Fred J. Genthner

Heterotrophic bacteria of the freshwater neuston and their ability to act as plasmid recipients under nutrient deprived conditions.

Significantly higher numbers of Gram-negative heterotrophic bacteria were present at the air-water interface (neston) of freshwater lakes than in the bulk water. Neuston bacteria were distinguished as a population distinct from bacteria in the bulk water by a higher incidence of pigmented colony types and significantly greater levels of multiple resistance to antibiotics and heavy metals. The incidence of plasmids in 236 neuston and 229 bulk water strains were similar (14 and 16.2%, respectively). Nine of 168 plasmid-free strains and 2 of 14 plasmid carrying strains, isolated from both bulk water and neuston, acted as recipients of plasmid R68.45 in plate matings with aPseudomonas aeruginosa donor strain PAO4032 at 21°C, but at frequencies below that of matings with a restriction-minus recipient strain ofP. aeruginosa, strain PAO1168. In a model system composed of nutrient-free synthetic lake water, plasmid R68.45 was shown to transfer betweenP. aeruginosa strains at frequencies between 10−3 and 10−5. Transconjugants were detected about 100 times more frequently at the interface than in the bulk water, which in part reflected a greater enrichment of the donor at this site. None of the aquatic isolates were able to act as recipients of plasmid R68.45 in this model system with strain PAO4032 as donor. The results suggest that under nutrient deprived conditions, the spread of plasmid R68.45 and similar plasmids by lateral transfer into this particular aquatic population would be a rare event.

Effects of light reduction on growth of the submerged macrophyte Vallisneria americana and the community of root-associated heterotrophic bacteria

Abstract A shading experiment was conducted over a growing season to measure the effects of light reduction on Vallisneria americana in Perdido Bay on the Florida–Alabama border and to determine the response of heterotrophic bacteria in the rhizosphere. Plants subjected to 92% light reduction showed the most pronounced effects in chlorophyll a concentration, above- and below-ground biomass, and leaf dimensions. These results further suggested that the V. americana life cycle, as exhibited in temperate waters, was impaired. Heterotrophic bacteria were enumerated and identified (i) from the roots and sediments of fully illuminated plants and from unvegetated sediments at three intervals and (ii) from the roots of plants that have been subjected to 92% light reduction for 3 months. Up to two orders of magnitude greater numbers of bacteria were enumerated from root samples than sediment samples on a dry weight basis. Bacteria enumerated from the roots of plants subjected to light reduction (1.3±1.1×10 8 CFU g −1 ) were significantly higher than numbers of bacteria enumerated from the roots of fully illuminated plants (4.8±1.8×10 7 g −1 in the summer) or sediment samples (1.4±0.03×10 6 g −1 ). This suggests the roots of seagrasses stressed by light reduction provided more nutrients for bacterial growth. Higher percentages of Gram-negative bacteria were isolated from roots (up to 85% in the fall) than sediments (0–15%). Examination of isolates for traits characteristic of rhizosphere bacteria (siderophore production, formation of the phytohormone indole-3-acetic acid, and antifungal activity) did not show a clear distinction between root-associated and sediment isolates. Taxonomic identifications of root-associated bacteria based on MIDI analysis of fatty acid methyl esters were consistent with bacteria known to be associated with other plants or found at oxic–anoxic interfaces. In addition, the bacterial identifications showed most species were associated with only roots or only sediments. These results support studies suggesting seagrass rhizospheres harbor distinct bacterial communities.

A rapid tetrazolium dye reduction assay to assess the bactericidal activity of oyster (Crassostrea virginica) hemocytes against Vibrio parahaemolyticus

