BIOL 2501 Chapter 2 Txtbk Notes + Lecture

Nutrient Transport This is review! Responsible to know. Facilitated Diffusion

* No energy needed, moves with a concentration gradient *Uses energy of one concentration gradient to drive movement of another particle against its concentration gradient

Gram-positive cells know how the cell wall differs from gram-negative bacteria. Know what teichoic acids are.

- thick peptidoglycan - teichoic acids Gram-Positive Cells Gram-positive bacteria have a thick cell wall with many overlapping strands of peptidoglycan (Figure 2.21). Beneath this structure is the periplasm, a thin space between the cell wall and the plasma membrane. The Gram-positive cell wall is exposed to the environment and provides an important protective function for the cell, but it is not impermeable. After all, nutrients must be able to get to the plasma membrane, where they can be taken into the cell. Peptidoglycan is the most abundant polymer in the Gram-positive cell wall, but a mixture of other polymers can constitute up to half of the dry weight of the cell wall, depending on the species. A charged polymer called teichoic acid (not found in gram-negative bacteria)

•Cell wall characteristics can help explain how the Gram-stain works.

-The alcohol decolorization step shrinks the large pores in the Gram-positive cell, helping to lock the crystal violet stain in. -The alcohol also may strip away some of the outer membrane lipids in the Gram-negative cells, making them more likely to lose the initial crystal violet stain.

Step 2: UDP-NAM associated with a transport lipid in the membrane In step 2 what are the molecules involved. What is bactoprenol? Is the second subunit added yet? bactoprenol holds the it on the cytoplasmic face of the membrane not yet.

-The second stage takes place at the cytoplasmic membrane -UDP-NAM-pentapeptide precursor is linked to the transport lipid (bactoprenol)

Bacterial Cell Envelope

-plasma membrane -cell wall -in some a second membrane* know what a cell envelope is and what structure are considered part of the bacterial cell envelope.

•What about nonflagellar motility?

•Gliding motility: smooth sliding over a surface, not well understood (myxobacteria, cyanobacteria) •Twitching motility: slow, jerky process using fibers (pili) that can be extended, attached to a surface, and pulled back to pull along a surface (N. meningitidis, P. aeruginosa) -Polymerization of actin in host cells for propulsion of bacteria into adjacent cells (Shigella dysenteriae, Listeria monocytogenes)

The Bacterial Cytoskeleton Review: What are the cytoskeleton molecules found in animal (human) cells? What are their equivalents in bacteria?

•Homologs of all 3 eukaryotic cytoskeletal elements have been identified in bacteria •Actin (microfilaments)-smaller •Tubulin (microtubulin) •Intermediate filaments •Functions are similar as in eukaryotes—So what are they?

3. Joining of subunits to existing cell Let's do this again- what does transglycosylation mean? Is this how is

-Transglycosidases are enzymes that link the two sugars in the backbone together. -Transglycosylation and transpeptidation reactions, resulting in the respective polymerization and crosslinking of the glycan strands via flexible peptides.

•What are the critical structural and functional properties of the bacterial cell envelope?

-But how do nutrients cross the PM? •Facilitated diffusion: using a protein channel to move particles with a concentration gradient (no energy) •Active transport: using energy to move particles against a concentration gradient -Co-transport mechanisms (symport/antiport) -ATP-binding cassette (ABC) transporters

•How can molecules get out of a Gram-negative cell's periplasmic space then?

-Some move from the periplasm to outside directly (these are known as autotransporters and are rare). -Type V (sec-dependent) -Some use single-step (never entering the periplasm) transport systems. (Type III secretion system) These are known as secretion systems. Type I: Sec-dependent Type III: Sec-independent and form ____________________. See with toxin delivery and related to proteins used in flagella formation! Know how flagella are made.

Gram-negative cells make a gram neg vs gram positive chart What molecule is found in most Gram negative bacteria? Make sure you know abbreviations and what they stand for. What type of molecule is LPS? (future)

-A varying-width periplasmic space containing a very thin layer of peptidoglycan -An outer membrane composed of lipopolysaccharide (LPS) A relatively thin layer of peptidoglycan surrounds Gram-negative bacteria. This slim cell wall may seem like scant protection, but it is supplemented by another structure, the outer membrane (Figure 2.22). For this reason, the plasma membrane of Gram-negative bacteria often is referred to as the inner membrane. The space between the inner and outer membranes is the periplasm. The width of the periplasm is not clear and probably fluctuates; the Escherichia coli periplasm is estimated to be 13-25 nm wide, with the peptidoglycan layer occupying roughly a third of that space. In addition to peptidoglycan assembly enzymes, the periplasm contains a roster of proteins that includes proteins to aid in nutrient uptake and components of protein export systems. The periplasm also can contain oligosaccharides that help the bacteria adjust to changes in the osmolarity of their environment.

•Aside from motility, what other stuff is still left on the outside of bacterial cells? What are the types of pili and what are they made from?

-Adherence molecules to stick to surfaces •Mediated by pili (s. pilus), fibers of pilin protein possess other proteins on their tips for sticking. •A sex pilus is a different structure used for conjugation (sending a DNA plasmid from one cell to another). •Some scientists prefer to use "pili" only for conjugation structures and "fimbriae" (s. fimbria) for adherence.

What do bacteria look like? Know not only the shape but examples of each Be able to identify them from pictures! I may not use these exact ones. Can you recognize the different shapes if given a gram stain or a drawing? If given a shape- what are examples of bacteria that would be classified by this morphology? For example: Treponema pallidum which causes syphilis is a spirochete. Now do this for other shapes.

-Bacteria can take many different shapes (or morphologies). >>Spherical (s.coccus, pl. cocci) >>Rod-shaped (s. bacillus, pl. bacilli) >>Comma-shaped or curved rods (s.vibrio, pl. vibrios) >>Spiral (s. spirillum, pl. spirilla) >>Pleiomorphic (varied shapes)

What is the purpose of a capsule? What type of bacteria more often express capsules and why? Hint: think about disease. Now you see why you need to replace your toothbrush every month or so!

-Capsules can help bacteria form biofilms. •Biofilms provide protection and enhanced survivability in harsh environments. •Examples of biofilms include dental plaque and mold on bathroom surfaces.

What is the purpose of a capsule? Can the same species of bacteria be both capsulated and non-capsulated? What term do we use to describe them? Can you see the capsule in Figure A?

-Capsules: thick layer of polysaccharides surrounding some cells •Can provide adhesion, defense against host immunity, protection against drying out (desiccation)

LPS from gram-negative cells can be harmful! You need to know what each part of LPS and how it contributes to bacteria's ability to induce immune responses. so is lyozyme in tears protective against G-bacteria?

-Lipid A portion induces a strong inflammatory response. -O (outer) side chain of polysaccharides can vary dramatically (and even be changed by the microbe to evade host immune responses). The structure of lipopolysaccharide The lipid A component, which is structurally conserved between Gram-negative species, contains the hydrocarbon chains composing the hydrophobic interior of the membrane. The sequence of the sugar building blocks in the core polysaccharide is less conserved between species, while the sugars in the outer O side chain can vary even more extensively in sequence, even between variants of a species. Representative sugars are shown here. We noted earlier that lysozyme is one of the human body's defenses against bacteria, but the outer membrane protects Gram-negative bacteria from its attack.

Bacterial flagella all have a common molecular structure a. Bacteria with polar flagella, such as Spirillum volutans, only have flagella emerging from the ends of the cell. b. Peritrichous flagella, as shown on this Salmonella enterica cell, are distributed across the cell surface. c. The flagellum is embedded in the cell wall and plasma membrane through the basal body. The flagellar motor, a protein complex embedded in the plasma membrane, rotates the flagellum

-Motility from flagella: spiral, hollow, rigid filaments extending from the cell surface -Locations and number vary from species to species. Bacterial cells sense many chemicals in their environment through receptors in the plasma membrane that communicate indirectly with the flagellar motor through intermediary proteins in the cytoplasm. This process of using chemical signals from the environment to direct motility is called chemotaxis (Figure 2.28). The cytoplasmic control system ensures that all of the motors in cells with multiple flagella tend to rotate in the same direction. If the chemoreceptors detect a higher attractant concentration as a bacterial cell moves, then the flagellar motors tend to continue rotating in the direction causing forward motion. The bacterium, in other words, moves up the gradient of the desired nutrient. This type of movement is referred to as a run. If the concentration of an attractant is not increasing or if the concentration of a repellent, a chemical the cell wants to avoid, is increasing, the flagellar motor switches more often to the direction that results in a tumble. The tumbling state is transient, and the cell sets off on a run in a new direction.

Step 1: Synthesis of precursors of NAG and NAM

-Peptidoglycan synthesis begins in the cytoplasm -Uridine diphosphate (UDP) is added to N-acetyl muramic acid

Stereoisomers are mirror images of each other d-Alanine and l-alanine have the same chemical formula. The arrangement of the bonds on the central carbon, however, differs. What is the relevance of knowing that NAMs have unusual forms of a peptide?

