2 Diana_358 (8-10) Isolation and Identification of Ice-Nucleating-Active Bacteria from Indonesian Edible Leafy Plant Poh-Pohan (Pilea glaberina) DIANA ELIZABETH WATURANGI1*, VICKY MEICY1, AND ANTONIUS SUWANTO2 1Faculty of Biotechnology, Universitas Katolik Indonesia Atma Jaya, Jalan Jenderal Sudirman 51, Jakarta 12930, Indonesia, 2Department of Biology, Faculty of Science and Mathematics, Institut Pertanian Bogor, Darmaga Campus, Bogor 16680, Indonesia Two ice-nucleating-active (INA) bacteria (isolates C and 6) were isolated from poh-pohan (Pilea glaberina), an Indonesian edible leafy plant (lalaban). The maximum nucleation temperature of aqueous suspensions of the two isolates is -5 °C. They were classified as a type II ice nucleator. Microscopic and morphological determination showed that these isolates had yellow pigmentation, rod shape, and were Gram negative. Biochemical analysis indicated that the isolates were exhibited catalase activity, but negative in oxidase and indole assays. DNA sequencing of 16SrRNA gene of isolate A3 showed a 94% similarity to Pseudomonas sp. while isolate A4 showed a 97% similarity to Xanthomonas campestris. To our knowledge, this is the first report of INA bacteria isolated from a tropical edible leafy plant. Keywords: Ice-nucleating bacteria, Indonesian edible leafy plant, Pilea glaberina _____________________________________________ ________________________ *Corresponding author, Phone: +62-21-5703306 ext. 335, Fax: +62-21-5719060, E-mail: diana.waturangi@atmajaya.ac.id Leaf microbial communities of leaves are diverse and include many different genera of bacteria, filamentous fungi, yeasts, algae, and less frequently, protozoa and nematodes (Andrews and Harris 2000) Bacteria are by far the most abundant inhabitants of the phyllosphere and ephiphytic bacterial populations differ sharply in size among and within plants of the same species (Lindow et al. 1978). One of the bacterial components of leaf microbial communities are ice- nucleation-active (INA) bacteria (Lindow and Brandl 2003). These bacteria have been shown to incite frost damage to some plants (Hirano and Upper 2000). The bacteria commonly found on plants comprise strains of several species (such as Pseudomonas syringae and Erwinia herbicola) which produce a protein able to induce ice nuclei at temperatures as high as -2°C. Leaf surface populations of these bacterial species limit supercooling in the plant parts on which they reside by initiating damaging ice formation at temperatures of -2 to -4°C. Since plants do not have intrinsic ice nuclei active at these temperatures, these bacteria play an important role in initiating frost damage (Lindow et al. 1978). While data of the presence of INA bacteria on the leaf surface are commonly found from subtropical areas, little is known about the presence of these bacteria in tropical areas. Riupassa et al. (2005) investigated the abundance of phyllosphere bacteria in a number of Indonesian edible leafy plants (lalaban) which could reach up to 3 x 108 CFU/g. In this study we are interested to focus on the isolation and identification of ice-nucleating active bacteria from an Indonesian edible leafy plant poh-pohan (Pilea glaberina). MATERIALS AND METHODS Sampling Technique. Isolations of INA bacteria were done from many edible leafy plants such as poh-pohan (P. glaberina), selada (Lactuca sativa), kemangi (Ocinum sanctum), and taoge kacang hijau (Vigna radiata). Samples were placed in sterile plastic bags and plated immediately upon return to the laboratory. Samples of 2 grams of leaves were cut into pieces and washed with sterile distilled water 3x. Samples were placed in sterile tubes containing 10 mM phosphate buffer (pH 8.0), vortexed for 2 minutes and then diluted to 100 fold. The suspensions were spread on King’s B medium agar and incubated at room