Comparative analyses of actinobacterial genomic fragments from Lake Kinneret Alon Philosof, Gazalah Sabehi and Oded Béjà* Faculty of Biology, Technion – Israel Institute of Technology, Haifa 32000, Israel. Summary The high genomic G+C group of Actinobacteria pos- sesses a variety of physiological and metabolic prop- erties, and exhibits diverse lifestyles and ecological distribution. In recent years, Actinobacteria have been found to frequently dominate samples obtained from freshwater samples. Furthermore, phylogenetic analy- ses have shown that 16S rRNA genes from uncultured actinobacterial freshwater samples cluster in four dis- tinct lineages. While these lineages are abundant, little is known about them and currently no pure-culture representatives or genomic fragments of them are available. In a screen of a genomic library from the moderately eutrophic freshwater Lake Kinneret, five fosmid clones containing actinobacterial genomic fragments were found. Three ~40 kb genomic frag- ments were chosen for sequencing. Fosmids K003 and K005 showed high similarity and were affiliated with the acIV actinobacterial freshwater lineage. Fosmid K004 was affiliated with the highly abundant acI lineage. A comparative genomic analysis revealed high synteny between the two freshwater clones K003 and K005 but a lower synteny between these two and the K004 fosmid. Fosmids K003 and K005 share an identical arrangement of arginine biosynthesis gene while K004 showed a slightly different arrangement by lacking the argF gene. Fosmid Ant4E12, an Antarctic actinobacterial clone, showed a higher synteny with K003/5 than K004 and a similar arginine operon, but in a different genomic context. The Clusters of Ortholo- gous Groups categories assignment of the three fosmids yielded genes that were mostly involved in amino acid and nucleotide metabolism, as well as transport and ribosomal RNA translation, structure and biogenesis. These genomic fragments represent the first sequences to be published from these lin- eages, providing a cornerstone for future work on this environmentally dominant group. Introduction Actinobacteria, a high G+C content Gram-positive phylum, ranging between 51% in some corynebacteria and more than 70% in Streptomyces and Frankia (Ventura et al., 2007), are abundant in diverse environ- ments, including extreme habitats, soil, marine and fresh- water (Glöckner et al., 2000; Rondon et al., 2000; Zwart et al., 2002; Cottrell et al., 2005; Ward and Bora, 2006). This phylum possesses a wide variety of physiological and metabolic properties, such as the production of extra- cellular enzymes and a variety of secondary metabolites, including many potent antibiotics (Goodfellow and Will- iams, 1983; Ward and Bora, 2006; Ventura et al., 2007). Furthermore, members of the Actinobacteria phylum exhibit diverse lifestyles, including pathogens, nitrogen- fixing symbionts, plant commensals, gastrointestinal tract inhabitants and more (Ventura et al., 2007). Actinobacteria are common in a variety of freshwater habitats and are distributed globally (Hiorns et al., 1997; Zwart et al., 2002; Sekar et al., 2003; Warnecke et al., 2005). In addition, Actinobacteria comprise the majority of bacterioplankton in many freshwater habitats (Glöckner et al., 2000; Hahn et al., 2003; Allgaier and Grossart, 2006). Phylogenetic analyses based on partial and nearly complete 16S rRNA gene sequences revealed distinct phylogenetic lineages of freshwater Actinobacteria (Zwart et al., 2002; Warnecke et al., 2004). The four lineages, acI-IV (Warnecke et al., 2004), appear to be diverse, con- taining sequences originating from different habitats such as rivers, lakes and estuaries (Warnecke et al., 2004; Allgaier and Grossart, 2006). Lineage acI, probably the most abundant of the four, is distributed globally in highly diverse environments and currently has no isolates in culture (Warnecke et al., 2004; 2005; Lindström et al., 2005; Allgaier and Grossart, 2006; Allgaier et al., 2007; Newton et al., 2007). This lineage is divided into sub- clusters, which appear to relate to ecophysiological differences (Newton et al., 2007). The acII lineage, also globally distributed, contains several ultramicrobial isolates in two of its subclusters (acII-B and acII-D) (Ludemann and Conrad, 2000; Hahn et al., 2003; Warnecke et al., 2004; Hahn and Pockl, 2005). Current sequences of the acIII lineage were obtained from hyper- saline, humic and soda lakes. (Humayoun et al., 2003; Warnecke et al., 2004; Newton et al., 2006; Wu et al., Received 25 November, 2008; accepted 26 June, 2009. *For corre- spondence. E-mail beja@tx.technion.ac.il; Tel. (+972) 4829 3961; Fax (+972) 4822 5153. Environmental Microbiology (2009) 11(12), 3189–3200 doi:10.1111/j.1462-2920.2009.02024.x © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd