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The regulation of cell cycle progression in normal mammalian cells is dependent on the presence of growth factors. In their absence, non-transformed cells will stop dividing and enter the quiescent state (G0). We show here that in Chinese hamster ovary cells, at least two serum-dependent points exist during G1 that lead to different cellular responses. The first point is located immediately after mitosis and is suggested to link with apoptosis. The second point is located late in G1, and probably corresponds with the 'classic' restriction point R. Cells depleted of serum after the first restriction point will not stop randomly in G1 but continue G1 progression until they reach the late restriction point, as marked by translocation of p42(MAPkinase) (ERK2) to the nucleus.

Hydrogen peroxide (H 2 O 2 ) induces a number of events, which are also induced by mitogens. Since the progression through the G1 phase of the cell cycle is dependent on mitogen stimulation, we were interested to study the effect of H 2 O 2 on the cell cycle progression. This study demonstrates that H 2 O 2 inhibits DNA synthesis in a dose-dependent manner when given to cells in mitosis or at different points in the G1 phase. Interestingly, mitotic cells treated immediately after synchronization are significantly more sensitive to H 2 O 2 than cells treated in the G1, and this is due to the inhibition of the cell spreading after mitosis by H 2 O 2 . H 2 O 2 reversibly inhibits focal adhesion activation and stress fiber formation of mitotic cells, but not those of G1 cells. The phosphorylation of MAPK is also reversibly inhibited in both mitotic and G1 cells. Taken together, H 2 O 2 is probably responsible for the inhibition of the expression of cyclin D1 and cyclin A observed in cells in both phases. In conclusion, H 2 O 2 inhibits cell cycle progression by inhibition of the spreading of mitotic CHO cells. This may play a role in pathological processes in which H 2 O 2 is generated.

Agrobacterium tumefaciens is known to transfer part of its tumor-inducing (Ti) plasmid to the filamentous fungus Aspergillus awamori by illegitimate recombination with the fungal genome. Here, we show that when this Ti DNA shares homology with the A. awamori genome, integration can also occur by homologous recombination. On the basis of this finding, we have developed an efficient method for constructing recombinant mold strains free from bacterial DNA by A. tumefaciens-mediated transformation. Multiple copies of a gene can be integrated rapidly at a predetermined locus in the genome, yielding transformants free of bacterial antibiotic resistance genes or other foreign DNA. Recombinant A. awamori strains were constructed containing up to nine copies of a Fusarium solani pisi cutinase expression cassette integrated in tandem at the pyrG locus. This allowed us to study how mRNA and protein levels are affected by gene copy number, without the influence of chromosomal environmental effects. Cutinase mRNA and protein were maximal with four gene copies, indicating a limitation at the transcriptional level. This transformation system will potentially stimulate market acceptance of derived products by avoiding introduction of bacterial and other foreign DNA into the fungi.

In yeast, glucanase extractable cell wall proteins are anchored to the plasma membrane at an intermediate stage in their biogenesis via a glycosylphosphatidylinositol (GPI) moiety before they become anchored to the wall glucan via a β1,6-glucan linkage. The mechanism of the membrane processing step of cell wall proteins is not known. Here, we report that Ascomycete filamentous fungi involved in food spoilage such as Aspergillus, Paecilomyces and Penicillium, also contain GPI membrane-anchored proteins some of which are processed by an endogenous phospholipase C activity. Furthermore, similar to the situation in yeast, their cell walls contain mannoproteins which are linked to the glucan backbone through a β1,6-glucan linkage. Interestingly, one mould which contains a significant amount of non covalently linked β1,6-glucosylated cell wall proteins, is much more sensitive towards β1,3-glucanases and membrane perturbing peptides than the others.

To increase the acceptability of food products containing genetically modified microorganisms it is necessary to provide in an early stage to the consumers that the product is safe and that the product provide a clear benefit to the consumer. To comply with the first requirement a systematic approach to analyze the probability that genetically modified lactic acid bacteria will transform other inhabitants of the gastro- intestinal (G/I) tract or that these lactic acid bacteria will pick up genetic information of these inhabitants has been proposed and worked out to some degree. From this analysis it is clear that reliable data are still missing to carry out complete risk assessment. However, on the basis of present knowledge, lactic acid bacteria containing conjugative plasmids should be avoided. Various studies show that consumers in developed countries will accept these products when they offer to them health or taste benefits or a better keepability. For the developing countries the biggest challenge for scientists is most likely to make indigenous fermented food products with strongly improved microbiological stability due to broad spectra bacteriocins produced by lactic acid bacteria. Moreover, these lactic acid bacteria may contribute to health.

