16S rRNA Restriction Fragment Length Polymorphism Analysis of Bacterial Diversity as a Biomarker of Ecological Health in Polluted Sediments from New Bedford Harbor, Massachusetts, USA

PII: S0025-326X(98)00199-4

Marine Pollution Bulletin Vol. 38, No. 8, pp. 663±675, 1999 Ó 1999 Published by Elsevier Science Ltd. All rights reserved Printed in Great Britain 0025-326X/99 $ - see front matter

16S rRNA Restriction Fragment Length Polymorphism Analysis of Bacterial Diversity as a Biomarker of Ecological Health in Polluted Sediments from New Bedford Harbor, Massachusetts, USA JONATHAN J. SORCI, JOSEPH D. PAULAUSKIS and TIMOTHY E. FORD* Department of Environmental Health, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115, USA

A polymerase chain reaction (PCR)-based method was developed to compare bacterial diversity among environmental sites with varying degrees of anthropogenic impact. New Bedford Harbor, MA, a US Environmental Protection Agency-designated Superfund hazardous waste site, was studied to assess changes in bacterial diversity resulting from long-term inputs of organic and inorganic pollutants. Total DNA was extracted from sur®cial sediments sampled from four sites along a transect of decreasing contamination (Upper and Lower Acushnet Estuary, New Bedford Harbor, and Buzzards Bay, respectively). Oligonnucleotide primers speci®c to conserved regions of the 16S rRNA gene were used to PCR-amplify sequences from DNA extracts. Restriction fragment length polymorphism (RFLP) analysis resulted in generation of a number of unique operational taxonomic units (OTUs). Cluster analysis of fragment pattern data using the computer program RESTSITE allowed for bacterial diversity estimations, which, in agreement with standard culture techniques, showed higher bacterial diversity in New Bedford Harbor sediments, relative to Buzzards Bay. By employing bacterial diversity as a sensitive indicator of environmental stress, the method has wide applicability to many environments for the assessment of anthropogenic impact on aquatic ecosystems. Ó 1999 Published by Elsevier Science Ltd. All rights reserved Keywords: biomarkers; ecological health; 16S ribosomal RNA (rRNA); polymerase chain reaction (PCR); restriction fragment length polymorphism (RFLP); New Bedford Harbor, Massachusetts, USA.

*Corresponding author. Tel.: +1-617-495-8351; fax: +1-617-4323349; e-mail: [email protected]

In ecological health assessment, a variety of organisms have been used to develop various biomarkers of both organic and inorganic contamination, including nematodes (Mutwakil et al., 1997), feral eels (van der Oost et al., 1996,1997), and mussels (Etxeberria et al., 1995; Sole et al., 1995; and Najimi et al., 1997). From these and other organisms, enzyme function has been used as biomarker of exposure to organic pollution in the environment (Kreamer et al., 1991; Livingstone, 1993; Goksoyr, 1995). Metallothionines, heat-shock, and other stress proteins are speci®cally induced by metals including Cd, Hg, Ag and Cu, and are used in both vertebrates and invertebrates as biomarkers of metal exposure (Sanders and Martin, 1993; Dallinger, 1994,1996; de Pomerai, 1996). Although many di€erent approaches have been taken to measure biomarkers of environmental stress, including molecular, physiological and behavioral (Depledge et al., 1995), a molecular approach using microorganisms has only recently been investigated (Sorci, 1998). Diversity of microbial communities has been demonstrated empirically to be a sensitive indicator of pollution in aquatic ecosystems (Atlas et al., 1991; Atlas and Bartha, 1993). Previous studies have measured bacterial diversity as a function of ocean surface water temperatures (Atlas, 1984), and oil exposure over time (Atlas et al., 1991). Using the Shannon±Wiener (S±W) Index of Diversity (H) and the plate culturing method, the latter study showed a decrease from H ˆ 3.5±2.1 over a six week exposure to oil from the Exxon Valdez oil tanker spill. New Bedford Harbour, Massachusetts, USA, a coastal marine environment and an EPA-designated Superfund hazardous waste site, o€ers a unique contaminant history and sediment pro®le for studying the e€ects of pollution on bacterial genetic diversity. 663

Marine Pollution Bulletin

Decades of industrial and municipal loading into the Acushnet River estuary and New Bedford Harbor have resulted in high sediment concentrations of organic and inorganic contaminants (Weaver, 1984). For example, polychlorinated biphenyl (PCB) concentrations exceed 10% of sediment by weight in an area de®ned as the ``hot spot'' located in the upper estuary. Metals, including Ag, As, Cd, Co, Cu, Cr, Hg, Mn, Ni, Pb and Zn, range from slightly above background (Buzzards bay) to percent levels in localized areas within the harbor (Pruell et al., 1990; Shine et al., 1995). Exposure primarily to PCBs and toxic metals, through ingestion of seafood and direct contact with sediments, has caused the most concern, as these pollutants have been known to be associated with adverse health e€ects, both acute and chronic (Williams and Weisberger, 1993). Molecular techniques to study microbial diversity and species evolution have been developed extensively over the past decade (Amann et al., 1995; Boivin-Jahns et al., 1995; Hugenholtz and Pace, 1996; Ward et al., 1990; Woese, 1987). A number of natural microbial communities have now been described by analysis of small-subunit (SSU) ribosomal RNA (rRNA) genes (Fuhrman et al., 1994; Giovannoni et al., 1990; Olsen et al., 1986; Pace et al., 1986; Schmidt et al., 1991; Ward et al., 1992). Characterization of groups of microorganisms, their relation to each other and their environment is now possible through detailed phylogenetic analysis of complete gene sequences (Britschgi and Giovannoni, 1991; Fuhrman et al., 1993; Neilan et al., 1995). However, given the potential genetic diversity in any particular environment, this approach is limited by the practical considerations of time and expense when comparing total diversity among many di€erent sites. An alternative, relatively inexpensive approach to characterizing bacterial genetic diversity is restriction fragment length polymorphism (RFLP) analysis of 16S rRNA gene sequences ampli®ed from sediment DNA extracts (Avaniss-Aghajani et al., 1994; DeLong et al., 1993; Moyer et al., 1994; Weidner et al., 1996). This study describes development of the RFLP approach to investigate di€erences in bacterial diversity resulting from sediment contamination in New Bedford Harbor. The study was designed to (1) develop an accurate and relatively inexpensive method of comparing bacterial genetic diversity among various environmental sites, (2) compare genetic diversity with bacterial diversity as determined by standard culture techniques, and (3) investigate the relationship of genetic diversity to speci®c environmental pollutants employing statistical correlation analysis.

Materials and Methods Site characterization and sampling procedure Representative sites from four locations were selected for sampling (Buzzards Bay [B], New Bedford Harbor [H], Lower [L] and Upper [U] Acushnet Estuary). 664

Fig. 1 Geographic site locations for Buzzards Bay [B], New Bedford Harbor [H], Lower Acushnet Estuary [L], and Upper Acushnet Estuary [U].

