Sucrose-grown Cells Research Articles

Resveratrol production from several types of saccharide sources by a recombinant Scheffersomyces stipitis strain

Resveratrol is a plant-derived aromatic compound with a wide range of beneficial properties including antioxidant and anti-aging effects. The resveratrol currently available on the market is predominantly extracted from certain plants such as grape and the Japanese knotweed Polygonum cuspidatum. Due to the unstable harvest of these plants and the low resveratrol purity obtained, it is necessary to develop a stable production process of high-purity resveratrol from inexpensive feedstocks. Here, we attempted to produce resveratrol from a wide range of sugars as carbon sources by a using the genetically-engineered yeast Scheffersomyces stipitis (formerly known as Pichia stipitis), which possesses a broad sugar utilization capacity. First, we constructed the resveratrol producing strain by introducing genes coding the essential enzymes for resveratrol biosynthesis [tyrosine ammonia-lyase 1 derived from Herpetosiphon aurantiacus (HaTAL1), 4-coumarate: CoA ligase 2 derived from Arabidopsis thaliana (At4CL2), and stilbene synthase 1 derived from Vitis vinifera (VvVST1)]. Subsequently, a feedback-insensitive allele of chorismate mutase was overexpressed in the constructed strain to improve resveratrol production. The constructed strain successfully produced resveratrol from a broad range of biomass-derived sugars [glucose, fructose, xylose, N-acetyl glucosamine (GlcNAc), galactose, cellobiose, maltose, and sucrose] in shake flask cultivation. Significant resveratrol titers were detected in cellobiose and sucrose fermentation (529.8 and 668.6 mg/L after 120 h fermentation, respectively), twice above the amount obtained with glucose (237.6 mg/L). Metabolomic analysis revealed an altered profile of the metabolites involved in the glycolysis and shikimate pathways, and also of cofactors and metabolites of energy metabolisms, depending on the substrate used. The levels of resveratrol precursors such as L-tyrosine increased in cellobiose and sucrose-grown cells. The results indicate that S. stipitis is an attractive microbial platform for resveratrol production from broad types of biomass-derived sugars and the selection of suitable substrates is crucial for improving resveratrol productivity of this yeast.

Mannitol Transport and Mannitol Dehydrogenase Activities are Coordinated in Olea europaea Under Salt and Osmotic Stresses

The intracellular accumulation of organic compatible solutes functioning as osmoprotectants, such as polyols, is an important response mechanism of several plants to drought and salinity. In Olea europaea a mannitol transport system (OeMaT1) was previously characterized as a key player in plant response to salinity. In the present study, heterotrophic sink models, such as olive cell suspensions and fruit tissues, and source leaves were used for analytical, biochemical and molecular studies. The kinetic parameters of mannitol dehydrogenase (MTD) determined in cells growing in mannitol, at 25°C and pH 9.0, were as follows: K(m), 54.5 mM mannitol; and V(max), 0.47 μmol h⁻¹ mg⁻¹ protein. The corresponding cDNA was cloned and named OeMTD1. OeMTD1 expression was correlated with MTD activity, OeMaT1 expression and carrier-mediated mannitol transport in mannitol- and sucrose-grown cells. Furthermore, sucrose-grown cells displayed only residual OeMTD activity, even though high levels of OeMTD1 transcription were observed. There is evidence that OeMTD is regulated at both transcriptional and post-transcriptional levels. MTD activity and OeMTD1 expression were repressed after Na+, K+ and polyethylene glycol (PEG) treatments, in both mannitol- and sucrose-grown cells. In contrast, salt and drought significantly increased mannitol transport activity and OeMaT1 expression. Taken together, these studies support that olive trees cope with salinity and drought by coordinating mannitol transport with intracellular metabolism.

