The replicated ftsQAZ and minB chromosomal regions of Escherichia coli segregate on average in line with nucleoid movement.

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 oriCs 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.

Cellular localization of oriC during the cell cycle of Escherichia coli as analyzed by fluorescent in situ hybridization.

The origin of replication of Escherichia coli, oriC, has been labeled by fluorescent in situ hybridization (FISH). The E. coli K12 strain was grown under steady state conditions with a doubling time of 79 min at 28 degrees C. Under these growth conditions DNA replication starts in the previous cell cycle at -33 min. At birth cells possess two origins which are visible as two separated foci in fully labeled cells. The number of foci increased with cell length. The distance of foci from the nearest cell pole has been measured in various length classes. The data suggest: i) that the two most outwardly located foci keep a constant distance to the cell pole and they therefore move apart gradually in line with cell elongation; and ii) that at the initiation of DNA replication the labeled origins occur near the center of prospective daughter cells.

Timing of FtsZ assembly in Escherichia coli.

The timing of the appearance of the FtsZ ring at the future site of division in Escherichia coli was determined by in situ immunofluorescence microscopy for two strains grown under steady-state conditions. The strains, B/rA and K-12 MC4100, differ largely in the duration of the D period, the time between termination of DNA replication and cell division. In both strains and under various growth conditions, the assembly of the FtsZ ring was initiated approximately simultaneously with the start of the D period. This is well before nucleoid separation or initiation of constriction as determined by fluorescence and phase-contrast microscopy. The durations of the Z-ring period, the D period, and the period with a visible constriction seem to be correlated under all investigated growth conditions in these strains. These results suggest that (near) termination of DNA replication could provide a signal that initiates the process of cell division.

Localization of cell division protein FtsQ by immunofluorescence microscopy in dividing and nondividing cells of Escherichia coli.

The localization of cell division protein FtsQ in Escherichia coli wild-type cells was studied by immunofluorescence microscopy with specific monoclonal antibodies. FtsQ could be localized to the division site in constricting cells. FtsQ could also localize to the division site in ftsQ1(Ts) cells grown at the permissive temperature. A hybrid protein in which the cytoplasmic domain and the transmembrane domain were derived from the gamma form of penicillin-binding protein 1B and the periplasmic domain was derived from FtsQ was also able to localize to the division site. This result indicates that the periplasmic domain of FtsQ determines the localization of FtsQ, as has also been concluded by others for the periplasmic domain of FtsN. Noncentral FtsQ foci were found in the area of the cell where the nucleoid resides and were therefore assumed to represent sites where the FtsQ protein is synthesized and simultaneously inserted into the cytoplasmic membrane.

Hybrid proteins of the transglycosylase and the transpeptidase domains of PBP1B and PBP3 of Escherichia coli.

The construction of hybrid proteins of PBP1B and PBP3 has been described. One hybrid protein (PBP1B/3) contained the transglycosylase domain of PBP1B and the transpeptidase domain of PBP3. In the other hybrid protein, the putative transglycosylase domain of PBP3 was coupled to the transpeptidase domain of PBP1B (PBP3/1B). The hybrid proteins were localized in the cell envelope in a similar way as the wild-type PBP1B. In vitro isolates of the strains containing the hybrid proteins had a transglycosylase activity intermediate between that of wild-type PBP1B-producing strain and that of a PBP1B overproducer. Analysis with specific antibiotics against PBP1A/1B and PBP3 and mutant analysis in strains containing PBP3/1B revealed no detectable effects in vivo compared with wild-type strains. The same was shown for PBP1B/3 when the experiments were performed in a recA background. The data indicate that the hybrid proteins cannot replace native penicillin-binding proteins. This finding suggests that functional high-molecular-weight penicillin-binding protein specificity is at least in part determined by the unique combination of the two functional domains.

Differences between inner membrane and peptidoglycan-associated PBP1B dimers of Escherichia coli.

