Genetically altered or tagged Vibrio fischeri strains can be observed in association with their mutualistic host Euprymna scolopes, providing powerful experimental approaches for studying this symbiosis. Two limitations to such in situ analyses are the lack of suitably stable plasmids and the need for a fluorescent tag that can be used in tandem with green fluorescent protein (GFP). Vectors previously used in V. fischeri contain the p15A replication origin; however, we found that this replicon is not stable during growth in the host and is retained by fewer than 20% of symbionts within a day after infection. In contrast, derivatives of V. fischeri plasmid pES213 were retained by ϳ99% of symbionts even 3 days after infection. We therefore constructed pES213-derived shuttle vectors with a variety of selectable and visual markers. To include a visual tag that can be used in conjunction with GFP, we compared seven variants of the DsRed2 red fluorescent protein (RFP): mRFP1, tdimer2(12), DsRed.T3, DsRed.T4, DsRed.M1, DsRed.T3_S4T, and DsRed.T3(DNT). The last variant was brightest, displaying >20-fold more fluorescence than DsRed2 in V. fischeri. RFP expression did not detectably affect the fitness of V. fischeri, and cells were readily visualized in combination with GFP-expressing cells in mixed infections. Interestingly, even when inocula were dense enough that most E. scolopes hatchlings were infected by two strains, there was little mixing of the strains in the light organ crypts. We also used constitutive RFP in combination with the luxICDABEG promoter driving expression of GFP to visualize the spatial and temporal induction of this bioluminescence operon during symbiotic infection. Our results demonstrate the utility of pES213-based vectors and RFP for in situ experimental approaches in studies of the V. fischeri-E. scolopes symbiosis.Vibrio fischeri is a useful model for studies of bioluminescence, quorum sensing, and beneficial animal-bacterium interactions. Genetic tools have advanced each of these fields and have been especially important for studying the light organ symbiosis between V. fischeri and the Hawaiian bobtail squid, Euprymna scolopes. This squid acquires V. fischeri from its surroundings after hatching, allowing reconstitution of this association with wild-type or genetically altered V. fischeri. In such experiments, mutant analyses have revealed colonization mechanisms (1, 8, 11, 14-16, 38, 42, 44) and visualization of wild-type cells tagged with green fluorescent protein (GFP) has elucidated early infection events (28,30,31). GFP also holds great promise as an in situ reporter of gene activity, because cells expressing GFP can be visualized in the light organ. However, despite the utility of genetic tools in this model symbiosis, researchers still lack suitably stable plasmids and a useful fluorescent tag that is compatible with GFP.To date, plasmids used to maintain genes in V. fischeri have contained the Escherichia coli plasmid p15A origin of replication (17,39,43). Although p15A-derived pl...

SummaryPeptidoglycan recognition proteins (PGRPs) are mediators of innate immunity and recently have been implicated in developmental regulation. To explore the interplay between these two roles, we characterized a PGRP in the host squid Euprymna scolopes (EsPGRP1) during colonization by the mutualistic bacterium Vibrio fischeri. Previous research on the squid-vibrio symbiosis had shown that, upon colonization of deep epithelium-lined crypts of the host light organ, symbiont-derived peptidoglycan monomers induce apoptosismediated regression of remote epithelial fields involved in the inoculation process. In this study, immunofluorescence microscopy revealed that EsPGRP1 localizes to the nuclei of epithelial cells, and symbiont colonization induces the loss of EsPGRP1 from apoptotic nuclei. The loss of nuclear EsPGRP1 occurred prior to DNA cleavage and breakdown of the nuclear membrane, but followed chromatin condensation, suggesting that it occurs during late-stage apoptosis. Experiments with purified peptidoglycan monomers and with V. fischeri mutants defective in peptidoglycan-monomer release provided evidence that these molecules trigger nuclear loss of EsPGRP1 and apoptosis. The demonstration of a nuclear PGRP is unprecedented, and the dynamics of EsPGRP1 during apoptosis provide a striking example of a connection between microbial recognition and developmental responses in the establishment of symbiosis.

