Liquid Shake Cultures Research Articles

Cutinase production byStreptomyces spp.

Forty-fiveStreptomyces strains, including representatives of the plant pathogensS. acidiscabies, S. scabies, andS. ipomoea, were screened for ability to produce enzymes (cutinases) capable of hydrolyzing the insoluble plant biopolyester cutin. Initially, all strains were tested for production of extracellular esterase in liquid shake (250 rpm) cultures at room temperature in defined (glycerol-asparagine) or complex (tryptone-yeast extract with or without addition of mannitol) broth media supplemented with either tomato or apple cutin. Esterase activity was determined by a spectrophotometric assay utilizing the model substratep-nitrophenyl butyrate. Of the five strains exhibiting highest esterase activity, four (S. acidiscabies ATCC 49003,S. “scabies” ATCC 15485 and IMRU 3018, andS. badius ATCC 19888) were confirmed to produce enzymes with cutin-degrading activity (cutinases). Confirmation of extracellular cutinase production was accomplished by use of a new high-performance liquid chromatography method for separation and quantification of released cutin monomers. Monomer identification was confirmed by GC/MS analyses. Cutinase production was induced 2- to 17-fold by inclusion of cutin in the media. To our knowledge this constitutes the first report of cutinase production byStreptomyces spp. other thanS. scabies.

Bud proliferation and plant regeneration in liquid-cultured philodendron treated with ancymidol and paclobutrazol

Philodendron plants propagated in liquid shake or bioreactor cultures proliferated profusely in the presence of paclobutrazol (PAC) and to a lesser extent in the presence of ancymidol (ANC). The growth retardants inhibited leaf development and induced the formation of bud clusters. Short transient treatments with low concentrations (1.7–3.4 μM) of the growth retardants limited leaf growth and proliferation to a lesser extent than higher concentrations (6.8–17 μM). The growth retardants had a carryover dwarfing effect in the semi-solid hardening medium, which was more pronounced at the higher concentrations or prolonged exposure periods. Regenerated plants resumed normal growth 3–6 weeks after transplanting. Treatment with growth retardants may become a useful method in the prevention of abnormal leaf growth in large-scale liquid cultures, as well as in enhancing bud proliferation.

Glucoamylase (exo-1,4- -d-glucan glucanohydrolase, EC 3.2.1.3) is the major starch-degrading enzyme secreted by the phytopathogenic fungus Colletotrichum gloeosporioides

Activity assays based on selective product detection were used to identify the classes of amylase present in culture filtrates of the phytopathogen Colletotrichum gloeosporioides. When transferred from a glucose medium to a medium containing starch as sole carbohydrate, C. gloeosporioides secreted amylolytic activity into liquid shake cultures starting 2-3 d after inoculation, and produced levels of activity up to 0.45 IU ml−1 (assayed by the p-hydroxybenzoic acid hydrazide — PAHBAH — reducing sugar method), about 20 times higher than the activity in comparable glucose-grown controls, suggesting that starch (or a starch degradation product) was required to induce amylase synthesis. In four separate experiments, the ratio of total reducing sugar production (assayed by the PAHBAH method) to glucose production (assayed by the glucose-specific glucose oxidase — GO — method) was about 1.0 in most samples of culture filtrate, clearly demonstrating that the major amylolytic enzyme secreted by C. gloeosporioides is a glucoamylase (exo-1,4-a-d-glucan glucanohydrolase, EC 3.2.1.3, synonym amylogluco-sidase). On one occasion only (a 7 d sample in one experiment) was evidence obtained for a-amylase (endo-1,4-a-d-glucan glucanohydrolase, EC 3.2.1.1) also being present in the culture filtrate (PAHBAH/GO ratio 1.37); the presence of a-amylase in this sample was confirmed by native polyacrylamide gel electrophoresis using a starch-iodine activity stain.

Precocious gladiolus corm formation in liquid shake cultures

Conditions were defined for precocious differentiation and improved growth of corms at the base of gladiolus shoots. Shoots were derived from explants cultured on agar solidified media, and corm regeneration was obtained in subsequent liquid shake cultures. Benzyladenine (BA), at 10-7 M, was found to have a stimulating effect mainly when provided to the shoots prior to manifestation of corm growth. Paclobutrazol and sucrose promoted corm formation when supplemented to the liquid media. Paclobutrazol, at 10 mg l-1, shifted assimilate allocation towards the growing corm. A differential promotion of corm development by sucrose was not observed, and the concentration of sucrose at which the sugar demand for maximal shoot and corm growth is satisfied (60 g l-1) was unaltered by the presence of paclobutrazol. The rate of corm growth on shoots cultured in a liquid medium supplemented with paclobutrazol and a saturating sucrose concentration, was a function of the length of the shoot's leaf blades, and was similar in light and in dark.

