Glucose-grown Cells Research Articles

Initial catabolism of sorbitol in Actinomyces naeslundii and Actinomyces viscosus.

The initial steps of sorbitol catabolism were studied in 4 strains of Actinomyces naeslundii and Actinomyces viscosus that had been isolated from human dental plaque. Cell-free extracts were prepared from cells grown in the presence of either sorbitol, xylitol or glucose. The extracts from all strains grown on sorbitol had nicotinamide adenine dinucleotide-linked dehydrogenase activities for sorbitol and xylitol and reduced nicotinamide adenine dinucleotide-linked reductase activities for fructose and xylulose. Two of the strains also exhibited these activities when grown in the presence of xylitol, and all glucose-grown cells lacked them. The results indicate that sorbitol metabolism in oral actinomyces involve oxidation of sorbitol to fructose by an inducible enzyme, nicotinamide adenine dinucleotide-linked sorbitol dehydrogenase. This step is followed by the phosphorylation of fructose with guanosine triphosphate as a main phosphoryl donor. Thus, the initial catabolic pathway of sorbitol in A. naeslundii and A. viscosus is different from those described for other oral bacteria.

Isolation and characterisation of the linked genes, FPS1 and QCR8, coding for farnesyl-diphosphate synthase and the 11 kDa subunit VIII of the mitochondrial bc 1-complex in the yeast Kluyveromyces lactis

The KlQCR8 gene of the yeast Kluyveromyces lactics encoding subunit VIII of the mitochondrial bc 1 complex is 70.2% identical to its counterpart in Saccharomyces cerevisiae (ScQCR8). As in S. cerevisiae, chromosomal linkage between the K. lactis QCR8 and FPS1 genes is conserved, the two genes being separated by only 292 bp. Disruption of the KlQCR8 gene results in a respiratory-deficient phenotype. Compared with S. cerevisiae, expression of the KlQCR8 gene in glucose-grown cells is relatively high, yet is significantly induced when the cells are grown on non-fermentable carbon sources. The QCR8 promoters regions of the two yeasts lack overall DNA sequence similarity, but share DNA-binding sites for the transcription factors ABF1, CPF1 and HAP2/3/4. Deletion from the KlQCR8 promoter of a 93 bp region containing these sites significantly lowers mRNA levels during growth on either glucose or ethanol/glycerol, with a consequent reduction of growth rate on ethanol/glycerol.

Regulation of phosphatidate phosphatase activity from the yeast Saccharomyces cerevisiae by nucleotides.

Regulation of Saccharomyces cerevisiae membrane-associated phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) activity by nucleotides was examined using pure enzyme and Triton X-100/phosphatidate-mixed micelles. Adenosine, guanosine, cytidine, and uridine nucleotides inhibited phosphatidate phosphatase activity in a dose-dependent manner. ATP and CTP were the most potent inhibitors of the enzyme. A kinetic analysis was performed to determine the mechanism of enzyme inhibition by nucleotides. The mechanism of inhibition by ATP and CTP with respect to phosphatidate (the substrate) was complex. The dependence of phosphatidate phosphatase activity on phosphatidate was cooperative, and nucleotides affected both Vmax and Km. ATP did not inhibit phosphatidate phosphatase activity by binding to the enzyme or to phosphatidate. Phosphatidate phosphatase dependence on Mg2+ ions (the cofactor) followed saturation kinetics, and the mechanism of nucleotide inhibition with respect to Mg2+ ions was competitive. Thus, the mechanism of enzyme inhibition by nucleotides was the chelation of Mg2+ ions. The inhibitor constant for ATP was lower than its cellular concentration in glucose-grown cells. However, the inhibitor constant for ATP was higher than its cellular concentration in glucose-starved cells. Changes in the cellular concentration of ATP affected the proportional synthesis of triacylglycerols and phospholipids. These results were consistent with the regulation of phosphatidate phosphatase activity by ATP through a Mg2+ ion chelation mechanism.

