Glycerol metabolism induces Listeria monocytogenes biofilm formation at the air-liquid interface.

Listeria monocytogenes is a food-borne pathogen that can grow as a biofilm on surfaces. Biofilm formation in food-processing environments is a big concern for food safety, as it can cause product contamination through the food-processing line. Although motile aerobic bacteria have been described to form biofilms at the air-liquid interface of cell cultures, to our knowledge, this type of biofilm has not been described in L. monocytogenes before. In this study we report L. monocytogenes biofilm formation at the air-liquid interface of aerobically grown cultures, and that this phenotype is specifically induced when the media is supplemented with glycerol as a carbon and energy source. Planktonic growth, metabolic activity assays and HPLC measurements of glycerol consumption over time showed that glycerol utilization in L. monocytogenes is restricted to growth under aerobic conditions. Gene expression analysis showed that genes encoding the glycerol transporter GlpF, the glycerol kinase GlpK and the glycerol 3-phosphate dehydrogenase GlpD were upregulated in the presence of oxygen, and downregulated in absence of oxygen. Additionally, motility assays revealed the induction of aerotaxis in the presence of glycerol. Our results demonstrate that the formation of biofilms at the air-liquid interface is dependent on glycerol-induced aerotaxis towards the surface of the culture, where L. monocytogenes has access to higher concentrations of oxygen, and is therefore able to utilize this compound as a carbon source.

Differential regulation of glyceroneogenesis by glucocorticoids in epididymal and retroperitoneal white adipose tissue from rats.

PURPOSE: Investigate the glycerol-3-phosphate generation pathways in epididymal (EPI) and retroperitoneal (RETRO) adipose tissues from dexamethasone-treated rats. METHODS: Rats were treated with dexamethasone for 7 days. Glycerol-3-phosphate generation pathways via glycolysis, glyceroneogenesis and direct phosphorylation of glycerol were evaluated, respectively, by 2-deoxyglucose uptake, phosphoenolpyruvate carboxykinase (PEPCK-C) activity and pyruvate incorporation into triacylglycerol (TAG)-glycerol, and glycerokinase activity and glycerol incorporation into TAG-glycerol. RESULTS: Dexamethasone treatment markedly decreased the body weight, but increased the weight and lipid content of EPI and RETRO and plasma insulin, glucose, non-esterified fatty acid and TAG levels. EPI and RETRO from dexamethasone-treated rats showed increased rates of de novo fatty acid synthesis (80 and 100%) and basal lipolysis (20%). In EPI, dexamethasone decreased the 2-deoxyglucose uptake (50%), as well as glyceroneogenesis, evidenced by a decrease of PEPCK-C activity (39%) and TAG-glycerol synthesis from pyruvate (66%), but increased the glycerokinase activity (50%) and TAG-glycerol synthesis from glycerol (72%) in this tissue. In spite of a similar reduction in 2-deoxyglucose uptake in RETRO, dexamethasone treatment increased glyceroneogenesis, evidenced by PEPCK activity (96%), and TAG-glycerol synthesis from pyruvate (110%), accompanied by a decrease in glycerokinase activity (50%) and TAG-glycerol synthesis from glycerol (50%). Dexamethasone effects on RETRO were accompanied by a decrease in p-Akt content and by lower insulin effects on the rates of glycerol release in the presence of isoproterenol and on the rates of glucose uptake in isolated adipocytes. CONCLUSION: Our data demonstrated differential regulation of glyceroneogenesis and direct phosphorylation of glycerol by glucocorticoids in EPI and RETRO from rats.

[The preparation and identification of diagnostic recombinant glycerol kinase].

