To determine whether the AlkS protein can regulate the expression of its own gene, we analysed the effect of introducing increasing copies of the alkS gene into the reporter strain PS16, which lacks alkS but contains in the chromosome a transcriptional fusion of the alkS promoter region to the lacZ reporter gene (positions − 344 to + 53 relative to the start site of PalkS1, the previously mapped σS-dependent promoter; Yuste et al., 1998; Canosa et al., 1999). Strain PS16 derives from Pseudomonas putida KT2442, in which the alk genes are correctly expressed, maintaining all known regulatory features (Yuste et al., 1998; Canosa et al., 1999). In fact, P. putida and the strain from which the OCT plasmid was originally isolated, P. oleovorans, are closely related species (Moore et al., 1996). The alkS gene was introduced into strain PS16 either with the help of a mini-Tn5 transposon, which allows the insertion of a single copy of alkS in the chromosome, or using the broad-host-range, high-copy-number plasmid pHCS1, which contains alkS (Yuste et al., 1998). Cells containing the PalkS–lacZ fusion and either no alkS (strain PS16), a single copy of alkS (strain PS16S1) or multiple copies of alkS (strain PS16/pHCS1) were grown in LB medium in the absence or presence of the non-metabolizable inducer dicyclopropylketone (DCPK), a water-soluble compound that mimics the effect of alkanes (Grund et al., 1975). Samples were obtained at different growth stages and processed to obtain total RNA. Transcripts arising from the alkS promoter located upstream from the lacZ reporter gene were analysed by S1 nuclease protection assays, using as probe a single-stranded DNA (ssDNA) whose labelled 5′ end hybridized to the start of the lacZ gene. In this way, only the transcripts arising from the PalkS–lacZ fusion were detected, whereas those arising from the promoter of the introduced alkS gene were not. As reported previously (Canosa et al., 1999), in the absence of alkS, expression of the PalkS–lacZ fusion was very low during exponential growth and increased considerably as cells entered the stationary phase, because expression occurs from a σS-dependent promoter (Fig. 2). Transcription from this promoter was very similar in both the absence or the presence of inducer. A weak additional signal, however, could be detected in stationary phase cells, suggesting the presence of a transcript 38 bp shorter than the former one. In the case of cells containing a single copy of alkS (strain PS16S1), the signal corresponding to the transcript arising from the σS-dependent promoter was considerably weaker in the absence of inducer (about 10-fold lower, as deduced from samples run in parallel in the same gel; not shown), and essentially undetectable when the inducer was present. At the same time, the inducer led to a considerable increase in the signal corresponding to the transcript 38 bp shorter than that arising from the σS-dependent promoter (Fig. 2). This new band was detectable only in stationary phase cells. When multiple copies of alkS and the inducer were present (strain PS16/pHCS1), the abundance of this transcript increased significantly, particularly in exponential phase. The transcript appeared as a set of four or five bands, each one being 1 bp shorter than the preceding one, suggesting heterogeneity in its 5′ end (this can be better appreciated in Fig. 6). A similar heterogeneity has been observed in several prokaryotic promoters whose start sites include a tract of three or more thymines (Harley et al., 1988; Guo and Roberts, 1990; Jacques and Susskind, 1990; Gülland and Hillen, 1992; Xiong and Reznikoff, 1993; Jin, 1994). At these promoters, the initial ribonucleotide pAAA or pUAAA slips against the template TTT, beginning a slippage cycle in which the transcript acquires a number of extra untemplated adenines at the 5′ end. Finally, an additional weak band about 15 bp shorter appeared in some assays; this band was not reproducible and was not considered further.
Figure 2. Expression of the promoters for the alkS gene.
A. P. putida strains PS16 (lacks alkS but contains a transcriptional fusion of the alkS promoter region to the lacZ reporter gene), PS16S1 (PS16 with a copy of alkS in the chromosome) and PS16/pHCS1 (PS16 bearing a high-copy-number plasmid containing alkS) were grown in duplicate flasks in LB medium. At an absorbance (A600) of about 0.08, the inducer DCPK was added to one of the flasks, leaving the other as a non-induced control. At different times after induction, aliquots were collected and processed to obtain total RNA. Transcripts arising from the promoter region of the PalkS::lacZ fusion were analysed by S1 nuclease protection assays. The gels show the transcripts observed at each cell density; samples were run in parallel with a DNA size ladder (M) obtained by chemical sequencing of the same ssDNA used as probe, as described by Maxam and Gilbert (1980). The transcription start sites for the two promoters observed are indicated.
