Bacteria

Most archaea (but not *Thermoplasma* and *Ferroplasma*) possess a cell wall. In most archaea the wall is assembled from surface-layer proteins, which form an S-layer. An S-layer is a rigid array of protein molecules that cover the outside of the cell (like chain mail). This layer provides both chemical and physical protection, and can prevent macromolecules from contacting the cell membrane. Unlike bacteria, archaea lack peptidoglycan in their cell walls.

* Methanobacteriales do have cell walls containing pseudopeptidoglycan, which resembles eubacterial peptidoglycan in morphology, function, and physical structure, but pseudopeptidoglycan is distinct in chemical structure; it lacks D-amino acids and N-acetylmuramic acid, substituting the latter with N-Acetyltalosaminuronic acid.

All gram-positive bacteria are bounded by a single-unit lipid membrane, and, in general, they contain a thick layer (20–80 nm) of peptidoglycan responsible for retaining the Gram stain.

* A number of other bacteria—that are bounded by a single membrane, but stain gram-negative due to either lack of the peptidoglycan layer, as in the Mycoplasmas, or their inability to retain the Gram stain because of their cell wall composition—also show close relationship to the Gram-positive bacteria.

For the bacterial cells bounded by a single cell membrane, the term "monoderm bacteria" or "monoderm prokaryotes" has been proposed.

In contrast all archetypical gram-negative bacteria are bounded by a cytoplasmic membrane and an outer cell membrane; they contain only a thin layer of peptidoglycan (2–3 nm) between these membranes. The presence of inner and outer cell membranes defines a new compartment in these cells: the periplasmic space or the periplasmic compartment. These bacteria have been designated as "diderm bacteria." Of these two structurally distinct groups of bacteria, monoderms are indicated to be ancestral.

* Based upon a number of observations including that the gram-positive bacteria are the major producers of antibiotics and that, in general, gram-negative bacteria are resistant to them, it has been proposed that the outer cell membrane in gram-negative bacteria (diderms) has evolved as a protective mechanism against antibiotic selection pressure.
* Some bacteria, such as *Deinococcus*, which stain gram-positive due to the presence of a thick peptidoglycan layer and also possess an outer cell membrane are suggested as intermediates in the transition between monoderm (gram-positive) and diderm (gram-negative) bacteria.

The diderm bacteria can also be further differentiated between

* simple diderms lacking lipopolysaccharide,
* the archetypical diderm bacteria where the outer cell membrane contains lipopolysaccharide,
* and the diderm bacteria where outer cell membrane is made up of mycolic acid.

## Archaea

Archaea and bacteria have generally similar cell structure, but cell composition and organization set the archaea apart. Like bacteria, archaea lack interior membranes and organelles. Like bacteria, the cell membranes of archaea are usually bounded by a cell wall and they swim using one or more flagella. Structurally, archaea are most similar to gram-positive bacteria. Most have a single plasma membrane and cell wall, and lack a periplasmic space; the exception to this general rule is *Ignicoccus*, which possess a particularly large periplasm that contains membrane-bound vesicles and is enclosed by an outer membrane.

### Flagella

Further information: Cell wall#Archaeal cell walls
Archaeal flagella are known as archaella, that operate like bacterial flagella – their long stalks are driven by rotatory motors at the base. These motors are powered by a proton gradient across the membrane, but archaella are notably different in composition and development. The two types of flagella evolved from different ancestors. The bacterial flagellum shares a common ancestor with the type III secretion system, while archaeal flagella appear to have evolved from bacterial type IV pili. In contrast with the bacterial flagellum, which is hollow and assembled by subunits moving up the central pore to the tip of the flagella, archaeal flagella are synthesized by adding subunits at the base.

### Membranes

Archaeal membranes are made of molecules that are distinctly different from those in all other life forms, showing that archaea are related only distantly to bacteria and eukaryotes. In all organisms, cell membranes are made of molecules known as phospholipids. These molecules possess both a polar part that dissolves in water (the phosphate "head"), and a "greasy" non-polar part that does not (the lipid tail). These dissimilar parts are connected by a glycerol moiety. In water, phospholipids cluster, with the heads facing the water and the tails facing away from it. The major structure in cell membranes is a double layer of these phospholipids, which is called a lipid bilayer.

