Biotransformation of octane by E. coli HB101[pGEc47] on defined medium: octanoate production and product inhibition

E. coli HB101[pGEc47], which is able to convert octane to octanoate, but cannot oxidize octanoate further, was grown on defined medium with glucose as carbon source in batch and continuous culture. The biomass yield on glucose decreased from 0.32 +/- 0.02 g g-1 in aqueous cultivations to 0.25 +/- 0.02 g g-1 in the presence of octane. Maximal octanoate productivities of 0.6 g L-1 h-1 were the same as found in cultivations on complex medium. The glucose-based carbon recovery in these experiments was 99 +/- 4% (in extreme, between 90% and 105%). An increase of the octane feed from 1% to 2% (v/v) or more led to washout of cells. This effect was reversible when the octane feed was decreased to its initial value of 1%. Analysis of experimental data by model simulation strongly suggested that washout was due to inhibition by octanoate only. Pulses of octanoate to a continuous culture grown on aqueous media were applied to analyze the inhibition further. Inhibition by acetate was not significant, but its presence in the medium reflected a physiological state that made the cells more sensitive to octanoate inhibition. Model simulation with linear inhibition kinetics could perfectly predict glucose consumption and the resulting glucose concentration. The linear type of inhibition was confirmed by a variety of batch experiments in the presence of different concentrations of octanoate. The glucose-based specific growth rate, mu, decreased linearly with increasing concentrations of octanoate and became zero at a threshold concentration pmax of 5.25 +/- 0.25 g L-1.     

Dimethyl sulfoxide reductase activity by anaerobically grown Escherichia coli HB101.

Escherichia coli grew anaerobically on a minimal medium with glycerol as the carbon and energy source and dimethyl sulfoxide (DMSO) as the terminal electron acceptor. DMSO reductase activity, measured with an artificial electron donor (reduced benzyl viologen), was preferentially associated with the membrane fraction (77 +/- 10% total cellular activity). A Km for DMSO reduction of 170 +/- 60 microM was determined for the membrane-bound activity. Methyl viologen, reduced flavin mononucleotide, and reduced flavin adenine dinucleotide also served as electron donors for DMSO reduction. Methionine sulfoxide, a DMSO analog, could substitute for DMSO in both the growth medium and in the benzyl viologen assay. DMSO reductase activity was present in cells grown anaerobically on DMSO but was repressed by the presence of nitrate or by aerobic growth. Anaerobic growth on DMSO coinduced nitrate, fumarate, and and trimethylamine-N-oxide reductase activities. The requirement of a molybdenum cofactor for DMSO reduction was suggested by the inhibition of growth and a 60% reduction in DMSO reductase activity in the presence of 10 mM sodium tungstate. Furthermore, chlorate-resistant mutants chlA, chlB, chlE, and chlG were unable to grow anaerobically on DMSO. DMSO reduction appears to be under the control of the fnr gene.     

Cell cycle analysis of plasmid-containing Escherichia coli HB101 populations with flow cytometry

The relationship between cell mass and cell number dynamics for bacteria such as Escherichia coli depends on the cell cycle parameters C and D. Effects of plasmid copy number on these cell cycle parameters have been studied for Escherichia coli HB101 containing pMB1 plasmids propagated at different copy numbers ranging from 12 to 122. Determination of cell cycle and cell size parameters was accomplished using flow cytometry data on single-cell light scattering and DNA content frequency functions in conjunction with a mathematical model of cell population statistics. Two independent methods for estimating C and D intervals based on flow cytometry were developed and applied with essentially identical results. The presence of plasmids decreases the C and D periods, mean cell sizes, and initiation masses for chromosome replication by 14, 24, 38, and 18%, respectively, relative to corresponding values for plasmid-free host cells. Plasmid copy number has a negligible influence on these parameters, suggesting that host-plasmid inter actions which determine these properties are centered on the single plasmid selected for replication according to the random selection model established for ColE1-type plasmids.     

