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Journal of Bacteriology, June 1999, p. 3666-3673, Vol. 181, No. 12
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Role in Cell Permeability of an Essential
Two-Component System in Staphylococcus aureus
Patrick K.
Martin,*
Tong
Li,
DongXu
Sun,
Donald P.
Biek, and
Molly
B.
Schmid
Microcide Pharmaceuticals, Inc., Mountain
View, California 94043
Received 2 February 1999/Accepted 5 April 1999
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ABSTRACT |
A temperature-sensitive lethal mutant of Staphylococcus
aureus was found to harbor a mutation in the uncharacterized
two-component histidine kinase (HK)-response regulator (RR) pair
encoded by yycFG; orthologues of yycFG could be
identified in the genomes of Bacillus subtilis and other
gram-positive bacteria. Sequence analysis of the mutant revealed a
point mutation resulting in a nonconservative change (Glu to Lys) in
the regulator domain of the RR at position 63. To confirm that this
signal transduction system was essential, a disrupted copy of either
the RR (yycF) or the HK (yycG) was constructed
with a set of suicide vectors and used to generate tandem duplications
in the chromosome. Resolution of the duplications, leaving an insertion
in either the yycF or the yycG coding region,
was achieved only in the presence of an additional wild-type copy of
the two open reading frames. Phenotypic characterization of the
conditional lethal mutant showed that at permissive growth conditions,
the mutant was hypersusceptible to macrolide and lincosamide
antibiotics, even in the presence of the ermB resistance
determinant. Other mutant phenotypes, including hypersensitivity to
unsaturated long-chain fatty acids and suppression of the conditional
lethal phenotype by high sucrose and NaCl concentrations, suggest that
the role of the two-component system includes the proper regulation of
bacterial cell wall or membrane composition. The effects of this point
mutation are strongly bactericidal at the nonpermissive temperature,
indicating that this pathway provides an excellent target for the
identification of novel antibiotics.
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INTRODUCTION |
The mounting problem of antibiotic
resistance among pathogenic bacteria, including Staphylococcus
aureus, has prompted renewed efforts toward the discovery of novel
antimicrobial agents. Useful targets for novel antimicrobial agents
will be found among genes that are essential for bacterial survival. In
an effort to identify genes essential for the growth of S. aureus, a collection of temperature-sensitive (ts)
mutants has been generated. One of the mutant strains, NT372, was found
to be complemented by genes encoding a novel two-component signal
transduction system.
Two-component signal transduction systems are widely distributed among
prokaryotes (6, 38) and mediate diverse adaptive responses
(52), including sporulation (18), elaboration of virulence factors (34), and chemotaxis (31).
Examples of structural homologues from eukaryotes have also been
reported (25). A prokaryotic two-component system usually
consists of a histidine kinase (HK) and a response regulator (RR).
Commonly, an environmental signal is sensed by the HK, resulting in a
change in the phosphorylation state of a conserved histidine residue in
the cytoplasmic kinase domain of the protein. This phosphohistidyl
residue serves as a source for phosphotransfer to a conserved aspartate
residue of the cognate RR, resulting in modulation of the activity of the RR. The activated RR can initiate subsequent actions, such as
transcriptional regulation or phosphorylation of other proteins, ultimately resulting in transduction of the primary signal.
Several essential two-component systems have now been described for
bacteria. An essential multicomponent pathway (60) in the
dimorphic bacterium Caulobacter crescentus requires the RR gene ctrA (44) to restrict DNA replication to the
stalked cell type, as well as to control the transcription of a number
of genes (43). The resDE system is required for
the proper expression of genes involved in respiration in
Bacillus subtilis (55), although strains bearing
insertional inactivations of either the HK or the RR are viable in the
presence of glucose or fructose. A recent report has offered a
preliminary characterization of the yycFG genes of B. subtilis (13); these genes are essential for the in
vitro growth of B. subtilis and are orthologues of the genes
described in this report.
The two-component system identified in this work is essential for the
in vitro viability of S. aureus. Several characteristics of
the genes and mutant phenotypes suggest that they will be uniquely suitable for serving as targets for novel antibacterial agents.
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MATERIALS AND METHODS |
Media and chemicals.
Antibiotics and medium supplements were
obtained from Sigma (St. Louis, Mo.). Commercially available media for
routine strain cultivation, including Trypticase soy broth (TSB),
Mueller-Hinton broth, and Luria-Bertani broth, were obtained from Difco
Laboratories (Detroit, Mich.). Chemically defined medium and dropout
media were prepared as described by Pattee and Neveln (41).
Oxyrase reagents and plates were purchased from Oxyrase, Inc.
(Mansfield, Ohio) and used to generate anoxic growth conditions in
combination with a GasPak chamber from BBL (Cockeysville, Md.).
