Purine-mediated growth inhibition caused by a pyrE mutation in Escherichia coli K-12.

A purine-sensitive phenotype results from a previously described mutation in the structural gene (pyrE) for orotate phosphoribosyltransferase (OPT) in Escherichia coli K-12. OPT from both the mutant and the wild-type was partially inhibited by adenine and adenosine, although other purine derivatives were not effective for this inhibition. The Km values of the mutant OPT were 580 and 760 microM for orotate and 5'-phosphoribosyl-1'-pyrophosphate (PRib-PP), respectively, whereas the corresponding values for the wild-type OPT were 40 and 60 microM. The intracellular level of PRib-PP was decreased to less than 15% of the normal level when purine derivatives were added to exponentially growing cultures of both the parent and mutant strains. However, this decrease of the PRib-PP level was not found in strains derived from the mutant, in which the purine-sensitive phenotype was suppressed by a secondary mutation. The purine-sensitive phenotype was caused by retardation of the pyrimidine de novo pathway, when the intracellular level of PRib-PP was diminished by exogenously supplied purine derivatives.     

Characterization of an Escherichia coli K-12 F-Con-mutant.

An Escherichia coli K-12 F-mutant defective in conjugation was isolated by means of a zygotic induction enrichment procedure. The recipient ability of the mutant was reduced about 50 times owing to a block in one of the first steps of the conjugation process. In the mutant, cell envelope alterations could not be observed. Sensitivity toward detergents, antibiotics, and phages was unaltered. The mutation appeared to be co-transducible with pyrD. The linkage order in the region of the mutation is origin KL 99-con-pyrD-aroA.     

Study of a pts-gene-linked pleiotropic mutation influencing expression of catabolyte-sensitive genes of Escherichia coli K-12

Properties of the pleiotropic mutation pts17 are described. This mutation is liked to pts1 gene, which specifies the synthesis of the enzyme I of phosphoenolpyruvate-dependent phosphotransferase system (PTS) in Escherichia coli K-12. Genetic analysis has shown that pts17 mutation is located between purC and pts1 markers and that the wild type allele pts17+ has transdominant character over the mutant allele pts17. The mutant strain J6217, isogenic to parent J62, shows normal growth properties in the minimal salt media with a number of carbohydrates used as a single carbon source. The pts17 mutations does not affect the enzyme I activity, but significantly suppresses the total PTS activity in the bacterial cell extracts. The intact mutant cells reveal the enhanced rate of accumulation and phosphorylation of alpha-methylglucoside. The pts17 bacteria show 3-fold enhanced phosphohydrolase activity with glucose-6-phosphate as substrate. It is established that pts17 mutation decreases the differential rate of the L-tryptophanase synthesis and makes the process of unductions resistant to glucose catabolite repression. It is suggested that this mutation affects the activity of the PTS factor III. One can suppose that the latter mediates the influence of ptsI and ptsH mutations upon the expression of catabolite-sensitive operons in E. coli.     

A novel dnaC mutation that suppresses priB rep mutant phenotypes in Escherichia coli K-12

The loading of a replisome in prokaryotic and eukaryotic cells at an origin of DNA replication and during replication restart is a highly ordered and regulated process. During replication restart in Escherichia coli, the PriA, PriB, PriC, DnaT and Rep proteins form multiple pathways that bind to repaired replication forks. These complexes are then recognized by DnaC as sites to load DnaB, the replicative helicase. Several dnaC mutations have been isolated that suppress phenotypes of some replication restart mutants. A new dnaC mutation (dnaC824) is reported here that efficiently suppresses priB rep mutant phenotypes. Furthermore, it is shown that dnaC824 will suppress phenotypes of priB priA300, rep priA300 and priB priC strains. Unlike other dnaC suppressors, it can only weakly suppress the absence of priA. Others have reported a different type of dnaC mutation, dnaC1331, is able to mimic priB mutant phenotypes. This is supported herein by showing that like dnaC1331, a priB mutation is synthetically lethal with a dam mutation and this can be rescued by a mutH mutation. Furthermore, priB dam lethality can also be suppressed by dnaC824. Like a priB mutation, a dnaC1331 mutation causes a priA2::kan-like phenotype when combined with priA300. Lastly, we show that dnaC824 is dominant to wild type and that dnaC1331 is recessive to wild type. Several models are discussed for the action of these mutant dnaC proteins in replication restart.     

Escherichia coli K-12 mutation that inactivates biodegradative threonine dehydratase by transposon Tn5 insertion.

