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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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(a)-(b) Multiple sequence alignment and phylogenetic relationship of PMM/PGM. (a) CLUSTALW analyses of PMM/PGM protein sequences displaying representative color of box which highlights conserved regions, that is, active site (red), Mg2+ binding site (green), and substrate binding site (blue). (b) Bootstrap consensus tree for PMM/PGM enzymes, constructed by MEGA v5.0 using the neighbor-joining (NJ) method (based on 1,000 replicates). Numbers on the nodes are bootstrap values and the bar represents scale of estimated evolutionary distance (20 substitutions at any amino acid position per 100 amino acid positions).
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fig4: (a)-(b) Multiple sequence alignment and phylogenetic relationship of PMM/PGM. (a) CLUSTALW analyses of PMM/PGM protein sequences displaying representative color of box which highlights conserved regions, that is, active site (red), Mg2+ binding site (green), and substrate binding site (blue). (b) Bootstrap consensus tree for PMM/PGM enzymes, constructed by MEGA v5.0 using the neighbor-joining (NJ) method (based on 1,000 replicates). Numbers on the nodes are bootstrap values and the bar represents scale of estimated evolutionary distance (20 substitutions at any amino acid position per 100 amino acid positions).

Mentions: To compare and evaluate the relatedness of A. baumannii AIIMS 7 algC encoded PMM/PGM, in silico analyses were performed. BLASTn results showed 100% similarity with the predicted PMM/PGM sequence in whole genome sequences of A. baumannii available in NCBI database. Alignment results also displayed significant similarity with PMM/PGM nucleotide sequences of other genera. 472 amino acid residues could be theoretically translated in one frame (5′–3′) of the DNA sequence. BLASTp showed similar results as in BLASTn alignment. The molecular weight of the protein was predicted to be 52.94 kDa with isoelectric point (pI) of 5.67. CLUSTALW alignment with selected bacterial and eukaryotic PMM/PGM sequences showed conserved regions in the protein as shown in Figure 4(a) (active site, Mg2+ binding site, and sugar binding site marked as colored rectangles) in the protein as shown. Figure 4(b) shows the phylogenetic tree indicating relatedness between PMM/PGM enzymes from pseudomonads and higher eukaryotes (rabbit and human). PMM/PGM sequence from A. baumannii had less than 25% identity with rabbit muscle PGM isoform 1, whereas it had a mere 10% identity with human PMM (Figure 4(b)).


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)

(a)-(b) Multiple sequence alignment and phylogenetic relationship of PMM/PGM. (a) CLUSTALW analyses of PMM/PGM protein sequences displaying representative color of box which highlights conserved regions, that is, active site (red), Mg2+ binding site (green), and substrate binding site (blue). (b) Bootstrap consensus tree for PMM/PGM enzymes, constructed by MEGA v5.0 using the neighbor-joining (NJ) method (based on 1,000 replicates). Numbers on the nodes are bootstrap values and the bar represents scale of estimated evolutionary distance (20 substitutions at any amino acid position per 100 amino acid positions).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4269089&req=5

fig4: (a)-(b) Multiple sequence alignment and phylogenetic relationship of PMM/PGM. (a) CLUSTALW analyses of PMM/PGM protein sequences displaying representative color of box which highlights conserved regions, that is, active site (red), Mg2+ binding site (green), and substrate binding site (blue). (b) Bootstrap consensus tree for PMM/PGM enzymes, constructed by MEGA v5.0 using the neighbor-joining (NJ) method (based on 1,000 replicates). Numbers on the nodes are bootstrap values and the bar represents scale of estimated evolutionary distance (20 substitutions at any amino acid position per 100 amino acid positions).
Mentions: To compare and evaluate the relatedness of A. baumannii AIIMS 7 algC encoded PMM/PGM, in silico analyses were performed. BLASTn results showed 100% similarity with the predicted PMM/PGM sequence in whole genome sequences of A. baumannii available in NCBI database. Alignment results also displayed significant similarity with PMM/PGM nucleotide sequences of other genera. 472 amino acid residues could be theoretically translated in one frame (5′–3′) of the DNA sequence. BLASTp showed similar results as in BLASTn alignment. The molecular weight of the protein was predicted to be 52.94 kDa with isoelectric point (pI) of 5.67. CLUSTALW alignment with selected bacterial and eukaryotic PMM/PGM sequences showed conserved regions in the protein as shown in Figure 4(a) (active site, Mg2+ binding site, and sugar binding site marked as colored rectangles) in the protein as shown. Figure 4(b) shows the phylogenetic tree indicating relatedness between PMM/PGM enzymes from pseudomonads and higher eukaryotes (rabbit and human). PMM/PGM sequence from A. baumannii had less than 25% identity with rabbit muscle PGM isoform 1, whereas it had a mere 10% identity with human PMM (Figure 4(b)).

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