Microbial Diversity Unit 1 Notes (NEP) PDF

Microbial biodiversity Microbial biodiversity is defined as the variability among microorganisms. Microbial biodiversity is all about how different kinds of microorganisms arose and why ; how exactly are microorganisms either similar to or differen...

Microbial biodiversity Microbial biodiversity is defined as the variability among microorganisms. Microbial biodiversity is all about how different kinds of microorganisms arose and why ; how exactly are microorganisms either similar to or different from each other; and evolution of microbial cells, The diversity of microorganisms we see today is the result of nearly 4 billion years of evolution. Microbial diversity can be seen in many ways besides phylogeny, including cell size and morphology (shape), physiology, motility, mechanism of cell division, pathogenicity, developmental biology, adaptation to environmental extremes and so on. With the laws of chemistry and physics. This enormous metabolic versatility has allowed prokaryotes to thrive in every potential habitat on Earth suitable for life. The diversity is measured by various perspective such as Morphology, structural, metabolic, ecology , behavioral and evolution. Origin of life, Evolution and origin of biodiversity: Earth is thought to have formed about 4.5 billion years ago, based on analyses of slowly decaying radioactive isotopes. Earth cooled much earlier than previously believed, with solid crust forming and water condensing into oceans perhaps as early as 4.3 billion years ago. The presence of liquid water implies that conditions could have been compatible with life within a couple of hundred million years after Earth was formed. Fossilized remains of procaryotic cells around 3.5 to 3.8 billion years old have been discovered in stromatolites (microbial mats consisting of layers of filamentous prokaryotes and trapped mineral materials) and sedimentary rocks. Microfossil evidence strongly suggests that microbial life was present within at least 1 billion years of the formation of Earth and probably somewhat earlier, and by that time, microorganisms had already attained an impressive diversity of morphological forms. Very likely the earliest procaryotes were anaerobic. Cyanobacteria and oxygen-producing photosynthesis probably developed 2.5 to 3.0 billion or more years ago. Microbial diversity increased greatly as oxygen became more plentiful. It appears likely that modern eucaryotic cells arose from prokaryotes about 1.4 billion years ago. There has been considerable speculation about how eucaryotes might have developed from procaryotic ancestors. It is not certain how this process occurred, and two hypotheses have been proposed. According to the first, nuclei, mitochondria, and chloroplasts arose by invagination of the plasma membrane to form double-membrane structures containing genetic material and capable of further development and specialization. The similarities between chloroplasts, mitochondria, and modern bacteria are due to conservation of primitive procaryotic features by the slowly changing organelles. According to the more popular endosymbiotic hypothesis, the first event was nucleus formation in the pro-eucaryotic cell. The ancestral eucaryotic cell may have developed from a fusion of ancient bacteria and archaea. Possibly a gram- negative bacterial host cell that had lost its cell wall engulfed an archaeon to form an endosymbiotic association. The archaeon subsequently lost its wall and plasma membrane, while the host bacterium developed membrane infolds. Eventually the host genome was transferred to the original archaeon, and a nucleus and the endoplasmic reticulum was formed. Both bacterial and archaeal genes could be lost during formation of the eucaryotic genome. It should be noted that many believe that the Archaea and Eucarya are more closely related than this hypothetical scenario implies. Vasantha B.S. They propose that the eucaryotic line diverged from the Archaea and then the nucleus formed, possibly from the Golgi apparatus.Mitochondria and chloroplasts appear to have developed later. The free-living, fermenting ancestral eucaryote with its nucleus established a permanent symbiotic relationship with photosynthetic bacteria, which then evolved into chloroplasts. Cyanobacteria have been considered the most likely ancestors of chloroplasts. EG:Prochloron lives within marine invertebrates and resembles chloroplasts in containing both chlorophyll a and b, but not phycobilins. The endosymbiotic hypothesis has received support from the discovery of an endosymbiotic cyanobacterium that inhabits the biflagellate protist Cyanophora paradoxa and acts as its chloroplast. This endosymbiont, called a cyanelle, resembles the cyanobacteria in its photosynthetic pigment system and fine structure and it is surrounded by a peptidoglycan layer. It differs from cyanobacteria in lacking the lipopolysaccharide outer membrane characteristic of gram-negative bacteria. The cyanelle may be a recently established endosymbiont that is evolving into a chloroplast. Further support is provided by rRNA trees, which locate chloroplast RNA within the cyanobacteria. At present both hypotheses have supporters. It is possible that new data may help resolve the issue to everyone’s satisfaction. However, these hypotheses concern processes that occurred in the distant past and cannot be directly observed. Thus a complete consensus on the matter may never be reached. Classification Systems The most desirable classification system, called a natural classification, arranges organisms into groups whose members share many characteristics and reflects as much as possible the biological nature of organisms. Linnaeus developed the first natural classification, based largely on anatomical characteristics, in the middle of the eighteenth century. It was a great improvement over previously employed artificial systems because knowledge of an organism’s position in the scheme provided information about many of its properties. For example, classification of humans as mammals denotes that they have hair, self-regulating body temperature, and milk-producing mammary glands in the female. There are two general ways in which classification systems can be constructed. Organisms can be grouped together based on overall similarity to form a phenetic system or they can be grouped based on probable evolutionary relationships to produce a phylogenetic system. Computers may be used to analyze data for the production of phenetic classifications. The process is called numerical taxonomy. Phenetic Classification  most natural classification is the one with the greatest information content or predictive value.  