New rfp- and pES213-Derived Tools for Analyzing Symbiotic Vibrio fischeri Reveal Patterns of Infection and lux Expression In Situ

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 ~99% 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.     

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.     

Osmotic shock induces expression of Vibrio fischeri lux genes in Escherichia coli cells

The effect of osmotic shock on the expression of genes in the lux regulon of marine bacteria Vibrio fischeri was studied in cells of Escherichia coli. Bioluminescence of cells was shown to drastically increase, when cells were exposed to osmotic shock at the early logarithmic growth phase. The expression of lux genes induced by osmotic shock is determined by the two-component regulatory system RcsC-RcsB. A nucleotide sequence in the regulatory region of the luxR gene homologous to the RcsB-box consensus of E. coli is assumed to be a primary site for this system.     

Synthesis of the lux gene autoinducer in Vibrio fischeri is positively autoregulated

The enzymes for luminescence in Vibrio fischeri are induced only after the accumulation of a sufficient concentration of a metabolic product (the autoinducer) generated by the bacteria themselves. Genetic analyses by others have previously suggested that biosynthesis of the autoinducer is catalyzed by a single gene product (autoinducer synthetase) presumably from precursors typically present in the bacterial cell. Also, the biosynthesis was predicted to be autocatalytic such that in the presence of autoinducer, more autoinducer synthetase should be produced. We have directly tested these predictions and found that autoinducer synthesis is indeed positively autoregulated. In addition, we have demonstrated autoinducer synthesis in vitro and have tentatively identified the substrates of autoinducer synthetase as S-adenosylmethionine and 3-oxohexanoyl coenzyme A.Abbreviations AdoMet S-adenosylmethionine - AI autoinducer, i.e. 3-oxohexsanoyl homoserine lactone - C-10 decanoyl homoserine lactone - HPLC high performance liquid chromatography - LM luminescence medium - LM-BT luminescence medium without tryptone - LU light units - 3-oxo 3-oxohexanoyl-coenzyme A - SWC sea water complete medium     

GroEL/GroES chaperone and Lon protease regulate expression of the Vibrio fischeri lux operon in Escherichia coli

Chaperone GroEL/GroES and Lon protease were shown to play a role in regulating the expression of the Vibrio fischeri lux operon cloned in Escherichia coli cells. The E. coli groE mutant carrying a plasmid with the full-length V. fischeri lux regulon showed a decreased bioluminescence. The bioluminescence intensity was high in E. coli cells with mutant lonA and the same plasmid. Bioluminescence induction curves lacked the lag period characteristic of lon ⁺ strains. Regulatory luxR of V. fischeri was cloned in pGEX-KG to produce the hybrid gene GST-luxR. The product of its expression, GST-LuxR, was isolated together with GroEL and Lon upon affinity chromatography on a column with glutathione-agarose, suggesting complexation of LuxR with these proteins. It was assumed that GroEL/GroES is involved in LuxR folding, while Lon protease degrades LuxR before its folding into an active globule or after denaturation.     

Osmotic Shock Induction of the Expression of Vibrio fischeri lux Genes in Escherichia coli Cells

The effect of osmotic shock on the expression of genes in the lux regulon of marine bacteria Vibrio fischeri was studied in cells ofEscherichia coli. Bioluminescence of cells was shown to drastically increase, when cells were exposed to osmotic shock at the early logarithmic growth phase, at far lower optic densities as compared to the critical optic density characteristic. The expression of lux genes induced by osmotic shock is determined by the two-component regulatory system RcsC–RcsB. A nucleotide sequence in the regulatory region of the luxR gene homologous to the RcsB-box consensus of E. coli is assumed to be a primary site for this system.     

The effects of the regulatory proteins RcsA and RcsB on the expression of the Vibrio fischeri lux operon in Escherichia coli

A study was made of the effect of RcsA and RcsB on the Vibrio fischeri lux expression in Escherichia coli. RcsA suppressed the LuxR activity and thereby inhibited expression of the lux genes coding for luciferase and reductase. In osmotic shock, RcsA-RcsB activated lux expression and, consequently, bioluminescence of E. coli cells in the early log phase.     

Vibrio fischeri lux genes play an important role in colonization and development of the host light organ

The bioluminescent bacterium Vibrio fischeri and juveniles of the squid Euprymna scolopes specifically recognize and respond to one another during the formation of a persistent colonization within the host's nascent light-emitting organ. The resulting fully developed light organ contains brightly luminescing bacteria and has undergone a bacterium-induced program of tissue differentiation, one component of which is a swelling of the epithelial cells that line the symbiont-containing crypts. While the luminescence (lux) genes of symbiotic V. fischeri have been shown to be highly induced within the crypts, the role of these genes in the initiation and persistence of the symbiosis has not been rigorously examined. We have constructed and examined three mutants (luxA, luxI, and luxR), defective in either luciferase enzymatic or regulatory proteins. All three are unable to induce normal luminescence levels in the host and, 2 days after initiating the association, had a three- to fourfold defect in the extent of colonization. Surprisingly, these lux mutants also were unable to induce swelling in the crypt epithelial cells. Complementing, in trans, the defect in light emission restored both normal colonization capability and induction of swelling. We hypothesize that a diminished level of oxygen consumption by a luciferase-deficient symbiotic population is responsible for the reduced fitness of lux mutants in the light organ crypts. This study is the first to show that the capacity for bioluminescence is critical for normal cell-cell interactions between a bacterium and its animal host and presents the first examples of V. fischeri genes that affect normal host tissue development.     

Luminescence control in the marine bacterium Vibrio fischeri: An analysis of the dynamics of lux regulation

A mathematical model has been developed based on the fundamental properties of the control system formed by the lux genes and their products in Vibrio fischeri. The model clearly demonstrates how the components of this system work together to create two, stable metabolic states corresponding to the expression of the luminescent and non-luminescent phenotypes. It is demonstrated how the cell can "switch" between these steady states due to changes in parameters describing metabolic processes and the extracellular concentration of the signal molecule N-3-oxohexanoyl-l-homoserine lactone. In addition, it is shown how these parameters influence how sensitive the switch mechanism is to cellular LuxR and N-3-oxohexanoyl-l-homoserine lactone and complex concentration. While these properties could lead to the collective phenomenon known as quorum sensing, the model also predicts that under certain metabolic circumstances, basal expression of the lux genes could cause a cell to luminesce in the absence of extracellular signal molecule. Finally, the model developed in this study provides a basis for analysing the impact of other levels of control upon lux regulation.     

Oxygen-utilizing reactions and symbiotic colonization of the squid light organ by Vibrio fischeri.

A major goal in microbiology is to understand the processes by which bacteria successfully colonize host tissue. Although a wealth of studies focusing on pathogenic microorganisms has revealed much about the rare interactions that result in disease, far less is known about the regulation of the ubiquitous, long-term, cooperative associations of bacteria with their animal hosts.