Module 3 - Species Concept and Taxonomic Methods.docx

Lesson 2 Part B: Microbial Phylogeny and Systematics (Species Concept and Taxonomic Methods) 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 05. Classification and Nomenclature (B)  Taxonomy and Describing New Species  Classification and nomenclature  Bergey’s Manual and the Prokaryotes (introduction)  Culture Collections 03. Species Concept  “Species are the fundamental units of biological diversity, and how we distinguish and classify species in microbiology greatly affects our ability to explain and assess the diversity of the microbial world.  At present, there is no universally accepted concept of a microbial species.  Microbial systematics combines phenotypic, genotypic, and sequence-based phylogenetic data within a framework of standards and guidelines for describing and identifying microorganisms in a taxonomic framework, but the issue of what actually constitutes a species remains controversial.  However, a working definition of a microbial species has been developed and widely used. “ A Phylogenetic Species Concept for Bacteria and Archaea Members of a species should be genetically and phenotypically cohesive, i.e. , they share distinct traits that are unique only to the species. A species should also be monophyletic, that is, the strains composing the species should all share a recent common ancestor to the exclusion of other species. Phylogenetic species concept: combines the two principles above, thus. a species is a group of strains that share certain characteristic traits and which are genetically cohesive, and share a unique recent common ancestor. This species concept requires that a majority of genes in the species have congruent phylogenies and share a recent common ancestor. It is NOT based on an evolutionary model of speciation, and thus species described in this way do not necessarily reflect meaningful units in terms of ecological or evolutionary processes. The phylogenetic species concept was developed to facilitate taxonomy, and species justifications derived from this concept are based largely on the expert judgment of taxonomists. Species of Bacteria and Archaea are defined operationally as a group of strains sharing a high degree of similarity in many traits and sharing a recent common ancestor for their SSU rRNA genes. Species characterization employs a polyphasic approach that considers a range of different traits in making taxonomic judgments. Traits currently considered most important for identifying species include genomic similarity based on DNA hybridization and comparisons of SSU rRNA sequences. The degree of DNA–DNA hybridization between the genomes of two organisms provides a measure of their genomic similarity. (see hybridization experiment in next slide) Genomic Hybridization as a Taxonomic Tool (a) Genomic DNA is isolated from the organisms to be compared and then sheared and denatured. Probe DNA is prepared from organism 1 by shearing, denaturing, and labeling the DNA (shown here as radioactive phosphate). share greater than 97% 16S rRNA gene sequence similarity and 70% genomic hybridization values, and thus, are likely members of the same species. Adapted from Rosselló-Mora, R., and R. Amann. 2001. FEMS Microbiol. Revs. 25: 39–67, and Stackebrandt, E., and J. Ebers. 2006. Microbiology Today. 11: 153–155. (b) Sheared single-stranded target DNA from each genome is immobilized on a membrane and then hybridized with the labeled probe DNA from organism 1. Radioactivity in the hybridized DNA is measured. To recap, what we understand so far operationally speaking….. What is a microbial “species”? Eukaryotic species = interbreeding populations. BUT Microbes are asexual! • >70% DNA hybridization • >97% rRNA sequence identity (Arbitrary boundary to avoid disrupting existing assignments, rather than on theoretical considerations) (c) Radioactivity in the control (organism 1 DNA hybridizing to itself) is taken as the 100% hybridization value. A value of 70% or less genomic hybridization and a difference in SSU rRNA gene sequence of 3% or more between two organisms is taken as evidence that the two are distinct species. Experimental data suggest that these criteria are valid, reliable, and consistent in identifying new microbial species for taxonomic purposes. Relationship between 16s rRNA gene sequence similarity and genomic DNA-DNA hybridization for pairs of organisms Pairs of microorganisms are compared on the basis of their 16S rRNA similarities and DNA–DNA hybridization values. Points in the upper right region represent pairs of strains that How many species of Bacteria and Archaea exist?  No firm estimate of the number of bacterial and archaeal species can be given at present, in part because of uncertainty about what defines a species in these domains. But this number is expected to be very large.  Analyses of environmental SSU rRNA gene sequences using the phylogenetic species concept indicate that over 10,000 different species can coexist in a single gram of soil!  