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PubMed2000

Adaptive amplification: an inducible chromosomal instability mechanism.

Adaptive mutation is an induced response to environmental stress in which mutation rates rise, producing permanent genetic changes that can adapt cells to stress. This contrasts with neo-Darwinian views of genetic change rates blind to environmental conditions. DNA amplification is a flexible, reversible genomic change that has long been postulated to be adaptive. We report the discovery of adaptive amplification at the lac operon in Escherichia coli. Additionally, we find that adaptive amplification is separate from, and does not lead to, adaptive point mutation. This contradicts a prevailing alternative hypothesis whereby adaptive mutation is normal mutability in amplified DNA. Instead, adaptive mutation and amplification are parallel routes of inducible genetic instability allowing rapid evolution under stress, and escape from growth inhibition.

Base Pair Mismatch↗
PubMed1996

Identification of new Fis binding sites by DNA scission with Fis-1,10-phenanthroline-copper(I) chimeras.

The chimeric nuclease Fis-OP has been used to identify novel Fis binding sites. Tethering the chemical nuclease OP-Cu+ to position 73 of the protein with a newly developed longer acetyl-beta-alanylamino spacer has facilitated the localization of two high-affinity Fis binding sequences in a 3 kb pUC19 plasmid. The shorter acetamido linker has allowed the chimeric nuclease to locate two strong Fis binding sites in the 50 kb phage lambda genome. All four sites reside in biologically interesting loci and have been confirmed by gel-retardation and DNase I footprint analyses. A newly discovered site resides in the lac operon of Escherichia coli. The binding of Fis to this site may antagonize repression by the LacI repressor. These studies demonstrate the feasibility of applying chimeric chemical nucleases to the task of identifying functional protein binding sites of biological interest within genomes without any assumption about their sequence preference.

Bacteriophage lambda↗
PubMed2007

Analysis of the mechanism and regulation of lactose transport and metabolism in Clostridium acetobutylicum ATCC 824.

Although the acetone-butanol-ethanol fermentation of Clostridium acetobutylicum is currently uneconomic, the ability of the bacterium to metabolize a wide range of carbohydrates offers the potential for revival based on the use of cheap, low-grade substrates. We have investigated the uptake and metabolism of lactose, the major sugar in industrial whey waste, by C. acetobutylicum ATCC 824. Lactose is taken up via a phosphoenolpyruvate-dependent phosphotransferase system (PTS) comprising both soluble and membrane-associated components, and the resulting phosphorylated derivative is hydrolyzed by a phospho-beta-galactosidase. These activities are induced during growth on lactose but are absent in glucose-grown cells. Analysis of the C. acetobutylicum genome sequence identified a gene system, lacRFEG, encoding a transcriptional regulator of the DeoR family, IIA and IICB components of a lactose PTS, and phospho-beta-galactosidase. During growth in medium containing both glucose and lactose, C. acetobutylicum exhibited a classical diauxic growth, and the lac operon was not expressed until glucose was exhausted from the medium. The presence upstream of lacR of a potential catabolite responsive element (cre) encompassing the transcriptional start site is indicative of the mechanism of carbon catabolite repression characteristic of low-GC gram-positive bacteria. A pathway for the uptake and metabolism of lactose by this industrially important organism is proposed.

Amino Acid Sequence↗
PubMed1991

Improved broad-host-range lac-based plasmid vectors for the isolation and characterization of protein fusions in Pseudomonas aeruginosa.

Several new broad-host-range vectors for the construction of protein fusions to the Escherichia coli lacZ gene have been developed. In all of the constructs, a multiple cloning site (MCS) containing unique restriction sites is located upstream of lac operon segments whose lacZ genes lack translational start signals. Some of the vectors (pPZ10, pPZ20 and pPZ30) also contain transcriptional terminators upstream of the MCS. The new vectors allow the fusion of genes to lacZ in all translational reading frames. Due to a higher copy number they allow direct screening in E. coli for weakly expressed foreign promoters. Their usefulness for gene analysis in Pseudomonas aeruginosa was demonstrated by construction and expression of a regA'::'lacZ-encoded protein fusion.

Amino Acid Sequence↗
PubMed1978

Isolation and characterization of ColE1-derived plasmid copy-number mutant.