The phospholipids of archaea are unusual in four ways:

* They have membranes composed of glycerol-ether lipids, whereas bacteria and eukaryotes have membranes composed mainly of glycerol-ester lipids. The difference is the type of bond that joins the lipids to the glycerol moiety; the two types are shown in yellow in the figure at the right. In ester lipids this is an ester bond, whereas in ether lipids this is an ether bond.
* The stereochemistry of the archaeal glycerol moiety is the mirror image of that found in other organisms. The glycerol moiety can occur in two forms that are mirror images of one another, called *enantiomers*. Just as a right hand does not fit easily into a left-handed glove, enantiomers of one type generally cannot be used or made by enzymes adapted for the other. The archaeal phospholipids are built on a backbone of *sn*-glycerol-1-phosphate, which is an enantiomer of *sn*-glycerol-3-phosphate, the phospholipid backbone found in bacteria and eucaryotes. This suggests that archaea use entirely different enzymes for synthesizing phospholipids as compared to bacteria and eukaryotes. Such enzymes developed very early in life's history, indicating an early split from the other two domains.
* Archaeal lipid tails differ from those of other organisms in that they are based upon long isoprenoid chains with multiple side-branches, sometimes with cyclopropane or cyclohexane rings. By contrast, the fatty acids in the membranes of other organisms have straight chains without side branches or rings. Although isoprenoids play an important role in the biochemistry of many organisms, only the archaea use them to make phospholipids. These branched chains may help prevent archaeal membranes from leaking at high temperatures.
* In some archaea, the lipid bilayer is replaced by a monolayer. In effect, the archaea fuse the tails of two phospholipid molecules into a single molecule with two polar heads (a bolaamphiphile); this fusion may make their membranes more rigid and better able to resist harsh environments. For example, the lipids in *Ferroplasma* are of this type, which is thought to aid this organism's survival in its highly acidic habitat.

## Gram-positive bacteria

**Gram-positive bacteria** are bacteria that give a positive result in the Gram stain test, which is traditionally used to quickly classify bacteria into two broad categories according to their cell wall.

Gram-positive bacteria take up the crystal violet stain used in the test, and then appear to be purple-coloured when seen through an optical microscope. This is because the thick peptidoglycan layer in the bacterial cell wall retains the stain after it is washed away from the rest of the sample, in the decolorization stage of the test.

In general, the following characteristics are present in gram-positive bacteria:

1. Cytoplasmic lipid membrane
2. Thick peptidoglycan layer
3. Teichoic acids and lipoids are present, forming lipoteichoic acids, which serve as chelating agents, and also for certain types of adherence.
4. Peptidoglycan chains are cross-linked to form rigid cell walls by a bacterial enzyme DD-transpeptidase.
5. A much smaller volume of periplasm than that in gram-negative bacteria.

Only some species have a capsule, usually consisting of polysaccharides. Also, only some species are flagellates, and when they do have flagella, have only two basal body rings to support them, whereas gram-negative have four. Both gram-positive and gram-negative bacteria commonly have a surface layer called an S-layer. In gram-positive bacteria, the S-layer is attached to the peptidoglycan layer. Gram-negative bacteria's S-layer is attached directly to the outer membrane. Specific to gram-positive bacteria is the presence of teichoic acids in the cell wall. Some of these are lipoteichoic acids, which have a lipid component in the cell membrane that can assist in anchoring the peptidoglycan.

## Peptidoglycan

From Wikipedia:Peptidoglycan

**Peptidoglycan** **(murein)** is a polymer consisting of sugars and amino acids that forms a mesh-like layer outside the plasma membrane of most bacteria, forming the cell wall. The sugar component consists of alternating residues of β-(1,4) linked *N*-acetylglucosamine (NAG) and *N*-acetylmuramic acid (NAM). Attached to the *N*-acetylmuramic acid is a peptide chain of three to five amino acids. The peptide chain can be cross-linked to the peptide chain of another strand forming the 3D mesh-like layer. Peptidoglycan serves a structural role in the bacterial cell wall, giving structural strength, as well as counteracting the osmotic pressure of the cytoplasm. Peptidoglycan is also involved in binary fission during bacterial cell reproduction.

