The LysR-type transcriptional regulator LeuO controls expression of several genes in Salmonella enterica serovar Typhi - PubMed (original) (raw)

The LysR-type transcriptional regulator LeuO controls expression of several genes in Salmonella enterica serovar Typhi

I Hernández-Lucas et al. J Bacteriol. 2008 Mar.

Abstract

LeuO is a LysR-type transcriptional regulator that has been implicated in the bacterial stringent response and in the virulence of Salmonella. A genomic analysis with Salmonella enterica serovar Typhi revealed that LeuO is a positive regulator of OmpS1, OmpS2, AssT, and STY3070. In contrast, LeuO down-regulated the expression of OmpX, Tpx, and STY1978. Transcriptional fusions supported the positive and negative LeuO regulation. Expression of ompS1, assT, and STY3070 was induced in an hns mutant, consistent with the notion that H-NS represses these genes; transcriptional activity was lower for tpx and STY1978 in an hns background, suggesting that this global regulatory protein has a positive effect. In contrast, ompS2 and ompX expression appeared to be H-NS independent. LeuO specifically bound to the 5' intergenic regions of ompS2, assT, STY3070, ompX, and tpx, while it was not observed to bind to the promoter region of STY1978, suggesting that LeuO regulates in direct and indirect ways. In this work, a novel set of genes belonging to the LeuO regulon are described; interestingly, these genes are involved in a variety of biological processes, suggesting that LeuO is a global regulator in Salmonella.

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Figures

FIG. 1.

FIG. 1.

Proteomic profiles of Salmonella serovar Typhi total protein extracts. (A) Salmonella serovar Typhi Δ_leuO_ containing pFM_Trc_12 (IMSS-32). (B) Salmonella serovar Typhi containing pFM_TrcleuO_-50 (IMSS-III). Cells were grown in MA broth to an OD595 of 0.6. Cultures were supplemented with 50 μM IPTG. The labeled spots were excised and identified using MALDI-TOF MS. The identities of circled proteins are shown in Table 1. More than three independent experiments were performed, and representative 2-DGE gels are shown.

FIG. 2.

FIG. 2.

Transcriptional profiles of the LeuO-dependent genes fused to the cat reporter gene. Bacterial strains IMSS-II (Salmonella serovar Typhi IMSS-1 harboring plasmid pFM_Trc_12) and IMSS-III (Salmonella serovar Typhi IMSS-1 harboring plasmid pFM_TrcleuO_-50) were independently transformed with fusions of each of the LeuO-regulated genes (pKK232-9 [STY3070], pKK232-9 [_assT_], pKK232-9 [_ompS1_], pKK232-9 [_ompS2_], pKK232-9 [_tpx_], pKK232-9 [_ompX_], and pKK232-9 [STY1978]) to generate individual strains containing the LeuO regulator, as well as the promoter region of the LeuO-dependent gene. To evaluate the effect of H-NS in the regulation of each of the LeuO-regulated genes, individual fusions were transformed into Salmonella serovar Typhi hns mutant STY_hns_99. The CAT specific activity of each strain was determined using samples collected at OD595 of 0.4, 0.6, 0.8, and 1 and at 12 h. For strains IMSS-II and IMSS-III 50 μM IPTG was used as an inducer. The bars indicate the means of three independent experiments performed in duplicate.

FIG. 2.

FIG. 2.

Transcriptional profiles of the LeuO-dependent genes fused to the cat reporter gene. Bacterial strains IMSS-II (Salmonella serovar Typhi IMSS-1 harboring plasmid pFM_Trc_12) and IMSS-III (Salmonella serovar Typhi IMSS-1 harboring plasmid pFM_TrcleuO_-50) were independently transformed with fusions of each of the LeuO-regulated genes (pKK232-9 [STY3070], pKK232-9 [_assT_], pKK232-9 [_ompS1_], pKK232-9 [_ompS2_], pKK232-9 [_tpx_], pKK232-9 [_ompX_], and pKK232-9 [STY1978]) to generate individual strains containing the LeuO regulator, as well as the promoter region of the LeuO-dependent gene. To evaluate the effect of H-NS in the regulation of each of the LeuO-regulated genes, individual fusions were transformed into Salmonella serovar Typhi hns mutant STY_hns_99. The CAT specific activity of each strain was determined using samples collected at OD595 of 0.4, 0.6, 0.8, and 1 and at 12 h. For strains IMSS-II and IMSS-III 50 μM IPTG was used as an inducer. The bars indicate the means of three independent experiments performed in duplicate.

FIG. 2.

FIG. 2.

Transcriptional profiles of the LeuO-dependent genes fused to the cat reporter gene. Bacterial strains IMSS-II (Salmonella serovar Typhi IMSS-1 harboring plasmid pFM_Trc_12) and IMSS-III (Salmonella serovar Typhi IMSS-1 harboring plasmid pFM_TrcleuO_-50) were independently transformed with fusions of each of the LeuO-regulated genes (pKK232-9 [STY3070], pKK232-9 [_assT_], pKK232-9 [_ompS1_], pKK232-9 [_ompS2_], pKK232-9 [_tpx_], pKK232-9 [_ompX_], and pKK232-9 [STY1978]) to generate individual strains containing the LeuO regulator, as well as the promoter region of the LeuO-dependent gene. To evaluate the effect of H-NS in the regulation of each of the LeuO-regulated genes, individual fusions were transformed into Salmonella serovar Typhi hns mutant STY_hns_99. The CAT specific activity of each strain was determined using samples collected at OD595 of 0.4, 0.6, 0.8, and 1 and at 12 h. For strains IMSS-II and IMSS-III 50 μM IPTG was used as an inducer. The bars indicate the means of three independent experiments performed in duplicate.

FIG. 3.

FIG. 3.

Transcriptional start sites and LeuO protected regions of genes belonging to the LeuO regulon. Primer extension and DNase I protection assays for the STY3070, assT ompS2, ompX, and tpx genes were performed. For STY1978, only the transcriptional initiation results are shown. (A) Transcription initiation in the presence (+) or absence (−) of LeuO. (B) LeuO sites protected from DNase I. The LeuO concentrations used are indicated at the top. D− and D+ indicate without and with DNase I, respectively. DHR, DNase I hypersensitive regions. (C) Schematic diagram of the regulatory region of the LeuO-dependent genes. The nucleotides corresponding to the transcription initiation site (formula image) the LeuO regions protected from DNase I (I and II), and the translation initiation sites (ATG) are indicated.

FIG. 3.

FIG. 3.

Transcriptional start sites and LeuO protected regions of genes belonging to the LeuO regulon. Primer extension and DNase I protection assays for the STY3070, assT ompS2, ompX, and tpx genes were performed. For STY1978, only the transcriptional initiation results are shown. (A) Transcription initiation in the presence (+) or absence (−) of LeuO. (B) LeuO sites protected from DNase I. The LeuO concentrations used are indicated at the top. D− and D+ indicate without and with DNase I, respectively. DHR, DNase I hypersensitive regions. (C) Schematic diagram of the regulatory region of the LeuO-dependent genes. The nucleotides corresponding to the transcription initiation site (formula image) the LeuO regions protected from DNase I (I and II), and the translation initiation sites (ATG) are indicated.

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