Molecular genetic analyses of Rhodobacter azotoformans sp. nov. and related species of phototrophic bacteria

System. Appl. Microbiol. 19, 168-177 (1996) © Gustav Fischer Verlag· Stuttgart· Jena . New York

Molecular Genetic Analyses of Rhodobacter azotoformans sp. nov. and Related Species of Phototrophic Bacteria AKIRA HIRAISHI"", KEIGO

MURAMATSU'~*, and

YOKO

UEDA'~''''

Laboratory of Environmental Biotechnology, Konishi Co., Sumida-ku, Tokyo 130, Japan Received January 8, 1996

Summary Genetic relationships of a new species of denitrifying phototrophic bacteria, Rhodobacter azotoformans sp. nov., to related species were determined by studying nucleotide sequences and restriction fragment length polymorphism (RFLP) of 16S ribosomal DNA (rDNA) and genomic DNA relatedness. The rDNA fragments from these bacteria were amplified by the polymerase chain reaction (PCR) and sequenced directly by a combined method consisting of cycle sequencing and automated fluorescence detection. A phylogenetic tree constructed on the basis of the 16S rDNA sequences showed that R. azotoformans formed a cluster with the previously known members of the genus Rhodobacter with R. sphaeroides as the nearest neighbor. The level of binary sequence similarity between R. azotoformans and R. sphaeroides of 98.3%, together with their phenotypic differences, allowed the placement of the two organisms in different species. RFLP patterns of PCR-amplified 16S rDNA were used to differentiate R. azotoformans from related species of phototrophic bacteria. The PCR-RFLP analysis with HaeIII, HhaI, MspI, and SmaI proved to be useful for the classification and identification of these bacteria at the species level. Genomic DNA-DNA hybridization studies supported the results of 16S rDNA sequence comparisons.

Key words: Rhodobacter - Rhodovulum - Rhodobacter azotoformans - Phototrophic bacteria - 165 rRNA - RFLP - Phylogeny

Introduction The genus Rhodobacter is an assemblage of facultatively anaerobic photoheterotrophic bacteria that have an ovoid to rod-shaped morphology and form vesicular intracytoplasmic membranes (except Rhodobacter blasticus, which contains lamellar membranes [Kawasaki et al., 1993]) together with bacteriochlorophyll a and carotenoids of the spheroidene series. Until recently, both freshwater and marine species were included in the genus

* Present address: A. Hiraishi, Central Research Laboratories,

Ajinomoto Co., Inc., Suzuki-cho 1-1, Kawasaki-ku, Kawasaki 210, Japan. Tel: +81-44-244-7181. Fax: +8144-246-2867 ** Present address: Keigo Muramatsu, Department of Biology, Tokyo Metropolitan University, Minamiohsawa, Hachioji, Tokyo 192-03, Japan *** Present address: Yoko Veda, Department of Pharmacology, University of Occupational and Environmental Health, School of Medicine, Iseigaoka 1-1, Yahatanishi-ku, Kitakyushu 807, Japan

Rhodobacter (Imhoff et al., 1984; Imhoff, 1989), but all marine species have been separated from this genus and transferred into the new genus Rhodovulum (Hiraishi and Veda, 1994). Members of the genus Rhodobacter are versatile in respect to their energy metabolism. They can acquire energy for growth not only by anaerobic photosynthesis but also by aerobic respiration in darkness. In addition, some Rhodobacter strains are capable of growing by anaerobic-dark respiration with nitrate, nitrous oxide, dimethylsulfoxide, or trimethylamine N-oxide as a terminal electron acceptor (Ferguson et al., 1987). Rhodobacter strains which are capable of complete denitrification have been isolated from biological wastewater treatment systems and some other environments (Satoh et al., 1976; Pellerin and Gest, 1983; Hiraishi et al., 1991; Shen and Hirayama, 1991), and most of these strains isolated were assigned to Rhodobacter sphaeroides. These photodenitrifiers may play an important role in the nitrogen cycle in these environments.

Molecular Analyses of Rhodobacter azotoformans sp. nov. In a previous study, we reported the characterization of new denitrifying strains of purple nonsulfur bacteria isolated from photosynthetic sludge for wastewater treatment (Hiraishi et aI., 1995b). The new denitrifying strains resembled R. sphaeroides in a number of characteristics including morphology, physiology, and denitrifying properties. On the basis of the phenotypic information and partial 16S ribosomal DNA (rDNA) sequence data, however, we concluded that these strains should be classified into a new species of the genus Rhodobacter. In this paper, we report molecular genetic analyses of the new Rhodobacter strains, for which we propose the name Rhodobacter azoto{ormans sp. nov., and of related species of the phototrophic bacteria. The molecular approaches to this study involved direct sequencing of polymerase chain reaction (PCR)-amplified 16S rDNA and genomic DNA-DNA hybridization, both of which are now widely used to provide a general framework for the classification of bacterial species and genera. Since restriction fragment length polymorphism (RFLP) analysis of PCR-amplified 16S rDNA have recently been used as a simple and rapid technique for identification and classification of various taxonomic groups of microorganisms (Gurtler et aI., 1991; Vaneechoutte et aI., 1992; Schneider et aI., 1993; Ralph et aI., 1993; Laguerre et aI., 1994; Ponsonnet and Nesme, 1994; Hiraishi et aI., 1995a, Nesme et aI., 1995), we also used this technique for the classification of the phototrophic bacteria.

