Transcriptional Organization of the Pseudomonas putida tol-oprL Genes
Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, 18008 Granada, Spain,1 Department of Molecular Microbiology, Utrecht University, Utrecht, The Netherlands2{l, http://www.100md.com
Received 15 July 2002/ Accepted 1 October 2002{l, http://www.100md.com
ABSTRACT{l, http://www.100md.com
Proteins of the Tol system play a key role in the maintenance of outer membrane integrity and cell morphology in gram-negative bacteria. In Pseudomonas putida, the seven genes, orf1, tolQ, tolR, tolA, tolB, oprL, and orf2, which encode the proteins of this complex, are clustered in a 5.8-kb region of chromosomal DNA. Analysis of polar mutations, reverse transcriptase PCR assays, and transcriptional fusion constructs with a promoterless lacZ gene revealed that the genes are arranged in two operons: orf1 tolQ tolR tolA tolB and oprL orf2. We were also able to find a transcript that was initiated at the orf1 promoter and covered the two operons in a single mRNA. On the basis of the OprL protein level, we surmised that this transcript contributed only about 10 to 15% of the total OprL protein. Primer extension analysis identified the oprL orf2 operon promoter within the tolB gene, and the -10 and -35 regions exhibited some similarity to those of {sigma} 70-recognized promoters. The transcription start point of orf1 was located 91 bp upstream of the orf1 start codon, and the -10/-35 region also exhibited {sigma} 70 -10/-35 recognition sequences. The expression from both promoters in rich and minimal media was constitutive and was very little influenced by the growth phase or iron-deficient conditions. In addition, analyses of the ß-galactosidase activities of different translational fusion constructs revealed that translation of tolA and orf2 genes was dependent on the translation of their corresponding upstream genes (tolR and oprL, respectively).
INTRODUCTIONr6, http://www.100md.com
The Pseudomonas putida tol-oprL system is composed of seven genes—orf1 (ybgC in Escherichia coli), tolQ, tolR, tolA, tolB, oprL (pal in E. coli), and orf2 (ybgF in E. coli)—clustered in a 5.8-kb DNA region. Except for Orf1, the proteins encoded by this gene cluster are located in the cell envelope, where they are believed to function in the maintenance of outer membrane integrity. This was deduced from the finding that mutations in the tolQ, tolR, tolA, tolB, or oprL gene alter cell morphology in P. putida and increase the sensitivity of this microorganism to a number of toxic compounds (28, 37). P. putida tol-oprL mutants release periplasmic proteins into the extracellular medium and form blebs at their cell surfaces (28). The tol-oprL (pal) systems have been studied mostly in E. coli (25, 27, 29), Pseudomonas aeruginosa (14, 16), Vibrio cholerae (22), and P. putida (28, 37), but homologues of the tol-oprL (pal) genes have been found in many other gram-negative bacteria (43). The phenotype exhibited by the tol-oprL (pal) mutants of E. coli (3), V. cholerae (22), and P. putida (28, 35) is consistent with the structural role proposed for this protein complex. In E. coli and P. aeruginosa there is also evidence that the Tol-OprL (PAL) proteins could be involved in outer membrane biogenesis (19, 25, 27). Interestingly, it has recently been shown that in E. coli the inner membrane protein TolA is able to undergo an energy-dependent change in conformation to form a complex with the outer membrane PAL protein; this process depends on the proton motive force and the TolQ and TolR proteins (9, 10, 20). These results clearly support the hypothesis that the Tol-PAL system forms a trans-envelope protein complex which links the inner and outer membranes, and they also suggest that this complex requires the proton motive force for its function (20, 29).
fig.ommitted7x+4*, http://www.100md.com
FI Physical organization of the tol-oprL gene cluster and scheme of the different transcriptional and translational fusions constructed. (A) Schematic map of the P. putida 5,800-bp Notl-SphI DNA fragment containing the tol-oprL gene cluster. Shaded arrows represent the different tol genes, their relative sizes, and the transcriptional directions. A putative rho-independent transcriptional terminator (G° = -19.2 kcal) is indicated downstream of orf2. Numbers below the map indicate the distance (in base pairs) between adjacent genes; negative numbers indicate that the genes overlap the indicated number of base pairs. Only relevant restriction sites are shown. Unique sites are boldfaced. The remaining sites are also present in other positions in the fragment. Bg, BglII; Bp, BlpI; E, EcoNI; N, NotI; Sg, SgrAI; Sp, SphI; X, XhoI. (B) Diagrams of the different transcriptional fusion constructs used for promoter analysis. Solid bars, DNA fragments from the tol-oprL gene cluster; hatched arrows, promoterless lacZ gene from plasmid pMP220. (C) Diagrams of the translational fusion constructs used in this study. Solid bars, DNA fragments from the tol-oprL gene cluster; dotted arrows, 'lacZ gene from plasmid pMLB1034. In all three panels, position 1 corresponds to the first G of the NotI restriction site (GCGGCCGC) located upstream of the orf1 gene (GenBank accession no. ).
Although the order of the tol-oprL (pal) genes is highly conserved among these microorganisms, their transcriptional organization seems to differ in those few cases in which it has been analyzed. In E. coli the tol-pal genes are arranged into two major transcriptional units (orf1-tolQ-tolR-tolA and tolB-oprL-orf2) (44), and for P. aeruginosa the existence of three transcriptional units (orf1-tolQ-tolR-tolA, tolB, and oprL-orf2) has been proposed (16). For these two species, the data were based mainly on analyses of ß-galactosidase activity using fusions of the putative promoter regions to lacZ, because numerous attempts to determine the transcriptional start sites of these genes in these two microorganisms by primer extension analyses had failed (16, 44). There is only one report on transcriptional analysis of the Haemophilus influenzae P6 (pal) gene, and it revealed—with the aid of Northern blotting and primer extension techniques—that the transcription initiation point of this gene was located within the upstream tolB gene (40).
In this study we analyzed the transcriptional organization of the P. putida tol-oprL gene cluster by primer extension and promoter fusion analysis. We found that, in contrast to their organization in E. coli and P. aeruginosa, the P. putida tol-oprL genes form two operons: one transcribed from a promoter located upstream of orf1 and comprising the orf1, tolQ, tolR, tolA, and tolB genes, and the other, consisting of oprL and orf2, transcribed from a promoter located within the tolB gene. We also show that translation of tolA and orf2 is dependent on the successful translation of the tolR and oprL genes, respectively.&@\.c, 百拇医药
MATERIALS AND METHODS&@\.c, 百拇医药
Bacterial strains, plasmids, culture media, and growth conditions. The bacterial strains and plasmids used in this study are listed in . Bacterial strains were routinely grown in liquid Luria-Bertani (LB) medium (38) at 30°C on a rotary shaker operated at 200 strokes per min. For experiments on the effect of iron concentration on tol-oprL expression, P. putida cells were grown in liquid M9 minimal medium without addition of ferric citrate (1), with benzoic acid (5 mM) as the sole carbon source, supplemented with either 50 µM FeCl3 (iron-rich medium) or 400 µg of ethylenediamine di(o-hydroxy) phenylacetic acid (EDDHA) per ml (iron-poor medium). When required, antibiotics were used at the following final concentrations (in micrograms per milliliter): ampicillin, 100; chloramphenicol, 30; kanamycin, 50; and tetracycline, 20.
fig.ommittedg, http://www.100md.com
Bacterial strains and plasmids used in this studyg, http://www.100md.com
Plasmids and cloning vectors are described in . Construction of plasmid pJBTOLi{Omega} Km is briefly described below. Plasmid pJBTOLi{Omega} Km is a pJB3Tc19 derivative (4) which carries the 5.7-kb SmaI-SphI insert from pTOL (28) containing the complete P. putida tol-oprL gene cluster. In addition, it also carries a 2.2-kb SmaI fragment from plasmid pHP45{Omega} Km containing the {Omega} -Km interposon (17) cloned into the SmaI site upstream of the orf1 gene to avoid any possible read-through from vector sequences. Plasmid pJBTOLi{Omega} Km was mobilized from E. coli JM109 into P. putida KT2440 by triparental mating using the E. coli HB101(pRK600) helper strain as described by de Lorenzo and Timmis (13).g, http://www.100md.com
Standard DNA methods. Standard molecular biology techniques were used for all DNA manipulations (38). DNA from the pTOL plasmid was sequenced by the dideoxy sequencing termination method (39) with T7 phage DNA polymerase (Sequenase 2.0 kit; Pharmacia). PCR amplifications were carried out as described previously (36). The sequences of the oligonucleotide primers used in this study for cloning, primer extension, and reverse transcriptase PCR (RT-PCR) will be made available upon request.
Construction of lacZ transcriptional and translational fusions and ß-galactosidase assays. Transcriptional fusions were constructed by cloning different DNA segments from the P. putida tol-oprL cluster, amplified by PCR as EcoRI-XbaI fragments with appropriate primers, into the EcoRI-XbaI sites of pMP220. To construct the translational fusions, the corresponding genes were amplified as BamHI fragments and cloned into the BamHI site of pMLB1034. All fusion constructs were confirmed by DNA sequencing. Transcriptional fusion constructs were assayed in P. putida KT2440, whereas translational fusion constructs were assayed in E. coli DH5. Transcriptional fusions were transferred from E. coli DH5 to P. putida by triparental mating using the helper plasmid pRK600 as described previously (13). ß-Galactosidase activity from fusion plasmids was measured as described previously (31). Each assay was run in duplicate at least three times, and the data given are averages. The standard deviation in all cases was below 15% of the value given.
