Selection of microbial mutants tolerant to extreme environmental stress using continuous culture-control design

The design of controllers for a continuous selection technique (BOICS; Brown and Oliver, 1982) is considered. This technique is used to obtain microbial mutants that are tolerant to extreme environmental stress. Applications of BOICS have been hampered by the problem of controller design. In this paper, a modified implementation of BOICS is considered which has a number of practical advantages. A model-based approach to controller design is taken. The case in which the stress is due to an inhibitory substance in the growth environment is considered. The analysis is intended to be applicable to any reasonable combination of organism and inhibitor. Conventional linear and time-invariant controllers are considered. Guidelines for the selection of controller parameters' values are suggested. The application of these guidelines requires that certain process parameters' values be identified. Methods by which these parameters' values can be identified are suggested. Simulation results indicate that the resulting controllers perform satisfactorily. This is confirmed by experimental data from a model selection experiment. A recipe for the design of controllers is a necessary part of a protocol for BOICS. It is hoped that the solution to the controller design problem that is offered in this paper will encourage further applications for the technique.     

Characterization of Escherichia coli mutants tolerant to bacteriocin JF246: two new classes of tolerant mutants

Several hundred independent bacteriocin-tolerant mutants have been isolated without mutagenesis from three strains of Escherichia coli. On the basis of patterns of sensitivity to eight different colicins, over 85% of these mutants could be grouped into four classes. Two classes of mutants, class A and class B, are equivalent to tolA and tolB type mutants. We found tolA and tolB mutants were sensitive to the antibiotic bacitracin. The other two classes of bacteriocin-tolerant mutants, class F and class G, are distinguished from other types of colicin-tolerant mutants on the basis of sensitivity to colicins, dyes, detergents, antibiotics, and chelating agents. The mutation in class F and class G mutants is located between 21 to 23 min on the E. coli chromosome. We propose to designate the loci of these mutations as tolF and tolG, respectively.     

An in vitro whole plant selection system: paraquat tolerant mutants in the fern Ceratopteris

Summary A whole plant selection system using the haploid gametophyte generation of the fern Ceratopteris richardii has been developed to select for mutations that confer resistance or tolerance to various selection pressures. The expression of the mutations can be analyzed and characterized in both the haploid gametophyte and diploid sporophyte generations. Genetic analyses are facilitated by the fern's rapid life cycle and the ease of manipulating the gametophyte generation. Selection for tolerance to the herbicide paraquat has yielded two mutants which have an increased tolerance to the herbicide in both the gametophyte and sporophyte generations. Both mutants exhibit single nuclear gene inheritance patterns and appear to be closely linked or allelic.     

Enrichment technique for the selection of catabolite repression-resistant mutants of Trichoderma as producers of cellulase

Abstract The enrichment technique for the preparation of catabolite repression-resistant producers of cellulase from Trichoderma reesei is based on the submerged cultivation of mutagenized conidia on 2% (w/v) cellobiose or carboxymethyl-cellulose and in the presence of 0.5% (w/v) 2-deoxyglucose as the catabolite repressor. Conidia that are resistant towards the catabolite repressor can produce enzymes necessary for hydrolysis of used substrates and grow under the given conditions. They can be separated from the ungerminated conidia by filtration and used for the production of new conidia which are already enriched with catabolite repression-resistant mutants.     

Alfalfa (Medicago sativa L.) clones tolerant to salt stress: in vitro selection

In order to quickly and efficiently evaluate the salt tolerance of alfalfa, salinity tests were conducted on Medicago sativa L. var. australis, var. icon, var. loi, and var. gea, under in vitro conditions. Pregerminated seeds of four varieties were subjected to five different NaCl concentrations (0, 50, 100, 150, 200 mM). The influence of saline stress was estimated on the basis of survival percentage, growth parameters, and electrolyte leakage. The seedlings surviving on the medium enriched with salt at the highest concentration were presumed to be tolerant and represented the mother plants for the production of in vitro clones. In the following step, the clones were evaluated in vitro to confirm the salt tolerance. The influence of mild salt stress (75 mM NaCl) on the growth parameters of selected clones was examined. At the end of this trial, the proline accumulation and sodium content in alfalfa shoots were also quantified. The results suggest an increased level of proline promotes salt tolerance. Medicago sativa L. var. icon is highly tolerant in comparison with the other varieties tested. In vitro selection of M. sativa L. varieties on salt-containing media allowed us to obtain clones with increased salinity tolerance.     

Plant responses to environmental stress.

Considerable progress is being made in identifying genes that are important for tolerance to abiotic stress and in defining stress-responsive gene promoters and signal-transduction pathways. Although genetically engineered crop plants with greater resistance to environmental stress have not yet been produced, research is at a turning point where correlative changes can now be tested for effectiveness in conferring stress tolerance.     

