Heme rescues a two-component system Leptospira biflexa mutant

Background Heme is typically a major iron source for bacteria, but little is known about how bacteria of the Leptospira genus, composed of both saprophytic and pathogenic species, access heme. Results In this study, we analysed a two-component system of the saprophyte Leptospira biflexa. In vitro phosphorylation and site-directed mutagenesis assays showed that Hklep is a histidine kinase which, after autophosphorylation of a conserved histidine, transfers the phosphate to an essential aspartate of the response regulator Rrlep. Hklep/Rrlep two-component system mutants were generated in L. biflexa. The mutants could only grow in medium supplemented with hemin or δ-aminolevulinic acid (ALA). In the pathogen L. interrogans, the hklep and rrlep orthologous genes are located between hemE and hemL genes, which encode proteins involved in heme biosynthesis. The L. biflexa hklep mutant could be complemented with a replicative plasmid harbouring the L. interrogans orthologous gene, suggesting that these two-component systems are functionally similar. By real-time quantitative reverse transcription-PCR, we also observed that this two-component system might influence the expression of heme biosynthetic genes. Conclusion These findings demonstrate that the Hklep/Rrlep regulatory system is critical for the in vitro growth of L. biflexa, and suggest that this two-component system is involved in a complex mechanism that regulates the heme biosynthetic pathway.


Background
Leptospires are spirochetes divided between saprophytic and pathogenic species that remain poorly understood because of the limited availability of genetic tools. For example, targeted gene inactivation is not feasible in pathogenic Leptospira species.
Iron is an essential nutrient for the growth of leptospires like for most of the bacteria and the ability to acquire iron certainly contributes to the virulence of pathogenic bacteria. We previously showed that leptospires can take up a wide diversity of iron sources including heme, which is the most abundant iron source in host [1]. We also showed that L. biflexa possesses genes encoding putative heme acquisition systems such as TonB-dependent receptors and ABC transporters and a putative heme oxygenase that could allow the release of iron from heme [2]. Heme is a ubiquitous molecule involved in many major cellular processes. For example, heme is a cofactor for catalases and peroxidases and is an integral component of the electron transport chain where it serves as an electron carrier for cytochromes. Leptospira spp. possess genes that encode enzymes for the heme biosynthetic pathway unlike the spirochetes Borrelia burgdorferi and Treponema pallidum. In a previous study [3], we showed that inactivation of hemH in L. biflexa, which encodes the ferrochelatase that introduces one iron molecule into the porphyrin ring, generates heme requirement. Taken together, our previous results argue that hemin is an iron source as well as an heme source for leptospires. Free heme is toxic to bacterial cells, so heme biosynthesis is usually tightly regulated. However, little is known about how organisms regulate both the acquisition of exogenous heme and synthesis of heme [4].
We used transposon mutagenesis to generate a mutant library in the saprophyte L. biflexa that we screened onto media with or without hemin supplementation [2]. A heme-requiring mutant was isolated with an insertion in a gene that was predicted to encode the regulator of a twocomponent system. Such systems regulate gene expression in many bacteria in order to support a physiological response. Two-component systems detect a wide variety of environmental signals, including iron availability [5,6]. These systems typically comprise a membrane-associated sensor kinase and a cytoplasmic transcription regulator. The sensor autophosphorylates in response to stimuli and then transfers the phosphate group to the response regulator, which binds to specific promoters and, thus, acts as a transcriptional regulator [7]. Two-component systems are involved in many processes such as motility, virulence gene expression, and oxidative stress response [7].
In this study, we investigated the biochemical and physiological properties of this two-component system of L. biflexa to elucidate its role in heme metabolism.