B. Sequence of the promoters for alkS and comparison with the promoter of the alkBFGHJKL operon, PalkB. Promoter PalkS1 (indicated as P1) is recognized by σS-RNAP (Canosa et al., 1999). The start site of promoter PalkS2 (indicated as P2) cannot be determined accurately because of the heterogeneity of its 5′ end (see text). The start site of PalkB corresponds to that reported in this work (see Fig. 3); the G residue marked with an asterisk denotes the start site proposed previously (Kok et al., 1989a). A highly homologous inverted repeat present upstream from the − 35 regions of PalkS2 and PalkB is indicated. The − 10 and − 35 regions of each promoter are underlined.
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The above results suggest that AlkS can repress the σS-dependent promoter in both the absence and the presence of inducer and that, when the inducer is present, it activates a new promoter located 38 bp downstream from the σS-dependent promoter. As shown in Fig. 2, we have named the σS-dependent promoter PalkS1 (or P1), and the new AlkS-activated promoter PalkS2 (or P2). Repression of PalkS1 and the simultaneous activation of PalkS2 in the presence of inducers could easily be explained if we assume that AlkS binds to PalkS1, a site at which the regulator would be adequately positioned to activate PalkS2 (see Fig. 2B). Interestingly, comparison of promoters PalkS2 and PalkB, both of which are activated by AlkS, reveals the presence of a highly homologous inverted repeat immediately upstream from the − 35 region (Fig. 2B). Accurate comparison of promoters PalkS2 and PalkB requires knowledge of the precise start site of PalkB, but the reported transcription initiation site for PalkB had been deduced by S1 protection assays using a set of end-labelled restriction fragments as size markers, which does not allow for precise mapping (Kok et al., 1989a). Using a DNA sequence ladder as size marker, we have now found that the start site for PalkB is 5 bp further upstream than previously proposed (see Figs 3 and 2B). Considering this new start site, the mentioned inverted repeat is centred at position − 41.5 at PalkB and at position − 42.5 at PalkS2, relative to their respective start sites (Fig. 2B). To investigate whether this inverted repeat could be the target for AlkS, we generated a series of PalkS2 derivatives in which the sequences upstream from position − 36 were deleted stepwise (see Fig. 4). The resulting promoters were fused to lacZ, introduced into a P. putida KT2442 derivative containing the alkS gene (strain PS1), and the ability of AlkS to activate transcription from each of them was analysed. As shown in Fig. 4, sequences upstream from position − 59 were dispensable for AlkS activation of promoter PalkS2. It should be noted, however, that deletion of sequences between positions − 120 and − 59 increased the basal expression levels of PalkS2 in the absence of inducer, although the presence of the inducer stimulated transcription significantly. Extending the deletion up to position − 42, which eliminates the left arm of the repeat, or to position − 36, which affects the right arm as well, rendered promoters that could not be activated by AlkS. Therefore, PalkS2 activation seems to require only the DNA region comprising the inverted repeat centred at position − 42.5.
Figure 4. Ability of AlkS to activate transcription from PalkS2 promoter derivatives harbouring serial deletions at the 5′ region. The activity of the transcriptional fusions to lacZ containing deletion derivatives of promoter PalkS2 (P. putida strains PS1S1, PS1S2, PS1S3, PS1S4, PS1S5 and PS1S6) was measured in cells grown in minimal salts medium containing citrate as carbon source, in the absence or presence of the inducer DCPK. The promoter present in strain PS16S1, which includes sequences up to position − 382 relative to PalkS2 start site, was considered as the wild type. The scheme shows the extent of the deletion for each mutant derivative, the location of the inverted repeat, the − 10 and − 35 regions of PalkS2 promoter (solid bars) and the transcription start site. Numbers on top denote the PalkS2 co-ordinates relative to the transcription start site. The β-galactosidase activity (in Miller units) observed in logarithmic cultures (A600 of 0.8, when PalkS1 activity is negligible) is shown on the right; the induction value corresponds to the Miller units observed in the presence of inducer relative to those measured in its absence. Values correspond to the average of three independent assays.
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It is worth noting that, when alkS was in monocopy, AlkS-dependent expression of promoter PalkS2 was detected only after cells entered the stationary phase of growth (A600 of 2 or 3). This behaviour is similar to that of promoter PalkS1, whose expression is much higher in stationary phase because it depends on σS-RNAP (Canosa et al., 1999). However, in the presence of multiple copies of alkS, activation of PalkS2 by AlkS was efficient both in the exponential and in the stationary phases of growth. This is reminiscent of the behaviour of the PalkB promoter in cells grown in LB medium and an inducer, a situation in which PalkB activation is silenced by catabolic repression during exponential growth unless AlkS is present in high amounts (Yuste et al., 1998). We therefore investigated whether the preferential expression of PalkS2 in stationary phase was caused by a dependence on σS or by a catabolite repression effect of the LB medium used to grow the cells.