Relationship between mutant amidases of Pseudomonas aeruginosa and hydroxyurea as an inhibitor.

Summary Hydroxyurea inhibited growth of Pseudomonas aeruginosa strain AI 3 on media containing either acetanilide (N-phenyl acetamide) or acetamide as sole carbon sources. Mutants resistant to hydroxyurea inhibition of growth on acetanilide (OUCH strains) and acetamide (AmOUCH strains) displayed altered growth properties on various amide media compared with the parent strain AI 3. AI 3 amidase, which catalyses the initial step in the metabolism of acetanilide and acetamide, was inhibited by hydroxyurea in a time-dependent reaction that was slowly reversible at pH 7.2 Compared with AI 3 amidase, amidases from the OUCH mutants were much less sensitive to inhibition by hydroxyurea and showed altered substrate specificities and pH/activity profiles; amidases from the AmOUCH mutants were more sensitive to hydroxyurea inhibition but showed increased activity towards acetamide. Association of resistance to hydroxyurea inhibition with a mutation in the amidase structural gene of strain OUCH 4 was confirmed by transduction.     

酵母过氧化物体生物合成缺陷突变株的诱变、筛选和鉴定

过氧化物体对生物的生长和发育非常重要,人类很多疾病就是由于过氧化物体生物合成缺陷引起。以解脂耶氏酵母E122为出发菌,采用硫酸二乙酯诱变,获得了两株过氧化物体生物合成缺陷突变株,其中一株为温度敏感的突变株。在正常生长条件下,突变株的免疫荧光分析显示弥散的染色模式,且在电镜下观察不到过氧化物体的形态结构。将克隆于表达载体pINA445上的目前所发现的与过氧化物体生物合成有关的基因转化这两株突变株,发现它们均不能恢复其在含油酸的培养基上的生长,表明这两个突变株是由与过氧化物体生物合成相关的新基因的突变引起。这两个突变株的获得为参与过氧化物体生物合成的新基因的发现奠定了基础。     

Infectivity and sensitivity to antibiotics of Rickettsia prowazekii mutants of the low-pathogenic strain E cultured in chick embryos

Selection of mutants of a low pathogenic strain E of R. prowazekii is a trend in genetic investigation of this Rickettsia species and one of the approaches to stabilizing the strain avirulent properties with a purpose of using in vaccine prophylaxis of typhus. The mutants of R. prowazekii, strain E selected by the authors earlier were characterized with respect to their infective capacity for chick embryos (CE) and antibiotic sensitivity. It was found that the infective capacity for CE of the erythromycin resistant mutant induced by nitroso guanidine (EErrI) was by ID50 2-3 logarithms lower than that of the initial strain E. The infective capacity for CE of the rifampicin resistant mutant induced by nitroso guanidine (ERifrI) and the spontaneous erythromycin resistant mutant was similar to that of strain E. The ERifrI strain differed from the initial strain E by higher sensitivity to tetracycline and erythromycin and the EErrI strain differed from the initial strain E by higher sensitivity to tetracycline and rifampicin. It was shown that the biological properties of the nitroso guanidine-induced mutants resistant to rifampicin and erythromycin differed from those of the initial strain E and the properties of the spontaneous erythromycin resistant mutant were similar to those of the initial strain E.     

A class of ompA mutants of Escherichia coli K12 affected in the interaction of ompA protein and the core region of lipopolysaccharide

Summary A group of ompA mutants of Escherichia coli K12 are described which were sensitive to bacteriophage K3 in a background wild-type for lipopolysaccharide (LPS). With mutant LPS in vivo (lacking some core sugar residues), however, the ompA mutations gave resistance to K3. Outer membrane levels of OmpA protein were normal or near-normal when the mutations resided in either wild-type or mutant LPS backgrounds. Strains in which the mutations occurred in a wild-type LPS background adsorbed K3 phage at the same initial rate and to the same extent as a wild-type strain, but the efficiency of plaquing of the adsorbed K3 was reduced to 25–50% of wild-type levels. Under conditions where a wild-type strain irreversibly adsorbed over 90% of available phage K3 within 3 min, double mutants (ompA mutant, LPS mutant) left 90% of the phage viable after 1h. The 10% of inactivated phage did not form plaques.     

