1 R&D Center, inBioNET Corporation, Daejeon 305-390, Korea
2 Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Korea
3 Department of Biotechnology, Yonsei University, Seoul 120-749, Korea
Correspondence
Do-Young Yum
dyyum{at}inbionet.com
Jung-Kee Lee
jklee{at}kribb.re.kr
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ABSTRACT |
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The GenBank accession numbers for the sequences reported in this paper are AY247743 (partial 16S rDNA sequence of Arthrobacter sp. IBN110), AF525800 (ahlD from Arthrobacter sp. IBN110) and AY222324 (ahlK from K. pneumoniae KCTC2241).
Present address: Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea.
Present address: Laboratory of Microbial Genomics, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Korea.
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INTRODUCTION |
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Since AHL-mediated signalling mechanisms are widespread and highly conserved in many pathogenic bacteria, they can be attractive targets for novel anti-infective therapies (Finch et al., 1998). Whitehead et al. (2001)
suggested that the inactivation of AHL itself is the most obvious strategy among intervention strategies for disrupting quorum sensing in bacteria. Recently, it was found that various Bacillus cereus groups are able to disrupt a quorum signal system through the enzymic degradation of the AHL molecules (Dong et al., 2000
, 2002
). Also, transgenic plants expressing AiiA, an N-acylhomoserine lactonase (AHLase) from Bacillus sp. 240B1, are protected from infection from the plant pathogen Erwinia carotovora (Dong et al., 2001
). In addition, our previous studies revealed that many subspecies of Bacillus thuringiensis have aiiA homologue genes, and a recombinant microbe producing the AiiA-like protein of B. thuringiensis can reduce the plant pathogenicity of Er. carotovora (Lee et al., 2002
). Similarly, the Gram-negative Variovorax parodoxus has been reported to metabolize AHL as the sole carbon and nitrogen source using an aminoacylase, whose AHL-degrading mechanism was apparently different from that of B. cereus groups (Leadbetter & Greenberg, 2000
). Interestingly, a recent report showed that an Arthrobacter isolate could utilize two signal breakdown products, homoserine lactone and acyl homoserine, rather than AHL as the sole carbon and nitrogen source (Flagan et al., 2003
).
As a source of AHL-degrading enzymes, micro-organisms are increasingly being investigated, and we have also been interested in isolating novel AHL-degrading bacteria for the purpose of disrupting and manipulating quorum-sensing signalling in agricultural pathogenic bacteria. In this study, we isolated an Arthrobacter strain that can utilize AHL as the sole carbon and nitrogen source, and also cloned a novel ahlD gene encoding AHLase from this strain. The existence of putative AhlD-like AHLases in several other bacteria is discussed.
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METHODS |
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Screening and isolation.
Fifty milligrams of soil was added to 2 ml of the minimal medium containing 2·5 mM OHHL. After 48 h cultivation at 30 °C, a 5 % (v/v) transfer was made to 200 µl of the minimal medium in a 96-well plate. After the third round of cultivation, turbid samples were spread on nutrient agar (NA) plates. To isolate bacteria growing on OHHL minimal medium, different type of colonies on NA plates were cultivated repeatedly in the minimal medium with and without OHHL in the 96-well plate. After 24 h cultivation, the optical densities and residual OHHL of the samples were measured using a SPECTRAMAX190 (Molecular Devices) and a bioassay plate, respectively.
Strain identification.
The 16S rDNA gene of strain IBN110 was PCR-amplified from a colony using the primers 9f (5'-AGAGTTTGATCCTGGCTCAG-3') and 926r (5'-CCGTCAATTCCTTTA/GAGTTT-3'). The PCR conditions involved denaturation at 94 °C for 5 min followed by 30 cycles at 94 °C for 30 s, 45 °C for 30 s and 72 °C for 60 s with a PCR Master (Roche). The PCR product of 899 bp was sequenced by an ABI3700 automatic sequencer (Applied Biosystems). Sequence identification was performed by use of the BLATAN facility of the National Center for Biotechnology Information and the sequence match facility of the Ribosomal Database Project (Maidak et al., 1999).
Growth experiments.
