John T. Singer

Additional Evidence that Juvenile Oyster Disease Is Caused by a Member of the Roseobacter Group and Colonization of Nonaffected Animals by Stappia stellulata-Like Strains

ABSTRACT Juvenile oyster disease (JOD) causes significant annual mortalities of hatchery-produced Eastern oysters, Crassostrea virginica, cultured in the Northeast. We have reported that a novel species of the α-proteobacteria Roseobacter group (designated CVSP) was numerically dominant in JOD-affected animals sampled during the 1997 epizootic on the Damariscotta River, Maine. In this study we report the isolation of CVSP bacteria from JOD-affected oysters during three separate epizootics in 1998. These bacteria were not detected in nonaffected oysters at the enzootic site, nor in animals raised at a JOD-free site. Animals raised at the JOD enzootic site that were unaffected by JOD were stably and persistently colonized by Stappia stellulata-like strains. These isolates (designated M1) inhibited the growth of CVSP bacteria in a disk-diffusion assay and thus may have prevented colonization of these animals by CVSP bacteria in situ. Laboratory-maintained C. virginica injected with CVSP bacteria experienced statistically significant elevated mortalities compared to controls, and CVSP bacteria were recovered from these animals during the mortality events. Together, these results provide additional evidence that CVSP bacteria are the etiological agent of JOD. Further, there are no other descriptions of specific marine α-proteobacteria that have been successfully cultivated from a defined animal host. Thus, this system presents an opportunity to investigate both bacterial and host factors involved in the establishment of such associations and the role of the invertebrate host in the ecology of these marine α-proteobacteria.

Broad-Host-Range Plasmids for Red Fluorescent Protein Labeling of Gram-Negative Bacteria for Use in the Zebrafish Model System

ABSTRACT To observe real-time interactions between green fluorescent protein-labeled immune cells and invading bacteria in the zebrafish (Danio rerio), a series of plasmids was constructed for the red fluorescent protein (RFP) labeling of a variety of fish and human pathogens. The aim of this study was to create a collection of plasmids that would express RFP pigments both constitutively and under tac promoter regulation and that would be nontoxic and broadly transmissible to a variety of Gram-negative bacteria. DNA fragments encoding the RFP dimeric (d), monomeric (m), and tandem dimeric (td) derivatives d-Tomato, td-Tomato, m-Orange, and m-Cherry were cloned into the IncQ-based vector pMMB66EH in Escherichia coli. Plasmids were mobilized into recipient strains by conjugal mating. Pigment production was inducible in Escherichia coli, Pseudomonas aeruginosa, Edwardsiella tarda, and Vibrio (Listonella) anguillarum strains by isopropyl-β-d-thiogalactopyranoside (IPTG) treatment. A spontaneous mutant exconjugant of P. aeruginosa PA14 was isolated that expressed td-Tomato constitutively. Complementation analysis revealed that the constitutive phenotype likely was due to a mutation in lacIq carried on pMMB66EH. DNA sequence analysis confirmed the presence of five transitions, four transversions, and a 2-bp addition within a 14-bp region of lacI. Vector DNA was purified from this constitutive mutant, and structural DNA sequences for RFP pigments were cloned into the constitutive vector. Exconjugants of P. aeruginosa, E. tarda, and V. anguillarum expressed all pigments in an IPTG-independent fashion. Results from zebrafish infectivity studies indicate that RFP-labeled pathogens will be useful for the study of real-time interactions between host cells of the innate immune system and the infecting pathogen.

Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta)

