Goodfellowia gen. nov., a new genus of the Pseudonocardineae related to Actinoalloteichus, containing Goodfellowia coeruleoviolacea gen. nov., comb. nov.

During the course of a phylogenetic evaluation of Saccharothrix strains held in the ARS Culture Collection, it was discovered that Saccharothrix coeruleoviolacea NRRL B-24058^T is unrelated to other species within this genus, and a polyphasic study was undertaken to clarify its taxonomic position. Strain NRRL B-24058^T is observed to be phylogenetically separate from the genus Saccharothrix and is most closely related to the genus Actinoalloteichus. The strain exhibits chemotaxonomic properties that distinguish it from members of Actinoalloteichus, including a whole-cell sugar pattern consisting of galactose and ribose as diagnostic sugars, phosphatidylethanolamine, phosphatidylethanolamine containing 2-OH fatty acids and diphosphatidylglycerol as the predominant polar lipids and MK-9(H₄) and MK-10(H₄) as the only menaquinones observed. Strain NRRL B-24058^T is distinct from other taxa within the suborder Pseudonocardineae and a new genus to be named Goodfellowia gen. nov. is proposed. The type species of this new genus is Goodfellowia coeruleoviolacea gen. nov., comb. nov., and the type strain is NRRL B-24058^T (=DSM 43935^T=INA 3564^T=JCM 9110^T=NBRC 14988^T=VKM Ac-1083^T).

---

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain NRRL B-24058^T is DQ093349.

Details of the reference sequences used in the generation of Fig. 1 are available as supplementary material in IJSEM Online.

During the course of a phylogenetic evaluation of described species of the genus Saccharothrix based on 16S rRNA gene sequences (Labeda & Kroppenstedt, 2000), it was noted that Saccharothrix coeruleoviolacea NRRL B-24058^T clearly was not within the genus Saccharothrix and appeared to represent a new taxon in the suborder Pseudonocardineae. This strain was originally described as Actinomadura coeruleoviolacea by Preobrazhenskaya et al. (1976) and was subsequently transferred to the genus Saccharothrix as Saccharothrix coeruleoviolacea by Kroppenstedt et al. (1990). A polyphasic investigation was undertaken to characterize this strain fully and to compare it with the phylogenetically closely related genera Actinoalloteichus, Crossiella, Kutzneria and Streptoalloteichus.

The strain was cultivated on NZamine medium (DSMZ medium no. 554; DSMZ, 2001) at 28 °C. Morphological observations were made on the media of Shirling & Gottlieb (1966) and DSMZ medium no. 554. Scanning electron microscopy was performed using a JEOL model JSM-4200 microscope on osmium tetroxide-fixed, dehydrated, critical point-dried and sputter-coated colonial growth.

Genomic DNA for sequencing of the 16S rRNA gene was isolated from growth on DSMZ medium no. 554 plates using UltraClean microbial DNA isolation kits (Mo Bio Laboratories), amplified and sequenced following previously described procedures (Labeda & Kroppenstedt, 2000). The sequence was aligned against sequences for taxa in the suborder Pseudonocardineae within ARB (Ludwig et al., 2004) and a phylogenetic tree was constructed according to the neighbour-joining method of Saitou & Nei (1987) and the stability of the groupings was estimated by bootstrap analysis (Felsenstein, 1989). Genomic DNA for determination of G+C content was isolated by the method of Marmur (1961) from biomass grown for 5 days in DSMZ medium no. 554. The mol% G+C content of the DNA was calculated from the T_m value by the method of Marmur & Doty (1962).

For the analyses of the fatty acids, about 40 mg cells was scraped from agar plates whereas, for the other chemical analyses, the cells were grown in liquid medium and harvested by centrifugation. Chemotaxonomic analysis of strains for polar lipids, menaquinones and fatty acids was performed using previously described methods (Grund & Kroppenstedt, 1989; Minnikin et al., 1984; Sasser, 1990).

