DNA amplification fingerprinting as a tool for checking genetic purity of strains in the cyanobacterial inoculum

Summary The electrophoretic patterns for 17 different cyanobacterial cultures derived from 6 different decamer primers were analysed to provide diagnostic fingerprints for each culture and their genetic distances based on RAPD markers.The primer OPB 09 produced a maximum of 24 amplified products. The primers OPB 09, OPG 04 and OPAH 02 generated markers specific for Nostoc cultures. Westiellopsis was found to be distinct from other cyanobacterial cultures in the RAPD profile obtained with the primer OPAH 02. The primer OPF 03 generated specific markers for Tolypothrix tenuis. Fischerella cultures could be identified with the primers OPB 09, OPAG 03 and OPF 05. The study revealed that these RAPD markers could be further used to identify and establish the genetic purity of the strains in the cyanobacterial inoculum. There was a similarity of 60–90% within Westiellopsis cultures. Nostoc cultures shared 50–80% similarity with Westiellopsis cultures. Anabaenacultures were similar to Westiellopsiscultures by 60–70. The markers produced for each culture were also applied to phylogenetic analysis to infer genetic relatedness in this group of prokaryotes. The dendrogram analysis clearly revealed that free-living cyanobacterial cultures are closely related to each other and are distinct from the symbiotic forms.     

Production of laccases by Pleurotus ostreatus in submerged fermentation in co‐culture with Trichoderma viride

Aims: To evaluate the production and stability of laccases by Pleurotus ostreatus in liquid co‐cultures with Trichoderma viride as a function of infection time and agitation rate. Methods and Results: Pleurotus ostreatus cultures were infected with T. viride spores at 30 and 48 h. Maximal laccase volumetric activity was seen after 48 h (control cultures) or 72 h (co‐cultures) of cultivation time. Only the cultures infected at 30 h showed an increased laccase volumetric activity compared to control cultures. After maximal laccase volumetric activity value was reached, a sharp decrease in it was observed in control cultures. Co‐cultures exhibited a comparatively lower loss of activity. The influence of P. ostreatus and/or T. viride on the stability of laccase volumetric activity and isoenzyme pattern was evaluated. Trichoderma viride induced changes in the laccase isoenzyme pattern. Agitated cultures increased biomass growth and specific productivity threefold and sevenfold, respectively, to the static cultures. Conclusions: The laccase volumetric activity is very likely the result of the balance between biosynthesis and degradation/biotransformation rates occurring during the cultures. The individual presence of P. ostreatus or T. viride in the culture negatively affected the volumetric laccase activity. Significance and Impact of the Study: The evaluation of culture parameters that could influence Trichoderma–basidomycetes interaction and laccase production during submerged fermentation has not been reported. This study showed how laccase production in co‐cultures of P. ostreatus and T. viride was influenced by the infection time and agitation/oxygenation conditions.     

Comparison of growth,acidification and productivity of pure and mixed cultures of Streptococcus salivarius subsp. thermophilus 404 and Lactobacillus delbrueckii subsp. bulgaricus 398

Pure and mixed controlled-pH batch cultures of Streptococcus salivarius subsp. thermophilus 404 and Lactobacillus delbrueckii subsp. bulgaricus 398 have been conducted. The characteristics of growth and acidification and the productivity of the cultures were compared. During the mixed cultures, the growth characteristics revealed a pronounced stimulation of S. thermophilus whereas L. bulgaricus metabolism was not significantly improved. The final total population was 1.4 to 4.9 higher than in pure cultures. The acidification characteristics were not enhanced by the mixed culture conditions. The productivity of mixed cultures was 1.7 to 2.4 times higher as compared to an equivalent mixing of pure cultures.Correspondence to: C. Béal     

Influence of bacteria on cell size development and morphology of cultivated diatoms

Vegetative cell division in diatoms often results in a decreased cell size of one of the daughter cells, which during long‐term cultivation may lead to a gradual decrease of the mean cell size of the culture. To restore the initial cell size, sexual reproduction is required, however, in many diatom cultures sexual reproduction does not occur. Such diatom cultures may lose their viability once the average size of the cells falls below a critical size. Cell size reduction therefore seriously restrains the long‐term stability of many diatom cultures. In order to study the bacterial influence on the size diminution process, we observed cell morphology and size distribution of the diatoms Achnanthidium minutissimum, Cymbella affiniformis and Nitzschia palea for more than two years in bacteria‐free conditions (axenic cultures) and in cultures that contain bacteria (xenic cultures). We found considerable morphological aberrations of frustule microstructures in A. minutissimum and C. affiniformis when cultivated under axenic conditions compared to the xenic cultures. These variations comprise significant cell length reduction, simplification and rounding of the frustule contour and deformation of the siliceous cell walls, features that are normally found in older cultures shortly before they die off. In contrast, the xenic cultures were well preserved and showed less cell length diminution. Our results show that bacteria may have a fundamental influence on the stability of long‐term cultures of diatoms.     

