Department of Microbiology, Cornell University, Ithaca, NY, 14853-8101, USA1
Biotechnology Center For Agriculture and the Environment, Cook College, Rutgers University, New Brunswick, NJ, 08901-8520, USA2
Author for correspondence: Eugene L. Madsen. Tel: +1 607 255 3086. Fax: +1 607 255 3904. e-mail: elm3{at}cornell.edu
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ABSTRACT |
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Keywords: microbial community, sediment, biodegradation, diversity, nahG, salA
b The GenBank accession number for the sequences of the tnpA-like gene, nahG and nahR of P. putida NCIB 9816-4 is AF491307. The GenBank accession numbers for the sequences of the nahRnahG intergenic region and the nahR homologue genes of strains Cg1, Cg2, Cg5, Cg7, Cg9, Cg11, Hg8 and N1 are AF491308AF491315, respectively. The GenBank accession numbers for the sequences of nahR from sediment-extracted DNA are AF491316AF491324.
a Present address: National Environmental Engineering Research Institute (NEERI), Nehru Marg, Nagpur 440 020, India.
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INTRODUCTION |
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The NahR protein, a LysR-type transcriptional regulator (LTTR), is necessary for activation of both the upper (genes nahA-F) and lower (genes nahG-M) operons on the NAH7 plasmid (Huang & Schell, 1991 ; Schell, 1985
, 1986
, 1993
; Schell et al., 1990
; Schell & Poser, 1989
; Schell & Wender, 1986
). Studies of the NAH7 plasmid in P. putida G7 have shown that NahR is constitutively expressed at low levels (Cebolla et al., 1997
). When bound to its inducer, salicylate, NahR activates both nah operons by interacting with DNA enhancer sequences upstream of the promoter region in upper and lower nah operons (Schell & Poser, 1989
). The 5' flanking region of known nahR genes is the divergently transcribed nahG-nahR promoter region. NahR binding in this -60 bp region upstream of the nahG gene transcription start site induces nahG gene transcription, but represses its own expression by negative autoregulation (Schell & Poser, 1989
; Schell & Wender, 1986
; Yen & Serdar, 1988
). The exact mechanism of autoregulation remains to be elucidated (Yen & Serdar, 1988
).
P. putida NCIB 9816 is another well-characterized bacterium capable of utilizing naphthalene as sole carbon and energy source (Cane & Williams, 1986 ; Kurkela et al., 1988
; Serdar & Gibson, 1989
; Simon et al., 1993
). This trait is conferred in P. putida NCIB 9816-4 by an 81 kb plasmid, pDTG1, that encodes key early enzymic steps in naphthalene degradation (Connors & Barnsley, 1980
; Kurkela et al., 1988
; Serdar & Gibson, 1989
; Simon et al., 1993
) The structural genes encoding naphthalene-degrading pathways in a variety of bacteria are highly conserved (Bosch et al., 1999
, 2000
; Goyal & Zylstra, 1996
; Serdar & Gibson, 1989
; Yen & Serdar, 1988
), although distinctive nahAcAd homologues (phn, nar) have been described (Fuenmayor et al., 1998
; Goyal & Zylstra, 1997
; Larkin et al., 1999
; Laurie & Lloyd-Jones, 1999
, 2000
; Saito et al., 2000
). The diversity and function of the regulatory region for naphthalene-catabolic operons is poorly explored. As of December 2001, GenBank listed nahR homologues from P. putida G7, P. stutzeri AN10 and Ralstonia sp. strain U2. Reports about nahR from environmental nucleic acid extracts are restricted to a single study (Silva et al., 1995
) which reported ethidium bromide- and hybridization-based detection of NahR amplified by PCR from four soil samples: amplification was sporadic and no sequence analysis was performed. Stuart-Keil et al. (1998)
showed by using Southern blot hybridization and RFLP analysis that plasmids carried by bacteria native to a coal-tar-contaminated site are closely related to pDTG1. With the above-described genetic information as a foundation, we investigated the degree to which the genetic regulatory system in the NAH7 plasmid applies to micro-organisms native to naphthalene-contaminated sediments and groundwater in South Glens Falls, NY, USA
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METHODS |
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Sediment samples from the contaminated study site.