-Several of the amino acids found associated with NAM in peptidoglycan are usually D forms. -D forms are stereoisomers (mirror images) of the L forms normally found in biological processes. We should note that several amino acids found in peptidoglycan are rarely found in proteins, including meso-diaminopimelic acid, and the D-stereoisomer of several amino acids.

The Nucleoid

-Usually not membrane bound -Location of chromosome and associated proteins -Usually 1 closed circular, double-stranded DNA molecule -Supercoiling is regulated by topoisomerase and nucleoid proteins (different from histones) aid in condensing DNA

Size of bacteria can vary greatly Be able to place microbes in order based on size and KNOW the exceptions. Look at the unites we use for size so be able to convert nm and micron!

-Usually smaller than eukaryal cells (bacteria are oftern .5 to .5 um in length) -Small eukaryal cells are usually >5 um in diameter. Have a general understanding of size of viruses (nm) to bacteria (microns) to what we can see with our own eyes (100 microns)

Gram-positive cells have know that this genera are spore formers (Anthrax) know what endospores are and why they are important. There are special stains that allows us to see endospores.

-a thick outer layer of peptidoglycan -a very narrow periplasmic space -teichoic acids in the peptidoglycan (negatively charged) Some Gram-positive bacteria, such as Bacillus and Clostridium species, can undergo an amazing process of cellular remodeling called endospore formation, which is initiated as a survival mechanism under stressful conditions, such as imminent starvation (Perspective 2.2). The endospores are largely metabolically inert structures that exhibit increased resistance to many harsh environmental conditions, such as desiccation, UV light exposure, and high temperatures.

Cell Wall Functions

-maintains shape of the bacterium •almost all bacteria have one -helps protect cell from osmotic lysis and toxic materials -may contribute to pathogenicity •Peptidoglycan (murein) -rigid structure lying just outside the cell plasma membrane -two types based on Gram stain •Gram-positive: stain purple; thick peptidoglycan •Gram-negative: stain pink or red; thin peptidoglycan and outer membrane

There are two main types of bacterial cell walls

1. Describe peptidoglycan structure 2. Compare and contrast the cell walls of typical Gram-positive and Gram-negative bacteria. 3. Relate bacterial cell wall structure to the Gram-staining reaction.

2.6 Fact Check

1. Identify the taxonomic groups used to classify bacteria. 2. Each bacterium's scientific name is binomial and based on which taxonomic categories?

2.4 Fact Check

1. What are the key components of the bacterial plasma membrane, and what are its functions? 2. Describe the structure and function of the bacterial cell wall. 3. Differentiate between Gram-positive and Gram-negative bacterial cell envelopes. 4. Describe the role of porins and TonB-dependent receptors in Gram-negative bacteria. 5. What is the type III secretion pathway?

2.5 Fact Check

1. What are the roles of bacterial flagella, and how do bacterial flagella differ from archaeal and eukaryal flagella? 2. Describe how chemoreceptors and flagella are involved in the process of chemotaxis. 3. Explain the processes of gliding motility and actin-based motility. Provide examples. 4. Differentiate between the terms pili and fimbriae. 5. Speculate on some potential benefits to bacteria of biofilm formation.

Bacteria can also assume multicellular organizations.

>>Hyphae (branching filaments of cells) >>Mycelia (tufts of hyphae) >>Trichomes (smooth, unbranched chains of cells)

Plasmids

>Extrachromosomal DNA -found in bacteria, archaea, some fungi >exist and replicate independently of chromosome -inherited during cell division >Several types of plasmids provide various functions -conjugative plasmids (these are passed via sex pili) -R plasmids (resistance against antibiotics)

Ribosomes

>Ribosomes are involved in protein synthesis -Facilitate joining of amino acids -Relative size expressed as S (Svedberg) -Reflects density: how fast they settle when centrifuged >Prokaryotic ribosomes are 70S -Made from 30S and 50S -16S small subunit -23S and 5S in large subunit >Eukaryotic ribosomes are 80S

You are responsible for knowing the abbreviations PMF. How are PMF used?

>The PM can also be used for capturing energy. •Embedded electron transport chains can help create proton motive force (PMF). •Can be used for respiration/photosynthesis (more in Chapters 6 and 13) •Can be used to derive energy for motion (flagella) >The PM can hold sensory systems. •Proteins in the PM can be used to detect environment changes. •The cell can use the detected changes to alter gene expression to respond.

Permeability of the Plasma Membrane to Water

A fundamental property of the plasma membrane is its differential or selective permeability. Small and uncharged molecules, such as O2 and CO2, can diffuse freely across a phospholipid bilayer. Larger compounds, or molecules that are more polar or charged, cross the membrane less readily. Although they are polar, water molecules are small enough to cross phospholipid bilayers. Their transit may be facilitated, and perhaps regulated, by protein channels called aquaporins. Water concentration differences between the interior and exterior of the cell create an osmotic gradient. The cell is in a hypotonic solution when the cytoplasm has a higher solute concentration than the external environment, causing water to move into the cell. Because phospholipid bilayers are quite flexible, the cell expands, like a balloon being inflated. Without a structure to provide stability, cells would risk explosive destruction. The cell wall prevents this outcome. Conversely, the cell is in a hypertonic solution when the cytoplasm has a lower solute concentration than the external environment, causing a net loss of water from the cell. Like a deflating balloon, cells risk structural collapse in a hypertonic solution. Once again, a protective cell wall helps to prevent this outcome. Animation: Osmosis

Surface Arrays ("S-layers")

A regular, crystalline-like layer of protein referred to as a surface array or S-layer (Figure 2.35) has been observed in many bacterial cells, both Gram-positive and Gram-negative. As you might imagine, making this protein coat represents a tremendous investment of resources by the cell. Surface arrays are generally thought to act like a microbial suit of armor, providing protective functions such as: Prevention of infection by bacteriophages (viruses that attack bacteria). Prevention of penetration by predatory bacteria such as Bdellovibrio. Prevention of attack by a host's immune system. As we can see from this brief list, the presence of an S-layer can be quite useful and worth the required expenditure of resources for its synthesis. When an S-layer is not needed, such as when bacteria are grown in pure culture, its synthesis often stops and it is lost

Test your Understanding the student would see all purple cells and not be able to identify the gram-negative cells clearly as they would look clear.

A student carried out the Gram stain on a culture of Gram-negative cells but forgot to add the safranin in the last step. What would the student see when she examined the stained cells under the microscope? Explain.

Endospores

A thick-walled protective spore that forms inside a bacterial cell and resists harsh conditions. Endospores have dramatically thickened cell envelopes (Figure B2.6), with additional layers of protein outside the peptidoglycan. This thick protective coat makes them resistant to desiccation and chemical attack. Endospores shut down their metabolism completely and compress chromosomal DNA tightly with protective proteins. As a consequence, they are incredibly durable and can remain viable for many years. Exactly how long endospores can survive is not known—thousands of years seems likely. Some research even suggests that endospores may be capable of survival for millions of years.

Section 2.1: What do bacterial cells look like?

Bacterial cells can have several distinct morphologies: spheres (cocci), rods (bacilli), curved rods (vibrios), and spirals (spirilla). Some bacterial species are pleiomorphic; they exhibit highly variable shapes. In many bacterial species, cells can stay attached after cell division. Within a given species, cells may form clusters, chains, and branching filaments. Some bacteria grow more complex multicellular arrangements, forming hyphae, mycelia, or trichomes. Bacterial cells range in size from 0.2 μm to 700 μm in diameter, but most are in the range of 0.5-5 μm.

fimbriae and pili: short, thin, hairlike, protein appendages

•Fimbriae allow attachment •Pili •Facilitate transfer of DNA from one cell to another •Gliding motility •Twitching motility •Sex pilus •used to join bacteria for DNA transfer •longer, thicker, less numerous

Section 2.3: What kinds of internal structures help to organize bacterial cells?

Bacterial cells contain cytoskeleton-like structures that are important for cell shape and division. The dynamic Z-ring, formed from the tubulin-like FtsZ protein, guides cell division. It is associated with the inner face of the plasma membrane and, as it undergoes depolymerization, causes the membrane to constrict. Actin-like proteins, such as MreB, form filaments that control cell shape in many bacteria. A related protein, ParM, ensures that plasmids are evenly distributed during cell division. Other cytoskeletal proteins may have roles in distribution of chromosomal DNA in bacteria.

What are the critical structural and functional properties of the bacterial cell envelope? What is a hopanoid? What is its function? What is the equivalent in eukaryotes? Fungi? Start making comparison tables between bacteria and eukaryotic organisms!

Bacterial membranes lack sterol lipids, such as cholesterol, which are major components of eukaryal membranes. Some bacteria produce sterol-like molecules called hopanoids (Figure 2.10). Like sterols, hopanoids are largely planar molecules that are thought to stabilize the plasma membrane. Although only about 10 percent of bacteria produce hopanoids, these molecules often are quite abundant in soils and sediments because they are very stable. The PM may have sterol molecules called "hopanoids" in it to help with stability across the temperature ranges. Bacteria membrane lack cholesterol but may contain hapanoids. Hopanoids, such as this bacteriohopanetetrol, are present in the membranes of some bacteria. These molecules are structurally similar to cholesterol and provide some of the same functions, strengthening the membrane.

doesn't actually look like a ring So Donze chemistry: does the beta lactam ring look like a ring? The "square" structure should always help you identify most beta-lactams!

