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Applied and Environmental Microbiology, February 2003, p. 1308-1314, Vol. 69, No. 2
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.2.1308-1314.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Cloning and Expression Analysis of the pcbAB-pcbC ß-Lactam Genes in the Marine Fungus Kallichroma tethys

Chi-fai Kim,1 Simon K. Y. Lee,1 Jackie Price,2 Ralph W. Jack,1,3,{dagger} Geoffrey Turner,2 and Richard Y. C. Kong1*

Department of Biology and Chemistry,1 Centre for Coastal Pollution and Conservation, City University of Hong Kong, Kowloon Tong, Hong Kong Special Administrative Region, People's Republic of China,3 Department of Molecular Biology and Biotechnology, Krebs Institute for Biomolecular Sciences, University of Sheffield, Sheffield S10 2TN, United Kingdom2

Received 2 July 2002/ Accepted 13 November 2002


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ABSTRACT
 
Here we report the identification of the ß-lactam biosynthesis genes pcbAB and pcbC from a cosmid genomic DNA library of the marine fungus Kallichroma tethys. A BLAST homology search showed that they share high sequence identity with the {delta}-(L-{alpha}-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetases and isopenicillin N synthases, respectively, of various fungal and bacterial ß-lactam producers, while phylogenetic analysis indicated a close relationship with homologous genes of the cephalosporin-producing pyrenomycete Acremonium chrysogenum. Expression analysis by reverse transciption-PCR suggested that both genes are highly regulated and are expressed in the late growth phase of K. tethys cultures. Complementation of an Aspergillus nidulans strain deficient in ACV synthetase suggested that at least pcbAB is functional, although attempts to isolate active antibiotic from K. tethys were unsuccessful.


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INTRODUCTION
 
The widely studied penicillins, cephalosporins, and cephamycins are ß-lactam-containing peptide antibiotics synthesized by nonribosomal peptide synthetases (for reviews, see references 15 and 16). The hydrophobic penicillins are produced solely by filamentous fungi, while the relatively more hydrophilic cephalosporins are produced by a variety of prokaryotic and eukaryotic microorganisms (15). Penicillins, cephalosporins, and cephamycins are synthesized by condensation of L-{alpha}-aminoadipic acid, L-cysteine, and L-valine and epimerization of the valine to form the linear tripeptide {delta}-(L-{alpha}-aminoadipyl)-L-cysteinyl-D-valine (ACV) (24), which is then cyclized to form isopenicillin N (IPN) (2). Previous studies have demonstrated that ACV synthetase and IPN synthase mediate these chemical transformations and that the enzymes are encoded by the genes pcbAB (7, 9) and pcbC (7, 22), respectively.

Terrestrial microorganisms are prodigious producers of ß-lactam antibiotics, and extensive studies on the molecular biology and regulation involved in this phenomenon have been reported (5, 15). Despite the fact that marine microorganisms have been shown to be a rich source of novel bioactive compounds (18), marine fungi appear to have been poorly explored as sources of novel antibiotics. Since marine fungi are exposed to different natural selective pressure compared to their terrestrial counterparts, it seems worthwhile to investigate their ability to synthesize novel pharmacologically active compounds. Thus, in our search for ß-lactam genes in marine fungi we investigated Kallichroma tethys, a wood-inhabiting marine fungus that occurs exclusively in tropical and subtropical waters (13) and is a member of the Hypocreales (21). Here we describe the cloning and expression analysis of the pcbAB-pcbC ß-lactam gene cluster from K. tethys.


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Screening for pcbAB-like sequences in marine fungi.
 
