Biomedical subjects

T Egli

Publications and source records attributed to T Egli.

At least 19 recordsLinked to original sources

PubMed2006

Efficacy of solar disinfection of Escherichia coli, Shigella flexneri, Salmonella Typhimurium and Vibrio cholerae.

AIMS: To determine the efficacy of solar disinfection (SODIS) for enteric pathogens and to test applicability of the reciprocity law. METHODS AND RESULTS: Resistance to sunlight at 37 degrees C based on F99 values was in the following order: Salmonella Typhimurium>Escherichia coli>Shigella flexneri>Vibrio cholerae. While F90 values of Salm. Typhimurium and E. coli were similar, F99 values differed by 60% due to different inactivation curve shapes. Efficacy seemed not to be dependent on fluence rate for E. coli stationary cells. Sensitivity to mild heat was observed above a temperature of 45 degrees C for E. coli, Salm. Typhimurium and Sh. flexneri, while V. cholerae was already susceptible above 40 degrees C. CONCLUSIONS: Salmonella Typhimurium was the most resistant and V. cholerae the least resistant enteric strain. The reciprocity law is applicable for stationary E. coli cells irradiated with sunlight or artificial sunlight. SIGNIFICANCE AND IMPACT OF THE STUDY: Escherichia coli might not be the appropriate indicator bacterium to test the efficacy of SODIS on enteric bacteria and the physiological response to SODIS might be different among enteric bacteria. The applicability of the reciprocity law indicates that fluence rate plays a secondary role in SODIS efficacy. Stating inactivation efficacy with T90 or F90 values without showing original data is inadequate for SODIS studies.

Disinfection↗

PubMed2001

Occurrence, synthesis and medical application of bacterial polyhydroxyalkanoate.

Polyhydroxyalkanoates (PHAs) comprise a large class of polyesters that are synthesized by many bacteria as an intracellular carbon and energy compound. Analysis of isolated PHAs reveal interesting properties such as biodegradability and biocompatibility. Research was focused only recently on the application of PHA in implants, scaffolds in tissue engineering, or as drug carriers. Such applications require that PHA be produced at a constant and reproducible quality. To date this can be achieved best through bacterial production in continuous culture where growth conditions are kept constant (chemostat). Recently, it was found that PHA producing bacteria are able to grow simultaneously limited by carbon and nitrogen substrates. Thus, it became possible to produce PHA at high yields on toxic substrate and also control its composition accurately (tailor-made synthesis). Finally, applications of PHA in medicine are discussed.

Acyltransferases↗

PubMed2001

Accumulation of poly[(R)-3-hydroxyalkanoates] in Pseudomonas oleovorans during growth in batch and chemostat culture with different carbon sources.

Pseudomonas oleovorans (ATCC 29347) was grown in batch and chemostat cultures with citrate, hexanoate, heptanoate, octanoate, and nonanoate as single carbon substrates. The growth medium for batch cultures was adjusted such that nitrogen (NH(4)(+)) limitation terminated the exponential-growth phase. During batch cultivation with octanoate or nonanoate the biomass continued to increase after depletion of ammonium due to the accumulation of medium-chain-length poly[(R)-3-hydroxyalkanoates] (mcl-PHAs). Additionally, a significant rate of mcl-PHA accumulation was also observed in the exponential-growth phase of batch cultures. It is well known that the accumulation of reserve materials is strongly dependent on the ratio of nutrients (here of carbon, C, and of nitrogen, N) and that in a batch culture the ratio of C:N is continuously changing. Therefore, we have also investigated the effect of defined ratios of C:N under constant cultivation conditions, namely at a fixed dilution rate (D) in a chemostat fed with different medium C:N ratios. These experiments were performed at a constant D of 0.2 h(-1). The concentration of the nitrogen source in the inflowing medium (N()) was kept constant, while its carbon concentration (C()) was increased stepwise, resulting in an increase of the medium carbon to nitrogen ratio (C()/N() ratio). The culture parameters and the cell composition of steady-state cultures were determined as a function of the C()/N() ratio in the feed medium. Mcl-PHA accumulation was detected during growth with the fatty acids, and three distinct regimes of growth limitation were discovered: In addition to carbon limitation at low, and nitrogen limitation at high C()/N() ratios, an intermediate growth regime of simultaneous limitation by carbon and nitrogen was detected where both substrates were used to completion. The width of this dual-nutrient-limited growth regime was dependent on the change in the yield factors for carbon and nitrogen (Y(X/C), Y(X/N)) measured during single-nutrient-limited growth.

