The core and O-polysaccharide structure of the Caulobacter crescentus lipopolysaccharide Michael D. Jones a , Evgeny Vinogradov b , John F. Nomellini a , John Smit a,⇑ a Department of Microbiology and Immunology, 2350 Health Sciences Mall, Life Sciences Centre, University of British Columbia, Vancouver, BC V6T 1Z3, Canada b National Research Council of Canada, 100 Sussex Drive, Building Sussex, Room 3079, Ottawa, Ontario K1A 0R6, Canada article info Article history: Received 7 July 2014 Received in revised form 10 September 2014 Accepted 10 October 2014 Available online 24 October 2014 Keywords: Lipopolysaccharides Extracellular polysaccharides Nuclear magnetic resonance abstract Here we describe the analysis of the structure of the lipopolysaccharide (LPS) from Caulobacter crescentus strain JS1025, a derivative of C. crescentus CB15 NA1000 with an engineered amber mutation in rsaA, lead- ing to the loss of the protein S-layer and gene CCNA_00471 encoding a putative GDP-L-fucose synthase. LPS was isolated using an aqueous membrane disruption method. Polysaccharide and core oligosaccha- ride were produced by mild acid hydrolysis and analyzed by nuclear magnetic resonance spectroscopy and chemical methods. Spectra revealed the presence of two polysaccharides, one of them, a rhamnan, could be removed using periodate oxidation. Another polymer, built from 4-amino-4-deoxy-D-rhamnose (perosamine), mannose, and 3-O-methyl-glucose, should be the O-chain of the LPS according to genetic data. The attribution of the rhamnan as a part of LPS or a separate polymer was not possible. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction Caulobacter crescentus is an aquatic alphaproteobacterium well known for a stalked, crescent cell morphology, asymmetric cell division, and a protein surface layer (S-layer). C. crescentus is a widely studied model organism for cell development and differen- tiation; despite this, the structure of its lipopolysaccharide (LPS) has not yet been fully determined. Interest in the LPS of C. crescentus is focused on its immunolog- ical profile 1 and its structural role as an anchor for the self-assem- bled, paracrystalline protein S-layer. 2 The LPS of C. crescentus possesses a much reduced immunogenic activity, most likely due to its lipid A structure, which is significantly different from that of LPS from enteric bacteria. The lipid A structure has been reported; 1 it is a unique molecule containing a di-diaminoglucose backbone (instead of di-glucosamine) and two galacturonate moi- eties that replace the canonical phosphates that are on each end of the disaccharide in most lipid A molecules. The C. crescentus S-layer non-covalently attaches to the O-polysaccharide (OPS). 2 However, the OPS’ structure has not been resolved. Genetic analyses have pointed toward the unusual N-acetylperosamine as being a major component. 3 A notable feature of this O-antigen is that it exists completely hidden beneath the S-layer, presumably inaccessible to the environment. 2 Carbohydrate structures from non- pathogenic bacterial LPS are rarely studied and an LPS that is sequestered beneath an S-layer is not represented in the literature. In the present study our data have determined the core oligo- saccharide structure from C. crescentus CB15 NA1000, (advancing an earlier report of core composition 4 ) as well as the central back- bone and non-reducing ends of its OPS. Unexpectedly, we identi- fied a previously unknown rhamnan polysaccharide. Along with previous reports on lipid A 1 and extracellular polysaccharide (EPS), 5 we believe that the major carbohydrate structures in C. crescentus’ cell envelope have now been solved. 2. Results 2.1. Initial assessment and component analysis The polysaccharide (PS) was released from the LPS by hydroly- sis with acetic acid. 1 H NMR spectrum of the PS (Fig. 1) contained a large number of partially overlapping signals of various intensities in the anomeric region. It was obviously not a regular polymer with well-defined repeating units. Attempts to separate this mate- rial by anion-exchange chromatography led to the isolation of a number of fractions from neutral to slightly retained, but all of them had virtually identical NMR spectra. Methylation of the polysaccharide led to the identification of 3- and 3,4-substituted mannopyranose, terminal glucopyranose (derived from side-chain http://dx.doi.org/10.1016/j.carres.2014.10.003 0008-6215/Ó 2014 Elsevier Ltd. All rights reserved. ⇑ Corresponding author. E-mail addresses: Mikedave@mail.ubc.ca (M.D. Jones), Evguenii.Vinogradov@ nrc-cnrc.gc.ca (E. Vinogradov), Nomellin@mail.ubc.ca (J.F. Nomellini), Jsmit@mail. ubc.ca (J. Smit). Carbohydrate Research 402 (2015) 111–117 Contents lists available at ScienceDirect Carbohydrate Research journal homepage: www.elsevier.com/locate/carres