Cold-tolerant alkane-degrading Rhodococcus species from Antarctica

Polar Biol (2000) 23: 100±105 Ó Springer-Verlag 2000 ORIGINAL PAPER Asim K. Bej á David Saul á Jackie Aislabie Cold-tolerant alkane-degrading Rhodococcus species from Antarctica Accepted: 6 September 1999 Abstract Bioremediation is a possible mechanism for clean-up of hydrocarbon-contaminated soils in the Antarctic. Microbes indigenous to the Antarctic are required that degrade the hydrocarbon contaminants found in the soil, and that are able to survive and maintain activity under in situ conditions. Alkanedegrading bacteria previously isolated from oil-contaminated soil from around Scott Base, Antarctica, grew on a number of n-alkanes from hexane (C6) through to eicosane (C20) and the branched alkane pristane. Mineralization of 14C-dodecane was demonstrated with four strains. Representative isolates were identi®ed as Rhodococcus species using 16S rDNA sequence analysis. Rhodococcus spp. strains 5/14 and 7/1 grew at )2°C but numbers of viable cells declined when incubated at 37°C. Both strains appear to have the major cold-shock gene cspA. Partial nucleotide sequence analyses of the PCRampli®ed cspA open reading frame from Rhodococcus spp. strains 5/14 and 7/1 were approximately 60% identical to cspA from Escherichia coli. Introduction Alkane-degrading bacteria have been isolated from oil-contaminated soils in the Antarctic (Kerry 1990; Aislabie 1997; MacCormack and Fraile 1997; Pruthi and A.K. Bej Department of Biology, University of Alabama at Birmingham, Birmingham, AL 35294-1170, USA D. Saul School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand J. Aislabie (&) Landcare Research, Private Bag 3127, Hamilton, New Zealand e-mail: aislabiej@landcare.cri.nz Tel.: +64-7-8583700, Fax: +64-7-8584964 Cameotra 1997). If these bacteria are to be useful for bioremediation in Antarctica they must degrade the contaminants that persist in the soil, and they need to survive and be active under in situ conditions. Chemical characterization of hydrocarbon contaminants from contaminated soil around Scott Base revealed predominantly n-alkanes with chain lengths from C9 to C14 (Aislabie et al. 1998). Previously we have demonstrated the potential for mineralization of C14-dodecane in oil-contaminated soils but not control soils, from around Scott Base (Aislabie et al. 1998) and alkane-degrading bacteria, presumptively identi®ed as Rhodococcus spp., were isolated (Aislabie 1997). Very little is known, however, about the physiological and genetic mechanisms by which these bacteria, and microbes in general, survive cold temperatures in Antarctica. While surface temperatures in soils around Scott Base may approach 20°C in summer, in winter they drop to )50°C (Campbell et al. 1998). Survival strategies for cold-temperature environments have been described in a number of bacterial species including Arthrobacter globiformis (Berger et al. 1996), Pseudomonas putida (Gumley and Inniss 1996) and Salmonella spp. (Je€reys et al. 1998). Increased expression of a major cold-shock protein, CS7.4, encoded by cspA, has been reported in response to low temperatures by some bacteria (Goldstein et al. 1990; Je€reys et al. 1998) and, recently, expression of a homolog of the cspA gene at a transcriptional level has been identi®ed in several bacteria isolated from the Schirmacher Oasis of Antarctica (Ray et al. 1994). Although cspA has been shown to respond by elevated expression following exposures to cold temperatures, and that the protein functions as an RNA chaperone (Jones et al. 1996; Thieringer et al. 1998), the exact role of this protein as a survival strategy in microorganisms is still to be determined. The major objectives of this study were to characterize alkane-degrading bacteria isolated from near Scott Base, Antarctica, for their biodegradative potential, identity, and tolerance to cold temperatures. A putative 101 cspA homolog, which codes for a major cold-shock protein, was identi®ed and partially sequenced from two isolates. Materials and methods Source of bacterial strains Alkane-degrading bacteria used for this study were isolated from oil-contaminated soils from around Scott Base, Antarctica (Aislabie 1997). The isolates have been deposited in the International Collection of Micro-organisms from Plants (ICMP, c/o Landcare Research, Private Bag 92170, Auckland, New Zealand). Strain identi®cation numbers are given in Table 1. 