- Volume 115, Issue 2, 1979

Pigmentation mutants of Mycobacterium aurum were isolated after chemical mutagenesis. Examination of the pigments extracted from these mutants indicated that at least 15 carotenoids were formed. β-Carotene was not detected and the major carotene of M. aurum appeared to be leprotene. The possible biosynthetic pathway is discussed on the basis of these results.

Butirosin, an aminoglycoside antibiotic, is produced by Bacillus circulans B-3312. Experiments using recombined ribosomal and supernatant fractions from this strain and from B. megaterium KM have shown that the ribosomes of both are sensitive to butirosin. The aminoglycoside 3′-phosphotransferase present in B. circulans modifies butirosin and neomycin in vitro but confers resistance only to the former in vivo. The phosphotransferase does not modify a detectable amount of extracellular butirosin while mediating resistance to the antibiotic. In vitro, however, the enzyme appears to protect against inhibition by butirosin by inactivating the bulk of the antibiotic in the system. An extrachromosomal element of unknown function has been detected in B. circulans.

Hyaluronidase from Propionibacterium acnes has been purified 13000-fold from the culture supernatant to homogeneity (as determined by polyacrylamide disc gel electrophoresis). The molecular weight of the purified enzyme was 85110 as determined by gel filtration. The purified enzyme had a pH optimum at 6·4, was stable between pH 5 and 5·8 and was completely inactivated after 15 min at 50 °C. Preliminary studies suggested that the enzyme is active against chondroitin 4- and 6-sulphates, but not against dermatan sulphate. Analysis by paper chromatography of the reaction products from the degradation of hyaluronic acid by bacterial, testicular and P. acnes enzymes suggested that the P. acnes enzyme is similar in its mode of action to other bacterial hyaluronate lyases. The enzyme from P. acnes may thus be tentatively classified as a hyaluronate lyase.

Eight mutants of Paracoccus denitrificans deficient in c-type cytochrome were detected by their inability to catalyse the conversion of α-naphthol plus dimethyl-p-phenylenediamine to indophenol. From the properties of two of these mutants which completely lacked spectrophotometrically detectable c-type cytochrome, evidence was provided for the presence of a respiratory pathway which terminates in a cytochrome o-like oxidase. The presence of this pathway allows mutants lacking c-type cytochrome to grow aerobically, and also explains many of the non-mitochondrial features of the aerobic respiratory chain of the wild-type. None of the mutants had significantly lower levels of b-type cytochromes compared with the parental strain, but two mutants were deficient in cytochrome aa 3. All of the mutants could reduce nitrate, but four of them were unable to denitrify and these showed poor growth with nitrate as added terminal electron acceptor.

Pili from Bacteroides nodosus were purified to greater than 99 % homogeneity by precipitation at pH 4·0 and in MgCl2 followed by chromatography on BioGel A150. The pili were composed entirely of one type of polypeptide subunit, pilin. No carbohydrates, nucleic acid, lipid, lipopolysaccharide or phosphate could be detected in purified pili preparations. The molecular weight of pilin from B. nodosus strains 91B and 198 was 18400 and from strain 80 was 19300. The isoelectric points of pili from B. nodosus strains 91B and 80 were both 4·5. The buoyant densities of pili from strains 91B, 80 and 198 were 1·287, 1·284 and 1·286 g ml−1, respectively. The three strains of B. nodosus did not cross-react in K-agglutination tests and produced pili which did not cross-react in immunodiffusion tests. Antiserum to highly purified pili caused a characteristic K-type agglutination reaction. It was concluded that pili are the K-agglutinogen.

A class of aggregation pattern mutants called ‘streamers’ have been isolated from Dictyostelium discoideum and analysed genetically. The streamer phenotype is the formation of very large streams of centripetally moving amoebae which are collected from abnormally large territories during the aggregation phase of this organism. Such mutants do not show the pleiotropic developmental defects seen with most other classes of aggregation mutants and after the abnormal aggregation phase they develop into normally differentiated stalk cells and spores. Twenty-four haploid streamers were isolated and assigned to seven complementation groups, stmA to stmG, after selecting diploids formed between pairs of the mutants. The complementation loci were assigned to the following linkage groups using parasexual genetic techniques: stmA and stmF, linkage group VII; stmB, stmD and stmG, linkage group II; stmC and stmE, linkage group III. Use was made of a new temperature sensitive for growth marker, tsgK21, which was assigned to linkage group VII. The total number of complementation groups giving the streamer phenotype is estimated from statistical calculation, based on the frequency of allelism, to be between seven and nine.

