Structure predictions and functional insights into Amidase_3 domain containing N-acetylmuramyl-L-alanine amidases from Deinococcus indicus DR1.

BACKGROUND: N-acetylmuramyl-L-alanine amidases are cell wall modifying enzymes that cleave the amide bond between the sugar residues and stem peptide in peptidoglycan. Amidases play a vital role in septal cell wall cleavage and help separate daughter cells during cell division. Most amidases are zinc metalloenzymes, and E. coli cells lacking amidases grow as chains with daughter cells attached to each other. In this study, we have characterized two amidase enzymes from Deinococcus indicus DR1. D. indicus DR1 is known for its high arsenic tolerance and unique cell envelope. However, details of their cell wall biogenesis remain largely unexplored. RESULTS: We have characterized two amidases Ami1Di and Ami2Di from D. indicus DR1. Both Ami1Di and Ami2Di suppress cell separation defects in E. coli amidase mutants, suggesting that these enzymes are able to cleave septal cell wall. Ami1Di and Ami2Di proteins possess the Amidase_3 catalytic domain with conserved -GHGG- motif and Zn2+ binding sites. Zn2+- binding in Ami1Di is crucial for amidase activity. AlphaFold2 structures of both Ami1Di and Ami2Di were predicted, and Ami1Di was a closer homolog to AmiA of E. coli. CONCLUSION: Our results indicate that Ami1Di and Ami2Di enzymes can cleave peptidoglycan, and structural prediction studies revealed insights into the activity and regulation of these enzymes in D. indicus DR1.

Genetic heterogeneity and respiratory chain enzyme analysis in pediatric Indian patients with mitochondrial disorder: Report of novel variants in POLG1 gene and their functional implication using molecular dynamic simulation.

Mitochondrial disorders are a heterogeneous group of disorders caused by mutations in the mitochondrial DNA or in nuclear genes encoding the mitochondrial proteins and subunits. Polymerase Gamma (POLG) is a nuclear gene and mutation in the POLG gene are one of the major causes of inherited mitochondrial disorders. In this study, 15 pediatric patients, with a wide spectrum of clinical phenotypes were screened using blood samples (n = 15) and muscle samples (n = 4). Respiratory chain enzyme analysis in the muscle samples revealed multi-complex deficiencies with Complex I deficiency present in (1/4) patients, Complex II (2/4), Complex III (3/4) and Complex IV (2/4) patients. Multiple large deletions were observed in 4/15 patients using LR-PCR. Whole exome sequencing (WES) revealed a compound heterozygous mutation consisting of a POLG1 novel variant (NP_002684.1:p.Trp261X) and a missense variant (NP_002684.1:p. Leu304Arg) in one patient and another patient harboring a novel homozygous POLG1 variant (NP_002684.1:p. Phe750Val). These variants (NP_002684.1:p. Leu304Arg) and (NP_002684.1:p. Phe750Val) and their interactions with DNA were modelled using molecular docking and molecular dynamics (MD) simulation studies. The protein conformation was analyzed as root mean square deviation (RMSD), root mean square fluctuation (RMSF) which showed local fluctuations in the mutants compared to the wildtype. However, Solvent Accessible Surface Area (SASA) significantly increased for NP_002684.1:p.Leu304Arg and decreased in NP_002684.1:p.Phe750Val mutants. Further, Contact Order analysis indicated that the Aromatic-sulfur interactions were destabilizing in the mutants. Overall, these in-silico analysis has revealed a destabilizing mutations suggesting pathogenic variants in POLG1 gene.

Targeting the Bottlenecks in Levan Biosynthesis Pathway in Bacillus subtilis and Strain Optimization by Computational Modeling and Omics Integration.

