Ecosystem condition assessments were conducted for 12 mangrove sites in the northern Gulf of Mexico. Nine sites were selected randomly; three were selected a priori based on best professional judgment to represent a poor, intermediate and good environmental condition. During single visits a visual assessment generated numerical scores for each site relative to the reference condition range. Relationships were examined among variables to evaluate relevance of visual observations and empirical data of environmental condition. Sites in poorest visual condition possessed low concentrations of soil contaminants as hydrological attributes that restricted contaminant inflow lowered visual scores. Bacterial abundance related to the greatest number of variables, and was the only indicator that correlated with the visually derived score (0.60, p < 0.05). Soil enzymatic activity ratios within classes provided estimates of microbial nutrient status. Low peptidases/phosphatase and glycosidases/phosphatase ratios supported the conclusion reached from the molar ratios of nutrients, that mangrove soils were phosphorous limited. A positive correlation (0.71, p < 0.05) observed between C:N ratios in leaf tissue and soil from all mangrove sites indicated that relative molar ratios of major nutrients in leaf tissue were reflective of limitations in the soil. Principal components analysis guided a reduction in variables retained for analysis, and provided an ordination of the sites which grouped into three clusters and two outliers. Grouping was primarily influenced by soil C:N. In future studies measuring major nutrients in soil, C:N:P, could possibly function as a single, cost effective indicator to validate rapid visual condition assessments of mangroves.

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.

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.

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.

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′).

The effects of large-scale poultry production operations on water quality and human health are largely unknown. Poultry litter is frequently applied as fertilizer to agricultural lands adjacent to large poultry farms. Run-off from the land introduces a variety of stressors into the surface waters including nutrients, antimicrobials and pathogenic bacteria. The Delaware, Maryland and Virginia (Delmarva) Peninsula has the highest concentration of broiler chickens per farm acre in the United States and provides an ideal location for studying the effects of stressors from poultry farms. We investigated potential effects by characterizing shifts in the structure of aquatic bacterial communities. DNA was isolated from microorganisms in water samples from streams and rivers at varying distances from, or having different frequencies of, litter applications. Fingerprints of 16S rDNA amplicons from bacteria in water samples collected during late summer 2001 to late spring 2002 were produced by denaturing gradient gel electrophoresis (DGGE). A statistical analysis of multiple fingerprints from each sampling location demonstrated that each site harboured a bacterial community significantly different from the communities at other sites. Similarly, the bacterial communities from each sampling time differed significantly from communities at other sampling times. Most importantly, a competitive, library-based analysis showed time of sampling (month) had a greater effect on community structure than did location.

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)-2H-tetrazolium (MTS), and phenyl-methasulfazone (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 degrees C in SM-augmented sea water; (2) growout of surviving bacteria in nutrient broth for 2 h at 37 degrees 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. (C) 1999 Elsevier Science B.V. All rights reserved.

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.

Sublethal heat shock of Salmonella typhimurium cells (60 min at 48°C, in 100 mM potassium phosphate buffer, pH 7·2 containing 50 mm EDTA) caused ribosome damage resulting in the formation of a 70S aggregate. This aggregate, lacking 16S ribosomal RNA, was formed by the non-specific binding of 30S ribosomal proteins to the 50S subunit. Several ribosomal proteins were present in reduced amounts on heat damaged ribosomes and in heat shocked cells. Protein synthesis, inhibited by puromycin during cell recovery from heat shock, was required for ribosomal subunit and ribosomal RNA maturation. Ribosomal proteins, preferentially synthesized during cell recovery, were used to reconstruct the damaged ribosome. Ribosomal proteins which had dissociated from the damaged ribosome following heat shock did not fully participate in ribosome repair.