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encodes resistance to ampicillin and tetracycline (2). Bacteria were grown in L broth (10 g of tryptone and 5 g of yeast extract per liter) supplemented with 1% ...

JOURNAL OF BACTERIOLOGY, Mar. 1996, p. 1699–1706 0021-9193/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 178, No. 6

The Pseudomonas putida Peptidoglycan-Associated Outer Membrane Lipoprotein Is Involved in Maintenance of the Integrity of the Cell Envelope ´ J. RODRI´GUEZ-HERVA, MARI´A-ISABEL RAMOS-GONZA ´ LEZ,† JOSE



Consejo Superior de Investigaciones Cientı´ficas, Department of Biochemistry and Molecular and Cellular Biology of Plants, Estacio ´n Experimental Zaidı´n, 18008 Granada, Spain Received 21 September 1995/Accepted 25 December 1995

Pseudomonas putida 14G-3, a derivative of the natural soil inhabitant P. putida KT2440, exhibited a chromosomal insertion of a mini-Tn5/*phoA transposon that resulted in reduced ability to colonize soil. In vitro characterization of P. putida 14G-3 revealed that it exhibited an altered cell morphology and envelope, as revealed by electron microscopy. The derived strain was sensitive to sodium dodecyl sulfate, deoxycholate, and EDTA, produced clumps when it reached high cell densities in the late logarithmic growth phase, and did not grow on low-osmolarity medium. The P. putida DNA surrounding the mini-Tn5/*phoA insertion was cloned and used as a probe to rescue the wild-type gene, which was sequenced. Comparison of the deduced peptide sequence with sequences in the Swiss-Prot database allowed the knocked-out gene to be identified as that encoding the peptidoglycan-associated lipoprotein (Pal or OprL) of P. putida. The protein was identified in coupled transcription and translation assays in vitro. natural soils (42). We report here the physiological characteristics of this isolate in vitro. The mutant strain was shown to exhibit an altered cell envelope; to be sensitive to saline shock, detergents, and EDTA; and to produce clumps at the end of the logarithmic growth phase. We cloned the mutated region, rescued the wild-type gene, and sequenced it. Alignments of the deduced polypeptide sequence with sequences of proteins in the Swiss-Prot database allowed the gene to be identified as that encoding the P. putida Pal (peptidoglycan-associated lipoprotein) protein, also called OprL.

Bacteria of the genus Pseudomonas constitute a broad group of ubiquitous microorganisms, including strains that are pathogenic for humans, animals, and plants (21, 31); phyllobacteria and rhizobacteria, which play a beneficial role by preventing plant diseases caused by fungi in the root system or leaves (51); and soil inhabitants, which play a key role in geochemical cycles (17). Many strains have been shown to be extremely tolerant to organic solvents (22, 38) and are considered useful biocatalysts for biotechnological purposes such as biotransformations in double-phase fermentors and bioremediation (37, 52). Interactions between Pseudomonas bacteria and their environments take place through cell surface structures. To understand how this group of microbes is able to colonize a wide variety of ecological niches, it is important to comprehend the organization and function of their cell envelope components. The cell surfaces of pathogenic Pseudomonas strains, such as strains of Pseudomonas aeruginosa, have been studied in detail (47). Most of the characterized proteins are porin-like proteins, some of which (e.g., OprF, the major general porin) also play a role in cell structure and growth at low osmolarity (19) and in tolerance to toluene (29). OprH is involved in resistance to polymyxin B, gentamicin, and EDTA. OprP is a phosphatespecific channel, and OprD is specific for the uptake of imipenem and basic amino acids (19). OprM and OprJ are involved in the expression of multidrug resistance (18). However, little is known of the surface of the natural soil inhabitant Pseudomonas putida. P. putida KT2440 (15), a restriction-negative strain which was derived from the natural soil inhabitant P. putida mt-2, has a wide catabolic potential and exhibits a striking ability to survive and establish in soils (39), including the plant rhizosphere (36). P. putida 14G-3 is a Tn5/9phoA mutant of the strain P. putida KT2440, which has exhibited an impaired ability to colonize