A good classification should bring order to biological diversity and may even clarify the function of a morphological structure. For example,: if motility and flagella are always associated in particular microorganisms, it is reasonable to suppose that flagella are involved in at least some types of motility. When viewed in this way, the best natural classification system may be a phenetic system, one that groups organisms together based on the mutual similarity of their phenotypic characteristics. Although phenetic studies can reveal possible evolutionary relationships, they are not dependent on phylogenetic analysis.. Organisms sharing many characteristics make up a single group or taxon. Numerical Taxonomy  Peter H. A. Sneath and Robert Sokal have defined numerical taxonomy as “the grouping by numerical methods of taxonomic units into taxa on the basis of their character states.”  Information about the properties of organisms is converted into a form suitable for numerical analysis and then compared by means of a computer.  The resulting classification is based on general similarity as judged by comparison of many characteristics, each given equal weight. This approach was not feasible before the advent of computers because of the large number Vasantha B.S. of calculations involved.  The process begins with a determination of the presence or absence of selected characters in the group of organisms under study.  A character usually is defined as an attribute about which a single statement can be made. Many characters, at least 50 and preferably several hundred, should be compared for an accurate and reliable classification. It is best to include many different kinds of data: morphological, biochemical, and physiological.  After character analysis, an association coefficient, a function that measures the agreement between characters possessed by two organisms, is calculated for each pair of organisms in the group. The simple matching coefficient (SSM), the most commonly used coefficient in bacteriology, is the proportion of characters that match regardless of whether the attribute is present or absent.  Sometimes the Jaccard coefficient (SJ) is calculated by ignoring any characters that both organisms lack. Both coefficients increase linearly in value from  0.0 (no matches) to 1.0 (100% matches).  The simple matching coefficients, or other association coefficients, are then arranged to form a similarity matrix. This is a matrix in which the rows and columns represent organisms, and each value is an association coefficient measuring the similarity of two different organisms so that each organism is compared to every other one in the table.  Organisms with great similarity are grouped together and separated from dissimilar organisms; such groups of organisms are called phenons (sometimes called phenoms).  The results of numerical taxonomic analysis are often summarized with a treelike diagram called a dendrogram.  The diagram usually is placed on its side with the X-axis or abscissa graduated in units of similarity.  Each branch point is at the similarity value relating the two branches.  The organisms in the two branches share so many characteristics that the two groups are seen to be separate only after examination of association coefficients greater than the magnitude of the branch point value. Below the branch point value, the two groups appear to be one. The ordinate in such a dendrogram has no special significance, and the clusters may be arranged in any convenient order.   The significance of these clusters or phenons in traditional taxonomic terms is not always evident, and the similarity levels at which clusters are labeled species, genera, and so on, are a matter of judgment.  Sometimes groups are simply called phenons and preceded by a number showing the similarity level above which they appear (e.g.,a 70-phenon is a phenon with 70% or greater similarity among its constituents). Phenons formed at about 80% similarity often are equivalent to species.  Numerical taxonomy has already proved to be a powerful tool in microbial taxonomy. Although it often has simply reconfirmed already existing classification schemes, sometimes accepted classifications are found wanting. Numerical taxonomic methods also can be used to compare sequences of macromolecules such as RNA and proteins. Phylogenetic Classification  Following the publication in 1859 of Darwin’s On the Origin of Species, biologists began trying to develop phylogenetic or phyletic classification systems.  These are systems based on evolutionary relationships rather than general resemblance (the term phylogeny [Greek phylon, tribe or race, and genesis, generation or origin] refers to the evolutionary development of a species).  This has proven difficult for procaryotes and other microorganisms, primarily because of the lack of a good fossil record.  The direct comparison of genetic material and gene products such as RNA and proteins overcomes many of these problems. Major Characteristics Used in Taxonomy Characteristics have been divided into two groups: classical and molecular. Classical Characteristics Vasantha B.S. Classical approaches to taxonomy make use of morphological, physiological, biochemical, ecological, and genetic characteristics.These characteristics have been employed in microbial taxonomy for many years. They are quite useful in routine identification and may provide phylogenetic information as well. Morphological Characteristics  Morphological features are important in microbial taxonomy fomany reasons. Morphology is easy to study and analyze, particularly in eucaryotic microorganisms and the more complex procaryotes.  Morphological comparisons are valuable because structural features depend on the expression of many genes, are usually genetically stable, and normally (at least in eucaryotes) these do not vary greatly with environmental changes. Thus morphological similarity often is a good indication of phylogenetic relatedness. Although the light microscope has always been a very important tool, its resolution limit of about 0.2 m reduces its usefulness in viewing smaller microorganisms and structures. The transmission and scanning electron microscopes, with their greater resolution, have immensely aided the study of all microbial groups. Physiological and Metabolic Characteristics  Physiological and metabolic characteristics are very useful because they are directly related to the nature and activity of microbial enzymes and transport proteins. Since proteins are gene products, analysis of these characteristics provides an indirect comparison of microbial genomes. Ecological Characteristics  Many properties are ecological in nature since they affect the relation of microorganisms to their environment. Often these are taxonomically valuable because even very closely related microorganisms can differ considerably with respect to ecological characteristics. Microorganisms living in various parts of the human body markedly differ from one another and from those growing in freshwater, terrestrial, and marine environments. Some examples of taxonomically important ecological properties are life cycle patterns; the nature of symbiotic relationships; the ability to cause disease in a particular host; and habitat preferences such as requirements for temperature,pH, oxygen, and osmotic concentration. Many growth requirements are also considered physiological characteristics. GENOTYPIC CLASSIFICATION  Transformation can occur between different procaryotic species but only rarely between genera. The demonstration of transformation between two strains provides evidence of a close relationship since transformation cannot occur unless the genomes are fairly similar. Transformation studies have been carried out with several genera: Bacillus, Micrococcus, Haemophilus, Rhizobium, and others. Despite transformation’s usefulness, its results are sometimes hard to interpret because an absence of transformation may result from factors other than major differences in DNA sequence.  