existence of an atmospheric super highway that transports bioaerosols from the south to the north of Europe.

Dry Sliding Wear Studies on Sillimanite and B4C Reinforced Aluminium Hybrid Composites Fabricated by Vacuum Assisted Stir Casting Process.

This paper presents the results of studies to understand the influence of hybridisation on mechanical and tribological behaviour as well as dry sliding wear of aluminium metal matrix composites. Sillimanite and boron carbide (B4C) were used as primary and secondary reinforcements and pure aluminium was used as the matrix material. The composite was fabricated by using a vacuum assisted stir casting process. Different research instruments were used, including a scanning electron microscope with EDX spectrometer, a surface measurement device, a thermal image analyser, as well as a tribotester. The results show that tensile, impact strength and hardness of the hybridised composites are superior (a step ahead) than unreinforced and primary composites. The wear behaviour of the fabricated specimens was tested for the dry sliding wear behaviour under the load range of 10-50 N with the steps of 20 N for the sliding velocities 0.75, 1.5 and 2.25 m/s over a distance of 1000 m. The wear rate increased with load and decreased as the wt.% of reinforcement increased. The wear rate of the composite with 10 wt.% Al2SiO5 was approximately 44% lower than that of the composite with 5 wt.% Al2SiO5. The same dependence was noted for hybrid composite (5 wt.% Al2SiO5 + 5 wt.% B4C)-the wear rate was approximately 50.8% lower than that of the composite with 5 wt.% Al2SiO5 under the same test condition. The friction coefficient decreased as the weight percentage of the reinforcement (Al2SiO5 and B4C) increased due to the uniform distribution of the reinforcement on the surface of the composites. The main wear mechanism of the studied materials was abrasion wear. The wear mechanism of the composite had tribochemical type. It involved the oxidation and transfer of the material, which formed protective tribolayers ensuring an additional sliding process. The mechanism that played the main role in the wear process of the composites was a combination of abrasive, adhesive and oxidative wear.

Influence of Epilepsy on the Quality of Life of Patients with Brain Tumors.

Epilepsy is a common consequence of brain tumors, occurring in 35 to 75% of cases. Here we evaluated the influence of epilepsy on the quality of life (QoL) of patients with malignant brain tumors (primary and metastatic) and assessed which areas of function are most affected by epilepsy and brain tumors. Sixty patients undergoing brain tumor surgery at the Neurosurgery Clinic of the 10th Military Research Hospital, Bydgoszcz, Poland (30 with epilepsy and 30 without epilepsy) were studied. Relationships between categorical variables were determined with Pearson's chi-squared test, while continuous data were analyzed with the Mann-Whitney U-test. A p value < 0.05 was considered statistically significant. A multiple regression model was used for multivariate analysis of QoL. Patients with epilepsy more frequently reported memory disorders as a problem in their daily life. There were trends towards greater impairments in social, professional, and family life, sports and recreational activities, and daily physical activities in brain tumor patients with epilepsy rather than those without epilepsy. Higher frequency and generalized seizures significantly and adversely influenced the ability of patients to leave home and drive vehicles, but a proportion of patients with frequent, generalized seizures continued to drive regardless. Patients with generalized seizures considered the adverse effects of taking medicines as significantly disruptive. Memory disorders significantly affect the QoL of patients with epilepsy, and the importance of stopping driving must be emphasized by all healthcare professionals.

Stereometric and Tribometric Studies of Polymeric Pin and Ceramic Plate Friction Pair Components.

Tw o complementary approaches should be used for the full characterisation of friction pair components. The first approach consists of stereometric studies of machined as well as worn surface topography of the friction components with multiple measurement methods used. The second approach, tribometric studies, enables the tribological characteristics of the friction pair. This work presents the complete characterisation of polymeric pin and ceramic plate friction pair components based on studies with the use of three research instruments: an interference microscope, a scanning electron microscope and a tribological tester. The results of the studies showed that the same treatment conditions used for different but similar ceramic materials did not provide exactly the same characteristics of both the machined and worn surface topography. Moreover, the results showed that the material properties and machined surface topography of the ceramic component significantly affected the friction coefficient and linear wear as well as the wear intensity of the polymeric component. Connecting the two approaches, stereometric studies and tribometric studies, allowed for a better identification of the wear mechanism of the polymeric pin (i.e., abrasion, fatigue and adhesion wear) and the kind of wear products (polymeric material).

