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At least 757 records · Page 42Linked to original sources
PubMed1984

Lactose inhibits the growth of Rhizobium meliloti cells that contain an actively expressed Escherichia coli lactose operon.

Expression of the Escherichia coli lactose operon in Rhizobium meliloti 104A14 made the cells sensitive to the addition of the beta-galactosides lactose, phenyl-beta-D-galactoside, and lactobionic acid. Growth stopped when the beta-galactoside was added and viability decreased modestly during the next few hours, but little cell lysis was observed and the cells appeared normal. Protein synthesis was not inhibited. Growth was inhibited only when beta-galactosidase expression was greater than 160 U. Lactose-resistant mutants had defects in the plasmid-carried E. coli beta-galactosidase or beta-galactoside permease and in the R. meliloti genome. We speculate that uncontrolled production of galactose by the action of the lactose operon proteins was responsible for growth inhibition.

Escherichia coli↗
PubMed2001

N-terminal truncations in the FhlA protein result in formate- and MoeA-independent expression of the hyc (formate hydrogenlyase) operon of Escherichia coli.

The formate hydrogenlyase complex of Escherichia coli catalyses the cleavage of formate to CO2 and H2 and consists of a molybdoenzyme formate dehydrogenase-H, hydrogenase 3 and intermediate electron carriers. The structural genes of this enzyme complex are activated by the FhlA protein in the presence of both formate and molybdate; ModE-Mo serves as a secondary activator. Mutational analysis of the FhlA protein established that the unique N-terminal region of this protein was responsible for formate- and molybdenum-dependent transcriptional control of the hyc operon. Analysis of the N-terminal sequence of the FhlA protein revealed a unique motif (amino acids 7-37), which is also found in ATPases associated with several members of the ABC-type transporter family. A deletion derivative of FhlA lacking these amino acids (FhlA9-2) failed to activate the hyc operon in vivo, although the FhlA9-2 did bind to hyc promoter DNA in vitro. The ATPase activity of the FhlA9-2-DNA-formate complex was at least three times higher than that of the native protein-DNA-formate complex, and this degree of activity was achieved at a lower formate level. Extending the deletion to amino acid 117 (FhlA167) not only reversed the FhlA(-) phenotype of FhlA9-2, but also led to both molybdenum- and formate-independence. Deleting the entire N-terminal domain (between amino acids 5 and 374 of the 692 amino acid protein) also led to an effector-independent transcriptional activator (FhlA165), which had a twofold higher level of hyc operon expression than the native protein. Both FhlA165 and FhlA167 still required ModE-Mo as a secondary activator for an optimal level of hyc-lac expression. The FhlA165 protein also had a twofold higher affinity to hyc promoter DNA than the native FhlA protein, while the FhlA167 protein had a significantly lower affinity for hyc promoter DNA in vitro. Although the ATPase activity of the native protein was increased by formate, the ATPase activity of neither FhlA165 or FhlA167 responded to formate. Removal of the first 117 amino acids of the FhlA protein appears to result in a constitutive, effector-independent activation of transcription of the genes encoding the components of the formate hydrogenlyase complex. The sequence similarity to ABC-ATPases, combined with the properties of the FhlA deletion proteins, led to the proposal that the N-terminal region of the native FhlA protein interacts with formate transport proteins, both as a formate transport facilitator and as a cytoplasmic acceptor.

ATP-Binding Cassette Transporters↗
PubMed1983

Mutations in the promoter regions of the malEFG and malK-lamB operons of Escherichia coli K12.