Physiological tests, including production of acid from carbohydrates, utilization of organic acids and hydrolysis and decomposition of adenine, guanine, hypoxanthine, tyrosine, xanthine, casein, aesculin, urea and hippurate, were evaluated by using the media of Gordon et al. (1974). Phosphatase activity was evaluated by using the method of Kurup & Schmitt (1973). The temperature range for growth was determined on slants of DSMZ medium no. 554.

Strain NRRL B-24058^T is phylogenetically separate from the genus Saccharothrix and appears to be most closely related to the genus Actinoalloteichus, as can be seen in Fig. 1. The strain exhibits 16S rRNA gene sequence similarity of 95.5, 95.4, 94.6, 94.5, 94.3 and 97.2 %, respectively, to Actinoalloteichus cyanogriseus IFO 14455^T, Actinoalloteichus spitiensis MTCC 6194^T, Kibdelosporangium aridum subsp. aridum DSM 43828^T, Kibdelosporangium aridum subsp. largum DSM 44150^T, Kibdelosporangium philippinense DSM 44226^T and Streptoalloteichus hindustanus IFO 15115^T.

(10K): Fig. 1. Phylogenetic dendrogram reconstructed for the genera of the suborder Pseudonocardineae calculated within ARB (Ludwig et al., 2004) from almost-complete 16S rRNA gene sequences using Kimura's evolutionary-distance methods (Kimura, 1980) and the neighbour-joining method of Saitou & Nei (1987) illustrating the taxonomic position of Goodfellowia coeruleoviolacea NRRL B-24058T relative to the other taxa within the suborder. The taxa and sequences included in the genus groups are detailed in Supplementary Table S1 available in IJSEM Online. Bar, 0.01 nucleotide substitutions persite.

The chemotaxonomic properties of the strain that distinguish it from members of Actinoalloteichus and other related taxa can be seen in Table 1. The whole-cell sugar pattern of only galactose and ribose differs from those of the other four genera, as does the lack of phosphatidylinositol in the phospholipid pattern and the presence of both phosphatidylmonomethylethanolamine and phosphatidylethanolamine containing 2-hydroxy fatty acids. The menaquinone pattern (Table 2) also appears to be distinct from the other related genera in that substantial quantities of MK-10(H₄) are present. The fatty acid profile of strain NRRL B-24058^T (Table 3) clearly distinguishes it from its nearest phylogenetic neighbouring genera by the presence of significant quantities of 17 : 0 10-methyl fatty acid and various hydroxylated fatty acids.

Table 1. Chemotaxonomic characteristics of Goodfellowia and related taxa All strains contain meso-diaminopimelic acid as the cell-wall diamino acid. Data for reference strains were taken from Tamura et al. (2000) (Actinoalloteichus cyanogriseus), Labeda (2001) (Crossiella cryophila), Tomita et al. (1993) (Kibdelosporangium albatum), Stackebrandt et al. (1994) (Kutzneria viridogrisea) and Tomita et al. (1987) (Streptoalloteichus hindustanus).

Table 2. Menaquinone content of Goodfellowia coeruleoviolacea and related taxa Strains: 1, Goodfellowia coeruleoviolacea DSM 43935T; 2, Actinoalloteichus cyanogriseus IFO 14455T (data from Tamura etal., 2000); 3, Crossiella cryophila NRRL B-16238T (Labeda, 2001); 4, Kibdelosporangium albatum ATCC 55061T (Tomita et al., 1993); 5, Kutzneria viridogrisea DSM 43850T (Stackebrandt et al., 1994); 6, Streptoalloteichus hindustanus IFO 15115T (Tomita et al., 1987). Values arepercentages of the total menaquinones; , not detected. Abbreviations are exemplified by MK-8(H4), a menaquinone which has two of the eight isoprene units hydrogenated.

Table 3. Fatty acid content of Goodfellowia coeruleoviolacea DSM 43935T (=NRRL B-24058T) and phylogenetically most closely related taxa Strains: 1, Goodfellowia coeruleoviolacea DSM 43935T; 2, Actinoalloteichus cyanogriseus DSM 43889T; 3, Streptoalloteichus hindustanus DSM 44523T; 4, Kibdelosporangium albatum ATCC 55061T (data from Tomita et al., 1993). Values are percentages of total fatty acids; , not detected. Abbreviations are exemplified by the following: 14 : 0 iso, isotetradecanoic acid or 12-methyl tridecanoic acid; 17 : 0 anteiso 2-OH, 2-hydroxy 14-methyl hexadecanoic acid.

