Subdomain organization and catalytic residues of the F factor TraI relaxase domain.
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TraI from conjugative plasmid F factor is both a "relaxase" that sequence-specifically binds and cleaves single-stranded DNA (ssDNA) and a helicase that unwinds the plasmid during transfer. Using limited proteolysis of a TraI fragment, we generated a 36-kDa fragment (TraI36) retaining TraI ssDNA binding specificity and relaxase activity but lacking the ssDNA-dependent ATPase activity of the helicase. Further proteolytic digestion of TraI36 generates stable N-terminal 26-kDa (TraI26) and C-terminal 7-kDa fragments. Both TraI36 and TraI26 are stably folded and unfold in a highly cooperative manner, but TraI26 lacks affinity for ssDNA. Mutational analysis of TraI36 indicates that N-terminal residues Tyr(16) and Tyr(17) are required for efficient ssDNA cleavage but not for high-affinity ssDNA binding. Although the TraI36 N-terminus provides the relaxase catalytic residues, both N- and C-terminal structural domains participate in binding, suggesting that both domains combine to form the TraI relaxase active site. (+info)
Characterizing the structural features of RNA/RNA interactions of the F-plasmid FinOP fertility inhibition system.
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F-like plasmid transfer is mediated by the FinOP fertility inhibition system. Expression of the F positive regulatory protein, TraJ, is controlled by the action of the antisense RNA, FinP, and the RNA-binding protein FinO. FinO binds to and protects FinP from degradation and promotes duplex formation between FinP and traJ mRNA, leading to repression of both traJ expression and conjugative F transfer. FinP antisense RNA secondary structure is composed of two stem-loops separated by a 4-base single-stranded spacer and flanked on each side by single-stranded tails. Here we show that disruption of the expected Watson-Crick base pairing between the loops of FinP stem-loop I and its cognate RNA binding partner, traJ mRNA stem-loop Ic, led to a moderate reduction in the rate of duplex formation in vitro. In vivo, alterations of the anti-ribosome binding site region in the loop of FinP stem-loop I reduced the ability of the mutant FinP to mediate fertility inhibition and to inhibit TraJ expression when expressed in trans at an elevated copy number. Alterations of intermolecular complementarity between the stems of these RNAs reduced the rate of duplex formation. Our results suggest that successful interaction between stem-loop I of FinP and stem-loop Ic of traJ mRNA requires that base pairing must proceed from an initial loop-loop interaction through the top portion of the stems for stable duplex formation to occur. (+info)
Spontaneous zygogenesis in Escherichia coli, a form of true sexuality in prokaryotes.
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A new type of mating, differing from classic conjugation and previously observed in a certain strain of Escherichia coli K-12, has also been found in strains derived from ordinary F- cells of E. coli K-12 exposed to an exogenous factor originating in an E. coli clinical isolate. Immunofluorescence and electron microscopy after single and double labelling of DNA were used to produce evidence in favour of a novel mating mechanism by cell contact at the poles of the bacterial rod. These findings are supported by genetic analyses indicating complete genetic mixing. Unstable complementing diploids were formed, which throw off phenotypically haploid cells, of which some showed a parental phenotype and some were true genetic recombinants. Recombination was observed even when one parent was a UV-inactivated F- RecA- strain. The name 'spontaneous zygogenesis' (Z-mating, for short) is proposed for this kind of mating. (+info)
Modulation of DNA supercoiling activity of Escherichia coli DNA gyrase by F plasmid proteins. Antagonistic actions of LetA (CcdA) and LetD (CcdB) proteins.
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The letA (ccdA) and letD (ccdB) genes of F plasmid contribute to stable maintenance of the plasmid in Escherichia coli cells; a product of the latter has a lethal effect on the host cell and that of the former neutralizes functions of the letD. In cells that overproduce the LetD (CcdB) protein, the plasmid DNA is extensively relaxed. Correspondingly, DNA supercoiling activity in a cell-free extract of the overproducing strain decreases to a level of less than 1% of that seen in normal cells. However, the extract does not inhibit DNA gyrase reconstituted from purified subunits, thereby indicating that the intrinsic DNA gyrase is inactivated in the overproducing strain. Upon addition of purified LetA (CcdA) protein to the extract of LetD overproducing cells, the DNA supercoiling activity was fully restored. Using this rejuvenation as an assay, we purified the "inactivated gyrase" and obtained evidence that the LetD protein formed an isolable complex with the A subunit of DNA gyrase. Thus, the LetD and the LetA proteins constitute an opposing pair in modulating the DNA supercoiling activity of gyrase, probably by direct interaction. (+info)
Random diffusion can account for topA-dependent suppression of partition defects in low-copy-number plasmids.
