Course Notes

Biology 140

Table of Contents Module One: Introduction 1 Module Two: The Diversity of Microorganisms 2 Module Three: Cell Structures and their Functions 4 Module Four: The Growth of Microorganisms 6 Module Five: Regulation 10 Module Six: Genetics and Molecular Biology 14 Module Seven: Microbial Taxonomy 21 Module Eight: The Bacteria (I) 25 Module Nine: The Bacteria (II) 29 Module Ten: The Archaea 33 Module Eleven: Controlling Microbial Growth 36

Module One: Introduction Learning Objectives: An introduction to (1) the historical origins of microbiology; (2) the concepts of cell structure and function (3) the concept of microbial diversity. (Please refer to textbook sections 1.1-1.6, 2.1-2.3)

Why Study Microbiology? The Historical Roots of Microbiology Make sure to understand the following: * Pasteur's experimental disproof of spontaneous generation. -see Figure 1.11 * Koch's postulates. -see Figure 1.12 An Introduction to Cell Structure and the Three Domains All living organisms, except viruses, are made up of cells (see Figure 2.1). Each cell is surrounded by a cell membrane, which separates the outside of the cell from the cytoplasm. Eukaryotic cells differ from prokaryotic cells in that they contain membrane enclosed structures (organelles). These include mitochondria, chloroplasts, and the nucleus. Prokaryotes do not have any of these structures. The comparison of ribosomal RNA sequences (Figure 2.6) has led to the development of an understanding of how different types of organisms are related to each other. This type of study is called phylogeny. We now know that there are two distinct lineages of prokaryotes, the Bacteria and the Archaea (Figure 2.7). The Archaea are actually more closely related to the Eukarya than they are to the Bacteria.

Module Two: The Diversity of Microorganisms Learning Objectives: An introduction to the diversity of microorganisms, cell morphology, and structure. (Please refer to textbook sections 2.4-2.6, 4.4-4.6, 4.8-4.9) The Physiological Diversity of Microorganisms Microbes make a living in many different ways. This is reflected in the metabolic diversity that is seen. In Figure 2.8 is outlined the metabolic options for obtaining energy: from organic chemicals, inorganic chemicals, or from light. Cells also require carbon. Some microorganisms obtain their carbon from organic compounds, and are called hetertrophic. Other microorganisms obtain their carbon from CO2; these are termed autotrophic. In the second half of the course, we will investigate the phylogenetic diversity of the microbial world in more detail. Figures 2.9 and 2.18 will serve as an introduction to the Bacteria and the Archaea. Cell Morphology Prokaryotes come in different shapes (=morphology). See Figure 4.11. Coccus, rod, spirillum, spirochaete, appendaged, filamentous. Prokaryotes come in different sizes. See Figure 4.13. Note the photomicrograph of the recently discovered giant prokaryote in Figure 4.12. For a discussion of large bacteria, see the recent review by Schulz and J¿rgensen published in Annual Review of Microbiology. Surface Area vs. Volume (see Figure 4.14). -Size affects rate of nutrient and waste transport across the cell membrane. -Small size results in more efficient exchange of nutrient and waste, support of higher metabolic rate.

The Cytoplasmic Membrane The cytoplasmic membrane surrounds the cell. It is made up of a phospholipid bilayer. Make sure that you understand the structure and composition of phospholipid bilayer, as illustrated in Figure 4.15 & 4.16. The cytoplasmic membrane also includes integral membrane proteins (see Figure 4.17). There are important differences between the lipids in Bacteria and the lipids in Archaea. As illustrated in Figure 4.19, archaeal lipids have ether linkages, while bacterial lipids have ester linkages. Please review the structure of lipids in Section 3.4. Functions of Cytoplasmic Membrane (Figure 4.21): * Permeability Barrier -- only small, uncharged, hydrophobic molecules can pass

through by diffusion (see Table 4.1). * Protein Anchor -- transport, generation of energy, chemotaxis. * Generation of proton motive force. Transport proteins are required for the accumulation of solutes within the cell against the concentration gradient (carrier-mediated transport). Specific transport proteins transport specific molecules or specific classes of molecules. In this course, you will not be responsible for knowing the different types of transport events and transport systems, but I encourage you to read about them in the textbook if you are interested. The Cell Wall of Prokaryotes The cell wall of bacteria is made of a thin sheet called peptidoglycan. It prevents the cell from lysing due to turgor pressure (see Figure 4.33). Peptidoglycan is only found in bacteria. Gram negative cells have an outer membrane exterior to the cell wall, while gram positive cells do not have an outer membrane. Peptidoglycan is composed of a glycan backbone (alternating molecules of N-acetylglucosamine and N-acetylmuramic acid) connected by peptide cross-links. Figures 4.30 & 4.31 will help you to understand the structure of peptidoglycan. Gram positive bacteria have teichoic acids, a type of acidic polysaccharide, embedded in the peptidoglycan layer. The structure of a particular teichoic acid is shown in Figure 4.32.

Although archaeal organisms do not have peptidoglycan walls, many have a wall constructed of a similar material called pseudopeptidoglycan. Pseudopeptidoglycan differs from peptidoglycan in that its backbone is composed of alternating molecules of N-acetylglucosamine and N-acetyltalosaminuronic acid which are connected by �-1,3 linkages rather than �-1,4 linkages as in bacterial peptidoglycan. Comparing Figures 4.34 & 4.30 will help you to understand the differences between peptidoglycan and pseudopeptidoglycan. Gram negative organisms have an outer membrane, exterior to the peptidoglycan cell wall. The space between the inner and outer membranes, which includes the peptidoglycan, is called the periplasm. The outer membrane consists of a phospholipid bilayer and lipopolysaccharide (Figure 4.35). Porins are proteins that span the outer membrane and allow small molecules to cross membrane.

Module Three: Cell Structures and their Functions Learning Objectives: A continuation of the introduction to cell structure. (Please refer to textbook sections 4.10-4-15) Motility Many prokaryotes have flagella, which they use to propel themselves. This ability to propel themselves is called motility. Some bacteria have polar flagella, which are attached at the end of the cell. Other bacteria have peritrichous flagella, which are distributed around the cell. See Figure 4.41 for a depiction of flagellar structure. As you can see, flagella are complex, elaborate structures. The long filament is made up of subunits of flagellin protein. Another type of protein forms the hook, which connects the filament to the basal body. The basal body is a motor that is embedded in the cytoplasmic membrane and cell wall, and rotates. Note that the flagellar rotation, like a propeller, rather than pulling like an oar, is what propels the cell. Counterclockwise rotation propels the cell forward in a run, while clockwise rotation causes the cell to tumble or propels the cell backward. This is shown in Figure 4.43. Chemotaxis Why are bacteria motile? Motility must confer a selective advantage, otherwise it would not be worth the effort to expend the energy that motility requires. It turns out that motile bacteria can respond to the presence of favourable or detrimental chemicals by moving toward or away from them. When bacteria respond in this way to chemicals, it is called chemotaxis. Bacteria sense a chemical gradient in a temporal manner, rather than a spatial manner. The Figure 4.46 shows how the chemical gradient can influence the duration of runs, resulting in biased net movement up or down the gradient. Besides chemotaxis, there are other types of taxes: * phototaxis - light * aerotaxis - oxygen * osmotaxis - osmotic strength Bacterial Cell Structures and Inclusions Many types of structures and cell inclusions are found only in certain types of bacteria. Some of these are described in Section 4.13 & 4.14.

* fimbriae - aid cells to adhere to surfaces * pili - conjugation, attachment to host cell * glycocalyx - polysaccharide layer outside cell, attachment to host cells, protection

from host immune system, resistance to dessication * polyhydroxyalkanoate deposits - intracellular carbon and energy store * polyphosphate - intracellular reserves * elemental sulfur - intracellular granules * magnetosomes - intracellular magnetite crystals * gas vesicles - cell buoyancy Bacterial Endospores Certain types of bacteria are able to differentiate into endospores, which are resistant to heat, radiation, acids, drying, and many chemicals. Endospores can remain dormant for very long periods of time. There are even recent claims of successfully reviving endospores that are over 250 million years old (see page 97, and the original paper). The stages of endospore formation are presented in Figure 4.63. These stages were defined by analysis of mutants that were blocked in sporulation. Sporulation is induced by conditions of nutrient limitation.

