antibiotics
Article
Ribosome Assembly as Antimicrobial Target
Rainer Nikolay 1, *,† , Sabine Schmidt 2,† , Renate Schlömer 2 , Elke Deuerling 2, * and
Knud H. Nierhaus 1
1
2
*
†
Institut für Medizinische Physik und Biophysik, Charité—Universitätsmedizin Berlin, 10117 Berlin,
Germany; [email protected]
Molecular Microbiology, University of Konstanz, Konstanz 78457, Germany;
[email protected] (S.S.); [email protected] (R.S.)
Correspondence: [email protected] (R.N.); [email protected] (E.D.);
Tel.: +49-30-450-524165 (R.N.); +49-7531-88-2647 (E.D.)
These authors contributed equally to this work.
Academic Editor: Claudio O. Gualerzi
Received: 31 March 2016; Accepted: 16 May 2016; Published: 27 May 2016
Abstract: Many antibiotics target the ribosome and interfere with its translation cycle. Since translation
is the source of all cellular proteins including ribosomal proteins, protein synthesis and ribosome
assembly are interdependent. As a consequence, the activity of translation inhibitors might indirectly
cause defective ribosome assembly. Due to the difficulty in distinguishing between direct and indirect
effects, and because assembly is probably a target in its own right, concepts are needed to identify
small molecules that directly inhibit ribosome assembly. Here, we summarize the basic facts of
ribosome targeting antibiotics. Furthermore, we present an in vivo screening strategy that focuses
on ribosome assembly by a direct fluorescence based read-out that aims to identify and characterize
small molecules acting as primary assembly inhibitors.
Keywords: protein synthesis as preferential target of antibiotics; ribosome as antibiotic target;
inhibitors of ribosome assembly; concepts for identifying assembly inhibitors
1. Introduction
Most antibiotics are microbial secondary metabolites mainly produced by actinomycetes.
The production is usually induced by unfavorable growth conditions, when the producer has turned
into the stationary or sporulation phase and its own metabolic activity is sharply reduced. Under these
conditions, antibiotics will block the growth of better adapted competitors and thus prevent a further
impairment of life conditions (for review, see [1]).
Antibiotics therefore improve the survival chance of the producer, which represents the selective
force for the natural development of antibiotics. Under these restricted conditions, synthesis of nucleic
acids and protein synthesis is strongly reduced in the producer cell, which explains that, in many cases,
the producer is not resistant against its own product.
Antibiotics are chemically extremely diverse. The synthesis by microorganisms and a molecular
weight less than 1000 Da are the only common features [2]. Nowadays, the term antimicrobials
(short for antimicrobial substances) is used, which includes both natural products (antibiotics)
and semi-synthetic or full-synthetic substances (chemotherapeutics). In addition to the wide
chemical spectrum, the target spectrum is also enormous: More than 160 cellular targets have been
described [3]. Examples are replication (e.g., nalidixic acid), transcription (e.g., rifamycin), translation
(e.g., chloramphenicol) and the synthesis of the cell wall (e.g., penicillin). Most of the antibiotics
inhibit the translating ribosome because, due to its complicated structure, it offers many interference
points. The Escherichia coli ribosome contains 57 different components, 54 proteins and three ribosomal
RNAs [4], and all of them are present in one copy per ribosome except the protein bL12, which is
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present
in four
copies.
Nevertheless, the binding sites of the antibiotics are concentrated at and2 of
around
Antibiotics
2016,
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13
functional hot spots of the small 30S and the large 50S subunit of the bacterial ribosome (Figure 1a,b).
copies. Nevertheless, the binding sites of the antibiotics are concentrated at and around functional
These sites are often dominated or exclusively built by elements of the rRNA (ribosomal RNA), and,
hot spots of the small 30S and the large 50S subunit of the bacterial ribosome (Figure 1a,b). These sites
importantly,
the multiplicity
of rrn operons
bacterial
species
aggravates
development
are often dominated
or exclusively
built by present
elementsinofmost
the rRNA
(ribosomal
RNA),
and, importantly,
of resistance
[5].
Mutations
in
only
one
gene
have
low
impact
and
multiple
mutations
are of low
the multiplicity of rrn operons present in most bacterial species aggravates development of resistance
probability
[6]. in only one gene have low impact and multiple mutations are of low probability [6].
[5]. Mutations
Figure
1. Antibiotic
targetsites
sitesofofbacterial
bacterial ribosomes.
ribosomes. (a)
target
sites
on on
the the
30S 30S
Figure
1. Antibiotic
target
(a)aafew
fewantibiotic
antibiotic
target
sites
subunit mentioned in the text. Tet, tetracycline; Neo, neomycin, Str, streptomycin; (b) some antibiotic
subunit mentioned in the text. Tet, tetracycline; Neo, neomycin, Str, streptomycin; (b) some antibiotic
target sites on the 50S subunit. Cam, chloramphenicol; Lin, lincomycin. (from [7], modified, courtesy
target sites on the 50S subunit. Cam, chloramphenicol; Lin, lincomycin. (from [7], modified, courtesy
of Daniel N. Wilson, Munich Gene Center).
of Daniel N. Wilson, Munich Gene Center).
Most of the natural antibiotics inhibit the peptidyltransferase center (PTC) on the 50S subunit
Most of
natural
antibiotics
the of
peptidyltransferase
on high
the 50S
subunit
(Figure
1a;the
[8-10]).
Reasons
for the inhibit
prevalence
the PTC as antibioticcenter
target (PTC)
are (i) the
number
of crevices
allowing
binding
of small
molecules
with
high
(ii)target
the fact
a of
(Figure
1a; [8–10]).