Interspecific competition among larvae ofHemipyrellia ligurriens (Calliphoridae) andBoettcherisca formosensis (Sarcophagidae) (Diptera)

1. The effects of larval rearing density and species relative proportions on life-history parameters of two necrophagous Diptera, Hemipyrellia ligurriens (Wiedemann) (Calliphoridae) and Boettcherisca formosensis Kirner and Lopes (Sarcophagidae), were investigated in mixed cultures. Larval rearing density had a significant effect on larval to adult survivorship, duration of immature development, adult size and relative performance (measured by the composite index of performance, r′) of both species. However, species relative proportions affected adult size of both flies and the duration of immature development of B. formosensis only.
2. B. formosensis had a higher survivorship than H. ligurriens in all mixed cultures and showed a similar survivorship pattern to that in pure cultures. By contrast, survivorship of H. ligurriens was lower in mixed than in pure cultures.
3. H. ligurriens adults reared from mixed cultures were smaller than those from pure cultures of comparable density, but B. formosensis adults from pure and mixed cultures were of similar size.
4. The results suggest that competition between B. formosensis and H. ligurriens larvae was asymmetric and the former was the superior competior.
5. At low larval densities in mixed cultures, the presence of H. ligurriens enhanced the performance (as measured by r′) of B. formosensis, a consequence of suspected interspecific facilitation of larval growth.

     

Continuous microbial leaching of a pyritic concentrate by Leptospirillum-like bacteria

Summary Continuous leaching of a pyritic flotation concentrate by mixed cultures of acidophilic bacteria was studied in a laboratory scale airlift reactor. Enrichment cultures adapted to the flotation concentrate contained Thiobacillus ferrooxidans and Thiobacillus thiooxidans. During the late stationary growth phase of these thiobacilli growth of Leptospirillum-like bacteria was observed, too. In discontinuous cultivation no significant influence of Leptospirillum-like bacteria on leaching rates could be detected. During continuous leaching at pH 1.5 Leptospirillum-like bacteria displaced Thiobacillus ferrooxidans. The iron leaching rate achieved by Leptospirillum-rich cultures was found to be up to 3.9 times higher than that by Leptospirillum-free cultures.     

Teratomas of <Emphasis Type="Italic">Drosera capensis</Emphasis> var. <Emphasis Type="Italic">alba</Emphasis> as a source of naphthoquinone: ramentaceone

Plants belonging to genus Drosera (family Droseraceae) contain pharmacologically active naphthoquinones such as ramentaceone and plumbagin. Hairy root cultures obtained following Agrobacterium rhizogenes-mediated transformation have been reported to produce elevated levels of secondary compounds as well as exhibit desirable rapid biomass accumulation in comparison to untransformed plants. The aim of this study was to establish hairy root or teratoma cultures of Drosera capensis var. alba and to increase the level of ramentaceone in transformed tissue by application of abiotic and biotic elicitors. The appearance of transformed tissues—teratomas but not hairy roots was observed 18 weeks after transformation. The transformation efficiency was 10% and all teratoma cultures displayed about 3 times higher growth rate than non-transformed cultures of D. capesis. The transformation was confirmed by PCR and Southern hybridization using primers based on the A. rhizogenes rolB and rolC gene sequences. HPLC analysis of ramentaceone content indicated 60% higher level of this metabolite in teratoma tissue in comparison to non-transformed cultures. Among the elicitors tested jasmonic acid (2.5 mg l⁻¹) turned out to be the most effective. The productivity of ramentaceone in elicited teratoma cultures was about sevenfold higher than in liquid cultures of D. capensis var. alba and amounted to 2.264 and 0.321 mg respectively during 4 weeks of cultivation. This is the first report on the transformation of Drosera plant with A. rhizogenes.     

