1 Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo Postal 510-3, Cuernavaca, Morelos 62250, Mexico
2 Programa de Ingeniería Metabólica, Centro de Investigación sobre Fijación de Nitrógeno, Universidad Nacional Autónoma de México, Mexico
Correspondence
Gloria Soberón-Chávez
gloria{at}biomedicas.unam.mx
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ABSTRACT |
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Present address: Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, UNAM, Apdo Postal 70-228, Ciudad de México, 04510, Mexico.
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INTRODUCTION |
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The LasR and RhlR proteins belong to the LuxR family of transcription regulators whose members bind to specific DNA sequences, called lux boxes (Fuqua et al., 2001). In the case of P. aeruginosa, these specific sequences have been called las boxes (Whiteley et al., 1999
), and the sequences determining whether a certain promoter is regulated by LasR, RhlR or both regulators (Whiteley et al., 1999
) are starting to be understood (De Kievit et al., 2002
; Whiteley & Greenberg, 2001
). LasR forms a multimer and binds las boxes only in the presence of 3O-C12-HSL (Kiratisin et al., 2002
), while RhlR dimerizes (Ventre et al., 2003
) and binds DNA (Medina et al., 2003b
) both in the presence and in the absence of C4-HSL.
The rhlR gene is encoded immediately downstream of the rhlAB operon and has been reported to be activated by LasR (Latifi et al., 1996; Pearson et al., 1997
). An rhlR-specific promoter has been predicted within the rhlBrhlR intergenic region (Ochsner et al., 1994b
). The role of LasR in rhlR transcription was concluded from two different studies (Latifi et al., 1996
; Pesci et al., 1997
). First, a P. aeruginosa lasR mutant did not express an rhlR : : lacZ fusion when grown on LuriaBertani (LB) medium and, second, Escherichia coli expressed an rhlR : : lacZ fusion at a low, but significant, level in the presence of LasR and 3O-C12-HSL.
Recently, the QS response has been studied using transcriptome analyses (Schuster et al., 2003; Wagner et al., 2003
). It is apparent from these reports that hundreds of P. aeruginosa genes, approximately 6 % of the organism's genome, are induced or repressed by the QS regulators LasR and RhlR and their respective coligands, 3O-C12-HSL and C4-HSL. These reports show that QS-regulated genes are mainly, but not exclusively, expressed at the early-stationary phase of growth and that this regulon is greatly influenced by environmental conditions. These results show that QS in P. aeruginosa is a very complex and fine-tuned genetic regulatory circuit that seems to be very important for both the pathogenesis of P. aeruginosa and its environmental life style.
The aim of this work was to characterize in detail the rhlR promoter region of the P. aeruginosa genome and to identify the different elements that participate in the regulation of expression of this region, determining whether environmental conditions modulate its transcription. One of the conditions studied was P. aeruginosa growth on a phosphate-limited peptone/glucose/ammonium salts (PPGAS) medium where rhamnolipid production is high (Zhang & Miller, 1992) we presumed that rhlR expression would also be high under this condition. Using primer extension analysis of RNA obtained from cells grown on PPGAS medium, we detected four rhlR transcription start sites, two of which were within the rhlB-coding region. It was also apparent from our results that cells grown on PPGAS medium expressed rhlR at a significant level in the absence of LasR and that different transcription activators, such as Vfr and RhlR itself, as well as the alternative sigma factor
54, participate in the expression of rhlR from these multiple promoters. However, when P. aeruginosa PAO1 was grown on LB medium, rhlR expression was found to be completely dependent on LasR and the gene was found to be transcribed solely from the previously predicted promoter (Ochsner et al., 1994b
). The role in rhlR transcriptional regulation of the two putative las boxes detected in the rhlR regulatory region was also explored.
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METHODS |
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Nucleic acid techniques.
DNA manipulations were performed following standard procedures (Sambrook et al., 1989). Plasmids were electroporated into P. aeruginosa as described by Smith & Iglewski (1989)
.
Primer extension analysis.
The different P. aeruginosa strains analysed were grown to an OD600 value of 1·5 and their total RNA was extracted using a GlassMAX RNA Microisolation Reagent Assembly (Life Technologies). Primer extension reactions were done using the two-step RT-PCR kit (Roche Diagnostics) according to the manufacturer's instructions. Reactions were carried out at 60 °C. These experiments were performed using two different oligonucleotides: Oligo-1 (5'-CATCTCGCTACGCAAACCGTCCCACC-3') is located at +25 bp with respect to the rhlR translational start site; Oligo-2 (5'-CGCGCATCCCCCTCCCTATGACAAC-3') is located at -146 bp with respect to the rhlR translation start site.
