- Volume 107, Issue 1, 1978

Envelopes of Thermus strains had a multilayered Gram-negative appearance. Peptidoglycan contained significant amounts of glycine, and ornithine was the dibasic amino acid. Lipopolysaccharide (LPS) did not contain heptose and 2-keto-3-deoxyoctonate was not positively identified. The polysaccharide from LPS contained glucose, glucosamine, galactose, galactosamine and, in two strains, mannose. The fatty acids identified in lipopolysaccharide were C14, C16 and C18, both saturated and unsaturated.

Both A1 and A2 mating types of Phytophthora cinnamomi, Phytophthora parasitica and Phytophthora palmivora formed oospores by selfing when they were paired with different mating types on opposite sides of polycarbonate membranes. The selfing of one mating type in the presence of the other mating type demonstrates the production of diffusible substances like plant hormones as found in related fungi. Young cultures and A2 isolates were better hormone producers, whereas old cultures and A1 isolates were more responsive to hormones in both intra- and interspecific pairings. The polycarbonate membrane method should facilitate identification and genetic studies of heterothallic species of Phytophthora.

The exposure of exponentially grown Escherichia coli k12 to 52 °C for 30 min in Tris/Mg2+ buffer resulted in a considerable loss of viability when plated on tryptone agar. When such heated bacteria were held at 37 °C for 2 h in tryptone broth before plating on tryptone agar, there was a significant increase in viability. Thus, heat damage was repaired in tryptone broth but not on tryptone agar. Recovery was greater in tryptone broth than in synthetic medium. In tryptone broth, recA or polA mutants also recovered but a lex mutant did not.

As a result of heating, the sensitivity of bacteria to ultraviolet radiation (u.v.), to mitomycin C and to plating on high salt medium was enhanced. After incubation for 2 h in tryptone broth at 37 °C, the bacteria regained their resistance to u.v. and mitomycin C and tolerance to high salt medium. Recovery of viability required RNA and protein synthesis, whereas recovery of u.v. resistance did not require protein synthesis. Heating for 30 min inhibited the release of acid-soluble material from DNA in all strains of E. coli used.

Chloramphenicol-resistant Myxococcus virescens were obtained by infecting myxococci with Escherichia coli specialized transducing phage P1CM. The drug-resistant myxococci were phenotypically unstable. They contained more than one type of plasmid; these plasmids were not found in the parent strain. Chloramphenicol-resistant E. coli were obtained by transformation with either a fraction of myxococcal DNA containing the plasmids or with P1CM prophage DNA. These transformants contained plasmids. Escherichia coli transformed by DNA from the myxococci contained both P1CM and myxococcal genes. Individual transformant clones differed in the genetic make-up of their plasmids. Among the myxococcal genes expressed in these plasmid-harbouring E. coli strains were a capacity for self-transmissibility and a pattern of phage sensitivity characteristic of R factor incompatibility group W. Escherichia coli transformed with P1CM prophage contained incomplete P1CM genomes; none of the chloramphenicol-resistant transformants produced P1CM phage particles. The significance of these findings for an understanding of mechanisms for the generation of R factors is discussed.

Conditions for highly efficient genetic recombination in Streptomyces by protoplast fusion are described. Protoplasts of S. fradiae and S. griseofuscus were formed by a modification of the glycine–lysozyme–lytic enzyme method (Okanishi, Suzuki & Umezawa, 1974). Regeneration of cells from protoplasts was monitored throughout the growth cycle and was most efficient when cells of either S. fradiae or S. griseofuscus were taken from the transition phase between the exponential and stationary growth phases. Fusion of protoplasts carrying different auxotrophic or chromosomal drug-resistance markers was achieved by treatment with polyethylene glycol, and high frequencies of stable genetic recombinants were obtained.

The sexuality of the wild four-spored Agaricus species, A. bitorquis, A. nivescens, A. macrosporus and A. silvicola, was investigated and compared with that of the two-spored commercially cultivated mushroom, A. bisporus. For each of the wild species, 20 single spore isolates were mated in all combinations. Heterokaryosis was considered to have occurred when novel mycelial growth was apparent at the junction zones. Unifactorial heterothallism was confirmed in A. bitorquis and shown to occur in A. nivescens and A. macrosporus. There was no indication that nuclear migration occurred. Additional alleles of the mating-type factor were detected in other stocks of these three species. In A. macrosporus, heterokaryosis was enhanced by the addition of activated charcoal to the culture medium.

Heterokaryosis was confirmed where possible by fruiting tests in conditions used for growing A. bisporus. Agaricus bitorquis and A. macrosporus fruited but A. nivescens did not. All matings of A. silvicola isolates were negative which, together with fruiting data, suggests that this species may be homothallic. There was no indication of heterokaryosis in interspecific matings amongst these species or with A. bisporus.

