J. Gen. Appl. Microbiol., 48, 335–343 (2002) Full Paper Tistrella mobilis gen. nov., sp. nov., a novel polyhydroxyalkanoate- producing bacterium belonging to a-Proteobacteria Bin-Hai Shi, Vullapa Arunpairojana,1 S. Palakawong,1 and Akira Yokota* Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113–0032, Japan 1 Thailand Institute of Scientific and Technological Research, 196, Phahonyothin Road, Chatuchak, Bangkok 10900, Thailand (Received October 2, 2001; Accepted November 13, 2002) Strain IAM 14872, isolated from wastewater in Thailand, is capable of producing polyhydrox- yalkanoate. This bacterium is Gram-negative, rod-shaped, strictly aerobic and highly motile with a single polar flagellum. Both oxidase and catalase activities are positive. The G؉C content of DNA is 67.5% and Q-10 is the major quinone. The major cellular fatty acids are C18:1w7c, 2-OH C18:0 and 3-OH C14:0. On the basis of the 16S rDNA sequence analysis and phenotypic properties, it is proposed that the strain IAM 14872 be classified in a new genus as Tistrella mobilis gen. .(TISTR 1108T؍) nov., sp. nov. The type strain is IAM 14872T Key Words——PHA and polyhydroxyalkanoate; a-Proteobacteria; 16S rDNA; taxonomy; Tistrella mo- bilis gen. nov., sp. nov. Introduction used for the microbial transformation of carbohydrate feedstock via PHA into chiral depolymerization prod- Polyhydroxyalkanoates (PHAs) are known to be pro- ucts (Seebach and Zuger, 1985) or small-molecule or- duced as intracellular granules by a variety of bacteria, ganic chemicals by pyrolysis (Anderson and Dawes, such as Alcaligenes eutrophus, Pseudomonas oleovo- 1990). So far, these biodegradable PHAs, however, rans, Rhodospirillum rubrum, etc. (Anderson and are not priced competitively mainly because of the Dawes, 1990). PHA accumulation usually occurs high cost, which lies in both the use of glucose as a under aerobic conditions when the carbon source is fermentation feedstock and the low product yield. present in excess but one or more other nutrients are For the purpose of isolating strains capable of pro- limited (Liu et al., 2000), and some phototrophic purple ducing PHA efficiently, we tried to isolate bacteria from sulfur bacteria accumulate PHAs when growing anaer- samples collected from various sources, including soil, obically in the light (Mas and Gemerden, 1995). Poly- compost, garbage, activated sludge, raw sewage, b-hydroxybutyrate (PHB) is the most common repre- freshwater, wastewater and estuarine environments. sentative of PHA. PHA is a biodegradable, biocompati- Nine strains of PHA-producing bacteria were isolated. ble and thermoplastic material, which has a potential Among these strains, strain IAM 14872T showed good role as a so-called biomass transducer, i.e., it can be ability in producing PHA efficiently. In this paper we report on the phenotypic and phylo- genetic characterization of strain IAM 14872T. On the * Address reprint requests to: Dr. Akira Yokota, Institute of Molecular and Cellular Biosciences, The University of Tokyo, basis of the results presented, we propose that strain T 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113–0032, Japan. IAM 14872 be classified as Tistrella mobilis gen. nov., E-mail: [email protected] sp. nov. 336 SHI, ARUNPAIROJANA, and YOKOTA Vol. 48 Materials and Methods al., 1994). PCR (Mullis and Faloona, 1987) was per- formed with Ex Taq polymerase (TaKaRa, Shiga, Bacterial strains and isolation.Twenty samples Japan) and the following primers: 8F 5Ј-AGAGT- were collected from soil, compost, garbage, activated TTGATCCTGGCTCAG-3Ј, 1510R 5Ј-GGCTACCT- sludge, raw sewage, freshwater, wastewater and estu- TGTTACGA-3Ј (according to Escherichia coli number- arine environments. Screenings were performed in ing). DNA was purified using GFXTM PCR DNA and order to isolate PHA-producing microorganisms from Gel Band Purification Kit (Amersham Pharmacia the 20 samples. In the screenings for PHA-producing Biotech, Inc., Piscataway, NJ, USA). Sequencing was strains, the mineral salt medium was prepared with carried out with the DNA Sequencing Kit (Applied TM 0.81 mM MgSO4, 0.58 mM CaSO4, 18 mM FeSO4, 1.0 mM Biosystems, Foster City, CA, USA) using ABI PRISM NaMoO4 in 5 mM potassium phosphate, 50 mM ferric cit- 310 Genetic Analyzer (Applied Biosystems). Se- rate, 3% glucose and 15 mM ammonium acetate (pH quences were aligned and the phylogenetic tree in the 7.1). The PHA content in bacterial colonies was deter- neighbor-joining (NJ) method (Saitou and Nei, 1987) mined qualitatively by observing the presence of visi- was constructed using the CLUSTAL W program ble, intracellular granules using a phase-contrast mi- (Thompson et al., 1994). The similarity values were croscope. To recognize PHA-rich colonies, colonies calculated using PAUP 3.1.1 package (Swofford, grown on nitrogen-deficient agar after 5-day incubation 1993). at 30°C were stained with sudanblack B (0.02% in Quinone system. Quinone system was determined 96% ethanol). The dye was removed after 20 min, and by the method of Uchino et al. (1998). Quinone frac- the plates were then