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Applied and Environmental Microbiology, September 2005, p. 5197-5207, Vol. 71, No. 9
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.9.5197-5207.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Novel Lectin-Like Bacteriocins of Biocontrol Strain Pseudomonas fluorescens Pf-5

Annabel H. A. Parret, Koen Temmerman, and René De Mot*

Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven, B-3001 Heverlee, Belgium

Received 30 November 2004/ Accepted 1 April 2005


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ABSTRACT
 
Bacteriocin LlpA, produced by Pseudomonas sp. strain BW11M1, is a peculiar antibacterial protein due to its homology to mannose-binding lectins mostly found in monocots (A. H. A. Parret, G. Schoofs, P. Proost, and R. De Mot, J. Bacteriol. 185:897-908, 2003). Biocontrol strain Pseudomonas fluorescens Pf-5 contains two llpA-like genes, named llpA1Pf-5 and llpA2Pf-5. Recombinant Escherichia coli cells expressing llpA1Pf-5 or llpA2Pf-5 acquired bacteriocin activity and secreted a 31-kDa protein cross-reacting with LlpABW11M1 antibodies. Antibacterial activity of the recombinant proteins was evidenced by gel overlay assays. Analysis of the antimicrobial spectrum indicated that LlpA1Pf-5 and LlpA2Pf-5 are able to inhibit P. fluorescens strains, as well as the related mushroom pathogen Pseudomonas tolaasii. LlpA-type bacteriocins are characterized by a domain structure consisting of tandem monocot mannose-binding lectin (MMBL) domains. Molecular phylogeny of these MMBL domains suggests that the individual MMBL domains within an LlpA protein have evolved separately toward a specific, as yet unknown, function or, alternatively, were acquired from different ancestral sources. Our observations are consistent with earlier observations, which hinted that MMBL-like bacteriocins represent a new family of antibacterial proteins, probably with a novel mode of action.


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INTRODUCTION
 
Bacteria living in a competitive environment are able to secrete proteinaceous toxins, known as bacteriocins, which can kill closely related bacterial competitors while causing little harm to the bacteriocinogenic cells. These bacterial inhibitors are produced by all major groups of Bacteria (53). Bacteriocin production has also been reported for Halobacteriaceae, a family of extremely halophilic Archaea (36). Bacteriocins constitute a structurally and functionally diverse group within the antimicrobials, ranging from small peptides, such as microcins of Enterobacteriaceae and antibiotics secreted by low-GC-content gram-positive bacteria (23, 31), to middle-sized polypeptides, such as colicins of Escherichia coli (34) and their counterparts in Pseudomonas aeruginosa (S pyocins) (39), to large phage tail-like multiprotein complexes, such as syringacin produced by Pseudomonas syringae (58) and R- and F-type pyocins of P. aeruginosa (39). Bacteriocin mode of killing can be either by membrane pore formation, nonspecific degradation of cellular DNA, cleavage of 16S rRNA or tRNA, or inhibition of peptidoglycan synthesis resulting in cell lysis (51).

Bacteriocins from lactic acid bacteria, such as nisin, have received considerable attention, given their potential as food preservatives (41, 42). Among bacteriocins of gram-negative bacteria, colicins are the most intensively studied. Colicins are produced by, and attack, strains of E. coli. These proteins, as well as the S pyocins, are organized in functional domains, namely, regions for cell attachment, translocation, and bactericidal activity. Colicins and S pyocins parasitize specific membrane-bound receptors on target cells, resulting in a rather narrow spectrum of activity. The host strain is protected from its own bacteriocin through the action of a cognate immunity protein that is coproduced with the bacteriocin (39, 51).

It has been proposed that bacteriocins may play a key role in bacterial population dynamics (52, 53). In a recent study, Kirkup and Riley demonstrated that the production of colicins by E. coli colonizing the mouse colon gives the colicinogenic strains a competitive advantage by killing colicin-sensitive E. coli sharing the same ecological niche (33). Possible applications of bacteriocinogenic strains in agriculture include their use in biological control of soilborne or phyllosphere-inhabiting bacterial plant pathogens. In this way, heterologous production of the peptide bacteriocin trifolitoxin by an avirulent Agrobacterium strain effectively enhances biological control of Agrobacterium vitis crown gall (24). Expression of the trifolitoxin genes from Rhizobium leguminosarum bv. trifolii T24 in Rhizobium etli CE3 increases bean nodulation competitiveness of the recombinant strain in the presence of indigenous rhizobia under agricultural conditions (54). A phage tail-like bacteriocin (serracin P) produced by Serratia plymithicum may be used for biological control of fire blight caused by Erwinia amylovora (30), while Xanthomonas campestris pv. glycines has antibacterial activity against phytopathogenic Xanthomonas species through the secretion of glycinecin A (26). Finally, a bacteriocin produced by P. syringae pv. ciccaronei inhibits P. syringae subsp. savastanoi, the causal agent of olive knot disease (35).

This study is part of an ongoing project examining bacteriocin production in rhizosphere-colonizing fluorescent Pseudomonas strains. Recently, we identified a novel type of Pseudomonas antibacterial protein (bacteriocin LlpA, lectin-like putidacin A) that is secreted by rhizosphere isolate Pseudomonas sp. BW11M1. LlpA is active against the rhizosphere-colonizing strain Pseudomonas putida GR12-2R3 as well as some phytopathogenic pseudomonads, including P. syringae pathovar strains (46). This novel bacteriocin exhibits remarkable similarity with a family of mannose-binding lectins, predominantly found in monocot plants. The sequence similarities of LlpA to these lectins are localized in two discrete regions, the so-called monocot mannose-binding lectin (MMBL) domains. Chen and coworkers identified such an MMBL domain in a 32-kDa bacteriocin (albusin B or AlbB) produced by the ruminal bacterium Ruminococcus albus strain 7. They showed that albusin B is a potent growth inhibitor of Ruminococcus flavescens strains (11). Albusin B and LlpABW11M1 are likely released from the bacteriocinogenic cells via a different route. LlpABW11M1 secretion is signal peptide independent (46), while albusin B was shown to be produced as a prepeptide containing a 49-amino-acid signal peptide (11). The mechanism by which this type of bacteriocin inhibits the growth of sensitive bacteria is still under investigation. Despite the presence of MMBL domains, LlpA and albusin B are unable to bind mannose (11, 46).

