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ABC Transporters Required for Hexose Uptake by Clostridium phytofermentans.

Journal of bacteriology (2019-05-22)
Tristan Cerisy, Alba Iglesias, William Rostain, Magali Boutard, Christine Pelle, Alain Perret, Marcel Salanoubat, Henri-Pierre Fierobe, Andrew C Tolonen

The mechanisms by which bacteria uptake solutes across the cell membrane broadly impact their cellular energetics. Here, we use functional genomic, genetic, and biophysical approaches to reveal how Clostridium (Lachnoclostridium) phytofermentans, a model bacterium that ferments lignocellulosic biomass, uptakes plant hexoses using highly specific, nonredundant ATP-binding cassette (ABC) transporters. We analyze the transcription patterns of its 173 annotated sugar transporter genes to find those upregulated on specific carbon sources. Inactivation of these genes reveals that individual ABC transporters are required for uptake of hexoses and hexo-oligosaccharides and that distinct ABC transporters are used for oligosaccharides versus their constituent monomers. The thermodynamics of sugar binding shows that substrate specificity of these transporters is encoded by the extracellular solute-binding subunit. As sugars are not phosphorylated during ABC transport, we identify intracellular hexokinases based on in vitro activities. These mechanisms used by Clostridia to uptake plant hexoses are key to understanding soil and intestinal microbiomes and to engineer strains for industrial transformation of lignocellulose.IMPORTANCE Plant-fermenting Clostridia are anaerobic bacteria that recycle plant matter in soil and promote human health by fermenting dietary fiber in the intestine. Clostridia degrade plant biomass using extracellular enzymes and then uptake the liberated sugars for fermentation. The main sugars in plant biomass are hexoses, and here, we identify how hexoses are taken in to the cell by the model organism Clostridium phytofermentans We show that this bacterium uptakes hexoses using a set of highly specific, nonredundant ABC transporters. Once in the cell, the hexoses are phosphorylated by intracellular hexokinases. This study provides insight into the functioning of abundant members of soil and intestinal microbiomes and identifies gene targets to engineer strains for industrial lignocellulosic fermentation.

Product Number
Product Description

D-(+)-Glucose, ACS reagent
D-(+)-Galactose, ≥99%
TRI Reagent®, for DNA, RNA and protein isolation
GenElute Bacterial Genomic DNA Kits, sufficient for 70 purifications
L-Lactic Dehydrogenase from rabbit muscle, Type II, ammonium sulfate suspension, 800-1,200 units/mg protein
Pefabloc® SC, analytical standard
D-(+)-Cellobiose, ≥98%
Glucose-6-phosphate Dehydrogenase from baker's yeast (S. cerevisiae), Type VII, ammonium sulfate suspension, ≥200 units/mg protein
Pyruvate Kinase from rabbit muscle, Type II, ammonium sulfate suspension, 350-600 units/mg protein
D-Galacturonic acid sodium salt, ≥95.0% (T)
Cellotetraose, ≥85% (HPLC)