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PubMed1975

Purification and properties of a soluble nicotinamide adenine dinucleotide-linked isocitrate dehydrogenase from Crithidia fasciculata.

A soluble NAD+-linked isocitrate dehydrogenase has been isolated from Crithidia fasciculata. The enzyme was purified 128-fold, almost to homogeneity, and was highly specific for NAD+ as the coenzyme. There is also a cytoplasmic NADP+-linked and a mitochondrial isocitrate dehydrogenase in the organism. Studies of the physical and kinetic properties of the soluble NAD+-isocitrate dehydrogenase from this organism showed that it resembled microbial NADP+-isocitrate dehydrogenases in general, all of which are cytoplasmic enzymes. The enzyme appeared not to be related to other NAD+-isocitrate dehydrogenases, which are found in the mitochondria of eukaryotic cells. The molecular weight of the soluble NAD+-isocitrate dehydrogenase was 105,000 which is within the range of the values for microbial NADP+-isocitrate dehydrogenases. Similar to the NADP+-isocitrate dehydrogenase in this organism, the enzyme was inhibited in a concerted manner by glyoxalate plus oxalacetate. Kinetic analysis revealed that Mn2+ was involved in the binding of isocitrate to the enzyme. Inhibition of the NAD+-linked isocitrate dehydrogenase by p-chloromercuribenzoate could be prevented by prior incubation of the enzyme with both Mn2+ and isocitrate; however, neither ion alone conferred protection. Free isocitrate, free Mn2+, and the Mn2+-isocitrate complex could all bind to the enzyme. Four different mechanisms with respect to the binding of isocitrate to the enzyme were tested. Of these, the formation of the active enzyme-Mn2+-isocitrate complex from (a) the random binding of Mn2+, isocitrate, and the Mn2+-isocitrate complex, or (b) the binding of Mn2+-isocitrate with free Mn2+ and isocitrate acting as dead-end competitors were both in agreement with these data.

Animals↗
PubMed2026

Unveiling microbial communities and biogeochemical cycles in Antarctic colored snow.

Snow cover, the extensive terrestrial habitat in Antarctica, sometimes exhibits vivid coloration, yet the structure and function of its microbial communities remain poorly characterized. Using metagenomic sequencing of red snow (RS) and green snow (GS) from the Fildes Peninsula, we found that bacterial, eukaryotic, and archaeal relative abundances were 85.82%, 13.52% and 0.16%, respectively. &#x3b2;-Diversity differed significantly between RS and GS across these three domains (P&#x2009;<&#x2009;0.05). Dominant bacterial phyla included Bacteroidota (RS: 62.61%; GS: 38.72%) and Pseudomonadota (RS: 32.80%; GS: 54.10%). Among eukaryotes, Chlorophyta (RS: 58.10%; GS: 52.98%) and Basidiomycota (RS: 14.80%; GS: 8.08%) were prevalent. Nanobdellota dominated archaea, with lower abundance in RS than GS. In the algal community, Sanguina, Gonium and Chloromonas were significantly enriched in red snow, while Chlorella and Micractinium were enriched in green snow (P&#x2009;<&#x2009;0.05). Marker genes associated with carbon (C), nitrogen (N), phosphorus (P) and sulfur (S) cycles were identified in green and red snow. Aerobic respiration and phosphate regulation were significantly enriched in red snow, while CO oxidation, fermentation, and denitrification were significantly enriched in green snow. Key microbial genera associated with these functional pathways also varied. In the denitrification of red snow, Stutzerimonas was the most abundant genus, while Janthinobacterium was abundant in green snow. Nitrification-related genes were detected only in red snow based on the present metagenomic data. The network of the red snow microbial community was potentially more complex and resistant based on topology, which not only benefited its own long-term survival but might also have potentially influenced the positive feedback effect of snowmelt by maintaining a low-albedo snow surface. This provided an ecological implication under climate warming: the expansion of red snow patches showed the potential to the increase nitrate runoff export, which would affect nitrogen nutrient levels in coastal Antarctic waters. Overall, this study used metagenomics to compare the multidomain (bacteria, archaea and eukaryotes) composition and diversity between red snow and green snow, and directly linked key microbial taxa with functional genes of biogeochemical cycles. This study provided new insights into the biological characteristics and functional potential of Antarctic colored snow.

