Life in an unusual intracellular niche a bacterial symbiont infecting the nucleus of amoebae Frederik Schulz, Ilias Lagkouvardos, Florian Wascher, Karin Aistleitner, Rok Kostanjšek, Matthias Horn Supplementary Information Supplementary Text S1 Description of Candidatus Nucleicultrix amoebiphila (Nu.cle.i.cul'trix. L. n. nucleus, a nut, and in biology a nucleus; L. fem. n. cultrix, inhabitant; a.mo.e.bi'phi.la. N.L. n. amoeba; N.L. fem. adj. phila, friend, loving; N.L. fem. adj. amoebiphila, amoeba-loving). Bacteria infecting and replicating in the nucleus of Hartmannella and Acanthamoeba species; the organism was originally discovered in a Hartmannella sp. isolate obtained from a denitrifying bioreactor; it has not been cultured in host-free media. The bacteria appear as coccoid rods of 0.5 to 1 µm in length and 0.3 to 0.4 µm in diameter and show a Gram-negative type cell wall. The organism is a member of the Alphaproteobacteria; its classification is based on 16S rrna and 23S rrna gene sequences (Genbank acc. numbers KF697195, KF697196) and fluorescence in situ hybridization with the 16S rrna-targeted oligonucleotide probe CBR125 (5 -TTCACTCTCAAGTCGCCC-3 ).
Figure S1. Phylogenetic relationship of Hartmannella sp. FS5. The tree is based on a 18S rrna alignment (Schmitz-Esser, 2008); the isolate Hartmannella sp. FS5 and selected relatives in Genbank/EMBL/DDBJ were added, and the tree was calculated with FastTree2 (Price et al., 2010). Hartmannella sp. FS5 is highlighted in grey. Accession numbers are provided in Table S1. 2
Figure S2. Identification and intranuclear location of Candidatus Nucleicultrix amoebiphila in the original amoeba isolate Hartmannella sp. FS5. Hartmannella sp. cells infected by Nucleicultrix were stained by DAPI (blue) and FISH using the general bacterial probe mix EUB338 I-III (labeled in red resulting in a pink color due to the overlay with the blue DAPI signal) and the eukaryotic probe EUK516 (shown in grey). Arrows indicate amoeba trophozoites with bacteria in the nuclear compartment. Bar, 5 µm. 3
Figure S3. 16S rrna-based maximum likelihood tree showing the relationship of Candidatus Nucleicultrix amoebiphila with other Alphaproteobacteria. Nucleicultrix and its closest relatives are highlighted in grey. Maximum likelihood bootstrap values and Bayesian posterior probabilities are indicated at the nodes. 4
Figure S4. 23S rrna-based maximum likelihood tree showing the relationship of Candidatus Nucleicultrix amoebiphila with other Alphaproteobacteria. Nucleicultrix and its closest relatives are highlighted in grey. Maximum likelihood bootstrap values and Bayesian posterior probabilities are indicated at the nodes. 5
Figure S5. Maximum likelihood tree showing the relationship of Candidatus Nucleicultrix amoebiphila with other Alphaproteobacteria based on concatenated 16S and 23S rrna. Nucleicultrix and its closest relatives are highlighted in grey. Maximum likelihood bootstrap values and Bayesian posterior probabilities are indicated at the nodes. 6
Figure S6. Escape of Candidatus Nucleicultrix amoebiphila from phagolysosomes. A. castellanii cells were incubated with LysoTracker-Yellow HCK-123 (Lifetechnologies; 2 µm) in 1xPAS for 1 hour and infected with Nucleicultrix as described. The cell suspension was then transferred to a chambered cover glass system (NalgeNunc Int.), and DAPI (0.2 µg/ml) was added. The infection process was monitored over a period of 6 hours with a confocal microscope (Leica SP8). The images shown here display the same amoeba trophozoites approximately 2 h post infection. (a) Nucleicultrix (blue, arrowhead) was initially located in phagolysosomes (shown in green). (b) The bacteria remained intact and 5 minutes later escaped into the cytoplasm. 7
Figure S7. Infection cycle of Candidatus Nucleicultrix amoebiphila in Acanthamoeba castellanii. The infection process was monitored over a course of 120 hours and visualized by FISH using the Nucleicultrix-specific probe CBR125 (shown in orange) and probe EUK516 targeting the amoeba host (shown in grey). Single bacteria colonize the nucleus within 6 hours p.i., the nucleoplasm is completely filled with bacteria after 72-96 hours, which is accompanied by significantly enlarged nuclei. Host cell lysis occurs from 72 hours p.i. on. Bar, 10 µm. 8
Figure S8. The presence of E. coli increases infection rate of Candidatus Nucleicultrix amoebiphila in Hartmannella sp. FS5. The percentage of infected amoebae 48 hours p.i. is shown in the absence or presence of E. coli (at two different cell numbers given in colony forming units, CFU). Error bars indicate standard deviation based on three replicate infection experiments. 9
Figure S9. Influence of Candidatus Nucleicultrix amoebiphila on Hartmannella sp. growth at different temperatures. 10 4 amoebae from either infected or uninfected cultures were seeded in 12-well plates, containing 1 ml PAS supplemented with E. coli tolc- and ampicillin (200 ng/ml). Cultures were incubated at three distinct temperatures (14 C, 24 C, and 37 C), and cells were detached and counted every 24 hours. As soon as stationary growth phase was reached, amoebae were harvested and fixed with 4% PFA. The percentage of infected amoebae was determined using FISH combined with DAPI staining. (a) Number of amoeba trophozoites with or without symbionts during incubation at 14 C, 24 C, and 37 C, respectively. (b) Percentage of infected amoebae at the beginning and end of the experiment. Error bars indicate standard deviation based on at least three replicate experiments. Differences in amoeba cell numbers between infected and uninfected cultures were not significant at all incubation temperatures (p>0.05; two-way ANOVA). 10
Figure S10. Environmental sequences similar to the 16S rrna of Candidatus Nucleicultrix amoebiphila. The number of amplicon sequences (> 200 nt) found in SRA and VAMPs using different similarity thresholds are shown and classified based on their environmental origin. Additional details are provided in Table S2. 11
Table S1. Genbank/EMBL/DDBJ accession numbers of 16S, 18S and 23S rrna sequences used for phylogenetic analysis. Provided as Excel file. Table S2. List of samples in SRA and VAMPS that include sequences similar to the 16S rrna gene of Candidatus Nucleicultrix amoebiphila. Provided as Excel file. References Price MN, Dehal PS, Arkin AP. (2010). FastTree 2--approximately maximum-likelihood trees for large alignments. PLoS One 5: e9490. Schmitz-Esser S, Toenshoff E, Haider S, Heinz E, Hoenninger VM, Wagner M, Horn M. (2008). Diversity of bacterial endosymbionts of environmental Acanthamoeba isolates. Appl Environ Microbiol 74: 5822 5831. 12