Tistrella mobilis gen. nov., sp. nov

J. Gen. Appl. Microbiol., 48, 335–343 (2002)

Materials and Methods

Bac terial strains and isolation .Twenty samples were collected from soil, compost, garbage, activated sludge, raw sewage, freshwater, wastewater and estu-arine environments. Screenings were performed in order to isolate PHA-producing microorganisms from the 20 samples. In the screenings for PHA-producing strains, the mineral salt medium was prepared with 0.81m M MgSO 4, 0.58m M CaSO 4, 18m M FeSO 4, 1.0m M NaMoO 4in 5m M potassium phosphate, 50m M ferric cit-rate, 3% glucose and 15m M ammonium acetate (pH 7.1). The PHA content in bacterial colonies was deter-mined qualitatively by observing the presence of visi-ble, intracellular granules using a phase-contrast mi-croscope. To recognize PHA-rich colonies, colonies grown on nitrogen-de?cient agar after 5-day incubation at 30°C were stained with sudanblack B (0.02% in 96% ethanol). The dye was removed after 20min, and the plates were then treated for 1min with 10ml of 96% ethanol. The colonies of PHA-rich cells retained the dye and appeared dark blue, whereas those of PHA-de?cient cells decolorized and appeared light gray. Strain I AM 14872T , isolated from a wastewater sample, showed good ability in the PHA-producing test. The composition of PHA was determined by gas chromatography.

Elec tron mic rosc opy .Flagellation was observed using a model JEM-100 SX scanning electron micro-scope (JEOL, Ltd., Tokyo, Japan) after negative-stain-ing with phosphotungstic acid. For ultra-thin section electron microscopy, cells were pre?xed in 3% glu-taraldehyde, ?xed in OsO 4, stained with uranyl ac-etate, embedded in Epon 812, sectioned, post-stained with lead acetate and examined with an electron mi-croscope, model JEOL 1210.

Physiological and biochemical test .The biochemi-cal properties were tested using the API 50CHE and API 20NE gallery methods (bioMérieux, Paris, France)according to the manufacturer’s instructions. Acetic acid production from ethanol was tested by the method of Asai et al. (1964). Oxidase activity was determined by oxidation of 1% tetramethyl-p -phenylenediamine on ?lter paper and catalase activity was determined by observing bubble production in a 3% hydrogen perox-ide solution.

16S rDNA sequenc ing and phylogenetic analysis .Cell lysate was made from 1ml culture suspension with Proteinase K solution and BL buffer (Hiraishi et

al., 1994). PCR (Mullis and Faloona, 1987) was per-formed with Ex Taq polymerase (TaKaRa, Shiga,Japan) and the following primers: 8F 5?-AGAGT-TTGATCCTGGCTCAG-3?, 1510R 5?-GGCTACCT-TGTTACGA-3?(according to Escherichia coli number-ing). DNA was puri?ed using GFX TM PCR DNA and Gel Band Puri?cation Kit (Amersham Pharmacia Biotech, Inc., Piscataway, NJ, USA). Sequencing was carried out with the DNA Sequencing Kit (Applied Biosystems, Foster City, CA, USA) using ABI PRISM TM 310 Genetic Analyzer (Applied Biosystems). Se-quences were aligned and the phylogenetic tree in the neighbor-joining (NJ) method (Saitou and Nei, 1987)was constructed using the CLUSTAL W program (Thompson et al., 1994). The similarity values were calculated using PAUP 3.1.1 package (Swofford,1993).

Quinone system .Quinone system was determined by the method of Uchino et al. (1998). Quinone frac-tions were extracted with chloroform-methanol (2:1,v/v) from the lyophilized cells. Then the fractions were separated with thin-layer chromatography (TLC) devel-oped with hexane :diethyl ether (85:15, v/v). Quinones were detected under UV light at 275nm. The spots of quinones were scraped off and quinones were ex-tracted with acetone. After concentration, the quinone samples were analyzed with a HPLC machine (Shi-madzu, Kyoto, Japan).

