Characterization of the first cultured free-living representative of Candidatus Izemoplasma uncovers its unique biology

Abstract

Candidatus Izemoplasma, an intermediate in the reductive evolution from Firmicutes to Mollicutes, was proposed to represent a novel class of free-living wall-less bacteria within the phylum Tenericutes. Unfortunately, the paucity of pure cultures has limited further insights into their physiological and metabolic features as well as ecological roles. Here, we report the first successful isolation of an Izemoplasma representative from the deep-sea methane seep, strain zrk13, using a DNA degradation-driven method given Izemoplasma's prominent DNA-degradation potentials. We further present a detailed description of the physiological, genomic and metabolic traits of the novel strain, which allows for the first time the reconstruction of the metabolic potential and lifestyle of a member of the tentatively defined Candidatus Izemoplasma. On the basis of the description of strain zrk13, the novel species and genus Xianfuyuplasma coldseepsis is proposed. Using a combined biochemical and transcriptomic method, we further show the supplement of organic matter, thiosulfate or bacterial genomic DNA could evidently promote the growth of strain zrk13. In particular, strain zrk13 could degrade and utilize the extracellular DNA for growth in both laboraterial and deep-sea conditions. Moreover, the predicted genes determining DNA-degradation broadly distribute in the genomes of other Izemoplasma members. Given that extracellular DNA is a particularly crucial phosphorus as well as nitrogen and carbon source for microorganisms in the seafloor, Izemoplasma bacteria are thought to be important contributors to the biogeochemical cycling in the deep ocean.

Fig. 1. DNA degradation-driven isolation strategy and morphology of X. coldseepsis zrk13.

A Diagrammatic scheme of enrichment and isolation of Izemoplasma bacteria. B and C TEM observation of strain zrk13. D TEM observation of the ultrathin section of strain zrk13. E TEM observation of the ultrathin section of a typical Gram-positive bacterium Clostridium sp. zrk8. CM cell membrane, PG peptidoglycan; Scale bars, 100 nm. F Phylogenetic analysis of X. coldseepsis zrk13. Phylogenetic tree of almost complete 16S rRNA gene sequences from Tenericutes and Firmicutes representatives. Some Actinobacteria members were used as the outgroup. The tree is inferred and reconstructed under the maximum likelihood criterion and nodes with >80% bootstrap support are labeled with a gray circle (expressed as percentages of 1000 replications). And the names indicated in gray in quotation represent taxa that are not yet validly published. Bar, 0.1 substitutions per nucleotide position.

Fig. 2. Organic nutrient significantly promotes the growth of X. coldseepsis zrk13.

A Growth assays of strain zrk13 in the oligotrophic and rich media. B Transcriptomics based heat map showing all up-regulated genes encoding glycosyl hydrolases. C Transcriptomics based heat map showing three up-regulated iron hydrogenase maturation genes (hydEFG). D Transcriptomics based heat map showing all up-regulated genes encoding NADH-quinone/ubiquinone oxidoreductase. E Transcriptomics based heat map showing all up-regulated genes encoding ATP synthase. “Oligo” indicates the oligotrophic medium; “Rich” indicates the rich medium.

Fig. 3. Thiosulfate significantly promotes the growth of X. coldseepsis zrk13.

A Growth assays of strain zrk13 in the modified rich medium supplemented without or with Na₂SO₄ (100 mM), Na₂S₂O₃ (100 mM), Na₂SO₃ (1 mM), and Na₂S (1 mM), respectively. B Transcriptomics based heat map showing an up-regulated gene cluster encoding Fe-S protein, nucleotide and amino acid metabolisms associated proteins. C Transcriptomics based heat map showing all up-regulated genes encoding glycosyl hydrolases. D Transcriptomics based heat map showing all up-regulated genes encoding sugar ABC transporter permease.

Fig. 4. X. coldseepsis zrk13 possesses a significant capability of degradation and utilization of extracellular DNA.

