Characterization of type IV pilus genes in plant growth-promoting Pseudomonas putida WCS358

In a search for factors that could contribute to the ability of the plant growth-stimulating Pseudomonas putida WCS358 to colonize plant roots, the organism was analyzed for the presence of genes required for pilus biosynthesis. The pilD gene of Pseudomonas aeruginosa, which has also been designated xcpA, is involved in protein secretion and in the biogenesis of type IV pili. It encodes a peptidase that processes the precursors of the pilin subunits and of several components of the secretion apparatus. Prepilin processing activity could be demonstrated in P. putida WCS358, suggesting that this nonpathogenic strain may contain type IV pili as well. A DNA fragment containing the pilD (xcpA) gene of P. putida was cloned and found to complement a pilD (xcpA) mutation in P. aeruginosa. Nucleotide sequencing revealed, next to the pilD (xcpA) gene, the presence of two additional genes, pilA and pilC, that are highly homologous to genes involved in the biogenesis of type IV pili. The pilA gene encodes the pilin subunit, and pilC is an accessory gene, required for the assembly of the subunits into pili. In comparison with the pil gene cluster in P. aeruginosa, a gene homologous to pilB is lacking in the P. putida gene cluster. Pili were not detected on the cell surface of P. putida itself, not even when pilA was expressed from the tac promoter on a plasmid, indicating that not all the genes required for pilus biogenesis were expressed under the conditions tested. Expression of pilA of P. putida in P. aeruginosa resulted in the production of pili containing P. putida PilA subunits.

Many Pseudomonas strains derived from the rhizosphere have been studied because of their plant growth-promoting properties (20,35). In the case of Pseudomonas putida WCS358, this property is based mainly on siderophore-mediated iron deprivation of plant-deleterious microorganisms (2,11). However, efficient root colonization is also essential for plant growth stimulation. It is assumed that bacterial cell surface structures are involved in adhesion to and colonization of plant roots. A P. putida WCS358 mutant lacking the O-antigenic side chain of the lipopolysaccharide was not impaired in adhesion (13). Other cell surface structures, such as flagella (12) or surface-exposed domains of outer membrane proteins like porin F (9), have been suggested to be involved in root colonization. We are investigating the possibility that pili, which have never been reported to be present on P. putida cells until now, and/or the production of exoproteins may have a role in the colonization process.
An organism closely related to P. putida, the opportunistic pathogen Pseudomonas aeruginosa, produces type IV cell surface pili (fimbriae), which are involved in adhesion to mammalian epithelial cells (14,51). In addition, these retractile pili are responsible for a primitive type of motion known as twitching motility, which enables bacterial translocation on solid surfaces (6,50). The pili are composed of identical subunits, encoded by the piLA gene. The N-terminal segments of type IV pilins produced by different bacteria are highly conserved (45). The pilins are synthesized as precursors containing a positively charged leader peptide 6 or 7 amino acid residues long, followed by a highly hydrophobic domain of
Plasmids and DNA manipulations. All plasmids used are listed in Table 2. Plasmid isolation, restriction endonuclease digestion, end filling with the Klenow fragment of E. coli DNA polymerase I, and ligation with T4 DNA ligase were performed by standard procedures (36). Plasmid pAG1 was constructed by cloning the 10-kb Hindlll fragment, which contains pilus genes of P. putida WCS358, from pPX851 into pUC18. Expression vector pUR6500HE is a derivative of pMMB67HE (19) carrying a kanamycin resistance gene inserted in the bla gene. The piLA gene was cloned in pUR6500HE in two steps. First, the 0.9-kb Stul fragment carrying pilA was cloned in the SmaI site of pEMBL18, resulting in pAG2, in which pilA is oriented in a direction opposite to that of the lac promoter. Subsequently, thepiLA gene was cloned as a BamHI-EcoRI fragment in pUR6500HE. In the resulting plasmid, pAG3, pilA is under control of the IPTG-inducible tac promoter. E. coli was transformed with plasmids as described elsewhere (36). Chromosomal DNA was isolated as described previously (37). Nylon membranes, Hybond-N for colony blotting and Hybond-N+ for Southern blotting, were used as recommended by the manufacturer (Amersham). DNA probes were labeled with digoxigenin by using a random-primed labeling kit (Boehringer Mannheim), and hybridizations were performed at 68°C as described by the manufacturer of the labeling kit. The final washings were done at 68°C in 2 x SSC (1 x SSC is 0.15 M NaCl, 0.015 M sodium citrate). Blots were developed by using a chemiluminescent detection kit as described by the manufacturer (Boehringer Mannheim).
