Viability of the nonculturable Vibrio cholerae O1 and O139.

Vibrio cholerae is capable of transforming into a viable but nonculturable (VBNC) state, and, in doing so, undergoes alteration in cell morphology. In the study reported here, Vibrio cholerae O1 and O139 cells were maintained in laboratory microcosms prepared with 1% Instant Ocean and incubated at 4 degrees C, i.e., conditions which induce the VBNC state. Cells were fixed at different stages during entry into the VBNC state and, when no growth was detectable on solid or in liquid media, the ultrastructure of these cells was examined, using both transmission and scanning electron microscopy. As shown in earlier studies, the cells became smaller in size and changed from rod to ovoid or coccoid morphology, with the central region of the cells becoming compressed and surrounded by denser cytoplasm. Because the coccoid morphology, indicative of the VBNC state is common for Vibrio cholerae in the natural environment, as well as in starved cells (Baker et al., 1983; Hood et al., 1986) viability of the coccoid, viable but nonculturable cell was investigated. The percentage of coccoid (VBNC) cells showing metabolic activity and retention of membrane integrity was monitored using direct fluorescence staining (LIVE/DEAD BacLight Bacterial Viability kit), with 75 to 90% of the viable but nonculturable coccoid cells found to be metabolically active by this test. Furthermore, the proportion of actively respiring cells, using the redox dye, 5-cyano-2, 3-ditolyl tetrazolium chloride (CTC), relative to total cells, the latter determined by DAPI staining, ranged from 10 to 50%. VBNC coccoid cells retained the antigenic determinants of Vibrio cholerae O1 and O139, respectively, evidenced by positive reaction with monoclonal fluorescent antibody. Viability was further established by susceptibility of the VBNC cells to chlorine, copper sulfate, zinc sulfate, and formaldehyde. Since retention of cell membrane integrity is a determining characteristic of viable cells, DNA was extracted from VBNC cells in microcosms maintained for two months and for one year. Conservation of cholera toxin and toxin-associated genes, ctxA, toxR, tcpA, and zot in chromosomal DNA of VBNC cells was demonstrated using PCR and employing specific primers. It is concluded that not only do VBNC V cholerae O1 and O139 retain viability up to one year, but genes associated with pathogenicity are retained, along with chromosomal integrity.

Idiomarina gen. nov., comprising novel indigenous deep-sea bacteria from the Pacific Ocean, including descriptions of two species, Idiomarina abyssalis sp. nov. and Idiomarina zobellii sp. nov.

Two bacterial strains, KMM 227T and 231T, were isolated from seawater samples collected from the north-western Pacific Ocean at a depth of 4000-5000 m and were characterized using polyphasic taxonomy. Both were Gram-negative, psychrotolerant, heterotrophic, aerobic and required NaCl for growth (0.6-15.0%). The temperature for growth was 4-30 degrees C. Both strains were rod-shaped, with a single flagellum. However, strain KMM 231T revealed a single long fimbrium. Cellular fatty acids detected in the isolates were predominantly odd-numbered and iso-branched, with 15 and 17 carbons (ca. 70%). Also present were saturated and monounsaturated straight-chain fatty acids. Results of phylogenetic analyses, employing three tree-making methods, strongly indicated that the two strains formed a distinct lineage within a clade containing the genera Alteromonas, Colwellia and Pseudoalteromonas, in the gamma-Proteobacteria. The two strains shared 16S rDNA sequence similarity of 96.9% and genomic DNA relatedness of 27%; the latter was determined by dot-blot hybridization. The strains were differentiated by the presence of fimbria, production of chitinase, ability to grow on 15% NaCl and BIOLOG profiles. Given the polyphasic evidence accumulated in this study, it is proposed that the two deep-sea isolates be classified in the genus Idiomarina gen. nov., as Idiomarina abyssalis sp. nov. (type strain is KMM 227T) and Idiomarina zobellii sp. nov. (type strain is KMM 231T).

A new species of Swingleus (Monogenea: Gyrodactylidae) from the mummichog Fundulus heteroclitus, in the Delaware Bay.

Swingleus ancistrus n. sp. (Monogenea: Gyrodactylidae) is described from the skin and fins of Fundulus heteroclitus from Canary Creek Marsh, Lewes, Delaware. Subsequent records from New Jersey, Maryland, Virginia, and North Carolina are reported. Swingleus ancistrus is differentiated from S. polyclithroides, the only other species of the genus, primarily by its size, haptoral morphology, host, and locality. Swingleus ancistrus is larger in almost every dimension and exhibits a distinct notch on the anterior border of the opisthaptor, and several features of the haptoral anatomy and peduncular bar are unique. The prevalence of infection in hosts collected from the type locality in August was 100%. The maximum number of S. ancistrus recovered from a single host was 134. The average intensity of infection was 24 S. ancistrus per host from the type locality.

Polyphasic taxonomy of a novel Halobacillus, Halobacillus thailandensis sp. nov. isolated from fish sauce.

