Restoration by the rac locus of recombinant forming ability in recB? and recC? merozygotes of Escherichia coli K-12

Summary The recombinant forming ability of recB ^– or recC ^– strains of E. coli K12 is almost totally recovered in merozygotes which are heterozygous for a genetic locus denoted rac which is located five minutes clockwise from trp on the genetic map. This transient recovery phenomenon only occurs when the donor strain is rac ⁺ (wild type) and the recipient strain is rac ^-. The recombinants derived from such crosses all have the normal phenotype characteristic of recB ^– (or recC ^–) strains, and they are almost always rac ^-. The results imply that the rac ⁺ locus (or loci) is zygotically expressed and excised from the chromosome in a manner which is analogous to the zygotic induction of a prophage.     

Red-mediated recombination of phage lambda in a recA? recB?host

Summary The lambda Red recombination system works poorly among unreplicated gam ⁺ lambda chromosomes in recA ^- cells compared to recA ⁺ cells. Recombination is not enhanced in recA ^- recB^-cells. Thus, the inability of Red to promote recombination in recA ^- replication-blocked cross is not due to the hypothetical destruction of recombination intermediates by the recB nuclease. This conclusion strengthens previous proposals that the products of the red genes can operate upon recombinational intermediates which require recA activity for their formation.     

Isolation of Rec? mutants from an F-prime merodiploid strain of Escherichia coli K-12

Summary This paper describes a method of screening mutagenised populations of an E. coli gal _A ^– gal _B ^– F-prime merodiploid for mutants defective in recombination. The method relies on scoring colonies on Eosin-Methylene Blue agar that have fewer than normal numbers of Gal⁺ papillae. With a suitable choice of gal ^–mutations most of the papillae arise by recombination and some of those colonies with less than normal numbers prove to be defective in some aspect of recombination or DNA repair. In addition to strains carrying mutations that can be ascribed to known loci, several novel mutant phenotypes were identified.     

Ethanol-hypersensitive and ethanol-dependent cdc? mutants in Schizosaccharomyces pombe

Ethanol-hypersensitive strains (ets mutants), unable to grow on media containing 6% ethanol, were isolated from a sample of mutagenized Schizosaccharomyces pombe wild-type cells. Genetic analysis of these ets strains demonstrated that the ets phenotype is associated with mutations in a large set of genes, including cell division cycle (cdc) genes, largely non-overlapping with the set represented by the temperature conditional method; accordingly, we isolated some ets non-ts cdc ^– mutants, which may identify novel essential genes required for regulation of the S. pombe cell cycle. Conversely, seven well characterized ts cdc ^– mutants were tested for their ethanol sensitivity; among them, cdc1–7 and cdc13–117 exhibited a tight ets phenotype. Ethanol sensitivity was also tested in strains bearing different alleles of the cdc2 gene, and we found that some of them were ets, but others were non-ets; thus, ethanol hypersensitivity is an allele-specific phenotype. Based on the single base changes found in each particular allele of the cdc2 gene, it is shown that a single amino acid substitution in the p34^cdc2 gene product can produce this ets phenotype, and that ethanol hypersensitivity is probably due to the influence of this alcohol on the secondary and/or tertiary structure of the target protein. Ethanol-dependent (etd) mutants were also identified as mutants that can only be propagated on ethanol-containing media. This novel type of conditional phenotype also covers many unrelated genes. One of these etd mutants, etd1-1, was further characterized because of the lethal cdc ^– phenotype of the mutant cells under restrictive conditions (absence of ethanol). The isolation of extragenic suppressors of etd1-1, and the complementation cloning of a DNA fragment encompassing the etd1 ⁺ wild-type gene (or an extragenic multicopy suppressor) demonstrate that current genetic techniques may be applied to mutants isolated by using ethanol as a selective agent.     

Acetate accumulation through alternative metabolic pathways in ackA?pta?poxB? triple mutant in E. coli B (BL21)

Individual deletions of acs and aceA genes in E. coli B (BL21) showed little difference in the metabolite accumulation patterns but deletion of the ackA gene alone or together with pta showed acetic acid gradually accumulated to 3.1 and 1.7 g/l, respectively, with a minimal extended lag in bacterial growth and a higher pyruvate formation. Single poxB deletion in E. coli B (BL21) or additional poxB deletion in the ackA-pta mutants did not change the acetate accumulation pattern. When the acetate production genes (ackA-pta-poxB) were deleted in E. coli B (BL21) acetate still accumulated. This may be an indication that perhaps acetate is not only a by-product of carbon metabolism; it is possible that acetate plays also a role in other cellular metabolite pathways. It is likely that there are alternative acetate production pathways.     

