Mechanisms supporting aminoadenine-based viral DNA genomes.

Bacteriophage genomes are the richest source of modified nucleobases of any life form. Of these, 2,6 diaminopurine, which pairs with thymine by forming three hydrogen bonds violates Watson and Crick's base pairing. 2,6 diaminopurine initially found in the cyanophage S-2L is more widespread than expected and has also been detected in phage infecting Gram-negative and Gram-positive bacteria. The biosynthetic pathway for aminoadenine containing DNA as well as the exclusion of adenine are now elucidated. This example of a natural deviation from the genetic code represents only one of the possibilities explored by nature and provides a proof of concept for the synthetic biology of non-canonical nucleic acids.

Genetic and biochemical characterization of Salmonella enterica serovar typhi deoxyribokinase.

We identified in the genome of Salmonella enterica serovar Typhi the gene encoding deoxyribokinase, deoK. Two other genes, vicinal to deoK, were determined to encode the putative deoxyribose transporter (deoP) and a repressor protein (deoQ). This locus, located between the uhpA and ilvN genes, is absent in Escherichia coli. The deoK gene inserted on a plasmid provides a selectable marker in E. coli for growth on deoxyribose-containing medium. Deoxyribokinase is a 306-amino-acid protein which exhibits about 35% identity with ribokinase from serovar Typhi, S. enterica serovar Typhimurium, or E. coli. The catalytic properties of the recombinant deoxyribokinase overproduced in E. coli correspond to those previously described for the enzyme isolated from serovar Typhimurium. From a sequence comparison between serovar Typhi deoxyribokinase and E. coli ribokinase, whose crystal structure was recently solved, we deduced that a key residue differentiating ribose and deoxyribose is Met10, which in ribokinase is replaced by Asn14. Replacement by site-directed mutagenesis of Met10 with Asn decreased the V(max) of deoxyribokinase by a factor of 2.5 and increased the K(m) for deoxyribose by a factor of 70, compared to the parent enzyme.

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

It was previously reported that the N-terminal domain of Azospirillum brasilense NifA was a negative regulator of the NifA activity and that the P(II) 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 P(II) for activation under nitrogen fixation conditions.

Azorhizobium caulinodans PII and GlnK proteins control nitrogen fixation and ammonia assimilation.

We herein report that Azorhizobium caulinodans PII and GlnK are not necessary for glutamine synthetase (GS) adenylylation whereas both proteins are required for complete GS deadenylylation. The disruption of both glnB and glnK resulted in a high level of GS adenylylation under the condition of nitrogen fixation, leading to ammonium excretion in the free-living state. PII and GlnK also controlled nif gene expression because NifA activated nifH transcription and nitrogenase activity was derepressed in glnB glnK double mutants, but not in wild-type bacteria, grown in the presence of ammonia.

Poly-beta-hydroxybutyrate turnover in Azorhizobium caulinodans is required for growth and affects nifA expression.

Azorhizobium caulinodans is able to fix nitrogen in the free-living state and in symbiosis with the tropical legume Sesbania rostrata. The bacteria accumulate poly-beta-hydroxybutyrate (PHB) under both conditions. The structural gene for PHB synthase, phbC, was inactivated by insertion of an interposon. The mutant strains obtained were devoid of PHB, impaired in their growth properties, totally devoid of nitrogenase activity ex planta (Nif-), and affected in nucleotide pools and induced Fix- nodules devoid of bacteria. The Nif- phenotype was the consequence of the lack of nifA transcription. Nitrogenase activity was partially restored to a phbC mutant by constitutive expression of the nifA gene. However, this constitutive nifA expression had no effect on the nucleotide content or on growth of the phbC mutant. It is suggested that PHB is required for maintaining the reducing power of the cell and therefore the bacterial growth. These observations also suggest a new control of nifA expression to adapt nitrogen fixation to the availability of carbon and reducing equivalents.

Characterisation of the glnK-amtB operon and the involvement of AmtB in methylammonium uptake in Azorhizobium caulinodans.

This work reports the characterisation of the Azorhizobium caulinodans amtB gene, the deduced protein sequence of which shares similarity to those of several ammonium transporters. amtB is located downstream from glnK, a glnB-like gene. It is cotranscribed with glnK from an NtrC- and sigma54-dependent promoter. glnK and amtB insertion mutant strains have been isolated. Methylammonium uptake was assayed in these strains and in other mutant strains in which the regulation of nitrogen metabolism is impaired. Our data suggest that the AmtB protein is an ammonium transporter, which is mainly regulated by NtrC in response to nitrogen availability.

