Identification of potential CepR regulated genes using a cep box motif-based search of the Burkholderia cenocepacia genome
© Chambers et al; licensee BioMed Central Ltd. 2006
Received: 31 August 2006
Accepted: 22 December 2006
Published: 22 December 2006
The Burkholderia cenocepacia CepIR quorum sensing system has been shown to positively and negatively regulate genes involved in siderophore production, protease expression, motility, biofilm formation and virulence. In this study, two approaches were used to identify genes regulated by the CepIR quorum sensing system. Transposon mutagenesis was used to create lacZ promoter fusions in a cepI mutant that were screened for differential expression in the presence of N-acylhomoserine lactones. A bioinformatics approach was used to screen the B. cenocepacia J2315 genome for CepR binding site motifs.
Four positively regulated and two negatively regulated genes were identified by transposon mutagenesis including genes potentially involved in iron transport and virulence. The promoter regions of selected CepR regulated genes and site directed mutagenesis of the cepI promoter were used to predict a consensus cep box sequence for CepR binding. The first-generation consensus sequence for the cep box was used to identify putative cep boxes in the genome sequence. Eight potential CepR regulated genes were chosen and the expression of their promoters analyzed. Six of the eight were shown to be regulated by CepR. A second generation motif was created from the promoters of these six genes in combination with the promoters of cepI, zmpA, and two of the CepR regulated genes identified by transposon mutagenesis. A search of the B. cenocepacia J2315 genome with the new motif identified 55 cep boxes in 65 promoter regions that may be regulated by CepR.
Using transposon mutagenesis and bioinformatics expression of twelve new genes have been determined to be regulated by the CepIR quorum sensing system. A cep box consensus sequence has been developed based on the predicted cep boxes of ten CepR regulated genes. This consensus cep box has led to the identification of over 50 new genes potentially regulated by the CepIR quorum sensing system.
Burkholderia cenocepacia, belongs to a group of nine related species with common phenotypes, but distinct genotypes collectively named the "Burkholderia cepacia complex" (Bcc) [1, 2]. The Bcc are opportunistic pathogens in immunocompromised and cystic fibrosis (CF) patients but have also been isolated from plant rhizopheres as well as urban and suburban soils [1–3].
The ability of bacteria to adapt to diverse environments is dependent on the coordinate regulation of factors required to survive and proliferate in each niche. The CepIR quorum sensing system is one regulatory network that contributes to the response of B. cenocepacia to environmental signals (reviewed in [4, 5]). Quorum sensing allows bacterial populations to coordinate gene expression in response to population density. CepIR belongs to a group of more than 50 quorum sensing systems that are homologous to the LuxIR system of Vibrio fishceri [6, 7]. LuxI homologs are N-acyl homoserine lactone (AHL) synthases that generate AHL signal molecules that are released into the environment. LuxR homologs are transcriptional regulators that complex with AHL and typically bind to a lux-box overlapping the -35 sequence of a promoter to regulate transcription. The lux-box consensus sequence recognized by LuxR homologs typically consists of an inverted repeat with significant consensus among quorum sensing systems [6, 8–10].
The CepIR system was originally identified in B. cenocepacia (formerly B. cepacia) K56-2  and has subsequently been shown to be widely distributed throughout the Bcc [12, 13]. CepI directs the synthesis of N-octanoyl homoserine lactone (OHL) and N-hexanoyl homoserine lactone (HHL) and cepR encodes for the transcriptional regulator [11–14]. CepR has been shown to negatively regulate its own expression, but positively regulate cepI expression at the transcriptional level . The cepIR genes are involved in the regulation of the pvdA gene required for ornibactin biosynthesis , the zmpA and zmpB extracellular metalloproteases [15, 16], the aidA gene involved in virulence in Caenorhabditis elegans [17–20], swarming motility and in at least some systems a functional CepIR quorum sensing system is necessary for biofilm formation [21–23]. The CepIR system has been shown to contribute to virulence in both plant and animal models. In B. cepacia ATCC 25416 mutations in cepI and cepR attenuated maceration in the onion-rot model . The contribution of CepIR to the severity of B. cenocepacia infections has been demonstrated in two different animal models, a chronic respiratory infection model in rats and an acute intranasal infection model in Cftr (-/-) mice . CepIR have also been shown to be important for virulence in C. elegans .
