The Electro-Optical Investigation of Suspensions of Escherichia coli K-12 Cells Metabolizing Glucose,Lactose, and Galactose

The electro-optical characteristics of suspensions of Escherichia coli K-12 cells metabolizing glucose, lactose, and galactose were studied by measuring the suspension turbidity as a function of cell alignment in an orienting electric field whose frequency was varied from 10 kHz to 10 MHz. In a frequency range of 10 kHz to 1 MHz, the orientational spectra of E. coli K-12 cells grown on glucose and lactose considerably changed after their incubation in the presence of the sugars. These changes likely reflect alterations in the polarizability of the cells induced by sugar metabolism.     

The effect of ampicillin on the electrophysical properties of Escherichia coli cells

The study of the effect of ampicillin on the electrophysical properties of Escherichia coli cells showed that this antibiotic influences the orientational spectra (OS) of the ampicillin-susceptible E. coli strains K-12 and XL-1 within the frequency range 10-1000 kHz of the orienting electric field and does not affect the OS of the ampicillin-resistant strains K-12(pUC-18) and XL-1(pHEN1). The change in the electrooptical signal of the ampicillin-susceptible cells was maximum at an ampicillin concentration of 50 microg/ml and did not depend on the exposure time. The conclusion is drawn that changes in the OS of cells can be used to evaluate their resistance to ampicillin.     

Salt tolerance of lactose-grown Vibrio parahaemolyticus carrying Escherichia coli lac genes

Lac- strains of Vibrio parahaemolyticus were converted to Lac+ on receiving a hybrid plasmid containing the lactose utilization genes of Escherichia coli K-12. A V. parahaemolyticus strain containing this hybrid plasmid exhibited optimal growth rates on glucose and other carbon sources in the presence of 0.2 to 0.4 M NaCl. Growth of the same strain on lactose was inhibited at similar concentrations of NaCl. The altered growth rate responses in lactose medium appeared to be attributable to effects of NaCl on the activity of lactose permease, and possibly on that of beta-galactosidase, rather than on the levels of these enzymes in V. parahaemolyticus cells.     

Salt tolerance of lactose-grown Vibrio parahaemolyticus carrying Escherichia coli lac genes.

Lac- strains of Vibrio parahaemolyticus were converted to Lac+ on receiving a hybrid plasmid containing the lactose utilization genes of Escherichia coli K-12. A V. parahaemolyticus strain containing this hybrid plasmid exhibited optimal growth rates on glucose and other carbon sources in the presence of 0.2 to 0.4 M NaCl. Growth of the same strain on lactose was inhibited at similar concentrations of NaCl. The altered growth rate responses in lactose medium appeared to be attributable to effects of NaCl on the activity of lactose permease, and possibly on that of beta-galactosidase, rather than on the levels of these enzymes in V. parahaemolyticus cells.     

Activation of the cryptic PhnE permease promotes rapid adaptive evolution in a population of Escherichia coli K-12 starved for phosphate

Escherichia coli K-12 suffers acetic acid stress during prolonged incubation in glucose minimal medium containing a limiting concentration of inorganic phosphate (0.1 mM P(i)), which decreases the number of viable cells from 6 × 10(8) to ≤10 CFU/ml between days 6 and 14 of incubation. Here we show that following two serial transfers into P(i)-limiting medium, evolved mutants survived prolonged incubation (≈10(7) CFU/ml on day 14 of incubation). The evolved strains that overtook the populations were generally PhnE(+), whereas the ancestral K-12 strain carries an inactive phnE allele, which prevents the transport of phosphonates. The switching in phnE occurred with a high frequency as a result of the deletion of an 8-bp repeated sequence. In a mixed culture starved for P(i) that contained the K-12 ancestral strain in majority, evolved strains grew through PhnE-dependent scavenging of probably organic phosphate esters (not phosphonates or P(i)) released by E. coli K-12 between days 1 and 3, before acetic acid excreted by E. coli K-12 reached toxic levels. The growth yield of phnE(+) strains in mixed culture was dramatically enhanced by mutations that affect glucose metabolism, such as an rpoS mutation inactivating the alternative sigma factor RpoS. The long-term viability of evolved populations was generally higher when the ancestral strain carried an inactive rather than an active phnE allele, which indicates that cross-feeding of phosphorylated products as a result of the phnE polymorphism may be essential for the spread of mutants which eventually help populations to survive under P(i) starvation conditions.     