Figure 1 shows the geographic location for each site. Selection criteria were based on previously measured sediment pollutant data (USEPA, 1990). Each site was relatively well-protected from physical disturbances such as wave action, storm events or boating/shipping trac and dredging activity. Preliminary sampling surveys had indicated homogeneity of New Bedford Harbor sediments sampled in depositional areas at a distance from its main, dredged, shipping channel. Sediment consistency has also increased due to lack of dredging activity over the past several years. Four sites were chosen to represent the maximum range of metal contamination, e.g. for Cu, a range of approximately 10 mg/kg dry sediment wt. in [B] to greater than 1000 mg/ kg in [U]. Triplicate core samples were taken at each site based on metals analyses showing less than ten percent standard deviation (USEPA, 1990). Depth of water in [B] and the hurricane barrier in [H] protect the sediments from disturbances to some degree. The [L] sampling site was in close proximity to a combined sewer over¯ow (CSO) outfall pipe. During November 1993 and August 1995, surface sediments (upper 2.5 cm) were sampled in triplicate from [L] and [U], and [H] and [B], respectively, using a gravity core sampler (Wildco, Saginaw, MI). Where possible, sites were located to within 30 m using the global positioning system (GPS) (Trimble Navigation, Sunnyvale, CA) Polycarbonate liners inserted into the corer were used to procure surface sediments and their associated overlying water. Asceptic techniques were used throughout the sampling procedures, and liners and sampling bottles were pre-sterilized by autoclaving. Samples from individual sites were preserved by

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storing on ice or at 4°C prior to analysis (within 24 h). Measured physico-chemical and hydrologic parameters included water depth, temperature, salinity, dissolved oxygen, conductivity, and pH using the HydroLabâ system (HydroLab, Austin, TX). Sediment samples were analyzed for percent carbon (C), hydrogen (H) and nitrogen (N) using a CHN analyzer (Perkin Elmer, Foster City, CA) calibrated with acetanilide (Shine et al., 1995). Carbonates were removed by exposure of sediments to concentrated HCL fumes for 48 h. Metals and PCB analysis of sediments For metals analysis, triplicate samples were combined and homogenized to form one composite. Sediment concentrations for Ag, Cd, Co, Cu, Cr, Mn, Ni, Pb and Zn were measured by microwave digestion and inductively coupled plasma ± mass spectrometry (ICPMS) as previously described (Shine et al., 1995). Additionally, As and Hg were analyzed by cold vapor atomic absorption spectrometry (AAS) as per EPA method 245.5 (USEPA, 1984). Secondary concentration of the above samples was performed using a Perkin Elmer (PE) 4100 AAS coupled with a gold amalgam and ¯ow injection analysis system. Total PCBs (quanti®ed as Arochlor 1254) were measured for each replicate by sediment sonication and 50:50 dichloromethane: acetone extraction, coupled with gas chromatography ± electron capture detection (USEPA, 1984). Bacterial plate cultures Standard plate culture techniques were used to initially determine bacterial diversity in surface sediments from Buzzards Bay and New Bedford Harbor. Samples were taken during the summer and winter, 1995, to compare seasonal e€ects on diversity. A 0.2±0.5 g aliquot of sediment was procured from cores sampled at the two sites [H] and [B]) by sterile swabbing the surface sediments (top 2.5 cm) into a series of dilution tubes containing 0.5 M NaCl and 0.05 M Tris-HCl (pH 7.8). Marine agar plates (DifCo, Ann Arbor, MI) were inoculated in triplicate with 100 lL aliquots from each dilution tube, then incubated at in situ temperature (approximately 10°C and 20°C for winter and summer samples, respectively) for 5±7 days, or until colonies were visually distinguishable. Individual colonies were counted and distinguished based on morphological characteristics (Boivin-Jahns et al., 1995), and data were used to calculate bacterial diversity by the S±W Diversity Index (Atlas and Bartha, 1993). Comparison of bacterial diversity, both seasonally and between [B] and [H], was accomplished by non-parametric Wilcoxon (Rank Sum) statistical analysis using the means and standard deviations for each site and each time point. Extraction and puri®cation of DNA from sediments A combination of methods was performed to recover DNA of sucient purity for ampli®cation via the polymerase chain reaction (PCR). DNA was extracted from

surface sediments using a modi®ed method of Tsai and Olson (1991). Total DNA recovery eciencies have been reported to exceed 90% using this method, yielding sucient quantities for ampli®cation by PCR (Tsai and Olson, 1991; Lee et al., 1996a; Zhou et al., 1996). The same sediment cores sampled in triplicate at each site for bacteriological plate culturing and diversity determinations were used for DNA extraction. Surface sediments from each of the three cores taken per site were mixed and homogenized to obtain a representative DNA sample. Brie¯y, a sediment sample (approximately 10 g wet weight) was washed twice in 30 mL of 120 mM sodium phosphate bu€er (pH 8.0) by shaking at 150 rpm for 15 min. The sediment slurry was pelleted by centrifugation at 6000 ´ g for 10 min. Resuspension of the pellet in 20 mL lysis solution (5 mg/ml lysozyme and 100 lg/ml proteinase K in 0.15 M NaCl, 0.1 M ethylenediaminetetraacetic acid (EDTA), pH 8) was followed by incubation at 37°C for 2 h with periodic agitation. Addition of 2 mL of 0.15 M NaCl, 0.1 M EDTA (pH 8), 20% sodium dodecyl sulfate was accompanied by three cycles of freezing in a ÿ70°C dry ice-ethanol bath and thawing at 65°C. Extraction of DNA was accomplished by addition of 15 mL 0.1 M Tris-HCl (pH 8.0)-saturated phenol and gentle inversion to form an emulsion. Imiscible layers were separated by centrifugation at 6000 ´ g for 10 min. The aqueous layer removed and further extracted with a 15 mL aliquot of phenol (pH 8.0)/chloroform/isoamyl alcohol (25:24:1). An additional extraction of the recovered aqueous layer using 15 mL chloroform resulted in a blood red, clear product containing nucleic acids, synthetic organic compounds and naturally occurring organic matter, e.g. humic acids. Humic acids are known to interfere with PCR at levels as low as 0.5 lg/mL (Tebbe and Vahjen, 1993). Treatment of the phenol/chloroform extracted supernatant with cetyltrimethyl ammonium bromide (CTAB) to remove humic acids (Sambrook et al., 1989) was followed by DNA precipitation overnight in 0.6 volumes isopropanol. DNA was pelleted by centrifugation at 12,000 ´ g for 20 min. The pellet was washed with 2 ml of 75% ethanol to remove excess salts, centrifuged at 12,000 ´ g for 10 min, air dried, and then resuspended in 4 ml of 20 mM Tris-HCL (pH 8.0), 1 mM EDTA (TE) solution. To each sample was added 4.3 g CsCl and 200 lL of 10 mg/mL ethidium bromide. Samples underwent ultracentrifugation through a CsCl gradient at 70,000 rpm (Rotor SW 55, Beckman Instruments, Palo Alto, CA) for 16 h at 15°C. The nucleic acid band was visualized using a hand-held ultraviolet (UV) light source, while a 12-gauge sterile needle was used to extract approximately 0.5 mL of the solution containing the band. Ethidium bromide was removed by sequential extractions with CsCl-saturated isopropanol, and samples were dialyzed overnight against 2 L TE bu€er. In order to completely eliminate interferences with the PCR reaction, it was found necessary to repeat the CTAB treatment. 665

