Identification of novel small RNAs and characterization of the 6S RNA of Coxiella burnetii

Abstract

Coxiella burnetii, an obligate intracellular bacterial pathogen that causes Q fever, undergoes a biphasic developmental cycle that alternates between a metabolically-active large cell variant (LCV) and a dormant small cell variant (SCV). As such, the bacterium undoubtedly employs complex modes of regulating its lifecycle, metabolism and pathogenesis. Small RNAs (sRNAs) have been shown to play important regulatory roles in controlling metabolism and virulence in several pathogenic bacteria. We hypothesize that sRNAs are involved in regulating growth and development of C. burnetii and its infection of host cells. To address the hypothesis and identify potential sRNAs, we subjected total RNA isolated from Coxiella cultured axenically and in Vero host cells to deep-sequencing. Using this approach, we identified fifteen novel C. burnetii sRNAs (CbSRs). Fourteen CbSRs were validated by Northern blotting. Most CbSRs showed differential expression, with increased levels in LCVs. Eight CbSRs were upregulated (≥2-fold) during intracellular growth as compared to growth in axenic medium. Along with the fifteen sRNAs, we also identified three sRNAs that have been previously described from other bacteria, including RNase P RNA, tmRNA and 6S RNA. The 6S regulatory sRNA of C. burnetii was found to accumulate over log phase-growth with a maximum level attained in the SCV stage. The 6S RNA-encoding gene (ssrS) was mapped to the 5' UTR of ygfA; a highly conserved linkage in eubacteria. The predicted secondary structure of the 6S RNA possesses three highly conserved domains found in 6S RNAs of other eubacteria. We also demonstrate that Coxiella's 6S RNA interacts with RNA polymerase (RNAP) in a specific manner. Finally, transcript levels of 6S RNA were found to be at much higher levels when Coxiella was grown in host cells relative to axenic culture, indicating a potential role in regulating the bacterium's intracellular stress response by interacting with RNAP during transcription.

Figure 1. Linkage maps showing CbSR loci on the C. burnetii chromosome (black line).

Red arrows indicate CbSRs and their relative orientation. Blue, grey and green arrows represent annotated, hypothetical ORFs and pseudogenes, respectively. CbSRs are classified into three groups based on their location relative to adjacent genes: A. Group I: CbSRs encoded within IGRs, B. Group II: CbSRs located antisense to identified ORFs and C. Group III: CbSRs that are ORF-derived.

Figure 2. Northern blot detection of CbSRs.

RNA was isolated from LCVs (3 dpi) and SCVs (21 dpi) grown in ACCM2. Hybridizations were performed at high stringency using biotinylated oligonucleotide probes specific to each CbSR. 3 µg RNA was used for all lanes. Apparent sizes of the CbSRs, as calculated from Northern blots, are indicated. (Note: intensity of bands is not comparable between panels, since exposure times for each panel have not been optimized).

Figure 3. Northern blots showing CbSRs up-regulated (≥2 fold) in host cells relative to ACCM2.

RNA was isolated from SCVs (3 dpi) grown in ACCM2 (A) and in Vero host cells (V). Hybridizations were performed at high stringency using biotinylated oligonucleotide probes specific to each CbSR. 3 µg RNA was used for all lanes. Apparent sizes of the CbSRs, as calculated from the Northern blots, are indicated. (Note: intensity of bands is not comparable between panels, since exposure times for each panel have not been optimized).

Figure 4. C. burnetii total RNA separated on a denaturing gel.

RNA isolated from C. burnetii LCVs (3 dpi) and SCVs (14 dpi) grown in Vero host cells, separated on a denaturing 8 M urea 8% acrylamide gel stained with ethidium bromide (5 µg RNA per lane). Arrow indicates the position of 6S RNA at ∼200 nucleotides. The number of nucleotides in RNA size standards (Std) is indicated to the left.

Figure 5. Linkage map showing the location of C. burnetii’s 6S RNA gene (ssrS).

ssrS is encoded in the 5′, untranslated region (UTR) of ygfA (encoding formyl tetrahydrofolate cyclo-ligase; CBU_0066). The gene immediately upstream (CBU_0067) encodes a hypothetical protein.

Figure 6. Northern blots showing 6S RNA levels of C. burnetii.

RNA was isolated from LCVs (3 dpi) and SCVs (SCV₁₄, 14 dpi; SCV₂₁, 21 dpi) grown in Vero host cells and ACCM2, respectively. Hybridizations were performed at high stringency using a 6S RNA-specific biotinylated oligonucleotide probe. 3 µg RNA was used for all lanes. The size of the signal is indicated to the left.

Figure 7. C. burnetii 6S RNA copies per genome over a 14-d infection period.

A. Number of C. burnetii genomes over a period of 14 d in infected Vero cells, as determined by qPCR with a primer set specific to rpoS. Values on graph represent the means ± standard deviations of the results of 6 independent determinations. B. Average number of copies of C. burnetii 6S RNAs per genome over a 14-d infection of Vero cells. The number of 6S RNA copies was determined by qRT-PCR using primers specific for 6S RNA and 1 µg total RNA from each time point using the same source cultures as panel A. Values represent the means ± standard deviations of the results of 6 independent determinations. Asterisks denote a significant difference relative to the 0-d sample (p<0.05 by student’s t test).

Figure 8. 6S RNA co-immunoprecipitates with C. burnetii RNAP.

A. Immunoblot showing IP reactions of a C. burnetii lysate and corresponding supernatant samples using various antibodies. IPs were performed with no antibody (lanes 2 and 6), rabbit anti-Coxiella Com1 antibody (lanes 3 and 7), pre-immune rabbit serum from the rabbit used to generate anti-RNAP antibodies (lanes 4 and 8) and rabbit anti-RNAP antibody (lanes 5 and 9). The presumed β/β’ and α subunits of RNAP are indicated. Molecular weight values from standards are given to the left in kDa. An asterisk indicates the IgG heavy chain band. B. RPAs performed on IP samples. Specific biotinylated probes were used to detect samples containing 6S RNA and 5S RNA. 43 ng of RNA and 4.3 pg probe were used in each RPA reaction, except IP-anti-Com1, where 22.8 ng RNA was used. Lanes 1 and 3 contain untreated 6S RNA and 5S RNA probes, respectively, while Lanes 2 and 4 contain 6S RNA and 5S RNA probes plus RNase, respectively. The RNase-protected portion of the 6S and 5S RNAs (6S’ and 5S’; respectively) are arrowed to indicate the presence or absence of corresponding signals in lanes 5–13.

Figure 9. Predicted secondary structure of C. burnetii’s 6S RNA as determined by Centroidfold .

The color scale at the bottom represents a heat color gradation from blue to red, corresponding to base-pairing probability from 0 to 1. The free energy of the structure is also shown.

Figure 10. The 4.2 region of E. coli RpoD and comparison to predicted, homologous regions of C. burnetii sigma factors.

E. coli (Ec) and C. burnetii (Cb) 4.2 regions of sigma factors RpoD, RpoS and RpoH are shown. Positively-charged amino acids of the E. coli sigma factor RpoD 4.2 region involved in binding 6S RNA are shown in red. Positively-charged residues in the predicted 4.2 region of C. burnetii sigma factors are shown in green. ClustalW alignment results are shown on the bottom line, where an asterisk indicates perfect identity, a colon indicates similar amino acids with conservation and a period indicates weakly similar amino acids with conservation.