Effect of various adamantane compounds on the reproduction of Sindbis virus. Isolation and properties of a resistant strain]

Rimantadine and its structural analogs, i. e. amide of 1-adamantane carboxylic acid (AACA) and 1-adamantane acetic acid amide, were shown to be able to inhibit reproduction of Sindbis virus in culture Vero cells. AACA had the maximum antiviral activity. Subcultures of the initial sensitive population of Sindbis virus in the presence of AACA led to formation of mutants resistant to AACA as well as to rimantadine, adamantane acetic acid amide and ammonium chloride. The Sindbis virus population was heterogenous in sensitivity to AACA, which was evident from isolation of separate clones with various levels of sensitivity to the above mentioned compounds from the population. It was found that reproduction of the AACA sensitive and resistant strains of Sindbis virus differed: the latent period of the resistant strain was 2 hours longer than that of the sensitive strain. The same effect was observed in the comparative study on synthesis of the virus-specific RNA.     

Transformability of Streptomycin-resistant Group H Streptococci

Several resistant mutants of a transformable group H streptococcus, strain Challis, were isolated from media containing high concentrations of streptomycin. Mutants SR5a and SR5 exhibited high and low transformability, respectively, when exposed to deoxyribonucleic acid (DNA) from a novobiocin-resistant Challis strain. With similar exposure, mutant SR30 exhibited loss of transformability. The mutants further differed from the parent strain in time of appearance of optimal competence, and, in the case of SR5 and SR30, total growth was somewhat less than that of the parent. The rapidity with which transformants appeared upon initial exposure to DNA was approximately the same in the mutants and the parent strain. The decrease or loss of transformability of mutants SR5 and SR30 was found to be due to an alteration in capacity to take up DNA. Mutant SR5a (highly transformable) was further differentiated from mutants SR5 and SR30 in that it was somewhat more sensitive to high concentrations of streptomycin. Transformants obtained by treating strain Challis with the three types of mutant DNA, on the other hand, exhibited similar degrees of resistance to increasing concentrations of streptomycin. The additional decrease in transforming ability of mutant SR5a and the loss of transforming ability of mutant SR5 after a second exposure to streptomycin may indicate a stepwise process in the change from transformability to nontransformability. Although streptomycin resistance may not be directly related to inability to transform, results indicate that streptomycin greatly increases the chances of selecting these mutants and also can be of value in serving as a marker in studies of this nature.     

Inhibition of citric acid accumulation by manganese ions in Aspergillus niger mutants with reduced citrate control of phosphofructokinase.

Mutant strains of Aspergillus niger with reduced citrate control of carbohydrate catabolism (cic mutants) grow faster than the parent strain on media containing 5% (wt/vol) citrate. The mutants tolerated a higher intracellular citrate concentration than the parent strain. One mutant (cic-7/3) contained phosphofructokinase activity significantly less sensitive towards citrate than the enzyme from the parent strain. When this mutant was grown under citrate-accumulating conditions, acidogenesis was far less sensitive to inhibition by Mn2+ than in the parent strain. Some of the cic mutants also showed altered citrate inhibition of NADP-specific isocitrate dehydrogenase.     

Selection of spontaneous mutants ofYarrowia lipolytica by inositol-less death

Summary A procedure was developed for the selection of spontaneous mutants of the yeastYarrowia lipolytica. An inositol-requiring mutant of a wild-typeY. lipolytica, YB 3-122, was derived by mutagenesis and screening. The mutant had a reversion frequency of less than 6×10^–9. A mutant selection procedure based on inositolless death was then developed using this mutant strain. The selection procedure killed growingY. lipolytica cells and enriched for mutants yielding cultures that consisted of 60–98% spontaneous mutants after two rounds of inositol-less death. The procedure enriched for four classes of mutants, strains that were auxotrophic, metabolite analog sensitive, temperature sensitive, or unable to grow on citric acid as the sole carbon source. Since strain YB 3-122 is now available to yeast researchers, inositol-less death will be useful for the routine isolation of spontaneous mutants ofY. lipolytica.     

Repair of methyl methane sulfonate-damaged phage by Haemophilus influenzae

Seven mutants of Haemophilus influenzae strain Rd (mmsA-) have been isolated that are more sensitive to methyl methane sulfonate (mms) than recombination-deficient (recA-) mutants. The mutations cotransformed about 25% with the strA locus while the five studied clustered tightly; they are all probably allelic. The mutants are not sensitive to ultraviolet radiation, X-rays, or nitrous acid. Mms-damaged phage HP1 plated very inefficiently on these mutants, indicating that they lack the first step in the excision repair of the lesion N3-methyladenine (m3A). Incubation of damaged phage at 30 degrees C in the absence of mms resulted in a steady decline of viability when the phage were plated on the wild mmsA+ host but an initial steep rise was seen when it was plated on an mmsA- mutant. The rise is explained by the assumption that m3A lesions hydrolyzed off the DNA giving rise to repairable apurinic sites by both the mmsA+ and mmsA- hosts. No decline in viability was observed when hydroxylamine was present in the medium. This compound is known to prevent or slow down beta-elimination. The delayed decline in viability is therefore explained by assuming that apurinic sites give rise to beta-elimination-induced single strand breaks in the phage DNA that cannot be repaired by either host. Marker rescue experiments indicated that these breaks did not interrupt injection of phage DNA.     

