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A plant MinD homologue rescues Escherichia coli HL1 mutant (DeltaMinDE) in the absence of MinE.

Zhang M, Hu Y, Jia J, Gao H, He Y - BMC Microbiol. (2009)

Bottom Line: AtMinD was localized to puncta at the poles of E. coli cells and puncta in chloroplasts without oscillation.AtMinD expressed in the HL1 mutant can cause a punctate localization pattern of GFP-EcMinC at cell ends.Yeast two hybrid and BiFC analysis showed that AtMinD can interact with EcMinC.

View Article: PubMed Central - HTML - PubMed

Affiliation: College of Life Science, Capital Normal University, Beijing 100037, PR China. zhangmin1689@gmail.com

ABSTRACT

Background: In E. coli, the Min operon (MinCDE) plays a key role in determining the site of cell division. MinE oscillates from the middle to one pole or another to drive the MinCD complex to the end of the cell. The MinCD complex prevents FtsZ ring formation and the subsequent cell division at cell ends. In Arabidopsis thaliana, a homologue of MinD has been shown to be involved in the positioning of chloroplast division site.

Results: To learn whether the MinD homologue in plants is functional in bacteria, AtMinD was expressed in E. coli. Surprisingly, AtMinD can rescue the minicell phenotype of E. coli HL1 mutant (DeltaMinDE) in the absence of EcMinE. This rescue requires EcMinC. AtMinD was localized to puncta at the poles of E. coli cells and puncta in chloroplasts without oscillation. AtMinD expressed in the HL1 mutant can cause a punctate localization pattern of GFP-EcMinC at cell ends. Yeast two hybrid and BiFC analysis showed that AtMinD can interact with EcMinC.

Conclusion: Similar to the MinD in Bacillus subtilis, AtMinD is localized to the polar region in E. coli and interacts with EcMinC to confine EcFtsZ polymerization and cell division at the midpoint of the cell.

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The phenotype of E. coli cells. (A) Wildtype, DH5α. (B) HL1 mutant (ΔMinDE). (C) HL1 mutant (ΔMinDE) complemented by pM1113-MinDE at 20 μM IPTG. (D) HL1 mutant (ΔMinDE) cannot be complemented by pM1113-AtMinD at 0 μM IPTG. (E) HL1 mutant (ΔMinDE) complemented by pM1113-AtMinD at 50 μM IPTG. (F) HL1 mutant (ΔMinDE) containing pM1113-MinD at 20 μM IPTG. (G) RC1 mutant (ΔMinCDE). (H) RC1 mutant (ΔMinCDE) containing pM1113-AtMinD at 50 μM IPTG. Arrows in (B, D, G and H) mark the minicells. The bar in (A to E, G and H) represents 10 μm; the bar in (F) represents 20 μm.
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Figure 1: The phenotype of E. coli cells. (A) Wildtype, DH5α. (B) HL1 mutant (ΔMinDE). (C) HL1 mutant (ΔMinDE) complemented by pM1113-MinDE at 20 μM IPTG. (D) HL1 mutant (ΔMinDE) cannot be complemented by pM1113-AtMinD at 0 μM IPTG. (E) HL1 mutant (ΔMinDE) complemented by pM1113-AtMinD at 50 μM IPTG. (F) HL1 mutant (ΔMinDE) containing pM1113-MinD at 20 μM IPTG. (G) RC1 mutant (ΔMinCDE). (H) RC1 mutant (ΔMinCDE) containing pM1113-AtMinD at 50 μM IPTG. Arrows in (B, D, G and H) mark the minicells. The bar in (A to E, G and H) represents 10 μm; the bar in (F) represents 20 μm.

