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N Nanninga

Publications and source records attributed to N Nanninga.

At least 19 recordsLinked to original sources
PubMed1992

Cross-linkage and cross-linking of peptidoglycan in Escherichia coli: definition, determination, and implications.

The glycan chains in peptidoglycan or murein are cross-linked by transpeptidation of the peptide side chains. To assess the fraction of side chains involved in cross-bridges, distinction has been made between cross-linkage and cross-linking. The first expression refers to the situation in unlabeled (or fully labeled) peptidoglycan, and the second refers to pulse-labeled peptidoglycan. It is argued that for the determination of the cross-linking value, the mode of insertion as denoted by the so-called acceptor/donor radioactivity ratio should be taken into account.

Cross-Linking Reagents↗
PubMed1991

Cell division and peptidoglycan assembly in Escherichia coli.

Research on bacterial cell division has recently gained renewed impetus because of new information about peptidoglycan assembly and about specific cell-division genes and their products. This paper concerns aspects of cell division that specifically concern the peptidoglycan. It is shown that upon division, peptidoglycan assembly switches from lateral wall location to the cell centre, that assembly takes place at the leading edge of the invaginating constriction, that the mode of glycan strand insertion changes from a single-stranded mode to a multi-stranded mode, and that the initiation of division (in contrast to its continuation) requires penicillin-insensitive peptidoglycan synthesis (PIPS). A membrane component X (possibly FtsQ) is proposed to coordinate PIPS with the cell division-initiating protein FtsZ. It is suggested that a largely proteinaceous macromolecular complex (divisome) at the leading edge of constriction encompasses three compartments (cytoplasm, membrane and periplasm). The composition of this complex is proposed to vary depending on whether division is being initiated or completed.

Bacterial Proteins↗
PubMed1991

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.

Antibodies, Monoclonal↗
PubMed1991

Amount of peptidoglycan in cell walls of gram-negative bacteria.

The amount of diaminopimelic acid (Dap) in the cell wall of Escherichia coli was measured in two ways. A radiochemical method first described by us in 1985 (F. B. Wientjes, E. Pas, P. E. M. Taschner, and C. L. Woldringh, J. Bacteriol. 164:331-337, 1985) is based on the steady-state incorporation of [3H]Dap during several generations. Knowing the cell concentration and the specific activity of the [3H]Dap, one can calculate the number of Dap molecules per sacculus. The second method measures the Dap content chemically in sacculi isolated from a known number of cells. With both methods, a value of 3.5 x 10(6) Dap molecules per sacculus was obtained. Combined with electron microscopic measurements of the surface area of the cells, the data indicate an average surface area per disaccharide unit of ca. 2.5 nm2. This finding suggests that the peptidoglycan is basically a monolayered structure.

Autoradiography↗
PubMed1990

Interaction of monoclonal antibodies with the enzymatic domains of penicillin-binding protein 1b of Escherichia coli.

Monoclonal antibodies (MAbs) against four different antigenic determinants of penicillin-binding protein (PBP) 1b were used to study the transglycosylase and transpeptidase activities of PBP 1b. Enzyme kinetics in the presence of and without the MAbs were determined, and the synthesized murein was analyzed. Two MAbs against the transglycosylase domain of PBP 1b appeared to inhibit this reaction. One MAb inhibited only the transpeptidase reaction, and one inhibited both enzymatic activities of PBP 1b. The latter two MAbs bound to the transpeptidase domain of PBP 1b. The following major conclusions were deduced from the results. (i) Transpeptidation is the rate-limiting step of the reaction cascade, and it is dependent on the product of transglycosylation. (ii) PBP 1b has only one type of transpeptidase activity, i.e., a penta-tetra transpeptidase activity. (iii) PBP 1b is probably a globular protein which has two intimately associated enzymatic domains.

Acyltransferases↗
PubMed1990

Topology of penicillin-binding protein 1b of Escherichia coli and topography of four antigenic determinants studied by immunocolabeling electron microscopy.

A method has been developed to study the orientation of proteins in the cytoplasmic membrane of Escherichia coli. Vesicles from sonicated cells were incubated in droplets on electron microscope support grids in sequence with a monoclonal antibody (MAb) against a protein with an unknown orientation (PBP 1b) followed by a MAb against a periplasmic component (peptidoglycan). The different MAbs were made visible with 5- and 10-nm gold-conjugated secondary antibodies, respectively. PBP 1b appeared to colabel with peptidoglycan. The labeling of PBP 1b in membrane vesicles with MAbs against four different epitopes was further used to estimate the number of PBP 1b molecules per cell. Approximately 1,400 PBP 1b molecules per cell grown in broth were labeled. The spatial distribution of the epitopes of the MAbs was studied by immunocolabeling of pairs of MAbs and by competitive antibody-binding inhibition. It could be tentatively concluded that the four epitopes form a cluster of antigenic determinants which occupy less than half of the surface of PBP 1b.

