Two novel bacteria of the phylum Proteobacteria were isolated during searches for amoebaresistant micro-organisms in natural and constructed water systems. Strain HT99 was isolated from amoebae found in the biofilm of an outdoor hot tub in Cookeville, Tennessee, USA, and strain CC99 was isolated from amoebae in the biofilm of a cooling tower in the same city. Both bacteria were Gram-stain-negative cocci to coccobacilli, unculturable on conventional laboratory media, and were found to be intranuclear when maintained in Acanthamoeba polyphaga. The genomes of both isolates were completely sequenced. The genome of CC99 was found to be 3.0 Mbp with a 37.9 mol% DNA G+C content, while the genome of HT99 was 3.6 Mbp with a 39.5 mol% DNA G+C content. The 16S rRNA gene sequences of the two isolates were 94 % similar to each other. Phylogenetic comparisons of the 16S rRNA, mip and rpoB genes, the DNA G+C content and the fatty acid composition demonstrated that both bacteria are members of the order Legionellales, and are most closely related to Coxiella burnetii. The phenotypic and genetic evidence supports the proposal of novel taxa to accommodate these strains; however, because strains HT99 and CC99 cannot be cultured outside of the amoeba host, the respective names 'Candidatus Berkiella aquae' and 'Candidatus Berkiella cookevillensis' are proposed.Free-living amoebae are natural environmental predators of bacteria and other micro-organisms found in soil and water, including anthropogenic systems such as drinking water systems, air conditioners and cooling towers, spas and swimming pools (Rodríguez-Zaragoza, 1994;Benkel et al., 2000;Greub & Raoult, 2004;Berk et al., 2006). Some bacteria only survive engulfment by amoebae whereas other bacteria not only survive but multiply within the amoebal host (Barker & Brown, 1994;Horn & Wagner, 2004;Molmeret et al., 2005). Recent reviews list over 40 genera of bacterial symbionts of amoebae that are human pathogens, making amoebae important environmental reservoirs of virulent bacteria (Lamoth & Greub, 2010;Moliner et al., 2010). Evidence also suggests that amoebae can aid in the selection of virulence traits and the adaptation of bacteria for intracellular survival in human macrophages and other cell types (Cirillo et al., 1994;Greub & Raoult, 2004;Evstigneeva et al., 2009). This report describes two bacterial isolates, HT99 and CC99, that were recovered from amoebae in biofilm samples from constructed aquatic environments (Berk et al., 2006). Both bacteria are amoebaresistant, intranuclear bacteria. We have determined, on the basis of both genotypic and phenotypic characteristics, that these two bacteria do not belong to known genera, justifying the proposal of novel taxa. However, due to the intracellular lifestyle of the bacteria and the lack of pure cultures, we propose the classification of these two bacteria as representatives of 'Candidatus Berkiella aquae' and 'Candidatus Berkiella cookevillensis' and in this report describe the characteristics of both novel bacteria.Bacter...

“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.

Background ‘ Candidatus Berkiella cookevillensis’ and ‘ Ca. Berkiella aquae’ have previously been described as intranuclear bacteria of amoebae. Both bacteria were isolated from amoebae and were described as appearing within the nuclei of Acanthamoeba polyphaga and ultimately lysing their host cells within 4 days. Both bacteria are Gammaproteobacteria in the order Legionellales with the greatest similarity to Coxiella burnetii. Neither bacterium grows axenically in artificial culture media. In this study, we further characterized ‘ Ca. B. cookevillensis’ by demonstrating association with nuclei of human phagocytic and nonphagocytic cell lines. Results Transmission electron microscopy (TEM) and confocal microscopy were used to confirm nuclear co-localization of ‘ Ca. B. cookevillensis’ in the amoeba host A. polyphaga with 100% of cells having bacteria co-localized with host nuclei by 48 h. TEM and confocal microscopy demonstrated that the bacterium was also observed to be closely associated with nuclei of human U937 and THP-1 differentiated macrophage cell lines and nonphagocytic HeLa human epithelial-like cells. Immunofluorescent staining revealed that the bacteria-containing vacuole invaginates the nuclear membranes and appears to cross from the cytoplasm into the nucleus as an intact vacuole. Conclusion Results of this study indicate that a novel coccoid bacterium isolated from amoebae can infect human cell lines by associating with the host cell nuclei, either by crossing the nuclear membranes or by deeply invaginating the nuclear membranes. When associated with the nuclei, the bacteria appear to be bound within a vacuole and replicate to high numbers by 48 h. We believe this is the first report of such a process involving bacteria and human cell lines. Electronic supplementary material The online version of this article (10.1186/s12866-019-1457-z) contains supplementary material, which is available to authorized users.

“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.