Characterization of a selenocystine-resistant carrot cell line : alterations in cystine and sulfate uptake

A selenocystine-resistant carrot cell line, C-1, was isolated from a haploid carrot (Daucus carota) cell culture, HA. The C-1 variant takes up cystine, but not cysteine, more slowly than does HA. The selenocystine resistance is maintained in culture in the absence of selection and is expressed in regenerated plants. Results based on chromatographic separation of sulfur metabolites from cells fed with [³⁵S]cystine suggest a block either in the uptake or reduction of cystine in the variant. Both lines can grow on cystine as sole sulfur source. Growth of the HA line on cystine suppressed the development of sulfate uptake capacity (Furner, Sung 1982 Proc Natl Acad Sci USA 79: 1149-1153), while cystine-grown C-1 cells have high levels of sulfate uptake capacity.

We suggest that the C-1 line, grown on cystine, accumulates an insufficient quantity of some sulfur metabolite, which is involved in the control of sulfate uptake, to suppress the uptake. C-1 grown on cystine is more sensitive than HA to growth inhibition by the sulfate analog selenate.

     

The study of mutagenesis inMycobacterium phlei

Results obtained when studying mutagenesis inMycobacterium phlei are summarized in this work. It was the aim of this paper to obtain an overall summary of mutation properties of this model in the selected genetic markers. Therefore, auxotrophic mutants, STM and INH resistant mutants and mutants with changed pigmentation induced by UV-radiation, ethyl methanesulfonate, nitrosoguanidine, nitrous acid, hydroxylamine and acriflavine were evaluated both qualitatively and quantitatively. Oceasional changes of morphology of rods and colonies and inactivating effects of the used mutagens were also considered.     

CgCYN1, a plasma membrane cystine-specific transporter of Candida glabrata with orthologues prevalent among pathogenic yeast and fungi

We describe a novel plasma membrane cystine transporter, CgCYN1, from Candida glabrata, the first such transporter to be described from yeast and fungi. C. glabrata met15Δ strains, organic sulfur auxotrophs, were observed to utilize cystine as a sulfur source, and this phenotype was exploited in the discovery of CgCYN1. Heterologous expression of CgCYN1 in Saccharomyces cerevisiae met15Δ strains conferred the ability of S. cerevisiae strains to grow on cystine. Deletion of the CgCYN1 ORF (CAGL0M00154g) in C. glabrata met15Δ strains caused abrogation of growth on cystine with growth being restored when CgCYN1 was reintroduced. The CgCYN1 protein belongs to the amino acid permease family of transporters, with no similarity to known plasma membrane cystine transporters of bacteria and humans, or lysosomal cystine transporters of humans/yeast. Kinetic studies revealed a K(m) of 18 ± 5 μM for cystine. Cystine uptake was inhibited by cystine, but not by other amino acids, including cysteine. The structurally similar cystathionine, lanthionine, and selenocystine alone inhibited transport, confirming that the transporter was specific for cystine. CgCYN1 localized to the plasma membrane and transport was energy-dependent. Functional orthologues could be demonstrated from other pathogenic yeast like Candida albicans and Histoplasma capsulatum, but were absent in Schizosaccharomyces pombe and S. cerevisiae.     

Developmental pattern of cystine transport in isolated rat renal tubules

Isolated renal cortical tubule fragments from rats ranging in age from less than 48 h to 15 weeks were used to examine the pattern of cystine uptake with development. Immature tubules took up cystine with a faster initial rate than mature tubules and did not reach a steady state by 60 min. By eight weeks of age, the timed uptake of cystine began to approach a steady state and between 8 and 11 weeks the uptake pattern achieved its adult form of reaching a steady state by 30 min of incubation. Analysis of the intracellular metabolism of the cystine taken up by the newborn tubules revealed that the majority had been reduced to cysteine with the formation of small amounts of reduced glutathione. Cystine entered the renal cortical tubule cell from the newborn via two saturable transport systems similar to the mature animal. The kinetic parameters of initial uptake of these two transport systems were similar in the mature and newborn animal except for a higher maximum transport velocity for the low K_m, low capacity system in the newborn. Lysine inhibited cystine uptake by newborn tubules and this inhibition appeared to occur on the low K_m, low capacity transport system similar to the adult. Cystine uptake was sodium dependent with an apparent affinity for sodium of 36 mequiv./l. From this data, the physiologic cystinuria of the immature animal does not appear to be referrable to a lower rate of influx as previously observed with the cortical slice. Other mechanisms should be sought to explain this phenomenon of immaturity.     

