“Host Shutoff” Function of Bacteriophage T7: Involvement of T7 Gene 2 and Gene 0.7 in the Inactivation of Escherichia coli RNA Polymerase

The "host shutoff" function of bacteriophage T7 involves an inactivation of the host Escherichia coli RNA polymerase by an inhibitor protein bound to the enzyme. When this inhibitor protein, termed I protein, was removed from the inactive RNA polymerase complex prepared from T7-infected cells by glycerol gradient centrifugation in the presence of 1 M KCl, the enzyme recovered its activity equivalent to about 70 to 80% of the activity of the enzyme from uninfected cells. Analysis of the activity of E. coli RNA polymerase from E. coli cells infected with various T7 mutant phages indicated that the T7 gene 2 codes for the inhibitor I protein. The activity of E. coli RNA polymerase from gene 2 mutant phage-infected cells, which was about 70% of that from uninfected cells, did not increase after glycerol gradient centrifugation in the presence of 1 M KCl, indicating that the salt-removable inhibitor was not present with the enzyme. It was found that the reduction in E. coli RNA polymerase activity in cells infected with T7(+) or gene 2 mutant phage, i.e., about 70% of the activity of the enzyme compared to that from uninfected cells after glycerol gradient centrifugation in the presence of 1 M KCl, results from the function of T7 gene 0.7. E. coli RNA polymerase from gene 0.7 mutant phage-infected cells was inactive but recovered a full activity equivalent to that from uninfected cells after removal of the inhibitor I protein with 1 M KCl. E. coli RNA polymerase from the cells infected with newly constructed mutant phages having mutations in both gene 2 and gene 0.7 retained the full activity equivalent to that from uninfected cells with or without treatment of the enzyme with 1 M KCl. From these results, we conclude that both gene 2 and gene 0.7 of T7 are involved in accomplishing complete shutoff of the host E. coli RNA polymerase activity in T7 infection.     

The binding site for ribosomal protein L11 within 23 S ribosomal RNA of Escherichia coli

Ribosomal protein L11 of Escherichia coli was bound to 23 S rRNA and the resultant complex was digested with ribonuclease T1. A single RNA fragment, protected by protein L11, was isolated from such digests and was shown to rebind specifically to protein L11. The nucleotide sequence of this RNA fragment was examined by two-dimensional fingerprinting of ribonuclease digests. It proved to be 61 residues long and the constituent oligonucleotides could be fitted perfectly between residues 1052 and 1112 of the nucleotide sequence of E. coli 23 S rRNA.     

A thermoresistant mutant of ribonuclease T1 having three disulfide bonds

Molecular-dynamic calculations predict that, if Tyr24 and Asn84 are each replaced by a Cys residue, it should be possible to form a third disulfide bond in ribonuclease T1 (RNase T1) between these residues, with only minimal conformational changes at the catalytic site. The gene encoding such a mutant variant of RNase T1 (Tyr24----Cys24, Asn84----Cys84) was constructed by the cassette mutagenesis method using a chemically synthesized gene. In order to reduce the toxic effect of the mutant enzyme (RNase T1S) on an Escherichia coli host, we arranged for the protein to be secreted into the periplasmic space by using a vector that harbors a gene for an alkaline phosphatase signal peptide under the control of the trp promoter. The nucleolytic activity of RNase T1S toward pGpC was approximately the same as that of RNase T1 at 37 degrees C (pH 7.5). Moreover, at 55 degrees C, RNase T1S retained nearly 70% of its activity while the activity of the wild-type enzyme was reduced to less than 10%. RNase T1S was also more resistant to denaturation by urea than the wild-type enzyme. However, unlike RNase T1, RNase T1S was irreversibly and almost totally inactivated by boiling at 100 degrees C for 15 min.     