The peptidoglycan layer is substantially thicker in Gram-positive bacteria (20 to 80 nanometers) than in Gram-negative bacteria (7 to 8 nanometers). Peptidoglycan forms around 90% of the dry weight of Gram-positive bacteria but only 10% of Gram-negative strains. Thus, presence of high levels of peptidoglycan is the primary determinant of the characterisation of bacteria as Gram-positive. In Gram-positive strains, it is important in attachment roles and serotyping purposes. For both Gram-positive and Gram-negative bacteria, particles of approximately 2 nm can pass through the peptidoglycan.

The peptidoglycan layer in the bacterial cell wall is a crystal lattice structure formed from linear chains of two alternating amino sugars, namely *N*-acetylglucosamine (GlcNAc or NAGA) and *N*-acetylmuramic acid (MurNAc or NAMA). The alternating sugars are connected by a β-(1,4)-glycosidic bond. Each MurNAc is attached to a short (4- to 5-residue) amino acid chain, containing L-alanine, D-glutamic acid, *meso*-diaminopimelic acid, and D-alanine in the case of *Escherichia coli* (a Gram-negative bacterium) or L-alanine, D-glutamine, L-lysine, and D-alanine with a 5-glycine interbridge between tetrapeptides in the case of *Staphylococcus aureus* (a Gram-positive bacterium). Peptidoglycan is one of the most important sources of D-amino acids in nature.

Cross-linking between amino acids in different linear amino sugar chains occurs with the help of the enzyme DD-transpeptidase and results in a 3-dimensional structure that is strong and rigid. The specific amino acid sequence and molecular structure vary with the bacterial species.

Penicillin binding protein forming cross-links in newly formed bacterial cell wall.
## Gram-negative bacteria

**Gram-negative bacteria** are bacteria that do not retain the crystal violet stain used in the Gram staining method of bacterial differentiation. They are characterized by their cell envelopes, which are composed of a thin peptidoglycan cell wall sandwiched between an inner cytoplasmic cell membrane and a bacterial outer membrane.

Their outer membrane protects them from many antibiotics (including penicillin); detergents that would normally damage the peptidoglycans of the (inner) cell membrane; and lysozyme, an antimicrobial enzyme produced by animals that forms part of the innate immune system. The periplasmic space (space between the two cell membranes) also contains enzymes which break down or modify antibiotics.

Gram-negative bacteria display these characteristics:

1. An inner cell membrane is present (cytoplasmic)
2. A thin peptidoglycan layer is present (This is much thicker in gram-positive bacteria)
3. Has outer membrane containing lipopolysaccharides (LPS, which consists of lipid A, core polysaccharide, and O antigen) in its outer leaflet and phospholipids in the inner leaflet
4. Porins exist in the outer membrane, which act like pores for particular molecules
5. Between the outer membrane and the cytoplasmic membrane there is a space filled with a concentrated gel-like substance called periplasm
6. The S-layer is directly attached to the outer membrane rather than to the peptidoglycan
7. If present, flagella have four supporting rings instead of two
8. Teichoic acids or lipoteichoic acids are absent
9. Lipoproteins are attached to the polysaccharide backbone
10. Some contain Braun's lipoprotein, which serves as a link between the outer membrane and the peptidoglycan chain by a covalent bond
11. Most, with very few exceptions, do not form spores

One of the several unique characteristics of gram-negative bacteria is the structure of the bacterial outer membrane. The outer leaflet of this membrane comprises a complex lipopolysaccharide (LPS) whose lipid portion acts as an endotoxin. If gram-negative bacteria enter the circulatory system, the LPS can cause a toxic reaction. This results in fever, an increased respiratory rate, and low blood pressure. This may lead to life-threatening septic shock.

### Lipopolysaccharide

From Wikipedia:Lipopolysaccharide

**Lipopolysaccharides** (**LPS**), also known as lipoglycans and **endotoxins**, are large molecules consisting of a lipid and a polysaccharide composed of O-antigen, outer core and inner core joined by a covalent bond; they are found in the outer membrane of Gram-negative bacteria.

The term *lipooligosaccharide* ("LOS") is used to refer to a low-molecular-weight form of bacterial lipopolysaccharides.

