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Plant lectin-like antibacterial proteins from phytopathogens Pseudomonas syringae and
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Xanthomonas citri
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Maarten G. K. Ghequire1, Wen Li1, Paul Proost2, Remy Loris3,4 and René De Mot1*
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Heverlee, Belgium
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3000 Leuven, Belgium
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Biotechnologie, 1050 Brussel, Belgium
Centre of Microbial and Plant Genetics, KU Leuven, Kasteelpark Arenberg 20, 3001
Laboratory of Molecular Immunology, Rega Institute for Medical Research, KU Leuven,
Molecular Recognition Unit, Department of Structural Biology, Vlaams Instituut voor
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1050 Brussel, Belgium
Structural Biology Brussels, Department of Biotechnology (DBIT), Vrije Universiteit Brussel,
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*For correspondence. E-mail rene.demot@biw.kuleuven.be; Tel. +32 (0)16 329681, Fax: +32
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(0)16 321963
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Running title: P. syringae and X. citri lectin-like bacteriocins
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Keywords: bacteriocin, MMBL lectin, LlpA, antibacterial activity
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Abstract
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The genomes of Pseudomonas syringae pv. syringae 642 and Xanthomonas citri pv.
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malvacearum LMG 761 each carry a putative homologue of the plant lectin-like bacteriocin
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(llpA) genes previously identified in the rhizosphere isolate Pseudomonas putida BW11M1
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and the biocontrol strain Pseudomonas fluorescens Pf-5. The respective purified
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recombinant proteins, LlpAPss642 and LlpAXcm761, display genus-specific antibacterial activity
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across species boundaries. The inhibitory spectrum of the P. syringae bacteriocin overlaps
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partially with those of the P. putida and P. fluorescens LlpAs. Notably, Xanthomonas
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axonopodis pv. citri str. 306 secretes a protein identical to LlpAXcm761. The functional
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characterization of LlpA proteins from two different phytopathogenic γ-proteobacterial
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species expands the lectin-like bacteriocin family beyond the Pseudomonas genus and
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suggests its involvement in competition among closely related plant-associated bacteria
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with different lifestyles.
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Introduction
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The ability for bacteria to produce antagonistic compounds targeting competing micro-
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organisms is an important asset for colonization of nutrient-proficient environments. In such
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highly competitive habitats the secretion of antagonistic molecules can provide the
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producing bacterium with a significant eco-evolutionary advantage. Characterization of
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antimicrobial compounds in bacteria has revealed an enormous diversity of the molecular
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armamentarium. Traditionally, these molecules are divided in two major classes: secondary
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metabolites (antibiotics) with broad-spectrum activity and ribosomally-synthesized peptides
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or proteins (bacteriocins) with narrow killing spectrum (Riley, 1998; Hibbing et al., 2010).
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Bacteriocins vary in structural complexity from small peptides to large multisubunit
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complexes acting as molecular machines (Ennahar et al., 2000; Michel-Briand and Baysse,
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2002; Papagianni, 2003). Their compound-specific mode of action ranges from, for example,
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membrane pore formation to inhibition of cell wall biosynthesis or degradation of nucleic
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acids.
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Among Gram-negative bacteria, next to colicins produced by E. coli strains, pyocins
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produced by Pseudomonas aeruginosa have been extensively studied (reviewed by Michel-
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Briand and Baysse, 2002). S-type pyocins are multi-domain toxins with a colicin-like modular
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structure (Denayer et al., 2007; Ling et al., 2010). Also colicin M-like bacteriocins have been
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identified in P. aeruginosa (Barreteau et al., 2009). The F- and R-type pyocins consist of
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multi-subunit particles resembling bacteriophage tails (Nakayama et al., 2000; Williams et
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al., 2008). Several reports have documented the ecological significance of pyocins for
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competitive interactions among P. aeruginosa strains (Inglis et al., 2009; Waite and Curtis,
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2009; Bakkal et al., 2010).
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An unusual type of Pseudomonas bacteriocin was found in the rhizosphere isolate
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Pseudomonas putida BW11M1 (LlpABW11M1; Parret et al., 2003) and biocontrol strain
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Pseudomonas fluorescens Pf-5 (LlpA1Pf-5 and LlpA2Pf-5; Parret et al., 2005). These proteins
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consist of a tandem of monocot mannose-binding lectin (MMBL) domains, sharing similarity
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with certain plant lectins. Both domains of the antibacterial lectins seem to have evolved
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independently but no specific function has yet been assigned to the individual domains. The
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phylogenetically unrelated Gram-positive species Ruminococcus albus produces albusin B, a
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bacteriocin with a single MMBL domain in addition to a second domain of unknown function
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(Chen et al., 2004). In recent years, several genes encoding hypothetical lectins with one or
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two MMBL domain(s), in some cases fused to additional unknown domains, have emerged
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from bacterial genomic sequencing projects.