The essential CDC25 gene product of Saccharomyces cerevisiae is the most upstream known component of the RAS/adenylate cyclase pathway. Cdc25 is a GTP-exchange protein involved in activating RAS in response to fermentable carbon sources. In this paper it is reported that the Cdc25 protein, in addition to its stimulatory role in the RAS/adenylate cyclase pathway, regulates glucose transport. Continuous culture studies and glucose uptake experiments showed that the cdc25-1 and the cdc25-5 temperature-sensitive mutants exhibit decreased glucose uptake activity at the restrictive temperature under both repressed and derepressed conditions as compared to the wild-type strain. Because the cdc25-1 mutant is not impaired in its cAMP metabolism, it is concluded that this effect on glucose transport is independent of cAMP levels. Furthermore, it is shown that the decrease in glucose uptake activity is not due to a decrease in protein synthesis or to an arrest in the G1 phase of the cell cycle. In addition to a defect in glucose uptake, the cdc25-5 mutant strain exhibited differences in glucose metabolism, probably due to the decreased cAMP level and hence decreased protein kinase A activity. Because the Cdc25 protein is localized at the membrane, these results indicate that Cdc25 is directly involved in glucose transport and may be in direct contact with the glucose transporters.

Saccharomyces cerevisiae was grown in a continuous culture at a single dilution rate with input ammonia concentrations whose effects ranged from nitrogen limitation to nitrogen excess and glucose limitation. The rate of ammonia assimilation (in millimoles per gram of cells per hour) was approximately constant. Increased extracellular ammonia concentrations are correlated with increased intracellular glutamate and glutamine concentrations, increases in levels of NAD-dependent glutamate dehydrogenase activity and its mRNA (gene GDH2), and decreases in levels of NADPH-dependent glutamate dehydrogenase activity and its mRNA (gene GDH1), as well as decreases in the levels of mRNA for the amino acid permease-encoding genes GAP1 and PUT4. The governing factor of nitrogen metabolism might be the concentration of ammonia rather than its flux.

A copy of the cutinase cDNA from Fusarium solani pisi was constructed starting from synthetic oligonucleotides. For this construction three separate cassettes were made, which were subsequently assembled to form the cutinase gene. Heterologous expression of the synthetic cutinase gene and the subsequent secretion of the recombinant enzyme was achieved in Saccharomyces cerevisiae and Aspergillus awamori.

This study describes a search for lactic acid bacteria capable of exo‐polysaccharide production or exhibiting antimicrobial or proteolytic activities. About 400 lactic acid bacteria were isolated from traditional fermented foods (sour dough, sausages, table olives, cheese and other dairy products). Together with almost 200 lactic acid bacterial strains obtained from culture collections, these strains were screened for exo‐polysaccharide production, bacteriocin production and proteolytic activity. Thirty strains producing exo‐polysaccharide (EPS) and 8 Lactococcus lactis strains producing a nisin‐like bacteriocin were selected. The proteolytic activities of all strains were determined, using a test based on the degradation of casein. As a result a division was made into 4 groups with increasing proteolytic activities. Among the EPS‐producing strains, 14 were isolated from various olives fermentations, all identified as Lactobacillus paracasei. EPS‐producing strains isolated from Belgian salami wer...

In order to determine whether the changes in the activities and mRNA levels of enzymes involved in intermediary carbon metabolism previously observed in glucose-limited continuous cultures (Sierkstra et al., 1992a) were glucose specific, we have analysed their regulation in a galactose-limited continuous culture of Saccharomyces cerevisiae. The Vmax of the galactose uptake system was shown to be dilution rate (D) dependent, comparable with the high-affinity glucose uptake. The maximum uptake was observed at D 0.2 h-1 (0.25 mmol min-1 per g) and the minimum uptake (0.1 mmol min-1 per g) at D 0.05 h-1 and 0.3 h-1. The aerobic fermentation of galactose occurred at D 0.275-0.3 h-1 which is identical to the results obtained in glucose-limited continuous cultures of this strain. Because galactose is not a repressing carbon source, this demonstrates that the Crabtree effect is not mediated by, or in any way related to glucose repression. Moreover, invertase and hexokinase I mRNA levels (both subject to glucose repression at the transcriptional level) were present when the yeast produced ethanol in galactose- and glucose-limited continuous cultures. In glucose-limited continuous cultures a decrease in alcohol dehydrogenase (I and II) mRNA levels and activity and phosphoglucomutase activity was observed with increasing dilution rates. In addition, at D 0.3 h-1, when the yeast produced ethanol, glucose-6-phosphate dehydrogenase and pyruvate decarboxylase were induced and a decrease in respiration was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

