Cell cycle parameters of slowly growing Escherichia coli B/r studied by flow cytometry

The cell cycle kinetics of Escherichia coli B/r A and B/r K cells were studied by flow cytometry. Three-dimensional histograms of cell cultures show the number of cells as a function of cellular DNA and protein contents and give detailed pictures of the cell cycle distribution with regard to these parameters. Histograms of slowly growing chemostat cultures showed that cell cycle periods B and C + D increase with a decreasing growth rate and that the B period occupies an increasing fraction of the cycle. The DNA replication patterns of B/r A and K were found to be quite similar. At extremely low growth rates (doubling time [T] = 17 h), B/r A cells had a B period of 0.8 T, a C period of 0.1 T, and a D period of 0.1 T, and B/r K cells (T = 16 h) had a B period of 0.6 T, a C period of 0.15 T, and a D period of 0.25 T. Mass increase, i.e., essentially protein synthesis, was seen in all three periods of the cell cycle. For B/r A cells, the average rate of mass increase was 11 times greater in the D period than in the B period, whereas for B/r K cells the rate of mass increase was twice as great in the D period as in the B period. The DNA and cell size distributions of batch cultures in exponential growth were found to vary with time, indicating that such cultures are not suitable for studies of cell cycle kinetics.     

Cell cycle kinetic analysis with flow cytometry in pea root meristem synchronized with aphidicolin

Meristematic cells in pea root were synchronized after treatment with aphidicolin. Flow cytometric analysis of DNA content variations revealed a variety of information: ca 50% of the cells were synchronized in the first part of the S phase; kinetic studies of the S-phase traverse revealed an increase of the DNA synthesis rate from early to late S; some of the cells in the meristem were non-cycling, arrested in Gl (19%) and G2 (12%). The possibility of using such a synchronized system in cytogenetic and biomolecular studies is discussed.     

Cell cycle analysis of asynchronous cell populations by flow cytometry using bromodeoxyuridine label and Hoechst-propidium iodide stain.

Continuous labelling of cells with deoxybromouridine (BrdUrd) followed by staining with a bis-benzimidazole (Hoechst 33258) and a phenanthridinium (propidium iodide or ethidium bromide) allows the cells to be separated by flow cytometry according to the extent of their DNA replication. This BrdUrd-Hoechst/PI method has been used mainly to observe perturbations of the cell cycle in synchronously growing cells. In this paper we demonstrate that, when the method is applied to asynchronously dividing cells, more extensive information can be derived about the effects of cytotoxic and other treatments on the kinetics of the cell cycle. The interpretation of the data is explained, the effects of different types of cytotoxic agent are described, and the method is compared briefly to other methods for following cell cycle kinetics.     

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 kinetic analysis of mixed populations using three-color fluorescence flow cytometry.

The development of antibodies to DNA-incorporated thymidine analogs has in turn led to the development of flow cytometric techniques for rapidly measuring cell kinetics parameters. More recently, these techniques have been applied to clinical tumor material. One problem with such measurements has been the difficulty of distinguishing malignant cells from coexistent normal cells in the biopsy material. In the present study, the feasibility of selecting out the desired malignant cell population for kinetic analysis from a mixture of cells was tested in vitro. An anticytokeratin antibody was used to discriminate between a mixture of tumor cells (cytokeratin positive) and normal cells (cytokeratin negative). The cell lines chosen for the study, A549 human lung carcinoma cells and Chinese hamster ovary (CHO) cells, were pulse labeled with iododeoxyuridine (IdUrd) and sampled every hour up to 16 hours. Selecting out cells from the mixture required the application of three-color fluorescence flow cytometry, which was carried out using the fluorochromes FITC (fluorescein isothionate, green fluorescence, IdUrd-DNA antibody), PE (phycoerythrin, orange fluorescence, cytokeratin antibody), and PI (propidium iodide, red fluorescence, DNA). This allowed single laser excitation. The staining procedure involved incubation with the IdUrd antibodies (specific antibody plus FITC-conjugated second antibody) followed by the cytokeratin antibodies (specific antibody plus PE-conjugated second antibody) and lastly by the DNA stain containing RNase. Two analysis methods of the IdUrd/DNA cytograms were applied: a mid-S window analysis and a relative movement (RM) analysis. Results of the analyses for cells selected out of mixtures were compared with results of cells stained and analyzed separately. A clear separation of the two cell lines could be obtained on the basis of orange fluorescence (cytokeratin content) despite a large overlap of their DNA histograms. By gating on high or low orange fluorescence, almost pure populations of the individual cell types could be selected out for further kinetic analysis. Little difference was seen, with both the mid-S and RM analyses, between cells gated from mixtures or stained separately. It is concluded that this technique is feasible for use on clinical material, provided good cell suspensions can be obtained, leading to the hope of increasing the accuracy of kinetic measurements on human tumors.     

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+.     

Detection of Escherichia coli in blood using flow cytometry

A rapid method for the detection of Escherichia coli in blood has been developed. The method employs blood cell lysis, staining of bacteria with ethidium bromide, and detection of stained bacteria using flow cytometry. The detection protocol requires less than 2 h sample handling time and is not dependent on bacterial growth. This method has been applied to human donor blood specimens seeded with various E. coli concentrations and to two rabbit model systems. Bacterial detection is evident from the in vitro human blood studies at levels of 10 E. coli/ml and from in vivo rabbit model studies at less than 100 E. coli/ml.     

