Identification of Quorum-Sensing-Regulated Genes of Burkholderia cepacia

Identification of CepR-C₈-HSL-regulated promoters of B. cepacia ATCC 25416.

The experimental strategy used to identify CepR-C₈-HSL-regulated genes was to insert B. cepacia genomic DNA fragments digested with different restriction enzymes (NruI, HaeIII, HincII, AluI, and Sau3AI) within the multiple cloning site of vector pSCR2 (Fig. 1A), scoring for active transcriptional fusions between the genomic fragments and the promoterless reporter gene lacZ. Vector pSCR2 also contains the luxR gene homologue cepR of B. cepacia ATCC 25146 that Aguilar et al. have recently shown to have activated gene transcription of a QSC promoter in a heterologous E. coli background when C₈-HSL (Fluka; Sigma-Aldrich) was exogenously provided (1). Consequently, clones were scored for β-galactosidase activity dependent upon the presence of C₈-HSL.

• Open in new tab
• Download powerpoint

FIG. 1.

(A) Schematic diagram (not to scale) of the cepR-based cloning plasmid used to identify QSC genes. Plasmid pSCR2 contains the B. cepacia ATCC 25146 cepR gene cloned in the broad-host-range plasmid pQF50 (12). The genetic components are as follows: cepR, the gene encoding the transcriptional activator CepR; lacZ, a promoterless β-galactosidase gene; T₀, a transcriptional trpA terminator; bla, an ampicillin resistance marker. The indicated restriction sites in the polylinker (N, NcoI; H, HincII; B, BamHI; S, SmaI; Hi, HindIII) were used to clone DNA fragments obtained from genomic DNA of B. cepacia ATCC 25416 digested with compatible-end restriction enzymes. Positive clones on selective plates in the presence of C₈-HSL were identified as blue colonies (see text for details). (B) E. coli clones were cross-streaked in close proximity to B. cepacia ATCC 25416 in LB plates containing 100 μg of ampicillin/ml and X-Gal and were incubated overnight at 30°C. The blue coloration developed only in proximity to the B. cepacia strain. pSCR2C and pSCon were the positive and negative controls, respectively. (C) E. coli clones were cross-streaked in close proximity to B. cepacia 25416-I on plates containing 100 μg of ampicillin/ml and X-Gal and were incubated overnight at 30°C. No blue coloration was observed either with clones or with the pSCR2C positive control, thus confirming the strict C₈-HSL-dependent expression of the reporter β-galactosidase gene.

Plasmid vector pSCR2 was constructed as follows: pBIR (1) (as the template) and two oligonucleotides (CEPRD2 [5′-ctccatgggtaacggtttcttgatcaac-3′] and CEPR-RA [5′-tggcatgccctcgttcgaggtcagggcg-3′]) were used to amplify the cepR gene as a 1,347-bp DNA fragment. Consequently, the PCR-amplified product was digested and cloned as an NcoI-SphI fragment into the corresponding sites of pQF50 (12), thus yielding pSCR2. Several restriction enzyme sites were maintained upstream from the promoterless lacZ gene (Fig. 1).

To test the ability of pSCR2 to detect CepR-C₈-HSL QSC gene promoters in E. coli, a 250-bp BamHI-HindIII fragment (obtained by PCR with pBIR as the template and the oligonucleotides CEPID [5′-ggtcgcgctcgaagctttcgttcgcc-3′] and CEPIR [5′-ccccgcggatccacgtcctgatcggcgtca-3′]) containing the promoter region of cepI, a known B. cepacia QSC gene (25), was cloned upstream from the promoterless lacZ gene, yielding pSCR2C. In this control experiment, we observed 15-fold-increased transcriptional activation in E. coli (pSCR2C) (measured as β-galactosidase activity) when C₈-HSL was externally provided (Table 1). On other hand, when the same PCR product, containing the cepI promoter, was cloned in the corresponding sites of vector pQF50, yielding pSCon, no β-galactosidase activity was detected regardless of the presence or absence of C₈-HSL, thus confirming the requirement of CepR for the activation of transcription of the lacZ gene in pSCR2 (Fig. 1B and Table 1).