A Gene arrangements of a putative DNA-degradation locus in strain zrk13. The alphabets shown in the X-axis indicate the code names of different genes within the gene cluster. B Detection of DNA degradation ability of strain zrk13 by agarose gel electrophoresis. Lane 1, lane 4 and lane 7 indicate 2 µg E. coli genomic DNA/RNA treated by the medium without cells for 5 min, 10 min and 15 min at 37 °C, respectively. Lane 2, lane 5 and lane 8 indicate 2 µg E. coli genomic DNA/RNA treated by the supernatant of a Clostridial bacterium for 5 min, 10 min and 15 min at 37 °C, respectively. Lane 3, lane 6 and lane 9 indicate 2 µg E. coli genomic DNA/RNA treated by the supernatant of strain zrk13 for 5 min, 10 min and 15 min at 37 °C, respectively. M, DL4500 molecular weight DNA marker. C Quantification of DNA degradation by strain zrk13. DNA concentrations after degradation by the medium and zrk13 supernatant as shown in (B) were determined by Nanodrop. Three replicates were performed. D Growth assays of strain zrk13 cultivated in the rich medium and basal medium supplemented with or without 1 µg/mL E. coli genomic DNA/RNA. E qRT-PCR detection of expression changes of genes shown in (A) when strain zrk13 was cultivated in the basal medium supplemented with or without 1 µg/mL E. coli genomic DNA/RNA. Three biological replicates were performed. The code names shown in the X-axis indicate the gene names shown in (A). “C” indicates control.

Fig. 5. Transcriptomics analysis of X. coldseepsis zrk13 incubated in the deep-sea cold seep.

Distant (A) and close (B) views of the in situ experimental apparatus in the deep-sea cold seep where distributed many mussels and shrimps. C Transcriptomics based heat map showing all up-regulated genes encoding enzymes degrading nucleic acids after a 10-day incubation of strain zrk13 in the deep-sea cold seep. D Transcriptomics based heat map showing an up-regulated gene cluster encoding Fe-S protein, nucleotide and amino acid metabolisms associated proteins. E Diagrammatic scheme of EMP glycolysis pathway. The gene numbers showing in this scheme are the same with those shown in (F). F Transcriptomics based heat map showing all down-regulated genes associated with EMP glycolysis pathway after a 10-day incubation of strain zrk13 in the deep-sea cold seep.

Fig. 6. Muti-omics based central metabolisms model of X. coldseepsis zrk13.

In this model, central metabolisms including DNA degradation, EMP glycolysis, oxidative pentose phosphate pathway, hydrogen production, electron transport system, sulfur metabolism and one carbon metabolism are shown. All the above items are closely related to the energy production in X. coldseepsis zrk13. In detail, strain zrk13 contains a complete set of genes related to DNA-degradation, which could catabolically exploit various sub-components of DNA, especially purine-based molecules. And further degradation into nucleotides and nucleosides by nucleases were required to facilitate the introduction of DNA sub-components into cells. Once imported into the cytoplasm, purine- and pyrimidine-deoxyribonucleosides were further broken down into different bases, whereby the respective bases may enter catabolic pathways. The metabolites of nucleosides catabolism were finally transformed into phosphoribosyl pyrophosphate (PRPP) and Ribose-1P, thus entering the oxidative pentose phosphate pathway, which is closely related to EMP glycolysis pathway. Sulfide generated by sulfur reduction from thiosulfate works together with the L-serine to form acetate and L-cysteine, which eventually enter the pyruvate synthesis pathway. Furthermore, the formate produced in one carbon metabolism was converted into CO₂/CO by formate dehydrogenase. Iron hydrogenases catalyze the reduction of protons to hydrogen for the energy production. A membrane-bound, Na⁺-transporting NADH: Ferredoxin oxidoreductase (RNF complex), the H⁺-transporting NADH: Quinone oxidoreductase (complex I) and F-type ATP synthase required for energy metabolism are present in strain zrk13 genome. Both complex I and the cytochrome bd oxidase interact with the quinone pool, which are associated with energy production.