The lipA gene encoding the extracellular lipase produced by Pseudomonas glumae PG1 was cloned and characterized. A sequence analysis revealed an open reading frame of 358 codons encoding the mature lipase (319 amino acids) preceded by a rather long signal sequence of 39 amino acids. As a first step in structure-function analysis, we determined the Ser-Asp-His triad which makes up the catalytic site of this lipase. On the basis of primary sequence homology with other known Pseudomonas lipases, a number of putative active site residues located in conserved areas were found. To determine the residues actually involved in catalysis, we constructed a number of substitution mutants for conserved Ser, Asp, and His residues. These mutant lipases were produced by using P. glumae PG3, from which the wild-type lipase gene was deleted by gene replacement. By following this approach, we showed that Ser-87, Asp-241, and His-285 make up the catalytic triad of the P. glumae lipase. This knowledge, together with information on the catalytic mechanism and on the three-dimensional structure, should facilitate the selection of specific modifications for tailoring this lipase for specific industrial applications.

A fusion of DNA sequences encoding the SPO2 promoter, the alpha-amylase signal sequence from Bacillus amyloliquefaciens, and the mature part of the alpha-galactosidase from Cyamopsis tetragonoloba (guar) was constructed on a Bacillus subtilis multicopy vector. Bacillus cells of the protease-deficient strain DB104 harboring this vector produced and secreted the plant enzyme alpha-galactosidase up to levels of 1,700 U/liter. A growth medium suppressing the residual proteolytic activity of strain DB104 was used to reach these levels in a fermentor. Purification of the secreted product followed by NH2-terminal amino acid sequencing showed that the alpha-amylase signal sequence had been processed correctly. The molecular mass of the product estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was slightly lower than that of the plant purified enzyme, which is most likely due to glycosylation of the latter. The alpha-galactosidase product was active both on the artificial substrate para-nitrophenyl-alpha-D-galactopyranoside and on the galactomannan substrate, guar gum. The activity of this Bacillus sp.-produced enzyme was similar to that of the glycosylated enzyme purified from guar seeds, indicating that glycosylation has no essential function for enzyme activity.

Impaired secretion of the hydrophobic CY028 cutinase invokes an unfolded protein response (UPR) in Saccharomyces cerevisiae cells. Here we show that the UPR in CY028-expressing S. cerevisiae cells is manifested as an aberrant morphology of the endoplasmic reticulum (ER) and as extensive membrane proliferation compared to the ER morphology and membrane proliferation of wild-type CY000-producing S. cerevisiae cells. In addition, we observed oxidative stress, which resulted in a 21-fold increase in carbonylated proteins in the CY028-producing S. cerevisiae cells. Moreover, CY028-producing S. cerevisiae cells use proteasomal degradation to reduce the amount of accumulated CY028 cutinase, thereby attenuating the stress invoked by CY028 cutinase expression. This proteasomal degradation occurs within minutes and is characteristic of ER-associated degradation (ERAD). Our results clearly show that impaired secretion of the heterologous, hydrophobic CY028 cutinase in S. cerevisiae cells leads to protein aggregation in the ER, aberrant ER morphology and proliferation, and oxidative stress, as well as a UPR and ERAD.

Knowledge of the human genome and other genomes, the selection of health-beneficial components, information and communication technology (ICT)-driven plant cultivation and small-scale processes will together change the agrofoods business from a rather low-tech to a high-tech (functional) foods business. ICT will provide consumers with information that in combination with their private genetic passport may be used to select those functional foods that are most beneficial for them.

