Genetic examination of the marine bacterium Pseudoalteromonas luteoviolacea and effects of its metamorphosis-inducing factors

Abstract

Pseudoalteromonas luteoviolacea is a globally distributed marine bacterium that stimulates the metamorphosis of marine animal larvae, an important bacteria-animal interaction that can promote the recruitment of animals to benthic ecosystems. Recently, different P. luteoviolacea isolates have been shown to produce two stimulatory factors that can induce tubeworm and coral metamorphosis; Metamorphosis-Associated Contractile structures (MACs) and tetrabromopyrrole (TBP) respectively. However, it remains unclear what proportion of P. luteoviolacea isolates possess the genes encoding MACs, and what phenotypic effect MACs and TBP have on other larval species. Here, we show that 9 of 19 sequenced P. luteoviolacea genomes genetically encode both MACs and TBP. While P. luteoviolacea biofilms producing MACs stimulate the metamorphosis of the tubeworm Hydroides elegans, TBP biosynthesis genes had no effect under the conditions tested. Although MACs are lethal to larvae of the cnidarian Hydractinia symbiologicarpus, P. luteoviolacea mutants unable to produce MACs are capable of stimulating metamorphosis. Our findings reveal a hidden complexity of interactions between a single bacterial species, the factors it produces and two species of larvae belonging to different phyla.

Fig 1.

Diverse Pseudoalteromonas luteoviolacea strains encode the biosynthesis genes for TBP and MACs. (A) MACs gene cluster from strain HI1. The filled arrows denote the genes interrogated by the blast and are previously shown to be necessary for MACs function. The red filled arrow represents the macB gene, which is knocked out to create the nonfunctional MACs (Shikuma et al., 2014) strain used in the biofilm metamorphosis assays. (B) Maximum likelihood phylogeny of 19 sequenced P. luteoviolacea genomes including Pseudoalteromonas sp. ATCC 29581 as an outgroup. The bootstrap values represent 100 re-samples. The banded boxes indicate highly conserved subgroups for which symbol representations apply throughout the group. The blue pentagon denotes strains that produce TBP. Hollow pentagons represent strains that encode some genes in the bmp gene cluster, but do not experimentally produce halogenated compounds. Purple arrows indicate the presence of MACs genes (macB, macS, macT, and mif1).

Fig 2.

Pseudoalteromonas luteoviolacea HI1 wild type produces TBP, while the Δbmp2 strain does not. (A) Genomic arrangement of the bmp gene cluster in strain HI1. Bolded genes bmp1–4 have previously been validated to code for TBP biosynthesis. The blue bolded gene bmp2 was deleted to create a nonfunctional TBP mutant in P. luteoviolacea. The gene outlined in red bmp8 is a pseudogene. (B) Representative ion chromatogram (EIC = 381.67) overlaid the comparison of an organic extraction of wild type and Δbmp2 strains to the TBP standard. Cultures were grown in MB and SWT media overnight in triplicate to quantify TBP production in P. luteoviolacea (see Fig. S1a).

Fig 3.

Pseudoalteromonas luteoviolacea stimulates Hydractinia metamorphosis in the absence of MACs. (A) Schematic of larval and metamorphosis phenotypes scored for Hydractinia larvae. All metamorphosis assays are performed in 96-well plates with either (B) the addition of synthesized TBP or (C) monoculture biofilms of P. luteoviolacea wild type or mutant strains. Phenotypic response of Hydractinia larvae to treatments containing (B) increasing concentrations of purified TBP and (C) P. luteoviolacea wild type or mutant biofilms. The bars represent the average of three biological replicates (n = 3). Values for the biological replicates were determined by averaging four technical replicates per treatment. The biological replicates were performed on different batches of larvae on different days. Error bars denote standard deviation. Asterisks above the bars denote statistical significance compared to the (B) ACN and (C) ASW controls. ACN is a 2% (v/v) acetonitrile solvent control. ASW is a filtered artificial seawater and is used as the negative control in all assays. DAG (1,2-Diocanoyl-sn-glycerol) is a chemical stimulant of metamorphosis and is used as a control for the competency of Hydractinia larvae at a concentration of 100 μM. (c) Statistical analyses were performed with a one-way ANOVA corrected for multiple comparisons by false discovery rate (FDR) using the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli (GraphPad Prism), where ***p = 0.0029, and ****p = 0.0006. No statistical difference was found between treatments ΔmacB and ΔmacBΔbmp2.

Fig 4.

Pseudoalteromonas luteoviolacea MACs stimulate the metamorphosis of Hydroides larvae. (A) Schematic of larval and metamorphosis phenotypes scored for Hydroides. Metamorphosis assays are performed as described previously (Shikuma et al., 2014). Phenotypic response of Hydroides larvae to treatments containing (B) increasing concentrations of purified TBP and (C) mutant biofilms. The bars represent the average of (B) five biological replicates (n = 5) and (C) three biological replicates (n = 3). A statistical power analysis aided with the determination for the appropriate number of biological replicates. Values for the biological replicates were found by averaging four technical replicates per treatment. The biological replicates were performed on different batches of larvae on different days. Error bars denote standard deviation. Asterisks above the bars denote statistical significance compared to the (B) ACN and (C) ASW controls. Statistical analyses were performed with a (B) Kruskal–Wallis ANOVA and (C) one-way ANOVA both corrected for multiple comparisons by FDR using the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli (GraphPad Prism), where ***p = 0.0054, and ****p < 0.0001.