The plasmid pBGP120 is a ColE1 derivative that contains elements of the Escherichia coli lac operon and the Tn3 transposon. We have selected and isolated a copy-number mutant of pBGP120. In exponentially growing cultures, the copy-number mutant, pOP1, represents approximately 30% of total intracellular DNA compared to about 5% for pBGP120. Plasmid-encoded beta-galactosidase monomer can represent 50% of newly synthesized protein in cells carrying pOP1. pOP1 is structurally unstable in certain genetic backgrounds and under certain growth conditions, breaking down to a smaller sized plasmid that retains the DNA overproducer phenotype and the Tn3 transposon. The smaller overproducer plasmid, pOP1delta6, is generated by a continuous deletion of sequences located between one end of the Tn3 transposon and a site about 630 nucleotides from the EcoRI site in the beta-galactosidase structural gene of pOP1. pOP1delta6 retains the ColE1 origin of replication but has lost the lac promotor and operator and most of the beta-galactosidase structural gene. pOP1delta6 exists at approximately 210 copies per chromosome in exponentially growing cells.

Bacteriocin Plasmids↗
PubMed1985

Locus affecting regulation of the colicin I receptor by iron.

Using a strain containing a cir-lac operon fusion and a selective medium, we isolated a regulatory mutant of the colicin I receptor, which we have designated cirR. Cells carrying the cirR mutation were defective in the transcriptional regulation of cir by iron, but synthesis of other iron-regulated proteins was unaffected. cirR was found to be cis dominant. This is in contrast to previously described mutations in iron regulation which are pleiomorphic and trans dominant. Temperature regulation of colicin I receptor production was unaffected by cirR.

Bacterial Proteins↗
PubMed1991

Genetic and physical analyses of the growth rate-dependent regulation of Escherichia coli zwf expression.

Growth rate-dependent regulation of the level of Escherichia coli glucose 6-phosphate dehydrogenase, encoded by zwf, and 6-phosphogluconate dehydrogenase, encoded by gnd, is similar during steady-state growth and after nutritional upshifts. To determine whether the mechanism regulating zwf expression is like that of gnd, which involves a site of posttranscriptional control located within the structural gene, we prepared and analyzed a set of zwf-lacZ protein fusions in which the fusion joints are distributed across the glucose 6-phosphate dehydrogenase coding sequence. Expression of beta-galactosidase from the protein fusions was as growth rate dependent as that of glucose 6-phosphate dehydrogenase itself, indicating that regulation does not involve an internal regulatory region. The level of beta-galactosidase in zwf-lac operon fusion strains and the level of zwf mRNA from a wild-type strain increased with increasing growth rate, which suggests that growth rate control is exerted on the mRNA level. The half-life of the zwf mRNA mass was 3.0 min during growth on glucose and 3.4 min during growth on acetate. Thus, zwf transcription appears to be the target for growth rate control of the glucose 6-phosphate dehydrogenase level.

Base Sequence↗
PubMed2001

Identification of an intragenic integration site for foreign gene expression in recombinant Streptococcus gordonii strains.

A new intragenic chromosomal integration site within the lacG gene of the lac operon has been identified in Streptococcus gordonii for use in the expression of foreign genes. Introduction of a portion of the Streptococcus pyogenes emm6 gene into the lacG locus resulted in the lactose-inducible surface expression of the S. pyogenes M6 protein. This result demonstrates the ability to modulate the in vitro or in vivo expression of a foreign gene in a S. gordonii recombinant using a biosynthetic metabolite.

Antigens, Bacterial↗
PubMed1979

Lactose metabolism involving phospho-beta-galactosidase in Klebsiella.

Klebsiella strain RE1755A is a Lac- Gal- mutant which has lost both of its lac operons, but possesses a gene specifying beta-galactosidase III, an enzyme which hydrolyzes o-nitrophenyl-beta-D-galactopyranoside but does not hydrolyze lactose. Selective pressure was applied to isolate mutants able to utilize lactose. The lactose-utilizing mutants obtained were shown to possess an unaltered beta-galactosidase III. Lactose utilization was shown to result from a pleiotropic mutation which also (i) permits galactose utilization and (ii) prevents induction of beta-galactosidase III synthesis by lactose. Evidence is presented suggesting that a phospho-beta-galactosidase enzyme is involved in lactose metabolism.

Galactosephosphates↗
PubMed1988

Ethanolamine utilization in Salmonella typhimurium.

Ethanolamine can serve as the sole source of carbon and nitrogen for Salmonella typhimurium if vitamin B12 is present to serve as a cofactor. The pathway for ethanolamine utilization has been investigated in order to understand its regulation and determine whether the pathway is important to the selective forces that have maintained the ability to synthesize B12 in S. typhimurium. We isolated mutants that are defective in ethanolamine utilization (eut mutants). These mutants defined a cluster of genes located between purC and cysA at 50 min on the Salmonella chromosome. A genetic map of the eut region was constructed. Included in the map are mutations which affect ethanolamine ammonia lyase, the first degradative enzyme, and mutations which affect the second enzyme in the pathway, acetaldehyde dehydrogenase. Transcriptional regulation of the eut genes was studied by using eut-lac operon fusions created by insertion of Mu d lac. Transcription is induced by the simultaneous presence of ethanolamine and B12 in the growth medium. The eut genes constitute a single unit of transcription. One class of mutations located at the promoter-distal end of the eut operon prevent induction of transcription.