Sequencing and high expression of aminopeptidase P gene from Escherichia coli HB101

A plasmid pAPP1 with a 4 kbp insert at the PstI site of pBR322, encoding aminopeptidase P gene of Escherichia coli HB101 (Yoshimoto et al. (1988) J. Biochem. 104, 730-734), was subcloned into pUC18 and pUC19. The transformant of E. coli JM83 harboring pAPP4 with a 1.9 kbp fragment showed more than 50-fold higher enzyme activity than that of the host, after cultivation at 37 degrees C for 40 h in LB-medium containing ampicillin. When the gene DNA was inserted reversely in pAPP4, the enzyme productivity decreased markedly. The whole nucleotide sequence of the inserted fragment of plasmid pAPP4 was clarified by the dideoxy chain-terminating method. Within this sequence, the mature enzyme protein-encoding sequence was found to start just after an ATG codon, as judged by comparison with amino-terminal protein sequencing. Eleven bases upstream from the proposed initiation codon was an AGGAGA sequence which seemed to be a ribosome binding site. Thirty-four bases upstream from the proposed start codon was the 6-base sequence TACAAA, the so-called -10 region or Pribnow box. Further, the 6-base sequence TTTACT around 77 bases upstream from the start codon was deduced to be a putative -35 region consensus sequence. The inverted repeat at 1334 was tentatively assumed to be a terminator. The molecular weight of the enzyme was estimated to be 49,650 from the nucleotide sequence. The purified enzyme contained 0.2 gram atom of zinc per subunit. The enzyme activity was inhibited by EDTA and activated 5-fold by Mn2+.     

Effect of growth rate on plasmid maintenance by Escherichia coli HB101(pAT153)

The effects of changing the composition of the growth medium, the dilution rate and the source of the bacterial host on maintenance of the plasmid pAT153 in Escherichia coli HB101 have been studied. In a medium supplemented with Casamino acids, the plasmid was maintained longer during phosphate-limited growth at a dilution rate of 0.3 h-1 than at 0.15 h-1. In contrast, phosphate-limited growth was not achieved when the Casamino acids were replaced by proline, leucine and thiamin to satisfy the auxotrophic requirements of the host. Although 100% of the bacteria were still ampicillin resistant after 72 generations of growth at a dilution rate of 0.15 h-1, the original plasmid had almost totally been replaced by a structurally modified plasmid which lacked a functional tet gene. Further experiments confirmed that neither the host nor the plasmid was retained unchanged in the minimal medium. The changes were highly reproducible and reflected periodic selection of sub-populations which were either plasmid-free or carried a structurally modified plasmid, which had reverted to Leu+ or Pro+, or had acquired other chromosomal mutations which gave them a selective advantage. We conclude that in complex media the plasmid is maintained longer by E. coli HB101 at a high than at a low growth rate and that different results reported from different laboratories are largely due to differences in analytical techniques and the growth medium rather than to differences in the bacterial host or the plasmid used. A fermenter-adapted strain was isolated which reproducibly maintained the plasmid longer during phosphate-limited continuous growth than the original strain which had been cultured on laboratory media.     

Subcellular distribution of alkalophilic Bacillus penicillinase produced by Escherichia coli HB101 carrying pEAP31

Summary The extracellular production of alkalophilic Bacillus penicillinase by Escherichia coli HB101 carrying pEAP31 was dependent on the cultivation temperature. Extracellular production occurred only above 26°C. The penicillinase produced by the organism grown at lower temperatures accumulated in the periplasm of the cells. At high temperature, the penicillinase accumulated transiently in the periplasm and then was released gradually from the cells. The penicillinase that accumulated in the periplasm of the organism grown at low temperature could also be released by shifting to a high temperature.     

Proton translocation coupled to dimethyl sulfoxide reduction in anaerobically grown Escherichia coli HB101.

