- Volume 140, Issue 11, 1994
Volume 140, Issue 11, 1994
- Physiology And Growth
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The effect of nutrient limitation on glycerol uptake and metabolism in continuous cultures of Pseudomonas aeruginosa
More LessPseudomonas aeruginosaNM48, a non-mucoid derivative of an alginate-producing strain isolated from a cystic fibrosis patient, was grown in batch culture with glycerol, glucose or succinate as carbon source, and in continuous culture (D0.05 h-1) under glycerol or glucose limitation. Glycerol uptake, glycerol kinase and glycerol-3-phosphate dehydrogenase were induced by glycerol, but not by glucose or succinate. Linear uptake of [14C]glycerol by washed cells (K m≤ 2 μM) was inhibited by unlabelled glycerol and glyceraldehyde, but not by cyanide or the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and was accompanied by substantial intracellular accumulation of glycerol-3-phosphate and/or dihydroxyacetone phosphate but not glycerol. Prolonged growth under glycerol limitation led to substantial increases in the activities and/or concentrations of the enzymes catalysing glycerol uptake and metabolism, together with a 48000 M router-membrane protein which was also over-expressed following prolonged growth under glucose limitation. The N-terminal amino acid sequence (AEAFSPN-) and electrophoretic properties of this protein were the same as those of the previously characterized glucose porin (OprB) from P. aeruginosa, indicating that this porin is active with both glucose and glycerol. It is concluded that during growth under glycerol limitation, glycerol is transported into P. aeruginosaNM48 via OprB and a high-affinity, binding-protein-independent facilitated-diffusion system.
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Use of a series of chemostat cultures to isolate ‘improved’ variants of the Quorne myco-protein fungus, Fusarium graminearumA3/5
More LessVariants (designated A23-S and A24-S) of the Quorne myco-protein fungus, Fusarium graminearumA3/5 were isolated from a series of glucose-limited cultures grown at a dilution rate of 0.18 h-1for a combined total of 109 d. These variants had unchanged mycelial morphologies but, when grown in mixed culture with the parental strain (A3/5) in glucose-limited chemostat culture at 0.18 h-1, A23-S and A24-S had selection coefficients of 0.013 and 0.017 h-1, respectively, and supplanted A3/5. When a monoculture of A23-S was grown in a glucose-limited culture at a dilution rate of 0.18 h-1, the appearance of highly branched (so-called colonial) mutants was delayed compared with their appearance in chemostat cultures of the parental strain. Furthermore, when a monoculture of A24-S was grown in glucose-limited culture at 0.18 h-1, the appearance of colonial mutants was delayed even further. Thus, it is possible to isolate advantageous (relative to A3/5) variants of F. graminearumA3/5 which have unchanged mycelial morphologies, but in which the appearance of colonial mutants is delayed.
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Evolution of Fusarium graminearumA3/5 grown in a glucose-limited chemostat culture at a slow dilution rate
More LessThe evolution of Fusarium graminearum A3/5 grown in a glucose-limited chemostat at a dilution rate of 0.05 h-1(doubling time of 13.9 h) was followed for 957 h or 69 generations. Periodic selection of advantageous mutants was monitored in the culture by determining increases and decreases in the concentration of cycloheximide-resistant macroconidia in the population. Six peaks in the concentration of cycloheximide-resistant macroconidia were observed representing five adaptive changes in the population; on average, an adaptive change occurred once every 148±22 h (mean±SE). The selection coefficient of strains present at the start of each increase in the concentration of cycloheximide-resistant macroconidia (i.e. after the establishment of a new advantageous strain) was determined relative to A3/5 and was found to increase progressively with time. When grown at a dilution rate of 0.05 h-1, the strain (A28-S) isolated from the last adaptive peak had a selection coefficient of 0.023 h-1relative to A3/5, but A28-S lost its selective advantage when grown at a dilution rate of about 0.11 h-1and was at a selective disadvantage when grown at a dilution rate higher than 0.11 h-1. The K mvalue (12±5 μM) for uptake of glucose by A28-S was significantly lower than that for A3/5. The spontaneous mutation rate from cycloheximide sensitivity to cycloheximide resistance was estimated to be 1.8 (±0.2) × 10-6h-1or 2.5 × 10-5generation-1. The culture initially contained about 1 × 106macroconidia ml-1but this decreased with time until, at about 800 h, the culture contained only about 1 × 104macroconidia ml-1. No highly branched (colonial) mutants were observed in glucose-limited cultures at dilution rates of 0.05 h-1even though the evolution of the population was followed for a further 1345 h in a second chemostat, making a total evolutionary period of 2207 h or 159 generations.
