Expression, secretion, and processing of staphylococcal nuclease by Corynebacterium glutamicum.
The gene for staphylococcal nuclease (SNase), an extracellular enzyme of Staphylococcus aureus, was introduced into Corynebacterium glutamicum. The heterologous gene was expressed in this host organism, and SNase was efficiently exported to the culture medium. Amino-terminal sequencing of SNase secreted by C. glutamicum revealed that the signal peptide was apparently cleaved off at precisely the same position as in the original host, S. aureus. As with S. aureus, a second smaller form of SNase (A form), whose appearance is presumably the result of a secondary processing step, was found in the culture medium of the recombinant C. glutamicum strain. The A form was one residue shorter than the mature nuclease A produced by S. aureus. Variation of the sodium chloride concentration in the growth medium had a marked influence on the location and the processing of SNase by C. glutamicum. In a complex growth medium containing 4% sodium chloride, SNase was exclusively located in the supernatant, but a significant amount of the enzyme remained cell associated if the strain was grown in a low-salt medium. Also, high salt concentrations seemed to inhibit processing of the high-molecular-weight form of SNase (B form) to the smaller A form. Similarities and differences in the export and modes of processing of SNase by three different, nonrelated gram-positive host organisms are discussed. Finally, a versatile Escherichia coli-C. glutamicum tac-lacIq expression shuttle vector was constructed. With this vector, it was possible to achieve isopropyl-beta-D-galactopyranoside (IPTG)-inducible overexpression and secretion of SNase in C. glutamicum, whereby the expression level was dependent on the concentration of the inducer.
Liebl
W
W
Lehrstuhl für Mikrobiologie, Technische Universität München, Germany.
Sinskey
A J
AJ
Schleifer
K H
KH
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003352:Corynebacterium / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D008836:Micrococcal Nuclease / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013045:Species Specificity
D011498:Protein Precursors; D011994:Recombinant Proteins; D012965:Sodium Chloride; D008836:Micrococcal Nuclease