Mannose accommodation of Vigna angularis cells on solid agar medium involves its possible conversion to sucrose mediated by enhanced phosphomannose isomerase activity

Mannose is an unusable carbon source for many plants. In our study we compared the effects of mannose and sucrose on growth and sucrose levels in azuki bean (Vigna angularis) cells grown in liquid media and in solid media. The suspension cells grew actively in a liquid medium containing 90 mM sucrose but not in that containing 90 mM mannose, where the intracellular sucrose levels were reduced to 20% or less of those in sucrose-grown cells. These results suggested that the limited conversion of mannose to sucrose resulted in cell growth inhibition. When sucrose-grown suspension cells (1 x 10(5)) were transferred onto agar medium containing mannose, they grew little initially, but, after a month lag period, they started to form many callus colonies at a high apparent variation rate (1.3 x 10(-3)). Time-course studies for sugar and enzyme analysis revealed that the mannose-accommodated cells were capable of converting mannose to sucrose, with enhanced phosphomannose isomerase activity. The mannose-accommodated cells actively grew in liquid medium with sucrose but lost their ability to grow with mannose again, suggesting a specific trait of callus culture for mannose utilization. The possible differences in the metabolic activities and other physiological characteristics are discussed between callus and suspension cells.

Utilization and Transport of Mannitol in Olea europaea and Implications for Salt Stress Tolerance

Mannitol is one of the primary photosynthetic products and the major phloem-translocated carbohydrate in Olea europaea L., an important crop in the Mediterranean basin. Uptake of mannitol in heterotrophic cell suspensions of O. europaea was shown to be mediated by a 1 : 1 polyol : H+ symport system with a Km of 1.3 mM mannitol and a Vmax of 1.3 nmol min(-1) mg(-1) DW. Dulcitol, sorbitol and xylitol competed for mannitol uptake, whereas glucose and sucrose did not. Reverse transcription-PCR (RT-PCR) performed on mRNA extracted from cultured cells exhibiting high mannitol transport activity allowed the cloning of a partial O. europaea mannitol carrier OeMaT1. The Vmax of mannitol uptake and the amount of OeMaT1 transcripts increased along with polyol depletion from the medium, suggesting that the mannitol transport system may be regulated by its own substrate. Addition of 100-500 mM NaCl to cultured cells enhanced the capacity of the polyol : H+ symport system and the amount of OeMaT1 transcripts, whereas it strongly repressed mannitol dehydrogenase activity. Measurements of cell viability showed that mannitol-grown cells remained viable 24 h after a 250 and 500 mM NaCl pulse, whereas extensive loss of cell viability was observed in sucrose-grown cells. OeMaT1 transcripts increased throughout maturation of olive fruits, suggesting that an OeMaT is involved in the accumulation of mannitol during ripening of olive. Thus, mannitol transport and compartmentation by OeMaT are important to allocate this source of carbon and energy, as well as for salt tolerance and olive ripening.

Regulation of expression of cellulosomal cellulase and hemicellulase genes in Clostridium cellulovorans.

The regulation of expression of the genes encoding the cellulases and hemicellulases of Clostridium cellulovorans was studied at the mRNA level with cells grown under various culture conditions. A basic pattern of gene expression and of relative expression levels was obtained from cells grown in media containing poly-, di- or monomeric sugars. The cellulase (cbpA and engE) and hemicellulase (xynA) genes were coordinately expressed in medium containing cellobiose or cellulose. Growth in the presence of cellulose, xylan, and pectin gave rise to abundant expression of most genes (cbpA-exgS, engH, hbpA, manA, engM, engE, xynA, and/or pelA) studied. Moderate expression of cbpA, engH, manA, engE, and xynA was observed when cellobiose or fructose was used as the carbon source. Low levels of mRNA from cbpA, manA, engE, and xynA were observed with cells grown in lactose, mannose, and locust bean gum, and very little or no expression of cbpA, engH, manA, engE, and xynA was detected in glucose-, galactose-, maltose-, and sucrose-grown cells. The cbpA-exgS and engE genes were most frequently expressed under all conditions studied, whereas expression of xynA and pelA was more specifically induced at higher levels in xylan- or pectin-containing medium, respectively. Expression of the genes (cbpA, hbpA, manA, engM, and engE) was not observed in the presence of most soluble di- or monosaccharides such as glucose. These results support the hypotheses that there is coordinate expression of some cellulases and hemicellulases, that a catabolite repression type of mechanism regulates cellulase expression in rapidly growing cells, and that the presence of hemicelluloses has an effect on cellulose utilization by the cell.

