ABSTRACT
In Liberibacter asiaticus, PrbP is an important transcriptional accessory protein that regulates gene expression through interactions with the RNA polymerase β-subunit and a specific sequence on the promoter region. The constitutive expression of prbP observed upon chemical inactivation of PrbP-DNA interactions in vivo indicated that the expression of prbP was not autoregulated at the level of transcription. This observation suggested that a modulatory mechanism via protein-protein interactions may be involved. In silico genome association analysis identified FerR (CLIBASIA_01505), a putative ferredoxin-like protein, as a PrbP-interacting protein. Using a bacterial two-hybrid system and immunoprecipitation assays, interactions between PrbP and FerR were confirmed. In vitro transcription assays were used to show that FerR can increase the activity of PrbP by 16-fold when present in the PrbP-RNA polymerase reaction mixture. The FerR protein-protein interaction surface was predicted by structural modeling and followed by site-directed mutagenesis. Amino acids V20, V23, and C40 were identified as the most important residues in FerR involved in the modulation of PrbP activity in vitro. The regulatory mechanism of FerR abundance was examined at the transcription level. In contrast to prbP of L. asiaticus (prbPLas), mRNA levels of ferR of L. asiaticus (ferRLas) are induced by an increase in osmotic pressure. The results of this study revealed that the activity of the transcriptional activator PrbPLas is modulated via interactions with FerRLas. The induction of ferRLas expression by osmolarity provides insight into the mechanisms of adjusting gene expression in response to host environmental signals in L. asiaticus.
IMPORTANCE The rapid spread and aggressive progression of huanglongbing (HLB) in the major citrus-producing areas have raised global recognition of and vigilance to this disease. As a result, the causative agent, Liberibacter asiaticus, has been investigated from various perspectives. However, gene expression regulatory mechanisms that are important for the survival and persistence of this intracellular pathogen remain largely unexplored. PrbP is a transcriptional accessory protein important for L. asiaticus survival in the plant host. In this study, we investigated the interactions between PrbP in L. asiaticus (PrbPLas) and a ferredoxin-like protein (FerR) in L. asiaticus, FerRLas. We show that the presence of FerR stabilizes and augments the activity of PrbPLas. In addition, we demonstrate that the expression of ferR is induced by increases in osmolarity in Liberibacter crescens. Altogether, these results suggest that FerRLas and PrbPLas may play important roles in the regulation of gene expression in response to changing environmental signals during L. asiaticus infection in the citrus host.
INTRODUCTION
The understanding of the physiology and regulatory mechanisms employed by the citrus pathogen Liberibacter asiaticus is progressing slowly in spite of the rigorous efforts of the research community. The main challenge remains the lack of reliable laboratory culturing conditions for L. asiaticus. The major hindrance has thwarted the use of traditional genetic and molecular approaches to elucidate mechanisms involved in Liberibacter pathogenicity (1–3). To circumvent the inability to axenically culture L. asiaticus, its mechanisms of pathogenesis have been investigated from various alternative perspectives. These include comparative genomics, biochemical analyses, heterologous pathogen gene expression in alternative hosts, as well as pathogen and host responses during infection by transOMIC techniques (4–14).
In 2009, the first metagenome sequence of L. asiaticus was published, providing dependable information that could be used to heterologously express L. asiaticus genes and study proteins in vitro (15). Since then, in silico analyses have been performed complementary to in vitro approaches to advance the knowledge of L. asiaticus infection mechanisms at the molecular level. Aiming to identify potential pathogenicity factors and select candidate proteins that can be used as drug targets in this bacterium, studies have focused on flagella, transporters, secreted proteins, and secretion systems (12–14, 16–19). Our approach instead was to investigate the regulatory mechanisms of gene expression that are necessary for the intracellular lifestyle of L. asiaticus in the citrus host (20–24). Genomic analyses indicated that L. asiaticus has a small genome compared to those of many other model microorganisms, most likely due to its highly adapted life within the host. Interestingly, the L. asiaticus genome contains genes encoding transcriptional factors that only account for 2% of the total genes (20), indicating that the transition of this bacterium from an insect symbiont to an intracellular plant pathogen, as well as the establishment of infection in plant hosts, relies on the regulation of gene expression by only a small number of transcription factors. Using a combination of biochemical assays and analyses with Liberibacter crescens, we have been able to determine that at least two of the transcription factors, LdtR and PrbP in L. asiaticus (PrbPLas), in L. asiaticus behave as global transcriptional regulators (20, 21, 23, 24).
PrbPLas is a transcriptional accessory protein that modulates gene expression via interactions with the RNA polymerase and a specific sequence on the promoter region. PrbPLas belongs to the CarD_CdnL_TRCF superfamily. Members of this protein family in Mycobacterium and Myxococcus spp. have been reported to be essential and shown to be necessary for stress responses, persistence, cell viability, and resistance to antibiotics (25–29). Recently, another member, LtpA, has been shown to be important for the enzootic cycle of the Lyme disease pathogen Borrelia burgdorferi (30). Our biochemical analyses identified tolfenamic acid (TA) as an inhibitor of PrbPLas binding to its cognate DNA sequence. In vitro, TA inhibited transcription initiation, while in vivo, the addition of TA to infected citrus plants significantly reduced survival of the pathogen in the citrus host (21, 24). At the molecular level, TA binds to the amino acid residues N107, G109, and E148 in the interface of the DNA and the RNA polymerase-interacting domains of PrbPLas (24). These results indicated that the activity of PrbPLas may be modulated by a yet-unknown small molecule or through modifications in protein-protein interactions triggered by the changing environment. Our previous results showed that PrbPLas does not bind to its own promoter region and that the expression of prbPLas was not significantly affected upon chemical inactivation of PrbPLas-DNA interactions (21).
In this study, we hypothesized that PrbPLas activity is regulated via protein-protein interactions. In silico analyses identified CLIBASIA_01505, a ferredoxin-like regulator protein (FerR) in L. asiaticus named FerRLas, as a potential interacting partner of PrbPLas. A bacterial two-hybrid system was utilized to validate the interactions between FerRLas and PrbPLas, followed by immunoprecipitation assays using L. crescens. The effect of FerRLas on PrbPLas DNA binding ability and transcriptional activity was evaluated using an electrophoresis mobility shift assay and in vitro transcription assay, respectively. In brief, this study shows that PrbPLas activity is modulated through direct interactions with FerRLas.