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In this study we report on the functional characterization of two novel tandem-MMBL
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bacteriocins in plant-associated -proteobacteria with a different lifestyle, namely pathovars
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of Pseudomonas syringae and Xanthomonas citri. This reveals a wider distribution of the
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LlpA family proteins, now including bacteriocins from strains belonging to two major groups
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of plant pathogens (Ryan et al., 2011; Silby et al., 2011).
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Results and discussion
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New candidate lectin-like bacteriocins
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Several hypothetical bacterial proteins with a tandem MMBL structure were identified by
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BlastP homology searches (http://blast.ncbi.nlm.nih.gov/Blast.cgi) with the known
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Pseudomonas LlpA sequences as amino acid queries. Hits were found for predicted gene
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products of P. syringae (two pathovars) as well as for several strains of non-pseudomonads:
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Xanthomonas axonopodis (-Proteobacteria), Burkholderia cenocepacia and Burkholderia
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ambifaria (-Proteobacteria), and Arthrobacter sp. (Actinobacteria). A phylogenetic tree was
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constructed to compare these hypothetical proteins with the three previously characterized
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LlpAs (Fig. 1). The protein from P. syringae pv. aptata str. DSM 50252 (Baltrus et al., 2011) is
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most similar to LlpABW11M1 (66% amino acid sequence identity), while the one from P.
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syringae pv. syringae 642 (Clarke et al., 2010) exhibits stronger divergence (< 50% identity to
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the known LlpAs). The lowest similarity is apparent for the Burkholderia proteins.
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Intermediate positions between the Pseudomonas and Burkholderia clusters are taken by
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the Arthrobacter and Xanthomonas sequences. The latter gene product (XAC0868) was
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fortuitously identified among extracellular proteins by Yamazaki et al. (2008) when studying
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HrpG-regulated protein secretion by Xanthomonas axonopodis pv. citri str. 306, but it was
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not further characterized. Using PCR primers based on the XAC0868-encoding gene
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(Supplementary Table S1) we identified an identical copy in X. axonopodis pv. malvacearum
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LMG 761. According to Schaad et al. (2006), these strains represent two distinct pathovars
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of Xanthomonas citri, of which only pv. citri causes bacterial cancer of citrus. The presence
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of a highly conserved xac0868 gene in both pathovars reflects their close phylogenetic
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relatedness, as apparent from multilocus sequence analysis (Young et al., 2008). No
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amplicon was detected for X. alfalfae subsp. alfalfae LMG 497 (previously X. axonopodis
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subsp. alfalfae) and X. axonopodis pv. manihotis LMG 784 (data not shown). It was decided
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to undertake functional characterization of the presumptive LlpA-like bacteriocin from
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Xanthomonas citri pv. malvacearum LMG 761 (LlpAXcm761), along with the putative one from
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P. syringae pv. syringae 642 (LlpAPss642).
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Expression of recombinant tandem MMBL lectins
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Sequence-verified PCR amplicons of llpAPss642 and llpAXcm761 were ligated in pET28a as
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described by Parret et al. (2004). Fusions were created to encode N-terminal His6-tags, since
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difficulties were encountered in obtaining active recombinant LlpA proteins with a C-
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terminal His-tag (unpublished data). Unlike the Pseudomonas LlpAs, the predicted LlpAXcm761
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precursor is preceded by a cleavable type-II secretion (T2SS) recognition sequence (SignalP
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analysis; http://www.cbs.dtu.dk/services/SignalP/). Consequently, two different constructs
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were made: one retaining the Xanthomonas T2SS signal sequence and one devoid of it. In
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the latter case the polyhistidine tag fusion was immediately adjacent to the expected start
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of the mature LlpAXcm761 protein. Induction of expression, cell harvest and disruption, and
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extraction of the recombinant proteins was performed as described previously (Parret et al.,
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2004).
