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

Confirmation of RNA-Seq data using RT-qPCR.RNA was isolated from cultures of strain Newman (saeSP), CYL11481 (saeSL) and CYL11771 (ΔsaeRS) for use in RT-qPCR assays. Values for the saeSL (open bars) and ΔsaeRS (grey bars) strains are expressed relative to wild type Newman, which was assigned an arbitrary value of 1. All assays were performed with at least two RNA preparations obtained from separate cultures.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123027.g006: Confirmation of RNA-Seq data using RT-qPCR.RNA was isolated from cultures of strain Newman (saeSP), CYL11481 (saeSL) and CYL11771 (ΔsaeRS) for use in RT-qPCR assays. Values for the saeSL (open bars) and ΔsaeRS (grey bars) strains are expressed relative to wild type Newman, which was assigned an arbitrary value of 1. All assays were performed with at least two RNA preparations obtained from separate cultures.

Mentions: To confirm the RNA-Seq results, we performed real-time RT-PCR of some select genes. Cultures of strains CYL5876, CYL11481, and CYL11771 were grown as for the RNA-Seq analysis. RNA was isolated from these cultures as described for RNA-Seq analysis except that rRNA depletion was not performed. As we are interested in genes that might influence biofilm formation, we decided to focus on genes predicted to affect the level of extracellular DNA (eDNA). We also verified expression of saeR in the three test strains (saeR in the saeSL strain was 4.2±1.2% of the wild type and undetectable in the saeRS strain). We measured expression of 6 saeRS-regulated genes, lytS, lrgA, atlR, atlA, arlR and aaa, that could potentially affect autolysis and eDNA release by strain Newman. The results shown in Fig 6 are in general agreement with the RNA-Seq results, although differences in gene expression between the strains were generally greater in the RNA-Seq analysis. As anticipated, expression levels of lytS, lrgA, arlR and atlR were highest in Newman and lowest in the deletion strain with expression in the saeSL strain being intermediate. The reverse pattern was observed for atlA and aaa; where the expression levels were greatest in ΔsaeRS≥saeSL>Newman. Most of these genes are probably indirectly regulated by saeRS. For example, lytS and atlR regulate expression of lrgAB and atlA, respectively, but neither lytS nor atlR has a predicted SaeR binding site in their promoter region [34]. The aaa gene does not appear to have a SaeR binding site either and may not be directly regulated by sae. SaeR has been shown to bind to the arlR promoter, suggesting direct regulation of this gene by saeRS. The arlRS 2-component system is involved in the negative regulation of the LytN autolysin [35] but an effect of sae on lytN was not apparent in our analysis.


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)

Confirmation of RNA-Seq data using RT-qPCR.RNA was isolated from cultures of strain Newman (saeSP), CYL11481 (saeSL) and CYL11771 (ΔsaeRS) for use in RT-qPCR assays. Values for the saeSL (open bars) and ΔsaeRS (grey bars) strains are expressed relative to wild type Newman, which was assigned an arbitrary value of 1. All assays were performed with at least two RNA preparations obtained from separate cultures.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123027.g006: Confirmation of RNA-Seq data using RT-qPCR.RNA was isolated from cultures of strain Newman (saeSP), CYL11481 (saeSL) and CYL11771 (ΔsaeRS) for use in RT-qPCR assays. Values for the saeSL (open bars) and ΔsaeRS (grey bars) strains are expressed relative to wild type Newman, which was assigned an arbitrary value of 1. All assays were performed with at least two RNA preparations obtained from separate cultures.
Mentions: To confirm the RNA-Seq results, we performed real-time RT-PCR of some select genes. Cultures of strains CYL5876, CYL11481, and CYL11771 were grown as for the RNA-Seq analysis. RNA was isolated from these cultures as described for RNA-Seq analysis except that rRNA depletion was not performed. As we are interested in genes that might influence biofilm formation, we decided to focus on genes predicted to affect the level of extracellular DNA (eDNA). We also verified expression of saeR in the three test strains (saeR in the saeSL strain was 4.2±1.2% of the wild type and undetectable in the saeRS strain). We measured expression of 6 saeRS-regulated genes, lytS, lrgA, atlR, atlA, arlR and aaa, that could potentially affect autolysis and eDNA release by strain Newman. The results shown in Fig 6 are in general agreement with the RNA-Seq results, although differences in gene expression between the strains were generally greater in the RNA-Seq analysis. As anticipated, expression levels of lytS, lrgA, arlR and atlR were highest in Newman and lowest in the deletion strain with expression in the saeSL strain being intermediate. The reverse pattern was observed for atlA and aaa; where the expression levels were greatest in ΔsaeRS≥saeSL>Newman. Most of these genes are probably indirectly regulated by saeRS. For example, lytS and atlR regulate expression of lrgAB and atlA, respectively, but neither lytS nor atlR has a predicted SaeR binding site in their promoter region [34]. The aaa gene does not appear to have a SaeR binding site either and may not be directly regulated by sae. SaeR has been shown to bind to the arlR promoter, suggesting direct regulation of this gene by saeRS. The arlRS 2-component system is involved in the negative regulation of the LytN autolysin [35] but an effect of sae on lytN was not apparent in our analysis.

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