Synthetic Growth Medium Research Articles

Effect of Nutrient Medium Composition on Laccase and Manganese Peroxidase Activity in Medicinal Mushrooms

Twenty strains belonging to 17 species and 14 genera (Bjerkandera, Cerrena, Coriolopsis, Fomes, Ganoderma, Grifola, Inonotus, Lentinus, Phellinus, Pleurotus, Pseudotrametes, Schizophyllum, Trametes, and Trichaptum) of higher Basidiomycetes were evaluated for their ability to produce laccase and manganese peroxidase (MnP) in submerged cultivation in glycerol and wheat bran-containing media. Screening results showed that the enzyme synthesis is species- and strain-dependent. The majority of the tested fungi expressed only traces or very low laccase and MnP activities during submerged growth in synthetic medium. Only Coriolopsis gallica 1184 accumulated high levels of laccase activity (19.3 U/mL) in the absence of any specific inductor. Wheat bran significantly promoted enzyme synthesis in the white-rot fungi. C. gallica 1184 followed by Ganoderma tsugae 1032 secreted very high levels of laccase activity (67.7 and 23.1 U/mL, respectively). Trichaptum biforme 117 and Trametes ochracea 1215 appeared to be the best producers of MnP (340−350 U/L). Lignocellulosic growth substrate significantly affected enzyme synthesis. Mandarin peels and kiwi were the most appropriate growth substrates for the laccase production by Coriolopsis gallica 1184 (91.8−95.9 U/mL), wheat bran followed by mandarin peels stimulated laccase production by Ganoderma tsugae 1032 (17.1 U/mL), whereas only banana peels significantly enhanced laccase secretion by Trametes ochracea 1215 (14.3 U/mL).

Production and Characterization of Fully Selenomethionine-Labeled Saccharomyces cerevisiae

This paper reports, for the first time, a quantitative replacement of methionine (Met) by selenomethionine (SeMet) at >98% substitution, with up to 4940 microg of SeMet/g of yeast obtained for the entire protein pool of a wild-type yeast grown on a SeMet-containing medium. The incorporation of selenium in yeast proteins, in the form of selenomethionine, and the influence of various organic and inorganic Se and S sources present in the media were monitored during the growth of a wild-type Saccharomyces cerevisiae , which allowed the optimization of the composition of a fully defined synthetic growth medium that ensured maximum SeMet incorporation. Quantitation of SeMet and Met was performed by species-specific isotope dilution GC-MS. The use of ascorbic acid and a minimum concentration of cysteine (5 microg/L) was found to be beneficial to achieve incorporation by limiting the oxidative stress due to the presence of selenium. Except for small amounts of cysteine, no other sources of sulfur were necessary to achieve yeast growth. In a medium containing Se(VI), the maximum replacement of Met with SeMet was 50%, which is considerably higher than that obtained with the current commercial Se yeast formulations. For yeast grown in a Met-free defined medium, which was supplemented with SeMet, nearly total replacement of Met with SeMet could be achieved. The fully Se-labeled yeast could be an important tool for the study of eukaryotic protein structures both by mass spectrometry and by X-ray crystallography through selenomethionine single- and multiple-wavelength anomalous dispersion (SAD and MAD) phasing. In addition, a particular yeast strain, BY4741, that cannot synthesize Met using inorganic sulfur (met15Delta0) was shown to produce SeMet in the presence of inorganic selenium. This might indicate that the incorporation of inorganic selenium salts [Se(VI) and Se(IV)] is obviously not occurring exclusively through the same biological pathways as for sulfur. The reduction of inorganic Se to hydrogen selenide (H(2)Se), its reactions with organic compounds present in the yeast or in the media, and the possible metabolization through unspecific enzymatic pathways (such as transsulfuration) could also be of considerable importance in the production of selenoamino acids during yeast growth.

Growth promotion of the freshwater microalga Chlorella vulgaris by the nitrogen-fixing, plant growth-promoting bacterium Bacillus pumilus from arid zone soils

Immobilization of Bacillus pumilus ES4 from arid land soils, a plant growth-promoting bacterium and the freshwater, green microalga Chlorella vulgaris enhanced microalgal growth only in the absence of combined nitrogen in synthetic growth medium (SGM), but not in medium with combined nitrogen. B. pumilus was able to fix nitrogen in N-free SGM and its growth yielded an accumulation of ammonium in the medium. On its own, B. pumilus is a poor agent for removing nitrogen and phosphorus from wastewater, while C. vulgaris is a capable microorganism. By jointly immobilizing the two microorganisms, the capacity to remove nitrogen and phosphorus from the medium by the microalgae culture was not enhanced, but, at the cell level, removal of these nutrients was significantly enhanced. It appears that growth promotion induced by B. pumilus on C. vulgaris is related to nitrogen fixation.

