On the mode of selection in escherichia coli

Selection experiments between a lac ⁺ wild type and a lac ^– mutant of Escherichia coli were conducted using a serial transfer prcedure at two levels of total sugar concentration, i.e., 0.750 g/l and 1.0 g/l. The relative frequencies of both genotypes were assayed every 48 hours, at the time of transfer and the frequency changes were followed for about four weeks. The frequency of lac ^– decreased steadily and it was eventually eliminated from the mixed populations in all replicate cultures. The regression of logit p_t was shown to be not linear but curvilinear, indicating that fitness differentials are frequency-dependent. The selection is also probably density-dependent.     

The interaction of an I-like R factor and transfer-deficient mutants of Flac in E. coli K 12

Summary Strains carrying an I-like R factor, R64, or its derepressed derivative, R64-11, together with an Flac episome mutant in one of ten cistrons determining transfer-proficiency, transferred the Flac mutant at a frequency equivalent to about 1% of the level of R factor transfer. Similarly, R64, R64-11 and transfer-deficient mutants of R64-11, were transferred at increased frequencies in the presence of wild-type Flac. Experiments using RecA^– strains showed that mobilisation by recA ⁺-promoted recombination was not involved, and others using strains carrying transfer-deficient mutants of both R64-11 and Flac suggested that even inefficient complementation between R64-11 and Flac transfer mutants did not occur. The transfer systems of the two plasmids seemed, therefore, to be unrelated, and plasmid-specific, although at a low frequency the entire transfer system of one, not just the pilus, could transfer a transfer-deficient mutant of the other.     

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.     

Growth and reactivation of single stranded DNA phage phiX174 in E. coli undergoing "thymineless death".

Summary Flac maintenance was aberrant at permissive temperature in a temperature-sensitive dnaC mutant of Salmonella typhimurium when the normally resident pLT2 plasmid was present. Flac was, however, efficiently transferred into the dnaC pLT2⁺ strain and the resulting Flac derivative was almost as efficient in transferring Flac as were dnaC ⁺ pLT2⁺ Flac strains indicating that aberrant Flac maintenance was not associated with appreciable inhibition of transfer replication. A range of F-like plasmids behaved like pLT2 in causing aberrant Flac maintenance when present in the dnaC pLT2^- strain. Flac was, however, stably maintained in the dnaC strain in the absence of other plasmids. Although the F-like plasmids destabilized Flac, each was stably maintained when introduced into strain 11G dnaC pLT2⁺ and pLT2 was also apparently stable under these conditions. The destabilizing effect of pLT2 and other fi ⁺ plasmids was not consequent upon their inhibiting the formation of a repressible F transfer component needed for Flac replication in the dnaC strain. Incompatibility between Flac and the other plasmids induced by the dnaC lesion also appeared unlikely to be a cause of the aberrant Flac maintenance. The possibility is discussed that the initiation of Flac replication differs from that of pLT2 and the F-like plasmids with F competing less effectively than the others for the DnaC gene product.     

Selection against homozygotes in the ADH polymorphic system of Drosophila melanogaster associated with the lethal factor l(2) Stm

In the natural populations ⁺Tüb, ⁺Prov, and ⁺Rov, similar Adh ^F allele frequencies occur (q _F=0.11, 0.18, and 0.08, respectively). However, there is a discrepancy in that the Adh ^F allele in ⁺Tüb is closely linked to the lethal factor 1(2)Stm, which reduces relative fitness of the F phenotype to zero. In spite of this, polymorphism is maintained also in ⁺Tüb, because the heterozygotes are superior to the homozygous S type (relative fitness=0.88). Under laboratory culture conditions, in ⁺Tüb the relative fitness of the S genotype further decreases to 0.6. After outcrossing the lethal factor, relative fitnesses for S, FS, and F become 0.6, 1, and 0.48, respectively, implying that fitness for S remains the same. Relative values for S, FS, and F in ⁺Prov, not affected by the lethal factor, are calculated by the maximum average fitness method to be 1, 1.2, and 0.2 under the assumption that heterozygous FS are similarly superior to S as in the natural ⁺Tüb population and all allele frequencies found are stable equilibrium values.     

NMR Investigation of Palindromic LAC Operator DNA Sequences of Varying Size: Influences of Complex Formation with the LAC Repressor Headpiece on the DNA 1H Resonances

Abstract

¹H-NMR studies on s3rmmetrical lac operators were performed to determine the minimum size of a lac operator for tight complex formation with the lac repressor headpiece. Four operators of varying size from 18 base pairs to 24 base pairs were chemically synthesized. The data obtained suggest that for a synthetic lac operator to form a tight complex with the lac headpiece it should be at least 22 base pairs long.     

