Estación Experimental del Zaidín, CSIC, Departamento de Bioquímica y Biología Molecular y Celular de Plantas, Apartado 419, E-18080 Granada, Spain1
Author for correspondence: Silvia Marqués. Tel: +34 958 121011. Fax: +34 958 129600. e-mail: silvia{at}eez.csic.es
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ABSTRACT |
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Keywords: 32, stress response, aromatic hydrocarbons, heat-shock response, downstream box
Abbreviations: DEPC, diethyl pyrocarbonate; DS box, downstream box; HS response, heat-shock response
The GenBank accession number for the sequence corresponding to the P. putida KT2440 chromosomal DNA fragment (2891 bp) cloned in pSH27, including the rpoH gene, is AF157048.
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INTRODUCTION |
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Pseudomonas putida is a soil-borne bacterium with significance in bioremediation and biodegradation. It is the natural host for several plasmids that confer the ability to mineralize toluene and other aromatic compounds (Worsey & Williams, 1975 ; Kunz & Chapman, 1981
; Ramos et al., 1997
). Bacteria have evolved adaptive mechanisms to survive various environmental stresses. In its natural habitat, P. putida is subjected to frequent changes in growth conditions probably periods of stress and starvation cycles. Exposure to aromatic hydrocarbons could be considered an environmental stress, and this bacterial strain has developed a stress-responsive mechanism to degrade and use these compounds as carbon sources for growth. Nothing is known about the HS response in P. putida, but it seems to play a key role in the performance of the strain when it is exposed to contaminants (Marqués et al., 1999
). We have identified, cloned and sequenced the rpoH gene from P. putida, and we have investigated its regulation at the transcriptional level. In addition, we have studied the effect of aromatic compounds on
32 transcription in P. putida.
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METHODS |
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ß-Galactosidase assays.
Overnight cultures grown on LB supplemented with tetracycline were diluted 1:100 in fresh medium and grown until the exponential phase was reached. Samples were taken to determine ß-galactosidase activity as described previously (Miller, 1972 ). Data are the mean of three independent assays.
Other techniques.
Standard molecular biology techniques were used for DNA manipulations (Sambrook et al., 1989 ). The rpoH DNA probe used for Southern blots was plasmid pAN4 (Calendar et al., 1988
), which contains the E. coli rpoH gene cloned into pBR322. DNA sequencing of the P. putida rpoH gene was carried out on both strands by using the dideoxy sequencing termination method involving dichloro-rhodamine (Drho Terminators TAQ FS 100 RXN kit; Perkin Elmer).
Prediction of RNA secondary structure.
Potential mRNA secondary structures for the 5'-ends of the different rpoH transcripts were predicted using MFOLD (Zucker et al., 1999), and the resulting structures were visualized with RNADRAW version 1.1 (Ole Matzura; available via an anonymous ftp at ftp.itc.univie.ac.at).
Peptide alignment.
Alignment of peptides was performed using the CLUSTAL W program.
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RESULTS AND DISCUSSION |
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A potential RBS (GGAGGU) was found six bases upstream of the potential initiation codon (Fig. 1a). In addition, a 15 bp region showing 80% complementarity with the P. putida 16S rRNA sequence between nucleotides 1456 and 1471 was found six bases downstream of the first base of the coding region (Fig. 1a
). The segment, called the downstream box (the DS box), has also been found in other Gram-negative bacteria, and seems to play a role in translation regulation (Nakahigashi et al., 1995
). It is worth noting that in P. putida, the anti-DS box sequence in the 16S rRNA is slightly shifted from that in other Gram-negative bacteria, as is the case in P. aeruginosa (Nakahigashi et al., 1995
).
Downstream of the stop codon, an inverted repeat sequence followed by a T(U) track was found (Fig. 1b). A similar hairpin sequence with the structure of a typical
-independent transcription terminator was also found downstream of the rpoH genes analysed so far (Naczynski et al., 1995
; Sahu et al., 1997
), although no experimental evidence for such a function has been reported. To analyse the possible role of this sequence in termination, we constructed pMPR, a terminator-probe vector able to replicate in Pseudomonas, in which lacZ gene expression was under the control of
p'R (see Methods). We inserted a 50 bp linker, with the inverted repeat and the T-track sequence located downstream of rpoH, between the
p'R promoter and the lacZ reporter gene, to produce pMPRH. As a control, we used pMPRA, the equivalent construction bearing a random sequence cloned in the same site. Fig. 2
shows that the presence of the inverted repeat downstream of
p'R reduced the ß-galactosidase activity to less than 10% of that of the construction without the insert; the presence of the random sequence had no effect. Similar results were obtained at 18, 30 and 42 °C (not shown). We therefore suggest that this sequence functions as a terminator for the rpoH transcription in P. putida.
