Crystal structure of GTPase YsxC from Staphylococcus aureus.

The YsxC protein from Staphylococcus aureus is a GTP-binding protein from the TRAFAC superfamily of the TrmE-Era-EngA-EngB-Septin-like GTPase class, EngB family of GTPases. Recent structural and biochemical studies of YsxC function show that it is an integral part of the pathogenic microorganism life cycle, as it is involved in the assembly of the large 50S ribosomal subunit. Structural studies of this protein with its specific functional features make it an attractive target for further development of new selective antimicrobials. In this study, we cloned the ysxC protein gene from S. aureus, overexpressed the protein in E. coli, and subsequently purified and crystallized it. Protein crystals were successfully grown using the vapor diffusion method, yielding diffraction data with a resolution of up to 2 Å. Comparative analysis of the structure of SaYsxC with known three-dimensional structures of homologs from other microorganisms showed the presence of structural differences for the apo form.

Mechanisms related to carbon nanotubes genotoxicity in human cell lines of respiratory origin.

Potential genotoxicity and carcinogenicity of carbon nanotubes (CNT), as well as the underlying mechanisms, remains a pressing topic. The study aimed to evaluate and compare the genotoxic effect and mechanisms of DNA damage under exposure to different types of CNT. Immortalized human cell lines of respiratory origin BEAS-2B, A549, MRC5-SV40 were exposed to three types of CNT: MWCNT Taunit-M, pristine and purified SWCNT TUBALL™ at concentrations in the range of 0.0006-200 µg/ml. Data on the CNT content in the workplace air were used to calculate the lower concentration limit. The genotoxic potential of CNTs was investigated at non-cytotoxic concentrations using a DNA comet assay. We explored reactive oxygen species (ROS) formation, direct genetic material damage, and expression of a profibrotic factor TGFB1 as mechanisms related to genotoxicity upon CNT exposure. An increase in the number of unstable DNA regions was observed at a subtoxic concentration of CNT (20 µg/ml), with no genotoxic effects at concentrations corresponding to industrial exposures being found. While the three test articles of CNTs exhibited comparable genotoxic potential, their mechanisms appeared to differ. MWCNTs were found to penetrate the nucleus of respiratory cells, potentially interacting directly with genetic material, as well as to enhance ROS production and TGFB1 gene expression. For A549 and MRC5-SV40, genotoxicity depended mainly on MWCNT concentration, while for BEAS-2B - on ROS production. Mechanisms of SWCNT genotoxicity were not so obvious. Oxidative stress and increased expression of profibrotic factors could not fully explain DNA damage under SWCNT exposure, and other mechanisms might be involved.

Yet Another Similarity between Mitochondrial and Bacterial Ribosomal Small Subunit Biogenesis Obtained by Structural Characterization of RbfA from S. aureus.

Ribosome biogenesis is a complex and highly accurate conservative process of ribosomal subunit maturation followed by association. Subunit maturation comprises sequential stages of ribosomal RNA and proteins' folding, modification and binding, with the involvement of numerous RNAses, helicases, GTPases, chaperones, RNA, protein-modifying enzymes, and assembly factors. One such assembly factor involved in bacterial 30S subunit maturation is ribosomal binding factor A (RbfA). In this study, we present the crystal (determined at 2.2 Å resolution) and NMR structures of RbfA as well as the 2.9 Å resolution cryo-EM reconstruction of the 30S-RbfA complex from Staphylococcus aureus (S. aureus). Additionally, we show that the manner of RbfA action on the small ribosomal subunit during its maturation is shared between bacteria and mitochondria. The obtained results clarify the function of RbfA in the 30S maturation process and its role in ribosome functioning in general. Furthermore, given that S. aureus is a serious human pathogen, this study provides an additional prospect to develop antimicrobials targeting bacterial pathogens.

Y98 Mutation Leads to the Loss of RsfS Anti-Association Activity in Staphylococcus aureus.

