Burkholderia cenocepacia is an important member of the Burkholderia cepacia complex, a group of closely related bacteria that inhabits a wide variety of environmental niches in nature and that also colonizes the lungs of compromised humans . Certain strains ofB . cenocepacia express peritrichous adherence organelles knownas cable pili, thought to be important in the colonization ofthe lower respiratory tract . The genetic locus required forcable pilus biogenesis is comprised of at least five genes,designated cblB, cblA, cblC, cblD, and cblS . In this study a transcriptional analysis of cbl gene expression was undertaken. The principal promoter, located upstream of the cbl locus, was identified and characterized . By using lacZ transcriptional fusions, the effects of multiple environmental cues on cbl gene expression were examined . High osmolarity, temperature of 37°C, acidic pH, and low iron bioavailability were found to inducecbl gene expression . Northern hybridization analysis of thecbl locus identified a single, stable transcript correspondingto cblA, encoding the major pilin subunit . Transcriptional fusionstudies combined with reverse transcription-PCR analysis indicatedthat the stable cblA transcript is the product of an mRNA processing event . This event may ensure high levels of expression of themajor pilin, relative to other components of the assembly pathway.Our findings lend further insight into the control of cablepilus biogenesis in B . cenocepacia and provide evidence for regulation of cbl gene expression on both the transcriptional and posttranscriptional levels.

 
Burkholderia cepacia is a complex of gram-negative bacteria widespread in nature that consists of at least nine genomicspecies, or genomovars [3, 6, 41]. Burkholderia cenocepacia,formerly genomovar III, has emerged as the dominant B . cepaciacomplex respiratory pathogen in compromised individuals andparticularly cystic fibrosis [CF] patients [12, 19] . B . cenocepacia colonization in individuals with CF generally leads to the establishment of chronic infection, causing a significant decrease in life expectancy . Colonization by B . cenocepacia can also lead to acute infections and fatal outcomes in CF patients [12, 19].Although several putative virulence factors have been identified,including the iron-scavenging siderophore ornibactin [35] anda type III secretion system [38], the molecular mechanisms facilitatingcolonization and pathogenesis of B . cenocepacia are still poorlyunderstood [22].

B . cenocepacia adherence to host cells and mucosal surfaces likely plays an important role in the initiation and establishmentof infection . The ability of B . cenocepacia to colonize theCF lung, as well as spread from patient to patient, has beenassociated with the expression of filamentous extracellularadherence organelles known as cable pili [4, 37] . These peritrichouslyexpressed structures derive their name from their unique cable-likeintertwined morphology [29] . Cable pili have been shown to facilitatebacterial binding to both mucin and CF respiratory epithelia,suggesting a direct role for cable pili in mediating colonization[27, 28] . Aside from the role of cable pili in adhesion, little is known about the mechanisms governing the expression and assembly of these structures in B . cenocepacia.

The DNA locus required for B . cenocepacia cable pilus biogenesis is comprised of at least five genes, designated cblB, cblA, cblC, cblD, and cblS [30] . The cblA gene encodes the major structuralsubunit of cable pili [29], while cblB, cblC, and cblD are predictedto encode the periplasmic chaperone, the outer membrane usher,and the minor pilus structural subunit, respectively . The fifthgene, designated cblS, is predicted to encode a new member ofthe sensor kinase superfamily of bacterial two-component systems.It has recently been demonstrated that the cblBACD locus is sufficient for heterologous expression of cable pili in Escherichia coli [30] . The B . cenocepacia cblBACD gene products share highhomology with the assembly machinery of the CS1 family of pili,elaborated by certain strains of human enterotoxigenic E . coli[ETEC] [30] . The CS1 family includes CS1, CS2, CS4, and CFA/Ipili [31], which have been implicated in colonization of thehuman small intestine and the establishment of infection byETEC [9, 17].

The genes required for CS1 pilus biogenesis, as well as the biogenesis of other pilus types, are typically organized asoperons [31] . Expression of CS1 and other pilus operons is subjectto both positive and negative regulation at the transcriptionallevel [15, 23] . A number of studies have examined the environmental regulation of pilus expression in ETEC and other pathogenic E . coli . These studies have drawn a correlation between stimuli resembling those encountered in vivo, including pH, osmolarity,and temperature, and transcriptional activation of pilus geneexpression [7, 10, 15, 20, 25, 42] . In contrast, far less isknown about the regulation of pilus gene expression in nonentericbacteria, including respiratory pathogens such as B . cenocepacia.

In addition to transcriptional control, expression of some pilus operons has been shown to be regulated at the posttranscriptional level . Specifically, mRNA processing and the various stabilitiesof the resulting mRNAs have been proposed as mechanisms forfacilitating differential expression of the various structuraland assembly components of pilus biogenesis pathways . PosttranscriptionalmRNA processing mechanisms have been shown to control CFA/Ipilus expression in ETEC, as well as expression of the F1845,Pap, and S fimbriae of pathogenic E . coli [1, 2, 14, 24].

