Quorum Sensing and Cyclic di-GMP Exert Control Over Motility of Vibrio fischeri KB2B1.

Courtney N Dial,Christopher J Corcoran,Cheryl A Whistler,Alice H Tischler,Randi Foxall,Robert M Bolz,Steven J Eichinger,Karen L Visick

doi:10.3389/fmicb.2021.690459

Courtney N Dial, Christopher J Corcoran + Show 6 more

Open Access

https://doi.org/10.3389/fmicb.2021.690459

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Abstract

Bacterial motility is critical for symbiotic colonization by Vibrio fischeri of its host, the squid Euprymna scolopes, facilitating movement from surface biofilms to spaces deep inside the symbiotic organ. While colonization has been studied traditionally using strain ES114, others, including KB2B1, can outcompete ES114 for colonization for a variety of reasons, including superior biofilm formation. We report here that KB2B1 also exhibits an unusual pattern of migration through a soft agar medium: whereas ES114 migrates rapidly and steadily, KB2B1 migrates slowly and then ceases migration. To better understand this phenomenon, we isolated and sequenced five motile KB2B1 suppressor mutants. One harbored a mutation in the gene for the cAMP receptor protein (crp); because this strain also exhibited a growth defect, it was not characterized further. Two other suppressors contained mutations in the quorum sensing pathway that controls bacterial bioluminescence in response to cell density, and two had mutations in the diguanylate cyclase (DGC) gene VF_1200. Subsequent analysis indicated that (1) the quorum sensing mutations shifted KB2B1 to a perceived low cell density state and (2) the high cell density state inhibited migration via the downstream regulator LitR. Similar to the initial point mutations, deletion of the VF_1200 DGC gene increased migration. Consistent with the possibility that production of the second messenger c-di-GMP inhibited the motility of KB2B1, reporter-based measurements of c-di-GMP revealed that KB2B1 produced higher levels of c-di-GMP than ES114, and overproduction of a c-di-GMP phosphodiesterase promoted migration of KB2B1. Finally, we assessed the role of viscosity in controlling the quorum sensing pathway using polyvinylpyrrolidone and found that viscosity increased light production of KB2B1 but not ES114. Together, our data indicate that while the two strains share regulators in common, they differ in the specifics of the regulatory control over downstream phenotypes such as motility.

Highlights

The symbiosis between Vibrio fischeri and its host, the squid Euprymna scolopes, is a well-established model system used to understand bacterial processes governing colonization [reviewed in Nyholm and McFall-Ngai (2004); Stabb (2006) and Bongrand and Ruby (2019)]
V. fischeri cells use chemotaxis and motility to migrate into pores, through ducts and chambers that are non-permissive for growth, to reach spaces deep inside; there, they proceed to multiply to a high cell density and produce luminescence, the product of the symbiosis (Ruby and Asato, 1993; Sycuro et al, 2006)
In comparative analyses that sought to identify in vitro phenotypic differences between the well-characterized wild-type squid symbiont ES114 and the recently isolated colonizationdominant strain KB2B1, we found that the latter strain exhibited unusual migration on soft agar medium (Figure 2 and Supplementary Videos 1, 2)

Summary

INTRODUCTION

The symbiosis between Vibrio fischeri and its host, the squid Euprymna scolopes, is a well-established model system used to understand bacterial processes governing colonization [reviewed in Nyholm and McFall-Ngai (2004); Stabb (2006) and Bongrand and Ruby (2019)]. Mutants defective for motility fail to colonize squid, while those defective for luminescence exhibit a defect in the overall colonization levels within 48 h and are lost from symbiosis over time (Graf et al, 1994; Visick et al, 2000; Lupp et al, 2003; Millikan and Ruby, 2003; Lupp and Ruby, 2005; Koch et al, 2014) These two phenotypes, luminescence and motility, are known to be linked in V. fischeri: certain regulators that control luminescence impact motility (Lupp and Ruby, 2005; Miyashiro et al, 2010, 2014; Cao et al, 2012). Our studies revealed that while KB2B1 shares regulators and regulatory mechanisms in common with ES114, it appears to exert control over downstream processes such as motility in a distinct manner

RESULTS

A Diguanylate Cyclase Gene Contributes to the KB2B1 Motility Phenotype

DISCUSSION