Population changes in a culture of Escherichia coli K12 1EA growing in a ribitol-limited chemostat

Summary The growth of the strain Escherichia coli K12 1EA in a chemostat was limited by ribitol as the source of carbon and energy. Specific activities of ribitol dehydrogenase and D-arabinitol dehydrogenase were assayed to measure expression of the two closely linked catabolic operons rbt and dal. Population changes occuring in a chemostat were analyzed by testing single colony isolates in batch cultures: double constitutive mutant 1EA-A was first selected while later hyperproducing strains of the type 11EA and 111EA synthesizing constitutively 4 times and 8 times more ribitol dehydrogenase, respectively, prevail in the chemostat.     

Ribitol dehydrogenase overproduction and maintenance of plasmids pBR322 and pACYC184 in chemostat cultures of Escherichia coli

Summary The maintenance of multicopy plasmids pBR322 and pACYC184 was studied in chemostat cultures subjected to limitation by glucose, ribitol or xylitol at D of 0.1 per h. While carbon source-dependent segregational stability of pBR322 was observed, no dependence and greater instability of pACYC184 was found. Plasmid-free and plasmid-bearing strains overproducing chromosomally coded ribitol dehydrogenase were found in pentitol-limited chemostat cultures.     

Growth characteristics and oxidative capacity of<Emphasis Type="Italic"> Acetobacter aceti</Emphasis> IFO 3281: implications for <Emphasis Type="SmallCaps">l</Emphasis>-ribulose production

We studied the growth characteristics and oxidative capacities of Acetobacter aceti IFO 3281 in batch and chemostat cultures. In batch culture, glycerol was the best growth substrate and growth on ethanol occurred only after 6 days delay, although ethanol was rapidly oxidized to acetic acid. In continuous culture, both glycerol and ethanol were good growth substrates with similar characteristics. Resting cells in a bioreactor oxidized ribitol to l-ribulose with a maximal specific rate of 1.2 g g^–1 h^–1). The oxidation of ribitol was inhibited by ethanol but not by glycerol. Biomass yield (Y_SX; C-mmol/C-mmol) on ethanol and glycerol was low (0.21 and 0.17, respectively). In the presence of ribitol the yield was somewhat higher (0.25) with ethanol but lower (0.13) with glycerol, with respectively lower and higher CO₂ production. In chemostat cultures the oxidation rate of ribitol was unaffected by ethanol or glycerol. Cell-free extract oxidized ethanol very slowly but not ribitol; the oxidative activity was located in the cell membrane fraction. Enzymatic activities of some key metabolic enzymes were determined from steady-state chemostat with ethanol, glycerol, or ethanol/glycerol mixture as a growth limiting substrate. Based on the measured enzyme activities, metabolic pathways are proposed for ethanol and glycerol metabolism.     

Selection ofEscherichia coli K12 1EA mutants with increased synthesis of ribitol dehydrogenase

Selection of an interspecific hybridEscherichia coli K 12 1EA in a chemostat on xylitol yielded a stable mutant synthesizing a four-fold amount of ribitol dehydrogenase (EC 1.1.1.56). Subsequent cultivation of the mutant under increased selection pressure resulted in an accumulation of a mutant with 12-fold higher level of ribitol dehydrogenase relative to the parent strain 1EA. A selection during which a UV-mutagenized population of the 1EA mutant was cultivated in a chemostat on xylitol was accompanied by monitoring the activities of ribitol dehydrogenase andD-arabinitol dehydrogenase (EC 1.1.1.11) of two adjacent catabolite operons. A several-fold increase in the activity of the two enzymes was followed by further increase in the activity of ribitol dehydrogenase and a concomitant drop in the activity ofD-arabinitol dehydrogenase. The two hyperproducing strains are compared with the parent mutant as to the rate of synthesis of the two dehydrogenases and growth parameters under the conditions of batch cultivation.     

