Abstract

The transient inactivation of factor regulatory proteins past their sequestration to the cytoplasmic membrane in response to cognate signals is an increasingly recognized machinery of gene regulation in bacteria. It remained to be shown, nonetheless, whether tethering to the membrane per se could be responsible for inactivation, i.e. whether such relocation leads to a spatial separation from the chromosome that results in inactivity or whether other mechanisms are involved. Nosotros, therefore, investigated the activity of Lac repressor artificially attached to the Escherichia coli cytoplasmic membrane. We demonstrate that this chimeric poly peptide perfectly represses transcription initiated at the tac operator–promoter present on a plasmid and even in the chromosome. Moreover, this repression is inducible as normal. The data suggest that proteins localized to the inner face of the cytoplasmic membrane in principle have unrestricted admission to the chromosome. Thus sequestration to the membrane in terms of physical separation from the chromosome cannot account alone for the inactivation of regulatory proteins. Other mechanisms, like induction of a conformational change or masking of binding domains are required additionally.

INTRODUCTION

In the past decade a novel manner of activity command of regulatory proteins has been revealed in bacteria. In this mode, transcription factors shuttle between their binding sites on the chromosome and the inner face of the cytoplasmic membrane, where they are held inactive. In response to a specific stimulus, these regulators are again released from the membrane and can demark back to their target sites on the chromosome [for a contempo review encounter ( 1 )].

The starting time case of such a mechanism was the regulation of the proline utilization ( put ) operon past PutA in Salmonella typhimurium ( 2 ). PutA has been shown to be a bi-functional poly peptide. In the absence of proline, PutA remains in the cytoplasm, where it binds to the put operators and represses put gene expression. When an backlog of proline is present, proline binds to PutA. Binding of proline, combined with reduction of the PutA protein by the membrane-associated electron transport chain, leads to release of PutA from its operators and its clan with the membrane. From this location it catalyzes the degradation of proline to glutamate.

Another elegant arrangement where membrane sequestration of a repressor leads to induction of its assigned genes is represented by the mlc regulon in Escherichia coli . The repressor Mlc controls expression of several genes involved in carbohydrate utilization [for reviews run across ( 1 , 3 )]. One of them is ptsG encoding the EII Glc send protein of the phosphotransferase system (PTS). Mlc is in turn regulated by the transport activity of EII Glc . In the absence of substrate for EII Glc , Mlc binds to its operator sites and represses transcription. When EII Glc is engaged in transport, it binds Mlc, thereby sequestering information technology to the membrane, which leads to concomitant de-repression of the Mlc-controlled genes. It has recently been shown that an artificial attachment of Mlc to the cytoplasmic membrane by fusion to the LacY permease causes de-repression of Mlc-controlled genes fifty-fifty in the absenteeism of EII Glc , suggesting that tethering of Mlc to the membrane alone, rather than binding to EII Glc , may lead to its inactivation as a repressor ( 4 ). Not simply repressors just also activator proteins were shown to be sequestered to the membrane in response to their cognate signals and concomitantly kept inactive. These include MalT of E.coli ( i , v ), TraR of Agrobacterium tumefaciens ( six ) and NifL of Klebsiella pneumoniae , in this case an inhibitor of a gene activator poly peptide ( seven ). In virtually of these cases the molecular reason for inactivity of the membrane-localized regulatory proteins is non clear, but it has been proposed that a spatial gap may be between the membrane and the chromosome with the consequence that the transcription factors have to 'shuttle' dorsum to the nucleoid to gain access to their binding sites in the chromosome ( 1 , 7 ). Notwithstanding, in one instance, the DNA binding gene activator protein ToxR of Vibrio cholerae , it has been demonstrated that this protein is naturally membrane-bound ( 8 ). In addition, we have previously shown that the transcriptional antiterminator protein BglG from E.coli that is regulated in its activity past a membrane spanning sugar ship protein tin be artificially tethered to the membrane without impairing its function. The membrane-jump BglG poly peptide is capable of interacting with its chromosomally encoded nascent RNA target sequence, catalyzing transcriptional read-through with virtually no difference to wild-type BglG, and it is perfectly regulated in its activity ( 9 ). These observations suggested that in principle in that location should be no mechanistic necessity for a regulatory poly peptide like BglG to leave the identify of its own regulation, i.e. the membrane, in order to interact with its target sequences.

Even so, for activator proteins like ToxR or BglG transient interactions with their cognate target sequences could be sufficient to stimulate gene expression. Furthermore, actively transcribed Deoxyribonucleic acid is believed to often reside close to the membrane, particularly when co-translational insertion of proteins into the membrane takes place. For efficient transcriptional repression, however, occupation of the operator binding site past the repressor protein for nearly of the time is required and no or little transcription should occur that could redirect the operator DNA to the membrane. It, therefore, was an open question whether repressor proteins could exist or be engineered that function from the cytoplasmic membrane. Here, nosotros addressed this question and artificially anchored the East.coli Lac repressor to the cytoplasmic membrane. We demonstrate that this membrane-attached LacI protein perfectly represses transcription initiation at a chromosomally located tac operator–promoter and that it tin can be released from repression by the addition of isopropyl-β- d -thiogalactopyranoside (IPTG) equally inducer. Our experiments propose that membrane-attached and soluble Lac repressor proteins find their operator site in the chromosome with similar efficiency. Together, our results advise that membrane sequestration of a repressor protein per se cannot exist the reason for its inactivation. Other mechanisms are required. The concept that membrane sequestration provides a spatial separation from and prevents access to the chromosome has thus to be revised.

MATERIALS AND METHODS

Plasmids, strains and growth conditions

The strains and plasmids used and their relevant characteristics are listed in Table 1 . For DNA cloning, standard procedures were followed ( 10 ). The right nucleotide sequences of PCR-derived fragments in the various plasmids were verified by DNA sequencing. Bacteria were routinely grown in 2YT broth supplemented with the appropriate antibiotics (kanamycin at 30 μg/ml, chloramphenicol at 10 μg/ml and ampicillin at 100 μg/ml, if not otherwise indicated).

