A DNA fragment of Serratia marcescens directing an extracellular serine protease (Mr, 41,000) Artificial frameshift mutations introduced into the codhig sequence for the assumed distal polypeptide after the C terminus of the piotease caused complete loss of the enzyme production. It was concluded that the Serratia serine protease is produced as a 112-kilodalton proenzyme and that its N-terminal signal peptide and a large C-terminal part are processed to cau4se excretion of the mature protease through the outer membrane of E. coli cells.

The Serratia marcescens serine protease, which is directed by the gene encoding a precursor composed of a typical NH2-terminal signal sequence, a mature enzyme domain, and a large COOH-terminal domain, was excreted through the outer membrane ofEscherichia coli. The precursor, with the expected molecular size (110 kilodaltons), was detected in an insoluble form in the periplasmic space of E. coli cells after induction with isopropyl-13-D-thiogalactopyranoside of the expression of the gene under the control of the tac promoter. Upon membrane fractionation of the disrupted cells by sucrose density gradient centrifugation, the precursor was recovered from a fraction slightly heavier than the outer membrane fraction but not from the inner membrane fraction. Conversion of the precursor into the mature form, which was accompanied by its excretion into the medium, was observed even in the absence of de novo protein synthesis caused by the addition of chloramphenicol. The mutated gene product lacking all of the COOH-terminal domain was localized in the periplasmic space only and was not excreted into the medium. Additional mutant genes were generated by site-directed mutagenesis to test the role of some amino acids in the excretion of this protease in E. coli. The mutant protein with no protease activity because of the change of the catalytic residue Ser-341 to Thr was still excreted into the medium but with abnormal processing. Both self-processing and host-dependent processing of the precursor seem to be involved in the excretion of the mature enzyme. Replacement of the four Cys residues, two in the mature enzyme and two in the COOH-terminal domain, with Ser in different combinations caused a distinct or complete loss of excretion, suggesting that a certain conformation possibly formed via disulfide bonding was important for the excretion of the S. marcescens protease.

No abstract

The DNA sequence of an 8-4kbp BamHI-EcoRI fragment of Marek's disease virus (MDV) strain GA was determined. Three of the predicted polypeptides are homologous to UL47, UL48 and UL49 encoding the major tegument proteins of herpes simplex virus type 1 (HSV-1), and four are homologous to HSV-1 UL45, UL46, UL49-5 and UL50. These seven genes are found in the long unique region of the MDV genome and are collinear with homologues in HSV-1 and varicella-zoster virus (VZV). Northern blot analysis revealed different transcriptional patterns from those of HSV-1 and VZV. MDV homologues of UL49.5, UL49 and UL47 lack a poly(A) signal immediately downstream of their coding regions. Amino acid conservation between MDV and HSV-1, and between MDV and VZV is as high as that between HSV-1 and VZV. The MDV homologue of UL48 shows 60% similarity to its HSV-1 counterpart. Amino acid sequence comparison reveals that the MDV homologue of UL48 lacks an acidic carboxyl terminus. This homologue, like the VZV homologue of UL48, may be involved in the trans-activation of immediate early genes and may function as an important component of the structural proteins.