SGM Journals
Actinobacteria

Nocardia iowensis sp. nov., an organism rich in biocatalytically important enzymes and nitric oxide synthase

Andrew S. Lamm, Arshdeep Khare, Patricia Conville, Peter C. K. Lau, Hélène Bergeron, John P. N. Rosazza

  • 1Center for Biocatalysis and Bioprocessing, 2501 Crosspark Road, Suite C100 Oakdale Research Park, University of Iowa, Iowa City, IA 52242-5000, USA
  • 2Microbiology Service, Department of Laboratory Medicine, Warren G. Magnuson Clinical Center, National Institutes of Health, US Department of Health and Human Services, Bethesda, MD 20892-1508, USA
  • 3Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Ave., Montreal, Quebec H4P 2R2, Canada
  • Correspondence
    John P. N. Rosazza
    john-rosazza{at}uiowa.edu

    • International Journal of Systematic and Evolutionary Microbiology 2009; 59(10):2408–2414 · https://doi.org/10.1099/ijs.0.007427-0

    View at publisher PubMed

    Abstract

    Nocardia strain NRRL 5646, isolated from a garden soil sample in Osceola, Iowa, USA, was initially of interest as an antibiotic producer. It contained biocatalytically important enzymes and represented the first described nitric oxide synthase enzyme system in bacteria. The present polyphasic taxonomic study was undertaken to differentiate strain NRRL 5646T from related species of the genus Nocardia. Chemotaxonomic analyses included determinations of the fatty acid methyl ester profile (C16 : 1ω6c/C16 : 1ω7c, C16 : 0, C18 : 1ω9c and C18 : 0 10-methyl as major components), quinone [cyclo MK-8(H4) as the major component], polar lipid (diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside as major components) and mycolic acid. These results supported its placement within the genus Nocardia. Biochemical testing and 16S rRNA, 65-kDa heat-shock protein (hsp65) and preprotein translocase (secA1) gene sequence analyses differentiated strain NRRL 5646T from recognized Nocardia species. Previous studies have demonstrated that other genetic sequences (carboxylic acid reductase, Nocardia phosphopantetheinyl transferase and GTP cyclohydrolase I) from strain NRRL 5646T can also be used to substantiate its uniqueness. The level of 16S rRNA gene sequence similarity between strain NRRL 5646T and the type strains of Nocardia tenerifensis and Nocardia brasiliensis was 98.8 %. However, strain NRRL 5646T could be clearly distinguished from these Nocardia species based on DNA–DNA hybridization data. Consequently, strain NRRL 5646T is considered to represent a novel species of the genus Nocardia, for which the name Nocardia iowensis sp. nov. is proposed. The type strain is NRRL 5646T (=UI 122540T=NRRL B-24671T=DSM 45197T).

    • The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA, secA1 and hsp65 gene sequences of strain NRRL 5646T are DQ925490, EU650382 and EU650383, respectively.

    Strain NRRL 5646T was initially identified as an antibiotic-producing bacterium isolated from a garden soil sample in Osceola, Iowa, USA (Hlavka & Bitha, 1977; Martin et al., 1977). This strain showed an extraordinary diversity of biotransforming enzymes and the first bacterial nitric oxide synthase (NOS) system with possible physiological importance, making its identification to the species level important. Strain NRRL 5646T converts many natural and synthetic organic compounds into valuable products, including flavonoids (Herath et al., 2006; Maatooq & Rosazza, 2005), vanillic acid (Dhar et al., 2007) and 4-vinylphenol (Lee & Rosazza, 2002). A novel ATP/NADPH-dependent carboxylic acid reductase was isolated from strain NRRL 5646T (Li & Rosazza, 1997) and subsequently cloned (car; GenBank accession number AY495697; He et al., 2004a). The reduction system required a phosphopantetheinyl transferase (npt, GenBank accession number DQ904035; Venkitasubramanian et al., 2007). Strain NRRL 5646T contains the first NOS system characterized in prokaryotes (Chen & Rosazza, 1995). As part of the NOS system, strain NRRL 5646T contains guanylate cyclase for cyclic GMP synthesis (Son & Rosazza, 2000), and GTP cyclohydrolase I (gch; GenBank accession number AY128670; He et al., 2004b) for tetrahydrobiopterin biosynthesis.

