The inside scoop: Comparative genomics of two intranuclear bacteria, “Candidatus Berkiella cookevillensis” and “Candidatus Berkiella aquae”

PLOS ONE RESEARCH ARTICLE The inside scoop: Comparative genomics of two intranuclear bacteria, “Candidatus Berkiella cookevillensis” and “Candidatus Berkiella aquae” Destaalem T. Kidane1,2, Yohannes T. Mehari3, Forest C. Rice2, Brock A. Arivett4, John H. Gunderson5, Anthony L. Farone1,2, Mary B. Farone ID1,2* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Molecular Biosciences Program, Middle Tennessee State University, Murfreesboro, TN, United States of America, 2 Department of Biology, Middle Tennessee State University, Murfreesboro, TN, United States of America, 3 Department of Biological Sciences, Auburn University, Auburn, AL, United States of America, 4 Division of Infectious Disease, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, United States of America, 5 Department of Biology, Tennessee Technological University, Cookeville, TN, United States of America * OPEN ACCESS Citation: Kidane DT, Mehari YT, Rice FC, Arivett BA, Gunderson JH, Farone AL, et al. (2022) The inside scoop: Comparative genomics of two intranuclear bacteria, “Candidatus Berkiella cookevillensis” and “Candidatus Berkiella aquae”. PLoS ONE 17(12): e0278206. https://doi.org/ 10.1371/journal.pone.0278206 Editor: Daniel E. Voth, University of Arkansas for Medical Sciences, UNITED STATES Received: August 5, 2022 Accepted: November 12, 2022 Published: December 30, 2022 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0278206 Copyright: © 2022 Kidane et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Genome sequence files of “Ca. B. cookevillensis” and “Ca. B. aquae” have been deposited at GenBank database under Abstract “Candidatus Berkiella cookevillensis” (strain CC99) and “Candidatus Berkiella aquae” (strain HT99), belonging to the Coxiellaceae family, are gram-negative bacteria isolated from amoebae in biofilms present in human-constructed water systems. Both bacteria are obligately intracellular, requiring host cells for growth and replication. The intracellular bacteria-containing vacuoles of both bacteria closely associate with or enter the nuclei of their host cells. In this study, we analyzed the genome sequences of CC99 and HT99 to better understand their biology and intracellular lifestyles. The CC99 genome has a size of 2.9Mb (37.9% GC) and contains 2,651 protein-encoding genes (PEGs) while the HT99 genome has a size of 3.6Mb (39.4% GC) and contains 3,238 PEGs. Both bacteria encode high proportions of hypothetical proteins (CC99: 46.5%; HT99: 51.3%). The central metabolic pathways of both bacteria appear largely intact. Genes for enzymes involved in the glycolytic pathway, the non-oxidative branch of the phosphate pathway, the tricarboxylic acid pathway, and the respiratory chain were present. Both bacteria, however, are missing genes for the synthesis of several amino acids, suggesting reliance on their host for amino acids and intermediates. Genes for type I and type IV (dot/icm) secretion systems as well as type IV pili were identified in both bacteria. Moreover, both bacteria contain genes encoding large numbers of putative effector proteins, including several with eukaryotic-like domains such as, ankyrin repeats, tetratricopeptide repeats, and leucine-rich repeats, characteristic of other intracellular bacteria. Introduction Free-living amoebae, found in natural and human-made aquatic environments, are predators of many bacteria, and thus play an important role in controlling microbial populations in the PLOS ONE | https://doi.org/10.1371/journal.pone.0278206 December 30, 2022 1 / 22 PLOS ONE Comparative genomics of two intranuclear bacteria accession numbers LKHV00000000 and LKAJ00000000, respectively. This project has been deposited at NCBI under the BioProject accession number PRJNA289553. Funding: This work was supported by the National Institute of General Medical Sciences at the National Institutes of Health (https://nigms.nih.gov/ ) grant R15GM131357-01 to MBF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. environment [1]. Some bacteria, however, have evolved to become resistant to predation by these protozoa. These amoeba resistant bacteria (ARB) have mechanisms that not only allow them to survive internalization and digestion by amoebae, but also allow them to replicate within the intra-amoebal environment [2, 3]. Both facultative and obligate intracellular ARB belonging to several evolutionary lineages, including alphaproteobacteria [4–6], betaproteobacteria [7], gammaproteobacteria [8, 9], Bacteroidetes [10] and Chlamydiae [11, 12] have been recovered from free-living amoebae. Among those identified, several ARB such as Legionella pneumophila and Mycobacterium avium are established human pathogens [13, 14] while others, including Parachlamydia acanthamoebae and the Legionella-like amoebal pathogens (LLAPs), have been designated as potential emerging human pathogens [15, 16]. “Candidatus Berkiella cookevillensis” (type strain CC99) and “Candidatus Berkiella aquae” (type strain HT99) are two ARB that were each isolated from an amoeba present in biofilm recovered from a hospital cooling tower and an outdoor hot tub spa, respectively [17]. Both bacteria are non-spore-forming, motile, obligate intracellular gram-negative bacteria. CC99 bacteria are coccoid shaped with diameters ranging from 0.30 to 0.60 μm whereas HT99 are coccobacilli with a width ranging from 0.30–0.55μm and length ranging from 0.45–0.65μm (Fig 1) [17]. Based on 16S rRNA gene phylogenetic analyses and cellular fatty acid composition Fig 1. Protozoa infected with “Ca. B. cookevillensis” (CC99) or “Ca. B. aquae” (HT99). Electron micrograph (13,000x magnification) of intracellular CC99 (arrow) exhibiting coccoid morphology following exposure of the BCV by the tape ripping technique (A). Micrograph of adherent HT99 (arrow) on the surface of A. polyphaga exhibiting coccobacillus morphology (20,000x magnification) (B). Giemsa staining of D. discoideum (strain AX2) infected with CC99 (C) and HT99 (D) showing bacteria (arrow; dark purple) associated with nuclei (pink). https://doi.org/10.1371/journal.pone.0278206.g001 PLOS ONE | https://doi.org/10.1371/journal.pone.0278206 December 30, 2022 2 / 22 PLOS ONE Comparative genomics of two intranuclear bacteria analyses, CC99 and HT99 have been classified as separat (...truncated)