Mechanisms of Activation of the Cryptic cel Operon of Escherichia coli K 12 Laura

The cel (cellobiose utilization) operon of Escherichia coli K12 is not expressed in the wild-type organism. However, mutants that can express the operon and thereby utilize the P-glucoside sugars cellobiose, arbutin and salicin are easily isolated. Two kinds of mutations are capable of activating the operon. The first involves mutations that allow the repressor to recognize the substrates cellobiose, arbutin and salicin as inducers. We have identified the sequence changes in five different active alleles and found those differences to be single base pair changes at one of two lysine codons in the repressor gene. The second kind of mutation involves the integration of the insertion sequences ISI, IS2 or IS5 into a 108-bp region 72-180 bp upstream of the start of transcription. Integration occurs at several different sites and in different orientations. Transcription of the cel operon begins at the same base pair in all mutants examined. Of 44 independent cel+ mutants, 27 were activated by point mutations and 17 were activated by insertion sequences. The preferred mechanism of activation appears to be strain dependent, since one of the parents yielded 94% insertionally activated alleles, while another yielded 100% point mutation activated alleles. C RYPTIC genes are silent genes that are not able to be expressed in the wild-type organism. Escherichia coli K12 possess a variety of genes for @-glucoside utilization, but most of these genes are cryptic in the wild-type organism. Mutations at several different loci that result in the expression of these cryptic genes have been investigated. The best characterized system is the bgl operon, a system that specifies all of the functions necessary for fermentation of the aryl-@glucoside sugars, arbutin and salicin (PRASAD and SCHAEFLER 1974). That operon consists of three structural genes, bgZG, bgZF and bgZB (SCHNETZ, TOLOCZYKI and RAK 1987; PRASAD and SCHAEFLER 1974). The regulatory region of the bgZ operon contains a 130-bp leader region where termination of transcription occurs in the absence of arbutin and salicin (MAHADEVAN, REYNOLDS and WRIGHT 1987; SCHNETZ and RAK 1988). In the presence of arbutin or salicin the bglG gene product acts as a positive regulatory element and prevents termination of transcription. The second gene of the operon, bgZF, encodes the @-glucoside specific phosphotransferase protein that both transports and phosphorylates the substrates. In the absence of arbutin and salicin, the product of bgZF interacts with the product of bgZG to prevent antitermination and thereby represses the operon (MAHADEVAN, REYNOLDS and WRIGHT 1987). The third gene, bglB, School, Boston, Massachusetts 021 11. ' Present address: Department of Physiology, Tufts University Medical ' Present address: Department of Biology, University of Rochester, Rochester, New York 14627. The publication costs of this article were partly defrayed by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. $1734 solely to indicate this fact. Genetics 1 2 4 473-482 (March, 1990) encodes a phospho-&glucosidase that is specific for aryl-@-glucosides. Even if arbutin and salicin are present the wild-type bgl operon does not permit fermentation of these sugars because specific changes in the promoter region are required to vercome the cryptic state of the system. It has been shown that insertions of IS1 or IS5 into a 47-bp region upstream of a nearby promoter are capable of activating the bgl operon (REYNOLDS, FELTON and WRIGHT 1981; REYNOLDS et al. 1985, 1986). It is known that the insertions increase the activity of this promoter (SCHNETZ and RAK 1988), however, the exact mechanism by which this happens is not known. It has been shown that ethyl methansulfonate induced single base changes in the cyclicAMP receptor protein binding site, and particular gyrA and gyrB mutations, are also capable of activating the bgl operon (REYNOLDS et al. 1985). The cryptic ceZ operon has also been studied extensively (KRICKER and HALL 1984, 1987; HALL, BETTS and KRICKER 1986; Parker and HALL 1990). Expression of the ceZ operon allows the utilization of cellobiose, salicin, and arbutin. The operon consists of five genes, celABCDF (PARKER and HALL 1990), in which celB and celC encode gene products that are required for phosphoenolpyruvate-dependent transport and phosphorylation of cellobiose, arbutin and salicin, ceZD encodes a repressor, and celF encodes a phospho-@glucosidase that acts on cellobiose, arbutin and salicin. The function of the celA gene product is unknown. In this report we show that activation of the cel operon can be accomplished two ways. One mechanism of 474 L. L. Parker and B. G. Hall activation involves mutations in celD that allow the repressor to recognize cellobiose, arbutin, and salicin as inducers. Another mechanism of activation involves the integration of the insertion sequences IS I , IS2 o r IS5 in a 108-bp region 72-180 bp upstream of the transcriptional initiation site. The insertions increase the basal level of gene expression, suggesting that these elements interfere with binding of the repressor