MATERIALS AND METHODS Bacterial strains, plasmids, and growth conditions. The strain P. putida EEZ40 is a streptomycin-resistant spontaneous mutant of P. putida KT2440 (40), which lacks alkaline phosphatase activity. P. putida 14G-3 is a PhoA1 derivative of P. putida EEZ40 which was obtained after mini-Tn5/9phoA mutagenesis (40). This clone bears an in-frame fusion of the oprL gene, which encodes an outer membrane lipoprotein, to the promoterless 9phoA gene. It was selected as a blue colony on medium supplemented with the chromogenic PhoA substrate 5-bromo-4-chloro-3-indolylphosphate (40). The Escherichia coli strains used were CC118lpir [D(ara-leu) araD139 DlacX74 galK galE phoA20 thi-1 rpsE rpoB argE recA1 lpir] (8), HB101 (F2 hsdS20 proA2 leu supE44 ara-14 galK2 lacY1 rpsL20 xyl-5 mtl-1 recA13) (4), and TG1 [supE hsdD5 thi D(lac-proAB) F9 (traD36 proAB1 lacIq lacZDM15)]. P. putida was grown at 308C, and E. coli strains were grown at 30 or 378C. Plasmid pLAFR3 is a tetracycline-resistant derivative of the cosmid pLAFR1 (16), modified to include a polylinker with multiple cloning sites, the Plac promoter, and the a peptide of 9lacZ. Plasmid pBR322 is a cloning vector that encodes resistance to ampicillin and tetracycline (2). Bacteria were grown in L broth (10 g of tryptone and 5 g of yeast extract per liter) supplemented with 1% (wt/vol) or 0.1% (wt/vol) NaCl and the appropriate antibiotics. The final concentrations of antibiotics (in micrograms per milliliter) were as follows: ampicillin, 100; chloramphenicol, 30; kanamycin, 25 to 50; piperacillin, 30 to 100; rifampin, 10; streptomycin, 50 to 100; and tetracycline, 15. When antibiotics were supplied on disk, their amounts per disk (in micrograms) were as follows: amikacin, 30; ampicillin, 10; chloramphenicol, 30; erythromycin, 15; nalidixic acid, 30; piperacillin, 100; rifampin, 30; streptomycin, 10; tetracycline, 30; and vancomycin, 30. Recombinant DNA techniques. Plasmid DNA was isolated by the alkaline lysis method (23). Digestion of DNA with restriction enzymes and ligations were done in KGB buffer (30). Agarose gel electrophoresis was done by standard methods (45). Restriction fragments for subcloning were recovered from agarose gels with the GeneClean kit (Bio 101, Inc., Vista, Calif.). Competent cells of E. coli were prepared according to Nishimura et al. (33). P. putida total DNA libraries were prepared in cosmid pLAFR3 (16). P. putida total DNA was isolated as previously

* Corresponding author. Mailing address: CSIC, Estacio ´n Experimental del Zaidı´n, Apdo Correos 419, 18008 Granada, Spain. † Present address: Department of Microbiology, Technical University of Denmark, Lyngby, Denmark. 1699



FIG. 1. Growth of P. putida EEZ40 and 14G-3 in L broth with different concentrations of NaCl. P. putida EEZ40 (circles) or P. putida 14G-3 (triangles) was grown overnight in L broth supplemented with 1% (wt/vol) NaCl. Cells were then harvested and resuspended in L broth supplemented with 1% (wt/vol) NaCl (open symbols) or with 0.1% (wt/vol) NaCl (closed symbols) to reach an initial cell density of about 0.03 at 660 nm. At the indicated times, the turbidities of the cultures were determined. OD660, optical density at 660 nm.

