A Nitric Oxide-Responsive Transcriptional Regulator NsrR Cooperates With Lrp and CRP to Tightly Control the hmpA Gene in Vibrio vulnificus

Abstract

Nitric oxide (NO) is an important antimicrobial effector produced by the host innate immune system to counteract invading pathogens. To survive and establish a successful infection, a fulminating human pathogen Vibrio vulnificus expresses the hmpA gene encoding an NO dioxygenase in an NO-responsive manner. In this study, we identified an Rrf2-family transcriptional regulator NsrR that is predicted to contain the Fe-S cluster coordinated by three cysteine residues. Transcriptome analysis showed that NsrR controls the expression of multiple genes potentially involved in nitrosative stress responses. Particularly, NsrR acts as a strong repressor of hmpA transcription and relieves the repression of hmpA upon exposure to NO. Notably, nsrR and hmpA are transcribed divergently, and their promoter regions overlap with each other. Molecular biological analyses revealed that NsrR directly binds to this overlapping promoter region, which is alleviated by loss of the Fe-S cluster, leading to the subsequent derepression of hmpA under nitrosative stress. We further found that a leucine-responsive regulatory protein (Lrp) negatively regulates hmpA in an NsrR-dependent manner by directly binding to the promoter region, presumably resulting in a DNA conformation change to support the repression by NsrR. Meanwhile, a cyclic AMP receptor protein (CRP) positively regulates hmpA probably through repression of nsrR and lrp by directly binding to each promoter region in a sequential cascade. Altogether, this collaborative regulation of NsrR along with Lrp and CRP enables an elaborate control of hmpA transcription, contributing to survival under host-derived nitrosative stress and thereby the pathogenesis of V. vulnificus.

Keywords: Vibrio vulnificus; gene regulation; nitric oxide; nitric oxide dioxygenase; nitrosative stress; stress response; transcriptional regulator.

Figure 1

Identification of NsrR and transcriptome analysis of its downstream genes. (A) The physical map of nsrR and hmpA on the V. vulnificus MO6-24/O genome. The open arrows represent the coding regions and transcriptional directions of the genes. (B) The amino acid sequences of various bacterial NsrRs were retrieved from the NCBI protein database and aligned using the T-Coffee alignment program (Notredame et al., 2000). Identical sequences and conserved sequences are shaded in black and gray, respectively. Dashed lines represent missing sequences. Conserved helix-turn-helix DNA-binding motif and three cysteine residues potentially involved in the Fe-S cluster ligation are indicated by a blue open box and red open boxes, respectively. VvNsrR, V. vulnificus NsrR; EcNsrR, E. coli NsrR; StNsrR, S. Typhimurium NsrR; ScNsrR, S. coelicolor NsrR. (C) The volcano plot depicting the genes differentially expressed by the nsrR deletion (fold change ≥ 2 with p < 0.05). The red dots and green dots represent the differentially up-regulated and down-regulated genes, respectively.

Figure 2

The effect of nitrosative stress and the nsrR mutation on hmpA transcription in vitro and ex vivo. The wild-type and ΔnsrR strains were grown aerobically to an A₆₀₀ of 0.5, and then exposed to 0.15 mg/ml NO/PPNPs in vitro (A) or NO-producing RAW 264.7 cells ex vivo in the presence or absence of L-NMMA (B) for 10 min. The hmpA transcript levels were determined by qRT-PCR, and the hmpA transcript levels in the wild-type strain exposed to PPNPs (A) or DMEM without L-NMMA (B) were set to 1. Error bars represent the SD. Statistical significance was determined by the Student's t-test (*p < 0.05; ***p < 0.0005; ns, not significant). WT, wild type; ΔnsrR, nsrR-deletion mutant.

Figure 3

The role of the three cysteine residues in NsrR on hmpA and nsrR transcription. (A,B) Total RNA and proteins were isolated from the parent and mutant strains grown aerobically to an A₆₀₀ of 0.5. (A) The hmpA transcript levels were determined by qRT-PCR, and the hmpA transcript level in the parent strain was set to 1. (B) The cellular HmpA, NsrR^FLAG or ${\text{NsrR}}_{3\text{CS}}^{\text{FLAG}}$, and DnaK (internal control) protein levels were determined by Western blot analysis. Molecular size markers (Bio-Rad) are shown in kDa. Parent, parent strain; ΔnsrR, nsrR-deletion mutant; nsrR_3CS, strain expressing apo-locked NsrR^FLAG. (C) A PCR fragment carrying the P_nsrR was cloned into pBBR-lux to create a reporter plasmid, pGR2025. The wild-type and mutant strains containing pGR2025 were grown aerobically to an A₆₀₀ of 0.5, and then used to measure the cellular luminescence. Error bars represent the SD. Statistical significance was determined by the Student's t-test (^****p < 0.00005; ns, not significant). RLU, relative luminescence unit; WT, wild type; ΔnsrR, nsrR-deletion mutant; nsrR_3CS, strain expressing apo-locked NsrR.

Figure 4

Sequence analysis of the nsrR-hmpA regulatory region. (A,B) The TSSs of hmpA (A) and nsrR (B) were determined by the primer extension of RNA isolated from the wild-type and ΔnsrR strains grown aerobically to an A₆₀₀ of 0.5. Lanes C, T, A, and G represent the nucleotide sequencing ladders. The asterisks indicate the TSSs. WT, wild type; ΔnsrR, nsrR-deletion mutant. (C) Double-stranded DNA sequence of the nsrR-hmpA regulatory region is shown. The TSS and putative translation start codon of nsrR are indicated by dashed bent arrows, and those of hmpA are indicated by solid bent arrows. The putative −10 and −35 regions are underlined with dashed lines for P_nsrR and solid lines for P_hmpA. The putative ribosome-binding sites (AGGA) are boldface. The binding sequences of NsrR (NSRRB; a white box), Lrp (LRPB; a gray box), and CRP (CRPB1, CRPB2, CRPB3; black boxes) were determined in the later parts of this study.