In this study we [i] undertook a transcriptional analysis ofB . cenocepacia cbl gene expression, [ii] identified and characterized the principal promoter upstream of the cbl locus, [iii] examined the environmental modulation of cbl gene expression, and [iv] characterized an mRNA processing event, predicted to resultin higher expression levels of the major structural subunitof cable pili, CblA, relative to the other components of thepilus biogenesis pathway . Our findings lend new insight intothe regulation of cable pilus gene expression in B . cenocepaciaand provide evidence for control at both transcriptional andposttranscriptional levels.

 
Bacterial strains, plasmids, and media. The bacterial strains and plasmids used in this study are listedin Table 1. B . cenocepacia [formerly B . cepacia complex genomovar III] strain BC7 was obtained from the Belgium Coordinated Collections of Microorganisms/Laboratorium Microbiologie Ghent [BCCM/LMG].Strain BC7 is a cable-piliated CF clinical isolate of B . cenocepacia [29] . E . coli strains were grown with aeration at 37°C inLuria-Bertani [LB] broth [32] or on LB agar plates supplementedwith ampicillin [100 µg/ml], tetracycline [12 µg/ml],or chloramphenicol [30 µg/ml] as necessary . B . cenocepacia strains were grown with aeration at 37°C in LB or in M9minimal medium [32], supplemented with 0.2% glucose and 0.3% Casamino Acids [wt/vol] . For propagation of B . cenocepacia strains harboring transcriptional fusion constructs, tetracycline was added to liquid media [25 µg/ml] and LB agar [500 µg/ml].

 
DNA manipulations. DNA-modifying enzymes, including restriction endonucleases,T4 polynucleotide kinase, T4 DNA ligase and T4 polymerase, wereobtained from either Promega or New England Biolabs . SuperscriptII was obtained from Invitrogen, and Taq polymerase was obtainedfrom Promega . Plasmid DNA was isolated by the boiling lysismethod or by using the QIAprep Spin Miniprep kit [QIAGEN Inc.].Recombinant plasmids were introduced into E . coli by electroporationwith a Gene Pulser II [Bio-Rad] . Genomic DNA from B . cenocepaciawas extracted by means of a PureGene kit [Gentra] . Southernblot hybridizations were generally performed as described bySambrook et al . [32] with Hybond N nitrocellulose membranesand probes labeled with [-³²P]dCTP [Amersham Pharmacia Biotech]by the random primer method.

Cloning and sequencing of the B . cenocepacia cbl locus. The B . cenocepacia cbl locus from strain BC7 was cloned and sequenced by two converging strategies . Initially, portionsof the locus harboring cblB, cblA, cblC, and the first 1,074 nucleotides of cblD were cloned from strain CM256, a cblA::cat derivative of the parental strain BC7 [40] . The last 90 nucleotidesof the cblD gene and the entire cblS gene were cloned from acblD-cross-hybridizing cosmid, designated p3A4, which was identifiedby probing a B . cenocepacia strain BC7 cosmid library, constructedas previously described by our laboratory [39] . Multiple subclonesof the cbl locus were generated, and their sequences were determinedon both strands . Nucleotide sequencing was performed by theAdvanced Genetic Analysis Center at the University of Minnesotaby using the dideoxy chain termination method and an ABI 1371ADNA sequencer [Applied Biosystems] . Oligonucleotide primersused for sequencing were standard forward and reverse [T3 andT7] pBluescript primers or custom oligonucleotides synthesizedby Integrated DNA Technologies . Double-stranded sequences werealigned and assembled by the EditSeq and SeqMan components ofa demonstration version of the Lasergene sequence analysis softwarepackage [DNASTAR Inc.] . Nucleotide and amino acid sequence searchesand analysis utilized the BLASTX and BLASTP programs at theNational Center for Biotechnology Information.

Growth conditions and measurement of ß-galactosidase activity. Transcriptional fusion constructs were generated in the low-copy-number vector pRKlac290, harboring a promoterless ß-galactosidase reporter gene, lacZ, and are described in Table 1 and Fig . 1.Transcriptional fusion constructs were introduced into B . cenocepaciastrain BC7 by conjugation, as previously described, by usingE . coli S17-1 as the donor strain [39] . B . cenocepacia cultureswere grown in the presence of tetracycline [25 µg/ml],in order to ensure maintenance of pRKlac290 and pRKlac290-derivedconstructs . For measurement of ß-galactosidase activity,B . cenocepacia strains harboring the plasmid-borne transcriptionalfusion constructs were grown to stationary phase for 17 h in3 ml of LB or M9 medium, and aliquots were used to inoculatefresh 3-ml volumes of the corresponding medium . Cultures weregrown for an additional 16 to 18 h, until an optical densityat 600 nm [OD₆₀₀] of 0.2 was reached, at which point the firstß-galactosidase measurements were taken . The ß-galactosidaseactivities were assayed throughout the growth phase as describedby Miller [21] . Assays were performed in triplicate with a minimumof two independent experiments for each transcriptional fusionconstruct and/or growth condition.

