Transformation of Methanococcus maripaludis and identification of a Pst I-like restriction system

Abstract An optimized polyethylene glycol (PEG) method of transformation was developed for Methanococcus maripaludis using the pKAS102 integration vector. The frequency of transformation with 0.8 μg of plasmid and 3×10⁹ cells was 4.8×10⁻⁵ transformants cfu⁻¹, or 1.8×10⁵ transformants μg⁻¹, which was four orders of magnitude greater than with the natural transformation method. A Pst I restriction activity in M. maripaludis was also identified. Methylation of the plasmid with Pst I methylase increased the methanococcal transformation frequency at least four-fold. Also, chromosomal DNA from M. maripaludis was resistant to digestion by the Pst I endonuclease.     

Electrotransformation of whole cells of Brevibacterium sp. R312 a nitrile hydratase producing strain: Construction of a cloning vector

Abstract: A rapid and effective method is described for electroporation of Brevibacterium sp. R312, a coryneform strain producing nitrile hydratase and amidase. The transformation efficiency of the method is 10⁸ transformants per μg of plasmid under optimal conditions. Parameters optimised included field strength (11.8 kV cm⁻¹), pulse length (2.4 ms), plasmid DNA concentration (0.25 μg ml⁻¹ and cell density (10¹⁰ cells ml⁻¹). Surprisingly, the transformation efficiency did not vary with the growth stage, in contrast to results in the literature. A shuttle vector was constructed containing several unique cloning sites down-stream of the SP6 RNA polymerase promoter.     

Species of Anguilla as indicators of mercury in the coastal rivers and lakes of Victoria, Australia

Mercury concentrations in the axial muscle tissue of most (243) of the 254 Anguilla australis and most (20) of the 27 A. reinhardtii collected from 30 sites in coastal rivers and lakes in Victoria, Australia, during 1975–78 were well below the Australian statutory health limit (0.5 μg g⁻¹ wet weight). For A. australis the mean mercury concentration was 0.17 μg g⁻¹ (±0.16 s.d. , range 0.01–1.60 μg g⁻¹); for A. reinhardtii the values were 0.37 ± 0.23 μg g⁻¹ (range 0.12–1.10 μg g⁻¹). Statistical analyses showed that variation in mercury concentration due to total length accounted for only 13% of the total variation in A. australis and 2% in A. reinhardtii whereas locality accounted for 54 and 68%, respectively. Both species are thus considered suitable as indicators of mercury pollution.     

The effects of water hardness upon the uptake, accumulation and excretion of zinc in the brown trout, Salmo trutta L.

The effects of water hardness (9 and 220 mgl⁻¹ as CaCO₃) upon zinc exchange in brown trout exposed to 0.77 μmol Zn 1⁻¹ have been investigated using artificial soft water (<49.9 μmol Ca ^l-1, <40.1 μmol Mg 1⁻¹) and mains hard water (1671.7 μmol Ca 1⁻¹, 493.6 μmol Mg 1⁻¹) of known composition. Both hard and soft water-adapted fish exhibited a bimodal pattern of net zinc influx. Net zinc influxes during both fast and slow uptake phases were significantly greater ( P <0.001) in soft (82.9 and 6.2 μmol Zn 100 g⁻¹ h⁻¹) than in hard water (46.3 and 2.4 μmol Zn 100 g h⁻¹). Zinc efflux (- 0.2 μmol Zn 100 g⁻¹ h⁻¹) was enhanced only in hard water during the slow net influx phase.
Brown trout exposed to zinc in hard water and placed in metal-free media exhibited a greater net efflux (- 25.6 μmol Zn 100 g⁻¹ h⁻¹) of the metal than did fish in soft water (-4.2 μmol Zn 100 g⁻¹ h⁻¹) treated in the same manner. Tissue ⁶⁵Zn activities reflected both the differences in uptake and excretion rates of the metal between hard and soft water fish. During zinc exposure (0.77 μmol Zn 1⁻¹) high water hardness reduced tissue burdens of the metal by reducing net branchial influx, and enhancing efflux of the metal in hard water fish.     

