Journal Article Citations and Abstracts

Culturable counts of antibiotic resistant, genetically engineered Pseudomonas fluorescens were determined on antibiotic-containing plate count agar during starvation in nutrient deficient water. Prior to starvation, colony counts obtained on all media separated into two groups. The mean of the colony counts on plate count agar with or without tetracycline (4.9 X 106 ml-1) was significantly higher than the mean colony counts on all other media tested (2.5 X 106 ml-1). After 20 days of starvation the highest mean colony counts continued to be obtained on plate count agar (7.2 X 106 ml-1) with slightly, but significantly, lower counts obtained on plate count agar containing either nalidixic acid (5.6 X 106 ml-1) or tetracycline (1.5 X 106 ml-1). In addition, a combination of nalidixic acid and tetracycline in plate count agar dramatically reduced colony counts (8.3 X 102 ml-1) after this starvation period. The addition of catalase to plate count agar containing nalidixic acid and tetracycline negated the effect caused by this combination of antibiotics. When colony counts obtained over the entire 20 day incubation were considered, the addition of MgSO4 to plate count agar containing nalidixic and tetracycline resulted in a significant increase in colony counts. Other antibiotics, added alone or in combination, did not inhibit colony formation of starved cells. Antibiotic resistant strains of P. putida and Escherichia coli also displayed sensitivity to the combination of nalidixic acid and tetracycline in plate count agar after starvation.

Middaugh, Douglas P., Michael J. Hemmer, Jonathan M. Shenker and Toru Takita. 1990. Laboratory Culture of Jacksmelt, Atherinopsis californiensis, and Topsmelt, Atherinops affinis (Pisces: Atherinidae), with a Description of Larvae. Calif. Fish Game. 76(1):4-13. (ERL,GB 646).

Embryonic and larval jacksmelt, Atherinopsis californiensis, and topsmelt, Atherinops affinis, were cultured in the laboratory. Larval A. californiensis were grown for 24 days at 10, 20 and 30 o/oo salinity. Survival, 80-91%, was highest at 10 o/oo salinity. Increases in standard length (SL) and wet weight were greatest for larvae cultured at 10 or 20 o/oo. Survival of larval A. affinis cultured at 10, 20 and 30 o/oo for 24 days ranged from 99-100%. Increases in SL and wet weight were greatest for larvae cultured at 20 or 30 o /oo salinity. Illustrations of day of hatch, 8-, and 24-day-old larvae are presented with morphometric descriptions for each species. Unique melanophore patterns provide a useful character for identification of these two closely related atherinid fishes which occur sympatrically in California bays and estuaries.

McKenney, Charles L., Jr. and Edward Matthews. 1990. Alterations in the Energy Metabolism of an Estuarine Mysid (Mysidopsis bahia) as Indicators of Stress from Chronic Pesticide Exposure. EPA/600/J-90/381. Mar. Environ. Res. 30(1):1-19. (ERL,GB 663). (Avail. from NTIS, Springfield, VA: PB91-163949)

Dose-response relationships were developed for several metabolic rate functions (oxygen consumption and ammonia excretion) and physiological indices (K2 values and O:N ratios) at different life stages of an estuarine mysid (Mysidopsis bahia) during a life-cycle exposure to fenthion, an organophosphate insecticide. Initial exposure to fenthion resulted in elevated respiration rates of juvenile mysids. As shown by lower net growth efficiency (K2 values), these increased metabolic demands reduced the amount of assimilated energy available for production of new tissue, resulting in retarded juvenile growth rates. O:N ratios indicated that juvenile mysids primarily utilized lipid substrates to support the elevated rates of oxidative metabolism, resulting in less lipid material being available for gamete production. These results, when compared with similar studies with two other pesticide classes, suggest that measurements of alterations in the energy metabolism of contaminated individuals from sensitive zooplankton populations (e.g. mysids) may be used as indicators of reductions in population performance from chronic exposure to toxic organics.

Hemmer, Michael J., Douglas P. Middaugh and James C. Moore. 1990. Effects of Temperature and Salinity on Menidia beryllina Embryos Exposed to Terbufos. EPA/600/J-90/375. Dis. Aquat. Org. 8:127-136. (ERL,GB 667). (Avail. from NTIS, Springfield, VA: PB91-163881)

Embryos of the inland silverside, Menidia beryllina, were exposed to the organophosphorus pesticide terbufos at nine combinations of temperature (20°, 25° and 30° C) and salinity (5, 12.5 and 20 o/oo). Nominal exposure concentrations were 12.5, 25, 50 and 100 µg terbufos l-1 with an acetone and seawater control for each temperature/salinity combination. Test durations were temperature dependent and ranged from 5 to 14 days. Endpoints were embryo survival, hatching and percentage of larvae with normal vertebrae. Embryo survival was significantly (a= 0.05) lower in tests conducted at 20° C for all salinities. Salinity affected survival only at combinations of 20 o/oo and 100 µg terbufos l-1. Both temperature and salinity affected the percentage hatch, with the lowest hatching occuring in 20° C tests, and in tests conducted at 20 o/oo. The percentage of larvae with normal vertebrae was significantly (a=0.05) reduced from controls at terbufos concentrations of 25 (7 to 32 %), 50 (44 to 62 %) and 100 µg l-1 (58 to 73 %) for the 3 temperatures tested, whereas salinity showed no significant effect. Anomalies in the development occurred across all temperature and salinity combinations, and were observed at concentrations as low as 12.5 µg terbufos l-1.

McKenney, Charles L., Jr. and Edward Matthews. 1990. Influence of an Insect Growth Regulator on the Larval Development of an Estuarine Shrimp. EPA/600/J-90/108. Environ. Pollut. 64:169-178. (ERL,GB 673). (Avail. from NTIS, Springfield, VA: PB90-264706)

The influence of methoprene, an insect growth regulator used in mosquito control, on larval development of the estuarine grass shrimp (Palaemonetes pugio) was examined in the laboratory. No grass shrimp larvae successfully completed metamorphosis when continuously exposed to 1000 µg methoprene litre-1 . Completion of larval metamorphosis was significantly reduced by exposure to 100 µg litre-1 of the isomeric mixture (R,S)-methoprene but not the single isomer formulation (S)-methoprene. No statistically significant difference was revealed, however, in ability to inhibit metamorphosis between these two isomeric types across the broad range of exposure concentrations from 0.1 to 1000.0 µg litre-1. The first two larval stages and the final premetamorphic larval stage were more sensitive to methoprene toxicity than intermediate larval stages. Methoprene exposure did not alter either the duration of total larval development or the total number of larval stages prior to metamorphosis.

Couch, John A. 1990. Pericyte of a Teleost Fish: Ultrastructure, Position, and Role in Neoplasia as Revealed by a Fish Model. EPA/600/J-90/366. Anat. Rec. 228(1):7-14. (ERL,GB 676). (Avail. from NTIS, Springfield, VA: PB91-163808)

The morphology and position of the pericyte, a periendothelial cell, is described for a teleost fish, Cyprinodon variegatus. This cell was found attached to the abluminal surfaces of capillaries, venules, and arterioles of the submucosa of the midgut of the fish. The cell was encompassed by a thin basal lamina, possessed numerous plasmalemmal vesicles, a "sole region" which contained thinner actin-like filaments and possibly thicker myosin-like filaments, and ranged in form from ovoid to stellate, with long cytoplasmic extensions that partially covered the endothelium of the associated microvessel. The pericyte of C. variegatus has been shown to give rise to hemangiopericytomas (experimentally induced with diethylnitrosamine) and possibly to pericytomas. In this regard and in regard to its normal ultrastructural morphology, and anatomical position, in relationship to microvasculature in this fish, the cell is very similar to other vertebrate pericytes. Limited evidence suggests that small fish species may be excellent study models for further elucidation of pericyte form, function, and role in disease.

Fournie, John W., Steven S. Foss, Lee A. Courtney and Albert H. Undeen. 1990. Testing of Insect Microsporidians (Microspora:Nosematidae) in Nontarget Aquatic Species. EPA/600/J-90/376. Dis. Aquat. Org. 8:137-144. (ERL,GB 680). (Avail. from NTIS, Springfield, VA: PB91-163899)

This paper reports results of tests with the mosquito microsporidian Nosema algerae and the orthopteran microsporidian N. locustae on nontarget aquatic organisms. Organisms tested were the freshwater grass shrimp Palaemonetes kadiakensis, the estuarine grass shrimp P. pugio, the marine rotifer Brachionus plicatilis, and the inland silverside Menidia beryllina. These organisms were exposed by intrahemocoelic injection, gavage, or ingestion. Infections did not develop in either the freshwater grass shrimp or the estuarine grass shrimp that were gavaged with N. algerae spores. However, infections did develop in both species of grass shrimp after intrahemocoelic injections with N. algerae spores. Infected tissues included the gills, antennal gland, eyes, skeletal muscle, heart, and gonads. Proof of infection was demonstrated ultrastructurally by the presence of mature spores and developmental stages in infected tissues. Infections did not develop in P. pugio following intrahemocoelic injections of N. locustae spores. N. algerae infections did not develop in P. pugio following intrahemocoelic injections of N. locustae spores. N. algerae infections did not develop in the marine rotifer after ingestion of spores or in inland silversides fed marine rotifers containing ingested spores.

Walsh, Gerald E., David E. Weber, Linda K. Brashers and Tasha L. Simon. 1990. Artificial Sediments for Use in Tests with Wetland Plants. EPA/600/J-90/387. Environ. Exp. Bot. 30(3):391-396. (ERL,GB 684). (Avail. from NTIS, Springfield, VA: PB91-164004)

Artificial sediments are described for use in studies on rooted plants. The sediments are formulated from commercially available sand, silt, clay and organic matter. Survival of seedlings of Echinochloa crusgalli var. crusgalli, Scirpus paludosus and Spartina alterniflora was the same in natural and artificial sediments. Average seedling weight of each species was greater in artificial than in natural sediment, probably, because of a more suitable pH in the artifical sediments. Particle size of sand, silt, or clay, percentage sand, silt or clay, percentage organic matter, and cation exchange capacity did not affect growth of E. crusgalli and S. alterniflora. Growth of S. paludosus was related to percentage organic matter in sediment and to interaction between particle size and percentage sand.

