The leuO Gene Product Has a Latent Ability To Relieve bgl Silencing in Escherichia coli

Abstract

The Escherichia coli bgl operon is of interest, since its expression is silent (phenotypically Bgl⁻), at least under standard laboratory conditions. Here we attempted to identify a trans-acting factor(s) that is presumably relevant to the regulation of bgl by a random insertion mutagenesis with mini-Tn10. These collected mutations, conferring the phenotype of Bgl⁺, were localized in three loci on the genetic map, two of which appeared to be hns and bglJ, which were previously implicated as the factors affecting the Bgl phenotype. The other locus at 1 to 2 min on the genetic map appeared to be a new one. In this case, the insertion mutation was found to be just in front of the leuO gene encoding a putative LysR-like DNA-binding protein. Genetic analyses revealed that overproduction of LeuO in the wild-type cells causes the phenotype of Bgl⁺. A leuO deletion mutant was also characterized in terms of expression of bgl. From these results, the possible function of LeuO in bgl expression will be discussed from an evolutionary and/or ecological point of view.

The bgl operon, located at about 84 min on the Escherichia coli K-12 genetic map, is involved in the utilization of certain β-glucosides, such as salicin and arbutin (9, 15). This operon is of interest in that, although it is intact in the genetic sense (bgl⁺), its expression is normally silent in the wild-type background (i.e., phenotypically Bgl⁻) but is enhanced upon the occurrence of various types of spontaneous mutations, resulting in a phenotype of Bgl⁺. Such Bgl⁺ mutations include insertion of IS1 or IS5 upstream of the bgl promoter, point mutations within a CRP (cyclic AMP receptor protein) binding site and deletions within a region upstream of the CRP binding site (10, 11, 13, 14). Interestingly, mutations outside the bgl operon can also result in Bgl⁺. Specifically, certain mutations in the genes encoding the subunits of DNA gyrase (gyrA and gyrB) or the H-NS nucleoid protein (hns [formerly called bglY]) can activate the bgl operon (1, 2). Recently, another trans-acting gene, named bglJ, was reported to affect bgl silencing, although its significance is less clear (3). In this context, the molecular mechanism underlying bgl silencing has previously been characterized to some extent, and a plausible model of Schnetz (12) postulated that a region of the bgl operon including its promoter is organized into a nucleoprotein structure, within which the activity of the bgl promoter is repressed somehow. Nevertheless, no clear picture with regard to such a “silencing nucleoprotein structure” has emerged.

Based on a number of current studies, it is believed that H-NS is directly implicated in the formation of such a presumed silencing nucleoprotein structure (12, 16). From our recent results, however, it is also evident that the DNA-binding ability of H-NS is dispensable for bgl silencing (21). That is, a carboxyl-terminally truncated form of H-NS, lacking the entire DNA-binding domain, is still able to fully repress bgl expression. This fact may suggest that other trans-acting DNA-binding protein(s) must also be involved in bgl silencing. In this study, we thus attempted to search for such a new trans-acting factor(s) that may be relevant to the underlying mechanism of bgl silencing.

Isolation of Bgl⁺ mutants.

A method of transposon mutagenesis with mini-Tn10cam (cam gene confers chloramphenicol resistance [Cm^r]) (7) was adopted by employing a specialized E. coli strain (BGL1), which was designed to carry not only the intact bgl operon on the chromosome but also a bgl-lacZ fusion gene at the λatt site (21). Note that this particular strain is phenotypically Bgl⁻ and LacZ⁻ due to bgl silencing. Cells of CSH26 [Δ(pro-lac) ara thi], which is the parental strain of BGL1, were extensively mutagenized with mini-Tn10cam. Then, P1 phage lysates from the mutagenized cells were used for P1 transduction into strain BGL1, yielding 3.6 × 10⁴ Cm^r cells. Among these cells, we searched for trans-acting mutants, which should exhibit simultaneously the phenotypes of Bgl⁺ and LacZ⁺. The former was screened on agar plates containing salicin and bromothymol blue, and the latter was screened on MacConkey-lactose plates containing salicin. After such an extensive double screening, we isolated 29 mutants that exhibited the desirable phenotypes of Cm^r, Bgl⁺, and LacZ⁺. They were assumed to carry a Tn10 insertion mutation in a certain trans-acting gene, thereby resulting in a relief of bgl silencing. The positions of these 29 mutations on the E. coli chromosome were roughly mapped by P1 transduction with the help of a large set of Tn10 insertions in the entire E. coli chromosome, which were previously constructed by Singer et al. (zxx series::Tn10) (20). This whole set of Tn10 insertions provided us with appropriate markers (Tet^r [tetracycline resistant]), whose positions are known and located evenly on the entire E. coli chromosome. The results of such analyses showed that 12 mutations (class A) were linked to zab-3051::Tn10 (1 to 2 min on the chromosome) (99% linkage), 11 mutations (class B) were linked to zci-506 (27 to 28 min) (80% linkage), and the remainder (class C) were linked to zji-202::Tn10 (98 to 99 min) (80% linkage). Together with the previous notions with regard to the trans-acting mutations affecting bgl silencing (see the introduction), we assumed that class A mutations most likely represent a novel mutation, while the other classes, B and C, correspond to each known gene, hns and bglJ, respectively. From these 29 mutants, we thus selected representative strains for each class (namely, CU305 for class A, CU306 for class B, and CU307 for class C).

