Detection of the Light Organ Symbiont, Vibrio fischeri, in Hawaiian Seawater by Using lux Gene Probes

Symbiotic bacteria that inhabit the light-emitting organ of the Hawaiian squid Euprymna scolopes are distinctive from typical Vibrio fischeri organisms in that they are not visibly luminous when grown in laboratory culture. Therefore, the abundance of these bacteria in seawater samples cannot be estimated simply by identifying them among luminous colonies that arise on nutrient agar plates. Instead, we have used luxR and polymerase chain reaction generated luxA gene probes to identify both luminous and non-visibly luminous V. fischeri colonies by DNA-DNA hybridization. The probes were specific, hybridizing at least 50 to 100 times more strongly to immobilized DNAs from V. fischeri strains than to those of pure cultures of other related species. Thus, even non-visibly luminous V. fischeri colonies could be identified among colonies obtained from natural seawater samples by their probe-positive reaction. Bacteria in seawater samples, obtained either within or distant from squid habitats, were collected on membrane filters and incubated until colonies appeared. The filters were then observed for visibly luminous V. fischeri colonies and hybridized with the lux gene probes to determine the number of total V. fischeri colonies (both luminous and non-visibly luminous). We detected no significant differences in the abundance of luminous V. fischeri CFU in any of the water samples observed (≤1 to 3 CFU/100 ml). However, probe-positive colonies of V. fischeri (up to 900 CFU/100 ml) were found only in seawater collected from within the natural habitats of the squids. A number of criteria were used to confirm that these probe-positive strains were indistinguishable from symbiotic V. fischeri. Therefore, the luxA and luxR gene probes were species specific and gave a reliable estimate of the number of culturable V. fischeri colonies in natural water samples.     

Dominance of Vibrio fischeri in Secreted Mucus outside the Light Organ of Euprymna scolopes: the First Site of Symbiont Specificity

Previous studies of the Euprymna scolopes-Vibrio fischeri symbiosis have demonstrated that, during colonization, the hatchling host secretes mucus in which gram-negative environmental bacteria amass in dense aggregations outside the sites of infection. In this study, experiments with green fluorescent protein-labeled symbiotic and nonsymbiotic species of gram-negative bacteria were used to characterize the behavior of cells in the aggregates. When hatchling animals were exposed to 10³ to 10⁶ V. fischeri cells/ml added to natural seawater, which contains a mix of approximately 10⁶ nonspecific bacterial cells/ml, V. fischeri cells were the principal bacterial cells present in the aggregations. Furthermore, when animals were exposed to equal cell numbers of V. fischeri (either a motile or a nonmotile strain) and either Vibrio parahaemolyticus or Photobacterium leiognathi, phylogenetically related gram-negative bacteria that also occur in the host's habitat, the symbiont cells were dominant in the aggregations. The presence of V. fischeri did not compromise the viability of these other species in the aggregations, and no significant growth of V. fischeri cells was detected. These findings suggested that dominance results from the ability of V. fischeri either to accumulate or to be retained more effectively within the mucus. Viability of the V. fischeri cells was required for both the formation of tight aggregates and their dominance in the mucus. Neither of the V. fischeri quorum-sensing compounds accumulated in the aggregations, which suggested that the effects of these small signal molecules are not critical to V. fischeri dominance. Taken together, these data provide evidence that the specificity of the squid-vibrio symbiosis begins early in the interaction, in the mucus where the symbionts aggregate outside of the light organ.     

Roles of Vibrio fischeri and Nonsymbiotic Bacteria in the Dynamics of Mucus Secretion during Symbiont Colonization of the Euprymna scolopes Light Organ

