Molecular biological taxonomy of some free-living nitrogen-fixing bacteria

A novel approach to the taxonomy of several free-living nitrogen-fixing bacteria is proposed on the basis of two DNA parameters. 1) DNA base composition, expressed as average molar (guanine + cytosine) content, determined by thermal denaturation and 2) DNA homology, determined by DNA hybridization with bothPseudomonas fluorescens andPseudomonas putida. The following taxonomic conclusions emerged:

1. 1.
   The existence ofBeijerinckia andDerxia as individual genera seems justified.     
   
   

Multiple roles of siderophores in free-living nitrogen-fixing bacteria

Free-living nitrogen-fixing bacteria in soils need to tightly regulate their uptake of metals in order to acquire essential metals (such as the nitrogenase metal cofactors Fe, Mo and V) while excluding toxic ones (such as W). They need to do this in a soil environment where metal speciation, and thus metal bioavailability, is dependent on a variety of factors such as organic matter content, mineralogical composition, and pH. Azotobacter vinelandii, a ubiquitous gram-negative soil diazotroph, excretes in its external medium catechol compounds, previously identified as siderophores, that bind a variety of metals in addition to iron. At low concentrations, complexes of essential metals (Fe, Mo, V) with siderophores are taken up by the bacteria through specialized transport systems. The specificity and regulation of these transport systems are such that siderophore binding of excess Mo, V or W effectively detoxifies these metals at high concentrations. In the topsoil (leaf litter layer), where metals are primarily bound to plant-derived organic matter, siderophores extract essential metals from natural ligands and deliver them to the bacteria. This process appears to be a key component of a mutualistic relationship between trees and soil diazotrophs, where tree-produced leaf litter provides a living environment rich in organic matter and micronutrients for nitrogen-fixing bacteria, which in turn supply new nitrogen to the ecosystem.     

Chemotaxis of free-living nitrogen-fixing bacteria towards maize mucilage

Summary A method for collecting sterile mucilage from maize root tips growing in sterile conditions has been devised.Enterobacter andAzospirillum strains were isolated from the rhizosphere of maize and rice using the spermosphere model method. To evaluate chemotaxis of these strains, a modification of Adler's microcapillary method was used. Under these conditions, the number of attracted bacteria was proportional to the concentration of mucilage. When comparing the chemotaxis ofA. lipoferum andE. cloacae from the rhizosphere of maize and from the rhizosphere of rice, it appeared that the strains isolated from maize were strongly attracted by maize mucilage whereas strains isolated from rice were not more attracted than the control (E. coli K12). Thus, bacteria of the same species are not equivalent in their chemotactic behaviour. This could imply that some degree of specificity exists in the establishment of plant-bacteria associations.     

DNA base composition and homology values in the classification of some Rhodococcus species

Similar DNA homology values were recorded when a modified S1 nuclease technique and a standard nitrocellulose membrane filter method were applied to representative strains of Rhodococcus. The DNA homology data showed that R. globerulus, R. luteus and R. sputi form distinct genomic species. The congruence between the DNA homology and earlier numerical phenetic data was good, but there was evidence that some strains had been misclassified in the previous studies. In particular, the type strains of R. obuensis and R. sputi belong to a single genomic species. The former name is thus a later, subjective synonym of the latter. The guanine plus cytosine content of the DNA of the rhodococci fell within the range 61 to 72 mol%.     

Biochemical and physiological studies with free-living, nitrogen-fixing bacteria

Summary 1. Rumen fluid from four sheep, one on a low nitrogen diet, showed slight acetylene reduction.Desulfotomaculum ruminis, a rumen anaerobe, fixes N₂ but the effective organisms in rumen samples seem to resembleClostridium pasteurianum; this organism can persist in the sheep rumen. In domestic sheep the contribution of rumen fixation to the animal's N-nutrition is probably negligible; other ruminants on various diets require study. 2. Respiration inAzotobacter species functions partly to protect nitrogenase from interference by oxygen. When such ‘respiratory protection’ of nitrogenase fails, the organisms reversibly ‘switch off’ nitrogenase activity, a process attributed to a change in the conformation of the nitrogenase components. When this ‘conformational protection’ fails, irreversible damage to the oxygen-sensitive protein 2 (Fe protein) of nitrogenase occurs and can be demonstrated with cell-free extracts. 3. Protein 1 (Mo-Fe protein) and protein 2 (Fe protein) ofKlebsiella pneumoniae nitrogenase, labelled with Fe⁵⁷, show M?ssbauer resonances tentatively assigned to ferrous and ferric iron. In mixtures, these are additive unless both ATP and Na₂S₂O₄ (the components necessary for enzymic activity) are present, when changes take place, including the appearance of a new doublet at −0.85 and +2.2 mm/sec. Permutation of labelled and unlabelled proteins indicates that the major change occurs in protein 1. N₂, C₂H₂, CN⁻ or CO altered the intensity of an absorption at +2.8 mm/sec attributable to protein 2. Hence activation of the N₂-ase complex involves changes in the environment of Fe but no resonances assignable to Fe-substrate binding appear.     

Production of amino acids by free-living heterotrophic nitrogen-fixing bacteria

Summary Interest of microbial production of amino acids has been increased greatly since development of biotechnological methods. These methods represent a perspective way applied in a future large-scale manufacture of inexpensive amino acids. In this context, the isolation of producing organisms that may be exploited in the desing of alternative methods for the production of amino acids could be of primary importance.In this review we will describe the liberation of amino acids (methionine, lysine, arginine, tryptophane and glutamic acid) byAzotobacter andAzospirillum during growth in culture media with different carbon sources under diazotrophic and adiazotrophic conditions. These organisms may be useful in developing new methods for the industrial production of amino acids.     