temperature (28-30°C) for 2 days. Detection of INA Colonies by Replica Freezing Assay. Colony growth from the isolations were picked and prepared for ice-nucleation assay by the method of Lindow et al. (1978). Escherichia coli DH5α carrying pJL1703 was used as a positive control. Ice nucleation activity was assessed with the drop-freezing assay. Frequencies of the ice nucleation activity of the isolates were measured using the formula described by Vali (1971). N(t) = (- ln f)/V, where N (t) is the nucleation frequency at temperature T, f is the proportion of droplets unfrozen and V is the volume of individual droplets. Biochemical Tests. Biochemical properties of these isolates were determined for indole, catalase, and oxidase assays. PCR Amplification, Cloning, and Sequencing of 16S rDNA. Genomic DNA was extracted and purified using the Wizard Genomic DNA Purification Kit (Promega, USA). The extracted DNA was used as a template for PCR amplification of the 16S rRNA gene using 63f (5’- CAGGCCTAACACA TGCAAGTC-3’) primer and 1387r primer (5’-GGGCGGWGTG TACAAGGC-3’) (Marchesi et al. 1998). The reactions were performed in a volume of 25 µl containing 1 µl of DNA template, 1 µl of each primer (25 pmol/µl) (Research Biolabs), 0.5 µl of dNTP mix (Reseearch Biolabs) and 0.5 µl (2.5 U) of Taq polymerase, 2.5 µl of 10 x buffer (New England Biolabs, Mass. USA). The total volume was adjusted to 25 µl with sterile double- distilled water. The following cycles were applied for PCR: 94°C for 5 minutes then 25 cycles: 94°C for 30 seconds denaturation, 55°C for 30 seconds annealing and 72°C for 1 minutes elongation. After the amplification, an 8 μl of amplified DNA was mixed with 3 µl of loading buffer and then loaded onto 1% (w/v) gel electrophoresis (Mini-Sub ISSN 1978-3477 Volume 2, Number 1, April 2008 p8-10 Cell GT; Biorad, Richmond, CA) which was run for 60 minutes at 55 V prior to visualization using a UV transiluminator. The resulting amplicons were purified using a QIAquick PCR Purification Kit (Qiagen, Germany) and then cloned using the pGMT easy vector System I (Promega, USA) and transformed into E. coli D H 5 á . E. coli transformants were selected on LB agar supplemented with X-gal (40 µg/ml). Recombinant plasmids were isolated using Plasmid Wizard Plus SV Minipreps DNA Purification System (Promega, USA). The cloned amplicons were sequenced (ABI Prism 3130x genetic analyser) on both strands using M13 reverse and forward primers. The sequences were compared to GenBank sequences by using a standard nucleotide basic local alignment search tool (BLAST) search. RESULTS We recovered two isolates of bacteria (isolate A3 and A4) which showed ice-nucleation activity from poh-pohan (P. glaberina) (Fig 1). The ice-nucleation-activity of isolate A4 is higher than isolate A3 with an ice-nucleation frequencies of 59.9/ml and 37.9/ml respectively (Table 1). Microscopic and morphological determination showed that these isolates had yellow pigmentation, rod shape and were Gram negative. Biochemical analysis indicated that the isolates were exhibited catalase activity, but negative in oxidase and indole assays. DNA sequencing of 16SrRNA gene of isolate A3 showed a 94% similarity to Pseudomonas sp while isolate A4 showed a 97% similarity to Xanthomonas campestris. The DNA sequences have been submitted to Genbank with the accession number EU563218 for isolate A3 and EU531514 for isolate A4. DISCUSSION There are two isolates (A3 and A4) from poh-pohan, Indonesian edible leafy plant, which were identified as ice- nucleation bacteria. These two isolates are categorized as type two ice-nucleation bacteria, having the maximum nucleation temperature of aqueous suspensions at -5°C. Turner et al. (1990) classified ice nucleation proteins into three classes: class A which showed the ice nucleation activity between