44 Introduction to bacteria

Structural components of bacterial cell walls

It is important to note that not all bacteria have a cell wall. However, most bacteria (about 90%) do have a cell wall and they typically have one of two types: a gram positive cell wall or a gram negative cell wall (Figure 9.8). The two different cell wall types can be identified in the lab by a differential stain known as the Gram stain. After this stain technique is applied the gram positive bacteria will stain purple, while the gram negative bacteria will stain pink. Originally, it was not known why the Gram stain allowed for such reliable separation of bacterial into two groups. Once the electron microscope was invented in the 1940s, it was found that the staining difference correlated with differences in the cell walls.

Overview of bacterial cell walls

A cell wall, not just of bacteria but for all organisms, is found outside of the cell membrane. It’s an additional layer that typically provides some strength that the cell membrane lacks, by having a semi-rigid structure.

Both gram positive and gram negative cell walls contain an ingredient known as peptidoglycan (also known as murein). This particular substance hasn’t been found anywhere else on Earth, other than the cell walls of bacteria. But both bacterial cell wall types contain additional ingredients as well, making the bacterial cell wall a complex structure overall, particularly when compared with the cell walls of eukaryotic microbes. The cell walls of eukaryotic microbes are typically composed of a single ingredient, like the cellulose found in algal cell walls or the chitin in fungal cell walls.

The bacterial cell wall performs several functions as well, in addition to providing overall strength to the cell. It also helps maintain the cell shape, which is important for how the cell will grow, reproduce, obtain nutrients, and move. It protects the cell from osmotic lysis, as the cell moves from one environment to another or transports in nutrients from its surroundings. Since water can freely move across both the cell membrane and the cell wall, the cell is at risk for an osmotic imbalance, which could put pressure on the relatively weak plasma membrane. Studies have actually shown that the internal pressure of a cell is similar to the pressure found inside a fully inflated car tire. That is a lot of pressure for the plasma membrane to withstand! The cell wall can keep out certain molecules, such as toxins, particularly for gram negative bacteria. And lastly, the bacterial cell wall can contribute to the pathogenicity or disease –causing ability of the cell for certain bacterial pathogens.

Structure of peptidoglycan

Let us start with peptidoglycan, since it is an ingredient that both bacterial cell walls have in common.

Peptidoglycan is a polysaccharide made of two glucose derivatives, N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), alternated in long chains (Figure 9.9). The chains are cross-linked to one another by a tetrapeptide that extends off the NAM sugar unit, allowing a lattice-like structure to form. The four amino acids that compose the tetrapeptide are: L-alanine, D-glutamine, L-lysine or meso-diaminopimelic acid (DPA), and D-alanine. Typically only the L-isomeric form of amino acids are utilized by cells but the use of the mirror image D-amino acids provides protection from proteases that might compromise the integrity of the cell wall by attacking the peptidoglycan. The tetrapeptides can be directly cross-linked to one another, with the D-alanine on one tetrapeptide binding to the L-lysine/ DPA on another tetrapeptide. In many gram positive bacteria there is a cross-bridge of five amino acids such as glycine (peptide interbridge) that serves to connect one tetrapeptide to another. In either case the cross-linking serves to increase the strength of the overall structure, with more strength derived from complete cross-linking, where every tetrapeptide is bound in some way to a tetrapeptide on another NAG-NAM chain.

While much is still unknown about peptidoglycan, research in the past ten years suggests that peptidoglycan is synthesised as a cylinder with a coiled substructure, where each coil is cross-linked to the coil next to it, creating an even stronger structure overall.

Classification of bacteria as gram positive or gram negative based on cell wall structure

Gram positive cell walls

The cell walls of gram positive bacteria are composed predominantly of peptidoglycan. In fact, peptidoglycan can represent up to 90% of the cell wall, with layer after layer forming around the cell membrane. The NAM tetrapeptides are typically cross-linked with a peptide interbridge and complete cross-linking is common. All of this combines together to create an incredibly strong cell wall.

