JOURNAL OF BACTERIOLOGY, Oct. 1996, p. 5836–5840 Vol. 178, No. 19 0021-9193/96/$04.00ϩ0 Copyright ᭧ 1996, American Society for Microbiology

Characterization of an OprL Null Mutant of putida

JOSE´-JUAN RODRI´GUEZ-HERVA AND JUAN L. RAMOS* Department of Biochemistry and Molecular and Cellular Biology of Plants, Consejo Superior de Investigaciones Cientı´ficas—Estacio´n Experimental del Zaidı´n, 18008 Granada, Spain

Received 12 March 1996/Accepted 11 July 1996

A Pseudomonas putida oprL null mutant was generated with reverse genetics by using an in vitro-truncated oprL::xylE construct and in vivo allelic exchange. The nature of the mutation introduced in P. putida was confirmed by Southern blotting. Western blots (immunoblots) of peptidoglycan-associated proteins revealed that the OprL protein was not made in the mutant strain, whereas it was detectable as a 19-kDa band in protein preparations of the wild-type strain. The P. putida oprL mutant exhibited altered cell morphology as revealed by electron microscopy and was more sensitive to sodium dodecyl sulfate, deoxycholate, and EDTA than the wild-type strain. The oprL gene was conserved in a wide variety of the Pseudomonas strains belonging to rRNA group I, which suggests that this gene is important for the maintenance of the cell envelope and cell morphology in this group of microorganisms.

The maintenance of integrity of the gram-negative cell en- the stationary phase, and did not grow on low-osmolarity me- velope appears to involve a complex series of interactions dium (23), a characteristic previously described for OprF among proteins inserted in the outer membrane, proteins in- Pseudomonas mutants (9, 29). The outer membrane of the serted in the inner membrane, periplasmic proteins, and the P. putida oprL::ЈphoA mutant was highly fragile, as revealed by peptidoglycan layer (5, 10). Although the minute details are the presence of discontinuities in the cell surface in the sta- not completely understood, it has been established that pepti- tionary phase (23). doglycan-associated lipoproteins (Pal in members of the family Given that in the oprL::ЈphoA P. putida mutant a hybrid Enterobacteriaceae and OprL in members of the family Pseudo- bulky protein was introduced into the outer membrane, it was monadaceae [see reference 23 for a sequence comparison]) of interest to create null OprL mutants of P. putida to deter- and the Tol proteins are required to maintain cell envelope mine whether the phenotypes described above were a conse- structure (15, 23). In Pseudomonas spp. the major porin OprF quence of the lack of functional OprL or of the insertion of a is also involved in cell membrane integrity (9, 29). foreign bulky protein in the outer membrane of this microor- In Escherichia coli the interplay between Pal proteins, Tol ganism. To this end we generated a null oprL mutant of P. proteins, and peptidoglycan has been partially determined. putida KT2440 and characterized it. The Pal protein is inserted in the outer membrane; the Cys-1 Construction of an oprL null mutant of P. putida by gene re- in the mature protein is bound via