1 Department of Microbiology, Changwon National University, Kyongnam 641-773, Korea
2 Department of Pharmacy and Research Center for Bioresource and Health, Chungbuk National University, Cheongju 361-736, Korea
3 Department of Microbiology, Chungbuk National University, Cheongju 361-736, Korea
Correspondence
Kyoung Lee
Klee{at}sarim.changwon.ac.kr
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ABSTRACT |
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Present address: Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yusong, Taejon 305-600, Korea.
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INTRODUCTION |
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In the present study, we isolated P. putida F1 cells adapted to grow on n-propylbenzene, n-butylbenzene, cumene and biphenyl as carbon and energy sources, and examined the biochemical and genetic backgrounds of the adapted F1 strains to better understand the mechanisms underlying the control of the expressions of the cym, cmt and tod catabolic genes. In addition, we compared the genetic backgrounds obtained from the strains adapted to grow on biphenyl and other aromatics at our laboratory with those from the natural isolate P. putida CE2010.
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METHODS |
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Molecular cloning and Southern blot analysis.
The standard techniques for molecular cloning were performed as described by Sambrook & Russell, (2001). Plasmids were isolated with a Bioneer plasmid extraction kit (Taejeon). DNA fragments from gels were purified using GFX PCR DNA and Gel Band Purification kit (Amersham Pharmacia Biotech). Southern blot analysis was performed with a Photogene Nucleic Acid Detection System version 2.0. (Gibco-BRL), using a biotin-labelled probe made using a BioPrime DNA labelling kit according to the manufacturer's protocol.
Mutational analysis of the todF, cymR and todS genes from adapted strains.
The todF and cymR genes from adapted strains were retrieved by PCR amplification. PCR fragments were cloned into a pGEM-T easy vector and the resulting plasmids were amplified in E. coli DH5. The plasmids purified from the E. coli recombinant strains were subjected to nucleotide sequence analysis. The mutation site(s) found was additionally confirmed by nucleotide sequence analysis from a second clone. For the mutational analysis of the todS gene from adapted strains, pTodS-B1, -B4, -C1, -C4 and -C6 (Table 1
) were used for nucleotide sequencing. Mutation(s) was also confirmed from the second clone. Nucleotide sequence analysis was carried out by Genotech using an ABI Prism 3700 DNA Analyser (Applied Biosystems).
Construction of P. putida F1(cymR : : Tcr).
The plasmid pEN-15 harboured in E. coli DH5 was transferred to a P. putida F1 recipient by conjugation with an E. coli HB101(pRK2013) helper strain as follows. Cells of E. coli DH5
(pEN-15), E. coli HB101(pRK2013) and P. putida F1, grown freshly overnight in LB, were suspended individually in saline to OD600 of 2. The suspensions were mixed in equal volumes and the mixture was placed on a nitrocellulose filter deposited on the surface of LB agar medium. The plate was then incubated at 28 °C for 12 h. The mated cells were spread, after serial dilution with saline, on MSB agar containing 0·5 % ethanol and tetracycline to select P. putida exoconjugants. pEN-15 could not replicate in P. putida F1 and thus the Tcr gene had to be incorporated into the chromosome for P. putida F1 strain to be resistant against tetracycline. The double cross-over recombination event was expected to yield a cymR : : Tcr mutation. The expected exconjugants were screened by PCR using the primers used for amplification of cymR. The mutation was also confirmed by Southern analysis using the Tcr gene as a probe. The right mutants were obtained at the probability of one in forty.
Measurements of the oxygen uptake and substrate preference of CmtE.
The rate of oxygen consumption of P. putida F1 or of its adapted strains grown in MSB on indicated carbon source(s) was determined with a Clark-type oxygen electrode (Rank Brothers) as described previously (Cho et al., 2000). The methods for IPTG induction of the cloned gene from recombinant E. coli cells, preparation of cell extracts from bacterial cells and the production of meta-cleavage products were described in a previous report (Cho et al., 2000
). The activities of CmtE were determined at 25 °C by measuring the absorbance decrease of each meta-cleavage product. Assay mixtures (1 ml total volume) contained 0·1 M potassium phosphate buffer (pH 7·5), meta-cleavage product (final 50 µM) and crude extract. The wavelengths (extinction coefficients in mM-1 cm-1) (Duggleby & Williams, 1986
; Seah et al., 1998
) used to monitor the change in concentration of the meta-cleavage products of catechol, 3-methylcatechol, 4-methylcatechol, 3-propylcatechol, 3-isopropylcatechol and 2,3-dihydroxybiphenyl were 376 (40), 389 (11·9), 382 (24·5), 383 (20), 395 (10·7) and 434 (19·8) nm, respectively. Protein was determined using the BCA protein assay (Pierce) with BSA as the standard. Specific enzyme activities are reported as µmol substrate utilized min-1 (mg protein)-1. UV/visible absorbance spectra were measured on a spectrophotometer (Scinco, model 2130). Oxygen uptake and CmtE activity assays were conducted in triplicate, and the initial rates of the assays were determined and used for calculation of means and standard deviations.