Sediment samples were obtained in South Glens Fall, NY (Herrick, 1995 ; Herrick et al., 1997
; Hohnstock et al., 2000
). Sediments representing a broad spectrum of locations and contaminant exposure histories were examined. Unless otherwise specified, all samples were transported from the site to the laboratory and stored at 4 °C until used. Uncontaminated aquifer and contaminated aquifer samples were obtained via aseptic coring techniques from near the site water table (Madsen et al., 1991
) in 1989 and were stored in core barrels. The contaminated source sample, very rich (>30 mg kg-1) in coal tar and naphthalene, was excavated from the site in 1991 and placed in a bucket; similarly clean sediment was excavated in 1991, but from outside the contaminated zone. Seep sediment was gathered in 1995, 400 m down gradient from the contamination source in a low elevation area where naphthalene (
10 mg kg-1) had accumulated in an organic matter-rich stream bed. On site frozen seep was gathered from the seep area in 1993, but immediately frozen in liquid nitrogen and stored at -20 °C after reaching the laboratory. Sediments were incubated three ways. In one pair of treatments, 2 g sediment samples were amended with 50 mg naphthalene crystals and distilled water (100 µl) with and without NH4NO3 (2·5 mg) plus K2HPO4 (2·5 mg) and incubated for 2 weeks at room temperature (22 °C). The third treatment replaced the above NH4NO3/K2HPO4 mixture with the full complement of inorganic nutrients (500 µl MSB medium; Stanier et al., 1966
) and the incubation was at 30 °C for 2 days.
Sediment DNA extraction and PCR amplification.
Nucleic acids from various sediment samples were extracted using a FastDNA spin kit for soil (Qbiogene) according to the manufacturers instructions. Sediment samples (0·5 g dry wt) and 100 µl elution buffer were used for each DNA extraction. For PCR amplification of the sediment-extracted DNA, it was diluted 1:10 with distilled water. The 50 µl PCR reaction mixture contained each primer set (onahR-F/inahR-R or onahR-F/onahR-R; 0·5 µM), 1xPCR buffer (Gibco-BRL), dNTP (50 µM), MgCl2 (1·5 mM) and 1 U Taq DNA polymerase (Gibco-BRL). A cycling regime of 94 °C for 5 min (1 cycle), 94 °C for 30 s, 43 °C for 1 min, 72 °C for 1 min (30 cycles) and 72 °C for 5 min (1 cycle) was employed (PTC-200 thermocycler; MJ Research).
Cloning and sequencing of nahR homologues.
The nahR-like genes were amplified with onahR-F and onahR-R primers. The intergenic regions between nahG-like and nahR-like genes were amplified using the degenerate primer set (pro-F/pro-R). Amplified PCR fragments were cloned into pCR2.1-TOPO, a ligation-ready vector containing kanamycin and ampicillin resistance genes, and M13 primer sites for sequencing (TOPO TA cloning kit; Invitrogen). Characterization of the cloned inserts was checked by both PCR and enzyme digestion with EcoRI. The constructed plasmids were introduced into E. coli INV- F' competent cells (One Shot; Invitrogen). In an attempt to optimize clone diversity, E. coli INV-
F' was incubated for only 45 min at 37 °C after transformation. Both directions of nahR-like genes were sequenced using M13 reverse and M13 forward primers and all sequencings were completed on an ABI model 377 instrument at the Biological Resource Center, Cornell University, NY. The DNA sequences were aligned by using the MEGALIGN program and all sequence comparisons were computed as percentage identity using CLUSTAL (DNASTAR).
