Moraxella catarrhalis expresses an unusual Hfq protein

Abstract

The Hfq protein is recognized as a global regulatory molecule that facilitates certain RNA-RNA interactions in bacteria. BLAST analysis identified a 630-nucleotide open reading frame in the genome of Moraxella catarrhalis ATCC 43617 that was highly conserved among M. catarrhalis strains and which encoded a predicted protein with significant homology to the Hfq protein of Escherichia coli. This protein, containing 210 amino acids, was more than twice as large as the Hfq proteins previously described for other bacteria. The C-terminal half of the M. catarrhalis Hfq protein was very hydrophilic and contained two different types of amino acid repeats. A mutation in the M. catarrhalis hfq gene affected both the growth rate of this organism and its sensitivity to at least two different types of stress in vitro. Provision of the wild-type M. catarrhalis hfq gene in trans eliminated these phenotypic differences in the hfq mutant. This M. catarrhalis hfq mutant exhibited altered expression of some cell envelope proteins relative to the wild-type parent strain and also had a growth advantage in a continuous flow biofilm system. The presence of the wild-type M. catarrhalis hfq gene in trans in an E. coli hfq mutant fully reversed the modest growth deficiency of this E. coli mutant and partially reversed the stress sensitivity of this E. coli mutant to methyl viologen. The use of an electrophoretic mobility shift assay showed that this M. catarrhalis Hfq protein could bind RNA derived from a gene whose expression was altered in the M. catarrhalis hfq mutant.

FIG. 1.

Comparison of the deduced amino acid sequence of the M. catarrhalis Hfq protein with the sequences of Hfq proteins from other bacteria. The deduced amino acid sequence of the M. catarrhalis ATCC 43617 Hfq protein (top sequence) was aligned to the amino acid sequences of seven other bacterial Hfq proteins. The asterisks indicate the amino acids predicted to form the nucleotide-binding pocket in other Hfq proteins (5, 49, 50, 69). This figure was generated by using the CLUSTAL W alignment program in MacVector (version 6.5).

FIG. 2.

Genetic organization of the M. catarrhalis hfq chromosomal locus. (A) Schematic representation of the M. catarrhalis hfq chromosomal locus in M. catarrhalis ATCC 43617, including the two genes flanking the hfq ORF. The relative positions of the different oligonucleotide primers used for PCR and RT-PCR are indicated by the arrows. (B) Photograph of an agarose gel showing the results of a RT-PCR experiment involving the hfq ORF and the two flanking genes. The primer pair used in each experiment is indicated on the top of the gel. Lane 1 contains DNA size markers. Lanes 2 and 5 contain samples from RT-PCRs where DNA was used as the template. Lanes 3 and 6 contain samples from RT-PCRs where RNA was used as the template but no reverse transcriptase was added [(−)RT]. Lanes 4 and 7 contain samples from RT-PCRs where RNA was used as the template and reverse transcriptase was added [(+)RT].

FIG. 3.

Construction of M. catarrhalis hfq mutants. (A and B) Schematic representation of the M. catarrhalis chromosomal locus containing the hfq gene and flanking regions in the hfq mutant O35E.hfq::kan (A) and the hfq deletion mutant O35EΔhfq (B). The relative positions of the different primers used for PCR are indicated by the arrows. (C) Western blot analysis using mouse polyclonal antibody raised against the recombinant M. catarrhalis O35E Hfq protein to probe WCL derived from the wild-type O35E strain (WT, lane 1), the O35EΔhfq mutant (Δhfq, lane 2), the O35EΔhfq mutant containing the pAA200 plasmid with the wild-type O35E hfq gene [Δhfq(pAA200), lane 3], and this same mutant containing only the plasmid vector [Δhfq(pWW115), lane 4].

FIG. 4.

Growth profiles of wild-type, mutant, and complemented mutant strains of M. catarrhalis. Cells of the wild-type strain O35E (⋄), the hfq deletion mutant O35EΔhfq (▪), the complemented hfq deletion mutant O35EΔhfq(pAA200) (▵), and the negative control O35EΔhfq(pWW115) (•) were suspended in BHI broth to an OD₆₀₀ of 1.0 and then diluted 1:20. These suspensions were allowed to grow with aeration at 37°C, and the growth was monitored by measuring the absorbance at 600 nm every hour.

FIG. 5.