Plasmid construction.
Plasmid pRD58-1, containing 288 bp of the rhlR upstream region fused to a promoterless lacZ gene, was constructed by isolating a 580 bp DNA fragment containing the 288 bp of the rhlR regulatory region and part of the coding region by PCR, using the oligonucleotides 5'-GCGCAAGCTTGTGGCGCTTGCTCGAGGACC-3' and 5'-CGCTCCAGACCACCATTTCC-3'. The PCR product was digested with HindIII and BamHI and cloned into the vector pMP220 (Spaink et al., 1987). Plasmid pRD58-2, which contains 498 bp of the rhlR regulatory region fused to the lacZ gene of the vector pMP220, was constructed by ligating into this vector a fragment of 741 bp which included the rhlR upstream region and part of the coding region, which was purified from a PstI/BamHI digestion of pUO58, a plasmid containing the entire rhlA, rhlB, rhlR and rhlI gene cluster (Ochsner et al., 1994a
).
Plasmid pMT1 contains the lasR gene under the control of the lac promoter; it was constructed by isolating a PCR fragment of 860 bp containing lasR, using pSB1075 (Winson et al., 1998) as template. The oligonucleotides 5'-CCGGAATTCTCGGACTGCCGTACAAC-3' and 5'-GAAGGGCGAATTCCCGATCGCCAG-3' were used for the amplification. The PCR product was cloned into pMOSblue (Amersham Life Science), which was later digested with KpnI and HindIII. The insert was then subcloned into pUCP20 (West et al., 1994b
).
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RESULTS AND DISCUSSION |
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Expression from the P1 and P4 rhlR transcription start sites is LasR-dependent
To correlate the detected transcription start sites with the level of rhlR expression, we measured -galactosidase expression from the rhlR : : lacZ fusion encoded on pPCS1002 by growing different P. aeruginosa strains on PPGAS and LB media (Fig. 5
).
It has been reported that rhlR transcription on LB medium depends on LasR activation (Latifi et al., 1996; Pesci et al., 1997
). In accordance with the reported results, we found that rhlR was expressed at a very low level in the lasR mutant PAOR1 when grown on LB medium (Fig. 5a
), even though we could still detect transcription from promoter P2 in strain PAOR1 at a level similar to that seen in P. aeruginosa PAO1 using primer extension analysis (Fig. 4
). These results suggest that rhlR expression is subject to post-transcriptional regulation.
In contrast to the results obtained with cells grown on LB medium, we found that rhlR was expressed at a significant level in the lasR mutant PAOR1 when it was grown on PPGAS medium (Fig. 5a). This result shows that, when P. aeruginosa is grown on PPGAS medium, rhlR expression can be activated in a LasR-independent manner.
We found that start sites P1 and P4 were not detected when the lasR mutant PAOR1 was grown on PPGAS medium (Figs 1a and 2a). Promoter P1 has a characteristic structure of the promoters activated by regulators of the LuxR family (Whiteley & Greenberg, 2001
), presenting the LasR-binding site (las box1) centred at -40 nucleotides from the transcription start site (Fig. 3a
). Primer extension analysis in mutant strain PAOR1 confirms that rhlR expression from this promoter is completely dependent on LasR. However, there is not a clear explanation for the LasR-dependent transcription from promoter P4, since the position of the putative las boxes relative to this start site is not consistent with its direct activation by LasR. It is possible that this promoter is activated by LasR in an indirect manner.
The P. aeruginosa PAO1 lasR mutant (PAOR1) is completely unable to produce rhamnolipid when grown on PPGAS medium (data not shown). This result could be due to the lack of C4-HSL, considering that rhlI expression is strongly regulated by LasR (De Kievit et al., 2002). To test this hypothesis, we grew strain PAOR1 on PPGAS supplemented with 10 µM C4-HSL and found that it did not produce rhamnolipids. Recently, we have reported that the presence of RhlR and C4-HSL is a necessary, but not sufficient, condition for rhlAB expression (Medina et al., 2003a
). It is therefore possible that, when P. aeruginosa PAOR1 is grown on PPGAS supplemented with 10 µM C4-HSL, the expression of the genes encoding rhamnosyltransferase 1 is limited by other factors. Another intriguing result that highlights the importance of additional regulatory factors in rhlAB expression, besides RhlR and C4-HSL, is the lack of rhamnolipid production by P. aeruginosa PAO1 grown on LB medium, even though rhlR is expressed (Figs 4 and 5
) and C4-HSL is produced in this culture medium (Diggle et al., 2002
).