Figure 5

Specific binding of NsrR to the nsrR-hmpA regulatory region. (A,B) A 393-bp DNA fragment of the nsrR-hmpA regulatory region (10 nM) was labeled with 6-FAM, and then incubated with increasing amounts of NsrR (A,B) or NsrR_3CS (B) as indicated. For the competition analysis, various amounts of the unlabeled DNA fragment were added as a self-competitor. B, bound DNA; F, free DNA. (C) The same DNA probe (40 nM) was incubated with increasing amounts of NsrR as indicated, and then digested with DNase I. The region protected by NsrR is indicated by a white box (NSRRB). Nucleotide numbers shown are relative to the TSS of hmpA.

Figure 6

The effect of the lrp mutation on hmpA transcription and the specific binding of Lrp to the nsrR-hmpA regulatory region. (A–D) Total RNA and proteins were isolated from the parent and mutant strains grown aerobically to an A₆₀₀ of 0.5. (A,C) The hmpA transcript levels were determined by qRT-PCR, and the hmpA transcript levels in the parent strain were set to 1. Error bars represent the SD. Statistical significance was determined by the Student's t-test (^****p < 0.00005; ns, not significant). (B,D) The cellular HmpA, NsrR^FLAG or ${\text{NsrR}}_{3\text{CS}}^{\text{FLAG}}$, Lrp, and DnaK (internal control) protein levels were determined by Western blot analysis. Molecular size markers (Bio-Rad) are shown in kDa. Parent, parent strain; Δlrp, lrp-deletion mutant; pJH0311, broad-host-range vector; pJH0311::lrp, pJH0311 carrying the lrp gene (pZW1818); nsrR_3CS, strain expressing apo-locked NsrR^FLAG; nsrR_3CSΔlrp, lrp-deletion mutant expressing apo-locked NsrR^FLAG. (E) A 393-bp DNA fragment of the nsrR-hmpA regulatory region (10 nM) was labeled with 6-FAM, and then incubated with increasing amounts of Lrp as indicated. For the competition analysis, various amounts of the unlabeled DNA fragment were added as a self-competitor. B, bound DNA; F, free DNA. (F) The same DNA probe (40 nM) was incubated with increasing amounts of Lrp as indicated, and then digested with DNase I. The region protected by Lrp is indicated by a gray box (LRPB). The nucleotides showing enhanced cleavage are indicated by asterisks. Nucleotide numbers shown are relative to the TSS of hmpA.

Figure 7

The effect of the crp mutation on hmpA and nsrR transcription, and the specific binding of CRP to the nsrR-hmpA regulatory region. (A–D) Total RNA and proteins were isolated from the parent and mutant strains grown aerobically to an A₆₀₀ of 0.5. (A,C) The hmpA transcript levels were determined by qRT-PCR, and the hmpA transcript levels in the parent strain were set to 1. Error bars represent the SD. Statistical significance was determined by the Student's t-test (*p < 0.05; ***p < 0.0005; ****p < 0.00005; ns, not significant). (B,D) The cellular HmpA, NsrR^FLAG or ${\text{NsrR}}_{3\text{CS}}^{\text{FLAG}}$, Lrp, CRP, and DnaK (internal control) protein levels were determined by Western blot analysis. Molecular size markers (Bio-Rad) are shown in kDa. Parent, parent strain; Δcrp, crp-deletion mutant; pJH0311, broad-host-range vector; pJH0311::crp, pJH0311 carrying the crp gene (pKK1502); nsrR_3CS, strain expressing apo-locked NsrR^FLAG; nsrR_3CSΔcrp, crp-deletion mutant expressing apo-locked NsrR^FLAG. (E) A 393-bp DNA fragment of the nsrR-hmpA regulatory region (10 nM) was labeled with 6-FAM, and then incubated with increasing amounts of CRP as indicated. For the competition analysis, various amounts of the unlabeled DNA fragment were added as a self-competitor. B, bound DNA; F, free DNA. (F) The same DNA probe (40 nM) was incubated with increasing amounts of CRP as indicated, and then digested with DNase I. The regions protected by CRP are indicated by block boxes (CRPB1, CRPB2, and CRPB3). The nucleotides showing enhanced cleavage are indicated by asterisks. Nucleotide numbers shown are relative to the TSS of nsrR.

Figure 8

A regulatory network controlling hmpA transcription and nitrosative stress defense systems in V. vulnificus. (A) A regulatory network comprising transcriptional regulators NsrR, Lrp, and CRP controls the hmpA transcription. NsrR directly represses hmpA and nsrR itself. Lrp indirectly represses hmpA presumably by enhancing the repression activity of NsrR through DNA structure remodeling. CRP indirectly activates hmpA possibly through the repression of nsrR and lrp in a sequential cascade. (B) Apo-NsrR alleviates the repression of hmpA and nsrR upon exposure to nitrosative stress. On the other hand, apo-IscR relieves the repression of the isc operon, and the resulting increased apo-IscR directly activates prx3 encoding 1-cysteine peroxiredoxin. The induced NO-decomposition proteins, HmpA and Prx3, would facilitate the survival of V. vulnificus under host-derived nitrosative stress. The gray dots represent the Fe-S cluster.