    The present polyphasic taxonomic study was used to identify strain NRRL 5646T to the species level. Phenotypic, biochemical, chemotaxonomic (fatty acid methyl esters, quinones, polar lipids, mycolic acids) and genotypic [16S rRNA, 65-kDa heat-shock protein (hsp65) and preprotein translocase (secA1) gene sequence analyses, DNA–DNA hybridization] characteristics were used to show that strain NRRL 5646T represents a novel species of the genus Nocardia.

    Strain NRRL 5646T was grown and maintained on sporulating agar slants (Li & Rosazza, 1997) at room temperature for 5 days and was maintained at 4 °C. Strain NRRL 5646T was grown in shaken flask cultures at 29 °C to provide cell pellets for analysis as described by Li & Rosazza (1997). The strain initially grew as a whitish mycelium on sporulating agar at 29 °C and after several days showed whitish aerial hyphae. Smears were prepared from the growth on sporulating agar and were Gram stained according to Gerhardt et al. (1994). Smears were also stained via the modified Ziehl-Neelsen method of Gordon (1967) to determine the degree of acid-fastness. Cells of strain NRRL 5646T stained as Gram-positive rods with branching characteristic of Nocardia species. The acid-fastness of strain NRRL 5646T was confirmed by using modified Kinyoun acid-fast staining (Brown-Elliott et al., 2006). For scanning electron microscopy analysis at the Central Electron Microscopy Research Facility at the University of Iowa, an agar block containing substrate and aerial mycelium was fixed with 25 % glutaraldehyde and subsequently stained with 1 % osmium tetroxide. The sample was rinsed with 0.1 M cacodylate buffer, then water and then progressively dehydrated with increasing concentrations of ethanol. The sample was critical-point dried, mounted on stubs and sputter coated, and scanning electron micrographs were taken with a Hitachi S-4800 scanning electron microscope. Micrographs revealed branching aerial hyphae.

    The cultural, morphological and phenotypic properties of strain NRRL 5646T were originally described by Martin et al. (1977) and Hlavka & Bitha (1977). Additional biochemical tests were done by using Nocardia ID QUAD plates (BD Microbiology Systems), bile aesculin agar and urea agar slants (BD Microbiology Systems). Hydrolysis of hypoxanthine and uric acid was determined as described by Isik et al. (1999). Duplicate plates containing the relevant substrates were inoculated from a glycerol stock and incubated at 29 °C for 2–4 weeks. Results of the physiological characterization are given in the species description below.

    Fatty acid and mycolic acid profiles of strain NRRL 5646T were determined by Microbial ID, Inc., Newark, Delaware, USA (), by using the Sherlock Microbial Identification System software (MIDI Inc.) following standard protocols (Miller, 1982; Sasser, 1990; Butler et al., 1986). Bacterial samples were grown at 29 °C on freshly prepared yeast-malt extract agar (Difco) prior to analysis. The major fatty acids of strain NRRL 5646T were C16 : 0 (28.2 % of the total), C18 : 1ω9c (27.9 %) and C18 : 0 10-methyl (18.4 %). A mixture of C16 : 1ω6c and C16 : 1ω7c fatty acids was also detected as a significant component (19.2 %). This profile most closely matches those of members of the genus Nocardia. Table 1⇓ provides a comparison of the fatty acid profiles of strain NRRL 5646T and the type strains of closely related Nocardia species.

    Table 1.

