MSE lec 4-5

• Rare Biosphere


  Microorganisms that are genetically diverse but are typically found in low abundance in various microbial communities
• The Rare Biosphere was firstly coined for the highly diverse pool of low relative abundance microbial populations reported in sample from deep water masses in North Atlantic
• Rare Microbial Biosphere


  Refers to a large number of rare species of microbial life, i.e. bacteria, archaea and fungi, that can be found in very low concentrations in an environment
• Rare microbial taxa


  • Many rare taxa can grow abundant under changing conditions and respond to perturbations
  • High diversity of low abundance taxa represents a large reservoir of genetic traits underpinning a wide spectrum of both known and potentially novel microbial functions
  • Members of the rare biosphere can be disproportionately active relative to their abundance for key functions
• Biotechnological applications of the Rare Microbial Biosphere

  • Bioremediation - use of living organisms for removal of contaminants, pollutants, toxins etc.
  • Bioprospecting - systemic and organized search for useful products from bioresources
• Relevance of the Rare Biosphere to Microbial Ecology

  • Community assembly theory - study of the processes that shape the identity and abundance of species within ecological communities
  • Host-microbiome associations - microorganisms occurring inside and on host surfaces, influence evolutionary, immunological, and ecological processes
  • Competitive interactions - interaction between two or more organisms of the same or different species where the species compete with each other for different resources
  • Microbial responses to climate change - climate change may alter metabolic activity of microbes
• Rare Biosphere represented as "Long Tail"


  All taxa are ordered from the most abundant to the least abundant ones
• Defining "rarity" is subjective - no clear consensus or proper method determining specific threshold from which populations should be considered low abundant or 'rare' within any given community—i.e. point where the 'long-tail' starts
• Vast majority of studies uses arbitrary threshold to label rare populations as 0.1% or 0.01% relative abundance/ sample
• Relative Abundance


  The proportion of the number of organisms from species A relative to the total number of organisms from species A, B and C, collected in the same sample
• Relative Abundance Formula: RA = Total number of Individuals species (Isi) / Total number of Species Population (∑ Nsi)
• Relative Abundance Example


  • Staphylococcus aureus (28.77%)
  • Escherichia coli (8.38%)
  • Clostridium kluyveri (9.02%)
  • Listeria monocytogenes (24.42%)
  • Streptococcus penumoniae (10.96%)
  • Pseudomonas fluorescens (14.58%)
  • Thiobacillus novellus (2.66%)
  • Salmonella Typhimurium (0.96%)
  • Mycobacterium tuberculosis (0.24%)
• Early definitions of rarity focused on geographic distribution and habitat specificity of microorganisms as indicators of frequency of occupancy
• The Prokaryotic Biosphere


  • Composed of cells in different metabolic states, including dormant but viable cells which supports an early hypothesis that the rare biosphere functions as a seed bank to leverage microbial community responses to environmental changes
  • Comprises a highly diverse genetic pool that encompasses both novel and already described prokaryotes
  • Recent evidence suggests that rare prokaryotes may equip abundant or growing prokaryotes with novel functions by means of horizontal gene transfer
  • Abundant populations have a more constant abundance and are composed of generalists, while the rare biosphere is more prone to respond to environmental fluctuations
• The Microeukaryotic Biosphere


  • Ecological mechanisms underlying the dynamics of the rare microeukaryotic and prokaryotic biospheres are similar
  • Both represent a genomic pool that can work as seed bank and respond to changing conditions
  • Composition and structure of the microeukaryotic rare biosphere found specific biogeographic and temporal patterns, suggesting responsiveness to environmental factors and metabolic activity, based on the ratio of 18S rRNA genes to 18S rRNA transcripts
• Diatom blooms


  Induced shifts in the structure and diversity of rare prokaryotic communities
• Cyanobacterial bloom


  Diversity of rare microeukaryotes significantly changed
• Viral Control of Rare Microbial Populations

  • Viral predation is dependent on the probability of encounter with the host, and this probability is lower for low abundance microbes, thus low abundance is an advantage against predators
  • The "kill the winner hypothesis" where viral predation increases with host density, thus favoring the lytic cycle in high host density situations
  • The "king of the mountain hypothesis" where increasing host abundance, factors such as host defense and horizontal transfer of resistance genes gain importance, providing a positive feedback loop favoring the most abundant populations
  • The "piggyback-the-winner hypothesis" where viruses can piggyback on abundant hosts to maintain their own populations
• Viral Control of Rare Microbial Populations - "Bottle Effect"


  • Near the wall of the receptacle used to store cells after water sample filtration, the viral particles bounce back and forth from the walls to the microorganisms, increasing the probability of encountering and infecting a host
• Rare Virosphere


  • Study is dependent on high throughput sequencing based approaches
  • Co-occurrence analysis of rare viral particles, prokaryotes and eukaryotes could lead to important new insights into these dynamics
  • Due to phage specificity, it is possible that rare phages follow the same patterns of their rare hosts, if able to coexist at low abundance, without extinction
• Rare Biosphere and Host Associations


  • Horizontally transmitted symbionts are taken up from the environment anew by each host generation, and vertically transmitted symbionts are most often transferred through the female germ line
  • Symbiont transmission can also be transferred during ontogeny (vertical symbiont transmission)
• Microbial species abundance distribution dynamics


  1. Near the wall of the receptacle used to store cells after water sample filtration, the viral particles bounce back and forth from the walls to the microorganisms