During light organ colonization of the squid Euprymna scolopes by Vibrio fischeri, host-derived mucus provides a surface upon which environmental V. fischeri forms a biofilm and aggregates prior to colonization. In this study we defined the temporal and spatial characteristics of this process. Although permanent colonization is specific to certain strains of V. fischeri, confocal microscopy analyses revealed that light organ crypt spaces took up nonspecific bacteria and particles that were less than 2 μm in diameter during the first hour after hatching. However, within 2 h after inoculation, these cells or particles were not detectable, and further entry by nonspecific bacteria or particles appeared to be blocked. Exposure to environmental gram-negative or -positive bacteria or bacterial peptidoglycan caused the cells of the organ's superficial ciliated epithelium to release dense mucin stores at 1 to 2 h after hatching that were used to form the substrate upon which V. fischeri formed a biofilm and aggregated. Whereas the uncolonized organ surface continued to shed mucus, within 48 h of symbiont colonization mucus shedding ceased and the formation of bacterial aggregations was no longer observed. Eliminating the symbiont from the crypts with antibiotics restored the ability of the ciliated fields to secrete mucus and aggregate bacteria. While colonization by V. fischeri inhibited mucus secretion by the surface epithelium, secretion of host-derived mucus was induced in the crypt spaces. Together, these data indicate that although initiation of mucus secretion from the superficial epithelium is nonspecific, the inhibition of mucus secretion in these cells and the concomitant induction of secretion in the crypt cells are specific to natural colonization by V. fischeri.     

Alterations in the Proteome of the Euprymna scolopes Light Organ in Response to Symbiotic Vibrio fischeri

During the onset of the cooperative association between the Hawaiian sepiolid squid Euprymna scolopes and the marine luminous bacterium Vibrio fischeri, the anatomy and morphology of the host's symbiotic organ undergo dramatic changes that require interaction with the bacteria. This morphogenetic process involves an array of tissues, including those in direct contact with, as well as those remote from, the symbiotic bacteria. The bacteria induce the developmental program soon after colonization of the organ, although complete morphogenesis requires 96 h. In this study, to determine critical time points, we examined the biochemistry underlying bacterium-induced host development using two-dimensional polyacrylamide gel electrophoresis. Specifically, V. fischeri-induced changes in the soluble proteome of the symbiotic organ during the first 96 h of symbiosis were identified by comparing the protein profiles of symbiont-colonized and uncolonized organs. Both symbiosis-related changes and age-related changes were analyzed to determine what proportion of the differences in the proteomes was the result of specific responses to interaction with bacteria. Although no differences were detected over the first 24 h, numerous symbiosis-related changes became apparent at 48 and 96 h and were more abundant than age-related changes. In addition, many age-related protein changes occurred 48 h sooner in symbiotic animals, suggesting that the interaction of squid tissue with V. fischeri cells accelerates certain developmental processes of the symbiotic organ. These data suggest that V. fischeri-induced modifications in host tissues that occur in the first 24 h of the symbiosis are independent of marked alterations in the patterns of abundant proteins but that the full 4-day morphogenetic program requires significant alteration of the host soluble proteome.     

A New Niche for Vibrio logei, the Predominant Light Organ Symbiont of Squids in the Genus Sepiola

Two genera of sepiolid squids—Euprymna, found primarily in shallow, coastal waters of Hawaii and the Western Pacific, and Sepiola, the deeper-, colder-water-dwelling Mediterranean and Atlantic squids—are known to recruit luminous bacteria into light organ symbioses. The light organ symbiont of Euprymna spp. is Vibrio fischeri, but until now, the light organ symbionts of Sepiola spp. have remained inadequately identified. We used a combination of molecular and physiological characteristics to reveal that the light organs of Sepiola affinis and Sepiola robusta contain a mixed population of Vibrio logei and V. fischeri, with V. logei comprising between 63 and 100% of the bacteria in the light organs that we analyzed. V. logei had not previously been known to exist in such symbioses. In addition, this is the first report of two different species of luminous bacteria co-occurring within a single light organ. The luminescence of these symbiotic V. logei strains, as well as that of other isolates of V. logei tested, is reduced when they are grown at temperatures above 20°C, partly due to a limitation in the synthesis of aliphatic aldehyde, a substrate of the luminescence reaction. In contrast, the luminescence of the V. fischeri symbionts is optimal above 24°C and is not enhanced by aldehyde addition. Also, V. fischeri strains were markedly more successful than V. logei at colonizing the light organs of juvenile Euprymna scolopes, especially at 26°C. These findings have important implications for our understanding of the ecological dynamics and evolution of cooperative, and perhaps pathogenic, associations of Vibrio spp. with their animal hosts.     

