Interference with the germination and growth of Ulva zoospores by quorum-sensing molecules from Ulva-associated epiphytic bacteria

Abstract

Ulva zoospores preferentially settle on N-acylhomoserine lactone (AHL) producing marine bacterial biofilms. To investigate whether AHL signal molecules also affect the success and rate of zoospore germination in addition to zoospore attraction, the epiphytic bacteria associated with mature Ulva linza were characterized and bacterial isolates representative of this community tested for the ability to produce AHLs. Two of these AHL-producing isolates, Sulfitobacter spp. 376 and Shewanella spp. 79, were transformed with plasmids expressing the Bacillus spp. AHL lactonase gene aiiA to generate AHL-deficient variants. The germination and growth of U. linza zoospores was studied in the presence of these AHL-deficient strains and their AHL-producing counterparts. This revealed that the AHLs produced by Sulfitobacter spp. and Shewanella spp. or the bacterial products they regulate have a negative impact on both zoospore germination and the early growth of the Ulva germling. Further experiments with Escherichia coli biofilms expressing recombinant AHL synthases and synthetic AHLs provide data to demonstrate that zoospores germinated and grown in the absence of AHLs were significantly longer than those germinated in the presence of AHLs. These results reveal an additional role for AHLs per se in the interactive relationships between marine bacteria and Ulva zoospores.

Figure 1

Effect of marine bacterial biofilms on Ulva zoospore germling growth.

1. A

   Average germling length of zoospores exposed to biofilms of AHL-expressing Shewanella sp. 79 pBBRIMCS (□) and non-AHL expressing Shewanella sp. 79 pMT01 (□) at 48 h incubation.

2. B

   Average germling length of zoospores exposed to biofilms of AHL expressing Sulfitobacter sp. 376 pBBRIMCS (□) and non-AHL expressing Sulfitobacter sp. 376 pMT01 (□) at 48 incubation. Error bars represent 95% confidence intervals.

Figure 2

Effect of recombinant E. coli biofilms on Ulva germling growth. Average Ulva germling length when exposed to biofilms of E. coli producing AHLs from plasmids expressing recombinant AHL synthases, in comparison with those from their E. coli counterparts harbouring the corresponding control vectors without the AHL syntheses after 24 h incubation. The plasmids used in this experiment are listed in Table S2. Error bars represent 95% confidence intervals and asterisks show those values significantly different to the controls (one-way ANOVA * = P ≤ 0.001).

Figure 3

Effect of exogenously added synthetic C12-HSL on Ulva germling growth. Average Ulva germling length when exposed to a range of C12-HSL concentrations at 48 h incubation. Error bars represent 95% confidence intervals and asterisks show those values that differed significantly from that of 0 μM AHL (one-way ANOVA * = P ≤ 0.001).