A LysR Family Transcriptional Regulator Modulates Burkholderia cenocepacia Biofilm Formation and Protease Production

ABSTRACT Quorum-sensing (QS) signals are widely employed by bacteria to regulate biological functions in response to cell densities. Previous studies showed that Burkholderia cenocepacia mostly utilizes two types of QS systems, including the N-acylhomoserine lactone (AHL) and cis-2-dodecenoic acid (BDSF) systems, to regulate biological functions. We demonstrated here that a LysR family transcriptional regulator, Bcal3178, controls the QS-regulated phenotypes, including biofilm formation and protease production, in B. cenocepacia H111. Expression of Bcal3178 at the transcriptional level was obviously downregulated in both the AHL-deficient and BDSF-deficient mutant strains compared to the wild-type H111 strain. It was further identified that Bcal3178 regulated target gene expression by directly binding to the promoter DNA regions. We also revealed that Bcal3178 was directly controlled by the AHL system regulator CepR. These results show that Bcal3178 is a new downstream component of the QS signaling network that modulates a subset of genes and functions coregulated by the AHL and BDSF QS systems in B. cenocepacia. IMPORTANCE Burkholderia cenocepacia is an important opportunistic pathogen in humans that utilizes the BDSF and AHL quorum-sensing (QS) systems to regulate biological functions and virulence. We demonstrated here that a new downstream regulator, Bcal3178 of the QS signaling network, controls biofilm formation and protease production. Bcal3178 is a LysR family transcriptional regulator modulated by both the BDSF and AHL QS systems. Furthermore, Bcal3178 controls many target genes, which are regulated by the QS systems in B. cenocepacia. Collectively, our findings depict a novel molecular mechanism with which QS systems regulate some target gene expression and biological functions by modulating the expression level of a LysR family transcriptional regulator in B. cenocepacia.

. In addition to AHL-type signals, there are many other kinds of QS signals. One of them is the diffusible signal factor (DSF)-type signal, which was first identified in Xanthomonas campestris (8,9).
Burkholderia cenocepacia is a major opportunistic pathogen that causes infection in cystic fibrosis and immunocompromised patients (10,11). It can produce biofilm and numerous virulence factors, including lipopolysaccharide, exopolysaccharide, protease, and toxin. B. cenocepacia possesses the CepIR QS system, which is a LuxIR-type QS system (12). CepI synthesizes two different AHL signals; one is N-octanoyl homoserine lactone (C 8 -HSL, OHL), and the other one is N-hexanoyl homoserine lactone (C 6 -HSL, HHL). CepR is a homolog of the LuxR protein; it contains two domains, the signal binding domain and the transcriptional activation domain, and uses the same regulatory mechanism as other LuxR-type regulators to control target gene expression (12)(13)(14)(15)(16). Furthermore, a fatty acid signal molecule synthesized by B. cenocepacia was identified as cis-2-dodecenoic acid, which was also called Burkholderia diffusible signal factor (BDSF) (17). Previous study showed that BDSF signal is biosynthesized by B. cenocepacia RpfF (RpfF BC ). It accumulates in a cell density-dependent manner and regulates the production of various virulence factors. With the bacterial cells gradually accumulating to a high density, BDSF signals bind to RpfR to enhance the phosphodiesterase activity of RpfR and decrease the intracellular cyclic diguanosine monophosphate (c-di-GMP) level, and then they increase the ability of the RpfR-GtrR complex to bind to the promoter DNA of target genes (18). The AHL-and BDSF-type QS systems coordinate to control virulence and physiological functions in B. cenocepacia. In addition, BDSF was also revealed to positively regulate AHL signal production (19).