Ribosomal silencing factor S (RsfS) is a conserved protein that plays a role in the mechanisms of ribosome shutdown and cell survival during starvation. Recent studies demonstrated the involvement of RsfS in the biogenesis of the large ribosomal subunit. RsfS binds to the uL14 ribosomal protein on the large ribosomal subunit and prevents its association with the small subunit. Here, we estimated the contribution of RsfS amino acid side chains at the interface between RsfS and uL14 to RsfS anti-association function in Staphylococcus aureus through in vitro experiments: centrifugation in sucrose gradient profiles and an S. aureus cell-free system assay. The detected critical Y98 amino acid on the RsfS surface might become a new potential target for pharmacological drug development and treatment of S. aureus infections.

Anti-Citrullinated Peptide Antibodies Control Oral Porphyromonas and Aggregatibacter species in Patients with Rheumatoid Arthritis.

Oral microbiome changes take place at the initiation of rheumatoid arthritis (RA); however, questions remain regarding the oral microbiome at pre-RA stages in individuals with clinically suspect arthralgia (CSA). Two cross-sectional cohorts were selected including 84 Tatarstan women (15 early-RA as compared to individuals with CSA ranging from CSA = 0 [n = 22], CSA = 1 [n = 19], CSA = 2 [n = 11], and CSA ≥ 3 [n = 17]) and 42 women with established RA (median: 5 years from diagnosis [IQ: 2-11]). Amplicon sequence variants (ASVs) obtained from oral samples (16S rRNA) were analyzed for alpha and beta diversity along with the abundance at the genus level. A decrease in oral Porphyromonas sp. is observed in ACPA-positive individuals, and this predominates in early-RA patients as compared to non-RA individuals irrespective of their CSA score. In the RA-established cohort, Porphyromonas sp. and Aggregatibacter sp. reductions were associated with elevated ACPA levels. In contrast, no associations were reported when considering individual, genetic and clinical RA-associated factors. Oral microbiome changes related to the genera implicated in post-translational citrullination (Porphyromonas sp. and Aggregatibacter sp.) characterized RA patients with elevated ACPA levels, which supports that the role of ACPA in controlling the oral microbiome needs further evaluation.

Structures and dynamics of hibernating ribosomes from Staphylococcus aureus mediated by intermolecular interactions of HPF.

In bacteria, ribosomal hibernation shuts down translation as a response to stress, through reversible binding of stress-induced proteins to ribosomes. This process typically involves the formation of 100S ribosome dimers. Here, we present the structures of hibernating ribosomes from human pathogen Staphylococcus aureus containing a long variant of the hibernation-promoting factor (SaHPF) that we solved using cryo-electron microscopy. Our reconstructions reveal that the N-terminal domain (NTD) of SaHPF binds to the 30S subunit as observed for shorter variants of HPF in other species. The C-terminal domain (CTD) of SaHPF protrudes out of each ribosome in order to mediate dimerization. Using NMR, we characterized the interactions at the CTD-dimer interface. Secondary interactions are provided by helix 26 of the 16S ribosomal RNA We also show that ribosomes in the 100S particle adopt both rotated and unrotated conformations. Overall, our work illustrates a specific mode of ribosome dimerization by long HPF, a finding that may help improve the selectivity of antimicrobials.

Antifungal Properties, Abiotic Stress Resistance, and Biocontrol Ability of Bacillus mojavensis PS17.

Plant-protecting Bacillus sp. strains used as biocontrol agents frequently produce metabolites inhibiting phytopathogenic fungi. Recently, the search for a novel biocontrol agent with a wide spectrum of disease control drew attention to Bacillus subtilis and their related species, including Bacillus mojavensis. In this study, we determined the antifungal properties of the endophytic B. mojavensis PS17 isolated from wheat seeds. Metabolites produced by B. mojavensis PS-17 inhibit the growth of Fusarium graminearum, Fusarium oxysporum, Fusarium chlamydosporum, Ascochyta pisi, Alternaria alternate, Sclerotinia sclerotiorum, Verticillium dahliaee, and Epicoccum nigrum strains. B. mojavensis strain PS17 produces several hydrolytic enzymes, such as chitinase, ß-glucanase, cellulase, lipase, and protease. Additionally, strain B. mojavensis PS17 demonstrates drought tolerance under osmotic pressure of -2.2 MPa and a moderate halotolerance in 5% (w/v) of NaCl. B. mojavensis PS17 on tomato seedlings was able to reduce lesions of Forl ZUM2407 by 48.11% ± 1.07, showing the potentials of B. mojavensis PS17 to be adapted as a biocontrol agent for agricultural use.