Ethanolamines↗
PubMed1970

Operon coordination in different bacterial hosts.

Coordination of gene expression in the lac operon was compared in Escherichia coli and Salmonella typhimurium as an approach to detecting possible differences in protein synthesis or membrane structure between organisms. Either a wild-type F' lac pro(AB) episome or the same episome with a polar mutation in one of the lac genes was introduced into pro(-) derivatives of the two strains of bacteria. Activity assays showed that the beta-galactosidase levels were only slightly lower in the S. typhimurium cells than in E. coli cells, whereas the transacetylase levels were significantly higher in S. typhimurium for all of the lac markers tested. Galactoside transport activities were always comparable in the two strains of bacteria; this latter result indicates that the cell envelopes of E. coli and S. typhimurium do not differ sufficiently to affect the membrane-associated lac transport system. It was found, however, that the specific transport activity is very sensitive to culture age in both bacteria, and decreases rapidly in cultures past the mid-exponential phase of growth.

Bacterial Proteins↗
PubMed1993

The use of lac-type promoters in control analysis.

For control analysis, it is necessary to modulate the activity of an enzyme around its normal level and measure the changes in steady-state fluxes or concentrations. We describe an improved method for effecting the modulation, as elaborated for Escherichia coli. The chromosomal gene, encoding the enzyme of interest, is put under the control of a lacUV5 or a tacI promoter. The alternative use of the two promoters leads to an expression range which should make it suitable for the use in control analysis of many enzymes. The lacUV5 promoter should be used when the wild-type expression level is low, the tacI promoter when the latter is high. The endogenous lac operon is placed under the control of a second copy of the lacUV5 promoter and a lacY7am mutation (eliminating lactose permease, the transport system for the inducer isopropyl-thio-beta-D- galactoside) is introduced. The method was demonstrated experimentally by constructing E. coli strains, in which the chromosomal atp operon is transcribed from the lacUV5 and the tacI promoter. We measured the concentration of the c subunit of H(+)-ATPase, and found that the expression of this enzyme could be modulated between non-detectable levels and up to five times the wild-type level. Thus, in the absence of inducer, no expression of atp genes could be detected when the atp operon was controlled by the lacUV5 promoter, and we estimate that the expression was less than 0.0025 times the wild-type level. We show that the introduction of a lacY mutation facilitated the attainment of steady induction levels of partially induced cells. The mutation also reduced positive cooperativity in the dependence of expression on the concentration of isopropyl-thio-beta-D-galactoside (the inducer) and shifted the concentration of inducer needed for half maximum induction to higher values. These properties should facilitate the experimental modulation of the enzyme activity by varying the concentration of the inducer.

Cloning, Molecular↗
PubMed1978

The transposon Tn9 generates a 9 bp repeated sequence during integration.

We performed a genetic and sequencing analysis of insertions of the transposon Tn9 into the lac operon of E. coli. Genetic mapping of 70 insertions into lacl and Z shows that starting from the same point on the chromosome, Tn9 goes to at least 50 different points in these two genes. Although there are preferred regions for insertion, these consist of multiple integration points within a small area, as demonstrated by pairwise crosses and restriction mapping. Sequence analysis of three Tn9 insertions reveals that Tn9 integration is associated with a direct repeat of 9 base pairs (bp) of host sequence. We show that these extra 9 nucleotide pairs are generated upon insertion and not brought in with the element.

Base Sequence↗
PubMed1980

Improved methods for maximizing expression of a cloned gene: a bacterium that synthesizes rabbit beta-globin.

In this paper we describe a method for constructing E. coli plasmids that direct efficient expression of genes that encode eucaryotic or procaryotic proteins. No functional assays for the proteins are needed, and they are produced in their native, unfused state. The only requirement is that the genes be isolable without intervening sequences. We describe as an example the construction of a plasmid that directs the synthesis of about 10,000-15,000 monomers per cell of rabbit beta-globin. The essential steps in a typical construction are as follows. --A region of the gene encoding the amino-terminal portion of the protein is fused to DNA encoding an enzymatically active carboxy terminal fragment of beta-galactosidase. The latter is carried on one of three plasmids designed to facilitate the fusion (the construction of these three plasmids is described in the Appendix). --A "portable promoter" of the lac operon is placed at many positions in front of the fused gene using nucleases in vitro. Those promoter placements that elicit efficient expression of the fused gene are identified by the beta-galactosidase activity that they express. (In the special case we describe, plasmids identified as directing efficient expression of beta-globin were found to bear "hybrid" ribosome binding sites consisting of the Shine-Dalgarno sequence carried on the promoter fragment and the ATG of the beta-globin gene.) --The gene of interest is reconstituted intact, with the portable promoter in place, by recombination in vitro or in vivo.