Reasons
for the
prevalence
of the
PTC
asaffinity;
antibiotic
arethat
(i) the
the PTC
highneeds
number
high
amount
of
structural
flexibility
and
any
interference
by
drug
binding
or
mutations
causing
crevices allowing binding of small molecules with high affinity; (ii) the fact that the PTC needs a high
resistance
might hamper
the and
speed
and/or
accuracyby
of translation,
which
leads to thecausing
final outcome
amount
of structural
flexibility
any
interference
drug binding
or mutations
resistance
that
any
rescuing
mutations
within
the
PTC
are
associated
with
high
fitness
costs
[10].
might hamper the speed and/or accuracy of translation, which leads to the final outcome that any
The most common target site of the 30S subunit is the decoding center of the A site, where the
rescuing mutations within the PTC are associated with high fitness costs [10].
large group of aminoglycosides interferes with the fidelity of aminoacyl-tRNA(aa-tRNA)·EF-Tu·GTP
The most common target site of the 30S subunit is the decoding center of the A site, where the
selection (Figure 1b). Examples of binding sites on the 30S part of the A site outside the decoding center
large group of aminoglycosides interferes with the fidelity of aminoacyl-tRNA(aa-tRNA)¨ EF-Tu¨ GTP
are the tetracyclines, which block stable binding of aa-tRNAs to the A site. The sketch of the ribosomal
selection
(Figure
1b). Examples
of binding
sites onpoints
the 30S
of the A
site outside
the decoding
functions
in Figure
2 summarizes
the interference
of part
the various
groups
of antibiotics
with
center
are
the
tetracyclines,
which
block
stable
binding
of
aa-tRNAs
to
the
A
site.
The
sketch of the
ribosomal functions. Some antibiotics act directly on translational G-proteins. One example is kirromycin,
ribosomal
in elongation
Figure 2 summarizes
the interference
of the
groups
of antibiotics
which functions
binds to the
factor EF-Tu·GTP
and blocks points
its switch
intovarious
the GDP
conformer
after
withdelivery
ribosomal
functions.
act directly
translational
of the
aa-tRNA Some
to the antibiotics
A site. The result
is that on
EF-Tu
remains onG-proteins.
the ribosomeOne
andexample
thus
blocks protein
synthesis.
example is fusidic
(FA)
acting
the second
elongation
is kirromycin,
which
bindsAnother
to the elongation
factor acid
EF-Tu¨
GTP
andonblocks
its switch
into factor
the GDP
G (EF-G).
EF-G
is responsible
for the translocation
of The
ribosomes
to that
the next
codon
of theon
mRNA
after
conformer
after
delivery
of the aa-tRNA
to the A site.
result is
EF-Tu
remains
the ribosome
a
decoding
step.
FA
also
blocks
the
conformational
switch
into
the
GDP
conformer
of
EF-G,
which
is
and thus blocks protein synthesis. Another example is fusidic acid (FA) acting on the second elongation
necessary
for
the
dissociation
of
this
factor
after
it
has
fulfilled
its
ribosomal
function.
factor G (EF-G). EF-G is responsible for the translocation of ribosomes to the next codon of the mRNA
after a decoding step. FA also blocks the conformational switch into the GDP conformer of EF-G,
which is necessary for the dissociation of this factor after it has fulfilled its ribosomal function.
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Kir, Str
Tet
Ksg
Ede
50S
Cam
Pmn
70S-scanning
initiation
Ery, Tel
FA
Cam
Pmn
FA
Neo
HygB
Ths
Vio
Figure 2. Overview of some antibiotics interfering with ribosomal functions. Kir, kirromycin; Str,
Figure 2. Overview of some antibiotics interfering with ribosomal functions. Kir, kirromycin; Str,
streptomycin, tet, tetracycline; Cam, chloramphenicol; Pmn, puromycin; FA, fusidic acid; Neo,
streptomycin, tet, tetracycline; Cam, chloramphenicol; Pmn, puromycin; FA, fusidic acid; Neo,
neomycin, HygB, hygromycin B; Ths, thiostrepton; Vio, viomycin; Ery, erythromycin, Tel,
neomycin, HygB, hygromycin B; Ths, thiostrepton; Vio, viomycin; Ery, erythromycin, Tel, telithromycin.
telithromycin. 70S-scanning initiation is a newly detected initiation mode in bacteria, which can
70S-scanning initiation is a newly detected initiation mode in bacteria, which can replace the RRF/EF-G
replace the RRF/EF-G dependent recycling phase. Modified, from [11].
dependent recycling phase. Modified, from [11].
Antibiotics are estimated to have originated between 2 billion. and 40 million years ago [12].
Antibiotics are
estimated
to have originated
billion.
and 40
million
yearsofago
[12].
Consequently,
resistance
mechanisms
have beenbetween
evolving2 for
the same
large
amount
time.
Consequently,
resistance mechanisms
have been
evolving for
the samesoil
large
amount of
time.
Recently,
Recently, 30,000-year-old
bacterial isolates
of Canadian
permafrost
confirmed
that
resistance
30,000-year-old
Canadian
permafrost
soil confirmed
that resistance
predated
predated the bacterial
industrialisolates
use of of
antibiotics
[12].
Five different
types of resistance
mechanisms
arethe
known: use of antibiotics [12]. Five different types of resistance mechanisms are known:
industrial
Mutations
membranecomponents
components affect
affect the permeability
transport
(1) (1)Mutations
ofofmembrane
permeabilitybarrier
barrierand,
and,alternatively,
alternatively,
transport
proteins
are
affected,shifting
shiftingthe
theimport:export
import:export ratio
ratio towards
proteins
are
affected,
towards the
theexport.
export.
Theantibiotic
antibiotictarget
targetisisaltered
altered by
by blocking
blocking binding
binding or
(2) (2)The
or modifying
modifyingthe
thebinding
bindingsite,
site,causing
causing
−6 to 10−8, i.e., one
insensitivity
to
the
drug.
Mutation
rates
for
types
1
and
2
are
in
the
range
of
10
´
6
insensitivity to the drug. Mutation rates for types 1 and 2 are in the range of 10 to 10´8 , i.e.,
bacterium out of 106 to 108 is resistant to the respective drug.
one bacterium out of 106 to 108 is resistant to the respective drug.
(3) Plasmids coding for enzymes that modify (acetylation, phosphorylation of adenylation) or
(3) Plasmids coding for enzymes that modify (acetylation, phosphorylation of adenylation) or
degrade the antibiotic [3].
degrade the antibiotic [3].
(4) Special factors remove the antibiotic from the target. For example, the EF-G derivative Tet(O)
(4) Special
factors
the antibiotic
from
the target.
ForInterestingly,
example, theresistance-types
EF-G derivative
protein
chasesremove
bound tetracycline
off the
ribosome
[13,14].
1–4Tet(O)
are
protein
chases
tetracycline
the ribosome [13,14]. Interestingly, resistance-types 1–4 are
known
for thebound
tetracycline
group off
[15].
for the tetracycline
group
[15].