Selection for the drug resistance markers ermC and
tetK was performed on Trypticase soy agar (TSA) plates (TSA
with erythromycin [TSA-Em] or TSA with tetracycline [TSA-Tc],
respectively) with 1 µg of antibiotic per ml for initial selection
and scoring on 10 µg/ml. To select for the presence of the rescue
plasmid, chloramphenicol (10 µg/ml) was added when required. Unless
otherwise noted, strains carrying the yycF1 mutation were
grown at 30°C as a permissive temperature; growth curves were
acquired in a microtiter format with a SpectraMAXplus plate
reader from Molecular Devices (Sunnyvale, Calif.). MICs were determined
by twofold broth dilution on microtiter plates; end points were
determined after overnight growth (35°C, 20 h).
Bacterial strains and plasmids.
The strains and vectors used
in this work are listed in Table 1; all
S. aureus strains used in this study were derivatives of
8325-4 (SAM23). The standard molecular biological procedures used are
described by Sambrook et al. (45). All enzymes were purchased from New England Biolabs (Beverly, Mass.); deoxynucleotides were obtained from Pharmacia (Piscataway, N.J.). Recombinant constructs were introduced into bacteria either by electroporation with a Gene
Pulser (BioRad, Inc.) or by transduction (47).
Mutant isolation and complementation.
Heat-sensitive mutant
NT372 was obtained by UV mutagenesis of S. aureus SAM23;
mutagenized cultures were diluted, plated onto TSA, and incubated
overnight (30°C, 18 h). The growing colonies were replica
printed in duplicate and incubated under either permissive (30°C) or
nonpermissive (43°C) growth conditions. Colonies growing at 30°C
but not at 43°C were isolated, and their temperature-sensitive (TS)
phenotypes were reconfirmed in a second round of plating. Genomic DNA
for library construction and for PCR was prepared from S. aureus by the method of Dyer and Iandolo (12). Genomic DNA from SAM23 was partially digested (Sau3AI), and
fragments (2 to 8 kb) were isolated by sucrose gradient centrifugation; the fragments were ligated into the pMP16 shuttle vector
(BamHI), and the mixture was electroporated into
Escherichia coli DH5
. The shuttle library plasmids were
introduced into S. aureus SAM13 by electroporation; the
pooled erythromycin-resistant (Emr) transformants were then
infected with bacteriophage
11 at a multiplicity of infection of
0.01. The resulting lysates were used to transduce the ts
mutant NT372 to temperature resistance for the selection of
complementing clones. Transducing lysates of each of the colonies
surviving incubation at 43°C were used to retransduce the original
NT372 mutant to confirm the complementation.
DNA sequence and analysis.
Complementing plasmids and PCR
amplicons were sequenced with a PRISM dye terminator kit from Applied
Biosystems, Inc. (Foster City, Calif.) and an ABI 373A automated DNA
sequencer. Oligonucleotides for DNA sequencing were produced on an ABI
392 synthesizer. All sequencing reactions were performed in multiple
passes from both directions. Multiple genomic PCR amplicons from the
NT372 mutant and the SAM23 parent were sequenced in parallel and
compared to differentiate genomic mutations from possible PCR-induced
artifacts. Version 8.0.1 of the Wisconsin Genetics Computer Group
programs (11) was used for sequence analysis. Potential open
reading frames (ORFs) were identified with Genemark (5) by
use of an S. aureus matrix; similarity searches were
performed with the BLASTX (1) program against the GenBank
database (release 110).
Strain construction.
To generate a transposon insertion near
the ts mutation in NT372, a Tn917lac transposon
insertion library from SAM23 was generated with pLTV1 (42).
A transducing lysate prepared from this insertion library was used to
transduce the NT372 mutant to a temperature-resistant phenotype
(26). In a modified protocol (see Results), the transduction mixture was plated on TSA-Em plates supplemented with sodium citrate (500 µg/ml) and Oxyrase (1:10 [vol/vol]). The plates were incubated under anoxic conditions until colonies began to appear (30°C, 48 h); colonies were reselected on TSA-Em plates at 43°C. A transducing lysate from one of the temperature-resistant derivatives, SAM970, was
used to establish a genetic linkage between the transposon and the
ts lesion (46). Genomic DNA flanking the
transposon insertion was cloned in E. coli as described
previously (62). Chromosomal digests (SmaI), used
to map Tn917lac insertions and plasmid integrants (see
below), were analyzed on a contour-clamped homogeneous electric field
(CHEF) gel system (Bio-Rad) according to the manufacturer's
directions. The Tn917lac insertion identified in SAM970 was
used to create the isogenic strains SAM1010 (yycF1) and
SAM1011 (yycF+) by backcrossing into SAM23 (wild type).
