We have previously demonstrated that the extracellular neutral metalloprotease (Npr) of Streptomyces cacaoi is synthesized as a 60-kDa preproenzyme (P60), then processed to the 35-kDa mature form (P35) (Chang, P. C., Kuo, T.-C., Tsugita, A., and Lee, Y.-H. W. (1990) Gene (Amst.) 88, 87-95). In this study, we investigated the active site and the mechanism involved in the maturation of the protease. Site-specific mutations at the putative zinc-binding ligands and active site of Npr at His202, Glu203, His206, and Glu240 led to complete abolishment of Npr activity and concomitant accumulation of a 57-kDa inactive protein (P57) which was secreted. Sequence analysis of the NH2 terminus indicated that P57 was derived from P60 after removal of the signal peptide and represented the proenzyme form of Npr (pro-Npr). Analysis of the zinc content of purified mutant P57 proteins revealed a dramatic loss of zinc atom as compared with the wild-type P35 protein. In vitro with the aid of exogenous active Npr, the mutant P57 protein could be converted to the mature inactive P35 with an identical NH2-terminal sequence and a molecular mass the same as that of the wild-type P35. From these studies, we conclude that these highly conserved residues (His202, Glu203, His206, and Glu240) are indispensable for zinc binding and protease activity, as well as processing of Npr. In addition, we have clearly demonstrated that maturation of Npr occurs extracellularly via an autocatalytic cleavage of the pro-Npr propeptide. This is the first report of such a maturation mechanism for an extracellular protease in streptomycetes which can serve as a model for further studies on the mechanism of secretion and processing of proteases from Gram-positive bacteria.

The nucleotide sequence of the 2.7-kb DNA fragment upstream of the structural gene of beta-lactamase in Streptomyces cacaoi was determined. Computer-aided "FRAME" analysis revealed four possible open reading frames (ORFs), three in one direction and one in the opposite direction. One of them (ORF1, BlaA) encoded an activator-regulator protein whose deduced amino acid sequence was similar to that of other activator-regulator proteins in bacteria. Insertion of an 8-bp BamHI linker into the BlaA region decreased the beta-lactamase activity sharply, from 50 U to 1 U/ml. This protein (BlaA) was found to bind to the nucleotide sequence between the bla (beta-lactamase structural gene) and blaA genes. Another ORF (ORF2, BlaB) in the same orientation had a couple of amino acid sequences similar to that of pBR322 beta-lactamase. However, insertion of the 8-bp BamHI linker indicated that this ORF was functional as an activator-regulator but not as a beta-lactamase. Therefore, there were two activator-regulator proteins in the upstream region of the structural gene of the beta-lactamase. Nuclease S1 mapping predicted that transcription for the activator proteins commenced at the translational initiation codon or within a few nucleotides from the translational start site. Transcription was in the opposite direction to that of the beta-lactamase structural gene.

We attempted to screen a series of Streptomyces subtilisin inhibitor-like (SIL) proteins among several Streptomyces strains by using a highly sensitive assay system established by us. Of six randomly tested strains, four were found to produce SIL inhibitors as their major secreted proteins, suggesting that they might be distributed in a high frequency among this genus. Three inhibitors exhibited inhibition of both subtilisin BPN' and trypsin. Comparison of the amino terminal sequences of these isolated proteins with those of other reported SIL inhibitors revealed that the beta 1- and beta 2-sheets in SSI were highly conserved.

Two beta-lactamase genes called blaL and blaU have been cloned independently in Li?ge and in Ume?, from Streptomyces cacaoi. Genes blaL and blaU were found to differ largely in their nucleotide sequences, although the encoded proteins both belonged to the class A of beta-lactamases (active-site serine penicillinases). DNA-hybridization and polymerase chain reaction assays have now demonstrated that both blaL and blaU genes were present in the S. cacaoi strains used in Li?ge and in Ume?.

The active-site serine of the extracellular beta-lactamases of Streptomyces cacaoi and Streptomyces albus G has been labelled with beta-iodopenicillanate. The determination of the sequence of the labelled peptides obtained after trypsin digestion of the denatured proteins indicate both enzymes to be class A beta-lactamases. Surprisingly the two Streptomyces enzymes do not appear to be especially homologous, and none of them exhibited a high degree of homology with the Streptomyces R61 DD-peptidase. Our data confirm that, as a family of homologous enzymes, class A is rather heterogeneous, with only a small number of conserved residues in all members of the class.

