Detection of differential gene expression in biofilm-forming versus planktonic populations of Staphylococcus aureus using micro-representational-difference analysis

Abstract

Microbial proliferation and biofilm formation on biologic or inert substrates are characteristics of invasive Staphylococcus aureus infections and is associated with phenotypic alterations such as reduced antimicrobial susceptibility. To identify genes which are typically expressed in biofilms, a micro-representational-difference analysis (micro-RDA) was adapted for gram-positive bacteria and used with cDNA derived from populations of S. aureus DSM 20231 growing in a biofilm or plankonically. In comparison to previously described cDNA RDA protocols, micro-RDA has the advantages that only minimal quantities of total RNA are needed and, most importantly, that total RNA can be used since the large amount of rRNA in total RNA does not interfere with the micro-RDA procedure. Using a series of spiked controls with various amounts of MS2 RNA in a background of total RNA from S. aureus, the equivalent of five copies of MS2 per cell were detectable after three rounds of subtractive enrichment. Five genes were identified as being differentially expressed in biofilm versus planktonic cultures. These genes revealed homology to a threonyl-tRNA synthetase, a phosphoglycerate mutase, a triosephosphate isomerase, an alcohol dehydrogenase I, and a ClpC ATPase. Differential levels of expression were subsequently confirmed by standard Northern blotting. In conclusion, micro-RDA is a sensitive and specific method to detect transcripts differentially expressed as a function of different S. aureus growth conditions.

FIG. 1

Schematic illustration of the micro-RDA protocol. Broken lines represent the driver population, and solid lines represent the tester fraction. Small solid boxes symbolize the Bgl adapter series. This diagram illustrates cDNA synthesis, amplicon synthesis (A), and the synthesis of DP 1 via subtractive hybridization and selective amplification (B). To generate DP 2 and DP 3, the adapters have to be changed and subtractive hybridization and selective amplification have to be repeated. ss, single stranded; ds, double stranded.

FIG. 2

Micro-RDA sensitivity test. Different amounts, ranging from 1 copy per cell up to 100 copies per cell, of a known RNA species (MS2 RNA) was mixed with total RNA of S. aureus. In the subsequent micro-RDA, this mixture was used as the tester and total S. aureus RNA was used as the driver. DP 3 was electrophoresed through a 2% agarose gel and stained with ethidium bromide. Fragments typical of the MS2 amplicon could be detected in the spike with a ratio of minimally 5 copies per cell. Lane 1, the S. aureus amplicon (MS2 RNA) at 100 copies/cell in RNA (3-h culture); lanes 2 to 6, DP 3 (MS2 RNA spike in RNA [3-h culture]) at 1 copy of MS2/cell and 5, 10, 50, and 100 copies of MS2/cell, respectively; lane 7, the MS 2 amplicon; lanes M, molecular size markers.

FIG. 3

Micro-RDA of sessile versus planktonic S. aureus populations grown in unsupplemented medium. The biofilm-forming and planktonic populations were grown in TSB-glucose medium. The amplicons and DPs were electrophoresed through a 2% agarose gel and stained with ethidium bromide. Lanes 1 to 3, amplicon and DPs of micro-RDA using biofilm-derived RNA as the tester and plankton-derived RNA as the driver; lanes 4 to 6, amplicon and DPs of micro-RDA using plankton-derived RNA as the tester and biofilm-derived RNA as the driver; lane 1, amplicon biofilm; lane 4, amplicon planktonic culture; lanes 2 and 5, DP 1; lanes 3 and 6, DP 2; lane M, molecular size markers.

FIG. 4

Northern blot analysis of S. aureus DSM 20231 RNA derived from biofilm (lanes 1 and 3) and planktonic (lanes 2 and 4) cultures probed with digoxigenin-labeled DPs (A to E) obtained by micro-RDA. Lanes 1 and 2, RNAs derived from cells cultivated in TSB-glucose medium; lanes 3 and 4, RNA derived from cells cultivated in TSB-glucose medium supplemented with 1% threonine. (A) DP 11/10 (fragment of the threonyl-tRNA synthetase); (B) DP P4 (with homology to the phosphoglycerate mutase of B. subtilis); (C) DP P9 (fragment of the triosephosphate isomerase); (D) DP 11/6 (with homology to the alcohol dehydrogenase of Zymomonas mobilis); (E) DP 11/1 (homology to clpC of B. subtilis).

FIG. 5

Micro-RDA of sessile versus planktonic S. aureus populations grown in threonine-supplemented medium. Lanes 1 to 4, amplicon and DPs of micro-RDA using biofilm-derived RNA as the tester and planktonic-culture-derived RNA as the driver. The amplicon and DPs were electrophoresed through a 2% agarose gel and stained with ethidium bromide. Lane 1, biofilm amplicon; lane 2, DP 1; lane 3, DP 2; lane 4, DP 3; lanes M, molecular size markers.