Growth with OHHL as the sole carbon source was determined in minimal medium containing 2·5 mM OHHL. Cell growth was monitored based on OD600. To measure the residual OHHL in the growth medium, duplicate culture supernatants (100 µl) were collected, and incubated at 95 °C for 5 min to stop the reaction. Appropriately diluted culture supernatants were loaded into a well in a plate overlaid with A. tumefaciens NT1(pDCI41E33), and the diameter of the blue zone on the bioassay plate was measured after incubation at 30 °C for 24 h. The relative amounts of OHHL were calculated using the following equation, obtained by Microsoft Excel regression analysis program: OHHL (pmol)=0·0335e1·3679x (R2=0·9952), where x is the diameter of the colour zone. The relationship was established by adding 0·1510 pmol OHHL to the bioassay plate and measuring the diameter of the resulting colour zone after incubation at 30 °C for 24 h.
Bioassay of AHL-degrading activity.
The AHL substrates N-butanoyl-L-homoserine lactone (BHL), N-octanoyl-L-homoserine lactone (OHL), N-decanoyl-L-homoserine lactone (dhl) and n-3-oxododecanoyl-L-homoserine lactone (OdDHL) were all purchased from Quorum Sciences Inc. and Fluka. N-Hexanoyl-L-homoserine lactone (HHL) and N-3-oxohexanoyl-L-homoserine lactone (OHHL) were synthesized in the authors' laboratory, as previously described (Zhang et al., 1993). For the whole-cell assay, Arthrobacter sp. IBN110 cultured overnight was harvested and resuspended in 100 mM Tris/HCl (pH 7·0). Then, 50 µl of the cell resuspension (OD600 1·0) and an equal volume of 40 µM AHL were mixed and incubated at 30 °C with gentle agitation. After boiling at 95 °C for 5 min to stop the reaction, the reaction mixtures were diluted to the appropriate concentration, and loaded into the well of plates overlaid with A. tumefaciens NT1(pDCI41E33) for OHHL, OHL, DHL and OdDHL detection, or of plates overlaid with C. violaceum CV026 for BHL and HHL detection. The residual amounts of AHL were calculated using relationship equations based on the colour zone size and known amounts of AHLs.
Co-culture of Er. carotovora N98 and Arthrobacter sp. IBN110.
Er. carotovora N98 and Arthrobacter sp. IBN110 were precultured separately in LB medium at 30 °C for 15 h. For co-culture, Er. carotovora N98 was inoculated in LB medium at 2x106 c.f.u. ml-1, whereas initial levels of Arthrobacter sp. IBN110 were 2x106 and 6x106 c.f.u. ml-1. Co-culture experiments of all combinations were done in triplicate. To determine the growth rate, viable cells were enumerated as c.f.u. by duplicate plating of serially diluted samples. After 10 h incubation (early stationary phase) at 30 °C, samples were withdrawn for determination of OHHL amounts and pectate lyase activities. Five-microlitre samples of cell-free culture supernatants were loaded into wells in plates overlaid with A. tumefaciens NT1(pDCI41E33) for detecting the amount of OHHL present during the co-culture. Extracelluar pectate lyase activity was determined as described by Laurent et al. (2000). One unit was defined as a 0·01 increase of A235 min-1 (ml supernatant)-1.
Partial purification of the AHL-degrading enzyme.
Arthrobacter sp. IBN110 cells harvested from the culture broth were resuspended in 10 mM Tris/HCl (pH 7·0) containing 0·1 mM PMSF, then disrupted three times using a French pressure cell (model FA-030; Aminco) at 260 MPa. The precipitated proteins were collected from a 4070 % saturation of ammonium sulfate, dissolved in 10 mM Tris/HCl (pH 7·0), and dialysed against the same buffer. The dialysate was applied to a DEAE-Sepharose CL-6B column (Pharmacia) and eluted with an NaCl gradient (01·0 M). The fractions exhibiting AHL-degrading activity were collected, concentrated using Centriprep-10 tubes (Millipore), and injected onto a 5 ml Hitrap Q-Sepharose column (Pharmacia). Finally, the proteins were eluted with an NaCl gradient (01·0 M) and the active fractions were collected.
Determination of amino-terminal amino acid sequence.