Beta-lactam ring

Remember the fluid mosaic model? All of this should be review. Keep this in mind when we talk about the environment in which you find mircobes.

Biological membranes are not static structures. Lipids move relatively freely within the two layers of the membrane. The fluidity of the membrane depends on the types of lipids present, environmental factors such as temperature, and the presence of other molecules associated with the membrane. Biological membranes are far from pure lipid structures; roughly half of the mass of the bacterial plasma membrane is protein. Many proteins either cross or are integrated into the membrane bilayer (see Figure 2.9). These proteins have hydrophobic surfaces that interact with the interior of the membrane, and hydrophilic domains exposed to the cytoplasmic or outside environments. Key functions of the plasma membrane proteins include: - The control of access of materials to the cytoplasm through differential permeability. - The capture and/or storage of energy through photosystems, oxidative electron transport, and maintenance of chemical and electrical gradients -Environmental sensing and signal transduction.

You need to know the steps in cell wall synthesis in detail! 1.Where does cell wall synthesis start? 2.What is UDP? What is its purpose? Does it look like (UTP? Hmmmm) 3.UDP gets added to________________. 4.This UDP-______________ gets added to bactoprenol. 5.What is unique about bactoprenol? 6.What is added next? 7.Then what uniquely happens that isn't understood? 8.Now where is the NAG-NAM subunit? 9.What do autolysins do? Why is this important? 10.What are the enzymes involved and what linkages are they responsible for? 11.What specific type of linkage is found between NAM and NAG sugar? 12.How are the layers of the cell wall connected?

CW Synthesis 1. Outside the cell plasma membrane in the cytoplasm. 2. UDP synthesizes NAM. 3. NAM 4. phosphate 5. holds it onto the cytoplasmic face of the membrane 6. NAG is added to NAM 7. bactoprenol flips sides. 8. in the hydrophobic periplasm 9. autolysins

Section 2.5: How do structures on the surface of bacterial cells allow for complex interactions with the environment?

Cell surface structures allow bacteria to interact with the environment in many important ways. These structures allow them to move, stick to surfaces, sense the environment, and acquire nutrients. Flagella are cell surface structures that propel bacterial cells through liquid environments. Bacteria with flagella may be monotrichous, lophotrichous, or peritrichous. Bacterial, archaeal, and eukaryal flagella are structurally and evolutionarily distinct. Chemotaxis is used to direct bacterial motility in response to concentration gradients of attractants and/or repellents and involves chemoreceptors. Other forms of taxis also exist. Some bacteria utilize non-flagellar-based motility, including pilus-mediated twitching, gliding motility across solid surfaces, and actin-based motility inside mammalian host cells. Adherence can be mediated by cell surface proteins, pili, stalks, fimbriae, or sex pili. Some bacterial cells are surrounded by a polysaccharide capsule, whereas other cells are surrounded by a less well-defined slime layer. The term glycocalyx can refer to both capsules and slime layers. Strains may differ in their ability to adhere. Surface adhesion can be the first step in the creation of biofilms, or adherent communities of microorganisms. Crystalline-like surface arrays, or S-layers, surround some bacterial cells.

Bacterial movement by straight line "runs" and random "tumbles" Peritrichous flagella rotating in one direction will bundle together and cause the bacterium to move in a straight direction. When the direction of rotation of the flagella is changed, the bacterium tumbles and randomly reorients. When flagellar rotation switches again, the cell takes off in a new direction. Bacterial cells with a single polar flagellum are pushed or pulled depending on the direction of flagellar rotation.

Chemotactic attractants are often molecules that can be productively metabolized (e.g., sugars and amino acids), whereas chemicals that could damage the cell (e.g., acids and alcohols) are more likely to be interpreted as repellents. Bacterial species often have many different chemoreceptors, receptors that detect these attractants and repellents. The different chemoreceptors, in turn, each have different specificities. They interact with, and thus signal a response to, different chemical signals.

Capsules know what capsules are made from and their purpose. what does capsules have to do with pathogenesis of bacteria? How do capsulated bacteria escape the immune response?

Colonies of some pathogenic bacteria, such as Streptococcus pneumoniae, a cause of bacterial pneumonia, and Neisseria meningitidis, a major cause of bacterial meningitis, have a smooth, glistening appearance. A thick layer of polysaccharides, called a capsule, surrounds these cells (Figure 2.33). Many pathogens use capsules to shield themselves from host defense systems, particularly phagocytic host cells that are capable of engulfing and destroying bacteria. Phagocytic cells often cannot recognize encapsulated microbes as foreign invaders worthy of destruction. Capsules also may serve other purposes. Because most capsules are made of hydrophilic material that retains water, capsules may be useful for surviving desiccation. Some species of bacteria have a less well-defined outer layer, known as the slime layer. Again, this structure typically consists of polysaccharides and can serve several purposes. The term glycocalyx can refer to both capsules and slime layers

KNOW this one! Why is this variation important? What does cross-linking provide to the bacteria cell wall? What happens if this were to be disrupted?

Crosslinking between the peptide chains is very important for the strength of the peptidoglycan network, and bacterial species can achieve this crosslinking through different mechanisms. For example, in E. coli and most other Gram-negative bacteria, the fourth amino acid attached to one NAM residue is directly linked to the third amino acid present on another NAM residue. In most Gram-positive species, the tetrapeptide chains are crosslinked via a short peptide interbridge. As shown in Figure 2.15, S. aureus uses a pentaglycine interbridge. The exact composition and length of the interbridge varies between species. >While the amino acids in the peptide on N A M can vary from species to species, the way the peptides are crosslinked in the CW can also vary.

Bacterial Cell Organization -Common Features

Draw out cells and label them- write out what each structure does and if it applies what makes up that structure. Doing this will also let you compare cells from different domains.

why crystal violet? because itstains all bacteria cells first iodine is known as a mordant- meaning it makes the crystal violet stick together stabilizing it what does alcohol do? decolorizes the cell. extracts the crystal violet from the cell. stain complex removed from gram negative but not gram-positive cells why would it be lost from gram negative and not gram positive? because gram-positive has a thicker cell wall why a thick cell wall? teichoic acids? what color does safranin leave behind and why don't you see it in Gram positive if they are also stained with it? in gram positive the crystal violet primary stains remains when applying alcohol (decolorizer) whereas the purple from gram-negative cells were removed (loses crystal violet) but it is stained with safranin which has a pink or light red color.

First, the specimen is placed on a microscope slide. Then the specimen is fixed, or attached, to the slide by heating the underside of the slide. The cells first are stained with crystal violet, the primary stain—virtually all bacteria are stained by crystal violet. The cells then are stained with iodine, which interacts with the crystal violet, forming a larger, more stable complex. The slide then is immersed in an alcohol, or an alcohol/acetone mixture, often referred to as a decolorizer. Exposure to this reagent results in the loss of the crystal violet-iodine complex from Gram-negative cells. Crystal violet-iodine complexes remain in Gram-positive cells because of their thick cell walls. The slide then is dipped into a counterstain, safranin. This final stain provides a light red or pink color to cells, thereby allowing the viewer to see cells from which the primary stain was removed. The stained cells can be viewed with a light microscope (Figure B2.5). Interpretation Gram-positive = Purple (retains the crystal violet primary stain) Gram-negative = Pink or light red (loses the crystal violet but is stained with safranin)

know sec III secretion system

For Gram-negative bacteria, secreted proteins require special export systems to cross the outer membrane. Research has revealed that several distinct mechanisms seem to be involved in secretion. Often the general secretory pathway (see Figure 2.13) is first used to transport the protein into the periplasm. Some proteins, called "autotransporters," then catalyze their own transit across the outer membrane, but most proteins seem to need the assistance of export systems.

Motility from Flagella you will need to be able to describe how flagella is made in bacteria. flagella are very different between all three domains. as such make sure you know the characteristics of each and how they differ. Know how flagella are organized and the terms used. Know the structure of the flagella. they have different types of movement: bacterial flagella rotate know the different names for how flagella are organized on bacteria

For active movement, bacteria commonly use flagella, spiral filaments that extend from the surface of the cell and rotate in order to propel the cell (Figure 2.26). Some bacteria have flagella only at the ends of the cell, in which case they are called polar flagella. Monotrichous bacteria have only a single polar flagellum (trich being Latin for "hair"). Lophotrichous (lopho being Greek for "tuft") bacteria have more than one flagellum at one or both ends of the cell, and peritrichous bacteria have multiple flagella spread all over the surface of the cell.

The shape of bacterial cells is determined by the organization of the cell wall, the semi-rigid structure surrounding the cell. Morphology is a fairly reliable feature of most bacterial species. For instance, Escherichia coli cells generally are straight rods, Vibrio cholerae cells are curved, Staphylococcus aureus cells are spherical, and Treponema pallidum cells are long, thin spirals. However, because many bacterial species have similar morphologies and because environmental conditions and stresses can sometimes cause changes in bacterial morphology, physical appearance is seldom conclusive for identifying bacterial species.

