Identification of PTSFru as the major fructose uptake system of Clostridium acetobutylicum

As a member of the saccharolytic clostridia, a variety of different carbohydrates like glucose, fructose, or mannose can be used as carbon and energy source by Clostridium acetobutylicum ATCC 824. Thirteen phosphoenolpyruvate-dependent phosphotransferase systems (PTS) have been identified in C. acetobutylicum, which are likely to be responsible for the uptake of hexoses, hexitols, or disaccharides. Here, we focus on three PTS which are expected to be involved in the uptake of fructose, PTS^Fru, PTS^ManI, and PTS^ManII. To analyze their individual functions, each PTS was inactivated via homologous recombination or insertional mutagenesis. Standardized comparative batch fermentations in a synthetic medium with glucose, fructose, or mannose as sole carbon source identified PTS^Fru as primary uptake system for fructose, whereas growth with fructose was not impaired in PTS^ManI and slightly altered in PTS^ManII-deficient strains of C. acetobutylicum. The inactivation of PTS^ManI resulted in slower growth on mannose whereas the loss of PTS^ManII revealed no phenotype during growth on mannose. This is the first time that it has been shown that PTS^Fru and PTS^ManI of C. acetobutylicum are directly involved in fructose and mannose uptake, respectively. Moreover, comprehensive comparison of the fermentation products revealed that the loss of PTS^Fru prevents the solvent shift as no butanol and only basic levels of acetone and ethanol could be determined.     

Relationship between pseudo-HPr and the PEP: fructose phosphotransferase system in Salmonella typhimurium and Escherichia coli

Summary We have studied in Salmonella typhimurium and Escherichia coli the properties of pseudo-HPr suppressor mutations. These mutations suppressed the defects in a ptsH mutant which lacks HPr, one of the enzymes of the phosphoenolpyruvate: carbohydrate phosphotransferase system. The suppressor mutation was mapped in S. typhimurium at 3 min, closely linked to leu. The corresponding chromosomal fragment of 1.7 kb from S. typhimurium and E. coli (extending clockwise from ilvH) was cloned. In a maxicell system a protein with an approximate molecular weight of 36,000 was synthesized. Pseudo-HPr suppressor mutations (fruR) and a deletion extending clockwise from leu resulted in the constitutive expression of the fru operon containing the genes for II^Fru (fruA), III^Fru (fruB), fructose 1-phosphate kinase (fruK) and pseudo-HPr (fruF). fruR probably codes for a repressor of the fru operon. Tn10 mutagenesis revealed the following order of genes in the fru operon: fruB-(fruK, fruF)-fruA. Pseudo-HPr activity could replace HPr in PEP-dependent phosphorylation of PTS carbohydrates. III^Fru could be phosphorylated both via HPr and pseudo-HPr, since mutants lacking pseudo-HPr activity were still able to phosphorylate fructose in the presence of added HPr. Both the pseudo-HPr suppressor mutations at 3 min and the deletion extending from leu had an additional phenotype. Introduction of these mutations or deletions was always accompanied by disappearance of PEP synthase activity. Complementation of such a mutant with the cloned fragments reversed both phenotypes at the same time. Possibly, the fruR gene product acts as an activator of the gene coding for PEP synthase.     

Two Uptake Systems for Fructose in Lactococcus lactis subsp. cremoris FD1 Produce Glycolytic and Gluconeogenic Fructose Phosphates and Induce Oscillations in Growth and Lactic Acid Formation

Fructose transport in lactococci is mediated by two phosphotransferase systems (PTS). The constitutive mannose PTS has a broad specificity and may be used for uptake of fructose with a fructose saturation constant (K_Fru) of 0.89 mM, giving intracellular fructose 6-phosphate. The inducible fructose PTS has a very small saturation constant (K_Fru, <17 μM), and the fructose 1-phosphate produced enters the Embden-Meyerhof-Parnas (EMP) pathway as fructose 1,6-diphosphate. Growth in batch cultures of Lactococcus lactis subsp. cremoris FD1 in a yeast extract medium with fructose as the only sugar is poor both with respect to specific growth rate and biomass yield, whereas the specific lactic acid production rate is higher than those in similar fermentations on other sugars metabolized via the EMP pathway, e.g., glucose. In fructose-limited chemostat cultures, the biomass concentration exhibits a strong correlation with the dilution rate, and starting a continuous culture at the end of a batch fermentation leads to large and persistent oscillations in the biomass concentration and specific lactic acid production rate. Two proposed mechanisms underlying this strange growth pattern follow. (i) Fructose transported via the fructose PTS cannot be converted into essential biomass precursors (glucose 6-phosphate or fructose 6-phosphate), because L. lactis subsp. cremoris FD1 is devoid of fructose 1,6-diphosphatase activity. (ii) The fructose PTS apparently produces a metabolite (presumably fructose 1-phosphate) which exerts catabolite repression of both mannose PTS and lactose PTS. Since the repressed mannose PTS and lactose PTS are shown to have identical maximum molar transport rates, the results indicate that it is the general PTS proteins which are repressed.     