In silico analysis indicated that the genome of Pseudomonas fluorescens Pf-5 contains two putative llpA homologues. This rhizosphere-inhabiting bacterium is a well-known biocontrol strain and produces an impressive array of secondary metabolites that inhibit fungal plant pathogens, including pyoluteorin, pyrrolnitrin, 2,4-diacetylphloroglucinol, and hydrogen cyanide (9, 40, 48). These properties have incited a genome sequencing project of this strain (http://www.ars-grin.gov/hcrl/Pf5genome/index.htm).

In this study, we show that both llpA homologues in strain Pf-5 are functional bacteriocin genes. As in the case for LlpA from Pseudomonas sp. BW11M1, these LlpA-like bacteriocins of Pf-5 have an antibacterial spectrum limited to Pseudomonas species.


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MATERIALS AND METHODS
 
Bacterial strains, growth media, and culture conditions.
Bacterial strains used and plasmids described in this study are listed in Table 1. Pseudomonas strains were routinely propagated in tryptic soy broth (BD Biosciences) at 30°C, with the exception of P. aeruginosa, which was cultured at 37°C. Luria-Bertani (LB) broth (55) was used for growing E. coli at 37°C. Rhodococcus erythropolis and Streptomyces coelicolor were grown in LB medium at 30°C. Agrobacterium tumefaciens and Azospirillum brasilense were cultivated at 30°C in YEP medium containing 1% Bacto peptone (BD Biosciences), 0.5% of NaCl, and 1% yeast extract (BD Biosciences). Bacillus subtilis was incubated at 30°C in TY medium (0.5% peptone and 0.3% yeast extract). Media were solidified with 1.5% agar (Invitrogen). Media were supplemented with antibiotics at the following concentrations: ampicillin, 100 µg/ml; tetracycline, 10 µg/ml; and kanamycin, 50 µg/ml. 5-Bromo-4-chloro-3-indolyl-ß-D-galactopyranoside and isopropyl ß-D-thiogalactoside (40 µg/ml) were added to detect the presence of insert DNA cloned in pUC18-derived plasmids in E. coli. Long-term storage of bacterial strains was at –80°C in the appropriate medium with 25% (wt/vol) glycerol added.


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  		TABLE 1. Bacterial and fungal strains, plasmids and PCR primers used in this study

DNA and cellular manipulations.
Standard methods were used for DNA electrophoresis, preparation of competent E. coli cells, and transformation of E. coli (55). Restriction enzymes were used as specified by the supplier (Roche Diagnostics). DNA fragments were recovered from agarose gels with a QIAquick Gel Extraction kit (QIAGEN). DNA ligations were performed using a Rapid DNA ligation kit (Roche Diagnostics). Pseudomonas total DNA was isolated from 5-ml early stationary phase cultures with a PureGene DNA purification kit (Gentra Systems). Plasmids were isolated using a QIAprep Spin Miniprep kit (QIAGEN). PCRs were performed in a Mastercycler Personal (Eppendorf) instrument.

Bacteriocin and antifungal assays.
Standard bacteriocin plate assays based on deferred antagonism were performed as previously described (46). The effect of UV irradiation on bacteriocin production by strain Pf-5 and recombinant E. coli TOP10F' expressing llpA1Pf-5 and llpA2Pf-5 was examined as in Parret et al. (46). To analyze the effect of mannose (0.01 M and 0.1 M) on the activity of recombinant bacteriocins produced by E. coli, the assay was carried out in sugar-supplemented LB medium. For detection of protein bands with antibacterial activity, a gel overlay assay was carried out (46) with the following modification. Instead of an overnight washing step for renaturation of bacteriocin bands, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels were washed twice for 90 min at 4°C in 50 mM MES (2-morpholinoethanesulfonic acid)-NaOH (pH 6) containing 0.1% (wt/vol) Triton X-100. For detection of antifungal activity, an agar plug containing fungal mycelium was placed in the middle of potato dextrose (BD Biosciences) agar plates in duplicate, and fungal mycelium growth was initiated by incubation for 3 days at room temperature. The exact incubation time depended upon the growth rate of each fungus. Subsequently, a 3-µl drop of an overnight culture of the bacterial strain to be tested was spotted at one-third of the radius of the plates. The plates were further incubated at 25°C. Suppression of fungal growth was evaluated macroscopically after 2 days and again after 4 days. When evaluating antibacterial and antifungal activity of recombinant E. coli TOP10F' or Pseudomonas strains expressing llpA genes, E. coli TOP10F' carrying pUC18 or Pseudomonas sp. harboring pJB3Tc20 were included as a negative control.

