A rod-shaped, gram-negative bacteria known as Aliivibrio fischeri (also known as Vibrio fischeri) is prevalent worldwide in maritime habitats. This species, which is bioluminescent, coexists mostly in symbiosis with other marine organisms like the Hawaiian bobtail squid. The Hawaiian bobtail squid is a tiny, nocturnal squid that lives in shallow coastal waters. It spends the daytime hiding in the sand and emerges at night to hunt for small shrimp and other prey. What is particularly intriguing is how a bacterium navigates the various, diverse surroundings that are presented during a successful infection of an animal host. The symbiotic relationship between Vibrio fischeri and its Hawaiian bobtail squid host, Euprymna scolopes, is one practicable model system to research how a bacterium manages a host's many conditions. (see here) From its initial interacting with the squid until its eventual colonization of the light organ, V. fischeri experiences a variety of host environments. For instance, V. fischeri recognizes the squid and creates a biofilm aggregate outside the light organ, which is necessary for effective colonization.
Figure1: Photo by David Slater
Complex populations of microorganisms called "biofilms" are enclosed in a self-produced matrix that offers protection and enables adhesion. (see here) In a laboratory context, the development of cohesive wrinkling colonies on solid agar, pellicles (a thin membrane) at the air-liquid interface in liquid cultures under static circumstances, and cell clumping in liquid cultures under shaking conditions are all indicators of biofilm formation. The stated in vitro biofilms are only created by strains that have undergone genetic manipulation, as opposed to the biofilms that develop spontaneously on the host (for instance by disrupting negative regulators and/or overexpressing positive biofilm regulators). These findings imply that V. fischeri has well regulated biofilm development. Currently, it is known that seven regulators govern how biofilms form. Six of these regulators are members of the vast class of regulators known as two-component signaling (TCS) regulators, while the final regulator's function is still unknown. Additionally, here, researchers identified calcium as a signal that encourages biofilm development by strains that are capable of forming them in environments where biofilms are not normally seen.
Fig 2: Model describing how V. fischeri controls the growth of biofilms
By controlling the development of the symbiosis polysaccharide (Syp-PS), the main constituent of the biofilm matrix, in V. fischeri, TCS affects biofilm formation both positively and negatively. When a signal is activated, RscS (labelled (2) in the figure above) starts a phosphorelay that activates SypG (labelled (5) in the figure above) via SypF's (labelled (3) in the figure above) Hpt (Histidine phosphotransferase) domain, increasing syp transcription and promoting the creation of biofilms. According to recent research, SypF's Hpt domain and HahK (labelled (4) in the figure above) appear to operate together to promote the production of biofilms. Biofilm development is also governed by two opposing regulators, BinK (labelled (1) in the figure above) and SypE (labelled (6) in the figure above). Strongly inhibiting syp transcription and the development of biofilms is the role of the hybrid SK BinK. SypE regulates SypA's (not shown in the above figure) phosphorylation status to limit biofilm at a level below transcription, albeit its activity is still unknown.
In this study, the researchers created double mutants of the two recognized negative regulators of biofilm formation, BinK and SypE, and evaluated biofilm formation in the absence of the calcium-dependent biofilm-inducing signal. It was found that SypF, a known positive regulator, as well as BinK and SypE, negatively regulate biofilms in these circumstances. In the absence of the calcium signal, the loss of BinK and SypE along with a disruption of SypF's inhibitory function allowed V. fischeri to form biofilms. Together, these findings show that SypF is effective as a biofilm inhibitor in typical laboratory settings and show that three regulators stop wild-type V. fischeri from forming biofilms. The SK BinK and the RR SypE are two recognized negative regulators of biofilm development that are encoded by V. fischeri. On Luria-Bertani salt (LBS) medium, the usual rich media used to cultivate this organism, some genetically altered strains of V. fischeri (for example, RscS-overexpressing strains) generate wrinkled colonies; nevertheless, mutation of either binK or sypE alone does not permit wrinkled colony formation. In order to allow the production of wrinkled colonies, it was thus postulated that both negative regulators may need to be disrupted.
In order to test this theory, the researchers created KV7856 by deleting binK from strain KV3299, a well-studied sypE mutant.
Figure 3: Investigation of a mutation that results in colonies with wrinkles
The sypE mutant formed smooth colonies under these circumstances, just like the wild-type strain ES114 (Fig. 3Ai) and as previously reported (Fig. 3Aii). Similar to this, a strain with the deletion of binK alone (∆binK) produced smooth colonies on LBS medium (Fig 3Aiii). KV7856, on the other hand, produced colonies that were wrinkled due to defects in the two well-known negative regulators (Fig. 3Aiv). These findings first suggested that BinK and SypE combined could prevent the development of biofilms. An independently created ∆binK ∆sypE mutant (∆binK1 sypE::Cm [where "Cm" represents chloramphenicol]) failed to produce robust wrinkled colonies when grown under the same conditions (Fig. 3Biii), and was instead smooth like the wild-type strain (Fig. 3Bi), indicating that this conclusion was only partially accurate. By introducing a marked ∆binK deletion (∆binK::Cm) into two ∆sypE deletion mutants, the original strain KV3299 (∆sypE1) and one that had a different derivation (∆sypE3), we were able to create two additional ∆binK ∆sypE mutants to investigate the underlying reason of the distinct indications. When compared to the original double mutant, the latter mutant formed smooth colonies (Fig. 3Bv), whereas the former produced wrinkled colonies (Fig. 3Bii and and iv). Together, their findings revealed that the wrinkled colony phenotype could not be produced by interrupting both binK and sypE. As a result, it appeared likely that KV3299, the sypE strain used to create the first double mutant, had a secondary mutation (marked by an asterisk in the above figure).
In conclusion, this study improves our understanding of how V. fischeri controls biofilm formation by discovering a trio of negative regulators whose coordinated actions inhibit biofilm growth in the absence of calcium supplementation. Additionally, SypF, a protein whose activity is necessary for biofilm formation, exhibits a negative regulatory activity according to this study. Together, these results highlight the significance of biofilm regulation for V. fischeri and make the circumstances evident in which signal transduction mechanisms can be studied. Whether it is for understanding bioluminiscence and quorum sensing in-depth, or even developing new antibiotics to combat the infections caused by V. fischeri, it can be useful to pursue research on signal trandsuction mechanisms.
About the Author:
Bineeta Debnath ’23 is a Biochemistry major from Dhaka, Bangladesh. After Mount Holyoke she is going to work as a Research Specialist at the Silhavy Lab in Princeton, NJ. She aspires to obtain a phD degree in Microbiology in the future. Outside of academics, she loves to dance and drink bubble tea.
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