DNA ligations between pSCR2 and the genomic restriction DNA fragments from B. cepacia (obtained using the restriction enzymes NruI, HaeIII, HincII, AluI, and Sau3AI independently) were transformed in E. coli DH5α and plated on selective medium containing 100 μg of ampicillin (Sigma-Aldrich)/ml, 20 μg of X-Gal (Sigma-Aldrich)/ml, and 100 nM C₈-HSL. From the various cloning experiments we obtained approximately 50,000 colonies, and of these, approximately 2,400 had turned blue in the selective plates, indicating that a promoter most probably had been cloned upstream from the promoterless lacZ gene in pSCR2. To identify cloned gene promoters that had been activated in a CepR-C₈-HSL-dependent way, all 2,400 clones were used in cross-streaking experiments on plates containing 100 μg of ampicillin/ml and 20 μg of X-Gal/ml in close proximity to the parent B. cepacia ATCC 25416 strain as an exogenous source of C₈-HSL. Of these 2,400 clones, 28 clones had turned blue only in close proximity to B. cepacia ATCC 25416, as shown in part in Fig. 1B. To confirm the C₈-HSL dependence of expression, the clones were also cross-streaked with the quorum-sensing mutant B. cepacia 25416-I, which does not produce HSL molecules (1), and as can be seen in Fig. 1C, no blue coloration was visible. Consequently, it was postulated that these 28 clones contained putative promoters of B. cepacia ATCC 25416 driving the transcription of the reporter lacZ gene in a CepR-C₈-HSL-dependent manner in the E. coli background. This was further confirmed for each clone by performing β-galactosidase assays (35, 49) in the presence or absence of C₈-HSL, and as shown in Table 1, there was a 5- to 25-fold activation of transcription when C₈-HSL was present in the medium. To confirm that transcriptional activities driven from the cloned DNA fragments were, apart from being C₈-HSL dependent, also CepR dependent, each DNA fragment containing the putative QSC promoters was cloned in the original pQF50 vector as a SalI/HindIII or BamHI/HindIII fragment. As shown in Table 1, the clones devoid of the CepR gene did not display activation of transcription in the presence of C₈-HSL. It was concluded that activation of transcription from the cloned DNA fragments in E. coli was dependent on the presence of C₈-HSL and the CepR protein, thus strongly indicating that they contain QSC promoters of B. cepacia.

Characterization of clones that activated transcription in a CepR-C₈-HSL-dependent way.

The sequence of the B. cepacia ATCC 25416 genomic DNA present in all the 28 clones was determined, and it was subsequently compared (using the BLASTN program) to sequences in the sequenced genome of B. cepacia J2315 (http://www.sanger.ac.uk/Projects/B_cepacia/ ). We observed that the majority (20 of 28) of the clones displayed very high (more than 80%) identity at the DNA level with a region of the genome. Moreover, we also observed that these clones included a part of an open reading frame (ORF) or were just upstream from one. In contrast, seven clones had rather small inserts (ranging from 57 to 294 bp) and it was not possible to detect any significant homologies with regions of the B. cepacia J2315 genome or any other DNA sequence available in data banks. It is therefore possible that these clones contained intergenic regions of DNA or represented promoters of QSC genes which were strain specific, and further characterization is necessary to determine which gene(s) these putative identified promoters regulate. Two identified clones appeared to represent intragenic regions; thus, they do not seem to have been proximal to promoter sequences. Some promoters in bacteria were found intragenically; however, the possibility that these two clones gave false-positive results cannot be excluded. A BLASTP analysis of the 20 putative ORFs postulated to be under the control of the cloned quorum-sensing-dependent promoters is shown in Table 1. The seven clones without significant homologies are indicated as unknown. In silico analysis of the 20 clones from Table 1 whose sequences displayed significant identity to regions of the B. cepacia J2315 genome is presented in Fig. 2. The size of the insert and the corresponding region (and adjacent loci) of high identity in the genome of B. cepacia J2315 (with the gene(s) identified [where possible] in that region) are depicted in that figure.

• Open in new tab
• Download powerpoint

FIG. 2.