Alkanes↗

PubMed2001

Short- and long-term changes in proteome composition and kinetic properties in a culture of Escherichia coli during transition from glucose-excess to glucose-limited growth conditions in continuous culture and vice versa.

To investigate the ability of Escherichia coli K12 MG1655 to cope with excess and limitation of a carbon and energy source, we studied the changes in kinetic properties and two-dimensional (2D) gel protein patterns of an E. coli culture. The population was transferred from glucose-excess batch to glucose-limited continuous culture (D = 0.3 h(-1)), in which it was cultivated for 500 h (217 generations) and then transferred back to glucose-excess batch culture. Two different stages to glucose-limitation were recognized: a short-term physiological adaptation characterized by a general effort in enhancing the cell's substrate scavenging ability and mutations resulting in a population exhibiting increased glucose affinity. Physiological short-term adaptation to glucose-limitation was achieved by upregulation of 12 proteins, namely MglB, MalE, ArgT, DppA, RbsB, YdcS, LivJ (precursor), UgpB (precursor), AceA, AldA, AtpA and GatY. Eight of these proteins are periplasmic binding proteins of ABC transporters. Most of them are not involved in glucose transport regulons, but rather in chemotaxis and transport of other substrates, whereas MalE and MglB have previously been shown to belong to transport systems important in glucose transport under glucose-limited conditions. Evolution under low glucose concentration led to an up to 10-fold increase in glucose affinity (from a K(s) of 366 +/- 36 microg l(-1) at the beginning to 44 +/- 7 microg l(-1)). The protein pattern of a "500-h-old" continuous culture showed a highly increased expression of MglB and MalE as well as of the regulator protein MalI. When adapted cells taken from the "500-h-old" continuous culture were transferred to batch culture, an increased expression of MalE was observed, compared with cells from un-adapted batch-grown cells. Otherwise, no significant changes were observed in the protein pattern of batch-grown populations before and after 500 h of evolution in the glucose-limited continuous culture.

Biological Evolution↗

PubMed2001

Isolation and characterization of a Pseudomonas putida strain able to grow with trimethyl-1,2-dihydroxy-propyl-ammonium as sole source of carbon, energy and nitrogen.

Trimethyl-1,2-dihydroxypropyl-ammonium (TM) originates from the hydrolysis of the parent esterquat surfactant, which is widely used as softener in fabric care. Based on test procedures mimicking complex biological systems, TM is supposed to degrade completely when reaching the environment. However, no organisms able to degrade TM were isolated nor has the degradation pathway been elucidated so far. We isolated a Gram-negative rod able to grow with TM as sole source of carbon, energy and nitrogen. The strain reached a maximum specific growth rate of 0.4(h-1) when growing with TM as the sole source of carbon, energy and nitrogen. TM was degraded to completion and surplus nitrogen was excreted as ammonium into the growth medium. A high percentage of the carbon in TM (68% in continuous culture and 60% in batch culture) was combusted to CO2 resulting in a low yield of 0.54 mg cell dry weight per mg carbon during continuous cultivation and 0.73 mg cell dry weight per mg carbon in batch cultures. Choline, a natural structurally related compound, served as a growth substrate, whereas a couple of similar other quaternary aminoalcohols also used in softeners did not. The isolated bacterium was identified by 165-rDNA sequencing as a strain of Pseudomonas putida with a difference of only one base pair to P. putida DSM 291T. Despite their high identity, the reference strain P. putida DSM 291T was not able to grow with TM and the two strains differed even in shape when growing on the same medium. This is the first microbial isolate able to degrade a quaternary ammonium softener head group to completion. Previously described strains growing on quaternary ammonium surfactants (decyltrimethylammonium, hexadecyltrimethylammonium and didecyldimethylammonium) either excreted metabolites or a consortium of bacteria was required for complete degradation.