(5)known
Rare mechanisms
are dilution
(overproduction)
of the target molecule or activation of alternative
(5) Rare
mechanisms
are known
dilutionfor
(overproduction)
of inhibits
the target
molecule orreductase
activation
of alternative
pathways.
Both are
trimethoprim that
dihydrofolate
[16].
pathways. Both are known for trimethoprim that inhibits dihydrofolate reductase [16].
Wasteful use of antibiotics in animal fattening and clinical applications have strengthened the
spread
of resistant
and often inmulti-resistant
germs
horizontal
gene transfer
[17], which isthe
Wasteful
use of antibiotics
animal fattening
andvia
clinical
applications
have strengthened
considered
one of
theoften
major
threads of medical
It is therefore
obvious
that
newisdrugs
and
spread
of resistant
and
multi-resistant
germstherapies.
via horizontal
gene transfer
[17],
which
considered
possibly
new
targets
are
needed
[5,18].
one of the major threads of medical therapies. It is therefore obvious that new drugs and possibly new
Facing
the enormous
targets are
needed
[5,18]. accumulation of knowledge about all aspects of antibiotics, it is astonishing
that
the
highly
complicated
process ofofassembly
of the
bacterial
ribosomes
does notitseem
to be a
Facing the enormous accumulation
knowledge
about
all aspects
of antibiotics,
is astonishing
major target for antibiotics [19]. One possible reason is the lack or scarcity of suitable screening
that the highly complicated process of assembly of the bacterial ribosomes does not seem to be a major
techniques to identify antibiotics specifically interfering with the ribosomal biogenesis. In this
target for antibiotics [19]. One possible reason is the lack or scarcity of suitable screening techniques
respect, some promising progress has been achieved in recent years, which we will consider in the
to identify antibiotics specifically interfering with the ribosomal biogenesis. In this respect, some
following section.
promising progress has been achieved in recent years, which we will consider in the following section.
Antibiotics 2016, 5, 18
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2. Ribosome Assembly as Attractive Target for New Antimicrobials
2. Ribosome Assembly as Attractive Target for New Antimicrobials
The bacterial ribosome is one of the most intricate structures in the bacterial cell, and its assembly
The bacterial ribosome is one of the most intricate structures in the bacterial cell, and its assembly
is a highly complex process. The fact that the small and large ribosomal subunits can be reconstituted
is a highly complex process. The fact that the small and large ribosomal subunits can be reconstituted
in vitro [20,21] indicates that the complete information for the assembly process is intrinsically present
in vitro [20,21] indicates that the complete information for the assembly process is intrinsically present
in the
sequences of ribosomal proteins and rRNAs [20]. With the help of the reconstitution technique,
in the sequences of ribosomal proteins and rRNAs [20]. With the help of the reconstitution technique,
important
mechanistic
stepscould
couldbebe
unraveled,
examples
are identification
of the
important
mechanisticassembly
assembly steps
unraveled,
andand
examples
are identification
of the two
twoassembly
assemblyinitiator
initiatorproteins
proteins
[22]
and
five
early
assembly
proteins
necessary
sufficient
for one
[22]
and
thethe
five
early
assembly
proteins
necessary
andand
sufficient
for one
of the
most
dramatic
conformational
changes
known,
which
occurs
during
the
assembly
of
precursor
of the most dramatic conformational changes known, which occurs during the assembly of precursor
intermediates
(a 33S
particle
becomes
a highly
compact
41S
particle;
intermediates
(a 33S
particle
becomes
a highly
compact
41S
particle;[23]).
[23]).Two
Tworibosomal
ribosomalproteins,
proteins,L24
andL24
L20,
could
mere
proteins
without
important
functions
in in
thethe
mature
and
L20,be
could
beassembly
mere assembly
proteins
without
important
functions
matureribosome
ribosome[24].
The analyses
cumulated
in assembly
maps
forthe
both
the small
and large
subunit
(Figure
The[24].
analyses
cumulated
in assembly
maps for
both
small
and large
subunit
(Figure
3a,b;3a,b;
[25,26];
[25,26];
for
review,
see
[27]).
for review, see [27]).
5'
(a)
3'
16S RNA
S20
S4
S18
S17
S16
S15
S8
S6
S12
S11
S7
S5
S10
S19
S9
S13
S14
S2
S3
S21
S17
S4
S15
S20
S16
S8
S12
S6:S18
S5
S11
S7
S9
S21
S13
S19
S10
S14
S3
S2
5'
(b)
3'
23S RNA
L24
L22
L21
L20
L4
L15
L1
L3
L13
L23
L17
L5
L18
L29
L34
L11
L33
L19
L32
L14
L2
L28
L9
L6
L36
L35
L25
L30
L27
L10
L7
L16
L31
L24
L20
L21
L22
L23
L17
L3
L13
L34
L5
L29
L19
L32
L2
L14
L6
L33
L28
L30
L10
L7/12
L36
L18
5S
L11
L35
L1
L15
L4
L9
L16
L27
L25
L31
Figure
3. Assembly
maps.
map
of the
small
subunit;
map
of the
large
subunit.
The
Figure
3. Assembly
maps.
(a)(a)
map
of the
small
30S30S
subunit;
(b)(b)
map
of the
large
50S50S
subunit.
The
maps
maps
of
Nomura
(a)
and
Nierhaus
(b)
are
color
coded
from
blue
to
red
according
to
their
appearance
of Nomura (a) and Nierhaus (b) are color coded from blue to red according to their appearance during
the process
assembly
in vivo, modified
from Reference
The assembly
maps showing
the
the during
assembly
inprocess
vivo, modified
from Reference
[28]. The[28].
assembly
maps showing
the assembly
assembly
dependencies
of
the
ribosomal
proteins
during
the
total
reconstitution
in
vitro
demonstrate
dependencies of the ribosomal proteins during the total reconstitution in vitro demonstrate an excellent
an excellent agreement with the in vivo data. Please check if there is copyright issue.
agreement with the in vivo data. Please check if there is copyright issue.
What are the advantages of ribosome assembly as a target for antimicrobials? The inhibition of
What are
the advantages
assembly
as a target
foroperating
antimicrobials?
The
inhibition
ribosome
assembly
precedes of
theribosome
interference
with functions
of the
ribosome.