Metabolic Fluxes in Corynebacterium glutamicum during Lysine Production with Sucrose as Carbon Source

Metabolic fluxes in the central metabolism were determined for lysine-producing Corynebacterium glutamicum ATCC 21526 with sucrose as a carbon source, providing an insight into molasses-based industrial production processes with this organism. For this purpose, ¹³C metabolic flux analysis with parallel studies on [1-¹³C^Fru]sucrose, [1-¹³C^Glc]sucrose, and [¹³C₆^Fru]sucrose was carried out. C. glutamicum directed 27.4% of sucrose toward extracellular lysine. The strain exhibited a relatively high flux of 55.7% (normalized to an uptake flux of hexose units of 100%) through the pentose phosphate pathway (PPP). The glucose monomer of sucrose was completely channeled into the PPP. After transient efflux, the fructose residue was mainly taken up by the fructose-specific phosphotransferase system (PTS) and entered glycolysis at the level of fructose-1,6-bisphosphate. Glucose-6-phosphate isomerase operated in the gluconeogenetic direction from fructose-6-phosphate to glucose-6-phosphate and supplied additional carbon (7.2%) from the fructose part of the substrate toward the PPP. This involved supply of fructose-6-phosphate from the fructose part of sucrose either by PTS^Man or by fructose-1,6-bisphosphatase. C. glutamicum further exhibited a high tricarboxylic acid (TCA) cycle flux of 78.2%. Isocitrate dehydrogenase therefore significantly contributed to the total NADPH supply of 190%. The demands for lysine (110%) and anabolism (32%) were lower than the supply, resulting in an apparent NADPH excess. The high TCA cycle flux and the significant secretion of dihydroxyacetone and glycerol display interesting targets to be approached by genetic engineers for optimization of the strain investigated.     

Molecular analysis of two ScrR repressors and of a ScrR–FruR hybrid repressor for sucrose and D-fructose specific regulons from enteric bacteria

The scr regulon of pUR400 and the chromosomally encoded scr regulon of Klebsiella pneumoniae KAY2026 are both negatively controlled by a specific repressor (ScrR). As deduced from the nucleotide sequences, both scrR genes encode polypeptides of 334 residues (85.5% identical base pairs, 91.3% identical amino acids), containing an N-terminal helix-turn-helix motif. Comparison with other regulatory proteins revealed 30.6% identical amino acids to FruR, 27.0% to Lacl and 28.1% to GaIR. Six scrR^s super-repressor mutations define the inducer-binding domain. The scr operator sequences were identified by in vivo titration tests of the sucrose repressor and by in vitro electrophoretic mobility shift assays. D-fructose, an intracellular product of sucrose transport and hydrolysis, and D-fructose 1-phosphate were shown to be molecular inducers of both scr regulons. An active ScrR–FruR hybrid repressor protein was constructed with the N-terminal part of the sucrose repressor of K. pneumoniae and the C-terminal part of the fructose repressor of Salmonella typhimurium, LT2. Gel retardation assays showed that the hybrid protein bound to scr-specific operators, and that D-fructose 1-phosphate, the inducer for FruR, was the only inducer. In vivo, neither the operators of the fru operon nor of the pps, operon, the natural targets for FruR, were recognized, but the scr operators were. These data and the data obtained from the super-repressor alleles confirm previous models on the binding of repressors of the Lacl family to their operators.     

