Uncovering the lignin-degrading potential of Serratia quinivorans AORB19: insights from genomic analyses and alkaline lignin degradation.

BACKGROUND: Lignin is an intricate phenolic polymer found in plant cell walls that has tremendous potential for being converted into value-added products with the possibility of significantly increasing the economics of bio-refineries. Although lignin in nature is bio-degradable, its biocatalytic conversion is challenging due to its stable complex structure and recalcitrance. In this context, an understanding of strain's genomics, enzymes, and degradation pathways can provide a solution for breaking down lignin to unlock the full potential of lignin as a dominant valuable bioresource. A gammaproteobacterial strain AORB19 has been isolated previously from decomposed wood based on its high laccase production. This work then focused on the detailed genomic and functional characterization of this strain based on whole genome sequencing, the identification of lignin degradation products, and the strain's laccase production capabilities on various agro-industrial residues. RESULTS: Lignin degrading bacterial strain AORB19 was identified as Serratia quinivorans based on whole genome sequencing and core genome phylogeny. The strain comprised a total of 123 annotated CAZyme genes, including ten cellulases, four hemicellulases, five predicted carbohydrate esterase genes, and eight lignin-degrading enzyme genes. Strain AORB19 was also found to possess genes associated with metabolic pathways such as the ß-ketoadipate, gentisate, anthranilate, homogentisic, and phenylacetate CoA pathways. LC-UV analysis demonstrated the presence of p-hydroxybenzaldehyde and vanillin in the culture media which constitutes potent biosignatures indicating the strain's capability to degrade lignin. Finally, the study evaluated the laccase production of Serratia AORB19 grown with various industrial raw materials, with the highest activity detected on flax seed meal (257.71 U/L), followed by pea hull (230.11 U/L), canola meal (209.56 U/L), okara (187.67 U/L), and barley malt sprouts (169.27 U/L). CONCLUSIONS: The whole genome analysis of Serratia quinivorans AORB19, elucidated a repertoire of genes, pathways and enzymes vital for lignin degradation that widens the understanding of ligninolytic metabolism among bacterial lignin degraders. The LC-UV analysis of the lignin degradation products coupled with the ability of S. quinivorans AORB19 to produce laccase on diverse agro-industrial residues underscores its versatility and its potential to contribute to the economic viability of bio-refineries.

Cisplatin and Starvation Differently Sensitize Autophagy in Renal Carcinoma: A Potential Therapeutic Pathway to Target Variegated Drugs Resistant Cancerous Cells.

Cisplatin, a powerful chemotherapy medication, has long been a cornerstone in the fight against cancer due to chemotherapeutic failure. The mechanism of cisplatin resistance/failure is a multifaceted and complex issue that consists mainly of apoptosis inhibition through autophagy sensitization. Currently, researchers are exploring ways to regulate autophagy in order to tip the balance in favor of effective chemotherapy. Based on this notion, the current study primarily identifies the differentially expressed genes (DEGs) in cisplatin-treated autophagic ACHN cells through the Illumina Hi-seq platform. A protein-protein interaction network was constructed using the STRING database and KEGG. GO classifiers were implicated to identify genes and their participating biological pathways. ClueGO, David, and MCODE detected ontological enrichment and sub-networking. The network topology was further examined using 12 different algorithms to identify top-ranked hub genes through the Cytoscape plugin Cytohubba to identify potential targets, which established profound drug efficacy under an autophagic environment. Considerable upregulation of genes related to autophagy and apoptosis suggests that autophagy boosts cisplatin efficacy in malignant ACHN cells with minimal harm to normal HEK-293 growth. Furthermore, the determination of cellular viability and apoptosis by AnnexinV/FITC-PI assay corroborates with in silico data, indicating the reliability of the bioinformatics method followed by qRT-PCR. Altogether, our data provide a clear molecular insight into drug efficacy under starved conditions to improve chemotherapy and will likely prompt more clinical trials on this aspect.

Validating potent anti-inflammatory and anti-rheumatoid properties of Drynaria quercifolia rhizome methanolic extract through in vitro, in vivo, in silico and GC-MS-based profiling.