Proteomics and promoter based approaches have been used to identify genes regulated by the CepIR quorum sensing system. Proteome analysis was used to compare the protein profiles of B. cenocepacia strain H111 and an H111 cepI mutant . Differences in expression were observed for 55 out of 585 proteins and partial N-terminal amino acid sequences were determined for peptide fragments of 11 proteins including AidA, FimA, and SodB. A promoter trap approach was used to identify positively regulated OHL-CepR dependent promoters in B. cepacia ATCC 25416 . A library of ATCC 25416 fragments cloned upstream of a promoterless lacZ gene in a vector that also contained cepR was screened in E. coli in the presence and absence of OHL. Twenty-eight clones with genes upregulated in the presence of OHL were identified. The genes belonged to several functional classes; however, the only overlap in genes identified between the two studies was aidA [17, 19]. Mutagenesis with a transposon containing a promoterless lacZ reporter was used to identify seven genes positively regulated by the cepIR quorum sensing system in B. cenocepacia strain K56-2, including cepI and aidA .
Identification of genes directly and indirectly regulated by CepR is a key step to understanding this regulatory system and the regulatory hierarchies that mediate the adaptation B. cenocepacia to the diverse environments it encounters. The above approaches search for genes regulated under defined in vitro conditions and therefore may not identify genes induced only in specific environmental niches including the plant or animal host. Only the study by Aguilar et al.  attempted to identify genes that are regulated by the direct interaction of CepR at the promoter.
LuxR homologs have been shown to bind to specific sequences referred to as lux boxes or the boxes for the gene designation of the respective luxR homolog such as tra boxes in the case of recognition sequences for Agrobacterium tumefaciens TraR [26–28]. These sequences have dyad symmetries and generally overlap the -35 RNA polymerase binding site. Lewenza et al. demonstrated that CepR was required for the expression of cepI in B. cenocepacia [11, 14] and identified a putative lux-box like sequence with imperfect repeats that overlapped the -35 region of the putative cepI promoter . Weingart et al.  demonstrated that CepR directly bound to a DNA fragment that contained the cepI promoter using electrophoretic mobility shift assays. They also mapped the transcriptional start site of cepI and using DNAase I footprinting experiments localized the CepR binding site to a region that closely corresponded to the cep box predicted by Lewenza et al. In the present study, we used a functional genomics approach to identify genes in the B. cenocepacia J2315 sequence with a cep box-like sequence in their promoters. We confirmed by site-directed mutagenesis the cep box sequence located upstream of the cepI gene that is necessary for cepI transcription. Using selected B. cenocepacia CepR regulated genes we predicted a consensus cep box motif sequence and used that motif to search the B. cenocepacia J2315 genome to identify promoters potentially regulated by CepR.
Functional analysis of the CepR binding site
Identification of CepR regulated genes by transposon mutagenesis
OHL responsive genes identified by Tn5-OT182 mutagenesis of K56-I2
Predicted start codonb
Location of insertb
OHL effect on expressiond
COG1835: Predicted acyltransferases
scpB: serine-carboxyl proteinase precursor
COG2860: predicted membrane protein
phuV: hemin specific ATP-binding protein
COG4774, Outer membrane receptor
Construction of the first generation cep box motif and search of the B. cenocepacia genome for match sequences
Identification of a cep box consensus motif.
