Enhancement of anti-cancer compounds in fungal elicited-Oldenlandia umbellata culture.

Our study focused on enhancing the production of anthraquinone derivatives in Oldenlandia umbellata using fungal elicitors. Aspergillus niger, Mucor prayagensis, and Trichoderma viride were used to elicit the anthraquinone derivatives in root cultures. The elicitation process led to an increase in the production of phytochemicals and secondary metabolites, with the highest total protein content observed in A. niger-elicited plants. We performed qualitative and quantitative phytochemical screening of the 80% methanol extract of the plants. Using reverse phase-ultra-fast liquid chromatography, we identified and quantified five anthraquinone compounds: aloe-emodin, rhein, emodin, chrysophanol, and alizarin. The in vitro root samples elicited with A. niger and M. prayagensis exhibited four and three anthraquinone derivatives, respectively, whereas those elicited with T. viride showed only two derivatives. Interestingly, chrysophanol content was the highest in A. niger-elicited root samples. We constructed a system pharmacology framework consisting of 40 nodes and 45 edges with 34 interacting genes. We also identified human proteins that interact with these derivatives, and inferred their roles in cancer-associated pathways. These anthraquinone derivatives interact with various proteins in multiple pathways, including apoptosis, human cytomegalovirus infection, proteoglycans in cancer, MAPK signaling, and hepatitis C, highlighting their potential therapeutic applications in cancer treatment.

Discovery of novel virulence mechanisms in Clostridium botulinum type A3 using genome-wide analysis.

OBJECTIVE: Clostridium botulinum type A is a neurotoxin-producing, spore-forming anaerobic bacterium that causes botulism in humans. The evolutionary genomic context of this organism is not yet known to understand its molecular virulence mechanisms in the human intestinal tract. Hence, this study aimed to investigate the mechanisms underlying virulence and pathogenesis by comparing the genomic contexts across species, serotypes, and subtypes. METHODS: A comparative genomic approach was used to analyze evolutionary genomic relationships, intergenomic distances, syntenic blocks, replication origins, and gene abundance with phylogenomic neighbors. RESULTS: Type A strains have shown genomic proximity to group I strains with distinct accessory genes and vary even within subtypes. Phylogenomic data showed that type C and D strains were distantly related to a group I and group II strains. Synthetic plots indicated that orthologous genes might have evolved from Clostridial ancestry to subtype A3 strains, whereas syntonic out-paralogs might have emerged between subtypes A3 and A1 through α-events. Gene abundance analysis revealed the key roles of genes involved in biofilm formation, cell-cell communication, human diseases, and drug resistance compared to the pathogenic Clostridia. Moreover, we identified 43 unique genes in the type A3 genome, of which 29 were involved in the pathophysiological processes and other genes contributed to amino acid metabolism. The C. botulinum type A3 genome contains 14 new virulence proteins that can provide the ability to confer antibiotic resistance, virulence exertion and adherence to host cells, the host immune system, and mobility of extrachromosomal genetic elements. CONCLUSION: The results of our study provide insight into the understanding of new virulence mechanisms to discover new therapeutics for the treatment of human diseases caused by type A3 strains.

Comparative genomic analysis of hyper-ammonia producing Acetoanaerobium sticklandii DSM 519 with purinolytic Gottschalkia acidurici 9a and pathogenic Peptoclostridium difficile 630.

Acetoanaerobium sticklandii DSM519 (CST) is a hype-ammonia producing non-pathogenic anaerobe that can use amino acids as important carbon and energy sources through the Stickland reactions. Biochemical aspects of this organism have been extensively studied, but systematic studies addressing its metabolic discrepancy remain scant. In this perspective, we have intensively analyzed its genomic and metabolic characteristics to comprehend the evolutionary conservation of amino acid catabolism by a comparative genomic approach. The whole-genome data indicated that CST has shown a phylogenomic similarity with hyper-ammonia producing, purinolytic, and proteolytic pathogenic Clostridia. CST has shown to common genomic context sharing across the purinolytic Gottschalkia acidurici 9a and pathogenic Peptoclostridium difficile 630. Genome syntenic analysis described that syntenic orthologs might be originated from the recent ancestor at a slow evolution rate and syntenic-out paralogs evolved from either CDF or CAC via α-event and ß-event. Collinearity of either gene orders or gene families was adjusted with syntenic out-paralogs across these genomes. The genome-wide metabolic analysis predicted 11 unique putative metabolic subsystems from the CST genome for amino acid catabolism and hydrogen production. The in silico analysis of our study revealed that a characteristic system for amino acid catabolism-directed biofuel synthesis might have slowly evolved and established as a core genomic content of CST.

