Physiology Of Plasmids

PHYSIOLOGY OF PLASMIDS


General Properties Of Plasmids

Plasmids are closed circular DNA molecules which replicate independently of the bacterial chromosome. They code for functions involved in their own life cycles and also for functions which affect the physiology of the host cell. Properties vary from plasmid to plasmid.

Maintenance is the ability of a plasmid to replicate in phase with the host chromosome, hence to survive in the host bacterium. Host range varies widely, some plasmids are restricted to a few closely related bacteria (e.g. F factor of E. coli and related enterobacteria) others have a wide host range (e.g. P-type plasmids can live in most gram negative genera tested).

Copy Number varies from 1 or 2 per chromosome (e.g. F, P-types) to 40 or 50 (e.g. ColEl). Number of copies has major effect on expression of plasmid-borne characters especially antibiotic resistance.

Size varies enormously. The F-factor is about 1% size of E. coli chromosome, whereas most high copy plasmids are much smaller (eg. ColEl about 1/10 size of F). Very large plasmids are sometimes found (eg. >10% of chromosome size) but are difficult to work with and few have been properly characterized.

Transferability: Many plasmids can transfer themselves from host to host (eg. F & P-types, most R-plasmids). In addition some (e.g. F) can also mobilize the bacterial chromosome. Others cannot self-transfer (e.g. ColEl).

Mobilizability: Many small plasmids such as ColEl cannot transfer themselves, but can be mobilized by transmissable plasmids like F or ColV. Not all non-self-transmissible plasmids can be mobilized.

Incompatibility: Two plasmids which belong to the same family cannot coexist in the same cell. Such incompatibility groups are designated by letters of alphabet, e.g. F-type plasmids include F, ColV, Rl; P-types include RPl and R751. Plasmids of a given incompatibility group are homologous in the DNA sequences involved in replication, maintenance etc. although the genes encoding other properties may be very different.

Cryptic plasmids confer no identifiable phenotype on the host cell. The properties of these cryptic plasmids are presumably of some use to the host cell but are still unknown.

Colicins and Bacteriocins are lethal proteins made by one bacterial strain to kill other bacteria. They are almost always plasmid-encoded.

Virulence Factors are proteins made by bacteria to promote their infection of animals or plants. Often plasmid-encoded.


ANTIBIOTIC RESISTANCE PLASMIDS

Most R-plasmids are of moderate to large size and present in 1-2 copies per chromosome. Most are self-transmissable at a low frequency, although derepressed mutants showing high transfer frequency are sometimes found. The original F-factor is such a "naturally-occurring mutant". R-plasmids belong to a wide range of incompatibility groups. Many carry resistances to one or more antibiotics and/or toxic heavy metals and may also carry genes for colicins, virulence factors etc. Examples of plasmid types:

F-family. The F-plasmid itself carries no resistance markers and is a transfer derepressed mutant. Many resistance plasmids belong to this group e.g. R100 codes for resistance to streptomycin, chloramphenicol, tetracycline, and sulfonamides; R1 carries resistance to beta-lactams and kanamycin/neomycin. ColV and ColVB colicin plasmids are in F-family. ColV plasmids often specify virulence factors in addition to colicin V. Hly (hemolysin) and Ent (enterotoxin) plasmids are usually F-type. F-types are self-transmissable, large, single copy plasmids and can mediate transfer of many small non-self-transmissable plasmids e.g. ColEl.

I-family. Includes many R-plasmids (e.g. R64, R144) and plasmids carrying colicin I. Some I-type plasmids carry genes for both antibiotic resistance and colicin production. Self-transmissible, can mobilize other plasmids, large, single copy.

P-family. Noted for wide host range. Can self-transfer to almost every gram-negative genus tested in contrast to most plasmids which are restricted to a cluster of closely related bacteria. RPI (same as RP4, RK2), 62Md, single to five copies depending on host, carries resistance to beta-lactam, neomycin/kanamycin and tetracycline and is widely used in research. R751 specifies sulfonamide and trimethoprim resistance.

X-family. Unusual in being both multicopy (10-15 per cell) and self-transmissable. e.g. R6K, 26Md, carries resistance to beta-lactams and streptomycin.