Selection of ribitol dehydrogenase hyperproducing strains in a chemostat culture of Escherichia coli 1EA at different dilution rates

Summary The growth of the strain Escherichia coli K12 1EA in a chemostat was limited by xylitol at dilution rates 0.05, 0.10 and 0.15 per h. Specific activities of ribitol dehydrogenase and D-arabinitol dehydrogenase were assayed to study the effect of dilution rate on the course of selection of ribitol dehydrogenase hyperproducing strains. It was found that relatively small differences in dilution rates lead to great differences in the outcome of selection experiments with respect to the level and basis of enzyme synthesis.     

Control of synthesis of wall teichoic acid in phosphate-starved cultures of Bacillus subtilis W23

CDP-glycerol pyrophosphorylase, CDP-ribitol pyrophosphorylase and poly(ribitol phosphate) synthetase activities have been measured in cultures of Bacillus subtilis W23 as they became phosphate-starved either in batch culture or during changeover from potassium limitation to phosphate limitation in a chemostat. The results indicated that repression of synthesis of all three enzymes occurred at the onset of phosphate starvation and that this was accompanied by inhibition of inactivation of CDP-glycerol pyrophosphorylase and poly(ribitol phosphate) synthetase. These results show that the initial response to phosphate starvation involves more than inhibition of one enzyme as proposed by Glaser and Loewy [Glaser L. and Loewy, A. (1979) J. Biol. Chem. 254, 2184-2186]. Synthesis of both linkage unit and poly(ribitol phosphate) are inhibited independently.     

Ribitol dehydrogenase of Klebsiella aerogenes. Sequence and properties of wild-type and mutant strains.

Evidence is presented for the sequence of 249 amino acids in ribitol dehydrogenase-A from Klebsiella aerogenes. Continuous culture on xylitol yields strains that superproduce 'wild-type' enzyme but mutations appear to have arisen in this process. Other strains selected by such continuous culture produce enzymes with increased specific activity for xylitol but without loss of ribitol activity. One such enzyme, ribitol dehydrogenase-D, has Pro-196 for Gly-196. Another, ribitol dehydrogenase-B, has a different mutation.     

Autonomous selection of differentiation mutants of Streptomyces lividans TK24 in continuous culture

Summary Streptomyces lividans TK24 strains transformed with different recombinat derivatives of the vector plasmid pIJ487 were continuously cultivated in a chemostat. The first plasmid derivative contained the determinant for human interferon-alpha-1 (IFN) and the expression-secretion-unit (ESU) for this protein. In the second one a nourseothricin resistance determinant (ntc) was inserted additionally. The chemostat operated mainly at glucose limitation and low dilution rates. The structural and functional stabilities of the plasmids were shown to depend on the selection pressure. The host mutants enriched in the chemostat differed from the parental strain with respect to the growth pattern of aerial and submerged mycelium, the spore formation, and the formation and secretion of pigments and enzymes. Some highly stable host-vector systems could be selected. The plasmids' genotype influenced the growth pattern of the host mutants enriched in the chemostat in dependence on the limitation conditions as well as the stability of plasmid inheritance, plasmid structure and pigment formation in these mutants.     

Mathematical modeling of population dynamics of unstable plasmid-containing bacteria during continuous cultivation in a chemostat

A structural approach to studying the regularities of the population dynamics of unstable recombinant bacterial strains in a chemostat was elaborated. The approach is based on the mathematical modeling of cell distribution in a population with different numbers of plasmid copies. The effect of decreased selective preference of plasmidless variants of the recombinant strain in the chemostat, which is related to a decrease in the number of plasmid copies in cells upon long-term incubation was analyzed. It is shown that the time of half-elimination of plasmids from the bacterial population in the steady state in the chemostat T1/2 does not depend on the maximum number of plasmid copies in cells N but is determined only by the mean time of generation g and the probability of the loss of one plasmid copy tau. The dependence of the preference of bacterial plasmidless variants on the efficiency of expression of genes cloned into plasmids in chemostat was analyzed using the recombinant strain E. coli Z905, whose plasmids pPHL-7 contain cloned genes for the luminescence system of marine luminescing bacteria Photobacterium leiognathi.     