Saccharomyces cerevisiae is often used to produce heterologous proteins that are preferentially secreted to increase economic feasibility. We used N-glycosylation as a tool to enhance protein secretion. Secretion of cutinase, a lipase, and llama VHH antibody fragments by S. cerevisiae or Pichia pastoris improved following the introduction of an N-glycosylation site. When we introduced an N-glycosylation consensus sequence in the N-terminal region of a hydrophobic cutinase, secretion increased fivefold. If an N-glycosylation site was introduced in the C-terminal region, however, secretion increased only 1.8-fold. These results indicate that the use of N glycosylation can significantly enhance heterologous protein secretion. Eukaryotic cells, such as yeasts, have several quality control systems to ensure that only correctly folded proteins are trans- ported along their secretory pathway (9, 13, 20). These quality control systems are necessary because protein folding in vivo is much more complex than protein folding in vitro. The nascent polypeptide chain emerges from the ribosome and is translo- cated across the endoplasmic reticulum (ER) membrane, thereby being gradually exposed to the lumen of the ER. Since

Mitogen-activated protein (MAP) kinases are serine/threonine protein kinases that are activated rapidly in cells stimulated by various extracellular signals. With stimulation of quiescent cells by growth factors, activated p42/p44 MAP kinases rapidly translocate to the nucleus, where they induce immediate early gene transcription. The MAP kinase signal transduction pathway represents an important mechanism by which growth factors regulate cellular events such as cell cycle progression or cell growth. In the present study, p42MAPK (ERK2) was studied during the ongoing cell cycle of Chinese hamster ovary cells synchronized by mitotic shake-off. We show that protein expression of p42MAPK increased in mid-G1 and that MAP kinase is phosphorylated during G1, as visualized by a gel-mobility shift and by the use of phosphospecific antibodies. This phosphorylation appeared to occur in the cytoplasm rather than at the plasma-membrane. In addition, phosphorylated p42MAPK was found to translocate to the nucleus during late/mid-G1. Treatment of cells with MEK inhibitor PD098059 prevented the phosphorylation and nuclear translocation of MAP kinase and DNA synthesis. Thus, nuclear translocation of p42MAPK is not restricted to the G0/G1 transition but occurs in every cell cycle and seems to be required for cell cycle progression.

Trehalose and glycogen accumulate in Saccharomyces cerevisiae when growth conditions deteriorate. It has been suggested that aside from functioning as storage factors and stress protectants, these carbohydrates may be required for cell cycle progression at low growth rates under carbon limitation. By using a mutant unable to synthesize trehalose and glycogen, we have investigated this requirement of trehalose and glycogen under carbon-limited conditions in continuous cultures. Trehalose and glycogen levels increased with decreasing growth rates in the wild-type strain, whereas no trehalose or glycogen was detected in the mutant. However, the mutant was still able to grow and divide at low growth rates with doubling times similar to those for the wild-type strain, indicating that trehalose and glycogen are not essential for cell cycle progression. Nevertheless, upon a slight increase of extracellular carbohydrates, the wild-type strain degraded its reserve carbohydrates and was able to enter a cell division cycle faster than the mutant. In addition, wild-type cells survived much longer than the mutant cells when extracellular carbon was exhausted. Thus, trehalose and glycogen have a dual role under these conditions, serving as storage factors during carbon starvation and providing quickly a higher carbon and ATP flux when conditions improve. Interestingly, the CO 2 production rate and hence the ATP flux were higher in the mutant than in the wild-type strain at low growth rates. The possibility that the mutant strain requires this steady higher glycolytic flux at low growth rates for passage through Start is discussed.

Several cutinase variants derived by molecular modelling and site-directed mutagenesis of a cutinase gene from Fusarium solani pisi are poorly secreted by Saccharomyces cerevisiae . The majority of these variants are successfully produced by the filamentous fungus Aspergillus awamori . However, the L51S and T179Y mutations caused reductions in the levels of extracellular production of two cutinase variants by A. awamori . Metabolic labelling studies were performed to analyze the bottleneck in enzyme production by the fungus in detail. These studies showed that because of the single L51S substitution, rapid extracellular degradation of cutinase occurred. The T179Y substitution did not result in enhanced sensitivity towards extracellular proteases. Presumably, the delay in the extracellular accumulation of this cutinase variant is caused by the enhanced hydrophobicity of the molecule. Overexpression of the A. awamori gene encoding the chaperone BiP in the cutinase-producing A. awamori strains had no significant effect on the secretion efficiency of the cutinases. A cutinase variant with the amino acid changes G28A, A85F, V184I, A185L, and L189F that was known to aggregate in the endoplasmic reticulum of S. cerevisiae , resulting in low extracellular protein levels, was successfully produced by A. awamori . An initial bottleneck in secretion occurred before or during translocation into the endoplasmic reticulum but was rapidly overcome by the fungus.

