Open access
18 February 2016

Draft Genome Sequences of Two Novel Amoeba-Resistant Intranuclear Bacteria, “Candidatus Berkiella cookevillensis” and “Candidatus Berkiella aquae”


Candidatus Berkiella cookevillensis” and “Candidatus Berkiella aquae” are obligate intranuclear endosymbionts of freshwater amoebae. Here, we present the draft genome sequences of these two bacteria, with total sizes of 2,990,361 bp and 3,626,027 bp, respectively.


Two novel bacteria were isolated from freshwater amoebae found in aquatic biofilms in Cookeville, TN, USA. “Candidatus Berkiella cookevillensis” strain CC99 was recovered from an infected amoeba from a cooling tower, and “Candidatus Berkiella aquae” strain HT99 was collected from an infected amoeba in an outdoor hot tub. The 16S rRNA gene sequences were 100% (CC99) and 96% (HT99) similar to sequences from uncultured organisms in the NCBI GenBank database. CC99 had 100% identity to an uncultured isolate from a South Korean oyster shell waste pile. Preliminary analyses showed 94% 16S rRNA sequence similarity between the two isolates and <92% similarity to Legionella or Coxiella species (1). Similar bacteria resembling Legionella spp. have been recovered from free-living amoebae, some of which are associated with respiratory disease (2). However, unlike Legionella spp., both bacteria invade and replicate in the host nucleus, resulting in complete lysis of the host cells within 2 to 4 days.
The bacteria were maintained in the laboratory by continuous passage in Acanthamoeba polyphaga. For genome sequencing, the bacteria were purified from infected amoebae by Renografin density gradient centrifugation, as described by Shannon and Heinzen (3). Total DNA was extracted using the DNeasy blood and tissue kit (Qiagen, Valencia, CA). Whole-genome sequencing was performed using a 250-bp paired-end MiSeq platform (Illumina, San Diego, CA) at the Institute for Genome Sciences (University of Maryland School of Medicine, Baltimore, MD). A total of 11,839,536 and 11,009,736 reads were generated for CC99 and HT99, respectively. The reads were subsampled to genome coverages of between 60 and 200× in 10× increments and assembled de novo with MaSuRCA (4). For “Ca. Berkiella cookevillensis” strain CC99, 44 contigs were generated, for a combined draft genome size of 2,990,285 bp, with a G+C content of 37.9%. The draft genome of “Ca. Berkiella aquae” strain HT99 contains 56 contigs, with a total length of 3,625,323 bp and a G+C content of 39.5%.
Prokka and the IGS Annotation Engine were used for structural and functional annotation of the sequences (5, 6), and Manatee was used to view and curate the annotations ( For isolate CC99, 2,598 genes were predicted, with 2,557 predicted coding sequences (CDSs). For HT99, 3,231 genes and 3,189 CDS regions were identified. Using RNAmmer and tRNAscan-SE, the CC99 genome is predicted to contain 3 rRNA and 40 tRNA genes, while the HT99 genome is predicted to contain 3 rRNA and 41 tRNA genes.
Also noteworthy is the existence of several of the dot or icm-type IV secretion system (T4SS) genes in both isolates, which is important for the pathogenesis of both Legionella pneumophila and Coxiella burnetii, bacteria similar to CC99 and HT99 (7, 8). The new draft genome sequences from both “Ca. Berkiella cookevillensis” and “Ca. Berkiella aquae” will allow for more comprehensive phylogenetic analyses of these novel bacteria and greater understanding of their intranuclear invasion and growth. The intranuclear lifestyles of the bacteria in amoebae might also enhance horizontal gene transfer between organisms, consistent with the idea of amoebae as evolutionary “melting pots” (9).

Nucleotide sequence accession numbers.

These whole-genome shotgun projects, associated with BioProject PRJNA289553 (“Novel amoeba-resistant bacteria”), have been deposited at DDBJ/EMBL/GenBank as Berkiella cookevillensis CC99 under the accession no. LKHV00000000 and Berkiella aquae HT99 under the accession no. LKAJ00000000. The versions described in this paper for “Ca. Berkiella cookevillensis” strain CC99 and “Ca. Berkiella aquae” strain HT99 are versions LKHV01000000 and LKAJ01000000, respectively.


This work has been supported by grants from the U.S. Environmental Protection Agency Science to Achieve Results (STAR) grant program (grants R827111 and R833102) and FRCAC grants from Middle Tennessee State University, and by continued support from the Biology Department at Middle Tennessee State University and the Department of Biology and Center for the Management, Utilization and Protection of Water Resources at Tennessee Technological University.
We thank the Institute for Genome Sciences Annotation Engine service at the University of Maryland School of Medicine for providing structural and functional annotation of the sequences.


Mehari YT, Hayes BJ, Redding KS, Mariappan PV, Gunderson JH, Farone AL, Farone MB. 2015. Description of “Candidatus Berkiella aquae” and “Candidatus Berkiella cookevillensis,” two intranuclear bacteria of freshwater amoebae. Int J Syst Evol Microbiol, in press.
Lamoth F, Greub G. 2010. Amoebal pathogens as emerging causal agents of pneumonia. FEMS Microbiol Rev 34:260–280.
Shannon JG, Heinzen RA. 2008. Infection of human monocyte-derived macrophages with Coxiella burnetii. Methods Mol Biol 431:189–200.
Zimin AV, Marçais G, Puiu D, Roberts M, Salzberg SL, Yorke JA. 2013. The MaSuRCA genome assembler. Bioinformatics 29:2669–2677.
Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068–2069.
Galens K, Orvis J, Daugherty S, Creasy HH, Angiuoli S, White O, Wortman J, Mahurkar A, Giglio MG. 2011. The IGS standard operating procedure for automated prokaryotic annotation. Stand Genomic Sci 4:244–251.
Segal G, Purcell M, Shuman HA. 1998. Host cell killing and bacterial conjugation require overlapping sets of genes within a 22-kb region of the Legionella pneumophila genome. Proc Natl Acad Sci USA 95:1669–1674.
Zamboni DS, McGrath S, Rabinovitch M, Roy CR. 2003. Coxiella burnetii express type IV secretion system proteins that function similarly to components of the Legionella pneumophila Dot/Icm system. Mol Microbiol 49:965–976.
Boyer M, Yutin N, Pagnier I, Barrassi L, Fournous G, Espinosa L, Robert C, Azza S, Sun S, Rossmann MG, Suzan-Monti M, La Scola B, Koonin EV, Raoult D. 2009. Giant Mareseillevirus highlights the role of amoebae as a melting pot in emergence of chimeric microorganisms. Proc Natl Acad Sci USA 106:21848–21853.

Information & Contributors


Published In

cover image Genome Announcements
Genome Announcements
Volume 4Number 125 February 2016
eLocator: 10.1128/genomea.01732-15


Received: 5 January 2016
Accepted: 6 January 2016
Published online: 18 February 2016



Yohannes T. Mehari
Department of Biology, Middle Tennessee State University, Murfreesboro, Tennessee, USA
Brock A. Arivett
Department of Biology, Middle Tennessee State University, Murfreesboro, Tennessee, USA
Anthony L. Farone
Department of Biology, Middle Tennessee State University, Murfreesboro, Tennessee, USA
John H. Gunderson
Department of Biology, Tennessee Technological University, Cookeville, Tennessee, USA
Mary B. Farone
Department of Biology, Middle Tennessee State University, Murfreesboro, Tennessee, USA


Address correspondence to Mary B. Farone, [email protected].

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