 
To examine the effect of pH on cbl gene expression, B . cenocepacia strains harboring either pRKlac290 [vector control] or the cblB transcriptional fusion construct pMT55 were grown in M9 medium [pH 7.0] or in M9 medium in which the pH was adjusted to either5.0 or 6.0, by using concentrated HCl, or pH 8.0, by using 10M NaOH . In order to determine the effect of osmolarity, M9 mediumwas prepared without NaCl or supplemented with 5 M NaCl to afinal concentration of either 100 or 200 mM . To examine theeffect of temperature, bacteria were grown with shaking at 250rpm at either 30°C or 37°C . In order to examine theeffect of iron, M9 minimal medium was supplemented with 50 µMFeCl₃ . Conversely, iron was chelated in LB broth by the additionof 100 µg of ethylenediamine-N,N'-diacetic acid [EDDA]per ml.

Primer extension. To determine the transcription initiation site of the cblB promoter,a synthetic oligonucleotide primer, cbl50, complementary tonucleotides -213 to -232 relative to the cblB start codon, wasused in primer extension reactions [Table 2] . In order to mapthe 5' end of the stable cblA transcript, primer extension analysiswas performed with primer cbl49, complementary to nucleotides+746 to +727, relative to the cblB translational start codon[Table 2] . The cbl49 and cbl50 primers were 5' end-labeled with[-³²P]ATP by using T4 polynucleotide kinase and were hybridizedto 9 or 21 µg, respectively, of total RNA isolated fromB . cenocepacia strain BC7, grown in M9 medium to an OD₆₀₀ of1.0 . Total bacterial RNA was isolated by using Trizol reagent[Invitrogen], according to the manufacturer's instructions.After a 5-min RNA denaturation at 70°C, primers were annealedat 45°C for 30 min, followed by reverse transcription [RT]with Superscript II [Invitrogen] at 37°C for 30 min . Theprimer extension products were precipitated with LiCl, extractedwith phenol:chloroform [1:1], and reprecipitated with ethanol.To precisely determine the 5' ends of transcripts, DNA sequencingreactions were carried out by means of a Thermo Sequenase cyclesequencing kit [Amersham Pharmacia Biotech] with the same 5'end-labeled primers as used in the primer extension reactions.The primer extension products and sequencing ladders were analyzedby denaturing electrophoresis on 6.5% polyacrylamide sequencinggels . After electrophoresis, the gels were dried and exposedto X-ray film [Kodak].

 
Northern hybridization analysis. B . cenocepacia strain BC7 was grown in M9 medium to an OD₆₀₀of 1.0, and total RNA was extracted by Trizol reagent [Invitrogen].Equivalent amounts of strain BC7 RNA were denatured, electrophoresedin formaldehyde agarose gels, and blotted onto Hybond N nitrocellulosemembranes [Amersham Pharmacia Biotech] . An RNA molecular sizemarker [Invitrogen] was also electrophoresed and used as a reference. Membranes were hybridized with DNA probes corresponding to eachof the five genes in the cblBACDS locus . The following DNA fragments were used as probes: a 0.8-kb cblA EcoRI fragment; a 0.6-kb cblB PstI fragment; a 1.4-kb cblC BamHI/PstI fragment; a 0.7-kbcblD HindIII/XhoI fragment; and a 0.9-kb cblS EcoRV fragment.All probes were labeled with [-³²P]dCTP [Amersham PharmaciaBiotech] by the random primer method [32].

RT-PCR. Total bacterial RNA was isolated from B . cenocepacia strainBC7 grown in M9 minimal medium to an OD₆₀₀ of 1.0 by using Trizolreagent . To ensure that the RNA was devoid of contaminatingDNA, the preparation was treated with RNase-free RQ1 DNase [Promega]for 1 h . The isolated RNA was used as a template in RT-PCRs,utilizing the SuperScript One-Step RT-PCR system [Invitrogen],generally according to the manufacturer's instructions . Briefly,the RT reaction was carried out at 45°C for 30 min in a thermal cycler [Hybaid], immediately followed by 40 cycles of PCR, consisting of 1 min at 94°C, 1 min at 52°C, and2 min 20 s at 68°C, ending with a 10-min incubation at 72°C.RT-PCR was performed in 40-µl reaction mixtures, with0.4 to 0.8 µg of total B . cenocepacia RNA and appropriateoligonucleotide primer pairs [see Fig . 7 and Table 2] . For RT-PCR amplification of all transcripts, 0.4 µg of total RNAwas used as a template, with the exception of the cblBA transcript,for which 0.8 µg of RNA was utilized . Reactions in whichthe reverse transcriptase Taq polymerase mix was replaced withTaq polymerase alone were also performed to confirm the absenceof contaminating DNA in the RNA sample . RT-PCR amplificationswere performed at least twice with total RNA preparations obtainedfrom a minimum of two independent extractions . The RT-PCR andPCR products were analyzed by agarose gel electrophoresis.