Gellan lyases-novel polysaccharide lyases

A number of bacterial strains capable of degrading the bacterial exopolysaccharide gellan have been isolated by standard enrichment procedures. They include several pink-pigmented Gram-negative rod-shaped bacteria. A red-pigmented Gram-positive bacillus earlier found to degrade the exopolysaccharide xanthan from Xanthomonas campestris also showed slight gellanase activity. All the Gram-negative bacteria are non-fermentative, motile and amylase-producing. The gellan degradation in each case is due to eliminase-type enzymes (lyases) which appear to be extracellular enzymes cleaving the sequence... beta-D-glucosyl-(1-->4)-beta-D-glucuronosyl... in the tetrasaccharide repeat unit of the substrate polysaccharides. Although in some isolates these enzymes appear to be exo-acting, it appears from the loss in viscosity of the alternative substrate deacetylated rhamsan that they are predominantly endoenzymes. The enzyme activity is inducible: it is almost absent from glucose-grown cells. Associated with the 'gellanase' activity, all the Gram-negative bacterial isolates possess intracellular alpha-L-rhamnosidase and beta-D-glucosidase activities apparently located in the periplasm. The enzymes are highly specific and fail to cause significant degradation of most of the other bacterial exopolysaccharides which have been shown to be structurally related to gellan. As well as acting on gellan, they exert similar degradative activity against the chemically deacylated form of polysaccharide S194 (rhamsan gum), which is effectively a gentiobiosylated form of gellan. The enzymes only have relatively slight activity against the natural, acylated gellan-like polysaccharides from the bacteria now designated as strains of Sphingomonas paucimobilis.

Growth rate influences MF alpha 1 promoter activity in MAT alpha Saccharomyces cerevisiae.

The signal sequences of the MF alpha 1 prepro alpha-factor gene are frequently used to direct secretion of heterologous proteins from Saccharomyces cerevisiae. They are often employed together with the MF alpha 1 promoter in secretion vectors, such that this promoter directs the transcription of many heterologous gene cassettes in yeast. Most of the existing literature indicates that the MF alpha 1 promoter is constitutive in MAT alpha cells, although some data suggests that it may be more active in respiratory or late logarithmic fermentative cultures. To identify whether there is a growth rate or medium control over MF alpha 1 promoter activity a strain was constructed with an integrated MF alpha 1 promoter-beta-galactosidase (lacZ) reporter gene fusion. Intracellular beta-galactosidase of this strain during batch culture on glucose, raffinose and acetate showed that MF alpha 1 promoter activity was higher during respiratory growth on acetate as compared to more rapid fermentative growth on glucose or raffinose, a result that might indicate this activity being inversely related to growth rate. Chemostat culture confirmed that growth rate does indeed influence MF alpha 1 promoter activity in glucose-grown cells, the activity of this promoter increasing 2- to 2.5-fold as dilution (growth) rates were reduced from maximal values to 0.2 h-1, but then decreasing with the further decreases in dilution rate needed for fully respiratory growth. Thus a promoter generally thought to be constitutive in MAT alpha cells is nevertheless subject to a complex growth rate control.

Growth and regulation of enzyme synthesis in the nitrilotriacetic acid (NTA)-degrading bacterium Chelatobacter heintzii ATCC 29600