Anti-bacterial activity of synthetic N-heterocyclic oxidizing compounds

Synthetic chlorochromate derivatives of pyridine and quinoline were active in vitro against type cultures of Escherichia coli (ATCC 128), Staphylococcus aureus (ATCC 14775), Pseudomonas aeruginosa (ATCC 10145) and Bacillus subtilis (NCTC 8236). The minimum inhibitory concentrations (MIC) were 125–250 μg ml⁻¹ and 250–500 μg ml⁻¹ for pyridinium chlorochromate and quinolinium chlorochromate, respectively. An established derivative of quinoline (Perfloxacin) had an MIC of 125–250 μg ml⁻¹. The extinction time for 10⁵ cfu in broth was 90 min for pyridinium chlorochromate and 120 min for quinolinium chlorochromate, except for B. subtilis which survived up to about 180 min and 360 min. A combination of the two compounds produced an antagonistic effect. The 50% lethal dose (LD₅₀ toxicity) in mice was estimated at 76 μg g⁻¹ and 33 μg g⁻¹ body weight for the quinolinium and pyridinium chlorochromates. The compounds also exhibited some potential for suppressing a simulated staphylococcal infection in mice at the dosage levels of ca 22 μg g⁻¹ for pyridinium chlorochromate and 45 μg g⁻¹ for quinolinium chlorochromate.     

Electrotransformation of Streptococcus agalactiae with plasmid DNA

Abstract A protocol for efficient electrotransformation of Streptococcus agalactiae (group B streptococcus) Lancefield's strain O90R (NTCT 9993) (an unencapsulated derivative of type Ia strain O90) was developed. The Escherichia coli - Streptococcus shuttle vector pDP28 (7.8 kb) carrying the ermB gene for resistance to erythromycin was used as donor DNA. Frozen 'electrocompetent' cells were prepared by repeated washes in 10% glycerol. A 50-μl aliquot containing about 5×10⁹ colony forming units of bacteria was subjected to the electric pulse. Optimal conditions for electrotransformation were determined using different media, harvesting cells at different points of the growth curve, and using different field strengths. The dose-response curve for transformation of S. agalactiae with pDP28 showed one-hit kinetics as donor DNA varied between 0.01 and 3 μg. The efficiency of electrotransformation for this range of amounts of donor DNA was 1.2×10⁴ cfu μg⁻¹. The transformation frequencies obtained with this electroporation protocol are high enough to allow both subcloning and shotgun cloning of streptococcal DNA in S. agalactiae .     

The cellular location and effect on nisin immunity of the NisI protein from Lactococcus lactis N8 expressed in Escherichia coli and L. lactis

Abstract Vibrio anguillarum and Pasteurella piscicida are Gram-negative bacteria which are pathogenic for marine fish and we report here the first successful transformation of these two bacteria by electroporation. The optimal conditions for electroporation included a field strength of 12.5 kV cm^t-1 and a time constant of 5 ms using 0.2-cm cuvettes. With these parameters, three plasmids (pSU2718, pCML, pEV3) with molecular sizes of 2.6, 5 and 13.7 kb, respectively were successfully transformed into both pathogens. V. anguillarum isolates belonging to serotypes O1 and O2 were transformed with greatest efficiency, 2.5 × 10³ transformants per μg DNA, being achieved in the serotype O2 strains using plasmid pCML. Strains of serotype O3 were not transformed. In the case of P. piscicida the maximum efficiency achieved was 9.8 × 10² transformants per μg pCML plasmid DNA. This optimized system will allow development of procedures for the genetic manipulation of these pathogens.     

