Identification of indolepyruvic acid as an intermediate of rebeccamycin biosynthesis

Summary Experimental evidence is presented to demonstrate that indolepyruvic acid is an intermediate in the rebeccamycin biosynthetic pathway. [3-¹⁴C]Indolepyruvic acid was prepared and efficiently incorporated (8%) into rebeccamycin bySaccharothrix aerocolonigenes.     

Particle-based production of antibiotic rebeccamycin with Lechevalieria aerocolonigenes

Rebeccamycin is a promising antibiotic synthesized by the filamentous bacterium Lechevalieria aerocolonigenes. To date, the main investigations were focused on the biological effect or the structure elucidation of rebeccamycin. The aim of this study was to develop an efficient cultivation process based on shake flask experiments. The relationship between morphology and productivity and the positive effects of micro- and macroparticle enhanced cultivation on rebeccamycin production with this strain in the pellet-like morphology was investigated. The addition of talc microparticles increased the rebeccamycin concentration to 120 mg L⁻¹ and 3-fold compared to the control without microparticles. An up to 9-fold increase in rebeccamycin concentration was achieved using surface modified talc particles. Through the addition of glass beads as macroparticles, the highest rebeccamycin concentration of approximately 120 mg L⁻¹ was achieved by the induction of mechanical stress. Cultivation with glass beads enhanced the rebeccamycin concentration 22-fold compared to the control without macroparticles. By further increasing the diameter of the glass beads and increasing mechanical stress, the morphology was driven toward mycelial growth and the productivity was decreased. An adjusted mechanical stress, through the addition of macro-shaped glass bead particles, led to the desired increase in productivity.     

Regulation of cephalosporin synthesis in Cephalosporium acremonium by phosphate and glucose

The regulation of cephalosporin synthesis in Cephalosporium acremonium was studied in a simple chemically-defined medium with glucose as the carbon source. Antibiotic synthesis depended on the phosphate content of the medium. At phosphate concentrations above 2.75 mM maximum antibiotic titres were not reached while glucose uptake and growth rates were increased. Phosphate exerted its effect indirectly by regulating the rate of glucose consumption. The negative effect of high phosphate concentrations could be overcome completely by controlling the sugar supply in fed-batch and chemostat experiments. High actual concentrations of phosphate or of glucose alone had no direct negative effect on antibiotic synthesis.     

Production of PHB and P (3HB-co-3HV) biopolymers by <Emphasis Type="Italic">Bacillus megaterium</Emphasis> strain OU303A isolated from municipal sewage sludge

Bacillus megaterium strain OU303A isolated from municipal sewage sludge was selected for the study of biosynthesis of polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-hydroxyvalerate P (HB-co-HV) copolymer. The strain yielded a maximum of 62.43% DCW polymer in the medium containing glycerol as carbon source, which was followed by 58.63% DCW polymer in glucose containing medium. We found that this strain was capable of producing 2.5% hydroxyvalerate copolymer from a single carbon substrate, glucose. The strain showed an increase in the amount of HV monomer content, when the precursor for the copolymer was included in the fermentation medium. The characterization of the biopolymers was carried out using FTIR, GC-MS, H¹ NMR and DSC. This is the first report of B. megaterium strain producing HV copolymer, without the addition of any precursor in the fermentation medium.     

Use of response surface methodology to examine chitinase regulation in the basidiomycete Moniliophthora perniciosa