Since 1977 more than 3.3 million SSU rRNA sequences have been generated and used to characterize the vast diversity of the microbial world.  The Ribosomal Database Project (RDP; http://rdp.cme.msu.edu ) contains an ever-growing collection of these sequences and provides computational programs for their analysis and for the construction of phylogenetic trees. 04. Taxonomic Methods in Systematics A polyphasic approach, that is, an approach that uses many different methods in combination, is used to identify and name species of Bacteria and Archaea in accordance with the currently accepted phylogenetic species concept. Next, we describe methods commonly used for characterizing microbial, and primarily prokaryotic, species. RDP provides quality-controlled, aligned and annotated Bacterial and Archaeal 16S rRNA sequences, and Fungal 28S rRNA sequences, and a suite of analysis tools to the scientific community. After browsing this page for familiarization, what new thing did you learn about rRNA? http://rdp.cme.msu.edu/ How do we reconstruct evolutionary relationships? 1. Observe 2. Biological Records a) Fossils b) Geology 3. Infer from data of current organisms (chemistry, gene sequence, protein sequence) Important! - Key word is infrence (not always correct!) - none of he organisms in the “tree of life” are ancient; they are all modern organisms - Some may have characteritics of ancient organisms a. Gene Sequence Analysis  Gene sequences are commonly determined from PCRamplified fragments of DNA, and the sequences are analyzed using phylogenetic analyses.  SSU rRNA gene sequences are highly conserved, and while they provide valuable phylogenetic information, they are not always useful for distinguishing closely related species.  Other highly conserved genes, such as recA, which encodes a recombinase protein and gyrB, which encodes DNA gyrase can be useful for distinguishing bacteria at the species level.  The DNA sequences of protein-encoding genes accumulate mutations more rapidly than rRNA genes.  Therefore, sequences from such genes can often distinguish prokaryotic species that cannot be resolved by rRNA gene sequence analyses alone. (see figure in next slide) Multigene phylogenetic analysis A phylogeny is shown for species in the genus Photobacterium. (a) 16S rRNA gene tree, showing the species to be poorly resolved. (b) Multigene analysis based on combined analysis of the 16S rRNA gene and gyrB and luxABFE genes in 21 isolates from three Photobacterium species. Multigene analysis clearly resolves the strains into three distinct phylogenetic species, P. phosphoreum, P. iliopiscarium, and P. kishitanii. The scale bar indicates the branch length equal to a total of 50 nucleotide changes. The type strain (Section 13.10) of each species is listed in bold. (All abbreviations are part of strain designations.) Phylogenetic analyses courtesy of Tory Hendy and Paul V. Dunlap, University of Michigan. (lifted from Fig, 13. 24 of Madigan et al. 2019) b. Multilocus sequence typing (MLST) MLST is a method that uses “housekeeping “genes from several related organisms , which are sequenced and the sequences used collectively to distinguish the organisms. Housekeeping genes encode essential functions in cells and are always located on the chromosome rather than on a plasmid. For each gene, an approximately 450-base-pair sequence is amplified and then sequenced. The alleles of each gene (variants that differ by at least one nucleotide) are each assigned a number. The strain being studied is then assigned an allelic profile, or multilocus sequence type, consisting of a series of numbers representing its particular combination of alleles. Ribotype results for four different lactic acid bacteria. DNA was taken from a strain of each bacterium, digested into fragments by restriction enzymes, separated by gel electrophoresis, and then probed with a 16S rRNA gene probe. Variations in position and intensity of the bands are important in identification.  Different microbial species can have different numbers of rRNA operons, ranging from 1 to 15, and the number of rRNA operons present in a microbial genome is a conserved feature of all strains of a species.  Changes in genome sequence between strains can cause the endonuclease enzyme to cut in different locations, producing variation in the lengths of the restriction fragments that are visualized. Hence, the size and number of bands generates a specific pattern, a kind of genome fingerprint called a ribotype, and this pattern can be compared with patterns of reference organisms in a computer database.  