Disruption and rescue constructs.
To generate integration
constructs, the 3.7-kb yycFG genomic insert from the
complementing plasmid (pMP373) was subcloned (EcoRI/HindIII) into pGEM3Zf(+), maintaining
a rare SmaI site that could be used to map the integrants by
CHEF gel analysis. A 2.1-kb fragment (HindIII)
containing the tetK gene from plasmid pT181 was then
introduced into the polylinker (pMP1008). A 1.2-kb fragment
(MspI/ClaI) containing the ermC gene
from plasmid pMIN164 was blunt ended (T4 DNA polymerase) and then
introduced into one of two sites (Fig.
1): insertion at the EcoRV
site created the yycF disruption (pMP626), and insertion at
the HpaI site created the yycG disruption
(pMP705).

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FIG. 1.
Partial chromosomal map detailing the local organization
around the yycFG locus. The location in the C-terminal
portion of purA of the Tn917lac insertion used in
the construction of the isogenic strains SAM1010 and SAM1011 is shown
( ), along with the HpaI (H) and EcoRV (V)
restriction sites used to create ermC insertions in either
yycF or yycG. Predicted tRNA sequences are
denoted by an inverted triangle. The relative locations of the original
complementing clone (pMP373), the clone rescued from SAM970 (pMP749),
and the two inactivation constructs are shown below the sequence. The
ORFs that have clear orthologues in B. subtilis are labeled;
the last ORF exhibits only limited sequence similarity to
yycH from B. subtilis (data not shown).
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Each plasmid was introduced into
S. aureus SAM13 by
electroporation and selected on TSA-Tc plates. Chromosomal integrants
were isolated and scored for tetracycline resistance; isolates
bearing
the
ermC marker were also scored for erythromycin
resistance.
Site-specific integration of each construct was confirmed
by CHEF
gel analysis of a
SmaI genomic digest; single-copy
integration
was confirmed by genomic PCR. Resolution of the tandem
duplication,
leaving a single disrupted copy of either ORF, was
attempted by
overnight growth in the absence of antibiotic selection.
Single
colonies were inoculated into TSB and grown to the
postexponential
phase (35°C, 24 h); the cultures were then
diluted, plated on
TSA, and allowed to grow overnight (35°C, 24 h). The resulting
colonies were replica printed onto a selective medium
(TSA-Tc);
any colonies that were identified as tetracycline sensitive
(Tc
s) were scored for retention of the
ermC insertion.
The rescue plasmid (pMP782) was constructed by linearizing pC194
(
HindIII) and ligating it into plasmid pMP621
(
HindIII).
The rescue plasmid was introduced by
electroporation into strains
bearing a tandem duplication of the
yycFG locus (containing either
a
yycF::
ermC or a
yycG::
ermC insertion in one copy) and
selected
on TSA containing chloramphenicol. Strains obtained in this
manner
were grown overnight (35°C, 18 h) in TSB supplemented
with erythromycin
(1 µg/ml) and then scored on TSA-Em plates.
Colonies were replica
plated onto TSA-Tc plates and examined for
Tc
s. The loss of the integration construct from the
chromosome, indicated
by the loss of the
tetK marker and the
loss of the additional
SmaI site from the integration
construct, was confirmed by CHEF
gel analysis and genomic PCR.
Transducing lysates were made from
strains bearing either chromosomal
disruption (
yycF::
ermC or
yycG::
ermC)
and used to attempt to
transduce SAM13, either with or without
the rescue plasmid, to
erythromycin
resistance.
Nucleotide sequence accession number.
The DNA sequence
corresponding to the complementing clone from pMP373 has been deposited
with GenBank under accession no. AF136709.
 |
RESULTS |
Mutant isolation and complementation.
The S. aureus
ts mutant NT372 was initially identified by its inability to form
colonies on TSA plates at 43°C; subsequent experiments showed that it
could not grow on any standard rich medium (solid or liquid) or in
liquid or on solid defined medium (chemically defined medium) at
temperatures above 40°C. A Tn917lac insertion linked to
the TS phenotype in NT372 was identified (see Materials and Methods)
and used to move the ts mutation into unmutagenized strain
backgrounds. The isogenic strains SAM1010 (yycF1) and
SAM1011 (yycF+) have the TS and
temperature-insensitive growth phenotypes, respectively. SAM1010 failed
to form colonies on rich and defined media at 43°C, demonstrating
that a single locus was solely responsible for the inability of NT372
to grow at a high temperature.
CHEF gel analysis of a
SmaI chromosomal digest followed by
Southern hybridization with a probe specific for Tn
917lac
localized
the transposon insertion to the
SmaI-G fragment
(data not shown).