G ?C content of DNA .DNA was extracted and pu-ri?ed according to the method of Saito and Miura (1963). The G ?C content of DNA was determined by HPLC as described by Mesbah et al. (1989).

Cellular fatty ac ids .Fatty acid methyl esters were prepared from biomass that was scraped from Tryptic Soy Agar medium (Difco, MD, USA), and incubated at 30°C for 2 days. Cellular fatty acids of the test strains were analyzed as methyl esters by GC according to the instructions of the Microbial I denti?cation System (MIDI, Inc., Newark, DE, USA).

Photosynthetic analysis .The phototrophic ability was determined by observing the growth under anaer-obic conditions with light. The detection of bacteri-ochlorophyll a was performed both in vivo and in vitro by the method of Uchino et al. (1998). In vivo spectra were determined on cell suspension in 60% sucrose solution from 3-day cultures grown aerobically in nutri-ent broth. I n vitro spectra were determined using methanol extracts of 3-day cultures grown aerobically on nutrient broth agar. With Rhodobac ter c apsulatus

336S HI , A RUNPAIROJANA , and Y OKOTA Vol.48

(I AM 14232T) as a positive control, the absorbance values both in vivo and in vitro were examined using a Shimadzu UV-3000 spectrophotometer.

Nucleotide sequence accession numbers.The ac-cession number of 16S rDNA sequence of strain IAM 14872T in DDBJ/EMBL/GenBank is AB071665. The other accession numbers of the published 16S rDNA sequences in this study are X74066 (Acetobacter aceti NCIB 8621T), D86512 (Acidisphaera rubrifaciens JCM 10600T), D30773 (A c idiphilium c ryptum ATCC 33463T), D30774 (Ac idoc ella fac ilis ATCC 35904T), X77468 (A c idomonas methanoli c a MB 58T), AB025928 (Asaia bogoresis JCM 10569T), Z29619 (Azospirillum lipoferum NC I MB 11861T), D85828 (Crauroc oc c us roseus JCM 9933T), X80725 (Esch-eric hia c oli ATCC 11775T), X75617 (Gluconacetobac-ter liquefac iens I FO 12388T), X73820 (Gluconobacter oxydans DSM 3503T), AB056321 (Kozakia baliensis Yo-3T), Y10109 (Magnetospirillum gryphisealdense DSM 6361T), Y10110 (Magnetospirillum magneto-tacticum DSM 3856T), D85827 (Paracraurococcus ruber NS 89T), D14433 (Phaeospirillum fulvum NCIMB 11762T), D12701 (Rhodocista centenaria IAM 14193T), D86513 (Rhodopila globiformis DSM 161T), X99671 (Rhodospira trueperi ATCC 700224T), D30777 (Rho-dospirillum photometricum E11), X87278 (Rhodospiril-lum rubrum DSM 107T), D14431 (Rhodothalassium salexigens ATCC 35888T), D14432 (Rhodovibrio sali-narum NCIMB 2243T), M59072 (Rhodovibrio sodomensis DS 1T), X72908 (Roseoc oc c us thiosulfatophilus RB 3T), AJ000989 (Roseospira mediosalina BN 280T), X90760 (Skermanella parooensis ACM 2042T) and AF358664 (Thalassospira lucentensis QMT2T).Results

Bacterial strains

Among nine strains of PHA-producing bacteria iso-lated from 20 samples, strain AM 14872T showed good PHA-producing ability. Fed-batch culture studies of strain IAM 14872T showed that the yield of PHA ac-counted for 19.6% cell dry weight under nitrogen limi-tation with the concentrations of NH

4

?(sole nitrogen source) and cane molasses (sole carbon source) being 0.1% w/v and 5% w/v, respectively. The analysis of PHA by gas chromatography compared to the stan-dard chromatograms of methyl-n-butyrate, methyl-n-valerate and methyl esters of eight- to twelve-carbon atoms fatty acid indicated that the biopolymer con-sisted of four-, ?ve- and eight- to ten-carbon monomers.

