Reading three

• Describe the basic enrichment process for Ca. Nitrospira inopinata?

  Inoculum (oil well associated thermophilic biofilm sample)

  Enrichment & cultivation (in a defined medium that contained ammonia and CO2, at high temperature of 46 because it’s thermophilic, added antibiotics such as vancomycin to kill of gram-positive bacteria, Nitrospira is gram negative)

  Dilution to extinction (I think to get a single colony?)

  • Composition of enrichment culture (Uing FISH, PCR of 16S rRNA)
• Describe how comammox was demonstrated and how it was correlated with growth of Ca. N. inopinata

  Through growth experiments. Inopinata is an ammonia-oxidizing lithotroph. Graphs showed that over time, as ammonia levels decreased, nitrate (NO3-) levels increased, as did the levels of inopinata. NH4+ to NO3- is the comammox rxn (both steps of nitrification)How did they measure the levels of inopinata?

  o They used SYBR green and qPCR of the amoA gene
• 23. Briefly discuss the evidence for HGT in the evolution of comammox Nitrospira?

  metagenome analysis – inopinata was a metagenome assembled genome.

  o Broke genome into fragments, sequenced it via NGS (illumine and nanopore) then pieced the reads together to form a metagenome-assembled genome

  • Amo and hao are the ammonia-oxidizing genes. These transposases did not surround the nitrite-reducing gene (nxr), thus nitrite oxidizer that acquired genes for ammonia reduction – the amo/hao gene base composition also varied from the rest of the inopinato genome suggesting HGT
• . Explain how the AmoA protein in comammox bacteria was determined to be unique compared to other AmoA protein superfamilies

  s Daim’s second approach with the AmoA proteiny used phylogenetic analysis to compare AmoA proteins.

  • AmoA is a metabolic gene, therefore is more subject to HGT, therefore this gene would never be used to make a phylogenetic tree of life, but phylogenetic analysis can be used to see if comammox evolved from another organism.Found 2 clades of comammox AmoA from Nitrospira, and possible HGT.
• Discuss the implications of the discovery of Comammox

  The discovery of Comammox microorganisms has implications for nitrogen cycling, wastewater treatment, ecology, biotechnology, and microbiology, offering new insights into their roles in different environments.wonder why others didn’t develop similar processes in other bacteria that only are able to do half the nitrification process

  • Many research papers with evidence explained by comammox were disregarded due to the dogma being comammox didn’t exist
• Briefly explain the parts of a phylogenetic tree and the information that each represents

  Root: the common ancestor of all the species or groups Point for tracing the evolutionary relationships

  • Branches: rep the lineages of organisms
  • Nodes: rep the hypothetical common ancestors of the lineages that split at that point

  o Most recent common ancestor connected by the branches emerging from the node
• The universal tree of life has evolved with the evolving science of phylogenetics.
• Woose was the first phylogenetic tree generated from SSU rRNA gene sequence analysis.
• Discovered archaea domain.
• Two domains still originated from the root.
• Protein sequences were aligned, concatenated to form over a thousand MAGs (metagenome assembled genomes).
• Bootstrapping (resampling data at random hundreds of times) was used to generate the tree.
• Eukarya was shown to have evolved from the archaeal domain.
• Only bacteria and archaea shared the root (furthest common ancestor).
• Describe the experimental approach developed by Woese and Fox and explain why it is so revolutionary

  First phylogenetic tree created. Wanted to see how prokaryotes were related to each other so they needed to compare something present in all of them that changed little over time, and was only subject to VGT not HGT. Therefore they used SSU rRNA gene sequence analysis. Discovered a third domain (archaea)
• At a very basic level, explain the major differences in phylogenetic analyses of Woese and Fox, Hug et al, Imachi et al, Zhu et al, and Gong et al. all differed and the resulting findings from each

  Woese (1985)

  o Phylogeny came from SSU gene comparisons

  o 3 domains on ToL

  Hug (2016)

  o Phylogeny from 16 aligned and concatenated ribosomal protein sequences

  o 2 domains on ToL

  Imachi (2019)

  o Phylogeny from 31 aligned and concatenated ribosomal protein sequences

  o 2 domains on ToL

  • Zhu (2019)

  o Phylogeny from over 300 non-concatenated marker genes

  o 2 domains: bacteria and archaea
• Describe the relevance of bootstrapping to phylogenetic trees and in particular, to polyphyletic and paraphyletic clades

  Bootstrapping phylogenetic trees allows researchers to verify whether their initial assessment of the tree was accurate

  • Paraphyletic and polyphyletic clades are groups that share taxa but not all common ancestors.

  o Bootstrapping helps provide insight into if the data is accurate – goal is to create monophyletic clades
• Briefly discuss why, for bacteria and archaea in particular, the phylogeny of an organism may be incongruent with the phylogeny of many of its genes


  Phylogeny of an organism may be incongruent with the phylogeny of many of its genes due to horizontal gene transfer, gene loss, or recombination/mutations
• Briefly discuss, using the following genes as examples, the fluidity of bacterial and archaeal genomes

  1. Rhodopsin

  Fluidity comes from horizontal gene transfer. 2 genes spread (rhodopsin gene + retinal enzyme gene). It is widespread in species with a cosmopolitan distribution (everywhere). In oligotrophic conditions, species can use this as a source of PMF when there aren’t enough nutrients for aerobic respiration to make the PMF.