Materials and Methods Bacterial strains and cultivation. The investigated strains of Rhodobacter azotoformans were KA25 T (superscript T = type strain), KA30, SA16, and SA29, all of which were isolated from photosynthetic sludge for wastewater treatment (Hiraishi et aI., 1995b). The reference organisms used in this study were: , Rhodobacter capsulatus strains DSM 1710T and C5; Rhodobacter sphaeroides strains DSM 158 T , KA38, and NR3; Rhodobacter blasticus ATCC 33485 T ; Rhodobacter veldkampii ATCC 35601 T ; Rhodovulum sulfidophilum DSM 1374T ; Rhodovulum adriaticum DSM 2781 T ; Rhodovulum euryhalinum DSM 4868 T . The strains with ATCC and DSM numbers were obtained, respectively, from the American Type Culture Collection (Rockville, USA) and from the DSM-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). The sources of all other investigated strains have been described previously (Hiraishi and Kitamura, 1984; Hiraishi and Ueda, 1994). MYS medium (Hiraishi and Kitamura, 1984) and modifications of this medium were used for cultivation of the organisms. The medium was modified by adding 2 mM cysteine (filter sterilized) for growth of Rhodobacter veldkampii. For Rhodovulum species, 0.5 mM sodium thiosulfate, 0.5 mM sodium sulfide, and 1 to 3% NaCl were supplemented to the medium. Cells were grown anaerobically at 30°C in screw-capped test tubes or bottles filled with medium under incandescent illumination (ca. 5,000 Ix). Cells were harvested by centrifugation from cultures at the mid-exponential phase of growth, washed with sterilized 1% saline, resuspended either in pure water (for PCR experiments) or in EDTA-saline (for DNA hybridization studies), and stored at -20°C until they were used. PCR amplification of 16S rDNA. From the stock cell suspensions, crude Iysates were prepared by protease digestion, heat

169

treatment, and centrifugation. 16S rDNA fragments that corresponded to positions 8 to 1510 (all positions for the 16S rRNA molecule refer to the Escherichia coli numbering system [Brosius et aI., 1978]) were amplified by PCR directly from the crude lysate. Detailed information on the PCR procedures used has been given elsewhere (Hiraishi, 1992; Hiraishi et aI., 1994). PCR products were extracted with chloroform-isoamylalcohol (24: 1 [vol/vol]), precipitated with ammonium acetate and ethanol, and then purified either by agarose gel electrophoresis with resin binding (Hiraishi, 1992) or by the polyethylene glycol precipitation method (Hiraishi et aI., 1995a). Sequencing and phylogenetic analysis. Gel-purified 16S rDNA was sequenced directly by the fluorescent cycle sequencing method, and the reactions were analyzed with a Pharmacia laser fluorescent DNA sequencer as previously described (Hiraishi et aI., 1994). Sequence data were compiled and binary sequence similarities were calculated with the GENETYX-MAC computer program (Software Development Co., Tokyo, japan). Nucleotide substitution rates (K nuc values) (Kimura, 1980) were calculated and a distance matrix tree was constructed by the neighbor-joining method (Saitou and Nei, 1987), using the program CLUSTAL V (Higgins et aI., 1992). Alignment positions with gaps and unidentified bases were not taken into consideration for the calculations. The topology of the phylogenetic tree was evaluated by bootstrap analysis (Felsenstein, 1985) with 1,000 bootstrapped trials. RFLP analysis. PCR products purified by the polyethylene glycol method were cut with restriction endonucleases, HaeIII, HhaI, MspI, and Sma!. Digested fragments were separated by using a Mupid mini-gel electrophoresis apparatus (Advantec Co., Tokyo, japan) with 2% MetaPhor agarose gels (FMC BioProducts, Rockland, ME, USA) and detected by staining with ethidium bromide as described previously (Hiraishi et aI., 1995a). RFLP patterns were photographed with Fuji instant films and analyzed with an image analyzer, Atto Densitograph (Atto Co., Tokyo, japan). DNA base composition and DNA-DNA hybridization. Genomic DNA was extracted and purified by the method of Marmur (1961). DNA base composition (mol% guanine [G] + cytosine [C]) was determined by high-performance liquid chromatography of nuclease Pl hydrolysates of genomic DNA with external standards obtained from Yamasa Shoyu Co. (Choshi, japan) (Katayama et aI., 1984; Hiraishi et aI., 1991). DNA-DNA paring studies were performed by the quantitative dot blot hybridization method with photobiotin labeling as described previously (Ezaki et aI., 1988; Hiraishi et aI., 1991), but the assay was modified by elevating hybridization temperature to 45°C in the presence of 45% formamide. Hybridized DNA was detected calorimetrically and measured with a Shimadzu model CS-9000 two-dimensional densitometer. Nucleotide sequence accession numbers. The 16S rDNA sequences determined in the present study have been depositied in the DDBj, EMBL, and GenBank nucleotide sequence databases under accession numbers D70846 and D70847.

