- Volume 127, Issue 2, 1981

Mycobacterium leprae separated from armadillo tissues stored at -80 0C is similar to that from human sources in its ability to take up 3H-labelled 3,4-dihydroxyphenylalanine (DOPA). Several inhibitors were studied which showed complete or partial inhibition of [3H]DOPA uptake. These findings suggest that M. leprae isolated from frozen tissue possesses an active uptake system for [3H]DOPA.

The isolation of cell surface mutants of Acinetobacter calcoaceticus RAG-1 was demonstrated by a method based on the differential immunoprecipitating properties of wild-type cells and mutants. In the presence of antiserum, about 99% of wild-type cells in suspension sedimented following overnight agglutination. The kinetics of antiserum-induced agglutination was quantified by measuring the time-dependent decrease in turbidity. For mutant isolation, a sample of the cells remaining in the supernatant fluid served as inoculum for a fresh culture. This enrichment procedure of sequential growth and agglutination was repeated until the supernatant fluid following the overnight agglutination step remained turbid. Of the 40 colonies isolated in this manner, 34 exhibited altered agglutination properties.

The agglutinability of wild-type cells varied with the phase of growth - cells harvested during the exponential phase were agglutinated better than those in stationary phase. In contrast, the antiserum-induced agglutination of most mutants was poor during the early phases of growth, but improved during late-stationary phase. One mutant failed to agglutinate in the presence of antiserum irrespective of the phase of growth.

Differentiation of Dictyostelium discoideum cells in suspension culture is reported, using a medium containing glucose, albumin, cyclic AMP, EDTA and streptomycin in a phosphate buffer. Production of UDPgalactose: polysaccharide transferase, an enzyme specifically present in prespore cells, and the formation of prespore-specific antigens in more than 60% of the cells, are demonstrated. Differentiation in this medium differs from that previously reported with other suspension systems in that (a) cells form only small, amorphous agglomerates, (b) there is an absolute requirement for cyclic AMP and (c) prior formation of loose cell mounds on a solid substratum is essential for subsequent differentiation in this medium. This last requirement indicates that the differentiation process, giving rise to the prespore-specific enzyme and antigen, can be resolved into two distinct stages, one requiring cell contact on a solid substratum and the other proceeding in small agglomerates incubated in the medium. This medium may be useful for elucidating the role of cyclic AMP and cell contact in slime mould development.

A multistage continuous culture system is described in which solutes are transferred between vessels in opposite directions simultaneously. The system, called a gradostat, produces opposing solute gradients and is a good laboratory model of many natural microbial ecosystems in which solute gradients are important. Theoretical predictions concerning solute transfer were confirmed under steady-state and non steady-state conditions, using a coloured dye. Paracoccus denitrificans grew anaerobically in the gradostat at the intersection between opposing gradients of succinate and nitrate. Opposing gradients of glucose and oxygen separated the growth of a Bacillus sp. (a facultative anaerobe) and Clostridium butyricum (an obligate anaerobe). Viable counts for both species fell exponentially away from their growth positions at the ends of the gradostat. The potential value of the gradostat and possible alternative conformations are discussed.

Gradients of nutrients are extremely common in nature, and this paper decribes changes in the physiology of Escherichia coli grown in the gradostat, a series of five linked vessels with opposing gradients of glucose and of oxygen plus nitrate. Most growth occurred at the aerobic and anaerobic ends of the system. High rates of respiration, high energy charge and high activities of various oxidative enzymes were seen in the two most aerobic vessels; however, oxygen provision was presumably poor, because nitrate reductase activities were also high in this region. Vessels 3 and 4 showed the lowest values for respiration rate, enzyme activity and energy charge, and cells here were both nutrient starved and possibly inhibited by nitrite. Vessel 5 was highly anaerobic, resulting in the presence of hydrogenase activity. It was concluded that cells found in different regions of the gradostat had undergone biochemical differentiation in spatial gradients of electron donors and acceptors.