For many bacterial species, like E. coli, individual cells typically remain separate from each other. The cells of other bacteria stay physically connected after they divide. For example, the rod-shaped cells of Bacillus anthracis, the cause of anthrax, and the spherical cells of Streptococcus pyogenes, the cause of strep throat, often are seen in long chains. In contrast, Staphylococcus cells tend to form clusters rather than chains (see Figure 2.1). Some bacteria do not exhibit regular shapes but may exhibit highly variable cell morphologies. These bacteria are referred to as pleiomorphic. Examples of pleiomorphic bacteria include members of the genus Mycoplasma, which do not make a cell wall and, as a result, do not have a regular shape (Figure 2.2).

Proteus mirabilis: A Swarming Bacterium

Gram-negative bacteria such as Caulobacter and Hyphomonas attach to surfaces using a stalk. Because the stalk extends the cell envelope and contains nutrient transport systems, it increases the surface-to-volume ratio of the cell and boosts nutrient uptake efficiency. Stalked bacteria are common in aquatic habitats, particularly where nutrient concentrations are very low. The stalks, researchers hypothesize, serve as extensions of the cell surface that can aid in nutrient acquisition due to their relatively high surface-to-volume ratio.

What is the nucleoid and how is the DNA condensed? What cellular processes are not impeded by a nuclear membrane? What are the benefits?

If an average-size bacterial chromosome were stretched out in linear form, it would be hundreds of times longer than the bacterial cell in which it resides. To pack the DNA into a manageable form, bacteria use several strategies. Cations, such as Mg2+, K+, or Na+, shield negative charges on the sugar-phosphate backbone of each strand of the DNA helix, allowing the DNA molecule to pack more closely. Also, molecules of small, positively charged proteins bind to the chromosome to help maintain the condensed structure of the nucleoid. Finally, the topology (the arrangement) of the DNA molecule is adjusted by topoisomerases, enzymes that encourage the chromosome to coil upon itself (supercoiling) in order to collapse it into a more compact mass.

how is bacteria named and ruled?

Important! Remember that most microbes still can't be cultured! oWhat we can grow, we name according to the standard binomial system. •Species: group of strains sharing common features while differing considerably from other strains •Genus: group of closely related species Above the genus level, we use family, order, class, phylum, and finally domain.

What are inclusion bodies? No Membranes!!!! Just areas that have certain functions. Often will show up using TEM or markers against enzymes as seen in this image. Do all bacteria have the same inclusion bodies? Look at the images I have given you-do all of these images look similar-why or why not?

In Prokaryotes ONLY. It is used to survive when nutrient is depleted. --NOT ALL HAVE •Microcompartments •Not bound by membranes but compartments for specific functions •Some have proteins that serve as boundaries

called a run counterclockwise

In this case, rotation of the flagellar motor in one direction (counterclockwise for E. coli and related Gram-negative bacteria) causes the flagella to bundle together and push the cell forward. Rotation in the other direction causes the flagella to fly apart and point in different directions, which causes the cell to tumble in place (Figure 2.27). This tumbling randomly reorients the cell so that when flagellar rotation is reversed once again, the cell sets off in a new direction.

What is Sec YEG? What is it and what does it do?

It is a membrane chennel complex along the plasma membrane and is the gate/exit/entrance (as long as SecA binds to it) letting the polypeptide out.

Discuss w/ secretion. Know what ABC stands for and what these transporters do.

One of the most common types of active transport involves the ABC transporters. ABC stands for "ATP-binding cassette," reflecting the fact that these proteins include a nucleotide-binding domain by which ATP is hydrolyzed to provide the energy for transport.

What kinds of internal structures help to organize bacterial cells? Ok- so many forget what eukaryotes use for the cytoskeleton- you may want to review those (ch3) to make connections. So what happens if you delete one of the these cytoskeletal proteins? Review: What are the cytoskeleton molecules found in animal (human) cells? What are their equivalents in bacteria?

Just as our skeleton provides a structural framework for organizing our organs and tissues, the cytoskeleton is important for the internal organization of cells. In bacteria, a few key proteins have been discovered that form filamentous structures that organize cell growth and division (Figure 2.8). The major cytoskeletal proteins discovered so far in bacteria are located in the cytoplasm, but the key role of these proteins involves interacting with the plasma membrane and the cell wall, structures that will be discussed in the next section. The cytoskeleton is a series of internal proteins that assist in keeping everything in (or moving it to) the right locations in cells.

know the LPS abbreviation. what does it do? which type of bacteria you find on it? know three parts of LPS!!!

LPS molecules have three distinct parts: lipid A, a core polysaccharide, and the O side chain. The hydrophobic portion of LPS constitutes the outer layer of the membrane bilayer, while the polysaccharide portion is exposed on the cell surface. The lipid A component, which is very similar in all Gram-negative bacteria, includes the hydrophobic hydrocarbon chains. The organization of the core polysaccharide also is conserved, although the identities and sequence of the sugar building blocks can vary somewhat between species. In contrast, the sugars in the O (outer) side chain can vary substantially, even between different variants of bacteria that clearly are members of the same species. Like the LTA of Gram-positive bacteria, LPS from invading Gram-negative bacteria, especially the lipid A component, triggers a powerful inflammatory response in the human body that is responsible for many of the symptoms of infections.

What if you delete ParM?

Like MreB, the ParM protein forms actin-like filaments. Experiments have shown that ParM filaments aligned with the long axis of the E. coli cell are responsible for moving copies of plasmids, extrachromosomal molecules of DNA, to opposite sides of the cell. This separation ensures that copies of the plasmid will be found on either side of the division site, so they will be passed along to each of the new cells (see Figure 2.8c).

Why does S. simulans carry this enzyme?

Lysostaphin, an enzyme discovered in Staphylococcus simulans, cuts the pentaglycine crossbridge of S. aureus and some related species (see Figure 2.18b). The cloned lysostaphin gene has been engineered experimentally into dairy cattle to generate cows that suffer much less frequently from mastitis, a painful udder infection usually caused by S. aureus. Such genetically engineered animals, resistant to various infectious organisms, may become common in our future.

Where is the lysozyme made in our bodies??

Lysozyme hydrolyzes the β-1,4-glycosidic bond between NAG and NAM, profoundly weakening the cell wall (Figure 2.18). If a bacterium encounters lysozyme under isotonic conditions (water leaves the cell at the same rate it enters), then it may survive as a protoplast, the term given to the generally fragile form of the cell with its protective cell wall removed.

How? What is the enzyme name? Why is the beta lactam ring structure important to inhibit cell wall synthesis?

One way for bacteria to become resistant to an antibiotic is to produce enzymes that modify or destroy the antibiotic. β-Lactamases hydrolyze the C—N bond in the β-lactam ring of antibiotics, such as penicillin and cephalosporin (see Figure 2.19A). This ring structure is necessary for these antibiotics to bind to their target. Cleavage of the ring inactivates the antibiotics. Soil fungi have produced these antibiotics for millions of years. In response, some bacteria long ago evolved β-lactamases to combat these fungal weapons.

adherance what structures are used for adherence?

Many bacteria have fibers called pili sticking out from the cell surface. These fibers are constructed from a single type of protein subunit, called pilin. As with flagella, some bacteria display pili only at the cell poles, while others have them spread across the entire cell surface. Although we mentioned pili in the context of motility above, the more common purpose of pili is to allow bacteria to attach to surfaces, including other cells. The tip of the pilus usually contains distinct proteins that serve as adhesins designed to bind specific molecules on target surfaces. Pili can be very important for pathogenic microorganisms because adherence to target cells in the host is often an early step in the infection process. Even in non-pathogens, pili that mediate surface attachment can be important for persistence in any environment where there is fluid flow. A special type of pilus, the sex pilus, or conjugal pilus, is used to connect bacterial cells for the transfer of plasmid DNA. Genes for constructing sex pili are found on some large plasmids that have evolved to move between bacteria. Both adhesive and conjugal pili may be found on the same E. coli cell. Some microbiologists prefer to use the term "pilus" exclusively for the structures involved in conjugation, and they assign the alternative term fimbria (plural: fimbriae) to cell surface fibers used for adhesion and lacking the associated intracellular machinery for conjugal plasmid transfer (Figure 2.31).

Connections

Many bacterial pathogens cause disease by secreting proteases, toxins, and other factors that damage host cells. As we will see in Section 21.1, some Gram-negative pathogens use the type III secretion system to deliver toxins directly into target cells.

proteins around nucleoid

Many other proteins are located around the nucleoid, including enzymes involved in replicating and transcribing the DNA (DNA and RNA polymerases, respectively) and proteins that control gene expression. As RNA polymerases produce messenger RNA, ribosomes assemble on the mRNA and begin producing polypeptide chains. Ribosomes are thus indirectly associated with the nucleoid in living bacterial cells,

What are the critical structural and functional properties of the bacterial cell envelope?