Transformation of Azospirillum brasilense Cd with an ACC deaminase gene from enterobacter cloacae UW4 fused to the Tet r gene promoter improves its fitness and plant growth promoting ability

It has been reported that PGPB, containing ACC deaminase, can cleave the plant ethylene precursor ACC and thereby lower ethylene concentration in a developing or stressed plant, protecting it against the deleterious effects of stress ethylene and facilitating the formation of longer roots. In a previous work we have demonstrated expression of the ACC deaminase gene (acdS) from Enterobacter cloacae UW4 under the control of the lac promoter in Azospirillum brasilense Cd. With the inference that a construct including the ACC deaminase gene under the control of a constitutive promoter weaker than the lac promoter might impose less metabolic load on Azospirillum and improve its fitness, it was decided to clone acdS under the control of a tetracycline resistance gene promoter. The ACC deaminase structural gene was fused to the Tet ^r gene promoter by overlap extension using PCR, cloned in pRK415, and transferred into A. brasilense Cd. The resulting transformants showed lower ACC deaminase activity than those with the lac promoter controlled acdS gene. However, acdS under the control of the Tet ^r gene promoter imposed lesser metabolic load on Azospirillum brasilense Cd. The result was significantly increased IAA synthesis and greater bacterial growth rate, as well as increased ability to survive on the surface of tomato leaves and to promote the growth of tomato seedlings.     

The DNA gyrase of Escherichia coli participates in the formation of a spontaneous deletion by recA-independent recombination in vivo

Summary A system for detecting a spontaneous deletion in Escherichia coli was developed and the role of DNA gyrase in deletion formation was studied. A derivative of plac5, AM36, was isolated in which whole pBR322 DNA was inserted in the lacZ gene and 227 by of the lac gene duplicated at both sides of the pBR322 DNA. E. coli lac ^–strains lysogenized by AM36 had a Lac^– phenotype and segregated Lac^– revertants. Sequence analyses showed that the revertant was formed by a deletion that eliminated the inserted pBR322 DNA and one copy of the duplicated segments. The frequency of lac ^– revertant formation was independent of recA function, was increased by oxolinic acid, an inhibitor of DNA gyrase, but was not increased in a lysogen of a nalidixic acid-resistant derivative. The reversion frequencies of temperature sensitive mutants of gyrA gene are 10 to 100 times lower than that of the wild-type strain. These results indicate that the DNA gyrase of E. coli participated in the in vivo deletion formation resulting from the direct repeats.     

The Effects of Genotypic Frequency and Population Density on Fitness Differentials in ESCHERICHIA COLI

Two strains of Escherichia coli K-12, a lac ⁺ wild type and a lac^- auxotroph, were grown both as pure and mixed cultures, using a serial transfer procedure. Four different growth media were employed, consisting of the same minimal salts solution, but different total concentrations of the sugars lactose, arabinose, and glucose (in proportions 5:4:1). Population densities and genotypic frequencies were assayed every 48 hours, at the time of transfer. Population density of the pure lac⁺ culture was greater than that of the pure lac^- culture for all media; this was expected, since the latter cannot utilize lactose. Mixed cultures quickly approached the same density as the corresponding lac⁺ controls, and the frequency of the lac⁺ genotype increased steadily for all media. Trajectories of Λ = log (P ÷ Q) were strictly nonlinear, indicating a dependence of the selective differential on population density and genotypic frequency. The rate of substitution decreased slightly with increasing sugar concentration, contrary to theoretical expectation. It was speculated that either the generation interval was longer for denser cultures (higher substrate concentrations) or that buildup of organic by-products reduced the selective differential in denser cultures. For a single medium, however, the behavior of competing genotypic strains was reasonably well predicted by theoretical models of frequency and density-dependent selection, the parameters of which may be related to the experimental inputs.     

