Use of Rhodotorula minuta live cells hosted in water-in-oil macroemulsion for biotrasformation reaction

A lecithin/water/isooctane water-in-oil (w/o) macroemulsion was used as a host system for biotransformation reactions. In particular, the hydrolytic activity of the yeast Rhodotorula minuta toward (+/-)-succinic acid bis-2-isopropyl-5-methylcyclohexyl ester and p-nitrophenyl butyrate is reported. Evidence that R. minuta entrapped in w/o macroemulsion is able to hydrolyze the p-nitrophenyl butyrate ester is presented. By performing the yeast-catalyzed hydrolysis of (+/-)-succinic acid bis-2-isopropyl-5-methylcyclohexyl ester, the synthesis of (-)-menthol was obtained, indicating that R. minuta retains its high stereoselectivity in the macroemulsion system. In addition, no significant differences were observed among the hydrolysis rates and yields obtained using yeast cells hosted into w/o macroemulsions containing different amounts of water. Optical microscopy studies on the distribution of diameters of the dispersed phase indicate that the macroemulsion system is stable in terms of polydispersity. The diameter of the w/o macroemulsion droplets is indeed constant irrespective of the addition of water and/or chemicals (involved in the biotransformation reaction) to the system hosting yeast cells. The reactor devised here might be applied to other interesting bioconversion processes.     

Interaction between colicinogenic factor V and the integrated F factor in an Hfr strain of Escherichia coli

Kahn, Phyllis L. (Princeton University, Princeton, N.J.), and Donald R. Helinski. Interaction between colicinogenic factor V and the integrated F factor in an Hfr strain of Escherichia coli. J. Bacteriol. 90:1276-1282. 1965.-The production of colicin V by strains of Escherichia coli is determined by a colicinogenic factor, colV. The colV factor possesses a genetic determinant of fertility, F(v). V(+)F(v) (+) cells are characteristically susceptible to a male-specific phage, mu, and able to transfer the colV factor and chromosomal markers to recipient cells. The present work describes an interaction of the colV factor with the chromosome of the Hfr strain, HfrH. A colV-containing HfrH strain, designated HfrHV(+)F(v) (+)(l), was isolated and shown to be insensitive to phage mu and impaired in its fertility properties. Loss of the colV factor by this strain, either spontaneous or induced by acridine orange, resulted in a further 10(3)- or 10(4)-fold loss in fertility. This additional loss of fertility was restored by reinfection of these strains with the colV factor. The colV interaction with the HfrH chromosome also can result in defects in the fertility properties of the colV factor. Altered colV factors were found in recombinants isolated from a cross between the HfrHV(+)F(v) (+)(l) strain and F(-) recipients. It is postulated that in the HfrHV(+)F(v) (+)(l) strain an interaction of the colV episome with the integrated F region of the chromosome occurs, with a resulting modification of the fertility properties of the HfrH strain. This interaction can also result in a defect in certain properties of the colV factor.     

Evolution of a Site of Specific Genetic Homology on the Chromosome of Escherichia coli

Integration of the factors F(v) and F into the chromosome of a substrain of Escherichia coli K-12 has been studied. The F(v) factor is a fertility factor derived from Col V, lacking the ability to govern the production of colicin V. The derivatives of an Hfr(v) (Hfr isolated from a V colicinogenic parent) strain, PK2 (initially isolated from C600 V(+)), were shown to retain a unique bidirectional sex factor affinity locus between recA and pheA. This site shows no affinity for the E. coli K-12 F factor as shown by inability to isolate Hfr strains with origins in this region from a parental strain containing a cytoplasmic F factor. However this area exhibits two regions of homology to the V colicinogenic factor. One gives rise to Hfr(v) strains identical to the original Hfr(v) strain, PK2, with an origin and polarity of transfer designated pheA-CC injecting markers in the order pheA-his-trp-pro. The second gives rise to strains apparently originating at the same site but with reverse polarity designated recA-C, transferring markers in the order recA-thyA-str-xyl. For strains possessing the F(v) factor only the second homology is apparent. A model for the evolution of these strains is presented.     

Factors Influencing the Survival and Revival of Heat-treated Escherichia coli

Maximal revival of heat-damaged Escherichia coli occurred in nutrient media containing 0.8 to 1.0% (w/v) of Difco yeast extract. Vitamins did not appear to be involved in the recovery process. The situation with amino acids was less clear-cut, and, although certain of these may be essential for revival, proof for this is as yet inconclusive. Replica plating, in which colonies (from cells which had survived a heating process) on a rich medium were replicated onto minimal agar, revealed that no auxotrophic mutants had been formed as a result of heat treatment. Bacteria which were heated in 1% (w/v) yeast extract were killed more slowly than those heated in water.     