16S rDNA sequencing and phylogenetic analysis Genomic DNA was isolated from colonies of strains 5/1, 5/14, 7/1, and 8/1 using the method of Woo et al. (1992). PCR and DNA sequencing were performed using primers described in Saul et al. (1993) and a Perkin-Elmer/Applied Biosystems 377 automated DNA sequencer. Full-length sequences were assembled from overlapping fragments covering both strands. Sequences were aligned by the program PILEUP using a low gap weight (Feng and Doolittle 1987). Sequence alignments were adjusted manually to accommodate RNA secondary structure. With the number of taxa used in the phylogeny, it was not possible to perform a de novo maximum likelihood (ML) analysis without ®rst limiting the search space. An initial heuristic search was carried out using 2000 random sequence additions with maximum parsimony as optimality criterion. This search found 30 most parsimonious (MP) trees, which were then used as starting trees for a more rigorous ML search. The Hasegawa-Kishino Yano (Hasegawa et al. 1985) model was used and likelihood parameters were estimated from the starting trees. Corynebacterium glutamicum was chosen as outgroup for the phylogeny (Rainey et al. 1995). used to inoculate 10 ml BH amended with 50 ll of the following substrates, supplied separately as sole carbon source; hexane (BDH), heptane (Sigma), undecane (Aldrich), dodecane (Sigma), tridecane (Aldrich), hexadecane (Sigma), eicosane (Aldrich), 1-dodecene (Aldrich), 2,2,4, trimethylpentane (Aldrich), pristane (Aldrich) and cyclohexane (BDH). Naphthalene (Merck) and toluene (BDH) were supplied as a vapour (Aislabie 1997). All media were sealed and incubated statically at 16°C for 1 month. Growth in liquid media was measured by spectrophotometric determination (Shimadzu UV-160A) at 600 nm. Tubes with an OD600nm>0.2 were scored as positive for growth. Inoculated substrate-free media served as negative controls. All substrates were >98% pure except for 1-dodecene and hexane, which were 95% pure. Mineralization of dodecane by Rhodococcus spp. strains 5/1, 5/14, 7/1, and 8/1 was determined in biometer ¯asks containing 30 ml BH supplemented with 20 ll of unlabeled substrate spiked with 0.022 ll [141-C]dodecane (0.4 lCi) (4.1 mCi mmol)1) (Sigma). Each ¯ask was inoculated with 108 cells grown on dodecane and incubated at room temperature (18±22°C) in the dark with shaking at 200 rpm. 1 M KOH was used as CO2 trapping agent and the accumulation of 14CO2 determined using standard methods. Survival at low temperatures To determine growth and survival at low temperatures, a 1-ml portion of an overnight culture of Rhodococcus sp. strain 5/14 or 7/1 grown on trypticase soy broth (TSB; Difco) at 15°C was inoculated into 300 ml TSB and incubated at 15°C until the OD450nm reached 0.35. At this stage, a 1 ml sample of the culture was diluted and plated onto trypticase soy agar (Difco) to determine the initial number of viable cells in the culture. Five aliquots (50 ml each) of the culture were then separated in ®ve sterile ¯asks and transferred to incubator shakers set at 37°C, 15°C, 4°C, and )2°C. At various time intervals, 1-ml samples of the cultures were removed, diluted and viable plate counts were determined. All agar plates were incubated at 15°C for 48±72 h. The purity of the cultures was determined by Gram stain of the cells followed by microscopic examination. PCR ampli®cation of the cspA open reading frame Substrate speci®city Isolates were screened for their ability to aerobically utilize aliphatic and aromatic compounds for growth. One hundred microliters of bacterial cultures grown on JP8 jet fuel (50 