OPEN The ISME Journal (2017) 11, 2334–2344 www.nature.com/ismej ORIGINAL ARTICLE Unexpected genomic features in widespread intracellular bacteria: evidence for motility of marine chlamydiae Astrid Collingro1, Stephan Köstlbacher1, Marc Mussmann1, Ramunas Stepanauskas2, Steven J Hallam3,4,5,6,7 and Matthias Horn1 1Department of Microbial and Ecosystems Science, Division of Microbial Ecology, University of Vienna, Vienna, Austria; 2Bigelow Laboratory for Ocean Sciences, East Boothbay, ME, USA; 3Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada; 4Genome Science and Technology Program, University of British Columbia, Vancouver, British Columbia, Canada; 5Graduate Program in Bioinformatics, University of British Columbia, Vancouver, British Columbia, Canada; 6Peter Wall Institute for Advanced Studies, University of British Columbia, Vancouver, British Columbia, Canada and 7ECOSCOPE Training Program, University of British Columbia, Vancouver, British Columbia, Canada Chlamydiae are obligate intracellular bacteria comprising important human pathogens and symbionts of protists. Molecular evidence indicates a tremendous diversity of chlamydiae particularly in marine environments, yet our current knowledge is based mainly on terrestrial representatives. Here we provide first insights into the biology of marine chlamydiae representing three divergent clades. Our analysis of single-cell amplified genomes revealed hallmarks of the chlamydial lifestyle, supporting the ancient origin of their characteristic developmental cycle and major virulence mechanisms. Surprisingly, these chlamydial genomes encode a complete flagellar apparatus, a previously unreported feature. We show that flagella are an ancient trait that was subject to differential gene loss among extant chlamydiae. Together with a chemotaxis system, these marine chlamydiae are likely motile, with flagella potentially playing a role during host cell infection. This study broadens our view on chlamydial biology and indicates a largely underestimated potential to adapt to different hosts and environments. The ISME Journal (2017) 11, 2334–2344; doi:10.1038/ismej.2017.95; published online 23 June 2017 Introduction extracellular elementary body (Greub and Raoult, 2003; Horn et al., 2004; Schmitz-Esser et al., 2004; Investigating environmental relatives of bacterial Abdelrahman and Belland, 2005; Collingro et al., pathogens has been fundamental for understanding 2011; Nunes and Gomes, 2014). Chlamydiae gener- their evolution and biology, and the emergence of ally possess reduced genomes, lack essential bio- virulence (Horn, 2008; Gordon et al., 2009; Robins synthesis pathways and thus rely on a variety of and Mekalanos, 2014; Khodr et al., 2016; Maury metabolites from their eukaryotic hosts (Omsland et al., 2016). The chlamydiae include some of the et al., 2014). most successful bacterial pathogens of humans, such No free-living chlamydia has been identified to as Chlamydia trachomatis causing over 100 million date, but chlamydiae are frequently found within infections worldwide each year (Wright et al., 2008; protists and arthropods that are ubiquitous in Darville, 2013; Roulis et al., 2013; Dal Conte et al., terrestrial environments (Horn, 2008; Taylor-Brown 2014; Knittler and Sachse, 2015). They likely et al., 2015). Chlamydiae have also been identified in represent the most ancient group of obligate intra- a number of marine animals, including fish in which cellular bacteria and are characterized by a biphasic developmental cycle consisting of the replicative they have been associated with the gill disease intracellular reticulate body and the infectious epitheliocystis (Karlsen et al., 2008; Draghi et al., 2010; Mitchell et al., 2010; Fehr et al., 2013; Stride et al., 2013; Nylund et al., 2015; Steigen et al., 2015; Correspondence: M Horn, Department of Microbial and Ecosys- Pawlikowska-Warych and Deptuła, 2016). Moreover, tems Science, University of Vienna, 1090 Vienna, Austria. metagenomics and amplicon sequencing data indi- E-mail: [email protected] Received 16 January 2017; revised 8 May 2017; accepted 12 May cates the existence of a vast, yet unexplored diversity 2017; published online 23 June 2017 of divergent chlamydiae, primarily in marine Marine chlamydiae encode flagella A Collingro et al 2335 environments (Pizzetti et al., 2012; Lagkouvardos sequenced with NextSeq 500 (Illumina) in 2 × 150 bp et al., 2014; Pizzetti et al., 2015; Vanthournout and mode using v.1 reagents. The obtained sequence Hendrickx, 2015; Bou Khalil et al., 2016). However, reads were quality-trimmed with Trimmomatic owing to their obligate intracellular lifestyle our v0.32 4 using the following settings: -phred33 knowledge about chlamydiae is based