Ecology of Plasmids
R-plasmids were in existence before therapeutic use of antibiotics but have become much more frequent in distribution since widescale use of antibiotics started. R-plasmids were discovered first in Japan in dysentery causing Shigella. Shown to be transferred to and from intestinal E. coli. Sulfonamide (Sul) resistance was found first, followed rapidly by tetracycline (Tet), chloramphenicol (Cam) and streptomycin (Str).
by 1952 - 80% of Shigella SulR
by 1960 - 11% of Shigella SulRCamRTetRStrR
by 1969 - 34% of Shigella SulRCamRTetRStrR

One outbreak of infant diarrhoea caused by enteropathogenic E. coli harbored a plasmid specifying resistance to b-lactams, streptomycin, chloramphenicol, tetracycline, erythromycin, neomycin/kanamycin and novobiocin and was eventually killed with gentamycin. In absence of antibiotic selection, R-plasmids tend gradually to be lost and resistant bacteria therefore decline in frequency. Major factor in R-plasmid spread is the practice of feeding animals (eg. pigs and chickens) antibiotics to increase yield. Recently some countries have banned use of human antibiotics in animal feed and there has been a major decline in frequency of R+ bacteria carried by farm animals.

Transposons (Tn)

Transposons are mobile genetic elements which can jump between plasmids, phages, chromosome etc. Responsible for rapid dissemination of resistance genes many of which occur on plasmids as part of a transposon. Commonest are:

Tn 1, 2, 3 beta-lactamase
Tn4 beta-lactamase
Tn5,6 neomycin/kanamycin phosphotransferase
Tn7 trimethoprim
Tn9 chloramphenicol
Tn10 tetracycline


Summary of Possible Mechanisms of Resistance:

The mechanism of R-plasmid encoded resistance is usually quite distinct from mechanisms observed in chromosomal mutants.

Antibiotic Chromosomal Resistance Plasmid Resistance

Beta-Lactams 1) loss of porins cleavage by
2) beta-lactamase beta-lactamase
3) altered PBP

Aminoglycosides 1) altered ribosomes modification
(Str, Neo, gentamycin) 2) defective transport

Chloramphenicol loss of porins acetylation

Tetracycline various but feeble impermeability

Erythromycin altered ribosomal proteins 1) altered rRNA
2) hydrolysis

Sulfonamides more target enzyme resistant enzyme

Trimethoprim 1) more target enzyme resistant enzyme
2) thymine auxotrophy

Nitrofurans loss of activating reductase not found

Rifamycins altered RNA polymerase not found


Beta-Lactams

b-Lactam antibiotics prevent cross-linking of the peptidoglycan of the bacterial cell envelope (see section on the cell envelope). This antibiotic family includes the well-known penicillins and cephalosporins, as well as newer types such as nocardicins, sulfazecins and clavulanic acid derivatives. A b-lactam structure is a four-membered ring containing an amide group.


Plasmid-encoded resistance to b-lactams is due to destruction of the antibiotics by the enzyme b-lactamase. This enzyme opens the b-lactam ring producing penicilloic acid from penicillins and cephalosporoic acid from cephalosporins. Cephalosporoic acid is unstable and decomposes spontaneously to complex products. Most b-lactamases prefer either penicillins or cephalosporins, though a few attack both equally well.



Gram positive b-lactamases are usually inducible. This causes a pronounced inoculum effect. If a small number of organisms is hit with a high concentration of antibiotic, death occurs before sufficient b-lactamase is produced to protect the bacteria. If inoculum is heavy or initial antibiotic level is relatively low, bacteria survive and b-lactamase is induced and its level rises to as much as 3% of total cell protein.

Gram negative b-lactamases fall into two main groups:
1) chromosomal; usually inducible, of low activity, prefer cephalosporins
2) plasmid encoded; constitutive, high activity, broad spectrum
Location is in periplasmic space - very efficient since only b-lactams entering the periplasmic-space can attack peptidoglycan synthesis. Chromosomal ampC mutants of E. coli which overproduce b-lactamase increase resistance level from about 1-2 mg/ml of ampicillin to around 10 mg/ml. Compare this with RPl which confers a resistance level of several mg/ml. Certain Pseudomonads carrying RPl can use ampicillin as sole carbon source!

Methicillin and cloxacillin have bulky side groups which make them resistant to almost all b-lactamases. They work well against b-lactam resistant gram positives but fail to penetrate the gram-negative outer membrane.


The original penicillin, benzyl-penicillin (= penicillin G) as well as methicillin, cloxacillin etc., penetrate the outer membrane of gram-negative bacteria very poorly. This is because they are too hydrophobic to diffuse through the water filled pores made by the porin proteins. Thus these antibiotics are only effective against gram-positives. Derivatives that are more hydrophilic were made by adding amino or carboxyl groups - ampicillin and carbenicillin respectively. These penetrate the pores of the outer membrane easily and are very active against gram-negative bacteria. Unfortunately they are readily broken down by b-lactamases.


