The multi-omics promise in context: from sequence to microbial isolate

Figures & data

Figure 1. A model depicting the positive feedback loop between multi-omics data generation and isolation of as yet uncultured microorganisms. The rise of multi-omics tools has led to a better understanding of microbial life in nature, the resource for novel biotechnological applications needed by our society. The tedious cultivation of microorganisms often represents the first milestone in novel biotechnological process development and facilitates testing of ecological hypotheses. Multi-omics information, curated by physiological characterization of already available microbial isolates, represents a huge pool of knowledge about the yet-uncultured microbial world. Hence, integrating multi-omics data directed at culturing novel environmental bacteria (dashed arrow) brings multi-omics data into context and has the potential to boost biotechnological innovation for the benefit of society and nature conservation.

Figure 2. Schematic depiction of selected environments discussed in this review according to the diversity of the residing microbial community.

Figure 3. Proposed workflow for integrating multi-omics data into microbial cultivation. Arrows indicate the flow of information, blue for multi-omics strategies and green for microbial cultivation (in correspondence to Figure 1). *The sampling strategy prior to metagenomic or single-cell genomics differs for low- and high-diversity environments, the latter requiring a larger number of samples for enhanced genome recovery.

Table 1. Summary of existing literature on the use of multi-omics for isolating as-yet uncultured microorganisms.

Organism (description)	Multi-omics strategy	Multi-omics-derived information	Cultivation medium development	Key medium component(s)	Problems and bottlenecks	Reference
Low-diversity environmental habitats
Xylella fastidiosa (Extracellular pathogen)	Empirical isolation, genome sequence	(Partial) Absence of amino acid biosynthesis pathways	5 different minimal media, growth measurements	Amino acids, myoinositol, vitamins	Not all biosynthetic pathways visible in genome	(Lemos et al. 2003)
Tropheryma whipplei (Intra-cellular pathogen)	Genome sequencing, metabolic models	(Partial) Absence of 9 amino acid biosynthesis pathways, absence of tricarboxylic acid cycle	Cell-culture medium supplemented with FCS, glutamine and human non-essential amino acids	Amino acids (histidine, tryptophan, leucine, arginine, proline, lysine, methionine, cysteine, asparagine, glutamine)	Distinct morphology and restriction profiles in axenic culture	(Renesto et al. 2003)
Coxiella burnetii (Intra-cellular pathogen)	Genome sequencing, metabolic models	Expression microarrays, genomic reconstruction, and metabolite typing	Replicating niche characteristics (low pH, salts)	Microaerobic conditions 2.5% oxygen), casamino acids and L-cysteine, high chloride, citrate buffer (pH ∼4.75), 3 complex nutrient sources (neopeptone, FBS and RPMI cell culture medium)	None mentioned	(Omsland et al. 2009)
Rikenella-like bacterium (extracellular symbiont)	Metatranscriptomics on leech gut content when organism was proliferating rapidly	Mucin and glycans as main carbon and energy source	Replacing glucose by mucin	Mucin	Initial goal of study was not microbial isolation.	(Bomar et al. 2011)
Leptospirillum ferrodiazotrophum (acid mine drainage biofilm)	Metagenome assembly and genome reconstruction	Single nitrogen fixation (nif) operon belonging to one uncultured community member	Nitrogen free medium replicating environmental conditions	Nitrogen free, low pH, metal rich, 37 °C, nitrogen fixing chemolithoautotrophs respiring ferrous iron	Plating impossible due to low pH. Multiple replicates needed to ensure isolate purity	(Tyson et al. 2005)
Entire acid mine drainage biofilm	Metabolic labelling-based quantitative proteomic analysis: autotrophic primary production and stress response monitoring	Stress response and oxidative damage repair protein expression, heat shock and osmotic shock protein expression (Hsp20), growth rate and primary production measurements	Extreme environment imitation, modifications of cultivation medium to even more natural conditions	40 °C, pH 1, higher medium recharge rate, lower N and P salt concentrations	Interpretation of protein abundance changes caused by media modification difficult for rare organisms	(Belnap et al. 2010)
Nanopusillus acidilobi (Nanoarchaeon)	Single-cell sorting of antibody-labelled cells and whole genome amplification	Physiological host-dependency, peptide and complex sugar degradation pathways	Replicating niche conditions and addition of casamino acids, sucrose and glycogen	Optical tweezer isolation and co-cultivation with its host archaeon	Obligate host-dependency	(Wurch et al. 2016)
High-diversity environmental habitats
Candidate Phylum TM7 Phylotype (human oral microbiome)	SNP in 16S rRNA sequence	Streptomycin resistance	Addition of streptomycin to enrichment culture	Streptomycin	Epibiotic parasitic organism, physically associated with Actinomyces host species	(He et al. 2015)
Succinivibrionaceae clade (Wallaby foregut microbiome)	Genome assembled from metagenome	Utilization of starch as sole C source, Urea as non-protein N source, antibiotic resistance	Defined minimal medium, semicontinuous batch culture enrichments	Starch, urea and the antibiotic bacitracin	CO2 concentration-dependent growth	(Pope et al. 2011)
45 novel candidate species	Identifying genomic signatures from publicly available metagenomes	Highly conserved sporulation and germination pathways	Ethanol pre-treatment of samples	Pre-selection of ethanol resistant spores	None mentioned	(Browne et al. 2016)
SAR11 strains	Dilution to extinction	Natural sea water medium	High abundance in sea water	Oligotrophic media, extended cultivation time	Natural niche conditions	(Rappé et al. 2002)
Pelagibacter ubique	Genome sequencing, metabolic models	Fragmented glycine and serine biosynthesis pathways	Multiple growth media, optimization for high OD	Micro- and macronutrients, glycine and pyruvate	Fine tuning of micronutrient media composition	(Carini et al. 2012)