BACKGROUND: The fronds of Drynaria quercifolia have traditionally been used in rheumatic pain management. The goal of the present study was to validate the potent anti-inflammatory and anti-rheumatoid properties of the methanolic-extract of its rhizome using in vitro, in vivo and in silico strategies. METHODS: The plant was collected and the methanolic extract was prepared from its rhizome. Protein denaturation test, hypotonicity and heat-induced haemolysis assays were performed in vitro. The in vivo anti-rheumatoid potential was assessed in Freund's complete adjuvant (FCA)-induced Wistar rat model through inflammatory paw-edema, haematological, biochemical, radiological and histopathological measurements. Moreover, metabolites of methanolic extract were screened by gas chromatography-mass spectrometry (GC-MS) and 3D molecular structures of active components were utilized for in silico docking study using AutoDock. RESULTS: In vitro results evinced a significant (p < 0.05) anti-inflammatory activity of the rhizome methanolic extract in a dose-linear response. Further, Drynaria quercifolia rhizome methanolic extract (DME) significantly ameliorated rheumatoid arthritis as indicated by the inhibition of arthritic paw-edema (in millimeter) in the rat rheumatoid arthritis models in both the low (57.71 ± 0.99, p < 0.01) and high dose groups (54.45 ± 1.30, p < 0.001) when compared to arthritic control. Treatment with DME also normalized the haematological (RBC, WBC, platelet counts and hemoglobin contents) and biochemical parameters (total protein, albumin, creatinine and ceruloplasmin) significantly (p < 0.05), which were further supported by histopathological and radiological analyses. Furthermore, GC-MS analysis of DME demonstrated the presence of 47 phytochemical compounds. Compounds like Squalene, Gamma Tocopherol, n-Hexadecanoic acid showed potent inhibition of cyclooxygenase-2 (COX-2), tumor necrosis factor (TNF-α), and interleukin (IL-6) in the docking analysis. CONCLUSION: Results from in vivo and in vitro studies indicated that DME possesses a potent anti-inflammatory and anti-arthritic activity. In silico studies delineated the emergent potent inhibitory effects of several bio-active components on the target inflammatory markers (COX-2, TNF-α and IL-6).

Pandrug-resistant Pseudomonas sp. expresses New Delhi metallo-ß-lactamase-1 and consumes ampicillin as sole carbon source.

OBJECTIVES: This study aims to investigate ampicillin catabolism in a pandrug-resistant strain, Pseudomonas sp. MR 02 of P. putida lineage. METHODS: The characterization of carbapenem resistance was done following the standard protocol. The broth macrodilution method was used to determine the MIC values of antimicrobial agents both in the presence and in the absence of phenylalanine-ß-naphthylamide. High MIC values (>10 000 mg/L) of ampicillin led to speculation that it may serve as a growth substrate, and thus minimal medium was used to evaluate ampicillin as a nutrient. The growth of MR 02 was measured in minimal medium in the presence or absence of 0.4 mM EDTA, supplemented with ampicillin as sole carbon, nitrogen and energy source. RNA-seq was used to generate expression profiles of genes in ampicillin or glucose-grown cells. The blaNDM-1 gene of MR 02 was cloned in the pHSG398 vector and expressed in Escherichia coli DH5α. RESULTS: Phenotypic analysis along with genome sequence data identifies Pseudomonas sp. MR 02 as a pandrug-resistant strain. Transcriptome data has revealed that blaNDM-1 was among the top 50 differentially expressed genes in ampicillin grown cells compared to the glucose grown cells in the minimal medium. Heterologous expression of blaNDM-1 gene in E. coli DH5α enabled its growth and subsistence on ampicillin as the sole source of carbon and energy. DISCUSSION: The ability of a pandrug-resistant Pseudomonas sp. MR 02 to consume ampicillin for growth has a huge implication in the bioremediation of ß-lactam residues in the environment.

Pradoshia eiseniae gen. nov., sp. nov., a spore-forming member of the family Bacillaceae capable of assimilating 3-nitropropionic acid, isolated from the anterior gut of the earthworm Eisenia fetida.

A Gram-stain-positive, spore-forming bacterium, EAG3T, capable of growing on 3-nitropropionic acid as the sole source of carbon, nitrogen and energy, was isolated from the anterior gut of an earthworm (Eisenia fetida) reared at the Centre of Floriculture and Agribusiness Management of the University of North Bengal at Siliguri (26.7072° N, 88.3558° E), West Bengal, India. The DNA G+C content of strain EAG3T was 42.5 mol%. Strain EAG3T contained MK-7 and MK-8 as predominant menaquinones. The predominant polar lipids were phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine. The major cellular fatty acids were 13-methyltetradecanoic acid, (9Z)-9-hexadecen-1-ol, 12-methyltetradecanoic acid and 14-methylpentadecanoic acid. The draft genome of strain EAG3T, distributed in 57 contigs, was found to be 3.8 Mb. A total of 3811 potential coding sequences or genes were predicted, including 3672 protein-coding and 108 RNA-coding ones together with 31 pseudogenes. One hundred and thirty-five genes encoded hypothetical proteins with no meaningful homologies with known proteins. The EAG3T genome encompassed two nitronate monooxygenase and one methylmalonate-semialdehyde dehydrogenase (CoA acylating) homologues. 16S rRNA gene sequence-based phylogeny revealed that the closest relative of strain EAG3T was Bacillus methanolicus NCIMB 13113T (95.7 % similarity). Phylogenetic, physiological and biochemical characteristics differentiated strain EAG3T from B. methanolicus, as well as from the other close taxonomic relatives Planococcus rifietoensis M8T, Bhargavaea cecembensis DSE10T, Planomicrobium flavidum ISL-41Tand Fermentibacilluspolygoni IEB3T, with which strain EAG3T had 93.3-94.2 % 16S rRNA gene sequence similarities. The new isolate, therefore, was considered as representing a novel genus of family Bacillaceae, for which the name Pradoshia eiseniae gen. nov., sp. nov. is proposed, with EAG3T (=LMG 30312T=JCM 32460T) as the type strain.