A new technique for selection of sensitive and auxotrophic mutants of E. coli: isolation of a strain sensitive to an excess of one-carbon metabolites.

Summary An E. coli strain deleted in the region malasd is used for the selection of conditional or auxotrophic mutants. Thermosensitive and auxotrophic strains have thus been isolated on plates. After selection in liquid medium, a strain has been isolated which is sensitive to excess one-carbon metabolites. It carries two mutations, smg A (near metA and argH), probably identical to rel C, and smg B (between asn and ilv), probably part of the E. coli membrane ATPase.Abbreviations dap 1 meso diaminopimelic acid - smg serine+methionine+glycine - 1:1 1 per weight     

Mapping and marker-assisted selection for a gene for extreme resistance to potato virus Y

The chromosomal location of the major gene Ry _adg controlling extreme resistance to potato virus Y (PVY) in Solanum tuberosum subsp. andigena was identified by RFLP analysis of a diploid potato population. A total of 64 tomato and potato RFLP markers were screened with the bulked segregant analysis (BSA) on segregants extremely resistant, hypersensitive or susceptible to PVY. Four markers TG508, GP125, CD17 and CT168 at the proximal end of chromosome XI showed close linkage with extremely resistant phenotypes. TG508 was identified as the closest marker linked with the Ry _adg locus with the maximum map distance estimated as 2.0 cM. The 4 markers linked with the Ry _adg locus were tested on independent tetraploid and diploid potato clones and were subsequently found useful for marker-assisted selection for plants containing Ry _adg . Received: 5 July 1996 / Accepted: 19 July 1996     

Shifts in population size structure for a drying‐tolerant fish in response to extreme drought

For freshwater systems, climate change‐induced alterations to drought regimes are a considerable threat to already threatened species. This is particularly poignant for kōwaro (or Canterbury mudfish, Neochanna burrowsius), a critically endangered fish largely restricted to drying‐prone waterways on the Canterbury Plains, New Zealand. By comparing three catchment‐wide surveys (2007, 2010, 2015) within the Waianiwaniwa Valley, we assessed the scale and magnitude of population change induced by 2 years of consecutive drought (2014–15), when compared to surveys during wetter conditions (2007, 2010). The droughts triggered a catchment‐wide switch from adult‐dominated populations to populations comprised of juveniles indicated by a significant reduction in median size (~95 mm during the wet to ~60 mm after drought). In comparison, population abundances were highly variable, indicated by no significant change in catch‐per‐unit‐effort. The large variation in catch rates and connection of median size to reproductive potential mean median size will be useful to measure in monitoring to infer potential changes to population resilience, particularly during extreme events. Furthermore, because N. burrowsius could be regarded as extremophile fish, already restricted to harsh habitats, they are likely to become increasingly threatened by climate change. Thus, tools that allow for insightful comparisons between populations, such as a population resilience framework based on both abundance and body size distribution, will be increasingly important for pragmatic decision‐making for targeted conservation measures.     

Use of continuous-culture techniques for determining the growth kinetics of a celluloytic Thermoactinomyces sp.

In order to better understand the kinetics of cellulose degradation by Thermoactinomyces sp., continuous-culture experiments were performed utilizing the various intermediates of cellulose degradation as the feed substrates. Steady-state data from the glucose runs suggest that this organism has a growth yield of 0.42 g cell/g glucose, and a specific maintenance of 0.24 g glucose/g cell/hr. The Monod equation did not seen to model the growth well, since a plot of 1/D vs. 1/S gave a maximum specific growth rate that was even lower than one of the steady-state dilution rates. A dynamic washout experiment suggested a maximum specific specific growth rate of 0.36 hr^?1 and indicated that glucose is only slightly growth inhibitory as the inhibition constant, K_i, is 19 g glucose/liter. An equation for substrate concentration for washout conditions was derived. This equation predicted the transient glucose concentration relatively well. A fill-and-draw technique was investigated for determination of the growth parameters. It was not successful because of difficulties in contamination and accurately monitoring the dissolved oxygen in the small highly agitated vessel. However, the technique could be useful in studying the growth characteristics of sludge in a waste treatment system where contamination is not a worry. One could cover the medium surface and use a nonsterilizable dissolved oxygen probe of high sensitivity membrane to overcome these difficulties.     