A bioelectrochemical study of a suspension of Escherichia coli cells metabolizing glucose and lactose

The metabolism of glucose and lactose in E. coli K-12 cells has been studied using a bioelectrochemical (BEC) approach. The magnitude and the duration of the response of a BEC anode were found to be functions of the composition of nutrient media and the concentration of bacterial cells. The amount of electricity that is generated enzymatically during the metabolism of a particular substrate depends on the activity of the relevant enzymes. This suggests that the BEC approach can be used for evaluating the activity of particular enzyme systems.     

Changes in electrophysical characteristics of Azospirillum brasilense Sp7 cells during their interaction with polyclonal antibodies

Electrooptical characteristics of Azospirillum brasilense Sp7 cells during their specific interaction with polyclonal rabbit antibodies were studied. Dependence of optical density of cell suspension during electroorientation of cells from frequency of orienting field in interval 10, 100, 250, and 500 kHz was evaluated. Itwas shown that electrooptical (EO) characteristics of bacterial suspensions change during interaction of A. brasilense cells with antibodies, and maximal changes occur when frequency of oriented field amounts 100-250 kHz. During interaction of A. brasilense Sp7 with strain-specific polyclonal antibodies in the presence of Escherichia coli K-12 and Pseudomonas putida C-11 decrease of amplitude of analytic signal was observed but detection of A. brasilense Sp7 cells was possible. Possibility of detection of microorganisms by EO analysis during their interaction with antibodies was shown.     

Electrooptical parameters of kanamycin-treated E. coli cell suspensions

The effect of kanamycin on the electrophysical parameters of cell suspensions of Escherichia coli K-12 and pMMB33 was investigated. Incubation of the sensitive K-12 strain with kanamycin resulted in significant changes in the orientation spectra (OS) of the cell suspensions; these changes were not revealed in the case of the resistant pMMB33 strain. In the case of the sensitive K-12 strain incubated with different kanamycin concentrations, changes in the OS of the cell suspensions occurred within the 10-1000 kHz frequency range of the orienting electrical field. The most pronounced change in the electrooptical signal was observed at 10 microg/ml of kanamycin. Control experiments were carried out by standard plating on nutrient media. Thus, the OS changes of suspensions in the presence of antibiotics may be used as a test for microbial resistance to such antibiotics.     

Possible mechanisms underlying the slow lactose fermentation phenotype in Shigella spp.

A Southern hybridization analysis revealed that the region homologous to Escherichia coli lacZ was present on the chromosomal DNAs of beta-galactosidase-positive Shigella strains, such as Shigella dysenteriae serovar 1 and Shigella sonnei strains, whereas this region was absent from chromosomal DNAs of beta-galactosidase-negative strains of Shigella flexneri and Shigella boydii. We found that the lacY-A region was deficient in S. dysenteriae serovar 1 and believe that this is the reason for the slow fermentation of lactose by this strain. S. sonnei strains possessed the region which hybridized with E. coli lacY-A despite their slow hydrolysis of lactose. The whole lactose-fermenting region was cloned from S. sonnei and compared with the cloned lac operon of E. coli K-12. Both clones directed the synthesis of beta-galactosidase in an E. coli K-12 strain lacking indigenous beta-galactosidase activity (strain JM109-1), and we observed no difference in the expression of beta-galactosidase activity in S. sonnei and E. coli. However, E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei exhibited the slow lactose fermentation phenotype like the parental strain. S. sonnei strains had no detectable lactose permease activities. E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei had a detectable permease activity, possibly because of the multicopy nature of the cloned genes, but this permease activity was much lower than that of strain JM109-1 harboring the lac operon of E. coli K-12. From these results we concluded that slow lactose fermentation by S. sonnei is due to weak lactose permease activity.     