Inhibition
of of
ribosome assembly precedes the interference with functions of the operating ribosome. Inhibition of
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early assembly events result in premature precursors, so called assembly dead ends, which, due to
early assembly events result in premature precursors, so called assembly dead ends, which, due to
their loose structures, are cleared by proteases and RNases [29]. While such a scenario is irreversible,
their loose structures, are cleared by proteases and RNases [29]. While such a scenario is irreversible,
inhibition of translation is not. As soon as the inhibitor is gone, translation can recommence [30]. In
inhibition of translation is not. As soon as the inhibitor is gone, translation can recommence [30].
addition, assembly differs significantly between bacterial and mitochondrial ribosomes [31]. This is
In addition, assembly differs significantly between bacterial and mitochondrial ribosomes [31]. This is
an important notion because the latter ones are frequent targets of adverse reactions as has been
an important notion because the latter ones are frequent targets of adverse reactions as has been shown
shown for chloramphenicol [32], linezolid [33] and aminoglycosides [34].
for chloramphenicol [32], linezolid [33] and aminoglycosides [34].
3.
3. Ribosome
RibosomeAssembly
Assemblyand
andTranslation
TranslationAre
AreCoupled
Coupledin
inBacteria
Bacteria
Facing
Facing both
both the
the highly
highly complicated
complicated process
process of
of ribosome
ribosome assembly
assembly and
and the
the fact
fact that
that the
the majority
majority
of
of antibiotics
antibiotics inhibit
inhibit functions
functions of
of the
the bacterial
bacterial ribosomes,
ribosomes, itit is
is surprising
surprising that
that hitherto
hitherto no
no antibiotics
antibiotics
have
specifically
block
ribosomal
biogenesis.
It is It
expected
that
have been
beendetected,
detected,which
whichprimarily
primarilyand
and
specifically
block
ribosomal
biogenesis.
is expected
translation
inhibitors
will will
affect
alsoalso
thethe
synthesis
of of
ribosomal
proteins
that translation
inhibitors
affect
synthesis
ribosomal
proteinsand
andthus
thusthe
the assembly
assembly
process
(Figure
4a).
Consequently,
assembly
and
translation
are
interdependent,
which
results
in in
a
process (Figure 4a). Consequently, assembly and translation are interdependent, which
results
chicken-and-egg
problem
[19].
a chicken-and-egg problem [19].
(a)
(b)
(c)
Figure 4. Scheme
Scheme illustrating
illustrating interconnectivity
interconnectivity of
of ribosome
ribosome assembly
assembly and
and translation.
translation. (a)
(a) ribosome
ribosome
Figure
assembly
is
the
prerequisite
for
translation,
which
in
turn
is
required
for
ribosome
assembly;
assembly is the prerequisite for translation, which in turn is required for ribosome assembly; (b)
(b) inhibition
of translation
a translation
inhibitor
the causative
mechanistic
element.
inhibition
of translation
by a by
translation
inhibitor
is the iscausative
mechanistic
element.
As a
As a consequence,
this results
in inhibition
(orleast
at least
reduction)
assembly
process,which,
which,ininturn,
turn,
consequence,
this results
in inhibition
(or at
reduction)
of of
thethe
assembly
process,
inhibits translation
translation completing
completing the
the negative
negative feedback
feedback loop.
loop. Both
Both processes
processes are
are clearly
clearly interconnected
interconnected
inhibits
in vivo
vivo and
and underlie
underlie complex
complex regulation;
regulation; (c)
(c) inhibition
inhibition of
of ribosomal
ribosomal subunit
subunit assembly
assembly by
by aa putative
putative
in
primary
assembly
inhibitor
acting
on
one
of
the
two
subunits
(cause)
will
result
in
inhibition
of
primary assembly inhibitor acting on one of the two subunits (cause) will result in inhibition of
translation
(consequence),
which
additionally
inhibits
assembly.
This
also
creates
a
negative
feedback
translation (consequence), which additionally inhibits assembly. This also creates a negative feedback
loop. Legend:
Legend:Black
Blacklines
lineswith
witharrows:
arrows:positive
positiveeffects;
effects;red
redlines:
lines:direct
direct(causative)
(causative)inhibitory
inhibitoryeffects;
effects;
loop.
orange
lines:
resulting
(consequent)
inhibitory
effects;
asterisks:
site
of
inhibition;
dark
gray
circle:
orange lines: resulting (consequent) inhibitory effects; asterisks: site of inhibition; dark gray circle:
large
ribosomal
subunit;
light
gray
circle:
small
ribosomal
subunit;
dark
gray
line:
mRNA.
large ribosomal subunit; light gray circle: small ribosomal subunit; dark gray line: mRNA.
For
to distinguish
distinguish between
between cause
cause and
and consequence.
consequence. Inhibition
For this
this reason, it is difficult to
Inhibition of
of
translation
a decrease
in translation
(consequence)
(Figure
4b). 4b).
However,
this
translation(cause)
(cause)would
wouldresult
resultinin
a decrease
in translation
(consequence)
(Figure
However,
is
difficult
to distinguish
fromfrom
a scenario
where
inhibition
of assembly
would
be be
thethe
cause
and
a
this
is difficult
to distinguish
a scenario
where
inhibition
of assembly
would
cause
and
decreased translation the consequence (Figure 4c). This interdependence was observed by Nomura
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Antibiotics 2016, 5, 18
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aand
decreased
translation
theconcluded
consequence
4c). Thisofinterdependence
was
observed
by Nomura
coworkers,
and they
that(Figure
any inhibition
translation forces
assembly
defects
due to
and
coworkers,
and
they
concluded
that
any
inhibition
of
translation
forces
assembly
defects
due to
the arising imbalance between rRNA and r-protein production [35].
the arising
imbalance
between
rRNA
and r-protein
[35].
Indeed,
macrolides
such as
erythromycin
haveproduction
been demonstrated
to block the assembly of the
Indeed,
macrolides
such
as
erythromycin
have
been
demonstrated
block
the assembly
of
large 50S ribosomal subunit [36]. Chloramphenicol and erythromycin havetobeen
shown
to decrease
the
large
50S
ribosomal
subunit
[36].
