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The production of extracellular proteins is regulated by ribonuclease III via two different pathways in Staphylococcus aureus.

Liu Y, Dong J, Wu N, Gao Y, Zhang X, Mu C, Shao N, Fan M, Yang G - PLoS ONE (2011)

Bottom Line: It was found that the extracellular proteins of Δrnc were decreased.We found during the lag phase of the bacterial growth cycle RNase III could influence the extracellular protein secretion via regulating the expression of secY2, one component of accessory secretory (sec) pathway.Our results suggest that RNase III could regulate the pathogenicity of S. aureus by influencing the level of extracellular proteins via two different ways respectively at different growth phases.

View Article: PubMed Central - PubMed

Affiliation: Beijing Institute of Basic Medical Sciences, Beijing, People's Republic of China.

ABSTRACT
Staphylococcus aureus ribonuclease III belongs to the enzyme family known to degrade double-stranded RNAs. It has previously been reported that RNase III cannot influence cell growth but regulates virulence gene expression in S. aureus. Here we constructed an RNase III inactivation mutant (Δrnc) from S. aureus 8325-4. It was found that the extracellular proteins of Δrnc were decreased. Furthermore, we explored how RNase III regulated the production of the extracellular proteins in S. aureus. We found during the lag phase of the bacterial growth cycle RNase III could influence the extracellular protein secretion via regulating the expression of secY2, one component of accessory secretory (sec) pathway. After S. aureus cells grew to exponential phase, RNase III can regulate the expression of extracellular proteins by affecting the level of RNAIII. Further investigation showed that the mRNA stability of secY2 and RNAIII was affected by RNase III. Our results suggest that RNase III could regulate the pathogenicity of S. aureus by influencing the level of extracellular proteins via two different ways respectively at different growth phases.

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Detection of RNase III inactive mutant.A: Verification of RNase III inactive mutant by RT-PCR. Total RNA of cells was extract and used as the template to amplify the rnc gene. In Δrnc strain, the rnc mRNA could not be detected like WT and rncR because the kana gene was inserted into the rnc gene of Δrnc genome. 16s rRNA was used as the internal control. WT (wild type, S. aureus 8325-4), Δrnc(an RNase III inactivation mutant from 8325-4) and rncR (the restoration of RNase III activity in Δrnc). B: The growth curves of S. aureus strains. There is no significant difference between WT and Δrnc. WT: wild type, S. aureus 8325-4; Δrnc: an RNase III inactivation mutant from 8325-4. The experiment has been repeated for three times. C: qRT-PCR quantification of the expression level of spa. The total RNA of the cells cultured for 6 h was extracted and the mRNA level of spa was detected by qRT-PCR. In the Δrnc strain, the level of spa mRNA was significantly increased compared with WT. WT: wild type, S. aureus 8325-4; Δrnc: an RNase III inactivation mutant from 8325-4; rncR: the restoration of RNase III activity in Δrnc. (**: P<0.01).
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pone-0020554-g001: Detection of RNase III inactive mutant.A: Verification of RNase III inactive mutant by RT-PCR. Total RNA of cells was extract and used as the template to amplify the rnc gene. In Δrnc strain, the rnc mRNA could not be detected like WT and rncR because the kana gene was inserted into the rnc gene of Δrnc genome. 16s rRNA was used as the internal control. WT (wild type, S. aureus 8325-4), Δrnc(an RNase III inactivation mutant from 8325-4) and rncR (the restoration of RNase III activity in Δrnc). B: The growth curves of S. aureus strains. There is no significant difference between WT and Δrnc. WT: wild type, S. aureus 8325-4; Δrnc: an RNase III inactivation mutant from 8325-4. The experiment has been repeated for three times. C: qRT-PCR quantification of the expression level of spa. The total RNA of the cells cultured for 6 h was extracted and the mRNA level of spa was detected by qRT-PCR. In the Δrnc strain, the level of spa mRNA was significantly increased compared with WT. WT: wild type, S. aureus 8325-4; Δrnc: an RNase III inactivation mutant from 8325-4; rncR: the restoration of RNase III activity in Δrnc. (**: P<0.01).

Mentions: We constructed an RNase III inactivation strain (Δrnc) in 8325-4 with allelic homologous recombination. Then the mutant was verified by RT-PCR (figure 1A) using the primers (rncV1 and rncV2) as listed in the Table 1. To further observe the phenotype of Δrnc, the growth curves of Δrnc and its parent strain were measured. The result showed that there were no obvious differences observed between the wild type and mutation strains (figure 1B). In previous reports, RNase III could degrade the target mRNAs (spa) of RNAIII [7], so we tested the mRNA level of spa by real-time quantitative PCR. Compared with its parent strain, the level of spa significantly increased in Δrnc (figure 1C).


