On the adaptive nature of the dissociation process in Bacillus thuringiensis

The process of dissociation into variants differing in colony morphology occurring in batch cloned cultures of two Bacillus thuringiensis strains belonging to different subspecies was studied at optimal and elevated temperatures. An increase in the cultivation temperature to 40 degrees C resulted in an increase in the fraction of R variants to 100% after 72 h of cultivation of either of the strains. This increase was not due to the selection of forms with greater resistance to elevated temperature. The level of resistance to elevated temperature was determined by the strain genotype and did not correlate with morphological characteristics of the colonies.     

Temperature range variants of Bacillus megaterium

Facultatively and obligately thermophilic variants were isolated from 3 out of 12 tested mesophilic Bacillus megaterium strains. The variants occurred at a frequency of 10^-8–10^-9. The ability to grow at elevated temperatures was cured by means of treatment with acridine orange. Stable revertants were isolated from facultatively and obligately thermophilic variants. An unknown type of megacin was produced by the facultative thermophiles. This megacin attacked mesophilic and obligately thermophilic strains. The thermophiles displayed a few divergent taxonomic characteristics but a close relationship between the strains was indicated by the megacin spectrum and sensitivity to phage. Arrhenius plots revealed that the strains could be considered as temperature range variants and that the temperature characteristic increased with growth at a higher temperature range. The case for a plasmid involvement in the phenomenon is discussed.Abbreviations M Mesophilic - Fp facultatively psychrophilic - Ft facultatively thermophilic - Ot obligately thermophilic     

Seasonal growth activity of local and foreign gracilarioid strains in Israel

Six gracilarioid strains originating from different climatical environments were cultured in two cultivation systems: a short-term indoor one with a cross gradiant table, and a long-term outdoor one. Seasonal growth performances of the different strains were determined. The growth results in the two culture systems showed similar trends. The tropical species.Gracilaria cornea andG. cornea mutant, showed highest growth rates during summer and no growth at all during winter. The temperate species,Gracilaria verrucosa andGracilariopsis lemaneiformis, showed best growth performances during winter with small fluctuations between seasons. The subtropical speciesGracilaria conferta (local) showed seasonal growth fluctuations all over the year. The foreign species definitely did not acclimate under local conditions, but successfully preserved their original response to temperature. Regression equations confirmed that temperature was the dominant environmental variable in most of the gracilarioid strains. The growth rate results obtained showed encouraging prospects for high algal productivity as compared to other cultivation systems. Seasonal cultivation strategy ofGracilaria spp. in Israel is discussed.     

Population heterogeneity of plasmid-bearing and plasmid-freeBacillus subtilis strains under different environmental conditions

The population heterogeneity of recombinant and plasmid-freeBacillus subtilis strains introduced into aquatic microcosms was studied. After introduction, the population of the plasmid-free strainB. subtilis 2335 in microcosms has long been represented by both vegetative cells and spores, whereas, already ten days after introduction, the population of the recombinant strainB. subtilis 2335/105 (Km^rInf⁺) was represented only by spores. The number of plasmid copies in the spore isolates of the recombinant strain was the same as before introduction, but the plasmid abundance in the vegetative isolates of this strain decreased. The isolates ofB. subtilis 2335/105 obtained from microcosms and the variants of this strain obtained by ten successive subcultures on M9 and 0. I× M9 media with and without kanamycin (Km) differed in the number of plasmid copies, Km resistance, and maximum biomass yield during batch cultivation. Irrespective of the presence of Km, more than 50% of the variants subcultured on M9 medium showed reduced plasmid abundance. At the same time, about 70% of the variants subcultured on 0.1 × M9 medium with Km and 90% of the variants subcultured on the same medium without Km retained the initial number of plasmid copies. The variants subcultured on media with Km retained the initial biomass level. In more than 70% of the variants isolated from media without Km, the biomass yield increased.     

Temperature resistant Nocardia biovarieties in Tucumán,Argentina?

