The formation of cyclopropane fatty acid (CFA) and its role in the acid shock response in Salmonella enterica serovar Typhimurium (S. typhimurium) was investigated. Data obtained by GC/MS demonstrated that the CFA level in S. typhimurium increased upon its entry to the stationary phase, as in other bacteria. The cfa gene encoding CFA synthase was cloned, and mutants of the cfa gene were constructed by allelic exchange. A cfa mutant could not produce CFA and was sensitive to low pH. Introduction of a functional cfa gene into a cfa mutant cell made the mutant convert all unsaturated fatty acids to CFAs and partially restored resistance to low pH. Interestingly, the alternative sigma factor RpoS, which was induced during the stationary phase, affected the production of C19 CFA but not C17 CFA. Western blotting analysis showed that the increase in expression of CFA synthase at early stationary phase was due to the alternative sigma factor RpoS.

No abstract

Gram-negative bacteria, including Salmonella enterica serovar Typhimurium, exploit type III secretion systems (T3SSs) through which virulence proteins are delivered into the host cytosol to reinforce invasive and replicative niches in their host. Although many secreted effector proteins and membrane-bound structural proteins in the T3SS have been characterized, the functions of many cytoplasmic proteins still remain unknown. In this study, we found that IacP, encoded by Salmonella pathogenicity island 1, was important for nonphagocytic cell invasion and bacterial virulence. When the iacP gene was deleted from several Salmonella serovar Typhimurium strains, the invasion into INT-407 epithelial cells was significantly decreased compared to that of their parental strains, and retarded rearrangements of actin fibers were observed for the iacP mutant-infected cells. Although IacP had no effect on the secretion of type III translocon proteins, the levels of secretion of the effector proteins SopB, SopA, and SopD into the culture medium were decreased in the iacP mutant. In a mouse infection model, mice infected with the iacP mutant exhibited alleviated pathological signs in the intestine and survived longer than did wild-type-infected mice. Taken together, IacP plays a key role in Salmonella virulence by regulating the translocation of T3SS effector proteins.

c Flagella are surface appendages that are important for bacterial motility and invasion of host cells. Two flagellin subunits in Salmonella enterica serovar Typhimurium, FliC and FljB, are alternatively expressed by a site-specific DNA inversion mechanism called flagellar phase variation. Although this inversion mechanism is understood at the molecular level, the key factor controlling the expression of the two flagellin subunits has not been determined. In this study, we found that a putative acyl carrier protein, IacP, affects flagellar phase variation in S. Typhimurium strain UK-1 under Salmonella pathogenicity island 1 (SPI1)-inducing conditions. Liquid chromatography-mass spectrometry analysis of the secreted proteins from S. Typhimurium determined that the amount of FljB secreted was significantly higher in the iacP mutant strain, a finding confirmed by Western blot analysis. Northern blotting, quantitative PCR, and microarray data showed that the level of FljB in the iacP mutant strain was regulated at the transcriptional level, although the transcription and expression of the fliC gene were independent of IacP. FljB production was abolished by the deletion of the Hin DNA invertase but could be restored by the introduction of a plasmid carrying the hin gene. We also found that in the iacP mutant strain, the orientation of the invertible H segment is in the FljB-expressing phase. Furthermore, electron microscopy observations indicated that the iacP mutant strain had more flagella per cell than the wildtype strain. These results suggest that IacP is associated with flagellar phase switching under SPI1-inducing conditions. F lagella play crucial roles in bacterial motility, chemotactic behavior, and host cell invasion as a virulence determinant (1). Salmonella enterica serovar Typhimurium has 5 to 10 flagella emanating from the bacterial surface. The individual flagellum is a complex structure composed of three basic parts: a basal body, a hook, and a filament (22). The flagellar filament consists of thousands of flagellin protein monomers, which are polymerized into a helical structure that can be 10 m long. S. Typhimurium possesses two genetically distinct flagellin subunits, FliC (phase 1 flagellin) and FljB (phase 2 flagellin), which are alternatively expressed via a site-specific DNA inversion process (19,30). When the 1-kb upstream region of the fljB gene (H segment) is inverted by the DNA invertase Hin, the promoter for the fljB gene is properly oriented for the transcription of the fljB-fljA operon. Because FljA is responsible for the posttranscriptional and posttranslational inhibition of FliC expression (2, 38), only FljB flagellin is produced. Although the molecular mechanism of the Hin-mediated DNA inversion mechanism has been studied intensively (6, 24, 28), the question of which cellular signals control the flagellinswitching mechanism and the frequency of DNA inversion remains unclear.Salmonella pathogenicity island 1 (SPI1) encodes a type III secretion system (T3SS) that is required for the ...