Folate-producing bifidobacteria: metabolism, genetics, and relevance.

Folate (the general term for all bioactive forms of vitamin B9) plays a crucial role in the evolutionary highly conserved one-carbon (1C) metabolism, a network including central reactions such as DNA and protein synthesis and methylation of macromolecules. Folate delivers 1C units, such as methyl and formyl, between reactants. Plants, algae, fungi, and many bacteria can naturally produce folate, whereas animals, including humans, must obtain folate from external sources. For humans, folate deficiency is, however, a widespread problem. Bifidobacteria constitute an important component of human and many animal microbiomes, providing various health advantages to the host, such as producing folate. This review focuses on bifidobacteria and folate metabolism and the current knowledge of the distribution of genes needed for complete folate biosynthesis across different bifidobacterial species. Biotechnologies based on folate-trophic probiotics aim to create fermented products enriched with folate or design probiotic supplements that can synthesize folate in the colon, improving overall health. Therefore, bifidobacteria (alone or in association with other microorganisms) may, in the future, contribute to reducing widespread folate deficiencies prevalent among vulnerable human population groups, such as older people, women at child-birth age, and people in low-income countries.

ELM-the Eukaryotic Linear Motif resource-2024 update.

Short Linear Motifs (SLiMs) are the smallest structural and functional components of modular eukaryotic proteins. They are also the most abundant, especially when considering post-translational modifications. As well as being found throughout the cell as part of regulatory processes, SLiMs are extensively mimicked by intracellular pathogens. At the heart of the Eukaryotic Linear Motif (ELM) Resource is a representative (not comprehensive) database. The ELM entries are created by a growing community of skilled annotators and provide an introduction to linear motif functionality for biomedical researchers. The 2024 ELM update includes 346 novel motif instances in areas ranging from innate immunity to both protein and RNA degradation systems. In total, 39 classes of newly annotated motifs have been added, and another 17 existing entries have been updated in the database. The 2024 ELM release now includes 356 motif classes incorporating 4283 individual motif instances manually curated from 4274 scientific publications and including >700 links to experimentally determined 3D structures. In a recent development, the InterPro protein module resource now also includes ELM data. ELM is available at: http://elm.eu.org.

Bifidobacterium mellis sp. nov., isolated from the honey stomach of the honey bee Apis mellifera.

A novel Bifidobacterium strain, Bin7NT, was isolated from the honey stomach of the honey bee Apis mellifera. Cells are Gram-positive, non-motile, non-sporulating, facultative anaerobic and fructose 6-phosphate phosphoketolase-positive. Their optimal growth is at 37 °C in anaerobiosis in MRS (De Man, Rogosa and Sharpe) added with cysteine. The honey bee microbiota was composed of several phylotypes of Bifidobacterium and Lactobacillus. Comparative analysis of 16S rRNA gene sequence similarity revealed that strain Bin7NT grouped with Bifidobacterium species originating from honey bees and was closely related to Bifidobacterium asteroides DSM 20089T (99.67 % similarity). However, the highest average nucleotide identity and digital DNA-DNA hybridization values of 94.88 and 60.6 %, respectively, were obtained with Bifidobacterium choladohabitans JCM 34586T. The DNA G+C content of the type strain is 60.8 mol%. The cell-wall peptidoglycan is of the A4ß l-Orn-d-Asp type. The main cellular fatty acids of strain Bin7NT are C18 : 1 ω9c, C16 : 0, C18 : 1 ω7c and C18 : 0. Phenotypic characterization and genotyping based on the genome sequences clearly show that this strain is distinct from the type strains of the so far recognized Bifidobacterium species. Thus, Bifidobacterium mellis sp. nov. (Bin7NT=DSM 29108T=CCUG 66113T) is proposed as novel Bifidobacterium species.

Insertions and deletions mediated functional divergence of Rossmann fold enzymes.