Proton translocation coupled to dimethyl sulfoxide (DMSO) reduction was examined in Escherichia coli HB101 grown anaerobically on glycerol and DMSO. Rapid acidification of the medium was observed when an anaerobic suspension of cells, preincubated with glycerol, was pulsed with DMSO, methionine sulfoxide, nitrate, or trimethylamine N-oxide. The DMSO-induced acidification was sensitive to the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (60 microM) and was inhibited by the quinone analog 2-n-heptyl-4-hydroxy-quinoline-N-oxide (5.6 microM). Neither sodium azide nor potassium cyanide inhibited the DMSO response. An apparent----H+/2e- ratio of 2.9 was obtained for DMSO reduction with glycerol as the reductant. Formate and H2(g), but not lactate, could serve as alternate electron donors for DMSO reduction. Cells grown anaerobically on glycerol and fumarate displayed a similar response to pulses of DMSO, methionine sulfoxide, nitrate, and trimethylamine N-oxide with either glycerol or H2(g) as the electron donor. However, fumarate pulses did not result in acidification of the suspension medium. Proton translocation coupled to DMSO reduction was also demonstrated in membrane vesicles by fluorescence quenching. The addition of DMSO to hydrogen-saturated everted membrane vesicles resulted in a carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone-sensitive fluorescence quenching of quinacrine dihydrochloride. The data indicate that reduction of DMSO by E. coli is catalyzed by an anaerobic electron transport chain, resulting in the formation of a proton motive force.     

Maximum yield of foreign lipase in Escherichia coli HB101 limited by duration of protein expression

When a microbial lipase was overexpressed in Escherichia coli HB101, the expression kinetics as represented by the expression rate, duration, and maximum yield of lipase were studied. Lipase synthesis, controlled by the tac promoter, continued for about 4h after IPTG induction. The duration of the expression phase was similar, irrespective of expression rate and yield, which were manipulated by using α-methyl glucose (α-MG), a competitive inhibitor of glucose. By measuring the specific oxygen uptake rate, specific CO₂ evolution rate, specific glucose uptake rate, intracellular protease level and the acetate concentration in the culture, the limited duration of the expression phase was found to be caused by metabolic stress arising from the rapid and massive production of the foreign protein under the strong promoter. Neither the total cell number nor the number of living cells increased substantially after induction, whereas the optical density of the culture gradually increased. The duration of the expression phase was reduced to less than 2 h by the addition of menadione, a redox cycling agent, seemingly due to an acceleration of the energetic flow of the host cells after induction. In contrast, the duration of the expression phase was extended to 8 h in the glucose-starved condition, although the maximum expression yield was much lower than that in the glucose-surplus condition. Therefore, it was suggested that the expression rate after induction determined the maximum expression yield of the foreign lipase gene in E. coli HB101 because of the restrained capacity of foreign protein production.     

Extracellular accumulation of recombinant protein by Escherichia coli in a defined medium

Extracellular accumulation of recombinant proteins in the culture medium of Escherichia coli is desirable but difficult to obtain. The inner or cytoplasmic membrane and the outer membrane of E. coli are two barriers for releasing recombinant proteins expressed in the cytoplasm into the culture medium. Even if recombinant proteins have been exported into the periplasm, a space between the outer membrane and the inner membrane, the outer membrane remains the last barrier for their extracellular release. However, when E. coli was cultured in a particular defined medium, recombinant proteins exported into the periplasm could diffuse into the culture medium automatically. If a nonionic detergent, Triton X-100, was added in the medium, recombinant proteins expressed in the cytoplasm could also be released into the culture medium. It was then that extracellular accumulation of recombinant proteins could be obtained by exporting them into the periplasm or releasing them from the cytoplasm with Triton X-100 addition. The tactics described herein provided simple and valuable methods for achieving extracellular production of recombinant proteins in E. coli.     