The Iron-Dependent Regulator Fur Controls Pheromone Signaling Systems and Luminescence in the Squid Symbiont Vibrio fischeri ES114

Bacteria often use pheromones to coordinate group behaviors in specific environments. While high cell density is required for pheromones to achieve stimulatory levels, environmental cues can also influence pheromone accumulation and signaling. For the squid symbiont Vibrio fischeri ES114, bioluminescence requires pheromone-mediated regulation, and this signaling is induced in the host to a greater extent than in culture, even at an equivalent cell density. Our goal is to better understand this environment-specific control over pheromone signaling and bioluminescence. Previous work with V. fischeri MJ1 showed that iron limitation induces luminescence, and we recently found that ES114 encounters a low-iron environment in its host. Here we show that ES114 induces luminescence at lower cell density and achieves brighter luminescence in low-iron media. This iron-dependent effect on luminescence required ferric uptake regulator (Fur), which we propose influences two pheromone signaling master regulators, LitR and LuxR. Genetic and bioinformatic analyses suggested that under low-iron conditions, Fur-mediated repression of litR is relieved, enabling more LitR to perform its established role as an activator of luxR. Interestingly, Fur may similarly control the LitR homolog SmcR of Vibrio vulnificus. These results reveal an intriguing regulatory link between low-iron conditions, which are often encountered in host tissues, and pheromone-dependent master regulators.     

Characterization of pES213, a small mobilizable plasmid from Vibrio fischeri

Most Vibrio fischeri strains isolated from the Euprymna scolopes light organ carry plasmids, often including both a large (>40kb) plasmid, and one or more small (<12kb) plasmids. The large plasmids share homology with pES100, which is the lone plasmid in V. fischeri type strain ES114. pES100 appears to encode a conjugative system similar to that on plasmid R721. The small plasmids lack extensive similarity to pES100, but they almost always occur in cells that also harbor a large plasmid resembling pES100. We found that many or all of these small plasmids share homology with pES213, a plasmid in strain ES213. We determined the 5501-bp pES213 sequence and generated selectable antibiotic resistance encoding pES213 derivatives, which enabled us to examine replication, retention, and transfer in V. fischeri. An 863-bp fragment of pES213 with features characteristic of theta-type replicons conferred replication without requiring any pES213 open reading frame (ORF). We estimated that pES213 derivatives were maintained at 9.4 copies per genome, which corresponds well with a model of random plasmid segregation to daughter cells and the approximately 10(-4) per generation frequency of plasmid loss. pES213 derivatives mobilized between V. fischeri strains at frequencies up to approximately 10(-4) in culture and in the host, apparently by employing the pES100 conjugative apparatus. pES213 carries two homologs of the putative pES100 origin of transfer (oriT), and V. fischeri strains lacking the pES100 conjugative relaxase, including a relaxase mutant, failed to serve as donors for transmission of pES213 derivatives. In other systems, genes directing conjugative transfer can function in trans to oriT, so it was noteworthy that ORFs adjacent to oriT, VFB51 in pES100 and traYZ in pES213, enhanced transfer 100- to 1000-fold when provided in cis. We also identified and disrupted the V. fischeri recA gene. RecA was not required for stable pES213 replication but surprisingly was required in donors for efficient transfer of pES213 derivatives. These studies provide an explanation for the prevalence and co-occurrence of pES100- and pES213-type plasmids, illuminate novel elements of pES213 mobilization, and provide the foundation for new genetic tools in V. fischeri.     

Characterization of Vibrio fischeri rRNA operons and subcloning of a ribosomal DNA promoter.

Analysis of rRNA genes in Vibrio fischeri indicates the presence of eight rRNA gene sets in this organism. It was found that the genes for 5S rRNA, 16S rRNA, and 23S rRNA are organized in operons in the following order: 5' end 16S rRNA 23S RNA 5S rRNA 3' end. Although the operons are homologous, they are not identical with regard to cleavage sites for various restriction endonucleases. A DNA library was constructed, and three ribosomal DNA clones were obtained. One of these clones contained an entire rRNA operon and was used as a source for subcloning. The promoter region which leads to plasmid instability was successfully subcloned into pHG165. The terminator region was subcloned into pBR322.     