Construction of chimeric lacI fusion genes

Plasmid pFDX4151 was the antecedent of plasmids conveying the different chimeric lacI derivatives. It was constructed past a three fragment ligation combining the ClaI–PstI- and the Psp1406I–ClaI-fragments of plasmid pFDX500 ( 11 ) with a PCR fragment which was amplified with primers #631 (5′-CGTATAACGTTACTGGTTTCATATGCACCACCCTGAATTG) and #632 (5′-GCCGGGAAGCTAGAG) from plasmid pFDX500 as template and digested with Psp1406I and PstI. This construction inverse the start codon of lacI from GTG to ATG and simultaneously created an NdeI site overlapping with the start codon that immune the insertion of foreign DNAs in-frame with lacI . For structure of the M13gene8 - lacI fusion gene, the M13gene8 was amplified by using primers #293 (five′-GGGATTTAAACATATGAAAAAGTCTTTAGTCCTCAAAGC) and #294 (five′-TTTAAAGTACTTGCTTTCGAGGTGAATTTCTTAAA) and plasmid M13mp18 as template. The PCR fragment was digested at the NdeI and Csp6I sites within the primers and inserted into the NdeI site of plasmid pFDX4151, resulting in plasmid pFDX4152. For the structure of plasmid pFDX4154 conveying the galK′-lacI fusion gene, the v′-part of galK encompassing the get-go 32 codons was amplified by using the primers #636 (v′-GATATACATATGGCTAG) and #637 (v′-CACTGCATTAATAGCAAGGACAGGC) and plasmid pFDX3225 ( 12 ) every bit template. The PCR fragment was digested at the NdeI and AseI sites within the primers and inserted into the NdeI site of plasmid pFDX4151. Plasmids pFDX4155 and pFDX4156 carrying the galK′ -λ cI′ - lacI and the gene8 -λ cI′ - lacI fusion genes, respectively, were synthetic in two steps. Beginning, codons 96–132 of the λ cI gene encoding a flexible linker were amplified by using primers #634 (5′-AAAGAGTATCATATGGACCCGTCACTT) and #635 (v′-CTCCTCATTAATGCTTACCCATCTCTC) and plasmid λdv1 ( 13 ) as template. The PCR fragment was digested at the NdeI and MseI sites within the primers and inserted into the NdeI site of plasmid pFDX4151 resulting in the intermediate construct pFDX4153 carrying a λ cI′ - lacI fusion gene. Next, the digested PCR fragments from above, encompassing the 5′-part of galK and gene8 , respectively, were inserted into the NdeI site of plasmid pFDX4153, resulting in plasmids pFDX4155 and pFDX4156. In gild to supplant the ribosomal binding site (RBS) in front of lacI and its various chimeric derivatives, the sequence upstream was amplified past using primers #646 (5′-GTTGCCATTGCTGCAGGC) and #647 (5′-GCATATCGCATATGTATATCTCCTTCTTGACTCTCTTCCGGGCGCTAT; contains the RBS of T7gene10 ) and plasmid pFDX4152 as template. The PCR fragment was digested at the PstI and NdeI sites inside the primers and used to replace the corresponding fragment in plasmids pFDX4151, pFDX4152, pFDX4154, pFDX4155 and pFDX4156 resulting in plasmids pFDX4165, pFDX4157, pFDX4158, pFDX4159 and pFDX4160, respectively.

Construction of a tacOP-lacZ reporter cassette and integration into the chromosomal attB site

Plasmid pFDX3551 carries a tacOP-lacZ cassette with the relatively weak RBS sequence of the bglG gene in front end of lacZ . Information technology was constructed by replacement of the HindIII–ClaI fragment of plasmid pFDX3157 ( 12 ) encompassing gene bglG by the HindIII–ClaI fragment of plasmid pFDX3549 ( 12 ) encompassing the lacZ cistron. Plasmid pFDX3552 is isogenic with plasmid pFDX3551 but carries the strong RBS sequence of gene10 of phage T7 in front of lacI . Its construction involved several steps, and details are available on request. Integration of the tacOP-lacZ cassette of plasmid pFDX3552 by site-specific recombination into the bacteriophage λ attachment site attB , was accomplished with the integration system provided by Diederich et al . ( xiv ) every bit described previously ( 12 ).

Decision of β-galactosidase activities

Enzyme assays were performed co-ordinate to the method of Miller ( 15 ) as described previously ( 16 ). The bacteria were grown in M9 minimal medium supplemented with i% (westward/v) glycerol, 20 μg/ml proline, 1 μg/ml thiamine, 0.66% (w/v) casamino acids and the advisable antibiotics. If not otherwise indicated, overnight cultures were inoculated to an OD 600 of 0.15 and grown for 1 h at 37°C. And then, ane mM IPTG was added where necessary and growth was continued for two h before the cultures were harvested. Samples were assayed in triplicate and each experiment was repeated three times using independent transformants. Enzyme activities are expressed in Miller units.

In vivo pulse-chase [ 35 S]methionine labeling of proteins

The pulse-hunt labeling of proteins with [ 35 Southward]methionine was carried out as described previously ( 9 ).

Subcellular fractionation of [ 35 S]methionine-labeled proteins

Separation of [ 35 Due south]methionine-labeled soluble and membrane proteins was performed past sucrose gradient centrifugation as described previously ( 9 ).

RESULTS

Construction, stability and subcellular localization of chimeric Lac repressor proteins

It has previously been shown that the 50 amino acrid residue major glaze protein of bacteriophage M13 encoded by M13gene8 is a suitable tool to stably anchor a protein to the cytoplasmic membrane. For this purpose the protein to be anchored is fused to the C -terminus of the coat protein ( 9 , 17 ). We fabricated use of this tool to attach the Lac repressor to the membrane. To this end, a translational fusion of the lacI gene encoding Lac repressor to the verbal 3′ finish of gene8 was synthetic and placed on a low copy plasmid nether command of the lacI promoter. In lodge to decide whether an Northward -concluding extension would by and large impair the function of Lac repressor, a control fusion comprising the beginning 32 codons of the galK factor fused to lacI was also constructed. The galK gene encodes galactokinase, a soluble protein ( ix ).

The Deoxyribonucleic acid bounden domain of Lac repressor encompasses the first 60 N -terminal amino acrid residues. It thus appeared conceivable that the tight tethering of this domain to the membrane could negatively interfere with its Dna binding power. We, therefore, constructed additional fusion genes in which codons 96–132 of the λ cI repressor gene encoding a flexible linker ( eighteen ) were placed between the lacI - and the gene8 - or galK -part of the respective fusion genes.

In order to allow investigation by gel electrophoresis of whether the fusion proteins are stable in vivo and indeed attached to the membrane, a second set of isogenic vectors was constructed in which the RBS of lacI was replaced by the stronger RBS of gene 10 of phage T7 , leading to a higher expression of the dissimilar Lac repressor derivatives. Transformants carrying the corresponding expression plasmids were analyzed past [ 35 Southward]methionine labeling and subsequent SDS–PAGE ( Effigy i ). Equally a control, the untransformed strain was as well employed. Labeled protein bands became visible, which were exclusively present in the extracts of the corresponding transformants expressing LacI (39.6 kDa), the φ(Gene8–λCI–LacI) fusion (49.6 kDa) or the φ(Gene8–LacI) fusion (45.3 kDa). The positions of these proteins on the gel fitted well with the calculated molecular weight of the respective LacI derivatives, indicating that they are properly expressed ( Figure ane , compare lanes 1, 7, xiii and nineteen; data non shown for the GalK–LacI and the GalK–λCI′–LacI fusion proteins).

In order to test the stability of the different LacI derivatives, the labeled cultures were chased with unlabeled methionine for unlike times prior to poly peptide extraction and SDS–PAGE. All LacI derivatives were stable over the tested menses of 30 min, as was the wild-type LacI protein ( Figure one , compare lanes 14–xviii and 20–24 with lanes 8–12; data not shown for the GalK–LacI and the GalK–λCI′–LacI fusion proteins).