Marine Pollution Bulletin

Ampli®cation of 16S rRNA genes Primer sequences, designated 27f and 1492r according to position in the 16S rRNA gene were 50 -AGAGTTTGATCMTGGCTCAG-30 and 50 respectively TACGGYTACCTTGTTACGACTT-30 , (M ˆ A or C, Y ˆ C or T) (Brosius et al., 1978; Lane, 1991). These primers are in highly conserved regions of the 16S rRNA gene in E. Coli and show high hybridization eciency to bacterial SSU rRNA gene sequences in the Ribosomal Database Project (Brunk et al., 1996). A PE 2400 Thermocycler was used to PCR-amplify 16S rRNA genes under the following conditions. 10 mM Tris-HCl (pH 8.8), 1.5 mM MgCl2 , 50 mM KCl, 0.1% Triton X-100, 200 lM each dATP, dCTP, dGTP and dTTP, 0.2 lM forward and reverse primers, 1.0 unit of recTbr thermostable DNA polymerase (AMRESCO, Solon, OH) and approximately 10 ng/lL template DNA (Bruce et al., 1992; Ste€an and Atlas, 1991). The temperature program was 95°C for 3 min, followed by 25 cycles at 94°C for 15 s, 50°C for 30 s and 72°C for 2 min. The ®nal cycle allowed extension for 30 min. The annealing temperature was set relatively high to avoid ampli®cation of spurious products. the number of cycles was kept as low as possible to minimize di€erential or selective ampli®cation of particular 16S rDNA which could bias genetic diversity determinations (Suzuki and Giovannoni, 1996). Generation of RFLP data PCR products were ®rst puri®ed by low melt agarose electrophoresis, excision of the 1500 base pair (bp) band using a sterile razor blade under 312 nm UV light, and application of the MagicPrepâ fragment puri®cation system as per manufacturerÕs protocol (Promega, Madison, WI). 16S rRNA genes were subcloned employing the TA Cloning Systemâ (Invitrogen, San Diego, CA). Clones were screened on ampicillin-amended LB agar (DifCo) coated with XGal for di€erential selection. Following incubation at 37°C for 24 h, white colonies were randomly picked, grown overnite in ampicillin-amended LB medium, and their plasmids recovered using the FlexiPrepâ system as per manufacturerÕs protocol (Pharmacia, Piscataway, NJ). Endonuclease digestion using EcoR I was performed on all plasmids to ensure the presence of the correct insert (1504 bp gene sequences). Subsequently, individual 16S rDNA sequences were ampli®ed away from the vector using M13 forward and reverse primers and PCR under the conditions described above, using 40 temperature cycles. Separate endonuclease digestions on ampli®ed sequences using Rsa I (50 -GT¯Ac-30 ) were performed to generate a minimum of 50 unique RFLP patterns and operational taxonomic units (OTUs) from each site for phylogenetic comparisons. RFLP patterns were resolved in Metaphorâ agarose gels (2%) (FMC Bioproducts, Rockland, ME). Gels were photographed using a 100 bp standard marker as a size reference. 666

Hybridization of RFLP patterns with E. Coli 16S rRNA probe To substantiate the presence of 16S rRNA genes or gene gragments in unique RFLP patterns, a a-32 P-dATP labelled probe was used as a control to hybridize select OTUs. The probe was constructed by PCR-amplifying the 16S rRNA gene directly from E. coli genomic DNA using the above primers, and labelling with a-32 P-dATP by a random priming method using Klenow DNA polymerase I (Life Technologies, Gaithersburg, MD). Agarose gels showing RFLP patterns from [B] and [H] OTUs were Southern blotted overnight onto nitrocellulose, cross-linked with UV, and hybridized with labelled probe following a standard method (Sambrook et al., 1989). The blot was washed under stringent conditions prior to autoradiography. Analysis of RFLP data by RESTSITEâ Fragment data were transformed from distances on gels to approximate lengths in base pairs by linear regression. The natural logarithm of distances (measured in mm) of a 100 bp DNA ladder was plotted against those of individual OTU fragments to determine fragment lengths. A total of 50 independent OTUs from each of the four environmental sites was selected for genetic distance comparisons. Some OTUs appeared identical by visual comparison of agarose gel pictures, yet mathematically determined fragment lengths yielded minor di€erences. Given the similarity of certain RFLP patterns, incorporation of variability in fragment length determinations was warranted. Thus, standard errors were calculated for fragment lengths as determined above, based on variability of a known DNA sequence (684 bp) from the pCRIIâ vector, present in all RFLP patterns, as determined by gel electrophoresis. This variability (3.0%) was applied to all fragment data using computer programs written in SASâ v. 6.11 (SAS Institute, Cary, NC) and STATAâ v. 5.0 (State Corporation, College Station, TX) statistical analysis software. Brie¯y, fragment length values from all 200 OTUs were placed in numerically ascending order. If the next highest value was within 3.0% of the preceding one, it was replaced with this value, otherwise left untreated. A similar treatment of variability in fragment patterns has been applied in diversity determinations of primates (Miller, 1991). The statistical programs generated a ®nal compressed data set which was then used in RESTSITEâ , a phylogenetic/cluster analysis program (Nei and Miller, 1990). The computer program analyzed DNA fragment data to determine genetic distance among the di€erent groups of DNA sequences. Final OTUs and fragment length data were input into the program to calculate the ^ between all individual average genetic distance …d† OTUs within a particular environmental site (based on shared fragments), as well as between the four sites, [B], [H], [L], and [U] (Nei, 1987). For the fragment data, d^ values were used to calculate distance matrices allowing

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for the determination of quantitative tree reconstructions to better visualize relationships between OTUs. The unweighted pair group method using arithmetic averages (UPGMA) of Sneath and Sokal (1973) was used to compare bacterial genetic distances among the four environmental sites. Comparison of bacterial diversity as determined by RESTSITEâ with plate culture diversity The results obtained for bacterial diversity using RESTSITEâ were compared with those calculated from the S±W Diversity Index using data obtained by plate culturing. In order to make meaningful comparisons, S± W Diversity Index calculations were performed on RFLP/cluster analysis data from the two sites, [B] and [H], using a range of percent similarities arbitrarily determined from the RESTSITEâ output and analyzed, based on the maximum number from [B]. By de®ning percent similarities (0±100%) along the distance matrix axis and determining number of ``species'', i.e. clustered OTUs, and number of individual OTUs within species, analogous S±W Diversity Indices were calculated at these arbitrarily de®ned similarity percentages. These calculated indices were plotted versus % similarity to compare with values obtained from standard plate culture techniques. Correlation of genetic distance with environmental parameters Bacterial genetic distance, as determined by RESTSITEâ using RFLP data, was correlated with speci®c environmental pollutants. A simple, parametric correlation (PearsonÕs correlation coecient) of the average genetic distances, d^ values, with each measured environmental parameter from the four sites was performed. Since the average genetic distance is an indicator of bacterial diversity within each environmental site, this metric could be used as a variable to assess correlations with various pollutants or sediment/water quality parameters. The logarithm of each variable was used to approximate a symmetric distribution of the data. In addition, similar analyses were performed using metal concentration data normalized to background levels based on crustal composition (Mason, 1966; Taylor, 1964). Normalization produced enrichment factors, de®ned as the ratio of speci®c metal concentration to aluminum concentration in the sediment sample, relative to the crustal ratios for the same elements. These were then correlated with average genetic distances from each environmental site.

Results A molecular approach was used to test the hypothesis that input of heavy metals and synthetic organic compounds has in¯uenced bacterial diversity in New Bedford Harbor sediments.

Site characterization Sediments were sampled in the New Bedford Harbor ecosystem along a linear transect into Buzzards Bay (see Figure 1). The results of measured environmental parameters for each representative site ([B], [H], [L], and [U] are presented in Table 1. Water chemistries were fairly uniform throughout the harbor and bay, indicating a well-mixed environment. Salinities varied only slightly, from 31.7 ppt in [B] to 27.4 ppt in [U], suggesting that the Acushnet Estuary could be considered a marine environment. This may largely be a function of low river water input and surface water runo€, which vary seasonally, but give rise to an average ¯ow rate of only 1 m3 /s (USEPA, 1990). Values for sediment % organic carbon (OC) ranged from a low of 1.66 in [B] to 9.39 in [U]. Although speci®c organic compounds, other than total PCBs, were not characterized in this study, sources have been documented to come from both industrial and municipal e‚uent (USEPA, 1990). All measured sediment metal concentrations, with the exception of Mn and Co, were signi®cantly higher in New Bedford Harbor than Buzzards Bay. Several metals with known toxicological properties (Cd, Cu, Hg, and Zn) had sediment concentrations ranging from background in [B] to several orders of magnitude above background, e.g. Cd (0.119 mg/kg dry wt. in [8] to 18.6 mg/kg dry wt. in [L]). The close proximity of site [L] to a CSO outfall pipe may explain the high values of contaminant concentrations relative to the [U] site. Although di€erences in sediment metal concentrations for Ag, As, Co, Cr, Ni, and Pb between Buzzards Bay and the other sites were not as pronounced as those mentioned above, their concentrations were still signi®cantly elevated above background. Total PCBs (quanti®ed as Arochlor 1254) were measured for each site, with concentrations ranging from background (<0.06 mg/kg dry wt.) in [B] to 115 mg/kg dry wt in [U]. Concentrations of all metals and PCBs decreased to background levels from the Upper Acushnet Estuary seaward into Buzzards Bay, and were consistent with previously reported concentrations (Pruell et al., 1990; Shine et al., 1995; USEPA, 1990; Weaver, 1984). It appears that sediment concentrations have not changed substantially over time, suggesting that changes in sediment contaminant concentrations should be minimal between the two sampling periods (November 1993 and August 1995). Bacterial plate cultures Standard plate culture techniques were used to determine bacterial diversity in surface sediments from Buzzards Bay and New Beford Harbor. Visually, very few bacterial colonies were similar between the two sites. However, bacterial concentrations between the two sites were comparable, averaging 5 ´ 106 CFU/gram wet sediment weight. Results comparing bacterial diversity between [B] and [H] in summer and winter, 1995, as determined by S±W Diversity Indices, are presented in 667