Module Four: The Growth of Microorganisms Learning Objectives: Following this module, you should have an understanding of how bacteria are cultured, the difference between defined and complex growth media, the concept of exponential growth, and how growth is measured in the laboratory. You should understand that microorganisms have preferred conditions, and that these differ according to the specific type of microorganism. Different microorganism have different optima for temperature, pH, water availability, and oxygen. (Please refer to textbook sections 5.1-5.3, 6.1-6.6, 6.8-6.13) Laboratory Culture of Microorganisms Before we learn about cell growth, it is necessary to briefly consider metabolism and nutrition. For cells to grow, they must convert chemical compounds into cellular matter. Nutrients from outside the cell are transformed into new cell material. This process is called anabolism or biosynthesis. The cell requires energy for biosynthesis, as well as for other processes (e.g. transport, motility). Energy is often derived from the oxidation of chemical compounds, in a process called catabolism. Energy Classes of Microorganisms * Phototrophs: light as energy source * Chemotrophs: chemicals as energy source

! chemoorganotrophs: organic chemicals ! chemolithotrophs: inorganic chemicals

Culture Media * Defined: the exact chemical composition is known -- purified ingredients are

used. * Complex: the exact chemical composition is not known. See Table 5.4 for a good demonstration of the different nutritional requirements of different bacteria. Cell Growth The microbiological concept of growth really refers to increase in cell number. This is achieved by division of a single cell into two new cells. Figure 6.1 illustrates the process of binary fission. An important thing to consider is how cell wall synthesis takes place. This is illustrated in Figures 6.3-6.5. During cell growth, the peptidoglycan layer must be opened up as new peptidoglycan is being deposited. The dissacharide pentapeptide is

transported to the periplasm from the cytoplasm by the lipid carrier bactoprenol, and inserted into the growing cell wall. The next step, transpeptidation, in which the peptide cross-links in the peptidoglycan are formed, is the target of penicillin. Population growth

Growth rate = change in cell # / time Generation time (g) = time for formation of two cells from one = doubling time If the generation time of a culture remains constant, the growth rate will change in an exponential fashion. This is called exponential growth (see Figure 6.6). The generation time (g) can be calculated if the number of cells in a culture at the beginning and end of a given time period are known (Figure 6.7).

Growth cycle of populations

A typical growth curve for a batch culture is shown in Figure 6.8. Lag phase: the time required for the organisms to adapt to new culture conditions or recover from injury. Exponential phase: exponential growth Stationary phase: no net change in cell number Death phase: some populations are unable to maintain stationary phase indefinitely, and there will then be a net reduction in cell number

Measurement of growth

The number of cells in a culture can be measured by either direct microscopic count (Figure 6.9) or viable count (Figures 6.10 & 6.11). One of the limitations of the direct method is that it does not distinguish living from dead cells. Another very useful method of estimating cell numbers is to measure the culture turbidity (Figure 6.12). In culture, there is a proportional relationship between turbidity and cell number.

The effect of environment on growth

So far, we have been considering growth only under ideal conditions. In the real world, most microorganisms are not growing in log phase (we know why that could not be possible!) and there are several factors, besides nutrient availability and growth inhibition by toxic substances, that limit growth. In this lecture, we will look at some of these factors.

Growth at Low and High Temperatures and pH Each type of microorganism has upper and lower temperature limits (Figure 6.16), beyond which they are not able to grow. Between these upper and lower limits, there is an optimum temperature at which growth is maximum. Maximum growth temperature -- inactivation of at least one important protein. Minimum growth temperature -- freezing of the cytoplasmic membrane. There is a wide variation in the temperature limits and optima for different microorganisms (Figure 6.17). * Psychrophiles: low temperature optima * Mesophiles: midrange temperature optima * Thermophiles: high temperature optima * Hyperthermophiles: very high temperature optima Low Temperature Growth * Psychrophiles: optimal growth temperature 15 C or lower, maximum growth

temperature below 20 C, minimal growth temperature 0 C or lower * Psychrotolerant: grow at 0 C, optimal growth temperature 20-40 C Many psychrophiles are actually killed by temperatures above 20 C. They are often very difficult to study, since they must always be kept below room temperature. Their intolerance to heat is due to the denaturation of some of their enzymes at even moderate temperatures. As expected, the cytoplasmic membranes of psychrophiles are more fluid at low temperatures, and this is due to a greater proportion of unsaturated fatty acids in the membrane phospholipids. High Temperature Growth * Thermophiles: optimal growth temperature above 45 C * Hyperthermophiles: optimal growth temperature above 80 C

Examples of hot environments-- soil surface in midsummer sun: 50 C compost piles: 65 C hot springs: boiling point of water ocean floor hydrothermal vents: 350 C hot water heaters Archaea are the most thermophilic organisms, followed by bacteria, and then eukaryotes (see Table 6.1). For thermophiles and hyperthermophiles to survive and thrive at high temperatures, their proteins must be resistant to thermal denaturation (i.e. thermostable). Their membrane phospholipids also have a high proportion of saturated fatty acids (for the bacterial thermophiles) or lipid monolayer (for the archaeal hyperthermophiles Figure 4.20d). Enzymes from thermophiles and hyperthermophiles are valued for their use in industrial processes, which are often preferentially carried out at high temperatures. Growth at Low or High pH * Acidophiles: pH optimum between 2 and 6 * Neutrophiles: pH optimum between 6 and 8 * Alkalophiles: pH optimum between 8 and 11 Salt Tolerance and the Effect of Oxygen Water availability is expressed at water activity (aw) (see Table 6.2). Solute concentration is an important contributor to this value. The higher the aw value, the easier it is for the microorganism to obtain the water. If the solute concentration outside of the cell is lower than inside (i.e. high water concentration outside) then the water tends to diffuse into the cell. If the solute concentration is higher outside the cell, then the water tends to diffuse out of the cell. In nature, water activity is commonly influenced by the NaCl concentration. * Halophiles: growth optimum above 1% NaCl, often requiring sodium for growth * Halotolerant: can tolerate the presence of solute, but grow better in the absence of solute * Osmophiles: can grow in presence of high sugar * Xerophiles: can grow in very dry conditions To cope with low water activity, many microorganisms are able to increase the internal solute concentration (Figure 6.24, Table 6.3). They do this by increasing the intracellular concentration of compatible solutes, which do not inhibit biochemical processes.

Oxygen Effects on Microbial Growth Please see Table 6.4 for an outline of how different microorganisms are affected by O2. Note that some organisms are very sensitive to O2, others require O2 for growth, and others can grow either in the presence or absence of O2. Note that reduction of O2 produces toxic byproducts, which are very good oxidizers. Organisms that are able to grow in the presence of O2 are usually able to detoxify these byproducts (Figure 6.28) using specific enzymes such as catalase, peroxidase, and superoxide dismutase. Since O2 is not very soluble in water, to culture aerobic microorganisms, it is necessary to aerate the culture by either shaking the tube or flask or by bubbling air into the culture medium. In contrast, growth of obligate anaerobes requires that O2 is excluded from the culture medium. Thioglycolate broth, along with the redox indicator dye resazurin, can be used to show the aerobic or anaerobic nature of a given microorganism (Figure 6.25).

Module Five: Regulation Learning Objectives: You should understand the concept of regulation, the different levels at which this regulation may occur, and some of the mechanisms for gene regulation. The different strategies employed by synthetic vs. degradation pathways will be discussed. Interaction of proteins with specific sequences of DNA is crucial to regulation at the level of transcription. You should be able to describe the process of attenuation, using the tryptophan operon as an example. Other examples of regulatory mechanisms that you should understand are quorum sensing, two component regulatory systems, and chemotaxis. Realize that these are examples of common mechanisms that are used in many different regulatory circuits within the cell. (Please refer to textbook sections 8.1-8.6, 8.8-8.11) Modes of Regulation Enzymes and other proteins are not all required by the cell at the same time, under the same conditions. Some are required only under certain conditions, while others are required under other conditions. The cell responds by regulating the activity or amount of a given enzyme in relation to the requirements of the cell. There are many levels at which this regulation may occur (Figure 8.1). The first level that we will look at is posttranslational regulation. This is the level at which response may be the most rapid. In a pathway of many steps, the final product, the allosteric effector, often inhibits the activity of the enzyme at the first unique step of the pathway (Figures 8.2 & 8.4), resulting in shut-down of the pathway. The inhibitory product binds to the enzyme at the allosteric site (Figure 8.3), changing the conformation of the enzyme so that the substrate is unable to bind at the active site. In other cases, regulation might be due to covalent modification such as phosphorylation or methylation. An example of regulation of enzyme activity by adenylation is shown in Figure 8.6. One of the most important levels of gene regulation is that of transcription. Regulation at this level ensures that enzymes are not synthesized unless they are needed by the cell. Transcription may be regulated by repression (Figure 8.10)or induction (Figure 8.11) by specific small molecules called effectors. Examples of Mechanisms of Repression or Induction of Enzyme Activity Repression

Genes for the production of biosynthetic enzymes are often regulated by repression, and the effectors are usually products of the pathway, while genes for the production of catabolic enzymes are often regulated by induction, and the effectors are usually substrates. See Figures 8.12 & 8.13 for examples of mechanisms of repression and induction. Note that in both of these examples,

effector molecules interact with allosteric repressor molecules to influence their binding of the operator region of the gene. Binding of the operator blocks the path of the RNA polymerase, thus inhibiting transcription. Both of these types of regulation involve repressors, and the term for this type of regulation is thus negative control.