Levan is a fructan polymer with many industrial applications such as the formulation of hydrogels, drug delivery, and wound healing, among others. To this end, metabolic systems engineering is a valuable method to improve the yield of a specific metabolite in a wide range of bacterial and eukaryotic organisms. In this study, we report a systems biology approach integrating genomics data for the Bacillus subtilis model, wherein the metabolic pathway for levan biosynthesis is unpacked. We analyzed a revised genome-scale enzyme-constrained metabolic model (ecGEM) and performed simulations to increase levan biopolymer production capacity in B. subtilis. We used the model ec_iYO844_lvn to (1) identify the essential genes and bottlenecks in levan production, and (2) specifically design an engineered B. subtilis strain capable of producing higher levan yields. The FBA and FVA analysis showed the maximal growth rate of the organism up to 0.624 hr-1 at 20 mmol gDw-1 hr-1 of sucrose intake. Gene knockout analyses were performed to identify gene knockout targets to increase the levan flux in B. subtilis. Importantly, we found that the pgk and ctaD genes are the two target genes for the knockout. The perturbation of these two genes has flux gains for levan production reactions with 1.3- and 1.4-fold the relative flux span in the mutant strains, respectively, compared to the wild type. In all, this work identifies the bottlenecks in the production of levan and possible ways to overcome them. Our results provide deeper insights on the bacterium's physiology and new avenues for strain engineering.

Surface exposed and charged residues drive thermostability in fungi.

Fungi, though mesophilic, include thermophilic and thermostable species, as well. The thermostability of proteins observed in these fungi is most likely to be attributed to several molecular factors, such as the presence of salt bridges and hydrogen bond interactions between side chains. These factors cannot be generalized for all fungi. Factors impacting thermostability can guide how fungal thermophilic proteins gain thermostability. We curated a dataset of proteins for 14 thermophilic fungi and their evolutionarily closer mesophiles. Additionally, the proteome of Chaetomium thermophilum and its evolutionarily related mesophile Chaetomium globosum was analyzed. Using eggNOG, we categorized the proteomes into clusters of orthologous groups (COGs). While the individual count of proteins is over-represented in mesophiles (for COGs S, G, L, and Q), there are certain features that are significantly enriched in thermophiles (such as charged residues, exposed residues, polar residues, etc.). Since fungi are known to be cellulolytic and chitinolytic by nature, we selected 37 existing carbohydrate-active enzymes (CAZyme) families in Eurotiales, Mucorales, and Sordariales. We looked at closely similar sequences and their modeled structures for further comparison. Comparing solvent accessibilities of thermophilic and mesophilic proteins, exposed and intermediate residues are observed higher in thermophiles whereas buried residues are observed higher in mesophiles. For specific five CAZYme families (GH7, GH11, GH18, GH45, and CBM1) we looked at position-specific substitutions between thermophiles and mesophiles. We also found that there are relatively more intramolecular interactions in thermophiles compared to mesophiles. Thus, we found factors such as surface exposed residues and charged residues that are highly likely to impart thermostability in fungi, and this study sets the stage for further studies in the area of fungal thermostability.

Key essential oil components delocalize Candida albicans Kar3p and impact microtubule structure.

BACKGROUND: Treatment of Candida albicans associated infections is often ineffective in the light of resistance, with an urgent need to discover novel antimicrobials. Fungicides require high specificity and can contribute to antifungal resistance, so inhibition of fungal virulence factors is a good strategy for developing new antifungals. OBJECTIVES: Examine the impact of four plant-derived essential oil components (1,8-cineole, α-pinene, eugenol, and citral) on C. albicans microtubules, kinesin motor protein Kar3 and morphology. METHODS: Microdilution assays were used to determine minimal inhibitory concentrations, microbiological assays assessed germ tube, hyphal and biofilm formation, confocal microscopy probed morphological changes and localization of tubulin and Kar3p, and computational modelling was used to examine the theoretical binding of essential oil components to tubulin and Kar3p. RESULTS: We show for the first time that essential oil components delocalize the Kar3p, ablate microtubules, and induce psuedohyphal formation with reduced biofilm formation. Single and double deletion mutants of kar3 were resistant to 1,8-cineole, sensitive to α-pinene and eugenol, but unimpacted by citral. Strains with homozygous and heterozygous Kar3p disruption had a gene-dosage effect for all essential oil components, resulting in enhanced resistance or susceptibility patterns that were identical to that of cik1 mutants. The link between microtubule (αß-tubulin) and Kar3p defects was further supported by computational modeling, showing preferential binding to αß-tubulin and Kar3p adjacent to their Mg2+-binding sites. CONCLUSION: This study highlights how essential oil components interfere with the localization of the kinesin motor protein complex Kar3/Cik1 and disrupt microtubules, leading to their destabilization which results in hyphal and biofilm defects.

