Description of ‘Candidatus Berkiella aquae’ and ‘Candidatus Berkiella cookevillensis’, two intranuclear bacteria of freshwater amoebae

Abstract

Two novel bacteria of the phylum <i>Proteobacteria</i> were isolated during searches for amoeba-resistant 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 <i>Acanthamoeba polyphaga</i>. 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, <i>mip</i> and <i>rpoB</i> genes, the DNA G+C content and the fatty acid composition demonstrated that both bacteria are members of the order <i>Legionellales</i>, and are most closely related to <i>Coxiella burnetii</i>. 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 '<i>Candidatus</i> Berkiella aquae' and '<i>Candidatus</i> Berkiella cookevillensis' are proposed.