The expression of sexuality in A. bitorquis, A. nivescens and A. macrosporus is contrasted with that in A. bisporus. It is suggested that A. bisporus evolved from a species of the macrosporus/nivescens type and that the constitutive heterokaryosis of A. bisporus spores may be leading to true homothallism.

A number of deoxyribonucleoside-requiring mutants (dns) of Bacillus subtilis were isolated and their growth characteristics and ribonucleotide reductase activities were compared with those of the wild type and of a dna mutant (tsA13). Both tsA13 and dns mutants required the presence of a mixture of deoxyribonucleosides for growth at 45 °C but not at 25 °C. All the mutant strains tested contained ribonucleotide reductase activity which showed heat sensitivity similar to that of the enzyme from a wild-type strain. The reductase in B. subtilis seemed to reduce ribonucleoside triphosphates in a similar manner to the enzyme in Lactobacillus leichmannii.

Mating procedures for detection of mobilization of the Proteus mirabilis chromosome were re-investigated. The chromosome was mobilized by plasmid D, the previously used hybrid between plasmids P-lac and Rldrdl9. About a 40-fold increase in recombinant recovery correlated with the absence of swarming during mating and a lower temperature of incubation. The modification introduced was that conjugation was allowed to proceed on a non-selective supplemented minimal medium at 30 °C before washing and plating on selective media. Final incubation was also at 30 °C. This technique enabled eight additional chromosomal markers to be mapped. Polarized transfer of the chromosome was shown by gradient of transmission experiments using a previously described marker as reference, by linkage analysis with reference to proximal and distal markers and (less successfully) by interrupted mating on solid medium. Markers of plasmid D transferred at high frequency to all recombinants. The plasmid was stable in recombinants and could transfer itself and chromosomal markers of the new hosts in further matings. Resulting recombination of markers occurred at usual frequencies. The marker order, his-1, ser-2, ura-2, pyrB1, trp-3, cysA1, ade-2, ilv-2, cysG1, gly-1, cysC1, argA2, metF2, nalA1, thr-1, leuB2, did not resemble the order of these markers in Escherichia coli.

Selenomonas ruminantium produced one mole of d(–)-lactate per mole of glucose used at all dilution rates in ammonia-limited continuous culture. In contrast, lactate production varied according to the dilution rate when glucose was the limiting nutrient. At dilution rates of less than 0·2 h–1, acetate and propionate were the main fermentation products and lactate production was low. At dilution rates above 0·2 h–1, the pattern changed to one of high lactate production similar to that under ammonia limitation. Experiments with cell-free extracts of S. ruminantium showed that d(–)-lactate dehydrogenase had sigmoidal kinetics consistent with homotropic activation of the enzyme by its substrate, pyruvate. This feature allows S. ruminantium to amplify the effects of relatively small changes in the intracellular concentration of pyruvate to cause much larger changes in the rate of production of lactate. Some confirmation that this mechanism of control occurs under physiological conditions was obtained in glucose-limited culture, in which the sigmoidal increase in lactate production was accompanied by a linear increase in pyruvate excretion as the dilution rate increased.

Pretreatment of Escherichia coli w3110 with levorphanol, a morphine analogue, reduced chemotaxis to serine, aspartic acid and galactose. This decreased chemotaxis was not due to decreased viability or motility. Pretreatment with 1·1 mm-levorphanol for 1 h, followed by washing to remove the drug prior to determination of chemotaxis, inhibited chemotaxis to each of the attractants by at least 80%. Pretreatment with dextrorphan, the enantiomorph of levorphanol, or levallorphan, the N-allyl analogue of levorphanol, resulted in a similar inhibition of chemotaxis. Reversal of the inhibition produced by pretreatment with levorphanol required a period of growth of at least one generation time.

Exopolysaccharide was produced by Azotobacter vinelandii NCIB 9068 grown under a variety of environmental conditions. The rate of exopolysaccharide synthesis from sucrose in continuous culture remained within the range 5 to 14 mg-atom C (g cell)–1 h–1 for specific growth rates of 0·05 to 0·25 h–1, for rates of sucrose utilization of 16 to 105 mg-atom C (g cell)–1 h–1 and for a range of growth-limiting nutrients, including sucrose.

A proteolytic mutant from Clostridium botulinum type E produced extracellular proteases after the end of exponential growth coinciding with the period of sporulation. Proteases were separated into four fractions by chromatography on a DEAE-cellulose column. One was a sulphydryl-dependent protease that also apparently required a divalent cation for enzyme activity since it was inhibited by EDTA. This enzyme hydrolysed synthetic amide and ester compounds containing an arginine residue, and showed some activity towards L-lysine methyl ester. It appeared that two of the other proteases were serine proteases and the fourth was a metal protease. These last three proteases did not require a thiol agent and did not hydrolyse any of the synthetic amides or esters examined. Only the sulphydryl-dependent protease could activate C. botulinum type B, E and F toxins. The ability of this enzyme to activate type B and E toxins was markedly lower than that of trypsin. The susceptibility of type B toxin to this protease was lower than that of type E toxin. C2 toxin was not activated by this enzyme. It is suggested that the sulphydryl-dependent protease in this proteolytic mutant of C. botulinum type E has properties similar to those of proteases from C. botulinum types B and F.