Nucleobase-containing coenzymes are hypothesized to be relics of an early RNA-based world that preceded the emergence of proteins. Despite the importance of coenzyme-protein synergisms, their emergence and evolution remain understudied. An excellent target to address this issue is the Rossmann fold, the most catalytically diverse and abundant protein architecture in nature. We investigated two main Rossmann lineages: the nicotinamide adenine dinucleotide phosphate (NAD(P)) and the S-adenosyl methionine (SAM)- binding superfamilies. To identify the evolutionary changes that lead to a coenzyme specificity switch on these superfamilies, we performed structural and sequence-based Hidden Markov model analysis to systematically search for key motifs in their coenzyme-binding pockets. Our analyses revealed that through insertions and deletions (InDels) and a residue substitution, the ancient ß1-loop-α1 coenzyme-binding structure of NAD(P) could be reshaped into the SAM-binding ß1-loop-α1 structure. To experimentally prove this obsevation, we removed three amino acids from the NAD(P)-binding pocket and solved the structure of the resulting mutant, revealing the characteristic loop features of the SAM-binding pocket. To confirm the binding to SAM, we performed isothermal titration calorimetry measurements. Molecular dynamics simulations also corroborated the role of InDels in abolishing NAD binding and acquiring SAM binding. Our results uncovered how nature may have utilized insertions and deletions to optimize the different coenzyme-binding pockets and the distinct functionalities observed for Rossmann superfamilies. This work also proposes a general mechanism by which protein templates could have been recycled through the course of evolution to adopt different coenzymes and confer distinct chemistries.

Structural dynamics shape the fitness window of alanine:glyoxylate aminotransferase.

The conformational landscape of a protein is constantly expanded by genetic variations that have a minimal impact on the function(s) while causing subtle effects on protein structure. The wider the conformational space sampled by these variants, the higher the probabilities to adapt to changes in environmental conditions. However, the probability that a single mutation may result in a pathogenic phenotype also increases. Here we present a paradigmatic example of how protein evolution balances structural stability and dynamics to maximize protein adaptability and preserve protein fitness. We took advantage of known genetic variations of human alanine:glyoxylate aminotransferase (AGT1), which is present as a common major allelic form (AGT-Ma) and a minor polymorphic form (AGT-Mi) expressed in 20% of Caucasian population. By integrating crystallographic studies and molecular dynamics simulations, we show that AGT-Ma is endowed with structurally unstable (frustrated) regions, which become disordered in AGT-Mi. An in-depth biochemical characterization of variants from an anticonsensus library, encompassing the frustrated regions, correlates this plasticity to a fitness window defined by AGT-Ma and AGT-Mi. Finally, co-immunoprecipitation analysis suggests that structural frustration in AGT1 could favor additional functions related to protein-protein interactions. These results expand our understanding of protein structural evolution by establishing that naturally occurring genetic variations tip the balance between stability and frustration to maximize the ensemble of conformations falling within a well-defined fitness window, thus expanding the adaptability potential of the protein.

Inter-paralog amino acid inversion events in large phylogenies of duplicated proteins.

Connecting protein sequence to function is becoming increasingly relevant since high-throughput sequencing studies accumulate large amounts of genomic data. In order to go beyond the existing database annotation, it is fundamental to understand the mechanisms underlying functional inheritance and divergence. If the homology relationship between proteins is known, can we determine whether the function diverged? In this work, we analyze different possibilities of protein sequence evolution after gene duplication and identify "inter-paralog inversions", i.e., sites where the relationship between the ancestry and the functional signal is decoupled. The amino acids in these sites are masked from being recognized by other prediction tools. Still, they play a role in functional divergence and could indicate a shift in protein function. We develop a method to specifically recognize inter-paralog amino acid inversions in a phylogeny and test it on real and simulated datasets. In a dataset built from the Epidermal Growth Factor Receptor (EGFR) sequences found in 88 fish species, we identify 19 amino acid sites that went through inversion after gene duplication, mostly located at the ligand-binding extracellular domain. Our work uncovers an outcome of protein duplications with direct implications in protein functional annotation and sequence evolution. The developed method is optimized to work with large protein datasets and can be readily included in a targeted protein analysis pipeline.

