Lipoprotein PssN of Rhizobium leguminosarum bv. trifolii: subcellular localization and possible involvement in exopolysaccharide export.
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 beta-structure; however, alpha-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.
Marczak
Małgorzata
M
Department of General Microbiology, Institute of Microbiology and Biotechnology, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland.
Mazur
Andrzej
A
Król
Jarosław E
JE
Gruszecki
Wiesław I
WI
Skorupska
Anna
A
D019677:Artificial Gene Fusion; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology; D001426:Bacterial Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology; D001692:Biological Transport; D019084:Fluorescent Antibody Technique, Indirect; D008074:Lipoproteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology; D008970:Molecular Weight; D033903:Periplasmic Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis / Q000235:genetics; D011135:Polysaccharides, Bacterial / Q000378:metabolism*; D021382:Protein Sorting Signals; D017433:Protein Structure, Secondary; D016961:Rhizobium leguminosarum / Q000737:chemistry* / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared; D013194:Staining and Labeling; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D008074:Lipoproteins; D033903:Periplasmic Proteins; D011135:Polysaccharides, Bacterial; D021382:Protein Sorting Signals; D010744:Phosphoric Monoester Hydrolases; D001616:beta-Galactosidase