Substitutions at Auxiliary Operator O3 Enhance Repression by Nitrate-Responsive Regulator NarL at Synthetic lac Control Regions in Escherichia coli K-12

Abstract

We constructed monocopy lac operon control regions in which the operators O1-lac and O3-lac were replaced by NarL and NarP binding sites from the nirB or napF operon control regions. The results support the hypothesis that DNA-bound dimers of phospho-NarL can participate in higher-order cooperative interactions.

Anaerobic respiratory gene expression in Escherichia coli K-12 is controlled in part by nitrate and nitrite, acting through the Nar regulatory system. The paralogous sensors NarX and NarQ respond to nitrate and nitrite to control phosphorylation of the paralogous response regulators NarL and NarP. Phosphorylation increases the affinity of the NarL and NarP proteins for their specific DNA binding sites, from whence they activate and repress target operon expression (for a review, see reference 39).

Most phospho-NarL and -NarP binding sites consist of inverted heptamer sequences (consensus sequence, TACYYMT, where Y = C or T and M = A or C) separated by 2 nucleotides (nt) (11, 24). Binding sites with this geometry are termed 7-2-7 heptamer pairs. Specificity determinants that discriminate between binding by one regulator or the other have not yet been defined, although most sites characterized to date appear to have greater affinity for the phospho-NarL protein (11, 37).

The control regions for many nitrate-regulated operons contain heptamer sequences in addition to an essential 7-2-7 heptamer pair (for a review, see reference 39). These extra heptamers are critical for NarL-dependent regulation of nrfA and fdnG operon expression (10, 11, 19, 20, 42). Evidence suggests that a dimer of the phospho-NarL protein bound to the 7-2-7 heptamer pair has cooperative interactions with additional phospho-NarL molecules, thereby promoting their binding to the extra heptamer sequences. By contrast, phospho-NarP dimers apparently interact weakly if at all and thus are restricted to binding single 7-2-7 heptamer pairs (10, 11).

Transcription of the lacZYA operon for lactose catabolism is repressed by the LacI protein. The primary operator (O1-lac) for LacI repressor binding consists of inverted half-sites centered at position 11 with respect to the transcription initiation site (Fig. 1). At least one of the two auxiliary operators is required for maximal repression (32). LacI repressor binds cooperatively to the primary operator and to one of the auxiliary operators, thereby increasing its local concentration (for a review, see reference 29).

FIG. 1.

Control region sequences. The modified lac operon control region sequence from O3 through O1 is shown at the top. The complete sequence for the lac operon control region used has been described previously (37). Sequences of O1 and O3 substitutions are indicated below the corresponding lac operators. The deletion and insertions were employed to make the spacing variants used for the experiments whose results are shown in Fig. 2 and Tables 2 to 4. The deletion is indicated by a vertical line, whereas inserted sequences are enclosed in boxes. The transcription initiation site is labeled +1. The arrowheads indicate centers of protein-binding half-sites.

Previously, we described construction of synthetic lac operon control regions in which the primary operator O1 is replaced by 7-2-7 heptamer pairs from different Nar-regulated operons (37). Transcription from these constructs is repressed by the phospho-NarL and -NarP proteins in response to added nitrate. Here, we describe additional constructs in which the auxiliary operator O3 was also replaced by different 7-2-7 heptamer pairs. In narL⁺ strains, these double-operator-substituted constructs exhibited increased repression, supporting the hypothesis that two dimers of the phospho-NarL protein bind cooperatively to specific DNA sites.

Strains.

Strains are listed in Table 1. Construction of the synthetic lac operon control region has been described previously (37). New versions reported here, with substitutions at operator O3, were constructed by the same methods. All constructs were based on lacZ gene fusion plasmid pVJS3253 (37) and were crossed into bacteriophage λInCh (5).

TABLE 1.

