Disrupted synthesis of a di- N -acetylated sugar perturbs mature glycoform structure and microheterogeneity in the O -linked protein glycosylation system of Neisseria elongata subsp. glycolytica .

The genus Neisseria includes three major species of importance to human health and disease ( Neisseria gonorrhoeae , N. meningitidis , and N. lactamica ) that express broad spectrum O -linked protein glycosylation systems. The potential for related pgl systems in other species in the genus, however, remains to be determined. Using a strain of N. elongata subsp. glycolytica , a unique tetrasaccharide glycoform consisting of di- N -acetylbacillosamine and glucose as the first two sugars followed by a rare sugar whose mass spectrometric fragmentation profile was most consistent with di- N -acetyl hexuronic acid and a N -acetylhexosamine at the non-reducing end has been identified. Based on established mechanisms for UDP-di- N -acetyl hexuronic acid biosynthesis found in other microbes, we searched for genes encoding related pathway components in the N. elongata subsp. glycolytica genome. Here, we detail the identification of such genes and the ensuing glycosylation phenotypes engendered by their inactivation. While the findings extend the conservative nature of microbial UDP-di- N -acetyl hexuronic acid biosynthesis, mutant glycosylation phenotypes reveal unique, relaxed specificities of the glycosyltransferases and oligosaccharyltransferases to incorporate pathway intermediate UDP-sugars into mature glycoforms. IMPORTANCE Broad-spectrum protein glycosylation ( pgl ) systems are well recognized in bacteria and archaea. Knowledge of how these systems relate structurally, biochemically as well as evolutionarily to one another and to others associated with microbial surface glycoconjugate expression is still incomplete. Here, we detail reverse genetic efforts towards characterization of protein glycosylation mutants in N. elongata subsp. glycolytica that define the biosynthesis of a conserved but relatively rare UDP-sugar precursor. The results show both a significant degree of intra- and trans- kingdom conservation in the utilization of UDP-di- N -acetyl-glucuronic acid and singular properties related to the relaxed specificities of the N. elongata subsp. glycolytica system.