Relationship between Spontaneous Aminoglycoside Resistance in Escherichia coli and a Decrease in Oligopeptide Binding Protein

Changes in the amount of oligopeptide binding protein (OppA) in spontaneous kanamycin-resistant mutants of Escherichia coli were investigated. Among 20 colonies obtained from 10⁸ cells cultured in the presence of 20 μg of kanamycin/ml, 1 colony had no detectable OppA and 7 colonies were mutants with reduced amounts of OppA. Sensitivity of wild-type cells to kanamycin increased slightly by transformation of the oppA gene, but the sensitivity of the mutants increased greatly by the transformation. A mutant with no OppA was found to be a nonsense mutant of the oppA gene at amino acid position 166. In a mutant having a reduced level of OppA, the reduction was due to the decrease in OppA synthesis at the translational level. These mutants were also resistant to other aminoglycoside antibiotics, including streptomycin, neomycin, and isepamicin. Isepamicin uptake activities decreased greatly in these two kinds of mutants. The results support the proposition that aminoglycoside antibiotics are transported into cells by the oligopeptide transport system, and that transport is an important factor for spontaneous resistance to aminoglycoside antibiotics.     

Genetic interaction observed in Bacillus subtilis

Summary Some evidence was obtained that genetic interaction occurs inBacillus subtilis K. A mixed inoculation of two doubly auxotrophic mutants onto approriate media yielded tiny colonies which seemed to be initiated by heterocaryons or heterozygotes. The tiny colonies contained not only a recombinant type which acquired two characters from one or another parent, but also some abnormal types having new characters which were not recognized in either parent. The phenomenon is similar to the genetic interaction found inStreptomyces.With 5 Figures in the Text     

A search for allelic recombination in Chinese hamster cell hybrids

Summary Mutants resistant to 6-thioguanine were selected from CHO cells which were either temperature sensitive or proline requiring. These mutants were stable and had low levels of hypoxanthine guanine phosphoribosyl transferase (HGPRT). Hybrids were selected which were heteroallelic at the hgprt locus and complementation between the mutants used was not observed. Interallelic recombination at this locus would generate hgprt ⁺ cells which could be selected in Littlefield's HAT medium. Selection experiments with hybrids containing three different pairs of mutants yielded no recombinants among populations of 4x10⁶-2x10⁷ cells. After treatment with the recombinagen mitomycin C, 3 putative recombinants were detected amongst 1.4x10⁷ surviving cells from one hybrid. One of these strains was examined and shown to have a normal level of HGPRT and its heterozygosity at this locus was demonstrated by the segregation of colonies resistant to 6-thioguanine. It cannot be excluded that the rare hgprt ⁺ colonies seen arose by mutation rather than by recombination. Mitotic allelic recombination therefore appears to be a much less frequent event in CHO cells than it is in lower eukaryotes. It is possible that mitotic recombination is effectively suppressed in mammalian cells to prevent the expression of deleterious recessive mutants.     

Characterisation of mutants of Escherichia coli K12, selected by resistance to streptozotocin

Summary From cultures of sensitive bacteria, treated with the antibiotic streptozotocin, two classes of resistant mutants can be isolated: 1) mutants, resistant under all the conditions tested to even the highest doses of the antibiotic. These are either pleiotropicdefective, pts-mutants, or more frequently, mutants lacking a transport system (enzyme II^Nag-complex of the PEP-dependent phosphotransferase system) encoded by the gene nagE. This gene is inducible by N-acetyl-glucosamine and seems to be part of the nag operon. The transport system in question is responsible for the uptake of N-acetyl-glucosamine, of D-glucosamine and of streptozotocin; 2) conditional resistant mutants which are unable to energize or to synthesize the streptozotocin transport system under certain growth conditions but do have the transport activity under other conditions. These include a) mutants auxotrophic for amino acids, vitamins, or nucleotides, b) mutants negative or sensitive to carbohydrates in the medium, and c) mutants with defects in energy metabolism such as PEP synthesis.     

Biosynthesis of the iron-molybdenum cofactor and the molybdenum cofactor in Klebsiella pneumoniae: effect of sulfur source.

