Cycling expression and cooperative operator interaction in the trp operon of Escherichia coli

Oscillatory behaviour in the tryptophan operon of an Escherichia coli mutant strain lacking the enzyme-inhibition regulatory mechanism has been observed by Bliss et al. but not confirmed by others. This behaviour could be important from the standpoint of synthetic biology, whose goals include the engineering of intracellular genetic oscillators. This work is devoted to investigating, from a mathematical modelling point of view, the possibility that the trp operon of the E. coli inhibition-free strain expresses cyclically. For that we extend a previously introduced model for the regulatory pathway of the tryptophan operon in Escherichia coli to account for the observed multiplicity and cooperativity of repressor binding sites. Thereafter we investigate the model dynamics using deterministic numeric solutions, stochastic simulations, and analytic studies. Our results suggest that a quasi-periodic behaviour could be observed in the trp operon expression level of single bacteria.     

Mutants in position 69 of the Trp repressor ofEscherichia coli K12 with altered DNA-binding specificity

Structural analysis by X-ray crystallography has indicated that direct contact occurs between Arg69, the second residue of the first helix of the helix-turn-helix (HTH) motif of the Trp repressor, and guanine in position 9 of the α-centred consensustrp operator. We therefore replaced residue 69 of the Trp repressor with Gly, Ile, Leu or Gln and tested the resultant repressor mutants for their binding to synthetic symmetrical α-or β-centredtrp operator variants, in vivo and in vitro. We present genetic and biochemical evidence that Ile in position 69 of the Trp repressor interacts specifically with thymine in position 9 of the α-centredtrp operator. There are also interactions with other bases in positions 8 and 9 of the α-centredtrp operator. In vitro, the Trp repressor of mutant RI69 binds to the consensus α-centredtrp operator and a similartrp operator variant that carries a T in position 9. In vivo analysis of the interactions of Trp repressor mutant RI69 with symmetrical variants of the β-centredtrp operator shows a change in the specificity of binding to a β-centred symmetricaltrp operator variant with a gua-nine to thymine substitution in position 5, which corresponds to position 9 of the α-centredtrp operator.     

On-off regulation of gene expression from tryptophan promoter with cross-flow filtration

Summary Recombinant plasmid containing β-galactosidase gene fused to trp promoter (pMCT98) and that containing cloned trp repressor gene (pRLK13) were introduced into Escherichia coli C600. The bacterium was cultivated in a jar-fermetor equipped with a cross-flow filtration apparatus to attain the on-off regulation of the gene expression by controlling tryptophan concentration in the medium. In logarithmic growth phase, the cross-flow filtration was started. Tryptophan concentration dropped to a low level within 1 h and an efficient expression of β-galactosidase gene was started. By this twostage cultivation, very high biomass was achieved (final OD₅₇₀: 150) and the amount of produced β-galactosidase was about 10% of total cellular proteins.     

In vivoandin vitroStudies of TrpR-DNA Interactions

The binding of tryptophan repressor (TrpR) to its operators was examined quantitatively usingin vitroandin vivomethods. DNA sequence requirements for 1:1 and tandem 2 :1 (TrpR : DNA) binding in various sequence contexts were studied. The results indicate that the optimal half-site sequence for recognition by one helix-turn-helix motif of one TrpR dimer is_{3′ CNTGA 5′}^{5′ GNACT 3′}, consistent with contacts observed by X-ray diffraction analysis of cocrystalline 1:1 and 2 :1 complexes. Half-sites can be paired to form a palindrome either by direct abutment, forming the nucleation site for a tandem 2 :1 complex, or with an 8-base-pair spacer, forming a 1:1 target. Dimethylsulfate (DMS) methylation-protection footprintingin vitroof 1:1 and 2 :1 complexes formed sequentially on the two unequal half-site pairs of thetrpEDCBA operator fromSerratia marcescensindicated an obligate hierarchy of site occupancy, with one half-site pair serving as the nucleation site for tandem binding. DMS footprinting ofEscherichia colioperatorsin vivoshowed that, over a wide range of intracellular TrpR concentration, thetrpEDCBA operator is occupied by three repressor dimers,aroH is occupied by two dimers, and the 1:1 binding mode is used on thetrpR operator. The coexistence of these distinct occupancy states implies that changes in protein concentration affect only the fractional occupancy of each operator rather than the binding mode, which is determined by the number of half-site sequences present in the operator region. Cooperativity of tandem complex formation measured by gel retardation using a symmetrized synthetic operator containing identical, optimal sites spaced as in natural operators was found to be modest, implying a maximum coupling free energy of ∼−2 kcal/mol. On other sequences the apparent degree of cooperativity, as well as the apparent affinity, varied with sequence and sequence context in a manner consistent with the structural models and which suggests compensation between affinity and cooperativity as a mechanism that allows tolerance of operator sequence variation.     

