Molecular Microbiology Laboratory, Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK
Correspondence
David J. Clarke
bssdjc{at}bath.ac.uk
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ABSTRACT |
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INTRODUCTION |
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Photorhabdus luminescens subsp. laumondii TT01 has been shown to produce a -lactam antibiotic, carbapenem, during the exponential phase of growth (Derzelle et al., 2002
). Members of Photorhabdus have also been reported to produce a variety of colicin-like antimicrobial proteins and both the carbapenem and the colicins have been shown to have activity against Gram-negative bacteria (Derzelle et al., 2002
; Sharma et al., 2002
). In addition, all strains of Photorhabdus tested have been shown to produce an antibiotic activity identified as 3,5-dihydroxy-4-isopropylstilbene (ST) (see Fig. 1
) (Hu et al., 1997
; Li et al., 1995
; Richardson et al., 1988
). The ST antibiotic is produced as the bacteria enter the stationary phase of growth [both in the insect (in vivo) and in nutrient broth (in vitro)] and it has been suggested that the role of the ST antibiotic is to protect the insect cadaver from predation and saprophytic attack (Hu & Webster, 2000
). In support of this, it has been demonstrated that ST has anti-bacterial, anti-fungal and anti-nematode activity (Akhurst, 1982
; Han & Ehlers, 1999
; Hu et al., 1999
). However, the biochemical pathway responsible for the production of this antibiotic has not yet been characterized.
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In this report, we describe the identification and characterization of a PAL from the bacterium P. luminescens TT01. This is the first report of PAL activity in a member of the Proteobacteria. We also demonstrate that cinnamic acid is required for the production of the ST antibiotic in P. luminescens and propose a model for the biochemical pathway leading to ST production.
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METHODS |
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DNA manipulations.
Chromosomal DNA was isolated from P. luminescens and E. coli strains using standard procedures (Sambrook et al., 1989). The chromosomal DNA flanking the transposon was identified, as previously described, by cloning into pBR322 and sequencing (Joyce & Clarke, 2003
).
Cloning of stlA into pTRC99a and pTRC99a-His.
The stlA gene was amplified from P. luminescens TT01 chromosomal DNA by PCR using oligonucleotides JW124 (5'-CGATCCCATGGAAGCTAAAGATGTTCAGCC-3') and JW125 (5'-CGATCTCTAGATTATTCTTCCAGCATGATTTCTGG-3'), for cloning into the NcoI and XbaI sites of pTRC99a (Amersham Pharmacia Biotech) and a derivative, pTRC99a-His (a gift from Dr Mark Blight, CNRS, Orsay, France), resulting in pBMM901 and pBMM902, respectively. The integrity and accuracy of all plasmid clones were confirmed by DNA sequencing.
TLC analysis of culture supernatants.
An overnight culture of the bacterial strain being tested was inoculated (1 : 100) into 100 ml LB and incubated at 28 °C for 48 h. When required, cinnamic acid (Sigma-Aldrich) was added to the LB to a final concentration of 33 µg ml1. The cells were removed by centrifugation at 12 000 r.p.m. for 10 min and the culture supernatant was aliquotted into four 25 ml samples. The supernatant was then extracted three times with 20 ml ethylacetate (HPLC grade) and the ethylacetate extractions were collected into a large round-bottomed flask. The sample was then dried down in a rotary evaporator using partial pressure and a temperature of approx. 5560 °C and resuspended in 500 µl methanol. The extract was transferred to a 1·5 ml Eppendorf tube and stored at 20 °C until required. A small aliquot (50 µl) of each extract was applied to a TLC plate [aluminium-backed silica gel 60 F254 (fluorescence wavelength) (Merck)] using a glass microsyringe. The TLC was run using a developing solvent of chloroform : methanol (98·5 : 1·5) and the plates were analysed under UV light (Hu et al., 1997). To overlay with M. luteus, the TLC plate was placed on the surface of an LB agar plate and covered with 10 ml soft agar containing 0·1 ml of an overnight culture of M. luteus. The agar plate was then incubated at 30 °C for 23 days and antibiotic activity was observed as an inhibition of growth over the stilbene band on the TLC plate.
Purification of HisStlA.