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Involvement of mitochondria in the assimilatory metabolism of anaerobic Saccharomyces cerevisiaecultures
The possible physiological role of mitochondria in anaerobically grown Saccharomyces cerevisiaewas investigated via enzyme localization and inhibitor studies. Almost all of the activity of citrate synthase (EC 4.1.3.7) was recovered in the mitochondrial fraction after differential centrifugation of spheroplast lysates. The enzyme exhibited a high degree of latency which was demonstrated by sonication of the mitochondrial fractions. Since citrate synthase is an important enzyme in anabolic reactions, a consequence of this localization is the requirement for transport of metabolites across the mitochondrial membranes. Such transport is likely to require energy which, as a result of anaerobiosis, cannot be supplied by respiration. It was therefore investigated whether ATP translocation into the mitochondria by an ADP/ATP translocase might be involved in anaerobic mitochondrial energy metabolism. It was shown that addition of the ADP/ATP translocase inhibitor bongkrekic acid to anaerobic cultures indeed inhibited growth, although only partially. It is concluded that mitochondria of S. cerevisiaefulfil a vital role in anaerobic sugar metabolism.
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Motility and chemosensory behaviour of the sulphur bacterium Thiovulum majus
More LessThe swimming track of the sulphur/sulphide-oxidizing bacterium Thiovulum majusis a left-handed helix. The cells modulate swimming speed by changing the tangential speed and/or the pitch and radius of the helix. Whether attached (to a mucous thread) or swimming, the spherical cells rotate around their anterior-posterior axis in a counter-clockwise direction when observed from the posterior pole. Swimming speeds may exceed 600 μm s-1, which is 5-6 times faster than recorded for any other bacterium. Thiovulumcells congregate at oxygen tensions of about 4% atmospheric saturation (0.85 kPa). Cells which accidentally leave the optimum zone make a U-turn within 150-200 μm, thus returning to where they came from. This represents a type of phobic response in which the eventual swimming direction is correlated with the initial direction; it is not a true chemotactic response in the sense that the cells can orient themselves in O2-gradients. The 180°-bend of the swimming path is probably accomplished by changes in the rotational velocity component which take place when the cells swim into an adverse environment. The U-turn response allows the bacteria to maintain the characteristic 100-200 μm thick veils which separate the sulphidic and the oxygenated zone in or above sediments. Evidence for a chemosensory response to sulphide could not be found.
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Growth temperature regulates the induction of β-lactamase in Pseudomonas fluorescensthrough modulation of the outer membrane permeation of a β-lactam-inducing antibiotic
More LessThe psychrotrophic bacterium, Pseudomonas fluorescensstrain MFO, is more sensitive to the β-lactam mezlocillin at a low growth temperature (i.e. 8°C) than at a higher growth temperature (28°C). An early effect of this antibiotic at all temperatures is bacterial filamentation, but this occurs later at 8°C than at 28 °C, which suggests a lower permeability of the cell envelopes to mezlocillin at low growth temperature. β-Lactamase production is later induced by mezlocillin, but the level of this induction also depends on the growth temperature, the overall induction being much less efficient at 8 °C. It is hypothesized that the periplasmic concentration of the drug might be too low at 8 °C to allow efficient β-lactamase induction; this hypothesis was confirmed by the demonstration that β-lactamase production is drastically enhanced in cells cultivated at 8°C permeabilized for 10 min by Na-EDTA. In addition, induction kinetic curves display a marked dependence upon growth temperature. A rapid saturation was evident when mezlocillin concentrations were increased at 8°C; this was not seen at 28 T°C at up to 1000 μg mezlocillin ml-1. The results are discussed in terms of two different routes of drug permeation, depending on the growth temperature.
