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Lipoprotein PssN of Rhizobium leguminosarum bv. trifolii: Subcellular Localization and Possible Involvement in Exopolysaccharide Export

Magorzata Marczak,¹ Andrzej Mazur,¹ Jarosaw E. Król,¹ Wiesaw I. Gruszecki,² and Anna Skorupska^1*

Department of General Microbiology, Institute of Microbiology and Biotechnology, Maria Curie-Skodowska University, Akademicka 19, 20-033 Lublin, Poland,¹ Department of Biophysics, Institute of Physics, Maria Curie-Skodowska University, Maria Curie-Skodowska Sq. 1, 20-031 Lublin, Poland²

Received 8 May 2006/ Accepted 15 July 2006

Surface expression of exopolysaccharides (EPS) in gram-negative bacteria depends on the activity of proteins found in the cytoplasmic membrane, the periplasmic space, and the outer membrane. pssTNOP genes identified in Rhizobium leguminosarum bv. trifolii strain TA1 encode proteins that might be components of the EPS polymerization and secretion system. In this study, we have characterized PssN protein. Employing pssN-phoA and pssN-lacZ gene fusions and in vivo acylation with [³H]palmitate, we demonstrated that PssN is a 43-kDa lipoprotein directed to the periplasm by an N-terminal signal sequence. Membrane detergent fractionation followed by sucrose gradient centrifugation showed that PssN is an outer membrane-associated protein. Indirect immunofluorescence with anti-PssN and fluorescein isothiocyanate-conjugated antibodies and protease digestion of spheroplasts and intact cells of TA1 provided evidence that PssN is oriented towards the periplasmic space. Chemical cross-linking of TA1 and E. coli cells overproducing PssN-His₆ protein showed that PssN might exist as a homo-oligomer of at least two monomers. Investigation of the secondary structure of purified PssN-His₆ protein by Fourier transform infrared spectroscopy revealed the predominant presence of ß-structure; however, -helices were also detected. Influence of an increased amount of PssN protein on the TA1 phenotype was assessed and correlated with a moderate enhancement of EPS production.