Morphological characteristics

The ultra-thin section electron micrograph (Fig. 1A) showed that the cells were short straight rods occur-ring in pairs or as single cells. PHAs were accumu-lated in the cytoplasm. I ntracytoplasmic membranes (ICMs) were not observed. The scanning electron mi-crograph (Fig. 1B) showed that strain IAM 14872T pos-sesses a single polar ?agellum.

Photosynthetic properties

The 16S rDNA sequence suggested that strain IAM 14872T is phylogenetically related to the genus Rho-dospirillum. Nevertheless, this strain did not show anoxic phototrophic growth, which is a signi?cant char-acteristic of the genus Rhodospirillum. I n the spectra

2002Tistrella mobilis gen. nov.337

Fig. 1.Electron micrographs of strain IAM 14872T.

(A) Ultrathin section electron micrograph. The large granules shown by the arrow are PHAs. Bar?0.5m m. (B) Scan-

ning electron micrograph shows a single polar ?agellum. Bar?0.5m m.

338S HI, A RUNPAIROJANA, and Y OKOTA Vol.48

Acid production from ethanol was negative. Both cata-lase and oxidase activities were positive.

Quinone system, cellular fatty acids and G?C content of DNA

Strain I AM 14872T contained ubiquinone Q-10 as the predominant component of the quinone system while menaquinone or rhodoquinone was not detected. Strain AM 14872T contained a large amount of a straight-chain unsaturated fatty acid, cis-7-octade-cenoic (C

18:1

w7c) (Table 2). Additionally, 3-hydroxy and 2-hydroxy fatty acids also exist and the major hy-droxy fatty acids are 3-hydroxytetradecanoic (3-OH

C

14:0) and 2-hydroxy octadecenoic (2-OH C

18:1

), re-

spectively. The G?C content of DNA of strain I AM 14872T was 67.5mol%, which is close to the values of the published genera of the Rhodospirillales order (Table 3).

Phylogenetic analysis

The almost complete 16S rDNA sequence of strain IAM 14872T, consisting of 1,453 nucleotides, has been determined. Additionally, the 16S rDNA sequence sim-ilarity values between strain IAM 14872T and the gen-era from the order Rhodospirillales have been calcu-lated, which were from 86.0% (Crauroc oc c us roseus JCM 9933T) to 90.1% (Phaeospirillum molisc hianum ATCC 14031T) and at a level widely accepted for dif-ferentiating between genera. Consistent with the simi-larity values, the NJ tree (Fig. 2) based on 16S rDNA sequenes shows that strain AM 14872T is located within the cluster of the Rhodospirillales. The topology, however, showed that strain IAM 14872T clustered nei-ther within the family Rhodospirillaceae nor within the family Acetobacteraceae of the order Rhodospirillales, suggesting that strain IAM 14872T probably belongs to a novel family of the order Rhodospirillales.

Discussion

Strain I AM 14872T is a non-photosynthetic bac-terium lacking bacteriochlorophyll a and intracytoplas-mic membranes. I n the a-Proteobacteria, the photo-synthetic genera are phylogenetically intermingled with some non-photosynthetic genera (Woese et al., 1984). Some non-photosynthetic genera are closely related to the photosynthetic ones, and phototrophic activity does not re?ect phylogeny (Kawasaki et al., 1993). To date, there are ten photosynthetic genera validly pub-lished in the order of the Rhodospirillales: Rhodopila (I mhoff et al., 1984), Rhodocista(Kawasaki et al., 1992), Rhodospira(Pfennig et al., 1997), Phaeospiril-lum, Rhodospirillum, Rhodothalassium, Rhodovibrio and Roseospira(mhoff et al., 1998), Roseococcus (Yurkov et al., 1994), and Thalassospira luc entensis (Lopez-Lopez et al., 2002). Strain I AM 14872T differs from these photosynthetic genera by the absence of anoxic phototrophic growth. I t differs from the spiral-shaped bacteria, Azospirillum and Magnetospirillum, by the absence of nitrogen-?xation and magnetite for-mation, respectively. The genus Skermanella differs from our isolate by the formation of conglomerate-cells. The optimal pH of growth of strain IAM 14872T is very different from those of the acidophilic bacteria. It differs from the genera, Acidiphilium, Acidisphaera, Acidocella, Acidomonas, Asaia, and Kozakia by the absence of acidophily. It differs from Acetobacter, Glu-conobacter and Gluconacetobacter by the absence of acetic acid production from ethanol. The absence of bacteriochlorophyll a in strain I AM 14872T differenti-ates it from Craurococcus and Paracraurococcus.

