Iones Ln 3+ dopados en Cs 2 NaYCl 6

Prokaryotic cells such as bacteria lack internal membrane-bound organelles.

They are much simpler than eukaryotic cells such as the cells of the human body that contain internal membrane-bound organelles (nucleus, mitochon-dria, endoplasmic reticulum; see below). Because bacteria are so simple, our first inclination to is believe that they should be simple in shape. In fact, most are either cylindrical (rod-shaped) or spherical (cocci), but they can also be shaped as spirals (spirilla and spirochetes), curves (vibrios), and even squares and triangles. The shape of the cell is determined by its wall.

Bacteria are also smaller than most eukaryotes. Some bacteria are as very small as 100 nm (Mycoplasma spp.); some are as large as 60 to 800 mm (Epulopisicum fishelsoni). The large size of E. fishelsoni contradicts the small size assumption by overcoming the hypothetical diffusion limits through a plasma membrane that is highly invaginated into the cytoplasm.

Bacterial organelles

A variety of structures exist within prokaryotes. Figure 2.1 illustrates a typical bacterial (Gram-positive) cell. Table 2.1 describes the functions of cell struc-tures. Not all bacteria are identical or contain all the structures shown.

In general, a cell wall and plasma (or cytoplasmic) membrane surround prokaryotic cells. Some prokaryotes lack cell walls but all have cytoplasmic membranes. The membrane can invaginate to form mesosomes and other internal membranous structures within the cytoplasm. In all prokaryotic cells, however, the cytoplasm is very simple and has no membrane-bound internal organelles. The genetic material (e.g., DNA) is in a region called the Figure 2.1 Typical Gram-positive bacterial cell.

Nucleoid Capsule Ribosome

Flagellum

Inclusion body

Peptidoglycan Cell membrane Mesosome 1453_C002.fm Page 25 Friday, March 10, 2006 5:26 AM

26 Cosmetic Microbiology: A Practical Approach

nucleoid. This region is not surrounded by a nuclear membrane in a nucleus as it is in eukaryotes. Ribosomes and inclusion bodies are found throughout the cytoplasm. One organelle that extends outside the cell wall is the flagel-lum. A capsule and pili or fimbriae surround the cell wall. These structures help a bacterium adhere to surfaces and form biofilms. They also allow bacteria to stick to each other and clump, especially when exposed to bio-cides or other adverse conditions.

Cell walls and cytoplasmic membranes. Nearly all prokaryotes have cell walls. The only exceptions are the mycoplasma and a few archaeobacteria.

A cell wall gives a bacterium its strength and shape. Without the rigid wall, a bacterium would lyse (explode) because of the effect of osmosis. Most bacteria exist in a dilute external environment. In fact, the environment inside the cell is more concentrated in solutes (dissolved materials) than the outside.

Therefore, water flows into the cell continuously. A whimsical analogy is the water balloon. If a balloon is placed into a rigid box before it is filled with water, it cannot be filled so full that it will pop. If filled outside a box, it will pop when filled too full.

Between the cell wall and cytoplasmic membrane is the periplasmic space — a gel-like area filled with hydrolytic enzymes and binding proteins that digest nutrients and transport them into the cell.

The next structure for consideration is the cytoplasmic membrane. Sim-plistically, a novice would claim that this structure is not all that different from any other cell membrane — eukaryotic or prokaryotic. After all, it has proteins and lipids and surrounds the cytoplasm. It is the main point of contact between the cell and its environment. The cytoplasmic membrane is selectively permeable and will allow only certain molecules to enter the cell.