The production of extracellular proteins is regulated by ribonuclease III via two different pathways in Staphylococcus aureus.

Liu Y, Dong J, Wu N, Gao Y, Zhang X, Mu C, Shao N, Fan M, Yang G - PLoS ONE (2011)

Detection of RNase III inactive mutant.A: Verification of RNase III inactive mutant by RT-PCR. Total RNA of cells was extract and used as the template to amplify the rnc gene. In Δrnc strain, the rnc mRNA could not be detected like WT and rncR because the kana gene was inserted into the rnc gene of Δrnc genome. 16s rRNA was used as the internal control. WT (wild type, S. aureus 8325-4), Δrnc(an RNase III inactivation mutant from 8325-4) and rncR (the restoration of RNase III activity in Δrnc). B: The growth curves of S. aureus strains. There is no significant difference between WT and Δrnc. WT: wild type, S. aureus 8325-4; Δrnc: an RNase III inactivation mutant from 8325-4. The experiment has been repeated for three times. C: qRT-PCR quantification of the expression level of spa. The total RNA of the cells cultured for 6 h was extracted and the mRNA level of spa was detected by qRT-PCR. In the Δrnc strain, the level of spa mRNA was significantly increased compared with WT. WT: wild type, S. aureus 8325-4; Δrnc: an RNase III inactivation mutant from 8325-4; rncR: the restoration of RNase III activity in Δrnc. (**: P<0.01).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105085&req=5

pone-0020554-g001: Detection of RNase III inactive mutant.A: Verification of RNase III inactive mutant by RT-PCR. Total RNA of cells was extract and used as the template to amplify the rnc gene. In Δrnc strain, the rnc mRNA could not be detected like WT and rncR because the kana gene was inserted into the rnc gene of Δrnc genome. 16s rRNA was used as the internal control. WT (wild type, S. aureus 8325-4), Δrnc(an RNase III inactivation mutant from 8325-4) and rncR (the restoration of RNase III activity in Δrnc). B: The growth curves of S. aureus strains. There is no significant difference between WT and Δrnc. WT: wild type, S. aureus 8325-4; Δrnc: an RNase III inactivation mutant from 8325-4. The experiment has been repeated for three times. C: qRT-PCR quantification of the expression level of spa. The total RNA of the cells cultured for 6 h was extracted and the mRNA level of spa was detected by qRT-PCR. In the Δrnc strain, the level of spa mRNA was significantly increased compared with WT. WT: wild type, S. aureus 8325-4; Δrnc: an RNase III inactivation mutant from 8325-4; rncR: the restoration of RNase III activity in Δrnc. (**: P<0.01).
Mentions: We constructed an RNase III inactivation strain (Δrnc) in 8325-4 with allelic homologous recombination. Then the mutant was verified by RT-PCR (figure 1A) using the primers (rncV1 and rncV2) as listed in the Table 1. To further observe the phenotype of Δrnc, the growth curves of Δrnc and its parent strain were measured. The result showed that there were no obvious differences observed between the wild type and mutation strains (figure 1B). In previous reports, RNase III could degrade the target mRNAs (spa) of RNAIII [7], so we tested the mRNA level of spa by real-time quantitative PCR. Compared with its parent strain, the level of spa significantly increased in Δrnc (figure 1C).

Bottom Line: It was found that the extracellular proteins of Δrnc were decreased.We found during the lag phase of the bacterial growth cycle RNase III could influence the extracellular protein secretion via regulating the expression of secY2, one component of accessory secretory (sec) pathway.Our results suggest that RNase III could regulate the pathogenicity of S. aureus by influencing the level of extracellular proteins via two different ways respectively at different growth phases.

View Article: PubMed Central - PubMed

Affiliation: Beijing Institute of Basic Medical Sciences, Beijing, People's Republic of China.

ABSTRACT
Staphylococcus aureus ribonuclease III belongs to the enzyme family known to degrade double-stranded RNAs. It has previously been reported that RNase III cannot influence cell growth but regulates virulence gene expression in S. aureus. Here we constructed an RNase III inactivation mutant (Δrnc) from S. aureus 8325-4. It was found that the extracellular proteins of Δrnc were decreased. Furthermore, we explored how RNase III regulated the production of the extracellular proteins in S. aureus. We found during the lag phase of the bacterial growth cycle RNase III could influence the extracellular protein secretion via regulating the expression of secY2, one component of accessory secretory (sec) pathway. After S. aureus cells grew to exponential phase, RNase III can regulate the expression of extracellular proteins by affecting the level of RNAIII. Further investigation showed that the mRNA stability of secY2 and RNAIII was affected by RNase III. Our results suggest that RNase III could regulate the pathogenicity of S. aureus by influencing the level of extracellular proteins via two different ways respectively at different growth phases.

Show MeSH
Related in: MedlinePlus