This study focuses on the different efficiencies of secretion of two fungal cutinases by Saccharomyces cerevisiae , a wild-type cutinase (CY000) and a hydrophobic mutant cutinase (CY028). Both cutinases are placed under control of the GAL7 promoter, by which the expression levels can be regulated. Wild-type cutinase was secreted at up to 25 mg per g (dry weight), while CY028 was secreted at a level of 2 mg per g (dry weight); this difference is nearly independent of the expression level. Pulse-chase experiments revealed that whereas CY000 cutinase is secreted, CY028 is irreversibly retained in the cell. Immunogold labelling followed by electron microscopy revealed colocalization of CY028 with immunoglobulin heavy-chain binding protein (BiP) in the endoplasmic reticulum (ER). The increase of wild-type cutinase expression did not result in higher levels of the molecular chaperone BiP, but BiP levels are raised by increased induction of the hydrophobic mutant cutinase. Immunoprecipitation studies showed that in contrast to the wild-type cutinase, the hydrophobic mutant cutinase interacts with BiP. These results indicate that the introduction of two exposed hydrophobic patches in cutinase results in a higher affinity for BiP which might cause the retention of this mutant cutinase in the ER.

We describe the cloning and characterisation of the BiP gene homologues of the filamentous fungi Aspergillus niger and Aspergillus awamori. The BiP genes of these black Aspergilli encode an identical protein of 672 amino acids, which has a high homology with the BiP protein from Saccharomyces cerevisiae and contains a putative signal sequence of 38 amino acids. The DNA sequences of the Aspergillus BiP genes diverge in particular in the three intronic sequences and the 5′- and 3′- noncoding regions. Sequences resembling Heat Shock Elements (HSE) and Unfolded Protein Response (UPR) elements, as found in the yeast KAR2 promoter, are present in the 5′ non-transcribed regions of both genes. The expression of the A. niger bipA gene is increased by heat shock and tunicamycin treatment.

The carboxyl-terminal regions of five cell wall proteins (Cwp1p, Cwp2p, Ag alpha 1p, Tip1p, and Flo1p) and three potential cell wall proteins (Sed1p, YCR89w, and Tir1p) all proved capable of immobilizing alpha-galactosidase in the cell wall of Saccharomyces cerevisiae. The fraction of the total amount of fusion protein that was localized to the cell wall varied depending on the anchor domain used. The highest proportion of cell wall incorporation was achieved with Cwp2p, Ag alpha 1p, or Sed1p as an anchor. Although 80% of these fusion proteins were incorporated in the cell wall, the total production of alpha-galactosidase-Ag alpha 1p was sixfold lower than that of alpha-galactosidase-Cwp2p and eightfold lower than that of alpha-galactosidase-Sed1p. Differences in mRNA levels were not responsible for this discrepancy, nor was an intracellular accumulation of alpha-galactosidase-Ag alpha 1p detectable. A lower translation efficiency of the alpha-galactosidase-AG alpha 1 fusion construct is most likely to be responsible for the low level of protein production. alpha-Galactosidase immobilized by the carboxyl-terminal 67 amino acids of Cwp2p was most effective in the hydrolysis of the high-molecular-weight substrate guar gum from Cyamopsis tetragonoloba. This indicates that the use of a large anchoring domain does not necessarily result in a better exposure of the immobilized enzyme to the exterior of the yeast cell.