Sequence analysis of a clone containing the
chromosomal DNA flanking
the Tn
917lac insertion localized
the insertion to the extreme
C-terminal end of the
purA
locus of
S. aureus, which has been
shown to reside in the
SmaI-G fragment (
40). These results position
the
ts mutation site near the
purA locus of
S. aureus.
Three independently isolated overlapping clones were selected from a
genomic library by complementation of the
ts defect of
the
NT372 mutant. The shortest complementing clone, pMP373, contained
an
insert of 3,731 nucleotides; sequence analysis identified two
complete
ORFs, predicted to encode proteins of 233 and 608 amino
acids. No ORFs
were predicted within the first 650 bp of this
clone, and two tRNA
consensus motifs were identified in the genomic
DNA proximal to the
yycFG locus (Fig.
1). The first ORF was preceded
by a
Shine-Dalgarno sequence (AGAGG) by 9 bp, and a predicted
promoter
region (positions

35 and

10) showing agreement with
the consensus
sequences recognized by
A from
B. subtilis
could be identified 215 bp upstream (TTGTCA
and ACTAAT,
respectively).
Searches against the GenBank database with the first putative ORF
revealed a high degree of similarity to a number of RR genes.
The
highest degree of similarity observed was to
yycF from the
B. subtilis genomic sequence database (
29); we
found 89% amino
acid similarity and 74% amino acid identity.
Extensive similarity
to PhoP (
48) from
B. subtilis was also noted, placing the product
of this ORF in the
OmpR-PhoB subfamily of RRs. Structural alignments
with orthologues of
YycF from other gram-positive organisms highlighted
conserved features
characteristic of RRs (Fig.
2). In
B. subtilis (
29) and in
S. aureus
(Fig.
1), the
yycFG locus is located immediately
downstream
of the
purA locus, in agreement with the chromosomal
mapping
data.

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FIG. 2.
Structural alignment of RRs with a high degree of
similarity to YycF. The conserved residues necessary for proper
phosphotransfer (56) in the regulatory domain are boxed and
highlighted with an inverted triangle. The location of the amino acid
substitution (E63K) in the variant form of the RR is highlighted by a
star. Abbreviations: Bsu, B. subtilis; Sau, S. aureus; Efa, E. faecalis; Spn, S. pneumoniae; Spy, Streptococcus pyogenes. The S. pneumoniae (type 4) unpublished genomic sequence data used to
identify the YycF orthologue were provided by The Institute for Genomic
Research (25a). The E. faecalis sequence (strain
V583) was obtained by designing PCR primers based on the unpublished
genomic sequence data provided by The Institute for Genomic Research
(25a) and then resolving frameshifts by sequencing
PCR-derived amplicons from strain ATCC 29212. The S. pyogenes sequence (strain M1 GAS) was provided by the University
of Oklahoma (44a).
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Searches with the second putative ORF revealed significant similarity
to a number of HK genes. The highest degree of similarity
observed was
to an ORF designated
yycG from
B. subtilis
(
29);
we found overall 69% similarity and 46% identity.
Hydrophobicity
analysis of the translated
yycG ORF predicted
two strongly hydrophobic
stretches in the N-terminal third of the
polypeptide, each consisting
of a stretch of approximately 25 amino
acids and bounding a hydrophilic
sequence of 145 amino acids. In the
absence of structural studies,
these predictions suggested two
membrane-spanning regions (TM1
and TM2) with an intervening cell
surface loop that could define
a sensory domain (
6); the
remaining hydrophilic C-terminal
portion of the polypeptide, which
contained sequence motifs characteristic
of the kinase domains of HKs
(
39), could define a cytoplasmic
signaling
domain.
Identification of the ts mutation in NT372.
To
determine the identity of yycF1, the genomic mutation
causing the TS phenotype of NT372, genomic fragments containing
yycFG were amplified by PCR from the chromosomal DNAs of
wild-type strain SAM23 and ts mutant strain NT372. Sequence
analysis of the PCR amplicons confirmed that a point mutation existed
in the regulatory domain of the RR at nucleotide 187 (G187A), resulting
in a predicted E63K variant polypeptide. The mutated residue (Lys)
represents a nonconservative change from the acidic residues (Asp and
Glu) usually occurring at this position (56). By comparison
of the translated regulatory domain of yycF with the
structurally characterized RR Spo0F from B. subtilis
(14), the mutation is predicted to occur in the third
helix of the RR. This residue is a solvent-exposed side chain and may
identify one point of interaction between this RR and its cognate HK.
No other mutations in the NT372 genomic sequence were identified within
the 3.7-kb region containing the yycFG locus.
Phenotypic effects of the yycF1 mutation.