Mentions: The E. coli HL1 mutant (ΔMinDE) has an apparent minicell phenotype with 30.5% of the cells are shorter than 2 μm and 38.1% of the cells are between 2 μm to 5 μm (Figure 1B and Table 1). Actually, most of the cells shorter than 2 μm are minicells that are usually shorter than 1.2 μm. In the wild-type DH5α, only 2.6% of the cells are smaller than 2 μm and 97.4% of the cells are between 2 μm to 5 μm (Figure 1A and Table 1). The mutant phenotype of HL1 mutant was complemented by a pM1113-MinDE plasmid with 20 μM IPTG (Figure 1C and Table 1), which was used for the induction of MinD and MinE. Because the homologues of MinD and MinE are involved in the division of chloroplasts in plants [9] and their function may still be conserved, we set up a bacterial system to study their function. Surprisingly, a pM1113-AtMinD plasmid can complement the mutant phenotype with 50 μM IPTG in the absence of EcMinE or AtMinE (Figure 1E, Table 1 and Table 2). We have also grown the E. coli HL1 mutant cells (ΔMinDE) containing pM1113-AtMinD with higher or lower concentration of IPTG, and found the mutant phenotype was recovered best with 50 μM IPTG (Figure 1E and our unpublished results). Minicells were reduced from 30.5% to 8.7% and the cells that are between 2 μm and 5 μm were increased from 38.1% to 87.4% (Table 1). Misplaced septa were also reduced from 55% to 6%, which is close to 3% in DH5α and 1% in the HL1 mutant rescued by EcMinD and EcMinE (Table 2). At higher IPTG concentration, the growth of cells was inhibited and the phenotype was not recovered so well (data not shown). Even without IPTG addition, the mutant phenotype was slightly rescued with a reduction of the cells that were 5–10 μm long from 29% to 5.6% (Table 1). This may be due to a leaky expression of AtMinD. As a control, HL1 mutant cells (ΔMinDE) transformed with a pM1113-EcMinD plasmid and grown with 20 μM IPTG showed a phenotype of long filaments but not minicells (Figure 1F and Table 1). This indicates that EcMinD is expressed and active but can not complement the mutant phenotype without EcMinE. To further understand the function of AtMinD in E. coli, AtMinD was expressed in RC1 mutant (Figure 1G and Table 1) that has a deletion of Min operon, i.e. MinCDE, with 50 μM IPTG. The RC1 mutant has a minicell phenotype similar to that of HL1 mutant. Expression of AtMinD in RC1 mutant couldn't rescue the mutant phenotype. These data suggest that the complementation of HL1 mutant by AtMinD requires the presence of EcMinC.


A plant MinD homologue rescues Escherichia coli HL1 mutant (DeltaMinDE) in the absence of MinE.

Zhang M, Hu Y, Jia J, Gao H, He Y - BMC Microbiol. (2009)

