Regulation of lactate metabolism in the acetogenic bacterium Acetobacterium woodii

Abstract

Acetogenic bacteria compete in an energy-limited environment by coupling different metabolic routes to their central metabolism of CO fixation. The underlying regulatory mechanisms are often still not understood. In this work, we analysed how lactate metabolism is regulated in the model acetogen Acetobacterium woodii. Construction of a ΔlctCDEF mutant and growth analyses demonstrated that the genes are essential for growth on lactate. Subsequent bridging PCR and quantitative PCR analyses revealed that the lctBCDEF genes form an operon that was expressed only during lactate metabolism. The lctA gene was cloned, expressed in Escherichia coli and purified. LctA bound to the intergenic DNA region between lctA and the lct operon in electromobility shift assays, and binding was revoked in the presence of lactate. Further restriction site protection analyses consolidated the lactate-dependent binding of LctA and identified the binding site within the DNA. Cells grew mixotrophically on lactate and another energy source and showed no diauxic growth. From these data, we conclude that the catabolic lactate metabolism is encoded by the lct operon and its expression is negatively regulated by the DNA-binding repressor LctA.

MtcB, a member of the MttB superfamily from the human gut acetogen Eubacterium limosum, is a cobalamin-dependent carnitine demethylase

“…7). L-Carnitine-dependent methylation of THF would be crucial for methylotrophic acetogenesis with L-carnitine as the major growth substrate but would not be required for the proposed pathway of acetogenesis from lactate (45) in which lactate is initially catabolized by a regulated lactate dehydrogenase (44). Correspondingly, MtcB is only abundant in cells grown on L-carnitine and not on lactate.…”

“…Abundant catabolic proteins specific to growth on each substrate were also identified (Table S2). For example, in lactate-grown cells, a lactate dehydrogenase and associated proteins (44,45) were abundant among the ;1630 proteins identified. These same proteins were at very low abundance or not detectable among the ;1400 proteins identified from cells grown on L-carnitine.…”

It does not always take two to tango: “Syntrophy” via hydrogen cycling in one bacterial cell

“…substrates were used at a pressure of 1.0 × 10 5 Pa. For growth experiments, concentrations of the used carbon sources were as follows: lactate, 80 mM; ethanol, 50 mM; formate, 100 mM. Minimal medium used for genetic manipulations was prepared as previously described [19] in which yeast extract was omitted and higher amounts of 0.2 g l −1 KH 2 PO 4 , 1.35 g l −1 NH 4 Cl, and 1.5 ml l −1 of selenite/tungstate solution were used and 10 µg ml −1 of D/L-pantothenate was added. For mutant selection, 1.7 µg ml −1 uracil and 1.5 mg ml −1 5-fluoroorotic acid were added.…”

Synthetic Biology on Acetogenic Bacteria for Highly Efficient Conversion of C1 Gases to Biochemicals

“…Nevertheless, plasmid-based gene expression methods developed for Clostridium species have been shown to be functional in other acetogens. In addition, many metabolic engineering efforts have recently been made using homologous recombination (HR) [ 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 ], ClosTron [ 28 , 36 , 37 , 38 , 39 ] and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) along with its CRISPR-associated (Cas) protein (CRISPR-Cas) system [ 32 , 40 , 41 , 42 , 43 , 44 , 45 ] to improve the production of value-added biochemicals from C1 gases.…”

“…Consequently, double-crossover mutants can be readily isolated in uracil-free medium as they restore uracil protrophy [ 74 ]. This approach has been employed in A. woodii [ 25 , 26 , 27 ], C. autoethanogenum [ 28 ], Moorella thermoacetica [ 33 ], and Thermoanaerobacter kivui [ 34 , 35 ]. For example, a uracil auxotrophic mutant, ∆ pyrE , was first generated in A. woodii to successfully delete rnf genes [ 25 ].…”