Effects of environmental stress on leaf hair density and consequences for selection

We explored how five different environmental stresses and a benign environment affect the phenotypic expression of leaf hairs, and the potential for evolutionary response in this trait. To address these questions we planted full-sib families that had been selected for stress tolerance in a factorial design in which selection history was fully crossed with the current environment (eight families × six selection histories × six current environments × three blocks=864 plants). Our data suggest that leaf hair density is a complex character composed of two separable traits: leaf area and the number of hairs initiated per leaf. Leaf size is primarily controlled by the growth environment, whereas leaf hair initiation shows more genetically based variation. In the control and low water environments there was a negative linear relationship between leaf size and leaf hair density. However, within the low light and high boron treatments, leaf hair density remained constant across a range of leaf sizes, suggesting that these stresses disrupt normal leaf hair development. Selection gradient analysis showed that leaf hair density and leaf size were significantly positively associated with fitness in 4/6 of our environments. Our results suggest that environmental variation may diminish the ability of herbivores and pathogens to cause directional selection on leaf hair density.     

Regulation of nitrogen metabolism in Escherichia coli and Klebsiella aerogenes: studies with the continuous-culture technique.

Ammonia-nitrogen-limited continuous cultures of Escherichia coli and Klebsiella aerogenes contain induced levels of glutamine synthetase that is deadenylyated (i.e., fully active). In the presence of excess ammonia or glutamate in glucose-limited cultures of E. coli, glutamine synthetase is repressed and adenylylated (inactive). The average state of adenylylation (n) is a linear function of the specific growth rate. At low specific growth rates, glutamine synthetase is adenylylated; as the specific growth rate increases, n decreases, approaching 0 to 2 at rapid growth rates. The average state of adenylylation correlates well with the intracellular concentrations and ratios of alpha-ketoglutarate and glutamine, which are key effectors in the adenylylation-deadenylylation systems. E. coli and K. aerogenes differ markedly in their growth yields, growth rates, and enzymatic composition during nitrogen limitation. The data suggest that, unlike K. aerogenes, E. coli W uses glutamate dehydrogenase to incorporate ammonia during nitrogen limitation. In E. coli, glutamate dehydrogenase is progressively induced during nitrogen limitation when mu (growth rate) approaches mumax. In contrast, in K. aerogenes glutamate dehydrogenase is repressed during nitrogen limitation, whereas glutamate synthase, an alternative supplier of glutamate to the cell, is induced. Data are presented that support the regulatory schemes proposed for the control of glutamine synthetase activity by induction-repression phenomena and adenylylation-deadenylylation reaction. We propose that the intracellular ratio of alpha-ketoglutarate to glutamine may be the most important physiological parameter in determining the activity of glutamine synthetase.     

Improved method of selection for mutants of Pseudomonas putida.

Optimum conditions for enrichment of mutants of Pseudomonas putida in liquid culture were established using a procedure which combines N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis with an improved D-cycloserine selection.     

Responses of neurons to extreme osmomechanical stress

Neurons are often regarded as fragile cells, easily destroyed by mechanical and osmotic insult. The results presented here demonstrate that this perception needs revision. Using extreme osmotic swelling, we show that molluscan neurons are astonishingly robust. In distilled water, a heterogeneous population of Lymnaea stagnalis CNS neurons swelled to several times their initial volume, yet had a ST₅₀ (survival time for 50% of cells) >60 min. Cells that were initially bigger survived longer. On return to normal medium, survivors were able, over the next 24 hr, to rearborize.Reversible membrane capacitance changes corresponding to about 0.7 F/cm² of apparent surface area accompanied neuronal swelling and shrinking in hypo- and hyperosmotic solutions; reversible changes in cell surface area evidently contributed to the neurons' ability to accommodate hydrostatic pressures then recover. The reversible membrane area/capacitance changes were not dependent on extracellular Ca²⁺.Neurons were monitored for potassium currents during direct mechanical inflation and during osmotically driven inflation. The latter but not the former stimulus routinely elicited small potassium currents, suggesting that tension increases activate the currents only if additional disruption of the cortex has occurred.Under stress in distilled water, a third of the neurons displayed a quite unexpected behavior: prolonged writhing of peripheral regions of the soma. This suggested that a plasma membrane-linked contractile machinery (presumably actomyosin) might contribute to the neurons' mechano-osmotic robustness by restricting water influx. Consistent with this possibility, 1 mM, N-ethylmaleimide, which inhibits myosin ATPase, decreased the ST₅₀ to 18 min, rendered the survival time independent of initial size, and abolished writhing activity.For neurons, active mechanical resistance of the submembranous cortex, along with the mechanical compliance supplied by insertion or eversion of membrane stores may account for the ability to withstand diverse mechanical stresses. Mechanical robustness such as that displayed here could be an asset during neuronal out-growth or regeneration.This work was supported by a NSERC Canada research grant to CEM.