1.1. Quorum Sensing in Vibrio fischeri
Quorum sensing (QS) describes the mechanism of intercellular communication by which bacteria can alter group behavior in accordance with population density [
1]. This process depends on the synthesis and diffusion of signaling molecules, called autoinducers, into the surrounding environment. Upon reaching a threshold concentration, autoinducers will trigger cellular responses, typically by altering gene expression across the entire population. QS controls a wide variety of processes in bacteria including bioluminescence production, sporulation, competence, biofilm formation and the synthesis of antibiotics and virulence factors [
2,
3].
QS was first discovered in the Gram-negative, marine bacterium
Vibrio fischeri as the mechanism that controls the induction of luminescence within growing cultures [
4].
V. fischeri belongs to the family
Vibrionaceae, which is comprised of many bacterial species that are commonly found in fresh and marine water habitats.
Vibrio fischeri was originally described as a member of the genus
Vibrio. More recently, however,
Vibrio fischeri, along with
Vibrio logei,
Vibrio salmonicida, and
Vibrio wodanis, has been placed in a new genus called
Aliivibrio, as these species form a monophyletic clade that can be differentiated based on phenotypic traits and biochemical tests from the other members of the genus [
5]. Because the majority of studies focusing on QS and host-microbe interactions use the
Vibrio nomenclature, we will continue its use in this review.
The proteins required for luminescence in
V. fischeri are encoded within the
luxICDABEG operon [
6,
7]. Light is released during the oxidation reaction by the enzyme luciferase, which is comprised of two subunits, α and β, encoded by
luxA and
luxB, respectively. The enzymatic reaction converts a long-chain fatty acid (RCHO), reduced flavin mononucleotide (FMNH
2), and O
2 into aliphatic acid (RCOOH) and FMN. A reductase complex composed of the proteins encoded by
luxCDE synthesizes the substrate RCHO [
8]. LuxG converts FMN to FMNH
2 [
9].
Multiple QS systems control luminescence in
V. fischeri (
Figure 1). Directly regulating expression of the
luxICDABEG operon is the LuxI-LuxR QS system. LuxI synthesizes the autoinducer
N-3-oxohexanoyl-homoserine lactone (3-oxo-C6-HSL) [
10] that, at a threshold concentration (100–200 nM), binds and activates the transcription factor LuxR [
11]. The LuxR/3-oxo-C6-HSL complex binds as a dimer upstream of the
luxICDABEG operon and recruits RNA polymerase to initiate transcription of the operon [
12]. Interestingly, while the overall luminescence output of a population of cells increases as the level of 3-oxo-C6-HSL increases, the luminescence levels of individual cells display significant heterogeneity [
13]. Whether this cell-cell heterogeneity in luminescence output has a biological role remains unclear at this time.
Two additional QS systems, AinS-AinR and LuxS-LuxP/Q, indirectly control luminescence by modulating
luxR transcription (
Figure 1). AinS synthesizes
N-octanoyl-homoserine lactone (C8-HSL), which is an autoinducer detected by the histidine kinase AinR. LuxS synthesizes autoinducer 2 (AI-2), which binds to the periplasmic protein LuxP [
14,
15]. Based on studies of the homologous system in
V. harveyi, LuxP forms a complex with LuxQ, a histidine kinase that dimerizes within the inner membrane [
15]. Binding of AI-2 to LuxP induces a rotational shift in the corresponding dimer of LuxQ that inhibits kinase activity. AinR and LuxP/Q act in parallel by controlling the phosphorylation state of LuxU, which serves as an intermediate step within the phosphorelay that dictates the phosphorylation state, and consequently, activity of the response regulator LuxO. Under conditions of low cell density,
i.e., in the absence of sufficient levels of autoinducers, the histidine kinases activate the phosphorelay, leading to high levels of phosphorylated LuxO. Phosphorylated LuxO activates transcription of
qrr1, which encodes a small regulatory RNA that post-transcriptionally represses the transcription factor LitR via the RNA chaperone Hfq [
16]. Under conditions of high cell density, the phosphorelay is reversed, which stabilizes
litR transcript. LitR enhances
luxR expression, thereby contributing to light production [
17].