Unlike penicillins which just have a single R-group, cephalosporins have two sites where modifying groups may be added. A wide variety of modified cephalosporins have been made, some of which are both resistant to b-lactamase and penetrate the outer membrane of gram-negatives reasonably well - e.g. cefuroxime and cefotaxime.

Another approach is to administer a mixture of a b-lactam antibiotic plus a b-lactamase inhibitor such as clavulanic acid or one of its derivatives. Clavulanic acid binds to b-lactamases and reacts forming a covalent bond to the protein that kills the enzyme. Clavulanic acid also penetrates well into gram-negative cells. Sulbactam is another b-lactamase inhibitor, but it doesn't penetrate gram-negatives as well.


Several new classes of b-lactam in addition to penicillins and cephalosporins have been discovered. Although the parent molecules are usually not of practical use, derivatives with assorted side chains are much more active.
Thienamycins -- sulfur in 5-membered ring is replaced by carbon; sulfur is found in a side chain attached to the 5-membered ring instead. These have a wide spectrum of activity and are relatively resistant to b-lactamases.
Nocardicins and Sulfazecins -- b-lactam ring is not fused to another ring at all. These are effective against gram negatives but have little activity against gram positives.

The origin of b-lactamases is uncertain but they were around before antibiotic therapy. Presumably b-lactamases were selected in environments where b-lactam producing fungi compete with bacteria for survival. Possibly they are mutant versions either of: a) transpeptidases which gained the ability to destroy the substrate analogs - the b-lactams or b) biosynthetic enzymes for b-lactam synthesis in the original producer organisms.


Aminoglycosides

This family of antibiotics consists of three (sometimes more) sugar rings, at least one of which (and ususally two or all three) has amino groups attached. They inhibit protein synthesis by binding to the small subunit of the ribosome. Streptomycin binds to a single site on the 30S subunit and distorts the A-site which prevents correct positioning of incoming aminoacyl-tRNAs. The gentamycin, tobramycin, kanamycin, neomycin group of aminoglycosides bind to multiple ribosomal sites and prevent the translocation step of protein synthesis.

Resistance is due to modification of the antibiotic by phosphorylation of -OH groups, adenylation (i.e. addition of AMP) of -OH groups or acetylation of -NH2 groups.
R + ATP Æ R-P + ADP
R + ATP Æ R-AMP + PP
R + Acetyl-CoA Æ R-Acetyl + CoA

A wide variety of modifying enzymes is found, including:
Streptomycin phosphotransferase
Neomycin/Kanamyin phosphotransferase
Streptomycin/Spectinomycin adenyltransferase
Gentamycin/Tobramycin/Kanamycin adenyltransferase
Aminoglycoside acetyl transferases (3 different but related enzymes, donate acetyl group to different positions) work on Neo, Kan, Gen and Tob to varying degrees.


Modified aminoglycosides can no longer induce the polyamine transport system by which they enter the cell and in addition they no longer inhibit their ribosomal target sites. There are many different aminoglycosides and a correspondingly wide range of modifying enzymes. Amikacin is a derivative of Kanamyin A with the l-NH2 group (the NH2 on the middle ring that gets acetylated) substituted with hydroxybutyrate. Amikacin is resistant to all modifying enzymes except one of the N-acetyl transferases and a recently discovered phosphotransferase specific for amikacin!

Aminoglycoside modifying enzymes probably came originally from Streptomyces strains which are the producer organisms. These strains contain modifying enzymes. Role of such enzymes in producer cell is 1) catalyse steps in biosynthesis and 2) self-protection.


Chloramphenicol

Chloramphenicol binds to the large subunit of the ribosome, probably to the 23S rRNA. It inhibits the peptidyl transferase reaction.


Chloramphenicol resistance is due to modification of the antibiotic by chloramphenicol acetyl transferase (CAT). Acetyl-CoA is used as a source of acetyl groups and two acetyl groups are added. Replacement of the terminal -OH of chloramphenicol with fluorine results in non-modifiable yet still antibacterially active derivatives.

Like b-lactamase, CAT is inducible in Staphylococcus but constitutive in E. coli. The induction process is slow due to fact that Cam is inhibiting protein synthesis conjointly with inducing synthesis of CAT (a protein!). Rapid induction can be achieved with the gratuitous inducer 3-deoxy-Cam which induces CAT but is not an enzyme substrate nor an inhibitor of protein synthesis. Gram positive CAT enzymes are highly homologous with each other and so are gram negative enzymes. The two groups differ greatly from each other except for sequence homology in the Cam-binding region.