E. coli EC100 containing the pBMM902 plasmid was cultured overnight in LB at 37 °C and inoculated (1 : 100) into 50 ml fresh LB. The cells were grown at 37 °C until an OD600 of 0·5 and 100 µM IPTG was added to induce expression of the stlA gene. After 3 h induction, the cells were harvested by centrifugation and the pellet was stored at 20 °C. The cell pellet was resuspended in 2 ml BugBuster solution (Novagen) containing 50 U Benzonase and 100 µg lysozyme. The cell suspension was left at room temperature for 30 min and cell debris was removed by centrifugation at 13 300 r.p.m. for 15 min at 4 °C. The clarified supernatant was mixed with 250 µl Ni-NTA resin (Novagen) and incubated at 4 °C with constant mixing for 60 min. At this stage, the mixture was loaded into a column and the resin was washed twice with 8 volumes of wash buffer (50 mM sodium phosphate buffer, pH 8·0, 300 mM NaCl, 20 mM imidazole). The HisStlA protein was then eluted in four 0·5 ml aliquots of elution buffer (50 mM sodium phosphate buffer, pH 8·0, 300 mM NaCl, 250 mM imidazole) and the fractions were analysed by SDS-PAGE. Fractions containing StlA were pooled and dialysed, using a Slide-A-Lyser (Pierce), against 50 mM sodium phosphate buffer (pH 8·0), 300 mM NaCl, to remove the imidazole. Glycerol was added to a final concentration of 20 % (v/v) and the sample was aliquotted and stored at 20 °C. The protein concentration was then determined with the Bradford reagent assay (Sigma) using BSA as the standard. Typical protein yields were approximately 1 mg purified HisStlA per 50 ml culture.
PAL enzyme assay.
PAL activity was assayed as previously described with some modifications (Alunni et al., 2003; Kyndt et al., 2002
). Whole-cell lysates were prepared by culturing P. luminescens in 500 ml LB broth for 24 h and harvesting the cells by centrifugation at 4000 g for 20 min. The cells were then resuspended in 1 ml 0·1 M sodium borate buffer (pH 8·8) and lysed by sonication. The concentration of protein in the lysate was determined by a Bradford reagent assay (Sigma) using BSA as the standard. To measure PAL activity, we added 0·1 M sodium borate buffer (pH 8·8) and 20 mM L-phenylalanine to an aliquot of the lysate to give a final reaction volume of 1 ml and the reaction tube was incubated at 30 °C. The production of cinnamic acid was detected by measuring the increase in A290 (Kyndt et al., 2002
). The specific activity of PAL in total cell extract was calculated using an absorbance coefficient for cinnamic acid of 10 000 M1 cm1. For the PAL assay using purified protein, the reaction (1 ml in total) was performed in 50 mM sodium phosphate buffer (pH 8·0), 300 mM NaCl at 30 °C with 10 mM L-phenylalanine as the substrate and 5 µg purified StlA protein. The Km and vmax of the purified HisStlA were calculated using the direct linear plot method described by Eisenthal & Cornish-Bowden (1974)
using the EnzPack software package.
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RESULTS |
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BMM901 is defective in ST production
It has previously been reported that ST can be visualized after TLC by using UV light (Hu et al., 1997). Therefore, to confirm that the lack of antibiotic production in BMM901 was due to an absence of the ST antibiotic, we carried out a TLC analysis of organic extracts of cell-free supernatants from overnight cultures of TT01 and BMM901. From this analysis, it is clear that two compounds were missing from the BMM901 supernatant (Fig. 2b
; UV). The major compound (Fig. 2b
; indicated with an arrow) migrated with an Rf of approximately 0·60 and this is very close to the Rf reported previously for ST (Rf=0·59) (Hu et al., 1997
). To confirm that this band was ST, we placed the TLC plate on the surface of an LB agar plate and covered it with 10 ml soft agar containing 0·1 ml of an overnight culture of M. luteus. The agar plate was then incubated at 30 °C for 23 days and antibiotic activity was determined by observing inhibition of bacterial growth over certain regions of the TLC plate (Fig. 2b
; Overlay). It is clear that the major compound present in TT01 supernatants and absent from BMM901 supernatants strongly inhibits the growth of M. luteus, confirming that this compound is ST. Although the nature of the minor compound has not yet been determined, it is possible that it is an intermediate of ST production. Furthermore, we did not detect any other compounds with anti-microbial activity in supernatants of TT01 cultures using the extraction protocol outlined in this study.
To confirm that the defect in ST production in BMM901 is entirely due to the mutation in plu2234, we cloned plu2234 into the vector pTRC99a and the resulting plasmid, pBMM901, was transformed into BMM901 and ST production was determined by TLC analysis (Fig. 2b). It is clear that the defect in ST production in BMM901 is entirely due to the mutation in plu2234. In addition to restoring production of ST, complementation of BMM901 with pBMM901 also restored the production of the minor compound shown to be absent from supernatants of BMM901.