an amide link to a palmitic placement. The mobilizable suicide plasmid pKNG101XYLE acid and via a sulfur ester to a glycerol molecule, which in turn is a pKNG101 derivative (13) bearing a truncated oprL gene in is linked to fatty acids (30). The C-terminal end of the Pal which an internal 400-bp fragment of the coding region (posi- protein faces the periplasm and is involved in interactions with tions 42 to 442, base 1 being the A of the first ATG) was peptidoglycan (4, 14, 16); in E. coli it also interacts with the removed and replaced by a promoterless xylE gene (Fig. 1A). TolB protein (1). In E. coli the TolA, TolQ, and TolR proteins In this construct and as a consequence of the xylE insertion, the are associated with the inner membrane (17, 20, 27). TolA open reading frame of oprL was shortened from 166 amino expands towards the periplasmic space and seems to interact acids to a 19-amino-acid polypeptide. Plasmid pKNG101 with outer membrane proteins and with the peptidoglycan XYLE was used to deliver the oprL::xylE mutation to the host layer (18). This interplay explains, at least in part, how the P. putida chromosome via homologous recombination because integrity of the cell envelope is maintained. this plasmid has the advantage of containing both a strepto- We previously isolated an oprL::ЈphoA mutant of Pseudomo- mycin resistance (Smr) gene as a selectable marker for the nas putida, called P. putida 14G-3 (23). The strain was selected cointegration event and the Bacillus subtilis sacB gene as a on media with BCIP (5-bromo-4-chloro-3-indolylphosphate) counterselectable marker to enhance the second step, that of as blue colonies and exhibited a fusion of the first 92 amino allelic exchange (8, 13). Plasmid pKNG101XYLE was mobi- acids of OprL and the bulky ЈPhoA protein (462 amino acids). lized from E. coli CC118␭pir, which bears the chlorampheni- Like Pal mutants of members of the family Enterobacteriaceae, col-resistant helper plasmid pRK600 (12), into the benzoate- the P. putida oprL mutant was more sensitive to sodium dode- utilizing P. putida strain KT2440 by conjugational mating. cyl sulfate (SDS), EDTA, and deoxycholate than the wild-type P. putida transconjugants bearing a cointegrate of the plasmid P. putida strain KT2440 (23). In addition, the Pseudomonas in the host chromosome were selected on M9 minimal medium mutant exhibited altered cell morphology, formed clumps in (24) with benzoic acid (10 mM) as the sole C source and 100 ␮g of streptomycin per ml. Smr transconjugants appeared at a Ϫ5 * Corresponding author. Mailing address: Consejo Superior de In- frequency of about 10 per recipient, and all turned bright vestigaciones Cientı´ficas—Estacio´n Experimental del Zaidı´n, Depart- yellow after being sprayed with a 0.5 M solution of catechol, ment of Biochemistry and Molecular and Cellular Biology of Plants, confirming the incorporation of xylE (catechol 2,3-dioxygen- Apdo. Correos 419, 18008 Granada, Spain. Phone: 34-58-121011. Fax: ase, encoded by xylE, converts catechol into the yellow 2-hy- 34-58-129600. droxymuconic acid semialdehyde). As expected from the in-