Due to the instability of the enzyme, we failed to fully purify the AHL-degrading enzyme by column chromatography. To determine the amino-terminal sequence of the AHL-degrading enzyme, the enzyme from the collected active fractions was first identified as follows. A partially purified sample was mixed with native gel electrophoresis sample buffer and separated by SDS-12 % PAGE. After electrophoresis, the duplicate gels were either stained with Coomassie brilliant blue R250 or renatured by washing three times with 10 mM Tris/HCl (pH 7·0) containing 2·5 % Triton X-100 for 15 min at room temperature with gentle agitation. The renatured gel was then sliced according to the protein bands on the stained gel. The slices were mixed with 20 µM OHL, and incubated at 30 °C with gentle agitation for 3 h. Thirty microlitres (3 pmol OHL) of the reaction mixtures diluted 200-fold was loaded into the well of a plate overlaid with A. tumefaciens NT1(pDCI41E33). The protein band with AHL-degrading activity was then transferred to a PVDF membrane (Bio-Rad) after SDS-PAGE, and sequenced by automatic Edman degradation (Applied Biosystems).
Cloning of the ahlD gene.
The total DNA of Arthrobacter sp. IBN110 was used as the template for the PCR. The PCR primers were designed based on the most conserved amino acid sequences of AHLases, AiiA and AttM (see Fig. 6). The sequences of the degenerate primers were 5'-GAGGATCCCA(C/T)CTICA(C/T)(T/G)(T/C)NGA(C/T)CA-3' and 5'-ATGAATTCGGIGT(G/A)TGNCCNGGNGT-3' (the BamHI and EcoRI sites are underlined). The PCR amplification was performed for 30 cycles consisting of 94 °C for 45 s, 50 °C for 45 s and 72 °C for 45 s. The PCR products were then subcloned into a pT7Blue T-vector (TaKaRa), sequenced, and used as a probe for colony hybridization, as described below.
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Expression of recombinant AhlD and its homologue in E. coli.
The ORF of the ahlD gene was amplified by a PCR using the following primers: EN1, 5'-ATGGAGTCATATGGAAAAAGATCA-3'; EN2, 5'-ATGGAGTGCCATGGAAAAAGATCA-3'; and EC, 5'-TCGTAGAAAGCTTCTGGTAGTGCC-3' (the NdeI, NcoI and HindIII sites are underlined). The PCR products amplified by the EN1 and EC primers were digested with NdeI and HindIII, and subcloned into the 6xHis tagging expression vector pET22b(+) (Novagen) between the NdeI and HindIII sites to give pAhlD. The PCR products amplified by the EN2 and EC primers were cloned into the NcoI and HindIII sites of pTrc99A (Amersham Pharmacia Biotech), resulting in pEAhlD. The expression vectors pAhlD and pEAhlD were then transformed into E. coli BL21(DE3) and E. coli JM109, respectively. The E. coli cells carrying the recombinant AhlD were cultivated for 3 h after IPTG induction at 30 °C. For the SDS-PAGE analysis, the cell extracts were sonicated in 10 mM Tris/HCl (pH 7·0) and loaded onto a polyacrylamide gel. The AHL-degrading activity of the IPTG-induced recombinant cells was measured using the bioassay described above.
To investigate whether the ahlK from K. pneumoniae KCTC2241 encodes an AHL-degrading enzyme, the gene containing promoter was amplified by PCR using the following primers: KF, 5'-CTGAATTCCTGAGTCAGGCTA-3'; KR, 5'-TTGAATTCTCAGCGAGGAATGAT-3' (the EcoRI sites are underlined). The PCR product of 855 bp was subcloned into the EcoRI site of pUC19, and sequenced. The resulting plasmid was named pUAhlK. The AHL-degrading activity of the E. coli JM109 harbouring pUAhlK was determined by whole-cell assay.
HPLC and ESI-MS analysis.
HHL digestion by AhlD was analysed using HPLC and electrospray ionization-mass spectrometry (ESI-MS). A 10 ml sample of the IPTG-induced recombinant cells was harvested and sonicated. Next, 490 µl of cell extract of E. coli carrying the recombinant pEAhlD was mixed with 10 µl HHL (2 mM final concentration), and incubated at 30 °C for 1 h with gentle shaking. After incubation, the mixture was extracted three times with ethyl acetate, and evaporated. For the HPLC analysis, the sample was dissolved in 0·1 ml 50 : 50 (v/v) methanol/water and analysed using a C18 reverse-phase column. The fractions were separated by eluting isocratically with 70 : 30 (v/v) methanol/water at a flow rate of 0·3 ml min-1. The ESI-MS was performed using a Mariner instrument (Perspective Biosystems) in the Korea Basic Science Institute (KBSI), where the sample was dissolved in 50 : 50 (v/v) methanol/water and ionized by a positive-ion electrospray.