Conjugation studies also yield taxonomically useful data, particularly with the enteric bacteria. For example, Escherichia can undergo conjugation with the genera Salmonella and Shigella but not with Proteus and Enterobacter. These observations fit with other data showing that the first three of these genera are more closely related to one another than to Proteus and Enterobacter  Plasmids are undoubtedly important in taxonomy because they are present in most bacterial genera, and many carry genes coding for phenotypic traits. Because plasmids could have a significant effect on classification if they carried the gene for a trait of major importance in the classification scheme, it is best to base a classification on many characters. When the identification of a group is based on a few characteristics and some of these are coded for by plasmid genes, errors may result. For example, hydrogen sulfide production and lactose Vasantha B.S. fermentation are very important in the taxonomy of the enteric bacteria, yet genes for both traits can be borne  on plasmids as well as bacterial chromosomes. One must take care to avoid errors as a result of plasmid-borne traits. Molecular methods are the most powerful approaches to taxonomy through the study of amino acid sequence of proteins and nucleic acid hybridization. NUCLEIC ACID HYBRIDIZATION  Microbial genomes can be directly compared, and taxonomic similarity between genomes can be compared more directly by the use of nucleic acid hybridization. A mixture of ssDNA formed by heating dsDNA is cooled and held a temperature of about 250c below the Tm (melting temperature), strands with complementary base sequence will reassociate to form a stable dsDNA and non-complementary strands will remain single. DNA-DNA HYBRIDIZATION This is the most widely used hybridization technique to study the closely related microbes. Using this technique a new isolate is compared with a known organism The nitrocellulose filters with bound non- radioactive DNA strands are incubated at appropriate temperature with single stranded DNA fragments which are made radioactive with 32P, 3H and 34C. After radioactive fragments are allowed to hybridize with the membrane bound ssDNA, the membrane is washed to remove any non-hybridized ssDNA and its radioactivity is measured. The quantity of radioactivity bound to the filter reflects the amount of hybridization and thus the similarity of the DNA sequence. The more the radioactivity bound, the greater the homology between DNA sequence. Membrane attached with experimental Non-radioactive DNA Incubated with radioactive Known DNA Membrane is washed The amount of radioactivity Bound to the membrane is measured RNA-DNA HYBRIDIZATION In this method more distantly related organisms are compared by carrying out DNA-RNA hybridization experiments using radioactive ribosomal or transfer RNA Distant relationships can be detected because rRNA and tRNA genes represent only a small portion of the total DNA genome Membrane bound experimental DNA is incubated with radioactive known rRNA The membrane is washed and counted An accurate measurement of homology is obtained by finding the temperature required to dissociate and remove half the radioactive rRNAfrom the membrane Vasantha B.S. The higher the temperature, the stronger the rRNA-DNA complex and the more similar the sequence  Nucleic Acid Sequencing  Genome structures can be directly compared only by sequencing DNA and RNA.  RNA sequencing has been used more extensively in microbial taxonomy.  Most attention has been given to sequences of the 5S and 16S rRNAs isolated from the 50S and 30S subunits, respectively, of procaryotic ribosomes.  Ribosomal RNAs can be characterized in terms of partial sequences by the oligonucleotide cataloging method as follows.  Purified, radioactive 16S rRNA is treated with the enzyme T1 ribonuclease, which cleaves it into fragments.  The fragments are separated, and all fragments composed of at least six nucleotides are sequenced.  The sequences of corresponding 16S rRNA fragments from different procaryotes are then aligned and compared using a computer, and association coefficients (Sab values) are calculated.  Complete rRNAs now are sequenced using procedures like the following. First, RNA is isolated and purified. Then, reverse transcriptase is used to make complementary DNA (cDNA) using primers that are complementary to conserved rRNA sequences. Next, the polymerase chain reaction amplifies the cDNA. Finally, the cDNA is sequenced and the rRNA sequence deduced from the results Taxonomic Ranks In preparing a classification scheme, one places the microorganism within a small, homogeneous group that is itself a member of larger groups in a nonoverlapping hierarchical arrangement. A category in any rank unites groups in the level below it based on shared properties In procaryotic taxonomy the most commonly used levels or ranks (in ascending order) are species, genera, families, orders, classes, and phyla. Microbial groups at each level or rank have names with endings or suffixes characteristic of that level. Microbiologists often use informal names in place of formal hierarchical ones. Typical examples of such names are purple bacteria, spirochetes, methane-oxidizing bacteria, sulfate-reducing bacteria, and lactic acid bacteria. The basic taxonomic group in microbial taxonomy is the species. Taxonomists working with higher organisms define the term species differently than do microbiologists. Species of higher organisms are groups of interbreeding or potentially interbreeding natural populations that are reproductively isolated from other groups. This is a satisfactory definition for organisms capable of sexual reproduction but fails with many microorganisms because they do not reproduce sexually. Procaryotic species are characterized by phenotypic and genotypic differences. A prokaryotic species is a collection of strains that share many stable properties and differ significantly from other groups of strains. This definition is very subjective and can be interpreted in many ways. The following more precise definition has been proposed by some bacterial taxonomists. A species (genomospecies) is a collection of strains that have a similar G _C composition and 70% or greater similarity as judged by DNA hybridization experiments. Ideally a species also should be phenotypically distinguishable from other similar species. A strain is a population of organisms that is distinguishable from at least some other populations within a particular taxonomic