Cell-cell interactions in conjugating Escherichia coli: Con? mutants and stabilization of mating aggregates

Summary Conjugation-deficient (Con^-) mutants of Escherichia coli K-12 have been previously described which were defective in recipient ability. Such Con^- mutants were obtained from several laboratories and retested by a standardized set of procedures. Many of the mutants did not satisfy minimal criteria for conjugation-deficiency and were discarded. The remaining mutants included 11 ConF^- mutants mutated in or near the ompA cistron, 3 ConF^- mutants synthesizing a heptose-deficient lipopolysaccharide and 1 ConI^- mutants synthesizing a defective lipopolysaccharide. This set of mutants was tested for resistance to a variety of bacteriophages and colicins; the only phenotype fully correlated with the ConF^- phenotype was that of resistance to colicin L. No simple correlation existed between the protein profile (on SDS polyacrylamide gel electrophoresis) of cell envelope outer membrane preparations and conjugation deficiency. However, many ConF^- mutants did not synthesize detectable levels of outer membrane protein II^* and protein II^* may have been nonfunctional in the remainder. All the ConF^- mutants were conjugation-deficient when matings were conducted in liquid but (with one exception) were conjugation-proficient on the surfaces of membrane filters. None of the ConF^- mutants formed stable mating aggregates in liquid with (Flac)⁺ donor cells although all bound purified F pili. The ConF^- phenotype associated with a II^*-deficient recipient could be mimicked by the addition of purified protein II^* (solubilized with lipopolysaccharide). In both cases, the formation of stable mating aggregates (analyzed with an improved Coulter counter technique) was inhibited whereas unstable mating aggregates were detected by electron microscopy. F pilus and wall to wall contacts were both observed under these conditions by electron microscopy. These results were used to define a stage in F-promoted conjugation, the stabilization stage, which requires the functional interaction of protein II^* and lipopolysaccharide in the outer membrane of the recipient cell.     

Assembly of nucleosomal DNA in a cell-free extract from wild-type and top1? strains ofUstilago maydis

An in vitro nucleosome assembly system has been established from cell-free extracts of the fungusUstilago maydis. The extract catalyzed DNA supercoiling in the absence of exogenously added co-factors such as ATP and MgCl₂ and was inhibited by moderate concentrations (200 mM) of KCl or NaCl. DNA supercoiling occurs via the formation of nucleosomes. Similar extracts, displaying the same activity, were prepared fromSaccharomyces cerevisiae andCandida albicans, suggesting that the extract preparation protocol may be useful for many lower eukaryotic systems. An extract prepared from a strain ofU. maydis lacking topoisomerase I failed to catalyze nucleosome assembly, clearly implicating this enzyme in this process. Addition of purified topoisomerase I, and, to a lesser extent, topoisomerase II, to the top1^– extract regenerated the supercoiling activity. Our results provide a method for preparing assembly extracts from organisms, that are particularly amenable to genetic manipulation.     

DnaK-dependent ribosome biogenesis in Escherichia coli? : competition for dominance between the alleles dnaK756 and dnaK ?+

The Escherichia coli chaperone DnaK is vital for many cellular functions, including ribosome biogenesis at high temperature. Thus, the dnaK756-ts (λ ^R ) mutant, at the non-permissive temperature, is inhibited at a late stage of ribosome assembly, yielding 21S, 32S and 45S precursor particles. This defect, unlike the λ resistance and thermosensitivity phenotypes, is not complemented by lysogenisation with a transducing phage λ dnaK ⁺ bearing the wild-type dnaK gene. However this dominant phenotype becomes recessive when dnaK ⁺ is expressed from a medium-copy-number plasmid. On the other hand, an excess of DnaK causes an unexpected dominant-lethal effect of the dnaK756 allele near non-permissive temperatures. This interplay between the dnaK ⁺ and dnaK756 alleles supports the idea of that DnaK oligomers form in the cell. Received: 28 April 1998 / Accepted: 24 July 1998     

Construction of a fusion gene comprising the Taka-amylase A promoter and the Escherichia coli β-glucuronidase gene and analysis of its expression in Aspergillus oryzae

Summary Northern blot analysis of glucose-grown and starch-grown mycelia of Aspergillus oryzae R11340 was conducted using the cloned Taka-amylase A (TAA) gene as a probe. The amount of mRNA homologous to the TAA gene was increased when this fungus was grown with starch as a sole carbon source. In order to analyze the induction mechanism, we inserted the Escherichia coli uidA gene encoding -glucuronidase (GUS) downstream of the TAA promoter and introduced the resultant fusion gene into the A. oryzae genome. Production of a functional GUS protein was induced by starch, but not by glucose. When the effects of various sugars on expression of the fusion gene were examined, the results suggested that the expression of the fusion gene was under control of the TAA gene promoter.     

