Biological potential of Ascophyllum nodosum extract on rhizobial diversity in nodules of mothbean Vigna aconitifolia Jacq. via Amplified Ribosomal DNA Restriction Analysis

Aim: The aim of this study was to use Ascophyllum nodosum for potentially increasing the growth and rhizobial diversity in nodulating rhizobia in Vigna aconitifolia. Methodology: Different concentrations of Ascophyllum nodosum extracts (0.01%, 0.02%, 0.05%, 0.10% and 0.50%) were applied via foliar spray and on roots of Vigna aconitifolia. Growth characteristics and Amplified Ribosomal DNA Restriction Analysis were conducted to detect the morphological and molecular changes in rhizobial diversity. The restriction profiles thus obtained were used to study the rhizobial communities via Cluster analysis and Dendrogram using NTSYS-PC program and UPGMA constructed. Results: Roots treated with 0.05% Ascophyllum nodosum extract showed best growth of plants. This concentration not only proved best for the aggregation of nodules but also for obtaining enormous rhizobial diversity. Interpretation: Ascophyllum nodosum is a modern, cheap, non-toxic natural biofertilizer and Amplified Ribosomal DNA Restriction Analysis represents a favorable alternative to culture dependent method for assessing rhizobial diversity in nodulating bacteria.

Persistent Bacterial Coinfection of a COVID-19 Patient Caused by a Genetically Adapted Pseudomonas aeruginosa Chronic Colonizer

Pseudomonas aeruginosa is a biofilm-forming opportunistic pathogen which causes chronic infections in immunocompromised patients and leads to high mortality rate. It is identified as a common coinfecting pathogen in COVID-19 patients causing exacerbation of illness. In our hospital, P. aeruginosa is one of the top coinfecting bacteria identified among COVID-19 patients. We collected a strong biofilm-forming P. aeruginosa strain displaying small colony variant morphology from a severe COVID-19 patient. Genomic and transcriptomic sequencing analyses were performed with phenotypic validation to investigate its adaptation in SARS-CoV-2 infected environment. Genomic characterization predicted specific genomic islands highly associated with virulence, transcriptional regulation, and DNA restriction-modification systems. Epigenetic analysis revealed a specific N6-methyl adenine (m6A) methylating pattern including methylation of alginate, flagellar and quorum sensing associated genes. Differential gene expression analysis indicated that this isolate formed excessive biofilm by reducing flagellar formation (7.4 to 1,624.1 folds) and overproducing extracellular matrix components including CdrA (4.4 folds), alginate (5.2 to 29.1 folds) and Pel (4.8–5.5 folds). In summary, we demonstrated that P. aeuginosa clinical isolates with novel epigenetic markers could form excessive biofilm, which might enhance its antibiotic resistance and in vivo colonization in COVID-19 patients.

Crystal structure and DNA cleavage mechanism of the restriction DNA glycosylase R.CcoLI from Campylobacter coli

While most restriction enzymes catalyze the hydrolysis of phosphodiester bonds at specific nucleotide sequences in DNA, restriction enzymes of the HALFPIPE superfamily cleave N-glycosidic bonds, similar to DNA glycosylases. Apurinic/apyrimidinic (AP) sites generated by HALFPIPE superfamily proteins are cleaved by their inherent AP lyase activities, other AP endonuclease activities or heat-promoted β-elimination. Although the HALFPIPE superfamily protein R.PabI, obtained from a hyperthermophilic archaea, Pyrococcus abyssi, shows weak AP lyase activity, HALFPIPE superfamily proteins in mesophiles, such as R.CcoLI from Campylobacter coli and R. HpyAXII from Helicobacter pylori, show significant AP lyase activities. To identify the structural basis for the AP lyase activity of R.CcoLI, we determined the structure of R.CcoLI by X-ray crystallography. The structure of R.CcoLI, obtained at 2.35-Å resolution, shows that a conserved lysine residue (Lys71), which is stabilized by a characteristic β-sheet structure of R.CcoLI, protrudes into the active site. The results of mutational assays indicate that Lys71 is important for the AP lyase activity of R.CcoLI. Our results help to elucidate the mechanism by which HALFPIPE superfamily proteins from mesophiles efficiently introduce double-strand breaks to specific sites on double-stranded DNA.

Phenetic and Molecular Diversity of Nitrogen Fixating Plant Growth Promoting Azotobacter Isolated from Semiarid Regions of India

In the present study, 24 Azotobacter strains were isolated from soils of different areas of southern Rajasthan and characterized at biochemical, functional, and molecular levels. The isolated Azotobacter strains were gram negative and cyst forming when viewed under the microscope. These strains were also screened for their plant growth promoting activities and the ability of these isolates to survive under abiotic stress conditions viz. salt, pH, temperature, and drought stress. All the isolates showed IAA, siderophore, HCN, and ammonia production, whereas seven Azotobacter strains showed phosphate solubilization. Amplified Ribosomal DNA Restriction Analysis (ARDRA) revealed significant diversity among Azotobacter strains and the dendrogram obtained differentiated twenty-four of the strains into two major clusters at a similarity coefficient of 0.64. Qualitative and quantitative N2 fixation abilities of these strains were also detrained, and the amounts of acetylene reduced by Azotobacter strains were in the range of 1.31 to 846.56 nmol C2H4 mg protein−1 h−1. The strains showing high nitrogen fixation ability with multiple PGP activities were selected for further pot studies, and these Azotobacter strains significantly increased the various plant growth parameters of maize plantlets. Furthermore, the best Azotobacter isolates were subjected to 16S rRNA sequencing and confirmed their identities as Azotobacter sp. The indigenous Azotobacter strains with multiple PGP activities could be further used for commercial production.