RNA preparation and primer extension analysis. Total bacterial RNA was isolated by the single-step extraction method of Chomczynski and Sacchi (12). The different P. putida tol-oprL mutants were grown in LB medium to the desired growth phase. Culture samples (10 ml) were then harvested by centrifugation at 4°C in tubes precooled in liquid nitrogen. After centrifugation, cell pellets were immediately immersed in liquid nitrogen and treated with a guanidinium isothiocyanate-phenol-chloroform reagent as described by Chomczynski (11). RNA preparations were then subjected to standard treatments with RNase-free DNase I and proteinase K (38), and after organic-solvent extraction and ethanol precipitation they were suspended in 20 to 30 µl of diethyl pyrocarbonate-treated water.\, 百拇医药
Primer extension analyses were done basically as described by Marqués et al. (30). The amount of total RNA template used in each reaction varied between 20 and 25 µg. About 105 cpm of 32P-labeled 5'-end oligonucleotides was used as primers in extension reactions. The cDNA products obtained after the reverse transcriptase reaction were separated and analyzed in urea-polyacrylamide sequencing gels. The gels were exposed to Amersham RPN-8 films for autoradiography.
RT-PCR analysis. RT-PCR analyses were performed by using the Titan One-Tube RT-PCR kit (catalog no. 1939823; Roche) in accordance with the manufacturer's recommendations. For each reaction, 1 µg of total RNA was used. The annealing temperature in each reaction was determined according to the composition of the primers included. As negative controls, RNA samples treated with RNase prior to the reaction were included. DNA contamination of the RNA samples was ruled out by PCR using Taq DNA polymerase without reverse transcriptase.!&php, 百拇医药
Protein analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblot analyses were performed as described previously (28). The P. putida OprL protein was detected with monoclonal antibody MA1-6, raised against the P. aeruginosa OprL protein (33). The polyclonal antibody against the TolA protein was raised by immunizing rabbits with a synthetic 18-amino-acid peptide (EEAKKKAAEDAKKKAAEE). This peptide was designed from a predicted highly antigenic and repetitive sequence of a putative -helical internal domain of the P. putida TolA protein. To increase antigenicity, the peptide was coupled through glutaraldehyde to the carrier protein keyhole limpet hemocyanin. To remove nonspecific antibodies, the final antibody preparation was purified by affinity chromatography with the synthetic peptide used in the immunization protocol.
Densitometric analyses. The intensities of the bands in the autoradiographs were recorded densitometrically and quantified as the band peak areas as described previously (2).x@, 百拇医药
Computer analysis. RNA secondary structure predictions were done by using the mfold, version 2.3, program, which is available at (46).x@, 百拇医药
RESULTSx@, 百拇医药
Analysis of P. putida OprL and TolA protein expression by Western blotting. To study the transcriptional organization of the P. putida tol-oprL gene cluster, we took advantage of the availability of P. putida tol-oprL polar mutants (28) and the specific anti-OprL and anti-TolA antibodies (33; also this study). We first determined the levels of the OprL protein in P. putida tol mutants. These mutants were grown in LB liquid medium and harvested in the exponential phase of growth. Expression of OprL was analyzed by Western blotting using monoclonal antibody MA1-6. P. putida strains Q{Omega} , R{Omega} , and A{Omega} expressed the OprL protein at similar levels, although the amount of protein was lower (about half as much) than that found in the wild-type strain . The amount of OprL in the B{Omega} mutant was, however, markedly reduced (about one-fifth of the level in the wild-type strain). In this mutant the {Omega} -Km interposon was inserted into the BglII site at the end of tolB . Because in E. coli the PAL protein (the equivalent to OprL in Enterobacteriaceae) has been shown to interact with TolB (5, 6), the reduced OprL level in the P. putida B{Omega} mutant might be due to protein instability in the absence of TolB. To rule out this possibility, we analyzed the expression of OprL in P. putida BX, a strain which carries a nonpolar tolB mutation. The level of OprL in this strain was similar (about 90%) to that found in the wild-type strain . These results suggested that the lower level of OprL in the B{Omega} strain was not due to the lack of TolB. Instead, it seemed that oprL could be transcribed from more than one promoter, one of which is located upstream of the BglII site in the tolB gene, while the other is located downstream of the BglII site where the {Omega} -Km interposon was placed.
fig.ommittedh8\tw's, 百拇医药
Immunodetection of OprL and TolA in whole-cell lysates of P. putida KT2440 and tol-oprL mutants. Cells were grown in LB liquid medium, and samples were harvested when turbidity at 660 nm reached 0.5 for treatment as described in Materials and Methods. About 108 cells were loaded on each lane, and proteins were separated by SDS-PAGE (10 or 12.5% [wt/vol] polyacrylamide) and immunodetected with either the anti-OprL antibody MA1-6 (A) or an anti-TolA polyclonal antibody (B). Western blot reactions were revealed by use of the peroxidase colorimetric method (38). The position of the 21.5-kDa (A) or 45-kDa (B) molecular size marker is indicated on the right, and the protein source is given at the top. WT, wild-type strain; Q, R, A, and B, P. putida mutants carrying polar insertions of the -Km interposon in the tolQ, tolR, tolA, and tolB genes, respectively; QX, RX, AX, BX, and DOT-OX2, P. putida tolQ, tolR, tolA, tolB, and oprL nonpolar mutants, respectively. As positive and negative controls for the TolA assay, we used E. coli JM109(pTOL) and E. coli JM109(pTOLAKm), respectively.h8\tw's, 百拇医药
We also studied the expression of the P. putida TolA protein in the wild-type P. putida strain KT2440 and in the different polar and nonpolar P. putida tol-oprL mutants . Cells were grown in LB liquid medium and harvested in the exponential phase, and the expression of TolA was examined by Western blotting with a polyclonal anti-TolA antibody. P. putida KT2440, BX, B, and DOT-OX2 showed similar amounts of the TolA protein. However, TolA was not detected in the P. putida tolQ::Km and tolR::Km strains . In principle, these results suggested that at least tolQ, tolR, and tolA were cotranscribed. However, we could not detect the TolA protein in the tolQ::xylE and tolR::xylE mutant strains . We hypothesized that this was due to possible instability of the TolA protein in the absence of TolQ or TolR; in that case, the lack of TolA expression in the tolQ::Km and tolR::{Omega} Km mutants could not be interpreted as evidence for their cotranscription. The possibility also remained that translation of tolA depended on the successful translation of the wild-type tolQ-tolR mRNA. To clarify these results, we decided to analyze both possibilities as detailed below.
Identification of the transcription initiation points of the different promoters of the P. putida tol-oprL gene cluster. To define the promoters of the P. putida tol-oprL genes, we first decided to locate the transcription initiation point of the oprL mRNA by primer extension analyses. Total RNA isolated from P. putida KT2440 was annealed to a 5'-labeled oligonucleotide complementary to the oprL gene, and primer extension experiments were carried out as described in Materials and Methods. The size of the cDNA product obtained with primer 5'-ACTTCAGCATTTCCATCGTG-3', where the first A is complementary to the nucleotide located at position 4330 of the tol-oprL sequence (as numbered in ), pointed to the presence of a transcriptional start site at an adenine residue located 174 bases upstream of the putative oprL translational start codon and 120 bases upstream of the tolB translational stop codon. The -10 region of this promoter exhibited some degree of similarity to the consensus sequence proposed to be recognized by RNA polymerase with sigma-70, although it should be noted that an extended -10 box, TGCTAATCT, could also operate in this promoter; the similarity in the -35 region with the consensus sigma-70 promoter was low . Total RNA isolated from a P. putida strain harboring a low-copy-number plasmid that carried the whole tol-oprL cluster (pJBTOLi{Omega} Km) was also used in the primer extension analysis. A cDNA product of a size identical to that found in the wild-type strain was detected . In agreement with the expected increase in the tol-oprL mRNA copy number, the intensity of this band was stronger than that of the band derived from the wild-type strain. This suggested that oprL is transcribed mainly from a promoter located within the tolB gene. To clarify the reason for the smaller amount of OprL protein present in P. putida tolB::{Omega} Km, primer extension analyses were done using total RNA isolated from this strain. A cDNA product whose size was identical to that of the parental strain was found; however, the amount of product was significantly lower than that found in the wild-type strain. This suggested that the insertion of the -Km interposon in the BglII site negatively influenced transcription from the downstream oprL promoter, although the reason for this effect is at present unknown.
fig.ommitted&7, http://www.100md.com
Primer extension analysis of oprL mRNA. Cells were grown in LB medium, and samples for total-RNA isolation were taken at a turbidity of 0.5. The autoradiogram shows the cDNA products obtained after reverse transcription of 20 µg of total RNA with the 5'-labeled oligonucleotide, as described in Materials and Methods. DNA from plasmid pTOL was sequenced by using the same primer, and the sequencing products were used as size markers (lanes A, C, G, and T). Lane 1, P. putida KT2440; lane 2, P. putida KT2440(pJBTOLiKm); lane 3, P. putida B. The DNA sequence of the putative oprL promoter region is shown on the left. The proposed +1 site (boldfaced) and the putative -35 and -10 promoter regions (shaded) are indicated.&7, http://www.100md.com
Primer extension analyses were also carried out to look for the presence of additional promoters upstream of the other genes of the tol-oprL cluster. Total RNA isolated from P. putida KT2440 was hybridized to 5'-end-labeled oligonucleotides complementary to the different tol-oprL gene sequences at about 50 nucleotides from the A of the first ATG of each gene. After primer extension, we obtained a definite cDNA band for the primer based on the orf1 gene, whereas in the rest of the cases, instead of a well-defined cDNA product, numerous long extension products were always observed, probably corresponding to degraded mRNA species forming part of a larger transcript coming from a distant promoter (data not shown). The 5' end of orf1 mRNA was identified as a thymine residue located 91 bases upstream of its translational start codon. Putative -35 and -10 sites resembling those recognized by RNA polymerase with sigma-70 were found . These results suggested that the orf1, tolQ, tolR, tolA, and tolB genes formed a transcriptional unit that was read from a single promoter located upstream of orf1.
