Regulated expression of the nifM of Azotobacter vinelandii in response to molybdenum and vanadium supplements in Burk's nitrogen-free growth medium.
Azotobacter is a diazotrophic bacterium that harbors three genetically distinct nitrogenases referred to as nif, vnf, and anf systems. The nifM is an accessory gene located in the nif gene cluster and is transcriptionally regulated by the NifA. However, Azotobacter mutants that lack NifA are known to synthesize functional NifM and this accessory protein is known to be needed for the activity of nitrogenase-2 and nitrogenase-3. To determine how the transcription of nifM is regulated when Azotobacter is grown under conditions in which nitrogenase-2 or nitrogenase-3 is expressed, we generated an Azotobacter vinelandii strain that carries a nifM:lacZ-kanamycin resistance gene cassette in its chromosome. In this strain the nifM open reading frame was disrupted by the presence of a lacZ-kanamycin resistance gene cassette so that it could not produce active NifM. Moreover, the lacZ gene was placed under the transcriptional control elements of the nifM gene so that the lacZ expression could be used as a marker to determine the extent of expression of the nifM gene under different growth conditions. Our results show that this strain was unable to grow in Burk's nitrogen-free medium supplemented with either molybdenum or vanadium or lacking both metals suggesting that in the absence of functional NifM none of the nitrogenases were active. It was also found that the nifM expression was differentially regulated when the A. vinelandii cells were grown under conditions that activate nitrogenase-2 and nitrogenase-3, as determined by liquid beta-galactosidase activity measurements. These results suggest that the transcriptional activators, VnfA and AnfA, may regulate the nifM expression.
Lei
S
S
Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio, 43403, USA.
Pulakat
L
L
Gavini
N
N
D016948:Azotobacter vinelandii / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003470:Culture Media / Q000494:pharmacology; D004269:DNA, Bacterial / Q000032:analysis; D004268:DNA-Binding Proteins / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D007613:Kanamycin Resistance / Q000235:genetics; D007763:Lac Operon; D008969:Molecular Sequence Data; D008982:Molybdenum / Q000494:pharmacology*; D005810:Multigene Family; D009591:Nitrogenase / Q000235:genetics* / Q000378:metabolism; D016366:Open Reading Frames; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014639:Vanadium / Q000494:pharmacology*
D001426:Bacterial Proteins; D003470:Culture Media; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C061338:NifA protein, Bacteria; D015534:Trans-Activators; D014157:Transcription Factors; C090095:VnfA protein, Azotobacter vinelandii; D014639:Vanadium; D008982:Molybdenum; D009591:Nitrogenase