The phenotype of E. coli cells. (A) Wildtype, DH5α. (B) HL1 mutant (ΔMinDE). (C) HL1 mutant (ΔMinDE) complemented by pM1113-MinDE at 20 μM IPTG. (D) HL1 mutant (ΔMinDE) cannot be complemented by pM1113-AtMinD at 0 μM IPTG. (E) HL1 mutant (ΔMinDE) complemented by pM1113-AtMinD at 50 μM IPTG. (F) HL1 mutant (ΔMinDE) containing pM1113-MinD at 20 μM IPTG. (G) RC1 mutant (ΔMinCDE). (H) RC1 mutant (ΔMinCDE) containing pM1113-AtMinD at 50 μM IPTG. Arrows in (B, D, G and H) mark the minicells. The bar in (A to E, G and H) represents 10 μm; the bar in (F) represents 20 μm.
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Figure 1: The phenotype of E. coli cells. (A) Wildtype, DH5α. (B) HL1 mutant (ΔMinDE). (C) HL1 mutant (ΔMinDE) complemented by pM1113-MinDE at 20 μM IPTG. (D) HL1 mutant (ΔMinDE) cannot be complemented by pM1113-AtMinD at 0 μM IPTG. (E) HL1 mutant (ΔMinDE) complemented by pM1113-AtMinD at 50 μM IPTG. (F) HL1 mutant (ΔMinDE) containing pM1113-MinD at 20 μM IPTG. (G) RC1 mutant (ΔMinCDE). (H) RC1 mutant (ΔMinCDE) containing pM1113-AtMinD at 50 μM IPTG. Arrows in (B, D, G and H) mark the minicells. The bar in (A to E, G and H) represents 10 μm; the bar in (F) represents 20 μm.
Mentions: The E. coli HL1 mutant (ΔMinDE) has an apparent minicell phenotype with 30.5% of the cells are shorter than 2 μm and 38.1% of the cells are between 2 μm to 5 μm (Figure 1B and Table 1). Actually, most of the cells shorter than 2 μm are minicells that are usually shorter than 1.2 μm. In the wild-type DH5α, only 2.6% of the cells are smaller than 2 μm and 97.4% of the cells are between 2 μm to 5 μm (Figure 1A and Table 1). The mutant phenotype of HL1 mutant was complemented by a pM1113-MinDE plasmid with 20 μM IPTG (Figure 1C and Table 1), which was used for the induction of MinD and MinE. Because the homologues of MinD and MinE are involved in the division of chloroplasts in plants [9] and their function may still be conserved, we set up a bacterial system to study their function. Surprisingly, a pM1113-AtMinD plasmid can complement the mutant phenotype with 50 μM IPTG in the absence of EcMinE or AtMinE (Figure 1E, Table 1 and Table 2). We have also grown the E. coli HL1 mutant cells (ΔMinDE) containing pM1113-AtMinD with higher or lower concentration of IPTG, and found the mutant phenotype was recovered best with 50 μM IPTG (Figure 1E and our unpublished results). Minicells were reduced from 30.5% to 8.7% and the cells that are between 2 μm and 5 μm were increased from 38.1% to 87.4% (Table 1). Misplaced septa were also reduced from 55% to 6%, which is close to 3% in DH5α and 1% in the HL1 mutant rescued by EcMinD and EcMinE (Table 2). At higher IPTG concentration, the growth of cells was inhibited and the phenotype was not recovered so well (data not shown). Even without IPTG addition, the mutant phenotype was slightly rescued with a reduction of the cells that were 5–10 μm long from 29% to 5.6% (Table 1). This may be due to a leaky expression of AtMinD. As a control, HL1 mutant cells (ΔMinDE) transformed with a pM1113-EcMinD plasmid and grown with 20 μM IPTG showed a phenotype of long filaments but not minicells (Figure 1F and Table 1). This indicates that EcMinD is expressed and active but can not complement the mutant phenotype without EcMinE. To further understand the function of AtMinD in E. coli, AtMinD was expressed in RC1 mutant (Figure 1G and Table 1) that has a deletion of Min operon, i.e. MinCDE, with 50 μM IPTG. The RC1 mutant has a minicell phenotype similar to that of HL1 mutant. Expression of AtMinD in RC1 mutant couldn't rescue the mutant phenotype. These data suggest that the complementation of HL1 mutant by AtMinD requires the presence of EcMinC.

Bottom Line: AtMinD was localized to puncta at the poles of E. coli cells and puncta in chloroplasts without oscillation.AtMinD expressed in the HL1 mutant can cause a punctate localization pattern of GFP-EcMinC at cell ends.Yeast two hybrid and BiFC analysis showed that AtMinD can interact with EcMinC.

View Article: PubMed Central - HTML - PubMed

Affiliation: College of Life Science, Capital Normal University, Beijing 100037, PR China. zhangmin1689@gmail.com

ABSTRACT

Background: In E. coli, the Min operon (MinCDE) plays a key role in determining the site of cell division. MinE oscillates from the middle to one pole or another to drive the MinCD complex to the end of the cell. The MinCD complex prevents FtsZ ring formation and the subsequent cell division at cell ends. In Arabidopsis thaliana, a homologue of MinD has been shown to be involved in the positioning of chloroplast division site.

Results: To learn whether the MinD homologue in plants is functional in bacteria, AtMinD was expressed in E. coli. Surprisingly, AtMinD can rescue the minicell phenotype of E. coli HL1 mutant (DeltaMinDE) in the absence of EcMinE. This rescue requires EcMinC. AtMinD was localized to puncta at the poles of E. coli cells and puncta in chloroplasts without oscillation. AtMinD expressed in the HL1 mutant can cause a punctate localization pattern of GFP-EcMinC at cell ends. Yeast two hybrid and BiFC analysis showed that AtMinD can interact with EcMinC.

Conclusion: Similar to the MinD in Bacillus subtilis, AtMinD is localized to the polar region in E. coli and interacts with EcMinC to confine EcFtsZ polymerization and cell division at the midpoint of the cell.

Show MeSH
Related in: MedlinePlus