fig.ommitted#mq, 百拇医药
Primer extension analysis of orf1 mRNA. Cells were grown in LB medium, and samples were taken when turbidity at 660 nm reached 0.5 for total RNA isolation. The autoradiogram shows the cDNA products obtained after reverse transcription of 20 µg of total RNA with the 5'-end-labeled oligonucleotide, as described in Materials and Methods. DNA from plasmid pTOL was sequenced by using the same primer, and the sequencing products were used as size markers (lanes A, C, G, and T). Lane 1, P. putida KT2440(pJBTOLiKm); lane 2, P. putida KT2440. The DNA sequence of the putative orf1 promoter region is shown on the left. Boldfaced nucleotides represent the proposed +1 site and the ruvB stop codon. The putative -35 and -10 promoter regions (shaded) and an SphI restriction site are indicated.#mq, 百拇医药
RT-PCR analysis. The data obtained by Western blotting and primer extension analysis suggested that the tol-oprL region of P. putida consisted of two operons, orf1-tolQRAB and oprL-orf2. To confirm this transcriptional organization and to investigate in further detail whether the transcription of the two operons is connected to any degree (as shown for the E. coli and P. aeruginosa tol-oprL [pal] genes [16, 44]), we carried out RT-PCR analysis of the RNA isolated from P. putida KT2440. Primer pairs that hybridized with the 3' and 5' ends of each two adjacent genes of the tol-oprL cluster were designed . We also checked for the presence of putative mRNA products extending beyond the orf2 gene, or which appeared as a consequence of read-through from ruvB, the gene located immediately upstream of orf1. No cDNA amplification products were obtained for the primer pair hybridizing with the ruvB and orf1 genes or for that hybridizing with the orf2 and orf3 genes ( lanes 7 and 8). These results demonstrated that the P. putida tol-oprL mRNA was clearly delimited by the orf1 and orf2 genes. RT-PCR products of the predicted sizes were, however, generated when the remaining primer pairs were used , lanes 1 to 6). Particularly important was the positive amplification obtained with the primers covering the region between tolB and oprL . Since the 5' end of primer 4045 is located 96 bp upstream of the oprL transcriptional start site, this result strongly suggested that some proportion of the expression of oprL-orf2 could also derive from the promoter located upstream of orf1. This would imply the existence of a single mRNA comprising the entire tol-oprL region, from orf1 to orf2, plus a second mRNA, initiated in the promoter located upstream of oprL, which would include the two 3'-most genes of the cluster.
fig.ommittedw?\h^, http://www.100md.com
Organization of the tol-oprL genes in P. putida KT2440. (A) Localization of the tol-oprL genes and positions of the primers used for mRNA amplification in RT-PCR assays. Numbers below the primers indicate their positions in the tol-oprL DNA sequence. Nucleotides are numbered as in . (B) Gel electrophoresis of the cDNA amplified with oligonucleotides 450 and 747 (lane 1), 1308 and 1461 (lane 2), 1777 and 1929 (lane 3), 2837 and 3049 (lane 4), 4045 and 4391 (lane 5), 4532 and 4918 (lane 6), 25 and 308 (lane 7), and 5510 and 5857 (lane 8). Positions of molecular size markers (in base pairs) are indicated on the left. Negative controls containing the same amounts of RNA, primers, and Taq polymerase, but no reverse transcriptase, were included in this assay (data not shown).w?\h^, http://www.100md.com
Effects of growth phase and iron concentration on the expression of the tol-oprL genes of P. putida KT2440. It has been suggested that in P. aeruginosa the expression of the tol-oprL gene cluster is under growth phase and iron limitation control (16, 24, 34). We cultured P. putida KT2440 in LB medium and harvested cells in the exponential-growth phase (optical density at 660 nm, 0.6) or in the stationary phase (optical density at 660 nm, 2) and estimated the levels of TolA and OprL by Western blot analysis as described in Materials and Methods. It was found that OprL and TolA levels decreased when cells reached the stationary-growth phase (data not shown). To determine whether the expression of the oprL and orf1 promoters was growth phase regulated, transcriptional fusion analyses were done . A 271-bp DNA fragment containing the region upstream of oprL was fused to a promoterless lacZ reporter gene on the pMP220 low-copy-number plasmid to generate plasmid pMPLB. A 224-bp DNA fragment upstream of orf1 bearing the promoter region was also fused to the pMP220 plasmid to generate plasmid pMP1B . These plasmids were then transferred to P. putida KT2440, and the expression of the corresponding fusion was determined by measuring ß-galactosidase activity. In the exponential phase, the ß-galactosidase activity of the fusion in pMPLB was about 20,000 Miller units, whereas in the stationary phase, the activity decreased to 15,000 Miller units . The level of expression from the orf1 promoter, quantified by measuring the ß-galactosidase activity of the pMP1B construct in cells grown in LB medium, was about 2,700 Miller units in the exponential phase and decreased to 900 Miller units in the stationary phase . These results confirmed the results obtained by Western blot analysis.
fig.ommitted9t-c}7z, 百拇医药
Transcriptional analysis of the oprL and orf1 promoters with lacZ fusions. (A) Effects of growth phase on expression of the oprL and orf1 promoters. P. putida cells bearing either pMP200 as a negative control (open circles), the Porf1::lacZ fusion in pMP1B (filled circles), or the PoprL::lacZ fusion in pMPLB (filled triangles) were grown in LB medium. (Insets) Growth curve of each strain used in the assay. (B) Effects of iron on expression from the Porf1 and PoprL promoters in P. putida KT2440. P. putida KT2440 cells bearing the indicated plasmids were grown in M9 minimal medium supplemented with 50 µM FeCl3 (hatched bars) or 400 µg of EDDHA ml-1 (open bars). Values are means from triplicate experiments.9t-c}7z, 百拇医药
Because some degree of iron regulation had previously been reported for the P. aeruginosa oprL and tolQRA genes (16, 24, 34), we decided to study whether the iron concentration in the medium affected P. putida oprL and orf1 expression. The expression levels of oprL and orf1 were analyzed in P. putida cells harboring plasmid pMPLB or pMP1B grown in M9 minimal medium under iron-rich (50 µM FeCl3) or iron-restricted (400 µg of EDDHA ml-1) conditions. The levels of expression from the oprL or orf1 promoter were similar under iron-rich and iron-restricted conditions . This expression pattern was growth phase independent (data not shown).
Analysis of P. putida tol-oprL translational lacZ fusions. Analysis of the DNA sequence upstream of tolA revealed a potential loop structure in the mRNA region, which contained the stop codon of tolR, as well as the potential ribosome-binding site and the start codon of tolA . Another potential mRNA loop structure was predicted between oprL and orf2 . We reasoned that if these loops existed in vivo, translation of these genes was likely to be coupled. To investigate this possibility, translational fusions to the lacZ reporter gene were constructed for every gene of the tol-oprL cluster and their ß-galactosidase activities were determined . Then the plasmids harboring the different tol-oprL translational fusions were digested at unique restriction sites, and after treatment with Klenow enzyme, they were religated. The addition of nucleotides by this treatment altered the reading frame of the gene where the restriction site was located, resulting in the appearance of premature stop codons. The effect of generating frameshift mutations in the genes upstream of each tol-oprL gene fusion was tested by measuring the ß-galactosidase activity . We found that when the expression of TolR was abolished by the presence of a frameshift mutation, the expression of the TolA protein was reduced drastically, to 5% of the control value compare ß-galactosidase values for pMLA::lacZ and pMLA-R). This suggested that translation of the tolA mRNA depended on translation of the preceding tolR mRNA. The same result was obtained for the oprL and orf2 genes. A frameshift mutation in oprL exerted a polar effect on the expression of the orf2 gene, reducing ß-galactosidase activity to less than 1% of the activity of the original fusion (pMLO2::lacZ versus pMLO2-P), which strongly suggested that these genes were also translationally coupled.
fig.ommitted9-{, 百拇医药
Predicted secondary structures of the tolR-tolA mRNA (A) and the oprL-orf2 mRNA (B). The start codon is shaded, whereas the predicted ribosome binding site is boxed. Boldfaced nucleotides represent translational stop codons. The most stable secondary structures and their G° were predicted using the mfold, version 2.3, program, with the default setting at 30°C. Nucleotides are numbered as in .9-{, 百拇医药
fig.ommitted9-{, 百拇医药
ß-Galactosidase activities of the lacZ translational fusion constructsa9-{, 百拇医药
On the other hand, because the RT-PCR assays suggested that a single mRNA containing the entire region from orf1 to orf2 could be transcribed from the promoter upstream of orf1, we decided to quantify the contribution of this promoter to the expression of the oprL and orf2 genes. To abolish the activity of the orf1 promoter, an -Km interposon was cloned into the SphI restriction sites of the plasmids harboring the oprL and orf2::lacZ fusions (constructions pMLP-P1Km and pMLO2-P1Km in ), and ß-galactosidase activities of the new fusion constructs were then determined and compared with those of the original fusions. With these constructions we found that approximately 85% of the ß-galactosidase activity was still present in the oprL and orf2::lacZ fusions when expression from the orf1 promoter was abolished . These results confirmed that most of the OprL and Orf2 proteins are expressed from an mRNA starting at the promoter upstream of oprL, and only a small portion (15%) of these proteins is synthesized from an mRNA derived from the promoter upstream of orf1.