Waste to drugs: identification of pyrolysis by-products as antifungal agents against Cryptococcus neoformans.

The fungi, Cryptococcus neoformans cause major infections such as cryptococcal meningitis and cryptococcosis. Therefore, we explored the use of Thioredoxin reductase (Trr1) from C. neoformans as a gene target for the development of novel antifungal agents. Trr1 plays an essential role in the survival in the oxidative environment of macrophages and is important for the virulence of C. neoformans. During the thermochemical conversion (pyrolysis) of lignocellulosic biomass (LCB), a cocktail of compounds is produced by the decomposition and degradation. In general, LCB-derived cocktail of compounds is a rich source of aromatic compounds that have been shown to be antifungal in nature. Usually, the aqueous phase produced during biomass pyrolysis is generally regarded as waste. Here, we used Parthenium hysterophorus biomass as the antifungal source and obtained the aqueous phase after pyrolysis. Using GC-MS analysis of the aqueous phase collected from P. hysterophorus biomass revealed the presence of a large number of aromatic and organic compounds. Using virtual screening, the compounds present in the aqueous phase were docked against Trr1 using GLIDE. Two promising candidates were analyzed further by performing molecular dynamics simulation using GROMACS, to establish stable interactions. We validated the computational results with clustering analysis. We report that 2,4-Di-tertbutyl phenol and 1H-Pyrazole, 4-ethyl-3,5-dimethyl have a potent antifungal property and we postulate that they could be a potent antifungal agent against Trr1 of C. neoformans.

Structural and functional mapping of ars gene cluster in Deinococcus indicus DR1.

Deinococcus indicus DR1 is a novel Gram-negative bacterium, isolated from the Dadri wetlands in Uttar Pradesh, India. In addition to being radiation-resistant, the rod-shaped, red-pigmented organism shows extraordinary resistance to arsenic. The proteins of the corresponding ars gene cluster involved in arsenic extrusion in D. indicus DR1 have not yet been characterized. Additionally, how these proteins regulate each other providing arsenic resistance is still unclear. Here, we present a computational model of the operonic structure and the corresponding characterization of the six proteins of the ars gene cluster in D. indicus DR1. Additionally, we show the expression of the genes in the presence of arsenic using qRT-PCR. The ars gene cluster consists of two transcriptional regulators (ArsR1, ArsR2), two arsenate reductases (ArsC2, ArsC3), one metallophosphatase family protein (MPase), and a transmembrane arsenite efflux pump (ArsB). The transcriptional regulators are trans-acting repressors, and the reductases reduce arsenate (As5+) ions to arsenite (As3+) ions for favourable extrusion. The proteins modelled using RoseTTAFold, and their conformationally stable coordinates obtained after MD simulation indicate their various functional roles with respect to arsenic. Excluding ArsB, all the proteins belong to the α + ß class of proteins. ArsB, being a membrane protein, is fully α-helical, with 12 transmembrane helices. The results show the degree of similarity or divergence of the mechanism utilized by these proteins of ars gene cluster in D. indicus DR1 to confer high levels of arsenic tolerance. This structural characterization study of the ars genes will enable new and deeper insights of arsenic tolerance.

'Targeting' the search: An upgraded structural and functional repository of antimicrobial peptides for biofilm studies (B-AMP v2.0) with a focus on biofilm protein targets.