Side by side, we wanted to verify that the Gene8 fusion proteins were indeed tightly fastened to the cytoplasmic membrane. We, therefore, performed a subcellular fractionation of [ 35 S]methionine-labeled transformants harboring the wild-type lacI cistron or the φ( gene8 - lacI ) and the φ( gene8 -λ cI ′ -lacI ) fusion genes, respectively, by sucrose gradient centrifugation, as described previously ( 9 ). The untransformed strain served as a control. The soluble and membrane fractions were analyzed past SDS–Folio forth with the unfractionated samples ( Effigy 2 ). It can be seen that both the φ(Gene8-LacI) besides as the φ(Gene8–λCI′–LacI) fusion proteins were nowadays in the membrane fraction ( Effigy ii , lanes 8 and 11) just not in appreciable amounts in the soluble fraction ( Figure ii , lanes 7 and 10). The wild-type LacI poly peptide, on the other hand, was simply visible in the soluble fraction, as expected ( Effigy 2 , lane 4). Its corporeality was, all the same, lower than that detected with the [ 35 Southward]methionine-labeled whole cell extracts analyzed in Figure 1 . Since the corporeality of LacI was similarly reduced in instance of the unfractionated command sample ( Effigy two , lane 6), it is feasible that this reduction was due to the TCA precipitation step which was performed with all samples analyzed in Effigy 2 but not with those presented in Figure i . This difference has, nevertheless, no touch on on the main conclusion that both, the φ(Gene8–LacI) too every bit the φ(Gene8–λCI′–LacI) fusion protein are stable and are anchored to the cytoplasmic membrane.

Membrane-attached Lac repressor is active in transcriptional repression

The of import question was whether or not the membrane-attached LacI proteins are functional in repression and thus in operator binding, and if then, whether or non repression past these proteins could be relieved by the add-on of IPTG, a gratis inducer for wild-type Lac repressor. To this end, strain R1279 in which the chromosomal lac operon is deleted, was co-transformed with the various plasmids carrying the dissimilar lacI derivatives under control of the wild-type lacI RBS and in addition with reporter plasmid pFDX3551. This plasmid carries a lacZ reporter gene under control of the tac promoter which is controlled by the main lac operator O 1 that overlaps with its transcriptional first site. When strain R1279 was transformed with this reporter plasmid alone, high β-galactosidase activities of ∼5000 U were produced which were unaffected by the absence or presence of IPTG, equally expected ( Figure 3 , bars 1). The boosted presence of plasmid pFDX500 delivering wild-type Lac repressor led to the synthesis of 51 U in the absence and of 1640 U in the presence of IPTG. Thus, the expression of lacZ was repressed 32-fold by wild-type Lac repressor in this reporter system ( Figure 3 , confined two). Nearly identical values were obtained when the LacI delivering plasmid pFDX4151 carrying an NdeI site that overlaps with the lacI offset codon was present ( Figure three , bars 3). Thus, the introduction of this site and the commutation of the GTG start codon confronting ATG had no appreciable outcome on repression. The expression of the φ(GalK–LacI) fusion protein yielded activities which were similar to those obtained with the wild-type LacI poly peptide ( Figure iii , compare bars iii and iv), indicating that the fusion of a strange polypeptide to the North-terminus of LacI may in general not take deleterious effects on repression and thus on binding of the Lac repressor to its operator. Expression of the membrane-anchored φ(Gene8–LacI) fusion protein led to the synthesis of 2250 U of β-galactosidase activities in the presence of IPTG which were reduced to 120 U in its absence. Thus, the membrane-anchored φ(Gene8–LacI) protein was indeed able to efficiently repress expression of the lacZ reporter cistron. Moreover, this repression could be relieved 19-fold by the improver of IPTG ( Effigy three , bars five). The presence of the flexible protein linker of the λCI protein had no outcome on the repression activity of the φ(GalK–λCI′–LacI) poly peptide when compared with the φ(GalK–LacI) protein without this linker ( Effigy 3 , compare bars four and vi). In contrast, the φ(Gene8–λCI′–LacI) poly peptide was less active in transcriptional repression when compared with the φ(Gene8–LacI) fusion protein lacking the λCI-linker. Therefore, induction was just 10-fold ( Figure 3 , compare confined seven and 5). Obviously, the λCI-linker negatively interfered with the operator binding ability of this membrane-attached Lac repressor protein.

The membrane-fastened LacI proteins regulate expression of a chromosomal gene

The experiments presented to a higher place ( Effigy 3 ) demonstrate that the membrane-attached Lac repressor proteins are indeed active in transcriptional repression. However, in these tests the lacO one target site was encoded on a plasmid. Although plasmids appear to be clustered and partitioned within a cell ( 19 ) they may be more readily attainable by membrane-localized Dna binding proteins than sequences within the nucleoid. The question was thus yet open up whether the membrane-located Lac repressor protein would have free access and tightly bind to a chromosomally located operator site. In order to address this question, the tacOP-lacZ reporter cassette nowadays in plasmid pFDX3551 was integrated into the λ attB site in the chromosome past site-specific recombination. All the same, this strain yielded very low β-galactosidase activities [<100 Miller units already in the absence of Lac repressor (data not shown)], making it difficult to precisely compare activities of the various Lac repressor derivatives. Therefore, the isogenic strain R2171 was constructed in which the more efficient ribosome binding site of gene ten of phage T7 was placed in front of lacZ . This strain yielded ∼4000 U of β-galactosidase, a convenient activity for the intended experiments. As expected, the presence of IPTG had no event on this activity ( Figure 4 , bars 1). Transformants of this strain harboring plasmids pFDX500 and pFDX4151, respectively, that both encode the wild-type Lac repressor yielded 30–40 U of β-galactosidase. These activities increased 70–80-fold in the presence of IPTG ( Figure 4 , bars ii and three), verifying that expression of the chromosomal lacZ reporter cassette is perfectly regulated past the wild-type repressor in an inducer-dependent manner. Very similar results were obtained in the presence of plasmids pFDX4154 and pFDX4155 encoding the φ(GalK–LacI) and the φ(GalK–λCI′–LacI) fusion proteins, respectively, demonstrating one time again that this fusion of a strange peptide to the N-terminus of LacI has no negative effect on Lac repressor function ( Effigy 4 , confined 4 and vi). When strain R2171 was transformed with plasmid pFDX4152 that encodes the membrane-anchored φ(Gene8-LacI) chimeric repressor, the transformants synthesized only 34 U of β-galactosidase. Thus, the membrane-anchored repressor is able to repress the tac promoter present in the chromosome as efficiently as the wild-type repressor. Addition of IPTG resulted in 3760 U or a 111-fold consecration ( Figure four , bars 5). Thus, the membrane-fastened LacI derivative is capable of regulating the expression of the chromosomal lacZ reporter cistron as efficiently as the wild-type repressor. Finally, the membrane-anchored φ(Gene8–λCI–LacI) fusion protein delivered by plasmid pFDX4156 was tested. Presence of this plasmid led to 53 U of β-galactosidase in the absenteeism of IPTG and to 3560 U in its presence, which is a 67-fold consecration ( Figure 4 , bars 7). This result corroborates the higher up observation ( Figure 3 , bars 7) that the presence of the λCI-linker may slightly impair operator binding of the membrane-anchored repressor.

