The β-Lactam Resistance Protein Blr, a Small Membrane Polypeptide, Is a Component of the Escherichia coli Cell Division Machinery

Abstract

In Escherichia coli, cell division is performed by a multimolecular machinery called the divisome, made of 10 essential proteins and more than 20 accessory proteins. Through a bacterial two-hybrid library screen, we identified the E. coli ␤-lactam resistance protein Blr, a short membrane polypeptide of 41 residues, as an interacting partner of the essential cell division protein FtsL. In addition to FtsL, Blr was found to associate with several other divisomal proteins, including FtsI, FtsK, FtsN, FtsQ, FtsW, and YmgF. Using fluorescently tagged Blr, we showed that this peptide localizes to the division septum and that its colocalization requires the presence of the late division protein FtsN. Although Blr is not essential, previous studies have shown that the inactivation of the blr gene increased the sensitivity of bacteria to ␤-lactam antibiotics or their resistance to cell envelope stress. Here, we found that Blr, when overproduced, restores the viability of E. coli ftsQ1(Ts) cells, carrying a thermosensitive allele of the ftsQ gene, during growth under low-osmotic-strength conditions (e.g., in synthetic media or in Luria-Bertani broth without NaCl). In contrast, the inactivation of blr increases the osmosensitivity of ftsQ1(Ts) cells, and blr ftsQ1 double mutants exhibit filamentous growth in LB broth even at a moderate salt concentration (0.5% NaCl) compared to parental ftsQ1(Ts) cells. Altogether, our results suggest that the small membrane polypeptide Blr is a novel component of the E. coli cell division apparatus involved in the stabilization of the divisome under certain stress conditions.

Small Proteins Can No Longer Be Ignored

“…blr was first identified as a stand-alone gene (40) that, when disrupted, caused increased sensitivity to β-lactam antibiotics (β-lactam resistance) (41) and, surprisingly, decreased sensitivity to cell envelope stress (42). Blr was later identified in a bacterial two-hybrid screen (43) for proteins that interacted with FtsL, an integral membrane protein with a very poorly understood function that is nonetheless a core component of the division machinery (30). The two-hybrid assay also suggested that Blr could associate with other components of the cell division machinery, such as FtsI, FtsK, FtsN, FtsQ, and FtsW (43).…”

“…Blr was later identified in a bacterial two-hybrid screen (43) for proteins that interacted with FtsL, an integral membrane protein with a very poorly understood function that is nonetheless a core component of the division machinery (30). The two-hybrid assay also suggested that Blr could associate with other components of the cell division machinery, such as FtsI, FtsK, FtsN, FtsQ, and FtsW (43). Consistent with these results, GFP-Blr was found to localize to the division septum of E. coli in a divisome-dependent manner.…”

Two novel heteropolymer-forming proteins maintain multicellular shape of the cyanobacteriumAnabaenasp. PCC 7120

“…Chemically competent E. coli BTH101 cells were co-transformed with 5 ng of plasmids carrying the respective T18 and T25 translational fusion constructs, plated onto LB plates supplemented with 200 µg ml −1 X-gal, 0.5 mM IPTG, Amp, Km and grown at 30°C for 24-36 h. Interactions were quantified by beta-galactosidase assays from three independent colonies. For this aim, cultures were either grown over night at 30 °C or for two days at 20 °C in LB Amp, Km, 0.5 mM IPTG and beta-galactosidase activity was recorded as described in the manufacturer’s instructions (Euromedex; BACTH System Kit Bacterial Adenylate Cyclase Two-Hybrid System Kit) in a 96 well plate according to Karimova, Davi and Ladant 71 .…”

“…Beta-galactosidase assays of E. coli BTH101 cells co-expressing indicated T25 and T18 translational fusions of all possible pair-wise combinations. E. coli cells carrying the respective plasmids were subjected to beta-galactosidase assay as described by Karimova et al 71 in triplicates from three independent colonies grown for 2 d at 20 °C. Quantity values are given in Miller Units per milligram LacZ of the mean results from three independent colonies.…”

Conditional Proteolysis of the Membrane Protein YfgM by the FtsH Protease Depends on a Novel N-terminal Degron

“…A possible explanation for this observation is that YfgM has several other interaction partners and that binding of these partners may be altered when the N terminus is changed. Most conspicuous in this context is the essential membranebound cell division protein FtsL, which was found as the interaction partner of YfgM in a bacterial two-hybrid screen (83).…”