Molecular cloning and expression in Escherichia coli of a cellodextrinase gene from Bacteroides succinogenes S85

A DNA fragment coding for a cellodextrinase of Bacteroides succinogenes S85 was isolated by screening of a pBR322 gene library in Escherichia coli HB101. Of 100,000 colonies screened on a complex medium with methylumbelliferyl-beta-D-cellobioside as the indicator substrate, two cellodextrinase-positive clones (CB1 and CB2) were isolated. The DNA inserts from the two recombinant plasmids were 7.7 kilobase pairs in size and had similar restriction maps. After subcloning from pCB2, a 2.5-kilobase-pair insert which coded for cellodextrinase activity was isolated. The enzyme was located in the cytoplasm of the E. coli host. It exhibited no activity on carboxymethyl cellulose, Avicel microcrystalline cellulose, acid-swollen cellulose, or cellobiose but hydrolyzed p-nitrophenyl-beta-D-cellobioside and p-nitrophenyl-beta-D-lactoside. The Km (0.1 mM) for the hydrolysis of p-nitrophenyl-cellobioside by the enzyme expressed in E. coli was similar to that reported for the purified enzyme from B. succinogenes. Expression of the cellodextrinase gene was subjected to catabolite repression by glucose and was not induced by cellobiose. The origin of the DNA insert from B. succinogenes was confirmed by Southern blot analysis. Western blotting (immunoblotting) using antibodies raised against the purified B. succinogenes cellodextrinase revealed a protein with a molecular weight of approximately 50,000 in E. coli clones which comigrated with the native enzyme isolated from B. succinogenes. These data indicate that the cellodextrinase gene expressed in E. coli is fully functional and codes for an enzyme with properties similar to those of the native enzyme.     

Molecular cloning and expression in Escherichia coli of a cellodextrinase gene from Bacteroides succinogenes S85.

A DNA fragment coding for a cellodextrinase of Bacteroides succinogenes S85 was isolated by screening of a pBR322 gene library in Escherichia coli HB101. Of 100,000 colonies screened on a complex medium with methylumbelliferyl-beta-D-cellobioside as the indicator substrate, two cellodextrinase-positive clones (CB1 and CB2) were isolated. The DNA inserts from the two recombinant plasmids were 7.7 kilobase pairs in size and had similar restriction maps. After subcloning from pCB2, a 2.5-kilobase-pair insert which coded for cellodextrinase activity was isolated. The enzyme was located in the cytoplasm of the E. coli host. It exhibited no activity on carboxymethyl cellulose, Avicel microcrystalline cellulose, acid-swollen cellulose, or cellobiose but hydrolyzed p-nitrophenyl-beta-D-cellobioside and p-nitrophenyl-beta-D-lactoside. The Km (0.1 mM) for the hydrolysis of p-nitrophenyl-cellobioside by the enzyme expressed in E. coli was similar to that reported for the purified enzyme from B. succinogenes. Expression of the cellodextrinase gene was subjected to catabolite repression by glucose and was not induced by cellobiose. The origin of the DNA insert from B. succinogenes was confirmed by Southern blot analysis. Western blotting (immunoblotting) using antibodies raised against the purified B. succinogenes cellodextrinase revealed a protein with a molecular weight of approximately 50,000 in E. coli clones which comigrated with the native enzyme isolated from B. succinogenes. These data indicate that the cellodextrinase gene expressed in E. coli is fully functional and codes for an enzyme with properties similar to those of the native enzyme.     

A novel thermostable branching enzyme from an extremely thermophilic bacterial species, Rhodothermus obamensis

A branching enzyme (EC 2.4.1.18) gene was isolated from an extremely thermophilic bacterium, Rhodothermus obamensis. The predicted protein encodes a polypeptide of 621 amino acids with a predicted molecular mass of 72 kDa. The deduced amino acid sequence shares 42-50% similarity to known bacterial branching enzyme sequences. Similar to the Bacillus branching enzymes, the predicted protein has a shorter N-terminal amino acid extension than that of the Escherichia coli branching enzyme. The deduced amino acid sequence does not appear to contain a signal sequence, suggesting that it is an intracellular enzyme. The R. obamensis branching enzyme was successfully expressed both in E. coli and a filamentous fungus, Aspergillus oryzae. The enzyme showed optimum catalytic activity at pH 6.0-6.5 and 65 degrees C. The enzyme was stable after 30 min at 80 degrees C and retained 50% of activity at 80 degrees C after 16 h. Branching activity of the enzyme was higher toward amylose than toward amylopectin. This is the first thermostable branching enzyme isolated from an extreme thermophile.     

