Nucleotide sequence of the uhp region of Escherichia coli.

The Escherichia coli uhp region encodes the transport system that mediates the uptake of a number of sugar phosphates as well as the regulatory components that are responsible for induction of this transport system by external glucose 6-phosphate. Four uhp genes have been identified by analysis of the complementation behavior and polypeptide coding capacity of plasmids carrying subcloned regions or transposon insertions. The nucleotide sequence of a 6.5-kilobase segment that contains the 3' end of the ilvBN operon and the entire uhp region was determined. Four open reading frames were identified in the locations expected for the various uhp genes; all were oriented in the same direction, counterclockwise relative to the genetic map. The properties of the polypeptides predicted from the nucleotide sequence were consistent with their observed features. The 196-amino-acid UhpA polypeptide has the composition characteristic of a soluble protein and bears homology to the DNA-binding regions of many regulatory activators and repressors. The 518-amino-acid UhpB and the 199-amino-acid UhpC regulatory proteins contain substantial segments of hydrophobic character. Similarly, the 463-amino-acid UhpT transporter is a hydrophobic protein with numerous potential transmembrane segments. The UhpC regulatory protein has substantial sequence homology to part of UhpT, suggesting that this regulatory protein might have evolved by duplication of the gene for the transporter and that its role in transmembrane signaling may involve sugar-phosphate-binding sites and transmembrane orientations similar to those of the transport protein.     

Deletions Generated by the Transposon Tn10 in the srl recA Region of the ESCHERICHIA COLI K-12 Chromosome

A negative regulatory gene for the srl operon (srlR) was recognized by the characteristics of an insertion mutation generated by the transposon Tn10 determining tetracycline resistance. This finding is discussed in light of previous hypotheses on the regulation of the srl genes, which mediate metabolism of glucitol (i.e., sorbitol). Mapping showed that the order of genes in this region is: srlR srlD srlC recA alaS. Using two different methods, five mutations of both srl and recA were detected. The phenotype conferred by these mutations, UV sensitivity and extreme recombination deficiency, is characteristic of standard recA point mutants. Three of the mutations were deletions that also removed the genes for tetracycline resistance of the nearby transposon. A fourth mutation ended at a distance from Tn10 sufficient to allow separation of the two by recombination following P1 transduction; our tests did not allow us to conclude whether this mutation was an inversion or a deletion. The fifth mutation was a deletion that seemed to end immediately adjacent to the boundary of Tn10, proximal to recA. Mechanisms for the generation of these srl recA mutations are discussed.     

Genetic Control of the Transport of Hexose Phosphates in Escherichia coli: Mapping of the uhp Locus

A number of mutations affecting the transport of hexose phosphates in Escherichia coli were ordered within the uhp locus. Three-point crosses by transduction or conjugation allowed the ordering of the alleles relative to the adjacent pyrE marker. The same linear map was obtained by both methods. This, combined with the regulatory properties of revertants of these mutants, allowed a tentative identification of two genes, one presumably coding for the transport system (uhpT) and the other(s) specifying a regulatory element (uhpR). The order of these is pyrE-uhpT-uhpR. Mutants exhibiting constitutive expression of the transport system were isolated. This behavior is genetically linked to the uhp locus, but more precise localization was not possible.     

Genetic analysis of the Escherichia coli K-12 srl region.

Specialized transducing lambda derivatives, deletion mapping, and Plkc transductional crosses have been used to analyze the genetic organization and regulation of the srl genes. Transducing phages obtained from a secondary site lambda insertion in srlA are of two types: lambdapsrlC1 and lambdaprecA are substituted in the b2 region of the lambda chromosome (galtype) and carry the srlC gene but not srlD; lambdapsrlD is substituted in the early region of the phage deoxyribonucleic acid (biotype) and carries the srlD gene but not srlC. The lambdapsrlC1 phage, which lysogenizes at attlambda, complements srlC mutants in trans, indicating that this gene codes for a diffusable positive regulatory element. The srl genes have been ordered relative to the cysC, recA, and alaS genes by two- and three-factor P1kc crosses. The order, cysC...srlD-srlA-srlC-recA-alaS, has been obtained. The srlA and srlD genes comprise an operon with srlD operator distal. From the secondary site lysogen, it has been possible to obtain deletion mutants of this region that are sensitive to ultraviolet light and are recombination deficient. Genetic evidence suggests that these deletions extend from srl into the recA gene.     

Location of the gene specifying hexose phosphate transport (uhp) on the chromosome of Escherichia coli.

The uhp gene, which specifies the uptake of hexose phosphates, and several other genes in the vicinity of minute 81 on the E. coli linkage map have been located by phage-mediated transductions. The order found is mtl-gpsA-pyre-gltc-uhp-tna-dnaa. Alleles specifying the Uhp- and Uhp+ characters were separated from that specifying constitutivity of hexose phosphate uptake (Uhpc). Although cotransduction frequencies between gltC and uhp as high as 90%, and between uhp and tna as high as 80%, were observed, these frequencies were unusually strongly dependent on which marker was selected. This may be due to the proximity of the uhp region to the point of origin of chromosome replication.     