The structured nature of microbial ecosystems makes their study difficult, and simple laboratory analogues are needed. Two gel-stabilized gradient systems are described in which solute transfer is by diffusion alone. In the first, organisms grow in a semi-solid agar gel located above a source layer of full-strength agar containing the diffusible solute, which was glucose in the experiments reported here. Changes in physicochemical parameters, various solutes and cell concentration have been monitored in cultures of Bacillus megaterium NCTC 10342 and Lactobacillus confusus NCIB 4037 grown in this system. In experiments with a range of bacteria, banded growth was noted for several strains. The microbiology of the water at the base of an oil storage tank was investigated in the second model, in which gas oil was poured over a semi-solid layer containing agar, a basal salts medium, cells and a steel plate. After incubation for up to 90 d the system had differentiated into aerobic and anaerobic regions, and activities included hydrocarbon catabolism, oxygen removal, sulphate reduction, and the growth of a large population of aerobic and anaerobic heterotrophs. The value of these models is discussed with reference to microbial ecology.

Mycobacterium leprae separated from armadillo tissues stored at −80 °C is similar to that from human sources in its ability to take up 3H-labelled 3,4-dihydroxyphenylalanine(DOPA). Several inhibitors were studied which showed complete or partial inhibition of[3H]DOPA uptake. These findings suggest that M. leprae isolated from frozen tissuepossesses an active uptake system for [3H]DOPA.

Mutants of Dictyostelium discoideum that developed huge aggregation streams in expanding clones were investigated using optical and biochemical techniques. Representatives of the six complementation groups previously identified (stmA-stmF) were found to be similar to the parental wild-type strain XP55 in both the extent and timing of their ability to initiate and relay chemotactic signals and in the formation of cyclic AMP receptors and phosphodiesterases. The mutants differed from the wild-type in producing an abnormal chemotactic (movement) response visible using both dark-field optics with synchronously aggregating amoebae on solid substrata and light scattering techniques with oxygenated cell suspensions. Mutants of complementation group stmF showed chemotactic movement responses lasting up to 520 s, rather than 100 s as seen in the parental and other strains. Measurements of cyclic GMP formed intracellularly in response to chemotactic pulses of cyclic AMP in stmF mutants showed that abnormally high concentrations of this nucleotide were formed within 10 s and were not rapidly degraded. A causal correlation between defective cyclic GMP metabolism and the altered chemotactic response is suggested, and a model is proposed that accounts for the formation of huge aggregation streams in clones of these mutants.

The association of the haemagglutinating activities of Salmonella typhimurium cultures with bacterial adhesion to HeLa cells, and the internalization of the bacteria by HeLa cells, was studied. Adhesion was not inhibited by α-methyl-d-mannoside (i.e. adhesion was mannoseresistant), and only four of the six strains tested produced type 1 fimbriae and the associated mannose-sensitive haemagglutinin (MSHA). The other two strains belonged to the non-fimbriate FIRN biogroup. Cultures of all six strains contained a mannose-resistant haemagglutinating (MRHA) activity when grown at 37 °C, but cultures of only one fimbriate and one non-fimbriate strain did so when grown at 18 °C. From the comparison of cultures grown at 18 °C and 37 °C, and of mutant strains with the phenotypes MRHA-negative/MSHA-positive, or MRHA-positive/MSHA-negative, it was concluded that the MRHA activity was responsible for the attachment of salmonellae to HeLa cells. Only bacterial adhesion that was resistant to mannose resulted in the internalization of the bacteria by the HeLa cells.

Three cutaneous propionibacteria - Propionibacterium acnes, P. avidum and P. granulosum - were grown in chemostats using semi-synthetic medium with various concentrations of glucose. Biomass rose with increasing glucose concentration up to 0·3–0·4% (w/v) and then remained constant. Propionibacterium granulosum had both a low yield and μ max when grown in the absence of glucose suggesting that this organism is essentially ‘saccharolyti’ in its nutrition. In contrast, P. acnes and P. avidum had higher growth yields than P. granulosum and showed their highest μ max values in the absence of glucose. Extracellular lipase, hyaluronidase (hyaluronate lyase) and acid phosphatase activities varied with different glucose concentrations, but in all cases were lowest at the highest glucose concentration tested (0·5%, w/v).