bp to ORFd
First Generation Motif Sequencesa
CTGTAA A AGT TAC CAGTT e
Second Generation Motif Sequencesf
C TGT A A A AGTT AC C A G T T g
Construction of the second generation cep box motif and search of the B. cenocepacia genome for potential cep boxes
B. cenocepacia J2315 genes identified using the second generation cep box motif
Gene/domain and predicted functione
(Adjacent downstream genes possibly in operon)
Cell Surface or Membrane
COG0793: Periplasmic protease; cell envelope biogenesis
phaP: phasin-like protein
COG0859, rfaF, LPS heptosyltransferase (rfa L,rfaG; LPS biosynthesis genes)
COG3468, autotransporter type V secretion, shdA homolog: adhesin
COG3203: Outer membrane protein
COG1680: ampC, β-lactamase class C
Conserved hypothetical protein
MST2020 (+) MST028 f
COG4104: conserved hypothetical protein (vgrG: vgr related protein)
hypothetical protein (Chemoreceptor mcpA)
Elongation factor Tu
COG0308: Aminopeptidase N
MST2007 (+) MST011
COG1250: fadB, 3-hydroxyacyl-CoA dehydrogenase; lipid metabolism
COG0183: paaJ, Probable beta-ketoadipyl CoA thiolase (caiD; lipid metabolism)
COG0525: valS, Valyl-tRNA synthetase
COG0644: fixC, electron transfer flavoprotein-ubiquinone oxidoreductase
Hypothetical signal peptide protein (COG3000: Sterol desaturase, lipid metabolism)
COG0556,uvrB: Helicase subunit of the DNA excision repair complex
COG1448, tyrB: aspartate/tyrosine/aromatic aminotransferase
COG0165,argH: Argininosuccinate lyase, arginine biosynthesis
MST2035 (+) MST052
COG0480, fusA: Translation elongation factor
COG0043, ubiD: 3-polyprenyl-4-hydroxybenzoate decarboxylase (rhtB, Putative threonine efflux or homoserine/homoserine lactone efflux)
MST2039(+) MST059 f
COG1960: caiA, acyl CoA dehydrogenase
COG0757: aroQ: 3-dehydroquinate dehydratase II
kbl homolog, AKB ligase
MST2046 (+) MST072 f
COG0654: ubiH or mhpA, hydroxylase
sacB: Levansucrase (sacC: Levanase precursor)
MST2059 (+) f
cepI: homoserine lactone synthase
repA: replication protein
COG0757: aroQ: 3-dehydroquinate dehydratase II (aroE: Shikimate 5-dehydrogenase)
COG3185: hppD, 4-hydroxyphenylpyruvate dioxygenase
menG: putative S-adenosylmethionine:2 demethylmenaquinone methyltransferase
Hypothetical proteins Mup46, Mup47 and Mup48 [phage tail protein]
COG2747, flgM: Negative regulator of flagellin synthesis (flgN; Flagellar biosynthesis/type III secretory pathway)
MST2007 (+) MST011 f
COG0583: LysR-type transcriptional regulator
COG3073: RseA; Negative regulator of sigma E activity (RseB or MucB, negative regulator for alginate biosynthesis)
COG3707, nasR: nitrate-and nitrite-responsive positive regulator
gltF: regulator of gltBDF operon, glutamate synthaseenzymes
COG3073, rseA: Negative regulator of sigma E activity (mucB/rseB, mucD)
MST2039 (+) MST059
COG1414: Transcriptional regulator, IclR family
COG1396: hipB homolog, Putative transcription regulator
MST2045 (+) MST068 f
COG1396: hipB homolog, Predicted transcriptional regulator
MST2046 (+) MST072
COG1309: ArcR domain: Bacterial regulatory proteins, tetR family
COG: aglR, HTH-type transcriptional regulator
COG1846: Transcriptional regulator, MarR family
cepR: Transcriptional regulator, LuxR family
COG0583: LysR type Transcriptional regulator
Secretion or secreted product
COG3671: Predicted membrane protein (tatA, tatB, tatC secretion pathway)
MST2004 (+) MST005 f
COG0457: TPR repeat, (evpA and evpB, evpC, evpE, evpF, and evpG virulence and possible secretion)
zmpA: extracellular zinc metalloprotease
COG0834: ABC-type amino acid transport/signal transduction systems
MST2066 (+) f
phuR: Haem/Haemoglobin uptake outer membrane receptor precursor (phuS, phuT, phuU phuV
COG1638,dctP homolog: TRAP-type C4-dicarboxylate transport system,
MST2004 (+) MST005
COG3521: Uncharacterized protein conserved in bacteria
COG2606: Uncharacterized conserved protein
Putative vgr-related protein (pldA: Phosphatidylserine/phosphatidylglycerophosphate/cardiolipin synthases)
no homology (COG1536: Flagellar motor switch protein)
aidA, intracellular protein of unknown function involved in nematode virulence; (second aid A)