Plasmid mobilization. Broad-host-range plasmids were transferred from E. coli 1046 to Pseudomonas strains by triparental mating by using helper plasmid pRK2013. P. aeruginosa and P. putida transconjugants were selected on Pseudomonas isolation agar and King's B medium, respectively, supplemented with the appropriate antibiotics.
DNA sequencing. DNA fragments were obtained from pAG1 and subcloned in pEMBL18 or -19 phagemids. Unidirectional deletions were made by using the double-stranded Nested Deletion Kit (Pharmacia). Single-stranded DNA was prepared from E. coli PC2494 carrying pEMBL derivatives after superinfection with helper phage VCSM13 (Stratagene). The nucleotide sequence of single-stranded DNA templates was determined by using the Gene-ATAQ Sequencing Kit (Pharmacia). The label used was [a-35S]dATPaS (Amersham).
In vivo expression. Expression of the genes cloned under the control of the T7 promoter in plasmid pJX3 was analyzed in P. putida and P. aeruginosa by inducing the expression of T7 RNA polymerase encoded by pJRD7pol. Cells were grown in L broth at 30°C to an optical density at 660 nm (OD660) of 0.8 (P. aeruginosa) or 1.5 (P. putida). Cells were washed and resuspended in minimal medium supplemented with 0.5% methionine assay medium (Difco) and 0.4% glucose and incubated for 1 h at 30°C. The synthesis of the T7 RNA polymerase was induced by shifting the temperature to 42°C. After 30 min, rifampin was added (final concentration, 200 pRg/ml) to inhibit the host RNA polymerase, and incubation was continued for 10 min. Next, the cultures were returned to 30°C and incubated for an additional 30 min. Proteins were labeled by adding [35S]methionine (5 ,uCi; Amersham) to 200 RI of cells for 5 min, after which the cells were harvested by centrifugation. Subcellular fractionation was performed as described elsewhere (4). Briefly, cells were broken by ultrasonic treatment, followed by centrifugation to pellet the membranes. The membranes were resuspended in 2% Sarkosyl to solubilize the  176,1994 inner membranes and then centrifuged to pellet the outer membranes. The inner membrane proteins were precipitated by adding 1 volume of chloroform and 2.4 volumes of methanol.
Enzyme assays and phage sensitivity test. Plate assays for elastase, lipase, and phospholipase C activity were performed as described previously (8). Sensitivity to the P. aeruginosa pilus-specific phage P04 (5) was tested as described previously (40).
Pilus preparations. Cells, grown on L broth agar plates, were resuspended in 10 ml of 1 x SSC to an OD660 of 2 to 4. Pili were sheared from the cells by passing the suspension three times through a 21-gauge needle (40). The cells were removed from the suspension by repeated centrifugation at 6,000 x g for 10 min until no pellet was visible. The pH of the supernatant was adjusted to 4.3 with 1 M citric acid (44). After precipitation at 4°C for 4 h, pili were pelleted at 6,000 x g for 15 min and resuspended in SDS-PAGE sample buffer.
Immunoblotting. Proteins were blotted from polyacrylamide gels onto nitrocellulose membranes (Schleicher & Schuell). The blots were blocked for 1 h in 0.5% nonfat dried milk (Protifar; Nutricia) in phosphate-buffered saline (PBS), pH 7.0, followed by incubation for 1 h with rabbit antiserum against PAO pilin in 0.1% Protifar in PBS. The blots were washed in both PBS and water for 10 min each. Subsequently, the blots were incubated with peroxidase-conjugated goat anti-rabbit immunoglobulin G in 0.1% Protifar in PBS for 1 h and then washed as before. Detection was performed by using 4-chloro-1-naphthol and H202 as substrate for peroxidase (30 mg of 4-chloro-1-naphthol dissolved in 9 ml of methanol, with addition of 51 ml of PBS and 30 ,ll of 35% H202).
Electron microscopy. Cells were grown on L broth agar, tryptic soy broth agar, or King's B medium agar plates overnight and prepared for electron microscopy as described elsewhere (44), except that staining was performed with 1% uranyl acetate.
Nucleotide sequence accession number. The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank, and DDBJ nucleotide sequence data bases under the accession number X74276.

