Physiological and genetic characterisation of osmosensitive mutants of Saccharomyes cerevisiae

The screening of 20,000 Saccharomyces cerevisiae random mutants to identify genes involved in the osmotic stress response yielded 14 mutants whose growth was poor in the presence of elevated concentrations of NaCl and glucose. Most of the mutant strains were more sensitive to NaCl than to glucose at the equivalent water activity (a_w) and were classified as salt-sensitive rather than osmosensitive. These mutants fell into 11 genetic complementation groups and were designated osr1–osr11 (osmotic stress response). All mutations were recessive and showed a clear 2⁺ : 2^– segregation of the salt-stress phenotype upon tetrad analysis when crossed to a wild-type strain. The complementation groups osr1, osr5 and osr11 were allelic to the genes PBS2, GPD1 and KAR3, respectively. Whereas intracellular and extracellular levels of glycerol increased in the wild-type strains when exposed to NaCl, all mutants demonstrated some increase in extracellular glycerol production upon salt stress, but a number of the mutants showed little or no increase in intracellular glycerol concentrations. The mutants had levels of glycerol-3-phosphate dehydrogenase, an enzyme induced by osmotic stress, either lower than or similar to those of the parent wild-type strain in the absence of osmotic stress. In the presence of NaCl, the increase in glycerol-3-phosphate dehydrogenase activity in the mutants did not match that of the parent wild-type strain. None of the mutants had defective ATPases or were sensitive to heat stress. It is evident from this study and from others that a wide spectrum of genes is involved in the osmotic stress response in S. cerevisiae. Received: 5 January 1998 / Accepted: 24 March 1998     

Penicillin production by glucose-derepressed mutants ofPenicillium chrysogenum

Summary Wild-type strains ofPenicillium chrysogenum produce lower penicillin V titers in media containing excess glucose. Two mutant strains were isolated and shown to produce normal penicillin V titers in the presence of excess glucose. These strains, designated as glucose-repression insensitive (GRI) mutants, produced higher penicillin V titers than the wild-type strain in media containing lactose as the main carbohydrate source. In lactose-based media, the production of penicillin V was depressed to a much lesser extent by in-cycle additions of glucose with the GRI mutants when compared to the wild-type strain. In short-term biosynthesis experiments using washed cells in a medium containing glucose as the sole carbon source, the GRI mutants produced penicillin V at a faster rate than the wild-type strain. In fed-batch fermentations in 14-liter fermentors, where glucose was fed continuously and pH controlled, both GRI mutants produced more than 10% higher penicillin V titers than the wild-type strain. These results suggest that isolation of GRI mutants is an effective way to select for higher producing strains and that the synthesis of penicillin synthesizing enzymes in GRI mutants may be less repressed by glucose than in wild-type strains.     

The improvement of riboflavin production in <Emphasis Type="Italic">Ashbya gossypii</Emphasis> via disparity mutagenesis and DNA microarray analysis

We generated a high riboflavin-producing mutant strain of Ashbya gossypii by disparity mutagenesis using mutation of DNA polymerase δ in the lagging strand, resulting in loss of DNA repair function by the polymerase. Among 1,353 colonies generated in the first screen, 26 mutants produced more than 3 g/L of riboflavin. By the second screen and single-colony isolation, nine strains that produced more than 5.2 g/L of riboflavin were selected as high riboflavin-producing strains. These mutants were resistant to oxalic acid and hydrogen peroxide as antimetabolites. One strain (W122032) produced 13.7 g/L of riboflavin in a 3-L fermentor using an optimized medium. This represents a ninefold improvement on the production of the wild-type strain. Proteomic analysis revealed that ADE1, RIB1, and RIB5 proteins were expressed at twofold higher levels in this strain than in the wild type. DNA microarray analysis showed that purine and riboflavin biosynthetic pathways were upregulated, while pathways related to carbon source assimilation, energy generation, and glycolysis were downregulated. Genes in the riboflavin biosynthetic pathway were significantly overexpressed during both riboflavin production and stationary phases, for example, RIB1 and RIB3 were expressed at greater than sixfold higher levels in this strain compared to the wild type. These results indicate that the improved riboflavin production in this strain is related to a shift in carbon flux from β-oxidation to the riboflavin biosynthetic pathway.     