PCR analysis.
Reaction mixtures (50 µl) contained chromosomal DNA (20 ng), ExTaq DNA polymerase (1 U), dNTP (0·2 mM each) and the primer set (0·5 µM each) in buffer supplied by the manufacturer (TaKaRa). PCR was carried out with a Bioneer thermal cycler (Taejeon) under the following conditions: 2 min at 94 °C, and then 30 cycles of 30 s at 94 °C, 30 s at 55 °C, variable times at 72 °C and a final 5 min at 72 °C. The times used for the DNA polymerization step at 72 °C were 1·5, 1·5, 1 and 4 min for the PCR products designed for pJHE-W1, pEN-9, pEN-14 and TodST-F1, respectively. Primers were synthesized by Bioneer. When required, the sequences were confirmed by nucleotide sequence analysis.
Fluorescence measurements.
Cells harvested by centrifugation at a specific incubation time were washed twice with saline and resuspended to OD600 0·2. The intensity of the fluorescence was measured using a spectrofluorophotometer (model RF-5391PC, Shimadza). Samples were prepared from three independent cultures. The excitation and emission wavelengths used were 393 and 509 nm, respectively, with each 3·0 nm of wavelength split. The specific fluorescence intensity of each sample was defined as the measured fluorescence intensity divided by the OD600.
Measurement of meta-cleavage product accumulation with a resting cell system.
P. putida F1 and its recombinant cells were cultured in a shaking incubator for 24 h with toluene as a carbon and energy source. Cells were harvested by centrifugation and suspended in 0·1 M potassium phosphate buffer (pH 7·5) to OD600 0·5. Twenty microlitres of 0·1 M chemical stock dissolved in methanol was added to 10 ml the cell suspension in 250 ml Erlenmeyer flasks. The reaction mixtures were incubated at 28 °C with shaking at 180 r.p.m. and the supernatants from 1 ml aliquots were obtained every hour by centrifugation. The accumulation of meta-cleavage product from n-propylbenzene, cumene and biphenyl was monitored at the wavelengths described above; that from n-butylbenzene was monitored at 388 nm.
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RESULTS |
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The adapted strains had a mutation in cymR that led to the constitutive expression of the cym and cmt operons
Early studies showed that the meta-cleavage products formed from n-propylbenzene, n-butylbenzene, cumene and biphenyl through consecutive actions of toluene dioxygenase, TodD and TodE enzymes could be hydrolysed inefficiently by TodF in the tod pathway (Cho et al., 2000; Gibson et al., 1968
). Therefore we believed that the todF gene might be altered in the adapted strains to act on a broad range of substrates. However, nucleotide sequence analysis showed no mutations in any of the PCR-cloned todF genes from the five adapted strains.
The second possibility for the hydrolysis of the meta-cleavage products was that the adapted strains employed CmtE, as the cmt pathway substrate (6-isopropyl-HOHD) of the enzyme is the same as that formed by cumene from the tod pathway (Fig. 1). The cmt operon is induced in the presence of p-cumate and regulated by a repressor CymR (Eaton, 1997
). For the adapted strains to express cmtE in the absence of an effector, the CymR protein has to be incapable of functioning. At first, the level of CmtE expression in P. putida F1, and in its adapted strains, was determined by measuring the promoter activity of the cmt operon based on the green fluorescent protein (GFP)-reporter vector (pEN-19). The results are shown in Table 2
. The cmt operon in P. putida F1 was not induced by succinate, n-propylbenzene, n-butylbenzene, cumene, biphenyl and toluene, but was induced by p-cymene and p-cumate. In contrast, the cmt operon was constitutively expressed in all five adapted strains at levels similar to that in P. putida F1(cymR : : Tcr). The constitutive expression of the catabolic cym operon in the adapted strains and P. putida F1(cymR : : Tcr) was also confirmed by oxygen consumption assays using p-cymene as a substrate (data not shown). These results indicate that the cym and cmt catabolic operons are constitutively expressed in all five adapted strains, and that this is probably achieved by the impairment of the repressor, CymR, resulting in weak or the non-binding of CymR to operator regions in both the cym and cmt promoters. Sequencing of the cymR gene from the adapted strains confirmed the presence of different types of mutations, which included point mutation in strains C1 (T284C), C4 (C35A), C6 (G528A) and B4 (G541A), an insertion in strain B1 (342TGATT343) and a deletion in strain B4 (C537). Numbers indicate nucleotide position of the DNA sequence GenBank accession no. U24215. Number 1 corresponds to 841 of the sequence accession number. Changes in nucleotides of the cymR gene yield mutations in the repressor as shown in Fig. 2
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The growth of adapted F1 strains on biphenyl and other new aromatic hydrocarbon substrates should be accompanied by the induction of the tod catabolic operon. Therefore, the expression of the tod operon was assessed by monitoring the expression of GFP from a transcriptional fusion todX : : gfp construct (pEN-12) and by determining the toluene-dependent oxygen uptake of P. putida F1 cells exposed to various aromatic hydrocarbons during growth in the presence of succinate. The results are shown in Fig. 3. As expected, the induction of GFP and toluene-dependent oxygen uptake were observed in the presence of benzene, toluene and ethylbenzene. In contrast, the expression of the tod catabolic operon was not detected in either analyses in response to other aromatic hydrocarbons such as n-propylbenzene, n-butylbenzene, cumene and biphenyl. In fact, GFP induction was not observed in the presence of the latter aromatic hydrocarbons during 5 day culture by 24 h testing.