Eighty-six regional strains of the pathogenic Nocardia species isolated from soil and human mycetoma were tested for their response to different incubation temperatures and for their tolerance to different temperatures. The aim was to assess whether growth temperature and tolerance to elevated temperatures are valuable criteria for the differentiation of pathogenic species of local strains based on the results obtained from a large number of strains. The results showed that 75.34% of all N. brasiliensis isolates from both sources grew at a temperature higher than 37 °C. 20% of the mycetoma strains and 11.32% of those from soil grew at 45 °C. 98.1% of N. brasiliensis from soil and 55.0% of the mycetoma strains tolerated 50 °C for 8 h and many isolates from both sources endured this temperature for an even longer time and tolerated yet higher temperatures. Both properties (growth temperature and temperature tolerance) are used to identify N. asteroides complex (N. farcinica) and N. otitidiscaviarum, and according to our results they are not suitable to differentiate regional strains of this species. The N. asteroides strains assayed showed an ability to grow at and tolerate elevated temperatures superior to those belonging to the other species. Although adaptation of local N. asteroides and N. otitidiscaviarum strains to temperature is important, it is more significant for N. brasiliensis, because this species is predominant in the Tucumán soil and responsible for the major number of diseases in the area.This revised version was published online in October 2005 with corrections to the Cover Date.     

Haplopappus gracilis cell strains resistant to pyrimidine analogues

Summary Strains of Haplopappus gracilis (Nutt.) Gray cells resistant to 6-azauracil have been isolated from cultures of diploid cells. These strains are also resistant to 8-azaguanine, as is their parent. The variants are 100- to 125-fold more resistant to 6-azauracil than their parent, and they exhibit different spectra of cross resistance to other pyrimidine analogues. The phenotype of each variant is stable in the absence of selection. The majority of cells in cultures of the variants are diploid; all others examined were tetraploid. Initial rates of uptake of uracil are not reduced in the variants. Fluorouracil, to which two variants are resistant, is taken up by one of them as well as by the parent. Responses of the other two to fluorouracil are not correlated with decreased ability to accumulate this analogue.     

Exposure of Fischerella [Mastigocladus] to high and low temperature extremes: strain evaluation for a thermal mitigation process

In conjunction with a proposed algal cultivation scheme utilizing thermal effluent, twelve Fischerella strains were tested for tolerance to temperatures above and below their growth range. Exposure to 65 °C or 70 °C for 30 min caused bleaching and death of most or all cells. Effects of 60 °C exposure for periods of up to 2 h ranged from undetectable to severe for the various strains. Chlorophyll a content typically decreased 21–22% immediately following 60 °C or 65 °C (1 h) exposure. However, the 60 °C-shocked cultures regained normal Chl a content after 24 h at 45 °C, whereas Chl a in 65 °C-shocked cultures immediately lost visible autofluorescence and was later degraded. Exposure to 15 °C virtually stopped growth of all strains during a 48 h exposure period. Most strains grew as rapidly as 45 °C controls when restored to 45 °C, while a few strains recovered more slowly. Comparison with dark-incubated controls indicated that photooxidative damage did not occur during cold shock. Certain strains exhibited relatively rapid recovery from both heat and cold exposure, thus meeting the temperature tolerance criteria for the proposed algal cultivation process.     

Effect of sugar beet genotype on the Beet necrotic yellow vein virus P25 pathogenicity factor and evidence for a fitness penalty in resistance-breaking strains

Beet necrotic yellow vein virus (BNYVV), vectored by Polymyxa betae, causes rhizomania in sugar beet. For disease control, the cultivation of hybrids carrying Rz1 resistance is crucial, but is compromised by resistance-breaking (RB) strains with specific mutations in the P25 protein at amino acids 67–70 (tetrad). To obtain evidence for P25 variability from soil-borne populations, where the virus persists for decades, populations with wild-type (WT) and RB properties were analysed by P25 deep sequencing. The level of P25 variation in the populations analysed did not correlate with RB properties. Remarkably, one WT population contained P25 with RB mutations at a frequency of 11%. To demonstrate selection by Rz1 and the influence of RB mutations on relative fitness, competition experiments between strains were performed. Following a mixture of strains with four RNAs, a shift in tetrad variants was observed, suggesting that strains did not mix or transreplicate. The plant genotype exerted a clear influence on the frequency of RB tetrads. In Rz1 plants, the RB variants outcompeted the WT variants, and mostly vice versa in susceptible plants, demonstrating a relative fitness penalty of RB mutations. The strong genotype effect supports the hypothesized Rz1 RB strain selection with four RNAs, suggesting that a certain tetrad needs to become dominant in a population to influence its properties. Tetrad selection was not observed when an RB strain, with an additional P26 protein encoded by a fifth RNA, competed with a WT strain, supporting its role as a second BNYVV pathogenicity factor and suggesting the reassortment of both types.     

