Microbial Physiology最新文献

Introduction: The transposase protein, InsAB', is the key cytoplastic determinant that catalyzes IS1 movement within a single DNA molecule or between two DNA molecules. Regulation of InsAB' synthesis, its structural characteristics, and its potent cis effect on IS1 transposition have been subjects of thorough investigation. However, limited efforts have been devoted to measuring the levels of IS1 transcripts and transposases made by native IS1 elements and examining their trans-acting function, particularly concerning their role in transposing otherwise immobile IS1 elements and other target DNA segments within the bacterial genome.

Methods: All constructs were made on the Escherichia coli chromosome using the lambda-Red system. IS1 transposition frequencies were determined using Bgl+ mutation assays, colony PCR and DNA sequencing. IS1 transcripts and transposases were quantitated using lacZ transcriptional and translational reporters.

Results: We first confirm that a native IS1 element, IS1E, can transpose at a dramatically elevated rate in the absence of both transcriptional and post-transcriptional regulations. Using lacZ reporters targeting the transposase gene insAB', we reveal that the InsA repressor moderately reduces insAB' transcription by as much as 16.8-fold. However, the combined action of InsA and ribosomal frameshifting leads to a much stronger reduction in InsAB' production, by up to 735-fold. We show that only a small percentage (roughly 1.6%) of IS1 transcripts are successfully translated into InsAB', highlighting the primary role of post-transcriptional regulation over transcriptional repression in governing IS1 transposition. We also demonstrate, for the first time, that high levels of InsAB' exhibit a strong trans effect, being capable of efficiently transposing not only normally non-mobile IS1 elements but also a larger miniIS1 cassette within the genome, with greater amounts of InsAB' leading to higher frequencies of transposition.

Conclusions: By dissecting and quantitating the roles of transcriptional and translational controls in transposase production, our study reveals that post-transcriptional regulation via ribosomal frameshifting is the central mechanism governing IS1 intragenomic transposition. Our findings demonstrate that IS1 transposases function in trans, mediating the movement of DNA segments specifically bordered by its inverted repeat at the left end and right end sequences. These findings advance our comprehension of how IS elements mobilize within bacterial genomes and provide potential strategies to counter IS1-mediated antibiotic resistance in clinical bacterial isolates.

Introduction: Viroporins are small multifunctional proteins that modify cellular membranes facilitating processes such as viral nucleic acid entry and the release of virions from infected cells. We are interested in studying the evolutionary relationships among these proteins, in particular their organization into families and superfamilies.

Methods: We applied a variety of computational strategies to perform comparative genomics analyses of 120 viral genomes, using the phylogenetic profile method. This allowed the identification of 12 families, organized into four functionally related groups. Additionally, we compiled a list of 40 families from the Transporter Classification Database (TCDB) with viroporin-like attributes (i.e., length ≤300 aas, similar topologies, and/or documented viroporin activities). We then used TCDB as a reference to search for evidence of homology among families. Our well-established bioinformatic pipeline for inference of homology included (1) sequence similarity, (2) compatibility of topology and hydropathy profiles, (3) similarity of family-based HMM profiles, (4) shared motifs, and (5) conserved domains.

Results: We were able to infer homology among 15 families, four of which (Vpu-C, p10 viroporin/GDU1, FAST, and R-FAST) expanded the established Influenza A/B Virus M2 Protein (M2) superfamily. The other families constituted three novel superfamilies: viroporin-1, consisting of three families (RVP10, NS3, and NSP4); viroporin-2, composed of two functionally linked families (SARS-VP and M-protein); and viroporin-3 composed of 3 functionally related families (viroporin E, IBV-E, and PRRSV).

Conclusion: The application of comparative genomics and remote homology identification strategies allowed the classification of homologous and functionally related viroporin-like families into superfamilies. These results will be useful in future functional, mechanistic, and evolutionary studies of viroporins.

The human microbiome is a dynamic, polymicrobial ecosystem that plays an essential role in nutrition, immune development, barrier integrity, and host physiology, acting as a mutualistic partner under balanced conditions. However, its ecological complexity, genetic adaptability through horizontal gene transfer, and interactions with other prokaryotes as well as protozoan and metazoan parasites can transform commensals into pathobionts, resulting in weakened host's barriers, immunity declines with the progression of age, and community composition shifts toward dysbiosis. Factors such as diet, genetics, aging, immune-senescence, impaired autophagy, and environmental exposure, all influence this delicate balance, determining whether the microbiome remains protective or becomes an opportunistic source of inflammation and disease. This review focuses on the study of the intestinal microbiome in humans. Maintaining microbiome homeostasis is promoted through (a) dietary diversity, (b) limited antimicrobial use, (c) use of probiotics, (d) support for gut barrier function, and (e) healthy lifestyle improvements. These actions and considerations are critical to prevent the emergence of pathogenic states and preserving the microbiome's vital role in host health throughout life.