  2. As a consequence, the probability of encountering and infecting a host increases, resulting in a wall-chain-reaction
• Study of rare virosphere


  • Dependent on high throughput sequencing based approaches
  • Co-occurrence analysis of rare viral particles, prokaryotes and eukaryotes could lead to important new insights into these dynamics
  • Due to phage specificity, it is possible that rare phages follow the same patterns of their rare hosts, if able to coexist at low abundance, without extinction
• Symbiont transmission


  1. Horizontally transmitted symbionts are taken up from the environment anew by each host generation

  2. Vertically transmitted symbionts are most often transferred through the female germ line

  3. Symbiont transmission can also be transferred during ontogeny (vertical symbiont transmission)
• Rare biosphere in host associations has been studied mostly in sponges and corals
• Most studies on the microbial rare biosphere have focused primarily on diversity assessments, although currently there is an increasing interest in the functional component of low abundance microbes in open and host-associated environments
• Marker gene high throughput sequencing based methods


  • Predominantly used for comprehensive analyses of the microbial rare biosphere
  • Prokaryotes- 16S rRNA gene sequences of specific regions (V4 hypervariable region routinely used in rare biosphere diversity assessments)
  • Eukaryotes- 18S rRNA gene(V9 region)

  
• Methodological approaches for studying the microbial rare biosphere

  • OTU- Operational Taxonomical Units
  • ASV - Exact amplicon variants
  • DNA Extraction
  • PCR Amplification
  • Metagenome Sequencing
  • Culture-dependent
  • MAGs-Metagenome Assembled Genomes
  • Single-cell sorting
  • Functional marker genes
• OTUs -Operational Taxonomical Units

  • Traditionally used, most common method
  • Based on a percentage identity (e.g. 97%)
  • OTUs can be made de novo or reference based
  • Created independently of an external reference, meaning that they can identify novel diversity
• Exact amplicon variants (ASV)


  • Does not cluster sequences based on percent identity
  • Instead, a score of the error rate is calculated and based on that value, sequences are grouped as the same variant or separated as a distinct one
  • The score basically, allows to distinguish true biological differences from random errors (in the process of sequencing and PCR amplification)
  • Using ASVs makes it possible to distinguish sequence variation by a single nucleotide change

  
• DNA extraction


  • Pros: tested protocols present more differences in rare rather than abundant populations (planktonic communities), in terms of diversity, composition, DNA quality and reproducibility, which suggests that direct comparison of datasets using different DNA extraction kits are safer for abundant bacteria
  • Cons: Contamination during sampling and/or DNA extraction
• PCR Amplification


  • Bias towards specific taxonomic groups, because PCR primers are designed based on well-known taxa
• Candidate Phyla Radiation


  • A large evolutionary radiation of bacterial lineages whose members are mostly uncultivated and only known from metagenomics and single cell sequencing
  • They have been described as nanobacteria (not to be confused with non-living nanoparticles of the same name) or ultra-small bacteria due to their reduced size (nanometric) compared to other bacteria
  • Genome is very small and it is thought that many are symbionts, based on the lack of essential metabolic pathways
  • Has a significant impact on the breadth of the tree of life
  • Several microbial lineages within the candidate phyla radiation belong in the rare biosphere of multiple ecosystems
• Evolutionary radiation


  • An increase in taxonomic diversity that is caused by elevated rates of speciation, that may or may not be associated with an increase in morphological disparity
  • Radiations may affect one clade or many, and be rapid or gradual; where they are rapid, and driven by a single lineage's adaptation to their environment, they are termed adaptive radiations
• Untargeted Metagenome Sequencing


  • Open, untargeted metagenome sequencing of total community DNA avoids the PCR bias, but the lack of an amplification step might as well miss rare sequences, reinforcing the need of high sequencing depth for the proper characterization of the rare biosphere

  
• Culture-Dependent: Microfluidic Streak Plate


  • Can identify members of the rare biosphere, including those not identified by molecular methods
  • High throughput culturing approaches Microfluidic streak plate
  • Based on cell sorting followed by cultivation, enabled consistent and comprehensive diversity assessments, including the detection of rare prokaryotes from soil
  • Approach was useful in the isolation of low abundance prokaryotes from deep sea sediments and soil samples
• Metagenome Assembled Genomes - MAGs


  • Rare biosphere's functional contribution on Ecosystem dynamics: combination of multiple methodologies was used to study rare prokaryotes in the sulfur cycle
  • MAGs from rare populations are difficult to obtain, but some strategies include selective sample enrichment to reduce community complexity before sequencing or differential coverage binning coupled with high sequencing power
  • Different studies have combined MAGs with phylogenetic markers to study the functions of the rare biosphere

  
• Single Cell Sorting - Whole Genome Amplification


  • Another alternative to retrieve genomes from rare prokaryotes, without the need of sequencing whole community metagenomes
• Functional Markers


  • Instead of (or in combination with) taxonomical marker genes
  • Useful in increasing our understanding of the rare biosphere impact on the soil nitrogen cycle under climate change
  • Expanding the knowledge of nitrifiers diversity in wastewater treatment plants using nxrB amplicons and in soils, using amoA amplicons
• Mere calculations of gene relative abundances is not necessarily informative of microbial activity. One approach to estimate whether members of the microbial rare biosphere are active is based on the ratio between rRNA gene and rRNA transcript counts