T Cells Expressing a TCR-Like Antibody Selected Against the Heteroclitic Variant of a Shared MAGE-A Epitope Do Not Recognise the Cognate Epitope

Antibodies-recognising peptides bound to the major histocompatibility complex (pMHC) represent potentially valuable and promising targets for chimeric antigen receptor (CAR) T cells to treat patients with cancer. Here, a human phage-Fab library has been selected using HLA-A2 complexed with a heteroclitic peptide variant from an epitope shared among multiple melanoma-associated antigens (MAGEs). DNA restriction analyses and phage ELISAs confirmed selection of unique antibody clones that specifically bind to HLA-A2 complexes or HLA-A2-positive target cells loaded with native or heteroclitic peptide. Antibodies selected against heteroclitic peptide, in contrast to native peptide, demonstrated significantly lower to even negligible binding towards native peptide or tumour cells that naturally expressed peptides. The binding to native peptide was not rescued by phage panning with antigen-positive tumour cells. Importantly, when antibodies directed against heteroclitic peptides were engineered into CARs and expressed by T cells, binding to native peptides and tumour cells was minimal to absent. In short, TCR-like antibodies, when isolated from a human Fab phage library using heteroclitic peptide, fail to recognise its native peptide. We therefore argue that peptide modifications to improve antibody selections should be performed with caution as resulting antibodies, either used directly or as CARs, may lose activity towards endogenously presented tumour epitopes.

Characterisations of Probiotic Lactobacillus Strains by Amplified Ribosomal DNA Restriction Analysis (ARDRA)

Twelve probiotic Lactobacillus strains for poultry were characterised by amplified ribosomal DNA restriction analysis (ARDRA) using Sau3AI, TaqI, HaeIII and AluI restriction endonucleases. Species-specific and strain-specific restriction patterns were observed from the bacterial strains. Numerical analysis of composite analysis of ARDRA exhibited D value of 0.8456. Whereas, the caculated D values of ARDRA patterns generated by Sau3AI, TaqI, HaeIII and AluI were 0.8309, 0.8382,0.8088 and 0.8088, repectively. Composite analysis of ARDRA was the most discriminative method when compared to the individual analysis. ARDRA could distinguished L. reuteri C 10 and L. panis C 17 into single strains. The 16S rRNA gene restriction patterns were also able to group L. gallinarum I 16 and I 26 into single strains. Lactobacillus brevis I 12, I 23, I 25, I 211 and I 218 seem to be multiple clones of the same bacterial strain as are L. reuteri C 1 and C 16. ARDRA is a valuable fingerprinting method to discriminate probiotic Lactobacillus strains.

Evaluation of Malolactic Bacteria Associated with Wines from Albariño Variety as Potential Starters: Screening for Quality and Safety

The biodiversity of lactic acid bacteria in musts and wines of Albariño variety has been studied. The identification of species was addressed through a combination of biochemical and genetic methods (API® 50 CHL test, 16S rDNA and recA gene sequences, Amplified Ribosomal DNA Restriction Analysis -ARDRA- and 16S-26S intergenic region analysis). The results grouped the isolates into six species predominating those of the genus Lactobacillus and showing a typical biogeographical distribution. Among sixteen strains evaluated, eight of them showed malolactic activity. The study of the presence of genes hdc, odc, and tdc, along with the LC/MS-MS analysis of biogenic amines in wine, showed five strains lacking aminogenic ability. The absence of the pad gene in the above-mentioned strains discards its ability to produce volatile phenols that may adversely affect the aroma. Finally, all malolactic strains showed β-glucosidase activity so that they could contribute to enhance and differentiate the aromatic profile of Albariño wines.

Chloroviruses

Chloroviruses are large dsDNA, plaque-forming viruses that infect certain chlorella-like green algae; the algae are normally mutualistic endosymbionts of protists and metazoans and are often referred to as zoochlorellae. The viruses are ubiquitous in inland aqueous environments throughout the world and occasionally single types reach titers of thousands of plaque-forming units per ml of native water. The viruses are icosahedral in shape with a spike structure located at one of the vertices. They contain an internal membrane that is required for infectivity. The viral genomes are 290 to 370 kb in size, which encode up to 16 tRNAs and 330 to ~415 proteins, including many not previously seen in viruses. Examples include genes encoding DNA restriction and modification enzymes, hyaluronan and chitin biosynthetic enzymes, polyamine biosynthetic enzymes, ion channel and transport proteins, and enzymes involved in the glycan synthesis of the virus major capsid glycoproteins. The proteins encoded by many of these viruses are often the smallest or among the smallest proteins of their class. Consequently, some of the viral proteins are the subject of intensive biochemical and structural investigation.