Physiological and proteomic analyses of Fe(III)‐reducing co‐cultures of Desulfotomaculum reducens MI‐1 and Geobacter sulfurreducens PCA

We established Fe(III)‐reducing co‐cultures of two species of metal‐reducing bacteria, the Gram‐positive Desulfotomaculum reducens MI‐1 and the Gram‐negative Geobacter sulfurreducens PCA. Co‐cultures were given pyruvate, a substrate that D. reducens can ferment and use as electron donor for Fe(III) reduction. G. sulfurreducens relied upon products of pyruvate oxidation by D. reducens (acetate, hydrogen) for use as electron donor in the co‐culture. Co‐cultures reduced Fe(III) to Fe(II) robustly, and Fe(II) was consistently detected earlier in co‐cultures than pure cultures. Notably, faster cell growth, and correspondingly faster pyruvate oxidation, was observed by D. reducens in co‐cultures. Global comparative proteomic analysis was performed to observe differential protein abundance during co‐culture vs. pure culture growth. Proteins previously associated with Fe(III) reduction in G. sulfurreducens, namely c‐type cytochromes and type IV pili proteins, were significantly increased in abundance in co‐cultures relative to pure cultures. D. reducens ribosomal proteins were significantly increased in co‐cultures, likely a reflection of faster growth rates observed for D. reducens cells while in co‐culture. Furthermore, we developed multiple reaction monitoring (MRM) assays to quantitate specific biomarker peptides. The assays were validated in pure and co‐cultures, and protein abundance ratios from targeted MRM and global proteomic analysis correlate significantly.     

Über O-Demethylierung und Decarboxylierung von Benzoesäuren in pflanzlichen Zellsuspensionskulturen

Summary Various benzoic acids ¹⁴C-labelled in para and meta methoxyl groups as well as (O-methyl-¹⁴C) p-methoxy cinnamic acid were tested for O-demethylation in cell suspension cultures of Phaseolus aureus Roxb. and Glycine max Merr. On the basis of ¹⁴CO₂-formation and product analyses the O-demethylation reactions were shown to be specific for para methoxyl groups. A vanillate-O-demethylase known from microbial sources seemed to be absent in the plant cell cultures.In this and in an earlier publication (Berlin et al., 1971) some twenty ¹⁴C-labelled aromatic compounds were tested for catabolic reactions in the cell cultures, and here we report on the product analyses and the general pattern of distribution of radioactivity. Finally, some indications for compartmentalisation in connection with catabolic studies of aromatic compounds in plant cell cultures are discussed.Decarboxylation of substituted benzoic acids in the cell cultures is restricted to aromatic acids possessing a hydroxyl group in the para position. Only trace amounts of labelled CO₂ were released from (carboxyl-¹⁴C)-anisic acid. This acid, however, was nearly quantitatively demethylated to p-hydroxybenzoic acid, which itself was decarboxylated to a considerable extent after being fed to cell suspension cultures. Similar differences in respect to decarboxylation were observed with syringic acid produced by demethylation of trimethoxybenzoic acid and syringic acid applied directly to the cell cultures.     

Comparison of two methods for detection of Mollicutes (Mycoplasmatales and Acholeplasmatales) in cell cultures in The Netherlands

A total of 1949 cell cultures was tested for contamination with mollicutes by cultivation on and in mycoplasma media, 25.7% of the cell cultures was positive, 243 strains of Mycoplasma hyorhinis were isolated. Furthermore, mainly M. arginini and M. orale were detected, less often Acholeplasma laidlawii, M. fermentans and M. pneumoniae. Optimal conditions for isolation were discussed. About one third of 217 hybridoma cultures and two third of 57 myeloma cultures proved to be contaminated, all with M. hyorhinis. A DNA fluorochrome staining method (DAPI-test) was compared to cultivation for testing 1039 cell cultures. The efficiency of the DAPI-test could be estimated to be about 96% that of cultivation about 89%, but cultivation is more specific. The highest assurance is obtained when both methods are applied.     

Influence of clay particles (Illite) on substrate utilization by sulfate-reducing bacteria

The presence of calcium-or iron-saturated illite had a positive effect on the conversion of ethanol and acetate by non-starved cultures of Desulfobacter postgatei D.A41, but had no effect on non-starved cultures of Desulfobulbus propionicus Lindhorst and Sesulfovibrio baculatus H.L21. Starvation of these cultures at room temperature induced adhesion of cells of D. baculatus H.L21 to the surface of the clay particles. No adhesion of cells of D. propionicus Lindhorst and D. postgatei D.A41 was ever observed. However, for the three strains studied, the presence of clay particles had a positive effect on conservation of the oxidative capacity of the cultures during starvation.     

