Discontinuity of homology of Escherichia coli and Salmonella typhimurium DNA in the lac region

Summary Partial homology of Salmonella typhimurium DNA to Escherichia coli DNA was demonstrated by Southern hybridization blots to exist on either side of the lac operon of E. coli but no homology was detected between S. typhimurium DNA and about 12 kb of E. coli DNA including the lac genes as well as about 5 kb of E. coli DNA between lac and proC. Thus portions of DNA seem to have been either added to the E. coli genome or deleted from the S. typhimurium genome since their divergence from a common ancestor. Although an IS1 element was located near the lac operon of E. coli, the insertional element was shown not to be near any of the junctures of discontinuity of E. coli - S. typhimurium homology near lac.     

THE INCREASED POTENTIAL FOR SELECTION OF THE LAC OPERON OF ESCHERICHIA COLI

The fitness effects of six lac operons from natural isolates of Escherichia coli were determined in chemostats, in a test of the idea that selective differences among natural alleles are greater in novel conditions than in the prevailing environment, resulting in a greater genetic variance in fitness in novel conditions. Fitnesses were determined in the common milk sugar lactose, the natural substrate of the lac operon, and in three rare β-galactosides, lactulose, galactosyl-arabinose, and methyl-galactopyranoside, that are novel for E. coli. Significantly greater fitness differences were observed among the lac alleles in each of the novel β-galactosides than in lactose. An alternative explanation of the experimental findings is discussed. General evolutionary causes and consequences of selection potentials are discussed, and an outline of the work necessary to further elucidate the physiological basis of the observed potential for selection of the lac operon of E. coli is presented.     

Transposition and insertion of intact, deleted and enlarged ampicillin transposon Tn3 from mini-R1 (Rsc) plasmids into transfer factors

Summary The miniR1-(Rsc)-plasmids which derive from the copy mutant R1drd-19B2 (pKN102) are nonconjugative extrachromosomal elements which can not be co-transferred by various transfer factors to recipient strains under standard mating conditions. The attempts to mobilize Rsc11 by F'lac lead to transconjugants carrying F'lac::Tn3 with Tn3 mainly inserted into the lac operon. In addition it can be shown that Rsc11 can become inserted as a complete unit into the transfer factor giving rise to rather unstable recombinant intermediates. Dissociation of these intermediates may lead to alterations of the original plasmids.The Tn3 part of Rsc13 can be enlarged or deleted by in vitro manipulations. In vitro insertion of EcoRI-fragments into an EcoRI⁺ site of Tn3 leads to new transposable units which can be transposed to the RTF part of R1. This new genetic entity can be stably integrated into the chromosome of E. coli by integrative suppression of a dnaA^ts-mutation.Deletions at one end or the central region of Tn3 abolish the capability of transposition. However, the Rsc-plasmids containing the deleted Tn3 can still be inserted into the transfer factor as complete units. The resulting recombinants are unstable leading after dissociation in some cases to new plasmids with altered properties.     

Inactivation of lacZ gene expression by UV light and bound DNA photolyase implies formation of extended complexes in the genomes of specific Escherichia coli strains

Summary In Escherichia coli strains WU and CS101, UV inactivation of lacZ gene expression is more effective when the cells contain amplified DNA photolyase, and flash photoreactivation (fPR) after 15 min of metabolism does not reverse inactivation by the photolyasedimer complexes. In other strains, also studied with or without amplified DNA photolyase, there is no differential UV inactivation and fPR reverses inactivation by the complexes regardless of continued metabolism. The irreparable condition in strain WU is not due to dysfunction of photolyase: during post-UV metabolism, fPR still restores viability and dimers are removed from the region of the lac operon. When the wild-type lac promoter is repalced by the UV5 promoter, making expression insensitive to relaxed supercoiling and catabolite repression, inactivation by dimers alone becomes more resistant, i.e. requires higher fluences, but inactivation in WU and CS101 is still exceptionally sensitive to photolyasedimer complexes. This indicates that dimers external to the wild-type lac operon may inhibit expression by altering supercoiling but that complexes must involve some other mechanism for their special effect in WU and CS101. The exceptionally efficient inactivation and irreparable condition are consistent with the idea that, in two specific laboratory strains, photolyase bound to dimers at a considerable distance from the lac operon may initiate an aggregation of DNA with other cellular molecules that extends to, and inactivates expression from, the operon.     

