Bioluminescent marine bacteria of the species Vibrio fischeri are the specific light organ symbionts of the sepiolid squid Euprymna scolopes. Although they share morphological and physiological characteristics with other strains of V. fischeri, when cultured away from the light organ association the E. scolopes symbionts depress their maximal luminescence over 1,000-fold. The primary cause of this reduced luminescence is the underproduction by these bacteria of luciferase autoinducer, a molecule involved in the positive transcriptional regulation of the V. fischeri lux operon. Such an absence of visible light production outside of the symbiotic association has not been previously reported among light organ symbionts of this or any other species of luminous bacteria. Levels of luminescence approaching those of the E. scolopes bacteria in the intact association can be restored by the addition of exogenous autoinducer to bacteria in laboratory culture and are affected by the presence of cyclic AMP. We conclude that some condition(s) specffic to the internal environment of the light organ is necessary for maximal autoinduction of luminescence in the symbionts of this squid-bacterial association.The emission of visible light by the marine bacterium Vibriofischeri is dependent upon the enzymatic activity of an inducible luciferase. Its synthesis is regulated at the transcriptional level by a diffusible, sensory autoinducer molecule (7,22,28). The autoinducer, which is believed to be species specific (7,13,22), has been chemically identified as N-(3-oxohexanoyl)homoserine lactone (8). Synthesis of this molecule appears to be continuous (22), and, because it is freely diffusible across the cell membrane, its internal concentration is directly related to the extent of its external accumulation (20). Thus, the autoinducer acts as the sensory component of the autoinduction process, enhancing transcription of the bacterial luminescent system only when V. fischeri cells achieve a high density in a confined environment (19,26,33). This self-regulation of light production results in visible luminescence when the cells exist either as colonies on the surfaces of decaying organic matter or as symbionts within species-specific light-emitting structures of certain marine fishes and cephalopods (13). Conversely, in seawater and other environments where V. fischeri concentrations are rarely more than a few cells per milliliter, autoinducer does not accumulate to a sufficient level for visible luminescence to be expressed (27).The symbiotic luminescent bacteria that are responsible for light emission by the marine sepiolid squid Euprymna scolopes are acquired by the animal early in its development (37). The bacterial culture that develops is maintained in a pair of internal light organs within the mantle cavity (17, 21), where it produces an easily visible luminescence. However, as we demonstrate herein, once removed from the animal, the bacteria become essentially nonluminous. This condition is in marked contrast to the bright lumin...

No 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 ␣-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 ␣-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 ␣-proteobacteria.Juvenile oyster disease (JOD) refers to a syndrome of unknown origin that results in seasonal mortalities of hatcheryproduced juvenile Crassostrea virginica raised in the northeastern United States (9,11,17). While the severity of the annual epizootics has been variable since they first appeared in the late 1980s, mortalities in some years have exceeded 90% of total production at JOD enzootic sites in Maine, Massachusetts, and New York (9,11,39,40). Typical external signs of JOD include a reduction in growth rate, the development of fragile and uneven shell margins, and cupping of the left valve. Internally, signs of JOD usually include mantle retraction and lesions and proteinaceous deposits (conchiolin) on the inner shell surfaces (9,11,17). Such signs usually appear within 4 to 6 weeks after deployment of seed at enzootic sites, and they immediately precede mortality events during which losses may exceed 50% of total production in a single week (3, 7).Several hypotheses concerning the etiology of JOD have been explored, and evidence indicates that the disease is infectious rather than due to nutritional and/or abiotic factors (9). Although no obvious agent has been identified in histological samples (9,15,39,40), the pathology and correlating environmental factors (e.g., warm temperatures and moderate salinity) have led to investigations of a possible bacterial (9, 18, 31) or protistan etiology (14,33,44)...

Vibrio fischeri is the specific light organ symbiont of the sepiolid squid species Euprymna scolopes and Euprymna morsei. Both species of squid are luminescent by virtue of their bacterial symbionts, but the natural symbionts of E. scolopes do not produce visible luminescence in laboratory culture. The primary cause of this depressed luminescence by E. scolopes symbionts in culture was found to be the production of relatively low levels of V. fischeri autoinducer, a positive transcriptional coregulator of the lux regulon, identified as N-(3-oxohexanoyl) homoserine lactone. Concentrations of autoinducer activity produced by these symbionts in culture were quantified and found to be at least 10-fold lower than those produced by E. morsei isolates (which are visibly luminous outside the association) and perhaps 10,000-fold lower than those of the brightest V. fischeri strains. Despite the differences in their symbiont strains, the intact light organs of the two species of squid contained comparable amounts of extractable autoinducer activity (between 100 and 200 pg per adult animal). The chromatographic behavior of this autoinducer activity on reverse-phase high-performance liquid chromatography was consistent with its presumptive identification as V. fischeri autoinducer. Within the 5-l volume of the epithelial core of the light organ in which the symbiotic V. fischeri strains are housed, these amounts would result in an effective autoinducer concentration of at least 100 nM. Because these levels are over 40-fold higher than the concentration needed for the induction of luminescence of bacteria in culture, we conclude that the inherent degree of autoinducer production by strains of V. fischeri may not influence their effectiveness as light organ symbionts. Furthermore, this study provides the first direct evidence that the phenomenon of cell density-dependent autoinduction, discovered and described first for laboratory cultures of V. fischeri but believed to be a general phenomenon in many species of host-associated symbionts and pathogens, is in fact a consequence of bacterial colonizations of host tissue.

Roseovarius crassostreae sp. nov., a member of the Roseobacter clade and the apparent cause of juvenile oyster disease (JOD) in cultured Eastern oysters