A DNA macroarray was developed and evaluated for its potential to distinguish variants of the dinitrogenase reductase (nifH) gene. Diverse nifH gene fragments amplified from a clone library were spotted onto nylon membranes. Amplified, biotinylated nifH fragments from individual clones or a natural picoplankton community were hybridized to the array and detected by chemiluminescence. A hybridization test with six individual targets mixed in equal proportions resulted in comparable relative signal intensities for the corresponding probes (standard deviation, 14%). When the targets were mixed in unequal concentrations, there was a predictable, but nonlinear, relationship between target concentration and relative signal intensity. Results implied a detection limit of roughly 13 pg of target ml(-1), a half-saturation of signal at 0.26 ng ml(-1), and a dynamic range of about 2 orders of magnitude. The threshold for cross-hybridization varied between 78 and 88% sequence identity. Hybridization patterns were reproducible with significant correlations between signal intensities of duplicate probes (r = 0.98, P < 0.0001, n = 88). A mixed nifH target amplified from a natural Chesapeake Bay water sample hybridized strongly to 6 of 88 total probes and weakly to 17 additional probes. The natural community results were well simulated (r = 0.941, P < 0.0001, n = 88) by hybridizing a defined mixture of six individual targets corresponding to the strongly hybridizing probes. Our results indicate that macroarray hybridization can be a highly reproducible, semiquantitative method for assessing the diversity of functional genes represented in mixed pools of PCR products amplified from the environment.

The relationships of the genus Azotobacter, Azomonas macrocytogenes and the genus Pseudomonas were revealed by comparative analysis of partial 16S rRNA and atpD, carA and recA gene sequences and as concatenated nucleotide and peptide sequences. Sequence similarities of Azotobacter species and Azomonas macrocytogenes indicated that these may be considered to be synonyms at the molecular level. In addition, these species show an intimate relationship with species of Pseudomonas, especially P. aeruginosa (the type species of the genus). In terms of the current circumscription of the genus Pseudomonas, Azotobacter and Azomonas macrocytogenes should be considered for amalgamation with Pseudomonas. Azotobacter and Azomonas comprise nitrogen-fixing strains with large pleomorphic cells that form cysts, and peritrichous flagella insertion; characteristics not included in the current circumscription of Pseudomonas. The data are discussed in the light of whether lateral transfer of genes could be involved in the determination of significant morphological characteristics, thus leading to a problem that may be encountered more frequently: how to resolve classification of taxa based on conserved sequences with those based on their phenotype. More fundamentally, the results illuminate problems that will increasingly be encountered: by what criteria can taxa be delineated, what are the most appropriate methods for classification, and what are the proper assumptions of bacterial classification?

The diversity of endophytic and epiphytic diazotrophs in different parts of rice plants has specificity to the niche (i.e. leaf, stem and root) of different genotypes and nutrient availability of the organ. Inoculation of the indigenous, polyvalent diazotrophs can facilitate and sustain production of non-leguminous crops like rice. Therefore, N2-fixing plant growth promoting bacteria (PGPB) were isolated from different parts of three Indian cultivated [Oryza sativa L. var. Sabita (semi deep/deep water)/Swarna (rain fed shallow lowland)/Swarna-Sub1(submergence tolerant)] and a wild (O. eichingeri) rice genotypes which respond differentially to nitrogenous fertilizers. Thirty-five isolates from four rice genotypes were categorized based on acetylene reduction assay on nitrogenase activity, biochemical tests, BIOLOG and 16S rRNA gene sequencing. The bacteria produced 9.36-155.83 nmole C2H4 mg(-1) dry bacteria h(-1) and among them nitrogenase activity of 11 potent isolates was complemented by nifH-sequence analysis. Phylogenetic analysis based on 16S rDNA sequencing divided them into five groups (shared 95-100 % sequence homology with type strains) belonging to five classes-alpha (Ancylobacter, Azorhizobium, Azospirillum, Rhizobium, Bradyrhizobium, Sinorhizobium, Novosphingobium, spp.), beta (Burkholderia sp.), gamma (Acinetobacter, Aeromonas, Azotobacter, Enterobacter, Klebsiella, Pantoea, Pseudomonas, Stenotrophomonas spp.) Proteobacteria, Bacilli (Bacillus, Paenibacillus spp.) and Actinobacteria (Microbacterium sp.). Besides, all bacterial strains possessed the intrinsic PGP traits of like indole (0.44-7.4 ?g ml(-1)), ammonia (0.18-6 mmol ml(-1)), nitrite (0.01-3.4 mol ml(-1)), and siderophore (from 0.16-0.57 �gmol ml(-1)) production. Inoculation of rice (cv. Swarna) seedlings with selected isolates had a positive impact on plant growth parameters like shoot and root elongation which was correlated with in vitro PGP attributes. The results indicated that the diverse polyvalent phytonic PGP bacteria, which may be exploited as bio-inoculants to improve rice production.

The nucleotide sequence spanning the nifH gene and part of the nifD gene encoding the molybdenum nitrogenase from Azotobacter chroococcum was determined. The transcription start point of the nifH promoter was mapped, and a potential transcriptional attenuator was located between the nifH and nifD genes.