Acyltransferases↗
PubMed1990

Mapping of conformational epitopes of monoclonal antibodies against Escherichia coli penicillin-binding protein 1B (PBP 1B) by means of hybrid protein analysis: implications for the tertiary structure of PBP 1B.

We have analyzed the location of the epitope areas of the four monoclonal antibody groups against penicillin-binding protein 1B (PBP 1B; T. den Blaauwen, F. B. Wientjes, A. H. J. Kolk, B. G. Spratt, and N. Nanninga, J. Bacteriol. 171:1393-1401). They could be specified by studying monoclonal antibody binding patterns to amino- and carboxy-terminal truncated PBP 1B molecules. Monoclonal antibodies against conformational epitopes, with the exception of one epitope area, did not recognize PBP 1B molecules that had not been translocated across the membrane. Apparently, translocation is required for PBP 1B to fully obtain its native conformation.

Antibodies, Monoclonal↗
PubMed1989

Three-dimensional imaging in fluorescence by confocal scanning microscopy.

The improved resolution and sectioning capability of a confocal microscope make it an ideal instrument for extracting three-dimensional information especially from extended biological specimens. The imaging properties, also with finite detection pinholes are considered and a number of biological applications demonstrated.

Animals↗
PubMed1989

Peptidoglycan synthesis during the cell cycle of Escherichia coli: composition and mode of insertion.

The composition and the mode of insertion of peptidoglycan synthesized during the cell cycle of Escherichia coli were determined. This was carried out on peptidoglycan that was periodically pulse-labeled in synchronously growing cultures. The chemical composition of the pulse-labeled (newly synthesized) peptidoglycan remained constant throughout the cell cycle, as judged from high-pressure liquid chromatography analysis of the muropeptide composition. The mode of insertion was deduced from the acceptor-donor radioactivity ratio in the bis-disaccharide tetratetra compound. The ratio was low in elongating cells and high in constricting cells. This indicates that during elongation, peptidoglycan was inserted as single strands, whereas during constriction, a multistranded (or sequential single-stranded) insertion occurred. Experiments with an ftsA division mutant suggested that the composition and mode of insertion of newly synthesized peptidoglycan remained the same throughout the constriction process. Our results imply that the changed mode of insertion rather than the chemical structure of the peptidoglycan might be responsible for the transition from cell elongation to polar cap formation.

Carbohydrate Sequence↗
PubMed1989

Preparation and characterization of monoclonal antibodies against native membrane-bound penicillin-binding protein 1B of Escherichia coli.

We prepared monoclonal antibodies against penicillin-binding protein 1B (PBP 1B) of Escherichia coli to study the membrane topology, spatial organization, and enzyme activities of this protein. The majority of the antibodies derived with PBP 1B as the immunogen reacted against the carboxy terminus. To obtain monoclonal antibodies recognizing other epitopes, we used PBP 1B lacking the immunodominant carboxy-terminal 65 amino acids as the immunogen. Eighteen monoclonal antibodies directed against membrane-bound PBP 1B were isolated and characterized. The epitopes recognized by those monoclonal antibodies were located with various truncated forms of PBP 1B. We could distinguish four different epitope areas located on different parts of the molecule. Interestingly, we could not isolate monoclonal antibodies against the amino terminus, although they were specifically selected for. This is attributed to its predicted extreme hydrophilicity and flexibility, which could make the amino terminus very sensitive to proteolytic degradation. All antibodies reacted against native PBP 1B in a dot-blot immunobinding assay. One monoclonal antibody also recognized PBP 1B in a completely sodium dodecyl sulfate-denatured form. This suggests that all the other monoclonal antibodies recognize conformational epitopes. These properties make the monoclonal antibodies suitable tools for further studies.

Antibodies, Monoclonal↗
PubMed1989

Rate and topography of peptidoglycan synthesis during cell division in Escherichia coli: concept of a leading edge.