Biodegradation, Environmental↗

PubMed2001

Environmental fate and microbial degradation of aminopolycarboxylic acids.

Aminopolycarboxylic acids (APCAs) have the ability to form stable, water-soluble complexes with di- and trivalent metal ions. For that reason, synthetic APCAs are used in a broad range of domestic products and industrial applications to control solubility and precipitation of metal ions. Because most of these applications are water-based, APCAs are disposed of in wastewater and reach thus sewage treatment plants and the environment, where they undergo abiotic and/or biotic degradation processes. Recently, also natural APCAs have been described which are produced by plants or micro-organisms and are involved in the metal uptake by these organisms. For the two most widely used APCAs, nitrilotriacetate (NTA) and ethylenediaminetetraacetate (EDTA), transformation and mineralisation processes have been studied rather well, while for other xenobiotic APCAs and for the naturally occurring APCAs little is known on their fate in the environment. Whereas NTA is mainly degraded by bacteria under both oxic and anoxic conditions, biodegradation is apparently of minor importance for the environmental fate of EDTA. Photodegradation of iron(III)-complexed EDTA is supposed to be mostly responsible for its elimination. Isolation of a number of NTA- and EDTA-utilising bacterial strains has been reported and the spectrum of APCAs utilised by the different isolates indicates that some of them are able to utilise a range of different APCAs whereas others seem to be restricted to one compound. The two best characterised obligately aerobic NTA-utilising genera (Chelatobacter and Chelatococcus) are members of the alpha-subgroup of Proteobacteria. There is good evidence that they are present in fairly high numbers in surface waters, soils and sewage treatment plants. The key enzymes involved in NTA degradation in Chelatobacter and Chelatococcus have been isolated and characterised. The two first catabolic steps are catalysed by a monooxygenase (NTA MO) and a membrane-bound iminodiacetate dehydrogenase. NTA MO has been cloned and sequenced and its regulation as a function of growth conditions has been studied. Under denitrifying conditions, NTA catabolism is catalysed by a NTA dehydrogenase. EDTA breakdown was found to be initiated by a MO also which shares many characteristics with NTA MO from strictly aerobic NTA-degrading bacteria. In contrast, degradation of [S,S]-ethylenediaminedisuccinate ([S,S]-EDDS), a structural isomer of EDTA, was shown to be catalysed by an EDDS lyase in both an EDTA degrader and in a NTA-utilising Chelatococcus strain. So far, transport of APCAs into cells has only been studied for EDTA and the results obtained give strong evidence for an energy-dependent carrier system and Ca(2+) seems to be co-transported with EDTA. Due to their metal-complexing capacities, APCAs occur in the environment mostly in the metal-complexed form. Hence, the influence of metal speciation on various degradation processes is of utmost importance to understand the environmental behaviour of these compounds. In case of biodegradation, the effect of metal speciation is rather difficult to assess at the whole cell level and therefore only limited good data are available. In contrast, the influence of metal speciation on the intracellular enzymatic breakdown of APCAs is rather well documented but no generalising pattern applicable to all enzymes was found.

Bacteria↗

PubMed2001

Biodegradation of metal-complexing aminopolycarboxylic acids.

