Differences in the Effect of Coal Pile Runoff (Low pH, High Metal Concentrations) Versus Natural Carolina Bay Water (Low pH, Low Metal Concentrations) on Plant Condition and Associated Bacterial Epiphytes of Salvinia minima.

Numerous wetlands and streams have been impacted by acid mine drainage (AMD) resulting in lowered pH and increased levels of toxic heavy metals. Remediation of these contaminated sites requires knowledge on the response of microbial communities (especially epiphytic) and aquatic plants to these altered environmental conditions. We examined the effect of coal pile runoff waters as an example of AMD in contrast to natural water from Carolina Bays with low pH and levels of metals on Salvinia minima, a non-native, metal accumulating plant and associated epiphytic bacteria. Treatments included water from two Carolina Bays, one AMD basin and Hoagland's Solution at two pH levels (natural and adjusted to 5.0-5.5). Using controlled replicated microcosms (N = 64) we determined that the combination of low pH and high metal concentrations has a significant negative impact (p < 0.05) on plant condition and epiphytes. Solution metal concentrations dropped indicating removal from solution by S. minima in all microcosms.

Patterns of Multi-Antibiotic-Resistant Escherichia Coli from Streams with No History of Antimicrobial Inputs.

A growing body of evidence suggests that contaminated environments may harbor a greater proportion of antibiotic-resistant microorganisms than unpolluted reference sites. Here, we report the screening of 427 Escherichia coli strains isolated from 11 locations on nine streams draining the US Department of Energy's Savannah River Site against a panel of five antibiotics. Streams were chosen to capture a wide range of watersheds from minimally disturbed to highly impacted. Overall, higher levels of resistance were found in waterborne E. coli that also generally exhibited low spatial variability. However, 3 of 11 locations also demonstrated elevated resistance levels in sediments. Two of these occurred in highly disturbed tributaries with no obvious sources of antimicrobials. To further investigate these patterns, we screened a subset of isolates obtained from three streams against 23 antibiotics or antibiotic combinations. A large proportion of these isolates (>40 %) demonstrated resistance to 10 or more antimicrobials, suggesting that environmental multi-antibiotic resistance may be prevalent in this bacterial commensal. Only 4 of 87 viable isolates were tested susceptible to all 23 antibiotics and combinations. Among these multi-antibiotic-resistant isolates, several demonstrated resistance to all structural classes of antimicrobial agents tested, including frontline antibiotics such as gatifloxacin and ciprofloxacin.

Multi-site analysis reveals widespread antibiotic resistance in the marine pathogen Vibrio vulnificus.

Vibrio vulnificus is a serious opportunistic human pathogen commonly found in subtropical coastal waters, and is the leading cause of seafood-borne mortality in the USA. This taxon does not sustain prolonged presence in clinical or agricultural settings, where it would undergo human-induced selection for antibiotic resistance. Therefore, few studies have verified the effectiveness of commonly prescribed antibiotics in V. vulnificus treatment. Here we screened 151 coastal isolates and 10 primary septicaemia isolates against 26 antimicrobial agents representing diverse modes of action. The frequency of multiple resistances to antibiotics from all sources was unexpectedly high, particularly during summer months, and a substantial proportion of isolates (17.3%) were resistant to eight or more antimicrobial agents. Numerous isolates demonstrated resistance to antibiotics routinely prescribed for V. vulnificus infections, such as doxycycline, tetracycline, aminoglycosides and cephalosporins. These resistances were detected at similar frequencies in virulent and non-virulent strains (PCR-based virulence typing) and were present in septicaemia isolates, underlying the public health implications of our findings. Among environmental isolates, there were no consistent differences in the frequency of resistance between pristine and anthropogenically impacted estuaries, suggesting natural rather than human-derived sources of resistance traits. This report is the first to demonstrate prevalent antibiotic resistance in a human pathogen with no clinical reservoirs, implying the importance of environmental studies in understanding the spread, evolution and public health relevance of antibiotic resistance factors.

Antibiotic resistance in the shellfish pathogen Vibrio parahaemolyticus isolated from the coastal water and sediment of Georgia and South Carolina, USA.