    Fatty acid methyl ester profiles of strain NRRL 5646T and the type strains of related Nocardia species

    Strains: 1, NRRL 5646T (N. iowensis sp. nov.); 2, N. tenerifensis DSM 44704T; 3, N. transvalensis DSM 43405T; 4, N. brasiliensis DSM 43758T; 5, N. farcinica DSM 43665T; 6. N. beijingensis DSM 44636T. Values are percentages of the total fatty acids. For unsaturated fatty acids, the position of the double bond is located by counting from the methyl (ω) end of the carbon chain; cis and trans isomers are indicated by the suffixes c and t, respectively. Data for reference strains were taken from Kämpfer et al. (2004). TSBA, Tuberculostearic acid. −, Not detected.

    Mycolic acid analysis also confirmed strain NRRL 5646T as a member of the genus Nocardia. MIS (Microbial Indentification System) library comparisons gave low similarity indices of 0.346, 0.313 and 0.262 for strain NRRL 5646T versus the type strains of Nocardia carnea, Nocardia brasiliensis and Nocardia asteroides, respectively. Thus, although strain NRRL 5646T was not present in the Microbial ID, Inc. databases, it has a mycolic acid composition similar to those of recognized members of the genus Nocardia.

    Polar lipids and menaquinone analyses of strain NRRL 5646T (performed by the DSMZ, Braunschweig, Germany; ), important classification tools for the identification of members of the genus Nocardia (Kämpfer et al., 2004), revealed the presence of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside as major polar lipids, and cyclo MK-8(H4) as the major quinone. These data also supported the affiliation of strain NRRL 5646T to the genus Nocardia.

    Near-full-length 16S rRNA gene sequences are considered to provide reliable criteria for identification of members of the genus Nocardia (Brown-Elliott et al., 2006; Roth et al., 2003; Wauters et al., 2005). Moreover, a variable region that exists within the first 500 bp of the gene is considered to be the basis for a reliable method for species differentiation among members of the genus (Conville et al., 2000). The nearly full-length 16S rRNA gene sequence of strain NRRL 5646T was determined by using eubacteria primers 27f and 1492R (Lane, 1991). A 1.4-kb fragment of the 16S rRNA gene from strain NRRL 5646T was amplified (Lee et al., 2007). Analysis of this sequence via the blast program (; Altschul et al., 1997) showed that it shared 98.8 % similarity to Nocardia tenerifensis DSM 44704T and 98.8 % to N. brasiliensis DSM 43758T [along with several other N. brasiliensis strains (GenBank accession numbers Z36935, X80608, AY245543, DQ659902 and X80591)]. This confirmed the designation of strain NRRL 5646T to the genus Nocardia but not to any recognized species as at least 99.2 % 16S rRNA gene sequence similarity is required for this level of classification (Conville et al., 2000). The 16S rRNA gene sequence of strain NRRL 5646T was aligned with corresponding nucleotide sequences of the type strains of recognized Nocardia species, retrieved from the GenBank/EMBL/DDBJ databases, by using clustal_x software (Thompson et al., 1997). Phylogenetic trees were inferred by using the least-squares (Fitch & Margoliash, 1967), maximum-likelihood (Felsenstein, 1981), maximum-parsimony (Kluge & Farris, 1969) and neighbour-joining (Saitou & Nei, 1987) algorithms from the phylip suite of programs (Felsenstein, 1993). Evolutionary distance matrices were generated as described by Kimura (1980). The resultant unrooted tree topologies were evaluated by bootstrap analysis (Felsenstein, 1985) of the neighbour-joining method based on 1000 resamplings, by using the seqboot and consense options within the phylip package. Strain NRRL 5646T formed a distinct phyletic lineage among other species of aerobic actinomycetes (Fig. 1⇓). Within one important variable region of the 16S rRNA gene sequence, strain NRRL 5646T (146–TTCCAGT–152) differed significantly from that of N. tenerifensis DSM 44704T (172–TCATGTC–178) and showed one base difference from N. brasiliensis DSM 43758T (149–TTTCAGT–155). (Nucleotide position numbers above refer to the original GenBank 16S rRNA gene sequences whereas comparisons were made based on clustal_x alignment.) This signature sequence also distinguished strain NRRL 5646T from the type strains of N. asteroides, Nocardia farcinica, Nocardia nova, Nocardia otitidiscaviarum, Nocardia pseudobrasiliensis and Nocardia transvalensis (Conville et al., 2006). On this basis, strain NRRL 5646T probably represents a novel Nocardia species. Strain NRRL 5646T differed from all other recognized species of the genus with regard to this variable region of the 16S rRNA gene.

