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Appl Environ Microbiol. 2008 October; 74(19): 5882–5890.
Published online 2008 August 8. doi:  10.1128/AEM.00882-08
PMCID: PMC2565972

Insertional Inactivation of Branched-Chain α-Keto Acid Dehydrogenase in Staphylococcus aureus Leads to Decreased Branched-Chain Membrane Fatty Acid Content and Increased Susceptibility to Certain Stresses[down-pointing small open triangle]

Abstract

Staphylococcus aureus is a major community and nosocomial pathogen. Its ability to withstand multiple stress conditions and quickly develop resistance to antibiotics complicates the control of staphylococcal infections. Adaptation to lower temperatures is a key for the survival of bacterial species outside the host. Branched-chain α-keto acid dehydrogenase (BKD) is an enzyme complex that catalyzes the early stages of branched-chain fatty acid (BCFA) production. In this study, BKD was inactivated, resulting in reduced levels of BCFAs in the membrane of S. aureus. Growth of the BKD-inactivated mutant was progressively more impaired than that of wild-type S. aureus with decreasing temperature, to the point that the mutant could not grow at 12°C. The growth of the mutant was markedly stimulated by the inclusion of 2-methylbutyrate in the growth medium at all temperatures tested. 2-Methylbutyrate is a precursor of odd-numbered anteiso fatty acids and bypasses BKD. Interestingly, growth of wild-type S. aureus was also stimulated by including 2-methylbutyrate in the medium, especially at lower temperatures. The anteiso fatty acid content of the BKD-inactivated mutant was restored by the inclusion of 2-methylbutyrate in the medium. Fluorescence polarization measurements indicated that the membrane of the BKD-inactivated mutant was significantly less fluid than that of wild-type S. aureus. Consistent with this result, the mutant showed decreased toluene tolerance that could be increased by the inclusion of 2-methylbutyrate in the medium. The BKD-inactivated mutant was more susceptible to alkaline pH and oxidative stress conditions. Inactivation of the BKD enzyme complex in S. aureus also led to a reduction in adherence of the mutant to eukaryotic cells and its survival in a mouse host. In addition, the mutant offers a tool to study the role of membrane fluidity in the interaction of S. aureus with antimicrobial substances.

Staphylococcus aureus is an aggressive bacterial pathogen that is responsible for a variety of diseases, ranging from pyogenic skin infections to complicated life-threatening diseases, such as bacteremia and endocarditis (20, 28). It is estimated that 32.4% of the population in the community and up to 90% of health care professionals in the United States are colonized with S. aureus (21). In this context, the spread of methicillin-resistant S. aureus strains, and the more recent emergence of resistance to vancomycin, poses a significant threat in terms of our ability to control infections by this major human pathogen.

S. aureus encounters a wide range of environments that include thermal fluctuations and a nutritionally restricted milieu and must adapt to these conditions in order to survive. Cytoplasmic membranes are important barriers between bacterial cells and the environment, and it is essential for the bacteria to regulate the fluidity of their cytoplasmic membrane for the proper functioning of various membrane-associated processes. S. aureus has a complex fatty acid composition comprised of straight-chain saturated fatty acids (SCFAs), unsaturated fatty acids, and branched-chain fatty acids (BCFAs) (30, 35). BCFAs account for about 55 to 65% of the total fatty acids, and anteiso C15:0 is the major BCFA. BCFAs in general, and anteiso C15:0 in particular, are major determinants of membrane fluidity in S. aureus.

In Bacillus subtilis, two enzymes are critical in the synthesis of BCFAs: branched-chain α-keto acid dehydrogenase (BKD) and β-ketoacyl-acyl carrier protein synthase III (FabH) (6, 19, 25). The synthesis of BCFAs begins with the transamination of isoleucine, valine, and leucine by branched-chain amino acid transaminase. The products of this reaction are subsequently decarboxylated by the BKD enzyme complex to produce short branched-chain acyl coenzyme A (acyl-CoA) derivatives 2-methylbutyryl-CoA, isobutyryl-CoA, and isovaleryl-CoA from isoleucine, valine, and leucine, respectively. These acyl-CoA precursors are then utilized by FabH to initiate BCFA biosynthesis.