A hijack mechanism of Indian SARS-CoV-2 isolates for relapsing contemporary antiviral therapeutics.

Coronavirus disease (COVID-19) rapidly expands to a global pandemic and its impact on public health varies from country to country. It is caused by a new virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It is imperative for relapsing current antiviral therapeutics owing to randomized genetic drift in global SARS-CoV-2 isolates. A molecular mechanism behind the emerging genomic variants is not yet understood for the prioritization of selective antivirals. The present computational study was aimed to repurpose existing antivirals for Indian SARS-CoV-2 isolates by uncovering a hijack mechanism based on structural and functional characteristics of protein variants. Forty-one protein mutations were identified in 12 Indian SARS-CoV-2 isolates by analysis of genome variations across 460 genome sequences obtained from 30 geographic sites in India. Two unique mutations such as W6152R and N5928H found in exonuclease of Surat (GBRC275b) and Gandhinagar (GBRC239) isolates. We report for the first time the impact of folding rate on stabilizing/retaining a sequence-structure-function-virulence link of emerging protein variants leading to accommodate hijack ability from current antivirals. Binding affinity analysis revealed the effect of point mutations on virus infectivity and the drug-escaping efficiency of Indian isolates. Emodin and artinemol suggested herein as repurposable antivirals for the treatment of COVID-19 patients infected with Indian isolates. Our study concludes that a protein folding rate is a key structural and evolutionary determinant to enhance the receptor-binding specificity and ensure hijack ability from the prevalent antiviral therapeutics.

Genomics insights of SARS-CoV-2 (COVID-19) into target-based drug discovery.

Coronavirus disease (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 is a global health emergency and no clinically approved vaccines or antiviral drugs available to date. Intensive research on SARS-CoV-2 is urgently warranted to understand its pathogenesis and virulence mechanisms and to discover target-based antiviral therapeutics. Among various research logics, current bioinformatics highlights novel testable hypotheses for systematic drug repositioning and designing against COVID-19. A total of 121 articles related to bioinformatics facets of this virus were collected from the PubMed Central. The content of each investigation was comprehensively reviewed, manually curated, and included herein. Interestingly, 109 COVID-19-related literature published in 2020 (January-June) were included in this review. The present article emphasizes novel resource development on its genome structure, evolution, therapeutic targets, drug designing, and drug repurposing strategies. Genome organization, the function of coding genes, origin, and evolution of SARS-CoV-2 is described in detail. Genomic insights into understanding the structure-function relationships of drug targets including spike, main protease, and RNA-dependent RNA polymerase of SARS-CoV-2 are discussed intensively. Several molecular docking and systems pharmacology approaches have been investigated some promising antiviral drugs against SARS-CoV-2 based on its genomic characteristics, pathogenesis mechanism, and host specificity. Perhaps, the present genomic insights of this virus will provide a lead to the researchers to design or repurpose of antiviral drugs soon and future directions to control the spread of COVID-19.

Comparative genomic analysis reveals starvation survival systems in Methanothermobacter thermautotrophicus ΔH.