temperature -2°C to -5°C, class B within the temperature range of -5°C to -8°C, while class C is below -8°C. Based on that system, these two isolates were categorized as class B. The presence of INA-bacteria on leaf surfaces could alter leaf habitats at subzero temperatures. Frost-sensitive plants are injured when ice forms within plant tissues. Without ice formation, such injury does not occur, and perhaps many ice-nucleation genes of INA-bacteria recovered from tropical leaf might not being expressed. This finding might raise question such as: What is the role of these bacteria on tropical leaves?; are these bacteria transmitted from subtropical habitats?; how is the distribution on plants in tropical areas? From the measurement of ice-nucleation frequency (Table 1), isolate A3 have ice-nucleation activity of 37.9/ml, while isolate A4 is 59.91/ml. Isolate A4 showed higher ice- nucleation activity than A3. Identification of the isolates through PCR amplification, cloning and DNA sequencing indicated that isolate A3 showed a 94% similarity to Pseudomonas sp. while isolate A4 showed a 97% similarity to Xanthomonas campestris. Loper and Lindow (1994) reported that the ice nucleation protein from Pseudomonas sp. was classified as IceC. While the ice nucleation protein from X. campestris was InaX (Turner et al. 1990; Edwards et al. 1994). However, all of the publication data were derived from subtropical areas. To our knowledge, this is the first finding of INA bacteria from a tropical edible leafy plant. Finding of Pseudomonas sp. and X. campestris on P. glaberina was interesting and we need more studies on characterization of the activity of the protein and their role in their natural habitats. ACKNOWLEDGEMENTS This research was supported by Atma Jaya Research Center. We also would like to give gratitude to Steven Lindow, University of California Berkeley, USA for the scientific discussion and positive control of INA Bacteria. REFERENCES Andrews JH, Harris RF. 2000. The ecology and biogeography of microorganisms on plant surfaces. Annu Rev Phytopathol 38:145- 180. Edwards AR, Ronald A, Wichman HA, Orser CS. 1994. Unusual pattern of bacterial ice nucleation gene evolution. Mol Biol Evol 11:911-920. Hirano SS, Upper CD. 2000. Bacteria in the leaf ecosystem with emphasis on Pseudomonas syringae a pathogen, ice nucleus, and epiphyte. Mol Microbiol Biol Rev 64:624-653. Lindow SE, Arny DC, Upper CD. 1978. Distribution of ice nucleation- active bacteria on plants in nature. Appl Environ Microbiol 36:831- 838. Lindow SE, Brandl MT. 2003. Microbiology of the phyllosphere. Appl Environ Microbiol 69:1875-1883. Loper JE, Lindow SE. 1994. A biological sensor for iron available to bacteria in their habitats on plants surface. Appl Environ Microbiol 60:1934-1941. Fig 1 Assay of ice nucleation activity. a, negative control; b, positive control E.coli DH5α carrying pJL1703; c, isolate A3; d, isolate A4. Table 1 Ice-nucleation activity of bacteria form poh-pohan (Pilea glaberina) Isolates f N(t) (-8 °C) A3 A4 17/20 19/20 37.9/ml 59.9/ml N(t) = Ice-nucleation activity per ml. Volume 2, 2008 Microbiol Indones 9 Marchesi JR, Sato T, Weightman AJ, Martin TA, Fry JC, Hiom SJ, Wade WG. 1998. Design and evaluation of useful bacterium- specific PCR primers that amplify genes coding for bacterial 16S rRNA. Appl Environ Microbiol 64:795-799. Riupassa PA, Suwanto A, Tjahjoleksono A. 2005. The abundance of phyllosphere bacteria isolated from some Indonesian fresh leafy salad (lalaban). Microbiol Indonesia 10:96-98. Turner MA, Arellano F, Kozloff LM. 1990. Three separate classes of bacterial ice nucleation structures. J Bacteriol 172:2521-2526. Vali G. 1971. Quantitative evaluation of experimental result on the heterogenous freezing nucleation of supercooled liquids. J Atmos Sci 28:402-409. 10 WATURANGI ET AL. Microbiol Indones