A synthetic derivative of the cutinase cDNA of Fusarium solani pisi was expressed in Aspergillus awamori using the A. awamori endoxylanase II (exlA) promoter and terminator. The influence of the origin of the pre-sequence and the presence of a pro-sequence on the efficiency of extracellular cutinase production was analysed in single-copy transformants containing an expression cassette integrated at the pyrG locus. Transformants containing a construct encoding a direct, in-frame fusion of the xylanase pre-peptide to the mature cutinase showed a 2-fold higher cutinase production level compared to strains containing constructs with an additional cutinase pro-peptide. The effect of multicopy integration of the expression cassette on cutinase production was analysed in strains with different numbers of a cutinase construct containing its own pre-prosequence. The multicopy strains showed a 6- to 12-fold increased production of extracellular cutinase relative to the single-copy strains. No linear dose response relation to the number of expression cassettes present in the strains was observed. The amount of active enzyme produced by the strains correlated with the amount of cutinase-specific mRNA, suggesting that cutinase overproduction is not limited at the level of translation or secretion.

Yeast has a rigid cell wall comprising an outer layer of glycoproteins and an internal skeletal layer of glucan; heterologous proteins can be targeted to the glycoprotein layer and become covalently linked to the glucan skeleton. Yeast is a eukaryote that has 'generally regarded as safe' (GRAS) status, and is easy to cultivate, so it seems ideally suited for applications including the manufacture of recyclable, immobilized, biocatalysts, whole-cell vaccines, the presentation of peptide or antibody libraries, and the presentation of adhesion or metal-binding proteins.

Cutinase from the fungus Fusarium solani pisi is a lipolytic enzyme able to hydrolyze both aggregated and soluble substrates. It therefore provides a powerful tool for probing the mechanisms underlying lipid hydrolysis. Lipolytic enzymes have a catalytic machinery similar to those present in serine proteinases. It is characterized by the triad Ser, His, and Asp (Glu) residues, by an oxyanion binding site that stabilizes the transition state via hydrogen bonds with two main chain amide groups, and possibly by other determinants. It has been suggested on the basis of a covalently bond inhibitor that the cutinase oxyanion hole may consist not only of two main chain amide groups but also of the Ser42 O gamma side chain. Among the esterases and the serine and the cysteine proteases, only Streptomyces scabies esterase, subtilisin, and papain, respectively, have a side chain residue which is involved in the oxyanion hole formation. The position of the cutinase Ser42 side chain is structurally conserved in Rhizomucor miehei lipase with Ser82 O gamma, in Rhizopus delemar lipase with Thr83 O gamma 1, and in Candida antartica B lipase with Thr40 O gamma 1. To evaluate the increase in the tetrahedral intermediate stability provided by Ser42 O gamma, we mutated Ser42 into Ala. Furthermore, since the proper orientation of Ser42 O gamma is directed by Asn84, we mutated Asn84 into Ala, Leu, Asp, and Trp, respectively, to investigate the contribution of this indirect interaction to the stabilization of the oxyanion hole. The S42A mutation resulted in a drastic decrease in the activity (450-fold) without significantly perturbing the three-dimensional structure. The N84A and N84L mutations had milder kinetic effects and did not disrupt the structure of the active site, whereas the N84W and N84D mutations abolished the enzymatic activity due to drastic steric and electrostatic effects, respectively.

The feasibility of stabilizing proteins towards proteolytic degradation was explored by engineering the primary proteolytic cleavage site(s). This novel approach does not require information on the 3-D structure of the native enzyme. As a model system, the extracellular lipase of Pseudomonas glumae was chosen, which is sensitive towards degradation by subtilisin-type proteases. The primary proteolytic cleavage in the lipase appeared to be located between amino acids serine 153 and histidine 154. Since subtilisins are known to show a preference towards amino acid residues surrounding the scissile bond, non-preferred amino acids were introduced in this area. Two concepts were tested: the introduction of arginine or glutamate residues (charge concept) and the introduction of proline residues (proline concept). Although the mutant lipases produced according to either of these concepts were still cleaved in the same area, they showed a considerably increased stability towards proteolytic degradation.

