The pcnB gene of Escherichia coli, which is required for ColE1 copy number maintenance, is dispensable

Abstract

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 new Escherichia coli cell division gene, ftsK

“…The TOE44 mutation was then mapped more precisely by P1 cotransduction with various transposons at known locations (Genetic Stock Center, National Institute of Genetics) and finally to between 20.1 and 20.3 min (on the collated map of K. E. Rudd, contributed to reference 37) by recombination with phages carrying overlapping chromosomal fragments (214 and 215 [30]). DNA from this region proved difficult to maintain in a plasmid vector except in a pcnB mutant host (35), in which the copy number is greatly reduced, but a single clone, containing part of the region common to both 214 and 215, was obtained by subcloning a 6.3-kbp BamHI fragment from 215 into pUC19. The TOE44 pcnB strain (C600 ftsK44 pcnB) is complemented by this plasmid (pSU44), so that colonies are formed on plates at 42ЊC, although cells growing at this temperature are somewhat longer than normal cells.…”

“…Chromosomal DNA for PCR analysis was obtained from TOE44, NM306 (F Ϫ thi-1 ilv-192 argH1 metB1 xyl-7 hisG1 lacY1 tnaA1 strA8,9 tsx-7 tonA2 supE44 trp uhp pyrE purA), and MM28-2 (F Ϫ argG6 asnA31 asnB32 his-1 leuB6 metB1 pyrE gal6 lacI lacY1 xyl-7 supE44 fhuA2 gyrA rpsL104 tsx-1 uhp pcnB recA56 tonA) (35).…”

Regulation of plasmid R1 replication: PcnB and RNase E expedite the decay of the antisense RNA, CopA

“…RNase E encoded by the rne-3071 allele is inactivated at 44ЊC. Strains N3433⌬pcnB and N3431⌬pcnB were constructed by P1 transduction of a pcnB deletion allele from strain MM38 (a Kan R cassette replaces pcnB ; Masters et al, 1993). For plasmid constructions, we used strain DH5␣ (Hanahan, 1985; Table 3).…”

“…The pcnB mutation also affects the copy number of the IncFII plasmid R1, although to a lesser extent (2-to 3-fold reduction; Masters et al, 1993) than it does that of ColE1 (Ϸ10-fold reduction; He et al, 1993;Masters et al, 1993). Furthermore, as is the case with RNA I in ColE1-containing cells, two forms of CopA are found in R1-containing cells, a full-length form of Ϸ90 nt, CopA-FL, and an Ϸ65-nt-long 3Ј segment, SL-E, which is a cleavage product previously hypothesized to be generated by RNase E (Blomberg et al, 1990).…”

Defects in RNA polyadenylation impair both lysogenization by and lytic development of Shiga toxin-converting bacteriophages

“…E. coli MG1655 strain (Jensen, 1993) and its isogenic DpcnB :: kan derivative (Masters et al, 1993;Wró bel et al, 1998) were used in this study. The following bacteriophages were used: l (papa), W24 B (Dstx2 :: cat) (Allison et al, 2003), 933WDtox (Dstx2 :: catGFP), 22Dtox (Dstx2 :: catGFP), 27Dtox (Dstx2 :: catGFP) and 32Dtox (Dstx2 :: catGFP); derivatives of phages 933W, 22, 27 and 32 were described previously (Gamage et al, 2004), they are devoid of the toxin genes (due to a safety concern) but have all other features of the original phages with cat and/or GFP markers; these are referred to as W24 B , 933W, P22, P27 and P32, respectively, in the text.…”