Nara Gavini

Role of C-terminal cytoplasmic domain of the AT2 receptor in ligand binding and signaling

A stop codon at position 322 was introduced to generate a truncated, C‐terminal‐deleted AT2 receptor. Expression studies in Xenopus oocytes showed that C‐terminal‐deleted AT2 had reduced affinity to [125I]angiotensin II (K d=1.7 nM) and enhanced binding of the AT2‐specific peptidic ligand [125I]CGP42112A (K d=0.097 nM). AT2 activation by angiotensin II resulted in reduction of cGMP levels in oocytes and this reduction was further enhanced by C‐terminal deletion, implying that the C‐terminus may have a negative effect on the AT2‐mediated cGMP reduction. Moreover, interaction of the AT2 with the ATP‐binding domain of the human ErbB3 receptor in yeast two‐hybrid assay was abolished by C‐terminal deletion. In summary, the C‐terminal cytoplasmic tail of AT2 modulates its ligand binding and signaling properties.

Peptidyl-Prolyl cis/trans Isomerase-Independent Functional NifH Mutant of Azotobacter vinelandii

Peptidyl-prolyl cis/trans isomerases (PPIases) play a pivotal role in catalyzing the correct folding of many prokaryotic and eukaryotic proteins that are implicated in a variety of biological functions, ranging from cell cycle regulation to bacterial infection. The nif accessory protein NifM, which is essential for the biogenesis of a functional NifH component of nitrogenase, is a PPIase. To understand the nature of the molecular signature that defines the NifM dependence of NifH, we screened a library of nifH mutants in the nitrogen-fixing bacterium Azotobacter vinelandii for mutants that acquired NifM independence. Here, we report that NifH can acquire NifM independence when the conserved Pro258 located in the C-terminal region of NifH, which wraps around the other subunit in the NifH dimer, is replaced by serine.

Functional Expression of a Fusion-dimeric MoFe Protein of Nitrogenase in Azotobacter vinelandii

The MoFe protein component of the complex metalloenzyme nitrogenase is an α2β2 tetramer encoded by thenifD and the nifK genes. In nitrogen fixing organisms, the α and β subunits are translated as separate polypeptides and then assembled into tetrameric MoFe protein complex that includes two types of metal centers, the P cluster and the FeMo cofactor. In Azotobacter vinelandii, the NifEN complex, the site for biosynthesis of the FeMo cofactor, is an α2β2 tetramer that is structurally similar to the MoFe protein and encoded as two separate polypeptides by the nifE and the nifNgenes. In Anabaena variabilis it was shown that a NifE-N fusion protein encoded by translationally fused nifE andnifN genes can support biological nitrogen fixation. The structural similarity between the MoFe protein and the NifEN complex prompted us to test whether the MoFe protein could also be functional when synthesized as a single protein encoded by nifD-Ktranslational fusion. Here we report that the NifD-K fusion protein encoded by nifD-K translational fusion in A. vinelandii is a large protein (as determined by Western blot analysis) and is capable of supporting biological nitrogen fixation. These results imply that the MoFe protein is flexible in that it can accommodate major structural changes and remain functional.

Isolation and characterization of nif DK::kanamycin and nitrogen fixation proficient Azotobacter vinelandii strain, and its implication on the status of multiple chromosomes in Azotobacter.

Several lines of experimental analyses on the ploidy status of Azotobacter vinelandii genome lead to the conclusion that it contains more than 40 copies of its chromosome and therefore it is a polyploid organism. The genetic evidence argues against the existence of polyploidy in these cells since the segregation pattern of genetic markers under lack of selection pressure mimic that of haploids. However, when A. vinelandii was made Nif− by inserting a kanamycin resistance marker gene in the nifDK sequence and the cells were selected for kanamycin resistance and Nif+ phenotype, we were able to score colonies that are both kanamycin resistant and Nif+. Therefore, when the cells were subjected to forced double selection of the same locus, they behaved as if they carried at least two chromosomes, one carrying the kanamycin resistance marker in the nifDK genes and the other carrying the intact nifDK genes. These analyses suggested that at least a diploidy status can be induced in these cells under selection pressure.

Functional expression of the FeMo-cofactor-specific biosynthetic genes nifEN as a NifE-N fusion protein synthesizing unit in Azotobacter vinelandii

The nifEN encodes an E2N2 tetrameric metalloprotein complex that serves as scaffold for assembly of the FeMo cofactor of nitrogenase. In most diazotrophs, the NifE and NifN are translated as separate polypeptides and then assembled into tetrameric E2N2 complex. However, in Anabaena variabilis which has two nif clusters that encode two different NifEN complexes, the NifEN2 is encoded by a single nifE-N like gene, which has high homology to the NifE at amino-terminus and to the NifN at the carboxy-terminus. These observations implied that a metalloprotein like NifEN can accommodate large variations in their amino acid composition and also in the way they are synthesized (as two separate proteins or as a single protein) and yet remain functional. In Azotobacter vinelandii NifE and NifN are synthesized separately. To test whether NifEN could retain its functionality when encoded by a single gene, we generated a translational fusion of the nifE and nifN genes of A. vinelandii that could encode a large NifE-N fusion protein. When expressed in the nifEN-minus strain of A. vinelandii, the nifE-N gene fusion could complement the NifEN function. Western blot analysis by using polyclonal NifEN antibodies revealed that the complementing nifEN product is a large NifE-N fusion protein unit. The fact that the gene fusion of nifE-N specifies a functional NifE-N fusion protein reflects that these metalloproteins can accommodate a wide range of flexibility in their gene organization, structure, and assembly.