described (43) and then partially digested with PstI to render 20- to 25-kb DNA fragments. About 0.5 mg of 25-kb chromosomal fragments was ligated to 0.15 mg of pLAFR3 linearized with PstI, packaged in lambda heads, and transfected in E. coli HB101 host cells (24). DNA was sequenced on both strands by the dideoxy sequencing termination method (46), with 35S-labelled nucleotide, T7 phage DNA polymerase, and universal or specific 20-mer oligonucleotides to prime synthesis. Screening of a P. putida library with a probe against the oprL gene. About 10,000 colonies of E. coli HB101 bearing the pLAFR3-derived gene library of P. putida was screened against a probe containing a 1.1-kb NotI-SphI fragment of pPRO63 with part of the oprL gene. Bacteria were grown on nylon membranes and lysed as described by Sambrook et al. (45). Then DNA was denatured and fixed to the membrane (45). Nonradioactive labelling of the probe and detection of hybrids were done with the Boehringer Mannheim kit (reference no. 1175041). The membranes were hybridized and washed under strict conditions (45). Detection of plasmid-encoded polypeptides in vitro. Plasmid-encoded proteins were detected with a prokaryotic DNA-directed transcription and translation kit (Amersham International plc, Little Chalfont, Buckinghamshire, United Kingdom) and L-[35S]methionine. The reactions were allowed to proceed in the presence of 1 U of the RNase inhibitor RNasin (Promega Corp., Madison, Wis.). 35 S-labelled polypeptides were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis as described by Laemmli (25). We used running gels of 17.5% (wt/vol) polyacrylamide and stacking gels of 5% (wt/vol) polyacrylamide. Electron microscopy. P. putida was grown in L broth supplemented with 1% (wt/vol) or 0.1% (wt/vol) NaCl, and cells were harvested by centrifugation, immediately fixed with 2% (vol/vol) glutaraldehyde plus 1% (vol/vol) formaldehyde in cacodylate buffer, postfixed with osmium tetroxide in the presence of 2% (wt/vol) potassium ferrocyanide, and embedded in Eponate 12. Thin sections were poststained with uranyl acetate and lead citrate and examined in a Zeiss transmission electron microscope at an accelerating voltage of 75 kV. Computer analysis. Proteins were aligned with the Pileup program provided with the Genetics Computer Group package (11). Analysis of DNA primary sequences and prediction of secondary structures of DNA and proteins were done with various programs included in the PC/GENE package (Intelligenetics). Nucleotide sequence accession number. The nucleotide sequence of the P. putida gene encoding OprL was submitted to the EMBL data bank under accession number X74218.

RESULTS Physiological characterization of P. putida clone 14G-3. P. putida EEZ40 grew in L broth with high (1% [wt/vol]) or low (0.1% [wt/vol]) concentrations of NaCl, with a logarithmic generation time of about 35 min (Fig. 1). P. putida 14G-3 grew


with a generation time similar to that of the parental strain in L broth with 1% (wt/vol) NaCl, but it did not grow in L broth with 0.1% (wt/vol) NaCl (Fig. 1). Both strains grew exponentially until they reached around 108 CFU/ml at the end of the logarithmic growth phase in L broth with 1% (wt/vol) NaCl. Afterwards, the mutant strain tended to produce clumps and the turbidity of the culture decreased (Fig. 1). In contrast, parental strain cells remained unassociated and suspended in the culture medium, and turbidity increased linearly with time, as expected from the limitation of one or more nutrients in the culture medium. Some E. coli and P. aeruginosa mutants with altered membrane protein compositions exhibit altered patterns of sensitivity against antibiotics, detergents, chelating agents, and other substances, in comparison with the wild type (18, 19, 26, 47). We assayed the sensitivities of P. putida 14G-3 to antibiotics to which the parental strain was resistant (ampicillin, chloramphenicol, erythromycin, streptomycin, and vancomycin) and antibiotics to which the wild-type strain was sensitive (amikacin, nalidixic acid, piperacillin, rifampin, and tetracycline). The pattern of resistance or sensitivity of the mutant strain was similar to that of the parental strain when the halo inhibition zones around antibiotics supplied on disks were determined. The responses of both strains to the detergents SDS, Triton X-100, and deoxycholate were assayed in L broth with 1% (wt/vol) NaCl as described by Sukupolvi et al. (48). The first detergent inhibited growth of the parental strain at a concentration of 3% (wt/vol) and inhibited growth of the mutant strain 14G-3 at a concentration of 1.8% (wt/vol). Deoxycholate inhibited growth of the parental strain at a concentration of 225 mM and at 60 mM inhibited growth of the mutant. Triton X-100 at concentrations up to 3% (wt/vol) did not inhibit the growth of either the wild-type or the mutant strain. Growth of the parental strain was inhibited when the broth was supplemented with 1.9 mM EDTA, whereas 1.4 mM EDTA inhibited growth of the mutant strain. Therefore, three phenotypic characteristics were associated with the mini-Tn5/9phoA insertion in mutant strain 14G-3: impaired growth in low-osmolarity medium; increased sensitivity to SDS, deoxycholate, and EDTA; and production of clumps once the culture reached high cell density. Ultrastructures of wild-type and mutant cells under different growth conditions. The ultrastructures of wild-type and 14G-3 strains were analyzed with cells grown in L broth with 1% (wt/vol) NaCl. Cells were harvested in either the midlogarithmic or the stationary phase, and subjected to electron microscopy as described in Materials and Methods. Regardless of the growth phase, thin sections of parental cells showed them as rod-shaped bacteria whose ultrastructural features were typical of members of the family Pseudomonadaceae (Fig. 2A and B): the outer and inner membranes were close together, and ribosomes were relatively abundant and well-defined. In contrast to parental bacteria, cells of mutant strain 14G-3 appeared relatively deformed (Fig. 2C), a feature which was more obvious in cells in the stationary phase (Fig. 2D). A number of cells of mutant 14G-3 in the stationary phase appeared as ghost bacteria (Fig. 2D). Although cells in the logarithmic phase had a well-defined inner membrane, the outer membrane showed discontinuities (Fig. 2C), which were especially evident in the stationary phase (Fig. 2D and E). This suggests that the mutant strain exhibits a more fragile outer membrane. Wild-type and mutant cells grown in L broth with 1% (wt/ vol) NaCl were diluted 10-fold in L broth, so that the concentration of NaCl decreased to 0.1% (wt/vol). After 30 min, cells were subjected to electron microscopy as described above. Whereas the morphological appearance of wild-type cells was

