M. Subba Rao*, G. Kishore and C. Rambabu
Department of Chemistry and Biochemistry, Acharya Nagarjuna University-
Dr.M.R.Appa Row Campus, Nuzvid-521 201, Krishna District, A.P. India.
*For Correspondence - email@example.com
Current trends in Biotechnology and Pharmacy April, 2010, Volume 4, No. 2
A large indigenous plasmid was found in the naphthalene degrading Pseudomonas sp. (NGK1). This plasmid, pND15 appears to contain complete genetic information for the degradation of naphthalene as the plasmid cured strain failed to utilize naphthalene as source of carbon. The E. coli transformed with pND15 showed ability to degrade naphthalene and growth pattern similar to that of wild type Pseudomonas sp. (NGK1).
Key words: Pseudomonas sp.NGK1, Plasmid, Naphthalene degradation
Polycyclic aromatic hydrocarbons (PAHs) such as naphthalene show potential toxicity to higher organisms (1-3). Owing to the bioaccumulation, inferred recalcitrance and genotoxicity, studies on the fate of naphthalene in the environment have acquired lot of importance. Attempts have also been made to find safe and reliable methods of disposing naphthalene wastes. Bioremediation is found to be the most acceptable and suitable method of decontaminating the polluted environments. However this technology mainly depends on isolation of the biological agents that either degrade or use the genobiotics as source of carbon and energy. Soil bacteria show immense potential of degrading the variety of recalcitrant chemicals that are found to be toxic to other forms of life. Naphthalene degrading soil bacteria that belong to different taxonomic groups have been isolated from polluted soil samples (4-13). In most of the cases indigenous plasmids have been implicated in the mineralization of naphthalene. These naphthalene degrading plasmids show considerable genetic diversity despite of having highly conserved naphthalene degrading genes (14-21). We have isolated naphthalene degrading Pseudomonas sp. NGK1 (10) from the effluent canals of a textile industry. This bacterial strain showed higher degradative efficiency and more tolerance to naphthalene than many naphthalene degrading strains reported till date. Therefore in the present study we attempted to understand the molecular basis of naphthalene degradation in Pseudomonas sp. (NGK1).
Materials and Methods
Media and growth conditions: The minimal-mineral salts medium contained (gm/L) K2HPO4 (0.38), MgSO4.7H2O (0.2), KNO3 (1.0), and FeCl3 (0.05). The pH was adjusted to 7 and the medium was supplemented with filter sterilized naphthalene (0.1% w/v). This medium was used for growth studies and naphthalene utilization. While isolating plasmid pND15 the cultures were grown in the medium containing (g/L) bacto-tryptone (10), yeast extract (5) and NaCl (5). The pH of the medium was adjusted to 7.5. When necessary 20 mg/mL mytomycin C was supplemented to the medium.
The minimal- mineral salts medium used for culturing Escherichia coli HB101 contained filter sterilized proline (166 mg/L), thiamine (0.166 mg/L) and leucine (41 mg/L). When required minimal medium plates were prepared by adding 2% agar to the minimal salts medium. The Pseudomonas sp. NGK1 culture was grown in the mineral salts medium supplemented with naphthalene (1%) as the sole source of carbon and energy. The growth of the bacterium was measured spectrophotometrically by monitoring the optical density at 660 nm at different incubation periods. Further cells were withdrawn from the 100 µl of the culture medium at different time intervals and the viable-cell population was counted by plating on nutrient-agar plates after performing the serial dilution. Detection of plasmid DNA: A single colony of Pseudomonas sp. NGK1 was taken from naphthalene mineral salts agar to inoculate 10 mL of LB medium and the culture was incubated for 18 h on a rotary shaker with vigorous shaking. Similarly Flavobacterium sp. ATCC27551 having indigenous plasmid pPDL2 (22) and E. coli cells without any plasmid were grown in LB medium. Plasmid preparations from these bacterial strains were isolated following the procedures described elsewhere (23) and analyzed on 0.8% agarose gel.
