A primary aim of microbial ecology is to determine patterns and drivers of community distribution, interaction, and assembly amidst complexity and uncertainty. Microbial community composition has been shown to change across gradients of environment, geographic distance, salinity, temperature, oxygen, nutrients, pH, day length, and biotic factors 1-6 . These patterns have been identified mostly by focusing on one sample type and region at a time, with insights extra polated across environments and geography to produce generalized principles. To assess how microbes are distributed across environments globally-or whether microbial community dynamics follow funda mental ecological 'laws' at a planetary scale-requires either a massive monolithic cross environment survey or a practical methodology for coordinating many independent surveys. New studies of microbial environments are rapidly accumulating; however, our ability to extract meaningful information from across datasets is outstripped by the rate of data generation. Previous meta analyses have suggested robust gen eral trends in community composition, including the importance of salinity 1 and animal association 2 . These findings, although derived from relatively small and uncontrolled sample sets, support the util ity of meta analysis to reveal basic patterns of microbial diversity and suggest that a scalable and accessible analytical framework is needed.The Earth Microbiome Project (EMP, http://www.earthmicrobiome. org) was founded in 2010 to sample the Earth's microbial communities at an unprecedented scale in order to advance our understanding of the organizing biogeographic principles that govern microbial commu nity structure 7,8 . We recognized that open and collaborative science, including scientific crowdsourcing and standardized methods 8 , would help to reduce technical variation among individual studies, which can overwhelm biological variation and make general trends difficult to detect 9 . Comprising around 100 studies, over half of which have yielded peer reviewed publications (Supplementary Table 1), the EMP has now dwarfed by 100 fold the sampling and sequencing depth of earlier meta analysis efforts 1,2 ; concurrently, powerful analysis tools have been developed, opening a new and larger window into the distri bution of microbial diversity on Earth. In establishing a scalable frame work to catalogue microbiota globally, we provide both a resource for the exploration of myriad questions and a starting point for the guided acquisition of new data to answer them. As an example of using this Our growing awareness of the microbial world's importance and diversity contrasts starkly with our limited understanding of its fundamental structure. Despite recent advances in DNA sequencing, a lack of standardized protocols and common analytical frameworks impedes comparisons among studies, hindering the development of global inferences about microbial life on Earth. Here we present a meta-analysis of microbial community samples collected by hundreds of r...

Minimal standards for describing new taxa within the aerobic endospore-forming bacteria are proposed, following Recommendation 30b of the Bacteriological Code (1990 Revision). These minimal standards are recommended as guidelines to assist authors in the preparation of descriptions for novel taxa. They encourage broad polyphasic characterization and the construction of descriptions that are practically useful in routine diagnostic laboratories. The proposals have been endorsed by the Subcommittee on the Taxonomy of the Genus Bacillus and Related Organisms of the International Committee on Systematics of Prokaryotes

During a survey of the occurrence of Azospirillum spp. in cereal roots, we obtained 119 isolates which could not be identified as members of one of the three previously described Azospirillum species. These strains formed a very homogeneous group of N2-fixing, microaerobic, motile, vibrioid, gram-negative rod-shaped organisms which formed a veillike pellicle in semisolid medium similar to that of Azospirillum spp. However, the new isolates differed from Azospirillum spp. by their smaller cell width (0.6 to 0.7 pm), variable flagellation (one to three flagella on one or both poles), moist brownish colonies, and broader pH and oxygen tolerance for nitrogenase activity. Organic acids were the preferred carbon sources, but glucose, galactose, L-arabinose, mannitol, sorbitol, and glycerol were also used. The guanine-plus-cytosine content of the deoxyribonucleic acid was slightly lower than the guanine-plus-cytosine contents of Azospirillum spp. (66 to 67 mol%). Deoxyribonucleic acid hybridization experiments with 17 strains of the group showed 50 to 100% complementarity, while the levels of hybridization with the type strains of Azospirillum brasilense, Azospirillum lipuferum, and Azospirillum amazonense were 23, 15, and 6%, respectively. For these new isolates we propose a new genus, Herbaspirillum (the name refers to the habitat of the orgahisms, the roots of cereals, which are herbaceous seed-bearing plants). The type species is named Herbaspirillum seropedicae after the place where it was first isolated. The type strain is strain 267, which has been deposited in the American Type Culture Collection as strain ATCC 35892.

