It was found that the protein of this gene displays 92% identity and 98% similarity
to the GlnB proteins from Azospirillum sp. B510 and A. brasilense, and 96% identity and 98% Momelotinib supplier similarity to the GlnB protein of R. centenum. The glnB gene is located upstream of the glnA gene (glutamine synthetase), the same genetic context observed in these bacteria (Figure 1). In A. brasilense, glnB has a key role in nitrogen fixation because its protein product regulates the activity of NifA, the transcriptional factor of nitrogen fixation [16, 17]. Furthermore, both of the GlnZ (GlnK-like homolog) and GlnB proteins are also implicated in MK-4827 datasheet the DraT/DraG system, which regulates dinitrogenase reductase activity by covalent modifications [15]. However, Fu et al. [18] verified that A. amazonense does not have the DraT/DraG system. Hence, in the near future, the interaction targets of the PII protein in A. amazonense should be determined to better understand their
roles in the nitrogen metabolism of this microorganism. Antibiotic minimum inhibitory concentration Most DNA manipulation is dependent on the use of vectors containing resistance markers to antibiotics [19, 20]. selleck kinase inhibitor In a previous work using antibiotic susceptibility test discs, Magalhães et al. (1983) [5] showed that A. amazonense is sensitive to kanamycin and gentamicin, tolerant to tetracycline, and resistant to penicillin. In this work, we determined the minimum inhibitory concentrations of A. amazonense to antibiotics that are normally used to provide a selective pressure for
vectors. The susceptibility of A. amazonense to kanamycin and gentamicin was confirmed, since no growth was observed in concentrations of these antibiotics of 0.25 μg/mL; therefore, vectors that contain selection markers for these compounds are appropriate for use. High concentrations of ampicillin (128 μg/mL) were required for complete growth inhibition, showing that A. amazonense is also resistant to this beta-lactam antibiotic. It is worth noting that the growth of A. amazonense was absent in a relatively high concentration of tetracycline (32 μg/mL), indicating that this species is, in fact, resistant to this antibiotic, instead of tolerant, as pointed out by Magalhães et al. Selleck Hydroxychloroquine [5]. These findings about the latter two antibiotics are relevant because they could be used in counter-selection procedures in conjugation experiments, as there is a variety of E. coli strains that are susceptible to them. Conjugation Conjugation mediated by E. coli is the standard DNA transfer technique of the Azospirillum genus [21]. Therefore, in this work the conjugation ability of A. amazonense was evaluated. Unlike A. brasilense, A. amazonense cannot grow in LB medium. Furthermore, E. coli cannot grow in M79 medium; therefore, the first concern was to establish a medium that provided appropriate growth conditions for the donor and recipient strains.
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