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

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.

INTRODUCTION

Quorum sensing (QS) is a cell-cell communication mechanism used by various bacteria (1–3). The first, most characterized QS system is the AHL-type system, which usually consists of two components, LuxI and LuxR proteins. The LuxI protein is an autoinducer synthetase that synthesizes the chemical signaling molecule N-acyl homoserine lactone (AHL). The LuxR protein is a cytoplasmic autoinducer receptor that contains a DNA-binding transcriptional activation domain. Autoinducers (AHLs) diffuse into the extracellular matrix and accumulate with the increasing number of cells. When the density of the signal reaches a threshold, the signal molecule will bind to the LuxR protein, and then the activated LuxR protein stimulates the expression of target genes (4–7). 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 (C8-HSL, OHL), and the other one is N-hexanoyl homoserine lactone (C6-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 (12–16). 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 (RpfFBC). 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 (5-bromo-4-chloro-3-indolyl β-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).

FIG 1

Effects of Bcal3178 on the QS-regulated phenotypes. (A) Genomic organization and domain structure analysis of Bcal3178 in B. cenocepacia H111 (domain structure was analyzed by using the SMART program at http://smart.embl-heidelberg.de). (B and C) Effects of Bcal3178 on biofilm formation (B) and protease activity (C). The data are means ± standard deviations from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (unpaired t test).

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 β-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).

FIG 2

Effects of Bcal3178 on a QS-controlled target gene. (A) The effects of Bcal3178 on the expression level of bclACB were measured by assessing β-galactosidase activity of the bclACB-lacZ transcriptional fusion in the B. cenocepacia H111 wild-type strain (●) and Bcal3178 deletion mutant strain (■). (B) SDS-PAGE of the GST-Bcal3178 protein. (C) EMSA detection of Bcal3178 binding to the promoter DNA of bclACB.

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 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 mutant, and cepI mutant strains. The β-galactosidase activity assays revealed that Bcal3178 expression levels were downregulated in both rpfF and cepI mutant strains, and the expression levels were restored with addition of 20 μM BDSF and AHL (OHL), respectively (Fig. 3B).

FIG 3

Effects of QS systems on the expression of Bcal3178. (A) The expression levels of Bcal3178 in the wild-type strain and ΔrpfF, ΔrpfR, ΔcepI, and ΔcepR mutant strains were analyzed by using qRT-PCR. The expression level of Bcal3178 in the wild-type strain was arbitrarily defined as 100% and used to normalize the expression ratios of Bcal3178 in the mutant strains. (B) The expression levels of Bcal3178 in the wild-type strain and QS signal-deficient mutant strains were measured by assessing β-galactosidase activity of the Bcal3178-lacZ transcriptional fusions. BDSF and AHL (OHL) were added at a final concentration of 20 μM. The data are means ± standard deviations from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (unpaired t test).

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 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 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.

FIG 4

Complementation of the QS signal-deficient mutants with Bcal3178. In trans expression of Bcal3178 complemented biofilm formation (A) and protease production (B) in the BDSF-deficient ΔrpfF mutant and AHL-deficient ΔcepI mutant. The data are means ± standard deviations from three independent experiments. ***, P < 0.001 (unpaired t test).

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 25- and 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.

FIG 5

Analysis of the binding between CepR and Bcal3178 promoters. (A) SDS-PAGE of the CepR protein. (B) EMSA detection of in vitro binding of CepR to the promoter of Bcal3178, in which a biotin-labeled Bcal3178 promoter DNA probe was used for protein-binding assays. (C) EMSA detection of in vitro binding of CepR to the promoter of Bcal3178 with the addition of different amounts of AHL (OHL).

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, 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 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.

FIG 6

qRT-PCR analysis of the genes that showed differential expression between the ΔrpfF, ΔcepI, ΔcepR, and ΔBcal3178 mutant strains and the wild-type strain. The data are based on three independent experiments, and error bars represent standard deviations.

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).

FIG 7

Schematic diagram of the QS signaling network in B. cenocepacia. The two-component system RqpSR positively regulates expression of the cepI and rpfF 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.

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 wild-type 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 μg/ml; kanamycin, 100 μg/ml; gentamicin, 50 μg/ml; and tetracycline, 20 μg/ml. The chromogenic substrate X-Gal (5-bromo-4-chloro-3-indolyl β-d-galactopyranoside) was used at 40 μg/ml. Bacterial growth was monitored spectrophotometrically by measuring the optical density at a wavelength of 600 nm (OD600).