LysR-type transcriptional regulators (LTTRs) are the most widespread transcriptional regulators in prokaryotes (20). Structures of this type of regulator are conserved and usually contain an N-terminal DNA-binding helix-turn-helix motif and a C-terminal coinducer-binding domain. The coinducing agents are mostly metabolic intermediate substances (21,22). Increasing evidence suggests that LTTR ShvR controls QS, protease, type II secretion, and colony morphology in B. cenocepacia (23,24). In this study, we demonstrated that a novel LysR family transcriptional regulator, Bcal3178, was positively controlled by both the AHL and BDSF systems. We also uncovered the regulatory mechanism of Bcal3178 to modulate the phenotypes and QS-regulated target genes in B. cenocepacia H111. In general, our results identify a novel downstream component of the QS systems that help us to further understand the QS signaling hierarchy in B. cenocepacia.

RESULTS
Bcal3178 controls QS-regulated phenotypes in B. cenocepacia. To further investigate the regulatory mechanism, especially the downstream signaling pathways, of the BDSF QS system, we constructed a library of Tn5 random insertion mutants. The bclACB operon is simultaneously regulated by the BDSF and CepIR QS systems in B. cenocepacia H111 (25,26). Taking advantage of this feature, the bclACB operon promoter lacZ was fused to a plasmid and transferred into the wild-type B. cenocepacia H111 strain. We screened and identified about 40,000 colonies of the mutant library of B. cenocepacia H111, and the light blue colonies grown in LB agar medium (5 g yeast extract, 10 g tryptone, 10 g NaCl, and 15 g agar per liter) supplemented with X-Gal (5bromo-4-chloro-3-indolyl b-D-galactopyranoside) were picked out for the identification of insertion sites. Among those identified genes (see Table S1 in the supplemental material), there was a LysR family transcriptional regulator (Bcal3178; I35_RS03450) whose homologues are widely present in many bacteria and play an important role in various physiological activities (27,28). However, the functions and regulatory mechanisms of Bcal3178 are unclear in B. cenocepacia. Domain structure analysis of Bcal3178 by using the SMART program (http://smart.embl-heidelberg.de) shows that it has an N-terminal DNA-binding helix-turn-helix motif and a C-terminal coinducer-binding domain (Fig.  1A). In-frame deletion of Bcal3178 caused a significant downregulation of biofilm formation and protease activity, which are the phenotypes controlled by the two different types of QS systems in B. cenocepacia, and the complemented strain exhibited restored phenotypes ( Fig. 1B and C).
Bcal3178 regulates the target genes by directly binding to the promoter. As an insertion mutation of Bcal3178 resulted in a light blue colony with a bclACB operon promoter-lacZ fusion plasmid, we supposed that Bcal3178 controls the expression level of the bclACB operon. To confirm this speculation, we constructed the PbclACB-lacZ reporter system in the Bcal3178 deletion mutant strain. The expression of bclACB in the Bcal3178 deletion mutant was remarkably lower than that in the wild-type B. cenocepacia H111 strain, as determined by measuring the b-galactosidase activity, suggesting that Bcal3178 positively regulates the expression of bclACB ( Fig. 2A). To further study whether transcriptional regulation of bclACB was achieved by direct binding of Bcal3178 to the promoter, we continued to perform electrophoretic mobility shift analyses (EMSAs) to identify the regulatory mechanism of Bcal3178. A 506-bp DNA fragment of the bclACB promoter was obtained by PCR amplification for use as the probe. Bcal3178 is composed of 327 amino acids and was purified using affinity chromatography (Fig. 2B). The EMSAs showed that the complex of Bcal3178 and bclACB probe migrated slower than the unbound probe, and the bclACB promoter probe that bound to Bcal3178 significantly increased with increasing amounts of Bcal3178 protein (Fig. 2C). Moreover, the amount of labeled probe bound to Bcal3178 decreased in the presence of unlabeled probe (Fig. 2C).