New phenolic antioxidants of PYA and PYE class increase the resistanceS. cerevisiae strain SP4, its SOD- and catalase-deficient mutants to lipophilic oxidants

Was demonstrate the protection ability against reactive oxygen species afforded toS. cerevisiae (wild-type strain SP4 and its mutants deficient in major antioxidant enzymes—catalase T and A, CuZnSOD) by PYA and PYE, new groups of phenolic antioxidants (quaternary ammonium salts of dihydrocinnamic acid amino esters with different alkyl chains; synthesized in this laboratory). The survival of strains exposed to the lipophilic oxidation inducerstert-butyl hydroperoxide (TBHP) and 1,1′-azobis(4-cyclohexane carbonitrile) (ACCN) with or without antioxidant pretreatment was determined by platingS. cerevisiae mutant deficient in SOD was found to be hypersensitive to TBHP and ACCN while the sensitivity of the strain deficient in catalase T and A was about the same as in the wild-type strain. A 1-h preincubation of cells of both the wild-type and the mutant strains with the phenolic antioxidants prior to exposure to TBHP or ACCN substantially increased the cell survival. The magnitude of protection depended on the strain and the length of the alkyl chain of the antioxidant; the best average protection against TBHP was provided by PYE and PYA compounds with 12-and 16-membered alkyl chains whereas PYE-8 and PYA-12 derivatives, afforded the best average protection against ACCN.     

The isolation and preliminary characterisation of auxotrophic and analogue resistant mutants of the moss, Physcomitrella patens

Summary Eighteen nutritional mutants have been isolated in the haploid, monoecious moss, Physcomitrella patens: five nicotinic acid auxotrophs, four p-aminobenzoic acid auxotrophs, four adenine auxotrophs, two amino acid requiring mutants and three nitrate non-utilising mutants. Seventeen of them were obtained using total isolation; one was isolated selectively. Strains resistant to the amino acid analogues, D-serine and p-fluorophenyl-alanine, and the purine analogue, 8-azaguanine, have been selected. Many of the auxotrophs are self-sterile. Crosses between auxotrophic strains have been effected and the progeny analysed. No linkage has been detected. Nicotinic acid auxotrophy has resulted from mutation in at least two genes. Self-sterility segregates as a pleiotropic effect of four mutations which produce nutritional dependence. A diploid strain has been obtained by aposporus regeneration from a hybrid sporophyte and the phenotypes of progeny resulting from the self-fertilisation of this strain have been analysed.     

Double deletion of <Emphasis Type="Italic">dtsR1</Emphasis> and <Emphasis Type="Italic">pyc</Emphasis> induce efficient <Emphasis Type="SmallCaps">l</Emphasis>-glutamate overproduction in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum strains are used for the fermentative production of l-glutamate. Five C. glutamicum deletion mutants were isolated by two rounds of selection for homologous recombination and identified by Southern blot analysis. The growth, glucose consumption and glutamate production of the mutants were analyzed and compared with the wild-type ATCC 13032 strain. Double disruption of dtsR1 (encoding a subunit of acetyl-CoA carboxylase complex) and pyc (encoding pyruvate carboxylase) caused efficient overproduction of l-glutamate in C. glutamicum; production was much higher than that of the wild-type strain and ΔdtsR1 strain under glutamate-inducing conditions. In the absence of any inducing conditions, the amount of glutamate produced by the double-deletion strain ΔdtsR1Δpyc was more than that of the mutant ΔdtsR1. The activity of phosphoenolpyruvate carboxylase (PEPC) was found to be higher in the ΔdtsR1Δpyc strain than in the ΔdtsR1 strain and the wild-type strain. Therefore, PEPC appears to be an important anaplerotic enzyme for glutamate synthesis in ΔdtsR1 derivatives. Moreover, this conclusion was confirmed by overexpression of ppc and pyc in the two double-deletion strains (ΔdtsR1Δppc and ΔdtsR1Δpyc), respectively. Based on the data generated in this investigation, we suggest a new method that will improve glutamate production strains and provide a better understanding of the interaction(s) between the anaplerotic pathway and fatty acid synthesis.     