Walsh, Gerald E. 1990. Anatomy of the Seed and Seedling of Spartina alterniflora Lois. (Poaceae). EPA/600/J-90/386. Aquat. Bot. 38:177-193. (ERL,GB 686). (Avail. from NTIS, Springfield, VA: PB91-163998)

Members of the genus Spartina are dominant macrophytes in many salt marshes of North and South America, Europe, and Africa. Although the genus is of great ecological importance, seeds and seedlings of its 16 species have not been described. The seed and seedling of an American species, Spartina alterniflora, are described here. The embryo is enclosed in a lemma, a palea, and two glumes. Vascularization of the embryo is panicoid. After germination, leaves arise by periclinal division of cells of the first tunica layer. Laminae assume the adult form by growth of ribs and formation of furrows on the adaxial surface. The panicoid anatomy of each furrow contains a vascular bundle surrounded by a mestome sheath and large, nucleated, parenchyma (Kranz) cells. Radiating chlorenchyma extends from the Kranz cells to the epidermis. Stomata were found only on the adaxial lamina surface and hydathodes on the adaxial and abaxial surfaces. Schlerenchyma is not present in the lamina of the seedling leaf, but layers of cells above and below the parenchymatous bundle sheath appear to be the precursors of schlerenchyma. The primary seminal root is composed of an epidermis, a cortex composed of an outer layer of thick-walled parenchyma and an inner layer of thin-walled parenchyma, and an endodermis that surrounds metaxylem vessels in a triarch configuration. The anatomy of S. alterniflora is that of a C4-Kranz plant adapted for living in the estuarine environment. The highly developed mesocotyl of the seed may function in osmoregulation.

Folsom, B.R., P.J. Chapman and P.H. Pritchard. 1990. Phenol and Trichloroethylene Degradation by Pseudomonas cepacia Strain G4: Kinetics and Interactions Between Substrates. EPA/600/J-90/110. Appl. Environ. Microbiol. 56(5):1279-1285. (ERL,GB 687). (Avail. from NTIS, Springfield, VA: PB90-264201)

Intact cells of Pseudomonas cepacia strain G4 completely degraded trichloroethylene (TCE) following growth with phenol. Degradation kinetics were determined for both phenol, used to induce requisite enzymes, and TCE, the target substrate. Apparent Ks and Vmax values for degradation of phenol by cells were 8.5 µM and 466 nmol/min per mg protein, respectively. At phenol concentrations above 50 µM, phenol degradation was inhibited, yielding an apparent kSI of 0.45 mM as modeled by the Haldane expression. A partition coefficient for TCE was determined to be 0.40 +/- 0.02, [TCE air]/[TCE water], consistent with Henry's law. To eliminate experimental problems associated with TCE volatility and partitioning, a no-headspace bottle assay was developed allowing for direct and accurate determinations of aqueous TCE concentration. By this assay procedure, apparent Ks and Vmax values determined for TCE degradation by intact cells were 5.6 µM and 7.9 nmol/min per mg protein, respectively. Following a transient lag period, P. cepacia G4 degraded TCE at concentrations of at least 300 µM TCE with no apparent retardation in rate. Consistent with Ks values determined for degradation, TCE significantly inhibited phenol degradation.

Barkay, Tamar, Mark Gillman and Cynthia Liebert. 1990. Genes Encoding Mercuric Reductases from Selected Gram-Negative Aquatic Bacteria Have a Low Degree of Homology with merA of Transposon 501. EPA/600/J-90/364. Appl. Environ. Microbiol. 56(6):1695-1701. (ERL,GB 689). (Avail. from NTIS, Springfield, VA: PB91-163782)

An investigation was conducted on the Hg2+ resistance mechanism of four freshwater and four coastal marine bacteria that did not hybridize with a mer operonic probe (T. Barkay, C. Liebert, and M. Gillman, Appl. Environ. Microbiol. 55, 1196, 1989). Inducible Hg2+ volatilization was demonstrated for all eight organisms and NADPH-dependent mercuric reductase activities were detected in crude cell extracts of six of the strains. Hybridization with a mer A probe, the gene encoding the mercuric reductase polypeptide, at a stringency permitting hybrid formation between evolutionary distant merA genes (as exists between gram positive and negative bacteria) detected merA sequences in the genomes of all tested strains. Because these strains represented random selections of bacteria from three aquatic environments, it is concluded that merA encodes a common molecular mechanism for Hg2+ resistance and volatilization in aerobic heterotrophic aquatic communities.

Mueller, James G., Peter J. Chapman, Beat O. Blattmann and P. Hap Pritchard. 1990. Isolation and Characterization of a Fluoranthene-Utilizing Strain of Pseudomonas paucimobilis. EPA/600/J-90/109. Appl. Environ. Microbiol. 56(4):1079-1086. (ERL,GB 691). (Avail. from NTIS, Springfield, VA: PB90-264698)

A soil bacterium capable of utilizing fluoranthene as the sole source of carbon and energy for growth was purified from a seven-member bacterial community previously isolated from a creosote waste site for its ability to degrade polycyclic aromatic hydrocarbons. By standard bacteriological methods, this bacterium was characterized taxonomically as a strain of Pseudomonas paucimobilis and was designated strain EPA505. Utilization of fluoranthene by strain EPA505 was demonstrated by increase in bacterial biomass, decrease in aqueous fluoranthene concentration, and transient formation of transformation products in liquid cultures where fluoranthene was supplied as the sole carbon source. Resting cells grown in complex medium showed activity toward anthraquinone, benzo[b]fluorene, biphenyl, chrysene, and pyrene as demonstrated by the disappearance of parent compounds or changes in their UV absorption spectra. Fluoranthene-grown resting cells were active against these compounds as well as 2,3-dimethylnaphthalene, anthracene, fluoranthene, fluorene, naphthalene, and phenanthrene. These studies demonstrate that organic compounds not previously reported to serve as growth substrates can be utilized by axenic cultures of microorganisms. Such organisms may possess novel degradative systems that are active toward other compounds whose biological degradation has been limited because of inherent structural considerations or because of low aqueous solubility.

Genthner, Barbara R. Sharak, G.T. Townsend and P.J. Chapman. 1990. Effect of Fluorinated Analogues of Phenol and Hydroxybenzoates on the Anaerobic Transformation of Phenol to Benzoate. EPA/600/J-90/374. Biodegradation. 1:65-74. (ERL,GB 692). (Avail. from NTIS, Springfield, VA:PB91-163873)

The effects of fluorinated analogues on the anaerobic transformation of phenol to benzoate were examined. At = or >µM 2- or 3-fluorophenol, phenol transformation was delayed. 2-Fluorophenol had no apparent effect on subsequent degradation of benzoate, but benzoate accumulated in the presence of = or >250 uM 3-fluorophenol. In contrast, 4-fluorophenol at =or >2mM had no effect on either phenol transformation or benzoate degradation. Phenol and 2-, or 3-fluorophenol were transformed simultaneously, but phenol was transformed more rapidly than either fluorophenol. Thus, fluorinated analogues of phenol did not prevent anaerobic transformation of phenol to benzoate. 2-Fluorophenol was converted to 3-fluorobenzoate, and phenol enhanced the rate and extent of its transformation. 3-Fluorophenol was transformed to 2-fluorobenzoate to a limited extent (approximately 3%) when phenol was present. 4-Fluorophenol was not transformed regardless of the presence of phenol. 3-Fluoro-4-hydroxybenzoate, a potential fluorinated intermediate product of para-carboxylation, was transformed rapidly to 2-fluorophenol and 3-fluorobenzoate, irrespective of the presence of phenol, indicating that both dehydroxylation and decarboxylation occurred. Initially, 2-fluorophenol and 3-fluorobenzoate were rapidly formed in an approximate molar ratio of 2:1. Once 3-fluoro-4-hydroxybenzoate was completely removed, the 2-fluorophenol, initially formed, was converted to 3-fluorobenzoate at a slower rate. Thus, phenol enhanced transformation of the fluorinated analogues, and the products of transformation suggested para -carboxylation. 3-Fluoro-2-hydroxybenzoate was not transformed in either the presence or absence of phenol, indicating that ortho-carboxylation did not occur.

Davis, William P., D. Scott Taylor and Bruce J. Turner. 1990. Field Observations of the Ecology and Habits of Mangrove Rivulus (Rivulus marmoratus) in Belize and Florida (Teleostei: Cyprinodontiformes: Rivulidae). EPA/600/J-90/371. Ichthyol. Explor. Freshwaters. 1(2):123-134. (ERL,GB 693). (Avail. from NTIS, Springfield, VA: PB91-163840)

This report provides a synopsis of field studies of Rivulus marmoratus from two population surveys of mangrove islands adjacent to the Belize barrier reef and observations made over fifteen years at several sites in South Florida. This small, cryptically colored killifish is the only known vertebrate selfing hermaphrodite. Florida populations consist nearly exclusively of hermaphrodites (>99%), while the Belize populations contained a significant proportion (10-25%) of males. Our combined observations demonstrate that this species is not "rare" as previously thought, but elusive and highly adapted to microhabitats within mangrove forests. Standard ichthyological collecting techniques are ineffective in this habitat and have previously failed to reveal the strength of the association of R. marmoratus with the mangral ecosystem.

Middaugh, Douglas P., John W. Fournie and Michael J. Hemmer. 1990. Vertebral Abnormalities in Juvenile Inland Silversides Menidia beryllina Exposed to Terbufos During Embryogenesis. EPA/600/J-90/382. Dis. Aquat. Org. 9(2):109-116. (ERL,GB 695). (Avail. from NTIS, Springfield, VA: PB91-163956)

Embryos of the inland silverside, Menidia beryllina, were exposed to a nominal concentration of 50 µg terbufos 1-1 during the first five days of embryogenesis. Silversides were maintained in clean dilute seawater until 37 days after hatching. Radiographs revealed compressed and fused vertebrae and dorsal-ventral misalignment of pre- and post-zygapophyseal processes. Histopathological examination of individuals exposed to terbufos during hyperostoses to almost complete fusion of some vertebrae.