Heterologous expression of llpA1Pf-5 and llpA2Pf-5.
The llpA1Pf-5 and llpA2Pf-5 genes were amplified by PCR with Platinum Pfx DNA polymerase (Invitrogen) using 100 ng of P. fluorescens Pf-5 genomic DNA as a template. The primers llpA1Pf-5-5'-BamHI and llpA1Pf-5-3'-SphI were utilized to amplify the llpA1Pf-5 region as a 1,086-bp fragment ranging from position 394669 to 395754 of contig 3337 (7,075,370 bp) (http://www.tigr.org/tdb/ufmg/, version 15 November 2004). For amplification of llpA2Pf-5, primer llpA2Pf-5-5'-BamHI and llpA2Pf-5-3'-SphI were chosen to amplify a 1,236-bp region ranging from position 1343328 to 1344427 of contig 3337. PCR conditions used included 30 cycles of annealing at 61°C (30 s), polymerization at 68°C (1 min), and denaturation at 94°C (1 min). The resulting PCR products were cloned into the pCRII-TOPO vector (TOPO TA cloning Dual Promoter kit; Invitrogen). Amplicons were checked by sequencing, digested with BamHI and SphI, and ligated into the cognate sites of pUC18 under control of the lacZ promoter, resulting in plasmids pCMPG6052 (llpA2Pf-5) and pCMPG6053 (llpA1Pf-5), respectively. Ligation mixes were transformed to TOP10F' cells for expression in E. coli. For heterologous expression in Pseudomonas, the BamHI/SphI inserts from vectors pCMPG6052 and pCMPG6053 were recloned into the cognate sites of broad-host-range vector pJB3Tc20, in sense with the lacZ promoter, giving pCMPG6057 (llpA2Pf-5) and pCMPG6058 (llpA1Pf-5). Similarly, a third construct (pCMPG6060) was prepared consisting of an 897-bp fragment amplified by Pfx PCR using primers llpABW11M1-5'-KpnI and llpABW11M1-3'-BamHI using pCMPG6012 as a template. This fragment, containing llpA from Pseudomonas sp. BW11M1, was cut with KpnI and BamHI, followed by cloning into vector pJB3Tc20. The three resulting bacteriocin constructs were propagated in the nonmethylating E. coli strain GM2163, and plasmid DNA was extracted for electroporation of Pseudomonas. Pseudomonas transformants were then checked for the presence of llpA constructs by miniprep and restriction analysis.

Protein electrophoresis and immunodetection.
E. coli TOP10F' cells harboring llpA constructs were grown to early stationary phase (optical density at 600 nm, 1.4). Proteins were phenol extracted from lyophilized cell-free culture supernatants as described by Parret et al. (46). Protein concentration was assayed using a 2D Quant kit (Amersham Biosciences) using bovine serum albumin (BSA; Sigma-Aldrich) as a standard. Protein fractions were separated on Criterion 12% ready gels (Bio-Rad) at 200 V for 60 min using a Bio-Rad mini-PROTEAN II electrophoresis apparatus. Proteins were visualized with Coomassie blue R-250. SDS-PAGE gels were dried with the DryEase Mini-Gel Drying System (Invitrogen) according to the manufacturer's instructions. Polyclonal rabbit antiserum against LlpABW11M1 was obtained from two New Zealand White rabbits by a standard immunization protocol carried out in the facilities of Eurogentec (Seraing, Belgium) using 800 µg of bacteriocin purified as described previously (47). Upon delivery, rabbit serum was aliquoted and then stored at –20°C. Separated supernatant proteins were transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore) with an LKB Multiphor II blotting unit (Amersham Biosiences), according to the manufacturer's instructions. Western hybridization was carried out as described by Harlow and Lane (21) with minor modifications. Blocking of aspecific sites was carried out by incubation at room temperature for 2 h in Tris-buffered saline (TBS) (20 mM Tris base, 0.5 M NaCl [pH 7.5]) supplemented with 1% (wt/vol) BSA. Subsequently, the membrane was washed three times for 10 min with TBS buffer before incubation (at least 1 h) with primary antibody solution (rabbit antiserum diluted 1:500 in 1% BSA-TBS). Unbound polyclonal antibodies were removed by washing the membrane three times for 5 min in TBS buffer supplemented with 0.1% (wt/vol) Tween. Secondary antibody solution (3% goat anti-rabbit immunoglobulin G [IgG] horseradish peroxidase conjugate [Sigma-Aldrich] in 0.1% Tween-TBS) was then applied, and the membrane was incubated for 1 h. Unbound secondary antibodies were removed by washing the membrane three times for 10 min in 0.1% Tween-TBS. Binding of secondary antibody to the polyvinylidene difluoride membrane was visualized with nitroblue tetrazolium chloride and BCIP (5-bromo-4-chloro-3-indoylphosphate toluidine salt) by immersing the membrane in a diluted solution of 200 µl of nitroblue tetrazolium-BCIP stock solution (Roche Diagnostics) in 10 ml of 0.1 M Tris-HCl (pH 9.5), 0.1 M NaCl. All procedures were carried out at room temperature with gentle agitation, with the exception of the developing step, which was carried out without shaking.

DNA and protein sequence analysis.
Nucleotide sequences were determined by the dideoxy chain terminator method using a BigDye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems) and universal M13 Forward and Reverse primers. Sequencing was carried out on an ABI 3100 Avant Genetic Analyzer (Applied Biosystems). The ClustalW program (62) was used to align DNA and amino acid sequences. The molecular weights and isoelectric points of proteins were estimated with the ProtParam tool available at the ExPASy molecular biology server (http://us.expasy.org/tools/protparam.html). Potential signal peptide cleavage sites were predicted using the SignalP World Wide Web Prediction Server (http://www.cbs.dtu.dk/services/SignalP).