Results 16S rDNA sequence comparisons and phylogenetic analysis

Almost complete sequences of the 16S rRNA gene from R. azoto{ormans strains KA25 T and SA16 were amplified in vitro and sequenced directly by a combined method consisting of cycle sequencing and automated fluorescence detection. The sequences of these strains were identical.

170

A. Hiraishi, K. Muramatsu, and Y. Veda

Figure 1 shows the primary structure of the R. azotoformans 165 rDNA with restriction sites with the four endonucleases, HaeIII, HhaI, MspI, and SmaI, which were used for RFLP analysis. The 165 rRNA molecule of R. 10

20

30

40

azotoformans deduced from the 165 rDNA sequence contained the definitive signatures of the alpha subclass of the Proteobacteria (Woese, 1987) and exhibited nucleotide deletions of 18, 11, 25, and 21 bases in loop and stem 50

60

70

80

90

aqaqtttqatcctqqctcaqAATGAACGCTGGCGGCA~TAACACATGCAAGTCGAGCGAAGTCTTCGGACTTAGCGGCGGACGGGTG

/HaeIIl 100 110 120 130 140 150 160 170 180 AGTAACGCGTGGGAACATGCCCAAAGGTACGGAATAGCCCCGGGAAACTGGGAGTAATACCGTATGTGCCCTTCGGGGGAAAGATTTATC Mspl//Smal 190 200 210 220 230 240 250 260 270 GCCTTTAGAT~CGCGTTGGATTAGGTAGTTGGTGGGGTAA~TACCAAGCCGACGATCCATAGCTGGTTTGAGAGGATGATCA

280

/Haelll 290

300

310

/Haelll 320

330

340

350

360

GCCACACTGGGACTGAGACAC~CAGACTCCTACGGGAGGCAGCAGTGGGGAATCTTAGACAAT~GCCTGATCTAGCCATG

370

380

/HaeIIl 390

/Hhal 400

410

420

430

440

450

CCGCGTGATCGATG~TTAGGGTTGTAAAGATCTTTCAGGTGGGAAGATAATGACGGTACCACCAGAAGAAGCC~TAACTCC

460

/Haelll 470

480

490

500

510

520

/Mspl 530

540

GTGCCAGCAGCCGCGGTAATACGGAGGGGGCTAGCGTTATTCGGAATTACTGGGCGT~CGTAGGCGGACTGGAAAGTCAGGGG

/Hhal 550

560

570

580

590

600

610

620

630

TGAAATCCCGGGGCTCAACC~CTGCCTTTGAAACTCCCAGTCTTGAGGTCGAGAGAGGTGAGTGGAATTCCGAGTGTAGAGGTGA

Mspl//Smal /Mspl 640 650 660 670 680 690 700 710 720 AATTCGTAGATATTCGGAGGAACACCAGTGGCGAAGGCGGCTCACTGGCTCGATACTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACA 730 740 750 760 770 780 790 800 810 GGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGCCAGTCGTCGGGCAGCATGCTGTTCGGTGACACACCTAACGGATTAAGC 820

830

840

850

860

870

880

890

900

ATTCCGCCTGGGGAGTAC~GCAAGGTTAAAACTCAAAGGAATTGAC~CGCACAAGCGGTGGAGCATGTGGTTTAATTCGAA

910

/Haelll 920 930

940

950

/Haelll 960

970

980

990

GCAAC~GAACCTTACCAACCCTTGACATGGCGATCGCGGTTCCAGAGATGGTTCCTTCAGTTCGGCTGGATCGCACACAGGTGCTG

/Hhal 1000

1010

1020

1030

1040

1050

1060

1070

1080

CATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTCGGTTAAGT~AAC~CCCACGTCCTTAGTTGCCAGCATTCAGTTGGG

1090

1100

1110

1120

/Mspl 1130

/ Hhal 1140

1150

1160

1170

CACTCTGGGGAAACT~TGATAAG~GGAAGGTGTGGATGACGTCAAGTCCTCA~CTTACGGGTTGGGCTACACACGTGC

/Mspl /Mspl /Haelll 1180 1190 1200 1210 1220 1230 1240 1250 1260 TACAATGGCAGTGACAATGGGTTAATTCCAAAAAGCTGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTTGGAATCGCTA 1270 1280 1290 1300 1310 1320 1330 1340 1350 GTAATCGCGTAACAGCATGACGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAATTGGTTCTACCCGAAGG Mspl/ /HaelII 1360