From a collection of kanamycin-resistant mutants of Escherichia coli K-12 isolated by transposon Tn5 mutagenesis, we have identified a mutant that lacks functional biodegradative threonine dehydratase (EC 4.2.1.16) by direct enzyme assay and by the loss of cross-reacting material with affinity-purified antibodies against the purified enzyme. Aerobic and anaerobic growth of this strain on various carbon sources failed to reveal a phenotype. Evidence for the insertional inactivation of threonine dehydratase by Tn5 was obtained by cloning the DNA segments flanking the Tn5 insertion site into pBR322 and hybridizing the cloned DNA to a synthetic oligodeoxynucleotide probe complementary to the DNA segment coding for a unique hexapeptide at the amino terminus end of the enzyme; the region of homology to the synthetic cDNA sequence appears to be located within about 500 nucleotides from one end of Tn5. Genetic analysis with the transposon element that caused insertional inactivation located the tdc gene at min 67 on the E. coli chromosome.     

Characterization of Pyruvate Uptake in Escherichia coli K-12

The monocarboxylate pyruvate is an important metabolite and can serve as sole carbon source for Escherichia coli. Although specific pyruvate transporters have been identified in two bacterial species, pyruvate transport is not well understood in E. coli. In the present study, pyruvate transport was investigated under different growth conditions. The transport of pyruvate shows specific activities depending on the growth substrate used as sole carbon source, suggesting the existence of at least two systems for pyruvate uptake: i) one inducible system and probably highly specific for pyruvate and ii) one system active under non-induced conditions. Using the toxic pyruvate analog 3-fluoropyruvate, a mutant was isolated unable to grow on and transport pyruvate. Further investigation revealed that a revertant selected for growth on pyruvate regained the inducible pyruvate transport activity. Characterization of pyruvate excretion showed that the pyruvate transport negative mutant accumulated pyruvate in the growth medium suggesting an additional transport system for pyruvate excretion. The here presented data give valuable insight into the pyruvate metabolism and transport of E. coli suggesting the presence of at least two uptake systems and one excretion system to balance the intracellular level of pyruvate.     

Characterization of V-Prime Factors in Escherichia coli K-12

An episome derived from an Hfr_v (Hfr isolated from a V colicinogenic parent) strain of Escherichia coli K-12 was isolated and characterized. The direction of gene transfer was inverted from that in the original parental strain.     

Chromosomal mutation for citrate utilization by Escherichia coli K-12.

A mutant strain of Escherichia coli K-12 that utilizes citrate as a sole source of carbon and energy was isolated. Citrate utilization arose as the consequence of two mutations in genes citA and citB, which are linked to the gal operon. The mutant strain expresses a semiconstitutive citrate transport system, and it utilizes both citrate and isocitrate as carbon and energy sources. It is capable of utilizing cis- and trans-aconitate, but only if it is preinduced by growth on citrate.     

Characterization of the pleiotropic phenotypes of rifampin-resistant rpoB mutants of Escherichia coli

We used our collection of 17 sequenced rifampin resistance alleles in rpoB to perform a systematic analysis of the phenotypes historically reported with this class of mutants, including growth phenotype, ability to support the growth of different bacteriophages, ability to maintain the F' episome, interaction with mutant alleles at other loci, sensitivity to uracil, inhibition by 5-fluorouridine, and dominance. We found that mutational changes leading to the same phenotype were often located together and that certain phenotypes were associated with one another.     

RecOR suppression of recF mutant phenotypes in Escherichia coli K-12.

The recF, recO, and recR genes form the recFOR epistasis group for DNA repair. recF mutants are sensitive to UV irradiation and fail to properly induce the SOS response. Using plasmid derivatives that overexpress combinations of the recO+ and recR+ genes, we tested the hypothesis that high-level expression of recO+ and recR+ (recOR) in vivo will indirectly suppress the recF mutant phenotypes mentioned above. We found that overexpression of just recR+ from the plasmid will partially suppress both phenotypes. Expression of the chromosomal recO+ gene is essential for the recR+ suppression. Hence we call this RecOR suppression of recF mutant phenotypes. RecOR suppression of SOS induction is more efficient with recO+ expression from a plasmid than with recO+ expression from the chromosome. This is not true for RecOR suppression of UV sensitivity (the two are equal). Comparison of RecOR suppression with the suppression caused by recA801 and recA803 shows that RecOR suppression of UV sensitivity is more effective than recA803 suppression and that RecOR suppression of UV sensitivity, like recA801 suppression, requires recJ+. We present a model that explains the data and proposes a function for the recFOR epistasis group in the induction of the SOS response and recombinational DNA repair.     