It comprises three parts:

1. O antigen (or O polysaccharide)
2. Core oligosaccharide
3. Lipid A

#### O-antigen

A repetitive glycan polymer contained within an LPS is referred to as the O antigen, O polysaccharide, or O side-chain of the bacteria.
The O antigen is attached to the core oligosaccharide, and comprises the outermost domain of the LPS molecule. The composition of the O chain varies from strain to strain. For example, there are over 160 different O antigen structures produced by different *E. coli* strains. The presence or absence of O chains determines whether the LPS is considered rough or smooth. Full-length O-chains would render the LPS smooth, whereas the absence or reduction of O-chains would make the LPS rough. Bacteria with rough LPS usually have more penetrable cell membranes to hydrophobic antibiotics, since a rough LPS is more hydrophobic. O antigen is exposed on the very outer surface of the bacterial cell, and, as a consequence, is a target for recognition by host antibodies.

#### Core

Main articles: Core oligosaccharide
The Core domain always contains an oligosaccharide component that attaches directly to lipid A and commonly contains sugars such as heptose and 3-Deoxy-D-manno-oct-2-ulosonic acid (also known as KDO, keto-deoxyoctulosonate). The LPS Cores of many bacteria also contain non-carbohydrate components, such as phosphate, amino acids, and ethanolamine substituents.

#### Lipid A

Main articles: Lipid A
Lipid A is, in normal circumstances, a phosphorylated glucosamine disaccharide decorated with multiple fatty acids. These hydrophobic fatty acid chains anchor the LPS into the bacterial membrane, and the rest of the LPS projects from the cell surface. The lipid A domain is responsible for much of the toxicity of Gram-negative bacteria. When bacterial cells are lysed by the immune system, fragments of membrane containing lipid A are released into the circulation, causing fever, diarrhea, and possible fatal endotoxic shock (also called septic shock). The Lipid A moiety is a very conserved component of the LPS. However Lipid A structure varies among bacterial species and Lipid A structure defines an overall host immune activation.

### Biological effects on hosts infected with Gram-negative bacteria

#### Immune response

LPS acts as the prototypical endotoxin because it binds the CD14/TLR4/MD2 receptor complex in many cell types, but especially in monocytes, dendritic cells, macrophages and B cells, which promotes the secretion of pro-inflammatory cytokines, nitric oxide, and eicosanoids.

As part of the cellular stress response, superoxide is one of the major reactive oxygen species induced by LPS in various cell types that express TLR (toll-like receptor).

LPS is also an exogenous pyrogen (fever-inducing substance).

Being of crucial importance to Gram-negative bacteria, these molecules make candidate targets for new antimicrobial agents.

Some researchers doubt reports of generalized toxic effects attributed to all lipopolysaccharides, in particular, for cyanobacteria.

LPS function has been under experimental research for several years due to its role in activating many transcription factors. LPS also produces many types of mediators involved in septic shock. Humans are much more sensitive to LPS than other animals (e.g., mice). A dose of 1 µg/kg induces shock in humans, but mice will tolerate a dose up to a thousand times higher. This may relate to differences in the level of circulating natural antibodies between the two species.
Said et al. showed that LPS causes an IL-10-dependent inhibition of CD4 T-cell expansion and function by up-regulating PD-1 levels on monocytes which leads to IL-10 production by monocytes after binding of PD-1 by PD-L1.

Endotoxins are in large part responsible for the dramatic clinical manifestations of infections with pathogenic Gram-negative bacteria, such as *Neisseria meningitidis*, the pathogens that causes meningococcal disease, including meningococcemia, Waterhouse–Friderichsen syndrome, and meningitis.

Bruce Beutler was awarded a portion of the 2011 Nobel Prize in Physiology or Medicine for his work demonstrating that TLR4 is the LPS receptor.

Portions of the LPS from several bacterial strains have been shown to be chemically similar to human host cell surface molecules; the ability of some bacteria to present molecules on their surface which are chemically identical or similar to the surface molecules of some types of host cells is termed molecular mimicry. For example, in *Neisseria meningitidis* L2,3,5,7,9, the terminal tetrasaccharide portion of the oligosaccharide (lacto-N-neotetraose) is the same tetrasaccharide as that found in paragloboside, a precursor for ABH glycolipid antigens found on human erythrocytes. In another example, the terminal trisaccharide portion (lactotriaose) of the oligosaccharide from pathogenic *Neisseria* spp. LOS is also found in lactoneoseries glycosphingolipids from human cells. Most meningococci from groups B and C, as well as gonococci, have been shown to have this trisaccharide as part of their LOS structure. The presence of these human cell surface 'mimics' may, in addition to acting as a 'camouflage' from the immune system, play a role in the abolishment of immune tolerance when infecting hosts with certain human leukocyte antigen (HLA) genotypes, such as HLA-B35.