The possible roles of residues 79 and 80 of the Trp repressor from Escherichia coli K-12 in trp operator recognition

We constructed mutants of the Trp repressor from Escherichia coli K-12 with all possible single amino acid exchanges at positions 79 and 80 (residues 1 and 2 of the recognition helix). We tested these mutants in vivo by measuring the repression of synthesis of β-galactosidase with symmetric variants of α- and β-centered trp operators, which replace the lac operator in a synthetic lac system. The Trp repressor carrying a substitution of isoleucine 79 by lysine, showed a marked specificity change with respect to base pair 7 of the α-centered trp operator. Gel retardation experiments confirmed this result. Trp repressor mutant IR79 specifically recognizes a trp operator variant with substitutions in positions 7 and 8. Another mutant, with glycine in position 79, exhibited loss of contact at base pair 7. We speculate that the side chain of Ile79 interacts with the AT base pairs 7 and 8 of the α-centered trp operator, possibly with the methyl groups of thymines. Replacement of thymine in position 7 or 8 by uracil confirms the involvement of the methyl group of thymine 8 in repressor binding. Several Trp repressor mutants in position 80 (i.e. AI80, AL80, AM80 and AP80) broaden the specificity of the Trp repressor for α-centered trp operator variants with exchanges in positions 3, 4 and 5.     

Direct identification of the amino acid changes in two mutant lac repressors.

Deletions extending into the trp operon at one terminus and the lacI control region at the other terminus have been examined. One of these, B116, ends within the trp leader sequence and eliminates the trp attenuator site, placing the synthesis of lac repressor under trp control. We have isolated and characterized the B116 repressor. The protein sequence of the aminoterminus of B116 shows that an additional 16 residues are added to the amino-terminal end of wild-type repressor. Moreover, a valine residue appears in place of methionine at position 17 (the original amino-terminal residue of the wild-type repressor). A comparison of the messenger RNA sequence of the trp leader region and of the I leader region demonstrates that the translation of the B116 repressor is initiated at an AUG codon within the trp leader sequence. The GUG initiation codon at the start point for translation of wild-type repressor is now read as valine, since it appears at an internal position (residue 17 of the altered repressor). The B116 repressor accumulates at levels as high as 1% of the soluble cell protein in trpR^? strains. The efficiency of the trp leader initiation codon in translation suggests that in wild-type strains this AUG is also active in directing protein synthesis, which would result in a polypeptide consisting of 14 amino acids. We have examined the physical properties of the B116 repressor, which shows a marked tendency to form higher aggregates. Other characteristics of B116 are also described.     

Displacement of a DNA binding protein by Dda helicase

Bacteriophage T4 Dda helicase has recently been shown to be active as a monomer for unwinding of short duplex oligonucleotides and for displacing streptavidin from 3′-biotinylated oligonucleotides. However, its activity for streptavidin displacement and DNA unwinding has been shown to increase as the number of Dda molecules bound to the substrate molecule increases. A substrate was designed to address the ability of Dda to displace DNA binding proteins. A DNA binding site for the Escherichia coli trp repressor was introduced into an oligonucleotide substrate for Dda helicase containing single-stranded overhang. Here we show that a Dda monomer is insufficient to displace the E.coli trp repressor from dsDNA under single turnover conditions, although the substrate is unwound and the repressor displaced when the single-stranded overhang is long enough to accommodate two Dda molecules. The quantity of product formed increases when the substrate is able to accommodate more than two Dda molecules. These results indicate that multiple Dda molecules act to displace DNA binding proteins in a manner that correlates with the DNA unwinding activity and streptavidin displacement activity. We suggest a cooperative inchworm model to describe the activities of Dda helicase.     

Complex structure of the membrane nuclease of Streptococcus pneumoniae revealed by two-dimensional electrophoresis

Close contacts between Escherichia coli RNA polymerase and specific purine residues in the tryptophan (trp) operon promoter of Salmonella typhimurium were revealed using the methylating agent dimethyl sulfate. RNA polymerase bound to trp promoter DNA caused alterations in the rate of methylation at seven specific sites; in the anti-sense strand, guanine residues at positions ?37, ?34 and ?2 showed enhanced methylation, while those at positions ?14, ?6 and +3 showed reduced methylation. In the sense strand, only the guanine residue at ?32 showed reduced methylation. No RNA polymerase contacts with adenine residues were observed. Using the same method, close interactions between E. coli trp repressor and purine residues in the trp operator of S. typhimurium were examined. Bound trp repressor alters the methylation rates of both guanine and adenine residues from positions ?25 to +3. The points of contact are distributed rather symmetrically on both DNA strands. Three points of close contact are shared by RNA polymerase and trp repressor, supporting previous models of trp repressor action.     

Inactivation of prophage lambda repressor in vivo.

