Microarray-based analysis of the Staphylococcus aureus sigmaB regulon

Abstract

Microarray-based analysis of the transcriptional profiles of the genetically distinct Staphylococcus aureus strains COL, GP268, and Newman indicate that a total of 251 open reading frames (ORFs) are influenced by sigmaB activity. While sigmaB was found to positively control 198 genes by a factor of > or =2 in at least two of the three genetic lineages analyzed, 53 ORFs were repressed in the presence of sigmaB. Gene products that were found to be influenced by sigmaB are putatively involved in all manner of cellular processes, including cell envelope biosynthesis and turnover, intermediary metabolism, and signaling pathways. Most of the genes and/or operons identified as upregulated by sigmaB were preceded by a nucleotide sequence that resembled the sigmaB consensus promoter sequence of Bacillus subtilis. A conspicuous number of virulence-associated genes were identified as regulated by sigmaB activity, with many adhesins upregulated and prominently represented in this group, while transcription of various exoproteins and toxins were repressed. The data presented here suggest that the sigmaB of S. aureus controls a large regulon and is an important modulator of virulence gene expression that is likely to act conversely to RNAIII, the effector molecule of the agr locus. We propose that this alternative transcription factor may be of importance for the invading pathogen to fine-tune its virulence factor production in response to changing host environments.

FIG. 1.

Expression pattern variation of ORFs influenced positively by σ^B. (A) Growth curves of S. aureus Newman (▪) and its mutant IK184 (▴). Time points of sampling are indicated by arrows. (B) Examples of expression pattern types of ORFs found to be influenced positively by σ^B in experiment 2. Transcript levels for Newman (□) and IK184 (▵) cells sampled at different time points of growth (x axis) are shown. Data points were plotted as relative intensity values (y axis).

FIG. 2.

Chromosomal distribution and orientation of ORFs upregulated by σ^B. ORFs and their respective orientations are represented by arrows. The origin of replication (oriC) and the borders of genome fragments 1 to 3 are indicated.

FIG. 3.

The yabJ-spoVG locus of S. aureus. (A) Schematic representation of the yabJ-spoVG operon of S. aureus N315 (GenBank accession no. AP003130). Proposed ORFs and promoter and terminator sequences are indicated. (B) Transcript levels for Newman (□) and IK184 (▵) cells sampled at different time points of growth (x axes). Data points were plotted as relative intensity values (y axes). (C) High-resolution S1 nuclease mapping of the transcriptional start point for yabJ in the E. coli two-plasmid system. The 5′ end-labeled DNA fragment was hybridized with 40 μg of RNA and treated with 100 U of S1 nuclease (as described in Materials and Methods). RNA was isolated from exponentially grown E. coli containing pSA3C and pAC7-sigB (lane1) and pSA3C and pAC7 (lane 2). The RNA-protected DNA fragments were analyzed on DNA sequencing gels together with G+A (lane A) and T+C (lane T) sequencing ladders derived from the end-labeled fragments. The horizontal arrow indicates the position of the RNA-protected fragment, and the vertical arrow indicates the nucleotide corresponding to tsp. Before assigning the tsp, 1.5 nucleotides were subtracted from the length of the protected fragment to account for the difference in the 3′ ends resulting from S1 nuclease digestion and the chemical sequencing reactions. The predicted −35 and −10 boxes are indicated.

FIG. 4.

Transcription profiles of ORFs influenced by σ^B. Transcript levels for Newman (□) and IK184 (▵) cells sampled at different time points of growth (x axes). Data points were plotted as relative intensity values (y axes). (A) Influence of σ^B on known regulatory elements. arlR, autolysis-related locus regulator protein (response regulator); arlS, autolysis-related locus sensor protein (histidine kinase); RNAIII, effector molecule of the agr locus; sarA, staphylococcal accessory regulator A; sarS, staphylococcal accessory regulator S. (B) Adhesions factors upregulated by σ^B. bbp, bone sialoprotein-binding protein; clfA, clumping factor A; ebpS, elastin binding protein S; fnbA, fibronectin binding protein A. (C) Exoproteins and toxins downregulated by σ^B. aur, zinc metalloprotease aureolysin; hla, α-hemolysin; hlgBC, γ-hemolysin components B and C; lip, lipase; lrgAB, holin-like proteins LrgA and LrgB; lukF, synergohymenotropic toxin precursor; lukM, leukocidin chain precursor; nuc, staphylococcal nuclease; plc, 1-phosphatidylinositol phosphodiesterase precursor; sak, staphylokinase precursor (protease III); scp, staphopain; splAB, serine proteases SplA and SplB; sspA, staphylococcal serine protease (V8 protease); sspB, cysteine protease SspB.

FIG. 5.

Transcription profiles of capA and asp23. Transcript levels for Newman (□) and IK184 (▵) cells sampled at different time points of growth (x axes). Data points were plotted as relative intensity values (y axes). The influence of σ^B on the expression of capA, encoding capsular polysaccharide synthesis enzyme A, and asp23, encoding alkaline shock protein 23, is shown.