RESEARCH ARTICLE Unique communities ofanoxygenic phototrophic bacteria in saline lakes of Salar de Atacama (Chile): evidence fora new phylogenetic lineage of phototrophic Gammaproteobacteria from pufLM gene analyses Vera Thiel1, Marcus Tank1, Sven C. Neulinger1, Linda Gehrmann1, Cristina Dorador2 & Johannes F. Imhoff1 1Leibniz-Institut f ¨urMeereswissenschaften (IFM-GEOMAR), Kiel, Germany; and 2Departamento de Acuicultura, Facultad de Recursos del Mar, Downloaded from https://academic.oup.com/femsec/article/74/3/510/586152 by guest on 30 September 2021 Universidad de Antofagasta, Antofagasta, Chile Correspondence: Johannes F. Imhoff, Abstract Leibniz-Institut f ¨urMeereswissenschaften, D¨usternbrooker Weg 20, D-24105 Kiel, Phototrophic bacteria are important primary producers of salt lakes in the Salar de Germany. Tel.: 149 431 600 4450; fax: 149 Atacama and at times form visible mass developments within and on top of the 431 600 4452; e-mail: jimhoff@ifm- lake sediments. The communities of phototrophic bacteria from two of these lakes geomar.de were characterized by molecular genetic approaches using key genes for the biosynthesis of the photosynthetic apparatus in phototrophic purple bacteria Received 6 July 2010; revised 6 August 2010; (pufLM) and in green sulfur bacteria (fmoA). Terminal restriction fragment length accepted 6 August 2010. polymorphism of the pufLM genes indicated high variability of the community Final version published online 24 September composition between the two lakes and subsamples thereof. The communities 2010. were characterized by the dominance of a novel, so far undescribed lineage of DOI:10.1111/j.1574-6941.2010.00966.x pufLM containing bacteria and the presence of representatives related to known halophilic Chromatiaceae and Ectothiorhodospiraceae. In addition, the presence of Editor: Riks Laanbroek BChl b-containing anoxygenic phototrophic bacteria and of aerobic anoxygenic bacteria was indicated. Green sulfur bacteria were not detected in the environ- Keywords mental samples, although a bacterium related to Prosthecochloris indicum was anoxygenic phototrophic bacteria; Salar de identified in an enrichment culture. This is the first comprehensive description of Atacama; pufL; pufM; fmoA; functional gene. phototrophic bacterial communities in a salt lake of South America made possible only due to the application of the functional pufLM genes. reviewed in Imhoff (2001). Also, green sulfur bacteria have Introduction been observed in various saline environments mainly based on It has been known for long that the strongly saline environ- microscopic and macroscopic observations (Giani et al., 1989; ment is primarily a domain of prokaryotes and the spectrum Caumette, 1993; Oren, 1993). Species of the genus Prostheco- of eukaryotic species in highly saline biotopes is rather chloris were obtained from marine and saline environments restricted. The dominant primary producers are halophilic and are recognized as halotolerant and moderately halophilic and halotolerant algae and cyanobacteria as well as anoxygenic organisms (Gorlenko, 1970; Imhoff, 2001, 2003; Vila et al., phototrophic bacteria (Imhoff et al., 1979; Truper¨ & Galinski, 2002; Alexander & Imhoff, 2006; Triado-Margarit´ et al., 2010). 1986; Imhoff, 1988, 2001, 2002). A variety of anoxygenic Highly saline lakes in the extremely arid Atacama Desert phototrophic bacteria has been isolated from different hyper- located in northern Chile are characterized by high UV saline habitats, such as marine salterns (Rodriguez-Valera radiation, high salt concentrations and wide diurnal tem- et al., 1985; Caumette et al., 1988, 1991; Caumette, 1993), perature variations. The Salar de Atacama is located at an MICROBIOLOGY ECOLOGY MICROBIOLOGY alkaline soda lakes in the Egyptian Wadi Natrun (Imhoff & altitude of 2300 m and is the largest evaporitic basin in Chile Truper,¨ 1977, 1981; Imhoff et al., 1978), in Siberia and (2900 km2). It has several permanent hypersaline lakes that Mongolia (Bryantseva et al., 1999, 2000) and from Solar Lake receive waters from the Andes Range (Risacher & Alonso, (Sinai) (Cohen & Krumbein, 1977; Caumette et al., 1997) as 1996). Like other hypersaline environments, the studied c 2010 Federation of European Microbiological Societies FEMS Microbiol Ecol 74 (2010) 510–522 Published by Blackwell Publishing Ltd. All rights reserved APB of Salar de Atacama using functional genes 511 lakes of the Salar de Atacama (Laguna Chaxa and Laguna associated in a trimeric structure (Fenna et al., 1974). Its Tebenquiche) are inhabited by only a few higher organisms unique occurrence in green sulfur bacteria and the recently such as brine shrimps, some copepods and surrounding described ‘Candidatus Chloracidobacterium thermophilum’ macrophytes (Zu´ niga˜ et al., 1991). Visually, the shallow (Bryant et al., 2007) makes fmoA an appropriate target to Laguna Tebenquiche and Laguna Chaxa exhibit the presence specifically analyze environmental communities of these of extensive red–purple-colored microbial mats on the sur- bacteria (Alexander & Imhoff, 2006). 