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1 Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA
2 Departamento de Zoologia and Centro de Neurociências, Universidade de Coimbra, 3004-517 Coimbra, Portugal
3 Departamento de Bioquímica and Centro de Neurociências, Universidade de Coimbra, 3001-401 Coimbra, Portugal
4 Department of Civil and Environmental Engineering, Louisiana State University, Baton Rouge, LA 70803, USA
Correspondence
William M. Moe
moemwil{at}lsu.edu
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ABSTRACT |
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-hydroxybutyrate storage granules are produced. Dominant fatty acids when grown in R2A and VM ethanol media for 72 h at 37 °C are C16 : 0, C16 : 1
7c, C17 : 0 cyclo, C10 : 0 3-OH, C18 : 17c, C12 : 0 and C15 : 0. DNA G+C content is 67.9 mol%. Phenotypic and phylogenetic data indicate that strain SUA2T is related to, but clearly differentiated from Azospira oryzae. Strain SUA2T is thus proposed as a novel species of the genus Azospira with the name Azospira restricta sp. nov. The description of the genus Azospira is emended to include the characteristics of this novel species. The type strain of Azospira restricta is SUA2T (=NRRL B-41660T=DSM 18626T=LMG 23819T).
Supplementary figures showing morphological features and whole-cell soluble protein patterns (SDS-PAGE), and a supplementary table of fatty acid composition data are available with the online version of this paper.
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MAIN TEXT |
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to accommodate a lineage of nitrogen-fixing bacteria within the Betaproteobacteria that could be phylogenetically and phenotypically differentiated from other strains originally assigned to the genus Azoarcus sensu lato (Reinhold-Hurek et al., 1993). At present, the genus Azospira includes a single validly published species, Azospira oryzae, strains of which have been isolated from surface-sterilized roots of gramineae such as Kallar grass (Leptochloa fusca) and rice (Oryza), or resting stages (sclerotia) of a basidiomycete (Reinhold-Hurek & Hurek, 2000). The description of Azospira oryzae sp. nov. was recently emended to accommodate perchlorate-reducing strains isolated from a swine-waste treatment lagoon that were originally described as Dechlorosoma suillum (Achenbach et al., 2001), but which were later shown to be phylogenetically and phenotypically similar to and represent a later subjective synonym of Azospira oryzae (Tan & Reinhold-Hurek, 2003).
During a study aimed at characterizing the microbial population in groundwater from an uncontaminated groundwater well located upgradient from a hazardous waste site near Baton Rouge, Louisiana, USA (Bowman et al., 2006), a novel bacterial strain, designated SUA2T, was isolated on R2A agar (Difco) incubated aerobically at 23 °C. Purity of the strain was verified by microscopy after multiple transfers.
Colony morphology was observed after aerobic growth on plates of VM ethanol medium (Reinhold-Hurek & Hurek, 2000) incubated at 37 °C for 4 days, or R2A (Difco) at 30 °C for 7 days. Cell morphology was observed by phase-contrast microscopy (Nikon Optiphot), differential interference contrast microscopy (Nikon microphot-Fxa) and transmission electron microscopy (JEOL 1000CX TEM). Electron micrographs were taken from cells negatively stained with uranyl acetate (2 %, v/v). Gram staining and catalase tests were performed as described by Smibert & Krieg (1981). Oxidase tests were performed as described by Tarrand & Gröschel (1982). NaCl tolerance was tested in liquid VM ethanol medium supplemented with 1.0 and 2.0 % (w/v) NaCl. The temperature range for growth was tested in liquid VM ethanol medium incubated at temperatures of 15, 20, 23, 30, 37 and 40 °C. Additional biochemical and physiological properties were determined using API 20 NE (bioMérieux) test kits, according to the manufacturer's instructions. Substrate metabolism was determined on Biolog GN and GP MicroPlates inoculated with cells grown in R2A liquid broth overnight at 23 °C. Cells were washed with a 0.9 % (w/v) NaCl solution, centrifuged and resuspended in the inoculation fluid provided with the kit. Substrate metabolism was determined by purple colour development after incubation of the plate at 30 °C for 85 h followed by measurement of A595 using an automated plate reader (Multiskan Ascent, version 4.0; Labsystems).