Pseudomonas aeruginosa strain 473 can grow aerobically with various n-alkanes as the sole source of carbon and energy. Anaerobic growth, with nitrate as the terminal electron acceptor, occurs with a variety of carbon substrates but not with n-alkanes. This bacterium has now been grown in continuous culture with malate, malate and octane, or octane as the carbon source. The effects of varying the dissolved oxygen tension on the cell yield, key enzyme activities, the cytochrome content and the ability to metabolize components of the medium were determined in order to assess whether alkane oxidation and nitrate dissimilation could occur concomitantly in poorly aerated cultures.

The activities of dissimilatory nitrate and nitrite reductases were maximal in bacteria which were grown anaerobically. However, during growth with malate at oxygen tensions between 2 and 3 mmHg, nitrate reductase activity was readily detected, but nitrite reductase activity and cytochrome cd were absent. It is therefore concluded that the synthesis and activity of nitrate and nitrite reductases are not coordinately regulated. In contrast, the activities of enzymes for octane oxidation were greatest in cultures grown aerobically. When octane was the sole carbon source, growth continued when the dissolved oxygen tension decreased to below 2 mmHg, but no growth occurred in the absence of oxygen. Although alkane oxidation occurred during growth in poorly aerated cultures, the cell yield on octane was higher in the absence of nitrate than in its presence.

Thiobacillus denitrificans was cultured chemolithotrophically under aerobic and anaerobic conditions in a chemostat with thiosulphate, nitrate or nitrite as limiting nutrient. Estimations of growth yields and maintenance coefficients showed that T. denitrificans grew more efficiently than other thiobacilli both aerobically and anaerobically. Relative growth yield data enabled the probable amounts of ATP generated during thiosulphate-limited aerobic growth and nitrate-limited anaerobic growth on thiosulphate to be calculated as, respectively, 6 to 7 and 4 to 5 mol ATP formed per mol thiosulphate oxidized. The energy available from tetrathionate oxidation was almost twice that from thiosulphate.

Thiobacillus denitrificans grew exclusively aerobically in thiosulphate-limited chemostat culture at all dissolved oxygen concentrations between 12 and 216 μ m. Nitrate reduction did not occur in aerobic cultures and nitrate and nitrite reductases only reached high levels under complete anaerobiosis. Growth yield was greatest [11.50 g dry wt (mol thiosulphate oxidized)–1] at the lowest dissolved oxygen concentration (12 μ m) and decreased at higher dissolved oxygen concentrations, indicating oxygen to be a growth-inhibitory substrate; the anaerobic yield was only 77 % of the maximum aerobic yield (all tested at a dilution rate of 0.08 h–1) in agreement with thermodynamic calculations. Low activities of thiosulphate-oxidizing enzyme and sulphur-oxidizing enzyme were detected in aerobic cultures, but activities were even lower in anaerobic cultures. Efficient energy coupling mechanisms with respect to sulphur and thiosulphate oxidations are indicated.

Enzyme activities of microsomal fractions have been used to examine the behaviour of cytoplasmic membranes during growth deceleration and encystment of Acanthamoeba castellanii. Rough and smooth microsomal protein content increased two- to threefold during the transition from exponential growth phase to stationary phase, but declined again as the organisms encysted. Total activities of rotenone-insensitive NADH- and NADPH-cytochrome c reductases, enzymes associated with endoplasmic reticulum, were two- to fivefold higher in stationary phase organisms than in exponentially growing organisms, but were 10- to 100-fold lower in mature cysts. Specific activities of these enzymes in homogenates and rough and smooth microsomal fractions showed the same pattern of change. Homogenate and microsomal activities of alkaline phosphatase, an enzyme known to be present on the contractile vacuole membrane, were highest in exponentially growing organisms, lower in stationary phase organisms and lower still, by at least eightfold relative to stationary phase cells, in mature cysts. The data indicate that during the transition to stationary phase there is extensive proliferation of cytoplasmic membranes which are subsequently broken down as the cells encyst. The changing activities of the cytochrome c reductases can be attributed to this pattern of membrane synthesis followed by breakdown, although there may also be changes in the abundance of functional enzyme molecules on the membranes. The alterations in alkaline phosphatase activity portray the changing status of the membrane system responsible for water expulsion and reflect a less critical need for osmotic control in the cyst than in the trophozoite.