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The NeuC Protein of Escherichia coli K1 Is a UDP N-Acetylglucosamine 2-Epimerase. Willie F. Vann, 2004.The K1 capsule is an essential virulence determinant of Escherichia coli strains that cause meningitis in neonates . Biosynthesis and transport of the capsule, an  -2,8-linked
polymer of sialic acid, are encoded by the 17-kb kps gene
cluster . We deleted neuC, a K1 gene implicated in sialic acid
synthesis, from the chromosome of EV36, a K-12-K1 hybrid, by allelic
exchange . Exogenously added sialic acid restored capsule expression
to the deletion strain ( neuC),
confirming that NeuC is necessary for sialic acid synthesis . The
deduced amino acid sequence of NeuC showed similarities to those of
UDP-N-acetylglucosamine (GlcNAc) 2-epimerases from both
prokaryotes and eukaryotes . The NeuC homologue from serotype III
Streptococcus agalactiae complements
neuC .
We cloned the neuC gene into an intein expression vector to
facilitate purification . We demonstrated by paper chromatography that
the purified neuC gene product catalyzed the formation of [2-14C]acetamidoglucal
and [N-14C]acetylmannosamine (ManNAc) from UDP-[14C]GlcNAc .
The formation of reaction intermediate 2-acetamidoglucal with
the concomitant release of UDP was confirmed by proton and phosphorus
nuclear magnetic resonance spectroscopy . NeuC could not use GlcNAc as
a substrate . These data suggest that neuC encodes an epimerase
that catalyzes the formation of ManNAc from UDP-GlcNAc via a
2-acetamidoglucal intermediate . The unexpected release of the glucal
intermediate and the extremely low rate of ManNAc formation likely
were a result of the in vitro assay conditions, in which a key
regulatory molecule or protein was absent .
Synthesis of Catalytically Active Form III Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase in Archaea. Michael W. Finn, 2003.Ribulose 1,5 bisphosphate carboxylase/oxygenase (RubisCO) catalyzes the biological reduction and assimilation of carbon dioxide gas to organic carbon; it is the key enzyme responsible for the bulk of organic matter found on earth . Until recently it was believed that there are only two forms of RubisCO, form I and form II . However, the recent completion of several genome-sequencing projects uncovered open reading frames resembling RubisCO in the third domain of life, the archaea . Previous work and homology comparisons suggest that these enzymes represent a third form of RubisCO, form III . While earlier work indicated that two structurally distinct recombinant archaeal RubisCO proteins catalyzed bona fide RubisCO reactions, it was not established that the rbcL genes of anaerobic archaea can be transcribed and translated to an active enzyme in the native organisms . In this report, it is shown not only that Methanococcus jannaschii, Archaeoglobus fulgidus, Methanosarcina acetivorans, and Methanosarcina barkeri possess open reading frames with the residues required for catalysis but also that the RubisCO protein from these archaea accumulates in an active form under normal growth conditions . In addition, the form III RubisCO gene (rbcL) from M . acetivorans was shown to complement RubisCO deletion strains of Rhodobacter capsulatus and Rhodobacter sphaeroides under both photoheterotrophic and photoautotrophic growth conditions . These studies thus indicate for the first time that archaeal form III RubisCO functions in a physiologically significant fashion to fix CO2 . Furthermore, recombinant M . jannaschii, M . acetivorans, and A . fulgidus RubisCO possess unique properties with respect to quaternary structure, temperature optima, and activity in the presence of molecular oxygen compared to the previously described Thermococcus kodakaraensis and halophile proteins . Thin Pilus PilV Adhesins of Plasmid R64 Recognize Specific Structures of the Lipopolysaccharide Molecules of Recipient Cells. Akiko Ishiwa, 2003.IncI1 plasmid R64 encodes a type IV pilus called a thin pilus, which includes PilV adhesins . Seven different sequences for the C-terminal segments of PilV adhesins can be produced by shufflon DNA rearrangement . The expression of the seven PilV adhesins determines the recipient specificity in liquid matings of plasmid R64 . Salmonella enterica serovar Typhimurium LT2 was recognized by the PilVA' and PilVB' adhesins, while Escherichia coli K-12 was recognized by the PilVA', PilVC, and PilVC' adhesins . Lipopolysaccharide (LPS) on the surfaces of recipient cells was previously shown to be the specific receptor for the seven PilV adhesins . To identify the specific receptor structures of LPS for various PilV adhesins, R64 liquid matings were carried out with recipient cells consisting of various S . enterica serovar Typhimurium LT2 and E . coli K-12 waa mutants and their derivatives carrying various waa genes of different origins . From the mating experiments, including inhibition experiments, we propose that the GlcNAc(  1-2)Glc and Glc(1-2)Gal structures of the LPS core of S . enterica serovar Typhimurium LT2 function as receptors for the PilVB' and PilVC' adhesins, respectively, while the PilVC' receptor in the wild-type LT2 LPS core may be masked . We further propose that the GlcNAc(ß1-7)Hep and Glc(1-2)Glc structures of the LPS core of E . coli K-12 function as receptors for the PilVC and PilVC' adhesins, respectively .
Dominance of Vibrio fischeri in Secreted Mucus outside the Light Organ of Euprymna scolopes: the First Site of Symbiont Specificity. Spencer V. Nyholm, 2003.Previous studies of the Euprymna scolopes-Vibrio fischeri symbiosis have demonstrated that, during colonization, the hatchling host secretes mucus in which gram-negative environmental bacteria amass in dense aggregations outside the sites of infection . In this study, experiments with green fluorescent protein-labeled symbiotic and nonsymbiotic species of gram-negative bacteria were used to characterize the behavior of cells in the aggregates . When hatchling animals were exposed to 103 to 106 V . fischeri cells/ml added to natural seawater, which contains a mix of approximately 106 nonspecific bacterial cells/ml, V . fischeri cells were the principal bacterial cells present in the aggregations . Furthermore, when animals were exposed to equal cell numbers of V . fischeri (either a motile or a nonmotile strain) and either Vibrio parahaemolyticus or Photobacterium leiognathi, phylogenetically related gram-negative bacteria that also occur in the host's habitat, the symbiont cells were dominant in the aggregations . The presence of V . fischeri did not compromise the viability of these other species in the aggregations, and no significant growth of V . fischeri cells was detected . These findings suggested that dominance results from the ability of V . fischeri either to accumulate or to be retained more effectively within the mucus . Viability of the V . fischeri cells was required for both the formation of tight aggregates and their dominance in the mucus . Neither of the V . fischeri quorum-sensing compounds accumulated in the aggregations, which suggested that the effects of these small signal molecules are not critical to V . fischeri dominance . Taken together, these data provide evidence that the specificity of the squid-vibrio symbiosis begins early in the interaction, in the mucus where the symbionts aggregate outside of the light organ .
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