Gellan Gum Plates Research Articles

Preculture in an enriched nutrient medium greatly enhances the Agrobacterium-mediated transformation efficiency in Arabidopsis T87 cultured cells.

The Arabidopsis T87 cell line has been widely used in both basic and biotechnological plant sciences. Agrobacterium-mediated transformation of this cell line was reported to be highly efficient when precultured in Gamborg's B5 medium for a few days. However, because we could not obtain the expected efficiency in our laboratory, we further examined the preculture conditions of Arabidopsis T87 cells in the Agrobacterium-mediated transformation. As a result, we found that preculture in an excess amount of Murashige and Skoog (MS) macronutrients before cultivation in the B5 medium enhanced the transformation efficiency up to 100-fold, based on the transformed callus number on selective gellan gum plates. In this study, transformants were labeled with green fluorescent protein (GFP), and we found multiple fluorescent spots on individual transgenic calli. Therefore, the actual number of transgenic clones seems much more than that of transgenic calli. In our MS macronutrient-rich culture condition, T87 cells tended to aggregate and formed bigger cell clumps, a change that might be related to the enhancement of transformation efficiency. Based on these results, we report an improved protocol of Agrobacterium-mediated transformation of Arabidopsis T87 cells with high efficiency.

Determination of available breaking stress of agar and gellan gum plate culture methods and the duration of bacterial culture under strong acidic conditions.

Several acidophilic bacteria have not been cultured, primarily owing to the lack of suitable culture methods under strong acidic conditions. This study aimed to quantitatively evaluate the strengths of the agar plates (AP) and gellan gum plates (GP), and optimal culture periods under strong acidic conditions. To define the lower limit of plate strength for bacterial isolation culture, the diameter of Escherichia coli K12 colonies and the breaking stress of plates at different concentrations of gelling agents, medium composition and pH conditions were determined. The lower limit of available strength of AP and GP was 19·6 and 14·8kPa, respectively. Medium composition slightly affected AP breaking stress, although GP with a high cationic concentration medium could not be prepared. Assessment of the strength limits of AP and GP revealed that AP is not suitable for prolonged bacterial culture (≥72h). Furthermore, GP was completely ineffective for bacterial culture under highly acidic conditions (≤pH 1·0). Our quantitative evaluation method based on breaking stress is a potentially valuable tool to understand the state and the suitable limit of plate culture methods in more detail under various conditions.

Construction of a Selectable Marker Recycling System and the Use in Epitope Tagging of Multiple Nuclear Genes in the Unicellular Red Alga Cyanidioschyzon merolae.

The nuclear genome of the unicellular red alga Cyanidioschyzon merolae can be modified by homologous recombination with exogenously introduced DNA. However, it is presently difficult to modify multiple chromosome loci because of the limited number of available positive selectable markers. In this study, we constructed a modified URA5.3 gene (URA5.3T), which can be repeatedly used for nuclear genome transformation, as well as two plasmid vectors for 3× FLAG- or 3× Myc-epitope tagging of nuclear-encoded proteins using URA5.3T. In the URA5.3T marker, the promoter region and open reading frame were located between directly repeated URA5.3 terminator sequences, and the URA5.3 gene can be eliminated by 5-fluoroorotic acid selection through homologous recombination. To demonstrate the utility of the constructed system, a 3× FLAG-tag and 3× Myc-tag were introduced at the C-termini of two of the six Rab proteins in C. merolae, CmRab18 and CmRab7, respectively, and the differential expression levels were successfully monitored by immunoblot analysis using these epitope tags. The URA5.3T marker's introduction and elimination cycle can be repeated. Thus, we have constructed a marker recycling system for C. merolae nuclear transformation. A novel procedure to obtain a high plating efficiency of C. merolae cells on solid gellan gum plates is also presented.

Culture-Dependent and Independent Analyses of the Microbial Communities Inhabiting the Giant Duckweed (Spirodela polyrrhiza) Rhizoplane and Isolation of a Variety of Rarely Cultivated Organisms within the Phylum Verrucomicrobia

The microbial communities of the rhizoplane, the surface part of roots, in aquatic plants are not understood at all. In this study, we analyzed microbial communities in the rhizoplane of a floating aquatic plant, giant duckweed (Spirodela polyrrhiza), based on cultivation-dependent and independent analyses. The cultivation-based analysis using agar and gellan gum plates revealed that the rhizoplane isolates were affiliated with four bacterial lineages; the Alphaproteobacteria, Betaproteobacteria, Bacteroidetes and Verrucomicrobia. Interestingly, microbes belonging to the phylum Verrucomicrobia accounted for 24% of all the isolates, suggesting that the rhizoplane of S. polyrrhiza forms a specific habitat for the organisms within this phylum. Culture-independent 16S rRNA gene cloning showed that the clonal sequences were affiliated with eight bacterial classes and phyla: the classes Alphaproteobacteria (14% total clones), Betaproteobacteria (45%), Gammaproteobacteria (2%) and Deltaproteobacteria (2%), and the phyla Bacteroidetes (11%), Verrucomicrobia (2%), Planctomycetes (2%) and Cyanobacteria (22%). Comparative analysis of the microbial communities in the rhizoplane between culture-dependent and independent methods revealed that 33% of the taxonomic groups of bacterial species detected in the molecular analysis were cultivable. Our findings suggest that the microbes in the rhizoplane of giant duckweed are comprised of a diverse array of readily cultured organisms including a variety of strains within the Verrucomicrobia, a well-known phylum that contains a number of yet-to-be cultivated organisms.