Possible mechanisms underlying the slow lactose fermentation phenotype in Shigella spp.

A Southern hybridization analysis revealed that the region homologous to Escherichia coli lacZ was present on the chromosomal DNAs of beta-galactosidase-positive Shigella strains, such as Shigella dysenteriae serovar 1 and Shigella sonnei strains, whereas this region was absent from chromosomal DNAs of beta-galactosidase-negative strains of Shigella flexneri and Shigella boydii. We found that the lacY-A region was deficient in S. dysenteriae serovar 1 and believe that this is the reason for the slow fermentation of lactose by this strain. S. sonnei strains possessed the region which hybridized with E. coli lacY-A despite their slow hydrolysis of lactose. The whole lactose-fermenting region was cloned from S. sonnei and compared with the cloned lac operon of E. coli K-12. Both clones directed the synthesis of beta-galactosidase in an E. coli K-12 strain lacking indigenous beta-galactosidase activity (strain JM109-1), and we observed no difference in the expression of beta-galactosidase activity in S. sonnei and E. coli. However, E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei exhibited the slow lactose fermentation phenotype like the parental strain. S. sonnei strains had no detectable lactose permease activities. E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei had a detectable permease activity, possibly because of the multicopy nature of the cloned genes, but this permease activity was much lower than that of strain JM109-1 harboring the lac operon of E. coli K-12. From these results we concluded that slow lactose fermentation by S. sonnei is due to weak lactose permease activity.     

大肠杆菌磷酸果糖激酶基因在极端嗜酸性氧化硫硫杆菌中的表达

构建了含大肠杆菌磷酸果糖激酶(EC 2.7.1.11)基因pfkA的重组质粒pSDK1,利用大肠杆菌pfk缺陷株筛选含目的基因的重组质粒,通过接合转移的方式将其导入氧化硫硫杆菌TtZ2中,接合转移频率达2.6×10-6。重组质粒在TtZ2中有较好的稳定性,在无选择压力条件下传代50次基本保持稳定(重组质粒保留68%以上)。酶活性测定、SDSPAGE及RTPCR结果表明,pfkA基因在氧化硫硫杆菌中得到表达,但其表达水平低于大肠杆菌。葡萄糖可促进含pSDK1的氧化硫硫杆菌TtZ2的生长,而对照菌株的生长则未受明显影响,说明重组菌可部分利用葡萄糖作为碳源生长。     

Dielectric analysis of Escherichia coli suspensions in the light of the theory of interfacial polarization.

Dielectric measurements of Escherichia coli suspensions were carried out over a frequency range from 10 kHz to 100 MHz, and marked dielectric dispersions having characteristic frequency of approximately 1 MHz were observed. On the basis of the cell model that a spheroid is covered with two confocal shells, a dielectric theory was developed to determine accurately four electrical parameters for E. coli cells such as the conductivity of the cell wall, the dielectric constant of the cell membrane, and the dielectric constant and the conductivity of the protoplasm. The observed data were analyzed by means of the procedure based on the dielectric theory to yield a set of plausible electrical parameters for the cells. By taking account of the size distribution of the cells and a dielectric relaxation of the protoplasm, the observed dispersion curves were successfully reconstituted by the present theory.     

