W. Harder

Highly-efficient electrotransformation of the yeast Hansenula polymorpha

A highly-efficient method for transformation of the methylotrophic yeast Hansenula polymorpha has been developed. Routinely, transformation frequencies of up to 1.7×106/μg plasmid DNA were obtained by applying an electric pulse of the exponential decay type of 7.5 kV/cm to a highly-concentrated cell mixture during 5 ms. Efficient transformation was dependent on: (1) pretreatment of the cells with the reducing agent dithiotreitol, (2) the use of sucrose as an osmotic stabilizer in an ionic electroporation buffer, and (3) the use of cells grown to the mid-logarithmic phase. Important parameters for optimizing the transformation frequencies were field strength, pulse duration, and cell concentration during the electric pulse. In contrast to electrotransformation protocols described for Saccharomyces cerevisiae and Candida maltosa, transformation frequencies (transformants per μg DNA) for H. polymorpha remained high when large amounts (up to 10μg) of plasmid DNA were added. This feature renders this procedure pre-eminently advantageous for gene cloning experiments when high numbers of transformants are needed.

The Significance of Peroxisomes in the Metabolism of One-Carbon Compounds in Yeasts

Publisher Summary This chapter reviews the role of peroxisomes in the metabolism of methanol and methylated amines in yeasts. It provides information regarding their biogenesis and turnover. The studies on the physiology and biochemistry of methanol oxidation by methylotrophic yeasts exhibit that adaptation of these organisms to growth on methanol is associated with the proliferation of large microbodies in the cells. To evaluate the unique function and structure of the microbodies during methylotrophic growth in yeasts it is necessary to consider pertinent biochemical and physiological aspects of methanol metabolism in these organisms. Microbodies of methanol-grown yeasts show a number of characteristic properties.They appear in clusters in the cell and exist in close association with strands of endoplasmic reticulum. Evidence that the microbodies of methanol-grown yeasts contain alcohol oxidase and catalase is obtained via cell fractionation and cytochemical studies. The information on various aspects of the role of peroxisomes in the metabolism of one-carbon compounds in yeasts clearly shows that these unicellular organisms offer an almost ideal model system for the study of function, morphogenesis, and turnover of these intriguing organelles.

Growth of Hansenula polymorpha in a methanol-limited chemostat

Hansenula polymorpha has been grown in a methanol-limited continuous culture at a variety of dilution rates. Cell suspensions of the yeast grown at a dilution rate of 0.16 h-1 showed a maximal capacity to oxidize excess methanol (QO2max) which was 1.6 times higher than the rate required to sustain the growth rate (QO2). When the dilution rate was decreased to 0.03 h-1, QO2max of the cells increased to a value of more than 20 times that of QO2. The enzymatic basis for this tremendous overcapacity for the oxidation of excess methanol at low growth rates was found to be the methanol oxidase content of the cells. The level of this enzyme increased from 7% to approximately 20% of the soluble protein when the growth rate was decreased from 0.16 to 0.03 h-1. These results were explained on the basis of the poor affinity of methanol oxidase for its substrates. Methanol oxidase purified from Hansenula polymorpha showed an apparent Kmfor methanol of 1.3 mM in air saturated reaction mixtures and the apparent Kmof the enzyme for oxygen was 0.4 mM at a methanol concentration of 100 mM.The involvement of an oxygen dependent methanol oxidase in the dissimilation of methanol in Hansenula polymorpha was also reflected in the growth yield of the organism. The maximal yield of the yeast was found to be low (0.38 g cells/g methanol). This was not due to a very high maintenance energy requirement which was estimated to be 17 mg methanol/g cells x h.

Methanol metabolism in yeasts: Regulation of the synthesis of catabolic enzymes

The regulation of the synthesis of four dissimilatory enzymes involved in methanol metabolism, namely alcohol oxidase, formaldehyde dehydrogenase, formate dehydrogenase and catalase was investigated in the yeasts Hansenula polymorpha and Kloeckera sp. 2201. Enzyme profiles in cell-free extracts of the two organisms grown under glucose limitation at various dilution rates, suggested that the synthesis of these enzymes is controlled by derepression — represion rather than by induction — repression. Except for alcohol oxidase, the extent to which catabolite repression of the catabolic enzymes was relieved at low dilution rates was similar in both organisms. In Hansenula polymorpha the level of alcohol oxidase in the cells gradually increased with decreasing dilution rate, whilst in Kloeckera sp. 2201 derepression of alcohol oxidase synthesis was only observed at dilution rates below 0.10 h−1 and occurred to a much smaller extent than in Hansenula polymorpha.Derepression of alcohol oxidase and catalase in cells of Hansenula polymorpha was accompanied by synthesis of peroxisomes. Moreover, peroxisomes were degraded with a concurrent loss of alcohol oxidase and catalase activities when excess glucose was introduced into the culture. This process of catabolite inactivation of peroxisomal enzymes did not affect cytoplasmic formaldehyde dehydrogenase.

Dimethyl sulphoxide and dimethyl sulphide as a carbon, sulphur and energy source for growth of Hyphomicrobium S

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.

