Recombination and the Escherichia coli K-12 sex factor F.

Recombination between two Flac tra minus elements to give Flac tra plus recombinants was measured in Rec plus and Rec minus strains of Escherichia coli K-12. Polar tra mutations were used to increase the proportion of tra plus recombinants among the parental Flac tra minus elements transferred by complementation. The kinetics, measured in a rec plus strain, showed that recombination began about 1 h after the initiation of mating and was completed about 1 h later. Recombination was abolished in a recA minus strain, reduced by two-thirds in a recF minus strain, and unaffected in recB minus and recC minus strains. It is proposed that the part not due to the RecF pathway results from a RecBC- and RecF-independent system for formation of single-stranded joins. One such join could be followed either by transfer and a site-specific recombination event, or by a second single-stranded join and then transfer: in either case replication and inheritance of the recombinant molecule would be dependent upon the F transfer replication system. Chromosome mobilization by an F' element was normal in a recB plus recF minus strain, and was reduced only fourfold in a recB minus recF plus strain: in the latter strain, both the RecF pathway and the system for single-stranded joins may have contributed to mobilization. Measurement of post-conjugational chromosomal recombination in exponential-phase recipient cells carrying surface exclusion-deficient Flac mutants indicated that F does not itself determine a generalized recombination system able to replace the RecA plus product or the RecBC and RecF pathways.     

cis-Dominant, transfer-deficient mutants of the Escherichia coli K-12 F sex factor.

Rare conjugational progeny formed by crossing each of five Hfr strains with a recA-F- strain have been characterized. Selection was made for a proximal Hfr marker, taking strict precautions to prevent transfer of recA+ to the zygotes. Most of the progeny were found to be F' strains containing deletion mutant plasmids. With two exceptions, these mutant plasmids have lost all of the tra genes, which are required to confer conjugational donor ability upon a host. In addition, all but the exceptional mutant plasmids were found to be very poorly transmissible from transient heterozygotes which also contain a wild-type F' plasmid. The poor transmissibility is a cis-dominant transfer-defective phenotype which may result from deletion of all or part of the origin of transfer replication (ori), or of a gene determining a cis-acting protein. The two exceptional mutant plasmids may carry short deletions of some of the tra genes or polar tra mutations. The remaining progeny were nonmutant F' strains and F- strains. The frequency with which the F- strains were recovered permits us to estimate that the maximum amount of recombination possible in a recA56 zygote is 10(-6) that of a recA+ zygote.     

Nucleotide sequence of the traD region in the Escherichia coli F sex factor

The complete nucleotide sequence has been determined of a 3635-bp region, extending from the HpaI site in traT, at F coordinate 90.3 kb, to beyond the end of traD, of the F sex factor plasmid of Escherichia coli K-12. This region contains the C-terminal coding part of traT and the entire traD gene. An open reading frame (ORF) of 2148 bp within the sequence confirms that traD encodes an 81.4-kDa cytoplasmic membrane protein. The TraD protein has several regions with an unusually high pI (greater than 10), suggesting that they may correspond to the DNA-binding domains. Several other ORFs were detected within the region including the gene (ORF1) for a 26.3-kDa protein and ORF2, probably corresponding to traI, which continues to the end of the sequence. An ORF for an 8.5-kDa protein preceded by an excellent promoter and ribosome-binding site is present in the region following traD but on the opposite strand. This promoter is thought to correspond to the major RNA polymerase binding site in this region, implying that traI does not have its own promoter. The lack of a typical terminator following traD and ORF1 and the translational coupling provided by overlapping stop and start codons is consistent with this conclusion.     

Five control systems preventing transfer of Escherichia coli K-12 sex factor F.

The transfer inhibition systems of 28 Fin+ plasmids have been characterized, using Flac mutants insensitive to inhibition by R100 or R62. All F-like plasmids (except R455) and one N group plasmid determined systems analogous to that of R100; this is designated the FinOP system. None of these plasmids could supply a FinP component of the transfer inhibitor able to replace that of F itself. In addition to the FinOP and R62 transfer inhibition systems described previously, new systems were encoded by the F-like plasmid R455, the I-like plasmid JR66a, and the group X plasmid R485. Besides inhibiting F transfer, JR66a also inhibited F pilus formation and surface exclusion, whereas R485 inhibited only pilus formation and R455 inhibited neither. All three R factors inhibited transfer of J-independent Flac elements, indicating that they act directly on one or more genes (or products) of the transfer operon, rather than directly via traJ. The tral products and transfer origin sequences of two Fin+ F-like plasmids, ColB2 and R124, appear to have similar specificities to those of F itself.     