Bacterial biofilms, often as multispecies communities, are recalcitrant to conventional antibiotics, making the treatment of biofilm infections a challenge. There is a push towards developing novel anti-biofilm approaches, such as antimicrobial peptides (AMPs), with activity against specific biofilm targets. In previous work, we developed Biofilm-AMP, a structural and functional repository of AMPs for biofilm studies (B-AMP v1.0) with more than 5000 structural models of AMPs and a vast library of AMP annotations to existing biofilm literature. In this study, we present an upgraded version of B-AMP, with a focus on existing and novel bacterial biofilm targets. B-AMP v2.0 hosts a curated collection of 2502 biofilm protein targets across 473 bacterial species, with structural protein models and functional annotations from PDB, UniProt, and PubMed databases. The biofilm targets can be searched for using the name of the source organism, and function and type of protein, and results include designated Target IDs (unique to B-AMP v2.0), UniProt IDs, 3D predicted protein structures, PDBQT files, pre-defined protein functions, and relevant scientific literature. To present an example of the combined applicability of both, the AMP and biofilm target libraries in the repository, we present two case studies. In the first case study, we expand an in silico pipeline to evaluate AMPs against a single biofilm target in the multidrug resistant, bacterial pathogen Corynebacterium striatum, using 3D protein-peptide docking models from previous work and Molecular Dynamics simulations (~1.2µs). In the second case study, we build an in silico pipeline to identify candidate AMPs (using AMPs with both anti-Gram positive and anti-Gram negative activity) against two biofilm targets with a common functional annotation in Pseudomonas aeruginosa and Staphylococcus aureus, widely-encountered bacterial co-pathogens. With its enhanced structural and functional capabilities, B-AMP v2.0 serves as a comprehensive resource for AMP investigations related to biofilm studies. B-AMP v2.0 is freely available at https://b-amp.karishmakaushiklab.com and will be regularly updated with structural models of AMPs and biofilm targets, as well as 3D protein-peptide interaction models for key biofilm-forming pathogens.

Coarse grained modelling highlights the binding differences in the two different allosteric sites of the Human Kinesin EG5 and its implications in inhibitor design.

Kinesins involved in mitotic cell division have gained prominence as promising chemotherapy targets. One such kinesin, EG5, a motor protein responsible for cell division, is a validated chemotherapy target with several compounds at various stages of clinical trials. EG5 has an active site and two different allosteric sites that are known to have ligand specificity. Upon ligand binding, EG5's motor domain will no longer undergo nucleotide-dependent conformational changes required to complete the catalytic cycle. However, there is a lack of in-depth knowledge on the mechanism of inhibitor binding to the two different allosteric sites. To understand the EG5's inhibition mechanism and interactions at allosteric sites and other functionally important regions, we generated two coarse-grained models, Gaussian Network Model (GNM) and Anisotropic Network Model (ANM), to identify the dynamics and its correlation to EG5's function. The first three slowest modes of GNM showed marked differences between the various models of EG5. In the first mode, when the inhibitor is bound at allosteric site 1, there is a presence of a hinge region around residue 166, which is not found when the inhibitor is bound at allosteric site 2 or allosteric sites 1 and 2. The third slowest mode showed a distinctive positively correlated region when the inhibitor is bound at allosteric site 2. These differences indicated that the mechanism of binding at allosteric site 1 and allosteric site 2 are unique. Further, it was observed that the simultaneous ligand binding at allosteric sites 1 and 2 shares structural dynamics and interactions that were found while ligand binds at allosteric sites 1 and 2 independently, leading to a new mechanism. Taken together, our observations suggest that there are different mechanisms at play in each inhibitor bound system considered.

DNA-Binding Properties of YbaB, a Putative Nucleoid-Associated Protein From Caulobacter crescentus.