Enhanced survival of plasmid-containing Escherichia coli in aquatic microcosms

The survival of Escherichia coli K-12 J62-1 containing the antibiotic-resistance plasmid R1 and an isogenic plasmid-free strain were studied in pond water microcosms. The number of plasmid-containing cells recovered from the microcosms remained constant over a sampling period of 31 days whereas plasmid-free cell numbers declined.     

Growth characteristics of Escherichia coli HB101[pGEc47] on defined medium

This paper shows that differences in growth behavior of Escherichia coli strain HB101 and strain HB101[pGEc47] can be related to yeast extract-enriched medium rather than plasmid properties. An optimal medium for growth of E. coli HB101[pGEc47] was designed based on the individual yield coefficients for specific medium components (NH4+ 6 g g-1, PO43- 14 g g-1, SO42- 50 g g-1). The yield coefficient for L-leucine depends on the glucose content of the medium (20 g g-1 for 3% glucose, 40 g g-1 for 1% glucose) and the yield coefficient for L-proline depends on the cultivation mode (20 g g-1 for batch cultivation, 44 g g-1 for continuous cultivation). Growth on defined medium after medium optimization is as rapid as on complex medium (0. 42-0.45 h-1). The critical dilution rate (DR) in the defined medium above which undesired production of acetic acid occurs is in the range of 0.23-0.26 h-1.     

Cell cycle analysis of synchronized chinese hamster cells using bromodeoxyuridine labeling and flow cytometry

Summary Chinese hamster ovary cells were synchronized into purified populations of viable G1-, S-, G2-, and M-phase cells by a combination of methods, including growth arrest, aphidicolin block, cell cycle progression, mitotic shake-off, and centrifugal elutriation. The DNA content and bromodeoxyuridine (BrdUrd) labeling index were measured in each purified fraction by dual-parameter flow cytometry. The cell cycle distributions determined from the DNA measurements alone (single parameter) were compared with those calculated from both DNA and BrdUrd data (dual parameter). The results show that highly purified cells can be obtained using these methods, but the assessed purity depends on the method of cell cycle analysis. Using the single versus dual parameter measurement to determine cell cycle distributions gave similar results for most phases of the cell cycle, except for cells near the transition from G1- to S-phase and S- to G2-phase. There the BrdUrd labeling index determined by flow cytometry was more sensitive for detecting small amounts of DNA synthesis. As an alternative to flow cytometry, a simple method of measuring BrdUrd labeling index on cell smears was used and gave the same result as flow cytometry. Measuring both DNA content and DNA synthesis improves characterization of synchronized cell populations, especially at the transitions in and out of S-phase, when cells are undergoing dramatic shifts in biochemical activity.     

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.     

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.     

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.     

Cell cycle analysis of F''lac replication in Escherichia coli B/r.

The timing and control of replication of an F'lac plasmid was investigated in two substrains of Escherichia coli B/r lac/F'lac growing at a variety of rates. The cellular content of covalently closed circular F'lac deoxyribonucleic acid and the cellular mass at the time of F'lac replication both increased as a function of growth rate. The timing of plasmid replication during the division cycle was determined by measuring the inducibility of beta-galactosidase in cells of different ages in exponentially growing cultures. At all growth rates, the rate of induced beta-galactosidase synthesis increased in a step-wise fashion during the division cycle, indicating that the F'lac plasmid replicated at a discrete time in the cycle. At growth rates greater than one doubling per h, the cell age at F'lac replication was indistinguishable from the cell age at chromosomal lac+ replication in an isogenic F- parent. The ratio of plasmids to chromosomal origins decreased from about 0.7 to 0.4 between growth rates of 1.0 to 2.5 doublings per h. These observations are all consistent with replication of F'lac at about the same time in the division cycle as replication of the homologous chromosomal region at these growth rates. This similarity in timing of replication of homologous deoxyribonucleic acid regions was not evident in slower-growing cells.     

Cell cycle parameters of Escherichia coli K-12.

A computer simulation routine was used to calculate the DNA distributions of exponentially growing cultures of Escherichia coli K-12. Simulated distributions were compared with distributions obtained experimentally by flow cytometry. Durations of the DNA replication period (C) and the postreplication period (D) were found by minimizing the difference between theoretical and experimental DNA histograms. It was demonstrated that the K-12 strains AB1157 and CM735 had C and D periods that differed widely from each other and from those of the previously measured strain B/rA, while strain MC1000 was shown to have the same durations of the C and D periods as strain B/rA. The variation between K-12 strains may explain the divergence in the literature regarding their C and D periods. Strains W3110 and AB1157 recA1 had DNA histograms that could not be adequately simulated by the classical Cooper-Helmstetter model, which is consistent with the asymmetrically located origin and terminus for W3110 and the asynchrony of initiation for AB1157 recA1.     

Induction studies with Escherichia coli expressing recombinant interleukin-13 using multi-parameter flow cytometry

The expression of interleukin-13 (IL13) following induction with IPTG in Escherichia coli results in metabolic changes as indicated by multi-parameter flow cytometry and traditional methods of fermentation profiling (O₂ uptake rate, CO₂ evolution rate and optical density measurements). Induction early in the rapid growth phase was optimal although this led to lower overall biomass concentrations and lower maximum specific growth rates. In contrast, induction in the mid-rapid growth phase was the most detrimental to cell quality as measured by cytoplamsic membrane depolarisation.