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FIG. 2. Ultrastructure of P. putida EEZ40 and 14G-3 under different growth conditions and in different growth states. The parental strain was grown in L broth with 1% NaCl. (A) Exponential phase. Magnification, 340,000. (B) Stationary phase. Magnification, 340,000. Mutant strain 14G-3 was also grown in L broth with 1% NaCl. (C) Exponential phase. Magnification, 320,000. (D) Stationary phase. Magnification, 316,000. (E) Detail of a cell of strain 14G-3 in the stationary phase. Magnification, 3100,000. (F) Mutant cells after hypotonic saline shock. Magnification, 312,000.

similar to that of cells grown on 1% (wt/vol) NaCl (not shown), the mutant cells appeared lysed (Fig. 2F). Cloning of the region containing the kanamycin marker in the mutant 14G-3 and recruitment of the wild-type allele. To clone the mutation in P. putida 14G-3, a gene bank of this

mutant in plasmid pLAFR3 was generated, and after packaging and transfection of E. coli HB101 under the conditions described in Materials and Methods, we searched for tetracycline- and kanamycin-resistant (Tcr Kmr) clones. A plasmid designated pPRO6 (Fig. 3A) had the kanamycin marker, the



FIG. 3. Restriction maps relevant to the cloning of the gene interrupted by mini-Tn5/9phoA in P. putida 14G-3 and the rescue of the wild-type gene. (A) Plasmid pPRO6 is a pLAFR3 derivative bearing mini-Tn5/9phoA and adjacent DNA from the P. putida 14G-3 chromosome. Plasmid pPRO63 is a pBR322 derivative bearing the 2.8-kb SphI fragment of pPRO6 in the SphI site of the cloning vector. (B) pPRO200 carries the wild-type P. putida DNA corresponding to the region interrupted by mini-Tn5/9phoA in strain 14G-3. The open triangle in pPRO200 denotes the insertion site of the mini-Tn5 in the P. putida 14G-3 DNA. Restriction sites are as follows: B, BstEII; C, ClaI; E, EcoRI; H, HindIII; K, KpnI; Nc, NcoI; Nt, NotI; and S, SphI. The arrows indicate the direction of transcription.