Tetracyclines

Tetracycline binds to the 16S rRNA of the small subunit and stops protein synthesis by preventing the binding of aminoacyl-tRNAs. In fact, tetracycline binds to both prokaryotic and eukaryotic ribosomes. Bacteria are more sensitive than animal cells because tetracyclines are taken up by an energy-dependent transport system in bacteria but not eukaryotes. In fact eukaryotic cells actively export tetracyclines - as do bacteria that carry tetracycline resistance genes on R-plasmids. Since there is no similarity between Tet and any known transportable nutrients, the purpose of the bacterial transport system that takes up tetracycline and its mechanism of operation are still baffling.

Plasmid coded tetracycline resistance is similar in operation in both gram positives and gram negatives. Tetracycline resistance is typically two-level. A basal constitutive level of resistance protects by 5-10 fold relative to sensitive bacteria. In addition, exposure to tetracycline induces a second higher resistance level. Both resistance levels are due to successive drops in tetracycline accumulation. Tetracycline resistance is due to production of proteins which are found in the cytoplasmic membrane and actively expel tetracycline from the cell. Tetracycline enters the cell as the protonated form by an obscure active transport system. Inside the cell it binds Mg2+. The Tet resistance protein is powered by the proton motive force and acts by proton antiport to expel the Tet-Mg2+ complex.

Tetracycline (in tetracycline itself R = H)

Lipophilic tetracylines e.g. monocycline, where R = (CH3)2N–, probably penetrate membranes by diffusion well enough to short circuit the usual uptake system -- and also avoid the waiting resistance proteins. Tet resistance plasmids therefore confer little resistance to minocycline. Chlorotetracycline, where R = Cl, is intermediate in properties between minocycline and tetracycline.


Erythromycin

Erythromycin binds to the 23S rRNA in the large subunit of the ribosome. Due to distortion of the ribosome, the growing peptidyl-tRNA is lost prematurely.

In gram positive bacteria, resistance to erythromycin is due to an inducible plasmid coded rRNA methylase that modifies the 23sRNA of the 50s ribosomal subunit by dimethylation of a specific adenine. The modified ribosomes are resistant to erythromycin. Gram negative bacteria are inherently resistant to erythromycin which cannot penetrate the outer membrane effectively.


Sulfonamides and Trimethoprim

Sulfonamides are synthetic antibiotics. They are analogs of p-aminobenzoic acid, a precursor of folic acid. Sulfonamides inhibit dihydropteroate synthetase, an enzyme in the synthertic pathway for folate. The reduced form of folate is tetrahydrofolate, a cofactor used in the synthesis of methionine and thymine and other metabolites whose synthesis involves adding a single carbon. Animal cells cannot make folic acid and require it as a vitamin. Sulfonamides are therefore only active against bacteria that make their own folate.

Trimethoprim is an analog of the pterin ring portion of the tetrahydrofolate cofactor. It inhibits dihydrofolate reductase, the enzyme that converts dihydrofolate to tetrahydrofolate, the active form of the cofactor. The dihydrofolate reductase enzyme varies in structure in different groups of organisms.
Trimethoprim inhibits dihydrofolate reductase in bacteria.
Pyrimethamine inhibits dihydrofolate reductase in fungi.
Methotrexate inhibits dihydrofolate reductase in animals.


Plasmid mediated resistance to sulfonamides and trimethoprim in both cases involves synthesis of a plasmid encoded alternative enyme which is resistant to the inhibitor. For example R-plasmid encoded dihydropteroate synthetase has the same affinity for p-aminobenzoic acid as the chromosomal enzyme but it is several thousand times (Type I) or almost totally (Type II) resistant to sulfonamides. R-plasmid encoded dihydrofolate reductase is similarly resistant to trimethoprim. Sulfonamides plus trimethoprim are often used in combination for double blockade of the folate pathway, however, R-plasmids carrying both sulfonamide and trimethoprim resistance now occur (e.g. R388, a W-type plasmid).

Bacteriocins


Bacteriocins are protein antibiotics sythesized by certain strains of bacteria and lethal to other, related but sensitive strains. Bacteriocins made by E. coli are called colicins, those made by Enterobacter cloacae are cloacins, etc. {Since most work is done on E. coli, bacteriocins from other bacteria are often referred to as colicins even though not strictly correct.} Antagonism between bacteria may be due to production of lactic acid, NH3, fatty acids, nucleotide analogs, etc. and is therefore not always due to colicin production. Colicins are usually plasmid specified.