Functioning of the lactose operon of E. coli K-12 under the influence of non-specific regulators]

A possible role played by cAMP in the stimulating action of ACTH and hydrocortisone on lactose E. coli K-12 operon was studied. It was shown that ACTH caused no effect in the E. coli WZ-78/F'lac (cya855) and E. coli CA8001 (L1) strains with destroyed positive cAMP control system of the lactose operon function, at the same time producing a stimulating effect on the lactose operon in the strains of wild type, i.e. E coli 200PS/F'lac and E. coli 3000. Hydrocortisone stimulated the lactose operon function both in E. coli 3000 and in the mutant E. coli CA8001 (L1). It was supposed that the accelerating effect of ACTH on the lactose operon was mediated through cAMP; as to hydrocortisone--it stimulated the lactose operon function independently of cAMP.     

Characterization of the gcd gene from Escherichia coli K-12 W3110 and regulation of its expression.

DNA sequence and expressional analyses of the gcd gene of Escherichia coli K-12 W3110 revealed that two promoters that were detected were regulated negatively by cyclic AMP and positively by oxygen. Sequence conservation of the gcd gene between E. coli K-12 W3110 and PPA42 suggests that glucose dehydrogenase is required for the E. coli cells, even though it ordinarily exists as an apoprotein.     

R-plasmid transfer frequencies from environmental isolates of Escherichia coli to laboratory and fecal strains.

Multiple-drug-resistant strains of Escherichia coli were isolated from the water at an estuarine site. They represented about 8.3% of the total E. coli population. Fifty-five strains, representing each of the 32 resistance patterns identified, were mated with an E. coli K-12 F- strain. Matings were performed on membrane filters, and the cells were washed to remove any colicins produced by the donors. Thirty-one strains, about 5% of the mean E. coli density in the samples, transferred drug resistance and, hence, posessed conjugative R plasmids. Of these, 80% transferred drug resistance at a frequency of about 10(-4) or less. Nine environmental R+ strains were mated with three fecal recipients. The R-plasmid transfer frequencies to the fecal strains from the environmental donors correlated well with those from a derepressed K-12 R+ laboratory donor. The R+ X K-12 F- lac- transconjugants from 16 environmental strains were "backcrossed" to a lac+ K-12 F- strain. All transfer frequencies were higher in the backcrosses than in the original matings from the environmental donor. Furthermore, 7 of 13 different transconjugants, which accepted plasmids at repressed frequencies of less than 10(-3), donated them at frequencies greater than 10(-2). This suggests that these were derepressed plasmids in a repressed host.     

Functional coupling between vanillate-O-demethylase and formaldehyde detoxification pathway

Pseudomonas putida vanillate-O-demethylase consisting of VanA and VanB was expressed in Escherichia coli strain K-12. Recombinant E. coli strain K-12 cells expressing VanAB efficiently converted vanillate into protocatechuate with glucose consumption. Mutant lacking either pgi or zwf showed higher or lower converting activity than the parental strain, respectively. Formaldehyde, which is the by-product of the demethylation, was converted into formate in the cellular reaction. Formate accumulation was blocked by gene disruption of the E. coli frmA that coded glutathione-dependent formaldehyde dehydrogenase.     

Mapping of sul, the suppressor of lon in Escherichia coli.

The suppressor sul, which is allele specific for the ultraviolet sensitivity gene lon, has been mapped by conjugation and transductional crosses in Escherichia coli K-12 and B/r. Previously, sul was reported to lie in the azi region of the Escherichia coli chromosome. Evidence is presented which positions sul close to and clockwise of fabA on the Escherichia coli map. Cotransductional frequencies of 31.3% were obtained between sul and pyrD, and frequencies of 82% were obtained between sul and fabA. Also, the mucoid phenotype of K-12 lon strains grown on minimal glucose agar plates at 37 C was not significantly effected in sul derivatives. No differences between the sul of Escherichia coli B/r and that of K-12 derivatives with regard to map location or effect on mucoid production were observed.     