Deoxyribonucleic acid homology experiments were performed among representative strains of Bacillus polymyxa, Bacillus macerans, and Bacillus azotofixans and other nitrogen-fixing Bacillus strains identified as B. polymyxa-like. All B . azotofixans strains showed less than 20% homology when B . polymyxa or B . macerans was used as a probe. The range of homology among B. azotofixans strains (strain P3L-5T [T = type strain] was used as the probe) varied from 54 to loo%, indicating that these strains comprise a relatively homogeneous species.Strain Hino appears to be a variant of B . azotofixans because it exhibited only 28 to 44% homology with this species. Strains P3L-2, CR5D-16, and CR5D-23 showed less than 20% homology with B. polymyxa, B . macerans, and B . azotofixans, indicating that these strains do not belong to these species. Additional data will be necessary to fully clarify the taxonomy of these nitrogen-fixing Bacillus strains. In this work, we performed deoxyribonucleic acid (DNA) homology experiments with representative strains of B. polymyxa and B. macerans and the strains reported to be B . azotojixans and B . polymyxa-like (10, 18) in an attempt to clarify the taxonomy of the nitrogen-fixing bacilli. MATERIALS AND METHODSBacterial strains. All of the strains used in this work are listed in Table 1.Propagation of cultures. Portions (2 ml) from 24-h cultures grown in liquid medium [glucose, 10 g; MgS04 -7H20, 0.2 g; NaCI, 0.1 g; yeast extract, 1 g; (NH&HP04, 1 g; K2HP04, 4 g; KH2P04, 2.8 g; distilled water, 1,000 ml; pH 6.51 were inoculated into 200 ml of the same medium and incubated at 32°C for 18 h.Isolation of chromosomal DNA. Cells from 200-ml cultures were concentrated in 5 ml of lysis buffer [25% sucrose, 0.05 M tris(hydroxymethyl)aminomethane (Tris), 0.001 M ethylenediaminetetraacetate, pH 8.01 and treated with 1 mg of lysozyme per ml for 30 min at 37°C. B . polymyxa strains were treated for 2.5 h at the same temperature. Sodium dodecyl sulfate was added to a final concentration of 1%, and the preparations were incubated for 30 min at 65°C. In order to purify the DNA, 1 mg of pronase (catalog no. P5147; Sigma Chemical Co.) per ml was added, and the preparations were incubated at 50°C for 2 h. The DNA was then precipitated with 2 volumes of cold ethanol, spooled with a glass stirring rod, dissolved in 0 . 1~ SSC (IX SSC is 0.15 M NaCI plus 0.015 M sodium citrate) (131, and shaken with an * Corresponding author. equal volume of phenol neutralized with TES buffer (50 mM NaCI. 30 mM Tris, 5 mM ethylenediaminetetraacetate) at pH 7.5 for 15 min. The phenol treatment was repeated several times until very little interface was observed. The aqueous phase containing the DNA was spooled after ethanol precipitation and suspended in 0 . 1~ SSC. Then, the preparations were treated with pancreatic ribonuclease (50 pglml) and ribonuclease TI (1 U/ml) for 30 min at 37°C. After two treatments with phenol the DNA was precipitated, spooled, and dissolved in 1 ml of DNA buffer (4 mM NaCI, 10 mM Tris, 0.1 mM ethyl...

