A site-specific recombinase is re colonization by Pseudomonas flu

A colonization mutant of the efficient rootcolonizing biocontrol strain Pseudomonasfluorescens WCS365 is described that is impaired in competitive root-tip colonization of gnotobiotically grown potato, radish, wheat, and tomato, indicating a broad host range mutation. The colonization of the mutant is also impaired when studied in potting soil, suggesting that the defective gene also plays a role under more natural conditions. A DNA fragment that is able to complement the mutation for colonization revealed a multicistronic transcription unit composed of at least six ORFs with similarity to lppL, lysA, dapF, orf235/233, xerC/sss, and the largely incomplete orf238. The transposon insertion in PCL1233 appeared to be present in the orj235/233 homologue, designated orf240. Introduction of a mutation in the xerC/sss homologue revealed that the xerC/sss gene homologue rather than orJ240 is crucial for colonization. xerC in Escherichia coli and sss in Pseudomonas aeruginosa encode proteins that belong to the A integrase family of site-specific recombinases, which play a role in phase variation caused by DNA rearrangements. The function of the xerC/sss homologue in colonization is discussed in terms of genetic rearrangements involved in the generation of different phenotypes, thereby allowing a bacterial population to occupy various habitats. Mutant PCL1233 is assumed to be locked in a phenotype that is not well suited to compete for colonization in the rhizosphere. Thus we show the importance of phase variation in microbe-plant interactions. The use of microorganisms, including fluorescent Pseudomonas spp., to protect plants against soil-borne diseases is an alternative for the use of chemical pesticides. The biocontrol activity of these strains usually results from the production of one or more antifungal factors. The application of fluorescent Pseudomonas spp. and other plant-growth-promoting rhizobacteria is hampered by inconsistency of performance in the field (1, 2). Although the mechanisms underlying biocontrol are complex and diverse, the need to bring the plant-growthpromoting rhizobacteria cells and their antifungal factors to the right sites at the right time is universal. The importance of this process, designated as root colonization, is underscored in two studies. Schippers et al. (1) showed that inadequate colonization leads to decreased biocontrol activity, and Bull et al. (3) reported an inverse relation between the number of bacteria present on the wheat root and the number of take-all lesions seen on the plant. For these and other reasons, root colonization is often considered the limiting factor for biocontrol in the rhizosphere (1, 2). Two approaches were used in our laboratory to identify traits involved in root colonization. The first approach is to guess which traits are involved in colonization, isolate mutants in these traits, and then test these mutants for colonization in The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. ?1734 solely to indicate this fact. ? 1998 by The National Academy of Sciences 0027-8424/98/957051-6$2.00/0 PNAS is available online at http://www.pnas.org. quired for competitive root torescens WCS365 DER FITS, AND BEN J. J. LUGTENBERG* assenaarseweg 64, 2333AL Leiden, The Netherlands n, WA, March 18, 1998 (received for review April 1, 1997) competition with the parental strain. With this approach, motility (4) and synthesis of the O-antigen of lipopolysaccharide (LPS) (5) were shown to be essential for colonization. Moreover, mutants auxotrophic for amino acids or vitamin B, (6) and mutants with a slightly increased generation time (7) also appeared to be reduced in colonization. Our second approach involves random transposon mutagenesis, using Tn5lacZ (8), of the efficient root colonizer Pseudomonas fluorescens WCS365 (9). Individual mutants were tested for their colonization ability in competition with the parental strain in a gnotobiotic system as described by Simons et al. (7). This approach enables us to obtain knowledge of bacterial colonization traits. In this paper, we describe such a mutant strain, PCL1233, and the results indicate the involvement of a site-specific recombinase in root colonization. MATERIALS AND METHODS Bacterial Strains and Culture Conditions. The characteristics of all bacterial strains and plasmids used in this work are described in Table 1. Wild-type P. fluorescens WCS365 cells and derivatives of this strain were grown overnight at 28?C on solidified King B medium (18) or in liquid King B or standard succinate medium (SSM) (19) with vigorous shaking. Various Escherichia coli strains were grown overnight at 37?C in liquid or on solidified Luria-Bertani medium (LBM) (20). When appropriate a final concentration of the following antibiotics was added to these media: nalidixic acid, 15 pg/ml; tetracyclin, 40 ptg/ml; streptomycin, 500 p.g/ml; spectinomycin, 200 p.g/ml; kanamycin 50 pg/ml; and carbenicillic acid, 100 pug/ml. To distinguish between the wild-type P. fluorescens WCS365 cells and its Tn5lacZ derivatives, 5-bromo-4-chloro3-indolyl ,3-D-galactoside was added to the medium to 40 p.g/ml. The ability to produce siderophores such as pyoverdin was tested on solidified LBM supplemented with 2.5 or 5 mM ZnSO4 (13). Addition of the salt was followed by adjustment of the pH of the medium. Plasmid Constructions. To isolate and study the DNA fragment from mutant PCL1233, which contains the Tn5lacZ (8) insertion, we isolated a flanking region of the transposon by using SalI to digest the chromosomal DNA of mutant PCL1233. Subsequently, ligation of this digested chromosomal DNA into pIC20H (17) and transformation to E. coli resulted in plasmid pMP5206 (Fig. 1 and Table 1). Plasmid pMP5206 was subsequently used to construct pMP5211 (Fig. 1 and Table 1). After introduction of pMP5211 into P. fluorescens WCS365 and selection for a single homologous recombination event, total DNA was isolated and digested with BamHI. Self-ligation and selection for kanamycin resistance (21) resulted in plasmid pMP5209, which contains an 8.5-kb EcoRI fragment of P. fluorescens WCS365 spanning the Tn5lacZ insertion (Table 1). Abbreviations: LPS, lipopolysaccharide; IHF, integration host factor. Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. Y12268). *To whom reprint requests should be addressed. e-mail: Lugtenberg@ rulbim.leidenuniv.nl.