Five strains of Streptococcus mutans were grown in continuous culture with either a limited supply or an excess of glucose. Proteins secreted into the extracellular fluid by strains C67–1, 3209 and K1 rapidly catalysed the synthesis of insoluble glucan from sucrose (mutansucrase activity). The culture fluid from strains Ingbritt or C67–25 catalysed the synthesis of soluble glucan (dextransucrase activity) and fructan, but little or no mutansucrase activity was detected. The strains which secreted active mutansucrase readily colonized a smooth hard surface during growth in batch culture and were more cariogenic in pathogen-free rats than those which secreted little mutansucrase activity. There was no similar correlation between fructosyltransferase, dextransucrase or total glucosyltransferase activity and either adherence or cariogenicity. We conclude that the ability to catalyse insoluble glucan synthesis is a major determinant of the cariogenicity of S. mutans strains.

Mitochondria from conidia of Neurospora crassa were isolated and purified by a simple and rapid technique. Electron micrographic analysis and isopycnic centrifugation on a sucrose density gradient revealed a heterogeneous population.

Succinate, citrate, 2-oxoglutarate, NADH and ascorbate + tetramethyl-p-phenylenediamine (TMPD) were oxidized by the mitochondria. Oxygen consumption was totally inhibited by antimycin A or cyanide. The electron transport chain was coupled to sites 1 and 2 of phosphorylation. Oligomycin, atractyloside and 2,4-dinitrophenol prevented energy coupling.

A Hyphomicrobium sp. capable of growth on dimethyl sulphide and dimethyl sulphoxide was isolated from aerobic enrichment cultures containing dimethyl sulphoxide as the carbon and energy source. Suspensions of cells taken from a dimethyl sulphoxide-limited chemostat oxidized dimethyl sulphide, methanethiol, formaldehyde, formate and thiosulphate. Enzyme studies indicated that the pathway of dimethyl sulphoxide metabolism involves an initial reduction to dimethyl sulphide, which is subsequently oxidized by an NADH-dependent mono-oxygenase to formaldehyde and methanethiol. Further oxidation of methanethiol is by a hydrogen peroxide-producing oxidase, again resulting in the production of formaldehyde. Extracts of dimethyl sulphoxide-grown cells also contained high levels of catalase as well as NAD+-dependent formaldehyde and formate dehydrogenases. The organism probably used the serine pathway for growth on dimethyl sulphoxide. This was indicated by the presence of high activities of hydroxypyruvate reductase in dimethyl sulphoxide-grown cells.

l-Proline entered both mycelial and yeast cells of Candida albicans by an active transport system of high specificity at low (< 0·1 mm) external concentrations of substrate. The apparent K m value of this system was 0·1 mm for both types of cells, while the V value was 4 nmol min−1 (mg dry wt)−1 for mycelial cells and 1·4 nmol min−1 (mg dry wt)−1 for yeast cells. At l-proline concentrations greater than 0·1 mm, the amino acid appeared to enter both morphological forms by diffusion as well as active transport. As saturation was approached diffusion became increasingly important. The higher uptake rate of mycelial cells seemed not to be the result of an inducible system. The optimal pH and temperature for transport of l-proline were 7·0 and 37 °C, respectively. Sodium azide and the proline analogues sarcosine and l-azetidine-2-carboxylic acid inhibited l-proline uptake, while l-thiazolidine-4-carboxylic acid was less effective. The active transport system was highly specific for l-proline since neither ammonium ions, which inhibit the general amino acid transport system of fungi, nor 16 different amino acids interfered substantially with uptake.