DISCUSSIONs, 百拇医药
The tol-oprL gene cluster of P. putida is organized in two operons. Although the gene organization of the tol-oprL (pal) systems is highly conserved among most gram-negative bacteria (43), their transcriptional organization has been studied in only a few species (16, 24, 32, 40, 44), and most of these studies were based on gene fusion analysis. Furthermore, most attempts to determine the transcriptional initiation points of the tol-oprL (pal) promoters in these microorganisms have failed; one of the reasons was the low level of transcription or the instability and rapid degradation of these mRNA species (16, 32, 44). Alternative approaches, such as analyzing the mRNA overexpressed from the tol-oprL genes cloned into plasmids, have usually resulted in misleading interpretations (16, 24). For this reason we decided to use total mRNA isolated directly from wild-type cells as a source for our analysis. We have shown that the P. putida tol-oprL genes are arranged into two operons, orf1-tolQ-tolR-tolA-tolB and oprL-orf2, and we have also identified the transcription start sites for the promoters of both operons. Within each operon the genes are tightly clustered, and the distance that separates the coding sequences of the two operons is 54 bp. This arrangement is almost identical to that found in P. aeruginosa, but surprisingly, the transcriptional organization differs in the two bacteria.
We have also analyzed the expression of the P. putida TolA protein by Western blotting in different P. putida tol-oprL mutants . Once the transcriptional and translational organization of the whole gene cluster was elucidated, it became clear that the lack of TolA protein in the tolQ::xylE mutant was probably due to the absence of the TolQ protein. It therefore follows that the P. putida TolQ protein is required for the stability of the TolA protein. TolQ may also be necessary for TolR stability, and TolR, rather than TolQ (or probably both), may be required for TolA stability in P. putida KT2440. In E. coli, TolA seems to be a key protein for the trans-envelope Tol-Pal system, since it is involved in a wide variety of protein interactions within this complex (26). For example, it interacts with TolQ and TolR (15, 23), with Pal (8, 9, 20), and also with TolB (29). Germon et al. (20) have observed that in E. coli, the lack of TolQ or TolR does not affect TolA stability; however, this protein is highly unstable in a tolB pal background.
In E. coli and P. aeruginosa a long mRNA transcript containing the entire tol-oprL (pal) region has been proposed to exist (16, 32, 44). In P. putida, the lack of obvious terminators within the gene cluster makes it possible that transcription from the orf1 promoter may read through oprL and orf2. The results of the RT-PCR and the ß-galactosidase assays support this possibility. As Vianney et al. (44) pointed out for E. coli, the ability to express oprL-orf2 from its own promoter allows orf1-tolQ-tolR-tolA-tolB to be separately modulated. In any case, the long orf1-orf2 mRNA transcript in these bacteria must be highly unstable, as suggested by the fact that only mRNA species smaller than those predicted were detected by Northern expression analysis (24, 32).j!r8|\, http://www.100md.com
The orf1 and oprL promoters exhibit features of promoters transcribed by RNA polymerase with sigma-70. The transcriptional start sites of P. putida orf1 and oprL promoters were determined by primer extension. Both promoters show features typical of promoters recognized by sigma-70 . The P. putida oprL promoter is located within the tolB gene; in this connection it is worth recalling that in H. influenzae, transcriptional analysis of the P6 (pal) gene revealed that its transcription initiation point was also within the region immediately upstream of the tolB gene (40). The -35 box of the oprL promoter exhibits low similarity to the -35 consensus box of promoters recognized by sigma-70. However, its -10 box shows a better match with the consensus than it does with the -10 box of the orf1 promoter , since the presence of the 5'-TG-3' sequence located 1 base upstream of the -10 hexamer could result in an extended -10 element . This may also explain the lower activity of the orf1 promoter in comparison to the oprL promoter and could be the reason for the differences in the expression levels of lacZ transcriptional fusions. The above results for P. putida are in agreement with those for P. aeruginosa and E. coli. In P. aeruginosa, the level of expression of an oprL::lacZ transcriptional fusion was higher than that observed for an orf1::lacZ fusion (16). This was also the case for E. coli, where the amount of orf1 mRNA detected by Northern blot analysis was lower than that of tolB mRNA (32). Cascales et al. (8) recently estimated that in E. coli, the Pal lipoprotein is present in larger amounts (30,000 to 40,000 copies per cell) than the TolA (400 to 800 copies) or TolR (2,000 to 3,000 copies) protein. These proportions correlate well with the above data.
Comparison of the ruvB-orf1 intergenic regions of the different Pseudomonas species sequenced so far showed a low level of sequence conservation in the positions where the P. putida orf1 promoter was found. In addition, in P. aeruginosa this region (52 bp) is considerably shorter than it is in the other three species (125 bp in P. putida, 134 bp in Pseudomonas syringae, and 138 bp in Pseudomonas fluorescens). However, comparison of the oprL promoter region revealed complete conservation of the positions located in the proposed -35 and -10 boxes . Nevertheless, it should also be pointed out that since this region is located within the coding sequence of the tolB gene, a stronger selective pressure will operate in maintaining the sequence conservation at these positions (although the conservation of the TolB proteins at the amino acid level in this region is not particularly high among the different gram-negative bacteria).84)0uu1, http://www.100md.com
fig.ommitted84)0uu1, http://www.100md.com
Alignment of the regions containing the P. putida tol-oprL promoters with the corresponding regions from P. fluorescens, P. aeruginosa, and P. syringae. Nucleotides that are conserved at least in three of the four positions are included in the consensus sequence. The predicted ruvB stop codons are boldfaced. The start positions of the P. putida mRNA transcripts (indicated by +1) are underlined. The proposed -10 and -35 promoter regions are boxed. (A) Alignment of the orf1 promoter region. The consensus motif for Fur binding is shown above the empirically determined Fur box for P. aeruginosa (34). Nucleotides that are identical in the two sequences are indicated by asterisks. (B) Alignment of the oprL promoter region.
Expression of the P. aeruginosa orf1-tolQ-tolR-tolA gene cluster is negatively regulated by the presence of iron in the culture medium (16). In this process the ferric uptake regulator (Fur) has been shown to be involved by directly binding to the orf1 promoter region, where a fur box motif is present (34). In P. putida KT2440 we have observed no influence of iron concentration on the expression of the tol-oprL genes, and we could find no recognizable fur box motif in the corresponding promoter regions . In the case of P. syringae and P. fluorescens, DNA sequences (matching 9 out of 19 bp in the Fur-binding consensus sequence) have been found in exactly the same location as in P. aeruginosa . Although these are only putative Fur boxes, it is noteworthy that DNA sequences exhibiting homology of only 10 bp to the consensus have been identified as being directly bound by P. aeruginosa Fur (34).l{p)f)[, 百拇医药
Translational coupling between oprL and orf2 and between tolR and tolA. We have also shown evidence of the existence of translational coupling between P. putida tolR and tolA, as well as between oprL and orf2 . A similar situation has also been reported for the E. coli tolQ and tolR genes (44). Translational regulation is potentially important for the balanced synthesis of these proteins, which is probably required to maintain the stoichiometry of this protein complex. Although we have not analyzed the molecular mechanism of these translational couplings in detail, the presence of recognizable ribosome-binding sites upstream of both tolA and orf2 suggests that translation is inhibited by making the ribosome-binding sites of these genes inaccessible by camouflaging them within stable mRNA secondary structures, as predicted, and thus making their translation dependent on the prior translation of the gene immediately upstream.