Methanothermobacter thermautotrophicus ΔH (MTH) is a thermophilic hydrogenotrophic methanogenic archaeon capable of reducing CO2 with H2 to produce methane gas. It is the potential candidate in the biomethanation of CO2 and CO in anaerobic reactors and biogas upgrading process. However, systematic studies addressing its genome conservation and function remain scant in this genome. In this study, we have evaluated its evolutionary resemblance and metabolic discrepancy, particularly in starvation survival systems by comparing the genomic contexts with Methanothermobacter marburgensis str. Marburg (MMG) and Methanobacterium formicicum DSM 1535 (MFO). The phylogenomic analysis of this study indicated that there was a strong phylogenomic signal among MTH, MMG, and MFO in the whole-genome tree. DNA replication machinery was conserved in the MTH genome and might have evolved at different evolution rates. Genome synteny analysis observed collinearity of either gene orders or gene families has to be maintained with syntenic blocks located in the syntenic out-paralogs. A genome-wide metabolic analysis identified some unique putative metabolic subsystems in MTH, which are proposed to determine its growth characteristics in diverse environments. MTH genome comprised of 93 unique genes-coding for starvation survival and stress-response proteins. These proteins confer its adaptation to nutritional deprivation and other abiotic stresses. MTH has a typical system to withstand its growth and cell viability during stable operation and recovery after prolonged starvation. Thus, the present work will provide an insight to improve the genome refinement and metabolic reconstruction in parallel to other closely related species.

Functional Prediction and Assignment of Methanobrevibacter ruminantium M1 Operome Using a Combined Bioinformatics Approach.

Methanobrevibacter ruminantium M1 (MRU) is a rod-shaped rumen methanogen with the ability to use H2 and CO2, and formate as substrates for methane formation in the ruminants. Enteric methane emitted from this organism can also be influential to the loss of dietary energy in ruminants and humans. To date, there is no successful technology to reduce methane due to a lack of knowledge on its molecular machinery and 73% conserved hypothetical proteins (HPs; operome) whose functions are still not ascertained perceptively. To address this issue, we have predicted and assigned a precise function to HPs and categorize them as metabolic enzymes, binding proteins, and transport proteins using a combined bioinformatics approach. The results of our study show that 257 (34%) HPs have well-defined functions and contributed essential roles in its growth physiology and host adaptation. The genome-neighborhood analysis identified 6 operon-like clusters such as hsp, TRAM, dsr, cbs and cas, which are responsible for protein folding, sudden heat-shock, host defense, and protection against the toxicities in the rumen. The functions predicted from MRU operome comprised of 96 metabolic enzymes with 17 metabolic subsystems, 31 transcriptional regulators, 23 transport, and 11 binding proteins. Functional annotation of its operome is thus more imperative to unravel the molecular and cellular machinery at the systems-level. The functional assignment of its operome would advance strategies to develop new anti-methanogenic targets to mitigate methane production. Hence, our approach provides new insight into the understanding of its growth physiology and lifestyle in the ruminants and also to reduce anthropogenic greenhouse gas emissions worldwide.

Functional prediction, characterization, and categorization of operome from Acetoanaerobium sticklandii DSM 519.

Acetoanaerobium sticklandii DSM 519 is a hyper-ammonia producing anaerobic bacterium that can be able utilizes amino acids as sole carbon and energy sources for its growth and energetic metabolism. A lack of knowledge on its molecular machinery and 30.5% conserved hypothetical proteins (HPs; operome) hinders the successful utility in biofuel applications. In this study, we have predicted, characterized and categorized its operome whose functions are still not determined accurately using a combined bioinformatics approach. The functions of 64 of the 359 predicted HPs are involved in diverse metabolic subsystems. A. sticklandii operome has consisted of 16% Rossmann fold and 46% miscellaneous folds. Subsystems-based technology has classified 51 HPs contributing to the small-molecular reactions, 26 in macromolecular reactions and 12 in the biosynthesis of cofactors, prosthetic groups and electron carriers. A generality of functions predicted from its operome contributed to the cell cycle, amino acid metabolism, membrane transport, and regulatory processes. Many of them have duplicated functions as paralogs in this genome. A. sticklandii has the ability to compete with invading microorganisms and tolerate abiotic stresses, which can be overwhelmed by the predicted functions of its operome. Results of this study revealed that it has specialized systems for amino acid catabolism-directed solventogenesis and acidogenesis but the level of gene expression may determine the metabolic function in amino acid fermenting niches in the rumina of cattle. As shown by our analysis, the predicted functions of its operome allow us for a better understanding of the growth and physiology at systems-scale.

Deciphering Molecular Virulence Mechanism of Mycobacterium tuberculosis Dop isopeptidase Based on Its Sequence-Structure-Function Linkage.

