1. Characterization of the acid phosphatase from Staphylococcus aureus strain 154 and its development as a reporter system

2. © CSIR 2007 www.csir.co.za Characterization of a phosphatase secreted by Staphylococcus aureus strain 154 ● S. aureus strain 154 was isolated from rotten vegetables while screening bacterial isolates for the presence of extracellular phosphatases ● Enzyme activity detected by: ● a cost effective plate screen ● quantitatively measured by a simple enzyme assay ● the size of the protein product estimated with zymography

3. © CSIR 2007 www.csir.co.za ● Molecular mass of the protein is 30 Kda ● Optimum activity at 40°C and pH 5 ● In a reconstitution assay MgCl 2 or Triton X-100 restored enzyme activity, but not CaCl 2 ● Enzyme activity was negatively affected by EDTA and sodium molybdate as determined with in vitro enzyme assays

4. © CSIR 2007 www.csir.co.za Sequence analysis of the putative acid phosphatase gene ● N-terminal sequence of the extracellular enzyme determined ● Sequence corresponded to amino acids 32-39 of a hypothetical outer membrane protein in S. aureus subsp. aureus N315 ● Putative sapS gene cloned and sequenced

5. © CSIR 2007 www.csir.co.za ● S ignificant homology to the bacterial class C family of non-specific acid phosphatases ● Low level of overall conservation between the proteins. Four conserved sequence motifs identified. ● Characterization of a phosphatase secreted by Staphylococcus aureus strain 154, a new member of the bacterial class C family of non-specific acid phosphatases published in Systematic and Applied Microbiology 25, 21-30 (2002).

6. © CSIR 2007 www.csir.co.za Saps as an enzymatic reporter system ● SapS gene has been cloned and expressed successfully in Escherichia coli, Bacilllus subtilis and B. halodurans from its own promoter and signal sequence. ● Saps gene expression from heterologous promoters. Transcriptional and translational fusions created.

7. © CSIR 2007 www.csir.co.za Zymographic analysis of acid phosphatase activity in E. coli, Bacillus and Staphylococcus aureus

8. Schematic presentation of the vector constructs for expression/secretion studies

9. © CSIR 2007 www.csir.co.za Acid phosphatase activity stain of the E. coli host strain harbouring the vector constructs Control (c), pNW33N; 1, pNW33N1; 2, pNW33N2; 3, pNW33N3; 4, pNW33N4; 5, pNW33N5; 6, pNW33N6; 7, pNW33N7; 8, pNw33N8; 9, pNW33N9 and 10, pNW33- SapS.

10. © CSIR 2007 www.csir.co.za Acid phosphatase activity stain of the Bacillus subtilis host strain harbouring the vector constructs Control (c), pNW33N; 1, pNW33N1; 2, pNW33N2; 3, pNW33N3; 4, pNW33N4; 5, pNW33N5; 6, pNW33N6; 7, pNW33N7; 8, pNw33N8; 9, pNW33N9 and 10, pNW33-SapS.

11. © CSIR 2007 www.csir.co.za Acid phosphatase activity stain of the Bacillus halodurans host strain harbouring the vector constructs Control (c), pNW33N; 1, pNW33N1; 2, pNW33N2; 3, pNW33N3; 4, pNW33N4; 5, pNW33N5; 6, pNW33N6; 7, pNW33N7; 8, pNw33N8; 9, pNW33N9 and 10, pNW33-SapS.

12. © CSIR 2007 www.csir.co.za Whole cell in vitro acid phosphatase activity results of E. coli harbouring the vector constructs Promoter constructs:1, pNW33N1; 2, pNW33N2; 3, pNW33N3; 4, pNW33N4; 5, pNW33N5; 6, pNW33N6; 7, pNW33N7; 8, pNw33N8; 9, pNW33N9 and 10, pMG36e-SapS.

13. © CSIR 2007 www.csir.co.za Whole cell in vitro acid phosphatase activity results of B. subtilis harbouring the vector constructs Promoter constructs:1, pNW33N1; 2, pNW33N2; 3, pNW33N3; 4, pNW33N4; 5, pNW33N5; 6, pNW33N6; 7, pNW33N7; 8, pNw33N8; 9, pNW33N9 and 10, pMG36e-SapS.

14. © CSIR 2007 www.csir.co.za Whole cell in vitro acid phosphatase activity results of B. halodurans harbouring the vector constructs Promoter constructs:1, pNW33N1; 2, pNW33N2; 3, pNW33N3; 4, pNW33N4; 5, pNW33N5; 6, pNW33N6; 7, pNW33N7; 8, pNw33N8; 9, pNW33N9

15. © CSIR 2007 www.csir.co.za Summary of results ● A phosphatase secreted by S. aureus characterized and identified as a new member of the bacterial class C non- specific acid phosphatases ● The strongest promoter for heterologous protein production in each of the host strains was identified, i.e. the E. coli lacZ promoter in E. coli, the B. halodurans alkaline protease promoter in B. subtilis and the B. halodurans σ D promoter in B. halodurans. ● First report on the development of a Class C acid phosphatase gene as a reporter gene for Gram-positive and Gram-negative host strains

16. © CSIR 2007 www.csir.co.za Summary of results ● A phosphatase secreted by S. aureus characterized and identified as a new member of the bacterial class C non- specific acid phosphatases ● The strongest promoter for heterologous protein production in each of the host strains was identified, i.e. the E. coli lacZ promoter in E. coli, the B. halodurans alkaline protease promoter in B. subtilis and the B. halodurans σ D promoter in B. halodurans. ● First report on the development of a Class C acid phosphatase gene as a reporter gene for Gram-positive and Gram-negative host strains