New insights into metabolic properties of marine bacteria encoding proteorhodopsins

Abstract

Proteorhodopsin phototrophy was recently discovered in oceanic surface waters. In an effort to characterize uncultured proteorhodopsin-exploiting bacteria, large-insert bacterial artificial chromosome (BAC) libraries from the Mediterranean Sea and Red Sea were analyzed. Fifty-five BACs carried diverse proteorhodopsin genes, and we confirmed the function of five. We calculate that proteorhodopsin-exploiting bacteria account for 13% of microorganisms in the photic zone. We further show that some proteorhodopsin-containing bacteria possess a retinal biosynthetic pathway and a reverse sulfite reductase operon, employed by prokaryotes oxidizing sulfur compounds. Thus, these novel phototrophs are an unexpectedly large and metabolically diverse component of the marine microbial surface water.

Figure 1. Phylogenetic Tree of PR Proteins from the Mediterranean and Red Seas along with PR Homologs in GenBank

The tree was divided into what we propose are distinct subfamilies of sequences, based on bootstrap values significance. The tree was constructed as follows: (i) All homologs of PR proteins were identified in GenBank including predicted proteins from the Sargasso Sea assemblies using BLASTp [36] searches with representatives of previously identified PR-like protein families as query sequences. (ii) All sequences greater than 300 nucleotides in length were aligned to each other using CLUSTALx [37], and a neighbor-joining phylogenetic tree was inferred using the neighbor programs of PAUP* [38]. Bootstrap resampling (1,000 pseudoreplications) of neighbor-joining and maximum parsimony trees were performed in all analyses to provide confidence estimation for the inferred topologies. Bootstraps values greater than 50% are indicated above the branches (neighbor-joining/maximum parsimony). The scale bar represents the number of substitutions per site. The sequences are colored according to the type of sample in which they were found: blue, cultured species; orange, sequences from uncultured organisms obtained using PCR-based methods; and red, BAC-derived sequences from uncultured species in the Mediterranean Sea and Red Sea (this study) or from previously reported Pacific, Antarctic, and Red Sea [1,7,8] BAC/fosmids. Black squares mark sequenced BACs in this study; red squares label BACs sequenced in previous reports. α, Alphaproteobacteria; γ, Gammaproteobacteria. Red circles mark the two abundant PR groups discussed in the manuscript.

Figure 2. Laser-Flash-Induced Absorbance Changes in Suspensions of E. coli Membranes Containing PR Proteins

(A–E) PR proteins are from BACs MED46A06, MED66A03, MED18B02, MED49C08, and MED13K09, respectively. A 532-nm pulse (6-ns duration, 40 mJ) was delivered at time 0, and absorption changes were monitored at wavelengths near the absorption maximum of the main absorption band in the visible range of the unphotolyzed pigment (520 nm for A and B, 480 nm for C–E) and the final photointermediate (the O intermediate) which is the longest-lived species in each of the photochemical reaction cycles (620 nm for A and B, 580 nm for C–E). 150–2,000 transients were collected at one to two flashes/sec and averaged for each trace as previously described [39]. The bar in each panel indicates the scale of the absorption change (× 10⁻³). Panel E exhibits greater noise because of the lower amplitudes of absorption changes due to lower expression level of the pigment. Insets: E. coli membranes containing PR apoproteins in 50 mM Tris-HCl (pH 9.0) were reconstituted with an ethanolic solution of 2 μM all-trans retinal. The retinal-reconstitution of PR pigments were recorded in a Cary 4000 spectrophotometer at room temperature. The spectra were taken 40 min after retinal addition, which produced between 0.035 to 0.078 absorption units at the absorption maxima indicated.

Figure 3. Dsr Operons in PR-Carrying BAC and Sargasso Sea Scaffolds

(A) Phylogenetic tree showing the affiliation of DsrAB from MED13K09. Alignment regions of insertions and deletions were omitted in DsrAB amino acid sequence analyses. Polytomic nodes connect branches for which a relative order could not be determined unambiguously by using distance-matrix (FITCH with the Dayhoff PAM matrix, global rearrangements, and randomized input order of species), maximum-parsimony, and maximum-likelihood (with JTT-f as the amino acid replacement model) methods. Maximum-parsimony bootstrap values (%) are indicated at each node (1,000 re-samplings). The bar represents 10% sequence divergence as estimated from distance-matrix analysis. α, Alphaproteobacteria; β, Betaproteobacteria; γ, Gammaproteobacteria. In total, nine Sargasso Sea shotgun clones contained complete (IBEA_CTG_1982486, AACY01045584; IBEA_CTG_2027414, AACY01063972) or partial (IBEA_CTG_UAAO864TF, AACY01493489; IBEA_CTG_SSBMN57TR, AACY01327066; IBEA_CTG_SKBEW15TR, AACY01199346; IBEA_CTG_2002781, AACY01059482; IBEA_CTG_1960714, AACY01122073; IBEA_CTG_2018072, AACY01005285; IBEA_CTG_UAAYT68TR, AACY01523913) dsrAB sequences that formed a monophyletic cluster with MED13K09 and A. vinosum. Whole-genome shotgun sequence data for Thiobacillus denitrificans, Magnetospirillum magnetotacticum, and Magnetococcus sp. MC-1 were produced by the US Department of Energy Joint Genome Institute (http://www.jgi.doe.gov/). The yet-uncompleted genome sequence of T. denitrificans contains a frame shift in dsrB. Dissimilatory (bi)sulfite reductase sequences of sulfate-/sulfite reducers were taken from Wagner et al.[40], Klein et al.[41], and Zverlov et al. [42]]. (B) Organization of the dsr operons on MED13K09, Sargasso Sea shotgun clones IBEA_CTG_2027414 and IBEA_CTG_1982486, and in A. vinosum, Chlorobium tepidum TLS, and the sulfate-reducer Archaeoglobus fulgidus. Asterisk indicates an authentic frame shift in the second copy of dsrB in the genome of C. tepidum.

Figure 4. Retinal Biosynthesis Pathways in PR-Carrying BACs

(A) Schematic comparison of different carotenoid biosynthesis gene clusters linked to PR genes. ORF marked in gray represent predicted carotenoid biosynthesis genes while PR is marked in black. (B) HPLC separation of the retinoids formed in the β-carotene producing E. coli and expressing the Blh protein. Left panel, extract from non-induced cells; right panel, after 60 min of induction (L-arabinose). Insights, absorption spectra of peaks 1 (β-carotene) and 2 (all-trans retinal). (C) Color shift due to the cleavage of β-carotene to retinal in E. coli cells. Color shift from orange (β-carotene; non induced) to almost white (retinal; L-arabinose induced cells) in β-carotene producing and accumulating E. coli cells caused by expression of the blh gene and, the same β-carotene producing cells co-expressing the blh and a PR gene; color shift to red (L-arabinose and IPTG induced cells).