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SaeRS-dependent inhibition of biofilm formation in Staphylococcus aureus Newman.

Cue D, Junecko JM, Lei MG, Blevins JS, Smeltzer MS, Lee CY - PLoS ONE (2015)

Bottom Line: The inhibitory protein was found to affect the attachment step in biofilm formation, but had no effect on preformed biofilms.Although a number of candidate proteins were identified, none were found to be the actual inhibitor.RNA-Seq results indicated that sae regulates many genes that may affect biofilm formation by Newman.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR, 72205, United States of America.

ABSTRACT
The SaeRS two-component regulatory system of Staphylococcus aureus is known to affect the expression of many genes. The SaeS protein is the histidine kinase responsible for phosphorylation of the response regulator SaeR. In S. aureus Newman, the sae system is constitutively expressed due to a point mutation in saeS, relative to other S. aureus strains, which results in substitution of proline for leucine at amino acid 18. Strain Newman is unable to form a robust biofilm and we report here that the biofilm-deficient phenotype is due to the saeSP allele. Replacement of the Newman saeSP with saeSL, or deletion of saeRS, resulted in a biofilm-proficient phenotype. Newman culture supernatants were observed to inhibit biofilm formation by other S. aureus strains, but did not affect biofilm formation by S. epidermidis. Culture supernatants of Newman saeSL or Newman ΔsaeRS had no significant effect on biofilm formation. The inhibitory factor was inactivated by incubation with proteinase K, but survived heating, indicating that the inhibitory protein is heat-stable. The inhibitory protein was found to affect the attachment step in biofilm formation, but had no effect on preformed biofilms. Replacement of saeSL with saeSP in the biofilm-proficient S. aureus USA300 FPR3757 resulted in the loss of biofilm formation. Culture supernatants of USA300 FPR3757 saeSP, did not inhibit biofilm formation by other staphylococci, suggesting that the inhibitory factor is produced but not secreted in the mutant strain. A number of biochemical methods were utilized to isolate the inhibitory protein. Although a number of candidate proteins were identified, none were found to be the actual inhibitor. In an effort to reduce the number of potential inhibitory genes, RNA-Seq analyses were done with wild-type strain Newman and the saeSL and ΔsaeRS mutants. RNA-Seq results indicated that sae regulates many genes that may affect biofilm formation by Newman.

No MeSH data available.


Related in: MedlinePlus

Zymograms of S. aureus murein hydrolase activity.Cell free extracts of strains Newman (wt), saeSL (CYL11481) and ΔsaeRS (CYL11771) were run on 10% acrylamide-SDS gels containing heat killed S. aureus RN4220 cells (leftmost panel) or heat killed Micrococcus luteus cells (middle panel). Numbers to the left of each gel indicate the molecular weights of size standards. Dark bands indicate regions of murein hydrolase activity. The large clear band migrating at approximately 60 kDa is presumably the Map protein, which is highly expressed in strain Newman. The rightmost panel is a Coomassie Blue-stained gel showing the same extracts used for the other panels. The large band migrating at approximately 60 kDa is presumably the Map protein.
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pone.0123027.g007: Zymograms of S. aureus murein hydrolase activity.Cell free extracts of strains Newman (wt), saeSL (CYL11481) and ΔsaeRS (CYL11771) were run on 10% acrylamide-SDS gels containing heat killed S. aureus RN4220 cells (leftmost panel) or heat killed Micrococcus luteus cells (middle panel). Numbers to the left of each gel indicate the molecular weights of size standards. Dark bands indicate regions of murein hydrolase activity. The large clear band migrating at approximately 60 kDa is presumably the Map protein, which is highly expressed in strain Newman. The rightmost panel is a Coomassie Blue-stained gel showing the same extracts used for the other panels. The large band migrating at approximately 60 kDa is presumably the Map protein.

Mentions: The transcriptional analysis of murein hydrolase genes summarized above, suggested that the ΔsaeRS and saeSL strains may produce higher levels of these enzymes than does wild type Newman. To test this possibility, zymographic analyses of the three strains were performed (Fig 7). Extracts of these strains were prepared and fractionated on gels containing either heat-killed S. aureus strain RN4220 cells or cells of Micrococcus luteus. As shown in Fig 7, increased murein hydrolase activity, relative to Newman, was associated with both the saeSL and ΔsaeRS strains. The differences were most prominent for protein bands of approximately 60 and 90 KDa, and somewhat less prominent for a protein migrating at approximately 30 kDa. Additionally, the ΔsaeRS strain was deficient in expression of 2 small hydrolases that migrated at the 17 to 20 kDa range.