L. biflexa hklep and rrlep mutants only grow with heme and heme precursor
Himar1 mutagenesis was previously used to identify genes involved in heme utilization by L. biflexa [2]. One of the transposon mutants, which was only able to grow in EMJH supplemented with hemin, but not in EMJH alone, exhibited the transposon into a putative gene encoding the response regulator of a two-component system ( Figure  1A). RT-PCR results showed that the two genes, rrlep and hklep, of this putative two-component system are co-transcribed as an operon in L. biflexa (data not shown). To investigate the functions of these two-component signalling genes in L. biflexa, we constructed rrlep and hklep deletion mutants by targeted gene replacement. Again, the resulting mutant strains were unable to grow on EMJH medium unless exogenous hemin was added ( Figure 2). These mutants were also able to grow in EMJH medium supplemented with haemoglobin and δ-aminolevulinic acid (ALA) a key intermediate in heme biosynthesis path-way, but not with other potential sources of iron, e.g. iron salts or desferrioxamine. These results then indicate that only a source of heme or a precursor could support the growth of the mutant strains in EMJH ( Figure 2).
Transformation of each mutant strain with the replicative plasmid harbouring the respective two-component signalling gene of L. biflexa restored a wild-type growth in EMJH medium (Figures 2 and 3). These results indicate that the growth defect was due to the inactivation of the two-component system and not to second-site mutations. It also suggests that there is no cross-talk between regulators and kinases of other cognate pairs of two-component systems in the bacterium. We concluded that the system has a crucial role in the survival of leptospires in EMJH and for heme supplying.

The homologous two-component system of the pathogen L. interrogans is genetically linked to heme biosynthesis genes
The complete genome sequences of four pathogenic Leptospira are available, namely L. interrogans serovar Lai, L. interrogans serovar Copenhageni, and two strains of L. borgpetersenii serovar Hardojobovis [8][9][10]. The L. biflexa two-component system was found in these four pathogenic strains and the proteins share 66% and 69% similarities with LB014 sensor and LB015 regulator of L. interrogans, respectively ( Figure 1B). Surprisingly, the twocomponent system found in the pathogens is genetically linked to the genes of the heme biosynthesis pathway, as it is also the case in the phylogenetically distant deltaproteobacteria Bdellovibrio bacteriovorus (data not shown). These genomic data may support a functional association between the two-component system and the biosynthesis of heme. The L. biflexa hklep mutant was complemented by the putative sensor gene of L. interrogans (LB014) carried on the shuttle vector ( Figure 3). However, expression of the regulator gene of L. interrogans (LB015) of both LB014 and LB015 (data not shown) in the L. biflexa rrlep mutant did not restore wild-type growth ( Figure 3). This may indicate that the heterologous expression of the L. interrogans regulator does not allow interactions with the L. biflexa sensor or L. biflexa target DNAs. In conclusion, the orthologous two-component systems in L. biflexa and L. interrogans are partially replaceable and functionally similar.

Signal transduction through Hklep/Rrlep relies on phosphorylation capabilities
Sequence analysis of the two-component system of both L. biflexa and L. interrogans reveals that it is a classical His/ Asp-dependent two-component system consisting of a histidine protein kinase and a response regulator with receiver and transmitter domains. The first reaction in the signalling cascade consists in the autophosphorylation of a highly conserved histidine residue of the histidine kinase. By incubating with [γ-32 P]ATP, the recombinant Hklep protein was rapidly phosphorylated (Figure 4a). The phosphorylation assays also allowed the observation of a secondary signal corresponding to twice the expected size of Hklep, suggesting that Hklep may form homodimers (Figures 4 and 5). Following the autophosphorylation of the sensor kinase, the phosphate group is typically transferred to an aspartyl residue of the conserved N-terminal receiver domain of the response regulator. By incubating Rrlep with the phosphorylated Hklep, we observed both phosphorylation of the regulator and reduction of the phosphosignal of the kinase ( Figure 4B). The amount of labelled Rrlep also decreased during the course of the experiment, possibly due to unstable phosphorylation.
Based on sequence alignments of other two-component systems, the histidine residue at position 98 in Hklep was predicted to be the auphosphorylation site, and the aspartate D53 of Rrlep the site of phosphotransfer (data not shown). To confirm the identity of the phosphate-donor and -receiver residues, we mutated the H98 and D53 of Hklep and Rrlep, respectively, by site-directed mutagenesis. In vitro phosphorylation assays showed that Hklep(H98A) was not able to autophosphorylate anymore (Figures 3 and 5) and that Rrlep(D53A) was not subject to phosphotransfer and did not stimulate the dephosphorylation of the kinase (Figures 3 and 5). In addition, the mutated alleles were unable to complement the hklep and rrlep mutant strains ( Figure 3). In conclusion, H98 and D53 are key residues for the phosphorylation mechanisms of the L. biflexa Hklep/Rrlep twocomponent system.

