Uncoupling Fermentative Synthesis of Molecular Hydrogen from Biomass Formation inThermotoga maritima

ABSTRACTWhen carbohydrates are fermented by the hyperthermophilic anaerobeThermotoga maritima, molecular hydrogen (H2) is formed in strict proportion to substrate availability. Excretion of the organic acids acetate and lactate provide an additional sink for removal of excess reductant. However, mechanisms controlling energy management of these metabolic pathways are largely unexplored. To investigate this topic, transient gene inactivation was used to block lactate production as a strategy to produce spontaneous mutant cell lines that overproduced H2through mutation of unpredicted genetic targets. Single-crossover homologous chromosomal recombination was used to disrupt lactate dehydrogenase (encoded byldh) with a truncatedldhfused to a kanamycin resistance cassette expressed from a native PgroESLpromoter. Passage of the unstable recombinant resulted in loss of the genetic marker and recovery of evolved cell lines, including strain Tma200. Relative to the wild type, and considering the mass balance of fermentation substrate and products, Tma200 grew more slowly, produced H2at levels above the physiologic limit, and simultaneously consumed less maltose while oxidizing it more efficiently. Whole-genome resequencing indicated that the ABC maltose transporter subunit, encoded bymalK3, had undergone repeated mutation, and high-temperature anaerobic [14C]maltose transport assays demonstrated that the rate of maltose transport was reduced. Transfer of themalK3mutation into a clean genetic background also conferred increased H2production, confirming that the mutant allele was sufficient for increased H2synthesis. These data indicate that a reduced rate of maltose uptake was accompanied by an increase in H2production, changing fermentation efficiency and shifting energy management.IMPORTANCEBiorenewable energy sources are of growing interest to mitigate climate change, but like other commodities with nominal value, require innovation to maximize yields. Energetic considerations constrain production of many biofuels, such as molecular hydrogen (H2) because of the competing needs for cell mass synthesis and metabolite formation. Here we describe cell lines of the extremophileThermotoga maritimathat exceed the physiologic limits for H2formation arising from genetic changes in fermentative metabolism. These cell lines were produced using a novel method called transient gene inactivation combined with adaptive laboratory evolution. Genome resequencing revealed unexpected changes in a maltose transport protein. Reduced rates of sugar uptake were accompanied by lower rates of growth and enhanced productivity of H2.

Investigation of themalEPromoter and MalR, a Positive Regulator of the Maltose Regulon, for an Improved Expression System in Sulfolobus acidocaldarius

ABSTRACTIn this study, the regulator MalR (Saci_1161) of the TrmB family fromSulfolobus acidocaldariuswas identified and was shown to be involved in transcriptional control of the maltose regulon (Saci_1660 to Saci_1666), including the ABC transporter (malEFGK), α-amylase (amyA), and α-glycosidase (malA). The ΔmalRdeletion mutant exhibited a significantly decreased growth rate on maltose and dextrin but not on sucrose. The expression of the genes organized in the maltose regulon was induced only in the presence of MalR and maltose in the growth medium, indicating that MalR, in contrast to its TrmB and TrmB-like homologues, is an activator of the maltose gene cluster. Electrophoretic mobility shift assays revealed that the binding of MalR tomalEwas independent of sugars. Here we report the identification of the archaeal maltose regulator protein MalR, which acts as an activator and controls the expression of genes involved in maltose transport and metabolic conversion inS. acidocaldarius, and its use for improvement of theS. acidocaldariusexpression system under the control of an optimized maltose binding protein (malE) promoter by promoter mutagenesis.

Obacunone Represses Salmonella Pathogenicity Islands 1 and 2 in anenvZ-Dependent Fashion

ABSTRACTObacunone belongs to a class of unique triterpenoids called limonoids, present inCitrusspecies. Previous studies from our laboratory suggested that obacunone possesses antivirulence activity and demonstrates inhibition of cell-cell signaling inVibrio harveyiandEscherichia coliO157:H7. The present work sought to determine the effect of obacunone on the food-borne pathogenSalmonella entericaserovar Typhimurium LT2 by using a cDNA microarray. Transcriptomic studies indicated that obacunone repressesSalmonellapathogenicity island 1 (SPI1), the maltose transporter, and the hydrogenase operon. Furthermore, phenotypic data for the Caco-2 infection assay and maltose utilization were in agreement with microarray data suggesting repression of SPI1 and maltose transport. Further studies demonstrated that repression of SPI1 was plausibly mediated throughhilA. Additionally, obacunone seems to repress SPI2 under SPI2-inducing conditions as well as in Caco-2 infection models. Furthermore, obacunone seems to represshilAin an EnvZ-dependent fashion. Altogether, the results of the study seems to suggest that obacunone exerts an antivirulence effect onS.Typhimurium and may serve as a lead compound for development of antivirulence strategies forS.Typhimurium.

Exposure to Glycolytic Carbon Sources Reveals a Novel Layer of Regulation for the MalT Regulon

Bacteria adapt to changing environments by means of tightly coordinated regulatory circuits. The use of synthetic lethality, a genetic phenomenon in which the combination of two nonlethal mutations causes cell death, facilitates identification and study of such circuitry. In this study, we show that theE.coli ompR malTcondouble mutant exhibits a synthetic lethal phenotype that is environmentally conditional. MalTcon, the constitutively active form of the maltose system regulator MalT, causes elevated expression of the outer membrane porin LamB, which leads to death in the absence of the osmoregulator OmpR. However, the presence and metabolism of glycolytic carbon sources, such as sorbitol, promotes viability and unveils a novel layer of regulation within the complex circuitry that controls maltose transport and metabolism.