Functional expression of a human GDP-L-fucose transporter in Escherichia coli.

OBJECTIVES: To investigate the translocation of nucleotide-activated sugars from the cytosol across a membrane into the endoplasmatic reticulum or the Golgi apparatus which is an important step in the synthesis of glycoproteins and glycolipids in eukaryotes. RESULTS: The heterologous expression of the recombinant and codon-adapted human GDP-L-fucose antiporter gene SLC35C1 (encoding an N-terminal OmpA-signal sequence) led to a functional transporter protein located in the cytoplasmic membrane of Escherichia coli. The in vitro transport was investigated using inverted membrane vesicles. SLC35C1 is an antiporter specific for GDP-L-fucose and depending on the concomitant reverse transport of GMP. The recombinant transporter FucT1 exhibited an activity for the transport of 3H-GDP-L-fucose with a Vmax of 8 pmol/min mg with a Km of 4 µM. The functional expression of SLC35C1 in GDP-L-fucose overproducing E. coli led to the export of GDP-L-fucose to the culture supernatant. CONCLUSIONS: The export of GDP-L-fucose by E. coli provides the opportunity for the engineering of a periplasmatic fucosylation reaction in recombinant bacterial cells.

Analysis of the intestinal microbiota using SOLiD 16S rRNA gene sequencing and SOLiD shotgun sequencing.

BACKGROUND: Metagenomics seeks to understand microbial communities and assemblages by DNA sequencing. Technological advances in next generation sequencing technologies are fuelling a rapid growth in the number and scope of projects aiming to analyze complex microbial environments such as marine, soil or the gut. Recent improvements in longer read lengths and paired-sequencing allow better resolution in profiling microbial communities. While both 454 sequencing and Illumina sequencing have been used in numerous metagenomic studies, SOLiD sequencing is not commonly used in this area, as it is believed to be more suitable in the context of reference-guided projects. RESULTS: To investigate the performance of SOLiD sequencing in a metagenomic context, we compared taxonomic profiles of SOLiD mate-pair sequencing reads with Sanger paired reads and 454 single reads. All sequences were obtained from the bacterial 16S rRNA gene, which was amplified from microbial DNA extracted from a human fecal sample. Additionally, from the same fecal sample, complete genomic microbial DNA was extracted and shotgun sequenced using SOLiD sequencing to study the composition of the intestinal microbiota and the existing microbial metabolism. We found that the microbiota composition of 16S rRNA gene sequences obtained using Sanger, 454 and SOLiD sequencing provide results comparable to the result based on shotgun sequencing. Moreover, with SOLiD sequences we obtained more resolution down to the species level. In addition, the shotgun data allowed us to determine a functional profile using the databases SEED and KEGG. CONCLUSIONS: This study shows that SOLiD mate-pair sequencing is a viable and cost-efficient option for analyzing a complex microbiome. To the best of our knowledge, this is the first time that SOLiD sequencing has been used in a human sample.

Dmbt1 does not affect a Western style diet-induced liver damage in mice.

In the last three decades the prevalence of non-alcoholic fatty liver disease has markedly increased. Results from epidemiologic studies indicate that not only a general overnutrition but rather a diet rich in sugar, fat and cholesterol (= Western style diet) maybe a risk factor for the development of non-alcoholic fatty liver disease. Concerning liver diseases, it is known that Deleted in malignant brain tumors 1 is amongst others related to liver injury and repair. In addition Deleted in malignant brain tumors 1 seems to play a role in regard to the maintenance of the intestinal homeostasis and the regulation of food intake. Starting from this background the aim of the present study was to investigate if Dmbt1 plays a role in Western style diet-induced non-alcoholic steatohepatitis in mice. Dmbt1 (+/+) and Dmbt1 (-/-) mice were fed a Western style diet or control diet ad libitum for 12 weeks. Both Western style diet fed groups gained significant more weight than the controls and developed a mild non-alcoholic steatohepatitis. The presence/absence of functional Deleted in malignant brain tumors 1 had no effect on parameters like food intake, weight gain, fasting glucose, and liver damage. These results suggest that Deleted in malignant brain tumors 1 plays a minor part on the development of a diet-induced liver damage in mice.

Catabolism of citronellol and related acyclic terpenoids in pseudomonads.

