Bacillus azotofixans sp. nov., a Nitrogen-Fixing Species from Brazilian Soils and Grass Roots

Abstract

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...

Paenibacillus borealis sp. nov., a nitrogen-fixing species isolated from spruce forest humus in Finland.

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