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DISCUSSION |
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It has been previously demonstrated that a natural isolate of P. putida CE2010 assimilates biphenyl in a CmtE and tod catabolic operon manner, via a mosaic of pathways (Ohta et al., 2001). Although the biochemical backgrounds of the growth of P. putida CE2010 and of the adapted F1 strains on biphenyl appear to be similar, the ways in which the expressions of the degradation enzymes are controlled appear to differ as follows. P. putida CE2010 differs by a single base in the cmt promoter-operator region from P. putida F1 in the DNA region from the 3' terminus of cymE to the 5' terminus of todF (GenBank accession number AB042508) (Fig. 1
). It was concluded that P. putida CE2010 first degrades biphenyl to 6-phenyl-HOHD using the tod pathway enzymes, and degraded further by CmtE (Fig. 1
), which is constitutively expressed by the less effective binding of the repressor CymR to the cmt operator region (Eaton, 1997
) due to the changed nucleotide. No base difference was found in cymR in P. putida strains F1 and CE2010. Although the involvement of CmtE in the hydrolysis of the ring-fission products in both P. putida CE2010 and the adapted F1 strains is the same, the expression levels of CmtE could differ. While in P. putida CE2010 CmtE was constitutively expressed at a low level, though inducible by biphenyl, in the adapted F1 strains CmtE was constitutively expressed at a high level, and was not inducible by biphenyl. Furthermore, it was interesting to find in the same study that when a P. putida CE2010-type cmt promoter region was artificially introduced into P. putida F1, the recombinant P. putida F1 could not grow on biphenyl as the sole carbon source (Ohta et al., 2001
). This result indicates that the constitutive expression of CmtE is not enough for P. putida F1 to achieve the biphenyl-dependent growth, and thereby suggests that the induction of the tod operon by biphenyl differs in two strains. Because P. putida CE2010 can grow on biphenyl, it is expected that the tod operon is inducible by biphenyl, and that this probably reflects the amino acid differences of TodSCE2010 and TodSF1, the latter of which is unaffected by biphenyl. The nucleotide sequence of TodSCE2010 is not available in the databases.
The level of induction of the tod catabolic operon in P. putida F1 by various aromatic hydrocarbons has been determined previously by examining the level of TodE expression and the presence of its mRNA (Cho et al., 2000). While n-propylbenzene and cumene induced the TodE activity at a level of one third that induced by toluene and also yielded mRNA signals, n-butylbenzene and biphenyl induced a basal level of TodE activity and gave negative mRNA signals. In the present study, measurements of the activity of a gfp-based reporter of the promoter of todX, and of toluene-dependent oxygen uptake, indicated that the expression of the tod catabolic operon is negligible in the presence of n-propylbenzene, n-butylbenzene, cumene or biphenyl (Fig. 3
). The discrepancy between the previous and the present work in induction of the tod catabolic operon by the chemicals may be due to the methods of cell culture applied. The cells used for the previous experiments were from plates whereas the cells used in this study were from liquid culture. In many aspects including availabilities of oxygen, water and other nutrients, the conditions imposed on the cells would be not the same in the two culture systems. Nevertheless, the induction of the tod catabolic operon even by n-propylbenzene and n-butylbenzene may be insufficient to support the growths of the recombinant strains P. putida F1(cymR : : Tcr) or P. putida F1(pJHE-W1), in which CmtE is expressed constitutively.