Aminopolycarboxylic acids (APCAs) are used in a broad range of household products and industrial applications to control the solubility and precipitation of metal ions. The two most widely used APCAs are ethylenediaminetetraacetate (EDTA) and nitrilotriacetate (NTA). Because their application is mostly water-based APCAs are disposed off in wastewater from where they reach the environment if not sufficiently well eliminated. A number of NTA and EDTA-degrading bacterial strains have been isolated. Many of them are strictly aerobic members of the alpha-2 subgroup of Proteobacteria. The two newly established NTA-degrading genera Chelatobacter and Chelatococcus appear to be well represented in most aerobic environments. The biochemistry of NTA and EDTA catabolism has been studied in considerable detail and recently, also information on the degradation of ethylenediaminedisuccinate [S,S]-EDDS, a structural isomer of EDTA of natural origin, was reported. For both, NTA and EDTA, a specific monooxygenase/NADH2:FMN oxidoreductase enzyme system has been purified and characterised and their regulation under environmentally relevant growth conditions has been studied. The genes for NTA MO and EDTA MO have been cloned and sequenced and they share many similarities. In addition, an oxygen-independent NTA dehydrogenase has been isolated from a denitrifying NTA-degrading bacterium and the breakdown of [S,S]-EDDS was found to be initiated by a lyase. Information on the transport of APCAs is presently limited to EDTA, where there is strong evidence for an energy-dependent carrier that can transport free and Ca2+-complexed EDTA. Metal speciation of APCAs strongly influences both transport and subsequent metabolism. However, from the data available for the intracellular metabolism of APCAs no generalising pattern with respect to influence of metal speciation on degradation can be deduced yet.

Journal Article↗

PubMed2000

Description of Sphingomonas xenophaga sp. nov. for strains BN6T and N,N which degrade xenobiotic aromatic compounds.

The taxonomic position of two bacterial strains, BN6T and N,N, with the ability to degrade xenobiotic aromatic compounds (naphthalenesulfonates or N,N-dimethylaniline) was investigated. The 165 rRNA gene sequence, the G+C content of the DNA (62-63 mol%) and the detection of ubiquinone Q-10, 2-hydroxymyristic acid and the sphingoglycolipid present clearly placed the two strains into the genus Sphingomonas. Both strains are representatives of one species according to the level of DNA relatedness (70.7%). The strains could be separated from all validly described taxa of the genus Sphingomonas, according to the 16S rRNA gene sequence (the highest sequence similarity observed was 96 % to Sphingomonas yanoikuyae), the pattern of the polar lipids and physiological characteristics. Therefore, the new species Sphingomonas xenophaga is proposed to accommodate strains BN6T (= DSM 6383T) and N,N (= DSM 8566).

Aniline Compounds↗

PubMed2000

Accumulation of Poly[(R)-3-hydroxyalkanoates] in Pseudomonas oleovorans during growth with octanoate in continuous culture at different dilution rates.

Pseudomonas oleovorans ATCC 29347 was grown in chemostat culture at different dilution rates with mineral media varying in their ratios of octanoate to ammonia (C(0)/N(0) ratio). At all dilution rates tested, three distinct growth regimes were observed: (i) carbon limitation with NH(4)(+) in excess at low C(0)/N(0) ratios, (ii) purely nitrogen-limited growth conditions at high C(0)/N(0) ratios with residual octanoate in the culture supernatant, and (iii) an intermediate zone of dual-nutrient-limited growth conditions where both the concentration of octanoate and that of ammonia were very low. The dual-nutrient-limited growth zone shifted to higher C(0)/N(0) ratios with decreasing dilution rates, and the extension of the dual-nutrient-limited growth zone was inversely proportional to the growth rate. The cells accumulated the storage compound medium-chain-length poly[(R)-3-hydroxyalkanoate] (mcl-PHA) during dual (C and N)-nutrient-limited and N-limited growth conditions. Within the dual-nutrient-limited growth zone, the cellular mcl-PHA contents increased when the C(0)/N(0) ratio in the feed was increased, whereas the cellular mcl-PHA level was independent from the feed C(0)/N(0) ratio during N-limited growth. The monomeric composition of the accumulated mcl-PHA was independent of both the dilution rate and the feed C(0)/N(0) ratio and consisted of 12 mol% 3-hydroxyhexanoic acid and 88 mol% 3-hydroxyoctanoic acid. Accumulation of mcl-PHA led to an increase in the cellular C/N ratio and to changes in elemental growth yields for nitrogen and carbon.