Functional divergence outlines the evolution of novel protein function in NifH/BchL protein family.

Biological nitrogen fixation is accomplished by prokaryotes through the catalytic action of complex metalloenzyme, nitrogenase. Nitrogenase is a two-protein component system comprising MoFe protein (NifD and K) and Fe protein (NifH). NifH shares structural and mechanistic similarities as well as evolutionary relationships with light-independent protochlorophyllide reductase (BchL), a photosynthesis-related metalloenzyme belonging to the same protein family. We performed a comprehensive bioinformatics analysis of the NifH/BchL family in order to elucidate the intrinsic functional diversity and the underlying evolutionary mechanism among the members. To analyse functional divergence in the NifH/ BchL family, we have conducted pair-wise estimation in altered evolutionary rates between the member proteins. We identified a number of vital amino acid sites which contribute to predicted functional diversity. We have also made use of the maximum likelihood tests for detection of positive selection at the amino acid level followed by the structure-based phylogenetic approach to draw conclusion on the ancient lineage and novel characterization of the NifH/BchL protein family. Our investigation provides ample support to the fact that NifH protein and BchL share robust structural similarities and have probably deviated from a common ancestor followed by divergence in functional properties possibly due to gene duplication.

Characterization of haemoglobin from actinorhizal plants--an in silico approach.

Plant haemoglobins (Hbs), found in both symbiotic and non-symbiotic plants, are heme proteins and members of the globin superfamily. Hb genes of actinorhizal Fagales mostly belong to the non-symbiotic type of haemoglobin; however, along with the non-symbiotic Hb, Casuarina sp. posses a symbiotic one (symCgHb), which is expressed specifically in infected cells of nodules. A thorough sequence analysis of 26 plant Hb proteins, currently available in public domain, revealed a consensus motif of 29 amino acids. This motif is present in all the members of symbiotic class II Hbs including symCgHb and non-symbiotic Class II Hbs, but is totally absent in Class I symbiotic and non-symbiotic Hbs. Further, we constructed 3D structures of Hb proteins from Alnus and Casuarina through homology modelling and peeped into their structural properties. Structure-based studies revealed that the Casuarina symbiotic haemoglobin protein shows distinct stereochemical properties from that of the other Casuarina and Alnus Hb proteins. It also showed considerable structural similarities with leghemoglobin structure from yellow lupin (pdb id 1GDI). Therefore, sequence and structure analyses point to the fact that symCgHb protein shows significant resemblance to symbiotic haemoglobin found in legumes and may thus eventually play a similar role in shielding the nitrogenase from oxygen as seen in the case of leghemoglobin.

Draft Genome Sequence of Frankia sp. Strain BMG5.12, a Nitrogen-Fixing Actinobacterium Isolated from Tunisian Soils.

Members of the actinomycete genus Frankia form a nitrogen-fixing symbiosis with 8 different families of actinorhizal plants. We report a draft genome sequence for Frankia sp. strain BMG5.12, a nitrogen-fixing actinobacterium isolated from Tunisian soils with the ability to infect Elaeagnus angustifolia and Myrica gale.

Draft Genome Sequence of Frankia sp. Strain BCU110501, a Nitrogen-Fixing Actinobacterium Isolated from Nodules of Discaria trinevis.

Frankia forms a nitrogen-fixing symbiosis with actinorhizal plants. We report a draft genome sequence for Frankia sp. strain BCU110501, a nitrogen-fixing actinobacterium isolated from nodules of Discaria trinevis grown in the Patagonia region of Argentina.

Contrasted evolutionary constraints on secreted and non-secreted proteomes of selected Actinobacteria.