RESULTS
FerRLas is a potential interacting partner of PrbPLas.It has been proposed that proteins that are functionally related tend to be encoded in close proximity to each other on the genome (31–35). Identifying genes in the neighborhood of the gene of interest that are conserved across various species can be a helpful inference of potential physical interactions and functional relationships among them (31–35). To identify a potential interacting partner of PrbPLas, the genomic context of prbPLas was examined using the Joint Genome Institute (JGI) IMG genome viewer and SEED (Markowitz et al. [36]). In L. asiaticus, CLIBASIA_01505, annotated as a putative ferredoxin-like protein-encoding gene (here named ferRLas), is located 316 bp upstream of prbPLas (Fig. 1A). Further analyses using the STRING database (37) showed that the synteny of these two genes is conserved in all the Alphaproteobacteria genera analyzed, whereas in Gammaproteobacteria/Deltaproteobacteria, only cooccurrence was observed. The cooccurrences of ferR and prbPLas were highly varied within Actinobacteria, Firmicutes, and Cyanobacteria (Fig. 1B).
Synteny of ferRLas and prbPLas homologs is conserved in Alphaproteobacteria. (A) Graphic representation of the prbPLas genomic context in representative genomes from Alphaproteobacteria. Shown are prbPLas and its homologous genes in L. asiaticus (GenBank accession no. NC_012985.3), L. crescens (GenBank accession no. NC_019907.1), Sinorhizobium meliloti (GenBank accession no. NC_003047.1), and Bartonella bacilliformis (GenBank accession no. NC_008783.1). Genomes were visualized in the JGI IMG genome viewer. (B) Taxonomy tree of microbial species containing ferRLas and its homologs. FerRLas homologs were identified by Protein BLAST. The taxonomy order was based on the NCBI taxonomy database. The CBP group is a bacterial group that primarily consists of members of Bacteroidetes and Chlorobi as well as putative environmental isolates. The cooccurrence and/or synteny of ferRLas and prbPLas were examined using STRING, JGI IMG genome, and SEED viewers. The cooccurrence of ferRLas and prbPLas is represented as discontinued lines in the gene representation figures, while synteny is represented as continuous lines.
To determine if ferRLas and prbPLas constitute an operon, assays of cotranscription by reverse transcription-PCR were performed. Total DNA or cDNA (synthesized from total RNA) extracted from citrus infected tissue were utilized as templates. Amplification of ferRLas and prbPLas fragments was observed in both genomic DNA and cDNA reactions, while amplification of ferRLas-prbPLas fragment was only observed with genomic DNA (see Fig. S1 in the supplemental material). These results indicate that ferRLas and prbPLas are not cotranscribed in L. asiaticus during infection of the citrus host.
FerRLas interacts with PrbPLas.To validate the in silico predictions that FerRLas is an interacting partner of PrbPLas, a bacterial two-hybrid system was utilized. The genes were fused to the β-galactosidase subunits truncations, Δα and Δɷ, by cloning the coding sequence into plasmids pB2HΔα and pB2HΔɷ, as described earlier (21, 38). The recombinant plasmids were transformed in different combinations into Escherichia coli JM109 (a β-galactosidase-deficient strain), which was used as the reporter strain (Table 1). The protein-protein interactions were followed by β-galactosidase activities at different points in the growth curve.
Strains and plasmids used in this study
At late-exponential phase, the highest β-galactosidase activities were detected in strain 2HB04 (carrying pB2HΔα-prbPLas and pB2HΔɷ-ferRLas; 19,664.67 ± 949.17 arbitrary units [AU]), while strain 2HB01 (carrying pB2HΔα and pB2HΔɷ) showed significantly lower values, at 7,274.96 ± 288.40 AU (P < 0.05). It was also observed that strain 2HB02 (carrying pB2HΔα and pB2HΔɷ-ferRLas) showed activity, albeit significantly lower (11,367.85 ± 2,973.37 AU) (P < 0.05) than 2HB04 (Fig. 2 and S2A). These results indicate that FerRLas directly interacts with PrbPLas.
PrbPLas and FerRLas interact in a bacterial two-hybrid system. β-Galactosidase activity was determined using E. coli JM109 as a reporter strain. The genetic constructs in the strains are as follows: 2HB01 carrying pB2HΔα and pB2HΔω, 2HB02 carrying pB2HΔα and pB2HΔω_ferRLas, 2HB03 carrying the pB2HΔα_prbPLas and pB2HΔω, and 2HB04 carrying pB2HΔα_prbPLas and the pB2HΔω_ferRLas. β-Galactosidase assays were performed at different stages during the exponential-growth phase (OD600, 0.3, 0.5, and 0.8). The growth curves of all the strains tested are shown in Fig. S2A. Enzymatic activities are shown as the average arbitrary units (AU) with the standard deviation (SD) from biological and technical triplicates. Statistical significance was determined as described in Materials and Methods. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
Due to the inability to culture L. asiaticus in an axenic culture, we used L. crescens to assess the interactions between FerR and PrbP. The genome of L. crescens shares an average 77% nucleotide identity with L. asiaticus and is widely used as the model organism for Liberibacter studies. The recombinant His-tagged PrbPLas, overexpressed and purified in E. coli, was used in immunoprecipitation assays to pull down the FerR homologous protein in L. crescens (FerRLcr). FerRLas and PrbPLas both share 79% identity with the corresponding homologous proteins in L. crescens. For this assay, purified recombinant PrbPLas was immobilized on magnetic protein G beads charged with anti-His antibody as the bait to capture potential binding partners from the cell extract of L. crescens, as described in Materials and Methods. As a control, protein G beads bound to anti-His (but without His-PrbPLas) were incubated with cell extract. After elution, samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Several proteins were identified as result of His-PrbPLas interactions, and among those was FerRLcr from L. crescens (Table 2). As expected, PrbPLas was also able to interact with the β-subunit of the RNA polymerase (21, 24). Other components of the RNA polymerase, such as β′- and α-subunits, were also identified. This is in agreement with previous findings that PrbPLas and its homologs interact with RNA polymerase (21, 25, 28, 39). In summary, the immunoprecipitation assays further confirmed the interaction between FerR and PrbP.