Marine Pollution Bulletin TABLE 1 Measured physico-chemical parameters of water and sediments from Buzzards Bay [B], New Bedford Harbor [H], Lower Acushnet Estuary [L].and Upper Acushnet Estuary [U]. Site Site location Depth (ft) Water Tem.°C pH Salinity (ppt) a Sediment %H2 O % Organic carbon % Organic nitrogen Metals (mg/kg dry wt.) Ag As Cd Co Cr Cu Hg Mn Ni Pb Zn b

Total PCBs (mg/kg dry wt.)

B

H

L

U

41°300 5100 N 70°500 0000 W 60 19.5 7.63 31.7 56 1.66 0.10

41°380 4200 N 70°540 5300 W 10 22.4 7.62 31.0 71 6.46 0.35

41°390 3900 N 70°550 090 0 W 5 10.0 8.27 30.6 53 7.51 0.25

41°400 1100 N 70°550 060 0 W 6 10.0 8.20 27.4 55 9.39 0.27

0.193 10.1 0.119 6.36 46.8 15.1 0.013 290 18.9 21.3 73.6

2.77 16.0 2.64 6.38 638 1150 1.88 297 55.3 231 417

5.32 18.2 18.6 9.68 873 1360 1.94 376 117 351 1120

4.71 17.1 16.1 11.6 520 902 1.92 347 152 441 1280

<0.06 ‹ 0.02

13.2 ‹ 2.2

83 ‹ 38

115 ‹ 19

[L] and [U] sampled November 1993; [B] and [H] sample August 1995. All analyses, except for PCBs, performed on homogenates of 3 independent core samples. PCB analyses performed on individual replicates (n ˆ 3). Results given as mean ‹ standard deviation.

a

b

Table 2. Results showed a statistically signi®cant (p ˆ 0.006) di€erence between the bacterial diversity of [B] and [H], with consistently higher diversity in the harbor relative to the bay, during both winter and summer. In addition, a signi®cant (p ˆ 0.025) di€erence was obtained in diversity both [B] and [H] between summer and winter. RFLP analysis To more accurately assess bacterial diversity, genomic DNA was extracted from the surface sediments of all four sites for PCR-ampli®cation of the 16S rRNA genes and subsequent RFLP analysis. Each sediment sample (approximately 10 g wet wt.) yielded approximately 10±20 lg total DNA per g of sediment. There was no indication of unlysed cells in the residual sediments, evidenced by acridine orange staining and UV¯uorescence microscopy. These results are consistent with similar methods (Gray and Herwig, 1996; Tsai and Olson, 1991), which reported greater than 95% cell lysis

and DNA yields on the order of 50 lg DNA per g of sediment (wet wt.). PCR-ampli®cation of 16S rRNA genes resulted in the generation of several hundred unique sequences, which were used for RFLP analysis of the four environmental sites. Randomly sampled clones (approximately 10%) yielded over 50 unique gene sequences per site, which were used to generate RFLP data and input into RESTSITEâ for cluster analysis (Miller, 1991). Rsa I was considered a suitable restriction enzyme for the purpose of generating RFLP patterns because it has only one unique recognition sequence. Additionally, use of this tetrameric endonuclease resulted in an average number of four fragments per 16S rRNA sequence (1504 bp length). It has been demonstrated that Rsa I is one of the most ecient endonucleases in detecting and di€erentiating bacterial small subunit rRNA genes, based on a restriction enzymesÕ ability to correctly classify OTUs (Moyer et al., 1996). Figure 2 shows several examples of unique RFLP patterns (OTUs) generated from DNA

TABLE 2 Bacterial diversity comparisons between Buzzards Bay [B] and New Bedford Harbor [H] using the Shannon-Weaner Diversity Index. Site

Sampling date

H H B B

Summer, 1995 Winter, 1995 Summer, 1995 Winter, 1995

a

a

S±W Diversity Index 1.71 ‹ 0.20 1.94 ‹ 0.18 1.46 ‹ 0.22 1.65 ‹ 0.28

(n ˆ 5) (n ˆ 9) (n ˆ 6) (n ˆ 9)

Wilcoxon (Rank Sums) comparisons H vs. B p ˆ 0.006 S vs. W p ˆ 0.025

Diversity indices given as mean ‹ standard deviations of replicates (maximum 3) from 3 core samples at each site and sampling date.

668

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Fig. 2 Examples of unique RFLP patterns generated from Buzzards Bay [B], New Bedord Harbor [H], Lower Acushnet Estuary [L], and Upper Acushnet Estuary [U] samples. M designates the marker/standard ± a 100 base pair ladder. Note: The 684 base pair fragment shown in all lanes was generated as a result of RSA I ± digestion of the pCRIIâ vector and used as an internal reference standard.

extracted from harbor and bay sediments. A sample of ten OTUs was arbitrarily selected from a total of 75 from each of the four sites. Each numbered lane (1±10) represents the RFLP pattern from an OTU, illustrating di€erences and similarities between sites. Hybridization of RFLP patterns with E. Coli 16S rRNA probe The a-32 P-dATP labelled probe was hybridized to randomly selected OTUs to ensure that 16S rRNA genes or gene fragments were present in the RFLP patterns generated from each site. Results of the autoradiogram, compared with photographs of ethidium bromide ± stained gels, showed hybridization of the probe only to fragments originating from the 16S rRNA gene sequences (data not shown). Analysis of RFLP data by RESTSITEâ Application of 3% error for fragment lengths to condense the number of DNA fragments in the total OTU data set resulted in an 82% reduction in unique fragments. Out of 200 OTUs with 803 total fragments (average 4 fragments per OTU, digested with Rsa I), initially, 421 unique fragment were reduced to only 77. Application of 4% and 5% errors to the fragment length data resulted in little further data reduction, i.e. no signi®cant decrease in the number of unique fragments.

