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Structural properties of the tubular appendage spinae from marine bacterium Roseobacter sp. strain YSCB
A. Bernadac1,a, L.-F. Wu2,8,a,*, C.-L. Santini2,8, C. Vidaud3, J. N. Sturgis4, N. Menguy5,8, P. Bergam1, 9, C. Nicoletti6 and T. Xiao7,8
1 Service de Microscopie Electronique; 2Laboratoire de Chimie Bactérienne, UMR 7283; Institut de Microbiologie de la Méditerranée, Aix-Marseille University, CNRS, 31 chemin Joseph Aiguier, 13402 Marseille cedex 20, France.
3Laboratory of target protein studies, Institut de Biologie Environnementale et de Biotechnologie, CEA Marcoule, BP 17171, F-30207 Bagnols-sur-Cèze, France.
4Laboratoire d'Ingénierie des Systèmes Macromoléculaires UMR 7255, Institut de Microbiologie de la Méditerranée, Aix-Marseille University, CNRS, 31 chemin Joseph Aiguier, 13402 Marseille cedex 20, France.
5IMPMC, UMR 7590 CNRS, Université Pierre et Marie Curie, Université Paris Diderot, IPGP, IRD, Case courrier 115, 4 Place Jussieu, 75252 Paris Cedex 05, France
6Aix Marseille Université, CNRS, ISM2, UMR-7313, 13397, Marseille cedex 20, France
7 Key Laboratory of Marine Ecology & Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China
8 Laboratoire International Associé de la Bio-Minéralisation et Nano-Structures (LIA-BioMNSL), CNRS, F-13402 Marseille cedex 20, France
9current address: Institut Curie, UMR 144 CNRS, Cell and Tissue Imaging Facility (IBiSA), F-75248, Paris, France
a: contributed equally to this work
* For correspondance: L.-F. Wu, LCB, CNRS, 31, chemin Joseph Aiguier, 13402 Marseille cedex 20. Tel: 33-4-9116-4157. Email: email@example.com
Fig. S1. Morphology and appendages of Roseobacter sp. YSCB strain. Negatively stained transmission electron microscopy micrographs of the R. spinae cells. Single polar flagella (A) or spinae (B) were observed on few cells when grown in normal 2216 Marine Broth liquid media. Spinae production was enhanced when the cells were incubated in half-diluted 2216 Marine Broth (C) and spinae were detached from cells by proteinase K treatment (D).
Fig. S2. N-terminal sequences and BlastP analysis of polypeptides in the spine extraction.
A: N-terminal sequences
Polypeptide band of 68 kDa: [SQ] G [GQ] [AE] [DL] [N?].
Polypeptide band of 50 kDa: M [RS] [IP] [NT] [AH] [GN] [I?].
Polypeptide band of 42 kDa: D V N G [?P] [?A] A T.
Polypeptide band of 36 kDa: Q S V L E R V L.
B: BlastP analysis
Using 36 kDa N-terminal sequence as a query
Using the N-terminal sequence of the 42 kDa polypeptide as a query
Fig. S3. Carbohydrate analysis of the spine extraction
Carbohydrate analysis of spine extraction fractions.
Panel A: Periodic acid-Schiff staining performed according to Zacharius et al. (1996, Anal. Biochem. 30: 148-152).
Panel B: lectin binding assay using lectins conjugated to biotin. Streptavidin-horseradish-peroxydase was then added on the membrane and the carbohydrates were visualized by chemoluminescence according instructions of provider (ECL, Pierce, France). B1: lectin from Triticum vulgaris, recognizing N-acetyl-D-glucosamine. B2: lectin from Wisteria floribunda detecting N-acetyl-D-galactosamine.
M: molecular mass markers of which the sizes are on left. Lanes 1 and 2 are spine extractions. Lane 1 is the same sample as those in Figure 3 of the main text whereas Lane 2 is another independently prepared sample. Lane 3 is a positive control of flagellar sheath of marine magnetotactic bacteria described in Lefèvre et al., 2010, Mol. Microbiol. 78:1304-1312. Lane 4 is a positive control of surface protein extracts from a marine bacterium.
Movie: Cryo-tomography of the spinae