In this study we used a computational genome screen and experimental approaches to identify cepR regulated genes in B. cenocepacia. Transposon mutagenesis was used to identify OHL responsive genes in an approach similar to that described by Weingart et al. Since we had previously determined that genes involved in production of the siderophore ornibactin were cepIR regulated , we performed our screen in low iron medium in an attempt to identify other iron regulated genes that were responsive to OHL. We also had previously determined that cepR could both positively and negatively regulate gene expression, and therefore, the transposon library was screened for insertion mutants in which β-galactosidase activity was either turned on or off in the presence of exogenous AHLs. Four unique positively regulated and three negatively regulated lacZ fusions were identified. We identified two genes potentially involved in iron transport, a putative outer membrane receptor (BCAM1187) and phuV, a hemin ATP binding protein (BCAM2630). Interestingly, expression of the outer membrane receptor gene was negatively influenced by OHL, whereas phuV expression was positively influenced.
In a screen of approximately 25,000 transposon mutants we only identified six loci with AHL responsive genes. The screening assay was dependent on the visual identification of colonies that were either blue or white in the presence or absence of AHL on medium with X-gal. Although we were able to detect as little as two-fold differences in expression with this assay, we would not detect gene fusions expressed in both the presence and the absence of AHL since we did not attempt to identify mutants with varying shades of blue. For example, although CepR positively regulates zmpA, the CepIR system is not required for its expression since zmpA is expressed at low levels in the absence of AHL and in cepI or cepR mutants . The lacZ fusions in the positively regulated genes identified with transposon insertions were only expressed at significant levels in the presence of OHL. The three negatively regulated fusions had very low expression in the presence of OHL (Fig. 2). It was surprising that we did not identify cepI since CepR tightly regulates cepI expression  and cepI appeared to be a hotspot for transposon insertions in the study by Weingart et al. . The aidA gene which is tightly regulated by cepIR was identified in both transposon screens, as well as the proteomics and promoter trap approaches [17, 19].
Lewenza et al  identified a putative CepR binding site in the cepI promoter. During the course of this current study it was reported that CepR directly interacted with a cep box that overlapped this region and directly bound to a cep box within the aidA promoter . We demonstrated using site directed mutagenesis of the cep box region that a 24 bp sequence that contained the cep box was required for cepI expression. All cepI::luxCDABE promoter fusions with mutations in the 24 bp cep box had levels of expression less than or equal to 20% of K56-2 (pCPI301). Similar mutations constructed flanking the cep box had either no effect or in one case increased transcription.
The use of bioinformatics to identify CepIR regulated genes has several advantages that are complementary to the experimental methods used to search for CepIR regulated genes. Procedures such as transposon mutagenesis, promoter libraries, microarray analysis or proteomics are dependent on the transcription and expression levels of the genes and on the conditions used in the study. Furthermore, the genes and proteins identified by these approaches may be regulated directly or indirectly by CepR. The use of a motif in a genome-wide search for CepIR regulated genes may identify niche specific genes that may only be expressed in certain conditions. Identification of a cep box motif may also be used to predict whether CepIR genes are directly regulated by interaction with CepR at the promoter or indirectly by CepR interaction with a promoter for an intermediate regulatory gene. In fact, 14 of the 55 putative cep boxes identified were in the predicted promoter regions for regulatory genes. We are currently characterizing some of these regulatory genes to confirm that they are cepR regulated and to determine their regulatory properties.