Dimerization of long hibernation promoting factor from Staphylococcus aureus: Structural analysis and biochemical characterization.

Staphylococcus aureus hibernation promoting factor (SaHPF) is responsible for the formation of 100S ribosome dimers, which in turn help this pathogen to reduce energy spent under unfavorable conditions. Ribosome dimer formation strongly depends on the dimerization of the C-terminal domain of SaHPF (CTDSaHPF). In this study, we solved the crystal structure of CTDSaHPF at 1.6 Šresolution and obtained a precise arrangement of the dimer interface. Residues Phe160, Val162, Thr171, Ile173, Tyr175, Ile185 andThr187 in the dimer interface of SaHPF protein were mutated and the effects were analyzed for the formation of 100S disomes of ribosomes isolated from S. aureus. It was shown that substitution of any of single residues Phe160, Val162, Ile173, Tyr175 and Ile185 in the SaHPF homodimer interface abolished the ribosome dimerization in vitro.

NMR and crystallographic structural studies of the Elongation factor P from Staphylococcus aureus.

Elongation factor P (EF-P) is a translation protein factor that plays an important role in specialized translation of consecutive proline amino acid motifs. EF-P is an essential protein for cell fitness in native environmental conditions. It regulates synthesis of proteins involved in bacterial motility, environmental adaptation and bacterial virulence, thus making EF-P a potential drug target. In the present study, we determined the solution and crystal structure of EF-P from the pathogenic bacteria Staphylococcus aureus at 1.48 Å resolution. The structure can serve as a platform for structure-based drug design of novel antibiotics to combat the growing antibiotic resistance of S. aureus.

In seroconverted rheumatoid arthritis patients a multi-reactive anti-herpes IgM profile is associated with disease activity.

Conflicting results have been reported regarding human herpes virus (HHV) reactivation in patients with rheumatoid arthritis (RA). To explore this link, 74 RA patients were selected and compared to 42 first degree relatives (FDR) from probands with RA and 25 healthy controls from the Tatarstan women cohort. The serological analysis was done by testing anti-HSV/CMV/EBV IgM, IgG, plus the IgG avidity index, and completed by evaluating HSV/CMV/EBV DNA by PCR. Results from these analyses reveal: (i) a long lasting infection of HHV in RA, FDR and healthy controls (IgG seroconversion >97%); (ii) an elevated IgM anti-HHV response in seroconverted RA patients which is related to HSV1/2 reactivation (HSV1/2 PCR+); and (iii) a multi-reactive IgM HHV burden profile associated with disease activity (DAS28). In conclusion, HSV1/2 reactivation in seroconverted RA patients is associated with an abnormal anti-HHV immune response, which was reflected in IgM HHV burden, and in activity disease profile.

Solution structure of the N-terminal domain of the Staphylococcus aureus hibernation promoting factor.

Staphylococcus aureus hibernation promoting factor (SaHPF) is a 22,2 kDa protein which plays a crucial role in 100S Staphylococcus aureus ribosome formation during stress. SaHPF consists of N-terminal domain (NTD) that prevents proteins synthesis by binding to the 30S subunit at the P- and A-sites, connected through a flexible linker with a C-terminal domain (CTD) that keeps ribosomes in 100S form via homodimerization. Recently obtained 100S ribosome structure of S. aureus by cryo-EM shown that SaHPF-NTD bound to the ribosome active sites, however due to the absence of SaHPF-NTD structure it was modeled by homology with the E. coli hibernation factors HPF and YfiA. In present paper we have determined the solution structure of SaHPF-NTD by high-resolution NMR spectroscopy which allows us to increase structural knowledge about HPF structure from S. aureus.

Identification and Aggressiveness of Fusarium Species Associated with Onion Bulb (Allium cepa L.) during Storage.