Significance Fossil evidence shows that red algae (Rhodophyta) are one of the most ancient multicellular lineages. Their ecological, evolutionary, and commercial importance notwithstanding, few red algal nuclear genomes have been sequenced. Our analyses of the Porphyra umbilicalis genome provide insights into how this macrophyte thrives in the stressful intertidal zone and into the basis for its nutritional value as human food. Many of the novel traits (e.g., cytoskeletal organization, calcium signaling pathways) we find encoded in the Porphyra genome are extended to other red algal genomes, and our unexpected findings offer a potential explanation for why the red algae are constrained to small stature relative to other multicellular lineages. Porphyra umbilicalis (laver) belongs to an ancient group of red algae (Bangiophyceae), is harvested for human food, and thrives in the harsh conditions of the upper intertidal zone. Here we present the 87.7-Mbp haploid Porphyra genome (65.8% G + C content, 13,125 gene loci) and elucidate traits that inform our understanding of the biology of red algae as one of the few multicellular eukaryotic lineages. Novel features of the Porphyra genome shared by other red algae relate to the cytoskeleton, calcium signaling, the cell cycle, and stress-tolerance mechanisms including photoprotection. Cytoskeletal motor proteins in Porphyra are restricted to a small set of kinesins that appear to be the only universal cytoskeletal motors within the red algae. Dynein motors are absent, and most red algae, including Porphyra, lack myosin. This surprisingly minimal cytoskeleton offers a potential explanation for why red algal cells and multicellular structures are more limited in size than in most multicellular lineages. Additional discoveries further relating to the stress tolerance of bangiophytes include ancestral enzymes for sulfation of the hydrophilic galactan-rich cell wall, evidence for mannan synthesis that originated before the divergence of green and red algae, and a high capacity for nutrient uptake. Our analyses provide a comprehensive understanding of the red algae, which are both commercially important and have played a major role in the evolution of other algal groups through secondary endosymbioses.

Use of a restriction-defective variant for the construction of stable attenuated strains of the marine fish pathogen Vibrio anguillarum

Abstract A method for the in vitro construction of stable attenuated strains of the marine fish pathogen Vibrio anguillarum 775 is described. A cloned gene for a membrane-associated polypeptide, p40, that is required for virulence of V. anguillarum , was inactivated by insertion of a 1.5-kb kan fragment. The mutagenized p40- kan DNA sequence was introduced into a restriction-defective recipient strain of virulent V. anguillarum 775 by conjugal mobilization of a pBR322-p40- kan derivative from an Escherichia coli HB101 donor. A homologous recombinant of V. anguillarum 775 was selected that lost pBR322 sequences but that retained p40- kan DNA sequences. This strain was > 10 5 -fold attenuated in virulence for rainbow trout, expressed a surface-exposed outer membrane protein, pOM2, that is also known to be required for virulence, and persisted in immunized fish for at least 9 days post-injection.

Cytotoxicity and genotoxicity of wood drying condensate from Southern Yellow Pine: an in vitro study

We tested condensates from Southern Yellow Pine for potential cytotoxicity and genotoxicity in CHO-WBL and human peripheral blood lymphocytes (PBL) in the absence of S-9 activation. Cytotoxicity was evaluated by the Trypan blue exclusion assay, mitotic index (MI) and proliferative rate index (PRI). Genotoxicity was measured by the chromosome aberration (CA) assay and sister chromatid exchange (SCE) analysis. Both cytotoxic and genotoxic effects were observed. Laboratory-generated Southern Yellow Pine condensate reduced the viability of CHO-WBL cells. The number of viable cells was roughly inversely proportional to dosage over a range of 100% to 31% in treated groups, in both experiments, as compared to 2.6 x 10(5) (100%) in the control. The MI data in both CHO cells and PBL also showed an inverse correlation. The highest scorable dose limited by toxicity was determined to be 1 ml of Southern Yellow Pine condensate in 10 ml total of medium. Lastly, a dose response curve was observed in CHO cells, as well as in PBL, using the CA assay and also with the SCE analysis. The present findings corroborate the results from Ames testing and represent the only information currently available on the genotoxic potential of these chemicals.

Virulence plasmid pJM1 prevents the conjugal entry of plasmid DNA into the marine fish pathogen Vibrio anguillarum 775.