Snow↗
PubMed2024

A distinct, high-affinity, alkaline phosphatase facilitates occupation of P-depleted environments by marine picocyanobacteria.

Marine picocyanobacteria of the genera Prochlorococcus and Synechococcus, the two most abundant phototrophs on Earth, thrive in oligotrophic oceanic regions. While it is well known that specific lineages are exquisitely adapted to prevailing in situ light and temperature regimes, much less is known of the molecular machinery required to facilitate occupancy of these low-nutrient environments. Here, we describe a hitherto unknown alkaline phosphatase, Psip1, that has a substantially higher affinity for phosphomonoesters than other well-known phosphatases like PhoA, PhoX, or PhoD and is restricted to clade III Synechococcus and a subset of high light I-adapted Prochlorococcus strains, suggesting niche specificity. We demonstrate that Psip1 has undergone convergent evolution with PhoX, requiring both iron and calcium for activity and likely possessing identical key residues around the active site, despite generally very low sequence homology. Interrogation of metagenomes and transcriptomes from TARA oceans and an Atlantic Meridional transect shows that psip1 is abundant and highly expressed in picocyanobacterial populations from the Mediterranean Sea and north Atlantic gyre, regions well recognized to be phosphorus (P)-deplete. Together, this identifies psip1 as an important oligotrophy-specific gene for P recycling in these organisms. Furthermore, psip1 is not restricted to picocyanobacteria and is abundant and highly transcribed in some &#x3b1;-proteobacteria and eukaryotic algae, suggesting that such a high-affinity phosphatase is important across the microbial taxonomic world to occupy low-P environments.

Alkaline Phosphatase↗
PubMed1976

Mutagenicity and DNA-modifying activity: a comparison of two microbial assays.

Aspects of the Salmonella mutagenesis and Escherichia coli DNA polymerase deficient (pol A1) assay procedures for detecting environmental mutagens are discussed. The chief limitation of the pol A1-- assay involves substances that do not diffuse rapidly in agar. This problem can be overcome by performing the test in suspension. A simple procedure for accomplishing this is described. Although the Salmonella assay is more flexible, under routine conditions it does not respond to several classes of substances which give positive responses in the pol A1- system. For optimal testing, it is recommended that the two microbial assays be used in tandem. The DNA-modifying properties of povidone-iodine for eukaryotic and prokaryotic cells are described. Even though this substance does not display mutagenic properties in the standard Salmonella assay, it does so in suspension culture. The basis of the mutagenic and DNA-modifying properties of povidone-iodine appears to involve the iodination of the cystosine residue of DNA.

Cells, Cultured↗
PubMed1979

Differences in the degradation of native collagen by two microbial collagenases.

The early stages of degradation of native collagen by two bacterial collagenases were studied by electron microscopy and by automatic Edman degradation. The purified collagenase from Clostridium histolyticum was shown to cleave native collagen at several sites, but not progressively from the N-terminus, as had been previously suggested. The homogeneous collagenase from Achromobacter iophagus cleaves native collagen preferentially at two sites corresponding to the interbands 33-34 and 41-42. The latter lies within the region cleaved by the eukaryotic collagenases.

Alcaligenes↗
PubMed1977

Mode of action of trichothecenes.

