Bacillus licheniformis penicillinase genes, penP and penl, are coded on a 4.2-kilobase EcoRI fragment of pTTE21 (T. Imanaka, T. Tanaka, H. Tsunekawa, and S. Aiba, J. Bacteriol. 147:776-186, 1981). The EcoRI fragment was subcloned in a low-copy-number plasmid pTB522 in Bacillus subtilis. B. subtilis carrying the recombinant plasmid pPTB60 (Tcr penP+ penI+) was chemically mutagenized. Of about 150,000 colonies, two penl(Ts) mutant plasmids, pPTB60D13 and pPTB60E24, were screened by the plate assay at 30 and 48°C for penicillinase. By constructing recombinant plasmids between wild-type and mutant plasmids, the mutation points were shown to be located in a 1.7-kilobase EcoRI-PstI fragment. The EcoRI-PstI fragments of the wild-type plasmid and two mutant plasmids were sequenced. A large open reading frame, composed of 384 bases and 128 amino acid residues (molecular weight, 14,983), was found. Since the mutation points were located at different positions in the protein coding region (Ala to Val for pPTB60D13 and Pro to Leu for pPTB6OE24), the coding region was concluded to be the pen! gene. A Shine-Dalgarno sequence was found 7 bases upstream from the translation start site (ATG). A probable promoter sequence which is very similar to the consensus sequence was also found upstream of the penP promoter, but in the opposite direction. A consensus twofold symmetric sequence (AAAGTATTA CATATGTAAGNTTT) which might have been used as a repressor binding region was found downstream and in the midst of the penP promoter and also downstream of the penl promoter. The regulation of penP and pen! by the repressor is discussed.

Protective effects of intracellular glutathione (GSH) against the cytotoxicity of human recombinant tumor necrosis factor (TNF) were investigated. Three tumor cell lines (L-M, B-16, HeLa) were used as target cells. Exposure of these cells to buthionine sulfoximine (BSO) or diethyl maleate (DEM) resulted in the depletion of intracellular GSH content to 5.2-43.0% of control values and enhancement of their susceptibility to TNF cytotoxicity. The hydroxyl radical production in L-M cells stimulated by TNF was increased by treatment with BSO or DEM. These results are consistent with the suggestion that intracellular GSH exerts its protective function against the cytocidal effect of TNF by inhibiting the hydroxyl radical production stimulated by TNF.

The penicillinase antirepressor gene, penJ, of Bacillus licheniformis ATCC 9945a was cloned in Escherichia coli by using pMB9 as a vector plasmid. The penicillinase gene, penP, its repressor gene, pen!, and penJ were encoded on the cloned 5.2-kilobase Hindlll fragment of the recombinant plasmid pTTE71. The penJ open reading frame was composed of 1,803 bases and 601 amino acid residues (molecular weight, 68,388). A Shine-Dalgarno sequence was found 7 bases upstream from the translation start site. Since this sequence was located in the 3'-terminal region of the penI gene, penJ might be transcribed together with penI as a polycistronic mRNA from the pen! promoter. Frameshift mutations of penJ were constructed in vitro from pTTE71, and the penJ mutant gene was introduced into B. licheniformis by chromosomal recombination. The transformant B. licheniformis U173 (penP+ penI+ penJ) turned out to be uninducible for penicillinase production, whereas the wild-type strain (penP+ penI+ penJ+) was inducible. Only when these three genes (penP, pen!, and penJ) were simultaneously subcloned in Bacillus subtilis did the plasmid carrier exhibit inducible penicillinase production, as did wild-type B. licheniformis. It was concluded that penJ is involved in the penicillinase induction. The regulation of penP expression by pen! and penJ is discussed.The penicillinase gene, penP, of Bacillus licheniformis 749/C has been cloned (3, 7) and sequenced (20). We have independently cloned penP and the repressor gene, pen!, from both the wild-type and constitutive strains of B. licheniformis ATCC 9945a (13) and determined the nucleotide sequence of penI and the flanking regions (8,12 DNA manipulations and analyses. Preparation of plasmid DNA, cleavage of DNA with restriction enzymes, repair and ligation of DNA, gel electrophoresis for DNA analysis, and isolation were all performed as described previously (11). Chromosomal DNA of B. licheniformis was prepared as mentioned previously (13). DNA was sequenced by the Maxam-Gilbert method (16) and the dideoxy method (18), with an M13 sequencing kit (Takara Shuzo Co., Kyoto, Japan). The sequencing was done on both strands, and all restriction sites were sequenced across.Penicillinase assay. Penicillinase was assayed by the iodometric method as described previously (13). The method of detecting penicillinase-positive colonies on LP plates (L agar containing 0.75% [wt/vol] polyvinyl alcohol) has been described previously (13). The inducibility of penicillinase production was examined by cultivating microbial cells either on LPC plates (LP plus 5 ,ug of cephalosporin C [an inducer] per ml) or in liquid medium.Computer analysis of hydropathic character of the protein. The AC program to assess hydropathy, as described by Kyte and Doolittle (14), was translated to BASIC and implemented on an NEC PC-8001 computer (Nippon Electric Co., Tokyo, Japan). The mean hydropathy of a protein was evaluated by a moving segment of 9 amino acid residues along the sequence as mentioned previously (15). RESULTS

We have constructed secretion vector plasmids that have unique BglII sites within or near the signal sequence of Bacillus licheniformis penicillinase, and have also constructed penicillinase cartridges that lack either one, two or three of the processing sites for the membrane-bound, exo-large and exo-small enzymes. Each of these penicillinase cartridges was cloned on secretion vectors in Bacillus subtilis, and enzyme production was examined. The presence of both the signal sequence and the three host-specific processing sites on the secretion vector was required for an effective expression of the enzyme in B. subtilis. The presence of any of the processing sites on the cartridge reduced the accumulation of penicillinase in the culture medium. When a vector plasmid lacking part of the hydrophobic region of the signal sequence and lacking the three processing sites was used, total penicillinase production decreased and enzyme accumulation in the medium was extremely low, despite the complete or incomplete presence of the processing sites on the cartridge. Molecular mass determination of these extracellular penicillinases suggested the existence of a new cleavage site for the enzyme.

Based on findings that the cytotoxic effects of tumor necrosis factor (TNF) are closely related to levels of intracellular oxygen radicals, and on the results of TNF gene transfection studies, the hypothesis was made that endogenous TNF (enTNF) acts as a protective factor against exogenous TNF by inducing inhibitors or scavengers of oxygen radicals. In order to test this hypothesis, we investigated the intracellular levels of manganous superoxide dismutase (MnSOD) and glutathione (GSH) in L-M(pNTnF) cells carrying a TNF gene induced by dexamethasone (DM). When L-M(pNTnF) cells were treated with DM they expressed enTNF, and acquired resistance to exogenous TNF. There was no change in the GSH concentration after enTNF induction, but a 1.9- to 3.9-fold increase in MnSOD levels was noted. Our findings suggest that enTNF exerts its protective function against the cytocidal effect of exogenous TNF by inducing MnSOD production.