- Volume 131, Issue 4, 1985

Predictions based on a general model of surface components involved in gliding motility of Cytophaga johnsonae along with characteristics of previously isolated nonspreading mutants of C. johnsonae led us to suspect that isolation of such mutants could be accomplished by selecting for nonadherent or nonhydrophobic mutants. Accordingly, conditions were devised to select for mutants that failed to attach to cheesecloth suspended in growth media and for mutants that failed to adhere to n-hexadecane droplets. Populations of cells obtained from both selection procedures were screened for mutants producing nonspreading colonies. Both techniques resulted in enrichment for nonspreading mutants that were classified by previously established criteria as MNS (motile nonspreading), CNS (conditional nonspreading) or TNM (truly nonmotile). When assayed for adherence and hydrophobicity, all TNM mutants were nonadherent and nonhydrophobic, compared with wild-type cells. Most mutants of the other two classes were unchanged with respect to these properties. Results from subjecting cells of four other strains of gliding bacteria to selection by the same procedures indicate that the methods will be broadly applicable for isolating nonspreading mutants of gliding bacteria.

An ammonium permease was derepressed when Rhizobium leguminosarum was grown in chemostat culture under conditions of nitrogen limitation. The ammonium permease was characterized by direct measurements with an ion-specific ammonium electrode. Cells grown under ammonia, nitrate, glutamate or methylamine limitation had permease activity, while those grown with excess (10 mm) ammonium chloride or glutamate did not. On transfer from N-limited to N-excess conditions, the permease disappeared rapidly, and all activity was lost within 18 h. Uptake by the permease was sensitive to azide, carbonyl cyanide m-chlorophenylhydrazone,2,4-dinitrophenol, nigericin and valinomycin. The apparent K m for was 0·015 mm; ammonium uptake had a narrow maximum around pH 7·5. The internal ammonia concentration of N-limited cells was 0·4 mm, with up to 60-fold gradients of was 0·015 mm; forming across the membrane within 30 min. Hydrazine and hydroxylamine strongly inhibited ammonium uptake, with methylamine, glutamine, aspartate and glycine less effective as inhibitors. Isolated pea bacteroids capable of transporting succinate did not possess the ammonium permease.

The uptake of succinate and malate has been compared in cultured cells and bacteroids of two species of slow-growing Rhizobium: R. japonicum (USDA I-110) and cowpea Rhizobium (USDA 3278). Cultured cells of both organisms actively accumulated both compounds, and uptake was abolished by KCN and 2,4-DNP, but not by arsenate. Kinetic studies using cultured cells showed that succinate competitively inhibited malate uptake, and vice versa, implying a common step in the uptake of these dicarboxylic acids. Uptake of both of these compounds was inhibited by osmotic shock and N-ethylmaleimide in cultured cells of both species. Purified bacteroids accumulated succinate in a process that was sensitive to 2,4-DNP and KCN, but at a rate significantly slower than for cultured cells. No detectable malate uptake was observed in purified symbiotic cells. Furthermore, succinate uptake was insensitive to osmotic shock in bacteroids of both strains. These results show that although bacteroids of both strains are competent in succinate uptake, significant differences exist in the expression and/or stability of dicarboxylate uptake systems between free-living and symbiotic cells.

Nikkomycin was found to be a potent growth inhibitor of Candida albicans through competitive inhibition of chitin synthase [K i = 0·16 μm (0·1 μg ml–l)]. The activity of the peptide-nucleoside drug was antagonized by both peptone and defined peptides. Transported dipeptides were effective antagonists while transported oligopeptides were not. A mutant of C. albicans resistant to the effects of nikkomycin through a transport defect was unable to transport dipeptides, while oligopeptide uptake was apparently unaffected. At least two peptide permeases are operational in this organism.

The metabolism of phospholipids in Vibrio costicola, a moderately halophilic bacterium, has been investigated in relation to sudden changes in salinity. Both the absolute and relative rates of biosynthesis of phosphatidylglycerol and phosphatidylethanolamine depend on the salt concentration of the medium; a sudden rise in salt concentration has an instantaneous inhibitory effect on phospholipid biosynthesis, but this inhibition lessens as. the bacteria adapt to the higher salinity. There is no turnover of phospholipids during isotonic growth, nor when the salt concentration is suddenly altered. The alterations in biosynthetic rates of phosphatidylglycerol and phosphatidylethanolamine that occur after sudden changes in salt concentration are consistent with the known compositional changes. We conclude that the mechanisms of changes in phospholipid composition during adaptation to raised or lowered salt concentrations are not necessarily the same.

Four species of the purple ‘non-sulphur’ bacteria (Rhodospirillaceae) were examined with respect to their dissimilatory sulphur metabolism. Under anaerobic conditions with CO2 as sole carbon source, cell suspensions of Rhodopseudomonas sulfoυiridis, Rhodobacter υeldkampii and Rhodo-bacter adriaticus, all dependent on reduced sulphur compounds for growth, oxidized sulphide to an intermediate compound (elemental sulphur, possibly polysulphides) that was converted either partly (R. υeldkampii) or completely into sulphate, whereas thiosulphate oxidation occurred without detectable intermediates. In contrast, Rhodobacter sulfidophilus oxidized sulphide as well as thiosulphate to sulphate. Both oxidations occurred with simultaneous excretion of sulphate and sulphite; the latter was subsequently also transformed into sulphate. In cell-free extracts the presence of a reverse sulphite reductase could not be proven. An adenosine 5′-phosphosulphate (APS) reductase could not be detected either, but all strains contained a membrane-bound sulphite oxidoreductase that is obviously responsible for sulphate production. This enzyme was solubilized and partly purified from R. adriaticus, Rps. sulfoυiridis and R. sulfidophilus.