9phoA gene fused to 59-adjacent P. putida DNA, and additional P. putida DNA 39 of the kanamycin gene. Within the mini-Tn5/ 9phoA transposon used for mutagenesis, a single SphI site was present between the 9phoA gene and the gene encoding kanamycin resistance (Fig. 3A). Digestion of pPRO6 with SphI therefore released the whole kanamycin gene plus 39-adjacent P. putida DNA. The kanamycin gene was found on a 2.8-kb SphI fragment, of which about 1.7 kb corresponded to the kanamycin gene and about 1.1 kb corresponded to P. putida DNA. The 2.8-kb SphI fragment of pPRO6 was subcloned into the single SphI site of pBR322, and Kmr ampicillin-resistant (Apr) clones were sought. A random clone (pPRO63) was selected (Fig. 3A). The 1.1-kb NotI-SphI fragment of pPRO63 bearing the DNA adjacent to the kanamycin gene was used as a probe to screen a parental P. putida library. Colony-screening hybridization showed that 1 of 10,000 clones screened was found to hybridize to the labelled probe. The plasmid borne by this clone was called pPRO50. Cosmid DNA was then prepared, digested with SphI, and hybridized to the 1.1 NotI-SphI fragment of pPRO63 in Southern blots. A positive signal was found with a 2.3-kb SphI fragment. Complementation of the mutation in P. putida 14G-3 by a cosmid bearing the rescued wild-type gene. The cosmid pPRO50 (Tcr) and the control parental plasmid pLAFR3 (Tcr) without P. putida DNA were transferred via triparental matings to P. putida 14G-3. Kmr Tcr transconjugants of this strain were selected in L broth solid medium with 1% (wt/vol) NaCl and the two above-mentioned antibiotics. Growth of P. putida 14G-3(pLAFR3) and P. putida 14G-3(pPRO50) was assayed in L broth with high-concentration (1% wt/vol) and low-concentration (0.1% wt/vol) NaCl. The strain bearing the pPRO50


plasmid was able to grow in both media and did not produce clumps, whereas the strain bearing the control plasmid pLAFR3, like the plasmidless strain, was unable to thrive in low-concentration-NaCl medium, and in high-concentrationNaCl medium it formed clumps in the stationary phase. In contrast with this observation, the merodiploid strain was as sensitive as the wild type to detergents, EDTA, and antibiotics. Therefore, pPRO50 was able to compensate for the induced mutation. Sequence analysis. The 2.3-kb SphI fragment of pPRO50 producing a positive hybridization signal with the DNA adjacent to the 9phoA gene was further subcloned into pUC18 to yield pPRO200, whose restriction map was determined (Fig. 3B). Comparison of the restriction pattern of pPRO63 and pPRO200 allowed us to identify the SphI site 39 of the kanamycin gene in pPRO63 as the SphI site proximal to the HindIII site in pPRO200. About 1.6 kb beginning with the SphI site proximal to the HindIII in pPRO200 was sequenced on both strands (Fig. 4). The position of the transposon was found by sequencing the insert of pPRO63 as far as the Tn5 end. The transposon was located between positions 454 and 455 in the sequence given in Fig. 4. The DNA sequence was found to contain 59% G1C, close to the percentage of G1C in P. putida DNA (60%). The sequence was analyzed by the algo-

FIG. 4. DNA sequence around the insertion point of mini-Tn5/9phoA in P. putida 14G-3 and deduced protein sequences corresponding to ORF1 and ORF2. Nucleotide 1608 is part of the SphI site proximal to the HindIII site in pPRO200. The vertical arrow shows the position of the active Pal-PhoA fusion generated by mini-Tn5/9phoA mutagenesis. /, signal peptide cleavage site. ppp, stop codon.

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FIG. 5. In vitro transcription and translation of pUC18, pPRO200, and pPRO200SN. About 1 mg of DNA from the plasmids mentioned above was used in transcription and translation assays with L-[35S]methionine as recommended by the manufacturer of the S-30 cell extracts. Lane M, molecular weight markers (in kilodaltons); lane 1, pUC18; lane 2, pPRO200; lane 3, pPRO200SN. The arrow indicates the 18-kDa protein.