Klebsiella pneumoniae nif-lac fusions are expressed in Agrobacterium tumefaciens C58

Summary Plasmids containing hybrid genes, in which different Klebsiella pneumoniae nif (nitrogen-fixation) promoters were fused with the structural part of the Escherichia coli lac operon, were introduced into a double auxotrophic derivative of Agrobacterium tumefaciens C58. A study of their expression in the new host was made simple by the inherent inability of A. tumefaciens C58 to produce -galactosidase unless provided with the wild-type lac operon of E. coli. As shown by quantitative measurements of the enzyme, all K. pneumoniae promoters were expressed well in A. tumefaciens C58, even under conditions known to repress them. It also has been shown that the activity of K. pneumoniae nif A is essential for the expression of nifHDK even when introduced into A. tumefaciens. After entering the new host the plasmids, the nif genes and the fusion alleles contained in them, remained stable. Possible mechanisms responsible for the constitutive behaviour of nif promoters in A. tumefaciens are discussed.     

Stringent regulation of human growth hormone expression in cultured murine C2C12 myoblasts by theE. coli lac repressor

Summary Gene transfer techniques can be used to encode the production of a polypeptide product, such as human growth hormone (hGH), that is missing in an acquired or inherited disease state such as growth hormone deficiency. In one model system, engineered C2C12 myoblasts are injected intramuscularly into a mouse and subsequently secrete hGH into the circulation. In this regard, a gene-expression regulatory system that functions in myoblasts would be of interest. We demonstrate that theEscherichia coli lac operon system can be used to stringently regulate the expression of hGH in engineered C2C12 myoblasts in tissue culture. A DNA segment encoding hGH was linked to a DNA segment containing an SV40 enhancer and promoter. The latter components were positioned between two syntheticlac operators.Lac repressor expression was driven by a simian cytomegalovirus promoter. In transient co-transfection assays, hGH expression from cultured C2C12 myoblasts could be modulated up to 60-fold (P = 0.002) with the inducing agent, isopropyl-β-d-thiogalactoside (IPTG). In the absence of IPTG, hGH expression was almost fully repressed. These results show that the components of theE. coli lac operon provide a stringent regulatory system for use in myoblasts. The system might prove to be useful for the regulation of transferred genes in animals.     

Lactose- and galactose-utilizing strains of poly(hydroxyalkanoic acid)-accumulating Alcaligenes eutrophus and Pseudomonas saccharophila obtained by recombinant DNA technology

Summary Transposon Tn 951-encoded -galactosidase was expressed in Pseudomonas saccharophila and enabled this bacterium to grow on lactose as sole carbon source. In contrast, -galactosidase was not expressed in Alcaligenes eutrophus even if the lacZ gene of Tn 951 was separated from the lacI gene. However, -galactosidase was expressed in A. eutrophus, if a DNA fragment, which was suspected to harbour the promoter of the A. eutrophus poly(3-hydroxybutyric acid)-synthetic genes, was ligated to the promoter probe vector pMC1403, which employs lac Z, Y as reporter genes. Plasmid pPL76, which harboured one of the promoter-lac fusions, enabled A. eutrophus not only to express -galactosidase but also to grow slowly on lactose (doubling time = 25–30 h). Subsequently, the promoter-lac fusion was ligated to Tn5 in pSUP5011 and was inserted into the genome of A. eutrophus H16 and of the glucose-utilizing mutant H16-G⁺1 by applying the suicide plasmid technique. Two recombinant strains, H16-cPL and H16-G⁺1-cPL, which grow with a doubling time of 16–23 h on lactose, were investigated in detail. The cells only utilized the glucose residue of lactose as a carbon source for grouth and excreted galactose into the medium. Only after the Escherichia coli gal operon had been cloned in vector pVK101 and had been mobilized to H16-cPL or H16-G⁺1-cPL, was lactose completely utilized; no galactose was detected in the medium and the growth yields increased twofold. Depending on the orientation of the gal operon in pVK101, the expression of galactokinase seems to be dependent either on the promoter of aminoglycoside phosphotransferase gene (kan) or on the promoter of the tetR gene. Offprint requests to: A. Steinbüchel     

