Proteorhodopsin light-enhanced growth linked to vitamin-B1 acquisition in marine Flavobacteria

Abstract

Proteorhodopsins (PR) are light-driven proton pumps widely distributed in bacterioplankton. Although they have been thoroughly studied for more than a decade, it is still unclear how the proton motive force (pmf) generated by PR is used in most organisms. Notably, very few PR-containing bacteria show growth enhancement in the light. It has been suggested that the presence of specific functions within a genome may define the different PR-driven light responses. Thus, comparing closely related organisms that respond differently to light is an ideal setup to identify the mechanisms involved in PR light-enhanced growth. Here, we analyzed the transcriptomes of three PR-harboring Flavobacteria strains of the genus Dokdonia: Dokdonia donghaensis DSW-1(T), Dokdonia MED134 and Dokdonia PRO95, grown in identical seawater medium in light and darkness. Although only DSW-1(T) and MED134 showed light-enhanced growth, all strains expressed their PR genes at least 10 times more in the light compared with dark. According to their genomes, DSW-1(T) and MED134 are vitamin-B1 auxotrophs, and their vitamin-B1 TonB-dependent transporters (TBDT), accounted for 10-18% of all pmf-dependent transcripts. In contrast, the expression of vitamin-B1 TBDT was 10 times lower in the prototroph PRO95, whereas its vitamin-B1 synthesis genes were among the highest expressed. Our data suggest that light-enhanced growth in DSW-1(T) and MED134 derives from the use of PR-generated pmf to power the uptake of vitamin-B1, essential for central carbon metabolism, including the TCA cycle. Other pmf-generating mechanisms available in darkness are probably insufficient to power transport of enough vitamin-B1 to support maximum growth of these organisms.

Figure 1

Venn diagram showing the genes shared by the Dokdonia strains DSW-1^T, MED134 and PRO95. Numbers indicate the genes that were unique for each organism as well as the ones shared by two or the three strains. The diagrams were made using the program eulerAPE (http://www.eulerdiagrams.org/eulerAPE/).

Figure 2

Genomic region where the vitamin-B₁ synthesis operon of PRO95 is located. Green arrow shows the thiamin pyrophosphate riboswitch and in red are the vitamin-B₁-synthesis genes. The genomic area around this operon is shared by the three Dokdonia strains although the synthesis genes are only present in PRO95.

Figure 3

Diagram of the de novo synthesis pathway of vitamin-B₁. Green, orange and red boxes indicate the presence of the specific genes in DSW-1^T, MED134 and PRO95, respectively. The red dashed lines indicate transport functions. Question marks indicate that those genes have not been found but would be expected to be present in the genomes.

Figure 4

Normalized mean mRNA read counts of the Dokdonia transcriptomes. (a) MED134, (b) DSW-1^T and (c) PRO95. In red are the reads that are significantly higher in light or dark (P-adj<0.05) regardless of the light/dark fold change value. Horizontal black lines denote the linear fold change levels of 2 and 0.5. Circled green and dark blue dots denote the PR and NaR transcript reads, respectively. Transcript sequence data were normalized by sequencing depth as described in the DESeq package (see Materials and methods).

Figure 5

(a) Total transcripts that were significantly higher in light (in yellow) or dark (in black) in DSW-1^T, MED134 and PRO95. Significantly higher expression was considered when the linear fold change in light vs dark transcripts was larger than 2 or lower than 0.5 and the P-adjusted values were<0.05. Venn diagrams show the number of transcripts that were significantly higher in light (b) or dark (c) for each strain exclusively, as well as the number of transcripts shared by two or the three organisms in the two treatments. The Venn diagrams were made using the program eulerAPE (http://www.eulerdiagrams.org/eulerAPE/).

Figure 6

(a) Gene expression levels of pmf-dependent processes. Gray shading denotes the dark treatment transcriptome samples. (b) Average number of transcripts within light and dark treatments of vitamin-B₁ (thiamin pyrophosphate)-dependent enzymes as well as vitamin-B₁ transport and synthesis in DSW-1^T, MED134 and PRO95. mRNA sequence data were normalized by sequencing depth as described in the DESeq package (see Materials and methods).

Figure 7

Hypothetical scenarios of vitamin-B₁ uptake and utilization in the context of PR phototrophy.