Membrane-attached and soluble LacI proteins showroom similar kinetics of binding to chromosomally located operator lacO1

The experiments above demonstrated that the membrane-attached φ(Gene8–LacI) fusion protein is capable of efficiently repressing expression of the lacZ reporter cistron and thus of binding to a chromosomally located operator, lacO1 . The addition of IPTG led to release from this repression. Moreover, repression also as inducibility were quite similar with its membrane-anchored fusion derivatives as compared with the wild-blazon repressor, suggesting that fifty-fifty the equilibrium constants for binding of the operator sequence are like. However, in the experiments described above, cultures were pregrown without IPTG, split up and afterwards grown in the presence or absence of IPTG before enzyme activities were determined. Thus, these information allow no conclusion nearly the initial rate of operator occupancy. To address this question, cultures of the transformants shown in Figure 4 were pregrown in the presence of IPTG. Subsequently, they were done gratis of IPTG to allow establishment of repression, and the kinetics of decline of β-galactosidase equally a function of time was analyzed ( Figure five ). In the absence of repressor, specific activity remained constant as expected ( Effigy v , diamonds). In all other cases, enzyme activities declined with fourth dimension as a result of re-establishment of repression. In these cases, the remaining activities represent the sum of dilution of enzyme activities past cell growth and of the basal enzyme synthesis under repressing conditions. As can be seen, the slopes of decline of enzyme activities are similar in all cases. The differences that can exist seen reflect the differences in repression every bit revealed by the data in Figure iv and support the conclusion that the membrane-anchored φ(Gene8–LacI) repressor protein represses the tac promoter at least as efficient as the wild-type repressor. Thus, not only the equilibrium constants but also the initial rate of finding the operator target appears to be like within the time frame of these kinetics for the membrane-localized and soluble repressor proteins. Taken together, it can be concluded that a membrane-attached Lac repressor protein tin can discover and bind its operator sequence present in the chromosome as efficient as the soluble wild-type LacI poly peptide.

DISCUSSION

In this study, nosotros have demonstrated that the E.coli Lac repressor retains its activeness when it is artificially fixed with its N-terminus to the inner confront of the cytoplasmic membrane. From this location information technology represses the expression of a tacOP-lacZ reporter cassette present on a plasmid and even in the chromosome with the same efficiency as the wild-type repressor. Moreover, add-on of IPTG as an inducer resulted in the same de-repression exhibited by the wild-type repressor ( Figures 3 and 4 ). The information point that the equilibrium constants for the occupation of chromosomal lac operator O i must be similar for the membrane-localized φ(Gene8–LacI) protein and the wild-type Lac repressor ( Figures 4 and v ). Pulse-hunt experiments verified that the chimeric φ(Gene8–LacI) protein is stable ( Figure 1 ). Subcellular fractionations demonstrated that the chimeric repressor protein is stably anchored to the membrane ( Figure two ).

Nevertheless, we did non demonstrate that the membrane-anchored fusion protein is capable of regulating the accurate lac operon. Indeed, when we replaced the wild-type lacI cistron past the gene8-lacI fusion factor inside the context of the authentic lac operon in the chromosome, expression of the operon was only weakly repressed. Withal, very like results were obtained when the lacI cistron was replaced past 1 of the galK-lacI fusion genes (data not shown). Thus, a fusion to the Northward-terminus of the Lac repressor encompassing its Dna binding domain generally leads to a defect in repressor function in its authentic context, the lac operon. In this context, total repression requires cooperative binding of LacI to ane of 2 auxiliary operators in addition to operator O 1 ( 20 ). This cooperative binding requires the tetramerization of LacI ( 21 ). It may be concluded from our data that a fusion of a foreign peptide to the North -terminus of LacI in general may negatively interfere with its binding capability to the main operator, O 1 , when auxiliary bounden sites are present.

Notwithstanding, our data clearly demonstrate that a membrane-anchored Lac repressor does take unrestricted access to the chromosomally located lacO1 operator. Thus, not only transcriptional antiterminator proteins like the E.coli BglG protein tin can exist engineered to work from the membrane ( ix ) but also transcriptional repressor proteins that have to occupy their binding sites for most of the time and be able to bind back fast in order to achieve efficient repression. Thus, a physical gap between membrane and chromosome does not be. It appears that either the chromosome already resides in the vicinity of the membrane, making a concrete movement of the regulator unnecessary, or that the chromosome dynamically rearranges very fast allowing costless admission to its sequences from the membrane. This view is in understanding with early on studies that reported the chromosome to be associated with the cytoplasmic membrane ( 22 , 23 ).

A major consequence of our results is that membrane sequestration of gene regulators in terms of their physical separation from the chromosome alone cannot account for their inactivation. A well-studied case of a gene activator that shuttles between the chromosome and the membrane is MalT of E.coli . Upon maltose transport MalT is released from the membrane-bound subunit MalK of the maltose transporter which is a prerequisite for MalT activity. Information technology has been known for a long time that even a soluble course of MalK inhibits mal gene expression in vivo , at to the lowest degree when overproduced ( 24 ). Recently, information technology has been demonstrated that soluble MalK is able to inhibit MalT action in a purified transcription system by blocking the binding of the inducer maltotriose to MalT ( 25 ). Thus, membrane sequestration of MalT appears to be only a secondary consequence as a consequence of its interaction with the membrane-localized MalK subunit rather than to be direct responsible for MalT inactivity. The quorum-sensing activator protein TraR of A.tumefaciens in the presence of the homoserine lactone signal exists as an active dimer in the cytoplasm, whereas in its absence TraR is sequestered to the membrane in an inactive, monomeric course ( half dozen ). It, thus, appears that the subcellular localization of TraR modulates its oligomerization state and thereby indirectly regulates the action of TraR.

In a recent written report, it was plant that different sites in the chromosome significantly differ in their accessibility to phage λ integrase catalyzed site-specific recombination. In fact, several loci were found that were fifty-fifty designated 'blackness holes' ( 26 ). On the other hand, the Due east.coli chromosome is more often than not accessible in vivo along its entire length to restriction enzymes suggesting that in that location are no extensive stably bound structural components that prevent their access ( 27 ). Our tests were performed with lac operator O 1 integrated into the λ attB site of the E.coli chromosome. We are currently investigating with the aid of a transposon borne tacOP-lacZ cassette whether differences in the accessibility to repression by the membrane-bound Lac repressor be within the chromosome. Our finding that membrane-jump Lac repressor tin can tightly demark to its operator should in master allow to tether nigh whatever DNA sequences containing lac operator to the cytoplasmic membrane and to study the consequences. For example, Dna topology or chromatin construction could be captured betwixt ii operator sites and released upon the improver of IPTG, and the consequences for recombination, gene expression or Dna replication could be studied.