In the present discussion, the author summarized the toxicological and biological features of thirty kinds of trichothecene mycotoxins which are produced by a wide range of Fusarium, Myrothecium and others. The 12, 13-epoxytrichothecenes induce nausea, emesis, vomiting, skin inflamation, leukopenia, diarrhea, hemorrhage in lung and brain, and destruction of bone marrow. Since these toxicological characteristics coincide with a major symptom of intoxicated humans and farm animals induced by consumption of moldy cereals and feeds, the red-mold toxicosis and bean-hulls poisoning in Japan, moldy corn toxicosis in U.S.A., A.T.A., stachybotryotoxicosis and dendrochiotoxicosis in Europe, are originated from a common toxicant, trichothecenes. Orally administered trichothecenes are rapidly absorbed and eliminated into the feces and urine upon deacetylation at C-4 by the microsomal esterase of liver. Biochemical approaches to the mode of action revealed that the trichothecenes are a potent inhibitor of protein and D.N.A. syntheses in eukaryotic cells. Bindings to the eukaryotic polysomes and ribosomes and the subsequent inactivation of ribosomal cycle is responsible for their inhibitory effect to initiation and termination reactions. Microbial approaches revealed that the trichothecenes are mutagenic to yeast cells, but are negative in D.N.A.-attacking ability to Bacillus subtilis and reversion assay with Salmonella typhimurium. Reactivity of the epoxide ring of trichothecenes with S.H.-group of proteins will be discussed in relation to the molecular mechanism of action.

Animals↗
PubMed2026

The anaerobic fungus Caecomyces churrovis produces H2 via a non-bifurcating NADH-dependent enzyme complex.

UNLABELLED: Hydrogenosomes are mitochondrion-derived organelles that produce ATP and H2 to support energy metabolism in anaerobic eukaryotes. H2 production allows reoxidation of reduced cofactors generated during fermentative metabolism; however, the metabolic mechanisms for H2 production in anaerobic eukaryotes remain incompletely understood. In particular, it remains unclear whether anaerobic fungi (AF) hydrogenosomes use a ferredoxin-dependent pathway or a distinct mechanism to regenerate NAD(P)+ and link electron transfer to H2 formation. Here, by combining genomic search, proteomic analysis, and enzymology, we reveal the molecular mechanism for H2 production in the AF Caecomyces churrovis. Our enzyme assays on the organelle fraction of C. churrovis revealed the activity of H2:NAD+ oxidoreductase but not pyruvate:ferredoxin oxidoreductase, which is usually linked to H2 formation. We identified genes encoding [FeFe] hydrogenase (Hyd) and NADH dehydrogenase subunits E and F (NuoE and NuoF) in C. churrovis and confirmed their expression in the isolated hydrogenosomal fractions by proteomic analysis. Combining the individually purified enzymes, we found Hyd and NuoEF proteins formed H2 directly from NADH independently of ferredoxin, functioning as a non-bifurcating NADH-dependent enzyme rather than an electron-bifurcating enzyme known from anaerobic prokaryotes. We identified homologs of hydrogenosomal NuoE, NuoF, and Hyd in many other AF, indicating this pathway is commonly shared among the AF. This work demonstrates the existence of a non-bifurcating NADH-dependent enzyme complex for H2 production in eukaryotes. Moreover, this complex could potentially be exploited as a target for controlling AF H2 production and altering fungal metabolism. IMPORTANCE: H2 production is a prominent feature of anaerobic energy metabolism, yet our understanding of eukaryotic mechanisms remains limited. Anaerobic fungi (AF) are key decomposers of lignocellulose and contribute to hydrogen flux in anaerobic environments. Although it has been more than 40 years since the H2 production in Neocallimastix was first reported, the molecular mechanism for hydrogenosomal H2 production and redox balance remains unclear. We demonstrate that AF produce H2 from NADH utilizing a non-bifurcating NADH-dependent enzyme complex rather than an electron-bifurcating, ferredoxin-dependent variant. We show that this enzyme complex is conserved across multiple AF lineages and thus demonstrate the occurrence of a non-bifurcating NADH-dependent enzyme in eukaryotes. This discovery expands our understanding of eukaryotic hydrogenosomal metabolism, reveals a previously unknown strategy for redox balancing, and highlights potential targets for manipulating H2 production. These insights have broad implications for microbial energy metabolism, anaerobic ecosystems, and bioengineering of H2-producing systems.

Hydrogen↗
PubMed2026

Glutathione acts as an exometabolite that promotes growth recovery in fission yeast with defects in amino acid metabolism and cell polarity.

