Isolation of Rhodococcus rhodochrous NCIMB13064 derivatives with new biodegradative abilities

MICROBIOLOGY LETTERS FEMS Microbiology Letters 145 (1996) 227-231

Isolation of Rhodococcus rhodochrous NCIMB 13064 derivatives 1 -1-1. with new biodegradative atxmes Anna N. Kulakova ajb, Karen A. Reid atb, Michael J. Larkin a,b, Christopher C.R. Allen ajb, Leonid A. Kulakov ajbj* a The Questor Centre, David Keir Building, Stranmillis Road, The Queens University of Belfnrt, Belfast BYIY 5AG. UK h &hoot of Biology and Biochemistry, Medical Bioiogy Centre. The Queen’s University of Beifast, Belfast BIP 7B.5, UK Received 11 September 1996; revised 27 September 1996; accepted 30 September 1996

AbStTSt Rhoabcoccus

rhodochrous

NCIMBl3064

can dehalogenate

and utilise a number of halogenated

aliphatic compounds

as sole

carbon and energy source. Mutants of NCIMB13064 can be easily isolated with an enlarged range of I-chloroalkane utilising ability. Dehalogenation of 1-chlorononane, I-chlorodecane and short-chain I-chloroalkanes (C&B) is encoded by the same plasmid pRTL1. However, a different genetic element(s) is required for the dehalogenation of 3-chloropropionic acid. Two derivatives (PZOOand P400) of R. rhodochrour NCIMB13064 were isolated which had acquired the ability to utilise naphthalene as sole carbon and energy source. Both strains lost the ability to utilise short-chain I-chloroalkanes and underwent some rearrangements Keywords:

associated

with pRTL1 plasmid.

Rhodococcus rhodochrous; I-Cbloroalkane degradation; Naphtbalene degradation

1. Introduction

The genus Rhodococcus is a diverse group of Gram-positive soil bacteria which demonstrates a remarkable ability to degrade a wide variety of xenobiotics, including aromatic and aliphatic compounds [l]. Rhodococcus rhodochrous NCIMB13064 can utilise a wide range of 1-haloalkanes as sole carbon and energy source. Different routes were shown to be present for the degradation of short-chain (C&s) and long-chain (Ci&is) 1-chloroalkanes [2]. Two

* Corresponding author. Tel.: +44 (1232) 335577; Fax: +44 (1232) 661462; E-mail: [email protected]

plasmids (pRTL1 and pRTL2) were detected in NCIMB13064 and pRTL1 (100 kb) was shown to be carrying at least some genes necessary for the dehalogenation of short-chain 1-chloroalkanes. No connection was found between the utilisation of lchloroalkanes with chain lengths of Ci2-C1s and the presence of plasmid pRTL1 in bacterial cells [3]. Three separate events were found to lead to the inability of NCIMB 13064 to dehalogenate short-chain 1-chloroalkanes: the complete loss of pRTL1, the integration of pRTL1 into the chromosome or deletion of a 20-kb fragment from this plasmid [3]. Although R rhodochrous NCIMB13064 was able to dehalogenate l-chlorononane and l-chlorodecane, it could not utilise these compounds as sole carbon and

0378-1097/96/%12.00 Copyright 0 1996 Federation of European Microbiological Societies. Published by Elsevier science B.V. PIISO378-1097(96)00414-4

228

A.N. Kulakova et al. IFEMS

Microbiology Letters 145 (1996) 227-231

energy sources. This strain was also unable to grow on aromatic compounds such as naphthalene. Here we describe the derivatives of R. rhodochrous NCIMB13064 which utilise chlorinated compounds. Two derivatives were isolated which were able to use naphthalene as sole carbon and energy source. The initial characterisation of these strains is presented.

2. Materials and methods 2.1. Bacteria and growth conditions Rhodococcus rhodochrous NCIMBl3064 which was used in this study has been described previously [2]. Isolation of dehalogenase minus (Dhll) derivatives of this strain was described in [3]. Bacteria were propagated in either a nutrient (2YT) or a minimal (M9) medium [4]. For the isolation of plasmid DNA, R. rhodochrous NCIMB13064 and its derivatives were grown in YEP medium [3]. Sodium pyruvate, n-butanol or 1-chloroalkanes (all at a final concentration of 10 mM) and solid naphthalene (1 g/l) were added to M9 medium. When the cultures were grown on minimal agar plates, short-chain chloroalkanes and naphthalene were supplied as vapours. To prepare cell-free extracts, cultures (1 litre) were harvested in mid-exponential phase after growing at 30°C in M9 medium.

