The second RNA chaperone, Hfq2, is also required for survival under stress and full virulence of Burkholderia cenocepacia J2315

Abstract

Burkholderia cenocepacia J2315 is a highly virulent and epidemic clinical isolate of the B. cepacia complex (Bcc), a group of bacteria that have emerged as important pathogens to cystic fibrosis patients. This bacterium, together with all Bcc strains and a few other prokaryotes, is unusual for encoding in its genome two distinct and functional Hfq-like proteins. In this work, we show results indicating that the 188-amino-acid Hfq2 protein is required for the full virulence and stress resistance of B. cenocepacia J2315, despite the presence on its genome of the functional 79-amino-acid Hfq protein encoded by the hfq gene. Similar to other Hfq proteins, Hfq2 is able to bind RNA. However, Hfq2 is unique in its ability to apparently form trimers in vitro. Maximal transcription of hfq was observed in B. cenocepacia J2315 cells in the early exponential phase of growth. In contrast, hfq2 transcription reached maximal levels in cells in the stationary phase, depending on the CepR quorum-sensing regulator. These results suggest that tight regulation of the expression of these two RNA chaperones is required to maximize the fitness and virulence of this bacterium. In addition, the ability of Hfq2 to bind DNA, not observed for Hfq, suggests that Hfq2 might play additional roles besides acting as an RNA chaperone.

FIG. 1.

Genetic organization of the hfq and hfq2 genes in B. cenocepacia J2315. (A) Genetic organization of the hfq gene locus showing the trimethoprim (Tmp) cassette insertion. (B) Genetic organization of the hfq2 gene locus. Open reading frames BCAL1535 (membrane protein with unknown function), BCAL1536 (σ⁵⁴-dependent transcriptional regulator), BCAL1537 (putatively exported lipoprotein), BCAL1538 (Hfq2 chaperone), BCAL1539 (putative exported protein), BCAL1540 (transmembrane lipoprotein), BCAL1541 (acyl-CoA synthetase), BCAL1542 (TetR family regulatory protein), and BCAL1543 (major facilitator superfamily protein) are represented in scale. The positions of the three nonsense stop signals introduced after nucleotide 211 are indicated by the filled flag (hfq2::Frt). Primers used in RT-PCR experiments are indicated by the letters F (Hfq2c_Fwd) and R (Hfq2c_Rev). The predicted CepR-dependent promoter is also indicated. (C) PCR amplification of the hfq and hfq2 genes from B. cenocepacia wild-type (WT) strain J2315 and mutant strains CJ1 (Δhfq) and CJ2 (Δhfq2).

FIG. 2.

Expression analysis of the hfq and hfq2 genes. (A) RT of hfq and hfq2 RNA samples obtained from cells of B. cenocepacia wild-type (WT) strain J2315 and the CJ1 (Δhfq) and CJ2′ (Δhfq2) mutants. Control experiments were carried out using chromosomal DNA. (B) Northern blot analysis of mRNA corresponding to hfq and hfq2 in B. cenocepacia J2315. Relative abundance was quantified for both hfq (black bars) and hfq2 (white bars) in B. cenocepacia J2315 using the RNA levels of the 5S rRNA as a control. Error bars represent the standard deviations of the means. (C) Detection of hfq and hfq2 mRNA levels in B. cenocepacia wild-type strain H111 and a cepR mutant strain by Northern blotting. Quantification of hfq mRNA in the B. cenocepacia wild-type H111 (black bars) and ΔcepR mutant (white bars) strains and of hfq2 transcripts in the B. cenocepacia wild-type H111 (gray bars) and ΔcepR mutant (dashed bars) strains was performed using the 5S rRNA mRNA levels as a control. All experiments were repeated at least three times.

FIG. 3.