Experiments designed to test the ability of NT372 cells to recover from
a nonpermissive temperature incubation showed that the loss of YycF
function had a bactericidal effect. Transient incubation of NT372 at a
nonpermissive growth temperature (43°C, 2 h), followed by
extended incubation at 30°C, resulted in less than 15% survival;
longer incubation times at the higher temperature (43°C, 4 h)
reduced the number of viable counts recovered at 30°C (<0.1%
survival). Microscopic examination of the yycF1 mutants did
not reveal gross changes in cell morphology or altered rates of
autolysis when liquid cell cultures were shifted to the nonpermissive temperature.
The
yycF1 mutation caused novel nutritional requirements in
chemically defined medium at permissive growth temperatures. An
apparent Ilv

(isoleucine, leucine, and valine) auxotrophy
was observed in
both
yycF1-containing strains (NT372 and
SAM1010) but not in the
corresponding wild-type strains (SAM23 and
SAM1011). Single additions
of isoleucine, leucine, or valine or of
metabolic intermediates
required for the biosynthesis of these amino
acids (
51) failed
to correlate this apparent auxotrophy with
a specific enzymatic
defect. However, the combination of valine and
pyruvate restored
growth to the
yycF1-containing strains,
suggesting that the defect
was related to branched-chain fatty acid
biosynthesis, perhaps
through improper regulation of 2-ketoisovalerate
levels, rather
than to an amino acid auxotrophy (
15,
33).
Additionally, this
apparent auxotrophy could be corrected by
supplementation with
pantothenate, which is also required for
branched-chain fatty
acid biosynthesis as a precursor for coenzyme A
(
22).
The possible connection between the apparent Ilv

phenotype of the
yycF1 mutants and membrane integrity was
investigated further.
Certain long-chain
cis unsaturated
fatty acids (UFAs) are known
to inhibit the growth of
S. aureus (
7), presumably by affecting
the permeability of
the membrane (
17). A strain carrying the
yycF1
mutation, SAM1010, was more susceptible to the bactericidal
action of
the long-chain
cis UFAs linoleic acid and oleic acid
than
the isogenic wild-type strain, SAM1011 (Table
2). The in
vitro interference of UFAs
specifically in HK autophosphorylation
has also been reported for KinA
from
B. subtilis (
53); whether
the observed
hypersusceptibility was due to direct inhibition
of YycG
autophosphorylation or was an indication of altered membrane
integrity
is not currently known. Other phenotypic characteristics
of
S. aureus that might depend upon the integrity of the phospholipid
bilayer, including the production of cell surface and secreted
proteins
(e.g., coagulase, lipase, and alpha-toxin), appeared
to be unaffected
in strains carrying the
yycF1 mutation (data
not shown).
The
yycF1 mutation completely prevented growth in a variety
of media at temperatures above 40°C; the rate of growth of the
mutant
was comparable to that of the wild type below 39°C. It
was found that
strain SAM1010 could grow under anoxic conditions
in liquid media (Fig.
3) at 41°C but not 43°C. The TS
phenotype
of SAM1010 could be suppressed by growth on solid media
supplemented
with NaCl (500 mM) or sucrose (1 M) at 43°C; however,
the addition
of NaCl (from 500 mM to 2 M) to liquid media did not
restore growth
at 43°C but did allow growth at 41°C. Although the
alteration
of some environmental conditions was found to permit the
growth
of the
ts mutant only on solid media at 43°C, no
conditions that
allowed the cells to survive when either ORF,
yycF or
yycG, was
insertionally inactivated were
found (see below). It is well established
that increased salt
concentrations can suppress the growth conditional
phenotypes of many
ts mutants, as well as mutants with defects
in cell wall and
membrane integrity (
27). In addition, these
high-salt
conditions are known to influence the fatty acid composition
of the
membrane of
S. aureus (
23).

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FIG. 3.
Comparison of the growth of isogenic strains SAM1010 and
SAM1011 at 41°C. (a) The SAM1010 mutant could not survive above
40°C in the presence of oxygen in a variety of liquid media,
including TSB. (b) The rate of growth of the yycF1 mutant
was only partially restored under anoxic conditions at a nonpermissive
temperature in the same liquid media. OD, optical density;
ts, temperature sensitive; wt, wild type.
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The yycF1 mutation affects resistance to
MLSB antibiotics.
