Growth-dependent activity of the cold shock cspA promoter + 5′ UTR and production of the protein CspA in Staphylococcus aureus Newman

Chandana K. Uppalapati, Kimberley D. Gutierrez, Gina Buss-Valley, Sam Katzif

Research involving the cold shock gene cspA of the medically important bacterium Staphylococcus aureus is steadily increasing as the relationships between the activity of this gene at 37 °C and a spectrum of virulence factors (e.g., biofilm formation, capsule production) as well as stress-related genes (e.g., alkaline shock protein, asp-23 and the alternative sigma factor, sigB) are distinguished. Fundamental to each of these discoveries is defining the regulation of cspA and the production of its protein product CspA. In this paper, primer extension analysis was used to identify a transcriptional start point at 112 bp upstream of the initiation codon of the cspA coding sequence from S. aureus Newman RNA collected at 37 °C. Based on the location of the putative -10 and -35 sites as well as putative cold shock protein binding sites, a 192 bp sequence containing an 80 bp promoter + a 112 bp 5' UTR was generated by polymerase chain reaction. The activity of this 192 bp sequence was confirmed in a pLL38 promoter::xylE reporter gene construct. In addition, Western blots were used to confirm the production of CspA at 37 °C and demonstrated that production of the protein was not constitutive but showed growth-dependent production with a significant increase at the 6 h time point. The results presented identify another regulatory region for the cold shock gene cspA of S. aureus and show growth-dependent activity of both this cspA regulatory sequence, presented as a 192 bp sequence of promoter + 5' UTR and the production of the CspA protein at 37 °C. The presence of two active transcription start points, a -112 bp sequence defined in this work and a second previously defined at -514 bp upstream of the cspA initiation codon, suggests the possibility of interactions between these two regions in the regulation of cspA. The growth-dependent production of the cold shock protein CspA supports the availability of this protein to be a modulator of virulence and stress factor genes at 37 °C.