Induction

In some cases, transcription is mediated by an activator protein that operates by increasing the affinity of the RNA polymerase to the promoter (Figure 8.14). Effector molecules bind to the activator protein, which changes the conformation so that the activator protein can bind to the activator binding site on the DNA. This binding of the activator protein to the DNA increases the affinity of the RNA polymerase to the promoter. The promoters for these types of genes are usually not very close to the consensus sequence (see Figure 7.27). This type of regulation is called positive control.

Interaction of the Activator Protein with the DNA Sequence An important characteristic of regulatory proteins that interact with DNA is their ability to bind DNA in a sequence-specific manner. Many of these regulatory proteins are dimers, and interact with inverted repeats of DNA (Figure 8.7). The helix-turn-helix (Figure 8.8) is a structure that is commonly seen in DNA binding proteins. Attenuation An elaborate type of regulatory mechanism that some genes use involves interaction of transcription and translation. This mechanism is called attenuation. Regulation by attenuation occurs after the initiation of transcription, but before transcription is completed. This regulation does not influence the rate of transcription initiation. Typically, if translation of a particular transcript is able to occur, then further transcription of that gene will be terminated after initiation. The model for this mechanism is the tryptophan operon of E. coli. Operons that use attenuation are often amino acid biosynthetic operons, for reasons that will soon become obvious. Attenuation of of the tryptophan biosynthetic operon involves a translated leader sequence immediately following the operator (Figure 8.20). The leader sequence contains two tandem tryptophan codons. * If tryptophan is abundant, it will be incorporated into the leader peptide chain from the corresponding aminoacyl-tRNA. * If tryptophan is scarce, the tryptophan aminoacyl-tRNA will be rare, and the leader peptide will not be completely synthesized.

If the leader peptide is fully translated, then transcription of the downstream genes in the tryptophan operon will be terminated. How does translation of a transcript affect further transcription of that same transcript? First, remember that in prokaryotes transcription and translation are coupled. So the 5' end of a transcript can be translated before synthesis of that transcript is complete. As shown in Figure 8.21, the tryptophan operon transcript is subject to strong intramolecular base-pairing. If translation of the leader peptide does occur, then the translating ribosome is able to continue along the transcript, and blocks the formation of the antitermination stem-loop structure. The termination stem-loop structure can then form, and this causes the transcript to be released by the RNA polymerase. If translation of the leader peptide does not occur, because of tryptophan starvation, then the ribosome pauses, and blocks the formation of the termination stem-loop structure. Transcription can then continue. The tryptophan operon is also subject to regulation by repression at the promoter. The attenuator control acts to fine-tune the level of expression given by repression control. Global Control Networks In many cases, the regulation of several genes in an organism will be influenced by a particular environmental change. There are many such global control systems (Table 8.1). It has been established that bacteria are able to sense the cell density within their population. This is called quorum sensing, and it is mediated by molecules called acylated homoserine lactones (AHL), which are produced by the bacteria. The higher the population, the higher the concentration of AHL. The AHL molecules are able to combine with an activator protein, and activate many different genes that are subject to regulation by population density. Another means of global control is the use of alternative sigma factors. Remember that transcription initiation of most genes is carried out by sigma 70, but some promoters are recognized by other sigma factors, such as sigma 32 (heat shock) or sigma 54 (nitrogen regulation). In the case of heat shock, sigma 32 protein is normally degraded very quickly after it is synthesized, but when the temperature rises, the sigma 32 protein is stabilized, and can therefore promote initiation of transcription at sigma 32 promoters. One of the most widespread regulatory mechanisms that bacteria use to respond to changes in their external environment is the use of two-component regulatory systems (Figure 8.23). As implied by the name, these systems typically consist of two proteins, one of which, the sensor kinase, senses the stimulus, and transduces the signal to the response regulator, which then regulates transcription. The way that the signal is transduced is through phosphorylation events. The sensor, which is usually a transmembrane protein, is phosphorylated in response to the presence of the signal. The phosphorylated sensor is then able to transfer the phosphoryl group to the response regulator. The response regulator is only able to regulate transcription when it is phosphorylated. Regulation may be by either activation or repression, depending on the

system. Some examples of two-component regulatory systems in E. coli are listed in Table 8.3. Regulation of Chemotaxis Chemotaxis is also carried out by a series of signal transduction events (Figure 8.24). The presence of attractant or repellent is first sensed by transmembrane sensory proteins called methyl-accepting chemotaxis proteins (MCPs). The MCPs interact with the cytoplasmic protein CheW, and modulate the level of autophosphorylation of the CheA sensor kinase (attractants decrease the level of phosphorylation, repellants increase the level of autophosphorylation). The phosphorylated CheA (CheA-P) transfers the phosphoryl group to the response regulator CheY. CheY-P differs from other response regulators in that it does not influence transcription of a gene, but rather determines the direction of flagellar rotation. CheY-P causes clockwise rotation, which means that the cells will tumble. There is another level of regulation of chemotaxis, adaptation, and this involves methylation. The protein CheR is able to methylate the MCPs. Another protein, CheB, is phosphorylated by CheA-P, and CheB-P is able to demethylate the MCPs. Thus, in the presence of a continually high level of attractant (resulting in lower level of CheA-P, CheY-P and CheB-P), the level of methylation will increase because of lack of CheB-P mediated demethylation. The level of methylation of the MCPs will affect their sensitivity to the attractant or repellant. Fully-methylated MCPs are not able to respond to attractant, resulting in, eventually, the phosphorylation of CheA and subsequent phosphorylation of CheB, and demethylation of the MCP.

Module Six: Genetics and Molecular Biology Learning Objectives: To learn some of the terminology of microbial genetics, and also to begin to understand the tremendous power of the genetic approach. You should understand how homologous recombination takes place, and be familiar with different ways in which DNA can enter the cell. We will also discuss plasmids, partly in preparation for the topic of conjugation. You should become familiar with the different parts of plasmids, plasmid conjugation, and chromosome conjugation mediated by integrated plasmids. Transposons, insertion sequences and restriction endonucleases will also be covered. You should understand what microbial genomics is, and the impact that it has had on the study of microbiology in recent years. (Please refer to textbook sections 10.1-10.12, 10.19, 15.2-15.3, 15.8-15.9) Introduction to the Terminology of Molecular Biology Microbial Genetics is of fundamental importance, for many reasons. First, cell function is determined by gene function and gene regulation. Also, since genetics of prokaryotic microorganisms is generally much more accessible than genetics of eukaryotic organisms (with the exception, perhaps, of yeast), microbial systems are very useful as experimental systems. Microbial Genetics gave rise to Molecular Biology, and most of the tools of molecular cloning are of prokaryotic origin. Terminology * Mutant: a strain carrying a mutation * Genotype: genetic description of a strain (e.g. hisC) * Phenotype: observable properties of a strain (e.g. His-) * Wild-type strain: the original isolate, from which mutant strains are derived The Phenotypes of Mutations: Selection vs. Screening Depending on their phenotype, mutations may be selectable or nonselectable. Selectable mutations confer on the strain the ability to grow under conditions that do not allow growth of a strain that does not carry the mutation. For example, an antibiotic resistance phenotype would be selectable, while an antibiotic sensitivity phenotype would be nonselectable. It is possible to select directly for the antibiotic resistance phenotype on antibiotic containing medium. In contrast, the antibiotic sensitivity phenotype cannot be selected for, but could be screened for. In Microbial Genetics, inappropriate use of the terms selection and screening is not tolerated. Replica plating (Figure 10.2) is a technique for screening a large number of colonies for a nonselectable phenotype. This technique is especially useful for the isolation of

auxotrophs, which are mutants that have a nutritional requirement (e.g. amino acid, vitamin). The non-mutant, parental strain is referred to as a prototroph. Penicillin selection is a method that can be very useful as a negative selection to enrich for nonselectable mutants. The key to the success of this method lies in the fact that penicillin only kills growing cells. The addition of penicillin to a population of mutated cells growing in a particular growth medium will result in the death of only cells that are able to grow in that growth medium. The mutants that are unable to grow because they require a growth factor which is absent from the growth medium will not be killed, and will thus be enriched in the population. Types of Mutations Mutations arise spontaneously. During DNA replication, the DNA polymerase will incorporate errors at a low frequency(10-7 - 10-11 per base-pair replicated). Point mutations are those mutations which involve the change of a single base pair by substitution, insertion or deletion (Figures 10.3 & 10.4). The effect of a substitution mutation within a protein coding region depends on the resulting codon change. In some cases, especially if the substitution is at the third base of a codon, the new codon might encode the same amino acid as the original codon, and the result will be no change in protein. This is called a silent mutation. A missense mutation is when the new codon encodes a different amino acid than the original codon. A nonsense mutation is when the new codon is a stop codon. Insertion and deletion mutations in protein coding sequence result in a reading frame shift (Figure 10.4). They are thus frameshift mutations. The correct reading frame can be restored by a second insertion or deletion mutation near the first mutation, and sometimes this will fully or partially restore activity of the protein. A revertant is a strain that has regained wild-type phenotype from mutant phenotype. It is easy to envision how a point mutation could revert back to the wild-type sequence, or at least a sequence that restores activity to the encoded protein. Mutations that restore the wild-type phenotype also sometimes arise at a different site from the original mutation, and these are called suppressor mutations. Some suppressor mutations arise in the same gene, such as the ones described above that restore the correct reading frame in a frameshift mutation. Suppressor mutations can also occur in other genes resulting in compensation for the loss of the protein activity caused by the original mutation. Other types of mutations include deletions, in which large segments of DNA can be removed, and insertions of large segments within the DNA. Some insertions are caused by insertion sequences, which are genetic elements that are able to transpose (="hop") from one part of the genome to another part of the genome.

Mutagens Although mutations arise spontaneously, there are many mutagens that increase the rate of mutation. Major chemical mutagens are listed in Table 10.2. Base analogs (Figure 10.5) can be incorporated in place of the correct nucleotide but do not base-pair properly. Some other chemical mutagens are able to chemically alter nucleotides within a DNA strand, resulting in changes to their base-pairing properties. Yet another class of chemical mutagens interferes with the spacing between adjacent base-pairs, causing errors during replication. Another important type of mutagen is radiation (Figure 10.6). Ultraviolet light is strongly absorbed by the nucleotide bases, with a peak at 260 nm. The UV light can cause the formation of pyrimidine dimers between adjacent pyrimidine bases on the same DNA strand, and this can disrupt replication, increasing the incorporation of errors. UV light is a common tool used for mutagenesis in the microbial genetics laboratory. The cell has mechanisms to repair certain types of DNA damage. However sometimes the DNA damage cannot be repaired to give the original sequence, and the cell must repair the DNA however it can, often incorrectly, or else die because the damage is too severe. Some of the DNA repair systems that are used as the last resort are error-prone. The genes of some of the DNA repair systems are expressed in response to DNA damage, regulated by the SOS regulatory system (Figure 10.7). The Ames Test The Ames Test is a measure of the mutagenicity of a compound, which is often correlated with carcinogenicity. It is based on the ability of the compound to cause reversion of an auxotrophic point mutation in Salmonella typhimurium or Escherichia coli, allowing the strain to grow in the absence of the nutritional supplement (Figure 10.8). This test has been standardized, and is an important first screen in determining the potential carcinogenicity of a compound. Genetic Recombination Homologous recombination refers to the exchange or crossing over between identical or nearly identical (=homologous) DNA sequences. The process of recombination (Figure 10.9) is very complex, and involves many different proteins. The most important protein involved in recombination in bacteria is RecA, which is encoded by recA. In lecture, we will go through the molecular events that take place during homologous recombination. Genetic crosses involving homologous recombination between genetically distinct sequences form the basis of classical genetics. How are genetic crosses involving recombination carried out in bacteria? First, DNA from a donor strain must enter the recipient cell (Figure 10.11). This can occur by

transformation, in which naked DNA is taken up by the recipientcell, transduction, in which a phage injects DNA from the donor cell into the recipient cell, or conjugation, in which plasmid or chromosomal DNA is transferred from the donor cell to the recipient cell as a single strand. After the DNA from the donor strain enters the recipient cell, homologous recombination might occur. In order to detect a recombination event, it is necessary that the presence of the genetically distinct but homologous sequences can be differentiated by phenotype of the recombinant organism. Use of selectable markers (Figure 10.11) allows the detection of recombination events that occur at even very low frequencies. Transformation and Transduction Some types of bacterial cells are naturally transformable by DNA, or competent. In organisms that are naturally competent, specific competence proteins aid in the transformation event (Figure 10.13). Some other types of bacterial cells can be made competent by physical or chemical treatment. For example, E. coli cells can be made competent by treatment with calcium ions, and this has made possible the development of E. coli as the preferred host for molecular biology and genetic engineering. Electroporation, a relatively new method in which electric fields are used to create small pores in the cell membranes through which DNA molecules can enter, is gaining in popularity as a method of transformation. Bacteriophage are sometimes able to transfer genes between bacterial strains by transduction. Generalized transduction (Figure 10.14) involves the transfer of DNA from any region of the chromosome. Sometimes, when the bacteriophage DNA is packaged into phage particles, some of the host cell's DNA can be accidentally packaged into some of the phage particles. These particles thus contain only host cell DNA, and no phage DNA. They are called transducing particles, because they can inject the packaged DNA from the host cell into an appropriate recipient cell, where it is free to recombine into the recipient cell's genome. Specialized transduction, in contrast to generalized transduction, only involves transfer of DNA from a specific region of the chromosome (Figure 10.15). Plasmids The replication of plasmids is generally carried out by host cell DNA polymerases. However, plasmids encode genes which are involved in the regulation of the rate of plasmid replication, and thus determine plasmid copy number within the cell. If the replication of two plasmids is controlled by the same genes, then those plasmids will not be able to be maintained together in the same cell, and they are said to be incompatible. Plasmids that are incompatible with one another are closely related, and belong to the same Inc group. One of the best studied plasmids is the F plasmid (Figure 10.17).

Conjugation Many plasmids are able to transfer from donor cell to recipient cell in a process called conjugation (Figure 10.18). This transfer process is mediated by the products of tra genes, some of which interact with the oriT region of the plasmid to initiate the transfer of a single strand of DNA, while others form structures such as pili which aid in the transfer of the DNA to the recipient cell. Besides the genes involved in replication control and conjugation, many other genes can be carried by plasmids (Table 10.3). Some of the first plasmids to be studied were those that encode antibiotic resistance. These are termed resistance plasmids, which is sometimes abbreviated to R plasmids or R factors. A variety of virulence factors are encoded by plasmids. Also, some plasmids encode peptides called bacteriocins that kill closely related bacterial strains that do not carry the plasmid. As well as being able to conjugate themselves between bacterial cells, plasmids are sometimes able to integrate into the bacterial chromosome and transfer part of the chromosome between bacterial cells. Donor cells are called "males", and recipient cells are called "females". Only the donor cells have the sex pilus, which is a structure that is encoded by some of the plasmid-encoded tra genes and helps to bring the donor and recipient cells together (Figure 10.20). Once the cells have been brought together, one strand of the plasmid is nicked at the oriT by a tra-encoded endonuclease, the resulting single strand is unwound from the helix, and is transferred into the recipient cell. The strand that is transferred is replaced in the donor cell by replication, and the complementary strand is also synthesized in the recipient cell (Figure 10.21). Under appropriate conditions, plasmids can spread rapidly within and between bacterial populations by conjugation.