NifQ- and Mol- mutants of Klebsiella pneumoniae show an elevated molybdenum requirement for nitrogen fixation. Substitution of cystine for sulfate as the sulfur source in the medium reduced the molybdenum requirement of these mutants to levels required by the wild type. Cystine also increased the intracellular molybdenum accumulation of NifQ- and Mol- mutants. Cystine did not affect the molybdenum requirement or accumulation in wild-type K. pneumoniae. Sulfate transport and metabolism in K. pneumoniae were repressed by cystine. However, the effect of cystine on the molybdenum requirement could not be explained by an interaction between sulfate and molybdate at the transport level. Cystine increased the molybdenum requirement of Mol- mutants for nitrate reductase activity by at least 100-fold. Cystine had the same effect on the molybdenum requirement for nitrate reductase activity in Escherichia coli ChlD- mutants. This shows that cystine does not have a generalized effect on molybdenum metabolism. Millimolar concentrations of molybdate inhibited nitrogenase and nitrate reductase derepression with sulfate as the sulfur source, but not with cystine. The inhibition was the result of a specific antagonism of sulfate metabolism by molybdate. The effects of nifQ and mol mutations on nitrogenase could be suppressed either by the addition of cystine or by high concentrations of molybdate. This suggests that a sulfur donor and molybdenum interact at an early step in the biosynthesis of the iron-molybdenum cofactor. This interaction might occur nonenzymatically when the levels of the reactants are high.     

Cystine reductase in the dimorphic fungus Histoplasma capsulatum.

Organo-sulfur compounds favor the transition of mycelia of Histoplasma capsulatum to the yeast form (6, 8). Investigation of the role of cystine in the transition revealed that the two phases concentrated this amino acid at comparable rates and that mutants defective in the uptake of cystine were still able to undergo the transition normally. Uptake of cystine is therefore probably not a requirement for transition to or maintenance of the yeast phase. Both phases contained a reduced nicotinamide adenine dinucleotide phosphate-dependent glutathione reductase; but a reduced nicotinamide adenine dinucleotide-dependent cystine reductase was detectable only in the yeast phase. The cystine reductase appeared early in the transition of mycelium to yeast. Treatment of mycelia with p-chloromercuriphenylsulfonic acid, which prevented the transition to yeast, had no effect on cystine uptake but strongly inhibited the cystine reductase. These results suggest that cystine reductase may provide reduced sulfhydryl groups involved in the transition of mycelium to yeast.     

Isolation and characterization of mutants of mesophilic methanogenic bacteria resistant to analogues of DNA bases and nucleosides

Spontaneous mutants of mesophilic Methanobacterium, Methanobrevibacter and Methanosarcina species resistant to 6-mercaptopurine, 5-fluorouracil, 8-azaguanine, 6-azauracil or 5-fluorodeoxyuridine were isolated. Low level resistant mutants were unstable but highly resistant strains (resistance factor greater than 10-fold) were stable and showed growth characteristics comparable to the parent. Wild type strains showed linear uptake of hypoxanthine and uracil into cells, but guanine uptake was only detected in Methanosarcina mazei. 6-Mercaptopurine-resistant clones of Methanobacterium and Methanobrevibacter species and 8-azaguanine-resistant clones of Methanosarcina mazei showed reduced uptake of hypoxanthine and guanine respectively, but no evidence for altered permeability of 5-fluoro-and 6-azauracil-resistant strains to uracil was obtained. Double resistant mutants of Methanobacterium sp. strain FR-2 were characterised. Although these generally exhibited reduced specific growth rates, several were selected which showed similar growth to the parent.Abbreviations DSM Deutsche Sammlung von Mikroorganismen, Federal Republic of Germany - MJC minimum inhibitory concentration - cfu colony forming unit - MP 6-mercaptopurine - FU 5-fluorouracil - FDU 5-fluorodeoxyuridine - AG 8-azaguanine - AU 6-azauracil - DA l-deazaadenosine     

Characterization of sugar transport in 2-deoxy-d-glucose resistant mutants of yeast

Summary A number of 2-deoxy-d-glucose (2-DOG) resistant mutants exhibiting resistance to glucose repression were isolated from variousSaccharomyces yeast strains. Most of the mutants isolated were observed to have improved maltose uptake ability in the presence of glucose. Fermentation studies indicated that maltose was taken up at a faster rate and glucose taken up at a slower rate in the mutant strains compared to the parental strains, when these sugars were fermented together. When these sugars were fermented separately, only the 2-DOG resistant mutant obtained fromSaccharomyces cerevisiae strain 1190 exhibited alterations in glucose and maltose uptake compared to the parental strain. Kinetic analysis of sugar transport employing radiolabelled glucose and maltose indicated that both glucose and maltose were transported with higher rates in the mutant strain. These results suggested that the high affinity glucose transport system was regulated by glucose repression in the parental strain but was derepressed in the mutant.     