SaeRS-dependent inhibition of biofilm formation in Staphylococcus aureus Newman.

Cue D, Junecko JM, Lei MG, Blevins JS, Smeltzer MS, Lee CY - PLoS ONE (2015)

Zymograms of S. aureus murein hydrolase activity.Cell free extracts of strains Newman (wt), saeSL (CYL11481) and ΔsaeRS (CYL11771) were run on 10% acrylamide-SDS gels containing heat killed S. aureus RN4220 cells (leftmost panel) or heat killed Micrococcus luteus cells (middle panel). Numbers to the left of each gel indicate the molecular weights of size standards. Dark bands indicate regions of murein hydrolase activity. The large clear band migrating at approximately 60 kDa is presumably the Map protein, which is highly expressed in strain Newman. The rightmost panel is a Coomassie Blue-stained gel showing the same extracts used for the other panels. The large band migrating at approximately 60 kDa is presumably the Map protein.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390220&req=5

pone.0123027.g007: Zymograms of S. aureus murein hydrolase activity.Cell free extracts of strains Newman (wt), saeSL (CYL11481) and ΔsaeRS (CYL11771) were run on 10% acrylamide-SDS gels containing heat killed S. aureus RN4220 cells (leftmost panel) or heat killed Micrococcus luteus cells (middle panel). Numbers to the left of each gel indicate the molecular weights of size standards. Dark bands indicate regions of murein hydrolase activity. The large clear band migrating at approximately 60 kDa is presumably the Map protein, which is highly expressed in strain Newman. The rightmost panel is a Coomassie Blue-stained gel showing the same extracts used for the other panels. The large band migrating at approximately 60 kDa is presumably the Map protein.
Mentions: The transcriptional analysis of murein hydrolase genes summarized above, suggested that the ΔsaeRS and saeSL strains may produce higher levels of these enzymes than does wild type Newman. To test this possibility, zymographic analyses of the three strains were performed (Fig 7). Extracts of these strains were prepared and fractionated on gels containing either heat-killed S. aureus strain RN4220 cells or cells of Micrococcus luteus. As shown in Fig 7, increased murein hydrolase activity, relative to Newman, was associated with both the saeSL and ΔsaeRS strains. The differences were most prominent for protein bands of approximately 60 and 90 KDa, and somewhat less prominent for a protein migrating at approximately 30 kDa. Additionally, the ΔsaeRS strain was deficient in expression of 2 small hydrolases that migrated at the 17 to 20 kDa range.

Bottom Line: The inhibitory protein was found to affect the attachment step in biofilm formation, but had no effect on preformed biofilms.Although a number of candidate proteins were identified, none were found to be the actual inhibitor.RNA-Seq results indicated that sae regulates many genes that may affect biofilm formation by Newman.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR, 72205, United States of America.

ABSTRACT
The SaeRS two-component regulatory system of Staphylococcus aureus is known to affect the expression of many genes. The SaeS protein is the histidine kinase responsible for phosphorylation of the response regulator SaeR. In S. aureus Newman, the sae system is constitutively expressed due to a point mutation in saeS, relative to other S. aureus strains, which results in substitution of proline for leucine at amino acid 18. Strain Newman is unable to form a robust biofilm and we report here that the biofilm-deficient phenotype is due to the saeSP allele. Replacement of the Newman saeSP with saeSL, or deletion of saeRS, resulted in a biofilm-proficient phenotype. Newman culture supernatants were observed to inhibit biofilm formation by other S. aureus strains, but did not affect biofilm formation by S. epidermidis. Culture supernatants of Newman saeSL or Newman ΔsaeRS had no significant effect on biofilm formation. The inhibitory factor was inactivated by incubation with proteinase K, but survived heating, indicating that the inhibitory protein is heat-stable. The inhibitory protein was found to affect the attachment step in biofilm formation, but had no effect on preformed biofilms. Replacement of saeSL with saeSP in the biofilm-proficient S. aureus USA300 FPR3757 resulted in the loss of biofilm formation. Culture supernatants of USA300 FPR3757 saeSP, did not inhibit biofilm formation by other staphylococci, suggesting that the inhibitory factor is produced but not secreted in the mutant strain. A number of biochemical methods were utilized to isolate the inhibitory protein. Although a number of candidate proteins were identified, none were found to be the actual inhibitor. In an effort to reduce the number of potential inhibitory genes, RNA-Seq analyses were done with wild-type strain Newman and the saeSL and ΔsaeRS mutants. RNA-Seq results indicated that sae regulates many genes that may affect biofilm formation by Newman.

No MeSH data available.


Related in: MedlinePlus