Tetracycline modulates the valine induced stringent response and decreases expressed RpoS in Escherichia coli

Abstract

The stringent response in Escherichia coli is a physiological response to stress characterized by accumulation of the alarmone (p)ppGpp and the down-regulation of various processes involved in cellular growth and protein synthesis. It has been previously shown that activation of the stringent response by amino acid starvation is able to confer protection against various classes of antibiotics, including tetracycline and ampicillin. However, no observations have been made on partial activation of stringency. This investigation attempted to repress the level of bacterial stringent response in E. coli CP78 through the use of tetracycline. Tetracycline is an antibiotic which has been shown to restrict the cellular accumulation of (p)ppGpp in CP78 Escherichia coli. The effectiveness of the starvation by valine was monitored by measuring turbidity. The modulation of stringent response was monitored by the level of RpoS, since RpoS production is known to be enhanced by activated stringent response. As measured by turbidity, we successfully established an amino acid starvation condition with valine treatment as seen in the inhibited growth of CP78 cells as compared to untreated cells. The valine inhibition of the growth of the bacteria appeared to be reduced by the addition of tetracycline. However, cultures treated with high concentrations of tetracycline seemed to become more turbid than the untreated control even though they had similar levels of protein. This suggested that the tetracycline might have been deteriorating and increasing the absorbance of the cultures. The increasing absorbance might have contributed to the observed turbidity readings. The expression of RpoS as determined by Western blots was barely detectable in the lysates; therefore, the effect of tetracycline on RpoS levels was difficult to interpret. However, slight upregulation of RpoS expression was observed at high tetracycline concentration when cells were treated for a prolonged period of time (2 hours), suggesting that tetracycline may be involved in modulating the expression of RpoS. Taken together, the results indicate that tetracycline may decrease the bacterial stringent response and that this model may prove to be effective to study antibiotic cross-resistance in the future.