Some Representative Colicins:

Properties of Plasmid
Colicin Tra size (Md) copies Action of Colicin Receptor Uptake

ColB + 70 single copy pore formation FepA TonB
ColD – 3.3 multicopy inhibits protein synth FepA TonB
ColE1 – 4.2 multicopy pore formation BtuB (Bfe) Tol
ColE2 – 5.0 multicopy DNase BtuB (Bfe) Tol
ColE3 – 5.0 multicopy RNase BtuB (Bfe) Tol
CloDF13 – 6 multicopy RNase IutA Tol
ColIa + single copy pore formation Cir TonB
ColIb + 62 single copy pore formation Cir TonB
ColK – 6 multicopy pore formation Tsx Tol
ColM inhibits synthesis of FhuA (TonA) TonB
LPS & peptidoglycan
ColV + 85 single copy pore formation Tsx TonB

Tra = the ability for self transfer.
CloDF13 is not a spelling mistake it is actually a cloacin.

Colicin plasmids

There are two general classes:
1) Large, single copy, self transferable plasmids (ColB, I, V). The larger Col plasmids may carry other properties, e.g. ColV encodes virulence factors enhancing pathogenicity of E. coli. ColIb encodes a UV repair system for DNA and many ColI factors carry antibiotic resistance(s). These plasmids may be eliminated by treating the host cell with acridine orange or sodium dodecylsulfate or by high temperature. When plasmids are lost, so is the ability to produce colicin.
2) Small, multicopy, non-self transferable plasmids (ColD, E, K, CloDF13). These may however be mobilized by F-class plasmids such as F, ColV or ColB.

Colicin Production

In a population of colicinogenic bacteria most cells do not produce colicin. At any given time around 1/1000 of the cells produces colicin. This is lethal for the producing cells. Colicin production is thus a sort of molecular kamikaze attack against sensitive bacteria. But note that it is not the colicin itself which kills the producer cell. Colicin negative mutants also die when induced. Some other property which resides on the Col factor is lethal. Treatment with UV light, mitomycin C or other agents damaging the host cell DNA induce colicin production by most cells in a Col+ population. These are the same sort of treatments which induce lytic growth of many lysogenic phages such as lambda and f80. Note that colicin production does not require replication of Col plasmid DNA whereas phage production does require replication of phage DNA. Some colicins, e.g. colicin V tend to remain attached to surface of producing strain whereas others e.g. colicins E, are released as freely soluble proteins.

Colicin Receptors and Entry

Sensitive cells carry receptors for colicins which are outer membrane proteins, e.g. BtuB (=Bfe) -protein is the receptor for vitamin B12, phage BF23 and colicins of the E type. {See notes on outer membrane}. A single hit by one colicin molecule is sufficient to kill a sensitive bacterium. However, for every colicin which enters successfully many others just bind to the cell envelope. Colicin entry only occurs when the receptor is in the correct position within the outer membrane which allows it to transfer the colicin to the inner membrane - probably via the inner membrane/outer membrane adhesion sites (i.e. Bayers patches).

Loss of receptors or uptake systems protects against colicins but interferes with outer membrane transport systems for nutrients and hence is counter-selected strongly. Osmotically shocked cells are often sensitive to colicins even in the absence of appropriate receptors since outer membrane damage allows entry - this has been shown for E3 and M colicins.

1) Colicin resistant cells - lost receptor - don't bind colicin.
2) Colicin tolerant cells - retain receptor - still bind colicin but do not transport it inside due to defects in the uptake system.

Two classes of colicins from receptor-point of view:
Group A (A, E1, E2, E3, K, L) - uptake needs the Tol system. These colicins are therefore inactive against tolA or tolB mutants but kill tonB mutants.
Group B (B, Ia, Ib, V, D, M) - uptake needs the TonB system. These colicins are therefore inactive against mutants in the tonB gene, but kill tolA or B mutants.

This classification is independent of the mode of action of the colicins. Both the TolAB system and the TonB system provide energy to transport material across the outer membrane, probably via the Bayer sites where the inner and outer membranes adhere. The two systems are similar in operation, but each energizes a different set of transport proteins (see Envelope section).

Note that many colicins are long thin molecules (axial ratios 10-20) and are long enough to penetrate the inner membrane while still attached to an outer membrane receptor. This is true for colicins A, D, E1, Ia, Ib and K all of which exert their killing effect on the inner membrane. In contrast, colicins E2 and E3 are shorter (axial ratio around 5) and act inside the cytoplasm after completely transiting the envelope. Thus membrane active colicins probably kill the cell while their outer end is still attached to the outer membrane. In fact, colicin E1 can kill cells while bound to sephadex resin whereas colicins E2 and E3 cannot. The N-terminus of colE1 binds to the receptor while the C-terminus is the lethal end. Treatment of inhibited cells with the protein-degrading enzyme trypsin reverses the effect of E1, K or Ia indicating that the colicin molecule remains exposed at the cell surface and can be degraded.

