The <i>Δrnc</i> was less pathogenic compared with its parent strain.

<p><b>A: Analysis of apoptosis and necrosis of MDBK cell after treatment with the supernatant from different strains.</b> Flow cytometric analysis was prepared to observe the apoptosis and necrosis of MDBK cell after treated with the supernatant of <i>S. aureus</i>. Comparing with its parent stain, the percentage of apoptosis and necrosis induced by the supernatant of <i>Δrnc</i> was significantly decreased. Data were from a representative experiment repeated for three times. (**: p<0.01). <b>B: Analysis of the production of the heat-stable toxins from different stains.</b> The heat-stable toxins were obtained as the described method and incubated with MDBK cell. The survival cell number was determined by MTT method. Comparing with its parent strain, the heat-stable toxins of <i>Δrnc</i> supernatant was decreased. Data were from a representative experiment repeated for three times. (**: p<0.01). <b>C: The detection of the pathogenicity of the different strains with the acute peritonitis animal model.</b> Groups of 10 Balb/c mice were injected intra-abdominally with 500 µl of <i>Δrnc</i> and its parent strain (1×10⁸ CFU). The number of the survival mice was recorded at different time points. The survival rate was calculated. The result showed that the pathogenicity of <i>Δrnc</i> was decreased.</p

Detection of the protein profile from different phases of WT and <i>Δrnc</i>.

<p>Equal number of <i>S. aureus</i> cells was harvested at the indicated time points. The total proteins of whole-cell and supernatant proteins were extracted. The results showed that the supernatant proteins of the <i>Δrnc</i> were decreased significantly compared with WT, while the total proteins of whole-cell did not show the same change as the supernatant proteins. The experiment has been repeated for three times. 1,4,7: WT, wild type, <i>S. aureus</i> 8325-4; 2,5,8: <i>Δrnc</i>; 3,6,9: rncR.</p

The decrease of <i>secY2</i> resulted in the inhibition of extracellular protein secretion in <i>Δrnc</i> at 1.5 h.

<p><b>A: Detection of the mRNA level of </b><b><i>secA1, secY1</i></b><b>,</b><b><i>secA2</i></b><b> and </b><b><i>secY2.</i></b> The mRNA levels of <i>secA1, secY1</i>,<i>secA2</i> and <i>secY2</i> were detected at 1.5 h by qRT-PCR. The results showed that the level of <i>secY2</i> was decreased in <i>Δrnc</i>. (**: P<0.01). Then the decrease of <i>secY2</i> mRNA was confirmed by Northern blot. 16s rRNA was used as the internal control. WT: wild type, <i>S. aureus</i> 8325-4; <i>Δrnc</i>: an RNase III inactivation mutant from 8325-4; rncR: the restoration of RNase III activity in <i>Δrnc</i>. <b>B: Detection of the profile of extracellular proteins and the expression of Efb in the different strains.</b> The pOS1-secY2 plasmid was constructed and transferred to <i>Δrnc</i> to recover the level of SecY2. At the same time, the double mutant <i>Δrnc/secY2</i> was constructed. And then the extracellular proteins were extracted. The results showed that the production of the extracellular proteins was significantly increased at 1.5 h after the recovery of the level of <i>secY2</i>. The mRNA level of <i>secY2</i> was measured by RT-PCR. 16s rRNA was used as the internal control. At the same time, the expression of Efb was determined by Western blot. The result showed that Efb was restored after the level of <i>secY2</i> was recovered in <i>Δrnc</i>. 1: wild type; 2: <i>Δrnc</i>; 3: secY2r(the <i>Δrnc</i> strain transferred with the plasmid pOS1-secY2); 4, <i>Δrnc/secY2;</i> 5. <i>ΔsecY2</i>.</p

RNAIII regulates the levels of extracellular proteins at 6 h and 12 h.

