Inhibition of cell wall synthesis and acylation of the penicillin binding proteins during prolonged exposure of growing Streptococcus pneumoniae to benzylpenicillin

Growing cultures of an autolysis-defective pneumococcal mutant were exposed to [3H]benzylpenicillin at various multiples of the minimal inhibitory concentration and incubated until the growth of the cultures was halted. During the process of growth inhibition, we determined the rates and degree of acylation of the five penicillin-binding proteins (PBPs) and the rates of peptidoglycan incorporation, protein synthesis, and turbidity increase. The time required for the onset of the inhibitory effects of benzylpenicillin was inversely related to the concentration of the antibiotic, and inhibition of peptidoglycan incorporation always preceded inhibition of protein synthesis and growth. When cultures first started to show the onset of growth inhibition, the same characteristic fraction of each PBP was in the acylated form in all cases, irrespective of the antibiotic concentration. Apparently, saturation of one or more PBPs with the antibiotic beyond these threshold levels is needed to bring about interference with normal peptidoglycan production and cellular growth. Although it was not possible to correlate the inhibition of cell wall synthesis or cell growth with the degree of acylation (percentage saturation) of any single PBP, there was a correlation between the amount of peptidoglycan synthesized and the actual amount of PBP 2b that was not acylated. In cultures exposed to benzylpenicillin concentrations greater than eight times the minimal inhibitory concentration, the rates of peptidoglycan incorporation underwent a rapid decline when bacterial growth stopped. However, in cultures exposed to lower concentrations of benzylpenicillin (one to six times the minimal inhibitory concentration) peptidoglycan synthesis continued at constant rate for prolonged periods, after the turbidity had ceased to increase. We conclude that inhibition of bacterial growth does not require a complete inhibition or even a major decline in the rate of peptidoglycan incorporation. Rather, inhibition of growth must be caused by an as yet undefined process that stops cell division when the rate of incorporation of peptidoglycan (or synthesis of protein) falls below a critical value.     

Human impact on vegetation at the Alpine tree-line ecotone during the last millennium: lessons from high temporal and palynological resolution

Three mires and a small lake in the Swiss and Austrian Alps were studied palynologically at high resolution, covering the last 1,000, 400, 50 and 1,200 years, respectively. Methodological lessons include: (1) Sub-decadal resolution in upper, little-decomposed peat layers reveals recurrent marked fluctuations in both percentages and influx of regional tree-pollen types, reflecting variations in pollen production rather than in plant-population sizes. (2) Intermittent, single-spectrum pollen maxima in samples of sub-decadal resolution indicate pollen transport in clumps. This type of pollen transport may remain unrecognized in sections with lower sampling resolution, which may then lead to inappropriate interpretation in terms of plant-population sizes. (3) The detection of short-lived phases of human impact in decomposed peat requires sampling intervals as close as 0.2 cm. (4) PAR (pollen influx) may reflect vegetation dynamics more faithfully than percentages. Reliable PAR, however, is difficult to achieve in Alpine mires due to past human impact on peat growth, even when complex depth–age modelling techniques are used. Critical comparison of PAR with percentages is therefore essential. (5) Careful consideration of spatial scales in pollen signals (local–regional and subdivisions) is essential for a realistic palaeo-ecological interpretation. Results in terms of past human impact on vegetation are summarized as follows: (1) Trends in pollen types reflecting regional human action are in general agreement with earlier findings for the western Swiss Alps, allowing for regional differences. (2) All mires in the Alps investigated here and in an earlier study experienced human impact during the last millennium. The studied small lake, lying in sub-alpine pasture, records forest dynamics at a lower elevation since a.d. 800.     

Differential effects of metal-binding agents on the D-glucose-6-phosphate phosphohydrolase activity of the rat liver.

The metal-binding agents (citrate, oxalate, bicarbonate, EDTA) exert dual effects on D-glucose-6-phosphate phosphohydrolase activity in the homogenate as well as in the subcellular fractions. The important differences of the effects are associated with the concentration of the chelator and with time of its addition. The small (appropriate) concentrations of the metal-binding agents stimulate and stabilize the enzyme activity. However, chelators used in higher concentrations exert the inhibitory influence on the activity of glucose-6-phosphatase. Stimulation of the reaction was observed only if the chelator was added before the enzyme-substrate complex formation has been started. The formation of the ternary complex: the enzyme(metal)-chelator substrate exerting a protective influence on the active centre has been suggested. The hypothesis of a similar action of the metal-binding agents and Pi on the glucose-6-phosphatase as a metaloproteid has been proposed.     

Teichoic acid-containing muropeptides from Streptococcus pneumoniae as substrates for the pneumococcal autolysin.

Pneumococcal cell walls in which the normal phosphorylcholine component of the wall teichoic acids is replaced with phosphorylethanolamine cannot absorb the homologous autolytic enzyme and are completely resistant to autolytic degradation (S. Giudicelli and A. Tomasz, J. Bacteriol. 158:1188-1190, 1984). We have now isolated and characterized soluble teichoic acid-containing muropeptides from such cell walls and tested them as substrates for the pneumococcal autolytic enzyme. Both choline- and ethanolamine-containing muropeptides were hydrolyzed to the same extent by the enzyme. Furthermore, free choline concentrations that totally inhibited the digestion of pneumococcal cell walls in vivo and in vitro were without effect when the soluble substrates were used.     