UNLABELLED: Microorganisms in nature form communities through diverse interactions, such as mutualism and competition, to adapt to their ecological environments. These interactions seem to be mediated by extracellular metabolites (exometabolites), yet the chemical and biological diversity underlying these processes remains largely unexplored. In this study, we examined the chemical basis of exometabolite-mediated interactions in the fission yeast Schizosaccharomyces pombe by a genome-wide screen employing 3,420 viable gene deletion mutants. We identified 37 strains that exhibited growth defects in monoculture on a minimal medium but exhibited growth recovery in the vicinity of wild-type colonies (co-culture), suggesting that exometabolites derived from wild-type cells compensated for the gene deletion. Both lipophilic and water-soluble fractions obtained by solvent partitioning of the wild-type culture supernatant promoted growth recovery. Among the 11 mutants rescued by the water-soluble fraction, 6 were cysteine auxotrophs, prompting analyses of thiol-containing metabolites by liquid chromatography-mass spectrometry (LC-MS), revealing the presence of glutathione (GSH) in the culture supernatant. GSH restored growth in most strains as a nutrient source. In contrast, GSH rescued cell morphology defects in the hob3&#x2206; mutant, lacking the Bin/amphiphysin/Rvs (BAR) adaptor protein Hob3, through a mechanism independent of nutrition. This research advances understanding of exometabolite-mediated interactions in S. pombe by identifying GSH as an exometabolite that influences cellular processes and potentially shapes microbial communities. IMPORTANCE: Microorganisms secrete a wide range of metabolites that control microbial community behavior. These extracellular metabolites (exometabolites) include not only well-studied signaling molecules but also diverse primary and secondary metabolites, suggesting complex interactions among microbes. However, the molecular basis of these interactions remains poorly understood, partly due to challenges in detecting them experimentally. In this study, we surveyed exometabolites involved in cell-cell interactions in the model eukaryotic microorganism Schizosaccharomyces pombe. S. pombe releases a wide variety of metabolites outside the cells, including previously reported nitrogen signaling factors (NSFs) and glutathione (GSH) identified in this work. By analyzing gene deletion mutants whose growth is supported by extracellular GSH, we provide new insights into how secreted primary exometabolites compensate for specific genetic defects and influence cell physiology in microbial populations.

exometabolite↗
PubMed2026

Metax enables accurate cross-domain taxonomic profiling of metagenomes.

Taxonomic profiling is fundamental to microbiome research, yet achieving high species-level accuracy remains challenging for complex communities that span bacteria, viruses, eukaryotes, and archaea, and these limitations are exacerbated in low-biomass, host-dominated samples. We introduce Metax, a cross-domain taxonomic profiler that integrates coverage-based probabilistic modeling with an expectation-maximization framework to distinguish true microbial signals from artifacts. Across >600 samples from host-associated, environmental, wastewater, and low-biomass clinical settings, including benchmarks with limited reference representation, Metax improved profiling accuracy, achieving on average 55% higher F1 scores and 45% lower Bray-Curtis dissimilarity than other methods. Moreover, this broad evaluation demonstrated that Metax resolved bacterial and viral signatures of peri-implantitis in oral microbiomes and revealed signals suggestive of reagent-borne contaminants and reference misassemblies in plasma-cell-free DNA. By leveraging genome-wide coverage evidence, Metax enables robust cross-domain profiling across diverse sample types and sequencing depths, including settings where reference databases are highly incomplete.

abundance estimation↗
PubMed1976

Quantitative effects of unsaturated fatty acids in microbial mutants. VI. Selective growth responses of yeast and bacteria to cis-octadecenoate isomers.

The full series of positional isomers of cis-octadecenoate were tested for their suitability in meeting the nutritional requirement for unsaturated fatty acids by mutants of Escherichia coli and Saccharomyces cerevisiae that were unable to synthesize unsaturated fatty acids. Quantitative comparisons of the efficiencies of the various isomers showed a range from 0-48 cells per femtomole for the prokaryotic cells and 0-5 for eukaryotic cells. The delta 5 isomer was much more effective than the delta 6 isomer with the bacterial cells whereas the reverse was true with the yeast cells. In general, isomers containing a cis ethylenic bond between carbons 7 and 12 were able to support extensive growth of either type of mutant. Since all of the various isomers were incorporated into cellular lipids by both types of microorganism, the different efficiencies observed in supporting growth were not a simple reflection of the inability of an acid to be esterified. The differences may reflect the suitability of the resultant esterified product to function as a normal membrane lipid. The contents of various fatty acids in the cellular phospholipids when growth ceases may have a linearly cumulative relationship to the degree of expansion of the acyl chains.