In summary, the Tol-OprL (Pal) system plays a key role in maintaining cell morphology and outer membrane integrity in gram-negative bacteria. The proteins are well conserved in this group of prokaryotes; however, the transcriptional organization of the gene clusters, and the levels of expression of the genes in response to different environmental conditions, differ among these microorganisms. These differences may be related to the microbe's preferred habitat and to the way in which expression of these gene clusters is integrated in global regulatory networks.k0dkgt|, http://www.100md.com
Finally, it is also interesting that the orf1 (ybgC) gene associated with the tol-pal gene cluster in H. influenzae has recently been shown to encode a protein that displays a significant level of thioesterase activity toward short-chain acyl coenzyme A thioesters. However, it is not yet clear how the thioesterase activity of this protein may be related to the function of the Tol-Pal system (45).k0dkgt|, http://www.100md.com
ACKNOWLEDGMENTS
We thank Silvia Marqués for helpful advice and discussions. We thank Ana Hurtado for assistance in DNA sequencing, Carmen Lorente and M. Mar Fandila for secretarial assistance, and K. Shashok for help with the language in the manuscript.r., 百拇医药
M. A. Llamas was the recipient of a fellowship from the Spanish Ministry of Education and Culture. This work was supported by grants from the Ministerio de Ciencia y Tecnología (FEDER 1FD97-1437) and the European Commission (QLK3-2000-00170).r., 百拇医药
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Received 15 July 2002/ Accepted 1 October 2002{l, http://www.100md.com
ABSTRACT{l, http://www.100md.com
Proteins of the Tol system play a key role in the maintenance of outer membrane integrity and cell morphology in gram-negative bacteria. In Pseudomonas putida, the seven genes, orf1, tolQ, tolR, tolA, tolB, oprL, and orf2, which encode the proteins of this complex, are clustered in a 5.8-kb region of chromosomal DNA. Analysis of polar mutations, reverse transcriptase PCR assays, and transcriptional fusion constructs with a promoterless lacZ gene revealed that the genes are arranged in two operons: orf1 tolQ tolR tolA tolB and oprL orf2. We were also able to find a transcript that was initiated at the orf1 promoter and covered the two operons in a single mRNA. On the basis of the OprL protein level, we surmised that this transcript contributed only about 10 to 15% of the total OprL protein. Primer extension analysis identified the oprL orf2 operon promoter within the tolB gene, and the -10 and -35 regions exhibited some similarity to those of {sigma} 70-recognized promoters. The transcription start point of orf1 was located 91 bp upstream of the orf1 start codon, and the -10/-35 region also exhibited {sigma} 70 -10/-35 recognition sequences. The expression from both promoters in rich and minimal media was constitutive and was very little influenced by the growth phase or iron-deficient conditions. In addition, analyses of the ß-galactosidase activities of different translational fusion constructs revealed that translation of tolA and orf2 genes was dependent on the translation of their corresponding upstream genes (tolR and oprL, respectively).
INTRODUCTIONr6, http://www.100md.com
The Pseudomonas putida tol-oprL system is composed of seven genes—orf1 (ybgC in Escherichia coli), tolQ, tolR, tolA, tolB, oprL (pal in E. coli), and orf2 (ybgF in E. coli)—clustered in a 5.8-kb DNA region. Except for Orf1, the proteins encoded by this gene cluster are located in the cell envelope, where they are believed to function in the maintenance of outer membrane integrity. This was deduced from the finding that mutations in the tolQ, tolR, tolA, tolB, or oprL gene alter cell morphology in P. putida and increase the sensitivity of this microorganism to a number of toxic compounds (28, 37). P. putida tol-oprL mutants release periplasmic proteins into the extracellular medium and form blebs at their cell surfaces (28). The tol-oprL (pal) systems have been studied mostly in E. coli (25, 27, 29), Pseudomonas aeruginosa (14, 16), Vibrio cholerae (22), and P. putida (28, 37), but homologues of the tol-oprL (pal) genes have been found in many other gram-negative bacteria (43). The phenotype exhibited by the tol-oprL (pal) mutants of E. coli (3), V. cholerae (22), and P. putida (28, 35) is consistent with the structural role proposed for this protein complex. In E. coli and P. aeruginosa there is also evidence that the Tol-OprL (PAL) proteins could be involved in outer membrane biogenesis (19, 25, 27). Interestingly, it has recently been shown that in E. coli the inner membrane protein TolA is able to undergo an energy-dependent change in conformation to form a complex with the outer membrane PAL protein; this process depends on the proton motive force and the TolQ and TolR proteins (9, 10, 20). These results clearly support the hypothesis that the Tol-PAL system forms a trans-envelope protein complex which links the inner and outer membranes, and they also suggest that this complex requires the proton motive force for its function (20, 29).
fig.ommitted7x+4*, http://www.100md.com
FI Physical organization of the tol-oprL gene cluster and scheme of the different transcriptional and translational fusions constructed. (A) Schematic map of the P. putida 5,800-bp Notl-SphI DNA fragment containing the tol-oprL gene cluster. Shaded arrows represent the different tol genes, their relative sizes, and the transcriptional directions. A putative rho-independent transcriptional terminator (G° = -19.2 kcal) is indicated downstream of orf2. Numbers below the map indicate the distance (in base pairs) between adjacent genes; negative numbers indicate that the genes overlap the indicated number of base pairs. Only relevant restriction sites are shown. Unique sites are boldfaced. The remaining sites are also present in other positions in the fragment. Bg, BglII; Bp, BlpI; E, EcoNI; N, NotI; Sg, SgrAI; Sp, SphI; X, XhoI. (B) Diagrams of the different transcriptional fusion constructs used for promoter analysis. Solid bars, DNA fragments from the tol-oprL gene cluster; hatched arrows, promoterless lacZ gene from plasmid pMP220. (C) Diagrams of the translational fusion constructs used in this study. Solid bars, DNA fragments from the tol-oprL gene cluster; dotted arrows, 'lacZ gene from plasmid pMLB1034. In all three panels, position 1 corresponds to the first G of the NotI restriction site (GCGGCCGC) located upstream of the orf1 gene (GenBank accession no. ).
Although the order of the tol-oprL (pal) genes is highly conserved among these microorganisms, their transcriptional organization seems to differ in those few cases in which it has been analyzed. In E. coli the tol-pal genes are arranged into two major transcriptional units (orf1-tolQ-tolR-tolA and tolB-oprL-orf2) (44), and for P. aeruginosa the existence of three transcriptional units (orf1-tolQ-tolR-tolA, tolB, and oprL-orf2) has been proposed (16). For these two species, the data were based mainly on analyses of ß-galactosidase activity using fusions of the putative promoter regions to lacZ, because numerous attempts to determine the transcriptional start sites of these genes in these two microorganisms by primer extension analyses had failed (16, 44). There is only one report on transcriptional analysis of the Haemophilus influenzae P6 (pal) gene, and it revealed—with the aid of Northern blotting and primer extension techniques—that the transcription initiation point of this gene was located within the upstream tolB gene (40).
In this study we analyzed the transcriptional organization of the P. putida tol-oprL gene cluster by primer extension and promoter fusion analysis. We found that, in contrast to their organization in E. coli and P. aeruginosa, the P. putida tol-oprL genes form two operons: one transcribed from a promoter located upstream of orf1 and comprising the orf1, tolQ, tolR, tolA, and tolB genes, and the other, consisting of oprL and orf2, transcribed from a promoter located within the tolB gene. We also show that translation of tolA and orf2 is dependent on the successful translation of the tolR and oprL genes, respectively.&@\.c, 百拇医药
MATERIALS AND METHODS&@\.c, 百拇医药
Bacterial strains, plasmids, culture media, and growth conditions. The bacterial strains and plasmids used in this study are listed in . Bacterial strains were routinely grown in liquid Luria-Bertani (LB) medium (38) at 30°C on a rotary shaker operated at 200 strokes per min. For experiments on the effect of iron concentration on tol-oprL expression, P. putida cells were grown in liquid M9 minimal medium without addition of ferric citrate (1), with benzoic acid (5 mM) as the sole carbon source, supplemented with either 50 µM FeCl3 (iron-rich medium) or 400 µg of ethylenediamine di(o-hydroxy) phenylacetic acid (EDDHA) per ml (iron-poor medium). When required, antibiotics were used at the following final concentrations (in micrograms per milliliter): ampicillin, 100; chloramphenicol, 30; kanamycin, 50; and tetracycline, 20.
fig.ommittedg, http://www.100md.com
Bacterial strains and plasmids used in this studyg, http://www.100md.com
Plasmids and cloning vectors are described in . Construction of plasmid pJBTOLi{Omega} Km is briefly described below. Plasmid pJBTOLi{Omega} Km is a pJB3Tc19 derivative (4) which carries the 5.7-kb SmaI-SphI insert from pTOL (28) containing the complete P. putida tol-oprL gene cluster. In addition, it also carries a 2.2-kb SmaI fragment from plasmid pHP45{Omega} Km containing the {Omega} -Km interposon (17) cloned into the SmaI site upstream of the orf1 gene to avoid any possible read-through from vector sequences. Plasmid pJBTOLi{Omega} Km was mobilized from E. coli JM109 into P. putida KT2440 by triparental mating using the E. coli HB101(pRK600) helper strain as described by de Lorenzo and Timmis (13).g, http://www.100md.com
Standard DNA methods. Standard molecular biology techniques were used for all DNA manipulations (38). DNA from the pTOL plasmid was sequenced by the dideoxy sequencing termination method (39) with T7 phage DNA polymerase (Sequenase 2.0 kit; Pharmacia). PCR amplifications were carried out as described previously (36). The sequences of the oligonucleotide primers used in this study for cloning, primer extension, and reverse transcriptase PCR (RT-PCR) will be made available upon request.
Construction of lacZ transcriptional and translational fusions and ß-galactosidase assays. Transcriptional fusions were constructed by cloning different DNA segments from the P. putida tol-oprL cluster, amplified by PCR as EcoRI-XbaI fragments with appropriate primers, into the EcoRI-XbaI sites of pMP220. To construct the translational fusions, the corresponding genes were amplified as BamHI fragments and cloned into the BamHI site of pMLB1034. All fusion constructs were confirmed by DNA sequencing. Transcriptional fusion constructs were assayed in P. putida KT2440, whereas translational fusion constructs were assayed in E. coli DH5. Transcriptional fusions were transferred from E. coli DH5 to P. putida by triparental mating using the helper plasmid pRK600 as described previously (13). ß-Galactosidase activity from fusion plasmids was measured as described previously (31). Each assay was run in duplicate at least three times, and the data given are averages. The standard deviation in all cases was below 15% of the value given.
