Mutation in Escherichia coli K-12 Mediating Spherelike Envelopes and Changed Tolerance to Ultraviolet Irradiation and Some Antibiotics

In a mutation experiment with a rough, ampicillin-resistant strain of Escherichia coli K-12, two smooth ampicillin-sensitive mutants were isolated. One of the mutants (with the envA gene) was recently described. The second mutant (strain D23) with the envB gene which has been mapped to a position close to streptomycin resistance (strA) at 64 min is described. The envB gene gives rise to spherelike cells. Electron microscopy revealed an abnormal septum formation and a circular distribution of the nuclear material. Strain D23 (with envB) showed a changed resistance to several antibiotics as well as an increased tolerance to ultraviolet irradiation. The envB gene decreased the ampicillin resistance mediated by the ampA gene (at 82 min), but no effect was found on episomally mediated penicillin resistance.     

Conditional transduction of Salmonella typhimurium envB mutations.

Joint transduction of the argR and envB genes was observed, at a frequency of 24.5%, when four envB strains were transduced to tetracycline resistance (Tetr) with bacteriophage P22 grown on an argR372::Tn10 envB+ donor. When round-cell argR372::Tn10 derivatives of those envB strains were used as donors, two of them did not produce envB transductants in wild-type LT2 and other envB+ recipients, even though large numbers of Tetr transductants were obtained. This apparent exclusion of envB mutations did not occur when mecillinam-resistant derivatives of those envB+ strains were used as recipients. Mutations conferring partial resistance to mecillinam were found, unlinked to the argR-envB region, in three of the four envB strains studied; envB+ derivatives of the four strains were competent to accept envB mutations excluded by wild-type recipients. It is suggested that some envB mutations are lethal in the absence of suppressor mutations, some of which increase resistance to mecillinam.     

Cloning, characterization, and DNA base sequence of the high-level streptomycin resistance gene strA1 of Haemophilus influenzae Rd.

The high-level streptomycin resistance strA1 gene of Haemophilus influenzae Rd was cloned in plasmid pAT4 as a 2.1-kbp EcoRI insert. It was later replaced in pAT4 by the wild-type strA+ gene. Plasmid pAT4 carrying the strA+ gene is highly unstable and renders chromosomally resistant recipients sensitive to streptomycin. The strA+ gene and the instability factor both reside on a 500-base HindIII-EcoRI subfragment. The two biological activities are also expressed in Escherichia coli. Both wild-type (strA+) and mutant (strA1) genes were sequenced. They show considerable nucleotide homology with the E. coli strA+ gene and its product.     

In vivo transfer of chromosomal mutations onto multicopy plasmids utilizing polA strains: Cloning of an ompR 2 mutation in Escherichia coli K-12

Abstract We have devised a simple in vivo scheme for moving chromosomal mutations onto multicopy plasmids in Escherichia coli K-12. A plasmid clone of the relevant wild-type gene is first integrated into the chromosome of a PolA⁻ strain carrying the desired mutation. The plasmid cointegrate formed is then resolved by P1 transduction to a PolA⁺ host. A certain fraction of these transductants will have the mutant allele on the plasmid. Employing this scheme we cloned an ompR 2 mutation onto a multicopy plasmid. To show that the plasmid actually contained the ompR 2 mutation, this allele was introduced back into the chromosome by the gene replacement technique of Gutterson and Koshland [1] and shown to be indistinguishable from the original ompR 2 by genetic mapping and phenotype.     

Mechanism of Action and Development of Resistance to a New Amidino Penicillin

The mechanism of killing of Escherichia coli by a novel beta-lactam antibiotic, an amidino penicillin, has been investigated. This compound converts E. coli to relatively stable spherical forms at low concentration. However, the amidino penicillin caused no alteration in any of those parameters of peptidoglycan synthesis which can be studied. Above 10 mug of the antibiotic per ml the cells began to lyse, and a second mode of killing appeared. Mutants resistant to the amidino penicillin were isolated and several were studied in detail. Three mutant phenotypes were distinguished: (i) spherical shape and hypersensitive to lysis by either amidino penicillin or ampicillin; (ii) spherical shape and normally sensitive to lysis; (iii) rod shape, converted to viable spheres by amidino penicillin and normally sensitive to lysis.     

