Deciphering the structural–functional dynamics of glucosyltransferases from novel Apilactobacillus isolates through in silico analysis

Genomic-Based Prediction of Exopolysaccharide Composition and Structure: Insights from Rhizobium and Sinorhizobium Species

Bacterial exopolysaccharides (EPS) are key components in biofilm formation, stress protection, and symbiosis in Rhizobiaceae. While EPS structural diversity is extensive, experimental characterization remains limited. In this study, we experimentally determined and compared four distinct EPS structures produced by ten Rhizobium alamii strains. Using genomic data, we bioinformatically identified supra-operonic clusters (SOCs) responsible for these EPS biosynthesis. We introduced a computational framework to predict, score, and compare EPS SOCs across 84 Rhizobium and Sinorhizobium species, linking gene content to structural and functional EPS diversity. A total of 743 EPS SOCs was selected for network analyses, allowing the identification of 36 major groups of orthologous EPS SOCs, successfully recovering all known EPS biosynthetic loci and two novels SOCs potentially encoding uncharacterized EPS (xEPS-I, xEPS-II). Profiles of EPS SOCs correlated with taxonomical groups, with a single EPS SOC conserved through all 84 genomes and distinct additional EPS SOCs depending on the group, but do not strictly explain symbiotic capacity. Genetic comparisons of transporters (Wzx, Wzy) and glycosyltransferase sequences indicated these proteins as key markers of EPS structure. Overall, this computational framework accurately identified and classified EPS SOCs, providing a scalable, genome-based method for predicting EPS biosynthetic potential in Rhizobiaceae and usable in other microbial genera. Author Summary We developed a computational approach to predict how beneficial soil bacteria produce natural exopolysaccharides (EPS) that help them survive and interact with plants. These macromolecules, which form protective coatings and enable partnerships with crops, are currently difficult and expensive to study using traditional laboratory methods. By examining the genetic blueprints of 84 bacterial species from the Rhizobiaceae, we identified the key gene clusters responsible for producing these EPS, including two that were previously unknown. Our findings show that while some genetic patterns are widely shared among many bacteria, others are unique to specific types, shaping their individual abilities and characteristics. This research provides a faster, more cost-effective way to explore the vast diversity of these important macromolecules across different bacterial species. These insights could enable enhanced bacterial treatments that boost crop growth and resilience, particularly against water stress. Additionally, our method offers a valuable template for studying similar processes in other beneficial microorganisms, ultimately advancing our understanding of how they contribute to plant health, soil fertility, and the development of more sustainable farming practices worldwide.

Comparative genomics reveals hidden biosynthetic diversity in Streptomyces spp. and metal-dependent regulatory features associated with untapped specialized metabolites

The genus Streptomyces is one of the richest sources of bioactive natural products; however, a substantial proportion of its biosynthetic gene clusters (BGCs) remain cryptic and their metabolic products are unresolved. Advances in genome mining and computational prediction now enable comprehensive exploration of this hidden biosynthetic repertoire. In this study, whole-genome sequencing and comparative genomic analyses were performed on three three newly isolated Streptomyces strains to evaluate their specialized metabolic potential. Genome assemblies were annotated and systematically analyzed using antiSMASH, DeepBGC, GECCO, and PRISM to identify, cross-validate, and functionally characterize BGCs while predicting their associated secondary metabolite scaffolds. Taxonomic analyses based on Average Nucleotide Identity (ANI), phylogenomics, and BLAST identified the isolates as Streptomyces thinghirensis, Streptomyces novocaesareae , and Streptomyces griseorubens . Applying the consensus framework across the three Streptomyces genomes yielded 43 cryptic BGCs, lacking close similarity to reference BGCs in the MIBiG database, of which 26 were classified as HIGH, 10 as MEDIUM, and 7 as LOW confidence. Notably, numerous BGCs exhibited low abundance to characterized reference clusters, indicating a high potential for previously undescribed biosynthetic pathways and novel metabolite scaffolds. Comparative analyses further revealed strain-specific biosynthetic architectures together with putative metal-responsive regulatory systems; Fur, Zur , and Nur , which were frequently associated with specialized metabolite biosynthetic loci. Collectively, these findings demonstrate the effectiveness of integrated genome-mining strategies for prioritizing cryptic biosynthetic gene clusters and highlight the remarkable biosynthetic potential of newly identified Streptomyces isolates as a source of novel natural products.