RESULTS
Prepilin peptidase activity in P. putida WCS358. In P. aeruginosa, the PilD (XcpA) prepilin peptidase is required both for type IV pilus biogenesis and for secretion of most proteins (3). It cleaves off the short leader peptide of the pilin precursor and of pilin-like components of the protein secretion machinery. Southern hybridization experiments have shown that P. putida WCS358 contains pilD (xcpA)-related DNA (8). To analyze whether a functional prepilin peptidase is produced, an in vivo expression experiment was performed.
Plasmid pJX3 contains the xcpS, -T, and -U genes of P. aeruginosa, cloned in vector pJRD253 under control of the inducible T7 promoter. After induction of the T7 promoter, only the pilin-like XcpT protein (16a) (and not XcpU as suggested in reference 4) is clearly detected. Bally et al. (4) have shown that this XcpT protein is processed in wild-type P. aeruginosa but not in apilD (xcpA) mutant. To detect possible processing activity in P. putida, pJX3 was mobilized into strain WCS358 and, as a control, into P. aeruginosa PA0222 (Xcp+) and KS904 (xcpAl). All strains also contained pJRD7pol encoding the T7 RNA polymerase. Strains were grown to an OD660 of 0.8 and pulse labeled as described in Materials and Methods. Both precursor and mature XcpT proteins could be detected in P. putida (Fig. 1A). However, only a small fraction of the total amount of XcpT protein was processed to the mature form under these conditions. Recently, it appeared that the expression of xcp genes in P. aeruginosa is induced at entry to the stationary phase of growth (1). Since this could also be the case for the pilD (xcpA) gene, the experiment was repeated, but this time P. putida cells were grown to an OD660 of 1.5. Under these conditions, processing was more efficient (Fig.  1B). Moreover, subcellular fractionation of the P. putida cells revealed that the precursor XcpT protein was present mainly in the fraction containing the cytoplasmic membrane proteins, whereas the mature product was located mainly in the outer membrane fraction (Fig. 1B). These different fractionation behaviors of precursor and mature XcpT were also found in the natural host, P. aeruginosa (4). The results indicate that P. putida produces a functional prepilin peptidase that is able to correctly process a heterologous prepilin-like protein.
Cloning of the pilD (xcpA) gene of P. putida WCS358. To clone pilD (xcpA) and possibly additional flanking pilus genes of P. putida, a gene bank of strain WCS358 in pLAFR3 in E. coli was screened by colony hybridization. A 1.1-kb EcoRI-HindIll fragment from pUP2, carrying the pilD (xcpA) gene of P. aeruginosa, was used as a DNA probe. Approximately 1,000 colonies were screened, and five positive clones were identified. Plasmid DNA was prepared from these positive clones and further analyzed by restriction endonuclease digestion and Southern hybridization. All clones had an approximately 10-kb HindIlI fragment in common which hybridized with the pilD (xcpA) probe. A similar fragment was present in HindlIldigested chromosomal DNA of P. putida (results not shown).
Complementation of apilD (xcpA) mutation in P. aeruginosa. To analyze whether the isolated cosmid clones, carrying DNA of P. putida which hybridized with the pilD (xcpA) probe, encode a functional prepilin peptidase, complementation experiments were performed. The P. aeruginosa xcpAl mutant KS904 is not piliated and is also affected in the secretion of most proteins, including elastase, lipase, and phospholipase C. The cosmid clones were mobilized into this xcpAl mutant, and the transconjugants were assayed for protein secretion and piliation. Elastase plate assays indeed revealed that secretion was restored, as shown for clones pPX816 and pPX851 (Fig. 2). J. BACTERIOL.
