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Characterization of the algC gene expression pattern in the multidrug resistant Acinetobacter baumannii AIIMS 7 and correlation with biofilm development on abiotic surface.

Sahu PK, Iyer PS, Barage SH, Sonawane KD, Chopade BA - ScientificWorldJournal (2014)

Bottom Line: Comparison revealed differential algC expression pattern with maximum 81.59-fold increase in biofilm cells versus 3.24-fold in planktonic cells (P < 0.05).Expression levels strongly correlated with specific biofilm stages (scale of 3 to 96 h), coinciding maximum at initial surface attachment stage (9 h) and biofilm maturation stage (48 h).Moreover, molecular dynamics analysis on the three-dimensional structure of PMM/PGM (simulated up to 10 ns) revealed enzyme structure as stable and similar to that in P. aeruginosa (synthesis of alginate and lipopolysaccharide core) and involved in constitution of biofilm EPS (extracellular polymeric substances).

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioinformatics and Biotechnology, University of Pune, Pune 411 007, India ; Ispat General Hospital, SAIL, Rourkela 769 005, India.

ABSTRACT
Relative quantification of algC gene expression was evaluated in the multidrug resistant strain Acinetobacter baumannii AIIMS 7 biofilm (3 to 96 h, on polystyrene surface) compared to the planktonic counterparts. Comparison revealed differential algC expression pattern with maximum 81.59-fold increase in biofilm cells versus 3.24-fold in planktonic cells (P < 0.05). Expression levels strongly correlated with specific biofilm stages (scale of 3 to 96 h), coinciding maximum at initial surface attachment stage (9 h) and biofilm maturation stage (48 h). Cloning, heterologous expression, and bioinformatics analyses indicated algC gene product as the bifunctional enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) of ∼ 53 kDa size, which augmented biofilms significantly in algC clones compared to controls (lacking algC gene), further localized by scanning electron microscopy. Moreover, molecular dynamics analysis on the three-dimensional structure of PMM/PGM (simulated up to 10 ns) revealed enzyme structure as stable and similar to that in P. aeruginosa (synthesis of alginate and lipopolysaccharide core) and involved in constitution of biofilm EPS (extracellular polymeric substances). Our observation on differential expression pattern of algC having strong correlation with important biofilm stages, scanning electron-microscopic evidence of biofilm augmentation taken together with predictive enzyme functions via molecular dynamic (MD) simulation, proposes a new basis of A. baumannii AIIMS 7 biofilm development on inanimate surfaces.

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Related in: MedlinePlus

Comparison of algC gene expression pattern in biofilm and planktonic A. baumannii AIIMS 7. Real-time quantitative PCR results showing relative quantification of algC gene expression levels (calculated according to ΔΔCt-method and represented as “fold over increase”) at corresponding time points. Expression levels were normalized with indigenous control (16SrRNA gene expression) in both biofilm and planktonic cells (calibrator: 24-hour biofilm sample, fold over increase = 1).
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fig1: Comparison of algC gene expression pattern in biofilm and planktonic A. baumannii AIIMS 7. Real-time quantitative PCR results showing relative quantification of algC gene expression levels (calculated according to ΔΔCt-method and represented as “fold over increase”) at corresponding time points. Expression levels were normalized with indigenous control (16SrRNA gene expression) in both biofilm and planktonic cells (calibrator: 24-hour biofilm sample, fold over increase = 1).

Mentions: Quantitative real-time PCR experiment showed significant variation in the threshold cycle (Ct) values in biofilm and planktonic mode of growth of A. baumannii, which described differential expressional patterns of the two modes of growth (Figure 1). Number of copies of algC mRNA were calculated using 16SrRNA gene as internal control at each time point (ΔCt). To evaluate the relative gene expression in biofilm and planktonic modes of growth over the temporal scale of 3 to 96 hours, the 24-hour biofilm sample was used as calibrator (ΔΔCt). Relative quantification (RQ) plot for gene expression at various time points from 3 to 96 hours showed low basal (almost linear) expression of algC in planktonic mode (maximum of 3.24-fold, 48 hours) whereas highly variable and elevated algC expression was seen in biofilm mode (maximum 81.59-fold, 48 hours) and displaying a definite pattern. The initial attachment stage (9 hours) and maturation stage (48 hours) of biofilm showed maximal expression of algC, which was in contrast and could be seen as steadily low expression at almost all time points in planktonic mode of growth (Figure 1). This observation affirms that the algC gene is highly up-regulated (at the transcription level) while the A. baumannii cells grow in a biofilm mode (i.e., attached to an abiotic surface) and follows a pattern as depicted (Figure 1); however in planktonic (free form, in a suspension, unattached to surface) the algC gene is transcribed at a basal rate.