treated for 1 min with 10 ml of tions were extracted with chloroform-methanol (2 : 1, 96% ethanol. The colonies of PHA-rich cells retained v/v) from the lyophilized cells. Then the fractions were the dye and appeared dark blue, whereas those of separated with thin-layer chromatography (TLC) devel- PHA-deficient cells decolorized and appeared light oped with hexane : diethyl ether (85 : 15, v/v). Quinones gray. Strain IAM 14872T, isolated from a wastewater were detected under UV light at 275 nm. The spots of sample, showed good ability in the PHA-producing quinones were scraped off and quinones were ex- test. The composition of PHA was determined by gas tracted with acetone. After concentration, the quinone chromatography. samples were analyzed with a HPLC machine (Shi- Electron microscopy. Flagellation was observed madzu, Kyoto, Japan). using a model JEM-100 SX scanning electron micro- GϩC content of DNA. DNA was extracted and pu- scope (JEOL, Ltd., Tokyo, Japan) after negative-stain- rified according to the method of Saito and Miura ing with phosphotungstic acid. For ultra-thin section (1963). The GϩC content of DNA was determined by electron microscopy, cells were prefixed in 3% glu- HPLC as described by Mesbah et al. (1989). taraldehyde, fixed in OsO4, stained with uranyl ac- Cellular fatty acids. Fatty acid methyl esters were etate, embedded in Epon 812, sectioned, post-stained prepared from biomass that was scraped from Tryptic with lead acetate and examined with an electron mi- Soy Agar medium (Difco, MD, USA), and incubated at croscope, model JEOL 1210. 30°C for 2 days. Cellular fatty acids of the test strains Physiological and biochemical test. The biochemi- were analyzed as methyl esters by GC according to cal properties were tested using the API 50CHE and the instructions of the Microbial Identification System API 20NE gallery methods (bioMérieux, Paris, France) (MIDI, Inc., Newark, DE, USA). according to the manufacturer’s instructions. Acetic Photosynthetic analysis.The phototrophic ability acid production from ethanol was tested by the method was determined by observing the growth under anaer- of Asai et al. (1964). Oxidase activity was determined obic conditions with light. The detection of bacteri- by oxidation of 1% tetramethyl-p-phenylenediamine on ochlorophyll a was performed both in vivo and in vitro filter paper and catalase activity was determined by by the method of Uchino et al. (1998). In vivo spectra observing bubble production in a 3% hydrogen perox- were determined on cell suspension in 60% sucrose ide solution. solution from 3-day cultures grown aerobically in nutri- 16S rDNA sequencing and phylogenetic analysis. ent broth. In vitro spectra were determined using Cell lysate was made from 1 ml culture suspension methanol extracts of 3-day cultures grown aerobically with Proteinase K solution and BL buffer (Hiraishi et on nutrient broth agar. With Rhodobacter capsulatus 2002 Tistrella mobilis gen. nov. 337 (IAM 14232T) as a positive control, the absorbance Results values both in vivo and in vitro were examined using a Shimadzu UV-3000 spectrophotometer. Bacterial strains Nucleotide sequence accession numbers. The ac- Among nine strains of PHA-producing bacteria iso- cession number of 16S rDNA sequence of strain IAM lated from 20 samples, strain IAM 14872T showed 14872T in DDBJ/EMBL/GenBank is AB071665. The good PHA-producing ability. Fed-batch culture studies other accession numbers of the published 16S rDNA of strain IAM 14872T showed that the yield of PHA ac- sequences in this study are X74066 (Acetobacter aceti counted for 19.6% cell dry weight under nitrogen limi- T ϩ NCIB 8621 ), D86512 (Acidisphaera rubrifaciens JCM tation with the concentrations of NH4 (sole nitrogen 10600T), D30773 (Acidiphilium cryptum ATCC source) and cane molasses (sole carbon source) 33463T), D30774 (Acidocella facilis ATCC 35904T), being 0.1% w/v and 5% w/v, respectively. The analysis X77468 (Acidomonas methanolica MB 58T), of PHA by gas chromatography compared to the stan- AB025928 (Asaia bogoresis JCM 10569T), Z29619 dard chromatograms of methyl-n-butyrate, methyl-n- (Azospirillum lipoferum NCIMB 11861T), D85828 valerate and methyl esters of eight- to twelve-carbon (Craurococcus roseus JCM 9933T), X80725 (Esch- atoms fatty acid indicated that the biopolymer con- erichia coli ATCC 11775T), X75617 (Gluconacetobac- sisted of four-, five- and eight- to ten-carbon ter liquefaciens IFO 12388T), X73820 (Gluconobacter monomers. oxydans DSM 3503T), AB056321 (Kozakia baliensis Yo-3T), Y10109 (Magnetospirillum gryphisealdense Morphological characteristics DSM 6361T), Y10110 (Magnetospirillum magneto- The ultra-thin section electron micrograph (Fig. 1A) tacticum DSM 3856T), D85827 (Paracraurococcus showed that the cells were short straight rods occur- ruber NS 89T), D14433 (Phaeospirillum fulvum NCIMB ring in pairs or as single cells. PHAs were accumu- 11762T), D12701 (Rhodocista centenaria IAM 14193T), lated in the cytoplasm. Intracytoplasmic membranes D86513 (Rhodopila globiformis DSM 161T), X99671 (ICMs) were not observed.