Construction from Mu d1 (lac Apr) lysogens of lambda bacteriophage bearing promoter-lac fusions: isolation of lambda ppheA-lac.

Bacteriophage Mu d1 (lac Aprr) was used to obtain strains of Escherichia coli K-12 in which the lac genes are expressed from the promoter of pheA, the structural gene for the enzyme chorismate mutase P-prephenate-dehydratase. A derivative of bacteriophage lambda which carries the pheA-lac fusion was prepared; the method used is generally applicable for the construction, from Mu dl lysogens, of specialized transducing lambda phage carrying the promoter-lac fusions. A restriction enzyme cleavage map of lambda ppheA-lac for the enzymes HindIII and PstI is presented.     

Isolation and analysis of aroFo mutants by using an aroF-lac operon fusion

The lac structural genes were fused to the regulatory region of the aroF-tyrA operon so that the expression of beta-galactosidase was regulated by the tyrR+ gene product. Transducing phage carrying the aroF-lac fusion were isolated, and a lambda aroF-lac lysogen was used to select for aroFo mutants. A plasmid vector was constructed onto which the aroFo mutations were transferred by recombination in vivo.     

Regulation of the ColV plasmid-determined iron (III)-aerobactin transport system in Escherichia coli.

Regulation by iron was studied in Escherichia coli strains whose iron supply was entirely dependent on the iron(III)-aerobactin system determined by the ColV plasmid. By the insertion of phage Mu (Ap lac) into the ColV plasmid, mutants were selected that could no longer grow in iron-limited media. The inserted Mu (Ap lac) strongly reduced the amount of aerobactin and he cloacin receptor protein formed by the cells. Their production was no longer subject to regulation by iron. The Mu (Ap lac) insertion apparently led to a polar effect on the expression of the presumably closely linked genes that control the synthesis of aerobactin and the cloacin receptor protein. The expression of the beta-galactosidase gene on the inserted phage genome came under the control of the iron state of the cells. Under iron-limited growth conditions, the amount of beta-galactosidase synthesized was, depending on the strain studied, 6 to 30 times higher than under iron-sufficient growth conditions. In fur mutants with an impaired iron regulation of ll iron supply systems studied so far, high amounts of beta-galactosidase were synthesized independent of the cells' iron supply. The results demonstrate an iron-controlled promoter on the ColV plasmid which is subject to regulation by the chromosomal fur gene.     

Infection of Salmonella typhimurium with coliphage Mu d1 (Apr lac): construction of pyr::lac gene fusions.

A procedure was developed for introducing the coliphage Mu d1 (Apr lac) into Salmonella typhimurium in order to construct gene fusions that place the structural genes of the lac operon under the control of the promoter-regulatory region of other genes. To introduce Mu d1 from Escherichia coli K-12 into S. typhimurium, which is normally not a host for Mu, we first constructed an E. coli double lysogen carrying the defective Mu d1 phage and a Mu-P1 hybrid helper phage (MuhP1) that confers the P1 host range. A lysate prepared from this strain was used to infect a P1-sensitive (i.e., galE), restriction-deficient, modification-proficient strain of S. typhimurium, and a double lysogen carrying Mu d1 and MuhP1 was isolated. Induction of the latter strain produced lysates capable of infecting and generating gene fusions in P1-sensitive strains of S. typhimurium. In this paper we describe the construction of pyr::lac fusions by this technique.     

hisT is part of a multigene operon in Escherichia coli K-12.

The Escherichia coli K-12 hisT gene has been cloned, and its organization and expression have been analyzed on multicopy plasmids. The hisT gene, which encodes tRNA pseudouridine synthase I (PSUI), was isolated on a Clarke-Carbon plasmid known to contain the purF gene. The presence of the hisT gene on this plasmid was suggested by its ability to restore both production of PSUI enzymatic activity and suppression of amber mutations in a hisT mutant strain. A 2.3-kilobase HindIII-ClaI restriction fragment containing the hisT gene was subcloned into plasmid pBR322, and the resulting plasmid (designated psi 300) was mapped with restriction enzymes. Complementation analysis with different kinds of hisT mutations and tRNA structural analysis confirmed that plasmid psi 300 contained the hisT structural gene. Enzyme assays showed that plasmid psi 300 overproduced PSUI activity by ca. 20-fold compared with the wild-type level. Subclones containing restriction fragments from plasmid psi 300 inserted downstream from the lac promoter established that the hisT gene is oriented from the HindIII site toward the ClaI site. Other subclones and derivatives of plasmid psi 300 containing insertion or deletion mutations were constructed and assayed for production of PSUI activity and production of proteins in minicells. These experiments showed that: (i) the proximal 1.3-kilobase HindIII-BssHII restriction fragment contains a promoter for the hisT gene and encodes a 45,000-dalton polypeptide that is not PSUI; (ii) the distal 1.0-kilobase BssHII-ClaI restriction fragment encodes the 31,000-dalton PSUI polypeptide; (iii) the 45,000-dalton polypeptide is synthesized in an approximately eightfold excess compared with PSUI; and (iv) synthesis of the two polypeptides is coupled, suggesting that the two genes are part of an operon. Insertion of mini-Mu d1 (lac Km) phage into plasmid psi 300 confirmed that the hisT gene is the downstream gene in the operon.     

