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PubMed1976

A plasmid cloning vehicle allowing regulated expression of eukaryotic DNA in bacteria.

We have constructed a plasmid cloning vehicle in which transcription of inserted heterologous DNA fragments can be regulated by a defined bacterial operator and promoter. The lambda plac 5 EcoRIDNA fragment containing the operator, promoter, and beta-galactosidase gene of the lactose operon was linked to the ColE1 derivative plasmid pSF2124, creating a plasmid designated pBGP100, pBGP100 contains one EcoRI site at the lac DNA/pSF2124 DNA junction and another at the lambda DAN/pSF2124 DNA junction. We deleted the latter EcoRI site to generate a plasmid (pBGP120) retaining a single EcoRI site at the lac DNA/nSF2124 DNA junction. To determine whether DNA introduced at the EcoRI site of pBGP120 was expressed under lactose control, we inserted the EcoRI fragment containing 28S ribosomal DNA of Xenopus laevis, creating the hybrid plasmid pBGP123. RNA-DNA hybridization of pulse-labeled RNA from cells containing pBGP123 showed that induction of the lac operon increases the percentage of labeled RNA complementary to Xenopus 28S DNA about 9-fold. This vehicle may be of use for production of eukaryotic gene products in bacteria.

Animals↗
PubMed1990

Homologous recombination and mutagenesis of gamma-irradiated plasmid DNA in Escherichia coli host cells.

Plasmid DNA was used to study gamma-radiation-induced recombination and mutagenesis in Escherichia coli host cells. Plasmid pBRP1, a derivative of pBR322 containing the lac operon of E. coli, was irradiated with 60Co gamma rays prior to transformation into E. coli strains of different recA and lac genotypes. Plasmid-chromosome recombination was assayed in lacY1 host cells, whereas plasmid mutagenesis was assayed in delta lac host cells lacking chromosomal sequences homologous to the plasmid. Both recombinant and mutant plasmids were identified by the phenotypic changes in lactose utilization, and confirmed by restriction analysis of isolated plasmids. Plasmid-chromosome recombination was induced to high levels (about 20% of survivors at 700 Gy) and was dependent on the host recA gene. Plasmid mutagenesis occurred at lower levels (about 1.5% of survivors at 600 Gy) and was relatively independent of the recA gene. Plasmid survival was unaffected by the presence or absence of host recA mutations or the potential for plasmid-chromosome recombination.

Cobalt Radioisotopes↗
PubMed1976

Transcriptional and post-transcriptional control of beta-galactosidase synthesis.

lac messenger ribonucleic acid synthesis is dissociated from its translation in certain lac operon mutants. More beta-galactosidase messenger ribonucleic acid (as determined by hybridization) is made in fully induced lacOc mutants and in trp-lac fusions than is eventually translated into enzyme. Consequently, beta-galactosidase synthesis may be post-transcriptionally controlled under these conditions.

Cyclic AMP↗
PubMed1987

Molybdenum-sensitive transcriptional regulation of the chlD locus of Escherichia coli.

The chlD gene in Escherichia coli is required for the incorporation and utilization of molybdenum when the cells are grown with low concentrations of molybdate. We constructed chlD-lac operon fusions and measured expression of the fusion, Mo cofactor, and nitrate reductase activities under a variety of growth conditions. The chlD-lac fusion was highly expressed when cells were grown with less than 10 nm molybdate. Increasing concentrations of molybdate caused loss of activity, with less than 5% of the activity remaining at 500 nM molybdate; when tungstate replaced molybdate, it had an identical affect on chlD expression. Expression of chlD-lac was increased in cells grown with nitrate. Strains with chlD-lac plus an additional mutation in a chl or nar gene were constructed to test whether the regulation of chlD-lac required the concerted action of gene products involved with Mo cofactor or nitrate reductase synthesis. Mutations in narL prevented the increase in activity in response to nitrate; mutations in chlB, narC, or narI resulted in partial constitutive expression of the chlD-lac fusion: the fusion was regulated by molybdate, but it no longer required the presence of nitrate for maximal activity. Mutations in chlA, chlE, or chlG which affect Mo cofactor metabolism, did not affect the expression of chlD-lac.

