- Volume 132, Issue 6, 1986

A reducible hydroperoxidase, haemoprotein b-590, has been purified 16-fold from a soluble fraction of Escherichia coli K12, grown anaerobically with glycerol and fumarate. The M r of the native protein, determined by gel filtration, was 331000 although a minor, smaller species with a M r of 188000 was also detected; both had catalase activities. Based on the subunit M r, determined from SDS gel electrophoresis to be 75000, the above species are tentatively identified as tetramers and dimers, respectively. The isoelectric point of both species was 4·4. The absorption spectrum of the isolated haemoprotein is typical of ferric, high-spin haem. The A 405/A 280 ratio never exceeded 0·27, a value half of that obtained for E. coli hydroperoxidase I. On reduction with dithionite, the ?, ?, and ? bands were at 441, 559 and 590 nm respectively, the ?-band being unusually distinct. Treatment of the reduced form with CO gave a sharp prominent ?-band at 426 nm and caused significant shifts of the ? and ? bands to shorter (574 and 545 nm) wavelengths.

The pyridine haemochrome spectra showed the haem to be protohaem IX; the spectra were featureless between 580 and 630 nm, thus excluding the presence of haem a. However, some features of the difference spectra of the haemoprotein were reminiscent of cytochrome a 1, notably the maxima in reduced minus oxidized spectra at 444 and 593 nm and the peaks and troughs in CO difference spectra at 426 and 446 nm respectively. The haemoprotein had high catalase activity: V max was 2·3 × 106 mol H2O2 (mol haem)?1 min?1 and the K m was 11 mM. At 10 mM-H2O2 the first order rate constant was 0·3 × 107 M?1 s?1. The haemoprotein was also a peroxidase with o-dianisidine or 2,3’,6-trichloroindophenol as substrates; for the latter substrate, the K m was 0·18 mM. It is concluded that haemoprotein b-590 strongly resembles the hydroperoxidase I purified by Claiborne & Fridovich (Journal of Biological Chemistry 254, 4245-4252, 1979) and that a similar haemoprotein was mistaken for a cytochrome a 1 b complex by Barrett & Sinclair (Abstracts of the 7th International Congress of Biochemistry, Tokyo, H-107, p. 907, 1967).

A mutant of Saccharomyces cerevisiae highly resistant to 2-amino-4-methyl-5-?-hydroxyethylthiazole (2-aminohydroxyethylthiazole), an antimetabolite of 4-methyl-5-?-hydroxyethylthiazole (hydroxyethylthiazole), has been isolated. Its resistance to 2-aminohydroxyethylthiazole was about 104 times that of the sensitive parent strain. The amount of thiamin synthesized in the cells of the resistant strain grown in minimal medium was less than half of that of the sensitive strain. The ability to synthesize thiamin from 2-methyl-4-amino-5-hydroxymethylpyrimidine (hydroxymethylpyrimidine) and hydroxyethylthiazole in the resistant strain was low compared with that of the sensitive strain. These results were found to be due to a deficiency of hydroxyethylthiazole kinase in the resistant strain: in sonic extracts of cells the enzyme activity was only 0·67% of that of the sensitive strain. Although the cells of the sensitive strain could accumulate exogenous hydroxyethylthiazole in the form of hydroxyethylthiazole monophosphate, no significant uptake of hydroxyethylthiazole by the cells of the resistant strain was observed. The possibilities that 2-aminohydroxyethylthiazole monophosphate may be the actual inhibitor of the growth of Saccharomyces cerevisiae, and that hydroxyethylthiazole may not be involved in the pathway of de novo synthesis of thiamin via hydroxyethylthiazole monophosphate, are discussed.

The structures of the mycolic acids from the cell wall skeleton of Rhodococcus lentifragmentus AN-115 were established. Field desorption mass spectrometry of underivatized mycolic acids showed that the molecular species of the mycolic acids were distributed from C38 to C50 and the major components were C44, C46 and C48 mono- and dienoic ?-hydroxy fatty acids. Pyrolysis gas chromatography-mass spectrometry confirmed that the mycolic acids consisted of a saturated straight C12 and C14 ?-chain and an unsaturated ?-chain, having mainly C29 and C31 carbons with one or two double bonds. The double bonds in the ?-chain were established as the cis isomers by nuclear magnetic resonance. The absolute configurations of the ?- and ?-asymmetric carbons were both established as R using molecular rotation data.