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Analysis of induced cell lysates by SDS-PAGE revealed the expected size (33 kDa observed
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versus 33,795 Da predicted) for recombinant His6-tagged LlpAPss642 (Fig. 2). The presence of
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the histidine-tag, with removal of the N-terminal methionine, was confirmed by N-terminal
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amino acid sequencing (according to Parret et al., 2003) and immunodetection by Western
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blot using polyhistidine-specific antibodies (Roche Diagnostics) (data not shown).
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For LlpAXcm761, no recombinant protein expression was observed with the construct lacking
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the cognate T2SS signal sequence, while the one retaining this sequence yielded a protein
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band (23 kDa) significantly smaller than expected for the His6-tagged product ( 30,244 Da)
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(Fig. 2). This protein was however not detected with the anti-polyhistidine antibodies. In line
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with this, N-terminal sequencing yielded QMLRANFPGQ, corresponding to the N-terminal
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sequence of the mature form of LlpAXcm761 following removal of the T2SS signal sequence
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and the preceding His6-tag. The same proteolytic processing was observed by Chen et al.
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(2004) for the LlpAXcm761 counterpart, protein XAC0868, upon secretion by X. axonopodis pv.
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citri, indicating the unexpected heterologous expression of the native form of LlpAXcm761.
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Purification of recombinant proteins
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Purification of recombinant His6-tagged LlpAPss642 was performed on Ni-NTA agarose by
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HPLC, using an Äkta Purifier (GE Healthcare Life Sciences, Amersham Bioscience) with a 5 ml
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Histrap column (GE Healthcare Life Sciences) using buffers, washing steps and elution
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conditions described previously (Parret et al., 2004). Due to the lack of the His6-tag in
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recombinant LlpAXcm761, it was purified by cation ion exchange chromatography (given the
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high predicted pI of mature LlpAXcm761: 8.66) instead of affinity chromatography, using a SP
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Sepharose HP column (XK 16/20, GE Healthcare Life Sciences) in combination with an Äkta
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Purifier. At pH 6.0 with malonic acid (50 mM) only contaminating proteins from the raw
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lysate were retained on the column. A second purification step with adsorption at pH 4.8
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(50 mM acetic acid) and salt gradient elution (flow rate 2 ml/min; 0 to 1 M NaCl in 100 min)
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resulted in elution of LlpAXcm761 without contaminating proteins at a NaCl concentration of
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approx. 300 mM.
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Both recombinant LlpAPss642 and LlpAXcm761 were dialyzed against bis-tris propane buffer (20
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mM, NaCl 200 mM, pH 7.0). As the N-terminal polyhistidine tag does not interfere with LlpA
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activity (Parret et al., 2004), it was not removed from LlpAPss642. The concentration of the
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purified proteins was determined by UV absorbance measurement (A280), using molar
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extinction coefficients of 60,850 M-1 cm-1 for His6-LlpAPss642 and 38,055 M-1 cm-1 for
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LlpAXcm761, calculated according to Pace et al. (1995).
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Bacteriocin activity of novel LlpAs
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Potential bacteriocin activity of His6-LlpAPss642 and LlpAXcm761 was assayed by applying 10 µl
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spots of pure protein (conc. 1-2 mg/ml) on TSB agar plates, overlaid with 5 ml soft agar
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(0.5%) seeded with 200 µl of an overnight culture (108 CFU/ml) of the respective indicator
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strains. Plates were incubated overnight at 30°C, except for P. aeruginosa (37°C). The
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following day, inhibitory activity was observed as a halo in a confluent lawn of bacterial cells
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(Fig. 3). Dialysis buffer was taken as negative control. The test panel consisted of
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representative Pseudomonas and Xanthomonas culture collections strains and fluorescent
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Pseudomonas rhizosphere isolates from our lab collection. In parallel, the sensitivity of
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these strains to purified LlpABW11M1 (Parret et al., 2003) and LlpA1Pf-5 (Parret et al., 2005) was
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determined. The results are summarized in Table 1 and Table S2.
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For LlpAPss642, a sensitive P. syringae, P. fluorescens and P. resinovorans strain, along with
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two partially inhibited strains, were identified. The sensitive P. syringae strain (rifampicin-
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resistant derivative of GR12-2) was until recently described as a P. putida strain but now
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reclassified by Blakney and Patten (2011). The LlpAPss642 activity indicates that LlpA-mediated
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killing is not restricted to strains of the same species, as observed before for LlpABW11M1
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(Parret et al., 2003) and LlpA1Pf-5 and LlpA2Pf-5 (Parret et al., 2005). The LlpAPss642 target
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strains are also sensitive to at least one other antibacterial lectin described before (Parret et
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al., 2003; Parret et al., 2005). Only a limited number of LlpAPss642-sensitive strains (about 4%
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of tested Pseudomonas strains) were identified, compared to the much more frequently
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found LlpABW11M1-inhibited strains (about 42%) and LlpA1Pf-5-inhibited strains (about 12%).