With these representatives, we first demonstrated that each mutant does indeed exhibit the expected phenotype, Bgl⁺, by monitoring expression of the bgl-lacZ fusion in these mutants (Fig. 1A). The levels of β-galactosidase activities, expressed in these mutants, increased in an inducer (β-methyl-d-glucoside)-dependent manner, as remarkably as in the case of the known hns deletion mutant. Thus, we succeeded in isolating at least one new class of Bgl⁺ mutants, in which the mini-Tn10cam insertion results in a relief of bgl silencing. We then focused our attention on this particularly new mutant, CU305, while the presumed hns and bglJ mutants, CU306 and CU307, were discarded.

FIG. 1.

(A) Expression of the bgl operon in the set of transposon insertion mutants. Cells of the indicated strains, all carrying a bgl-lacZ transcriptional fusion on the chromosome, were grown at 37°C to mid-logarithmic phase in TB medium (19) in the absence (−) and presence (+) of 5 mM β-methyl-d-glucoside, an inducer for the bgl operon. β-Galactosidase activities expressed by these cells were measured by the method of Miller (8). Each value is the mean ± standard deviation from four independent assays. (B) Expression of the bgl operon in the cells in which LeuO is overproduced. Transformants of BGL1 with pTO3 (see FIG. 2C) or pUSI2 (a control vector) were grown, and then β-galactosidase activities expressed by these cells were measured, as described above.

Derepression of bgl expression in CU305 is due to overproduction of LeuO.

By employing standard techniques of recombinant DNA, we clarified the structure of the chromosomal region of CU305 where mini-Tn10cam had been inserted (Fig. 2A). The inserted element was found to be 19 bp upstream of the known open reading frame (ORF), named leuO, which is located at 1.8 min on the E. coli genetic map, and between the leuABCD and ilvIH operons. The leuO gene specifies a putative DNA-binding protein, which appears to be a member of the LysR family of regulatory proteins (6). Based on its location on the map (Fig. 2A), it has previously been speculated that LeuO might be implicated in the regulation of the leuABCD operon, but no evidence for such a role has been reported (e.g., in fact, the ΔleuO strain constructed in this study is not leucine auxotrophic). In CU305, this leuO ORF is not interrupted by the mini-Tn10cam insertion. Then, the question of how this particular insertion causes the Bgl⁺ phenotype arose. Among several explanations envisaged, we first tested the idea that overproduction of LeuO, due to the mini-Tn10cam insertion upstream of the coding sequence, may result in the Bgl⁺ phenotype. This was found to be the case, as demonstrated below. The leuO gene was cloned, and placed under the isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible tac promoter in a versatile expression vector (named pUSI2) (18), to yield pTO3 (Fig. 2C). When this plasmid was introduced into the wild-type background (BGL1), it was found that expression of bgl-lacZ was greatly enhanced in a manner dependent on the presence of IPTG (Fig. 1B). The level of bgl expression was more or less the same as in the case of CU305. These results collectively supported the view that overexpression of LeuO somehow results in the phenotype of Bgl⁺. In this respect, it may be noted that, although it was previously reported that overproduction of LeuO negatively affects cadBA expression (17), the result described here demonstrated that overproduction of LeuO also has a somewhat positive effect on expression of this particular operon (bgl).

FIG. 2.

Schematic representations of leuO mutations on the chromosome and of the cloned leuO gene on the plasmid. (A) Structure of the chromosomal region encompassing the leuO gene is shown schematically, in which the position of the mini-Tn10cam insertion in CU305 was indicated. Each polygonal symbol shows the indicated ORFs as well as their relative directions of transcription (the letter P shows each putative promoter). (B) Construction of the ΔleuO::cam deletion mutation used in this study. The BstXI-ApaI DNA region encompassing the leuO coding sequence was replaced by the DNA fragment carrying the cam (Cm^r) gene on the chromosome of TO2. (C) Construction of the plasmid pTO3 that can overproduce LeuO. The EcoT22I-VspI fragment encompassing the entire region of the leuO gene was cloned under the tac promoter on pUSI2 (17), to yield pTO3.

Overproduction of LeuO affects bgl expression in H-NS-independent manner.