INVOLVEMENT OF INDOLE-3-ACETIC ACID PRODUCED BY THE GROWTH-PROMOTING BACTERIUM AZOSPIRILLUM SPP. IN PROMOTING GROWTH OF CHLORELLA VULGARIS(1).

Involvement of indole-3-acetic acid (IAA), produced by the microalgae-growth-promoting bacteria Azospirillum brasilens and A.lipoferum, in promoting growth of the microalga Chlorella vulgaris Beij. was studied. Four wildtype strains of Azospirillum and their IAA-deficient mutants were co-immobilized with C.vulgaris in alginate beads. Cultures were grown in synthetic growth medium supplemented with tryptophan. Growth promotion of microalgae and production of exogenous IAA by Azospirillum spp. were monitored. All wildtype Azospirillum spp. produced significant but varying amounts of IAA, while their mutant forms produced significantly less. The results demonstrated a significant growth promotion in Chlorella cultures when immobilized with the four wildtype strains of Azospirillum, while very low or no enhanced growth was induced by the four IAA-deficient mutants, compared to when C.vulgaris is immobilized alone. A complementation experiment, where an IAA-attenuated mutant (A.brasilense SpM7918) was supplemented with IAA produced by its parental wildtype strain (A.brasilense Sp6), restored growth promotion in the microalgae-mutant culture.

Algorithm-based design of novel synthetic media forMetarhizium anisopliaesimulating its nutritional conditions in the environment

The objective of this study was to develop a novel synthetic growth medium for increased conidium production of the entompathogenic fungus Metarhizium anisopliae. The medium should simulate natural nutritional conditions. Macro- and micronutrients were selected in concentrations found in insects or from sources which are believed to be advantageous for virulence. Medium development was guided by a problem-oriented genetic algorithm (GA) implemented with 'mutation' and 'recombination' operators specific for this optimization problem. Following five optimization steps and a total of 181 medium variations, 19 synthetic media led to increased conidium production as compared with the standard Sabouraud dextrose agar medium: increases in conidia yields of up to 120% and a 17-fold higher conidium production per square centimetre of mycelium were achieved. Rapid germination of conidia, conidial C : N ratio, as well as spore-bound Pr1 protease production were monitored as proposed quality control markers for a virulent inoculum. It was demonstrated that our problem-oriented GA is a powerful tool for the design of chemically defined cultivation media with special/enhanced properties for anamorphic fungi. This is the first study, which demonstrates the advantage of the use of a GA for the rapid design of solid synthetic media for maximum production of virulent conidia.

Growth rate and medium composition strongly affect folate content in Saccharomyces cerevisiae

Folate content in a Saccharomyces cerevisiae strain was monitored during aerobic batch fermentation in synthetic growth medium, yeast peptone dextrose medium, and a molasses based medium. During growth in the synthetic medium large differences in intracellular folate content was observed at different phases. Specific folate levels, expressed per unit biomass, were highest during respiro-fermentative growth (120 μg/g) and decreased during the respiratory and stationary phases. Thus, the physiological state of the cells clearly affects the folate content. This was confirmed in chemostat cultures where total intracellular folate content increased linearly with increasing growth rate ( r 2 = 0.998), indicating high growth rate i.e. respiro-fermentative growth to be most favourable to obtain high specific folate content. In complex media however, much lower folate content (15–40 μg/g) was found throughout the batch growth. Only minor growth-phase related differences were detected. This shows the impact of cultivation medium on folate content in yeast. To further investigate which components that influence folate content, batch experiments in synthetic medium with addition of specific components were performed. Adding a raw mixture of peptides and amino acids (peptone) decreased folate levels extensively (90%) whereas adding amino acids one-by-one only had minor effects on the intracellular folate content. Furthermore, supplementing synthetic medium with pABA, folate or nucleotides did not change the intracellular folate content. This work constitutes the first steps towards an optimised process for production of natural folates for fortification purposes, as well as an effort to gain fundamental understanding of folate requirements in yeast in relation to environmental conditions.

