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.     

L-arabinose transport systems in Escherichia coli K-12.

Mutations in the arabinose transport operons of Escherichia coli K-12 were isolated with the Mu lac phage by screening for cells in which beta-galactosidase is induced in the presence of L-arabinose. Standard genetic techniques were then used to isolate numerous mutations in either of the two transport systems. Complementation tests revealed only one gene, araE, in the low-affinity arabinose uptake system. P1 transduction placed araE between lysA (60.9 min) and thyA (60.5 min) and closer to lysA. The operon of the high-affinity transport system was found to contain two genes: araF, which codes for the arabinose-binding protein, and a new gene, araG. The newly identified gene, araG, was shown by two-dimensional gel electrophoresis to encode a protein which is located in the membrane. Only defects in araG could abolish uptake by the high-affinity system under the conditions we used.     

Stringent control of peptidoglycan biosynthesis in Escherichia coli K-12.

[3H]Diaminopimelic acid (Dap) was incorporated exclusively into peptidoglycan by Escherichia coli strains auxotrophic for both lysine and Dap. The rate of [3H]Dap incorporation by stringent (rel+) strains was significantly decreased when cells were deprived of required amino acids. The addition of chloramphenicol to amino acid-starved rel+ cultured stimulated both peptidoglycan and ribonucleic acid synthesis. In contrast, a relaxed (relA) derivative incorporated [3H]Dap at comparable rates in the presence or absence of required amino acids. Physiologically significant concentrations of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) inhibited the in vitro synthesis of both carrier lipid-linked intermediate and peptidoglycan catalyzed by a particulate enzyme system. The degree of inhibition was dependent on the concentration of ppGpp in the reaction mixture. Thus, the results of in vivo and in vitro studies indicate that peptidoglycan synthesis is stringently controlled in E. coli.     

Cloning and analysis of the sfrB (sex factor repression) gene of Escherichia coli K-12.

The sfrB gene of Escherichia coli K-12 and the rfaH gene of Salmonella typhimurium LT2 are homologous, controlling expression of the tra operon of F and the rfa genes for lipopolysaccharide synthesis. We have determined a restriction map of the 19-kilobase ColE1 plasmid pLC14-28 which carries the sfrB gene of E. coli. After partial Sau3A digestion of pLC14-28, we cloned a 2.5-kilobase DNA fragment into the BamHI site of pBR322 to form pKZ17. pKZ17 complemented mutants of the sfrB gene of E. coli and the rfaH gene of S. typhimurium for defects of both the F tra operon and the rfa genes. pKZ17 in minicells determines an 18-kilodalton protein not determined by pBR322. A Tn5 insertion into the sfrB gene causes loss of complementing activity and loss of the 18-kilodalton protein in minicells, indicating that this protein is the sfrB gene product. These data indicate that the sfrB gene product is a regulatory element, since the single gene product elicits the expression of genes for many products for F expression and lipopolysaccharide synthesis.     

Promoter-distal region of the tra operon of F-like sex factor R100 in Escherichia coli K-12.

The distal region of the tra (transfer) operon of F-like plasmid R100 was investigated, using small plasmids derived from R100, primarily the plasmid pSM6. The transposon Tn5 (which confers kanamycin resistance) was inserted at different positions into pSM6, and the transposition derivatives were tested for ability to complement defined tra mutants of the F sex factor. Thus, the tra genes traH, G, T, and D were localized on the plasmid R100. A restriction map of pSM6 was constructed, and the locations of the insertions were mapped, using restriction endonuclease digestion of the plasmid DNA and exploiting the fact that several restriction sites are localized in the inverted repeat regions of the transposon. The gene products of the genes traG, S, T, and D were identified by radioactive labeling of proteins synthesized in minicells carrying the various insertion plasmids followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of another transfer gene, traI, was inferred from these data. Another protein, the r2-A protein, was also identified, and its gene was mapped. On the basis of the data, a best-fit physical map of this region of the tra operon of R100 was constructed. The results confirmed that the general order and size of the distal transfer genes is as in the F sex factor, but showed that differences exist with respect to all of the gene products. The significance of these differences are discussed in the light of the genetic and physical homology (Manning et al., J. Bacteriol. 150:76-88) of the transfer regions.     

Multiplicity of leucine transport systems in Escherichia coli K-12

The major component of leucine uptake in Escherichia coli K-12 is a common system for l-leucine, l-isoleucine, and l-valine (LIV-I) with a Michaelis constant (K(m)) value of 0.2 muM (LIV-I system). The LIV-binding protein appears to be associated with this system. It now appears that the LIV-I transport system and LIV-binding protein also serve for the entry of l-alanine, l-threonine, and possibly l-serine. A minor component of l-leucine entry occurs by a leucine-specific system (L-system) for which a specific leucine-binding protein has been isolated. A mutant has been obtained that shows increased levels of the LIV-I transport activity and increased levels of both of the binding proteins. Another mutant has been isolated that shows only a major increase in the levels of the leucine-specific transport system and the leucine-specific binding protein. A third binding protein that binds all three branched-chain amino acids but binds isoleucine preferentially has been identified. The relationship of the binding proteins to each other and to transport activity is discussed. A second general transport system (LIV-II system) with a K(m) value of 2 muM and a relatively low V(max) can be observed in E. coli. The LIV-II system is not sensitive to osmotic shock treatment nor to growth of cells in the presence of leucine. This high K(m) system, which is specific for the branched-chain amino acids, can be observed in membrane vesicle preparations.     