Vfr activates the expression of the P4 rhlR promoter
It has been reported that Vfr, a P. aeruginosa Crp homologue that regulates the expression of different virulence-associated traits (West et al., 1994a), directly activates lasR expression (Albus et al., 1997
). However, primer extension experiments done with RNA extracted from the PAO1-derived vfr mutant PAO9001 show that P1, the LasR-dependent transcription start site, is fully expressed (Fig. 1
), while the P4 transcription start site is not (Fig. 2
). Accordingly, with these results we found that rhlR expression is reduced in the vfr mutant PAO9001 (Fig. 5a
).
Upstream of promoter P3 and overlapping the P4 transcription start site we detected a putative Vfr-binding site (Figs 3 and 6a). The relative position of this Vfr-binding site with respect to promoter P4, which is the detected point of Vfr regulation of rhlR transcription, is uncommon for a promoter activated by a Crp-like regulator. The Crp-activated promoters present a binding site centred at -40 or -60 nucleotides from the transcription start site, and when this protein binds downstream from the start site it acts as a repressor. It is possible, however, that even though Vfr and Crp have sequence homology and recognize similar consensus sequences, they have a different mode of transcriptional activation. To address this matter, additional evidence is needed, via Vfr-binding and footprinting analyses, before it can be concluded that Vfr directly activates the P4 rhlR promoter.
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To study the influence of 54 on rhlR expression, we used the P. aeruginosa PAK-derived rpoN mutant PAKN1 (Ishimoto & Lory, 1989
) (Table 1
). We found that strain PAKN1, when grown on PPGAS medium, expressed the rhlR : : lacZ fusion at a significantly lower level than the wild-type strain PAK (Fig. 5b
), demonstrating that
54 is involved in rhlR expression.
By comparing the rhlR transcription start sites present in strains PAK and PAKN1 grown on PPGAS medium, we found that the only promoter that was completely dependent on 54 was P3 [Fig. 1
; promoter P4 was not affected by
54 activity (data not shown)]. Upstream of the P3 transcription start site we found sequences with the nucleotides and positions required for this promoter to be recognized by the RNA polymerase containing a
54 subunit (Fig. 3a
).
All 54-dependent promoters are activated by proteins belonging to the NtrC family. The activator protein of promoter P3 and its DNA-binding site remain to be identified.
Expression from the P2 rhlR transcription start site is repressed by RhlR
We detected an increased level of rhlR : : lacZ expression in the rhlR mutant 65E12 (Ochsner et al., 1994b; Table 1
) compared to the PG201 wild-type strain, when grown on LB medium (Fig. 5c
). This enhanced expression, however, could not be visualized as an enhanced level of transcription from promoter P2 using primer extension analysis (Fig. 4
). These data suggest that RhlR plays a negative role in the expression of the P2 transcription start site of its own gene, rhlR.
When mutant strain 65E12 was grown on PPGAS medium, we detected increased transcription starting from promoter P2 during the stationary phase of growth (Fig. 1), but this increment was not reflected in an increased level of expression of the rhlR : : lacZ fusion (Fig. 5c
). These data suggest that RhlR also regulates its own gene expression when cells are grown on PPGAS medium by reducing the level of transcription from promoter P2. It seems, though, that the contribution of promoter P2 to the expression of rhlR, when cells are grown on PPGAS medium, is not as important as that of the other transcription start sites.
It has been proposed that RhlR binds to the lasB OP2 las box and represses its LasR-dependent expression (Anderson et al., 1999). We found that rhlR las box1 is similar to this lasB las box (Fig. 6
a). It is possible that RhlR exerts its negative auto-regulation on promoter P2 by binding to las box1. This possibility remains to be validated experimentally.