Addition of ammonia or amino acids to a nitrogen-depleted medium affects gene expression patterns in yeast cells during alcoholic fermentation

Yeast cells require nitrogen and are capable of selectively using good nitrogen sources in preference to poor ones by means of the regulatory mechanism known as nitrogen catabolite repression (NCR). Herein, the effect of ammonia or amino acid addition to nitrogen-depleted medium on global yeast expression patterns in yeast cells was studied using alcoholic fermentation as a system. The results indicate that there is a differential reprogramming of the gene expression depending on the nitrogen source added. Ammonia addition resulted in a higher expression of genes involved in amino acids biosynthesis while amino acid addition prepares the cells for protein biosynthesis. Therefore, a high percentage of the genes regulated by the transcription factors involved in the regulation of amino acid biosynthesis are more expressed during the first hours after ammonia addition compared with amino acid addition. The opposite occurs for those genes regulated by the transcription factor Sfp1p, related to ribosome biosynthesis. Although both additions include rich nitrogen sources, most NCR-regulated genes are more expressed after adding ammonia than amino acids. One of the differentially expressed genes, YBR174W, is required for optimal growth in synthetic medium.

Flow-cytometric total bacterial cell counts as a descriptive microbiological parameter for drinking water treatment processes

There are significantly more microbial cells in drinking water than what can be cultured on synthetic growth media. Nonetheless, cultivation-based heterotrophic plate counts (HPCs) are used worldwide as a general microbial quality parameter in drinking water treatment and distribution. Total bacterial cell concentrations are normally not considered during drinking water treatment as a design, operative or legislative parameters. This is mainly because easy and rapid methods for quantification of total bacterial cell concentrations have, up to now, not been available. As a consequence, the existing lack of data does not allow demonstrating the practical value of this parameter. In this study, we have used fluorescence staining of microbial cells with the nucleic acid stain SYBR ® Green I together with quantitative flow cytometry (FCM) to analyse total cell concentrations in water samples from a drinking water pilot plant. The plant treats surface water (Lake Zürich) through sequential ozonation, granular active carbon (GAC) filtration and membrane ultrafiltration (UF). The data were compared with adenosine tri-phosphate (ATP) measurements and conventional HPCs performed on the same water samples. We demonstrated that the impact of all three major treatment steps on the microbiology in the system could accurately be described with total cell counting: (1) ozonation caused chemical destruction of the bacterial cells; (2) GAC filtration facilitated significant regrowth of the microbial community; and (3) membrane UF physically removed the bacterial cells from the water. FCM typically detected 1–2 log units more than HPC, while ATP measurements were prone to interference from extracellular ATP released during the ozonation step in the treatment train. We have shown that total cell concentration measured with FCM is a rapid, easy, sensitive and importantly, a descriptive parameter of several widely applied drinking water treatment processes.

Detection of constitutive molecules onHistoplasma capsulatumyeasts through single chain variable antibody fragments displayed in M13 phages

A nonimmune library, containing single chain variable fragments (scFv) of immunoglobulin human genes displayed on the surface of M13 filamentous phages, was used to recognize molecules exposed on Histoplasma capsulatum yeasts' surface, during their growth in synthetic medium. The scFv clones were checked in their consistency by Dot-ELISA using HRP/anti-M13 conjugate, and they were tested to recognize molecules on H. Capsulatum yeasts' surface by ELISA in plates. Three out of 80 scFv cones (C2, C6, and C52) reacted consistently with H. capsulatum molecules, and they recognized molecules from both H. capsulatum morphologic phases. However, C6 and C52 clones reacted better with molecules on the surface of whole yeasts, with molecules from the yeasts' cell-wall extract, and with molecules released to the supernatant of the yeast culture. Mycelial supernatants from other fungi, as well as from a Mycobacterium filtrate, were not recognized by scFv phage monoclones. Monoclones C2, C6, and C52 recognized yeast molecules irrespective of the H. capsulatum strains used; the C6 clone revealed a specific immunohistochemistry reaction when tested against homologous and heterologous fungal infected tissues. The scFv clones isolated will be a useful toll to define the role of their target molecules in the host-parasite relationship of histoplasmosis.