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RESULTS |
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With OHHL as the sole carbon source, the doubling time of Arthrobacter sp. IBN110 was 2·1 h. As the cell density increased, the residual amount of OHHL in the culture medium significantly decreased. Most of the OHHL in the culture medium was consumed after 10 h of cultivation (Fig. 1). No growth was observed in an OHHL-depleted minimal medium. It has been previously reported that the half-life of an AHL is <3 h under alkaline conditions (pH 8·0) (Leadbetter & Greenberg, 2000
; Yates et al., 2002
). During 10 h of cultivation, the pH of the culture medium changed from 6·5 to 6·8. Thus, the dramatic decrease of OHHL in the medium is unlikely to have been due to alkaline pH. In minimal medium containing 0·2 % glucose as the carbon source and 0·5 mM OHHL as the sole nitrogen source, Arthrobacter sp. IBN110 grew exponentially with a doubling time of 1·6 h, and utilized most of the OHHL during the exponential phase (data not shown). Thus Arthrobacter sp. IBN110 was able to utilize OHHL as both the sole carbon source and the sole nitrogen source.
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In general, the genes that metabolize various organic compounds often appear as a gene cluster in Arthrobacter species. Part of a divergently transcribed ABC-transporter-like gene was identified 244 bp upstream of the ahlD, while 435 bp downstream of ahlD and transcribed in the opposite orientation to ahlD, we identified a putative transcription regulator of the lacI type involved in carbon catabolite repression (data not shown).
Expression of recombinant AhlD in E. coli
The E. coli clone that contained the 2·3 kb DNA fragment showed relatively lower AHL-degrading activity than Arthrobacter sp. IBN110. To confirm that the ahlD gene indeed encodes an AHL-degrading enzyme, it was expressed under control of the T7 promoter in E. coli. Upon induction of E. coli BL21(DE3)(pAhlD) with IPTG, a His-tagged protein with a molecular mass of about 36 kDa was produced, yet most of the protein was expressed in an insoluble form (Fig. 4a). Although most of the AhlD protein was produced as inclusion bodies, 1 ml of the recombinant E. coli expressing AhlD effectively degraded the AHLs HHL, OHHL, OHL, DHL and OdDHL, while E. coli carrying pET22b, as the control, was unable to degrade any of the tested AHLs (Fig. 4b
). It was also observed that the recombinant E. coli degraded less OHHL than OHL and OdDHL, which was in good agreement with the AHL degradation pattern of Arthrobacter sp. IBN110 (Fig. 2
). Accordingly, these results reconfirm that the ahlD gene of Arthrobacter sp. IBN110 encodes an AHL-degrading enzyme. In order to effectively express the AhlD enzyme, the ahlD gene was cloned into a pTrc99A expression vector, resulting in pEAhlD. E. coli JM109(pEAhlD) induced with IPTG exhibited a higher AHL-degrading activity than that observed with E. coli BL21(DE3)(pAhlD), although the expression level of AhlD was low (data not shown). Therefore, recombinant AhlD from E. coli JM109(pEAhlD) was used for an HHL product analysis.
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Consequently, a further search for AhlD-like enzymes was conducted using the microbial genome PEDANT (http://pedant.gsf.de/) and GenBank databases. Besides AiiA and AttM, six AhlD homologues were found, all of which included the conserved motif HXDHH
D and exhibited 2030 % identity in their amino acid sequence with that of AhlD from Arthrobacter sp. IBN110. Among these homologues, two AhlD homologues, from Bacillus stearothermophilus 10 and Klebsiella pneumoniae MGH78578, were aligned with the other AHLases shown in Fig. 6
. The Orf40 from the unfinished genome of B. stearothermophilus 10 exhibited 27 % identity with AhlD, which was slightly higher than the identities with the other AHL-degrading enzymes. AhlD shared 2023 % identity with proteins of an unknown function, annotated as an AttM-like protein from Bradyrhizobium japonicum, K. pneumoniae MGH78578 and Thermoplasma volcanium GSS1. In particular, the Orf99 from the unfinished genome of K. pneumoniae MGH78578 exhibited 58·6 % identity with the AttM of A. tumefaciens for the amino acid sequences, indicating that the strain may contain a putative AHL-degrading enzyme. The AHL-degrading activity was tested using B. stearothermophilus KCTC3067 and K. pneumoniae KCTC2241. Indeed, AHL-degrading activities were observed in B. stearothermophilus KCTC3067 and K. pneumoniae KCTC2241 when using a whole-cell assay (data not shown). Recombinant E. coli expressing AhlK, an Orf99 homologue from K. pneumoniae KCTC2241, also exhibited AHL-degrading activity, indicating that ahlK and the Orf99 gene encode AHL-degrading enzymes (Fig. 7
). The AhlK was different from Orf99 in only two amino acid residues (Ala68
Thr; Leu96
Ile). Therefore, these results suggest the possibility that AhlD-like AHLases could exist in many other micro-organisms, including thermophilic bacteria.
