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ABSTRACT: In this study, we characterized the essentiality of enolase for growth of Staphylococcus aureus in vitro by using a TetR-regulated antisense RNA expression technology. The induced enolase antisense RNA dramatically decreased
the production of enolase, which in turn inhibited the growth of S. aureus. In addition, we found that the down-regulation of eno expression can effectively inhibit Triton X-100-induced lysis and alleviate penicillin-caused cell lysis. To further confirm
the specific effect of enolase on autolysis, we constructed an enolase over-expression system and demonstrated that the over-expression
of enolase enhances both Triton X-100 and penicillin-induced cell lysis without increasing cell growth rate. We also performed
hydrolase induced autolysis and zymographic assays and found that enolase had no impact on either bacterial sensitivity to
hydrolase or hydrolase activity. Moreover, we found that the down-regulating expression of enolase selectively increased bacterial
sensitivity to phosphomycin. Taken together, the above results suggest that the enolase is essential for S. aureus and involved in the process of bacterial autolysis.
Keywords
S. aureus
–Enolase (eno)–Autolysis–Antisense
World Journal of Microbiology and Biotechnology 04/2012; 27(4):897-905. · 1.53 Impact Factor
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ABSTRACT: Our previous studies revealed that the staphylococcal protein Gcp is essential for bacterial growth; however, the essential function of Gcp remains undefined. In this study, we demonstrated that Gcp plays an important role in the modulation of the branched-chain amino acids biosynthesis pathway. Specifically, we identified that the depletion of Gcp dramatically elevated the production of key enzymes that are encoded in the ilv-leu operon and responsible for the biosynthesis of the branched-chain amino acids isoleucine, leucine, and valine (ILV) using proteomic approaches. Using qPCR and promoter-lux reporter fusions, we established that Gcp negatively modulates the transcription of the ilv-leu operon. Gel-shift assays revealed that Gcp lacks the capacity to bind the promoter region of ilv. Moreover, we found that the depletion of Gcp did not influence the transcription level of CodY, a known repressor of the ilv-leu operon, while induced the transcription of CcpA, a known positive regulator of the ilv-leu operon. In addition, the depletion of Gcp decreased the biosynthesis of N(6)-threonylcarbamoyladenosine (t6A). To elucidate whether the essentiality of Gcp is attributable to its negative modulation of ILV biosynthesis, we determined the impact of the ilv-leu operon on the requirement of Gcp for growth, and revealed that the deletion of the ilv-leu operon did not affect the essentiality of Gcp. Taken together, our results indicate that the essentiality of Gcp isn't attributable to its negative regulation of ILV biosynthesis in S. aureus. These findings provide new insights into the biological function of the staphylococcal Gcp.
PLoS ONE 01/2012; 7(10):e46836. · 4.09 Impact Factor
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ABSTRACT: Previous studies have demonstrated that the novel protein Gcp is essential for the viability of various bacterial species including Staphylococcus aureus; however, the reason why it is required for bacterial growth remains unclear. In order to explore the potential mechanisms of this essentiality, we performed RT-PCR analysis and revealed that the gcp gene (sa1854) was co-transcribed with sa1855, yeaZ (sa1856) and sa1857 genes, indicating these genes are located in the same operon. Furthermore, we demonstrated that Gcp interacts with YeaZ using a yeast two-hybrid (Y2H) system and in vitro pull down assays. To characterize the Gcp-YeaZ interaction, we performed alanine scanning mutagenesis on the residues of C-terminal segment of Gcp. We found that the mutations of the C-terminal Y317-F322 region abolished the interaction of Gcp and YeaZ, and the mutations of the D324-N329 and S332-Y336 regions alleviated Gcp binding to YeaZ. More importantly, we demonstrated that these key regions of Gcp are also necessary for the bacterial survival since these mutated Gcp could not complement the depletion of endogenous Gcp. Taken together, our data suggest that the interaction of Gcp and YeaZ may contribute to the essentiality of Gcp for S. aureus survival. Our findings provide new insights into the potential mechanisms and biological functions of this novel essential protein.