Complementation Complementation analysis can be used to determine whether two parental strains have mutations in the same gene. For example (as shown in Figure 10.27) if DNA from one Trp- mutant strain is introduced into another Trp- mutant strain, and Trp+ progeny are recovered, then this means that the two trp mutations are in different genes. If no Trp+ progeny are recovered, then the two trp mutations are in the same gene. Transposons and Insertion Sequences How is it that plasmids can integrate into the chromosome and mediate conjugation of part of the chromosome? Some plasmids, such as F, have insertion sequences (IS) which are specific DNA sequences that are also found on the chromosome, and homologous recombination can therefore occur between the plasmid and the chromosome at these sequences (Figure 10.22). Strains that have F integrated in the chromosome are called Hfr (for "high frequency of recombination"). A given Hfr strain will be able to transfer a

particular gene at a characteristic frequency. Genes that are nearest the site of insertion in the direction of transfer are transferred at the highest frequency (Figure 10.24). This is because the genes nearest the oriT of the integrated F (Figure 10.23) will be most likely to be transferred into the recipient cell before the DNA strand is broken. The sequential nature of gene transfer can be demonstrated using interrupted mating experiments (Figure 10.26), and this property can be used to map genes. In order to detect a chromosome conjugation event between donor and recipient cells, it is necessary to use appropriate markers and selection. Usually, this entails the use of a combination of antibiotic resistance and auxotrophic markers (Figure 10.25). The conditions for selection must be such that the desired transconjugant organisms are able to grow, but the donor and recipient are not able to grow. Sometimes, an integrated F plasmid is able to excise from the chromosome, and sometimes when it does this, it brings along part of the chromosome as well. This results in a plasmid that carries chromosomal genes. Such a plasmid is called a F' plasmid. Some genes are able to move from one part of the genome to another at low frequencies. This process is called transposition, and is carried out by transposable elements. Insertions sequences, such as those found on F, are transposable elements, as are transposons. These elements contain genes that encode transposase enzymes which recognizes terminal inverted repeat sequences (Figure 10.28). The transposition process is illustrated in Figures 10.29, 10.30. 10.31. You should note that although transposition is a recombination process, it does not involve homologous recombination, but rather site-specific recombination. Transposition is actually a very useful tool for mutagenesis. If a transposon inserts within a gene, it will disrupt the function of that gene (Figure 10.32). It is possible to set up a transposition experiment where a transposon that confers antibiotic resistance is introduced into a recipient cell, and all selected antibiotic resistant colonies contain a transposon insertion at a unique location. The insertion containing colonies can then be screened for the desired phenotype. Restriction Enzymes Microbial cells often take up foreign DNA from the environment, or are injected with DNA by bacteriophage. Some of this DNA could potentially harm the cell, especially if it encodes functions that are detrimental to cellular metabolism. Many prokaryotes are able to deal with foreign DNA by recognizing it as foreign and destroying it. How do these cells recognize and destroy foreign DNA? They use what are called restriction-modification systems. Incoming foreign DNA is destroyed by restriction enzymes, which cleave DNA at sites with specific DNA sequence (often palindromes), creating a double-stranded break. How is the restriction enzyme prevented from cleaving the cell's own DNA? The corresponding modifying enzyme methylates the DNA that is

synthesized in that cell at the same specific DNA sequence that is recognized by the restriction enzyme. The methylation prevents the restriction enzyme from cleaving at the modified sites (Figure 10.34). Restriction enzymes are very important for molecular biology and molecular genetics research. Hundreds of these enzymes, with different specificities, are available commercially. They enable the cleavage of DNA molecules at specific restriction sites. Resulting restriction fragments with compatible end sequences can be ligated together (using DNA ligase enzyme), resulting in recombinant DNA molecules. Mapping Genomes Genetic maps of bacteria were first constructed using techniques such as Hfr-mediated conjugation, transformation and transduction of auxotrophic and other markers. In E. coli, the most commonly used techniques were Hfr mapping and, for more precise mapping, transduction using P1 phage (10.48). It is now possible to completely sequence an entire genome. This type of work is called genomics. Microbial Genomics Since the complete sequences of the genomes of many prokaryotes have now been determined (Table 15.1), molecular genetic techniques are becoming ever more important in microbial genetics. It is incredible that even with an organism as intensively studied as E. coli, 1/3 - 1/2 of the genes uncovered by the genome sequence are of unknown function (Table 15.2). The determination of the functions of these unknown genes is called functional genomics, and involves the construction of mutants and the analysis of the biochemical and physiological effects of the mutations. Functional genomics also involves analysis of the structure, function and regulation of proteins (proteomics) and the analysis of the expression of all of the transcripts in the genome at once (microarray analysis). For updated information on the genomes that have been sequenced or are currently being sequenced, please follow these web sites-- Genomes Online Database (http://www.genomesonline.org) US Department of Energy Microbial Genome Program (http://www.ornl.gov/microbialgenomes) The Sanger Institute (http://www.sanger/ac.uk/Projects/Microbes/) National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/PMGifs/Genomes/bact.html)

One of the most popular techniques for sequencing bacterial genomes is called shotgun sequencing. This involves cutting the genome into small pieces, cloning the pieces into plasmid vectors, and then sequencing the cloned DNA randomly. The sequences that are obtained are then analyzed using computational methods. The computer finds all of the overlapping sequences, and uses these to order the sequences so that the entire genome is covered. Since the clones are sequenced at random, without prior knowledge of the part of the genome that they carry, as much as ten-fold coverage is usually needed to ensure that the entire genome sequence can be pieced together. Once the genome sequence has been completed, it is necessary to identify the coding sequences. Open reading frames (ORFs) can be predicted by locating potential Shine-Dalgarno sequences preceeding start codons that would result in uninterrupted translation until a stop codon is reached. Genomics, coupled with the necessary computational analysis, or bioinformatics, is revolutionizing the study of microbial genetics, microbial physiology, microbial ecology and microbial evolution. This makes it a very exciting time to be a microbiologist!

Module Seven: Microbial Taxonomy Learning Objectives: In this module, we will consider how life on Earth might have originated, how eukaryotes might have evolved from prokaryotes, and how to measure how organisms are related in evolution. (Please refer to textbook sections 11.1-11.12) Theories on the Origins of Life How did life originate on a sterile Earth? As we have discussed in earlier lectures, microorganisms were the first organisms to arise on Earth, and have been present on Earth for much of its history. Microorganisms have been around for at least 3.8 billion of the 4.6 billion years since the Earth was formed. Multicellular eukaryotic organisms (= metazoans) have evolved comparatively recently, and have been around for less than 1 billion years. The atmosphere of the early Earth was quite different than the present atmosphere. It was the physiology of the existing microorganisms thatcreated the environmental conditions conducive to the rise of multicellular organisms, which then evolved from microorganisms. Conditions on early Earth: * little O2, reducing atmosphere * significant amounts of H20, CH4, CO2, N2, NH3 * temperature >100 C * energy input from various sources, including UV light It has been shown experimentally that UV irradiation of gaseous mixtures resembling the atmosphere of early Earth can result in the formation of organic macromolecules. Is it possible that macromolecules that were formed under these conditions gained the ability to polymerize to make polypeptides, polynucleotides and other polymers, and then gained the ability to direct this polymerization process? It is now thought by many scientists that the first life was RNA-based (Figure 11.4). There are many examples of ribosome molecules (ribozymes) that possess catalytic properties. The early life forms could have thus been composed of RNA only, and no DNA or proteins. The RNA would have been the genetic material, as well as providing the catalytic functions necessary for the replication of the genetic material. Eventually, the coding material in the cell might have developed the ability to direct peptide synthesis, and the resulting peptides might have had catalytic activity and replaced the catalytic function of some of the RNA molecules. The genomic function of RNA would have had to be eventually replaced by DNA, with the RNA continuing to play an important role. There is some discussion in the textbook about the higher fidelity of DNA

replication compared to RNA replication, but I wonder how much of this is a property of the different polymerase enzymes rather than the different types of macromolecules. So far, we have focused on the origin of the genetic system. What about the origin of the metabolic system? Replication, and the activities that support replication, would require an input of energy. How did energy generating systems arise? An example of a hypothetical early, simple energy-generating system is outlined in Figure 11.5. Note that this system could function in the absence of O2, as would be required in the anoxic conditions of early Earth (see Figure 11.6). We will see later on in the course that similar reactions are carried out by some existing prokaryotes. Although the first photosynthetic organisms were anoxygenic (i.e. did not produce O2), photosynthetic cyanobacteria that did produce O2 arose quite early. As more and more oxygenic photosynthesis took place, eventually O2 began to accumulate in the atmosphere. The transition of the Earth's atmosphere from an anoxic, reducing environment to an oxic environment is thus due to the physiology of prokaryotic organisms. This paved the way for the evolution of metazoan organisms with aerobic metabolism. Once the atmosphere had been primed by the physiological activities of prokaryotes, how did the eukaryotes evolve to take advantage of this new environment? We know that the eukaryotes arose by evolution of the prokaryotic genetic material, but the whole story is not so simple. Remember that eukaryotes, except the most primitive ones, have organelles such as mitochondria and chloroplasts, and these organelles contain DNA. The endosymbiotic theory postulates that an archaeal organism whose genetic material eventually became the nuclear DNA of the eukaryotic cell "swallowed" an aerobic bacterial organism, which set up shop and provided the archaeal cell with energy, while receiving a steady supply of nutrients in return (Figure 11.7). Similarly, an archaeal organism taking up an oxygenic cyanobacterial type organism would result in another successful symbiosis. Eventually, the endosymbiotic organisms became specialized according to their new environment within another cell, and lost the functions that were not related to their new role. What is the evidence for the endosymbiotic theory? Among other things, it has been observed that many of the eukaryotic organisms that do not contain organelles (note that these types of organisms are not very common) are also very near the root of the evolutionary tree. In addition, the ribosomes within the mitochondria and chloroplasts are of the bacterial type, and are inhibited by antibiotics that inhibit bacterial ribosomes but not eukaryotic ribosomes. Their rRNA also has more sequence similarity to bacterial rRNA than to eukaryotic rRNA.