The ribotype of a particular organism can be unique and diagnostic, allowing rapid identification of different species and even different strains of a species. Steps in MLST leading to a similarity phenogram are shown. Strains 1–5 are virtually identical, whereas strains 6 and 7 are distinct from one another and from both strains 1–5 and the new strain. (Lifted from Fig 13.25 of Madigan et al. 2019) MLST has sufficient resolving power to distinguish among even very closely related strains of a given species. In practice, strains can be discriminated on the basis of a single nucleotide change in just one of the analyzed genes. c. Genome fingerprinting  A rapid approach for evaluating polymorphisms between strains of a species. The fingerprints are generally fragments of DNA generated from individual genes or whole genomes.  Polymorphism, as related to genomics, refers to the presence of two or more variant forms of a specific DNA sequence that can occur among different individuals or populations. The most common type of polymorphism involves variation at a single nucleotide (also called a single-nucleotide polymorphism, or SNP).  e.g. Ribotyping is a form of genome fingerprinting based on the localization of SSU rRNA genes on genome fragments. In this method, genomic DNA from an organism is digested by a restriction enzyme and the fragments are separated by gel electrophoresis, transferred to a nylon membrane, and labeled with an SSU rRNA gene probe DNA fingerprinting with rep-Pcr.  Other common genome fingerprinting methods include repetitive extragenic palindromic PCR (rep-PCR) and amplified fragment length polymorphism (AFLP).   The rep-PCR method is based on the presence of highly conserved repetitive DNA elements interspersed randomly around the bacterial chromosome. The number and positions of these elements differ between strains of a species. Genomic DNAs from five strains (1–5) of a single species of bacteria were PCR-amplified using specific primers called rep (repetitive extragenic palindromic). The PCR products were separated in an agarose gel on the basis of size to generate DNA fingerprints. Arrows indicate some of the differing bands. Lanes 6 and 7 are 100-bp and 1kbp DNA size markers, respectively, used for estimating sizes of the DNA fragments. Other common genome fingerprinting methods include repetitive extragenic palindromic PCR (rep-PCR) and amplified fragment length polymorphism (AFLP). AFLP is based on the digestion of genomic DNA with one or two restriction enzymes and selective PCR amplification of the resulting fragments, which are then separated by agarose gel electrophoresis. Strain-specific banding patterns similar to those of rep-PCR or other DNA fingerprinting methods are generated, with the large number of bands giving a high degree of discrimination between strains within a species. e. Phenotypic Analyses The phenotype of a bacterium provide many traits that can be used to differentiate species. Typically, several phenotypic traits are determined routinely when describing a new microorganism. These phenotypic results are then compared to the phenotypes of organisms that have been described previously. The phenotypic traits that are determined will depend on the type of organism being described. A wide range of sequence analyses can be performed on entire genomes, providing insights into microbial physiology and microbial evolution. These analyses have also provided important insights into the large role that horizontal gene exchange has played in microbial evolution and on the highly dynamic nature of microbial genomes. The table in the below lists the general categories and examples of some phenotypic traits used in identifications and species descriptions. d. Multigene and Whole Genome Analyses The use of multiple genes and entire genomes for the identification and description of bacteria is becoming increasingly common as DNA sequencing capacities improve and costs decline. A wide range of sequence analyses can be performed on entire genomes, providing insights into microbial physiology and microbial evolution. These analyses have also provided important insights into the large role that horizontal gene exchange has played in microbial evolution and on the highly dynamic nature of microbial genomes. Shared orthologs (homologous genes that share the same function, can be aligned and examined using phylogenetic methods to determine the average nucleotide identity of these genes.  Genes separated by speciation are called orthologs. Genes separated by gene duplication events are called paralogs. Comparative analyses of gene content (presence or absence of genes), synteny (the order of genes in the genome), and genome GC content provide further insights into relationships between strains. Entire genome sequences can also be used for metabolic reconstruction and characterization of a cell’s genetic capacities. Several methods in comparative genomics and population genomics have been developed for use in systematic analyses. General categories and examples of some phenotypic traits used in identification and species descriptions Fatty acid profile: example of a phenotypic trait  types and proportions of fatty acids present in cytoplasmic membrane lipids and the outer membrane lipids of gramnegative bacteria are often used in taxonomic analysis  FAME (fatty acid methyl ester): method used to identify these fatty acids; widely used in clinical, public health, and food- and water inspection laboratories where pathogens routinely must be identified.  