Table 2.1 Functions of Various Prokaryotic Cell Structures

Structure Function

Capsule/slime layer Resistance to phagocytosis; adherence to surfaces Fimbria/pilus Attachment to surfaces; bacterial conjugation

Flagellum Movement

Cell wall Provides shape and protection from lysis in dilute solutions

Plasma membrane Serves as selectively permeable barrier and boundary of cell; location of respiration and photosynthesis; serves as receptor for chemotaxis

Periplasmic space Site of hydrolytic enzymes and proteins for nutrient uptake and processing

Ribosome Protein synthesis

Inclusion body Storage

Gas vacuole Provides buoyancy for certain bacteria Nucleoid Site of genetic material (DNA)

Endospore Allows survival under harsh environments for certain bacteria

1453_C002.fm Page 26 Friday, March 10, 2006 5:26 AM

chapter two: Biology of microbes 27 It transports large molecules that cannot diffuse and also carries on metabolic functions such as respiration, photosynthesis, and some biosyntheses. This membrane is composed of lipids that arrange into a bilayer (because of a lipid molecule’s amphipathic nature — one end is hydrophilic and the other is hydrophobic). However, the few differences that do exist between eukary-otic and prokaryeukary-otic membranes are significant. The bacterial membranes of prokaryotes do not contain cholesterol as eukaryotic cells do. Their proteins are different. Although membranes have a common basic design, they differ widely in their various structural and functional capacities.

The model of a membrane that is widely accepted by most scientists is called the fluid mosaic model. One can think of this model as a layer of oil (lipid membrane) on top of an ocean (the cytoplasm) into which are inter-spersed ships of protein, except that our model includes dilute water on top of the oil layer (the external environment of the cell) as well. Like ships on the ocean, the proteins move laterally around the surface. The protein ships are peripheral (loosely connected to the membrane and soluble in water) or integral (hard to extract from the membrane and insoluble in water).

Peptidoglycans. The cytoplasmic membrane of any cell is delicate and prone to rupture unless the cell exists in an isotonic state. This is why physi-ological saline is used instead of distilled water to replace body fluids: to prevent rupture of red blood cells. However, bacteria rarely have the luxury of floating in an isotonic environment unless they are living inside a blood-stream. Most of the time, they exist in a hypotonic environment where water pours into cells. Peptidoglycan makes up the structure of the cell wall to provide it with rigidity and strength and combat the hypotonic environment.

Peptidoglycan (also known as murein) is a net-like structure comprising the cell wall. It is a polymer composed of two amino sugars (N-acetylglu-cosamine and N-acetylmuramic acid) and several amino acids (some unique only to prokaryotes and not even found in most eukaryotic proteins). Figure 2.2 shows the structure of peptidoglycan.

Cell walls of Gram-negative and Gram-positive bacteria exhibit con-siderable differences. One of the major differences is the type of pepti-doglycan. A Gram-positive cell has a pentaglycine bridge between the d-alanine and l-lysine of the tetrapeptide that comes off the N-acetylmu-ramic acid (see Figure 2.2). Gram-negative cells exhibit a direct link between the alanine and the lysine.

The definition of Gram-positive and Gram-negative cell walls relates to two concepts: the color of the cell after the Gram stain and the physiological structure of the wall. Christian Gram developed a technique that allowed some bacteria to stain pink and others purple. When Gram devised the method, he did not know the reason for the staining. As a result of this differential staining, microbiologists began calling purple-stained bacteria

“positive” and pink-stained bacteria “negative.” Later on, they found out that the purple-staining Gram-positive bacteria usually had thicker pepti-doglycan layers and the pink-staining Gram-negative bacteria had thinner

1453_C002.fm Page 27 Friday, March 10, 2006 5:26 AM

28 Cosmetic Microbiology: A Practical Approach

peptidoglycan layers. We will explore additional differences between Gram-positive and Gram-negative bacteria next.

Gram-positive walls. Gram-positive bacteria have very thick pepti-doglycan layers. The peptipepti-doglycan has a pentaglycine bridge between the d-alanine and the l-lysine of the tetrapeptide coming off the N-acetylmu-ramic acid (NAM). The NAM is polymerized to N-acetylglucosamine (NAG). Gram-positive cell walls also contain teichoic acids (Figure 2.3).