Phylogenetic analysis of LlpA domains.
Searches to compare protein sequences against the nonredundant protein sequence database (version 19 October 2004) at the National Center for Biotechnology Information (NCBI) were accomplished using the protein-BLAST (blastp) and translated-BLAST (tblastn) programs (version 2.2.10) (1). llpA homologues in unfinished genomes were identified using the tblastn program provided at NCBI (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi). Detection of conserved protein domains was performed with the Pfam (Protein family database) search tool (version 15) on the Sanger Center Internet server (http://www.sanger.ac.uk/Software/Pfam). Known or predicted mannose-binding lectin domains (Pfam accession no. PF01453: B_Lectin) of similar protein sequences were truncated to the appropiate length (103 to 125 amino acids). The alignment was further adapted manually according to secondary structure predictions (Jpred2, http://jura.ebi.ac.uk:8888/) using the GeneDoc program (http://www.psc.edu/biomed/genedoc/). The wEMBOSS program clustalnj provided by Belgian EMBnet Node was used to infer the tree topology by phylogenetic distance analysis (Kimura correction) with a neighbor-joining algorithm.

Sequence data for P. fluorescens Pf-5, Mycobacterium smegmatis mc2-155, and R. albus strain 8 were obtained from The Institute for Genomic Research website at http://www.tigr.org (version 15 November 2004). Mycobacterium marinum sequence data were produced by the M. marinum Sequencing Group at the Sanger Institute and were retrieved from ftp://ftp.sanger.ac.uk/pub/pathogens/mm (version 8 Nov. 2004). Sequence data for Ralstonia metallidurans strain CH34 were produced by the U.S. Department of Energy Joint Genome Institute (http://www.jgi.doe.gov/, version 25 November 2003).


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RESULTS
 
The P. fluorescens Pf-5 genome contains two llpA homologues.
To identify possible homologues of the lectin-like bacteriocin LlpA produced by Pseudomonas sp. BW1M1 (LlpABW11M1) in other bacteria, the LlpA protein sequence was queried against the NCBI nonredundant database and against microbial genomes using both the blastp and tblastn programs. The tblastn search revealed two open reading frames in the unannotated genome of the rhizosphere strain P. fluorescens Pf-5 with significant sequence similarity to llpABW11M1. These hypothetical genes, designated llpA1Pf-5 and llpA2Pf-5, were located at positions 394751 to 395590 and 1343588 to 1344427 of the Pf-5 unfinished genome (contig 3337), respectively. Both genes would encode 280-amino-acid proteins with predicted isoelectric points of 8.74 and calculated molecular masses of 31,032 Da (LlpA1Pf-5) and 31,104 Da (LlpA2Pf-5). A SignalP scan did not indicate the presence of possible N-terminal signal peptides. A multiple protein alignment of LlpA1Pf-5, LlpA2Pf-5, and LlpABW11M1 reveals strong homology between LlpA1Pf-5 and LlpA2Pf-5 (264 identical amino acids out of 280) and significant homology of each protein with LlpABW11M1 (46% amino acid sequence identity). Analysis of the genomic contexts of the llpA paralogues of P. fluorescens Pf-5 revealed that llpA1Pf-5 is located about 300 bp upstream of the conserved gene cluster ygdA-recA-recX (on the same DNA strand). The llpA2Pf-5 downstream region contains, on the opposite strand, a gene encoding a hypothetical protein related to P. aeruginosa PA3270 and other acetyltransferases of the GNAT (GEN5-containing N-acetyltransferase) family (Pfam accession no. PF00583). Both llpA genes contain in their upstream region a gene with similarity to the bacteriophage Mu protein Com (22), but these paralogues are located at different intergenic distances (9 bp from llpA1Pf-5 and 233 bp from llpA2Pf-5), suggesting that these genes do not constitute a conserved operon with llpA.

Heterologous expression of llpA1Pf-5 and llpA2Pf-5 confers bacteriocin activity to E. coli and Pseudomonas.
To circumvent possible interference by other antagonistic compounds (including other bacteriocins) produced by P. fluorescens Pf-5, the bacteriocinogenic potential of the two llpA homologues was investigated using heterologous expression. The llpA1Pf-5 and llpA2Pf-5 open reading frames were amplified from P. fluorescens Pf-5 genomic DNA and cloned into pUC18, creating plasmids pCMPG6053 (llpA1Pf-5) and pCMPG6052 (llpA2Pf-5), which were used to transform E. coli TOP10F' cells. To allow expression from its own promoter, an upstream region of 260 bp was included for llpA2Pf-5. The llpA1Pf-5 gene was expressed without the upstream region due to the presence of a predicted phage-like gene immediately upstream of the bacteriocin gene. The resulting E. coli transformants containing pCMPG6052 or pCMPG6053 were tested for growth-inhibitory activity against a set of strains representing all major groups of pseudomonads. Growth inhibition patterns obtained for each E. coli transformant were compared to the bacteriocin phenotype of P. fluorescens strain Pf-5 (Table 2). These bacteriocin activity assays indicated that E. coli TOP10F' cells carrying pCMPG6053 or, to a lesser extent, TOP10F' cells carrying pCMPG6052 were able to inhibit the growth of some pseudomonads by expressing the llpA1Pf-5 and llpA2Pf-5 genes, respectively. No such growth inhibitory effects were observed in case of the control E. coli TOP10F' cells carrying pUC18 (Fig. 1). The apparent difference in expression levels of recombinant llpA1Pf-5 compared to llpA2Pf-5 could result from their vector-driven expression. Alternatively, lower expression of llpA2Pf-5 could be explained by the presence of some negative regulatory elements in its promoter region.


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  		TABLE 2. Antimicrobial activity of P. fluorescens Pf-5 and E. coli TOP10F' carrying pCMPG6053 (llpA1Pf-5) or pCMPG6052 (llpA2Pf-5)



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  		FIG. 1. Growth inhibition of P. fluorescens LMG 1794 by strain Pf-5 and by E. coli TOP10F' harboring pCMPG6052 (llpA2Pf-5) and pCMPG6053 (llpA1Pf-5). E. coli TOP10F' carrying pUC18 was included as a negative control.