A New Fungal Metabolite and Root Growth-stimulating Activity of Its Methyl Ester

Escherichia coli rodA mutant AOS151 grows as round cells at 30 and 42°C (H. Matsuzawa, K. Hayakawa, T. Sato, and K. Imahori, J. Bacteriol., 115, 436–442 (1973)). The mutant was found to be resistant to mecillinam at both temperatures. lip⁺ transductants were prepared by Pl phage transduction via strain AOS151, the cotransduction frequency of round morphology (Rod^?) at 42°C with the lip gene being about 90%. At 42°C all 54 Rod^? transductants tested were resistant to mecillinam. At 30°C all but two of these Rod^? (at 42°C)-type transductants were rod-shaped, and all were sensitive to mecillinam; the two strains grew as ovoid cells. The original rodA mutant AOS151 probably involves an additional mutation(s), that expresses the round cell shape at lower temperature, whereas the rodA51 mutation alone seems to result in temperature-sensitive expression of round cell morphology and mecillinam resistance. rodA mutant cells cultured at either 30 or 42°C had wild-type penicillin-binding protein 2, judging from penicillin-binding activity, electrophoretic mobility, and thermosensitivity.     

SLOB, a SLOWPOKE channel binding protein, regulates insulin pathway signaling and metabolism in Drosophila

There is ample evidence that ion channel modulation by accessory proteins within a macromolecular complex can regulate channel activity and thereby impact neuronal excitability. However, the downstream consequences of ion channel modulation remain largely undetermined. The Drosophila melanogaster large conductance calcium-activated potassium channel SLOWPOKE (SLO) undergoes modulation via its binding partner SLO-binding protein (SLOB). Regulation of SLO by SLOB influences the voltage dependence of SLO activation and modulates synaptic transmission. SLO and SLOB are expressed especially prominently in median neurosecretory cells (mNSCs) in the pars intercerebralis (PI) region of the brain; these cells also express and secrete Drosophila insulin like peptides (dILPs). Previously, we found that flies lacking SLOB exhibit increased resistance to starvation, and we reasoned that SLOB may regulate aspects of insulin signaling and metabolism. Here we investigate the role of SLOB in metabolism and find that slob null flies exhibit changes in energy storage and insulin pathway signaling. In addition, slob null flies have decreased levels of dilp3 and increased levels of takeout, a gene known to be involved in feeding and metabolism. Targeted expression of SLOB to mNSCs rescues these alterations in gene expression, as well as the metabolic phenotypes. Analysis of fly lines mutant for both slob and slo indicate that the effect of SLOB on metabolism and gene expression is via SLO. We propose that modulation of SLO by SLOB regulates neurotransmission in mNSCs, influencing downstream insulin pathway signaling and metabolism.     

A heat shock-resistant mutant of Saccharomyces cerevisiae shows constitutive synthesis of two heat shock proteins and altered growth

A heat shock-resistant mutant of the budding yeast Saccharomyces cerevisiae was isolated at the mutation frequency of 10(-7) from a culture treated with ethyl methane sulfonate. Cells of the mutant are approximately 1,000-fold more resistant to lethal heat shock than those of the parental strain. Tetrad analysis indicates that phenotypes revealed by this mutant segregated together in the ratio 2+:2- from heterozygotes constructed with the wild-type strain of the opposite mating type, and are, therefore, attributed to a single nuclear mutation. The mutated gene in the mutant was herein designated hsr1 (heat shock response). The hsr1 allele is recessive to the HSR1+ allele of the wild-type strain. Exponentially growing cells of hsr1 mutant were found to constitutively synthesize six proteins that are not synthesized or are synthesized at reduced rates in HSR1+ cells unless appropriately induced. These proteins include one hsp/G0-protein (hsp48A), one hsp (hsp48B), and two G0-proteins (p73, p56). Heterozygous diploid (hsr1/HSR1+) cells do not synthesize the proteins constitutively induced in hsr1 cells, which suggests that the product of the HSR1 gene might negatively regulate the synthesis of these proteins. The hsr1 mutation also led to altered growth of the mutant cells. The mutation elongated the duration of G1 period in the cell cycle and affected both growth arrest by sulfur starvation and growth recovery from it. We discuss the problem of which protein(s) among those constitutively expressed in growing cells of the hsr1 mutant is responsible for heat shock resistance and alterations in the growth control.     

delta-Aminolevulinic acid dehydratase deficiency can cause delta-aminolevulinate auxotrophy in Escherichia coli