Ribosomal RNA as an Evolutionary Chronometer How can we reliably measure the evolutionary distance or phylogenetic relationship between two organisms? First, remember that genomes are continually mutating and evolving. This involves continual changes in DNA sequence. If two organisms had a common ancestor, the difference in their genome sequence would be an indication of their evolutionary relatedness. It is not practical to compare organisms at the level of the entire genome, but fortunately comparison of entire genomes is not necessary to infer evolutionary relatedness. It is sufficient to compare homologous sequences, or molecular chronometers, that are found in both of the organisms. Genes encoding rRNA (Figure 11.8) are some of the best such sequences, in large part because all organisms have rRNA, and rRNA has the same function in all organisms. Although 5S rRNA, 16S rRNA and 23S rRNA molecules can each be used for the determination of phylogenetic relationship between organisms, it is the 16S rRNA (= small subunit rRNA) molecule that is used most often. Within the length of ~1500 nucleotides there are both highly conserved and highly variable regions. The 16S rRNA sequences have been determined for several thousand organisms, and these sequences are catalogued in databases that are publicly accessible over the internet. The determination of 16S rRNA sequence followed by comparison to the sequences in the online database is become a fairly standard procedure in the phylogenetic characterization of microorganisms. Microbial Phylogeny from Ribosomal RNA So how does one obtain the 16S rRNA sequence of a given organism? In actuality, what is sequenced is almost always the 16S rDNA, the gene which encodes the 16S rRNA. First, primers to conserved regions of the 16S gene are used in a PCR reaction to amplify the gene from genomic DNA of the organism (Figure 11.9). That PCR product can then be sequenced directly. The use of rRNA sequences is revolutionizing the science of microbiology. First, it has re-ordered our understanding of how the different life forms on Earth are related. Second, we are now able to probe the microbial community structure of environments without first culturing the organisms. Through such studies, it has become very apparent that almost all environments contain microorganisms that have never been cultured and described before. Oligonucleotide probes have been designed for small subunit RNA signature sequences (Table 11.1). These can be designed to differentiate between sequences at different levels, from domain to genus or even sometimes species. These probes can be used to detect organisms in situ (Figures 11.11 & 11.12), using fluorescent label, or to analyze community structure after first isolating total community DNA and then amplifying the small subunit genes by PCR, cloning the amplfied fragment, followed by sequencing.

Many of the sequences obtained do not match the sequences currently in the database, and thus represent previously unknown species. Taxonomy and the Species Concept Biologists had previously organized organisms into five kingdoms, including one kingdom that included all the prokaryotes.It has now been shown that the five kingdom system does not reflect the true evolutionary relatedness between organisms. Molecular phylogeny based on comparisons of small subunit rRNA sequences has revealed that there are in fact three domains, or evolutionary lineages (Figure 11.13). Two of these domains are prokaryotic (Bacteria and Archaea), while the third, the Eukarya, includes all other organisms. There are many more than five kingdoms within the three domains of the universal phylogenetic tree. Although small subunit rRNA sequencing is providing a relatively clear picture of phylogeny, it is not so simple. Phylogeny determined using other gene sequences is sometimes at odds with the phylogeny determined using the small subunit rRNA. This is evidence that there has been a certain amount of lateral gene transfer, where genes have been transferred between organisms. The universal phylogenetic tree confirms that mitochondria and chloroplasts arose from endosymbiotic bacteria. It is interesting that the mitochondria are most closely related to organisms that are capable of living within eukaryotic cells. We had previously considered some of the structural differences between members of the three domains. These include cell wall structure and nature of lipids. In many ways, protein synthesis in the Archaea shares more with the Eukarya than with Bacteria. While the Archaea do have ribosome binding sites like the Bacteria, protein synthesis in Archaea is inhibited by antibiotics and toxins that inhibit protein synthesis in the Eukarya, but not by antibiotics that inhibit protein synthesis is Bacteria (Table 11.2). A summary of features that allow differentiation of members of the three domains is listed in Table 11.3. There is still controversy over whether the Archaea are more closely related to the Eukarya or to the Bacteria. It is clear that the new molecular phylogeny methods have changed the the science of Taxonomy, which is concerned with the classification of organisms. Traditionally, taxonomy relied on phenotypic analysis. This is relatively difficult with microorganisms, which have few structural characteristics to compare. Many of the microbial phenotypic analyses thus include biochemical properties as well as structural traits. Although these conventional methods might not result in phylogenetically valid groupings, they are nevertheless quite useful for describing and differentiating microorganisms using a dichotomous key (Figure 11.17). Comparison of the GC base ratio can often be used to differentiate between organisms that are phenotypically similar but are not phylogenetically related (Figure 11.18). However, organisms that have similar GC ratios are not necessarily closely related. A measure of relatedness can be obtained by genomic hybridization experiments (Figure 11.19). Hybridization values above 70% indicate same species, while hybridization values above 30% indicate same genus. Note, however, that

the species concept in clonal organisms such as bacteria and archaea is not the same as for eukaryotic organisms that have a sexual stage. While plants and animals of a given species are defined based on their reproductive isolation from other species, this can not be done for prokaryotes. Nevertheless, the taxonomy of prokaryotes involves a concept of species. In addition to genomic hybridization, direct comparison of small subunit rRNA sequence is sometimes used. Organisms of the same species should be at least 97% identical in this sequence. There is a formal classification system for bacteria, including nomenclature that includes genus and species names. In order for a newly isolated organism to be designated as a new species, it must be fully described, shown to be sufficiently different from known species, and a culture must be deposited in an approved culture collection from which it is made available to the research community. Taxonomic information for described species is cataloged in Bergey's Manual of Systematic Bacteriology.

Module Eight: The Bacteria (I) Learning Objectives: To look at some of the incredible diversity of Bacterial life. We will see where these organisms live, and how they make their living. (Please refer to textbook sections 12.1-12.2, 12.6-12.7, 12.9-12.11, 19.21-19.22) The Proteobacteria We will consider the phylogenetic groupings, starting with the Proteobacteria, which are gram-negative organisms. Overview Purple Phototrophic Bacteria * Anoxygenic photosynthesis * O2 inhibits photosynthesis, but some can grow aerobically using respiration * Bacteriochlorophylls and carotenoid pigments (Figure 12.2) determine which

wavelengths of light are used for photosynthesis Purple Sulfur Bacteria (Table 12.2) * Photoautotrophs, oxidize H2S to S0 during photosynthetic CO2 reduction * S0 is stored in periplasm * Found in anoxic zones of lakes where H2S present Purple Nonsulfur Bacteria (Table 12.3) * Only very low levels of H2S oxidation * Photoheterotrophs (can use light for energy and organic compound for carbon) * N2 fixers Methanotrophs (Tables 12.7 & 12.8) * These are the only organisms which are able to oxidize methane and other C1

compounds which lack carbon-carbon bonds * Are unable to utilize compounds which contain carbon-carbon bonds * Contain the enzyme methane monooxygenase, which is used to convert methane

to methanol * Obligate aerobes * In nature, often found at the thermocline between anoxic zones, where methane is

formed, and oxic zones, where O2 is available for respiration

* Some methanotrophs are symbionts of marine mussels that live near geological sources of methane (Figure 12.16), and are found in mussel gill tissue

Pseudomonads (Tables 12.10 & 12.11) * Very heterogeneous group, taxonomy has been revised recently * Aerobic chemoorganotrophs, nutritionally versatile

! Inhabit many different environments (soil, water, animal pathogens, plant pathogens)

Free Living Aerobic N2-Fixing Bacteria (Table 12.12) * e.g. Azotobacter * Strict aerobes * Able to fix N2 aerobically, although the nitrogenase enzyme, which reduces N2 to

NH3, is irreversibly inactivated by O2 * Have thick slime layer and very high rate of respiration, which together keep the

intracellular O2 concentration low during N2 fixation Neisseria and the Enteric Bacteria Neisseria, Chromobacterium, and Relatives (Table 12.13) * Uniquely gram-negative cocci * Non-motile, aerobic * Some are animal pathogens Enteric Bacteria (Table 12.14) * e.g. Escherichia coli, Salmonella * All within the gamma Proteobacteria * More closely related than justified by genus-level groupings * Peritrichous flagella, facultative, oxidase negative, simple nutritional

requirements (Table 12.14) * Includes many pathogens, therefore very well studied * Genera can be separated by diagnostic tests (Tables 12.15 & 12.16, Figure 12.25) The Rhizobiaceae The members of the Rhizobiaceae are defined by their interaction with plant hosts. The best known examples are Agrobacterium, which form tumours and hairy roots with dicot plants, and the root nodule bacteria, which form N2 fixing symbioses with legume plants.