E. coli K-12 strains

Strain	Genotype	Reference
VJS632	F− λ− prototroph	38
VJS676	As VJS632 but Δ(argF-lacIZYA)U169	38
VJS8364	As VJS632 but ΔlacZ	21
Derivatives of strain VJS676
VJS7449	λ− Δ(attλ-lom)::bla [O3-lac O1-napF lacZ+]	37
VJS7475	λ− Δ(attλ-lom)::bla [O3-lac O1-napF lacZ+]/narL215::Tn10	37
VJS7476	λ− Δ(attλ-lom)::bla [O3-lac O1-napF lacZ+]/narP253::Tn10d(Cm)	37
VJS7446	λ− Δ(attλ-lom)::bla [O3-nirB O1-napF lacZ+]	This study
VJS7473	λ− Δ(attλ-lom)::bla [O3-nirB O1-napF lacZ+]/narL215::Tn10	This study
VJS7474	λ− Δ(attλ-lom)::bla [O3-nirB O1-napF lacZ+]/narP253::Tn10d(Cm)	This study
VJS7902	λ− Δ(attλ-lom)::bla [O3-nirB O1-napF (95 nt) lacZ+]	This study
VJS7922	λ− Δ(attλ-lom)::bla [O3-nirB O1-napF (102 nt) lacZ+]	This study
VJS7923	λ− Δ(attλ-lom)::bla [O3-nirB O1-napF (106 nt) lacZ+]	This study
VJS7929	λ− Δ(attλ-lom)::bla [O3-nirB O1-napF (110 nt) lacZ+]	This study
VJS8206	λ− Δ(attλ-lom)::bla [O3-nirB O1-napF (95 nt) lacZ+]/narL215::Tn10	This study
VJS8207	λ− Δ(attλ-lom)::bla [O3-nirB O1-napF (95 nt) lacZ+]/narP253::Tn10d(Cm)	This study
Derivatives of strain VJS8364
VJS8880	λ− Δ(attλ-lom)::bla [O3-lac O1-napF lacZ+]	This study
VJS8881	λ− Δ(attλ-lom)::bla [O3-lac O1-napF lacZ+]/ΔnarL261	This study
VJS8882	λ− Δ(attλ-lom)::bla [O3-lac O1-napF lacZ+]/ΔnarP262	This study
VJS8883	λ− Δ(attλ-lom)::bla [O3-napF O1-napF lacZ+]	This study
VJS8884	λ− Δ(attλ-lom)::bla [O3-napF O1-napF lacZ+]/ΔnarL261	This study
VJS8885	λ− Δ(attλ-lom)::bla [O3-napF O1-napF lacZ+]/ΔnarP262	This study
VJS8886	λ− Δ(attλ-lom)::bla [O3-lac O1-lac lacZ+]	This study
VJS8899	λ− Δ(attλ-lom)::bla [O3-napF O1-napF (87 nt) lacZ+]	This study
VJS9000	λ− Δ(attλ-lom)::bla [O3-napF O1-napF (87 nt) lacZ+]/ΔnarP262	This study
VJS9001	λ− Δ(attλ-lom)::bla [O3-napF O1-napF (87 nt) lacZ+]/ΔnarL261	This study
VJS9002	λ− Δ(attλ-lom)::bla [O3-napF O1-lac (87 nt) lacZ+]	This study
VJS9003	λ− Δ(attλ-lom)::bla [O3-napF O1-lac lacZ+]	This study
VJS9004	λ− Δ(attλ-lom)::bla [O3-lac O1-napF lacZ+]/ΔnarL261 ΔnarP262	This study
VJS9005	λ− Δ(attλ-lom)::bla [O3-napF O1-napF lacZ+]/ΔnarL261 ΔnarP262	This study
VJS9006	λ− Δ(attλ-lom)::bla [O3-napF O1-napF (87 nt) lacZ+]/ΔnarL261 ΔnarP262	This study

Genetic crosses were performed by bacteriophage P1kc-mediated generalized transduction (26). Null alleles of nar regulatory genes (Table 1) have been described previously (21, 33). Standard methods were used for restriction endonuclease digestion, ligation, transformation, and PCR amplification of DNA (22).

Culture media and conditions.

Defined, complex, and indicator media for genetic manipulations were used as described previously (22). The defined medium used to grow cultures for enzyme assays was buffered with 3-(N-morpholino)propanesulfonic acid (MOPS) as described previously (38). The medium used for overnight cultures arrested in the mid-exponential phase (13) contained glucose (6 mM) or glucose plus NaNO₃ (4 and 10 mM, respectively) (37). The concentrations were determined empirically to support growth to the mid-exponential phase (about 35 to 40 Klett units). Isopropyl-β-d-thiogalactoside (IPTG) was added as indicated to inactivate the LacI repressor in cultures of O1-lac derivatives.

Cultures were grown at 37°C. Culture densities were monitored with a Klett-Summerson photoelectric colorimeter (Klett Manufacturing Co., New York, NY) equipped with a number 66 (red) filter. Anaerobic cultures used for enzyme assays were grown in screw-cap tubes as described previously (38).

Enzyme assay.

β-Galactosidase activities were determined at room temperature (approximately 21°C) by monitoring the hydrolysis of o-nitrophenyl-β-d-galactoside in CHCl₃-sodium dodecyl sulfate-permeabilized cells. Specific activities are expressed in arbitrary units (26). All cultures were assayed in duplicate, and the reported values are averages from at least two independent experiments.