Proteins identified by LC-MS/MS in the immunoprecipitation assaysa
FerRLas increases PrbPLas activity as a transcriptional activator.We have previously shown that PrbPLas binds specifically to the promoter region of rplK and interacts with the β-subunit of the RNA polymerase (RNAP) to activate transcription. Using in vitro transcription assays, it was found that PrbPLas acts as a transcription activator to increase the concentration of the rplK transcript in a dose-dependent manner (21, 24). To evaluate the role of FerRLas on PrbPLas activity, recombinant FerR protein was overexpressed in E. coli, purified, and tested in in vitro transcription assays. Low concentrations of PrbPLas were used to allow basal transcription activation. The addition of increasing concentrations of FerRLas (from 1 to 4 μM) to a constant concentration of PrbPLas (at 2 μM) resulted in a significant increase (P < 0.05) in the rplK transcript (Fig. 3A). The highest transcription rate was found at 4 μM, with an increase up to 32-fold in transcript concentration, while a 15-fold increase was observed at equimolar concentrations of FerRLas and PrbPLas. The addition of PrbPLas or FerRLas alone resulted in 3.1-fold and 2.8-fold increases, respectively, in the rplK transcript concentration (Fig. 3B). The increase of transcription observed in the in vitro transcription assays may be the result of the stabilization of the PrbPLas-DNA-RNAP complex. Alternatively, FerRLas may have an effect on the interactions of PrbPLas with DNA. To elucidate the role of FerRLas on PrbPLas binding stability, electrophoresis mobility shift assays (EMSAs) were performed using the promoter region of rplK, as described earlier (21). Control assays were performed with PrbPLas and FerRLas added alone. It was found that PrbPLas binds DNA in a concentration-dependent manner, as described earlier (Fig. 3C and S3) (21). However, FerRLas was also found to bind to PrplK independently, albeit at lower affinity than PrbPLas (Fig. 3C and S3). The combined addition of PrbPLas and FerRLas increased the amount of the shifted DNA. However, the quantification of the shifted bands revealed that the effect observed is additive (Fig. 3C and S4). In summary, FerRLas does not significantly affect the DNA binding activity of PrbPLas.
FerRLas increases PrbPLas activity as a transcriptional activator. (A) In vitro transcription assays using increasing concentrations of FerRLas (0, 1, 2, and 4 μM) and/or 2.0 μM PrbPLas added to the RNA polymerase-containing reactions as indicated at the top. (B) ImageJ was utilized to quantify the amount of the PrplK transcript obtained in the in vitro transcription. The fold change was calculated by the band intensity normalized to the transcript level in the reactions performed in the absence of PrbPLas and FerRLas. The quantification was based on observations from at least three replicates. Statistical significance was determined as described in Materials and Methods. Different letters on top of the bars denote statistical significance of at least a P value of <0.05. (C) The effects of FerRLas on PrbPLas-DNA binding were evaluated using electrophoresis mobility shift assays. The reaction mixture contained 1.0 ng of the biotinylated Prplk probe and 2.5 μM PrbPLas or 1 to 5 μM FerRLas, as indicated at the top of each lane. The first lane has no protein added.
FerRLas and PrbPLas interactions are not modulated by oxidizing/reducing conditions in vitro.The analyses of conserved domains within the sequence of FerRLas revealed the presence of a PreA/ferredoxin domain in the N-terminal end, while in the C terminus, a conserved domain with unknown function, DUF3470, was found. Many proteins containing 4Fe-4S and/or 3Fe-4S clusters are involved in electron transfer and/or sensing of oxidative stress conditions (40, 41). To evaluate the role of oxidizing/reducing conditions on FerRLas activity, the protein was pretreated with increasing concentrations of dithiothreitol (DTT) or diamide (up to 60-fold stressor-to-protein ratio) to mimic reducing and oxidizing conditions, respectively. The effect of DTT or diamide on FerRLas was confirmed by titration of free thiol groups in treated and nontreated recombinant FerRLas using Ellman’s reagent (data not shown) (42).
First, the effect of oxidation/reduction conditions on the ability of FerRLas to bind DNA was evaluated. It was found that treated proteins behaved similarly to the untreated proteins in the EMSA (Fig. 4A). The effect of FerRLas’s redox state on PrbPLas activity was then investigated using in vitro transcription assays (Fig. 4B). No significant effect on transcription of the PrplK fragment was observed by the addition of DTT or diamide to FerRLas or to PrbPLas-containing reaction mixtures. These results suggest that the physical interactions between FerRLas and PrbPLas may be the primary determinant in modulating PrbPLas activity.
FerRLas activity is not modulated by oxidizing/reducing conditions. (A) Electrophoresis mobility shift assay of PrbPLas and FerRLas with PrplK; the reaction mixture contained 1.0 ng of the biotinylated Prplk probe, 2.5 μM PrbPLas, 2.5 μM FerRLas, and increasing concentrations (0 to 500 μM) of DTT or diamide, as indicated at the top. The first lane has no protein added. (B) The effects of oxidizing and reducing agents were tested on FerRLas activity using in vitro transcription assays. PrbPLas and FerRLas (0 to 2.5 μM) were added to the reactions, as indicated at the top. Increasing concentrations of DTT or diamide were added at the indicated concentrations. All reactions contained the same amount of RNA polymerase (0.5 μM).
Identification of the PrbPLas-interacting interface in FerRLas.To identify the role of specific residues on FerRLas-PrbPLas interactions, the PrbPLas-interacting interface in FerRLas was predicted in silico. A structural model of FerRLas was built using the SWISS-MODEL server (43). Eight models were obtained based on crystalized structures of FerRLas homologs with coverage higher than 90%. No significant difference was observed among the eight models. The three-dimensional (3D) model based on PDB ID 1F5B from Azotobacter vinelandii (44) was selected because of its overall best quality parameters (92% coverage; global model quality estimation [GMQE], 0.72; QMEAN, −2.29). This FerRLas model is named FerRLas-SD and was used to predict amino acids involved in protein-protein interaction using the meta-PPISP server (45). Amino acids that are buried inside the structure or predicted to be negative in protein-protein interactions were excluded from further analyses. Positive hits with a prediction score higher than 0.5 were selected and manually examined in the FerRLas-SD model using PyMOL (46). It has been suggested that the protein-protein interaction surface usually has a high number of hydrophobic residues surrounded by a rim of polar residues (47–49). Based on these observations, hydrophobicity and surface electrostatic potential analyses were performed with FerRLas-SD (50, 51). It was found that residues Y3, V20, C21, V23, D24, C40, D42, C43, and V45 form a major hydrophobic patch that is surrounded by negatively charged polar residues in FerRLas-SD (Fig. 5). Furthermore, these residues are highly conserved throughout FerRLas homologs, suggesting important roles in FerRLas function.