Identification of cellular intermediates unveils unique enzymes for flagellar glycan biosynthesis in Clostridioides difficile.

Glycosylation of bacterial surface proteins, such as flagellin (FliC), is important for their function and is often involved in virulence of pathogens. Glycans can be further modified by so-called postglycosylation modifications (PGMs), often resulting in exclusive molecular structures. In Clostridioides difficile, a unique glycan structure (Type A) decorates FliC (which forms the flagellar filament) that consists of an O-linked N-acetyl-β-d-glucosamine (GlcNAc) modified with an N-methyl-L-threonine via a phosphodiester linkage. This PGM is synthesized by a set of four enzymes encoded in one operon (ftaABCD), but the exact biosynthesis pathway and biosynthetic intermediates remain unknown. In this study, we chemically synthesized two hitherto undescribed biosynthetic intermediates that we predicted based on bioinformatic analyses, CDP-threonine and CDP-N-methylthreonine. We showed that they are involved in the Type A PGM biosynthesis, as evidenced by mass spectrometric analyses of extracts of a set of C. difficile mutant strains. Furthermore, we characterized FtaC to be a SAM-dependent CDP-threonine N-methyltransferase that installs the methyl group on CDP-threonine prior to transfer of the PGM to GlcNAc-FliC, and we revealed FtaD as the CDP-N-methylthreonine:GlcNAc N-methylthreoninephosphotransferase. Finally, using recombinantly expressed FtaC and FtaD in combination with synthetic CDP-threonine, we reconstituted the biosynthesis pathway of the Type A PGM in vitro. Overall, our results open avenues to explore these unique biosynthesis enzymes in molecular detail to provide new points of entry for the development of biosynthesis inhibitors and tools to study the role of this PGM in virulence and flagellar function.

Whole-Genome Sequence Dataset of Rhodococcus qingshengii IEGM 267—Terpenoid Biotransformer Toward Genetic Functional Annotation

Background/Objectives: Microbial biotransformation of monoterpenoids is a promising approach for obtaining bioactive compounds. Rhodococcus species are attractive biocatalysts due to their metabolic versatility and ability to transform hydrophobic substrates. In this study, we investigated the catalytic potential of Rhodococcus qingshengii IEGM 267 toward carveol isomers and explored genomic features that may underlie this activity. Methods: The strain was cultivated in mineral medium supplemented with (−)-trans-carveol. Biotransformation products were analyzed by TLC and GC–MS. The draft genome was sequenced, assembled, taxonomically assigned, and annotated using standard bioinformatics tools. Results: Rhodococcus qingshengii IEGM 267 efficiently converted (−)-trans-carveol to carvone. Genome analysis confirmed the taxonomic assignment of the strain and revealed a large repertoire of oxidoreductases, including monooxygenases, hydroxylases, and dehydrogenases. Seven genes encoding cytochrome P450-dependent oxygenases were identified as candidate enzymes potentially involved in carveol oxidation. Conclusions: R. qingshengii IEGM 267 is an efficient and stereoselective biocatalyst for (−)-trans-carveol oxidation. The results of bioinformatics analysis suggest an alternative enzymatic basis for this transformation and provide a foundation for future functional characterization.

Identification of Novel Polysaccharide from Myxococcus xanthus and its Function in Developmental Sporulation

Bioinformatics analysis of extracellular glycosyl hydrolases mined from the genome of Halophilic Meridianimaribacter sp. CL38.

Insights are provided into putative glycosyl hydrolase candidates for efficient lignocellulosic waste pre-treatment and significant predicted halophilic and thermostable properties are revealed, suggesting these putative enzymes may endure industrial conditions.