    Figure image not available in archive
    Fig. 1.

    Unrooted neighbour-joining tree (Saitou & Nei, 1987), based on nearly complete 16S rRNA gene sequences, showing the position of strain NRRL 5646T among representatives of closely related Nocardia species. Phylogenetic data were recovered by using the maximum-likelihood (Felsenstein, 1981) and maximum-parsimony (Kluge & Farris, 1969) tree-making algorithms, respectively. Numbers at nodes indicate levels of bootstrap support based on a neighbour-joining analysis of 1000 resampled datasets; only values >50 % are given. Asterisks indicate branches that were also found in trees constructed based on hsp65 and secAI gene sequences (see Figs 2 and 3). Bar, 0.01 changes per nucleotide position.

    The use of hsp65 gene sequences (but not hsp65 PCR-restriction enzyme pattern analysis) containing sufficient polymorphism was found to be as reliable as 16S rRNA gene sequence analysis for comprehensive Nocardia species identification (Rodriguez-Nava et al., 2006). Additionally, secA1 gene sequences have been shown to be useful in this regard (Conville et al., 2006). As a result, public sequence repositories such as GenBank have a growing number of both hsp65 and secA1 gene sequences.

    The hsp65 and secA1 genes of strain NRRL 5646T were amplified and sequenced according to Conville et al. (2000, 2006), respectively. Levels of hsp65 and secA1 gene sequence analysis were again determined by performing blast searches. Evaluation of hsp65 gene sequence multiple alignment data placed the novel strain in the genus Nocardia with closest matches to the type strains of Nocardia ninae (95.9 % similarity), N. tenerifensis (95.7 %), Nocardia arthritidis (94.9 %) and N. brasiliensis (94.6 %). The neighbour-joining phylogenetic tree based on hsp65 gene sequences is shown in Fig. 2⇓. The secA1 gene sequence alignment data also placed strain NRRL 5646T within the genus Nocardia with closest matches to the type strains of Nocardia pneumoniae (95.5 % similarity), N. brasiliensis (95.3 %), Nocardia beijingensis (95.3 %) and N. asteroides (95.3 %). The neighbour-joining phylogenetic tree based on secA1 gene sequences is shown in Fig. 3⇓.

    Figure image not available in archive
    Fig. 2.

    Unrooted neighbour-joining tree (Saitou & Nei, 1987), based on complete hsp65 gene sequences, showing the position of strain NRRL 5646T among representatives of closely related Nocardia species. Phylogenetic data were recovered by using the maximum-likelihood (Felsenstein, 1981) and maximum-parsimony (Kluge & Farris, 1969) tree-making algorithms, respectively. Numbers at nodes indicate levels of bootstrap support based on a neighbour-joining analysis of 1000 resampled datasets; only values >50 % are given. Asterisks indicate branches that were also found in trees constructed based on 16S rRNA and secAI gene sequences (see Figs 1 and 3). Bar, 0.01 changes per nucleotide position.

    Figure image not available in archive
    Fig. 3.

    Unrooted neighbour-joining tree (Saitou & Nei, 1987), based on complete secA1 gene sequences, showing the position of strain NRRL 5646T among representatives of closely related Nocardia species. Phylogenetic data were recovered by using the maximum-likelihood (Felsenstein, 1981) and maximum-parsimony (Kluge & Farris, 1969) tree-making algorithms, respectively. Numbers at nodes indicate levels of bootstrap support based on a neighbour-joining analysis of 1000 resampled datasets; only values >50 % are given. Asterisks indicate branches that were also found in trees constructed based on 16S rRNA and hsp65 gene sequences (see Figs 1 and 2). Bar, 0.01 changes per nucleotide position.

