- Volume 108, Issue 1, 1978

Over 60% of streptomycete phage, isolated from soil, were adsorbed by unsubstituted or sodium-treated kaolin, when suspended in nutrient broth, but less phage were adsorbed by sodium and calcium montmorillonite or by calcium- and aluminium-treated kaolin. The montmorillonites did, however, partially protect one of the phage from damage during experimental manipulations. Adsorption to kaolin was not influenced by pH and most of the adsorbed phage retained their infectivity. Attempts to obtain free infectious phage by desorption were unsuccessful. The pH stabilities of adsorbed and free phage differed: 50% inactivation of phage adsorbed to kaolin occurred at pH 6·1 whereas for free phage 50% inactivation occurred at pH 4·9. This suggested that ΔpH (pHb–pHs) was about 1·2 units, i.e. similar to previous estimates based on the activity of enzymes adsorbed to kaolin.

Following u.v.-irradiation and plating on a selective chlorate medium, about 200 chlorate-resistant isolates of Chlamydomonas reinhardii were obtained. These were divided into three groups all of which grew well with ammonium as nitrogen source. One group grew well with nitrate under all conditions tested. The second, and largest group, grew well with nitrate only in the absence of acetate. The third group could not grow with nitrate as sole nitrogen source. Isolates in this group were further subdivided according to their ability to grow with hypoxanthine or nitrite as sole nitrogen source. Three mutants (14/15, 17/4 and 0/8) were examined genetically in some detail. Each differed from wild-type (strain 2192) by a mutation in a single gene. Isolates 14/15 and 17/4 could grow with hypoxanthine as a nitrogen source. The mutant genes in these two isolates were shown to be located in different linkage groups and have been designated nitA (in 17/4) and nitB (in 14/15). There is evidence that nitA may be in linkage group VI. Mutant 0/8 resembles the cnx mutants of Aspergillus in that it would not grow on hypoxanthine. The mutation in 0/8 is designated nitC; it, too, may be in linkage group VI. Strain 137c was also analysed. It appears to be unable to grow on nitrate because of mutations in two loci.

Two mutants of Chlamydomonas reinhardii that cannot grow with nitrate as nitrogen source have been examined biochemically. Each mutant differs from the wild-type strain by a single mutation in a Mendelian gene. One mutant (14/15) carries a mutation in a gene designated nitB. This organism took up nitrate and converted nitrate-nitrogen to insoluble-nitrogen; nevertheless no enzymic activities associated with nitrate reductase (NADH-nitrate reductase, reduced benzyl viologen nitrate reductase or inducible NADH-cytochrome c reductase) could be demonstrated in frozen/thawed cells or cell-free extracts. At high nitrate concentrations (5 mM) the rate of nitrate uptake by this mutant [V max 59 nmol (mg dry wt)–1 h–1] was considerably lower than that of wild-type [V max 920 nmol (mg dry wt)–1 h–1]. It is concluded that nitB is probably a regulatory gene for nitrate reductase. The other mutant (17/4) has a mutation in a gene designated nitA. This organism did not take up nitrate and had no NADH-nitrate reductase or inducible NADH-cytochrome c reductase activities. Frozen/thawed cells and cell-free extracts had reduced benzyl viologen nitrate reductase activity. The enzyme from the nitA mutant eluted from a Sepharose 4B column later than wild-type enzyme; it was therefore probably of lower molecular weight. The nitA mutation of C. reinhardii closely resembles the nit-3 mutation of Neurospora crassa and, like this mutation, it probably results in loss of the NADH combining sub-unit of nitrate reductase. Strain 137c of C. reinhardii had no enzymic activities associated with nitrate reductase and did not take up nitrate. This organism may have mutations in both the nitA and nitB loci.

A variant of Caulobacter crescentus cb15, strain GU1, was isolated on the basis of its resistance to phage ϕCbK. Strain GU1 was also resistant to phages ϕCb12r and ϕCb13, developed filaments in liquid culture and showed altered sensitivity to the inhibitors methylene blue, sodium deoxycholate and ampicillin. Analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the membranes of strains cb15 and gu1 showed major differences. Partial revertants of the gu1 phenotype were isolated in which only the inhibitor sensitivities were changed. The origin of these partial revertants was investigated by the fluctuation analysis of Luria & Delbruck (1943) . The results suggest that these partial revertants, which were stably inherited, were non-random in origin.