BKD is a multisubunit enzyme complex that has been studied for several bacteria, e.g., Pseudomonas aeruginosa (22), Pseudomonas putida (34), B. subtilis (18), and Listeria monocytogenes (38). The purified enzyme complex is composed of four polypeptides, a dehydrogenase (E1α), a decarboxylase (E1β), a dihydrolipoamide acyltransferase (E2), and a dihydrolipoamide dehydrogenase (E3) (8, 38). Genes encoding these four polypeptide components are organized in a cluster and are coregulated. A comprehensive search of the S. aureus genome sequence (http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=610) identified a locus consisting of four genes which encode proteins with significant sequence homology with the four subunit proteins of the BKD enzyme complex of B. subtilis and L. monocytogenes. To investigate the roles of this locus in S. aureus physiology, a BKD-deficient strain of S. aureus was created by insertional inactivation of the first gene (dihydrolipoamide dehydrogenase; lpd) of the bkd locus. Further studies with this mutant demonstrated that the lack of a functional BKD enzyme complex in S. aureus leads to an alteration in membrane fatty acid composition, decreased membrane fluidity, enhanced susceptibility to alkaline pH and hydrogen peroxide stressors, reduced adherence to eukaryotic cells, and reduced survival in a murine host.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions.

The bacterial strains and plasmid constructs used in this study are shown in Table Table1.1. S. aureus was grown in tryptic soy broth or agar (TSB or TSA; Becton Dickinson) or in brain heart infusion (BHI) medium (Becton Dickinson). Escherichia coli cells were grown in Luria-Bertani broth or agar. When needed, ampicillin (50 μg ml−1), kanamycin (30 μg ml−1 in the case of E. coli; 100 μg ml−1 in the case of S. aureus), and chloramphenicol (10 μg ml−1) were added to the growth medium.

TABLE 1.
Bacterial strains and plasmids used in this study

DNA manipulation and analysis.

Plasmid DNA was isolated using a Qiaprep kit (Qiagen, Inc.); chromosomal DNA was isolated using a DNAzol kit (Molecular Research Center) from lysostaphin-treated S. aureus cells, per the manufacturer's instructions. All restriction and modification enzymes were purchased from Promega. DNA manipulation and Southern blot analysis were carried out using standard procedures. PCR was performed with a PTC-200 Peltier thermal cycler (MJ Research). Oligonucleotide primers were obtained from Sigma Genosys.

Construction of the mutation in the first gene of the S. aureus BKD gene cluster.

A functional BKD enzyme is generated by the association of polypeptides encoded by individual genes of the four-gene BKD cluster (8, 38). To inactivate the BKD enzyme complex in S. aureus, the lpd gene, which is the first gene of the S. aureus BKD gene cluster, was disrupted by the insertion of a kanamycin resistance cassette in the coding region. To generate this mutant, two primers (forward primer 5′-CTACCGGTGAACTTTGAGA-3′ and reverse primer 5′-GGCAGAGAAAATGCGAGA-3′) were used to amplify a 2,213-bp DNA fragment using genomic DNA from S. aureus strain SH1000 as the template. This amplicon represents a DNA fragment starting 409 nt upstream and spans the entire lpd gene of the bkd gene cluster. This amplicon was cloned in vector pGEM-T Easy (Promega) to generate the construct pGEM-lpd. The cloned fragment contained a unique HindIII restriction site into which a kanamycin resistance cassette (11) was cloned. The resulting construct, pGEM-lpd-kan, was used as a suicide plasmid to transform S. aureus strain RN4220 (a restriction-minus strain) by electroporation (29). The transformants were selected on TSA plates containing kanamycin. The selection resulted in a single crossover and integration of the entire construct into the S. aureus chromosome. Phage 80[proportional, variant] was propagated on these transformants and used to resolve the mutation in the lpd gene in S. aureus by performing transduction outcrosses as described previously (32, 33). The transductants were confirmed for a mutation in the lpd gene using PCR and Southern blotting. For genetic complementation studies, the entire bkd locus starting 225 nt upstream of the first gene (lpd) and terminating 133 bp after the fourth gene (bkdB) was PCR amplified (5,058-bp amplicon) using appropriate primers (forward primer 5′-TGCATTCAACCATGTTGATT-3′ and reverse primer 5′-TCAGGTGCAAGTGTTACTG-3′) and S. aureus SH1000 genomic DNA as the template. PCR was carried out using Stratagene EXL DNA polymerase (Stratagene, CA) per the manufacturer's instructions. This amplicon was cloned in vector pGEM-T from which it was subcloned in vector pCU1 (2). The lpd mutant of S. aureus strain SH1000 was subsequently transformed with this construct.