rithm of Fickett (14) to detect open reading frames (ORFs) encoding polypeptides. Two ORFs were found and called ORF1 and ORF2. Their translated sequences are also shown in Fig. 4. Downstream of ORF2 and between nucleotides 1463 and 1485, a hairpin structure (DGo5 219.2 kcal [280.3 kJ]) typical of the Rho-independent transcription termination was found. ORF1 was located between nucleotides 124 and 621. This ORF probably encoded a 166-amino-acid polypeptide with a predicted molecular weight of 17,883. The predicted peptide was relatively rich in charged amino acids, with Asp, Glu, Arg, Lys, and His representing 27% of the total. The predicted 166-amino-acid polypeptide contained a 21-amino-acid signal peptide, which contained two positively charged residues (Lys), a hydrophobic central region, and a motif similar to that recognized by lipoprotein signal peptidase II (50). The proposed cut point lies between residues 21 and 22, and the sequence around it was A V G-21 C-22 S S. We therefore predicted that the mature protein in vivo is made of 145 amino acids and has a molecular weight of 15,681. In mutant P. putida 14G-3, a fusion of the first 110 amino acids to the 9PhoA protein occurred as a consequence of the Tn5/9phoA insertion. ORF2 was located between positions 631 and 1434, and probably encoded a 268-residue polypeptide with a theoretical



molecular weight of 28,731. According to the 23,21 rule of von Heijne (50), this peptide should exhibit a signal peptide between residues 21 and 22. The charged amino acid content was 21%, and the Ala content was 16%. To confirm that the predicted peptides were indeed synthesized, we performed in vitro coupled transcription-translation assays with pPRO200 and the corresponding cloning vector (pUC18). Figure 5 shows a band of about 18 kDa in the lane corresponding to pPRO200; this band was absent from the products made from pUC18. No extra band of about 29 kDa was found. To confirm that the 18-kDa protein band was indeed the product of ORF1, plasmid pPRO200 was cut at the single NcoI site within the ORF1 DNA, filled in, and ligated to generate pPRO200SN. This was expected to result in the introduction of a premature stop codon such that the ORF would encode a 28-amino-acid peptide. Figure 5 shows that pPR200SN did not produce the 18-kDa polypeptide. Comparison of the ORF1 and ORF2 translated sequences. The amino acid sequence corresponding to ORF1 was compared with all the entries in the Swiss-Prot database (August 1995) by the algorithm of Pearson and Lipman (34) as described in the FastA program provided with the Genetics Computer Group computer package. The bank sequences that showed the greatest homology were four precursors for the peptidoglycan-associated lipoprotein Pal in four gram-negative bacteria: Haemophilus influenzae (7), Legionella pneumophila (13), E. coli (5, 27), and Brucella abortus (49). Porin-like proteins such as OmpA from different microbial sources and OprF from several Pseudomonas strains also gave high-score homologies (9, 53). For further comparison, these proteins were compared with each other by the algorithm of Dayhoff (6). This algorithm enables the calculation of an alignment score (A), which is the number of standard deviations by which the maximum scores of the real sequences exceed the mean of the maximum scores for permutations. An A value higher than 3 is significant for homology. According to the results obtained, two groups were distinguished (Fig. 6). Group I includes the four Pal proteins and the peptide deduced from P. putida ORF1. A comparison of the deduced amino acid sequences corresponding to these proteins with the sequence of ORF1 gave an A score higher than 10. Group II includes OmpA and OprF proteins; the A score for the ORF1 deduced amino acid sequence was between 3.0 and 6.7. This was reflected in the individual alignment of the deduced protein sequence of ORF1 with those of the Pal proteins and the OmpA/OprF proteins. In fact, with the Pal proteins, which were about 153 to 176 amino acids long, the

FIG. 6. Relatedness among the members of the Pal/OmpA family of proteins. Proteins were compared with each other by the algorithm of Dayhoff as described in the PCompare program. The maximum score (S) obtained by the alignment of two sequences is compared with the distribution of maximum scores obtained with 100 randomized sequences of the same length and composition. The mean (Sr) and standard deviation (SDr) of these randomized comparisons are calculated. The given alignment score (A) is the number of standard deviations by which the maximum scores of the real sequences exceed the mean of the maximum scores for permutations according to the equation A 5 (S 2 Sr)/SDr. A value higher than 3.0 is considered significant for homology. Abbreviations: B.a., B. abortus; E.c., E. coli; H.i., H. influenzae; L.p., L. pneumophila; P.a., P. aeruginosa; and P.p., P. putida. The sources of the proteins were E. coli OmpA (1), P. aeruginosa OprF (12), B. abortus Pal (49), E. coli Pal (5), L. pneumophila Pal (13), and H. influenzae Pal (7).



