- Volume 136, Issue 12, 1990

Very low concentrations of CuSO4 induced a massive leakage of K+ from Pseudomonas syringae ATCC 12271 cells suspended in distilled water, Ca2+/PIPES or Na+/PIPES. Cell suspensions in distilled water, Ca2+/ or Mg2+/PIPES, treated with Cu2+, showed appreciable increases in optical density, whereas suspensions in Na+/PIPES were unaffected. The addition of monovalent cations to suspensions in distilled water, Ca2+/ or Mg2+/PIPES prevented the optical density increase induced by Cu2+, whereas the addition of Ca2+ or Mg2+ to suspensions in distilled water did not have this effect. Cells suspended in Na+/PIPES and treated with Cu2+ showed no major ultrastructural alterations, but cells treated with Cu2+ in distilled water showed pronounced plasmolysis. At all Cu2+ concentrations, two types of cells were observed, normal and heavily plasmolysed. An increase in Cu2+ concentration resulted in an increase in the percentage, but not in the degree of plasmolysis, of the plasmolysed cells. Cells suspended in distilled water or Na+/PIPES bound significant amounts of copper. Cu2+ concentrations that induced leakage of most of the unbound K+ did not saturate the copper-binding sites in the cells. These results indicated that plasmolysis is a direct consequence of the massive K+ leakage from the cells, in agreement with the notion that K+ is the main osmolyte in bacteria grown under normal conditions. Monovalent (but not divalent) cations prevented plasmolysis induced by copper by entry into the cells after the release of K+ ions. Na+ and Li+ probably replaced resident K+ ions in the neutralization of negative charges of cytoplasmic constituents. K+ efflux and plasmolysis, induced by Cu2+, appeared to be essentially ‘all-or-nothing’ effects.

The lectin in Rhizoctonia solani is a major protein, although its function was previously unknown. An enzyme-linked immunosorbent assay was developed to quantify the lectin content in mycelium, sclerotia and culture filtrate during the development of R. solani grown in a synthetic medium. Lectin content was low in young mycelium but increased dramatically at the onset of sclerotium formation, reaching a maximum in adult sclerotia. Upon myceliogenic germination, the lectin content of the sclerotia rapidly decreased. It thus appears that the lectin is a developmental regulated sclerotium-specific protein accumulating during sclerotium formation and disappearing during germination. Its relative abundance and the pattern of developmental control suggests that the lectin is probably a storage protein. Lectin accumulation was also influenced by the composition of the medium. Addition of supplementary carbohydrate (d-glucose) caused a marked reduction in lectin content whereas increased nitrogen in the form of l-asparagine led to a higher lectin content. However, when the l-asparagine concentration was too high, autolysis occurred and part of the lectin was released into the medium.

In medium of low or high osmolality, bacteroids isolated from Medicago sativa nodules induced by Rhizobium meliloti 102F34 rapidly catabolized [14C]choline. Trimethylamine was never detected and glycine betaine was the predominant product within the ethanol-soluble fraction of bacteroids subjected to salt stress (0·4 m-NaCl). Both choline oxidase and glycine betaine aldehyde dehydrogenase activities were characterized; the apparent K m values for choline and glycine betaine aldehyde were 2·0 and 0·4 mm, respectively. A 3 h incubation of the bacteroids in medium of high osmolarity, supplemented or not with choline, did not significantly modify the specific activity of the two enzymes. Similarly, salinization of the host plants for 2 weeks with 0·15 m-NaCl had only a slight effect on both enzymic activities. Thus, the choline-glycine betaine biosynthetic pathway was not modulated by the external osmotic pressure. Glycine betaine itself was actively degraded by bacteroids suspended in low-osmolarity medium, but the demethylation process producing sarcosine and glycine was extremely slow in bacteroids subjected to salt stress. Thus, high concentration of glycine betaine can be maintained in salt-stressed bacteroids.

In standard liquid medium containing 5g mannitol l−1and 1g glutamic acid l−1, Rahizobium meliloti SU-47 cells accumulated 350 mg cyclic 1,2-β-glucans (g protein)−1. The cyclic glucans were 36% glycerol-1-phosphatesubstituted and 64% were uncharged. In the same medium with 10 g mannitol l−1, repeating units of succinoglycan (1110 mg l−1) were found as extracellular carbohydrates, and only low amouns of the succinoglycan polymer (up to 300 mg l−1) were excreted. By raising the omotic pressure of the medium by the addition of NaCl or other ionic and non-ionic osmolytes, succinoglycan production could be stimulated: up to 2·4 g l−1at 0·2 m-NaCl was produced at the expense of the repeating units. Above 0·2 m-NaCl growth was slowed down, and succinoglycan excretion diminished. At 1 m-NaCl growth stopped completely. In standard medium containing 0·6 m-NaCl the amount of cellular cyclic 1,2-β-glucans was lowered to 150 mg (g protein)−1out of which the glycerol-1-phosphatesubstituted glucan fraction was reduced to 15%. Instead, high amounts of oliogosaccharides were synthesized as osmoprotectants, with trehalose as the major component [up to 200 mg (g protein)−1]. Glycogen synthesis was completely suppressed at this salt conenctration, while poly β-hydroxyutyric acid syntheis was unaffected.