Characterization of identified clones containing putative CepR-C₈-HSL gene promoters. The DNA sequence of each identified clone was compared (using the BLASTN program [http://www.sanger.ac.uk/Projects/B_cepacia ]) to sequences in the genome of B. cepacia J2315. For those clones that showed a high degree of identity at the DNA level (see text), a region of the genome surrounding 2 kbp of each sequence was subsequently annotated. In the majority of the cases, it was possible to identify a homologue of a gene coding for a protein of known function. In such cases, the names of the respective genes are indicated inside of the putative ORFs (represented as thick gray arrows). A black arrow represents the size, position, and orientation of the identified insert in each clone (in scale with each clone). Within some genetic regions shown, hypothetical proteins of unknown function were found (here designated ORF). Such was the case with P67 (ORF represents what was probably a lysR family transcriptional regulator from R. solanacearum), P80 (ORF represents a 4-hydroxybenzoate octaprenyl transferase from Xanthomonas axonopodis), P114 (ORF1 represents a hypothetical protein from B. fungorum; ORF2 represents a probable transcriptional regulator, PA1627, from P. aeruginosa), P111 (ORF2 represents a short chain dehydrogenase-reductase from Pseudomonas putida, KT2440; ORF1 represents a hypothetical protein, PA1829, from P. aeruginosa), P79 (ORF represents a protein of unknown function), P103 (ORF represents a protein of unknown function), P56 (ORF1 represents a probable efflux membrane protein from Mesorhizobium loti; ORF2 represents a probable nucleoside triphosphate pyrophosphohydrolase from Agrobacterium tumefaciens), P57 (ORF1 represents a probable porin transmembrane protein from R. solanacearum; ORF2 represents a putative lipoprotein from R. solanacearum), P105 (ORF represents a hypothetical protein from R. solanacearum), and P135 (ORF1 represents a human RNA binding protein; ORF2 represents a putative small heat shock protein from R . solanacearum). See text for details of all other ORFs mentioned.

To further verify whether the cloned fragments contained promoters and whether they were QSC in the parent strain, we carried out mapping (using total mRNA from wild-type B. cepacia ATCC 25416 and cepI knockout mutant derivative B. cepacia 25416-I) of the mRNA 5′ end by reverse transcription of clones P67, P53, and P110. A primer was designed near the far end of the clone (see above) and also served to generate the corresponding sequence ladder (Fig. 3). Primer extension analysis (as depicted in Fig. 3) revealed the presence of a transcript initiation point which was in part dependent on the quorum-sensing system, since the transcript abundance decreased significantly in the cepI mutant, demonstrating that the QSC loci identified in E. coli are also QSC in B. cepacia.

• Open in new tab
• Download powerpoint

FIG. 3.

Primer extension analysis of three identified QSC promoters. Primer extension mapping of the mRNA 5′ end of clones P67 (A), P53 (B), and P110 (C) is shown. Total RNA was isolated from B. cepacia ATCC 25416 (WT) and the cepI mutant derivative (25416-I). Primer extensions were performed using 15 μg of RNA, and extension products were run adjacent to the sequencing ladder generated with the same primer used for reverse transcription. The approximate position of the mRNA 5′ end is depicted for each clone and indicated by an arrow.

As described above and depicted in Fig. 2, several ORFs are postulated to be under CepR-C₈-HSL control. Among the findings pertaining to such ORFs were the following. (i) Sequence analysis of clone P67 revealed the presence of a malate synthase, an enzyme belonging to the glyoxylate cycle (44) which has been associated with virulence in Mycobacterium tuberculosis and Candida albicans (29, 33). (ii) Sequence analysis of clone P80 did not reveal the presence of any putative ORF; however, a DpsA homologue was found in the genome of B. cepacia J2315 around 200 bp downstream from the identified sequence (Fig. 2). DpsA is a ferritin which is induced under stress conditions such as periods of nutrient starvation or oxidative stress (28, 42). (iii) Clone P53 contained the promoter of the aidA homologue, which encodes AidA (autoinducer dependent), a protein of unknown function whose expression has been shown to be regulated by quorum sensing in Ralstonia solanacearum (15) and shown recently also to be regulated by quorum sensing in B. cepacia H111 (43). (iv) Clone P91 contained the promoter of a gene encoding a peptidyl-prolyl cis/trans isomerase (44) which has been described as an essential virulence factor for Legionella pneumophila (14, 30) and the expression of which has been associated with the response in Pseudomonas aeruginosa under extreme stress conditions (13). (v) Clone P96 was found to contain a promoter controlling an ORF similar to that for CeoA protein AAB58160 (believed to be member of a putative efflux operon associated with multiple antibiotic resistance). (vi) Clone P57 is believed to contain a promoter regulating the expression of a porin gene (44). (vii) Clone P55 appeared to have a promoter upstream from an ORF encoding an ABC transmembrane transporter of sugars, and clone P110 appeared to have a promoter upstream from an ORF encoding a transporter of amino acids (44). (viii) A catechol 1,2-dioxygenase homologue (51) and its promoter were detected in clone P103. (ix) A promoter controlling an ORF homologue of ribonucleoside reductase 1 (44) was observed in clone P121. (x) A promoter for an acetaldehyde dehydrogenase II (41) was present in clone P56 and one for short chain dehydrogenase-reductase (38) was present in clone P111. (xi) In clone P69 a promoter controlling the expression of an ExbB homologue, which is a member of the cytoplasmic membrane complex TonB, ExbB, and ExbD (involved in the transport of iron siderophores, haem-haemin, transferrin, and vitamin B₁₂ in various gram-negative bacteria), was identified (39). Interestingly, an ExbB homologue in P. aeruginosa has also recently been described as QSC (47). (xii) In clones P79 and P135, the identified sequences were apparently driving the transcription of an ORF sharing some identity with a DNA (34) and with RNA binding protein BAA83713, respectively (Table 1). (xiii) In the case of clone P68, the DNA did not share a high level of identity with the genomic DNA of B. cepacia J2315, so the whole fragment (686 bp) was analyzed and a putative promoter was found to be followed by a putative ORF sharing partial (39%) identity with TrkA potassium uptake protein from Bacillus subtilis (50) (data not shown). (xiv) DNA downstream from clone P15 was localized to an ORF coding for a PilA homologue (48) required for virulence and twitching motility in P. aeruginosa (17) and R. solanacearum (27); interestingly, pilA in P. aeruginosa is not regulated by quorum sensing (2a). (xv) In clone P38, a putative promoter was found controlling a YwnB homologue, a protein of unknown function in B. subtilis (11). Interestingly, downstream from this sequence there was a homologue of MdeR, a protein member of the Lrp family of transcriptional regulators (22).