Initially we screened the marine fungal strains Dactylspora haliotrepha PP3609, Halosarpheia trullifera PP4268 and PP7297, Halosarpheia viscosa PP3043, K. tethys PP320, Kallichroma glabrum PP406, Lignincola laevis PP3236, and the fungal positive control Penicillium chrysogenum 26518 (all of which were generous gifts from E. B. G. Jones, BIOTEC, Bangkok, Thailand), as well as the bacterial positive control Streptomyces clavuligerus ATCC 27064. Fungal cultures were grown in 2% (wt/vol) malt extract (Oxoid) prepared in filter-sterilized natural seawater at 25°C with shaking (100 rpm) for periods of 4 weeks to 3 months depending on the strain being cultured, while S. clavuligerus was grown at 30°C on nutrient agar (Oxoid). Genomic DNA, extracted as described previously (14), was used as a template and was screened by PCR using the primers pcbAB1F and pcbAB1R (Table 1), which were designed against consensus sequences derived from multiple alignment of Aspergillus nidulans (X54854), P. chrysogenum (X54296), and Acremonium chrysogenum (E05192) pcbAB sequences. Both of the control strains yielded a single 1.3-kb product (expected size). Although PCR amplification of the marine fungal DNA yielded products of various sizes, only K. tethys and K. glabrum produced a 1.3-kb product (data not shown); these last fragments were subsequently cloned into pUC18 and sequenced. A database search using BLASTN showed that the products shared high degrees of identity (55 to 68%) with the published pcbAB sequences of various microbial species. Southern blots of the PCR products from all of the strains tested showed that the 1.3-kb pcbAB gene fragment hybridized only with the products of the two control strains as well as with K. tethys and K. glabrum (data not shown), suggesting that the additional PCR products obtained from the other marine fungi were not related to pcbAB. This suggestion was further confirmed by direct sequencing of those products.


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  		TABLE 1. PCR primers used in this study


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Construction and screening of a cosmid DNA library.
 
Genomic DNA extracted from lyophilized K. tethys mycelia (14) was partially digested with Sau3AI. DNA fragments larger than 9.5 kb were ligated into BamHI-digested Supercos-1 (Stratagene) and packaged into Escherichia coli XL1-Blue MR cells. The cosmid library was plated onto Luria-Bertani plates containing ampicillin at 50 µg ml-1, and colony hybridization was performed by use of the 1.3-kb PCR fragment from K. tethys as a probe (Fig. 1). One strongly hybridizing cosmid clone, D7221, was further characterized by restriction mapping and Southern blot analyses. A 1.0-kb pcbC gene fragment derived from the control P. chrysogenum strain was also used as a probe to screen D7221 DNA digested with various endonucleases by Southern hybridization. Appropriate fragments that showed positive hybridization with both probes were cloned into pUC18 and sequenced on both strands, and gaps in the sequences were filled by primer walking. A contiguous stretch of ca. 20 kb of genomic sequence was obtained (Fig. 1).



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  		FIG. 1. Genomic organization of the pcbAB, pcbC, and orf1 gene cluster of K. tethys. Arrows indicate the ORFs and their orientation shows the direction of transcription. Hybridization probes are represented by open boxes. The potential transcription start sites for pcbAB and pcbC are indicated by vertical arrows ({downarrow}) and are labeled TSAB and TSC, respectively. The ATG start codons of pcbAB and pcbC are labeled accordingly. Coding regions are represented by shaded boxes; unshaded portions represent 5'-UT domains. The hatched region delineates the bidirectional core promoter. Restriction site abbreviations: B, BamHI; E, EcoRI; H, HindIII; P, PstI. Not all restriction sites for BamHI, EcoRI, HindIII, and PstI are indicated.


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Nucleotide sequence analysis.
 
Sequence analysis revealed the following three open reading frames (ORFs): pcbAB, pcbC, and orf1 (Fig. 1). The ORFs of pcbAB and pcbC are oriented in opposite directions separated by a 704-bp intergenic spacer, and a promoter arrangement similar to that found in the ß-lactam-producing fungi, such as P. chrysogenum (2) and Acremonium chrysogenum (9), was also identified. A further gene, orf1, is located 853 bp downstream from pcbC and, interestingly, is encoded on the opposite strand. Sequencing of a further 2.0 kb downstream of orf1 failed to reveal the presence of any additional genes, such as the penDE gene typically associated with the penicillin-producing filamentous fungi Aspergillus nidulans and P. chrysogenum (28).

