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.     

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.     

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.     

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.     

Characterization of a ts β′ mutant RNA polymerase ofEscherichia coli

Summary RNA polymerase isolated from ts XH56, a conditional lethal mutant unable to grow and synthesize RNA at 42°, was found to be temperature sensitive in vitro. The mutation affects the subunit as determined by mixed reconstitution of isolated subunits from wild-type and mutant enzyme. The mutant RNA polymerase is unstable; addition of glycerol stabilizes the enzyme and increases its activity on native DNA. In addition, the mutant enzyme is sensitive to high ionic strength. Both high temperature and high ionic strength do not affect chain elongation; thus, the mutation renders the enzyme unable either to bind to or melt out promotor sites. From these data we conclude that the subunit plays an important role in promotor selection.     

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.     

lamB mutations in E. coli K12: Growth of λ host range mutants and effect of nonsense suppressors

Summary Over sixty EMS induced mutations affecting gene lamB, presumably the structural gene for the receptor in Escherichia coli K12, were examined for growth of host range mutants and effect of nonsense suppressors. By the first criterion the mutations could be grouped in three classes. Bacteria with class I mutations allow growth of mutants with extended host range (noted h) of the type already described (Appleyard, Mac Gregor and Baird, 1956). Bacteria with class II mutations allow growth of h mutants with still more extended host range (noted hh ^*). No host range mutants of could be found which would grow on bacteria with class III mutations. Using nonsense suppressors it was found that class I and II consist of missense mutations, while class III consists of nonsense mutations. Exceptions are likely to exist (especially in class III) but were not found among the mutations tested. These observations are briefly discussed in terms of outer membrane protein integration and of phage receptor interaction.     

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.     

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     

Expression of the plasmid pKM101-determined DNA repair system in recA? and lex? strains of Escherichia coli

Summary pKM101, a plasmid R factor of the N compatibility group increases methylmethane sulfonate mutagenesis and diminishes UV-killing in recA ⁺ lex ⁺ and recA ⁺ lex ^– strains, but not in recA ^– lex ⁺ strains. The induction of a reclex dependent colicin is not present in lex ^– strains carrying the pKM101 factor. These facts indicate that pKM101 acts through an error-prone DNA repair system, which is recA ⁺ dependent, but not lex ⁺ dependent.This paper is published on the occasion of Dr. C. Callerio's seventy-fifth birthday     

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.     

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.     

2-Aminopurine and 5-bromouracil induce alleviation of type I restriction in Escherichia coli: Mismatches function as inducing signals?

Summary The EcoK restriction of unmodified phage is 1000-fold alleviated in Escherichia coli grown in the presence of base analogs 2-aminopurine (2AP) and 5-bromouracil (5BU). 2AP treatment of bacteria affects specificially the type I restriction systems (EcoA, EcoB, EcoD and EcoK) and does not influence type II (EcoRI) and type III (EcoP1) restriction. 2AP-induced alleviation of restriction occurs in bacteria which are deficient in the SOS response (recA and lexA) and mismatch repair (mutH, mutL and mutS) and can be distinguished from the alleviation of restriction observed in dam ^- strains. We suggest that mismatches induced by 2AP and 5BU may function as an inducing signal for the alleviation of restriction observed in the presence of base analogs.     

σs-dependent overexpression offtsZ in anEscherichia coli K-12 rpoB mutant that is resistant to the division inhibitors DicB and DicF RNA

TheEscherichia coli genesdicF anddicB encode division inhibitors, which prevent the synthesis and activity, respectively, of the essential division protein FtsZ. A mutation at the C-terminal end of the RNA polymerase subunit renders cells resistant to both inhibitors. In the mutant strain the level of theftsZ gene product is higher than in the wild type. Disruption ofrpoS, which encodes the stationary phase sigma factor ^S, lowers FtsZ protein levels in the mutant, and partially restores sensitivity to the inhibitors.     

Genetic complementation of rhizobial nod mutants with Frankia DNA: artifact or reality?

Two divergent reports have been published on the genetic complementation of rhizobial nod mutants using Frankia DNA. In 1991 putative Frankia cosmid library clones were reported to restore normal nodulation properties to Rhizobium leguminosarum biovar viciaenodD::Tn5, but no supporting sequence data were published. In 1992 a second group reported a failure to find any evidence of functional complementation of various rhizobial nod mutants by Frankia DNA (nodA, nodB and nodC). Complementation tests of nine Nod⁻ R. leguminosarum bv. viciae or Sinorhizobium meliloti Tn5 mutants (nodA ⁻ , nodB ⁻ , nodC ⁻ , nodD ⁻ , nodF  ⁻ , nodL ⁻ , nodH ⁻ ) were thus performed using a Frankia gene library in pLAFR3 to clarify this situation. Rhizobial transconjugants obtained by tri-parental matings were screened for restoration of the nodulation phenotype on their host plants, Vicia sativa subsp. nigra or Medicago sativa. Nodulation was observed on plants inoculated with transconjugants of the R. leguminosarum bv. viciaenodC::Tn5 mutant. The Nod⁺ rhizobial transconjugants were isolated and analysed. The Nod⁺ phenotype of these transconjugants was found to be due to Tn5 excision/transposition. No functional complementation was found with any of the mutants used, suggesting that rhizobial complementation of nod mutants with Frankia DNA is unlikely to occur. Received: 17 April 1998 / Accepted: 22 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.