 
Nucleotide sequence accession number. The DNA sequence of the cbl locus has been deposited in theGenBank database under accession number AY114293.

 
Cloning and sequencing of the B . cenocepacia cbl locus. Our efforts to clone and sequence the B . cenocepacia cbl locus were initiated prior to the recent publication of the sequenceof the cbl locus of strain BC7 by Sajjan et al . [30] . Our sequenceof the cblBACDS locus is 99% identical to that published bySajjan et al . and is 100% identical to the nucleotide sequenceof the cbl locus in the closely related B . cenocepacia strainJ2315, whose genome has recently been sequenced by the SangerCentre [http://www.sanger.ac.uk/Projects/B_cenocepacia].

Transcriptional fusion analysis of the cbl locus. The cable pilus biogenesis locus is comprised of at least fivegenes, designated cblB, cblA, cblC, cblD, and cblS . The tandemarrangement of the cblBACDS genes suggested an operonic structurewith transcription initiating from an A+T-rich region identifiedupstream of cblB [Fig . 1] . In order to delimit the upstreamsequences required for expression of cblB and possibly othercbl genes, a series of transcriptional fusion constructs wasgenerated . DNA fragments were inserted into the multiple cloningsite of vector pRKlac290 to generate transcriptional fusionsto the lacZ reporter gene . Constructs were introduced into B.cenocepacia strain BC7, and ß-galactosidase activitywas measured in either rich [LB] or minimal [M9] medium throughoutgrowth phase . Preliminary studies showed that B . cenocepaciastrain BC7 does not exhibit intrinsic ß-galactosidaseactivity [data not shown] and that introduction of the pRKlac290vector into B . cenocepacia strain BC7 results in only low-levelß-galactosidase activity [Fig . 1].

Transcriptional fusion constructs pMT58 and pMT55, containing approximately 900 and 381 nucleotides upstream of the predicted cblB start codon, respectively, exhibited indistinguishable ß-galactosidase activities in both LB and M9 and inall phases of growth [Fig . 1 and data not shown] . In contrast,a third deletion derivative [pMT95], encompassing 127 nucleotides upstream of the cblB start codon, exhibited ß-galactosidase activities similar to the levels of the pRKlac290 vector controlin both LB and M9 media [Fig . 1] . Together, these results demonstratethat the cis-acting DNA elements required for maximal expressionof cblB are located between nucleotides -381 and -127, relativeto the cblB start codon.

To determine whether additional promoters may be responsiblefor the transcription of genes downstream of cblB, transcriptional fusion constructs encompassing the intergenic regions betweencblB and cblA [pMT59], cblA and cblC [pMT92], cblC and cblD[pMT93], and cblD and cblS [pMT62] were generated [Fig . 1].Only construct pMT93, encompassing the cblC-cblD intergenicregion, exhibited ß-galactosidase activity above thebackground level of the vector control [Fig . 1] . The activityof this transcriptional fusion, however, was less than twofoldhigher than the activity of the vector control and significantlylower than that of the cblB transcriptional fusions pMT58 andpMT55 in both LB and M9 media.

Identification of the cblB promoter transcriptional initiation site. Primer extension analysis was performed in order to precisely determine the transcriptional initiation site of the cblB-proximal promoter [Materials and Methods] . A single primer extension product was consistently obtained, corresponding to a single transcriptional initiation site, located 303 nucleotides upstreamof the predicted cblB translational start codon [Fig . 2] . Totalcellular RNA was also hybridized to a second primer, designated cbl8, complementary to nucleotides -49 to -69 with respect to the cblB translational start site . Analysis of the primer extension product obtained with this primer identified the same transcriptional initiation site that is identified with primer cbl50 [data not shown].

 
Analysis of the DNA region upstream of the cblB transcriptional initiation site revealed the presence of several A+T-rich tracts, as well as partial direct and inverted repeats . The putative -35 [TATATT] and -10 [CAAAAT] promoter regions share only weak homology with known consensus sequences . Four out of six nucleotidesin the -10 region match the conventional ⁷⁰ consensus, whilefour out of seven match the E . coli consensus sequence for thestationary phase-specific factor, RpoS.