In the aerobic bacterium Chelatobacter heintzii, growth and regulation of enzymes involved in nitrilotriacetic acid (NTA) degradation have been investigated in chemostat culture during cultivation with glucose, NTA or mixtures thereof. In batch culture mu max with NTA was 0.18 h-1 and with glucose 0.22 h-1. Growth yields for both substrates were reduced at low dilution rates. During growth with NTA specific activity of the NTA monooxygenase (NTA-MO) exhibited a maximum at D = 0.03 h-1 and gradually decreased with increasing dilution rates. In glucose-grown cells the specific activity as well as immunologically detectable NTA-MO protein was always close to the detection limit. During cultivation with different mixtures of NTA and glucose at a dilution rate of 0.06 h-1, both substrates were utilized simultaneously, irrespective of the NTA/glucose ratio and the presence of excess ammonia. Synthesis of both NTA-MO and iminodiacetic acid dehydrogenase became induced when NTA contributed to more than approximately 1-3% of the total carbon in the substrate mixture supplied. However, NTA was also degraded when the proportion of NTA in the mixture was lower than 1%, which is consistent with the low constitutive level of expression for NTA-MO observed. Results are discussed with respect to NTA biodegradation during sewage treatment and in ecosystems.

Mechanism of maltose uptake and glucose excretion in Lactobacillus sanfrancisco

Lactobacillus sanfrancisco LTH 2581 can use only glucose and maltose as sources of metabolic energy. In maltose-metabolizing cells of L. sanfrancisco, approximately half of the internally generated glucose appears in the medium. The mechanisms of maltose (and glucose) uptake and glucose excretion have been investigated in cells and in membrane vesicles of L. sanfrancisco in which beef heart cytochrome c oxidase had been incorporated as a proton-motive-force-generating system. In the presence of ascorbate, N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), and cytochrome c, the hybrid membranes facilitated maltose uptake against a concentration gradient, but accumulation of glucose could not be detected. Similarly, in intact cells of L. sanfrancisco, the nonmetabolizable glucose analog alpha-methylglucoside was taken up only to the equilibration level. Selective dissipation of the components of the proton and sodium motive force in the hybrid membranes indicated that maltose is transported by a proton symport mechanism. Internal [14C]maltose could be chased with external unlabeled maltose (homologous exchange), but heterologous maltose/glucose exchange could not be detected. Membrane vesicles of L. sanfrancisco also catalyzed glucose efflux and homologous glucose exchange. These activities could not be detected in membrane vesicles of glucose-grown cells. The results indicate that maltose-grown cells of L. sanfrancisco express a maltose-H+ symport and glucose uniport system. When maltose is the substrate, the formation of intracellular glucose can be more rapid than the subsequent metabolism, which leads to excretion of glucose via the uniport system.

Microbial oxidation of cumene by octane-grown cells

Previously, we reported that eight glucose-grown microbial cultures out of 1229 screened oxidize the alkyl side-chain of 2-phenylpropane (cumene) stereospecifically. Now, we have adapted these cultures to grow on n-octane and found that their cumene oxidation activities increased more than 30 times. We also found an additional 11 cultures (ten bacteria, one actinomycete) that oxidized cumene when grown on octane but not on glucose. In general, octane-grown cells were more active in cumene oxidation than glucose-grown cells. Rhodococcus rhodochrous NRRL B-2153 showed the best conversion yield (2-phenyl-1-propanol plus 2-phenyl-1-propionic acid was 5.5%) at 25°C, pH 8.0, 250 rpm, and 12 h of reaction. Structures of the reaction products were confirmed by gas chromatography (GC)/mass spectrometry and GC/infrared analyses. Products contained 84% ee (enantiomeric excess) of the R(−) isomer, as analyzed with a GC cyclodextrin chiral column. Strain B-2153 oxidized alkylbenzenes in the following order of reaction rate: ethylbenzene >amylbenzene > butylbenzene > cumene > propylbenzene > sec-butylbenzene. tert-Butylbenzene was not oxidized.

Expression of the Saccharomyces cerevisiae CYT2 gene, encoding cytochrome c1 heme lyase.