Rescue of abortive T7 gene 2 mutant phage infection by rifampin.

Infection of Escherichia coli with T7 gene 2 mutant phage was abortive; concatemeric phage DNA was synthesized but was not packaged into the phage head, resulting in an accumulation of DNA species shorter in size than the phage genome, concomitant with an accumulation of phage head-related structures. Appearance of concatemeric T7 DNA in gene 2 mutant phage infection during onset of T7 DNA replication indicates that the product of gene 2 was required for proper processing or packaging of concatemer DNA rather than for the synthesis of T7 progeny DNA or concatemer formation. This abortive infection by gene 2 mutant phage could be rescued by rifampin. If rifampin was added at the onset of T7 DNA replication, concatemeric DNA molecules were properly packaged into phage heads, as evidenced by the production of infectious progeny phage. Since the gene 2 product acts as a specific inhibitor of E. coli RNA polymerase by preventing the enzyme from binding T7 DNA, uninhibited E. coli RNA polymerase in gene 2 mutant phage-infected cells interacts with concatemeric T7 DNA and perturbs proper DNA processing unless another inhibitor of the enzyme (rifampin) was added. Therefore, the involvement of gene 2 protein in T7 DNA processing may be due to its single function as the specific inhibitor of the host E. coli RNA polymerase.     

Expression and purification of recombinant rat protein disulfide isomerase from Escherichia coli.

Rat liver protein disulfide isomerase (PDI) catalyzes the oxidative folding of proteins containing disulfide bonds. We have developed an efficient method for its overproduction in Escherichia coli. Using a T7 RNA polymerase expression system, isolated yields of 15-30 mg/liter of recombinant rat PDI are readily obtained. Convenient purification of the enzyme from E. coli lysates involves ion-exchange (DEAE) chromatography combined with zinc chelate chromatography. The recombinant PDI shows catalytic activity identical to that of PDI isolated from bovine liver in both the reduction of insulin and the oxidative folding of ribonuclease A. The enzyme is expressed in E. coli as a soluble, cytoplasmic protein. After complete reduction and denaturation in 6 M guanidinium hydrochloride, PDI regains complete activity within 3 min after removal of the denaturant, implying that disulfide bonds are not essential for the maintenance of PDI tertiary structure. Both the protein isolated from E. coli and the protein isolated from liver contained free cysteine residues (1.8 +/- 0.2 and 1.4 +/- 0.3 SH/monomer, respectively).     

Active-site-directed inactivation of Aspergillus oryzae beta-galactosidase with beta-D-galactopyranosylmethyl-p-nitrophenyltriazene

beta-D-Galactopyranosylmethyl-p-nitrophenyltriazene (beta-GalMNT), a specific inhibitor of beta-galactosidase, was isolated as crystals by HPLC and its chemical and physicochemical characteristics were examined. Aspergillus oryzae beta-galactosidase was inactivated by the compound. We studied the inhibition mechanism in detail. The inhibitor was hydrolyzed by the enzyme to p-nitroaniline and an active intermediate (beta-galactopyranosylmethyl carbonium or beta-galactopyranosylmethyldiazonium), which inactivated the enzyme. The efficiency of inactivation of the enzyme (the ratio of moles of inactivated enzyme to moles of beta-GalMNT hydrolyzed by the enzyme) was 3%; the efficiency of Escherichia coli beta-galactosidase was 49%. In spite of the low efficiency, the rate of inactivation of A. oryzae enzyme was not very different from that of the E. coli enzyme, because the former hydrolyzed beta-GalMNT faster than the latter did. A. oryzae beta-galactosidase was also inactivated by p-chlorophenyl, p-tolyl, and m-nitrophenyl derivatives of beta-galactopyranosylmethyltriazene. However, E. coli beta-galactosidase was not inactivated by these triazene derivatives. The results showed that the inactivation of A. oryzae and E. coli beta-galactosidases by beta-GalMNT was an enzyme-activated and active-site-directed irreversible inactivation. The possibility of inactivation by intermediates produced nonenzymatically was ruled out for E. coli, but not for the A. oryzae enzyme.     

