Bacterial protein glycosylation systems from varying species have been functionally reconstituted in Escherichia coli. Both N- and O-linked glycosylation pathways, in which the glycans are first assembled onto lipid carriers and subsequently transferred to acceptor proteins by an oligosaccharyltransferase (OTase), have been documented in bacteria. The identification and characterization of novel OTases with different properties may provide new tools for engineering glycoproteins of biotechnological interest. In the case of OTases involved in O-glycosylation (O-OTases), there is very low sequence homology between those from different bacterial species. The Wzy_C signature domain common to these enzymes is also present in WaaL ligases; enzymes involved in lipopolysaccharide biosynthesis. Therefore, the identification of O-OTases using solely bioinformatic methods is problematic. The hypothetical proteins BTH_I0650 from Burkholderia thailandensis E264 and VC0393 from Vibrio cholerae N16961 contain the Wzy_C domain. In this work, we demonstrate that both proteins have O-OTase activity and renamed them PglL(Bt) and PglL(Vc), respectively, similar to the Neisseria meningitidis counterpart (PglL(Nm)). In E. coli, PglL(Bt) and PglL(Vc) display relaxed glycan and protein specificity. However, effective glycosylation depends upon a specific combination of the protein acceptor, glycan and O-OTase analyzed. This knowledge has important implications in the design of glycoconjugates and provides novel tools for use in glycoengineering applications. The codification of enzymatically active O-OTase in the genomes of members of the Vibrio and Burkholderia genera suggests the presence of still unknown O-glycoproteins in these organisms, which might have a role in bacterial physiology or pathogenesis.

SummaryAeromonas hydrophila transports extracellular protein toxins via the type II secretion system, an export mechanism comprised of numerous proteins that spans both the inner and outer membranes. Two components of this secretion system, ExeA and ExeB, form a complex in the inner membrane that functions to locate and/or assemble the ExeD secretin in the outer membrane. In the studies reported here, twocodon insertion mutagenesis of exeA revealed that an insertion at amino acid 495 in the C-terminal region of ExeA did not alter ExeAB complex formation yet completely abrogated its involvement in ExeD secretin assembly and thus rendered the bacteria secretion negative. In silico analysis of protein motifs with similar amino acid profiles revealed that this amino acid is located within a putative peptidoglycan (PG) binding motif in the periplasmic domain of ExeA. Substitution mutations of three highly conserved amino acids in the motif were constructed. In cells expressing each of these mutants, the ability to assemble the ExeD secretin or secrete aerolysin was lost, while ExeA retained the ability to form a complex with ExeB. In in vivo cross-linking experiments, wild-type ExeA could be cross-linked to PG, whereas the three substitution mutants of ExeA could not. These data indicate that PG binding and/or remodelling plays a role in the function of the ExeAB complex during assembly of the ExeD secretin.

Campylobacter curvus was isolated from blood cultures of a patient with liver abscesses. Bacterial identification involved Gram staining, biochemical analysis, gas-liquid chromatography, and 16S rRNA sequencing. The difficulty in isolation, identification, and growth of the species confirms previous work that these organisms may be overlooked by conventional detection methods. CASE REPORTA 52-year-old carpenter with a history of smoking and diverticulitis, but otherwise generally healthy, presented with general malaise and pyrexia. A chest X-ray image revealed minor changes suggestive of pneumonia, and the patient was treated with intravenous cefuroxime. He improved and was discharged.