Fed-batch xylitol production with two recombinant Saccharomyces cerevisiae strains expressing XYL1 at different levels, using glucose as a cosubstrate: a comparison of production parameters and strain stability

Xylitol production with two recombinant Sacharomyces cerevisiae strains expressing the XYL1 gene, coding for xylose reductase (XR), at different levels, the 'low XR strain' at 0.51 U/mg and the 'high XR strain' at 10.8 U/mg, was compared in batch and fed-batch culture. Xylose was not consumed in the presence of high glucose concentrations, because both sugars are transported by the glucose transport system, which has a higher affinity for glucose than for xylose. When glucose was fed gradually to the culture, high concentrations were avoided, and xylose was converted to xylitol with a specific productivity of 0.10 g g(-1) h(-1) attained with the low XR strain and 0.19 g g(-1) h(-1) with the high XR strain, indicating that factors other than the XR-activity control the rate of xylose conversion.The overproduction of XR put a substantial protein burden on the high XR strain, contributing to a 50% decrease in specific growth rate and reduced biomass yield compared with the low XR strain. Despite the use of selective medium, the stability of the high XR strain was poor in long fed-batch and chemostat cultures, whereas the low XR strain was stable. The high XR strain lost its XR activity almost completely in some fed-batch cultures and in chemostat culture. In chemostat cultivation, part of the population lost the plasmid harboring the XR gene. This was due to the fact that leucine was released into the broth from plasmid containing cells, which enabled some cells to grow without the plasmid containing the LEU2 auxotrophic complementation selection marker. Furthermore, isolation and analysis of plasmids from a population that had lost its XR activity, showed that in addition to the original plasmid, a rearranged form of the plasmid, retaining the selection marker but not the expression of active XR, was present. However, these observations could only partly explain the decrease in XR activity. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 391-399, 1997.     

COEVOLUTION IN BACTERIAL-PLASMID POPULATIONS

Evolutionary changes are described in plasmid-containing strains isolated after approximately 800 generations of growth in glucose-limited chemostat culture. The reproductive fitness increased dramatically over this period. Genetic changes associated with the increases in fitness were localized to both the bacterial and the plasmid chromosomes. In addition, some of the genetic changes on the bacterial and the plasmid chromosomes interact to minimize the deleterious effect of the plasmid. Thus, the changes observed may be considered coevolutionary. Reductions in the deleterious effects of the plasmid were shown to be associated with a decrease in plasmid copy number and an increase in the rate of segregational loss of the plasmid.     

The nature of the competitive ability of spontaneous staphylocoagulase-negative mutants of Staphylococcus aureus with respect to growth of the parent strains in continuous culture

During prolonged cultivation of S. aureus strains 104 and NCTC 8178 in continuous culture, staphylocoagulase-negative mutants arose and accumulated progressively in increasing proportions. The resulting loss of production of staphylocoagulase was accompanied by a simultaneous loss of production of -haemolysin and PV-leucocidin. Characterization of the strains revealed no further differences in biotype, exoenzymes, phage pattern and plasmid content.Cultivation in batch cultures showed that the maximal specific growth rates and specific oxygen-consumption rates of the mutant strains were slightly higher than those of the parent strains, whereas the production of total extracellular protein of the mutant strains had decreased significantly.From competition experiments between parent and mutant strains in chemostat cultures at different dilution rates and cultivation temperatures, it was concluded that the underlying mechanism of accumulation of staphylocoagulase-negative mutants in the chemostat is based on differences in affinity for the limiting substrate(s) rather than on differences in the production rates of total extracellular proteins. The complete repression of three exoenzymes, a partial repression of the total extracellular protein production, and an increased affinity for the limiting substrate(s) suggested that a mutation in a regulatory gene is involved. The possible role of a transposon in this mutation is discussed.     