Both LlpA bacteriocins can inhibit the growth of P. fluorescens strains LMG 1794 and Pf0-1, as well as Pseudomonas tolaasii LMG 2344, while no inhibitory activity was observed against nonpseudomonad bacteria or toward a selection of fungi. Importantly, the exposure of E. coli cells expressing llpA1Pf-5 or llpA2Pf-5 to UV radiation did not result in larger inhibition halos (results not shown), whereas the production of LlpABW11M1 was previously shown to be induced upon DNA-damaging treatment (46).

To explore possible expression of the llpA genes in other Pseudomonas species, the inserts from pCMPG6052 and pCMPG6053 were transferred to the broad-host-range vector pJB3Tc20. In this way, llpA1Pf-5 and llpA2Pf-5 were successfully expressed in Pseudomonas after electroporation of the constructs pCMPG6058 and pCMPG6057, respectively, into P. aeruginosa strain PA01, P. putida strain KT2440, and P. fluorescens strain OE 28.3.

Screening of 69 strains from an in-house collection of rhizosphere-colonizing fluorescent Pseudomonas isolates identified five additional rhizosphere strains that were inhibited by E. coli and Pseudomonas cells expressing llpA1Pf-5 or llpA2Pf-5. These strains originated from the rhizosphere of wheat (Pseudomonas sp. strains OE 32.1, OE 39.2, and OE 45.2) and maize (Pseudomonas sp. strains OE 50.2 and PGSB 7716). These isolates had been tentatively characterized as belonging to the P. fluorescens species by metabolic profiling (68). Their tentative assignment to the genus Pseudomonas sensu stricto was confirmed by partial 16S rDNA sequencing (data not shown).

In view of the homology of LlpA proteins with MMBL-type mannose-binding lectins, the effect of mannose on bacteriocin activity was analyzed for recombinant LlpA1Pf-5 and LlpA2Pf-5. Inclusion of mannose (up to 0.1 M) did not affect halo formation when E. coli TOP10F' cells carrying pCMPG6053 or pCMPG6052 were assayed against representative indicator strains (P. fluorescens OE 50.2 and LMG 1794). This lack of interference of mannose with activity of MMBL-like bacteriocins is consistent with previous reports in which no binding of mannose could be demonstrated (11, 46).

Expression of two llpA genes broadens the activity range of bacteriocinogenic Pseudomonas strains.
A combinatory approach was performed in order to evaluate the effect of expression of several llpA genes in one Pseudomonas strain. To this end, P. fluorescens Pf-5 cells were electrotransformed with plasmid pCMPG6060 (llpABW11M1). Cells transformed with pJB3Tc20 plasmid DNA were included as a negative control. Bacteriocin plate assays showed that strain Pf-5 carrying pCMPG6060 also inhibited the growth of Pseudomonas sp. GR12-2R3, a strain routinely used as an indicator to detect LlpABW11M1 activity (46). In addition, electrotransformation of Pseudomonas sp. BW11M1 was carried out with pCMPG6057 or pCMPG6058, respectively. The resulting transformants also showed growth inhibitory activity toward previously identified indicator strains for LlpA1Pf-5 and LlpA1Pf-5.

Detection of LlpA1Pf-5 and LlpA2Pf-5 in culture supernatant of E. coli transformants.
Culture supernatants of E. coli TOP10F' cells containing pCMPG6052 and pCMPG6053 were collected and concentrated 25-fold by lyophilization. Coomassie staining of an SDS-PAGE gel (Fig. 2) of phenol-extracted total supernatant proteins from E. coli TOP10F' harboring llpAPf-5 constructs revealed the presence of many bands with no detectable difference compared to the negative control. This is probably due to low-level expression and/or comigration of the bacteriocins with an E. coli protein. We therefore attempted to demonstrate the presence of recombinant LlpA1Pf-5 and LlpA2Pf-5 proteins in culture supernatant by performing a gel overlay assay. An unstained SDS-PAGE gel run with concentrated supernatant proteins of recombinant E. coli was washed and then a lawn seeded with an indicator strain was applied. After overnight incubation, clearing zones indicative of bacteriocin activity were observed at positions corresponding to migration of the expected 31-kDa recombinant proteins (Fig. 2). The clearing zone was most obvious in the case of the protein sample from E. coli TOP10F' expressing llpA1Pf-5. Pure recombinant LlpABW11M1 protein (47) was used to generate polyclonal antibodies in New Zealand White rabbits. The cross-reaction of the anti-LlpABW11M1 antibodies was tested against concentrated supernatant proteins obtained from overnight cultures of E. coli TOP10F' carrying pCMPG6053 (llpA1Pf-5) or pCMPG6052 (llpA2Pf-5). Immunoblotting revealed a single band of the expected size (31 kDa) per supernatant culture, while no band was visible in the control supernatant (fraction of E. coli TOP10F' carrying pUC18) (Fig. 3). The strongest immunoreactive band was observed for E. coli TOP10F' expressing llpA1Pf-5.



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  		FIG. 2. Separation of E. coli supernatant proteins by SDS-PAGE (left panel) and gel overlay bacteriocin assay using P. fluorescens LMG 1794 as an indicator strain embedded in 0.5% tryptic soy agar (right panel). Total E. coli protein amounts were 20 µg (SDS-PAGE) and 100 µg (gel overlay). Lane 1, prestained low-range SDS PAGE protein markers; lane 2, E. coli TOP10F' carrying pUC18; lane 3, E. coli TOP10F' carrying pCMPG6052 and expressing llpA2Pf-5; lane 4, E. coli TOP10F' carrying pCMPG6053 and expressing llpA1Pf-5. The arrowheads indicate active bacteriocin bands. Due to the reproduction of the image, the vague inhibition zone (indicated by the dotted ellipse) generated by LlpA2Pf-5 is poorly visible.