Terpenes are a huge group of natural compounds characterised by their predominantly pleasant smell. They are built up by isoprene units in cyclic or acyclic form and can be functionalised by carbonyl, hydroxyl or carboxyl groups and by presence of additional carbon-carbon double bonds (terpenoids). Currently, much more than 10,000 terpenoid compounds are known, and many thereof are present in different iso- and stereoforms. Terpenoids are secondary metabolites and can have important biological functions in living organisms. In many cases, the biological functions of terpenoids are not known at all. Nevertheless, terpenoids are used in large quantities as perfumes and aroma compounds for food additives. Terpenoids can be also precursors and building blocks for synthesis of complex chiral compounds in chemical and pharmaceutical industry. Unfortunately, only few terpenoids are available in large quantities at reasonable costs. Therefore, characterisation of suited biocatalysts specific for terpenoid compounds and development of biotransformation processes of abundant terpenoids to commercially interesting derivates becomes more and more important. This minireview summarises knowledge on catabolic pathways and biotransformations of acyclic monoterpenes that have received only little attention. Terpenoids with 20 or more carbon atoms are not a subject of this study.

PQQ-dependent alcohol dehydrogenase (QEDH) of Pseudomonas aeruginosa is involved in catabolism of acyclic terpenes.

Growth of Pseudomonas aeruginosa on acyclic terpene alcohols such as geraniol depends on the presence of the atuRABCDEFGH gene cluster and a functional acyclic terpene utilisation (Atu) pathway. The proteins encoded by the atu gene cluster are necessary but not sufficient for growth on acyclic terpenes. Comparative 2-dimensional polyacrylamide gel electrophoresis of soluble P. aeruginosa proteins revealed the presence of an additional spot (besides Atu proteins) that is specifically expressed in geraniol cells but is absent in isovalerate-grown cells. The spot was identified as PA1982 gene product a pyrroloquinoline quinone (PQQ) dependent ethanol oxidoreductase (QEDH). Inactivation of PA1982 by insertion mutagenesis resulted in inability of the mutant to utilise ethanol and in reduced growth on geraniol. Growth on ethanol was restored by transferring an intact copy of the PA1982 gene into the mutant. The PA1982 gene product was purified from recombinant Escherichia coli and revealed PQQ-dependent oxidoreductase activity with a variety of substrates including acyclic terpene derivates at comparable V(max)-values. Our results show that QEDH participates in oxidation of acyclic terpene derivates in addition to the well-known function in ethanol metabolism.

Flavonoid glucuronides and a chromone from the aquatic macrophyte Stratiotes aloides.

The first phytochemical analysis of the aquatic macrophyte Stratiotes aloides afforded two new flavonoid glucuronides, luteolin 7-O-beta-D-glucopyranosiduronic acid-(1-->2)-beta-D-glucopyranoside (1) and chrysoeriol 7-O-beta-D-glucopyranosiduronic acid-(1-->2)-beta-D-glucopyranoside (2), as well as the new 2-(2-hydroxypentyl)-5-carboxy-7-methoxychromone (5) and chrysoeriol 7-O-beta-(6-O-malonyl)glucopyranoside (3), which has been assigned via NMR data for the first time. Additionally, free amino acids such as tryptophan, arginine, leucine, isoleucine, phenylalanine, and tyrosine along with choline, cis-aconitic acid, the phenolic glycoside alpha-arbutine, the chlorophyll derivative phaeophorbide a, and the flavonoid glycoside luteolin 7-O-beta-(6-O-malonyl)glucopyranoside (4) were isolated. Despite the low quantities obtained in some cases (between 50-300 microg), the structures of all compounds were unambiguously elucidated by extensive NMR and MS experiments. With a delay of 2 days compound 1 (10 and 50 microM test concentration) strongly inhibited the growth of human SH-SY5Y neuroblastoma cells in a dose-dependent manner, whereas only a moderate growth inhibition of human Patu 8902 carcinoma cells could be observed. Compounds 1 and 2 showed no activities against the bacteria Escherichia coli BW25113, Pseudomonas pudida KT2440, and Enterobacter cloacae subsp. dissolvens.

Biochemical characterization of isovaleryl-CoA dehydrogenase (LiuA) of Pseudomonas aeruginosa and the importance of liu genes fora functional catabolic pathway of methyl-branched compounds.