Analysis of fatty acid composition in wild-type and lpd mutant S. aureus.

To determine the membrane fatty acid composition, cultures of the lpd mutant and its isogenic S. aureus parent strain were grown in 100 ml BHI medium at 37°C. Cells were harvested in the mid-exponential phase (optical density at 600 nm [OD600] of 0.5 to 0.7), and the cell pellet was washed three times with distilled water. The fatty acids in the bacterial cells (30 to 40 mg [wet weight]) were saponified, methylated, and extracted as described previously (38). The resulting methyl ester mixtures were separated by an Agilent 5890 dual-tower gas chromatograph. Fatty acids were identified by a Midi microbial identification system (Sherlock 4.5). This analysis was performed at Microbial ID, Inc. (Newark, DE).

Growth kinetics of wild-type S. aureus and its isogenic lpd mutant.

Mid-exponential phase cultures (OD600 = 0.6) were diluted 50-fold in an Erlenmeyer flask containing 50 ml fresh BHI medium with a flask-to-medium volume ratio of 6:1. All supplements (0.1 mM 2-methylbutyrate, isobutyrate, or isovalerate) were added to the medium as filter-sterilized solutions. In parallel flasks, the following stress conditions were imposed through the appropriate modifications of BHI medium: 8.8 mM hydrogen peroxide (H2O2), low pH (pH 5.5), high pH (pH 9.5), and NaCl (1.5 M). Bacterial growth was subsequently monitored by incubating the flask in a shaking incubator (250 rpm) and measuring the turbidity of the liquid culture at OD600 by using a Beckman DU-70 spectrophotometer.

Determination of membrane fluidity of the lpd mutant and its isogenic wild-type strain SH1000.

Membrane fluidity was measured as recently described (3, 4). In brief, overnight grown cultures were used to inoculate fresh TSB and incubated at 37°C with shaking (250 rpm) until an OD600 of 0.6 ± 0.05 was reached. The bacteria were pelleted by centrifugation at 4°C and washed twice with 0.85% NaCl. The cells were resuspended in 0.85% NaCl containing 2 μM 1,6-diphenyl-1,3,5-hexatriene (DPH; Sigma, MO) to an OD600 of 0.3 ± 0.05. DPH specifically labels and fluoresces within the hydrophobic regions of the lipid bilayer but does not fluoresce in aqueous environments (3, 4). Fluorescence polarization was subsequently measured using an SLM Aminco 8000C spectrofluorometer (SLM Aminco, SLM Instruments, Inc., IL). Excitation of the fluorescent probe was accomplished with vertically polarized monochromatic light at 360 nm for DPH, with emission intensity quantified at 426 nm, using a detector oriented either parallel to or perpendicular to the direction of the polarized excitation source. The experiment was performed three times, and the mean polarization values were compared for statistically significant differences by using the Student t test.