    DNA–DNA hybridization experiments (performed by the DSMZ) between strain NRRL 5646T and itself, N. tenerifensis DSM 44704T and N. brasiliensis DSM 43758T revealed levels of relatedness of 92.1 (94.9), 5.4 (7.4) and 19.9 % (15.8 %), respectively (values in parentheses are the results of duplicate measurements). Based on the recommended threshold value of 70 % DNA–DNA relatedness for species delineation (Wayne et al., 1987), strain NRRL 5646T represents a species distinct from N. tenerifensis and N. brasiliensis.

    In addition, blast searches and analysis of car, npt and gch gene sequences of strain NRRL 5646T revealed closest matches to N. farcinica IFM 10152, with maximum similarity values of 74, 84 and 81 %, respectively. However, N. farcinica IFM 10152 is the only Nocardia taxon for which the entire genome sequence has been established (Ishikawa et al., 2004).

    Based on the above physiological, biochemical and chemotaxonomic analyses, strain NRRL 5646T is considered to represent a novel species of the genus Nocardia, for which the name Nocardia iowensis sp. nov. is proposed.

    Description of Nocardia iowensis sp. nov.

    Nocardia iowensis (i.o.wen′sis. N.L. fem. adj. iowensis after the state of Iowa, from where the type strain was isolated).

    Initial growth on sporulating agar at 29 °C is as a whitish mycelium followed by whitish aerial hyphae after incubation for 5 days. Cells are Gram-positive branching rods that are acid-fast. Moderate growth occurs on yeast extract, asparagine glucose, Benedict's, Bennett's potato starch and Weinstein agars; light growth occurs on Hickey and Tresner's, tomato paste and oatmeal agars; and only trace growth occurs on inorganic salts-starch, Kustner's oatflake, Czapek's solution and rice agars. No growth occurs with l-rhamnose, adonitol, l-arabinose, melibiose or d-xylose. Predominant fatty acids are C16 : 0, C18 : 1ω9c and C18 : 0 10-methyl, plus a mixture of C16 : 1ω6c and C16 : 1ω7c. The major menaquinone is cyclo MK-8(H4). Polar lipids present are diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside. No soluble pigments are produced; no liquefaction of gelatin. Nitrates are reduced to nitrites in 7 days. Can tolerate >4 but <7 % NaCl in yeast extract agar. Catalase-positive. Hydrolyses aesculin, urea and starch. Degrades xanthine, tyrosine, hypoxanthine and uric acid but not casein. Optimum temperature for growth is 29 °C. Utilization of carbon sources and additional phenotypic properties are given in Table 2⇓.

    Table 2.

    Differential physiological characteristics of strain NRRL 5646T and the type strains of Nocardia species grouped in the same cluster on the basis of 16S rRNA gene sequence analysis

    Strains: 1, NRRL 5646T (N. iowensis sp. nov.); 2, N. tenerifensis DSM 44704T; 3, N. brasiliensis DSM 43758T; 4, N. beijingensis DSM 44636T; 5, N. takedensis DSM 44801T. +, Positive; (+), weakly positive; −, negative; nd, not determined. Data for reference strains were taken from Kämpfer et al. (2004), Wang et al. (2001) and Yamamura et al. (2005). Data for growth on carbon sources for strain NRRL 5646T were taken from Martin et al. (1977) and Hlavka & Bitha (1977).

    The type strain, NRRL 5646T (=UI 122540T=NRRL B-24671T=DSM 45197T), was isolated from soil in Osceola, Iowa, USA.

    Acknowledgments

    We acknowledge the assistance of Claude Pujol and Jean Ross at the University of Iowa. This work was supported by the Iowa Biotechnology Byproducts Consortium, through the Cooperative State Research, Education and Extension Service, US Department of Agriculture, under Agreement nos 2002-34188-12035 and 2004-34188-15067. Any opinions, findings, conclusions or recommendations expressed in this publication are those of the authors and do not necessarily reflect the views of the US Department of Agriculture. In addition, this research was supported in part by the Intramural Research Program of the NIH Warren G. Magnuson Clinical Center. The views expressed herein are those of the authors and should not be construed as those of the US Department of Health and Human Services.

