Role of nucleoid-associated proteins Hha and H-NS in expression of Salmonella enterica activators HilD, HilC, and RtsA required for cell invasion

Abstract

The coordinate expression of Salmonella enterica invasion genes on Salmonella pathogenicity island 1 is under the control of the complex circuits of regulation that involve the AraC/XylS family transcriptional activators HilD, HilC, and RtsA and nucleoid-associated proteins. Single-copy transcription fusions were used to assess the effects of nucleoid-associated proteins Hha and H-NS on hilD, hilC, and rtsA expression. The data show that all three genes, hilD, hilC, and rtsA, were repressed by H-NS and/or Hha. The repression of rtsA was the highest among tested genes. The level of rtsA-lac was equally elevated in hns and hha mutants and was further enhanced in the hns hha double mutant under low-osmolarity conditions. Electrophoretic mobility shift experiments showed that H-NS and Hha directly bind to the rtsA promoter. In addition to the negative control that was exerted by H-NS/Hha under low-osmolarity conditions, the homologous virulence activators HilD, HilC, and RtsA (Hil activators) induced rtsA-lac expression in a high-salt medium. A DNase footprinting assay of the rtsA promoter revealed one common DNA-binding site for all three Hil activators centered at position -54 relative to the transcriptional start site. In the absence of Hha and H-NS, however, osmoregulation of the rtsA promoter was lost, and Hil activators were not required for rtsA transcription. These results taken together suggest that the HilD, HilC, and RtsA proteins induce the transcription of the rtsA promoter by counteracting H-NS/Hha-mediated repression.

FIG. 1.

The effect of deletions of hns and hha regulators on rtsA, hilC, and hilD transcription. (A) The expression of chromosomal reporters was examined for the following strains (ATCC 14028 background): the wild-type (WT) rtsA-lac strain (JS324), the hha rtsA-lac mutant (IO949), the hns rtsA-lac mutant (IO954), the hha hns rtsA-lac mutant (IO955), the WT hilC-lac strain (JS487), the hha hilC-lac mutant (IO969), the hns hilC-lac mutant (IO971), the hha hns hilC-lac mutant (IO973), the WT hilD-lac strain (JS488), the hha hilD-lac mutant (IO959), the hns hilD-lac mutant (IO961), and the hha hns hilD-lac mutant (IO963). The level of β-galactosidase was determined following growth in LB medium with 1% NaCl (+NaCl) or no added NaCl (−NaCl). Data are representative of three independent experiments. (B) The transcription of the rtsA and marA genes on the chromosome was monitored by RT-PCR following the growth of the WT (IO908) and the hha hns mutant (IO923) in LB medium without NaCl. The experiment was performed three times, and typical data are shown.

FIG. 2.

A schematic version of the STM4316-rtsA intergenic region. The rtsA promoter-specific transcript is indicated with an arrow, and the sequence of the start site is marked as +1. The nucleotide sequence of the rtsA promoter region is shown below. The rtsA promoter “−10” and “−35” elements are underlined. The rtsA transcription start site was determined by primer extension as described in Materials and Methods.

FIG. 3.

EMSAs of the binding of H-NS and MBP-Hha to the rtsA promoter. The proteins were incubated with a ³²P-labeled DNA fragment representing a portion of the rtsA promoter at positions −56 to +169. Samples were resolved by nondenaturing polyacrylamide gel electrophoresis, and the positions of the radioactive bands were detected by use of a PhosphorImager.

FIG. 4.

EMSA of the binding of MBP-RtsA to the rtsA, hilC, hilD, and hilA promoter fragments. The MBP-RtsA protein in concentrations of 0, 37, 75, 150, and 300 nM was incubated with ³²P-labeled DNA fragments representing portions of the four promoters, as indicated at the left. Samples were resolved by nondenaturing polyacrylamide gel electrophoresis, and the positions of the radioactive bands were detected by use of a PhosphorImager.

FIG. 5.

EMSA of the binding of HilC, HilD, and MBP-RtsA to portions of the rtsA promoter. Fragments are identified at the top. Fragments were labeled with ³²P as described in Materials and Methods. The proteins added in each lane are as follows: lanes 1, no added protein; lanes 2 and 5, 75 nM HilC; lanes 3 and 6, 75 nM HilD; lanes 4 and 7, 75 nM MBP-RtsA. Unlabeled rtsA promoter DNA (10 ng) was included in reactions 5 to 7.

FIG. 6.

DNase I protection assay of HilC, HilD, and MBP-RtsA binding to the rtsA promoter. The DNase I digestions represent the noncoding strand of the ³²P-labeled rtsA fragment from −173 to +29 incubated with increasing amounts of purified proteins. Protected bands are indicated on the left. Hypersensitive sites are shown by arrows. Lane 1 has the products of the A+G sequencing reaction of the same DNA fragment. The added proteins in each lane are as follows: lane 2, no added protein; lane 3, 150 nM HilC; lane 4, 300 nM HilC; lane 5, 150 nM HilD; lane 6, 300 nM HilD; lane 7, 150 nM MBP-RtsA; lane 8, 300 nM MBP-RtsA.

FIG. 7.

Comparison of HilC/HilD binding sites in the rtsA, hilC, hilD, and hilA promoters. (A) Alignment of the HilC/HilD binding sites defined by DNase I footprinting here and previously (37). The common positions are indicated by shading. The proposed consensus is shown in the bottom. The arrows indicate two direct repeats of CNATTNNT (shown in bold). Predominant bases (5/5 or 4/5) in the consensus sequence are indicated by uppercase letters, bases identical in 3/5 of the HilC/HilD binding sites are indicated by lowercase letters, and degenerate bases are marked by the letter N. The inverted positions of hilC C2 and hilA A1 sites in alignment compared to regions in hilD, rtsA promoters and hilA A2 site are marked by asterisks. (B) Positions and directions of the HilD/HilC consensus in the rtsA, hilC, hilD, and hilA promoters relative to the transcription start point, indicated as +1.

FIG. 8.

The effect of the deletions of hns and hha on the expression of the chromosomal transcriptional fusion rtsA-lac in the absence of hilCD. The expression of the rtsA-lac reporter was examined for the following strains (ATCC 14028 background): the WT rtsA-lac strain (JS324), the ΔhilCD rtsA-lac mutant (IO976), the hha ΔhilCD rtsA-lac mutant (IO997), the hns ΔhilCD rtsA-lac mutant (IO999), and the hha hns ΔhilCD rtsA-lac mutant (IO1000). The level of β-galactosidase was determined following growth in LB medium with 1% NaCl (+NaCl) or no added NaCl (−NaCl). Data represent three independent experiments.

FIG. 9.

A model of SPI1 osmoregulation by nucleoid-associated proteins H-NS/Hha and activators HilD/HilC/RtsA. H-NS/Hha negatively regulate the hilA and rtsA genes under low-osmolarity conditions. HilD/HilC/RtsA derepress the rtsA gene. Under high-osmolarity conditions, HilD, HilC, and RtsA activate SPI1 genes directly and indirectly through hilA. For clarity, the proposed autoregulation of HilA, HilD, and HilC is not shown. Repression is indicated as “−” and activation as “+.”