Nar-dependent repression of lacZ gene expression from O1 substitution control regions.

Previously, we used site-specific mutagenesis to replace the primary operator O1-lac with 7-2-7 heptamer pairs from different Nar-responsive control regions (37). One of the constructs, the O1-nirB construct, exhibits about 100-fold repression of lacZ gene expression in response to nitrate, whereas the O1-napF construct exhibits only about 10-fold repression. However, the O1-nirB construct displays only about fivefold repression in a narP⁺ narL null strain, indicating that the phospho-NarP protein binds relatively poorly at O1-nirB. By contrast, the O1-napF construct displays similar repression in both narL⁺ narP null and narP⁺ narL null strains, indicating that the phospho-NarL and -NarP proteins bind equally well at O1-napF (37).

For the experiments described below, we used constructs with the O1-napF substitution so that (i) enhanced repression could be more readily detected and (ii) relative levels of repression by the phospho-NarL and -NarP proteins could be compared directly.

Enhanced repression by the O3-nirB substitution.

The LacI repressor consists of two DNA-binding dimers assembled as a homotetramer (for reviews, see references 7, 18, 29, and 47). Full repression of lac operon expression requires not only the high-affinity primary operator O1-lac but also at least one of two lower-affinity auxiliary operators, O2-lac (within the lacZ gene) or O3-lac (32). Occupancy of the operator O1-lac by one dimer is increased through cooperative binding of the other dimer to an auxiliary operator (6, 32, 34), resulting in a more stable repressor complex (14, 17, 46). This cooperative binding accounts for the nonlinear response of lacZYA operon transcription to an inducer (30).

Mutant LacI repressors that form essentially normal dimers but fail to assemble into tetramers have been described (1, 2, 6, 8, 32). Mutant dimeric LacI proteins and wild-type LacI protein both repress transcription from O1-lac to about the same extent. However, repression by a dimeric repressor is not enhanced by the presence of an auxiliary operator (27, 32), because the repressor does not bind cooperatively to two operators (6, 8, 23, 32). Thus, specific interaction between two dimers, each bound at a separate operator, enhances the overall stability of the repression complex. Analogous observations have been made with other repressors (9, 35), including the GalR repressor (15, 23, 36).

Therefore, we reasoned that the lac operon control region might provide a means to study specific interactions between phospho-NarL dimers (23). Accordingly, we used site-specific mutagenesis to replace the auxiliary operator O3-lac (positions −92 through −72 [Fig. 1]) with 7-2-7 heptamer pairs from the nirB or napF control regions. These operators were placed in the O1-napF, O1-nirB, O1-fdnG, and O1-lac constructs (Fig. 1), forming various combinations of O3- and O1-substituted lac control regions.

In a narL⁺ narP⁺ background, the O3-lac O1-napF construct exhibited about 10-fold repression of lacZ gene expression in response to nitrate, whereas the O3-nirB construct exhibited about 30-fold repression (Table 2). Similar results were observed with the congruent O1-nirB and O1-fdnG constructs (data not shown). Thus, placing a Nar 7-2-7 heptamer pair in operator O3 resulted in a modest enhancement of nitrate repression.

TABLE 2.

Interoperator spacing influences O3-nirB enhancement of NarL-mediated repression at O1-napF

Strain	Construct	Spacinga	Genotype		LacZ sp act (arbitrary units)b		Repression (fold)
			narL	narP	No NO3−	NO3−
VJS7449	O3-lac O1-napF	91	+	+	850	66	13
VJS7476	O3-lac O1-napF	91	+	−	850	71	12
VJS7475	O3-lac O1-napF	91	−	+	920	100	9.2
VJS7446	O3-nirB O1-napF	91	+	+	790	24	33
VJS7474	O3-nirB O1-napF	91	+	−	970	20	49
VJS7473	O3-nirB O1-napF	91	−	+	950	100	10
VJS7902	O3-nirB O1-napF	95	+	+	800	4	200
VJS8207	O3-nirB O1-napF	95	+	−	830	4	208
VJS8206	O3-nirB O1-napF	95	−	+	820	70	12

^aInteroperator spacing (see Fig. 1).

^bStrains were cultured overnight in MOPS defined medium with limiting glucose.

We next introduced narL or narP null alleles into the strains. The O3-nirB-dependent enhanced repression of lacZ gene expression was eliminated in the narP⁺ narL null strain but increased to nearly 50-fold in the narL⁺ narP null strain (Table 2). Thus, the O3-nirB-dependent enhanced repression of lacZ gene expression required the narL⁺ gene and appeared to be slightly inhibited by the narP⁺ gene.