Caprylates↗

PubMed2000

Growth kinetics of Escherichia coli with galactose and several other sugars in carbon-limited chemostat culture.

Kinetic models for microbial growth describe the specific growth rate (mu) as a function of the concentration of the growth-limiting nutrient (s) and a set of parameters. A typical example is the model proposed by Monod, where mu is related to s using substrate affinity (Ks) and the maximum specific growth rate (mu max). The preferred method to determine such parameters is to grow microorganisms in continuous culture and to measure the concentration of the growth-limiting substrate as a function of the dilution rate. However, owing to the lack of analytical methods to quantify sugars in the microgram per litre range, it has not been possible to investigate the growth kinetics of Escherichia coli in chemostat culture. Using an HPLC method able to determine steady-state concentrations of reducing sugars, we previously have shown that the Monod model adequately describes glucose-limited growth of E. coli ML30. This has not been confirmed for any other sugar. Therefore, we carried out a similar study with galactose and found steady-state concentrations between 18 and 840 micrograms.L-1 for dilution rates between 0.2 and 0.8.h-1, respectively. With these data the parameters of several models giving the specific growth rate as a function of the substrate concentration were estimated by nonlinear parameter estimation, and subsequently, the models were evaluated statistically. From all equations tested, the Monod model described the data best. The parameters for galactose utilisation were mu max = 0.75.h-1 and Ks = 67 micrograms.L-1. The results indicated that accurate Ks values can be estimated from a limited set of steady-state data when employing mu max measured during balanced growth in batch culture. This simplified procedure was applied for maltose, ribose, and fructose. For growth of E. coli with these sugars, mu max and Ks were for maltose 0.87.h-1, 100 micrograms.L-1; for ribose 0.57.h-1, 132 micrograms.L-1, and for fructose 0.70.h-1, 125 micrograms.L-1.

Culture Media↗

PubMed1998

Kinetic models for the growth of Escherichia coli with mixtures of sugars under carbon-limited conditions.

In natural environments, heterotrophic microorganisms encounter complex mixtures of carbon sources, each of which is present only at very low concentrations. Under such conditions no significant growth could be expected if cells utilized only one of the available carbon compounds as suggested by the principle of diauxic growth. Indeed, there is much evidence that microbial cells utilize many carbon sources simultaneously. In order to predict bacterial growth under such conditions we developed a model describing the specific growth rate as a function of the individual concentrations of several simultaneously utilized carbon substrates. Together with multisubstrate models previously published, this model was evaluated for its ability to describe growth of Escherichia coli during the simultaneous utilization of mixtures of sugars in carbon-limited continuous culture. Using the micromax and Ks constants determined for single substrate growth with six different sugars, the model was able for most experiments to adequately describe the specific growth rate of the culture, i.e., the experimentally set dilution rate, from the measured concentrations of the individual sugars. The model provides an explanation why bacteria can still grow relatively fast under environmental conditions where the concentrations of carbon substrates are usually extremely low.

Culture Media↗

PubMed1998

Influence of carbon source on nitrate removal by nitrate-tolerant Klebsiella oxytoca CECT 4460 in batch and chemostat cultures.

