Interaction network among Escherichia coli membrane proteins involved in cell division as revealed by bacterial two-hybrid analysis

Abstract

Formation of the Escherichia coli division septum is catalyzed by a number of essential proteins (named Fts) that assemble into a ring-like structure at the future division site. Several of these Fts proteins are intrinsic transmembrane proteins whose functions are largely unknown. Although these proteins appear to be recruited to the division site in a hierarchical order, the molecular interactions underlying the assembly of the cell division machinery remain mostly unspecified. In the present study, we used a bacterial two-hybrid system based on interaction-mediated reconstitution of a cyclic AMP (cAMP) signaling cascade to unravel the molecular basis of septum assembly by analyzing the protein interaction network among E. coli cell division proteins. Our results indicate that the Fts proteins are connected to one another through multiple interactions. A deletion mapping analysis carried out with two of these proteins, FtsQ and FtsI, revealed that different regions of the polypeptides are involved in their associations with their partners. Furthermore, we showed that the association between two Fts hybrid proteins could be modulated by the coexpression of a third Fts partner. Altogether, these data suggest that the cell division machinery assembly is driven by the cooperative association among the different Fts proteins to form a dynamic multiprotein structure at the septum site. In addition, our study shows that the cAMP-based two-hybrid system is particularly appropriate for analyzing molecular interactions between membrane proteins.

FIG. 1.

Detection of membrane protein associations with the BACTH system. (a) Proteins of interest X and Y are genetically fused to the two complementary fragments, T25 and T18, from the catalytic domain of B. pertussis AC and coexpressed in E. coli cya cells. (b) Interaction between the two hybrid proteins results in functional complementation between the T25 and T18 fragments, leading to cAMP synthesis. cAMP, upon binding to the catabolite activator protein (a transcriptional regulator) triggers the expression of E. coli catabolic operons, allowing the bacteria to utilize sugars such as lactose and maltose. Efficiencies of complementation can be determined by measuring β-galactosidase activities in the transformed cells.

FIG. 2.

BACTH analysis of interactions between Fts proteins. The efficiencies of functional complementation between the indicated hybrid proteins were quantified by measuring β-galactosidase (beta-Gal) activities in suspensions of toluene-treated E. coli DHM1 cells harboring the corresponding plasmids, as described in Materials and Methods. Each bar represents the mean value from results for at least three independent cultures. In all cases, standard deviations were within 20% of the mean except in the assays involving the T18-FtsQ hybrid, for which higher standard deviations (up to 40%) were observed due to the instability of the pUT18C-ftsQ plasmid. The two membrane proteins MalF and MalG of the E. coli maltose ABC transporter (20) were used as controls. Under the same assay conditions, functional complementation between T25-MalF and T18-MalG and between T25-MalG and T18-MalF yielded 10,000 ± 1,200 and 1,000 ± 150 U of β-galactosidase/mg (dry weight) of bacteria, respectively.

FIG. 3.

Mapping of the FtsQ interacting domains. (a) Schematic representation of FtsQ and its truncated variants. The cytosolic part (CP), transmembrane domain (TM), and periplasmic part (PP) of FtsQ are indicated. (b) Functional complementation between the indicated hybrid proteins was quantified by measuring β-galactosidase (beta-Gal) activities in suspensions of toluene-treated E. coli DHM1 cells harboring the corresponding plasmids, as described in Materials and Methods. Each bar represents the mean value from results for at least three independent cultures. Standard deviations were within 40% of the mean for the wild-type T18-FtsQ assays (due to pUT18C-ftsQ instability) and below 20% of the mean for all other assays (plasmids carrying the T18-truncated FtsQ derivatives were stable). FtsQ_wt, wild-type FtsQ.

FIG. 4.

Mapping of the FtsI interacting domains. (a) Schematic representation of FtsI and its truncated variants. The cytosolic part (CP), transmembrane domain (TM), and periplasmic part (PP), including the catalytic domain (CD), of FtsI are indicated. (b) Functional complementation between the indicated hybrid proteins was quantified as described in the legend to Fig. 3. Each bar represents the mean value from results for at least three independent cultures, with standard deviations below 20% of the mean. beta-Gal, β-galactosidase; FtsI_wt, wild-type FtsI.

FIG. 5.

Three-hybrid experiments. (a) Schematic maps of pUT18C-ftsB and pUT18C-ftsB/ftsL plasmids show the locations of the T18 gene-ftsB fusion and the ftsL and bla (ampicillin resistance) genes, the ColE1 origin of replication (ori ColE1), the Shine-Dalgarno sequence (SD), and the lac promoter (Pr lac). DHM1 cells were cotransformed with the pKT25 derivative expressing the indicated T25-Fts protein fusion, either plasmid pUT18C-ftsB, expressing the T18-FtsB fusion, or plasmid pUT18C-ftsB/ftsL, coexpressing the T18-FtsB fusion and the wild-type FtsL polypeptide. beta-Gal, β-galactosidase. (b) Schematic maps of pUT18C-ftsI and pUT18C-ftsI/ftsL plasmids show locations of the T18 gene-ftsI fusion and the ftsL and bla (ampicillin resistance) genes, the ColE1 origin of replication, the Shine-Dalgarno sequence, and the lac promoter. DHM1 cells were cotransformed with the indicated pKT25 plasmid, either pUT18C-ftsI, expressing the T18-FtsI fusion, or pUT18C-ftsB/ftsL, coexpressing the T18-FtsI fusion and the wild-type FtsL polypeptide. Efficiencies of the interactions were monitored as described in the legend to Fig. 3. Each bar represents the mean value from results for at least three independent cultures, with standard deviations below 20% of the mean.