Most bacterial cells also contain a semi-rigid cell wall made of peptidoglycan and some bacteria contain a second membrane, the outer membrane. Collectively, these layers are referred to as the cell envelope. The plasma membrane, also referred to as the cell membrane or the cytoplasmic membrane, is a bilayer composed primarily of phospholipids. The structure of these phospholipids is amphipathic, meaning that they have a polar portion and a non-polar portion. Most cellular membranes contain mixtures of phospholipids that can vary in the chemical structure of the polar head group, as well as in the length of the non-polar hydrocarbon tail and the frequency and position of double bonds within the hydrocarbon chain (see Figure 2.9). The polar head is hydrophilic and thus interacts with water inside and outside the cell, whereas the non-polar hydrocarbon chains associate to form the interior of the membrane. All cells have a plasma membrane (PM). -Separates the interior of the cell from the external environment -Usually composed of a phospholipid bilayer with embedded proteins

Step 3: NAG is added to the peptidoglycan subunit on the NOW the second subunit is added and then the most amazing biology-defying thing occurs...

NAG is then added to the NAM. -Bactoprenol flips sides.

So the CW is critical, but are all CW structures the same? useful in lab know the steps of gram staining and what would happen if you accidently didn't do one step then it would all look purple or clear-- bacteria wouldn't be clearly identified

NO! -A stain method developed in 1884 by Hans Christian Gram can separate many microbes into one of two classes. 1. Gram Negative 2. Gram Positive The Gram stain is an excellent example of a differential stain; bacterial species differ in their response to the staining procedure, depending on the structure of the cell envelope. This staining method classifies bacteria into two major groups: Gram-positive and Gram-negative. gram-positive is purple gram-negative is pink

Which subunit do you find this on? What is its function in forming the cell wall. How is this structure unique between types of bacteria? Know what NAM and NAG are. What types of molecules are NAG and NAM? BIOL 1201: What type of chemical bonds link two sugars? BIOL 1201: What type of chemical bonds link amino acid?

Peptidoglycan forms a net-like structure composed of a glycan backbone made up of alternating molecules of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), connected by β-1,4-glycosidic bonds. The N-acetylmuramic acid carries a short peptide chain that is used to crosslink peptidoglycan strands, creating the protective network surrounding the cell. The sequence of amino acids in the peptide chain can vary somewhat between species. Escherichia coli, for example, has a slightly different sequence than Staphylococcus aureus (Figure 2.15). In most bacteria, the first amino acid of this peptide chain is L-alanine (L-Ala), whereas the fourth and fifth amino acids typically are D-alanine (D-Ala). As we will see later in this section, the fifth amino acid is removed when crosslinking occurs. Gram-negative bacteria generally contain D-isoglutamate (D-Glu) in the second position, whereas Gram-positive bacteria generally contain D-isoglutamine (D-GluNH2) in this position. Amino acids at the third position vary more widely. >Each peptidoglycan disaccharide subunit is •N-acetylmuramic acid (N A M) with a small peptide chain >The peptide varies by species. •N-acetylglucosamine (N A G)

what are biofilms?

Polysaccharide coatings on microorganisms can aid in adherence of the microbes to surfaces. In wet habitats, microorganisms can form attached communities called biofilms (Figure 2.34). Biofilms often contain more than one kind of microorganism. A biofilm may be initiated by a bacterium sticking to a surface using pili or some other attachment mechanism. The number of adherent cells grows as a result of division or aggregation from the surrounding environment. As the cells pile up, extracellular polysaccharides form a matrix holding the biofilm together. Over time, the biofilm may get deeper and spread outward, developing into a complex structure with distinct microenvironments. Biofilms represent complex communities in which various members of the community benefit from the presence of the other members. We will examine in Section 14.1 how individual members of a biofilm community contribute to and benefit from the association with other members.

What is Sec A? What does it do? Can you start putting the Sec-pathway in an order yet? So what is SecA really- another general name? If SecA was missing- what would happen to the protein? Where would you find it. Ask this of all of the components for the Sec pathway.

SecA binds to SecYEG, a membrane channel complex and facilitates movement of the polypeptide through SecYEG channel. SecA uses energy deprived from hydrolysis of ATP

What is Sec B? What is the general term for molecules who escort proteins? What does it bind to and why?

SecB brings to the nascent polypeptide as it leaves the ribosome, preventing it from folding into the cytoplasm and delivering it to SecA.

Magnetosomes: organelle associated with direction finding Be sure to read the extra information in Perspective Box 2.1 to better understand magnetosomes and their formation in bacteria!

Several examples of membrane-bound organelles also exist within bacteria. Magnetosomes, the magnetite-containing particles are a rare example of a membrane-enclosed cytoplasmic organelle in a bacterial cell. Some bacteria in the Planctomycetes phylum contain an intracellular membrane-bound compartment, the anammoxosome, where ammonium is oxidized for energy. Magnetosomes are cool! Only certain species of bacteria have them. They are membrane enclosed.

produce air-filled gas vesicles that provide buoyancy to the cells -- this is an example of a cell structure that would not be found for example in routine soil bacteria In addition to what each inclusion body they have- if a bacteria lives in a certain environment- what cytoplasmic structure would they need. Think about cyanobacteria (aquatic and produce oxygen)

Some aquatic bacteria, for example, produce air-filled gas vesicles that provide buoyancy to the cells. These gas vesicles can regulate the cell's position in a water column in response to light or nutrient levels. Cyanobacteria also can produce carboxysomes that contain the key enzymes involved in the conversion of inorganic carbon into organic matter. cytoplasmic organelles- are NOT membrane enclosed

Wait a minute-- What about antibiotic resistance? What is the name of the enzyme that destroys beta-lactam rings?

Some bacteria can produce an enzyme to destroy the critical B-lactam ring structure. But we can add a second drug to inhibit that enzyme and restore the first drug's efficiency!

non-flagellar motility what type causes twitching? (not all do.)

Some forms of bacterial motility do not depend on flagella. During gliding motility, some non-flagellated bacteria, such as myxobacteria and cyanobacteria, slide smoothly over surfaces. The mechanism of gliding motility is not well understood, and there may be more than one mechanism. Other bacteria that exhibit surface-dependent motility rely on fibers called pili that help the bacteria move along surfaces by a slow, jerky process called twitching motility. The pili bind to the surface and appear to be capable of rapid retraction to pull the cell body along. Neisseria meningitidis and Pseudomonas aeruginosa can use this type of motility, although P. aeruginosa usually relies on flagella for motility. Only some pili-forming bacteria are capable of twitching motility, suggesting that this action is not an intrinsic property of all pili.

Peptidoglycan Synthesis Know the steps. How do you think Penicillin binding proteins got their name? What is their other name? transpeptidase

Synthesis of peptidoglycan, the major constituent of the bacterial cell wall, takes place in three stages that occur at three different locations in the cell 1. Synthesis of NAM and NAG UDP-precursors in cytoplasm 2. Transferring precursors to bactoprenol and joining NAG to NAM. 3. Adding new NAG-NAM subunits to cell wall via penicillin binding proteins

Variation in Bacterial Cell Envelope

The Gram stain, a technique for staining bacterial cells developed by Hans Christian Gram in 1884, allows us to differentiate two types of bacteria: Gram-positive and Gram-negative (Toolbox 2.1). Electron microscopic examinations of bacteria have revealed that these two categories of bacteria differ in the organization of the cell wall and the presence or absence of an additional outer membrane. In this section, we will explore these structural differences more fully.

Also, how can nutrients get through the cell walls? What are the purpose of pores? You do not have to know Ton B but only that it is a specialized pore.

The Gram-positive peptidoglycan layer has large pores throughout its matrix. -The Gram-negative cell has porin and TonB proteins in its outer membrane to transfer molecules into the periplasmic space. > Once there, active transport mechanisms can more the molecule into the cytoplasm.

What processed is actin involved in (Hint: action actin) What does this tell you? What shape of bacteria would it then be found in more often if not cocci?

The MreB protein is another important cytoskeletal component in bacteria. MreB is evolutionarily related to actin, a eukaryal cytoskeletal protein. MreB polymerizes into filaments that look strikingly similar to actin filaments, the heart of cytoskeletal structures called microfilaments in eukaryal cells. In bacteria, MreB forms long helical bands underlying the plasma membrane (see Figure 2.8b). MreB is nearly universal in non-spherical bacteria but is rarely, if ever, present in cocci. Experiments with E. coli and Bacillus subtilis suggest that MreB helps guide cell wall formation to produce an elongated cylinder rather than a spherical shape. Scientists are still figuring out how bacterial cytoskeletal proteins help to guide the synthesis and organization of the cell wall, and there is considerable interest in how such processes might be modified in bacteria that form more elaborate shapes, such as vibrios, spirilla (e.g., Magnetospirillum), and hyphae.

There will be a lot here - why do i make you know this? Always think about how are bacterial cells different from outs. This means that we can target difference with antimicrobials to eliminate this infection. What is the alternative name for the cell wall. What is one of its major functions? What happens if the wall is disrupted? What molecules are used to build different types of cell walls. What is the process for building the wall in Gram positive bacteria. How are things transported in and out of the cell wall? You will need to know details here-pay-close attention and write out the steps of cell wall syntehsis. Know the different between cell envelope and cell membrane. What is another name for peptidoglycan?

