Prevotella enzymes involved in mucin oligosaccharide degradation and evidence for a small operon of genes expressed during growth on mucin.

Mucin desulfation is believed to be a rate-limiting step in mucin degradation by colon bacteria. The activities of enzymes hydrolysing nine linkages found in mucin oligosaccharide chains were measured using model substrates, in extracts of two mucin-degrading bacteria, Prevotella strain RS2 and Bacteroides fragilis. Sulfatases desulfating N-acetylglucosamine-6-sulfate, galactose-6-sulfate and galactose-3-sulfate were found. The genomic DNA downstream from the gene encoding the mucin-desulfating sulfatase (N-acetylglucosamine-6-sulfatase) in Prevotella was sequenced, and two putative genes identified which are likely to be coexpressed with this sulfatase, though their activities are unknown. Northern and Western analyses showed that expression of this short operon of three genes is increased during growth on mucin.

Cloning of a mucin-desulfating sulfatase gene from Prevotella strain RS2 and its expression using a Bacteroides recombinant system.

A gene encoding the mucin-desulfating sulfatase in Prevotella strain RS2 has been cloned, sequenced, and expressed in an active form. A 600-bp PCR product generated using primers designed from amino acid sequence data was used to isolate a 5,058-bp genomic DNA fragment containing the mucin-desulfating sulfatase gene. A 1,551-bp open reading frame encoding the sulfatase proprotein was identified, and the deduced 517-amino-acid protein minus its signal sequence corresponded well with the published mass of 58 kDa estimated by denaturing gel electrophoresis. The sulfatase sequence showed homology to aryl- and nonarylsulfatases with different substrate specificities from the sulfatases of other organisms. No sulfatase activity could be detected when the sulfatase gene was cloned into Escherichia coli expression vectors. However, cloning the gene into a Bacteroides expression vector did produce active sulfatase. This is the first mucin-desulfating sulfatase to be sequenced and expressed. A second open reading frame (1,257 bp) was identified immediately upstream from the sulfatase gene, coding in the opposite direction. Its sequence has close homology to iron-sulfur proteins that posttranslationally modify other sulfatases. By analogy, this protein is predicted to catalyze the modification of a serine group to a formylglycine group at the active center of the mucin-desulfating sulfatase, which is necessary for enzymatic activity.

In Vivo and in Vitro Studies of Glucose-6-Phosphate Dehydrogenase from Barley Root Plastids in Relation to Reductant Supply for NO2- Assimilation.

Pyridine nucleotide pools were measured in intact plastids from roots of barley (Hordeum vulgare L.) during the onset of NO2- assimilation and compared with the in vitro effect of the NADPH/NADP ratio on the activity of plastidic glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) from N-sufficient or N-starved roots. The NADPH/NADP ratio increased from 0.9 to 2.0 when 10 mM glucose-6-phosphate was supplied to intact plastids. The subsequent addition of 1 mM NaNO2 caused a rapid decline in this ratio to 1.5. In vitro, a ratio of 1.5 inactivated barley root plastid G6PDH by approximately 50%, suggesting that G6PDH could remain active during NO2- assimilation even at the high NADPH/NADP ratios that would favor a reduction of ferredoxin, the electron donor of NO2- reductase. Root plastid G6PDH was sensitive to reductive inhibition by dithiothreitol (DTT), but even at 50 mM DTT the enzyme remained more than 35% active. In root plastids from barley starved of N for 3 d, G6PDH had a substantially reduced specific activity, had a lower Km for NADP, and was less inhibited by DTT than the enzyme from N-sufficient root plastids, indicating that there was some effect of N starvation on the G6PDH activity in barley root plastids.

Bacterial glycosulphatases and sulphomucin degradation.

The presence of a high bacterial population in a region of the gastrointestinal tract is usually associated with the secretion of sulphomucins into the mucus gel covering that region. The term 'sulphomucin' is a histochemical description of the staining properties of mucin. At present this term can only be qualitatively related to the percentage of sulphate in the mucin molecule, which makes the term difficult to use in a biochemical and functional sense. Sulphomucins are thought to carry out the normal functions attributed to mucins; in addition, heavy sulphation rate-limits the degradation of mucins by bacterial mucin-degrading glycosidases. A number of mucin-specific glycosulphatases have been reported in bacteria, although only two such enzymes have been purified. These enzymes remove part of the sulphate content from sulphomucins and make them more susceptible to further enzymic degradation. The variety of chain locations and sugar attachment sites of sulphate esters on the mucin oligosaccharides, taken together with the data on the enzymes, suggest there will be a spectrum of bacterial glycosulphatases, with different properties, cellular locations and substrate specificities. Bacterial glycosulphatases have the potential to modify sulphated glycoconjugates at mucosal surfaces and should prove useful as biochemical tools for the study of sulphated glycoconjugates.

Analytical survey of N-nitroso contaminants in pesticide products.

1. Levels of N-nitroso dialkylamines in dinitroaniline derivatives were high (up to 153 mg/kg) in the past, but have been decreased considerably in current production, due to process changes. 2. Butralin and pendimethalin contain significant amounts of N-nitroso parent compounds. 3. Most dimethylamine salts of phenoxyalkanoic acids presently contain low or non-detectable levels of NDMA. 4. Quaternary ammonium compounds contain NDMA levels up to 16.8 mg/kg. 5. Two morpholine derivatives appear to contain N-nitrosomorpholine. 6. The triethanolamine salt of dinoseb contains over 200 mg/kg N-nitrosodiethanolamine. 7. A large number of amides, carbamates, organophosphates, triazines, urea derivatives and miscellaneous nitrogen-containing pesticides do not contain N-nitrosamines above 1 mg/kg. 8. Most N-nitroso contaminants in pesticides can be avoided by simple process changes and the elimination of nitrite salts in the formulation.