The pcnB gene product of Escherichia coli is required for copy number maintenance of plasmids related to ColEl and also for that of the IncFII plasmid R1. Because PcnB is similar to the tRNA-binding protein tRNA nucleotidyltransferase, we have suggested that the protein would be required only for processes in which an RNA is a prominent regulatory component. This appears to be so; strains deleted for pcnB, although defective in ColEl and R1 plasmid maintenance, maintain the iteron-regulated plasmids F and P1 normally. We also find that strains deleted for pcnB grow normally, demonstrating that PcnB has no essential cellular role under the conditions tested and suggesting that regulation by antisense RNAs similar to RNAI has no critical role in any essential host process. We confirm by immunological tests that PcnB is likely to be the commercially available enzyme poly(A) polymerase.The Eschenchia coli pcnB gene product is required for normal copy number maintenance of plasmids related to either ColEl or R1 (13, 14, 16, 17). Replication frequency in each of these plasmid groups is controlled, in part, by an antisense RNA (for a review, see reference 5). RNAI binding prevents productive folding of the ColEl replication primer, while copA RNA prevents translation of the message for the essential R1 replication protein, RepA. PcnB shares homology with the product of the E. coli cca gene, tRNA nucleotidyltransferase, a protein which binds tRNA (17). It has therefore been suggested that PcnB may also be an RNAbinding protein and may expedite plasmid replication by interfering with the inhibitory sense-antisense RNA interactions which limit it. Although several host processes have been described in which RNA interactions are believed to affect gene expression (for reviews, see references 6 and 26), none of these appear to be of critical importance to the cell, and it is thus possible that the principal role of PcnB is the modulation of plasmid replication. To investigate this question further, we have constructed a strain from which the pcnB gene has been deleted and report its properties here. We find pcnB to be a dispensable gene. MATERIALS AND METHODSBacterial strains, plasmids, and genetic techniques. The bacterial strains used are described in Table 1. Linear transformations were performed as described previously (25). The plasmids used in curing experiments, pBR325, the mini-R1 plasmid pGW71, the mini-F plasmids pSC138, pXX325, and pXX327, the mini-P1 plasmids pAX274 and pAX275, and pRE1-1, in which pcnB is expressed from a A PL, are also described in Table 1. pRE1-1 is maintained in MZ1, which is lysogenic for XcI857. Plasmids used for fusion protein production are described below. Other transformations, transductions, and conjugations were performed by using standard techniques, and media used were as previously described (19). Antibiotic concentrations used for routine growth were as follows: ampicillin, 10 ,ug/ml (mini-F * Corresponding author. and mini-P1 plasmids) or 50 ,ug/ml (ColEl-based plasmids); tetrac...

A gene, pcnB, affecting the copy number of ColE1-related plasmids has been cloned and mapped to 3.6 min on the Escherichia coli chromosome between panD and fhu. The gene encodes a previously undescribed 48 kD protein. Several independently isolated mutants exhibiting the same phenotype, reduced copy number, have been shown to be pcnB-.