FIG. 2. Plate assay demonstrating elastase secretion. P. aeruginosa PA0222 (Xcp+ Pil+) and prepilin peptidase mutant KS904 (xcpAl) were mobilized with cloning vector pLAFR3 or with cosmid clone pPX816 or pPX851 carrying pilD (xcpA) of P. putida WCS358. A halo around the colonies shows the degradation of elastin in the plates. Also note the compact size of the colony of the xcpAl mutant carrying vector pLAFR3 compared with the sizes of the other colonies, which are more spread, probably because of twitching motility. Similar results were found in plate assays for lipase and phospholipase C (results not shown). Figure 2 also indicates that piliation was restored. Because of the twitching motility caused by the pili, the colony of the wild-type P. aeruginosa is flat and spread on the surface of the plate, whereas the xcpAl mutant, which does not assemble pili, grows as a compact, dome-shaped colony. Introduction of the pilD (xcpA) gene of P. putida in the xcpAl mutant resulted in wild-type colony morphology (Fig. 2). The presence or absence of the retractile pili was also analyzed by using the P. aeruginosa pilus-specific phage P04. The xcpAl mutant was resistant, whereas the wild type and the xcpA1 mutant containing pilD (xcpA) of P. putida were sensitive (results not shown). Hence, the cloned pilD (xcpA) gene of P. putida encodes a functional prepilin peptidase and restores type IV pilus biogenesis and protein secretion in the P. aeruginosa xcpAl mutant.
Nucleotide sequence analysis. The 10-kb Hindlll fragment of pPX851, containing the pilD (xcpA) gene of P. putida WCS358, was subcloned in pUC18, resulting in pAG1. The location of the pilD (xcpA) gene was determined more accurately by restriction mapping and Southern hybridizations (results not shown). Subsequently, appropriate DNA fragments were cloned in pEMBL18 or -19 to determine the nucleotide sequence. The pilD (xcpA) gene was found to encode a protein of 288 amino acid residues (Fig. 3B), with 63.9% identity to the 290-amino-acid-residue-long PilD (XcpA) protein of P. aeruginosa (Fig. 4). The pilD (xcpA) gene of P. aeruginosa is preceded by two additional genes required for pilus biogenesis, i.e., pilB and pilC (Fig. 3A). Moreover, upstream of pilB in the opposite transcriptional orientation, the pilA gene encoding the pilin subunit is located (Fig. 3A). To identify possible homologs in P. putida, the region adjacent to pilD (xcpA) was analyzed. The nucleotide sequence of a DNA fragment of a total of 3.6 kb, determined on both strands, is shown in Fig. 3B and C. Immediately upstream of pilD (xcpA), a pilC homolog was identified (Fig. 3B). The gene encodes a protein 401 amino acid residues long that is homologous (43.1% identity) over its entire length with the 406amino-acid-residue-long PilC protein of P. aeruginosa (Fig. 5).
Remarkably, the nucleotide sequence upstream of pilC did not reveal an open reading frame homologous to pilB. However, separated by an intergenic region of 218 bp, a pilin structural gene, piL4, was identified, oriented in the opposite direction to pilC and pilD (xcpA) (Fig. 3C). The 136-aminoacid-residue-long PilA protein has the characteristics of type IV prepilins; namely, a positively charged leader peptide 6 amino acid residues long followed by a stretch of hydrophobic residues which is highly conserved in type IV pilins of different gram-negative bacteria (Fig. 6). In P. aeruginosa and other species, it has been shown that the leader peptide is cleaved off after the glycine and that the new N-terminal phenylalanine is methylated (18,45). In P. putida, the first amino acid after the cleavage site is isoleucine (Fig. 6). Furthermore, the 130amino-acid-residue-long mature PilA contains the invariant glutamate at position 5 and two cysteine residues close to the C terminus, which possibly form a disulfide bridge (Fig. 6).
Downstream of piU, a putative threonine tRNA gene including the promoter was found (Fig. 3C). The tRNA molecule is predicted to be 76 bases long and contains a CGU anticodon. A highly homologous tRNAThr (CGU) gene has been identified downstream of piL4 in P. aeruginosa (23). Downstream of the tRNA gene, an imperfect inverted repeat followed by four T's that possibly functions as a transcriptional terminator can be discerned (Fig. 3C).
It was noticed that the stop codon of pilD (xcpA) overlaps with a start codon of an open reading frame, designated orfX (Fig. 3B). The protein potentially encoded by orfX contains a putative ATP-binding site close to the N terminus. Comparison with the P. aeruginosa sequence downstream ofpilD (xcpA) (reference 2a and our own unpublished results) revealed strong homology (results not shown). Whether the product of this putative gene is involved in pilus biogenesis and/or protein secretion in P. putida and P. aeruginosa requires further investigation. It should be noted, however, that the products of the piL4, -B, -C, and -D (xcpA) genes are probably not sufficient for pilus biogenesis, since additional genes have been shown to be required for the assembly of type IV pili in V. cholerae (42).