The pcbAB ORF is ca. 11.2 kb in length and shares high sequence similarity at both the DNA and deduced protein levels with the Acremonium (65 and 72%, respectively), Penicillium (58 and 62%, respectively), and Aspergillus (56 and 61%, respectively) pcbAB genes. The second ORF, pcbC, is 996 bp in size and also shares a high degree of sequence similarity at both the DNA and protein levels with the Acremonium (78.6 and 90.4%, respectively), Penicillium (71.4 and 86%, respectively), and Aspergillus (70 and 86%, respectively) pcbC genes. The third ORF, orf1, is 1,065 bp in size but shares little or no sequence similarity with known ß-lactam-related genes in the GenBank/EMBL/Swissprot databases, including penDE, cefD, and cefE. However, Orf1 does show high similarity (60 to 71%) to a number of different epimerases and/or racemases from various eukaryotic or prokaryotic species (data not shown). In addition to the observed sequence similarities, pcbAB and pcbC of K. tethys are single genes without introns and are of comparable length to those of terrestrial fungi, and the deduced proteins have predicted molecular masses (415,881 Da for PcbAB and 37, 581 Da for PcbC) rather similar to those of numerous previously described pcbAB and pcbC gene products.

Together, these results suggest that pcbAB and pcbC represent the genes encoding an ACV synthetase and an IPN synthase in K. tethys, respectively. To date, ß-lactam production has been reported only for terrestrial fungi; to our knowledge, this is the first report that a marine filamentous fungus may be equipped to produce a ß-lactam antibiotic. At least from our limited screening, it is also interesting that the presence of pcbAB-pcbC-related genes does not appear to be widespread among the marine fungi.

Further analysis of the deduced pcbAB sequence showed the presence of three repeat modules (1, amino acids 286 to 1087; 2, amino acids 1371 to 2161; and 3, amino acids 2439 to 3225) and a thioesterase domain (amino acids 3587 to 3594) which are conserved in other fungal and bacterial ACV synthetases (12). Moreover, all three repeat modules contain a consensus AMP-binding motif and an acyl carrier protein domain, while modules 1 and 2 also contained putative phosphopantatheine-binding motifs (data not shown). Analysis of conserved residues in the PcbC sequences from a variety of organisms suggests that two histidine, one aspartate, and one glutamine residue are essential for binding of iron (23, 26); in K. tethys PcbC, the histidine and aspartate residues appear to be conserved, although the glutamine has probably been replaced by a further histidine residue at position 318. In addition, the two cysteine residues proposed to bind the peptide substrate (19) are also conserved at positions 104 and 255 in the deduced K. tethys PcbC sequence (data not shown). The conservation of known functional modules and specific side chain groups suggests that the putative gene products may represent functional analogues of described PcbAB and PcbC proteins.


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Identification of transcriptional start sites and promoter analyses.
 
To identify the pcbAB and pcbC transcription start sites in the 704-bp pcbAB-pcbC intergenic region, we used reverse transcription (RT)-PCR, since conventional primer extension experiments were unsuccessful, probably as a result of the low expression levels of these genes. For each gene, four different sense primers spanning potential transcription start sites upstream of the ATG start codon were separately used for RT-PCR with a common antisense primer that is complementary to the coding sequence of the gene. The locations of the primers used are shown in Fig. 2A. RT-PCR was performed on first-strand cDNAs that were reverse transcribed from DNase I-treated total RNA by use of Thermoscript reverse transcriptase (Invitrogen) and either reverse primer AB-R1 for pcbAB or C-R1 for pcbC (Table 1); control reactions were performed on the same RNA but without the addition of reverse transcriptase. PCR mixtures (in 100 µl) contained 0.2 µM (each) primer, 0.2 mM (each) deoxynucleoside triphosphates, 1.5 mM MgCl2, and 5 U of Taq DNA polymerase (Invitrogen). The PCR program consisted of predenaturation at 94°C for 2 min, followed by 35 cycles of amplification (denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 30 s) and a final extension at 72°C for 10 min in a Gene Cycler (Bio-Rad, Richmond, Calif.). Forward primers that yielded RT-PCR products of the expected size would indicate priming within a 5'-untranslated (UT) region, while those that failed to do so would indicate priming within a promoter region. As shown in Fig. 2B, the pcbAB-specific primer pair AB-R1-AB-F6 yielded a 390-bp product, while AB-R1-AB-F5 produced no detectable signal, indicating that the 5'-UT region of pcbAB is at least 236 bp in length. Using the pcbC-specific primer pair C-R1-C-F5, a 310-bp product was obtained, but no product was observed with either primer pair C-R1-C-F6 or C-R1-C-F7, indicating that the 5'-UT region of pcbC is at least 141 bp in length.