Mobilization of plasmid pHSV106 from Escherichia coli HB101 in a laboratory-scale waste treatment facility

The mobilization of plasmid pHSV106 from Escherichia coli HB101, in a laboratory model waste treatment facility, by both laboratory and indigenous wastewater strains of E. coli was monitored by transfer of antibiotic resistance characteristics and by detection of pHSV106 DNA sequences in recipient cells. The mobilization was demonstrated to occur under several different treatment conditions, such as different media composition, microbial concentrations, and waste flow rates. The herpes simplex virus thymidine kinase gene was used as a hybridization marker to confirm the occurrence of the transfer. The use of the HB101 (recA mutant) host cell implies that recA functions are unnecessary for the gene transfer.     

Fermentation conditions for high-level expression of the tac-promoter-controlled calf prochymosin cDNA in Escherichia coli HB101

Escherichia coli HB101 harboring an expression plasmid that bears the calf prochymosin gene controlled by the tac promoter was cultivated under different conditions in order to find an optimal fermentation arrangement that would lead to maximal prochymosin yield. Our results indicate that it is advantageous to use lactose in the double role of inducer and carbon/energy source when foreign gene expression is controlled by the tac promoter and the gene product is only moderately toxic owing to its accumulation in the form of an intracellular body. Glucose, on the other hand, may be used when expression should be repressed. Growth temperature substantially influenced the specific rate of prochymosin and beta-lactamase gene expression and the plasmid copy number. Three phases were distinguished in the time course of the fermentation on lactose: exponential growth practically without prochymosin synthesis, linear growth with prochymosin synthesis, and prochymosin synthesis without growth of biomass. The synthesis of prochymosin in the form of intracellular inclusion body was accompanied by the loss of respiratory activity of the cell and the loss of its ability to multiply. Sixteen hours cultivation at 37 degrees C in a complex medium with lactose as inducer and carbon/energy source resulted in up to 30% of the volume and 48% of the total protein of biomass being accumulated for as prochymosin inclusion bodies. The concentration of extractable enzymatically active chymosin in the culture reached 12 mg/L.     

A single-use luciferase-based mercury biosensor using Escherichia coli HB101 immobilized in a latex copolymer film

A single-use Hg(II) patch biosensor has been developed consisting of 1.25-cm diameter patches of two acrylic vinyl acetate copolymer layers coated on polyester. The top layer copolymer was 47 μm thick whereas the bottom layer of copolymer plus E. coli cells was 30 μm thick. The immobilized E. coli HB101 cells harbored a mer-lux plasmid construct and produced a detectable light signal when exposed to Hg(II). The immobilized-cell Hg(II) biosensor had a sensitivity similar to that of suspended cells but a significantly larger detection range. The levels of mercury detected by the patches ranged from 0.1 nM to 10 000 nM HgCl₂ in pyruvate buffer, and luciferase induction as a function of Hg(II) concentration was sigmoidal. Luciferase activity was detected in immobilized cells for more than 78 h after exposure of the cells to HgCl₂. Addition of 1 mM D-cysteine to the pyruvate buffer increased luciferase induction more than 100-fold in the immobilized cell patches and 3.5-fold in a comparable suspension culture. The copolymer patches with immobilized cells were stable at −20°C for at least 3 months, and the Hg(II)-induced luciferase activity after storage was similar to that of samples assayed immediately after coating. Patches stored desiccated at room temperature for 2 weeks showed lower mercury-induced luciferase activity when compared to freshly prepared patches, but they still had a considerable detection range of 1 to 10 000 nM HgCl₂. Received 05 November 1998/ Accepted in revised form 08 April 1999     

Coenzyme Q production by metabolic engineered Escherichia coli strains in defined medium

Bioprocess and Biosystems Engineering - Coenzyme Q (CoQ) plays an important role as an electron transporter in the respiratory chain. It is formed from a benzoquinone ring and an isoprenoid chain...     