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The maintenance of partition-defective (Par-) mini-P1 and mini-F plasmids was studied in topA strains of Escherichia coli, which are defective in topoisomerase I activity. The partition defects were substantially but not completely suppressed in broth-grown cultures. This suppression was not due to a large increase in copy number. However, the absolute number of copies of Par- mini-P1 plasmids per average dividing cell is sufficiently high to account for the modest stability observed if a random distribution of the copies to daughter cells is assumed. The similar number of Par- plasmid copies in wild-type cells are distributed in a considerably worse-than-random fashion. Thus, it is unnecessary to propose, as was suggested previously, that an active, par-independent pathway operates in topA strains to ensure proper segregation of the plasmids to daughter cells. Rather, it seems likely that the lack of topoisomerase I activity aids the random distribution of the partition-defective plasmids, perhaps by facilitating their separation after replication. The results of studies carried out at reduced growth rates were consistent with this view; when topA cells containing Par- mini-P1 plasmids were cultured in minimal medium, in which the copy number of the plasmids per average cell is sharply reduced, very little suppression of the partition defect was observed. (+info)
Transfer of Tn916 between Lactococcus lactis subsp. lactis strains is nontranspositional: evidence for a chromosomal fertility function in strain MG1363.
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Lactococcus lactis subsp. lactis MG1363 can act as a conjugative donor of chromosomal markers. This requires a chromosomally located fertility function that we designate the lactococcal fertility factor (Laff). Using inter- and intrastrain crosses, we identified other L. lactis strains (LMO230 and MMS373) that appear to lack Laff. The selectable marker in our crosses was Tcr, carried by Tn916, a transposon present on the chromosome. The transfer of Tcr was not due to Tn916-encoded conjugative functions, because (i) L. lactis cannot act as a donor in Tn916-promoted conjugation (F. Bringel, G. L. Van Alstine, and J. R. Scott, Mol. Microbiol. 5:2983-2993, 1992) and (ii) transfer occurred when the Tcr marker was present in a Tn916 derivative containing a mutation, tra-641, that prevents Tn916-directed conjugation in any host. In addition, we isolated a strain in which Tn916 appears to be linked to Laff; this strain should be useful for further analysis of this fertility factor. In this strain, Tn916 is on the same 600-kb SmaI fragment as Clu, a fertility factor previously shown to promote lactose plasmid transfer in L. lactis. Thus, it is possible that Clu and Laff are identical. (+info)
Further evidence that transposition of Tn5 in Escherichia coli is strongly enhanced by constitutively activated RecA proteins.
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We have shown that excision and transposition of Tn5 in Escherichia coli are greatly increased by recA(Prtc) genes, which encode constitutively activated RecA proteins (C.-T. Kuan, S.-K. Liu, and I. Tessman, Genetics 128:45-57, 1991). Contrary results, showing a significant decrease in Tn5 transposition under SOS conditions, were subsequently reported (M. D. Weinreich, J. C. Makris, and W. S. Reznikoff, J. Bacteriol. 173:6910-6918, 1991). We have extended our studies to examine the following: (i) transposition of Tn5 from sites in the phoA, phoB, proC, trpD, and ilvD genes; (ii) the effect of gene transcription; (iii) the comparative effect of dinD+ and dinD(Def) alleles; (iv) the use of a mating-out assay of transposition; (v) the effect of a recA(Prtc) allele located at the normal chromosomal site; and (vi) the effect at 41.5 degrees C of the recA441(Prtc) allele. The new results fully confirm our previous conclusions, including the fact that the frequency of Tn5 transposition under constitutive SOS conditions is site dependent. (+info)
Filamentation promotes F'lac loss in Escherichia coli K12.
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The stability of plasmid F'lac in Escherichia coli strain SP45 (a temperature conditional mutant which grows as spherical cells at 42 degrees C and as a rod at 30 degrees C) was studied. F'lac elimination was demonstrated when bacteria exposed to subinhibitory concentrations of various chemicals were induced to form filaments. No plasmid loss was found when spherical cells were subjected to the same treatments. Plasmid loss was also observed in dnaA46 and lexA41 mutants when cell filamentation was induced at 42 degrees C, but not when they were cultured at 30 degrees C. Nalidixic acid promoted F'lac elimination at 0.25 micrograms ml-1 in a recA13 mutant and at 1.5 micrograms ml-1 in the recA+ counterpart. A marked difference was found in the rate of F'lac elimination from thermosensitive DNA gyrase mutants [gyrA43(Ts) and gyrB41(Ts)] between rods and their spherical (rodA51) derivatives growing at semipermissive temperature (36.5 degrees C). Plasmids carrying the ccd segment of F in DNA gyrase mutants were lost after 2.5 generations from rods and after 6 generation from spherical cells. Plasmid segregation into non-viable minicell-like elements was found after induction of filaments. These data suggest that plasmid stability is correlated with cell shape and that curing is more easily achieved when bacteria can elongate normally. (+info)