Chloramphenicol
and
erythromycin
have
been
shown
to
the production of some proteins (among others r-proteins). Precursor particles isolated upon
decrease
production
of found
some proteins
Precursor
particles
isolated
upon
antibioticthe
treatment
were
to have (among
reducedothers
levels r-proteins).
of exactly those
proteins
that were
reduced
antibiotic
treatment
were
found
to
have
reduced
levels
of
exactly
those
proteins
that
were
reduced
in
in production [37,38]. Similarly, it has been described that the macrolide erythromycin and the
production
[37,38]. Similarly,
has been
described
that thepolypeptide
macrolide erythromycin
and
ketolide
ketolide telithromycin
do not it
block
the passage
of nascent
chains through
thethe
exit
tunnel
telithromycin
do
not
block
the
passage
of
nascent
polypeptide
chains
through
the
exit
tunnel
completely
completely but rather in a case-dependent manner. Biosynthesis of a number of polypeptides
but
rather ina anewly
case-dependent
manner. Biosynthesis
of a number
of and
polypeptides
containing
a newly
containing
identified N-terminal
signal sequence
did occur
was interpreted
as a strategy
identified
N-terminal
signal
sequence
did In
occur
was interpreted
as a strategy
to deregulate
cellular
to deregulate
cellular
pathways
[39].
thatand
sense,
ribosome assembly
defects
and metabolic
pathways
[39].
In
that
sense,
ribosome
assembly
defects
and
metabolic
dysregulation
would
be
indirect
dysregulation would be indirect effects caused by translation inhibitors.
effects
caused byassembly
translation
Likewise,
ofinhibitors.
the small 30S subunit is inhibited by aminoglycosides such as
Likewise,or
assembly
of the
30S subunit
is inhibited
aminoglycosides
as streptomycin
streptomycin
neomycin
[40].small
However,
it is likely
that theby
primary
target of thesuch
drugs
are functions
or
neomycin
[40].
However,
it
is
likely
that
the
primary
target
of
the
drugs
are
functions
of the mature
of the mature ribosome [37].
ribosome
[37].
Taken
together, it seems that identification of selective inhibitors of assembly requires the ability
Taken
together,
it seems
of face
assembly
requires is
thetoability
to distinguish
between
causethat
andidentification
consequence.ofAselective
possibleinhibitors
strategy to
that problem
focus
to
distinguish
between
cause
and
consequence.
A
possible
strategy
to
face
that
problem
is
to focus
directly on subunit assembly.
directly on subunit assembly.
4. Specific Readouts for Assembly Are Needed
4. Specific Readouts for Assembly Are Needed
A first step towards a specific readout for subunit assembly has been described recently. Two
A first step towards a specific readout for subunit assembly has been described recently.
reporter strains have been generated possessing one r-protein of the large and the small subunit fused
Two reporter strains have been generated possessing one r-protein of the large and the small subunit
with green or red fluorescent proteins, respectively. While the reporter strain termed MCrg [41]
fused with green or red fluorescent proteins, respectively. While the reporter strain termed MCrg [41]
harbors ribosomes with two labeled late assembly proteins (Supplementary Figure S1), in a second
harbors ribosomes with two labeled late assembly proteins (Supplementary Figure S1), in a second
reporter strain MCrg* [42], two early assembly proteins were labeled (Supplementary Figure S2).
reporter strain MCrg* [42], two early assembly proteins were labeled (Supplementary Figure S2).
Consequently, in MCrg, only fully assembled subunits are fluorescently marked, which allows for
Consequently, in MCrg, only fully assembled subunits are fluorescently marked, which allows for
using that strain for identification of assembly inhibitors in small molecule screenings by comparing
using that strain for identification of assembly inhibitors in small molecule screenings by comparing the
the fluorescence ratio (green/red) of untreated with treated reporter cells. In contrast, in MCrg*, both
fluorescence ratio (green/red) of untreated with treated reporter cells. In contrast, in MCrg*, both fully
fully assembled and incomplete subunits are fluorescently marked, allowing for monitoring of
assembled and incomplete subunits are fluorescently marked, allowing for monitoring of assembly
assembly landscapes when ribosome profiles are analyzed fluorometrically [42]. Given the necessity
landscapes when ribosome profiles are analyzed fluorometrically [42]. Given the necessity to not only
to not only detect but also quantify subunit specific assembly defects, two new strains were created,
detect but also quantify subunit specific assembly defects, two new strains were created, which are
which are the logical consequence of the two predecessors MCrg and MCrg*. The new strains possess
the logical consequence of the two predecessors MCrg and MCrg*. The new strains possess either
either large (MCrgL) or small subunits (MCrgS) labeled with fluorescent proteins. In both strains, one
large (MCrgL) or small subunits (MCrgS) labeled with fluorescent proteins. In both strains, one early
early assembly protein (uL1 or uS15) is labeled with mCherry and one late assembly protein (bL19 or
assembly protein (uL1 or uS15) is labeled with mCherry and one late assembly protein (bL19 or uS2)
uS2) with mAzami. Both phenotypic and biochemical characterization of MCrgL and MCrgS,
with mAzami. Both phenotypic and biochemical characterization of MCrgL and MCrgS, respectively,
respectively, revealed that the strains show no defects at 37 or 42 °C and only a mild growth defect
revealed that the strains show no defects at 37 or 42 ˝ C and only a mild growth defect at very low
at very low temperature. In summary, we conclude that their growth properties and translation
temperature. In summary, we conclude that their growth properties and translation apparatuses are
apparatuses are wild type-like (Figure 5).
wild type-like (Figure 5).
(a)
A)
20 °C
30 °C
37 °C
MC4100
MCrgL
MCrgS
Figure
Figure 5.
5. Cont.
Cont.
42°C
Antibiotics 2016, 5, 18
Antibiotics 2016, 5, 18
7 of 13
7 of 13
37°C
20°C
37°C
42°C
42°C
1.0
0.8
180
130
100
75
M
ribosomes
lysate
ribosomes
uS2-mAzami
uL1-mCherry
63
0.6
uS15-mCherry
uS2
uL1
degradation
products
MC
MC
41
MC
rg
MC
rg
10
MC
4
bL19-mAzami
*
rgS
MC
41
00
MC
rg L
MC
rg
S
28
00
0
rg L
35
S
0.2
L
48
0
0.4
MC
normalized growth rate
20°C
lysate
41
M C 00
rg
L
MC
rg
MC S
4
M C 100
rg
MC L
rg
MC S
41
M C 00
rg
L
MC
rg
MC S
4
M C 100
rg
MC L
rg
S
(c)
C)
MC
(b)
B)
17
bL19
uS15
10
SDS-PAGE
WB: αL1, αL19, αS2, αS15
Figure
biochemical
characterization
of strains
MCrgLMCrgL
and MCrgS.