BACKGROUND: Actinobacteria have adapted to contrasted ecological niches such as the soil, and among others to plants or animals as pathogens or symbionts. Mycobacterium genus contains mostly pathogens that cause a variety of mammalian diseases, among which the well-known leprosy and tuberculosis, it also has saprophytic relatives. Streptomyces genus is mostly a soil microbe known for its secondary metabolites, it contains also plant pathogens, animal pathogens and symbionts. Frankia, a nitrogen-fixing actinobacterium establishes a root symbiosis with dicotyledonous pionneer plants. Pathogens and symbionts live inside eukaryotic cells and tissues and interact with their cellular environment through secreted proteins and effectors transported through transmembrane systems; nevertheless they also need to avoid triggering host defense reactions. A comparative genome analysis of the secretomes of symbionts and pathogens allows a thorough investigation of selective pressures shaping their evolution. In the present study, the rates of silent mutations to non-silent mutations in secretory proteins were assessed in different strains of Frankia, Streptomyces and Mycobacterium, of which several genomes have recently become publicly available. RESULTS: It was found that secreted proteins as a whole have a stronger purifying evolutionary rate (non-synonymous to synonymous substitutions or Ka/Ks ratio) than the non-secretory proteins in most of the studied genomes. This difference becomes statistically significant in cases involving obligate symbionts and pathogens. Amongst the Frankia, secretomes of symbiotic strains were found to have undergone evolutionary trends different from those of the mainly saprophytic strains. Even within the secretory proteins, the signal peptide part has a higher Ka/Ks ratio than the mature part. Two contrasting trends were noticed amongst the Frankia genomes regarding the relation between selection strength (i.e. Ka/Ks ratio) and the codon adaptation index (CAI), a predictor of the expression rate, in all the genes belonging to the core genome as well as the core secretory protein genes. The genomes of pathogenic Mycobacterium and Streptomyces also had reduced secretomes relative to saprophytes, as well as in general significant pairwise Ka/Ks ratios in their secretomes. CONCLUSION: In marginally free-living facultative symbionts or pathogenic organisms under consideration, secretory protein genes as a whole evolve at a faster rate than the rest and this process may be an adaptive life-strategy to counter the host selection pressure. The higher evolutionary rate of signal peptide part compared to mature protein provides an indication that signal peptide parts may be under relaxed purifying selection, indicative of the signal peptides not being secreted into host cells. Codon usage analysis suggests that in actinobacterial strains under host selection pressure such as symbiotic Frankia, ACN, FD and the pathogenic Mycobacterium, codon usage bias was negatively correlated to the selective pressure exerted on the secretory protein genes.

Draft genome sequence of Frankia sp. strain CN3, an atypical, noninfective (Nod-) ineffective (Fix-) isolate from Coriaria nepalensis.

We report here the genome sequence of Frankia sp. strain CN3, which was isolated from Coriaria nepalensis. This genome sequence is the first from the fourth lineage of Frankia, strains of which are unable to reinfect actinorhizal plants. At 10 Mb, it represents the largest Frankia genome sequenced to date.

Draft genome sequence of Frankia sp. strain QA3, a nitrogen-fixing actinobacterium isolated from the root nodule of Alnus nitida.

Members of the actinomycete genus Frankia form a nitrogen-fixing symbiosis with 8 different families of actinorhizal plants. We report a high-quality draft genome sequence for Frankia sp. strain QA3, a nitrogen-fixing actinobacterium isolated from root nodules of Alnus nitida.

Identification of TTA codon containing genes in Frankia and exploration of the role of tRNA in regulating these genes.

The TTA codon, one of the six available codons for the amino acid leucine, is the rarest codon among the high GC genomes of Actinobacteria including Frankia. This codon has been implicated in various regulatory mechanisms involving secondary metabolism and morphological development. TTA-mediated gene regulation is well documented in Streptomyces coelicolor, but that role has not been investigated in other Actinobacteria including Frankia. Among the various Actinomycetes with a GC content of more than 70%, Frankia genomes had the highest percentages of TTA-containing genes ranging from 5.2 to 10.68% of the genome. In contrast, TTA-bearing genes comprised 1.7, 3.4 and 4.1% of the Streptomyces coelicolor, S. avermitilis and Nocardia farcinia genomes, respectively. We analyzed their functional role, evolutionary significance, horizontal acquisition and the codon-anticodon interaction. The TTA-bearing genes were found to be well represented in metabolic genes involved in amino acid transport and secondary metabolism. A reciprocal Blast search reveal that many of the TTA-bearing genes have orthologs in the other Frankia genomes, and some of these orthologous genes also have a TTA codon in them. The gene expression level of TTA-containing genes was estimated by the use of the codon adaption index (CAI), and the CAI values were found to have a positive correlation with the GC3 (GC content at the 3rd codon position). A full-atomic 3D model of the leucine tRNA recognizing the TTA (UUA) codon was generated and utilized for in silico docking to determine binding affinity in codon-anticodon interaction. We found a proficient codon-anticodon interaction for this codon which is perhaps why so many genes hold on to this rare codon without compromising their translational efficiency.