Elevated Mutagenesis Does Not Explain the Increased Frequency of Antibiotic Resistant Mutants in Starved Aging Colonies

The frequency of mutants resistant to the antibiotic rifampicin has been shown to increase in aging (starved), compared to young colonies of Eschierchia coli. These increases in resistance frequency occur in the absence of any antibiotic exposure, and similar increases have also been observed in response to additional growth limiting conditions. Understanding the causes of such increases in the frequency of resistance is important for understanding the dynamics of antibiotic resistance emergence and spread. Increased frequency of rifampicin resistant mutants in aging colonies is cited widely as evidence of stress-induced mutagenesis (SIM), a mechanism thought to allow bacteria to increase mutation rates upon exposure to growth-limiting stresses. At the same time it has been demonstrated that some rifampicin resistant mutants are relatively fitter in aging compared to young colonies, indicating that natural selection may also contribute to increased frequency of rifampicin resistance in aging colonies. Here, we demonstrate that the frequency of mutants resistant to both rifampicin and an additional antibiotic (nalidixic-acid) significantly increases in aging compared to young colonies of a lab strain of Escherichia coli. We then use whole genome sequencing to demonstrate conclusively that SIM cannot explain the observed magnitude of increased frequency of resistance to these two antibiotics. We further demonstrate that, as was previously shown for rifampicin resistance mutations, mutations conferring nalidixic acid resistance can also increase fitness in aging compared to young colonies. Our results show that increases in the frequency of antibiotic resistant mutants in aging colonies cannot be seen as evidence of SIM. Furthermore, they demonstrate that natural selection likely contributes to increases in the frequency of certain antibiotic resistance mutations, even when no selection is exerted due to the presence of antibiotics.     

Temperature response,pathogenicity, seed infection and mutant evaluation against Macrophomina phaseolina causing charcoal rot disease of sesame

Charcoal rot caused by Macrophomina phaseolina is a serious disease of sesame in Pakistan. M. phaseolina sesame isolate was subjected to growth rate test at 10, 15, 20, 25, 30, 35 and 40°C. The optimum temperature for fungal growth and microsclerotia production was found to be 30–35°C. Gray to black, radial fungal colonies with intermediate mycelial growth and jet black oval to round microsclerotia were observed at this optimum range. M. phaseolina was found to be pathogenic against all the 18 tested plant species and this pathogenicity proved its necrophytic behavior. Seed infection efficiency of M. phaseolina was 100% with significant reduction in seed index. For two consecutive years 21 mutants/varieties were screened in the field for their reactions to charcoal rot disease. During 2007 three mutants NS11704S1, NS11304S2 and NS26004 were ranked as resistant while others were moderately resistant to highly susceptible. During 2008 all mutants showed a susceptible to highly susceptible reaction with variable disease reactions. All over screening results revealed that four mutants viz, NS13P1, NS163-1, NS270P1 and NS26004 showed about 50% stand with consistent performance during both years under optimum disease conditions and can be used to manage the disease following the disease management strategies, however in the future improvement for high seed yield along with resistance is a prerequisite for sustainable high production.     

Selection of Daucus carota somatic hybrids using drug resistance markers and characterization of their mitochondrial genomes

Summary Protoplasts from different Daucus carota L. cell strains carrying resistance to glyphosate, 5-methyltryptophan, sodium selenate or selenocystine were fused in three combinations using dextran. Clones were selected for both of the resistances carried by the individual parental strains in medium with both inhibitors. No doubly resistant colonies formed from unfused controls or from protoplasts from each individual parental strain alone. Suspension cultures from the selected clones contained predominantly the additive chromosome numbers of the parental strains. Apparently the four resistances used are expressed dominantly in fusion hybrids. Analysis of mitochondrial DNA showed that recombination occurred in one fusion combination since the mitochondrial DNA in the hybrid cells was different from that of either parent as shown by restriction endonuclease fragment analysis. Mitochondrial DNA in the other two somatic hybrid combinations was parental. Thus, a dominant, nuclear resistance marker system has been developed to select efficiently for somatic hybrids in which mitochondrial DNA recombination can be studied.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - FW fresh weight - Glp Glyphosate - mt mitochondrial - MX Murashige and Skoog (1962) medium containing 0.4 mg/l 2,4-D - 5MT 5-methyltryptophan - MXG MX containing (5% Glucose) - SC selenocystine - SS sodium selenate     

Morphology-related effects on gene expression and protein accumulation of the yeast Arxula adeninivorans LS3