Recently, a new study published has discovered that LPS can be sensed directly by hematopoietic stem cells (HSCs) through the bonding with TLR4, causing them to proliferate in reaction to a systemic infection. This response activate the TLR4-TRIF-ROS-p38 signaling within the HSCs and through a sustained TLR4 activation can cause a proliferative stress, leading to impair their competitive repopulating ability. Infection in mice using *S. typhimurium* showed similar results, validating the experimental model also in vivo.

#### Effect of variability on immune response

O-antigens (the outer carbohydrates) are the most variable portion of the LPS molecule, imparting the antigenic specificity. In contrast, lipid A is the most conserved part. However, lipid A composition also may vary (e.g., in number and nature of acyl chains even within or between genera). Some of these variations may impart antagonistic properties to these LPS. For example, Rhodobacter sphaeroides diphosphoryl lipid A (RsDPLA) is a potent antagonist of LPS in human cells, but is an agonist in hamster and equine cells.

It has been speculated that conical Lipid A (e.g., from *E. coli*) are more agonistic, less conical lipid A like those of *Porphyromonas gingivalis* may activate a different signal (TLR2 instead of TLR4), and completely cylindrical lipid A like that of *Rhodobacter sphaeroides* is antagonistic to TLRs.

LPS gene clusters are highly variable between different strains, subspecies, species of bacterial pathogens of plants and animals.

Normal human blood serum contains anti-LOS antibodies that are bactericidal and patients that have infections caused by serotypically distinct strains possess anti-LOS antibodies that differ in their specificity compared with normal serum. These differences in humoral immune response to different LOS types can be attributed to the structure of the LOS molecule, primarily within the structure of the oligosaccharide portion of the LOS molecule.
In *Neisseria gonorrhoeae* it has been demonstrated that the antigenicity of LOS molecules can change during an infection due to the ability of these bacteria to synthesize more than one type of LOS, a characteristic known as phase variation. Additionally, *Neisseria gonorrhoeae*, as well as *Neisseria meningitidis* and *Haemophilus influenzae*, are capable of further modifying their LOS *in vitro*, for example through sialylation (modification with sialic acid residues), and as a result are able to increase their resistance to complement-mediated killing or even down-regulate complement activation or evade the effects of bactericidal antibodies. Sialylation may also contribute to hindered neutrophil attachment and phagocytosis by immune system cells as well as a reduced oxidative burst. *Haemophilus somnus*, a pathogen of cattle, has also been shown to display LOS phase variation, a characteristic which may help in the evasion of bovine host immune defenses.
Taken together, these observations suggest that variations in bacterial surface molecules such as LOS can help the pathogen evade both the humoral (antibody and complement-mediated) and the cell-mediated (killing by neutrophils, for example) host immune defenses.

#### Non-canonical pathways of LPS recognition

Recently, it was shown that in addition to TLR4 mediated pathways, certain members of the family of the transient receptor potential ion channels recognize LPS. LPS-mediated activation of TRPA1 was shown in mice and *Drosophila melanogaster* flies. At higher concentrations, LPS activates other members of the sensory TRP channel family as well, such as TRPV1, TRPM3 and to some extent TRPM8.
LPS is recognized by TRPV4 on epithelial cells. TRPV4 activation by LPS was necessary and sufficient to induce nitric oxide production with a bactericidal effect.

### Health effects

#### Endotoxemia

The presence of endotoxins in the blood is called endotoxemia. It can lead to septic shock, if the immune response is severely pronounced.

Moreover, endotoxemia of intestinal origin, especially, at the host-pathogen interface, is considered to be an important factor in the development of alcoholic hepatitis, which is likely to develop on the basis of the small bowel bacterial overgrowth syndrome and an increased intestinal permeability.