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  		FIG. 3. Immunodetection of recombinant LlpA homologues in culture supernatant from E. coli. Lane 1, MagicMark protein marker; lane 2, 10 ng of pure LlpABW11M1His6; lane 3, 5 µg of E. coli TOP10F' carrying pUC18; lane 4, 5 µg of E. coli TOP10F' carrying pCMPG6052 and expressing llpA2Pf-5; lane 5, 5 µg of E. coli TOP10F' carrying pCMPG6053 and expressing llpA1Pf-5. Proteins were detected using polyclonal antibodies raised against His6-tagged LlpABW11M1.

Tandem domain structure of LlpA1Pf-5 and LlpA2Pf-5 bacteriocins.
A Pfam search for conserved protein domains showed that LlpA1Pf-5 and LlpA2Pf-5 possess a domain structure consisting of two tandemly arranged MMBL domains linked by 10 amino acids, namely MMBL_1 ranging from amino acids 13 to 139 and MMBL_2 ranging from amino acids 147 to 255. MMBL_1 and MMBL_2 within each protein are quite dissimilar (16% identity and 28% similarity). A multiple protein alignment of each of the MMBL domains of LlpA1Pf-5 and LlpA2Pf-5 with MMBL domains of the well-characterized mannose-binding garlic lectin ASAI (66) reveals the presence of two perfect or close matches to the characteristic mannose-binding motif Q-X-D-X-N-X-V-X-Y in each MMBL domain of the Pf-5 bacteriocins (Fig. 4). The equivalent of the third motif (in MMBL_2) and in particular the counterpart of the second motif (in MMBL_1) are poorly conserved in LlpA1Pf-5 and LlpA2Pf-5. However, three highly conserved Trp (W) residues were detected in each MMBL domain. These hydrophobic residues are situated at the center of each of the monocot MMBL subunits, close to the threefold axis, and form a stabilizing hydrophobic core (10). The C-terminal MMBL domains of LlpA1Pf-5 and LlpA2Pf-5 display 30% amino acid identity to the N-terminal MMBL domain of albusin B (AlbB) (Fig. 4). The 290-amino-acid putative homologue of AlbB (AlbB2) was predicted from unfinished genome sequence data of R. albus strain 8 (contig 3858, retrieved from TIGR).



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  		FIG. 4. Multiple alignment of LlpABW11M1 (LlpA), LlpA1Pf-5 (LlpA1), LlpA2Pf-5 (LlpA2), the N-terminal region (amino acids 50 to 200) of albusin B from R. albus strain 8 (AlbB_N), and its predicted homologue in R. albus strain 7 (AlbB2_N, amino acids 50 to 199) with mature ASAI, a mannose-specific lectin found in bulbs of garlic (Allium sativum). The signal peptides of albusin B (49 amino acids), as well as the predicted signal peptide of AlbB2 (49 amino acids), are not included in the alignment. Gaps used to optimize the alignment are indicated by dashes. Predicted MMBL domains are enclosed in boxes. Amino acids involved in mannose binding in ASAI are indicated by filled circles when present in LlpA1Pf-5 and LlpA2Pf-5 or by open circles when not conserved in LlpA1Pf-5 and LlpA2Pf-5. The labeled tryptophan residues (asterisks) are part of a stabilizing hydrophobic core structure and are conserved in nearly all homologous proteins. Cysteine residues typical for plant MMBL domains are also marked (inverted open triangles).

MMBL domains are found in diverse bacterial proteins.
Following the identification of LlpABW11M1 comprised of two MMBL domains, we described a similar tandem arrangement of MMBL domains in other bacterial MMBL proteins, namely, in the hypothetical protein XAC0868 of Xanthomonas axonopodis pv. citri and in a hypothetical protein from R. metallidurans (46). With the advent of more and more genome sequences, novel hypothetical bacterial MMBL-containing proteins emerge. The genome of Chromobacterium violaceum encodes a 113-amino-acid putative protein (protein CV3134 with an N-terminal extension of 34 amino acids) comprising a single MMBL domain. Additional MMBL domains were detected in the 212-amino-acid protein NFA49840 from Nocardia farcinica and in a putative 207-amino-acid protein from M. marinum. In both proteins, the MMBL domain is located at the N terminus and is connected to a putative LysM domain by a proline-rich linker sequence. A similar domain organization was described for a putative protein from another actinomycete M. smegmatis (46). The LysM domain is one of the most common modules in bacterial cell surface proteins. It occurs most often in cell wall-degrading enzymes, where it probably acts to anchor the catalytic domains to their substrates (4). Additional MMBL domains were identified in putative proteins from environmental isolates from the Sargasso Sea (67). One of these protein sequences is truncated at the N terminus. Both putative proteins are composed of two C-terminally located tandemly organized MMBL domains. In the nontruncated protein, a Pfam search identified a peptidase domain as found in a large family of serine proteases (subtilisins). In S. coelicolor SCO3053, an MMBL domain is also fused to a protease domain (trypsin).