1370

1380

1390

1400

1410

1420

CGG~CAACCTCGCAAGAGGAGGCAGCCGACCACGGTAGGATCAGTGACTGGGGTGaaqtcqtaacaaqqtaace

/Hhal Fig. 1. Primary structure of the 16S rDNA of Rhodobacter azotoformans with restriction sites for HaeIII, HhaI, MspI, and Sma!. Sequences indicated by lower case letters correspond to those of the peR primers used.

I

Rba. azotoformans Rba. sphaeroides Rba. capsulatus Rba. blasticus Rba. veldkampii P. denitrificans P. versutus P. thiocyanatus P. aminophilus P. aminovorans P. alcaliphilus P. kocurii Rdv. sulfidophilum Rdv. strictum Rdv. adriaticum Rdv. euryhalinum Rsb. denitrificans Rsp. rubrum

0.0471 0.0510 0.0495 0.0682 0.0740 0.0681 0.0665 0.0716 0.0739 0.0632 0.0730 0.0762 0.0656 0.0696 0.0924 0.1468

98.3

-

0.0166 0.0567 0.0542 0.0518 0.0665 0.0706 0.0738 0.0689 0.0748 0.0771 0.0705 0.0746 0.0762 0.0704 0.0648 0.0923 0.1462

2

1

0.0510 0.0527 0.0664 0.0640 0.0714 0.0681 0.0631 0.0838 0.0754 0.0689 0.0813 0.083] 0.0762 0.099] 0.1496

94.5 95.4

3

-

0.0559 0.0762 0.0737 0.0837 0.0804 0.0795 0.0888 0.0770 0.0745 0.0680 0.0838 0.0713 0.]0] 7 0.1483

94.5 94.7 94.9

4

-

0.0632 0.0640 0.0623 0.0494 0.0623 0.0697 0.0688 0.0631 0.0663 0.0714 0.0647 0.0764 0.1466

94.6 95.0 94.7 94.6

5

-

0.0083 0.0329 0.0369 0.0415 0.0422 0.0439 0.0797 0.0958 0.0831 0.0822 0.0856 0.1447

93.4 93.1 93.3 92.6 93.7

6

-

0.0360 0.0401 0.0376 0.0470 0.0463 0.0857 0.1002 0.0908 0.0881 0.0890 0.1475

93.2 93.0 93.9 93.1 93.8 99.]

7

-

0.0283 0.0344 0.0328 0.0368 0.0889 0.1043 0.0855 0.0965 0.0822 0.1472

92.4 92.9 92.6 91.5 93.6 96.7 96.4

8

0.0377 0.0344 0.0479 0.0831 0.0975 0.0796 0.0888 0.0772 0.1334

93.0 93.1 93.4 92.1 94.9 96.2 95.9 97.0

9

0.0312 0.0535 0.0813 0.0923 0.0925 0.0889 0.0805 0.1499

-

92.3 92.6 94.2 92.1 93.6 95.7 96.5 96.5 96.2

10

0.0407 0.0748 0.0907 0.0797 0.0967 0.0838 0.1454

-

92.3 92.6 92.1 91.3 92.8 95.7 95.4 96.5 96.4 96.8

11

0.0730 0.0889 0.0730 0.09]5 0.0907 0.]372

-

93.1 93.7 92.6 92.6 93.2 95.5 95.4 96.1 94.8 94.4 95.7

12

0.0344 0.0447 0.0542 0.1035 0.1397

-

92.8 93.1 93.2 92.6 93.8 92.1 91.6 91.5 91.8 92.4 93.1 92.6

13

Level of sequence similarity (%) or evolutionary disrance (Knuc )

-

92.9 93.6 91.9 91.7 92.9 91.9 91.5 91.5 91.9 91.5 92.6 92.7 95.5 94.2

15

94.0 93.6 93.0 93.6 94.2 92.4 92.0 90.8 92.2 91.5 90.9 91.3 94.7 96.8 93.7

16

91.1 91.3 90.1 90.3 92.5 91.3 91.1 92.0 92.4 92.1 91.8 91.8 90.2. 89.7 89.7 90.4

17

0.0649 0.0321 0.0673 0.1191 0.]052 0.1103 0.]471 0.1413 0.1530 0.1590

93.3 93.5 92.6 94.1 94.3 90.9 90.8 90.2 91.2 90.9 91.2 91.5 96.3

14

-

86.5 86.6 86.9 86.7 86.7 86.8 86.6 86.1 87.3 86.0 86.4 87.1 87.6 87.7 87.5 87.4 85.6

