Identification of Francisella tularensis genes encoding exported membrane-associated proteins using TnphoA mutagenesis of a genomic library.
Francisella tularensis, the causative agent of tularemia, is a highly infectious pathogen of humans and animals, yet little is known about the surface proteins of this organism that mediate mechanisms of pathogenicity. lambdaTnphoA was used to generate random alkaline phosphatase gene fusions in a F. tularensis subsp. tularensis (strain Schu S4) genomic library to identify genes encoding exported extracytoplasmic proteins. Eleven genes encoding membrane-associated proteins were identified by this method and their respective signal peptides were characterized. Three of the genes encoded conserved 'housekeeping' enzymes, while the other eight genes were unique to F. tularensis, encoding proteins with molecular masses ranging from 11 to 78kDa as deduced from the amino acid sequences. Two genes putatively encoded lipoproteins based on the presence of characteristic signal peptidase II cleavage sites. Four selected proteins were found associated with outer membranes from Schu S4 and LVS strains by Western blotting. Indirect immunofluorescence of strain Schu S4 cells also showed evidence of protein localization to the outer membrane. Protein database searches produced significant alignments with proteins from other bacteria involved in carbohydrate transport, lipid metabolism, and cell envelope biogenesis, thereby providing clues for putative functions. These findings demonstrated that TnphoA mutagenesis can be used in conjunction with F. tularensis genome sequence data to provide a foundation for studies to identify and define cellular surface protein virulence factors of this pathogen.
Gilmore
Robert D
RD
Bacterial Zoonoses Branch, Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, P.O. Box 2087, Rampart Road, Foothills Campus, Fort Collins, CO 80522, USA. rbg9@cdc.gov
Bacon
Rendi Murphree
RM
Sviat
Steven L
SL
Petersen
Jeannine M
JM
Bearden
Scott W
SW
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D005455:Fluorescent Antibody Technique; D005604:Francisella tularensis / Q000235:genetics* / Q000378:metabolism*; D005798:Genes, Bacterial; D017930:Genes, Reporter; D015151:Immunoblotting; D008074:Lipoproteins / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016254:Mutagenesis, Insertional; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology; D021381:Protein Transport; D017422:Sequence Analysis, DNA; D017385:Sequence Homology
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D008074:Lipoproteins; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D000469:Alkaline Phosphatase