The yycF1 mutation was
also found to increase the susceptibility of S. aureus to
macrolides and lincosamides, although not to other classes of
antibiotics, including
-lactams, quinolones, aminoglycosides, and
glycopeptides (Table 3). In addition, the Tn917lac insertions present in the isogenic strains SAM1010
and SAM1011, which bear the ermB resistance determinant
(50), conferred different levels of Emr (Table
3). The NT372 mutant strain was fourfold more susceptible to
erythromycin than was the unmutagenized parent, SAM23 (data not shown),
suggesting that the difference might be indicative of a specific defect
in the yycF1 strains. The mechanism of
erm-encoded resistance to
macrolide-lincosamide-streptogramin B (MLSB) antibiotics is
well studied (58) and involves control by translational
attenuation (2); accordingly, no difference in
ermB mRNA levels was observed in samples from SAM1010 and
SAM1011 (data not shown).
The differences in the susceptibilities of the SAM1010 mutant to the
lincosamide antibiotics lincomycin hydrochloride, clindamycin
hydrochloride, and clindamycin phosphate were large; the SAM1010
mutant
was highly susceptible to lincomycin and clindamycin hydrochloride
but
not to clindamycin phosphate (Table
3). Since the YycF protein
exhibits
a high degree of similarity to other RRs of the PhoB
subfamily, the
effect of added P
i on the MICs of erythromycin
and
lincomycin was examined. A simple addition of P
i (100 µM)
to the assay medium substantially increased the MIC of lincomycin
from
0.25 µg/ml to >1,024 µg/ml for SAM1010; similar differences
were
noted for the two commercial preparations of clindamycin.
No change in
susceptibility to erythromycin was noted; since the
lincosamides are
more hydrophilic, factors governing susceptibility
to the more
hydrophobic erythromycin may be different (e.g., partitioning
into the
membrane [
10]). Indeed, the addition of low levels
of
the UFA linoleic acid (2 ng/ml) to Mueller-Hinton medium significantly
affected the MIC of erythromycin for SAM1010 (lowered it from
16 µg/ml to 0.25 µg/ml) but not for SAM1011 (>1,024 µg/ml under
both
conditions).
These differences in macrolide and lincosamide susceptibility were
influenced by the same conditions that influenced the growth
of the
yycF1 mutant SAM1010 at the semipermissive temperature;
for
example, the MIC of erythromycin for SAM1010 increased from
16 µg/ml
to 256 µg/ml when the strain was grown under anoxic conditions
at a
neutral pH. It is interesting to note that this correction
was only
partial and that growth under anoxic conditions allowed
the
ts mutant to grow at 41°C but not at 43°C. Changes in
the
fatty acid compositions of membrane lipid pools have been observed
to occur in
S. aureus transitioning between aerobic growth
and
anaerobic growth (
59); it is possible that an altered
balance
of fatty acids in this example led to the measured increase in
resistance to erythromycin under anoxic growth
conditions.
Attempted insertional inactivation of yycFG.
Two
chromosomal integration constructs were created to achieve insertional
disruption of either the RR (yycF) or the HK
(yycG) ORF. By using the suicide plasmid pMP1008, the
ermC gene was inserted either at the EcoRV site
to disrupt the RR at amino acid 53, creating pMP626, or at the
HpaI site to disrupt the HK at amino acid 344, creating
pMP705 (Fig. 1). Both of these constructs were integrated separately
into the chromosome of SAM13, creating a tandem duplication consisting
of a wild-type copy of the yycFG locus, an intervening tetK gene, and an additional copy of the locus with a
disruption of either yycF (SAM1156) or yycG
(SAM1157). Successful integration of these constructs added a rare
SmaI site; CHEF gel analysis of the SmaI genomic
digests of both of the integrants (data not shown) confirmed the
addition of a SmaI site in the "G" fragment. Both
tandem-duplication constructs were stable, although the strain bearing
the insertion in the second copy of the RR grew more slowly under
antibiotic selection than the corresponding strain bearing the
insertion in the second copy of the HK.
For comparison, the original suicide plasmid (pMP1008) was also
integrated into the chromosome of SAM13 at the
SmaI-G
fragment,
resulting in two complete copies of the
yycFG
locus with an intervening
tetK marker. The resulting
merodiploid strain, SAM1443, did not
exhibit growth-related phenotypes
distinct from those of the parental
strain. In the absence of selection
during overnight growth, resolution
of the
yycFG duplication
was first identified by replica plating
for the Tc
s
phenotype and then confirmed by genomic PCR and CHEF gel analysis.
When
this procedure was used, Tc
s segregants arose from SAM1443
at 21 in 5,000 CFU. This result
is in contrast to that for either of
the tandem-duplication constructs.
Attempts to resolve the tandem
duplication and retain the chromosomal
insertion in
yycF or
yycG were not successful in the absence of
a wild-type copy
of the gene; this finding held true for either
disruption construct
(SAM1156 and SAM1157). For both strains,
resolution of the chromosomal
tandem duplications was initially
identified by the loss of the
tetK marker and then scored for
retention of the
ermC insertion. In the absence of the rescue
plasmid,
Tc
s segregants arose at 18 in 10,000 CFU; none of these
resulting
colonies (0 of 18) was Em
r, indicating that the
duplications had resolved without retaining
the
ermC
insertion (Fig.