<p><b>A: The expression level of RNAIII was analyzed by Northern blot.</b> The level of RNAIII in different strains at 6 h and 12 h was detected by Northern blot. 16s rRNA was used as the internal control. WT: wild type, <i>S. aureus</i> 8325-4; <i>Δrnc</i>: an RNase III inactivation mutant from 8325-4; rncR: the restoration of RNase III activity in <i>Δrnc</i>. <b>B: Detection of the extracellular proteins of WT and </b><b><i>ΔRNAIII</i></b><b> at the different time points.</b> The extracellular proteins from the equal number of cells were extracted at the indicated time points. The results of SDS-PAGE showed that the extracellular proteins of <i>ΔRNAIII</i> were decreased in comparing with WT at 6 h and 12 h. 1,4,7: wild type; 2,5,8: <i>ΔRNAIII</i> (RNAIII deletion mutant); 3,6,9: ΔRNAIIIR (the restoration of RNAIII in <i>ΔRNAIII</i>). <b>C: Detection of the extracellular proteins from different strains.</b> The pOS1-RNAIII plasmid was constructed to recover the level of RNAIII in <i>Δrnc</i>. At the same time, the double mutant <i>Δrnc/RNAIII</i> was constructed. Then the extracellular proteins were extracted. The results showed that the extracellular proteins were increased at 6 h and 12 h after the level of RNAIII was recovered in <i>Δrnc</i>. The level of RNAIII was measured by RT-PCR. 16s rRNA was used as the internal control. 1,5: WT, wild type; 2,6: <i>Δrnc</i>; 3,7: RNAIIIr(the <i>Δrnc</i> strain transferred with the plasmid pOS1-RNAIII); 4,8, <i>Δrnc/RNAIII</i>. The experiment has been repeated for three times.</p

Sequences of forward and reverse primers used in this study.

<p>Sequences of forward and reverse primers used in this study.</p

Stability of RNAs.

<p>Half-lives of <i>secY2</i> mRNA and RNAIII were determined in the presence of rifampicin (500 µg ml^-1) in the WT and <i>Δrnc</i> strains. Percentage of RNA was calculated normalizing with 5s rRNA.</p

The secretion of the proteins in <i>Δrnc</i> was inhibited at 1.5 h.

<p><b>A: qRT-PCR quantification of the level of </b><b><i>efb</i></b><b> mRNA.</b> The level of <i>efb</i> mRNA in the different strains was detected at 6 h. The results showed that the expression of <i>efb</i> was not regulated by RNAIII. WT: wild type; <i>ΔRNAIII</i>: RNAIII deletion mutant; ΔRNAIIIR: the restoration of RNAIII in <i>ΔRNAIII</i>. <b>B: Detection of the expression of Efb in the extracellular proteins from the different </b><b><i>S. aureus</i></b><b> strains by Western blot at 1.5 h.</b> The extracellular proteins from same number of cells were extracted from different <i>S. aureus</i> strains. The expression of Efb was tested with the specific antibodies of Efb (prepared by ourselves) by Western blot. The result showed that Efb couldn't be detected in the supernatant of <i>Δrnc</i>. 1: WT, wild type, <i>S. aureus</i> 8325-4; 2: <i>Δrnc,</i> an RNase III inactivation mutant from 8325-4; 3: rncR, the restoration of RNase III activity in <i>Δrnc</i>. <b>C: qRT-PCR quantification of the mRNA level of </b><b><i>efb</i></b><b> at 1.5 h</b>. The quantity of <i>efb</i> mRNA from different strains was measured by qRT-PCR at 1.5 h. The result showed that the level of <i>efb</i> mRNA wasn't changed in <i>Δrnc</i>. WT: wild type, <i>S. aureus</i> 8325-4; <i>Δrnc</i>: an RNase III inactivation mutant from 8325-4; rncR: the restoration of RNase III activity in <i>Δrnc</i>. <b>D: The schematic diagram of construction of the reporter vectors.</b> Uefb::lacZ: the promoter and 5′UTR of <i>efb</i> were fused with Lacz; UefbSP: the promoter, 5′UTR and the signal peptides of <i>efb</i> were fused with LacZ. <b>E: Detection of the β-galactosidase activity of different strains.</b> The Uefb::lacZ reporter vector was separately transferred into <i>Δrnc</i> and its parent strains. Then the β-galactosidase activity of different strains was measured at 1.5 h and expressed by miller units. There was no significant difference observed. The results represented a mean of three independent experiments. WT: wild type, <i>S. aureus</i> 8325-4; <i>Δrnc</i>: an RNase III inactivation mutant from 8325-4. <b>F: </b><b>Detection of the β-galactosidase activity of the cultured medium from different strains</b>.The UefbSP::lacZ reporter vector was separately transferred into <i>Δrnc</i> and its parent strains. Then the β-galactosidase activities of the cultured medium were measured and expressed by miller units. Comparing with it parent stain, the β-galactosidase activitiy of the cultured medium from the <i>Δrnc</i> was decreased at 1.5 h. The results represented a mean of three independent experiments. WT: wild type, <i>S. aureus</i> 8325-4; <i>Δrnc</i>: an RNase III inactivation mutant from 8325-4. (**: p<0.01).</p

Bacterial strains and plasmids.

<p>Bacterial strains and plasmids.</p

Detection of RNase III inactive mutant.

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