iBet uBet web content aggregator. Adding the entire web to your favor.
iBet uBet web content aggregator. Adding the entire web to your favor.



Link to original content: https://pubmed.ncbi.nlm.nih.gov/20305030/
Constitutive expression of the proteorhodopsin gene by a flavobacterium strain representative of the proteorhodopsin-producing microbial community in the North Sea - PubMed Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 May;76(10):3187-97.
doi: 10.1128/AEM.02971-09. Epub 2010 Mar 19.

Constitutive expression of the proteorhodopsin gene by a flavobacterium strain representative of the proteorhodopsin-producing microbial community in the North Sea

Thomas Riedel  1 , Jürgen Tomasch, Ina Buchholz, Jenny Jacobs, Mario Kollenberg, Gunnar Gerdts, Antje Wichels, Thorsten Brinkhoff, Heribert Cypionka, Irene Wagner-Döbler
Affiliations

Constitutive expression of the proteorhodopsin gene by a flavobacterium strain representative of the proteorhodopsin-producing microbial community in the North Sea

Thomas Riedel et al. Appl Environ Microbiol. 2010 May.

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.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Rarefaction analysis (A) and Chao richness estimation (B) for PR sequences retrieved from the North Sea near the island of Helgoland. All 117 nucleotide sequences were grouped into OTUs based on 0% divergence (unique sequences) and 6 and 15% divergence cutoffs. Rarefaction curves and Chao richness estimation curves are labeled according to the cutoff value.
FIG. 2.
FIG. 2.
Neighbor-joining tree based on PR protein sequences isolated from North Sea surface water near the island of Helgoland showing the phylogenetic distribution of PRs in the North Sea. Bootstrap values (for 1,000 replicates) of >80% are indicated by filled circles at the branches. The scale bar shows the number of amino acid substitutions per site. For comparison, PR sequences from isolates and clones from the North Atlantic and Pacific Oceans and the Baltic, Mediterranean, Red, and Arctic Seas have been included. Clone and isolate names are colored according to the relationship of the corresponding PRs to PRs from Bacteroidetes, Alphaproteobacteria, and Gammaproteobacteria. Clusters of more than 8 sequences within these groups are highlighted. The outer ring shows the predicted absorption of green or blue light as inferred from the amino acid sequence. The GenBank accession numbers of the sequences used for the alignment are as follows: Helgoland (HEL) PR clones (HEL1 to HEL161), FJ560751 to FJ560867; Halobacterium salinarum, BAA01801.1; Polaribacter dokdonensis, ZP_01054176.1; Polaribacter irgensii, ZP_01117885.1; Psychroflexis torquis, ZP_01253360.1; D. donghaensis MED134, ZP_01049273; EBACHOT4E07 (SAR86 clade), AAT38609.1; HOT2C01, AAR05342.1; HOT75m4, AAK30179.1; HOT0m1, AAK30176.1; Photobacterium sp. SKA34, ZP_01161099.1; EBAC20E09 (SAR86 clade), AAS73014.1; EBAC31A08, AAG10475.1; PalE6, AAK30200.1; EBACmed18B02 (SAR11 clade), AAY82751.1; MEDeBAC46A06 (SAR11 clade), AAY82845.1; “Ca. Pelagibacter ubique,” AAZ21446.1; RED23, AAO21449.1; HTCC2207 (SAR92 clade), ABO88140.1; BTC1A0mJuly, ACB55057.1; Gsee1D, ACB55102.1; NASB75, ABU49527.1; NASB90, ABU49536.1; NASB58, ABU49514.1; NASB18, ABU49502.1; NASB64, ABU49517.1; NASB41, ABU49506.1; NASB71, ABU49523.1; NASB91, ABU49537.1; NASB33, ABU49505.1; NASB28, ABU49498.1; NASB31, ABU49455.1; NASB20, ABU49456.1; MS0242A, ABM91108.1; MedPR13e07, AAY68041.1; and PRO95, ACM89772.1. Flavo, flavobacteria.
FIG. 3.
FIG. 3.
