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ABSTRACT 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 marginalislacked serine hydroxymethyltransferase (SHMT), andMethylobacterium variabileexhibited both isozymes of SHMT, suggesting its potential to utilize a broader range of carbon sources. Notably,Methylobrevissp. 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.Methylovirgulasp. 4MZ18 stood out by acquiring genes from both glyoxylate and EMC pathways, andMethylocapsasp. 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 forMethylovirgulasp. 4M-Z18 andMethylocapsasp. 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. IMPORTANCEMethylotrophs 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 forMethylovirgulasp. 4M-Z18 andMethylocapsasp. 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. 

Noncoding RNAs (ncRNAs) play key roles in the regulation of important pathways, including cellular growth, stress management, signaling, and biofilm formation. Sulfate-reducing bacteria (SRB) contribute to huge economic losses causing microbial-induced corrosion through biofilms on metal surfaces. To effectively combat the challenges posed by SRB, it is essential to understand their molecular mechanisms of biofilm formation. This study aimed to identify ncRNAs in the genome of a model SRB, Oleidesulfovibrio alaskensis G20 (OA G20). Three in silico approaches revealed genome-wide distribution of 37 ncRNAs excluding tRNAs in the OA G20. These ncRNAs belonged to 18 different Rfam families. This study identified riboswitches, sRNAs, RNP, and SRP. The analysis revealed that these ncRNAs could play key roles in the regulation of several pathways of biosynthesis and transport involved in biofilm formation by OA G20. Three sRNAs, Pseudomonas P10, Hammerhead type II, and sX4, which were found in OA G20, are rare and their roles have not been determined in SRB. These results suggest that applying various computational methods could enrich the results and lead to the discovery of additional novel ncRNAs, which could lead to understanding the “rules of life of OA G20” during biofilm formation. 

In the present study, a thermophilic strain designated CamBx3 was isolated from the Campanario hot spring, Chile. Based on 16S rRNA gene sequence, phylogenomic, and average nucleotide identity analysis the strain CamBx3 was identified asBacillus paralicheniformis. Genome analysis ofB. paralicheniformisCamBx3 revealed the presence of genes related to heat tolerance, exopolysaccharides (EPS), dissimilatory nitrate reduction, and assimilatory sulfate reduction. The pangenome analysis of strain CamBx3 with eightBacillusspp. resulted in 26,562 gene clusters, 7,002 shell genes, and 19,484 cloud genes. The EPS produced byB. paralicheniformisCamBx3 was extracted, partially purified, and evaluated for its functional activities.B. paralicheniformisCamBx3 EPS with concentration 5 mg mL⁻¹showed an optimum 92 mM ferrous equivalent FRAP activity, while the same concentration showed a maximum 91% of Fe²⁺chelating activity.B. paralicheniformisCamBx3 EPS (0.2 mg mL⁻¹) demonstratedβ-glucosidase inhibition. The EPS formed a viscoelastic gel at 45°C with a maximum instantaneous viscosity of 315 Pa.s at acidic pH 5. The present study suggests thatB. paralicheniformisCamBx3 could be a valuable resource for biopolymers and bioactive molecules for industrial applications. 