We report here the first analysis of chitinase regulation in Moniliophthora perniciosa, the causal agent of the witches' broom disease of cacao. A multivariate statistical approach was employed to evaluate the effect of several variables, including carbon and nitrogen sources and cultivation time, on M. perniciosa non-secreted (detected in mycelium, i.e. in symplasm and cell wall) and secreted (detected in the culture medium) chitinase activities. Non-secreted chitinase activity was enhanced by peptone and chitin and repressed by glucose. Chitinase secretion was increased by yeast extract alone or in combination with other nitrogen sources, and by N-acetylglucosamine, and repressed in presence of chitin. The best cultivation times for non-secreted and secreted chitinase activities were 30 and 20 d, respectively. However, chitinase activity was always higher in the mycelium than in the culture medium, suggesting a relatively poor chitinase secretion activity. Conversely, higher mycelial growth was observed when the activity of the non-secreted chitinase was at its lowest, i.e. when the fungus was grown on glucose and yeast extract as sources of carbon and nitrogen, respectively. Conversely, the induction of non-secreted chitinase activity by chitin decreased the mycelium growth. These results suggest that the culture medium, by the induction or repression of chitinases, affected the hyphal growth. Thus, as an essential component of M. perniciosa growth, chitinases may be a potential target for strategies to control disease.     

Effect of simple and complex carbon sources,low temperature culture and complex carbon feeding policies on poly-3-hydroxybutyric acid (PHB) content and molecular weight (Mw) from Azotobacter chroococcum 6B

The molecular weight (M _w) of poly-3-hydroxybutyrate (PHB), produced by shake-flask culture of Azotobacter chroococcum showed little variation with increasing glucose concentration as carbon source (being in the range of 400–500 kDa), while M _w increased from 300–400 to 640 kDa when grown with increasing concentration of sugar cane molasses. Molecular weight increased nearly 30% from 48 to 72 h culture time when 5% molasses as carbon source was used, while with glucose the highest M _w was reached at 48 h. Under fermentor cultivation A. chroococcum produced PHB with a relatively high M _w of 1590 kDa at 53 h culture time when grown in modified Burk's medium with glucose as carbon source at an initial C/N ratio (molar basis) of 69 under fermentor cultivation. A batch glucose-grown ammonium-limited fermentor culture was repeatedly fed with sugar cane molasses (initial C/N ratio 69) and it was observed that PHB content curve decreased at a slower rate than in the fed-batch culture in which glucose and sucrose were not consumed in the culture medium after the feed.     

Regulatory flexibility of methylotrophic yeasts in chemostat cultures: Simultaneous assimilation of glucose and methanol at a fixed dilution rate

The influence of the composition of methanol/glucose-mixtures as only sources of carbon and energy on growth and regulation of the synthesis of enzymes involved in methanol-dissimilation was studied under chemostat conditions at a fixed dilution rate with the methylotrophic yeasts Hansenula polymorpha and Kloeckera sp. 2201. Both carbon sources were found to be utilized completely independently of the composition of the C₁/C₆ mixture. Using mixtures of ¹⁴C-labelled methanol and glucose the growth yield for glucose was found to be constant for all C₁/C₆-mixtures tested and both yeasts. The growth yield for methanol, however, was reduced by up to 25% when the proportion of methanol in the inflowing medium was lower than 20% (w/w with respect to glucose) for H. polymorpha and 50% (w/w with respect to glucose) for Kloeckera sp. 2201 respectively. During growth with C₁/C₆-mixtures containing higher C₁-proportions of methanol regular growth yields for methanol were recorded which corresponded to the growth yields found with methanol as the only carbon source.The regulation of the synthesis of the enzymes of the dissimilatory pathway for methanol was found to be under multiple control. Although glucose was present in the medium methanol had a positive effect on the synthesis of these enzymes. Thus, in addition to derepression induction by methanol was also observed. This inductive effect was found to increase with increasing proportions of methanol in the mixture. Depending on the enzyme, 10–40% methanol in the mixture resulted in a maximal induction with enzyme specific activities equal to those found in cells grown with methanol as the only carbon source. No further enhancements in enzyme specific activities were observed during growth on mixtures containing more than 40% methanol.Abbreviations and terms C₁ Methanol - C₆ glucose - C₁/C₆ mixture compositions are given in % (w/w) - C₀ concentration of ¹⁴C in the inflowing medium (DPM ml^-1) - C(t) concentration of ¹⁴C incorporated in cells as a function of time t (DPM ml^-1) - d dilution rate (h^-1) - DPM disintegrations per minute - q _s q _C1 and q _C6 are specific rates of consumption of substrate, methanol and glucose respectively [g (g cell dry weight)^-1 h^-1] - q _O2 and q _CO2 are the specific rates of oxygen consumption and carbon dioxide release [mmol (g cell dry weight)^-1 h^-1] - RQ respiration quotient (q _CO2 q _O2 ^-1) - s _C1 and s _C6 are the residual concentrations of methanol and glucose in the culture liquid (g l^-1) - s _O/C1 and s _O/C6 are the concentrations of methanol and glucose in the inflowing medium (g l^-1) - Sp.A. enzyme specific activity - x cell dry weight concentration (g l^-1) - Y _X/C1 and Y _X/C6 are growth yields on methanol and glucose respectively (g cell dry weight (g substrate)^-1 - Y _C/C1 growth yield with methanol with respect to carbon (g carbon assimilated (g carbon supplied)^-1 - _m maximum specific growth rate (h^-1)     

