Genetic and Molecular Analysis of GogB, a Phage- encoded Type III-secreted Substrate in Salmonella enterica Serovar Typhimurium with Autonomous Expression from its Associated Phage Brian K. Coombes 1 , Mark E. Wickham 1 , Nat. F. Brown 1 Sebastien Lemire 2 , Lionello Bossi 2 , William W. L. Hsiao 3 Fiona S. L. Brinkman 3 and B. Brett Finlay 1,4,5 * 1 Michael Smith Laboratories University of British Columbia Vancouver, BC, Canada V6T 1Z3 2 Centre de Ge ´ne ´tique Mole ´culaire, Centre National de la Recherche Scientifique 91198 France 3 Department of Molecular Biology and Biochemistry Simon Fraser University Burnaby, BC, Canada V5A 1S6 4 Department of Biochemistry and Molecular Biology University of British Columbia Vancouver, BC, Canada V6T 1Z3 5 Department of Microbiology and Immunology, University of British Columbia, Vancouver BC, Canada V6T 1Z3 Salmonella enterica serovar Typhimurium is lysogenized by several temperate bacteriophages that encode lysogenic conversion genes, which can act as virulence factors during infection and contribute to the genetic diversity and pathogenic potential of the lysogen. We have investigated the temperate bacteriophage called Gifsy-1 in S. enterica serovar Typhimurium and show here that the product of the gogB gene encoded within this phage shares similarity with proteins from other Gram-negative pathogens. The amino-terminal portion of GogB shares similarity with leucine-rich repeat-containing virulence-associated proteins from other Gram-negative pathogens, whereas the carboxyl-terminal portion of GogB shares similarity with uncharacterized proteins in other pathogens. We show that GogB is secreted by both type III secretion systems encoded in Salmonella Pathogenicity Island-1 (SPI-1) and SPI-2 but translocation into host cells is a SPI-2-mediated process. Once translocated, GogB localizes to the cytoplasm of infected host cells. The genetic regulation of gogB in Salmonella is influenced by the transcriptional activator, SsrB, under SPI-2- inducing conditions, but the modular nature of the gogB gene allows for autonomous expression and type III secretion following horizontal gene transfer into a heterologous pathogen. These data define the first autonomously expressed lysogenic conversion gene within Gifsy-1 that acts as a modular and promiscuous type III-secreted substrate of the infection process. q 2005 Elsevier Ltd. All rights reserved. Keywords: Salmonella; Gifsy; lambdoid phage; GogB; type III secretion *Corresponding author Introduction Salmonella enterica are facultative intracellular Gram-negative bacteria that are important enteric pathogens for humans and commercial livestock. Infection with these organisms causes a self-limit- ing enterocolitis that can progress to a serious systemic disease in ruminants and humans called enteric fever. The infecting S. enterica serovar and the host species influence systemic disease pro- gression. For example, serovar Typhi is a human host-restricted pathogen, whereas serovar Typhi- murium exhibits a wide host range including humans, mice, cattle and chickens. Two major virulence determinants that contribute to Salmonella pathogenesis are encoded in separate chromosomal pathogenicity islands called Salmonella Patho- genicity Island: SPI-1 1 and SPI-2. 2,3 Each encodes a suite of molecules that assemble into a type III secretion system that can translocate effector pro- teins directly from the bacterial cell into target host cells. Effector proteins translocated by the SPI-1 type III secretion system influence early cyto- skeletal and membrane rearrangements involved 0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. Abbreviations used: SPI-1, Salmonella Pathogenicity Island-1; SARC11, Salmonella Reference Collection C-11; LRR, leucine-rich repeat; HMM, hidden Markov model; LEE, locus of enterocyte effacement; FRT, FLP recognition target; RLU, relative light units. E-mail address of the corresponding author: bfinlay@interchange.ubc.ca doi:10.1016/j.jmb.2005.03.024 J. Mol. Biol. (2005) 348, 817–830