Actinorhodopsin genes discovered in diverse freshwater habitats and among cultivated freshwater Actinobacteria

Abstract

Microbial rhodopsins are membrane proteins that utilize a retinal chromophore to harvest sunlight for energetic and photosensory functions. Recently, a group of novel rhodopsin sequences named 'actinorhodopsins' (ActRs) was hypothesized to exist among uncultured planktonic Actinobacteria. ActRs were discovered by mining metagenomic data obtained during the Venter Institute's Global Ocean Sampling expedition, from a hypersaline lagoon, two estuaries and a freshwater lake. On the basis of these findings, and many studies that show Actinobacteria are common inhabitants of lakes, we predicted that ActR genes would likely be present in other freshwater habitats and among the genomes of cultivated Actinobacteria. Using degenerate polymerase chain reaction primers, we discovered an ActR gene present in an actinobacterial isolate of the family Microbacteriaceae. Isolate MWH-Uga1 was cultivated prior to this study from a freshwater pond in Uganda and belongs to a group of Actinobacteria previously identified in freshwater ecosystems. ActR genes were also discovered present in numerous mixed cultures containing freshwater Actinobacteria and among environmental DNA samples obtained from three freshwater sources; a small woodland pond and the Laurentian Great Lakes Superior and Erie. An analysis of small subunit ribosomal RNA genes from metagenomic DNA samples harboring ActR genes suggests that organisms belonging to the acI lineage, an uncultured group of Actinobacteria commonly present in fresh waters, may utilize rhodopsins. The co-occurrence of an acI organism with a specific ActR variant in a mixed culture supports our hypothesis. The ISME Journal (2009) 3, 726-737; doi:10.1038/ismej.2009 published online 26 February 2009 Subject Category: microbial ecology and functional diversity of natural habitats

Comparative single-cell genomics reveals potential ecological niches for the freshwater acI Actinobacteria lineage

“…The latter possibility is contradicted by the apparent inability of acI to utilize carboxylic acids (see above), a major product of photochemical DOC reactions. Instead, previous work suggested that actinorhodopsins are broadly distributed in freshwater Actinobacteria and provide the potential for phototrophy (Sharma et al, 2008(Sharma et al, , 2009Martinez-Garcia et al, 2012;Garcia et al, 2013;Salka et al, 2014). We confirmed the presence of actinorhodopsin in all but three of the eleven acI SAGs (AAA023-J06, AAA028-A23 and AB141-P03) making this gene a likely part of the broadly shared genes of the acI lineage.…”

“…We identified the top 25 COGs that are overrepresented in the acI SAGs as compared with the 424 Actinomycetales reference genomes available in the IMG database in March 2013 (Table 3). Bacteriorhodopsin (COG5524) was the most overrepresented COG in this analysis, and represents the putative light-harvesting protein previously identified as actinorhodopsin (Sharma et al, 2008(Sharma et al, , 2009Martinez-Garcia et al, 2012;Wurzbacher et al, 2012). The aforementioned cyanophycinase (COG4242) was another COG overrepresented in the acI lineage.…”

Winter diversity and expression of proteorhodopsin genes in a polar ocean

“…Six primer pairs from Koh et al (2010) were used in combination with a seventh pair using the additional reverse primer (Supplementary Information; Table 1). It should be noted that sequences representative of the actinorhodopsin-like LG1 and LG2 groups (Sharma et al, 2008(Sharma et al, , 2009) present a TWXXYP region at the 3 0 end and would not be amplified by our set of reverse primers that are based on GWXXYP (Atamna-Ismaeel et al, 2008) and SWXFY regions. PCRs were performed with the hot start Platinum Taq DNA Polymerase (Invitrogen, Madrid, Spain).…”

“…Although degenerate primers can capture a wide range of PRs, other types of microbial rhodopsins may not be amplified (see Materials and Methods section). For example, our primers did not target a diverse group of rhodopsins, the actinorhodopsins, associated with Actinobacteria (Sharma et al, 2008(Sharma et al, , 2009.…”

GenGIS: A geospatial information system for genomic data

“…The initial publication ) analyzed 44 metagenome samples (0.1-0.8 mm fraction) collected from 41 sites, including Sargasso Sea sites examined previously by Venter et al (2004). Data from these locations have been analyzed to reveal an immense set of novel proteins and breadth of taxonomic and functional diversity in different habitats Yutin et al 2007;Zhang and Gladyshev 2008;Sharma et al 2009). …”