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yciM, one of the uncharacterized ORFs in Escherichia coli, located next to the DNA replication terminus terA, was found to suppress a suppressor sensitive crpmutant in 2 copies, one placed in trans in a single copy plasmid and the other on the chromosome in cis, but not in single copy on the chromosome. Moreover, it seems to be lethal to the host in multiple copies, because it could not be subcloned in a multicopy vector. The suppression sensitive crp mutation was leaky and had been credted by the insertion of an 152 element between the crp promoter and its coding region. Introduction The completion of the E. coli genome project has left a large number of putative ORFs (about 38%) whose function or expression remained unknown (ref. 1) . What types of gene products do they code for? Are they of the same types of genes as those described heretofore but slipped through preceding studies, or do they contain new categories difficult to be discovered by the conventional methods? yciM, located next to the DNA replication terminus as indicated in the DNA database, is one of these uncharacterized ORFs. Although there is an annotation in the database identifying it as a "putative heat shock protein", in spite of considerable effort we have not been able to track down the source of this information. In a previous paper (ref. 2) we have reported on a crp — mutant of Escherichia coli (ref. 3) caused by the insertion of an IS 2 element between the promoter and the coding region. This mutant was leaky and presented crp + phenotype in multicopy plasmids but was too weak in a single copy to do so; it was suppressible by a mutation elsewhere in the genome. During the work to identify the suppressor, we have found that yciM gene cloned in a single copy vector strongly suppressed the crp — mutant. This gene seems to be involved in dosage dependent suppression of the crp — mutant Result and Discussion Genomic DNA of such a suppressor mutant R 1-2-1 was treated with BamHI and ligated in the BamHI site of a single copy plasmid pMF 3 (ref. 4). The linearized pMF 3 had been treated with alkaline phosphatase to avoid selfligation. The ligation mixture was electroporated into the E. coli strain DOO crp — (ref. 2). About 7000 colonies were screened on EMB lactose plates, most of which were white, except 3 colonies which were black (Fig. 1 a). Plamids from these colonies (pS011, pS012, pS013) were isolated and sequenced with the primers placed at both ends of the Barn HI site in pMF 3 (Table 1 and Fig. 2). All three plasmids had the same E. coli BamHI fragment derived from around 28 min in the genetic map of E. coli, two in one orientation, the other in the other. It was impossible to redone this fragment in the multicopy plasmids pDELTA 2 (GIBCO BRL) or pKF 3 (ref. 5), presumably because it was lethal to the host in multicopies (see Table 2). EZ::TN transposition insertion was performed on one (pS011) of these plasmids. In all the transposition products which showed crp — phenotype (that is, those that produced white colonies) that we examined (we sequenced 12 out of about 150 such colonies), EZ::TN had been inserted in the gene yciM, except one which had it in yciS . yciS and yciM are adjacent ORFs probably transcribed together from the same two putative promoters upstream of yciS (annotation to the database) (Fig. 2). Thus an EZ::TN insertion in yciS would also result in abolition of yciM transcription. On the other hand, in the plasmids which showed crp+ phenotype (we sequenced 12 out of about 1850), sites of EZ::TN were not in yciS or yciM but elsewhere in the 28 min fragment (Fig. 2), or in the vector pMF 3 (data not shown). We conclude that the suppressor activity resides in the yciM gene. To learn the state of the yciM ORF in the recipient host DOO crp—, four primers were synthesized (Table 1 and Fig. 2) and used for direct PCR sequencing of the region including yciS and yciM for both the host E. coli genomic DNA and the plasmid pS011 (control). Their sequences were completely identical between E. coli DNA coordinate 1338089 and 1339896, which include the two promoters, the yciS and the yciM ORFs; neither do they show any difference from the database sequence. These results indicate that the difference in dosage of the yciM gene is responsible for the suppression of the crp phenotype in the transformant (see Table 2). As far as we know this is the first report of such an activator that depends on the gene dosage, (and therefore presumably on the dosage of the gene product) in the sense that a single copy of the suppressor gene is inactive as suppressor but two copies are. In this connection it is interesting that the yciM gene is located in the E. coli genetic map next to the DNA replication terminus, where its DNA is duplicated only near the end of each replication cycle. Thus it could be involved in signaling the termination of a DNA replication cycle, and/or triggering the inception of a cell division step. If such is the case, it would be