The TonB3 system in the human pathogen Vibrio vulnificus is under the control of the global regulators Lrp and cyclic AMP receptor protein

Abstract

TonB systems transduce the proton motive force of the cytoplasmic membrane to energize substrate transport through a specific TonB-dependent transporter across the outer membrane. Vibrio vulnificus, an opportunistic marine pathogen that can cause a fatal septicemic disease in humans and eels, possesses three TonB systems. While the TonB1 and TonB2 systems are iron regulated, the TonB3 system is induced when the bacterium grows in human serum. In this work we have determined the essential roles of the leucine-responsive protein (Lrp) and cyclic AMP (cAMP) receptor protein (CRP) in the transcriptional activation of this system. Whereas Lrp shows at least four very distinctive DNA binding regions spread out from position -59 to -509, cAMP-CRP binds exclusively in a region centered at position -122.5 from the start point of the transcription. Our results suggest that both proteins bind simultaneously to the region closer to the RNA polymerase binding site. Importantly, we report that the TonB3 system is induced not only by serum but also during growth in minimal medium with glycerol as the sole carbon source and low concentrations of Casamino Acids. In addition to catabolite repression by glucose, l-leucine acts by inhibiting the binding of Lrp to the promoter region, hence preventing transcription of the TonB3 operon. Thus, this TonB system is under the direct control of two global regulators that can integrate different environmental signals (i.e., glucose starvation and the transition between "feast" and "famine"). These results shed light on new mechanisms of regulation for a TonB system that could be widespread in other organisms.

Fig 1

Organization of the tonB3 operon and the regulatory region. (A) Genetic arrangement of the tonB3 operon. The gene names are based on homology to the counterpart of the TonB2 systems of V. vulnificus and other vibrios. (B) Close-up of the regulatory region analyzed in this work. The different lines indicate the approximate locations of the probes used throughout this work. The length of the promoter region and the sizes of the probes in this panel are not drawn to scale. (C) Sequence of the promoter region from position −551 (relative to the transcription start site [+1]) to position +81 of the partial coding sequence of the VV1_0842 gene. The likely −10 and −35 elements are underlined, and the identified start point of the transcription and the predicted start codon are shown in bold. The extents of the protection identified by DNase I footprinting for each protein are indicated with black (Lrp) and gray (CRP) boxes, and the locations of the predicted binding sites as determined by similarity to consensus motifs (13, 17, 28) are indicated in bold. Binding site positions that could not be delimited by DNase I footprinting are indicated with dotted lines. The positions of the primers used for amplification of the probes used in EMSA and DNase I footprintings as well as those used in the construction of the lacZ fusions are indicated with double underlines. Relevant nucleotide positions relative to the transcription start site are also indicated.

Fig 2

Competitive indices for tonB mutant strains. Mixtures of the strains analyzed (0.1 ml) were injected s.c. in iron-overloaded animals as described in Materials and Methods. When animals showed signs of the disease, organs were removed and homogenized and bacterial cells plated on TSBS plus 1.5% agar (TSAS)–X-Gal plates. Bars represent the geometric mean CI value for each organ. Experiments were performed twice. ***, P < 0.001 based on one-way ANOVA with Bonferroni's posttest.

Fig 3

Expression of various tonB3 promoter deletions in human serum. (A) The deletions of the promoter region of the TonB3 operon were cloned in front of the promoterless lacZ gene in the pTL61T vector and plasmids conjugated into the ΔlacZ strain. Cells were grown in human serum plus FAC (200 μg/ml) and samples taken at different times to determine β-galactosidase activities (white bars [T2], 2 h; gray bars [T4], 4 h; and black bars [T6], 6 h after inoculation). Due to the turbidity of the human serum, the OD₆₀₀ was not recorded and activity was normalized to 10⁶ CFU ml⁻¹. The results shown are the means from three to seven independent experiments with standard deviations. Those bars showing no significant difference are labeled with the same letters, and those showing significant difference (P < 0.05) are labeled with different letters, based on one-way ANOVA with Tukey's posttest. (B) Expression of the tonB3 gene in the wild-type (wt) and Δ498 strains growing in HS-FAC as determined by qRT-PCR. RNA extracted from the wild-type and Δ498 strains growing in HS-FAC was reverse transcribed and cDNAs used in qRT-PCR as described in Materials and Methods. Relative expression of the tonB3 gene was determined by calculating 2^−ΔΔCT using the 23S rRNA gene as an internal control. ***, P < 0.001 based on one-way ANOVA with Bonferroni's posttest for values for the wild type compared to the mutant strain.