Transcriptional expression of Bcal3178 is positively regulated by both the BDSF and AHL QS systems. As bclACB operon expression, biofilm formation, and protease activity are regulated by both the BDSF and AHL systems in B. cenocepacia H111, we continued to investigate the relationship between Bcal3178 and the QS systems. We first measured the expression levels of Bcal3178 in the wild-type H111, BDSF-deficient mutant (rpfF BC deletion mutant), rpfR deletion mutant, AHL-deficient mutant (cepI deletion mutant), and cepR deletion mutant strains by using quantitative reverse transcription-PCR (RT-PCR) analysis. The results showed that the expression levels of Bcal3178 of the mutant strains were lower than that of the wild-type H111 strain (Fig.  3A). We then constructed the Bcal3178-lacZ reporter system in the wild-type H111, rpfF BC mutant, and cepI mutant strains. The b-galactosidase activity assays revealed that Bcal3178 expression levels were downregulated in both rpfF BC and cepI mutant strains, and the expression levels were restored with addition of 20 mM BDSF and AHL (OHL), respectively (Fig. 3B).
Bcal3178 is a downstream component of the BDSF and AHL QS systems. Based on the facts that Bcal3178 controlled QS-regulated phenotypes and its expression was modulated by QS systems at the transcriptional level, we then expressed Bcal3178 in trans in the rpfF BC mutant and cepI mutant strains. It was shown that in trans, the expression of Bcal3178 in the two mutant strains increased biofilm formation to 75% and 87% of the wild-type strain level (Fig. 4A), and the in trans expression of Bcal3178 in the rpfF BC mutant and cepI mutant strains can almost fully restore protease production to the wild-type strain level (Fig. 4B). These results suggested that Bcal3178 is a downstream component of the QS signaling network in B. cenocepacia.
CepR regulates Bcal3178 expression by directly binding to the promoter. To study how the QS systems control Bcal3178, we investigated whether transcriptional regulation of Bcal3178 is achieved by direct binding of regulators of QS systems to the target gene promoter. Since GtrR and CepR are the regulators of the BDSF and AHL systems, respectively, in B. cenocepacia, we used EMSAs to test whether GtrR and CepR can bind the Bcal3178 promoter. A 223-bp DNA fragment of the Bcal3178 promoter was obtained by PCR amplification for use as the probe. CepR and GtrR, which are  composed of 239 and 463 amino acids, respectively, were purified by affinity chromatography ( Fig. 5A and Fig. S1A). It was shown that GtrR did not bind to the probe (Fig.  S1B), and the expression levels of Bcal3178 showed no detectable difference in the wild-type and gtrR deletion mutant strains ( Fig. S1C and D). CepR, which was purified in the presence of OHL signal, formed a stable DNA-protein complex with the Bcal3178 promoter DNA fragment, and the migration rate of the complex was slower than that of the unbound probe (Fig. 5B). The amount of labeled probe that bound to CepR increased with increasing amounts of CepR and decreased in the presence of both 25and 50-fold unlabeled probe (Fig. 5B). Moreover, the binding of CepR to the Bcal3178 promoter probes was enhanced in the presence of OHL signal (Fig. 5C). These results suggested that CepR is responsible for modulating Bcal3178 expression. Intriguingly, it was reported that CepR usually binds to the sequence called lux-box, which contains the conserved sequence NCTGTNNNGATCNNNCAGNN (12,15,27). However, we analyzed the promoter sequence of Bcal3178 and found no lux-box but only a similar sequence, TTCGATACGAGAGCGAAC, in the promoter of Bcal3178. Deletion of this fragment from the promoter region of Bcal3178 did not affect the binding of CepR to the promoter of Bcal3178 (Fig. S2), suggesting that there is a new binding site for CepR in the promoter region of Bcal3178.
