THE BACTERIA

Metabolism-Antibiotic Sensitivity
Table of Contents
 Educational Objectives
 Microbial Metabolism Overview
 Bacterial Cell Wall Biosynthesis
 Cytoplasmic Membrane
 DNA Replication
 Protein Synthesis
 Competitive Antagonistic Anitbiotics
 Summary

Specific educational objectives (terms and concepts upon which you will be tested)

• Aminoglycoside antibiotics
• Antibiotic mode of action
• b-lactam antibiotics
• Cell wall inhibitors
• Competitive antagonistic antibiotics
• Macrolide antibiotics
• Protein synthesis inhibitors
• Quinolone antibiotics

Microbial Metabolism as Related to Sensitivity to

Antibiotics-Overview
Many metabolic activities of the bacterial cell differ significantly from those in the human
cell. At least theoretically these differences can be exploited in the development of
chemotherapeutic agents. Ideally, an antimicrobial agent should have its maximal effect on
the bacterial cell and have little or no effect on the human cell. In reality there is almost
always some effect on the human be it induction of hypersensitivity or liver or kidney
toxicity. Despite some adverse reactions in the human, effective antibiotics have been
developed that have one ore more of these modes of action on the bacterial cell:

A. Inhibition of cell wall synthesis

B. Alteration of cell membranes

C. Inhibition of protein synthesis

D. Inhibition of nucleic acid synthesis

E. Antimetabolic activity or competitive antagonism

Bacterial Cell Wall Biosynthesis
Since bacteria have a cell wall made up of repeating units
of peptidoglycan and human cells lack this feature, it
would seem that the bacterial cell wall presents an ideal
target for chemotherapy. Indeed, this has been the case;
the following antibiotics have been developed as
inhibitors of cell wall synthesis:

A. β -lactam antibiotics

1. Penicillins

Penicillin G Oxacillin Ampicillin Amoxicillin Cloxaciillin

Penicillin V Nafcillin Ticarcillin Carbenicillin Dicloxacillin
Methicillin Piperacillin
2. Cephalosporins

First Generation Second Generation Third Generation Fourth

Generation

Cefadroxil * Cefaclor * Cefdinir Cefepime

Cefazolin Cefamandole Cefoperaxone
Cefelixin * Cefonicid Cefotaxime
Cephalothin Ceforanide Ceftazidime
Cephaprin Cefotetan Ceftibuten
Cephradine * Cefoxitin Ceftizoxime
Cefuroxime Ceftriaxone

* Oral Agent
3. Monobactams
4. Thienamycins
5. β -lactamase inhibitors (e.g., clavulanic acid)
B. Cycloserine, Ethionamide, Isoniazid
C. Fosfomycin (Phosphonomycin)
D. Vancomycin
E. Bacitracin
F. Ristocetin
G. Fosphomycin (Phosphonomycin)
 The biosynthesis of peptidoglycan consists of three stages, each of which occurs at a different site in
the cell.

Stage 1 occurs in the cytoplasm. In this stage the recurring units of the backbone structure of murein,
N-acetylglucosamine and N-acetyl-muramylpentapeptide are synthesized in the form of their uracil
diphosphate (UDP) derivatives.

The only antibiotic that affects this stage of cell wall metabolism is D-cycloserine. D-cycloserine is a
structural analog of D-alanine; it binds to the substrate binding site of two enzymes, thus being
extremely effective in preventing D-alanine from being incorporated into the N-acetylmuramylpeptide.

Structural relationship between cycloserine (left) and D-ala-nine (right).

Stage 2 of peptidoglycan synthesis occurs on the inner surface of the cytoplasmic membrane where N-
cetylmuramylpeptide is transferred from UDP to a carrier lipid and is then modified to form a complete
nascent peptidoglycan subunit. The nature of the modification depends upon the organism.

This stage terminates with translocation of the completed subunit to the exterior of the cytoplasmic
membrane. The only antibiotic that affects this stage of cell wall synthesis is bacitracin. Bacitracin is an
inhibitor of the lipid phosphatase.

Bacitracin A. One of a group of polypeptide antibiotics containing a thiazoline ring structure.

Stage 3 occurs in the periplasmic space (in gram-negative bacteria) and in the growing peptidoglycan of
the cell wall. This is a complex metabolic sequence which offers multiple targets for chemotherapeutic
agents. The earliest acting of these are vancomycin and ristocetin.