The bacterial cell wall consists of a highly crosslinked polysaccharide-peptide matrix called peptidoglycan (Figure 2.14). The cell wall is necessary for bacteria to resist damage from osmotic pressure, mechanical forces, and shearing. The organization of peptidoglycan also gives bacterial cells their characteristic shapes. In contrast to the plasma membrane, the cell wall is not a permeability barrier. A few bacteria, most notably the mycoplasmas (see Figure 2.2), survive without peptidoglycan, but these bacteria generally live inside eukaryal host cells where they are protected from osmotic stress. -The bacterial cell wall (CW) is a crucial structure. -It is composed of crosslinked strands of peptidoglycan subunits forming a matrix (similar to a chain-link fence). -It gives the cells their shape and protection from osmotic lysis/mechanical forces.

Section 2.6: How are bacteria categorized and named?

The evolution of bacteria over 4 billion years has resulted in extensive physical, biochemical, physiological, and genetic diversity. A regulated system of nomenclature ensures that bacteria are described and named effectively. The classification system for bacteria is a hierarchical taxonomic system, in which the basic taxonomic level of species refers to groups of strains that share common physical, metabolic, and genetic features. Taxon refers to a named grouping within the classification system. Phyla within the domain Bacteria contain many diverse species that have sequence similarities and may exhibit several common properties. Bacterial taxonomy is dynamic, accommodating newly identified taxa and phylogenetic relationships.

Section 2.2: What is in the cytoplasm of bacterial cells?

The nucleoid, which is primarily composed of chromosomal DNA and associated proteins, is the most massive component of the cytoplasm. The nucleoid is not partitioned from the cytoplasm by a membrane. Chromosomal DNA is compacted significantly, through the action of topoisomerases, which cause supercoiling of the DNA, and interaction with packaging proteins. Ribosomes produce proteins within the cytoplasm. The cytoplasm contains many different soluble organic metabolites and inorganic ions and may contain large inclusion bodies storing various nutrients. Other structures, such as sulfur globules, gas vesicles, carboxysomes, and magnetosomes, exist in the cytoplasm of some bacteria.

Nutrient Transport Through the Outer Membrane gram-negative: porin channels + TonB proteins gram-positive: peptidoglycan

The outer membrane of Gram-negative bacteria is not completely impervious. Various nutrients, for instance, must cross this barrier and then the plasma membrane to be used by the cell. To allow the entry of these essential molecules, the outer membrane of Gram-negative bacteria contains numerous channels. The most common of these channels are the porins. Porin proteins typically form trimeric, or three-subunit, pores through the outer membrane. These pores allow diffusion of small polar molecules, including some nutrients, across the outer membrane from the external environment into the periplasm, where they are available to plasma membrane transport systems (Figure 2.24). For example, glucose enters the periplasm by passing through a porin channel in the outer membrane of E. coli. Once inside the periplasm, glucose is transported into the cytoplasm via an active transport system. Gram-positive bacteria, which have no protective outer membrane, are generally susceptible to vancomycin. Some antibiotics are small enough to pass through outer membrane pores, but under the selective pressure of antibiotic exposure, bacteria containing mutant porins have been isolated. In these cases, the altered porins are apparently even more exclusive, keeping the antibiotics out that normally would have entered the periplasm. Bacteria then rely on high-affinity transporters. In addition to porins, the outer membrane contains proteins called TonB-dependent receptors that bind scarce nutrients (such as iron and vitamin B12) with high affinity and deliver them into the periplasm by active transport.

Know this pathway in detail and write it out as steps. What happens if one of the molecules in this pathway is altered? Later we will discuss other secretion pathways (Type III) that are specific to excreting toxins and such. This is the normal way bacteria secrete molecules they make. •What are the critical structural and functional properties of the bacterial cell envelope? What process lead to protein production? Where are ribosomes found in bacteria? Why is the signal peptide hydrophobic if the cytoplasm is hydrophilic? KNOW PATHWAY DIAGRAM!!! It is a pathway that has to occur in a certain order therefore think about what happens if you remove each component. Delete SecB, delete SecA, delete SecY, delete the signal peptide, delete the peptidase.

The plasma membrane contains proteins for the general secretory pathway, allowing the transmembrane movement of proteins that are needed outside the cytoplasm (Figure 2.13). -Protein secretion and the PM: making proteins and shipping them outside the cell -Uses ATP energy! -Toxins, siderophores, enzymes, etc. To ensure that only the correct proteins exit the cytoplasm, proteins targeted to this secretory pathway are identified by a signal peptide, a short sequence of largely hydrophobic amino acids at the amino-terminal end of the protein. The SecB protein binds to the nascent polypeptide as it leaves the ribosome, preventing it from folding in the cytoplasm and delivering it to SecA. In turn, SecA associates with SecYEG, a membrane channel complex. Using energy derived from the hydrolysis of ATP, SecA facilitates movement of the polypeptide through the SecYEG channel. Once the polypeptide crosses the plasma membrane, a signal peptidase removes the signal peptide and the protein assumes its functional conformation. What does process does the abbreviations sec refer to? What are the first sets to initiate this process? What is the signal peptide? What is a chaperone protein? What does Sec B do? What is a pre-protein? In the cell the preprotein goes from _______________ to __________________________? What are the components of the membrane channel used in the general secretion pathway? Which component is an ATPase? What is the final step which generates a protein that can be folded?

Why can bacteria concentrate protons outside of the plasma membrane? What structure allows this to occur?

The plasma membrane hosts critical parts of the energy-capturing machinery of many bacterial cells. These include cytochromes and other membrane-soluble electron carriers that form the electron transport system of microorganisms. These molecules are crucial components of two important processes: cellular respiration and photosynthesis. In cellular respiration, organic molecules are oxidized and the released energy is used to drive the synthesis of ATP. In photosynthesis, light energy is captured and used to reduce inorganic carbon, forming organic molecules. Both processes rely on the movement of protons from the cytoplasm, across the plasma membrane. As they accumulate outside the plasma membrane, these protons create concentration and charge gradients that combine to create a proton motive force. We will discuss the importance of this proton gradient more fully in Chapter 6. For now, let's note that the proton gradient is a very useful energy source.

Section 2.4: What are the critical structural and functional properties of the bacterial cell envelope?

The plasma membrane is a phospholipid bilayer in which many proteins and, in some species, hopanoids are embedded. This membrane mediates many critical cellular processes, including nutrient transport, energy metabolism, environmental sensing, and protein secretion. Protein channels called aquaporins regulate the transit of water across the plasma membrane. Active transport systems, like the ABC transporters, aid in transporting nutrients across the membrane. These molecules may cross the membrane through symport or antiport mechanisms. The plasma membrane contains crucial components of two energy-capturing processes, cellular respiration and photosynthesis. Protein secretion involves a general secretory pathway and the presence of signal peptides on proteins targeted for secretion. The bacterial cell wall, which lies outside the plasma membrane, is composed of a cross-linked network of peptidoglycan that determines shape and provides mechanical strength and protection for bacterial cells. Lysozyme, an enzyme found in human tears and mucus, hydrolyzes specific bonds within the peptidoglycan. Lysostaphin, an enzyme produced by Staphylococcus simulans, affects the peptidoglycan of Staphylococcus aureus. Antibiotics in the β-lactam family also attack peptidoglycan. β-Lactamases destroy these antibiotics, providing bacteria with resistance to them. There are two major types of cell envelopes in bacteria, which can be distinguished by the Gram stain. Gram-positive bacteria have a thick cell wall composed of multiple layers of peptidoglycan, along with teichoic and lipoteichoic acids. Some Gram-positive bacteria form structurally modified, metabolically inactive cells called endospores under stressful conditions. Gram-negative bacteria have an additional outer membrane outside a relatively thin layer of peptidoglycan. The space between the plasma and outer membranes of Gram-negative cells is called the periplasm. The outer membrane of Gram-negative cells contains lipopolysaccharide (LPS) and transport systems such as porins and TonB-dependent transporters for nutrient passage. The type III secretion system is used to transport some materials across the outer membrane

We will discuss this with a very specific examples like chemotaxis and biofilms. It is the receptors in the membrane (integral proteins that does this)

The plasma membrane senses the environment around bacterial cells with sensor proteins that transduce signals to cytoplasmic response systems.

How does the CW actually form? what is UDP and why use it? Bactoprenol is a unique lipid. You need to know the name and what it does. follow the order + write it down this is so cool-think about it. A lipid flips a large hydrophilic molecule across the hydrophobic membrane So here is the chicken and egg- it is attached to an existed cell wall. So pay attention to the other enzymes and what has to happen to make room for growth and then to incorporate the new subunit.