The ability of SUA2T cells to use nitrate or perchlorate as alternative electron acceptors was tested using anoxic DSMZ medium 908 (www.dsmz.de/media/med908.htm) with acetate replaced by lactate (10 mM) and supplemented with either sodium nitrate (10 mM) or sodium perchlorate (10 mM) as potential electron acceptors. For both perchlorate and nitrate, electron acceptor utilization was assessed by comparing growth of cells in the perchlorate- or nitrate-containing medium with control cultures prepared in media without the supplemental electron acceptor after 8 days incubation at 30 °C. Nitrate reduction and denitrification activities were further assessed as described by Smibert & Krieg (1981).
Nitrogen-fixing ability was determined by growth and acetylene reduction in semi-solid (0.2 % w/v, agar), nitrogen-free SM medium (Reinhold et al., 1986), in which malate was replaced by 10 mM lactate. Cells grown on R2A for 3 days were inoculated vertically into 17 ml Hungate tubes containing 10 ml of the semi-solid SM medium using a sterile loop. The headspace was supplemented with 0.2 ml acetylene prior to incubation at 30 °C for 8 days. Uninoculated tubes served as negative controls. Acetylene reduction (ethylene production) was determined by analysis of 0.1 ml gas headspace samples using a gas chromatograph (HP6890; HP) equipped with a capillary column (GS-Gaspro, 60 mx0.32 mm i.d.; J&W P/N 113-4362) and a flame-ionization detector. Helium was used as carrier gas at a flow rate of 45 ml min–1. The injector and detector temperature was 225 °C. The oven temperature was increased from 50 to 200 °C at a rate of 50 °C min–1. A PCR-based assay using the PolF/PolR PCR primer set and a PCR protocol as described by Poly et al. (2001) was used to detect presence of the nifH gene.
For the preparation of soluble whole-cell proteins, cells were cultured in liquid R2A medium overnight and washed with 20 mM potassium phosphate buffer (pH 7.0). Cells were disrupted by sonication (Fisher Scientific Sonic Dismemberator) at 20 kHz in an ice bath. After removal of cell debris by centrifugation at 16 000 g for 15 min, soluble proteins were separated by SDS-PAGE on a 12.5 % (w/v) polyacrylamide gel (30 : 1, w/w, acrylamide/bisacrylamide). Protein bands were visualized by staining with Coomassie brilliant blue R250. For comparison purposes, Azospira oryzae strains 6a3T (=LMG 9096T), obtained from BCCM/LMG (the Belgian Co-ordinated Collections of Microorganisms/Laboratorium voor Microbiologie) and PS (syn. Dechlorosoma suillum), kindly provided by Dr John Coates of the University of California at Berkeley, were also analysed. These two reference strains were grown and processed in exactly the same manner as strain SUA2T.
Cellular fatty acids were extracted, saponified and methylated according to the protocol of the Sherlock Microbial Identification System (MIDI) after growth in R2A (this study) and VM ethanol (DSMZ Identification Service) media for 72 h at 37 °C. The fatty acids were analysed by a gas chromatograph equipped with the Microbial Identification software package (Sasser, 1990). Genomic DNA G+C content was determined by HPLC as described by Mesbah et al. (1989).
Poly--hydroxybutyrate (PHB) extraction, collection and conversion to crotonic acid were performed according to the protocol of Smibert & Krieg (1981). Strain SUA2T and Azospira oryzae strains 6a3T and PS were grown in liquid R2A medium for 5 days at 30 °C on a rotary shaker prior to biomass collection for this analysis. The presence of crotonic acid was detected spectrophotometrically at 235 nm (Smibert & Krieg, 1981).
Extraction of genomic DNA, PCR amplification and sequencing of 16 rRNA genes was carried out as described by Rainey et al. (1996). Purified sequencing-reaction products were electrophoresed using a model 3100 DNA sequencer (Applied Biosystems). The 16S rRNA gene sequence of strain SUA2T was aligned against previously determined sequences from the NCBI database (www.ncbi.nlm.nih.gov) using the BioEdit (version 4.7.8; Hall, 1999) and CLUSTAL_X programs (Thompson et al., 1997). Phylogenic analyses were performed using components of the MEGA 3.1 package (Kumar et al., 2004). The neighbour-joining algorithm was used to build the phylogenetic tree from distance matrices calculated with Jukes–Cantor correction (Jukes & Cantor, 1969). Bootstrap analysis was performed using PHYLIP 3.62 (Felsenstein, 2004; http://evolution.genetics.washington.edu/phylip.html) with 1000 resamplings.