Tabular array i

Strains and plasmids used in this study

Genotype or relevant structures Source or reference
Strains
    R1279 CSH50 Δ( pho-bgl )201 Δ( lac-pro ) ara thi ( 16 )
    R2171 Every bit R1279 but attB ::[ tacOP - lacZ , bla ]; RBS of phage T7gene10 in front of lacZ pFDX3552→R1279
Plasmids
    M13mp18 M13 phage cloning vector ( 28 )
    pFDX500 lacI , neo , ori p15A ( 11 )
    pFDX3225 φ( galK ′ -bglG ) under control of Ptac , tet , ori p15A ( 12 )
    pFDX3401 λ int under control of λP R , λ cI 857 ( true cat ; ori pSC101- rep TS ) ( 12 )
    pFDX3551 pLDR10 derivative for the insertion of a tacOP - lacZ cassette into the attB site; RBS of gene bglG in front of lacZ , bla , ori pMB1 This study
    pFDX3552 Equally pFDX3551, but RBS of phage T7gene10 in front end of lacZ This study
    pFDX4151 As pFDX500, but with a NdeI site overlapping with the lacI ATG start codon This report
    pFDX4152 Equally pFDX4151, but lacI fused to M13gene8 This study
    pFDX4153 As pFDX4151, only lacI fused to λ cI ′ (codons 96–132) This report
    pFDX4154 As pFDX4151, but lacI fused to galK ′ (codons 1–32) This study
    pFDX4155 Equally pFDX4151, but lacI fused to galK ′-λ cI ′ This study
    pFDX4156 Every bit pFDX4151, but lacI fused to M13gene8 -λ cI ′ This study
    pFDX4157 As pFDX4152, but RBS of phage T7gene10 in front of φ( M13gene8 - lacI ) This study
    pFDX4158 Equally pFDX4154, just RBS of phage T7gene10 in front of φ( galK ′- lacI ) This study
    pFDX4159 As pFDX4155, just RBS of phage T7gene10 in front of φ( galK ′-λ cI ′ -lacI ) This study
    pFDX4160 Every bit pFDX4156, but RBS of phage T7gene10 in forepart of φ( M13gene8 -λ cI ′- lacI ) This study
    pFDX4165 As pFDX4151, only RBS of phage T7gene10 in front of lacI This study
    pLDR10 Multiple cloning site, λ attP , bla , cat , ori pMB1 ( 14 )
Genotype or relevant structures Source or reference
Strains
    R1279 CSH50 Δ( pho-bgl )201 Δ( lac-pro ) ara thi ( 16 )
    R2171 As R1279 only attB ::[ tacOP - lacZ , bla ]; RBS of phage T7gene10 in front of lacZ pFDX3552→R1279
Plasmids
    M13mp18 M13 phage cloning vector ( 28 )
    pFDX500 lacI , neo , ori p15A ( 11 )
    pFDX3225 φ( galK ′ -bglG ) nether control of Ptac , tet , ori p15A ( 12 )
    pFDX3401 λ int nether command of λP R , λ cI 857 ( cat ; ori pSC101- rep TS ) ( 12 )
    pFDX3551 pLDR10 derivative for the insertion of a tacOP - lacZ cassette into the attB site; RBS of gene bglG in front of lacZ , bla , ori pMB1 This study
    pFDX3552 Every bit pFDX3551, but RBS of phage T7gene10 in front of lacZ This written report
    pFDX4151 Every bit pFDX500, just with a NdeI site overlapping with the lacI ATG start codon This study
    pFDX4152 Every bit pFDX4151, but lacI fused to M13gene8 This report
    pFDX4153 As pFDX4151, but lacI fused to λ cI ′ (codons 96–132) This study
    pFDX4154 As pFDX4151, but lacI fused to galK ′ (codons ane–32) This study
    pFDX4155 Every bit pFDX4151, only lacI fused to galK ′-λ cI ′ This study
    pFDX4156 As pFDX4151, only lacI fused to M13gene8 -λ cI ′ This report
    pFDX4157 As pFDX4152, but RBS of phage T7gene10 in front end of φ( M13gene8 - lacI ) This study
    pFDX4158 As pFDX4154, merely RBS of phage T7gene10 in front of φ( galK ′- lacI ) This study
    pFDX4159 As pFDX4155, just RBS of phage T7gene10 in forepart of φ( galK ′-λ cI ′ -lacI ) This study
    pFDX4160 Equally pFDX4156, merely RBS of phage T7gene10 in front of φ( M13gene8 -λ cI ′- lacI ) This study
    pFDX4165 As pFDX4151, but RBS of phage T7gene10 in forepart of lacI This study
    pLDR10 Multiple cloning site, λ attP , bla , cat , ori pMB1 ( 14 )

Table i

Strains and plasmids used in this report

Genotype or relevant structures Source or reference
Strains
    R1279 CSH50 Δ( pho-bgl )201 Δ( lac-pro ) ara thi ( sixteen )
    R2171 As R1279 just attB ::[ tacOP - lacZ , bla ]; RBS of phage T7gene10 in front end of lacZ pFDX3552→R1279
Plasmids
    M13mp18 M13 phage cloning vector ( 28 )
    pFDX500 lacI , neo , ori p15A ( eleven )
    pFDX3225 φ( galK ′ -bglG ) nether control of Ptac , tet , ori p15A ( 12 )
    pFDX3401 λ int nether command of λP R , λ cI 857 ( cat ; ori pSC101- rep TS ) ( 12 )
    pFDX3551 pLDR10 derivative for the insertion of a tacOP - lacZ cassette into the attB site; RBS of gene bglG in front end of lacZ , bla , ori pMB1 This written report
    pFDX3552 As pFDX3551, only RBS of phage T7gene10 in front of lacZ This study
    pFDX4151 As pFDX500, simply with a NdeI site overlapping with the lacI ATG kickoff codon This study
    pFDX4152 Equally pFDX4151, but lacI fused to M13gene8 This study
    pFDX4153 As pFDX4151, but lacI fused to λ cI ′ (codons 96–132) This study
    pFDX4154 As pFDX4151, but lacI fused to galK ′ (codons i–32) This written report
    pFDX4155 Every bit pFDX4151, but lacI fused to galK ′-λ cI ′ This written report
    pFDX4156 As pFDX4151, but lacI fused to M13gene8 -λ cI ′ This written report
    pFDX4157 As pFDX4152, merely RBS of phage T7gene10 in front of φ( M13gene8 - lacI ) This study
    pFDX4158 As pFDX4154, simply RBS of phage T7gene10 in front of φ( galK ′- lacI ) This study
    pFDX4159 Equally pFDX4155, but RBS of phage T7gene10 in front of φ( galK ′-λ cI ′ -lacI ) This study
    pFDX4160 As pFDX4156, simply RBS of phage T7gene10 in front of φ( M13gene8 -λ cI ′- lacI ) This written report
    pFDX4165 Equally pFDX4151, but RBS of phage T7gene10 in forepart of lacI This report
    pLDR10 Multiple cloning site, λ attP , bla , cat , ori pMB1 ( xiv )
Genotype or relevant structures Source or reference
Strains
    R1279 CSH50 Δ( pho-bgl )201 Δ( lac-pro ) ara thi ( 16 )
    R2171 Equally R1279 simply attB ::[ tacOP - lacZ , bla ]; RBS of phage T7gene10 in front of lacZ pFDX3552→R1279
Plasmids
    M13mp18 M13 phage cloning vector ( 28 )
    pFDX500 lacI , neo , ori p15A ( xi )
    pFDX3225 φ( galK ′ -bglG ) under command of Ptac , tet , ori p15A ( 12 )
    pFDX3401 λ int nether control of λP R , λ cI 857 ( cat ; ori pSC101- rep TS ) ( 12 )
    pFDX3551 pLDR10 derivative for the insertion of a tacOP - lacZ cassette into the attB site; RBS of gene bglG in front of lacZ , bla , ori pMB1 This study
    pFDX3552 As pFDX3551, but RBS of phage T7gene10 in front of lacZ This study
    pFDX4151 As pFDX500, simply with a NdeI site overlapping with the lacI ATG get-go codon This study
    pFDX4152 Equally pFDX4151, but lacI fused to M13gene8 This written report
    pFDX4153 Every bit pFDX4151, just lacI fused to λ cI ′ (codons 96–132) This study
    pFDX4154 Equally pFDX4151, but lacI fused to galK ′ (codons one–32) This study
    pFDX4155 As pFDX4151, only lacI fused to galK ′-λ cI ′ This written report
    pFDX4156 As pFDX4151, simply lacI fused to M13gene8 -λ cI ′ This study
    pFDX4157 As pFDX4152, but RBS of phage T7gene10 in front of φ( M13gene8 - lacI ) This study
    pFDX4158 Equally pFDX4154, only RBS of phage T7gene10 in forepart of φ( galK ′- lacI ) This written report
    pFDX4159 As pFDX4155, only RBS of phage T7gene10 in front of φ( galK ′-λ cI ′ -lacI ) This study
    pFDX4160 As pFDX4156, but RBS of phage T7gene10 in front of φ( M13gene8 -λ cI ′- lacI ) This study
    pFDX4165 Equally pFDX4151, only RBS of phage T7gene10 in front of lacI This written report
    pLDR10 Multiple cloning site, λ attP , bla , cat , ori pMB1 ( fourteen )