Involvement of nitric oxide synthase in sucrose-enhanced hydrogen peroxide tolerance of Rhodococcus sp. strain APG1, a plant-colonizing bacterium

Hydrogen peroxide (H 2O 2) tolerance of Rhodococcus sp. strain APG1, previously isolated from the aquatic fern Azolla pinnata, was examined in relation to nitric oxide (NO) production by cells cultured on a variety of C sources. Cells inoculated onto A. pinnata fronds established a surface-sterilant resistant density of 2–4 × 10 7 cells g −1 without causing disease. Compared to cultures containing glucose, fructose, mannitol, or glycerol, those provided only with sucrose displayed, on a per C basis, substantially lower (<10%) growth yields and higher resistance to H 2O 2. NO, a positive regulator of catalase synthesis in bacteria, was produced in larger amounts in sucrose-grown cells as evidence by eightfold greater per cell accumulations in the medium of nitrite (NO 2 −), a stable oxidation product of NO. Addition to cells of l-arginine, the substrate for nitric oxide synthase (NOS), stimulated production of NO, detected both by fluorometric reaction with diaminofluorescein-FM diacetate (DAF-FM DA) and by increased levels of NO 2 − in the culture medium. These results suggest that sucrose may enhance H 2O 2 tolerance of Rhodococcus APG1 by increasing cellular NO producing capacity. We propose a regulatory role for NOS in promoting tolerance of Rhodococcus APG1 to oxidative stress in the phyllosphere.

Metabolism of fructooligosaccharides by Lactobacillus paracasei 1195.

Fermentation of fructooligosaccharides (FOS) and other oligosaccharides has been suggested to be an important property for the selection of bacterial strains used as probiotics. However, little information is available on FOS transport and metabolism by lactic acid bacteria and other probiotic bacteria. The objectives of this research were to identify and characterize the FOS transport system of Lactobacillus paracasei 1195. Radiolabeled FOS was synthesized enzymatically from [(3)H]sucrose and purified by column and thin-layer chromatography, yielding three main products: glucose (G) alpha-1,2 linked to two, three, or four fructose (F) units (GF(2), GF(3), and GF(4), respectively). FOS hydrolysis activity was detected only in cell extracts prepared from FOS- or sucrose-grown cells and was absent in cell supernatants, indicating that transport must precede hydrolysis. FOS transport assays revealed that the uptake of GF(2) and GF(3) was rapid, whereas little GF(4) uptake occurred. Competition experiments showed that glucose, fructose, and sucrose reduced FOS uptake but that other mono-, di-, and trisaccharides were less inhibitory. When cells were treated with sodium fluoride, iodoacetic acid, or other metabolic inhibitors, FOS transport rates were reduced by up to 60%; however, ionophores that abolished the proton motive force only slightly decreased FOS transport. In contrast, uptake was inhibited by ortho-vanadate, an inhibitor of ATP-binding cassette transport systems. De-energized cells had low intracellular ATP concentrations and had a reduced capacity to accumulate FOS. These results suggest that FOS transport in L. paracasei 1195 is mediated by an ATP-dependent transport system having specificity for a narrow range of substrates.

Acid diffusion through extracellular polysaccharides produced by various mutants of Streptococcus mutans

Mutants of Streptococcus mutans V403, constructed by allelic exchange and altered in their capacity to produce enzymes involved in the production of extracellular polysaccharides from sucrose, were used to study the role of glucans and fructans in the diffusion of ions through cell concentrates. A 4.0 mm diameter, 0.75 mm deep diffusion chamber with an ion-sensitive field-effect transistor electrode positioned at the base was used to monitor the diffusion of hydronium ions from a sodium lactate buffer using cell concentrates prepared from bacteria grown in various concentrations of sucrose and glucose. The wild-type strain V403 produced at least seven times as much water-insoluble glucan (ISG) from sucrose as mutants deficient in various combinations of glucosyltransferase B (GTF B), GTF C, GTF D and fructosyltransferase. The fastest diffusion of hydronium ions occurred with sucrose-grown cell concentrates of strain V403, and the time of diffusion to the bottom of the chamber was approximately 2.3 times longer when this strain was grown in glucose. The speed of diffusion with glucose-grown V403 was similar to that obtained with each of the mutants. When cells of strain V403 grown in sucrose and glucose were mixed, increases in diffusion speed were found to be directly related to the proportion of sucrose-grown cells. The mixing of ISG with several strains of S. mutans revealed that increases in diffusion speed were directly related to the quantity of ISG added.

Metabolism of sucrose and its five isomers by Fusobacterium mortiferum.