In silico prediction of the PrbPLas-interacting interface in FerRLas. (A) Hydrophobicity analysis in the 3D model FerRLas-SD. The color gradient from red to white illustrates hydrophobic potential to neutral, while the black dashed line circle encloses the predicted hydrophobicity patch in FerRLas-SD. (B) Surface electrostatic potential analysis in FerRLas-SD. The color gradient from red to blue illustrates negative to positive surface charges, while the black dashed line circle encloses the predicted hydrophobicity patch in FerRLas-SD. (C) Cartoon representation of the FerRLas-SD model with stick representation of the amino acid residues that form the predicted hydrophobicity patch. (D) Hydrophobic amino acid residues in the predicted patch are shown in red.
The role of each of these residues in the interactions with PrbPLas was tested using the bacterial two-hybrid system described above. All nine residues identified were replaced with alanine in the FerRLas wild type (WT) using site-directed mutagenesis. The reporter strains containing pB2HΔα_prbPLas and the pB2HΔω_ferRLas (or single alanine replacements in FerRLas) were constructed (Table 1). The β-galactosidase activities were followed at different stages during exponential phase, and values obtained were normalized to the highest-background control strains (carrying pB2HΔα and pB2HΔω_ferRLas-specific mutant). It was found that residues Y3, C21, D24, D42, C43, and V45 showed nonsignificant decreases in β-galactosidase activity compared to the values obtained with strain HB04 containing wild-type FerRLas (Fig. S2C and S5). In contrast, reporter strains 2HB08, 2HB12, and 2HB16 containing mutations FerRLas V20A, FerRLas V23A, and FerRLas C40A showed a significant decrease (P < 0.05) in β-galactosidase activity compared to that with wild-type FerRLas (Fig. 6A and S2B). These results suggest that residues V20, V23, and C40 in FerRLas are important for interactions with PrbPLas. Based on these results, FerRLas V20A, V23A, and C40A mutants were selected for further experiments.
FerRLas:PrbPLas interactions are mediated by residues V20, V23, and C40 in FerRLas. (A) Mutations V20A, V23A, and C40A in FerRLas decrease interactions with PrbPLas in a bacterial two-hybrid system. β-Galactosidase activity was performed in E. coli JM109 (β-galactosidase deficient). The reporter strains used were 2HB04 carrying pB2HΔα_prbPLas and pB2HΔω_ferRLas, 2HB08 carrying pB2HΔα_prbPLas and pB2HΔω_ferRLas V20A, 2HB12 carrying pB2HΔα and pB2HΔω_ferRLas V23A, and 2HB16 carrying pB2HΔα_prbPLas and pB2HΔω_ferRLas C40A. β-Galactosidase assays were performed at different stages during the exponential-growth phase (OD600, 0.3, 0.5, and 0.8). The growth curves of all the strains tested are shown in Fig. S2B. The activities were normalized to the highest background strains of 2HB02 carrying pB2HΔα and pB2HΔω_ferRLas, 2HB07 carrying pB2HΔα and pB2HΔω_ferRLas V20A, 2HB11 carrying pB2HΔα and pB2HΔω_ferRLas V23A, and 2HB15 carrying pB2HΔα and pB2HΔω_ferRLas C40A and are shown as arbitrary units (AU). Statistical significance was determined as described in Materials and Methods. *, P < 0.05; **, P < 0.01. (B) The mutations in V20, V23, and C40 reduced FerRLas activity on PrbPLas. In vitro transcription assays were performed with PrbPLas, FerRLas WT, or the FerRLas V20A, V23A, and C40A mutants. Each protein was added at a concentration of 2.5 μM, as indicated at the top; all reactions contained the same amount of RNA polymerase (0.5 μM). The image has been cropped and rearranged for presentation. (C) ImageJ was utilized to quantify the amount of the PrplK transcripts obtained in the in vitro transcription assay. The activity fold change was calculated by the band intensity normalized to the transcript level in the reactions performed in the presence of PrbPLas and FerRLas. The quantification was based on observations from at least three replicates. Statistical significance was determined as described in Materials and Methods. *, P < 0.05; **, P < 0.01.
Residues V20, V23, and C40 in FerRLas modulate PrbPLas activity.To investigate the role of the V20, V23, and C40 residues in FerRLas in modulating the activity of PrbPLas, in vitro transcription assays were performed. PrbPLas activity as a transcriptional activator was determined in the presence or in the absence of FerRLas WT and FerRLas V20A, FerRLas V23A, and FerRLas C40A mutants. As previously observed, the addition of FerRLas WT to the reaction with PrbPLas significantly increased the transcript concentration (Fig. 6B). The stimulation of PrbPLas transcriptional activity by the addition of FerRLas V20A, FerRLas V23A, and FerRLas C40A decreased to 81% ± 5%, 59% ± 2%, and 39% ± 17% (P < 0.05), respectively, compared to FerRLas WT (Fig. 6C). The decrease in activity observed was in agreement with the decrease in interactions observed in the two-hybrid system, where a mutation in residue C40 had the strongest effect on interactions with PrbPLas. These results support the important role of residues V20, V23, and C40 in mediating interactions with PrbPLas to modulate its activity as a transcriptional activator.
To rule out the possibility that the decrease in PrbPLas activity is due to the decrease in DNA binding ability caused by the point mutations in FerRLas, the DNA binding ability of the FerRLas mutants were evaluated by EMSA. No differences in DNA binding were observed between the FerRLas mutants and FerRLas WT (Fig. S3). The results obtained indicate that the decrease in PrbPLas activity observed in the in vitro transcription assays is not due to a decrease in FerRLas binding to DNA. Altogether, the results obtained suggest that the interactions of PrbPLas with the RNA polymerase are stabilized through specific interactions with FerRLas.