These are ribitol and glycerol phosphate polymers. Coming off the ribitol and glycerol may be amino acids or sugars. The teichoic acids attach to Figure 2.2 Peptidoglycan subunit. The crosslink between two NAG–NAM strands is direct via d-alanine and diaminopimelic acid (DAP) for Gram-negative peptidogly-can. In Gram-positive cell walls, the crosslink is indirect via a pentaglycine bridge.

NAG = N-acetylglucosamine. NAM = N-acetyl muramic acid. A tetrapeptide is attached to NAM.

Figure 2.3 Gram-positive cell wall.

NAM CH₂OH

CH₂OH NAG CH₂

CH₂ O

O

O

O

H₂C-CH-C=O

NH-C=O

NH-C=O

OH

L-alanine

D-glutamic acid

meso-Diaminopimelic acid

D-alanine

1453_C002.fm Page 28 Friday, March 10, 2006 5:26 AM

chapter two: Biology of microbes 29

the peptidoglycan layer and extend to the outside of the cell where they give the cell a negative charge. They can even extend all the way down into the cell membrane and attach to lipids (lipoteichoic acids). They are like tie rods that hold the peptidoglycan to the membrane, but their true function has not been entirely clarified yet. They are not present in Gram-negative cell walls.

Gram-negative walls. A Gram-negative bacterium has a thin pepti-doglycan where the tetrapeptide coming off the NAM is directly linked to the one coming off a NAM on an adjacent strand of NAG–NAM polymer.

The complexity of Gram-negative cell walls is astounding. They have outer membranes on the outsides of their thin peptidoglycan layers. Linking this outer membrane to the peptidoglycan is a compound called Braun’s lipo-protein. It is covalently linked to the peptidoglycan with its hydrophobic end stuck in the lipids of the underlying surface of the outer membrane (Figure 2.4).

The outer membrane of a Gram-negative bacterium is made of a lipid bilayer, as is the case with most classical membranes. Its uniqueness comes from the lipopolysaccharides (LPSs) that extend from the outer layer of the outer membrane into the environment. LPSs are very large molecules made of lipids and carbohydrates (see Figure 2.5). The O-region of the LPS elicits the most antibody response during an infection and the O-region rapidly changes to avoid antibody attack. The LPS also confers a negative charge to the bacterial surface and helps stabilize the outer membrane. It is also the component that acts as the endotoxin.

The outer membrane of Gram-negative cell walls really does not act as a selectively permeable membrane in the same way that a cytoplasmic mem-brane does. Instead, it offers protection and slows entry of toxic substances Figure 2.4 Gram-negative cell wall.

O-specific side chains

Lipopolysaccharide Braun’s lipoprotein Porin

Periplasmic space and peptidoglycan Outer membrane

Phospholipids

Integral protein Peptidoglycan Plasma membrane 1453_C002.fm Page 29 Friday, March 10, 2006 5:26 AM

30 Cosmetic Microbiology: A Practical Approach

into the cell. It does, however, allow small molecules such as monosaccha-rides to pass to the cell membrane via porin proteins. The porins form a channel through which these smaller molecules (<700 daltons) pass. Large molecules are transported by specific carrier proteins. The outer membrane of a Gram-negative cell wall has some selective permeability about it, but should never be thought of as another cytoplasmic membrane.

Gram stain. When performing a Gram stain, start by spreading a thin suspension of bacteria on a glass slide, let the suspension dry on the slide, and heat it gently to fix the bacteria onto the slide. The next step is to put a crystal violet solution on the slide for about a minute and rinse the slide with water. The bacteria absorb the dye and turn purple. Then put Gram’s iodine on the slide. The iodine acts as a mordant that “sets” the purple dye. Next, rinse with an acetone–alcohol mixture to try to remove the set dye. Finally, counterstain with safranin, a pink dye. If the crystal violet dye cannot be removed with the acetone–alcohol mixture, then the safranin counterstain is not even seen; the bacterium is so dark purple that Figure 2.5 Lipopolysaccharide from Salmonella species. Abe – abequose. Gal = galac-tose. Glc = glucose. GlcN = glucosamine. Hep = heptulose. KDO = 2-keto-3-deoxy octonate. Man = mannose. NAG = N-acetyl glucosamine. P = phosphate. Rha = L-rhamnose.