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

The 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.

Characterization of Azorhizobium caulinodans glnB and glnA genes: involvement of the P(II) protein in symbiotic nitrogen fixation.

The nucleotide sequence and transcriptional organization of Azorhizobium caulinodans ORS571 glnA, the structural gene for glutamine synthetase (GS), and glnB, the structural gene for the P(II) protein, have been determined. glnB and glnA are organized as a single operon transcribed from the same start site, under conditions of both nitrogen limitation and nitrogen excess. This start site may be used by two different promoters since the expression of a glnB-lacZ fusion was high in the presence of ammonia and enhanced under conditions of nitrogen limitation in the wild-type strain. The increase was not observed in rpoN or ntrC mutants. In addition, this fusion was overexpressed under both growth conditions, in the glnB mutant strain, suggesting that P(II) negatively regulates its own expression. A DNA motif, similar to a sigma54-dependent promoter consensus, was found in the 5' nontranscribed region. Thus, the glnBA operon seems to be transcribed from a sigma54-dependent promoter that operates under conditions of nitrogen limitation and from another uncharacterized promoter in the presence of ammonia. Both glnB and glnBA mutant strains derepress their nitrogenase in the free-living state, but only the glnBA mutant, auxotrophic for glutamine, does not utilize molecular nitrogen for growth. The level of GS adenylylation is not affected in the glnB mutant as compared to that in the wild type. Under symbiotic conditions, the glnB and glnBA mutant strains induced Fix- nodules on Sesbania rostrata roots. P(II) is the first example in A. caulinodans of a protein required for symbiotic nitrogen fixation but dispensable in bacteria growing in the free-living state.

Azorhizobium caulinodans uses both cytochrome bd (quinol) and cytochrome cbb3 (cytochrome c) terminal oxidases for symbiotic N2 fixation.

Azorhizobium caulinodans employs both cytochrome bd (cytbd; quinol oxidase) and cytcbb3 (cytc oxidase) as terminal oxidases in environments with very low O2 concentrations. To investigate physiological roles of these two terminal oxidases both in microaerobic culture and in symbiosis, knockout mutants were constructed. As evidenced by visible absorbance spectra taken from mutant bacteria carrying perfect gene replacements, both the cytbd- and cytcbb3- mutations were null alleles. In aerobic culture under 2% O2 atmosphere, Azorhizobium cytbd- and cytcbb3- single mutants both fixed N2 at 70 to 90% of wild-type rates; in root nodule symbiosis, both single mutants fixed N2 at 50% of wild-type rates. In contrast, Azorhizobium cytbd- cytcbb3-double mutants, which carry both null alleles, completely lacked symbiotic N2 fixation activity. Therefore, both Azorhizobium cytbd and cytcbb3 oxidases drive respiration in environments with nanomolar O2 concentrations during symbiotic N2 fixation. In culture under a 2% O2 atmosphere, Azorhizobium cytbd- cytcbb3- double mutants fixed N2 at 70% of wild-type rates, presumably reflecting cytaa3 and cytbo (and other) terminal oxidase activities. In microaerobic continuous cultures in rich medium, Azorhizobium cytbd- and cytcbb3- single mutants were compared for their ability to deplete a limiting-O2 sparge; cytbd oxidase activity maintained dissolved O2 at 3.6 microM steady state, whereas cytcbb3 oxidase activity depleted O2 to submicromolar levels. Growth rates reflected this difference; cytcbb3 oxidase activity disproportionately supported microaerobic growth. Paradoxically, in O2 limited continuous culture, Azorhizobium cytbd oxidase is inactive below 3.6 microM dissolved O2 whereas in Sesbania rostrata symbiotic nodules, in which physiological, dissolved O2 is maintained at 10 to 20 nM, both Azorhizobium cytbd and cytcbb3 seem to contribute equally as respiratory terminal oxidases.

Modulation of NifA activity by PII in Azospirillum brasilense: evidence for a regulatory role of the NifA N-terminal domain.