Diacetyl production and growth of Lactobacillus rhamnosus on multiple substrates

Lactobacillus rhamnosus is a heterolactic acid bacterium, which can be used to produce flavour compounds like diacetyl and acetoin. Various startegies have been applied to improve the growth rate and diacetyl yield. The use of multiple substrates affected growth as well as the yield of diacetyl. Growth on a medium containing glucose demonstrated a diauxic growth profile, with the second phase of growth being on the product, lactic acid. L. rhamnosus also grew on a medium containing citrate. Growth on medium containing glucose+citrate demonstrated simultaneous utilization of carbon sources. L. rhamnosus did not grow in a medium containing acetate and also did not co-metabolize it with glucose. Maximum specific growth rate ( _max) was found to increase in the case of simultaneous utilization of glucose+citrate (0.38 h^–1) as compared to glucose as the sole carbon source (0.28 h^–1). The yields of diacetyl were also found to increase for glucose + pyruvate and glucose + citrate (0.10 and 0.05 g g^–1 of glucose, respectively) as compared to glucose alone (0.01 g g^–1 of glucose). The productivity of diacetyl on medium containing glucose and citrate was double that of a medium containing only citrate, although the yields were comparable.     

Regulation of endo-1,4-β-glucanase secretion fromTermitomyces clypeatus by carbon catabolic product(s)

Secretion of CMCase byTermitomyces clypeatus was only observed in the presence of a gluconeogenic amino acid, a citrate-cycle acid, maleate, subinhibitory concentrations of glucosamine, or fluoride in the medium. The enzyme was not secreted in the presence of caffeine or IBMX or theophylline, and these phosphodiesterase inhibitors lowered the secretion of CMCase by glutamate. The presence of both glucosamine and glutamate in a cellulose medium were, however, antagonistic to CMCase secretion. In a growth medium, xylose and glucose were equivalent carbon source for the fungus while succinate was a poor source and strongly repressed growth at higher concentrations. Growth ofT. clypeatus was highly favored in media containing xylose/glucose with succinate/glutamate. During growth ofT. clypeatus in a glucose medium, the intracellular glucose level was stabilized by the presence of succinate, glutamate or glucosamine in the medium. All these observation suggested that a negative cellular regulation, mediated by carbon catabolic product(s), existed inT. clypeatus which regulated the secretion of CMCase. A transient but significant increase of intracellular cAMP and cGMP levels was observed at the onset of mycelial growth in glucose and glucose/maleate media, respectively.     