Heat Shock–Induced Changes in the Respiration of the Yeast Saccharomyces cerevisiae

The incubation of Saccharomyces cerevisiaeat elevated temperature (45°C) stimulated the respiration of yeast cells and decreased their survival rate. The respiration-deficient mutant of this yeast was found to be more tolerant to the elevated temperature than the wild-type strain. At the same time, the cultivation of the wild-type strain in an ethanol-containing medium enhanced the respiration, catalase activity, and thermotolerance of yeast cells, as compared with their growth in a glucose-containing medium. It is suggested that the enhanced respiration of yeast cells at 45°C leads to an intense accumulation of reactive oxygen species, which may be one of the reasons for the heat shock–induced cell death.     

Biochemical and physiological characteristics of Xenorhabdus species,symbiotically associated with entomopathogenic nematodes including Steinernema kushidai and their pathogenicity against Spodoptera litura (Lepidoptera: Noctuidae)

The symbiotic bacterium strain, SK-1 isolated from Steinernema kushidai, a new species of entomopathogenic nematode, was compared with other strains of Xenorhabdus species. Like other Xenorhabdus nematophilus strains, this new strain is gram-negative, facultatively anaerobic, peritrichously flagellated rod and negative for catalase and nitrate reduction. It can be distinguished from the other Xenorhabdus spp. by differences in reactions to phenylalanine deaminase, no acid production from myo-inositol and utilizations of inosine, dl-malate, formate and methanol. Intra-haemocoelic injection of actual cells or liquid culture supernatant into sixth instar larvae of Spodoptera litura for either Phase I or II variants were not pathogenic. Other strains of X. nematophilus showed pathogenicity for whole cell injections. The supernatants of strain D-1 and ATCC 19061, which are symbionts of Steinernema carpocapsae were pathogenic, however pathogenicity decreased and then terminated by increases in temperature.     

Construction of a new vector for the expression of foreign genes inZymomonas mobilis

Summary Broad host range plasmids have previously been shown to be suitable as vectors to introduce antibiotic resistance genes intoZ. mobilis. However, attempts to use these vectors to carry other genes with enteric promoters and controlling elements have resulted in limited success due to poor expression. Thus we have constructed a promoter cloning vector in a modified pBR327 and used this vector to isolated 12 promoters fromZ. mobilis which express various levels of -galactosidase inEscherichia coli. Four of these were then subcloned into pCVD 305 for introduction intoZ. mobilis. All expressed -galactosidase inZ. mobilis with activities of 100 to 1800 Miller units. One of these retained aBamHl site into which new genes can be readily inserted immediately downstream from theZ. mobilis promoter. Genetic traits carried by pCVD 305 were initially unstable but spontaneous variants were produced during sub-culture in which the plasmid was resistant to curing at elevated temperature. One of these variants was examined in some detail. The increased stability of this variant appears to result from an alteration in the plasmid rather than a chromosomal mutation or from chromosomal integration.     

Somaclonal variants resistant to sugarcane mosaic virus and their agronomic characterization

Summary The sugarcane mosaic virus is a pathogen that causes severe disease to sugarcane. New varieties resistant to insects and pathogens have been developed in the last 70 yr through sugarcane breeding programs, but this takes between 10 and 15 yr. Tissue culture techniques are used as an aid for sugarcane improvement to increase desirable agronomic characteristics, such as disease resistance. In the present work, we report the generation of somaclonal variants from sugarcane (Saccharum spp.) cultivar PR62258 susceptible to Sugarcane Mosaic Virus, by somatic embryogenesis. These new variants identified as AT626 and BT627 are resistant to Sugarcanes Mosaic Virus strains A and B, respectively. We established an indirect enzyme linked inmunosorbent assay (ELISA) to test the presence of the viral particles in plants, and its was demonstrated that the leaves of resistant somaclones do not contain viral particles. The field performance of the somaclones AT626 and BT627 was similar to the field performance of the mother plant PR62258.     

