JOURNAL OF BACTERIOLOGY, Oct. 1996, p. 5836–5840 Vol. 178, No. 19 0021-9193/96/$04.0010 Copyright q 1996, American Society for Microbiology Characterization of an OprL Null Mutant of Pseudomonas 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::9phoA 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::9phoA 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::9phoA 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 9PhoA protein (462 amino acids). lized from E. coli CC118lpir, 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 mg of streptomycin per ml. Smr transconjugants appeared at a 25 * 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 (428C, 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 bacteria 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.