Growth of Pseudomonas aeruginosa on acyclic terpene alcohols (citronellol) and on other methyl-branched compounds such as leucine or isovalerate requires a functional leucine/isovalerate utilization (Liu) pathway. In this study, we investigated the liuABCDE gene cluster by insertion mutant analysis, heterologous expression of liuA in Escherichia coli and by biochemical characterization of purified LiuA protein. Mutants with insertion in any of the liu genes were unable to utilize acyclic terpenes or leucine/isovalerate and confirmed the importance of the liu genes for catabolism of methyl-branched compounds. An insertion mutant in liuA was complemented by a liuA copy in trans, indicating that possible polar downstream effects of the insertion are not essential for growth. LiuA purified from recombinant E. coli revealed acyl-CoA dehydrogenase activity with isovaleryl-CoA (KM 2.3 microM) and butyryl-CoA as substrates. Other acyl-CoA compounds such as isobutyryl-CoA, 3-hydroxybutyryl-CoA, octanoyl-CoA, citronellyl-CoA or 5-methyl-hex-4-enoyl-CoA were not utilized. Experimental evidence for expression and essential functions of other Liu proteins in metabolism of methyl-branched compounds is provided.

Identification and characterization of the acyclic terpene utilization gene cluster of Pseudomonas citronellolis.

The catabolism of citronellol and geraniol [acyclic terpene utilization (Atu) pathway] was investigated in Pseudomonas citronellolis. A 13.3-kb genomic DNA fragment was cloned and harboured a putative regulator gene atuR and a gene cluster consisting of eight genes (atuABCDEFGH). Sequence analysis of the atu gene products showed a high degree of amino acid similarity (78-91% identity) to products of a similar gene cluster previously identified in Pseudomonas aeruginosa. Insertion mutagenesis in atuA resulted in inability of the bacteria to utilize acyclic terpenes as a sole source of carbon and energy and confirmed the involvement of atuA in the Atu pathway. Western blot analysis of wild-type and atuA mutant cells of P. citronellolis and P. aeruginosa for biotin-containing proteins enabled the identification of geranyl-CoA carboxylase (GCase), which is the key enzyme of the Atu pathway. GCase subunits were encoded by atuC and atuF. Putative functions for the other Atu proteins in the catabolic pathway of acyclic terpenes are discussed.

Malate:quinone oxidoreductase (MqoB) is required for growth on acetate and linear terpenes in Pseudomonas citronellolis.

Mini-transposon-induced mutants with defects in utilization of linear terpenes such as citronellol and citronellic acid were isolated from Pseudomonas citronellolis. Mutants with strongly reduced growth on citronellol and citronellic acid (class I) were obtained together with mutants growing normally on citronellic acid but with impairment in growth on citronellol (class II) and auxotroph mutants (class III). The transposon carrying DNA fragments of two class I mutants were cloned and malate:quinone oxidoreductase gene (mqoB) was identified as the transposon insertion site in both mutants. The mqoB genes of P. aeruginosa and of P. citronellolis wild types were cloned. Conjugative transfer of the mqoB genes to the two P. citronellolis mutants increased the strongly reduced levels of MqoB activity in cell extracts of the mutants to the level of the wild type and restored the ability of the mutants to grow on citronellol and citronellic acid. Physiological analysis of the wild type and of mutants showed that MqoB is part of the glyoxylate cycle in P. citronellolis and is necessary for growth on C(2)-compounds and linear terpenes such as citronellol or citronellic acid.

Bifidobacterium adolescentis protects from the development of nonalcoholic steatohepatitis in a mouse model.

To investigate the hypothesis that an oral supplementation of Bifidobacterium adolescentis protects against a diet-induced nonalcoholic steatohepatitis in a mouse model, C57BL/6 mice were fed either a Western-style or a control diet±tap water fortified with B. adolescentis (5×10(7) cfu/ml) ad libitum for 12 weeks. Mice fed a Western-style diet gained significantly more weight than mice fed a control diet and developed a mild steatohepatitis. Western-style diet fed groups concomitantly treated with B. adolescentis had significantly decreased liver damage, whereas portal endotoxin levels and toll-like receptor-4 protein levels as well as myeloid differentiation factor 88 mRNA were increased in livers of both Western-style diet fed groups. The protective effects of the B. adolescentis were associated with a significant attenuation of the formation of reactive oxygen species, activation of nuclear factor κB (NFκB) and induction of markers of inflammation in the liver. Taken together, our data suggest that an oral supplementation of the B. adolescentis attenuates diet-induced steatohepatitis, and this effect is associated with prevention from lipid peroxidation, NFκB activation and finally inflammation in the liver.

AtuR is a repressor of acyclic terpene utilization (Atu) gene cluster expression and specifically binds to two 13 bp inverted repeat sequences of the atuA-atuR intergenic region.