Magnesium promotes flagellation of Vibrio fischeri

The bacterium Vibrio fischeri requires bacterial motility to initiate colonization of the Hawaiian squid Euprymna scolopes. Once colonized, however, the bacterial population becomes largely unflagellated. To understand environmental influences on V. fischeri motility, we investigated migration of this organism in tryptone-based soft agar media supplemented with different salts. We found that optimal migration required divalent cations and, in particular, Mg2+. At concentrations naturally present in seawater, Mg2+ improved migration without altering the growth rate of the cells. Transmission electron microscopy and Western blot experiments suggested that Mg2+ addition enhanced flagellation, at least in part through an effect on the steady-state levels of flagellin protein.     

Entry of Vibrio harveyi and Vibrio fischeri into the viable but nonculturable state

AIMS: Physiological responses of marine luminous bacteria, Vibrio harveyi (ATCC 14216) and V. fischeri (UM1373) to nutrient-limited normal strength (35 ppt iso-osmolarity) and low (10 ppt hypo-osmolarity) salinity conditions were determined. METHODS AND RESULTS: Plate counts, direct viable counts, actively respiring cell counts, nucleoid-containing cell counts, and total counts were determined. Vibrio harveyi incubated at 22 degrees C in nutrient-limited artificial seawater (ASW) became nonculturable after approximately 62 and 45 d in microcosms of 35 ppt and 10 ppt ASW, respectively. In contrast, V. fischeri became nonculturable at approximately 55 and 31 d in similar microcosms. Recovery of both culturability and luminescence of cells in the viable but nonculturable state was achieved by addition of nutrient broth or nutrient broth supplemented with a carbon source, including luminescence-stimulating compounds. Temperature upshift from 22 degrees C to 30 degrees C or 37 degrees C did not result in recovery from nonculturability. CONCLUSIONS: The study confirms entry of V. harveyi and V. fischeri into the viable but nonculturable state under low-nutrient conditions and demonstrates nutrient-dependent resuscitation from this state. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms loss of luminescence of V. harveyi and V. fischeri on entry into the viable but nonculturable state and suggests that enumeration of luminescent cells in water samples may be a rapid method to deduce the nutrient status of a water sample.     

Natural transformation of Vibrio fischeri requires tfoX and tfoY

Recent evidence has indicated that natural genetic transformation occurs in Vibrio cholerae, and that it requires both induction by chitin oligosaccharides, like chitohexaose, and expression of a putative regulatory gene designated tfoX. Using sequence and phylogenetic analyses we have found two tfoX paralogues in all sequenced genomes of the genus Vibrio. Like V. cholerae, when grown in chitohexaose, cells of V. fischeri are able to take up and incorporate exogenous DNA. Chitohexaose-independent transformation by V. fischeri was observed when tfoX was present in multicopy. The second tfoX paralogue, designated tfoY, is also required for efficient transformation in V. fischeri, but is not functionally identical to tfoX. Natural transformation of V. fischeri facilitates rapid transfer of mutations across strains, and provides a highly useful tool for experimental genetic manipulation in this species. The presence of chitin-induced competence in several vibrios highlights the potential for a conserved mechanism of genetic exchange across this family of environmentally important marine bacteria.     

Recognition between symbiotic Vibrio fischeri and the haemocytes of Euprymna scolopes

The light organ crypts of the squid Euprymna scolopes permit colonization exclusively by the luminous bacterium Vibrio fischeri. Because the crypt interior remains in contact with seawater, the squid must not only foster the specific symbiosis, but also continue to exclude other bacteria. Investigation of the role of the innate immune system in these processes revealed that macrophage-like haemocytes isolated from E. scolopes recognized and phagocytosed V. fischeri less than other closely related bacterial species common to the host's environment. Interestingly, phagocytes isolated from hosts that had been cured of their symbionts bound five times more V. fischeri cells than those from uncured hosts. No such change in the ability to bind other species of bacteria was observed, suggesting that the host adapts specifically to V. fischeri . Deletion of the gene encoding OmpU, the major outer membrane protein of V. fischeri , increased binding by haemocytes from uncured animals to the level observed for haemocytes from cured animals. Co-incubation with wild-type V. fischeri reduced this binding, suggesting that they produce a factor that complements the mutant's defect. Analyses of the phagocytosis of bound cells by fluorescence-activated cell sorting indicated that once binding to haemocytes had occurred, V. fischeri cells are phagocytosed as effectively as other bacteria. Thus, discrimination by this component of the squid immune system occurs at the level of haemocyte binding, and this response: (i) is modified by previous exposure to the symbiont and (ii) relies on outer membrane and/or secreted components of the symbionts. These data suggest that regulation of host haemocyte binding by the symbiont may be one of many factors that contribute to specificity in this association.     

