From genome to evolution: investigating type II methylotrophs using a pangenomic analysis.

A comprehensive pangenomic approach was employed to analyze the genomes of 75 type II methylotrophs spanning various genera. Our investigation revealed 256 exact core gene families shared by all 75 organisms, emphasizing their crucial role in the survival and adaptability of these organisms. Additionally, we predicted the functionality of 12 hypothetical proteins. The analysis unveiled a diverse array of genes associated with key metabolic pathways, including methane, serine, glyoxylate, and ethylmalonyl-CoA (EMC) metabolic pathways. While all selected organisms possessed essential genes for the serine pathway, Methylooceanibacter marginalis lacked serine hydroxymethyltransferase (SHMT), and Methylobacterium variabile exhibited both isozymes of SHMT, suggesting its potential to utilize a broader range of carbon sources. Notably, Methylobrevis sp. displayed a unique serine-glyoxylate transaminase isozyme not found in other organisms. Only nine organisms featured anaplerotic enzymes (isocitrate lyase and malate synthase) for the glyoxylate pathway, with the rest following the EMC pathway. Methylovirgula sp. 4MZ18 stood out by acquiring genes from both glyoxylate and EMC pathways, and Methylocapsa sp. S129 featured an A-form malate synthase, unlike the G-form found in the remaining organisms. Our findings also revealed distinct phylogenetic relationships and clustering patterns among type II methylotrophs, leading to the proposal of a separate genus for Methylovirgula sp. 4M-Z18 and Methylocapsa sp. S129. This pangenomic study unveils remarkable metabolic diversity, unique gene characteristics, and distinct clustering patterns of type II methylotrophs, providing valuable insights for future carbon sequestration and biotechnological applications. IMPORTANCE: Methylotrophs have played a significant role in methane-based product production for many years. However, a comprehensive investigation into the diverse genetic architectures across different genera of methylotrophs has been lacking. This study fills this knowledge gap by enhancing our understanding of core hypothetical proteins and unique enzymes involved in methane oxidation, serine, glyoxylate, and ethylmalonyl-CoA pathways. These findings provide a valuable reference for researchers working with other methylotrophic species. Furthermore, this study not only unveils distinctive gene characteristics and phylogenetic relationships but also suggests a reclassification for Methylovirgula sp. 4M-Z18 and Methylocapsa sp. S129 into separate genera due to their unique attributes within their respective genus. Leveraging the synergies among various methylotrophic organisms, the scientific community can potentially optimize metabolite production, increasing the yield of desired end products and overall productivity.

Biochemical unravelling of the endoxylanase activity in a bifunctional GH39 enzyme cloned and expressed from thermophilic Geobacillus sp. WSUCF1.

The glycoside hydrolase family 39 (GH39) proteins are renowned for their extremophilic and multifunctional enzymatic properties, yet the molecular mechanisms underpinning these unique characteristics continue to be an active subject of research. In this study, we introduce WsuXyn, a GH39 protein with a molecular weight of 58 kDa, originating from the thermophilic Geobacillus sp. WSUCF1. Previously reported for its exceptional thermostable ß-xylosidase activity, WsuXyn has recently demonstrated a significant endoxylanase activity (3752 U·mg-1) against beechwood xylan, indicating towards its bifunctional nature. Physicochemical characterization revealed that WsuXyn exhibits optimal endoxylanase activity at 70 °C and pH 7.0. Thermal stability assessments revealed that the enzyme is resilient to elevated temperatures, with a half-life of 168 h. Key kinetic parameters highlight the exceptional catalytic efficiency and strong affinity of the protein for xylan substrate. Moreover, WsuXyn-mediated hydrolysis of beechwood xylan has achieved 77 % xylan conversion, with xylose as the primary product. Structural analysis, amalgamated with docking simulations, has revealed strong binding forces between xylotetraose and the protein, with key amino acid residues, including Glu278, Tyr230, Glu160, Gly202, Cys201, Glu324, and Tyr283, playing pivotal roles in these interactions. Therefore, WsuXyn holds a strong promise for biodegradation and value-added product generation through lignocellulosic biomass conversion.

Enhancement of Methane Catalysis Rates in Methylosinus trichosporium OB3b.