TABLE 1

Bacterial strains and plasmids used in this study

Strain or plasmid	Phenotype and/or characteristicsa	Reference or source
B. cenocepacia
H111	Wild-type strain, genomovar III of the B. cepacia complex	34
ΔrpfFBC	BDSF-minus mutant derived from H111 with rpfFBC deleted	17
ΔrpfR	Deletion mutant with rpfR deleted	38
ΔcepI	Deletion mutant with cepI deleted	19
ΔcepR	Deletion mutant with cepR deleted	12
ΔgtrR	Deletion mutant with gtrR deleted	18
ΔBcal3178	Deletion mutant with Bcal3178 deleted	This study
ΔrpfFBC(Bcal3178)	ΔrpfFBC mutant harboring expression construct pBBR1-mcs2-Bcal3178	This study
ΔcepI(Bcal3178)	ΔcepI mutant harboring expression construct pBBR1-mcs2-Bcal3178	This study
ΔBcal3178(Bcal3178)	ΔBcal3178 mutant harboring expression construct pBBR1-mcs2-Bcal3178	This study
H111(PbclACB-lacZ)	H111 harboring reporter construct PbclACB-lacZ	18
ΔBcal3178(PbclACB-lacZ)	ΔBcal3178 mutant harboring reporter construct PbclACB-lacZ	This study
H111(PBcal3178-lacZ)	H111 harboring reporter construct PBcal3178-lacZ	This study
ΔcepI(PBcal3178-lacZ)	ΔcepI mutant harboring reporter construct PBcal3178-lacZ	This study
ΔrpfFBC(PBcal3178-lacZ)	ΔrpfFBC mutant harboring reporter construct PBcal3178-lacZ	This study
ΔgtrR(PBcal3178-lacZ)	ΔgtrR mutant harboring reporter construct PBcal3178-lacZ	This study
E. coli
DH5α	supE44 lacU169(80lacZM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 pir	Laboratory collection
BL21	F− ompT hsdS (rB−mB−) dcm+ Tetr gal(DE3) endA	Stratagene
Plasmids
pBBR1-mcs2	Broad-host-range cloning vector; Kanr	Laboratory collection
pBBR1-mcs2-Bcal3178	pBBR1-mcs2 containing Bcal3178	This study
pK18	pK18, sacB+; gene replacement vector; Kanr	Laboratory collection
pK18-Bcal3178	pK18 containing fragments flanking Bcal3178 and Gm-resistant fragment; Kanr, Gmr	This study
pRK2013	RK2 derivative, mob+ tra+ ori ColE1; Kanr	39
pME2-lacZ	Transcriptional level reporter vector; Tetr	Laboratory collection
PbclACB-lacZ	pME2-lacZ containing promoter of bclACB	18
PBcal3178-lacZ	pME2-lacZ containing promoter of Bcal3178	This study
pGEX-6p-1	Expression vector; Ampr	Amersham
pGEX-Bcal3178	pGEX-6p-1 containing Bcal3178	This study
pDBHT2	Expression vector; Kanr	Laboratory collection
pDBHT2-cepR	pDBHT2 containing cepR	This study
pDBHT2-gtrR	pDBHT2 containing gtrR	This study

Kanr, Tetr, Ampr, and Gmr indicate resistance to kanamycin, tetracycline, ampicillin, and gentamicin, respectively.

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 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 ΔBcal3178 deletion mutant using triparental mating with pRK2013 as the mobilizing plasmid. The construct was also conjugated into B. cenocepacia H111 ΔrpfF and ΔcepI deletion mutants using the same methods (33).