Bacterial degradation of polyester polyurethane

An account is presented of work undertaken to investigate the ability of sixteen bacteria to degrade polyester polyurethane. The results of liquid shake culture experiments using a mineral salts medium, incorporating the polyurethane as the sole carbon source, indicated that none of the bacteria were able to degrade the polyurethane over a 12-week period. The addition of supplementary organic nutrients at a low level, achieved by the addition of 1 ml aliquots of overnight broth cultures of the bacteria to the shake cultures, was found to be sufficient to effect significant degradation in two isolates; a species of Corynebacterium and Pseudomonas aeruginosa (ATCC 13388) over a 12-week incubation period. The provision of higher levels of organic nutrients in the form of yeast extract (1% w/v) to the liquid shake cultures was found to effect significant degradation by a further five bacterial isolates. Results are presented which indicate that four bacterial isolates could degrade the polyester polyurethane within 2 weeks in the presence of yeast extract.

Survey ofFusarium sambucinum(Gibberella pulicaris) for Mating Type, Trichothecene Production, and Other Selected Traits

The bisexual heterothallic ascomycete Gibberella pulicaris (anamorph=Fusarium sambucinum) is an important trichothecene-producing plant pathogen that has been used to study the genetics of trichothecene synthesis and pathogenicity by analysis of naturally occurring variants. To obtain additional useful natural variants, 53 strains, identified as F. sambucinum, were tested for their ability to make trichothecenes and to form fertile crosses. Eight new fertile strains were identified. These eight strains, plus 40 of the nonfertile strains, made detectable trichothecenes in liquid shake culture (...)

Oncorhyncolide, a novel metabolite of a bacterium isolated from seawater

A novel metabolite, oncorhyncolide ( 1), has been isolated from liquid shake cultures of the bacterial isolate #157 obtained from seawater. The structure of oncorhyncolide has been elucidated by spectroscopic analysis.

Bioconversion of Aflatoxin G1 to Aflatoxin B3 by selected fungi

Aflatoxin G1 (AFG1) was transformed into aflatoxin B3 (AFB3) by the fungiRhodotorula sp,Sporobolomyces sp,Rhizopus oryzae NRRL395,Pythium ultimum, Aspergillus terreus, A clavatus and Penicillium frequentans grown in a medium containing AFG1, Difco potato dextrose broth, yeast extract, and peptone both in liquid shaken cultures and in solid static cultures at 25°C in the dark. A maximum rate of transformation of 10 % was obtained after 2 to 3 weeks of incubation. The transformation was correlated with an increase in the pH of the media from 5.7 -5.9 to 8.3 - 8.8.Saccharomyces cerevisiae also transformed AFG1 into AFB3, but at a slower rate; the pH of the media did not reach above 8.0 until 5 weeks after incubation. No transformation was observed whenA niger andP chrysogenum were tested; in both cases, no increase in pH was noticed. However, some transformation of AFG1 to AFB3 by both fungi was observed when the initial pH of the media was adjusted to 9.0. The rate of transformation increased to 15 - 20% in the static culture where the same medium was adsorbed onto vermiculite andRhizopus andAspergilli gave the highest increase in AFB3 yield.

Differences in cerato‐ulmin production between the EAN, NAN and non‐aggressive subgroups of Ophiostoma ulmi

A test of comparative in vitro cerato‐ulmin wilt toxin production in the aggressive and non‐aggressive subgroups of the Dutch elm disease pathogen Ophiostoma ulmi was carried out by turbidity and ELISA methods. Ten non‐aggressive, ten EAN aggressive and ten NAN aggressive isolates were tested from a range of geographical sources. In liquid shake cultures the non‐aggressive isolates produced the greatest and the NAN aggressives the least mean biomass. Despite considerable variation in cerato‐ulmin production by individual isolates in three separate experiments, both the turbidity and ELISA methods showed a clear separation of the non‐aggressive and aggressive subgroups. Non‐aggressive isolates produced little or no cerato‐ulmin (ELISA range of means 0–56.0 ng/ml) and EAN and NAN aggressive isolates moderate to high levels (EAN 1.6–89.0 × 104 ng/ml and NAN 0.2–300 × 104 ng/ml). In the aggressive isolates no correlation was detected between cerato‐ulmin production and either biomass or pathogenicity to clonal Commelin elm. The role of cerato‐ulmin in the pathogenicity of O. ulmi is discussed.

A new technique for the culture of fern gametophytes

Fern gametophytes initiated from spores grew successfully when immobilised in reticulate polyurethane foam in liquid shake cultures. Colonisation of this matrix was complete seven days after medium inoculation and appeared to be a completely passive process. Yield (dry weight) was greater than that from liquid grown cultures.