Current cyanobacterial model organisms were not selected for their growth traits or potential for the production of renewable biomass, biofuels, or other products. The cyanobacterium strain BL0902 emerged from a search for strains with superior growth traits. Morphology and 16S rRNA sequence placed strain BL0902 in the genus Leptolyngbya. Leptolyngbya sp. strain BL0902 (hereafter Leptolyngbya BL0902) showed robust growth at temperatures from 22°C to 40°C and tolerated up to 0.5 M NaCl, 32 mM urea, high pH, and high solar irradiance. Its growth rate under outdoor conditions rivaled Arthrospira (“pirulina” strains. Leptolyngbya BL0902 accumulated higher lipid content and a higher proportion of monounsaturated fatty acids than Arthrospira strains. In addition to these desirable qualities, Leptolyngbya BL0902 is amenable to genetic engineering that is reliable, efficient, and stable. We demonstrated conjugal transfer from Escherichia coli of a plasmid based on RSF1010 and expression of spectinomycin/streptomycin resistance and yemGFP reporter transgenes. Conjugation efficiency was investigated in biparental and triparental matings with and without a “elper”plasmid that carries DNA methyltransferase genes, and with two different conjugal plasmids. We also showed that Leptolyngbya BL0902 is amenable to transposon mutagenesis with a Tn5 derivative. To facilitate genetic manipulation of Leptolyngbya BL0902, a conjugal plasmid vector was engineered to carry a trc promoter upstream of a Gateway recombination cassette. These growth properties and genetic tools position Leptolyngbya BL0902 as a model cyanobacterial production strain.

The light-organ symbiont Vibrio fischeri releases N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramylalanyl-␥-glutamyldiaminopimelylalanine, a disaccharide-tetrapeptide component of peptidoglycan that is referred to here as "PG monomer." In contrast, most gram-negative bacteria recycle PG monomer efficiently, and it does not accumulate extracellularly. PG monomer can stimulate normal light-organ morphogenesis in the host squid Euprymna scolopes, resulting in regression of ciliated appendages similar to that triggered by infection with V. fischeri. We examined whether the net release of PG monomers by V. fischeri resulted from lytic transglycosylase activity or from defects in AmpG, the permease through which PG monomers enter the cytoplasm for recycling. An ampG mutant displayed a 100-fold increase in net PG monomer release, indicating that AmpG is functional. The ampG mutation also conferred the uncharacteristic ability to induce light-organ morphogenesis even when placed in a nonmotile flaJ mutant that cannot infect the light-organ crypts. We targeted five potential lytic transglycosylase genes singly and in specific combinations to assess their role in PG monomer release. Combinations of mutations in ltgA, ltgD, and ltgY decreased net PG monomer release, and a triple mutant lacking all three of these genes had little to no accumulation of PG monomers in culture supernatants. This mutant colonized the host as well as the wild type did; however, the mutant-infected squid were more prone to later superinfection by a second V. fischeri strain. We propose that the lack of PG monomer release by this mutant results in less regression of the infection-promoting ciliated appendages, leading to this propensity for superinfection.Microbe-associated molecular patterns (MAMPs) are recognized by hosts in a variety of pathogenic and symbiotic relationships. MAMP is an umbrella term for a variety of semiconserved bacterial molecules, including lipopolysaccharide, lipoproteins, flagella, and peptidoglycan (PG), that are sensed by conserved host surveillance mechanisms (e.g., the innate immune system), triggering context-dependent reactions to bacterial colonization. Mounting evidence shows that PG-derived MAMPs play important and previously underappreciated roles in host-bacterium interactions (11).The PG layer of gram-negative bacteria is a rigid network in the periplasm that protects against osmotic lysis and helps to determine cell size and shape while still allowing diffusion of molecules into the cell (14). In PG, repeated subunits of Nacetylglucosamine and N-acetylmuramic acid are connected to a short pentapeptide side chain of L-alanyl-D-␥-glutamyl-mesodiaminopimelyl-D-alanyl-D-alanine (Ala-Glu-DAP-Ala-Ala). Adjacent peptides are cross-linked through Ala-DAP or DAP-DAP peptide bonds, and side chains are converted to tetra-, tri-, and dipeptides through the action of carboxypeptidases in the periplasm (19,49).Despite its mechanical stability, PG is a dynamic structure that undergoes remodeling and recycling. Murein hydrola...

