VapC-1 of nontypeable Haemophilus influenzae is a ribonuclease - PubMed (original) (raw)

VapC-1 of nontypeable Haemophilus influenzae is a ribonuclease

Dayle A Daines et al. J Bacteriol. 2007 Jul.

Abstract

Nontypeable Haemophilus influenzae (NTHi) organisms are obligate parasites of the human upper respiratory tract that can exist as commensals or pathogens. Toxin-antitoxin (TA) loci are highly conserved gene pairs that encode both a toxin and antitoxin moiety. Seven TA gene families have been identified to date, and NTHi carries two alleles of the vapBC family. Here, we have characterized the function of one of the NTHi alleles, vapBC-1. The gene pair is transcribed as an operon in two NTHi clinical isolates, and promoter fusions display an inverse relationship to culture density. The antitoxin VapB-1 forms homomultimers both in vitro and in vivo. The expression of the toxin VapC-1 conferred growth inhibition to an Escherichia coli expression strain and was successfully purified only when cloned in tandem with its cognate antitoxin. Using total RNA isolated from both E. coli and NTHi, we show for the first time that VapC-1 is an RNase that is active on free RNA but does not degrade DNA in vitro. Preincubation of the purified toxin and antitoxin together results in the formation of a protein complex that abrogates the activity of the toxin. We conclude that the NTHi vapBC-1 gene pair functions as a classical TA locus and that the induction of VapC-1 RNase activity leads to growth inhibition via the mechanism of mRNA cleavage.

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Figures

FIG. 1.

FIG. 1.

NTHi strains R2866 and 86-028NP express vapBC-1 as an operon. Reverse transcriptase PCRs with a forward primer in vapB-1 and a reverse primer in vapC-1 electrophoresed on a 0.8% agarose gel. Lanes 1 and 4, 1-kb DNA ladder; lanes 2 and 5, R2866 and 86-028NP with reverse transcriptase (+RT); lanes 3 and 6, R2866 and 86-028NP without reverse transcriptase (−RT).

FIG. 2.

FIG. 2.

Expression of vapBC-1 over the cell cycle. (A) The β-galactosidase activity of the R2866 vapBC-1 promoter fusion (curve) is inversely related to culture density (bars). (B) The 86-028NP vapBC-1 promoter fusion (curve) displays lower initial β-galactosidase activity than the R2866 fusion but mirrors the trend toward decreasing expression with increasing culture density (bars). OD, optical density.

FIG. 3.

FIG. 3.

VapB-1 forms multimers in vitro. (A) Coomassie-stained 12% SDS-PAGE separation of a typical purified pTrc::VapB-1 construct (2 μg) from DH5α. (B) An identical immunoblot probed with anti-Xpress-HRP monoclonal antibody. Note the multiple bands resulting from apparent homo-interactions.

FIG. 4.

FIG. 4.

VapB-1 homodimerizes in vivo. With no protein fused to the LexA DBD, the repressor cannot form a dimer, and transcription of the lacZ reporter gene is constitutive (gray bar). However, when the LexA DBD is fused to VapB-1, competent dimers are formed, and the chimeras can bind to the LexA operator sequence, repressing transcription of the reporter gene (black bar). This level of repression indicates strong VapB-1 homo-interaction.

FIG. 5.

FIG. 5.

VapC-1 causes growth inhibition in vivo. When grown in LB broth without IPTG, the pTrcHisA vector promoter allowed a small amount of transcription of pTrc::VapC-1, which conferred growth inhibition to the DH5α expression strain and made this construct unsuitable for protein isolation. No significant growth effects were observed with the pTrc::VapB-1 fusion or the vector alone. OD, optical density.

FIG. 6.

FIG. 6.

VapC-1 was successfully purified via tandem cloning into pET24b and expression in BL21(DE3). (A) The vapBC-1 operon was fused to pET24b such that vapB-1 was in frame with the vector's ATG start codon at the N terminus, and vapC-1 was in frame with the C-terminal polyhistidine tag, creating pDD686. Induction of the construct with IPTG resulted in no significant growth inhibition of the expression strain. (B) Coomassie-stained 12% SDS-PAGE separation of a typical purified pET::VapC-1 construct (3.5 μg). Two bands are visible: one at the calculated molecular mass of pET::VapC-1 (16.6 kDa) and one at the size of pET::VapB-1 (10.5 kDa). (C) Identical immunoblot probed with anti-His C-terminal HRP-linked monoclonal antibody. Note that only a single band is apparent.

FIG. 7.

FIG. 7.

VapC-1 is an RNase toxin. (A) Total RNA from E. coli K-12 or H. influenzae strain 86-028NP was used as the substrate in RNase activity assays with increasing amounts of the purified VapC-1 toxin. Lanes 1 and 4, MagneHis protein elution buffer control; lanes 2 and 5, 0.35 μg of VapC-1; lanes 3 and 6, 0.7 μg of VapC-1. (B) The Cat protein was cloned into pET24b and purified in the same manner as VapC-1 as a control for any copurifying RNase activity. Lane 1, MagneHis protein elution buffer control; lane 2, 0.35 μg of Cat protein; lane 3, 0.35 μg of VapC-1. Densitometry indicated that the observed RNase activity was specific to VapC-1. Ave, average.

FIG. 8.

FIG. 8.

VapB-1 forms nontoxic complexes with VapC-1 in vitro. The antitoxin VapB-1 was cloned into pET24b as a single gene and purified using the MagneHis native protein purification protocol. Various amounts of purified VapB-1 were incubated with a constant amount of VapC-1 for 30 min prior to the addition of the total RNA substrate in RNase activity assays. A 4:1 ratio of VapB-1 to VapC-1 abrogates the RNase activity of VapC-1. Lanes 1 and 6, MagneHis protein elution buffer control; lanes 2 and 7, 0.2 μg of VapB-1; lanes 3 and 8, 0.4 μg of VapB-1 plus 0.1 μg of VapC-1 (4:1 ratio); lanes 4 and 9, 0.2 μg of VapB-1 plus 0.1 μg of VapC-1 (2:1 ratio); lanes 5 and 10, 0.1 μg of VapC-1 alone. For these assays, H. influenzae strain R2866 total RNA was used. Note the natural 23S fragmentation pattern, which differs from that of strain 86-028NP.

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