Fisher, D.J. and J.R. Clark. 1990. Bioaccumulation of Kepone by Grass Shrimp (Palaemonetes pugio): Importance of Dietary Accumulation and Food Ration. EPA/600/J-90/373. Aquat. Toxicol. 17(2):167-186. (ERL,GB 698). (Avail. from NTIS, Springfield, VA: PB91-163865)

The relative extent of Kepone uptake from food (dietary accumulation) and water (bioconcentration) by grass shrimp (Palaemonetes pugio) was evaluated quantitatively at food rations of 4% and 8% of the average wet weight of the shrimp. [14C]Kepone was utilized to determine bioconcentration and dietary accumulation separately, while [14C]Kepone-contaminated food (grass shrimp) and unlabeled Kepone in water were used to determine accumulation from both sources simultaneously. Grass shrimp and their food were exposed to the same aqueous Kepone concentration (0.04 µg/l). A first-order pharmacokinetic equation was used to model Kepone accumulation kinetics during the 16-day uptake and 21-day clearance phases. A doubling of contaminated food ration caused a significant (c=0.05) increase in the whole-body Kepone concentration in the shrimp. Shrimp fed either a 4% or 8% ration of uncontaminated food and exposed to 0.04 µg/l Kepone in water bioconcentrated Kepone equally. When shrimp were exposed to contaminated water and food, Kepone accumulation from each source was additive. Food ration, however, was very important in determining final Kepone body burdens. The dietary source of Kepone represented approximately 24% and 33% of the total body burden accumulated by shrimp fed 4% and 8% food rations, respectively, but assimilation efficiencies of Kepone from the food source were low. The laboratory results suggest that dietary accumulation of Kepone by grass shrimp may play an important role in determining final Kepone body burdens in feral organisms, especially during periods of high food consumption.

Henis, Yigal and Martin Alexander. 1990. Effect of Organic Amendments on Bacterial Multiplication in Lake Water. EPA/600/J-90/092. Antonie Leeuwenhoek. 57:9-20. (ERL,GB X577). (Avail. from NTIS, Springfield, VA: PB90-245580)

In Cayuga Lake water amended with 30 ug of glucose or amino acids per ml, an added strain of Pseudomonas fluorescens and indigenous bacteria grew extensively. Pseudomonas sp. B4 and two rhizobia multiplied at a moderate extent, and introduced Escherichia coli and Klebsiella pneumoniae multiplied but to only a slight degree. The amendments did not enhance growth of Micrococcus flavus and Arthrobacter citreus, and an asporogenous strain of Bacillus subtilis decreased in numbers. The pseudomonads, rhizobia, E. coli and K. pneumoniae multiplied extensively when inoculated into sterile lake water amended with amino acids. A. citreus grew to a slight extent, but the numbers of M. flavus and B. subtilis did not change appreciably. In nonsterile lake water amended with 30 ug of Trypticase soy broth per ml, the indigenous bacteria greatly increased in abundance, the pseudomonads, rhizobia, and E. coli developed to a lesser extent, the numbers of K. pneumoniae, A. citreus and M. flavus showed little increase, and B. subtilis decreased in numbers. Tests in pure culture containing 2 to 64 ug of Trypticase soy broth per ml demonstrated good growth of P. fluorescens, Pseudomonas sp. B4, and the rhizobia at all concentrations; an initial decline followed by growth of E. coli, K. pneumoniae and R. leguminosarum biovar phaseoli at low concentrations; little or no growth or decline at the low levels but multiplication at the high levels by A. citreus and B. subtilis; and decline of M. flavus. It is proposed that the apparent Ks value, umax value, length of lag phase and resistance to stress can be used to predict behavior of bacteria in lake water receiving low levels of organic nutrients.

Kelly, John R., David T. Rudnick, R. Dana Morton, Linda A. Buttel, Suzanne N. Levine and Kelly A. Carr. 1990. Tributyltin and Invertebrates of a Seagrass Ecosystem: Exposure and Response of Different Species. EPA/600/J-90/378. Mar. Environ. Res. 29(4):245-276. (ERL,GB X613). (Avail. from NTIS, Springfield, VA:PB91-163915)

14C-labeled tributyltin-chloride (TBT-Cl) was delivered to the water column of seagrass microcosms held in the laboratory under flow-through conditions. Benthic macroinvertebrate abundances across a three treatment, logarithmic dose gradient were compared to untreated control microcosms. Within 3 to 6 weeks, statistically significant mortality appeared in the high treatment. Sensitive species included surface deposit feeders of several phyla, as well as a suspension feeding mollusc. Results suggest that effects can arise because TBT is rapidly accumulated in surface sediments, as well as in Thalassia tissues. Concentration of tracer in plant tissues, animals, and sediments suggests that measurement of TBT (and total butylin) in these components of seagrass beds would provide a better indicator of exposure regimes than occasional measurements in the water. A propensity for accumulation, along with biological vulnerability, suggests a sentinel role for seagrass ecosystems in some shallow coastal areas. Experimental findings demonstrate concern for some key invertebrates within beds proximal to TBT sources, and ecological risks could radiate through coastal food webs dependent on these productive vegetated shallows.

Kelly, John R., Suzanne N. Levine, Linda A. Buttel, Kelly A. Carr, David T. Rudnick and R. Dana Morton. 1990. Effects of Tributyltin Within a Thalassia Seagrass Ecosystem. Estuaries. 13(3):301-310. (ERL,GB X614).

Flow-through seagrass core microcosms were used to examine responses of species and processes to a logarithmic gradient of dosing with 14C-labeled tributyltin-chloride (TBT-Cl). Experiments involved delivery of TBT-Cl to the water column of replicate cores of a treatment (n=16) once per week; one-half of the cores were sacrificed after 3 wk of dosing, the others were dosed for 6 wk. Initial water column concentrations for the three treatments averaged 0.205, 2.23, and 22.221 ugl-1, expressed as the TBT+ cation, but these concentrations dropped rapidly. Retained 14C tracer, an estimate of total organotin species, was distributed to sediments, plants, and other biological tissues, all of whose tracer concentrations increased with time. Measures to indicate responses of both autotrophic and heterotropic organisms were made; in general, treatment effects were demonstrable statistically only at the highest dose level. Accumulation of chlorophyll and biomass on glass slides was highest when suspended for the entire experiment in the water of the highest treatment; this unexpected result was perhaps an indirect effect related to reduced grazing activity in the microcosms. The highest dose of TBT-Cl resulted in virtual population mortality of a few macrobenthic species and decreased loss of plant material in litter bags, both demonstrated within the first 3 wk of dosing. Reduced litter loss was coincident with mortality of an amphipod (Cymadusa compta) capable of shredding plant material, and a causal relation between the two effects is plausible. Thus, if concentrated to similar levels in a Thalassia bed, TBT+ may have direct species-level effects and process-level effects, potentially causing ecosytem change via disruption of a species-process linkage influential in seagrass detrial food web dynamics.

Barnthouse, Lawrence W., Glenn W. Suter, II and Aaron E. Rosen. 1990. Risks of Toxic Contaminants to Exploited Fish Populations: Influence of Life History, Data Uncertainty and Exploitation Intensity. EPA/600/J-90/513. Environ. Toxicol. Chem. 9(3):297-311. (ERL,GB X623). (Avail. from NTIS, Springfield, VA: PB91-199984)

Use of toxicity test data for population-level risk assessment was investigated as follows: (1) the influence of life history characteristics of menhaden and striped bass on vulnerability of contaminant-induced stress, (2) the importance of test data availability, and (3) the influence of exploitation intensity. Menhaden and striped bass differed in terms of their capacity to sustain the same level of contaminant-induced mortality. Changes in exploitation intensity affect the responses of both populations to the same level of additional contaminant-induced mortality. However, the quantitative effects of both factors were small compared to the uncertainty associated with estimating long-term effects from short-term tests or QSARs.

Ogunseitan, Oladele A., Gary S. Sayler and Robert V. Miller. 1990. Dynamic Interactions of Pseudomonas aeruginosa and Bacteriophages in Lake Water. EPA/600/J-90/369. Microb. Ecol. 19:171-185. (ERL,GB X632). (Avail. from NTIS, Springfield, VA: PB91-163832)

The persistence and interaction between newly isolated strains of Pseudomonas aeruginosa and resident bacteriophages indigenous to a freshwater environment was monitored over 45 days in lake water microcosms. The interaction between susceptible and resistant bacteria with pure phage (UT1) particles, or a mixed phage population (M1) was investigated by following temporal changes in host density, phage to bacteria ratio (PBR), and the appearance of apparent prophage carriers within the host population. Decay rates of the phage (UT1) ranged from 0.054 h-1 in natural water to 0.027 h-1 in filtered lake water. About 45% of sensitive bacteria incubated with phage UT1 were pseudo-lysogenic within 12 h of incubation in natural lake water. This process was delayed until 72 h in the sterile lake water control, suggesting that host-phage interaction is promoted in the presence of a viable natural microbial community. Phage UT1 appeared to stabilize the density of host bacteria in lake water at a level of 10 to the fourth power cfu ml-1. Bacterial coexistence with the mixed phage (M1) population resulted in an oscillating equilibrium with the (PBR) stabilizing at about 3. The presence of extraneous homoimmune phages appeared to be detrimental to the stability of the pseudo-lysogens, which were maintained at a lower population density than prophage-free cells in lake water containing the mixed phage (M1) population.

Haugland, Richard A., Udaykumar M.X. Sangodkar and A.M. Chakrabarty. 1990. Repeated Sequences Including RS1100 from Pseudomonas cepacia AC1100 Function as IS Elements. Mol. Gen. Genet. 220(2):222-228. (ERL,GB X633).