While they obtain benefit from their intimate interactions with the plant host, they are also well adapted to life in the soil. The Rhizobiaceae are alpha Proteobacteria. Agrobacterium General Concepts

! Trans-kingdom genetic transfer (Bacteria to Plant). o a natural example.

! Gene regulation in response to environmental signals.

o Expression of bacterial virulence genes induced by signals from wounded plant.

o Genes only expressed when susceptible host plant is available.

! Design and creation of habitat in which it survives best. o Not only does Agrobacterium colonize a specific habitat, but it

actually creates (or enhances) that habitat.

! Sophisticated relationship between 3 players. a) Oncogenic plasmid. b) Agrobacterium cell. c) Plant.

! Use of Agrobacterium for genetic engineering of plants.

Infection Process

1. Wounding required. 2a. Attachment of Agrobacterium to plant cells. 2b. vir gene induction in response to signals from plant. phenolics monosaccharides acidic pH (temperature) 3. T-DNA processing. 4. T-DNA transfer into plant cell. 5. Integration of T-DNA into plant nuclear genome and expression of genes. 6. Tumour formation, opine synthesis.

Root Nodule Forming Bacteria Legume Root Nodules There are over 15,000 species in the Leguminosae family, ranging from forage legumes to grain legumes to trees. Many of these plants have organs on their roots called nodules. These nodules are packed full of bacteria called “rhizobia”. The rhizobia can live either within the nodule or in the soil, but they can only fix N2 while they are inside the nodule. Inside the nodule, the rhizobia are provided with carbon and energy in the form of photosynthate, and the oxygen-sensitive nitrogenase enzyme is protected.

What are Rhizobia?

The rhizobia are all members of the same branch of the Gram negative phylogenetic tree. While all legume root nodulating bacteria were originally placed in the same genus, Rhizobium, there are currently several recognized genera : Rhizobium, Bradyrhizobium, Azorhizobium, Photorhizobium, Sinorhizobium, Mesorhizobium. The best known rhizobia are those that form root nodules on agriculturally important crop plants such as pea, bean, soybean, alfalfa and clover. Their are certainly many types of rhizobia waiting to be discovered.

The Root Nodule Formation Process

How is a Root Nodule Formed?

Root nodule formation is actually induced by the rhizobia. The colonization of a legume root by the appropriate rhizobia results in controlled meristematic activity within the root tissue. As the nodule is developing, the rhizobia infect through plant cell wall invaginations which form tubes called infection threads. The rhizobia are then released into the plant cells and differentiate into N2-fixing cells called bacteroids.

How is Nodulation Studied?

The most dramatic advances in the understanding of nodule formation and function have been made in the last fifteen years. Genetic studies have indicated that specific bacterial genes (nod genes) are required for nodule formation to take place. Signal molecules are involved in the communication between the bacterium and the plant. The nod genes are only expressed in the presence of flavonoid compounds that indicate the presence of a suitable plant host. The products of the nod genes are enzymes that carry out the formation of lipo-chito-oligosaccharides, which then signal nodule induction in the plant. Genetic and biochemical studies have also revealed that C4-dicarboxylic acids such as malate are used by the bacteroid to generate energy and reducing power. Many other aspects of nodulation are being studied using bacterial mutants, and, more recently, plant mutants.

Module Nine: The Bacteria (II) Learning Objectives: To continue our look at some examples of the incredible diversity of Bacterial life. We will see where these organisms live, and how they make their living. After covering additional gram-negative bacteria, we will then consider the gram-positive bacteria. These organisms fall into two distinct phylogenetic groups: low GC and high GC. However, as with Proteobacteria, there are many examples of where traditional taxonomic groupings are at odds with phylogeny. (Please refer to textbook sections 12.12-12.14, 12.16-12.17, 12.19-12.21, 12.23-12.25) Vibrio and Photobacterium * Gram-negative, facultatively aerobic rods, mostly polarly flagellated, oxidase

positive * Aquatic, freshwater or marine * Vibrio cholerae is an important human pathogen, associated with poor water

sanitation * Some are bioluminescent, associated with fish (see Figure 12.28) * Bioluminescence requires luciferase enzyme and a long chain aliphatic aldehyde

Luciferase production is subject to regulation by autoinduction, which involves the production of particular N-acyl-homoserine lactone molecules by members of the population. When the concentration of these molecules reaches a critical point, indicating that the population has reached a certain density, the synthesis of the luciferase enzyme is induced at the transcriptional level.

Rickettsias (Table 12.17) * Obligate intracellular parasites * Closely related to mitochondria * Cause diseases such as Rocky Mountain spotted fever, Q fever, * Very restricted energy metabolism * Often transmitted by arthropod vectors Spirilla (Table 12.18) * Phylogenetically and physiologically diverse group * Spiral shape

One of the more interesting members of the Spirilla is Bdellovibrio (Figures 12.33 & 12.34). Bdellovibrio cells prey on other gram-negative bacteria.They stick to the surface of the "victim" cells, then penetrate into the periplasmic space where they replicate. Bdellovibrio cells do not grow well outside of the host cell, but variants have been isolated that are able to grow in pure culture.

Budding and Prosthecate/Stalked Bacteria (Table 12.19) * Physiologically heterogeneous group * Cytoplasmic extrusions * Unequal cell division (Figure 12.38) results in daughter cell that is distinct from

mother cell * An example of a budding cell is Hyphomicrobium (Figure 12.39)

* Mother cell is often attached to solid substrate * Bud forms at end of hypha * Daughter cell is a swarmer cell

* An example of a stalked cell is Caulobacter (Figure 12.42) * Mother cell has stalk, attached to solid substrate * Cell division by unequal binary fission * Daughter cell is swarmer cell

Gliding Myxobacteria (Table 12.20) * Complex behaviour and development (Figure 12.47) * Very large chromosome (9.5 Mb) * Vegetative cells exhibit gliding motility over surfaces, obtain nutrients by lysing

other bacterial cells * Under conditions of nutrient limitation, differentiate to form multicellular,

pigmented fruiting bodies which are visible to the naked eye * Fruiting bodies are filled with myxospores Low GC, Gram Positive Bacteria Nonsporulating (Tables 12.22 & 12.23) Staphylococcus and Micrococcus * Aerobic, catalase positive * Resistant to drying and high salt * Often pigmented * Staphylococci commonly found on animals (including human skin) * Micrococcus is actually a high GC organism Sarcina * Obligate anaerobes, cell division results in "packets" of eight cells, acid tolerant * Some species are among the few organisms that can grow in acid environment of

the human stomach

Lactic Acid Bacteria * Rods and cocci * Produce lactic acid as major fermentation product * No electron transport system, therefore no aerobic respiration -- substrate level

phosphorylation only * Anaerobes, but are aerotolerant (not sensitive to O2) * Most can only catabolize sugars * Complex nutritional requirements, limited biosynthetic capacity (fastidious) * Heterofermenters differ from homofermenters * Involved in many processes that are of human interest

* Pathogens, fermented products, dental health Endospore-Forming, Low GC (Table 12.25) This group includes all endospore forming bacteria. Their natural environment is the soil, and they are usually not pathogenic. Bacillus (Table 12.26) * Facultative or obligate aerobes * Can break down polymers (contrast with Pseudomonads, which cannot) * Many produce antibiotics * Some species produce crystal protein toxins (Figure 12.57) that kill insect larvae -

