The average cellular positions of the ftsQAZ region (2 min) and the minB region (26.5 min) during the cell cycle was determined by fluorescent in situ hybridization using the position of oriC as a reference point. At the steady‐state growth conditions used, newborn cells had replicated about 50% of the chromosome. By measuring the distances of the labelled oriC s with respect to mid‐cell, we found two well‐separated average oriC positions in cells of newborn length. These average oriC positions moved further apart along with cell elongation. The cellular position of the ftsQAZ gene region resembled the position of oriC , although its average position was closer to mid‐cell. In contrast, a single minB focus was observed at cell birth. Separated minB foci appeared towards the end of DNA replication. The average positions of oriC , ftsQAZ and minB relative to each other fitted a model in which DNA replication takes place in the cell centre and subsequent gene regions pass sequentially through this centre. We have interpreted the polarized orientation of the studied gene regions as a consequence of the mode of DNA segregation.
Summary The murein (peptidoglycan) sacculus is an essential polymer embedded in the bacterial envelope. The Escherichia coli class B penicillin‐binding protein (PBP) 3 is a murein transpeptidase and essential for cell division. In an affinity chromatography experiment, the bifunctional transglycosylase‐transpeptidase murein synthase PBP1B was retained by PBP3‐sepharose when a membrane fraction of E. coli was applied. The direct protein–protein interaction between purified PBP3 and PBP1B was characterized in vitro by surface plasmon resonance. The interaction was confirmed in vivo employing two different methods: by a bacterial two‐hybrid system, and by cross‐linking/co‐immunoprecipitation. In the bacterial two‐hybrid system, a truncated PBP3 comprising the N‐terminal 56 amino acids interacted with PBP1B. Both synthases could be cross‐linked in vivo in wild‐type cells and in cells lacking FtsW or FtsN. PBP1B localized diffusely and in foci at the septation site and also at the side wall. Statistical analysis of the immunofluorescence signals revealed that the localization of PBP1B at the septation site depended on the physical presence of PBP3, but not on the activity of PBP3. These studies have demonstrated, for the first time, a direct interaction between a class B PBP (PBP3) and a class A PBP (PBP1B) in vitro and in vivo , indicating that different murein synthases might act in concert to enlarge the murein sacculus during cell division.
A high-molecular-weight band has been detected in Western immunoblots of nonboiled Escherichia coli samples incubated with polyclonal antiserum against penicillin-binding protein 1B (PBP 1B). This band was shown to be a dimer of PBP 1B. The dimer was more strongly associated with the envelope than the monomer, and it was still able to bind penicillin G. Analysis of the binding of fusion proteins of PBP 1B and beta-lactamase showed that the part of PBP 1B necessary for complex formation lies in the amino-terminal half of the protein.
In an attempt to distinguish between the UDP-glucose: flavonol 3-O-glucosyltransferase (3GT) and the UDP-glucose: anthocyanidin 3-O-glucosyltransferase in flower buds of Petunia hybrida, several properties of these activities were determined. The 3-glucosylation of anthocyanidin had a pH-activity optimum of 7.2, that of flavonol pH 9.2 to 9.5. Anthocyanidin 3GT activity was lowered in the presence of EDTA or β-mercaptoethanol, but this was due to an effect on the anthocyanidin substrate. The two 3-glucosylating activities were to a similar extent inhibited by an increasing ionic strength in the enzyme assay and showed an identical iso-electric point (5.2) as determined by chromatofocusing. Molecular weights were identical: 26000, 52000 or 78000 daltons as determined by gel-filtration. Antiserum raised against partially purified 3GT gave identical immunoprecipitation curves with flavonol 3GT and anthocyanidin 3GT. Special attention was given to the 3-O-glucosyltransferase in mutants with low levels of 3GT activity. These mutants are unable to form significant amounts of anthocyanins but contain wild- type amounts of flavonols. The enzyme of such mutants had the same iso-electric point and identical titration-curves with antiserum as the enzyme from wildtype plants. 3GT from wildtype or mutant plants glucosylated flavonols at higher rates than anthocyanidins.