Azospirillum brasilense NifA, which is synthesized under all physiological conditions, exists in an active or inactive from depending on the availability of ammonia. The activity also depends on the presence of PII, as NifA is inactive in a glnB mutant. To investigate further the mechanism that regulates NifA activity, several deletions of the nifA coding sequence covering the amino-terminal domain of NifA were constructed. The ability of these truncated NifA proteins to activate the nifH promoter in the absence or presence of ammonia was assayed in A. brasilense wild-type and mutant strains. Our results suggest that the N-terminal domain is not essential for NifA activity. This domain plays an inhibitory role which prevents NifA activity in the presence of ammonia. The truncated proteins were also able to restore nif gene expression to a glnB mutant, suggesting that PII is required to activate NifA by preventing the inhibitory effect of its N-terminal domain under conditions of nitrogen fixation. Low levels of nitrogenase activity in the presence of ammonia were also observed when the truncated gene was introduced into a strain devoid of the ADP-ribosylation control of nitrogenase. We propose a model for the regulation of NifA activity in A. brasilense.

The expression of nifA in Azorhizobium caulinodans requires a gene product homologous to Escherichia coli HF-I, an RNA-binding protein involved in the replication of phage Q beta RNA.

We report the characterization of a mutant of Azorhizobium caulinodans, isolated after ethyl methanesulfonate mutagenesis. This Nod+ Nif- Fix- mutant is unable to synthesize 10 of 15 polypeptides normally induced under conditions of nitrogen fixation. By using lacZ fusions it was shown that nifA and nifA-regulated genes were not expressed in this strain. The mutation was complemented by a constitutively expressed nifA gene or by a 1.1-kb DNA fragment from the wild-type strain, whose nucleotide sequence revealed a single open reading frame of 255 bp coding for an 85-amino acid polypeptide. The deduced amino acid sequence is similar to that of HF-I, an RNA-binding protein of Escherichia coli, which is required for replication of bacteriophage Q beta RNA. The similarity can be extended to the function since hfq, the structural gene for HF-I, complemented the A. caulinodans mutant. The corresponding gene in A. caulinodans was termed nrfA (for nif regulatory factor). Inactivation of nrfA in the mutant was due to a missense mutation resulting in the replacement of a cysteine residue by arginine. A null mutant, constructed by disruption of nrfA, exhibited the same phenotype as the missense mutant. Thus, an additional factor can be added to the already complex system of nifA regulation in A. caulinodans.

Functional analysis of the fixNOQP region of Azorhizobium caulinodans.

The deduced amino acid sequences of four open reading frames identified upstream of the fixGHI region in Azorhizobium caulinodans are very similar to the putative terminal oxidase complex coded by the fixNOQP operons from Rhizobium meliloti and Bradyrhizobium japonicum. The expression of the A. caulinodans fixNOQP genes, which was maximal under microaerobiosis, was positively regulated by FixK and independent of NifA. In contrast to the Fix- phenotype of B. japonicum and R. meliloti fixN mutants, an A. caulinodans fixNO-deleted mutant strain retained 50% of the nitrogenase activity of the wild type in the symbiotic state. In addition, the nitrogenase activity was scarcely reduced under free-living conditions. Analysis of membrane fractions of A. caulinodans wild-type and mutant strains suggests that the fixNOQP region encodes two proteins with covalently bound hemes, tentatively assigned to fixO and fixP. Spectral analysis showed a large decrease in the c-type cytochrome content of the fixN mutant compared with the wild type. These results provide evidence for the involvement of FixNOQP proteins in a respiratory process. The partial impairment in nitrogen fixation of the fixN mutant in planta may be due to the activity of an alternative terminal oxidase compensating for the loss of the oxidase complex encoded by fixNOQP.

Role of the fixGHI region of Azorhizobium caulinodans in free-living and symbiotic nitrogen fixation.

A 19-kb DNA region containing genes sharing homology with Rhizobium meliloti fixNOQP and fixGHI was isolated from a genomic library of Azorhizobium caulinodans. Identity of fixG was confirmed by partial nucleotide sequencing. Mutant strains in the fixGHI region were constructed by deletion or Tn5 insertions. In contrast with the situation in R. meliloti, the mutants still displayed a significant nitrogenase activity in symbiosis.

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

We 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.

Identification of a nifA-like regulatory gene of Azospirillum brasilense Sp7 expressed under conditions of nitrogen fixation and in the presence of air and ammonia.