Abstract An assay was developed to assess the ability of oyster, Crassostrea virginica , hemocytes to kill the human pathogenic bacterium, Vibrio parahaemolyticus (ATCC 17802). Bacterial killing was estimated colorimetrically by the enzymatic reduction of a tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H -tetrazolium (MTS), and phenylmethasulfazone (PMS). The assay proposed here provides an indicator of immunocompetence of oysters against V. parahaemolyticus . The assay involved: (1) exposure of plasma-free oyster hemocytes to a streptomycin (SM)-resistant mutant strain of V. parahaemolyticus in a 96-well plate for 3 h at 17°C in SM-augmented sea water; (2) growout of surviving bacteria in nutrient broth for 2 h at 37°C; (3) addition of MTS and PMS; and (4) measurement of MTS/PMS reduction product (formazan) at 490 nm using a microplate reader. Advantages of this assay include the absence of radio-isotopes used in some killing assays and requirement of low volumes of plasma and numbers of hemocytes. In addition, we demonstrated greater precision than traditional, plate counting methods for bacterial estimation. This technique has the potential to evaluate oyster capacity to eliminate microbial agents and to assess effects of environmental changes and pollutant stress on defense capabilities of oysters.

Gene Mapping and Phylogenetic Analysis of the Complete Genome from 30 Single-Stranded RNA Male-Specific Coliphages (Family Leviviridae)

ABSTRACT Male-specific single-stranded RNA (FRNA) coliphages belong to the family Leviviridae. They are classified into two genera (Levivirus and Allolevivirus), which can be subdivided into four genogroups (genogroups I and II in Levivirus and genogroups III and IV in Allolevivirus). Relatively few strains have been completely characterized, and hence, a detailed knowledge of this virus family is lacking. In this study, we sequenced and characterized the complete genomes of 19 FRNA strains (10 Levivirus strains and 9 Allolevivirus strains) and compared them to the 11 complete genome sequences available in GenBank. Nucleotide similarities among strains of Levivirus genogroups I and II were 75% to 99% and 83 to 94%, respectively, whereas similarities among strains of Allolevivirus genogroups III and IV ranged from 70 to 96% and 75 to 95%, respectively. Although genogroup I strain fr and genogroup III strains MX1 and M11 share only 70 to 78% sequence identity with strains in their respective genogroups, phylogenetic analyses of the complete genome and the individual genes suggest that strain fr should be grouped in Levivirus genogroup I and that the MX1 and M11 strains belong in Allolevivirus genogroup III. Strains within each genus share >50% sequence identity, whereas between the two genera, strains have <40% nucleotide sequence identity. Overall, amino acid composition, nucleotide similarities, and replicase catalytic domain location contributed to phylogenetic assignments. A conserved eight-nucleotide signature at the 3′ end of the genome distinguishes leviviruses (5′ ACCACCCA 3′) from alloleviviruses (5′ TCCTCCCA 3′).

A reverse transcription-PCR assay to distinguish the four genogroups of male-specific (F+) RNA coliphages.

Goals of reducing fecal contamination in recreational, drinking, shellfishing and other waters and accurately assessing risk from exposure can best be attained if tools to distinguish between sources of pollution are available. The male-specific RNA coliphage (FRNA) genogroups display a trend of source specificity. Reverse transcription-PCR (RT-PCR) can be effectively used for genotyping if specific primer sets are designed to be capable of identifying all members within each genogroup. In this study genogroup-specific primer sets were designed using a minimum of 5 to a maximum of 10 complete phage genome sequences from strains in each genogroup. With these primers and employing a heat-release procedure that eliminated the need for RNA purification an RT-PCR method for genotype identification of FRNA phages was developed. The four genogroup-specific primer sets generated discrete PCR amplicon sizes from a variety of environmental FRNA phage strains. Limits of detection, cross-reactivity and/or non-specific binding to strains from other genogroups were evaluated.

Design and assessment of a real time reverse transcription-PCR method to genotype single-stranded RNA male-specific coliphages (Family Leviviridae).

A real-time, reverse transcription-PCR (RT-qPCR) assay was developed to differentiate the four genogroups of male-specific ssRNA coliphages (FRNA) (family Leviviridae). As FRNA display a trend of source-specificity (human sewage or animal waste) at the genogroup level, this assay provides a tool to help identify the origin of fecal contamination. Primers and probes were designed using complete genomic sequences from 29 FRNA phages. The final selection of primer/probe sets were based on (i) ability to amplify a single, specific product, (ii) genogroup specificity, (iii) lack of cross-reactivity, and (iv) experimental reproducibility and sensitivity over a range of target concentrations. Assay time was reduced by using heat-released viral RNA rather than purified RNA. For quality assurance, a custom RNA molecule was employed as an internal, non-competitive control. The usefulness of this method to identify sources of fecal contamination was tested on a total of 49 FRNA phages isolated from various warm-blooded animals, sewage and combined sewage overflow. FRNA phages from animal wastes were genotyped as 86% I, 4% III Q-like and 9% IV. Two sewage isolates typed to genogroup I and combined sewage overflow isolates genotyped as 40% II and 52% III. Primer specificity designed from this comprehensive sequence database may better discriminate FRNA from different sources.