4).

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|
FIG. 4.
Resolution frequencies for chromosomal duplications. The
ability to resolve a tandem duplication of the yycFG locus
with an intervening tetK marker (hatched box) was compared
for strains either bearing a complete duplication (left) or bearing an
ermC insertion (shaded box) within the second copy of
yycG (right). Not shown is the parallel insertion in
yycF.
|
|
To demonstrate further the essential nature of the
yycFG
locus, attempts were made to transduce the chromosomal disruption
of
either ORF to a new set of strains either lacking or bearing
a separate
copy of the wild-type
yycFG locus. To do this, a rescue
plasmid (pMP782) constructed with the complementing clone from
pMP373
(Fig.
1) was used to aid in the generation of derivatives
of SAM1156 or
SAM1157 that had resolved the tandem duplication
and retained the
ermC insertion in the chromosome. In the presence
of the
rescue plasmid, retention of the
ermC insertion in either
SAM1156 or SAM1157 was selected by overnight growth in liquid
cultures
in the presence of erythromycin (1 µg/ml); resulting
colonies that
were Tc
s and Em
r could be identified at 6 in
10,000 CFU. Successful resolution
of the tandem duplication in either
case, resulting in one genomic
copy of the disrupted locus, was
confirmed by genomic PCR and
CHEF gel analysis (data not shown).
Transducing lysates were prepared
from the resulting strains, SAM1158
(
yycF::
ermC/pMP782) and SAM1159
(
yycG::
ermC/pMP782). The recipient
strain was either SAM13 or
SAM1061 (SAM13 with rescue plasmid pMP782).
In the presence of
the rescue plasmid, Em
r transductants
were isolated successfully (on average, 200 CFU
per 10
9 PFU
of transducing lysate); genomic PCR and CHEF gel analysis
confirmed the
presence of a single disrupted chromosomal copy
of either of the ORFs
in the transductants. In the absence of
the rescue plasmid, no
Em
r transductants (0 CFU) were observed; no altered growth
conditions,
including selection with high salt, high sucrose, or low
oxygen
levels, that could be used to select Em
r
transductants of SAM13 were
found.
 |
DISCUSSION |
This work identifies a ts mutation in a two-component
system from S. aureus that is essential for survival in
vitro. An isogenic pair of strains, SAM1010 and SAM1011, was
constructed from the original mutant (NT372) for further
characterization by use of a closely linked Tn917lac
chromosomal insertion. Strains carrying the yycF1 ts
mutation exhibited attenuated virulence in two different in vivo models
(4), implying that this system could be defined as essential
for in vivo survival as well. Given the in vitro and in vivo effects of
a ts mutation in the yycFG locus on the proliferation of S. aureus, this two-component system is an
attractive target for antimicrobial compound screening.
The yycF1 mutation causes hypersensitivity to
macrolides.
The yycF1 strain SAM1010 was affected in
the phenotypic expression of MLSB resistance; this partial
resistance could be influenced by the same environmental factors that
influenced the TS phenotype. The observed differences in susceptibility
to macrolides between SAM1010 and SAM1011 could be an indication either
of increased permeability or of decreased efflux in the mutant
(36). For example, macrolide susceptibility in S. aureus has been observed to increase markedly for strains bearing
an insertional inactivation in either femAB (30)
or lysC (3), both of which contribute to the
structural integrity of the cell envelope. Conditional lethal alleles
of genes involved in phospholipid biosynthesis in S. aureus,
including pgsA (phosphatidylglycerol phosphate synthase; EC
2.7.8.5) and cdsA (phosphatidate cytidylyl transferase; EC 2.7.7.41), have also been found to confer hypersusceptibility to
macrolides (32). Alternatively, disruption of the
norA efflux pump in S. aureus results in
hypersusceptibility to a number of antimicrobial compounds
(21). Since the SAM1010 mutant is not broadly
hypersusceptible to antibiotics, and those UFAs that are incorporated
into membrane lipid pools at subinhibitory concentrations (16) also affect the erythromycin susceptibility of this
mutant, it is proposed that the observed differences in susceptibility are due to a defect in the permeability barrier of the mutant.
The yycFG locus encodes an essential HK-RR pair.
