著者

Ohnishi Yasuo Beppu Teruhiko Horinouchi Sueharu

出版者

The Japanese Biochemical Society

雑誌

The Journal of Biochemistry (ISSN:0021924X)

巻号頁・発行日

vol.121, no.5, pp.902-913, 1997

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A serine protease (SSP) of <i>Serratia marcescens</i> is one of the extracellular enzymes secreted from this Gram-negative bacterium. SSP is produced as a large precursor and converted to a mature protein by cleavages removing an NH₂-terminal signal sequence and a COOH-terminal pro-region. This COOH-terminal pro-region is integrated into the outer membrane and has a functional role for the export of the mature protein across the outer membrane. Southern hybridization analysis with a DNA fragment encoding the COOH-terminal pro-region as the probe showed a wide distribution of nucleotide sequences encoding SSP exporter-like proteins among <i>Serratia</i> species. Moreover, S. marcescens IFO 3046, from which the ssp gene had been cloned, was found to contain two ssp homologues (<i>ssp-h1</i> and <i>ssp-h2</i>). They were cloned and their nucleotide sequences were determined. The two ssp homologues were found to exist in tandem on the genome and their amino acid sequences showed 81% identity to each other. Both of them showed 55% identity in amino acid sequence to preproSSP. In addition, both showed end-to-end similarity to the 100 kDa serotype-specific antigen (Ssa1) of Pasteurella haemolytica. Escherichia coli JM105 containing <i>ssp-h1</i> gene produced a 53 kDa protein corresponding to the NH₂terminal portion and a 49 kDa protein corresponding to the COOH-terminal portion, both of which were rigidly integrated in the outer membrane. Consistent with the significant similarity of the COOH-terminal portions of the homologues to that of SSP, they showed the ability to translocate the mature SSP part across the outer membrane into the medium. Furthermore, the NH₂-terminal portion of the homologue was not translocated into the outer membrane without its COOH-terminal part. All of these data show that the SSP homologues are outer membrane proteins that are translocated into the outer membrane with the aid of the translocator function of their COOH-terminal part.

著者

PARK Jae-Seon HORINOUCHI Sueharu BEPPU Teruhiko

出版者

社団法人日本農芸化学会

雑誌

Agricultural and Biological Chemistry (ISSN:00021369)

巻号頁・発行日

vol.55, no.7, pp.1745-1750, 1991-07-23

被引用文献数

8

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An endo-type semi-alkaline cellulase (CMCase I) produced by an alkalophilic Streptomyces strain has an extraordinarily long leader peptide of about 70 amino acids (aa), which can be grouped into four distinct regions, an NH2-terminal region (13aa), an Arg-cluster region (13aa), a hydrophobic region (23aa), and an Ala/Pro-repeat region (12aa). For identification of the function of each part of the leader peptide for secretion of the enzyme, mutations in the leader peptide were generated by site-directed mutagenesis using the cloned gene, and the mutant genes were expressed in a cellulase-negative mutant strain, Streptomyces lividans HN1. Although all the alterations in the leader peptide, except for one case, decreased secretion to various extents, in S. lividans, the following conclusions were obtained. Comparison of the intra- and extra-cellular enzyme activities of the mutants suggested that the Arg-cluster region was essential in secretion of the cellulase. Deletion of 8 amino acids rich in threonine and proline in the NH_2-terminal region enhanced the secretion to a small extent. Deletion of the Ala/Pro repeat region had almost no effect on secretion.