Phylogenetic analysis of MMBL domains.
To further explore the apparent similarity of the Pf-5 LlpA-like bacteriocins with the superfamily of monocot mannose-binding lectins and other diverse proteins containing MMBL domains, we performed a molecular phylogenetic analysis of its MMBL domains. In addition to known and predicted MMBL domains from green plants and bacteria, we enclosed representative eukaryotic sequences from proteins that were identified from BLAST searches. Of particular interest are the pufflectins isolated from skin and intestinal mucus of the Japanese pufferfish Takifugu rubripes (63). Pufflectins are homodimers composed of two noncovalently associated subunits of 13 kDa. Each of these subunits contains one MMBL domain. Tsuitsui and coworkers demonstrated mannose-sensitive pufflectin binding to the surface of the parasitic trematode Heterobothrium okamotoi, suggesting a role of pufflectin in host defense mechanisms (63). The genome of another pufferfish, Tetraodon nigroviridis, encodes a 116-amino-acid pufflectin homologue (32). MMBL domains were also detected in fungal proteins, namely, in hypothetical proteins from Aspergillus oryzae and Gibberella zeae.

From a multiple alignment of MMBL_1 and MMBL_2 of each LlpA from Pf-5 with known or predicted MMBL domains, an unrooted phylogenetic tree was inferred by neighbor-joining distance analysis (Fig. 5). Four prominent clusters are indicated encompassing MMBL domains found in green plants, animal proteins, fungi, and hypothetical proteins from actinomycetes. MMBL domains of bacteriocins are found in two divergent clusters. The N-terminal MMBL domains from the LlpA bacteriocins are clustered with the N-terminal MMBL domain of a hypothetical bacteriocin from X. axonopodis pv. citri. On the other hand, C-terminal MMBL domains from Pseudomonas LlpA-type bacteriocins cluster with the C-terminal MMBL domains of the hypothetical bacteriocin from X. axonopodis pv. citri and the single N-terminal MMBL domain of bacteriocin AlbB from R. albus. It should be noted that tandem MMBL domains detected in multidomain proteins, such as the hypothetical proteins from R. metallidurans and environmental isolates from the Sargasso Sea, are much less divergent in comparison to tandem MMBL domains from bacteriocins.



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  		FIG. 5. Molecular phylogeny of MMBL domains and domain structures of bacterial and selected eukaryotic proteins containing one or two MMBL domains. An unrooted neighbor-joining distance tree was constructed from a multiple alignment of amino acid sequences of known and inferred MMBL domains found in eukaryotic and bacterial proteins. The extensions "_1" and "_2" refer to tandem MMBL domains present in certain polypeptides. MMBL domains present in proteins containing another domain are indicated with an asterisk. The scale bar represents 0.2 substitutions per site. A selection of MMBL domains from lectins found in monocot plants belonging to 11 different families is included. Monocot lectins with a two-domain precursor are exemplified by lectins from Allium sativum (Allsat; GenBank accession no. S23495), Crocus vernus (Crover; P49329) and Arum maculatum (Arumac; AAC48997). Lectins from Hyacinthoides hispanica (Hyahis; AAD16403), Galanthus nivalis (Galniv; AAA33345), Ananas comosus (Anacom; AAM28277), Tulipa sp. (Tulipa; AAC49386), Dioscorea polystachya (Diopol; BAD67183), Aloe arborescens (Aloarb; P49329) and Polygonatum multiflorum (Polmul; AAC49413) represent proteins containing a single MMBL domain. Gastrodianin, an antifungal protein from Gastrodia elata with mannose-binding capacity (Gasela; AAG52664) and the actin-binding mannose-specific protein from Dictyostelium discoideum (Dicdis; Q03380) are also included. Liverwort (Marchantia polymorpha) contains two MMBL-like proteins, Marpola-1 (Marpol1; AU08173) and Marpola-3 (Marpol3; C95861). MMBL domains were also detected in the MleA lectin of Aspergillus oryzae (Aspory; MleA lectin BAC56173) and in hypothetical proteins FG03689.1 (Gibzea1; EAA71502) and FG03828.1 (Gibzea2; EAA71530) from Gibberella zeae. Fish proteins containing MMBL domains are T. rubripes pufflectins (Takrub1 from intestinal mucus, BAC57044; Takrub2 from skin mucus, BAC57043) and a hypothetical protein from T. nigroviridis (Tetnig; CAG10253). Bacterial proteins containing MMBL domains are LlpA1 and LlpA2 from P. fluorescens Pf-5, LlpA from Pseudomonas sp. BW11M1 (AAM95702), R. albus strain 7 bacterial inhibitor (Rumalb; AAL73547), a protein from C. violaceum (Chrvio; a truncated version of this protein, without the first 34 residues, is referred to in GenBank as a bulb-type mannose-specific lectin [AAQ60810]), X. axonopodis pv. citri hypothetical protein XAC0868 (Xanaxo; NP_641220), hypothetical protein NFA49840 from N. farcinica (Nocfar; YP_121200), the precursor of a putative antifungal protein from M. smegmatis strain mc2155 (Mycsme; GSMORF3668, retrieved from TIGR), putative secreted hydrolase SCO3053 from S. coelicolor (Strcoe; NP_627274), and two putative proteins encoded by environmental DNA samples isolated from the Sargasso Sea (Sargas1, EAK32154; Sargas2, EAD21447). Additional proteins with MMBL domains homologous to the LlpA MMBL domains were predicted from unfinished genome sequences of R. albus strain 8 (Rumalb2; contig 3859, positions 5864 to 4848; retrieved from TIGR), M. marinum (Mycmar, contig mar813f05.p2k1095, positions 653229 to 653849; retrieved from Sanger) and R. metallidurans strain CH34 (Ralmet, revised contig 67, gene 2519; retrieved from JGI; a truncated version of this protein, without the first 244 residues, is found in GenBank [ZP_00022141]).