The mechanisms by which cellular oscillators keep time and transmit temporal information are poorly understood. In cyanobacteria, the timekeeping aspect of the circadian oscillator, composed of the KaiA, KaiB, and KaiC proteins, involves a cyclic progression of phosphorylation states at Ser431 and Thr432 of KaiC. Elucidating the mechanism that uses this temporal information to modulate gene expression is complicated by unknowns regarding the number, structure, and regulatory effects of output components. To identify oscillator signaling states without a complete description of the output machinery, we defined a simple metric, Kai-complex output activity (KOA), that represents the difference in expression of reporter genes between strains that carry specific variants of KaiC and baseline strains that lack KaiC. In the absence of the oscillator, expression of the class 1 paradigm promoter P kaiBC was locked at its usual peak level; conversely, that of the class 2 paradigm promoter P purF was locked at its trough level. However, for both classes of promoters, peak KOA in wild-type strains coincided late in the circadian cycle near subjective dawn, when KaiC-pST becomes most prevalent (Ser431 is phosphorylated and Thr432 is not). Analogously, peak KOA was detected specifically for the phosphomimetic of KaiC-pST (KaiC-ET). Notably, peak KOA required KaiB, indicating that a KaiBC complex is involved in the output activity. We also found evidence that phosphorylated RpaA (regulator of phycobilisome associated) represses an RpaA-independent output of KOA. A simple mathematical expression successfully simulated two key features of the oscillator-the time of peak KOA and the peak-to-trough amplitude changes.bioluminescence | chronobiology | transcription regulation C ircadian biological clocks are recognized as endogenous 24-h timers that evolved through the selective fitness advantage they confer in anticipation of daily environmental variations and that generate rhythms in metabolic and behavioral processes (1-3). Both the ability to keep 24-h time and the mechanism by which such a clock regulates cellular processes are only partially understood in any organism. In the oxygenic photosynthetic bacteria known as cyanobacteria, the oscillator mechanism is a posttranslational protein interaction loop, and the nature of its temporal output signal is more easily addressable than in eukaryotic models. The recent report of a posttranslational circadian system that is shared among the kingdoms of life suggests a more universal role of posttranslational oscillators in nature (4, 5). Among the prokaryotic cyanobacteria, Synechococcus elongatus PCC 7942 is the prevalent model system for circadian studies due to its genetic manipulability and small (2.7 Mb) fully sequenced genome (6). The ability to monitor the circadian regulation of gene expression in vivo, achieved by fusing the promoter of a gene of interest to a bioluminescence reporter gene (7,8), provides a tool for investigating the circadian clock and its connections with m...

Vibrio fischeri, a bioluminescent marine bacterium, exists in an exclusive symbiotic relationship with the Hawaiian bobtail squid, Euprymna scolopes, whose light organ it colonizes. Previously, it has been shown that the lipopolysaccharide (LPS) or free lipid A of V. fischeri can trigger morphological changes in the juvenile squid's light organ that occur upon colonization. To investigate the structural features that might be responsible for this phenomenon, the lipid A from V. fischeri ES114 LPS was isolated and characterized by multistage mass spectrometry (MS n ). A microheterogeneous mixture of mono-and diphosphorylated diglucosamine disaccharides was observed with variable states of acylation ranging from tetra-to octaacylated forms. All lipid A species, however, contained a set of conserved primary acyl chains consisting of an N- The bioluminescent bacterium Vibrio fischeri exists in a symbiotic relationship with the Hawaiian bobtail squid, Euprymna scolopes. Colonization of the juvenile squid's light organ by V. fischeri begins within hours of hatching (1). From the complex bacterial community present in seawater, V. fischeri, which represents less than 1% of the bacterial population, is exclusively recruited by E. scolopes in a multistep winnowing process (2). As the colonization of the juvenile's light organ progresses, a series of developmental changes occur in the tissues. The most dramatic of these morphogenetic events is the loss of a superficial ciliated field of cells important for the initial recruitment of V. fischeri. This process occurs over the ϳ96 -120 h following initial colonization by the symbiont (2) and does not occur without interactions with the symbiont in the deep crypt regions of the organ. Once the colonization is established, the squid continues to maintain a population of V. fischeri in its light organ, with cycles of daily flushing and repopulation by residual bacteria.The selection process that leads to the exclusive symbiotic relationship between V. fischeri and E. scolopes involves the interaction of bacterial surface components with host tissues. Previously, we showed that bacterial lipopolysaccharide (LPS) and lipid A can induce early stage apoptosis in the cells of the ciliated field of the juvenile squid's light organ (3). However, the effect was not species-specific, suggesting that a conserved portion of the lipid A may be the responsible component of the LPS structure (3). In later stages of the colonization process, bacterial peptidoglycan acts synergistically with LPS to induce most, if not all, of light organ morphogenesis (4).How the developmental signals of bacterial LPS and peptidoglycan, which are presented by the symbionts in the deep crypts of the light organ, are conveyed to the superficial tissue remains unknown, but one piece of the puzzle has recently been discovered. At hatching, the light organ has high levels of nitric oxide (NO). Following symbiont colonization of the crypts, the levels of both NO and the enzyme that catalyzes its production, nitric-o...