A new species, Bacillus azotojixans, is described. This taxon is based upon 16 soil and root-associated strains that exhibit acetylene-reducing ability and nitrogen-fixing Bacillus sp. strain Hino. B . azotojixans is phenotypically similar to Bacillus polymyxa and Bacillus macerans. However, 13 tests (nitrate reduction; production of acid and gas from xylose, arabinose, lactose, ribose, and glycerol; resistance to lysozyme; liquefaction of gelatin; starch hydrolysis; decomposition of casein and pectin; production of dihydroxyacetone; susceptibility to B . polymyxa phages) differentiate it from B. polymyxa, and 12 characteristics (spore position; Voges-Proskauer test; nitrate reduction; production of acid and gas from xylose, arabinose , lactose, ribose, and glycerol; growth at 45°C; hydrolysis of starch; decomposition of pectin; formation of crystalline dextrins) differentiate it from B. macerans. The guanine-plus-cytosine contents of five strains ranged from 47.9 to 52.5 mol%. All strains reduced acetylene much more efficiently than B. polymyxa or B . macerans. In four strains, nitrogen fixation was confirmed by micro-Kjeldahl analysis of acetylene-reducing cultures. Acetylene reduction was not inhibited by nitrate and was not dependent on yeast extract or thiamin plus biotin. The proposed type strain of B. azotojixans is strain P3L-5 (= ATCC 35681).Classically, all Bacillus strains capable of fixing molecular nitrogen were found to belong to Bacillus polymyxa (7, 9) or Bacillus macerans (23). In later work, other (nonsymbiotic) nitrogen-fixing Bacillus isolates were tentatively identified as strains of Bacillus circulans (10). To date, no strains of other Bacillus species have been accepted with certainty as nitrogen fixers, although recently it was claimed that several nitrogen-fixing bacilli resembled Bacillus cereus or Bacillus licheniformis (1). Most of the recently isolated nitrogenfixing bacilli have been identified clearly as B. polymyxa (16)(17)(18)(19)(20) or B. macerans strains (17) or as B . polymyxa-like (24) organisms. The original work of Hino and Wilson (9) on nitrogen fixation by a Bacillus strain was done with an isolate that has been classified as an atypical B. polymyxa strain (4, 14, 19). However, the deoxyribonucleic acid (DNA) of this strain revealed a guanine-plus-cytosine (G + C) content beyond the range reported for B. polymyxa, which is typical of B . macerans (14).In a previous report (20), we described 18 acetylenereducing B . polymyxa strains from soil and 6 other strains with this property that were tentatively identified as "variants" of B . polymyxa. Since then, 10 other acetylenereducing bacilli have been isolated in our laboratory from washed or surface-sterilized roots of different grasses; these strains had properties that were remarkably similar to those of the six aforementioned isolates. In this paper, we describe the characteristics of all of the strains belonging to this group of nitrogen-fixing bacilli. MATERIALS AND METHODSBacterial strains. The strains used and their...

The hypothesis that sweet potato genotypes containing different starch yields in their tuberous roots can affect the bacterial communities present in the rhizosphere (soil adhering to tubers) was tested in this study. Tuberous roots of field-grown sweet potato of genotypes IPB-149 (commercial genotype), IPB-052, and IPB-137 were sampled three and six months after planting and analyzed by denaturing gradient gel electrophoresis (DGGE) and pyrosequencing analysis of 16S rRNA genes PCR-amplified from total community DNA. The statistical analysis of the DGGE fingerprints showed that both plant age and genotypes influenced the bacterial community structure in the tuber rhizosphere. Pyrosequencing analysis showed that the IPB-149 and IPB-052 (both with high starch content) displayed similar bacterial composition in the tuber rhizosphere, while IPB-137 with the lowest starch content was distinct. In comparison with bulk soil, higher 16S rRNA gene copy numbers (qPCR) and numerous genera with significantly increased abundance in the tuber rhizosphere of IPB-137 (Sphingobium, Pseudomonas, Acinetobacter, Stenotrophomonas, Chryseobacterium) indicated a stronger rhizosphere effect. The genus Bacillus was strongly enriched in the tuber rhizosphere samples of all sweet potato genotypes studied, while other genera showed a plant genotype-dependent abundance. This is the first report on the molecular identification of bacteria being associated with the tuber rhizosphere of different sweet potato genotypes.

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