Abstract The phase changes of soil water or porous media have a crucial influence on the performance of natural and man-made infrastructures in cold regions. While various methods have been explored to address the impacts of frost-action arising from these phase changes, conventional approaches often rely on chemicals, mechanical techniques, and the reuse of waste materials, which often exhibit certain limitations and environmental concerns. In contrast, certain organisms produce ice-binding proteins (IBPs) or antifreeze proteins (AFPs) to adapt to low temperatures, which can inhibit ice crystal growth by lowering the freezing point and preventing ice crystallization without the need for external intervention. This study explores the potential of three psychrophilic microbes:Sporosarcina psychrophile,Sporosarcina globispora, andPolaromonas hydrogenivorans, to induce non-equilibrium freezing point depression and thermal hysteresis in order to control ice lens growth in frost-susceptible soils. We hypothesize that the AFPs produced by psychrophiles will alter the phase changes of porous media in frost-susceptible soils. The growth profiles of the microbes, the concentration of released proteins in the extracellular solution, and the thermal properties of the protein-mixed soils are monitored at an interval of three days. The controlled soil showed a freezing point of − 4.59 °C and thermal hysteresis of 4.62 °C, whereas protein-treated soil showed a maximum freezing point depression of − 8.54 °C and thermal hysteresis of 7.71 °C. Interestingly, except for the controlled sample, all the protein-treated soil samples were thawed at a negative temperature (minimum recorded at − 0.85 °C). Further analysis showed that the treated soils compared to porous media mixed soil freeze (1.25 °C vs. 0.51 °C) and thaw (2.75 °C vs. 1.72 °C) at extensive temperature gap. This freezing and thawing temperature gap is the temperature difference between the beginning of ice core formation and completed frozen, and the beginning of ice core thawing and completed thawed for the treated soil samples selected from different incubation days. Overall, this study presents a novel bio-mediated approach using psychrophilic microbes to control ice formation in frost-susceptible soils. 

Over the past decade, copper (Cu) has been recognized as a crucial metal in the differential expression of soluble (sMMO) and particulate (pMMO) forms of methane monooxygenase (MMO) through a mechanism referred to as the “Cu switch”. In this study, we used Methylosinus trichosporium OB3b as a model bacterium to investigate the range of Cu concentrations that trigger the expression of sMMO to pMMO and its effect on growth and methane oxidation. The Cu switch was found to be regulated within Cu concentrations from 3 to 5 µM, with a strict increase in the methane consumption rates from 3.09 to 3.85 µM occurring on the 6th day. Our findings indicate that there was a decrease in the fold changes in the expression of methanobactin (Mbn) synthesis gene (mbnA) with a higher Cu concentration, whereas the Ton-B siderophore receptor gene (mbnT) showed upregulation at all Cu concentrations. Furthermore, the upregulation of the di-heme enzyme at concentrations above 5 µM Cu may play a crucial role in the copper switch by increasing oxygen consumption; however, the role has yet not been elucidated. We developed a quantitative assay based on the naphthalene–Molisch principle to distinguish between the sMMO- and pMMO-expressing cells, which coincided with the regulation profile of the sMMO and pMMO genes. At 0 and 3 µM Cu, the naphthol concentration was higher (8.1 and 4.2 µM, respectively) and gradually decreased to 0 µM naphthol when pMMO was expressed and acted as the sole methane oxidizer at concentrations above 5 µM Cu. Using physical protein–protein interaction, we identified seven transporters, three cell wall biosynthesis or degradation proteins, Cu resistance operon proteins, and 18 hypothetical proteins that may be involved in Cu toxicity and homeostasis. These findings shed light on the key regulatory genes of the Cu switch that will have potential implications for bioremediation and biotechnology applications. 

Natural polysaccharides being investigated for use in the field of drug delivery commonly require the addition of sugars or pretreated biomass for fabrication. Geobacillus sp. strain WSUCF1 is a thermophile capable of secreting natural polymers, termed exopolysaccharides (EPSs), cultivated from cost-effective, non-treated lignocellulosic biomass carbon substrates. This preliminary investigation explores the capabilities of a 5% wt/wt amikacin-loaded film constructed from the crude EPS extracted from the strain WSUCF1. Film samples were seen to be non-cytotoxic to human keratinocytes and human skin-tissue fibroblasts, maintaining cell viability, on average, above 85% for keratinocytes over 72-h during a cell viability assay. The drug release profile of a whole film sample revealed a steady release of the antibiotic up to 12 h. The amikacin eluted by the EPS film was seen to be active against Staphylococcus aureus, maintaining above a 91% growth inhibition over a period of 48 h. Overall, this study demonstrates that a 5% amikacin-EPS film, grown from lignocellulosic biomass, can be a viable option for preventing or combating infections in clinical treatment. 

