- Volume 131, Issue 5, 1985

Protoplast fusion of Clostridium acetobutylicum P262 auxotrophs produced stable recombinants and segregating biparentals at frequencies of 0·3–2·0% and 1·4–8·3% respectively. Two novel classes of biparentals, partially-complementing and zero non-complementing, were observed.

Pseudomonas putida MT15 contains a large plasmid, pWW15, of about 250 kbp, which encodes the genes for toluene and xylene catabolism. Growth on benzoate selects strongly against the wild-type and results in the segregation of three phenotypically distinguishable mutant types. (1) B1 mutants, which have lost the complete plasmid. (2) B3 mutants, in which the plasmid has undergone a large deletion of about 90 kbp which appears to affect the regulation of the catabolic enzymes; these mutants retain the ability to grow on m-xylene and toluene (Mxy+ Tln+) but no longer grow on the metabolite of m-xylene, m-toluate (Mtol–). (3) A novel class not previously described, the B5 mutants, which still grow well on toluene but grow very poorly on m-xylene and do not grow on m-toluate (Mxy– Tln+ Mtol–). The B5 mutants appear to share the regulatory lesion of the B3 mutants but in addition do not express the xylF and xylG gene products, 2-hydroxymuconic semialdehyde hydrolase and 2-hydroxymuconic semialdehyde dehydrogenase. The plasmids in the B5 mutants have also undergone a deletion of about 90 kbp similar to, but distinguishable from, that in the B3 mutants.

Both B3 and B5 mutants can revert to growth on m-toluate. The revertants all show elevated constitutive levels of catechol 2,3-oxygenase, 2-hydroxymuconic semialdehyde dehydrogenase and 2-hydroxymuconic semialdehyde hydrolase which are not further induced by m-toluate. The reversion is accompanied by the tandem amplification of a region of 23–28 kbp on either side of the original deletion. As a result of Southern hybridizations, it was shown that the amplified region contains the structural genes of some of the enzymes which metabolize m-toluate but not the enzymes which convert m-xylene to m-toluate.

The successive steps in the isolation of mitochondria from chemostat-grown Candida utilis have systematically been investigated for their effects on organelle integrity.Growth rate had a profound effect on the susceptibility of carbon-limited cells towards Zymolyase, whereas the nature of the carbon source had no effect. Stabilization of spheroplasts with at least 2m-sorbitol was required to prevent premature lysis. This was concluded from the amounts of glucose-6-phosphate dehydrogenase liberated during Zymolyase treatments. The influence of the method for disruption of spheroplasts on the quality of the mitochondria was analysed with particular emphasis on respiratory control values and the distribution of marker enzymes among the cell fractions. Disruption by osmotic shock resulted in mitochondria without respiratory control and a high degree of solubilization of NADH and NADPH dehydrogenase activities. Only a gradual decrease of the osmotic value of the medium, preferably by dialysis against a hypotonic buffer, in combination with mechanical disruption with a Potter–Elvehjem homogenizer yielded mitochondria with high respiratory control values and a high retention of NADH dehydrogenase in the organelle. It is concluded that, for the quality of mitochondrial preparations from yeasts, the distribution of NADH dehydrogenase among the cell fractions is a more reliable measure than that of the usual marker enzymes.

Mitochondria were isolated from Candida utilis CBS 621 grown in carbon-limited continuous cultures on glucose, gluconate, xylose, ethanol or acetate as the carbon source and ammonia or nitrate as the nitrogen source. In all cases mitochondria were isolated which could oxidize exogenous NADH and NADPH via a cyanide- and antimycin A-sensitive but rotenone-insensitive respiratory chain. Oxidation of NADH and NADPH was coupled to energy conservation as evidenced by high respiratory control values. Different respiratory control values of mitochondria with NADH and NADPH as well as variations in the ratio of NADH and NADPH oxidase activities indicate that separate systems exist for the oxidation of exogenous redox equivalents by mitochondria of C. utilis.

Variation of the NADPH requirement for biomass formation by applying different growth conditions did not result in significant changes in NADPH oxidase activities of mitochondria. It is concluded that in C. utilis NADPH can be used in dissimilatory processes for the generation of ATP.

Three d-glucan-hydrolysing enzymes from Bacteroides oralis Ig4a have been isolated. Two of them are dextranases which hydrolyse (1→6) but not (1→3) linked α-d-glucans; one (EC 3.2.1.11,1,6-α-d-glucan 6-glucanohydrolase) is localized in the periplasm, and the other, which is an exo-enzyme (EC 3.2.1.70,1,6-α-d-glucan glucohydrolase), in the cytoplasm. The third is a mutanase (EC 3.2.1.59, 1,3-(1,3;1,4)-α-d-glucan 3-glucanohydrolase) that hydrolyses (1→3) but not (1→6) linked α-d-glucans, and is present only in the cytoplasm.

A selection procedure for uracil catabolism mutant strains involving indicator dye plates was developed. Using this method, a strain defective in uracil catabolism has been isolated in Salmonella typhimurium that was temperature-sensitive at 42°C where it required low concentrations of N-carbamoyl-β-alanine, β-alanine or pantothenic acid for growth. An extract of the mutant strain degraded uracil at 37°C at a significantly diminished rate compared to that observed for the wild-type strain under the same growth conditions. The conversion of dihydrouracil to N-carbamoyl-β-alanine was blocked at all temperatures examined in the mutant strain. By means of genetic analysis, the mutant strain was determined to be defective at two genetic loci. Transduction studies with bacteriophage P22 indicated that the panD gene is mutated in this strain, accounting for its β-alanine requirement. Episomal transfers between Escherichia coli and the mutant strain provided evidence that the defect in uracil catabolism was located in another region of the S. typhimurium chromosome.

Immunofluorescence staining with fimbria- specific antibodies was used to study the organization of fimbriate cells in colonies of Escherichia coli strain 3040. The strain has both type-1 and S fimbriae and shows fast phase variation between the fimbrial types. Colonies stained in sectors whose length and number per colony were dependent on the fimbrial phase of progeny cells. It is proposed that such sectors result from fimbrial phase variation.