The rate at which the peptidoglycan of Escherichia coli is synthesized during the division cycle was studied with two methods. One method involved synchronization of E. coli MC4100 lysA cultures by centrifugal elutriation and subsequent pulse-labeling of the synchronously growing cultures with [meso-3H]diaminopimelic acid ([3H]Dap). The second method was autoradiography of cells pulse-labeled with [3H]Dap. It was found that the peptidoglycan is synthesized at a more or less exponentially increasing rate during the division cycle with a slight acceleration in this rate as the cells start to constrict. Apparently, polar cap formation requires synthesis of extra surface components, presumably to accommodate for a change in the surface-to-volume ratio. Furthermore, it was found that the pool size of Dap was constant during the division cycle. Close analysis of the topography of [3H]Dap incorporation at the constriction site revealed that constriction proceeded by synthesis of peptidoglycan at the leading edge of the invaginating cell envelope. During constriction, no reallocation of incorporation occurred, i.e., the incorporation at the leading edge remained high throughout the process of constriction. Impairment of penicillin-binding protein 3 by mutation or by the specific beta-lactam antibiotic furazlocillin did not affect [3H]Dap incorporation during initiation of constriction. However, the incorporation at the constriction site was inhibited in later stages of the constriction process. It is concluded that during division at least two peptidoglycan-synthesizing systems are operating sequentially.

Autoradiography↗
PubMed1989

Three-dimensional chromosome arrangement of Crepis capillaris in mitotic prophase and anaphase as studied by confocal scanning laser microscopy.

To estimate the extent of ordering of chromosomes, confocal scanning laser microscopy was used to make three-dimensional images from optical sections. For Crepis capillaris, which has 2n = 6 easily recognizable chromosomes, a statistically significant sample of 75 Feulgen-stained root tip anaphases was analysed. A comparison of the observed chromosome ordering and the expected random distribution showed a significant surplus of one of the arrangements with a juxtaposition of the two chromosomes with a nucleolus organizer region. Two of the arrangements with these chromosomes in opposite positions were never observed in our material. Another analysis of 30 mithramycin A-stained prophases and 30 meta- and anaphases showed partly different patterns of non-random chromosome distribution in the two stages of mitosis. A preference for an association of the homologues was observed for all pairs of chromosomes in prophase cells, whereas in meta- and anaphase the association only persisted for the nucleolus organizer chromosomes. This indicates that there may be some relocation of the chromosome positions during the transition from prophase to metaphase. In meta- and anaphase one of the arrangements with juxtaposed NOR chromosomes was preferred, i.e. the ordering in which chromosomes 1 and 3 occupied alternate positions. Probably, the nucleolus is an important factor in producing a non-random distribution, but there could be other factors that influence chromosome ordering as well. A comparison of the anaphase chromosome ordering in C. capillaris plants from very different localities, indicated that the observed non-random distribution was independent of the origin of the material. Existing models of chromosome disposition are not sufficient to explain the observed non-random chromosome ordering in C. capillaris.

Cell Division↗
PubMed1988

Changes in activities of several enzymes involved in carbohydrate metabolism during the cell cycle of Saccharomyces cerevisiae.

Activity changes of a number of enzymes involved in carbohydrate metabolism were determined in cell extracts of fractionated exponential-phase populations of Saccharomyces cerevisiae grown under excess glucose. Cell-size fractionation was achieved by an improved centrifugal elutriation procedure. Evidence that the yeast populations had been fractionated according to age in the cell cycle was obtained by examining the various cell fractions for their volume distribution and their microscopic appearance and by flow cytometric analysis of the distribution patterns of cellular DNA and protein contents. Trehalase, hexokinase, pyruvate kinase, phosphofructokinase 1, and fructose-1,6-diphosphatase showed changes in specific activities throughout the cell cycle, whereas the specific activities of alcohol dehydrogenase and glucose-6-phosphate dehydrogenase remained constant. The basal trehalase activity increased substantially (about 20-fold) with bud emergence and decreased again in binucleated cells. However, when the enzyme was activated by pretreatment of the cell extracts with cyclic AMP-dependent protein kinase, no significant fluctuations in activity were seen. These observations strongly favor posttranslational modification through phosphorylation-dephosphorylation as the mechanism underlying the periodic changes in trehalase activity during the cell cycle. As observed for trehalase, the specific activities of hexokinase and phosphofructokinase 1 rose from the beginning of bud formation onward, finally leading to more than eightfold higher values at the end of the S phase. Subsequently, the enzyme activities dropped markedly at later stages of the cycle. Pyruvate kinase activity was relatively low during the G1 phase and the S phase, but increased dramatically (more than 50-fold) during G2. In contrast to the three glycolytic enzymes investigated, the highest specific activity of the gluconeogenic enzyme fructose-1, 6-diphosphatase 1 was found in fractions enriched in either unbudded cells with a single nucleus or binucleated cells. The observed changes in enzyme activities most likely underlie pronounced alterations in carbohydrate metabolism during the cell cycle.

Alcohol Dehydrogenase↗