Fig 4

The lrp gene is essential for tonB3 induction in HS-FAC. (A) Wild-type, Δlrp, and Δlrp pMMlrp strains harboring plasmid p632 containing the tonB3-lacZ fusion were grown in HS-FAC. Samples were removed at various time points and analyzed as described for Fig. 3A. (B) Constructs p548 and pdint were conjugated into the V. vulnificus ΔlacZ strain, samples were removed at various times, and β-galactosidase activity was measured as described above. The bars represent means ± standard deviations (n = 3). Bars showing no significant difference are labeled with the same letters, and those showing a significant difference (P < 0.01 for panel A and P < 0.001 for panel B) are labeled with different letters, based on one-way ANOVA with Tukey's posttest.

Fig 5

Analysis of the binding of the Lrp(His)₆ protein to the tonB3 promoter region. (A and B) EMSA with different promoter regions of this operon that were amplified, labeled as described in Materials and Methods (probe concentration, 4 nM), and incubated with increasing concentrations of Lrp(His)₆ as indicated in each panel. (A) Probe A amplified with primers 42-632 and 42-477Bam; (B) probe B amplified with primers 42-498 and 42-300Rev and probe C amplified with primers 42-300 and 42Bam. Probes are illustrated in Fig. 1B. In each panel it is indicated when 10 mM l-leucine was added to the binding buffer as well as when unlabeled probe DNA (400 nM) was used for competition. Free labeled DNA and Lrp(His)₆-DNA complexes are indicated. (C, D, and E) DNase I footprinting analysis of the tonB3 promoter with purified Lrp(His)₆. End-labeled probes D (C and D) and C (E) were incubated with various concentrations of Lrp(His)₆ as indicated in each panel and treated with DNase I. Gels were calibrated using G+A sequencing reactions (G+A), and relevant positions are indicated. The locations of DNA binding sites for Lrp(His)₆ are shown by black lines, and putative binding sites with intrinsic resistance to DNase I are shown with dotted lines. Hypersensitive sites due to Lrp(His)₆ binding are labeled with asterisks.

Fig 6

Effects of l-leucine and glucose on tonB3 promoter expression. (A) The V. vulnificus ΔlacZ strain harboring the p632 construct was grown in HS-FAC or in the same medium with the addition of 10 mM l-leucine or 0.5% glucose. Samples were removed at various times, and β-galactosidase activity was measured as described for Fig. 3A. Values obtained at 6 h after the inoculation are shown. (B) The V. vulnificus ΔlacZ strain harboring the p632 construct was grown in M9 with 0.5% glycerol and various concentrations of CAA in the presence or absence of 10 mM l-leucine up to an OD₆₀₀ of ∼0.7, samples were removed, and β-galactosidase activity was measured as described in Materials and Methods. (C) Wild-type, Δlrp, and Δlrp pMMlrp strains harboring plasmid p632 containing the tonB3-lacZ fusion were grown in M9–0.5% glycerol–0.02% CAA in the presence or absence of 10 mM l-leucine, and β-galactosidase activity was measured as described above. (D) The V. vulnificus ΔlacZ strain harboring the p632 construct was grown in M9–0.5% glycerol–0.02% CAA or in the same medium with the addition of 10 mM l-leucine. When indicated, 200 μg/ml FAC was added. Samples were removed at an OD₆₀₀ of ∼0.7 and β-galactosidase activity measured as described above. (E) V. vulnificus ΔlacZ strains harboring the p300, p400, p498, or p632 construct were grown in M9–0.5% glycerol–0.02% CAA or in the same medium with the addition of 10 mM l-leucine. Samples were removed at an OD₆₀₀ of ∼0.7 and β-galactosidase activity measured as described above. The bars represent means ± standard deviations (n = 3). Those bars showing no significant difference are labeled with the same letters, and those showing a significant difference (P < 0.001 for panels A, C, D, and E and P < 0.05 for panel B) are labeled with different letters, based on one-way ANOVA with Tukey's posttest.

