Natural Strain Variation Reveals Diverse Biofilm Regulation in Squid-Colonizing Vibrio fischeri

Abstract

The mutualistic symbiont Vibrio fischeri builds a symbiotic biofilm during colonization of squid hosts. Regulation of the exopolysaccharide component, termed Syp, has been examined in strain ES114, where production is controlled by a phosphorelay that includes the inner membrane hybrid histidine kinase RscS. Most strains that lack RscS or encode divergent RscS proteins cannot colonize a squid host unless RscS from a squid symbiont is heterologously expressed. In this study, we examine V. fischeri isolates worldwide to understand the landscape of biofilm regulation during beneficial colonization. We provide a detailed study of three distinct evolutionary groups of V. fischeri and find that while the RscS-Syp biofilm pathway is required in one of the groups, two other groups of squid symbionts require Syp independent of RscS. Mediterranean squid symbionts, including V. fischeri SR5, colonize without an RscS homolog encoded by their genome. Additionally, group A V. fischeri strains, which form a tightly related clade of Hawaii isolates, have a frameshift in rscS and do not require the gene for squid colonization or competitive fitness. These same strains have a frameshift in sypE, and we provide evidence that this group A sypE allele leads to an upregulation in biofilm activity. Thus, this work describes the central importance of Syp biofilm in colonization of diverse isolates and demonstrates that significant evolutionary transitions correspond to regulatory changes in the syp pathway. IMPORTANCE Biofilms are surface-associated, matrix-encased bacterial aggregates that exhibit enhanced protection to antimicrobial agents. Previous work has established the importance of biofilm formation by a strain of luminous Vibrio fischeri bacteria as the bacteria colonize their host, the Hawaiian bobtail squid. In this study, expansion of this work to many natural isolates revealed that biofilm genes are universally required, yet there has been a shuffling of the regulators of those genes. This work provides evidence that even when bacterial behaviors are conserved, dynamic regulation of those behaviors can underlie evolution of the host colonization phenotype. Furthermore, this work emphasizes the importance of investigating natural diversity as we seek to understand molecular mechanisms in bacteria.A fundamental question in studying host-associated bacterial communities is understanding how specific microbial taxa assemble reproducibly in their host. Key insights into these processes were first obtained by studying plant-associated microbes, and the discovery and characterization of Nod factors in rhizobia were valuable to understanding how partner choice between microbe and host could be mediated at the molecular level (1, 2). There are complex communities in humans and other vertebrate animals, yet metagenomic and imaging analyses of these communities have Citation Rotman ER,

Multiplexed competition in a synthetic squid light organ microbiome using barcode-tagged gene deletions

“…Plasmids are listed in Table 2, and DNA oligonucleotides are listed in Table S2. The unmarked deletion of copA in MJM1100 was made by allelic exchange as described previously (73). Briefly, 1.6 kb upstream (US) and 1.6 kb downstream (DS) sequences of copA were amplified by PCR using oligos HB44 and HB45, and HB46 and HB47, respectively, and were cloned using Gibson Assembly (NEBuilder HiFi DNA assembly cloning kit) into the linearized vector pEVS79 (linearized using oligos HB52 and HB53) ( Table S2).…”

“…The middle dsDNA fragment containing the erm cassette flanked by FRT sites and the randomized barcode was obtained by PCR with Phusion Hot Start Flex 2X master mix (NEB; M0536L) and pHB1 as template, which contains the LL-FRT-erm-FRT-spacer sequences and was built as described previously (73), and oligos HB42 and HB154. Oligo HB154 is a reverse primer and contains the RL sequence, 18 bp of randomized sequence composed of 6 trimers of 'NNB' to prevent formation of stop codons (results in 'VNN' codons in the forward direction), and the spacer sequence (Table S2) Fig.…”

“…Previous studies have shown that multiple strains can co-colonize the squid LO and that they do so at different rates(70)(71)(72). More recent studies have focused on deciphering the specific mechanisms that result in differing colonization behavior(73,74). However, the large number of V. fischeri strains make the study of the evolution of colonization mechanisms labor-intensive and prohibitive.…”

“…The barcode-tagged mutagenesis method presented here can be applied to generate uniquely-tagged WT or mutant strains of the various phylogenetically-distinct V.fischeri strains and assayed in multiplexed format during squid colonization using BarSeq. We have already successfully used our mutagenesis approach to make deletions in the ancestral strain SR5, showing that this method is applicable to V. fischeri strains that are evolutionarily distant to the frequently used ES114 strain(73).BarSeq can also be used in directed evolution experiments to test the function of genetic variation of colonization factors. Directed evolution experiments have recently been applied to study colonization factors in V. fischeri(75).…”

Reports from a Healthy Community: the 7th Conference on Beneficial Microbes

“…S. praecaptivus is also able to colonize tsetse flies through a mechanism that requires quorum sensing, suggesting that quorum sensing regulation of host colonization is likely a conserved trait. On the topic of connecting natural diversity to evolving host interactions, Ella Rotman (Northwestern University, Chicago, IL) and Katherine Bultman (University of Wisconsin-Madison, Madison, WI) presented a poster on surprising diversity in symbiotic biofilm regulation, entitled "Distinct Biofilm Regulatory Strategies across the Vibrio fischeri Evolutionary Tree" (28).…”