Increase in osmolarity induces ferRLcr expression level in L. crescens.Our previous work on LdtR, a MarR family transcriptional regulator from L. asiaticus, showed that it is involved in tolerance to osmotic stress. Further RNA sequencing (RNA-seq) determined that the chemical inactivation of LdtRLcr in L. crescens resulted in the activation of 131 and repression of 121 genes, respectively. Among them, ferRLcr was downregulated in the absence of an active LdtRLcr. Additionally, in L. asiaticus genome, an LdtRLas transcription activator binding motif on the ferRLas promoter region was identified and verified by EMSAs. The chemical inactivation of LdtR modulating ferRLas expression level in huanglongbing (HLB)-infected citrus leaves was also confirmed by quantitative reverse transcription-PCR (qRT-PCR) (23). However, the role of osmotic pressure in ferR expression was not evaluated. The expression levels of ferRLcr and prbPLcr in L. crescens (B488_01730 and B488_01720, respectively), grown in absence of or with increasing concentrations of sucrose, were determined by qRT-PCR. The relative changes in gene expression were normalized to gyrase subunit A (gyrA) gene expression. The expression level of ferRLcr from L. crescens cells grown with 50 mM sucrose did not show significant change, while a 1.9-fold increase (P < 0.05) in expression was found from L. crescens grown with 100 mM sucrose compared to the controls (Fig. 7). In contrast, each condition resulted in no significant change in prbPLcr expression levels (Fig. 7). The induced expression of ferRLcr by increased osmolarities is in agreement with the previous findings that ferR expression is under the control of LdtR (23). The constitutive expression of prbPLcr under the tested conditions provides further supporting evidence the idea that prbPLas is not regulated at the level of transcription. Taken together, our results suggest that the availability of FerR is regulated at the transcriptional level by the change in osmolarity. The increase in FerR stabilizes PrbP to augment its activity in the host, where changes in osmolarity are encountered.
Increase of osmolarity induces ferRLcr expression level in L. crescens. The expression levels of ferRLcr (B488_01730) and prbPLcr (B488_01720) were determined in L. crescens in the prescence or absence of increasing concentrations of sucrose (50 to 100 mM) by qRT-PCR. The relative expression value of each gene was normalized to the expression levels of gyrase subunit A gene (gyrA). In black, control (Ctrl) condition in BM7 medium; light gray, BM7 medium supplemented with 50 mM sucrose; dark gray, BM7 medium supplemented with 100 mM sucrose. L. crescens cells were collected when the OD600 was 0.3. The experiment was performed using 4 biological replicates. The black horizontal line indicates statistical significance between connecting data bars. Statistical significance was determined as described in Materials and Methods. *, P < 0.05.
DISCUSSION
Our previous studies on the transcriptional accessory protein PrbPLas delivered an example of regulatory gene expression in L. asiaticus (21, 24); however, the mechanism behind the regulation of PrbPLas activity remained obscured. The analysis of prbPLas mRNA in the presence of tolfenamic acid suggested that the expression of prbPLas is not autoregulated at the level of transcription. While exploring potential posttranscriptional regulatory mechanisms, the ferredoxin-like protein FerRLas was examined as a potential regulatory protein for PrbPLas activity due to the genomic association between the two encoding genes (ferRLas and prbPLas, respectively). Interestingly, despite FerRLas homologs being distributed throughout bacterial and archaeal domains and PrbPLas homologs being widely distributed in Alphaproteobacteria, Actinobacteria, and Firmicutes (52), the synteny of the two genes is almost exclusively conserved in Alphaproteobacteria (Fig. 1B). The conserved genomic association suggests a functional connection between the two proteins. This observation also warrants further examination of FerRLas homologs encoded in Alphaproteobacteria, as they may mediate the regulation of protein activity by protein-protein interactions, rather than through modification of redox states as reported in other bacterial ferredoxins.
Annotated as a ferredoxin-like protein with one 3Fe-4S and one 4Fe-4S cluster, FerRLas was found to interact with PrbPLas and promote its transcriptional activity in vitro. Efforts to clarify the molecular mechanism revealed that FerRLas does not affect the DNA binding properties of PrbPLas. However, FerRLas was found to interact with the promoter region of rplK when examined independently (Fig. 3C and S3). Bacterial ferredoxins are generally considered small iron-sulfur clusters containing proteins that mediate electron transfer but lack independent enzymatic activity (40, 41). Due to the redox activity conferred by the iron-sulfur clusters, the biological functions of ferredoxins were first recognized in metabolic reactions that require electron transportation, including H2 metabolism, N2 and CO2 fixation, photosynthesis, and respiration. Interactions between bacterial ferredoxins and RNA have only been reported in Desulfovibrio vulgaris (53), and interactions with DNA have only been hypothesized for the Azotobacter vinelandii ferredoxin I (AvFdI) (54). The interactions we observed between FerRLas and DNA suggest that ferredoxins and ferredoxin-like proteins, such as FerRLas, have the potential to function as regulatory proteins.
Based on the conserved PreA/ferredoxin domain, our hypothesis was that FerRLas may modulate PrbPLas interactions with DNA and subsequently regulate PrbPLas activity in a redox-dependent manner. However, the oxidation state of FerRLas was not found to be correlated with PrbPLas activity under the conditions tested (Fig. 4A and B). Based on these results, the activity of PrbPLas appears to be regulated by the presence of FerRLas, irrespective of oxidative status.
FerRLas was modeled to the closest crystal structure of the 7Fe ferredoxin AvFdI (fdxA). As one of the most well-studied bacterial ferredoxins, AvFdI was shown to lack activity in the classic phosphoroclastic assay (40, 41). Although first believed to function as an electron donor to nitrogenase (55, 56), molecular and genetic experiments suggest that the primary function of AvFdI is to modulate expression of an NADPH:ferredoxin reductase (FPR) via protein-protein interactions with FPR and the pyruvate dehydrogenase E1 subunit (PDHE1) that binds specifically to the fpr promoter region (57–62). The importance of FerRLas in the regulation of PrbPLas activity was confirmed by site-directed mutagenesis studies and in vitro assays, where decreased stimulation of PrbPLas activity was observed with FerRLas mutants that had lower affinity for PrbPLas than did the FerRLas WT (Fig. 6).