Investigation of the synthesis of xylose/glucose isomerases in sixArthrobacter sp. strains

The substrate specificity of isomerases produced by six strains ofArthrobacter sp. was studied. The role of utilizable carbon sources in controlling enzyme biosynthesis was established. All of the strains studied were found to produce xylose isomerases efficiently, converting D-xylose into D-xylulose and D-glucose into D-fructose. All but A.ureafaciens B-6 strains showed low activity toward D-ribose,Arthrobacter sp. B-5 was slightly active toward L-arabinose, andA. ureafaciens B-6 andArthrobacter sp. B-2239, toward L-rhamnose. InArthrobacter sp. B-5, the synthesis of xylose/glucose isomerase was constitutive (i.e., it was not suppressed by readily metabolizable carbon sources. The synthesis of xylose/glucose isomerase induced by D-xylose inArthrobacter sp. strains B-2239, B-2240, B-2241, and B-2242 and by D-xylose and xylitol inA. ureafaciens B-6 was suppressed by readily metabolizable carbon sources in a concentration-dependent manner. The data obtained suggest that D-xylose and/or its metabolites are involved in the regulation of xylose/glucose isomerase synthesis in theArthrobacter sp. strains B-5, B-2239, B-2240, and B-2241.     

Interactions of abscisic acid and sugar signalling in the regulation of leaf senescence

Leaf senescence can be triggered by a high availability of carbon relative to nitrogen or by external application of abscisic acid (ABA). Most Arabidopsis mutants with decreased sugar sensitivity during early plant development are either ABA insensitive (abi mutants) or ABA deficient (aba mutants). To analyse the interactions of carbon, nitrogen and ABA in the regulation of senescence, wild-type Arabidopsis thaliana (L.) Heynh. and aba and abi mutants were grown on medium with varied glucose and nitrogen supply. On medium containing glucose in combination with low, but not in combination with high nitrogen supply, senescence was accelerated and sucrose, glucose and fructose accumulated strongly. In abi mutants that are not affected in sugar responses during early development (abi1-1 and abi2-1), we observed no difference in the sugar-dependent regulation of senescence compared to wild-type plants. Similarly, senescence was not affected in the sugar-insensitive abi4-1 mutant. In contrast, the abi5-1 mutant did exhibit a delay in senescence compared to its wild type. As ABA has been reported to induce senescence and ABA deficiency results in sugar insensitivity during early development, we expected senescence to be delayed in aba mutants. However, the aba1-1 and aba2-1 mutants showed accelerated senescence compared to their wild types on glucose-containing medium. Our results show that, in contrast to sugar signalling in seedlings, ABA is not required for the sugar-dependent induction of leaf senescence. Instead, increased sensitivity to osmotic stress could have triggered early senescence in the aba mutants.Abbreviations ABA Abscisic acid - aba Abscisic acid deficient - abi Abscisic acid insensitive - F_v/F_m Maximum efficiency of photosystem II photochemistry     

Regulation of the synthesis of methanol oxidizing enzymes inKloeckera sp. 2201 andHansenula polymorpha,a comparison

A comparative study was made of the regulation of the synthesis of methanol dissimilating enzymes inkloeckera sp. 2201 andHansenula polymorpha using chemostat and batch growth conditions and methanol or glucose as carbon sources. During growth in methanol-limited chemostat cultures similar enzyme patterns for alcohol oxidase, catalase, formaldehyde dehydrogenase and formate dehydrogenase in the two yeasts were found. When growing in batch culture with glucoseH. polymorpha, but notKloeckera sp. 2201, was found to produce ethanol which might affect the synthesis of these enzymes.     

Enhancement of phenol biodegradation by Ochrobactrum sp. isolated from industrial wastewaters

Ochrobactrum sp., was tested with regard to its phenol degradation capacity at different pH levels, and with different carbon sources (mineral salt medium with glucose (MSG) and the same medium with 0.5%, 1%, and 2% (v/v) molasses (MSM)) and phenol concentrations. The highest degradation was in mineral salt medium with 1% (v/v) molasses (45.9%), while degradation was 21.1% in mineral salt medium with 5 g l⁻¹ glucose. These data show that the addition of molasses to mineral salt medium enhanced phenol degradation by Ochrobactrum sp. The bacterium can be used effectively to treat wastewaters containing phenol.     