A gene bank of Azospirillum lipoferum Br17 constructed in the vector lambda GEM11 was screened with a Bradyrhizobium japonicum nifA gene probe. A 7.3 kb EcoRI fragment carrying a nifA-like gene was thereby isolated and subsequently used to screen a gene bank of Azospirillum brasilense Sp7 constructed in pUC18. Two EcoRI fragments of 5.6 kb and 3.6 kb covering the nifA-homology region were found. Mutants with Nif- phenotype were obtained by site-directed Tn5 mutagenesis of the 5.6 kb fragment and subsequent recombination into the A. brasilense Sp7 genome. The mutations were clustered into two loci located at each extremity of the fragment. One of these loci corresponded to nifA and the other to nifB. The nucleotide sequence of nifA of A. brasilense Sp7 was determined. Comparison of the deduced amino acid sequences of NifA of A. brasilense Sp7 and NifA of B. japonicum, Rhizobium leguminosarum biovar trifolii and Klebsiella pneumoniae confirmed that it was a nifA-like gene. Construction of a nifA-lacZ fusion and mapping of the RNA transcriptional start site showed that the nifA-like gene was expressed from an unidentified promoter, under conditions of nitrogen fixation and in the presence of oxygen and ammonia.

Regulation of nitrogen fixation in Azorhizobium caulinodans: identification of a fixK-like gene, a positive regulator of nifA.

The nucleotide sequence of a 1 kb fragment upstream of Azorhizobium caulinodans fixL was established. An open reading frame of 744 bp was identified as a fixK homologue. A kanamycin cartridge was inserted into the cloned fixK-like gene and recombined into the host genome. The resulting mutant was Nif-Fix-, suggesting that FixK was required for nitrogen fixation both in symbiotic conditions and in the free-living state. Using a pfixK-lacZ fusion, the FixLJ products were shown to control the expression of fixK. Using a pnifA-lacZ fusion, the FixK product was shown to regulate positively the transcription of nifA in bacteria grown in the free-living state. In addition, a double ntrC-fixL mutant was constructed and was shown to be completely devoid of nitrogenase activity. A model of regulation, based on these data, is presented and might explain the unusual ability of A. caulinodans to fix nitrogen both under symbiotic conditions and in the free-living state.

Involvement of fixLJ in the regulation of nitrogen fixation in Azorhizobium caulinodans.

A gene bank of Azorhizobium caulinodans DNA constructed in the bacteriophage lambda GEM11 was screened with Rhizobium meliloti fixL and fixJ genes as probes. One positive recombinant phage, ORS lambda L, was isolated. The nucleotide sequence of a 3.7 kb fragment was established. Two open reading frames of 1512bp and 613bp were identified as fixL and fixJ. Kanamycin cartridges were inserted into the cloned fixL and fixJ genes and recombined into the host genome. The resulting mutants were Nif- Fix-, suggesting that the two genes were required for symbiotic nitrogen fixation and for nitrogen fixation in the free-living state. Using pnifH-lacZ and pnifA-lacZ fusions, it was shown that the FixLJ products controlled the expression of nifH and nifA in bacteria grown in the free-living state.

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.

The 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.

Characterization of the fixABC region of Azorhizobium caulinodans ORS571 and identification of a new nitrogen fixation gene.

The fast growing strain, Azorhizobium caulinodans ORS571, isolated from stem nodules of the tropical legume Sesbania rostrata, can grow in the free-living state at the expense of molecular nitrogen. Five point mutants impaired in nitrogen fixation in the free-living state have been complemented by a plasmid containing the cloned fix-ABC region of strain ORS571. Genetic analysis of the mutants showed that one was impaired in fixC, one in fixA and the three others in a new gene, located upstream from fixA and designated nifO. Site-directed Tn5 mutagenesis was performed to obtain Tn5 insertions in fixB and fixC. The four genes are required for nitrogen fixation both in the free-living state and under symbiotic conditions. The nucleotide sequence of nifO was established. The gene is transcribed independently of fixA and does not correspond to fixX, recently identified in Rhizobium meliloti and R. leguminosarum. Biochemical analysis of the five point mutants showed that they synthesized normal amounts of nitrogenase components. It is unlikely that fixA, fixC and nifO are involved in electron transport to nitrogenase. FixC could be required for the formation of a functional nitrogenase component 2.