Plasmid curing from Pseudomonas sp. NGK1: The indigenous plasmid found in Pseudomonas sp. NGK1 is cured by using mytomycin C (24). The LB medium containing 20mg/mL of mytomycin C is inoculated with the over night culture of Pseudomonas sp. NGK1 and incubated for 18 h at 30oC. Serial dilutions were prepared from these cultures and plated on LB medium to develop single colonies. After formation of colonies, the individual colonies were patch plated on naphthalene - mineral salts agar plate. The colonies which failed to grow on naphthalene - mineral salts agar were selected from the master plate and examined for the presence of plasmid DNA.
Molecular size determnination of plasmid: The E.coli stains containing plasmids of known molecular weights were grown in LB medium and plasmid preparations from those cultures were made and analyzed on 0.8% agarose gels along with the indigenous plasmid pND15 of Pseudomonas sp. NGK1 and its molecular weight was determined by following the procedure (24).
Transformation : The competent Escherichia coli HB101 cells and the plasmid cured Pseudomonas sp. NGK1, Nah-, Sal- were prepared and transformed them with plasmid pND15 following the procedures described in Promega protocol and application guide (25). The transformants were selected by plating on naphthalene/salicylate mineral salts agar plates amended with filter sterilized proline, thiamine and leucine. The colonies were then sub-cultured and were screened for the presence of plasmid DNA following the methods described elsewhere.
Growth Behaviour of transformed Escherichia coli HB101: The growth behaviour of transformed Escherichia coli HB101 was carried out by incubating actively growing culture (5% v/v) into 250 mL Erlenmeyer flask containing 100 mL mineral salts medium amended with naphthalene (0.1% w/v ), filter sterilized proline (166 mg/L),thiamine (0.166 mg/L) and leucine (41 mg/L). The culture was incubated on a rotary shaker and the growth of the E.coli cells with and without plasmid pND15 was measured.
Enzyme assays: The cell free extract was prepared from freshly grown cells of Pseudomonas sp. NGK1 and transformed Escherichia coli HB101. The obtained cell free extract was used for different enzymatic assays. Naphthalene 1, 2- dioxygenase, 1, 2‑dihydroxynaphthalene dioxygenase, salicylate hydroxylase, catechol 1, 2 dioxygenase and catechol 2, 3 -dioxygenase were assayed according to the methods described elsewhere (7). The concentration of the protein in the cell free extract was determined spectroscopically (26). The specific activity of enzyme was expressed as mmol substrate converted or product formed/min/mg of protein.
Results and Discussion
A naphthalene degrading Pseudomonas sp. strain NGK1 was used to study its ability to utilize naphthalene as a sole source of carbon and energy. The growth behaviour of the bacterium is presented in fig. 2. It was shown that the growth of the bacterium was increased with increase in the incubation time and after 18 h the growth entered the stationary phase. The initial viable cell population of 2x106 cfu/mL has increased to 5x1010 cfu/mL after 24 h of incubation in the mineral salt medium supplemented with naphthalene (0.1% w/v). To gain better insight into the molecular mechanism of naphthalene degradation in Pseudomonas sp. strain NGK 1, an attempt was made to detect the presence of indigenous plasmid in this bacterium. While attempting to isolate the plasmid from Pseudomonas sp. strain NGK 1 we have also used Flavobacterium sp. ATCC 27551 having well characterized indigenous plasmid as positive control and plasmid less E. coli HB101 as negative control. The plasmid DNA was found only in Pseudomonas sp.NGK1 and Flavobacterium sp. ATCC 27551 (Fig. 1) whereas no plasmid band was found in plasmid preparations made from plasmid less E.coli cells. These results clearly indicate existence of indigenous plasmid in Pseudomonas sp. NGK1 and it is named as pND15.
Fig 1. Plasmid preparations from E.coli HB101 (lane 1) and Pseudomonas sp.NGK1 (lane 2). Large indigenous plasmid is found in lane 2
Fig 2. Growth behaviour of Pseudomonas sp. strain NGK1 (-♦-) and plasmid pND15 transformed Escherichia coli (-¦-) in the mineral salts medium containing
naphthalene (7.8 mM) as the sole source of carbon.