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DISCUSSION
 
The results described here show that, in addition to an arsenal of antifungal biosynthetic genes, the rhizosphere bacterium P. fluorescens Pf-5 is also equipped with at least two functional antibacterial genes. Expression of each of these genes in E. coli and Pseudomonas results in the production of 31-kDa bacteriocins that are released in the growth medium. We named these bacteriocins LlpA1Pf-5 and LlpA2Pf-5 based on their similarity (chromosomally located structural gene, size, and domain structure) to LlpABW11M1, a plant lectin-like bacteriocin previously identified in rhizosphere isolate Pseudomonas sp. BW11M1 (46). Although displaying differences in activity spectrum, their killing activity is limited to fluorescent Pseudomonas strains. Finally, these LlpA-type bacteriocins can be produced in heterologous hosts (E. coli and other Pseudomonas species) without the need of a signal peptide for secretion or a specific immunity protein, in contrast to the "classical" bacteriocins of gram-negative bacteria such as pyocins (P. aeruginosa) and colicins (Enterobacteriaceae). It is very likely that these LlpA-type bacteriocins share a common mode of action that differs from those of previously characterized bacteriocins. We propose that LlpA-type bacteriocins interact with sensitive bacterial cells by binding to a specific glycosylated outer membrane component, possibly lipopolysaccharide, resulting in cell death through a yet unknown mechanism.

Similar to LlpABW11M1, LlpA1Pf-5 and LlpA2Pf-5 are composed of two MMBL-like domains connected by a short linker sequence. These domains are conserved among mannose-specific lectins of monocots (57, 65). The in silico comparative analysis presented in this work, together with other data (11, 61), clearly establishes that MMBL domains have a much wider distribution than previously assumed. In this study, we identified MMBL domains not only in various (putative) proteins encoded by fungi and fish genomes but also in phylogenetically unrelated bacteria such as C. violaceum and several actinomycetes. In some cases, these bacterial MMBL domains were found to be linked to other protein domains with predicted bacterial cell wall-degrading or protease activity. However, the XAC0868 hypothetical protein from X. axonopodis pv. citri displays an identical tandem domain structure as the LlpA-type bacteriocins of Pseudomonas sp. BW11M1 and P. fluorescens Pf-5. Moreover, its domains constitute monophyletic groups with the cognate MMBL domains of the LlpA-type bacteriocins. This strongly suggests that this Xanthomonas protein is also an LlpA-type bacteriocin.

From the MMBL domain tree topology it is not obvious whether N-terminal and C-terminal MMBL domains in LlpA bacteriocins have evolved from a common ancestral protein by speciation or whether these MMBL domains have evolved toward specialized functions following a gene duplication event. Conceivably, one domain could be involved in target binding, while the other might be responsible for the actual killing. As monocot mannose-binding lectins are the sole MMBL-containing proteins that have been crystallized and analyzed by X-ray diffraction (3, 25, 38, 70), we expect that ongoing studies to resolve the three-dimensional structure of recombinant LlpABW11M1 crystals will ultimately provide more insight into the specific role of N- and C-terminal MMBL domains in LlpA-type bacteriocin activity (47).

Biological control of soilborne crop diseases by the introduction of beneficial microorganisms into the rhizosphere has been proposed as an environmentally friendly alternative to the use of synthetic chemicals. Certain plant-associated bacteria, particularly fluorescent Pseudomonas spp., have been exploited for suppression of crop diseases by direct antagonism between the bacteria and soilborne pathogens (7, 49), by iron depletion through the production of siderophores (13), or by the induction of systemic resistance (29, 60). Exploiting fluorescent pseudomonads to improve crop yield has tremendous potential, but field trials yield inconsistent results. Introduction of these bacteria in the field often fails because the organisms are not able to recolonize the roots, or they colonize the roots but do not produce biocontrol metabolites in the new niche. This inconsistent biocontrol activity could be due, in part, to bacteriocins produced by endogenous pseudomonads active against the introduced strain. We have previously explored the potential of plant-associated P. fluorescens and P. putida to produce a large variety of pyocin S-like bacteriocins with nuclease activity (44, 45). While a similar in silico approach suggests that P. fluorescens Pf-5 does not produce S pyocins (data not shown), the current study hints that this strain does produce LlpA-like bacteriocins in the rhizosphere. Upon investigation of the antibacterial spectrum of LlpA1Pf-5 and LlpA2Pf-5 toward several fluorescent pseudomonads, no significant qualitative differences could be observed. However, this does not exclude the possibility that these bacteriocins act on different Pseudomonas strains in the rhizosphere. Alternatively, their expression may be triggered by different environmental signals. Various stress response pathways influence the expression of LlpA in Pseudomonas sp. BW11M1 (17). Bacteriocin production by biocontrol strains may contribute to their rhizosphere competence (64). If so, recombinant biocontrol strains equipped with (several) bacteriocin genes may perform better as plant growth-promoting inocula by outcompeting rhizosphere-inhabiting pseudomonads.


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ACKNOWLEDGMENTS
 
A.P. is the recipient of a postdoctoral fellowship (PDM 03/197) from the Research Council of the Katholieke Universiteit Leuven. This work was supported by a grant (Onderzoeksproject G.0303.04 to R.D.M.) from the Fund for Scientific Research (F.W.O.-Vlaanderen).

The authors thank Joyce Loper (USDA-ARS-Horticultural Crops Research Unit, Corvallis, Oreg.) for providing a culture of P. fluorescens Pf-5.


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FOOTNOTES
 
* Corresponding author. Mailing address: Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Heverlee, Belgium. Phone: 32 16 329681. Fax: 32 16 321966. E-mail: rene.demot{at}biw.kuleuven.be. Back


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Applied and Environmental Microbiology, September 2005, p. 5197-5207, Vol. 71, No. 9
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.9.5197-5207.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.




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