The putative Escherichia coli dehydrogenase YjhC metabolises two dehydrated forms of N-acetylneuraminate produced by some sialidases

Homologues of the putative dehydrogenase YjhC are found in operons involved in the metabolism of N-acetylneuraminate (Neu5Ac) or related compounds. We observed that purified recombinant YjhC forms Neu5Ac from two dehydrated forms of this compound, 2,7-anhydro-N-acetylneuraminate (2,7-AN) and 2-deoxy-2,3-didehydro-N-acetylneuraminate (2,3-EN) that are produced during the degradation of sialoconjugates by some sialidases. The conversion of 2,7-AN into Neu5Ac is reversible and reaches its equilibrium when the ratio of 2,7-AN to Neu5Ac is ≈1/6. The conversion of 2,3-EN is irreversible, leading to a mixture of Neu5Ac and 2,7-AN. NMR analysis of the reaction catalysed by YjhC on 2,3-EN indicated that Neu5Ac was produced as the α-anomer. All conversions require NAD+ as a cofactor, which is regenerated in the reaction. They appear to involve the formation of keto (presumably 4-keto) intermediates of 2,7-AN, 2,3-EN and Neu5Ac, which were detected by liquid chromatography-mass spectrometry (LC-MS). The proposed reaction mechanism is reminiscent of the one catalysed by family 4 β-glycosidases, which also use NAD+ as a cofactor. Both 2,7-AN and 2,3-EN support the growth of Escherichia coli provided the repressor NanR, which negatively controls the expression of the yjhBC operons, has been inactivated. Inactivation of either YjhC or YjhB in NanR-deficient cells prevents the growth on 2,7-AN and 2,3-EN. This confirms the role of YjhC in 2,7-AN and 2,3-EN metabolism and indicates that transport of 2,7-AN and 2,3-EN is carried out by YjhB, which is homologous to the Neu5Ac transporter NanT

The metalloprotein YhcH is an anomerase providing N-acetylneuraminate aldolase with the open form of its substrate

N-acetylneuraminate (Neu5Ac), an abundant sugar present in glycans in vertebrates and some bacteria, can be used as an energy source by several prokaryotes, including Escherichia coli. In solution, more than 99% of Neu5Ac is in cyclic form (≈92% beta-anomer and ≈7% alpha-anomer), whereas <0.5% is in the open form. The aldolase that initiates Neu5Ac metabolism in E. coli, NanA, has been reported to act on the alphaanomer. Surprisingly, when we performed this reaction at pH 6 to minimize spontaneous anomerization, we found NanA and its human homolog NPL preferentially metabolize the open form of this substrate. We tested whether the E. coli Neu5Ac anomerase NanM could promote turnover, finding it stimulated the utilization of both beta and alpha-anomers by NanA in vitro. However, NanM is localized in the periplasmic space and cannot facilitate Neu5Ac metabolism by NanA in the cytoplasm in vivo. We discovered that YhcH, a cytoplasmic protein encoded by many Neu5Ac catabolic operons and belonging to a protein family of unknown function (DUF386), also facilitated Neu5Ac utilization by NanA and NPL and displayed Neu5Ac anomerase activity in vitro. YhcH contains Zn, and its accelerating effect on the aldolase reaction was inhibited by metal chelators. Remarkably, several transition metals accelerated Neu5Ac anomerization in the absence of enzyme. Experiments with E. coli mutants indicated that YhcH expression provides a selective advantage for growth on Neu5Ac. In conclusion, YhcH plays the unprecedented role of providing an aldolase with the preferred unstable open form of its substrate

The nitrogen dioxide increases Pseudomonas fluorescens biofilm formation: identification of the bacterial response to air pollutant

International audienceBackgroundNitrogen dioxide is an air pollutant of increasing interest in biology. Exposure of animals to NO2results in several toxic effects, mostly lung injury.ObjectivesAware ofthe effect of NO2on pseudomonalbiofilm formation, we looked for the mechanismsof bacterialresponsethat could explain their resistance to NO2.MethodsThe confocal microscopy was used for biofilm studying, completed by motility assays. In a second time, in silicoand in vitrogenomics tools were used to narrow our research scope. Additionally, as a strategy to characterize the mechanism of P. fluorescens resistanceto NO2pollutionthese data were completed with proteomics and lipidomics studies. For this purpose, the MALDI-TOF MS Imaging was coupled to HPTLCand compiled with the traditional GC-MS.ConclusionsWhen the NO2increases P. fluorescens biofilm formation, the bacterial motility decreases. In coherence, the level of cyclic di-GMP evolves in NO2exposed cells. The lipidic study shows no drastic change in membrane charge and its composition in phospholipids and fatty acids. This suggests that NO2free radical could freely pass through membrane.In contrast, NO2promotes an extensive modification in protein production, notably the over-production of the proteins of stress response, involved in oxidative stress tolerance and iron transport/metabolism. Thus both iron up-regulation and C-di-GMP level could be signals for biofilm development.This knowledge should probably offer therapeutical solutions in antibacterial treatment

The nitrogen dioxide increases Pseudomonas fluorescens biofilm formation: identification of the bacterial response to air pollutant

International audienceBackgroundNitrogen dioxide is an air pollutant of increasing interest in biology. Exposure of animals to NO2results in several toxic effects, mostly lung injury.ObjectivesAware ofthe effect of NO2on pseudomonalbiofilm formation, we looked for the mechanismsof bacterialresponsethat could explain their resistance to NO2.MethodsThe confocal microscopy was used for biofilm studying, completed by motility assays. In a second time, in silicoand in vitrogenomics tools were used to narrow our research scope. Additionally, as a strategy to characterize the mechanism of P. fluorescens resistanceto NO2pollutionthese data were completed with proteomics and lipidomics studies. For this purpose, the MALDI-TOF MS Imaging was coupled to HPTLCand compiled with the traditional GC-MS.ConclusionsWhen the NO2increases P. fluorescens biofilm formation, the bacterial motility decreases. In coherence, the level of cyclic di-GMP evolves in NO2exposed cells. The lipidic study shows no drastic change in membrane charge and its composition in phospholipids and fatty acids. This suggests that NO2free radical could freely pass through membrane.In contrast, NO2promotes an extensive modification in protein production, notably the over-production of the proteins of stress response, involved in oxidative stress tolerance and iron transport/metabolism. Thus both iron up-regulation and C-di-GMP level could be signals for biofilm development.This knowledge should probably offer therapeutical solutions in antibacterial treatment