In the case of clone P81, it was established that the identified clone contained highly identical DNA sequences within an ORF coding for a putative penicillin binding transmembrane protein from R. solanacearum (44) (Table 1) and, interestingly, MurE and MurF homologues were found downstream from this sequence. A cluster in P. aeruginosa with similar organization has been described that is part of a peptidoglycan biosynthesis pathway (2, 26). It is not known whether there is a functional promoter in this clone; future work will determine whether a gene promoter is present within this putative ORF. Similarly, in clone P105 the identified putative promoter was localized on the basis of homology searches inside an ORF with identity to the virulence factor MviN (32) followed by searches inside another ORF encoding a putative protein of unknown function (Fig. 2).

Using the screening performed in the present study, we identified 28 different putative promoters of genes positively regulated by quorum sensing; to our knowledge, this is the first report of a molecular approach aimed at characterizing the quorum-sensing regulon in B. cepacia. We do not believe that this number represents all the promoters in the quorum-sensing regulon of B. cepacia; in fact, known CepR-C₈-HSL promoters such as cepI (1) were not identified in this screening. Further investigations are needed to confirm the total number of possible QSC loci; the results presented here are highly suggestive that quorum sensing is a global regulatory system in B. cepacia. This latter observation has also recently been made by Riedel et al. (43), whose use of proteomics determined that about 6% of the proteins in B. cepacia H111 are subjected to control by quorum sensing. Similarly, identification of QSC genes via the use of microarrays has determined that in P. aeruginosa about 6% (above 200) of the genes present in the chromosome are affected either positively or negatively by quorum sensing (47, 54). It must be stressed that for the complete identification and characterization of a regulon in bacteria several different approaches must be conducted, since every methodology has limitations, as sensibility or growth conditions can possibly mask the expression of genes that otherwise could be easily detected (7). We tried to search for a putative cep box representing a putative lux box-like binding sequence in which CepR-C₈-HSL binds and regulates gene expression; however, this proved difficult, as these boxes do not have many nucleotides which are conserved and to date there is no CepR binding region yet established. Interestingly, the methodology used in the present work does not depend on the expression level of the gene of interest in the B. cepacia background and it detects CepR-C₈-HSL directly activated gene promoters in the E. coli background. The results of the investigations of CepR-C₈-HSL-dependent expression in E. coli and the cross-streaking experiments with B. cepacia offer convincing evidence that in the identified fragments there is a quorum-sensing-modulated promoter. One limitation is that only positive regulation by quorum sensing is easily detectable using this screening system. Future work will also need to focus on the possible negative regulation of gene expression by quorum sensing in B. cepacia. Finally, since CepR homologues among the B. cepacia complex share 93 to 97% amino acid identity (data not shown), it is very likely that the same pSCR2 vector can probably be used in a way similar to that described in this study to obtain information about QSC genes (CepR-C₈-HSL dependent) for strains of the different genomovars in the B. cepacia complex and possibly also for other bacterial species which produce HSL molecules that can be recognized by CepR.