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<br> Previous work using spectrophotometric assays reported that E. coli YjhC showed a weak interaction with Neu5Ac, with an apparent Km of 68.8 mm (20). Here, we used ESI-MS to assess the activity of YjhC against 2,7-anhydro-Neu5Ac or Neu5Ac. The glycan array binding data provide independent support of AAV1 interaction with α2,3 and α2,6 trisaccharides. Our genetic data suggest that YjhB could transport 2,7-anhydro-Neu5Ac but not Neu5Ac, where the previously characterized Neu5Ac transporter NanT was not able to transport 2,7-anhydro-Neu5Ac. Based on its sequence, NanX (YjhB) is classified in the MFS class of sugar transporters and is a close homologue of NanT. If you adored this information and you would such as to receive even more info regarding sialic acid powder factory kindly check out our web site. For example, whereas R. gnavus possesses the full complement of genes to produce and utilize 2,7-anhydro-Neu5Ac, including the IT-sialidase (RgNanH), the 2,7-anhydro-Neu5Ac SAT2 transporter, and the oxidoreductase (RgNanOx) within the otherwise canonical Nan cluster, E. coli harbors a transporter with specificity for 2,7-anhydro-Neu5Ac (NanX) and the a NanOx homolog (NanY) but does not express an IT-sialidase.<br>
<br> A, Neu5Ac lyase; nanK, N-acetylmannosamine kinase; nanE, N-acetylmannosamine-6-phosphate epimerase; nanC, Neu5Ac outer membrane channel; nanM, Neu5Ac mutarotase; nanS, N-acetyl-9-O-acetylneuraminate esterase; nagB, glucosamine-6-phosphate deaminase; GNAT, GCN5-related N-acetyltransferase; Reg, regulator (please note that GNAT family proteins and regulator proteins, while recurrent within clusters, may belong to different clades and thus function differently in each organism); SAT2, 2,7-anhydro-Neu5Ac transporter of the ABC superfamily; siaPQM, Neu5Ac transporter of the TRAP family; satABCD, Neu5Ac transporter of the ABC superfamily (SAT); nanUVW (SAT3), Neu5Ac transporter of the ABC superfamily (also named satABC); nanT, Neu5Ac transporter of the MFS superfamily; siaT, Neu5Ac transporter of the SSS family; nanX (yjhB), 2,7-anhydro-Neu5Ac transporter (nanT-like) of the MFS superfamily ABC; MFS, major facilitator superfamily; SSS, sodium solute symporter family; GPH, glycoside-pentoside-hexuronide:cation symporter family; SBP, solute-binding protein; TMD, transmembrane domain; NBD, nucleotide-binding domain. Our bioinformatics provide striking evidence for two additional families of secondary transporters having evolved to recognize 2,7-anhydro-Neu5Ac, namely those of the SSS and GPH families, bringing the total number of transporter families for 2,7-anhydro-Neu5Ac to four. Streptococcus pneumoniae strains, on the other hand, may express up to three sialidases (neuraminidases), NanA, NanB, and NanC, of which the first two are part of a universally conserved nan gene cluster (42), whereas the third one is part of an additional locus present in some strains but not others (50). The conserved nan cluster is well-studied in strain D39 (42, 51) and is divided into three operons that include operon I (nanA monocistronic), operon II (the nanB locus), and operon III (the nanE locus carrying the catabolic genes) (51). The transcriptomic response of S. pneumoniae D39 to Neu5Ac clearly demonstrated that NanR acts as a transcriptional activator of the nan operons I and III in the presence of Neu5Ac, but not of operon II, for which regulation mechanisms remained unknown (51). Because NanB has been functionally characterized as an IT-sialidase in S. pneumoniae (52) and the nan operon II also contains a gene encoding an oxidoreductase and a SAT2 ABC transporter (as in the case of R. gnavus), our results strongly suggest that the nan operon II is dedicated to 2,7-anhydro-Neu5Ac utilization.<br>