Isolation and Some Properties of Cell Envelope Altered Mutants of Escherichia coli

Mutants of Escherichia coli which have a defect in their permeability barrier were selected. The technique used was to employ a strain of E. coli having a deletion in the gene for lactose permease and to select for mutants which can grow on lactose at 40 C. Twenty such mutants were isolated and six of these were found to be more sensitive to actinomycin D, sodium deoxycholate, and sodium dodecyl sulfate than was the parental strain. They were also more sensitive to the antibiotics vancomycin and bacitracin, which inhibit peptidoglycan biosynthesis. These mutants were no more sensitive to several different colicins or phages than was the wild-type strain. One of the mutants selected by this technique has an abnormal morphology when grown on certain carbon sources in minimal medium, and this mutant is more extensively studied in the accompanying paper.     

Toxic effects of chlorinated and brominated alkanoic acids on Pseudomonas putida PP3: selection at high frequencies of mutations in genes encoding dehalogenases

Mutant strains of Pseudomonas putida PP3 capable of utilizing monochloroacetate (MCA) and dichloroacetate (DCA) as the sole sources of carbon and energy were isolated from chemostat cultures. The mutants differed from the parent strain in that they could grow on products of MCA and DCA dehalogenation (catalyzed by inducible dehalogenases I and II) and were resistant to growth inhibition by the two substrates. The growth inhibition of strain PP3 by MCA, DCA, and other halogenated alkanoic acids was studied. Sensitivity to dehalogenase substrates was related to the expression of the dehalogenase genes. For example, mutants producing elevated levels of one or both of the dehalogenases were sensitive to 2-monochloropropionate and 2-monochlorobutanoate at concentrations which did not affect the growth of strain PP3. P. putida PP1, the parent of strain PP3, was resistant to the inhibitory effects of MCA and DCA. Spontaneous mutants of strain PP3, also resistant to MCA and DCA, were selected at high frequency, and four different classes of these strains were distinguished on the basis of dehalogenase phenotype. All dehalogenase-producing mutants were inducible; no constitutive mutant has yet been isolated. Most of the resistant mutants examined did not produce one or both of the dehalogenase, and over half of those tested failed to revert back to the parental (strain PP3) phenotype, indicating that the observed mutations involved high-frequency deletion of DNA base sequences affecting expression of genes encoding dehalogenases and associated permease(s).     

The roles of different excision-repair mechanisms in the resistance of Micrococcus luteus to UV and chemical mutagens

M. luteus mutants showing increased sensitivity to both UV and 4-NQO were isolated after the treatment of parental ATCC4698 strain with MNNG. The mutants were also highly sensitive to mitomycin C, cis-platinum, 8-methoxypsoralen (8-MOP) plus near-UV and angelicin plus near-UV in various degrees. The endonuclease activity specific for pyrimidine dimers in UV-irradiated DNA was normally detected in extract of the mutants. With regard to host-cell reactivation ability the mutants fell into two groups. The hcr- mutants lacked the ability to reactivate UV-damaged N6 phage and were resistant to X-rays. The incision of DNA did not occur during incubation after the treatment with angelicin plus near-UV in the hcr- mutants, whereas it occurred in the parental strain. The facts indicate that the hcr- mutants are defective in the incision mechanism which has a wide substrate specificity, similar to the UVRABC nuclease of E. coli. On the other hand, the incision of DNA and the removal of UV-induced thymine dimers from DNA occurred in the hcr- mutants as well as in the parental strain, which is ascribed to the UV endonuclease activity. Compared with the hcr- mutants, hcr+ mutants were highly sensitive to X-rays, like recA- mutants of E. coli.     