Effigy one

Synthesis and stability of LacI derivatives. Strain R1279 was transformed with plasmid pFDX4160 carrying the φ( gene8 -λ cI ′ -lacI ) fusion gene (lanes 13–18) or with plasmid pFDX4157 carrying the φ( gene8 - lacI ) fusion gene (lanes 19–24) under control of the wild-type lacI promoter, respectively. For comparison, a transformant with the isogenic plasmid pFDX4165 carrying wild-type lacI (lanes 7–12) as well as the untransformed strain (lanes 1–6) were also employed. Aliquots of the cultures were pulse-labeled with [ 35 S]methionine and subsequently chased with unlabeled methionine for the times indicated in the figure. Proteins were separated by SDS–PAGE and analyzed by autoradiography and phospho-imager analysis. The positions of molecular weight standards are given on the left (in kDa).

Synthesis and stability of LacI derivatives. Strain R1279 was transformed with plasmid pFDX4160 carrying the φ( gene8 -λ cI ′ -lacI ) fusion cistron (lanes 13–18) or with plasmid pFDX4157 carrying the φ( gene8 - lacI ) fusion gene (lanes 19–24) under control of the wild-type lacI promoter, respectively. For comparison, a transformant with the isogenic plasmid pFDX4165 carrying wild-type lacI (lanes vii–12) as well equally the untransformed strain (lanes 1–half-dozen) were as well employed. Aliquots of the cultures were pulse-labeled with [ 35 S]methionine and subsequently chased with unlabeled methionine for the times indicated in the figure. Proteins were separated past SDS–PAGE and analyzed by autoradiography and phospho-imager analysis. The positions of molecular weight standards are given on the left (in kDa).

Effigy 1

Synthesis and stability of LacI derivatives. Strain R1279 was transformed with plasmid pFDX4160 carrying the φ( gene8 -λ cI ′ -lacI ) fusion gene (lanes 13–18) or with plasmid pFDX4157 carrying the φ( gene8 - lacI ) fusion gene (lanes 19–24) under control of the wild-type lacI promoter, respectively. For comparison, a transformant with the isogenic plasmid pFDX4165 carrying wild-type lacI (lanes 7–12) as well as the untransformed strain (lanes 1–6) were also employed. Aliquots of the cultures were pulse-labeled with [ 35 S]methionine and subsequently chased with unlabeled methionine for the times indicated in the figure. Proteins were separated by SDS–PAGE and analyzed by autoradiography and phospho-imager analysis. The positions of molecular weight standards are given on the left (in kDa).

Synthesis and stability of LacI derivatives. Strain R1279 was transformed with plasmid pFDX4160 conveying the φ( gene8 -λ cI ′ -lacI ) fusion gene (lanes 13–18) or with plasmid pFDX4157 conveying the φ( gene8 - lacI ) fusion gene (lanes 19–24) under control of the wild-type lacI promoter, respectively. For comparing, a transformant with the isogenic plasmid pFDX4165 carrying wild-type lacI (lanes vii–12) likewise equally the untransformed strain (lanes i–6) were besides employed. Aliquots of the cultures were pulse-labeled with [ 35 S]methionine and subsequently chased with unlabeled methionine for the times indicated in the figure. Proteins were separated past SDS–Folio and analyzed by autoradiography and phospho-imager analysis. The positions of molecular weight standards are given on the left (in kDa).

Figure ii

Subcellular fractionation demonstrating that both, the φ(Gene8–LacI) as well as the φ(Gene8–λCI′–LacI) fusion proteins, are membrane-anchored. The untransformed strain R1279 as well as its various transformants ( Figure 1 ) were labeled with [ 35 S]methionine. Subsequently the cells were gently lysed, and half of the lysates were subjected to sucrose gradient centrifugation to separate soluble (S) and membrane (M) proteins. The remaining halves were kept on ice during the fractionation procedure and served as samples representing total protein (T). The samples were separated by SDS–PAGE, followed by autoradiography and phospho-imager analysis. Lanes 1–3, strain R1279 (untransformed); lanes 4–6, R1279/pFDX4165 (wild-type lacI ); lanes 7–9, R1279/pFDX4157 [φ( gene8-lacI )]; lanes 10–12, R1279/pFDX4160 [φ( gene8 -λ cI ′ -lacI )]. The positions of molecular weight standards are given on the left (in kDa), and the positions of the respective LacI derivatives are indicated by arrows on the right.

Subcellular fractionation demonstrating that both, the φ(Gene8–LacI) equally well as the φ(Gene8–λCI′–LacI) fusion proteins, are membrane-anchored. The untransformed strain R1279 every bit well as its various transformants ( Figure one ) were labeled with [ 35 Due south]methionine. Subsequently the cells were gently lysed, and half of the lysates were subjected to sucrose gradient centrifugation to separate soluble (S) and membrane (M) proteins. The remaining halves were kept on ice during the fractionation process and served as samples representing total poly peptide (T). The samples were separated past SDS–Page, followed by autoradiography and phospho-imager analysis. Lanes 1–3, strain R1279 (untransformed); lanes 4–6, R1279/pFDX4165 (wild-type lacI ); lanes seven–9, R1279/pFDX4157 [φ( gene8-lacI )]; lanes 10–12, R1279/pFDX4160 [φ( gene8 -λ cI ′ -lacI )]. The positions of molecular weight standards are given on the left (in kDa), and the positions of the corresponding LacI derivatives are indicated by arrows on the right.