on a limited LEADING:0 TRAILING:5 SLIDINGWINDOW:4:15 number of representatives, originating exclusively MINLEN:36. Human DNA (⩾95% identity to the from non-marine environments. Homo sapiens reference assembly GRCh38) and low Single-cell sorting in combination with genome complexity reads (containing o5% of any nucleo- amplification and sequencing has been used suc- tide) were removed. The remaining reads were cessfully to explore yet uncultured microbes digitally normalized with kmernorm 1.05 (http:// (Stepanauskas, 2012; Rinke et al., 2013). Here, we sourceforge.net/projects/kmernorm) and then applied this approach to study the biology of marine assembled with SPAdes v.3.0.0. Each end of the chlamydiae. Our findings suggest that while basic obtained contigs was trimmed by 100 bp, and then features of the chlamydial lifestyle are well con- only contigs longer than 2000 bp were retained. The served, marine chlamydiae show unique adapta- accuracy of the resulting assemblies was assessed by tions, indicating that the biology of chlamydiae is applying the same workflow on previously cultured much more variable than currently recognized. and sequenced strains of Prochlorococcus marinus and Escherichia coli and comparing the obtained benchmark SAG assemblies against reference gen- Materials and methods omes with QUAST (Gurevich et al., 2013), see public benchmark data sets on the SCGC website. North Sea Sampling and sequencing of single-cell amplified SAGs were screened for 16S rRNA genes by PCR genomes using primers 341 f and 907rev (Muyzer et al., 1993; Samples were collected from 100, 150 and 185 m Muyzer and Smalla, 1998). For the two SAGs depth intervals in Saanich Inlet a seasonally anoxic affiliated with chlamydiae (AG-110-P3 and AG-110- fjord on Vancouver Island, British Columbia (Station M15) 500 bp shotgun libraries were created with the S3 48°35′18.0″N, 123°30′13.2″W), on 9 August 2011. NEBNext Ultra DNA Library Prep Kit for Illumina Sample collection and biochemical measurements (NEB, Frankfurt, Germany). These libraries were were performed as previously described (Zaikova sequenced with a HiSeq2500 (Illumina) in et al., 2010; Roux et al., 2014). Water column redox 2 × 250 bp rapid mode at the Max Planck Genome conditions were typical for stratified summer Centre (MP-GC; Cologne, Cologne, Germany, http:// months. One milliliter aliquots were amended with mpgc.mpipz.mpg.de/home/). Reads were trimmed 5% glycerol and 1 × TE buffer (all final concentra- and adaptors were removed with bbduk (BBMap- tions), and stored at − 80 °C until further processing. 35.07 suite, https://sourceforge.net/projects/bbmap) Sediment samples were collected in October 2014 using a minimum quality cutoff of 25 and checked from the upper 5 cm of sublittoral sediment off the for quality with FastQC (Version 0.11.3, http://www. island of Sylt (North Sea, Germany, 55°05′38.9″N, 8° bioinformatics.bbsrc.ac.uk/projects/fastqc). Reads 15′48.8″E). After immediate transfer to the lab, the were assembled de novo using SPAdes 3.6.2 sediment was mixed and 1 ml was transferred to a (Bankevich et al., 2012) in single-cell mode (k-mer 50 ml plastic tube. After adding 3 ml of sterile- sizes: 21, 35, 43, 55, 63, 71, 81, 91, 99). Then all reads filtered seawater, slurries were vortexed at maximum were remapped against the genome scaffold (mini- speed for 3 min. Sand grains were allowed to settle mum sequence identity of 97%) and reassembled and the supernatant was filtered through a 5 μm iteratively with SPAdes 3.6.2. (Bankevich et al., pore-size membrane. The cell extracts were cryopre- 2012) as described previously (Mußmann et al., served with N,N,N-trimethylglycine (‘glycine 2017). Contigs larger than 2000 bp were retained betaine’) (Sigma-Aldrich, St Louis, MO, USA) at a and assembly quality was assessed with QUAST 3.0 final concentration of 4% according to Cleland et al. (Gurevich et al., 2013). (2004), and stored at − 80 °C until further processing. Single-cell sorting and whole-genome multiple displacement amplification were performed at the Genome annotation and analysis Bigelow Laboratory Single Cell Genomics Center For genome analysis only contigs 42 kb were used (https://scgc.bigelow.org; SCGC) as described pre- unless explicitly mentioned. The quality and con- viously (Swan et al., 2011). For Saanich Inlet, a total tamination of all three assembled SAGs was con- of 315 single cells per sample were subjected to trolled with CheckM 1.0.6 (Parks et al., 2015). multiple displacement amplification and the taxo- Automatic genome annotation was performed with nomic identity of single amplified genomes (SAGs) ConsPred 1.24 (Weinmaier et al., 2016). The chla- was determined by directly sequencing 16S rRNA mydial origin