Wild-type Saccharomyces cerevisiae and a strain carrying a deletion in the glucose-6-phosphate-isomerase gene (pgi1) were grown in carbon-limited continuous cultures on a mixture of fructose and galactose. Pulses of glucose, fructose and galactose were given to these cultures to investigate whether the pgi1 strain was capable of normal glucose repression. Glucose and galactose pulses inhibited fructose consumption and thus glycolysis in the pgi1 strain by a combination of competition between glucose and fructose at the uptake and/or phosphorylation level and inhibition of fructose uptake and/or phosphorylation by glucose 6-phosphate. Fructose pulses administered to the pgi1 strain transiently decreased the glycolytic flux downstream of fructose-1,6-bisphosphate. Transcriptional induction of the PDC1 gene (encoding pyruvate decarboxylase) was observed after glucose or galactose pulses were applied to the pgi1 strain, demonstrating that metabolism of these sugars beyond glucose 6-phosphate is dispensable for PDC1 induction. Fructose also induced PDC1 transcription, indicating that intracellular sugars could act as trigger for PDC1 induction or, alternatively, that two inductors are present. In contrast to the wild-type transcriptional inhibition of the glucose-repressible genes, HXK1 and GAL10 (encoding hexokinase isoenzyme 1 and uridine diphosphoglucose-4-epimerase, respectively) did not occur upon addition of glucose or fructose to the pgi1 mutant. Transcriptional repression was observed after application of the fructose pulse when the yeast had resumed metabolism of fructose. These results demonstrate that the initial signal for catabolite repression is not generated by high sugar concentrations or high concentrations of intermediates; moreover a simple role for the hexokinases can also be excluded. The absence of an increased glycolytic flux in the pgi1 mutant after administration of the sugar pulses while the concentrations of sugar and glycolytic intermediates were high, suggests that the initial signal for glucose repression could be linked to an increased glycolytic flux. The occurrence of PDC1 induction in the pgi1 strain while GAL10/HXKI repression is absent, demonstrates that the initial signals for catabolite induction and catabolite repression are different.

Saccharomyces cerevisiae SU50B and Hansenula polymorpha 8/2, both carrying a multicopy integrated guar alpha-galactosidase, have been cultivated in continuous cultures, using various mixtures of carbon sources and cultivation conditions. Both S. cerevisiae SU50B and H. polymorpha 8/2 are stable and produce high levels of extracellular alpha-galactosidase in continuous cultures for more than 500 h. For these expression systems the strong inducible promoter systems GAL7 and methanol oxidase, respectively, were used. The induction of alpha-galactosidase synthesis by galactose in SU50B is limited by the low galactose uptake. Apart from that, at high dilution rates, the glucose repression is substantial, and a maximal expression level of 28.6 mg of extracellular alpha-galactosidase.g (dry weight) of biomass-1 could be obtained. In H. polymorpha, the induction of alpha-galactosidase synthesis, in addition to methanol oxidase synthesis using formaldehyde, is very effective up to 42 mg of extracellular alpha-galactosidase.g (dry weight) of biomass-1. Productivities in terms of specific production rate enable a good comparison with those of other heterologous expression systems in the literature. The productivities found with S. cerevisiae SU50B and H. polymorpha, 3.25 and 5.5 mg of alpha-galactosidase.g of biomass-1.liter-1.h-1, respectively, rank among the highest reported in the literature. Enzyme production and secretion in H. polymorpha are more complex. A two-peaked optimum is found in enzyme production. No clear explanation of this phenomenon can be given.

A strain of the methylotrophic yeast Hansenula polymorpha, A16, has been developed that expresses the guar alpha-galactosidase gene to 22.4 mg/g dry cell weight in chemostat cultures at a dilution rate of 0.1 h(-1). This corresponds to more than 13.1% of soluble cell protein, of which 56-62% is secreted into the medium. The alpha-galactosidase gene was flanked by the promoter and terminator sequences of the H.polymorpha mox gene, which can direct expression of the mox gene itself more than 30% of total cell protein under methanol growth. The expression cassette (pUR3510) based on the Saccharomyces cerevisiae plasmid, YEp13, was integrated into the genome. Such transformants were stable in chemostat cultures and exhibited 100% stability for both alpha-galactosidase+ and leu+ phenotypes. Chemostat cultures produced higher levels of alpha-galactosidase with higher specific productivities expressed as mg alpha-galactosidase g(-1) h(-1) compared to batch cultures.