In this paper we examine the expression of the Saccharomyces cerevisiae CYT2 gene, which encodes cytochrome c1 heme lyase. This enzyme is required for covalent attachment of heme to apocytochrome c1, a subunit of the mitochondrial respiratory chain. Transcription of the 1-kb CYT2 mRNA initiates at four prominent sites at a distance of 52-225 bp in front of the AUG start codon. The level of CYT2 mRNA is not influenced by the presence or absence of oxygen or of heme, but it is subject to carbon-source control. The concentration of the CYT2 mRNA is significantly reduced in glucose-grown cells as compared to cells grown under non-repressing conditions. Neither the HAPp activator proteins nor MIG1p, a repressor protein involved in glucose repression, seem to mediate this effect.

Genetic regulation of fructosyltransferase in Streptococcus mutans

Streptococcus mutans possesses several extracellular sucrose-metabolizing enzymes which have been implicated as important virulence factors in dental caries. This study was initiated to investigate the genetic regulation of one of these enzymes, the extracellular fructosyltransferase (Ftf). Fusions were constructed with the region upstream of the S. mutans GS5 Ftf gene (ftf) and a promoterless chloramphenicol acetyltransferase (CAT) gene. The fusions were integrated at a remote site in the chromosome, and transcriptional activity in response to the addition of various carbohydrates to the growth medium was measured. A significant increase in CAT activity was observed when glucose-grown cells were shifted to sucrose-containing medium. Sucrose-induced expression was repressed immediately upon addition of phosphoenolpyruvate phosphotransferase system sugars to the growth media. Deletion analysis of the ftf upstream region revealed that an inverted repeat structure was involved in the control of ftf expression in response to carbohydrate. However, the control of the level of ftf transcription appeared to involve a region distinct from that mediating carbohydrate regulation. CAT gene fusions also were constructed with the ftf upstream region from S. mutans V403, a fructan-hyperproducing strain which synthesizes increased levels of Ftf. Sequence analysis of the upstream ftf region in this strain revealed several nucleotide sequence changes which were associated with high-level ftf expression. Comparison of the GS5 and V403 ftf expression patterns suggested the presence of a trans-acting factor(s) involved in modulation of ftf expression in response to carbohydrate. This factor(s) was either absent or altered in V403, resulting in the inability of this organism to respond to the presence of carbohydrate. The sequences of the ftf regions from three additional fructan-hyperproducing strains were determined and compared with that of V403. Only one strain displayed nucleotide changes similar to those of V403. Two additional strains did not have these changes, suggesting that several mechanisms for up-regulation of ftf expression exist.

Identification of the Maltose Transport Protein of Saccharomyces cerevisiae

Identification of the maltose transport protein of Saccharomyces cerevisiae was attempted by searching for maltose-inducible proteins in isolated plasma membranes. Membranes from maltose-grown cells contained two proteins that were absent in glucose-grown cells. The proteins differed in size, but peptide sequence analysis indicated a high degree of homology. The amino-terminal and internal sequences of the largest protein, with an apparent molecular mass of 64 kDa, were determined. These sequences were identical to predicted amino acid sequences in the MAL61 gene product. It is concluded that this protein is the inducible maltose permease of Saccharomyces cerevisiae.

Microbial oxidation of cumene

A total of 1229 cultures, including 230 actinomycetes, 508 other bacteria, 12 fungi and 479 yeasts were screened for their ability to oxidize the isopropyl side chain of 2-phenyl propane (cumene). Four strains of actinomycetes and six strains of bacteria but no yeasts were found positive in converting 2-phenyl propane to its oxygenated products. Eight strains oxidized cumene through the alkyl side chain producing 2-phenyl-1-propanol. TwoBacillus strains oxidized cumene to an oxygenated product.Pseudomonas oleovorans NRRL B-3429 exhibited the highest alkyl side chain oxidation activity. The optimum reaction conditions for strain B-3429 are: 25 °C, pH 6.5 and 48 h of reaction. Octane-grown cells of strain B-3429 produced higher product yields (about 7.2-fold) than the glucose-grown cells. Prolonged incubation resulted in an increase in 2-phenyl-1-propionic acid production at the expense of 2-phenyl-1-propanol. The yield of 2-phenyl-1-propanol plus 2-phenyl-1-propionic acid was 5.1%. Reaction in the presence of methanol favored the accumulation of 2-phenyl-1-propionic acid and also increased the total yield. (The yield of 2-phenyl-1-propanol plus 2-phenyl-1-propionic acid was 14.9%.) Structures of the reaction products were confirmed by GC/MS and GC/IR analyses. Products contained 92% R(−) isomer.