Animals↗
PubMed1977

A third beta-galactosidase in a strain of Klebsiella that possesses two lac genes.

Klebsiella strain RE1544 contains two lac operons, one on the chromosome and one on a lac plasmid. A mutant of RE1544, in which the lacZ genes of both operons produce no active enzyme, was found to synthesize a beta-galactosidase that hydrolyzes ortho-nitrophenyl-beta-D-galactopyranoside but not lactose. Synthesis of this beta-galactosidase (BGase-III) is induced by lactose but not by isopropyl-1-thio-beta-D-galactopyranoside or methyl-beta-D-thiogalactopyranoside. In both the regulation of synthesis and substrate specificity, BGase-III strongly resembles the ebg0 enzyme of Escherichia coli. Nevertheless, by the criteria of immunological cross-reactivity and subunit molecular weight, BGase-III is not related to the ebg0 enzyme.

Cross Reactions↗
PubMed1986

Nuclease activity of 1,10-phenanthroline-copper: sequence-specific targeting.

The nuclease activity of 1,10-phenanthroline-copper ion can be targeted to specific DNA sequences by attachment of the ligand to the 5' end of complementary deoxyoligonucleotides via a phosphoramidate linkage. To synthesize the adduct, the phosphorimidazolide of the deoxyoligonucleotide is prepared using a water-soluble carbodiimide and is then coupled to 5-glycylamido-1,10-phenanthroline. After hybridization to the target DNA, sequence-specific cleavage is observed upon the addition of cupric ion and 3-mercaptopropionic acid. Two methods of assaying the cutting of the operator sequence of the lac operon have been employed using the oligonucleotide 5'-AATTGTTATCCGCTCACAATT-3' representing sequence positions 21-1 of the template strand. In the first, the single-stranded DNA of the phage M13mp8 was the target, and cuts were detected using a primer-extension assay. In the second, the substrate was an EcoRI fragment 3' labeled in the nontemplate strand. After denaturation and reannealing to the oligonucleotide-1,10-phenanthroline adduct, cupric ion and 3-mercaptopropionic acid were added, and the products were analyzed directly on a sequencing gel. With the phenanthroline moiety attached to position 21 of the oligonucleotide carrier, cutting was observed at positions 20-25 using both assays.

Base Sequence↗
PubMed1996

Expression of the tolQRA genes of Escherichia coli K-12 is controlled by the RcsC sensor protein involved in capsule synthesis.

The tolQRABpal cluster of Escherichia coli K-12 encodes proteins involved in the maintenance of cell-envelope integrity. In addition, tol/pal mutations result in a mucoid colony phenotype at low temperature. The synthesis of capsular polysaccharides by the cps genes is controlled by the positive regulator RcsA and the two-component RcsC/RcsB system. It was shown that the mucoid phenotype of the tol/pal mutants was due to an rcsCB-dependent activation of the cps genes. Furthermore, we have identified a mutation in the rcsC gene that decreased the activity of a tolA-lac operon fusion independently of RcsA and partially independently of RcsB activators. The corresponding rcsC338 mutation resulted in a Glu to Lys substitution at residue 338 of RcsC. This mutation induced mucoidy even at high temperature. We propose that RcsC modulates the phosphorylated forms of RcsB and an uncharacterized regulatory protein involved in the control of the tolQRA genes in an opposite manner. Moreover, our findings strengthen the previous suggestion that RcsC senses some alterations in the cell surface such as those induced by tol, pal or rfa mutations, and activates capsule synthesis to protect the cell against deleterious agents.

Alleles↗
PubMed1982

Expression of transforming region of Moloney murine sarcoma virus in Escherichia coli as a fusion protein with small tumor antigen of polyoma virus.

Bacterial expression of the transforming region of Moloney murine sarcoma virus, designated mos, was obtained as a fusion protein with a portion of the small tumor antigen of polyoma virus. This was accomplished by fusing the entire mos open reading frame, encoding a 41,000-dalton protein, with a plasmid that expresses a beta-galactosidase-polyoma fusion protein under lac operon control. The resulting plasmid directed synthesis of the predicted polyoma antigen-sarcoma virus fusion protein of 59,000 daltons. This protein was immunoprecipitated by an anti-polyoma tumor antigen antiserum that recognized polyoma determinants at the NH2 terminus of the hybrid protein. This protein was also immunoprecipitated by an antiserum directed against a synthetic peptide containing the 12 COOH-terminal amino acids encoded by the mos open reading frame. This work confirms the existence of a long open reading frame in the mos gene and resolves a discrepancy between different nucleotide sequences for its COOH-terminal coding region.

Animals↗