Superoxide dismutases of Azotobacter vinelandii and other aerobic, free-living nitrogen-fixing bacteria

Abstract Cell-free extracts obtained from nitrogen-fixing cells of organisms of the family Azotobacteriaceae were analyzed for superoxide dismutases (SOD). For all the representative organisms examined, unusually high specific activities for SOD were recorded. Single Fe-containing SOD were found in Azotobacter vinelandii (5 strains), A. chroococcum (2 strains), A. beijerinckii (1 strain), Azomonas macrocytogenes (2 strains), and Derxia gummosa (2 strains). Highly active, single Mn-containing SOD were found in all of 6 Beijerinckia strains examined ( B. indica, B. lacticogenes, B. mobilis, B. fluminensis, B. derxii and B. venezuelae ). Multiple SOD were found for Azomonas agilis (Mn-SOD and 2 Fe-SOD), A. insignis (Mn-SOD, Fe-SOD) and Azospirillum lipoferum (2 Fe-SOD) and Azospirillum brasilense (Mn-SOD and 2 Fe-SOD).     

Diversity and activity of free-living nitrogen-fixing bacteria and total bacteria in organic and conventionally managed soils

Agricultural soils are heterogeneous environments in which conditions affecting microbial growth and diversity fluctuate widely in space and time. In this study, the molecular ecology of the total bacterial and free-living nitrogen-fixing communities in soils from the Nafferton Factorial Systems Comparison (NFSC) study in northeast England were examined. The field experiment was factorial in design, with organic versus conventional crop rotation, crop protection, and fertility management factors. Soils were sampled on three dates (March, June, and September) in 2007. Total RNA was extracted from all soil samples and reverse transcribed. Denaturing gradient gel electrophoresis (DGGE) and quantitative PCR (qPCR) were used to analyze nifH and 16S rRNA genes in order to study free-living diazotrophs and the total bacterial community, respectively. Crop rotation was shown to have a significant effect on total bacterial diversity (and that of free-living N fixers) (P ≤ 0.001). On all three dates, nifH activity was higher in the conventional crop rotation. In contrast, qPCR analysis of free-living N fixers indicated significantly higher levels of activity in conventionally fertilized plots in June (P = 0.0324) and in plots with organic crop protection in September (P = 0.0143). To our knowledge, the effects of organic and conventional farming systems on free-living diazotrophs have never been studied. An increased understanding of the impacts of management practices on free-living N fixers could allow modifications in soil management practices to optimize the activity of these organisms.     

Accumulation of ppGpp in symbiotic and free-living nitrogen-fixing bacteria following amino acid starvation

Following amino acid or ammonium starvation, ppGpp is accumulated by Rhizobium meliloti strain 1021 but not by R. meliloti strain 41 or Rhizobium tropici. Azorhizobium caulinodans ORS571 produced ppGpp following amino acid deprivation; however, the free-living nitrogen-fixing bacteria Azotobacter vinelandii and Azomonas agilis did not produce ppGpp. Western blot analysis using anti-RelA antibody demonstrated that R. meliloti strain 1021, Azotobacter vinelandii and Azorhizobium caulinodans cross-reacted under conditions that detected RelA in Escherichia coli CF1648. Cross-reaction was not observed in R. meliloti strain 41, R. tropici, or Azomonas agilis. All strains that accumulated ppGpp also produced high intracellular levels of ATP. Received: 28 August 1998 / Accepted: 11 November 1998     

DNA homology in various strains of nitrogen-fixing bacteria

Melting temperature and GC content were evaluated for DNA of some nitrogen-fixing bacteria of Rhizobium leguminosarum and Onobrychis spp. (Adans). The degree of homology between strains of the same species was determined. A combination of thermal denaturing and molecular hybridization can serve as a rapid test for evaluating the genome homology of the organisms compared.     

Amplified ribosomal DNA restriction analysis for the characterization of Azotobacteraceae: a contribution to the study of these free-living nitrogen-fixing bacteria

A 16S rRNA gene-based fingerprinting method was developed for the identification of Azotobacteraceae and tested onto 48 soil isolates and 28 reference strains belonging to the free-living nitrogen-fixing bacterial group and to the most common species found in soil samples. According to this method, the 16S rRNA gene was amplified using universal primers for Eubacteria and PCR products were subsequently digested with RsaI, HhaI, HpaII, FnuDII, and AluI. The analysis of the restriction profiles obtained showed that the method is able to define a unique species-specific phylotype (SSP) for each of the eight Azotobacteraceae species tested. Cluster analysis was successfully employed for the identification of members of the family Azotobacteraceae, being assignation into species of the isolates confirmed by means of partial 16S rRNA gene sequencing.     

DNA base composition, nature of intracellular DNA, morphology, and classification of bacteriophages infecting Micrococcus luteus

Ten bacteriophages infecting Micrococcus luteus have been characterized. All phages contain double-stranded DNA, of 64.3--73.5 mol% guanine plus cytosine (GC). The DNA of phage N7 has the highest GC content reported for any bacterial virus. No unusual bases have been found. The intracellular replicating DNAs of six phages are covalently closed circular molecules. All 10 phages have isometric, probably icosahedral, heads and long, flexible, noncontractile tails and can be sorted into two morphological groups based on size and presence or absence of a collar. Host-range studies indicate six host-range groups.