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Tools for genetic manipulation of the plant growth-promoting bacterium Azospirillum amazonense.

Sant'anna FH, Andrade DS, Trentini DB, Weber SS, Schrank IS - BMC Microbiol. (2011)

Bottom Line: Finally, a promoter analysis protocol based on fluorescent protein expression was optimized to aid genetic regulation studies on this bacterium.In this work, genetic tools that can support the study of A. amazonense were described.These methods could provide a better understanding of the genetic mechanisms of this species that underlie its plant growth promotion.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9500, Campus do Vale, Porto Alegre, RS, Brazil.

ABSTRACT

Background: Azospirillum amazonense has potential to be used as agricultural inoculant since it promotes plant growth without causing pollution, unlike industrial fertilizers. Owing to this fact, the study of this species has gained interest. However, a detailed understanding of its genetics and physiology is limited by the absence of appropriate genetic tools for the study of this species.

Results: Conjugation and electrotransformation methods were established utilizing vectors with broad host-replication origins (pVS1 and pBBR1). Two genes of interest--glnK and glnB, encoding PII regulatory proteins--were isolated. Furthermore, glnK-specific A. amazonense mutants were generated utilizing the pK19MOBSACB vector system. Finally, a promoter analysis protocol based on fluorescent protein expression was optimized to aid genetic regulation studies on this bacterium.

Conclusion: In this work, genetic tools that can support the study of A. amazonense were described. These methods could provide a better understanding of the genetic mechanisms of this species that underlie its plant growth promotion.

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Related in: MedlinePlus

Electrical parameters tested for the A. amazonense electroporation. A - Effect of electrical field strength on the transformation efficiency of A. amazonense. Competent cells were electroporated at the electric field strengths indicated with the pHRGFPGUS vector, with the GenePulser apparatus set at 200 Ω and 25 μF. B - Effect of the pulse length on the transformation efficiency of A. amazonense. Competent cells were electroporated with different pulse lengths, using 50 ng of the pHRGFPGUS vector and with the GenePulser apparatus set at 12.5 kV/cm and 25 μF. The pulse lengths 2.2 ms, 4.3 ms, 7.3 ms and 10.5 ms are obtained setting the GenePulser apparatus at 100 Ω, 200 Ω, 400 Ω and 600 Ω, respectively.
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Figure 2: Electrical parameters tested for the A. amazonense electroporation. A - Effect of electrical field strength on the transformation efficiency of A. amazonense. Competent cells were electroporated at the electric field strengths indicated with the pHRGFPGUS vector, with the GenePulser apparatus set at 200 Ω and 25 μF. B - Effect of the pulse length on the transformation efficiency of A. amazonense. Competent cells were electroporated with different pulse lengths, using 50 ng of the pHRGFPGUS vector and with the GenePulser apparatus set at 12.5 kV/cm and 25 μF. The pulse lengths 2.2 ms, 4.3 ms, 7.3 ms and 10.5 ms are obtained setting the GenePulser apparatus at 100 Ω, 200 Ω, 400 Ω and 600 Ω, respectively.

Mentions: Different electroporation parameters were tested. The increase in electrical field strength had a positive effect on electroporation efficiency (Figure 2A). The highest electrical field strength tested was 12.5 kV/cm, and this condition was found to be the most efficient, generating about 8000 transformants/μg of pHRGFPGUS (Figure 2A). The effect of pulse length on electroporation efficiency was also investigated (Figure 2B). A pulse length of 4.3 ms (electroporation apparatus set at 200Ω) was the most efficient. The pulse lengths of 7.3 ms (400 Ω) and 10.5 ms (600 Ω) had a dramatic negative effect on transformation efficiency, where only few transformants were obtained (Figure 2B). These conditions are in agreement with the general parameters of bacterial electroporation [22-24].


Tools for genetic manipulation of the plant growth-promoting bacterium Azospirillum amazonense.

Sant'anna FH, Andrade DS, Trentini DB, Weber SS, Schrank IS - BMC Microbiol. (2011)

Electrical parameters tested for the A. amazonense electroporation. A - Effect of electrical field strength on the transformation efficiency of A. amazonense. Competent cells were electroporated at the electric field strengths indicated with the pHRGFPGUS vector, with the GenePulser apparatus set at 200 Ω and 25 μF. B - Effect of the pulse length on the transformation efficiency of A. amazonense. Competent cells were electroporated with different pulse lengths, using 50 ng of the pHRGFPGUS vector and with the GenePulser apparatus set at 12.5 kV/cm and 25 μF. The pulse lengths 2.2 ms, 4.3 ms, 7.3 ms and 10.5 ms are obtained setting the GenePulser apparatus at 100 Ω, 200 Ω, 400 Ω and 600 Ω, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3111374&req=5

Figure 2: Electrical parameters tested for the A. amazonense electroporation. A - Effect of electrical field strength on the transformation efficiency of A. amazonense. Competent cells were electroporated at the electric field strengths indicated with the pHRGFPGUS vector, with the GenePulser apparatus set at 200 Ω and 25 μF. B - Effect of the pulse length on the transformation efficiency of A. amazonense. Competent cells were electroporated with different pulse lengths, using 50 ng of the pHRGFPGUS vector and with the GenePulser apparatus set at 12.5 kV/cm and 25 μF. The pulse lengths 2.2 ms, 4.3 ms, 7.3 ms and 10.5 ms are obtained setting the GenePulser apparatus at 100 Ω, 200 Ω, 400 Ω and 600 Ω, respectively.
Mentions: Different electroporation parameters were tested. The increase in electrical field strength had a positive effect on electroporation efficiency (Figure 2A). The highest electrical field strength tested was 12.5 kV/cm, and this condition was found to be the most efficient, generating about 8000 transformants/μg of pHRGFPGUS (Figure 2A). The effect of pulse length on electroporation efficiency was also investigated (Figure 2B). A pulse length of 4.3 ms (electroporation apparatus set at 200Ω) was the most efficient. The pulse lengths of 7.3 ms (400 Ω) and 10.5 ms (600 Ω) had a dramatic negative effect on transformation efficiency, where only few transformants were obtained (Figure 2B). These conditions are in agreement with the general parameters of bacterial electroporation [22-24].

Bottom Line: Finally, a promoter analysis protocol based on fluorescent protein expression was optimized to aid genetic regulation studies on this bacterium.In this work, genetic tools that can support the study of A. amazonense were described.These methods could provide a better understanding of the genetic mechanisms of this species that underlie its plant growth promotion.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9500, Campus do Vale, Porto Alegre, RS, Brazil.

ABSTRACT

Background: Azospirillum amazonense has potential to be used as agricultural inoculant since it promotes plant growth without causing pollution, unlike industrial fertilizers. Owing to this fact, the study of this species has gained interest. However, a detailed understanding of its genetics and physiology is limited by the absence of appropriate genetic tools for the study of this species.

Results: Conjugation and electrotransformation methods were established utilizing vectors with broad host-replication origins (pVS1 and pBBR1). Two genes of interest--glnK and glnB, encoding PII regulatory proteins--were isolated. Furthermore, glnK-specific A. amazonense mutants were generated utilizing the pK19MOBSACB vector system. Finally, a promoter analysis protocol based on fluorescent protein expression was optimized to aid genetic regulation studies on this bacterium.

Conclusion: In this work, genetic tools that can support the study of A. amazonense were described. These methods could provide a better understanding of the genetic mechanisms of this species that underlie its plant growth promotion.

Show MeSH
Related in: MedlinePlus