The respiration of young coho Oncorhynchus kisutch at gradually declining oxygen levels and during recovery from oxygen deprivation

The respiration of coho salmon, Oncorhynchus kisutch , weighing between 15 and 50 g was measured at gradually declining oxygen levels and at temperatures ranging between 14 and 17°C. The maximum and minimum oxygen concentrations tested were 250 and 40 μmol L⁻¹, respectively. Respiration rates were measured for 1 h periods before oxygen concentration was lowered by 12.5 or 25.0 μmol oxygen L⁻¹. At the end of these endurance tests the oxygen level was returned to normoxic conditions and respiration rates were determined for the recovery period. Under normoxic conditions (> 200 μmol L⁻¹) the respiration of coho levelled around 5.1 μmol g⁻¹ wet weight h⁻¹. At intermediate levels between 150 and 200 μmol oxygen L⁻¹, the average rate increased to 5.8 μmol g⁻¹ h⁻¹, which could be attributed to higher spontaneous activity of the test animals. At low oxygen levels (< 150 μmol⁻¹) average respiration rates dropped to values between 5.5 and 5.7 μmol g⁻¹ h⁻¹, reaching a minimum of 3.8 μmol g⁻¹ h⁻¹ at oxygen levels below 50 μmol L^μ. First mortality was observed in this range. After exposure to reduced oxygen levels the fish maintained a higher respiration rate when again exposed to normoxic oxygen levels above 200 μmol L⁻¹. Increased respiration rates were observed for a recovery period of 6 h.     

Transformation of Bacillus thuringiensis by electroporation

Abstract A simple and reliable method of transforming Bacillus thuringiensis is described. This protocol, based on high-voltage electro-transformation (electroporation) in the presence of polyethylene glycol, allows introduction of plasmid DNA in most of the Bacillus thuringiensis strains tested. Efficiencies vary between 10² and 10⁵ transformants per μg DNA, depending on the strain or the replicon used.     

Transformation of an entomopathic strain of Bacillus sphaericus by high voltage electroporation

Abstract High voltage electroporation, which permits cellular uptake of plasmid DNA by inducing a transient permeability of the cell membrane, was employed to transform the entomocidal microorganism Bacillus sphaericus . Electroporation of B. sphaericus cell suspensions routinely produced 10⁵ to 10⁶ transformants/μg plasmid DNA.     

Endogenous levels of polyamines and abscisic acid in pepper fruits during growth and ripening

The levels of polyamines (PA) and abscisic acid (ABA) in the pericarp of California variety pepper fruit ( Capsicum annuum L.) were analyzed during development and ripening. Putrescine level was 2.75 μmol g⁻¹ fresh weight 7 days after fruit set and fell during the exponential stage of growth to 1.05 μmol g⁻¹ fresh weight. During the second growth stage. PA and ABA levels remained stable and fell sharply at the beginning of maturation. The levels of spermidine and spermine decreased throughout fruit development and maturation from 0.61 to 0.05 and 0.31 to 0.02 μmol g⁻¹ fresh weight, respectively, but no changes were associated with the onset of maturation. ABA levels remained high (0.70-0.80 μg g⁻¹ fresh weight) during the stages of fruit growth and fell at the beginning of maturation to 0.12 μg g⁻¹ fresh weight, before rising again during the last stages of maturation and senescence. The decrease in putrescine and ABA levels and the subsequent increase in the latter may be responsible for controlling the processes of ripening in pepper fruit.     

Ethylene synthesis and growth in embryogenic tissue of Norway spruce: Effects of oxygen, 1-aminocyclopropane-1-carboxylic acid, benzyladenine and 2,4-dichlorophenoxyacetic acid

Ethylene production from an embryogenic culture of Norway spruce ( Picea abies L.) was generally low. ca 2.5 nl g⁻¹ h⁻¹, whereas 1-aminocyclopropane-1 -carboxylic acid (ACC) concentration was high, fluctuating between 50 and 500 nmol g⁻¹ during the 11-day incubation period. Hypoxia (2.5 and 5 kPa O₂) rapidly inhibited ethylene production without subsequent accumulation of ACC. Exogenous ACC (1, 10 and 100 μ M ) did not increase ethylene production, but the highest concentrations inhibited tissue growth. Ethylene (7 μl I⁻¹) did not inhibit growth either when supplied as ethephon in the medium or in a continuous flow system. Benzyladenine (BA) had little effect on ethylene production, although it was necessary for sustaining the ACC level. Omission of 2.4-dichloro-phenoxyacetic acid (2.4-D) from the medium caused ethylene production to increase from about 2.5 to 7 nl g⁻¹ h⁻¹ within the 11-day incubation period. Although 2.4-D did not specifically alter the endogenous level of ACC, the lowest ACC level, 33 nmol g⁻¹, was observed in tissue treated with 2.4-D (22.5 μ M ) and no BA for 11 days. Data from this treatment were used to estimate the kinetic constants for ACC oxidase, the apparent K_m was 50 μ M and V_max 2.7 nl g⁻¹ h⁻¹. Growth of the tissue was strongly inhibited by 2.4-D in the absence of BA, but weakly in the presence of BA (4.4 μ M ). The results suggest that ethylene or ACC may be involved in the induction of embryogenic tissue and in the early stages of embryo maturation.     