Monounsaturated but not polyunsaturated fatty acids are required for growth of the deep-sea bacterium Photobacterium profundum SS9 at high pressure and low temperature

There is considerable evidence correlating the production of increased proportions of membrane unsaturated fatty acids (UFAs) with bacterial growth at low temperatures or high pressures. In order to assess the importance of UFAs to microbial growth under these conditions, the effects of conditions altering UFA levels in the psychrotolerant piezophilic deep-sea bacterium Photobacterium profundum SS9 were investigated. The fatty acids produced by P. profundum SS9 grown at various temperatures and pressures were characterized, and differences in fatty acid composition as a function of phase growth, and between inner and outer membranes, were noted. P. profundum SS9 was found to exhibit enhanced proportions of both monounsaturated (MUFAs) and polyunsaturated (PUFAs) fatty acids when grown at a decreased temperature or elevated pressure. Treatment of cells with cerulenin inhibited MUFA but not PUFA synthesis and led to a decreased growth rate and yield at low temperature and high pressure. In addition, oleic acid-auxotrophic mutants were isolated. One of these mutants, strain EA3, was deficient in the production of MUFAs and was both low-temperature sensitive and high-pressure sensitive in the absence of exogenous 18:1 fatty acid. Another mutant, strain EA2, produced little MUFA but elevated levels of the PUFA species eicosapentaenoic acid (EPA; 20:5n-3). This mutant grew slowly but was not low-temperature sensitive or high-pressure sensitive. Finally, reverse genetics was employed to construct a mutant unable to produce EPA. This mutant, strain EA10, was also not low-temperature sensitive or high-pressure sensitive. The significance of these results to the understanding of the role of UFAs in growth under low-temperature or high-pressure conditions is discussed.     

Properties of the cured oncogenic strain 37400 ofAgrobacterium tumefaciens

The properties of the 37400 oncogenic strain ofAgrobacterium tumefaciens are described. This strain was derived from the VI lysogenic strain originally isolated by Hamilton from aZinnia elegans tumour. Strain 37400 has a number of properties which render it suitable for quantitative and genetic studies. It is cured of prophages and can serve as a universal sensitive indicator for a number of phages isolated from various lysogenic strains ofAgrobacterium tumefaciens. Its good growth properties in synthetic media and at elevated temperatures enable the isolation of auxotrophic mutants and temperature sensitive phage mutants. Preliminary experiments show that strain 37400 will serve as suitable starting material for conjugation experiments under defined conditions.     

Regulation of hydrogenase in Rhizobium japonicum: analysis of mutants altered in regulation by carbon substrates and oxygen

The synthesis of the H2 uptake system in free-living Rhizobium japonicum SR is repressed both by oxygen and by carbon substrates. Mutants selected for the ability to express hydrogenase in 10.0% partial pressure O2 were also less sensitive than the wild type to repression by carbon substrates such as arabinose, glycerol, gluconate, and succinate. The H2 uptake system in another class of mutants, previously shown to be hypersensitive to repression by O2, is also more sensitive to repression by carbon substrates. The oxygen- and carbon-insensitive mutants express the hydrogen uptake system during heterotrophic growth in the absence of hydrogen and thus can be considered constitutive (Hupc). The amount of cytochromes in the Hupc mutants is similar to that in the wild-type strain; however, the Hupc mutants contain greater methylene blue-dependent and O2-dependent hydrogenase activity, both as free-living cells and as bacteroids. Two-dimensional polyacrylamide gel electrophoresis revealed that during heterotrophic growth the Hupc mutant strain SR470 synthesized at least six peptides not found in the wild-type strain. The concentrations of cyclic AMP and guanosine tetraphosphate were similar in strain SR and the Hupc mutants during heterotrophic growth.     

Ribonucleic acid polymerase mutation affecting glutamate synthase activity in and sporulation of Bacillus subtilis.

Spore formation of 15 rifampin-resistant (Rifr) mutants of Bacillus subtilis strain 168 was examined. As a pleiotropic effect of a Rifr mutation, glutamate synthase activity was lost in these mutants. Twelve of the 15 mutants examined formed as many spores as the parent, but the remaining 3 formed significantly fewer (1%) spores. One of the latter mutants characterized further (RF301) was blocked in its sporulation process at stage 0. Thus, it was concluded that a certain modification of ribonucleic acid polymerase may affect specifically the gene expression of glutamate synthase and also the sporulation process at the initial stage.     

Yeast genome-wide expression analysis identifies a strong ergosterol and oxidative stress response during the initial stages of an industrial lager fermentation

Genome-wide expression analysis of an industrial strain of Saccharomyces cerevisiae during the initial stages of an industrial lager fermentation identified a strong response from genes involved in the biosynthesis of ergosterol and oxidative stress protection. The induction of the ERG genes was confirmed by Northern analysis and was found to be complemented by a rapid accumulation of ergosterol over the initial 6-h fermentation period. From a test of the metabolic activity of deletion mutants in the ergosterol biosynthesis pathway, it was found that ergosterol is an important factor in restoring the fermentative capacity of the cell after storage. Additionally, similar ERG10 and TRR1 gene expression patterns over the initial 24-h fermentation period highlighted a possible interaction between ergosterol biosynthesis and the oxidative stress response. Further analysis showed that erg mutants producing altered sterols were highly sensitive to oxidative stress-generating compounds. Here we show that genome-wide expression analysis can be used in the commercial environment and was successful in identifying environmental conditions that are important in industrial yeast fermentation.     