PLoS ONE 01/2011; 6(5):e20163. · 4.09 Impact Factor
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ABSTRACT: Our previous studies demonstrated that a putative Staphylococcus aureus glycoprotease (Gcp) is essential for bacterial survival, indicating that Gcp may be a novel target for developing antibacterial agents. However, the biological function of Gcp is unclear. In order to elucidate the reason that Gcp is required for growth, we examined the role of Gcp in bacterial autolysis, which is an important biological process for bacterial growth. Using both a spacp-regulated gcp expression strain and a TetR-regulated gcp antisense expression strain, we found that the down-regulation of gcp expression can effectively inhibit Triton X-100-induced lysis, eliminate penicillin- and vancomycin-caused cell lysis, and dramatically increase tolerance to hydrolases. Moreover, we determined whether resistance to lysis is due to a defect in murein hydrolase activity by using a zymogram analysis. The results showed that the cell lysate of a down-regulated gcp expression mutant displayed several bands of decreased murein hydrolytic activity. Furthermore, we explored the potential mechanism of Gcp's involvement in autolysis and demonstrated that Gcp may function independently from several key autolysins (Atl, LytM, and LytN) and regulators (ArlRS, Mgr/Rat, and CidA). Taken together, the above results indicate that the essential Gcp is involved in the modification of substrates of murein hydrolases as well as in the regulation of expression and/or activity of some murein hydrolases, which, in turn, may play important roles in bacterial viability.
Journal of Bacteriology 05/2007; 189(7):2734-42. · 3.83 Impact Factor
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ABSTRACT: The microarray has shown tremendous potential for investigating gene expression profiles and expression levels in comparative biology; exploring the regulation mechanisms of gene expression; and evaluating target gene for developing new chemotherapeutic agents, vaccine, and diagnostic methods. In this chapter, we provide a detailed protocol for scientists who wish to investigate gene expression profiles by performing a microarray analysis, including different methods of RNA purification, decontamination, cDNA synthesis, fragmentation, and biotin labeling for hybridization using Affymetrix Staphylococcus aureus chips.
Methods in molecular biology (Clifton, N.J.) 02/2007; 391:169-78.
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ABSTRACT: We previously demonstrated that regulated antisense RNA technology enables us to validate and identify the mode of action
for some antibiotics. In this study, we have expanded the application of the regulated antisense approach to track the mode
of action for a novel inhibitor of polypeptide deformylase (Pdf), which is an attractive target for the development of novel
classes of antibacterial agents. We created a pdf antisense isogenic strain in Staphylococcus aureus using a TetR-regulated expression system. We demonstrated that the partial inhibition of pdf expression significantly increased the susceptibility of S. aureus to Pdf-specific inhibitor. This result provides further evidence that the TetR-regulated antisense technology is a robust
tool for tracking the mode of action of novel antibacterial agents.
World Journal of Microbiology and Biotechnology 02/2006; 22(3):299-303. · 1.53 Impact Factor
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ABSTRACT: Two-component signal transduction systems play an important role in the ability of bacteria to adapt to various environments by sensing changes in their habitat and by altering gene expression. In this study, we report a novel two-component system, YhcSR, in Staphylococcus aureus which is required for bacterial growth in vitro. We found that the down-regulation of yhcSR expression by induced yhcS antisense RNA can inhibit and terminate bacterial growth. Moreover, without complementary yhcS or yhcR, no viable yhcS or yhcR gene replacement mutant was recoverable. Collectively, these results demonstrated that the YhcSR regulatory system is indispensable for S. aureus growth in culture. Moreover, induced yhcS antisense RNA selectively increased bacterial susceptibility to phosphomycin. These data suggest that YhcSR probably modulates the expression of genes critical for bacterial survival and may be a potential target for the development of novel antibacterial agents.
Journal of Bacteriology 12/2005; 187(22):7876-80. · 3.83 Impact Factor
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ABSTRACT: Staphylococcus aureus expresses various cell wall-associated and extracellular virulence factors, coordinately controlled by different two-component signal transduction systems and transcriptional regulators. In this study, we used microarray technology to identify the genes regulated by ArlR. The microarray data indicate that ArlR functions as a positive regulator and also as a negative repressor to directly and/or indirectly mediate the expression of at least 114 genes involved in different functions, including autolysis, cell division, growth, and pathogenesis.
Journal of Bacteriology 09/2005; 187(15):5486-92. · 3.83 Impact Factor
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ABSTRACT: The emergence of multi-drug resistant bacterial pathogens is generating enormous public health concern, and highlights an urgent need for new, alternative agents for treating multi-drug-resistant pathogens. The gene products essential for bacterial growth in vitro and survival during infection constitute an initial set of protein targets for the development of antibacterial agents. In this study, we employed regulated gene expression approaches and demonstrated that a putative glycoprotease (Gcp) is required for staphylococcal growth in the culture. We found that Staphylococcus aureus becomes more sensitive to the Zn(2+) ion under the downregulation of Gcp expression in vitro. Bioinformatic analyses demonstrated that Gcp is conserved in many Gram-positive pathogens and exists in a variety of Gram-negative pathogens. Our results indicate that Gcp is a potential novel target for the development of antimicrobials against S. aureus infection.
FEMS Microbiology Letters 05/2005; 245(2):279-85. · 2.04 Impact Factor