Fate of Bacillus sphaericus 2362 spores following ingestion by nontarget invertebrates

Elimination of Bacillus sphaericus spores ingested by midge larvae, snails, and oysters was most rapid among midge larvae. Spores remained in oysters up to 21 days and in snails up to 49 days. Viable spores were recovered in snail and oyster feces for these same periods. There was no indication of actively growing B. sphaericus in the animals. Passage through oyster gut detoxified the B. sphaericus mosquito larval toxin, but there was a 33% retention of toxicity following snail gut passage. Midge larvae reared to adults in spore-containing water carried spores in/on the adult body. This suggests that these animals could carry the bacteria to sites beyond the application area.

Anomalies in the enumeration of starved bacteria on culture media containing nalidic acid and tetracycline.

Culturable counts of antibiotic resistant, genetically engineeredPseudomonas fluorescens were determined on antibiotic-containing plate count agar during starvation in 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 × 106 ml−1) was significantly higher than the mean colony counts on plate count agar containing either nalidixic acid or nalidixic acid plus tetraclycline (2.5×106 ml−1). After 20 days of starvation the highest mean colony counts continued to be obtained on plate count agar (7.2 × 106 ml−1) with slightly, but significantly, lower counts obtained on plate count agar containing either nalidixic acid (5.6 × 106 ml−1) or tetraclycline (1.5×106 ml−1). A combination of nalidixic acid and tetracycline in plate count agar, however, dramatically reduced colony counts (8.3 × 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 acid and tetracycline resulted in a significant increase in colony counts. Other combinations of antibiotics, nalidixic acid+carbenicillin, nalidixic acid+kanamycin, streptomycin+tetracycline, streptomycin+carbenicillin, rifampicin+tetracycline, rifampicin+carbenicillin, and rifampicin+kanamycin, did not inhibit colony formation of starved cells. Antibiotic resistant strains ofP. putida andEscherichia coli also displayed sensitivity to the combination of nalidixic acid and tetracycline in plate count agar after starvation.

Genomic Sequences of two Novel Levivirus Single-Stranded RNA Coliphages (Family Leviviridae): Evidence for Recombinationin Environmental Strains

Bacteriophages are likely the most abundant entities in the aquatic environment, yet knowledge of their ecology is limited. During a fecal source-tracking study, two genetically novel Leviviridae strains were discovered. Although the novel strains were isolated from coastal waters 1130 km apart (North Carolina and Rhode Island, USA), these strains shared 97% nucleotide similarity and 97–100% amino acid similarity. When the novel strains were compared to nine Levivirus genogroup I strains, they shared 95–100% similarity among the maturation, capsid and lysis proteins, but only 84–85% in the RNA-dependent RNA polymerase gene. Further bioinformatic analyses suggested a recombination event occurred. To the best of our knowledge, this is the first description of viral recombinants in environmental Leviviridae ssRNA bacteriophages.

Bacillus sphaericus mosquito pathogens in the aquatic environment

The fate of Bacillus sphaericus spores in the aquatic environment was investigated by suspending spores in dialysis bags in fresh and seawater. Spore viability was lost more rapidly in seawater. Neither B. sphaericus nor B. thuringiensis israelensis (B.t.i.) spores mixed with pond sediment appeared to attach to the sediment. However, rapid decrease in B.t.i. toxicity suggested attachment of parasporal bodies to sediment. B. sphaericus toxin settled more slowly and less completely. B. sphaericus spores fed to larvae of four aquatic invertebrates were mostly eliminated from the animal gut in less than one week. An exception was the cranefly (Tipula abdominalis) where spores persisted in the posterior gut for up to five weeks.