Cell Division↗
PubMed2026

Metagenomic analysis of the midgut microbiome in Dermacentor abaensis ticks at different feeding states.

Ticks are blood-sucking ectoparasites of humans and animals, ranking second only to mosquitoes as vectors of diseases. Dermacentor abaensis is distributed in Sichuan, Qinghai, and Gansu, China. Because D. abaensis harbors several pathogens, it poses a threat to public health and livestock production. However, the midgut microbiota of D. abaensis at distinct feeding states remains poorly characterized. Adult D. abaensis ticks at various feeding states were collected from yaks in Gansu Province, China. Genomic DNA was extracted from midguts and midgut contents of unfed, partially fed, and fully engorged female D. abaensis. A metagenomic sequencing approach was employed to profile the midgut microflora among three groups. A total of 83 phyla, 908 genera, and 1857 species were annotated across the three groups. At the phylum level, Pseudomonadota, Mucoromycota, and Ascomycota were the most abundant. At the species level, common bacterial species such as Klebsiella pneumoniae and Anaplasma phagocytophilum, alongside viruses and eukaryotes, were detected in all three groups. Unique microorganisms were also observed in each group: unfed (n = 305), partially fed (n = 59), and fully engorged (n = 20). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis suggested that the D. abaensis microbiome contains a relatively high abundance of functional genes involved in lipid and amino acid metabolism across the three different feeding states. These findings indicate that while core microbial taxa are shared in the midgut of female D. abaensis, observable trends suggest variations in microbial diversity and composition as blood-feeding progresses. The present study provides a descriptive baseline of the midgut microbial composition of D. abaensis, which may inform future studies on tick biology and the ecology of tick-borne pathogens.

Animals↗
PubMed1978

Chicken ovalbumin is synthesized and secreted by Escherichia coli.

By recombinant DNA methods, the chicken ovalbumin structural gene has been fused to Escherichia coli lac transcriptional and translational control regions. When a plasmid containing the hybrid gene was introduced into E. coli, a protein identified as ovalbumin by immunoreactivity and sodium dodecyl sulfate/polyacrylamide gel electrophoresis was synthesized. The chicken ovalbumin made in bacteria was full length (43,000 daltons) and constituted 1.5% of the cellular protein. In addition, the microbially synthesized ovalbumin was secreted through the cell membrane into the periplasmic space of E. coli. The ability of the E. coli secretory apparatus to recognize chicken ovalbumin, which is normally synthesized and secreted in hen oviducts, suggests that common features exist in the secretion-recognition mechanisms found in these two organisms. The bacterial synthesis of significant amounts of chicken ovalbumin demonstrates that the E. coli cellular machinery may be utilized to synthesize a higher eukaryotic protein which is relatively stable in the bacterial intracellular environment.

Alkaline Phosphatase↗
PubMed1979

[Effect of polyene antibiotics and their perhydrovderivatives on intact cells and protoplasts of yeast Candida guilliermondii].

Perhydroderivatives of polyene antibiotics have a much lower activity against eukaryotic cells than the polyene antibiotics itself. Bacterial cells are normally resistant against most polyene antibiotics and their perhydroderivatives. In earlier experiments with wall less L-form cells of Escherichia coli we have shown that the bacterial cell wall may be responsible for the resistance of the intact bacterial cells against polyene antibiotics and their perhydroderivatives by masking internal target sites. In the present paper we studied the effect of polyene antibiotics and their perhydroderivatives on intact cells and protoplasts of Candida guilliermondii. Our experiments have shown that most of the perhydroderivatives studied had a lower activity against intact cells as well as protoplasts than the corresponding polyene antibiotics. This means that in the case of eukaryotic cells the cell wall as a penetration barrier cannot mainly be responsible for the low activity of perhydroderivatives. The results are compared with those obtained previously with intact cells and protoplast type L-form cells of E. coli.