<br> This ability to utilise multiple sialic acid derivatives contrasts with R. gnavus strains, which can only grow on 2,7-anhydro-Neu5Ac but not on Neu5Ac (19) and is consistent with E. coli being able to integrate diverse sialic acids into its core catabolic pathway (33). Beyond E. coli, our bioinformatics analyses revealed RgNanOx homologues across many bacterial species that also co-occurred with predicted sialic acid transporters. For example, Bacteroides thetaiotaomicron VPI-5482 encodes a sialidase and can release free Neu5Ac but lacks the nan operon required to metabolize the liberated monosaccharide (47). On the other hand, most C. difficile and S. typhimurium subsp. 48) and benefit from sialidase-producing organisms such as B. thetaiotaomicron to acquire this nutrient from the mucosal environment (49). Our bioinformatics analyses suggest a similar diversity for 2,7-anhydro-Neu5Ac metabolism across bacterial species. From an ecological point of view, because R. gnavus is the only strain reported to produce 2,7-anhydro-Neu5Ac in the gut, the strict specificity of its sialic acid transporter may give it a nutritional advantage while maintaining its keystone status in the mucus niche by providing an important nutrient to the microbial community. E. coli can transport and catabolize the common sialic acid, Neu5Ac, as a sole source of carbon and nitrogen but also related sialic acids, N-glycolylneuraminic acid (Neu5Gc) and 3-keto-3-deoxy-d-glycero-d-galactonononic acid (KDN), which are transported via the sialic acid transporter NanT and catabolized using the sialic acid aldolase NanA (33). Here, we showed that E. coli BW25113 strain was able to grow on 2,7-anhydro-Neu5Ac as a sole carbon source and that the two-gene NanR-regulated operon nanXY (yjhBC) encodes both the transporter and oxidoreductase enzyme required for E. coli to uptake and catabolize 2,7-anhydro-Neu5Ac. This also now completes the functional characterization of all NanR-regulated genes in E. coli (25), giving us a broader picture of the sialic acid molecules it likely encounters in its natural environment.<br>
<br> For oligosaccharides, e.g., sialylated products, purity can be determined using, e.g., thin layer chromatography, HPLC, or mass spectroscopy. Those of skill are aware that insertion of a nucleic acid into a chromosome can occur, e.g., by homologous recombination. In some embodiments, the microorganism is a bacterium, e.g., E. coli. Given the structural resemblance of RgNanOx to YjhC, it is likely that the E. coli oxidoreductase also uses the same mechanism of action for the reversible conversion of 2,7-anhydro-Neu5Ac to Neu5Ac. This analysis supported the earlier findings that YjhC could act on Neu5Ac (20) but also revealed that the enzyme was able to utilize 2,7-anhydro-Neu5Ac as a substrate in the same manner as RgNanOx. The existence of multiple transporters with different specificities for sialic acid derivatives within the same species (e.g. E. coli NanT/YjhB) or restricted to 2,7-anhydro-Neu5Ac (e.g. R. gnavus SAT2) points toward divergent evolution of a common ancestor. 200:1 preference for cleavage of terminal α(2,3)-linked sialic acids (24), and one from Clostridium perfringens, which exhibits only a slight preference for α(2,3)-linked sialic acids over α(2,6)-linked sialic acids (3, 4). It is important to note, however, that one must be cautious in oversimplification regarding neuraminidase specificity because said specificity is dependent on both the core oligosaccharide and the protein and lipid structures that the oligosaccharides are attached to; subtle differences can dramatically influence the rate of release of different glycosidic linkages (4). Furthermore, O-acetylation is one of the most common modifications that occurs on sialic acids, and it has been demonstrated that monoacetylation of the 7, 8, or 9 position of a sialic acid largely attenuates the effectiveness of neuraminidase hydrolysis; diacetylation completely abrogates the hydrolytic ability of neuraminidases from Clostridium perfringens and Vibrio cholerae (15). Cells were treated with 1 U/ml neuraminidase for 2 and 5 h, followed by staining with fluorescently tagged lectins: FITC-tagged MAA to observe α(2,3)-linked sialic acids, and FITC-tagged SNA to observe α(2,6)-linked sialic acids.<br>

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