Nucleoid-associated proteins (NAPs) or histone-like proteins (HLPs) are DNA-binding proteins present in bacteria that play an important role in nucleoid architecture and gene regulation. NAPs affect bacterial nucleoid organization via DNA bending, bridging, or forming aggregates. EbfC is a nucleoid-associated protein identified first in Borrelia burgdorferi, belonging to YbaB/EbfC family of NAPs capable of binding and altering DNA conformation. YbaB, an ortholog of EbfC found in Escherichia coli and Haemophilus influenzae, also acts as a transcriptional regulator. YbaB has a novel tweezer-like structure and binds DNA as homodimers. The homologs of YbaB are found in almost all bacterial species, suggesting a conserved function, yet the physiological role of YbaB protein in many bacteria is not well understood. In this study, we characterized the YbaB/EbfC family DNA-binding protein in Caulobacter crescentus. C. crescentus has one YbaB/EbfC family gene annotated in the genome (YbaB C c ) and it shares 41% sequence identity with YbaB/EbfC family NAPs. Computational modeling revealed tweezer-like structure of YbaB C c , a characteristic of YbaB/EbfC family of NAPs. N-terminal-CFP tagged YbaB C c localized with the nucleoid and is able to compact DNA. Unlike B. burgdorferi EbfC protein, YbaB C c protein is a non-specific DNA-binding protein in C. crescentus. Moreover, YbaB C c shields DNA against enzymatic degradation. Collectively, our findings reveal that YbaB C c is a small histone-like protein and may play a role in bacterial chromosome structuring and gene regulation in C. crescentus.

A low CO2-responsive mutant of Setaria viridis reveals that reduced carbonic anhydrase limits C4 photosynthesis.

In C4 species, ß-carbonic anhydrase (CA), localized to the cytosol of the mesophyll cells, accelerates the interconversion of CO2 to HCO3-, the substrate used by phosphoenolpyruvate carboxylase (PEPC) in the first step of C4 photosynthesis. Here we describe the identification and characterization of low CO2-responsive mutant 1 (lcr1) isolated from an N-nitroso-N-methylurea- (NMU) treated Setaria viridis mutant population. Forward genetic investigation revealed that the mutated gene Sevir.5G247800 of lcr1 possessed a single nucleotide transition from cytosine to thymine in a ß-CA gene causing an amino acid change from leucine to phenylalanine. This resulted in severe reduction in growth and photosynthesis in the mutant. Both the CO2 compensation point and carbon isotope discrimination values of the mutant were significantly increased. Growth of the mutants was stunted when grown under ambient pCO2 but recovered at elevated pCO2. Further bioinformatics analyses revealed that the mutation has led to functional changes in one of the conserved residues of the protein, situated near the catalytic site. CA transcript accumulation in the mutant was 80% lower, CA protein accumulation 30% lower, and CA activity ~98% lower compared with the wild type. Changes in the abundance of other primary C4 pathway enzymes were observed; accumulation of PEPC protein was significantly increased and accumulation of malate dehydrogenase and malic enzyme decreased. The reduction of CA protein activity and abundance in lcr1 restricts the supply of bicarbonate to PEPC, limiting C4 photosynthesis and growth. This study establishes Sevir.5G247800 as the major CA allele in Setaria for C4 photosynthesis and provides important insights into the function of CA in C4 photosynthesis that would be required to generate a rice plant with a functional C4 biochemical pathway.

AMPing Up the Search: A Structural and Functional Repository of Antimicrobial Peptides for Biofilm Studies, and a Case Study of Its Application to Corynebacterium striatum, an Emerging Pathogen.

Antimicrobial peptides (AMPs) have been recognized for their ability to target processes important for biofilm formation. Given the vast array of AMPs, identifying potential anti-biofilm candidates remains a significant challenge, and prompts the need for preliminary in silico investigations prior to extensive in vitro and in vivo studies. We have developed Biofilm-AMP (B-AMP), a curated 3D structural and functional repository of AMPs relevant to biofilm studies. In its current version, B-AMP contains predicted 3D structural models of 5544 AMPs (from the DRAMP database) developed using a suite of molecular modeling tools. The repository supports a user-friendly search, using source, name, DRAMP ID, and PepID (unique to B-AMP). Further, AMPs are annotated to existing biofilm literature, consisting of a vast library of over 10,000 articles, enhancing the functional capabilities of B-AMP. To provide an example of the usability of B-AMP, we use the sortase C biofilm target of the emerging pathogen Corynebacterium striatum as a case study. For this, 100 structural AMP models from B-AMP were subject to in silico protein-peptide molecular docking against the catalytic site residues of the C. striatum sortase C protein. Based on docking scores and interacting residues, we suggest a preference scale using which candidate AMPs could be taken up for further in silico, in vitro and in vivo testing. The 3D protein-peptide interaction models and preference scale are available in B-AMP. B-AMP is a comprehensive structural and functional repository of AMPs, and will serve as a starting point for future studies exploring AMPs for biofilm studies. B-AMP is freely available to the community at https://b-amp.karishmakaushiklab.com and will be regularly updated with AMP structures, interaction models with potential biofilm targets, and annotations to biofilm literature.