Measurement of solvent tolerance.

Solvent tolerance was determined using the plate overlay method as previously described (24). Briefly, 20 μl of overnight-grown cultures (~106 cells) of strain SH1000 and the lpd mutant were spotted separately onto BHI agar and BHI agar supplemented with 0.1 mM 2-methylbutyrate in glass petri dishes. The plates were incubated at 37°C, and the spots were allowed to dry for 45 min. Around 5 ml of toluene was directly pipetted onto the top of each agar plate surface in a well-ventilated laboratory area. The plates were incubated at room temperature for 8 h, and toluene was then poured off from the plates. The plates were inverted, and different sets were incubated at 25°C and 37°C for 24 h.

Autolysis assays.

Autolysis assays were performed as previously described (15, 26). Briefly, wild-type and lpd mutant cultures of S. aureus SH1000 were grown to an OD600 of 1.0 at 37°C in PYK medium (0.5% Bacto peptone, 0.5% yeast extract, 0.3% K2HPO4 [pH 7.2]). After one wash with cold water (8,500 × g, 4°C, 15 min), cells were suspended in 0.05 M Tris-HCl buffer, pH 7.2, containing 0.05% Triton X-100 to a OD600 of 1.0. The flasks were incubated at 37°C with shaking (150 rpm), and the subsequent decline in the turbidity of the bacterial cell suspension was measured spectrophotometrically at 600 nm every 30 min for 10 h. Autolysis of the lpd mutant was also analyzed in terms of total and membrane-bound autolysin in the mutant compared to that for wild-type S. aureus strain SH1000. The total autolysins were extracted after the bead beating of bacterial cells in 0.25 M phosphate buffer (pH 7.0) using a BioSpec Mini-Beadbeater after growth in PYK medium to an OD600 of 1.0. The membrane autolysins were extracted by exposing the bacterial cells to five freeze-thaw cycles in 0.25 M phosphate buffer (pH 7.0). The cells were vortexed vigorously after each thaw. The samples were analyzed for the presence of autolysins by a zymographic method using autoclaved S. aureus 8325-4 cells as described previously (15, 26).

Adherence assays.

The relative adherence of S. aureus strain SH1000 and its derivative lpd mutant were determined with a mixed infection of A549 human lung epithelial cells (ATCC CCL 185) as recently described (17). Wild-type S. aureus strain SH1000 and its derivative lpd mutant were grown in BHI medium to an OD600 of 0.3. Bacterial cells were washed three times in PBS and mixed in F-12K medium (ATCC). The resulting mixture was biased for mutant cells to better determine if there was any appreciable decrease in the adherence of the mutant compared to that of wild-type cells. A549 cells were cultured in F-12K medium supplemented with 10% heat-inactivated calf serum at 37°C in a humidified 5% CO2 atmosphere. For adherence assays, approximately 5 × 105 bacterial cells were added to the monolayers of A549 cells (~2 × 105 cells/well) to give an approximate multiplicity of infection of 2.5:1 (bacteria/A549 cells). The plate was centrifuged at 100 × g for 5 min to facilitate contact between bacteria and A549 cells. After 1 h of incubation, nonadherent bacterial cells were removed by washing the epithelial cell monolayer three times with warm sterile PBS. Next, epithelial cells were dispersed by the addition of 150 μl of 0.25% trypsin-1 mM EDTA (Sigma) and then lysed by the addition of 400 μl of 0.025% Triton X-100. The numbers of bacterial CFU adhering to the epithelial cells were determined by plating of diluted epithelial cell lysates on TSA plates with and without kanamycin. The fraction of lpd mutants that adhered to the A549 cells was then calculated and compared to the fraction of lpd mutant cells in the mixed culture used for adherence assays. Each experiment was conducted in triplicate.

Survival of the wild-type and S. aureus lpd mutant cells in a murine systemic infection model.