5836 VOL. 178, 1996 NOTES 5837

FIG. 1. Strategy for the gene replacement method. (A) Steps. In step 1, the initial recombination event occurs between 1-kb homologous flanking sequences of the oprL::xylE construct on plasmid pKNG101XYLE and the P. putida host chromosome; it results in the formation of a cointegrate. In step 2, a second recombination event results in successful allelic exchange, leading to an oprL mutant. P, PstI; Hc, HincII; C230, catechol 2,3-dioxygenase. (B) Southern blot analysis of the oprL locus, demonstrating gene replacement. Total DNA was prepared from P. putida DOT-OX1, which bears a pKNG100XYLE-host chromosome cointegrate (lane 1), the wild-type strain (lane 2), P. putida DOT-OX2 (lane 3), pKNG101 (lane 4), and pKNG101XYLE (lane 5). DNA samples were digested with PstI, and after agarose gel electrophoresis, they were transferred to a nylon hybridization membrane. The probe was the 0.65-kb PstI-HincII fragment from pPRO200 (23), containing 146 bp from the oprL coding sequence shown in panel A. The DNA probe was randomly labeled with digoxigenin-dUTP according to the manufacturer’s instructions (Boehringer Mannheim). For DNA prehybridization and hybridization, high-stringency conditions (42ЊC, 50% [vol/vol] formamide) were used. The digoxigenin-labeled hybrid DNA was detected by using an enzyme immunoassay according to the manufacturer’s instructions (Boehringer Mannheim). corporation of the sacB gene too, all Smr were unable digested with PstI and used as a control. The hybridization to grow on Luria-Bertani (LB) medium with 5% (wt/vol) su- patterns with oprL are shown in Fig. 1B. As expected, the oprL crose. Figure 1A illustrates the organization in one of the gene was identified in the wild-type strain as a 0.9-kb band, clones, called P. putida DOT-OX1. Total DNA of strain DOT- while in the mutant strain a 2.4-kb band was found. DNA OX1 was isolated and digested with PstI, and after Southern prepared from strain DOT-OX2 hybridized as expected with blotting and hybridization with the oprL gene probe, two bands the xylE gene in the 2.4-kb PstI fragment (data not shown) and of 0.9 and 2.4 kb, corresponding to the wild-type oprL and the did not hybridize against probes prepared from plasmid oprL::xylE insertion, respectively, were detected (Fig. 1B). To pKNG101 (data not shown). Therefore, it was clear that in complete the allelic exchange and therefore the replacement of P. putida DOT-OX2 the wild-type oprL gene had been re- the wild-type oprL gene with the oprL::xylE mutation, a second placed by the oprL::xylE construction. crossover event was needed. The desired event would result To further confirm that the OprL protein was absent in the in the loss of the Smr marker and the sacB gene (Fig. 1A), so DOT-OX2 strain, peptidoglycan-associated proteins were pre- that the resulting P. putida strain should be Sms, sucrose tol- pared from the wild-type strain and oprL mutants. After gel erant (Sucr), and positive in the catechol 2,3-dioxygenase test. electrophoresis it was found that the wild-type strain produced P. putida DOT-OX1 was grown overnight in streptomycin-free a peptide of about 19 kDa, whereas this protein was absent liquid L broth medium (10 g of tryptone,5gofyeast extract) from the OprL mutants (Fig. 2A). This protein was recognized with 1% (wt/vol) NaCl and was then diluted 1/1,000 in the by monoclonal antibody (MAb) MA1-6 raised against the same medium and incubated for an additional 12 h. Serial OprL protein (Fig. 2B). Furthermore, dilutions were plated on the same medium, supplemented with other known peptidoglycan-associated proteins, e.g., OprF sucrose or unsupplemented. Sucrose-resistant colonies ap- (38.5 kDa), were present in the wild-type and mutant strains peared at a frequency of about 10Ϫ3. Sucr clones resulting from (Fig. 2A). the appropriate homologous recombination event turned yel- Phenotypic analysis of the OprL null mutant of P. putida. low after catechol spraying. Enterobacteriaceae pal mutants and the P. putida oprL::ЈphoA Among the Sucr, catechol 2,3-dioxygenase-positive clones, mutant exhibited a pattern of resistance and sensitivity to an Sms clone was randomly chosen for further characterization EDTA and certain detergents that was different from the pat- and was called P. putida DOT-OX2. First we confirmed the terns shown by the corresponding wild-type strains (15, 23). gene replacement event in this clone. Total DNA from P. pu- The abilities of wild-type P. putida KT2440 and of P. putida tida DOT-OX2 was isolated, digested with PstI, and analyzed DOT-OX2 to grow in L broth solid medium with 1% (wt/vol) by Southern blotting. DNA from the wild-type strain was also NaCl containing, in addition, EDTA, deoxycholate, or SDS 5838 NOTES J. BACTERIOL.