Identification of nsSNPs of transcription factor E2F1 predisposing individuals to lung cancer and head and neck cancer.

E2Fs transcription factors family is involved in the G1/S transition and DNA replication and their deregulated expression have been reported in various human cancers. Studies have shown that the genetic variants of E2F1 family members play an important role in head and neck carcinogenesis. In this study, we predicted six highly deleterious nsSNPs (C227F, R252H, V295D, C298Y, R56W, and Y59C) of E2F1 gene through in silico analyses. The latter was based on protein structure, function, and amino acid conservation. Molecular dynamics studies showed a deviation of the structures of the mutant proteins from the global protein parameters. Further, a case-control study that included total 535 samples (305 cancer patients and 230 controls) was conducted to find the association of the predicted SNPs with the susceptibility to lung cancer (LC) and head and neck cancer (HNC). The genotyping was done applying in-house artificial-RFLP method. Statistical analysis showed that the mutant alleles/genotypes of rs3213172 (R252H) were found to increase ∼ 2-5 fold risk of LC and HNC in all the genetic models. These results suggest that the rs3213172C/T polymorphism of the E2F1 gene could be used as an effective biomarker for genetic susceptibility to LC and HNC in our population.

Genome-Wide Mutagenesis of Hepatitis C Virus Reveals Ability of Genome To Overcome Detrimental Mutations.

To gain insight into the impact of mutations on the viability of the hepatitis C virus (HCV) genome, we created a set of full-genome mutant libraries, differing from the parent sequence as well as each other, by using a random mutagenesis approach; the proportion of mutations increased across these libraries with declining template amount or dATP concentration. The replication efficiencies of full-genome mutant libraries ranged between 71 and 329 focus-forming units (FFU) per 105 Huh7.5 cells. Mutant libraries with low proportions of mutations demonstrated low replication capabilities, whereas those with high proportions of mutations had their replication capabilities restored. Hepatoma cells transfected with selected mutant libraries, with low (4 mutations per 10,000 bp copied), moderate (33 mutations), and high (66 mutations) proportions of mutations, and their progeny were subjected to serial passage. Predominant virus variants (mutants) from these mutant libraries (Mutantl, Mutantm, and Mutanth, respectively) were evaluated for changes in growth kinetics and particle-to-FFU unit ratio, virus protein expression, and modulation of host cell protein synthesis. Mutantm and Mutantl variants produced >3.0-log-higher extracellular progeny per ml than the parent, and Mutanth produced progeny at a rate 1.0-log lower. More than 80% of the mutations were in a nonstructural part of the mutant genomes, the majority were nonsynonymous, and a moderate to large proportion were in the conserved regions. Our results suggest that the HCV genome has the ability to overcome lethal/deleterious mutations because of the high reproduction rate but highly selects for random, beneficial mutations.IMPORTANCE Hepatitis C virus (HCV) in vivo displays high genetic heterogeneity, which is partly due to the high reproduction and random substitutions during error-prone genome replication. It is difficult to introduce random substitutions in vitro because of limitations in inducing mutagenesis from the 5' end to the 3' end of the genome. Our study has overcome this limitation. We synthesized full-length genomes with few to several random mutations in the background of an HCV clone that can recapitulate all steps of the life cycle. Our study provides evidence of the capability of the HCV genome to overcome deleterious mutations and remain viable. Mutants that emerged from the libraries had diverse phenotype profiles compared to the parent, and putative adaptive mutations mapped to segments of the conserved nonstructural genome. We demonstrate the potential utility of our system for the study of sequence variation that ensures the survival and adaptation of HCV.

Random insertion transposon mutagenesis of Mycobacterium fortuitum identified mutant defective in biofilm formation.