Propagation of Indian Rhubarh (Rheum emodi Wall.) using shoot-tip and leaf explant culture

Shoot-tip explants of Rheum emodi Wall. (Polygonaceae) gave rise to multiple shoots when cultured on a Murashige and Skoog (1962) medium (MS) with 2.0 mg/l 6-benzylaminopurine (BAP) and 1.0 mg/l indole-3-butyric acid (IBA). Also, shoot buds developed from leaf explants using MS medium with 2.0 mg/l BAP and 0.25 to 1.0 mg/l indole-3-acetic acid (IAA) or IBA. Roots were induced when the resulting shoots were placed on MS medium with 1.0 mg/l IBA. Both regeneration procedures gave rise to healthy plantlets that were established in soil under glasshouse conditions at 80% frequency after hardening phase of two weeks. Regenerated plants showed a constant chromosome number of 2n=2x=22, same as the parent plant. The use of liquid shake cultures minimized the time and culture medium requirements for propagation. This procedure can be applied for the conservation and utilization of elite clones of R. emodi.

Actinomycete morphology in shaken culture

The morphology of 49 Streptomyces strains and eight other actinomycetes was studied in shaken liquid culture, using a range of spore inocula in a complex medium. Results showed that each strain would produce one, two, or more different gross morphological types throughout the spore inoculum range tested. The morphological forms that were observed included compact pellets, spiky or fluffy pellets, oblong pellets, flakes, hydrophobic rafts, aggregates, dispersed mycelium, fragmented mycelium, or combinations of these forms. Two strains of S. viridochromogenes developed different ranges of morphology in the test conditions.Key words: Streptomyces, pellets, spore inoculum.

Distinctions between cellulase isoenzyme patterns of five plant-pathogenic Verticillium species

Cellulase isoenzymes produced in liquid shake cultures by isolates of five species of Verticillium were separated by isoelectric focusing. Each species gave a distinctive pattern (or patterns) of isoenzymes which could be used for identification purposes. There were no differences between the isoenzyme patterns of isolates of the same fungal species even from different host species or showing different degrees of virulence. Filtrates from cultures of different ages did not show any qualitative changes in isoenzyme patterns. The same species-specific patterns of cellulase isoenzymes were produced in media containing either a simple carbon source (carboxymethyl cellulose) or a more complex one (solvent extracted plant tissues).

Versicolorin synthesis by Aspergillus parasiticus: Regulation by temperature and zinc

A mutant strain of Aspergillus parasiticus (ATCC 36537) was grown in shaken liquid culture at 23 or 37°C. The polyketide secondary metabolite versicolorin A was produced at the lower but not the higher temperature. Temperature upshift (23 → 37°C) arrested versicolorin synthesis; temperature downshift (37 → 23°C) allowed versicolorin synthesis to occur after a 6-h lag. Versicolorin synthesis after temperature downshift was blocked by addition of cycloheximide or actinomycin D at the time of temperature shift. Zn 2+ is known to be required for versicolorin synthesis, and Zn 2+ uptake must occur before temperature downshift. We suggest that a transcriptional event required for versicolorin synthesis occurs only in the presence of Zn 2+ and at temperatures below 37°C.

Control of Precocious Gladiolus Corm and Cormel Formation in Tissue Culture

Gladiolus propagates vegetatively by virtue of corm formation at the base of the shoot and by cormel differentiation on tips of stolons. The precocious generation of both organs was accomplished in vitro on plantlets incubated for one month in a liquid shake culture supplemented with 1.0–10.0 mg/1 paclobutrazol. Paclobutrazol-dependent corm and cormel development could be prevented by addition of GA3 to a paclobutrazol-containing medium, thereby indicating that paclobutrazol mediates its influence by reducing the endogenous GA level. BA was also found to inhibit corm formation. The dose-response relations for the influence of paclobutrazol, BA and GA3 on corm development are presented. Paclobutrazol-induced cormels, when excised from the culture-derived plant, germinated and started to regenerate adventitious shoots upon transfer to an agar-solidified medium with 0.3 μM BA and 0.1 μM NAA.

Degradation of a non-phenolic arylglycerol β-aryl ether by Streptomyces cyaneus

A non-phenolic β- O-4 lignin substructure model compound, 1,3-dihydroxy-2-(2-methoxyphenoxy)-1-(4-ethoxy-3-methoxyphenyl)propane, was degraded by streptomyces cyaneus in liquid shaken cultures. 4-Ethoxy-3-methoxybenzoic acid could be identified by TLC and GC/MS as the main product, indicating the cleavage of the CαCβ bond of the aryl glycerol-β-aryl ether. In addition, 4-ethoxy-3-methoxybenzaldehyde and guaiacol were identified in culture extracts as further degradation products.

Plasmid profiles as indicators of the source of contamination of Staphylococcus aureus endemic within poultry processing plants.