PssJ Is a Terminal Galactosyltransferase Involved in the Assembly of the Exopolysaccharide Subunit in Rhizobium Leguminosarum bv. Trifolii.

Rhizobium leguminosarum bv. trifolii produces exopolysaccharide (EPS) composed of glucose, glucuronic acid, and galactose residues at a molar ratio 5:2:1. A majority of genes involved in the synthesis, modification, and export of exopolysaccharide are located in the chromosomal Pss-I region. In the present study, a ΔpssJ deletion mutant was constructed and shown to produce EPS lacking terminal galactose in the side chain of the octasaccharide subunit. The lack of galactose did not block EPS subunit translocation and polymerization. The in trans delivery of the pssJ gene restored the production of galactose-containing exopolysaccharide. The mutant was compromised in several physiological traits, e.g., motility and biofilm production. An impact of the pssJ mutation and changed EPS structure on the symbiotic performance was observed as improper signaling at the stage of molecular recognition, leading to formation of a significant number of non-infected empty nodules. Terminal galactosyltransferase PssJ was shown to display a structure typical for the GT-A class of glycosyltransferases and interact with other GTs and Wzx/Wzy system proteins. The latter, together with PssJ presence in soluble and membrane protein fractions indicated that the protein plays its role at the inner membrane interface and as a component of a larger complex.

Engineering the specificity of Streptococcus pyogenes sortase A by loop grafting.

Sortases are a group of enzymes displayed on the cell-wall of Gram-positive bacteria. They are responsible for the attachment of virulence factors onto the peptidoglycan in a transpeptidation reaction through recognition of a pentapeptide substrate. Most housekeeping sortases recognize one specific pentapeptide motif; however, Streptococcus pyogenes sortase A (SpSrtA WT) recognizes LPETG, LPETA and LPKLG motifs. Here, we examined SpSrtA's flexible substrate specificity by investigating the role of the ß7/ß8 loop in determining substrate specificity. We exchanged the ß7/ß8 loop in SpSrtA with corresponding ß7/ß8 loops from Staphylococcus aureus (SaSrtA WT) and Bacillus anthracis (BaSrtA WT). While the BaSrtA-derived variant showed no enzymatic activity toward either LPETG or LPETA substrates, the activity of the SaSrtA-derived mutant toward the LPETA substrate was completely abolished. Instead, the mutant had an improved activity toward LPETG, the preferred substrate of SaSrtA WT.

Contamination of the urban environment with excrements of companion animals as an underestimated source of Staphylococcus species posing a threat to public health.

The aim of the study was to assess the incidence, resistance, virulence, and genotypic characteristics of Staphylococcus spp. residing in the gastrointestinal tract of dogs and cats, as a group of animals causing potential contamination of the urban space. A high percentage of strains resistant to penicillin (58%), oxacillin (9%) and tetracycline (60%) were found. All isolates resistant to penicillin, kanamycin, or chloramphenicol carried genes responsible for individual resistance (blaZ, aph(3')-IIIa, and cat (pC194)/cat (pC223), respectively. The mecA gene was detected in 45% of the oxacillin-resistant Staphylococcus pseudintermedius strains. The amplification of DNA fragments surrounding rare restriction sites analysis demonstrated high heterogeneity of genotypic profiles correlating with phenotypic resistance profiles. Multilocus sequence typing analysis classified the methicillin-resistant S. pseudintermedius strains as ST71, ST890, and the totally new ST1047. The presence of a high level of resistance among Staphylococcus strains may suggest a potential risk of transfer of these bacteria between companion animals and humans.

A series of 10 Polish patients with thromboembolic events and antithrombin deficiency: two new c.1154-1 G>C and c.1219-534 A>G SERPINC1 gene splicing mutations.

