Genetic characterization of a multicomponent signal transduction system controlling the expression of cable pili in Burkholderia cenocepacia

Abstract

Cable pili are peritrichous organelles expressed by certain strains of Burkholderia cenocepacia, believed to facilitate colonization of the lower respiratory tract in cystic fibrosis patients. The B. cenocepacia cblBACDS operon encodes the structural and accessory proteins required for the assembly of cable pili, as well as a gene designated cblS, predicted to encode a hybrid sensor kinase protein of bacterial two-component signal transduction systems. In this study we report the identification of two additional genes, designated cblT and cblR, predicted to encode a second hybrid sensor kinase and a response regulator, respectively. Analyses of the deduced amino acid sequences of the cblS and cblT gene products revealed that both putative sensor kinases have transmitter and receiver domains and that the cblT gene product has an additional C-terminal HPt domain. Mutagenesis of the cblS, cblT, or cblR gene led to a block in expression of CblA, the major pilin subunit, and a severe decrease in cblA transcript abundance. Using transcriptional fusion analyses, the decrease in the abundance of the cblA transcript in the cblS, cblT, and cblR mutants was shown to be due to a block in transcription from the cblB-proximal promoter, located upstream of the cblBACDS operon. Furthermore, ectopic expression of either cblS or cblR in wild-type B. cenocepacia strain BC7 led to a significant increase, while ectopic expression of cblT resulted in a dramatic decrease, in abundance of the CblA major pilin and the cblA transcript. Our results demonstrate that the B. cenocepacia cblS, cblT, and cblR genes are essential for cable pilus expression and that their effect is exerted at the level of transcription of the cblBACDS operon. These findings are consistent with the proposed function of the cblSTR gene products as a multicomponent signal transduction pathway controlling the expression of cable pilus biosynthetic genes in B. cenocepacia.

FIG. 1.

Physical map of the B. cenocepacia cblBACDSTR locus. The arrows denote the direction of transcription. The deletion in the cblS gene in strain CM543 is indicated with the Δ symbol. The solid gray box denotes the site of the tmp cassette insertion in the cblT gene in strain CM506, and the hatched box denotes the site of insertion of the cat cassette in the cblR gene in strain CM434. The predicted functions of the deduced gene products are indicated below. Abbreviations: B, BamHI; Bg, BglII; E, EcoRI; H, HindIII; X, XhoI.

FIG. 2.

Domain architecture of the predicted cblS, cblT, and cblR gene products. The amino (N) and carboxyl (C) termini are denoted. The conserved His (H) and Asp (D) residues in the transmitter, receiver, and HPt domains are indicated. The locations of the ATP-binding H, N, G1, F, and G2 boxes in the transmitter domains of CblS and CblT are shown in black. Abbreviations: HPt, histidine phosphotransfer domain; HTH, helix-turn-helix domain; PBPb, bacterial periplasmic substrate-binding protein domain; SS, signal sequence.

FIG. 3.

Effects of inactivation of B. cenocepacia cblS, cblT, or cblR on CblA major pilin and cblA transcript abundance. (A) Immunoblot of whole-cell extracts from strain BC7 and the isogenic cblS (CM543), cblT (CM506), and cblR (CM434) mutants and the mutant strains with either pVN3 (carrying cblS), pMT100 (carrying cblT), or pMT66 (carrying cblR), probed with CblA-specific antiserum. An equal amount of protein was loaded in each lane. The arrow indicates the position of the CblA protein band. (B) RNA dot blot of total RNA extracted from strain BC7 and the isogenic cblS (CM543), cblT (CM506), and cblR (CM434) mutants, hybridized with a probe specific for cblA.

FIG. 4.

Effects of the B. cenocepacia cblS, cblT, and cblR mutations on cable pilus expression. Transmission electron micrographs of wild-type strain BC7 (A), the cblS null strain CM543 (B), the cblS null strain transcomplemented with pVN3 (C), the cblT null strain CM506 (D), the cblR null strain CM434 (E), and the cblR null strain transcomplemented with pMT66 (F) are shown. Bars = 0.5 μm.

FIG. 5.

Effects of the B. cenocepacia cblS, cblT, and cblR mutations on activity of the cblB promoter. The β-galactosidase activities of the pRKlac290 vector control in wild-type strain BC7 or the cblB transcriptional fusion construct pMT58 in the wild-type strain BC7 and the cblS, cblT, or cblR mutants, grown in minimal M9 medium, were measured throughout the growth phase at 2-h intervals. (A) β-Galactosidase activity measurements in Miller units on the y axis and time on the x axis. The corresponding growth curves are shown in panel B.

FIG. 6.

Effects of ectopic expression of cblS, cblT, or cblR on CblA major pilin and cblA transcript abundance in wild-type B. cenocepacia strain BC7. (A) Immunoblot of whole-cell extracts from strain BC7 with or without pMR4 (vector control), pVN3 (carrying cblS), pMT100 (carrying cblT), or pMT66 (carrying cblR), probed with the CblA-specific antiserum. Equal amounts of protein were loaded in each lane. The arrow indicates the position of the CblA protein band. (B) RNA dot blot of total RNA extracted from strain BC7 with or without pMR4 (vector control), pVN3 (carrying cblS), pMT100 (carrying cblT), or pMT66 (carrying cblR), hybridized with the cblA-specific probe. (C) Quantification of cblA transcript levels. The levels of the cblA transcript in each strain were normalized to the level of the cblA transcript in wild-type strain BC7, which was arbitrarily set to 100%. The asterisks denote P values of <0.04.

FIG. 7.

Effects of ectopic expression of cblS, cblT, and cblR in the wild-type B. cenocepacia strain BC7 on cable pilus expression. Transmission electron micrographs of wild-type strain BC7 (A) or BC7 with pVN3 (carrying cblS) (B), pMT100 (carrying cblT) (C), or pMT66 (carrying cblR) (D). Bars = 0.5 μm.

FIG. 8.

A working model for the CblSTR signal transduction pathway. (i) Upon receiving a signal from the environment via their periplasmic domains, the CblS and/or CblT hybrid sensor kinases undergo autophosphorylation at the histidine H322 or H344 residues, respectively, catalyzed by hydrolysis of ATP by the transmitter domain. It is also possible that CblS and CblT form homodimers and/or heterodimers, which may lead to cross-phosphorylation. (ii) Phosphotransfer reactions (indicated by arrows and circled P) are carried out between the transmitter, receiver, and HPt domains of CblS and CblT. The transfers of phosphoryl groups may occur intramolecularly and/or intermolecularly. (iii) The aspartate D60 in the receiver domain of the CblR response regulator is phosphorylated through interactions with the CblT HPt domain, leading to activation of CblR and transcription of the cblBACDS operon, possibly by directly binding the cblB promoter. Abbreviations: HPt, histidine phosphotransfer domain; HTH, helix-turn-helix domain; PBPb, bacterial periplasmic substrate-binding protein domain; Cp, cytoplasm; CM, cytoplasmic membrane; Pp, periplasm; OM, outer membrane.