Expression of luciferases from Vibrio harveyi and Vibrio fischeri in filamentous cyanobacteria.

Shuttle vectors that had previously been shown to replicate both in Escherichia coli and in strains of Anabaena spp. were used to transfer the lux genes from Vibrio harveyi and Vibrio fischeri into Anabaena spp. The level of expression of luciferase in the cyanobacteria (up to 7,000 quanta cell-1 s-1) makes these genes good candidates for use as promoter probes during the differentiation of certain cells in a filament into heterocysts.     

Role for cheR of Vibrio fischeri in the Vibrio-squid symbiosis

Upon hatching, the Hawaiian squid Euprymna scolopes is rapidly colonized by its symbiotic partner, the bioluminescent marine bacterium Vibrio fischeri . Vibrio fischeri cells present in the seawater enter the light organ of juvenile squid in a process that requires bacterial motility. In this study, we investigated the role chemotaxis may play in establishing this symbiotic colonization. Previously, we reported that V.?fischeri migrates toward numerous attractants, including N-acetylneuraminic acid (NANA), a component of squid mucus. However, whether or not migration toward an attractant such as squid-derived NANA helps the bacterium to localize toward the light organ is unknown. When tested for the ability to colonize juvenile squid, a V. fischeri chemotaxis mutant defective for the methyltransferase CheR was outcompeted by the wild-type strain in co-inoculation experiments, even when the mutant was present in fourfold excess. Our results suggest that the ability to perform chemotaxis is an advantage during colonization, but not essential.     

Aspects of Light Production by Photobacterium fischeri

Studies of luminescence in growing cultures of Photobacterium fischeri revealed the characteristic kinetics of light emission, including a minimal phase of bacterial light output. A dialyzable substance present in the nutrient broth medium caused this transient inhibition in light production, although this substance did not affect culture growth. Experiments were carried out to determine the mechanism of action and the chemical properties of the inhibitor. The results suggest that the inhibitor may be binding directly to the luciferase molecules.     

Characterization of a Vibrio fischeri aminopeptidase and evidence for its influence on an early stage of squid colonization

Vibrio fischeri cells are the sole colonists of a specialized light organ in the mantle cavity of the sepiolid squid Euprymna scolopes. The process begins when the bacteria aggregate in mucus secretions outside the light organ. The cells eventually leave the aggregate, enter the light organ, and encounter a rich supply of peptides. The need to dissociate from mucus and presumably utilize peptides led us to hypothesize that protease activity is integral to the colonization process. Protease activity associated with whole cells of Vibrio fischeri strain ES114 was identified as the product of a putative cell membrane-associated aminopeptidase (PepN). To characterize this activity, the aminopeptidase was cloned, overexpressed, and purified. Initial steady-state kinetic studies revealed that the aminopeptidase has broad activity, with a preference for basic and hydrophobic side chains and k(cat) and K(m) values that are lower and smaller, respectively, than those of Escherichia coli PepN. A V. fischeri mutant unable to produce PepN is significantly delayed in its ability to colonize squid within the first 12 h, but eventually it establishes a wild-type colonization level. Likewise, in competition with the wild type for colonization, the mutant is outcompeted at 12 h postinoculation but then competes evenly by 24 h. Also, the PepN-deficient strain fails to achieve wild-type levels of cells in aggregates, suggesting an explanation for the initial colonization delay. This study provides a foundation for more studies on PepN expression, localization, and role in the early stages of squid colonization.