(a) Serial(a)
dilutions
Figure 5.5.Phenotypic
Phenotypicand
and
biochemical
characterization
of strains
and MCrgS.
Serial
of
MC4100,
MCrgL
and
MCrgS
were
spotted
on
LB-agar
plates
and
incubated
at
temperatures
dilutions of MC4100, MCrgL and MCrgS were spotted on LB-agar plates and incubated as
at
indicated;
(b) LB
cultures of(b)
theLB
indicated
were
inoculated
a start
OD600 of to
0.05
and cultured
temperatures
as indicated;
culturescells
of the
indicated
cellstowere
inoculated
a start
OD600 of
at 20, 37 and 42 ˝ C for 7 h in triplicates. Growth rates were calculated and normalized; (c) cell
0.05 and cultured at 20, 37 and 42 °C for 7 h in triplicates. Growth rates were calculated and
lysates and purified ribosomes obtained from the indicated strains were subjected to SDS-PAGE
normalized; (c) cell lysates and purified ribosomes obtained from the indicated strains were subjected
(left panel) or SDS-PAGE with subsequent immunoblotting (right panel). The SDS-PAGE was stained
to SDS-PAGE (left panel) or SDS-PAGE with subsequent immunoblotting (right panel). The SDSwith Coomassie-brilliant blue and the Western blot membrane was probed with the indicated primary
PAGE was stained with Coomassie-brilliant blue and the Western blot membrane was probed with
antibodies in combination with appropriated secondary antibodies. Proteins of interest are labeled.
the indicated primary antibodies in combination with appropriated secondary antibodies. Proteins of
WB, Western blot; αL1, antibody against ribosomal protein uL1.
interest are labeled. WB, Western blot; αL1, antibody against ribosomal protein uL1.
To
To test
test their
their capability
capability to
to analyze
analyze subunit
subunit specific
specific assembly
assembly defects,
defects, in both
both strains,
strains, conditional
knock
outs
of
ribosomal
protein
genes
were
introduced.
The
absence
of
the
L-protein
uL3uL3
(encoded
by
knock outs of ribosomal protein genes were introduced. The absence of the L-protein
(encoded
rplC)
or the
S-protein
uS17
(encoded
byby
rpsQ)
by rplC)
or the
S-protein
uS17
(encoded
rpsQ)would
wouldrender
rendereither
eitherlarge
largeor
orsmall
smallsubunit
subunit assembly
defective, as described previously [41,43,44]. While in MCrgL large subunit,
subunit, mono- and
and polysomes
polysomes
contain red and green fluorescence (Figure
6c),
in
MCrgS
small
subunit,
monoand
polysomes
show
(Figure
red and green
green fluorescence
fluorescence (Figure
(Figure 6d).
6d). In MCrgL, the absence of uL3 causes defects in the large
subunit as becomes evident by a decrease of the green signal and an additional red fluorescence peak
(Figure 6e). Defective
Defective small
small subunit
subunit assembly, in the absence of uS17, cannot directly be detected
detected
fluorometrically.
fluorometrically. However,
However, directly
directly caused
caused defects
defects in
in small
small subunit
subunit assembly
assembly seem to result in slight
but detectable assembly defects of the large subunit as indicated by asterisks (Figure 6g). MCrgS, on
the other
which
were
caused
byby
depletion
of
other hand,
hand, allows
allowsdetection
detectionofofsmall
smallsubunit
subunitassembly
assemblydefects,
defects,
which
were
caused
depletion
uS17,
directly
(Figure
6f).6f).
While
thethe
green
fluorescence
signal
within
the the
30S30S
region,
which
reflects
the
of uS17,
directly
(Figure
While
green
fluorescence
signal
within
region,
which
reflects
intact
portion
of theofsubunit,
is decreased,
the redthe
fluorescence
signal issignal
broader
and of higher
intensity,
the intact
portion
the subunit,
is decreased,
red fluorescence
is broader
and of
higher
clearly
indicating
small subunit
assembly
defect.
In thedefect.
absence
uL3absence
(Figureof
6h),
defective
intensity,
clearly aindicating
a small
subunit
assembly
Inofthe
uL3
(Figurelarge
6h),
subunit
cannot
be detected
fluorometrically.
However, againHowever,
the subunit
not the
affected
by
defectiveassembly
large subunit
assembly
cannot
be detected fluorometrically.
again
subunit
gene
depletion,
in
this
case
the
small
subunit,
shows
a
decrease
in
green
fluorescence
and
a
broadened
not affected by gene depletion, in this case the small subunit, shows a decrease in green fluorescence
red
a left-sided
shoulder.
Quantitation
of the fluorescence
confirms the
andfluorescence
a broadenedpeak
red with
fluorescence
peak
with a left-sided
shoulder.
Quantitationprofiles
of the fluorescence
observed
tendencies
6i–j).
profiles confirms
the(Figure
observed
tendencies (Figure 6i–j).
Taken together,
together,fluorescence
fluorescenceanalyses
analyses
of sucrose
density
gradient
fractions
from
of sucrose
density
gradient
fractions
derivedderived
from MCrgL
MCrgL
and enable
MCrgSdetection
enable detection
and quantitation
of assembly
defectsthe
within
large or
small
and MCrgS
and quantitation
of assembly
defects within
largethe
or small
subunit,
subunit,
respectively.
respectively.
Antibiotics 2016, 5, 18
Antibiotics 2016, 5, 18
8 of 13
8 of 13
Figure
gradient centrifugation
centrifugation and
and fluorescence
fluorescence detection.
detection.
Figure 6.
6. Ribosome
Ribosome profiles
profiles after
after sucrose
sucrose density
density gradient
(a,b)
schematic
drawing
of
MCrgL
and
MCrgS*,
respectively.