Rapid method for altering bacterial ribosome-binding sequences for overexpression of proteins in Escherichia coli.

In an Escherichia coli expression system, two genes, one from an anaerobic intestinal bacterium and one from E. coli, were overexpressed following the alteration of ribosome-binding (Shine-Dalgarno) sequences. For both genes, the polymerase chain reaction (PCR) was used to modify the ribosome-binding sequence and, at the same time, provide restriction endonuclease sequences at each end of the gene. These restriction endonuclease sequences were used for inserting the DNA into the E. coli plasmid vector pGEM2, which has the T7 promoter upstream from its multiple cloning sites. Each chimeric plasmid, made by ligating the PCR product into pGEM2, was transformed into E. coli strain HMS174(DE3) which, when induced, produces T7 RNA polymerase for regulated overexpression. The gene isolated from the anaerobic intestinal bacterium, a 27-kDa polypeptide gene from Eubacterium sp. strain 12708, when expressed using this system, produced about one-third of the total cell protein as measured in Coomassie-stained protein gels and confirmed by Western blots with rabbit antibody. The E. coli enzyme, a 28.4-kDa tRNA methylation enzyme, was increased fivefold in activity of cell extracts over that of the best previous strain.     

A new member of the bacterial ribonuclease inhibitor family from Saccharopolyspora erythraea

We have identified Sti, the gene of a ribonuclease inhibitor from Saccharopolyspora erythraea, by using a T7 phage display system. A specific phage has been isolated from a genome library by a biopanning procedure, using RNase Sa3, a ribonuclease from Streptomyces aureofaciens, as bait. Sti, a protein of 121 amino acid residues, with molecular mass 13059 Da, is a homolog of barstar and other microbial ribonuclease inhibitors. To overexpress its gene in Escherichia coli, we optimized the secondary structure of its mRNA by introducing a series of silent mutations. Soluble protein was isolated and purified to homogeneity. Inhibition constants of complex of Sti and RNase Sa3 or barnase were determined at pH 7 as 5 x 10(-12) or 7 x 10(-7), respectively.     

Rapid isolation of homogeneous cloned T7 gene 5 protein and T7 DNA polymerase by affinity chromatography on immobilized thioredoxin.

Phage T7 DNA polymerase consists of a strong 1:1 complex of T7 gene 5 protein (80 kDa) and the reduced form of Escherichia coli thioredoxin (12 kDa). Immobilization of E. coli thioredoxin on the agarose matrix Affi-Gel retained both its redox activity and its ability to bind T7 gene 5 protein. This was used to develop a simple and fast high-yield purification method. Cloned T7 gene 5 protein, expressed in a thioredoxin-negative host cell, was isolated in pure and highly active form after elution from Affi-Gel--thioredoxin with a pH gradient from 10 to 12. This purification step separated gene 5 protein from variable amounts of two sets of reconstituting large polypeptide fragments without catalytic activity. Proteolytic cleavage in vivo probably gave rise to the fragments, the generation of which was mimicked by trypsin cleavage of pure gene 5 protein. The gene 5 protein preparation had an inherent low DNA polymerase and double-stranded 3'-exonuclease activity, which was stimulated at least 30-fold by the presence of reduced thioredoxin. Highly active and pure T7 DNA polymerase was obtained by reconstitution of gene 5 protein with thioredoxin and was isolated by phosphocellulose or FPLC Mono Q chromatography. The gene 5 protein and T7 DNA polymerase preparations are suitable for further physicochemical characterization and as reagents in DNA sequencing.     

Evidence for rapid association-dissociation of ribonuclease T1 from a recombinant strain of Escherichia coli

On the basis of photon correlation experiments and computer simulations, we provide evidence for a rapid dimerization of the enzyme ribonuclease T1 isolated from an Escherichia coli overproducing strain. An attractive potential in addition to the usual repulsive hardcore and electrostatic potentials was found to be necessary for interpreting the concentration dependence of the diffusion coefficient of the enzyme. Computer searches of surface complementarity suggest that dimer formation of ribonuclease T1 takes place due to an extensive surface contact of approximately 700 A2. Energy minimization of the ribonuclease T1 dimer shows that large conformational changes are not induced upon self-association of the enzyme. The two molecules in the dimer are orientated back-to-back, and this is expected to lead to an active enzyme form.     