category. It is considered to have descended from a single organism or pure culture isolate. Strains within a species may differ slightly from one another in many ways. Biovars are variant Vasantha B.S. procaryotic strains characterized by biochemical or physiological differences, morphovars differ morphologically, and serovars have distinctive antigenic properties. One strain of a species is designated as the type strain. It is usually one of the first strains studied and often is more fully characterized than other strains; however, it does not have to be the most representative member. The type strain for the species is called the type species and is the nomenclatural type or the holder of the species name. A nomenclatural type is a device to ensure fixity of names when taxonomic rearrangements take place. For example, the type species must remain within the genus of which it is the nomenclatural type. Only those strains very similar to the type strain or type species are included in a species. Each species is assigned to a genus, the next rank in the taxonomic hierarchy. A genus is a well-defined group of one or more species that is clearly separate from other genera. In practice there is considerable subjectivity in assigning species to a genus, and taxonomists may disagree about the composition of genera. Microbiologists name microorganisms by using the binomial system of the Swedish botanist Carolus Linnaeus. The Latinized, italicized name consists of two parts. The first part, which is capitalized, is the generic name, and the second is the uncapitalized specific epithet (e.g., Escherichia coli). The specific epithet is stable; the oldest epithet for a particular organism takes precedence and must be used. In contrast, a generic name can change if the organism is assigned to another genus because of new information. For example, the genus Streptococcus has been divided to form two new genera, Enterococcus and Lactococcus based on rRNA analysis and other characteristics. Thus Streptococcus faecalis is now Enterococcus faecalis. Often the name will be shortened by abbreviating the genus name with a single capital letter, for example E. coli. Approved lists of bacterial names were published in 1980 in the International Journal of Systematic Bacteriology, and new valid names are published periodically. Bergey’s Manual of Systematic Bacteriology contains the currently accepted system of procaryotic taxonomy and will be discussed later in the chapter. Domain (super kingdom) :, A domain also dominion, also called superkingdom, , is the highest taxonomic rank of all organisms taken together. Kingdom: A kingdom is the second highest taxonomic rank, just below domain. Kingdoms are divided into smaller groups called phyla. ERNST HAECKEL THREE-KINGDOM SYSTEM  Ernst Haekel suggested the three-kingdom system in 1866. In which he added the Protista as a new kingdom that contained most microscopic organisms.  One of the major divisions of Protista was called Moneres. Haeckel’s Moneres included known bacterial groups such as Vibrio.  Haeckel’s Protista kingdom also included eukaryotic organisms now classified as Protista..  The idea of a third kingdom lay dormant for almost a century.  Opposition to three-kingdoms heralded the start of a tradition which is still evident, involving the ‘Lumpers’ who favour a two-kingdom scheme and ‘Splitters’ who support three or more kingdoms. by the 1960s the unchallenged position of the two kingdoms had ended and a three-kingdom system were widely used and appeared in many biology texts. WHITTAKER’S FIVE-KINGDOM SYSTEM OF CLASSIFICATION In 1969, Robert Whittaker of Cornell University proposed a comprehensive system of classification based on nutrition and absorption of food materials by the living organisms. He proposed that all living beings can be classified under five kingdoms. Principle: The classification is based on three levels of cellular organization which evolved to accommodate three main methods of nutrition Photosynthesis – Plantae were mostly multicellular autotrophs Absorption - Fungi multicellular heterotrophs Ingestion - Animalia multicellular heterotrophs The remaining two kingdoms, Protista and Monera included Unicellular and simpler cellular colonies Criteria of classification: According to Whittaker all living beings can be accommodated in five kingdoms namely, Kingdom Monera – all procaryotes Kingdom Protista - unicellular eucaryotes Kingdom Fungi - all fungi Kingdom Plantae - all eukaryotic multicellular plants Kingdom Animalia–heterotrophic multicellular organisms Five kingdoms Property Monera Protista Fungi Plantae Animalia Cell type Prokaryotic Eukaryotic Eukaryotic Eukaryotic Eukaryotic Cell wall Non Present Present Non Absent cellulosic cellulosic Nuclear Present Present Present Present Present membrane Organization Unicellular Unicellular Multicellular Tissue/organ Tissue/organ/or gan system Mode of Autotrophic Autotrophic Heterotrophic Autotrophic Heterotrophic nutrition & heterotrophi c This system of classification looks more scientific and natural because of the following considerations: Separation of prokaryotes into independent kingdoms is justifiable because they differ from all other organisms in their general organization. Grouping of all unicellular eukaryotes under the kingdom Protista has solved many problems, particularly related to the position of organisms like Euglena. Elevation of the group of fungi to the status of a kingdom is justifiable since fungi totally differ from other primitive eukaryotes like algae and protozoans.  The kingdom Metaphyta and Metazoa are now more homogenous groups than they were in two kingdom classification as it shows the phylogeny in different life styles. The five-kingdom classification gives a clear indication of cellular organization and mode of nutrition, the characters which appeared very early in the evolution of life. However, the five-kingdom classification has certain drawbacks / demerits also, particularly with reference to the lower forms of life. The kingdoms Monera and Protista include diverse, heterogeneous forms of life. In both the kingdoms there are photosynthetic as well as non-photosynthetic organisms Both the kingdoms include organisms which have cells with cell wall as well as without cell wall. None of the three higher kingdoms include a single ancestor of all its forms. Multicellular lines have originated from Protista several items (phylogenetic). Unicellular green algae like Volvox and Chlamydomonas have not been included under Protista because of their resemblance to other green algae. Slime moulds differ totally from other members of Protista in their general organization. Viruses have not been given proper place in this system of classification. Figure 7: The five-kingdom classification by Whittaker ( Whittaker RH, Science 163:150–160, 1969. doi:10.1128/9781555818517.ch2. CARL R WOESE 3 DOMAINS CLASSIFICATION  The three-domain is a biological classification introduced by Carl R Woese was an American microbiologists and Biophysicist. In 1977  Woese divided cellular life forms into archaea, bacteria and eukaryote domains. This theory emphasizes the separation of prokaryotes into two groups as Eubacteria (now Bacteria) and Archaebacteria (now Archaea) on the basis of difference in 16s rRNA genes, these two groups and the eukaryotes each arose separately from an ancestor with poorly developed genetic machinery, often called progenote.  