    References

    1. Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res 25, 3389–3402.
    2. Brown-Elliott, B. A., Brown, J. M., Conville, P. S. & Wallace, R. J., Jr (2006). Clinical and laboratory features of the Nocardia spp. based on current molecular taxonomy. Clin Microbiol Rev 19, 259–282.
    3. Butler, W. R., Ahearn, D. G. & Kilburn, J. O. (1986). High-performance liquid chromatography of mycolic acids as a tool in the identification of Corynebacterium, Nocardia, Rhodococcus, and Mycobacterium species. J Clin Microbiol 23, 182–185.
    4. Chen, Y. & Rosazza, J. P. N. (1995). Purification and characterization of nitric oxide synthase (NOSNoc) from a Nocardia species. J Bacteriol 177, 5122–5128.
    5. Conville, P. S., Fischer, S. H., Cartwright, C. P. & Witebsky, F. G. (2000). Identification of Nocardia species by restriction endonuclease analysis of an amplified portion of the 16S rRNA gene. J Clin Microbiol 38, 158–164.
    6. Conville, P. S., Zelazny, A. M. & Witebsky, F. G. (2006). Analysis of secA1 gene sequences for identification of Nocardia species. J Clin Microbiol 44, 2760–2766.
    7. Dhar, A., Lee, K.-S., Dhar, K. & Rosazza, J. P. N. (2007). Nocardia sp. vanillic acid decarboxylase. Enzyme Microb Technol 41, 271–277.
    8. Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17, 368–376.
    9. Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.
    10. Felsenstein, J. (1993). phylip (phylogeny inference package), version 3.6. Distributed by the author. Department of Genome Sciences, University of Washington, Seattle, USA.
    11. Fitch, W. M. & Margoliash, E. (1967). Construction of phylogenetic trees: a method based on mutation distances as estimated from cytochrome c sequences is of general applicability. Science 155, 279–284.
    12. Gerhardt, P., Murray, R. G. E., Wood, W. A. & Krieg, N. R. (1994). Methods for General and Molecular Bacteriology. Washington, DC: American Society for Microbiology.
    13. Gordon, R. E. (1967). The taxonomy of soil bacteria. In The Ecology of Soil Bacteria, pp. 293–321. Edited by T. R. G. Gray & D. Parkinson. Liverpool: Liverpool University Press.
    14. He, A., Li, T., Daniels, L., Fotheringham, I. & Rosazza, J. P. N. (2004a). Nocardia sp. carboxylic acid reductase: cloning, expression and purification of a new oxidoreductase family. Appl Environ Microbiol 70, 1874–1881.
    15. He, A., Simpson, D. R., Daniels, L. & Rosazza, J. P. N. (2004b). Cloning, expression, purification and characterization of Nocardia sp. GTP cyclohydrolase I. Protein Expr Purif 35, 171–180.
    16. Herath, W., Mikell, J. R., Hale, A. L., Ferreira, D. & Khan, A. I. (2006). Microbial metabolism. Part 6. Metabolites of 3- and 7-hydroxyflavones. Chem Pharm Bull (Tokyo) 54, 320–324.
    17. Hlavka, J. J. & Bitha, P. (1977). Alkylated derivatives of antibiotic BM123γ. United States Patent 4,048,431.
    18. Ishikawa, J., Yamashita, A., Mikami, Y., Hoshino, Y., Kurita, H., Hotta, K., Shiba, T. & Hattori, M. (2004). The complete genomic sequence of Nocardia farcinica IFM 10152. Proc Natl Acad Sci U S A 101, 14925–14930.
    19. Isik, K., Chun, J., Hah, Y. C. & Goodfellow, M. (1999). Nocardia salmonicida nom. rev., a fish pathogen. Int J Syst Bacteriol 49, 833–837.
    20. Kämpfer, P., Buczolits, S., Jäckel, U., Grün-Wollny, I. & Busse, H.-J. (2004). Nocardia tenerifensis sp. nov. Int J Syst Evol Microbiol 54, 381–383.