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  		FIG. 2. Transcriptional start site mapping. (A) DNA sequence of the K. tethys pcbAB-pcbC intergenic region. The coding sequences of the pcbAB and pcbC genes are in bold and the direction of transcription is indicated with an arrow. Numbers on the right refer to nucleotide positions. The primers used in RT-PCR for transcription start site mapping are underlined with dashes. (B) Mapping the transcription start sites of pcbAB and pcbC by RT-PCR. First-strand cDNA templates were prepared from total RNA of 10-week-old K. tethys cultures. Negative control reactions were performed with total RNA samples without reverse transcriptase. Lanes for pcbAB-specific RT-PCR: 1, primers AB-R1 and AB-F4; 2, primers AB-R1 and AB-F5; 3, primers AB-R1 and AB-F6; 4, primers AB-R1 and AB-F7. Lanes for pcbC-specific RT-PCR: 5, primers C-R1 and C-F4; 6, primers C-R1 and C-F5; 7, primers C-R1 and C-F6; 8, primers C-R1 and C-F7. M, DNA ladder (100 bp).

The divergent promoter sequences between the two transcription start sites were subsequently examined for putative transcription factor binding sites using MatInspector (20). In the case of pcbAB, we identified two putative binding sites (CCAAT) for PENR1 complexes (27), two putative AbaA binding sites (3) and two putative Aspergillus stunted protein (StuAp) binding sites (11). In contrast, analysis of the promoter sequence of pcbC revealed four consensus binding sites (GATA) for the nitrogen regulatory protein (NRE) (10) and two putative binding sites for StuAp. Whether these sites functionally bind proteins in K. tethys to regulate pcbAB and pcbC transcription remains to be determined, however their putative identification does suggest that the ß-lactam biosynthetic genes so-far identified in K. tethys may be regulated by environmetal factors such as nitrogen concentration.


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Temporal expression of K. tethys ß-lactam genes.
 
In general, even on rich media marine fungi are extremely slow growing and could take up to 6 months to reach the stationary growth phase. In order to determine the temporal expression pattern of pcbAB and pcbC in K. tethys, total RNA for RT-PCR analysis was extracted by use of the RNeasy Plant kit (Qiagen) from K. tethys that had been growing in sterile natural seawater supplemented with malt extract broth for 4 to 10 weeks. First-strand cDNA reactions performed with Thermoscript reverse transcriptase (Invitrogen) were used as templates for subsequent PCR; for pcbAB, primers S22 and S43 were used, for pcbC, primers S9 and S30 were used, and for control RT-PCRs, fungal-specific 28S rRNA primers JS5 and JS8 were used (Table 1). As shown in Fig. 3A, a pcbAB-specific RT-PCR product was only detected in cultures that had been grown for 10 weeks. In contrast, specific products from RT-PCR analysis of pcbC were detected at weeks 8, 9, and 10 of culture (Fig. 3B), indicating that the two genes are not coordinately regulated and that pcbC expression begins up to 2 weeks before that of pcbAB. To ensure that the total RNA samples for all time points tested were intact, RT-PCR amplification of the 28S rRNA gene was performed. In each case, the RNA was found to be not degraded and was present in approximately equal amounts (Fig. 3C).