In order to establish an efficient and low-cost production procedure of recombinant glycerol kinase (r-GK), we expressed the r-GK gene at high level in E. coli by induction with lactose on a large-scale fermentation of 300L. The results showed that the biomass concentration reached OD600 of 42 and the expression of r-GK in E. coli accounted for about 30% of total soluble protein. The cell-free extract was processed by selective thermo-denaturation and then purified with Ni sepharose FF column chromatography. Finally, highly purified r-GK was obtained and its purity reached 97% by using analysis on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), polyacrylamide gel electrophoresis (PAGE) and gradient polyacrylamide gel electrophoresis (Gradient PAGE). Further identification study showed that the molecular weight of r-GK was 120kDa with two subunit of 58kDa. Contaminants of NADH oxidase and catalase were not detected in the sample pool of r-GK. The purified r-GK was able to retain about 85% of its initial activity at 4 degrees C for 30 days. After lyophilized, it can retain 93% of its initial activity at 4 degrees C for one year.

Aquaporin 7 expression in postimplantation mouse uteri: a potential role for glycerol transport in uterine decidualization.

The aquaglyceroporin aquaporin 7 (AQP7) is dynamically expressed in mouse uteri undergoing decidualization after implantation. The expansion of AQP7 during uterine decidualization is associated with elevated uterine glycerol accumulation and glycerol kinase expression, suggesting that glycerol might be a potential energy substrate involved in the process of decidualization.

Bovine oocytes and early embryos express mRNA encoding glycerol kinase but addition of glycerol to the culture media interferes with oocyte maturation.

Glycerol plays multi-functional roles in cellular physiology. Other than forming the backbone molecule for glycerophospholipid and triglyceride (TG), glycerol acts as an energy substrate for glycolysis. Spermatozoa are known to utilize glycerol for energy production, but there are no reports of this in oocytes. In this study, the value of glycerol as an energy substrate for bovine oocyte maturation (Exp. 1) and the gene expression of glycerol kinase (GK), an enzyme crucial for cellular glycerol utilization, in bovine oocytes and early embryos (Exp. 2) were examined. In Exp. 1, in vitro maturation (IVM) was conducted using synthetic oviduct fluid supplemented with/without glucose (1.5 mM) and/or glycerol (1.0 mM), and maturation rate, degree of cumulus expansion, glucose consumption and lactate production by cumulus-oocyte complexes (COC) were examined. In Exp. 2, to examine the developmental expression of GK mRNA, cumulus cells, oocytes and embryos at the 2-, 8- and 16-cell, morula, expanded blastocyst and hatched blastocyst stages were obtained in separate experiments, and the expression of GK mRNA was quantified using a real-time PCR. Glycerol did not support oocyte maturation or cumulus expansion. Addition of glycerol to glucose-supplemented media significantly decreased the maturation rate. Expression of GK mRNA was very low in cumulus cells, whereas an appreciable level of the transcript was observed in the oocytes. GK mRNA was detected in embryos at all the stages examined, and its expression significantly increased at the morula stage. These results indicate that glycerol, at least at the present concentration, is not beneficial as a constituent of the medium for bovine oocyte maturation. However, the appreciable levels of GK mRNA found in the oocyte and embryo imply a physiological role for glycerol in bovine oocyte maturation and embryo development.

Effects of modulation of glycerol kinase expression on lipid and carbohydrate metabolism in human muscle cells.

Glycerol is taken up by human muscle in vivo and incorporated into lipids, but little is known about regulation of glycerol metabolism in this tissue. In this study, we have analyzed the role of glycerol kinase (GlK) in the regulation of glycerol metabolism in primary cultured human muscle cells. Isolated human muscle cells exhibited lower GlK activity than fresh muscle explants, but the activity in cultured cells was increased by exposure to insulin. [U-(14)C]Glycerol was incorporated into cellular phospholipids and triacylglycerides (TAGs), but little or no increase in TAG content or lactate release was observed in response to changes in the medium glycerol concentration. Adenovirus-mediated delivery of the Escherichia coli GlK gene (AdCMV-GlK) into muscle cells caused a 30-fold increase in GlK activity, which was associated with a marked rise in the labeling of phospholipid or TAG from [U-(14)C]glycerol compared with controls. Moreover, GlK overexpression caused [U-(14)C]glycerol to be incorporated into glycogen, which was dependent on the activation of glycogen synthase. Co-incubation of AdCMV-GlK-treated muscle cells with glycerol and oleate resulted in a large accumulation of TAG and an increase in lactate production. We conclude that GlK is the limiting step in muscle cell glycerol metabolism. Glycerol 3-phosphate is readily used for TAG synthesis but can also be diverted to form glycolytic intermediates that are in turn converted to glycogen or lactate. Given the high levels of glycerol in muscle interstitial fluid, these finding suggest that changes in GlK activity in muscle can exert important influences on fuel deposition in this tissue.

