J. Gen. Appl. Microbiol., 48, 309–319 (2002) Full Paper Proposal of Pseudorhodobacter ferrugineus gen. nov., comb. nov., for a non-photosynthetic marine bacterium, Agrobacterium ferrugineum, related to the genus Rhodobacter Yoshihito Uchino,* Tohru Hamada,1 and Akira Yokota Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113–0032, Japan 1 Kamaishi Laboratories, Marine Biotechnology Institute, Kamaishi 026–0001, Japan (Received August 2, 2001; Accepted October 30, 2002) The marine gram-negative non-photosynthetic bacterium, Agrobacterium ferrugineum IAM 12616T forms one cluster with the species of the photosynthetic genus Rhodobacter in phyloge- netic trees based on molecules of 16S rRNA, 23S rRNA and DNA gyrases. Agrobacterium ferru- gineum and Rhodobacter species are similar in that growth occurs without NaCl in the culture medium (optimal NaCl concentration for growth of P. ferrugineus is 1%) and their major hydroxy fatty acid compositions are 3-hydroxy decanoic acids (3-OH 10:0) and 3-hydroxy tetradecanoic acids (3-OH 14:1). However, A. ferrugineum differs from Rhodobacter species in G؉C content (58 mol% in A. ferrugineum versus 64–73 mol% in Rhodobacter species), in having an insertion in its 16S rRNA gene sequence, and in lacking photosynthetic abilities, bacteriochlorophyll a and intracytoplasmic membrane systems. Furthermore, experiments using PCR and Southern hy- bridization show that A. ferrugineum does not have puhA gene and puf genes localized near the opposite ends of the photosynthesis gene cluster of Rhodobacter capsulatus. It suggests that A. ferrugineum may not have any genes for photosynthesis. We propose the transfer of A. ferru- gineum IAM 12616T to the genus Pseudorhodobacter gen. nov. as Pseudorhodobacter ferru- gineus comb. nov. Although Pseudorhodobacter ferrugineus disturbs the phylogenetic mono- phyly of the genus Rhodobacter, this taxonomic proposal seems adequate until it has been clari- fied whether P. ferrugineus possesses an incomplete photosynthetic apparatus. Key Words——Agrobacterium ferrugineum; bacterial taxonomy; DNA gyrase sequence; Pseudorhodobacter ferrugineus gen. nov., comb. nov.; Rhodobacter; 16S rRNA sequence; 23S rRNA sequence Introduction photosynthetic bacteria, and that many photosynthetic genera were heterogeneous (Stackebrandt et al., In the past phylogenetic analyses based on various 1996). To date, the polyphyletic photosynthetic genera molecules indicated that the photosynthetic bacteria of have been divided and reorganized into several gen- the class Proteobacteria were intermingled with non- era, and consequently, almost all the present pho- totrophic genera are monophyletic. * Address reprint requests to: Dr. Yoshihito Uchino, NITE Bio- Rhodobacter is one of the photosynthetic genera logical Resource Center, National Institute of Technology and which was newly proposed by Imhoff et al. (1984), and Evaluation, 2–5–8 Kazusakamatari, Kisarazu, Chiba 292–0812, later reorganized by Hiraishi and Ueda (1994). Hiraishi Japan. E-mail: [email protected] and Ueda (1994) divided the genus Rhodobacter; the 310 UCHINO, HAMADA, and YOKOTA Vol. 48 species whose habitats were seawater were trans- medium containing 1% polypeptone, 0.2% yeast ex- ferred to the new genus Rhodovulum, and, as a result, tract and 0.1% MgSO4 (PY medium). Rhodobacter the genus Rhodobacter involved only species whose veldkampii DSM 11550T was grown phototrophically at habitats were freshwater. At present Rhodobacter is a 27°C in SA medium (Kawasaki et al., 1992) to which a monophyletic genus in the Proteobacteria a-3 sub- sulfide solution had been added, and Rhodovulum group. strictum JCM 9220T was grown phototrophically at As described in previous reports, the marine non- 27°C in MMYS-II (Hiraishi and Ueda, 1995). Tests to photosynthetic bacterium Agrobacterium ferrugineum determine NaCl ranges for growth were performed IAM 12616T formed one cluster with the species of the with PY medium containing different concentrations of genus Rhodobacter in phylogenetic trees based on nu- NaCl (0–20%). cleotide sequences of 16S rRNA (Uchino et al., 1997, 23S rDNA sequencing. Fragments including the 1998). Agrobacterium ferrugineum did not grow pho- total 23S rRNA genes of A. ferrugineum IAM 12616T, totrophically under anoxic conditions, and the bacteri- Rba. azotoformans IAM 14814T, Rba. blasticus DSM ochlorophyll a (BChl a), intracytoplasmic membrane 2131T and Rba. veldkampii DSM 11550T were ampli- systems and genes for photosynthesis, e.g., puf L and fied by PCR using conserved primers 16SF01 (5Ј-AC- puf M coding subunits L and M of the photosynthetic CGCCCGTCACACC-3Ј) and 5SR01 (5Ј-SYGTTCG- reaction center could not be detected. Hence, the GRAWGGGA-3Ј) (Ludwig et al., 1992). The PCR