Depicted
are
the
positions
(a,b) schematic drawing of MCrgL and MCrgS*, respectively. Depicted are the positions of
of the
the
fluorescent
proteinsand
andtheir
theircoding
coding
sequences.
Dark
gray:
ribosomal
subunit;
light small
gray:
fluorescent proteins
sequences.
Dark
gray:
largelarge
ribosomal
subunit;
light gray:
small
ribosomal
subunit;
red barrel:
mCherry;
gray mRNA;
line: mRNA;
curved
ribosomal
subunit;
green green
barrel:barrel:
EGFP;EGFP;
red barrel:
mCherry;
gray line:
curved
blackblack
line:
line:
bacterial
chromosome
with
red
and
green
strips
symbolizing
coding
sequences
of
mCherry
bacterial chromosome with red and green strips symbolizing coding sequences of mCherry and
and
mAzami,
mAzami, respectively;
respectively; (c–h)
(c–h) Ribosome
Ribosome profiles
profiles derived
derived from
from MCrgL
MCrgL (c),
(c), MCrgL∆lC
MCrgLΔlC (d),
(d), MCrgL∆sQ
MCrgLΔsQ
(e),
profiles are given in black lines, mCherry and
254 profiles are given in black lines, mCherry and
(e), MCrgS
MCrgS (f),
(f), MCrgS∆sQ
MCrgSΔsQ (g)
(g) and
and MCrgS∆lC
MCrgSΔlC (h);
(h); A
A254
mAzami
mAzami specific
specific fluorescence
fluorescence intensities
intensities are
are given
given as
as red
red or
or green
green bars,
bars, respectively.
respectively. Fluorescence
Fluorescence
intensities
were
normalized
to
the
first
polysome
peak.
Red
and
green
fluorescence
intensities were normalized to the first polysome peak. Red and green fluorescence intensities
intensities were
were
calculated
to to
each
other,
(i,j). (i,j).
Total Total
mCherry
and mAzami
specific fluorescence
emission
calculated and
andcompared
compared
each
other,
mCherry
and mAzami
specific fluorescence
of each strain’s profile is given in bar charts. mCherry signals were set to 100% and mAzami signals
emission of each strain’s profile is given in bar charts. mCherry signals were set to 100% and mAzami
are given as relative values. Error bars show S.D. of two independent experiments. One asterisk
signals are given as relative values. Error bars show S.D. of two independent experiments. One
indicates additional peak or shoulder within the subunit with the primary defect, two asterisks indicate
asterisk indicates additional peak or shoulder within the subunit with the primary defect, two
additional peak or shoulder within the subunit was not targeted for assembly defect.
asterisks indicate additional peak or shoulder within the subunit was not targeted for assembly defect.
Antibiotics 2016, 5, 18
9 of 13
5. Materials and Methods
5.1. Media, Buffers, Antibodies and Antibiotics
LB-Medium (0.5% (w/v) yeast extract, 1% (w/v) tryptone, 86 mM NaCl; for growth on solid media
additionally 1.5% (w/v) bacto agar); 5ˆ M9 salts (63 mM Na2 HPO4 ¨ 7H2 O, 110 mM KH2 PO4 , 43 mM
NaCl, 94 mM NH4 Cl); M9 medium (1ˆ M9 salts, 2 mM MgSO4 , 0.1 mM CaCl2 , 0.4% (w/v) glucose);
PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2 HPO4 , 1.8 mM KH2 PO4 , pH 7.4). Horseradish peroxidase
(HRP)-conjugated rabbit anti-sheep (CodeNo: 313-035-003; LotNo: 106383) and donkey anti-rabbit
(CodeNo: 711-035-152; LotNo: 103871) and donkey anti-rabbit (CodeNo: 711-035-152; LotNo: 103871)
secondary antibodies were from Jackson ImmunoResearch (West Grove, Pennsylvania, USA). HRP
substrate for detection: 1 mL solution A + 100 µl solution B + 1 µL solution C (solution A: 0.1 mM TRIS
(pH 8.6), 25 mg Luminol, 100 mL distilled H2 O; solution B: 11 mg p-hydroxycoumaric acid in 10 mL
DMSO; solution C: 30% H2 O2 (w/v)). Antibiotics were used in concentrations as indicated: Ampicillin
100 µg/mL (Applichem-A0839, Darmstadt, Germany), kanamycin 50 µg/mL (Carl Roth-T832.4,
Karlsruhe, Germany) and chloramphenicol 7 µg/mL (Sigma-C0378, St. Louis, MO, USA).
5.2. Plasmids and Bacterial Strains
rpsQ and rplC were amplified from genomic E. coli DNA, brought into DH5α-Z1 and isolated as
described earlier [41].
MC4100 (F-[araD139]B/r ∆(argF-lac)169 lambda-e14-flhD5301 ∆(fruK-yeiR)725 (fruA25) relA1
rpsL150(strR) rbsR22 ∆(fimB-fimE)632(::IS1) deoC1); DY330 (W3110 ∆ lacU169 gal490 λcI857 ∆
(cro-bioA)) [45], DH5α-Z1 (F endA1 hsdR17(rk mk+) supE44 thi-1 recA1 gyrA relA1 ∆ (lacZYAargF)
U169 deoR Φ80 lacZ∆ M15 LacR TetR and Spr).
5.3. λ-Red Recombineering
Fusion proteins of the ribosomal proteins with the FPs mAzami green and mCherry were
generated by λ red recombineering and brought into strains of interest using P1-phage transduction
as described previously. Genomic integration was confirmed by colony-PCR and DNA-sequencing.
Gene deletions of rpsQ and rplC were also accomplished as described earlier [41].
5.4. Cell Growth Analyses
For growth on solid media, E. coli cells were grown at 37 ˝ C until stationary phase in LB medium
and diluted to a cell density of OD600 = 0.025. Serial dilutions with a dilution factor of 0.2 were
prepared and transferred onto LB agar plates using a plating stamp. Plates were incubated at 20 ˝ C,
30 ˝ C, 37 ˝ C and 42 ˝ C until single colonies were visible.
For growth in liquid media, stationary E. coli cells were grown until stationary phase and diluted
to an initial OD600 = 0.025 for incubation at 42 ˝ C and to OD600 = 0.05 for incubation at 37 ˝ C and 20 ˝ C.