Coenzymes↗
PubMed1998

Flk couples flgM translation to flagellar ring assembly in Salmonella typhimurium.

The hook-basal body (HBB) is a key intermediate structure in the flagellar assembly pathway in Salmonella typhimurium. The FlgM protein inhibits the flagellum-specific transcription factor sigma28 in the absence of the intact HBB structure and is secreted out of the cell following HBB completion. The flk gene encodes a positive regulator of the activity of FlgM at an assembly step just prior to HBB completion: at the point of assembly of the P- and L-rings. FlgM inhibition of sigma28-dependent class 3 flagellar gene transcription was relieved in P- and L-ring assembly mutants (flgA, flgH, and flgI) by introduction of a null mutation in the flk gene (J. E. Karlinsey et al., J. Bacteriol. 179:2389-2400, 1997). In P- and L-ring mutant strains, recessive mutations in flk resulted in a reduction in intracellular FlgM levels to those seen in wild-type (Fla+) strains. The reduction in intracellular FlgM levels by mutations in the flk gene was concomitant with a 10-fold increase in transcription of the flgMN operon compared to that of the isogenic flk+ strain, while transcription of the flgAMN operon was unaffected. This was true for both direct measurement of the flgAMN and flgMN mRNA transcripts by RNase T2 protection assays and for lac operon fusions to either the flgAMN or flgMN promoter. Loss of Flk did not allow secretion of FlgM through basal-body structures lacking the P- and L-rings. Intracellular FlgM was stable to proteolysis, and turnover occurred primarily after export out of the cell. Loss of Flk did not result in increased FlgM turnover in either P- or L-ring mutant strains. With lacZ translational fusions to flgM, a null mutation in flk resulted in a significant reduction of flgM-lacZ mRNA translation, expressed from the class 3 flgMN promoter, in P- and L-ring mutant strains. No reduction in either flgAMN or flgMN mRNA stability was measured in the absence of Flk in Fla+, ring mutant, or HBB deletion strains. We conclude that the reduction in the intracellular FlgM levels by mutation in the flk gene is only at the level of flgM mRNA translation.

Alleles↗
PubMed1985

[Effect of mutation changes in RNA-polymerase and transcription termination factor rho on expression of various operons in E. coli].

Six mutations, impairing DNA polymerase of E. coli in combination with the wild type gene for rho factor or ts-mutation rho 15 have been studied in relation to the expression of seven operons having different types of regulation. The expression of genes for glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase is shown to be constitutive and resistant to mutationally altered RNA polymerase and rho factor. The expression of genes for adenine phosphoribosyltransferase and of deo operon is regulated by rho dependent attenuators with attenuation being lifted incomplete medium. Mutation rho 15 decreases the level of enzymes of thr and lac operons independent of mRNA levels of these operons. Mutation rho 15 effect on posttranscriptional level is modified by mutations damaging RNA polymerase. The data obtained suppose RNA polymerase to affect all stages of realization of genetic information, beginning with promoter recognition and RNA synthesis and including the protein synthesis on mRNA.

DNA Restriction Enzymes↗
PubMed1988

Gene expression in E. coli after treatment with streptozotocin.