Studies involving the introduction of cloned homologous genes into Vibrio anguillarum revealed that several plasmids could not be conjugally introduced into V. anguillarum 775(pJM1), but were transmissible to the pJM1-cured derivative H775-3. Recombinant pBR322 plasmids containing V. anguillarum genomic DNA inserts were mobilized from Escherichia coli donors, using pRK2013, into V. anguillarum H775-3 recipients at frequencies of 10(-6) to 10(-5) per recipient. When identical matings were performed with V. anguillarum 775(pJM1) recipients, the infrequent exconjugants recovered carried the pBR322-based plasmid but had lost the large virulence plasmid pJM1. Similar studies were carried out with plasmid RP4 and with recombinant derivatives of the closely related broad-host-range plasmid pRK290. While RP4 was transmissible from E. coli to V. anguillarum H775-3 at frequencies of 6.7 x 10(-2) per recipient, transmission to V. anguillarum 775(pJM1) recipients occurred at frequencies of only 2.5 x 10(-7). When pRK290 contained V. anguillarum DNA inserts, the only exconjugants recovered had lost pJM1, or contained pJM1 and a deletion derivative of the recombinant pRK290 plasmid where all of the DNA insert had been deleted. The use of Dam-, Dcm-, or EcoK- methylation-deficient E. coli donor strains failed to result in appreciable numbers of V. anguillarum 775(pJM1) exconjugants that contained the desired transferred plasmids. Following UV mutagenesis, a derivative of V. anguillarum 775(pJM1) was isolated that would accept conjugally transferred plasmid DNAs at frequencies similar to those observed when using V. anguillarum H775-3 recipients. These data suggest that virulence plasmid pJM1 mediates a restriction system that prevents conjugal transmission of plasmid DNA from E. coli donors into V. anguillarum 775(pJM1). This putative restriction system appears not to be directed towards Dam-, Dcm-, or EcoK-methylated DNA, and appears not to involve a Type II restriction endonuclease.

Expression of Capsid Proteins from Infectious Pancreatic Necrosis Virus (IPNV) in the Marine Bacterium Vibrio Anguillarum

V. anguillarum is a marine fish pathogen with serotype O1 strains comprising the major disease-causing serotype (Crosa et al., 1980). Two components of a high-affinity iron uptake system encoded by the virulence plasmid pJM1 are the secreted siderophore, anguibactin, and the outer membrane protein receptor for iron-siderophore complexes, OM2. Because formalin-inactivated serotype O1 strains are used commercially as vaccines for the prevention of vibriosis in salmonids, we explored the use of V. anguillarum as an expression host for protective epitopes of the major capsid protein VP2 from infectious pancreatic necrosis virus (IPNV). IPNV causes primarily a disease of salmonid fry, but survivors become life-long carriers and serve as reservoirs of future infection (Dobos & Roberts, 1983). Segment A of the bisegmented dsRNA IPNV genome is 2.9 kb and is translated from a single open reading frame into a polyprotein which is subsequently cleaved into three proteins, VP2, NS and VP3 (Huang et al., 1986). The major capsid protein of the virus, VP2 (59 kDa), contains most of the neutralization epitopes (Caswell-Reno et al., 1986). NS is the non-structural polyprotein protease and VP3 is a minor structural protein. We show here that IPNV cDNA in a translational fusion with V. anguillarum DNA results in production of IPNV and OM2 hybrid proteins expressed in V. anguillarum.

Microbiological methods for the detection of mutagens and cytotoxic components in wood drying condensates from Douglas fir, red oak, southern yellow pine, yellow poplar and eastern white pine

Abstract Three microbiological methods of testing for genotoxic chemicals in uncharacterized wood drying condensates were compared. A commercially successful version of the SOS Chromotest, carried out in microtiter plates, was compared with the original assay that was modified to be performed in soft agar overlays. While the microtiter plate version was quantitative and could be read by an automated microtiter plate reader, the soft agar overlay adaptation was at least six times more sensitive with control chemicals, and was more easily carried out. Each version of the test was able to detect cytotoxic effects of wood drying condensates, but neither test was sensitive enough to routinely detect unknown mutagenic components present in wood drying condensates. The Ames Salmonela/ microsome assay, while more laborious, revealed cytotoxic dose-response relationships with wood drying condensates from yellow poplar, Douglas fir, white pine, and southern yellow pine; but none of the condensates was mutagenic in the absence of metabolic activation by mammalian liver enzymes. In the presence of rat liver S-9 extracts, yellow poplar condensate resulted in the highest single mutagenic response, yielding over 5,300 revertants/ml of condensate, but was mutagenic for only one of the four Ames test strains. Douglas fir and white pine condensates were mutagenic for three of four Ames strains, and exhibited significant cytotoxicity in the presence and absence of S-9 extract. Southern yellow pine condensate was mutagenic for two of the four Ames strains, but was far less mutagenic and cytotoxic than condensates from Douglas fir or white pine. Condensate from red oak was cytotoxic, but not mutagenic. The failure of the SOS Chromotest to detect mutagenic components in wood drying condensates was unexpected since over 80% of all compounds tested thus far give similar results in both the Ames test and SOS Chromotest. It appears that mutagenic components present in wood drying condensates are poor inducers of DNA repair, and are not detected by the SOS Chromotest or similar test that rely on SOS induction.