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The BamHI restriction-modification system contains a third gene, bamHIC, which positively regulates bamHIR. Similar small genes from other systems were tested in vivo for their ability to cross-complement. C.BamHI protein was identified, purified, and used to raise polyclonal antibodies. Attempts to detect other C proteins in cell extracts by cross-reactivity with C.BamHI antibodies proved unsuccessful.

BamHI, from Bacillus amyloliquefaciens H, is a type II restriction-modification system recognizing and cleaving the sequence G--GATCC. The BamHI restriction-modification system contains divergently transcribed endonuclease and methylase genes along with a small open reading frame oriented in the direction of the endonuclease gene. The small open reading frame has been designated bamHIC (for BamHI controlling element). It acts as both a positive activator of endonuclease expression and a negative repressor of methylase expression of BamHI clones in Escherichia coli. Methylase activity increased 15-fold and endonuclease activity decreased 100-fold when bamHIC was inactivated. The normal levels of activity for both methylase and endonuclease were restored by supplying bamHIC in trans. The BamHI restriction-modification system was transferred into Bacillus subtilis, where bamHIC also regulated endonuclease expression when present on multicopy plasmid vectors or integrated into the chromosome. In B. subtilis, disruption of bamHIC caused at least a 1,000-fold decrease in endonuclease activity; activity was partially restored by supplying bamHIC in trans.

Endogenous chromosomal DNA amplifications with associated tetracycline resistance (Tcr) in Bacillus subtilis were first described by C. R. Wilson and A. E. Morgan (J. Bacteriol. 163:445-453, 1985). We have confirmed and extended their results, and we show that fusion of protoplasts from Tcs B. subtilis 168 trpC2 with polyethylene glycol and regeneration on medium containing 20 micrograms of tetracycline per ml induces Tcr regenerants that contain amplified DNA. This phenomenon appeared to be recE dependent and requires the addition of polyethylene glycol. Along with three regenerants kindly provided by Wilson and Morgan (RAD1, RAD6, and RAD7), we characterized three strains (CLI20, CLI22, CLI30) isolated in this laboratory. All six contain an amplified region of DNA which was independently cloned on plasmid pCIS7. Integration of pCIS7 into the wild-type (Tcs) B. subtilis chromosome and amplification of the plasmid sequences generated a Tcr phenotype, even though the DNA on pCIS7 was cloned from Tcs B. subtilis KS162 (Ives and Bott, J. Bacteriol. 171:1801-1810, 1989). The amplified DNA also showed homology (through hybridization analysis) with pAM alpha 1 delta 1, a gram-positive Tcr plasmid, indicating that B. subtilis normally contains a silent integrated copy of the gene whose amplification confers Tcr. The amplifications were determined to lie between purA and gyrB on the B. subtilis chromosome, and the endpoints were mapped. RAD6 and CLI30 may share the same left-hand endpoint, but the other endpoints are different in each isolate. The amplified DNAs of RAD1, RAD6, CLI20, and CLI30 end near known DNA membrane binding sites. The number of amplified units of DNA was determined through dot blot analysis to do approximately 80 to 100 copies per cell, with corresponding increases in transcription of RAD1, RAD6, CLI20, CLI22, and CLI30.

Plasmid pCIS7, containing 11.5 kilobases (kb) of Bacillus subtilis DNA, was isolated from a Tn917 transposon insertion in tetracycline-sensitive B. subtilis KS162. When integrated into the chromosome of B. subtilis 168, this plasmid conferred tetracycline resistance upon reiteration of the plasmid DNA sequences in the chromosome. Deletions and subclones of pCIS7 were constructed and introduced into an Escherichia coli in vitro transcription-translation system. A 72-kilodalton protein was localized to a 3.1-kb PstI-EcoRI fragment of the plasmid. Amplification of the 3.1-kb PstI-EcoRI fragment was required for expression of tetracycline resistance in B. subtilis 168. By hybridization to previously characterized clones, the 11.5-kb fragment was localized to the origin region of the chromosome. Through contour-clamped homogeneous electric field electrophoresis, this cluster of clones was shown to reside on a 200-kb NotI fragment bridging SfiI fragments of 150 and 250 kb and was oriented with respect to the purA and guaA loci, developing an accurate physical map of the region surrounding the origin of replication.