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DISCUSSION |
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Although Arthrobacter sp. IBN110 was initially isolated based on its OHHL-utilizing ability (Fig. 1), the strain was also found to effectively degrade different kinds of AHLs that regulate the virulence factors in Erwinia (Kievit & Iglewski, 2000
; De Jones et al., 1993
), Agrobacterium (Piper et al., 1993
; Zhang et al., 1993
), and the human pathogen P. aeruginosa (Pearson et al., 1994
, 1997
) (Fig. 2
). This ability to degrade various AHLs was also confirmed in recombinant E. coli expressing AhlD (Fig. 4
). Furthermore, the co-culture of Arthrobacter sp. IBN110 and Er. carotovora resulted in a decreased OHHL concentration and OHHL-mediated pectate lyase activity by Er. carotovora in the medium without any change in the cell growth of Er. carotovora (Table 1
), indicating the possibility of applying Arthrobacter sp. IBN110 and its ahlD gene in the control of AHL-producing Gram-negative bacteria.
For V. paradoxus, it has been reported that an aminoacylase may be involved in AHL metabolism by cleaving the intact lactone from the AHL acyl side-chain (Leadbetter & Greenberg, 2000). Recently, an AHL acylase was cloned from a Ralstonia isolate capable of using BHL and OdDHL as its carbon source (Lin et al., 2003
). It was predicted that Arthrobacter sp. IBN110 growing on OHHL may also produce a putative aminoacylase for utilizing AHL. However, AhlD from Arthrobacter sp. IBN110 was found to contain the conserved motif, HXDH
H
D, the essential motif for AHLase activity (Dong et al., 2002
) (Fig. 6
). In addition, HPLC and MS analyses showed that AhlD produced acylhomoserine by hydrolysing AHL, indicating that AhlD is an AHLase (Fig. 5
). Therefore, the current study shows that the quorum signal molecule AHL could be disrupted and utilized by the AHLase of Arthrobacter sp. IBN110, in contrast to the acylase of V. paradoxus and the Ralstonia isolate, suggesting that the metabolic pathway for utilizing AHL may be different between Arthrobacter sp. IBN110 and V. paradoxus. However, further study is required to determine the metabolic fate of AHL.
To date, two AHLase genes have been identified in A. tumefaciens and in B. cereus groups including B. thuringiensis. The identification of AhlD from Arthrobacter sp. IBN110 indicates that the conserved motif, HXDHH
D, is required for AHLase activity, even though the overall identities among the AHLases AiiA, AttM and AhlD were <26 % (Fig. 6
). From a homology search based on the conserved motif, putative AhlD-like homologues were found in six different bacteria. Interestingly, AHL-degrading activity was detected in the thermophilic bacterium B. stearothermophilus (data not shown). Also, it was observed that recombinant E. coli expressing AhlK from K. pneumoniae KCTC2241 showed AHL-degrading activity (Fig. 7
). Accordingly, the current findings may be helpful in uncovering AHLases in natural ecosystems. Putative AhlD-like homologues were distributed in diverse bacteria, including two Archaea, Thermoplasma volcanium GSS1 and Sulfolobus solfataricus P2, which grow optimally at 60 and 80 °C, respectively, and the symbiotic bacterium B. japonicum, where nodulation genes were regulated by quorum sensing using a bradyoxetin, rather than AHL (Loh et al., 2002
). In addition, an AhlD-like homologue was also found in Burkholderia fungorum LB400 with quorum-sensing genes (data not shown), suggesting that it is involved in a signal turnover system, as in A. tumefaciens (Zhang et al., 2002
). The actual roles of AhlD and putative AhlD-like homologues in bacteria and natural environment remain unclear. However, the current findings indicate that AhlD-like lactonases are widespread in diverse bacteria, although whether or not these homologues exhibit AHL-degrading enzyme activities remains to be elucidated.
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ACKNOWLEDGEMENTS |
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Received 29 January 2003;
revised 12 March 2003;
accepted 17 March 2003.