13 face of the lake sediments. Based on low d C(HCO3) values of À 1.38 for Laguna Chaxa measured in previous studies, biological productivity in these lakes is expected to be high Materials and methods (Boschetti et al., 2007). However, the content of chlorophyll a was shown to be rather low in previous studies (Demer- Study area Downloaded from https://academic.oup.com/femsec/article/74/3/510/586152 by guest on 30 September 2021 gasso et al., 2008), leading to the assumption of a consider- The Salar de Atacama is a closed saline basin within the pre- able impact of anoxygenic phototrophic bacteria on the Andean depression of the Atacama Desert located at 2000300S primary productivity in these habitats. Despite the visual and 6800150W in northern Chile and covers approximately indication, phototrophic bacteria have not been specifically 2900 km2 (Zu´ niga˜ et al., 1991; Demergasso et al., 2004). The studied and almost nothing is known about the diversity Atacama Desert is characterized by extreme aridity and is and composition of the communities of anoxygenic photo- considered to be one of the driest places on earth (Rech trophic bacteria in these lakes. Main studies of the micro- et al., 2006). The average amount of precipitation in the biology of the Salar de Atacama have been focused on the desert and the Salar area reaches o 3 and 25–50 mm yearÀ1, cultivable diversity. Heterotrophic strains of moderately respectively. The low precipitation together with an excep- halophilic bacteria have been analyzed by numerical taxon- tionally high evaporation of 1800–3200 mm yearÀ1 leads to a omy (Prado et al., 1991; Valderrama et al., 1991) and hyperarid ecosystem (Risacher et al., 2003; Boschetti et al., chemotaxonomic analysis (Marquez et al., 1993) and domi- 2007). Solar radiation is high, especially UV-B radiation, nated the isolation-based studies (Ramos-Cormenzana, which is 20% increased compared with that at sea level 1993; Campos, 1997). The only phototrophic bacteria so (Cabrera et al., 1995). far described in Laguna Tebenquiche (Salar de Atacama) The saline basin of the Salar de Atacama is covered with a were oxygenic cyanobacteria represented by Oscillatoria thick halite crust of several hundred meters (Bobst et al., (Zu´ niga˜ et al., 1991). Recently, the bacterial diversity in 2001). At its edges and in its interior, there are small ponds water samples of Laguna Tebenquiche has been studied by and a number of shallow lakes with high concentrations of ribosomal gene library analysis (Demergasso et al., 2008). salts, which receive streams of fresh subsurface water. However, sequences related to anoxygenic phototrophic Laguna Chaxa and Laguna Tebenquiche (the largest of these bacteria were not recovered, except for a single clone related lakes) are two of these hypersaline lakes receiving freshwater to the aerobic phototrophic purple bacteria of the Roseobac- from the subsurface (Zu´ niga˜ et al., 1991). The pH was only ter clade (Demergasso et al., 2008) with no evidence for any slightly alkaline (Table 1) and despite the shallow character phototrophic potential and activity. of the lakes, low dissolved oxygen concentrations have been In order to specifically study the communities of photo- measured in this (1.2 mg LÀ1) and in previous studies trophic prokaryotes of these habitats, we used molecular (0.6 mg LÀ1,Zu´ niga˜ et al., 1991; Boschetti et al., 2007). genetic analyses with group-specific primers for functional Sodium and chloride are the dominating ions, followed by genes (pufLM, fmoA), which target phototrophic bacterial sulfate (Risacher & Alonso, 1996). communities. Because they represent a physiological group of polyphyletic origin, it is not possible to recover the diversity of phototrophic communities using 16S rRNA Samples gene sequences. The pufLM genes encode for the light (L) and medium (M) subunit of the photosynthetic reaction Sediment samples were taken from two lakes at the Salar de center type II structural proteins of phototrophic purple Atacama, Laguna Tebenquiche and Laguna Chaxa located in bacteria including purple sulfur bacteria, purple nonsulfur the north and in the east of the Salar, respectively (Fig. 1). bacteria and aerobic anoxygenic phototrophic bacteria, as Four sediment samples were taken from Laguna Teben- well as Chloroflexaceae. These genes have been used pre- quiche (23.13S 68.24W; samples SAT1–SAT5) and six from viously to access phototrophic bacteria