In Mycobacterium spp., the PrbPLas homolog CarD was found to work simultaneously with another transcription regulator, RbpA, to costabilize the transcription initiation complex (63). While RbpA functions in a different mechanism, which involves binding to sigma factors and interacting with the promoter DNA, the structural and kinetic studies of RbpA and CarD with the Mycobacterium transcription initiation complex suggest that RbpA and CarD promote transcription activation in a cooperative manner (63, 64). The L. asiaticus genome lacks genes encoding an RbpA homolog, but a protein, such as FerRLas, could potentially function to achieve a similar effect. If prbPLas is constitutively expressed, and interactions between FerRLas, PrbPLas, and promoter DNA regulate PrbPLas activity, it is rational to postulate that mechanisms controlling interactions between FerRLas and PrbPLas are likely to exist. Our previous studies suggest that ferRLas expression is under the control of LdtR (20, 23), a transcription factor that mediates osmotic stress tolerance. In this study, we showed that ferRLcr expression is induced by increased osmolarity in L. crescens. Taken together, we propose that upon the entry of L. asiaticus cells into the phloem, the expression of ferRLas is induced in response to the increased osmotic pressure due to the presence of excessive phloem contents. Consequently, the elevated ferRLas expression level promotes interactions between FerRLas and PrbPLas, which increase PrbPLas activity in stabilizing the promoter open complex formation of genes that are necessary for adaptation to the new living conditions (Fig. 8).
Proposed model of FerRLas modulation of PrbPLas activity. We propose that under insect symbiont living conditions, ferRLas and prbPLas expression is maintained at basal level. When L. asiaticus is exposed to osmotic pressure, i.e., there were high sucrose contents during entrance of this bacterium into the citrus phloem, LdtRLas activates mRNA expression of ferRLas. Consequently, elevated concentrations of FerRLas promote interactions between FerRLas and PrbPLas, which increase PrbPLas activity by stabilizing the promoter open complex formation of genes that are necessary for adaptation to the host environment. The figure does not represent accurate scale and molecular ratio.
In summary, FerRLas is a novel example of how ferredoxins or ferredoxin-like proteins may serve a role as regulatory factors through protein-protein interactions and/or nucleotide binding (53, 54, 57, 58, 62, 65–67). The DNA binding ability of FerRLas and its relevance to PrbPLas activity modulation remain open questions. Further investigation into FerRLas:DNA interactions, especially whether or not the FerRLas interactions with DNA are important for PrbPLas selection and recognition of the promoter regions, is necessary.
Overall, our study describes a posttranscriptional regulation mechanism of PrbPLas activity. Our findings provide important insights into the regulation network of gene expression in L. asiaticus and a novel example of regulatory function by a ferredoxin-like protein.
MATERIALS AND METHODS
Bioinformatics.The STRING and SEED databases were used to predict interacting partners and to analyze prbPLas gene associations (37). The JGI IMG genome viewer was utilized to examine the genomic context of prbPLas (36). The FerRLas sequence was retrieved from the NCBI protein database, and FerRLas homologs were identified using Protein BLAST. The taxonomy tree consists of bacterial and archaeal species that contain FerRLas homologs. The tree was constructed based on the NCBI taxonomy database and visualized and rendered using iTOL (68). To perform multiple-sequence alignments, FerRLas homolog sequences were retrieved from the NCBI protein database, and alignments were performed using Clustal Omega (69), with default parameters. Structural modeling was performed under automated mode using SWISS-MODEL (70). Prediction of the protein-protein interaction sites in FerRLas was done through meta-PPISP (45), using default parameters. The amino acids predicted as positive were manually examined using PyMOL (46). The protein hydrophobicity patch was visualized using the Color h script based on the Eisenberg hydrophobicity scale (50).
Strains and DNA manipulation.Escherichia coli DH5α was used to maintain and replicate all plasmids used for cloning, protein purification, and site-directed mutagenesis. Escherichia coli JM109 was used to construct two-hybrid system strains and perform β-galactosidase assays. E. coli ArcticExpress (DE3) RIL (Agilent) was used for overexpression and purification of recombinant FerRLas proteins. The cells were grown at 200 rpm in lysogeny broth (LB) medium at 37°C under aerobic conditions. When necessary, the culture medium was supplemented with ampicillin (100 μg/ml), kanamycin (30 μg/ml), and chloramphenicol (34 μg/ml). All antibiotics and chemicals were purchased from Sigma-Aldrich and Fisher Scientific.
Liberibacter crescens BT-1 was cultured at 26°C with moderate agitation (200 rpm) in modified BM7 medium, as described in a previous study (20). All chemicals were purchased from Sigma-Aldrich.
Chromosomal DNA was isolated with the Qiagen DNeasy kit, and plasmid extractions were achieved with the QIAprep Spin miniprep kit (Qiagen). Standard molecular protocols described in Molecular Cloning (71) were used to perform PCR, restriction enzyme digestion, construction of recombinant DNA molecules, and cell transformations. Q5 high-fidelity DNA polymerase master mix (NEB) was used for PCRs. Site-directed mutagenesis was performed using the QuikChange site-directed mutagenesis kit (Agilent Technologies), following the manufacturer’s protocol. All predicted residues were mutated to alanine using plasmid p15TV-L, pB2Hα, or pB2Hω carrying ferRLas wild type (WT) as the template. Plasmids pB2Hα and pB2Hω carrying prbPLas WT were obtained from previous studies (21, 24).
All cloned DNA fragments described herein were verified by DNA sequencing. The strains, plasmids, and primers used in this study are listed in Tables 1 and 3, respectively.
Primers used in this study
Total RNA extraction and cDNA synthesis.Total RNA was extracted from 75 mg of homogenized infected citrus leaf tissue. Extractions were carried out using the Isolate II RNA plant kit (Bioline), following the manufacturer’s instructions. Zirconia beads (0.1 mm) were used during lysis to aid in the disruption of bacterial cells. Purified RNA was eluted with 30 μl of RNase/DNase-free water and subsequently treated with Turbo DNA-free DNase (Thermo Scientific) to eliminate trace amounts of DNA. Purified RNA samples were quantified using a NanoDrop ND 1000 spectrophotometer (Thermo Scientific) and stored at −80°C. cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad) with 0.5 μg of RNA. The cDNA products were stored at −80°C.