Plant pathogens present a major challenge to crop production, leading to decreased yield and quality during growth and storage. During long-term storage, healthy onions can develop diseases from latent pathogen infections. This poses a challenge for onion growers because infected bulbs without visible symptoms can lead to significant crop losses during the growing season. In this study, we aimed to isolate and identify Fusarium species from yellow onion bulbs (Allium cepa L.) that developed disease symptoms during storage. The aggressiveness of these strains against onion bulbs and seedlings was also evaluated. The isolated strains were further subjected to morphological and molecular differentiation. The results revealed that all 16 isolated strains belonged to the Fusarium complex species incarnatum-equiseti and Fusarium fujikuroi, namely, F. proliferatum (98%), F. oxysporum (1%), and Fusarium sp. (1%). Koch's postulate analysis of isolated strains revealed varying aggressiveness on onion bulbs and plants depending on fungal species. Disease symptoms developed more slowly on plants than on onion bulb plants according to Koch's postulates. Moreover, the results revealed that Fusarium strains that can infect onion plants were less pathogenic to onion bulbs and vice versa. In addition, three isolates were found to be non-pathogenic to onions. Furthermore, the in vitro control of Fusarium species through Bacillus velezensis KS04-AU and Streptomyces albidoflavus MGMM6 showed high potential for controlling the growth of these pathogenic fungi. These results may contribute to the development of environmentally friendly approaches for controlling onion spoilage caused by pathogens during storage.

Structural aspects of RimP binding on small ribosomal subunit from Staphylococcus aureus.

Ribosome biogenesis is an energy-intense multistep process where even minimal defects can cause severe phenotypes up to cell death. Ribosome assembly is facilitated by biogenesis factors such as ribosome assembly factors. These proteins facilitate the interaction of ribosomal proteins with rRNA and correct rRNA folding. One of these maturation factors is RimP which is required for efficient 16S rRNA processing and 30S ribosomal subunit assembly. Here, we describe the binding mode of Staphylococcus aureus RimP to the small ribosomal subunit and present a 4.2 Å resolution cryo-EM reconstruction of the 30S-RimP complex. Together with the solution structure of RimP solved by NMR spectroscopy and RimP-uS12 complex analysis by EPR, DEER, and SAXS approaches, we show the specificity of RimP binding to the 30S subunit from S. aureus. We believe the results presented in this work will contribute to the understanding of the RimP role in the ribosome assembly mechanism.

Is L-arabinose important for the endophytic lifestyle of Pseudomonas spp.?

Twenty endophytic bacteria were isolated from surface-sterilized stems and roots of cucumber plants. After removal of potential siblings and human pathogens, the remaining seven strains were identified based on their 16S rDNA as Pseudomonas fluorescens (2 strains) and P. putida (5 strains). Three strains, namely P. fluorescens CS1, P. fluorescens CR2 and P. putida CR3, were able to suppress tomato foot and root rot (TFRR). Special attention was paid to the characterization of the BIOLOG carbon oxidation profiles of the isolated pseudomonads in order to identify nutrients which might be important for their endophytic lifestyle. Comparative analysis of the profiles of these seven strains with those of seven rhizospheric Pseudomonas spp. revealed that endophytes were able to oxidize L-arabinose and 2,3-butanediol significantly more often than the rhizospheric group. An independent growth experiment performed in tubes using L-arabinose and 2,3-butanediol as sole carbon sources showed the same results as seen using BIOLOG for L-arabinose, but not for 2,3-butanediol. Since L-arabinose is one of the most abundant sugars in xylem of cucumber plants and was not detected in their rhizosphere, our data suggest that utilization of L-arabinose might be a trait contributing to the endophytic lifestyle of the isolated Pseudomonas endophytes.

Characterization of a Novel Bacillus glycinifermentans Strain MGMM1 Based on Full Genome Analysis and Phenotypic Properties for Biotechnological Applications.

