Transfer ofF′ fromEscherichia coli K 12 toEscherichia coli B and to strains ofParacolobacter andKlebsiella

The transfer of theF episome fromEscherichia coli K 12 toE. coli B,Paracolobacter andKlebsiella was studied. The frequency of transfer of the episomal markers toE. coli B was very low. The large majority ofE. coli B cells which had received the episomal markerslac ⁺ orgal ⁺ were F^–, which indicates that the episomal markers were stably integrated on the chromosome. Recombinants from K 12 F⁺ × B F^– crosses were mostly F^–. These results suggest that the multiplication of theF-factor ofE. coli K 12 is restricted inE. coli B. The transfer of theF-lac ⁺ Ad ⁺ episome fromE. coli K 12 toParacolobacter andKlebsiella strains was in most cases only possible when donor and acceptor strain were plated together on selective media. Stable incorporation of episomal markers was also found withParacolobacter coliforme. Paracolobacter aerogenoides andKlebsiella aerogenes strains could be infected withF-lac ⁺ Ad ⁺. The episomal markers were not incorporated and the episomes were easily lost, which indicates that these strains contained theF factor in the autonomous state.     

Effects of puromycin aminonucleoside on excision repair and recombination repair inescherichia coli

Ultraviolet (UV) lethality was increased when puromycin aminonucleoside (PAN) (3.0 mM) was added to the postirradiation medium ofEscherichia coli strains. The extent of repair inhibition differed greatly for strains WP-2hcr ⁺, B/r()hcr ⁺, WP-2hcr ^–, and Bs-1hcr ^–. The interaction between PAN and UV was synergistic in thehcr ⁺ strains. PAN enhanced UV lethality in strain B/r () to a greater degree than in WP-2hcr ⁺. There was no UV lethality enhancement by PAN (3.0 mM) in thehcr ^– strains, but the interaction of PAN (8.0 mM) with UV was synergistic. PAN decreased plaque formation of T1 UV-irradiated phage plated onE. coli Bhcr ⁺ but had no effect on phage plated on Bs-1 or WP-2hcr ^– strains. These results suggest that PAN interferes with thehcr function in UV-irradiated bacteria.     

A direct comparison of approaches for increasing carbon flow to aromatic biosynthesis inEscherichia coli

Different approaches to increasing carbon commitment to aromatic amino acid biosynthesis were compared in isogenic strains ofEscherichia coli. In a strain having a wild-type PEP: glucose phosphotransferase (PTS) system, inactivation of the genes encoding pyruvate kinase (pykA andpykF) resulted in a 3.4-fold increase in carbon flow to aromatic biosynthesis. In a strain already having increased carbon flow to aromatics by virtue of overexpression of thetktA gene (encoding transketolase), thepykA and/orpykF mutations had no effect. A PTS glucose⁺ mutant showed a 1.6-fold increase in carbon flow to aromatics compared to the PTS⁺ control strain. In the PTS^– glucose⁺ host background, overexpression oftktA caused a further 3.7-fold increase in carbon flow, while inactivation ofpykA andpykF caused a 5.8-fold increase. When all of the variables tested (PTS^– glucose⁺,pykA, pykF, and overexpressedtktA) were combined in a single strain, a 19.9-fold increase in carbon commitment to aromatic biosynthesis was achieved.     

The action of nitrosoguanidine and other DNA-inactivating agents on Streptococcus pyogenes K56 strains with normal and reduced dark repair ability

Summary The response pattern for ultraviolet light, nitrogen mustard, and ethyl methane sulphonate of Hcr⁺ and Hcr^- strains ofStreptococcus pyogenes K 56 is similar to that observed for analogous strains ofE. coli, whereas repair-apt streptococcal strains are much more sensitive to nitrosoguanidine and mitomycin C thanE. coli. Theuvr gene(s) appear(s) to be without effect upon survival, prophage induction, and mutation to streptomycin resistance caused by nitrosoguanidine and only of little influence on repair of mitomycin C damage.     

The influence of pH and external H+ concentration on caesium toxicity and accumulation inEscherichia coli andBacillus subtilis

Summary Toxicity screening ofEscherichia coli NCIB 9484 andBacillus subtilis 007, NCIB 168 and NCIB 1650 has shown Cs⁺ to be the most toxic Group 1 metal cation. However, toxicity and accumulation of Cs⁺ by the bacteria was affected by two main external factors; pH and the presence of other monovalent cations, particularly K⁺. Over the pH range 6–9 bothE. coli andB. subtilis showed increasing sensitivity towards caesium as the pH was raised. The presence of K⁺ and Na⁺ in the laboratory media used lowered caesium toxicity and lowered acumulation of the metal. In order to assess accurately Cs⁺ toxicity towards the bacterial strains it was therefore necessary to define the K⁺:Cs⁺ ratio in the external medium. The minimum inhibitory K⁺:Cs⁺ concentration ratio for theBacillus strains tested was in the range 12–13 whileE. coli had a minimum inhibitory K⁺:Cs⁺ concentration ratio of 16.     