嗜热毁丝霉高通量培养技术及其应用

【背景】丝状真菌是一类重要的工业发酵生产宿主菌,如何进行高通量纯菌培养和高效检测筛选性能优异的菌株是工业丝状真菌研究的重要方向。【目的】研究建立丝状真菌的高通量培养技术并测试应用效果。【方法】通过对丝状真菌培养过程中的制种、接种、培养和检测研究,建立基于孔板的高通量培养技术,并以嗜热毁丝霉为例对该技术进行验证。【结果】与传统的平板制种和摇瓶接种培养方式相比,高通量孔板的培养方式将制种通量提高24倍,单位面积产孢子能力提高350%,液体培养转接效率提高10-40倍,并建立96孔板测定乙醇含量的高通量检测技术。【结论】将丝状真菌的培养和检测通量提高1-2个数量级,为快速检测丝状真菌改造过程产生的大量性状不同菌株并获得目标菌株奠定基础,为丝状真菌高通量筛选研究提供应用指导价值。     

Evaluation of host and pathogen responses for screening soil amendments to control Sclerotium rolfsii

Three strains of Bradyrhizobium, 280A, 2209A and 32H1, that nodulated peanuts (Arachis hypogaea L.), were tested for their ability to grow and survive at elevated temperatures of up to 42°C in laboratory culture. Strain 32H1 was unable to grow at 37°C and was more sensitive to elevated temperatures than the other two strains. All three produced heat-shock proteins of molecular weights 17 kDa and 18 kDa. Two greenhouse experiments were conducted to determine the effect of high root temperature on nodulation, growth and nitrogen fixation of peanut. Two peanut varieties (Virginia cv NC7 and Spanish cv Pronto) were inoculated and exposed to root temperatures of 30°, 37° and 40°C. Nodulation and nitrogen fixation were strongly affected by root temperature but there was no variety × temperature interaction. At a constant 40°C root temperature no nodules were formed. Nodules were formed when roots were exposed to this temperature with diurnal cycling but no nitrogen fixation occurred. Highest plant dry weight, shoot nitrogen content and total nitrogen were observed at a constant root temperature of 30°C. Increasing root temperature to 37°C reduced average nitrogen content by 37% and total nitrogen by 49% but did not reduce nodulation. The symbiotic performance of the strains corresponded to their abilities to grow and survive at high temperature in culture.     

Keratinase Production by Newly Isolated Antarctic Actinomycete Strains

Summary The ability of actinomycete strains newly isolated from Antarctic soils to produce keratinolytic enzymes during growth on sheep wool waste was investigated. The strains which displayed highest keratinase activity and identified as Streptomyces flavis 2BG (mesophilic) and Microbispora aerata IMBAS-11A (thermophilic) were selected for a more detailed analysis. The addition of starch to the growth medium affected keratinase secretion by both strains. After 5 days of cultivation, a 6-fold increase in keratinase activity of strain 11A was observed in the presence of 11 g starch/l and a 9-fold increase in keratinase activity of the strain 2BG in the presence of 5 g starch/l. The results obtained showed that both newly isolated strains are very promising for effective processing of native keratinous wastes. To our knowledge, this is the first report of Antarctic actinomycete strains that were able to grow on keratin-containing wastes by producing keratinolytic enzymes.     

A comparison of two strains of the anaerobic ciliate Trimyema compressum

Two strains of the anaerobic ciliate Trimyema compressum, isolated from different habitats, were compared. The cytoplasm of the ciliates contained hydrogenosome-like microbodies and methanogenic bacteria; the latter were lost during continued cultivation. In addition both strains harbored a non-methanogenic endosymbiont, which was lost in strain K. The ciliates lacked cytochromes, cytochrome oxidase and catalase but contained superoxide dismutase. Hydrogenase activity could be demonstrated only in strain N. In monoxenic culture strain K needed sterols as growth factors. The cells of both strains reacted similarly with respect to oxygen tolerance (up to 0.5 mg O₂/l), inhibition of growth by cyanide and azide, and resistance to antimycin A. Only cells of strain N showed growth inhibition by chloramphenicol. It is concluded that Trimyema compressum is an anaerobic, microaerotolerant organism, its microbodies show more resemblance to hydrogenosomes than to mitochondria.     