RNA preparation and primer extension analysis. Total bacterial RNA was isolated by the single-step extraction method of Chomczynski and Sacchi (12). The different P. putida tol-oprL mutants were grown in LB medium to the desired growth phase. Culture samples (10 ml) were then harvested by centrifugation at 4°C in tubes precooled in liquid nitrogen. After centrifugation, cell pellets were immediately immersed in liquid nitrogen and treated with a guanidinium isothiocyanate-phenol-chloroform reagent as described by Chomczynski (11). RNA preparations were then subjected to standard treatments with RNase-free DNase I and proteinase K (38), and after organic-solvent extraction and ethanol precipitation they were suspended in 20 to 30 µl of diethyl pyrocarbonate-treated water.\, 百拇医药
Primer extension analyses were done basically as described by Marqués et al. (30). The amount of total RNA template used in each reaction varied between 20 and 25 µg. About 105 cpm of 32P-labeled 5'-end oligonucleotides was used as primers in extension reactions. The cDNA products obtained after the reverse transcriptase reaction were separated and analyzed in urea-polyacrylamide sequencing gels. The gels were exposed to Amersham RPN-8 films for autoradiography.
RT-PCR analysis. RT-PCR analyses were performed by using the Titan One-Tube RT-PCR kit (catalog no. 1939823; Roche) in accordance with the manufacturer's recommendations. For each reaction, 1 µg of total RNA was used. The annealing temperature in each reaction was determined according to the composition of the primers included. As negative controls, RNA samples treated with RNase prior to the reaction were included. DNA contamination of the RNA samples was ruled out by PCR using Taq DNA polymerase without reverse transcriptase.!&php, 百拇医药
Protein analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblot analyses were performed as described previously (28). The P. putida OprL protein was detected with monoclonal antibody MA1-6, raised against the P. aeruginosa OprL protein (33). The polyclonal antibody against the TolA protein was raised by immunizing rabbits with a synthetic 18-amino-acid peptide (EEAKKKAAEDAKKKAAEE). This peptide was designed from a predicted highly antigenic and repetitive sequence of a putative -helical internal domain of the P. putida TolA protein. To increase antigenicity, the peptide was coupled through glutaraldehyde to the carrier protein keyhole limpet hemocyanin. To remove nonspecific antibodies, the final antibody preparation was purified by affinity chromatography with the synthetic peptide used in the immunization protocol.
Densitometric analyses. The intensities of the bands in the autoradiographs were recorded densitometrically and quantified as the band peak areas as described previously (2).x@, 百拇医药
Computer analysis. RNA secondary structure predictions were done by using the mfold, version 2.3, program, which is available at (46).x@, 百拇医药
RESULTSx@, 百拇医药
Analysis of P. putida OprL and TolA protein expression by Western blotting. To study the transcriptional organization of the P. putida tol-oprL gene cluster, we took advantage of the availability of P. putida tol-oprL polar mutants (28) and the specific anti-OprL and anti-TolA antibodies (33; also this study). We first determined the levels of the OprL protein in P. putida tol mutants. These mutants were grown in LB liquid medium and harvested in the exponential phase of growth. Expression of OprL was analyzed by Western blotting using monoclonal antibody MA1-6. P. putida strains Q{Omega} , R{Omega} , and A{Omega} expressed the OprL protein at similar levels, although the amount of protein was lower (about half as much) than that found in the wild-type strain . The amount of OprL in the B{Omega} mutant was, however, markedly reduced (about one-fifth of the level in the wild-type strain). In this mutant the {Omega} -Km interposon was inserted into the BglII site at the end of tolB . Because in E. coli the PAL protein (the equivalent to OprL in Enterobacteriaceae) has been shown to interact with TolB (5, 6), the reduced OprL level in the P. putida B{Omega} mutant might be due to protein instability in the absence of TolB. To rule out this possibility, we analyzed the expression of OprL in P. putida BX, a strain which carries a nonpolar tolB mutation. The level of OprL in this strain was similar (about 90%) to that found in the wild-type strain . These results suggested that the lower level of OprL in the B{Omega} strain was not due to the lack of TolB. Instead, it seemed that oprL could be transcribed from more than one promoter, one of which is located upstream of the BglII site in the tolB gene, while the other is located downstream of the BglII site where the {Omega} -Km interposon was placed.
fig.ommittedh8\tw's, 百拇医药
Immunodetection of OprL and TolA in whole-cell lysates of P. putida KT2440 and tol-oprL mutants. Cells were grown in LB liquid medium, and samples were harvested when turbidity at 660 nm reached 0.5 for treatment as described in Materials and Methods. About 108 cells were loaded on each lane, and proteins were separated by SDS-PAGE (10 or 12.5% [wt/vol] polyacrylamide) and immunodetected with either the anti-OprL antibody MA1-6 (A) or an anti-TolA polyclonal antibody (B). Western blot reactions were revealed by use of the peroxidase colorimetric method (38). The position of the 21.5-kDa (A) or 45-kDa (B) molecular size marker is indicated on the right, and the protein source is given at the top. WT, wild-type strain; Q, R, A, and B, P. putida mutants carrying polar insertions of the -Km interposon in the tolQ, tolR, tolA, and tolB genes, respectively; QX, RX, AX, BX, and DOT-OX2, P. putida tolQ, tolR, tolA, tolB, and oprL nonpolar mutants, respectively. As positive and negative controls for the TolA assay, we used E. coli JM109(pTOL) and E. coli JM109(pTOLAKm), respectively.h8\tw's, 百拇医药
We also studied the expression of the P. putida TolA protein in the wild-type P. putida strain KT2440 and in the different polar and nonpolar P. putida tol-oprL mutants . Cells were grown in LB liquid medium and harvested in the exponential phase, and the expression of TolA was examined by Western blotting with a polyclonal anti-TolA antibody. P. putida KT2440, BX, B, and DOT-OX2 showed similar amounts of the TolA protein. However, TolA was not detected in the P. putida tolQ::Km and tolR::Km strains . In principle, these results suggested that at least tolQ, tolR, and tolA were cotranscribed. However, we could not detect the TolA protein in the tolQ::xylE and tolR::xylE mutant strains . We hypothesized that this was due to possible instability of the TolA protein in the absence of TolQ or TolR; in that case, the lack of TolA expression in the tolQ::Km and tolR::{Omega} Km mutants could not be interpreted as evidence for their cotranscription. The possibility also remained that translation of tolA depended on the successful translation of the wild-type tolQ-tolR mRNA. To clarify these results, we decided to analyze both possibilities as detailed below.
Identification of the transcription initiation points of the different promoters of the P. putida tol-oprL gene cluster. To define the promoters of the P. putida tol-oprL genes, we first decided to locate the transcription initiation point of the oprL mRNA by primer extension analyses. Total RNA isolated from P. putida KT2440 was annealed to a 5'-labeled oligonucleotide complementary to the oprL gene, and primer extension experiments were carried out as described in Materials and Methods. The size of the cDNA product obtained with primer 5'-ACTTCAGCATTTCCATCGTG-3', where the first A is complementary to the nucleotide located at position 4330 of the tol-oprL sequence (as numbered in ), pointed to the presence of a transcriptional start site at an adenine residue located 174 bases upstream of the putative oprL translational start codon and 120 bases upstream of the tolB translational stop codon. The -10 region of this promoter exhibited some degree of similarity to the consensus sequence proposed to be recognized by RNA polymerase with sigma-70, although it should be noted that an extended -10 box, TGCTAATCT, could also operate in this promoter; the similarity in the -35 region with the consensus sigma-70 promoter was low . Total RNA isolated from a P. putida strain harboring a low-copy-number plasmid that carried the whole tol-oprL cluster (pJBTOLi{Omega} Km) was also used in the primer extension analysis. A cDNA product of a size identical to that found in the wild-type strain was detected . In agreement with the expected increase in the tol-oprL mRNA copy number, the intensity of this band was stronger than that of the band derived from the wild-type strain. This suggested that oprL is transcribed mainly from a promoter located within the tolB gene. To clarify the reason for the smaller amount of OprL protein present in P. putida tolB::{Omega} Km, primer extension analyses were done using total RNA isolated from this strain. A cDNA product whose size was identical to that of the parental strain was found; however, the amount of product was significantly lower than that found in the wild-type strain. This suggested that the insertion of the -Km interposon in the BglII site negatively influenced transcription from the downstream oprL promoter, although the reason for this effect is at present unknown.
fig.ommitted&7, http://www.100md.com
Primer extension analysis of oprL mRNA. Cells were grown in LB medium, and samples for total-RNA isolation were taken at a turbidity of 0.5. The autoradiogram shows the cDNA products obtained after reverse transcription of 20 µg of total RNA with the 5'-labeled oligonucleotide, as described in Materials and Methods. DNA from plasmid pTOL was sequenced by using the same primer, and the sequencing products were used as size markers (lanes A, C, G, and T). Lane 1, P. putida KT2440; lane 2, P. putida KT2440(pJBTOLiKm); lane 3, P. putida B. The DNA sequence of the putative oprL promoter region is shown on the left. The proposed +1 site (boldfaced) and the putative -35 and -10 promoter regions (shaded) are indicated.&7, http://www.100md.com
Primer extension analyses were also carried out to look for the presence of additional promoters upstream of the other genes of the tol-oprL cluster. Total RNA isolated from P. putida KT2440 was hybridized to 5'-end-labeled oligonucleotides complementary to the different tol-oprL gene sequences at about 50 nucleotides from the A of the first ATG of each gene. After primer extension, we obtained a definite cDNA band for the primer based on the orf1 gene, whereas in the rest of the cases, instead of a well-defined cDNA product, numerous long extension products were always observed, probably corresponding to degraded mRNA species forming part of a larger transcript coming from a distant promoter (data not shown). The 5' end of orf1 mRNA was identified as a thymine residue located 91 bases upstream of its translational start codon. Putative -35 and -10 sites resembling those recognized by RNA polymerase with sigma-70 were found . These results suggested that the orf1, tolQ, tolR, tolA, and tolB genes formed a transcriptional unit that was read from a single promoter located upstream of orf1.
