Strategies Shaping the Transcription of Carbohydrate-Active Enzyme Genes in Aspergillus nidulans

Understanding the coordinated regulation of the hundreds of carbohydrate-active enzyme (CAZyme) genes occurring in the genomes of fungi has great practical importance. We recorded genome-wide transcriptional changes of Aspergillus nidulans cultivated on glucose, lactose, or arabinogalactan, as well as under carbon-starved conditions. We determined both carbon-stress-specific changes (weak or no carbon source vs. glucose) and carbon-source-specific changes (one type of culture vs. all other cultures). Many CAZyme genes showed carbon-stress-specific and/or carbon-source-specific upregulation on arabinogalactan (138 and 62 genes, respectively). Besides galactosidase and arabinan-degrading enzyme genes, enrichment of cellulolytic, pectinolytic, mannan, and xylan-degrading enzyme genes was observed. Fewer upregulated genes, 81 and 107 carbon stress specific, and 6 and 16 carbon source specific, were found on lactose and in carbon-starved cultures, respectively. They were enriched only in galactosidase and xylosidase genes on lactose and rhamnogalacturonanase genes in both cultures. Some CAZyme genes (29 genes) showed carbon-source-specific upregulation on glucose, and they were enriched in β-1,4-glucanase genes. The behavioral ecological background of these characteristics was evaluated to comprehensively organize our knowledge on CAZyme production, which can lead to developing new strategies to produce enzymes for plant cell wall saccharification.

Intracellular copper storage and delivery in a bacterium

A family of cytosolic copper (Cu) storage proteins (the Csps) are widespread in bacteria. The Csps can bind large quantities of Cu(I) via their Cys-lined four-helix bundles, and the majority are cytosolic (Csp3s). This is inconsistent with the current dogma that bacteria, unlike eukaryotes, have evolved not to maintain intracellular pools of Cu due to its potential toxicity. Sporulation in Bacillus subtilis has been used to investigate if a Csp3 can store Cu(I) in the cytosol for a target enzyme. The activity of the Cu-requiring endospore multi-Cu oxidase BsCotA (a laccase) increases under Cu-replete conditions in wild type B. subtilis, but not in the strain lacking BsCsp3. Cuprous ions readily transfer from BsCsp3, but not from the cytosolic copper metallochaperone BsCopZ, to BsCotA in vitro producing active enzyme. Both BsCsp3 and BsCotA are upregulated during late sporulation. The hypothesis we propose is that BsCsp3 acquires and stores Cu(I) in the cytosol for BsCotA.

A GH89 human α-N-acetylglucosaminidase (hNAGLU) homologue from gut microbe Bacteroides thetaiotaomicron capable of hydrolyzing heparosan oligosaccharides

Carbohydrate-Active enZYme (CAZY) GH89 family enzymes catalyze the cleavage of terminal α-N-acetylglucosamine from glycans and glycoconjugates. Although structurally and mechanistically similar to the human lysosomal α-N-acetylglucosaminidase (hNAGLU) in GH89 which is involved in the degradation of heparan sulfate in the lysosome, the reported bacterial GH89 enzymes characterized so far have no or low activity toward α-N-acetylglucosamine-terminated heparosan oligosaccharides, the preferred substrates of hNAGLU. We cloned and expressed several soluble and active recombinant bacterial GH89 enzymes in Escherichia coli. Among these enzymes, a truncated recombinant α-N-acetylglucosaminidase from gut symbiotic bacterium Bacteroides thetaiotaomicron ∆22Bt3590 was found to catalyze the cleavage of the terminal α1–4-linked N-acetylglucosamine (GlcNAc) from a heparosan disaccharide with high efficiency. Heparosan oligosaccharides with lengths up to decasaccharide were also suitable substrates. This bacterial α-N-acetylglucosaminidase could be a useful catalyst for heparan sulfate analysis.

Genomic analysis of family UBA6911 (Group 18 Acidobacteria) expands the metabolic capacities of the phylum and highlights adaptations to terrestrial habitats

Approaches for recovering and analyzing genomes belonging to novel, hitherto unexplored bacterial lineages have provided invaluable insights into the metabolic capabilities and ecological roles of yet-uncultured taxa. The phylum Acidobacteria is one of the most prevalent and ecologically successful lineages on earth yet, currently, multiple lineages within this phylum remain unexplored. Here, we utilize genomes recovered from Zodletone spring, an anaerobic sulfide and sulfur-rich spring in southwestern Oklahoma, as well as from multiple disparate soil and non-soil habitats, to examine the metabolic capabilities and ecological role of members of the Family UBA6911 (group18) Acidobacteria. The analyzed genomes clustered into five distinct genera, with genera Gp18_AA60 and QHZH01 recovered from soils, Genus Ga0209509 from anaerobic digestors, and genera Ga0212092 and UBA6911 from freshwater habitats. All genomes analyzed suggested that members of Acidobacteria group 18 are metabolically versatile heterotrophs capable of utilizing a wide range of proteins, amino acids, and sugars as carbon sources, possess respiratory and fermentative capacities, and display few auxotrophies. Soil-dwelling genera were characterized by larger genome sizes, higher number of CRISPR loci, an expanded carbohydrate active enzyme (CAZyme) machinery enabling de-branching of specific sugars from polymers, possession of a C1 (methanol and methylamine) degradation machinery, and a sole dependence on aerobic respiration. In contrast, non-soil genomes encoded a more versatile respiratory capacity for oxygen, nitrite, sulfate, trimethylamine N-oxide (TMAO) respiration, as well as the potential for utilizing the Wood Ljungdahl (WL) pathway as an electron sink during heterotrophic growth. Our results not only expand our knowledge of the metabolism of a yet-uncultured bacterial lineage, but also provide interesting clues on how terrestrialization and niche adaptation drives metabolic specialization within the Acidobacteria. Importance Members of the Acidobacteria are important players in global biogeochemical cycles, especially in soils. A wide range of Acidobacterial lineages remain currently unexplored. We present a detailed genomic characterization of genomes belonging to the Family UBA6911 (also known as group 18) within the phylum Acidobacteria. The genomes belong to different genera and were obtained from soil (genera Gp18_AA60 and QHZH01), freshwater habitats (genera Ga0212092 and UBA6911), and anaerobic digestor (Genus Ga0209509). While all members of the family shared common metabolic features, e.g. heterotrophic respiratory abilities, broad substrate utilization capacities, and few auxotrophies; distinct differences between soil and non-soil genera were observed. Soil genera were characterized by expanded genomes, higher numbers of CRISPR loci, larger carbohydrate active enzyme (CAZyme) repertoire enabling monomer extractions from polymer side chains, and methylotrophic (methanol and methylamine) degradation capacities. In contrast, non-soil genera encoded more versatile respiratory capacities for utilizing nitrite, sulfate, TMAO, and the WL pathway, in addition to oxygen as electron acceptors. Our results not only broaden our understanding of the metabolic capacities within the Acidobacteria, but also, provide interesting clues on how terrestrialization shaped Acidobacteria evolution and niche adaptation.

Correction to: Combined whole cell wall analysis and streamlined in silico carbohydrate‑active enzyme discovery to improve biocatalytic conversion of agricultural crop residues

An amendment to this paper has been published and can be accessed via the original article.