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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

SaePQRS regulates biofilm formation by S. aureus strain Newman.(A) Stationary phase cultures of the indicated strains were diluted to an OD660 of 0.05 and inoculated into wells of a microtiter plate. The microtiter plate wells had been precoated with human plasma. After 16 h, biofilms were washed, fixed, and stained with crystal violet. The wells labeled “medium” indicates wells that contained sterile biofilm medium. (B) Inhibition of UAMS-1 biofilm formation by culture supernatants. Stationary phase culture supernatants of strain Newman and its derivatives were harvested, filter sterilized and added to microtiter plate wells preinoculated with S. aureus UAMS-1 suspended in biofilm medium. The source of each culture supernatant is listed to the right of the picture. The wells labeled “medium” indicates sterile TSB-0G medium was added in place of an exogenous culture supernatant. Plates were incubated and processed as described for A.
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pone.0123027.g001: SaePQRS regulates biofilm formation by S. aureus strain Newman.(A) Stationary phase cultures of the indicated strains were diluted to an OD660 of 0.05 and inoculated into wells of a microtiter plate. The microtiter plate wells had been precoated with human plasma. After 16 h, biofilms were washed, fixed, and stained with crystal violet. The wells labeled “medium” indicates wells that contained sterile biofilm medium. (B) Inhibition of UAMS-1 biofilm formation by culture supernatants. Stationary phase culture supernatants of strain Newman and its derivatives were harvested, filter sterilized and added to microtiter plate wells preinoculated with S. aureus UAMS-1 suspended in biofilm medium. The source of each culture supernatant is listed to the right of the picture. The wells labeled “medium” indicates sterile TSB-0G medium was added in place of an exogenous culture supernatant. Plates were incubated and processed as described for A.

Mentions: The capacity of S. aureus Newman to form a biofilm has been addressed by several laboratories. Some have reported that Newman can form a biofilm whereas others, including our laboratory, have concluded that strain Newman is a poor biofilm producer [12,15,26,27]. As stated above, strain Newman constitutively expresses the SaeRS two-component system. To determine if constitutive activation of SaeRS in Newman contributes to the biofilm-deficient phenotype, strains Newman, Newman saeSL, Newman deleted for sae (ΔsaePQRS::kan or ΔsaeRS) and the ΔsaeRS complemented with either saePQRSP or saePQRSL, were assessed in a static microtiter plate biofilm assay. Strikingly, the saeSL, ΔsaePQRS::kan and ΔsaeRS strains formed robust biofilms compared to Newman wild-type (Fig 1A). Introduction of the saePQRSP, but not saePQRSL, bearing plasmid into the ΔsaeRS mutant resulted in restoration of the Newman wild-type, biofilm-deficient phenotype (Fig 1A).


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)

SaePQRS regulates biofilm formation by S. aureus strain Newman.(A) Stationary phase cultures of the indicated strains were diluted to an OD660 of 0.05 and inoculated into wells of a microtiter plate. The microtiter plate wells had been precoated with human plasma. After 16 h, biofilms were washed, fixed, and stained with crystal violet. The wells labeled “medium” indicates wells that contained sterile biofilm medium. (B) Inhibition of UAMS-1 biofilm formation by culture supernatants. Stationary phase culture supernatants of strain Newman and its derivatives were harvested, filter sterilized and added to microtiter plate wells preinoculated with S. aureus UAMS-1 suspended in biofilm medium. The source of each culture supernatant is listed to the right of the picture. The wells labeled “medium” indicates sterile TSB-0G medium was added in place of an exogenous culture supernatant. Plates were incubated and processed as described for A.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123027.g001: SaePQRS regulates biofilm formation by S. aureus strain Newman.(A) Stationary phase cultures of the indicated strains were diluted to an OD660 of 0.05 and inoculated into wells of a microtiter plate. The microtiter plate wells had been precoated with human plasma. After 16 h, biofilms were washed, fixed, and stained with crystal violet. The wells labeled “medium” indicates wells that contained sterile biofilm medium. (B) Inhibition of UAMS-1 biofilm formation by culture supernatants. Stationary phase culture supernatants of strain Newman and its derivatives were harvested, filter sterilized and added to microtiter plate wells preinoculated with S. aureus UAMS-1 suspended in biofilm medium. The source of each culture supernatant is listed to the right of the picture. The wells labeled “medium” indicates sterile TSB-0G medium was added in place of an exogenous culture supernatant. Plates were incubated and processed as described for A.
Mentions: The capacity of S. aureus Newman to form a biofilm has been addressed by several laboratories. Some have reported that Newman can form a biofilm whereas others, including our laboratory, have concluded that strain Newman is a poor biofilm producer [12,15,26,27]. As stated above, strain Newman constitutively expresses the SaeRS two-component system. To determine if constitutive activation of SaeRS in Newman contributes to the biofilm-deficient phenotype, strains Newman, Newman saeSL, Newman deleted for sae (ΔsaePQRS::kan or ΔsaeRS) and the ΔsaeRS complemented with either saePQRSP or saePQRSL, were assessed in a static microtiter plate biofilm assay. Strikingly, the saeSL, ΔsaePQRS::kan and ΔsaeRS strains formed robust biofilms compared to Newman wild-type (Fig 1A). Introduction of the saePQRSP, but not saePQRSL, bearing plasmid into the ΔsaeRS mutant resulted in restoration of the Newman wild-type, biofilm-deficient phenotype (Fig 1A).

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