Bacillus species have gained much attention based on their phenotypic characteristics and their genetic architecture as biological control agents and plant growth-promotor with bioremediation potential. In this study, we analyzed the whole genome of a novel strain, Bacillus glycinifermentans MGMM1, isolated from the rhizosphere of a weed plant (Senna occidentalis) and assayed its phenotypic characteristics, as well as antifungal and biocontrol ability. The whole genome analysis of MGMM1 identified 4259 putative coding sequences, with an encoding density of 95.75% attributed to biological functions, including genes involved in stimulating plant growth, such as acetolactate synthase, alsS, and genes involved in the resistance to heavy metal antimony (arsB and arsC). AntiSMASH revealed the presence of biosynthetic gene clusters plipastatin, fengycin, laterocidine, geobacillin II, lichenysin, butirosin A and schizokinen. Tests in vitro confirmed that MGMM1 exhibited antifungal activity against Fusarium oxysporum f.sp. radicis-lycopersici (Forl) ZUM2407, Alternaria alternata, F. graminearum and F. spp. and produce protease, lipase amylase and cellulase. Bacillus glycinifermentans MGMM1 demonstrated proteolytic (4.82 ± 1.04 U/mL), amylolytic (0.84 ± 0.05 U/mL) and cellulosic (0.35 ± 0.02 U/mL) enzymatic activities, as well as indole-3-acetic acid production (48.96 ± 1.43 µg/mL). Moreover, the probiotic strain MGMM1 demonstrated a high biocontrol potential of inhibiting (up to 51.45 ± 8.08%) the development of tomato disease caused by Forl ZUM2407. These results suggest that B. glycinifermentans MGMM1 has significant potential as a biocontrol, plant growth-promoting agent in agriculture.

Biotechnological Key Genes of the Rhodococcus erythropolis MGMM8 Genome: Genes for Bioremediation, Antibiotics, Plant Protection, and Growth Stimulation.

Anthropogenic pollution, including residues from the green revolution initially aimed at addressing food security and healthcare, has paradoxically exacerbated environmental challenges. The transition towards comprehensive green biotechnology and bioremediation, achieved with lower financial investment, hinges on microbial biotechnology, with the Rhodococcus genus emerging as a promising contender. The significance of fully annotating genome sequences lies in comprehending strain constituents, devising experimental protocols, and strategically deploying these strains to address pertinent issues using pivotal genes. This study revolves around Rhodococcus erythropolis MGMM8, an associate of winter wheat plants in the rhizosphere. Through the annotation of its chromosomal genome and subsequent comparison with other strains, its potential applications were explored. Using the antiSMASH server, 19 gene clusters were predicted, encompassing genes responsible for antibiotics and siderophores. Antibiotic resistance evaluation via the Comprehensive Antibiotic Resistance Database (CARD) identified five genes (vanW, vanY, RbpA, iri, and folC) that were parallel to strain CCM2595. Leveraging the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) for biodegradation, heavy metal resistance, and remediation genes, the presence of chlorimuron-ethyl, formaldehyde, benzene-desulfurization degradation genes, and heavy metal-related genes (ACR3, arsC, corA, DsbA, modA, and recG) in MGMM8 was confirmed. Furthermore, quorum-quenching signal genes, critical for curbing biofilm formation and virulence elicited by quorum-sensing in pathogens, were also discerned within MGMM8's genome. In light of these predictions, the novel isolate MGMM8 warrants phenotypic assessment to gauge its potential in biocontrol and bioremediation. This evaluation extends to isolating active compounds for potential antimicrobial activities against pathogenic microorganisms. The comprehensive genome annotation process has facilitated the genetic characterization of MGMM8 and has solidified its potential as a biotechnological strain to address global anthropogenic predicaments.

Genomic Insights into the Microbial Agent Streptomyces albidoflavus MGMM6 for Various Biotechnology Applications.

Microbial biotechnology plays a crucial role in improving industrial processes, particularly in the production of compounds with diverse applications. In this study, we used bioinformatic approaches to analyze the genomic architecture of Streptomyces albidoflavus MGMM6 and identify genes involved in various metabolic pathways that have significant biotechnological potential. Genome mining revealed that MGMM6 consists of a linear chromosome of 6,932,303 bp, with a high G+C content of 73.5%, lacking any plasmid contigs. Among the annotated genes, several are predicted to encode enzymes such as dye peroxidase, aromatic ring-opening dioxygenase, multicopper oxidase, cytochrome P450 monooxygenase, and aromatic ring hydroxylating dioxygenases which are responsible for the biodegradation of numerous endogenous and xenobiotic pollutants. In addition, we identified genes associated with heavy metal resistance, such as arsenic, cadmium, mercury, chromium, tellurium, antimony, and bismuth, suggesting the potential of MGMM6 for environmental remediation purposes. The analysis of secondary metabolites revealed the presence of multiple biosynthesis gene clusters responsible for producing compounds with potent antimicrobial and metal-chelating activities. Furthermore, laboratory tests conducted under controlled conditions demonstrated the effectiveness of MGMM6 in inhibiting phytopathogenic microbes, decolorizing and degrading aromatic triphenylmethane dyes, particularly Blue Brilliant G250, from wastewater by up to 98 ± 0.15%. Overall, the results of our study highlight the promising biotechnological potential of S. albidoflavus MGMM6.