Gene induction by the methylating agents streptozotocin (STZ), N-methyl-N-nitrosourea (MNU), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was evaluated in E. coli fusion mutants. These mutants have fusions of the lac operon to genes induced by treatment with sublethal levels of alkylating agents and were previously selected from random insertions of the Mu-dl (Apr lac) phage by screening for induction of beta-galactosidase activity in the presence of methyl methanesulfonate or MNNG. The results demonstrate that STZ differs from MNNG and MNU in failing to induce aidC expression. Further, expression of aidC after exposure to MNU and MNNG occurs only in nonaerated cultures; aeration blocks the induction. Induction of aidD, alkA, aidB, and sfiA expression occurs with all 3 agents although at markedly lower concentrations of MNNG and STZ compared to MNU. alkA and to a lesser extent aidD mutants of E. coli strains were more sensitive to these agents, while no differences were evident between wild-type and aidB or aidC fusion mutants.

Alkylating Agents↗
PubMed1974

Mutations in the L-arabinose operon of Escherichia coli B-r that result in hypersensitivity to catabolite repression.

Two independent mutants resistant to l-arabinose inhibition only in the presence of d-glucose were isolated from an l-arabinose-sensitive strain containing the araD139 mutation. Preliminary mapping studies indicate that these mutations are closely linked to the araIOC region. Addition of d-glucose to growing cultures of these mutants results in a 95 to 98% repression of ara operon expression, as compared to a 50% repression of the parental control. Since cultures of both mutant and parental strains undergo a 50% repression of lac operon expression upon addition of glucose, the hypersensitivity to catabolite repression exhibited by these mutants is specific for the ara operon. Addition of cyclic adenosine monophosphate reverses the catabolite repression of the ara operon in both mutant and parent strains to 70 to 80% of the control. It is suggested that in these mutants the affinity of the ara operon initiator region for the cAMP-catabolite-activator protein complex may have been altered.

Arabinose↗
PubMed1988

Gene expression caused by alkylating agents and cis-diamminedichloroplatinum(II) in Escherichia coli.

Previous work has demonstrated heterogeneous effects of methylating agents on induction of DNA damage inducible genes in Escherichia coli. These studies employed E. coli mutants that have fusions of the lac operon to genes induced by treatment with sublethal levels of alkylating agents. These mutants were selected from random insertions of the Mu-dl (Apr lac) phage by screening for induction of beta-galactosidase activity in the presence of methylmethanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine. The current report extends these findings by analyzing gene expression caused by mechlorethamine, chloroethylnitrosoureas and cis-diamminedichloroplatinum(II) (cis-DDP). The results demonstrate heterogeneous effects by these agents on gene expression. While 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea induces alkA, other nitrosoureas, mechlorethamine, and cis-DDP do not cause expression of this gene. Further, while all nitrosoureas caused expression of aidC, mechlorethamine and cis-DDP did not. Lastly, cis-DDP caused marked expression of a sulA fusion mutant while not inducing any of the other E. coli fusion mutants.

Alkylating Agents↗
PubMed1984

Studies on the structure and expression of Escherichia coli pyrC, pyrD, and pyrF using the cloned genes.

The Escherichia coli pyrC, pyrD and pyrF genes were cloned on multicopy plasmids derived from pBR322 and analysed by means of restriction endonucleases. It was found that the pyrC gene is destroyed by cutting with the restriction endonuclease BamHI, that the entire pyrD gene can be isolated on a 1300-base pairs DNA fragment generated by EcoRI cleavage and that cutting with EcoRI removes the promotor and probably also the translational start site from the pyrF gene. More details on the restriction maps are presented. Further, it was found that the presence of a pyr gene in multiple copies on a plasmid does not significantly interfere with the activity of the chromosomal pyr genes. Using the 'minicell' technique, the polypeptides encoded by the three cloned pyr genes were identified. The relative molecular masses for the pyrC-encoded and pyrD-encoded polypeptides are 38 000-40 000 and 36 000-38 000, respectively. Thus in their native form, dihydroorotase and dihydroorotate oxidase appear to be dimeric proteins. The 'minicell' experiments positively identified a protein chain of Mr 23 000-24 000 as being a subunit of OMP decarboxylase encoded by pyrF. Moreover, the coding frame for this polypeptide seems to be expressed as the first gene in the operon with the coding frame for another protein chain of Mr 13 000-14 000. Since, however, the native OMP decarboxylase during sedimentation and gel filtration behaves as a protein of Mr 45 000 +/- 4000, this latter polypeptide (Mr 13 000-14 000) is hardly a component of the enzyme. Pyr-lac+ operon fusions were constructed by the Mu d1 procedure. By integrating an F'lac episome into the lac part of the fusions and determining the direction of chromosomal transfer from the resultant Hfr strains, the direction of pyrC transcription was found to be counter-clockwise, while pyrD and pyrF were found to be transcribed in a clockwise direction.

