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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

In silico sigma 70 promoter analysis. The upstream sequences of the genes were analyzed by Patser software using an S. meliloti sigma 70 factor weight matrix [33]. aat - upstream region of the aat gene; glnB - upstream region of the glnB gene; glnK - upstream region of the glnK gene; lac - lac promoter; W/P - negative control, 500 bp upstream of the eyfp gene of the plasmid pHREYFP. The S. meliloti promoter consensus is the first sequence. Nucleotides that match the S. meliloti consensus are in red, and those that match the most conserved residues of the S. meliloti promoter consensus (relative frequencies above 0.8) are in bold. Gaps were inserted to preserve the alignment at the regions of the promoters.
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Figure 4: In silico sigma 70 promoter analysis. The upstream sequences of the genes were analyzed by Patser software using an S. meliloti sigma 70 factor weight matrix [33]. aat - upstream region of the aat gene; glnB - upstream region of the glnB gene; glnK - upstream region of the glnK gene; lac - lac promoter; W/P - negative control, 500 bp upstream of the eyfp gene of the plasmid pHREYFP. The S. meliloti promoter consensus is the first sequence. Nucleotides that match the S. meliloti consensus are in red, and those that match the most conserved residues of the S. meliloti promoter consensus (relative frequencies above 0.8) are in bold. Gaps were inserted to preserve the alignment at the regions of the promoters.

Mentions: In silico analysis using a Sinorhizobium meliloti sigma 70 promoter weight matrix revealed that the genes aat, glnK, and glnB of A. amazonense have putative promoter sequences in their upstream regions (Figure 4). In E. coli, sigma 70 is considered to be the vegetative sigma factor, as it is responsible for the expression of the majority of genes [32,33]. Therefore, one could expect that these putative A. amazonense sigma 70 promoters could act under standard laboratory growth conditions (aerobic environment, 35°C and M79 medium). Consequently, different vectors were constructed to determine the activity of the upstream regulatory sequences of A. amazonense genes in the expression of EYFP.


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)

In silico sigma 70 promoter analysis. The upstream sequences of the genes were analyzed by Patser software using an S. meliloti sigma 70 factor weight matrix [33]. aat - upstream region of the aat gene; glnB - upstream region of the glnB gene; glnK - upstream region of the glnK gene; lac - lac promoter; W/P - negative control, 500 bp upstream of the eyfp gene of the plasmid pHREYFP. The S. meliloti promoter consensus is the first sequence. Nucleotides that match the S. meliloti consensus are in red, and those that match the most conserved residues of the S. meliloti promoter consensus (relative frequencies above 0.8) are in bold. Gaps were inserted to preserve the alignment at the regions of the promoters.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: In silico sigma 70 promoter analysis. The upstream sequences of the genes were analyzed by Patser software using an S. meliloti sigma 70 factor weight matrix [33]. aat - upstream region of the aat gene; glnB - upstream region of the glnB gene; glnK - upstream region of the glnK gene; lac - lac promoter; W/P - negative control, 500 bp upstream of the eyfp gene of the plasmid pHREYFP. The S. meliloti promoter consensus is the first sequence. Nucleotides that match the S. meliloti consensus are in red, and those that match the most conserved residues of the S. meliloti promoter consensus (relative frequencies above 0.8) are in bold. Gaps were inserted to preserve the alignment at the regions of the promoters.
Mentions: In silico analysis using a Sinorhizobium meliloti sigma 70 promoter weight matrix revealed that the genes aat, glnK, and glnB of A. amazonense have putative promoter sequences in their upstream regions (Figure 4). In E. coli, sigma 70 is considered to be the vegetative sigma factor, as it is responsible for the expression of the majority of genes [32,33]. Therefore, one could expect that these putative A. amazonense sigma 70 promoters could act under standard laboratory growth conditions (aerobic environment, 35°C and M79 medium). Consequently, different vectors were constructed to determine the activity of the upstream regulatory sequences of A. amazonense genes in the expression of EYFP.

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