Lipid A may cause uncontrolled activation of mammalian immune systems with production of inflammatory mediators that may lead to septic shock. This inflammatory reaction is mediated by Toll-like receptor 4 which is responsible for immune system cell activation. Damage to the endothelial layer of blood vessels caused by these inflammatory mediators can lead to capillary leak syndrome, dilation of blood vessels and a decrease in cardiac function and can lead to septic shock. Pronounced complement activation can also be observed later in the course as the bacteria multiply in the blood. High bacterial proliferation triggering destructive endothelial damage can also lead to disseminated intravascular coagulation (DIC) with loss of function of certain internal organs such as the kidneys, adrenal glands and lungs due to compromised blood supply. The skin can show the effects of vascular damage often coupled with depletion of coagulation factors in the form of petechiae, purpura and ecchymoses. The limbs can also be affected, sometimes with devastating consequences such as the development of gangrene, requiring subsequent amputation. Loss of function of the adrenal glands can cause adrenal insufficiency and additional hemorrhage into the adrenals causes Waterhouse-Friderichsen syndrome, both of which can be life-threatening.
It has also been reported that gonococcal LOS can cause damage to human fallopian tubes.

#### Auto-immune disease

The molecular mimicry of some LOS molecules is thought to cause autoimmune-based host responses, such as flareups of multiple sclerosis. Other examples of bacterial mimicry of host structures via LOS are found with the bacteria *Helicobacter pylori* and *Campylobacter jejuni*, organisms which cause gastrointestinal disease in humans, and *Haemophilus ducreyi* which causes chancroid. Certain *C. jejuni* LPS serotypes (attributed to certain tetra- and pentasaccharide moieties of the core oligosaccharide) have also been implicated with Guillain–Barré syndrome and a variant of Guillain–Barré called Miller-Fisher syndrome.

#### Link to obesity

Epidemiological studies have shown that increased endotoxin load, which can be a result of increased populations of endotoxin-producing bacteria in the intestinal tract, is associated with certain obesity-related patient groups. Other studies have shown that purified endotoxin from *Escherichia coli* can induce obesity and insulin-resistance when injected into germ-free mouse models. A more recent study has uncovered a potentially contributing role for *Enterobacter cloacae* B29 toward obesity and insulin resistance in a human patient. The presumed mechanism for the association of endotoxin with obesity is that endotoxin induces an inflammation-mediated pathway accounting for the observed obesity and insulin resistance. Bacterial genera associated with endotoxin-related obesity effects include *Escherichia* and *Enterobacter*.

### Laboratory research and biotechnology production systems

Lipopolysaccharides are frequent contaminants in plasmid DNA prepared from bacteria or proteins expressed from bacteria, and *must* be removed from the DNA or protein to avoid contaminating experiments and to avoid toxicity of products manufactured using industrial fermentation.

Also, ovalbumin is frequently contaminated with endotoxins. Ovalbumin is one of the extensively studied proteins in animal models and also an established model allergen for airway hyper-responsiveness (AHR). Commercially available ovalbumin that is contaminated with LPS can fully activate endothelial cells in an in-vitro assay of the first step of inflammation, and it falsifies research results, as it does not accurately reflect the effect of sole protein antigen on animal physiology.

In pharmaceutical production, it is necessary to remove all traces of endotoxin from drug product containers, as even small amounts of endotoxin will cause illness in humans. A depyrogenation oven is used for this purpose. Temperatures in excess of 300 °C are required to break down this substance. A defined endotoxin reduction rate is a correlation between time and temperature. Based on primary packaging material as syringes or vials, a glass temperature of 250 °C and a holding time of 30 minutes is typical to achieve a reduction of endotoxin levels by a factor of 1000.

The standard assay for detecting presence of endotoxin is the Limulus Amebocyte Lysate (LAL) assay, utilizing blood from the Horseshoe crab (*Limulus polyphemus*). Very low levels of LPS can cause coagulation of the limulus lysate due to a powerful amplification through an enzymatic cascade. However, due to the dwindling population of horseshoe crabs, and the fact that there are factors that interfere with the LAL assay, efforts have been made to develop alternative assays, with the most promising ones being ELISA tests using a recombinant version of a protein in the LAL assay, Factor C.

## References

1. ↑ Cite error: Invalid `<ref>` tag; no text was provided for refs named `Sleytr2014`