Bcal3178 controls a wide range of QS-regulated genes. The BDSF and AHL QS systems control numerous genes and many physiological functions in B. cenocepacia (26). As Bcal3178 is a downstream component of the QS signaling network, we continued to test whether Bcal3178 controls the genes regulated by the BDSF and AHL QS systems (26). Quantitative real-time fluorescence PCR (qRT-PCR) results showed that at least 25 genes were decreased in the rpfF BC , cepI, cepR, and Bcal3178 mutant strains compared with their expression levels in the wild-type H111 strain (Fig. 6). These differentially expressed genes are involved in a range of biological functions (Table S2). However, the expression levels of rpfF BC and cepI showed no detectable differences between the wild-type and Bcal3178 mutant strains (Fig. S3). These findings suggested that Bcal3178 controls at least a subset of target genes of the QS systems.

DISCUSSION
In this study, we identified that a LysR family transcriptional regulator, Bcal3178, is a new global transcriptional regulator controlling various gene expression and biological functions (Fig. 1, 2, and 6). Intriguingly, both of these genes and biological functions are coregulated by the BDSF and AHL systems in B. cenocepacia. Moreover, the expression levels of Bcal3178 were significantly downregulated in the QS-deficient mutant strains compared to the wild-type B. cenocepacia H111 strain (Fig. 3), while Bcal3178 exhibited no detectable effect on the expression levels of BDSF or AHL signal synthase-encoding genes (Fig. S3). Previous studies showed that another LysR family transcriptional regulator, ShvR, was controlled by the AHL QS system in B. cenocepacia K56-2 (23,24). ShvR also influenced the production of a set of virulence factors and AHL signal production in B. cenocepacia K56-2 (23, 24), suggesting the distinguishing roles of Bcal3178 from other LysR family transcriptional regulators in B. cenocepacia.
It was already demonstrated that the AHL and BDSF QS systems are not completely independent and form a signaling network (19). The two QS systems coregulate various genes and phenotypes. BapA (encoded by BCAM2143) is a large surface protein that plays a vital role in biofilm formation (29,30). A cluster of three genes, bclACB (BCAM0184 to -0186), encode lectins, which are also needed for biofilm structural development (26,29). ZmpB is a zinc metalloprotease that is a vital component of proteolytic activity (31). All of these genes were significantly downregulated in both the BDSF-deficient and AHL-deficient mutant strains, as previously reported (26), and were downregulated in the Bcal3178 deletion mutant strain compared to the wild-type H111 strain (Fig. 6). In addition, the deletion of Bcal3178 impaired biofilm formation and protease production, while in trans expression of Bcal3178 restored the biofilm formation and protease production of both the QS signal-deficient mutants and the Bcal3178 deletion mutant ( Fig. 1 and 4). Moreover, the substantially overlapping genes controlled by both the BDSF and AHL systems were regulated by Bcal3178 (Fig. 6). Taken together, these findings support that Bcal3178 is a novel key downstream component of the QS signaling network in B. cenocepacia (Fig. 7).
Several previous reports showed that LTTR was controlled by the AHL-dependent QS system, but the regulatory mechanism is unclear (23,27). In this study, we discovered that CepR, the receptor of the AHL system, directly bound to the promoter of Bcal3178, and OHL signal enhanced the binding activity ( Fig. 5B and C). From these results, we can propose a new regulatory mechanism in which the AHL signals accumulate to the threshold and bind to CepR, and then the activated CepR regulates Bcal3178 by directly binding to the promoter of Bcal3178, thereby controlling target gene expression as well as biofilm formation and protease phenotypes (Fig. 7). Nevertheless, a new issue is that we did not find an obvious lux-box sequence in the promoter region of Bcal3178. Furthermore, the BDSF QS system usually regulates target genes through the BDSF-RpfR-GtrR complex (18), but the EMSA result showed that there was no binding between GtrR and the Bcal3178 promoter (see Fig. S1B in the supplemental material), and the expression levels of Bcal3178 were similar in the wildtype and gtrR deletion mutant strains (Fig. S1C and D), suggesting that BDSF employs another novel regulator or the AHL QS system to regulate Bcal3178 expression, which needs to be investigated further (Fig. 7).