18

The values on the upper right are levels of sequence similarity, and the values on the lower left are corrected evolutionary distances. Organisms and sequences used for phylogenetic analysis: ], Rhodobacter azotoformans (070846); 2, Rhodobacter sphaeroides (016425); 3, Rhodobacter capsulatus (0] 6428); 4, Rhodobacter blasticus (016429); 5, Rhodobacter veldkampii (016421); 6, Paracoccus denitrificans (X69159); 7, Paracoccus versutus (032243); 8, Paracoccus thiocyanatus (032242); 9, Paracoccus aminophilus (032239); ]0, Paracoccus aminovorans (D32240); 11, Paracoccus alcaliphilus (032238); 12, Paracoccus kocurii (032241); 13, Rhodovulum sulfidophilum (016423); ]4, Rhodovulum strictum (016419); 15, Rhodovulvum adriaticum (0]6418); 16, Rhodovulum euryhalinum (016426); 17, Roseobacter denitrificans (M59063); and 18, Rhodospirillum rubrum (030778)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Organism

Table 1. Levels of sequence similarities and evolutionary distances for 165 rDNAs of Rhodobacter azotoformans and related species of phototrophic and nonphototrophic bacterial

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structures at positions 74 to 94, 201 to 215, 452 to 479, and 1258 to 1278, respectively. The 165 rRNA of R. azotoformans also contained a larger stem-loop around position 1,450 than the E. coli 165 rRNA. These features .are characteristic of species belonging to group 3 of the alpha subclass of the Proteobacteria (Drynden and Ka·plan, 1990; Hiraishi and Veda, 1994; Katayama et aI., 1995). The sequence of R. azotoformans was aligned and compared with a data set which consisted of 18 sequences from phototrophic and nonphototrophic relatives belonging to the alpha-3 subgroup of the Proteobacteria and from the alpha-1 phototrophic bacterium Rhodospirillum rubrum as an outgroup reference. We calculated evolutionary distance values on the basis of the 1,340 positions that could be aligned in the entire sequence set (Table 1), and constructed a neighbor-joining phylogenetic tree using the distance matrix data (Fig. 2). The tree showed that R. azotoformans formed a cluster with other members of the genus Rhodobacter, with R. sphaeroides as the nearest relative, and that the cluster of the genus Rhodobacter was separated clearly from the related genera Paracoccus, Roseobacter, and Rhodovulum.

The level of binary sequence similarity between R. azotoformans and the type strain of R. sphaeroides was 98.3% (distance = 0.0166). This level seems to be low enough to separate the former organism from the latter at the species level. A similar level of 165 rDNA sequence divergence (98.2%) was found between R. azotoformans and a representative of denitrifying R. sphaeroides, strain ILl06 (DDBJ accession number D16424). RFLP analysis

Based on the 165 rDNA data, R. azotoformans had a relatively close relationship to R. sphaeroides but was phylogenetically distinguishable from the latter species by a nucleotide substitution rate of 1.66% (per 100 nucleotides) and from other established species of the phototrophic and nonphototrophic relatives belong to group 3 of the alpha subclass of the Proteobacteria. We further studied the applicability of RFLP analysis of the PCRamplified 165 rDNA as a simple technique for the differentiation of R. azotoformans from other members of the genera Rhodobacter and Rhodovulum (Hiraishi and Veda, 1994; 1995).

Rhodovulum strictum (Dl6419)

0.01

Rhodovulum euryhalinum (D16426)

798 995

Rhodovulum sulfidophilum (D 16423) Rhodovulum adriaticum (D16418) Rhodobacter azotoformans (D70846)

1000

Rhodobacter sphaeroides (016425)

866 628

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Rhodobacter capsulatus (D 16428)

-

Rhodobacter blasticus (D16429) Rhodobacter veldkampii (016421) Paracoccus aminovorans (D3224O) Paracoccus alcaliphilus (D32241)

567 382 1000

Paracoccus denitrificans (X69159) Paracoccus versutus (D32243)

989

-

25

210

~234

Paracoccus thiocyanatus (D32242) Paracoccus kocurii (D32241) Paracoccus aminophilus (032239) Roseobacter denitrificans (M59063)

0.10217---------------

Rhodospirillum rubrum (D30778)

Fig. 2. Distance matrix tree showing phylogenetic relationships between Rhodobacter azotoformans and phototrophic and nonphototrophic relatives belonging to group 3 of the alpha subclass of the Proteobacteria. The sequence of Rhodospirillum rubrum was used to root the tree. Bootstrap confidence values obtained with 1,000 bootstrap trials are given at branching points. Scale bar represents 1% nucleotide substitution per 100 nucleotides.