Rapid detection of Escherichia coli O157:H7 by using green fluorescent protein-labeled PP01 bacteriophage

A previously isolated T-even-type PP01 bacteriophage was used to detect its host cell, Escherichia coli O157:H7. The phage small outer capsid (SOC) protein was used as a platform to present a marker protein, green fluorescent protein (GFP), on the phage capsid. The DNA fragment around soc was amplified by PCR and sequenced. The gene alignment of soc and its upstream region was g56-soc.2-soc.1-soc, which is the same as that for T2 phage. GFP was introduced into the C- and N-terminal regions of SOC to produce recombinant phages PP01-GFP/SOC and PP01-SOC/GFP, respectively. Fusion of GFP to SOC did not change the host range of PP01. On the contrary, the binding affinity of the recombinant phages to the host cell increased. However, the stability of the recombinant phages in alkaline solution decreased. Adsorption of the GFP-labeled PP01 phages to the E. coli cell surface enabled visualization of cells under a fluorescence microscope. GFP-labeled PP01 phage was not only adsorbed on culturable E. coli cells but also on viable but nonculturable or pasteurized cells. The coexistence of insensitive E. coli K-12 (W3110) cells did not influence the specificity and affinity of GFP-labeled PP01 adsorption on E. coli O157:H7. After a 10-min incubation with GFP-labeled PP01 phage at a multiplicity of infection of 1,000 at 4 degrees C, E. coli O157:H7 cells could be visualized by fluorescence microscopy. The GFP-labeled PP01 phage could be a rapid and sensitive tool for E. coli O157:H7 detection.     

Purification and properties of T4 phage thymidylate synthetase produced by the cloned gene in an amplification vector

We have introduced the T4 thymidylate synthetase gene, resident in a 2.7-kilobase EcoRI restriction fragment, into an amplification plasmid, pKC30. By regulating expression of this gene from the phage lambda pL promoter within pKC30 in a thyA host containing a temperature-sensitive lambda repressor, the T4 synthetase could be amplified about 200-fold over that after T4 infection. At this stage, a 20-fold purification was required to obtain homogeneous enzyme, mainly by an affinity column procedure. The purified plasmid-amplified T4 synthetase appeared to be identical with the T2 phage synthetase purified from phage-infected Escherichia coli in molecular weight, amino end group analysis, and immunochemical reactivity. The individual nature of the phage and host proteins was revealed by the fact that neither the T2 nor the T4 enzyme reacted with antibody to the E. coli synthetase, nor did antibody to the phage enzymes react with the E. coli synthetase. These differences were corroborated by DNA hybridization experiments, which revealed the absence of apparent homology between the T4 and E. coli synthetase genes. The techniques and genetic constructions described support the feasibility of employing similar amplification methods to prepare highly purified thymidylate synthetases from other sources.     

Genetic analysis of gene 1.2 of bacteriophage T7: isolation of a mutant of Escherichia coli unable to support the growth of T7 gene 1.2 mutants.

The product of gene 1.2 of bacteriophage T7 is not required for the growth of T7 in wild-type Escherichia coli since deletion mutants lacking the entire gene 1.2 grow normally (Studier et al., J. Mol. Biol. 135:917-937, 1979). By using a T7 strain lacking gene 1.2, we have isolated a mutant of E. coli that was unable to support the growth of both point and deletion mutants defective in gene 1.2. The mutation, optA1, was located at approximately 3.6 min on the E. coli linkage map in the interval between dapD and tonA; optA1 was 92% cotransducible with dapD. By using the optA1 mutant, we have isolated six gene 1.2 point mutants of T7, all of which mapped between positions 15 and 16 on the T7 genetic map. These mutations have also been characterized by DNA sequence analysis, E. coli optA1 cells infected with T7 gene 1.2 mutants were defective in T7 DNA replication; early RNA and protein synthesis proceeded normally. The defect in T7 DNA replication is manifested by a premature cessation of DNA synthesis and degradation of the newly synthesized DNA. The defect was not observed in E. coli opt+ cells infected with T7 gene 1.2 mutants or in E. coli optA1 cells infected with wild-type T7 phage.     