Genetic analysis ofCochliobolus heterostrophus polyoxin-resistant mutants

Nine polyoxin-resistant mutants ofCochliobolus heterostrophus were isolated after ethyl methanesulphonate mutagenesis. All were highly resistant to polyoxin (MIC≥1,600 ppm). Crosses between the mutants and a wild-type strain revealed that the resistance trait was inherited to the offsprings in different fashions. Four of the mutant strains inherited polyoxin resistance in a 1∶1 segregation ratio, indicating that the phenotypes in these strains were due to alteration at a single locus. Allelism tests revealed four new loci,Pol1, Pol2, Pol3 andPol4, for polyoxin resistance in these mutant strains. The genes responsible for the phenotypes of the other five mutant strains were not determined, because of extremely slow growth of progenies in one cross, sterility in another cross, and inexplicable responses to polyoxin of the progenies in the other crosses. No linkage was detected between the genes for polyoxin resistance and mating type.     

Effects of oxidative stress on the production of carotenoid pigments byPhaffia rhodozyma (Xanthophyllomyces dendrorhous)

The resistance to killing by free radicals of two mutants ofPhaffia rhodozyma was determined. Mutant 5–7 did not produce astaxanthin but produced β-carotene, while mutant 3–4 did not produce any carotenoid pigments. The resistance of mutant 5–7 was the same as that of the wild type but mutant 3–4 was rapidly killed. Carotenoid pigments increased the resistance to killing by free radicals. We investigated the effects of free radicals, generated by H₂O₂ and Fe²⁺ added to the medium, on wild-type cells and mutants ofP. rhodozyma. Unpigmented mutants of basidiomycetous yeasts (Rhodotorula spp. and others) are more susceptible to killing by UV-irradiation than the pigmented, wild-type strains. Therefore, we investigated the effect of free radicals on a similar basidiomycetous yeast,P. rhodozyma, a species of economic importance, in the biological production of astaxanthin.     

Mutants of Pseudomonas putida affected in poly-3-hydroxyalkanoate synthesis

The generation and characterization of Pseudomonas putida KT2442 mutants affected in poly-3-hydroxyalkanoate (PHA) synthesis are reported. The mutants from P. putida KT2442 carrying several copies of the PHA-polymerase-encoding gene (phaC) were isolated via N-methyl-N′-nitro-N-nitrosoguanidine chemical mutagenesis and contained mutation(s) on genes that are involved in PHA accumulation other than the phaC genes. No PHA-free mutants were obtained, suggesting that there must be various routes for the synthesis of PHA polymerase precursors. One of the isolated mutants (GPp120) accumulated more PHA than the parental strain, and there was virtually no down-regulation of PHA formation by growth in non-limiting amounts of nitrogen, which normally block or reduce formation of PHA. Compared to the parental strain, GPp120 exhibited significant changes in physiology and morphology when grown in minimal medium: the growth rate was reduced more than twofold and cells formed filaments. The other four groups of isolated mutants, with P. putida strains GPp121 to GPp124 as characteristic type strains, exhibited morphological characteristics similar to those of the parental strain. However, they showed reduced PHA production compared to the parental PHA⁺ strain, and especially GPp121 and GPp122 showed PHA formation tightly controlled by nutrient conditions. All of these mutants provide starting points for genetically dissecting the biosynthesis and regulation of PHA precursors. Received: 10 November 1997 / Received revision: 6 February 1998 / Accepted: 6 February 1998     

Enhanced production ofd(−)-lactic acid by mutants ofLactobacillus delbrueckii ATCC 9649