2

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Rba. capsulatus C5

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Rba. capsulatus DSM 1710T

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Rba. azotoformans (4 strains)

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Rba. veldkampii

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Rdv. sulfidophilum

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Rba. sphaeroides (3 strains)

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(-) (-) (-) (-)

(+) (+) (+) (+) (+) (+)

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(-)

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(-)

(-) (-)

(+d)

(+)

(-)

(+)

(-)

Rdv. strictum

(-)

(-) (-) (-)

(+) (+d)

(-) (-)

(+)

(-)

Rba. blasticus

Symbols: +, fragment present; +d, duplicate or more fragments present; -, fragment absent. Test strains are given in the same order as shown in Fig. 3. For Rba. blasticus and Rdv. strictum, eletrophoretic analysis of fragments was not performed and only computer-predicted data (parenthesized) are shown Fragment sizes determined by image analysis of RFLP patterns on agarose electrophoresis. Numbers in parentheses indicates computer-predicted fragment sizes on the basis of the available 16S rONA sequences

Smal (CCCGGG) 1250 (1297) 1150 (1167-1169) 740 (749-751) (677) 543-550 (549) (529) 415 (418) 130 (131, 128-130)

Mspl (CCGG) 598-591 (594-596) (568) 508 (508) 484 (485) 470 (472-474) 442-435 (439) 401 (403) 310-304 (308-309) 269-261 (263-264) 188-186 (189) 132-128 (131, 129-130) 108 (109)

Hhal (GCGC) (582) 520-511 (512-513) 397-390 (393-395) 340-338 (341) 313-309 (311-312) 171-169 (172) 138-135 (139-137)

HaeIlI (GGCC) 488-484 (486) 455-447 (452) 297 (300) 283-276 (278-281) (199) 158-151 (158-157, 154) 129 (130) 125-123 (125-127) 110-109 (110)

Restriction enzyme used and size (bp) of fragments determined 2

Table 2. Fragment size of the endonuclease-digested 165 rONA from Rhodobacter azotoformans and related species of phototrophic bacterial

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As shown in Fig.3 and Table 2, digestion of the PCRamplified 165 rDNA with the four enzymes used gave RFLP patterns which were the same as predicted on the basis of the available 165 rDNA sequences (for R. azotoformans, d. Fig.1 and Table 2). Combination of

1 2 3 4 5 6 7 8 9 10 11121314 15

bp

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c

o 500

Fig. 3. RFLP analysis of 16S rDNAs of Rhodobacter azotoformans and related phototrophic bacteria with different endonucleases. A, HaeIII; B, HhaI; C; MspI; D, Sma!. Lane: 1 and 15, 100 ~ ladder marker (Pharmacia); 2, Rba. azotoformans KA25 ; 3, Rba. azotoformans KA30; 4, Rba. azotoformans SA16; 5, Rba. azotoformans SA29; 6, Rba. capsu/atus DSM 1710T ; 7, Rba. capsu/atus C5; 8, Rba. ve/dkampii ATCC 35605 T ; 9, Rba. sphaeroides DSM 158 T ; 10, Rba. sphaeroides KA38; 11, Rba. sphaeroides NR3; 12, Rdv. sulfidophi/um DSM 1374 T j 13, Rdv. adriaticum DSM 2781 T ; 14, Rdv. euryhalinum T

DSM 4686 . Digested fragments were separated by agarose gel electrophoresis (2% MetaPhor agarose), stained with ethidium bromide and photographed.

these RFLP profiles was useful for the identification of the investigated strains at the species level. In particular, the strains could be separated at the species level by the MspIinvolved RFLP profiles alone. The four strains of R. azotoformans were identical in RFLP profiles, suggesting that all these strains have the same 165 rRNA structure. Differences between R. azotoformans and its phylogenetic neighbor R. sphaeroides were noted in the RFLP patterns with MspI and Sma!.

Genomic DNA relatedness As described above, nucleotide sequence comparison and RFLP analysis of PCR-amplified 165 rDNA demonstrated that R. azotoformans was phylogenetically distinguishable from all of the previously known species of the genus Rhodobacter and their phototrophic and nonphototrophic relatives. To obtain more definitive information on the taxonomic position of R. azotoformans as a distinct species, we studied the DNA base composition of and DNA-DNA relatedness between R. azotoformans and related species of the phototrophic bacteria (Table 3). The investigated strains of R. azotoformans had a narrow range of the G + C content of the genomic DNA (69.5 to 70.2 mol%) which seemed to be the highest among species of group 3 of the alpha subclass of the Proteobacteria. Genomic DNA pairing studies showed that all tested strains of R. azotoformans were closely related to strain KA25 T at a hybridization level of more than 89%. Among the Rhodobacter strains tested, the R. sphaeroides strains had relatively high levels of hybridization to R. azotoformans, but these levels were less than 50%. All other investigated species of the genus Rhodobacter showed much lower relationships to R. azotoformans at a hybridization level of less than 20%.

