Azospirillum Genomes Reveal Transition of Bacteria from Aquatic to Terrestrial Environments

Fossil records indicate that life appeared in marine environments ∼3.5 billion years ago (Gyr) and transitioned to terrestrial ecosystems nearly 2.5 Gyr. Sequence analysis suggests that “hydrobacteria” and “terrabacteria” might have diverged as early as 3 Gyr. Bacteria of the genus Azospirillum are associated with roots of terrestrial plants; however, virtually all their close relatives are aquatic. We obtained genome sequences of two Azospirillum species and analyzed their gene origins. While most Azospirillum house-keeping genes have orthologs in its close aquatic relatives, this lineage has obtained nearly half of its genome from terrestrial organisms. The majority of genes encoding functions critical for association with plants are among horizontally transferred genes. Our results show that transition of some aquatic bacteria to terrestrial habitats occurred much later than the suggested initial divergence of hydro- and terrabacterial clades. The birth of the genus Azospirillum approximately coincided with the emergence of vascular plants on land

The control of Azorhizobium caulinodans nifA expression by oxygen, ammonia and by the HF‐I‐like protein, NrfA

International audienceThe control of Azorhizobium caulinodans nifA expression in response to oxygen and ammonia involves FixLJ, FixK, NtrBC, NtrXY and the HF-I-like protein NrfA. The regulation is thus complex and possibly involves post-transcriptional regulation by NrfA. The coding region of nifA was determined using a translational lacZ fusion and by site-directed mutagenesis to identify which of four in frame AUG codons was used. The major NifA protein is translated from the second AUG codon and is predicted to consist of 613 amino acids. Primer extension analysis showed a major transcript starting 34 bp downstream from the anaerobox in wild-type, nifA, rpoN, ntrC and nrfA strains, but not in a fixK mutant. FixK- and oxygen-dependent transcription of nifA was confirmed by the analysis of four transcriptional nifA-lacZ fusions with fusion junctions at positions +1, +47, +110 and +181 with respect to the start site. Regulation by ammonia was independent of FixK and RpoN, NtrC being only partially required. Thus, there may be another type of nitrogen control that does not involve NtrC in A. caulinodans. NrfA is not required for the initiation of nifA transcription but, most probably, has an effect on nifA mRNA stability and/or translation. NrfA also restores the defect in rpoS translation to an Escherichia coli hfq mutant, indicating that HF-I and NrfA have similar activities in both A. caulinodans and E. coli

Nucleotide sequence of the fixABC region of Azorhizobium caulinodans ORS571: similarity of the fixB product with eukaryotic flavoproteins, characterization of fixX, and identification of nifW

International audienceThe nucleotide sequence of a 4.1 kb DNA fragment containing the fixABC region of Azorhizobium caulinodans was established. The three gene products were very similar to the corresponding polypeptides of Rhizobium meliloti. The C-terminal domains of both fixB products displayed a high degree of similarity with the alpha-subunits of rat and human electron transfer flavoproteins, suggesting a role for the FixB protein in a redox reaction. Two open reading frames (ORF) were found downstream of fixC. The first ORF was identified as fixX on the basis of sequence homology with fixX from several Rhizobium and Bradyrhizobium strains. The second ORF potentially encoded a 69 amino acid product and was found to be homologous to a DNA region in the Rhodobacter capsulatus nif cluster I. Insertion mutagenesis of the A. caulinodans fixX gene conferred a Nif- phenotype to bacteria growth in the free-living state and a Fix- phenotype in symbiotic association with the host plant Sesbania rostrata. A crude extract from the fixX mutant had no nitrogenase activity. Furthermore, data presented in this paper also indicate that the previously identified nifO gene located upstream of fixA was probably a homologue of the nifW gene of Klebsiella pneumoniae and Azotobacter vinelandii

Nitrogen-fixing bacteria associated with leguminous and non-leguminous plants

Nitrogen is generally considered one of the major limiting nutrients in plant growth. The biological process responsible for reduction of molecular nitrogen into ammonia is referred to as nitrogen fixation. A wide diversity of nitrogen-fixing bacterial species belonging to most phyla of the Bacteria domain have the capacity to colonize the rhizosphere and to interact with plants. Leguminous and actinorhizal plants can obtain their nitrogen by association with rhizobia or Frankia via differentiation on their respective host plants of a specialized organ, the root nodule. Other symbiotic associations involve heterocystous cyanobacteria, while increasing numbers of nitrogen-fixing species have been identified as colonizing the root surface and, in some cases, the root interior of a variety of cereal crops and pasture grasses. Basic and advanced aspects of these associations are covered in this review

Control of Azospirillum brasilense NifA activity by P II : effect of replacing Tyr residues of the NifA N-terminal domain on NifA activity

