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

"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.

ABSENCE OF BATRACHOCHYTRIUM SALAMANDRIVORANS IN A GLOBAL HOTSPOT FOR SALAMANDER BIODIVERSITY.

Batrachochytrium salamandrivorans (Bsal) is an emerging fungal pathogen that affects salamander and newt populations in Asia and Europe. In the Western Hemisphere, Bsal represents a major threat to endemic amphibian populations, which have not evolved resistance to infection, and which could experience local extinction events such as those observed in European fire salamanders (Salamandra salamandra). We report findings of a survey focusing specifically on wild lungless salamanders in the southeastern US, the most biodiverse location for salamander species globally. Between May 2016 and July 2018, we conducted 25 surveys at 10 sites across three ecoregions in Tennessee, US. Using quantitative (q)PCR, we screened water samples and skin swabs from 137 salamanders in five plethodontid genera. Although single replicates of six samples amplified during qPCR cycling, no samples could be confirmed as positive for the presence of Bsal with 28S rRNA PCR and independent laboratory screening. It is probable that we found false positive results, as reported by other researchers using the same assay. We offer recommendations for future monitoring efforts.

Infection and nuclear interaction in mammalian cells by 'Candidatus Berkiella cookevillensis', a novel bacterium isolated from amoebae.

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.

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.

Description of 'Candidatus Berkiella aquae' and 'Candidatus Berkiella cookevillensis', two intranuclear bacteria of freshwater amoebae.

Two novel bacteria of the phylum Proteobacteria 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 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.

Amoeba stages in the deepest branching heteroloboseans, including Pharyngomonas: evolutionary and systematic implications.

The taxon Heterolobosea (Excavata) is a major group of protists well known for its diversity of life stages. Most are amoebae capable of transforming into flagellates (amoeboflagellates), while others are known solely as flagellates or solely as amoebae. The deepest-branching heterolobosean taxon confirmed previously, Pharyngomonas, was generally assumed to be a pure flagellate, suggesting that the amoeba form arose later in the evolution of Heterolobosea sensu lato. Here we report that multiple isolates of Pharyngomonas are actually amoeboflagellates that also have cyst stages, with only amoebae transforming into cysts. The amoeba form of Pharyngomonas showed heterolobosean characteristics (e. g. eruptive movement), but also possessed unusual morphological features like slow-flowing crenulated hyaline crescents with conical subpseudopodia, finger-like projections and branching posterior extensions. Furthermore, phylogenetic analyses of 18S ribosomal RNA gene sequences that included two undescribed species of amoebae showed that Pharyngomonas is not the only deep-branching heterolobosean to possess an amoeba stage. These results suggest that possession of an amoeba stage was ancestral for Heterolobosea, unifying this taxon as a group of species with amoeba stages in their lifecycle or derived from organisms with such stages.

Interactions between food-borne pathogens and protozoa isolated from lettuce and spinach.

The survival of Salmonella enterica was recently shown to increase when the bacteria were sequestered in expelled food vacuoles (vesicles) of Tetrahymena. Because fresh produce is increasingly linked to outbreaks of enteric illness, the present investigation aimed to determine the prevalence of protozoa on spinach and lettuce and to examine their interactions with S. enterica, Escherichia coli O157:H7, and Listeria monocytogenes. Glaucoma sp., Colpoda steinii, and Acanthamoeba palestinensis were cultured from store-bought spinach and lettuce and used in our study. A strain of Tetrahymena pyriformis previously isolated from spinach and a soil-borne Tetrahymena sp. were also used. Washed protozoa were allowed to graze on green fluorescent protein- or red fluorescent protein-labeled enteric pathogens. Significant differences in interactions among the various protist-enteric pathogen combinations were observed. Vesicles were produced by Glaucoma with all of the bacterial strains, although L. monocytogenes resulted in the smallest number per ciliate. Vesicle production was observed also during grazing of Tetrahymena on E. coli O157:H7 and S. enterica but not during grazing on L. monocytogenes, in vitro and on leaves. All vesicles contained intact fluorescing bacteria. In contrast, C. steinii and the amoeba did not produce vesicles from any of the enteric pathogens, nor were pathogens trapped within their cysts. Studies of the fate of E. coli O157:H7 in expelled vesicles revealed that by 4 h after addition of spinach extract, the bacteria multiplied and escaped the vesicles. The presence of protozoa on leafy vegetables and their sequestration of enteric bacteria in vesicles indicate that they may play an important role in the ecology of human pathogens on produce.

Multiple strains of the parasitic dinoflagellate Amoebophrya exist in Chesapeake Bay.

Small subunit rRNA sequences were amplified from Amoebophrya strains infecting Karlodinium micrum, Gymnodinium instriatum and an unidentified Scrippsiella species in Chesapeake Bay. The alignable parts of the sequences differed from each other and from the previously reported rRNA sequence of the Amoebophrya strain infecting Akashiwo sanguinea in Chesapeake Bay by 4 to 10%. This is a greater degree of difference than sometimes found between sequences from separate genera of free-living dinoflagellates. These sequence differences indicate that the Amoebophrya strains parasitizing dinoflagellates in Chesapeake Bay do not all belong to the same species. In spite of their relative dissimilarity, the sequences do group together into a single clade with high bootstrap support in phylogenetic trees constructed from the sequences.