Ball, Timothy Keith (1990-05). Regulation of expression and excretion of a Serratia marcescens nuclease. Doctoral Dissertation. Thesis uri icon


  • The extracellular nuclease of Serratia marcescens was used as a model for studying extracellular protein localization in Gram-negative bacteria. The gene encoding the nuclease was cloned from S. marcescens SM6. The nuclease gene was expressed in Escherichia coli and the gene product was detected in the supernatant fraction in the absence of cell lysis, suggesting that the nuclease was excreted from E. coli as it is in S. marcescens. The DNA sequence of the gene was determined and predicts a precursor protein containing a signal peptide of 21 amino acids that, when cleaved, yields a mature protein of 29.5 kDa. The gene product was purified and used to raise antisera specific for the nuclease. Supernatants of E. coli and S. marcescens expressing the gene from plasmids were used to confirm the size of the mature peptide by western blot analysis. The rate and route of excretion of the nuclease in S. marcescens was determined by immunoprecipitation of radiolabeled proteins. The protein is synthesized and transferred to the periplasm, where it remains at least one hour prior to release from the cell. A mode of regulation of expression of this and other genes encoding extracellular proteins from S. marcescens was identified. The 5. marcescens recA gene was shown to be involved in the expression of at least the nuclease. The S. marcescens recA gene was cloned and sequenced and compared to similar genes in other Gram-negative bacteria. This regulatory gene product was about 90% homologous to the E. coli RecA protein, and was required for expression of the nuclease. It was determined that the E. coli recA gene could complement recA defective mutants of S. marcescens. The nuclease gene was found to be induced to high levels of expression following exposure of S. marcescens to SOS inducing agents. The synthesis and release of nuclease from the cell was found to follow a growth-phase-dependent manner which is superimposed upon SOS inducibility. The growth-phase and SOS-inducible modes of regulation did not function in E. coli, indicating that other functions are involved in the regulation of expression of nuclease in S. marcescens. Models for regulation of expression and excretion in S. marcescens are proposed.

publication date

  • December 1990