Structural characterization of two lipopolysaccharide O-antigens produced by the endofungal bacterium Burkholderia sp. HKI-402 (B4) Alba Silipo a , Maria R. Leone a , Rosa Lanzetta a , Michelangelo Parrilli a , Gerald Lackner b , Benjamin Busch b , Christian Hertweck b,c , Antonio Molinaro a,⇑ a Dipartimento di Chimica Organica e Biochimica, Università di Napoli Federico II, via Cintia 4, 80126 Napoli, Italy b Department of Biomolecular Chemistry, Leibniz Institute for Natural Product Research and Infection Biology, HKI, Beutenbergstr. 11a, 07745 Jena, Germany c Chair for Natural Product Chemistry, Friedrich Schiller University, Jena, Germany article info Article history: Received 12 September 2011 Received in revised form 21 October 2011 Accepted 25 October 2011 Available online 4 November 2011 Keywords: Burkholderia rhizoxinica O-antigen Lipopolysaccharide Endosymbiontic bacteria abstract Two different polysaccharides were isolated and identified from the lipopolysaccharide fraction of endofungal bacterium Burkholderia sp. HKI-402 (B4). The complete structure was elucidated by chemical analysis and 2D NMR spectroscopy as the following: Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction The rice seedling blight fungus Rhizopus microsporus forms a un- ique type of mutualism with bacterial endosymbionts that produce a phytotoxin. 1,2 Specifically, the antimitotic agent rhizoxin is not produced by the fungus, but by bacteria belonging to the genus Burkholderia, 3,4 as evidenced by genetic, microbial and chemical analyses. 5–8 All rhizoxin-producing endosymbionts investigated to date form a monophyletic group of bacterial species termed the Burholderia rhizoxinica complex, including B. rhizoxinica HKI- 454 (B1), Burkholderia endofungorum (B5), 9 and Burkholderia sp. HKI-402 (B4). 10 Apart from the various implications of toxin pro- duction in agriculture, nutrition and health, 11,12 this mutualistic life form is intriguing since vegetative reproduction of the fungus has become totally dependent of the endofungal bacteria. 13 In this regard, there are many open questions concerning factors govern- ing the establishment, stability and persistence of the symbiosis. Recently, we have shown that apart from acquired host resis- tance, 14 and a crucial type III secretion system, 15 the B. rhizoxinica HKI-454 (B1) lipopolysaccharide (LPS) plays a pivotal role for the stability of the endosymbiosis. 16 LPSs are the major components of the outer membrane of Gram-negative bacteria, greatly contributing to the structural integrity of the bacteria and to the protection of the bacterial cell envelope. 17 Owing to their external location on outer bacterial membrane, the LPSs are responsible for many biological interac- tions of bacteria with the external environment. In particular, they are pivotal in many processes of host–bacteria interaction like adhesion, recognition, pathogenesis, symbiosis, etc. 17 Moreover, LPSs are also called endotoxins because they are cell-bound and, once released, play a key role in the pathogenesis of Gram-negative infections owing to the ability to trigger the immune system in a wide range of eukaryotic organisms. 17 In order to understand the molecular basis for bacterium–host interaction, it is important to elucidate the structure of LPS and to identify how the bacterium modifies this structure in response to different environments. We have recently demonstrated that LPS may not only be required to establish a disease (pathogens), but also to produce a beneficial outcome in the Rhizopus–Burkholderia interaction. 16 Here we report the chemical characterization of the LPS O-antigen portion of Burkholderia sp. HKI-402 (strain B4) that proved to be a mixture of two glycopolymers. 2. Results 2.1. Isolation and chemical analysis of the O-antigen portion The LPS extraction was carried out on dried cells with hot phe- nol/water method. 18 The SDS–PAGE analysis revealed that the LPS 0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2011.10.038 ⇑ Corresponding author. Tel.: +39 081 674 123; fax: +39 081 674393. E-mail address: molinaro@unina.it (A. Molinaro). Carbohydrate Research 347 (2012) 95–98 Contents lists available at SciVerse ScienceDirect Carbohydrate Research journal homepage: www.elsevier.com/locate/carres