Characterization of an operon required for growth on cellobiose in Clostridioides difficile

Cellobiose metabolism is linked to the virulence properties in numerous bacterial pathogens. Here, we characterized a putative cellobiose PTS operon of Clostridiodes difficile to investigate the role of cellobiose metabolism in C. difficile pathogenesis. Our gene knockout experiments demonstrated that the putative cellobiose operon enables uptake of cellobiose into C. difficile and allows growth when cellobiose is provided as the sole carbon source in minimal medium. Additionally, using reporter gene fusion assays and DNA pulldown experiments, we show that its transcription is regulated by CelR, a novel transcriptional repressor protein, which directly binds to the upstream region of the cellobiose operon to control its expression. We have also identified cellobiose metabolism to play a significant role in C. difficile physiology as observed by the reduction of sporulation efficiency when cellobiose uptake was compromised in the mutant strain. In corroboration to in vitro study findings, our in vivo hamster challenge experiment showed a significant reduction of pathogenicity by the cellobiose mutant strain in both the primary and the recurrent infection model – substantiating the role of cellobiose metabolism in C. difficile pathogenesis.

Characterization of an operon required for growth on cellobiose in Clostridioides difficile

Cellobiose metabolism is linked to the virulence properties in numerous bacterial pathogens. Here, we characterized a putative cellobiose PTS operon of Clostridiodes difficile to investigate the role of cellobiose metabolism in C. difficile pathogenesis. Our gene knockout experiments demonstrated that the putative cellobiose operon enables uptake of cellobiose into C. difficile and allows growth when cellobiose is provided as the sole carbon source in minimal medium. Additionally, using reporter gene fusion assays and DNA pull-down experiments, we show that its transcription is regulated by CelR, a novel transcriptional repressor protein, which directly binds to the upstream region of the cellobiose operon to control its expression. We have also identified cellobiose metabolism to play a significant role in C. difficile physiology as observed by the reduction of sporulation efficiency when cellobiose uptake was compromised in the mutant strain. In corroboration to in vitro study findings, our in vivo hamster challenge experiment showed a significant reduction of pathogenicity by the cellobiose mutant strain in both the primary and the recurrent infection model- substantiating the role of cellobiose metabolism in C. difficile pathogenesis.

Dissecting cellobiose metabolic pathway and its application in biorefinery through consolidated bioprocessing in Myceliophthora thermophila

Background Lignocellulosic biomass has long been recognized as a potential sustainable source for industrial applications. The costs associated with conversion of plant biomass to fermentable sugar represent a significant barrier to the production of cost-competitive biochemicals. Consolidated bioprocessing (CBP) is considered a potential breakthrough for achieving cost-efficient production of biomass-based fuels and commodity chemicals. During the degradation of cellulose, cellobiose (major end-product of cellulase activity) is catabolized by hydrolytic and phosphorolytic pathways in cellulolytic organisms. However, the details of the two intracellular cellobiose metabolism pathways in cellulolytic fungi remain to be uncovered. Results Using the engineered malic acid production fungal strain JG207, we demonstrated that the hydrolytic pathway by β-glucosidase and the phosphorolytic pathway by phosphorylase are both used for intracellular cellobiose metabolism in Myceliophthora thermophila, and the yield of malic acid can benefit from the energy advantages of phosphorolytic cleavage. There were obvious differences in regulation of the two cellobiose catabolic pathways depending on whether M. thermophila JG207 was grown on cellobiose or Avicel. Disruption of Mtcpp in strain JG207 led to decreased production of malic acid under cellobiose conditions, while expression levels of all three intracellular β-glucosidase genes were significantly up-regulated to rescue the impairment of the phosphorolytic pathway under Avicel conditions. When the flux of the hydrolytic pathway was reduced, we found that β-glucosidase encoded by bgl1 was the dominant enzyme in the hydrolytic pathway and deletion of bgl1 resulted in significant enhancement of protein secretion but reduction of malate production. Combining comprehensive manipulation of both cellobiose utilization pathways and enhancement of cellobiose uptake by overexpression of a cellobiose transporter, the final strain JG412Δbgl2Δbgl3 produced up to 101.2 g/L and 77.4 g/L malic acid from cellobiose and Avicel, respectively, which corresponded to respective yields of 1.35 g/g and 1.03 g/g, representing significant improvement over the starting strain JG207. Conclusions This is the first report of detailed investigation of intracellular cellobiose catabolism in cellulolytic fungus M. thermophila. These results provide insights that can be applied to industrial fungi for production of biofuels and biochemicals from cellobiose and cellulose.

Genetics of the lac-PTS system ofKlebsiella

SummaryThe isolation of a temperature sensitivepts Imutant which fails to utilize lactose provides strong evidence thatKlebsiellastrain CT-1 utilizes lactose via a phosphoenolpyruvate dependent lactose-phosphotransferase system (PTS-lac). We designate this lactose utilization systemeluforevolved lactose utilization. Analysis of a series ofLac−mutants identifies two genes,eluAandeluB, whose function is required for lactose utilization by this pathway. The functions specified by these genes are not known, but neither locus specifies the hydrolytic enzyme phospho-β-galactosidase. A mutant of CT-1, strain RPD-2, exhibits a half-maximal growth rate at a lactose concentration 40 fold lower than that of strain CT-1; and it has aKmfor lactose uptake that is 40 fold lower than that of strain CT-1. That mutation defines the locuseluC, which is assumed to specify the enzyme Il(lac) of the PTS-lactose system. From the observations that (i) cellobiose induces the phospho-β-galactosidase enzyme, (ii) pregrowth in cellobiose dramatically reduces the growth lag when cells are shifted into lactose minimal medium, (iii)eluBmutants exhibit a growth lag when shifted into cellobiose minimal medium, and (iv) lactose induces a phospho-β-glucosidase enzyme; we speculate that the phospho-β-glucosidase enzyme is the same enzyme as the phospho-β-glucosidase that normally functions in cellobiose metabolism. We conclude that the original mutation that allowed CT-1 to utilize lactose was a regulatory mutation that permitted inducible expression of theeluCgene.