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Glycine betaine transport by Staphylococcus aureus: evidence for feedback regulation of the activity of the two transport systems
More LessThe regulation of glycine betaine accumulation by Staphylococcus aureuswas investigated. The accumulation of glycine betaine was regulated by the osmotic pressure of the medium and the low affinity transport system played the major role in this regulation. Mutants were isolated that lack the low affinity, osmotically activated glycine betaine/proline transport system. Such mutants accumulated glycine betaine via the high affinity system but the glycine betaine pool was smaller and responded poorly to osmotic pressure changes. The regulation of glycine betaine transport has revealed that at the steady state net influx is reduced and that this is achieved by inhibition of both the low affinity and the high affinity transport systems. Cells pre-loaded with glycine betaine exhibited a reduced V maxfor both systems: the low affinity system was reduced in activity fivefold and the high affinity system was reduced 10-fold and became virtually undetectable. Although glycine betaine transport at the steady state is reduced, retention of the compatible solute is an active process since addition of an uncoupler provokes rapid release of the accumulated material. These data suggest that feedback regulation of the activity of the uptake systems is a major mechanism for controlling the level of compatible solute accumulation.
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Evidence for feedback (trans) regulation of, and two systems for, glycine betaine transport by Staphylococcus aureus
More LessPrevious reports are in conflict as to the number of transport systems for glycine betaine in Staphylococcus aureus.Cells grown in complex medium exhibited a single transport system of moderate affinity. Cells grown in defined medium in the absence of glycine betaine showed a high affinity and a low affinity transport system. Cells grown in the presence of glycine betaine in the presence of osmotic stress in either complex or defined media accumulated large pools of internal glycine betaine. Smaller, but still significant, amounts of glycine betaine were accumulated by cells grown in its presence in either complex or defined media in the absence of osmotic stress. Cells grown in defined medium in the presence of glycine betaine in the presence or absence of osmotic stress showed lower rates of glycine betaine transport than cells grown in its absence. This suggests that glycine betaine transport is subject to feedback or transinhibition by internal glycine betaine. This can explain the difference in observed kinetics in cells grown in complex or defined media, the high affinity system being predominantly inhibited in cells grown in complex medium.
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Monitoring of peroxisome induction and degradation by flow cytometric analysis of Hansenula polymorphacells grown in methanol and glucose media: cell volume, refractive index and FITC retention
More LessCell refractive index has been used to monitor peroxisome behaviour in the yeast Hansenula polymorphaby means of flow cytometry. Peroxisomes are inducible organelles which may occupy a large fraction of the cell volume when yeast cells are growing in methanol media. These organelles harbour a catalase that decomposes the hydrogen peroxide produced in methanol oxidation by alcohol oxidase, a peroxisomal enzyme whose subunits are arranged to form a regular crystalloid. Peroxisomes undergo a degradation process mediated by vacuoles whenever they and their enzymes become metabolically redundant (e.g. during growth on glucose). Flow cytometric analyses of side scattered light (depending on cell volume, morphology and structure) and fluorescein isothiocyanate retention (due to the vacuole) were made on two wild-type strains of H. polymorphaduring exponential growth in glucose and methanol media and during nutritional shifts from one carbon source to the other. The same parameters were also analysed for a mutant strain only partially repressed by glucose. We show that both the parameters are substrate-dependent and appear to reflect peroxisome development in the cells. The data reported correlate well with the known cytological and biochemical data, showing the possibility of using flow cytometry, a fast and sensitive technique, to analyse the dynamics of peroxisome proliferation and degradation in response to environmental as well as genetic factors.