Defined media optimization for growth of recombinant Escherichia coli X90

An optimized, defined minimal medium was developed to support balanced growth of Escherichia coli X90 harboring a recombinant plasmid. Foreign protein expression was repressed in these studies. A pulse injection technique was used to identify the growth responses to nutrients in a chemostat. Once the nutrients essential for growth had been identified, the yield coefficients for individual medium components. These yield coefficients were used to develop an optimized, glucose-limited defined minimal medium that supports balanced cell growth in chemostat culture. The biomass and substrate concentrations follow the Monod chemostat model. The maximum specific growth rate determined in a washout experiment is 0.87 h(-1) for this strain in the optimized medium. the glucose yield factor is 0.42 g DCW/g glucose and the maintenance coefficient is zero in the glucose-limited chemostat culture. (c) 1993 John Wiley & Sons, Inc.     

Stability and dynamics of R-plasmids in Escherichia coli populations in continuous cultivation

The stability of the conjugative plasmid RP4 and the nonconjugative plasmid pBS94 in Escherichia coli C600 cells containing both plasmids was studied in continuous cultivation under chemostat and pH-stat conditions. The plasmids remained stable in the cells of the bacterial population for 100 generations, and no cells were found without the plasmids. The competition between strains with and without the plasmids in a mixed culture resulted in the removal of the plasmid-free strain from the population. In these experiments, conjugative transfer of plasmids into the plasmid-free strain was observed, and co-transfer of both plasmids was more effective under the pH-stat conditions.     

The isolation of strains of Bacillus subtilis showing improved plasmid stability characteristics by means of selective chemostat culture

A pUB110-derived plasmid encoding chloramphenicol resistance, kanamycin resistance and high-temperature alpha-amylase showed a high degree of segregational instability when inserted into Bacillus subtilis. In an attempt to obtain stable derivatives, the organism was grown in chemostat culture in the presence of chlorampheniol. It was periodically found necessary to increase the concentration of chloramphenicol in the medium feed in order to avoid plasmid loss. Strains were isolated after 19 and 160 generations, which showed high levels of plasmid stability. This characteristic appeared to be genotypic. No detectable difference in plasmid copy number was found between the original and the improved strains. The stability characteristics resided in the host, rather than in the plasmid. Stable isolates possessed elevated MICs for both chloramphenicol and kanamycin. Their maximum specific growth rates were higher than that of the original strain, and similar to that of the plasmid-free parent strain.     

Optimization and stability of glucoamylase production by recombinant strains of Aspergillus niger in chemostat culture

When grown on a medium containing 5 g maltodextrin L-1, Aspergillus niger transformant N402[pAB6-10]B1, which has an additional 20 copies of the glucoamylase (glaA) gene, produced 320 +/- 8 mg (mean +/- S.E.) glucoamylase (GAM) L-1 in batch culture and 373 +/- 9 mg GAM L-1 in maltodextrin-limited chemostat culture at a dilution rate of 0.13 h-1. These values correspond to specific production rates (qp) of 5.6 and 16.0 mg GAM [g biomass]-1 h-1, respectively. In maltodextrin-limited chemostat cultures grown at dilution rates from 0.06 to 0.14 h-1, GAM was produced by B1 in a growth-correlated manner, demonstrating that a continuous flow culture system operated at a high dilution rate is an efficient way of producing this enzyme. In chemostat cultures grown at high dilution rates, GAM production in chemostat cultures was repressed when the limiting nutrient was fructose or xylose, but derepressed when the limiting nutrient was glucose (qp, 12.0), potassium (6.2), ammonium (4.1), phosphate (2.0), magnesium (1.5) or sulphate (0.9). For chemostat cultures grown at a dilution rate of 0.13 h-1, the addition of 5 g mycopeptone L-1 to a glucose-mineral salts medium resulted in a 64% increase in GAM concentration (from 303 +/- 12 to 496 +/- 10 mg GAM L-1) and a 37% increase in specific production rate (from 12.0 +/- 0.4 to 16.4 +/- 1.6 mg GAM [g biomass]-1 h-1). However, although recombinant protein production was stable for at least 948 h (191 generations) when A. niger B1 was grown in chemostat culture on glucose-mineral salts medium, it was stable for less than 136 h (27 generations) on medium containing mycopeptone. The predominant morphological mutants occurring after prolonged chemostat culture were shown to have selective advantage in the chemostat over the parental strain. Compared to their parental strains, two morphological mutants had similar GAM production levels, while a third had a reduced production level. Growth tests and molecular analysis revealed that the number of glaA gene copies in this latter strain (B1-M) was reduced, which could explain its reduced GAM production. Shake-flask cultures carried out with the various morphological mutants revealed that in batch culture all three strains produced considerably less GAM than their parent strains and even less than N402. We show that physiological changes in these morphological mutants contribute to this decreased level of GAM production.     