In order to speed up fish sauce production, a more complete understanding of the microorganisms associated with the fermentation was needed. This study was undertaken to meet that need. A bacterium was isolated from a fish sauce production line containing 25% NaCl. It is a Gram-positive, rod-shaped bacillus with pointed ends, occurring as single cells, pairs, or short chains. Endospores are produced on a low nutrient medium and, in old cultures, the cells round up, even when undergoing division. The cell wall is relatively amorphous and similar to that of Gram-positive bacteria in structure and composition. Cells grown in a medium containing 10-20% salt possess thicker cell walls than those grown in a medium with 3% salt. Based on 16S rRNA sequence and DNA/DNA hybridization data, we conclude that the bacterium is a species of Halobacillus. This bacterium shares 99.2% and 97.2% 16S rRNA similarity with Halobacillus litoralis and Halobacillus halophilus respectively and DNA/DNA homology was lower than 70%, considered indicative of species similarity. Three highly expressed extra-cellular proteolytic enzymes with M(r) of approximately 100 kDa, 42 kDa and 17 kDa, respectively, were detected by gelatin-polyacrylamide gel electrophoresis. Activity of the 100 kDa and 17 kDa proteases was inhibited by phenylmethanesulphonyl fluoride (PMSF), without being affected by L-trans epoxysuccinyl-leucylamide 4-guanidino-butane (E-64), pepstatin, EDTA, or 1, 10-phenanthroline, leading to the conclusion that these enzymes are serine proteases. The 42-kDa protease was inhibited by EDTA and 1,10-phenanthroline, but not by PMSF, thus, being classified a metalloprotease. The strain has been successfully employed to improve fermentation in industrial production of fish sauce in Thailand.

Annual Viral Expression in a Sea Slug Population: Life Cycle Control and Symbiotic Chloroplast Maintenance.

In a few well-known cases, animal population dynamics are regulated by cyclical infections of protists, bacteria, or viruses. In most of these cases, the pathogen persists in the environment, where it continues to infect some percentage of successive generations of the host organism. This persistent re-infection causes a long-lived decline, in either population size or cycle, to a level that depends upon pathogen density and infection level (1-4). We have discovered, on the basis of 9 years of observation, an annual viral expression in Elysia chlorotica, an ascoglossan sea slug, that coincides with the yearly, synchronized death of all the adults in the population. This coincidence of viral expression and mass death is ubiquitous, and it occurs in the laboratory as well as in the field. Our evidence also suggests that the viruses do not re-infect subsequent generations from an external pathogen pool, but are endogenous to the slug. We are led, finally, to the hypothesis that the viruses may be involved in the maintenance of symbiotic chloroplasts within the molluscan cells.

On the Giant Octopus (Octopus giganteus) and the Bermuda Blob: Homage to A. E. Verrill.

We have obtained samples of two large carcasses. One washed up on a beach in St. Augustine, Florida, in 1896 and has been occasionally attributed to a species of gigantic octopus (Octopus giganteus). The other carcass washed up on Bermuda in 1988 and has remained unidentified, although its gross morphology, except for a much smaller total mass, was remarkably similar to the Florida carcass. We have subjected both samples to electron microscopic and biochemical analyses. Our results show that both carcasses are masses of virtually pure collagen. Furthermore, neither sample has the biochemical characteristics of invertebrate collagen, nor the collagen fiber arrangement of octopus mantle. Instead, they are large pieces of vertebrate skin, the Bermuda sample from a poikilotherm and the Florida sample from a huge homiotherm. We conclude that there is no evidence to support the existence of Octopus giganteus.

Morphological correlate of regional partitioning of integumental water absorption in terrestrial slugs.

An ultrastructural study of the foot surface of the terrestrial mollusc, Limax maximus, has revealed a correlation of epithelial cell type with the functional partitioning of the surface. The lateral absorptive bands of the foot are comprised exclusively of microvillar epithelial cells, while those of the medial locomotor band are all ciliated. Thus, there is a clear partitioning of epithelial cell types between areas of the foot surface with distinct functional roles. Consistent with the proposed role for paracellular absorption, varying states of hydration are shown to affect the extent of the intercellular spaces, but not the intracellular architecture.

Pressure-induced depolymerization of spindle microtubules. III. Differential stability in HeLa cells.

Evidence from light microscopy (principally polarization microscopy) has demonstrated that hydrostatic pressure can reversibly inhibit mitosis by rapidly depolymerizing the spindle fiber microtubules. We have confirmed this finding in ultrastructural studies of mitotic HeLa cells incubated at 37 degrees C and pressurized at 680 atm (10,000 psi). Althouth there are many spindle microtubules in the cells at atmospheric pressure, electron micographs of cells pressurized for 10 min (and fixed while under pressure in a Landau-Thibodeau chamber) show few microtubules. Pressure has a differential effect on the various types of spindle microtubules. Astral and interpolar MTs appear to be completely depolymerized in pressurized cells, but occasional groups of kinetochore fiber microtubules are seen. Surprisingly, the length and density of microtubules of the stem bodies and midbody of telophase cells appear unchanged by pressurization. In cells fixed 10 min after pressure was released, microtubules were again abundant, the density often appearing to be higher than in control cells. Reorganization seems incomplete, however, since many of the microtubules are randomly oriented. Unexpectedly, kinetochores appeared diffuse and were difficult to identify in sections of pressurized cells. Even after 10 min of recovery at atmospheric pressure, their structure was less distinct than in unpressurized cells.