In vivo survival experiments were carried out as described recently (33). Briefly, S. aureus strain SH1000 and its isogenic lpd mutant were grown to mid-log phase (OD600 = 0.6) in BHI medium and subsequently washed three times with BHI medium. Wild-type and the lpd mutant cells were subsequently combined in BHI medium (72%:28% mixture of mutant/wild-type), and 0.25 ml of this suspension, containing 1.15 × 107 bacteria, was injected into the peritoneal cavity of Swiss white Hla(ICR)CVF female mice (16 to 20 g) with a 26-gauge needle fitted to a 1-ml syringe. At 4, 8, and 18 h, three mice were euthanized by CO2 asphyxiation. The liver and spleen were aseptically removed and homogenized in 2 and 1 ml of BHI medium, respectively, using a glass tissue grinder fitted with a glass pestle. Tissue homogenates were serially diluted, plated on TSA plates with or without kanamycin and allowed to grow overnight by incubation at 37°C. The bacterial colonies growing in the presence of kanamycin were used to calculate the fraction of lpd mutants relative to wild-type bacteria in the infected tissues and compared to the fraction of lpd mutants in the mixed suspension that was used to inject mice.

RESULTS

Genetic organization of the bkd gene cluster and construction of a BKD-null mutant in S. aureus.

The genetic organization of the bkd gene cluster in S. aureus is shown in Fig. Fig.1A.1A. A comparison of the bkd gene cluster in three gram-positive bacterial species indicates a similar genetic organization of the bkd structural genes but presents interesting differences in the genes upstream of this cluster (Fig. (Fig.1A).1A). In B. subtilis, the bkd gene cluster contains seven genes, in contrast to what appears to be only four genes in the cases of S. aureus and L. monocytogenes (38). The seven-gene bkd cluster in B. subtilis is cotranscribed, and the expression of this locus is under the control of a regulatory protein, BkdR (8). Expression of bkdR itself is under the control of an alternative sigma factor, SigL (8). A bkdR-like gene is not present upstream of the bkd locus in the case of S. aureus. Rather, a gene (recN) encoding a putative DNA repair protein is present in the S. aureus chromosome immediately upstream of the first gene (lpd) of the bkd locus. To characterize the function of the S. aureus BKD, a kanamycin resistance cassette was inserted within the first gene of the bkd cluster to inactivate this enzyme complex. The production of a larger PCR product when genomic DNA from the resulting mutant was used as the template (Fig. (Fig.1B,1B, lane 3) confirmed the insertion of a kanamycin gene cassette in the lpd open reading frame. Products of all four individual genes of the bkd cluster associate to form the functional BKD enzyme. It is speculated that a mutation in the lpd gene will have a polar effect on the expression of the entire bkd locus. Even if the mutant generated in this study is “leaky” for the expression of lpd downstream genes, the mutant bacteria will fail to generate an active BKD enzyme complex due to the lack of a functional Lpd.

FIG. 1.
(A) Schematic organization of the bkd gene cluster in three gram-positive bacteria. argR, arginine repressor; recN, DNA repair protein; ptb, phosphate acetyl/butyryltransferase family protein; bcd, leucine dehydrogenase; buk, butyrate kinase; bkdA1/bkdAA ...

Analysis of fatty acid composition in the wild type and the lpd mutant of S. aureus.