Mode of Action

There are two major classes with respect to mode of action:
1) Intracellular target, e.g. DNA or ribosomal RNA or protein synthesis.
2) Membrane target; usually inner membrane, rarely peptidoglycan.

Receptor Class MembraneTarget Intracellular Target
A (TolAB) E1, A, K, L E2, E3, DF13
B (TonB) Ia, Ib, B, V, M D
Pesticin A1122

Cytoplasmic Acting Colicins:
a) Colicins E3 and DF13 are ribonucleases which cleave the 16s rRNA of the 30S robosomal subunit, releasing a fragment of 49 nucleotides from the 3'-terminus. This inactivates protein synthesis. The E3.I3 and DF13.IDF13 complexes are inactive against ribosomes (I3 & IDF3 are the immunity proteins - see below).
b) Colicin E2 is an endonuclease which destoys target cell DNA. Again, E2.I2 complex is enzymatically inactive.
c) Colicins D inhibits prtoein synthesis

For all of E2, E3 and DF13 mild proteolysis gives a C-terminal fragment, about 25% of the colicin, which possesses nuclease activity, binds immunity protein and is very basic (for binding to negatively charged nucleic acids). The N-terminal 25% is hydrophobic and probably involved in translocation across membrane. The central region of these colicin molecules interacts with the outer membrane receptor.


Colicin E2 and E3 are very similar in the N-terminal 75% of molecule - they share the same receptor. They differ in the C-terminus and have different nuclease specificities and immunity proteins. Although their respective immunity proteins (I2 and I3) are both very acidic and of the same approximate size they show no obvious structural relatedness.

Membrane Active Colicins
ColE1, A, B, Ia, Ib, K and V form ion-permeable channels in the cytoplasmic membrane or in artificial lipid vesicles. Primary event is collapse of proton motive force. Many secondary effects follow, e.g. drop in ATP levels, loss of K+ and Mg2+ from cell, cessation of macromolecular synthesis, etc. The cell membrane is not destroyed and most solutes do not escape. The colicin forms an ion-specific channel for protons and K+. A single colicin molecule is sufficient to depolarize a whole cell in a few minutes. Membrane active colicins have long thin molecules. The 18Kd C-terminal fragment of ColE1 is enriched in nonpolar aminoacids and is effective in depolarizing membrane vesicles or liposomes. The N-terminal fragment (40kd) contains the receptor recognition region and is necessary only for penetrating the cell envelope.


Colicin M and Pesticin A1122 destroy the peptidoglycan rather than depolarizing the cytoplasmic membrane. These colicins need penetrate only as far as outer surface of cytoplasmic membrane, i.e. to site of peptidoglycan assembly. Cell lysis results in medium of low osmotic pressure whereas spheroplasts are formed at high OP. Pesticin A1122 is made by Yersinia pestis and kills Y. pseudotuberculosis, Y. enterocolitica, non-pesticinogenic Y .pestis and many E. coli but not E. coli K12. Pesticin hydrolyses the b-1,4 bond between N-acetyl glucosamine and N-acetyl muramic acid in peptidiglycan. Peptidoglycan from resistant mutants can still be degraded, so resistance is due to inability of the pesticin to penetrate OM.

Megacin A216 is a phospholipase made by Bacillus megaterium which hydrolyzes phosphatidylcholine to lyso-PC (a "lyso" phospholipid is missing one fatty acid). Meg+ cells are protected by an immunity protein, which is specific for megacin but does not protect against the action of any other phospholipases.

Bacteriophage Related Bacteriocins

The R-type pyocins made by Pseudomonas aeruginosa are very different from other bacteriocins:
1) they are chromosomally encoded
2) the pyocins are similar in structure to a contractile bacteriophage tail with sheath, core, and fibers.
3) they contain over 20 different proteins

Several P. aeruginosa strains produce phages which show immunological cross reactions to R-type pyocins. Furthermore, isolated tails from such phages show pyocin actovity. Thus pyocins are the remains of lysogenic phages rather than "true" colicins. Pyocins act by damaging the cytoplasmic membrane. Marcescin A (from Serratia marcescens) is also a phage tail structure as are a few bacteriocins from bacteria, such as Clostridium and Vibrio. Few have been well characterized.