Discussion Molecular phylogenetic studies on the basis of 165 rRNA sequence information have in recent years provided a basis for the improvement of the taxonomy of the phototrophic bacteria and their nonphototrophic relatives, including members of the genera belonging to group 3 of the alpha subclass of the Proteobacteria (Kawasaki et al., 1993; Ludwig et al., 1993; Hiraishi and Veda, 1994, 1995; Katayama et al., 1995). The results of these studies have demonstrated that a combined use of 165 rRNA sequence data with phenotypic criteria is of much help for the delineation of a genus and a species of the phototrophic bacteria as well as of other prokaryotes. In the present study, we determined nearly complete sequences of the 165 rRNA gene of the new photodenitrifier R. azotoformans, and compared the 165 rDNA sequences, RFLP patterns, and DNA-DNA similarities of this organism with those of related species of the phototrophic bacteria. The results of our molecular approaches

showed that R. azotoformans formed a lineage with members of the genus Rhodobacter with R. sphaeroides as the nearest phylogenetic relative. Among members of the

Molecular Analyses of Rhodobacter azotoformans sp. nov. Table 3. Genomic DNA relatedness between Rhodobacter azotoformans and orher species of the genus Rhodobacter

G+C content of DNA (mol%)

Organism

R. azotoformans KA25 T R. azotoformans KA30 R. azotoformans SA16 R. azotoformans SA29 R.sphaeroides DSM 158 T R.sphaeroides NR3 R.sphaeroides KA38 R. capsulatus DSM 1710T R. blasticus ATCC 33485 T R.veldkampii ATCC 35601 T 1

2

70.0 69.9 69.5 70.2 69.2 1 69.5 69.3 66.0 1 65.6 2 66.5 2

175

% Hybridization with labeled DNA from: KA25 T

DSM 158 T

100 100 89 90 41 50 49 12 16

43 44 40 42 100 69 85

DSM 1710T 19 12 19 14

100

9

Cited from Katayama et al. (1955) Cited from Hiraishi and Ueda (1994)

genus Rhodobacter, denitrifying strains have been found mainly in R. sphaeroides, as represented by strain IL 106 (Satoh et aI., 1976). In this respect, there are some similarities between R. azotoformans and denitrifying R. sphaeroides. However, the sequence comparisons and RFLP analyses of 165 rDNA reported in this study have demonstrated that R. azotoformans is phylogenetically distinguishable from R. sphaeroides and all other species of the genus Rhodobacter. The results of DNA-DNA hybridization studies were consistent with those of 165 rDNA sequence comparisons. The genomic DNA relatedness between R. azotoformans and the phylogenetic neighbor R. sphaeroides were 40 to 50%, and these hybridization levels are low enough to justify separation of the former as a distinct genospecies from the latter following the genetic criterion for delineation of bacterial species (Wayne et aI., 1987). Previous studies showed that denitrifying and nondenitrifying strains of R. sphaeroides are genetically coherent at the species level (De Bont et aI., 1981; Hiraishi and Ueda, 1994). 5ince 165 rDNA sequence comparisons revealed that denitrifying and nondenitrifying strains of R. sphaeroides showed the same evolutionary distance to R. azotoformans, we did not study genomic DNA relatedness between R. azotoformans and denitrifying R. sphaeroides itself. However, the two organisms represent different species on the basis of genetic and phenotypic data. Previously, we found that there were major phenotypic differences between R. azotoformans and R. sphaeroides in photoassimilation of some organic compounds including glycolate, tartrate, and dulcitol and anaerobic growth with dimethylsulfoxide and trimethylamine N-oxide as a terminal electron acceptor. Our concurrent studies on the phenotypic characteristics of R. azotoformans confirmed and expanded the previous results, as mentioned below in the species description (also see Table 4). R. azotoformans can be differentiated from all other established species of the genus Rhodobacter by some basic phenotypic characteristics (Table 4). Thus, the results of the phenotypic and genetic studies reported here and elsewhere (Hiraishi et aI.,

1995b) justify our proposal for the creation of R.

azotoformans as a new species of the genus Rhodobacter.

RFLP analysis of PCR-amplified 165 rDNA has begun to be recognized as a rapid and simple technique for the classification and identification of microorganisms. In the present study, we applied this technique to the classification of the phototrophic proteobacteria belonging to group 3 of the alpha subclass (i.e., members of the genera Rhodobacter and Rhodovulum) and found that the RFLP patterns provide information of value in the classification of these bacteria at the species level. Members of the two photosynthetic genera are classified primarily on the basis of their requirement to NaCl for optimum growth, but are morphologically and physiologically similar to each other. Only on the basis of phenotypic information, it is not so easy to identify these bacteria rapidly and accurately. To ensure reliable identification of the phototrophic proteobacteria of the alpha-3 group at the species level, the PCR-RFLP analysis is an appropriate taxonomic technique compared to conventional phenotypic methods.

