- Volume 133, Issue 2, 1987

The role of cell-surface proteins in the sexual cell-cell interactions between haploid cells of two mating types of Tremella mesenterica was studied. Gamete cells (mating-pheromone-treated cells) of the two mating types immediately associated to form large sexual agglutination complexes in a mating-type-specific manner. Vegetative cells, on the other hand, agglutinated after a lag period that corresponded to the time required for mating tube formation. Sexual agglutination was inhibited by the presence of various proteases. Polypeptides with binding activity specific for gamete cells of the opposite mating type were released from gamete cells of the two mating types by digestion with thermolysin.

Leucine and histidine biosynthetic genes from Streptomyces rochei HP1 that complemented auxotrophic mutations in S. lividans TK54 were cloned in pIJ61. DNA from one leucine recombinant plasmid was subcloned into pBR322. From the latter, a recombinant plasmid was obtained that complemented the leuA mutation in Escherichia coli CV512 but not other leucine markers in E. coli. Analysis of this and several subclones, including mutant plasmids constructed in vitro, established that the cloned S. rochei gene was expressed in E. coli from the tetracycline promoter of pBR322 to produce a polypeptide of 67 kDa; the corresponding coding region was shown to be within a 1·7 kbp DNA fragment. Blot hybridization revealed corresponding homologous genes in several other streptomycetes.

A repeated DNA sequence in the genome of Bordetella pertussis has been demonstrated. At least 20 copies of this sequence could be observed in either BamHl or EcoRI restriction enzyme digests of chromosomal DNA; fragments carrying the repeated DNA sequence ranged in size from about 1·5 to 20 kbp. The repeated DNA sequence was cloned from two separate regions of the B. pertussis genome, as shown by restriction enzyme site maps of the two clones and by hybridization studies. A small number of differences in the pattern of hybridization of the repeated DNA sequence to chromosomal DNA from several strains of B. pertussis was observed. No repeated DNA sequences were observed in one strain each of B. parapertussis and B. bronchiseptica, and there was no hybridization of B. pertussis DNA to Escherichia coli chromosomal DNA. The repeated DNA sequence was subcloned on a 2·54 kbp BamHl fragment from one of the two original clones. Restriction enzyme digests and hybridization studies showed that the repeated DNA sequence was about 1 kbp in size and had a single, internal ClaI site.

Two genes, designated rcs A (regulation of capsule synthesis) and rcsB, that had been cloned from the chromosome of Klebsiella aerogenes (K. pneumoniae) capsular serotype K21 were capable of activating expression of colanic acid capsular polysaccharide in Escherichia coli K12. The Klebsiella rcsA gene encoded a polypeptide of 23 kDa that was required for the induction of a mucoid phenotype at ≤30 ° but not at ≥37 °. The Klebsiella rcsB locus encoded no apparent polypeptides and was not capable by itself of causing the overproduction of colanic acid. However, when present in the same cell with rcs A, either in cis or in trans, rcsB caused expression of mucoidy in E. coli at all growth temperatures. These findings are best explained if the Klebsiella rcs A gene product acts as a positive regulator of colanic acid biosynthesis in E. coli and that activity of this protein is in turn subject to regulation by Lon protease. The Klebsiella rcsB locus may exert its effect by preferentially binding a negative regulator of capsular biosynthesis, possibly Lon itself. DNA sequences homologous to the Klebsiella K21 b rcs A and rcsB genes were found in the genomes of all other capsular serotypes of klebsiellae examined, including K2, K12, K36 and K43. However, there was no homology between such genes and the chromosome of E. coli. The ability of these rcs genes to induce a mucoid phenotype explains the apparent conjugative transfer from klebsiellae to E. coli of the ability to produce K21 or other Klebsiella capsular polysaccharides that are structurally and antigenically related to colanic acid.