Ethylmethane sulfonate-induced mutants of several Escherichia coli strains that required delta-aminolevulinic acid (ALA) for growth were isolated by penicillin enrichment or by selection for respiratory-defective strains resistant to the aminoglycoside antibiotic kanamycin. Three classes of mutants were obtained. Two-thirds of the strains were mutants in hemA. Representative of a third of the mutations was the hem-201 mutation. This mutation was mapped to min 8.6 to 8.7. Complementation of the auxotrophic phenotype by wild-type DNA from the corresponding phage 8F10 allowed the isolation of the gene. DNA sequence analysis revealed that the hem-201 gene encoded ALA dehydratase and was similar to a known hemB gene of E. coli. Complementation studies of hem-201 and hemB1 mutant strains with various hem-201 gene subfragments showed that hem-201 and the previously reported hemB1 mutation are in the same gene and that no other gene is required to complement the hem-201 mutant. ALA-forming activity from glutamate could not be detected by in vitro or in vivo assays. Extracts of hem-201 cells had drastically reduced ALA dehydratase levels, while cells transformed with the plasmid-encoded wild-type gene possessed highly elevated enzyme levels. The ALA requirement for growth, the lack of any ALA-forming enzymatic activity, and greatly reduced ALA dehydratase activity of the hem-201 strain suggest that a diffusible product of an enzyme in the heme biosynthetic pathway after ALA formation is involved in positive regulation of ALA biosynthesis. In contrast to the hem-201 mutant, previously isolated hemB mutants were not ALA auxotrophs and had no detectable ALA dehydratase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of a temperature-sensitive dnaK mutant of Escherichia coli B.

A temperature-sensitive dnaK mutant (strain MT112) was isolated from Escherichia coli B strain H/r30RT by thymineless death selection at 43 degrees C. By genetic mapping, the mutation [dnaK7(Ts)] was located near the thr gene (approximately 0.2 min on the may). E. coli K-12 transductants of the mutation to temperature sensitivity were assayed for their susceptibility to transducing phage lambda carrying the dnaK and/or the dnaJ gene. All of the transductants were able to propagate phage lambda carrying the dnaK gene. When macromolecular synthesis of the mutant was assayed at 43 degrees C, it was observed that both deoxyribonucleic acid and ribonucleic acid syntheses were severely inhibited. Thus, it was suggested that the conditionally defective dnaK mutation affects both cellular deoxyribonucleic acid and ribonucleic acid syntheses at the nonpermissive temperature in addition to inability to propagate phage lambda at permissive temperature.     

Correction of a deletion mutant by gene targeting with an adenovirus vector.

The usefulness of adenovirus type 5 as a vector for homologous recombination was examined in CHO cells by using the adenine phosphoribosyltransferase (aprt) gene. Infection of a hemizygous CHO APRT- cell line containing a 3-bp deletion in exon 5 of the aprt gene with a recombinant adenovirus containing the wild-type gene resulted in restoration of the APRT+ phenotype at a frequency of 10(-5) to 10(-6) per infected cell. A relatively high frequency (approximately 6 to 20%) of the transductants appears to result from a homologous recombination event. The mutation on the chromosomal aprt gene is corrected in the homologous recombinants, and APRT expression is restored to a normal hemizygous level. Neither adenovirus nor exogenous promoter sequences are detected in the homologous recombinants. The remaining transductants result from random integration of the aprt gene with the adenovirus sequence. A number of adenovirus vectors containing different promoter sequences linked to the hamster aprt gene were constructed. A possible role for the promoter region in the homologous recombination event was indicated by the lack of homologous recombination in constructs lacking an active promoter.     

Genetic analysis of a streptomycin-resistant oligosporogenous Bacillus subtilis mutant

Strain SRB15T+, a streptomycin-resistant, oligosporogenous mutant of Bacillus subtilis, contains two mutations, fun and strR. These mutations were mapped by PBS-1 mediated transduction and by transformation to two different sites in the cysA-linked region of the B. subtilis chromosome. The fun mutation mapped very close to rpsLl, a classic strA mutation, whereas strR mapped to a site distal to rpsE. The effects of these mutations on growth, sporulation, and streptomycin resistance in vivo and in vitro were determined. The fun mutation gave a different phenotype than did the rpsLl mutation and caused altered migration of a ribosomal protein which was identified as S12, the protein encoded by rpsL. It therefore appears that fun is an allele of the rpsL gene.     