Use of lac Gene Fusions to Study Regulation of Tyramine Oxidase,Which is Involved in Derepression of Latent Arylsulfatase in Escherichia coli

Strains with lac fused to each of the arylsulfatase (ats) and tyramine oxidase (tyn) operons in Escherichia coli were isolated. Synthesis of β-galactosidase in strains with tyn:: lac fusions was induced by tyramine, histamine, tryptamine, dopamine and octopamine, and the induction of the tyn operon was subject to catabolite and ammonium repressions. These repressions were relieved when the cells were grown with a poor carbon or nitrogen source. No arylsulfatase activity is detected in E. coli strains. Synthesis of β-galactosidase in strains with ats:: lac fusions was repressed by sulfur compounds. The repression was relieved by monoamine compounds, which induced tyramine oxidase synthesis. The inhibition of tyramine oxidase activity by cysteine resulted in a decrease of the derepressed synthesis of β-galactosidase in the ats:: lac fusion. Repressing and derepressing conditions for the tyn operon prevented and stimulated, respectively, expression of the ats operon. Thus, the expression of latent arylsulfatase in E. coli seems to be regulated by expression of the tyn operon.     

Binding of lac repressor to the secondary lac operator in Escherichia coli.

Summary In the lac operon, the existence of a secondary repressor binding site, inside Z gene, had been inferred from in vitro binding studies (Reznikoff et al., 1974; Gilbert et al., 1975).A serie of deletions have been constructed from a lac transducing bacteriophage. Some of those deleted bacteriophages have still the property of derepressing a chromosomal lac operon, even though they do not contain any more the lac operator. This phenomenon is an indication that the secondary repressor binding site is also active in vivo.     

A Modified Consumer Inkjet for Spatiotemporal Control of Gene Expression

This paper presents a low-cost inkjet dosing system capable of continuous, two-dimensional spatiotemporal regulation of gene expression via delivery of diffusible regulators to a custom-mounted gel culture of E. coli. A consumer-grade, inkjet printer was adapted for chemical printing; E. coli cultures were grown on 750 µm thick agar embedded in micro-wells machined into commercial compact discs. Spatio-temporal regulation of the lac operon was demonstrated via the printing of patterns of lactose and glucose directly into the cultures; X-Gal blue patterns were used for visual feedback. We demonstrate how the bistable nature of the lac operon''s feedback, when perturbed by patterning lactose (inducer) and glucose (inhibitor), can lead to coordination of cell expression patterns across a field in ways that mimic motifs seen in developmental biology. Examples of this include sharp boundaries and the generation of traveling waves of mRNA expression. To our knowledge, this is the first demonstration of reaction-diffusion effects in the well-studied lac operon. A finite element reaction-diffusion model of the lac operon is also presented which predicts pattern formation with good fidelity.     

Detection of developmentally regulated genes of the myxobacterium Stigmatella aurantiaca with the transposon Tn5lacZ

Stigmatella aurantiaca is a prokaryotic organism that undergoes a multicellular cycle of development resulting in the formation of a fruiting body. Insertional mutations were introduced at random sites into the Stigmatella aurantiaca genome with the promotor probe Tn5lacZ derived from Tn5lac by deleting non-essential sequences. 638 transconjugants were obtained with a frequency of 1×10^-7. In 260 of the transconjugants isolated the -glactosidase gene of Tn5lacZ is fused to vegetative promotors of Stigmatella aurantiaca. In 65 of the strains -galactosidase is induced by starvation; in 14 of the transconjugants -galactosidase activity is observed after chemical induction of sporulation by 3-methyl-indole. Thirtytwo of the mutants are affected in fruiting body formation and morphology.     

Involvement of a gene of the chl E locus in the regulation of the nitrate reductase operon

Summary A strain of E. coli carrying a Mudl insertion leading to chlorate resistance was found to lack nitrate reductase and formate dehydrogenase activities, but to synthesize b-type cytochrome constitutively. Introduction of this insertion mutation into a strain bearing a fusion between the nitrate reductase operon (chl C, chl I) and the lac structural genes resulted in the constitutive expression of the lac genes of this last fusion. Identical results were found when the Mudl was eliminated promoting a deletion in the original insertion site. This mutation was located midway between gal and aro A, at the chl E locus. Study of a chl E strain already described revealed similar behaviour. Absence of nitrate reductase activity in these strains which constitutively express the structural genes of the nitrate reductase operon was tentatively attributed to the simultaneous lack of a cofactor of the nitrate reductase terminal enzyme, possibly cofactor Mo-X, and of a repressor of the operon.     