Vibrio fischeri isolates from Euprymna scolopes are dim in culture but bright in the host. We found the luminescence of V. fischeri to be correlated with external osmolarity both in culture and in this symbiosis. Luminescence enhancement by osmolarity was independent of the lux promoter and unaffected by autoinducers or the level of lux expression, but the addition of an aldehyde substrate for luciferase raised the luminescence of cells grown at high and low osmolarities to the same high level. V. fischeri culture media have lower osmolarities than are typical in seawater or in cephalopods, partially accounting for the bacterium's low light output in culture.The light organ symbiosis between the bioluminescent bacterium Vibrio fischeri and the Hawaiian bobtailed squid Euprymna scolopes has been developed as a model for studying mutualistic animal-bacterium interactions (15,21). In establishing this system in our laboratory, we discovered that E. scolopes juveniles infected with V. fischeri in artificial seawater (Instant Ocean; Aquarium Systems, Mentor, Ohio) lost luminescence after 3 days, but remained colonized, if they were kept in diluted seawater (700 to 850 mosM) rather than seawater mixed to marine concentrations (975 to 1,025 mosM). Figure 1 illustrates this phenomenon with the results of one representative experiment. Squid were infected as previously described (16), and their luminescence was measured with a model LS 6500 counter (Beckman Coulter, Fullerton, Calif.). Although marine organisms are sometimes maintained at relatively low osmolarities, our data highlight the fact that this can perturb their natural biology in important ways.These data were intriguing because luminescence contributes to colonization persistence (20), and V. fischeri isolates from E. scolopes are unusual in that they are dim in culture (even dense culture) and bright only in the host (1, 10). Our data show wild-type cells exhibiting a dimness like that seen in culture in fully colonized squid, suggesting that we had mimicked culture conditions by placing the animals in dilute seawater. A simple explanation for our observations of symbiotic luminescence was that the squid, which maintain hyperosmotic tissues, lost the ability to osmoregulate against an unnaturally steep gradient and that the luminescence of the V. fischeri symbionts was dependent on the osmolarity of their surroundings (e.g., the light organ crypts). Consistent with the latter part of this model, in 1950 Farghaly reported a correlation between osmolarity and luminescence in culture for a bacterium that was probably a planktonic V. fischeri isolate (7).We therefore tested the relationship between osmolarity and luminescence in a wild-type E. scolopes isolate, V. fischeri strain ES114 (1). Medium osmolarity was assessed using a freezingpoint depression-automated osmometer (Osmette A; Precision Systems Inc., Natick, Mass.). The optical density at 595 nm (OD 595 ) was determined with a BioPhotometer (Brinkmann Instruments Inc., Westbury, N.Y.) by measuring the cu...

Bacterial lipid A is an important mediator of bacterium-host interactions, and secondary acylations added by HtrB and MsbB can be critical for colonization and virulence in pathogenic infections. In contrast, Vibrio fischeri lipid A stimulates normal developmental processes in this bacterium's mutualistic host, Euprymna scolopes, although the importance of lipid A structure in this symbiosis is unknown. To further examine V. fischeri lipid A and its symbiotic function, we identified two paralogs of htrB (designated htrB1 and htrB2) and an msbB gene in V. fischeri ES114 and demonstrated that these genes encode lipid A secondary acyltransferases. htrB2 and msbB are found on the Vibrio "housekeeping" chromosome 1 and are conserved in other Vibrio species. Mutations in htrB2 and msbB did not impair symbiotic colonization but resulted in phenotypic alterations in culture, including reduced motility and increased luminescence. These mutations also affected sensitivity to sodium dodecyl sulfate, kanamycin, and polymyxin, consistent with changes in membrane permeability. Conversely, htrB1 is located on the smaller, more variable vibrio chromosome 2, and an htrB1 mutant was wild-type-like in culture but appeared attenuated in initiating the symbiosis and was outcompeted 2.7-fold during colonization when mixed with the parent. These data suggest that htrB2 and msbB play conserved general roles in vibrio biology, whereas htrB1 plays a more symbiosis-specific role in V. fischeri.Vibrio fischeri is a bioluminescent bacterium that forms a mutualistic symbiosis with Euprymna scolopes, the Hawaiian bobtail squid. E. scolopes supports the growth and bioluminescence of V. fischeri in a specialized light organ (40), and the bacterial luminescence is apparently used by the squid in a camouflaging behavior (23). V. fischeri is more abundant in E. scolopes habitats, reinforcing the belief that the relationship is beneficial for both partners (28). Among the hundreds of bacterial species present in Hawaiian waters, only V. fischeri is able to colonize the E. scolopes light organ. This specific colonization triggers developmental changes in light organ tissue, and it is clear that E. scolopes identifies and responds to signals from V. fischeri (32). Although the mechanisms underlying the specificity, signaling, and persistence of this symbiosis are not entirely clear, previous studies suggested that a key molecule in some of these symbiotic processes is V. fischeri's lipopolysaccharide (LPS) (13).LPS comprises 70% of the outer membrane of gram-negative bacteria and has the following three main components: (i) O antigen, which is a variable hydrophilic polysaccharide that projects into the environment; (ii) a relatively conserved central core of sugars; and (iii) lipid A, which anchors LPS into the outer membrane. The innate immune systems of many animals recognize lipid A through LPS-binding protein and Toll-like receptors (20,21), which stimulate inflammation to attract more host immune cells to clear pathogenic infections (8,33). Ind...