Binding of single-mutant epidermal growth factor (EGF) ligands alters the stability of the EGF receptor dimer and promotes growth signaling.

The epidermal growth factor receptor (EGFR) is a membrane-anchored tyrosine kinase that is able to selectively respond to multiple extracellular stimuli. Previous studies have indicated that the modularity of this system may be caused by ligand-induced differences in the stability of the receptor dimer. However, this hypothesis has not been explored using single-mutant ligands thus far. Herein, we developed a new approach to identify residues responsible for functional divergence by selecting residues in the epidermal growth factor (EGF) ligand that are conserved among orthologs yet divergent between paralogs. Then, we mutated these residues and assessed the mutants' effects on the receptor using a combination of molecular dynamics (MD) and biochemical techniques. Although the EGF mutants had binding affinities for the EGFR comparable with the WT ligand, the EGF mutants showed differential patterns of receptor phosphorylation and cell growth in multiple cell lines. The MD simulations of the EGF mutants indicated that mutations had long-range effects on the receptor dimer interface. This study shows for the first time that a single mutation in the EGF is sufficient to alter the activation of the EGFR signaling pathway at the cellular level. These results also support that biased ligand-receptor signaling in the tyrosine kinase receptor system can lead to differential downstream outcomes and demonstrate a promising new method to study ligand-receptor interactions.

CNTNAP2 mutations and autosomal dominant epilepsy with auditory features.

Autosomal dominant epilepsy with auditory features (ADEAF) is clinically characterized by focal seizures with prominent auditory or aphasic auras and absence of structural brain abnormalities. Mutations in LGI1 and RELN genes account for the disorder in about 50% of ADEAF families. In a recent paper, a heterozygous intragenic deletion in the CNTNAP2 gene has been associated to ADEAF in a single family. We screened 28 ADEAF families for mutations in CNTNAP2 by next generation sequencing and copy number variation analyses and found no likely pathogenic mutations segregating with the disease. CNTNAP2 should be screened in genetically unsolved ADEAF families, but causative mutations are expected to be infrequent in this gene.

Bifidobacterium callitrichidarum sp. nov. from the faeces of the emperor tamarin (Saguinus imperator).

Three Gram-stain-positive, non-spore-forming, microaerophilic and fructose-6-phosphate phosphoketolase positive strains were isolated from a faecal sample of an adult subject of the emperor tamarin (Saguinus imperator). Given that the isolates revealed identical BOX PCR profiles, strain TRI 5T was selected as a representative and characterized further. Comparative analysis of 16S rRNA gene sequence similarity revealed that strain TRI 5T was closely related to Bifidobacterium saguini DSM 23967T (96.4 %) and to Bifidobacterium longum subsp. longum ATCC 15708 (96.2 %). Multilocus sequence analyses of five housekeeping genes showed the close phylogenetic relatedness of this strain to Bifidobacterium breve DSM 20213T (hsp60 94.1 %), Bifidobacterium saguini DSM 23967T (clpC 91 %), Bifidobacterium avesanii DSM 100685T (dnaG 80.3 %), Bifidobacterium longumsubsp. infantis ATCC 15697T (dnaJ 85.3 %) and Bifidobacterium longumsubsp. longum ATCC 15708 (rpoB 93 %), respectively. The peptidoglycan type was A3ß, with an interpeptide bridge comprising l-Orn (Lys) - l-Ser - l-Ala - l-Thr - l-Ala. The DNA G+C content of strain TRI 5T was 60.9 mol%. Based on the data provided, strain TRI 5T represents a novel species of the genus Bifidobacterium for which the name Bifidobacteriumcallitrichidarum sp. nov. is proposed. The type strain is TRI 5T (=DSM 103152T=JCM 31790T).