Cytochemical Studies on the Localization of Methanol Oxidase and Other Oxidases in Peroxisomes of Methanol-Grown Hansenula polyrnorpha

The localization of methanol oxidase activity in cells of methanol-limited chemostat cultures of the yeast Hansenula polymorpha has been studied with different cytochemical staining techniques. The methods were based on enzymatic or chemical trapping of the hydrogen peroxide produced by the enzyme during aerobic incubations of whole cells in methanol-containing media. The results showed that methanol-dependent hydrogen peroxide production in either fixed or unfixed cells exclusively occurred in peroxisomes, which characteristically develop during growth of this yeast on methanol. Apart from methanol oxidase and catalase, the typical peroxisomal enzymes d-aminoacid oxidase and l-α-hydroxyacid oxidase were also found to be located in the peroxisomes. Urate oxidase was not detected in these organelles. Phase-contrast microscopy of living cells revealed the occurrence of peroxisomes which were cubic of form. This unusual shape was also observed in thin sections examined by electron microscopy. The contents of the peroxisomes showed, after various fixation procedures, a completely crystalline or striated substructure. It is suggested that this substructure might represent the in vivo organization structure of the peroxisomal enzymes.

Development of Crystalline Peroxisomes in Methanol-Grown Cells of the Yeast Hansenula polymorpha and Its Relation to Environmental Conditions

The development of peroxisomes has been studied in cells of the yeast Hansenula polymorpha during growth on methanol in batch and chemostat cultures. During bud formation, new peroxisomes were generated by the separation of small peroxisomes from mature organelles in the mother cells. The number of peroxisomes migrating to the buds was dependent upon environmental conditions. Aging of cells was accompanied by an increase in size of the peroxisomes and a subsequent increase in their numbers per cell. Their ultimate shape and substructure as well as their number per cell was dependent upon the physiological state of the culture. The change in number and volume density of peroxisomes was related to the level of alcohol oxidase in the cells. Development of peroxisomes in cells of batch cultures was accompanied by an increase in size of the crystalline inclusions in the organelles; they had become completely crystalline when the cells were in the stationary phase. Peroxisomes in cells from methanol-limited chemostat cultures were completely crystalline, irrespective of growth rate. Results of biochemical and cytochemical experiments suggested that alcohol oxidase is a major component of the crystalline inclusions in the peroxisomes of methanol-grown Hansenula polymorpha. Possible mechanisms involved in the ultrastructural changes in peroxisomes during their development have been discussed.

Characterization of Peroxisomes in Glucose-Grown Hansenula polymorpha and Their Development after the Transfer of Cells into Methanol-containing Media

Cells of Hansenula polymorpha growing exponentially on glucose generally contained a single peroxisome of small dimension, irregular in shape and located in close proximity to the cell wall. Crystalline inclusions in the peroxisomal matrix were not observed. Associations of the organelles with one or more strands of endoplasmic reticulum were evident. In stationary phase cells the size of the peroxisomes had increased considerably. They were more cubical in form and showed a partly or completely crystalline matrix.After the transfer of cells growing exponentially on glucose into media containing methanol, large peroxisomes with a partly crystalline matrix developed in the cells within 6 h. These organelles originated from the small peroxisomes in the glucose-grown cells. De novo synthesis of peroxisomes was not observed. Prolonged cultivation in the presence of methanol resulted in a gradual increase in the number of peroxisomes by means of separation of small peroxisomes from mature organelles. During growth of peroxisomes associations with the endoplasmic reticulum remained evident.The increase in volume density of peroxisomes in stationary phase cells grown on glucose and in methanol-grown cells was accompanied by the synthesis of the peroxisomal enzymes alcohol oxidase and catalase. Cytochemical staining techniques revealed that alcohol oxidase activity was only detected when the peroxisomes contained a crystalloid inclusion. Since in peroxisomes of an alcohol oxidase-negative mutant of Hansenula polymorpha crystalline inclusions were never detected, it is concluded that the development of crystalloids inside peroxisomes is due to the accumulation of alcohol oxidase in these organelles.

Competition of marine psychrophilic bacteria at low temperatures

The occurrence of obligately and facultatively psychrophilic bacteria in the marine environment suggests that environmental conditions exist which can favour each of these groups in competitive processes. Differences were found in the way in which temperature affected the growth rates of obligate and facultative psychrophiles. Maximum specific growth rates of a number of obligately and facultatively psychrophilic bacteria were determined in batch culture and competition experiments were carried out in a chemostat at growth-limiting substrate concentrations. From the results the relation between the specific growth rate and the concentration of the growth-limiting substrate for both types of organisms at different temperatures was deduced. Both at low and high substrate concentrations obligate psychrophiles grew faster than facultative psychrophiles at the lower temperature extreme (⩽ 4 C). These results suggest that obligately psychrophilic chemoorganotrophs are responsible for mineralization processes in cold natural environments such as ocean waters and the arctic and antarctic regions. In these environments they can successfully compete with facultative psychrophiles because they can grow faster.

A rapid and specific enrichment procedure forHyphomicrobium spp.

An enrichment procedure for the isolation of stalked bacteria of the genusHyphomicrobium is described. The method is based on the use of an organic C1 compound as carbon and energy source for growth together with anaerobic incubation in the presence of nitrate as an electron acceptor. Optimal conditions for the growth of a number ofHyphomicrobium isolates have been investigated. Applying these conditions,Hyphomicrobium spp. have been enriched from a wide range of natural habitats within 1–2 weeks.