Woese initially used the term “Kingdom” to refer to three primary phylogenic groupings now referred to as “Domains” until the later term was coined in 1990. Evolutionary tree of microorganisms The evolutionary history of a group of organisms is called its phylogeny, and a major goal of evolutionary analysis is to understand phylogenetic relationships. Phylogenetic Trees- Reconstructing evolutionary history from observed nucleotide sequence differences includes construction of a phylogenetic tree, which is a graphic depiction of the relationships among sequences of the organisms under study, much like a family tree. A phylogenetic tree is composed of nodes and branches The tips of the branches represent species that exist now and from which the sequence data were obtained. The nodes are points in evolution where an ancestor diverged into two new organisms, each of which then began to evolve along its separate pathway. The branches define both the order of descent and the ancestry of the nodes, whereas the branch length represents the number of changes that have occurred along that branch. Vasantha B.S.  In its most basic form, a phylogenetic tree is a depiction of lines of descent, and the relationship between two organisms. therefore should be read in terms of common ancestry. That is, the more recently two species shared a common ancestor, the more closely related they are. The rooted trees in Fig.- 6 b and c illustrate this point. Species 2 is more closely related to species 3 than it is to species 1 because 2 and 3 share a more recent common ancestor than do 2 and 1. Figure. 6: : Phylogenetic trees. Unrooted (a) and rooted (b, c) forms of a phylogenetic tree are shown. 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. Tree Construction Modern evolutionary analysis uses character-state methods, also called cladistics, for tree construction. Character-state methods define phylogenetic relationships by examining changes in nucleotides at particular positions in the sequence, using those characters that are phylogenetically informative. 16S ribosomal RNA (16S rRNA), a molecule used for phylogenetic analyses, has both highly conserved and highly variable regions, primers specific for the 16S rRNA gene from various taxonomic groups can be synthesized. These may be used to survey different groups of organisms in any specific habitat. This technique is in widespread use in microbial ecology and has revealed the enormous diversity of the microbial world The characters that define a monophyletic group; that is, a group that has descended from one ancestor (fig. 8) describes how phylogenetically informative characters are recognized in aligned sequences. Computer-based analysis of these changes generates a phylogenetic tree, or cladogram. A widely used cladistic method is parsimony, which is based on the assumption that evolution is most likely to have proceeded by the path requiring fewest changes. Biologists previously grouped living organisms into five kingdoms: plants, animals, fungi, protists, and bacteria (fig. 7). DNA sequence-based phylogenetic analysis, on the other hand, has revealed that the five kingdoms do not represent five primary evolutionary lines. Life on Earth has evolved along three primary lineages, called domains. Two of these domains, the Bacteria and the Archaea, are exclusively composed of prokaryotic cells. The Eukarya contains the eukaryotes including the plants, animals, fungi, and protists  Figure. 8: Universal phylogenetic tree as determined from comparative SSU rRNA gene sequence analysis. Only a few key organisms or lineages are shown in each domain. At least 80 lineages of Bacteria have now been identified although many of these have not yet been cultured. LUCA, last universal common ancestor. Bergey’s Manual of Systematic Bacteriology  In 1923, David Bergey, professor of bacteriology at the University of Pennsylvania, and four colleagues published a classification of bacteria that could be used for identification of bacterial species, the Bergey’s Manual of Determinative Bacteriology.  This manual is now in its ninth edition. The first edition of Bergey’s Manual of Systematic Bacteriology, a more detailed work that contains descriptions of all procaryotic species currently identified, also is available. The First Edition of Bergey’s Manual of Systematic Bacteriology  Because it has not been possible in the past to classify procaryotes satisfactorily based on phylogenetic relationships, the system given in the first edition of Bergey’s Manual of Systematic Bacteriology is primarily phenetic.   General shape and morphology, Gram-staining properties, oxygen relationship, motility, the presence of endospores, the mode of energy production, and so forth. Procaryotic groups are divided among the four volumes in the following manner:  gram –negative bacteria of general, medical, or industrial importance; (2) gram-positive bacteria other than actinomycetes; (3) gram-negative bacteria with distinctive properties, cyanobacteria, and archaea; and (4) actinomycetes (gram-positive filamentous bacteria). The Second Edition of Bergey’s Manual of Systematic Bacteriology  There has been enormous progress in procaryotic taxonomy since 1984, the year the first volume of Bergey’s Manual of Systematic Bacteriology was published. In particular, the sequencing of rRNA, DNA, and proteins has Vasantha B.S. made phylogenetic analysis of procaryotes feasible.  As a consequence, the second edition of Bergey’s Manual will be largely phylogenetic rather than phenetic and thus quite different from the first edition.   The second edition will be published in five volumes. It will have more ecological information about individual taxa.  The second edition will not group all the clinically important procaryotes together as the first edition did. Instead, pathogenic species will be placed phylogenetically and thus scattered throughout the following five volumes.  Volume 1—The Archaea, and the Deeply Branching and Phototrophic Bacteria  Volume 2—The Proteobacteria  Volume 3—The Low G C Gram-Positive Bacteria  Volume 4—The High G C Gram-Positive Bacteria  Volume 5—The Planctomycetes, Spirochaetes, Fibrobacteres, Bacteroidetes, and Fusobacteria (Volume 5 also will contain a section that updates descriptions and phylogenetic arrangements that have been revised since publication of volume 1.) A CONCISE ACCOUNT OF CLASSIFICATION  Volume 1 contains a wide diversity of procaryotes in two domains: the Archaea and the Bacteria.. Archaea : At present, they are divided into two phyla based on rRNA sequences o The phylum Crenarchaeota contains thermophilic and hyperthermophylic sulfur-metabolizing organisms of the orders Thermoproteales, Desulfurococcales, and Sulfolobales. o phylum, the Euryarchaeota, contains primarily methanogenic procaryotes and halophilic procaryotes; thermophilic, sulfur-reducing organisms (the thermoplasmas and thermococci) also are in this phylum. o The two phyla are divided into eight classes and 12 orders. ( Methanobacteria, Methanococci, Halobacteria, Thermoplasmata, Thermococci, Archeoglobi and Methanopyri.)  