Are formae speciales pathogens really host specific? A broadened host specificity in Fusarium oxysporum f.sp. radicis-cucumerinum.

Phytopathogenic strains of Fusarium oxysporum Schlecht exhibit clear host specificity, which appears to be a persistent characteristic and a dependable base for the forma specialis system of these pathogens. Here, we report an altered host specificity of the F. oxysporum f.sp. radicis-cucumerinum strain V03-2 g (Forc V03-2 g) - a causative agent of cucumber root-rot, the clonal derivates of which acquired the ability to infect tomato plants. Since the clonal derivates of Forc V03-2 g with transformed host specificity preserved their ability to parasitize on cucumber plants, the changes that occurred can be classified as broadening of host specificity. To our knowledge, this is the first observation of pathogenicity changes in formae speciales of F. oxysporum. The clonal derivates acquired could be used to trace genetic determinants of the host specificity of phytopathogenic strains of F. oxysporum.

Backbone and side chain NMR assignments for the ribosome maturation factor P (RimP) from Staphylococcus aureus.

The ribosomal maturation factor (RimP) is a 17.7 kDa protein and is the assembly factor of the 30S subunit. RimP is essential for efficient processing of 16S rRNA and maturation (assembly) of the 30S ribosome. It was suggested that RimP takes part in stabilization of the central pseudoknot at the early stages of the 30S subunit maturation, and this process may occur before the head domain assembly and later stages of the 30S assembly, but the mechanism of this interaction is still not fully understood. Here we report the assignment of the 1H, 13C and 15N chemical shift in the backbone and side chains of RimP from Staphylococcus aureus. Analysis of chemical shifts of the main chain using TALOS + suggests that the RimP contains eight ß-strands and three α-helices with the topology α1-ß1-ß2-α2- ß3- α3- ß4- ß5- ß6- ß7- ß8. Structural studies of RimP and its complex with the ribosome by integrated structural biology approaches (NMR spectroscopy, X-ray diffraction analysis and cryoelectron microscopy) will allow further screening of highly selective inhibitors of the translation of S. aureus.

Persistence as a Constituent of a Biocontrol Mechanism (Competition for Nutrients and Niches) in Pseudomonas putida PCL1760.

Competition for nutrients and niches (CNN) is known to be one of the mechanisms for biocontrol mostly exhibited by Pseudomonas strains. Phenotypic and full genome analysis revealed Pseudomonas putida PCL1760 controlling tomato foot and root rot (TFRR) solely through CNN mechanism. Although the availability of nutrients and motility are the known conditions for CNN, persistence of bacteria through dormancy by ribosomal hibernation is a key phenomenon to evade both biotic and abiotic stress. To confirm this hypothesis, rsfS gene knockout mutant of PCL1760 (SB9) was first obtained through genetic constructions and compared with the wild type PCL1760. Primarily, relative expression of rsfS in PCL1760 was conducted on tomato seedlings which showed a higher expression at the apical part (1.02 ± 0.18) of the plant roots than the basal (0.41 ± 0.13). The growth curve and persistence in ceftriaxone after the induction of starvation with rifampicin were performed on both strains. Colonization on the tomato root by CFU and qPCR, including biocontrol ability against Fusarium, was also tested. The growth dynamics of both PCL1760 and SB9 in basal and rich medium statistically did not differ (p ≤ 0.05). There was a significant difference observed in persistence showing PCL1760 to be more persistent than its mutant SB9, while SB9 (pJeM2:rsfS) was 221.07 folds more than PCL1760. In colonization and biocontrol ability tests, PCL1760 was dominant over SB9 colonizing and controlling TFRR (in total, 3.044 × 104 to 6.95 × 103 fg/µL and 55.28% to 30.24%, respectively). The deletion of the rsfS gene in PCL1760 reduced the persistence and effectiveness of the strain, suggesting persistence as one important characteristic of the CNN.