Hfq2 is highly conserved within the Bcc. (A) Alignment of the B. cenocepacia J2315 Hfq and Hfq2 proteins with other Hfq-like proteins showing the predicted secondary structure of Hfq2 above the amino acid residues. The Sm1 and Sm2 motifs are indicated. RNA binding domains are represented by blue boxes. Residues also predicted to be involved in RNA binding are highlighted in yellow. The DNA binding domain is within the gray box. Boxed sequences denote the identified repeats. Identical amino acid residues are marked with an asterisk, and conserved and semiconserved substitutions are marked with double and single dots, respectively. Abbreviations: Bcen, B. cenocepacia; Bviet, B. vietnamensis; Bmul, B. multivorans; Nmening, Neisseria meningitidis; Mcatar, Moraxella catarrhalis. (B) Superimposition of the monomeric predicted three-dimensional structures of Hfq and Hfq2 from B. cenocepacia J2315. The structural alignment is highlighted in yellow, with a root mean square deviation of 0.910 Å.

FIG. 4.

Hfq2 forms trimeric structures in vitro. (A) Far UV CD spectrum of Hfq2 from B. cenocepacia J2315. The regions of the spectrum corresponding to the α-helix and β-sheet are indicated. (B) Discontinuous native PAGE showing a predominant Hfq2 band at ∼63 kDa compatible with a trimeric form of the protein. The monomeric form is also visible. Possible higher-order complexes (marked by an asterisk) can be seen. BSA, bovine serum albumin.

FIG. 5.

B. cenocepacia J2315 Hfq2 is able to bind sRNAs. (A) EMSA experiments using 0, 0.2, 1, and 2 μM purified, His-tagged [Hfq2]₃ and 400 nM FITC-12-UTP 5′-end-labeled mtvR sRNA. (B) With increasing [Hfq]₆, additional Hfq multimers bind to the mtvR sRNA. [Hfq2]₃, trimeric form of Hfq2; [Hfq]₆, hexameric form of Hfq; R-H, complexes formed by [Hfq2]₃ and the mtvR sRNA; R^.H, complexes formed by [Hfq]₆ and the mtvR sRNA (R).

FIG. 6.

B. cenocepacia J2315 Hfq2 is able to bind DNA. The ability of Hfq2 to bind DNA was evaluated by band shift assays. The Pbr transcription regulator (28) was used as a positive control. All binding kinetic parameters were calculated by assuming a fixed-term single-site binding isotherm.

FIG. 7.

Both Hfq and Hfq2 are required for stress resistance. The susceptibility of the indicated B. cenocepacia strains to various stresses was tested by spot inoculating serially diluted bacterial suspensions with an initial OD₆₄₀ of 1.0. wt, wild type.

FIG. 8.

B. cenocepacia J2315 Hfq and Hfq2 are able to complement an E. coli hfq mutation. The ability of the B. cenocepacia hfq or hfq2 gene to complement the hfq mutation in E. coli GS081 was evaluated by subjecting the indicated E. coli strains to the stresses indicated. phfq, E. coli GS081 transformed with pSAS3; phfq2, E. coli GS081 transformed with pCGR9; wt, wild type.

FIG. 9.

Hfq and Hfq2 are both required for full B. cenocepacia J2315 virulence in the nematode C. elegans. (A) Abilities of B. cenocepacia wild-type strain J2315 (wt, black bars), J2315 harboring pSAS3 (wt + phfq, dashed bars) or pCGR9 (wt + phfq2, squared bars), and CJ3 (hfq^sil Δhfq2, dotted bars) to kill the nematode C. elegans DH26. (B) Abilities of B. cenocepacia hfq mutant CJ1 (Δhfq, gray bars) and CJ1 harboring pSA3 (Δhfq + phfq, dashed gray bars) or pCGR9 (Δhfq + phfq2, squared gray bars) to kill the nematode C. elegans DH26. (C) Abilities of the B. cenocepacia hfq2 mutant CJ2 (Δhfq2, white bars) and CJ2 harboring pSAS3 (Δhfq2 + phfq, dashed bars) or pCGR9 (Δhfq + phfq2, squared bars) to kill the nematode C. elegans DH26. (D) Abilities of B. cenocepacia J2315 (wt, black bars), CJ1 (Δhfq, gray bars), CJ2 (Δhfq2, white bars), CJ1 harboring pSA3 (Δhfq + phfq, dashed gray bars), and CJ2 harboring pCGR9 (Δhfq + phfq2, squared bars) to colonize the nematode's digestive tract, expressed as the number of CFU/worm. (E) The health status of worms after infection for 24, 48, or 72 h with the indicated strains was assessed by visual inspection. Worms in panel E were randomly chosen and photographed.