The effect of bacteria on interspecific relationships between the euryhaline rotifer Brachionus rotundiformis and the harpacticoid copepod Tigriopus japonicus

Inconsistent results have been obtained on the population growth of Brachionus rotundiformis and Tigriopus japonicus, when results from single-species and two-species mixed cultures are compared. Bacteria growth was not regulated in these experiments, which could be the cause for this. In order to test this possibility, we conducted similar experiments under axenic and synxenic (with presence of one species of bacteria) conditions. The population growth of B. rotundiformis was suppressed by the presence of T. japonicus in axenic cultures. T. japonicus could not persist in axenic cultures, but its population increased when grown in synxenic cultures. T. japonicus used RT bacteria strain as a food source, while these bacteria were toxic to B. rotundiformis. These results suggest that bacteria can modify the interspecific relationship between B. rotundiformis and T. japonicus.     

Comparison of Liquid and Agar-Solidified Defined Media Regarding the Physiological Mechanism by which β-2-Thienylalanine Inhibits Growth of Escherichia,Shigella, and Salmonella Cultures

Growth comparisons were made, using Shigella, Escherichia, and Salmonella cultures, in liquid and agar-solidified defined media containing β-2-thienylalanine (β-2-t). The comparisons were performed to determine the nature of growth inhibition by β-2-t under different physical growth conditions. In a plate assay, with increasing β-2-t mixed into the agar, inhibition of Escherichia and Shigella increased. However, Salmonella cultures were not inhibited even at the highest β-2-t concentrations used. With β-2-t added to liquid cultures, however, dose-response growth relationships were exhibited by all three genera. The differences occurring in β-2-t inhibition between liquid and plate assay conditions were not due to composition of culture plates, time of challenge of cultures with β-2-t, availability of oxygen and associated differences in ratios of volume of media to available surface area, selection of mutants in the plate assay, or to extractable substances from the agar. However, when β-2-t diffusion into the liquid medium was delayed by using agar plug diffusion cultures, a physiological mechanism was demonstrable which largely protected Salmonella cultures, but not Escherichia and Shigella cultures, from growth inhibition.     

Identification of Rhizobium strains on antibiotic concentration gradients

Antibiotic concentration gradients were used to characterise several cultures of R. phaseoli and Rhizobium spp. (isolated from Cicer arietinum) by differences in intrinsic antibiotic resistance (IAR). Differentiation between cultures was facilitated by use of cluster analyses. The method permitted 15/16 cultures of R. phaseoli to be distinguished on 14 antibiotics. Two cultures which exhibited similar IAR patterns were shown to be the same strain obtained from different collections. The validity of the technique for strain identification was demonstrated by fluorescent antibody tests which gave corresponding identity for 50 nodule isolates from plants inoculated with a mixture of three strains of R. phaseoli. The method was less suitable for characterising cultures of the slow-growing Rhizobium spp. because several antibiotics produced growth lacking a clearly defined boundary between resistance and susceptibility. Although 15/16 cultures of Rhizobium spp. could be differentiated, several isolates were distinguishable only by a difference on a single antibiotic. Similarity between stock cultures and derivative nodule isolates indicated that IAR on gradient plates was a stable property unaffected by plant passage.     

Regulation of Iron on Bacterial Growth and Production of Thermostable Direct Hemolysin by Vibrio parahaemolyticus in Intraperitoneal Infected Mice

Pathogenesis of Vibrio parahaemolyticus is not clearly understood. Effects of iron on the bacterial proliferation and production of thermostable direct hemolysin (TDH) in intraperitoneal infected mice were studied. Injection of bacterial culture in the presence of ferric ammonium citrate (100 μg/ml) significantly enhanced the lethality for mice, and simultaneously activated bacterial proliferation in vivo. The iron-limited cultures showed better proliferation than those iron-rich cultures in response to the addition of supplementary iron source. Production of TDH by the hemolytic strains ST550 and D62 was higher in the iron-limited cultures than the iron-rich cultures. Production of TDH by both the iron-limited or iron-rich cultures was inhibited by the addition of iron. In conclusion, the virulence enhancement effect of iron in V. parahaemolyticus was probably by activating bacterial proliferation in vivo and not by stimulating the production of TDH. V. parahaemolyticus precultured in iron-limited condition may be more adaptable to in vivo environment.     