Isolation and Characterization of Mutations Affecting the Transport of Hexose Phosphates in Escherichia coli

Mutants of Escherichia coli defective in the hexose phosphate transport system were isolated. Negative selection by penicillin treatment or positive selection with phosphonomycin was employed. These mutants grew normally on all carbon sources other than hexose phosphates. The map location of the mutations in 18 independently isolated mutant strains was investigated by transduction crosses. All of the mutations were found to lie in the same region of the chromosome, in the region represented by min 72 on the Taylor map. The order of the genes in this region was found to be mtl-cysE-pyrE-uhp-bgl-ilv. Revertants of some of the mutants exhibited altered regulatory control of this transport system.     

A Genetic Characterization of the Nadc Gene of Salmonella Typhimurium

The nadC gene of Salmonella encodes the pyridine biosynthetic enzyme PRPP-quinolinate phosphoribosyltransferase. Using a combination of genetic techniques, a deletion map for the Salmonella nadC gene has been generated which includes over 100 point mutants and 18 deletion intervals. The nadC alleles obtained by hydroxylamine mutagenesis include those suppressed by either amber, ochre, or UGA nonsense suppressors as well as alleles suppressed by the missense suppressor, sumA. Deletions were obtained by three separate protocols including spontaneous selection for loss of the nearby aroP gene, recombination between aroP::MudA and nadC::MudA insertion alleles, and selection for spontaneous loss of tetracycline resistance in a nearby guaC::Tn10dTc insertion mutant allele. The nadC mutants comprise one complementation group and the nadC+ allele is dominant to simple, nadC auxotrophic mutant alleles. Intragenic complementation of two nadC alleles, nadC493 and nadC494, mapping to deletion intervals 17 and 18, respectively, suggests that nadC encodes a multimeric enzyme. Both nadC and the nearby aroP locus are transcribed counterclockwise on the standard genetic map of Salmonella, in opposite orientation to the direction of chromosome replication.     

Analysis of tetracycline resistance encoded by transposon Tn10: deletion mapping of tetracycline-sensitive point mutations and identification of two structural genes

Deletions in the tet genes derived from Tn10 were formed from different tet::Tn5 insertion mutations by removing DNA sequences located between a HindIII site in Tn5 and a HindIII site adjacent to the tet genes. Tetracycline-sensitive point mutations were mapped in recombination tests with the deletions and were thus aligned with the genetic and physical map of the tet region. Plasmids carrying point mutations were tested for complementation with derivatives of pDU938, a plasmid carrying cloned tet genes derived from Tn10 which had been inactivated by Tn5 insertions. Complementation occurred between promoter-proximal tet point mutations and distal tet::Tn5 insertions, suggesting the existence of two structural genes, tetA and tetB. These results, together with the analysis of polypeptides in minicells harboring pDU938tet::Tn5 mutants, suggested that tetA and tetB are expressed coordinately in an operon. The tetB gene encodes the previously characterized 36,000-dalton cytoplasmic membrane TET protein, but the product of tetA was not identified. Point mutations in either tetA or tetB led to the defective expression of the resistance mechanism involving tetracycline efflux. It is suggested that the tetA and tetB products interact cooperatively in the membrane to express resistance.     

Fine-structure map of the histidine transport genes in Salmonella typhimurium.

Afine-structure genetic map of the histidine transport region of the Salmonella typhimurium chromosome was constructed. Twenty-five deletion mutants were isolated and used for dividing the hisJ and hisP genes into 8 and 13 regions respectively. A total of 308 mutations, spontaneous and mutagen induced, have been placed in these regions by deletion mapping. The histidine transport operon is presumed to be constituted of genes dhuA, hisJ, and hisP, and the regulation of the hosP and hisJ genes by dhuA is discussed. The orientation of this operon relative to purF has been established by three-point crosses as being: purF duhA hisJ hisP.     

Fine Structure Genetic and Physical Map of the Phage P22 Tail Protein Gene

Bacteriophage P22 which are incapable of making functional tail protein can be propagated by the addition of purified mature tail protein trimers to either liquid or solidified medium. This unique in vitro complementation condition has allowed us to isolate 74 absolute lethal tail protein mutants of P22 after hydroxylamine mutagenesis. These phage mutants have an absolute requirement for purified P22 tail protein to be present in a soft agar overlay in order to form plaques and do not grow on any nonsense suppressing strains of Salmonella typhimurium. In order to genetically map and physically locate these mutations we have constructed two complementary sets of fine structure deletion mapping strains using a collection of Tn1 insertions in gene 9, the structural gene for the tail protein. Fourteen bacteriophage P22 strains carrying unique Tn1 transposon insertions (Ap phage) in gene 9 have been crossed with Ap phage carrying Tn1 insertions in gene 20. Phage carrying deletions that arose from homologous recombination between the Tn1 elements were isolated as P22 lysogens. The deletion prophage were shown to be missing all genetic information bracketed by the parental Tn1 elements and thus form a set of deletions into gene 9 from the 5' end of the gene. From the frequency of production of these deletion phage the orientation of the Tn1 insertions in gene 9 could be deduced. The genetic end points of the deletions in gene 9 and thus the order of Tn1 insertions were determined by marker rescue experiments using the original Ap phage. The genetic end points of the deletions in gene 20 were determined in similar experiments using nonsense mutations in gene 20. To locate the physical end points of these deletions in gene 9, DNA containing the Tn1 element has been cloned from each of the original Ap phage into plasmids. The precise point of insertion of Tn1 into gene 9 was determined by restriction enzyme mapping and DNA sequencing of the relevant portions of each of these plasmids. In vitro deletion of different 3' gene 9 sequences in the plasmid clones was accomplished through the use of unique restriction endonuclease sites in Tn1. The resulting plasmids form a set of deletions extending into the 3' end of the gene which are complementary compared to the deletion lysogens.(ABSTRACT TRUNCATED AT 400 WORDS)     