    21. Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120.
    22. Kluge, A. G. & Farris, F. S. (1969). Quantitative phyletics and the evolution of anurans. Syst Zool 18, 1–32.
    23. Lane, D. J. (1991). 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics, pp. 115–175. Edited by E. Stackebrandt & M. Goodfellow. Chichester: Wiley.
    24. Lee, K.-S. & Rosazza, J. P. N. (2002). Biocatalytic oxidation of 4-vinylphenol by Nocardia. Can J Chem 80, 582–588.
    25. Lee, S., Bergeron, H., Lau, P. C. K. & Rosazza, J. P. N. (2007). Thiols in a nitric oxide synthase-containing Nocardia sp. strain NRRL 5646. Appl Environ Microbiol 73, 3095–3097.
    26. Li, T. & Rosazza, J. P. N. (1997). Purification, characterization and properties of an aryl aldehyde oxidoreductase from Nocardia sp. strain NRRL 5646. J Bacteriol 179, 3482–3487.
    27. Maatooq, G. T. & Rosazza, J. P. N. (2005). Metabolism of daidzein by Nocardia species NRRL 5646 and Mortierella isabellina ATCC 38063. Phytochemistry 66, 1007–1011.
    28. Martin, J. H. E. J., Tresner, H. D. & Porter, J. N. (1977). Antibiotic BM123 and production thereof. United States Patent 4,007,167.
    29. Miller, L. T. (1982). Single derivatization method for routine analysis of bacterial whole-cell fatty acid methyl esters, including hydroxy acids. J Clin Microbiol 16, 584–586.
    30. Rodriguez-Nava, V. A., Couble, A., Devulder, G., Flandrois, J.-P., Boiron, P. & Laurent, F. (2006). Use of PCR-restriction enzyme pattern analysis and sequencing database for hsp65 gene-based identification of Nocardia species. J Clin Microbiol 44, 536–546.
    31. Roth, A., Andrees, S., Kroppenstedt, R. M., Harmsen, D. & Mauch, H. (2003). Phylogeny of the genus Nocardia based on reassessed 16S rRNA gene sequences reveals underspeciation and division of strains classified as Nocardia asteroides into three established species and two unnamed taxons. J Clin Microbiol 41, 851–856.
    32. Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.
    33. Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDI Inc.
    34. Son, J.-K. & Rosazza, J. P. N. (2000). Cyclic GMP and biopteridine biosynthesis in Nocardia. J Bacteriol 182, 3644–3648.
    35. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
    36. Venkitasubramanian, P., Daniels, L. & Rosazza, J. P. N. (2007). Reduction of carboxylic acids by Nocardia aldehyde oxido-reductase requires a phosphopantetheinylated enzyme. J Biol Chem 282, 478–485.
    37. Wang, L., Zhang, Y., Lu, Z., Shi, Y., Liu, Z., Maldonado, L. & Goodfellow, M. (2001). Nocardia beijingensis sp. nov., a novel isolate from soil. Int J Syst Evol Microbiol 51, 1783–1788.
    38. Wauters, G., Avesani, V., Charlier, J., Janssens, M., Vaneechoutte, M. & Delmee, M. (2005). Distribution of Nocardia species in clinical samples and their routine rapid identification in the laboratory. J Clin Microbiol 43, 2624–2628.
    39. Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & other authors (1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.
    40. Yamamura, H., Hayakawa, M., Nagakawa, Y., Tamura, T., Kohono, T., Komatsu, F. & Imura, Y. (2005). Nocardia takedensis sp. nov., isolated from moat sediment and scumming activated sludge. Int J Syst Evol Microbiol 55, 433–436.