Genes,enzymes and membrane proteins of the nitrate respiration system ofEscherichia coli

Summary A method was devised to isolate mutants carrying deletions through several genetic loci (chlD ⁺ andchlA ⁺) which are involved in the membrane-bound nitrate respiratory complex ofEscherichia coli. Specific transducing phages were used to reintroduce these genes. Comparisons of membrane fractions from these transduced strains showed five membrane proteins that are necessary for the formation of an active nitrate respiration system. Two particular bacterial genes (chlD ⁺ andchlA ⁺) were shown to control these five membrane proteins.Three of the proteins specified bychlA ⁺, appear to be constitutively controlled and always present in the membrane ofE. coli irrespective of growth conditions, while the other two proteins, specified bychlD ⁺, appear to be induced byanaerobic growth in the presence of nitrate.     

The bacterial phleomycin resistance geneble as a dominant selectable marker inChlamydomonas

A chimeric gene composed of the coding sequence of theble gene fromStreptoalloteichus hindustanus fused to the 5 and 3 untranslated regions of theChlamydomonas reinhardtii nuclear geneRBCS2 has been constructed. Introduction of this chimeric gene into the nuclear genome ofC. reinhardtii by co-transformation with theARG7 marker yields Arg⁺ transformants of which approximately 80% possess theble gene. Of these co-transformants, approximately 3% display a phleomycin-resistant (Pm^R) phenotype. Western blot analysis using antibodies against theble gene product confirms the presence of the protein in the Pm^R transformants and genetic analysis demonstrates the co-segregation of theble gene with the phenotype in progeny arising from the mating of a Pm^R transformant to wild-type strains. Direct selection of Pm^R transformants was achieved by allowing an 18-h period for recovery and growth of transformed cells prior to selection. This work represents the first demonstration of stable expression and inheritance of a foreign gene in the nuclear genome ofC. reinhardtii and provides a useful dominant marker for nuclear transformation.     

Characterization of intracellular polygalacturonic acidtrans-eliminase fromKlebsiella oxytoca,Yersinia enterocolitica,andErwinia chrysanthemi

Intracellular polygalacturonic acidtrans-eliminase (PATE) was purified and characterized fromKlebsiella oxytoca andYersinia enterocolitica, enterobacteria unable to macerate plant tissue. The well-studied PATE from a strain ofErwinia chrysanthemi, a phytopathogen able to macerate plant tissue and cause soft-rot disease, was included for comparison. PATE from all strains displayed endo-splitting activity with pH optima between pH 8.5 and 9.0E. chrysanthemi had three isozymes (pls at pH 9.4, 9.0, and 7.8),K. oxytoca had two isozymes (pIs at pH 5.9 and 5.3), andY. enterocolitica had one isozyme (pI at pH 5.8). Molecular weights for theKlebsiella andYersinia PATEs were 71,000 and 55,000, respectively, compared with 33,000 for theErwinia PATE. Unlike theErwinia enzyme, theKlebsiella andYersinia PATEs did not require divalent cations for activity and could not macerate plant tissue without addition of pectinmethylesterase. The polygalacturonic acid-degrading enzymes found inK. oxytoca andY. enterocolitica appear to represent a separate type of PATE enzyme. It is unlikely that these organisms are phytopathogens; however, their ability to degrade polygalacturonic acid is probably advantageous to their survival in environments containing decomposing plant residues.     

Plasmid replication and Hfr formation in strains of Escherichia coli carrying seg mutations.