Earlier studies revealed that PBP1B of Escherichia coli occurred as a monomeric as well as a dimeric form (C.A.L. Zijderveld, M.E.G. Aarsman, T. den Blaauwen, and N. Nanninga, J. Bacteriol. 173:5740-5746, 1991). In this study, the dimer of PBP1B was further analyzed. It appeared that the dimeric form could be divided into two classes. One class, which cofractionated with the cell wall fraction, could be artificially cross-linked to peptidoglycan, indicating a close association with the latter. This class of PBP1B dimers was sensitive to beta-mercaptoethanol. The second class, like the monomeric form of PBP1B, could be isolated with the inner membrane fraction. This dimeric form dissociated in the presence of zinc in combination with beta-mercaptoethanol.

Penicillin-binding protein 1B of Escherichia coli exists in dimeric forms.

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.

Influence of autonomic agonists on the in vitro incorporation of [3H]leucine and N-acetyl[14C]mannosamine into submandibular action of the mouse.

The influence of isoproterenol and pilocarpine on the in vitro incorporation of [3H]leucine and N-acetyl[14C]mannosamine into the proteins of the submandibular glands of the mouse has been investigated during a 10 h period. The total uptake of both labelled precursors into the glands was hardly affected by isoproterenol and pilocarpine during the first 2 h of incubation, thereafter both agonists decreased the uptake slightly. The incorporation of [3H]leucine into secreted proteins was largely similar for the control, isoproterenol and pilocarpine during an incubation of 10 h. [14C]ManNAc incorporation showed a lag period of about 2 h and could be observed in the secreted proteins after 2 h. Particularly after 6 h a strong increase was observed for the control and isoproterenol, whereas pilocarpine showed a much lower increase. The secreted protein components were separated by electrophoresis to study the incorporation of the labelled precursors in separate secretory proteins such as submandibular mucin. Apparently, both agonists increased the incorporation of [14C]ManNAc relative to [3H]leucine into submandibular mucin of the mouse. During a period of 10 h the [14C]ManNAc incorporation into the mucin was enhanced 2-3-fold by isoproterenol and 3-4-fold by pilocarpine. A non-radioactive experiment in vitro showed that the molar ratio of the sugar residues did not change. However, the total amount of sugars relative to the amino acids increased by 50%, pointing to an increase in the degree of glycosylation. This suggests that both adrenergic and cholinergic agonists regulate the total number of carbohydrate chains attached to one and the same polypeptide core of the submandibular mucin of the mouse.

On the origin of anthocyanin methyltransferase isozymes of Petunia hybrida and their role in regulation of anthocyanin methylation.

Four genes controlling anthocyanin methylation in flowers of Petunia hybrida have been described. Three of them, Mt2, Mf1 and Mf2, caused a dosage effect on anthocyanin methyltransferase activity and degree of methylation of anthocyanins. Antiserum raised against partially purified Mf2-enzyme precipitated three of the four anthocyanin methyltransferases. In two subspecies of one of the ancestral species of P. hybrida: Petunia integrifolia, different anthocyanin methyltransferases were found as determined by immunoprecipitation. The methyltransferase isozymes showed no differences in subcellular or tissue location, and had no physiologically important difference in time course of activity during bud development. The methylation-system in Petunia is discussed with regard to anthocyanin methylation in other plant species.

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 Mg(2+), 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.

Properties and genetic control of anthocyanin 5-O-glucosyltransferase in flowers of Petunia hybrida.

An anthocyanin 5-O-glucosyltransferase from flowers of Petunia hybrida was purified about 30-fold. Using uridine 5'-diphosphoglucose as glucose donor (Km 0.22 mM), the enzyme glucosylated the 3-(p-coumaroyl)-rutinoside derivatives of delphinidin and petunidin (Km 3 µM), isolated from pollen of Petunia. Delphinidin 3-rutinoside, cyanidin 3-rutinoside and delphinidin 3-glucoside did not serve as substrates. The glucosylation of petunidin 3-(p-coumaroyl)-rutinoside showed a pH-activity optimum at pH 8.3 and was neither stimulated by Mg(2+) or Ca(2+), nor inhibited by ethylenediaminetetraacetic acid. After separating the 5-O-glucosyltransferase from the anthocyanidin 3-O-glucosyltransferase by means of chromatofocusing, it was shown that both enzymes exhibit a high degree of positional specificity. The 5-O-glucosyltransferase activity was correlated with the gene An1, but not with the gene Gf.