MATERIALS AND METHODS
Bacteria strains and growth conditions. All the strains used in this study are listed in Table 1. B. cenocepacia H111 strains and Escherichia coli were grown at 37°C in LB medium (5 g yeast extract, 10 g tryptone, and 10 g NaCl per liter; solid medium also contains 15 g agar per liter). In this work, antibiotics were used at the following concentrations: ampicillin, 100 mg/ml; kanamycin, 100 mg/ml; gentamicin, 50 mg/ml; and tetracycline, 20 mg/ml. The chromogenic substrate X-Gal (5-bromo-4-chloro-3-indolyl b-Dgalactopyranoside) was used at 40 mg/ml. Bacterial growth was monitored spectrophotometrically by measuring the optical density at a wavelength of 600 nm (OD 600 ).
Screening and identification of mutants in which Tn5 was randomly inserted. A mini-Tn5 transposon with a gentamicin resistance gene was transformed into B. cenocepacia H111 with the bclACB operon promoter-lacZ fusion by triparental mating. The transformants were selected on LB plates supplemented with X-Gal and gentamicin. The light blue colonies were picked out for the identification of The two-component system RqpSR positively regulates expression of the cepI and rpfF BC genes, which are required for the synthesis of the BDSF and AHL signals, respectively. Binding of BDSF to the receptor RpfR substantially increases its c-di-GMP degradation activity and results in a reduced intracellular c-di-GMP level and, consequently, affects cepI transcriptional expression. CepR, activated by AHL signals, directly binds to the promoter of Bcal3178 and enhances the expression of Bcal3178, which finally controls some QS-regulated target gene expression and biological functions. Solid arrows indicate regulation or signal transduction. insertion sites. High-efficiency thermal PCR was used to identify DNA flanking sequences at the insertion site of the Tn5 transposon as previously described (32).
Construction of in-frame deletion mutant and complemented strains. B. cenocepacia H111 was used as the parental strain to construct the Bcal3178 deletion mutant by following previously described methods (17). The upstream and downstream fragments of Bcal3178 were generated by using the two PCR primer pairs listed in Table 2. For the generation of complementation, the coding region of Bcal3178 was amplified and cloned into the plasmid pBBR1-MCS2. The resulting construct was conjugated into the B. cenocepacia H111 DBcal3178 deletion mutant using triparental mating with pRK2013 as the mobilizing plasmid. The construct was also conjugated into B. cenocepacia H111 DrpfF BC and DcepI deletion mutants using the same methods (33).
Biofilm formation and protease activity assays. The bacterial cells were cultured overnight and diluted to an OD 600 of 0.01 by using minimal medium [per liter, 2 g glycerin, 2 g mannitol, 10.5 g For in trans expression and reporter (Continued on next page) promoters were 39-end labeled with biotin according to the manufacturer's instructions (Thermo). The biotin-labeled probes and proteins were prepared for the DNA-protein binding reactions by following the manufacturer's instructions (Thermo). A 5% polyacrylamide gel was used to separate the DNA-protein complexes from the unbound probes by following methods described previously (33). After UV cross-linking, the biotin-labeled probes were detected in the membrane, with different mobilities for the bound probes and unbound probes. Quantitative real-time fluorescence PCR. The bacterial cells were collected by centrifuging at 13,000 rpm for 2 min after growth to an OD 600 of 1.0. The total RNA was prepared using an RNA extraction kit (Promega). Reverse transcription-PCR was performed using a cDNA synthesis kit (Promega) according to the manufacturer's instructions. The qRT-PCR assays were performed using a SYBR green qPCR master mix (Thermo Scientific) and a 7300Plus real-time PCR system (Applied Biosystems). recA was used as the control. The relative expression levels of different target genes were analyzed by following the quantitation-comparative threshold cycle (DDC T ) method as described previously (37).

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 0.7 MB.
ACKNOWLEDGMENT This work was supported by a grant from the Guangdong Natural Science Funds for Distinguished Young Scholars (no. 2014A030306015), China.