ATP-dependent fructose uptake system in <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

The bacterial phosphoenolpyruvate (PEP)-dependent group translocation system (PTS) requires the presence of both membrane-bound and cytoplasmic components to phosphorylate and translocate sugar. Deinococcus radiodurans has a functional fruA gene coding for the membrane-bound components of the fructose-specific PTS. However, fruB gene coding for the fructose-specific cytosolic components of PTS is a pseudogene. Yet, this bacterium metabolized fructose readily. In vitro studies showed that both cell membranes and cytoplasmic fractions of the cells were needed for fructose phosphorylation. Further studies showed that fructose phosphorylation required ATP, not PEP, as the phosphate donor. Unlike most PEP-dependent PTS systems, fructose phosphorylation is sensitive to sodium fluoride, a kinase inhibitor. Fructose phosphorylation was also inhibited in the presence of antiserum against a kinase phosphorylation site. Rhodobacter capsulatus has a functional fruA–fruB system. Complementation assays by reconstituting the membrane fraction of D. radiodurans to the cytoplasmic fraction of R. capsulatus resulted in a PEP-dependent fructose phosphorylation, whereas mixing the membranes of R. capsulatus and the deinococcal cytosol in vitro resulted in an ATP-dependent fructose phosphorylation.     

From fruitless to sex: On the generation and diversification of an innate behavior

Male sexual behavior in Drosophila melanogaster, largely controlled by the fruitless (fru) gene encoding the male specific Fru^M protein, is among the best studied animal behaviors. Although substantial studies suggest that Fru^M specifies a neuronal circuitry governing all aspects of male sexual behaviors, recent findings show that Fru^M is not absolutely necessary for such behaviors. We propose that another regulatory gene doublesex encoding the male-specific Dsx^M protein builds a core neuronal circuitry that possesses the potential for courtship, which could be either induced through adult social experience or innately manifested during development by Fru^M expression in a broader neuronal circuitry. Fru^M expression levels and patterns determine the modes of courtship behavior from innate heterosexual, homosexual, bisexual, to learned courtship. We discuss how Fru^M expression is regulated by hormones and social experiences and tunes functional flexibility of the sex circuitry. We propose that regulatory genes hierarchically build the potential for innate and learned aspects of courtship behaviors, and expression changes of these regulatory genes among different individuals and species with different social experiences ultimately lead to behavioral diversification.     

Genetic and Biochemical Characterization of the Phosphoenolpyruvate:Glucose/Mannose Phosphotransferase System of Streptococcus thermophilus

In most streptococci, glucose is transported by the phosphoenolpyruvate (PEP):glucose/mannose phosphotransferase system (PTS) via HPr and IIAB^Man, two proteins involved in regulatory mechanisms. While most strains of Streptococcus thermophilus do not or poorly metabolize glucose, compelling evidence suggests that S. thermophilus possesses the genes that encode the glucose/mannose general and specific PTS proteins. The purposes of this study were to determine (i) whether these PTS genes are expressed, (ii) whether the PTS proteins encoded by these genes are able to transfer a phosphate group from PEP to glucose/mannose PTS substrates, and (iii) whether these proteins catalyze sugar transport. The pts operon is made up of the genes encoding HPr (ptsH) and enzyme I (EI) (ptsI), which are transcribed into a 0.6-kb ptsH mRNA and a 2.3-kb ptsHI mRNA. The specific glucose/mannose PTS proteins, IIAB^Man, IIC^Man, IID^Man, and the ManO protein, are encoded by manL, manM, manN, and manO, respectively, which make up the man operon. The man operon is transcribed into a single 3.5-kb mRNA. To assess the phosphotransfer competence of these PTS proteins, in vitro PEP-dependent phosphorylation experiments were conducted with purified HPr, EI, and IIAB^Man as well as membrane fragments containing IIC^Man and IID^Man. These PTS components efficiently transferred a phosphate group from PEP to glucose, mannose, 2-deoxyglucose, and (to a lesser extent) fructose, which are common streptococcal glucose/mannose PTS substrates. Whole cells were unable to catalyze the uptake of mannose and 2-deoxyglucose, demonstrating the inability of the S. thermophilus PTS proteins to operate as a proficient transport system. This inability to transport mannose and 2-deoxyglucose may be due to a defective IIC domain. We propose that in S. thermophilus, the general and specific glucose/mannose PTS proteins are not involved in glucose transport but might have regulatory functions associated with the phosphotransfer properties of HPr and IIAB^Man.