Mirjam E. G. Aarsman

Maturation of the Escherichia coli divisome occurs in two steps

Cell division proteins FtsZ (FtsA, ZipA, ZapA), FtsE/X, FtsK, FtsQ, FtsL/B, FtsW, PBP3, FtsN and AmiC localize at mid cell in Escherichia coli in an interdependent order as listed. To investigate whether this reflects a time dependent maturation of the divisome, the average cell age at which FtsZ, FtsQ, FtsW, PBP3 and FtsN arrive at their destination was determined by immuno‐ and GFP‐fluorescence microscopy of steady state grown cells at a variety of growth rates. Consistently, a time delay of 14–21 min, depending on the growth rate, between Z‐ring formation and the mid cell recruitment of proteins down stream of FtsK was found. We suggest a two‐step model for bacterial division in which the Z‐ring is involved in the switch from cylindrical to polar peptidoglycan synthesis, whereas the much later localizing cell division proteins are responsible for the modification of the envelope shape into that of two new poles.

Interaction between two murein (peptidoglycan) synthases, PBP3 and PBP1B, in Escherichia coli.

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.

Penicillin-binding protein PBP2 of Escherichia coli localizes preferentially in the lateral wall and at mid-cell in comparison with the old cell pole.

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.

R174 of Escherichia coli FtsZ is involved in membrane interaction and protofilament bundling, and is essential for cell division

We investigated the interaction between FtsZ and the cytoplasmic membrane using inside‐out vesicles. Comparison of the trypsin accessibility of purified FtsZ and cytoplasmic membrane‐bound FtsZ revealed that the protruding loop between helix 6 and helix 7 is protected from trypsin digestion in the latter. This hydrophobic loop contains an arginine residue at position 174. To investigate the role of R174, this residue was replaced by an aspartic acid, and FtsZ‐R174D was fused to green fluorescent protein (GFP). FtsZ‐R174D‐GFP could localize in an FtsZ and in an FtsZ84(Ts) background at both the permissive and the non‐permissive temperature, and it had a reduced affinity for the cytoplasmic membrane compared with wild‐type FtsZ. FtsZ‐R174D could also localize in an FtsZ depletion strain. However, in contrast to wild‐type FtsZ, FtsZ‐R174D was not able to complement the ftsZ84 mutation or the depletion strain and induced filamentation. In vitro polymerization experiments showed that FtsZ‐R174D is able to polymerize, but that these polymers cannot form bundles in the presence of 10 mM CaCl2. This is the first description of an FtsZ mutant that has reduced affinity for the cytoplasmic membrane and does not support cell division, but is still able to localize. The mutant is able to form protofilaments in vitro but fails to bundle. It suggests that neither membrane interaction nor bundling is a requirement for initiation of cell division.

Structural Determinants Required To Target Penicillin-Binding Protein 3 to the Septum of Escherichia coli

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.

Functional Analysis of the Cell Division Protein FtsW of Escherichia coli

Site-directed mutagenesis experiments combined with fluorescence microscopy shed light on the role of Escherichia coli FtsW, a membrane protein belonging to the SEDS family that is involved in peptidoglycan assembly during cell elongation, division, and sporulation. This essential cell division protein has 10 transmembrane segments (TMSs). It is a late recruit to the division site and is required for subsequent recruitment of penicillin-binding protein 3 (PBP3) catalyzing peptide cross-linking. The results allow identification of several domains of the protein with distinct functions. The localization of PBP3 to the septum was found to be dependent on the periplasmic loop located between TMSs 9 and 10. The E240-A249 amphiphilic peptide in the periplasmic loop between TMSs 7 and 8 appears to be a key element in the functioning of FtsW in the septal peptidoglycan assembly machineries. The intracellular loop (containing the R166-F178 amphiphilic peptide) between TMSs 4 and 5 and Gly 311 in TMS 8 are important components of the amino acid sequence-folding information.

Properties and genetic control of four methyltransferases involved in methylation of anthocyanins in flowers of Petunia hybrida

Four S-adenosyl-l-methionine:anthocyanin-3′,5′-O-methyltransferases in flowers of Petunia hybrida were separated using the chromatofocusing technique. Each methyltransferase is controlled by one of the methylation genes Mt1, Mt2, Mf1 or Mf2. Molecular weight, pH-activity optimum, isoelectric point, several kinetic properties and the behaviour in the presence of Mg2+, ethylenediaminetetraacetic acid and S-adenosyl-l-homocysteine of each of the four enzymes were determined. The methylation in vitro of delphinidin 3-(p-coumaroyl)-rutinosido-5-glucoside reflected the accumulation patterns of methylated anthocyanins in vivo and established the regulatory role of methyltransferases in vivo.

Methylation of anthocyanins by cell-free extracts of flower buds of Petunia hybrida

Abstract An O -methyltransferase activity which catalyses the methylation of anthocyanins was extracted from flowerbuds of Petunia hybrida . The methyltransferase uses S -adenosyl- l -methionine as methyl donor. Only anthocyanidin 3( p -coumaroyl)rutinosido-5-glucoside was methylated. No methylating activity towards anthocyanidins, anthocyanidin 3-glucosides, anthocyanidin 3-rutinosides, caffeic acid or p -coumaric acid could be detected.

A Novel in vivo Cell‐Wall Labeling Approach Sheds New Light on Peptidoglycan Synthesis in Escherichia coli

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 (ΔamiABC) 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.

Pre-replication assembly of E. coli replisome components

The localization of SeqA, thymidylate synthase, DnaB (helicase) and the DNA polymerase components α and τ, has been studied by immunofluorescence microscopy. The origin has been labelled through GFP‐LacI bound near oriC. SeqA was located in the cell centre for one replication factory (RF) and at 1/4 and 3/4 positions in pre‐divisional cells harbouring two RFs. The transition of central to 1/4 and 3/4 positions of SeqA appeared abrupt. Labelled thymidylate synthetase was found all over the cell, thus not supporting the notion of a dNTP‐synthesizing complex exclusively localized near the RF. More DnaB, α and τ foci were found than expected. We have hypothesized that extra foci arise at pre‐replication assembly sites, where the number of sites equals the number of origins, i.e. the number of future RFs. A reasonable agreement was found between predicted and found foci. In the case of multifork replication the number of foci appeared consistent with the assumption that three RFs are grouped into a higher‐order structure. The RF is probably separate from the foci containing SeqA and the hemi‐methylated SeqA binding sites because these foci did not coincide significantly with DnaB as marker of the RF. Co‐labelling of DnaB and oriC revealed limited colocalization, indicating that DnaB did not yet become associated with oriC at a pre‐replication assembly site. DnaB and τ co‐labelled in the cell centre, though not at presumed pre‐replication assembly sites. By contrast, α and τ co‐labelled consistently suggesting that they are already associated before replication starts.