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RESULTS AND DISCUSSION |
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The degenerate primers designed from these nahR sequences successfully amplified the entire nahR-like genes (921 bp; using primers onahR-F and onahR-R) and internal fragments (315 bp; using onahR-F and inahR-R) from five wild-type strains, Cg1, Cg2, Cg5, Cg7, Cg9, (Fig. 2, lanes 9, 10; 13, 14; 17, 18; 21, 22; and 25, 26; respectively) from our coal tar waste study site in Glens Falls, NY (Herrick, 1995
; Herrick et al., 1997
; Hohnstock et al., 2000
). P. putida NCIB 9816-4 and P. putida G7 (isolated originally from soil from Bangor, Wales and Berkeley, CA, respectively) were used as positive controls (Fig. 2
, lanes 1, 2 and 5, 6). P. putida F1 (toluene-degrading bacterium), E. coli DH5
, E. coli JM109, plasmid pVIK112 and water were successfully used as negative controls (data not shown). The amplicon with onahR-F/inahR-R and onahR-F/onahR-R was not found in any plasmid-cured strain derived from the type bacteria (P. putida G7 and NCIB 9816-4) or fived cured site isolates (Fig. 2
, lanes 3, 4; 7, 8; 11, 12; 15, 16; 19, 20; 23, 24; 27, 28; sporadic traces of non-specific amplicons were considered insignificant). This shows that the nahR-like gene is present only on the naphthalene-catabolic plasmid in the micro-organisms examined. The amplicons were also found in two additional naphthalene-degrading strains, P. fluorescens N1 and Nd9 (isolated originally from soil from Richland, WA and Glen Falls, NY, respectively, data not shown). It is worth noting that Silva et al. (1995)
failed to amplify an nahR-like sequence by PCR in P. fluorescens Nd9; this undoubtedly reflected the design characteristics of the primers used. These patterns in PCR amplification of nahR suggest that it is highly conserved in pure cultures of naphthalene-utilizing bacteria despite the fact that some naphthalene-degrading strains were isolated from geographically diverse locations.
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Comparison of nahG-nahR intergenic regions and NahR binding motifs in naphthalene-degrading bacterial isolates
nahR-like genes in strains Cg1, Cg2, Cg5, Cg7, Cg9, Cg11, P. putida strain NCIB 9816-4 as well as N1 and Hg8 were cloned and sequenced. All deduced proteins exhibited a highly conserved helixturnhelix motif and a putative enhancer-binding region in the N-terminal domain. nahG from pDTG1 (NCIB-nahG) was identified via a sequence homology search with known nahG genes from P. putida G7 and P. stutzeri AN10. When amino acid and nucleotide sequences were compared, NCIB-nahG had 92 and 92·4% identities, respectively, with nahG from P. putida G7. Corresponding identities between NCIB-nahG and nahG from P. stutzeri AN10 were 78·7 and 81·3%.
The nahG promoter-like region from four site-derived strains was cloned and sequenced. Alignment of nahG gene promoter-like regions from P. putida G7, P. stutzeri AN10, P. putida NCIB 9816-4, Cg1, Cg2, Cg5 and Cg 7 revealed 50·4100% identity (Fig. 3a). Surprisingly, amplified intergenic regions from naphthalene-degrading micro-organisms native to our study site were 100% identical to that of the pDTG1 plasmid, while the nahR coding regions were not. Conservation in the intergenic region may possibly be explained by recent acquisition of an nahG-nahR cassette. A high degree of identity was observed around the putative -35 box, and the putative -10 box also showed little variation (Fig. 3a
). The nahG and nahA promoter regions from naphthalene-degrading bacteria revealed the consensus NahR binding sites (5'-ATTCACGCTN2TGAT-3') that consists of imperfect inverted repeat sequences (Fig. 3b
). The upstream sequences of nagAa also showed putative NahR binding sites (Zhou et al., 2001
). These data support previously published RFLP analyses suggesting that naphthalene-catabolic plasmids in isolates from our coal-tar-contaminated site are closely related to the pDTG1 plasmid in P. putida NCIB 9816-4, despite the fact that P. putida NCIB 9816-4 was originally isolated in Wales (Stuart-Keil et al., 1998
). The highly conserved sequences of nahR and its 5' flanking region suggest that this regulatory gene and promoter region may behave as an intact gene cassette on pDTG1-like plasmids and may have evolved independently from the structural genes for the catabolic enzymes. It is also possible that the regulator, nahR, and the entire lower operon could together be a conserved cassette.