Molecular cloning of a cysteine synthase cDNA from Citrullus vulgaris (watermelon) by genetic complementation in an Escherichia coli Cys? auxotroph

We have isolated cDNA clones encoding cysteine synthase (CSase, EC 4.2.99.8), which catalyzes the terminal step in cysteine biosynthesis, by direct genetic complementation of a Cys^– mutation in Escherichia coli with an expression library of Citrullus vulgaris (watermelon) cDNA. The library was constructed from 8-day-old etiolated seedlings of C. vulgaris in the ZAPII vector, converted to a plasmid library by in vivo excision, and then used for transformation of cysteine auxotroph E. coli NK3, which lacks the cysK and cysM loci. The complementing cDNA containing a 560 by 5-untranslated region encodes a polypeptide of 325 amino acids of M_r 34342. The translational product reacted with an antibody raised against CSase A of Spinacia oleracea. CSase and -pyrazolealanine synthase activities were demonstrated in vitro in extracts from E. coli cells expressing the cDNA. Genomic DNA blot analysis indicated the presence of a single copy of the gene, designated cysA, in the C. vulgaris genome. RNA blot hybridization indicated constitutive expression of cysA in cotyledons, hypocotyls and radicles of green and etiolated seedlings. These data suggested that this cDNA clone encodes CSase A the homolog of which in spinach is localized in the cytoplasm. The molecular phylogenetic tree of the amino acid sequences of CSaes from plants and bacteria suggested that there are three families in the CSase superfamily; the plant CSase A family, the plant CSase B family and the bacterial CSase family. The proteins in the plant CSase A family are the most conserved relative to the ancestral CSase protein.     

Thiolutin resistant mutants of Escherichia coli are they RNA chain initiation mutants?

Summary Four mutants of Escherichia coli KL16 resistant to the antibiotic Thiolutin have been isolated. This drug was earlier reported to be an inhibitor of RNA chain elongation. The first mutant, TL^rI, is resistant only in rich or partially rich media: it can, however, grow in minimal medium containing the drug with a very long doubling time. The other mutants TL^rII, TL^rIIIa and TL^rIIIb are resistant in rich as well as minimal media. -galactosidase could not be induced in TL^rI and TL^rII in the presence of thiolutin whereas the enzyme is constitutively synthesised in TL^rIIIa and TL^rIIIb irrespective of the drug.The mutants do not support the development of phage T4 in presence of the drug, if the drug is added along with the phage, but escape the inhibition if phage development is allowed to proceed for some time before the addition of the drug. The time of this escape is characteristic of the mutant. Even in a sensitive strain, T7 growth escapes inhibition very soon after infection, around the time the phage-specific RNA polymerase is synthesised. In the parent strain the kinetics of inhibition of -galactosidase induction resembles more the inhibition caused by rifampicin than by streptolydigin. It is proposed that thiolutin could be an inhibitor of RNA chain initiation and resistance might be due to mutation in the subunit(s)/factor(s) involved in initiation.     

Mutagenic specificity of N′-methyl-N′-nitro-N-nitrosoguanidine in the gpt gene on a chromosome of Chinese hamster ovary cells and of Escherichia coli cells

Summary DNA base sequence changes induced by N-methyl-N-nitro-N-nitrosoguanidine (MNNG) mutagenesis have been determined for the Escherichia coli gpt gene stably incorporated in a chromosome of Chinese hamster ovary cells and in the chromosome of both growing and starving E. coli cells, instead of on a plasmid as in most previous studies. In the three cases, nearly all mutations were G: C to A: T transitions, with a 2-to 4-fold higher mutation rate, compared to other sites, at guanines flanked on the 5 side by another guanine. Mutagenic hot spots in these experiments were less prominent than in published results for MNNG mutagenesis of gpt and of other genes. A suggested explanation involves repair of O⁶meG. At low levels of mutagenic products, most are repaired and even small differences in the repair rates leads to large differences in the relative amounts of residual O⁶meG at various sites; in contrast, at high levels of mutagenic products there is little effect of repair on the distribution.Abbreviations MNNG N-methyl-N-nitro-N-nitrosoguanidine - MNU N-methyl-N-nitrosourea - O⁶meG O⁶-methylguanine - N7meG N7-methylguanine - CHO Chinese hamster ovary     

Escherichia coli K-12 F? mutants that form mating aggregates but form transconjugants with low frequencies

