Limits...
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

Extracellular (e) DNA in culture supernatants.Culture supernatants from strain Newman and its derivatives were filter sterilized, extracted with phenol-chloroform and ethanol precipitated. Precipitates were suspended in H2O. The volumes of water were varied to compensate for slight differences in OD660 of the initial cultures. (A) Agarose gel of isolated eDNAs. (B) qPCR results. qPCR was performed with isolated eDNAs using oligonucleotide primers specific for the hu gene. Data are expressed relative to the amount of eDNA in the saeSL strain supernatant, which was arbitrarily assigned a value of 1. Data are means from two independent cultures of each strain. Δnuc indicates a nuclease deficient mutant of Newman. TSB indicates material isolated from sterile culture medium (TSB-0G).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4390220&req=5

pone.0123027.g008: Extracellular (e) DNA in culture supernatants.Culture supernatants from strain Newman and its derivatives were filter sterilized, extracted with phenol-chloroform and ethanol precipitated. Precipitates were suspended in H2O. The volumes of water were varied to compensate for slight differences in OD660 of the initial cultures. (A) Agarose gel of isolated eDNAs. (B) qPCR results. qPCR was performed with isolated eDNAs using oligonucleotide primers specific for the hu gene. Data are expressed relative to the amount of eDNA in the saeSL strain supernatant, which was arbitrarily assigned a value of 1. Data are means from two independent cultures of each strain. Δnuc indicates a nuclease deficient mutant of Newman. TSB indicates material isolated from sterile culture medium (TSB-0G).

Mentions: In order to determine if SaeRS does impact eDNA accumulation, we quantified the amount of DNA present in culture supernatants (Fig 8). A mutant (Δnuc) strain that does not express the secreted nuclease was included in our analysis as a positive control for eDNA accumulation. The amount of eDNA was over 10-fold higher in cultures of the ΔsaeRS strain than the saeSL strain and eDNA was essentially undetectable for wild type Newman. These results are consistent with the proposal that saeSP is a negative regulator of autolysis and eDNA production. The fact that eDNA was higher in the ΔsaeRS strain than in the Δnuc strain suggests that factors other than nuclease affect eDNA accumulation.


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)

Extracellular (e) DNA in culture supernatants.Culture supernatants from strain Newman and its derivatives were filter sterilized, extracted with phenol-chloroform and ethanol precipitated. Precipitates were suspended in H2O. The volumes of water were varied to compensate for slight differences in OD660 of the initial cultures. (A) Agarose gel of isolated eDNAs. (B) qPCR results. qPCR was performed with isolated eDNAs using oligonucleotide primers specific for the hu gene. Data are expressed relative to the amount of eDNA in the saeSL strain supernatant, which was arbitrarily assigned a value of 1. Data are means from two independent cultures of each strain. Δnuc indicates a nuclease deficient mutant of Newman. TSB indicates material isolated from sterile culture medium (TSB-0G).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123027.g008: Extracellular (e) DNA in culture supernatants.Culture supernatants from strain Newman and its derivatives were filter sterilized, extracted with phenol-chloroform and ethanol precipitated. Precipitates were suspended in H2O. The volumes of water were varied to compensate for slight differences in OD660 of the initial cultures. (A) Agarose gel of isolated eDNAs. (B) qPCR results. qPCR was performed with isolated eDNAs using oligonucleotide primers specific for the hu gene. Data are expressed relative to the amount of eDNA in the saeSL strain supernatant, which was arbitrarily assigned a value of 1. Data are means from two independent cultures of each strain. Δnuc indicates a nuclease deficient mutant of Newman. TSB indicates material isolated from sterile culture medium (TSB-0G).
Mentions: In order to determine if SaeRS does impact eDNA accumulation, we quantified the amount of DNA present in culture supernatants (Fig 8). A mutant (Δnuc) strain that does not express the secreted nuclease was included in our analysis as a positive control for eDNA accumulation. The amount of eDNA was over 10-fold higher in cultures of the ΔsaeRS strain than the saeSL strain and eDNA was essentially undetectable for wild type Newman. These results are consistent with the proposal that saeSP is a negative regulator of autolysis and eDNA production. The fact that eDNA was higher in the ΔsaeRS strain than in the Δnuc strain suggests that factors other than nuclease affect eDNA accumulation.

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