Fusobacterium mortiferum utilizes sucrose [glucose-fructose in alpha(1-->2) linkage] and its five isomeric alpha-D-glucosyl-D-fructoses as energy sources for growth. Sucrose-grown cells are induced for both sucrose-6-phosphate hydrolase (S6PH) and fructokinase (FK), but the two enzymes are not expressed above constitutive levels during growth on the isomeric compounds. Extracts of cells grown previously on the sucrose isomers trehalulose alpha(1-->1), turanose alpha(1-->3), maltulose alpha(1-->4), leucrose alpha(1-->5) and palatinose alpha(1-->6) contained high levels of an NAD+ plus metal-dependent phospho-alpha-glucosidase (MalH). The latter enzyme was not induced during growth on sucrose. MalH catalysed the hydrolysis of the 6'-phosphorylated derivatives of the five isomers to yield glucose 6-phosphate and fructose, but sucrose 6-phosphate itself was not a substrate. Unexpectedly, MalH hydrolysed both alpha- and beta-linked stereomers of the chromogenic analogue p-nitrophenyl glucoside 6-phosphate. The gene malH is adjacent to malB and malR, which encode an EII(CB) component of the phosphoenolpyruvate-dependent sugar:phosphotransferase system and a putative regulatory protein, respectively. The authors suggest that for F. mortiferum, the products of malB and malH catalyse the phosphorylative translocation and intracellular hydrolysis of the five isomers of sucrose and of related alpha-linked glucosides. Genes homologous to malB and malH are present in both Klebsiella pneumoniae and the enterohaemorrhagic strain Escherichia coli O157:H7. Both these organisms grew well on sucrose, but only K. pneumoniae exhibited growth on the isomeric compounds.

Metabolism of Sucrose and Its Five Linkage-isomeric α-d-Glucosyl-d-fructoses by Klebsiella pneumoniae: PARTICIPATION AND PROPERTIES OF SUCROSE-6-PHOSPHATE HYDROLASE AND PHOSPHO-α-GLUCOSIDASE

Klebsiella pneumoniae is presently unique among bacterial species in its ability to metabolize not only sucrose but also its five linkage-isomeric alpha-d-glucosyl-d-fructoses: trehalulose, turanose, maltulose, leucrose, and palatinose. Growth on the isomeric compounds induced a protein of molecular mass approximately 50 kDa that was not present in sucrose-grown cells and which we have identified as an NAD(+) and metal ion-dependent 6-phospho-alpha-glucosidase (AglB). The aglB gene has been cloned and sequenced, and AglB (M(r) = 49,256) has been purified from a high expression system using the chromogenic p-nitrophenyl alpha-glucopyranoside 6-phosphate as substrate. Phospho-alpha-glucosidase catalyzed the hydrolysis of a wide variety of 6-phospho-alpha-glucosides including maltose-6'-phosphate, maltitol-6-phosphate, isomaltose-6'-phosphate, and all five 6'-phosphorylated isomers of sucrose (K(m) approximately 1-5 mm) yet did not hydrolyze sucrose-6-phosphate. By contrast, purified sucrose-6-phosphate hydrolase (M(r) approximately 53,000) hydrolyzed only sucrose-6-phosphate (K(m) approximately 80 microm). Differences in molecular shape and lipophilicity potential between sucrose and its isomers may be important determinants for substrate discrimination by the two phosphoglucosyl hydrolases. Phospho-alpha-glucosidase and sucrose-6-phosphate hydrolase exhibit no significant homology, and by sequence-based alignment, the two enzymes are assigned to Families 4 and 32, respectively, of the glycosyl hydrolase superfamily. The phospho-alpha-glucosidase gene (aglB) lies adjacent to a second gene (aglA), which encodes an EII(CB) component of the phosphoenolpyruvate-dependent sugar:phosphotransferase system. We suggest that the products of the two genes facilitate the phosphorylative translocation and subsequent hydrolysis of the five alpha-d-glucosyl-d-fructoses by K. pneumoniae.

Regulation of sucrose-6-phosphate hydrolase activity in Streptococcus mutans: characterization of the scrR gene.