Summary Several conditional-lethal mutations that do not permit the replication of F-factors ofEscherichia coli K-12 are located at a site calledseg. This gene is located on theE. coli chromosome betweenserB andthr. It is unrelated to other known genes involved in DNA replication. Strains carryingseg mutations were unable to replicate F-lac⁺, several F-gal⁺s, F-his⁺ and bacteriophage at 42°. However, neither phage T4, ColE1, nor any of the R factors tested were prevented from replicating at 42°C.When the kinetics of the loss of F-primes is studied inseg strains, it is found that the rate of curing depends on the size of the plasmid, larger F factors curing faster than smaller ones, and that Hfrs are formed at high frequencies. The Hfrs showed both F-genote enlargement and normal transfer of chromosomal markers. The F-genotes are unstable and segregate chromosomal markers at high frequencies. Some orthodox Hfrs were examined, and two that were known to revert to the F⁺ condition relatively frequently were found to generate enlarged F-genotes on mating, whereas two strains that were very stable with respect to reversion to the F⁺ state did not show F-genote formation.F-genote formation fromseg Hfr strains is dependent of a functionalrecA gene, as F-genote formation was not seen with aseg-2, recA-1 Hfr. This is in contrast to F-genote enlargement shown by both orthodox Hfrs and an Hfr strain constructed by integration of a temperature-sensitive F-gal⁺, whose F-genote enlargement is Rec-independent. Thus there may be more than one mechanism for the formation of enlarged F-genotes.     

Phenocopies ofBithorax mutants

Summary Exposure to ether of wild-type embryos of different strains ofDrosophila melanogaster causes phenocopies of different alleles of thebithorax system. Clonal analysis of the phenocopy spots has shown that the transformation caused by the treatment is maintained by cell heredity. Embryos heterozygous for several recessive mutant alleles ofbithorax show the same frequency of phenocopies as wild-type homozygous sib controls. The same holds for embryos heterozygous for the dominant mutant allelesCbx andUbx ¹ which are point mutants in thecis-regulatory region of the system. However, for dominant mutants which have breakpoints in this region (Ubx ⁸⁰,Ubx ¹³⁰ andHm) the frequency of phenocopies is about twice that of their sib controls. Embryos with increasing numbers of copies (from 1 to 4) of thebithorax system show a decreasing frequency of phenocopies. A model is proposed that explains bithorax phenocopies as resulting from disturbances in the distribution of positional information signals for segments (inductor molecules) which compete with the product of a regulator gene (repressor) and thecis-regulatory region of thebithorax system. On this model, the initiation of a metathoracic developmental pathway would result from the derepression of thebithorax system.     

Biotransformation of glucose to 5-keto-<Emphasis Type="SmallCaps">d</Emphasis>-gluconic acid by recombinant<Emphasis Type="Italic"> Gluconobacter oxydans</Emphasis> DSM 2343

For the conversion of glucose to 5-keto-d-gluconate (5-KGA), a precursor of the industrially important l-(+)-tartaric acid, Gluconobacter strains were genetically engineered. In order to increase 5-KGA formation, a plasmid-encoded copy of the gene encoding the gluconate:NADP-5 oxidoreductase (gno) was overexpressed in G. oxydans strain DSM 2434. This enzyme is involved in the nonphosphorylative ketogenic oxidation of glucose and oxidizes gluconate to 5-KGA. As the 5-KGA reductase activity depends on the cofactor NADP⁺, the sthA gene (encoding Escherichia coli transhydrogenase) was cloned and overexpressed in the GNO-overproducing G. oxydans strain. Growth of the sthA-carrying strains was indistinguishable from the G. oxydans wild-type strain and therefore they were chosen for the coupled overexpression of sthA and gno. G. oxydans strain DSM 2343/pRS201-gno-sthA overproducing both enzymes showed an enhanced accumulation of 5-KGA.     

Transposon-generated Tn10 insertion mutations at thearo genes ofEscherichia coli K-12

We have obtained a set ofEscherichia coli K-12 derivatives with transposon-generated Tn10 insertion mutations at thearo genes of their aromatic biosynthetic pathway. Bacteriophage NK561 (Tn10) has been used for transposon mutagenesis ofE. coli, strain BW545. Tetracycline (Tc)-resistant derivatives were screened by their Aro^– phenotype by growth on a minimal medium with adequate requirements. Sixaro mutant types were mapped; two strains werearoA, twoaroD, onearoB oraroE, and onearoC. A selective medium and ad-cycloserine enrichment in the presence of tetracycline were used to select for Aro^–, Tc-sensitive derivatives. The reversion index to aromatic-independent colonies of some derivatives was less than 2 × 10^–11 per bacterium per generation. P1 transduction experiments transferred an aroA::Tn10 insertion fromE. coli BW545 to an enterotoxigenicE. coli strain from porcine origin. Derivatives of this strain beingaro, Tc-sensitive and not reverting toaro ⁺ at a detectable frequency, and many others transduced at will, may prove their usefulness as live vaccines.     