Fig 7

Expression of the tonB3 promoter with various carbon sources. The V. vulnificus ΔlacZ strain harboring the p632 construct was grown in M9 with various carbon sources (0.5% each) and 0.02% CAA in the presence or absence of 10 mM l-leucine and β-galactosidase activity measured as described in Materials and Methods. The bars represent means ± standard deviations (n = 3). Those bars showing no significant difference are labeled with the same letters, and those showing a significant difference (P < 0.01) are labeled with different letters, based on one-way ANOVA with Tukey's posttest.

Fig 8

Expression of the tonB3, lrp, and crp genes under various conditions. Total RNA was extracted from the different strains growing under the indicated conditions. The mRNA levels (represented by 2^−ΔΔCT values) of various genes in the different strains then were measured by qRT-PCR and expressed as the fold change from that measured in the wild-type strain growing in M9–glycerol–0.02% CAA without the addition of l-leucine. (A) tonB3; (B) lrp; (C) crp. The bars represent means ± standard deviations (n = 3). The significance of differences was analyzed by one-way ANOVA with Bonferroni's posttest. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 9

Role of CRP in tonB3 promoter expression. (A) Wild-type, Δcrp, and Δcrp pMMcrp strains harboring plasmid p632 containing the tonB3-lacZ fusion were grown in HS-FAC. Samples were removed at various times as indicated and analyzed as described for Fig. 3A. (B) The V. vulnificus ΔlacZ strain harboring the p632 construct was grown in M9 with 0.5% glucose and 0.02% CAA in the presence or absence of 10 mM l-leucine. When indicated, 1 mM or 2 mM cAMP was also added to the medium. Samples were removed at the indicated OD₆₀₀ and β-galactosidase activity measured as described in Materials and Methods. The bars represent means ± standard deviations (n = 3). Those columns showing no significant difference are labeled with the same letters, and those showing a significant difference (P < 0.05) are labeled with different letters, based on one-way ANOVA with Tukey's posttest.

Fig 10

EMSA analysis of the promoter region of the TonB3 operon with CRP(His)₆. The different promoter regions of this operon were amplified and labeled as described in Materials and Methods and incubated with increasing concentrations of CRP(His)₆ in the presence of 0.2 mM cAMP as indicated in each panel. (A) Probe C (42-300/42Bam); (B) probes Ci (42-300/42-206Rev) and Cii (42-206-For/42Bam). Probes are illustrated in Fig. 1B. Indicated are free labeled DNA and CRP(His)₆-DNA complexes. Probes were used at 4 nM, and in each panel it is indicated when unlabeled probe DNA (400 nM) was used for competition.

Fig 11

DNase I footprint analysis of the tonB3 promoter region with purified CRP(His)₆ and Lrp(His)₆ proteins. End labeled probe C was incubated with various concentrations of CRP(His)₆ (A) or CRP(His)₆ and Lrp(His)₆ (B) as indicated and treated with DNase I. Gels were calibrated using G+A sequencing reactions (G+A), and relevant positions are indicated. The locations of DNA binding sites for CRP(His)₆ are shown by full black lines, while those that correspond to Lrp(His)₆ are shown by dotted lines. Hypersensitive sites produced by CRP(His)₆ binding are labeled with black stars, and those due to Lrp(His)₆ binding are labeled with white stars.