Genetic control of anthocyanin-O-methyltransferase activity in flowers of Petunia hybrida.

The relation between four methylation genes (Mt1, Mt2, Mf1 and Mf2) in flowers of Petunia hybrida and anthocyanin-methyltransferase activity was investigated in vitro. All genes controlled methyltransferase activity. This activity was measured with cyanidinnd petunidin-derivatives as substrates. A cross provided evidence that the Mf-genes regulate methyltransferases which are distinct from those controlled by the Mtgenes. Different effects of the two Mf-genes in vivo are shown. The results suggest that the four methylationgenes control four different methyltransferases.

Influence of isoproterenol on the incorporation in vitro of [3H]-leucine and n-acetyl-[14C]-mannosamine into proteins and glycoproteins of the parotid glands of the mouse.

The in-vitro incorporation of [3H]-Leu in murine parotid glands started rapidly and continued during the whole incubation period of 10 h and was not stimulated significantly by isoproterenol, except during the first half hour. Secretion of [3H]-Leu incorporated was already observed after 30 min and continued up to 10 h. Isoproterenol stimulated the secretion of 3H-labelled protein during the whole period, but maximally after 4 h. Incorporation of [14C]-ManNAc proceeded at a much slower rate than that of [3H]-Leu. It was at least 1 h before incorporated [14C]-ManNAc appeared in the incubation medium. The secretion of [14C]-ManNAc incorporated was increased by isoproterenol between 2 and 6 h. A major part of [3H]-Leu was incorporated in amylase. The electrophoretically slowest moving isoenzyme of amylase incorporated substantial amounts of [14C]-ManNAc, in agreement with the presence of sialic acid in the isoenzyme. The onset of the [14C]-ManNAc uptake in amylase was slow compared to [3H]-Leu uptake. The [14C]-label could be detected in the secreted amylase after at least 4 h of incubation. It is suggested that isoproterenol decreased the incorporation of [14C]-ManNAc relative to that of [3H]-Leu into the secreted amylase.

Electrophoretic isolation and partial characterization of a major secretory glycoprotein from the submandibular glands of the mouse.

After either cholinergic or adrenergic stimulation of the submandibular glands of the mouse, a major protein of the incubation medium could be isolated by electrophoresis, designated the AM2 protein. About 5 per cent of the secreted proteins and 2.4 per cent of the secreted protein-bound sialic acid was recovered as the purified AM2 protein. The AM2 protein appeared to be electrophoretically pure in 7.5% polyacrylamide gel both at pH 8.9 and at pH 4.3. In sodium dodecyl sulfate-electrophoresis the molecular weight was estimated to be about 80 000 for the major component and about 40 000 for the minor component. By isoelectric focusing the isoelectric point has been determined to be 4.7. The amino acid analysis indicated Glx, Asx, Leu and Ala as the major amino acids, comprising 15.0, 10.6, 9.2 and 9.1 per cent of the amino acid residues, respectively. The ratio of the acidic amino acids and their amides (Glx plus Asx) to the basic amino acids (Lys plus Arg) was 2.2. The sugar analysis showed that the AM2 glycoprotein consists of 17.3 per cent of carbohydrate, with as major carbohydrate component glucosamine. The molar ratio of the sugars was Man : Gal : Glc : GlcNH2 : sialic acid = 2.3 : 1.0 : 4.7 : 9.8 : 2.9. Galactosamine could be detected as a trace component and fucose was not detectable.