Particulate methane monooxygenase (pMMO), a membrane-bound enzyme having three subunits (α, ß, and γ) and copper-containing centers, is found in most of the methanotrophs that selectively catalyze the oxidation of methane into methanol. Active sites in the pMMO of Methylosinus trichosporium OB3b were determined by docking the modeled structure with ethylbenzene, toluene, 1,3-dibutadiene, and trichloroethylene. The docking energy between the modeled pMMO structure and ethylbenzene, toluene, 1,3-dibutadiene, and trichloroethylene was -5.2, -5.7, -4.2, and -3.8 kcal/mol, respectively, suggesting the existence of more than one active site within the monomeric subunits due to the presence of multiple binding sites within the pMMO monomer. The evaluation of tunnels and cavities of the active sites and the docking results showed that each active site is specific to the radius of the substrate. To increase the catalysis rates of methane in the pMMO of M. trichosporium OB3b, selected amino acid residues interacting at the binding site of ethylbenzene, toluene, 1,3-dibutadiene, and trichloroethylene were mutated. Based on screening the strain energy, docking energy, and physiochemical properties, five mutants were downselected, B:Leu31Ser, B:Phe96Gly, B:Phe92Thr, B:Trp106Ala, and B:Tyr110Phe, which showed the docking energy of -6.3, -6.7, -6.3, -6.5, and -6.5 kcal/mol, respectively, as compared to the wild type (-5.2 kcal/mol) with ethylbenzene. These results suggest that these five mutants would likely increase methane oxidation rates compared to wild-type pMMO.

Transcriptomics and Functional Analysis of Copper Stress Response in the Sulfate-Reducing Bacterium Desulfovibrio alaskensis G20.

Copper (Cu) is an essential micronutrient required as a co-factor in the catalytic center of many enzymes. However, excess Cu can generate pleiotropic effects in the microbial cell. In addition, leaching of Cu from pipelines results in elevated Cu concentration in the environment, which is of public health concern. Sulfate-reducing bacteria (SRB) have been demonstrated to grow in toxic levels of Cu. However, reports on Cu toxicity towards SRB have primarily focused on the degree of toxicity and subsequent elimination. Here, Cu(II) stress-related effects on a model SRB, Desulfovibrio alaskensis G20, is reported. Cu(II) stress effects were assessed as alterations in the transcriptome through RNA-Seq at varying Cu(II) concentrations (5 µM and 15 µM). In the pairwise comparison of control vs. 5 µM Cu(II), 61.43% of genes were downregulated, and 38.57% were upregulated. In control vs. 15 µM Cu(II), 49.51% of genes were downregulated, and 50.5% were upregulated. The results indicated that the expression of inorganic ion transporters and translation machinery was massively modulated. Moreover, changes in the expression of critical biological processes such as DNA transcription and signal transduction were observed at high Cu(II) concentrations. These results will help us better understand the Cu(II) stress-response mechanism and provide avenues for future research.

Metagenomics and Culture Dependent Insights into the Distribution of Firmicutes across Two Different Sample Types Located in the Black Hills Region of South Dakota, USA.

Firmicutes is almost a ubiquitous phylum. Several genera of this group, for instance, Geobacillus, are recognized for decomposing plant organic matter and for producing thermostable ligninolytic enzymes. Amplicon sequencing was used in this study to determine the prevalence and genetic diversity of the Firmicutes in two distinctly related environmental samples-South Dakota Landfill Compost (SDLC, 60 °C), and Sanford Underground Research Facility sediments (SURF, 45 °C). Although distinct microbial community compositions were observed, there was a dominance of Firmicutes in both the SDLC and SURF samples, followed by Proteobacteria. The abundant classes of bacteria in the SDLC site, within the phylum Firmicutes, were Bacilli (83.2%), and Clostridia (2.9%). In comparison, the sample from the SURF mine was dominated by the Clostridia (45.8%) and then Bacilli (20.1%). Within the class Bacilli, the SDLC sample had more diversity (a total of 11 genera with more than 1% operational taxonomic unit, OTU). On the other hand, SURF samples had just three genera, about 1% of the total population: Bacilli, Paenibacillus, and Solibacillus. With specific regard to Geobacillus, it was found to be present at a level of 0.07% and 2.5% in SURF and SDLC, respectively. Subsequently, culture isolations of endospore-forming Firmicutes members from these samples led to the isolation of a total of 117 isolates. According to colony morphologies, and identification based upon 16S rRNA and gyrB gene sequence analysis, we obtained 58 taxonomically distinct strains. Depending on the similarity indexes, a gyrB sequence comparison appeared more useful than 16S rRNA sequence analysis for inferring intra- and some intergeneric relationships between the isolates.

