However, the transcriptional response of GBS to changing growth conditions has not been fully analyzed, only single reports were recently published [16]. GBS is an important human and cow pathogen, responsible for thousands of severe invasive infections in man and large economic loss attributable
to bovine mastitis (see [17, 18] and references therein). One of the best examples of sequential gene regulation is bacterial growth in ACY-1215 complex medium and activation of stationary phase genes. During growth, bacteria utilize available nutrients, presumably from simple to more complex, and alter their environment (e.g. decrease or Smoothened Agonist mw increase in pH) as a result of metabolic byproduct release. Therefore, stationary phase can be considered the acid/alkali stress, depending on the type of metabolism and nutrients utilized. GAS grown to stationary phase sequentially expresses genes involved in various aspects of GAS physiology, metabolism and virulence, many genes activated or repressed Selleck U0126 during the transition to stationary phase have also been shown to play a role in GAS virulence [19]. The purpose of the present study was to identify growth phase-regulated
genes in GBS, with a special interest in providing new information about virulence factor gene expression. Methods Sample collection for microarray analysis GBS strain NEM316 [7] was grown as three static cultures (3 biological replicates) in liquid Todd Hewitt medium with 0.5% yeast extract in the 5% CO2 atmosphere at 37°C [12]. Samples were collected at OD600 approximately 0.5, 1.0, 2.0 and 2.5, representing mid-logarithmic (ML), late-logarithmic (LL), early stationary (ES) and stationary (S, about 3 h from entering the phase) growth phases, respectively. Growth curve of bacterial cultures used for data collection is presented as Figure 1. Five ml of each sample were immediately mixed after collection with 10 ml of RNAProtect (Qiagen), centrifuged and stored at -80°C until processing. Figure 1 Growth curve of NEM316 in
THY medium. Arrows denote points of sample collection. Glucose content of the medium at the beginning and end of the culture was measured using Optium Xido glucometer (Abbot) and pH was checked using pH test strips (Macherey Nagel). RNA isolation GBS cells were mechanically opened by shaking with glass beads (Lysing Methocarbamol Matrix B, MPBio) and TRIZOL (Invitrogen). RNA was isolated according to Chomczynski and Sacchi [20], with an additional purification step using RNeasy columns (Qiagen). Targets for hybridization with the array were prepared according to array manufacturer (Affymetrix) as described previously [12]. Array hybridization and data acquisition The custom expression array manufactured by Affymetrix [21] contained redundant sets of probes representing 1,994 open reading frames (ORFs) of previously sequenced GBS strain NEM316 [7]. Arrays were hybridized and scanned according to the manufacturers instructions.
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