Man Abe Rha Gal Man Abe Rha Gal Glc NAG Gal Glc Gal Hep

Hep P P ethanolamine

ethanolamine KDO

KDO KDO P

P GlcN GlcN P

Fatty acids

Fatty acids 1453_C002.fm Page 30 Friday, March 10, 2006 5:26 AM

chapter two: Biology of microbes 31 the pink dye does not contribute to the color. If the crystal violet is removed, the bacteria are colorless until the pink safranin counterstain is added. Bac-teria that stain purple are called Gram-positive (positive because they retain the first dye). Bacteria that stain pink are designated Gram-negative.

The key to understanding how the Gram stain works is in the cell wall.

One hypothesis is that the alcohol shrinks the molecular pores of the thick peptidoglycan of Gram-positive cells where the crystal violet is trapped. In the Gram-negative cells, the thin peptidoglycan is not highly cross-linked.

The pores are bigger and more permeable, allowing the crystal violet to leak out quicker when decolorized. The alcohol also dissolves the lipid of the outer membrane to allow the stain to escape.

Cytoplasm, mesosomes, ribosomes, and other inclusions. A bacterial cell minus its wall is a protoplast. A protoplast includes the plasma membrane, the cytoplasm, and everything within it. The prokaryotic cytoplasm, how-ever, does not have typical unit membrane-bound internal organelles. Within the cytoplasm is the nucleoid where the DNA genetic material is localized.

Also, within the cytoplasm are the enzymes needed for growth and metab-olism, the machinery for manufacturing those enzymes (ribosomes), and some internal membrane structures called mesosomes. Mesosomes are actu-ally invaginations of the plasma membrane. Finactu-ally, some bacteria also con-tain inclusion bodies consisting of polyphosphate, cyanophycin, and glyco-gen. These inclusions are not usually membrane-bound. Other bacteria have inclusions bound by a single-layered nonunit membrane. These consist of poly-b-hydroxybutyrate, sulfur, carboxysomes, hydrocarbons, and gas vac-uoles.

Mesosomes are rather enigmatic. Their exact function remains uncertain despite their presence in both Gram-positive and Gram-negative bacteria.

They are often found close to the septa of dividing cells; sometimes they even appear to be attached to chromosomes. Perhaps they are involved in cell wall formation or play a role in chromosome separation during cell division. This role would make them somewhat analogous to spindle fibers in eukaryotic cell mitosis. Maybe they are involved in secretory processes.

Some microbiologists dismiss them entirely, claiming they are artifacts of the fixation process used in preparing samples for electron microscopy. Since we also see them in freeze etches where few or no artifacts develop from chemical fixation, it is doubtful that this concept will prevail.

Other more complex membrane systems of invaginated plasma mem-branes are seen in the complex and extensive foldings within cyanobacteria, nitrifying bacteria, and purple bacteria. The current thinking is that these organisms require the larger surface area that these infoldings provide in order to carry on greater metabolic activity.

Ribosomes are packed into the cytoplasm; some are loosely attached to the plasma membrane. They are made of both ribonucleic acid and protein and serve as sites where RNA is translated into protein after it is transcribed from DNA. In bacterial systems and in mitochondria, ribosomes are

com-1453_C002.fm Page 31 Friday, March 10, 2006 5:26 AM

32 Cosmetic Microbiology: A Practical Approach posed of two components: 50S and 30S subunits (the S indicates a Svedberg unit — a measure of sedimentation based on a particle’s volume, shape, and weight). Together, the 50S and 50S subunits comprise a single ribosome and together weigh 70S (the numbers are not supposed to add to an expected total because a Svedberg unit is based on more than weight alone).

