Module 2 - Microbial Phylogeny and Systematics

Lesson 2A Microbial Phylogeny and Systematics Topic Outline 01. Molecular Phylogeny (Part A)  Obtaining DNA sequences  Sequence alignment 02. Phylogenetic Trees (Part A)  Composition and construction  Limitations 03. Species concept in Microbiology (Part B)  Phylogenetic concept 04. Taxonomic Methods in Systematics (Part B)  Gene sequence analysis  Multilocus sequence typing  Genome fingerprinting  Multigene and whole genome analysis  Phenotypic analysis (n ee dy )  Phenetic system: groups organisms together based on similar phenotypic characteristics Classical physiological descriptions of microbes constitute a taxonomy, but do not provide relationships (except as might be inferred subjectively). Methods such as G+C ratios, FAME, DNA-DNA hybridization, or REP PCR establish relationships, but only if close, i.e., they are not sufficiently general to be broadly applicable. All these methods require pure-cultivation of organisms for characterization, but we can't cultivate much of what is out there. Microbial Taxonomy  Traditional taxonomy or the classification through identification and nomenclature of microbes, both "prokaryote" and eukaryote, has been in a mess – we were stuck with it for traditional reasons.  A "natural" taxonomy would be based on evolutionary relatedness: thus, organisms in same "genus" (a collection of "species") would have similar properties in a fundamental sense.  A natural taxonomy of macrobes has long been possible: Large organisms have many easily distinguished features (e.g., body-plans and developmental processes, that can be used to describe hierarchies of relatedness).  Microbes usually have few distinguishing properties that relate them, so a hierarchical taxonomy mainly has not been possible. And also, the problem of unculturability…… Recent advances in molecular phylogeny have changed this picture. We now have a relatively quantitative way to view biodiversity, in the context of phylogenetic maps or evolutionary trees. Slowly evolving molecules (e.g. rRNA) used for large-scale structure; "fast- clock" molecules for fine-structure. The literature language (e.g. "species") and formal nomenclature, however, remain solidly rooted in the tradition of Linnaeus at this time. (You have to call them something!) Methods fo Microbial Taxonomy and Determination of Evolutionary Relationships  Phenetic System  More in-depth methods may establish relationships, but only if organisms are closely related. Not applicable on broad evolutionary landscapes  %GC (G+C ratios)  DNA:DNA Hybridization  Fatty Acid methyl ester analysis (FAME)   Ribotyping Phyletic System:  Compares organisms based on evolutionary relationships  rRNA sequence comparison  Multilocus sequence typing (MLST) Microbial taxonomy (particularly bacterial taxonomy) has evolved into a field that utilizes a combination of methods for the identification and description of new species. This polyphasic approach to taxonomy uses three kinds of methods—phenotypic, genotypic, and phylogenetic.  Phenotypic analysis = examines the morphological, metabolic, physiological, and chemical characteristics of the cell.  Genotypic analysis considers characteristics of the genome. These two kinds of analysis categorize organisms based on similarities.  Both methods are complemented by phylogenetic analysis, which seeks to place organisms within an evolutionary framework using molecular sequence data. Principle  Molecular sequences provide a record of past evolutionary events and can be used to build phylogenetic trees, which are diagrams used to depict evolutionary history.  Mutations in the genetic material of all cells (DNA) can accumulate over time and lead to natural variations that allow evolution.  The difference in nucleotide sequence between the DNA of any two organisms will be a function of the number of mutations that have accumulated since they shared a common ancestor.  As a result, differences in DNA sequences can be used to infer evolutionary relationships. rRNA sequencing and the Tree of Life Phyletic system: compares organisms based on evolutionary relatedness rRNA methods: NOT culture-based. Ribosomal RNAs (rRNAs) and its respective genes (rDNA) are excellent descriptors of microbial taxa based on phylogeny. Certain molecules are “molecular chronometers”: differences in nt or aa sequences of homologous molecules are a function of their evolutionary distance. They are: -universally distributed among all living organisms (essential for even the most primitive cells) -functionally homologous -lack horizontal gene transfer that could confound phylogenetic analysis Can’t accumulate many mutations in such an important macromolecule… so, evolutionary distance between rRNAs reflects evolutionary distance between organisms. Molecular chronometers let us look deep into the evolutionary past. Useful features of molecular chronometers: -regions of sequence conservation so DNA can be aligned -sequence change should reflect evolutionary change in organism as a whole Examples of molecular chronometers: rRNA, ATPase, RecA, DNA polymerase, etc. rRNA is the most widely used. A huge database of rRNA sequences exists. For example, the Ribosomal Database Project (RDP) contains a large collection of such sequences, now numbering over 100,000. Sequences used for molecular phylogeny 1. DNA  Typically, DNA is extracted from a culture (pure) for organisms that can be grown in the laboratory. The DNA isolated is also called genomic DNA or gDNA.  genomic DNA can be sequenced directly or used for PCR amplification. = genome sequencing. genome sequencing a standard tool employed in analyses of microbial phylogeny 2. RNA  sequence analysis of small subunit (SSU) ribosomal RNA (rRNA) genes is the basis of much of microbial phylogeny  SSU rRNA genes are highly conserved, present in all cellular organisms, and easily sequenced and analyzed Remember!  