Vibrio parahaemolyticus is a gram-negative pathogen commonly encountered in estuarine and marine environments, and a common cause of seafood-related gastrointestinal infections. We isolated 350 V. parahaemolyticus strains from water and sediment at three locations along the Atlantic coast of Georgia and South Carolina during various seasons. These isolates were tested for susceptibility to 24 antibiotics. Isolate virulence was determined through PCR of tdh and trh genes. The breadth of resistance to antibiotics was unexpectedly high, with 24% isolates demonstrating resistance to 10 or more agents. A significant fraction of isolates were resistant to diverse beta-lactams, aminoglycosides, and other classes of antibiotics. Fifteen of the 350 strains possessed virulence genes, with no apparent correlation between virulence and site, sample type, or season of isolation. Antibiotic resistance was slightly reduced among the virulent strains. This study represents one of the largest surveys to date of the virulence and antibiotic resistance in environmental V. parahaemolyticus strains. The observed antibiotic susceptibility patterns suggest that current guidelines for the antibiotic treatment of non-cholerae Vibrio should be reevaluated and extended.

Coselection for microbial resistance to metals and antibiotics in freshwater microcosms.

Bacterial resistances to diverse metals and antibiotics are often genetically linked, suggesting that exposure to toxic metals may select for strains resistant to antibiotics and vice versa. To test the hypothesis that resistances to metals and antibiotics are coselected for in environmental microbial assemblages, we investigated the frequency of diverse resistances in freshwater microcosms amended with Cd, Ni, ampicillin or tetracycline. We found that all four toxicants significantly increased the frequency of bacterioplankton resistance to multiple, chemically unrelated metals and antibiotics. An ampicillin-resistant strain of the opportunistic human pathogen Ralstonia mannitolilytica was enriched in microcosms amended with Cd. Frequencies of antibiotic resistance were elevated in microcosms with metal concentrations representative of industry and mining-impacted environments (0.01-1 mM). Metal but not antibiotic amendments decreased microbial diversity, and a weeklong exposure to high concentrations of ampicillin (0.01-10 mg l-1) and tetracycline (0.03-30 mg l-1) decreased microbial abundance only slightly, implying a large reservoir of antibiotic resistance in the studied environment. Our results provide first experimental evidence that the exposure of freshwater environments to individual metals and antibiotics selects for multiresistant microorganisms, including opportunistic human pathogens.

Co-selection of antibiotic and metal resistance.

There is growing concern that metal contamination functions as a selective agent in the proliferation of antibiotic resistance. Documented associations between the types and levels of metal contamination and specific patterns of antibiotic resistance suggest that several mechanisms underlie this co-selection process. These co-selection mechanisms include co-resistance (different resistance determinants present on the same genetic element) and cross-resistance (the same genetic determinant responsible for resistance to antibiotics and metals). Indirect but shared regulatory responses to metal and antibiotic exposure such as biofilm induction also represent potential co-selection mechanisms used by prokaryotes. Metal contamination, therefore, represents a long-standing, widespread and recalcitrant selection pressure with both environmental and clinical importance that potentially contributes to the maintenance and spread of antibiotic resistance factors.

Elevated microbial tolerance to metals and antibiotics in metal-contaminated industrial environments.

To test the hypothesis that industrial metal contaminants select for microorganisms tolerant to unrelated agents, such as antibiotics, we analyzed metal and antibiotic tolerance patterns in microbial communities in the intake and discharge of ash settling basins (ASBs) of three coal-fired power plants. High-throughput flow-cytometric analyses using cell viability probes were employed to determine tolerances of entire bacterioplankton communities, avoiding bias toward culturable versus nonculturable bacteria. We found that bacterioplankton collected in ASB discharges were significantly more tolerant to metal and antibiotic exposures than bacterioplankton collected in ASB intakes. Optical properties of microorganisms collected in ASB discharges indicated no defensive physiological adaptations such as formation of resting stages or excessive production of exopolymers. Thus, it is likely that the elevated frequency of metal and antibiotic tolerances in bacterioplankton in ASB discharges were caused by shifts in microbial community composition, resulting from the selective pressure imposed by elevated metal concentrations or organic toxicants present in ASBs.

Methylosarcina fibrata gen. nov., sp. nov. and Methylosarcina quisquiliarum sp.nov., novel type 1 methanotrophs.