Regulation of cbl gene transcription in response to environmental cues. To determine if expression of B . cenocepacia cbl genes is regulatedin response to environmental signals, we utilized the transcriptionalfusion construct pMT55 . We initially examined the ß-galactosidaseactivity throughout growth in rich [LB] or minimal [M9] medium,as these media are known to either repress or induce, respectively,pilus gene expression in E . coli [43] . When B . cenocepacia strainBC7 harboring pMT55 was grown in either rich [LB] or minimal[M9] medium, transcriptional activity increased approximatelytwofold during the mid-late exponential phase, with peak activitiesobserved in stationary phase [Fig. 3] . No differences in growthwere observed between strain BC7 harboring pRKlac290 and BC7harboring pMT55 in either LB or M9 [data not shown] . Overall,the cblB transcriptional fusion pMT55 exhibited four- to fivefoldhigher activity in the minimal medium, suggesting that the growthenvironment and growth phase can significantly influence cblgene expression.

 
Given the known role of pH in regulating pilus gene expressionin E . coli [33, 43], we tested the effect of pH on cbl genetranscription . B . cenocepacia strain BC7 harboring the cblBtranscriptional fusion construct pMT55 was grown in M9 medium,ranging in pH from 5.0 to 8.0 . Due to the inability of B . cenocepaciastrain BC7 to grow in M9 at pH 9.0, the most basic pH in whichß-galactosidase activity was measured was 8.0 . ThepH values of the cultures were monitored during growth and werefound to remain constant throughout the experiment . The highestoverall ß-galactosidase activity was measured at pH 6.0, with an approximately twofold increase compared to levels of activity measured at pH 7.0 [Fig . 4A] . The ß-galactosidase activity was lowest at pH 5.0 and only slightly higher at pH 8.0 . Based on these results, it appears that the optimal pHfor cblB expression is between 6.0 and 7.0.

 
There is evidence that the airway surface liquid [ASL] in theCF lung may have a higher concentration of Na⁺ and Cl^- ions than normal ASL [44] . The high concentration of NaCl in theCF lung could create a hyperosmotic environment that may influenceB . cenocepacia gene expression during the course of infection.The effect of osmolarity on cbl gene expression was examinedin M9 medium with concentrations of NaCl ranging from 0 to 200mM . An increase in osmolarity led to a corresponding increase in ß-galactosidase activity of B . cenocepacia harboring the cblB transcriptional fusion construct pMT55 [Fig . 4B] . Atwo- to threefold increase in activity was measured when M9 medium was supplemented with 200 mM NaCl, compared to medium without NaCl . At a concentration of 100 mM NaCl, the measured ß-galactosidase activity was intermediate, demonstratinga dose-dependent induction of cblB expression in response to increased concentration of NaCl.

Temperature has been shown to play an important role in controlling CFA/I pilus gene expression in ETEC . CFA/I pili are expressed at 37°C, the physiological temperature of the human body,but not at 20°C, suggesting that temperature may be a cuesensed by E . coli to distinguish between the in vivo and exvivo environments [15] . The effect of temperature on B . cenocepaciacbl gene expression was examined by measuring the ß-galactosidaseactivity of B . cenocepacia strain BC7 harboring pMT55 when grownin M9 medium at either 30°C or 37°C . Initially, theß-galactosidase activities were similar at both temperatures[Fig . 4C] . However, the induction of ß-galactosidaseexpression consistently observed at 37°C was absent at 30°C.The highest level of ß-galactosidase activity measuredat 37°C was twofold greater than that achieved at 30°C.These results suggest that transcription from the cblB-proximalpromoter is a temperature-dependent process and that growthat 37°C is required for induction of cbl gene expression.

Iron is both an essential and limiting nutrient in vivo, andiron starvation has been shown to activate expression of a numberof bacterial virulence factors, including the ETEC CFA/I fimbriae[16] . In order to determine if iron availability plays a rolein cbl gene expression, ß-galactosidase activitieswere determined for B . cenocepacia harboring the cblB transcriptionalfusion construct pMT55, grown under both iron-replete and iron-deplete conditions . To examine the effect of increasing iron concentration, M9 medium, normally containing only trace amounts of the metal,was supplemented with 50 µM FeCl₃ . Initially, the measured ß-galactosidase activities of the cblB transcriptionalfusion in both M9 medium and M9 medium supplemented with ironwere indistinguishable [Fig . 5A] . The exponential-phase induction of cblB promoter activity was observed in both M9 medium and M9 medium supplemented with FeCl₃ . However, the induction was delayed by approximately 4 h when the medium was supplemented with FeCl₃ . Furthermore, the ß-galactosidase activitydid not reach the same level in M9 medium supplemented withiron as it did in M9 medium alone, suggesting that iron maylead to repression of the mid-exponential phase induction ofcbl gene expression . To further examine the role of iron incbl gene expression, B . cenocepacia strain BC7 harboring pMT55was grown in LB medium, an iron-rich medium, or LB medium supplementedwith 100 µg of the iron chelator EDDA per ml . The measuredß-galactosidase activities in the presence of EDDAwere approximately twofold higher than activities in LB mediumalone [Fig . 5B], indicating that limiting iron bioavailabilityleads to induction of cbl gene expression . Together, our resultssuggest that iron starvation is a signal that leads to an increasein cbl gene expression.