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  		FIG. 3. Temporal expression pattern of pcbAB and pcbC. First-strand cDNA templates were prepared from total RNA of K. tethys that were cultured for 4 to 10 weeks in liquid medium. (A) RT-PCR of pcbAB transcripts was performed using primers S22 and S42 to produce a 335-bp product. (B) RT-PCR of pcbC transcripts was performed using primers S9 and S30 to produce a 328-bp product. (C) RT-PCR of 28S rRNA was performed using primers JS5 and JS8 to produce a 1.2-kb product. Negative control reactions were performed with total RNA samples without reverse transcriptase, and no RT-PCR product was obtained (data not shown). Lane 1, 4-week culture; lane 2, 5-week culture; lane 3, 6-week culture; lane 4, 7-week culture; lane 5, 8-week culture; lane 6, 9-week culture; lane 7, 10-week culture; lane M, 100-bp DNA ladder.


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Phylogenetic relationship of K. tethys proteins to other ß-lactam producers.
 
The K. tethys PcbAB and PcbC proteins were found to be most similar to homologues from Acremonium chrysogenum; PcbAB aligned with 72% similarity while PcbC aligned with 88% similarity. The higher similarity generally observed among the PcbC proteins may reflect stronger evolutionary constraints on this protein to maintain enzyme function. When we used PAUP version 4.0b (25), phylogenetic analysis of the K. tethys PcbAB protein and those from other fungal and prokaryotic (gram-positive and gram-negative) sources produced a single, parsimonious tree (Fig. 4A). The tree shows that the sequences may be divided into three separate clades with 100% bootstrap support and are formed of (i) the bacterial sequences, (ii) fungal penicillin producers (Aspergillus and Penicillium), and (iii) the two pyrenomycetes K. tethys and Acremonium chrysogenum (a cephalosporin producer). Alignment and parsimony analysis of various PcbC sequences revealed a similar phylogenetic tree, also well supported by bootstrap analysis (Fig. 4B), with the exception that four clades could be identified. Interestingly, these phylogenetic comparisons suggested both that K. tethys PcbAB and PcbC may be most closely related to similar genes from another pyrenomycete, Acremonium chrysogenum, and that these two sequence sets form a separate clade from other eukaryotic ß-lactam producers. It is noteworthy that Acremonium chrysogenum is a cephalosporin producer (1), perhaps suggesting that K. tethys could also produce a ceph-3-em-based ß-lactam derivative. It is also noteworthy that we did not identify a penDE gene, normally associated with penicillin production (7, 17), in the vicinity of pcbC but rather a putative epimerase (orf1), although the role played by orf1 (if any) remains to be determined.



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  		FIG. 4. Phylogenetic comparison of pcbAB and pcbC. (A) An unrooted tree depicting the evolutionary relatedness of the PcbAB proteins from seven different microorganisms. The translated products obtained from the GenBank/EMBL databases (accession numbers are in parentheses) were from the following species: Acremonium chrysogenum (P25464), Aspergillus nidulans (P27742), P. chrysogenum AS-P-78 (P19787), P. chrysogenum CMI314652 (P26046), Lysobacter lactamgenus (BAA08846), and Nocardia lactamdurans (P27743). (B) An unrooted tree depicting the evolutionary relatedness of the pcbC genes from 11 microorganisms. The coding sequences obtained from GenBank (accession numbers are in parentheses) were from the following species: Acremonium chrysogenum (M33522), Aspergillus nidulans (M21882), P. chrysogenum (M15083), Flavobacterium spp. (X17355), L. lactamgenus (X56660), Streptomyces cattleya (D78166), Streptomyces clavuligerus (M19421), Streptomyces griseus (X54609), S. jumonjinensis (M36687), and Streptomyces lipmanii (M22081). The bootstrap support for each branch (100 replications) is shown.


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Complementation of an Aspergillus nidulans ACV synthetase-deficient mutant with the K. tethys ß-lactam genes restores function.
 
