No abstract

Addition of sialic acid residues in the human pathogen Trypanosoma cruzi glycoconjugates is mediated by a trans-sialidase and not by a CMP-sialic acid : glycoconjugate sialyltransferase. Incubation of trans-sialidase with N-[galact~se-'~C]acetyllactosamine and 0-linked oligosaccharides, Nlinked glycopeptides (both obtained from fetuin) or sialyllactose showed that the last three compounds were donors of sialic acid residues to the first one. Moreover, N-and 0-linked oligosaccharides in asialofetuin and asialomucin, respectively, served as acceptors of sialic acid units. Gangliosides GM3, GD,, and GT,, but not GM2, GM,, nor GD,, donated sialic acid units to N-acetyllactos amine when incubated with trans-sialidase. This showed that only sialic acid units bound to terminal galactosyl residues were transferred. GMl, was converted to GD,,, and GD,, to GT,, when incubated with the appropriate donor. The fact that asialo-GM,, was converted to a ganglioside migrating as GD,, on thin-layer chromatography suggested that sialic acid units may be transferred to internal galactosyl residues, although once linked to those residues they can not be further transferred to other glycoconjugates. Sialic acid residues linked a2,3-but not a2,6-or a2,8-were transferred by the trans-sialidase. Methyl b-galactoside but not methyl a-galactoside served as acceptor of sialic acid units, thus suggesting that terminal a-linked galactosyl units in T cruzi and mammalian glycoproteins are not sialylated by the enzyme. As the trans-sialidase employed in these experiments has been shown to be located on the external surface of the parasite and to be shed to the medium, the relatively broad specificity shown by the enzyme with respect to protein-and lipid-linked oligosaccharides strongly suggests that infection by T cruzi might alter the sialic acid distribution in glycoproteins and glycolipids of the mammalian host.The human pathogen Trypanosoma cruzi differs from other eukaryotic and prokaryotic cells in the mechanism by which it adds sialic acid units to its glycoconjugates. Transfer of the above-mentioned residues appears to be mediated by a trans-sialidase activity and not by a CMP-sialic acid : glycoconjugate sialytransferase [l -31. It was reported that sialylation of a 2: cruzi structure by the trans-sialidase was required for the invasion of mammalian cells [3]. The activity has been detected in crude extracts of the parasite and found to recognize fetuin and short oligosaccharides as sugar donors and glycoproteins, short oligosaccharides and glycolipids as sialic acid acceptors but it was unknown if the enzyme recognized glycolipids as donor substrates or if it discriminated between N-, 0-and different lipid-linked oligosaccharides as acceptor and donor substrates. The activity assayed in crude preparations mainly required a2,3-linked sialic acid units as donors when the transfer to short oligosaccharides was tested but a2,6-linked sialic acid residues were about

Phenylacetic acid (PhAcOH) and 4‐hydroxyphenylacetic acid (4HOPhAcOH) are catabolized in Pseudomonas putida U through two different pathways. Mutation carried out with the transposon Tn5 has allowed the isolation of several mutants which, unlike the parental strain, are unable to grow in chemically defined medium containing either PhAcOH or 4HOPhAcOH as the sole carbon source. Analysis of these strains showed that the ten mutants unable to grow in PhAcOH medium grew well in the one containing 4HOPhAcOH, whereas four mutants handicapped in the degradation of 4HOPhAcOH were all able to utilize PhAcOH. These results show that the degradation of these two aromatic compounds in P. putida U is not carried out as formerly believed through a single linear and common pathway, but by two unrelated routes. Identification of the blocked point in the catabolic pathway and analysis of the intermediate accumulated, showed that the mutants unable to utilize 4HOPhAcOH corresponded to two different groups: those blocked in the gene encoding 4‐hydroxyphenylacetic acid‐3‐hydroxylase; and those blocked in the gene encoding homoprotocatechuate‐2,3‐dioxygenase. Mutants unable to use PhAcOH as the sole carbon source have been also classified into two different groups: those which contain a functional PhAc‐CoA ligase protein; and those lacking this enzyme activity.

Pseudomonas putida U does not degrade D-glucose through the glycolytic pathway but requires (i) its oxidation to D-gluconic acid by a peripherally located constitutive glucose dehydrogenase (insensitive to osmotic shock), (ii) accumulation of D-gluconic acid in the extracellular medium, and (iii) the induction of a specific energy-dependent transport system responsible for the uptake of D-gluconic acid. This uptake system showed maximal rates of transport at 30 "C in 50 mM potassium phosphate buffer, pH 7.0. Under these conditions the Km calculated for D-gluconic acid was 6 7 pM. Furthermore, a different transport system, specific for the uptake of glucose, was also identified. It is active and shows maximal uptake rates at 35 "C in 50 mM potassium phosphate buffer, pH 60, with a Km value of 8 3 pM.

Sununary:The capsular polysaccharide of Escherichia coli K92 consists of a linear polymer of NeuSAc with alternating a(2-8) and a(2-9) linkages. It accumulates when the bacterium is grown at 37 °C in a defined medium containing D-xylose and L-asparagine as carbon and nitrogen sources. Release of the capsular polymer into the medium was maximal (450 g x ml l ) in the stationary phase of growth (76 h). This medium could be useful for obtaining sufficient polymer to develop effective vaccines. Zusammenfassung: Das Kapsel-Polysaccharid vonEscherichia coli besteht aus einem linearen Polymer von NeuSAc mit alternierenden a(2-8)-und a(2-9)-Bindungen. Es reichert sich an, wenn das Bakterium bei 37 °C in einem definierten Medium mit D-Xylose und L-Asparagin als Kohlenstoff-bzw. Stickstoffquelle gezüchtet wird. Die Ausscheidung des KapselPolymeren ins Medium erreichte in der stationären Wachstumsphase (nach 76 h) ein Maximum (450 ^g x m/" 1 ). Dieses Medium könnte sich zur Herstellung ausreichender Mengen des Polymers für die Entwicklung eines wirksamen Impfstoffes eignen. Das Enzym CMP-Neu5Ac-Synthetase war nicht nachweisbar, wenn die Zellen bei 20 °C gezüchtet wurden. Das Fehlen dieses Enzyms erklärt den Ausfall der Synthese, wenn das Bakterium bei 20 °C gezüchtet wird.