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This is probably due to the biased species distribution in the test panel that is dominated by
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environmental isolates related to P. putida and P. fluorescens while only few strains of the
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producer’s taxonomic group (P. syringae) were included.
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Growth of none of the Xanthomonas strains tested was affected by any of the Pseudomonas
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LlpAs. However, several of these strains (6 out of 16) were sensitive to recombinant
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LlpAXcm761. These target strains represent six different Xanthomonas species, including a
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pathovar of X. axonopodis. Much like the antagonism among Pseudomonas, LlpA-mediated
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inhibition is not confined to Xanthomonas strains belonging to the same species as the
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bacteriocin producer. In line with the intragenus-specific killing of the Pseudomonas LlpAs,
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none of the tested Pseudomonas strains was inhibited by LlpAXcm761.
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Concluding remarks
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Although bacteriocin production is also common among plant-associated bacteria, relatively
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few bacteriocins have been characterized at the molecular level (reviewed by De Mot,
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2007). Bacteriocins LlpAPss642 and LlpAXcm761 identified in this work are unrelated to other
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bacteriocins produced by P. syringae strains (such as phage-like syringacins and colicin M-
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like bacteriocins: Smidt and Vidaver, 1986; Lavermicocca et al., 2002; Barreteau et al., 2009;
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Sisto et al., 2010) or by Xanthomonas species (glycinecin A: Heu et al., 2001; Pham et al.,
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2004).
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Present results assigning a role as antibacterial molecules to the tandem-MMBL lectin-like
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proteins in the γ-Proteobacteria P. syringae and X. citri, add to the diversity of the LlpA
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family with respect to producers and targets. In line with previous results for LlpA proteins
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from P. putida and P. fluorescens strains, the antagonism is strain-specific and difficult to
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predict but appears to be confined to the cognate genus, albeit not limited by species
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boundaries. The Pseudomonas LlpA proteins share some target strains but display distinct
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inhibitory spectra. It will be of interest to characterize additional, more divergent LlpA family
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members, like the proteins from Burkholderia (β-Proteobacteria) and Arthrobacter
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(Actinobacteria) to verify whether the intra-genus but species border-crossing activity
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applies more widely. In addition, this information may provide more insight in the
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evolutionary origin of these antibacterial proteins and their relatedness to the MMBL-type
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lectins broadly distributed in monocot plants. Conceivably, the ancestral antibacterial
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activity may have evolved following acquisition of such eukaryotic gene by a plant-
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colonizing, possibly phytopathogenic bacterium.
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Our current research aims at elucidation of the role played by the respective MMBL
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domains in overall structure and toxic activity and at identification of target specificity
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determinants. Of additional interest is the apparent difference in secretion pathways that is
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inferred from the presence of a T2SS signal sequence in some LlpAs (Xanthomonas,
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Burkholderia) but not in others (Pseudomonas, Arthrobacter). For the latter, alternative
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export routes may be involved. Recently, delivery of antibacterial effector molecules by the
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type-VI secretion machinery has been reported for several bacteria including P. aeruginosa
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(Russell et al., 2011). In X. citri pv. citri str. 308 (previously X. axonopodis pv. citri), secretion
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of the counterpart of LlpAXcm761 (XAC0868) is apparently coordinated with HrpG-dependent
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type III secretion, which plays a crucial role in infection and pathogenicity (Yamazaki et al.,
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2008). This suggests that the antibacterial activity of this protein may be important for
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competition with other Xanthomonas in the host plant environment prior to or during
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establishment of the infection.
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Acknowledgements
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This work was supported by grant G.0393.09N from FWO-Vlaanderen (to R.D.M. and R.L.).