The QS network of
V. fischeri is significantly different than those of other
Vibrionaceae members [
18]. For instance, the LuxI-LuxR system is exclusively present in the
Aliivibrio clade of the
Vibrionaceae family, which includes the fish pathogen
Aliivibrio salmonicida as well as
Vibrio fischeri [
19]. In non-
Aliivibrio members of
Vibrionaceae that are bioluminescent, the luminescence genes are directly regulated by the corresponding LitR homologue instead of a LuxI-LuxR system. As a result, a distinguishing feature of the
V. fischeri QS network is that, in contrast to parallel QS networks in
V. harveyi and
V. cholerae, it is both parallel and hierarchal. In other words, in all
Vibrionaceae members, histidine kinases, like AinS-AinR and LuxS-LuxP/Q in
V. fischeri, are arranged in parallel within the network and converge on the regulator LitR. The
V. fischeri network is also hierarchal because the LuxI-R system acts downstream of LitR, and therefore depends, in part, on the activities of the parallel QS systems. Another significant difference among the QS systems of different
Vibrionaceae members is the number of
qrr genes within their genomes. In particular,
V. fischeri possesses a single
qrr gene encoding Qrr1 that is sufficient to repress
litR expression [
16], whereas multiple Qrr sRNAs regulate the expression of the LitR homologues in
V. harveyi (LuxR
VH) and
V. cholerae (HapR) [
20,
21].
In
V. fischeri, the QS network also appears to have crosstalk among the different autoinducers: namely, C8-HSL, the autoinducer synthesized by AinS, can also directly bind to LuxR [
14,
22]. However, the LuxR/C8-HSL complex is not as effective as the LuxR/3-oxo-C6-HSL complex in activating transcription of the
lux operon [
22,
23]. As a result, the two autoinducers show competitive binding to LuxR that alters the transcriptional level of the
lux operon and resulting luminescence. At high concentration, C8-HSL acts as a competitive inhibitor and suppresses luminescence; however, the suppression can be overcome by sufficiently high concentrations of 3-oxo-C6-HSL [
14,
22]. Interestingly, the impact of deleting
ainS on luminescence depends on the particular strain of
V. fischeri, highlighting the complexity by which C8-HSL affects luminescence [
14,
22]. The crosstalk by autoinducers does not appear to affect the heterogeneity in the luminescence output observed among individual cells [
24].
The QS network of
V. fischeri also contains feedback loops that ultimately impact its overall dynamics. For instance, the LuxI-derived autoinducer 3-oxo-C6-HSL enhances its own synthesis as the
luxI gene is upregulated by the LuxR/3-oxo-C6-HSL. Interestingly, a positive feedback loop is also involved in QS by C8-HSL, as
ainS expression is apparently controlled through LitR [
25]. How different architectures of various QS networks can impact the magnitudes and temporal profiles of QS-regulated phenotypes is well documented [
2,
3]. As described throughout this review, the particular intricacies of the QS network of
V. fischeri, which experiences both free-living and symbiotic conditions, has evolved to accommodate such disparate life styles.
Notably, the QS network of
V. fischeri regulates processes in addition to bioluminescence. Flagella-based motility is controlled, in part, by the LuxO-Qrr1-LitR pathway, so that motility is enhanced under the conditions of low cell density that repress bioluminescence. Phosphorylation of LuxO, or equivalently expression of
qrr1, results in enhanced motility. Epistasis experiments have demonstrated that LitR is the transcription factor that mediates the effect on motility by LuxO and Qrr1 [
26]. AinS also regulates via LitR the expression of
acs, which encodes acetyl-CoA synthetase and, as a result, can control acetate metabolism [
27]. More recently, the LuxP/Q complex was shown to impact biofilm formation by
V. fischeri, which is a process mediated by an 18-gene, symbiosis polysaccharide (
syp) locus [
28]. Interestingly, the mechanism may involve direct phosphotransfer between LuxU and the response regulator SypG, although this has yet to be shown.
