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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).
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 (http://www.microbialid.com), 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.
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; http://www.dsmz.de), 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 (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi; 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.
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.
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.
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.
The type strain, NRRL 5646T (=UI 122540T=NRRL B-24671T=DSM 45197T), was isolated from soil in Osceola, Iowa, USA.
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.
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.