ll) in 10 ml Bushnell Haas media (BH; Difco) for 7 days at 16°C were Genetic manipulations were performed as described by Ausubel et al. (1987). Total genomic DNA from Rhodococcus spp. strains 5/14 and 7/1 was puri®ed and 1 lg used for PCR ampli®cation using oligonucleotide primers, L-CSPA (5¢-atgtccggtaaaatgactggt3¢) and R-CSPA (5¢-ttacaggctggttacgttacc-3¢), which target the Table 1 Substrate speci®cites of alkane-degrading bacteria isolated from soil around Scott Base, Antarctica () no growth, + growth) Isolate identi®cation ICMP no. Colony pigmentation 4/36 4/38 5/1 5/4 5/11 5/14 6/1 6/6 7/1 7/5 7/25 8/1 8/5 13753 13754 13755 13756 13757 13758 13759 13760 13761 13762 13763 13765 13764 Bu€ Bu€ Orange Orange Orange Orange Bu€ Bu€ Bu€ Orange Orange Bu€ Bu€ Hydrocarbon tested for growth Straight chain alkanes C6-hexane ) C8-heptane + C11-undecane + C12-dodecane + C13-tridecane + C16-hexadecane + C20-eicosane + Straight chain alkene C12-1-dodecane + Branched alkanes 2,2,4-trimethylpentane ) Pristane + + + + + + + + ) + + + + + ) ) ) + + + + + ) ) + + + + + ) ) + + + + + + + + + + + ) ) + + + + + + ) + + + + + ) ) ) + + + + + ) ) ) + + + ) + + + + + + + + + + + + + + + + ) + + + + ) + + + + ) + ) + ) + ) + ) + ) + ) + ) + ) + ) + ) + ) + 102 open reading frame (ORF) of the cspA gene (Goldstein et al. 1990). In each PCR reaction, 1 ´ PCR reaction bu€er (Perkin Elmer), 200 lM of each of the dNTPs, 2 lM of each of the two oligonucleotide primers and 3 U UlTma (Perkin Elmer) DNA polymerase were used in a 25-ll reaction volume (Je€reys et al. 1998). The PCR cycling parameters were as follows: (94°C, 3 min) ´ 1, (94°C, 30 s; 40°C, 30 s; 72°C, 60 s) ´ 30. PCR products were con®rmed by gel electrophoresis using the 123 bp DNA ladder size marker (Gibco BRL) and PCR-ampli®ed cspA gene from Escherichia coli. Cloning and nucleotide sequence analysis of the cspA ORF DNA segment PCR-ampli®ed DNA consisting of the cspA ORF from Rhodococcus spp. strains 5/14 and 7/1 was inserted directly into the TOPO TA cloning vector, pCR2.1 (Invitrogen), and used to transform competent cells of E. coli Top10 (Invitrogen). Positive clones were screened on agar plates supplemented with 50 lg kanamycin per ml, 0.1 mg per ml 5-bromo-4-chloro-b-galactoside (´ -gal) (Sigma) and 5-bromo-4-chloro-indolyl-galactopyranoside (IPTG) (Sigma). Ten white colonies were selected and plasmid DNA was puri®ed from these using a Qiagen plasmid MiniPrep kit (Qiagen). The puri®ed DNA was digested with EcoRI and the fragments visualized by gel electrophoresis to con®rm the presence of an insert. Puri®ed plasmid (1 lg) carrying the Rhodococcus putative cspA ORF from ®ve of the ten clones was sequenced, both strands, using an ABI automated DNA sequencer with M13 forward and reverse primer. The nucleotide sequence and the deduced amino acid residues were compared and aligned with the E. coli cspA ORF using the BLAST program (NCBI) and DNA Star computer software. The sequences of the cspA gene for Rhodococcus spp. strains 5/14 and 7/1 are available from GenBank under the accession numbers AF171925 and AF171926, respectively. Results Fig. 1 Maximum likelihood derived phylogenetic tree of selected Rhodococcus species. Numbers above the branches are the level of bootstrap support from 1000 maximum parsimony bootstrap replicates. Where no number is shown, bootstrap support is less than 50%. The scale bar shows the maximum likelihood estimate of the inferred number of changes per site. Numbers in square brackets are the GenBank accession numbers of the sequences Identity of the isolates Alkane-degrading bacteria used for this study were isolated from oil-contaminated soils from around Scott Base, Antarctica. The bacteria were Gram-positive, rodshaped organisms and had been presumptively identi®ed as Rhodococcus spp. as described in Aislabie (1997). The bacteria were of two morphological colony types and formed either small orange-pigmented colonies or large mucoid bu€-coloured colonies on R2A agar plates (Difco) (Table 1). To con®rm their