TfoX-Based Genetic Mapping Identifies Vibrio fischeri Strain-Level Differences and Reveals a Common Lineage of Laboratory Strains

Abstract

Bacterial strain variation exists in natural populations of bacteria and can be generated experimentally through directed or random mutation. The advent of rapid and cost-efficient whole-genome sequencing has facilitated strain-level genotyping. Even with modern tools, however, it often remains a challenge to map specific traits to individual genetic loci, especially for traits that cannot be selected under culture conditions (e.g., colonization level or pathogenicity). Using a combination of classical and modern approaches, we analyzed strain-level variation in Vibrio fischeri and identified the basis by which some strains lack the ability to utilize glycerol as a carbon source. We proceeded to reconstruct the lineage of the commonly used V. fischeri laboratory strains. Compared to the wild-type ES114 strain, we identify in ES114-L a 9.9-kb deletion with endpoints in tadB2 and glpF; restoration of the missing portion of glpF restores the wild-type phenotype. The widely used strains ESR1, JRM100, and JRM200 contain the same deletion, and ES114-L is likely a previously unrecognized intermediate strain in the construction of many ES114 derivatives. ES114-L does not exhibit a defect in competitive squid colonization but ESR1 does, demonstrating that glycerol utilization is not required for early squid colonization. Our genetic mapping approach capitalizes on the recently discovered chitin-based transformation pathway, which is conserved in the Vibrionaceae; therefore, the specific approach used is likely to be useful for mapping genetic traits in other Vibrio species.I dentifying relevant differences in bacterial strains is fundamental to determining the genetic basis of microbial phenotypes. In many cases, the number of polymorphisms between strains is so high that elucidating which locus or loci contribute to specific phenotypes cannot be achieved simply by determining the genome sequence of the isolates. This challenge is especially pronounced in identifying loci that contribute to colonization and/or pathogenicity phenotypes. The study of genomic islands has made it clear that the acquisition of large regions of DNA can profoundly influence a bacterium's ability to engage with a eukaryotic host (1, 2). Recently, it has become increasingly apparent that defined genetic changes in bacteria at individual loci, single genes, or even nucleotide changes have led to dramatic effects in the evolution of colonizing bacteria. As some examples, the acquisition of the nil locus in Xenorhabdus nematophila contributed to the species-specific association with the worm host Steinernema carpocapsae, inactivation of the RscA biofilm regulator was critical in the evolution of Yersinia pestis from Yersinia pseudotuberculosis, and acquisition of the biofilm regulation of RscS facilitated colonization of north Pacific squid by Vibrio fischeri (3-8).In most cases, identification of factors that contribute to host colonization specificity has relied first on identifying the factor as being necessary for host colonization by standard g...

Modernized Tools for Streamlined Genetic Manipulation and Comparative Study of Wild and Diverse Proteobacterial Lineages

“…Laboratory domestication is commonly used to improve genetic tractability or help discern specific bacterial strains within complex polymicrobial environments (e.g., the vertebrate intestine, water, or soil). However, domestication can alter bacterial physiology and impair behaviors involved in host colonization ( https://doi.org/10.6084/m9.figshare.7040267 [Text S1]) ( 62 – 64 ). Exemplifying this, some laboratory-derived lineages of Vibrio fischeri , which is the light organ symbiont of the Hawaiian bobtail squid ( Euprymna scolopes ), have lost the ability to utilize glycerol as a carbon source (possibly the unintended result of prolonged growth on rich media) and display attenuated host colonization (as a consequence of antibiotic-based domestication) ( 64 ).…”

Host-selected mutations converging on a global regulator drive an adaptive leap towards symbiosis in bacteria

“…This purified product was cloned into pCR2.1 TOPO and transformed into TOP10 cells (Invitrogen, Waltham, MA), following the manufacturer’s protocol. Putative clones were sequenced by the Sanger method with primers M13 F, M13 R, KanINF, KanINR ( Table 4 ) at Genewiz in South Plainfield, NJ before the construct, RF2B7, was used for allelic exchange with a modified chitin competence protocol ( Brooks et al, 2015 ). Briefly, V. fischeri cells were grown in minimal media with a chitin derivative (n-acetyl glucosamine) until they reached OD 600 0.2.…”

Genetic Manipulation of Vibrio fischeri

“…To introduce large PCR‐amplified fragments into the bacterium, a multistep mutagenesis can introduce individual (selectable) pieces of DNA, or the DNA can be introduced on a plasmid via conjugation. Although large pieces of chromosomal DNA (>10 kb) can be taken up and incorporated by V. fischeri , an exact upper limit has not been empirically determined (Brooks, Gyllborg, Kocher, Markey, & Mandel, 2015).…”