As plu2234 is the first gene shown to be required for the production of the stilbene antibiotic in P. luminescens, we propose to rename plu2234 as stlA (stilbene A).
DNA sequence analysis of the stlA locus
To determine whether any of the genes in the vicinity of stlA might be involved in antibiotic production, we carried out an in silico analysis of the proteins that are predicted to be encoded by the genes surrounding stlA, using the BLASTP algorithm available at http://www.ncbi.nlm.nih.gov/blast/ (Fig. 3). The gene plu2233, immediately upstream from stlA, is predicted to encode a protein with oxidoreductase activity [best BLASTP hit, 77 % identity with probable oxidoreductase in Yersinia pseudotuberculosis (YP_070712); score=e107]. Oxidoreductases are often involved in the detoxification of molecules and these genes may encode proteins involved in resistance to the ST antibiotic. Interestingly, plu2236 is also predicted to encode an FAD-dependent oxidoreductase (best BLASTP hit, 31 % identity to FAD-dependent oxidoreductase from Rubrobacter xylanophilus DSM 9941). The genes plu2232 and plu2231 are predicted to encode proteins of unknown function with homologues in other enteric bacteria [best BLASTP hit for plu2232, 62 % identity with hypothetical protein from Y. pseudotuberculosis (YP_070711); score=7e31; best BLASTP hit for plu2231, 70 % identity with hypothetical protein from Y. pseudotuberculosis (YP_070710); score=7e17]. Therefore, plu2231plu2233 have homologues in Y. pseudotuberculosis (YP_07010YP_07012), a bacterium that is not reported to produce any stilbene-based molecules, suggesting that these genes are unlikely to be involved in ST production in P. luminescens. Immediately downstream from stlA is a gene predicted to encode a chitinase (plu2235; best BLASTP hit, 49 % identity to a predicted chitinase from Burkholderia fungorum; score=e144). As P. luminescens is an insect pathogen, it is not surprising that this bacterium produces chitinase activity, although it is unlikely that the chitinase is directly involved in ST production. Therefore, from our analysis, it would appear likely that stlA is the only gene encoding a protein with a role in ST production in this region of the P. luminescens genome.
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Recent work in the actinomycete Streptomyces maritimus has identified a protein, EncP, that, despite strong homology with prokaryotic HALs, has been shown to have PAL activity (Xiang & Moore, 2002). Although the active sites of PALs and HALs are reported to be very similar, they are predicted to differ in some key residues (Calabrese et al., 2004
; Xiang & Moore, 2002
). In particular, Met382 and Glu414 in the HAL of Pseudomonas putida are highly conserved in other HALs but are replaced by Lys and Gln, respectively, in PALs (Calabrese et al., 2004
). These residues are predicted to be involved in substrate loading and would be important in distinguishing between L-histidine and L-phenylalanine. Therefore, to determine whether StlA could be a PAL, we compared the amino acid sequences of StlA with the predicted HAL proteins from Pseudomonas putida, S. enterica, Y. pestis and P. luminescens TT01 using the CLUSTAL W algorithm in DNASTAR (Fig. 4
). All of the key active-site residues shared by PALs and HALs are present in StlA, suggesting that StlA is either a HAL or a PAL (data not shown). However, it is clear from Fig. 4
that StlA is predicted to have a Lys (Lys407) residue in place of Met382 and a Gln (Gln440) in place of Glu414. Therefore, StlA has the sequence signature expected from a protein with PAL activity.
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To confirm that stlA encodes PAL, we cloned stlA into pTRC99a-His, a derivative of pTRC99a that places a 6xHis fusion onto the N terminus of the expressed protein, resulting in pBMM902 (see Methods). His-tagged StlA (HisStlA) was overproduced in E. coli and purified using Ni-NTA chromatography. The eluted protein was assessed for purity by SDS-PAGE and we observed a single major protein band migrating with a molecular mass of approximately 50 kDa, slightly smaller than the predicted molecular mass of StlA (57·7 kDa) (data not shown). The purified protein was then used in PAL enzyme assays as described in Methods. From these assays, it is clear that HisStlA was able to produce cinnamic acid from L-phenylalanine and, using the direct linear method, we calculated the Km (320±35 µM) and the vmax [13·2±0·02 pmol s1 (µg protein)1] of HisStlA. Therefore, the kcat for HisStlA is 0·8 s1, a value that falls within the range of kcat values (0·13·2 s1) recorded for some plant PAL enzymes (Cochrane et al., 2004). It should also be noted that PAL has high sequence identity to tyrosine ammonia-lyase (TAL), an enzyme that produces coumaric acid from tyrosine. However, we could not detect any TAL activity with HisStlA (data not shown).