Anti-Bacterial Agents↗
PubMed1979

Qualitative and quantitative aspects of labeling cell surface carbohydrates using lectins as probes.

Lectins are proteins which bind mono- and oligosaccharides with great specificity. Many polysaccharides, glycoproteins and glycolipids which are important constituents of cell walls and surface membranes of prokaryotic and eukaryotic cells, contain sugar moieties with which lectins can interact. As a result lectins have been extensively used for the study of cell surface and membrane structure of their labeling. This paper reviews and evaluates the available methods for the preparation of fluorescent, electron-dense and radioactive lectin derivatives. The procedures involved in the visualization of lectin binding under light and electron microscopy are described. In addition, the methods for quantitative evaluation of the fluorescence on labeled cells and for analysis of the number of cell surface lectin binding sites using radioactively-labeled lectins are outlined. Examples are given for the application of fluorescent lectin derivatives for the detection of specific saccharide-containing molecules on the surfaces of living or fixed microbial cells and various normal and neoplastic cells. The use of lectins to demonstrate the dynamic nature of cell membranes and to detect changes in membrane structure or organization which occur or organization which occur during differentiation, development or after neoplastic transformation is discussed.

Carbohydrates↗
PubMed2025

Opsins are Phospholipid Scramblases in All Domains of Life.

Opsins are highly abundant retinal proteins in the membranes of photoheterotrophic bacteria. However, some microbial genomes encode an opsin but lack the gene for the final enzyme in retinal synthesis. To account for this paradox, we hypothesized that bacterial opsins play a role in membrane structure and/or biogenesis independent from their potential for light-driven signaling or proton pumping. After purifying actinorhodopsin from a cell-free expression system and from E. coli membranes upon overexpression, we demonstrated both in vitro and in silico that actinorhodopsin from Nanopelagicus ca. is a phospholipid scramblase, serving in its pentameric state as a retinal-independent phospholipid diffusion channel. Phospholipid headgroups move along a transbilayer path between actinorhodopsin protomers, to equilibrate lipid content in the inner and outer leaflets. Two profound activities, membrane biosynthesis and capture of light energy, are thus facilitated by one ancient bacterial polypeptide. Light-dependent activity and light-independent phospholipid scrambling are shared functions of eukaryotic, archaeal, and bacterial rhodopsins.

ActR↗
PubMed2026

Experimental insights in taxon-specific functional responses to droughts in glacier-fed stream biofilms.

BACKGROUND: Glacier-fed streams are predicted to face increasingly frequent and intense droughts. However, the impacts of drought events on benthic biofilm, including bacteria, eukaryotes, and viruses, the dominating life form in glacier-fed streams, remain poorly understood. RESULTS: Using streamside flume mesocosms in the Swiss Alps, we grew glacier-fed stream biofilms over 103&#xa0;days and exposed them to three droughts. Using a multi-omics approach (metagenomics, metatranscriptomics, and metaproteomics), we assessed the effects of a series of droughts on the taxonomy and metabolic activity of bacterial, eukaryotic, and viral metagenome-assembled genomes (MAGs). We found that the first drought (6&#xa0;h) caused only minor changes, including mild upregulation of heterotrophic metabolism and signs of stress in diatoms. In contrast, the second drought (24&#xa0;h) significantly altered both the composition and functionality of the microbiome, shifting phototrophic dominance from diatoms to Cyanobacteriota, while maintaining overall phototropic biomass and further upregulating the heterotrophic metabolism. Interestingly, a third 24&#xa0;h drought had no detectable transcriptomic effect between pre- and post-drought conditions, suggesting a certain level of adaptive responses to droughts, but with the low diatom abundance being maintained. CONCLUSIONS: These findings indicate that glacier-fed biofilm microorganisms initially resisted short-term drought, but a second longer drought caused important shifts in their community structure, activity, and function. Climate-induced increases in drought frequency or duration may therefore have a lasting impact on microbial ecosystem functioning in glacier-fed streams. Video Abstract.

Biofilms↗