FocA belongs to the formate-nitrite transporter (FNT) superfamily of pentameric membrane proteins, which translocate small, monovalent anions across the cytoplasmic membrane of bacteria, archaea and certain protists. FocA translocates formate anions or formic acid bidirectionally through a hydrophobic pore present in each protomer. This pore has two highly conserved amino acid residues, threonine 91 and histidine 209 that are proposed to protonate the anion during the translocation process. Current evidence suggests that different mechanisms control efflux and influx of formate. Determination of changes in extracellular and intracellular formate levels were used to characterize new amino acid variants of FocA in which H209 was exchanged for cysteine or serine. While the FocAH209S mutant excreted formic acid very efficiently, the mutant synthesizing FocAH209C translocated formic acid out of the cell poorly. These different efflux efficiencies of formic acid through FocA clearly suggest that the reactivity of the sulfur atom in cysteine accounts for the inefficient translocation of formic acid by the FocAH209C variant. Mutants synthesizing the FocAH209S or FocAH209C variants were incapable to importing formate, or its toxic chemical analogue hypophosphite, a phenotype similar to previously identified H209-exchange variants. Notably, a mutant lacking a functional formate hydrogenlyase (FHL-1) complex, which under physiological conditions disproportionates formate to H2 and CO2, retained sensitivity to hypophosphite, but accumulated formate externally. Our findings indicate that, while coupling between FocA and FHL-1 controls formate import, the import of hypophosphite is not dependent on FHL-1. Further, our data support a model in which two mechanisms for formate import exist, depending on the external formate concentration: at low concentration, protonation of formate or hypophosphite by H209 facilitates anion translocation; at high concentration, formic acid is directed to FHL-1 where it is disproportionated to H2 and CO2.

D-Xylose and L-arabinose are major components of plant biomass and as such also important carbon sources for most fungi and attractive compounds for biotechnology. Most fungi use the pentose catabolic pathway to convert these sugars through several redox reactions. One of the steps in this pathway is the conversion of L-xylulose to xylitol, catalyzed by L-xylulose reductase (LXR). Genetic studies in Aspergillus niger revealed the involvement of two LXR-encoding genes, lxrA and lxrB. In this study, we compared the corresponding enzymes, LxrA and LxrB, with respect to substrate specificity and kinetic properties, which revealed significant differences between them. Evaluation of these genes and their homologs from A. niger and selected other fungi revealed high diversity at the level of number of homologs per species, phylogenetic relationship and expression profiles, suggesting species-specific adaptations in fungal sugar metabolism. This study therefore not only provides more detailed insights into an ecologically and biotechnologically important fungal metabolic pathway, but also demonstrates the high diversity of sugar metabolism in fungi. This is especially relevant when knowledge from one species is transferred to another, e.g., for the engineering of fungal cell factories.

A key factor for the survival of an organism in its habitat is its ability to quickly adapt to changes in its environment on the protein level. One fast and efficient mechanism to influence protein abundance is the regulation of mRNA stability by ribonucleases. In the prokaryotic model organism Bacillus subtilis, the membrane-anchored RNase Y performs a crucial regulatory role by degrading and maturating mRNA. Previous studies have shown that RNase Y acts in concert with three proteins forming the Y-complex. In addition to its role as specificity factor for RNase Y, ribonuclease-independent functions have been proposed for the Y-complex during natural competence, biofilm formation, as well as sporulation. In previous work, using single-molecule tracking, we showed that the Y-complex is highly dynamic and present in multiple compositions in vivo. Using biochemical analysis, recruitment of the Y-complex to RNase Y was shown to be mediated by YaaT whereas YlbF and YmcA did not display any direct interactions. Here we employ 3D- structured illumination microscopy (SIM) super resolution and bimolecular fluorescence complementation (BiFC) to further characterize RNase Y/ Y-complex localizations and interactions. By visualizing the Y-complex proteins and RNase Y using structured illumination microscopy (SIM), we provide additional evidence that YaaT localizes differently than YmcA and YlbF, in that the fraction of YaaT is localized membrane-proximal appears to be higher than the one observed for YmcA and YlbF. We also show that the strength of YaaT membrane association is culture medium dependent. Evidence for membrane-proximal interaction of the Y-complex proteins with RNase Y is provided through the use of bimolecular fluorescence complementation (BiFC). Taken together, our data support a model where the Y-complex is exclusively tethered to RNase Y by YaaT and where the composition of the Y-complex is fluctuating potentially in a function-dependent manner (Figure1).