Biochemical characterization of nonintegrated plasmid-folded chromosome complexes: sex factor F and the Escherichia coli nucleoid.

The existence of nonintegrated plasmid-chromosome complexes has been deduced in previous work from the cosedimentation of covalently closed, circular plasmids with host folded chromosomes. In the present work, it is shown that about 70 to 90% of the covalently closed, circular F deoxyribonucleic acid could be released in vitro from chromosome complexes by ribonuclease treatment but not by protease, Sarkosyl, or ethidium bromide. Consistent with the in vitro studies, Escherichia coli cells treated for 5 min with rifampin, an inhibitor of ribonucleic acid initiation, released upon lysis 90% of their plasmid deoxyribonucleic acid as freely sedimenting molecules.     

Bending of the bacteriophage lambda attachment site by Escherichia coli integration host factor

Escherichia coli integration host factor (IHF) is a small basic protein that is required for efficient integrative recombination of bacteriophage lambda. IHF binds specifically to sequences within attP, the site in bacteriophage lambda that undergoes recombination. It has been suggested that the binding of IHF creates bends in DNA so as to help attP condense into a compact structure that is activated for recombination. In this work we show that IHF binding to either of two sites found within attP does indeed produce bending of DNA. In contrast, the other recombination protein needed for integrative recombination, Int, does not appreciably bend the DNA to which it is bound. In agreement with the proposal that IHF bending is important for creating a condensed attP, bending by IHF persists in the presence of bound Int. Our conclusions about protein-directed bends in DNA are based on the study of the electrophoretic mobility of a set of permuted DNA fragments in the presence or absence of IHF and/or Int. To facilitate this study, we have constructed a novel vector that simplifies the generation of permuted fragments. This vector should be useful in studying the bending of other DNA sequences by specific binding proteins.     

Suppression by thymidine-requiring mutants of Escherichia coli K-12.

Thymidine-requiring strains of Escherichia coli isolated by trimethoprim selection often simultaneously acquire the ability to suppress bacteriophage T4 nonsense mutations. Suppression is lost in Thy+ revertants and recombinants, but is sometimes retained in thyA plasmid-bearing transformants. Suppression is restricted in Strr derivatives of the Thy- mutants, indicating that suppression occurs at the level of translation.     

Growth phase variation of integration host factor level in Escherichia coli.

We have measured the intracellular abundance of integration host factor (IHF), a site-specific, heterodimeric DNA-binding protein, in exponential- and stationary-phase cultures of Escherichia coli K-12. Western immunoblot analysis showed that cultures that had been growing exponentially for several generations contained 0.5 to 1.0 ng of IHF subunits per microgram of total protein and that this increased to 5 to 6 ng/microgram in late-stationary-phase cultures. IHF is about one-third to one-half as abundant in exponentially growing cells as HU, a structurally related protein that binds DNA with little or no site specificity. Wild-type IHF is metabolically stable, but deletion mutations that eliminated one subunit reduced the abundance of the other when cells enter stationary phase. We attribute this reduction to the loss of stabilizing interactions between subunits. A mutation that inactivates IHF function but not subunit interaction increased IHF abundance, consistent with results of previous work showing that IHF synthesis is negatively autoregulated. We estimate that steady-state exponential-phase cultures contain about 8,500 to 17,000 IHF dimers per cell, a surprisingly large number for a site-specific DNA-binding protein with a limited number of specific sites. Nevertheless, small reductions in IHF abundance had significant effects on several IHF-dependent functions, suggesting that the wild-type exponential phase level is not in large excess of the minimum required for occupancy of physiologically important IHF-binding sites.     

Suppression of recJ mutations of Escherichia coli by mutations in translation initiation factor IF3.