The bacteria are an extraordinarily diverse assemblage of procaryotes that have been divided into 23 phyla In volume 1 are placed deeply branching bacterial groups and phototrophic bacteria. The more important phyla are described here. 1.Phylum Aquificae:The phylum Aquificae contains autotrophic bacteria such as Aquifex and Hydrogenobacter that can use hydrogen for energy production. Aquifex (meaning “water maker”) actually produces water by using hydrogen to reduce oxygen. This group contains some of the most thermophilic organisms known and is the deepest or earliest branch of the bacteria. 2. Phylum Thermotogae. This phylum is composed of one class and five genera. Thermotoga and other members of the class Thermotogae are anaerobic, thermophilic, fermentative, gram-negative bacteria that have unusual fatty acids and resemble Aquifex with respect to their etherlinked lipids. Eg:Thermotoga, Geotoga and Fervidobacterium Vasantha B.S. 3. Phylum “Deinococcus Thermus.” The order Deinococcales contains bacteria that are extraordinarily radiation resistant. The genus Deinococcus is gram positive. It has high concentrations of carotenoid pigments, which may protect it from radiation, and unique lipids.  4.Phylum Chloroflexi: The phylum Chloroflexi has one class and two orders. Many members of this gram-negative group are called green nonsulfur bacteria. Chloroflexus carries out anoxygenic photosynthesis and is a gliding bacterium; in contrast, Herpetosiphon is a nonphotosynthetic, respiratory gliding bacterium. Both genera have unusual peptidoglycans and lack lipopolysaccharides in their outer membranes. 5.Phylum Cyanobacteria. The oxygenic photosynthetic bacteria are placed in the phylum Cyanobacteria, which contains the class Cyanobacteria and five subsections. Cyanobacteria have chlorophyll a and almost all species possess phycobilins. These bacteria can be unicellular or filamentous, either branched or unbranched. The cyanobacteria in the subsections differ from each other in general morphological characteristics and reproduction. Cyanobacteria incorporate CO2 photosynthetically through use of the Calvin cycle just like plants and many purple photosynthetic bacteria. 6. Phylum Chlorobi. The phylum Chlorobi contains anoxygenic photosynthetic bacteria known as the green sulfur bacteria. They can incorporate CO2 through the reductive tricarboxylic acid cycle rather than the Calvin cycle and oxidize sulfide to sulfur granules, which accumulate outside the cell. Volume 2: of the second edition is devoted completely to the gram-negative proteobacteria, often called the purple bacteria. The phylum Proteobacteria is a large and extremely complex group that currently contains over 1,300 species in 384 genera. The phylum is divided into five classes based on rRNA data 1. Class I—Alphaproteobacteria. The –proteobacteria include most of the oligotrophic forms (those capable of growing at low nutrient levels). Rhodospirillum and other purple nonsulfur bacteria are photosynthetic. Some genera have unusual metabolic modes: methylotrophy (e.g.,Methylobacterium), chemolithotrophy (Nitrobacter), and nitrogen fixation (Rhizobium). Rickettsia and Brucella are important pathogens. About half of the microbes in this group have distinctive morphology such as prosthecae (Caulobacter, Hyphomicrobium). 2. Class II—Betaproteobacteria. The -proteobacteria overlap the subdivision metabolically. However the - proteobacteria tend to use substances that diffuse from organic decomposition in the anaerobic zone of habitats. Some of these bacteria use such substances as hydrogen (Alcaligenes), ammonia (Nitrosomonas), methane (Methylobacillus), or volatile fatty acids (Burkholderia). 3. Class III—Gammaproteobacteria. The –proteobacteria compose a large and complex group of thirteen orders and 20 families. They often are chemoorganotrophic, facultatively anaerobic, and fermentative. However, there is considerable diversity among the -proteobacteria with respect to energy metabolism. Vasantha B.S. Some important families such as Enterobacteriaceae, Vibrionaceae, and Pasteurellaceae Pseudomonadaceae and Azotobacteriaceae. A few are photosynthetic e.g., Chromatium and Ectothiorhodospira), methylotrophic (Methylococcus), or sulfur-oxidizing (Beggiatoa). 4. Class IV—Deltaproteobacteria. The -proteobacteria contain seven orders, and 17 families. Many of these bacteria can be placed in one of three groups. Some are predators on other bacteria as the class name implies (e.g., Bdellovibrio). The order Myxococcales contains the fruiting myxobacteria such as Myxococcus, Stigmatella, and Polyangium. The myxobacteria often also prey on other bacteria. Finally, the class has a variety of anaerobes that generate sulfide from sulfate and sulfur while oxidizing organic nutrients (Desulfovibrio).  5. Class V—Epsilonproteobacteria. This section is composed of only one order, Campylobacterales, and two families. Despite its small size two important pathogenic genera are proteobacteria: Campylobacter and Helicobacter. Volume 3 of Bergey’s Manual surveys the gram-positive bacteria with low G C content in their DNA, which are members of the phylum Firmicutes. The dividing line is about 50% G C; bacteria with a mol% lower than this value are in volume 3. However because of their close relationship to low G C gram-positive bacteria, the mycoplasmas are placed here even though they lack cell walls and stain gram negative. The phylum contains three classes 1. Class I—Clostridia. This class contains three orders and 11 families. Although they vary in morphology and size, the members tend to be anaerobic. Genera such as Clostridium, Desulfotomaculum, and Sporohalobacter form true bacterial endospores; many others do not. Clostridium is one of the largest bacterial genera. 2. Class II—Mollicutes. The class Mollicutes contains five orders and six families. Members of the class often are called mycoplasmas. These bacteria lack cell walls and cannot make peptidoglycan or its precursors. Because mycoplasmas are bounded by the plasma membrane, they are pleomorphic and vary in shape from cocci to helical or branched filaments. They are normally nonmotile and stain gram negative because of the absence of a cell wall. In contrast with almost all other bacteria, most species require sterols for growth. The genera Mycoplasma and Spiroplasma contain several important animal and plant pathogens. 