Several lines of evidence were obtained that the repeated sequence RS1100 of Pseudomonas cepacia strain AC1100 undergoes transposition events. DNA sequences flanking the chlorohydroxy hydroquinone (CHQ) degradative genes of this organism were examined from sources including several independently isolated cosmid clones from an AC1100 genomic library and genomic DNA's of two independently maintained wild type AC1100 isolates. Hybridization and restriction endonuclease mapping studies revealed these sequences to be similar except for their numbers and distributions of RS1100 copies. A recombinant plasmid containing the immediate chq gene region and excluding any copies of RS1100 was conjugated into an AC1100 mutant shown to have undergone a deletion of its corresponding DNA. Hybridization and restriction mapping analyses of several reisolated plasmids revealed the presence of RS1100 sequences at different positions within either their vector or insert portions. One such plasmid contained tandem copies of RS1100 with an intervening DNA sequence also of AC1100 origin. Similar experiments involving introduction of the promoter probe plasmid pKT240 into wild type AC1100 cells resulted in the acquisition of high concentration streptomycin resistance by a number of recipients. Plasmids reisolated from these organisms in most cases also conferred streptomycin resistance to E. coli transformants and in each case were found to contain insertions close to the upstream portion of the aphC structural gene. These insertions alternatively contained RS1100 sequences or a newly identified 3400 bp repeated sequence from AC1100

Saye, Dennis J., O.A. Ogunseitan, G.S. Sayler and Robert V. Miller. 1990. Transduction of Linked Chromosomal Genes Between Pseudomonas aeruginosa During Incubation In Situ in a Freshwater Habitat. EPA/600/J-90/512. Appl. Environ. Microbiol. 56(1):140-145. (ERL,GB X636). (Avail. from NTIS, Springfield, VA: PB91-199976)

Both transduction of single chromosomal loci and co-transduction of closely linked loci were observed between lysogenic and non-lysogenic strains of Pseudomonas aeruginosa in a freshwater habitat. Transductants were recovered at frequencies of 10-6 to 10-5 transductants/CFU. Transductants of lysogenized strains were recovered ten to 100-fold more frequently than were transductants of non-lysogenic parents. Lysogens thus are capable of introducing phages capable of mediating generalized transduction into the natural microbial community and serving as recipients of transduced DNA. It would appear that lysogeny has the potential of increasing the size and flexibility of the gene pool available to natural populations of bacteria. The ability to generate and select new genetic combinations through phage-mediated exchange can be significant in the face of a continually changing environment and may contribute to the apparent fitness of the lysogenic state in natural ecosystems.

Jeffrey, Wade H. and John H. Paul. 1990. Natural Transformation of a Marine Vibrio Species by Plasmid DNA. EPA/600/J-90/377. Microb. Ecol. 19(3):259-268. (ERL,GB X637). (Avail. from NTIS, Springfield, VA: PB91-163907)

Vibrio sp. DI9, recently isolated from Tampa Bay, FL, has been found to be naturally transformed by the broad host range plasmid pKT230 in both filter transformation assays and sterile sediment microcosms. This is the first report of natural transformation by plasmid DNA of a Vibrio sp. and of a marine bacterial isolate. Transformation frequencies ranged from 0.3 to 3.1 x 10-8 transformants per recipient. Transformants were detected by both plating and by selection for growth in liquid medium in the presence of streptomycin and kanamycin and confirmed by probing of southern transfers. Transformation was enhanced by multimeric forms of the plasmid. A technique using sediment microcosms, mixed populations of Vibrio sp. DI9 and another antibiotic resistant organism, and enrichment in liquid media has been developed which allows detection of transformation at frequencies too low to be detected by plating. These techniques may serve as a model for the detection of natural transformation in the environment. These results suggest that natural transformation may be one mechanism of horizontal plasmid transfer in the marine environment, and may provide the methodology with which to detect this process in natural populations of bacteria.

Stewart, Gregory J. and Christopher D. Sinigalliano. 1990. Detection of Horizontal Gene Transfer by Natural Transformation in Native and Introduced Species of Bacteria in Marine and Synthetic Sediments. EPA/600/J-92/219. Appl. Environ. Microbiol. 56(6):1818-1824. (ERL,GB X638). (Avail. from NTIS, Springfield, VA: PB92-195767)

Both naturally occurring marine sediments and artifical sediments were used as supports for natural transformation of marine bacteria. While transformation was not detected in cells of Pseudomonas stutzeri strain ZoBell suspended in artificial seawater, when recipient cells and rifampin resistant DNA were loaded onto sterile sediment columns transformation could be detected at frequencies four to twenty times that for spontaneous resistance. Treatment of these columns with DNase I reduced transformation frequencies to levels comparable to spontaneous resistance frequencies. Sediments containing higher organic content supported higher frequencies of transformation than those with lower amounts of organic matter. Transformation was also detected when recipient cells and DNA were loaded on columns prepared from non-sterile sediments, although the frequencies of transformation were lower in these cases than when sterile sediments were employed. Finally, non-sterilized sediments that were unamended with laboratory strains did not support detectable levels of transformation in sediment columns, but when these same sediments were transferred to filters and placed on complex media, transformation was detected at a frequency three times that for spontaneous resistance. This transformation frequency was partially reduced to levels near that for spontaneous resistance by the addition of DNase I to sediment filters. These results indicate that marine sediments facilitate the uptake and expression of exogenous DNA by transformable marine bacteria, and that sediments are a more likely niche for natural transformation than the water column in the marine environment.

Wood, M.S., C. Lory and T.G. Lessie. 1990. Activation of the lac Genes of Tn951 by Insertion Sequences from Pseudomonas cepacia. EPA/600/J-90/111. J. Bacteriol. 172(4):1719-1724. (ERL,GB X645). (Avail. from NTIS, Springfield, VA: PB90-264193)

We have identified several transposable gene-activating elements from Pseudomonas cepacia on the basis of their ability to increase expression of the lac genes of the broad-host-range plasmid pGC91.14. When introduced into auxotrophic derivatives of P. cepacia 249 (ATCC 17616), this plasmid failed to confer ability to utilize lactose or lactulose (4-O-beta-galactopyranosyl-D-fructose). The lac genes of the Tn951 element on pGC91.14 were poorly expressed in P. cepacia and were not inducible by IPTG. Lac+ and lactulose+ variants of the pGC91.14 containing transcipients were isolated which expressed the lac genes of TN951 constitutively as a consequence of transposition of insertion sequences from the P. cepacia genome to sites upstream of the activated genes. Element IS408 inserted into lacZ and conferred ability to utilize lactulose by increasing lacY function. Certain of the elements also increased gene expression in other bacteria. For example, IS407 strongly activated the lacZ gene of Tn951 in Pseudomonas aeruginosa and Escherichia coli, and IS406 (but not IS407) did so in Zymomonas mobilis. The results indicate that IS elements from P. cepacia have potential for turning on the expression of foreign genes in a variety of gram-negative bacteria.

Levine, S.N., D.T. Rudnick, J.R. Kelly, R.D. Morton, L.A. Buttel and K.A. Carr. 1990. Pollutant Dynamics as Influenced by Seagrass Beds: Experiments with Tributyltin in Thalassia Microcosms. EPA/600/J-90/516. Mar. Environ. Res. 30(4):297-322. (ERL,GB X649). (Avail. from NTIS, Springfield, VA: PB91-206888)

Seagrass beds are highly productive ecosystems whose leaves and sediments provide considerable surface area for interactions with seawater; thus, they may be foci for the sorption, accumulation and degradation of pollutants. The fate of the potent biocide tributyltin (TBT) in water that passes through seagrasses and over sediments was studied in marine microcosms containing sediment cores from a subtropical seagrass bed (including Thalassia testudinum and associated fauna) and seawater. Over 3 or 6 weeks, 48 of these microcosms were dosed weekly for 24 h with 14 C-labelled TBT at three different doses (initial concentrations of 0-2, 2 and 20 ug TBT+ liter-1) and flushed with flowing seawater between dose periods. The TBT was rapidly removed from the water column (half times of 10-20 h), primarily through adsorption onto sediments and seagrass leaves. By contrast, 12 microcosms that received similar TBT doses but that contained only seawater had TBT removal half times of 2-7 days. Accumulation of TBT in sediments and grasses was temporary, however; at harvest, the seagrass microcosms contained just 20-30% of the 14C that had been adsorbed or assimilated during dose periods, and half of this label was in degradation products. The principal mechanism of TBT loss from solids was degradation followed by desorption of degradation products (largely monobutyltin and CO2, which are more soluble than TBT). Despite relatively rapid TBT degradation, TBT accumulated in fauna; at harvest, 2-6 of the 14C in microcosms was in invertebates. Thus seagrass beds can be viewed as foci for the concentration of TBT, as processors of TBT to less toxic degradation products, and as vectors for distribution of TBT through coastal food chains.

Simonson, C. Sue, Tyler A. Kokjohn and Robert V. Miller. 1990. Inducible UV Repair Potential of Pseudomonas aeruginosa PAO. J. Gen. Microbiol. 136:1241-1249. (ERL,GB X651).

Pseudomonas aeruginosa PAO lacks UV-inducible Weigle reactivation and Weigle mutagenesis of UV-damaged bacteriophages. This lack of UV-inducible, error-prone DNA repair appears to be due to the absence of efficiently expressed umuDC-like genes in this species. When the P. aeruginosa recA gene is introduced into a a recA(Def) mutant of Escherichia coli K12, the P. aeruginosa recA gene product is capable of mediating UV-induced mutagenesis, indicating that it could participate in a recA-lexA-like regulatory network and function in inducible DNA repair pathways if such existed in P. aeruginosa. The presence of the IncP9, UV-resistance plasmid R2 in RecA+ strains of P. aeruginosa PAO allows UV-inducible, mutagenic DNA repair of UV-irradiated bacteriophages. R2 also greatly stimulates the ability of UV radiation to induce mutagenesis of the bacterial chromosome. When R2 is introduced into P. aeruginosa strains containing either the recA908 or recA102 mutation, plasmid-mediated UV resistance and Weigle reactivation are not observed. These observations suggest that the increased protection afforded to P. aeruginosa by R2 is derived from a RecA-mediated, DNA-damage-inducible, error-prone DNA repair system which complements the lack of a chromosomally encoded umuDC-like operon.