FIG. 7. Multiple alignments of the P. putida ORF1 sequence to homologous sequences from the Swiss-Prot database (May 1995 release). Alignment with Pal proteins of members of the Enterobacteriaceae is shown. The sequences of the Pal proteins of E. coli (E.c.), L. pneumophila (L.p.), H. influenzae (H.i.), and B. abortus (B.a.) were from Chen and Henning (5), Engleberg et al. (13), Deich et al. (7), and Tibor et al. (49), respectively. The P. putida (P.p.) ORF1 sequence was determined in this study. An amino acid was chosen for the consensus if it was present in at least three of the proteins.

ORF1 sequence exhibited homology all along the sequence. In contrast, with the 250- to 350-amino-acid-long OmpA and OprF proteins, homology was mainly observed in the C-terminal region, which included the peptidoglycan-binding domain(s) (references 9 and 32 and data not shown). Next, a multiple alignment of ORF1 with the Pal proteins was performed with the Pileup program, and the results obtained are shown in Fig. 7. The overall identity of the deduced P. putida peptide sequence to the Pal proteins mentioned above was about 40%. Overall similarity among the four proteins was about 80%; similarity was as low as 60% in the amino end half and as great as 90% in the carboxy end half. The putative gene product of ORF2 showed homology to a peptide encoded downstream of the pal gene in E. coli and L. pneumophila (EMBL accession numbers X65796 and X60543, respectively), but no further homology was found. DISCUSSION After mini-Tn5/9phoA mutagenesis in P. putida, we isolated a mutant called P. putida 14G-3 that exhibited a reduced ability to colonize soils (42). In this study, the mutant was characterized in detail in vitro and the DNA surrounding the insertion site was cloned and used to rescue the wild-type gene. This DNA was sequenced and translated into a protein sequence; the deduced polypeptide sequence was compared against sequences in the Swiss-Prot database. Our comparisons showed that the ORF disrupted by the mini-Tn5/9phoA insertion encoded a protein of about 18 kDa, which showed the highest homology to the Pal proteins of enterobacteria, including the proteins from E. coli, L. pneumophila, H. influenzae, and B. abortus (5, 7, 13, 49). On the basis of the sequence alignment data, we suggest that this ORF encodes the Pal protein in P. putida (Fig. 6 and 7). This polypeptide most likely corresponds


