Uncovering the hidden bacterial ghost communities of yeast and experimental evidences demonstrates yeast as thriving hub for bacteria

Abstract

Our major concern was to address “yeast endobacteria” which was based on a few reports in the recent past where bacteria may find yeast as a niche for survival. In this study, we report the microbiota of twenty-nine axenic yeast cultures recovered from different habitats based on their 16S rRNA gene-amplicon metagenomes. Yeasts were identified based on D1/D2 or ITS gene sequences. Bacterial diversity was widespread, varied and rich among all yeasts except for four strains. Taxa belonging to the phylum Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes and the genera; Streptococcus, Propionibacterium were common to all the yeasts. Candida tropicalis was used as a model organism to confirm bacteria through fluorescence in situ hybridization (FISH), isolating and re-introducing the isolated bacteria into the yeast. FISH analysis confirmed the endobacteria of C. tropicalis and we have successfully isolated four bacteria only after lysis and disruption of yeast cells. These bacteria were identified as species of Pseudomonas, Chryseobacterium, Lysinibacillus and Propionibacterium. Guestimates indicate 95% of bacterial species of C. tropicalis are yet-to-be-cultivated. We have successfully reintroduced mCherry tagged Pseudomonas into C. tropicalis. Also, auto-fluorescent Prochlorococcus and Rhodopseudomonas could be introduced into C. tropicalis while mCherry tagged E. coli or Salmonella could not be introduced. FISH analysis confirmed the presence of both native and infected bacterial cells present in C. tropicalis. Our findings unveil the insights into the ghost microbiota associated with yeast, which otherwise are considered to be axenic cultures. Their inherent occurrence, together with co-cultivation experiments under laboratory conditions suggests that yeasts are a thriving hub for bacterial communities.

Endosymbiotic bacteria within the nematode-trapping fungus Arthrobotrys musiformis and their potential roles in nitrogen cycling

“…A Leica DM4000B microscope (Leica Microsystems, Wetzlar, Germany) with a Luminera camera and 100-W mercury arc lamp was used for fluorescent imaging. Visible fluorescence of bacterial nucleic acids within living hyphae, which could be distinguished with fungal mitochondrial or nuclear DNA according to the size and motility as described by previous studies ( Hoffman and Arnold, 2010 ; Arendt et al, 2016 ; Shaffer et al, 2016 ; Indu et al, 2021 ), provided evidence of viable endosymbiotic bacteria ( Stewart and Deacon, 1995 ).…”

Characterization of metagenome-assembled genomes of two endo-archaea of Candida tropicalis

“…Details of isolation and identification of Candida tropicalis strain JY101 are given by Indu et al (2021). The organism was grown on yeast peptone dextrose (YPD) agar (HIMEDIA, M1363) at 30°C with an incubation period of 4 to 5 days.…”

“…Before the DNA extraction, the purity of the yeast culture was checked by observing yeast cells under a phase contrast microscope and under the confocal microscope after staining the cells with DAPI (4′,6-diamidino-2-phenylindole) as mentioned by Indu et al, 2021. The genomic DNA of C. tropicalis was isolated from cells grown in YPD broth (at 30°C for five days and harvested by centrifuging at 6000xg for 10 min) by following the manufacturer's protocol of NucleoSpin Kit. DNA was stored at -20°C until further use.…”

“…In addition, they are amenable to genetic manipulation and thus for testing hypotheses. Recent studies have shown that yeasts are rich reservoirs of endobacteria (Deveau et al, 2018;Indu et al, 2021). Although bacteria-fungi interactions are well studied and tested (Hoffman et al, 2013;Uehling et al, 2017), the same is yet to be carried out in unicellular fungi like yeasts.…”

“…MAGs approaches can recover populations-variable genes and core genes, which can further assist in providing valuable insights related to population genetics, microbial-host interactions, and their survival strategies (Meziti et al, 2021). The present study attempts to extend existing information (Indu et al, 2021) on prokaryote-yeast interaction with the fundamental approach of using metagenome-assembled genomes (MAGs).…”