The dimorphism of the yeast Arxula adeninivorans LS3 is regulated by cultivation temperatures. Up to 42 degrees C the yeast grows as budding cells, which turn to mycelia at higher temperatures. To test whether the dimorphism is exclusively induced by high temperatures or also by other conditions, mutants were selected with an altered behaviour with respect to dimorphism. After mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, five of 25,000 colonies formed a very rough surface consisting of mycelia at 30 degrees C, in contrast to the wild-type. These mutants allow temperature-mediated and morphology-related effects on gene expression and protein accumulation to be distinguished. Budding cells and mycelia showed different expression of genes encoding secretory proteins at the same temperature. Mycelia secreted two-fold more protein than budding cells, including the enzymes glucoamylase and invertase. This indicated that morphology, rather than temperature, is the decisive factor in the analysed processes.     

A new experimental system for study on adaptive mutations

A super-repressed mutant of purR (purR^S), which encodes a repressor protein controlling expression of purine biosynthetic genes inSalmonella typhimurium, grew very slowly on NCE medium with 10 μg/mL Ade and lactose as sole carbon source (cannot form colonies). However, a phenomenon of late-arising mutations was observed when purR^S mutants were spread on NCE+lactose plates and subjected to a prolonged non-lethal selection. The reconstruction experiments of revertants showed that the late-arising “lac⁺” mutants are not slow growing mutants. Statistical analysis indicated that the distribution of late-arising mutants is Poisson distribution, showing that reversion occurred after plating. The result of co-transductional analysis preliminarily showed that late-arising mutation occurred at selected genepurR or 16 bp PUR box,cis element of structural genepurD. The above results suggest that the phenomenon of late-arising mutation observed by our system is a result of adaptive mutations which are different from random mutations. This is the first time to extend target genes at which adaptive mutations could occur from structural genes involved in carbon metabolism and amino acid biosynthesis totrans regulatory gene coding repressor protein. Our results have provided not only a new proof for generality of adaptive mutations but also a new system for study on adaptive mutations.     

A new experimental system for study on adaptive mutations

A super-repressed mutant of purR (purRs), which encodes a repressor protein controlling expression of purine biosynthetic genes in Salmonella typhimurium, grew very slowly on NCE medium with 10 μg/mL Ade and lactose as sole carbon source (cannot form colonies). However, a phenomenon of late-arising mutations was observed when purRs mutants were spread on NCE+lactose plates and subjected to a prolonged non-lethal selection. The reconstruction experiments of revertants showed that the late-arising "lac+" mutants are not slow growing mutants. Statistical analysis indicated that the distribution of late-arising mutants is Poisson distribution, showing that reversion occurred after plating. The result of co-transductional analysis preliminarily showed that late-arising mutation occurred at selected gene purR or 16 bp PUR box, cis element of structural gene purD. The above results suggest that the phenomenon of late-arising mutation observed by our system is a result of adaptive mutations which are different     

Characterization of bromoethanesulfonate-resistant mutants of Methanococcus voltae: evidence of a coenzyme M transport system.

Mutants of Methanococcus voltae were isolated that were resistant to the coenzyme M (CoM; 2-mercaptoethanesulfonic acid) analog 2-bromoethanesulfonic acid (BES). The mutants displayed a reduced ability to accumulate [35S]BES relative to the sensitive parental strain. BES inhibited methane production from CH3-S-CoM in cell extracts prepared from wild-type sensitive or resistant strains. BES uptake required the presence of both CO2 and H2 and was inhibited by N-ethylmaleimide and several reagents that are known to disrupt energy metabolism. The mutants showed normal uptake of isoleucine and were not cross-resistant to either azaserine or 5-methyltryptophan and, thus, were neither defective in general energy-dependent substrate transport nor envelope permeability. Both HS-CoM and CH3-S-CoM prevented the uptake of BES and protected cells from inhibition by it. We propose that M. voltae has an energy-dependent, carrier-mediated uptake system for HS-CoM and CH3-S-CoM which can also mediate uptake of BES.     

Regulation of sulfate assimilation in Cytophaga johnsonae

We have studied the regulation of sulfate assimilation by the gliding bacterium Cytophaga johnsonae in which 20% of the total sulfur is in the sulfornate moiety of sulfonolipid. Added cystine inhibited sulfate uptake and growth with cystine as sulfur source resulted in a repression of sulfate uptake. However, low concentrations of cystine preferentially repressed the terminal reactions of sulfate assimilation responsible for cysteine synthesis while allowing the transport and activation of sulfate for sulfonolipid synthesis. The significance of this novel pattern of regulation in bacteria is discussed.