Nucleoids. The nucleoid is another structure that is not surrounded by a membrane. Eukaryotes always contain their genetic material within a nucleus surrounded by a membrane; they have two or more linear chromo-somes. A prokaryote has only one circular chromosome located within a region called a nucleoid. The nucleoid is apparently attached to the cell membrane that may be involved in cell division via the aid of mesosomes.

Bacteria also contain plasmids. These are extrachromosomal pieces of DNA (also circular) that replicate independently of the chromosome. They can also be incorporated into the chromosome. They rarely have genes that are absolutely required by the organism for growth and metabolism, but often carry genes that are very useful for survival: resistance genes that make them able to withstand antibiotics, genes that allow the organism to produce a toxin, or genes that provide some other selective advantage.

Organelles outside the wall. Flagella, fimbriae, and pili. Fimbriae (or pili) are thin protein hairs on the outer surfaces of Gram-negative bacteria that cause the bacteria to stick to surfaces. If it were not for pili, bacteria would be less able to cause infections by attaching to host tissues. They would not be as able to form biofilms on pipes of water systems to create reservoirs of contamination or endotoxins. They would not be able to attach to ship hulls to produce further corrosion. Some pili are even involved in providing a passage for DNA to travel from one bacterium to another during conjuga-tion.

Flagella allow bacteria to move. Unlike eukaryotic flagella, the prokary-otic flagellum rotates rather than moving from side to side. Also, it is a relatively simple thread-like appendage of protein extending from the plasma membrane and cell wall (about 20 mm long and 20 nm thick). This portion is the filament. Compared to the 9 + 2 filament arrangement of eukaryotic flagella, this structure is simple.

A little more complexity appears when we look at the basal body of the bacterial flagellum. It is embedded within the bacterial cell wall and consists of a number of rings that vary, depending on whether the bacterium is Gram-negative or Gram-positive. A hook links the filament and the basal body. The basic difference between Gram-negative and Gram-positive fla-gella is the number of basal body rings. Gram-negative bacteria usually have four basal body rings. The first two rings are attached to the outer membrane (L-ring) and peptidoglycan layer (P-ring). The inner two rings contact the periplasmic space (S-ring) and the plasma membrane (M-ring). The Gram-positive flagellum has only two rings: one attached to the plasma membrane and the other attached to the peptidoglycan.

1453_C002.fm Page 32 Friday, March 10, 2006 5:26 AM

chapter two: Biology of microbes 33 Capsules and slime layers. Outside the cell wall, a bacterium may have a layer of material that can be fairly well-organized and not easily washed away. This layer is known as the capsule. If the material is easily washed off and not really organized, the layer is a slime. Many scientists prefer to not worry about these distinctions and so refer to both types of layers as the glycocalyx. In both cases, the layers are made from polysaccha-rides or polyamino acids (in some bacteria) surrounding the outer cell walls.

The capsule helps a bacterium resist phagocytosis when it infects a host.

Encapsulated bacterial pathogens are usually much more virulent than vari-eties without capsules. The capsule also helps the bacterium avoid drying, acts as a biofilm to aid it in attachment to surfaces, helps it avoid predation from zooplankton, and protects it from detergent and biocide actions. From a practical standpoint, encapsulated bacteria are more likely to develop tolerance to preservatives and biocides in manufacturing conditions.

Metabolism

In order to understand metabolism fully, the student should understand the basics of thermodynamics, chemical reactions coming to equilibrium, and oxidation–reduction reactions. He or she should also understand the roles

In order to understand metabolism fully, the student should understand the basics of thermodynamics, chemical reactions coming to equilibrium, and oxidation–reduction reactions. He or she should also understand the roles