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.     

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.     

A novel cloning vector for the direct selection of recombinant DNA in E. coli

A new plasmid cloning vector (pHE3) is described carrying dominant p-fluorophenylalanine-sensitivity (pheS) and chloramphenicol-resistance markers. This vector is used in combination with a p-fluorophenylalanine-resistant Escherichia coli recipient (strain RR28). Foreign DNA can be cloned into pHE3 leading to insertional inactivation of pheS. Transformation of RR28, and plating on minimal medium with chloramphenicol plus p-fluorophenylalanine (pfp) directly selects for colonies containing recombinant DNA.     

Insertion of DNA carrying ribosomal protein genes of Escherichia coli into Charon vector phages.

DNA fragments from lambdaspc1 and lambdafus2, carrying ribosomal protein genes from Escherichia coli, were inserted into lambda phage vectors Charon 3 and Charon 4. Eight of the resulting clones were characterized by agarose gel electrophoresis of EcoRI digests, analytical CsCl equilibrium centrifugation, and electron micrographic analysis of heteroduplexes. In each case, the identity, order, and orientation of each cloned fragment was determined. In all, 8 of the 12 EcoRI fragments of lambdafus2 were cloned in various arrangements. In the accompanying paper, genes for 15 ribosomal and related proteins and three bacterial promoters were detected in these phages. In addition, four of the hybrid phages carried fragments of lambda-DNA including the phage origin of replication (ori), the late promoter, PR', and the cohesive ends (cos site) in both orientations. The latter phages yield a circularly permuted collection of DNA molecules.     

Probable detection of kil peptide derived from colicin E1 plasmid in the envelope fraction of Escherichia coli HB101 carrying pEAP31.

Escherichia coli carrying plasmid pEAP31 produces extracellularly alkalophilic Bacillus penicillinase encoded on the plasmid. The extracellular production has been suggested to be caused by activation of dormant colicin E1 kil gene. Two peptides that could be respectively precursor and mature products of colicin E1 kil gene were detected on an SDS/polyacrylamide gel. One of the peptides (Mr 4800), which was probably a precursor peptide, was detected in the inner-membrane fraction from the organism when envelope proteins were subjected to differential solubilization. The other (Mr 3500), which was a mature peptide, was detected in the outer-membrane fraction of the organism. The mature peptide was only detected in the envelope of cells releasing the penicillinase transiently accumulated in the periplasm into the culture medium.     

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.     

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.     

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+.     

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.     

Construction of a promoter-probe shuttle vector for Escherichia coli and brevibacteria

We constructed a promoter-probe vector, pJUP05, for brevibacteria and Escherichia coli based on the promoterless neomycin-resistance (neoR) gene from Tn5. This gene confers resistance to the aminoglycosides, kanamycin and neomycin. The promoter of the neoR gene was deleted and replaced by a suitable multiple cloning site. There are translation stop codons in all three reading frames upstream from the neoR gene. The plasmid contains functional origins of DNA replication for both brevibacteria and E. coli, and permits selection for chloramphenicol- and/or ampicillin-resistance markers.     

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     

Construction of a high-copy "ATG vector" for expression in Escherichia coli.

We report the construction of an inducible, high-copy plasmid for the expression of foreign proteins in Escherichia coli. This plasmid, pPB1, combines the trc promoter, beta-galactosidase translation start site, and polylinker of pKK233-2 with the origin of replication region of pUC19. Replacement of the origin of replication of pKK233-2 results in a threefold increase in plasmid copy number of pPB1 compared with pKK233-2. Subclones of the cDNA for rabbit muscle fructose-1,6-bisphosphate aldolase (E.C. 4.1.2.13) in the two expression plasmids exhibit a comparable difference in copy number. An increase in protein expression measured by SDS-PAGE and aldolase specific activities reflects the increased copy number. Specific activities of aldolases in bacterial extracts differ approximately sixfold between the two expression plasmids in E. coli JM83. Aldolase A can compose up to 40% of the total protein in E. coli JM83 when expressed in pPB1, from which more than 100 mg of purified enzyme can be obtained per liter culture.     

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.     

A simplified procedure for the rapid identification of recombinant pAT153 plasmids in Escherichia coli HB101 cells

Insertion of foreign DNA into the unique HindIII site of the high copy number plasmid pAT153 reduces but does not completely abolish the resistance of Escherichia coli HB101 cells to tetracycline. Recombinant DNA-containing colonies could then be phenotypically differentiated from non-recombinant ones by their smaller size on nutrient agar plates with ampicillin and tetracycline at a final concentration of 50 and 4 micrograms/ml, respectively. A wide variety of human cytomegalovirus DNA fragments have been found in pAT153 molecules propagated by the ampicillin-resistant tetracycline-sensitive bacteria selected.     

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).     

Highly efficient positive selection of recombinant plasmids using a novel rglB-based Escherichia coli K-12 vector system

We have developed pBR328-derived vectors which allow highly efficient positive selection of recombinant plasmids. The system is based on the rglB-coded restriction activity of Escherichia coli K-12 directed against 5-methylcytosine (5mC)-containing DNA. The vectors code for cytosine-specific, temperature-sensitive DNA methyltransferases (ts-Mtases), whose specificity elicits RglB restriction. 5mC-free vector DNA - a prerequisite to allow establishment of such plasmids in cells expressing the RglB nuclease activity - can be prepared from cultures grown at 42 degrees C. At 30 degrees C the vector plasmids are vulnerable to RglB restriction due to the expression of suicidal Mtase activity. Cloning a DNA fragment into the ts-Mtase-coding gene disrupts the lethal methylation and thus permits selection of such recombinant plasmids at 30 degrees C. The standard vector used, pBN73, contains unique recognition sites for nine restriction enzymes within the ts-Mtase-coding gene, which can be used independently or in combination for the construction of recombinant plasmids selectable by the rglB-coded activity. Plasmid pBN74, which carries the determinants for both the ts-Mtase and the RglB nuclease, contains seven unique sites within the ts-Mtase-coding gene. While selection of recombinant plasmids derived from pBN73 obligatorily requires the employment of rglB+ strains, selection of pBN74 derivatives can be performed independent of the E. coli-host genotype. It remains to be elucidated whether positive selection of pBN74-derived recombinant plasmids can also be achieved in hosts other than E. coli. Plasmids pBN73, pBN74 and the recombinants are structurally stable. Generally applicable procedures, as developed during the establishment of this vector system, are described; they allow the isolation of ts-Mtases and facilitate the cloning of genes coding for nucleases directed against 5mC-containing DNA.     

Development of a novel vector system for programmed cell lysis in Escherichia coli

Although widely used as a host for recombinant protein production, Escherichia coli is unsuitable for massive screening of recombinant clones, owing to its poor secretion of proteins. A vector system containing T4 holin and T7 lysozyme genes under the control of the ptsG promoter derivative that is inducible in the absence of glucose was developed for programmed cell lysis of E. coli. Because E. coli harboring the vector grows well in the presence of glucose, but is lysed upon glucose exhaustion, the activity of the foreign gene expressed in E. coli can be monitored easily without an additional step for cell disruption after the foreign gene is expressed sufficiently with an appropriate concentration of glucose. The effectiveness of the vector was demonstrated by efficient screening of the amylase gene from a Bacillus subtilis genomic library. This vector system is expected to provide a more efficient and economic screening ofbioactive products from DNA libraries in large quantities.