Adaptive Enrichment of a Thermophilic Bacterial Isolate for Enhanced Enzymatic Activity.

The mimicking of evolution on a laboratory timescale to enhance biocatalyst specificity, substrate utilization activity, and/or product formation, is an effective and well-established approach that does not involve genetic engineering or regulatory details of the microorganism. The present work employed an evolutionary adaptive approach to improve the lignocellulose deconstruction capabilities of the strain by inducing the expression of laccase, a multicopper oxidase, in Geobacillus sp. strain WSUCF1. This bacterium is highly efficient in depolymerizing unprocessed lignocellulose, needing no preprocessing/pretreatment of the biomasses. However, it natively produces low levels of laccase. After 15 rounds of serially adapting this thermophilic strain in the presence of unprocessed corn stover as the selective pressure, we recorded a 20-fold increase in catalytic laccase activity, at 9.23 ± 0.6 U/mL, in an adapted yet stable strain of Geobacillus sp. WSUCF1, compared with the initial laccase production (0.46 ± 0.04 U/mL) obtained with the unadapted strain grown on unprocessed corn stover before optimization. Chemical composition analysis demonstrated that lignin removal by the adapted strain was 22 wt.% compared with 6 wt.% removal by the unadapted strain. These results signify a favorable prospect for fast, cost competitive bulk production of this thermostable enzyme. Also, this work has practical importance, as this fast adaptation of the Geobacillus sp. strain WSUCF1 suggests the possibility of growing industrial quantities of Geobacillus sp. strain WSUCF1 cells as biocatalysts on reasonably inexpensive carbon sources for commercial use. This work is the first application of the adaptive laboratory evolution approach for developing the desired phenotype of enhanced ligninolytic capability in any microbial strain.

A systems biology approach for elucidating the interaction of curcumin with Fanconi anemia FANC G protein and the key disease targets of leukemia.

Fanconi anemia (FA) is an autosomal recessive disorder with a high risk of malignancies including acute myeloid leukemia and squamous cell carcinoma. There is a constant search out of new potential therapeutic molecule to combat this disorder. In most cases, patients with FA develop haematological malignancies with acute myeloid leukemia and acute lymphoblastic leukemia. Identifying drugs which can efficiently block the pathways of both these disorders can be an ideal and novel strategy to treat FA. The curcumin, a natural compound obtained from turmeric is an interesting therapeutic molecule as it has been reported in the literature to combat both FA as well as leukemia. However, its complete mechanism is not elucidated. Herein, a systems biology approach for elucidating the therapeutic potential of curcumin against FA and leukemia is investigated by analyzing the computational molecular interactions of curcumin ligand with FANC G of FA and seven other key disease targets of leukemia. The proteins namely DOT1L, farnesyl transferase (FDPS), histone decetylase (EP3000), Polo-like kinase (PLK-2), aurora-like kinase (AUKRB), tyrosine kinase (ABL1), and retinoic acid receptor alpha (RARA) were chosen as disease targets for leukemia and modeled structure of FANC G protein as the disease target for FA. The docking investigations showed that curcumin had a very high binding affinity of -8.1 kcal/mol with FANC G protein. The key disease targets of leukemia namely tyrosine kinase (ABL1), aurora-like kinase (AUKRB), and polo-like kinase (PLK-2) showed that they had the comparable binding affinities of -9.7 k cal/mol, -8.7 k cal/mol, and -8.6 k cal/mol, respectively with curcumin. Further, the percentage similarity scores obtained from PAM50 using EMBOSS MATCHER was shown to provide a clue to understand the structural relationships to an extent and to predict the binding affinity. This investigation shows that curcumin effectively interacts with the disease targets of both FA and leukemia.