Unexpected presence of defective glpR alleles in various strains of Escherichia coli.

Alleles of glpR associated with the same GlpR(-) phenotype produce substitutions in different conserved portions of the glycerol 3-phosphate repressor which are not part of the helix-turn-helix motif. Analysis of the effects on growth and enzyme expression show that glucose repression of glycerol utilization is not dependent on a functional repressor.

Changes in protein synthesis during the adaptation of Bacillus subtilis to anaerobic growth conditions.

After a shift of Bacillus subtilis from aerobic to anaerobic growth conditions, nitrate ammonification and various fermentative processes replace oxygen-dependent respiration. Cell-free extracts prepared from wild-type B. subtilis and from mutants of the regulatory loci fnr and resDE grown under aerobic and various anaerobic conditions were compared by two-dimensional gel electrophoresis. Proteins involved in the adaptation process were identified by their N-terminal sequence. Induction of cytoplasmic lactate dehydrogenase (LctE) synthesis under anaerobic fermentative conditions was dependent on fnr and resDE. Anaerobic nitrate repression of LctE formation required fnr-mediated expression of narGHJI, encoding respiratory nitrate reductase. Anaerobic induction of the flavohaemoglobin Hmp required resDE and nitrite. The general anaerobic induction of ywfl, encoding a protein of unknown function, was modulated by resDE and fnr. The ywfl gene shares its upstream region with the pta gene, encoding the fermentative enzyme acetyl-CoA:orthophosphate acetyltransferase. Anaerobic repression of the synthesis of a potential membrane-associated NADH dehydrogenase (YjlD, Ndh), and anaerobic induction of fructose-1,6-bisphosphate aldolase (FbaA) and dehydrolipoamide dehydrogenase (PhdD, Lpd) formation, did not require fnr or resDE participation. Synthesis of glycerol kinase (GlpK) was decreased under anaerobic conditions. Finally, the effect of anaerobic stress induced by the immediate shift from aerobic to strictly anaerobic conditions was analysed. The induction of various systems for the utilization of alternative carbon sources such as inositol (IoIA, IoIG, IoIH, IoII), melibiose (MeIA) and 6-phospho-alpha-glucosides (GIvA) indicated a catabolite-response-like stress reaction.

Expression of GUT1, which encodes glycerol kinase in Saccharomyces cerevisiae, is controlled by the positive regulators Adr1p, Ino2p and Ino4p and the negative regulator Opi1p in a carbon source-dependent fashion.

In Saccharomyces cerevisiae glycerol utilization is mediated by two enzymes, glycerol kinase (Gut1p) and mitochondrial glycerol-3-phosphate dehydrogenase (Gut2p). The carbon source regulation of GUT1 was studied using promoter-reporter gene fusions. The promoter activity was lowest during growth on glucose and highest on the non-fermentable carbon sources, glycerol, ethanol, lactate, acetate and oleic acid. Mutational analysis of the GUT1 promoter region showed that two upstream activation sequences, UAS(INO) and UAS(ADR1), are responsible for approximately 90% of the expression during growth on glycerol. UAS(ADR1) is a presumed binding site for the zinc finger transcription factor Adr1p and UAS(INO) is a presumed binding site for the basic helix-loop-helix transcription factors Ino2p and Ino4p. In vitro experiments showed Adr1 and Ino2/Ino4 protein-dependent binding to UAS(ADR1) and UAS(INO). The negative regulator Opi1p mediates repression of the GUT1 promoter, whereas the effects of the glucose repressors Mig1p and Mig2p are minor. Together, the experiments show that GUT1 is carbon source regulated by different activation and repression systems.