Rrlep/Hklep may regulate the expression of heme biosynthesis genes
The C-terminal region of Rrlep (residues 110 to 227) is predicted to contain a helix-turn-helix DNA-binding motif. The response regulator may bind to target DNAs via this domain while being phosphorylated. Furthermore, two-component response regulators typically autoregulate their own expression. Quantitative Reverse Transcrip- tion-PCR (qRT-PCR) assays showed that inactivation of rrlep gene in L. biflexa led to a four-fold decrease in transcript level of hklep (data not shown). The expression of this two-component system is therefore likely to be autoregulated by the response regulator. No significant change was observed in the relative expression of the twocomponent signalling genes in the wild-type strain cultivated with or without hemin or δ-aminolevulinic acid (data not shown). Taken together, these results suggest that another regulator is involved in the regulation of this two-component system.

Genetic organization of the Hklep/Rrlep two-component system in Leptospira spp
Since the L. interrogans two-component system (LB014-LB015) is clustered with heme biosynthetic genes ( Figure  1), we investigated the expression of L. biflexa hem genes in response to heme availability by qRT-PCR. We compared the expression of hemL, leading to δ-aminolevulinic acid synthesis, hemE, hemA and hemH in L. biflexa wildtype and mutant strains in media supplemented with low (2 μM) or high (10 μM) level of hemin ( Figure 6). Our results showed that hemAELH expression was 2 to 4 fold higher with hemin than without hemin in the wild-type strain of L. biflexa (data not shown). Expression of hemAEL was significantly decreased, from 2 to 10 fold, in hklep and rrlep mutants relative to the wild-type strain. Conversely, hklep and rrlep mutations displayed weaker effects on hemH expression. We concluded that heme biosynthesis genes may be regulated in response to heme availability. The Hklep/Rrlep two-component system may regulate hemAEL genes but not, or differently, hemH, which is transcribed in the opposite direction and then apart from the heme biosynthetic genes.

The periplasmic loop of the kinase is not essential but the cytoplasmic region is not sufficient to ensure physiological function
Hklep is a sensor histidine kinase with two predicted Nterminally located transmembrane segments that may anchor the protein to the cytoplasmic membrane of L. biflexa. The extracytoplasmic region between transmembrane segments is usually involved in the detection of environmental stimuli in other sensor kinases. Since we assumed that the periplasmic loop region of Hklep is involved in the detection of the environmental signal, the corresponding 32 amino acids (residues 31 to 62) were replaced by site-directed mutagenesis. The resulting plasmid construct was then used to complement the L. biflexa hklep mutant. The hklep gene lacking the original periplasmic loop was still able to complement the hklep mutant, i.e. it restores a wild-type growth in EMJH without hemin (Figure 3). This suggests that the periplasmic loop is not involved in the recognition of the signal. Nevertheless, L. biflexa hklep mutant could not be complemented with a Hklep protein lacking both the predicted transmembrane segments and the periplasmic loop ( Figure 3).

Discussion
To isolate hemin-requiring mutants, we used transposon mutagenesis in L. biflexa and identified the Hklep/Rrlep two-component system. In this study, we showed that Hklep is a histidine kinase which, after autophosphorylation of H98, transfers the phosphate to D53 of the response regulator Rrlep.
The mutants of this two-component system can only grow in medium supplemented with hemin or δ-aminolevulinic acid (ALA) but not with other iron sources, suggesting that the mutants need to acquire exogenous hemin as an heme source instead of an iron source. This also suggests that Hklep/Rrlep contributes to the regula- tion of steps leading to ALA synthesis. Although significant differences were detected in the relative expression of the hemAEL genes in the mutant versus the wild-type, we do not have conclusive evidence for direct regulation of hem genes by the two-component system. We postulate that the two-component response regulator may be connected in interacting networks and cascades of regulation.
Most bacteria possess many two-component systems, and the L. interrogans genome is predicted to encode 25 [11], that are required to control the activity of many genes in response to a variety of environmental signals. A key aspect of any regulatory system is the identity of the environmental signal to which the system responds. Our data suggest that the Hklep/Rrlep system senses environmental and/or intracellular heme. Two-component system kinases are supposed to detect environmental signals through their periplasmic domain [7]. Our results showed that the predicted periplasmic loop is not essential for the function purpose of the system. However, it remains possible that the N-terminal domain of Hklep, including the putative membrane-spanning regions, plays a role in signal detection.
Biosynthesis of porphyrins and related compounds proceeds via a common set of intermediates from ALA through the first cyclic tetrapyrrole, uroporphyrinogen III, at which point the pathway splits into two branches, one leading to reduced products such as siroheme and vitamin B12 and the other leading to oxidized end products, including hemes, bilins, and bacteriochlorophylls. The expression of hem genes is usually transcriptionally regulated in response to exogenous heme concentration [4]. In L. biflexa, the transcription of the hem genes that we studied is significantly increased on hemin addition. Hklep/ Rrlep may be, at least indirectly, responsible for the regulation of several hem genes.
The orthologous two-component systems in pathogenic Leptospira spp. are arranged in a novel fashion, distinct from that in typical bacterial heme biosynthetic loci, and this provides genetic evidence for hem reorganization in