The additional component in a gram positive cell wall is teichoic acid, a glycopolymer, which is embedded within the peptidoglycan layers (Figure 9.10). Teichoic acid is believed to play several important roles for the cell, such as generation of the net negative charge of the cell, which is essential for development of a proton motive force. Teichoic acid contributes to the overall rigidity of the cell wall, which is important for the maintenance of the cell shape, particularly in rod-shaped organisms. There is also evidence that teichoic acids participate in cell division, by interacting with the peptidoglycan biosynthesis machinery. Lastly, teichoic acids appear to play a role in resistance to adverse conditions such as high temperatures and high salt concentrations, as well as to β-lactam antibiotics. Teichoic acids can either be covalently linked to peptidoglycan (wall teichoic acids or WTA) or connected to the cell membrane via a lipid anchor, in which case it is referred to as lipoteichoic acid.

Since peptidoglycan is relatively porous, most substances can pass through the gram positive cell wall with little difficulty. But some nutrients are too large, requiring the cell to rely on the use of exoenzymes. These extracellular enzymes are made within the cell’s cytoplasm and then secreted past the cell membrane, through the cell wall, where they function outside of the cell to break down large macromolecules into smaller components.

Gram negative cell walls

The cell walls of gram negative bacteria are more complex than that of gram positive bacteria, with more ingredients overall. They do contain peptidoglycan as well, although only a couple of layers, representing 5-10% of the total cell wall. What is most notable about the gram negative cell wall is the presence of a plasma membrane located outside of the peptidoglycan layers, known as the outer membrane (Figure 9.11). This makes up the bulk of the gram negative cell wall. The outer membrane is composed of a lipid bilayer, very similar in composition to the cell membrane with polar heads, fatty acid tails, and integral proteins. It differs from the cell membrane by the presence of large molecules known as lipopolysaccharide (LPS), which are anchored into the outer membrane and project from the cell into the environment. LPS is made up of three different components: 1) the O-antigen or O-polysaccharide, which represents the outermost part of the structure , 2) the core polysaccharide, and 3) lipid A, which anchors the LPS into the outer membrane. LPS is known to serve many different functions for the cell, such as contributing to the net negative charge for the cell, helping to stabilize the outer membrane, and providing protection from certain chemical substances by physically blocking access to other parts of the cell wall. It is also known that LPS can play a role in the response that a host (think human or other animal) develops to an infection by a pathogenic gram negative bacterium. Specifically, the O-antigen portion of LPS triggers an immune response in the host, causing the generation of protective antibodies that identify a particular O-antigen (think of E. coli O157: H7). It is also known that lipid A can act as a toxin, specifically an endotoxin, causing general symptoms of illness such as fever and diarrhoea. A large amount of lipid A released into the bloodstream can trigger endotoxic shock, a body-wide inflammatory response which can be life-threatening.

The outer membrane does present an obstacle for the cell. While there are certain molecules it would like to keep out, such as antibiotics and toxic chemicals, there are nutrients that it would like to let in and the additional lipid bilayer presents a formidable barrier. Large molecules are broken down by enzymes, in order to allow them to get past the LPS. Instead of exoenzymes (like the gram positive bacteria), the gram negative bacteria utilise periplasmic enzymes that are stored in the periplasm. Where is the periplasm, you ask? It is the space located between the outer surface of the cell membrane and the inner surface of the outer membrane, and it contains the gram negative peptidoglycan. Once the periplasmic enzymes have broken nutrients down to smaller molecules that can get past the LPS, they still need to be transported across the outer membrane, specifically the lipid bilayer. Gram negative cells utilise porins, which are transmembrane proteins composed of a trimer of three subunits, which form a pore across the membrane. Some porins are non-specific and transport any molecule that fits, while some porins are specific and only transport substances that they recognize by use of a binding site. Once across the outer membrane and in the periplasm, molecules work their way through the porous peptidoglycan layers before being transported by integral proteins across the cell membrane.

The peptidoglycan layers are linked to the outer membrane by the use of a lipoprotein known as Braun’s lipoprotein. At one end the lipoprotein is covalently bound to the peptidoglycan while the other end is embedded into the outer membrane via its polar head. This linkage between the two layers provides additional structural integrity and strength.