Base Composition↗
PubMed2000

SOS mutator activity: unequal mutagenesis on leading and lagging strands.

A major pathway of mutagenesis in Escherichia coli is mediated by the inducible SOS response. Current models of SOS mutagenesis invoke the interaction of RecA and UmuD'(2)C proteins with a stalled DNA replication complex at sites of DNA lesions or poorly extendable terminal mismatches, resulting in an (error-prone) continuation of DNA synthesis. The precise mechanisms of SOS-mediated lesion bypass or mismatch extension are not known. Here, we have studied mutagenesis on the E. coli chromosome in recA730 strains. In recA730 strains, the SOS system is expressed constitutively, resulting in a spontaneous mutator effect (SOS mutator) because of reduced replication fidelity. We investigated whether during SOS mutator activity replication fidelity might be altered differentially in the leading and lagging strand of replication. Pairs of recA730 strains were constructed differing in the orientation of the lac operon relative to the origin of replication. The strains were also mismatch-repair defective (mutL) to facilitate scoring of replication errors. Within each pair, a given lac sequence is replicated by the leading-strand machinery in one orientation and by the lagging-strand machinery in the other orientation. Measurements of defined lac mutant frequencies in such pairs revealed large differences between the two orientations. Furthermore, in all cases, the frequency bias was the opposite of that seen in normal cells. We suggest that, for the lacZ target used in this study, SOS mutator activity operates with very different efficiency in the two strands. Specifically, the lagging strand of replication appears most susceptible to the SOS mutator effect.

Alleles↗
PubMed1990

Chemical synthesis and characteristics of a hybrid phage T5-lac promoter.

A new P1O1R9 inducible promoter (where P1 is a promoter sequence analogous to that of the phage T5 early promoter; O1 is lac-operator; and R9 is a ribosome binding site) was synthesized. We studied the efficiency of this promoter in controlling and inducible gene expression using two model genes: human calcitonin tetrameric (hCT[4]) and human interferon alpha 1 (hIFN alpha 1). The synthetic lac-operator O1 was found to repress P1 activity in media free of lac-operon inducers which was derepressed in the presence of IPTG. The levels of expression of both genes (evaluated by quantitating mRNA and protein) in the presence of lac-inducers were close to those obtained with the P1R9 constitutive promoter.

Base Sequence↗
PubMed1992

Leucine-responsive regulatory protein controls the expression of both the pap and fan pili operons in Escherichia coli.

The methylation blocking factor gene (mbf) in Escherichia coli is required for specific methylation inhibition of two DNA GATC sites upstream of the papBA pilin promoter and transcriptional activation of pap. Complementation and mutational analysis using pap-lac and ilvIH-lac operon fusions indicates that the mbf gene is identical to a recently described global regulatory gene lrp (leucine-responsive regulatory protein) that acts as a positive regulator of some genes and a negative regulator of others in E. coli. DNA sequence analysis of an mbf::mTn10 insertion showed that the mbfDNA sequence was identical to lrp. Thus Lrp inhibits DNA methylation at specific GATC sites. We also show that Lrp positively regulates transcription of the fan operon, which encodes K99 pili of diarrheagenic E. coli. Purified Lrp was found to bind to DNA fragments encompassing the pap and fan promoters, which is consistent with previous results indicating that Lrp controls gene expression by binding to regulatory DNA sites. Exogenous leucine significantly reduced fan transcription and K99 pili expression, similar to results obtained with the ilvIH operon. However, pap gene expression was unresponsive to leucine, which distinguishes pap from other lrp-regulated genes whose expression is modulated by leucine.