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Characterization of a brown Nostocspecies from Java that is resistant to high light intensity and UV
More LessA brown Nostocspecies was isolated from a soil sample collected in Java, Indonesia. It has three physiological types of light-absorbing compounds: the photosynthetic pigments (chlorophyll a, carotenoids, phycocyanin, phycoerythrin and allophycocyanin), a brown pigment(s) and three UV-absorbing compounds. The organism undergoes complementary chromatic adaptation by varying its phycobilin content. It is resistant to photobleaching at high intensities of white light, and also to UV-C radiation, under which conditions a green-coloured Nostocspecies (ATCC 27895) was killed. Synthesis of the brown pigment and UV-absorbing compounds was not observed at low oxygen levels. A brown pigment was released suddenly from cells during exponential growth, resulting in a deep brown-coloured medium. In addition, the absorption spectrum of the medium after pigment release had maxima in the UV region, at 256 nm, 314 nm and 400 nm. The appearance of the UV-absorbing compounds coincided with the brown pigment release and with the disappearance from the vegetative cells of peripheral granules. The Nostocspecies reduced acetylene, a measure of nitrogen fixation, in both the light and the dark periods when grown on a 12/12 h light/dark cycle. On a 4/20 h light/dark cycle fixation occurred predominantly in the light period. When grown on this restricted light regime, or at very low light intensity, the cells did not produce the brown pigment or the UV-absorbing compounds.
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- Genome Analysis
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Conservation of gene arrangement and an unusual organization of rRNA genes in the linear chromosomes of the Lyme disease spirochaetes Borrelia burgdorferi, B. gariniiand B. afzelii
More LessPhysical maps of the chromosomes of the Lyme disease spirochaetes Borrelia gariniiand Borrelia afzeliihave been elucidated for the enzymes Cspl, SgrAI, I-CeuI, SmaI, EagI, BssHII, MluI and Apal by two-dimensional pulsed-field gel electrophoresis techniques. The maps contain 42 sites for B. gariniiand 32 for B. afzelii.The mapping studies showed that the two chromosomes are linear DNA molecules of 953 and 948 kbp, respectively. A comparison of the physical maps of B. gariniiand B. afzeliiand the published map of the other Lyme disease spirochaete, Borrelia burgdorferi[Davidson, B. E., MacDougall, J. & Saint Girons, I. (1992) J Bacteriol174, 3766-3774] revealed that the three chromosomes have few endonuclease sites in common, apart from a cluster in rrl(encoding 23S rRNA) and rrs(encoding 16S rRNA). Cloned borrelial genes were used as specific hybridization probes to construct genetic maps, using the physical maps as a basis. The resulting maps contain 41 genetic loci for B. burgdorferi, 39 for B. garinii, and 33 for B. afzelii.In contrast to the physical maps, the three genetic maps are closely related, with no detectable differences in gene order along the entire length of the chromosome. It is concluded that the chromosomes of these three borrelial species have undergone no major rearrangements, deletions or insertions during their evolution from a common ancestor. Detailed mapping of the region of the B. gariniiand B. afzeliichromosomes that encodes rRNA revealed that each chromosome contains one copy of rrsseparated by 5 kbp from two copies each of rrland rrf(encoding 5S rRNA). The gene order is rrs rrIA rrfA rrIB rrfB. B. burgdorferiis the only other member of the eubacteria for which this particular rRNA gene arrangement has been observed. A DNA length polymorphism in the region of the borrelial rRNA genes was shown to be due to the presence of 2.2 kbp more DNA between rrsand rrIAin B. gariniiand B. afzeliithan in B. burgdorferi.
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Mosaic structure of large regions of the Lactococcus lactissubsp. cremorischromosome
More LessLactococcus lactissubsp. lactisand Lactococcus lactissubsp. cremorisare closely related phenotypically and genetically. Here we report that certain regions of their chromosomes diverge considerably more than others. Conserved regions differ by less than 20%, whilst variable regions differ by more than 60%. This mosaic structure may have arisen by horizontal gene transfer from distantly related bacteria since in a particular region of the L. lactissubsp. cremorischromosome the G + C content and the codon bias are not typical for lactococci. Such an exchange, which conserves the function of the gene and cannot be achieved under selective pressure, may be of considerable importance in the evolution of bacteria.
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Sequence and organization of the lactococcal prolate-headed bIL67 phage genome
More LessbIL67 is a broad-host-range prolate-headed phage that is active against Lactococcuscells. The complete phage genome sequence of 22195 bp was established. Thirty-seven open reading frames (ORFs) organized in two clusters were identified. Functions were assigned to the putative products of six of the ORFs on the basis of comparison of the deduced amino acid sequences to known proteins, analysis of structural features of the proteins and search for conserved motifs. These were a DNA polymerase, a protein involved in recombination, a lysin, a terminase subunit, a structural protein and a holin.
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