3. Class III—Bacilli. This large class comprises a wide variety of gram positive, aerobic or facultatively anaerobic, rods and cocci. The class Bacilli has two orders, Bacillales and Lactobacillales, and 16 families. some genera (e.g., Bacillus, Sporosarcina, Paenibacillus, and Sporolactobacillus) form true endospores Volume 4 is devoted to the high G C gram positives, those bacteria with mol% values above 50 to 55%. All bacteria in this volume are placed in the phylum Actinobacteria and class Actinobacteria. There is enormous morphological variety among these procaryotes. Some are cocci, others are regular or irregular rods. complex branching hyphae. Although none of these bacteria produce true endospores, many genera do form a variety of asexual spores and some have complex life cycles. There is considerable variety in cell wall chemistry among the high G C gram positives.For example, the composition of peptidoglycan varies greatly. The taxonomy of these bacteria is very complex. There are five subclasses, six orders, 14 suborders, and 40 families. Genera such as Actinomyces, Arthrobacter, Corynebacterium, Micrococcus, Mycobacterium, and Propionibacterium were placed in volume 2 of the first edition. They are now in the new volume 4 within the suborders Actinomycineae, Micrococcineae, Corynebacterineae, and Propionibacterineae because rRNA studies have shown them to be actinobacteria. The largest and most complex genus is Streptomyces, which contains over 500 species. Volume 5 describes an assortment of nine phyla that are located here for convenience. The inclusion of these groups in volume 5 does not imply that they are directly related. Although they are all gram-negative bacteria, there is considerable variation in morphology, physiology, and life cycle pattern. Several genera are of considerable biological or medical importance. We will briefly consider four of the nine phyla. 1. Phylum Planctomycetes. The planctomycetes are related to the chlamydias according to their rRNA sequences. The phylum contains only one order, one family, and four genera. Planctomycetes are coccoid to ovoid or pearshaped cells that lack peptidoglycan. Some have a membrane-enclosed nucleoid. Although they are normally unicellular, the genus Isosphaera will form chains. They divide by budding and may produce nonprosthecate appendages called stalks. Planctomycetes grow in aquatic habitats, and many move by flagella or gliding motility. Vasantha B.S. 2. Phylum Chlamydiae. This small phylum contains one class, one order, and four families. The genus Chlamydia is by far the most important genus. Chlamydia is an obligately intracellular parasite with a unique life cycle involving two distinctive stages: elementary bodies and reticulate bodies. These bacteria do resemble planctomycetes in lacking peptidoglycan. They are small coccoid organisms with no appendages. Chlamydias are important pathogens and cause many human diseases. 3. Phylum Spirochaetes. This phylum contains helically shaped, motile, gram-negative bacteria characterized by a unique morphology and motility mechanism. The exterior boundary is a special outer membrane that surrounds the protoplasmic cylinder, which contains the cytoplasm and nucleoid. Periplasmic flagella lie between the protoplasmic cylinder and the outer membrane. The flagella rotate and move the cell even though they do not directly contact the external environment. These chemoheterotrophs can be free living, symbiotic, or parasitic. For example, the genera Treponema and Borrelia contain several important human pathogens. The phylum has one class, Spirochaetes, three families, and 13 genera. 4. Phylum Bacteroidetes. This phylum has three classes (Bacteroides, Flavobacteria, and Sphingobacteria), three orders, and 12 families. Some of the better-known genera are Bacteroides, Flavobacterium, Flexibacter, and Cytophaga. The gliding bacteria Flexibacter and Cytophaga are ecologically significant and will be discussed later. STUDY AND MEASURES OF MICROBIAL DIVERSITY MICROBIAL DIVERSITY is the measure of the number or relative abundance of microbial species in a local area or region. Measuring microbial diversity may involve counting individual species, numbers of functional groups, or units operationally defined by the particular method being used.  Understanding microbial biodiversity may translate into benefits for biotechnology; management of agricultural, forest, and natural ecosystems; biodegradation of pollutants; reclamation of damaged lands; waste treatment systems; and biological control systems.  Measuring the biodiversity of microbial communities is important for immediately practical and more fundamental considerations. The existence of such an immense variety of organisms, combined with the fact that microorganisms are too small to see without magnification, makes the task of measuring microbial biodiversity challenging and even daunting.  The unit of measurement used in biodiversity studies of macroscopic organisms is usually a taxonomic one: the species.  Traditionally in microbiology, a species was phenotypically characterized with organisms classified into taxonomic groups based on morphology, physiology, and metabolism. Recently, however, phenotypic classification has been replaced or supplemented by the use of genotypic analysis. Nucleic acid sequence information derived from the small subunit of the ribosome [16S ribosomal RNA (rRNA) for prokaryotes or 18S rRNA for eukaryotes] is used to determine the degree of similarity among groups of organisms and the evolutionary relationships of microorganisms and all other life-forms.  Although genetic sequencing for individual microbes is labor-intensive, particularly when characterizing complex microbial systems, some microbial ecologists maintain that an in-depth understanding of recently described systems is not possible until this information is gathered  Far more species of bacteria, fungi, and other microorganisms exist than have been described to date.  Numerous methods are available for characterizing microbial communities. Determining which approaches to use is a critical step and depends on the objectives of the study  Enrichment/Isolation versus Direct Measurement of Microbial Communities: A culture-based approach therefore probably offers a skewed perspective of microbial diversity in many environments. Consequently, there has been a shift in studies of microbial diversity toward the use of molecular biology and other biochemical and molecular tools that do not require culturing of organisms METHODS FOR MEASURING MICROBIAL DIVERSITY  Methods can be divided into those that involve the counting of cells, measurement of cellular constituents, or determination of activity. Vasantha B.S.  