Previous results have implicated an important role for the enzyme IIScr, the sucrose-specific permease, in the transport of sucrose by cariogenic Streptococcus mutans. The product of the scrB gene, sucrose-6-phosphate hydrolase (Suc-6PH), is required for the metabolism of phosphorylated sucrose. The results from the utilization of scrB::lacZ fusions in S. mutans GS-5 have suggested that sucrose-grown cells have higher levels of scrB gene expression than do cells grown with glucose or fructose. Northern blot analysis of scrB transcripts has also confirmed the relative strengths of expression as sucrose>glucose>fructose. Immediately downstream from the scrB gene, an open reading frame with homology to regulatory proteins of the GalR-LacI family as well as to ScrR proteins from several other bacteria has been identified. In addition, this gene appears to be transcribed in the same operon as scrB. Inactivation of this gene, scrR, did not alter the relative expression of the scrB gene in the presence of sucrose or fructose but did increase SUC-6PH levels in the presence of glucose to that observed with sucrose. Furthermore, the S. mutans ScrR homolog appears to bind to the scrB promoter region as determined from the results of gel shift assays. These results suggest that the scrR gene is involved in the regulation of scrB, and likely scrA, expression. However, it is not clear whether sucrose acts as an inducer of expression of these genes or, alternatively, whether glucose and fructose act as repressors.

Identification and Analysis of a Collagenolytic Activity in Streptococcus mutans

Streptococcus mutans is an important pathogen in coronal caries and is implicated in dental root decay by its ability to bind collagen from various sources. In the present study, electron microscopic analysis demonstrated the ability of S. mutans to bind and to disrupt collagen fibrils of the amniotic membrane. The synthetic peptide FALGPA, which is similar in structure to collagen, was degraded by S. mutans, with a lower level of FALGPA hydrolytic activity observed in sucrose-grown cells compared with cells grown in the absence of sucrose. Inhibition studies of FALGPA hydrolytic activity showed a pattern characteristic of collagenase activity, with inhibition by 1,10-phenanthroline and EDTA, but not by phenylmethylsulfonyl fluoride (PMSF). Additionally, immunological cross-reactivity was observed between proteins from disrupted cells of S. mutans and antiserum to collagenase from Clostridium histolyticum. Gelatinolytic activity was demonstrated by gelatin zymogram analysis. These findings suggest that collagenolytic activity by S. mutans may be an important virulence factor in dental root decay.

The cellular location of Prevotella ruminicola beta-1,4-D-endoglucanase and its occurrence in other strains of ruminal bacteria.

Prevotella ruminicola B(1)4, TC1-1, TF1-3, and TS1-5 all produced immunologically cross-reacting 88- and 82-kDa carboxymethyl cellulases (CMCases). P. ruminicola 23, 118B, 20-63, and 20-78 had much lower CMCase activities, and Western blots (immunoblots) showed no cross-reaction with the B(1)4 CMCase antiserum. Fibrobacter succinogenes S85 and Selenomonas ruminantium HD4 and D produced CMCase, but these enzymes were smaller and did not cross-react with the B(1)4 CMCase antiserum. The B(1)4 CMCase antiserum inhibited the B(1)4, TC1-1, TF1-3, and TS1-5 CMCase activities and agglutinated these cells, but it had no effect on the other strains or species. On the basis of these results, the B(1)4 CMCase is a strain-specific enzyme that is located on the outside surface of the cells. P. ruminicola B(1)4 cultures, grown on sucrose, did not have significant CMCase activity, but these cells could bind purified 88- and 82-kDa CMCase but not 40.5-kDa CMCase. Because the 40.5-kDa CMCase is a fully active, truncated form of the CMCase, it appears that the N-terminal domain of the 88-kDa B(1)4 CMCase anchors the CMCase to the cells. Cells grown on cellobiose produced at least 10-fold more CMCase than the sucrose-grown cells, and the cellobiose-grown cells could only bind 15% as much CMCase as sucrose-grown cells. Virtually all of the CMCase activity of exponentially growing cultures was cell associated, but CMCase activity was eventually detected in the culture supernatant. On the basis of the observation that the 88-kDa CMCase was gradually converted to the 82-kDa CMCase when cultures reached the stationary phase without a change in specific activity, it appears that the 82-kDa protein is probably a proteolytic degradation product of the 88-kDa CMCase.