Recombination between an Flac and a mini-FKm plasmid deleted for an origin of replication

The mini-F plasmid specifying resistance to kanamycin (Km), pML31, contains an origin of replication at kilobase coordinate 42.6 in the F DNA sequences. In previous research we found that this origin could be deleted by recombinant DNA techniques without the loss of plasmid maintenance functions. In this report we show that the deleted plasmid, designated pMF21, has normal incompatibility properties and a recA⁺-dependent ability to form cointegrates with an Flac plasmid. By comparison, pML31 does not form cointegrates with the Flac plasmid at a detectable frequency. The frequency for spontaneous loss of the Lac⁺ phenotype in strains containing pMF21:Flac cointegrates resembles that of the Flac plasmid; however, in some Lac⁻ variants the Km^r phenotype is retained. Examination of the plasmid DNA in four of these Lac⁻Km^r clones revealed two with normal pMF21 plasmids and two with plasmids intermediate in size between pMF21 and the Flac.     

pHG276: A multiple cloning site pBR322 copy number vector expressing a functional lacα peptide from the bacteriophage lambda PR promoter

The bacteriophage lambda early promoter P_R has been used to direct the synthesis of lacα in a plasmid containing the multiple cloning site of pUC8. The copy number of the plasmid is controlled by the rop(rom) gene and the plasmid incorporates the cI⁸⁵⁷ gene for temperature regulation of lacα expression. Isolation of recombinant derivatives with DNA inserts in the multiple cloning region can be identified by insertional inactivation of lacα and consequently, a Lac⁻ phenotype in a host carrying the M15 deletion of lac. The potential of pHG276 as a fully regulated expression vector is examined.     

Two-Dimensional 1H, 15N NMR investigation of uniformly 15N-labeled Lac Repressor Headpiece

Abstract

¹⁵N uniformly labeled lac repressor and lac repressor headpiece were prepared. ¹⁵N NMR spectra of lac repressor were shown resolution inadequate for detailed study while the data showed that the ¹⁵N labeled N-terminal part of the protein is quite suitable for this type of study allowing future investigation of the specific interaction of the lac repressor headpiece with the lac operator. We report here the total assignment of proton ¹H and nitrogen ¹⁵NH backbone resonances of this headpiece in the free state. Assignments of the ¹⁵N resonances of the protein were obtained in a sequential manner using heteronuclear multiple quantum coherence (HMQC), relayed HMQC nuclear Overhauser and relayed HMQC-HOHAHA spectroscopy. More than 80 per cent of residues were assigned by their ¹⁵NH(i)-N¹H(i+1) and ¹⁵NH(i)-N¹(i-1) connectivities. Values of the ³J_NHα splitting for 39 of the 51 residues of the headpiece were extracted from HMQC and HMQC-J. The observed ¹⁵NH(i)-C^βH cross peaks and the ³J_NHα coupling constants values are in agreement with the three α-helices previously described [Zuiderweg, E.R.P., Scheek, R.M., Boelens, R., van Gunsteren, W.F. and Kaptein, R., Biochimie 67, 707 (1985)]. The ³J_NHα coupling constants can be now used for a more confident determination of the lac repressor headpiece. From these values it is shown that the geometry of the ends of the second and third α-helices exhibit deviation from the canonical α-helix structure. On the basis of NOEs and ³J_NHα values, the geometry of the turn of the helix-turn-helix motif is discussed.     

Cloning of a lac+, bamHI fragment into transposon Tn3 and transposition of the Tn3[lac] element

By use of recombinant DNA techniques, we have inserted the lac⁺ operon into a transposon (Tn3). We constructed the recombinant in such a way that the essential step in assaying for transposition consisted of screening for bacteria with a thermostable Lac⁺ phenotype. Our results showed that transposition of the Tn3[lac⁺] element occurred and that its frequency was derepressed compared to frequencies reported by others for wild-type Tn3 transposition.     

Isolation and characterization of a plaque-forming lac-transducing phage phi80plac with special reference to its integration and excision

Summary From a double lysogen for 80dlac type II (Beckwith and Signer, 1966) and 80, we isolated a plaque-forming lac-transducing coliphage 80plac after selecting a strain with a suitable deletion in the 80 prophage. The lac region of the phage is i ⁺ o ⁺ z ⁺ y ⁺ a ^- and supposed to be located between genes 15 (N) and imm (CI). The phage showed feckless phenotype indicating deletion of genes of the red system. The phage is also deleted for int or att function, and integrates exclusively at the host lac region, largely dependent on the host rec system. Excision of the prophage upon UV-irradiation or by mating the male lysogen with a non-lysogenic female was efficient and largely dependent on the host rec system. But a considerable amount of rec-independent excision was observed at least in the case of zygotic induction, which was not likely to be caused by int-xis, red or ter system of the phage. 80plac/o ^e phage was also isolated by incorporation of o ^e1 mutation from strain 2000o ^e.     