The atuR-atuABCDEFGH gene cluster is essential for acyclic terpene utilization (Atu) in Pseudomonas aeruginosa and Pseudomonas citronellolis. The cluster encodes most proteins of the Atu pathway including the key enzyme, geranyl-CoA carboxylase. AtuR was identified as a repressor of the atu gene cluster expression by (1) amino acid similarity to TetR repressor family members, (2) constitutive expression of Atu proteins in the atuR insertion mutant and (3) specific binding of purified AtuR homodimers to the atuR-atuA intergenic region in electrophoretic mobility shift assay (EMSA). Two 13 bp inverted repeat sequences separated by 40 bp in the atuA operator/promoter region were identified to represent two sites of AtuR binding by EMSA. Changing of two or more bases within the inverted repeat sequences abolished the ability of AtuR to bind to its target. All EMSA experiments were sufficiently sensitive with ethidium bromide-stained DNA fragments after polyacrylamide gel electrophoresis.

Biochemical characterization of AtuD from Pseudomonas aeruginosa, the first member of a new subgroup of acyl-CoA dehydrogenases with specificity for citronellyl-CoA.

The atuRABCDEFGH gene cluster is essential for acyclic terpene utilization (Atu) in Pseudomonas aeruginosa. The biochemical functions of most Atu proteins have not been experimentally verified; exceptions are AtuC/AtuF, which constitute the two subunits of geranyl-CoA carboxylase, the key enzyme of the Atu pathway. In this study we investigated the biochemical function of AtuD and of the PA1535 gene product, a protein related to AtuD in amino acid sequence. 2D gel electrophoresis showed that AtuD and the PA1535 protein were specifically expressed in cells grown on acyclic terpenes but were absent in isovalerate- or succinate-grown cells. Mutant analysis indicated that AtuD but not the product of PA1535 is essential for acyclic terpene utilization. AtuD and PA1535 gene product were expressed in recombinant Escherichia coli and purified to homogeneity. Purified AtuD showed citronellyl-CoA dehydrogenase activity (V(max) 850 mU mg(-1)) and high affinity to citronellyl-CoA (K(m) 1.6 microM). AtuD was inactive with octanoyl-CoA, 5-methylhex-4-enoyl-CoA or isovaleryl-CoA. Purified PA1535 gene product revealed high citronellyl-CoA dehydrogenase activity (V(max) 2450 mU mg(-1)) but had significantly lower affinity than AtuD to citronellyl-CoA (K(m) 18 microM). Purified PA1535 protein additionally utilized octanoyl-CoA as substrate (V(max), 610 mU mg(-1); K(m) 130 microM). To our knowledge AtuD is the first acyl-CoA dehydrogenase with a documented substrate specificity for terpenoid molecule structure and is essential for a functional Atu pathway. Potential other terpenoid-CoA dehydrogenases were found in the genomes of Pseudomonas citronellolis, Marinobacter aquaeolei and Hahella chejuensis but were absent in non-acyclic terpene-utilizing bacteria.

Identification of genes and proteins necessary for catabolism of acyclic terpenes and leucine/isovalerate in Pseudomonas aeruginosa.

Geranyl-coenzyme A (CoA)-carboxylase (GCase; AtuC/AtuF) and methylcrotonyl-CoA-carboxylase (MCase; LiuB/LiuD) are characteristic enzymes of the catabolic pathway of acyclic terpenes (citronellol and geraniol) and of saturated methyl-branched compounds, such as leucine or isovalerate, respectively. Proteins encoded by two gene clusters (atuABCDEFGH and liuRABCDE) of Pseudomonas aeruginosa PAO1 were essential for acyclic terpene utilization (Atu) and for leucine and isovalerate utilization (Liu), respectively, as revealed by phenotype analysis of 10 insertion mutants, two-dimensional gel electrophoresis, determination of GCase and MCase activities, and Western blot analysis of wild-type and mutant strains. Analysis of the genome sequences of other pseudomonads (P. putida KT2440 and P. fluorescens Pf-5) revealed candidate genes for Liu proteins for both species and candidate genes for Atu proteins in P. fluorescens. This result concurred with the finding that P. fluorescens, but not P. putida, could grow on acyclic terpenes (citronellol and citronellate), while both species were able to utilize leucine and isovalerate. A regulatory gene, atuR, was identified upstream of atuABCDEFGH and negatively regulated expression of the atu gene cluster.