The nitrate-tolerant organism Klebsiella oxytoca CECT 4460 tolerates nitrate at concentrations up to 1 M and is used to treat wastewater with high nitrate loads in industrial wastewater treatment plants. We studied the influence of the C source (glycerol or sucrose or both) on the growth rate and the efficiency of nitrate removal under laboratory conditions. With sucrose as the sole C source the maximum specific growth rate was 0.3 h-1, whereas with glycerol it was 0.45 h-1. In batch cultures K. oxytoca cells grown on sucrose or glycerol were able to immediately use sucrose as a sole C source, suggesting that sucrose uptake and metabolism were constitutive. In contrast, glycerol uptake occurred preferentially in glycerol-grown cells. Independent of the preculture conditions, when sucrose and glycerol were added simultaneously to batch cultures, the sucrose was used first, and once the supply of sucrose was exhausted, the glycerol was consumed. Utilization of nitrate as an N source occurred without nitrite or ammonium accumulation when glycerol was used, but nitrite accumulated when sucrose was used. In chemostat cultures K. oxytoca CECT 4460 efficiently removed nitrate without accumulation of nitrate or ammonium when sucrose, glycerol, or mixtures of these two C sources were used. The growth yields and the efficiencies of C and N utilization were determined at different growth rates in chemostat cultures. Regardless of the C source, yield carbon (YC) ranged between 1.3 and 1.0 g (dry weight) per g of sucrose C or glycerol C consumed. Regardless of the specific growth rate and the C source, yield nitrogen (YN) ranged from 17.2 to 12.5 g (dry weight) per g of nitrate N consumed. In contrast to batch cultures, in continuous cultures glycerol and sucrose were utilized simultaneously, although the specific rate of sucrose consumption was higher than the specific rate of glycerol consumption. In continuous cultures double-nutrient-limited growth appeared with respect to the C/N ratio of the feed medium and the dilution rate, so that for a C/N ratio between 10 and 30 and a growth rate of 0.1 h-1 the process led to simultaneous and efficient removal of the C and N sources used. At a growth rate of 0.2 h-1 the zone of double limitation was between 8 and 11. This suggests that the regimen of double limitation is influenced by the C/N ratio and the growth rate. The results of these experiments were validated by pulse assays.

Culture Media↗

PubMed1998

Growth kinetics of suspended microbial cells: from single-substrate-controlled growth to mixed-substrate kinetics.

Growth kinetics, i.e., the relationship between specific growth rate and the concentration of a substrate, is one of the basic tools in microbiology. However, despite more than half a century of research, many fundamental questions about the validity and application of growth kinetics as observed in the laboratory to environmental growth conditions are still unanswered. For pure cultures growing with single substrates, enormous inconsistencies exist in the growth kinetic data reported. The low quality of experimental data has so far hampered the comparison and validation of the different growth models proposed, and only recently have data collected from nutrient-controlled chemostat cultures allowed us to compare different kinetic models on a statistical basis. The problems are mainly due to (i) the analytical difficulty in measuring substrates at growth-controlling concentrations and (ii) the fact that during a kinetic experiment, particularly in batch systems, microorganisms alter their kinetic properties because of adaptation to the changing environment. For example, for Escherichia coli growing with glucose, a physiological long-term adaptation results in a change in KS for glucose from some 5 mg liter-1 to ca. 30 microg liter-1. The data suggest that a dilemma exists, namely, that either "intrinsic" KS (under substrate-controlled conditions in chemostat culture) or micromax (under substrate-excess conditions in batch culture) can be measured but both cannot be determined at the same time. The above-described conventional growth kinetics derived from single-substrate-controlled laboratory experiments have invariably been used for describing both growth and substrate utilization in ecosystems. However, in nature, microbial cells are exposed to a wide spectrum of potential substrates, many of which they utilize simultaneously (in particular carbon sources). The kinetic data available to date for growth of pure cultures in carbon-controlled continuous culture with defined mixtures of two or more carbon sources (including pollutants) clearly demonstrate that simultaneous utilization results in lowered residual steady-state concentrations of all substrates. This should result in a competitive advantage of a cell capable of mixed-substrate growth because it can grow much faster at low substrate concentrations than one would expect from single-substrate kinetics. Additionally, the relevance of the kinetic principles obtained from defined culture systems with single, mixed, or multicomponent substrates to the kinetics of pollutant degradation as it occurs in the presence of alternative carbon sources in complex environmental systems is discussed. The presented overview indicates that many of the environmentally relevant apects in growth kinetics are still waiting to be discovered, established, and exploited.