Table 1 Utilisation

of chlorinated

substrates

and dehalogenation

activities in R.

2.2. Preparation

of cell-free extracts

Naphthalene or pyruvate grown cells were harvested by centrifugation and washed once with 50 mM Tris-HCl buffer, pH 7.8, containing 10% (v/v) ethanol, 10% (v/v) glycerol and 0.5 mM dithiothreito1 (TEG buffer). The cells were disrupted by sonication for 20 min at -8°C using a MSE Soniprep 150 sonicator at 16 kHz. The homogenate was then centrifuged at 38 000 X g for 1 h at 4°C and the supernatant analysed for enzyme activities. The protein concentration was determined using the BCA assay (Pierce, USA). 2.3. Enzyme assays Dehalogenation by resting cells was measured using a Corning Chloride Analyser 926 as described earlier [2]. Assays for naphthalene dioxygenase activity were performed by the method of Ensley and Haigler [5] and naphthalene cis-diol dehydrogenase according to Pate1 and Gibson [6]. (lR,2S) cis-1,2dihydroxy-1,2_dihydronaphthalene (naphthalene cisdiol) and (1 S,2R)-naphthalene cis-diol were obtained using a biotransformation method [7]. Salicylaldehyde dehydrogenase activity was assayed spectrophotometrically [8]. Salicylate hydroxylase was measured using an oxygen electrode [9]. Catechol-2,3and catechol-1 ,Zdioxygenase activities were assayed spectrophotometrically [lo]. Gentisate-l,%-dioxygen-

rhodochrous

NClMB13064

and mutant

strains

Strain

Utilisation SCh

LCh

ClCs

ClCIO

3CPA

SCh

LCh

ClCs

ClClO

3CPA

NCIMB s21 SC10 P200 P400

+ _

+ + + + +

_ _

_ _

+ _ _

+ _ _

_ -

100 0 90 0 0

10 10 10 10 9

30 0 35 0 0

25 0 30 0 0

60 60 60 60 60

+ _ _

Dehalogenation

SCh, short-chain 1-chloroalkanes: I-chloropropane, I-chlorobutane, 1-chloroheptane, 1-chlorohexane, 1-chlorooctane; LCh, long-chain: I-chlorododecane, 1-chlorotetradecane and I-chlorohexadecane; ClCs, 1-chlorononane; ClC 10, 1-chlorodecane; 3CPA, 3-chloropropionic acid were used as growth substrates. NCIMB, NCIMB13064. Dehalogenation was measured as umole chloride ion released when 1.O mM substrate was incubated with 10 ml of resting cells (lo7 cells ml-’ in 50 mM Tris/HsSOd buffer pH 8.0). Corrections were made for abiotic chloride release and dehalogenation was expressed as a percentage of the chloride ion released when NCIMB13064 was incubated with l-chlorobutane (CIC?) as a substrate. Dehalogenation of short-chain and long-chain I-chloroalkanes is presented as average of the values obtained in at least three separate experiments for each of the corresponding substrates,

A.N. Kulakova et al. IFEMS Microbiology Letters 145 (1996) 227-231

ase activity was measured using an oxygen electrode VUThe presence of salicylate in acidified ethyl acetate extracts was tested by GC-MS analysis [12]. 2.4. DNA techniques Plasmid DNA from R. rhodochrous NCIMB13064 and its derivatives was isolated as described by Schreiner et al. [13] with the only difference being that cells were grown in YEP medium. Isolation of total DNA and the analysis of DNA preparations were performed as described earlier [3].

