Effect of iacP mutation on flagellar phase variation in Salmonella enterica serovar typhimurium strain UK-1

Abstract

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 wild-type strain. These results suggest that IacP is associated with flagellar phase switching under SPI1-inducing conditions.

Fig 1

FljB secretion is increased in the iacP mutant strain. (A) Secreted proteins in the wild-type strain, the iacP mutant strain, and the iacP-complemented strain grown under SPI1-inducing conditions were analyzed by silver staining. To identify differentially secreted proteins in the iacP mutant strain, two bands were excised (band 1 with an increased expression level; band 2 with an unchanged expression level) from the gel and were analyzed by LC-MS. (B) The secretion profiles of the iacP mutant strain were compared with those of the iacP fljB double mutant strain. Secreted proteins from culture supernatants were subjected to SDS-PAGE and were visualized by silver staining. (C) Amino acid sequences of FljB. Peptide sequences identified by LC-MS analysis are indicated by white letters on a black background. Sequences specific to FljB, but not to FliC, are boxed.

Fig 2

IacP repressed FljB expression under SPI1-inducing conditions. (A) To induce IacP expression, l-arabinose was added to the cultures at the indicated concentrations during mid-log phase for 1 h before harvesting of the cells. (B) Growth curves of the S. Typhimurium wild-type strain (filled circles), the iacP mutant strain (filled triangles), and the iacP-complemented strain (filled squares) in LB broth containing 0.3 M NaCl at 37°C. Data are means for three independent experiments. (C and D) Whole-cell lysates and secreted proteins were prepared from the cultures of the wild-type strain, the iacP mutant strain, and the iacP mutant strain carrying the wild-type or point-mutated iacP gene on a plasmid. All strains were grown under SPI1-inducing conditions (0.3 M NaCl) or SPI1-repressing conditions (no NaCl) for 3 h. Western blot analyses were conducted by separating samples on an SDS-PAGE gel and immunoblotting with anti-FljB and anti-FliC antibodies. Anti-SipB and anti-DnaK antibodies were used as an SPI1 substrate and as a loading control for cytoplasmic proteins, respectively.

Fig 3

FljB is regulated at the transcriptional level by IacP. (A and B) Total RNAs were isolated from the wild-type strain, the iacP mutant strain, and the iacP-complemented strain grown under SPI1-inducing conditions for 3 h. The levels of fliC and fljB mRNAs were determined by Northern blot analysis (A) and RT-PCR experiments (B). Band intensities were quantified by densitometry and normalized to the level of the 16S rRNA and 5S rRNA, respectively. Experiments were performed on three independent samples, and representative results are shown. Graphs represent the relative mRNA levels for the fljB, fliC, fljA, and hin genes. (C) For the real-time PCR data, expression values were normalized to the 5S rRNA levels and were expressed relative to the expression level in the wild-type strain. Error bars represent the means ± standard deviations for three independent experiments. Asterisks indicate statistically significant differences by Student's t test (P < 0.05).

Fig 4

The orientation of the invertible H segment was determined in S. Typhimurium grown under SPI1-inducing conditions. (A) The hin genes in the wild-type and iacP mutant strains were disrupted, and hin-encoding plasmids were transformed into the respective deletion strains. All strains were grown under SPI1-inducing conditions for 3 h. Western blot analysis was conducted by separating samples on an SDS-PAGE gel and immunoblotting with anti-FljB and anti-FliC antibodies. An anti-DnaK antibody was used as a loading control. The data shown are representative of three experiments performed independently. (B) The transcription and expression of FljB in the hin mutant, the iacP hin double mutant, and the iacP-complemented strain were assessed by qPCR (left) and Western blotting (right). (C) To determine the orientation of the FliC- or FljB-expressing cells, two hix sites for the off orientation (top) or the on orientation (bottom) were PCR amplified from the genomic DNA of the wild-type strain, the iacP mutant strain, and the iacP-complemented strain grown under SPI1-inducing conditions. The expected sizes of PCR products were as follows: in the off orientation, 543 bp for hixL and 1,769 bp for hixR; in the on orientation, 1,125 bp for hixL and 1,188 bp for hixR.

Fig 5

FliC expression in the iacP mutant strain is repressed by plasmid-encoded FljA. The wild-type strain and the iacP mutant strain carrying either a FljA-encoding plasmid (pYKJ297) or an empty vector (pBAD24) were grown under SPI1-inducing conditions. For the activation of the P_BAD promoter, l-arabinose was added to bacterial cultures at a concentration of 0.1% (wt/vol). Western blot analysis was conducted by separating samples on an SDS-PAGE gel and immunoblotting with anti-FliC, anti-FljB, and anti-FLAG antibodies.

Fig 6

The iacP mutant strain possesses more flagellar filaments than the wild-type strain. (A) Bacteria and surface flagella were negatively stained with uranyl acetate and were visualized by transmission electron microscopy. Bars, 2 μm. Images are representative of three independent experiments. (B) For each strain, the number of flagella per cell was quantified from at least 50 cells. Error bars represent means ± standard deviations for three independent experiments. (C) After incubation for 6 h at 30°C on semisolid agar, the zones of motility were measured for the wild-type and iacP mutant strains. The fliGHI::Tn10 mutant was used as a nonmotile control. Analysis by Student's t test indicated that the differences were statistically significant (*, P < 0.05).