Cloning and expression of aminopeptidase P gene from Escherichia coli HB101 and characterization of expressed enzyme

The aminopeptidase P gene in Escherichia coli HB101 was cloned into the plasmid pBR322. Introduction of the hybrid plasmid, pAPP01, into the E. coli DH1 resulted in an 8-fold increase of aminopeptidase P activity as compared with that of the host. The enzyme was purified by series of chromatographies on DEAE-Sephadex, QAE-Sephadex, and hydroxyapatite. The purified enzyme was homogeneous as judged by disc-gel and SDS-gel electrophoreses. the enzyme was inhibited strongly by EDTA and slightly by p-chloromercuribenzoate, but was not affected by diisopropyl phosphorofluoridate, E-64, or iodoacetic acid. The optimum pH of the enzyme was 8.5. The enzyme was stable at pH 8 to 9. After incubation for 30 min at pH 8.0, 50% remaining activity was observed at 50 degrees C. The enzyme was activated 3-fold by the addition of 5 microM Mn2+. The molecular weight of the enzyme was estimated to be 50,000 and 200,000 by SDS-PAGE and gel filtration, respectively. The amino terminal amino acid was identified to be serine by Edman degradation, indicating that the enzyme is composed of a homo-tetramer. The enzyme hydrolyzed X-Pro bonds (X = amino acid) of peptides. These characteristics suggest that cloned aminopeptidase P is identical to APP-II reported by Yoshimoto et al. (Agric. Biol. Chem. 52(8), in press (1988].     

Mobilization of plasmid pHSV106 from Escherichia coli HB101 in a laboratory-scale waste treatment facility.

The mobilization of plasmid pHSV106 from Escherichia coli HB101, in a laboratory model waste treatment facility, by both laboratory and indigenous wastewater strains of E. coli was monitored by transfer of antibiotic resistance characteristics and by detection of pHSV106 DNA sequences in recipient cells. The mobilization was demonstrated to occur under several different treatment conditions, such as different media composition, microbial concentrations, and waste flow rates. The herpes simplex virus thymidine kinase gene was used as a hybridization marker to confirm the occurrence of the transfer. The use of the HB101 (recA mutant) host cell implies that recA functions are unnecessary for the gene transfer.     

Expression of a cloned 987P adhesion-antigen fimbrial determinant in Escherichia coli K-12 strain HB101

The properties of three independent enterotoxigenic Escherichia coli isolates known to express 987P adhesion fimbriae in a manner subject to phase variation were examined. Phase variation could not be correlated with any major changes in the plasmid DNA content of these strains or with readily detectable changes in any other tested phenotypic markers. The 987P genetic determinant from one of these strains, E. coli 987, was cloned into the non-fimbriated E. coli K-12 strains HB101, and expressed, using the cosmid vector system. 987P fimbriae produced by cells harbouring these recombinant plasmids (987P+ phenotype) could not be distinguished from 987P fimbriae produced by strain 987. Expression of 987P fimbriae from some recombinant plasmids was unstable but none of the recombinants exhibited the phase variation phenotype displayed by the parental strain. One recombinant plasmid, pPM200, contained an insert of strain 987 DNA of ca. 33 kb. The HB101[pPM200] displayed a rather stable 987P+ phenotype, but this was not true for several hosts, since pPM200 acquired approx. 20-kb deletions following transformations of E. coli K-12 strains other than HB101. The deletions mapped to the same region of pPM200 irrespective of the host strain transformed. Cells harbouring the deleted plasmids did not express 987P fimbriae (987P- phenotype).     

霍乱弧菌脂多糖基因在大肠杆菌中的克隆

利用柯斯质粒pHC 79为载体,构建了霍乱弧菌178(埃尔托生物型,小川血清型)染色体基因文库。经血清凝集试验及菌落固相ELISA检测,从基因文库中筛选到13株能够表达霍乱弧菌脂多糖O抗原的阳性克隆。经热酚水法从转化于中提取并纯化的脂多糖能与霍乱弧菌抗血清发生特异性结合。针对重组柯斯质粒PMM—VO 38进行了多种酶切分析,测定其分子量为46kb。