Characterization of the algC gene expression pattern in the multidrug resistant Acinetobacter baumannii AIIMS 7 and correlation with biofilm development on abiotic surface.

Sahu PK, Iyer PS, Barage SH, Sonawane KD, Chopade BA - ScientificWorldJournal (2014)

Comparison of algC gene expression pattern in biofilm and planktonic A. baumannii AIIMS 7. Real-time quantitative PCR results showing relative quantification of algC gene expression levels (calculated according to ΔΔCt-method and represented as “fold over increase”) at corresponding time points. Expression levels were normalized with indigenous control (16SrRNA gene expression) in both biofilm and planktonic cells (calibrator: 24-hour biofilm sample, fold over increase = 1).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4269089&req=5

fig1: Comparison of algC gene expression pattern in biofilm and planktonic A. baumannii AIIMS 7. Real-time quantitative PCR results showing relative quantification of algC gene expression levels (calculated according to ΔΔCt-method and represented as “fold over increase”) at corresponding time points. Expression levels were normalized with indigenous control (16SrRNA gene expression) in both biofilm and planktonic cells (calibrator: 24-hour biofilm sample, fold over increase = 1).
Mentions: Quantitative real-time PCR experiment showed significant variation in the threshold cycle (Ct) values in biofilm and planktonic mode of growth of A. baumannii, which described differential expressional patterns of the two modes of growth (Figure 1). Number of copies of algC mRNA were calculated using 16SrRNA gene as internal control at each time point (ΔCt). To evaluate the relative gene expression in biofilm and planktonic modes of growth over the temporal scale of 3 to 96 hours, the 24-hour biofilm sample was used as calibrator (ΔΔCt). Relative quantification (RQ) plot for gene expression at various time points from 3 to 96 hours showed low basal (almost linear) expression of algC in planktonic mode (maximum of 3.24-fold, 48 hours) whereas highly variable and elevated algC expression was seen in biofilm mode (maximum 81.59-fold, 48 hours) and displaying a definite pattern. The initial attachment stage (9 hours) and maturation stage (48 hours) of biofilm showed maximal expression of algC, which was in contrast and could be seen as steadily low expression at almost all time points in planktonic mode of growth (Figure 1). This observation affirms that the algC gene is highly up-regulated (at the transcription level) while the A. baumannii cells grow in a biofilm mode (i.e., attached to an abiotic surface) and follows a pattern as depicted (Figure 1); however in planktonic (free form, in a suspension, unattached to surface) the algC gene is transcribed at a basal rate.

Bottom Line: Comparison revealed differential algC expression pattern with maximum 81.59-fold increase in biofilm cells versus 3.24-fold in planktonic cells (P < 0.05).Expression levels strongly correlated with specific biofilm stages (scale of 3 to 96 h), coinciding maximum at initial surface attachment stage (9 h) and biofilm maturation stage (48 h).Moreover, molecular dynamics analysis on the three-dimensional structure of PMM/PGM (simulated up to 10 ns) revealed enzyme structure as stable and similar to that in P. aeruginosa (synthesis of alginate and lipopolysaccharide core) and involved in constitution of biofilm EPS (extracellular polymeric substances).

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioinformatics and Biotechnology, University of Pune, Pune 411 007, India ; Ispat General Hospital, SAIL, Rourkela 769 005, India.

ABSTRACT
Relative quantification of algC gene expression was evaluated in the multidrug resistant strain Acinetobacter baumannii AIIMS 7 biofilm (3 to 96 h, on polystyrene surface) compared to the planktonic counterparts. Comparison revealed differential algC expression pattern with maximum 81.59-fold increase in biofilm cells versus 3.24-fold in planktonic cells (P < 0.05). Expression levels strongly correlated with specific biofilm stages (scale of 3 to 96 h), coinciding maximum at initial surface attachment stage (9 h) and biofilm maturation stage (48 h). Cloning, heterologous expression, and bioinformatics analyses indicated algC gene product as the bifunctional enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) of ∼ 53 kDa size, which augmented biofilms significantly in algC clones compared to controls (lacking algC gene), further localized by scanning electron microscopy. Moreover, molecular dynamics analysis on the three-dimensional structure of PMM/PGM (simulated up to 10 ns) revealed enzyme structure as stable and similar to that in P. aeruginosa (synthesis of alginate and lipopolysaccharide core) and involved in constitution of biofilm EPS (extracellular polymeric substances). Our observation on differential expression pattern of algC having strong correlation with important biofilm stages, scanning electron-microscopic evidence of biofilm augmentation taken together with predictive enzyme functions via molecular dynamic (MD) simulation, proposes a new basis of A. baumannii AIIMS 7 biofilm development on inanimate surfaces.

Show MeSH
Related in: MedlinePlus