15.7 Synthetic growth medium and cartilage explants - a safe and improved method of culturing chondrocytes for Autologous Chondrocyte Implantation (ACI).

15.7 Synthetic growth medium and cartilage explants - a safe and improved method of culturing chondrocytes for Autologous Chondrocyte Implantation (ACI).

Kinetic responses of activated sludge to individual and joint nickel (Ni(II)) and cobalt (Co(II)): An isobolographic approach

The effects of Ni(II) and Co(II) on the activated sludge growth rate have been assessed for a batch growth system, for a range of concentrations between 0 and 320 mg L −1. The activated sludge was not acclimatized to the above metallic species, while a synthetic rich growth medium was used as substrate throughout out the experimental trials. Ni(II) and Co(II) have been found to stimulate microbial growth at concentrations approximately below 27 and 19 mg L −1, with maximum stimulation concentrations 10 and 5 mg L −1, respectively. The lethal concentrations (zero growth) for both species have been found to lie between 160 and 320 mg L −1, with Co(II) identified as more potent growth inhibitor compared to Ni(II). The behaviour of activated sludge was also tested at the presence of three Ni(II) and Co(II) quotas, at various concentrations (75%Ni–25%Co (w/w), 50%Ni–50%Co (w/w) and 25%Ni–75%Co (w/w)). All the mixtures stimulated more drastically the activated sludge growth at relatively small concentrations, compared with the stimulation of equal concentrations of single species, whilst they also acted as more potent inhibitors at relatively high concentrations. Based on the isobole method, the data indicated that Ni(II) and Co(II) acted synergistically at the increasing stimulation and at the intoxication zones, whilst an antagonistic relation determined at the decreasing stimulation zone. Under the light of the present study, it is obvious that interactions (particularly synergism) between different metallic species should be taken into account in the methodologies used to establish criteria for tolerance levels in the environment.

Isotopic labeling of recombinant proteins expressed in the protozoan host Leishmania tarentolae

Isotope labeling of recombinant proteins is a prerequisite for application of nuclear magnetic resonance spectroscopy (NMR) for the characterization of the three-dimensional structures and dynamics of proteins. Overexpression of isotopically labeled proteins in bacterial or yeast host organisms has several drawbacks. In this work, we tested whether the recently described eukaryotic protein expression system based on the protozoa Leishmania tarentolae could be used for production of amino acid specific 15N-labeled recombinant proteins. Using synthetic growth medium we were able to express in L. tarentolae and purify to homogeneity (15)N-valine labeled Enchanced Green Fluorescent Protein (EGFP) with the final yield of 5.7 mg/liter of suspension culture. NMR study of isolated EGFP illustrated the success of the labeling procedure allowing identification of all 18 valine residues of the protein in the HSQC spectrum. Our results demonstrate the suitability of the L. tarentolae expression system for production of isotopically labeled proteins.

Cultivation factors and population size control the uptake of nitrogen by the microalgae Chlorella vulgaris when interacting with the microalgae growth-promoting bacterium Azospirillum brasilense

Growth of and the capacity to take up nitrogen in the freshwater microalgae Chlorella vulgaris were studied while varying the concentrations of ammonium and nitrate, the pH and the source of carbon in a synthetic wastewater growth medium when co-immobilized in alginate beads with the microalgae growth-promoting bacterium Azospirillum brasilense. Analyses of 29 independent experiments showed that co-immobilization of the microalgae with A. brasilense could result in two independent phenomena directly affected by cultivation factors, such as nitrogen species, pH and presence of a carbon source. First, growth of the microalgal population increased without an increase in the capacity of the single cells to take up nitrogen, or second, the capacity of cells to take up nitrogen increased without an increase of the total microalgal population. These phenomena were dependent on the population density of the microalgae, which was in turn affected by cultivation factors. This supports the conclusion that the size of the microalgal population controls the uptake of nitrogen in C. vulgaris cells – the higher the population (regardless the experimental parameters), the less nitrogen each cell takes up.