The pupylation pathway marks proteins for prokaryotic ubiquitin-like protein (Pup)-proteasomal degradation and survival strategy of mycobacteria inside of the host macrophages. Deamidase of Pup (Dop) plays a central role in the pupylation pathway. It is still a matter of investigation to know the function of Dop in virulence of mycobacterial lineage. Hence, the present study was intended to describe the sequence-structure-function-virulence link of Dop for understanding the molecular virulence mechanism of Mycobacterium tuberculosis H37Rv (Mtb). Phylogenetic analysis of this study indicated that Dop has extensively diverged across the proteasome-harboring bacteria. The functional part of Dop was converged across the pathogenic mycobacterial lineage. The genome-wide analysis pointed out that the pupylation gene locus was identical to each other, but its genome neighborhood differed from species to species. Molecular modeling and dynamic studies proved that the predicted structure of Mtb Dop was energetically stable and low conformational freedom. Moreover, evolutionary constraints in Mtb Dop were intensively analyzed for inferring its sequence-structure-function relationships for the full virulence of Mtb. It indicated that evolutionary optimization was extensively required to stabilize its local structural environment at the side chains of mutable residues. The sequence-structure-function-virulence link of Dop might have retained in Mtb by reordering hydrophobic and hydrogen bonding patterns in the local structural environment. Thus, the results of our study provide a quest to understand the molecular virulence and pathogenesis mechanisms of Mtb during the infection process.

A human-food web-animal interface on the prevalence of food-borne pathogens (Clostridia and Enterococcus) in mixed veterinary farms.

In the present work, we addressed the impact of a human-food web-animal interface on the prevalence of food-borne pathogens in mixed farms of Tamil Nadu, India. We have isolated and identified six strains of Clostridium sp. and five strains of Enterococcus sp. from food and animal sources disposed near to the veterinary and poultry farms. Phylogenetic relationships of these strains were inferred from their homologies in 16S rDNA sequences and rRNA secondary structures. The strain PCP07 was taxonomically equivalent to C. botulinum confirmed by neurotoxin-specific PCR primers, followed by mouse bioassay. Other Clostridial and Enterococcal isolates have shown a phylogenetic similarity to the C. bifermentans and E. durans isolated from veterinary farms, respectively. Results of our study revealed that a human-food web-animal interface has influenced the disease incidence and prevalence of these isolates in the poultry to veterinary farms, where human food acted as a likely transmittance vehicle for their infections.

A metabolic study to decipher amino acid catabolism-directed biofuel synthesis in Acetoanaerobium sticklandii DSM 519.

Acetoanaerobium sticklandii DSM 519 is a hyper-ammonia-producing anaerobe. It has the ability to produce organic solvents and acids from protein catabolism through Stickland reactions and specialized pathways. Nevertheless, its protein catabolism-directed biofuel production has not yet been understood. The present study aimed to decipher such growth-associated metabolic potential of this organism at different growth phases using metabolic profiling. A seed culture of this organism was grown separately in metabolic assay media supplemented with gelatin and or a mixture of amino acids. The extracellular metabolites produced by this organism were qualitatively analyzed by gas chromatography-mass spectrometry platform. The residual amino acids after protein degradation and amino acids assimilation were identified and quantitatively measured by high-performance liquid chromatography (HPLC). Organic solvents and acids produced by this organism were detected and the quantity of them determined with HPLC. Metabolic profiling data confirmed the presence of amino acid catabolic products including tyramine, cadaverine, methylamine, and putrescine in fermented broth. It also found products including short-chain fatty acids and organic solvents of the Stickland reactions. It reported that amino acids were more appropriate for its growth yield compared to gelatin. Results of quantitative analysis of amino acids indicated that many amino acids either from gelatin or amino acid mixture were catabolised at a log-growth phase. Glycine and proline were poorly consumed in all growth phases. This study revealed that apart from Stickland reactions, a specialized system was established in A. sticklandii for protein catabolism-directed biofuel production. Acetone-butanol-ethanol (ABE), acetic acid, and butyric acid were the most important biofuel components produced by this organism. The production of these components was achieved much more on gelatin than amino acids. Thus, A. sticklandii is suggested herein as a potential organism to produce butyric acid along with ABE from protein-based wastes (gelatin) in bio-energy sectors.