Effects of an anti-beta monoclonal antibody on the interaction of the Escherichia coli RNA polymerase with the lac and TAC promoters

The effects of an inhibitory monoclonal antibody (mAb) raised against the beta subunit of the Escherichia coli RNA polymerase were determined on the kinetics and structural interactions during formation of the open promoter complex (RPo). Analysis of the kinetics of abortive initiation on linear and supercoiled templates of the lac and TAC16 promoters showed that abortive synthesis by mAb 210E8-RNA polymerase varied as a function of DNA topology. A kinetic analysis of RPl formation on the supercoiled lac UV5 promoter showed that mAb 210E8 effected a slight alteration in the isomerization rate and no effect on the initial rate of RNA polymerase binding to the promoter. The potent inhibition of initiation with linear promoters by mAb 210E8 was not apparent when the promoters were assayed in their supercoiled forms. Abortive synthesis with the TAC16 promoter was accompanied by an mAb 210E8 induced hindrance of ApUpU but not UpGpU synthesis. The data indicate that the inhibition by mAb 210E8 with the supercoiled TAC16 promoter is further alleviated when the spacer length is shifted from 16 base pairs (ApUpU formation) to 18 base pairs (UpGpU formation). When DNase I and dimethyl sulfate were used to probe DNA structure, mAb 210E8 was found to alter polymerase interactions with the lac promoter. DNase I footprinting indicated that the structural interactions for lac P+ promoter-RNA polymerase complexes were slightly altered in the presence of mAb 210E8. Treatment of the RNA polymerase-lac UV5 complex with dimethyl sulfate revealed an alternate mode of RNA polymerase interaction with essential guanine contacts which was intermediate between a fully protected and free promoter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutational analysis of the lac regulatory region: second-site changes that activate mutant promoters.

Second-site mutations that restored activity to severe lacP1 down-promoter mutants were isolated. This was accomplished by using a bacteriophage f1 vector containing a fusion of the mutant E. coli lac promoters with the structural gene for chloramphenicol acetyltransferase (CAT), so that a system was provided for selecting phage revertants (or pseudorevertants) that conferred resistance of phage-infected cells to chloramphenicol. Among the second-site changes that relieved defects in mutant lac promoters, the only one that restored lacP1 activity was a T----G substitution at position -14, a weakly conserved site in E. coli promoters. Three other sequence changes, G----A at -2, A----T at +1, and C----A at +10, activated nascent promoters in the lac regulatory region. The nascent promoters conformed to the consensus rule, that activity is gained by sequence changes toward homology with consensus sequences at the -35 and -10 regions of the promoter. However, the relative activities of some promoters cannot be explained solely by consideration of their conserved sequence elements.     

The regulatory elements of araBAD operon,contrary to lac‐based expression systems,afford hypersynthesis of murine,and human interferons in Escherichia coli

The overexpression of four different interferons, i.e., murine interferon α1 and human interferons α1, α8, and α21 was challenged in Escherichia coli. Synthetic genes coding for these interferons were designed, assembled, and cloned into the vector pET9a (using the NdeI and BamHI sites), placing interferon expression under the control of phage T7 promoter. Despite an intensive screening for optimal culture conditions, no interferon synthesis was observed using overexpression systems based on the regulatory elements of lac operon (e.g., in E. coli BL21DE3). On the contrary, high levels of interferon expression were detected in E. coli BL21AI, which chromosome contains the gene coding for phage T7 RNA polymerase under the control of the araBAD promoter. To analyze the reasons of this striking difference, the molecular events associated with the lack of interferon expression in E. coli BL21DE3 were studied, and murine interferon α1 was chosen as a model system. Surprisingly, it was observed that this interferon represses the synthesis of T7 RNA polymerase in E. coli BL21DE3 and, in particular, the expression of lac operon. In fact, by determining β‐galactosidase activity in E. coli BL21AI, a significantly lower LacZ activity was observed in cells induced to interferon synthesis. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Bacteriophage f1 DNA replication genes. II. The roles of gene V protein and gene II protein in complementary strand synthesis