Figure 2

Subcellular fractionation demonstrating that both, the φ(Gene8–LacI) as well as the φ(Gene8–λCI′–LacI) fusion proteins, are membrane-anchored. The untransformed strain R1279 as well as its various transformants ( Figure 1 ) were labeled with [ 35 S]methionine. Subsequently the cells were gently lysed, and half of the lysates were subjected to sucrose gradient centrifugation to separate soluble (S) and membrane (M) proteins. The remaining halves were kept on ice during the fractionation procedure and served as samples representing total protein (T). The samples were separated by SDS–PAGE, followed by autoradiography and phospho-imager analysis. Lanes 1–3, strain R1279 (untransformed); lanes 4–6, R1279/pFDX4165 (wild-type lacI ); lanes 7–9, R1279/pFDX4157 [φ( gene8-lacI )]; lanes 10–12, R1279/pFDX4160 [φ( gene8 -λ cI ′ -lacI )]. The positions of molecular weight standards are given on the left (in kDa), and the positions of the respective LacI derivatives are indicated by arrows on the right.

Subcellular fractionation demonstrating that both, the φ(Gene8–LacI) likewise as the φ(Gene8–λCI′–LacI) fusion proteins, are membrane-anchored. The untransformed strain R1279 as well as its various transformants ( Figure 1 ) were labeled with [ 35 South]methionine. Subsequently the cells were gently lysed, and half of the lysates were subjected to sucrose gradient centrifugation to dissever soluble (S) and membrane (M) proteins. The remaining halves were kept on ice during the fractionation process and served as samples representing full protein (T). The samples were separated by SDS–PAGE, followed past autoradiography and phospho-imager assay. Lanes ane–3, strain R1279 (untransformed); lanes 4–6, R1279/pFDX4165 (wild-blazon lacI ); lanes seven–9, R1279/pFDX4157 [φ( gene8-lacI )]; lanes x–12, R1279/pFDX4160 [φ( gene8 -λ cI ′ -lacI )]. The positions of molecular weight standards are given on the left (in kDa), and the positions of the corresponding LacI derivatives are indicated by arrows on the right.

Figure 3

The membrane-anchored LacI fusion proteins regulate expression of a plasmid encoded tacOP-lacZ reporter cassette in an inducer-dependent manner. Strain R1279 was transformed with reporter plasmid pFDX3551 which carries the tacOP-lacZ cassette and in addition with one of various plasmids delivering LacI or its fusion derivatives. β-Galactosidase activities of these transformants were determined in the absence or presence for 2 h of IPTG, as indicated. Bars 1: R1279/pFDX3551 (single transformant); bars 2: R1279/pFDX3551/pFDX500 (wild-type lacI ); bars 3: R1279/pFDX3551/pFDX4151 (wild-type lacI with ATG-start codon and NdeI site); bars 4: R1279/pFDX3551/pFDX4154 [φ( galK-lacI )]; bars 5: R1279/pFDX3551/pFDX4152 [φ( gene8-lacI )]; bars 6: R1279/pFDX3551/pFDX4155 [φ( galK -λ cI ′ -lacI )]; and bars 7: R1279/pFDX3551/pFDX4156 [φ( gene8 -λ cI ′ -lacI )]. The reason for the high enzyme activities seen in the absence of a Lac repressor delivering plasmid (bars 1) as compared with those obtained in its presence when IPTG as inducer was additionally present is unclear. Similar high activities were seen when the vector plasmid without a lacI gene was present (data not shown). One reason could be that induction by IPTG is incomplete.

The membrane-anchored LacI fusion proteins regulate expression of a plasmid encoded tacOP-lacZ reporter cassette in an inducer-dependent manner. Strain R1279 was transformed with reporter plasmid pFDX3551 which carries the tacOP-lacZ cassette and in addition with one of various plasmids delivering LacI or its fusion derivatives. β-Galactosidase activities of these transformants were determined in the absence or presence for 2 h of IPTG, as indicated. Bars ane: R1279/pFDX3551 (single transformant); confined ii: R1279/pFDX3551/pFDX500 (wild-type lacI ); bars iii: R1279/pFDX3551/pFDX4151 (wild-type lacI with ATG-start codon and NdeI site); bars 4: R1279/pFDX3551/pFDX4154 [φ( galK-lacI )]; bars v: R1279/pFDX3551/pFDX4152 [φ( gene8-lacI )]; confined half-dozen: R1279/pFDX3551/pFDX4155 [φ( galK -λ cI ′ -lacI )]; and bars 7: R1279/pFDX3551/pFDX4156 [φ( gene8 -λ cI ′ -lacI )]. The reason for the high enzyme activities seen in the absence of a Lac repressor delivering plasmid (bars ane) as compared with those obtained in its presence when IPTG as inducer was additionally nowadays is unclear. Like loftier activities were seen when the vector plasmid without a lacI gene was present (information not shown). 1 reason could be that induction by IPTG is incomplete.

Effigy 3

The membrane-anchored LacI fusion proteins regulate expression of a plasmid encoded tacOP-lacZ reporter cassette in an inducer-dependent manner. Strain R1279 was transformed with reporter plasmid pFDX3551 which carries the tacOP-lacZ cassette and in addition with one of various plasmids delivering LacI or its fusion derivatives. β-Galactosidase activities of these transformants were determined in the absence or presence for 2 h of IPTG, as indicated. Bars 1: R1279/pFDX3551 (single transformant); bars 2: R1279/pFDX3551/pFDX500 (wild-type lacI ); bars 3: R1279/pFDX3551/pFDX4151 (wild-type lacI with ATG-start codon and NdeI site); bars 4: R1279/pFDX3551/pFDX4154 [φ( galK-lacI )]; bars 5: R1279/pFDX3551/pFDX4152 [φ( gene8-lacI )]; bars 6: R1279/pFDX3551/pFDX4155 [φ( galK -λ cI ′ -lacI )]; and bars 7: R1279/pFDX3551/pFDX4156 [φ( gene8 -λ cI ′ -lacI )]. The reason for the high enzyme activities seen in the absence of a Lac repressor delivering plasmid (bars 1) as compared with those obtained in its presence when IPTG as inducer was additionally present is unclear. Similar high activities were seen when the vector plasmid without a lacI gene was present (data not shown). One reason could be that induction by IPTG is incomplete.