Substrate-imprinted docking of Agrobacterium tumefaciens uronate dehydrogenase for increased substrate selectivity.

Agrobacterium tumefaciens uronate dehydrogenase (AtuUdh) belongs to the short-chain dehydrogenase superfamily, specifically those acting on the CH-OH group of donor with NAD+ or NADP+ as acceptor. It is apparently required for the production of D-glucaric acid. AtuUdh-catalyzed reaction is reversible with dual substrate-specific activity (D-galacturonic acid and D-glucuronic acid) in nature. In our study, 34 mutants were pre-screened from 155 mutants generated from AtuUdh (wild-type) and selected 10 structurally stable mutants with increased substrate selectivity. The specificity, efficiency, and selectivity of these mutants for different substrates and cofactors were predicted from 121 docked models using a substrate-imprinted docking approach. Q14F, S36L, and S75T mutants have shown a high binding affinity to D-glucuronic acid and its substrate intermediates such as D-glucaro-1,4-lactone and D-glucaro-1,5-lactone. These mutants exhibited a low binding affinity to the substrate and cofactor required for D-galactaric acid. D34S, N112E and S165E mutants found to show a high selectivity of D-galacturonic acid and its substrate intermediates for D-galactaric acid production. Ser75, Ser165, and Arg174 are active residues playing an imperative role in the substrate selectivity and also contributed in the conjecture the mechanism of transition state stabilization catalyzed by AtuUdh mutants. The present approach was used to reveal the substrate binding mechanism of AtuUdh mutants for a better understanding of the structural basis for selectivity and function.

Cloning and expression of immunogenic Clostridium botulinum C2I mutant proteins designed from their evolutionary imprints.

C2 toxin produced from Clostridium botulinum serotypes C and D has a potential role in many pathophysiological mechanisms in birds and animals. It has encompassed an ADP ribosyltransferase subunit (C2I) and a translocation/binding subunit (C2II). In the present study, we intended to produce C2I mutant proteins as recombinant subunit vaccines by using glutathione-S-transferase-gene fusion system. The mutants of this study were previously evaluated from their evolutionary imprints and suggested as suitable candidates for subunit vaccines. A synthetic C2 gene was inserted in a pGEX-2T vector, cloned and expressed in Escherichia coli BL21 host. The expressed mutant proteins were purified by using glutathione-agarose column and then examined for their ADP ribosyltransferase activity and vaccinogenic characteristics. The pGEX-2T-C2I constructs with Y298F, S347A and S350A substitutions have shown effective transformation efficiencies in E. coli XL10 competent cells but their mutagenesis efficiency was relatively low. Gene expression analysis indicated the rate of gene expression was depended on the fused mutant genes. A high-level expression was achieved for Y298F, S347A and S350A mutant proteins. All purified protein exhibited a molecular mass of 49 kDa. C2I mutant proteins exhibited a reduced ADP ribosyltransferase activity with retained immunogenic and vaccinogenic characteristics compared to the wild-type C2I subunit. The overall analysis of our study suggested the recombinant C2I proteins (S197A and Y298F) are the most promising candidates for the development of subunit vaccine or immunogen for C2 mutants mediated diseases in birds and animals.

Kinetic modeling of Stickland reactions-coupled methanogenesis for a methanogenic culture.

Studying amino acid catabolism-coupled methanogenesis is the important standpoints to decipher the metabolic behavior of a methanogenic culture. L-Glycine and L-alanine are acted as sole carbon and nitrogen sources for acidogenic bacteria. One amino acid is oxidized and another one is reduced for acetate production via pyruvate by oxidative deamination process in the Stickland reactions. Herein, we have developed a kinetic model for the Stickland reactions-coupled methanogenesis (SRCM) and simulated objectively to maximize the rate of methane production. We collected the metabolic information from enzyme kinetic parameters for amino acid catabolism of Clostridium acetobutylicum ATCC 824 and methanogenesis of Methanosarcina acetivorans C2A. The SRCM model of this study consisted of 18 reactions and 61 metabolites with enzyme kinetic parameters derived experimental data. The internal or external metabolic flux rate of this system found to control the acidogenesis and methanogenesis in a methanogenic culture. Using the SRCM model, flux distributions were calculated for each reaction and metabolite in order to maximize the methane production rate from the glycine-alanine pair. Results of this study, we demonstrated the metabolic behavior, metabolite pairing while mutually interact, and advantages of syntrophic metabolism of amino acid-directed methane production in a methanogenic starter culture.