Mycobacterium fortuitum has emerged as a nosocomial infectious agent and biofilm formation attributed for the presence of this bacterium in hospital environment. Transposon random mutagenesis was used to identify membrane-proteins for biofilm formation in M. fortuitum. Ten mutants were shortlisted from a library of 450 mutants for examine their biofilm forming ability. Comparative biofilm ability with respect to wild type M. fortuitum ATCC 6841 showed an altered and delayed biofilm formation in one mutant namely, MT721. Sequence analysis revealed mutation in anthranilate phosphoribosyl transferase (MftrpD), which is associated with tryptophan operon. Functional interaction study of TrpD protein through STRING showed its interaction with chorismate utilizing proteins, majorly involved in synthesis of aromatic amino acid and folic acid, suggesting that biofilm establishment and maintenance requires components of central metabolism. Our study indicates important role of MftrpD in establishment and maintenance of biofilm by M. fortuitum, which may further be explored for drug discovery studies against mycobacterial infections.

PreDSLpmo: A neural network-based prediction tool for functional annotation of lytic polysaccharide monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs), a family of copper-dependent oxidative enzymes, boost the degradation of polysaccharides such as cellulose, chitin, and others. While experimental methods are used to validate LPMO function, a computational method that can aid experimental methods and provide fast and accurate classification of sequences into LPMOs and its families would be an important step towards understanding the breadth of contributions these enzymes make in deconstruction of recalcitrant polysaccharides. In this study, we developed a machine learning-based tool called PreDSLpmo that employs two different approaches to functionally classify protein sequences into the major LPMO families (AA9 and AA10). The first approach uses a traditional neural network or multilayer percerptron-based approach, while the second employs bi-directional long short-term memory for sequence classification. Our method shows improvement in predictive power when compared with dbCAN2, an existing HMM-profile-based CAZyme predicting tool, on both validation and independent benchmark set.

Comprehensive evaluation of chitosan nanoparticle based phage lysin delivery system; a novel approach to counter S. pneumoniae infections.

Cpl-1, an endolysin derived from Cp-1 phage has been found to be effective in a number of in-vitro and in-vivo pneumococcal infection models. However its lower bioavailability under in-vivo conditions limits its applicability as therapeutic agent. In this study, Cpl-1 loaded chitosan nanoparticles were set up in order to develop a novel therapeutic delivery system to counter antibiotic resistant S. pneumoniae infections. Interactions of chitosan and Cpl-1 were studied by in-silico docking analysis. Chitosan nanoparticles and Cpl-1 loaded chitosan nanoparticles were prepared by using ionic gelation method and the process was optimized by varying chitosan:TPP ratio, pH, stirring time, stirring rate and Cpl-1 concentration. Chitosan nanoparticles and Cpl-1 loaded chitosan nanoparticles were characterized to ascertain successful formation of nanoparticles and entrapment of Cpl-1 into nanoparticles. Chitosan nanoparticles and Cpl-1 loaded nanoparticles were also evaluated for nanoparticle yield, entrapment efficiency, in-vitro release, stability, structural integrity of Cpl-1, in-vitro bioassay, swelling studies, in-vitro biodegradation and heamolysis studies. Mucoadhesion behavior of chitosan nanoparticles and Cpl-1 loaded nanoparticles was explored using mucous glycoprotein assay and ex-vivo mucoadhesion assay, both preparations exhibited their mucoadhesive nature. Cellular cytotoxicity and immune stimulation studies revealed biocompatible nature of nanoparticles. The results of this study confirm that chitosan nanoparticles are a promising biocompatible candidate for Cpl-1 delivery with a significant potential to increase bioavailability of enzyme that in turn can increase its in-vivo half life to treat S. pneumoniae infections.

Cinnamate-CoA ligase is involved in biosynthesis of benzoate-derived biphenyl phytoalexin in Malus × domestica 'Golden Delicious' cell cultures.