Unusual and wall-less bacteria

Having emphasised the important of a cell wall and the ingredient peptidoglycan to both the gram positive and the gram negative bacteria, it does seem important to point out a few exceptions as well. Bacteria belonging to the phylum Chlamydiae appear to lack peptidoglycan, although their cell walls have a gram negative structure in all other regards (i.e. outer membrane, LPS, porin, etc). It has been suggested that they might be using a protein layer that functions in much the same way as peptidoglycan. This has an advantage to the cell in providing resistance to β-lactam antibiotics (such as penicillin), which attack peptidoglycan.

Bacteria belonging to the phylum Tenericutes lack a cell wall altogether, which makes them extremely susceptible to osmotic changes. They often strengthen their cell membrane somewhat by the addition of sterols, a substance usually associated with eukaryotic cell membranes. Many members of this phylum are pathogens, choosing to hide out within the protective environment of a host.

Bacterial cell components and function

Internal components

We have already covered the main internal components found in all bacteria, namely, cytoplasm, the nucleoid, and ribosomes. Remember that bacteria are generally thought to lack organelles, those bilipid membrane-bound compartments so prevalent in eukaryotic cells. But bacteria can be more complex, with a variety of additional internal components to be found that can contribute to their capabilities. Most of these components are cytoplasmic but some of them are periplasmic, located in the space between the cytoplasmic and outer membrane in gram negative bacteria.

Cytoskeleton

It was originally thought that bacteria lacked a cytoskeleton, a significant component of eukaryotic cells. In the last 20 years, however, scientists have discovered bacterial filaments made of proteins that are analogues to the cytoskeletal proteins found in eukaryotes. It has also been determined that the bacterial cytoskeleton plays important roles in cell shape, cell division, and integrity of the cell wall.

Inclusions

Bacterial inclusions are generally defined as a distinct structure located either within the cytoplasm or periplasm of the cell. They can range in complexity, from a simple compilation of chemicals such as crystals, to fairly complex structures that start to rival that of the eukaryotic organelles, complete with a membranous external layer. Their role is often to store components as metabolic reserves for the cell when a substance is found in excess, but they can also play a role in motility and metabolic functions as well.

Carbon storage

Carbon is the most common substance to be stored by a cell, since all cells are carbon based. In addition, carbon compounds can often be broken down quickly by the cell, so they can serve as energy sources as well. One of the simplest and most common inclusions for carbon storage is glycogen, in which glucose units are linked together in a multi-branching polysaccharide structure.

Another common way for bacteria to store carbon is in the form of poly-β-hydroxybutyrate (PHB), a granule that forms when β-hydroxybutyric acid units aggregate together. This lipid is very plastic-like in composition, leading some scientists to investigate the possibility of using them as a biodegradeable plastic. The PHB granules actually have a shell composed of both protein and a small amount of phospholipid. Both glycogen and PHB are formed when there is an excess of carbon and then broken down by the cell later for both carbon and energy.

Inorganic storage

Often bacteria need something other than carbon, either for synthesis of cell components or as an alternate energy reserve. Polyphosphate granules allow for the accumulation of inorganic phosphate (PO43-), where the phosphate can be used to make nucleic acid or ATP.

Other cells need sulphur as a source of electrons for their metabolism and will store excess sulphur in the form of sulphur globules, which result when the cell oxidises hydrogen sulphide (H2S) to elemental sulphur (S0), resulting in the formation of refractile inclusions.

Non-storage  functions

There are times when a bacterium needs to do something beyond simple storage of organic or inorganic compounds for use in metabolism and there are inclusions to help with these non-storage functions. One such example is gas vacuoles, which are used by the cell to control buoyancy in a water column, providing the cell with some control over where it is in the environment. It is a limited form of motility, on the vertical axis only. Gas vacuoles are composed of conglomerations of gas vesicles, cylindrical structures that are both hollow and rigid. The gas vesicles are freely permeable to all types of gases by passive diffusion and can be quickly constructed or collapsed, as needed by the cell to ascend or descend.