CmtE hydrolyses 6-phenyl-HOHD (Ohta et al., 2001) as well as 6-isopropyl-HOHD (Eaton, 1996
). In the present study, we further characterized the substrate preference of CmtE. As pointed out by Ohta et al. (2001)
, the level of amino acid sequence identity between CmtE and the known hydrolases identified from aromatic degradation pathways is relatively low. In the web databases, CmtE was found to have a maximum sequence identity of 34 % with Gram-positive dibenzofuran-degraders Terrabacter sp. strain DBF63 (Habe et al., 2002
) and Rhodococcus sp. strain YK2 (Iida et al., 2002
), which indicated that CmtE is probably separated from the groups of known hydrolases. In this respect, it may be that CmtE has different substrate preference compared to the well-characterized BphDLB400, which is also known to hydrolyse 6-isopropyl- and 6-phenyl-HOHD. The latter enzyme has been identified in the biphenyl degradation pathway of Burkholderia sp. LB400 (Hofer et al., 1993
; Mondello, 1989
). CmtE showed higher activity toward 6-isopropyl-HOHD than 6-phenyl-HOHD and showed relatively high activity toward 6-methyl-HOHD (Table 3
), whereas BphDLB400 showed higher activity toward 6-phenyl-HOHD than 6-isopropyl-HOHD, and little activity to 6-methyl-HOHD (Seah et al., 1998
). This substrate preference of CmtE seems to lie between that of BpbDLB400 and TodF, which showed highest specificity for HOHDs with small substituents at C-6 (Seah et al., 1998
).
An early study showed that the introduction of the bphD gene obtained from the biphenyl degrader Pseudomonas pseudoalcaligenes KF707 into P. putida F1, enabled its growth on biphenyl (Furukawa et al., 1993). However, as biphenyl could not induce the tod catabolic operon in P. putida F1, the recombinant strain could not grow on biphenyl. In the study, however, it is likely that the selected recombinant strains might contain additional mutation(s) to allow biphenyl recognition as an effector. During this study, we found that P. putida F1(pJHE-W1), which was isolated initially from LB with kanamycin and contained the expected plasmid, could not grow on biphenyl as a sole carbon and energy source. However, a few colonies were formed with increased incubation time (23 weeks) due to additional mutation(s).
The involvement of a two-component signal transduction system as a transcriptional regulatory unit in the tod pathway is intriguing, because many other degradation pathways of biphenyl and other aromatics employing similar cis-dihydrodiol pathways use single component transcriptional regulators (Diaz & Prieto, 2000). The situation is complicated by the fact that TodS is composed of multiple protein domains as shown in Fig. 2
. The presence of a receiver domain and two PAS domains in TodS implies that the sensor may recognize different environmental and/or intracellular signals. In this regard, both TodS and TodT proteins are known to be required for the chemotaxis to aromatic hydrocarbons in P. putida F1 (Parales et al., 2000
), indicating that the catabolism and microbial behaviour are co-ordinately regulated, and the chemotaxis is probably controlled at the transcriptional level. Similar two-component signal transduction systems have been identified in the pathways of aerobic styrene degradation (O'Leary et al., 2002
; Panke et al., 1999
; Santos et al., 2000
; Velasco et al., 1998
), in other forms of aerobic toluene degradation (Mosqueda et al., 1999
), and in anaerobic toluene degradation (Coschigano & Young, 1997
; Leuthner & Heider, 1998
). The sensor proteins found in anaerobic toluene degradation are shorter than TodS and contain two PAS domains and one histidine kinase domain. However, the functions of each domain from the aromatic hydrocarbon sensors have not been identified with the exception of that of the leucine zipper dimerization motif from TodS in P. putida F1 (Lau et al., 1997
). In the present study, we were able to identify amino acids in TodS responsible for the recognition of new and the original effectors. The mutations were found in the receiver domain, the second PAS domain and in their boundaries (Fig. 2
). Mutations found in strains C1 and B1 increased the hydrophobicities of the amino acid residues, and those in strains C6, B4 and C4 introduced charge changes. The exact functions of those amino acids used for effector binding or for the activation of TodS require further investigation.
Finally, this study provides a means to determine the important amino acid residues of CymR in terms of its structural stability or DNA binding, and the residues required by TodS for effector binding. These modified proteins could be obtained from F1 strains adapted to assimilate n-propylbenzene, n-butylbenzene, cumene and biphenyl.
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ACKNOWLEDGEMENTS |
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Received 9 October 2002;
revised 27 November 2002;
accepted 27 November 2002.