TABLE 2

PCR primers used in this study

Primer	Sequencea (5′–3′)
For deletion
Bcal3178 L-F	CTATGACATGATTACGAATTCCGCTCGTTGATTAGGTGGTGT
Bcal3178L-R	TTCCACGGTGTGCGTCCACTGCGCGCGTCAGCCATCGGA
Bcal3178R-F	TAAATTGTCACAACGCCGCCGGCTTGCGTATTTCTGGCC
Bcal3178R-R	CTGCCGTTCGAATCCCACGGCGCAGCGAACTGA
Bcal3178GM-F	AGTGGACGCACACCGTGGAAA
Bcal3178GM-R	GGCGGCGTTGTGACAATTT
Bcal3178IN-F	GTACTGGCGGTTCGGATAGA
Bcal3178IN-R	CACCTGAACACACGGCTGAT
Bcal3178OUT-F	TCCGCTCGTTGATTAGGTGG
Bcal3178OUT-R	ATGAGGAAAGGAAGTGCCCG
For in trans expression and reporter
Bcal3178C-F	GGGGTACCATGAACCAGATTCAGACCATGCG
Bcal3178C-R	GCTCTAGATTACTGCAGGCCCGTGACG
PbclACB-F	CCGCTCGAGCGGAATCTGGCGCTTCAGGAAAGAA
PbclACB-R	CCCAAGCTTGGGGCGGTTGGATGACGTTTGAGA
PBcal3178-F	CCCAAGCTTATATTCGAATACCGCGACGG
PBcal3178-R	CCGCTCGAGATTGGACACGCCGAGATGGT
For EMSA
EMSA-bclACB-F	GATGTCGGTCCTCGGTCT
EMSA-bclACB-R	CGAACATGAATAGGGCCT
EMSA-Bcal3178-F	TGCTGCATTGCAACCTTA
EMSA-Bcal3178-R	GGCTTGCGTATTTCTGGCC
For recombinant protein
Bcal3178-GST-F	CGGGATCCATGAACCAGATTCAGACCATGCG
Bcal3178-GST-R	CGGAATTCTTACTGCAGGCCCGTGACG
cepR-HIS-F	CGGGATCCATGGAACTGCGCTGGCAG
cepR-HIS-R	CGGAATTCTCAGGGTGCTTCGATGAG
gtrR-HIS-F	CGGGATCCATGAGAAATACGCCCGCAAT
gtrR-HIS-R	CGGAATTCTTACTCGCTTTCGCGGGTCT
For RT-qPCR
Bcal3178-F	CATGCGTGTATTCGTCTGCG
Bcal3178-R	TGGATCAGCCGTGTGTTCAG
cepI-F	AGTTCGATCGCGACGATACC
cepI-R	AGCGACTTCAGCAGATACGG
rpfFBC-F	CACGTTCGACTTCTGGGTGA
rpfFBC-R	CCGAAGCCCGTGTAGATCTC
recA-F	GTACGATCAAGCGCACGAAC
recA-R	GATCCGGCGGATATCGAGAC
BCAL0124-F	ACCTGTCGTACCTCCTCCTC
BCAL0124-R	CGTGATCATCGAAGCGGAAG
BCAL0831-F	TCCGTATTTGCCCCCGAAAA
BCAL0831-R	TTGCAGGTTGAGTTCGACGA
BCAL0833-F	TAGTCGTCACGTATTCGCCG
BCAL0833-R	CTTCTCGATGCATTGCTGGC
BCAL1063-F	ACAACGACGTGATCTCGGTC
BCAL1063-R	TGAACAGGTACGACGTCACC
BCAL2353-F	CTGTTCCGCTCGGTGATGAA
BCAL2353-R	AGCAGGAAGTGGTCGTCATG
BCAL3285-F	ACATCACCGCTGACCAGATG
BCAL3285-R	TGCGTCTGGTTGAACAGGTT
BCAM0184-F	CAACCCTTTACCCACGACGA
BCAM0184-R	CGTATTGCGGCAGTTTCTCG
BCAM0185-F	CCCTCCTTTCGGCTTCGATT
BCAM0185-R	GCGATCGCGAAATAGATGCC
BCAM0186-F	CTCAAACGTCATCCAACCGC
BCAM0186-R	GCTGTCGCCGATGAACAATT
BCAM0191-F	TGACCGATTCGACGCTTCAA
BCAM0191-R	GAAATACTCGGCCGCGTAGA
BCAM0192-F	CGTGTGGGATTTCATGTCGC
BCAM0192-R	AGGTACAGGTCGTAGTGGCT
BCAM0193-F	GCACGACTACCACGAGGAAG
BCAM0193-R	GAAGTAGCTGCCTTCCCGAT
BCAM0194-F	TTCCTGCGCGAATACCTGAG
BCAM0194-R	TGACGATCATCGGATGCTGG
BCAM0195-F	ACGTCGTCGCGTTCTATCTC
BCAM0195-R	GATAGCCGAAATGCGCATCG
BCAM0196-F	GCTCGACCATACCGACATGA
BCAM0196-R	CGACGTATGGATCAGGCTCC
BCAM0835-F	GTGAACCGCATCTCGATTGC
BCAM0835-R	CAGCGTCGTATGGATCAGCA
BCAM1005-F	GAACACGCCGATGTCGAATG
BCAM1005-R	GTAGACGGTGTAGCTGACGG
BCAM1010-F	TGTCGGGCATCATCGAGAAG
BCAM1010-R	GCTTGCGCAGATGATCGAAG
BCAM1745-F	CCGACATCATCCTGCTCGAA
BCAM1745-R	TGGCCGTCATGTTCAGGTAC
BCAM1871-F	CTCGAACGACAGGTTGACGA
BCAM1871-R	GTATTTGCTGCGCATCTCCG
BCAM2060-F	GTGCTGTACGTGAACCAGGA
BCAM2060-R	GTTGAGCAGGAACAGGTCGA
BCAM2140-F	AATTCTCGACGAAGCTCGCA
BCAM2140-R	GATGTCTTTCACGATGCCGC
BCAM2141-F	CGATCATTTCGGCAAGCAGG
BCAM2141-R	GACGAACGGGATGTCGATCA
BCAM2142-F	GAACCGTGAAAGCCTCGAGA
BCAM2142-R	GCGGTCACTTTCTCGTAGCT
BCAM2143-F	GACGATCCAGGTCGATGGTC
BCAM2143-R	GTATCCACCACGATCCCCAC
BCAM2169-F	GTACACGTGGTACCGCATCA
BCAM2169-R	GTTTCCGTATAGCCGTCGGT
BCAM2227-F	ACAGGAAGGCTTGTCGGAAG
BCAM2227-R	CGTCCCAGTTGTAGACCCAG
BCAM2307-F	GATGGACAAGGCGTTCCTGA
BCAM2307-R	GTGCAGCTCTTGTTGTACGC
BCAM2308-F	GCCTACTCTGAAACCGACCC
BCAM2308-R	CATCGATGCGTTGAAGCTGG
BCAS0292-F	GTCTGGTGTTCGTTGCGATG
BCAS0292-R	CAAAGAGCCGGTTGTCGTTG
BCAS0293-F	ATGTCACGCGTTACCGATGT
BCAS0293-R	GACATAGCGCCAGTCGATCA