Uroporphyrin-accumulating mutant of Escherichia coli K-12.

An uroporphyrin III-accumulating mutant of Escherichia coli K-12 was isolated by neomycin. The mutant, designated SASQ85, was catalase deficient and formed dwarf colonies on usual media. Comparative extraction by cyclohexanone and ethyl acetate showed the superiority of the former for the extraction of the uroporphyrin accumulated by the mutant. Cell-free extracts of SASQ85 were able to convert 5-aminolevulinic acid and porphobilinogen to uroporphyrinogen, but not to copro- or protoporphyrinogen. Under the same conditions cell-free extracts of the parent strain converted 5-aminolevulinic to uroporphyringen, coproporphyrinogen, and protoporphyrinogen. The conversion of porphobilinogen to uroporphyrinogen by cell-free extracts of the mutant was inhibited 98 and 95%, respectively, by p-chloromercuribenzoate and p-chloromercuriphenyl-sulfonate, indicating the presence of uroporphyrinogen synthetase activity in the extracts. Spontaneous transformation of porphobilinogen to uroporphyrin was not detectable under the experimental conditions used [4 h at 37 C in tris(hydroxymethyl)aminomethane-potassium phosphate buffer, pH 8.2]. The results indicate a deficient uroporphyrinogen decarboxylase activity of SASQ85 which is thus the first uroporphyrinogen decarboxylase-deficient mutant isolated in E. coli K-12. Mapping of the corresponding locus by P1-mediated transduction revealed the frequent joint transduction of hemE and thiA markers (frequency of co-transduction, 41 to 44%). The results of the genetic analysis suggest the gene order rif, hemE, thiA, metA; however, they do not totally exclude the gene order rif, thiA, hemE, metA.     

Genetic control of recombination exchange frequency in Escherichia coli K-12.

The frequency of recombination exchanges per unit length of DNA (Freuld) can be estimated by measuring the scale of the genetic map that is the mean statistical distance between two neighboring crossovers. The scales appear to be equal for the alternative pathways of recombination, RecBCD (wild-type cells) or RecF (recBC- sbcB- sbcC- genotypes). The absolute value of the scale depends on specific experimental conditions. recR, recQ, ruv, recJ and recN genes of the RecF pathway of recombination (recBC- sbcBC- cell genotypes) do not appear to be silent in wild-type cells where the RecBCD pathway predominates. On the contrary, these genes are responsible for the Freuld. The list recF504::Kmr greater than recQ61::Tn3 greater than ruv-54 greater than recJ284::Tn10 shows decreasing efficiency in inhibiting recombination exchanges by these mutations. The recN264 mutation gives a small, but opposite effect of increasing the frequency of recombination exchanges. The effect of the recF and recQ mutations appears to be additive, but that is not the case in combinations of ruv-54 with recF504::Kmr or recQ61::Tn3.     

RNA-polymerase binding sites within the tra region of the F factor of Escherichia coli K-12

Chimeric plasmids containing the tra operon of the Escherichia coli K-12 F factor were used to map by electron microscopy the RNA polymerase binding sites within the contiguous F EcoRI restriction fragments f6, f16, f1, f17, f19 and f2. [These fragments have been previously cloned in the EcoRI site of pSC101 to give the chimeric plasmids pRS27 (f6, f15), pRS29 (f15, f1) and pRS31 (f17, f19 and f2)]. The results may reflect the presence of a number of previously unrecognized promoters within the traY----Z operon.     

F factor-mediated immunity to lethal zygosis in Escherichia coli K-12

Recipient (F(-)) cells of Escherichia coli are sensitive to an excess of Hfr donor cells. This phenomenon of lethal zygosis is associated with conjugation and is observed as a continuous fall in F(-) viable cells during liquid mating, or as inhibition of F(-) growth on solid media. One class of survivors, which arose in the zones of inhibition on solid media, was no longer sensitive to lethal zygosis and exhibited the following properties: sensitivity to male-specific phage, donor ability, and surface exclusion. Since these characteristics were sensitive to acridine orange treatment, the strains carry an F factor extrachromosomally. They are, however, defective in some way since they retain sensitivity to female-specific phage. Temporary sensitivity to lethal zygosis in these and in standard F(+) strains can be induced by the formation of F(-) phenocopies. We have suggested that there is an immunity to lethal zygosis (Ilz(+)) associated with the F factor and discuss the results in terms of this hypothesis.