Scanning electron microscopy (SEM) techniques have been extensively performed to image and study bacterial cells with high-resolution images. Bacterial image segmentation in SEM images is an essential task to distinguish an object of interest and its specific region. These segmentation results can then be used to retrieve quantitative measures (e.g., cell length, area, cell density) for the accurate decision-making process of obtaining cellular objects. However, the complexity of the bacterial segmentation task is a barrier, as the intensity and texture of foreground and background are similar, and also, most clustered bacterial cells in images are partially overlapping with each other. The traditional approaches for identifying cell regions in microscopy images are labor intensive and heavily dependent on the professional knowledge of researchers. To mitigate the aforementioned challenges, in this study, we tested a U-Net-based semantic segmentation architecture followed by a post-processing step of morphological over-segmentation resolution to achieve accurate cell segmentation of SEM-acquired images of bacterial cells grown in a rotary culture system. The approach showed an 89.52% Dice similarity score on bacterial cell segmentation with lower segmentation error rates, validated over several cell overlapping object segmentation approaches with significant performance improvement. 

Sulfate-reducing bacteria (SRB) are anaerobic bacteria that form biofilm and induce corrosion on various material surfaces. The quorum sensing (QS) system that employs acyl homoserine lactone (AHL)-type QS molecules primarily govern biofilm formation. Studies on SRB have reported the presence of AHL, but no AHL synthase have been annotated in SRB so far. In this computational study, we used a combination of data mining, multiple sequence alignment (MSA), homology modeling and docking to decode a putative AHL synthase in the model SRB, Desulfovibrio vulgaris Hildenborough (DvH). Through data mining, we shortlisted 111 AHL synthase genes. Conserved domain analysis of 111 AHL synthase genes generated a consensus sequence. Subsequent MSA of the consensus sequence with DvH genome indicated that DVU_2486 (previously uncharacterized protein from acetyltransferase family) is the gene encoding for AHL synthase. Homology modeling revealed the existence of seven α-helices and six β sheets in the DvH AHL synthase. The amalgamated study of hydrophobicity, binding energy, and tunnels and cavities revealed that Leu99, Trp104, Arg139, Trp97, and Tyr36 are the crucial amino acids that govern the catalytic center of this putative synthase. Identifying AHL synthase in DvH would provide more comprehensive knowledge on QS mechanism and help design strategies to control biofilm formation. 

The growth and survival of an organism in a particular environment is highly depends on the certain indispensable genes, termed as essential genes. Sulfate-reducing bacteria (SRB) are obligate anaerobes which thrives on sulfate reduction for its energy requirements. The present study usedOleidesulfovibrio alaskensisG20 (OA G20) as a model SRB to categorize the essential genes based on their key metabolic pathways. Herein, we reported a feedback loop framework for gene of interest discovery, from bio-problem to gene set of interest, leveraging expert annotation with computational prediction. Defined bio-problem was applied to retrieve the genes of SRB from literature databases (PubMed, and PubMed Central) and annotated them to the genome of OA G20. Retrieved gene list was further used to enrich protein–protein interaction and was corroborated to the pangenome analysis, to categorize the enriched gene sets and the respective pathways under essential and non-essential. Interestingly, thesatgene (dde_2265) from the sulfur metabolism was the bridging gene between all the enriched pathways. Gene clusters involved in essential pathways were linked with the genes from seleno-compound metabolism, amino acid metabolism, secondary metabolite synthesis, and cofactor biosynthesis. Furthermore, pangenome analysis demonstrated the gene distribution, where 69.83% of the 116 enriched genes were mapped under “persistent,” inferring the essentiality of these genes. Likewise, 21.55% of the enriched genes, which involves specially the formate dehydrogenases and metallic hydrogenases, appeared under “shell.” Our methodology suggested that semi-automated text mining and network analysis may play a crucial role in deciphering the previously unexplored genes and key mechanisms which can help to generate a baseline prior to perform any experimental studies.