Procedures are described for the selection of Escherichia coli mutants that constitutively take up and phosphorylate fructose, and convert it to fructose 1,6-bisphosphate. The phenotype of such mutants is described. The altered regulatory gene, fruC, is highly co-transducible with leu and other markers located at min 2 on the genome. In merozygotes, fruC + is dominant to fruC. Mutants can be readily isolated that are fruC at 42 °C but fruC + at 30 °C; moreover, the integration of a Tn 10 transposon in the genome at min 2converts fruC + strains to fruC. It is therefore likely that the fruC + regulatory gene specifies a repressor protein.

The electrophoretic karyotypes of five Candida albicans isolates and of five other Candida species have been determined, using orthogonal field alternating gel electrophoresis (OFAGE). None of the C. albicans isolates had the same electrophoretic karyotype. By comparing all five strains, we arrived at a chromosome number of nine to ten, but since the organism is diploid, we cannot distinguish genetically different chromosomes from homologues which resolve. We determined minimal chromosome numbers of 9 for Candida stellatoidea, 10 for C. glabrata and 6 for C. guilliermondii.

The partially purified monodehydroascorbate reductase (EC 1.6.5.4) from Euglena gracilis Z showed a pH optimum of 7·0 and a temperature optimum of 41 °C, and had an M r of 52000. Activity was threefold higher with NADPH than with N ADH; the K m values for N ADPH and NADH were 7 and 210 μm, respectively. The partially purified dehydroascorbate reductase (EC 1.8.5.1) had a pH optimum of 7·0, a temperature optimum of 38 °C and an M r of 28000. The enzyme was specific for reduced glutathione as an electron donor, with a K m of 0·85 mm, and for dehydroascorbate, with a K m of 0·26 mm. The enzyme reaction proceeded in either an ordered or a random manner. Both reductases were markedly inhibited by thiol inhibitors, and the inhibition was overcome by thiol compounds such as 2-mercaptoethanol and dithiothreitol. Both reductases were cytosolic. The activities of monodehydroascorbate and dehydroascorbate reductases could account for the regeneration of ascorbate from monodehydroascorbate and dehydroascorbate produced by ascorbate peroxidase (EC 1.11.1.11) for scavenging hydrogen peroxide.

Germination and growth of six fungi, Basipetospora halophila, Polypaecilum pisce, Aspergillus penicilloides, A. wentii, Eurotium repens and Exophiala werneckii, isolated from salt fish, and two new undescribed species from other sources, a Geomyces sp. and a Eurotium sp., were examined on media in which water activity (a w) was controlled over a wide range by glucose/fructose, glycerol or NaCl. Three species, B. halophila, P. pisce and Ex. werneckii, grew more rapidly on media containing NaCl in comparison with glucose/fructose, and hence can be considered to be halophilic. Two other species, A. penicilloides and A, wentii, were also unusually tolerant of NaCl: A. penicilloides could grow on media saturated with NaCl, while A. wentii grew more rapidly on NaCl-based media down to 0·82a w than any other xerophile examined so far. In contrast, the Geomyces sp. was intolerant of salt. The Eurotium sp. was obligately xerophilic, with growth confined to the range 0·935–0·68 a w.

The distribution and properties of the cellular water in sporangiospores of Phycomyces blakesleeanus were investigated using proton-NMR. In dormant spores different classes of water were characterized by a difference in their transverse relaxation times (T 2). The amount of cytoplasmic water was estimated to be as low as about 700 mg (g dry wt)−1 and its small T 2 (18·2 ms) indicated a very limited mobility. About 10 min after induction of germination (by a heat shockor by addition of 0·1 m-acetate), both the content and the mobility of the cytoplasmic water increased sharply. These changes coincided with a rapid breakdown of most of the cellular trehalose and with the production (and leakage from the spores) of large amounts of glycerol. The role of these biochemical changes is discussed in relation to the water status of the spores.