tem- monophyly of the phototrophic members of Rhodobac- perature control was done by using the following ther- ter was disturbed by non-photosynthetic Agrobac- mal profile: after initial denaturation at 94°C for 2 min, terium ferrugineum, and the genus Rhodobacter be- a total of 35 cycles of amplification were performed came a paraphyletic group. In a previous report, we with template DNA denaturation at 94°C for 1.5 min, deferred an official nomenclatural proposal for A. fer- primer annealing at 50°C for 1 min and primer exten- rugineum until more phenotypic information was avail- sion at 72°C for 2.5 min, and final extension at 72°C able. for 10 min. The PCR products were purified using Ul- In this study, we tried to detect genes for photosyn- trafree-MC centrifugal filter units (Millipore Corp., Bed- thesis in A. ferrugineum using PCR and Southern ford, MA, USA). Sequencing was carried out using an hybridization methods. The target genes were puf L ABI PRISMTM Big-Dye terminator Cycle Sequencing and puf M genes, and puhA gene coding subunit H Ready Reaction Kit (Perkin-Elmer Co., Foster City, of the reaction center. If these genes can be found in CA, USA) and a model ABI 310 Genetic Analyzer A. ferrugineum, it may suggest that A. ferrugineum is (Perkin-Elmer Co.) according to the manufacturer’s a variant of the photosynthetic bacterium, and we instructions. The sequencing primers used are listed can propose A. ferrugineum as species of genus in Table 1. The primers in this study included degener- Rhodobacter. ated primers (the mixture of some kinds of oligonu- In this study, we performed the multigene phyloge- cleotides). netic analyses based on nucleotide sequences of 23S gyrB sequencing. The protocol for determining the rRNA and amino acid sequences of subunit B of DNA gyrB sequences was almost the same as that gyrase adding the information based on the 16S rRNA described by Yamamoto and Harayama (1995). The to clarify the phylogeny of the organisms. gyrB fragments of A. ferrugineum IAM 12616T, P. aminophilus IAM 14245T, Rba. capsulatus ATCC T T Materials and Methods 11166 , Rba. sphaeroides ATCC 11167 , Rdv. strictum JCM 9220T, and Rsb. litoralis IFO 15278T were ampli- Bacterial culture conditions. Agrobacterium ferru- fied by PCR using primers UP-1E (Yamamoto et al., gineum IAM 12616T and Roseobacter litoralis IFO 1999) and UP-2r (Yamamoto and Harayama, 1995). 15278T were cultured aerobically at 27°C in Difco Ma- The gyrB fragments of Rba. azotoformans IAM rine broth 2216 medium. Paracoccus aminophilus IAM 14814T, Rba. blasticus DSM 2131T and Rba. veld- 14245T, Rhodobacter azotoformans IAM 14814T, kampii DSM 11550T were amplified by PCR using Rhodobacter blasticus DSM 2131T, Rhodobacter cap- primers gy21A (5Ј-CAGGAAACAGCTATGACCAAR- sulatus ATCC 11166T and Rhodobacter sphaeroides MGICCNGSIATGTAYATHGG-3Ј) and gyQTKr (5Ј-AC- ATCC 11167T were cultured aerobically at 27°C in a SAGCTTGTCCTTGGTYTG-3Ј). The primer gy21A 2002 Pseudorhodobacter ferrugineus gen. nov., comb. nov. 311 Table 1. Primer sequences of 23S ribosomal RNA gene (Perkin-Elmer Co.) and a model ABI 377 DNA Se- for PCR and sequence reaction. quencer (Perkin-Elmer Co.) according to the manufac- turer’s instructions. The sequencing primers used Ј Ј 16SF01 5 -ACCGCCCGTCACACC OH-3 were UP-1E, UP-2r, gyQTKr and M13R (5Ј-CAGGAA- 23SF01 5Ј-CCGAATGGGGAAACCC OH-3Ј ACAGCTATGACC-3Ј) –21M13 (5Ј-TGTAAAACGACG- 23SF02 5Ј-AGTAGTGGCGAGCGAA OH-3Ј Ј 23SF03 5Ј-AGTACCGTGAGGGAAAG OH-3Ј GCCAGT-3 ) universal primer. 23SF04 5Ј-AGCTGGTTCTCCGCGAAA OH-3Ј Phylogenetic analysis. The phylogenetic analysis 23SF05 5Ј-GCGTAACAGCTCACT OH-3Ј based on nucleotide sequences of 16S rRNA and 23S 23SF06 5Ј-GTAGCGAAATTCCTTGTCG OH-3Ј rRNA and amino acid sequences of DNA gyrase B 23SF07 5Ј-CCTCGATGTCGGCTC OH-3Ј subunit were performed. The 16S rDNA sequences of 23SR01 5Ј-CTTTCCCTCACGGTACT OH-3Ј the following 33 species were newly obtained from the Ј Ј 23SR02 5 -TTTCGCGGAGAACCAGCT OH-3 DNA data bank, and used with the previous data 23SR03 5Ј-TCAGGGTTGTTTCCCT OH-3Ј (Uchino et al., 1998). 23SR04 5Ј-CCACCTGTGTCGGTTT OH-3Ј Antarctobacter heliothermus (accession number: 23SR05 5Ј-CTTAGATGCCTTCAGC OH-3Ј 23SR06 5Ј-ACTWAGATGTTTCAGTTC OH-3Ј Y11552), Brucella melitensis (AF220147), Caulobacter 23SR07 5Ј-CCTTCTCCCGAAGTTACGG OH-3Ј crescentus (NC_002696), Hyphomonas adhaerens 5SR01 5Ј-SYGTTCGGRAWGGGA OH-3Ј (AF082790), Hyphomonas hirschiana (AF082794), Hy- phomonas johnsonii (AF082791), Hyphomonas neptu- The abbreviation R meant adenine or guanine; S, cytosine or nium (AF082798), Hyphomonas oceanitis (AF082797), guanine; W, adenine or thymine; and Y, cytosine or thymine, re- Hyphomonas polymorpha (AF082796), Hyphomonas spectively. rosenbergii (AF082795), “Marinosulfonomonas methyl- otropha” (U62894),