Bacterial two-hybrid system.The two-hybrid system was used as previously described (38). Briefly, candidate interacting proteins were fused to β-galactosidase truncations in the α- and ω-subunits, where the level of interactions between proteins tested is directly correlated with the complemented β-galactosidase activity detected. The ferRLas and prbPLas genes were cloned into the NotI and BamHI restriction sites on the pB2Hα and pB2Hω vectors. The fusion of the genes was verified by sequencing, and the recombinant genes in pB2Hα and pB2Hω were cotransformed into E. coli JM109, a β-galactosidase mutant strain. Strains carrying the empty vectors or single fusion plasmids were used as controls to determine baseline activity.
β-Galactosidase assays.E. coli cells were grown at 37°C in LB medium. Cells were collected at optical density at 600 nm (OD600) of 0.3, 0.5, and 0.8, suspended in Z-buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 50 mM β-mercaptoethanol; Miller [72]), and lysed by adding chloroform and 0.1% SDS. Expression of the fusion proteins was induced by the addition of 0.5 mM isopropyl-thio-β-d-galactopyranoside (IPTG). β-Galactosidase activity was determined by following the catalytic hydrolysis of chlorophenol red-β-d-galactopyranoside (Sigma-Aldrich). The absorbance at 570 nm was read every minute for 30 min using a Synergy HT 96-well plate reader (BioTek). The β-galactosidase activity was calculated using the slope of the absorbance curve normalized by the sample cell density and expressed as arbitrary units (AU). Each reaction was performed with three biological and technical replicates.
Protein purification.FerRLas was purified under denaturing conditions. Cells were grown in LB broth at 37°C to an OD600 of 0.6, and gene expression was induced with 0.5 mM IPTG. Induction was performed at 37°C for 3 h with shaking. The cells were harvested by centrifugation and resuspended in lysis buffer (500 mM NaCl, 5% glycerol, 50 mM HEPES, 5 mM imidazole [pH 7.5]). The buffer was supplemented with 1× Halt protease inhibitor cocktail, EDTA-free (Thermo Fisher Scientific). Cells were lysed by using a French pressure cell and passing the cells through 3 times at 1,500 lb/in2. The lysates were centrifuged at 17,000 × g for 45 min at 4°C, and the pellets were collected and washed in 1× BugBuster reagent (Novagen) 3 times by gentle pipetting and centrifugation at 5,000 × g for 20 min. The wash process was repeated 3 times using 0.1× BugBuster reagent and then 3 times using lysis buffer. The pellets obtained were solubilized in lysis buffer containing 6 M guanidine hydrochloride by incubation on ice overnight. The solution obtained was centrifuged at 14,000 × g for 20 min before the supernatant was applied to a HisPur nickel-nitrilotriacetic acid (Ni-NTA) resin spin column (Thermo Fisher Scientific) and washed extensively with wash buffer (lysis buffer containing 6 M guanidine hydrochloride and 25 mM imidazole, 50× column volume). The proteins were eluted from the column in elution buffer (lysis buffer containing 6 M guanidine hydrochloride and 250 mM imidazole). The eluted proteins were dialyzed against dialysis buffer containing decreasing concentrations of guanidine hydrochloride (250 mM NaCl, 2.5% glycerol, 200 μM FeCl2, and 10 mM HEPES [pH 7.5], with 6 to 0 M guanidine hydrochloride). The entire dialysis was performed at 4°C with gentle spinning. After dialysis, proteins were aliquoted and stored at −80°C.
Purification of PrbPLas was performed as described in a previous study (21). The 6×His-tagged fusion PrbPLas was overexpressed in E. coli BL21(DE3). Cells were grown in LB broth at 37°C to an OD600 of 0.6, and gene expression was induced with 0.5 mM IPTG. The induced cells were incubated at 17°C for 16 h with shaking. The cells were harvested and resuspended in lysis buffer and lysed as described above, except 0.5 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added to the cells immediately before lysing. The lysate was centrifuged, and the supernatant was applied to His60 Ni Superflow resin (Clontech). The column was washed extensively with lysis buffer containing 25 mM imidazole, and the proteins were eluted from the column in elution buffer (lysis buffer with 250 mM imidazole). The purified proteins were dialyzed against 10 mM HEPES (pH 7.5), 500 mM NaCl, 2.5% glycerol, and 0.5 mM TCEP, and then aliquoted and stored at −80°C.
Purification of the L. asiaticus RNAP holoenzyme was performed as described in a previous study (24); however, the buffers were modified as follows: the binding, wash, and elution buffers contained 500 mM NaCl, 5% glycerol, 50 mM Tris-HCl (pH 8) with 5, 15, and 250 mM imidazole, respectively. The purified protein complex was dialyzed against 10 mM Tris-HCl (pH 8), 250 mM NaCl, 50% glycerol, 0.1 mM EDTA, and 0.5 mM TCEP, and then aliquoted and stored at −80°C.
Immunoprecipitation assays.L. crescens BT-1 cells were cultured in modified BM7 medium until an OD600 of 0.8 was reached. Cells were collected by centrifugation at 8,000 rpm for 20 min at 4°C and washed with 50 mM Tris-HCl (pH 8) and 150 mM NaCl. The washed cells were collected by centrifugation, and the pellet was suspended in lysis buffer (50 mM Tris-HCl [pH 8], 150 mM NaCl, 2 mM MgCl2, 0.1% Triton X-100, and 1× EDTA-free Halt protease inhibitor cocktail). Two volumes of prewashed 0.1-mm zirconia/silica beads were added to 1 volume of the suspended cells in a 2-ml Eppendorf tube. Tubes were vortexed rigorously for 30 s and cooled down for 15 s on ice. This vortex-cool down process was repeated 6 times. The lysate was centrifuged at 17,000 × g for 10 min. The supernatant was recovered and centrifuged at 17,000 × g for another 15 min. For 1 immunoprecipitation reaction, 50 μl of Dynabeads protein G magnetic beads (Life Technologies) was charged with 10 μg of monoclonal anti-polyhistidine antibody (Sigma-Aldrich), following the manufacturer’s protocol. A total of 750 μl of sample containing 50 μg of purified recombinant PrbPLas from L. asiaticus and 500 μg of cell-free L. crescens extract was incubated with charged magnetic beads and incubated at 4°C with gentle rotation for 2 h. The tubes were placed on the magnet to remove supernatant. Residual unbound proteins were removed by washing the beads three times in 200 μl of washing buffer. The washed Dynabeads-Ab-Ag complexes were eluted in 20 μl of elution buffer. The eluted samples were run on a 15% SDS-PAGE gel for 1.5 cm, and the entire lanes were excised and sent for LC-MS/MS analysis. Independent duplicate reactions were performed, and reactions without recombinant PrbPLas were used as controls.