identity, representative isolates of each colony type were subjected to 16S rDNA phylogenetic analysis. A single ML tree was found (Fig. 1). Strains 5/1 and 5/14 group with high bootstrap support with Rhodococcus sp. strain JoyN, Rhodococcus sp. strain JoyP, and R. fascians. The 16S rDNA sequence of strain 7/1 is identical to that of R. erythropolis and Rhodococcus sp. strain AO7 (Fig. 1). These three sequences form a well-supported group with the known alkane degraders Rhodococcus sp. strain Q15 and Rhodococcus sp. strain PR4. One thousand and twenty-four base pairs of strain 8/1 16S rDNA were obtained and found to be identical to the corresponding sequence of strain 7/1. For this reason this sequence was not included in the phylogeny. Most of the branching order in the rest of the tree has low bootstrap support, which indicates that 16S rDNA sequences may only provide a poor resolution for inferring the full phylogeny of this genus. Degradative activity The bacteria utilized a number of saturated alkanes from hexane (C6) through to eicosane (C20) as sole source of carbon and energy (Table 1). Some isolates (4/38, 8/1, 8/5) degraded all the aliphatic compounds provided, whereas others degraded a narrower range of saturated alkanes (7/25). In addition, all isolates grew on pristane, and all but one utilized 1-dodecene for growth. None of the isolates grew on 2,2,4-trimethylpentane, cyclohexane, or the aromatic hydrocarbons, toluene and naphthalene (results not shown). The ability of isolates 5/14 and 7/1 to mineralize 14C-dodecane to 14CO2 was con®rmed (Fig. 2), and similar results were obtained for 5/1 and 8/1. 103 Fig. 2 14CO2 evolution from 14C-labeled dodecane for cultures 5/14 (+) and 7/1 (d). 14CO2 evolution from uninoculated and killed cell control ¯asks was less than 1%. The bars represent standard errors for three replicate ¯asks Survival at low temperatures Viable plate counts for Rhodococcus sp. strain 5/14 culture increased slightly during the ®rst 2 h of incubation at 37°C, followed by a steady decline in numbers during the next 39 h (Fig. 3A). After 41 h incubation at this temperature and subsequent plating up to 121 h, no viable cells were detected. Cultures maintained at 15°C exhibited relatively rapid growth, whereas those cultures incubated at 4°C or )2°C entered lag phase initially, but did eventually reach the same number of viable cells as cultures incubated at 15°C. Similar results were obtained for Rhodococcus sp. strain 7/1 (Fig. 3B). PCR ampli®cation, cloning, and nucleotide sequence analysis of the cspA ORF DNA segment PCR ampli®cation using the oligonucleotide primers encompassing the cspA ORF produced a DNA fragment for Rhodococcus spp. strains 5/14 and 7/1. Nucleotide sequence analysis showed the product to be 213 bp in length. Both sequences had approximately 60% nucleotide identity to the cspA gene sequence from E. coli (Goldstein et al. 1990) (data not shown). The di€erences were distributed throughout the cspA ORF with most non-silent changes located in the downstream segment. The deduced amino acid sequence of the Rhodococcus spp. cspA ORF di€ers from the E. coli CS7.4 protein at various locations, with 11 conservative and 16 nonconservative changes for strain 5/14, and 14 conservative and 14 non-conservative changes for strain 7/1 (Fig. 4). Among those 11 amino acids of the CS7.4 essential for binding with the DNA and RNA (Newkirk et al. 1994), there were 2 conservative changes (Tyr12, Trp42) from Fig. 3 Viable plate counts of Rhodococcus sp. strain 5/14 (A) and Rhodococcus sp. strain 7/1 (B) incubated at various temperatures. Cultures were maintained at 15°C (e); cultures transferred from 15°C to 37°C (h) and maintained at this temperature; cultures transferred from 15°C to 4°C (s) and maintained at this temperature; and cultures transferred from 15°C to )2°C (n) and maintained at this temperature. Each data point is an average of three identical experiments treated similarly and the error bars represent one standard error the E. coli sequence in Rhodococcus