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The authors wish to thank B. Vinatzer (Department of Plant Pathology, Virginia Tech, USA)
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for supplying genomic DNA of Pseudomonas syringae pv. syringae 642, M. Höfte and J. Xu
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(Laboratory of Phytopathology, Ghent University, Belgium) for assisting in antagonistic
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testing with different Xanthomonas oryzae pv. oryzae strains, J. Desair (CMPG, KU Leuven)
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for assisting in purification of the recombinant Xanthomonas lectin, and the Interfaculty
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Centre for Proteomics and Metabolomics (ProMeta, KU Leuven) for MALDI-TOF analysis.
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Table legends
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Table 1
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Antibacterial activity of purified recombinant LlpABW11M1, LlpA1Pf-5, LlpAPss642 and LlpAXcm761.
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Protein samples were spotted on a TSB agar plate overlaid with soft agar seeded with an
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indicator bacterium. In total, 118 Pseudomonas and 16 Xanthomonas strains were tested for
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growth inhibition. Only selected Pseudomonas strains are listed. The sensitivity patterns of
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additional Pseudomonas strains are compiled in Table S2.
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Figure legends
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Figure 1
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Phylogenetic analysis of LlpA-like proteins with tandem MMBL domains. PhyML (JTT matrix;
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Guindon and Gascuel, 2003) implemented in Geneious Pro 5.4 (Drummond et al., 2011) was
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used to construct an unrooted maximum-likelihood tree from a multiple amino acid
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alignment (Supplementary Figure S1) of LlpA homologues from P. putida BW11M1 (Parret et
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al., 2003), P. fluorescens Pf-5 (LlpA1 = PFL_1229, LlpA2 = PFL_2127; Parret et al., 2005) and
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P. syringae pv. syringae 642 (LlpA Pss642; Psyrps6_010100009383, ZP_07263221.1) with a
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Xanthomonas axonopodis pv. citri extracellular protein of unknown function (XAC0868,
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NP_641220.1; Yamazaki et al., 2008) and hypothetical proteins from Arthrobacter sp. FB24
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(Arth_4524, YP_829274.1), Burkholderia ambifaria MEX-5 (Bamb_0926, ZP_02905572.1),
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Burkholderia cenocepacia AU 1054 (Bcen_1091, ABF75998.1; Bcen_1092, ABF75999.1), and
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P. syringae pv. aptata DSM 50252 (PSYAP_13445, EGH77666.1). Orthologues (99-100%
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amino acid identity) of Bcen_1091 and Bcen_1092 are equally encoded by tandem gene
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pairs in B. cenocepacia strains MC0-3 and HI2424 (sequences not included). N-terminal
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signal peptide sequences predicted by SignalP (http://www.cbs.dtu.dk/services/SignalP)
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were removed before alignment (names marked with asterisk). The amino acid sequences
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of XAC0868 and bacteriocin LlpAXcm761 from Xanthomonas citri subsp. malvacearum LMG
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761 are identical. The scale bar represents 0.3 substitutions per site. Bootstrap values
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(percentage of 1000 replicates) are shown at the branches.
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Figure 2
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Analysis of LlpA expression and purification. Proteins present in the lysate soluble fraction of
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E. coli BL21(DE3) cells, IPTG-induced for 16h, were separated by SDS-PAGE (12%, Coomassie
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Brilliant Blue-stained). Lane 1, Kaleidoscope protein ladder; lane 2, lysate of induced E. coli
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BL21(DE3) carrying pCMPG6122 (encoding LlpAPss642); lane 3, purified LlpAPss642; lane 4,
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lysate of induced E. coli BL21(DE3) carrying pCMPG6123 (encoding LlpAXcm761 with T2SS
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signal peptide); lane 5, purified LlpAXcm761; lane 6, lysate of induced E. coli BL21(DE3)
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carrying pCMPG6124 (encoding LlpAXcm761 without T2SS signal peptide); lane 7, lysate of
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induced E. coli BL21(DE3) carrying pET28a. Bands immunodetected with anti-His antibodies
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are circled. The 15 kDa band corresponds to lysozyme added to facilitate cell lysis of E. coli
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cells before sonication. Primers used to construct pCMPG6122, pCMPG6123, pCMPG6124
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are listed in Table S1.
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Figure 3
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Growth inhibition of representative strains by purified bacteriocins LlpABW11M1 (A), LlpAPss642
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(B-E), LlpAXcm761 (F-I), applied to lawn of indicator cells. (A,B) GR12-2R3, (C) PGSB 7716, (D)
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KT2440, (E) F113, (F) LMG 784, (G) LMG 7411, (H) LMG 582, (I) LMG 737, (J) Negative
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control: LMG 784 with dialysis buffer.
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