fig.ommitted#mq, 百拇医药
Primer extension analysis of orf1 mRNA. Cells were grown in LB medium, and samples were taken when turbidity at 660 nm reached 0.5 for total RNA isolation. The autoradiogram shows the cDNA products obtained after reverse transcription of 20 µg of total RNA with the 5'-end-labeled oligonucleotide, as described in Materials and Methods. DNA from plasmid pTOL was sequenced by using the same primer, and the sequencing products were used as size markers (lanes A, C, G, and T). Lane 1, P. putida KT2440(pJBTOLiKm); lane 2, P. putida KT2440. The DNA sequence of the putative orf1 promoter region is shown on the left. Boldfaced nucleotides represent the proposed +1 site and the ruvB stop codon. The putative -35 and -10 promoter regions (shaded) and an SphI restriction site are indicated.#mq, 百拇医药
RT-PCR analysis. The data obtained by Western blotting and primer extension analysis suggested that the tol-oprL region of P. putida consisted of two operons, orf1-tolQRAB and oprL-orf2. To confirm this transcriptional organization and to investigate in further detail whether the transcription of the two operons is connected to any degree (as shown for the E. coli and P. aeruginosa tol-oprL [pal] genes [16, 44]), we carried out RT-PCR analysis of the RNA isolated from P. putida KT2440. Primer pairs that hybridized with the 3' and 5' ends of each two adjacent genes of the tol-oprL cluster were designed . We also checked for the presence of putative mRNA products extending beyond the orf2 gene, or which appeared as a consequence of read-through from ruvB, the gene located immediately upstream of orf1. No cDNA amplification products were obtained for the primer pair hybridizing with the ruvB and orf1 genes or for that hybridizing with the orf2 and orf3 genes ( lanes 7 and 8). These results demonstrated that the P. putida tol-oprL mRNA was clearly delimited by the orf1 and orf2 genes. RT-PCR products of the predicted sizes were, however, generated when the remaining primer pairs were used , lanes 1 to 6). Particularly important was the positive amplification obtained with the primers covering the region between tolB and oprL . Since the 5' end of primer 4045 is located 96 bp upstream of the oprL transcriptional start site, this result strongly suggested that some proportion of the expression of oprL-orf2 could also derive from the promoter located upstream of orf1. This would imply the existence of a single mRNA comprising the entire tol-oprL region, from orf1 to orf2, plus a second mRNA, initiated in the promoter located upstream of oprL, which would include the two 3'-most genes of the cluster.
fig.ommittedw?\h^, http://www.100md.com
Organization of the tol-oprL genes in P. putida KT2440. (A) Localization of the tol-oprL genes and positions of the primers used for mRNA amplification in RT-PCR assays. Numbers below the primers indicate their positions in the tol-oprL DNA sequence. Nucleotides are numbered as in . (B) Gel electrophoresis of the cDNA amplified with oligonucleotides 450 and 747 (lane 1), 1308 and 1461 (lane 2), 1777 and 1929 (lane 3), 2837 and 3049 (lane 4), 4045 and 4391 (lane 5), 4532 and 4918 (lane 6), 25 and 308 (lane 7), and 5510 and 5857 (lane 8). Positions of molecular size markers (in base pairs) are indicated on the left. Negative controls containing the same amounts of RNA, primers, and Taq polymerase, but no reverse transcriptase, were included in this assay (data not shown).w?\h^, http://www.100md.com
Effects of growth phase and iron concentration on the expression of the tol-oprL genes of P. putida KT2440. It has been suggested that in P. aeruginosa the expression of the tol-oprL gene cluster is under growth phase and iron limitation control (16, 24, 34). We cultured P. putida KT2440 in LB medium and harvested cells in the exponential-growth phase (optical density at 660 nm, 0.6) or in the stationary phase (optical density at 660 nm, 2) and estimated the levels of TolA and OprL by Western blot analysis as described in Materials and Methods. It was found that OprL and TolA levels decreased when cells reached the stationary-growth phase (data not shown). To determine whether the expression of the oprL and orf1 promoters was growth phase regulated, transcriptional fusion analyses were done . A 271-bp DNA fragment containing the region upstream of oprL was fused to a promoterless lacZ reporter gene on the pMP220 low-copy-number plasmid to generate plasmid pMPLB. A 224-bp DNA fragment upstream of orf1 bearing the promoter region was also fused to the pMP220 plasmid to generate plasmid pMP1B . These plasmids were then transferred to P. putida KT2440, and the expression of the corresponding fusion was determined by measuring ß-galactosidase activity. In the exponential phase, the ß-galactosidase activity of the fusion in pMPLB was about 20,000 Miller units, whereas in the stationary phase, the activity decreased to 15,000 Miller units . The level of expression from the orf1 promoter, quantified by measuring the ß-galactosidase activity of the pMP1B construct in cells grown in LB medium, was about 2,700 Miller units in the exponential phase and decreased to 900 Miller units in the stationary phase . These results confirmed the results obtained by Western blot analysis.
fig.ommitted9t-c}7z, 百拇医药
Transcriptional analysis of the oprL and orf1 promoters with lacZ fusions. (A) Effects of growth phase on expression of the oprL and orf1 promoters. P. putida cells bearing either pMP200 as a negative control (open circles), the Porf1::lacZ fusion in pMP1B (filled circles), or the PoprL::lacZ fusion in pMPLB (filled triangles) were grown in LB medium. (Insets) Growth curve of each strain used in the assay. (B) Effects of iron on expression from the Porf1 and PoprL promoters in P. putida KT2440. P. putida KT2440 cells bearing the indicated plasmids were grown in M9 minimal medium supplemented with 50 µM FeCl3 (hatched bars) or 400 µg of EDDHA ml-1 (open bars). Values are means from triplicate experiments.9t-c}7z, 百拇医药
Because some degree of iron regulation had previously been reported for the P. aeruginosa oprL and tolQRA genes (16, 24, 34), we decided to study whether the iron concentration in the medium affected P. putida oprL and orf1 expression. The expression levels of oprL and orf1 were analyzed in P. putida cells harboring plasmid pMPLB or pMP1B grown in M9 minimal medium under iron-rich (50 µM FeCl3) or iron-restricted (400 µg of EDDHA ml-1) conditions. The levels of expression from the oprL or orf1 promoter were similar under iron-rich and iron-restricted conditions . This expression pattern was growth phase independent (data not shown).
Analysis of P. putida tol-oprL translational lacZ fusions. Analysis of the DNA sequence upstream of tolA revealed a potential loop structure in the mRNA region, which contained the stop codon of tolR, as well as the potential ribosome-binding site and the start codon of tolA . Another potential mRNA loop structure was predicted between oprL and orf2 . We reasoned that if these loops existed in vivo, translation of these genes was likely to be coupled. To investigate this possibility, translational fusions to the lacZ reporter gene were constructed for every gene of the tol-oprL cluster and their ß-galactosidase activities were determined . Then the plasmids harboring the different tol-oprL translational fusions were digested at unique restriction sites, and after treatment with Klenow enzyme, they were religated. The addition of nucleotides by this treatment altered the reading frame of the gene where the restriction site was located, resulting in the appearance of premature stop codons. The effect of generating frameshift mutations in the genes upstream of each tol-oprL gene fusion was tested by measuring the ß-galactosidase activity . We found that when the expression of TolR was abolished by the presence of a frameshift mutation, the expression of the TolA protein was reduced drastically, to 5% of the control value compare ß-galactosidase values for pMLA::lacZ and pMLA-R). This suggested that translation of the tolA mRNA depended on translation of the preceding tolR mRNA. The same result was obtained for the oprL and orf2 genes. A frameshift mutation in oprL exerted a polar effect on the expression of the orf2 gene, reducing ß-galactosidase activity to less than 1% of the activity of the original fusion (pMLO2::lacZ versus pMLO2-P), which strongly suggested that these genes were also translationally coupled.
fig.ommitted9-{, 百拇医药
Predicted secondary structures of the tolR-tolA mRNA (A) and the oprL-orf2 mRNA (B). The start codon is shaded, whereas the predicted ribosome binding site is boxed. Boldfaced nucleotides represent translational stop codons. The most stable secondary structures and their G° were predicted using the mfold, version 2.3, program, with the default setting at 30°C. Nucleotides are numbered as in .9-{, 百拇医药
fig.ommitted9-{, 百拇医药
ß-Galactosidase activities of the lacZ translational fusion constructsa9-{, 百拇医药
On the other hand, because the RT-PCR assays suggested that a single mRNA containing the entire region from orf1 to orf2 could be transcribed from the promoter upstream of orf1, we decided to quantify the contribution of this promoter to the expression of the oprL and orf2 genes. To abolish the activity of the orf1 promoter, an -Km interposon was cloned into the SphI restriction sites of the plasmids harboring the oprL and orf2::lacZ fusions (constructions pMLP-P1Km and pMLO2-P1Km in ), and ß-galactosidase activities of the new fusion constructs were then determined and compared with those of the original fusions. With these constructions we found that approximately 85% of the ß-galactosidase activity was still present in the oprL and orf2::lacZ fusions when expression from the orf1 promoter was abolished . These results confirmed that most of the OprL and Orf2 proteins are expressed from an mRNA starting at the promoter upstream of oprL, and only a small portion (15%) of these proteins is synthesized from an mRNA derived from the promoter upstream of orf1.