Production of sophorose lipid by Candida (Torulopsis) apicola grown on glucose

The production of extracellular sophorose lipid by Candida apicola IMET 43747 was studied after growth on glucose as sole carbon source. In the presence of citrate and after 120 h of cultivation, the microcrystalline lactonic sophorose lipid started to appear in the medium. The lipid was shown to accumulate in the cell wall and only a minor part was liberated into the medium. By means of NMR studies it was shown that the cell wall-bound and the liberated lactonic sophorose lipid were identical and had a hydroxy Δ 8-hexadecenoic acid backbone. Biotransformation of d-[1- 13C]glucose revealed that in glucose-grown cells glucose was degraded to trioses which were rearranged and flow backward via the gluconeogenic pathway into the glycolipid as documented by scrambling of the 13C label from the C-1 position into the d-[6- 13C]glucose of sophorose. A further part of the trioses was used for de novo synthesis of the glycolipid fatty acid moiety via acetyl-CoA. By use of a mixed substrate, i.e., glucose and hexadecane, part of the glucose added was directly incorporated into the carbohydrate moiety of the lipid. These results are discussed in terms of the physiological significance of the sophorose lipid to act as extracellular carbon storage material.

Occurrence of fatty alcohol oxidase in alkane-and fatty-acid-utilising yeasts and moulds

Preparations of membrane fractions from 16 yeasts and three moulds were assayed for long-chain fatty alcohol oxidase (FAOD) activities after being grown on hexadecane or glucose and, in nine cases, on oleic acid. The enzyme was usually repressed in glucose-grown cells but in Candida bombicola ATCC 22 214 and Debaryomyces hansenii NCYC 33 appeared to be constitutive. Highest activities occurred in C. tropicalis and D. polymorphus (about 0.8 unit/mg protein) grown on hexadecane. Growth of yeasts on oleic acid partially induced FAOD activity but not with the moulds. In two strains of Yarrowia lipolytica (DSM 3286 and CBS 2076) no activity of FAOD was found but this could have been due to the known photo-lability of the enzyme. FAOD from different species shared similar characteristics with respect to substrate specificity and pH optimum.

Xylose and Glucose Utilization by Bacteroides xylanolyticus X5-1 Cells Grown in Batch and Continuous Culture

During cultivation on a mixture of xylose and glucose, Bacteroides xylanolyticus X5-1 showed neither diauxic growth nor a substrate preference. Xylose-limited continuous-culture cells were able to consume xylose and glucose both as single substrates and as mixed substrates without any lag phase. When glucose was the growth-limiting substrate, the microorganism was unable to consume xylose. However, in the presence of a small amount of glucose or pyruvate, xylose was utilized after a short lag phase. In glucose-limited cells, xylose isomerase was present at low activity but xylulose kinase activity could not be detected. On addition of a mixture of xylose and glucose, xylose isomerase was induced immediately and xylulose kinase was induced after about 30 min. The induction of the two enzymes was sensitive to chloramphenicol, showing de novo synthesis. Xylose uptake in glucose-grown cells was very low, but the uptake rate could be increased when incubated with a xylose-glucose mixture. The increase in the uptake rate was not affected by chloramphenicol, indicating that a constitutive uptake system had to be activated. The inability of B. xylanolyticus X5-1 cells undergoing glucose-limited continuous culture to induce the xylose catabolic pathway after the addition of only xylose probably was caused by energy limitation.

Physiological aspects of disinfection resistance in Pseudomonas cepacia.

