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Cellular responses during morphological transformation in Azospirillum brasilense and Its flcA knockout mutant.

Hou X, McMillan M, Coumans JV, Poljak A, Raftery MJ, Pereg L - PLoS ONE (2014)

Bottom Line: Comparison of 2-DE protein profiles of wild-type (Sp7) and a flcA deletion mutant (Sp7-flcAΔ) revealed a total of 33 differentially expressed 2-DE gel spots, with 22 of these spots confidently separated to allow protein identification.Analysis of these spots by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MASCOT database searching identified 48 proteins (≥10% emPAI in each spot).This work is a step forward in connecting the Azospirillum phenome and genome.

View Article: PubMed Central - PubMed

Affiliation: School of Science and Technology, University of New England, Armidale, New South Wales, Australia; Department of Microbiology and Immunology, Shanxi Medical University, Taiyuan, Shanxi, China.

ABSTRACT
FlcA is a response regulator controlling flocculation and the morphological transformation of Azospirillum cells from vegetative to cyst-like forms. To understand the cellular responses of Azospirillum to conditions that cause morphological transformation, proteins differentially expressed under flocculation conditions in A. brasilense Sp7 and its flcA knockout mutant were investigated. Comparison of 2-DE protein profiles of wild-type (Sp7) and a flcA deletion mutant (Sp7-flcAΔ) revealed a total of 33 differentially expressed 2-DE gel spots, with 22 of these spots confidently separated to allow protein identification. Analysis of these spots by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MASCOT database searching identified 48 proteins (≥10% emPAI in each spot). The functional characteristics of these proteins included carbon metabolism (beta-ketothiolase and citrate synthase), nitrogen metabolism (Glutamine synthetase and nitric oxide synthase), stress tolerance (superoxide dismutase, Alkyl hydroperoxidase and ATP-dependent Clp protease proteolytic subunit) and morphological transformation (transducer coupling protein). The observed differences between Sp7 wild-type and flcA- strains enhance our understanding of the morphological transformation process and help to explain previous phenotypical observations. This work is a step forward in connecting the Azospirillum phenome and genome.

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Related in: MedlinePlus

Differential protein expression in Sp7 and Sp7-flcAΔ analyzed by 2-DE.The left section shows the normalized expression volume of the spot in wild type (Sp7) and flcA knock out strain (Sp7-flcAΔ) under flocculation conditions; the relative fold change is shown above each column (Nx indicates that relative fold change could not be calculated as the protein was only detected in either Sp7 or Sp7-flcAΔ). The right section is a 3D representation of the area of interest as provided by PDquest software. Arrows indicate spots with relatively higher expression.
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pone-0114435-g004: Differential protein expression in Sp7 and Sp7-flcAΔ analyzed by 2-DE.The left section shows the normalized expression volume of the spot in wild type (Sp7) and flcA knock out strain (Sp7-flcAΔ) under flocculation conditions; the relative fold change is shown above each column (Nx indicates that relative fold change could not be calculated as the protein was only detected in either Sp7 or Sp7-flcAΔ). The right section is a 3D representation of the area of interest as provided by PDquest software. Arrows indicate spots with relatively higher expression.

Mentions: Five of the excised protein spots had higher expression levels in wild-type Sp7 than in flcA deletion strain Sp7-flcAΔ, indicating that these proteins were up-regulated in the presence of FlcA (Table 3; Fig. 4). Three of these proteins, (UTP-glucose-1-phosphate uridylyltransferase (GalU), chemotaxis signal transduction protein (CheW) and citrate synthase (GltA)) were selected for further analysis by qRT-PCR, which confirmed an increase in galU, cheW and gltA expression in wild-type Sp7 compared to Sp7-flcAΔ under flocculation conditions (Fig. 5).


Cellular responses during morphological transformation in Azospirillum brasilense and Its flcA knockout mutant.

Hou X, McMillan M, Coumans JV, Poljak A, Raftery MJ, Pereg L - PLoS ONE (2014)

Differential protein expression in Sp7 and Sp7-flcAΔ analyzed by 2-DE.The left section shows the normalized expression volume of the spot in wild type (Sp7) and flcA knock out strain (Sp7-flcAΔ) under flocculation conditions; the relative fold change is shown above each column (Nx indicates that relative fold change could not be calculated as the protein was only detected in either Sp7 or Sp7-flcAΔ). The right section is a 3D representation of the area of interest as provided by PDquest software. Arrows indicate spots with relatively higher expression.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4264754&req=5

pone-0114435-g004: Differential protein expression in Sp7 and Sp7-flcAΔ analyzed by 2-DE.The left section shows the normalized expression volume of the spot in wild type (Sp7) and flcA knock out strain (Sp7-flcAΔ) under flocculation conditions; the relative fold change is shown above each column (Nx indicates that relative fold change could not be calculated as the protein was only detected in either Sp7 or Sp7-flcAΔ). The right section is a 3D representation of the area of interest as provided by PDquest software. Arrows indicate spots with relatively higher expression.
Mentions: Five of the excised protein spots had higher expression levels in wild-type Sp7 than in flcA deletion strain Sp7-flcAΔ, indicating that these proteins were up-regulated in the presence of FlcA (Table 3; Fig. 4). Three of these proteins, (UTP-glucose-1-phosphate uridylyltransferase (GalU), chemotaxis signal transduction protein (CheW) and citrate synthase (GltA)) were selected for further analysis by qRT-PCR, which confirmed an increase in galU, cheW and gltA expression in wild-type Sp7 compared to Sp7-flcAΔ under flocculation conditions (Fig. 5).

Bottom Line: Comparison of 2-DE protein profiles of wild-type (Sp7) and a flcA deletion mutant (Sp7-flcAΔ) revealed a total of 33 differentially expressed 2-DE gel spots, with 22 of these spots confidently separated to allow protein identification.Analysis of these spots by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MASCOT database searching identified 48 proteins (≥10% emPAI in each spot).This work is a step forward in connecting the Azospirillum phenome and genome.

View Article: PubMed Central - PubMed

Affiliation: School of Science and Technology, University of New England, Armidale, New South Wales, Australia; Department of Microbiology and Immunology, Shanxi Medical University, Taiyuan, Shanxi, China.

ABSTRACT
FlcA is a response regulator controlling flocculation and the morphological transformation of Azospirillum cells from vegetative to cyst-like forms. To understand the cellular responses of Azospirillum to conditions that cause morphological transformation, proteins differentially expressed under flocculation conditions in A. brasilense Sp7 and its flcA knockout mutant were investigated. Comparison of 2-DE protein profiles of wild-type (Sp7) and a flcA deletion mutant (Sp7-flcAΔ) revealed a total of 33 differentially expressed 2-DE gel spots, with 22 of these spots confidently separated to allow protein identification. Analysis of these spots by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MASCOT database searching identified 48 proteins (≥10% emPAI in each spot). The functional characteristics of these proteins included carbon metabolism (beta-ketothiolase and citrate synthase), nitrogen metabolism (Glutamine synthetase and nitric oxide synthase), stress tolerance (superoxide dismutase, Alkyl hydroperoxidase and ATP-dependent Clp protease proteolytic subunit) and morphological transformation (transducer coupling protein). The observed differences between Sp7 wild-type and flcA- strains enhance our understanding of the morphological transformation process and help to explain previous phenotypical observations. This work is a step forward in connecting the Azospirillum phenome and genome.

Show MeSH
Related in: MedlinePlus