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DISCUSSION |
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PAL activity is ubiquitous in plants and the production of cinnamic acid is the starting point for the biosynthesis of a variety of plant phenylpropanoids, including some phytoalexins shown to have beneficial affects on human health, e.g. resveratrol (Dixon, 2001; Pervaiz, 2003
). However, PAL activity is extremely rare in prokaryotes and, in addition to StlA, the only other PAL identified in bacteria is EncP from the actinomycete Streptomyces maritimus (Xiang & Moore, 2002
). Interestingly, stlA and encP are predicted to encode proteins of similar sizes (532 and 522 amino acids, respectively). Moreover, the proteins encoded by stlA and encP are also predicted to be similar in size to prokaryotic HALs (see Fig. 4
). However, StlA and EncP are notably smaller than their eukaryotic PAL counterparts. The model plant Arabidopsis thaliana has genes encoding four isoforms of PAL (PAL1PAL4) and the predicted size of these proteins ranges from 695 to 723 amino acids (Cochrane et al., 2004
). This discrepancy in size suggests that the genes encoding the prokaryotic PALs do not have their origin in plants. Rather, it seems more likely that stlA and encP arose through a duplication of the hutH gene followed by subsequent mutation of the key active-site residues (see Fig. 4
).
In Streptomyces maritimus, encP was shown to be part of a larger gene cluster that contains all of the genes required for the biosynthesis of the antibiotic enterocin (Xiang & Moore, 2002, 2003
). However, from our analysis of the stlA locus (Fig. 3
), it appears that stlA is not part of a large ST biosynthetic gene cluster, suggesting that the other genes involved in the production of the ST antibiotic are located elsewhere on the TT01 genome. This may also suggest that the hutH duplication occurred independently in P. luminescens and Streptomyces maritimus.
The unlinked nature of the genes involved in ST production makes it difficult to speculate on the nature of the biochemical pathway involved in the production of this antibiotic. However, the ST antibiotic produced by Photorhabdus is very similar in structure to pinosylvin (3,5-dihydroxystilbene), a phytoalexin produced by pine trees (Raiber et al., 1995; Schanz et al., 1992
). The only difference between ST and pinosylvin is the presence of an isopropyl group at position 4 of the stilbene ring of ST (Fig. 1
). Biosynthesis of pinosylvin is predicted to occur through the action of a type III polyketide synthase (PKS) called pinosylvin synthase (Schanz et al., 1992
). Using a variety of methods, including degenerate PCR, Southern blotting and in silico homology searches, we could not find any gene encoding a protein with significant homology to pinosylvin synthase (or any other type III PKS) in the genome of P. luminescens TT01 (J. S. Williams, unpublished data). Moreover, it has been shown that ST antibiotic production in Photorhabdus requires the activity of a phosphopantetheinyl (P-pant) transferase, NgrA (Ciche et al., 2001
). Type III PKS action is independent of P-pant transferase activity and, therefore, it is unlikely that ST production involves a type III PKS. However, there are two other types of PKS (type I and type II), and both type I and type II PKSs do require P-pant transferase activity. Type I PKSs are large, multifunctional enzymes that contain a set of distinct, non-iteratively acting activities that are required for the production of the polyketide molecule, e.g. acetyltransferase (AT), keto-synthase (KS) (Hopwood, 1997
). Type II PKSs, on the other hand, are multienzyme complexes that carry a single set of iteratively acting activities (Hopwood, 1997
). Because of their large size and modular arrangement, it is relatively straightforward to identify genes encoding type I PKSs. Moreover, as the domains act non-iteratively, it is often possible to predict the molecule being produced by the PKS based on the number and arrangement of the active domains. We have used well-characterized AT and KS domains as queries in BLAST searches of the P. luminescens TT01 genome and we could not find a type I PKS with the arrangement of domains required to synthesize the ST antibiotic (J. S. Williams, unpublished data). Therefore, by default, we suggest that the production of ST requires an as-yet-unidentified type II PKS. This suggests that, although ST production in Photorhabdus involves the same starting molecule as used in plants, the production of the antibiotic probably requires a novel biochemical pathway. We are currently undertaking further genetic studies to identify other genes involved in the biosynthesis of this broad-spectrum antibiotic.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 20 April 2005;
revised 24 May 2005;
accepted 27 May 2005.
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