Gurijala, Koteswara R. and Martin Alexander. 1990. Effect of Growth Rate and Hydrophobicity on Bacteria Surviving Protozoan Grazing. EPA/600/J-90/368. Appl. Environ. Microbiol. 56(6):1631-1635. (ERL,GB X656). (Avail. from NTIS, Springfield, VA: PB91-163824)

Measurements were made of the predation by Tetrahymena thermophila on several bacterial species in media containing heat-killed Escherichia coli cells to serve as an alternative prey. If grazing pressure was initially not intense on a mixture of bacterial species, the species that survived protozoan feeding at greater densities were those that grew quickly before the onset of active predation. If members of several species were incubated individually at similar initial densities with actively grazing T. thermophila, some species survived at ca. 10 to the fourth power per ml, some at ca. 10 to the second power per ml, and others were eliminated. Members of the first two groups but not the third group were able to multiply in the medium in the absence of the protozoan, but the growth rates in the protozoan-free medium did not correlate with the number of survivors. However, the species that persisted at the higher densities possessed highly hydrophobic cell surfaces. The size of the surviving population of four bacterial species whose growth was prevented by chloramphenicol correlated with the initial cell density that was incubated with T. thermophila. It is concluded that the individual species surviving predation on a mixture of species is related to the capacity of the bacterium to grow, the hydrophobicity of its cell surface, and the population density of the species before the onset of intense grazing.

Miller, Robert V. and Tyler A. Kokjohn. 1990. General Microbiology of recA: Environmental and Evolutionary Significance. EPA/600/J-90/383. Annu. Rev. Microbiol. 44:365-394. (ERL,GB X657). (Avail. from NTIS, Springfield, VA: PB91-163964)

The RecA protein, a molecule of 38,000 Mr (125,126), is a multifunctional polypeptide directing a number of activities, none of which is completely understood in detail. It's central role in homologous recombination and DNA-damage repair has created considerable interest. Numerous studies in the last eight years which identify and characterize this gene have revealed the widespread distribution and evolutionary conservation of recA. In this review, authors explore the current knowledge of the general microbiology of recA and its protein product.

Coffin, Richard B., David J. Velinsky, Richard Devereux, William Allen Price and Luis A. Cifuentes. 1990. Stable Carbon Isotope Analysis of Nucleic Acids to Trace Sources of Dissolved Substrate Used by Estuarine Bacteria. EPA/600/J-90/388. Appl. Environ. Microbiol. 56(7):2012-2020. (ERL,GB X658). (Avail. from NTIS, Springfield, VA: PB91-164012)

The natural abundance of stable carbon isotopes measured in bacterial nucleic acids extracted from estuarine bacterial concentrates was used to trace sources of organic matter for bacteria in aquatic environments. The stable carbon isotope ratios of Pseudomonas aeruginosa and nucleic acids extracted from cultures resembled those of the carbon source on which bacteria were grown. The carbon isotope discrimination between the substrate and total cell carbon from bacterial cultures averaged 2.3% plus or minus 0.6% (n=13). Furthermore, the isotope discrimination between the substrate and nucleic acids extracted from bacterial cultures was 2.4% plus or minus 0.4% (n=10), not significantly different from the discrimination between bacteria and substrate. Estuarine water samples were prefiltered through 1-um-pore-size cartridge filters. Bacterium-sized particles in the filtrates were concentrated with tangential-flow filtration and centrifugation, and nucleic acids were then extracted from these concentrates. Hybridization with 16S rRNA probes showed that approximately 90% of the nucleic acids extracted on two sample dates were of eubacterial origin. Bacteria and nucleic acids from incubation experiments using estuarine water samples enriched with dissolved organic matter from Spartina alterniflora and Cyclotella caspia had stable carbon isotope values similar to those of the substrate sources. In a survey that compared diverse estuarine environments, stable carbon isotopes of bacteria grown in incubation experiments ranged from -31.9 to -20.5%. The range in isotope values of nucleic acids extracted from indigenous bacteria from the same waters was similar, -27.9 to -20.2%. Generally, the lack of isotope discrimination between bacteria and nucleic acids that was noted in the laboratory was observed in the field. Exceptions to this generalization were due to changes in bacterial substrate sources as a result of the incubation experiments that were used to obtain bacteria for isotope analysis. Our results from work in the field and laboratory indicate that this approach is useful for tracing sources of dissolved organic matter in aquatic environments and describing the bacterial role in its cycling.

Gurijala, Koteswara R. and Martin Alexander. 1990. Explanation for the Decline of Bacteria Introduced into Lake Water. EPA/600/J-90/517. Microb. Ecol. 20(3):231-244. (ERL,GB X661). (Avail. from NTIS, Springfield, VA: PB91-206896)

The sizes of the populations of individual bacterial species diminished following their addition to water from lakes with different trophic levels at temperatures of 5, 10, 15, and 30 degrees C. Some species persisted after their initial reduction in cell numbers, but others were undetectable after 3 to 5 days. The decline of these introduced bacteria was not a result of their inoculation at higher densities than are found in nature. The death of most of the test species was not the result of starvation, abiotic factors, bdellovibrios, or bacteriophages. Despite the presence of lytic bacteria, the lake water did not have lytic activity against the test species. Protozoan predation was a significant factor in the fall in bacterial population sizes because protozoa increased in numbers as the bacterial density fell, the suppression of protozoa led to the elimination or delay of the decline of the bacteria, and the addition of protozoa to lake water in which indigenous protozoa were suppressed produced the same pattern of bacterial elimination as in untreated lake water.

Vogelbein, Wolfgang K., John W. Fournie, Peter A. Van Veld and Robert J. Huggett. 1990. Hepatic Neoplasms in the Mummichog Fundulus heteroclitus from a Creosote-Contaminated Site. EPA/600/J-90/385. Cancer Res. 50(18):5978-5986. (ERL,GB X681). (Avail. from NTIS, Springfield, VA: PB91-163980)

High prevalences of idiopathic hepatic lesions were found in mummichog, Fundulus heteroclitus, from a site in the southern branch of the Elizabeth River, VA, contaminated with polycyclic aromatic hydrocarbons. Grossly visible hepatic lesions occurred in a total of 93% of the individuals from this site and 33% of these fish had hepatocellular carcinomas. Hepatic lesions were not detected in fish from two less contaminated sites. Lesions included foci of cellular alteration, hepatocellular adenoma, early and advanced hepatocellular carcinomas, and cholangiocellular proliferative lesions. Advanced carcinomas exhibited several distinct cellular patterns and some livers contained multiple neoplasms occupying up to 80% of the hepatic parenchyma. Sediments from the contaminated site contained extremely high concentrations (2200 mg/kg dry sediment) of polycyclic aromatic hydrocarbons, which are believed to originate from an adjacent wood treatment facility that has used creosote. Concentrations were 730- and 35-fold higher than those at the two other sites. These findings indicate a strong positive association between exposure to creosote-contaminated sediments and the high prevalence of hepatic neoplasms in a feral population of mummichog and support the putative role of polycyclic aromatic hydrocarbons in fish hepatocarcinogenesis. Additionally, they suggest that the mummichog may be a useful indicator of exposure to carcinogens in aquatic environments.

Turner, Bruce J., John F. Elder, Jr., Thomas F. Laughlin and William P. Davis. 1990. Genetic Variation in Clonal Vertebrates Detected by Simple-Sequence DNA Fingerprinting. EPA/600/J-90/384. Proc. Natl. Acad. Sci. U.S.A. 87(15):5653-5657. (ERL,GB X682). (Avail. from NTIS, Springfield, VA: PB91-163972)

The measurement of clonal heterogeneity is central to understanding the evolutionary and population genetics of the roughly 50 species of vertebrates which lack effective genetic recombination. Simple-sequence DNA fingerprinting with oligonucleotide probes (CAG)5 and (GACA)4 is a sensitive and efficient means of detecting this heterogeneity in natural populations of two clonal fishes, Poecilia formosa, an apomictic unisexual, and Rivulus marmoratus, a selfing hermaphrodite. The fingerprints are clonally stable for at least three generations. The technique clearly differentiates allozymically identical laboratory lines of R. marmoratus that were previously distinguishable only by histocompatibility analysis. The technique also reveals the first documented cases of apparent clonal turnover in a natural population of each species. Clonal variation in most natural populations is quite high. For example, a sample of 19 specimens of P. formosa from one station on the Rio Soto la Marina contained 16 clones (average clonal frequency .07). This level of clonal diversity implies that mutation, subsequent to the founding of clonal lineages, is an important source of variation in these populations. It also suggests that chance (sampling error) has a major role in determining the clonal composition of populations even though some of the clones may be divergent in biologically significant features.

Chandler, G. Thomas. 1990. Effects of Sediment-Bound Residues of the Pyrethroid Insecticide Fenvalerate on Survival and Reproduction of Meiobenthic Copepods. EPA/600/J-90/093. Mar. Environ. Res. 29(1):65-76. (ERL,GB X684). (Avail. from NTIS, Springfield, VA: PB90-245572)

Pure, microcosm-cultured populations of benthic copepods were established from pristine or pesticide-impacted Spartina marsh creeks and used as efficient bioassay groups to assess lethal and sublethal effects of sediment-bound residues. Naturally-weathered sediments contaminated with the synthetic pyrethroid insecticide fenvalerate were collected by traps moored in a tidal creek receiving major pesticide-laced runoff from an agricultural watershed, and used as dosing material. Silty sediments with fenvalerate residues reaching 100 ppb were trapped and then diluted with uncontaminated sediments to achieve an exposure range of 0, 25, 50 and 100 ppb (i.e. no dilution). Despite a broad database showing extreme sensitivity to water-solubilized fenvalerate by many marine invertebrates and fishes, a 7-day exposure to sediment-bound residues as high as 100 ppb caused no significant mortality for any life stages (i.e. nauplii, copepodites or adults) of the benthic harpacticoid copepods Microarthridion littorale or Paronychocamptus wilsoni, and no mortality for adults of Enhydrosoma propinquum. However, sediment-bound residues as low as 25 ppb significantly depressed egg production (50-100% reduction) and mean clutch sizes (40-100% reduction) of fertile M. littorale and P. wilsoni. If sedimenting fenvalerate depresses copepod reproduction in the field, then lowered recruitment of new individuals will lead inevitably to a decline in population growth.

Straube, W.L., M. O'Brien, K. Davis and R.R. Colwell. 1990. Enzymatic Profiles of 11 Barophilic Bacteria Under In Situ Conditions: Evidence for Pressure Modulation of Phenotype. Appl. Environ. Microbiol. 56(3):812-814. (ERL,GB X685).