sp. 5/14, and 3 conservative changes (Tyr12, Leu21, Trp42) in 7/1. At these positions in E. coli, the following amino acids residues are found: Phe12, Ile21, and Tyr42. Discussion Alkane-degrading bacteria isolated from soil from around Scott Base, Antarctica, are able to degrade alkanes with chain lengths from C6 to C20, typical of the hydrocarbon contaminants that persist in Antarctic 104 Fig. 4 Deduced amino acid sequence of the putative CspA from Rhodococcus spp. strains 5/14 and 7/1 aligned with Escherichia coli CS7.4. Amino acids encoded by the PCR primers have been omitted for the Rhodococcus species. Conservative changes in the amino acid residues are indicated by + and the non-conservative changes are kept blank. Eleven amino acid residues in E. coli that are essential for binding with the RNA are underlined and numbered consecutively in the bottom panel. STOP indicates the translational stop, 5¢-TAA-3¢, at the end of the ORF soil (Aislabie et al. 1998). The isolates were identi®ed as members of the genus Rhodococcus by 16S rDNA sequence analysis. Members of this genus, considered to be a signi®cant part of the soil community, are recognized for their wide range of metabolic capabilities (Warhurst and Fewson 1994). They are K-strategists, in that they exhibit slow growth, but with their high substrate anity and great persistence in the environment (Warhurst and Fewson 1994) they seem ideally suited for remediation of oil-contaminated Antarctic soils. Phylogenetic analyses indicate that the Rhodococcus species isolated in this study are similar to other alkanedegrading bacteria from cold climates including Rhodococcus sp. Q15 from Canada (Whyte et al. 1996) and Rhodococcus sp. JoyN and Rhodococcus spp. JoyP/ B from the deep sea (Coloquhoun et al. 1998). As Rhodococcus sp. JoyN and Rhodococcus spp. JoyP/B were isolated on paran-containing medium, we have assumed that they degrade alkanes also. The Rhodococcus species isolated were psychrotolerant because, while they could grow at low temperatures, their optimum temperature for growth was greater than 15°C. Temperature survival studies of Rhodococcus spp. strains 5/14 and 7/1 suggest that these microorganisms can adapt rapidly to the subzero temperature of )2°C and retain metabolic activity for growth and survival at this temperature. Investigations of their cold-tolerance mechanisms indicate that Rhodococcus spp. strains 5/14 and 7/1 appear to have the major cold-shock gene that codes for CS7.4, as found in E. coli (Goldstein et al. 1990) and salmonellae (Je€reys et al. 1998). Western blot analysis using E. coli CS7.4 rabbit polyclonal antibody, however, did not result in the positive detection of the major cold-shock protein in either of the two Rhodococcus species investigated (data not shown). Therefore it is not clear from this study whether the CspA protein is expressed in these microorganisms. It is possible that the putative major cold-shock protein encoded by the cspA gene in these Rhodococcus species has di€erent antigenic properties from that of CS7.4 from E. coli. Alternatively, the experimental conditions employed may not have been appropriate for induction of the cspA gene in Rhodococcus spp. strains 5/14 and 7/1, when compared to the extreme temperature regimes existing in Antarctic soil from which they were isolated. The observed di€erences in the deduced amino acid residues of the CspA from Rhodococcus spp. strains 5/14 and 7/1 compared to that of E. coli warrant further investigation with regard to the structure, expressional controls, and possible physiological role this protein may play in the survival of these hydrocarbon-degrading bacteria in Antarctica. Acknowledgements This work was supported by funding from the Foundation for Research, Science and Technology, New Zealand (C09818). Antarctica New Zealand provided logistic support. We thank Rhonda Fraser for technical assistance, and M. Inouye for providing us with the CspA polyclonal antibody. 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