UDP-galactose 4-epimerase. Phosphorus-31 nuclear magnetic resonance analysis of NAD+ and NADH bound at the active site

The phosphorus atoms of NAD+ bound within the active site of UDP-galactose 4-epimerase from Escherichia coli exhibit two NMR signals, one at delta = -9.60 +/- 0.05 ppm and one at delta = -12.15 +/- 0.01 ppm (mean +/- standard deviation of four experiments) relative to 85% H3PO4 as an external standard. Titration of epimerase.NAD+ with UMP causes a UMP-dependent alteration in the chemical shifts of the resulting exchange-averaged spectra, which extrapolate to delta = -10.51 ppm and delta = -11.06 ppm, respectively, for the fully liganded enzyme, with an interconversion rate between epimerase.NAD+ and epimerase.NAD+.UMP of at least 490 s-1. Conversely, the binding of 8-anilinonaphthalene-1-sulfonate, which is competitive with UMP, causes a significant sharpening of the epimerase.NAD+ resonances but very little alteration in their chemical shifts, to delta = -9.38 ppm and delta = -12.16 ppm, respectively. UMP-dependent reductive inactivation by glucose results in the convergence of the two resonances into a single signal of delta = -10.57 ppm, with an off-rate constant for UMP dissociation from the epimerase.NADH.UMP complex estimated at 8 s-1. Reductive inactivation by borohydride under anaerobic conditions yields a single, broad resonance centered at about delta = -10.2 ppm. The data are consistent with, and may reflect, the activation of NAD+ via a protein conformational change, which is known from chemical studies to be driven by uridine nucleotide binding. Incubation of epimerase.NAD+ with UMP in the absence of additional reducing agents causes a very slow reductive inactivation of the enzyme with an apparent pseudo-first-order rate constant of 0.013 +/- 0.001 h-1, which appears to be associated with liberation of inorganic phosphate from UMP.     

Buoyancy regulation in light-limited continuous cultures of Microcystis aeruginosa

Microcystis aeruginosa was grown in light-limited continuouscultures at different growth rates on light-dark cycles at variousphotopenods. Due to the strength of the gas vesicles the organismwas not able to collapse its gas vesicles by turgor pressure.Below the maximal growth rate, the organism was buoyant dueto its high gas vesicle content. The results suggested thatthe rate of gas vesicle synthesis was not regulated. Upon atransition to high irradiance it took several hours before thecells lost their buoyancy due to polyglucan accumulation. Theresults are interpreted in an ecological context and it is suggestedthat Microcystis is an epilimnetic species due to its buoyancyregulation.     

Interaction of the antitumor antibiotic mitomycin C with Z-DNA

Mitomycin C (MC), an antitumor antibiotic, alkylated Z-DNAs such as poly(dG-dC)/Co(NH3)3+(6), poly(dG-m5dC)/Mg2+ and brominated poly(dG-dC) upon reductive activation. Computer-generated energy-minimized molecular models indicated that monofunctional alkylation of Z-DNA at the N2-position of guanine by MC did not distort Z-DNA geometry, but bifunctional alkylation, leading to interstrand crosslinks between two N2-positions of guanine was sterically unfavorable. The above three Z-DNA's were exposed both to monofunctionally and bifunctionally activated MC in separate experiments and the resulting covalent MC-polynucleotide complexes were examined for conformation and for covalent MC-adducts, by circular dichroism (CD) spectroscopy and HPLC analysis of nuclease digests, respectively. Monofunctionally activated MC alkylated all three polynucleotides in their Z-forms, resulting in the same monofunctional N2-guanine adduct as that known to be formed with B-DNA. Upon bifunctional activation of MC, poly(dG-dC/Co(NH3)3+(6) reverted to the B-form and bifunctional (cross-link) adducts were detected, identical again with those formed with B-DNA. Poly(dG-m5dC), however, remained in the Z-form after the alkylation and only a monofunctional adduct could be detected. It was concluded that Z-DNA is subject to monofunctional alkylation by MC but cannot be cross-linked. The latter process occurs only when the Z-DNA is labile enough [as is in the case of poly(dG-dC)] to have some B-form in equilibrium at the site of the first formed monolinked adduct; the cross-linking then occurs at such local B-sites, pulling the overall B in equilibrium Z equilibrium irreversibly to the left. These results are in accord with the predictions from the above modeling. The irreversible "lock" by the MC cross-link on B-DNA may be exploited for probing Z-DNA intermediacy in various DNA functions.     

Autolysis-resistant peptidoglycan of anomalous composition in amino-acid-starved Escherichia coli.

Nongrowing Escherichia coli deprived of an essential amino acid continued to produce peptidoglycan at a rate approximately 30% of that of growing cells. The composition of this peptidoglycan was very different from that of growing cells and resembled that of peptidoglycan left undegraded during partial autolysis of the bacteria. Synthesis of this peptidoglycan of anomalous composition began at once upon the removal of the amino acid from the medium. Fifteen minutes of amino acid deprivation was sufficient to virtually completely prevent penicillin-induced autolytic wall degradation in vivo. During this time, although the specific activities of soluble and membrane-bound hydrolytic transglycosylases and endopeptidases remained high, the peptidoglycan produced showed decreased sensitivity to degradation in vitro. After more extensive (2-h) starvation, triggering of autolysis by chaotropic agents was also blocked. Autolysis in growing cells may be selective for peptidoglycan representing the cylindrical portion of the sacculus. It is suggested that at least part of the mechanism of the well-known lysis resistance of nongrowing E. coli is related to the deposition of structurally anomalous and relatively autolysin-resistant peptidoglycan at some strategically located sites on the bacterial surface.