No detectable effect of RNA-binding protein Hfq absence in Staphylococcus aureus

Abstract

Background: The RNA-binding protein Hfq is involved in stress and virulence of several pathogens, probably due to its role as mediator in small RNA (sRNA)-mRNA interactions. In this study, we investigate the function of Hfq in the Gram-positive pathogen Staphylococcus aureus, by constructing hfq null mutant derivatives.

Results: We report that unexpectedly, in S. aureus, Hfq does not seem to play a crucial role in stress response, RNAIII or spa mRNA quantity and exoprotein expression, as tested in three virulent genetic backgrounds. Moreover, a global analysis of the RN6390 hfq mutant, which tests approximately 2000 phenotypes, supports our results concerning the non-implication of Hfq in stress response, and shows that Hfq is also not involved in resistance to several chemical agents and antibiotics and does not seem to be implicated in metabolic pathways.

Conclusion: Our data suggest that although sRNA-mRNA interactions in S. aureus are decisive for gene expression regulation, they do not require the RNA-chaperone protein Hfq. These interactions possibly require an RNA-chaperone protein other than Hfq, which remains to be found.

Figure 1

Inactivation of hfq in S. aureus (1A) and expression of hfq in S. aureus RN6390 (1B). The complete hfq ORF is removed and replaced by the cat gene (1A). Genes directly up-(mia) and downstream (gpxA₁) of hfq are shown, and primers used for RT-PCR experiments are indicated by arrows. Positions of ORFs related to the N315 annotated genome are respectively: mia (13022047-1302982); hfq (1302997-1303230), gpxA₁ (c1303928-1303452). Total RNAs from post-exponential growth phase cultures of RN6390 WT and hfq mutant strains were first retro-transcribed using random hexamer primers. PCR amplification specific to the hfq ORF was realized from the cDNAs, obtained from random retro-transcription (RT) (1B). Chromosomal DNA of WT (WT chrom. DNA) was used as positive control of PCR amplification. PCR amplification of the spa gene was also used as a positive control of cDNA quality from the RN6390 hfq strain. Negative controls of RT-PCR reactions were also shown (neg. ctl).

Figure 2

Northern blot analysis of RNAIII (2A) or spa mRNA (2B) expression in S. aureus WT and Δhfq strains. Total RNA was extracted from post-exponential phase cultures of RN6390, Newman and COL, WT and Δhfq strains. 15 μg of total RNA of RN6390 and Newman strains were analysed by northern blot, using an RNAIII-specific probe (2A). Total RNA from RN6390, Newman and COL strains was also compared for spa mRNA expression, using a spa-specific probe (2B). Results from three independent experiments were identical.

Figure 3

PM comparative analysis of RN6390 WT and hfq strains. The WT and the hfq mutant were subject to PM analysis of ~ 2000 phenotypes. Incubation and time course curves for respiration (tetrazolium color formation) in specific conditions were generated with Omnilog-PM software (see for more details [42]). The PM kinetics shows consensus data comparing the RN6390 hfq mutant (green) to its respective WT strain (red). Red indicates a stronger response by the parental strain and green indicates a stronger response by the hfq mutant. When the two strains have equivalent metabolism or sensitivity to a stress or chemical agent, the red and green kinetic graphs overlap and are yellow. Boxes surrounding a specific condition indicate a significant difference in response. Values of gain or loss of phenotypes are in Table 1 of supplementary data.