Barophilic bacteria are microorganisms that grow preferentially (facultative barophiles) or exclusively (obligate barophiles) under elevated hydrostatic pressure. Barophilic bacteria have been isolated from a variety of deep-sea environments. Attempts to characterize these organisms have been hampered by a lack of appropriate methodologies. A colorimetric method for the detection of 19 constitutively expressed enzymes under in situ conditions of pressure and temperature has been devised, using a simple modification of the commercially available API ZYME enzyme assay kit. By using this method, enzyme profiles of 11 barophilic isolates, including an obligate barophile, were determined. Nine of the 10 facultatively barophilic isolates examined exhibited a change of phenotype in at least one enzyme reaction when tested at 1 atm (1 atm=101.29 kPa), compared with results obtained under in situ pressure. The assay is simple and rapid and allows for direct determination of enzyme activity under conditions of high pressure and low temperature.

Leahy, Joseph G. and Rita R. Colwell. 1990. Microbial Degradation of Hydrocarbons in the Environment. Microbiol. Rev. 54(3):305-315. (ERL,GB X686).

The ecology of hydrocarbon degradation by microbial populations in the natural environment is reviewed, emphasizing the physical, chemical, and biological factors which contribute to the biodegradation of petroleum and individual hydrocarbons. Rates of biodegradation depend greatly on the composition, state, and concentration of the oil or hydrocarbons, with dispersion and emulsification enhancing rates in aquatic systems, and absorption by soil particulates being the key feature of terrestrial ecosystems. Temperature, and oxygen and nutrient concentrations are important variables in both types of environments. Salinity and pressure may also affect biodegradation rates in some aquatic environments, and moisture and pH may limit biodegradation in soils. Degradation of hydrocarbons is accomplished primarily by bacteria and fungi. Adaptation by prior exposure of microbial communities to hydrocarbons increases hydrocarbon degradation rates. Adaptation is brought about by selective enrichment of hydrocarbon-utilizing microorganisms and amplification of the pool of hydrocarbon-catabolizing genes. The latter phenomenon can now be monitored through the use of DNA probes. Increases in plasmid frequency may also be associated with genetic adaptation. Seeding to accelerate rates of biodegradation has been shown to be effective in some cases, particularly when employed under controlled conditions, such as in fermentors or chemostats.

Knight, Ivor T., Stacey Shults, Charles W. Kaspar and Rita R. Colwell. 1990. Direct Detection of Salmonella spp. in Estuaries by Using a DNA Probe. Appl. Environ. Microbiol. 56(4):1059-1066. (ERL,GB X687).

A method for direct detection of Salmonella spp. in water was developed by using a commercially available DNA probe. Particulate DNA was extracted from 500- to 1,500-ml water samples collected from New York Harbor and Chesapeake Bay and used as a substrate for a salmonella-specific DNA probe in dot blot assays. The method detected salmonellae in water samples from 12 of 16 sites, including 6 sites where salmonellae could not be cultured. The specificity of the probe was evaluated, and cross-hybridization, although negligible, was used to set detection limits for the assay. Salmonella DNA bound the probe quantitatively, and from these results Salmonella DNA in the total particulate DNA in environmental samples could be estimated. The data obtained in this study indicate that Salmonella spp. often are not detected in water samples by culture methods, even when they are present in significant numbers.

Haugland, R.A., D.J. Schlemm, R.P. Lyons, III, P.R. Sferra and A.M. Chakrabarty. 1990. Degradation of the Chlorinated Phenoxyacetate Herbicides 2,4-Dichlorophenoxyacetic Acid and 2,4,5-Trichlorophenoxyacetic Acid by Pure and Mixed Bacterial Cultures. Appl. Environ. Microbiol. 56(5):1357-1362. (ERL,GB X688).

Combined cell suspensions of the 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)-metabolizing organism Pseudomonas cepacia AC1100, and the 2,4-dichlorophenoxyacetic acid (2,4-D)-metabolizing organism Alcaligenes eutrophus JMP134 were shown to effectively degrade either of these compounds provided as single substrates. These combined cell suspensions, however, poorly degraded mixtures of the two compounds provided at the same concentrations. Growth and viability studies revealed that such mixtures of 2,4-D and 2,4,5-T were toxic to AC1100 alone and to combinations of AC1100 and JMP134. High-pressure liquid chromatography analyses of culture supernatants of AC1100 incubated with 2,4-D and 2,4,5-T revealed the accumulation of chlorohydroquinone as a apparent dead-end catabolite of 2,4-D and the subsequent accumulation of both 2,4-dichlorophenol and 2,4,5-trichlorophenol. JMP134 cells incubated in the same medium did not catabolize 2,4,5-T and were also inhibited in initiating 2,4-D catabolism. A new derivative of strain AC1100 was constructed by the transfer into this organism of the 2,4-D-degradative plasmid pJP4 from strain JMP134. This new strain, designated RHJ1, was shown to efficiently degrade mixtures of 2,4-D and 2,4,5-T through simultaneous metabolism of these compounds.

Kaphammer, Bryan, Jerome J. Kukor and Ronald H. Olsen. 1990. Regulation of tfdCDEF by tfdR of the 2,4-Dichlorophenoxyacetic Acid Degradation Plasmid pJP4. J. Bacteriol. 172(5):2280-2286. (ERL,GB X689).

The closely linked structural genes tfdCDEF borne on the 2,4-dichlorophenoxyacetic acid (TFD) catabolic plasmid, pRO101, were cloned into vector pRO2321 as a 12.6-kilobase-pair BamHI C fragment and designated pRO2334. The first gene in this cluster, tfdC, encodes chlorocatechol 1,2-dioxygenase and was expressed constitutively. Chlorocatechol 1,2-dioxygenase expression by pRO2334 was repressed in trans by the negative regulatory element, tfdR, on plasmid pRO1949. Depression of tfdC was achieved when Pseudomonas aeruginosa PAO4032 containing both plasmids pRO2334 and pRO1949 was grown in minimal glucose medium containing TFD, 2,4-dichlorophenol, or 4-chlorocatechol, suggesting that TFD and other pathway intermediates can act as inducing compounds. Genetic organization of the tfdCDEF cluster was established by deletion of the tfdC gene, which resulted in the loss of tfdD and tfdE activity, suggesting that genes tfdCDEF are organized in an operon transcribed from the negatively regulated promoter of tfdC. Deletion subcloning of pRO1949 was used to localize tfdR to a 1.2-kilobase-pair BamHI-XhoI region of the BamHI E fragment of plasmid pRO101. The tfdR gene product was shown not to regulate the expression of tfdB, which encodes 2,4-dichlorophenol hydroxylase.

Tartera, C., K. Davis and R.R. Colwell. 1990. Production of Escherichia coli-Specific Hybridomas by Using Gnotobiotic Mice. Appl. Environ. Microbiol. 56(5):1397-1399. (ERL,GB X690).

Monoclonal antibodies provide a rapid and specific means of direct detection of microorganisms in water and food samples. However, monoclonal antibodies specific for some bacteria are difficult to obtain; a good example of such a bacterium is Escherichia coli. Gnotobiotic BALB/c mice immunized with whole-cell preparations of heat-treated strains of E. coli and subjected to high-frequency antigen injection showed a significant increase in the number of specific hybridomas produced. Fusions obtained by using regular BALB/c mice immunized by using standard immunization protocols produced nonspecific hybridomas. Twenty-one stable hybridomas that did not cross-react with Klebsiella pneumoniae ATCC 13883 or Citrobacter freundii 1604770 were obtained from gnotobiotic mice. The bacterial strains were selected for the specificity tests because of their high cross-reactivity, which has been deleted in previous fusion experiments. The method of immunization described here offers the potential of improving the production of highly specific hybridomas for bacteria which have been difficult to obtain.

Desmonts, Catherine, Jacques Minet, Rita Colwell and Michel Cormier. 1990. Fluorescent-Antibody Method Useful for Detecting Viable but Nonculturable Salmonella spp. in Chlorinated Wastewater. Appl. Environ. Microbiol. 56(5):1448-1452. (ERL,GB X691).

An indirect fluorescent-antibody (IFA) technique, which employed adsorbed Behring polyvalent I O antiserum, was used to detect Salmonella spp. in environmental water systems. The IFA method used in this study detected 95% of Salmonella serotypes encountered in human infections in France, with a sensitivity threshold of 7.5 x 10 to the third power bacteria ml of wastewater. Specificity was assessed by testing IFA against Salmonella-free seawater and a variety of bacteria other than Salmonella spp. When used to examine raw and chlorinated wastewater over a 2-month period, the IFA method was successful in detecting Salmonella spp. in all 12 of the samples examined, with total numbers determined to be 4.5 x 10 to the fifth power to 3.3 x 10 to the seventh power salmonellae per 100 ml. In comparison, for the same samples, enumeration by culture, using the most-probable-number technique, was effective in detecting Salmonella spp. in only four of eight raw-water samples and one of four chlorinated water samples tested. Three samples were further tested by using the direct viable count procedure combined with IFA and results showed that 5 to 31.5% of the Salmonella spp. enumerated by this method in chlorinated water were substrate responsive.

Straube, W.L., J.W. Deming, C.C. Somerville, R.R. Colwell and J.S. Baross. 1990. Particulate DNA in Smoker Fluids: Evidence for Existence of Microbial Populations in Hot Hydrothermal Systems. Appl. Environ. Microbiol. 56(5):1440-1447. (ERL,GB X693).

As part of an interdisciplinary study of hydrothermal vents on the Endeavour Segment of the Juan de Fuca Ridge, we used the submersible ALVIN to collect 57 fluid samples in titanium syringes and Go Flo Niskin bottles from 17 different hot vents (smokers and flanges) and their environs for the purpose of extracting particulate DNA. The relative purity of the vent fluids collected was determined by Mg content as an indicator of seawater entrainment. Particulate material concentrated from these samples was lysed enzymatically (enz) and by a combination of enzyme and French press treatment (fp). Concentrations of partially purified DNA recovered from these lysates were determined spectrofluorometrically by using the dye Hoechst 33258. Ambient seawater surrounding the vents was found to contain low DNA concentrations, 0.18 to 0.32 ng of DNA per ml (n = 4;mean enz = 0.23 plus or minus 0.05; mean fp = 0.26 plus or minus 0.05), while low-temperature vent samples yielded significantly higher concentrations of 0.37 to 2.12 ng of DNA per ml (n = 4; mean enz = 0.97 plus or minus 0.68; mean fp = 1.05 plus or minus 0.54). Although DNA recovery values from superheated (210 to 345 degrees C) flange samples (mean enz = 0.14 plus or minus 0.10; mean fp = 0.12 plus or minus 0.14) were not significantly different from ambient seawater values, most of the superheated (175 to 357 degrees C) smoker fluid samples contained particulate DNA in concentrations too high to be attributable to entrained seawater. Detailed sampling at one smoker site demonstrated not only the existence of significant levels of particulate DNA in the superheated smoker fluids but also the presence of an elevated microbial population in the buoyant plume 20 to 100 m above the smoker. These results underscore the heterogeneity of smoker environments within a given hydrothermal vent field and indicate that microorganisms exist in some superheated fluids.