Interaction of Œnococcus œni and oak wood compounds

The purpose of this work was to study the effect of wood extract on Œnococcus œni growth in synthetic media and its influence on the concentration of volatile compounds when lactic acid bacteria (LAB) are present. Analytical results and sensorial consequences are described when malolactic fermentation was performed in tank or barrels. Using gas chromatography-mass spectrometry, the modifications in oaklactone, eugenol, isoeugenol and vanillin levels were monitored in the presence of Œ. œni. These volatile compounds are known to have powerful characteristic odors (woody, spicy, smoky and vanillin) and to play a role in wine aroma. LAB cultures were performed in synthetic media supplemented with oak wood shavings or extracts. The bacterial growth rate was lower in the absence of heated shavings. This study shows that LAB, especially Œ. œni, are able to interact with wood and form volatile compounds. In laboratory medium, the heating of wood favored bacterial viability more than the simple addition of wood shavings. This is the first demonstration of the capacity of wine Œ. œni to enhance volatile compounds from wood.

Isolation of four aquatic streptomycetes strains capable of growth on organochlorine pesticides

Ninety-three wild-type isolates identified as actinomycetes were tested against 11 organochlorine pesticides (OPs): aldrin, chlordane, DDD, DDE, DDT, dieldrin, heptachlor, and heptachlor epoxides, lindane, and methoxychlor. Qualitative screening agar assays displayed 62–78% tolerance of strains to OPs. Four strains designed M4, M7, M9 and M15 were selected based on multi-OP-tolerance, and identified as members of the streptomycetes group. Different growth profiles were observed in cultures of the four selected streptomycetes cultured in synthetic medium containing 5–50 μg l −1 aldrin or chlordane or lindane. Increase of aldrin removal by the selected microorganisms was concomitant with the 4.8–36.0 μg l −1 pesticide concentration range. After 72 h of streptomycete M7 growth in synthetic medium containing 48.0 μg l −1 aldrin, the remaining OP concentration in the supernatant was approximately 10% of the initial concentration. Also, in stationary growth phase less than 2.5 μg l −1 aldrin residual concentration was detected in the medium.

Two-component systems in Haemophilus influenzae: a regulatory role for ArcA in serum resistance.

Knockout mutations were constructed in the arcA gene of a virulent type b strain of Haemophilus influenzae, and the behavior of the resulting mutants was investigated in a number of conditions that mimicked distinct steps in the natural infection pathway. In arcA mutants, synthesis of capsule and lipooligosaccharide (LOS) and growth in synthetic media were unaltered compared to synthesis of capsule and LOS and growth in synthetic media in the wild-type H. influenzae type b parent strain. However, the virulence of the arcA mutants for BALB/c mice was significantly reduced. Upon exposure to human blood or serum, the arcA mutants showed markedly reduced survival compared with the survival of its wild-type parent. Serum resistance could be fully restored by complementation in cis with the H. influenzae arcA gene but not by complementation in cis with the homologous gene from Escherichia coli. The proteomes of wild-type and mutant bacteria were markedly different, especially under anaerobic conditions, underscoring the global regulatory role of ArcAB in H. influenzae. Evaluation of antibody titers and classical complement activities in various serum samples pointed to complement-mediated bactericidal activity as the factor that distinguishes between the arcA mutant and wild-type phenotypes. Comparative analysis of the membrane fractions of the arcA mutants and the wild-type strain revealed several ArcA-regulated proteins, some of which may be implicated in the serum hypersensitivity phenotype.

Biosorption of heavy metals by Rhizopus arrhizus and Aspergillus niger

Biosorption of four heavy metals Pb, Cd, Ni and Cr by Rhizopus arrhizus and Aspergillus niger during their active growth was observed to develop strains to remove heavy metals from industrial waste waters. Among four metals tested for biosorption from synthetic growth media containing 1 mg/I of the metals, R. arrhizus showed maximum biosorption (45.54%) in case of Pb whereas A. niger showed maximum biosorption (59.67%) in case of Cd. A strain of R. arrhizus adapted to 100 mg/l Pb absorbed 57.77% of Pb and that of A. niger adapted to 100 mg/l Cd absorbed 91.59% of Cd during active growth. EDTA extraction studies showed that 78-85% of Pb was associated with cell surface of R. arrhizus and 60-65% of Cd associated with the cell surface of A. niger.

Oxygen requirements of the food spoilage yeast Zygosaccharomyces bailii in synthetic and complex media.