Population dynamics of a recombinant culture in a chemostat under prolonged cultivation

The dynamics of a chemostat culture of Escherichia coli K12 harboring plasmid pBR322 under prolonged cultivation with a nonselective complex medium were studied. The ability of the culture to form colonies on plates supplemented with different ampicillin concentrations was monitored. It was observed that almost all cells sampled were able to grow on a high concentration of ampicillin at the beginning of the experiment. However, a subpopulation which formed colonies on intermediate-concentration (500-1000 mg/L) plates, but not on a high-concentration (2000 mg/L) plate, was detected just before the appearance of the plasmid-free cells. As time progressed, the percentage of this subpopulation increased, reached a maximum, then decreased toward the end of experiment. At this time the culture was dominated by a subpopulation which could not form colonies on the 100 mg/L ampicillin plates. These results indicate that three major processes may occur in the chemostat: a gradual shift of the higher plasmid copy number population toward a relatively lower copy number population; the complete shedding of the plasmid due to faulty segregation of plasmids during cell division; and growth competition among the subpopulations. A previously derived model is extended to account for all subpopulations. The model agrees qualitatively with the experimental results.     

Isolation and characterization of Escherichia coli mutants able to utilize the novel pentose L-ribose.

Wild-type strains of Escherichia coli were unable to utilize L-ribose for growth. However, L-ribose-positive mutants could be isolated from strains of E. coli K-12 which contained a ribitol operon. L-ribose-positive strains of E. coli, isolated after 15 to 20 days, had a growth rate of 0.22 generation per h on L-ribose. Growth on L-ribose was found to induce the enzymes of the L-arabinose and ribitol pathways, but only ribitol-negative mutants derived from strains originally L-ribose positive lost the ability to grow on L-ribose, showing that a functional ribitol pathway was required. One of the mutations permitting growth on L-ribose enabled the mutants to produce constitutively an NADPH-linked reductase which converted L-ribose to ribitol. L-ribose is not metabolized by an isomerization to L-ribulose, as would be predicted on the basis of other pentose pathways in enteric bacteria. Instead, L-ribose was metabolized by the reduction of L-ribose to ribitol, followed by the conversion to D-ribulose by enzymes of the ribitol pathway.     

Excretion of Ribitol and Sucrose by Green Algae into the Culture Medium

Among 8 strains of algae grown with C¹⁴O₂ as a sole source of carbon, two species of Trebouxia produced appreciable amounts of two photosynthetic products in the culture medium. One of them was identified as sucrose by cochromatography and by acid hydrolysis. The other compound was identified as ribitol by paper chromatography, paper electrophoresis, periodic acid oxidation, recrystallization with authentic ribitol and finally by the enzymatic method with ribitol dehydrogenase.