H. K. Das

The Azotobacter vinelandii nifL-like gene: nucleotide sequence analysis and regulation of expression

SummaryThe nucleotide sequence of the Azotobacter vinelandii nifL-like gene (Av-nifL) was determined. The 1.9 kb sequence shows an open reading frame (ORF) of 1557 by which encodes a polypeptide of 519 amino acids, with a calculated molecular weight of 57 793. Av-nifL has about 50 % homology with the Klebsiella pneumoniae nifL gene (Kp-nifL) at the nucleotide level and a little more than 52% homology at the amino acid level. The N-terminal regions show more homology than the C-terminal regions. As is the case in K. pneumoniae, Av-nifL is located just upstream of the A. vinelandii nifA gene (Av-nifA) and both genes constitute an operon. The expression of Av-nifL, however, seems to be independent of NtrA and NtrC. Furthermore, Av-nifL expression is not autogenously regulated by NifA, unlike the case in K. pneumoniae. The expression of an Av-nifL: lacZ fusion in A. vinelandii is inhibited by novobiocin and coumermycin A, which are inhibitors of DNA gyrase.

Isolation and characterization of a locus from Azospirillum brasilense Sp7 that complements the tumorigenic defect of Agrobacterium tumefaciens chvB mutant

The chromosomal virulence gene chvB of Agrobacterium tumefaciens is required for pathogenesis. A DNA fragment from the chvB locus can hybridize to DNA from Azospirillum brasilense Sp7. This DNA fragment could restore the tumorigenic activity of the chvB mutant strain A. tumefaciens A1011 towards leaf disks of Nicotiana tabacum. An NH2-terminal open reading frame, 480 codons long, was most likely responsible for the restoration of the tumorigenic activity. The A. brasilense sequence showed good homology with the NH2-terminal region of the ndvB gene of Rhizobium meliloti.

Determination of the size of the Azotobacter vinelandii chromosome

The chromosome of Azotobacter vinelandii UW was digested separately with the rape cutter restriction endonucleases Swal (5′-ATTTAAAT), PmeI (5′GTTTAAAC) and Pacl (5′-TTAATTAA) and the products were separated by pulsed-field gel electrophoresis. The size of the chromosome was determined to be approximately 4.5 megabase pairs (Mb) based on the sum of the sizes of the restriction fragments. This is almost the same as the size of the chromosome of Escherichia coli. The inability of the undigested DNA to enter the gel has led us to infer that the chromosome is circular.

The ORF encoding a putative ferredoxin-like protein downstream of the vnfH gene in Azotobacter vinelandii is involved in the vanadium-dependent alternative pathway of nitrogen fixation

SummaryAn open reading frame (ORF) in the same operon as, but downstream of, vnfH in Azotobacter vinelandii can code for a ferredoxin-like protein. The role this ORF may play in the vnf (vanadium-dependent alternative) pathway of nitrogen fixation was investigated. Site-directed mutagenesis was used to alter one base in each of the codons specifying amino acids 18 and 19 generating a unique BglII site. A kanamycin resistance cartridge was cloned into the BglII site. This construct was mobilized into A. vinelandii CA12 (Δ nifHDK) strain by conjugation and the mutation was introduced into the genome by marker exchange. The resulting mutant was unable to fix nitrogen under conditions in which the vnf pathway of nitrogen fixation operates. This suggests that this ORF is functional and is essential for the vanadium-dependent alternative pathway of nitrogen fixation in A. vinelandii.

Identification of a Positive Transcription Regulatory Element within the Coding Region of the nifLA Operon in Azotobacter vinelandii

ABSTRACT Nitrogen fixation in Azotobacter vinelandii is regulated by the nifLA operon. NifA activates the transcription of nif genes, while NifL antagonizes the transcriptional activator NifA in response to fixed nitrogen and molecular oxygen levels. However, transcriptional regulation of the nifLA operon of A. vinelandii itself is not fully understood. Using the S1 nuclease assay, we mapped the transcription start site of the nifLA operon, showing it to be similar to the σ54-dependent promoters. We also identified a positive cis-acting regulatory element (+134 to +790) of the nifLA operon within the coding region of the nifL gene of A. vinelandii. Deletion of this element results in complete loss of promoter activity. Several protein factors bind to this region, and the specific binding sites have been mapped by DNase I foot printing. Two of these sites, namely dR1 (+134 to +204) and dR2 (+745 to +765), are involved in regulating the nifLA promoter activity. The absence of NtrC-like binding sites in the upstream region of the nifLA operon in A. vinelandii makes the identification of these downstream elements a highly significant finding. The interaction of the promoter with the proteins binding to the dR2 region spanning +745 to +765 appears to be dependent on the face of the helix as introduction of 4 bases just before this region completely disrupts promoter activity. Thus, the positive regulatory element present within the BglII-BglII fragment may play, in part; an important role in nifLA regulation in A. vinelandii.

The size of the chromosome of Azotobacter chroococcum

The DNA of Azotobacter chroococcum M4 was digested with rare-cutter restriction endonucleases Asel and Dral and restriction fragments were separated by pulsed-field gel electrophoresis. The chromosome size was estimated to be around 5300 kb by summing the sizes of the resolved restriction fragments.

An Environmentally Friendly Engineered Azotobacter Strain That Replaces a Substantial Amount of Urea Fertilizer while Sustaining the Same Wheat Yield

ABSTRACT 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.

TheAzotobacter vinelandii chromosome

The chromosome ofAzotobacter vinelandii was digested with the restriction endonucleasesSpeI (5’-ACTAGT),DraI (5’-TTTAAA) andAsel (5’-ATTAAT) and the products were separated by pulsed-field gel electrophoresis. The sum of the sizes of the restriction fragments comes to around 4.5 megabasepairs. Our earlier studies had revealed the presence of about 80 copies ofnifH, nifD, nifK andleuB genes in a log-phase cell ofA. vinelandii. To determine whether there are multiple identical chromosomes inA. vinelandii or one large chromosome with identical segments joined in tandem, we have subjected gamma-irradiated DNA ofA. vinelandii andEscherichia coli to pulsed-field gel electrophoresis. The results suggest thatA. vinelandii chromosomes contain multiple identical chromosomes of about the same size as that ofE. coli.

Non-coordinate regulation of RNA synthesis in escherichia coli exposed to 0°C

A non-coordinate mode of regulation of RNA synthesis is observed inEscherichia coli cells during exposure to 0°C. The stable RNA synthesis is preferentially inhibited with simultaneous accumulation of messenger RNA. The species of RNA synthesized at 0°C was determined by several criteria such as sedimentation value in sucrose gradients, DNA-RNA hybridization, half life measurements, protein synthesizing capacity and its functional rate of decay. The mode of regulation of RNA synthesis at 0°C is unique and is distinct from the non-coordinate regulation observed during amino acid starvation under stringent control.