Conclusion
The in vivo characterization of L. biflexa hklep and rrlep mutants argues for a role for this regulatory system in heme metabolism. However, the precise role of this twocomponent system remains unclear and further investigations will be necessary for identifying both its signal and target. Further investigation should also include the study of biochemical properties and transcriptional regulation of the homologous two-component system in the pathogens. As heme transport, storage, and metabolism are important for pathogenic bacteria [12], this system might contribute to the pathogenicity potential of leptospires.

DNA/RNA manipulations
Genomic DNA of leptospires was isolated as previously described [15]. Plasmid DNA was purified using the Qiagen Plasmid Miniprep Kit (Qiagen GmbH, Hilden, Germany).
For transcription studies, RNAs were isolated from exponential phase cultures of L. biflexa grown in EMJH, EMJH with 2 or 10 μM hemin or EMJH after an overnight incubation of the cells with 0.25 mM 2,2'-dipyridyl. Total RNAs were extracted using Tri-reagent solution and DNase I treatment following the manufacturer recommendations (Ambion Inc.). Absence of DNA contamination was checked by PCR. RNA concentrations were measured by spectrophotometry at 260 nm. RNAs were analyzed by RT-PCR using SuperScript One Step RT-PCR with Platinium Taq (Invitrogen). Quantitative reversetranscription PCR was performed in two steps as previously described [16]. Data from amplifications were analysed with the quantification program RelQuant (Roche). The relative expression of the target genes was normalized to the 23S rRNA. Quantitative expressions were evaluated in triplicate from three independent RNA extractions.

Recombinant protein expression and purification
To overproduce the proteins in E. coli, the full-length rrlep and the truncated hklep (region corresponding to residues 90-300) coding sequences from L. biflexa were amplified by PCR (nucleotide sequences of primer pairs available on request) and cloned into the HindIII and XhoI restriction sites of pET30a(+) (Novagen), generating pETrrlep and pEThklep, respectively. E. coli RosettaBlue(DE3) strain (Novagen) harbouring the pET30a(+)-based constructs were grown in LB supplemented with 25 μg chloramphenicol ml -1 and 50 μg kanamycin ml -1 at 37°C to a OD 600 of 0.6-0.8. IPTG (isopropyl-β-D-thiogalactopyranoside) was added at a final concentration of 1 mM and the cultures were further incubated for several hours. E. coli cells were washed into lysis buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole), lyzed by freezing and thawing followed by sonication, then centrifuged at 10,000 × g for 20 min at 4°C. The supernatant was recovered and incubated with Ni 2+ -agarose beads (Qiagen). In every case, the purity of the studied proteins was > 90 %, as judged by Coomassie-stained SDS-PAGE gels. Protein concentration was determined by Bradford protein assay (Biorad). To differentiate the proteins on gel, we produced Hklep with a N-terminal His-tag (31 kDa) and Rrlep without a His-tag (27 kDa). His-tagged proteins were eluted using a 250 mM imidazole-containing buffer. His-tag was removed by incubation with enterokinase as recommended by the manufacturer (Novagen).
Relative gene expression of L. biflexa hemA, hemE, hemL, and hemH genes measured by real-time quantitative RT-PCR Figure 6 Relative gene expression of L. biflexa hemA, hemE, hemL, and hemH genes measured by real-time quantitative RT-PCR. The levels of specific mRNA transcript of hemAELH genes were quantified in wild-type (bars in white), rrlep mutant (bars in grey), and hklep mutant (bars in black) grown in EMJH supplemented with 2 (left bar) or 10 μM (right bar) hemin. The amounts of mRNA transcript are shown relative to the quantity of that particular mRNA transcript in EMJH condition (without hemin). As an endogenous control, the 23S rRNA (23S rRNA = 1.0) was used for normalization of transcript levels. Experiments were performed in triplicate from distinct cultures to establish standard deviations.
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