Scanning electron microscopic observations of colony growth on several different media have not revealed the presence of sporangia and the substrate mycelium appears to fragment into coccoidal rod elements. (Fig. 2). Spore chains typical of those observed for Actinoalloteichus species were not observed.

(151K): Fig. 2. Scanning electron micrograph of 21-day growth of Goodfellowia coeruleoviolacea NRRL B-24058T on yeast extract-malt extract agar. Note that spores are produced by fragmentation of the vegetative mycelium. Bar, 10 µm.

Strain NRRL B-24058^T therefore appears distinct from other taxa within the suborder Pseudonocardineae and a new genus is proposed, to be named Goodfellowia gen. nov., to accommodate Saccharothrix coeruleoviolacea.

Description of Goodfellowia gen. nov.
Goodfellowia (Good.fel.low'i.a. N.L. fem. n. Goodfellowia named for Michael Goodfellow, a microbiologist at the University of Newcastle, in recognition of his contributions to microbial systematics).

Aerobic. Gram-positive, non-acid-fast, non-motile actinomycetes. Branched substrate mycelium (approx. 0.5 µm in diameter) and, on some media, aerial mycelia are produced. Ovoid conidia are produced by fragmentation of substrate mycelium. Catalase-positive. Contain meso-diaminopimelic acid as the diamino acid. The whole-cell sugar pattern consists of galactose and ribose. The phospholipid pattern consists of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylethanolamine containing hydroxylated fatty acids and traces of phosphatidylinositol and phosphatidylinositol mannosides. The predominant menaquinones are MK-9(H₄) and MK-10(H₄). Have a fatty-acid profile rich in branched-chain and saturated components including 10-methyl-branched heptadecanoic acid and anteiso-branched 2-hydroxy fatty acids. Phylogenetically nearest neighbour is the genus Actinoalloteichus. Type species is Goodfellowia coeruleoviolacea.

Description of Goodfellowia coeruleoviolacea comb. nov.
Basonym: Actinomadura coeruleoviolacea Preobrazhenskaya and Terekhova 1987.

Other synonym: Saccharothrix coeruleoviolacea (Preobrazhenskaya and Terekhova 1987) Kroppenstedt et al. 1991.

Vegetative mycelium is pale yellow to dark brownish yellow, depending on medium; white aerial hyphae are produced on most media, becoming blue in colour on several media including inorganic salts-starch (ISP-4) agar and yeast extract-malt extract (ISP-2) agar. Pale-violet soluble pigment produced on inorganic salts-starch agar and bluegreen soluble pigment produced on yeast extract-malt extract agar. Degrades or hydrolyses casein, aesculin, gelatin, hypoxanthine, starch, tyrosine and urea. No degradation of adenine, allantoin or xanthine. Weakly reduces nitrates. Assimilates acetate, citrate, malate, oxalate, propionate and succinate; does not assimilate benzoate, lactate, mucate or tartrate. Acid is produced from adonitol, arabinose, cellobiose, dextrin, erythritol, fructose, galactose, glucose, glycerol, inositol, lactose, maltose, mannitol, mannose, melibiose, methyl α-D-glucoside, methyl β-xyloside, raffinose, rhamnose, salicin, sorbitol, sucrose, trehalose and xylose; no acid produced from dulcitol or melezitose. Temperature range for growth 1545 °C with an optimum around 30 °C. Grows weakly in the presence of 4 % NaCl and not at all at higher salt concentrations. G+C content of the DNA is 68.2 mol% (T_m method).

The type strain is NRRL B-24058^T (=DSM 43935^T=INA 3564^T=JCM 9110^T=NBRC 14988^T=VKM Ac-1083^T), isolated from a soil sample from Russia.