High levels of manganese-containing superoxide dismutase and thermally induced DNA disruption in a dnaK7(Ts) mutant of Escherichia coli K12

Summary IndnaK7(Ts) mutant cells, scission of DNA strands occurred after temperature shift up. When cells at 30°C were labeled with [³H]-thymidine and then shifted to 46° or 49°C for 20 min, the profiles of sedimentation of thier cellular DNA in an alkaline sucrose gradient revealed a decrease in the size of DNA to a quarter of that at 30°C in the mutant, but not in wild-type cells. The level of manganese-containing superoxide dismutase (MnSOD) in the mutant was about twice that in wild-type cells, even at the permissive temperature, implying increased production of superoxide radical anion, which may cleave DNA strands directly or indirectly in the mutant. Moderate increase in the MnSOD level on temperature shift up was observed in both strains. These results indicated that some components of the DnaK protein participate in protection of cellular membrane functions from thermal damage resulting from elevated production of the superoxide anion radical.     

Co-evolution of the legume-Rhizobium association

Summary A number of examples is given demonstrating the co-existence of pea genotypes and their specific Rhizobium, strains isolated within the same region.R. leguminosarum strains compatible with the cultivated pea have a narrow symbiotic range and they are widely distributed in European soils. This is presumably due to the narrow genetic base of the cultivated pea and its wide-spread cultivation in European soils. Rhizobium strains capable of nodulating a primitive pea line from Afghanistan were only found in soils of the Middle East and Central Asia. A more restricted distribution of specific Rhizobium strains was found for fulvum peas from Israel. Rhizobium strains effective with the fulvum pea were found in Israeli soils. A good example of co-evolution due to geographical isolation was found in south Turkey. Here a pea line was found which can form an effective symbiosis with local Rhizobium strains but not with strains from other parts of Turkey.     

镉胁迫对姬松茸生长和镉吸收累积的影响

利用外源镉添加的袋栽试验,系统研究了镉对姬松茸J37和J1两菌株农艺性状、子实体产量、氨基酸和镉含量的影响,以求为低富集镉的姬松茸品种选育和安全栽培提供科学依据。结果表明外源镉添加对姬松茸J37和J1品种子实体的农艺性状的影响各异,两菌株单个子实体重量、子实体高度、菌盖直径、菌盖厚度和菌柄直径等均与外源镉浓度具有显著的负相关性,其中子实体重量与外源镉浓度间的相关性最大;15 mg/kg镉水平是两菌株农艺性状发生显著变化的敏感点。不同镉水平下J37菌株单个子实体重量比J1菌株高10.9%—36.6%,表现为较强镉耐受性。姬松茸J37与J1菌株子实体产量、氨基酸和镉含量均随着潮次的增加而降低,子实体产量和氨基酸含量随着外源镉水平增加而降低,而镉含量随外源镉水平增加而增加。与外源镉0mg/kg处理相比,当外源镉水平达15 mg/kg时,J1和J37子实体总产量分别下降23.4%和13.4%以上,J1和J37子实体镉含量分别提高51.5%、13.7%以上,且J1子实体镉含量已达到食品卫生标准的临界值。当外源镉水平达到35 mg/kg时,J1和J37子实体产量分别下降45%、32%以上,J1和J...     

Characterization of proteases produced by newly isolated and identified proteolytic microorganisms from fermented fish (Budu)

Eight different strains ofBacillus were isolated from fermented fish (Budu) and their proteolytic enzyme activities were determined after 18 h cultivation at room temperature (35° C). Four isolates possessed high protease activities. Optimum pH for these enzymes was between 7.0 and 8.0 and the optimal temperature was 55° C. The proteases retained 40% of their original activity after 20 min at 55° C but lost all activity at 65° C. Three of the four isolates were identified asBacillus subtilis, the fourth asBacillus licheniformis.