We then asked the question of what might be the possible mechanism underlying bgl derepression caused by LeuO overproduction. We focused our attention particularly on a possible linkage between the functions of H-NS and LeuO, since H-NS is a major trans-acting factor, so far known to be implicated in the bgl silencing (1, 16). Based on the fact that expression of the bgl operon is fully derepressed in a Δhns background (Fig. 1A), one can assume that overproduction of LeuO might cause inactivation of H-NS either directly or indirectly, which in turn should result in bgl derepression. However, we found by an immunoblotting analysis with an anti-H-NS antiserum that the cellular content of H-NS in CU305 is essentially the same as that in its parental cells (data not shown). We also confirmed that H-NS in strain CU305 is fully functional, because the expression of the proVWX operon is not derepressed by the LeuO overproduction (data not shown). Note that whether H-NS is functional can be monitored sensitively by examining whether the proVWX operon is depressed, as demonstrated previously (21). Thus, the above assumption was dismissed.

The further question was what would happen if the leuO gene on the wild-type chromosome were knocked out. To see this consequence with special reference to bgl silencing, a region on the BGL1 chromosome was replaced by a chloramphenicol-resistant cassette, so as to yield a ΔleuO::cam mutant (Fig. 1B). The resultant strain, named TO2, was examined for bgl silencing by monitoring bgl-lacZ expression, showing that no effect was seen (Fig. 3). This result suggested that LeuO is not essential to keep the bgl operon to be silent. We then needed to see the phenotype of the leuO deletion mutation in appropriate genetic backgrounds, under which bgl silencing had been relieved. To do so, the Δhns mutation was again concerned, since expression of the bgl operon is fully derepressed in this background (21). A strain with both the Δhns and ΔleuO mutations was constructed and examined for bgl expression. Again, no effect was seen (Fig. 3), suggesting that LeuO is not essential for bgl transcription at least under certain conditions.

FIG. 3.

Expression of the bgl operon in the leuO deletion mutant. A set of strains including BGL1 (wild type), TO2 (ΔleuO), BGL1 hns::neo (an hns::neo derivative of BGL1), and TO3 (an hns::neo derivative of TO2) were assayed for their expression of bgl-lacZ. The cells were grown at 37°C to the mid-logarithmic phase in TB medium (19) in the absence (−) and presence (+) of 5 mM β-methyl-d-glucoside, and then β-galactosidase activities expressed were measured by the method of Miller (8). Each value is the mean ± standard deviation from four independent assays.

LeuO is a LysR-like protein, and thus, it is most likely a DNA-binding protein. In this study, we found that LeuO is able to lead the bgl operon to its derepressed state (i.e., Bgl⁺), even in the presence of H-NS, provided that LeuO was overexpressed under certain conditions. It is not known at present how LeuO can do this task. However, we demonstrated that LeuO appears not to exert its function through H-NS, which is a major contributor for bgl silencing. It was also found that LeuO is not required for bgl activation caused by the Δhns mutation. These results suggest that LeuO exhibits its effect on the bgl operon in an H-NS-independent manner, that is, LeuO can fully antagonize the negative effect of H-NS on the bgl operon. Thus, LeuO may function as an activator for the bgl operon through its direct binding to this target. Alternatively, LeuO may have the ability to disrupt the presumed silencing nucleoprotein structure on the bgl promoter. Of course, more complex explanations cannot be excluded at present. In any case, it will be of interest to see whether LeuO can bind directly to a region of the bgl operon.

Implications.

It is not clear at present whether the single-copy leuO gene is relevant to the regulation of the bgl operon, because we have not so far succeeded in finding a phenotype for the leuO deletion mutant. However, it can be assumed that there may be certain natural conditions under which the expression of LeuO is enhanced. Under such a presumed circumstance, the bgl operon would be inducibly activated. In this context, it is also worth mentioning that these features of LeuO, described here, are very similar to those of BglJ (3). Overproduction of BglJ, caused by an IS insertion, was also reported to result in the phenotype of Bgl⁺. This protein belongs to yet another type of DNA-binding protein, a NarL/RcsB-like protein with a potential helix-turn-helix motif. These facts suggest that E. coli has at least two potential activators, which can relieve the tight bgl silencing. If they can exert a redundant effect on the bgl operon, then the leuO deletion mutation alone would not show any noticeable phenotype with regard to the bgl operon. It will be thus of interest to construct a ΔleuO ΔbglJ double mutant in order to see the Bgl phenotype.

In connection with a physiological significance of our finding, the issue should finally be addressed of why E. coli must keep the bgl operon, which can be activated (or used) only upon fortuitous mutations, and is otherwise useless. Why should E. coli have such proteins, like LeuO, which possesses a powerful but latent ability to activate the bgl operon? As pointed out by Giel et al. (3), one might expect that the bgl operon in E. coli would be lost eventually. Instead, the silent bgl operon is present in a variety of E. coli strains isolated from different geographical locations (4). Hall et al. (5) have proposed that the presence of the bgl operon, which can be activated solely by mutation, may confer a selective advantage to those organisms that have maintained this cryptic system (i.e., the bgl-leuO system). If so, with the onset of an urgent situation in nature, potential trans-acting genes for the bgl operon, like leuO and bglJ, may serve as a secret weapon, which can be triggered by naturally occurring IS insertions. From an evolutionary and/or ecological point of view, this may be one of the positive physiological functions of leuO.