Summary Chemical mutagenesis with ethyl methanesulfonate (EMS) was used to develop strains ofLactobacillus delbrueckii (ATCC 9649) that tolerated increased lactic acid concentrations while continuously producing the acid. Three mutants (DP2, DP3 and DP4) were compared with wild-typeL. delbrueckii by standing fermentations with different glucose concentrations. All three mutants produced higher levels of lactic acid than the wild-type. In pH-controlled (pH 6.0) stirred-tank-batch fermentations, mutant DP3 in 12% glucose, 1% yeast extract/mineral salt/oleic acid medium produced lactic acid at a rate that was more than 2-times faster than the wild-type. Mutant DP3 also produced 77 g/l lactic acid compared with 58 g/l for the wild-type. Overall, compated with wild-type, the mutants DP2 and DP3 exhibited faster specific growth rates, shorter lag phases, greater lactic acid yields, tolerated higher lactic acid concentrations, and produced as much as 12% lactic acid in 12% glucose, 3% yeast extract/mineral salt/oleic acid medium which required an additional 9% glucose when the residual glucose concentration decreased to 3%. Mutant DP3 was stable for over 1.5 years (stored freeze dried). The strain development procedure was very successful; mutants with enhanced lactic acid-producing capacity were obtained each time the procedure was employed.Journal Paper No. J-14087 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA. Projects No. 2889 and 0178.     

Cytochrome mutants of bradyrhizobium induced by transposon tn5

Transposon Tn5 was used to mutate Bradyrhizobium japonicum USDA 61N. From over 5000 clones containing Tn5, 12 were selected and purified using a chemical reaction to identify oxidase-deficient clones. Four classes of mutants were identified based on the alterations in cytochromes. Most of the mutants had alterations in more than one cytochrome. Southern hybridization analysis of restricted genomic DNA of a representative strain of each class demonstrated that each mutant had a single Tn5 insert. Thus a single Tn5 insert produced pleiotropic effects on cytochromes. One class, which was totally deficient in cytochromes aa₃ and c, produced ineffective nodules on soybeans. Most of the strains representing the other classes produced effective nodules but exceptions were observed in each class. Bacteroids of the wild-type strain contained cytochrome aa₃. Bacteroids from one class of mutants were totally devoid of cytochrome aa₃. Several of these strains produced effective symbioses indicating that cytochrome aa₃ is not required for an effective symbiosis in this DNA homology group II strain which normally has this terminal oxidase in bacteroids.     

Evaluation of colors in green mutants isolated from purple bacteria as a host for colorimetric whole-cell biosensors

The change in carotenoid-based bacterial color from yellow to red can be applied to whole-cell biosensors. We generated several green mutants to emphasize the color change in such biosensors. The blue-green crtI-deleted mutant, Rhodopseudomonas palustris no.711, accumulated the colorless carotenoid precursor, phytoene. Green Rhodovulum sulfidophilum M31 accumulated neurosporene, a downstream product of phytoene. Another green mutant, Rhodobacter sphaeroides Ga, accumulated neurosporene and chloroxanthin, which are both downstream products of phytoene. All green mutants accumulated bacteriochlorophyll a. Photosynthetic membrane obtained from the green mutants all exhibited decreased absorption of wavelength range at 510–570 nm. Therefore, these indicate that the greenish bacterial colors were mainly caused by the existence of bacteriochlorophyll a and the changes in carotenoid composition in photosynthetic membrane. The colors of the green mutants and their wild-type strains were plotted in the CIE-L*a*b* color space, and the color difference (ΔE*ab) values between a green mutant and its wild type were calculated. ΔE*ab values were higher in the green mutants than in Rdv. sulfidophilum CDM2, the yellowish host strain of reported biosensors. These data indicate that change in bacterial color from green to red is more distinguishable than that from yellow to red as a reporter signal of carotenoid-based whole-cell biosensors.     

Ethanol and glycerol production under aerobic conditions by wild-type,respiratory-deficient mutants and a fusion product of Saccharomyces cerevisiae

Summary Experiments were performed to investigate growth, ethanol and glycerol production by wild-type strains (RHO) and respiratory-deficient (rho) mutants of Saccharomyces cerevisiae. Furthermore protoplasts were fused in order to enhance the fermentation capacity of a flocculent strain. At high substrate conditions, 150 g/l of saccharose, there is no difference in cell growth. However, at a glucose concentration of 10–20 g/l the mutants grow much slower. After 3 days of incubation at 28° C in a complete medium the viability of the two strains is the same. In minimal medium on the other hand the number of viable cells of the mutant is 100-fold reduced. All mutants tested showed a higher specific activity of alcohol dehydrogenase (ADH I) and an enhanced production of glycerol compared with the wild-type strain. By protoplast fusion a modified flocculent strain was obtained with higher specific activity of ADH I and a reduced biosynthesis of glycerol. However, the yields of ethanol (75–78%) are about the same for the wild-type strain and the rho mutants under aerobic conditions in absence of catabolite repression.     

