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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

Analysis of EYFP expression controlled by different A. amazonense promoters. WT- A. amazonense without plasmid; W/P - negative control, A. amazonense harboring the pHREYFP vector (without promoter); P glnK - A. amazonense harboring the pHRPKEYFP vector (promoter of glnK gene); P glnB - A. amazonense harboring the pHRPBEYFP vector (promoter of glnB gene); P aat - A. amazonense harboring the pHRAATEYFP vector (promoter of aat gene); P lac (Z) - A. amazonense harboring the pPZPLACEYFP vector (lac promoter); P lac (H) - A. amazonense harboring the pHRLACEYFP vector (lac promoter). The error bars represent the confidence interval of 95%, calculated from seven independent experiments (excepting the P lac (H), where four experiments were performed). Asterisks indicate activities that do not differ statistically in the Tukey HSD test (P < 0.01).
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Figure 5: Analysis of EYFP expression controlled by different A. amazonense promoters. WT- A. amazonense without plasmid; W/P - negative control, A. amazonense harboring the pHREYFP vector (without promoter); P glnK - A. amazonense harboring the pHRPKEYFP vector (promoter of glnK gene); P glnB - A. amazonense harboring the pHRPBEYFP vector (promoter of glnB gene); P aat - A. amazonense harboring the pHRAATEYFP vector (promoter of aat gene); P lac (Z) - A. amazonense harboring the pPZPLACEYFP vector (lac promoter); P lac (H) - A. amazonense harboring the pHRLACEYFP vector (lac promoter). The error bars represent the confidence interval of 95%, calculated from seven independent experiments (excepting the P lac (H), where four experiments were performed). Asterisks indicate activities that do not differ statistically in the Tukey HSD test (P < 0.01).

Mentions: The lac promoter had the best score in the in silico analysis from among the promoters detected, and, as expected, the highest fluorescence levels were observed in the lac constructions (Figure 5). The difference in the fluorescence levels between the pHRLACEYFP and pPZPLACEYFP transformants could be a product of the difference in the copy number between these vectors.


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)

Analysis of EYFP expression controlled by different A. amazonense promoters. WT- A. amazonense without plasmid; W/P - negative control, A. amazonense harboring the pHREYFP vector (without promoter); P glnK - A. amazonense harboring the pHRPKEYFP vector (promoter of glnK gene); P glnB - A. amazonense harboring the pHRPBEYFP vector (promoter of glnB gene); P aat - A. amazonense harboring the pHRAATEYFP vector (promoter of aat gene); P lac (Z) - A. amazonense harboring the pPZPLACEYFP vector (lac promoter); P lac (H) - A. amazonense harboring the pHRLACEYFP vector (lac promoter). The error bars represent the confidence interval of 95%, calculated from seven independent experiments (excepting the P lac (H), where four experiments were performed). Asterisks indicate activities that do not differ statistically in the Tukey HSD test (P < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Analysis of EYFP expression controlled by different A. amazonense promoters. WT- A. amazonense without plasmid; W/P - negative control, A. amazonense harboring the pHREYFP vector (without promoter); P glnK - A. amazonense harboring the pHRPKEYFP vector (promoter of glnK gene); P glnB - A. amazonense harboring the pHRPBEYFP vector (promoter of glnB gene); P aat - A. amazonense harboring the pHRAATEYFP vector (promoter of aat gene); P lac (Z) - A. amazonense harboring the pPZPLACEYFP vector (lac promoter); P lac (H) - A. amazonense harboring the pHRLACEYFP vector (lac promoter). The error bars represent the confidence interval of 95%, calculated from seven independent experiments (excepting the P lac (H), where four experiments were performed). Asterisks indicate activities that do not differ statistically in the Tukey HSD test (P < 0.01).
Mentions: The lac promoter had the best score in the in silico analysis from among the promoters detected, and, as expected, the highest fluorescence levels were observed in the lac constructions (Figure 5). The difference in the fluorescence levels between the pHRLACEYFP and pPZPLACEYFP transformants could be a product of the difference in the copy number between these vectors.

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