Amino Acid Sequence↗
PubMed2003

Evolution of the bacterial species Lactobacillus delbrueckii: a partial genomic study with reflections on prokaryotic species concept.

The species Lactobacillus delbrueckii consists at present of three subspecies, delbrueckii, lactis and bulgaricus, showing a high level of DNA-DNA hybridization similarity but presenting markedly different traits related to distinct ecological adaptation. The internal genetic heterogeneity of the bacterial species L. delbrueckii was analyzed. Phenotypic and several genetic traits were investigated for 61 strains belonging to this species. These included 16S rDNA sequence mutations, expression of beta-galactosidase and of the cell wall-anchored protease, the characterization of the lactose operon locus and of the sequence of lacR gene, galactose metabolism, and the distribution of insertion sequences. The high genetic heterogeneity of taxa was confirmed by every trait investigated: the lac operon was completely deleted in the subsp. delbrueckii, different mutation events in the repressor gene of the operon led to a constitutive expression of lacZ in the subsp. bulgaricus. Structural differences in the same genetic locus were probably due to the presence of different IS elements in the flanking regions. The different expression of the cell wall-anchored protease, constitutive in the subsp. bulgaricus, inducible in the subsp. lactis, and absent in the subsp. delbrueckii was also a consequence of mutations at the gene level. The galT gene for galactose metabolism was found only in the subsp. lactis, while no specific amplification product was detected in the other two subspecies. All these data, together with the absence of a specific IS element, ISL6, from the major number of strains belonging to the subsp. bulgaricus, confirmed a deep internal heterogeneity among the three subspecies. Moreover, this evidence and the directional mutations found in the 16S rDNA sequences suggested that, of the three subspecies, L. delbrueckii subsp. lactis is the taxon closer to the ancestor. Limitations of the current prokaryotic species definition were also discussed, based on presented evidences. Our results indicate the need for an accurate investigation of internal heterogeneity of bacterial species. This study has consequences on the prokaryotic species concept, since genomic flexibility of prokaryotes collides with a stable classification, necessary from a scientific and applied point of view.

Amino Acid Sequence↗
PubMed1979

Transcriptional regulation of Escherichia coli K-12 major outer membrane protein 1b.

Eleven independent insertion mutations were isolated that prevented expression of major outer membrane protein 1b. Seven of the mutations were Mucts insertions located at ombP. These ompB::Mucts strains fell into two phenotypic classes with regard to expression of proteins 1a and 1b. The remaining four mutants were comprised of one Tn5 and three Mucts insertions mapping at par. The Mucts insertions at par were used to construct fusions of the lac operon to the par promoter. Expression of beta-galactosidase in these fusion strains reflected known regulatory properties of protein 1b. When an ompB allele was introduced into the par-lac fusion strains, beta-galactosidase activity was reduced 14- to 31-fold. Transcriptional regulation of the par gene and the existence of two functions at ompB are discussed. The results suggest that par is the structural gene for protein 1b and that an ompB gene product is a diffusible, positive regulatory element controlling expression of par.

Bacterial Proteins↗
PubMed1990

Characterization of the lactose-specific enzymes of the phosphotransferase system in Lactococcus lactis.