Two novel species of obligate methane-oxidizing bacteria, isolated from landfill soil, were characterized. Both strains were unusual in that some members of the population grew in irregularly shaped, refractile cell packets that resembled sarcina-like clusters. Electron microscopy revealed that the cell packets were covered with a slime layer and the cells contained many large granular inclusion bodies. The individual cells of each strain were sometimes motile and had differing morphologies. Isolate AML-C10T was always coccoidal in shape, and the cells were covered with extracellular fibrils. Isolate AML-D4T was pleomorphic, changing from rod to coccal form, sometimes exhibiting an unusual fusiform morphology. AML-D4T lacked the extensive fibrillar matrix observed with AML-C10T. Both strains utilized only methane and methanol as carbon sources. In stationary phase, the cells of each strain swelled in size and formed cysts. Aside from morphological differences, strains could also be distinguished from each other by cellular protein patterns, as well as by temperature and pH tolerances. 16S rDNA phylogenetic analysis showed that these are type I methanotrophs (family: Methylococcaceae) most closely related to the Methylobacter/Methylomicrobium clade, although they form a monophyletic grouping supported by moderately high bootstrap values. By 16S rDNA database searches, the most similar species to both isolates were Methylobacter spp. However, partial particulate methane monooxygenase sequence analysis suggested that these bacteria might be more closely related to Methylomicrobium than Methylobacter. Furthermore, cellular fatty acid profiles of the strains more closely resemble those of Methylomicrobium, although the absence of significant levels of 16:1omega5c argues for the uniqueness of these two strains. On the basis of the results described here, it is proposed that a new genus should be created, Methylosarcina gen. nov., harbouring two species, Methylosarcina fibrata sp. nov. (type species) and Methylosarcina quisquiliarum sp. nov. The type strains are AML-C10T (= ATCC 700909T = DSM 13736T) and AML-D4T (= ATCC 700908T = DSM 13737T), respectively.

Molecular genetic markers provide no evidence for reproductive isolation among retreat building phenotypes of the net-spinning caddisfly Macrostemum carolina.

Larvae of the stream-dwelling, filter-feeding caddisfly Macrostemum carolina construct silken catchnets within protective retreats. In the Savannah River, M. carolina individuals make three different retreats, each with a distinct water entrance hole: (i) at the end of a silken tube; (ii) with a approximately 180 degrees silken backstop; and (iii) flush with the top of the retreat. To resolve whether these different retreats represent alternative behavioural phenotypes within a single panmictic population or fixed phenotypes within three genetically distinct populations or species, we compared the allele frequencies at three polymorphic nuclear loci (allozyme electrophoresis for Gpi, Mpi and Pgm) and the mitochondrial DNA (mtDNA) haplotype frequencies among individuals displaying the three retreat morphs. We also calculated pairwise exact tests of population differentiation using the allozyme and mtDNA allele frequencies. No significant genetic differentiation was detected among caddisflies exhibiting the different retreat morphs. Therefore, these morphs apparently represent a single panmictic population in the Savannah River. Consequently, additional study is required to assess whether this retreat polymorphism is a phenotypically plastic trait under conditional control, or is mediated by alternative alleles at a Mendelian gene or genes (or a combination of the two).

Aquatic Actinomycete-Fungal Interactions and Their Effects on Organic Matter Decomposition: A Microcosm Study.

The role of fungi in the decomposition of organic matter in streams has been well examined, although the role of bacterial antagonists in such processes has gained little attention. To examine bacterial-fungal interactions, experiments involving pairwise combinations of four actinomycete isolates (A1+ and A2+ could remove chitin from chitin-containing media, and A1? and A2? could not) and two fungal isolates (F+ a true fungus, F? an oomycote) were conducted. For each bacterial-fungal combination, 250-ml microcosms were sampled at 8 day intervals for 32 days. Microbial biomass and organic matter, as well as the activities of five extracellular enzymes, were measured. Each experiment consisted of a control group and four treatment groups. Controls comprised sterilized stream water and macrophytes. The first treatment was inoculated with only actinomycetes (~103 cells ml-1), the second treatment was inoculated with only fungi (~102 cells ml-1), the third group was inoculated simultaneously with actinomycetes and fungi, and the fourth group was inoculated with actinomycetes 2 days after fungal establishment. For all combinations, the lowest rates of organic matter decomposition were expected in the controls, as a result of only physical degradation. In contrast, the greatest rates of organic matter decomposition were predicted in treatments inoculated with F+ 2 days prior to A1? or A2?. Greater than 50% of the organic matter was decomposed in each of the fungal treatments. Fungal-actinomycete interactions resulted in reduced fungal biomass relative to the fungal-only treatments. However, when inoculated 2 days apart, combinations of F? and actinomycetes resulted in enhanced rates of organic matter decomposition, as well as greater levels of extracellular enzyme activities. These results demonstrate that actinomycete-fungal interactions and their colonization dynamics affect the accumulation of biomass, extracellular enzyme activities, and rates of organic matter decomposition.