Effect of the hfq mutation on the sensitivity of M. catarrhalis to methyl viologen. Bacterial cells of each strain were suspended to the same OD₆₀₀ and then serially diluted and spotted onto BHI agar (A) and BHI agar containing 40 μM methyl viologen (B). Four strains were tested: wild-type O35E strain (WT), the O35EΔhfq mutant (Δhfq), the O35EΔhfq mutant containing the pAA200 plasmid with the wild-type O35E hfq gene [Δhfq(pAA200)], and this same mutant containing only the plasmid vector [Δhfq(pWW115)]. The plates were dried and incubated overnight, and the resultant growth was photographed.

FIG. 6.

Effect of the hfq mutation on the sensitivity of M. catarrhalis to increased salt concentration. Bacterial cells of the same four strains described in Fig. 5 were suspended to the same OD₆₀₀ and then serially diluted and plated onto BHI agar containing 1× NaCl (5 mg/ml; A and C) and onto BHI agar containing 8× NaCl (40 mg/ml; B and D) and incubated for 24 h (A and B) and 48 h (C and D) at 37°C.

FIG. 7.

Effects of the hfq mutation on protein expression by M. catarrhalis. Proteins present in WCL (A) and outer membrane vesicles (OMV) (B) from the wild-type strain O35E and the hfq mutant O35EΔhfq were resolved by SDS-PAGE and stained with Coomassie blue. The positions of some of the bands that were more abundant in the hfq mutant are indicated by the black arrows. Proteins contained in the four selected bands were subjected to N-terminal amino acid sequence analysis. Protein molecular mass position markers (in kilodaltons) are indicated on the left side of each panel.

FIG. 8.

The M. catarrhalis hfq mutant exhibits a growth advantage in a biofilm system. Cells of M. catarrhalis O35E-Sm^r (▪) and O35E.hfq::kan (□) were mixed together (input) and used to inoculate MH broth in a flask (A) and a Sorbarod filter in silicone tubing that was supplied with a continuous flow of MH broth (B). The cultures were allowed to grow overnight at 37°C and then harvested (output), serially diluted, and plated onto agar media containing the appropriate antibiotics to determine the relative percentages of each strain in the mixture. The data presented are the means of three independent experiments.

FIG. 9.

Complementation of an E. coli hfq mutant with the M. catarrhalis Hfq protein. (A) Growth curves of wild-type, mutant, and recombinant E. coli strains. The wild-type strain MC4100 (⋄), the hfq mutant GS081 (▪), the hfq mutant with the M. catarrhalis hfq gene provided in trans [GS081(pAA105)] (▴), and the hfq mutant containing the negative control plasmid [GS081(pAA105B)] (○) were suspended in LB broth to an OD₆₀₀ of 1.0 and then diluted 1:20. These cultures were allowed to grow with aeration at 37°C, and the growth was monitored by measuring the absorbance at 600 nm every hour. (B and C) Cells of the wild-type, mutant, and recombinant E. coli strains were suspended to the same OD₆₀₀ and then serially diluted and spotted onto BHI agar (B) and onto BHI agar containing 40 μM methyl viologen (C). The plates were dried and incubated overnight at 37°C.

FIG. 10.

The M. catarrhalis Hfq protein binds RNA. (A) Real-time RT-PCR analysis expression of the ORF 1068 gene that encodes the LysM motif-containing protein. Total RNA isolated from the wild-type O35E strain (□) and the O35EΔhfq mutant (▪) was used for real-time RT-PCR with oligonucleotide primers specific for the ORF 1068 gene and 16S RNA. The data are presented as the fold increase using the normalized level of the wild-type O35E ORF 1068 mRNA as the calibrator. These data represent the mean of two independent experiments (each performed with samples in triplicate), and the error bars represent the standard deviations. (B and C) EMSA. A radiolabeled RNA probe containing part of the ORF 1068 mRNA was incubated with increasing concentrations of the purified M. catarrhalis Hfq protein in the presence of 100 ng of E. coli tRNA/μl (B) or in the presence of 100 ng of E. coli tRNA/μl and 25 ng of unlabeled RNA probe (C). The binding reactions were then subjected to electrophoresis in a native 6% (wt/vol) polyacrylamide gel. The bands were visualized by exposing the gel to a storage phosphor intensifying screen that was then scanned by using a phosphorimager.