The isolation of effective and ineffective mutants of cowpea rhizobium

Summary A strain of cowpea Rhizobium was mutagenised and two ineffective mutants, M1 and M2, and an effective mutant, M3, were isolated. M1 produced more, but smaller nodules than the wild-type; these nodules lacked leghaemoglobin. M2 and the parental strain had similar nodulation characteristics, both forming large pink nodules. Plants inoculated with M3, nodulated earlier, produced more nodules (58%), had increased dry weights (26%) and the excised roots expressed greater acetylene (C₂H₂) reducing activity (39%) than plants inoculated with the wild-type. When competing with an indigenous population of effective rhizobia for nodule sites, M3 produced a higher proportion of the nodules (70–80%) than the parental strain (40–53%).M3 and the parental strain exhibited comparable rates of asymbiotic C₂H₂ reduction when grown on a defined medium, whereas M1 and M2 were inactive.The symbiotic properties of the mutants were unchanged after their reisolation following plant passage.     

Selection for Growth on 3-Nitrotoluene by 2-Nitrotoluene-Utilizing Acidovorax sp. Strain JS42 Identifies Nitroarene Dioxygenases with Altered Specificities

Acidovorax sp. strain JS42 uses 2-nitrotoluene as a sole source of carbon and energy. The first enzyme of the degradation pathway, 2-nitrotoluene 2,3-dioxygenase, adds both atoms of molecular oxygen to 2-nitrotoluene, forming nitrite and 3-methylcatechol. All three mononitrotoluene isomers serve as substrates for 2-nitrotoluene dioxygenase, but strain JS42 is unable to grow on 3- or 4-nitrotoluene. Using both long- and short-term selections, we obtained spontaneous mutants of strain JS42 that grew on 3-nitrotoluene. All of the strains obtained by short-term selection had mutations in the gene encoding the α subunit of 2-nitrotoluene dioxygenase that changed isoleucine 204 at the active site to valine. Those strains obtained by long-term selections had mutations that changed the same residue to valine, alanine, or threonine or changed the alanine at position 405, which is just outside the active site, to glycine. All of these changes altered the regiospecificity of the enzymes with 3-nitrotoluene such that 4-methylcatechol was the primary product rather than 3-methylcatechol. Kinetic analyses indicated that the evolved enzymes had enhanced affinities for 3-nitrotoluene and were more catalytically efficient with 3-nitrotoluene than the wild-type enzyme. In contrast, the corresponding amino acid substitutions in the closely related enzyme nitrobenzene 1,2-dioxygenase were detrimental to enzyme activity. When cloned genes encoding the evolved dioxygenases were introduced into a JS42 mutant lacking a functional dioxygenase, the strains acquired the ability to grow on 3-nitrotoluene but with significantly longer doubling times than the evolved strains, suggesting that additional beneficial mutations occurred elsewhere in the genome.     

Production of indole-3-acetic acid by mutant strains of Ustilago maydis (maize smut / huitlacoche)

Production of indole-3-acetic acid (IAA) by four strains of the maize pathogen Ustilago maydis was analyzed. The fungus induces gall formation on its host plant and IAA production by  U. maydis may be required as a pathogenicity or virulence factor. The study included the FB2 wild-type strain and the 103, 130FZ and 130FT mutants. Results show that treatment with clofibric acid, alone or in combination with UV light, can be used to obtain  U. maydis strains with defective IAA production in vitro, as quantified with the Salkowski reagent and by HPLC. The strain with the lowest production was 130FT, and its peak IAA level represented only 16% of the highest value obtained for the FB2 wild-type strain (124 μg/ml). Received: 11 April 1996 / Received last revision: 5 September 1997 / Accepted: 11 September 1997     