Role of the two putative rhlR las box sequences
The analysis of the sequence of the rhlR regulatory region shows two putative las boxes (Figs 3a and 6). LasR and RhlR activate gene expression by binding to sequences located around 40 nucleotides upstream of the transcription start site (Whiteley & Greenberg, 2001
). Only las box1 is located in the expected position to bind a transcription activator acting on promoter P1 (Fig. 3
). As mentioned above, we found that this las box (Fig. 6
) is similar to one (OP2) of the two present in the regulatory region of P. aeruginosa lasB. Both the lasB OP2 las box and rhlR las box1 have 11 nucleotides between the invariable CT and AG sequences (Fig. 6
). It has been shown that the lasB OP2 las box is a suboptimal LasR3O-C12-HSL-responsive sequence (Anderson et al., 1999
). Our results (Figs 1 and 5
) are compatible with the rhlR las box1 being the LasR3O-C12-HSL-recognized sequence for activation of rhlR transcription from promoter P1.
The great majority of las boxes, including rhlR las box2, present 12 nucleotides between the invariable CT and AG sequences (Whiteley et al., 1999; Whiteley & Greenberg, 2001
). It has been reported that on these canonical las boxes an A is present at position 8 and a T is present at position 13 on genes activated by both QS regulators (Whiteley & Greenberg, 2001
), even though they might show some specificity towards one of them (Anderson et al., 1999
; De Kievit et al., 2002
; Pearson et al., 1997
; Pesci et al., 1997
). We found that none of the rhlR las boxes has the sequence requirements to be recognized by both LasR and RhlR (Fig. 6
).
It has been reported that LasR is sufficient to activate rhlR expression in an E. coli background (Latifi et al., 1996; Pearson et al., 1997
). We used this heterologous host to determine whether the LasR-dependent rhlR expression was affected by the presence of las box2. The LasR-dependent rhlR expression in the E. coli DH5
background was determined using pRD58-1 and pRD58-2 (Table 1
), both of which have a transcriptional rhlR : : lacZ fusion containing the four detected promoters, but the former plasmid lacks the most distal las box2. To do these experiments, LasR was expressed from pMT1 (Table 1
) and 3O-C12-HSL was supplemented at 10 µM. It was found that strain DH5
(pRD58-1) showed nearly 50 % more rhlR : : lacZ expression than strain DH5
(pRD58-2) (22·1±2·07 Miller Units in the former case vs 14·7±1·34 Miller Units in the latter). This difference in expression levels shows that the presence of las box2 in pRD58-2 has a significant negative effect on LasR-dependent rhlR expression in the E. coli background. However, this result also suggests that LasR3O-C12-HSL binds to both rhlR las boxes.
Negative regulation by binding of transcriptional regulators to DNA-binding sites that are distant from the promoters has been reported in the case of Vibrio fischeri luxR (Shadel & Baldwin, 1992). One of the possible mechanisms for the negative effect of LasR binding to las box2 is the formation of multimers between the LasR molecules bound to this box and LasR molecules attached to las box1 that interfere with transcription initiation or elongation. LasR multimerization and gene activation have been shown to be dependent on binding of 3O-C12-HSL (Kiratisin et al., 2002
), but it is not known whether this regulator can form dimers or higher level multimers and whether this is a regulated process. The precise role of las box2 in P. aeruginosa rhlR expression remains to be determined.
In summary, we have shown that rhlR is subject to a complex transcriptional regulation that is greatly influenced by media composition. This gene presents multiple promoters and regulatory sequences in its upstream region (Fig. 3c). Its expression is tightly regulated by environmental stimuli and is not only dependent on bacterial density, as has also been shown to occur with the P. aeruginosa QS response studied as a whole by microarray analysis (Schuster et al., 2003
; Wagner et al., 2003
). The expression of rhlR is LasR-dependent when cells are grown on LB medium, but its expression is much less dependent on this QS regulator when they are grown on PPGAS medium. The transcription from some of the rhlR promoters is positively regulated by LasR (P1 and P4) and by Vfr (P4) and repressed by RhlR (P2), and one of the rhlR promoters (P3) is
54-dependent (Fig. 3c
). To our knowledge, this is the most complex pattern of transcriptional regulation described for any of the genes encoding members of the LuxR family of transcriptional regulators.
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ACKNOWLEDGEMENTS |
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Received 4 February 2003;
revised 3 July 2003;
accepted 13 August 2003.
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