Kaphammer, Bryan and Ronald H. Olsen. 1990. Cloning and Characterization of tfdS, the Repressor-Activator Gene of tfdB, from the 2,4-Dichlorophenoxyacetic Acid Catabolic Plasmid pJP4. EPA/600/J-90/552. J. Bacteriol. 172(10):5856-5862. (ERL,GB X694). (Avail. from NTIS, Springfield, VA: PB92-129634)

Plasmid pR101, a derivative of plasmid pJP4, which contains Tn1721 inserted into a non-essential region, is inducible for 2,4-dichlorophenol hydroxylase (DCPH) encoded by tfdB. Plasmid pRO103, which has a deletion in the BamHI-F:BamHI-E region of plasmid pRO101, has elevated basal levels of DCPH but is uninducible. The regulatory gene for tfdB, designated tfdS, was cloned as an 8.3 kbp EcoRI-E fragment. When the cloned tfdS gene was in trans with plasmid pRO103 the baseline DCPH levels were repressed to normal uninduced levels and fully induced when this strain was grown in the presence of 2,4-dichlorophenoxyacetic acid, 2,4-dichlorophenol, or 4-chlorocatechol (4CC). However, when tfdS was in trans with tfdB, in the absence of tfdCDEF, tfdB was repressed but could not be induced. When tfdS and tfdC1, which encodes chlorocatechol 1,2-dioxygenase, are in trans with tfdB, tfdB remained uninduced, indicating that a downstream metabolite of chloro-cis,cis-muconate, either 2-cis-chlorodiene lactone or chloromaleyacetic acid, is the effector. Collectively, these data demonstrate that the gene product of tfdS acts as a repressor of tfdB in the absence of an effector and as an activator of tfdB when an effector is present.

Kukor, Jerome J. and Ronald H. Olsen. 1990. Molecular Cloning, Characterization, and Regulation of a Pseudomonas pickettii PKO1 Gene Encoding Phenol Hydroxylase and Expression of the Gene in Pseudomonas aeruginosa PAO1c. EPA/600/J-90/379. J. Bacteriol. 172(8):4624-4630. (ERL,GB X698). (Avail. from NTIS, Springfield, VA: PB91-163923)

A 26 kilobase BamHI restriction endonuclease DNA fragment has been cloned from Pseudomonas pickettii PKO1, a strain isolated from a soil microcosm that had been amended with benzene, toluene, and xylene. This DNA fragment, cloned into vector plasmid pRO1727 and designated pRO1957, allowed P. aeruginosa PAO1c to grow on phenol as sole source of carbon. Physical and functional restriction endonuclease maps have been derived for the cloned DNA fragment. Two DNA fragments carried in trans and derived from subclones of pRO1957 show phenol hydroxylase activity in cell-free extracts of P. aeruginosa. Deletion and subcloning analyses of these fragments indicated that the gene encoding phenol hydroxylase is positively regulated. Phenol and m-cresol were shown to be inducers of the enzyme. o-Cresol and p-cresol did not induce enzymatic activity, but could be metabolized by cells that had been previously exposed to phenol or m-cresol, moreover the enzyme exhibited a rather broad substrate specificity and was sensitive to thiol-inhibiting reagents. A novel polypeptide with an estimated molecular mass of 80,000 daltons was detected in extracts of phenol-induced cells of P. aeruginosa carrying plasmid pRO1959.

Leahy, Joseph G., Charles C. Somerville, Kelly A. Cunningham, Grammenos A. Adamantiades, Jeffrey J. Byrd and Rita R. Colwell. 1990. Hydrocarbon Mineralization in Sediments and Plasmid Incidence in Sediment Bacteria from the Campeche Bank. Appl. Environ. Microbiol. 56(6):1565-1570. (ERL,GB X700).

Rates of degradation of radiolabeled hydrocarbons and incidence of bacterial plasmid DNA were investigated in sediment samples collected from the Campeche Bank, Gulf of Mexico, site of an offshore oil field containing several petroleum platforms. Overall rates of mineralization of [14C]hexadecane and [14C]phenanthrene measured for sediments were negligible: less than 1% of the substrate was converted to CO2 in all cases. Low mineralization rates are ascribed to nutrient limitations and to lack of adaptation by microbial communities to hydrocarbon contaminants. Plasmid frequency data for sediment bacteria similarly showed no correlation with proximity to the oil field, but, instead, showed correlation with water column depth at each sampling site. Significant differences between sites were observed for proportion of isolates carrying single or multiple plasmids and mean number of plasmids per isolate, each of which increased as a function of depth.

Amann, Rudolf I., Brian J. Binder, Robert J. Olson, Sallie W. Chisholm, Richard Devereux and David A. Stahl. 1990. Combination of 16S rRNA-Targeted Oligonucleotide Probes with Flow Cytometry for Analyzing Mixed Microbial Populations. Appl. Environ. Microbiol. 56(6):1919-1925. (ERL,GB X701).

Fluorescent oligonucleotide hybridization probes were used to label bacterial cells for analysis by flow cytometry. The probes, complementary to short sequence elements within the 16S rRNA common to phylogenetically coherent assemblages of microorganisms, were labeled with tetramethylrhodamine and hybridized to suspensions of fixed cells. Flow cytometry was used to resolve individual target and nontarget bacteria (1 to 5 um) via probe-conferred fluorescence. Target cells were quantified in an excess of nontarget cells. The intensity of fluorescence was increased additively by the combined use of two or three fluorescent probes complementary to different regions of the same 16S rRNA.

Vannelli, Todd, Myke Logan, David M. Arciero and Alan B. Hooper. 1990. Degradation of Halogenated Aliphatic Compounds by the Ammonia-Oxidizing Bacterium Nitrosomonas europaea. EPA/600/J-90/112. Appl. Environ. Microbiol. 56(4):1169-1171. (ERL,GB X702). (Avail. from NTIS, Springfield, VA: PB90-264185)

Suspensions of Nitrosomonas europaea catalyzed the ammonia-stimulated aerobic transformation of the halogenated aliphatic compounds dichloromethane, dibromomethane, trichloromethane (chloroform), bromoethane, 1,2-dibromoethane (ethylene dibromide), 1,1,2-trichloroethane, 1,1,1-trichloroethane, monochloroethylene (vinyl chloride), gem-dichloroethylene, cis- and trans-dichloroethylene, cis-dibromoethylene, trichloroethylene, and 1,2,3-trichloropropane. Tetrachloromethane (carbon tetrachloride), tetrachloroethylene (perchloroethylene), and trans-dibromoethylene were not degraded.

Devereux, Richard, Shao-Hua He, Carolyn L. Doyle, Silvia Orkland, David A. Stahl, Jean LeGall and William B. Whitman. 1990. Diversity and Origin of Desulfovibrio Species: Phylogenetic Definition of a Family. EPA/600/J-90/372. J. Bacteriol. 172(7):3609-3619. (ERL,GB X705). (Avail. from NTIS, Springfield, VA: PB91-163857)

The different nutritional properties of several Desulfovibrio desulfuricans strains suggest that either the strains are misclassified or there is a high degree of phenotypic diversity within the genus Desulfovibrio. The results of partial 16S rRNA and 23S rRNA sequence determinations demonstrated that Desulfovibrio desulfuricans ATCC 27774 and 'Desulfovibrio multispirans' are closely related to the type strain (strain Essex 6) and that strains ATCC 7757, Norway 4, and El Agheila Z are not. Therefore, these latter three strains of Desulfovibrio desulfuricans are apparently misclassified. A comparative analysis of nearly complete 16S rRNA sequences in which we used a least-squares analysis method for evolutionary distances, an unweighted pair group method, a signature analysis method, and maximum parsimony was undertaken to further investigate the phylogeny of Desulfovibrio species. The species analyzed were resolved into two branches with origins deep within the beta subdivision of the purple photosynthetic bacteria. One branch contained five deep lineages, which were represented by (i) Desulfovibrio salexigens and Desulfovibrio desulfuricans El Agheila Z; (ii) Desulfovibrio africanus; (iii) Desulfovibrio desulfuricans ATCC 27774, Desulfomonas pigra, and Desulfovibrio vulgaris; (iv) Desulfovibrio gigas; and (v) Desulfomicrobium baculatus (Desulfovibrio baculatus) and Desulfovibrio desulfuricans Norway 4. A correlation between 16S rRNA sequence similarity and percentage of DNA relatedness showed that these five deep lineages are related at levels below the minimum genus level suggested by Johnson (in Bergey's Manual of Systematic Bacteriology, vol. 1, 1984). We propose that this branch should be grouped into a single family, the Desulfovibrionaceae. The other branch includes other genera of sulfate-reducing bacteria (e.g., Desulfobacter and Desulfococcus) and contains Desulfovibrio sapovorans and Desulfovibrio baarsii as separate, distantly related lineages.

Byrd, Jeffrey J. and Rita R. Colwell. 1990. Maintenance of Plasmids pBR322 and pUC8 in Nonculturable Escherichia coli in the Marine Environment. Appl. Environ. Microbiol. 56(7):2104-2107. (ERL,GB X708).