The P-1 incompatibility group plasmid R772 can mobilize the chromosome of Proteus mirabilis strain pm5006. The decreasing gradient of recombinant recovery frequencies found for markers which were increasingly distal to O min with plasmid D donors was not found with R772. Instead, it produced recombinants for all markers at frequencies of about 5 × 10–5 per donor. This is about 10-fold lower than the plasmid transfer frequency. Recombinants were stable and recombination was only detected over short segments of the chromosome which corresponded to about 10 min on the D plasmid map of the chromosome. All recombinants had inherited R772 and expressed all properties of the plasmid. Attempts to isolate variant plasmids with increased frequencies of recombinant formation were unsuccessful.

R plasmids pMG1, R2, R931 and pMG15 increased the survival of Pseudomonas aeruginosa exposed to ultraviolet radiation (u.v.) in the wild type, and uvr and polA mutants but did not alter the u.v.-response of a recA mutant. The R plasmid RPL11 reduced u.v.-survival in the wild type, and uvr and polA mutants but did not alter the u.v.-response of a recA host. All the plasmids enhanced the level of spontaneous and u.v.-induced back mutation (Trp+) in a trpB1 strain.

The effect of a sublethal concentration of sodium arsenite following u.v.-irradiation was examined. It was concluded that in strains trpB1 (pMG1) and trpB1 (R931), u.v.-protection is determined by a recA +-dependent, arsenite-sensitive repair pathway, whereas in strains trpB1 (R2) and trpB1 (pMG15), u.v.-protection is determined by a recA +-dependent, arsenite-insensitive step in DNA repair.

The plasmids R711b (at present IncX) and F0 lac (IncFV) both determine pili morphologically like those of F (IncFI), and confer sensitivity to the F-specific filamentous bacterio-phages, but not to the F-specific isometric RNA phages. Detailed serological studies show that the two pilus types are unrelated, and that neither is related to any of the previously defined F pilus serotypes. Adsorption of the isometric RNA phage MS2 to R711b pili occurs in the presence but not in the absence of formalin, which presumably prevents elution of reversibly adsorbed virions. No adsorption occurs with F0 lac pili. MS2 multiplication, as measured by titre increase tests in liquid medium, is found with neither plasmid. The two plasmids are not incompatible. These observations indicate that R711b and F0 lac are different both from one another and from the plasmids belonging to the incompatibility groups IncFI–IV.

A new technique, the double strip method, for studying the chemotaxis of myxomycete plasmodia is described. Physarum polycephalum was attracted by the aldohexoses d-glucose, d-galactose and d-mannose and their derivatives 2-deoxy-d-glucose and maltose, thresholds ranging from 0·25 mm (d-glucose) to 5 mm (d-mannose). These sugars competed with each other, a uniform background of one of them inhibiting taxis to the others. Other attractants were N-acetyl-d-glucosamine and mannitol, with thresholds at 1 mm, and fucose (6-deoxy-d-galactose). Although in general only those carbohydrates which could support growth were attractants, there were exceptions such as 2-deoxy-d-glucose; hence metabolism of a compound was not necessary for attraction. In addition, some compounds, such as fructose, could be metabolized but did not attract. At high concentrations (about 100 mm) all the compounds tested, including attractants, could under appropriate conditions cause repulsion, probably through osmotic effects.

A selection technique based on low-speed centrifugation of an exponentially growing culture and a starvation induction method for obtaining synchronous cultures of Acanthamoeba castellanii were compared. The selection method gave the highest levels of division and synchrony. Amoebae which were induced to encyst at different times during the growth cycle by the addition of MgCl2 to the cultures differed in their ability to initiate encystation, indicating that there may be a relationship between the position of a cell in the growth cycle and its competence to initiate differentiation. The patterns of protein and ribose synthesis which were observed during synchronous growth indicate that the changes which occur in the levels of these materials during encystation may not be unique to the differentiation process.

Growth of Saccharomyces cerevisiae on poor nitrogen sources such as allantoin or proline was totally inhibited by addition of a non-degradable basic amino acid to the medium. Cells treated with lysine contained greatly reduced quantities of histidine and arginine. Conversely, lysine and histidine were severely reduced in arginase-deficient cells treated with arginine. When all three basic amino acids were present in the culture medium, growth was normal suggesting that synthesis of all three basic amino acids was decreased by an excess of any one of them. Inhibition of growth was accompanied by a fivefold increase in the observed ratio of budded to unbudded cells. These morphological changes suggested that DNA synthesis was inhibited. Consistent with this suggestion, addition of a basic amino acid to the culture medium substantially reduced the ability of the cells to incorporate [14C]uracil into alkali-resistant, trichloroacetic acid-precipitable material. RNA and protein synthesis, although decreased, were less sensitive to the effects of such additions.