Cloning and expression of the yeast plasma membrane ATPase in Escherichia coli

The yeast plasma membrane ATPase gene PMA1 was cloned into Escherichia coli using the high expression tac and T7 promoters. The gene product is toxic to the bacterial cell leading to very low expression levels and arrested growth of the host cell within minutes of induction. The expressed protein is immunologically cross-reactive with the yeast ATPase, comigrates with the original protein in sodium dodecyl sulfate-polyacrylamide gels, and is isolated in the E. coli membrane fraction. The partially purified protein exhibits ATPase activity.     

D-Xylulose kinase from Saccharomyces cerevisiae: isolation and characterization of the highly unstable enzyme, recombinantly produced in Escherichia coli

The Saccharomyces cerevisiae gene encoding xylulose kinase (XKS1) was over-expressed to an abundance of ≥ 10% intracellular protein in Escherichia coli. Instability of XKS1, not pointed out in previous reports of the enzyme, prevented isolation of active enzyme in native or "tagged" form under a wide range of purification conditions. A fusion protein haboring C-terminal Strep-tag II (XKS1-Strep) displayed activity (～20 U/mg) as isolated. However, the half-life time of purified XKS1-Strep was only ～1.5h at 4°C and could not be enhanced substantially by an assortment of extrinsic stabilizers (osmolytes, protein, substrates). Peptide mass mapping and N-terminal sequencing showed that the recombinant protein was structurally intact, ruling out proteolytic processing and chemical modifications as possible factors to compromise the stability of the enzyme as isolated. Partial functional complementation of a largely inactive XKS1 preparation by the high-molecular mass fraction (≥ 10kDa) of cell extract prepared from an E. coli BL21 (DE3) expression host suggests a possible role for heterotropic protein-XKS1 interactions in conferring activity/stability to the enzyme. Michaelis-Menten constants of XKS1-Strep were determined: d-xylulose (210 ± 40 μM) and Mg(2+)-ATP (1.70 ± 0.10 mM).     

Increase in nucleolytic activity of ribonuclease T1 by substitution of tryptophan 45 for tyrosine 45

The preparation and analysis of a mutant ribonuclease (RNase) T1 which possesses higher nucleolytic activity than the wild-type enzyme are described. The gene for the mutant RNase T1 (Tyr45----Trp45), in which a single amino acid at the binding site of the guanine base has been changed, was constructed by the cassette mutangenesis method using a chemically synthesized gene [Ikehara, M. et al. (1986) Proc. Natl Acad. Sci. USA 83, 4695-4699]. In order to reduce the nucleolytic activity of the enzyme in vivo, this gene was expressed in Escherichia coli as a fused protein connected through methionine residues to other proteins at both the N- and C-termini. After liberation from the fused protein by cleavage with cyanogen bromide at the methionine junctions, the mutant RNase T1 was purified by column chromatography. The nucleolytic activity toward pGpC increased to 120% of that of wild-type RNase T1. The kinetic parameters of the mutant enzyme demonstrate that this higher nucleolytic activity is due to a higher affinity for the substrate, probably because of an increased stacking effect in the binding pocket for the guanine base. This mutant enzyme also possessed a higher nucleolytic activity against pApC than wild-type RNase T1.     

Cloning and expression of the Escherichia coli D-xylose isomerase gene in Bacillus subtilis

A DNA fragment containing the Escherichia coli D-xylose isomerase gene and D-xylulokinase gene had been isolated from an E. coli genomic bank constructed by Clarke and Carbon. The D-xylose isomerase gene coding for the synthesis of an important industrial enzyme, xylose isomerase, was subcloned into a Bacillus-E. coli bifunctional plasmid. It was found that the intact E. coli gene was not expressed in B. subtilis, a host traditionally used to produce industrial enzymes. An attempt was then made to express the E. coli gene in B. subtilis by fusion of the E. coli xylose isomerase structural gene downstream to the promoter of the penicillinase gene isolated from Bacillus licheniformis. Two such fused genes were constructed and they were found able to be expressed in both B. subtilis and E. coli.