Genetic recombination of bacterial plasmid DNA: effect of RecF pathway mutations on plasmid recombination in Escherichia coli.

Tn5 insertion mutations in the recN gene, and in what appears to be a new RecF pathway gene designated recO and mapping at approximately 55.4 min on the standard genetic map, were isolated by screening Tn5 insertion mutations that cotransduced with tyrA. The recO1504::Tn5 mutation decreased the frequency of recombination during Hfr-mediated crosses and increased the susceptibility to killing by UV irradiation and mitomycin C when present in a recB recC sbcB background, but only increased the sensitivity to killing by UV irradiation when present in an otherwise Rec+ background. The effects of these and other RecF pathway mutations on plasmid recombination were tested. Mutations in the recJ, recO, and ssb genes, when present in otherwise Rec+ E. coli strains, decreased the frequency of plasmid recombination, whereas the lexA3, recAo281, recN, and ruv mutations had no effect on plasmid recombination. Tn5 insertion mutations in the lexA gene increased the frequency of plasmid recombination. These data indicate that plasmid recombination events in wild-type Escherichia coli strains are catalyzed by a recombination pathway that is related to the RecF recombination pathway and that some component of this pathway besides the recA gene product is regulated by the lexA gene product.     

Unequal crossing-over in conjugated crossings in Escherichia coli K-12

A genetic model has been developed allowing to study non-equal crossingover in conjugative crossings of Escherichia coli. The model is based on obtaining tandem duplications in the region of the deo operon of E. coli in conjugative crosses Hfr x Hfr. It was shown that when using transposon (Tn5, Tn10) genetic markers the majority of recombinant progeny phenotypically corresponding to rare 2-crossover and 4-crossover recombinants for the deo operon constitute direct tandem duplications resulting from non-equal interchromosomal exchange. Apart from the deo operon, the duplications obtained cover, in some cases, linked thr and leu loci. In the crosses, where point mutations for genes of the deo operon served as genetic markers, the frequency of duplications' formation was lower than that of rare 2-crossover and 4-crossover recombinants' formation. Evolutionary role of the non-equal crossingover phenomenon in E. coli is discussed.     

The transposon Tn9 generates a 9 bp repeated sequence during integration.

We performed a genetic and sequencing analysis of insertions of the transposon Tn9 into the lac operon of E. coli. Genetic mapping of 70 insertions into lacl and Z shows that starting from the same point on the chromosome, Tn9 goes to at least 50 different points in these two genes. Although there are preferred regions for insertion, these consist of multiple integration points within a small area, as demonstrated by pairwise crosses and restriction mapping. Sequence analysis of three Tn9 insertions reveals that Tn9 integration is associated with a direct repeat of 9 base pairs (bp) of host sequence. We show that these extra 9 nucleotide pairs are generated upon insertion and not brought in with the element.     

Genetic analysis of mutations that compensate for loss of Escherichia coli DNA topoisomerase I

A transposon Tn10 insertion in topA, the structural gene of Escherichia coli DNA topoisomerase I, behaves as an excluded marker in genetic crosses with many strains of E. coli. However, derivative strains that accept this mutant topA allele are readily selected. We show that many of these topA mutant strains contain additional mutations that compensate for the loss of DNA topoisomerase I. Genetic methods for mapping and manipulating such compensatory mutations are described. These methods include a plate-mating test for the ability of strains to accept a topA::Tn10 allele and a powerful indirect selection for transferring compensatory mutations from male strains into non-compensatory female strains. One collection of spontaneous compensatory mutants is analyzed in detail and is shown to include compensatory mutations at three distinct loci: gyrA and gyrB, the genes that encode the subunits of DNA gyrase, and a previously unidentified locus near tolC. Mutations at this third locus, referred to as toc (topoisomerase one compensatory) mutations, do not behave as point mutations in transductional crosses and do not result in lowered DNA gyrase activity. These results show that wild-type strains of E. coli require DNA topoisomerase I, and at least one class of compensatory mutations can relieve this requirement by a mechanism other than reduction of DNA gyrase activity.