The membrane-anchored LacI fusion proteins regulate expression of a plasmid encoded tacOP-lacZ reporter cassette in an inducer-dependent manner. Strain R1279 was transformed with reporter plasmid pFDX3551 which carries the tacOP-lacZ cassette and in add-on with 1 of various plasmids delivering LacI or its fusion derivatives. β-Galactosidase activities of these transformants were adamant in the absenteeism or presence for 2 h of IPTG, every bit indicated. Confined 1: R1279/pFDX3551 (single transformant); confined 2: R1279/pFDX3551/pFDX500 (wild-type lacI ); bars iii: R1279/pFDX3551/pFDX4151 (wild-type lacI with ATG-offset codon and NdeI site); bars 4: R1279/pFDX3551/pFDX4154 [φ( galK-lacI )]; bars 5: R1279/pFDX3551/pFDX4152 [φ( gene8-lacI )]; bars 6: R1279/pFDX3551/pFDX4155 [φ( galK -λ cI ′ -lacI )]; and bars 7: R1279/pFDX3551/pFDX4156 [φ( gene8 -λ cI ′ -lacI )]. The reason for the high enzyme activities seen in the absence of a Lac repressor delivering plasmid (bars one) every bit compared with those obtained in its presence when IPTG as inducer was additionally present is unclear. Similar loftier activities were seen when the vector plasmid without a lacI gene was present (data not shown). One reason could be that induction past IPTG is incomplete.

Figure four

Repression and de-repression of a chromosomal tacOP-lacZ reporter cassette by the membrane-anchored LacI fusion proteins. Strain R2171 carrying a tacOP-lacZ reporter cassette integrated into the λ attB site in the chromosome was transformed with the various plasmids carrying wild-type lacI and its derivatives, respectively. Subsequently, β-galactosidase activities were determined as in Figure 3 . Bars 1: R2171 (untransformed strain); bars 2: R2171/pFDX500 (wild-type lacI ); bars 3: R2171/pFDX4151 (wild-type lacI with ATG-start codon and NdeI site); bars 4: R2171/pFDX4154 [φ( galK-lacI )]; bars 5: R2171/pFDX4152 [φ( gene8-lacI )]; bars 6: R2171/pFDX4155 [φ( galK -λ cI ′ -lacI )]; and bars 7: R2171/pFDX4156 [φ( gene8 -λ cI ′ -lacI )].

Repression and de-repression of a chromosomal tacOP-lacZ reporter cassette by the membrane-anchored LacI fusion proteins. Strain R2171 carrying a tacOP-lacZ reporter cassette integrated into the λ attB site in the chromosome was transformed with the various plasmids carrying wild-type lacI and its derivatives, respectively. Afterward, β-galactosidase activities were adamant as in Effigy 3 . Bars 1: R2171 (untransformed strain); confined 2: R2171/pFDX500 (wild-type lacI ); bars 3: R2171/pFDX4151 (wild-type lacI with ATG-outset codon and NdeI site); confined iv: R2171/pFDX4154 [φ( galK-lacI )]; confined 5: R2171/pFDX4152 [φ( gene8-lacI )]; bars 6: R2171/pFDX4155 [φ( galK -λ cI ′ -lacI )]; and bars 7: R2171/pFDX4156 [φ( gene8 -λ cI ′ -lacI )].

Effigy iv

Repression and de-repression of a chromosomal tacOP-lacZ reporter cassette by the membrane-anchored LacI fusion proteins. Strain R2171 carrying a tacOP-lacZ reporter cassette integrated into the λ attB site in the chromosome was transformed with the various plasmids carrying wild-type lacI and its derivatives, respectively. Subsequently, β-galactosidase activities were determined as in Figure 3 . Bars 1: R2171 (untransformed strain); bars 2: R2171/pFDX500 (wild-type lacI ); bars 3: R2171/pFDX4151 (wild-type lacI with ATG-start codon and NdeI site); bars 4: R2171/pFDX4154 [φ( galK-lacI )]; bars 5: R2171/pFDX4152 [φ( gene8-lacI )]; bars 6: R2171/pFDX4155 [φ( galK -λ cI ′ -lacI )]; and bars 7: R2171/pFDX4156 [φ( gene8 -λ cI ′ -lacI )].

Repression and de-repression of a chromosomal tacOP-lacZ reporter cassette by the membrane-anchored LacI fusion proteins. Strain R2171 carrying a tacOP-lacZ reporter cassette integrated into the λ attB site in the chromosome was transformed with the various plasmids conveying wild-type lacI and its derivatives, respectively. Subsequently, β-galactosidase activities were determined equally in Figure 3 . Bars 1: R2171 (untransformed strain); confined ii: R2171/pFDX500 (wild-type lacI ); bars 3: R2171/pFDX4151 (wild-type lacI with ATG-start codon and NdeI site); bars 4: R2171/pFDX4154 [φ( galK-lacI )]; bars 5: R2171/pFDX4152 [φ( gene8-lacI )]; bars 6: R2171/pFDX4155 [φ( galK -λ cI ′ -lacI )]; and bars seven: R2171/pFDX4156 [φ( gene8 -λ cI ′ -lacI )].

Effigy 5

Kinetics of establishment of repression by the membrane-anchored LacI fusion proteins at the chromosomal tacOP-lacZ cassette. Strain R2171 and its various transformants ( Figure 4 ) were pregrown in the presence of 1 mM IPTG. Subsequently, the cells were washed free of IPTG and growth was continued in IPTG-free medium. Samples were taken after the removal of IPTG at the times indicated and β-galactosidase activities were determined.

Kinetics of establishment of repression past the membrane-anchored LacI fusion proteins at the chromosomal tacOP-lacZ cassette. Strain R2171 and its various transformants ( Effigy 4 ) were pregrown in the presence of ane mM IPTG. Subsequently, the cells were washed complimentary of IPTG and growth was connected in IPTG-free medium. Samples were taken after the removal of IPTG at the times indicated and β-galactosidase activities were adamant.

Effigy 5

Kinetics of establishment of repression by the membrane-anchored LacI fusion proteins at the chromosomal tacOP-lacZ cassette. Strain R2171 and its various transformants ( Figure 4 ) were pregrown in the presence of 1 mM IPTG. Subsequently, the cells were washed free of IPTG and growth was continued in IPTG-free medium. Samples were taken after the removal of IPTG at the times indicated and β-galactosidase activities were determined.

Kinetics of establishment of repression by the membrane-anchored LacI fusion proteins at the chromosomal tacOP-lacZ cassette. Strain R2171 and its various transformants ( Figure 4 ) were pregrown in the presence of 1 mM IPTG. Subsequently, the cells were washed free of IPTG and growth was continued in IPTG-free medium. Samples were taken afterwards the removal of IPTG at the times indicated and β-galactosidase activities were determined.

Present addresses: Boris Görke, Abteilung für Allgemeine Mikrobiologie, Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, Grisebachstrasse viii, D-37077 Göttingen, Federal republic of germany

Jana Reinhardt, CellGenix Technologie Transfer GmbH, Am Fughafen 16, D-79108 Freiburg, Deutschland

The authors wish it to be known that, in their opinion, the first two authors should exist regarded as joint First Authors

We give thanks Chiliad. Igloi for Deoxyribonucleic acid sequencing. This work was supported by grants from the Landesschwerpunkt Baden-Württemberg, the Graduiertenkolleg 'Biochemie der Enzyme' (DFG) and the Fonds der Chemischen Industrie to B.R. Funding to pay the Open Admission publication charges for this article was provided by the Land Baden-Württemberg.

Disharmonize of interest statement . None alleged.

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