Presence and relative abundance of Helgoland PR clusters in global metagenomic databases. Sampling sites represented in the GOS, Antarctica, Botany Bay, and Monterey Bay metagenomic databases are ordered and color coded by geographic location as indicated on the vertical axis. A detailed description of the sampling sites can be found at http://camera.calit2.net/. The relative abundance of PRs [log2(number of PR sequences/Gb)] from Helgoland clusters (horizontal axis) is visualized by the sizes of the circles. Note the data for the nonmarine Panama Channel and the hypersaline Galapagos Islands lake sample, where the only PR is the HTTC2255 PR-like protein (highlighted by arrows).
FIG. 4.
FIG. 4.
Phylogenetic positions of strains PRO95 and PRO100 within the Flavobacteriaceae based on 16S rRNA gene comparisons. Phylogenetic analyses of 16S rRNA gene sequences were performed with the ARB software package (24). Only sequences longer than 1,400 bp were considered in the calculations. The tree was generated using the neighbor-joining method. Bootstrap values indicated at the nodes were derived from 1,000 replicates. Only values higher than 50% are shown. Filled circles indicate nodes also recovered reproducibly with maximum likelihood. Selected members of the Gammaproteobacteria were used as an out-group (data not shown). Bar, 0.05 substitutions per nucleotide position.
FIG. 5.
FIG. 5.
Alignment of the PR of PRO95 with the PR of D. donghaensis MED134 and cloned PR sequences. Predicted transmembrane helices are marked by boxes. Gray shading indicates positions of conserved residues. Key amino acids for PR functionality (listed herein with EBAC31A08 numbering) are marked by colors: Lys131 (K) binds retinal, and Asp97 (D) and Glu108 (E) function as Schiff base proton acceptor and donor, respectively. Position 105 (§) plays a role in spectral tuning. Gln at position 105 (blue box) leads to absorption maxima at ∼490 nm. Met, Leu, Val, and Ala at this position (green box) result in absorption maxima at 518 to 535 nm. The GenBank accession numbers of the sequences used for the alignment are as follows: D. donghaensis MED134, ZP_01049273; EBAC31A08, AAG10475.1; MEDeBAC46A06 (SAR11 clade), AAY82845.1; “Ca. Pelagibacter ubique,” AAZ21446.1; REDr6a5a6, AAO21455.1; RED23, AAO21449.1; MEDeBAC49C08, AAY82659.1; EBACmed18B02 (SAR11 clade), AAY82751.1; HOT75m4, AAK30179.1; and PalE6, AAK30200.1.
FIG. 6.
FIG. 6.
Effects of carbon concentration and light on PR expression in PRO95 as determined by qRT-PCR. Bacteria were cultivated under the conditions indicated. Nutrients were peptone and yeast extract in the ratio 5:1. RNA was extracted, cDNA was generated, and the abundance of PR transcripts in relation to that of the 16S rRNA gene was determined (see Materials and Methods for details). Means and standard deviations for two to four independent biological replicas are shown. The standard error for technical replicas was ∼1% (data not shown).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410. - PubMed
    1. Atamna-Ismaeel, N., G. Sabehi, I. Sharon, K. P. Witzel, M. Labrenz, K. Jurgens, T. Barkay, M. Stomp, J. Huisman, and O. Beja. 2008. Widespread distribution of proteorhodopsins in freshwater and brackish ecosystems. ISME J. 2:656-662. - PubMed
    1. Beja, O., L. Aravind, E. V. Koonin, M. T. Suzuki, A. Hadd, L. P. Nguyen, S. B. Jovanovich, C. M. Gates, R. A. Feldman, J. L. Spudich, E. N. Spudich, and E. F. DeLong. 2000. Bacterial rhodopsin: evidence for a new type of phototrophy in the sea. Science 289:1902-1906. - PubMed
    1. Beja, O., E. N. Spudich, J. L. Spudich, M. Leclerc, and E. F. DeLong. 2001. Proteorhodopsin phototrophy in the ocean. Nature 411:786-789. - PubMed
    1. Bowman, J. P., S. A. McCammon, M. V. Brown, D. S. Nichols, and T. A. McMeekin. 1997. Diversity and association of psychrophilic bacteria in Antarctic sea ice. Appl. Environ. Microbiol. 63:3068-3078. - PMC - PubMed

Publication types

Associated data

LinkOut - more resources