NagC represses N-acetyl-glucosamine utilization genes in Vibrio fischeri within the light organ of Euprymna scolopes

Abstract

Bacteria often use transcription factors to regulate the expression of metabolic genes in accordance to available nutrients. NagC is a repressor conserved among γ-proteobacteria that regulates expression of enzymes involved in the metabolism of N-acetyl-glucosamine (GlcNAc). The polymeric form of GlcNAc, known as chitin, has been shown to play roles in chemotactic signaling and nutrition within the light organ symbiosis established between the marine bacterium Vibrio fischeri and the Hawaiian squid Euprymna scolopes. Here, we investigate the impact of NagC regulation on the physiology of V. fischeri. We find that NagC repression contributes to the fitness of V. fischeri in the absence of GlcNAc. In addition, the inability to de-repress expression of NagC-regulated genes reduces the fitness of V. fischeri in the presence of GlcNAc. We find that chemotaxis toward GlcNAc or chitobiose, a dimeric form of GlcNAc, is independent of NagC regulation. Finally, we show that NagC represses gene expression during the early stages of symbiosis. Our data suggest that the ability to regulate gene expression with NagC contributes to the overall fitness of V. fischeri in environments that vary in levels of GlcNAc. Furthermore, our finding that NagC represses gene expression within the squid light organ during an early stage of symbiosis supports the notion that the ability of the squid to provide a source of GlcNAc emerges later in host development.

Keywords: Euprymna scolopes; N-acetyl-glucosamine; NagC; Vibrio; symbiosis.

FIGURE 1

Model of gene regulation by NagC in V. fischeri. NagC represses the expression of genes, including nagE, nagA, and nagB. GlcNAc-6P, the intracellular form of GlcNAc, inhibits DNA binding by NagC through allosteric regulation.

FIGURE 2

Response of nagA expression to GlcNAc in V. fischeri. Transcriptional response of various genes to GlcNAc in WT (ES114) harboring the reporter plasmids for nagA (pTM355) or luxI (pTM280) grown in TMM with the indicated level of GlcNAc. GFP/mCherry levels are normalized by WT cells grown in the absence of GlcNAc. Graphical points and error bars represent averages and SD of triplicate biological replicates, respectively. Experiment was performed three times, with similar results.

FIGURE 3

Impact of NagC on fitness in minimal medium. Relative competitive indices (RCI) of test strains WT (ES114; black), ΔnagC (WPK100; red), and nagCE241A (TIM381, blue) with control strain TIM302 (ES114 gfp). Each line represents an individual culture. Each experiment was performed twice, with similar results. (A) TMM. (B) TMM + 10 mM GlcNAc. (C) LBS. (D) TMM + 5 mM [GlcNAc]₂.

FIGURE 4

Response of nagA expression to [GlcNAc]₂ in V. fischeri. Transcriptional response of nagA to GlcNAc and (GlcNAc)₂ in WT (ES114) harboring the reporter plasmid for nagA (pTM355) grown in TMM. GFP/mCherry levels are normalized by WT cells grown without inducer. Graphical points and error bars represent averages and SD of triplicate biological replicates, respectively.

FIGURE 5

Impact of NagC on fitness in filter-sterilized seawater. RCI of test strains WT (ES114; black), ΔnagC (WPK100; red), and nagCE241A (TIM381, blue) with control strain TIM302 (ES114 gfp). Each line represents an individual culture. Each experiment was performed twice, with similar results. (A) FSSW. (B) FSSW + 10 mM GlcNAc.

FIGURE 6

Impact of NagC on chemotaxis toward GlcNAc and [GlcNAc]₂. Motility of WT (ES114); (A,D), ΔnagC (WPK100); (B,E), and nagCE241A (TIM381); (C,F) on TB-IO plates incubated at 28°C. Top panels show motility rings prior to placement of 5-μl volume of indicated compound. Bottom panels show migration 45 min after placement of compounds in the positions indicated by the dots. Arrows indicate the perturbation of the ring as a result of the supplemented compound.

FIGURE 7

NagC regulation of gene expression in squid light organ. (A) (L to R) DIC, GFP, mCherry, and merged images of squid light organ colonized with ΔnagC harboring the luxI transcriptional reporter plasmid pTM280. Bar represents 200 μm. (B) Binary mask image generated by applying a thresholding algorithm to the mCherry image in (A). The white pixels represent mCherry-expressing V. fischeri populations within the light organ shown in (A). (C) Gene expression levels in squid colonized by WT (ES114), ΔnagC (WPK100), and ΔnagC::nagCE241A (TIM381) harboring transcriptional reporter plasmids for nagA (pTM355) and luxI (pTM280). For each promoter, the expression levels of each sample are normalized by wild-type levels. Graphical and error bars represent averages and SD of at least four light organs, respectively. Experiment was performed twice, with similar results.

FIGURE 8

Model for NagC function in symbiosis. In nutrient poor conditions, including seawater, NagC represses gene expression. During initial colonization of the squid light organ, NagC continues to repress gene expression, despite the presence of a COS gradient that facilitates chemotaxis into the light organ pores (Mandel et al., 2012). Within the mature light organ, populations of V. fischeri use NagC to regulate gene expression in accordance with a diel rhythm involving COS (Wier et al., 2010; Schwartzman et al., 2015).