The effects of hypersaline treatment (osmotic upshock) on cell water relations were examined in the Gram-positive bacterium Bacillus subtilis using particle size analysis. Application of the Boyle-van't Hoflf relationship (cell volume versus reciprocal of external osmolality) permitted direct determination of turgor pressure, which was approximately 0·75 osmol kg−1 (1·9 MPa) in exponentially growing bacteria in a defined medium. The abolition of turgor pressure immediately after upshock and the subsequent recovery of turgor were investigated. Recovery of turgor was K+ dependent. Calculation of turgor by an alternative method involving spectrophotometric analysis of shrinkage gave somewhat lower estimates of turgor pressure.

The effects of hypersaline treatment (osmotic upshock) on solute accumulation have been studied in the Gram-positive bacterium Bacillus subtilis. Natural abundance 13C NMR spectroscopy studies revealed only proline as a major organic osmoticum in cells grown in defined medium (no exogenous organic solutes) and this finding was confirmed by amino acid analysis. Intracellular concentrations of both K+ and proline rose markedly after osmotic upshock. K+ influx from the medium was rapid (< 1 h) but proline synthesis was a slower process (5–9 h). Proline synthesis appeared to be dependent on the prior accumulation of K+ and it is possible that K+ serves in some manner as the signal for increased proline synthesis. In cells upshocked in medium enriched in glycine betaine the endogenous synthesis of proline was repressed and glycine betaine served as the sole organic osmoticum. K+ was also accumulated under these conditions.

Influx of 45Ca2+ into Saccharomyces cerevisiae was measured under experimental conditions which enabled measurements of initial rate of transport across the plasma membrane, without interference by the vacuolar Ca2+ transport system. Addition of glucose or glycerol to the cells, after pre-incubation in glucose-free medium for 5 min, caused a rapid, transient increase in 45Ca2+ influx, reaching a peak at 3–5 min after addition of substrate. Ethanol, or glycerol added with antimycin A, had no effect on 45Ca2+ influx. We have shown previously that this increase is not mediated by an effect of the substrates on intracellular ATP levels. Changes in membrane potential accounted for only a part of the glucose-stimulated 45Ca2+ influx. The roles of intracellular acidification and changes in cellular cAMP in mediating the effects of glucose on 45Ca2+ influx were examined. After a short preincubation in glucose-free medium addition of glucose caused a decrease in the intracellular pH, [pH]i, which reached a minimum value after 3 min. A transient increase in the cellular cAMP level was also observed. Addition of glycerol also caused intracellular acidification, but ethanol or glycerol added with antimycin A had no effect on [pH]i. Artificial intracellular acidification induced by exposure to isobutyric acid or to CCCP caused a transient rise in Ca2+ influx but the extent of the increase was smaller than that caused by glucose, and the time-course was different. We conclude that intracellular acidification may be responsible for part of the glucose stimulation of Ca2+ influx. The role of the increase in cAMP level on Ca2+ influx was examined by measuring the effect of glucose and of artificial intracellular acidification on Ca2+ influx in a cyr1 strain which lacks adenylate cyclase activity. In this strain, addition of glucose or isobutyric acid still led to a transient increase in Ca2+ transport. Therefore, we concluded that at least part of the increase in Ca2+ influx in response to glucose is cAMP-independent.

Streptococcus sanguis P4A7 was grown in glucose limited conditions in continuous culture at pH 7·0 in a chemically defined medium containing either free amino acids or casein as the organic nitrogen source. Apart from aspartate and threonine, which were poorly utilized at the higher dilution rates, all amino acids in the free-amino-acid medium were utilized to various extents. At the higher dilution rates, aspartate actually increased in concentration, probably due to deamidation of asparagine. The amino acid most utilized at all dilution rates was arginine, with up to 99% of the amino acid being consumed. Both casein and its α s1-casein fraction supported growth at a level only slightly lower than that obtained with the free-amino-acid medium, provided that either cysteine or thioglycollate was present. With the exception of tyrosine, nearly all of the amino acyl residues of α s1-casein were utilized to some degree. In general, the higher the concentration of each amino acid in the medium (whether free or as part of α s1-casein) the higher the extent of utilization by S. sanguis P4A7. Only 50% of the arginyl residues (0·16 mm) of α s1-casein were utilized compared with 99% of free arginine (1·5 mm) under similar conditions, suggesting that only 50% of the as1-casein arginine was accessible to the organism. From a comparison of the amino acid composition of α s1-casein with that of the high M r fraction of the culture supernatant it was concluded that leucine, phenylalanine, lysine, histidine, arginine and valine were specifically released from α s1-casein by the endo- and exopeptidase activity of S. sanguis P4A7.

A wild-type strain of the fungus Aphanocladium album was mutagenized by UV irradiation in order to obtain chitinase-overproducing mutants. Mutants were screened on agar medium containing colloidal chitin and selected for their ability to produce large clearing zones around the colonies. Two mutant strains, designated E3 and E12, showed respectively a 26- and a 2·5-fold increase in maximal extracellular chitinase activity, determined in liquid medium with crystalline chitin as sole carbon source, compared to the wild-type strain. This is believed to be the first report on the induction of stable chitinase-overproducing mutants in a filamentous fungus. Regulation of enzyme activity was investigated in mutant E3 and the wild-type strain.