 
Northern hybridization analysis of cbl gene transcripts. Northern hybridization analysis was performed to further characterize the transcriptional organization of the cblBACDS locus . The tandem arrangement of genes in the cbl locus indicated that the cbl genes are transcribed as an operon . Furthermore, the lack of detectable promoter activity from the intergenic regions downstream of the cblB-proximal promoter, with the exception of weak activity within or adjacent to the cblC-cblD intergenic region, supported the conclusion that the cbl genes are cotranscribedas a polycistronic operon . We therefore expected to detect asingle polycistronic transcript, corresponding to the entire B . cenocepacia cblBACDS locus . Surprisingly, only one transcript of 0.7 kb consistently hybridized to a cblA-derived probe [Fig. 6] . Furthermore, under the same conditions, cblB-, cblC-, cblD-,and cblS-derived probes did not reproducibly hybridize to anytranscripts [Fig . 6], suggesting that mRNAs corresponding tothese genes are of low abundance and/or may be unstable . However,as reported above, the cblB-proximal promoter was successfullymapped by primer extension, indicating that transcripts correspondingto the cblB gene are generated.

 
RT-PCR analysis. To further investigate the transcriptional organization of thecbl locus, we utilized RT-PCR analysis using total RNA extractedfrom B . cenocepacia strain BC7 grown under the same conditionsas for the isolation of RNA used in the primer extension andNorthern hybridization analyses . We initially aimed to use RT-PCRto amplify the stable cblA transcript, identified by Northernhybridization analysis . With primers cbl13 and cbl15, an abundant0.2-kb product was obtained, corresponding to the predictedsize of a portion of the cblA transcript [Fig . 7A].

Additional primer sets were used to determine whether polycistronic transcripts, corresponding to other genes in the locus, could also be amplified by RT-PCR . Portions of transcripts correspondingto cblBA, cblBAC, cblCD, and cblDS were successfully amplified[Fig . 7B through E], confirming the operonic organization ofthe cbl locus . The RT-PCR products obtained from these reactions,however, were significantly less abundant than the 0.2-kb cblA-amplifiedproduct . Repeated attempts to amplify transcripts correspondingto the entire cblBACDS gene cluster or to cblBACD were unsuccessful. This result may be due to low transcript abundance, high G+Ccontent, mRNA secondary structure, transcript size limitation,or any combination thereof.

Lack of promoter activity immediately upstream of the cblA gene. The identification of a single 0.7-kb transcript hybridizing to the cblA-derived probe in Northern hybridization analysis, potentially encompassing all 501 nucleotides of the cblA gene, suggested two possible explanations for its origin: [i] the cblA transcript is initiated from a promoter immediately upstream of the cblA gene, within the cblB coding region, or [ii] a posttranscriptionalevent leads to the processing of a larger transcript, initiatedfrom the cblB-proximal promoter . To examine the former possibility,a DNA fragment encompassing the cblB-cblA intergenic regionwas cloned into vector pRKlac290, generating a transcriptionalfusion to lacZ [pMT59] [Fig . 1] . No significant difference in ß-galactosidase activity was measured between B . cenocepacia harboring pMT59 or the vector control in either LB or M9 medium throughout growth [Fig . 1 and data not shown], suggesting that the cblA gene is not transcribed from an independent promoter located within the cblB coding region.

Mapping the cblA mRNA processing site. A second hypothesis to account for the origin of the stable0.7-kb cblA transcript is that it is generated by the processingof a larger mRNA, initiated at the cblB-proximal promoter . Aregion of dyad symmetry, predicted to form a stem-loop structurein the corresponding transcript, was identified immediatelydownstream of the cblA gene . The stem-loop, followed by sixuracyl residues in the transcript, constitutes a strong Rho-independent transcriptional terminator and indicates the position of the3' end of the 0.7-kb cblA transcript . Furthermore, terminationof transcription at the stem-loop would position the 5' endof the 0.7-kb cblA transcript approximately 200 nucleotidesupstream of the cblA start codon and within the cblB coding region . To investigate this further, primer extension was utilizedto identify the 5' end of the stable 0.7-kb cblA transcript. Total bacterial RNA was isolated from B . cenocepacia strain BC7 and hybridized to the 5' end-labeled primer cbl49, complementary to the region immediately upstream of the cblA gene . Three predominantprimer extension products were identified [Fig. 8], correspondingto nucleotides TAT [UAU in the corresponding mRNA], locatedat positions +952, +953, and +954 relative to the cblB transcriptionalinitiation site . Mapping of the 5' end of the stable cblA transcriptconfirms that the 0.7-kb mRNA originates from within the cblBcoding region and also indicates that the 3' end of the 0.7-kbtranscript is immediately downstream of the cblA translationalstop codon . Together with the transcriptional fusion, Northernhybridization, and RT-PCR analyses, the primer extension resultssuggest that the cblA gene is cotranscribed with cblB on a dicistronic transcript, which may be posttranscriptionally cleaved to yieldan abundant, stable 0.7-kb cblA transcript and an unstable, truncated cblB transcript.