The modifier protein (M-protein) for methanol dehydrogenase (MDH) of the obligate methylotroph Methylophilus methylotrophus was purified almost to homogeneity (98%pure), characterized and shown to be similar to that previously described in the facultative methylotroph Pseudomonas AM1. It was a dimer of M r 140000, its isoelectric point was 5·6 and it showed no spectral absorbance above 320 nm. In the dye-linked MDH assay system, the M-proteins facilitated oxidation of alcohols not normally oxidized (1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol and 3-methylbutanol) by increasing the affinity of MDH for these alcohols. The effect of M-protein on the oxidation of formaldehyde was to decrease the affinity of MDH for formaldehyde by more than 97%, thus preventing any significant oxidation of formaldehyde. The M-protein also exerted its effect on the activity of MDH in the cytochrome-linked system of M. methylotrophus. The usual function of M-protein in methylotrophs is apparently to prevent formaldehyde oxidation. A dye-linked aldehyde dehydrogenase of wide specificity was partially purified and characterized; it failed to oxidize formaldehyde at a significant rate but it oxidized lactaldehyde at a rate sufficient to account for the oxidation of propanediol to lactate in whole bacteria.

Yeasts and mycelia of the pathogen Candida albicans grown in the presence of polyoxin D, a competitive inhibitor of chitin synthase, formed chains of swollen bulbous cells as observed by fluorescence microscopy. Wheat germ agglutinin (WGA) complexed to colloidal gold (Au) was used as a specific label at the ultrastructural level to visualize chitin in walls of control and polyoxin-treated cells. In control cells, Au-WGA labelling was preferentially localized in the innermost wall layers and was predominant at bud scars and septa. After 4·5 h in 4 mM-polyoxin D, budding in yeasts and lateral wall growth in mycelia continued, but primary septa failed to form and no Au-WGA labelling was detected in the walls. These results demonstrated that the morphological alterations caused by polyoxin D were due to the absence of chitin, a wall component important for formation of primary septa and for maintenance of structural integrity during morphogenesis.

Rhodotorucine A is a mating pheromone produced by mating type A cells of Rhodosporidium toruloides which induces mating tube formation in mating type a cells. Rhodotorucine A induced conspicuous changes in Ca2+ fluxes in the target cells which consisted of a very rapid, transient Ca2+ influx immediately after pheromone addition and a gradual Ca2+ accumulation during mating tube elongation. The magnitude of the pheromone-induced changes of Ca2+ fluxes was correlated with the efficiency with which the pheromone elicits biological responses in mating type a cells. Compared to vegetative growth, the response to the pheromone was highly sensitive to trifluoperazine, an inhibitor of calmodulin. Trifluoperazine inhibited exclusively the emergence and the elongation of mating tubes but not the signalling reaction.

Self-sporulating diploid and aneuploid cells were identified in the life cycle of Rhodosporidium toruloides by microscopic observation of nuclear behaviour, microphotometry of DNA content, and genetic characterization of progeny. From a sexual cross between haploid A- and a-type strains, diploid progeny with both A and a mating-type loci and aneuploid progeny with either A or a mating-type locus were isolated in addition to haploid progeny. The diploid isolates propagated by budding in yeast form and eventually developed monokaryotic hyphae. The diploid hyphae formed uninucleate teliospores and blastospores from which diploid yeast cells were isolated (self-sporulating). Aneuploids carrying the A or a mating-type isolated from a sexual cross and from self-sporulating diploids entered the sexual cycle like haploid cells. The life cycle consisting of sexual and self-sporulating cycles is presented.

Colonies of Microcystis in Abbots Pool, Avon, UK, were found to regulate their buoyancy according to light (photon flux density). The autumnal decline of the population was associated with an increase in the proportion of colonies that were non-buoyant, and with declining temperatures in the pond. Non-buoyant colonies taken from the pond regained buoyancy in the dark rapidly at 20°C but only slowly at 12°C and below. A laboratory strain of Microcystis behaved in a similar manner. Comparisons of the behaviour of this organism placed at 8°C and 20°C were made; in high photon flux density buoyancy was lost at both temperatures due to accumulation of dense carbohydrate. When transferred to the dark cells at 20°C became buoyant again as carbohydrate was utilized and more gas vesicles were made; at 8°C much less carbohydrate was used and no increase in gas-vacuolation occurred. The failure to regain buoyancy in the dark at low temperatures accounts for the loss of buoyancy and sedimentation of the Microcystis in Abbots Pool.

Summary: Some characteristics of the lytic development of the temperate phage ϕC31 in Streptomyces coelicolor A3(2) were studied using a thermoinducible lysogen. The physiological state of the host and the culture medium influenced the production of progeny virus after induction. The latent period lasted 45 min and the rise period 20-30 min. RNA synthesis in induced cultures was reduced with respect to controls. This reduction was restricted to cellular transcription as evidenced by: (i) no stable RNA being synthesized in induced cultures, and (ii) the proportion of phage specific RNA increasing from 0·5% before induction to more than 30% in induced cultures. Host RNA synthesis proceeded throughout the lytic cycle. Protein synthesis was also reduced in induced cultures, although to a lesser extent than RNA synthesis. Phage DNA synthesis started at around 10 min postinduction, marking the division between the early and late periods of phage development. Host DNA synthesis occurred during the first 20 min after induction, and gradually decreased later.