3. Results 3.1. Utilisation of chlorinated compounds by derivatives of NCIMBl3064

Although NCIMB13064 could not utilise l-chlorononane and I-chlorodecane as sole carbon and energy sources, a significant level of dehalogenase activity against these substrates was observed (Table 1). Derivatives using 1-chlorononane and 1-chlorodecane as sole carbon and energy sources arose spontaneously in the NCIMB13064 cell populations with the frequency of between 10m6 and 10m7. lChlorononane utilising mutants (ClCg+) always utilised 1-chlorodecane (ClCio+) and vice versa and mutants showed dehalogenase activity against a range of substrates that was characteristic of the parental strain (e.g. mutant X10; Table 1). Derivatives of NCIMB13064 which were selected on the basis of the loss of short-chain 1-chloroalkane utilisation (i.e. loss of pRTL1 plasmid - S21, or loss of 20 kb of pRTL1 - P200) were not able to dehalogenate or utilise 1chlorononane and 1-chlorodecane (Table 1). These results indicate that the same dehalogenase gene is likely to be involved in the dehalogenation of 1-chloroalkanes with chain lengths from Cs to Cio. Dehalogenation of 3-chloropropionic acid (3CPA) by NCIMB13064 was shown to be effective, although the wild-type (WT) bacterium could not use this compound as a growth substrate [2]. All NCIMB13064 Dhl- derivatives retained the ability to dehalogenate 3CPA (Table 1) and this suggests the presence of at least two inde-

229

pendent dehalogenation systems in NCIMB 13064. Mutants of NCIMB 13064 which were able to utilise 3CPA as the sole source of carbon and energy arose spontaneously at a frequency of approximately lo+ and all retained their ability to grow on all l-chloroalkanes. 3.2. Degradation of naphthalene by R rhodochrous NCIMB13064

derivatives

A number of derivatives of NCIMB13064 were isolated which had lost the ability to dehalogenate and utilise short-chain 1-chloroalkanes (Dhl-) [3]. Two of these (P200 and P400) had unexpectedly acquired the ability to use naphthalene as sole carbon and energy source whilst retaining the ability to utilise long-chain chloroalkanes and dehalogenate 3CPA. Both derivatives grew well at 25-30°C on minimal M9 plates in naphthalene vapour and in minimal liquid medium supplemented with naphthalene. Salicylate was tested as the growth substrate for these strains in concentrations ranging from 1 mM to 10 mM. Both P200 and P400 showed no or very slight growth on salicylate either in liquid medium or on plates. The presence of salicylate was not detected by GC-MS analysis in supernatants of P200 or P400 strains when grown on naphthalene. The Nab+ phenotype in strains P200 and P400 appeared to be fairly stable, however, Dhl+ re-

Table 2 Specific activities of enzymes of naphthalene metabolism in cell extracts of Rhodococcus rhodochrous strains P200 and P400 Strain

Substrate

Specific activity (nmole/min/mg NO

P200 P200 P400 P400

Naphthalene Pyruvate Naphthalene Pyruvate

0.14 i 0.01 0.34 co.01

NCDa 220.0
C230 8.8
protein) Cl20 3.3 0.9 5.0 7.1

Cells of strains P200 and P400 were grown on naphthalene or pyruvate as the sole carbon and energy source. Specifk activities of the enzymes of naphthalene metabolism were measured in cellfree extracts. The results are expressed as an average of two experiments. NO, naphthalene dioxygenase; C230, catechol-2,3-dioxygenase; C120, catechol-1,2_dioxygenase; NCD, (lR,2S)-naphthalene cisdihydrodiol dehydrogenase. *Activity was also measured with (1 S,ZR)-naphthalene cis-dihydrodiol as substrate, no activities were detected in this case.