Lemos EGDM, Alves LMC, Campanharo JC. 2003. Genomics-based design of defined growth media for the plant pathogen Xylella fastidiosa. FEMS Microbiol Lett. 219:39–45.

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Renesto P, Crapoulet N, Ogata H, Scola BL, Vestris G, Claverie JM, Raoult D. 2003. Genome-based design of a cell-free culture medium for Tropheryma whipplei. Lancet. 362:447–449.

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Omsland A, Cockrell DC, Howe D, Fischer ER, Virtaneva K, Sturdevant DE, Porcella SF, Heinzen RA. 2009. Host cell-free growth of the Q fever bacterium Coxiella burnetii. Proc Natl Acad Sci USA. 106:4430–4434.

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Bomar L, Maltz M, Colston S, Graf J. 2011. Directed culturing of microorganisms using metatranscriptomics. MBio. 2:1–8.

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Tyson GW, Lo I, Baker BJ, Allen EE, Hugenholtz P, Banfield JF. 2005. Genome-directed isolation of the key nitrogen fixer Leptospirillum ferrodiazotrophum sp. nov. from an acidophilic microbial community. Appl Environ Microbiol. 71:6319–6324.

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Belnap CP, Pan C, VerBerkmoes NC, Power ME, Samatova NF, Carver RL, Hettich RL, Banfield JF. 2010. Cultivation and quantitative proteomic analyses of acidophilic microbial communities. ISME J. 4:520–530.

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Wurch L, Giannone RJ, Belisle BS, Swift C, Utturkar S, Hettich RL, Reysenbach AL, Podar M. 2016. Genomics-informed isolation and characterization of a symbiotic Nanoarchaeota system from a terrestrial geothermal environment. Nat Commun. 7:12115.

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He X, McLean JS, Edlund A, Yooseph S, Hall AP, Liu SY, Dorrestein PC, Esquenazi E, Hunter RC, Cheng G, et al. 2015. Cultivation of a human-associated TM7 phylotype reveals a reduced genome and epibiotic parasitic lifestyle. Proc Natl Acad Sci USA. 112:244–249.

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Pope PB, Smith W, Denman SE, Tringe SG, Barry K, Hugenholtz P, McSweeney CS, McHardy AC, Morrison M. 2011. Isolation of Succinivibrionaceae implicated in low methane emissions from Tammar Wallabies. Science. 333:646–648.

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Browne HP, Forster SC, Anonye BO, Kumar N, Neville BA, Stares MD, Goulding D, Lawley TD. 2016. Culturing of “unculturable human microbiota reveals novel taxa and extensive sporulation”. Nature. 533:543–546.

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Rappé MS, Connon SA, Vergin KL, Giovannoni SJ. 2002. Cultivation of the ubiquitous SAR11 marine bacterioplankton clade. Nature. 418:630–633.

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Carini P, Steindler L, Beszteri S, Giovannoni SJ. 2012. Nutrient requirements for growth of the extreme oligotroph “Candidatus Pelagibacter ubique” HTCC1062 on a defined medium. IMSE J. 7:592–602.

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