Retrotransposition of glycerol kinase-related genes from the X chromosome to autosomes: functional and evolutionary aspects.

Glycerol kinase catalyzes the metabolism of endogenously derived and dietary glycerol. GyK is a member of a small group of kinases termed ambiquitous enzymes, which are found either in the cytosol or as membrane-bound complexes associated with the voltage-dependent anion channel of the mitochondrial outer membrane. In Homo sapiens, the GyK gene family consists of an X-encoded locus and several X-linked and autosomal intronless retroposons, which, apparently, comprise both functional genes and processed pseudogenes. To study the role of the autosomal genes in mammalian physiology, we have isolated two murine GyK-like genes, determined their structures and chromosomal locations, and examined their functions. These sequences are intronless retroposons, which appear to be paralogues of the X-encoded, brain-specific GyK isoform and are expressed only in the testes. Though both retrotransposition events appear to have occurred prior to the primate-rodent divergence of some 65-80 million years ago, only one of the retrotransposed murine gene sequences, based upon its chromosomal location, is conserved with modern H. sapiens. To test the hypothesis that the murine GyK-like genes encode functional GyK activity, transient transfection of the gene sequences into COS7 cells was carried out. While in vitro translation confirmed that the transcripts could direct the synthesis of proteins of the appropriate size, no GyK activity was detected. Such data suggest that the autosomal GyK-like genes have evolved novel, testis-specific functions. A comparison of the human and mouse GyK-like gene sequences demonstrates the evolutionary relationships between each autosomal isoform and its corresponding X-linked ancestral locus.

Cloning, sequencing, high expression, and crystallization of the thermophile Thermus aquaticus glycerol kinase.

Glycerol kinase (EC 2.7.1.30) is a key enzyme of glycerol uptake and metabolism in bacteria. Using PCR, we amplified and cloned a glycerol kinase gene, glpK, from Thermus aquaticus. The complete gene has 1488 base pairs, coding for a protein of 496 amino acids with a predicted molecular weight of 54,814. The amino acid sequence deduced from T. aquaticus glpK was found to have identities of 97 and 81%, respectively, with those of Thermus flavus and Bacillus subtilis glpK genes. After overproduction in Escherichia coli, the expressed enzyme was easily purified to homogeneity by DEAE-Toyopearl chromatography. The purified enzyme has been crystallized by the hanging drop vapor diffusion method at 22 degrees C. Comparison of the amino acid sequence with that of the B. subtilis enzyme showed that Ser and Lys are replaced by Ala and Arg, as was seen in mesophile and thermophile enzymes.

Structure and gene-polypeptide relationships of the region encoding glycerol diffusion facilitator (glpF) and glycerol kinase (glpK) of Pseudomonas aeruginosa.

The glycerol facilitator is one of the few known examples of bacterial solute transport proteins that catalyse facilitated diffusion across the cytoplasmic membrane. A second protein, glycerol kinase, is involved in entry of external glycerol into cellular metabolism by trapping glycerol in the cytoplasm as sn-glycerol 3-phosphate. Evidence is presented that glycerol transport in Pseudomonas aeruginosa is mediated by a similar transport system. The genes encoding the glycerol facilitator, glpF, and glycerol kinase, glpK, were isolated on a 4.5 kb EcoRI fragment from a chromosomal mini-library by functional complementation of an Escherichia coli glpK mutant after establishing a map of the chromosomal glpFK region with the help of a PCR-amplified glpK segment. The nucleotide sequence revealed that glpF is the promoter-proximal gene of the glpFK operon. The glycerol facilitator and glycerol kinase were identified in a T7 expression system as proteins with apparent molecular masses of 25 and 56 kDa, respectively. The identities of the glycerol facilitator and glycerol kinase amino acid sequences with their counterparts from Escherichia coli were 70 and 81%, respectively; this similarity extended to two homologues in the genome sequence of Haemophilus influenzae. A chromosomal delta glpFK mutant was isolated by gene replacement. This mutant no longer transported glycerol and could no longer utilize it as sole carbon and energy source. Two ORFs, orfX and orfY, encoding a putative regulatory protein and a carbohydrate kinase of unknown function, were located upstream of the glpFK operon.