Protoplast cotransformation of the Aspergillus nidulans strain JK2 (8), defective in both pcbAB and pyr4 (uridine biosynthesis), with plasmid pRG4 (29) and the cosmid D7221 was achieved by use of protoplast transformation as previously described (4). Transformants were selected for uridine prototrophy, and 10 were randomly picked and tested for the ability to produce penicillin-like antibiotic activity as previously described (6). Three of those tested produced zones of inhibition on Bacillus calidolactis in a well diffusion assay after 24 and 48 h of incubation, and quantitation of the activity suggested production of ca. 0.2 µg of penicillin per ml by two of these transformants (data not shown). Culture supernatants of the negative control, untransformed Aspergillus nidulans JK2, did not produce any zone of inhibition at either 24 or 48 h, while culture supernatants of the positive control (Aspergillus nidulans wild type) produced ca. 1.7 µg of penicillin per ml after both 24 and 48 h of fermentation, suggesting that the transformants produced less antibiotic activity than the wild-type control. This reduced production may be due to inefficient recognition of K. tethys promoters in Aspergillus nidulans or to related differences in regulation and expression of the genes between strains. Interestingly, prior treatment of culture supernatants with ß-lactamase (Sigma-Aldrich) resulted in abolition of antibiotic activity, suggesting that the inhibition of bacterial growth resulted from a ß-lactam-based antibiotic (data not shown).


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Bioassay for ß-lactam production by K. tethys cultures.
 
Supernatants of K. tethys cultures grown at 22°C for 10 weeks were either extracted with ethyl acetate and concentrated or directly lyophilized in order to concentrate any possible ß-lactam antibiotics present; in both cases the concentration factor was approximately 400. These were assayed using the disk diffusion method on agar plates (Antibiotic Test medium; Difco) into which cultures of E. coli ATCC 10536, Staphylococcus aureus ATCC 6538P, or Micrococcus luteus ATCC 25619 had been preseeded. With the exception of the controls (penicillin G and cephalosporin C; Sigma-Aldrich), none of the extracts at the concentrations assayed showed any inhibitory activity against the test strains (data not shown).

Although we were unable to directly detect ß-lactam production by K. tethys despite evidence that the genes pcbAB and pcbC are expressed at the level of mRNA and that at least pcbAB can complement lesions in a pcbAB-deficient Aspergillus nidulans mutant, there may be several explanations for this result. First, the putative ß-lactam produced by this marine fungus may be chemically different from those produced by its terrestrial counterparts. As a result, it may not be extractable in sufficient quantities or active against the test organisms selected at the concentrations employed in the bioassay, or it may be chemically unstable under the conditions employed in this study. Second, while temporal expression analyses showed that both pcbAB and pcbC were expressed after 10 weeks, it is conceivable that other, as yet unidentified, ß-lactam-related genes require longer times for expression before an active product can be synthesized. Alternatively, additional genes required for ß-lactam biosynthesis may be regulated by unknown factors absent from the growth medium employed in our study. Finally, it is equally possible that K. tethys is deficient in ß-lactam production despite possession and expression of genes related to the biosynthesis of such antibiotics, perhaps due to defects in one or more of the gene products.

Despite not directly detecting ß-lactam production by K. tethys, the localization of apparently functional ß-lactam-related genes, together with temporal studies demonstrating expression of the genes in question, suggests that this fungus may also produce a ß-lactam antibiotic(s). This, in turn, raises the tantalizing possibility that K. tethys produces novel ß-lactam antibiotics and/or that it produces antibiotics in the marine environment. With this in mind, we are now searching for other ß-lactam-related genes in this strain in an attempt to gain insight into the likely structure of the putative antibiotic produced by K. tethys.


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Nucleotide sequence accession numbers.
 
The 12,905-bp sequence containing pcbAB and pcbC has been deposited in GenBank under accession number AF335329 and the sequence of orf1 has been deposited in GenBank under accession number AY125466.


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ACKNOWLEDGMENTS
 
This work was supported by a grant (project no. 9040391) from the Research Grants Council of the Hong Kong Special Administrative Region, People's Republic of China.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Ave., Kowloon Tong, Kowloon, Hong Kong SAR, People's Republic of China. Phone: 852-2788-7794. Fax: 852-2788-7406. E-mail: bhrkong{at}cityu.edu.hk. Back

{dagger} Present address: Department of Microbiology, University of Otago, Dunedin, New Zealand. Back


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Applied and Environmental Microbiology, February 2003, p. 1308-1314, Vol. 69, No. 2
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.2.1308-1314.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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