Analysis of the genome of Azotobacter vinelandii revealed the presence of two genetically distinct group II introns on the chromosome.

Azotobacter vinelandii belongs to the γ subdivision of eubacteria and has one of the highest respiratory rates. It is considered to be among the probable progenitors of mitochondria. Group II introns were originally identified on organelle genomes. Analysis of the A. vinelandii genome for the presence of group II introns using a deduced group II intron consensus sequence identified two putative introns. The first intron (AV1) which was found to be inserted in the groEL, an essential gene, was already characterized. Our study identified another group II intron (AV2) in A. vinelandii genome. This intron is inserted in a mobile genetic element, similar to most of the group II introns in bacteria, which in this case is a transposase like gene, tnpA1. This putative TnpA1 protein is 52% identical to TnpA, the transposase of bacteriophage Lambda, and 85% identical to TnpA1 of Pseudomonas stutzeri. Sequence analysis showed that this intron encodes a reverse transcriptase (RT) like motif in domain IV, similar to other group II introns. The RT of this intron open reading frame (ORF) is 53% homologous with that of AV1 intron and 66% homologous with that of Pseudomonas putida(Tn5041c) intron. Secondary structure analysis showed that this intron has the typical sub-group IIB1 structure, but the EBS2–IBS2 interaction appears to be missing. Using the RNA generated by in vitro transcription of the intron sequence with its flanking exons, in vitro splicing experiments were performed. It was found that the AV2 intron is functional, despite of lacking the EBS2–IBS2 interaction that plays a role in exon recognition.

Genome of Azotobacter vinelandii: counting of chromosomes by utilizing copy number of a selectable genetic marker

Studies utilizing several physical, biochemical and spectroscopic methods have suggested that Azotobacter vinelandii contains multiple copies (40–80) of its chromosome per cell, whereas genetic analysis indicated that these cells function like haploid cells. To further verify if A. vinelandii indeed contains 40–80 copies of its chromosome per cell, we have developed an ‘in vivo chromosome counting’ technique. The basic principle of this technique is to introduce the same genetic marker on the chromosome and on an extrachromosomal element of known copy number into the bacterium. The copy number of the chromosome can be determined by comparing the intensity of the hybridization signal generated by the DNA fragment carrying the chromosomal marker with that of the extrachromosomal marker when the total DNA isolated from this strain is hybridized with a probe made of the same genetic marker DNA. To do this we used an A. vinelandii BG102 strain which carries a kanamycin resistance marker gene integrated into the nifY locus on its chromosome(s). The plasmids pRK293 and pKT230, which can replicate in A. vinelandii and carry the kanamycin resistance gene (similar to the one present on the chromosome of A. vinelandii BG102), served as the extrachromosomal elements with known copy number. Southern blotting and hybridization analysis of the total DNA, isolated from A. vinelandii BG102 containing these plasmids, with a probe made of the kanamycin resistance gene clearly indicated that the copy number of A. vinelandii chromosome is slightly lower than the copy number of the low-copy plasmid pRK293 and about 21-fold lower than the copy number of the high copy plasmid pKT230. We believe that this ‘In vivo chromosome counting’ technique can be used for determination of the copy number of the chromosome in other cells with appropriate modifications in the nature of the extrachromosomal element and the genetic marker.

Activation of vanadium nitrogenase expression in Azotobacter vinelandii DJ54 revertant in the presence of molybdenum

Azotobacter vinelandii carries three different and genetically distinct nitrogenase systems on its chromosome. Expression of all three nitrogenases is repressed by high concentrations of fixed nitrogen. Expression of individual nitrogenase systems is under the control of specific metal availability. We have isolated a novel type of A. vinelandii DJ54 revertant, designated A. vinelandii BG54, which carries a defined deletion in the nifH gene and is capable of diazotrophic growth in the presence of molybdenum. Inactivation of nifDK has no effect on growth of this mutant strain in nitrogen‐free medium suggesting that products of the nif system are not involved in supporting diazotrophic growth of A. vinelandii BG54. Similar to the wild type, A. vinelandii BG54 is also sensitive to 1 mM tungsten. Tn5‐B21 mutagenesis to inactivate the genes specific to individual systems revealed that the structural genes for vnf nitrogenase are required for diazotrophic growth of A. vinelandii BG54. Analysis of promoter activity of different nif systems revealed that the vnf promoter is activated in A. vinelandii BG54 in the presence of molybdenum. Based on these data we conclude that A. vinelandii BG54 strain utilizes vnf nitrogenase proteins to support its diazotrophic growth.