A Pseudomonas cepacia population was isolated which had reduced susceptibility to iodine and maintained resistance when subcultured several times in phosphate buffer. This population was also resistant to iodine after growth in a minimal medium containing glycerol but not glucose. Addition of cAMP to glucose-grown cells caused increased resistance to iodine. Iodine-resistant cultures also demonstrated reduced susceptibility to chlorination but not to heat or metals (Cu/Ag). The results indicate that halogen resistance can be expressed in varying degrees, dependent on the carbon source, and cAMP may promote this expression. Thus, a catabolite repression-like mechanism may cause resistant cultures grown in some media to become more sensitive to halogens.

Chromatin structure and regulation of the eukaryotic regulatory gene GAL80.

The chromatin structure around the 5' end of the yeast regulatory gene GAL80 has been determined. The chromatin organization is very similar to that on the 5' regions of the GAL1-10 structural genes: a constitutive hypersensitive region containing the upstream activating sequence (UAS) element, and nucleosomes around this hypersensitive region. The downstream nucleosome, which is a positioned nucleosome, covers the TATA and transcription start sites. The nucleosome upstream of the hypersensitive region undergoes significant change when cells are grown in galactose, where GAL80 gene expression is induced to maximal levels. The change may be related to the induction process. GAL4 protein binds strongly to the GAL80 UAS in galactose-grown cells, less strongly in glycerol-grown cells, and not at all in glucose-grown cells. These data and published gene expression data are used to develop a model for the regulation of the GAL80 regulatory gene.

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.

Separation of outer and cytoplasmic membranes of Fibrobacter succinogenes and membrane and glycogen granule locations of glycanases and cellobiase

The outer membrane (OM) of Fibrobacter succinogenes was isolated by a combination of salt, sucrose, and water washes from whole cells grown on either glucose or cellulose. The cytoplasmic membrane (CM) was isolated from OM-depleted cells after disruption with a French press. The OM and membrane vesicles isolated from the extracellular culture fluid of cellulose-grown cells had a higher density, much lower succinate dehydrogenase activity, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles different from those of the CM. The OM from both glucose- and cellulose-grown cells and the extracellular membrane vesicles from cellulose-grown cultures exhibited higher endoglucanase, xylanase, and acetylesterase activities than the CM and other cell fractions. Endoglucanase 2 was absent from the isolated OM fractions of glucose- and cellulose-grown cells and from the extracellular membrane vesicles of cellulose-grown cells but was present in the CM and intracellular glycogen granule fractions, while endoglucanase 3 was enriched in the OM. Cellobiosidase was located primarily in the periplasm as previously reported, while cellobiase was mainly present in the glycogen granule fraction of glucose-grown cells and in a nongranular glycogen and CM complex in cellulose-grown cells. The cellobiase was not eluted from glycogen granules by cellobiose, maltose, and maltotriose nor from either the granules or the cell membranes by nondenaturing detergents but was eluted from both glycogen granules and cell membranes by high concentrations of salts. The eluted cellobiase rebound almost quantitatively when diluted and mixed with purified glycogen granules but exhibited a low affinity for Avicel cellulose. Thus, we have documented a method for isolation of OM from F. succinogenes, identified the OM origin of the extracellular membrane vesicles, and located glycanases and cellobiase in membrane and glycogen fractions.

Glucose uptake by Cellulomonas fimi

Glucose uptake by whole-cell suspension of the facultative anaerobe Cellulomonas fimi, which was two-fold higher under aerobic conditions than under N2 or H2, was inhibited by inhibitors of electron transport and ATP synthesis and, particularly, by proton and metal ion ionophores. A variety of sugars, including 2-deoxyglucose, did not inhibit glucose uptake but cellobiose was a non-competitive inhibitor. Cells grown on cellobiose medium transported glucose at one half the rate of glucose-grown cells. Cellulomonas fimi has a highly specific active system for glucose transport.