The characteristics of strain SUA2T are provided in the emended genus and species description (Table 1, and Supplementary Fig. S1, available with the online version of this paper). Strain SUA2T grew in semi-solid, nitrogen-free medium, forming pellicles that moved up to the surface, indicating that strain SUA2T was able to fix N2 gas as a nitrogen source. After 8 days incubation in nitrogen-free medium, reduction of acetylene (C2H2) to ethylene (C2H4) was observed via GC, further indicating that strain SUA2T exhibits nitrogenase activity. The presence of the nifH gene was detected by gel electrophoresis following PCR amplification using the PolF/PolR PCR primer set described by Poly et al. (2001). The gene sequence was verified by sequencing of the PCR product.
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) showed large differences in the major fatty acids detected. In the description of the genus Azospira, which was based on Azospira oryzae 6a3T, the major fatty acids were described as C10 : 1 3-OH, C16 : 0, C16 : 19c and C18 : 0. However, reanalysis of the fatty acids of strains 6a3T (=LMG 9069T) and PS showed that three of the four fatty acids, namely C10 : 1 3-OH, C16 : 19c and C18 : 0, described as major fatty acids in the description of the genus Azospira (Reinhold-Hurek & Hurek, 2000) are in fact not detected in the Azospira oryzae strains (Table 1
). These data were reanalysed in two independent laboratories after the cells had been grown on VM ethanol medium under the conditions described by Reinhold-Hurek & Hurek (2000) and also on R2A medium (Supplementary Table S1, available with the online version of this paper).
Nearly complete 16S rRNA gene sequences were used to reconstruct a phylogenetic dendrogram showing the relationship between strain SUA2T, Azospira oryzae strains, other genera in the family Rhodocyclaceae and environmental 16S rRNA gene sequences (Fig. 1). Sequence similarity between strain SUA2T and the closest relative for which a sequence is available from the public databases, the uncultured groundwater bacterium 249ds10 (AY212697; Simpson et al., 2004), was 98.1 %. 16S rRNA gene sequence similarities between strain SUA2T and the four closest cultured relatives, Azospira oryzae strains 6a3T, PS, GR-1 and R-25019 (AF011347, AF170348, AY277622 and AM084030, respectively), were in the range 95.7–95.8 %, indicating that strain SUA2T represents a distinct, but related lineage to this species. High bootstrap values support the novelty of the lineage represented by strain SUA2T. Strain SUA2T is the first cultured member of this lineage which is currently composed of environmental 16S rRNA gene sequences from groundwater and activated sludge samples. The 16S rRNA gene sequence of strain SUA2T shares between 97.1 and 98.1 % similarity with the sequences of these as yet uncultured organisms. The clear differentiation of strain SUA2T from Azospira oryzae at the gene level was also seen in the comparison of the partial nifH gene sequence which showed only 81.5 % nucleotide similarity over 269 positions compared to those of Azospira oryzae strains 6a3T and PS. The highest similarity between the nifH gene sequence of strain SUA2T was to that of the unrelated taxon Azonexus caeni (DQ029203) at 94 %. The DNA G+C content of strain SUA2T was 67.9 mol%.
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Morphological characteristics and some phenotypic properties of strain SUA2T confer with those described for the genus Azospira (Reinhold-Hurek & Hurek, 2000). These phenotypic features include the Gram-negative reaction, heterotrophic metabolism, non-spore-forming, curved rods with cells ranging from 0.4 to 0.6 µm in width and from 1.1 to 2.5 µm in length, motility by means of a single polar flagellum, production of catalase and oxidase, ability to grow well at 37 °C, absence of growth in the presence of 2 % (w/v) NaCl, capacity for nitrogen (N2) fixation and reduction of acetylene to ethylene. Strain SUA2T also matches the phenotypic feature of the Azospira genus description in that no growth occurs on mono- or disaccharides, nor on most amino acids.
However, some of the phenotypic characteristics determined for strain SUA2T do not confer with the current description of the genus Azospira. Specifically, strain SUA2T is unable to utilize nitrate as a terminal electron acceptor and does not grow on organic acids such as L-malate, succinate, fumarate, acetate, 2-oxoglutarate, n-caproate and propionate, nor on ethanol. In addition, the G+C content of genomic DNA from strain SUA2T, 67.9 mol%, is outside of the range of 65–66 mol% for the genus.