Restriction enzyme sites are underlined.

Biofilm formation and protease activity assays.

The bacterial cells were cultured overnight and diluted to an OD600 of 0.01 by using minimal medium [per liter, 2 g glycerin, 2 g mannitol, 10.5 g K2HPO4, 4.5 g KH2PO4, 2 g(NH4)2SO4, 0.2 g MgSO4·7H2O, 0.005 g FeSO4, 0.01 g CaCl2, 0.002 g MnCl2]. Biofilm formation in 96-well polypropylene microtiter dishes was performed as described previously (34). For analyzing the protease activity, bacteria were cultured in NYG medium (per liter, 3 g yeast extract, 5 g peptone, 20 g glycerin) overnight at 37°C with shaking at 200 rpm, diluted to an OD600 of 0.01 in NYG, and then cultured at 37°C with shaking at 200 rpm for 18 h. Protease activity was determined by following previously published methods (35).

Construction of reporter strains and measurement of β-galactosidase assays.

The bclACB reporter was introduced into the B. cenocepacia H111 and ΔBcal3178 mutant strains by electroporation. The Bcal3178 reporter was introduced into the B. cenocepacia H111, ΔrpfF, ΔgtrR, and ΔcepI strains by triparental mating. The transconjugants were selected on LB agar plates supplemented with ampicillin, tetracycline, and X-Gal. For measurement of β-galactosidase activities, the overnight-cultured bacteria were diluted to the same cell densities (OD600, 0.01) in LB medium supplemented with ampicillin and tetracycline. The inoculated cultures were then incubated at 37°C with shaking at 200 rpm and harvested to measure β-galactosidase activities by following previously described methods (36).

Protein expression and purification assays.

The coding regions of Bcal3178, gtrR, and cepR were amplified with the primers listed in Table 2 and ligated to the expression vectors pGEX-6p-1 and pDBHT2, as indicated. The resulting constructs were transformed into E. coli BL21. The bacteria were cultured in LB medium with ampicillin and kanamycin, respectively, and the strain with pDBHT2-cepR was cultured with the addition of OHL (50 nM) (15). Affinity purifications of GST-Bcal3178, HIS-GtrR, and HIS-CepR fusion proteins were performed by following methods described previously (33). The fusion proteins were eluted and verified by SDS-PAGE.

EMSA.

The DNA probes used for electrophoretic mobility shift assay (EMSA) were harvested by PCR amplification using the primer pairs listed in Table 2. The purified PCR products of bclACB and Bcal3178 promoters were 3′-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 OD600 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 (ΔΔC) method as described previously (37).