Constitutive Expression of the Proteorhodopsin Gene by a Flavobacterium Strain Representative of the Proteorhodopsin-Producing Microbial Community in the North Sea

Abstract

Proteorhodopsin (PR), a photoactive proton pump containing retinal, is present in approximately half of all bacteria in the ocean, but its physiological role is still unclear, since very few strains carrying the PR gene have been cultured. The aim of this work was to characterize PR diversity in a North Sea water sample, cultivate a strain representative of North Sea PR clusters, and study the effects of light and carbon concentration on the expression of the PR gene. A total of 117 PR sequences, of which 101 were unique, were obtained from a clone library of PCR-amplified PR gene fragments. Of the North Sea PRs, 97% were green light absorbing, as inferred from the amino acid at position 105; 67% of the PR protein fragments showed closest similarity to PRs from Alphaproteobacteria, 4% showed closest similarity to PRs from Gammaproteobacteria, and 29% showed closest similarity to PRs from "Bacteroidetes"/Flavobacteria. The dominant PR cluster (comprising 18% of all PRs) showed a high degree of similarity to the PR from the cultivated Roseobacter strain HTCC2255. The relative abundances of the North Sea PR clusters were confirmed by quantitative PCR. They were detected in metagenomic fragments from coastal oceans worldwide with various degrees of abundance. Several hundred bacterial strains from the North Sea water sample were cultivated on oligocarbophilic media. By screening with degenerate primers, two strains carrying the PR gene were identified. Their 16S rRNA gene sequences were identical and affiliated with a Bacteroidetes subcluster from the North Sea. The PR sequence of isolate PRO95 was completed by chromosomal walking. It was 76% identical to that of Dokdonia donghaensis MED134 and was functional, as indicated by the signature amino acids. PRO95 expressed its PR gene in liquid media containing between 9.7 and 121 mM carbon, both in the light and in the dark. Growth was not enhanced by light. Thus, the detection of the physiological role of PR may require more sensitive methods.

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

“…However, previous light incubation experiments using this organism did not detect a light-enhanced growth response (Riedel et al, 2010;. The PR peptide of DSW-1 T differs from that of MED134 in only one amino acid while the identity with that of PRO95 is 74%.…”

“…Similarly to MED134, this bacterium grows better in the light compared with dark in a low organic matter medium (Yoshizawa et al, 2014). In contrast to these two Dokdonia (S1-08 and MED134), another strain of the same genus, PRO95, has not yet shown enhanced growth or increased PR gene expression in the light compared to the dark (Riedel et al, 2010). PRO95 also has more than one rhodopsin type and, according to sequence similarities, one functions to pump H + and the other one to pump Na + ions (Yoshizawa et al, 2014, Bertsova et al, 2015.…”

Integrated metatranscriptomic and metagenomic analyses of stratified microbial assemblages in the open ocean

“…Notably, PR genes were found most highly expressed in the 75 m sample (collected at 22 h), followed by the 25 m and 125 m samples (collected at 3 h and 6 h, respectively) (Supplementary Figure S5A; also see the Pelagibacter genome-wide gene expression analysis below), suggesting that PR genes may be constitutively expressed in the photic zone. Laboratory studies of PR-containing isolates, as well as a recently reported microcosm experiment have reported inconsistent observations, some suggesting constitutive PR expression (Giovannoni et al, 2005a;Riedel et al, 2010), whereas others suggesting light regulation of PR expression (Gó mez- Consarnau et al, 2007;Lami et al, 2009;Poretsky et al, 2009). Higher-resolution metatranscriptomic studies are necessary to provide further insight into light effects on PR gene expression in different taxa, and in different oceanographic provinces.…”

Sizing up metatranscriptomics

“…Similar arguments can be made regarding the assumption that protein abundance is a reliable proxy for cellular rates, as post-translational regulation of protein activity and concentration of substrate both strongly affect catalysis rates. For instance, expression of bacterial enzymes can be constitutive and therefore unlinked to environmental signals (for example, proteorhodopsin in marine Flavobacteria and SAR11, and DMSP lyases in marine roseobacters; Curson et al, 2008;Riedel et al, 2010;Steindler et al, 2011); induced proteins may outlive the resources they were synthesized to exploit (for example, in microscale substrate patches or plumes that dissipate in within minutes; Stocker et al, 2008); and proteins expressed in response to a scarcity may actually be most abundant when reaction rates are lowest (for example, ammonium or phosphate transporters during nutrient starvation; Gyaneshwar et al, 2005;Sowell et al, 2009). Even for pure cultures growing under laboratory conditions, systems biology-based analyses typically show that protein levels cannot be readily correlated with metabolic flux (Ovacik and Androulakis, 2008).…”