Summary We have isolated Escherichia coli F^– mutants which, when mated with either Hfr or F, can form stable mating aggregates well but produce transconjugants with reduced frequencies. Selection procedure and other tests rule out the possibility that they are Rec^– strains. These mutants can be classified into two types: type I mutants can induce conjugal DNA replication in the donor, yet form transconjugants poorly; whereas, type II mutants induce conjugal DNA replication with poor efficiencies in the donor. Further tests indicate that type I mutants are very sensitive to lethal zygosis and their membranes, both inner and outer, show alterations in protein composition, whereas type II mutants are insensitive to lethal zygosis, and have an obvious alteration in the protein composition of their outer membrane. These results suggest that type I is defective in transconjugant formation primarily due to a change in the inner membrane, whereas type II is defective in generating a mating signal, which induces donor conjugal DNA replication, due to an alteration in the outer membrane.     

Rifampicin-induced protein synthesis: A pre-requisite for increased expression of the ββ′ operon in Escherichia coli

Summary In a rif ^S/rif^R heterodiploid strain of E. coli, a 4 minute pulse of rifampicin can induce a prolonged (>60 min) increase in the rate of synthesis of the RNA polymerase subunits, and . The application of a constraint on the fidelity of protein synthesis during, but not after, the rifampicin pulse partially arrests the development of this capacity for subunit synthesis. I discuss the implications of these findings in relation to the control of the operon in E. coli.     

Altered synthesis and stability of RNA polymerase holoenzyme subunits in mutants of Escherichia coli with mutations in the β or β′ subunit genes

Summary Bacteria with specific temperature sensitive lethal mutations in the gene for the subunit of RNA polymerase synthesize both the and subunits at a several fold higher rate at 42°C than wildtype cells relative to total protein. Synthesis of the and subunits proceeds at essentially the wild-type rates under these conditions. In contrast, a mutant with a temperature sensitive lethal mutation in the subunit gene synthesizes and at 42°C at slightly lower rates than wild-type, while and synthesis is not significantly altered. In all of the mutants at 42°C, newly synthesized subunits are stable, while the , and subunits are rapidly degraded. The apparent uncoupling of from subunit synthesis seen in the mutants at 42°C might suggest that the synthesis of these subunits is at least in part controlled by different mechanisms.     

Recombinant human hemoglobin: Expression and refolding of β-globin fromEscherichia coli

A plasmid analogous to the one described by Nagai and Thogersen (Nature,309, 810–812, 1984) has been constructed for the expression of globins inE. coli. Induction with nalidixic acid produces high yields of a fusion protein, NS1-FX--globin, where NS1 represents 81 residues of a flu virus protein and FX represents a blood-clotting Factor Xa recognition sequence, Ile-Glu-Gly-Arg. This fusion protein is readily solubilized in 50 mM NaOH and remains in solution when thepH is adjusted to 8.6. Under these conditions, the fusion protein is hydrolyzed by activated Factor X, giving authentic -globin which can be folded in the presence of cyanohemin and native -chains to produce a tetrameric hemoglobin with the functional properties of natural human hemoglobin.     

A ple?otropic acid phosphatase-deficient mutant of Escherichia coli shows premature termination in the dsbA gene. Use of dsbA :: phoA furions to localize a structurally important domain in DsbA

An Arabidopsis thaliana cDNA library was used to complement Saccharomyces cerevisiae pyrimidine auxotrophic mutants. Mutants in all but one (carbamylphosphate synthetase) of the six steps in the de novo pyrimidine biosynthetic pathway could be complemented. We report here the cloning, sequencing and computer analysis of two cDNAs encoding the aspartate transcarbamylase (ATCase; EC 2.1.3.2) and orotate phosphoribosyltransferase-orotidine-5-phosphate decarboxylase (OPRTase-OMP-decase; EC 2.4.2.10, EC 4.1.1.23) enzymes. These results confirm the presence in A. thaliana of a bifunctional gene whose product catalyses the last two steps of the pyrimidine biosynthetic pathway, as previously suggested by biochemical studies. The ATCase encoding cDNA sequence (PYRB gene) shows an open reading frame (ORF) of 1173 by coding for 390 amino acids. The cDNA encoding OPRTase-OMPdecase (PYRE-F gene) shows an ORF of 1431 by coding for 476 amino acids. Computer analysis of the deduced amino acid sequences of both cDNAs shows the expected high similarity with the ATCase, ornithine transcarbamylase (OTCase; EC 2.1.3.3), OPRTase and OMPdecase families. This heterospecific cloning approach increases our understanding of the genetic organization and interspecific functional conservation of the pyrimidine biosynthetic pathway and underlines its usefulness as a model for evolutionary studies.