procaryotes = 16S rRNA  eucaryotes = 18S rRNA Ribosomal RNA (rRNA)  Primary and secondary structure of 16S rRNA from Escherichia coli (Bacteria). The 16S rRNA from Archaea is similar in secondary structure (folding) but has numerous differences in primary structure (sequence). The molecule is composed of conserved and variable regions (V1–V9). The approximate positions of the variable regions are indicated in color.  The rRNA gene can have different levels of phylogenetic specificity to target discrete species, genera, phyla, or domains, and there are even “universal” primers that will amplify the SSU rRNA gene from any organism. Why ribosomal RNAs?  Found among all living organisms (for 3.8 of the last 4.5 billion years).  Integral part of protein synthesis machinery (and therefore highly conserved among organisms).  Cell component analyses provide culture-independent means of investigating questions in microbial ecology (lack of morphology).  rRNAs offer a type of sequence information that makes them excellent descriptors of an organism's evolutionary history.  No detectable horizontal gene transfer, especially important for the prokaryotes.  Large and growing database; RDP contains ~100K SSU rRNAs. (more about RDP in Module 4) Bacterium Archaeobacterium Fungus Signature sequences can be obtained at any taxon level In 197, Carl Woese and George Fox: Identified Archaea (Greek: “Ancient ones”), separate branch of life from eubacteria Demonstrated that differenes in rRNA sequences usefully eflect evolutionary relationships Following DNA isolation (1) primers complementary to the ends of the 16S rRNA are used to PCR-amplify the 16S rRNA gene from the genomic DNA of five different unknown bacterial strains (2) , and the products are run on an agarose gel (3) The bands of amplified DNA are approximately 1465 nucleotides in length (4) Positions of DNA kilobase size markers are indicated at the left. Excision from the gel and purification of these PCR products is followed by sequencing (5) and analysis to identify the bacteria Sequence Alignment: Principle  Phylogeny can be inferred only from genes that have homology or genes that have been inherited from a common ancestor  genes that have been inherited from a common ancestor  homology is a binary trait; sequences are either homologous or they are not  Similarity and homology are NOT interchangeable  Similarity is a continuous trait defined as a percentage of nucleotide positions shared between any two sequences  Sequence similarity is used to infer homology, but a similarity value can be calculated between any two sequences regardless of their function or evolutionary relationship  Phylogenetic analyses typically focus on analysis of orthologous genes that have similar function.  Review: orthologs have the same function and originate from a single ancestral gene in a common ancestor  paralogs have evolved to have different functions as a result of gene duplication  Genes separated by speciation are called orthologs. Genes separated by gene duplication events are called paralogs.  Phylogenetic analyses estimate evolutionary changes from the number of sequence differences across a set of homologous nucleotide positions.  Some mutations introduce nucleotide insertions or deletions, leading to differences in sequence length, and making it necessary to align nucleotide positions prior to phylogenetic analysis of gene sequences.  sequence alignment is done to add gaps to molecular sequences in order to establish positional homology, i.e., to be sure that each position in the sequence was inherited from a common ancestor of all organisms under consideration An Example PCR amplification of the 16s rRNA gene    Alignment of DNA sequences A. Sequences for a hypothetical region of a gene are shown for three species before alignment and after alignment. A sequence alignment should display homologous positions in vertical columns. Sequence alignment is achieved by adding gaps, indicated by hyphens, to maximize local sequence similarity between the species in the alignment. B. The distance matrices show the number of sequence differences that would be inferred for each species pair both before and after alignment.  Composition and Construction     Unrooted and rooted examples of phylogenetic trees. The tips of the branches are species (or strains) and the nodes are ancestors. Ancestral relationships are revealed by the branching order in rooted trees.  