Error-corrected OTUs and fragment length data were input into the RESTSITEâ program as binary ®les to calculate average genetic distances and reconstruct dendograms comparing bacterial genetic distance among sites [B], [H], [L], and [U]. Both intra- and intersite similarity were calculated by the program to estimate the diversity of sediment bacterial populations. Figure 3 shows the tree reconstructions for the four environmental sites, as determined by the UPGMA cluster analysis method. The RESTSITEâ program ®rst computed genetic distances for all OTUs within and between sites. Then, cluster analysis was performed on the distance matrices until all environmental sites were clustered into one grouping or family. Branch lengths of the tree represented average genetic distances between two sites, with nodes indicating points at which sites were clustered together. These data were used to generate % similarities for diversity calculations. Comparison of bacterial diversity as determined by RESTSITEâ with plate culture diversity Results obtained for bacterial diversity for sites [B] and[H] using RESTSITEâ were compared with those generated from plate culturing by calculating S±W Diversity Indices (DIs) for both methods (Figure 4). Calculated DI values for both sites, using di€erent levels of resolution, de®ned as percent similarities (see Methods section) form the cluster analysis RFLP output, ranged 669

Marine Pollution Bulletin

Fig. 3 Phylogenetic tree comparing bacterial genetic diversity from the four sites- Buzzards Bay [B], New Bedford Harbor [H], Lower Acushnet Estuary [L], and Upper Acushnet Estuary [U], using the RESTSITEâ program. The tree was generated using the Unweighted Pair Group Method using Arithmetic Averages (Sneath and Sokol, 1973).

Fig. 4 Comparison of plate culture technique and genetic analysis in measuring bacterial diversity using the Shannon±Wiener Diversity Index from Buzzards Bay [B] and New Bedford Harbor [H] samples. Horizontal lines denote Shannon-Wiener Diversity Indices as determined by standard plate culturing on marine agar.

from 4.3 at 0% similarity to 0.0 at 100% similarity, i.e. every OTU would be considered di€erent at a resolution requiring 100% similarity. The axis of percent similarity represents a stringency of resolution, whereby distinctions between clustered OTUs becomes less clear as one moves toward 0% similarity, i.e. the stringency of resolving unique OTUs is relaxed. It is noteworthy that with both the cell culture diversity and the modeled, 16S 670

rRNA genetic diversity, New Bedford Harbor showed consistently higher levels of diversity over Buzzards Bay. As mentioned above. DIs obtained from standard plate culture techniques for [B] during the summer and winter were 1.46 and 1.65, respectively; and those for the [H] were correspondingly 1.71 and 1.94. These values are represented as horizontal lines in Figure 4, and intersect the sloping genetic distance DI values at approximately

Volume 38/Number 8/August 1999 TABLE 3 Correlation of bacterial genetic diversity (d) and % organic carbon (%OC) with individual environmental pollutants. Environmental parameter

da PearsonÕs correlation coecient (p value)

%OCa Pearson's correlation coecient (p value)

% Organic carbon % Organic nitrogen

ÿ0.91 (0.95) ÿ0.95 (0.046)

1.0 (0.00) 0.91 (0.085)

Ag As Cd Co Cr Cu Hg Ni Pb Zn

ÿ0.85 (0.15) ÿ0.82 (0.18) ÿ0.76 (0.24) ÿ0.43 (0.57) ÿ0.85 (0.15) ÿ0.89 (0.11) ÿ0.91 (0.086) ÿ0.76 (0.24) ÿ0.88 (0.12) ÿ0.79 (0.21)

0.99 (0.011) 0.98 (0.023) 0.97 (0.034) 0.70 (0.30) 0.96 (0.044) 0.97 (0.031) 0.98 (0.018) 0.95 (0.046) 0.99 (0.0014) 0.97 (0.028)

PCBs

ÿ0.84 (0.16)

0.99 (0.0088)

a

Correlation analysis performed using logarithms of variable values.

40% similarity for the winter samples and 50% similarity for the summer samples.

Correlation of Average Genetic Distance with Environmental Parameters A simple correlation of average genetic distance with speci®c environmental pollutants was obtained by linear regression of RESTSITEâ output (d^ values, see Methods section) with individual average contaminant concentrations for each environmental site. Parametric (PearsonÕs correlation coecients) correlations are reported in Table 3. Although no correlations were signi®cant at p < 0.05 (except % organic nitrogen), correlation coecients were consistently negative between environmental parameters and d^ values. In addition, %OC was signi®cantly associated with several metals, as might be expected based on a sorption mechanism of metals to OC-coated particles (Shine et al., 1995).

Discussion The greater bacterial diversity noted in New Bedford Harbor relative to Buzzards Bay may be a function of available carbon and energy sources in this ecosystem. The relatively high %OC concentrations in [H] sediments may provide a more abundant and varied source of labile compounds for metabolism. In contrast, Buzzards Bay has a much lower percentage of available sediment OC. The increased diversity in NBH appears to contradict the general ecological theory that diversity decreases with increased environmental stress (Atlas et al., 1991; Atlas and Bartha, 1993). However, given the long history of pollution in New Bedford Harbor, diverse bacterial communities may have adapted to these stressed conditions. The toxic e€ects of certain pollutants, e.g. metals, may explain a higher measured diversity in both sites

during the winter, as determined by plate culture methodology. Increased microbial activity and bioturbation by oligochaetes or other benthic organisms during summer months may lead to enhanced bioavailability of toxic metals (Peterson et al., 1996). This in turn may select for microorganisms capable of resisting toxicity, thus reducing the sediment bacterial diversity (Ford, 1994). Metal-resistant bacteria have been isolated form the [H] site (Ford et al., 1994); however, the limitations of culturability inherent in this technique make extended comparisons dicult (Amann et al., 1995). A direct comparison between plate culture diversity and bacterial diversity determined from cluster analysis of RFLP output is dicult. Part of the diculty not only stems from issues of culturability, but also how a bacterial species is genetically de®ned. Bacterial species have been de®ned on the basis of hybridization and DNA reassociation (Stackebrandt and Goebel, 1994). The researchers suggested a value of 70% similarity for the total genome, and 97% similarity for the 16S rRNA genes as a minimum for distinguishing unique bacterial species. More recently, 95% 16S rRNA similarity was proposed for the de®nition of a bacterial species (Amann et al., 1995). At 95% similarity, 69 out of 72 OTUs (96%) were di€erent for the [B] site, and 70 out of 72 OTUs (97%) were di€erent for the [H] site. However, a direct comparison to these numbers is not possible because the values were derived by di€erent means. The results presented here do suggest a large amount of bacterial genetic diversity in these environmental sites. As expected, RFLP analysis yielded far greater diversity for all environmental sites relative to traditional plate culturing. The limits of the technique were not reached, as very few RFLP patterns were identical both within and between environmental sites, suggesting more diversity would be revealed had more unique RFLP patterns been generated. The total bacterial genetic diversity per site remains unknown at this point, as it is uncertain what percentage of the unique 16S rRNA gene 671

Marine Pollution Bulletin

sequences were recovered from the total population of DNA extracted from the sediments. The rationale for using RFLP data is that the degree of genetic divergence between two DNA sequences is correlated with the proportion of DNA fragments shared by them (Nei, 1987). The RESTSITEâ program analyzed DNA fragment data to determine average bacterial genetic distance among di€erent groups of DNA sequences for each of the four sites using the length-di€erence method (Nei and Li, 1979). The following assumptions were made regarding genetic distance calculations: all four nucleotide types (A,T,C, and G) are randomly arranged in each DNA sequence; all four nucleotide sites have equal probability of substitution; and rates of substitution between the four nucleotides are the same. With small values of average genetic distance, these assumptions are rarely violated. The program only considered nucleotide substitutions based on polymorphisms and did not treat mutations resulting in deletions or insertions. However, it must be acknowledged that a signi®cant proportion of evolutionary/genetic change can occur via these mechanisms (Nei, 1987; Brunk et al., 1996). UPGMA appeared to work well as long as the assumption of constant rate of nucleotide substitutions holds and the distances among the taxa are large, i.e. d^ > 0:1 (Holsinger and Jansen, 1993; Nei, 1987). Average genetic distances for each of the four environmental sites were typically between 0.10 and 0.15. In each case, the relationship of bacterial genetic distance among the sites could be easily visualized from the UPGMA dendogram. There was a clear separation shown between all four environmental sites. Also, the [H], [L] and [U] sites were positioned in an order that re¯ected both their geographical relationship, as well as their contaminant pro®les. There appears to be both a relatively high degree of bacterial genetic diversity as re¯ected in the large number of OTUs recovered from each of the four environmental sites, as well as a certain degree of relatedness among them. This might be expected due to the water bodies connecting each environment. The RESTSITEâ program has been used to compare phylogenetic diversity between a variety of organisms, including plants using chloroplast (cp) DNA (Doyle, 1991; Doyle and Doyle, 1993; Mes and Hart, 1996) and mitochondrial (mt) DNA (Hervieu et al., 1994; Tarr and Fleischer, 1993; Vermeulen et al., 1994), mammals using nuclear and mtDNA (Riddle et al., 1993; Rogers and Kidd, 1996) and mtDNA (Lee et al., 1996b), arthropods using mtDNA (Tam et al., 1996), and reptiles using mtDNA (Phillips, 1994; Phillips et al., 1996). For microorganisms, the program has been employed using rDNA to relate strains of yeast (LeGuennec et al., 1995; Lewicka et al., 1995), Psuedomonas species (Laguerre et al., 1994), and carbofuran-degrading soil bacteria (Parekh et al., 1995). This is the ®rst application, however, of this program to RFLP/cluster analysis of PCRampli®ed 16S rRNA genes in DNA extracted from 672