The effects of water activity on colony radial growth rate, specific growth rate, growth yield and blastospore production of Paecilomyces farinosus were studied. The compound (PEG 200) used to adjust water activity was not used for growth either when it was the sole carbon source in the medium or when the medium also contained glucose. Daily additions of water were made to cultures ‘compensated’ cultures) to compensate for evaporation; when this was not done the water activity of the medium decreased during incubation and a water activity at which growth ceased was eventually attained. A correlation was observed between the inhibitory effects of water activity on specific growth rate (µ) in ‘compensated’ shake flask cultures and on colony radial growth rate (K r) on solid medium. Thus, K r can be used to assess the effects of water activity on mould growth. Growth yield decreased linearly with decrease in water activity, and a decrease in water activity from 0·985 to 0·958 caused an approximately sixfold increase in the yield of blastospores per unit biomass dry weight.

The changes in bacterial density, total extracellular protein and activities of three extracellular enzymes were monitored during growth of wild-type Staphylococcus simulans biovar staphylolyticus, a representative pleiotropic variant that produced decreased levels of the three extracellular enzymes, and a variant that produced only 5 of the 14 extracellular proteins secreted by the wild-type organism. Both variants produced less total extracellular protein than did the parental organism. SDS-PAGE of the proteins secreted by these hypoproducing variants showed that all of the extracellular proteins were produced in decreased amounts. No pleiotropic compensation in extracellular protein production was observed for these hypoproducing variants of S. simulans biovar staphylolyticus.

Growth of Clostridium pasteurianum was inhibited in media of high solute content. At equal osmolarities, permeant solutes (glycerol and acetamide) were much less growth-inhibitory than non-permeant solutes (KC1 and xylitol). Glycolysis by washed cell suspensions was inhibited by these solutes in parallel with growth. However, in their inhibition of glucose 6-phosphate dissimilation by permeabilized cells the distinction between permeant and impermeant solutes was significantly less marked. The glucose phosphotransferase system (PTS) of intact cells was much more strongly inhibited by non-permeant than by permeant solutes. It was concluded that the predominant inhibitory effects on this organism of media of high solute content are due not to the low water activity of such media per se, but to the creation of an osmotic pressure across the bacterial cytoplasmic membrane, which acts to inhibit the glucose PTS by which the organism effects glucose uptake. Parallel measurements of the effects of xylitol on both glycolysis and the activity of the glucose PTS suggested that despite this correlation between the osmotic inhibition of growth, glycolysis and the PTS, the flux-control coefficient of the PTS on glycolysis did not exceed 0.2 under the conditions used.

Four coryneform bacteria were isolated from enrichments with 1-chlorobutane, 1-chloropentane or 1-chlorohexane as sole source of carbon and energy. One organism, strain HA1, was identified as an Arthrobacter sp. It could utilize at least 18 1-chloro-, 1-bromo- and 1-iodoalkanes, but not the 1-fluoroalkane tested. Substrate utilization was quantitative and growth yields of about 5.5 g protein (mol C)−1 were observed for hexanol and 1-chlorohexane with specific growth rates of 0·19 and 0·14 h−1, respectively. The organohalogen atom was released quantitatively as the halide ion. Cells grown on 1-chlorobutane contained an inducible enzyme(s) that dehalogenated haloalkanes. The enzyme was soluble, required no cofactors, membrane components or oxygen for activity, and released halide and the corresponding n-alcohol from the 1-haloalkane. The halidohydrolase(s) in crude extract had a specific activity of about 0·7 mkat (kg protein)−1 and dehalogenated a much wider spectrum of compounds (at least 29, including mid-chain and α,ω-dichlorosubstituted alkanes) than supported growth.

The methane-oxidizing bacterium Methylosinus trichosporium OB3b retains the capacity to express two types of methane mono-oxygenase (MMO) during growth on methanol in continuous culture. Soluble MMO predominates during copper-limited growth whilst particulate (membrane-bound) MMO activity is expressed fully only in copper-sufficient cultures. Organisms containing soluble MMO oxidized ethylbenzene (an artificial substrate for soluble but not particulate MMO) but this capacity was progressively lost when additional copper was made available to the organism, and MMO activity was transferred to the particulate fraction of cell-free extracts. Although intracellular location of MMO is apparently regulated by copper availability during growth on both methane and methanol, whole-cell MMO activities are appreciably higher during growth on the former (natural) carbon source.