Spatial patterns in antibiotic resistance among stream bacteria: effects of industrial pollution.

The spatial distribution of antibiotic resistance to streptomycin and kanamycin was examined in natural bacterial communities of two streams. The proportion of resistant bacteria was substantially higher (P < 0.05) in the midreaches of an industrially perturbed stream, but no such pattern was apparent in an undisturbed reference stream. The highest relative frequency of resistance was found at the confluence of a tributary draining a nuclear reactor and industrial complex. Antibiotic resistance increased with distance upstream from the confluence and was positively correlated (r(2) = 0. 54, P = 0.023) with mercury concentrations in the sediments. When the data for two years were compared, this pattern was stable for streptomycin resistance (paired t test, P < 0.05) but not for kanamycin resistance (P > 0.05). Our results imply that heavy metal pollution may contribute to increased antibiotic resistance through indirect selection.

Methanotroph diversity in landfill soil: isolation of novel type I and type II methanotrophs whose presence was suggested by culture-independent 16S ribosomal DNA analysis.

The diversity of the methanotrophic community in mildly acidic landfill cover soil was assessed by three methods: two culture-independent molecular approaches and a traditional culture-based approach. For the first of the molecular studies, two primer pairs specific for the 16S rRNA gene of validly published type I (including the former type X) and type II methanotrophs were identified and tested. These primers were used to amplify directly extracted soil DNA, and the products were used to construct type I and type II clone libraries. The second molecular approach, based on denaturing gradient gel electrophoresis (DGGE), provided profiles of the methanotrophic community members as distinguished by sequence differences in variable region 3 of the 16S ribosomal DNA. For the culturing studies, an extinction-dilution technique was employed to isolate slow-growing but numerically dominant strains. The key variables of the series of enrichment conditions were initial pH (4. 8 versus 6.8), air/CH(4)/CO(2) headspace ratio (50:45:5 versus 90:9:1), and concentration of the medium (1x nitrate minimal salts [NMS] versus 0.2x NMS). Screening of the isolates showed that the nutrient-rich 1x NMS selected for type I methanotrophs, while the nutrient-poor 0.2x NMS tended to enrich for type II methanotrophs. Partial sequencing of the 16S rRNA gene from selected clones and isolates revealed some of the same novel sequence types. Phylogenetic analysis of the type I clone library suggested the presence of a new phylotype related to the Methylobacter-Methylomicrobium group, and this was confirmed by isolating two members of this cluster. The type II clone library also suggested the existence of a novel group of related species distinct from the validly published Methylosinus and Methylocystis genera, and two members of this cluster were also successfully cultured. Partial sequencing of the pmoA gene, which codes for the 27-kDa polypeptide of the particulate methane monooxygenase, reaffirmed the phylogenetic placement of the four isolates. Finally, not all of the bands separated by DGGE could be accounted for by the clones and isolates. This polyphasic assessment of community structure demonstrates that much diversity among the obligate methane oxidizers has yet to be formally described.

Bacterial diversity of a Carolina bay as determined by 16S rRNA gene analysis: confirmation of novel taxa.

Carolina bays are naturally occurring shallow elliptical depressions largely fed by rain and shallow ground water. To identify members of the domain Bacteria which inhibit such an environment, we used PCR to construct a library of 16S rRNA genes (16S rDNAs) cloned from DNA extracted from the sediments of Rainbow bay, located on the Savannah River Site, near Aiken, S.C. Oligonucleotides complementary to conserved regions of 16S rDNA were used as primers for PCR, and gel-purified PCR products were cloned into vector pGEM-T. Partial sequencing of the cloned 16S rDNAs revealed an extensive amount of phylogenetic diversity within this system. Of the 35 clones sequenced, 32 were affiliated with five bacterial groups: 11 clustered with the Proteobacteria division (including members of the alpha, beta, and delta subdivisions), 8 clustered with the Acidobacterium subdivision of the Fibrobacter division (as categorized by the Ribosomal Database Project's taxonomic scheme, version 5.0), 7 clustered with the Verrucomicrobium subdivision of the Planctomyces division, 3 clustered with the gram-positive bacteria (Clostridium and relatives subdivision), and 3 clustered with the green nonsulfur bacteria. One sequence branched very deeply from the Bacteria and was found not to be associated with any of the major divisions when phylogenetic trees were constructed. Two clones did not consistently cluster with specific groups and may be chimeric sequences. None of the clones exhibited an exact match to any of the 16S rDNA sequences deposited in the databases, suggesting that most of the bacteria in Rainbow Bay are novel species. In particular, the clones related to the Acidobacterium subdivision and the Verrucomicrobium subdivision confirm the presence of novel taxa discovered previously in other molecular surveys of this type.