Selection procedure for deregulated iron transport mutants (fur) in Escherichia coli K 12: fur not only affects iron metabolism

Summary A selection procedure using Mn²⁺ is described. A high percentage of the Mn²⁺ resistant mutants had constitutive iron transport systems. By P1 transduction, and complementation with the cloned fur gene it could be shown that nearly all the mutants constitutive in the expression of the operon fusion fiu::placMu were only defective in fur. High concentrations of manganese inhibited the derepression of an iron-regulated lac operon fusion. In another iron-regulated lac operon fusion that was inducible by iron, manganese also induced the production of -galactosidase. Most of the fur mutants isolated (80%) were not able to grow on succinate, fumarate or acetate. After transformation with a fur ⁺ plasmid all 39 mutants tested were able to grow on succinate. In fur mutants the presence of succinate in the growth medium reduced succinate uptake rates by 50%–70%. Succinate dehydrogenase activity was reduced to 10% of that of the parent strain.     

The appearance of the UmuD'C protein complex in Escherichia coli switches repair from homologous recombination to SOS mutagenesis

The process of SOS mutagenesis in Escherichia coli requires (i) the replisome enzymes, (ii) RecA protein, and (iii) the formation of the UmuD'C protein complex which appears to help the replisome to resume DNA synthesis across a lesion. We found that the UmuD'C complex is an antagonist of RecA-mediated recombination. Homologous recombination in an Hfr x F^- cross decreased as a function of the UmuD'C cell concentration; this effect was challenged by increasing RecA concentration. Recombination of a u.v.-damaged F-lac with the lac gene of an F^- recipient was reduced by increasing the UmuD'C concentration while lac mutagenesis increased, showing an inverse relationship between recombination and SOS mutagenesis. We explain our data with the following model. The kinetics of appearance of the UmuD'C complex after DNA damage is slow, reaching a maximum after an hour. Within that period, excision and recombinational repair have had time to occur. When the UmuD'C concentration relative to the number of residual RecA filaments, not resolved by recombinational repair, becomes high enough, UmuD'C proteins provide a processive factor for the replisome to help replication bypass and repel the standing RecA filament. Thus, at a high enough concentration, the UmuD'C complex will switch repair from recombination to SOS mutagenesis.     

Metabolic burden in recombinant CHO cells: effect ofdhfr gene amplification andlacZ expression

Foreign protein production levels in two recombinant Chinese hamster ovary (CHO) cell lines were compared in cells transfected with different expression vectors. One vector pNL1 contained the gene for neomycin resistance (neo ^r ) and thelacZ gene which codes for intracellular -galactosidase, with both genes controlled by the constitutive simian virus (SV40) promoter. The other vector CDG contained the amplifiabledhfr gene andlacZ gene, controlled by the constitutive SV40 and cytomegalovirus (CMV) promoters, respectively. Cell growth and -galactosidase expression were compared quantitatively after cells were selected in different concentrations of the neomycin analog G418 and methotrexate, respectively. A 62% reduction in growth rate occurred in recombinant CHO cells in which thelacZ anddhfr genes were highly amplified and expressed. In contrast, the combined effects of the unamplifiedneo ^r gene andlacZ gene expression on the growth kinetics were small. Any metabolic burden caused bylacZ gene expression, which was evaluated separately from the effect ofneo ^r gene expression, must be negligible, as higher expression of -galactosidase (1.5×10^–6 units/cell) occurred in unamplified cells compared to the cells in whichlacZ was amplified by thedhfr-containing vector (3×10^–7 units/cell). Thus, the main factor causing severe growth reduction (metabolic burden) in cells containing the amplifieddhfr gene system was not overexpression of -galactosidase butdhfr andlacZ gene co-amplification anddhfr gene expression.     

Reversion and immobilization of phage Mud1 cts (Apr lac) insertion mutations in Salmonella typhimurium

Summary Phage Mud1 cts (Ap^r lac), or Mud1, insertion mutations may be accompanied by adjacent deletion formation which can complicate use of lac fusions generated with this phage for gene regulatory studies. As for phage Mu insertion mutations, phage Mud1 insertions fail to revert at significant frequency (whether or not accompanied by an adjacent deletion). We describe isolation of revertible (X mutant) derivatives of phage Mud1 in Salmonella typhimurium. The X mutant derivatives allow use of reversion as a simple test to determine whether a Mud1 insertion has occurred precisely without an adjacent deletion that may have fused the lac genes to a promoter outside of the gene of interest. In addition, a simple method for stabilizing Mud1 generated lac fusions against subsequent transposition is described.