DISCUSSIONs, 百拇医药
The tol-oprL gene cluster of P. putida is organized in two operons. Although the gene organization of the tol-oprL (pal) systems is highly conserved among most gram-negative bacteria (43), their transcriptional organization has been studied in only a few species (16, 24, 32, 40, 44), and most of these studies were based on gene fusion analysis. Furthermore, most attempts to determine the transcriptional initiation points of the tol-oprL (pal) promoters in these microorganisms have failed; one of the reasons was the low level of transcription or the instability and rapid degradation of these mRNA species (16, 32, 44). Alternative approaches, such as analyzing the mRNA overexpressed from the tol-oprL genes cloned into plasmids, have usually resulted in misleading interpretations (16, 24). For this reason we decided to use total mRNA isolated directly from wild-type cells as a source for our analysis. We have shown that the P. putida tol-oprL genes are arranged into two operons, orf1-tolQ-tolR-tolA-tolB and oprL-orf2, and we have also identified the transcription start sites for the promoters of both operons. Within each operon the genes are tightly clustered, and the distance that separates the coding sequences of the two operons is 54 bp. This arrangement is almost identical to that found in P. aeruginosa, but surprisingly, the transcriptional organization differs in the two bacteria.
We have also analyzed the expression of the P. putida TolA protein by Western blotting in different P. putida tol-oprL mutants . Once the transcriptional and translational organization of the whole gene cluster was elucidated, it became clear that the lack of TolA protein in the tolQ::xylE mutant was probably due to the absence of the TolQ protein. It therefore follows that the P. putida TolQ protein is required for the stability of the TolA protein. TolQ may also be necessary for TolR stability, and TolR, rather than TolQ (or probably both), may be required for TolA stability in P. putida KT2440. In E. coli, TolA seems to be a key protein for the trans-envelope Tol-Pal system, since it is involved in a wide variety of protein interactions within this complex (26). For example, it interacts with TolQ and TolR (15, 23), with Pal (8, 9, 20), and also with TolB (29). Germon et al. (20) have observed that in E. coli, the lack of TolQ or TolR does not affect TolA stability; however, this protein is highly unstable in a tolB pal background.
In E. coli and P. aeruginosa a long mRNA transcript containing the entire tol-oprL (pal) region has been proposed to exist (16, 32, 44). In P. putida, the lack of obvious terminators within the gene cluster makes it possible that transcription from the orf1 promoter may read through oprL and orf2. The results of the RT-PCR and the ß-galactosidase assays support this possibility. As Vianney et al. (44) pointed out for E. coli, the ability to express oprL-orf2 from its own promoter allows orf1-tolQ-tolR-tolA-tolB to be separately modulated. In any case, the long orf1-orf2 mRNA transcript in these bacteria must be highly unstable, as suggested by the fact that only mRNA species smaller than those predicted were detected by Northern expression analysis (24, 32).j!r8|\, http://www.100md.com
The orf1 and oprL promoters exhibit features of promoters transcribed by RNA polymerase with sigma-70. The transcriptional start sites of P. putida orf1 and oprL promoters were determined by primer extension. Both promoters show features typical of promoters recognized by sigma-70 . The P. putida oprL promoter is located within the tolB gene; in this connection it is worth recalling that in H. influenzae, transcriptional analysis of the P6 (pal) gene revealed that its transcription initiation point was also within the region immediately upstream of the tolB gene (40). The -35 box of the oprL promoter exhibits low similarity to the -35 consensus box of promoters recognized by sigma-70. However, its -10 box shows a better match with the consensus than it does with the -10 box of the orf1 promoter , since the presence of the 5'-TG-3' sequence located 1 base upstream of the -10 hexamer could result in an extended -10 element . This may also explain the lower activity of the orf1 promoter in comparison to the oprL promoter and could be the reason for the differences in the expression levels of lacZ transcriptional fusions. The above results for P. putida are in agreement with those for P. aeruginosa and E. coli. In P. aeruginosa, the level of expression of an oprL::lacZ transcriptional fusion was higher than that observed for an orf1::lacZ fusion (16). This was also the case for E. coli, where the amount of orf1 mRNA detected by Northern blot analysis was lower than that of tolB mRNA (32). Cascales et al. (8) recently estimated that in E. coli, the Pal lipoprotein is present in larger amounts (30,000 to 40,000 copies per cell) than the TolA (400 to 800 copies) or TolR (2,000 to 3,000 copies) protein. These proportions correlate well with the above data.
Comparison of the ruvB-orf1 intergenic regions of the different Pseudomonas species sequenced so far showed a low level of sequence conservation in the positions where the P. putida orf1 promoter was found. In addition, in P. aeruginosa this region (52 bp) is considerably shorter than it is in the other three species (125 bp in P. putida, 134 bp in Pseudomonas syringae, and 138 bp in Pseudomonas fluorescens). However, comparison of the oprL promoter region revealed complete conservation of the positions located in the proposed -35 and -10 boxes . Nevertheless, it should also be pointed out that since this region is located within the coding sequence of the tolB gene, a stronger selective pressure will operate in maintaining the sequence conservation at these positions (although the conservation of the TolB proteins at the amino acid level in this region is not particularly high among the different gram-negative bacteria).84)0uu1, http://www.100md.com
fig.ommitted84)0uu1, http://www.100md.com
Alignment of the regions containing the P. putida tol-oprL promoters with the corresponding regions from P. fluorescens, P. aeruginosa, and P. syringae. Nucleotides that are conserved at least in three of the four positions are included in the consensus sequence. The predicted ruvB stop codons are boldfaced. The start positions of the P. putida mRNA transcripts (indicated by +1) are underlined. The proposed -10 and -35 promoter regions are boxed. (A) Alignment of the orf1 promoter region. The consensus motif for Fur binding is shown above the empirically determined Fur box for P. aeruginosa (34). Nucleotides that are identical in the two sequences are indicated by asterisks. (B) Alignment of the oprL promoter region.
Expression of the P. aeruginosa orf1-tolQ-tolR-tolA gene cluster is negatively regulated by the presence of iron in the culture medium (16). In this process the ferric uptake regulator (Fur) has been shown to be involved by directly binding to the orf1 promoter region, where a fur box motif is present (34). In P. putida KT2440 we have observed no influence of iron concentration on the expression of the tol-oprL genes, and we could find no recognizable fur box motif in the corresponding promoter regions . In the case of P. syringae and P. fluorescens, DNA sequences (matching 9 out of 19 bp in the Fur-binding consensus sequence) have been found in exactly the same location as in P. aeruginosa . Although these are only putative Fur boxes, it is noteworthy that DNA sequences exhibiting homology of only 10 bp to the consensus have been identified as being directly bound by P. aeruginosa Fur (34).l{p)f)[, 百拇医药
Translational coupling between oprL and orf2 and between tolR and tolA. We have also shown evidence of the existence of translational coupling between P. putida tolR and tolA, as well as between oprL and orf2 . A similar situation has also been reported for the E. coli tolQ and tolR genes (44). Translational regulation is potentially important for the balanced synthesis of these proteins, which is probably required to maintain the stoichiometry of this protein complex. Although we have not analyzed the molecular mechanism of these translational couplings in detail, the presence of recognizable ribosome-binding sites upstream of both tolA and orf2 suggests that translation is inhibited by making the ribosome-binding sites of these genes inaccessible by camouflaging them within stable mRNA secondary structures, as predicted, and thus making their translation dependent on the prior translation of the gene immediately upstream.
In summary, the Tol-OprL (Pal) system plays a key role in maintaining cell morphology and outer membrane integrity in gram-negative bacteria. The proteins are well conserved in this group of prokaryotes; however, the transcriptional organization of the gene clusters, and the levels of expression of the genes in response to different environmental conditions, differ among these microorganisms. These differences may be related to the microbe's preferred habitat and to the way in which expression of these gene clusters is integrated in global regulatory networks.k0dkgt|, http://www.100md.com
Finally, it is also interesting that the orf1 (ybgC) gene associated with the tol-pal gene cluster in H. influenzae has recently been shown to encode a protein that displays a significant level of thioesterase activity toward short-chain acyl coenzyme A thioesters. However, it is not yet clear how the thioesterase activity of this protein may be related to the function of the Tol-Pal system (45).k0dkgt|, http://www.100md.com
ACKNOWLEDGMENTS
We thank Silvia Marqués for helpful advice and discussions. We thank Ana Hurtado for assistance in DNA sequencing, Carmen Lorente and M. Mar Fandila for secretarial assistance, and K. Shashok for help with the language in the manuscript.r., 百拇医药
M. A. Llamas was the recipient of a fellowship from the Spanish Ministry of Education and Culture. This work was supported by grants from the Ministerio de Ciencia y Tecnología (FEDER 1FD97-1437) and the European Commission (QLK3-2000-00170).r., 百拇医药
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