230

A.N. Kulakova et al. I FEMS Microbiology Letters 145 (1996) 227-231

vertants which arose at a frequency of between 10-j and lop8 had all lost the ability to degrade naphthalene. Naphthalene dioxygenase and naphthalene cis-diol dehydrogenase activities were detected in cell-free extracts from both P200 and P400 strains when grown on naphthalene. The activities of these enzymes were not detected in pyruvate grown cells indicating that naphthalene degradation was inducible in these derivatives (Table 2). We have not been able to detect activity of salicylaldehyde dehydrogenase and salicylate hydroxylase even using assays optimised for another Rhodococcus strain [9]. Also, there was no gentisate oxygenase activity found in the cell-free extracts of P200 and P400 strains, Both catechol2,3- and catechol-1,2-dioxygenase activities were detected in the cell-free extracts of naphthalene grown P200 and P400 strains. However, catechol-1,2-dioxygenase activity was also detected in pyruvate grown cells. In fact, for P400 this activity was higher in pyruvate grown than naphthalene grown cells. This suggests that catechol-1,2-dioxygenase may not be associated with naphthalene degradation. To characterise strain P400, its plasmid and total DNA were isolated and analysed with restriction endonucleases. This analysis revealed that P400 had lost both pRTL1 and pRTL2 plasmids detected in the original NCIMB13064 strain [3]. Strain P200 which had lost a 20-kb fragment of pRTL1 was described earlier [3]. Dhl+ Nab- revertants of P400 regained the plasmid, the DNA of which was indistinguishable from that of the WT NCIMB13064 strain.

4. Discussion The ability of NCIMB13064 to give rise to derivatives which are able to utilise new chlorinated substrates has been demonstrated in this study. The dehalogenation of 1-chlorononane and 1-chlorodecane appears to depend on the same enzymatic system as that for dehalogenating short-chain 1-chloroalkanes. Different enzymatic systems seem to be involved in the dehalogenation of 3CPA and 1-chloroalkanes. The most important result of this study was the isolation of NCIMB13064 derivatives which acquired the ability to utilise naphthalene as the sole

carbon and energy source although this bacterium was originally detected for growth on 1-chlorobutane [2]. These indicate the presence of a ‘silent pathway’ for the degradation of aromatic compounds in R. rhodochrous NCIMB13064. Silent genes of the catechol meta-cleavage pathway were previously found on Pseudomonas putida plasmids for naphthalene degradation [14]. However, there have been no reports of silent genes encoding the initial steps of naphthalene oxidation. Both Nab+ strains were found amongst derivatives of NUMB13064 which had lost the ability of short-chain 1-chloroalkane degradation, but retained the long-chain l-chloroalkane degradation phenotype. In both strains pRTL1, which controls short-chain 1-chloroalkane degradation has been affected; it has been lost from strain P400 and undergone a 20-kb deletion in P200 (deleted fragment was shown to be integrated in bacterial chromosome [3]). Preliminary results (not shown) indicate that rearrangement of the Rhodococcus genome in P400 associated with integration of pRTL1 plasmid into chromosome may be responsible for the switching between Nab+ and Dhl+ phenotypes. It is worth noting that the other Dhl- strains described in a previous paper [3], which had lost pRTL1, were unable to utilise naphthalene. There are few reports available on naphthalene degradation in Rhodococcus spp. [9,11]. Degradation of naphthalene by strains P200 and P400 occurs with the expression of catechol-2,3-dioxygenase activity. At the same time we have not been able to detect activities of salicylaldehyde dehydrogenase and salicylate hydroxylase in cell-free extracts of these strains. Although it is possible that these enzymes are extremely unstable or require unusual cofactors, salicylate was not detected in supernatants from naphthalene grown cells. This means that either salicylate was not accumulated in detectable quantities in P200 and P400 cultures or the naphthalene degradation pathway of these strains differs from that known for Pseudomonas. It is interesting to note that only degradation of naphthalene via the gentisate pathway has been previously reported for Rhodococcus strains [9,1 l] and this is the first indication of naphthalene degradation through the meta-pathway of catechol oxidation in this genus. An unexpected finding was also the simultaneous presence of catechol-2,3- and catechol-1,2-dioxygen-

A. N. Kulakova et al. I FEMS Microbiology Letters 145 (1996) 227-231

ase activities in the naphthalene grown P200 and P400 cells. Although unusual, the simultaneous presence of these activities has been previously reported for some Pseudomonas strains 1151.At the same time results presented suggest that only catechol-2,3-oxy genase activity is associated with the degradation of naphthalene. Cloning of the naphthalene degradation genes from R. rhodochrous NCIMB13064 is now under way.

Acknowledgments

This work was supported by The Queen’s University Environmental Science and Technology Research Centre. The authors would like to acknowledge the assistance of Mr. D.K. Clements with the GS-MS analysis.

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