Maintenance of plasmids pBR322 and pUC8 in Escherichia coli that was nonculturable after exposure to seawater was studied. E. coli JM83 and JM101, which contained plasmids pBR322 and pUC8, respectively, were placed in sterile artificial seawater for 21 days. Culturability was determined by plating on both nonselective and selective agar, and plasmid maintenance was monitored by direct isolation of plasmid nucleic acid from bacteria collected on Sterivex filters. E. coli JM83 became nonculturable after incubation for 6 days in seawater yet maintained plasmid pBR322 for the entire period of the study, i.e., 21 days. E. coli JM101 was nonculturable after incubation in seawater for 21 days and also maintained plasmid pUC8 throughout the duration of the microcosm experiment. Direct counts of bacterial cells did not change significantly during exposure to seawater, even though plate count yielded no viable (i.e., platable) cells. We concluded that E. coli cells are capable of maintaining high-copy-number plasmids, even when no longer culturable, after exposure to the estuarine or marine environment.

TeBeest, D.O. and G.J. Weidemann. 1990. Preparation and Regeneration of Protoplasts of Colletotrichum gloeosporioides f. sp. aeschynomene. EPA/600/J-92/216. Mycologia. 82(2):249-255. (ERL,GB X710). (Avail. from NTIS, Springfield, VA: PB92-195734)

Protoplasts were produced from conidia of Colletotrichum gloeosporioides f. sp. aeschynomene, a fungal plant pathogen of Aeschynomene virginica, during treatment with Novozym 234 or a mixture of chitinase and beta-glucuronidase after pretreatment with 2-mercaptoethanol. Protoplasts were optimally stablized in 1.2 M mannitol after release from conidia but regenerated and reverted to hyphae optimally on 0.7 M sucrose. Approximately 84% of the protoplasts regenerated cell walls and reverted to hyphal colonies on 0.7 M sucrose. The osmotic stablizer and molarity of the stabilizer affected regeneration and reversion to colonies. Microscopic studies of the nuclear content of conidia protoplasts showed that the number of nuclei in protoplasts was similar to the number of nuclei in conidia from which they were produced. Of the 209 colonies grown from reverted protoplasts, all were as pathogenic to A. virginica as the wild-type parent, and all resembled the wild-type strain from which they were produced. The development of an efficient technique to produce protoplasts enables future research on the genetics of this fungus.

Simonsen, Lone. 1990. Dynamics of Plasmid Transfer on Surfaces. EPA/600/J-90/367. J. Gen. Microbiol. 136(6):1001-1007. (ERL,GB X727). (Avail. from NTIS, Springfield, VA: PB91-163816)

A protocol was developed to study the dynamics of growth and plasmid transfer in surface populations of bacteria. This method allows for quantitative estimates of cell population densities over time, as well as microscopic observations of colony growth and interactions. Using this 'surface slide system' (SSS), the dynamics of the plasmid R1 and its permanently derepressed mutant R1drd19 in surface cultures of Escherichia coli K12 was examined. In surface culture, the stationary-phase cell densities were constant over a wide range of initial cell density (=colony density) and comparable to those obtained in liquid culture. For high initial cell densities, where the cells formed a confluent layer at stationary phase, the kinetics of growth and plasmid transfer were similar to that obtained in liquid culture, and the relative yields of R1drd19 and R1 transconjugants were similar in the two habitats. In surface culture, however, R1drd19 transconjugant yield was profoundly affected, and R1 transfer to a lesser extent, by colony density. In contrast, liquid matings were virtually unaffected by initial cell density. The transfer advantage of the permanently derepressed over the repressed plasmid was much less apparent for lower colony densities. I propose a hypothesis for plasmid transfer between colonies that explains these observations as a consequence of the geometry of the surface habitat and the effect of transitory derepression of the synthesis of pili.

Simonsen, L., D.M. Gordon, F.M. Stewart and B.R. Levin. 1990. Estimating the Rate of Plasmid Transfer: an End-Point Method. EPA/600/J-90/551. J. Gen. Microbiol. 136(11):2319-2325. (ERL,GB X730). (Avail. from NTIS, Springfield, VA: PB92-129626)

We describe a method for determining the rate parameter of conjugative plasmid transfer that is based on single estimates of donor, recipient and transconjugant densities and the growth rate in exponential phase of the mating culture. The formula for estimating the plasmid transfer rate, Y, was derived from a mathematical model describing cell growth and plasmid transfer in batch culture. Computer simulations were used to explore the sensitivity of this method to the realities of bacterial life, such as growth rate differences, plasmid segregation and transitory derepression of pilus synthesis. As predicted by the theory, mating experiments with the plasmid R1 in E. coli K12 demonstrated that the estimate Y is unaffected by cell density, donor:recipient ratio and mating time. Unlike previous techniques, our method allows us to investigate the effect of environmental factors on plasmid transfer rates when these factors also influence population growth rates. To illustrate this, we examined the effect of temperature on the rate of plasmid transfer.

Miller, Robert V. 1990. Increasing Levels of Environmental Mutagens: Potential for Affecting Viral Evolution and Pathogenicity: A Speculative Review. EPA/600/J-90/514. J. Environ. Sci. Health Part C Environ. Carcinog. Rev. C8(1):89-137. (ERL,GB X731). (Avail. from NTIS, Springfield, VA: PB91-206862)

The author examines available data concerning the ways in which information contained in viral genomes is altered. Mechanisms of damage and repair of nucleic acids are discussed. Information available on the rates of evolution of various viruses is summarized.

Short, Kevin A., Ramon J. Seidler and Ronald H. Olsen. 1990. Survival and Degradative Capacity of Pseudomonas putida Induced or Constitutively Expressing Plasmid-Mediated Degradation of 2,4-Dichlorophenoxyacetate (TFD) in Soil. EPA/600/J-90/511. Can. J. Microbiol. 36(12):821-826. (ERL,GB X732). (Avail. from NTIS, Springfield, VA: PB91-182196)

Survival of genetically altered Pseudomonas putida strains harboring an inducible plasmid, pRO101, or a constitutive plasmid, pRO103, was compared. These plasmids encoded for the degradation of 2,4-dichlorophenoxyacetate (TFD) to 2-chloromaleylacetate, and the maintenance of either plasmid did not alter survival of P. putida PPO301 (pRO101) or PPO301 (pRO103) in an unamended agricultural soil. In a parallel study, Raphanus sativus (radish) seeds failed to germinate in uninoculated and PPO301-inoculated soil amended with 500 ppm TFD. Seed germination was 53 and 80% in soils inoculated with PPO301 (pRO101) and PPO301 (pRO103), respectively (P less than 0.001). However, the difference in the rate of TFD degradation between the native soil and soil inoculated with plasmid-bearing P. putida was probably related to the relatively high inoculum density of P. putida strains (10 to the eighth power cfu) and the relatively low population density of TFD metabolizers indigenous to the soil.

Summers, J. Kevin, William A. Richkus, C. Foster Stroup and Louis J. Rugolo. 1990. Influence of Natural and Anthropogenic Environmental Change on White Perch Stock Status in the Choptank River, Maryland. Fish. Res. 9(3):255-268. (ERL,GB X752).

A categorical time series regression model was developed to evaluate the importance of natural and anthropogenic environmental changes to the determination of white perch stock abundance in the Choptank River, Maryland. Ninety-one percent of the variability in historical stock size for the period 1929-1985 could be explained by a combination of April and May freshwater discharge level, parental stock size, and sewage loadings lagged 2-3 and 9-10 years. Clearly most of the annual variablity was associated with changes in natural environmental factors, but a significant, albeit, small portion of the variability in stock size was attributable to sewage loadings in the Choptank River.

McLean, Richard I. and J. Kevin Summers. 1990. Evaluation of Transport and Storage of 60Co, 134Cs, 137Cs and 65Zn by River Sediments in the Lower Susquehanna River. Environ. Pollut. 63:137-153. (ERL,GB X753).

The Peach Bottom Atomic Power Station (PBAPS) has contributed measurable quantities of radioactivity to Conowingo Reservoir, an impoundment of the lower Susquehanna River. As part of an ongoing radiological assessment program, concentrations of plant-related radionuclides in sediments have been monitored in spring and fall since 1980. Mass balance estimates derived from grab samples of surface sediments (less than 10 cm) indicate that less than 20% of reactor relased 60Co, 65Zn, 134Cs and 137Cs is present in these sediments. Significant seasonal variations in radionuclide trapping efficiency by the reservoir are not apparent. Deep core samples (c.200 cm) confirm that some, but not all, of this surface sediment radionuclide inventory remains within the reservoir--trapped in discrete locations by subsequent sediment accumulation. The remaining radionuclide mass, in dissolved or particle-associated form, appears to be transported downstream, through Conowingo Dam, to upper Chesapeake Bay. The detection of PBAPS-derived radionuclides in the sediments of upper Chesapeake Bay, primarily the Susquehanna Flats, confirms the transport of these radionuclides from the lower Susquehanna River.

Katsuwon, Jirasak and Anne J. Anderson. 1990. Catalase and Superoxide Dismutase of Root-Colonizing Saprophytic Fluorescent Pseudomonads. EPA/600/J-93/220. Appl. Environ. Microbiol. 56(11):3576-3582. (ERL,GB X773). (Avail. from NTIS, Springfield, VA: PB93-205037)

Root-colonizing, saprophytic fluorescent pseudomonads of the Pseudomonas putida-P. fluorescens group express similar levels of catalase and superoxide dismutase activities during growth on a sucrose- and amino acid-rich medium. Increased specific activities of catalase but not superoxide dismutase were observed during growth of these bacteria on components washed from root surfaces. The specific activities of both enzymes were also regulated during contact of these bacteria with intact bean roots. Increased superoxide dismutase and decreased catalase activities were observed rapidly, by 10 min upon inoculation of cells onto intact bean roots. Catalase specific activity increased with time to peak at 12 h before declining. By 48 h. the cells displayed this low catalase but maintained high superoxide dismutase specific activities. Catalase with a low specific activity and a high superoxide dismutase activity also were present in extracts of cells obtained from 7-day-old roots colonized from inoculum applied to seed. This specific activity of superoxide dismutase of root-contacted cells was about fourfold-higher in comparison to cells grown on rich medium, whereas the specific activity for catalase was reduced about fivefold. A single catalase isozyme, isozyme A, and one isozyme of superoxide dismutase, isozyme 1, were detected during growth of the bacteria on root surface components and during exposure of cells to intact bean roots for 1 h. An additional catalase, isozyme B, was detected from bacteria after exposure to the intact bean roots for 12 h. Catalase isozyme A and superoxide dismutase isozyme 1 were located in the cytoplasm and catalase band B was located in the membrane of P. putida.