Introduction: Phosphoglucomutases (PGM) are crucial for bacterial fitness, environmental survival, pathogenicity, and cell envelope stability, making them potential new targets for combating bacterial infection and transmission. PGM functionality relies on initial phosphorylation by the activator glucose-1,6-bisphosphate (glucose-1,6-BP). While the origin of glucose-1,6-BP in vertebrates is well investigated, a bacterial glucose-1,6-BP synthase was only recently identified in the cyanobacterium Synechocystis. In this photoautotroph, a secondary PGM (SynPGM2) efficiently catalyzes glucose-1,6-BP synthesis from fructose-1,6-bisphosphate (fructose-1,6-BP) and glucose-1-phosphate or glucose-6-phosphate . A homologous PGM from the heterotrophic Bacteroides salyersiae, belonging to the same conserved domain subfamily (cd05800) as SynPGM2, exhibited similar activity, suggesting that bacterial glucose-1,6-BP synthesis is a feature of this specific subfamily.

Methods: To investigate the specificity and regulation of various PGM enzymes from different heterotrophic bacteria, recombinant enzymes were purified and analyzed using enzymatic assays and HPLC-MS.

Results: We demonstrate that glucose-1,6-BP synthesis extends beyond the cd5800 subfamily to the cd05801, cd05799, and cd03089 subfamilies. PGMs from Escherichia coli (cd05801 and cd03089), Enterococcus faecium (cd05799), Yersinia enterocolitica (cd05801), and Candidatus Gastranaerophilales (cd05800) catalyze the same fructose-1,6-BP-dependent synthesis reaction of glucose-1,6-BP as SynPGM2. Notably, fructose-1,6-BP, a known inhibitor of some PGM, does not inhibit these bacterial PGMs. Moreover, E. faecium PGM, belonging to the same subfamily as the mammalian glucose 1,6 BP synthase, efficiently catalyzes the mammalian-type 1,3-bisphosphoglycerate-dependent glucose 1,6-BP synthesis reaction.

Conclusion: All investigated heterotrophic bacteria appear to use their primary PGM for both PGM activity and activator synthesis, suggesting a more versatile and less specialized role for PGMs in heterotrophic bacteria.

Introduction: The low yield of bioflocculants has been a bottleneck problem that limits their industrial applications. Understanding the metabolic mechanism of bacteria that produce bioflocculants could provide valuable insights and strategies to directly regulate their yield in future.

Methods: To investigate the change of metabolites in the process of bioflocculant production by a biomass-degrading bacterium, Pseudomonas boreopolis GO2, an untargeted metabolome analysis was performed.

Results: The results showed that metabolites significantly differed during the fermentation process when corn stover was used as the sole carbon source. The differential metabolites were divided into four co-expression modules based on the weighted gene co-expression network analysis. Among them, a module (yellow module) was closely related to the flocculating efficiency, and the metabolites in this module were mainly involved in carbohydrate, lipid, and amino acid metabolism. The top 30 metabolites with the highest degree in the yellow module were identified as hub metabolites for bioflocculant production. Finally, 10 hub metabolites were selected to perform the additional experiments, and the addition of L-rhamnose, tyramine, tryptophan, and glutaric acid alone all could significantly improve the flocculating efficiency of GO2 strain.

Conclusion: These results indicated that the hub metabolites were key for bioflocculant production in GO2 strain, and could help guide the improvement of high-efficiency and low-cost bioflocculant production.

Background: For many years, scientists have accepted Darwin's conclusion that "Survival of the Fittest" involves successful competition with other organisms for life-endowing molecules and conditions.

Summary: Newly discovered "partial" organisms with minimal genomes that require symbiotic or parasitic relationships for growth and reproduction suggest that cooperation in addition to competition was and still is a primary driving force for survival. These two phenomena are not mutually exclusive, and both can confer a competitive advantage for survival. In fact, cooperation may have been more important in the early evolution of life on earth before autonomous organisms developed, becoming large genome organisms.

Key messages: This suggestion has tremendous consequences with respect to our conception of the early evolution of life on earth as well as the appearance of intercellular interactions, multicellularity and the nature of interactions between humans and their societies (e.g., social Darwinism).