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Sequencing and alignment of nahR-like genes from sediment-extracted DNA
The amplified PCR products of the entire nahR gene from the enriched contaminated source DNA (Fig. 4c; lane 3) were cloned. Seven randomly chosen clones were sequenced and compared to the corresponding sequences from site-derived and reference pure cultures. Three main clades were found (Fig. 5a
). All four distinctive sediment DNA clone sequences (designated contaminated source DNA-1 to -4) fell into the uppermost clade of Fig. 5(a)
, along with sequences from seven site-derived pure cultures and the type strain, P. putida NCIB 9816-4. In two instances (contaminated source DNA-1 and -3), more than one DNA clone had identical sequences (Fig. 5a
). Scrutiny of nahR homologues revealed that the sequences from strain N1 and P. putida G7 cluster together (Fig. 5a
). Furthermore, the chromosomally encoded nahR(S) is quite different from any other of the plasmid-encoded NahR homologues (Fig. 5a
). The sequence of nahR from strain Hg8 showed that 2 nt are missing in the C-terminal region near the stop codon. However, like the terminal nucleotide sequences of nahR in some Cg strains, Hg8 featured two out-of-frame 5'-CTGATTGA-3' stop codons. This may compensate for the two missing nucleotides. Thus, nahR of strain Hg8 has 298 instead of 300 aa but retains a high level of identity to other nahR genes. The nahR-like sequences from cultured and uncultured micro-organisms featured a nucleotide identity range of 74·6100% and an amino acid identity range of 81·4100%. Thus, although subtle sequence variations were found in the nahR-like sequences displayed in Fig. 5(a)
, the dendrogram generally confirms that nahR-like genes are highly conserved, regardless of their varied geographic origins or their cultivated versus non-cultivated hosts (Fig. 5a
).
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Speculation about selective pressure on nahR and operon evolution
Interestingly, the upstream region of nahG gene in pDTG1 has tnpA (transposase)-like sequences (Fig. 1a). The truncated 414 bp nucleotide sequence of the tnpA-like gene from pDTG1 has 100% identity with tnpA of Pseudomonas strain CA10 (a carbazole-degrading bacterium) and 87% DNA identity with tnpA3 of P. strain O1G3 (an alkylbenzene-catabolic strain). This observation suggests that regulatory genes might in the past have been transferred in the form of a composite transposon. Bosch et al. (2000)
have shown that nahW (salicylate hydroxylase) in P. stutzeri AN10 is located between tnpA2 and tnpA3 and its position is adjacent to a lower pathway operon where nahR(S) and an additional copy of nahG (salicylate hydroxylase) reside. It has been suggested (Bosch et al., 1999
, 2000
), based on gene organization of the naphthalene-catabolic operon in P. stutzeri AN10, that the first step in assembling a naphthalene-degradation pathway may have been acquisition of a catechol meta-cleavage pathway (conferring catechol mineralization). The next theoretical step in operon assembly may be the acquisition of nahG (to form the lower pathway) and then acquisition of the upper pathway operon. Incorporation of the nahR regulatory gene may have been the last step.
Current paradigms suggest that biodegradative operons evolved from constitutive expression to substrate-dependent control by acquiring genetic regulatory components (Cases & de Lorenzo, 2001 ; de Lorenzo & Perez-Martin, 1996
; van der Meer et al., 1992
; Williams & Sayers, 1994
). This theory is supported partly by the fact that one type of regulatory protein can activate many types of operons. A major conclusion of the present study is that nahR shows remarkably little sequence variation. As acknowledged above, this apparent conservation may reflect the PCR-based methodology. But if we accept the results as reflecting the true disposition of nahR in soil microbial communities, we have license to speculate about selective pressures that act on regulatory versus structural genes in catabolic operons. It is obvious that the various forms of aromatic substrates (e.g. toluene, biphenyl, naphthalene, phenol) require substantial evolutionary malleability in structural catabolic genes. Drift at the sequence level is translated into gradual refinement of enzyme recognition sites that accommodate needs to bind, destabilize and catalyse electron transfer and oxygenase attack of aromatic rings (van der Meer et al., 1992
; Williams & Sayers, 1994
). It is possible that the DNA sequences encoding the regulatory components (nahR-type genes) of such catabolic operons have been optimized during multiple recruitment events. Progress towards explaining high conservancy of nahR, recruitment of nahR and other issues in operon evolution may occur through continued comparative genetic analysis of catabolic operons.
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ACKNOWLEDGEMENTS |
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Received 19 December 2001;
revised 22 March 2002;
accepted 18 April 2002.