di€erent environmental sites. Relating genetic sequences within a population and comparing them to di€erent sites is a novel application of this program for the characterization of bacterial genetic diversity. Other methods have been developed recently to describe bacterial community composition in various ecosystems. Capillary electrophoresis was used to analyze ¯uorescently-labelled small subunit rRNA genes which had been PCR-ampli®ed and then digested with restriction enzymes to determine fragment size di€erences (Avaniss-Aghajani et al., 1994). The researchers were able to identify a number of di€erent bacterial species contained in a complex environmental mixture. A non-radioactive method based on single-strand-conformation polymorphism analysis and PCR compared 16S rRNA gene sequences from di€erent environmental samples (Lee et al., 1996c). This technique is sensitive in detecting bacterial populations that comprise less than 1.5% of the total bacterial community. Noble et al (1997) described natural microbial community compositions by creating a back-propagating neural network and performing cluster analysis of 5S rRNA to interpret complex banding patterns generated from environmental samples. The method could relate diverse assemblages of bacterial species in di€erent environments. The method developed and presented here o€ers several advantages in that RFLP fragment data from a number of environmental sites can be used to relate genetic diversity within and among sites. The RESTSITEâ program can be expanded to analyze any number of OTUs. Using bacterial community composition as a biomarker of environmental stress Although attempts to correlate average genetic distance with environmental parameters did not produce signi®cant correlations at the 95% con®dence level, the analysis suggested two areas for discussion and further research: 1. d^ values generated from the RESTSITEâ analysis of RFLP data provide a useful parameter for comparing average genetic distance with environmental variables. In theory, diversity indices calculated form cluster analyses could also be used in these comparisons, if appropriate numbers of RFLP patterns could be generated to provide sucient numbers of identical patterns to allow generation of an index at the 95% (Amann et al., 1995), or otherwise appropriate, similarity level. 2. Consistent negative correlations between d^ values and environmental parameters suggest there may be increased genetic relatedness at contaminated sites versus less contaminated sites. An increase in diversity and genetic relatedness at contaminated sites could suggest a more constrained ecological niche, supporting a greater diversity of more closely related bacteria, e.g. many species of the same genus, growing on organically rich sediments with high levels of toxic contaminants.

Volume 38/Number 8/August 1999

In future research, generation of more RFLP patterns (only 10% of clones were analyzed), and a larger number of environmental sites sampled could strengthen these associations.

Conclusions A PCR-based method was developed to compare bacterial average genetic distance among di€erent environmental sites with varying degrees of anthropogenic impact. New Bedford Harbor, MA, an EPA-designated Superfund hazardous waste site, was studied to assess changes in microbial diversity resulting from long-term inputs of di€erent organic and inorganic compounds. Total DNA was extracted from sur®cial sediments sampled from sites along a transect of decreasing contamination (Upper and Lower Acushnet Estuary, New Bedford Harbor, and Buzzards Bay, respectively). RFLP analysis of PCR-ampli®ed 16S rRNA genes resulted in generation of a number of unique OTUs. Phylogenetic analyses of fragment pattern data using the computer program RESTSITEâ allowed for comparisons of overall bacterial genetic distance among these sites. The procedure described above is relatively rapid had has the advantage of recovering large numbers of OTUs, while not requiring the time and expense of full sequence analysis. Future work should allow inter-site comparisons in bacterial genetic distance to be correlated with concentration di€erences for speci®c environmental contaminants. Furthermore, since bacteria possess relatively high generation rates and simple genomes, and are susceptible to mutations and genetic exchange, bacterial average genetic distance may be a sensitive indicator of environmental stress. The authors wish to gratefully acknowledge the counsel and advice of the following individuals. Drs. Joseph Harrington, Stuart Lipsitz, Martin Poltz, James Shine, Stephen Tay, Ji-Dong Gu, Carol Barford, Tony and Mary Farone, George OÕToole and Steve Finkel; and technical support from Beth Kay, Liz LaPointe, Ravi Ika, Suzanne Michaud, Cristin Corless, and Sid Atwood. This publication was made possible by grant number 5 P42 ES-05947 from the National Institute of Environmental Health Sciences, NIH, with funding provided by EPA. Its contents are solely the responsibility of the authors and do not necessarily represent the ocial views of the NIEHS, NIH, or EPA. Amann, R., Ludwig, W. and Schleifer, H.-H. (1995) Phylogenetic identi®cation and in-situ detection of individual microbial cells without cultivation. Microbiological Reviews 59, 143±169. Atlas, R. M. (1984) Diversity of microbial communities. Advances in Microbial Ecology 7, 1±47. Atlas, R. M., Horowitz, A., Krichevsky, M. I. and Bej, A. K. (1991) Response of microbial populations to environmental stress. Microbial Ecology 22, 249±256. Atlas, R. M. and Bartha. R. (1993) Microbial communities and ecosystems. In Microbial Ecology: Fundamentals and Applications. Benjamin Cummings, New York, pp. 140±145. Avaniss-Aghajani, E., Jones, K., Chapman, D. and Brunk, C. (1994) A molecular technique for identi®cation of bacteria using small subunit ribosomal RNA sequences. Bio Techniques 17, 144±149. Boivin-Jahns, V., Bianchi, A., Ruimy, R., Garci, J., Daumass, S. and Christen, R. (1995) Comparison of phenotypical and molecular methods for the identi®cation of bacterial strains isolated from a deep subsurface environment. Applied and Environmental Microbiology 61, 3400±3406.