The plasmid-encoded lactose genes of the Lactococcus lactis phosphotransferase system encoding Enzyme IIIlac (lacF) and Enzyme IIlac (lacE) have been identified and cloned in Escherichia coli and L. lactis. Nucleotide sequence and transcription analysis showed that these genes are organized into a lactose-inducible operon with the gene order lacF-lacE-lacG-lacX, the latter two genes encoding phospho-beta-galactosidase and a 34-kDa protein with an unknown function, respectively. The lac-operon is immediately followed by an IS element that is homologous to ISS1. Enzyme IIIlac was purified from L. lactis and determination of its NH2-terminal sequence demonstrated that the lacF gene starts with a TTG codon and encodes a 105 amino acid protein (Mr = 11416). Cross-linking studies with the purified enzyme showed that Enzyme IIIlac is active as a trimer. A mutant lacF gene was identified in strain YP2-5 and appeared to encode Enzyme IIIlac containing the missense mutation G18E. The lacF gene could be expressed under control of vector-located promoter sequences resulting in overproduction of Enzyme IIIlac in E. coli and complementation of the L. lactis lacF mutant YP2-5. The deduced amino acid sequence of Enzyme IIlac consists of 586 amino acids (Mr = 61562) and shows the characteristics of a hydrophobic, integral membrane protein. The deduced primary structures of the L. lactis Enzyme IIIlac and Enzyme IIlac are homologous to those of Staphylococcus aureus (72 and 71% identity, respectively) and Lactobacillus casei (48 and 47% identity, respectively). In contrast, the organization of the lactose genes differs significantly between those Gram-positive bacteria. Heterogramic homology in specific domains was observed between the derived amino acid sequences of the lactose-specific enzymes and that of E. coli Enzyme IIIcel and Enzyme IIcel, which suggest a common function in the transport and phosphorylation of these structurally related beta-glucosides.

Amino Acid Sequence↗
PubMed1978

Involvement of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system in regulation of transcription of catabolic genes.

Synthesis of catabolite-sensitive enzymes is repressed in mutants defective in the general proteins (enzyme I and HPr) of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system (ptsI and ptsH mutations). To elucidate the mechanism of this phenomenon we constructed isogenic strains carrying pts mutations as well as different lesions of regulation of the lac operon or mutations affecting adenylate cyclase activity (cya mutation) and synthesis of cyclic AMP-receptor protein (crp mutation) Measurements of the differential rate of beta-galactosidase synthesis in these strains showed that the repressive effect of pts mutations was revealed in lac+, lacI, lacOc and cya bacteria, but it was lost in lacP and crp strains. It was concluded that mutational damage to the general components of the phosphoenolpyruvate-dependent phosphotransferase system diminishes activity of the lac promoter. The results obtained led to the conclusion that pts gene products (apparently phospho approximately HPr) are necessary for the initiation of transcription of catabolite-sensitive operons in E. coli.

DNA-Directed RNA Polymerases↗
PubMed1989

Transposable elements for efficient manipulation of a wide range of gram-negative bacteria: promoter probes and vectors for foreign genes.

We describe here the construction and use of a series of modified transposons based on the insertion sequence IS1. Like their parent, omegon-Km [Fellay et al., Gene 76 (1989) 215-226], these elements permit efficient insertional mutagenesis of a variety of Gram-negative bacteria. The presence of a functional pBR322 origin of replication within the transposable element facilitates subsequent cloning of the mutated gene. The omegon-Km system was previously shown to function in Pseudomonas putida, Rhizobium leguminosarum and Paracoccus denitrificans. The results we present here demonstrate that its use can be extended to Xanthomonas campestris, a plant pathogen, and to the microaeroduric Zymomonas mobilis. Derivative transposons carrying unique restriction sites for ScaI, NdeI, XbaI and XhoI have been constructed, allowing the cloning and introduction of foreign genes. We have also constructed two derivatives which can be used to generate operon fusions upon insertion and are thus useful for isolating and characterising indigenous promoters. One carries a promoterless chloramphenicol acetyl-transferase (CAT)-encoding gene (cat) and the second, the entire promoterless Escherichia coli lac operon. We demonstrate the utility of the cat promoter probe in X. campestris to target conditional promoters inducible by high salt or subject to repression by glucose.

Base Sequence↗