Description of Rhodobacter azotoformans sp. nov. Rhodobacter azotoformans (a.zo.to.for'mans. French n. azote nitrogen; L. part. adj. formans forming; azotoformans nitrogen forming). The characteristics described below are based on the information from Hiraishi et al.

(1995b) and this study. Cells are ovoid to rod-shaped, 0.6 to 1.0 !tm wide and 0.9 to 1.5 !tm long. Pleomorphic and swollen cells occur when growing in the presence of peptone or yeast extract at a concentration of 0.1 % or more. Motile by means of single polar flagella. Cells divide by binary fission. Neither zigzag arrangement of cells in chains nor rosette formation is found. Gram negative. Facultatively anaerobic phototrophs which can grow both under anaerobic conditions in the light and under aerobic conditions in the dark at full atmospheric oxygen tension. Phototrophically grown cells form vesicular intracytoplasmic membranes together with bacteriochlorophyll a (esterified with phytol) and carotenoids of the spheroidene

176

A. Hiraishi, K. Muramatsu, and Y. Ueda

Table 4. Differential characteristics of Rhodobacter azotoformans sp.nov. and related species of phototrophic bacterial Characteristic

Rhodobacter azotoformans

Rhodobacter blasticus

Rhodobacter capsulatus

Rhodobacter sphaeroides

Rhodobacter veldkampii

Cell diameter (l-tm) Motility Intracytoplasmic membrane Slime production Growth in the presence of 3% NaCI Sulfate assimilated Oxidation products of sulfide

0.6-1.0 + vesicle + + + SO

0.6-1.0

0.5-0.8 + vesicle +/-

0.6-1.2

+ SO

0.7-1.0 + vesicle + + + SO

+ + t (b, n)

-/+ + + b, n, t

Anaerobic growth with: Nitrate Dimethylsulfoxide Trimethylamine N-oxide Vitamins required Utilization of: Dulcitol Mannitol Tartrate Thiosulfate Mol% G+C of DNA 1

lamellae +

+ b, n, t

b, n, t, Bl2

+ +

+

69.5-70.2

66.3-66.6

+ + 65.3-66.8

68.4-69.9

vesicle

sulfate

b, p-ABA, t

+ 64.4-67.5

Symbols and abbreviations; +, positive; -, negative; +/- and -1+, variable reactions (the first sign indicates the most frequent result); So, elemental sulfur; b, biotin; n, niacin; p-ABA; p-aminobenzoate; t, thiamine Bl2 , vitamin Bl2 ; (b, t), biotin and thiamine required by a few strains only. Information from Eckersley and Dow (1980); Schmidt and Bowien (1983); Hansen and Imhoff (1985); Imhoff (1989); Hiraishi et al. (1995b)

series. The color of phototrophic cultures is yellow-green to yellow-brown, while aerobic cultures are pink- to red. Cell extracts of phototrophically grown cells have absorption maxima at 376, 449, 476, 510, 589, 800 and 850 nm. Mesophilic and neutrophilic. Optimum growth occurs at 30 to 35°C and at pH 7.0 to 7.5. Sodium ion is not required for optimum growth, but growth is possible up to 5% NaC!. Anaerobic growth in darkness occurs with nitrate (denitrification positve) but not with dimethylsulfoxide or trimethylamine N-oxide as a terminal electron acceptor. Biotin, niacin, and thiamine are required as growth factors. Slime is produced under phototrophic growth conditions. Photoorganotrophy with various organic compounds is the preferred mode of growth. Good carbon sources are acetate, pyruvate, lactate, succinate, fumarate, malate, D-xylose, D-fructose, D-glucose, D-mannose, Dmannitol, L-alanine, L-asparagine, L-glutamate, peptone, Casamino Acids, and yeast extract. Growth also occurs with formate, propionate, butyrate, malonate, D-dulcitol, D-sorbitol, glycerol, and L-Ieucine. Not utilized are caprylate, tartrate, glycolate, benzoate, and methanol. Low concentrations of sulfide (less than 0.5 mM) but not. thiosulfate is utilized under phototrophic conditions. Elemental sulfur is the end product of sulfide oxidation. Ammonium salts and glutamate are used as nitrogen source. Nitrogen fixation is positive and nitrogenase-dependent photoevolution of hydrogen gas is found under ammonium-limited conditions. Sulfate is assimilated. Ubiquinone-lO is the major quinone. The major fatty acid is C 18 : 1 . The G + C content of genomic DNA ranges from 69.5 to 70.2 mol% (measured by HPLC). Source: photo-

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A. Hiraishi, Central Research Laboratories, Ajinomoto Co., Inc., Suzuki-cho 1-1, Kawasaki-ku, Kawasaki 210, Japan. Tel: +81-44244-7181. Fax: +81-44-246-2867