The soil bacterium Streptomyces cacaoi produces an extracellular beta-lactamase. The beta-lactamase expression could be induced by the beta-lactam compound 6-amino penicillinoic acid (6-APA). In liquid cultures, a 50-fold increase in beta-lactamase expression was observed within the first three hours after addition of 6-APA. Using the cloned beta-lactamase gene as a probe, it was shown that this increase was mediated at the level of transcriptional initiation. The start point of the induced beta-lactamase transcript was determined, and the nucleotide sequence of the promoter region was analysed. No noticeable homology was found to control regions of inducible beta-lactamase genes of other bacteria. A striking feature was the presence of six direct repeats (ten base pairs each) upstream of the promoter region. Thus, an example of an inducible regulatory gene system in this Gram-positive microorganism is presented. Also, the primary structure of the beta-lactamase was deduced, showing a high degree of homology with class A beta-lactamases.

Plasmids were prepared by inserting genomic DNA fragments from Streptomyces cacaoi within the mel gene of plasmid pIJ702. The inserted DNA fragments contain the beta-lactamase-encoding bla gene and upstream nucleotide sequences of various lengths. The transcription start point of bla was identified by nuclease S1 mapping. Upstream nucleotide sequences of sufficient lengths had an enhancing effect on beta-lactamase production by the Streptomyces host. The dot blot hybridization assay revealed that this effect was exerted at the transcriptional level. Experimental evidence strongly suggests that the underlying mechanism involves, at least in part, one or several trans-acting elements. In one of the constructs, in which the upstream nucleotide sequence was reduced to 0.3 kb, the bla promoter was present but the bla gene was expressed by readthrough from a promoter, possibly the mel promoter, of the pIJ702 vector.

Protein proteinase inhibitors showing sequence homology with Streptomyces subtilisin inhibitor (SSI) have been found to be distributed widely in Streptomyces species, and accordingly have been named SSI-like (SIL) proteins. SIL1 from S. cacaoi was the first of these proteins to be isolated and to be given a serial number. To study the structure-function relationship of SIL proteins, we determined the primary structure of SIL1 and measured its inhibitory activities. It was found to be composed of 110 amino acids and to exist in dimer form. The amino-acid sequence of SIL1 was unique among other characterized SIL proteins in having a one-residue deletion in two regions and a three-residue insertion in the flexible loop region. Sequence comparison indicated that SIL1 was distantly related to other members of the SSI family, and that amino-acid replacements had occurred not only on the surface of the SIL1 molecule but also in the beta-sheet region. The reactive site of SIL1 was considered to be Arg70-Glu71 from sequence alignment with other SSI-family inhibitors. SIL1 inhibited subtilisin BPN' strongly with an inhibitor constant (Ki) of 2.8 x 10(-11) M, like other members of the SSI family possessing an Arg residue at the P1 site. In contrast, SIL1 exhibited weak inhibition toward trypsin with a Ki value of 5.5 x 10(-8) M, possibly as a consequence of insertion of the three residues in the flexible loop region near the reactive site. This contrast seems to be due to the difference in the subsite structure of the two proteinases.

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The gene (npr) encoding an extracellular neutral metalloprotease (Npr) from Streptomyces cacaoi YM15 was cloned in Streptomyces lividans using pIJ702 as a vector. The nucleotide (nt) sequence of npr was determined. The deduced open reading frame encoded 550 amino acids (aa) (60 kDa) with a putative signal sequence of 34 aa at the N terminus. High-resolution S1 mapping identified the transcriptional start point at about 132-134 nt upstream from the start codon. The nt sequences at both -10 and -35 regions resemble the consensus sequence of typical Escherichia coli promoters and a fragment containing the promoter was functional in an E. coli promoter probe plasmid. In vitro transcription and translation of the cloned npr sequence revealed a 60-kDa protein product, correlated with the sequence data but not with the size (35 kDa) of the extracellular Npr. The N-terminal aa sequence in conjunction with the aa composition analyses on the purified mature Npr led to the conclusion that it was processed from the 60-kDa pre-proenzyme form encoded by npr. The Npr protease contained putative zinc ligand-binding regions and two repeated motifs, Asp-Ser-Gly, similar to the active site residues of the aspartic acid and retroviral proteases.