t is concluded, therefore, that strain I AM 14872T can be separated from other described and related bacterial genera by using a combination of phenotypic characteristics. Thus, we propose the creation of a new genus, Tistrella gen. nov., containing strain I AM 14872T as Tistrella mobilis sp. nov. The new genus be-longs to the order Rhodospirillales of a-Proteobacteria.

Descriptions

Description of Tistrella gen. nov.

Tistrella(Tistr.el’la. M. L. dim. fem. ending -ella; N. L. fem. n. Tistrella arbitrary name formed from the acronym of the Thailand I nstitute of Scienti?c and Technological Research, TISTR, where the isolation of strain IAM 14872T was performed.)

Cells are rod-shaped, highly motile by means of a single polar ?agellum and Gram-negative. Strictly aer-obic chemoorganotrophic bacterium. Accumulates polyhydroxyalkanoates. Has no salt requirement. Non-photosynthetic. Lacks intracytoplasmic membrane sys-tems and bacteriochlorophyll a. Both catalase and oxi-dase activities are positive. Contains ubiquinone Q-10 as the major quinone. The major cellular fatty acid is C

18:1

w7c. Both 2-hydroxy and 3-hydroxy fatty acids are present, and the major hydroxy fatty acids are 2-OH

C

18:0

and 3-OH C

14:0

. The G?C content of DNA is

2002Tistrella mobilis gen. nov.339

340S HI, A RUNPAIROJANA, and Y OKOTA Vol.48

67.5%. The phylogenetic position is the order Rho-dospirillales of a-Proteobacteria.

The type species is Tistrella mobilis.

Description of Tistrella mobilis sp. nov.

Tistrella mobilis(mo’bi.lis. L. fem. adj. mobilis motile.)

Cells are rod-shaped, 0.7–1.0m m wide, highly motile by means of a single polar ?agellum and Gram-nega-tive. Shows binary ?ssion. Strictly aerobic chemoorgan-otrophic bacterium. Accumulates polyhydroxyalka-noates. Has no salt requirement. Growth occurs at a temperature of 20–40°C and a pH of 5.0–9.0. The opti-mal growth temperature and pH are 30°C and pH 7.4, respectively. Non-photosynthetic. Lacks intracytoplas-mic membrane systems and bacteriochlorophyll a. Uti-lizes L-arabinose, D-mannitol, N-acetylglucosamine, adipic acid and malic acid as carbon sources. Acid production was observed from L-arabinose, esculin,

maltose, melezitose, D-tagatose, L-fucose, D-arabitol, gluconate, and 5-keto-gluconate. Produces indole, re-duces nitrate to nitrite but not to nitrogen, hydrolyzes esculin, gelatin and arginine. Both catalase and oxi-dase activities are positive. Contains ubiquinone Q-10 as the major quinone. The major cellular fatty acid is C

18:1

w7c. Both 2-hydroxy and 3-hydroxy fatty acids are present, and the major hydroxy fatty acids are 2-OH

C

18:0

and 3-OH C

14:0

. The G?C content of DNA is 67.5%.

The type strain I AM 14872T(?TI STR 1108T) was isolated from wastewater in Thailand.

Acknowledgments

We would like to thank Dr. Aiko Hirata for help with the trans-mission electron micrograph.

2002Tistrella mobilis gen. nov.341

Fig. 2.Phylogenetic tree derived from 16S rDNA sequences for Tistrella mobilis and the members from the Rho-

dospirillales order with Escherichia coli as the outgroup. Bootstrap values (%) obtained with 1,000 bootstrap resam-

plings are shown on the branches and only those above 50% are shown. Bar?2 nucleotide substitutions per 100 nu-

cleotides.

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