Heterologous expression of P. putida pilA in P. aeruginosa. The nucleotide sequence analysis revealed the presence of type IV pilus genes in P. putida. To detect pili, cells were examined by electron microscopy. However, after growth on plates of several standard laboratory media (L broth agar, tryptic soy broth agar, and King's B medium agar), pili were not detected on the cell surface. Similarly, introduction of plasmid pAG3, carrying the pilU gene of P. putida under control of the tac promoter, into strain WCS358 did not result in the detection of pili (results not shown). To identify the product encoded by the P. putida pilA gene and to determine whether it can assemble into pili, heterologous expression experiments with P. aeruginosa were performed. It has been shown that multipiliated P. aeruginosa strains are able to assemble type IV pili composed of pilin subunits from other gram-negative bacteria (24). Plasmid pAG3 was mobilized into the multipiliated strain DB2 and into the normally piliated strain PA0222. Transconjugants were grown on plates containing IPTG, and pili were isolated as described in Materials and Methods. Isolated pili were analyzed by SDS-PAGE and immunoblotting (Fig. 7). In the case of the strains containing the cloning vector, only the host PilA protein was detected (Fig. 7, lanes 1 and 3 in both panels). An additional protein, which was not recognized by the antiserum against PAO pilin (Fig. 7B, lane 2  case of strain DB2 containing pAG3 (Fig. 7A, lane 2). This additional protein, which most likely represents PiLA of P. putida, migrated faster than the host PilA protein. This is consistent with the sequencing results, since the mature PilA of P. putida is predicted to be 130 amino acid residues long, whereas PilA of P. aeruginosa PAO is composed of 143 residues. When strain PA0222 was used, no P. putida PilA protein was detected and the biogenesis of host pili seemed to be inhibited to a large extent by the presence of the P. putida piL4 gene (Fig. 7A, lane 4).

DISCUSSION
The potato root-isolated P. putida WCS358 has been characterized as a plant growth-promoting bacterium (2,20). This phenomenon requires efficient root colonization, the molecular basis of which is still unclear. Bacterial cell surface structures such as pili, as well as secreted proteins, may have a role in this process. Hybridization experiments have shown that P. putida contains DNA related to the pilD (xcpA) gene of P. aeruginosa (8). The pilD (xcpA) gene encodes a prepilin peptidase that is required for the assembly of type IV pili and for protein secretion (3,40,41). Here, we characterized the pilD (xcpA) gene of P. putida WCS358, which was found to encode a functional product. Furthermore, upstream of pilD (xcpA), two additional genes connected with type IV pilus production appeared to be present; namely, pilC and piL4. The pilC gene encodes a hydrophobic protein and is probably located in the cytoplasmic membrane. The pilA gene encodes the pilin subunit and could be heterologously expressed and assembled into pili in P. aeruginosa (Fig. 7). The PilA protein has the characteristics of a type IV pilin subunit: it contains a positively charged leader peptide 6 amino acid residues long, M-NPSIR--LYAWQGTNADGLAVSGQMAGRSPAYVRAGLLRQGILVARLR -47 .. .. II 11.1I I 1 LGSLI -401 II ..
LGNVVG followed by a stretch of hydrophobic amino acids, that is highly conserved among type IV pilins of different gram-negative bacteria (28,45). The first amino acid after the predicted cleavage site is isoleucine. In most gram-negative bacteria producing type IV pili, the first residue of the mature pilin subunit is a phenylalanine, which becomes methylated after processing. However, the first residue of the mature pilin in V. cholerae is a methylated methionine (29). Moreover, substitution of the N-terminal phenylalanine in pilin of P. aeruginosa by other amino acids did not affect cleavage and assembly into pili, indicating that an N-terminal phenylalanine is not absolutely required (47). The mature pilin of P. putida contains a glutamate residue at position 5, which has been shown to be required for methylation and pilin assembly in P. aeruginosa (43,47). Like other type IV pili, but unlike the pilin-like components of the secretion machinery, the C terminus of PilA contains two cysteine residues (residues 120 and 133 in precursor PilA) (Fig. 6) that probably form a disulfide bridge. It has been shown that the C terminus of P. aeruginosa PilA contains an epithelial cell-binding domain, a function that is enhanced by the formation of an intrachain disulfide bond (25). Similarly, the C terminus of the P. putida pilin might contain a domain involved in adhesion. The P. putida pilin contains two additional cysteine residues, located toward the central part of the protein (residues 78 and 95 in precursor PilA) (Fig. 6). As far as we know, this is the first time that type  (38), and N. gonorrhoeae (NGO) (39). Residues identical in at least four proteins are shown boxed. The two cysteine residues that (probably) form a disulfide bond are indicated by asterisks. The prepilin peptidase cleavage site is indicated by an arrowhead.