Hydrogen-uptake (Hup) activity in Azotobacter chroococcum depends upon a cluster of genes spread over 13,687 bp of the chromosome. Six accessory genes of the cluster, hupABYCDE, begin 4.8 kb downstream of the structural genes, hupSL, and are required for the formation of a functional [NiFe] hydrogenase. The sequencing of the intervening 4.8 kb of hup-specific DNA has now been completed. This revealed eight additional closely linked ORFs, which we designated hupZ, hupM, hupN, hupO, hupQ, hupR, hupT and hupV. These genes potentially encode polypeptides with predicted masses of 27.7, 22.3, 11.4, 16.2, 31.3, 8.1, 16.2 and 36.7 kDa, respectively. All eight genes are transcribed from the same strand as hupSL and hupABYCDE. A chroococcum, therefore, has a total of 16 contiguous genes affecting hydrogenase activity beginning with hupS and ending with hupE. The amino acid sequence deduced from hupZ has the characteristics of a b-type cytochrome. Insertion mutagenesis of hupZ resulted in a mutant incapable of supporting O2-dependent H2 oxidation. The deduced amino acid sequence of hupR shares high homology with bacterial rubredoxins. HupZ and HupR may both be involved in transferring electrons from hydrogenase to the electron transport chain. A mutation in hupV knocked out hydrogenase activity entirely; this gene may be involved in processing the large subunit of hydrogenase. It is now clear that the genes controlling [NiFe] hydrogenase activity in many bacteria including Azotobacter chroococcum, Alcaligenes eutrophus, Rhizobium leguminosarum, Rhodobacter capsulatus and Escherichia coli are highly conserved, organized in much the same manner, and likely derived from a common ancestor.

The results presented extend previous investigations on the genetics of nitrogen fixation in Azotobacter chroococcum and indicate that nif- and fix-like DNA is located in at least five different regions of the genome. Region I contains functional copies of nifS,V and M, as well as nifH, D and K, all of which complemented mutants of Klebsiella pneumoniae. In addition, nifE- and/or nifN-like and nifU-like DNA is located in this region. The organization of the nif cluster in region I closely resembles that of K. pneumoniae. though spread over 22 kb as compared with 14 kb. Region II contains a functional nifB gene, which complemented a K. pneumoniae nifB mutant, and seems to be adjacent to ap nifA-like gene. Region III harbours nifH*, encoding a second nitrogenase Fe-protein. Region IV contains a reiteration of nifE- on and/or nifN-like sequences, and DNA homologous to Rhizobium meliloti fixABC is present in region V. The apparent complexity of nifDNA in A. chroococcum is probably related to the two systems for N2-fixation pr present in this organism.

In our endeavor to improve the nitrogen fixation efficiency of a soil diazotroph that would be unaffected by synthetic nitrogenous fertilizers, we have deleted a part of the negative regulatory gene nifL and constitutively expressed the positive regulatory gene nifA in the chromosome of Azotobacter chroococcum CBD15, a strain isolated from the local field soil. No antibiotic resistance gene or other foreign gene was present in the chromosome of the engineered strain. Wheat seeds inoculated with this engineered strain, which we have named Azotobacter chroococcum HKD15, were tested for 3 years in pots and 1 year in the field. The yield of wheat was enhanced by ?60% due to inoculation of seeds by A. chroococcum HKD15 in the absence of any urea application. Ammonium only marginally affected acetylene reduction by the engineered Azotobacter strain. When urea was also applied, the same wheat yield could be sustained by using seeds inoculated with A. chroococcum HKD15 and using ?85 kg less urea (?40 kg less nitrogen) than the usual ?257 kg urea (?120 kg nitrogen) per hectare. Wheat plants arising from the seeds inoculated with the engineered Azotobacter strain exhibited far superior overall performance, had much higher dry weight and nitrogen content, and assimilated molecular 15N much better. A nitrogen balance experiment also revealed much higher total nitrogen content. Indole-3-acetic acid (IAA) production by the wild type and that by the engineered strain were about the same. Inoculation of the wheat seeds with A. chroococcum HKD15 did not adversely affect the microbial population in the field rhizosphere soil.IMPORTANCE Application of synthetic nitrogenous fertilizers is a standard agricultural practice to augment crop yield. Plants, however, utilize only a fraction of the applied fertilizers, while the unutilized fertilizers cause grave environmental problems. Wild-type soil diazotrophic microorganisms cannot replace synthetic nitrogenous fertilizers, as these reduce atmospheric nitrogen very inefficiently and almost none at all in the presence of added nitrogenous fertilizers. If the nitrogen-fixing ability of soil diazotrophs could be improved and sustained even in the presence of synthetic nitrogenous fertilizers, then a mixture of the bacteria and a reduced quantity of chemical nitrogenous fertilizers could be employed to obtain the same grain yield but at a much-reduced environmental cost. The engineered Azotobacter strain that we have reported here has considerably enhanced nitrogen fixation and excretion abilities and can replace ?85 kg of urea per hectare but sustain the same wheat yield, if the seeds are inoculated with it before sowing.