Escherichia coli F1-ATPase can use GTP-nonchaseable bound adenine nucleotide to synthesize ATP in dimethyl sulfoxide.

Escherichia coli F1-ATPase contained 3 mol of tightly-bound adenine nucleotide/mol enzyme. A further 3 mol could be loaded by incubation of the enzyme with ATP. The unloaded enzyme was designated as a F1[2,1] type on the basis of the ability of GTP to displace 1 mol of adenine nucleotide/mol of F1 [Kironde, F.A.S., & Cross, R.L. (1986) J. Biol. Chem. 261, 12544-12549]. The loaded enzyme was designated F1[3,3] since GTP could displace 3 of the 6 mol of bound adenine nucleotide/mol of F1. Incubation of F1[2,1], F1[2,0], and F1[3,0] with phosphate in the presence of 30% (v/v) dimethyl sulfoxide led to the synthesis of ATP from endogenous bound ADP. Hydrolysis of newly synthesized ATP occurred on transfer of the F1 from 30% (v/v) dimethyl sulfoxide to an entirely aqueous medium. Thus, synthesis and hydrolysis of ATP can occur at GTP-nonchaseable adenine nucleotide binding sites, and these sites in dimethyl sulfoxide are not necessarily equivalent to noncatalytic sites.     

The recovery of heat-stressed Escherichia coli in lake water microcosms

C.-H. LIM AND K.P. FLINT. 1995. Escherichia coli was heat stressed at 55, 60 or 65°C in sterile flasks of lake water. After 6 h at these temperatures the viable count on nutrient agar had dropped below the limits of detection (1 colony in 100 ml). The flasks were transferred to a 15°C incubator and left for 7 d. Recovery of the stressed E. coli was shown to occur within 48 h at this temperature. Recovery also occurred in microcosms amended with 5o (v/v) synthetic sewage. The stressed E. coli multiplied in the amended but not in the unamended microcosms.     

No genetic barriers between Salmonella enterica serovar typhimurium and Escherichia coli in SOS-induced mismatch repair-deficient cells

Conjugational crosses trigger SOS induction in Escherichia coli F(-) cells mated with Salmonella enterica serovar Typhimurium Hfr donors. Using an epigenetic indicator of SOS induction, we showed that a strong SOS response occurring in a subpopulation of mated mismatch repair-deficient cells totally abolishes genetic barriers between these two genera.     

The in vivo synthesis of plant sesquiterpenes by Escherichia coli.

Three plant genes encoding (+)-delta-cadinene, 5-epi-aristolochene, and vetispiradiene cyclases were expressed in Escherichia coli to evaluate the potential of this bacterium to synthesize sesquiterpenes in vivo. Various growth temperatures, carbon sources, and host strains were examined to optimize terpene production. The highest levels of sesquiterpene production occurred when the enzymes were expressed in strain DH5alpha from the trc promoter (Ptrc) of the high-copy plasmidpTrc99A in M9 medium supplemented with 0.2% (v/v) glycerol at 30 degrees C for 5-epi-aristolochene and vetispiradiene and 37 degrees C for (+)-delta-cadinene. The highest concentrations of sesquiterpenes observed were 10.3 microg of (+)-delta-cadinene, 0.24 microg of 5-epi-aristolochene (measured as (+)-delta-cadinene equivalents), and 6.4 microg of vetispiradiene (measured as (+)-delta-cadinene equivalents) per liter of culture. These sesquiterpene production levels are >500-fold lower than carotenoid production, both of which are synthesized from endogenous trans-farnesyl diphosphate (FDP) in E. coli. Based on these results, we conclude that the limiting factor for sesquiterpene synthesis in E. coli is the poor expression of the cyclase enzyme and not supply of the FDP precursor.     

