ABC Transporters Required for Hexose Uptake by Clostridium phytofermentans

Abstract

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.

A Targetron-Recombinase System for Large-Scale Genome Engineering of Clostridia

“…Conjugal transfer of lox71 and lox66 targetron plasmids into C. phytofermentans consistently yielded 10 to 20 transconjugant colonies, of which 80% to 100% contained the expected targetron insertion, indicating that the efficiency of delivery and integration of lox -containing targetrons is similar to that in previous targetrons studies in C. phytofermentans (10, 26, 27). Following sequential delivery and curing of pQlox71R.2944 and pQlox66F.2993, PCR amplification of cphy2944 and cphy2993 showed targetron insertions in both genes (Fig.…”

“…The absence of additional, off-site genomic targetron insertions in strains DI-SS, DI-AS, and int1575 was confirmed by inverse PCR (27). To this end, genomic DNA was extracted and purified from 5 ml of log-phase culture using the GenElute Bacterial Genomic DNA kit (NA2110; Sigma).…”

“…We refer to this double insertion strain as DI-AS (Table 1). Off-site targetron insertions are common (27), which would result in unexpected recombinations. Thus, we applied an inverse PCR assay (27) using primers that anneal to the targetron in order to quantify the number and sequence of genomic targetron insertions.…”

“…Off-site targetron insertions are common (27), which would result in unexpected recombinations. Thus, we applied an inverse PCR assay (27) using primers that anneal to the targetron in order to quantify the number and sequence of genomic targetron insertions. This assay confirmed that DI-AS contains both expected genomic targetrons without any additional off-site insertions (Fig.…”

Handling Several Sugars at a Time: a Case Study of Xyloglucan Utilization by Ruminiclostridium cellulolyticum

“…Nevertheless, this scheme cannot apply to (hemi)cellulolytic bacteria whose selected strategy to utilize plant cell wall polysaccharides involves the uptake of rather large oligosaccharides (7,8), often composed of different monosaccharides (9)(10)(11)(12)(13). Once their cytosolic depolymerization is achieved, the bacterium faces the simultaneous intracellular release of different sugars requiring different metabolic pathways.…”