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To test if the presence of aromatic compounds could modify the transcription of rpoH, cultures of P. putida 2440(pWW0) growing on succinate were supplemented with either 3-methylbenzoate (to concentrations up to 10 mM) or toluene in the vapour phase, and total RNA was isolated after 2 h induction. In a control experiment, a culture of the same strain growing on succinate was subjected for 5 min to a heat shock at 42 °C. The rpoH-derived mRNAs were analysed as above. No difference in the pattern of bands was observed under any conditions (not shown), suggesting that in P. putida, rpoH transcription is not regulated by the presence of aromatics. In addition, it does not change after a heat shock; this is similar to what happens in most Gram-negative bacteria, in which 32 transcription is not autoregulated. Caulobacter crescentus is the only strain described so far in which
32 is autoregulated at the level of transcription (Wu & Newton, 1997
).
The presence, in P. putida, of three promoters controlling rpoH gene expression contrasts with the situation in E. coli, in which four promoters have been found upstream of rpoH. Three of them (P1, P4 and P5) are transcribed by RNA polymerase E70, while the fourth (P3) is transcribed by E
24 (Erickson & Gross, 1989
; Nagai et al., 1990
; Wang & Kaguni, 1989
). In P. aeruginosa, only two promoters have been found, one of them being
24-dependent (Naczynski et al., 1995
).
Analysis of rpoH mRNA secondary structure
The secondary structure of the 5'-proximal region of rpoH mRNA from different Gram-negative bacteria was predicted to contain conserved patterns (Nakahigashi et al., 1995 ; Morita et al., 1999
). The secondary structure is assumed to repress translation of the mRNA at low temperatures, through base pairing that would block access to the initiation codon and the DS box (Yuzawa et al., 1993
). We have analysed the potential secondary structure of rpoH mRNAs produced from the three promoters described above, using the algorithm of Zuker et al. (1999)
. The structures obtained (Fig. 5
) were different from the structure predicted for the E. coli rpoH mRNA. It is worth noting that although this structure was supported by analysis of point mutations (Nagai et al., 1991
; Yuzawa et al., 1993
), these authors did not consider, in their prediction, the different 5'-ends of the mRNA produced from the four promoters described for E. coli.
In the predicted structure of mRNAs originating from P2 and P3 of P. putida rpoH, some important features are conserved: (i) the first codon is blocked by the DS box in a similar folded structure (Fig. 5), and (ii) the RBS is partially blocked by base pairing. However, the situation predicted for the mRNA derived from the P1 promoter is different: the 5'-end of the transcript is completely blocked by the DS box in an almost perfect base pairing, whereas the first codon of the ORF is located within a loop. The RBS in this last folded structure has a level of obstruction by base pairing similar to that of P2- and P3-derived mRNAs. This suggests that translation efficiency could be a key factor in the regulation of
32 synthesis in P. putida. Although the strength of the three promoters detected was very similar in all conditions tested, the translation of the three resulting mRNAs is probably different, according to the mRNA structures predicted. In addition, the half-lives of the different mRNAs could be an additional step in P. putida
32 regulation.
Concluding remarks
Our results show that the rpoH gene of P. putida conserves the structural features of the rpoH genes described previously. Experimental data indicate that transcription of the rpoH gene in P. putida remains unchanged under the stress conditions assayed. Therefore, control may take place at a post-translational level because of the structural features found in the rpoH mRNAs, as observed in E. coli (Yura et al., 1993 ). It has been shown previously that the addition of aromatics provokes a typical HS response (van Dyck et al., 1995; Marqués et al., 1999
); this response is not due to an increase in rpoH transcription in P. putida.
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ACKNOWLEDGEMENTS |
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Received 14 August 2000;
revised 17 January 2001;
accepted 5 February 2001.