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The essential peptidoglycan glycosyltransferase MurG forms a complex with proteins involved in lateral envelope growth as well as with proteins involved in cell division in Escherichia coli.

Mohammadi T, Karczmarek A, Crouvoisier M, Bouhss A, Mengin-Lecreulx D, den Blaauwen T - Mol. Microbiol. (2007)

Bottom Line: In view of this, the loss of rod shape of DeltamreBCD strain could be ascribed to the loss of MurG membrane localization.Consequently, this could prevent the localized supply of the lipid II precursor to the peptidoglycan synthesizing machinery involved in cell elongation.A model representing the first complex is proposed.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 316, 1098 SM Amsterdam, PO Box 194062, 1090 GB Amsterdam, The Netherlands.

ABSTRACT
In Escherichia coli many enzymes including MurG are directly involved in the synthesis and assembly of peptidoglycan. MurG is an essential glycosyltransferase catalysing the last intracellular step of peptidoglycan synthesis. To elucidate its role during elongation and division events, localization of MurG using immunofluorescence microscopy was performed. MurG exhibited a random distribution in the cell envelope with a relatively higher intensity at the division site. This mid-cell localization was dependent on the presence of a mature divisome. Its localization in the lateral cell wall appeared to require the presence of MreCD. This could be indicative of a potential interaction between MurG and other proteins. Investigating this by immunoprecipitation revealed the association of MurG with MreB and MraY in the same protein complex. In view of this, the loss of rod shape of DeltamreBCD strain could be ascribed to the loss of MurG membrane localization. Consequently, this could prevent the localized supply of the lipid II precursor to the peptidoglycan synthesizing machinery involved in cell elongation. It is postulated that the involvement of MurG in the peptidoglycan synthesis concurs with two complexes, one implicated in cell elongation and the other in division. A model representing the first complex is proposed.

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Immunoblotting (IB) analysis of the protein complex with anti-MurG, anti-MreB and anti-β-lactamase under non-reducing conditions. The membranes extracted from BW25113ΔmraY/pMAKmraYec strain (MraY–β-lactamase–His expressing strain) were used for the IP (with cross-linking) with anti-MurG. The blot was then probed with anti-MurG (lanes 1 and 2), anti-MreB (lanes 3 and 4) or anti β-lactamase (lanes 5 and 6). In parallel the same IP was performed without the membrane fraction (lanes 1, 3 and 5). A cross-linked product with a molecular weight of about 120 kDa is visible in all samples except in control samples (lanes 1, 3 and 5). The faint band in lane 2 is MurG, which is not completely cross-linked in the protein complex. The band in lane 6 with the molecular weight of approximately 70 kDa corresponds to the MraY–β-lactamase–His protein that is not completely cross-linked in the protein complex.
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fig05: Immunoblotting (IB) analysis of the protein complex with anti-MurG, anti-MreB and anti-β-lactamase under non-reducing conditions. The membranes extracted from BW25113ΔmraY/pMAKmraYec strain (MraY–β-lactamase–His expressing strain) were used for the IP (with cross-linking) with anti-MurG. The blot was then probed with anti-MurG (lanes 1 and 2), anti-MreB (lanes 3 and 4) or anti β-lactamase (lanes 5 and 6). In parallel the same IP was performed without the membrane fraction (lanes 1, 3 and 5). A cross-linked product with a molecular weight of about 120 kDa is visible in all samples except in control samples (lanes 1, 3 and 5). The faint band in lane 2 is MurG, which is not completely cross-linked in the protein complex. The band in lane 6 with the molecular weight of approximately 70 kDa corresponds to the MraY–β-lactamase–His protein that is not completely cross-linked in the protein complex.

Mentions: Probing the immunoblot with anti-MurG resulted in cross-linked products among a smeared background. The prominent protein band of these products had a size of about 120 kDa (Fig. 4A, lane 2 and Fig. 5, lane 2). This band was also detected with anti-MreB (Fig. 4, lane 2 and Fig. 5, lane 4). Performing the analysis with a polyclonal antibody directed against β-lactamase (to detect the MraY of E. coli in fusion with β-lactamase) revealed the presence of a band with a molecular weight of around 120 kDa in addition to a 70 kDa band that corresponded to the molecular weight of MraY–β-lactamase–His fusion (Fig. 5, lane 6). As the complex was analysed under non-reducing conditions on SDS-PAGE, it might be of importance to mention that the molecular weight of the complexes might slightly diverge from the genuine mass of the separate proteins forming the complex. The 250 kDa complex could also be detected although the intensity of this band is much less than the cross-linked product in wild-type LMC500 strain and almost indistinguishable from the background. This difference might be attributed to the fact that LMC500 and BW25113ΔmraY/pMAKmraYec are distinct strains. Control experiments where the same IP procedure was carried out without the membrane fractions yielded no products (Fig. 5, lanes 1, 3 and 5) indicating that MurG and MraY interact. As a whole these findings suggest that MurG, MreB and MraY were cross-linked in the same protein complex.