Deciphering structure, function and mechanism of Plasmodium IspD homologs from their evolutionary imprints.

Malaria is a life-threatening mosquito-borne blood disease caused by infection with Plasmodium parasites. Anti-malarial drug resistance is a global threat to control and eliminate malaria and therefore, it is very important to discover and evaluate new drug targets. The 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD) homolog is a second in vivo target for fosmidomycin within isoprenoid biosynthesis in malarial parasites. In the present study, we have deciphered the sequence-structure-function integrity of IspD homologs based on their evolutionary imprints. The function and catalytic mechanism of them were also intensively studied by using sequence-structure homology, molecular modeling, and docking approach. Results of our study indicated that substrate-binding and dimer interface motifs in their structures were extensively conserved and part of them closely related to eubacterial origins. Amino acid substitutions in their coiled-coil regions found to bring a radical change in secondary structural elements, which in turn may change the local structural environment. Arg or Asp was identified as a catalytic site in plasmodium IspD homologs, contributing a direct role in the cytidylyltransferase activity similar to bacterial IspD. Results of molecular docking studies demonstrated how anti-malarial drugs such as fosmidomycin and FR-900098 have competitively interacted with the substrate-binding site of these homologs. As shown by our analysis, species-specific evolutionary imprints in these homologs determine the sequence-structure-function-virulence integrity and binding site alterations in order to confer anti-malarial drug resistance.

Functional annotation of operome from Methanothermobacter thermautotrophicus ΔH: An insight to metabolic gap filling.

Methanothermobacter thermautotrophicus ΔH (MTH) is a potential methanogen known to reduce CO2 with H2 for producing methane biofuel in thermophilic digesters. The genome of this organism contains ~50.5% conserved hypothetical proteins (HPs; operome) whose function is still not determined precisely. Here, we employed a combined bioinformatics approach to annotate a precise function to HPs and categorize them as enzymes, binding proteins, and transport proteins. Results of our study show that 315 (35.6%) HPs have exhibited well-defined functions contributing imperative roles in diverse cellular metabolism. Some of them are responsible for stress-response mechanisms and cell cycle, membrane transport, and regulatory processes. The genome-neighborhood analysis found five important gene clusters (dsr, ehb, kaiC, cmr, and gas) involving in the energetic metabolism and defense systems. MTH operome contains 223 enzymes with 15 metabolic subsystems, 15 cell cycle proteins, 17 transcriptional regulators and 33 binding proteins. Functional annotation of its operome is thus more fundamental to a profound understanding of the molecular and cellular machinery at systems-level.

Molecular evolution and functional divergence of IspD homologs in malarial parasites.

Malaria is one of the leading parasitic diseases to humans caused by Plasmodium falciparum. It is imperative to discover novel targets for the development of antimalarial drugs. The 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD) in 2-C-methyl-D-erythritol-4-phosphate pathway has been considered as a second in vivo off-target for antimalarial drugs discovery as its essentiality in malarial parasites and devoid in mammals. Our study was intended to reveal the molecular basis of its functional parts by inferring diversity, origin and evolution across important malarial parasites. Phylogenetic analyses revealed its conservation probability and sequence homology among bacterial IspD homologs. It also indicated that Plasmodium IspD homologs were distantly related to each other and their functional counterparts originated from different progenitor genes. Nucleotide-diphospho-sugar transferase fold and conserved domain of them might have evolved from green sulphur bacteria, whereas coiled-coil region and apicoplast targeting signal derived from protozoal origins. These homologs contained prospectively definable motifs subject to neutral or nearly neutral evolution on a scale that were diverged radically and subsequently converged in making spatial structural arrangements. Our genetic diversity analysis has shown a constructive signal for identifying the evolutionary constraints, which has imposed on their functional divergence in malarial parasites. Thus, this study provides a novel insight into our understanding of the molecular basis of the origin and evolution history of IspD homologs across apicomplexa.