Colicin Immunity

Col plasmids specity immunity to the colicins which they produce. Hence a Col+ cell is immune to colicins produced by other Col+ cells of the same type. Immunity overload may occur if a massive dose of colicin is applied to an immune cell. Immunity due to synthesis of immunity protein which binds and inactivates colicin. If too much colicin for immunity protein-cell killed. Note that, unlike colicin which is only produced by a few members of Col+ population, the immunity protein is continually produced by all Col+ bacteria (Obviously - otherwise they would be dead!).





For membrane active colicins (E1, Ia, Ib, K, A), immunity is due to a plasmid coded inner membrane protein. For example, the Ia immunity protein of MW 14,500 protects membranes against colicin Ia but not against the closely related colicin Ib even though Ia and Ib share the same receptor, have same mode of action, and have extensive sequence homology. Thus cells immune to Ia can be depolarized by Ib or E1 but not by Ia (and of course vice versa).

Cytoplasmic acting colicins (E2, E3, CloDF13) - upon synthesis these colicins react with corresponding cytoplasmic immunity protein to give inactive complex. The colicin/immune protein complex is eventually released into the medium. How the colicin is activated is uncertain - probably the immunity protein is lost during attachment of the colicin to the receptor of the target bacterium. This not yet confirmed experimentally, although colicin/immunity protein complexes have been purified from culture fluids. Such complexes kill sensitive cells but are inactive against the biochemical target in vitro; this implies that the immunity protein is lost upon entry into the sensitive cell.

Colicin Ecology

Purpose of colicins is presumably to kill related bacteria which compete for the same ecological niche. Difficult to demonstrate experimentally though certain mixed infection experiments have indicated that Col+ bacteria can destroy colicin sensitive strains in the intestine. Development of resistance to colicins is counterselected by loss of outer membrane transport ability.
TOXINS

Toxins are proteins which damage eukaryotic cells in contrast to colicins which kill other prokaryotes. Same basic principle of delivery system plus kill system on same molecule. However, if the toxin attacks a target which is different in eukaryotic and prokaryotic cells there is no need for bacterium to synthesise an immunity protein to protect itself. Many toxins and virulence factors are plasmid coded, but this is not such a general rule as for colicins. In fact the same or closely related toxins may be chromosomal in one strain and plasmid borne in others.

Toxin & Bacteria Structure Mechanism

Choleratoxin AB5 ADP-ribosylation of G-protein of adenylate cyclase;
Vibrio cholerae elevated cyclic AMP and severe watery diarrhea

Heat-labile enterotoxin AB5 ADP-ribosylation of G-protein of adenylate cyclase; E. coli elevated cyclic AMP and severe watery diarrhea

Petussis toxin A"B"5 (Note: the 5 B subunits are not identical)
Bordetella pertussis ADP-ribosylation of G-protein of adenylate cyclase;

Shiga toxin AB5 removal of an adenine in 28S rRNA; inhibition Shigella of protein synthesis, cell death, diarrhea, dysentery

Shiga-like toxin of E. coli AB5 as for Shiga toxin

Diphtheria toxin A-B (A = N-terminal domain) ADP-ribosylation of EF2;
Corynebacterium diphtheriae inhibition of protein synthesis and cell death,

Exotoxin A A-B (A = C-terminal domain) ADP-ribosylation of EF2;
Pseudomonas aeruginosa inhibition of protein synthesis and cell death,

Tetanus toxin A-B activation of protein kinase C; inhibition of
Clostridium tetani presynaptic transmission in spinal cord motor neurons

Botulinum toxin C1 A-B blocks release of acetylcholine in peripheral nerves
Clostridium botulinum


Choleratoxin

Choleratoxin (chromosomal) and the heat-labile enterotoxins of enteropathogenic E. coli (Ent-plasmid coded) are variants of the same toxin. Mechanism essentially the same for both. Effect is loss of water and ions from eukaryotic cells due to massive overproduction of cyclic AMP.

Structure: 3 proteins A1, A2 and B. A1 and A2 are derived from a single precursor A protein by proteolytic processing. A1 (23.5 Kd) and A2 (5.5 Kd) remain linked by a single disulfide bond. Five B (11.6 Kd) are non-covalently attached to the A subunits to give a ring-like structure, containing one each of A1 and A2 sticking through the middle of the ring of five B-subunits.