IV pilus genes have been detected in a nonpathogenic bacterium.
The P. putida PilA protein was found to be assembled into pili when expressed in the multipiliated P. aeruginosa DB2 but not when expressed in the normally piliated strain PA0222 (Fig. 7). Similar results have been described by Pasloske et al. (44), who found that a mutant pilin of P. aeruginosa PAK was assembled in the P. aeruginosa PAO-derived, multipiliated strain DB2 but not in a normally piliated PAO strain. These observations might be explained by the fact that the type IV pili are retractile by disassembly of the pili. In the normally piliated strains, disassembly results in a flow of pilin subunits back into a pool of subunits within the cell envelope. If assembly of host pilin subunits is favored over assembly of foreign pilins, pili composed of only host pilin subunits would be assembled. In the multipiliated strains, the pili are not disassembled and the host pilin subunits remain in the pilus structures. Consequently, a relatively high concentration of heterologous subunits will accumulate in the pool of subunits in the cell envelope, thus favoring their assembly into the pilus structures.
Although P. putida possesses type IV pilus genes, electron microscopy studies did not reveal the presence of pili on the cell surface. This suggests that the expression of the pil genes is strongly regulated. The demonstration that the prepilin-like XcpT protein of P. aeruginosa is processed when expressed in P. putida indicates that the pilD (xcpA) gene and probably also pilC and putative additional genes in the same operon are expressed. However, whenpilA was expressed in P. putida from an inducible promoter on a plasmid, pili were still not detected, indicating that additional genes required for pilus biogenesis are absent or not expressed. One of these genes might bepilB, which is not present upstream of pilC in the P. putida gene cluster, in contrast to the genetic organization in P. aeruginosa (Fig. 3). However, it seems unlikely that P. putida WCS358 has lost the pilB gene but maintained otherpil genes, includingpiLA and pilC, in its genome. Therefore, we favor the hypothesis that a pilB gene is present in P. putida but at a different chromosomal position. Alternatively, since pilB and xcpR are highly homologous, the XcpR homolog of P. putida might have a dual role and might, like PilD (XcpA), be involved both in pilus biogenesis and in protein secretion. The first hypothesis is supported by the cloning of two different DNA fragments by hybridization with a probe carrying the xcpR gene of P. aeruginosa (unpublished results). Preliminary DNA sequencing results showed that one clone contained xcpR and other xcp genes whereas the second clone may contain pilB (unpublished results). It should be noted that Koga et al. (31) recently reported that the pilB gene of P. aeruginosa, although located directly upstream of pilC and pilD (xcpA), does not form a transcriptional unit with these genes. Furthermore, it was recently reported that in Neisseria gonorrhoeae also, a pilB homolog is not arranged in tandem with pilD (xcpA)-and pilC-related genes (32).
In P. aeruginosa, transcription of pilA, but not of pilB, -C, and -D (xcpA), requires the sigma 54 RNA polymerase initiation factor RpoN (26,31). The transcription is controlled by PilS and PilR, belonging to the family of two-component transcriptional regulatory systems (22,27). The P. putida DNA sequence upstream of piU4 does not show homology to promoters that are transcribed by RpoN-containing RNA polymerase (GG-N1O-GC), suggesting that expression is regulated differently in this bacterium. The results of the experiment demonstrating processing of XcpT ( Fig. 1) suggest that the expression of pilD (xcpA) is growth phase dependent, since more processing was detected in the late logarithmic phase or at the beginning of stationary phase. Similarly, growth phasedependent expression was detected for the xcp genes in P. aeruginosa (1). Another interesting possibility is that the expression of pil genes in P. putida is induced by compounds present in plant root exudate. The use of transcriptional fusions with reporter genes may be helpful in finding the conditions that induce pilus synthesis. The pili and the exoproteins of P. putida may have a role in adhesion to and colonization of the plant roots and therefore may contribute to the plant growth stimulation. This possibility will be investigated by inactivating the pil genes on the chromosome and studying the effect of these mutations on colonization and plant growth promotion. .iLd --r ---rrr-,I-