The essential peptidoglycan glycosyltransferase MurG forms a complex with proteins involved in lateral envelope growth as well as with proteins involved in cell division in Escherichia coli.

Mohammadi T, Karczmarek A, Crouvoisier M, Bouhss A, Mengin-Lecreulx D, den Blaauwen T - Mol. Microbiol. (2007)

Immunoblotting (IB) analysis of the protein complex with anti-MurG, anti-MreB and anti-β-lactamase under non-reducing conditions. The membranes extracted from BW25113ΔmraY/pMAKmraYec strain (MraY–β-lactamase–His expressing strain) were used for the IP (with cross-linking) with anti-MurG. The blot was then probed with anti-MurG (lanes 1 and 2), anti-MreB (lanes 3 and 4) or anti β-lactamase (lanes 5 and 6). In parallel the same IP was performed without the membrane fraction (lanes 1, 3 and 5). A cross-linked product with a molecular weight of about 120 kDa is visible in all samples except in control samples (lanes 1, 3 and 5). The faint band in lane 2 is MurG, which is not completely cross-linked in the protein complex. The band in lane 6 with the molecular weight of approximately 70 kDa corresponds to the MraY–β-lactamase–His protein that is not completely cross-linked in the protein complex.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2170320&req=5

fig05: Immunoblotting (IB) analysis of the protein complex with anti-MurG, anti-MreB and anti-β-lactamase under non-reducing conditions. The membranes extracted from BW25113ΔmraY/pMAKmraYec strain (MraY–β-lactamase–His expressing strain) were used for the IP (with cross-linking) with anti-MurG. The blot was then probed with anti-MurG (lanes 1 and 2), anti-MreB (lanes 3 and 4) or anti β-lactamase (lanes 5 and 6). In parallel the same IP was performed without the membrane fraction (lanes 1, 3 and 5). A cross-linked product with a molecular weight of about 120 kDa is visible in all samples except in control samples (lanes 1, 3 and 5). The faint band in lane 2 is MurG, which is not completely cross-linked in the protein complex. The band in lane 6 with the molecular weight of approximately 70 kDa corresponds to the MraY–β-lactamase–His protein that is not completely cross-linked in the protein complex.
Mentions: Probing the immunoblot with anti-MurG resulted in cross-linked products among a smeared background. The prominent protein band of these products had a size of about 120 kDa (Fig. 4A, lane 2 and Fig. 5, lane 2). This band was also detected with anti-MreB (Fig. 4, lane 2 and Fig. 5, lane 4). Performing the analysis with a polyclonal antibody directed against β-lactamase (to detect the MraY of E. coli in fusion with β-lactamase) revealed the presence of a band with a molecular weight of around 120 kDa in addition to a 70 kDa band that corresponded to the molecular weight of MraY–β-lactamase–His fusion (Fig. 5, lane 6). As the complex was analysed under non-reducing conditions on SDS-PAGE, it might be of importance to mention that the molecular weight of the complexes might slightly diverge from the genuine mass of the separate proteins forming the complex. The 250 kDa complex could also be detected although the intensity of this band is much less than the cross-linked product in wild-type LMC500 strain and almost indistinguishable from the background. This difference might be attributed to the fact that LMC500 and BW25113ΔmraY/pMAKmraYec are distinct strains. Control experiments where the same IP procedure was carried out without the membrane fractions yielded no products (Fig. 5, lanes 1, 3 and 5) indicating that MurG and MraY interact. As a whole these findings suggest that MurG, MreB and MraY were cross-linked in the same protein complex.

Bottom Line: In view of this, the loss of rod shape of DeltamreBCD strain could be ascribed to the loss of MurG membrane localization.Consequently, this could prevent the localized supply of the lipid II precursor to the peptidoglycan synthesizing machinery involved in cell elongation.A model representing the first complex is proposed.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 316, 1098 SM Amsterdam, PO Box 194062, 1090 GB Amsterdam, The Netherlands.

ABSTRACT
In Escherichia coli many enzymes including MurG are directly involved in the synthesis and assembly of peptidoglycan. MurG is an essential glycosyltransferase catalysing the last intracellular step of peptidoglycan synthesis. To elucidate its role during elongation and division events, localization of MurG using immunofluorescence microscopy was performed. MurG exhibited a random distribution in the cell envelope with a relatively higher intensity at the division site. This mid-cell localization was dependent on the presence of a mature divisome. Its localization in the lateral cell wall appeared to require the presence of MreCD. This could be indicative of a potential interaction between MurG and other proteins. Investigating this by immunoprecipitation revealed the association of MurG with MreB and MraY in the same protein complex. In view of this, the loss of rod shape of DeltamreBCD strain could be ascribed to the loss of MurG membrane localization. Consequently, this could prevent the localized supply of the lipid II precursor to the peptidoglycan synthesizing machinery involved in cell elongation. It is postulated that the involvement of MurG in the peptidoglycan synthesis concurs with two complexes, one implicated in cell elongation and the other in division. A model representing the first complex is proposed.

Show MeSH
Related in: MedlinePlus