Carbon Metabolism Enzymes of Rhizobium tropici Cultures and Bacteroids

We determined the activities of selected enzymes involved in carbon metabolism in free-living cells of Rhizobium tropici CFN299 grown in minimal medium with different carbon sources and in bacteroids of the same strain. The set of enzymatic activities in sucrose-grown cells suggests that the pentose phosphate pathway, with the participation of the Entner-Doudoroff pathway, is probably the primary route for sugar catabolism. In glutamate- and malate-grown cells, high activities of the gluconeogenic enzymes (phosphoenolpyruvate carboxykinase, fructose-6-phosphate aldolase, and fructose bisphosphatase) were detected. In bacteroids, isolated in Percoll gradients, the levels of activity for many of the enzymes measured were similar to those of malate-grown cells, except that higher activities of glucokinase, glucose-6-phosphate dehydrogenase, and NAD-dependent phosphogluconate dehydrogenase were detected. Phosphoglucomutase and UDP glucose pyrophosphorylase showed high and constant levels under all growth conditions and in bacteroids.

1004 THE DUAL ROLE OF MANNITOL AS OSMOPROTECTANT AND PHOTOASSIMILATE IN CELERY

Mannitol, a six carbon sugar alcohol, is widely distributed in nature and is a major phloem-translocated photoassimilate in celery. II may also function as a compatible osmolyte providing stress tolerance. Until recently, little was known about the route of mannitol catabolism in sink tissues of higher plants. An enzyme. mannitol dehydrogenase. (MDH) that oxidizes mannitol to mannose utilizing NAD as the electron acceptor was discovered (Arch. Biochem. Biophys. 1991. 298:612-619) in “sink” tissues of celery and celeriac plants. The activity of the enzyme is inversely related to tissue mannitol concentration in various parts of celery plants suggesting a role for the enzyme in mannitol catabolism. In osmostressed celery plants, the activity of the enzyme in sink tissues decreases as mannitol accumulates. Celery cells growing heterotrophically in suspension culture utilize either sucrose or mannitol as the sole carbon source and grow equally well on either carbohydrate. Mannitol-grown cells contain more MDI-I activity than sucrose-grown cells, and the activity of the enzyme is correlated with the rate of depletion of mannitol from the culture medium. Cells growing on mannitol contain an internal pool of mannitol but little sugar. Cells growing on sucrose contain internal sugar pools but no mannitol. Mannitol-grown cells are also more salt tolerant than cells grown on sucrose. Our laboratory is involved in studies of the physiological role of the mannitol oxidizing enzyme in regulating mannitol utilization and the role of the enzyme in regulating mannitol pool size during salt and osmostress in both celery plants and celery suspension cultures. Current studies on the molecular control of expression of the enzyme will be discussed.

Effect of Different Carbon Sources on Relative Growth Rate, Internal Carbohydrates, and Mannitol 1-Oxidoreductase Activity in Celery Suspension Cultures.

Little information exists concerning the biochemical route of mannitol catabolism in higher plant cells. In this study, the role of a recently discovered mannitol 1-oxidoreductase (MDH) in mannitol catabolism was investigated. Suspension cultures of celery (Apium graveolens L. var dulce [Mill.] Pers.) were successfully grown on nutrient media with either mannitol, mannose, or sucrose as the sole carbon source. Cell cultures grown on any of the three carbon sources did not differ in relative growth rate, as measured by packed cell volume, but differed drastically in internal carbohydrate concentration. Mannitol-grown cells contained high concentrations of mannitol and extremely low concentrations of sucrose, fructose, glucose, and mannose. Sucrose-grown cells had high concentrations of sucrose early in the growth cycle and contained a substantial hexose pool. Mannose-grown cells had a high mannose concentration early in the cycle, which decreased during the growth cycle, whereas their internal sucrose concentrations remained relatively constant during the entire growth cycle. Celery suspension cultures on all three carbon substrates contained an NAD-dependent MDH. Throughout the growth cycle, MDH activity was 2- to 4-fold higher in mannitol-grown cells compared with sucrose- or mannose-grown cells, which did not contain detectable levels of mannitol, indicating that MDH functions pre-dominantly in an oxidative capacity in situ. The MDH activity observed in celery cells was 3-fold higher than the minimum amount required to account for the observed rate of mannitol utilization from the media. Cultures transferred from mannitol to mannose underwent a decrease in MDH activity over a period of days, and transfer from mannose to mannitol resulted in an increase in MDH activity. These data provide strong evidence that MDH plays an important role in mannitol utilization in celery suspension cultures.