Magnetosomes are inclusions that contain long chains of magnetite (Fe3O4), which are used by the cell as a compass in geomagnetic fields, for orientation within their environment. Magnetotactic bacteria are typically microaerophilic, preferring an environment with a lower level of oxygen than the atmosphere. The magenetosome allows the cells to locate the optimum depth for their growth. Magenetosomes have a true lipid bilayer, reminiscent of eukaryotic organelles, but it is actually an invagination of the cell’s plasma membrane that has been modified with specific proteins.

Microcompartments

Bacterial microcompartments (BMCs) are unique from other inclusions by virtue of their structure and functionality. They are icosahedral in shape and composed of a protein shell made up of various proteins in the BMC family. While their exact role varies, they all participate in functions beyond simple storage of substances. These compartments provide both a location and the substances (usually enzymes) necessary for particular metabolic activities.

Chlorosomes

Found in some phototrophic bacteria, a chlorosome is a highly efficient structure for capturing low light intensities. Lining the inside perimeter of the cell membrane, each chlorosome can contain up to 250,000 bacteriochlorophyll molecules, arranged in dense arrays. Harvested light is transferred to reaction centers in the cell membrane, allowing the conversion from light energy to chemical energy in the form of ATP. The chlorosome is bounded by a lipid monolayer.

Plasmids

A plasmid is an extrachromosomal piece of DNA that some bacteria have, in addition to the genetic material found in the nucleoid. It is composed of double-stranded DNA and is typically circular, although linear plasmids have been found. Plasmids are described as being “non-essential” to the cell, where the cell can function normally in their absence. But while plasmids have only a few genes, they can confer important capabilities for the cell, such as antibiotic resistance. Plasmids replicate independently of the cell and can be lost (known as curing), either spontaneously or due to exposure to adverse conditions, such as UV light, thymine starvation, or growth above optimal conditions. Some plasmids, known as episomes, can be integrated into the cell chromosome where the genes will be replicated during cell division.

Bacterial survival

Endospore

Then there is the endospore, a marvel of bacterial engineering. This is located under the heading “bacterial internal components,” but it is important to note that an endospore isn’t an internal or external structure so much as a conversion of the cell into an alternate form. Cells start out as a vegetative cell, doing all the things a cell is supposed to do (metabolising, reproducing, mowing the lawn…). If they get exposed to hostile conditions (dessication, high heat, an angry neighbour…) and they have the ability, they might convert from a vegetative cell into an endospore. The endospore is actually formed within the vegetative cell (doesn’t that make it an internal structure?) and then the vegetative cell lyses, releasing the endospore (does that make it an external structure?). Endospores are only formed by a few gram positive genera and provide the cell with resistance to a wide variety of harsh conditions, such as starvation, extremes in temperature, exposure to drying, UV light, chemicals, enzymes, and radiation. While the vegetative cell is the active form for bacterial cells (growing, metabolising, etc), the endospore can be thought of as a dormant form of the cell. It allows for survival of adverse conditions, but it does not allow the cell to grow or reproduce.

Structure

In order to be so incredibly resistant to so many different substances and environmental conditions, many different layers are necessary. The bacterial endospore has many different layers, starting with a core in the centre (Figure 9.12). The core is the location of the nucleoid, ribosomes, and cytoplasm of the cell, in an extremely dehydrated form. It typically contains only 25% of the water found in a vegetative cell, increasing heat resistance. The DNA is further protected by the presence of small acid-soluble proteins (SASPs), which stabilise the DNA and protect it from degradation. DNA stabilisation is increased by the presence of dipicolinic acid complexed with calcium (Ca-DPA), which inserts between the DNA bases. The core is wrapped in an inner membrane that provides a permeability barrier to chemicals, which is then surrounded by the cortex, a thick layer consisting of peptidoglycan with less cross-linking than is found in the vegetative cell. The cortex is wrapped in an outer membrane. Lastly are several spore coats made of protein, which provide protection from environmental stress such as chemicals and enzymes.