The production of peptidoglycan starts in the cytoplasm, where enzymes first link NAM and uridine diphosphate (UDP). This molecule then is linked to a pentapeptide (Figure 2.17). The UDP-NAM-peptide complex is coupled to a lipid carrier called bactoprenol, which holds it on the cytoplasmic face of the membrane. A second sugar, N-acetylglucosamine, then is added to form the basic disaccharide-peptide unit. This complex then pulls a remarkable trick, still not well understood, in which the bactoprenol carrier flips the disaccharide-peptide complex to the other side of the membrane. Only there can the new subunit be attached to the terminal sugar of a growing peptidoglycan chain. Other enzymes, most notably transpeptidase, then crosslink the pentapeptide precursor to another strand. This transpeptidation reaction results in the removal of the terminal D-alanine of the pentapeptide precursor leaving a tetrapeptide. Process Diagram: Synthesis of peptidoglycan Synthesis begins in the cytoplasm, where NAM is linked to UDP, and then coupled to a short peptide chain. This complex associates with the integral membrane protein bactoprenol via two phosphates (P). NAG then is linked to the NAM, forming the NAM-NAG disaccharide. This disaccharide flips across the membrane, transporting the NAM-NAG complex across the plasma membrane. The NAM-NAG disaccharide is linked to the growing polysaccharide chain. The peptide chain of the newly added NAM-NAG subunit is crosslinked by a transpeptidase enzyme to another strand of peptidoglycan to form the final structure3

What is in the cytoplasm of bacterial cells? Make sure you Know the different types of inclusion bodies. Think about the types of bacteria in which these are inclusion bodies are found. Where might you find those bacteria?

The remainder of the cytoplasm is a stew of macromolecules (tRNA, rRNA, mRNA, proteins, etc.) >Inclusion bodies may also be present •Polyhydroxybutyrate granules: carbon storage •Sulfur globules: sulfur storage •Gas vesicles: buoyancy control •Carboxysomes: location of carbon fixation reactions Magnetosomes: organelle associated with direction finding Know the function and what type of bacteria would have these structures.

You need to know example of beta lactams, what structure do they have in which you can easily identify them? Why do they have to be growing bacterial cells? why?

The β-lactam family, for example, is represented in Figure 2.19. The three-dimensional structure of the β-lactam ring mimics the terminal D-alanine of peptidoglycan precursors well enough to fool peptidoglycan crosslinking enzymes. These enzymes bind to β-lactam antibiotics and attempt to catalyze the crosslinking reaction, but instead attach the antibiotic molecule covalently to their active site, permanently destroying the enzyme. This inhibition of the enzyme causes big problems for growing bacterial cells, as they try to integrate new strands of peptidoglycan into their cell wall.The cell wall becomes ever weaker, and osmotic pressure can eventually burst the cell, as it does when lysozyme attacks. A key difference is that only growing cells that must add new peptidoglycan are sensitive to β-lactam drugs, whereas lysozyme, which directly cleaves peptidoglycan chains, also attacks non-growing cells.

Although most bacteria are less than 5 μm in diameter, some amazingly large bacteria have been discovered in recent years (Figure 2.5). The spherical bacterium Thiomargarita namibiensis can be up to 700 μm in diameter, and the cigar-shaped Epulopiscium fishelsoni (Microbes in Focus 2.1) can be over 600 μm long. At the other extreme, some atypically small bacteria exist, too. Mycoplasmas, as we noted previously, are only 0.2 μm in diameter. In early 2015, researchers reported the identification of even smaller bacteria. These ultra-small bacteria, which have not yet been cultivated, appear to have a volume of approximately 0.009 μm3. Just know that more are being discovered!

There are exceptions to the general size of bacterial cells. >> Thiomargarita namibiensis: up to 700 um in diameter! >> Epulopiscium fishelsoni: 200 to 700 um x 80 um! >> Some mycoplasma cells are only 0.2 um in diameter! (no cell wall; cause aneumonia) ^ AKA GIANTS of the bacterial world Study tip: Think about easy name associations to remember these! These are the exceptions to the average size that you need to know. We will discuss mycoplasma a lot because they are very different bacteria.

this mechanism is similar to type III secretions systems. Why is this important? It is more direct way from cytoplasm to outside of bacteria know steps in flagellar system

These proteins cross the plasma membrane and outer membrane in a single step, with no periplasmic intermediate. One of these single-step export systems, called the type III secretion system, has a particularly unusual evolutionary history. The secretory apparatus resembles a hypodermic syringe projecting from the surface of the bacterium (Figure 2.25). This form is appropriate as it literally injects proteins, such as toxins, into host cells. Several of the components of the type III secretion system are evolutionarily related to proteins involved in making flagella. Flagellar proteins and proteins exported through the type III secretion system appear to be secreted by a similar mechanism, entering a pore at the base of the flagellum or type III needle, then moving through a channel in the core of the flagellar filament or secretory syringe to emerge at the other end.

In 1974, Richard Blakemore, a graduate student at the University of Massachusetts-Amherst, was examining sediment samples with his microscope. He noticed something curious. Within this muddy microbial menagerie, some bacteria consistently accumulated on one side of the slide. More interestingly, a magnet placed near the microscope stage altered the movement of these bacteria. Placing one pole of the magnet directly next to the stage would cause them to swim toward or away from it, depending on which pole of the magnet was closest to the stage (Blakemore's bacteria turned out to be north-seeking). It was not simply magnetic attraction pushing or pulling the bacteria (dead bacteria aligned along their long axis in a magnetic field, like tiny compass needles, but otherwise were not active). The live bacteria used flagella—tiny propeller structures common in bacteria—to power their movement and somehow used a magnetic field for steering. Blakemore called this phenomenon "magnetotaxis."

This is cool and will help you understand why some bacteria can orient themselves. What types of bacteria would that be? Magnetotaxis provided a simple way for Blakemore to capture these bacteria. He put mud in a container with a magnet next to one side, allowed the magnetotactic bacteria to accumulate, collected the cells, and then repeated this process until he had a nearly pure population of magnetic bacteria. With this basic strategy, plus some trial and error to figure out how to grow the bacteria, he eventually isolated pure cultures of these magnetotactic bacteria. Closer analysis of the cells found them to be rich in iron due to chains of membrane-enclosed magnetite particles (dubbed "magnetosomes") lined up parallel to the long axis of the cell. The force exerted by the magnetosome chain, as it aligns with Earth's magnetic field, orients the bacterium with respect to the magnetic field.

•Some cytoskeleton proteins are involved in cell wall synthesis during cell division (FtsZ and MreB).

What is the homologue to FtsZ? What is the homologue for MreB? Remembering what you learned in BIOL 1201, does it make sense they are involved in cell wall synthesis and cell division?

Examples of Bacterial Cytoskeleton Molecules

•FtsZ - many bacteria •Involved in cell division •forms ring during septum formation in cell division •tubulin homolog (FtsZ protein:What is the equivalent in eukaryotes? What does it do in bacteria? If you delete it- what happens to the bacteria?) •MreB - many rods •Maintains shape •Chromosome alignment •Polarity •Actin homolog •CreS - rare, maintains curve shape -lamin/keratin homolog

Can the cell wall structure be degraded? where is lysozyme made in our bodies? know what a protoblast is but also have an understanding where an isotonic solutions? Do our cells exist in this state? so let's have a though here. If Gram Negative protect themselves why bother to have lysosome in tears or saliva? Hint: what bacteria are more likely to be found in our skin?

YES! -Naturally by lysosome and lysostaphin secretions -artificially from B-lactam antibiotics -these work by preventing peptidoglycan crosslinking, weakening the cell wall structure. Many potential enemies of bacteria, including bacteriophages and animals, encode or produce the enzyme lysozyme, which degrades peptidoglycan. Lysozyme hydrolyzes the β-1,4-glycosidic bond between NAG and NAM, profoundly weakening the cell wall (Figure 2.18). If a bacterium encounters lysozyme under isotonic conditions (water leaves the cell at the same rate it enters), then it may survive as a protoplast, the term given to the generally fragile form of the cell with its protective cell wall removed If a bacterium encounters lysozyme under hypotonic conditions, then water may enter the protoplast, swelling it until it bursts. Lysozyme in human tears and other secretions helps to control bacterial populations in and on our body. We will see later in this chapter that some bacteria (Gram-negative bacteria) cover their peptidoglycan with another membrane, effectively shielding the cell wall from lysozyme.

Can the cell wall structure be degraded? So what happens when you weaken the CW?

YES! -Naturally by lysosyme and lysostaphin secretions A. Chemical action of lysozyme B. Chemical action of lysostaphin A. B-Lactam antibiotics The call can't resist osmotic pressure change C. Effects of lysozyme on the cell

How are bacteria organized chart

You should know the terms for pairs, chains and clusters. Words using myces often originate from myco (fungal). So, hyphae and mycelium are used dominantly discussing fungi.

what makes this different from penicillin?

augmentin

The periplasm contains enzymes needed to continue wall synthesis What are peptides and what are the building blocks? What is a peptidase? Why is this called "trans" peptidase? Which subunit has a peptide bridge? Are you starting to make connections? If not see me in office hours.

autolysins- autolysins* degrade the existing peptidoglycan wall to allow new NAM-NAG subunits to be incorporated. transpeptidase (penicillin binding proteins) link peptidoglycan strands via the pentapeptides and catalyze controlled degradation for new growth -NAM subunits contain the peptides - this connects layers of cell wall together

You will need to know the inclusion bodies and what types do what!

depending on the environment and growth conditions, bacterial cells sometimes store extra carbon, nitrogen, or phosphorus in inclusion bodies large enough to be seen by microscopy as granules within the cytoplasm. A separate membrane does not surround these inclusion bodies. As an example, polyhydroxybutyrate, or PHB, is a lipid polymer used for carbon storage. PHB granules can compose over 50 percent of the cell's dry weight. PHB is an example of a broader group of polymers called polyhydroxyalkanoates that have been the subject of much industrial interest in recent years due to their ability to substitute for plastics that are derived from petrochemicals (see Section 16.4 for a discussion of these polyhydroxyalkanoate bioplastics).