When searching the genome using the first generation cep box motif we identified some sequences that were not identified with the refined motif used in the second screen of the genome (data not shown). It is possible that these genes are regulated by CepR but have less conserved cep box sequences. Of the eight promoter-lux fusions constructed from sequences identified in the first generation search, six were determined to have cepR regulated expression. There was no difference between the expression of the pMST112 in K56-R2 and K56-2; however, luminescence was increased in K56-dI2 in medium with OHL. The MST112 motif was not detected in the second cep box motif, suggesting that this BCAM1943 may not be cepR regulated. Mutations in cepI or cepR did not influence the expression of pMST052 (BCAL2739). Although this promoter region was excluded from the group used to generate the second motif, this potential cep box was also detected in the second search (MST2035). It is possible that BCAL2739 is, in fact, CepR regulated in different medium or growth conditions.
Interestingly, the MEME program identified a cep box motif farther upstream of the aidA cepR binding site identified by Weingart et al. . It is possible that there is more than one CepR binding site upstream of aidA. The additional site might contribute to its tight regulation by CepR and dependency on OHL for expression, features that may have resulted in aidA being detected in all of the approaches to date to identify CepR regulated genes.
We identified a cep box in the cepR promoter region that contains all of the most conserved bases. We have previously shown that cepR negatively regulates itself . This is the first confirmed negatively regulated gene identified in the motif search.
It is difficult to compare the extent of overlap between the genes identified using the bioinformatics approach to those identified by Aguilar et al.  and Weingart et al.  since the same annotation of the J2315 sequence was not used, although Aguilar et al. identified in addition to aidA, a lysR regulator and a putative short chain dehydrogenase which may be the same ones we identified. Concurrent with this study, we employed a random promoter library approach to identify promoter::lux fusion clones that were differentially expressed in the presence or absence of OHL in K56-dI2 . Of the 86 promoter clones identified, surprisingly only 4 genes overlapped between the two approaches, BCAM0009, BCAM0010, cepI and zmpA. A putative cep box was identified in the promoter regions of 30/89 OHL responsive genes from the promoter library, but generally with only 50–60% identity to the cep box consensus identified in this study. Therefore, these would not have been identified with the stringency employed in the search. It is surprising that more genes that were identified using the cep box motif were not found in the promoter library, although the promoter library also lacks other known CepR regulated genes indicating that it is not complete. Some of the genes with cep boxes may not be expressed in the conditions used to screen the library.
Strains of B. cenocepacia, including K56-2, that contain the cenocepacia island (cci) have a second set of quorum sensing genes . CciI is an AHL synthase that produces predominantly HHL and small amounts of OHL. CciR is the transcriptional regulator. CciIR are co-transcribed and cepR is required for cciIR expression . Little is currently known about the regulatory targets of cciIR, although the zinc metalloproteases zmpA and zmpB are regulated by cciIR, and CciR negatively regulates cepI [35, 36]. There is no apparent cep box upstream of cciIR; however, there is one located within the coding sequence 13 bp downstream of the predicted start codon. This putative cep box TTGCTGAAGTTGTTCGGT lacks the conserved A in position 6 present in all the sequences in Table 3 but contains the other conserved bases. It is currently unknown whether CciR binds to a similar site as CepR, but we have determined that some cepR regulated genes are not regulated by cciIR (data not shown). It is possible that some of the cep boxes we have identified might be CciR binding sites. Further studies are in progress to explore the regulatory relationships between these two quorum sensing systems in B. cenocepacia.
We have identified several new CepR regulated genes using transposon mutagenesis and lux promoter fusions. We have also used a cep box consensus sequence to identify several genes or operons potentially regulated by CepR. To confirm that these genes are regulated by cepR or possibly cciR, experimental approaches such as transcriptional fusions, microarrays, or demonstration of direct binding of CepR to their promoter regions will be required. These studies reveal a significant number of genes that may be further studied to increase our understanding of the CepR regulon.
Reagents, bacterial strains and culture conditions
Bacterial strains and plasmids used in this study.