 
In this study we investigated the transcriptional organizationof the cblBACDS locus, encoding components of the B . cenocepacia cable pilus biogenesis pathway . A promoter upstream of the cblB gene was identified and characterized, and the effects of multiple environmental cues on cbl gene expression were investigated. Our studies have also provided evidence for posttranscriptional control of cable pilus gene expression through differentialstability of cbl transcripts . This mechanism may ensure a highlevel of expression, relative to the other components of theassembly pathway, of the major structural subunit of cable pili.

The cblB promoter was found to be four- to fivefold more active in minimal M9 medium than in rich LB medium . Growth in rich media has also been found to repress the transcription of thepap, daa, and fan operons, encoding Pap pili, F1845, and K99 fimbriae in E . coli, respectively [43] . We then began to dissectthe role of individual environmental stimuli in cbl gene expression.The activity of the cblB promoter was sensitive to pH, inducedby acidic conditions [pH 6.0], and repressed in more acidic[pH 5.0] or basic [pH 8.0] environments . Our findings indicatethat the expression of cbl genes is maximal under slightly acidicconditions, with the optimal pH being between 6.0 and 7.0 . Thisrange correlates well with the known pH of the human ASL, whichhas been determined to be 6.78 ± 0.2 [13] . The proposedincreased acidity of the CF ASL [5] may have an additional inducingeffect on cbl gene expression.

Although the ionic content of the CF ASL has been a matter of debate, there is evidence for increased levels of Cl^- ions compared to normal ASL [44] . We found that increasing the NaCl concentrationhad a positive effect on cbl gene expression, with the lowestlevels of expression measured in the NaCl-free M9 medium . Severalstudies have determined the concentrations of both Na⁺ and Cl^- in the ASL to be approximately 100 mM each [13], which is inthe range of the NaCl concentrations tested in this study . Growthtemperature also had a significant effect on cbl gene expression,with up to twofold higher levels at 37°C compared to expressionlevels at 30°C . Our findings suggest that the cable pilus expression may be increased at the physiological temperatureof the human body.

Iron limitation had an inducing effect on cbl gene expression. Iron is a scarce nutrient in the human body, with the majority of the metal sequestered inside host cells or by transport and storage proteins [26] . Sokol and coworkers have demonstrated that secretion of ornibactin, an iron-scavenging siderophore, is essential for virulence of B . cenocepacia in both chronic and acute models of infection [35] . Furthermore, the B . cenocepaciafur gene has recently been identified, encoding a homolog ofthe pleiotropic iron-responsive transcriptional repressor [18].The B . cenocepacia Fur protein may directly or indirectly leadto a partial repression of cbl genes under iron-rich conditions,which is counteracted by derepression in iron-limiting environments.Although the consensus Fur-binding sequence 5'-GATAATGATAATCATTATC-3'[8] was not identified within the cblB promoter region, thereare multiple tracts of A+T nucleotides proximal to the cblB transcriptional initiation site, which Fur may interact withto mediate repression of cbl genes under iron-replete conditions. Our results indicate that acidic pH, high osmolarity, temperatureof 37°C, and iron limitation are all inducing conditionsfor cbl gene expression and may be sensed by B . cenocepaciain the CF lung, resulting in induction of cable pilus expression.

Several of the environmental conditions examined in this studyhad an effect on the growth rate of B . cenocepacia strain BC7. However, there was no direct correlation between growth rateand cbl gene expression . For example, incubation of B . cenocepacia strain BC7 both at 30°C or in the presence of EDDA resultedin a reduced growth rate . However, these conditions had oppositeeffects on cbl gene expression, repressing or inducing expression, respectively [Fig . 4C and Fig . 5B] . These observations indicatethat growth rate per se is not a direct indicator of the levelof cbl gene expression.

In addition to the cblB-proximal promoter characterized in this study, only one other region of the cbl locus, located within or adjacent to the cblC-cblD intergenic region, gave rise to transcriptional activity above levels of the vector control [Fig . 1] . While the measured activity was significantly lower than that of the cblB-proximal promoter, we cannot rule out the possibility that a weak promoter within this region also contributes to the expression of cblD and/or cblS . We also cannot exclude the formal possibility that additional promoters, which have yet to be identified, may be active under growth conditions other than those examined in this study.

By Northern hybridization analysis, we were unable to detect transcripts hybridizing to probes other than cblA . Similar findings have been reported for transcripts corresponding to the genes encoding the ETEC CFA/I usher and minor pilin, homologs of theB . cenocepacia cblC and cblD gene products, respectively [14]. However, using RT-PCR, we were able to amplify transcripts corresponding to portions of the cblBA, cblBAC, cblCD, and cblDS genes, whichalong with the transcriptional fusion studies strongly arguesthat all five genes are expressed and cotranscribed.