The RecE protein of Bacillus subtilis, known to be required for induction of the SOS response and of ϕ105 prophage, was shown to be involved in mitomycin C induction of B. subtilis diploid lysogens carrying a silent ϕ105 prophage in their unexpressed chromosome. These stable non-complementing diploid lysogens, formed by protoplast fusion and regeneration, did not synthesize repressor, so that the induction observed must have resulted from RecE-dependent activation of the prophage rather than from RecE-dependent inactivation of repressor. Mitomycin C treatment does not induce permanent expression of the silent chromosome, so the activation seems to be temporary, perhaps reflecting the action of an SOS function under RecE control.

Twenty-four thermophilic bacteriophages have been isolated from diverse sources such as compost, soil, silage and rotting straw. Although considerable individual host specificity was observed, the phages were able to infect most of the major taxonomic groups of Bacillus thermophiles. The phages varied considerably in morphology and size; the phage heads were either cylindrical or polyhedral with tails varying in length between 15 and 500 nm. Most of the phages were stable at 50°C for 4-5 h but at 70°C the plaque-forming units decreased by between 102- and 107-fold in 2 h. The DNA of morphologically similar phages was examined by restriction enzyme analysis, and some differences in the DNA fragment patterns were found. Efficiency of plating data indicated that ‘B. caldotenax’ has a restriction and modification system. These phages may be valuable for the study of the genetics of thermophilic bacilli: transduction of ‘B. caldotenax’ and ‘B. caldovelox’ by phage JS017 has been observed.

The majority of multiresistant Staphylococcus aureus strains isolated in Australian hospitals since 1970 carry a chromosomally-encoded minocycline and tetracycline resistance determinant. By using DNA-DNA hybridization, some of these multiresistant strains were shown to possess also a tetracycline resistance plasmid, equivalent to pT181, integrated into a unique site in the chromosome. By relating the hybridization data to the map of pT181, the site of integration on this plasmid was established to be between the genes for replication and tetracycline resistance.

The Aspergillus nidulans argB gene coding for ornithine carbamoyltransferase (OTCase) is not expressed in Escherichia coli. However, E. coli OTCase-deficient strains transformed with plasmids carrying the argB gene from A. nidulans reverted to prototrophy at a high frequency. In these derivatives the argB gene became functional due to DNA rearrangements upstream of the coding sequence. Two types of rearrangement were characterized. One was identified as an insertion of IS2. The second was a deletion that resulted in transcription of the argB gene from the TcR gene promoter and translation from a newly created ribosome-binding site formed at the junction between the A. nidulans and vector DNA sequences.

The Escherichia coli lacZ gene was stably introduced into ϕC31-based phage cloning vectors in Streptomyces lividans. However, lacZ could not be stably introduced into S. lividans on the plasmid vectors pIJ702 or pIJ41. Studies of the expression of lacZ in S. lividans and S. coelicolor were facilitated by the use of mutants and/or growth conditions in which endogenous β-galactosidase activity was low or absent. Plaques and lysogens of °C31::lacZ constructs involving transcriptional fusions to the pBR322 tet gene contained β-galactosidase activity. Activities were markedly higher in S. lividans than in S. coelicolor. Insertion of the major transcriptional terminator of coliphage fd between the tet promoter and lacZ reduced lacZ expression more in lysogens than in lytically infected cultures, suggesting the existence of a phage-specified anti-termination function. Several ϕC31 derivatives suitable for the construction of different kinds of transcriptional and translational fusions in Streptomyces were derived. The expression of lacZ in one transcriptional fusion vector (KC659) was increased both in plaques and in lysogens by the appropriately positioned insertion of a previously characterized promoter from the aph gene of S. fradiae. When ϕC31 DNA fragments were inserted into KC659, at least one recombinant phage gave high β-galactosidase activity in lytic infections, but not in lysogens. The potential usefulness of lacZ fusions in Streptomyces is discussed.

The genes encoding both subunits of the succinyl-CoA synthetase of Escherichia coli have been identified as distal genes of the suc operon, which also encodes the dehydrogenase (Elo; sucA) and succinyltransferase (E20; sucB) components of the 2-oxoglutarate dehydrogenase complex. The newly defined genes express polypeptides of 41 kDa (sucC) and 31 kDa (sucD), corresponding to the β and α subunits of succinyl-CoA synthetase, respectively. The genes are thus located at 16·8 min in the E. coli linkage map, together with the citrate synthase (gltA) and succinate dehydrogenase (sdh) genes, in a cluster of nine citric acid cycle genes: gltA-sdhCDABE-sucABCb. Four deletion strains lacking all of these citric acid cycle enzymes were characterized. The succinyl-CoA synthetase activities of strains harbouring plasmids containing the sucC and sucD genes were amplified some fourfold. Further emzymological studies indicated that expression of succinyl-CoA synthetase is coordinately regulated with 2-oxoglutarate dehydrogenase.