Genetic regulation of theAchaete-scute complex ofDrosophila melanogaster

Summary Mutants in two loci,hairy (h ⁺) andextramacrochaetae (emc ⁺), produce phenotypes corresponding to an excess of function of theachaete-scute complex (AS-C), that is, they cause the appearance of extra chaetae. These mutants, although recessive in normal flies, become dominant in the presence of extra doses of AS-C. Here we study the interactions between these three genes, in an attempt to elucidate their relationships. The results show that the insufficiency produced byh oremc mutants can be titrated by altering the number of copies of AS-C. Moreover, excess of function of AS-C produced by derepression mutants within the complex (Hairy-wing) can also be titrated by altering the number of wild type copies of⁺ oremc ⁺. These specific interactions indicate that bothh ⁺ andemc ⁺ code for repressors of AS-C that interact with theachaete andscute region of the complex respectively.     

Isolation and Genetic Study of Erwinia Mutants Devoid of Common Components of the Phosphoenolpyruvate-Dependent Phosphotransferase System

Mutants of bacteria belonging the genus Erwinia(Erwinia chrysanthemi andErwinia carotovora) with pleiotropic disturbances in the utilization of many substrates were obtained through chemical and transposon mutagenesis. Genetic studies revealed that these mutants had defective ptsI or ptsH genes responsible for the synthesis of common components of the phosphoenolpyruvate-dependent phosphotransferase system, enzyme I and the HPr protein, respectively. The ptsI ⁺ allele in both Erwinia species was cloned in vivo. Mapping of obtained mutations indicated that theptsIand ptsH genes ofE. chrysanthemi do not constitute a linkage group. The ptsI gene is located at 100 min of the chromosomal map, whereas theptsH gene is located at 175 min. Sequencing of a portion of theE. chrysanthemi ptsI gene showed that a product of the cloned DNA region had up to 68% homology with the N terminus of Escherichia coli enzyme I.     

A suitable method for construction and cloning hybrid plasmids containing EcoRI-fragments of E. coli genome.

Summary A convenient procedure for the isolation of specificEcoRI-fragments ofE. coli genome and their amplification on Km-resistance plasmid vector CK 11 is described. The hybrid molecules were constructedin vitro usingEcoRI-digestion, followed by ligation. Then appropriatedE. coli strain was transformed with ligated DNA mixture and hybrid plasmids CK 11-arg ⁺, CK 11-his ⁺, CK 11-thr ⁺ and CK 11-leu ⁺ containing loci ofE. coli genome were selected by molecular cloning. The hybrid plasmids obtained consisted of oneEcoRI-fragment of initial plasmid CK 11 and one respective specific portion ofE. coli genome.     

Synergistic effects of ethanol and temperature on yeast mitochondria

At temperatures lower than 37°C, the ethanol inhibition constant (Ki) for growth or fermentation inrho ⁺ cells of theSaccharomyces cerevisiae strain S288C was always higher (1.1M) than inrho ^– mutants (0.7M). At 37°C these differences disappeared, and both strains were equally inhibited by ethanol (Ki=0.7m). Mitochondrial activity can be inhibited by high ethanol concentration and temperature. In fact, the stronger inhibition by ethanol of therho ⁺ strain at 37°C was due to the fact that, under these conditions, this strain loses the advantage conferred by mitochondrial activity since the induction ofrho ^– cells in the population is very high. This does not result in an increase in the frequency ofrho ^– mutants because of the poor viability of these mutants in conditions of high temperature and ethanol. In consequence, S288C strain becomes as strongly inhibited by ethanol as therho ^– mutant strains. Differences in viability were not related to the fatty acids and ergosterol composition of the strain. In the presence of ethanol, bothrho ⁺ andrho ^– strains modified their lipids in the same way, but these changes did not improve their ethanol tolerance. They were not due to differences in adaptation to ethanol either, since after successive transfers in ethanol, growth () and fermentation () rates in therho ^– mutants were increasingly inhibited with time, whereas in the S288C strain inhibition of and by ethanol remained unaltered. Rather,rho ^– mutants are less viable thanrho ⁺ cells because of the inability of the former to respire. At 37°C the Ki increased to 0.9M ethanol either when mitochondrial from highly ethanol-tolerant wine yeasts were transferred torho ^– mutants of the strain S288C or when the mitochondria of strain S288C were preadapted by growing the strain in glycerol instead of glucose before it was cultivated in ethanol.     