A small defective generalized transducing bacteriophage (CA3) has been detected in culture supernatants of the nitrogen-fixing hydrogen bacterium Xanthobacter autotrophicus GZ29. This phage had a head diameter of 37 to 43 nm, and it had a low specific density of 1·349 g cm−3, probably due to a small DNA molecule of 3·3 ×106 molecular weight. The phage did not form plaques on any of the X. autotrophicus strains tested and therefore was detectable only by its transducing activity and by electron microscopy. All genetic markers tested were transducible at frequencies of about 10−4 per marker per phage particle. No cotransduction of markers was detected. Due to the high transduction rate and the small size of the DNA molecule, it is assumed that CA3 particles contain mainly or exclusively chromosomal DNA.

A new temperate phage, R4, of Streptomyces was isolated from soil on a restriction- deficient mutant of S. albus G. In its morphology, adsorption properties and growth kinetics R4 resembled other temperate phages of Streptomyces though its requirements for Ca2+ and Mg2+ were higher than usual. It was unable to form plaques above 34·5 °C. R4-mediated transduction was not detected. Unlike other Streptomyces temperate phages, R4 had a wide host-range, which correlated better with the absence of detectable class II restriction enzymes than with conventional taxonomic divisions. Many of the sensitive strains [but not, apparently, S. coelicolor A3(2)] could be lysogenized.

With the wild-type R4, plaques were obtained on S. albus G only after growth on a restriction-deficient, modification-proficient mutant, and then only at a very low efficiency of plating. All of these plaques were of a mutant type (R4G) which (unlike the parental R4 phage) showed conventional patterns of restriction-modification in the S. albus G (Sal GI) and S. albus P (5a/PI) systems. R4G mutants, but not R4, were sensitive to a restriction- modification system present in two S. rimosus strains (2251 and NRRL 2234). DNA from SWGI-unmodified (but not from modified) R4 or R4G was cleaved by SalGI into more than 30 fragments (mean size 1·35 kilobases; summed molecular weight 30·02 × 106). R4 DNA was cleaved at one site by EcoRI, at one site by SalPI (=PstI), and not at all by HindIII or BamHI.

The mating reaction in Tetrahymena thermophila includes a starvation period and two distinct cell interactions, co-stimulation and cell pairing, before the cells are cytoplasmically joined as conjugants. A selection procedure for harvesting mutants unable to mate at a restrictive temperature has been developed. A conjugant pair consisting of one cycloheximide-resistant cell and one wild-type cell (cycloheximide-sensitive) was itself sensitive to the drug. By adding cycloheximide and nutrient medium to a cross made at the restrictive temperature, only cycloheximide-resistant cells that did not successfully conjugate could survive and grow. Repetition of the selection procedure enriched for cells unable to conjugate at the restrictive temperature. The selected cells were able to grow at 38 °C and could conjugate at 28 °C. This procedure may be narrowed to select specifically for cell interaction mutants.

Using N-methyl-N′-nitro-N-nitrosoguanidine, ultraviolet irradiation, ethyl methanesul- phonate or 4-nitroquinoline-1-oxide mutagenesis and an enrichment method for the isolation of auxotrophs, 25 mutants with defects in the adA locus were obtained after screening 41376 colonies. One of these, adA24, did not complement with any of the other adA mutants, had a very high reversion rate and had some other properties which usually characterize strains carrying nonsense mutations. All revertants of adA24 carried dominant suppressor mutations. A group of adA24 suppressors was tested for allele and locus specificity. They were found to suppress only some adA alleles, and at the same time, some mutations in the methG, methH, argB and proA loci. It is proposed that the allele specific and locus nonspecific adenine suppressors are suppressors of nonsense mutations.