The gene (lacA) encoding galactoside acetyltransferase fromLactococcus lactis

Summary This paper reports the cloning and characterization of a gene encoding galactoside acetyltransferase from a strain ofLactococcus lactis. AP stI library ofL. lactis strain ATCC7962 DNA was constructed in plasmid pUC18. A clone harbouring a 10 kbp DNA fragment containing part of thelac operon was isolated using a labelled probe generated by PCR. DNA sequence analysis revealed the presence of a gene encoding a protein with 64.5% similarity to the galactoside acetyltransferase fromEscherichia coli. The codon usage pattern of this gene was not typical of lactococcal genes. The lactococcallac operon organization appears to be different to that of other organisms.     

Transposon Tn951 (TnLac) is defective and related to Tn3

Summary Tn951 is flanked by two perfect inverted repeats of 41 bp which include the 38 bp sequence of the IR of Tn3. Tn951 also contains the last 100 bp of the tnpA gene but with at least two mutations. However, beyond nucleotide 137 the sequences diverge and hybridization experiments show that Tn951 lacks at least the first two thirds of the tnpA gene.In agreement with these observations Tn951 does not transpose by itself at a detectable frequency but can be complemented by the tnpA gene of Tn801 or Tn3. Tn501, Tn1721 and gamma delta do not complement Tn951 transposition.Transposition of Tn951 duplicates 5 bp of target DNA sequence.     

The Effect of the lacY Gene on the Induction of IPTG Inducible Promoters, Studied in Escherichia coli and Pseudomonas fluorescens

The role of the Escherichia coli lacY gene product (the lactose permease) in the induction of isopropyl-β-D-thiogalactopyranoside (IPTG) inducible promoters was studied in E. coli and P. fluorescens. This was done by comparing strains containing a lacIPOZYA chromosomal insert with newly constructed strains containing inserts without the lacY gene (lacIPOZ). The lactose operon inserts were introduced as single-copy chromosomal inserts to eliminate differences in expression caused by differences in copy number. Comparison between the two types of inserts showed that the lactose permease was essential to allow growth on lactose by both bacteria and that the lactose permease plays an important role in transporting the inducer IPTG across the membrane of P. fluorescens. The use of a functional lactose permease allows expression of β-galactosidase to increase more than fivefold from a wild-type lac promoter in P. fluorescens SS1001. We suggest that an increase in the rate of protein synthesis from lac-type promoters could be enhanced if an active lactose permease is present as well. Received: 29 October 1997 / Accepted: 8 December 1997     

A derivative of Tn5 with direct terminal repeats can transpose

The 5.7 kb⁴ transposable kanamycin resistance determinant Tn5 contains 1.5 kb terminal inverted repeats which we here call arms. Tn5's arms contain the genes and sites necessary for Tn5 transposition, and are not homologous to previously described transposable elements. To determine whether one or both arms is a transposable (IS) element, we transposed Tn5 to pBR322 and used restriction endonuclease digestion and ligation in vitro to generate plasmid derivatives designated pTn5-DR1 and pTn5-DR2 in which Tn5's arms were present in direct rather than in inverted orientation. Analysis of transposition products from dimeric forms of the pTn5-DR1 plasmid to phage λ showed that the outside and inside termini of right and of left arms could function in transposition. We conclude that both of Tn5's arms are transposable elements and name them IS50L (left) and IS50R (right). IS50R, which encodes transposase, was used several-fold more frequently than IS50L, which contain an ochre mutant allele of transposase: this implies that Tn5's transposase acts preferentially on the DNA segment which encodes it. Analysis of transpositions of the amp^rkan^r element Tn5-DR2 to the lac operon showed that Tn5-DR2, like Tn5 wild-type, exhibits regional preference without strict site specificity in the choice of insertion sites.