Strain SUA2T is clearly discriminated from Azospira oryzae, the only validly published species of the genus Azospira, by substrate utilization, inability to utilize nitrate or perchlorate as electron acceptors and colony colour when grown on VM ethanol medium (Table 1). Fatty acid composition also distinguishes strain SUA2T from Azospira oryzae strains, based on the presence of C17 : 0 cyclo, C10 : 0 3-OH, C12 : 0 and C10 : 0 in the former and absence or presence of only trace quantities in the latter when grown at 37 °C on R2A medium. Whole-cell soluble protein patterns analysed by SDS-PAGE (supplementary Fig. S1) demonstrated that strain SUA2T is distinct from Azospira oryzae in overall protein profiles, and nifH gene sequences also differ, supporting the classification of strain SUA2T as a novel species.
On the basis of phylogenetic, chemotaxonomic and phenotypic features obtained in this study, strain SUA2T is clearly discriminated from the one validly published species of the genus Azospira. Considering the lack of genus-level characteristics for differentiation of strain SUA2T from Azospira oryzae as well as the relatively high 16S rRNA gene sequence similarities, we propose strain SUA2T as a novel species which we have named Azospira restricta sp. nov. In addition, we have emended the genus description for Azospira to include the characteristics of this novel species and to clarify the fatty acid content.
Emended description of the genus Azospira Reinhold-Hurek & Hurek 2000; Tan & Reinhold-Hurek 2003
The emended description is based on the results of Reinhold-Hurek & Hurek (2000), Tan & Reinhold-Hurek (2003) and this study. Gram-negative, non-spore-forming, curved rods. Motile by means of a single polar flagellum. Aerobic, capable of microaerophilic growth in semi-solid, N-free media. Variable with respect to use of nitrate and perchlorate as terminal electron acceptors. Heterotrophic. Oxidase-positive. Catalase-positive. Capable of nitrogen fixation. Growth does not occur on most amino acids, nor on mono-or disaccharides. Optimum growth temperature about 37 °C. Produces polyhydroxybutyrate storage granules. Neutral pH optimal for growth. Major cellular fatty acids are C16 : 17c, C16 : 0 and C18 : 17c. G+C content of DNA is 65–68 mol%. Type species is Azospira oryzae.
Description of Azospira restricta sp. nov.
Azospira restricta (res.tric'ta. L. fem. part. adj. restricta limited, restricted, referring to the restricted spectrum of metabolized substrates).
On VM ethanol agar colonies are 1–2 mm diameter, translucent, greenish-yellow, smooth, convex with entire margins. On R2A agar colonies are cream-coloured, smooth, convex with entire margins. Straight or curved rods, 0.5x1.2–2.5 µm. Inclusion granules are observed. Growth occurs from 20 to 40 °C with optimal growth at 37 °C. Growth occurs at neutral pH. No growth is observed in the presence of 2 % NaCl (w/v). Aesculin hydrolysis, gelatin hydrolysis, H2S production and acetoin production do not occur. Negative for -galactosidase, arginine dihydrogenase and lysine decarboxylase. Strain SUA2T metabolizes -hydroxybutyrate, 
-hydroxybutyrate, D,L-lactate and putrescine as growth substrates, but does not metabolize other substrates in Biolog GN and GP microplates. Nitrate and perchlorate are not used as electron acceptors. Growth occurs on -hydroxybutyric acid, -hydroxybutyric acid, D,L-lactic acid and putrescine. Dominant cellular fatty acids are C16 : 0, C16 : 17c, C17 : 0 cyclo, C10 : 0 3-OH, C18 : 17c, C12 : 0 and C15 : 0. DNA G+C content is 67.9 mol%.
The type strain SUA2T (=NRRL B-41660T=DSM 18626T=LMG 23819T) was isolated from groundwater.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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-ketoglutaric acid, D-lactose, lactulose, D-maltose, D-mannose, D-melibiose, L-phenylalanine, L-proline, D-raffinose, L-rhamnose, D-ribose, D-sorbitol, sucrose, D-tagatose, D-trehalose, turanose, xylitol or D-xylose.