Temporal Variation in Genetic Diversity and Structure of a Lotic Population of Burkholderia (Pseudomonas) cepacia.

The genetic structure and temporal patterns of genetic diversity in a population of Burkholderia (Pseudomonas) cepacia, isolated from a southeastern blackwater stream, were investigated by multilocus enzyme electrophoresis. Allelic variation in seven structural gene loci was monitored at a single stream location at 0, 6, 12, and 24 h and at 2, 4, 8, 16, and 32 days. Over the length of the study, 217 isolates were collected, from which 65 unique electrophoretic types (ETs) were identified. Most of these ETs were present at only one or two time periods and were considered transients; however, one resident ET was particularly abundant (64 of the 217 isolates [29.4%]) and was found at all time points except day 32. The mean genetic diversity of the entire population was 0.520, and the index of association (a measure of multilocus linkage disequilibrium) was 1.33. These results, taken in conjunction with a previous study focusing on spatial patterns of genetic diversity in lotic B. cepacia, show that these bacterial populations exhibit greater variability among sites than within a site over time, suggesting relative stability over short time periods.

Genetic structure of a lotic population of Burkolderia (Pseudomonas) cepacia.

The genetic structure of a population of Burkholderia (Pseudomonas) cepacia isolated from a southeastern blackwater stream was investigated by using multilocus enzyme electrophoresis to examine the allelic variation in eight structural gene loci. Overall, 213 isolates were collected at transect points along the stream continuum, from both the sediments along the bank and the water column. Multilocus enzyme electrophoresis analysis revealed 164 distinct electrophoretic types, and the mean genetic diversity of the entire population was 0.574. Genetic diversity values did not vary spatially along the stream continuum. From a canonical discriminant analysis, Mahalonobis distances (measurements of genetic similarity between populations) revealed significant differences among the subpopulations at the sediment sampling points, suggesting bacterial adaptation to a heterogeneous (or patchy) microgeographical environment. Multilocus linkage disequilibrium analysis of the isolates revealed only limited association between alleles, suggesting frequent recombination, relative to binary fission, in this population. Furthermore, the dendrogram created from the data of this study and the allele mismatch distribution are typical of a population characterized by extensive genetic mixing. We suggest that B. cepacia be added to the growing list of bacteria that are not obligatorily clonal.

Identification of aquatic Burkholderia (Pseudomonas) cepacia by hybridization with species-specific rRNA gene probes.

Burkholderia (Pseudomonas) cepacia is a common environmental bacterium which can be pathogenic for plants and humans. In this study, four strategies were used to identify aquatic isolates: API test strips, hybridization with species-specific DNA probes for the 16S and 23S rRNA genes, fatty acid methyl ester (FAME) profiles, and growth on selective medium (TB-T agar [C. Hagedorn, W. D. Gould, T. R. Bardinelli, and D. R. Gustarson, Appl. Environ. Microbiol. 53:2265-2268, 1987]). Only 59% of the isolates identified as B. cepacia with the API test strips were confirmed as B. cepacia by using fatty acid profiles. The 23S rRNA probe generated a few false-positive results but dramatically underestimated the number of B. cepacia isolates (i.e., 40% of the colonies that did not hybridize to the probe were B. cepacia, as determined by FAME). The 16S rRNA probe generated more false-positive results than the 23S rRNA probe but was effective in identifying the majority of the B. cepacia isolates. The selective medium was only partially successful in recovering B. cepacia. Use of the B. cepacia-specific 16S rRNA probe was the most efficient and accurate way of identifying B. cepacia.

Comparison of methods of DNA extraction from stream sediments.

