Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for "consolidated bioprocessing" (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts.

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Microbial cellulose utilization: fundamentals and biotechnology.

Bile salt biotransformations by human intestinal bacteria.

https://www.cell.com/cell-metabolism/pdf/S1550-4131(16)30223-6.pdf

Intestinal Crosstalk between Bile Acids and Microbiota and Its Impact on Host Metabolism.

Short-chain dehydrogenases/reductases (SDR) constitute a large protein family. Presently, at least 57 characterized, highly different enzymes belong to this family and typically exhibit residue identities only at the 15-30% level, indicating early duplicatory origins and extensive divergence. In addition, another family of 22 enzymes with extended protein chains exhibits part-chain SDR relationships and represents enzymes of no less than three EC classes. Furthermore, subforms and species variants are known of both families. In the combined SDR superfamily, only one residue is strictly conserved and ascribed a crucial enzymatic function (Tyr 151 in the numbering system of human NAD(+)-linked prostaglandin dehydrogenase). Such a function for this Tyr residue in SDR enzymes in general is supported also by chemical modifications, site-directed mutagenesis, and an active site position in those tertiary structures that have been characterized. A lysine residue four residues downstream is also largely conserved. A model for catalysis is available on the basis of these two residues. Binding of the coenzyme, NAD(H) or NADP(H), is in the N-terminal part of the molecules, where a common GlyXXXGlyXGly pattern occurs. Two SDR enzymes established by X-ray crystallography show a one-domain subunit with seven to eight beta-strands. Conformational patterns are highly similar, except for variations in the C-terminal parts. Additional structures occur in the family with extended chains. Some of the SDR molecules are known under more than one name, and one of the enzymes has been shown to be susceptible to native, chemical modification, producing reduced Schiff base adducts with pyruvate and other metabolic keto derivatives. Most SDR enzymes are dimers and tetramers. In those analyzed, the area of major subunit contacts involves two long alpha-helices (alpha E, alpha F) in similar and apparently strong subunit interactions. Future possibilities include verification of the proposed reaction mechanism and tracing of additional relationships, perhaps also with other protein families. Short-chain dehydrogenases illustrate the value of comparisons and diversified research in generating unexpected discoveries.

Short-chain dehydrogenases/reductases (SDR).

Bile acids are increasingly being appreciated as complex metabolic integrators and signalling factors and not just as lipid solubilizers and simple regulators of bile-acid homeostasis. It is therefore not surprising that a number of bile-acid-activated signalling pathways have become attractive therapeutic targets for metabolic disorders. Here, we review how the signalling functions of bile acids can be exploited in the development of drugs for obesity, type 2 diabetes, hypertriglyceridaemia and atherosclerosis, as well as other associated chronic diseases such as non-alcoholic steatohepatitis.

Targeting bile-acid signalling for metabolic diseases

Southern blot analysis indicated that the gene encoding the constitutive, NADP-linked bile acid 7 alpha-hydroxysteroid dehydrogenase of Eubacterium sp. strain VPI 12708 was located on a 6.5-kb EcoRI fragment of the chromosomal DNA. This fragment was cloned into bacteriophage lambda gt11, and a 2.9-kb piece of this insert was subcloned into pUC19, yielding the recombinant plasmid pBH51. DNA sequence analysis of the 7 alpha-hydroxysteroid dehydrogenase gene in pBH51 revealed a 798-bp open reading frame, coding for a protein with a calculated molecular weight of 28,500. A putative promoter sequence and ribosome binding site were identified. The 7 alpha-hydroxysteroid dehydrogenase mRNA transcript in Eubacterium sp. strain VPI 12708 was about 0.94 kb in length, suggesting that it is monocistronic. An Escherichia coli DH5 alpha transformant harboring pBH51 had approximately 30-fold greater levels of 7 alpha-hydroxysteroid dehydrogenase mRNA, immunoreactive protein, and specific activity than Eubacterium sp. strain VPI 12708. The 7 alpha-hydroxysteroid dehydrogenase purified from the pBH51 transformant was similar in subunit molecular weight, specific activity, and kinetic properties to that from Eubacterium sp. strain VPI 12708, and it reached with antiserum raised against the authentic enzyme on Western immunoblots. Alignment of the amino acid sequence of the 7 alpha-hydroxysteroid dehydrogenase with those of 10 other pyridine nucleotide-linked alcohol/polyol dehydrogenases revealed six conserved amino acid residues in the N-terminal regions thought to function in coenzyme binding.

