TEXT

Top Abstract Text References

The bgl operon, located at about 84 min on the Escherichia coli K-12 genetic map, is involved in the utilization of certain -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 att site (21). Note that this particular strain is phenotypically Bgl and LacZ due to bgl silencing. Cells of CSH26 [(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).

View larger version (27K): [in this window] [in a new window]   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 -methyl-D-glucoside, an inducer for the bgl operon. -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 -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 leuO 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--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).

View larger version (19K): [in this window] [in a new window]   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 leuO::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 (Cmr) 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 hns 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.

View larger version (33K): [in this window] [in a new window]   FIG. 3.   Expression of the bgl operon in the leuO deletion mutant. A set of strains including BGL1 (wild type), TO2 (leuO), 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 -methyl-D-glucoside, and then -galactosidase activities expressed were measured by the method of Miller (8). Each value is the mean ± standard deviation from four independent assays.

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 leuO bglJ double mutant in order to see the Bgl phenotype.