Sporulation: conversion from vegetative cell to endospore

Sporulation, the conversion of vegetative cell into the highly protective endospore, typically occurs when the cell’s survival is threatened in some way. The actual process is very complex and typically takes several hours until completion (Figure 9.13). Initially the sporulating cells replicates its DNA, as if it were about to undergo cell division. A septum forms asymmetrically, sequestering one copy of the chromosome at one end of the cell (called the forespore). Synthesis of endospore-specific substances occurs, altering the forespore and leading to the development of the layers specific for an endospore, as well as dehydration. Eventually the “mother cell” lyses, allowing for the release of the mature endospore into the environment.

Conversion from endospore to vegetative cell

The endospore remains dormant until environmental conditions improve, causing a chemical change that initiates gene expression. There are three distinct stages in the conversion from an endospore to the metabolically active vegetative cells: 1) activation, a preparation step that can be initiated by the application of heat; 2) germination, when the endospore becomes metabolically active and begins to take on water; 3) outgrowth, when the vegetative cell fully emerges from the endospore shell.

Layers outside the cell wall

A bacterial capsule is a polysaccharide layer that completely envelopes the cell. It is well organised and tightly packed, which explains its resistance to staining under the microscope. The capsule offers protection from a variety of different threats to the cell, such as desiccation, hydrophobic toxic materials (i.e. detergents), and bacterial viruses. The capsule can enhance the ability of bacterial pathogens to cause disease and can provide protection from phagocytosis (engulfment by white blood cells known as phagocytes). Lastly, it can help in attachment to surfaces.

Slime layer

A bacterial slime layer is similar to the capsule in that it is typically composed of polysaccharides and it completely surrounds the cell. It also offers protection from various threats, such as desiccation and antibiotics. It can also allow for adherence to surfaces. So, how does it differ from the capsule? A slime layer is a loose, unorganised layer that is easily stripped from the cell that made it, as opposed to a capsule which integrates firmly around the bacterial cell wall (Figure 9.14).

S-layer

Some bacteria have a highly organised layer made of secreted proteins or glycoproteins that self-assemble into a matrix on the outer part of the cell wall. This regularly structured S-layer is anchored into the cell wall, although it is not considered to be officially part of the cell wall in bacteria. S-layers have very important roles for the bacteria that have them, particularly in the areas of growth and survival, and cell integrity.

S layers help maintain overall rigidity of the cell wall and surface layers, as well as cell shape, which are important for reproduction. S layers protect the cell from ion/pH changes, osmotic stress, detrimental enzymes, bacterial viruses, and predator bacteria. They can provide cell adhesion to other cells or surfaces. For pathogenic bacteria they can provide protection from phagocytosis.

Structures outside the cell wall

Fimbriae (sing. fimbria)

Fimbriae are thin filamentous appendages that extend from the cell, often in the tens or hundreds. They are composed of pilin proteins and are used by the cell to attach to surfaces. They can be particularly important for pathogenic bacteria, which use them to attach to host tissues.

Pili (sing. pilus)

Pili are very similar to fimbriae in that they are thin filamentous appendages that extend from the cell and are made of pilin proteins. Pili can be used for attachment as well, to both surfaces and host cells, such as the Neisseria gonorrhea cells that use their pili to grab onto sperm cells, for passage to the next human host.

So, why would some researchers bother differentiating between fimbriae and pili?

Pili are typically longer than fimbriae, with only 1-2 present on each cell, but that hardly seems enough to set the two structures apart. It really boils down to the fact that a few specific pili participate in functions beyond attachment. The conjugative pili participate in the process known as conjugation, which allows for the transfer of a small piece of DNA from a donor cell to a recipient cell. The type IV pili play a role in an unusual type of motility known as twitching motility, where a pilus attaches to a solid surface and then contracts, pulling the bacterium forward in a jerky motion.