Strain or plasmid
Description and relevant genotype
φ80dlacZ ΔM15 (lacZYA-argF) recA1 endA gyrA96 thi -1 hsdR17 supE44 relA1 deoR U169
Mobilizing strain, RP4 tra genes integrated in the chromosome, Kmr
F-mcrA Δ(mrr-hds RMS-mcr BC) φ80dlac ZΔM15 Δlac X74 end A1 rec A1 deo R Δ(ara, leu)7697 ara D139 gal U gal K nup G rps L λ-
end A1 rec A1 gyr A96 thi hsd R17 (rk- mk+) rel A1 sup E44 Δ(lac-pro AB) [F' tra D36 pro AB laq IqZΔM15]
Cystic fibrosis respiratory isolate
cepR:: Tn5-OT182 derivative of K56-2, Tcr
cepI::tp derivative of K56-2, Tpr
cepI deletion mutant of K56-2
cepI::tp::Tn5-OT182 derivative of K56-2, Tcr, Tpr
BCAM03092:: Tn5-OT182 derivative of K56-I2, Tcr
BCAM0957:: Tn5-OT182 derivative of K56-I2, Tcr
BCAS0293:: Tn5-OT182 derivative of K56-I2, Tcr
BCAM2631:: Tn5-OT182 derivative of K56-I2, Tcr
BCAM2630:: Tn5-OT182 derivative of K56-I2, Tcr
Tn5-OT182 derivative of K56-I2, Tcr
BCAM1187:: Tn5-OT182 derivative of K56-I2, Tcr
PCR cloning vector, pUC ori, Plac, lacZα, KmR ApR
pSUP102(GM)::Tn5-OT182, Cmr, Tcr, Gmr, Apr
mutagenesis plasmid, Tcr
pUCP26 with 1.5 kb Sph I-Kpn I fragment containing the cepI gene, Tcr
pCR®2.1 TOPO with a 266 bp fragment containing the cepI promoter, Apr, Kmr
pAlter®-Ex 1 with the Bam HI-Xba I fragment from pCPI101, Tcr
Broad host range vector with promoterless luxCDABE operon, Tpr, Kmr
pMS402 with the Bam HI-Xho I fragment containing the wild type cepI promoter region from pCPI101, Tpr, Kmr
pMS402 containing the Bam HI-Xho I fragments containing the cepI promoter region with the cep box mutations designated 303-313, Tpr, Kmr
ColE1 Tra (RK2)+, Kmr
pMS402 containing the phuR promoter region
pMS402 containing the acyltransferase promoter region
pMS402 containing the scpB promoter region
pMS402 containing the aidA promoter region
pMS402 containing the MST005 promoter region
pMS402 containing the MST011 promoter region
pMS402 containing the MST028 promoter region
pMS402 containing the MST052 promoter region
pMS402 containing the MST059 promoter region
pMS402 containing the MST068 promoter region
pMS402 containing the MST112 promoter region
AHL extraction and OHL purification
AHLs were extracted from culture supernatants of K56-2 as previously described . The extract from 50 ml culture fluid was resuspended in 1 ml distilled water and 20 μl aliquots of this stock solution were spread onto agar plates to screen for mutants in which lacZ expression was altered in the presence of AHL. This quantity of AHL extract was found to restore wild-type protease activity to B. cenocepacia K56-I2 as indicated by the zones of clearing observed on skim milk plates. OHL was purified from culture supernatants of B. cenocepacia K56-I2 (pSLS225), a strain that carries the cepI gene in trans as previously described .
Molecular biology and sequence analysis
DNA manipulations were performed generally as described by Sambrook et al. . T4 DNA ligase was purchased from Promega Corporation (Madison, WI) and New England Biolabs Inc. (Beverly, MA). Custom oligonucleotides were synthesized by Invitrogen Life Technologies. DNA sequencing was performed at the Univeristy of Calgary Core DNA Services (Calgary, Canada) using an ABI1371A DNA sequencer or at Macrogen Inc. (Seoul, Korea) on an ABI3730 XL automatic DNA sequencer.