Isolation of an Escherichia coli K-12 dnaE mutation as a mutator.

Using a papillation method, a large number of Escherichia coli K-12 mutator mutations have been isolated. Only one of these (out of 1,250) mutator mutations has proved to be conditionally lethal at high temperatures. In vivo complementation tests indicated that this mutation, dnaE9, lies in dnaE, the structural gene for DNA polymerase III. The dnaE9 polymerase was not thermolabile in vitro; however, it showed a slow decline in specific activity in vivo at the nonpermissive temperature. Cultures of this mutant exhibited a comparably slow shutoff of DNA synthesis on shift to a nonpermissive temperature. dnaE9 showed temperature-sensitive mutator activity, which is not dependent on recA.     

Characterization of lexB mutations in Escherichia coli K-12.

Two mutations have been located at the recA locus and phenotypically characterized along with a third one, previously called rec-34. The three mutants behaved similarly to lexA mutants. They were sensitive to ultraviolet (UV) light and X rays, and lambdaFec- phages were able to plate on them. The three mutations were called lexB because they could be distinguished from recA mutations by the last property. lexB mutants were less sensitive to UV and X irradiations than were recA mutants and were, to various degrees, recombination proficient. UV light failed to induce prophage lambda in all three lexB lysogens. In contrast, thymine starvation induced lexB31 and lexB34 lysogens. In lexB34 mutants, but not in lexB30 and lexB31 mutants, UV reactivation occurred at a low level. In Escherichia coli K-12, the recA gene has basic functions in the repair of deoxyribonucleic acid lesions, deoxyribonucleic acid recombination, and prophage induction. The three lexB mutations alter unequally and independently the three functions. This suggests that the recA and lexB mutations affect the same gene.     

Over-representation of Chi sequences caused by di-codon increase in Escherichia coli K-12

Chi sequences (5'-GCTGGTGG-3') are cis-acting 8 bp sequence elements that enhance homologous recombination promoted by the RecBCD pathway in Escherichia coli. The genome of E. coli K-12 MG1655 contains 1009 Chi sequences and this frequency far exceeds the expected value for occurrence of an 8 bp sequence in a genome of this size. It is generally thought that the over-representation of Chi sequences indicates that they have been selected for during evolution because of their function in recombination. The genes from three E. coli strains (K-12, O157 and CFT) were classified into three categories (island, match to other E. coli, and backbone). Island genes have a different base composition and codon usage in comparison with those in the backbone genes, therefore they were relatively new and not yet adapted to the base composition patterns and codon usage typical of the recipient genome. The over-representation of Chi sequences was examined by comparing Chi frequencies and codon frequencies between island and backbone genes. The difference in the CTGGTG di-codon frequency between the backbone and island genes was correlated with the frequency of Chi sequences which were translated in the Leu-Val (-G/CTG/GTG/G-) reading frame in the K-12 strain. These results suggest that the main reading frame of Chi sequences increased as a result of the di-codon CTG-GTG increasing under a genome-wide pressure for adapting to the codon usage and base composition of the E. coli K-12 strain, and that the RecBCD recombinase might adjust its recognition sequence to a frequently occurring oligomer such as G-CTG-GTG-G.     

Stepwise selection of defective nitrogen-fixing phenotypes in Escherichia coli K-12 by dimethyl sulfoxide.

Storage in dimethyl sulfoxide of Escherichia coli K-12 hybrids carrying Klebsiella pneumoniae nif+ genes can result in selection of a defective nitrogen-fixing phenotype. Dimethyl sulfoxide appears to affect particular inner membrane proteins associated with energy metabolism in E. coli K-12.     

Trans-dominant mutation affecting restriction and modification in Escherichia coli K-12

Summary We have analysed the mechanism of action of a ts mutation in E. coli, which has an effect on the expression of the restriction and modification phenotype. The frequencies of recombinants obtained in transduction experiments support the idea that the temperature sensitive mutation is located outside the hsd operon in the gene denoted hsd. X. Complementation experiments demonstrated the trans-dominant nature of the temperature sensitive mutation. The possible role of the hsd.X product in the formation of EcoR.K and EcoM.K complexes and their interaction with the recognition site on the DNA is discussed.     

Identification of a mutation affecting an alanine-alpha-ketoisovalerate transaminase activity in Escherichia coli K-12.

Summary A mutation affecting alanine--ketoisovalerate transaminase activity has been shown to be cotransducibe with the ilv gene cluster. The transaminase deficiency results in conditional isoleucine auxotrophy in the presence of alanine.