Fig 3. Restriction of pattern of plasmid pND15. Lane 1, Molecular Weight markers, Lane 2 represents uncut plasmid pND15, Lane 3 to 1 are digests of pND15
with EcoRI, PstI, Bam HI Bgl II, Hind III
Subsequently, the involvement of pND15 in the degradation of naphthalene is assessed. The plasmid DNA from Pseudomonas sp. NGK1 was cured by using mytomycin C and the cured strain was tested for its ability to grow on naphthalene containing medium. Evidently the plasmid cured strain could not grow on naphthalene containing minimal medium indicating the presence of naphthalene degrading genes on plasmid pND15. Further, the cell free extracts prepared from this plasmid cured strain showed no naphthalene degrading enzyme activities that are otherwise found in the wild type strain of Pseudomonas sp. This further strengthens the involvement of pND15 in the degradation of naphthalene. Similarly the E.coli cells transformed with pND15 showed the presence of complete set of enzymes involved in naphthalene degradation. Interestingly the E.coli HB101 cells having plasmid pND15 and wild type Pseudomonas sp. NGK1 have shown similar growth pattern (Fig.2). Several naphthalene-degrading plasmids were found in Pseudomonas strains isolated from diversified geographical regions. These plasmids show maximum genetic diversity despite of having highly conserved naphthalene degrading genes (16). In most of the cases the size of the plasmid along with other characteristics such as restriction pattern, hybridization profile is taken as an important feature to draw the comparisons among various degradative plasmids. Therefore the molecular size of pND15 is determined; from this the size of the plasmid pND15 was determined to be approximately 150Kb. In order to assess the similarity of the pND15 with other naphthalene degrading plasmids the plasmid pND15 was digested with several restriction endonucleases such as EcoRI, PstI, Bam HI Bgl II, Hind III and the restriction pattern thus obtained was compared with the restriction maps of known naphthalene degrading plasmids (Fig 3). Surprisingly none of the known naphthalene degrading plasmids shows restriction profile that matches with the restriction pattern of pND15.
The plasmid pND15, when transformed into E.coli encodes necessary enzymes for degrading naphthalene (Table 1). It was observed that the growth of E.coli in the mineral salts medium showed an initial lag phase and then entered into the exponential phase that lasted for another 50 h. The viable-cell population from 107 cfu/ml increased to 109 cfu/mL after 120 h incubation. Further the complete degradation of naphthalene was observed at 140 hr incubation. The cell free extracts of Escherichia coli HB101 also showed the accumulation of a- hydroxymuconic semialdehyde. The enzyme activities of various enzymes involved in naphthalene degradation were found in the cell free extracts of Pseudomonas sp.strain NGK1 and transformed Escherichia coli. These results clearly demonstrate the existence of complete genetic mechanism for degradation of naphthalene on indigenous plasmid pND15. Further we have tested the wild type strain of Pseudomonas sp. NGK1 to assess its ability to degrade several other monocyclic, dicyclic and substituted hydrocarbons. This strain besides efficiently degrading anthracene and phenanthrene shows high tolerance towards naphthalene and hence can be a good candidate to treat the industrial effluents containing high concentrations of aromatic hydrocarbons.
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Table 1. Activities of various enzymes in the crude cell free extracts of the cells of Pseudomonas sp. strain NGK1 and Escherichia coli(pND15)grown in the mineral salts medium containing naphthalene.
Sl.No. Enzyme Pseudomonas sp. Escherichia coli
1. NDO 0.99 0.12
2. 1, 2 – DHND 4.76 0.30
3. SALH 0.60 0.09
4. C23O 0.48 0.10
5. C12O 0.10 0.08
Enzyme activity: mmol substrate converted or product formed min-1mg-1 protein.
NDO- (Naphthalene-1, 2-dioxygenase), 1, 2-DHND- (1, 2-dihydroxynaphthalene dehydrogenase), SALH-(Salicylate hydroxylase), C23O-(Catechol –2, 3-dioxygenase) and C12O- (Catechol 1, 2-dioxygenase).