This protocol was developed to label proteins in bacterial cells with antibodies conjugated to a fluorophore for fluorescence microscopy imaging. The procedure is optimized to minimize morphological changes and also to minimize the amount of antibodies needed for the staining. The protocol can also be used with primary antibodies conjugated to a fluorophore. The method has been verified extensively (van der Ploeg et al., 2013), but it should be noted that one case in Caulobacter crescentus (Hocking et al., 2012) has been reported in which the localization of a protein changed upon fixation by formaldehyde/glutaraldehyde. However, the localization of the same protein in E. coli did not change.
Abstract Peptidoglycan synthesis and turnover in relation to cell growth and division has been studied by using a new labeling method. This method involves the incorporation of fluorescently labeled peptidoglycan precursors into the cell wall by means of the cell‐wall recycling pathway. We show that Escherichia coli is able to import exogenous added murein tripeptide labeled with N ‐7‐nitro‐2,1,3‐benzoxadiazol‐4‐yl (AeK–NBD) into the cytoplasm where it enters the peptidoglycan biosynthesis route, resulting in fluorescent labels specifically located in the cell wall. When wild‐type cells were grown in the presence of the fluorescent peptide, peptidoglycan was uniformly labeled in cells undergoing elongation. Cells in the process of division displayed a lack of labeled peptidoglycan at mid‐cell. Analysis of labeling patterns in cell division mutants showed that the occurrence of unlabeled peptidoglycan is dependent on the presence of FtsZ, but independent of FtsQ and FtsI. Accumulation of fluorescence at the division sites of a triple amidase mutant (Δ amiABC ) revealed that AeK–NBD is incorporated into septal peptidoglycan. AmiC was shown to be involved in the rapid removal of labeled peptidoglycan side chains at division sites in wild‐type cells. Because septal localization of AmiC is dependent on FtsQ and FtsI, this points to the presence of another peptidoglycan hydrolase activity directly dependent on FtsZ.
Summary The localization of penicillin‐binding protein 2 (PBP2) in Escherichia coli has been studied using a functional green fluorescent protein (GFP)–PBP2 fusion protein. PBP2 localized in the bacterial envelope in a spot‐like pattern and also at mid‐cell during cell division. PBP2 disappeared from mid‐cell just before separation of the two daughter cells. It localized with a preference for the cylindrical part of the bacterium in comparison with the old cell poles, which are known to be inert with respect to peptidoglycan synthesis. In contrast to subunits of the divisome, PBP2 failed to localize at mid‐cell when PBP3 was inhibited by the specific antibiotic aztreonam. Therefore, despite its dependency on active PBP3 for localization at mid‐cell, it seems not to be an integral part of the divisome. Cells grown for approximately half a mass doubling time in the presence of the PBP2 inhibitor mecillinam synthesized nascent cell poles with an increased diameter, indicating that PBP2 is required for the maintenance of the correct diameter of the new cell pole.
In Escherichia coli, cell division is mediated by the concerted action of about 12 proteins that assemble at the division site to presumably form a complex called the divisome. Among these essential division proteins, the multimodular class B penicillin-binding protein 3 (PBP3), which is specifically involved in septal peptidoglycan synthesis, consists of a short intracellular M1-R23 peptide fused to a F24-L39 membrane anchor that is linked via a G40-S70 peptide to an R71-I236 noncatalytic module itself linked to a D237-V577 catalytic penicillin-binding module. On the basis of localization analyses of PBP3 mutants fused to green fluorescent protein by fluorescence microscopy, it appears that the first 56 amino acid residues of PBP3 containing the membrane anchor and the G40-E56 peptide contain the structural determinants required to target the protein to the cell division site and that none of the putative protein interaction sites present in the noncatalytic module are essential for the positioning of the protein to the division site. Based on the effects of increasing production of FtsQ or FtsW on the division of cells expressing PBP3 mutants, it is suggested that these proteins could interact. We postulate that FtsQ could play a role in regulating the assembly of these division proteins at the division site and the activity of the peptidoglycan assembly machineries within the divisome.