A taxonomic study of soil yeasts

Summary Eighty-four samples of Minnesota soils were collected in the spring of the year. All samples yielded yeasts, and a total of 180 cultures were isolated of which 117 were studied taxonomically. Twenty-five cultures were black yeasts. Approximately one-third of the isolates were spore forming yeasts and members of the oxidative and film forming generaHansenula, Pichia, andDebaryomyces, of which a new species (D. mrakii) has been described. Not a single culture ofSaccharomyces was isolated. Perhaps later in the season when fruit was abundant members of this genus would have been present.The greatest number of imperfects belonged to the genusCandida (32 cultures), althoughPullularia (25 cultures),Rhodotorula (20 cultures), andTorulopsis (16 cultures) were well represented.The cultures ofPullularia darkened very slowly if at all, in the asbence of excess sugar. Under these circumstances they were very similar toT. pullulans.A single culture ofTrichisporon cerebriformis was isolated. The culture isolated formed both blastospores and arthrospores.I would like to express my appreciation to Dr.C. E. Skinner of Washington State College, for his help and guidance throughout this entire problem. I would also like to thank Dr.E. M. Mark, of the University of California, Berkeley, for the privilege of working under his direction, and in his laboratory during the study.     

Nitrate assimilation in Candida nitratophila and other yeasts

Nitrate assimilation has been studied in four species of yeasts; Candida nitratophila, Candida utilis, Hansenula anomala and Rhodotorula glutinis. Ammonium-grown cultures of these organisms did not assimilate nitrate but acquired the capacity to do so after a 3 h period of nitrogenstarvation. Ammonium inhibited nitrate assimilation completely in nitrate-grown cultures of R. glutinis. With Candida spp. ammonium and nitrate were assimilated simultaneously but each was assimilated at a lower rate than when either was supplied alone. Nitrogen-starved cultures of C. nitratophila contained enough nitrate reductase activity to sustain high rates of nitrate assimilation. Results indicate that the high levels of nitrate reductase in nitrate-grown cultures of C. nitratophila do not limit nitrate assimilation. Nitrate assimilation appears to be limited by nitrate uptake and/or the supply of reducing equivalents for nitrate reduction in these cultures.     

Use of bacteria for rapid,pH-neutral,hydrolysis of the model hydrophobic carboxylic acid ester p-nitrophenyl picolinate

Abstract

Caulobacter crescentus, Escherichia coli and Bacillus subtilis cultures promote the hydrolysis of the model ester p-nitrophenyl picolinate (PNPP) at neutral pH with high efficiency. Hydrolysis is related to cell concentration, while the interaction of PNPP with both bacterial cells and their extracellular molecules is required for a maximum rate of PNPP hydrolysis in C. crescentus cultures. Furthermore, C. crescentus cultures hydrolyse PNPP at concentrations useful in synthetic chemistry.     

Monoxenic cultivation of the rotifer Brachionus calyciflorus in a defined medium

Summary Techniques are described for the initiation and maintenance of axenic cultures of Euglena gracilis strain Z and monoxenic cultures of Brachionus calyciflorus variety pala with the Euglena, using in both the same defined, buffered medium. The medium, which is inorganic—except for the citrate chelating agent, the buffer, and vitamins B₁ and B₁₂ — has been used for the axenic cultuvation of the Euglena for more than 13 months. The monoxenic Brachionus cultures, established by inoculating rotifers into Euglena cultures, have been maintained for more than 8 months. Contamination tests on the rotifer cultures were performed frequently in three different test media.Mictic females, males, and resting eggs of Brachionus were observed in the monoxenic cultures, and considerable variation in the length of the posterolateral spines was noted.The compatibility of a rotifer to a defined medium which sustains the axenic culture of its food organism is a feature of this system which is convenient, useful, and unique to date in synxenic rotifer culture work.Supported by National Science Foundation Grant No. GB 7717.     

Effect of Coronatine on Plant Cell Cultures

The effect of the phytotoxin coronatine, formed by pathovars of Pseudomonas syringae, on the plant cell cultures of Lycopersicon peruvianum, Lycopersicon esculentum, Chenopodium album and Solanum tuberosum was investigated. The parameters studied were growth, triphenyltetrazolium chloride activity (test for reductases), cell protein content and activities of polyphenol oxidase and peroxidase, both within the cells and in the culture liquid. The cultures of Lycopersicon peruvianum and Lycopersicon esculentum cultures were more sensitive than Chenopodium album and Solanum tuberosum. The more resistant cultures possess a high level of extracellular polyphenol oxidase.