Transcriptional and posttranscriptional control of cable pilus gene expression in Burkholderia cenocepacia

Abstract

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 of B. cenocepacia express peritrichous adherence organelles known as cable pili, thought to be important in the colonization of the lower respiratory tract. The genetic locus required for cable 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 degrees C, acidic pH, and low iron bioavailability were found to induce cbl gene expression. Northern hybridization analysis of the cbl locus identified a single, stable transcript corresponding to cblA, encoding the major pilin subunit. Transcriptional fusion studies combined with reverse transcription-PCR analysis indicated that the stable cblA transcript is the product of an mRNA processing event. This event may ensure high levels of expression of the major pilin, relative to other components of the assembly pathway. Our findings lend further insight into the control of cable pilus biogenesis in B. cenocepacia and provide evidence for regulation of cbl gene expression on both the transcriptional and posttranscriptional levels.

FIG. 1.

Physical map of the B. cenocepacia cbl locus and transcriptional fusions generated in this study. The arrows indicate the direction of transcription. DNA fragments used to generate transcriptional fusion constructs to the β-galactosidase reporter gene are shown as black bars under the physical map of the cbl locus. Levels of β-galactosidase activity in B. cenocepacia strain BC7 harboring the various transcriptional fusions are shown to the right. Representative β-galactosidase activities (± standard error) measured in either LB or M9 medium, taken at 10 h, are presented in Miller units. Abbreviations: B, BamHI; H, HindIII; E, EcoRI; P, PstI; X, XhoI. Parentheses indicate the nonendogenous restriction endonuclease sites that were introduced during subcloning.

FIG. 2.

Primer extension analysis of the cblB promoter. (A) Lanes G, A, T, and C denote the corresponding sequencing reactions, and the primer extension product was loaded in lane 1. The arrow indicates the single primer extension product obtained with primer cbl50. (B) Nucleotide sequence of the cblB promoter region. The horizontal bars indicate the location of primers cbl8 and cbl50, used in the primer extension analysis. The transcriptional initiation site corresponding to the primer extension product is designated +1 and shown in bold. The numbers to the left of the sequence indicate the positions of the nucleotides relative to the cblB transcriptional initiation site. Putative −35 and −10 promoter elements, ribosomal binding site, and the deduced amino acid sequence of the N terminus of CblB are also indicated in bold letters.

FIG. 3.

Regulation of cbl gene expression in rich (LB) and minimal (M9) media. B. cenocepacia strains harboring transcriptional fusion constructs were grown in LB (A) or M9 (B) medium. β-Galactosidase measurements were taken at 2-h intervals and are shown in Miller units. The bars indicate the standard errors of the measurements.

FIG. 4.

Analysis of the effects of environmental cues on cbl gene expression. B. cenocepacia strain BC7 harboring the cblB transcriptional fusion construct pMT55 was grown in standard or modified M9 medium, as indicated. β-Galactosidase measurements were taken at 2- or 4-h intervals and are shown in the graphs on the left in Miller units. The bars indicate the standard errors of the measurements. The corresponding growth curves are shown in the accompanying graphs on the right. (A) Analysis of the effect of pH on cbl gene expression. (B) Analysis of the effect of osmolarity on cbl gene expression. (C) Analysis of the effect of temperature on cbl gene expression.

FIG. 5.

Effect of iron bioavailability on cbl gene expression. (A) Analysis of the effect of iron supplementation on cbl gene expression. (B) Analysis of the effect of iron chelation on cbl gene expression. β-Galactosidase activity was monitored at 2-h intervals throughout growth. The bars indicate the standard errors of the measurements.

FIG. 6.

Northern hybridization analysis of cbl gene expression. Total bacterial RNA was extracted from B. cenocepacia strain BC7 grown in M9 minimal medium to an OD₆₀₀ of 1.0. Radiolabeled DNA fragments derived from the cblB, cblA, cblC, cblD, or cblS genes were used as probes in hybridizations, as described in Materials and Methods. The DNA fragments used as probes are shown as gray bars under the physical map of the B. cenocepacia cbl locus. The results of Northern hybridization analyses using probes corresponding to cblB, cblA, cblC, cblD, and cblS are shown below the gray bars. The positions of the bands in the RNA ladder are indicated on the left. The arrow indicates a 0.7-kb transcript hybridizing to the cblA probe. Abbreviations: B, BamHI; H, HindIII; P, PstI; V, EcoRV; X, XhoI.

FIG. 7.

RT-PCR analysis of cbl gene expression. Reactions were performed as described in Materials and Methods. The horizontal arrow (P) upstream of the cblBACDS genes indicates the position of the cblB-proximal promoter. The black bars and arrows below the physical map of the cbl locus indicate the designations and locations of the oligonucleotide primer pairs used and the predicted sizes of RT-PCR products. RT-PCRs corresponding to cblA (A), cblBA (B), cblBAC (C), cblCD (D), and cblDS (E) were analyzed by agarose gel electrophoresis. In each of the five panels A through E, the positions of molecular size markers (kb) are indicated to the left. The presence (+) or absence (−) of the RT enzyme in the amplification reactions is indicated. The arrows indicate the RT-PCR products obtained. X, XhoI.

FIG. 8.

Primer extension analysis of the 5′ end of the cblA mRNA. (A) Lanes G, A, T, and C denote the corresponding sequencing reactions, and the primer extension reaction was loaded in lane 1. The black arrows indicate the three predominant primer extension products obtained with primer cbl49. (B) Nucleotide sequence of the cblB-cblA intergenic region. The numbers to the left of the sequence indicate the positions of the nucleotides relative to the cblB transcriptional initiation site. The horizontal bar indicates the location of primer cbl49, used for the primer extension. The vertical arrows indicate nucleotides +952, +953, and +954, corresponding to the three predominant 5′ end nucleotides of the cblA mRNA. The putative ribosomal binding site and the deduced amino acid sequence of CblB and CblA are indicated in bold letters.

FIG. 9.

A model for transcriptional and posttranscriptional regulation of cbl gene expression. In response to environmental signals, the cbl genes are cotranscribed from the cblB-proximal promoter (P). Transcription is preferentially terminated downstream of cblA by a Rho-independent transcriptional termination mechanism, facilitated by the stem-loop structure (  |○) downstream of cblA. Thus, the stem-loop functions as an attenuator, reducing the expression of cblC, cblD, and cblS. A cblBA dicistronic transcript is processed within the cblB coding region by an as yet unknown mechanism, yielding a truncated cblB mRNA and the stable 0.7-kb cblA transcript. Since the truncated cblB mRNA does not encode a full-length CblB protein, the cblBA mRNA processing event effectively negatively regulates CblB expression. The 0.7-kb cblA mRNA is stabilized by the 3′ end stem-loop structure, leading to high-level expression of the major structural subunit of cable pili, relative to other components of the assembly pathway. In contrast, the truncated cblB mRNA is rapidly degraded. Low-level transcription through the terminator downstream of cblA allows transcription of the cblC, cblD, and cblS genes. A weak promoter within or adjacent to the cblC-cblD intergenic region may also contribute to the expression of cblD and/or cblS. Nucleotides in the cblBA transcript, shown in bold, indicate the mRNA processing site.