to the proposed OprL protein of fluorescent members of the family Pseudomonadaceae, according to the proposal of Hancock et al. (20) for outer membrane Pseudomonas proteins. These five proteins exhibit a signal peptide and the consensus cleavage site of those proteins recognized by lipoprotein peptidase (35). In enterobacteria, cysteine 1 in the mature Pal protein is the site to which a glycerol molecule is bound and to which lipids are linked. It has been suggested that this domain interacts with the outer membrane, whereas the C-terminal region interacts with the peptidoglycan layer. The main physiological role of the Pal protein in members of the family Enterobacteriaceae is probably to help maintain the integrity of the cell envelope structure by bridging the outer membrane and the peptidoglycan network. In connection with this role, Lazzaroni and Portalier (27) suggested that the positively charged residues located between positions 101 and 116 of the mature E. coli Pal protein were involved in the interaction with peptidoglycan. This domain is homologous to a region of OmpA, which likely interacts with peptidoglycan (9). Recently, Bouveret et al. (3) have suggested that in E. coli the Pal protein also interacts with the periplasmic protein TolB. In E. coli, the TolA, TolQ, and TolR proteins have also been implicated in the maintenance of the cell envelope. Derouiche et al. (10) have suggested that the N-terminal domain of TolA interacts with TolQ and TolR proteins within the inner membrane. TolQ is an integral membrane protein, whereas TolA and TolR are anchored to the inner membrane by a single transmembrane domain, and their C-terminal domain facing the periplasm seems to be involved in interactions with the peptidoglycan layer (10, 28). Therefore, the interplay of proteins linked to the outer or inner membrane, with projections of one end of the protein to the periplasm, allows interactions with the peptidoglycan layer and non-membrane-bound periplasmic proteins, and as a consequence all of these elements participate in the maintenance of the cell envelope. In this study we have shown that the cell morphology of the P. putida oprL (pal) mutant is altered with respect to the morphology of the wildtype strain. The insertion of 9phoA into the P. putida oprL (pal) gene probably generates a hybrid protein containing the first 89 amino acids of the mature OprL (Pal) protein and then the 9PhoA protein. According to Lazzaroni and Portalier (27) this hybrid protein lacks the proposed peptidoglycan interaction domain. It is therefore likely that the lack of appropriate bridges between the Pal protein and the peptidoglycan layer (and/or TolB if this protein is also located in the periplasm of P. putida) is responsible for the alterations in the cell morphology and cell envelope (Fig. 2C and D). This may explain why mutant 14G-3 cells exhibited a fragile outer membrane, especially in the stationary phase. External damage to the cell envelope would account for sensitivity to EDTA, SDS, and deoxycholate (as previously described for pal mutants among Enterobacteriaceae). A difference between phenotypes of P. putida and those of Pal mutants in the family Enterobacteriaceae is that the Pseudomonas mutants formed clumps at high cell densities. Cell adhesion may result from debris formed from lysed cells (e.g., DNA, polysaccharides, etc.). Another difference is that the Pseudomonas oprL (pal) mutant described here showed impaired ability to grow in a low-NaClcontent medium (Fig. 1) and was hypersensitive to hypotonic saline shock (Fig. 2F). These characteristics have been associated with the lack of the OprF protein in P. aeruginosa (53). In this context it is worth noting that the pal mutants of E. coli released periplasmic proteins into the extracellular medium (26). Although we did not study this property in detail, the highly antigenic P40 protein (which probably corresponds to OprF, although this has not been proved) in the oprL (pal)


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mutant of P. putida (41) was not immunologically detectable by enzyme-linked immunosorbent assays or Western blot (immunoblot) assays (40). This may well have been due to the protein being released from the outer membrane into the culture medium, which would explain why P. putida bearing an insertion of the bulky OprL::9PhoA protein exhibited impaired ability to grow on low-osmolarity medium. Most of the observed phenotypes for the oprL::9phoA mutant reverted to those of the wild type after the wild-type oprL gene within cosmid pPRO50 was transferred into the mutant strain. The P. putida oprL (pal) gene seems to form part of an operon, as an ORF named ORF2 was found 39 before any transcriptional stop signal appeared. This ORF2 is homologous to an ORF found downstream of the pal gene in E. coli and L. pneumophila (13) and probably encodes a polypeptide of about 29,000 Da. The role of the polypeptide made by ORF2 is unknown. A partial ORF was also found upstream from the pal gene in E. coli (44). In E. coli, the tolB gene was located immediately upstream of the pal gene, but whether tolB and pal genes are transcribed as a single operon in this microorganism is unknown (27). In L. pneumophila, another ORF was found upstream of the pal gene, but the operon structure is unknown (13). Our preliminary results suggest the oprL (pal) gene of P. putida is within an operon, since primer extension analysis with oligonucleotide primers complementary to mRNA of the oprL gene yielded cDNA products which were larger than would be expected if the mRNA had originated from a promoter proximal to the 59 end of the oprL (pal) gene (44). Further studies of the organization of this operon are being undertaken to shed light on the role of operon gene products in the alterations of the P. putida cell envelope.




11. 12.

13. 14. 15.

16. 17. 18.


ACKNOWLEDGMENTS This work was supported by research grants from the Biotechnology Program of the EC (BIOTECH BIO2-CT92-0084) and by grants from the Comisio ´n Interministerial de Ciencia y Tecnologı´a of Spain (AMB1524/94-CE and AMB1038-CO2-01). We thank C. Engleberg for communication of the OrfD-deduced protein sequence of L. pneumophila and express our appreciation to the University of Granada Technical Services for assistance with electron microscopic observations. J.J.R.-H. and M.-I.R.-G. contributed equally to the experimental work.

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