Our deletion analysis of the cblB-proximal promoter revealed that 78 base pairs upstream of the cblB-proximal promoter transcriptionalinitiation site are both required and sufficient for full activityin both rich and minimal media [Fig . 1] . This region of DNAmay be responsible for binding transcriptional regulator[s]of cbl gene expression . Downstream of the cblS gene, we haverecently identified an open reading frame predicted to encodea protein with high sequence homology to the DNA-binding responseregulators of bacterial two-component signal transduction systems.The putative response regulator, designated CblR, along with the CblS putative sensor kinase and possibly additional components of the signal transduction pathway, may be involved in the transcriptional control of cbl gene expression.

Analysis of the DNA sequence immediately downstream of the cblA gene identified a region of dyad symmetry, predicted to forma stem-loop structure in the corresponding mRNA, through interactions between nine G+C base pairs [Fig . 9] . The stem-loop structure is followed by a stretch of six uracyl nucleotides in the mRNA, which together may constitute a strong Rho-independent transcriptional terminator . It is likely that transcriptional termination preferentially occurs downstream of the cblA gene, resulting in a cblBA dicistronictranscript . Termination of transcription at the putative stem-loopstructure is consistent with the size of the processed 0.7-kbcblA transcript, whose 5' end was mapped by primer extension.Furthermore, under the same RT-PCR conditions, the molar amountof the amplified cblA transcript was significantly higher thanthat of the cblBAC product [Fig . 7A and C] . Since the 3' endoligonucleotide primer used to amplify the cblBAC product ispositioned downstream of the transcriptional terminator, theamount of the RT-PCR product obtained is reflective of the relativeefficiency of transcription continuing past the stem-loop . Ourresults suggest that termination at the stem-loop structureoccurs in approximately 80% of transcription events . Althoughour RT-PCR analysis was semiquantitative, the significantlyhigher abundance of the amplified cblA transcript compared tothe amount of cblBAC transcript suggests that termination oftranscription at the stem-loop structure is highly efficient.This transcriptional termination mechanism would result in reducedtranscription of the cblCDS genes relative to the cblBA genes.Additionally, stem-loop structures at the 3' ends of mRNAs havebeen shown to stabilize transcripts, protecting them from 3'to 5' exonuclease activities of RNases [36] . Therefore, thestem-loop structure may also act to stabilize the cblA transcript,generated by mRNA processing . A model for transcriptional andposttranscriptional control of cbl gene expression is presentedin Fig . 9.

 
We have mapped the 5' end of the 0.7-kb cblA transcript and have found that it originates from within the cblB coding region. The 5' end of the stable 0.7-kb cblA transcript corresponds to the cblBA mRNA processing site . The pattern of three major products obtained by primer extension suggests imprecise cleavage of cblBA mRNA, which is consistent with the known activity of RNases [1, 14] . RNA processing and differential stability aremechanisms known to control pilus gene expression in other systems[1, 2, 14, 24] . Although we did not consistently detect cblB-hybridizingmRNAs by Northern hybridization analysis, the mapping of thetranscriptional initiation site of the cblB-proximal promoterdemonstrates the presence of cblB transcript[s] . The transcriptmapped by primer extension is either a dicistronic cblBA mRNA,a polycistronic transcript, a truncated cblB RNA, or any combinationthereof . It is clear, however, that the cblB transcript[s] aresignificantly less abundant than the 0.7-kb cblA mRNA, as overtwofold more total B . cenocepacia RNA was used to map the transcriptional initiation site of the cblB-proximal promoter than was usedto map the 5' end of the stable cblA transcript . Moreover, the RT-PCR product corresponding to cblBA mRNA was significantly less abundant than the product corresponding to the processedcblA mRNA . Together, our results strongly suggest that cblBAmRNA processing is a highly efficient event.

Pilus gene expression in E . coli is known to be regulated at the posttranscriptional level through mRNA processing and differential stability . Our study is the first to provide evidence for similar posttranscriptional control of a pilus operon in a nonenteric pathogen . The results presented here suggest that regulationof pilus gene expression on the posttranscriptional level maybe more widespread in bacteria than previously appreciated.Studies are currently under way to identify elements, both cisand trans, controlling cbl gene expression on the transcriptional and posttranscriptional levels.

 
This work was supported by grant MOHR02G0 from the Cystic Fibrosis Foundation.

We thank Sandra Armstrong for critical reading of the manuscript, Victoria Nichols for assistance with generating subclones, andTim Leonard for technical assistance.

 
* Corresponding author . Mailing address: Department of Microbiology, University of Minnesota, Minneapolis, MN 55455-0312 . Phone: [612] 625-7104 . Fax: [612] 626-0623 . E-mail: mohr@lenti.med.umn.edu .

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