LC-MS/MS analyses.All MS/MS data were analyzed using Mascot (version 2.4.1; Matrix Science) with searches of the NCBI database (Bacteria domain) assuming complete digestion with trypsin. The false-discovery rate (FDR) was specified at ≤0.1% using the automatic decoy database search in Mascot. Fragment ion mass tolerance was 0.8 Da, and parent ion tolerance was 10 ppm. Scaffold (version 4.3.4; Proteome Software, Inc.) was used to validate MS/MS-based peptide and protein identifications. Protein identifications were accepted if they were established at greater than 70% probability and contained at least one identified unique peptide, as assigned by the ProteinProphet algorithm.
Electrophoresis mobility shift assays.Electrophoresis mobility shift assays (EMSAs) were performed as described earlier (21, 24). Briefly, a fragment of the rplK promoter region was amplified by PCR using prelabeled 5′-biotin primers. The reaction mixture contained 1 ng of 5′-biotin-labeled DNA probe, 10 mM HEPES (pH 7.5), 250 mM NaCl, 5% glycerol, and 12.5 ng/μl of nonspecific competitor DNA poly(dI-dC), purified PrbPLas WT, FerRLas WT, or FerRLas mutant protein (0 to 5 μM). When indicated, diamide and dithiothreitol (DTT) were added. Following incubation at 37°C for 20 min, the samples were analyzed by electrophoresis using 6% acrylamide-bisacrylamide nondenaturing gels in ice-cold 0.5× Tris-borate-EDTA (TBE) buffer (pH 8.3). The samples were then transferred from the polyacrylamide gel to a Hybond-N+ membrane (GE Healthcare) by electroblotting at 250 mA for 45 min using a semidry transfer blot (Fisher Scientific). The transferred DNA was UV-cross-linked, and the biotin-labeled DNA was detected using the Phototope-Star detection kit (NEB). Membranes were exposed to Kodak X-ray films. Vehicle controls were included in all assays.
In vitro transcription runoff assays.In vitro transcription runoff experiments were conducted as described earlier (24). The recombinant plasmid pMiniT-PrplK was linearized using the restriction enzyme NdeI. All proteins were diluted to the working concentration with transcription buffer (40 mM Tris-HCl [pH 8.0], 50 mM KCl, 10 mM MgCl2, 0.1 mM EDTA). Each 20-μl reaction mixture contained template DNA (5 nM) and purified proteins (0 to 5 μM). DTT or diamide was added when indicated. The mixtures were preincubated for 10 min at 37°C prior to adding the partially purified RNA polymerase. A second incubation was then performed at 37°C for 5 min. The transcription reaction was then initiated by the addition of nucleoside triphosphates (NTPs; 2 mM each ATP, GTP, and CTP, 1.5 mM UTP, and 0.5 mM biotin-11-UTP). The reactions were terminated by adding 10 mM EDTA after 30 min of incubation at 37°C. The transcripts were purified and concentrated by ethanol precipitation and analyzed using 6% acrylamide-bisacrylamide 7 M urea gels, in ice-cold 0.5× TBE buffer, at 100 V for 2.5 h. Transcripts were transferred to a Hybond-N+ membrane (GE Healthcare) by electroblotting at 380 mA for 40 min in a semidry transfer blot (Fisher Scientific). The transferred transcripts were UV-cross-linked and detected using the Phototope-Star detection kit (New England BioLabs). The membranes were exposed to Kodak X-ray films to visualize the transcription products. A biotinylated small RNA (sRNA) ladder (KeraFAST) was used as a molecular weight marker.
qRT-PCR analysis.qRT-PCR analyses were performed as described previously (23). L. crescens BT-1 cells were cultured in BM7 medium modified with sucrose (50 mM and 100 mM) or no additive as a control. The cells were collected by centrifugation at 8,000 rpm for 20 min at 4°C when the OD600 was 0.3. The total RNAs were extracted with the RiboPure-Bacteria kit (Life Technologies), following the manufacturer’s protocol. Concentrations of the RNAs were determined using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). cDNAs were synthesized using the iScript cDNA synthesis kit (Bio-Rad). qRT-PCR assays were performed in duplicate for each sample obtained from four biological replicates. The PowerUp SYBR green master mix was used as recommended by the manufacturer, and reactions were carried out in a QuantStudio 6 suite (Life Technologies). The changes in expression (threshold cycle [CT] values) between the samples from different treatments were determined using the ΔΔCT method. Amplification of the gyrA was used as the internal control. The primers used during the qRT-PCR experiments are described in Table 3.
Statistical analysis.Normality of the data was assessed by a D’Agostino-Pearson test. The statistical analysis of the β-galactosidase activity from the bacterial two-hybrid assays, the quantification of the in vitro transcription assays, and the data from qRT-PCR were assessed by one-way analysis of variance (ANOVA) and Tukey’s HSD post hoc test. Results with a P value of <0.05 were considered statistically significant.
ACKNOWLEDGMENTS
This work was supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture (http://nifa.usda.gov/; award 2015-70016-23029).
The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the granting agencies.
We thank Kaylie Padgett and Christopher Gardner for critical reading of the manuscript.
FOOTNOTES
- Received 27 October 2018.
- Accepted 8 December 2018.
- Accepted manuscript posted online 14 December 2018.
Supplemental material for this article may be found at https://doi.org/10.1128/AEM.02605-18.
- Copyright © 2019 American Society for Microbiology.