Polysaccharide production by a reduced pigmentation mutant of the fungusAureobasidium pullulans

A reduced pigmentation mutant was isolated from Aureobasidium pullulans ATCC 42023 by chemical mutagenesis and was subsequently characterized. The pigment melanin was present not only in A. pullulans cells but also contaminated the elaborated polysaccharide and thus, was measured in both fractions. Cellular and polysaccharide melanin levels of the mutant strain were at least 11-fold and 18-fold reduced, respectivelu, compared toits parent strain after 7 days of growth at 30°C whether sucrose or glucose served as the carbon source in the culture medium. Polysaccharide and cell dry weight levels of the mutant were very similar to those observed for the parent after growth on sucrose or glucose as the source of carbon over a period of 7 days at 30°C. The pullulan content of the polysaccharide produced by the parent or mutant strain was lower for sucrose-grown cells than for glucose-grown cells. It was also noted that the pullulan content of the polysaccharide elaborated by the mutant strain was slightly higher than that of the polysaccharide produced by the parent strain after growth on either sucrose or glucose.

Biotransformation of chloromethane to methanethiol

Monochloromethane (MeCl) was biotransformed by a mixed culture of anaerobic bacteria which had been enriched to utilize dichloromethane as the sole source of carbon and energy. Cells grown in dichloromethane-salts medium catalyzed the transformation, whereas sucrose-grown cells were inactive. MeCl disappeared almost totally, and the methyl group was recovered in a yield of 80-90% as methanethiol and traces of dimethyl sulfide, whose identities were confirmed by GC-MS. The methyl transfer was heat-labile, and free aquocobalamin did not promote the reaction, which was apparently enzymically catalyzed. 37 refs., 1 fig., 2 tabs.

Glucose and sucrose fermenting capacity of homofermentative lactic acid bacteria used as starters in fermented salads

Salads, i.e., vegetables and/or meat in an oil-in-water emulsion with a low pH due to the addition of acids, are popular in western countries. The shelf life of these salads is influenced mainly by storage be an interesting novel approach to salad preparation. As part of an investigation on fermented salads, lactic acid bacteria from the genera Lactobacillus, Pediococcus and Streptococcus were screened for their capacity to ferment glucose and sucrose in a model sysem and in a standard sald. The temperature optimum of these strains was in the range of 34–48°C. Most of these strains produced about equal amounts of d(+)- and d(−)- lactic acid. To determined the specific sugar fermenting capacity (mmol lactic acid produced per min per g dry weight) of non-growing cells of lactic acid bacteria, a semi-automated model system was used, based on monitoring the pH decrease in a phosphate buffer during conversion of sugars to lactic acid. The strains tested showed acid production rates on glucose varying between 0.03 and 0.39 mmol of acid produced per min per g dry weight (average 0.23). These glucose-grown cells showed varying, but lower fermentation rates on sucrose (0–0.25 mmol/min/g d.w., average 0.04) than sucrose-grown cells (0–0.33 mmol/min/g d.w., average 0.19). Results obtained with the model system were in good agreement with those of fermentation tests, carried out with a standard salad. Inhibition of spoilage bacteria in the standard salad could be achieved by using starter cultures with high fermenting capacity.

Carbon catabolism in continuous cultures and bacteroids of Rhizobium leguminosarum MNF 3841

Bacteroids of R. leguminosarum MNF3841 isolated from pea nodules using Percoll gradients had activities of TCA cycle enzymes up to 6-fold higher than those measured in free-living cells grown on fumarate or sucrose. Activities of sugar catabolic enzymes on the other hand were 2–14-fold lower in isolated bacteroids than in sucrose-grown free-living cells. In continuous culture, cells of strain MNF3841 grown on sucrose under Pi limitation had 2–3-fold higher activities of invertase, glucose-6-phosphate dehydrogenase, the Entner-Doudoroff enzymes and 6-phosphogluconate dehydrogenase, than cells grown on fumarate. With one exception O2 limited cultures had similar activities of the carbon catabolic enzymes to Pi-limited cultures grown in the same substrate. Glucose-6-phosphate dehydrogenase in O2-limited cells grown of fumarate was 50% lower than in Pi-limited cells. Co-utilization of fumarate and sucrose occurred with chemostat cultures supplied with both under a variety of conditions.