Identification of Genes Controlled by the Essential YycFG Two-Component System Reveals a Role for Biofilm Modulation in Staphylococcus epidermidis

Abstract

Biofilms play a crucial role in the pathogenicity of Staphylococcus epidermidis, while little is known about whether the essential YycFG two-component signal transduction system (TCS) is involved in biofilm formation. We used antisense RNA (asRNA) to silence the yycFG TCS in order to study its regulatory functions in S. epidermidis. Strain 1457 expressing asRNA _yycF exhibited a significant delay (~4-5 h) in entry to log phase, which was partially complemented by overexpressing ssaA. The expression of asRNA _yycF and asRNA _yycG resulted in a 68 and 50% decrease in biofilm formation at 6 h, respectively, while they had no significant inhibitory effect on 12 h biofilm formation. The expression of asRNA _yycF led to a ~5-fold increase in polysaccharide intercellular adhesion (PIA) production, but it did not affect the expression of accumulation-associated protein (Aap) or the release of extracellular DNA. Consistently, quantitative real-time PCR showed that silencing yycF resulted in an increased transcription of biofilm-related genes, including icaA, arlR, sarA, sarX, and sbp. An in silico search of the YycF regulon for the conserved YycF recognition pattern and a modified motif in S. epidermidis, along with additional gel shift and DNase I footprinting assays, showed that arlR, sarA, sarX, and icaA are directly regulated by YycF. Our data suggests that YycFG modulates S. epidermidis biofilm formation in an ica-dependent manner.

Keywords: Staphylococcus epidermidis; YycFG; antisense RNA; biofilm; two-component signal transduction system.

Figure 1

Representation of structure of yyc operon and design of asRNA as well as primers. The locations of the primers are indicated at the approximate locations on each gene. P, promoter of yyc operon; R, ribosome binding site.

Figure 2

Detection of asRNA_yycF and its effects on yycF mRNA. SE1457 and its transformants with different plasmids were grown in BM for 6 or 12 h, with or without addition of ATc to 250 ng/ml. Total RNA was extracted and expression levels of asRNA (A) and mRNAs (B) were examined by qRT-PCR.

Figure 3

Effects of asRNA on growth and morphology. (A) Expression of yycF or yycG asRNA on bacterial growth. S. epidermidis 1457 with plasmids were grown in BM medium at 37°C, and growth was monitored every hour by measuring the turbidity of the cultures at OD₆₀₀. (B) Effects of overexpression of ssaA on growth inhibition by asRNA_yycF. The initial inoculation of each strain was 1:1,000 to optimize the effect of the asRNA. Similar results were obtained in three independent experiments. ATc, anhydrotetracycline (added to a final concentration of 250 ng/ml). (C) Transmission electron microscopy of effects from silencing of yycF and YycF target genes. SE 1457 strains were incubated in BM containing 10 μg/ml CM and 250 ng/ml ATc until an OD 600 of 0.6–0.8 was reached. From cells in pMX6, pMXyycF, and pMXssaA (first column on the left), cells with abnormal appearances from pMXyycF and pMXssaA were shown in the right 3 columns, while none was found in pMX6, which is the plasmid control. The white patches inside of some normal cells were probably due to insufficient penetration of EP612 resin into cell walls of gram positive bacteria.

Figure 4

Effects of yycF and asRNA_yycG on biofilm formation. The strains were resuspended in TSB (1:200 dilution) and incubated in 96-well plates for 6 or 12 h. (A) Biofilm formation and (B) growth were measured by detecting the absorbance at 570 and 600 nm, respectively. (C) Primary attachment of the S. epidermidis 1457 strains to polystyrene surfaces. Overnight cultures grown to an OD₆₀₀ of 0.6 were adjusted to an OD₆₀₀ of 0.1 in PBS and inoculated into 6-well plates (2 ml/well). After 2 h at 37°C, the primary attachment at the bottom of the plates was observed under microscopy using a 40-fold objective lens.

Figure 5

Effects of asRNA_yycF on EPS production, Aap expression, and autolysis. (A) Detection of PIA synthesis by S. epidermidis after silencing yycF. Serial dilutions of the PIA extractions detected using spot assays. The data represent one of three independent experiments. (B) Detection of Aap synthesis after silencing yycF. Aap expression was detected using western blotting with MAb25C11 (1 ng/mL). After separation of the proteins using 7% SDS-PAGE, the gel sections carrying high-molecular-weight proteins (>130 kDa) were excised for the western blot assay, and the remaining gel was stained using Coomassie brilliant blue as the endogenous control. (C) Extracellular DNA quantification. Extracellular DNA was isolated from the supernatants of each culture. Q-PCRs of four chromosomal loci were performed for eDNA quantification. (D) Detection of effects of asRNA_yycF on autolysis. Cultures grown to an OD₆₀₀ of 0.6 were re-adjusted to an OD₆₀₀ of 1. Autolysis induced by Triton X-100 at 30°C in the presence of 0.1% Triton X-100. The lysis percentage was calculated as follows: [(OD_t0 − OD_tx /OD_t0) × 100%]. Experiments were performed three times independently.

Figure 6

Effects of asRNA_yycF on the expression of biofilm-related genes. The expression of the genes in Table 2 was detected using qRT-PCR, with gyrB as an internal control. The experiment was carried out in triplicate and the expression ratios of the biofilm-related genes are represented as means with standard deviations.

Figure 7

Binding of r-YycF to the YycF regulon and biofilm-related genes. Electrophoretic mobility shift assay using purified r-YycF with promoter regions of YycF target genes. DNA segments were amplified from the promoters of predicted YycF target genes and biofilm-related genes. For ica, all 164 nt between the coding sequence of icaA and icaR were used. For sarX, the promoter of serp3220, which is located upstream of sarX in the same operon, was amplified. A segment of the yycF coding sequence was used as a negative control.

Figure 8

Identification of the YycF-protected cis-elements in the promoter regions of (A) arlR and (B) ica using DNase I footprinting assays. The regions protected by r-YycF are marked with frames of dashed lines. The DNA sequences of the protected regions are provided, and the sequences that are consistent with the previously reported YycF binding pattern are underlined. The arrows on these lines indicate the direction of the corresponding genes of the promoters.

Figure 9

A graphic depiction of an extended motif for YycF to recognize target genes in S. epidermidis. The conservative property of each base is indicated with heights of each letter.

Figure 10

Schematic representation of the roles of YycFG in PIA-dependent biofilm formation in S. epidermidis. The cytoplasmic cell membrane is represented by a thin border, in which YycG, ArlR, IcaA, IcaD, and icaC are embedded. The cell wall is represented by bold border, in which IcaB and PIA are embedded. The phosphorylation of YycF and ArlR by YycG and ArlS is signified by blue arrows. The genes are shown as brown arrows with an associated promoter (light blue arrows). The gray arrows represent the translation into protein. The induction and repression of transcription are represented by green arrows and red broken lines, respectively, while the question mark indicates that the regulation of transcription is indirect or that it occurs via an unclear mechanism.