[Biochemical response of recombinant Hansenula polymorpha strains to oversynthesis of homologous dioxyacetone kinase and bacterial beta-galactosidase]. / Biokhimicheskii otvet rekombinantnykh shtammov Hansenula polymorpha na sverkhsintez gomoloichnoi dioksiatsetonkinazy i bakterial'noi beta-galaktozidazy.

Changes in the activities of key enzymes responsible for utilization of methanol by recombinant strains of methylotrophic yeasts H. polymorpha R22-2B and H. polymorpha LAC-56 grown in a chemostat are described. The strain R22-2B displaying a high activity of dioxyacetone kinase had also a high activity of formaldehyde dehydrogenase, which increased the rate of dissimilation of formaldehyde. There was a decrease in ATP concentration in the strain LAC-56 oversynthesizing beta-galactosidase from Escherichia coli; this effect decreased the rate of assimilation of formaldehyde.

Physiological studies on regulation of glycerol utilization by the phosphoenolpyruvate:sugar phosphotransferase system in Enterococcus faecalis.

In vitro studies with purified glycerol kinase from Enterococcus faecalis have established that this enzyme is activated by phosphorylation of a histidyl residue in the protein, catalyzed by the phosphoenolpyruvate-dependent phosphotransferase system (PTS), but the physiological significance of this observation is not known. In the present study, the regulation of glycerol uptake was examined in a wild-type strain of E. faecalis as well as in tight and leaky ptsI mutants, altered with respect to their levels of enzyme I of the PTS. Glycerol kinase was shown to be weakly repressible by lactose and strongly repressible by glucose in the wild-type strain. Greatly reduced levels of glycerol kinase activity were also observed in the ptsI mutants. Uptake of glycerol into intact wild-type and mutant cells paralleled the glycerol kinase activities in extracts. Glycerol uptake in the leaky ptsI mutant was hypersensitive to inhibition by low concentrations of 2-deoxyglucose or glucose even though the rates and extent of 2-deoxyglucose uptake were greatly reduced. These observations provide strong support for the involvement of reversible PTS-mediated phosphorylation of glycerol kinase in the regulation of glycerol uptake in response to the presence or absence of a sugar substrate of the PTS in the medium. Glucose and 2-deoxyglucose were shown to elicit rapid efflux of cytoplasmic [14C]lactate derived from [14C]glycerol. This phenomenon was distinct from the inhibition of glycerol uptake and was due to phosphorylation of the incoming sugar by cytoplasmic phosphoenolpyruvate. Lactate appeared to be generated by sequential dephosphorylation and reduction of cytoplasmic phosphoenolpyruvate present in high concentrations in resting cells. The relevance of these findings to regulatory phenomena in other bacteria is discussed.

An inducible phosphoenolpyruvate: dihydroxyacetone phosphotransferase system in Escherichia coli.

A phosphoenolpyruvate: dihydroxyacetone phosphotransferase was induced in Escherichia coli grown on dihydroxyacetone as sole carbon source or in its presence. This is the first example of a triose which can be acted upon by the membrane complex to provide a central intermediate in glycolysis. The presence of this system explains the ability of a mutant, in which the ATP-dependent glycerol kinase is genetically replaced by a glycerol: NAD 2-oxidoreductase, to grow on glycerol.

Glycerol catabolic enzymes and their regulation in wild-type and mutant strains of Streptomyces coelicolor A3(2).