Jacob &; Monod (1961) postulated that prophage A induction results from the inactivation of the λ repressor by a cellular inducer. Although it has been shown that the phage A repressor is inactivated by the recA gene product in vitro (Roberts et al., 1978), we wanted to determine the action of the “cellular inducer” in vivo. Our results have led to a new model, which defines the relationship between the “cellular inducer” and the recA gene product.In order to quantitate the action of the cellular inducer on the λ repressor, we made use of bacteria with elevated cellular levels of the λ repressor (hyperimmune lysogens). We determined the kinetics of repressor inactivation promoted by three representative inducing treatments: ultraviolet light irradiation, thymine deprivation and temperature shift-up of tif-1 mutants.The kinetics of repressor decay in wild-type monolysogens indicate that repressor inactivation is a relatively slow cellular process that takes a generation time to reach completion. Incomplete inactivation of the repressor without subsequent prophage development may occur in a cell. We call this phenomenon detected at the biochemical level “subinduction”. In hyperimmune lysogens. subinduction is always the case.A high cellular level of A repressor that prevents prophage λ induction does not prevent induction of a heteroimmune prophage such as 434 or 80. Although the cellular inducer does not seem specific for any inducible prophage, it does not inactivate two prophage repressors present in a cell in a random manner. We have called this finding “preferential repressor inactivation”. Preferential repressor inactivation may be accounted for by considering that the intracellular concentration of a repressor determines its susceptibility to the action of the inducer.In bacteria with varying repressor levels, a fixed amount of repressor molecules is inactivated per unit of time irrespective of the initial repressor concentration. The rate of repressor inactivation depends on the catalytic capacity of the cellular inducer that behaves as a saturated enzyme. In wild-type bacteria the cellular inducer seems to be produced in a limited amount, to have a weak catalytic capacity and a relatively short half-life. The amount of the inducer formed after tif-1 expression is increased in STS bacteria overproducing a tif-1-modified RecA protein. This result is an indication that a modified form of the RecA protein causes repressor inactivation in vivo.From the results obtained we propose a model concerning the formation of the cellular inducer. We postulate that the cellular inducer is formed in a two-step reaction. The is model visualises how the RecA protein can be induced to high cellular concentrations, even though the RecAp protease molecules remain at a low concentration. The latter accounts for the limited proteolytic activity found in vivo.     

On-off regulation of the tryptophan promoter in fed-batch culture

Fundamental properties of the trp promoter were investigated in fed-batch culture using a recombinant containing the lacZ gene controlled by this promoter. In tryptophan-deficient conditions, the amount of β-galactosidase accumulated in the cell was 10% of total cellular proteins. In the presence of the amino acid, it was repressed at a lower level, but considerable expression was observed in the later stages of cultivation. Although increasing concentration of tryptophan seemed to repress the promoter more completely, it strongly inhibited the bacterial growth. On-off regulation of the promoter was achieved by controlling the tryptophan level during fed-batch culture.     

Thr region between the operator and first structural gene of the tryptophan operon of Escherichia coli may have a regulatory function

Among a collection of 34 independent mutants with internal deletions in the trp operon of Escherichia coli we found six that fail to recombine with any known point mutant in trpE, the first gene in the operon. These six deletion mutants are regulated normally by tryptophan and thus appear to have the trp operator region intact. However, four of these deletions result in alterations in the maximum level of expression of the trpC, B and A genes when compared with wild type or with an internal deletion of similar length which retains a small operatorproximal segment of trpE. Two of these deletion mutants, trpΔED1 and trpΔED12, have lower levels of the protein products of trpB and trpA than the control strains. In contrast, deletions trpΔED2 and trpΔED102 both markedly increase the levels of the trpB and trpA polypeptides. Deletion mutant trpΔED2 has 3 to 3.5 times and mutant trpΔED102 has seven to eight times as much tryptophan synthetase β₂ and α proteins as the wild-type or deletion control strains. The increase in tryptophan synthetase β₂ and α proteins seen is a consequence of an increase in the level of trp mRNA directing the synthesis of these enzymes. The rate of synthesis of trpBA mRNA is increased in trpΔAED2 about twofold, and in trpΔED102 about four- to sixfold over the control strain. The left-hand deletion end-points of both trpΔED2 and trpΔAED102 have been shown to map to the right of a known trp operator-constitutive mutation and appear to lie before the first translation start codon in trpE (M. Bronson, C. Squires &; C. Yanofsky, unpublished results). We propose that these deletions alter a region between the earliest known trpE point mutation and the trp operator which influences the maximum rate of synthesis of trp operon mRNA.     

A new way of stabilizing recombinant plasmids

A new method to stabilize recombinant plasmids extremely well was exploited using Escherichia coli Tna (trpAEI trpR tnaA) and pSC101trpI15-14 (tetracycline resistance, whole trp operon) as a model system. We mutagenized the Tna strain carrying pSC101trpI15-14 and isolated a mutant 6F484 that stably maintained the recombinant plasmid for 100 generations. From 6F484, plasmid-free cells (tetracycline sensitive) were screened for on selective agar plates containing fusaric acid. The host strain FA14 was found to have lost the ability for active transport of tryptophan, in addition to the phenotype of Trp⁻. Therefore, strain FA14 could not grow normally even in a complete medium. However, when the strain was transformed with the trp operon recombinant plasmid, its growth rate was almost restored to the original level. These results suggest that the recombinant plasmid is indispensable for the normal growth of host cells like FA14. Even if plasmid-free segregants appear during the cultivation, they cannot grow so rapidly and are diluted as a minority in total population. Consequently, owing to the deficiency of both the biosynthesis and uptake of tryptophan in host strain, the trp operun recombinant plasmid can be stably maintained.