The heat resistance of Salmonella senftenberg 775 W, NCTC 9959, has been determined in distilled water pH 6.5 at sucrose concentrations up to 2.20 mol l−1 at temperatures between 63 and 70°C. Surviving cells were counted on minimal and enriched agar media to investigate the influence of the various nutrients on the recovery of heat injured cells. At various sucrose concentrations and temperatures multiphasic exponential parts of inactivation curves were found. Systematic differences between the recovery media depended on sucrose concentration, temperature and phase of exponential inactivation. At 60°C and sucrose concentrations between 0.52 and 1.82 mol l−1 the relationship between inactivation rate and sucrose concentration could be described by the equation ln k5=ln k0-αT [sucrose]. The activation energy of thermal inactivation reactions, substantially decreased when sucrose (1.82 mol l−1) was added to the heating menstruum. The activation energies in different recovery agars were of the same order, which suggests that the critical sites in heat inactivation are not key enzymes of the synthetic pathways of amino-acids and nucleotides. The differences between activation energies, calculated for cells of the various exponential phases of inactivation in both non-sucrose and 1.82 mol sucrose per 1 heating media, were also small, further suggesting that these critical sites are the same in cells from the various phases. Compared to published data on the heat resistance of S. senftenberg 775 W, we found a decreased resistance in a non-sucrose medium but an equal or increased resistance, depending on the phase of exponential inactivation, at a sucrose concentration of 1.82 mol l−1.

The heat resistance ofCitrobacter freundii NCTC 9750 between 45–65°C in media with various water activities has been determined. At a water activity of nearly 1.00, the Arrhenius plot of the death rate shows a sharp breakpoint at 56.5°C, suggesting the existence of at least two different thermal inactivation processes causing lethality of the bacterial cell. The activation energy below 56.5°C is 0.4186 MJ/mol (100 000 cal/mol), above 56.5°C it is 0.1863 MJ/mol (44 500 cal/mol). After addition of sucrose (1.8 mol/l) or NaCl (0.77 mol/l) to the heating medium, such a breakpoint is not observed. The activation energy for these processes are, for sucrose; 0.2097 MJ/mol, for NaCl; 0.3641 MJ/mol. However, at an NaCl concentration of 1.54 mol/l there is a breakpoint at 53.3°C. The influence of the sucrose concentration on the heat resistance can be described by the formula: ln kS=ln kO−a [sucrose]. Such a simple correlation does not exist for the influence of NaCl or glycerol. The heat inactivation of whole cells ofC. freundii was also measured with a differential scanning calorimeter. The first irreversible conformation change took place at 323 K, the main conformation change at 343 K.

In many cases, water-in-oil emulsions appear to be microbiologically more stable against the growth of non-lipolytic microorganisms than the isolated water phase itself. The two main reasons for this intrinsic stability are that only a small fraction of the droplets of the emulsion is occupied by microorganisms originating from the water phase and that the size of these droplets limits the outgrowth of microorganisms. It is possible to give a quantitative description of the intrinsic stability of a water-in-oil emulsion, using the yield coefficient of different microorganisms grown in different media and the size-distribution of the water droplets in the emulsion. Relationships are given between the amount and nature of growth compounds in a water droplet of an emulsion and the growth and fate of microorganisms as a function of storage time.

The microbial stability of water-in-oil emulsions is calculated as a function of the initial contamination, the concentration of carbon and energy sources, the size-distribution of the water droplets and of the storage time. The calculated values appear to be in good agreement with the experimental data.

The heat resistance of Klebsiella pneumoniae, an organism of widespread occurrence in nature has been determined in media containing various amounts of sucrose at temperatures between 47° and 59°C.

Strains of Clostridium botulinum type A, type E and both non-proteolytic and proteolytic types B and F were characterized by their electrophoretic protein patterns. As the protein pattern changes during sporulation, special attention was paid to the prevention of sporulation by selecting an appropriate medium (Strasdine's medium plus 1% w/v glucose) and a scheme of repeated subculturing. Ribosomal proteins, evolutionarily conservative and hence relatively similar in all types of bacteria, were removed to optimize the resolving power of the electrophoretic technique. Protein patterns were compared by computing correlation coefficients of normalized densitometric tracings. The method is highly reproducible and its resolving power is high: all protein patterns found were specific. The strains tested fall into two main groups: the proteolytic and the non-proteolytic cluster. Type A strains form a separate subgroup within the proteolytic cluster, the same applies to type E strains within the non-proteolytic group. Although time-consuming for spore-forming bacteria, this method is, to our knowledge, the only technique that recognizes individual strains of Cl. botulinum. For non-spore-forming micro-organisms the method is certainly much simpler and hence even more valuable.