They act by binding to the D-alanyl-D-alanine peptide termini of the nascent peptidoglycan-lipid carrier.
This inhibits the enzyme transglycosylase.
Stage 3 of biosynthesis continues with
transpeptidation and the binding of
soluble uncrosslinked, nascent
peptidoglycan to the preexisting,
crosslinked, insoluble cell wall
peptidoglycan matrix.

The b-lactam antibiotics are structural

analogs of the D-alanyl-D-alanine end
of the peptidoglycan strand.

In the cell wall there are as many as

seven enzymes (depending on the
bacterial species) which bind
peptidoglycan units via their D-alanyl-
D-alanine residues.

The b-lactams fill these substrate

binding sites and thus prevent the
binding of D-alanyl-D-alanine residues. Enzymes binding b-lactam antibiotics are known as penicillin-
binding proteins.
 The Cytoplasmic Membrane as the Site of Antibiotic Action
The cytoplasmic membrane of bacteria is
only affected by two clinically-used
antibiotics.

These are polymyxin B and polymyxin E

(colistin).

They act by competitively replacing Mg2+

and Ca2+ from negatively charged
phosphate groups on membrane lipids. The
result is disruption of the membrane.
 DNA Replication as the Site of Antimicrobic Action
The major group of antibacterial agents that act by blocking DNA synthesis/activity is the quinolone group.

Metronidazole represents as an antibiotic active

against DNA in a different way. This antibiotic,
upon being partially reduced, causes the
fragmentation of DNA in an, as yet, undefined
way. The antibiotic is only effective against
anaerobic bacteria and some parasites.

The quinolones all act by blocking the A subunit

of DNA gyrase and inducing the formation of a
relaxation complex analogue.

DNA gyrase introduces negative superhelical

turns into duplex DNA, using the energy of ATP.
This is the crucial enzyme that maintains the
negative superhelical tension of the bacterial
chromosome.

The sign-inversion mechanism for DNA gyrase.

The quinolones include:

nalidixic acid - first generation

norfloxacin, ciprofloxacin - second generation

 Protein Synthesis as the Site of Antimicrobic Action
Protein synthesis is the end result of two major processes, transcription and translation. An antibiotic that
inhibits either of these will inhibit protein synthesis.

Transcription
During transcription, the genetic information in
DNA is transferred to a complementary
sequence of RNA nucleotides by the DNA-
dependent RNA polymerase. This enzyme is
composed of 5 subunits, ß, ß', a, a' and σ .

Antibiotics that either alter the structure of the

template DNA or inhibit the RNA polymerase
will interfere with the synthesis of RNA, and
consequently with protein synthesis.

Actinomycin D binds to guanine in DNA,

distorting the DNA, and thus blocking
transcription.

Rifampin (Rifampicin or Rifamycin) inhibits

protein synthesis by selective inhibiting the
DNA-dependent RNA
polymerase. It does this by
binding to the ß subunit in a non-
covalent fashion.

Translation
In bacterial cells, the translation
of mRNA into protein can be
divided into three major phases:
initiation, elongation, and
termination of the peptide chain.
Protein synthesis starts with the
association of mRNA, a 30S
ribosomal subunit, and formyl-
methionyl-transfer RNA (fMet-
tRNA) to form a 30S initiation
complex. The formation of this
complex also requires guanosine
triphosphate (GTP) and the participation of three protein initiation factors. The codon AUG is the initiation
signal in mRNA and is recognized by the anticodon of fMet-tRNA. A 50S ribosomal subunit is
subsequently added to form a 70S initiation complex, and the bound GTP is hydrolyzed.
In the elongation phase of protein synthesis, amino acids are added one at a time to a growing
polypeptide in a sequence dictated by mRNA. It is this phase that is most susceptible to inhibition by a
number of antibiotics. For many of these the ribosome is the target site. There are two binding sites on the
ribosome, the P (peptidyl or donor site) and the A (aminoacyl) site. At the end of the initiation stage, the
fMet-tRNA molecule is empty. In the first step of the elongation cycle, an aminoacyl-tRNA is inserted into
the vacant A site on the ribosome. The particular species inserted depends on the mRNA codon that is
positioned in the A site. Protein elongation factors and GTP are required for polypeptide chain elongation.