Most yeast species can ferment sugars to ethanol, but only a few can grow in the complete absence of oxygen. Oxygen availability might, therefore, be a key parameter in spoilage of food caused by fermentative yeasts. In this study, the oxygen requirement and regulation of alcoholic fermentation were studied in batch cultures of the spoilage yeast Zygosaccharomyces bailii at a constant pH, pH 3.0. In aerobic, glucose-grown cultures, Z. bailii exhibited aerobic alcoholic fermentation similar to that of Saccharomyces cerevisiae and other Crabtree-positive yeasts. In anaerobic fermentor cultures grown on a synthetic medium supplemented with glucose, Tween 80, and ergosterol, S. cerevisiae exhibited rapid exponential growth. Growth of Z. bailii under these conditions was extremely slow and linear. These linear growth kinetics indicate that cell proliferation of Z. bailii in the anaerobic fermentors was limited by a constant, low rate of oxygen leakage into the system. Similar results were obtained with the facultatively fermentative yeast Candida utilis. When the same experimental setup was used for anaerobic cultivation, in complex YPD medium, Z. bailii exhibited exponential growth and vigorous fermentation, indicating that a nutritional requirement for anaerobic growth was met by complex-medium components. Our results demonstrate that restriction of oxygen entry into foods and beverages, which are rich in nutrients, is not a promising strategy for preventing growth and gas formation by Z. bailii. In contrast to the growth of Z. bailii, anaerobic growth of S. cerevisiae on complex YPD medium was much slower than growth in synthetic medium, which probably reflected the superior tolerance of the former yeast to organic acids at low pH.

Biomethylation of mercury(II) adsorbed on mineral colloids common in freshwater sediments

The effects of freshwater sediment components such as kaolinite, montmorillonite and birnessite (δ-MnO 2 ) on the biomethylation of mercury(II) in a synthetic growth medium (M-IIY) were assessed. Additions of kaolinite or montmorillonite to media containing mercuric nitrate [Hg(NO 3 ) 2 ; 12 μg Hg ml -1 ] had no significant effect on either bacterial growth or the production of methylmercury (CH 3 Hg + ). However, whereas the addition of birnessite resulted in only a small (ca 4%) increase in bacterial growth, it also produced a significant decrease (ca 50%) in the production of CH 3 Hg + . Further, it was demonstrated that, with the exception of kaolinite, adsorption of mercury(II) onto the sediment components before they were added to the M-IIY medium decreased its bioavailability, i.e., the amounts of CH 3 Hg + produced from the adsorbed mercury(II) were significantly lower than those produced from equivalent concentrations of Hg(NO 3 ) 2 in the absence of the mineral colloids. In the case of montmorillonite, CH 3 Hg + production was decreased by 21% relative to the control system. Most striking was the case of birnessite, in which no CH 3 Hg + was detected after a 25 h incubation period and only very small quantities of CH 3 Hg + (3-7 ng l -1 ) were present in the medium after 336 h. These data demonstrate that mineral colloids common in freshwater sediments significantly influence the extent of biomethylation of mercury(II) adsorbed on their surfaces. Birnessite, in particular, is a very effective inhibitor of the biomethylation of surface-bound mercury(II). Therefore, it may be possible to reduce the severity of mercury pollution in some aquatic environments by adding a reactive manganese oxide, such as birnessite, to the system and thereby to inhibit the transformation (methylation) of inorganic mercury(II) into the much more toxic CH 3 Hg + species.

Bacillus subtilis develops competence for uptake of plasmid DNA when growing in milk products

Transformation with plasmid DNA of naturally competent cells of Bacillus subtilis 168 in milk products was studied. Plasmid pMG36enpr, a broad host-range lactococcal vector carrying an erythromycin resistance and the B. subtilis npr gene encoding neutral protease, was taken up by B. subtilis cells grown in UHT chocolate milk. Under these conditions competence was optimal during transition from exponential to stationary growth phase, resulting in 9 x 10(1) transformants per 0.01 microgram DNA. No manipulation of the cells was necessary for competence to develop. When cells were pregrown in synthetic medium, higher transformation rates were obtained in assays, where the subsequent transformation experiments were either done in chocolate milk diluted 1:1 (v/v) with synthetic growth medium (up to 8 x 10(2) transformants) or in undiluted chocolate milk (1 x 10(2) transformants). The number of transformants was reduced to 4 x 10 (1), when diluted milk or flavored milks were used. No transformants were obtained in diluted yoghurt. Controls, in which both the preculturing and the transformation assays were done in synthetic medium, gave the maximum number of transformants (4 x 10(3) transformants per 0.01 microgram DNA).