We have isolated genetic suppressors of mutations in the recJ gene of Escherichia coli in a locus we term srjA. These srjA mutations cause partial to complete alleviation of the recombination and UV repair defects conferred by recJ153 and recJ154 mutations in a recBC sbcA genetic background. The srjA gene was mapped to 37.5 min on the E. coli chromosome. This chromosomal region from the srjA5 strain was cloned into a plasmid vector and was shown to confer recJ suppression in a dominant fashion. Mutational analysis of this plasmid mapped srjA to the infC gene encoding translation initiation factor 3 (IF3). Sequence analysis revealed that all three srjA alleles cause amino acid substitutions of IF3. Suppression of recJ was shown to be allele specific: recJ153 and recJ154 mutations were suppressible, but recJ77 and the insertion allele recJ284::Tn10 were not. In addition, growth medium-conditional lethality was observed for strains carrying srjA mutations with the nonsuppressible recJ alleles. When introduced into recJ+ strains, srjA mutations conferred hyperrecombinational and hyper-UVr phenotypes. An interesting implication of these genetic properties of srjA suppression is that IF3 may regulate the expression of recJ and perhaps other recombination genes and hence may regulate the recombinational capacity of the cell.     

Properties of new Escherichia coli Hfr strains constructed by integration of pSC101-derived conjugative plasmids.

Conjugative temperature-sensitive plasmids were derived from pSC101. These plasmids are useful in genetic analysis for two reasons: (i) they render possible the construction of new Hfr lines by plasmid integration at predetermined chromosomal loci via Tn10 inverse transposition, and (ii) the Hfr characters are transducible via bacteriophage P1. We also showed that replication from pSC101 origin is deleterious for the plasmid-chromosome fusion.     

Control of cell division by sex factor F in Escherichia coli. III. Participation of the groES (mopB) gene of the host bacteria

Cell division of F+ bacteria is coupled to DNA replication of the F plasmid. Two plasmid coded genes, letA (ccdA) and letD (ccdB) are indispensable for this coupling. To investigate bacterial genes that participate in this coupling, we attempted to identify the target of the division inhibitor (the letD gene product) of the F plasmid. Two temperature-sensitive growth defective mutants were screened from bacterial mutants that escaped the letD product growth inhibition that occurs in hosts carrying an FletA mutant. Phage P1-mediated transduction and complementation analysis indicated that the temperature-sensitive mutations are located in the groES (mopB) gene, which is essential for the morphogenesis of several bacteriophages and also for growth of the bacteria. The nucleotide sequence of the promoter region of the gene in which the temperature-sensitive mutations had occurred was virtually identical with that of the groES gene of Escherichia coli; furthermore the sequence of the first five amino acid residues and the overall amino acid composition predicted from the nucleotide sequence of the gene match those of the purified GroES protein. The temperature-sensitive mutants did not allow the propagation of phage lambda at 28 degrees C and formed long filamentous structures without septa at 41 degrees C, as is observed in the case of groES mutants. Growth of the two groES mutants tested was not inhibited by the F plasmid with the letA mutation. These observations suggest to us that the morphogenesis gene groES plays a key role in coupling between replication of the F plasmid and cell division of the host cells.     

Stabilization of bacteriophage Mu repressor-operator complexes by the Escherichia coli integration host factor protein

All of the previously described effects of integration host factor (IHF) on bacteriophage Mu development have supported the view that IHF favours transposition-replication over the alternative state of lysogenic phage growth. In this report we show that, consistent with a model in which Mu repressor binding to its operators requires a particular topology of the operator DNA, IHF stimulates repressor binding to the O1 and O2 operators and enhances Mu repression. IHF would thus be one of the keys, besides supercoiling and the H-NS protein, that lock the operator region into the appropriate topological conformation for high-affinity binding not only of the phage transposase but also of the phage repressor.     

Outer membrane of Escherichia coli: properties of the F sex factor traT protein which is involved in surface exclusion.

The traT protein (TraTp) of the F sex factor is the product of one of the two genes involved in surface exclusion. Several detergents were examined under different conditions in order to determine their ability to solubilize TraTp from membrane vesicles. These experiments showed that TraTp behaved similar to a number of peptidoglycan-associated outer membrane proteins and that it existed in multimeric aggregates within the membrane. However, unlike other major outer membrane proteins, the amount of TraTp incorporated into the membrane was not affected by lipopolysaccharide-deficient mutants, even when mutants totally lacking the neutral sugars in their lipopolysaccharide backbone were used. TraTp wqs also examined by two-dimensional gel electrophoresis, where it ran as a discrete spot with a very basic isoelectric point. By coupling cyanogen bromide-activated dextran onto whole cells and by labeling whole cells with 125I (via lactoperoxidase), it was shown that TraTp was exposed on the cell surface. TraTp in a membrane environment was also insensitive to proteolytic attack by trypsin.