Flagella (sing. flagellum)

Bacterial motility is typically provided by structures known as flagella (Figure 9.15). The bacterial flagellum differs in composition, structure, and mechanics from the eukaryotic flagellum, which operates as a flexible whip-like tail utilising microtubules that are powered by ATP. The bacterial flagellum is rigid in nature, operates more like the propeller on a boat, and is powered by energy from the proton motive force.

There are three main components to the bacterial flagellum:

1. the filament – a long thin appendage that extends from the cell surface. The filament is composed of the protein flagellin and is hollow. Flagellin proteins are transcribed in the cell cytoplasm and then transported across the cell membrane and cell wall. A bacterial flagellar filament grows from its tip (unlike the hair on your head), adding more and more flagellin units to extend the length until the correct size is reached. The flagellin units are guided into place by a protein cap.
2. the hook – this is a curved coupler that attaches the filament to the flagellar motor.
3. the motor – a rotary motor that spans both the cell membrane and the cell wall, with additional components for the gram negative outer membrane. The motor has two components: the basal body, which provides the rotation, and the stator, which provides the torque necessary for rotation to occur. The basal body consists of a central shaft surrounded by protein rings, two in the gram positive bacteria and four in the gram negative bacteria. The stator consists of Mot proteins that surround the ring(s) embedded within the cell membrane.

Bacterial Movement

Swimming

Rotation of the flagellar basal body occurs due to the proton motive force, where protons that accumulate on the outside of the cell membrane are driven through pores in the Mot proteins, interacting with charges in the ring proteins as they pass across the membrane. The interaction causes the basal body to rotate and turns the filament extending from the cell.

Corkscrew Motility

Some spiral-shaped bacteria, known as the Spirochetes, utilise a corkscrew-motility due to their unusual morphology and flagellar conformation. These gram negative bacteria have specialized flagella that attach to one end of the cell, extend back through the periplasm and then attach to the other end of the cell. When these endoflagella rotate they put torsion on the entire cell, resulting in a flexing motion that is particularly effective for burrowing through viscous liquids.

Gliding Motility

Gliding motility is just like it sounds, a slower and more graceful movement than the other forms covered so far. Gliding motility is exhibited by certain filamentous or bacillus bacteria and does not require the use of flagella. It does require that the cells be in contact with a solid surface, although more than one mechanism has been identified. Some cells rely on slime propulsion, where secreted slime propels the cell forward, where other cells rely on surface layer proteins to pull the cell forward.

Chemotaxis

Now that we have covered the basics of the bacterial flagellar motor and mechanics of bacterial swimming, let us combine the two topics to talk about chemotaxis. Chemotaxis refers to the movement of an organism towards or away from a chemical. You can also have phototaxis, where an organism is responding to light. In chemotaxis, a favourable substance (such as a nutrient) is referred to as an attractant, while a substance with an adverse effect on the cell (such as a toxin) is referred to as a repellant. In the absence of either an attractant or a repellant a cell will engage in a “random walk,” where it alternates between tumbles and runs, in the end getting nowhere in particular. In the presence of a gradient of some type, the movements of the cell will become biased, resulting over time in the movement of the bacterium towards an attractant and away from any repellants.

How does this happen?

First, let us cover how a bacterium knows which direction to go. Bacteria rely on protein receptors embedded within their membrane, called chemoreceptors, which bind specific molecules. Binding typically results in methylation or phosphorylation of the chemoreceptor, which triggers an elaborate protein pathway that eventually impacts the rotation of the flagellar motor. The bacteria engage in temporal sensing, where they compare the concentration of a substance with the concentration obtained just a few seconds (or microseconds) earlier. In this way they gather information about the orientation of the concentration gradient of the substance. As a bacterium moves closer to the higher concentrations of an attractant, runs (dictated by CCW flagellar rotation) become longer, while tumbling (dictated by CW flagellar rotation) decreases. There will still be times that the bacterium will head off in the wrong direction away from an attractant since tumbling results in a random reorientation of the cell, but it won’t head in the wrong direction for very long. The resulting “biased random walk” allows the cell to quickly move up the gradient of an attractant (or move down the gradient of a repellant).