Fatty acid composition of Bacteria (domain) varies from species to species in chain length and in the presence or absence of double bonds, rings, branched chains, or hydroxy groups In FAME analyses, fatty acids extracted from cells grown in culture under standardized conditions are analyzed by gas chromatography. A chromatogram showing the types and amounts of fatty acids from the unknown bacterium is then compared with a database containing the fatty acid profiles of thousands of reference bacteria grown under the same conditions. Fatty acid profiles of an organism, like many other phenotypic traits, can vary as a function of temperature, growth phase (exponential versus stationary), and growth medium. Hence, for comparative results to be valid, it is necessary to grow the unknown organism on a specific medium and at a specific temperature. For many organisms this is impossible, of course, limiting the applicability of FAME analyses as a taxonomic tool. In addition, the extent of variation in FAME profiles among strains of a species, a necessary consideration in studies to discriminate between species, is a work in progress. Phenotype analysis: limitations  Phenotypic characteristics of strains are generally highly dependent on growth conditions, and phenotypes observed in the laboratory environment may not well represent phenotypes present in the natural environment; thus care must be taken in using phenotypic characteristics in systematic analyses. Only a single example is given of each class; however, more than 200 structurally distinct fatty acids are known from bacterial sources. A methyl ester contains a methyl group (CH3) in place of the proton on the carboxylic acid group (COOH) of the fatty acid. Lifted from Madigan et al. 2019. FAME, Procedure. Each peak from the gas chromatograph is due to one particular fatty acid methyl ester, and the peak height is proportional to the amount.  In addition, the value of different phenotypic characteristics for reaching a firm systematics conclusion can vary with the taxonomic groups being examined. 05. Classification and Nomenclature: taxonomy and describing new species  science of taxonomy – how microbes are named and classified  Information are presented on culture collections, which are repositories for scientific deposition of live microbial cultures; on some key taxonomic resources available for microbiology; and on the procedures for naming new microbial species.  The formal description of a new microbial species and the deposition of cultures into a culture collection form an important foundation of the systematics of prokaryotic species. Taxonomy and describing new species  Classification is the organization of organisms into progressively more inclusive groups on the basis of either phenotypic similarity or evolutionary relationship.  A species is made up of one to several strains, and similar species are grouped into genera (singular, genus). Similar genera are grouped into families, families into orders, orders into classes, up to the domain, the highest-level taxon based on a collection of phenotypic and genotypic information.   Nomenclature is the actual naming of organisms and follows the binomial system of nomenclature devised by the Swedish medical doctor and botanist Carl Linnaeus and used throughout biology - organisms are given genus names and species epithets. The names are Latin or Latinized Greek derivations, often descriptive of some key property of the organism, and are printed in italics. (or underlined if hand-written). The creation of new taxa of Bacteria and Archaea must follow the rules described in the International Code of Nomenclature of Bacteria (the Bacteriological Code). This source presents the formal framework by which Bacteria and Archaea are to be officially named and the procedures by which existing names can be changed, for example, when new data warrant taxonomic rearrangements. Taxonomic hierarchy for Allochromatium warmingii the purple sulfur bacterium In each issue, the IJSEM also publishes an approved list of newly validated names. By providing validation of newly proposed names, publication in IJSEM paves the way for their inclusion in taxonomic reference sources. Two websites provide listings of valid, approved bacterial names: List of Prokaryotic Names with Standing in Nomenclature (http://www.bacterio.net ), and Prokaryotic Nomenclature Up-to-Date (http://www.dsmz.de). (Make sure to visit these websites and familiarize yourself with the contents) It is possible to use molecular and genomic techniques to characterize the phenotypic and genotypic characteristics of