The malB region of Escherichia coli is composed of two operons, malEFG and malK-lamB, transcribed divergently from a control region located between the malE and malK genes. Expression of the malB operons is under the positive control of the malT gene product (MalT) and maltose and of the crp gene product (CRP) and cyclic AMP. Strains in which the lac genes have been fused to malE or malK are unable to use lactose as carbon source if they have been deleted for malT or crp. Mutations in the malB region allowing such fusion strains to grow on lactose have been isolated. These and previously isolated mutations were genetically characterized. As regards the malEp promoter mutations, malEp9, malEp1 and malEp6 create new promoters that are MalT and CRP independent. malEp9 and malEp1 change residues -1 and -2, respectively, of malEp without altering its activity. malEp6 duplicates six base-pairs between residues -22 and -23. malEp3 improves the -10 region hexamer. malEp5 deletes residues -29 to -62. It creates a new promoter that is MalT independent, CRP dependent, likely by fusing together functional regions of malEp that are normally apart. malEp5 also reduces the expression of malK-lamB, suggesting the existence of a link between the malEp and malKp promoters. As regards the malKp mutations, malKp6 changes residue -81 of malKp without altering its activity. It creates a new promoter, which is MalT independent, CRP dependent, likely by using a pre-existing cyclic AMP/CRP binding site. malKp102 changes residue -36, two bases upstream of the -35 region hexamer. It decreases the activity of malKp by at least four orders of magnitude and likely alters the MalT binding site. These results are discussed in terms of regulatory interactions within the malB control region.

Base Sequence↗
PubMed1993

Expression of the Escherichia coli ftsZ gene: trials and tribulations of gene fusion studies.

The ftsZ gene of Escherichia coli, which codes for an essential cell division protein, is subjected to multiple regulation, as shown in part with studies using an ftsZ::lacZ operon fusion located on phage lambda JFL100. Using this same fusion, we sought to isolate regulatory mutants overexpressing ftsZ by selecting mutants able to grow on lactose. One Lac+ mutant was obtained which overexpressed the ftsZ::lacZ fusion 70-fold. The mutation responsible for the overexpression lies in a new gene, cot, located near 56 min on the E. coli genetic map. The cot mutation probably affects the transcription of a chromosomal open reading frame, ORF1, lying downstream of the bioA gene and adjacent to the ftzZ::lacZ fusion of the lambda JFL100 prophage integrated at att lambda. Using an ftsZ84(Ts) strain, in which there was a double selection for overexpression of both ftsZ::lacZ and ftsZ+, no Lac+Tr mutants were obtained from 3.6 x 10(10) bacteria; the introduction of a mutL allele, increasing spontaneous base substitution mutation rates 75-fold, did not permit us to isolate such a mutant. We conclude that Lac+ ftsZ-constitutive mutations cannot be obtained in lambda JFL100 lysogens by a single base substitution.

Bacterial Proteins↗
PubMed1981

A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system.

The use of gel electrophoresis for quantitative studies of DNA-protein interactions is described. This rapid and simple technique involves separation of free DNA from DNA-protein complexes based on differences in their electrophoretic mobilities in polyacrylamide gels. Under favorable conditions both unbound DNA and DNA associated with protein can be quantified. This gel method is applied to the study of the E. coli lactose operon regulatory system. At ionic strengths in the physiological range, the catabolite activator protein (CAP) is shown to form a long-lived complex with the wild type lac promotor, but not with a CAP-insensitive mutant. Formation of a stable "open" or "melted-in" complex of RNA polymerase with the wild type promoter requires the participation of CAP and cyclic AMP. Further, it is demonstrated that even when pre-formed in the presence of CAP-cAMP, the polymerase-promoter open complex becomes unstable if CAP is then selectively removed.

Carrier Proteins↗
PubMed1998

[Cloning and analysis of the regulatory gene fragment induced with seed extract in Rhizobium huakuii strain 7653R].

The large plasmids of strain 7653R were digested with restriction enzyme EcoRI. Their DNA fragments were cloned into the expression vector pMP220 to construct a lacZ fusion pool, which were transferred into the recipient strain 7653R. Tri-transconjugants were selected onto plates containing X-gal and seed extract. Five blue colonies were assayed of their beta-galactosidase activity after incubation with or without seed extract. A positive induced strain HN18 was obtained. Hybridization of nodDABC probe on the recombinant plasmid pHN18 showed a 1.7 kb positive band. The evidence makes a deduction that the pHN18 contains a promoter of nod operon.