Based on dry weight, first-order kinetics adequately describe the net decrease of RNA in Dictyostelium discoideum nc-4 until culmination, when a cessation of RNA degradation occurs. Between aggregation and sorocarp formation, the cells lose 40 % of their RNA, or 19 mm-glucose equivalents expressed in terms of cell volume at aggregation. In sorocarps the spores contain a disproportionately large amount of RNA. Net RNA contents in sorocarps and in spores are unaffected by the availability of exogenous glucose. The relationship of dry weight, cell number and packed cell volume during differentiation is given.

The transition from yeast-like to chlamydospore cells was induced by growing Pullularia pullulans on an unbuffered glucose medium with a limiting nitrogen source. The kinetics of cell growth and extracellular polymer production were determined. Changes in cell morphology were apparent during the whole process. Yeast-like cells became progressively enlarged before producing chlamydospores. The proportion of sugars increased at the expense of proteins and nucleic acids during the transition. Quantitative and qualitative changes in phospholipids were observed. Fatty acid unsaturation decreased as the cells grew older. Enzymic cell-wall analysis also revealed differences in the wall structures of the different forms. Protoplasts were easily prepared from yeast-like cells, but could not be produced from other cell forms.

In nocardioform organisms related to the ‘rhodochrous’ taxon (Nocardia restricta, Nocardia or Jensenia canicruria), morphogenesis was affected by nutritional conditions, especially the nature of the carbon source. With glucose, growth was slow and the bacteria divided at each doubling of the cell mass. With succinate or acetate, growth was much more rapid during the exponential phase and cells grew as long rods. Later, when growth slowed down, septa, which were clearly visible in thin sections examined by electron microscopy, gradually appeared and divided the organism into smaller cells. During the stationary phase of growth, cells recovered their original spherical shape. Synchronous growth of these bacteria was achieved in liquid synthetic medium by inoculating fresh medium with spherical cells taken from an advanced stationary phase culture. The synchrony was shown by the periodicity of DNA replication and protein synthesis.

The fragmentation of the rods was studied during incorporation of labelled diamino-pimelic acid into cell walls. Septa did not form simultaneously but in distinct phases. The regular periodicity of their formation suggested that this process is coordinated in time and is related to the organism’s growth rate. The data are interpreted using Cooper and Helm-stetter’s model for cell division in prokaryotes.

The unusual sensitivity of a Pythium ultimum isolate to three inhibitors of prokaryote protein synthesis [tetracycline (TET), chloramphenicol (CAM), erythromycin (ERY)] was investigated. TET inhibited growth in shake culture by 40% at 10 μg ml–1 and 99% at 100 μg ml–1 within 5 h (one doubling period). CAM inhibited growth by 40% at 500 μg ml–1 and 70%, at 1 mg ml–1. Cycloheximide (CHI), a eukaryote protein synthesis inhibitor, blocked growth completely at 1 μg ml–1. These agents reduced incorporation of leucine and phenylalanine into protein by 40 to 50% (100 μg TET ml–1, 500 μg CAM ml–1) or 93% (1 μg CHI ml–1) within 20 min. This rapid inhibitory effect could not be attributed to impaired mitochondrial energy generation. TET inhibition increased to 90% by 60 min, but CAM inhibition did not change for 5 h. TET inhibited both the transport and protein synthetic (cytoplasmic) components of amino acid incorporation, but CAM impaired only the transport activity. CHI blocked protein synthesis, but did not inhibit transport de-tectably. In contrast to these results, growth inhibition by ERY did not increase with dose (40% inhibition at 10 to 500 μg ml–1), and ERY at 500 μg ml–1 inhibited neither amino acid transport nor protein synthesis in 5 h. The results indicate that the sensitivity of P. ultimum to these prokaryote inhibitors reflects unusual permeability properties, which account for impaired transport (CAM, TET) and allow access of the drugs to sensitive intracellular sites, the cytoplasmic (TET) and mitochondrial (ERY) protein synthetic systems.

A reproducible, simple method for assessing nitrogenase activity by the acetylene reduction test on agar plates is described. The effects of phosphate, succinate and arabinose on nitrogenase activity and slime production by R. japonicum strain 61-a-101, growing on a solid medium with leucine or glutamine as sole source of fixed nitrogen, were investigated. Slime production varied with different media but there was no simple correlation between slime production and high nitrogenase activity. Arabinose greatly enhanced nitrogenase activity of bacteria growing on glutamine as sole nitrogen source, especially if the amino acid concentration was 1 mm or less. Without arabinose, the optimum concentration of glutamine for nitrogenase activity was 2 to 2·5 mm, but 1 mm-glutamine plus 50 mm-arabinose gave twice the activity [8 to 10 nmol ethylene produced h–1 (mg protein)–1].