Transformation of Bacillus amyloliquefaciens protoplasts with plasmid DNA

Abstract A method for efficient polyethylene glycol (PEG)-mediated transformation of Bacillus amyloliquefaciens protoplasts with plasmid DNA is described. The best conditions found for protoplast regeneration included using 0.45 M sucrose both during the cultivation of the cells and (as an osmotic stabilizer) during their treatment with lysozyme, whereas 0.25 M sodium-succinate was added to the regeneration plates. Under these conditions about 5–10% of input cells regenerated. The highest transformation frequency with plasmid DNA was obtained with a PEG 6000 concentration of 22.5% (w/v). Transforming B. amyloliquefaciens strains with the plasmid pUB110 isolated from B. amyloliquefaciens resulted in 2–4 · 10⁵ transformants/μg DNA, 100–1 000-times as high as with DNA from Bacillus subtilis , suggesting a restriction barrier between the two species. Transformation of B. amyloliquefaciens with plasmids pC194 or pE194 cop -6 gave poor yields and no restriction barrier could be demonstrated for these plasmids. However, by curing pC194 from one of the transformants, a mutant strain compatible to both the plasmids could be isolated, yielding 2–3·10⁴ transformants/μg DNA. Both laboratory and industrial B. amyloliquefaciens strains could be transformed with the procedure.     

Liposome-enhanced transformation of streptococcus lactis and plasmid transfer by intergeneric protoplast fusion of streptococcus lactis and bacillus subtilis

Abstract An efficient protoplast transformation system and a procedure of plasmid transfer by means of protoplast fusion is described for Streptococcus lactis . Protoplasts of S. lactis IL1403 and S. lactis MG1363 were transformed by pGK12 [2.9 MDa erythromycin resistance (Em^r)] with an efficiency of 3 × 10⁵ transformants per μg plasmid DNA. This high efficiency was obtained by the inclusion in the transformation mixture of liposomes composed of cardiolipin and phosphatidyl choline in a molar ratio of 1 to 6 in the presence of 22.5% polyethylene glycol (PEG). This paper also reports an efficient plasmid transfer method between lactic and streptococci and Bacillus subtilis by means of protoplast fusion. When S. lactis and B. lactis protoplasts undergo fusion mediated by exposure to 37.5% polyethylene glycol, plasmid pGKV21 (3.2 MDa; Em^r) was transfered from one host to the other with a frequency of 10⁻³−10⁻⁵ transformants per regenerating recipient protoplast.     

Electrotransformation of Streptococcus pneumoniae

Conditions of electroporation to transform encapsulated strains of Streptococcus pneumoniae with plasmid DNA have been defined. For a heavily encapsulated strain, an electroporation solution of 10–20% (v/v) glycerol and 3.2 kV cm^-1 field strength, 1000 Ω resistance and 25 μF capacitance were optimal. For lightly encapsulated or non-encapsulated strains, optimal conditions were a sucrose-based electroporation solution and 12.5 kV cm^-1 field strength, 200 Ω resistance and 25 μF capacitance.     

Transformation of group A streptococci by electroporation

Abstract The introduction, via electroporation, of free plasmid DNA into three strains of Streptococcus pyogenes is described. The method is very simple and rapid and efficiencies vary from 1 × 10³ to 4 × 10⁴ per μg of DNA. The method was also used to introduce an integrative plasmid and transformants were obtained, albeit at a somewhat lower frequency (2 × 10²). Some of the plasmids used in this study are derivatives of the Lactococcus lactis subsp. cremoris Wg2 plasmid pWV01. These broad host range vectors replicate in Gram-positives as well as Gram-negatives (viz. Escherichia coli ). Here we show that they also replicate in S. pyogenes and S. sanguis .     