International audienceIt was previously reported that the N-terminal domain of Azospirillum brasilense NifA was a negative regulator of the NifA activity and that the PII protein prevented this inhibition under nitrogen fixing conditions. Here, we show that a mutation of a single Tyr residue at position 18 of the N-terminal domain of NifA led to an active NifA protein that did not require PII for activation under nitrogen fixation conditions

Transcriptional analysis of the fix ABCXORF1 region of Azorhizobium caulinodans suggests post-transcriptional processing of the fix ABCXORF1 mRNA

International audienceWe report here the transcriptional analysis of the fixABCXORF1 region of Azorhizobium caulinodans. This led to the identification of a 0.9 kb transcript covering fixX and ORF1, which was synthesized only under conditions of nitrogen fixation. The 5' end of this transcript was mapped by primer extension and S1 nuclease protection analyses and shown to be located 70 +/- 1 nucleotides upstream of the fixX start codon. By means of transcriptional fixX- and ORF1-lacZ fusions, it was shown that fixX and ORF1 were most probably transcribed from the fixA promoter and that expression of fixX and ORF1 was dependent on NifA activation. This suggests that the 0.9 kb mRNA results from post-transcriptional processing of a large mRNA covering fixA,B,C,X and ORF1. In addition, ORF1 mutants were constructed and were shown not to be impaired in nitrogenase activity

A Transcriptional Regulator of the LuxR-UhpA Family, FlcA, Controls Flocculation and Wheat Root Surface Colonization by 'Azospirillum brasilense' Sp7

Genetic complementation of a spontaneous mutant, impaired in flocculation, Congo red binding, and colonization of root surface, led to the identification of a new regulatory gene in 'Azospirillum brasilense' Sp7, designated 'flcA'. The deduced amino acid sequence of 'flcA' shared high similarity with a family of transcriptional activators of the LuxR-UhpA family. The most significant match was with the AgmR protein, an activator for glycerol metabolism in 'Pseudomonas aeruginosa'. Derivatives of Sp7 resulting from site-directed Tn5 mutagenesis in the 'flcA' coding sequence were constructed by marker exchange. Characterization of the resulting mutant strains showed that 'flcA' controls the production of capsular polysaccharides, the flocculation process in culture, and the colonization of the root surface of wheat. This study provides new information on the genetic control of the mechanism of plant root colonization by 'Azospirillum'

A cheZ-Like Gene in Azorhizobium caulinodans Is a Key Gene in the Control of Chemotaxis and Colonization of the Host Plant

Chemotaxis can provide bacteria with competitive advantages for survival in complex environments. The CheZ chemotaxis protein is a phosphatase, affecting the flagellar motor in Escherichia coli by dephosphorylating the response regulator phosphorylated CheY protein (CheY similar to P) responsible for clockwise rotation. A cheZ gene has been found in Azorhizobium caulinodans ORS571, in contrast to other rhizobial species studied so far. The CheZ protein in strain ORS571 has a conserved motif similar to that corresponding to the phosphatase active site in E. coli. The construction of a cheZ deletion mutant strain and of cheZ mutant strains carrying a mutation in residues of the putative phosphatase active site showed that strain ORS571 participates in chemotaxis and motility, causing a hyperreversal behavior. In addition, the properties of the cheZ deletion mutant revealed that ORS571 CheZ is involved in other physiological processes, since it displayed increased flocculation, biofilm formation, exopolysaccharide (EPS) production, and host root colonization. In particular, it was observed that the expression of several exp genes, involved in EPS synthesis, was upregulated in the cheZ mutant compared to that in the wild type, suggesting that CheZ negatively controls exp gene expression through an unknown mechanism. It is proposed that CheZ influences the Azorhizobium-plant association by negatively regulating early colonization via the regulation of EPS production. This report established that CheZ in A. caulinodans plays roles in chemotaxis and the symbiotic association with the host plant. IMPORTANCE Chemotaxis allows bacteria to swim toward plant roots and is beneficial to the establishment of various plant-microbe associations. The level of CheY phosphorylation (CheY similar to P) is central to the chemotaxis signal transduction. The mechanism of the signal termination of CheY similar to P remains poorly characterized among Alphaproteobacteria, except for Sinorhizobium meliloti, which does not contain CheZ but which controls CheY similar to P dephosphorylation through a phosphate sink mechanism. Azorhizobium caulinodans ORS571, a microsymbiont of Sesbania rostrata, has an orphan cheZ gene besides two cheY genes similar to those in S. meliloti. In addition to controlling the chemotaxis response, the CheZ-like protein in strain ORS571 is playing a role by decreasing bacterial adhesion to the host plant, in contrast to the general situation where chemotaxis-associated proteins promote adhesion. In this study, we identified a CheZ-like protein among Alphaproteobacteria functioning in chemotaxis and the A. caulinodans-S. rostrata symbiosis