TheSalmonella entericaSerovar TyphitsxGene, Encoding a Nucleoside-Specific Porin, Is Essential for Prototrophic Growth in the Absence of Nucleosides

Abstract

The Salmonella enterica serovar Typhi tsx gene encodes a porin that facilitates the import of nucleosides. When serovar Typhi is grown under anaerobic conditions, Tsx is among the outer membrane proteins whose expression increases dramatically. This increase in expression is due, at least in part, to increased transcription and is dependent on Fnr but not on ArcA. A mutant derivative of serovar Typhi strain STH2370 with a deletion of the tsx gene is an auxotroph that requires either adenosine or thymidine for growth on minimal medium. In contrast, an otherwise isogenic nupG nupC double mutant, defective in the inner membrane nucleoside permeases, is a prototroph. Because anaerobic growth enhances the virulence of serovar Typhi in vitro, we assessed the role that the tsx gene plays in pathogenicity and found that the serovar Typhi STH2370 ⌬tsx mutant is defective in survival within human macrophage-like U937 cells. To understand why the ⌬tsx mutant is an auxotroph, we selected for insertions of minitransposon T-POP in the ⌬tsx genetic background that restored prototrophy. One T-POP insertion that suppressed the ⌬tsx mutation in the presence of the inducer tetracycline was located upstream of the pyrD gene. The results of reverse transcription-PCR analysis showed that addition of the inducer decreased the rate of pyrD transcription. These results suggest that the Tsx porin and the balance of products of the tsx and pyrD genes play critical roles in membrane assembly and integrity and thus in the virulence of serovar Typhi.

The Cotranscribed Salmonella enterica sv. Typhi tsx and impX Genes Encode Opposing Nucleoside-Specific Import and Export Proteins

“…In contrast, in three independent isolates of S. Typhi, a Dtsx nonpolar mutation confers an auxotrophic requirement for the nucleosides adenosine or thymidine (Bucarey et al 2005).…”

“…Previously, we have shown that complementation of an S. Typhi Dtsx mutant with a derivative of the moderate-copy-number plasmid pSU19 carrying the cloned tsx gene yields a strain that is prototrophic, yet cannot grow in the presence of high concentrations of adenoside or thymidine, suggesting that overproduction of the Tsx porin results in an imbalance of membrane proteins that renders S. Typhi sensitive to adenosine or thymidine (Bucarey et al 2005). This is the same phenotype exhibited by the S. Typhi DimpX mutant.…”

“…Surprisingly, we found that Tsx is essential for the prototrophic growth of S. Typhi in the absence of nucleosides. On the basis of these and other results, we have proposed that the Tsx porin plays a critical role in the assembly and integrity of the S. Typhi membrane, and thus in the virulence of S. Typhi (Bucarey et al 2005). In this article, we show that a short open reading frame (ORF) adjacent to the S. Typhi tsx gene, impX (STY0450), encodes an additional product that mediates nucleoside transport across the S. Typhi membrane.…”

“…This Phenotypic analysis of S. Typhi mutants: A modification of the disk diffusion assay (Bauer et al 1966) was used to determine the auxotrophic requirements of mutant strains, as well as their sensitivities to nucleosides. Assays to measure the efficiencies of plating of wild-type and mutant strains in the presence of nucleosides were performed as described previously (Bucarey et al 2005) and in the text. Efficiencies of plating are expressed as the titers of colony-forming units on media with or without nucleosides, divided by the titer of the wild-type strain on media without nucleosides.…”

Comparative Proteomic Analysis of the Haemophilus ducreyi Porin-Deficient Mutant 35000HP::P2AB

“…The loss of classical porin expression in gram-negative pathogens often results in decreased fitness in both in vitro and in vivo environments (1,6,(8)(9)(10)41). However, 35000HP:: P2AB did not exhibit any loss of viability or demonstrate any growth defect in vitro or in vivo in the human challenge model (20).…”