They were incubated in baffled flasks in a water bath incubator with a shaking frequency of 200 rpm
until stationary or exponential phase was reached. Cell density was measured using a photometer
(Ultrospec 3000, GE Healthcare, Little Chalfont, UK) and growth rates were calculated for periods of
exponential growth. Growth analyses in liquid media were conducted in biological triplicates.
5.5. Purification of Ribosomes by Sucrose Cushion Centrifugation
E. coli cells were cultured at 37 ˝ C in LB medium until stationary phase was reached. Pre-cultures were
diluted to an OD600 = 0.5 and incubated at 37 ˝ C until an OD600 = 0.8. 250 µg/mL chloramphenicol
was added to stop translation 5 min before harvesting. For this purpose, cultures were incubated on
ice for 10 min and sedimented for 10 min at 4400 rpm and 4 ˝ C. Pellets were resuspended in lysis
buffer I (100 mM TRIS, 10 mM MgCl2 , 100 mM NaCl, 15% (w/v) sucrose, 100 µg/mL chloramphenicol,
pH 7.5), snap frozen in liquid nitrogen and stored at ´80 ˝ C. For further preparation, the frozen
Antibiotics 2016, 5, 18
10 of 13
cell pellets were thawed on ice and resuspended in 3ˆ volumes of lysis buffer II (10 mM MgCl2 ,
100 mM NaCl). Cells were then lysed using FastPrep® -24 (MP Biomedicals, Eschwede, Germany).
Protein concentrations of the cleared lysates were determined by Bradford assay. In addition,
300 µL of cleared lysates were loaded on 700 µL of 20% sucrose cushion (20 mM TRIS, 10 mM
MgCl2 , 100 mM KCl, 5 mM β-mercaptoethanol, 20% sucrose (w/v), pH 7.5) and sedimented for 1 h
20 min at 65,000 rpm at 4 ˝ C in an S140-AT rotor (Thermofischer Scientific, Waltham, MA, USA).
Pellets harboring the ribosomes were resuspended in buffer III (10 mM TRIS, 12 mM MgCl2 , 30 mM
NaCl, 4 mM β-mercaptoethanol, pH 7.5). A260 values were determined using a spectrophotometer
(NanoVueTM Plus UV/Visible Spectrophotometer (GE Healthcare).
5.6. Sucrose Gradient Centrifugation
E. coli cells of the different strains were cultured at 37 ˝ C until stationary phase. The pre-cultures
were washed 3 times and diluted in M9 minimal medium to OD600 = 0.05 and cultured to OD600 of
about 0.3. Five minutes before harvesting, chloramphenicol (250 µg/mL) was added. The pellets were
sedimented, snap frozen in liquid nitrogen and stored at ´80 ˝ C. For further preparation, pellets were
resuspended in lysis buffer (10 mM TRIS, 10 mM MgCl2, 100 mM NH4 Cl, 250 µg/mL chloramphenicol,
0.5 mM DTT, 1 mM PMSF, 1x TM CompleteTM (05056489001, (Roche-05056489001, Basel, Swiss) pH7.5)
and lyzed using FastPrep® -24. A260 values of the cleared lysates were determined, the concentrations
were adjusted to A260 = 20. In addition, 100 µL were then loaded on 10%–40% sucrose gradients
and centrifuged at 4 ˝ C and 41,000 rpm for 2 h 40 min using a Sorvall TH-641 rotor (Thermofischer
Scientific, Waltham, MA, USA).
5.7. Polysome Analysis and Fluorometric Analysis of the Sucrose Fractions
Separated ribosomal populations were analyzed using a Teledyne Isco gradient reader
(Teledyne ISCOr, Lincoln, Nebraska, USA). A254 profiles were recorded and the obtained fractions
were collected in 96-well plates (5 drops per well) for the following fluorometric analyses. mAzami
and mCherry specific fluorescence was determined using Infinite F500 (Tecan, Männedorf, Swiss)
fluorescence microplate reader (filter combinations 485/535 nm and 535/612 nm, respectively).
The fluorescence intensities were normalized to the first polysome peak.
6. Conclusions
The intricate assembly of the macromolecular complex “ribosome” offers many distinct
interference points. Even though the structure of ribosomes is highly conserved across domains,
a number of specific features of the biogenesis of the bacterial ribosomes exist. Only recently suitable
methods have been developed to trace assembly inhibitors. We can expect that the near future will
reveal inhibitors specifically blocking ribosome assembly in bacteria, which might pave the way for
development of urgently needed new therapeutics against bacterial infections.
Supplementary Materials: The following are available online at www.mdpi.com/2079-6382/5/2/18/s1.
Figure S1: Ribosome profile analysis using MCrg, Figure S2: Ribosome profile analysis using MCrg*.
Acknowledgments: We thank Daniel N. Wilson for help and discussion (Gene Center, University of Munich,
Germany). This work was supported by a Human Frontier Science Program Organization (HFSP-Ref.
RGP0008/2014) to K.H.N. and a grant from the Deutsche Forschungsgemeinschaft DFG (A01, SFB969) to E.D.
Author Contributions: R.N., S.S. and E.D. conceived and designed the experiments; S.S., R.S. and R.N. performed
the experiments; R.N., S.S., E.D. and K.H.N. analyzed the data; R.N., S.S., E.D. and K.H.N. wrote the paper.
Conflicts of Interest: Engineered bacterial strains described in this study are part of a patent application
(application No: 13175775.9-1410) that is pending.
Antibiotics 2016, 5, 18
11 of 13
Abbreviations
The following abbreviations are used in this manuscript:
Da
EGFP
LB
LB agar
rRNA
PTC
tRNA
TRIS
aa-tRNA
GTP
GDP
EF-Tu
EF-G
FA
mAzami
mCherry
OD600
S.D.
SDS PAGE
molecular mass in Dalton
Enhanced green fluorescent protein
Lysogeny broth (medium)
LB and agar containing breeding grounds
ribosomal RNA
peptidyl transferase center
transfer RNA
Tris(hydroxymethyl)-aminomethan
aminoacyl tRNA
guanosin triphosphate
guanosin diphosphate
elongation factor thermo-unstable
elongation factor G
fusidic acid
Monomeric green fluorescent protein
Monomeric red fluorescent protein
Optical density at 600 nm wavelength
standard deviation
Sodium dodecyl sulfate poly acrylamide gel electrophoresis
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