In the next step of the elongation phase, the formylmethionyl residue of the fMet-tRNA located at the
peptidyl donor site is released from its linkage to tRNA, and is joined with a peptide bond to the α -amino
group of the aminoacyl-tRNA in the acceptor site to form a dipeptidyl-tRNA. The enzyme catalyzing this
peptide formation is peptidyl transferase, which is part of the 50S ribosomal subunit.

Following the formation of a peptide bond, an uncharged tRNA occupies the P site, whereas a dipeptidyl
tRNA occupies the A site. The final phase of the elongation cycle is translocation, catalyzed by elongation
factor EF-G and requiring GTP. It consists of three movements:

(1) the removal of the discharged tRNA from the P site

(2) the movement of fMet-aminoacyl-tRNA from the acceptor site to

the peptidyl donor site

(3) the movement or translocation of the ribosome along the mRNA

from the 5' toward the 3' terminus by the length of three
nucleotides.

After translocation, the stage is prepared for the binding of the next aminoacyl residue to the fMet-
aminoacyl-tRNA, each addition
requiring aminoacyl-tRNA binding,
peptide bond formation, and
translocation. Peptidyl-tRNAa replace
the fMet-tRNA in the second and in all
subsequent cycles.

The polypeptide chain grows from the

amino terminal toward the carboxyl
terminal amino acid and remains linked
to tRNA and bound to the mRNA-
ribosome complex during elongation of
the chain. When completed it is
released during chain termination.

Termination is triggered when a chain

termination signal (UAA, UAG, or
UGA) is encountered at the A site of
the ribosome.

Protein release factors bind to the

terminator codons triggering hydrolysis
by the peptidyl transferase. The
polypeptide is released, and the
messenger-ribosome-tRNA complex
dissociates.
Several medically important antibiotics owe their selective antimicrobial action to a specific attack on the
70S ribosome of bacteria, with mammalian 80S ribosomes left unaffected. Those that act on the 30S
ribosome are:

•
Amikacin
•
Gentamycin
•
Kanamycin
•
Neomycin
•
Streptomycin
•
Tobramycin
Macrolides:
Azithromycin Dirithromycin
Clarithromycin Erythromycin

A
Intibiotics that act on the 50S portion of the ribosome include:

• Chloramphenicol
• Clindamycin
• Furadantin
• Fusidic acid
• Lincomycin
• Nitrofuran
• Puromycin
• Quinopristin/Dalfopristin
• Spectinomycin
• Tetracycline

Lincomycin Clindamycin
Linezolid

Puromycin
 Competitive Antagonistic Antibiotics
Inhibitors of metabolic pathways via competitive antagonism include:

Isoniazid - Inhibits mycolic acid synthesis

 Trimethoprim - Inhibit folic acid biosynthesis

Summary
1. Antibiotics that are active against the cell wall of bacteria include the β -lactams, cycloserine,
ethionamide,
isoniazid, phosphomycin, vancomycin, bacitracin and ristocetin.
2. The β -lactam antibiotics are related structurally in that they all contain a β -lactam ring.
These are the penicillins, cephalosporins, monobactams and thienamycins. They are all analogs
of d-alanyl-d-alanine.

3. Antibiotics that are active against the bacterial cytoplasmic membrane are polymyxin B and
E (colistin).

4. Antibiotics that are active against bacterial DNA are the quinolones (nalidixic acid,
norfloxacin and ciprofloxacin), which inhibit DNA gyrase, and metronidazole, which fragments
DNA.

5. Antibiotics that block transcription in bacteria are actinomycin D and rifampin.

6. Antibiotics that block translation in bacteria by binding to the 30S ribosome are the
aminoglycosides, nitrofurans, spectinomycin and the tetracyclines.

7. The aminoglycoside antibiotics are related structurally in that they all contain a unique
aminocyclitol ring
structure. These include amikacin, gentamycin, kanamycin, neomycin, streptomycin and
tobramycin.

8. Antibiotics that block translation by binding to the 50S ribosome include chloramphenicol,
erythromycin,
clarithromycin, lincomycin, clindomycin, puromycin, fusidic acid and quinopristin/dalfopristin.

9. The macrolide antibiotics are related structurally in that they all contain a macrocyclic lactone
ring of 12-22 carbon atoms, to which one or more sugars are attached. These include
erythromycin, clarithromycin, azithrmycin and dirithromycin.

10. Antibiotics that act by inhibiting folic acid biosynthesis include the sulfonamides and
trimethoprim.