- many are specific for a particular type of insect. * Some species can infect humans and other animals (e.g. Bacillus anthracis). Clostridium (Table 12.27) * Strict anaerobes -- no electron transport system * Diversity of anaerobic fermentation (substrate and product) * Many industrially important products * Some fix N2 * Some produce toxins that cause human disease Low GC, The Mycoplasmas (Table 12.28) * No cell walls (no peptidoglycan), therefore resistant to antibiotics that inhibit cell

wall synthesis * Very small cells, as small as 0.2 µm, do not take a definite shape (Figure 12.62) * Colonies have "fried egg" appearance (Figure 12.63) * Very small genomes (as small as 0.5 Mb, 1/10 the size of the E. coli chromosome) * Sterols (see Figure 4.18) stabilize the cytoplasmic membrane, prevent osmotic

lysis * Range from strict aerobes to obligate anaerobes

High GC, Mycobacterium (Table 12.29) * Contain lipids called mycolic acids (Figure 12.69) in the cell wall, * Exhibit acid-fastness, a property in which the dye basic fuchsin is not removed by

acid-alcohol wash due to interaction of mycolic acids with the dye * Many are human pathogens * Resistant to many chemicals because of high lipid content of cell wall * Many contain pigments (carotenoids) High GC, Actinomycetes (Table 12.30)

These nutritionally versatile organisms are filamentous, and form branching mycelia. Many of them form spores called conidia (Figures 12.74 & 12.75). These conidia are formed in sporophores, aerial filaments that are important in the classification of the organisms. Perhaps the best studied genus is Streptomyces, members of which are found mostly in the soil and produce many of the most important antibiotics (Table 12.31).

Cyanobacteria (Table 13.33) * Oxygenic, usually obligate, phototrophs * Gliding motility, no flagella * Contain chlorophyll A, which is the same type of chlorophyll found in

chloroplasts * Morphologically diverse, ranging from unicellular to filamentous (Figure 12.78) * Some filaments contain differentiated cells called heterocysts distributed along

the filament, which lack the O2-evolving photosystem II and in which N2 fixation takes place (Figure 12.80)

* Heterocysts have thick cell wall that slows the diffusion of O2 into the cell.

Module Ten: The Archaea Learning Objectives: To look at some examples of the diversity of the Archaeal organisms. We will see where these organisms live, and how they make their living. (Please refer to textbook sections 13.1, 13.3-13.8) Overview of the Archaea The Archaea have long been recognized as being structurally and physiologically distinct from the Bacteria (review Table 11.3), and now it has been shown that they are indeed phylogenetically distinct from the Bacteria. Perhaps because Archaea are not known to cause diseases of animals or plants, they have not been studied as intensively as have some of the Bacteria. However, with modern molecular methods, we are now finding Archaea (many of them newly-described organisms) in places where they were not known previously. It is clear that they play important roles in the environment, and some of them do live in association with animals and other eukaryotic organisms. So far, the domain Archaea has been divided into three kingdoms (Figure 13.1) -- Euryarchaeota, Crenarchaeota and Korarchaeota. Some of the branches of the Archaeal phylogenetic tree, such as the Marine Euryarchaeota, the Marine Crenarchaeota, and the entire Korarchaeota kingdom, are represented only by genetic material isolated from environmental samples -- no organisms have yet been cultured. Euryarchaetoa Extremely Halophilic Archaea (Table 13.2) * Require very high salt concentration environment (Table 13.1), at least 1.5 M

NaCl (~9%). * Can grow at 5.5 M NaCl (saturated) * Chemoorganotrophs, obligate aerobes * Halobacterium has been most extensively studied * Requires sodium ion specifically, helps to stabilize the glycoprotein cell wall,

which contains acidic amino acids * Uses potassium as compatible solute inside the cell (Table 13.3) * Cytoplasmic proteins require high concentration of potassium ions for stability * Under low O2 conditions, some can use light to generate ATP, using the protein

bacteriorhodopsin and the carotenoid pigment retinal (Figure 13.4) which work together to set up a proton gradient

Methanogens (Table 13.5) * Obligate anaerobes, usually mesophilic

* Phylogenetically diverse * Range of habitats (Table 13.4) * A range of substrates can be converted to methane (Table 13.6) in energy yielding

reactions Thermoplasmatales * Thermophilic (optimum 55ºC), acidophilic chemoorganotrophs, facultative * Phylogenetically related * Thermoplasma lacks cell walls, cytoplasmic membrane has chemically unique

structure (Figure 13.11) * Picrophilus can grow below pH 0, optimum pH 0.7 Thermococcales * Hyperthermophiles (optimum >80ºC)

* Thermococcus grows from 70-95ºC * Pyrococcus grows from 70-106ºC

* Obligately anaerobic chemoorganotrophs * Use Sº as terminal electron acceptor, reducing to H2S * Motile, tuft of flagella Archaeoglobales * From hot marine sediments, near hydrothermal vents * Oxidation of H2 or organic compounds coupled to reduction of sulfate to sulfide * Irregular cocci, optimal growth ~80C * Crenarchaeota The cultured examples of crenarchaeotes are almost all obligate anaerobes, and they are either chemoorganotrophs or chemolithotrophs (Table 13.8). These organisms include some of the most hyperthermophilic organisms known (Table 13.9). Indeed, most of the crenarchaeotes that have been cultured are hyperthermophiles, and they have been isolated from some interesting habitats (Table 13.7). In addition to these organisms, environmental sampling has revealed that there are crenarchaeotes living in some of the coldest environments on Earth, such as the Antarctic (Table 13.7). These organisms are known only from their 16S rRNA sequences, however, and nothing is known of their physiology or biochemistry because they have not yet been cultured.

Module Eleven: Controlling Microbial Growth Learning Objectives: Earlier in the course, we considered the growth characteristics of microorganisms. Now, we’ll look at how to inhibit their growth or kill them. The methods that are used to inhibit growth or sterilize take advantage of cell sensitivity to heat, radiation or chemicals, or involve removal of the cells by filtration. (Please refer to textbook sections 20.1-20.5) Heat Sterilization The components of microbial cells are denatured if the temperature rises high enough, resulting in loss of cell viability (Figure 20.1). A given organism will have a characteristic sensitivity to temperature, with death occurring more rapidly at higher temperatures. There is an exponential relationship between time of heating and extent of killing. Decimal reduction time is the time required for a ten-fold reduction in the population density, and is not dependent on the initial cell concentration. Endospores are much more resistant to heat than are vegetative cells. For this reason, heat sterilization is aimed at ensuring that endospores are killed. At the autoclave temperature of 121ºC, endospores have a decimal reduction time of 4-5 minutes. Moist heat is more effective than dry heat, since it can penetrate the object being sterilized more effectively. The high temperature within the autoclave, above the boiling point of water, is achieved under pressure (Figure 20.3). A very common method of reducing the numbers of microorganisms in foods is pasteurization. The goal of pasteurization is not to sterilize, but rather to reduce the cell numbers so that the incidence of pathogenic microorganisms and/or the likelihood of spoilage is reduced. Pasteurization involves raising the temperature for brief periods of time so that the microbial cell numbers are decreased while minimizing the adverse effects on the product. For example, milk can be pasteurized by treatment at 71ºC for 15 seconds. Radiation Sterilization Many different types of radiation can be used for sterilization. The mechanisms vary, depending on the type of radiation. Microorganisms also vary in their sensitivity to radiation (Table 20.1). There is an exponential relationship between radiation dose and survival (Figure 20.5). Sterilization by radiation has many applications, especially for objects that are heat sensitive or would be destroyed by heat sterilization. Some examples are spices, pharmaceuticals, and medical equipment. Filter Sterilization

Filtration is a very effective method for sterilizing liquids and gases. The cells are removed based on size, rather than being destroyed. The filters that are used for sterilization are designed to remove all cells, but do not remove virus-sized particles. Membrane filters are commonly used in the microbiology laboratory to sterilize solutions or growth media (Figure 20.8). Chemical Growth Control Many chemical agents are available for killing or inhibiting the growth of microorganisms. These are called antimicrobial agents. A bacteriostatic agent would only inhibit the growth, while a bacteriocidal agent would kill (Figure 20.9). A bacteriolytic agent kills the cells by causing cell lysis. The sensitivity of a microorganism to a particular antimicrobial agent can be expressed as the minimal inhibitory concentration (MIC) (Figure 20.10). Sensitivity can also be determined by the agar diffusion method (Figure 120.11). Antiseptics: antimicrobial agents that can be applied to living tissue Disinfectant: antimicrobial agents that are used on non-living objects Please see examples in Tables 20.3 & 20.4.

Module Twelve: A Microbial Research Example Learning Objectives: To appreciate the application of microbial techniques to real-world research example. Bioplastics from Uncultivated Bacteria