Bacteria↗

PubMed1997

Cultivation of Escherichia coli with mixtures of 3-phenylpropionic acid and glucose: steady-state growth kinetics.

The fate of pollutants in the environment is affected by the presence of easily degradable carbon sources. As a step towards understanding these complex interactions, a model system was explored: the degradation of mixtures of glucose (i.e., an easily degradable substrate) and 3-phenylpropionic acid (3ppa) (a model pollutant) by Escherichia coli ML 30 was studied systematically in carbon-limited continuous culture. The two substrates were always consumed simultaneously regardless of the dilution rate applied. Even at dilution rates higher than the maximum specific growth rate for 3ppa (0.35 +/- 0.05 h-1), the two carbon substrates were utilized together. When cells were grown at a constant dilution rate with different mixtures of 3ppa and glucose, in which 3ppa contributed between 5 and 90% of carbon substrate in the feed medium, the steady-state concentrations of 3ppa and glucose were approximately proportional to the ratio of the two substrates in the feed medium. When cells were cultivated at different dilution rates with a 1:1 mixture (based on carbon) of glucose and 3ppa, an overall maximum specific growth rate of 0.90 +/- 0.05 h-1 and a Monod substrate saturation constant for 3ppa (Ks) of 600 to 700 micrograms liter-1, similar to that measured during growth with 3ppa alone, fitted the experimentally determined steady-state 3ppa concentrations. However, due to the highly differing substrate affinity constants for 3ppa and glucose (Ks approximately 30 to 70 micrograms liter-1), the total steady-state carbon concentration in the culture at a constant dilution rate was determined mainly by the steady-state 3ppa carbon concentration, and it increased with increasing proportions of 3ppa in the feed medium.

Biomass↗

PubMed1997

Identification and characterization of the two-enzyme system catalyzing oxidation of EDTA in the EDTA-degrading bacterial strain DSM 9103.

In a gram-negative isolate (DSM 9103) able to grow with EDTA as the sole source of carbon, nitrogen, and energy, the first two steps of the catabolic pathway for EDTA were elucidated. They consisted of the sequential oxidative removal of two acetyl groups, resulting in the formation of glyoxylate. An enzyme complex that catalyzes the removal of two acetyl groups was purified and characterized. In the reaction, ethylenediaminetriacetate (ED3A) was formed as an intermediate and N,N'-ethylenediaminediacetate was the end product. The enzyme complex consisted of two components: component A' (cA'), most likely a monooxygenase, which catalyzes the cleavage of EDTA and ED3A while consuming oxygen and reduced flavin mononucleotide (FMN)-H2, and component B' (cB'), an NADH2:FMN oxidoreductase that provides FMNH2 for cA'. cB' could be replaced by other NADH2:FMN oxidoreductases such as component B of the nitrilotriacetate monooxygenase or the NADH2:FMN oxidoreductase from Photobacterium fischeri. The EDTA-oxidizing enzyme complex accepted EDTA as a substrate only when it was complexed with Mg2+, Zn2+, Mn2+, Co2+, or Cu2+. Moreover, the enzyme complex catalyzed the removal of acetyl groups from several other aminopolycarboxylic acids that possess three or more acetyl groups.

Edetic Acid↗

PubMed1996

Dynamics of Substrate Consumption and Enzyme Synthesis in Chelatobacter heintzii during Growth in Carbon-Limited Continuous Culture with Different Mixtures of Glucose and Nitrilotriacetate.