Influence of medium components on growth kinetics of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>

Starting with the standard complex medium HL-5C, the influence of different medium components on the growth behavior of Dictyostelium discoideum was investigated. For this purpose, each component was individually deleted from the complex medium HL-5C, and the overall concentration of all components as well as the kinds and concentrations of carbon source were varied. The effects of the supplementation with major components of synthetic medium like vitamins, trace metals, inorganic salts were also investigated. When glucose being the carbon source was deleted from the standard HL-5C medium, the maximal cell density then had a drastic drop. Deletion of yeast extract also caused lower maximal cell density. Of all the carbohydrates investigated only maltose and glucose led to higher final cell concentration. Adding inorganic salts such as CaCl₂ and MgCl₂ to HL-5C medium significantly improved the cell growth, whereas addition of vitamins or trace metals had little effect on cell growth. A semi-empirical model is employed to simulate the cell growth.     

Regulation of amino acid and glucose dissimilation in so-called ammonifiers and in other soil microorganisms

Pseudomonas acidovorans and P. putida, isolated from an enrichment culture with casein hydrolysate, and Agrobacterium radiobacter and Torulopsis sp., isolated from a glucose enrichment, were compared with respect to the physiology of ammonification. Decreasing ammonifying ability as well as increasing repression of the synthesis of amino acid degrading enzymes by glucose were found in the above order of organisms. In degradation sequences, observed with P. putida and A. radiobacter as test organisms, substances dissimilated prior to others had both, enhancing and repressing effects on the oxidation of the other compounds. This fact was parallelled by the observation, that in these two bacteria, glucose and single amino acids, when added to the same medium, exerted mutual repression of the synthesis of catabolic enzymes of their partners. The ecological significance of this type of regulation has been discussed.     

Frontiers in mycoplasma pathogenicity

Four different strains ofLactobacillus delbrueckii subsp.bulgaricus (Ss₁ and Yop₁₂) andStreptococcus salivarius subsp.thermophilus (Ss₂ and Yop₉) were isolated from two different yogurt sources in Argentina. In medium containing different carbon sources: lactose, fructose, sucrose or glucose plus fructose, the growth of a mixed culture (Yop₁₂+Ss₂) shows stimulation ofS. thermophilus and inhibition ofL. bulgaricus with respect to pure cultures. Both microorganisms in mixed culture grew less well on glucose plus galactose. However, in medium with glucose or galactose, both microorganisms were stimulated.     

Production of polyesters consisting of medium chain length 3-hydroxyalkanoic acids by Pseudomonas mendocina 0806 from various carbon sources

Pseudomonas mendocina strain 0806 was isolated from oil-contaminated soil and found to produce polyesters consisting of medium chain length 3-hydroxyalkanoates (mclPHAs). The monomers of mclPHAs contained even numbers of carbon atoms, such as 3-hydroxyhexanoate (HHx or C₆), 3-hydroxyoctanoate (HO or C₈), and/or 3-hydroxydecanoate (HD or C₁₀) as major components when grown on many carbon sources unrelated to their monomeric structures, such as glucose, citric acid, and carbon sources related to their monomeric structures, such as myristic acid, octanoate, or oleic acid. On the other hand, PHA containing both even and odd numbers of hydroxyalkanoates (HA) monomers was synthesized when the strain was grown on tridecanoic acid. The molar ratio of carbon to nitrogen (C/N) had a significant effect on PHA composition: the strain produced PHAs containing 97–99% of HD monomer when grown in a glucose ammonium sulfate medium of C/N<20, and 20% HO, and 80% of the HD monomer when growth was conducted in media containing C/N>40. It was demonstrated that the HO/HD ratio in the polymers remained constant in media with a constant C/N ratio, regardless of the glucose concentration. Up to 3.6 g/L cell dry weight containing 45% of PHAs was produced when the strain was grown for 48 h in a medium containing 20 g/L glucose with a C/N ratio of 40.