In Upper Three Runs Creek (Aiken, S.C.) and many other environments, less than 1% of bacteria visible microscopically can be cultured. Exploitation of molecular biology techniques has led to development of new methods, such as extraction of nucleic acids from soils or sediments, to study the dominant, nonculturable bacteria. The purpose of this study was to compare three published methods of DNA extraction that fall into two general categories: those in which cells are lysed in sediments (the Ogram and Tsai and methods [A. Ogram, G. S. Sayler, and T. Barkay, J. Microbiol. Methods 7:57-66, 1987; Y. L. Tsai and B. H. Olson, Appl. Environ. Microbiol. 57:1070-1074, 1991]) and those in which cells are removed from sediments prior to lysis (the Jacobsen method [C. S. Jacobsen and O. S. Rasmussen; Appl. Environ. Microbiol. 58:2458-2462, 1992]). DNA yield varied with extraction method; the Ogram method had a significantly higher yield than the other methods. However, DNA extracted via the Ogram method was badly sheared and contained a smaller proportion of eubacterial DNA. The Tsai method was less time consuming than the other methods, but DNA samples were of lower purity. If DNA purity is of paramount concern (as would be the case if PCR was to be performed) and quantity is not important, the Jacobsen method is recommended because of the low concentration of contaminants. If DNA is to be used directly in DNA-DNA hybridizations, the Ogram method is recommended since it gives maximal yields.(ABSTRACT TRUNCATED AT 250 WORDS)

The relative importance of refugia in determining the drift and habitat selection of predaceous stoneflies in a sandy-bottomed stream.

Patch structure in sandy, compared to rocky streams, is characterized by isolated snags that can only be colonized by drifting. By measuring drift from patches (snags) of various quality we determined the factors that influence habitat selection and drift of the predaceous stoneflies Acroneuria abnormis and Paragnetina fumosa. The presence of refugia (loose bark and leaf packs) was more important than hunger level and modified the effects of increased predator densities and aggressive interactions. Stoneflies concentrated to 8x natural densities with access to refugia remained longer on snags than a single stonefly without access to refugia. During periods of activity, refugia were defended with larger stoneflies always displacing smaller nymphs. During long periods of inactivity, two and sometimes three nymphs would rest side-by-side sharing the same refuge. Hunger level (starved versus satiated stoneflies), an indirect measure of a predator's response to prey availability, had no significant effect on drift or habitat selection regardless of the presence of refugia. Stonefly predators had a uniform distribution while their prey were clumped. Drift was deliberate and almost always delayed until night, usually at dusk or dawn. An examination of previous research plus the results of this study suggest that non-predatory intra- and interspecific interactions can be an important mechanism causing drift in streams.

Detection of Tn5-like sequences in kanamycin-resistant stream bacteria and environmental DNA.

Resistance to kanamycin and neomycin in the bacterial assemblage of a coastal plain stream was detected by growth of colonies on media containing antibiotics. Three of 184 kanamycin-resistant colonies hybridized with a probe containing the nptII gene from transposon Tn5; the nptII gene encodes the enzyme neomycin phosphotransferase and conveys resistance to kanamycin and neomycin. In one of these isolates, the homologous gene was cloned and shown to confer resistance to a kanamycin-sensitive Escherichia coli strain. Since enumeration of bacteria by acridine orange direct counts revealed that less than 0.2% of the bacteria present were cultivated, direct examination of environmental DNA was used to assess abundance of sequences that hybridize to the nptII gene. To examine the resistance potential of bacteria that were not cultured, total DNA was extracted from environmental samples and hybridized with specific probes. The relative amount of eubacterial DNA in each sample was determined by using a eubacterial specific rDNA probe. Then, the abundance of sequences that hybridize to the eubacterial neomycin phosphotransferase gene was determined by hybridization and expressed relative to the total eubacterial DNA in the assemblage. Relative gene abundance was significantly different among assemblages from different habitats (leaves, midchannel sediments, and bank sediments) but did not differ among stream sites.

Genetic diversity in natural populations of a soil bacterium across a landscape gradient.

Genetic diversity in natural populations of the bacterium Pseudomonas cepacia was surveyed in 10 enzymes from 70 clones isolated along a landscape gradient. Estimates of genetic diversity, ranging from 0.54 to 0.70, were higher than any previously reported values of which we are aware and were positively correlated with habitat variability. Patterns of bacterial genetic diversity were correlated with habitat variability. Findings indicate that the source of strains used in genetic engineering will greatly affect the outcome of planned releases in variable environments. Selection of generalist strains may confer a large advantage to engineered populations, while selection of laboratory strains may result in quick elimination of the engineered strains.