Counting Microorganisms Microscopy : 1. Microscopy Major microbial groups, such as bacteria and fungi, can be distinguished by their morphologies as observed under a light or fluorescence microscope after staining the cells. The  microscope provides a quick but relatively superficial survey of microbial diversity based on the sizes, shapes, and staining properties of microorganisms. The relatively low number of morphotypes among bacteria limits descriptions of diversity. To some extent, fungal diversity can be investigated by examining hyphal and fruiting body structures and spore morphology, if present. Taxonomic resolution can be improved by using dyes tagged with fluorescent monoclonal antibodies specific to groups or strains, which permit selective visualization of members of the group to which the antibody binds. Recently, development of fluorescent in situ hybridization (FISH) with specific DNA probes has made the microscope a powerful and highly quantitative tool for use in studies of diversity. The antibody and DNA tagging methods require initial characterization of the organism or group of interest in order to develop the antibody or probe. Plate Counts:. Plate Counts Plate counts exploit the fact that individual microscopic cells quickly grow into a colony visible to the unaided eye if provided with suitable growth conditions. Microbial diversity has been assessed by culturing microorganisms on solid nutrient media to which substances are added to specifically promote the growth of target organisms and/or inhibit the growth of unwanted groups. For example, media containing complex organic substances combined with a low pH tend to select for fungi instead of bacteria. Colonies that grow on the plates are counted and may be further differentiated based on their color or other morphological properties. The diversity of culturable microorganisms is estimated by counting the number of different colonies present on agar media inoculated with a dilution series of the microbial community. Diversity measured based on this technique is unlikely to yield comprehensive, ecologically relevant information about the microbial community as a whole. An advantage of using plating techniques is that strains that grow on plates can often be easily isolated, characterized, and identified by traditional methods of microbiology. . MPN Methods: MPNs make use of specifically designed culturing conditions to estimate the number of microorganisms in a community able to carry out specific functions. The MPN medium is designed to select a specific trophic group by providing carbon, energy, nutrients, and environmental conditions needed to support the growth of that group. Thus, iron reducers are selected by providing anaerobic conditions, a carbon source, ferric iron, and other nutrients. A series of dilutions of an environmental sample are inoculated into the MPN medium. Measurements of turbidity, substrate utilization, or product formation confirm cell growth or activity. The number of organisms capable of carrying out the particular function being investigated is estimated from cell counts from the series of dilutions and with standard statistical calculations. MPN estimation of numbers is obviously a culture-dependent method, biased toward those organisms able to grow and compete under Vasantha B.S. laboratory conditions.  Analysis of Cellular Constituents  1. Nucleic Acids Methods : Extraction of DNA or RNA from environmental samples. One approach is to first extract cells from environmental samples (usually from aquatic environments) and then extract cellular DNA or perform analyses on cells collected on filters (e.g., using in situ hybridization). In situ approaches are quantitative because taxonomic information can be correlated with actual cell numbers within the community.  The other approach, direct extraction of DNA without first extracting cells, is more commonly employed in complex environmental media such as soil and sediment There is usually a trade-off between speed of extraction and quality of extracted DNA (e.g., with respect to purity of the DNA).  Nucleic acid-based approaches can be divided into those that employ PCR and those that do not. - Summary of Nucleic Acid Methods -PCR-Dependent Methods : o The polymerase chain reaction amplifies the signal of microbial DNA from environmental samples. o PCR enables detection of microbial populations from very small sample sizes and from extreme environments containing unculturable microorganisms.  o Denaturing/Thermal Gradient Gel Electrophoresis (D/TGGE) Small subunit rDNA fragments of the same length are separated based nucleotide sequence to produce fingerprint patterns. Isolated fragments may be excised and cloned for sequence analysis. o Intergenic Transcribed Spacer (ITS) Analysis Fragments between the small and large rDNA genes are separated based on length producing fingerprint patterns. Isolated fragments may be excised to differentiate strains by sequence analysis. o Restriction Fragment Length Polymorphism (RFLP) Small subunit rDNA PCR products are cut into smaller fragments with restriction enzymes and separated based on length to produce patterns that differentiate simple communities or strains. o Cloning and Sequencing DNA PCR products are inserted in plasmid vectors which are taken up by bacterial cells. The cells are then cloned to generate many copies for sequence analysis and phylogenetic classification. PCR-Independent Methods o Hybridization techniques have all the specificity of PCR-based techniques, are without PCR bias, and have the additional advantage of a means to quantify microorganisms. o Solution Hybridization This method takes community double stranded DNA through thermal dissociation and reassociation process to estimate community DNA complexity. o Membrane Hybridization Group or species-specific probes hybridize with community DNA or RNA immobilized on a membrane to produce estimates of relative abundance. o Fluorescent In-Situ Hybridization (FISH) Fluorescently labeled group or species-specific probes hybridize with RNA in intact cells. Individual cells may be counted and types of organisms quantified by microscopy. o Oligonucleotide array Group, species-specific, or functional gene probes are immobilized on a solid support. Hybridization between probes and community DNA is detected by laser technology. Measurement of Process or Function  Substrate Utilization Patterns : The measurement of substrate utilization patterns is commonly used to evaluate functional diversity of bacterial populations.  The commercially available Biolog system, which contains 95 carbon and energy sources on one microtiter plate, is widely used because of its convenience.  The plate is inoculated directly with a sample of a microbial community (e.g., a dilution of a soil or sediment suspension) and then incubated under aerobic conditions.  Substrate utilization leads to the development of a blue color originating from a redox sensitive dye.  Comparison of well color development patterns among different microbial communities coupled with multivariate statistical analysis may yield information on the functional relatedness of the bacterial populations in the sample  Enzyme Assays  Enzymes are usually measured based on the type and amount of activity they catalyze under controlled conditions in response to added substrates. ‘‘Potential’’ rather than actual activity is commonly measured.  Some enzymes are present in all organisms (protease and hydrogenase) and give general information about the intensity of biological activity in a sample. Other enzymes catalyze more specific reactions and provide information about the diversity of processes that potentially can be carried out by microbial populations within a given environment