A total of 530 strains of Staphylococcus aureus were isolated from the defeathering machinery of a chicken processing plant and from neck skin samples of carcasses at different stages of processing in two visits 4 weeks apart. Eleven different plasmid profiles were detected in the isolates, eight being common to both visits. The plasmid profiles of the strains forming the majority of the population on the freshly slaughtered birds were rarely present in the strains isolated from the pluckers (except at the entry to the first plucker) and were present in only a small proportion of the strains isolated from carcasses after plucking. However, the profiles from the strains isolated from the pluckers on both visits were different from those forming the majority of the population on the incoming birds but formed the major part of the carcass flora after plucking, suggesting that such strains were endemic. These strains were found as a small proportion of the isolates made from the incoming birds, suggesting that this was the route by which the endemic strains were introduced into the plant. Such endemic strains exhibited a clumping growth, even in liquid shake culture, which may have made it easier for them to become established on the pluckers and to resist cleaning and disinfection. This clumping phenotype was correlated with the presence of a 7.5-megadalton plasmid.

Somatic embryogenesis and plant regeneration from cell suspension cultures of Cucumis sativus L.

A procedure for the regeneration of whole cucumber plants (Cucumis sativus L. cv. Poinsett 76) by embryogenesis from cell suspension cultures is described. Embryogenic callus was initiated from the primary leaves of 14-17 day old plants. Suspension cultures of embryogenic cells were grown in liquid Murashige and Skoog basal medium containing 5 uM 2,4,5-trichlorophenoxyacetic acid and 4 uM 6-benzylaminopurine. Suspension cultures were composed of a population of cells that were densely cytoplasmic and potentially embryogenic. Differentiation of embryos was enhanced by washing the suspension culture cells with MS basal medium containing 0.5% activated charcoal and twice with MS basal medium followed by liquid shake cultures in MS basal medium. Sixty to 70 percent of the embryos prewashed with activated charcoal germinated into plantlets with normal morphology. Embryos obtained from suspension cultured cells without prewashing with activated charcoal organized into plantlets with abnormal primary leaves. Morphologically normal plantlets were obtained by excising the shoot tips and transferring them to fresh medium.

Biotransformation of coal by ligninolytic Streptomyces

The chemistry of coal liquefaction by Streptomyces viridosporus, Streptomyces setonii and Streptomyces badius was studied. These ligninolytic actinomycetes solubilized lignite and bituminous coals when coal particles were placed on the surfaces of mycelial lawns on agar culture media. On agar, liquefaction proceeded with the formation of clear to black water-soluble products. Up to 40% of some coals was solubilized in 3 weeks. Lignite coals were also solubilized in liquid shake flask cultures. Coal solubilization by S. viridosporus was oxidative, as shown by Fourier transform infrared spectroscopy and elemental analysis. Compared to the starting coals, the soluble products were enriched in oxygen, nitrogen, sulfur, and ash, but were lower in hydrogen relative to carbon. Insoluble coal residues were less altered than the liquefied products, but they were higher in ash content than the starting coals. Chemical analysis of culture filtrates showed that some low molecular weight products were released, but most of the soluble products were acid precipitable and probably polymeric. Lignite coal solubilization by the S. viridosporus did not require induction and in liquid media solubilization was always accompanied by a significant increase in medium pH. When medium pH was not allowed to become basic, extracellular coal solubilizing activity was negligible. Extracellular solubilizing activity was heat stable, and ultrafiltration experiments showed that the solubilizing agent was of less than 10 000 molecular weight. Coal solubilizing activity of S. viridosporus culture supernatants could be eliminated by lowering their pH, but the activity could be recovered by raising the pH back up to above neutral. Phosphate and Tris buffers of varying strength and pH showed that nonbiological solubilization of lignite coal occurred under basic conditions. The combined data show that biological lignite coal solubilization by these Streptomyces is mediated by alkaline products that the cells release into the growth medium. These findings confirm and expand an earlier report by others that coal solubilization by Streptomyces setonii may be nonenzymatic.

Relationship between the virulence of hop isolates of Verticillium albo-atrum and their in vitro secretion of cell-wall degrading enzymes

The in vitro production of extracellular polygalacturonase, pectin lyase, and cellulase by hop isolates of Verticillium albo-atrum was studied. A total of 24 isolates belonging to the two pathotypes commonly described as fluctuating or progressive, according to the symptoms produced in certain susceptible hop cultivars, were grown in liquid shake cultures. The media used contained glucose, pectin or acetone-extracted hop tissue as a carbon source. The production of all three enzymes was significantly correlated with pathotype when the medium contained hop tissue. Pectin lyase production was also correlated with pathotype when pectin was used as the carbon source. The influence of carbon source on enzyme output and possible reasons for some exceptions to the correlations observed are discussed.