Role of hydration in the binding of lac repressor to DNA

The osmotic stress technique was used to measure changes in macromolecular hydration that accompany binding of wild-type Escherichia coli lactose (lac) repressor to its regulatory site (operator O1) in the lac promoter and its transfer from site O1 to nonspecific DNA. Binding at O1 is accompanied by the net release of 260 +/- 32 water molecules. If all are released from macromolecular surfaces, this result is consistent with a net reduction of solvent-accessible surface area of 2370 +/- 550 A. This area is only slightly smaller than the macromolecular interface calculated for a crystalline repressor dimer-O1 complex but is significantly smaller than that for the corresponding complex with the symmetrical optimized O(sym) operator. The transfer of repressor from site O1 to nonspecific DNA is accompanied by the net uptake of 93 +/- 10 water molecules. Together these results imply that formation of a nonspecific complex is accompanied by the net release of 165 +/- 43 water molecules. The enhanced stabilities of repressor-DNA complexes with increasing osmolality may contribute to the ability of Escherichia coli cells to tolerate dehydration and/or high external salt concentrations.     

Elimination of Sex Factors in Escherichia coli by Urea

Eliminatory action of urea on the sex factor (F) in Escherichia coli K-12 strains is reported. Growth of E. coli harboring F or F'8 (F-gal) factors in Penassay Broth containing urea led to the loss of these genetic elements and yielded F(-) cells. Appearance of F(-) cells among survivors was already observed when the culture was in the very early stage of exponential phase. However, frequencies of F(-) cells formed did not increase much as a function of the incubation time. Unusual F(+) or F'8 cells which retained the ability of genetic transfer but showed resistance to M12 phage were also isolated. Addition of sucrose to broth with urea led to the favorable growth of cells in the culture and the increase, if little, of elimination frequencies of F factors by urea. These findings, coupled with other observations, suggest that urea has two separate actions in enhancing the frequency of F(-) bacteria, namely, (i) to inactivate F by direct action, such as mutation, and (ii) to select the F(-) variants by differentially inhibiting the growth of F(+).     

Genetic transformation of the ruminal bacteria Butyrivibrio fibrisolvens and Streptococcus bovis by electroporation

Electroporation methods for introduction of plasmid DNA into the ruminal bacteria Butyrivibrio fibrisolvens and Streptococcus bovis were developed. Electroporation of the strictly anaerobic B. fibrisolvens was carried out in an anaerobic glovebox with a buffer of 10% (v/v) glycerol and 1 mM MgCl₂ in distilled water. Streptococcus bovis electroporation could be carried out aerobically with a buffer of 10% (v/v) glycerol in distilled water. The Escherichia coli/Bacillus subtilis shuttle vector pBS42 could be transformed into B. fibrisolvens strain H17c, selecting for chloramphenicol resistance. The Streptococcus sanguis/E. coli shuttle vector pVA838 could replicate and express erythromycin resistance in Strep. bovis. Both vectors were stable in each organism in the absence of antibiotic selection. While the efficiency was low (<10²/μg DNA), the results demonstrate a means to introduce cloned genes into these organisms.     

Photosynthetic activity of plant cells solubilized in water-in-oil microemulsions

With the aim of possibly extending plant microbiology and photosynthesis beyond the usual applicability in aqueous solution, we investigated the solubilization of plant cells inorganic media with the help of water-in-oil microemulsions. Cells isolated from leaves of Rumex obtusifolius were solubilized in a water/2-ethyl-hexyl-sodiumsulfosuccinate/isooctane system, containing 20% water (v:v) and 240 mM surfactant, and the oxygen evolution/consumption was measured polarographically. Although no oxygen evolution was detectable in the organic medium, the cells were able to carry out photosynthetic oxygen consumption at the expense of ascorbate. To a lesser extent, photosynthetic oxygen consumption was measured using N, N, N', N'-tetramethyl-p-phenylenediamine as electron donor. The rate of ascorbate photooxidation was linearly related to the concentration of cells.     

Effect of the delta subunit on assembly and proton permeability of the F0 proton channel of Escherichia coli F1F0 ATPase.

During the assembly of the Escherichia coli proton-translocating ATPase, the subunits of F1 interact with F0 to increase the proton permeability of the transmembrane proton channel. We tested the involvement of the delta subunit in this process by partially and completely deleting uncH (delta subunit) from a plasmid carrying the genes for the F0 subunits and delta and testing the effects of those F0 plasmids on the growth of unc+ and unc mutant E. coli strains. We found that the delta subunit was required for inhibition of growth of unc+ cells. We also tested membranes isolated from unc-deleted cells containing F0 plasmids for F1-binding ability. In unc-deleted cells, these plasmids produced F0 in amounts comparable to those found in normal unc+ E. coli cells, while having only small effects on cell growth. These studies demonstrate that the delta subunit plays an important role in opening the F0 proton channel but that it does not serve as a temporary plug of F0 during assembly, as had been previously speculated (S. Pati and W. S. A. Brusilow, J. Biol. Chem. 264:2640-2644, 1989).     