Cloning, Molecular↗
PubMed1995

Construction of a food-grade host/vector system for Lactococcus lactis based on the lactose operon.

A plasmid-based food-grade vector system was developed for Lactococcus lactis by exploiting the genes for lactose metabolism. L. lactis MG5267 is a plasmid-free strain containing the entire lactose operon as a chromosomal insertion. The lacF gene was deleted from this strain by a double cross-over homologous recombination event. The lacF-deficient strain produced a Lac- phenotype on indicator agar. A cloned copy of the lacF gene expressed on a plasmid was capable of complementing the lacF-deficient strain resulting in a Lac+ phenotype. This stably maintained system fits the requirements of a self-selecting vector system and has the potential to be exploited in the food industry.

Base Sequence↗
PubMed1989

The use of lambda plac-Mu hybrid phages in Klebsiella pneumoniae and the isolation of stable Hfr strains.

Klebsiella pneumoniae 1033-5P14 and its P1-sensitive derivative KAY2026 were found to be resistant to lambda although they contained a LamB protein, active as a maltoporin. Sensitive derivatives could only be obtained after introduction of the pTROY9 plasmid which expresses lamB and the corresponding lambda receptor from Escherichia coli K12 at high levels. Lysogenic derivatives from such strains were shown to carry the phage at secondary att sites and to give high titer lysates when induced. The use of lambda plac-Mu hybrid phages allowed the isolation from several operons of lacZ fusions orientated in, or against, the direction of transcription. Such insertions could subsequently be used to isolate stable Hfr strains by allowing homologous recombination to take place between the lac genes in the inserted hybrid phages and those of plasmid F' ts114 lac+ zzf20::Tn10. The Hfr strains were able to transfer K. pneumoniae chromosomal genes and allowed the mapping of such genes. Characteristic differences between this conjugation system and that of Escherichia coli K12 are discussed. The insertions also allowed determination of the direction of transcription of the gut gene, the newly mapped scr gene and of the sor gene cluster encoding enzymes for the metabolism of D-glucitol, sucrose and L-sorbose.

Bacteriophage lambda↗
PubMed1997

Development of a bifunctional xylosidase/arabinosidase gene as a reporter gene for the gram-negative anaerobes Bacteroides and Porphyromonas, and Escherichia coli.

Members of the genera Bacteroides and Porphyromonas are common inhabitants of the human intestinal and oral microflora, and certain species are capable of causing disease states in humans. Genetic studies of these organisms are important for determining factors involved in the development of such diseases. A reporter gene for transcriptional fusions may prove useful for studies of gene regulation in these organisms. Bacteroides ovatus is a normal inhabitant of the human intestinal tract and is one of the few Bacteroides species capable of degrading xylan, a major component of fiber in the diet. A gene encoding for a bifunctional xylosidase/arabinosidase (XA) enzyme was previously cloned in our laboratory from B. ovatus V975 as part of a xylan-inducible operon. The XA gene was isolated by polymerase chain reaction and subcloned into the E. coli plasmid pBluescript II KS+. The XA gene is under transcriptional regulation in E. coli by the lac promoter, and both activities can be induced with isopropylthio-beta-galactoside (IPTG). The XA gene was subcloned into E. coli/Bacteroides shuttle vectors and introduced by conjugation into different Bacteroides species and Porphyromonas gingivalis. The results of transcriptional fusions in Bacteroides species and E. coli were evaluated. The characteristics of the XA reporter system are the low background or total lack of arabinosidase and xylosidase activities in most Bacteroides species, P. gingivalis, and E. coli, and the ease of enzymatic assays. In addition, bacterial colonies can be screened directly on agar plates by fluorescence with methylumbelliferyl derivatives as substrates for either enzymatic activity.

Artificial Gene Fusion↗