Production of tylosin in solid-state fermentation by Streptomyces fradiae NRRL-2702 and its gamma-irradiated mutant (γ-1)

Aims:  To develop solid-state fermentation system (SSF) for hyper production of tylosin from a mutant γ-1 of Streptomyces fradiae NRRL-2702 and its parent strain.
Methods and Results:  Various agro-industrial wastes were screened to study their effect on tylosin production in SSF. Wheat bran as solid substrate gave the highest production of 2500 μg of tylosin g⁻¹ substrate by mutant γ-1 against parent strain (300 μg tylosin g⁻¹ substrate). The tylosin yield was further improved to 4500 μg g⁻¹ substrate [70% moisture, 10% inoculum (v/w), pH 9·2, 30°C, supplemental lactose and sodium glutamate on day 9]. Wild-type strain displayed less production of tylosin (655 μg of tylosin g⁻¹ substrate) in SSF even after optimization of process parameters.
Conclusion:  The study has shown that solid-state fermentation system significantly enhanced the tylosin yield by mutant γ-1.
Significance and Impact of the Study:  This study proved to be very useful and resulted in 6·87 ± 0·30-fold increase in tylosin yield by this mutant when compared to that of wild-type strain.     

Transformation of the entomopathogenic fungus, Metarhizium flavoviride strain CG423 to benomyl resistance

Metarhizium flavoviride strain CG423 is being developed as a mycoinsecticide against grasshoppers. This strain has been transformed to resistance to the fungicide benomyl by a polyethylene glycol (PEG)-mediated procedure using a mutant tubulin gene from Neurospora crassa . Transformation frequencies of up to 84 transformants per microgram of transforming DNA were achieved. Benomyl-resistant transformants were obtained that could tolerate greater than 30 μg ml⁻¹ benomyl. Southern blot analysis of genomic DNA reveals that the mechanism of genetic transformation of all transformants was by homologous gene replacement of the β-tubulin allele.     

The content of prostaglandins and their precursors in Mortierella and Cunninghamella species

Five strains of filamentous fungi belonging to the genera Mortierella and Cunninghamella were examined for the content of dihomo-γ-linolenic, arachidonic, eicosapentaenoic acids and prostaglandins (type E₂ and F _2α). Prostaglandins were detected using an ELISA method in mycelia of all tested strains (range 50–4800 ng g⁻¹ of PGE₂ and 6–30 ng g⁻¹ of PG F _2α). Several micro-organisms also produced prostaglandins in the culture medium (2·2–137·6 μg l⁻¹ for PGE₂ and 0·4–7·8 μg l⁻¹ for PG F _2α).     

Soil microbial carbon uptake characteristics in relation to soil management

Abstract The kinetics of glucose uptake by soil microbial communities in 16 different soild (7 under monocultures and 9 under crop rotations) differing in microbial biomass content, % C_org, pH and clay content were investigated at 22°C. The V _max value of microbial bimasses under monoculture, was o.27 μg C_gluc · μg⁻¹ C_mic · h⁻¹ (range 0.18–0.44), twice as high as the mean value of V _max of microbial biomasses under rotations (0.13 μg C_gluc, range 0.07–0.19). Mean values of K _m were 714 μg C_gluc and 290 μg C_gluc · g⁻¹ soil, respectively.
These differences were highly significant ( P =0.001, based on SE) and could not be relate to particle size distribution of the soils, pH or C_org. A Michaelis-Menten type uptake response was apparent over the total range of glucose concentrations used (45.4–1453.3 μg C_gluc · g⁻¹ soil) for microbial biomasses under rotation while the majority of microbial biomasses under monocultures showed a similar response only at low glucose concentrations. A different uptake mechanism appeared to be involved at higher glucose concentrations (similar to diffusion) in monoculture soils.