Highly polymorphic DNA markers to specify strains of the ectomycorrhizal basidiomycete Tricholoma matsutake based on σ marY1 , the long terminal repeat of gypsy-type retroelement marY1

The ectomycorrhizal basidiomycete Tricholoma matsutake produces commercially valuable fruit bodies—matsutake—in Pinus sp. forest. Here we report that PCR with outward facing primers designed based on sequences comprising _marY1, the long terminal repeat of the gypsy-type retroelement marY1, specifies strains of T. matsutake. PCR with a primer based on the 22-bp sequence conserved at the 5-end of _marY1 conferred 73 reliable bands overall whose profiles depend upon strains of T. matsutake and T. magnivelare, the latter known as American matsutake. This PCR system gave no detectable band in any other species of Tricholoma tested, including T. bakamatsutake and T. fulvocastaneum, symbionts closely related to T. matsutake, as well as a host plant, Pinus densiflora. Similarly, PCR with a set of primers based on 26-bp and 28-bp sequences at bp 48–73 and bp 281–308 of _marY1, internal regions that are mutated in a variant of marY1, conferred 90 reliable bands only in strains of T. matsutake. Theoretically, PCR with the 22-bp primer would allow generation of 2⁷³, or 9.4×10²¹, types of polymorphism, and PCR with a combination of 26- and 28-bp primers, 2⁹⁰, or 1.2×10²⁷ types. The probability of falsely specifying two different isolates as the same strain is <1/10²¹. While polymorphisms conferred by the primer based on the 5 end of _marY1 rather exhibit genetic conservation of a group of T. matsutake, those resulting from primers based on the internal sequences more clearly demonstrate intra-specific diversification. Both systems revealed that T. matsutake is divergent within the species. Ectomycorrhizas formed between P. densiflora and T. matsutake were identified by the PCR systems developed in the present study. This method, using _marY1 as a genetic marker, is useful in analyzing the diversity of T. matsutake, monitoring the behavior of individual mycorrhizas, and specifying the ecological background of fruit bodies traded in markets.     

Properties of Mitomycin C-sensitive Mutants of Escherichia coli K-12

Strains hypersensitive to mitomycin C (MC) were isolated from Escherichia coli K-12 after treatment with nitrosoguanidine. Of 43 MC-sensitive strains tested for their ultraviolet light (UV) sensitivity and for their ability to reactivate UV-inactivated λ phage, 38 were found to be insensitive to UV irradiation and to be able to reactivate UV-irradiated bacteriophage λ. Some properties of the MC-sensitive, uvr⁺ mutants were analyzed. Synthesis of deoxyribonucleic acid (DNA) in MC-sensitive, uvr⁺ mutants was inhibited at a lower concentration of MC than in the wild-type strain. Mutant cells, labeled with ³H-thymidine and then exposed to MC, released radioactivity as low molecular weight compounds. The amount of radioactivity released was the same as that from the wild-type strain. MC-sensitive, uvr⁺ mutants, as well as the corresponding wild-type strain, were equally susceptible to induction of prophage 80 by UV irradiation. However, MC induction of prophage was achieved in MC-sensitive, uvr⁺ mutants at a lower concentration of the antibiotic than in the wild-type strain. Genetic experiments indicated that a gene controlling MC sensitivity is located close to that determining lactose fermentation of E. coli. It is situated on episome F′13, and the wild type is dominant to the MC-sensitive allele.     

Mapping of theHox-3.1 andMyc-1.2 genes on chromosome 15 of the mouse by restriction fragment length variations

Restriction endonuclease fragment length variations (RFLV) were detected by use of the cDNA probeHox-3.1 for the homeo box-3.1 gene and also thec-myc oncogene probe for exon 2. RFLV ofHox-3.1 were found inHindIII restriction patterns, and RFLV of theMyc-1.2 gene inEcoRV patterns. From the RFLV, theHox-3.1 andMyc-1.2 genes were mapped on chromosome 15. Three-point cross test data showed that the frequency of recombination is 26.4% betweenMyc-1.2 andGpt-1, 30.2% betweenGpt-1 andGdc-1, and 9.4% betweenGdc-1 andHox-3.1. The following order of these genes is proposed,Myc-1.2—Gpt-1—Gdc-1—Hox-3.1. All laboratory strains carry theHox-3.1 ^a andMyc-1.2 ^a alleles. Among strains of wild origin,domesticus strains carry only theHox-3.1 ^a andMyc-1.2 ^a alleles, as do the laboratory strains. One strain ofbrevirostris carries theHox-3.1 ^a andMyc-1.2 ^b alleles. Other wild subspecies from Europe and Asia,M. m. musculus, M. m. castaneus, M. m. molossinus, Chinese mice of wild origin, andM. m. yamashinai carry theHox-3.1 ^b andMyc-1.2 ^b alleles.