naturally lethal in multicopies, but may be tolerated in 2 copies (see the small size of black colonies seen in Fig. 1 b as compared with white colonies). We confirmed the crp phenotypes of pS011 and its EZ:: TN derivatives with other sugars; they reacted to these plasmids just as D-lactose. These results suggest that yciM does affect the lac gene expression via the crp gene rather than by acting directly on the lac gene itself. To confirm this point, the plasmid containing yciM was also electroporated into E. coli strain CA 8445-1 ( rel A 1 rpsL 136 A crp — 45 spoT 1 thi-1 cya :ref. 6,7), which has lost most of the crp gene by deletion: we plated the transformants on EMB lactose plates containing cyclic AMP (because this strain is cya 1. The colour of these colonies was white, suggesting that without the intervention of the CRP system yciM gene product cannot stimulate the lac gene; the CA 8445-1 strain also had an intact yciS-yciM region, which we have shown by PCR sequencing of its genomic DNA (Fig. 3). Does the present result imply that the suppressor mutation in the revertant R 1-2-1 was a duplication of a DNA segment including yciM, or there was a different suppressor mutation in that mutant? This question remains open at present; perhaps we will have to repeat the experiment using some other restriction enzymes than Bam HI. Thus, in a sense, yciM is a positive regulatory gene of another regulatory gene, i.e., crp . The case of yciM suggests that many, or at least some, of the uncharacterized ORFs of E. coli code for proteins involved in complicated interactions with other genes. A suppressor sensitive system of an activator-dependent type, like the crp mutant used here, may help to elucidate the functions of other such uncharacterized ORFs. METHODS Growth of cells. E. coli strains were grown at 37°C in L-broth (1% polypepton, 0.5% yeast extract, and 0.5% NaCl) or on L agar (L-broth added with 1.5% agar). EMB lactose agar (and some other sugar) plate (1% polypepton, 0.1% yeast extract, 0.5% NaCl, 0.4 g/1 eosin Y, 0.06 g/1 methylene blue, 0.2% K2 HP01 , 1% lactose, 1.5% agar) was used to check the crp phenotype. Media were supplemented with ampicillin (30 i g/ ml ) , kanamycin (50 if g/m1), or thymine (10 i g/ml) as required. Purification of DNA. Chromosomal DNA was isolated Table 1 • Direction, Location and Sequence of Synthesized Primers Location of primers yci 1 to yci4 are indicated by local coordinates of the positions of the 5' ends on the 28 min fragment (see Fig. 2). Directions refer to the usually presented orientation (increasing order of coordinate) of the E. coli genome DNA. (see Fig. 2 also.) Table 2 • Relation between dosage of yciM and phenotype Fig. 1. Photographs of colonies on EMB lactose plates two days after plating at 37'C . a. The arrow indicates the clone that contained the plasmid with the 28 min fragment pS011 . b. Transformants of EZ::TN reaction products derived from pS011 . Fig. 2. Insertion sites of EZ::TN on the BamHI fragment of E . coli DNA located at 28 min on the map . The horizontal line represents the Barn HI fragment of E. coli DNA. The local coordinates are shown above the E . coli genome coordinates. Arrows show genes and their transcription directions . The EZ::TN insertion sites for white colonies shown by upward spikes were always located in yciM , except one which was on yciS . Those for black colonies shown by downward spikes were located in the other genes or in the vector . Six triangles show the l ocation of primers. Fig. 3 A schematic representation of the interactions of crp (IS 2), yciM and lac genes in DOO crp— derivatives. The larger circle indicates the E. coli chromosome and the smaller circle indicates the plasmid. Arrows indicate the stimulatory effect. as described previously (ref. 8). Plamids were isolated using QIAGEN Plasmid Kit (QIAGEN). Closing. pMF 3 was cut by BamHI and BAP-treated according to the manufacturer's instruction . Chromosomal DNA restricted by BamHI was mixed with T 4 ligase and buffer purchased from TaKaRa. The ligation reaction was done at 16°C overnight. Determination of the up phenotypes. The phenotypes were determined by fermentation of various sugars including D-lactose, D-xylose and D-mannitol. For routine determination EMB lactose plate was used as the indicator plate when no ambiguity was expected. When a cya strain was used, cyclic AMP was externally supplied in the medium at 0.5 mM. Electrophoresis. Gel electrophoreses of plasmid DNA or restricted DNA was performed in agarose gel in Trisborate buffer at pH 8.3. Size markers for linear or covalently closed circular DNA were KiloBase DNA Marker purchased from Pharmacia or Supercoiled DNA ladder (Gibco-BRL) purchased from Life TechnologiesOriental Inc., respectively. PCR. PCR was performed as described (ref. 9) with slight modifications. Briefly, about 300 ng chromosomal DNA was used as template for amplification. The amplification was done in Gene Amp

Dosage Dependent Suppression of a crp - Mutant by yciM Gene Product in Escherichia coli