Tetrapyrrole biosynthesis in Rhodobacter capsulatus is transcriptionally regulated by the heme-binding regulatory protein, HbrL

Abstract

We demonstrate that the expression of hem genes in Rhodobacter capsulatus is transcriptionally repressed in response to the exogenous addition of heme. A high-copy suppressor screen for regulators of hem gene expression resulted in the identification of an LysR-type transcriptional regulator, called HbrL, that regulates hem promoters in response to the availability of heme. HbrL is shown to activate the expression of hemA and hemZ in the absence of exogenous hemin and repress hemB expression in the presence of exogenous hemin. Heterologously expressed HbrL apoprotein binds heme b and is purified with bound heme b when expressed in the presence of 5-aminolevulinic acid. Electrophoretic gel shift analysis demonstrated that HbrL binds the promoter region of hemA, hemB, and hemZ as well as its own promoter and that the presence of heme increases the binding affinity of HbrL to hemB.

FIG. 1.

Effect of hemin addition on hemZ reporter activity. (A) Wild-type expression of the hemZ reporter under semiaerobic conditions in the presence of various concentrations of exogenous hemin is shown. (B) Wild-type expression of hem reporters under semiaerobic conditions in the presence and absence of 30 μmol hemin is shown. Values presented for each reporter are the means and standard deviations of at least three independent assays and are reported as micromoles of ONPG hydrolyzed per minute per milligram of cellular protein.

FIG. 2.

HbrL coding region. (A) Schematic representation of plasmid pJS147, a 2.5-kb genomic BamHI fragment containing HbrL and neighboring genes. Open reading frames are depicted as filled arrows, and a partial restriction map is given. Triangles depict the locations of transposon insertions in HbrL and orf218. Subclones of pJS147 used for the construction of transposon insertions are depicted below. (B) BLAST matches for open reading frames (ORF) in plasmid pJS147.

FIG. 3.

HbrL alignment. (A) Schematic representation of conserved domain features of LTTRs (42). (B) Alignment of HbrL orthologs prepared using hierarchical clustering (11). The consensus sequence is shown using symbols defined previously (16); the regions of primary sequence corresponding to LTTR domain features are shaded as in A. X. axonopodis, Xanthomonas axonopodis; X. campestris, Xanthomonas campestris; P. syringae, Pseudomonas syringae; P. fluorescens, Pseudomonas fluorescens; A. vinelandii, Azotobacter vinelandii; P. aeruginosa, Pseudomonas aeruginosa; R. metallidurans, Ralstonia metallidurans; P. putida, Pseudomonas putida; E. carotovora, Erwinia carotovora; P. profundum, Photobacterium profundum; S. oneidensis, Shewanella oneidensis; V. parahaemolyticus, Vibrio parahaemolyticus.

FIG. 4.

Expression of hem reporters in wild-type and hbrL strains in the presence and absence of exogenous hemin. Expression of hem reporters in the wild type (black bars) and hbrL strain JS163K (white bars) under semiaerobic conditions in the absence (-) and presence (+) of 30 μM hemin is shown. Values presented for each reporter represent triplicate measurements of biological replicates, with error bars representing standard deviations. Units are micromoles of ONPG hydrolyzed per minute per milligram of cellular protein. MU, Miller units.

FIG. 5.

Overexpression of HbrL protein. (A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of uninduced (lane 1) and induced (lane 2) HbrL-H6 whole-cell lysate and affinity-purified HbrL-H6 protein (lane 3). Electrophoretic mobility of protein standards is indicated. (B) Absorbance spectrum of HbrL overexpression lysate (solid line) and affinity-purified HbrL (dashed line) grown in the presence of 5-aminolevulinic acid. (C) Pyridine hemochrome spectrum of affinity-purified HbrL protein. The wavelengths of characteristic heme b maxima and minima are indicated.

FIG. 6.

Titration of HbrL by exogenous addition of hemin. Affinity-purified HbrL-H6 (6.4 μM) was titrated by the addition of hemin (64 μM in DMSO) in 0.2 mol equivalents. Spectra were collected for HbrL-H6 and a control sample of lysozyme (6.4 μM); data presented are the difference spectra (HbrL minus lysozyme).

FIG. 7.

Electrophoretic mobility shift. Electrophoretic mobility shift assay of HbrL-H6 using hemA, hemB, hemZ, hbrL, hemCE, and hemH probes is shown. (A) Electrophoretic mobility of hemB probe with increasing amounts of HbrL lysate added in the presence and absence of 7 μM hemin is shown. Ten micrograms of Bl21(DE3) lysate was added to lanes marked “0.” (B) Electrophoretic mobility of hemA probe with increasing amounts of HbrL lysate added in the presence and absence of 7 μM hemin is shown. (C) Electrophoretic mobility of hemZ probe with increasing amounts of HbrL lysate added in the presence and absence of 7 μM hemin is shown. (D) Electrophoretic mobility of hbrL probe in the presence of 7 μM hemin with increasing amounts of HbrL lysate added is shown. (E) Electrophoretic mobility of hemCE probe in the presence of 7 μM hemin with increasing amounts of HbrL lysate added is shown. (F) Electrophoretic mobility of hemH probe in the presence of 7 μM hemin with increasing amounts of HbrL lysate added is shown.