Filamentous phage gene V, which encodes a single-stranded DNA binding protein, has been cloned and placed under control of the lac promoter. Cells bearing the clone are refractory to filamentous phage infection if the expression of the gene is induced with isopropyl-1-thio-beta-D-galactoside. The inhibition of infection is shown to occur at an early stage, and can be reversed if the cells express gene II in addition to gene V protein. These observations support the hypothesis that gene II protein, in addition to its role in nicking and facilitating the synthesis of phage viral (+) strand DNA, functions to prevent the gene V-mediated inhibition of complementary (-) strand synthesis. We proposed a model in which the absolute and relative concentrations of the products of genes II, X and V determine whether a single strand is to be exported as phage or incorporated into double-stranded replicative form DNA.     

Methionine aminopeptidase gene of Escherichia coli is essential for cell growth.

We localized the methionine aminopeptidase (map) gene on the Escherichia coli chromosome next to the rpsB gene at min 4. Genetically modified strains with the chromosomal map gene under lac promoter control grew only in the presence of the lac operon inducer isopropyl-beta-thiogalactoside. Thus, methionine aminopeptidase is essential for cell growth.     

Studies on the control of the genes for transcription and translation in Escherichia coli K12 I. tufB and rplA, K have separate promoters.

A spontaneous deletion, ΔM9, removing part of argCBH and extending through rnnB was isolated. Its endpoints were located by genetic crosses and restriction enzyme analysis of a λ transducing phage carrying ΔM9. Studies on this phage in ultraviolet-irradiated cells showed that ΔM9 places synthesis of EF-Tu, the tufB gene product, under arg control. By contrast the products of rplA and rplK (ribosomal proteins L1 and L11) remain independent of arg control. Thus the promoter for tufB is located counterclockwise of the structural genes on the Excherichia coli chromosome and the adjacent genes rplA and rplK have their own promoter.     

Use of chlC-lac fusions to determine regulation of gene chlC in Escherichia coli K-12.

Gene fusions between the lac structural genes and the chlC locus were isolated, and the regulation of lac gene expression was studied. The fused lac genes were induced by nitrate anaerobically and repressed by the presence of oxygen.     

Catabolite repression of the lac operon. Effect of mutations in the lac promoter

1. Several lac diploid strains of Escherichia coli were constructed and tested to discover whether mutations in the lac promoter alleviate catabolite repression. 2. In each of these diploids the chromosome carries one of the promoter mutations, L8, L29 or L1; so that the rate of synthesis of the enzymes of the lac operon is only 2-6% of the fully induced wild-type. Each diploid harbours the episome F'lacM15 that specifies the synthesis of thiogalactoside transacetylase under the control of intact regulator, promoter and operator regions, but has a deletion in the structural gene for beta-galactosidase. In each diploid more than 90% of the thiogalactoside transacetylase is synthesized from the episome, and 100% of the beta-galactosidase is synthesized from the chromosome, and comparison of the extent of catabolite repression that the two enzymes suffered indicated whether the chromosomal promoter mutation relieves catabolite repression. 3. In the strains in which the promoter carries either of the point mutations L8 or L29 the enzymes were equally repressed, suggesting that neither L8 nor L29 affects catabolite repression. 4. In a diploid strain harbouring the same episome but carrying deletion L1 on the chromosome, synthesis of beta-galactosidase suffered much less repression than that of thiogalactoside transacetylase. 5. In a diploid strain in which the chromosome carries L1 and also a second mutation that increases the rate of expression of lac to that permitted by L8 or L29, the synthesis of beta-galactosidase again suffered much less repression than the synthesis of thiogalactoside transacetylase. 6. The effect of L1 (which deletes the boundary between the i gene and the lac promoter) is ascribed to its bringing the expression of lac under the control of the promoter of the i gene. 7. Even in strains carrying L1, some catabolite repression persists; this is not due to a trans effect from the episome since it occurs equally in a haploid strain with L1.