Methanobacterium formicicum as a target rumen methanogen for the development of new methane mitigation interventions: A review.

Methanobacterium formicicum (Methanobacteriaceae family) is an endosymbiotic methanogenic Archaean found in the digestive tracts of ruminants and elsewhere. It has been significantly implicated in global CH4 emission during enteric fermentation processes. In this review, we discuss current genomic and metabolic aspects of this microorganism for the purpose of the discovery of novel veterinary therapeutics. This microorganism encompasses a typical H2 scavenging system, which facilitates a metabolic symbiosis across the H2 producing cellulolytic bacteria and fumarate reducing bacteria. To date, five genome-scale metabolic models (iAF692, iMG746, iMB745, iVS941 and iMM518) have been developed. These metabolic reconstructions revealed the cellular and metabolic behaviors of methanogenic archaea. The characteristics of its symbiotic behavior and metabolic crosstalk with competitive rumen anaerobes support understanding of the physiological function and metabolic fate of shared metabolites in the rumen ecosystem. Thus, systems biological characterization of this microorganism may provide a new insight to realize its metabolic significance for the development of a healthy microbiota in ruminants. An in-depth knowledge of this microorganism may allow us to ensure a long term sustainability of ruminant-based agriculture.

Amino acid catabolism-directed biofuel production in Clostridium sticklandii: An insight into model-driven systems engineering.

Model-driven systems engineering has been more fascinating process for the microbial production of biofuel and bio-refineries in chemical and pharmaceutical industries. Genome-scale modeling and simulations have been guided for metabolic engineering of Clostridium species for the production of organic solvents and organic acids. Among them, Clostridium sticklandii is one of the potential organisms to be exploited as a microbial cell factory for biofuel production. It is a hyper-ammonia producing bacterium and is able to catabolize amino acids as important carbon and energy sources via Stickland reactions and the development of the specific pathways. Current genomic and metabolic aspects of this bacterium are comprehensively reviewed herein, which provided information for learning about protein catabolism-directed biofuel production. It has a metabolic potential to drive energy and direct solventogenesis as well as acidogenesis from protein catabolism. It produces by-products such as ethanol, acetate, n-butanol, n-butyrate and hydrogen from amino acid catabolism. Model-driven systems engineering of this organism would improve the performance of the industrial sectors and enhance the industrial economy by using protein-based waste in environment-friendly ways.

Molecular Evolutionary Constraints that Determine the Avirulence State of Clostridium botulinum C2 Toxin.

Clostridium botulinum (group-III) is an anaerobic bacterium producing C2 toxin along with botulinum neurotoxins. C2 toxin is belonged to binary toxin A family in bacterial ADP-ribosylation superfamily. A structural and functional diversity of binary toxin A family was inferred from different evolutionary constraints to determine the avirulence state of C2 toxin. Evolutionary genetic analyses revealed evidence of C2 toxin cluster evolution through horizontal gene transfer from the phage or plasmid origins, site-specific insertion by gene divergence, and homologous recombination event. It has also described that residue in conserved NAD-binding core, family-specific domain structure, and functional motifs found to predetermine its virulence state. Any mutational changes in these residues destabilized its structure-function relationship. Avirulent mutants of C2 toxin were screened and selected from a crucial site required for catalytic function of C2I and pore-forming function of C2II. We found coevolved amino acid pairs contributing an essential role in stabilization of its local structural environment. Avirulent toxins selected in this study were evaluated by detecting evolutionary constraints in stability of protein backbone structure, folding and conformational dynamic space, and antigenic peptides. We found 4 avirulent mutants of C2I and 5 mutants of C2II showing more stability in their local structural environment and backbone structure with rapid fold rate, and low conformational flexibility at mutated sites. Since, evolutionary constraints-free mutants with lack of catalytic and pore-forming function suggested as potential immunogenic candidates for treating C. botulinum infected poultry and veterinary animals. Single amino acid substitution in C2 toxin thus provides a major importance to understand its structure-function link, not only of a molecule but also of the pathogenesis.