A recombination repair gene of Schizosaccharomyces pombe, rhp57, is a functional homolog of the Saccharomyces cerevisiae RAD57 gene and is phylogenetically related to the human XRCC3 gene

To identify Schizosaccharomyces pombe genes involved in recombination repair, we identified seven mutants that were hypersensitive to both methyl methanesulfonate (MMS) and gamma-rays and that contained mutations that caused synthetic lethality when combined with a rad2 mutation. One of the mutants was used to clone the corresponding gene from a genomic library by complementation of the MMS-sensitive phenotype. The gene obtained encodes a protein of 354 amino acids whose sequence is 32% identical to that of the Rad57 protein of Saccharomyces cerevisiae. An rhp57 (RAD57 homolog of S. pombe) deletion strain was more sensitive to MMS, UV, and gamma-rays than the wild-type strain and showed a reduction in the frequency of mitotic homologous recombination. The MMS sensitivity was more severe at lower temperature and was suppressed by the presence of a multicopy plasmid bearing the rhp51 gene. An rhp51 rhp57 double mutant was as sensitive to UV and gamma-rays as an rhp51 single mutant, indicating that rhp51 function is epistatic to that of rhp57. These characteristics of the rhp57 mutants are very similar to those of S. cerevisiae rad57 mutants. Phylogenetic analysis suggests that Rhp57 and Rad57 are evolutionarily closest to human Xrcc3 of the RecA/Rad51 family of proteins.     

Biological properties of antibiotic-resistant mutants of the mildly pathogenic Rickettsia prowazekii E strain

The use of R. prowazekii strain E with low pathogenicity as live vaccine against exanthematous typhus is limited by its high specific reactogenicity, which is probably due to the reversion of the virulence of the strain. One of the approaches to the stabilization of the avirulent properties of strain E is obtaining its mutants with stable decreased pathogenic properties. The article presents the data on the infectious properties of R. prowazekii antibiotic-resistant strain E mutants obtained in earlier experiments, in respect of chick embryos and laboratory animals, as well as the capacity of this strain for producing immunity to challenge with R. prowazekii virulent strain in guinea pigs. The study has revealed that the erythromycin-resistant mutant of R. prowazekii strain E, induced by nitrosoguanidine (NG), has lower infective capacity for chick embryos, guinea pigs, cotton rats and white mice. The infective capacity of the NG-induced rifampicin-resistant and spontaneous erythromycin-resistant mutants of R. prowazekii strain E is similar to the infective capacity of the initial strain. The rifampicin-resistant and spontaneous erythromycin-resistant mutants of R. prowazekii strain E possess immunogenicity similar to that of the initial strain E, and the NG-induced erythromycin-resistant mutant possesses lower, but sufficiently pronounced immunogenicity despite its decreased infective capacity for guinea pigs.     

Isolation and characterization of vanadate-resistant mutants of Saccharomyces cerevisiae

Cellular vanadium metabolism was studied in Saccharomyces cerevisiae by isolating and characterizing vanadate [VO4(3-), V(V)]-resistant mutants. Vanadate growth inhibition was reversed by the removal of the vanadate from the medium, and vanadate resistance was found to be a recessive trait. Vanadate-resistant mutants isolated from glucose-grown cells were divided into five complementation classes containing more than one mutant. Among the vanadate-resistant mutants isolated in maltose medium, the majority of mutants were found in only two complementation groups. Three of the classes of vanadate-resistant mutants were resistant to 2.5 mM vanadate but sensitive to 5.0 mM vanadate in liquid media. Two classes of vanadate-resistant mutants were resistant to growth in media containing up to 5.0 mM vanadate. Electron spin resonance studies showed that representative strains of the vanadate-resistant complementation classes contained more cell-associated vanadyl [VO2+, V(IV)] than the parental strains. 51 Vanadium nuclear magnetic resonance studies showed that one of the vanadate resonances previously associated with cell toxicity (G. R. Willsky, D. A. White, and B. C. McCabe, J. Biol. Chem. 259:13273-132812, 1984) did not accumulate in the resistant strains compared with the sensitive strain. The amount of vanadate remaining in the media after growth was larger for the sensitive strain than for the vanadate-resistant strains. All of the strains were able to accumulate phosphate, vanadate, and vanadyl.