Effect of gelling agent on colony formation in solid cultivation of microbial community in lake sediment

Since Robert Koch and colleagues found agar to be an effective gelling agent over a century ago, the pure culture method using agar plates has long been a standard of microbiology. Agar is undoubtedly easy to handle and useful for culture of microorganisms, but recent discovery of the ubiquity of microorganisms that cannot be cultured on agar raises a question: is agar really the best agent? In this study, we investigated the effect of two gelling agents, agar and gellan gum, on colony formation of a diverse array of microorganisms (total 108 strains) newly isolated from freshwater sediments and a representative microorganism as a slow grower on agar medium, Gemmatimonas aurantiaca, to clarify (i) whether they can grow on both agar and gellan gum plates, and (ii) the difference in time required for colony formation between the two gelling agents. Interestingly, 22 of 108 isolates showed no ability to form any visible colonies on the agar medium but did so on the gellan gum medium, and showed low 16S rRNA gene sequence similarities to their closest species. The remaining 86 isolates grew on both agar and gellan gum, but 52 of them grew much faster on gellan gum than on agar. Moreover, gellan gum also significantly stimulated the colony formation of the representative slow-growing microorganism G. aurantiaca. Our results demonstrate that the gelling agent is a crucial factor for the growth of bacteria on plate media, and that alternatives to agar will be very important for increasing the culturability of yet-to-be cultured microorganisms.

High Rate of N 2 Fixation by East Siberian Cryophilic Soil Bacteria as Determined by Measuring Acetylene Reduction in Nitrogen-Poor Medium Solidified with Gellan Gum

For evaluating N(2) fixation of diazotrophic bacteria, nitrogen-poor liquid media supplemented with at least 0.5% sugar and 0.2% agar are widely used for acetylene reduction assays. In such a soft gel medium, however, many N(2)-fixing soil bacteria generally show only trace acetylene reduction activity. Here, we report that use of a N(2) fixation medium solidified with gellan gum instead of agar promoted growth of some gellan-preferring soil bacteria. In a soft gel medium solidified with 0.3% gellan gum under appropriate culture conditions, bacterial microbiota from boreal forest bed soils and some free-living N(2)-fixing soil bacteria isolated from the microbiota exhibited 10- to 200-fold-higher acetylene reduction than those cultured in 0.2% agar medium. To determine the N(2) fixation-activating mechanism of gellan gum medium, qualitative differences in the colony-forming bacterial components from tested soil microbiota were investigated in plate cultures solidified with either agar or gellan gum for use with modified Winogradsky's medium. On 1.5% agar plates, apparently cryophilic bacterial microbiota showed strictly distinguishable microbiota according to the depth of soil in samples from an eastern Siberian Taiga forest bed. Some pure cultures of proteobacteria, such as Pseudomonas fluorescens and Burkholderia xenovorans, showed remarkable acetylene reduction. On plates solidified with 1.0% gellan gum, some soil bacteria, including Luteibacter sp., Janthinobacterium sp., Paenibacillus sp., and Arthrobacter sp., uniquely grew that had not grown in the presence of the same inoculants on agar plates. In contrast, Pseudomonas spp. and Burkholderia spp. were apparent only as minor colonies on the gellan gum plates. Moreover, only gellan gum plates allowed some bacteria, particularly those isolated from the shallow organic soil layer, to actively swarm. In consequence, gellan gum is a useful gel matrix to bring out growth potential capabilities of many soil diazotrophs and their consortia in communities of soil bacteria.

Improvement of culture conditions and evidence for nuclear transformation by homologous recombination in a red alga, Cyanidioschyzon merolae 10D.

Although the nuclear genome sequence of Cyanidioschyzon merolae 10D, a unicellular red alga, was recently determined, DNA transformation technology that is important as a model plant system has never been available thus far. In this study, improved culture conditions resulted in a faster growth rate of C. merolae in liquid medium (doubling time = 9.2 h), and colony formation on gellan gum plates. Using these conditions, spontaneous mutants (5-fluoroortic acid resistant) deficient in the UMP synthase gene were isolated. The lesions were then restored by introducing the wild-type UMP synthase gene into the cells suggesting DNA transformation by homologous recombination.

Effect of high-CO2 on the growth of the thermophilic cyanobacterium on plates

Cultivation on solid media is an essential technique for the isolation of desired strains or mutants of various organisms. As cyanobacteria especially thermophilic species generally require longer time for growth than bacteria, acceleration of growth is desirable. Growth of the thermophilic cyanobacterium, Synechococcus elongatus, was examined under different CO2 concentrations with plates of different gels at 48°C. The cyanobacterial colonies showed faster growth in their diameters by 3, 4 and 2 times under 2% CO2 than under air, on the agar, agarose and gellan gum plates, respectively. The cells in the colonies were competent to grow after 2 months stock at room temperature under dim light, while the cells cultivated in the liquid medium were not.

Feasibility study of disk diffusion susceptibility tests with Mueller-Hinton broth solidified with Gelrite, an agar substitute.

Feasibility studies were done to determine whether a new agar substitute, Gelrite gellan gum, could be used to prepare a solid Mueller-Hinton medium for disk diffusion susceptibility tests. Mueller-Hinton broth was combined with 0.43% of the gellan gum and 0.75% KCI. The resulting medium had performance characteristics similar to those of Mueller-Hinton agars; however, zones on the gellan gum media tended to be a little larger. Significant differences among Mueller-Hinton broths and among Mueller-Hinton agars from different manufacturers were documented: zones on different lots of the gellan gum were more consistent. The Mueller-Hinton broth-gellan gum medium appears to represent a satisfactory alternative to agar media. However, because somewhat larger zones were seen on the gellan gum plates, further study will be needed to develop quality control limits and interpretive zone size standards for tests on this new medium.