Britschgi, T. B. and Giovannoni, S. J. (1991). Phylogenetic analysis of a natural marine bacterioplankton population by rRNA gene cloning and sequencing. Applied and Environmental Microbiology 57, 1707±1713. Brosius J., Palmer M. L., Kennedy P. J. and Noller H. F. (1978) Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli. Proceedings of the National Academy of Sciences, USA 75, 4801±4805. Bruce, K. D., Hiorns, W. D., Hobman, J. L., Osborn, A. M., Strike, P. and Ritchie, D. A. (1992) Ampli®cation of DNA from native populations of soil bacteria by using the polymerase chain reaction. Applied and Environmental Microbiology 58, 3413±3416. Brunk, C. F., Avaniss-Aghjani, E. and Brunk, C. A. (1996) A computer analysis of primer and probe hybridization potential with bacterial small-subunit rRNA sequences. Applied and Environmental Microbiology 62, 872±879. Dallinger, R. (1994) Invertebrate organisms as biological indicators of heavy metal pollution. Applied Biochemistry and Biotechnology 48, 27±31. Dallinger, R. (1996) Metallothionein research in terrestrial invertebrates: synopsis and perspectives. Comparative Biochemistry and Physiology, Part C Pharmacology, Toxicology, and Endocrinology 113, 125±133. DeLong, E. F., Franks, D. G. and Alldredge, A. L. (1993) Phylogenetic diversity of aggregate-attached versus free-living marine bacterial assemblages. Limnology and Oceanography 38, 924±934. Depledge, M. H., Aagaard, A. and Gyorkos, P. (1995) Assessment of trace metal toxicity using molecular, physiological and behavioural biomarkers. Marine Pollution Bulletin 31, 19±27. De Pomerai, D. (1996) Heat-shock proteins as biomarkers of pollution. Human Experimental Toxicology 15, 279±285. Doyle, J. J. (1991) Evolution of higher-plant glutamine systhetase genes: Tissue speci®city as a criterion for predicting orthology. Molecular Biology and Evolution 8, 366±377. Doyle, J. J. and Doyle, J. L. (1993) Chloroplast DNA phylogeny of the papilionoid legume tribe phaseoleae. Systematic Botany 18, 309±327. Etxeberria, M., Gajaraville, M. P. and Marigomez, I. (1995) Changes in digestive cell lysosomal structure in mussels as biomarkers of environmental stress in the Urdaibai Estuary (Biscay Coast, Iberian Peninsula). Marine Pollution Bulletin 31, 599±603. Ford, T. E. (1994) Pollutant e€ects on the microbial ecosystem. Environmental Health Perspectives 102, 45±48. Ford, T. E., Sorci, J. J. and Shine, J. J. (1994) Microbial transport of toxic metals. In Trace Substances, Environment and Health, ed C. R. Cothern. Science Reviews, Northwood, UK, pp. 9±20. Fuhrman, J. A., Lee, S. H., Masuchi, Y., Davis, A. A. and Wilcox, R. M. (1994) Characterization of marine prokaryotic communities via DNA and RNA. Microbial Ecology 28, 133±145. Fuhrman, J. A., McCallum, K. and Davis, A. A. (1993) Phylogenetic diversity of subsurface marine microbial communities from the Atlantic and Paci®c Oceans. Applied and Environmental Microbiology 59, 1294±1302. Giovannoni, S. J., Britschgi, T. B., Moyer, C. L. and Field, K. G. (1990) Genetic diversity in Sargasso Sea bacterioplankton. Nature 345, 60±63. Goksoyr, A. (1995) Use of cytochrome P450 1A (CYP1A) in ®sh as a biomarker of aquatic pollution. Archives of Toxiocology Supplement 17, 80±95. Gray, J. P. and Herwig, R. P. (1996) Phylogenetic analysis of the bacterial communities in marine sediments. Applied and Environmental Microbiology 62, 4049±4059. Hervieu, F., Bannerot, H. and Pelletier, G. (1994) A unique cytoplasmic male sterility (CMS) determinant is present in three Phaseolus species characterized by di€erent mitochondrial genomes. Theoretical and Applied Genetics 88, 314±320. Holsinger, K. E. and Jansen, R. K. (1993) Phylogenetic analysis of restriction site data. Methods in Enzymology 224, 439±455. Hugenholtz, P. and Pace, N. R. (1996) Identifying microbial diversity in the natural environment: A molecular phylogenetic approach. Trends in Biotechnology 14, 190±197. Kreamer, G. L., Squibb, K., Gioeli, D., Garte, S. J. and Wirgin, I. (1991) Cytochrome P450IA mRNA expression in feral Hudson River tomcod. Environmental Research 55, 64±78. Laguerre, G., Rigottier-Gois, L. and Lemanceau, P. (1994) Fluorescent Pseudomonas species categorized by using polymerase chain reaction (PCR)/restriction fragment analysis of 16S rDNA. Molecular Ecology 3, 479±487.

673

Marine Pollution Bulletin Lane, D. (1991) 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics, eds E. Stackebrandt and M. Goodfellow. Wiley, New York, p. 133. Lee, S.-Y., Bollinger, D. and Ogram, A. (1996a) Estimation of the abundance of an uncultured soil bacterial strain by a competitive quantitative PCR method. Applied and Environmental Microbiology 62, 3787±3793. Lee, T. E., Riddle, B. R. and Lee, P.L. (1996b) Speciation in the desert pocket mouse (Chaetodipus penicillatus Woodhouse). Journal of Mammology 77, 58±68. Lee, D. H., Zo, Y. G. and Kim, S. J. (1996c) Nonradioactive method to study genetic pro®les of natural bacterial communities by PCRsingle-strand-conformation polymorphism. Applied and Environmental Microbiology 62, 3112±3120. Le Guennec, R., Reynes, J., Mallie, M., Pujol, C., Janbon, F. and Bastide, J.-M. (1995) Fluconazole- and intraconazole-resistant Candida albicans strains from AIDS patients: Multilocus enzyme electrophoresis analysis and antifungal susceptibilities. Journal of Clinical Microbiology 33, 2732±2737. Lewicka, K., Mallie, M. and Bastide, J.-M. (1995) Genetic variability in the Saccharomyces sensu stricto complex revealed by multilocus enzyme electrophoresis. International Journal of Systematic Bacteriology 45, 538±543. Livingstone, D. R. (1993) Biotechnology and pollution monitoring: Use of molecular biomarkers in the aquatic environment. Journal of Chemical Technology and Biotechnology 57, 195±211. Mason, B. H. (1966) Composition of the earth. Nature 211, 616±618. Mes, T. H. M. and Hart, H. T. (1996) The evolution of growth-forms in the Macaronesian genus Aeonium (Crassulaceae) inferred from chloroplast DNA RFLPs and morphology. Molecular Ecology 5, 351±363. Miller, J. C. (1991) RESTSITE: A phylogenetic program that sorts raw restriction data. Journal of Heredity 82, 262±263. Moyer, C. L., Dobbs, F. C. and Karl, D. M. (1994) Estimation of diversity and community structure through restriction fragment length polymorphism distribution analysis of bacterial 16S rRNA genes from a microbial mat at an active, hydrothermal vent system, Loihi Seamount, Hawaii. Applied and Environmental Microbiology 60, 871±879. Moyer, C. L., Tiedje, J. M., Dobbs, F. C. and Karl, D. M. (1996) A computer-simulated restriction fragment length polymorphism analysis of bacterial small-subunit rRNA genes: Ecacy of selected tetrameric restriction enzymes for studies of microbial diversity in nature. Applied and Environmental Microbiology 62, 2501±2507. Mutwakil, M. H. A. Z., Reader, J. P., Holdich, D. M., Smithurst, P. R., Candido, E. P. M., Jones, D., Stringham, E. G. and de Pomerai, D. I. (1997) Use of stress-inducible transgenic nematodes as biomarkers of heavy metal pollution in water samples from an English river system. Archives of Environmental Contamination and Toxicology 32, 146±153. Najimi, S., Bouhaimi, A., Daubeze, M., Zekhnini, A., Pellerin, J., Narbonne, J. F. and Moukrim, A. (1997) Use of acetylcholinesterase in Perna perna and Mytilus galloprovincialis as a biomarker of pollution in Agadir Marine Bay. Bulletin of Environmental Contamination and Toxicology 58, 901±908. Nei, M. 1987. Phylogenetic trees. In Molecular Evolutionary Genetics. Columbia University Press, New York, pp. 292±313. Nei, M. and Li, W.-H. (1979) Mathematical model for studying genetic variation in terms of restriction endonucleases. Proceedings of the National Academy of Sciences USA 76, 5269±5273. Nei, M. and Miller, J. C. (1990) A simple method for estimating average number of nucleotide substitutions within and between populations from restriction data. Genetics 125, 873±879. Neilan, B. A., Jacobs, D. and Goodman, A. E. (1995) Genetic diversity and phylogeny of toxic cyanobacteria determined by DNA polymorphisms within the Phycocyanin locus. Applied and Environmental Microbiology 61, 3875±3883. Noble, P. A., Bidle, K. D. and Fletcher, M. (1997) Natural microbial community compositions compared by a back-propagating neural network and cluster analysis of 5S rRNA. Applied and Environmental Microbiology 63, 1762±1770. Olsen, G. J., Lane, D. L., Giovannoni, S. J. and Pace, N. R. (1986) Microbial ecology and evolution: A ribosomal RNA approach. Annual Review of Microbiology 40, 337±365. Pace, N. R., Stahl, D. A., Lane, D. J. and Olsen, G. J. (1986) The analysis of natural microbial populations by ribosomal RNA sequences. Advances in Microbial Ecology 9, 1±55.