SDS, 1.9 mM EDTA, or 225 mM deoxycholate. Furthermore, P. putida DOT-OX2 was as sensitive as strain 14G-3 to SDS and EDTA but much more sensitive to deoxycholate, as shown by the fact that strain 14G-3 grew on plates supplemented with a 60 mM concentration of this detergent. These results show that the oprL null mutant was more sensitive than the parental strain to SDS, deoxycholate, and EDTA. The patterns of antibiotic resistance and sensitivity of P. putida DOT-OX2 and of the wild-type strain were assayed by the agar diffusion method (25). P. putida DOT-OX2 was slightly more sensitive than the wild-type strain and P. putida 14G-3 to nalidixic acid (30 ␮g per disk), piperacillin (100 ␮g per disk), rifampin (30 ␮g per disk), and tetracycline (30 ␮g per disk), as the inhibition zone halo with the mutant strain was 1.28- to 1.71-fold larger than that obtained with the latter strains. On the other hand, the oprL null mutant was as resis- tant as the parental strain and P. putida 14G-3 to ampicillin (10 ␮g per disk), chloramphenicol (30 ␮g per disk), erythromycin (15 ␮g per disk), and vancomycin (30 ␮g per disk), which did not inhibit growth of the parental strain, as determined by the inhibition zone halo. The growth rate of P. putida DOT-OX2 in L broth medium with 1% (wt/vol) NaCl (42 Ϯ 3 min) was similar to the growth rates of the wild-type strain (36 Ϯ 3 min) and P. putida 14G-3 (43 Ϯ 3 min). It was previously reported that, in contrast with the wild-type strain, strain 14G-3 produced clumps at the end of the exponential phase (23). The oprL null mutant generated in this study did not produce clumps at any time along the FIG. 2. SDS-polyacrylamide gel electrophoresis of the peptidoglycan-associ- growth curve. P. putida DOT-OX2 and the wild-type strain had ated proteins of Pseudomonas strains. P. putida and P. aeruginosa strains were similar generation times in low-osmolarity medium (L broth grown on L broth medium with 1% (wt/vol) NaCl until the late logarithmic phase with 0.1% [wt/vol] NaCl); this also contrasted with the inability (an optical density at 660 nm of approximately 1), and peptidoglycan-associated proteins were isolated as described by Fuchs et al. (7). (A) Coomassie blue of strain 14G-3 to grow in this medium. Thus, insertion of the staining of peptidoglycan-associated proteins. Lane M, molecular mass markers OprL-PhoA hybrid protein on the outer membrane of P. (in kilodaltons); lane 1, P. aeruginosa; lane 2, P. putida KT2440 (wild type); lane putida seems to give rise to additional phenotypes. 3, P. putida DOT-OX2; lane 4, P. putida 14G-3. (B) Western blot of SDS extracts Morphological appearance of OprL-deficient cells. Electron against MAb MA1-6. Peptidoglycan-associated proteins were run in polyacryl- amide gels, transferred to a nitrocellulose membrane, and immunodeveloped microscopy of thin sections of P. putida DOT-OX2 showed with MAb MA1-6 as recommended by Hancock’s laboratory (21). Lane 1, that the bacteria had well-defined inner and outer membranes. P. aeruginosa; lane 2, P. putida KT2440 (wild type); lane 3, P. putida DOT-OX2; lane However, some differences were apparent with respect to the 4, P. putida 14G-3. The arrow indicates the immunodeveloped 19-kDa OprL protein. wild-type morphology. The wild-type bacteria were rod-shaped bacteria with smooth envelopes (Fig. 3A), whereas the mutant were tested as described by Sukupolvi et al. (25). Growth of the strain appeared deformed (Fig. 3B) and its outer membrane null oprL P. putida mutant was impaired at 1.9% (wt/vol) SDS, was wavy (Fig. 3C). 1.5 mM EDTA, or 25 mM deoxycholate, whereas growth of the It was previously reported that a number of P. putida 14G-3 wild-type strain was impaired on plates containing 3% (wt/vol) cells looked like ghost bacteria under electron microscopy,

FIG. 3. Ultrastructure of P. putida KT2440 and DOT-OX2. Cells were grown on L broth medium with 1% (wt/vol) NaCl and prepared for electron microscopy as previously described (23). (A) Wild-type strain in the exponential phase. Magnification, ϫ16,000. (B) P. putida DOT-OX2 in the exponential phase. Magnification, ϫ16,000. (C) Detail of a P. putida DOT-OX2 cell in the exponential phase. Magnification, ϫ100,000. VOL. 178, 1996 NOTES 5839 particularly when they were in the stationary phase (23). This role in the correct assembly of lipids and proteins at the cell characteristic was also found in the null oprL mutant (not envelopes. It follows that if a component of the Pal (OprL)-Tol shown). system is missing, the link between the membranes and the These results suggest that OprL is involved in the mainte- peptidoglycan may break down and the envelope may be de- nance of cell morphology, although it is not essential for sur- stabilized. vival; a similar function was hypothesized previously for the Pal (OprL) protein in E. coli. This work was supported by an EC Biotech grant (BIO2-CT-92- Conservation of the oprL gene in members of the Pseudo- 0084) and by grants from the Spanish Comisio´n Interministerial de monadaceae belonging to different rRNA groups. The P. putida Ciencia y Tecnologı´a (PETRI PTR94-084 and AMB 1038-C02-1). OprL protein belongs to the class of gram-negative outer mem- We thank Robert Hancock for providing MAb MA1-6 and Silvia Marque´s for critical reading of the manuscript. brane lipoproteins known as Pal (23). 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