Effects of mutations in acetate metabolism on high-cell-density growth of Escherichia coli

To study the role played by acetate metabolism during high-cell-density growth of Escherichia coli cells, we constructed isogenic null mutants of strain W3100 deficient for several genes involved either in acetate metabolism or the transition to stationary phase. We grew these strains under identical fed-batch conditions to the highest cell densities achievable in 8 h using a predictive-plus-feedback-controlled computer algorithm that maintained glucose at a set-point of 0.5 g/l, as previously described. Wild-type strains, as well as mutants lacking the s^s subunit of RNA polymerase (rpoS), grew reproducibly to high cell densities (44–50 g/l dry cell weights, DCWs). In contrast, a strain lacking acetate kinase (ackA) failed to reach densities greater than 8 g/l. Strains lacking other acetate metabolism genes (pta, acs, poxB, iclR, and fadR) achieved only medium cell densities (15–21 g/l DCWs). Complementation of either the acs or the ackA mutant restored wild-type high-cell-density growth. On a dry weight basis, poxB and fadR strains produced approximately threefold more acetate than did the wild-type strain. In contrast, the pta, acs, or rpoS strains produced significantly less acetate per cell dry weight than did the wild-type strain. Our results show that acetate metabolism plays a critical role during growth of E. coli cultures to high cell densities. They also demonstrate that cells do not require the s^s regulon to grow to high cell densities, at least not under the conditions tested. Journal of Industrial Microbiology & Biotechnology (2000) 24, 421–430. Received 12 November 1999/ Accepted in revised form 24 February 2000     

Mutagenesis at the ad-3A and ad-3B loci haploid UV-sensitive strains of Neurospora crassa. III. Comparison of dose-response curves for inactivation and mutation induced by gamma-rays

Gamma-Ray-induced inactivation and induction of mutations at the ad-3A and ad-3B loci of Neurospora crassa have been compared among 6 different UV-sensitive strains and a standard wild-type strain. The 6 strains show varying degrees of sensitivity to gamma-ray-induced inactivation, with the relative sensitivity at 37% survival being uvs-6 greater than upr-1 greater than uvs-2 greater than uvs-3 greater than wild-type greater than uvs-5 greater than uvs-4. Studies on the induction of ad-3 mutants by gamma-rays show that when the dose-response curve (expressed in terms of ad-3 mutants among the surviving colonies) of the UV-sensitive strains are compared with wild-type, the 2 excision-repair-deficient mutants uvs-2 and upr-1 exhibit enhanced ad-3 mutant frequencies, uvs-3 exhibits reduced ad-3 mutant frequencies whereas both uvs-4 and uvs-5 show lower mutant frequencies than wild-type.     

Competitiveness of Rhizobium sp. [Leucaena leucocephala (Lam.) Dewit] strains and their genetically marked derivatives

D.M. SWELIM, L.D. KUYKENDALL, F.M. HASHEM, S.M. ABDEL-WAHAB AND N.I. HEGAZI. 1996. The competitiveness of wild-type strains of Rhizobium sp. ( Leucaena ) and their genetically marked double mutants was examined in mixed infection experiments in the greenhouse. Antibiotic resistance markers were selected for use in strain identification, but these genetic markers apparently lowered both competitiveness and effectiveness, except in the case of strain DS 144/2 where the genetically marked derivative was evidently superior to the wild-type parent strain in effectiveness. Four wild-type strains and their genetically marked derivatives were carefully evaluated using double reciprocal pairs, the results of which nevertheless allowed the formulation of some conclusions. Strains DS 65 and DS 78 were more competitive than strain DS 144/2; only strain DS 78 was more competitive than DS 158; and strains DS 158 and DS 65 were equally competitive. There was no correlation between nodule number and competitiveness. Shoot dry weight and nitrogen mass, as well as nitrogenase activity, decreased with some strain mixtures indicating that relatively ineffective symbioses had formed, as compared with single-strain inoculations using symbiotically competent strains.