Phylogenetic tree is a diagram that depicts the evolutionary history of an organism and bears some resemblance to a family tree. Phylogenetic trees allow us to make hypotheses about an organism’s characteristics since organisms that share a recent ancestor are likely to share characteristics. Phylogenetic trees are also of great use in taxonomy and species identification. A phylogenetic tree is composed of nodes and branches Rooted trees show the position of the ancestor of all organisms being examined. Unrooted trees depict the relative relationships among the organisms under study but do not provide evidence of the most ancestral node in the tree. The nodes represent a past stage of evolution where an ancestor diverged into two new lineages. The branch length represents the number of changes that have occurred along that branch. In a phylogenetic tree, only the position of nodes and the branch lengths are informative; rotation around nodes has no effect on the tree’s topology Phylogenetic analysis uses molecular sequence data in an attempt to identify the one correct tree that accurately represents the evolutionary history of a set of sequences. Building phylogenetic trees. The number of nucleotide differences between gene sequences can be used to build a phylogenetic tree. In the sequence alignment (a) we can count the number of differences between each pair of sequences to build a distance matrix (b). This distance matrix can be used to build a tree (c) where the cumulative lengths of the horizontal branches (labeled with a red “1”) between any two species in the tree are proportional to the number of nucleotide differences between these species. Three equivalent versions of the same phylogenetic tree are shown. The only difference between the trees is that their nodes have been rotated at the points indicated by red arrows. The vertical position of species is different between the trees but the pattern of ancestry (the nodes shared by each species) remains unchanged.    The structure of a phylogenetic tree is inferred by applying either an algorithm or some set of optimality criteria. An  algorithm is a programmed series of steps designed to construct a single tree. Algorithms used to build phylogenetic trees include the unweighted pair group method with arithmetic mean (UPGMA) and the neighbor-joining method. Methods that employ optimality criteria include parsimony, maximum likelihood, and Bayesian analyses. These evaluate many possible trees and select the one tree that has the best optimality score, that is, they select the tree that best fits the sequence data given a discrete model of molecular evolution. Optimality scores are calculated on the basis of evolutionary models that describe how molecular sequences change over time. Limitations of Phylogenetic Trees 1. difficult to choose the true tree based on available sequence data if several different trees fit the data equally well. Bootstrapping, a statistical method in which information is resampled at random, is an approach used to deal with uncertainty in phylogenetic trees.  Bootstrap values indicate the percentage of the time that a given node in a phylogenetic tree is  supported by the sequence data.  High bootstrap values indicate that a node in the tree is likely to be correct, while low bootstrap values indicate that the placement of a node cannot be accurately determined given the available data. 2. Homoplasy, or convergent evolution, occurs when organisms share a trait that was not inherited from a common ancestor. This occurs in sequences also.  when similar sequence positions result from recurrent mutation rather than inheritance from a common ancestor  as a result of homoplasy, the reconstruction of accurate phylogenetic trees gets more difficult when sequence divergence between organisms is very high. 3. prevalence of horizontal gene transfer also creates complications for evolutionary history of microorganisms.  In general, genes encoding SSU rRNAs appear to be transferred horizontally at very low frequencies , and rRNA gene phylogenies agree largely with those prepared from other genes that encode genetic informational functions.  Thus, SSU rRNA gene sequences are generally considered to provide an accurate record of organismal phylogeny. (note gene phylogeny depicts evolutionary history of a gene, while organismal phylogeny depicts evolutionary history of a cell).  Many microbial genomes contain genes that have been acquired by horizontal gene transfer at some point in their evolutionary history. Horizontal gene transfer. The horizontal transfer of a gene will cause it to have a different evolutionary history from the rest of the genome. (a) Genes are transferred horizontally between distantly related microorganisms. Colors are used to match microorganisms with their genomes. (b) As a result of the horizontal transfer events in part a, different phylogenetic trees for gene 1, gene 2, and gene 3 are obtained. Only the gene tree for gene 1, which was not transferred, remains congruent with the organismal phylogeny. (lifted from Madigan et all 2019, Brock Biology of Microorganisms) Challenge Questions Set 2 1. For the following sequences, construct the phylogenetic tree that best depicts their evolutionary relationships. Taxon 1: GTAAAACCCGAT Taxon 2: GTTCCCTTA Taxon 3: GAAAAACCCTAT Taxon 4: CTTCCCTTT Taxon 5: GTTCGGTAT 2. What is the difference between taxonomy and phylogeny? Word limit: 50 words max 3. How does horizontal gene transfer complicate evolutionary history or phylogenetic studies of microorganisms ? Answer in not more than 100 words