1.2. The Euprymna scolopes-Vibrio fischeri Symbiosis
Recent studies of the human gut microbiome have highlighted the general importance of beneficial bacteria on animal physiology and development [
29].
V. fischeri forms monospecific, beneficial symbioses in many marine animals, including various squid and fish [
30,
31]. Of these hosts,
E. scolopes, a species of bobtail squid found in the offshore waters of the Hawaiian Islands, is the most studied. The symbiosis is highly specific, such that the squid is colonized exclusively by only certain strains of
V. fischeri [
31,
32]. In addition, bacterial transmission is horizontal,
i.e.,
V. fischeri cells are acquired each generation by juveniles from seawater. Because of these features, the symbiosis offers an excellent model to study the establishment, development, and maintenance of a beneficial bacterial infection along an epithelial surface [
33]. The nocturnal squid uses bioluminescence emitted by
V. fischeri for camouflage by shining light downwards to disrupt its shadow within the water column [
34]. To harness this bioluminescence,
E. scolopes houses populations of
V. fischeri within an organ referred to as the light organ that is located in the middle of the mantle cavity, just inside of the ventral surface of the mantle (
Figure 2). The light organ possesses bilateral symmetry and, in juvenile squid, exhibits on each side two appendages that are surrounded by a field of ciliated epithelia [
35]. At the base of the appendages on either side of the light organ are three pores, which lead through ciliated ducts to crypt spaces deep within the organ, where bacteria reside extracellularly during the lifetime of the host.
The initial process of colonization begins within 2–4 h after juvenile squid hatch from their eggs into seawater containing as little as 1000
V. fischeri CFU/mL [
36]. The first event in establishing the symbiosis is the direct interaction of bacterial cells, including
V. fischeri, to host cilia that are associated with surface epithelial cells [
37,
38]. Beating of these cilia collect bacteria in aggregates, which are comprised of up to a few hundred cells within host-derived mucus that is secreted by surface epithelial cells of the light organ [
37]. Interestingly, hyper-motile mutants of
V. fischeri show a delay in aggregate formation, which highlights the importance of proper regulatory control over motility throughout the colonization process [
39]. Although mucus secretion is a general host response to the presence of bacteria [
32], the aggregate is dominated by
V. fischeri cells [
40]. The mechanism by which
V. fischeri dominates the aggregate despite its low abundance in seawater is largely unknown. It is unlikely that
V. fischeri cells within the aggregate break down the mucus for nutrients more efficiently than others as the cells do not multiply in the aggregate. Chemotaxis towards
N-acetylneuraminic acid, a component of squid mucus, suggests that
V. fischeri has evolved to respond to host-derived compounds [
41]. After 2–4 h within the aggregate,
V. fischeri migrates through the mucus towards the pores. Non-motile
V. fischeri and bacterial-sized particles do not display this behavior, suggesting that
V. fischeri actively participates in the process [
37]. Chemotaxis to host-derived compounds does not play a role during these steps, since a
cheA transposon-insertion mutant of
V. fischeri migrates to the pores in similar fashion to wild-type cells [
42].
Once
V. fischeri has entered a pore, it travels through ciliated ducts to gain access to deep crypt spaces, where it begins to multiply and establishes a stable association [
33]. Within each duct, a gradient of chitin-derived oligosaccharides, which are chemoattractants for
V. fischeri, recruit potential symbionts into the crypt spaces [
42]. As described above, the effect of this chemotactic gradient seems to be important only for entry into the pores and travelling through the ducts, not for the initial migration through the mucus [
42]. Within the deep crypts,
V. fischeri grows rapidly for the first 12 h, until the population size reaches about 10
5 CFU [
43]. This growth is concomitant with luminescence, which can be detected as early as 7 h and achieves steady-state levels by 12 h [
43].