Signal sequence mutants of beta-lactamase

The function of the NH2-terminal signal peptide in the translocation of beta-lactamase across the inner membrane of Escherichia coli has been studied by characterization of 15 signal sequence mutants. Three amino acid substitutions (Pro 20 to Ser, Pro 20 to Phe, and Cys 18 to Tyr) in the 23-amino acid signal sequence each cause, to varying degrees, a defect in the proteolytic processing of pre-beta-lactamase, abnormal growth of the host strain, and a severe reduction in the expression of beta-lactamase in vivo but not in vitro. The results are consistent with a model for protein secretion in E. coli that parallels the pathway proposed for translocation across the endoplasmic reticulum in eucaryotic cells.     

Cloning of chloroplast psbA and rbcL-genes from cotton Gossipium hirsutum

The fragments of cotton Gossipium hirsutum c.v. 108-f chloroplast genome were cloned in Escherichia coli cells. The cloned psbA and rbcL genes have been selected using the heterologous probes from spinach. The preliminary attempts to clone the complete psbA gene in pUC19 vector failed, probably, due to the toxicity of its product to Escherichia coli cells, and its 5'- and 3'-ends were cloned separately. Reconstruction experiments revealed that while the complete psbA gene was unable to be stably inherited by Escherichia coli cells, its structural part lacking the promoter region could be readily cloned in the bacterial cells.     

Protein Z is not an endonuclease of the recF recombination pathway in Escherichia coli K12

A 74 kD protein was extracted from Escherichia coli cells and purified under the physiological conditions. The protein is able to catalyze the reactions of endonucleolytic degradation of plasmid DNA. The genetic determinant coding for the 74 KD protein synthesis has been localized between 17 and 27 min on Escherichia coli chromosomal map. The endonuclease previously described as a recF gene dependent "protein Z" (Krivonogov S. V., Novitskaja V. A. Mol. Gen. Genet., 1982, v, 187, p. 302) is shown to be independent of the integrity of Escherichia coli recF gene.     

Purification of plasmid DNA with polymer-salt aqueous two-phase system: optimization using response surface methodology

An experimental design was used to optimize plasmid purification from an alkaline lysate of Escherichia coli cells using PEG-sodium citrate aqueous two-phase systems (ATPS), and to evaluate the influence of pH, PEG molecular weight, tie line length, phase volume ratio, and lysate load. To build the mathematical model and minimize the number of experiments for the design parameters, response surface methodology (RMS) with an orthogonal rotatable central composite design was defined based on the conditions found for the highest purification by preliminary tests. The adequacy of the calculated models for the plasmid recovery and remaining RNA were confirmed by means of variance analysis and additional experiments. Analysis of contours of constant response as a function of pH, PEG molecular weight, tie line length, and cell lysate load for three different phase volume ratios revealed different effects of these five factors on the studied parameters. Plasmid recovery of 99% was predicted for a system with PEG 400, pH 6.9, tie line length of 38.7%, phase volume ratio of 1.5, and lysate load of 10% (v/v). Under these conditions the predicted RNA removal was 68%.     

Viability of Escherichia coli O157:H7 in natural river water determined by the use of flow cytometry

The enzymatic activity and viability of Escherichia coli O157:H7 in natural river water was determined by flow cytometry. River water was collected at two sites (an agricultural area and an industrial area) on the Aigawa River (Osaka, Japan). To facilitate estimation of the physiology of E. coli O157 in natural river water, bacterial cells in the water were stained with 6-carboxyfluorescein diacetate (6CFDA) and propidium iodide (PI). The cells were sorted into two populations, using a flow cytometer, based on their esterase activity. Each population was stained with E. coli O157:H7 fluorescent antibody (FA), and E. coli O157:H7 cells were observed in the esterase-active population. River water samples collected at the same points were incubated with yeast extract containing antibiotics to prevent cell division, and bacterial cells in the incubated samples were stained with PI and FA. Escherichia coli O157:H7 existed in both the viable (elongated and/or fattened) and inactive bacterial population determined by flow cytometry. These results indicate that E. coli O157:H7 may retain metabolic activity and growth potential in the natural aquatic environment.