Apple (Malus sp.) and other genera belonging to the sub-tribe Malinae of the Rosaceae family produce unique benzoic acid-derived biphenyl phytoalexins. Cell cultures of Malus domestica cv. 'Golden Delicious' accumulate two biphenyl phytoalexins, aucuparin and noraucuparin, in response to the addition of a Venturia inaequalis elicitor (VIE). In this study, we isolated and expressed a cinnamate-CoA ligase (CNL)-encoding sequence from VIE-treated cell cultures of cv. 'Golden Delicious' (M. domestica CNL; MdCNL). MdCNL catalyses the conversion of cinnamic acid into cinnamoyl-CoA, which is subsequently converted to biphenyls. MdCNL failed to accept benzoic acid as a substrate. When scab-resistant (cv. 'Shireen') and moderately scab-susceptible (cv. 'Golden Delicious') apple cultivars were challenged with the V. inaequalis scab fungus, an increase in MdCNL transcript levels was observed in internodal regions. The increase in MdCNL transcript levels could conceivably correlate with the pattern of accumulation of biphenyls. The C-terminal signal in the MdCNL protein directed its N-terminal reporter fusion to peroxisomes in Nicotiana benthamiana leaves. Thus, this report records the cloning and characterisation of a cinnamoyl-CoA-forming enzyme from apple via a series of in vivo and in vitro studies. Defining the key step of phytoalexin formation in apple provides a biotechnological tool for engineering elite cultivars with improved resistance.

Amino Acid-Dependent Alterations in Cell Wall and Cell Morphology of Deinococcus indicus DR1.

Deinococcus radiodurans exhibits growth medium-dependent morphological variation in cell shape, but there is no evidence whether this phenomenon is observed in other members of the Deinococcaceae family. In this study, we isolated a red-pigmented, aerobic, Deinococcus indicus strain DR1 from Dadri wetland, India. This D. indicus strain exhibited cell-morphology transition from rod-shaped cells to multi-cell chains in a growth-medium-dependent fashion. In response to addition of 1% casamino acids in the minimal growth medium, rod-shaped cells formed multi-cell chains. Addition of all 20 amino acids to the minimal medium was able to recapitulate the phenotype. Specifically, a combination of L-methionine, L-lysine, L-aspartate, and L-threonine caused morphological alterations. The transition from rod shape to multi-cell chains is due to delay in daughter cell separation after cell division. Minimal medium supplemented with L-ornithine alone was able to cause cell morphology changes. Furthermore, a comparative UPLC analysis of PG fragments isolated from D. indicus cells propagated in different growth media revealed alterations in the PG composition. An increase in the overall cross-linkage of PG was observed in muropeptides from nutrient-rich TSB and NB media versus PYE medium. Overall our study highlights that environmental conditions influence PG composition and cell morphology in D. indicus.

Ribosomal maturation factor (RimP) is essential for survival of nontuberculous mycobacteria Mycobacterium fortuitum under in vitro acidic stress conditions.

Mycobacterium fortuitum is an important human pathogenic NTM, which resists stress conditions inside macrophages by exploitation of specific genes. TnphoA-based transposon mutagenesis was employed to identify membrane genes responsible for survival of M. fortuitum under such stress conditions. A library of about 450 mutants was constructed after electroporation of vector pRT291 into wild-type M. fortuitum. On the basis of blue color development and alkaline phosphatase assay, 20 mutants were shortlisted to screen for growth and survival under acidic stress at pH 6.5, 5.5, 4.5, and 3.5. Mutant MT727 showed reduced growth and survival under acidic stress. The acid susceptible mutant MT727 was subjected to other in vitro stress conditions prevalent inside macrophages including oxidative, nutrient starvation and nitrosative stress. However, the mutant showed no appreciable difference in growth behavior under oxidative, nutrient starvation and nitrosative stress conditions in comparison to the wild type. Genomic and bioinformatics analysis of MT727 led to identification of putative ribosomal maturation factor RimP of M. fortuitum to be affected by mutagenesis, showing closest homology to M. abscessus RimP. In silico functional interaction of RimP protein using STRING database showed its interaction with proteins of ribosomal assembly and maturation. Results indicate role of rimP gene in survival of M. fortuitum under acidic stress conditions which may be further explored for use as a potential drug target against M. fortuitum and other mycobacterial infections.