674

Parekh, N. R., Hartmann, A., Charnay, M.-P. and Fournier, J.-C. (1995) Diversity of carbofuran-degrading soil bacteria and detection of plasmid-encoded sequences homologous to the mcd gene. FEMS Micobial. Ecology 17, 149±160. Peterson, G. S., Ankley, G. T. and Leonard, E. N. (1996) E€ect of bioturbation on metal-sul®de oxidation in sur®cial freshwater sediments. Environmental Toxicology and Chemistry 15, 2147±2155. Phillips, C. A. (1994) Geographic distribution of mitochondrial DNA variants and the historical biogeography of the spotted salamander. Ambystoma maculatum. Evolution 48, 597±607. Phillips, C. A., Dimmick, W. W. and Carr, J. L. (1996) Conservation genetics of the common snapping turtle (Chelydra serpentina). Conservation Biology 10, 397±405. Pruell, R. J., Norwood, C. B., Bowen, R. D., Boothman, W. S., Rogerson, P. F., Hackett, M. and Butterworth, B. C. (1990) Geochemical study of sediment contamination in New Bedford Harbor, Massachussetts. Marine Environmental Research 29, 77±101. Riddle, B. R., Honeycutt, R. L. and Lee, P. L. (1993) Mitochondrial DNA phylogeography in northern grasshopper mice (Onychomys leucogaster) ± the in¯uence of Quaternary climatic oscillations on populations dispersion and divergence. Molecular Ecology 2, 183± 193. Rogers, J. and Kidd, K. K. (1996) Nucleotide polymorphism, e€ective population size, and dispersal distances in the yellow baboons (Papio hamadryas cynocephalus) of Mikumi National Park, Tanzania. American Journal of Primatology 38, 157±168. Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Sanders, B. M. and Martin, L. S. (1993) Stress proteins as biomarkers of contaminant exposure in archived environmental samples. Science of the Total Environmental 139±140, 459±470. Schmidt, T. M., DeLong, E. F. and Pace, N. R. (1991) Analysis of a marine picoplankton community by 16S rRNA gene cloning and sequencing. Journal of Bacteriology 173, 4371±4378. Shine, J. P., Ika, R. V. and Ford, T. E. (1995) Multivariate statistical examination of spatial and temporal patterns of heavy metal contamination in New Bedford Harbor marine sediments. Environmental Science and Technology 29, 1781±1788. Sneath, P.H.A. and Sokal, R.R. 1973. Numerical Taxonomy. W.H. Freeman, San Fransisco, CA, pp. 230±234. Sole, M., Porte, C. and Albaiges, J. (1995) The use of biomarkers for assessing the e€ects of organic pollution in mussels. Science of the Total Environment 159, 147±153. Sorci, J.J. 1998. Bacterial diversity and heavy metal gene expression in polluted marine sediments from New Bedford Harbor, Massachusetts. Doctoral Dissertation, Harvard University. Stackebrandt, E. and Goebel, B. M. (1994) Taxonomic note: A place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species de®nition in bacteriology. International Journal of Systematic Bacteriology 44, 846±849. Ste€an, R. J. and Atlas, R. M. (1991) Polymerase chain reaction: applications in environmental microbiology. Annual Review of Microbiology 45, 137±161. Suzuki, M.T. and Giovannoni, S.J. (1996) Bias caused by template annealing in the ampli®cation of mixtures of 16S rRNA genes by PCR. Applied and Environmental Microbiology 62, 625±630. Tam, Y. K., Korn®eld, I. and Ojeda, F. P. (1996) Divergence and zoogeography of mole crabs, Emerita spp (Decapoda: Hippidae), in the Americas. Marine Biology 125, 489±497. Tarr, C. L. and Fleishcer, R. C. (1993) Mitochondrial-DNA variation and evolutionary relationships in the Amakihi complex. Auk 110, 825±831. Taylor, S. R. (1964) Abundance of chemical elements in the continental crust ± a new table. Geochimica et Cosmochimica Acta 28, 1273± 1285. Tebbe, C. C. and Vahjen, W. (1993) Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast. Appied and Environmental Microbiology 59, 2657±2665. Tsai, Y. and Olson, B. H. (1991) Rapid method for direct extraction of DNA from soils and sediments. Applied and Environmental Microbiology 57, 1070±1074. US Environmental Protection Agency, (1984) Guidelines establishing test procedures for the analysis of pollutants; ®nal regulations. EPA methods 608, 3540/3550, and 8080. Federal Register, Vol. 49, No. 209, pp. 89±104.

Volume 38/Number 8/August 1999 U.S. Environmental Protection Agency. 1990. New Bedford Harbor NPL site estuary and lower harbor/bay operable unit: Administrative Record, Vol. VI, p. 5-5. van der Oost, R., Goksoyr, A., Celander, M., Heida, H. and Vermeulen, N. P. E. (1996) Biomonitoring of aquatic pollution with ferral eel (Anguilla anguilla). I Biomarkers: Pollution-induced biochemical responses. Aquatic Toxicology 36, 189±222. van der Oost, R., Vindimian, E., van den Brink, P. J., Satumalay, K., Heida, H. and Vermeulen, N. P. E. (1997) Biomonitoring of aquatic pollution with ferral eel (Anguilla anguilla). III Statistical analyses of relationships between contaminant exposure and biomarkers. Aquatic Toxicology 39, 45±75. Vermeulen, A., Desprez, B., Lancelin, D. and Bannerot, H. (1994) Relationships among Cichorium species and related genera as determined by analysis of mitochondrial RFLPS. Theoretical and Applied Genetics 88, 159±166. Ward, D. M., Bateson, M. M., Weller, R. and Ru€-Roberts, A. L. (1992) Ribosomal RNA analysis of microorganisms as they occur in nature. Advances in Microbial Ecology 12, 219±257.

Ward, D. M., Weller, R. and Bateson, M. M. (1990) 16S rRNA sequences reveal uncultured inhabitants of a well-studied thermal community. FEMS Microbiological Reviews 75, 105±116. Weaver, G. (1984) PCB Contamination in and around New Bedford, Mass. Environmental Science and Technology 18, 22A±27A. Weidner, S., Arnold, W. and Puhler, A. (1996) Diversity of uncultured microorganisms associated with the seagrass Halophila stipulacea estimated by restriction fragment length polymorphism analysis of pcr-ampli®ed 16S rRNA genes. Applied and Environmental Microbiology 62, 766±771. Williams, G.M. and Weisburger, J.H. 1993. Chemical Carcinogenesis. In Casarett and Doull's Toxicology: The Basic Science of Poisons, eds M. O. Amdur, J. Doull, and C. D. Klaassen, pp.127±210. Pergamon Press, New York. Woese, C. R. (1987) Bacterial evolution. Microbiological Reviews 51, 221±271. Zhou, J., Bruns, M. A. and Tiedje, J. M. (1996) DNA recovery from soils of diverse compositon. Applied and Environmental Microbiology 62, 316±322.

675