Bacterial Cell Walls

grampositive e has thick wall, peptidoglycan gram-negavtive has thin wall, lipooplypeptide

Classification and Nomenclature

know the basic taxonomy oClassification depends on many features: •Size/shape •Gram type •Colony morphology •Presence of structures such as capsules/endospores •Physiologic/metabolic traits (see Ch. 13) •DNA sequence data (in more recent years) •Once classified, microbes are deposited in at least two culture collections. oThe World Federation for Culture Collections maintains a database of more than 500 collections from over 60 countries. oThese are pure, maintained cultures made available to scientists for research purposes.

What are the terms used to describe the flagellar arrangement? Can you draw them? Can you recognize them on a bacterium?

monotrichous - one flagellum amphitrichous - one flagellum at each end of cell lophotrichous - cluster of flagella at one or both ends peritrichous - spread over entire surface of cell

flagella functions what is swarming? Colonies growing on agar plates rapidly spread over the entire surface of the agar in the course of a few hours. The cells coordinate swarming with the expression of other genes needed for infection, suggesting that this mode of motility is useful during infection, perhaps because it facilitates movement in host tissues.

motility and swarming behavior •Rotate flagella to run or tumble •Move toward or away from stimuli (taxis) may be virulence factors •Flagella proteins are H antigens (e.g., E. coli O157:H7)

2.5 The Bacteria Cell Surface

not all bacteria have them

•Aside from motility, what other stuff is still left on the outside of bacterial cells? what is stalk or holdfast?

oAdherence molecules to stick to surfaces •Some microbes (Caulobacter, Hyphomonas) will use an extension of the cell envelope tipped by a "holdfast" of polysaccharides. •These extensions provide extra surface area for nutrient absorption as well as the adherence capability.

What makes the S-layer unique? What is its purpose? What kind of molecules make the S-layer

oSurface arrays (S-layers) •Crystalline array of interlocking proteins •Found in Gram-positive and Gram-negative cells •Can act as armor, protecting a cell against predation or infection with bacteriophages

What is in the cytoplasm of bacterial cells? What is the nucleoid. What type of DNA? How is it condensed?

the aqueous environment within the plasma membrane, contains a diverse array of components. The largest single entity in the cytoplasm in the nucleoid, a convoluted mass of DNA (usually a single, circular chromosome) coated with proteins and RNA molecules still in the process of being synthesized. In contrast to the nucleus of eukaryal cells, a membrane does not surround the nucleoid of bacterial cells (Figure 2.6). The largest area is the nucleoid, a convoluted mass of DNA (usually a single, circular chromosome) coated with proteins and RNA molecules still in the process of being synthesized.

Sec-dependent pathway

the major pathway for all bacteria for transporting proteins across the plasma membrane >also called general secretion pathway -highly conserved in all domains >secreted proteins synthesized as preproteins having amino-terminal signal peptide -signal peptide delays protein folding -chaperone proteins (SecB) keep preproteins unfolded

gram-negative bacteria

the outer membrane of gram-negative bacteria is a bilayer, but only contains phospholipids in the inner leaf of the bilayer. Outer leaf is composed of a molecule called lipopolysaccharide (LPS). -thin peptidoglycan -outermembrane -lipopolysaccharides

Atypical Cell Walls

•Acid-fast cell walls •Like gram-positive cell walls •Waxy lipid (mycolic acid) bound to peptidoglycan •Mycobacterium •Nocardia •Mycoplasmas •Lack cell walls •Sterols in plasma membrane •Archaea •Wall-less, or •Walls of pseudomurein (lack NAM and D-amino acids)

CONCLUSION

•Bacterial cells exhibit great differences in composition, size, and shape. However, at their core, all bacteria still depend on the same DNA → RNA → protein system that is a common underlying theme to all cellular life on Earth. We will explore this theme more in subsequent chapters.

Make sure you can determine if bacteria are in the presence of a chemoattractant or repellent.

•Chemotaxis •move toward chemical attractants such as nutrients, away from harmful substances • •Move in response to temperature, light, oxygen, osmotic pressure, and gravity

Flagellar Synthesis You should know how bacteria make their flagella. How do they get the flagellin proteins outside of the cells. How does this flagella grow?

•Complex process involving many genes/gene products •New flagellin molecules transported through the hollow filament using Type III-like secretion system • •Filament subunits self-assemble with help of filament cap at tip, not base

LPS (lipopolysaccharide) Gram-negative bacteria have an outer membrane In Gram-negative bacteria, an outer membrane surrounds a thin peptidoglycan layer. The presence of this second membrane creates the periplasm as a separate compartment. The outer membrane contains phospholipids on its inner surface and lipopolysaccharide (LPS) on its outer surface. Like the plasma membrane, it also contains protein

•Consists of three parts •O side chain (O antigen) •core polysaccharide •lipid A (toxin) •O polysaccharide antigen •e.g., E. coli O157:H7 •Extends out prevents phagocytosis •Lipid A embedded in OM •Induces inflammation

flagellar movement

•Energy to spin flagella derived from proton motive force (PMF)

external structures

•Extend beyond the cell envelope in bacteria •Function in protection, attachment to surfaces, horizontal gene transfer, cell movement >pili and fimbriae >flagella

flagella What are the proteins that make up flagella in bacteria? How are these different from the other domains?

•Filament •extension outside cell wall •hollow, rigid cylinder of flagellin protein • •Hook •Attaches filament to the basal body • •Basal body •disk-like structure that produces torque on filament to turn it like a propeller

bacterial evasion of immune response

•Motility from flagella -By using chemoreceptor proteins to sense changes in concentrations of attractants or repellents, cells can produce more runs to move in a particular direction. Another fascinating mechanism of motility has been observed in some bacteria that invade eukaryal cells. Shigella dysenteriae, a Gram-negative, rod-shaped bacterium that causes bloody diarrhea (dysentery), and Listeria monocytogenes, a Gram-positive bacterium that can cause serious food poisoning, have evolved a strategy for hijacking the mammalian actin cytoskeleton. When these bacteria invade cells, they induce actin polymers to form at one end of the bacterium. Polymerization of actin propels the bacterium through the cytoplasm of the host cell (Figure 2.30), and the force generated can actually shoot the bacterial cell through the host plasma membrane into an adjacent cell, which may be the real purpose of this actin-based motility. When they take up residence within intestinal cells, these bacteria are protected from antibodies produced by the immune system. By spreading directly from one cell to another, they effectively evade the host immune response, thereby taking full advantage of this sheltered habitat.

the bacterial cell surface

•Motility from flagella -Composed of three basic pieces: •Filament •multiple flagellin proteins, •5 to 10 μm long •Hook •protein portion that connects filament to basal body •Basal body: •disk-like structure that produces torque on filament to turn it like a propeller

know run vs tumble

•Motility from flagella -Energy to spin flagella derived from proton motive force (PMF) -Complex structures with up to 40 different proteins -Spinning one way produces a "run" (directional movement) while spinning the other way produces a "tumble" (nondirectional movement)

Know an example of a spirochete and how the flagella is arranged.

•Motility from flagella oNot all cells have external flagella! oSome spirochetes have flagella in the periplasm. oAs they spin, they rotate the entire cell body like a corkscrew.

•What are the critical structural and functional properties of the bacterial cell envelope? How do items cross the PM and get into a cell? I will not go over this material on diffusion, osmosis and the types of transport but you are responsible for it on an exam in an applied context! You will see this when we discuss the formation of flagella and chemotaxis

•O2 and CO2 are small and can diffuse across readily. •H2O is helped across by aquaporin protein channels. •Osmosis is the flow of water across the PM toward the side with a higher solute (particle) concentration. •Osmosis can cause a cell to swell with water or shrivel as water leaves, but a strong cell wall can help keep a bacterial cell alive during these hardships.

Other cytoskeletal proteins are involved in moving internal items (e.g., plasmids, magnetosomes). New molecules: What is CreS ? What types of bacteria express it? What it's homologue and what would happen if it was mutated or deleted?

•ParM forms actin-like filaments that direct plasmid movement to either side of the cell, ensuring plasmid segregation. (Partition) •MamK forms actin-like filaments and is responsible for Magnetosome organization

Polyhydroxybutyrate granules (PHB): carbon storage Know what PHB stand for and under what circumstance you would see as many as you see above!

•Poly-3-hydroxybutyrate (PHB) is a biological polyester present in bacteria •PHB is used as an alternative carbon source for metabolism

Peptidoglycan Biosynthesis

•Transglycosylation: Glycolases catalyze glycosidic bond formation forming a unique b 1-4 glycosidic bond •Transpeptidation: final step in cell wall synthesis -Forms the peptide cross-links between muramic acid residues in adjacent glycan chains -Inhibited by the antibiotic penicillin