Suppression of spectinomycin resistance in a mutant of Escherichia coli K-12.

A mutant of Escherichia coli has been isolated which exhibits partial suppression of spectinomycin resistance. The site of mutation is in the streptomycin (strA) region and is closely linked to the spcA gene. However, this gene, which we propose to call mod, is phenotypically distinguishable from both the neomycin-kanamycin (nek) and the ribosomal ambiguity gene (ram). The relative gene order is mod spcA strA. In a cell-free protein synthesizing system, altered ribosomes appear to be responsible for the suppression of spectinomycin resistance caused by mod.     

Increased production of a knotted form of plasmid pBR322 DNA in Escherichia coli DNA topoisomerase mutants

Plasmid pBR322 prepared from Escherichia coli strains carrying deletion of the DNA topoisomerase I gene (delta topA) with a compensatory mutation of the DNA gyrase gene (gyrA or gyrB) and from their TopA+ transductants was analyzed by agarose gel electrophoresis followed by electron microscopy, and compared with that from isogenic wild-type strains. It was found that about 1% of the plasmid DNA molecules was a knotted species in the topA+ gyr+ strains W3110 and DM4100, while strains DM750 (delta topA gyrA224), DM800 (delta topA gyrB225), SD275 (topA+ gyrA224) and SD108 (topA+ gyrB225) produced six to ten times as much knotted DNA as the topA+ gyr+ controls. The results suggest that the increased production of knotted pBR322 DNA is closely related to mutations of the gyrase genes.     

Chromosomal Recombination in HAEMOPHILUS INFLUENZAE

Haemophilus influenzae cultures doubly lysogenic for defective phage HP1, with a prophage marker sequence +b+/a+c, always contained some free wild-type phage. Single ultraviolet-irradiated cells produced either no wild-type phage or large numbers of them. This suggested that the phage was not released by the original double lysogen but by internal recombinants, i.e., by double lysogens with altered prophage marker sequence such as +++/abc or +b+/++c. Thirty-one wild-type phage-producing clones have been isolated independently from cultures of this double lysogen and identified. They fell in five classes. Two classes, still possessing all three prophage markers, can be explained by Campbell's (1963) prophage recombination model. The other classes had lost one or more markers. They can be explained by interchromosomal double-strand DNA breakage and rejoining. A single-DNA-strand gene conversion model is discussed in view of the fact that genetic transformation involves single-DNA-strand exchanges. A number of potentially interesting mutants has been analyzed of which only the derivatives of rec1 mutant DB117 (obtained from Dr. J. Setlow) were incapable of internal recombination.     

Disruption of sucA, which encodes the E1 subunit of alpha-ketoglutarate dehydrogenase, affects the survival of Nitrosomonas europaea in stationary phase

Although Nitrosomonas europaea lacks measurable alpha-ketoglutarate dehydrogenase activity, the recent completion of the genome sequence revealed the presence of the genes encoding the enzyme. A knockout mutation was created in the sucA gene encoding the E1 subunit. Compared to wild-type cells, the mutant strain showed an accelerated loss of ammonia monooxygenase and hydroxylamine oxidoreductase activities upon entering stationary phase. In addition, unlike wild-type cells, the mutant strain showed a marked lag in the ability to resume growth in response to pH adjustments in late stationary phase.     

A mutation in the gene encoding the vaccinia virus 37,000-M(r) protein confers resistance to an inhibitor of virus envelopment and release.

Plaque formation in vaccinia virus is inhibited by the compound N1-isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine (IMCBH). We have isolated a mutant virus that forms wild-type plaques in the presence of the drug. Comparison of wild-type and mutant virus showed that both viruses produced similar amounts of infectious intracellular naked virus in the presence of the drug. In contrast to the mutant, no extracellular enveloped virus was obtained from IMCBH-treated cells infected with wild-type virus. Marker rescue experiments were used to map the mutation conferring IMCBH resistance to the mutant virus. The map position coincided with that of the gene encoding the viral envelope antigen of M(r) 37,000. Sequence analysis of both wild-type and mutant genes showed a single nucleotide change (G to T) in the mutant gene. In the deduced amino acid sequence, the mutation changes the codon for an acidic Asp residue in the wild-type gene to one for a polar noncharged Tyr residue in the mutant.