microorganisms without the need for the cultivation of an isolated strain. However, in the absence of an isolate that can be deposited in two international culture collections, it is not possible to validly name a new species of microorganism under the Bacteriological Code. When an organism is well characterized but not yet cultured or not yet obtained in pure culture, a provisional taxonomic name can be applied. The moniker Candidatus is appended to candidate taxonomic ranks. For example, “Candidatus Pelagibacter ubique” is a globally widespread and wellcharacterized marine bacterium that is difficult to grow in laboratory media, and so has not been formally named under the Bacteriological Code. By contrast, the bacterium “Candidatus Heliomonas lunata” can be grown in laboratory culture but the culture is not pure so this bacterium also retains Candidatus status. Taxonomy and describing new species When a new isolate of Bacteria or Archaea is isolated from nature and thought to be unique, a decision must be made as to whether it is sufficiently different from established taxa to be described as a new taxon. To achieve formal validation of taxonomic standing as a new genus or species, a detailed description of the organism’s characteristics and distinguishing traits, along with its proposed name, must be published, and viable cultures of the organism must be deposited in at least two international culture collections. The manuscript describing and naming a new taxon undergoes peer review before publication. A major vehicle for the description of new taxa is the International Journal of Systematic and Evolutionary Microbiology (IJSEM), the official publication of record for the taxonomy and classification of Bacteria, Archaea, and microbial eukaryotes. The International Committee on the Systematics of Prokaryotes (ICSP) oversees the nomenclature and taxonomy of Bacteria and Archaea. The ICSP also oversees the publication of IJSEM and the International Code of Nomenclature of Bacteria and gives guidance to several subcommittees that meet to establish and revise standards for the description of new species in the different groups of Bacteria and Archaea. Bergey’s Manual and The Prokaryotes The classification system most widely accepted by microbiologists is that of Bergey’s Manual of Systematic Bacteriology, a major taxonomic treatment of Bacteria and Archaea. (domains). Bergey’s Manual has served microbiologists since 1923 as a compendium of information on all recognized prokaryotic species. Each chapter, written by experts, contains tables, photos, figures, and other systematic information useful for identification purposes. A second major source describing the physiology, ecology, phylogeny, enrichment, isolation, and cultivation of Bacteria and Archaea is The Prokaryotes. This work is available online by subscription through university libraries. Collectively, Bergey’s Manual and The Prokaryotes offer microbiologists both the concepts and the details of the biology of Bacteria and Archaea as we know them today and are the primary resources for microbiologists characterizing newly isolated organisms. Culture Collections National microbial culture collections (some listed in table below) are an important foundation of microbial systematics. These permanent collections catalog and store microorganisms and provide cultures upon request (for a fee) to researchers in academia, medicine, and industry. The collections play an important role in protecting microbial biodiversity, just as museums do in preserving plant and animal specimens for future study. However, unlike museums, which maintain collections of chemically preserved or dried, dead specimens, microbial culture collections store microorganisms as viable cultures, typically frozen or in a freeze-dried state. These storage methods maintain the cells indefinitely in a living state and prevent genetic changes that might occur if the organisms were continually subcultured. A related and key role of culture collections is as repositories for type strains. When a new species of bacteria is described in a scientific journal, a strain is designated as the type strain and this strain serves as the nomenclatural type of the taxon for future taxonomic comparison with other strains of that species Deposition of this type strain in the national culture collections of at least two countries— thereby making the strain publicly available internationally—is a prerequisite for validation of the new species name. Challenge Questions Set 3 (30 pt) Answer the following in 50 words or less each. Place your word count at the end of your answer. 1. Why is a polyphasic approach necessary in bacterial taxonomy and systematics? 2. Why might viable cell cultures be of more use in microbial taxonomy than preserved specimens? 3. What are some key criteria from the phylogenetic species concept used to determine whether two strains belong to the same species?