Assay procedure were developed for a soluble glycerol kinase (apparent Km (glycerol) 9 microM) and a probably membrane-associated, NAD-independent L-glycerol-3-phosphate dehydrogenase [apparent Km (L-glycerol 3-phosphate) 7 mM] present in Streptomyces coelicolor A3(2). Both enzymes were cold sensitive. They were co-ordinately induced (about 35-fold) by addition of glycerol to cultures growing on arabinose as sole carbon source. Induction was rifampicin sensitive. The dehydrogenase was absent from glycerol-sensitive mutants, and both kinase and dehydrogenase were absent from glycerol non-utilizing (but glycerol-resistant) mutants, demonstrating that the two enzymes are part of the major pathway of glycerol catabolism in S. coelicolor. Circumstantial evidence suggested that their inducer is glycerol 3-phosphate rather than glycerol. The enzymes were subject to co-ordinate repression by various carbon sources, of which glucose exerted the strongest effect (a fivefold repression). Previously described mutants resistant to 2-deoxyglucose, shown here to have very low glucose kinase activity, were defective in glucose repression of the glycerol enzymes.

Lack of adenosine-3',5'-monophosphate receptor protein and apparent lack of expression of adenosine-3',5'-monophosphate functions in Mycobacterium smegmatis CDC 46.

The presence of adenosine-3',5'-monophosphate (cAMP), adenylate cyclase and the effect of glucose on cAMP levels in Mycobacterium spp have been reported earlier. To understand the role(s) of cAMP in these organisms, the induction of various enzyme systems was studied. Beta-galactosidase and L-tryptophanase were present at low levels of activity and could not be induced. Glycerokinase was inducible but the induction was not affected by glucose. The fructose uptake system was inducible, and the induction was lowered in the presence of glucose, but cAMP could not reverse the inhibition. cAMP binding protein was not detectable under a variety of conditions. On the basis of the lack of active cAMP binding protein, a model has been proposed to explain the apparent lack of expression of cAMP function in Mycobacterium smegmatis CDC 46.

Isolation and characterization of Saccharomyces cerevisiae mutants defective in glycerol catabolism.

Mutants of the yeast Saccharomyces cerevisiae that are defective in the catabolism of glycerol were isolated, and two types of mutants were obtained. One type was deficient in glycerol kinase activity, whereas the other type was deficient in sn-glycerol 3-phosphate dehydrogenase activity. Genetic analysis indicated that each mutant strain owed its phenotype to a single nuclear mutation, and that the two mutations were complementary. The mutations were not linked to each other or to any of 10 loci tested. In addition, neither mutation was centromere linked. Possible mechanisms for the regulation of these enzymes were tested by growing the parental strain in the presence of various carbon sources.

Induction of enzymes of the glycerophosphate pathway in leu-5 mutants of Neurospora crassa.

The inducible cytosolic glycerokinase and mitochondrial glycerol-3-phosphate dehydrogenase have been examined during the glycerol-specific induction in Neurospora crassa. Although both the fully induced levels and the respective rates of synthesis of these two enzymes were less than observed with wild-type cells, there were no major differences in the relative rates of induction of the glycerol-3-phosphate dehydrogenase at either permissive or restrictive temperatures. These results indicate that the processes involved in the assembly of this enzyme into the mitochondrial inner membrane are normal in a mutant lacking the mitochondrial leucyl tRNA synthetase and suggest that the functions of the mitochondrial synthetase may be replaced by those of the cytosolic leucyl tRNA synthetase.

Genetic control of the glp system in Bacillus subtilis.

In pleiotropic negative glycerol utilization mutants (GlpPI mutants) of Bacillus subitilis, glycerol kinase and sn-glycerol 3-phosphate (G3P) dehydrogenase are noninducible. GlpPI mutants also fail to take up exogenous [14C]G3P. To study the regulation of the glp system in B. subtilis phenotypically, Glp+ revertants were isolated from GlpPI mutants. Four classes of revertants were identified: phenotypically, wild type; R1 type, which contains an informational suppressor, R2 type, which produced G3P dehydrogenase constitutively; and R3 type, with a temperature-sensitive Glp phenotype producing G3P dehydrogenase constitutively at permissive temperature (32 degrees C). The properties of the revertants indicate that the glpPI locus codes for a protein with a positive regulatory function.