Tracking the cargo of extracellular symbionts into host tissues with correlated electron microscopy and nanoscale secondary ion mass spectrometry imaging.

Extracellular bacterial symbionts communicate biochemically with their hosts to establish niches that foster the partnership. Using quantitative ion microprobe isotopic imaging (nanoscale secondary ion mass spectrometry [NanoSIMS]), we surveyed localization of 15 N-labelled molecules produced by the bacterium Vibrio fischeri within the cells of the symbiotic organ of its host, the Hawaiian bobtail squid, and compared that with either labelled non-specific species or amino acids. In all cases, two areas of the organ's epithelia were significantly more 15 N enriched: (a) surface ciliated cells, where environmental symbionts are recruited, and (b) the organ's crypts, where the symbiont population resides in the host. Label enrichment in all cases was strongest inside host cell nuclei, preferentially in the euchromatin regions and the nucleoli. This permissiveness demonstrated that uptake of biomolecules is a general mechanism of the epithelia, but the specific responses to V. fischeri cells recruited to the organ's surface are due to some property exclusive to this species. Similarly, in the organ's deeper crypts, the host responds to common bacterial products that only the specific symbiont can present in that location. The application of NanoSIMS allows the discovery of such distinct modes of downstream signalling dependent on location within the host and provides a unique opportunity to study the microbiogeographical patterns of symbiotic dialogue.

Transcriptional patterns in both host and bacterium underlie a daily rhythm of anatomical and metabolic change in a beneficial symbiosis.

Mechanisms for controlling symbiont populations are critical for maintaining the associations that exist between a host and its microbial partners. We describe here the transcriptional, metabolic, and ultrastructural characteristics of a diel rhythm that occurs in the symbiosis between the squid Euprymna scolopes and the luminous bacterium Vibrio fischeri. The rhythm is driven by the host's expulsion from its light-emitting organ of most of the symbiont population each day at dawn. The transcriptomes of both the host epithelium that supports the symbionts and the symbiont population itself were characterized and compared at four times over this daily cycle. The greatest fluctuation in gene expression of both partners occurred as the day began. Most notable was an up-regulation in the host of >50 cytoskeleton-related genes just before dawn and their subsequent down-regulation within 6 h. Examination of the epithelium by TEM revealed a corresponding restructuring, characterized by effacement and blebbing of its apical surface. After the dawn expulsion, the epithelium reestablished its polarity, and the residual symbionts began growing, repopulating the light organ. Analysis of the symbiont transcriptome suggested that the bacteria respond to the effacement by up-regulating genes associated with anaerobic respiration of glycerol; supporting this finding, lipid analysis of the symbionts' membranes indicated a direct incorporation of host-derived fatty acids. After 12 h, the metabolic signature of the symbiont population shifted to one characteristic of chitin fermentation, which continued until the following dawn. Thus, the persistent maintenance of the squid-vibrio symbiosis is tied to a dynamic diel rhythm that involves both partners.

The symbiosis regulator rscS controls the syp gene locus, biofilm formation and symbiotic aggregation by Vibrio fischeri.

Successful colonization of a eukaryotic host by a microbe involves complex microbe-microbe and microbe-host interactions. Previously, we identified in Vibrio fischeri a putative sensor kinase, RscS, required for initiating symbiotic colonization of its squid host Euprymna scolopes. Here, we analysed the role of rscS by isolating an allele, rscS1, with increased activity. Multicopy rscS1 activated transcription of genes within the recently identified symbiosis polysaccharide (syp) cluster. Wild-type cells carrying rscS1 induced aggregation phenotypes in culture, including the formation of pellicles and wrinkled colonies, in a syp-dependent manner. Colonies formed by rscSl-expressing cells produced a matrix not found in control colonies and largely lost in an rscSl-expressing sypN mutant. Finally, multicopy rscS1 provided a colonization advantage over control cells and substantially enhanced the ability of wild-type cells to aggregate on the surface of the symbiotic organ of E. scolopes; this latter phenotype similarly depended upon an intact syp locus. These results suggest that transcription induced by RscS-mediated signal transduction plays, a key role in colonization at the aggregation stage by modifying the cell surface and increasing the ability of the cells to adhere to one another and/or to squid-secreted mucus.

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.

Effect of cadmium(II), chromium(VI), and arsenic(V) on long-term viability- and growth-inhibition assays using Vibrio fischeri marine bacteria.

As a complement to previous results obtained using the standard Microtox acute-toxicity test, which is based on measuring the rapid decrease of bioluminescence (5 to 30 minutes of exposure) in Vibrio fischeri bacteria in the presence of toxicants, the long-term effects of Cd(II), Cr(VI), and As(V) were studied on growth rate and viability assays of the same bacteria adapted to longer-lasting cultures, i.e., 48 or 72 hours instead of 5 or 30 minutes. Effects on viability or growth, as studied by establishing dose- and time-response curves, confirmed that these poisonous chemicals were not particularly toxic to these bacteria. Nevertheless, in the case of Cr(VI), the viability-inhibition assay appeared to be more sensitive than the Microtox acute-toxicity test. Interestingly, it was possible to observe a clear hormesis phenomenon, especially for Cd(II), under the conditions of both viability- and growth-inhibition assays.