: Inherited antithrombin (AT) deficiency, with prevalence in the general population ranging 0.02-0.17%, is an autosomal dominant disorder associated with a high risk of venous thromboembolism. In most cases, deficiency is caused by mutations in the AT-coding gene (SERPINC1). Only 24 splicing defects have been described causing AT deficiency, all affecting exon flanking regions. The aim of the current study was to characterize the mutations underlying AT deficiency in 10 venous thromboembolism Polish patients aged 42.9 (14-63) years. Whole SERPINC1 gene sequencing was done by next generation sequencing methods. Eight cases had mutations previously described. However, we identified two new intronic mutations that might affect the correct splicing of exon 6 according to in-silico predictions: c.1154-1 G>C, which strongly disturbs the acceptor sequence and c.1219-534 A>G, a deep intronic mutation that might generate a cryptic donor sequence; both might compete with the wild-type donor sequence and explain the associated moderate AT deficiency of carriers. In conclusion, we show the molecular base of AT deficiency in 10 new Polish patients, including two novel SERPINC1 gene mutations potentially affecting splicing.

Mgl2 Is a Hypothetical Methyltransferase Involved in Exopolysaccharide Production, Biofilm Formation, and Motility in Rhizobium leguminosarum bv. trifolii.

In this study, functional characterization of the mgl2 gene located near the Pss-I exopolysaccharide biosynthesis region in Rhizobium leguminosarum bv. trifolii TA1 is described. The hypothetical protein encoded by the mgl2 gene was found to be similar to methyltransferases (MTases). Protein homology and template-based modeling facilitated prediction of the Mgl2 structure, which greatly resembled class I MTases with a S-adenosyl-L-methionine-binding cleft. The Mgl2 protein was engaged in exopolysaccharide, but not lipopolysaccharide, synthesis. The mgl2 deletion mutant produced exopolysaccharide comprised of only low molecular weight fractions, while overexpression of mgl2 caused overproduction of exopolysaccharide with a normal low to high molecular weight ratio. The deletion of the mgl2 gene resulted in disturbances in biofilm formation and a slight increase in motility in minimal medium. Red clover (Trifolium pratense) inoculated with the mgl2 mutant formed effective nodules, and the appearance of the plants indicated active nitrogen fixation. The mgl2 gene was preceded by an active and strong promoter. Mgl2 was defined as an integral membrane protein and formed homodimers in vivo; however, it did not interact with Pss proteins encoded within the Pss-I region. The results are discussed in the context of the possible involvement of the newly described potential MTase in various metabolic traits, such as the exopolysaccharide synthesis and motility that are important for rhizobial saprophytic and symbiotic relationships.

Sortase mutants with improved protein thermostability and enzymatic activity obtained by consensus design.

Staphylococcus aureus sortase A (SaSrtA) is an enzyme that anchors proteins to the cell surface of Gram-positive bacteria. During the transpeptidation reaction performed by SaSrtA, proteins containing an N-terminal glycine can be covalently linked to another protein with a C-terminal LPXTG motif (X being any amino acid). Since the sortase reaction can be performed in vitro as well, it has found many applications in biotechnology. Although sortase-mediated ligation has many advantages, SaSrtA is limited by its low enzymatic activity and dependence on Ca2+. In our study, we evaluated the thermodynamic stability of the SaSrtA wild type and found the enzyme to be stable. We applied consensus analysis to further improve the enzyme's stability while at the same time enhancing the enzyme's activity. As a result, we found thermodynamically improved, more active and Ca2+-independent mutants. We envision that these new variants can be applied in conjugation reactions in low Ca2+ environments.

Identification of potential antivirulence agents by substitution-oriented screening for inhibitors of Streptococcus pyogenes sortase A.

Antimicrobial resistance resulting in ineffective treatment of infectious diseases is an increasing global problem, particularly in infections with pathogenic bacteria. In some bacteria, such as Streptococcus pyogenes, the pathogenicity is strongly linked to the attachment of virulence factors. Their attachment to the cellular membrane is a transpeptidation reaction, catalyzed by sortase enzymes. As such, sortases pose an interesting target for the development of new antivirulence strategies that could yield novel antimicrobial drugs. Using the substitution-oriented fragment screening (SOS) approach, we discovered a potent and specific inhibitor (C10) of sortase A from S. pyogenes. The inhibitor C10 showed high specificity towards S. pyogenes sortase A, with an IC50 value of 10 µM and a Kd of 60 µM. We envision that this inhibitor could be employed as a starting point for further exploration of sortase's potential as therapeutic target for antimicrobial drug development.