A strain of the gas-vacuolate cyanobacterium Microcystis was found to float in cultures grown at low light intensities and to sink in those grown at high intensities. The loss of buoyancy that occurred within 1 to 5 h on increasing the photon flux density from 10 to l00 μmol m–2 s–1 was investigated by centrifuging the cell suspensions in a horizontally placed capillary with a rectangular cross-section, and then separately counting the floating cells under the upper tube surface and sinking cells on the lower surface. Buoyancy loss was not accompanied by loss of gas vesicles, as occurs in some other planktonic cyanobacteria, but was caused by a relative increase in dry matter, principally carbohydrate, without a corresponding increase in gas vesicles. The increase in light intensity gave an increase in cell turgor pressure but this was insufficient to collapse the strong gas vesicles present in this strain, which had a median critical pressure of 0·75 MPa (7·5 bar).

When subcultures of the aux-2 and aux-4 strains of Physarum polycephalum, which had been grown for more than four years in axenic shake culture, were transferred to non-axenic surface culture they displayed progressively shorter lifespans (older axenic surface cultures yield shorter lived non-axenic cultures). Similar subcultures transferred to axenic agar medium also underwent senescent-like events. These subcultures, after a period of vigorous growth, displayed a slower growth rate, reduced cytoplasmic streaming, loss of yellow pigment, and eventually they fragmented into a number of small spherical structures with the concomitant lysis of most of the plasmodium. In non-axenic culture these structures quickly degenerated (and disappeared from the culture); however, in axenic culture they revived and after several days produced new vigorous plasmodia. Following a period of vigorous growth the plasmodium again underwent senescent-like events. This cycle of senescence and growth was repeated a number of times before death finally occurred.

Germinating spores of Streptomyces viridochromogenes excreted a substance into the surrounding medium which inhibited germination of another sample of the spores. The germination inhibitor (GI) was produced during submerged culture after exponential growth had ceased. The GI was purified 51-fold following extraction from growth liquor with chloroform. It was soluble in alcohol and water and had a molecular weight of less than 1000. The GI blocked growth and respiration of some Gram-positive bacteria and was an inhibitor of the membrane bound, but not solubilized, calcium-dependent ATPase of germinated spores and mycelia of the producing organism. Several sodium–potassium activated ATPases were also inhibited. All four activities (respiration, growth, germination inhibition. ATPase) co-purified during column and thin-layer chromatography. The GI activities released during germination and produced during growth were identical. A role for the GI antibiotic in regulation of dormancy of spores of the producing organism is discussed.

Maximum molar growth yields on oxygen ( ) and succinate ( ) were not affected when carbon-limited Acinetobacter calcoaceticus NCIB 8250 was changed to oxygen limitation.→H+/O quotients for cells grown with succinate- or ammonium-limitation did not differ significantly from those obtained with oxygen-limited cells. The levels of cytochromes b and o remained constant at 0‧13 and 0‧05 nmol (mg protein)–1 respectively in crude cell-free extracts of succinate-limited organisms grown at high (80 mmHg) or low (5–10 mmHg) dissolved oxygen tension respectively. Their levels increased in oxygen-limited conditions, when cytochrome a 2(d) was additionally synthesized and organisms exhibited increased resistance to cyanide inhibition of respiration. It is concluded that the respiratory efficiency of A. calcoaceticus NCIB 8250 is unaffected by oxygen concentration under the conditions studied. Theoretical calculations are presented for maximum molar growth yields on succinate and oxygen and these are compared with experimental values. Although the effective P/O ratio is only about unity, energy is probably conserved at two sites of oxidative phosphorylation and this is independent of the de novo synthesis of cytochrome a 2 (d) under conditions of oxygen limitation. Possible reasons for the observed low molar growth yields are discussed.

Acinetobacter calcoaceticus NCIB 8250 was grown in batch culture on 46 separate sources of carbon + energy and the molar growth yields were measured. The organism was also grown in continuous culture on l-mandelate, phenylglyoxylate and succinate and the results used to calculate maximum molar growth yields. The elemental composition of the bacteria was determined. Growth equations for each substrate were constructed so that the amount of oxygen consumed per mol of substrate could be calculated. Growth yields on oxygen (Y O2 ) were then estimated. The Y O2 values were low [averaging about 19 g dry wt (mol O2)–1]. The most likely explanation for the low yields is that the effective P/O ratio is only about one, although there are probably at least two sites of oxidative phosphorylation.

Hybridization of various Streptomyces cattleya aerial mycelium negative (Amy–) mutants with a probe containing the gene for argininosuccinate synthetase (pTG17) has revealed the presence of two different types of mutants (stable and unstable). Stable mutants appear to have lost all or part of the region covered by the probe, while the unstable mutants demonstrate no detectable changes in this region. In one group of stable mutants (those demonstrating a partial loss of sequences hybridizing to the probe), a 4·17 kb extrachromosomal element was detected, which hybridized with the pTG 17 probe. The significance of this finding is discussed with reference to the genetic instability of the genus Streptomyces.