Mutagenesis of B. cenocepacia K56-I2 (Tpr) with Tn5-OT182 was performed as described by Lewenza et al. . Tn5-OT182 is a self-cloning transposon with a promoterless lacZ gene that is transcribed from the promoter of a host gene when it is fused in the direction of transcription . Transposon insertion mutants were picked using a robot (Norgren Systems, Palo Alto, CA) into Becton Dickenson microtest flat bottom polystyrene 96 well microtiter plates containing 200 μl medium per well and grown overnight at 37°C with shaking at 200 rpm. Cultures were stamped onto TSBD-C (200 μg/ml tetracycline, 100 μg/ml trimethoprim and 40 μg/ml X-gal) agar with and without the addition of AHL extract and grown for 48 hours at 37°C. β-galactosidase expression was visually monitored at 24 and 48 hours for differences in blue color. Approximately 25,000 tetracycline and trimethoprim resistant transposon insertion mutants from five independent mutagenesis experiments were screened. Positively regulated insertion mutants appeared blue in the presence of AHL and X-gal and white in the absence of AHL. The reverse is true in the case of negatively regulated genes. Nine mutants exhibiting reproducible differences in AHL dependent β-galactosidase expression were chosen for further characterization. The DNA flanking the Tn5-OT182 insertions was self-cloned from Xho I or Eco RI digests of genomic DNA and sequenced using oligonucleotides OT182-LT and OT182-RT .
Construction of cepI promoter mutations
Restriction Site or size of product (bp)
oligonucleotides used to clone promoters
position of 5' basea
CCTGTAAGAG TTACCAGTTAAGATCT CCTC GTGCCGCGCG CTG
CAGCGCGCGG CACGAGGAGATCT TAACTGG TAACTCTTAC AGG
In vitro transcription assays
Putative promoters identified in this study were PCR amplified using the primers listed in Table 5 from K56-2 genomic DNA and cloned into the vector PCR2.1®-TOPO. The promoters were excised from the PCR2.1®-TOPO clones using Bam HI-Xho I and ligated into pMS402 to create plasmids pCPI301, pPHU301, pAYL301, pSCP301 and pAID301, respectively. The eight promoters identified in the first genome search for cep box motifs were cloned using the primers listed in Table 5 for each MST promoter as described above and named pMST005, pMST011, pMST028, pMST052, pMST059, pMST068, pMST072, and pMST112, respectively.
Five ml overnight cultures of K56-2, K56-dI2 and K56-R2 hosting the luxDCABE fusions were grown in TSB supplemented with 100 μg/ml trimethoprim to maintain pMS402. Overnight cultures were diluted with TSB to an A600 of 0.05 and aliquots of 150 μl were placed in wells of 96 well clear bottom plates (Costar, Corning Incorporated, Corning, NY). The plates were covered and incubated at 37°C with shaking and the luminescence and absorbance was measured in a Victor2™ multilabel counter at various intervals for 24 hours. Each strain was assayed at least three times in triplicate.
Nucleotide sequence obtained from DNA flanking the transposon insertions was used with BLASTN to determine the location of the insertion in the unpublished genome sequence of B. cenocepacia J2315 , a strain of the same lineage as K56-2. Homologues of open reading frames were predicted using BLASTP . Potential promoter elements were identified using BPROM . The cep box consensus sequence was predicted by analyzing the promoter regions of selected positively regulated genes with the motif discovery tool MEME . The MEME program  represents motifs as position-dependent letter-probability matrices which describe the probability of each possible letter at each position in the pattern. The output from the MEME program provides a position-specific scoring matrix (PSSM) for the predicted motif. The PSSM for the predicted cep box consensus sequence was used to search the B. cenocepacia J2315 genome with the motif alignment search tool MAST [45, 47]. The cep box motifs identified by MAST were also aligned using Multalin [48, 49].
This study was supported by a grant from the Canadian Cystic Fibrosis Foundation to PS. The authors thank J. Parkhill and M. Holden at the Welcome Trust Institute for access to the annotation data of B. cenocepacia J2315 genome sequence prior to publication.
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