Efficient ethanol production from glucose, lactose, and xylose by recombinant Escherichia coli

Lactose and all of the major sugars (glucose, xylose, arabinose, galactose, and mannose) present in cellulose and hemicellulose were converted to ethanol by recombinant Escherichia coli containing plasmid-borne genes encoding the enzymes for the ethanol pathway from Zymomonas mobilis. Environmental tolerances, plasmid stability, expression of Z. mobilis pyruvate decarboxylase, substrate range, and ethanol production (from glucose, lactose, and xylose) were compared among eight American Type Culture Collection strains. E. coli ATCC 9637(pLO1297), ATCC 11303(pLO1297), and ATCC 15224(pLO1297) were selected for further development on the basis of environmental hardiness and ethanol production. Volumetric ethanol productivities per hour in batch culture were 1.4 g/liter for glucose (12%), 1.3 g/liter for lactose (12%), and 0.64 g/liter for xylose (8%). Ethanol productivities per hour ranged from 2.1 g/g of cell dry weight with 12% glucose to 1.3 g/g of cell dry weight with 8% xylose. The ethanol yield per gram of xylose was higher for recombinant E. coli than commonly reported for Saccharomyces cerevisiae with glucose. Glucose (12%), lactose (12%), and xylose (8%) were converted to (by volume) 7.2% ethanol, 6.5% ethanol, and 5.2% ethanol, respectively.     

Phenomenon of transient repression in Escherichia coli

Paigen, Kenneth (Roswell Park Memorial Institute, Buffalo, N.Y.). Phenomenon of transient repression in Escherichia coli. J. Bacteriol. 91:1201-1209. 1966.-A family of mutants has been obtained in Escherichia coli K-12 in which beta-galactosidase is not inducible for approximately one cell generation after the cells are transferred to glucose from other carbon sources. After that period; the enzyme can be induced at the level appropriate to glucose-grown cultures of the parent cells. Among a wide variety of carbon sources, the only one capable of eliciting a state of transient repression is glucose. Conversely, transient repression occurs when cells are transferred to glucose from any of a variety of other carbon sources. The only exceptions to this so far discovered are lactose, gluconate, and xylose. Susceptibility to transient repression in mutants can also be induced in glucose-grown cells by a period of starvation. Mutant cells which have become susceptible to transient repression lose susceptibility in the presence of glucose only when they are under conditions which permit active protein synthesis. The presence of an inducer of beta-galactosidase is not required during this time, nor does pre-induction for beta-galactosidase diminish the susceptibility of mutants. At least two other catabolite repression-sensitive enzymes (galactokinase and tryptophanase) are also sensitive to transient repression, and the two phenomena are probably related. The absolute specificity of glucose and the pattern of response seen after growth in different carbon sources suggest that the endogenous metabolite which produces these repressions is far more readily derived from glucose in metabolism than it is from any other exogenous carbon source.     

Efficient ethanol production from glucose, lactose, and xylose by recombinant Escherichia coli.

Lactose and all of the major sugars (glucose, xylose, arabinose, galactose, and mannose) present in cellulose and hemicellulose were converted to ethanol by recombinant Escherichia coli containing plasmid-borne genes encoding the enzymes for the ethanol pathway from Zymomonas mobilis. Environmental tolerances, plasmid stability, expression of Z. mobilis pyruvate decarboxylase, substrate range, and ethanol production (from glucose, lactose, and xylose) were compared among eight American Type Culture Collection strains. E. coli ATCC 9637(pLO1297), ATCC 11303(pLO1297), and ATCC 15224(pLO1297) were selected for further development on the basis of environmental hardiness and ethanol production. Volumetric ethanol productivities per hour in batch culture were 1.4 g/liter for glucose (12%), 1.3 g/liter for lactose (12%), and 0.64 g/liter for xylose (8%). Ethanol productivities per hour ranged from 2.1 g/g of cell dry weight with 12% glucose to 1.3 g/g of cell dry weight with 8% xylose. The ethanol yield per gram of xylose was higher for recombinant E. coli than commonly reported for Saccharomyces cerevisiae with glucose. Glucose (12%), lactose (12%), and xylose (8%) were converted to (by volume) 7.2% ethanol, 6.5% ethanol, and 5.2% ethanol, respectively.