Sequence analysis of the genomic DNA of the mutant confirmed that a
point mutation existed in the genomic copy of the RR (encoded by
yycF). The Glu-63 residue that occurs in the regulatory
domain of the wild-type RR may contribute to the proper interaction
with its cognate HK, whereas the Lys-63 variation in the mutant may destabilize the interaction of this HK-RR pair at a high temperature. Thus, a temperature shift to a nonpermissive temperature (43°C) may
result in premature dissociation of the variant RR from the HK before
an essential phosphotransfer event can occur. If the RR requires
phosphorylation for activity (i.e., transcriptional activation), the
conditional lethal phenotype would be recessive, as we have reported
here, and thus suppressible in trans by a complete wild-type
copy of the HK-RR pair.
An attempt was made to disrupt the genomic copy of the
yycFG
locus to demonstrate its essential nature in vitro; toward this
end,
insertional inactivation constructs of either ORF were first
established as tandem duplications in the chromosome. The inability
to
resolve the duplication while retaining the
ermC insertion
was used as an initial measure of the essential nature of this
locus.
Attempts to disrupt either the HK or the RR in the chromosome
failed in
the absence of a wild-type copy of the locus. In the
presence of a
plasmid-borne copy of the wild-type locus, it was
possible to identify
strains with a disrupted copy of either ORF
in the chromosome. Once the
resolution of the duplications was
achieved, the inactivated loci could
be transduced only to a recipient
strain bearing a separate, functional
copy of the locus. Importantly,
environmental conditions that
influenced the temperature sensitivity
of mutant strains carrying
yycF1, such as 0.5 M NaCl or exclusion
of oxygen, did not
allow the viability of strains with an insertional
inactivation of
yycF or
yycG. Thus we believe that these
conditions
allow partial suppression of the effects of the variant
YycF1
protein but do not bypass the essential function of the
protein.
The yycFG system regulates an essential process.
Based upon unusually high peptide-level similarities (~89%), there
are clear orthologues of this HK-RR pair in the genomes of other
low-G+C-content gram-positive bacteria, including B. subtilis, Enterococcus faecalis, and
Streptococcus pneumoniae. Similarly, these genomes also
appear to contain distinct orthologues of genes encoding other members
of the OmpR-PhoB subfamily of HK-RR pairs, including phoPR
(48, 49) and resDE (55). Since phoPR and resDE have been demonstrated to
interact in B. subtilis (54) and the modulation
of at least one related environmental condition (i.e., anoxic growth
conditions) significantly influences the growth of the ts
mutant described here, it is possible that the yycFG system
also plays a role in this regulatory network. The sensing of limiting
phosphate levels by phoPR and how it might relate to the
regulatory role of yycFG in S. aureus remain
unclear; the response of S. aureus to phosphate limitation
has only recently received extensive attention (57), and
genetic studies characterizing the PHO regulon in this organism have
not been reported. Studies characterizing the in vitro and in vivo
pathway(s) regulated by yycFG and possible relationships to
other members of the PHO regulon in S. aureus are currently
under way.
Several features of these genes make them uniquely suitable as targets
for new antibiotic agents. A point mutation in the
genomic copy of the
RR confers a strongly bactericidal phenotype
at a nonpermissive
temperature, suggesting that inhibitors of
this pathway would also have
bactericidal effects. The mutant
is also hypersusceptible to the
bactericidal action of the UFAs
linoleic acid and oleic acid, which are
found at the sites of
S. aureus infections (
9,
61); it is possible that an inhibitor
of this specific
two-component system may render the bacteria
more vulnerable to killing
by the host. The essential nature of
this system in the soil bacterium
B. subtilis has been reported
(
13,
24); in
addition to the findings reported here with
S. aureus, this
system may also play an essential role in other pathogens
(e.g.,
E. faecalis) (Fig.
2), providing the opportunity for the
development of an antibacterial compound active against a range
of
pathogenic gram-positive bacteria. The existence of multiple
HK-RR
proteins in prokaryotes provides the possibility that a
single agent
might inhibit more than one signal transduction pathway.
Furthermore,
inhibition of the activity of the YycF protein causes
hypersensitivity
to macrolides, suggesting that YycF inhibitors
would show synergy with
macrolide
antibiotics.
 |
ACKNOWLEDGMENTS |
This work was supported in collaboration with the Robert Wood
Johnson Pharmaceutical Research Institute.
We acknowledge helpful discussions with Laura McDowell and Jerry Buysse
and the technical assistance of Kelly Winterberg, Henry Fang, and Skip Bond.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Microcide
Pharmaceuticals, Inc., 850 Maude Ave., Mountain View, CA 94043. Phone:
(650) 428-1550. Fax: (650) 428-3550. E-mail:
martin{at}microcide.com.
Present address: PharmaNet, Inc., Blue Bell, PA 19422.
Present address: Iconix Pharmaceuticals, Inc., Mountain View,
CA 94043.
 |
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