Interoperator spacing affects enhanced repression by the O3-nirB substitution.

Spacing between the O3-lac and O1-lac operators is critical for effective repression, because the repressor-operator interaction depends on the helical phase of the two operators in order to allow formation of the intervening DNA loop (14, 17, 28, 29, 31). Loop formation (and therefore cooperative repressor binding) is also influenced by factors such as DNA superhelicity (12, 16, 34, 40, 44, 45) and host proteins (3, 27).

The wild-type interoperator spacing between operators O1-lac and O3-lac is nearly optimal for cooperative LacI repression (28). (Our constructs have a slightly shorter interoperator space, 91 versus 92.5 nt [Fig. 1].) We wished to determine the optimal spacing for cooperative phospho-NarL repression. We therefore varied the spacing between the O3-nirB and O1-napF operators over the range from 95 to 110 nt (Fig. 1). The variant with 95-nt spacing was constructed by filling in the 4-nt BglII overhang with Klenow polymerase, and the remaining variants were constructed by consecutive rounds of site-specific mutagenesis.

The interoperator spacing strongly affected O3-nirB-dependent enhanced repression of lacZ gene expression (Fig. 2). For the O3-nirB O1-napF constructs with 91-nt (native) and 102-nt spacing, nitrate repression of lacZ gene expression was about 30-fold (Table 2), whereas for the constructs with 95- and 106-nt spacing, nitrate repression of lacZ gene expression was about 100-fold (Fig. 2). The level of nitrate repression of lacZ gene expression for the construct with 110-nt spacing was intermediate, about 60-fold. These results, based on the limited number of spacing variants examined (28), are consistent with the notion that the helical phase influences the magnitude of the O3-nirB-dependent enhanced nitrate repression of lacZ gene expression (for a review, see reference 25).

FIG. 2.

Interoperator spacing influences O3-nirB enhancement of repression at O1-napF. The fold repression of lacZ gene expression by nitrate is plotted against the distance between the centers of operator elements O1-napF and O3-nirB. The dashed line indicates the ≤15-fold repression observed for the O3-lacZ O1-napF construct (see Table 2). Strains carry both narL⁺ and narP⁺ alleles.

The experiments described above indicated that the native O3-O1 spacing is the least optimal spacing for monitoring O3-dependent enhanced repression of lacZ gene expression. We therefore examined the influence of narL and narP null alleles on enhanced nitrate repression of lacZ gene expression in the O3-nirB O1-napF construct with 95-nt spacing. In this experiment, the nitrate repression of lacZ expression was more than 200-fold in the narL⁺ narP null strain, whereas the repression was only 12-fold in the narP⁺ narL null strain (Table 2).

Enhanced repression by the O3-napF substitution.

For the experiments described above, we used the nirB 7-2-7 heptamer pair substituted at operator O3. Our intention was to use a site that binds phospho-NarL strongly, in an attempt to maximize repression resulting from cooperative interactions between operators O1 and O3 (28, 31). Indeed, with nearly optimal interoperator spacing, the O3-nirB sequence increased NarL-dependent repression by roughly 10-fold (Fig. 2 and Table 2). As noted above, however, the O1-nirB operator substitution yields feeble NarP-dependent repression, suggesting that it bound phospho-NarP weakly. Thus, the O3-nirB substitution constructs did not permit conclusions regarding cooperative binding by the phospho-NarP protein.

To examine this point, we made constructs with O3-napF operator substitutions (Table 3). In narL⁺ narP null strains, the O3-napF substitution resulted in repression that was threefold greater than that observed in O3-lac strains. The enhancement was even more pronounced (more than fivefold) with a construct in which the interoperator spacing was decreased to 87 nt (constructed by deleting the GATC overhang resulting from BglII digestion). By contrast, repression in the narP⁺ narL null strains was increased less than twofold in the presence of the O3-napF operator substitution (Table 3). This indicates that the phospho-NarP protein has only weak cooperative interactions that result in enhanced O3-dependent repression.

TABLE 3.