Cloning, sequencing, and expression of the gene coding for bile acid 7 alpha-hydroxysteroid dehydrogenase from Eubacterium sp. strain VPI 12708.

We previously cloned a genomic DNA fragment from the serogroup O11 Pseudomonas aeruginosa strain PA103 that contained all genes necessary for O-antigen synthesis and directed the expression of serogroup O11 antigen on recombinant Escherichia coli and Salmonella. To elucidate the pathway of serogroup O11 antigen synthesis, the nucleotide sequence of the biosynthetic genes was determined. Eleven open reading frames likely to be involved in serogroup O11 O-antigen biosynthesis were identified and are designated in order as wzzPaO111 (wzz from P. aeruginosa serogroup O11), wzxPaO11, wbjA, wzyPaO11, wbjB to wbjF, wbpLO11 and wbpMO11 (wbpL and wbpM from serogroup O11). Consistent with previous descriptions of O-antigen biosynthetic gene loci, the entire region with the exception of wbpMO11 has a markedly reduced G+C content relative to the chromosomal average. WzyPaO11 shows no significant similarity at the protein or DNA sequence level to any database sequence and is very hydrophobic, with 10 to 12 putative transmembrane domains, both typical characteristics of O-antigen polymerases. A nonpolar chromosomal insertion mutation in wzyPaO11 in P. aeruginosa PA103 confirmed the identity of this gene. There is striking similarity between WbjBCDE and Cap(5/8)EFGL, involved in type 5 and type 8 capsule biosynthesis in Staphylococcus aureus. There is nearly total identity between wbpMO11 and wbpMO5, previously shown by others to be present in all 20 P. aeruginosa serogroups. Using similarity searches, we have assigned functions to the proteins encoded by the PA103 O-antigen locus and present the potential steps in the pathway for the biosynthesis of P. aeruginosa serogroup O11 O antigen.

Characterization of the serogroup O11 O-antigen locus of Pseudomonas aeruginosa PA103.

https://www.gastrojournal.org/article/S0016-5085(01)27404-6/pdf

Expression of sterol 12α-hydroxylase alters bile acid pool composition in primary rat hepatocytes and in vivo

A cholate-inducible, NADH-dependent flavin oxidoreductase from the intestinal bacterium Eubacterium sp. strain VPI 12708 was purified 372-fold to apparent electrophoretic homogeneity. The subunit and native molecular weights were estimated to be 72,000 and 210,000, respectively, suggesting a homotrimeric organization. Three peaks of NADH:flavin oxidoreductase activity (forms I, II, and III) eluted from a DEAE-high-performance liquid chromatography column. Absorption spectra revealed that purified form III, but not form I, contained bound flavin, which dissociated during purification to generate form I. Enzyme activity was inhibited by sulfhydryl-reactive compounds, acriflavine, o-phenanthroline, and EDTA. Activity assays and Western blot (immunoblot) analysis confirmed that expression of the enzyme was cholate inducible. The first 25 N-terminal amino acid residues of purified NADH:flavin oxidoreductase were determined, and a corresponding oligonucleotide probe was synthesized for use in cloning of the associated gene, baiH. Restriction mapping, sequence data, and RNA blot analysis suggested that the baiH gene was located on a previously described, cholate-inducible operon > or = 10 kb long. The baiH gene encoded a 72,006-Da polypeptide containing 661 amino acids. The deduced amino acid sequence of the baiH gene was homologous to that of NADH oxidase from Thermoanaerobium brockii, trimethylamine dehydrogenase from methylotrophic bacterium W3A1, Old Yellow Enzyme from Saccharomyces carlsbergensis, and the product of the baiC gene of Eubacterium sp. strain VPI 12708, located upstream from the baiH gene in the cholate-inducible operon. Alignment of these five sequences revealed potential ligands for an iron-sulfur cluster, a putative flavin adenine dinucleotide-binding domain, and two other well-conserved domains of unknown function.

C V Franklund

Papers

Cloning, sequencing, and expression of the gene coding for bile acid 7 alpha-hydroxysteroid dehydrogenase from Eubacterium sp. strain VPI 12708.

Characterization of the serogroup O11 O-antigen locus of Pseudomonas aeruginosa PA103.

Expression of sterol 12α-hydroxylase alters bile acid pool composition in primary rat hepatocytes and in vivo

Characterization of the baiH gene encoding a bile acid-inducible NADH:flavin oxidoreductase from Eubacterium sp. strain VPI 12708.

Mechanism of intestinal 7 alpha-dehydroxylation of cholic acid: evidence that allo-deoxycholic acid is an inducible side-product.