Choleratoxin binds to ganglioside GM1 in eukaryotic cell membrane, via B protein. Each B subunit binds to the galactose end of a ganglioside molecule. Free B-protein protects eukaryotic cells against toxin by competing for GM1 binding sites. Structure of GM1 (sphingosine is a base):



A1 protein activates adenylate cyclase. It must first be released from A2 by reduction of S-S bond after binding of A1-S-S-A2/B5 complex to the cytoplasmic membrane of target cell. A1 can then enter the cell. Cyclase activation requires GTP and NAD. Choleratoxin hydrolyses NAD to nicotinamide and ADP-ribose. It was originally thought that the toxic effect of NAD-using toxins (both Cholera and Diptheria - see below) was due to destruction of NAD. However, in vivo these toxins actually transfer the ADP-ribose fragment of NAD to another acceptor molecule and this effect is responsible for lethal event. Choleratoxin A1 protein can ADP-ribosylate a variety of acceptors including arginine and its derivatives as well as many proteins. A1 protein can ADP-ribosylate itself and when so modified its activity increases by 50%.


The genuine in vivo lethal target is a specific GTPase involved in regulation of adenylate cyclase. This GTPase is a membrane protein which acts as a regulatory subunit (G-protein) for adenylate cyclase. ADP-ribosylation of an arginine residue on the GTPase results in inactivation of this protein. The cyclase is then freed from regulation and is permanently activated. The presence of GTP or its nonhydrolysable analog GMP-P-NH-P is necessary for cyclase activation. GTP hydrolysis allows the release of regulatory subunit and deactivation. ADP-ribosylation prevents hydrolysis of GTP and hence jams GTPase in its GTP-binding state. GTP analogs, like GMP-P-NH-P, which cannot be hydrolyzed show similar effects. Cyclic-AMP production rises several hundred fold in cells which are affected. Intestinal cells lose sodium and then water. Clinical symptom of Cholera is death by dehydration following loss of body fluids by masive diarrhea.

The virulence proteins of Vibrio cholerae include choleratoxin together with a pilus for binding to the intestinal cells and a special outer membrane protein, OmpU. Synthesis of these is co-regulated by the ToxR protein found in the inner membrane. It detects that the bacterium is inside an animal intestine and activates the virulence protein genes, ctxAB (toxin A and B subunits), tcpA (pilin) and ompU. The inside domain of ToxR protein binds directly to the promoters of these genes.






Diptheria Toxin

Diphtheria-toxin is encoded by tox genes carried on lysogenic phage rather than plasmids, however the principle is similar - they are not "genuine" chromosomal genes. Corynebacterium diptheriae strains which lose the phage also lose the ability to produce toxin and, conversely, non-pathogenic strains may be made toxin producers by lysogeny with the appropriate phage. The phages b-tox and w-tox have linear DNA with cohesive ends like lambda. They form single, double, or rarely, triple lysogens and the amount of toxin produced corresponds to the gene dosage. There are two chromosomal att sites (attB1 and attB2) into which phage integrate at random to give single or double lysogens. Rarely, tandem lysogeny at one of these sites gives an overall triple lysogen. The related phage, g-tox, is defective in toxin production.

About 100 nanograms/kg is a lethal dose for most animals, except mice and rats which need 1000 fold more. Toxin is a single 63Kd protein which is enzymatically inactive. It is activated by a serine protease which gives A (24Kd) and B (39Kd) fragments, linked by a single disulfide bridge. Fragment A is enzymatically active, B is needed for entry into the target cell.

Fragment A hydrolyses NAD to nicotinamide and ADP-ribose. Lethal event is transfer of the ADP-ribose fragment to elongation factor EF-2, a translation factor found in eukaryotic cells. The result is inhibition of protein synthesis. EF-2 is a GTP hydrolyzing protein required for moving the peptidyl-tRNA from aminoacyl-site to peptidyl-site on ribosome and shifting the mRNA by one codon in a reaction requiring GTP hydrolysis. ADP-ribosylated EF-2 still binds GTP but cannot hydrolyse it or translocate.

Modification occurs at a diphthamide residue of EF-2. Diphthamide is derived from histidine by post-translational modification and is found in eukaryotes and archebacteria only on EF-2 in part of the amino acid sequence which is highly conserved. The corresponding eubacterial factor, EF-G, does not contain diphthamide (nor do any eubacterial proteins). Archebacterial elongation factor EF-2 is ADP-ribosylated by diptheria toxin but much less efficiently than eukaryotic EF-2.



Bacteriophage Toxins

Several bacteriophages can ADP-ribosylate bacterial proteins, using NAD, in a manner analogous to cholera and diptheria toxins. Usually several bacterial proteins are modified and which is the intended target is therefore uncertain. The purpose may be to inactivate host metabolism by killing key enzymes, or to modify host polymerases so increasing their activity with phage DNA or RNA. Examples include phage T4 which ADP-ribosylates host RNA-polymerase which thereupon loses much of its ability to transcribe E. coli DNA but still works well with T4 DNA.