Interoperator spacing influences O3-napF enhancement of NarL-mediated repression at O1-napF

Strain	Construct	Spacinga	Genotype		LacZ sp act (arbitrary units)b		Repression (fold)
			narL	narP	No NO3−	NO3−
VJS8880	O3-lac O1-napF	91	+	+	1,190	100	12
VJS8882	O3-lac O1-napF	91	+	−	1,170	110	11
VJS8881	O3-lac O1-napF	91	−	+	1,020	140	7.3
VJS9004	O3-lac O1-napF	91	−	−	1,010	650	1.6
VJS8883	O3-napF O1-napF	91	+	+	1,150	55	21
VJS8885	O3-napF O1-napF	91	+	−	1,200	35	34
VJS8884	O3-napF O1-napF	91	−	+	1,100	130	8.5
VJS9005	O3-napF O1-napF	91	−	−	1,100	720	1.5
VJS8899	O3-napF O1-napF	87	+	+	870	23	38
VJS9001	O3-napF O1-napF	87	+	−	980	17	58
VJS9000	O3-napF O1-napF	87	−	+	1,100	100	11
VJS9006	O3-napF O1-napF	87	−	−	970	620	1.6

^aInteroperator spacing (see Fig. 1).

^bStrains were cultured overnight in MOPS defined medium with limiting glucose.

The level of repression with the O3-napF operator substitution was lower in narL⁺ narP⁺ strains than in narL⁺ narP null strains but was similar in the O3-lac strain (Table 3). This indicates that the phospho-NarP protein, which competes with the phospho-NarL protein for binding to the napF site, inhibits the enhanced repression resulting from phospho-NarL cooperative interactions. This further suggests that the phospho-NarP protein has weak, if any, cooperative interactions.

Control experiments.

In the lacZYA operon control region, the auxiliary operators cannot mediate repression in the absence of the primary operator, O1-lac (27, 32). To document this for the synthetic lac control regions described here, we made O3-napF O1-lac constructs with 91-nt (native) and 87-nt interoperator spacing. Neither construct exhibited Nar-dependent repression (Table 4). This established that the repression observed with O3 substitution constructs (Tables 2 and 3; Fig. 2) was wholly dependent upon substitution also at operator O1.

TABLE 4.

Neither O3-napF nor interoperator spacing influences expression from O1-lac

Strain	Construct	Spacinga	Genotype		LacZ sp act (arbitrary units)b		Repression (fold)
			narL	narP	No NO3−	NO3−
VJS8886	O3-lac O1-lac	91	+	+	3,290	2,050	1.6
VJS9003	O3-napF O1-lac	91	+	+	2,680	2,210	1.2
VJS9002	O3-napF O1-lac	87	+	+	2,770	2,200	1.3

^aInteroperator spacing (see Fig. 1).

^bStrains were cultured overnight in MOPS defined medium with limiting glucose and 0.1 M IPTG.

Analysis of NarL-NarP chimeras.

The NarL protein dimerizes through its carboxyl-terminal DNA-binding domain (24). Since the phospho-NarL and -NarP proteins differ in the ability to have cooperative interactions, we hypothesized that this is a property of the corresponding amino-terminal receiver domains. As one attempt to examine this, we constructed chimeric NarL-NarP proteins by fusing the receiver domain from one protein to the DNA-binding effector domain of the other protein. We made four chimeras with all possible combinations of receiver-linker-effector arrangements, based on a study of OmpR-PhoB chimeras (43). As observed in other studies of chimeric response regulators (4, 41, 43), transcription control by these NarL-NarP chimeras responded poorly or not at all to inducing signal, compromising analysis of enhanced repression at O3 substitution constructs. Although the results (data not shown) were consistent with the notion that the NarL receiver domain mediates cooperative interaction, the overall magnitude of the response was weak.

Concluding remarks.

This study exploited well-documented observations that cooperative interactions between LacI dimers, mediated by formation of a DNA loop, can enhance repression of lacZYA operon transcription by about 50-fold (32). We were curious to determine whether the lac control region could provide an artificial means to examine cooperative interactions between DNA-binding proteins unrelated to the LacI repressor. Previous studies revealed cooperative binding by phospho-NarL dimers to immediately adjacent sites in the fdnG and nrfA operon control regions (10, 11, 19, 20, 42). Accordingly, binding to the O1 and O3 operator substitutions, spaced approximately 90 nt apart, represents a nonnative context for the Nar regulators. On the other hand, the Nar system allows internal comparison of otherwise similar proteins that differ in the ability to form cooperative interactions.

The operator substitution O3-napF enhanced NarL-dependent repression from an O1-napF synthetic lac control region up to fivefold, whereas NarP-dependent repression was virtually unaltered (Table 3). This result provides independent support for the hypothesis that phospho-NarL dimers have cooperative interactions in binding to specific DNA sites, whereas the phospho-NarP protein has poor or no cooperative interactions. Additional analysis with the operator substitution O3-nirB revealed NarL-dependent repression that was enhanced up to 15-fold (Table 2) and was dependent on the spacing between the two operators (Fig. 2). Thus, the results mimic those obtained for the native lac system and suggest that the lac control region may provide a general assay for analyzing cooperative protein-DNA interactions.