Regulation of nitrilotriacetate (NTA) degradation and expression of NTA monooxygenase (NTA-MO) in the NTA-degrading strain Chelatobacter heintzii ATCC 29600 in continuous culture at a dilution rate of 0.06 h(sup-1) under transient growth conditions when the feed was switched between media containing NTA, glucose, or different mixtures thereof as the sole carbon and energy sources was investigated. A transition from NTA to glucose was accompanied by a rapid loss of NTA-MO. A transition from glucose to NTA resulted in a lag phase of some 25 h until NTA-MO expression started, and approximately 100 h was needed before a steady state for NTA-MO specific activity was reached. This transient lag phase was markedly shortened when mixtures of NTA plus glucose were supplied instead of NTA only; for example, when a mixture of 90% glucose and 10% NTA was used, induction of NTA-MO was detected after 30 min. This suggests a strong positive influence of alternative carbon substrates on the expression of other enzymes under natural environmental conditions. Regulation of NTA-MO expression and the fate of NTA-MO were also studied during starvation of both glucose-grown and NTA-grown cultures. Starvation of NTA-grown cells led to a loss of NTA-MO protein. No synthesis of NTA-MO (derepression) was observed when glucose-grown cells were starved.

Journal Article↗

PubMed1996

Kinetics of the simultaneous utilization of sugar mixtures by Escherichia coli in continuous culture.

In natural environments heterotrophic microorganisms encounter complex mixtures of carbon sources, each of which is present at a concentration of a few micrograms per liter or even less. Under such conditions no significant growth would be expected if cells utilized only one of the available carbon compounds, as suggested by the principle of diauxic growth. Indeed, there is much evidence that microbial cells utilize many carbon compounds simultaneously. Whereas the kinetics of single-substrate and diauxic growth are well understood, little is known about how microbial growth rates depend on the concentrations of several simultaneously utilized carbon sources. In this study this question was answered for carbon-limited chemostat growth of Escherichia coli fed with mixtures of up to six sugars; the sugars used were glucose, galactose, maltose, ribose, arabinose, and fructose. Independent of the mixture composition and dilution rate tested, E. coli utilized all sugars simultaneously. Compared with growth with a single sugar at a particular growth rate, the steady-state concentrations were consistently lower during simultaneous utilization of mixtures of sugars. The steady-state concentrations of particular sugars depended approximately linearly on their contributions to the total carbon consumption rate of the culture. Our experimental data demonstrate that the simultaneous utilization of mixtures of carbon sources enables heterotrophic microbes to grow relatively fast even in the presence of low environmental substrate concentrations. We propose that the observed reductions in the steady-state concentrations of individual carbon sources during simultaneous utilization of mixtures of carbon sources by heterotrophic microorganisms reflect a general kinetic principle.

Carbohydrate Metabolism↗

PubMed1996

Temperature-dependent growth kinetics of Escherichia coli ML 30 in glucose-limited continuous culture.

Detailed comparison of growth kinetics at temperatures below and above the optimal temperature was carried out with Escherichia coli ML 30 (DSM 1329) in continuous culture. The culture was grown with glucose as the sole limiting source of carbon and energy (100 mg liter(-1) in feed medium), and the resulting steady-state concentrations of glucose were measured as a function of the dilution rate at 17.4, 28.4, 37, and 40 degrees C. The experimental data could not be described by the conventional Monod equation over the entire temperature range, but an extended form of the Monod model [mu = mu(max) x (s - s(min))/(Ks + s - s(min))], which predicts a finite substrate concentration at 0 growth rate (s(min)), provided a good fit. The two parameters mu(max) and s(min) were temperature dependent, whereas, surprisingly, fitting the model to the experimental data yielded virtually identical Ks values (approximately 33 microg liter(-1)) at all temperatures. A model that describes steady-state glucose concentrations as a function of temperature at constant growth rates is presented. In similar experiments with mixtures of glucose and galactose (1:1 mixture), the two sugars were utilized simultaneously at all temperatures examined, and their steady-state concentrations were reduced compared with to growth with either glucose or galactose alone. The results of laboratory-scale kinetic experiments are discussed with respect to the concentrations observed in natural environments.

Cell Division↗