  Example organism: pelagibacter ubique – had a small genome potentially due to oligotrophic conditions
• Briefly discuss, using the following genes as examples, the fluidity of bacterial and archaeal genomes

  b. amoCAB and hao in Comammox nitrospira

  Ammonia-oxidizing genes (amo, hao) were obtained via HGT. Determine via the transposase genes flanking these two genes in the genome. The base composition of amo gene also different from the rest of the genome
• Briefly discuss, using the following genes as examples, the fluidity of bacterial and archaeal genomes?

  c. the pangenome of L. pneumophila and pathogenic strains

  pathogenicity of this species came from HGT – core genome was small, accessory genome (varies among strains) much larger – thus each strain had different virulence factors as there were several different pathogenicity islands in the large accessory genome
• define genome reduction and briefly describe how this phenomenon was identified in Prochlorococcus

  genome reduction: the organism is able to reduce its genome under certain circumstances.

  e.g. constant environment, less nutrients available

  In the case of prochlorococcus:

  Its genome is different sizes at different depths (ecotypes)

  • The more constant the environment, the smaller the genome
• Discuss the concept of “species” in bacteria and archaea

  The concept of "species" in bacteria and archaea can be challenging due to their unique biology, including horizontal gene transfer (HGT) and high genetic diversity

  Concept of "Species" in Bacteria and Archaea:

  • In bacteria and archaea, defining a species is complex due to their asexual reproduction and extensive HGT.
  • Traditional species definitions based on interbreeding or morphological criteria don't apply.
  • Instead, molecular methods, such as DNA sequencing, are often used to delineate species boundaries.
• pros and cons of using 16S rRNA for speciation, and

  • Universal Marker: 16S rRNA is a conserved gene found in all bacteria and archaea
  • Highly Conserved Regions: It contains both conserved regions for primer design
  • Widely Used: It has been extensively used for bacterial and archaeal taxonomy and phylogenetics.
  • Cons:
  • Limited Resolution: especially for closely related strains within a species.
  • Horizontal Gene Transfer: making it challenging to define species boundaries accurately.
  • Cryptic Diversity: with distinct genetic lineages not distinguishable by 16S rRNA alone.
• pros and cons of using 16S rRNA for speciation, and the connection between the concept of a species, and a “core genome”

  • the "core genome" refers to the set of genes that are present in all strains of a particular species.
  • shared genomic characteristics, including a core genome.
  • The core genome represents the genetic traits that are essential for defining a species and distinguishing it from other species.
  • Genomic approaches, can provide a more accurate and fine-grained speciation method than 16S rRNA, as they consider a broader range of genetic information.
• Explain, using the example of Legionella pneumophila, the concept of a pangenome of “dispensable” genes

  The pangenome is a set of all genes present in a species

  the core genome is one all strains will have

  the accessory genes are those that are not needed for essential function
• core vs pangenome of different hypothetical strains

  • is core genes that are conserved across all the strains of an organism most of these genes are involved in crucial processes like metabolism, replication, and cell structure.
  • the pangenome is includes the core genome plus all the unique genes of both strains ( both core and accessory genes)
• Explain how different approaches to phylogenetics may give rise to a 2-domain tree or a 3-domain tree of life

  Woese and Hug depicted a 3 domain tree. Woese used SSU ribosomal gene analysis to generate the tree. Therefore, there was not discrimination at the species level. Hug used ribosomal protein sequences, aligned, concatenated, then generated metagenome-assembled genoms.

  • Imachi used ribosomal protein sequences, but did not generate MAGs. Instead they used bootstrapping to generate a tree. Their tree only had 2 domains.
• Discuss the relevance of findings from Hug et al., and Imachi et al., regarding the Entangle-engulf-endogenize model of eukaryogenesis

  1. Hug et al.:

  • Revealed a greater diversity within the Archaea domain than previously known.
  • Identified close evolutionary relationships between certain archaeal lineages and eukaryotes.

  1. Imachi et al.:

  • Discovered the Asgard archaea group, which shares many genetic features with eukaryotes.
  • Highlighted the presence of complex cellular process genes in Asgard archaea, similar to those in eukaryotes.

  Determined that eukaryotes evolved from archaea.
• the statement that with phylogenomics, “we are getting closer to the goal of accurately depicting the phylogeny of life”

  1. Zhu et al. Study:

  • Provided an extensive genomic analysis of bacteria and archaea.
  • Offered a more detailed evolutionary tree for these domains.

  1. Imachi et al. Study:

  • Revealed eukaryotic-like genes in Asgard archaea, supporting theories of eukaryotic evolution from an archaeal lineage.
  • Contributed significantly to clarifying the early evolution of complex life forms.