Efficient and reproducible cell lysis and the isolation of plasmid DNA from nine phytopathogenic Corynebacterium species is described. One to three plasmids, ranging from 23 to 77 megadaltons, were recovered from each strain, as detected by comparative agarose gel electrophoresis. Corynebacterium michiganense showed considerable plasmid diversity. Other species, such as C. nebraskense and C. insidiosum, did not. Bacteriocin production, colony morphology, pigmentation and virulence were not correlated with the presence of plasmids. Sedimentation through neutral and alkaline sucrose gradients and electrophoretic separation on agarose gels gave similar values for plasmid molecular weights. Log-linear alkaline sucrose gradients proved useful for molecular weight determinations and also for the isolation of purified plasmids on a preparative scale.

The cyanobacterium Anacystis nidulans grown in light-limited and CO2-limited chemostat cultures showed varying rates of CO2 fixation with peaks at dilution rates of 0·10 to 0·12 h−1. The specific activities of a number of enzymes of the reductive and oxidative pentose phosphate and glycolytic pathways and the tricarboxylic acid and glyoxylate cycles varied significantly as a function of the growth environment (substrate limitation) and organism growth rate. Ribulose-1,5-bisphosphate carboxylase varied 15-fold under CO2-limited conditions but did not change under light-limited conditions. With the exception of phosphoribulokinase, all enzymes which showed a change in specific activity increased with decreasing dilution rate. The specific activities of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, hexokinase, ribulose-1,5-bisphosphate carboxylase and malate dehydrogenase were significantly higher in organisms grown under CO2-limited conditions than under light-limited conditions. Fructose-1,6-bisphosphate aldolase and phosphoribulokinase specific activities were similar at all growth rates and under both limitations. Isocitrate lyase was the only enzyme examined which showed higher specific activities under light-limited conditions. Thus Anacystis nidulans can selectively express different enzymes, possibly by transcriptional control.

The outcome of competition in thiosulphate-limited chemostat culture between the obligate chemolithotroph Thiobacillus neapolitanus and the versatile facultative autotroph Thiobacillus A2 was in part a function of pH. In pure culture T. neapolitanus grew faster than Thiobacillus A2 at pH values up to pH 7·6, but in competition Thiobacillus A2 dominated at pH 7·35 and 7·6. At pH 7·1, T. neapolitanus dominated, although a significant steady state population of Thiobacillus A2 persisted, apparently growing on organic nutrients excreted by T. neapolitanus. Coexistence of both organisms occurred under all chemolithotrophic growth conditions tested with the dominant organism comprising 85 to 99% of the population, indicating that competition was not the sole interaction between the species. At pH 7·1, the inclusion of glucose in the thiosulphate medium resulted in rapid domination of the culture by Thiobacillus A2, with the virtual elimination of T. neapolitanus. It is concluded that the capacity for mixotrophy is a selective advantage to a facultative thiobacillus in competition with an obligately chemolithotrophic species.

The spd 1 mutants of Saccharomyces cerevisiae are affected in the initiation of sporulation since they sporulate under conditions in which the wild-type does not. They are unable to grow on a range of non-fermentable carbon substrates, but can grow on ethanol. On others, including acetate, glycerol, lactate and pyruvate, the mutants sporulate abundantly, even in rich media. The mutation does not affect the uptake of glycerol or the synthesis of the enzymes concerned with its entry into general metabolic pathways. It probably affects a central metabolic function concerned with the metabolism of some, but not all, non-fermentable carbon sources. The spd 1 mutation is also expressed in haploids. When haploid or diploid cells are transferred to media containing only non-fermentable carbon substrates they become arrested in the G1 phase of the cell cycle before, or at, the execution point for the cdc 28 mutation. In diploids homozygous for the spd 1 gene these conditions also lead to a lowering of the repression by the nitrogen source. The spd 1 mutants are therefore probably not affected directly in events unique to the initiation of sporulation, but in areas of metabolism closely connected with both the arrest of cells in the G1 phase of the cell cycle and the control of induction of sporulation.