The fatty acid compositions of strain SH1000 and its derivative lpd mutant are shown in Table Table2.2. The fatty acid composition of strain SH1000 was typical for S. aureus, showing a complex mixture of BCFAs and SCFAs with anteiso C15:0 (29.4%) and C18:0 (15.7%) as the major fatty acids. Sixty-three percent of the total fatty acids were BCFAs, and 32% were SCFAs; the anteiso/iso fatty acid ratio was 1.4. The lpd mutant strain was deficient in BCFAs (35.4%), and the percentage of SCFAs rose to compensate (61.5%). The anteiso/iso fatty acid ratio was 0.8 in the lpd mutant. When the lpd mutant strain was grown in the presence of 0.1 mM 2-methylbutyrate (a precursor of odd-numbered anteiso BCFAs), the BCFAs were restored to 50% of the total, with a major increase in anteiso C15:0 from 14.1% in the mutant grown in the absence of 2-methylbutyrate to 37.1% in its presence. The anteiso/iso fatty acid ratio was increased to 4.9 in the presence of 2-methylbutyrate. Interestingly, 2-methylbutyrate increased the BCFAs in SH1000 to 68.8%, anteiso C15:0 to 40.1%, and the anteiso/iso fatty acid ratio to 2.6. These results clearly show that that the lpd mutant is deficient in BCFAs. In the growth experiments (see below), isobutyrate and isovalerate were less effective than 2-methylbutyrate in restoring the growth defect of the lpd mutant, consistent with the lower impact of iso than anteiso BCFAs on increasing membrane fluidity. Isobutyrate stimulated the production of the even-numbered iso fatty acids iso C14:0 and iso C16:0 in the lpd mutant (data not shown). The effects of 2-methylbutyrate on fatty acid composition are consistent with its effects on growth (see below). When cells of either SH1000 or the lpd mutant were grown at 25°C, the major changes in fatty acid composition of both strains were increases in anteiso C15:0 and total BCFAs and decreases in C18:0 and total SCFAs (data not shown). Similar findings were reported by Joyce et al. (13).

TABLE 2.
Fatty acid profiles of wild-type SH000, the lpd mutant strain, and a complemented strain

The lpd mutant showed reduced growth at low temperatures that could be alleviated with short BCFA precursors.

A comparison of the growth kinetics shows that the growth of the lpd mutant was somewhat slower at 37°C than that of the wild-type strain SH1000 (Fig. (Fig.2C).2C). However, growth of the lpd mutant was significantly slower at 20°C than that of wild-type S. aureus (Fig. (Fig.2B).2B). When growth was monitored at 12°C, the wild-type SH1000 was able to grow after a long lag, but the lpd mutant failed to grow even after 312 h of incubation at this temperature in medium without added fatty acid precursor (Fig. (Fig.2A).2A). These results suggest a critical role for the BKD enzyme complex during growth and survival of S. aureus, particularly at lower temperatures. To evaluate whether this defect in growth during cold conditions was indeed due to lack of a functional BKD, the media were supplemented with 2-methylbutyrate, isobutyrate, or isovalerate at 0.1 mM concentrations. Supplementation with these short-chain fatty acids allows the mutant bacteria to bypass the biochemical step that requires BKD activity and results in the synthesis of BCFAs. When supplemented with 0.1 mM 2-methylbutyrate, a precursor of odd-numbered anteiso fatty acids, the lpd mutant bacteria were able to grow at markedly increased rates at all temperatures (Fig. 2A to C) and especially at 20°C or 12°C. It is interesting that the growth of the wild-type S. aureus strain was also enhanced in the presence of 0.1 mM 2-methylbutyrate (Fig. 2A to C), which implies that the BHI growth medium may not provide an ideal environment for the synthesis of BCFA or that BKD activity is usually expressed at a low level in S. aureus. However, at 0.1 mM concentrations, neither isobutyrate nor isovalerate, which are precursors of even-numbered and odd-numbered iso fatty acids, respectively, were as effective in restoring the growth of the lpd mutant as 2-methylbutyrate (Fig. 2D and E). The combination of all three fatty acid precursors had a strong stimulatory effect on the growth of wild-type S. aureus at 20°C (Fig. (Fig.2F2F).

FIG. 2.
Growth kinetics of the wild-type S. aureus strain SH1000 and its derivative lpd mutant with and without short-chain keto acid precursors (2MB, 2-methylbutyrate; IB, isobutyrate; IV, isovalerate). Growth temperatures and the precursors used are indicated. ...

A nonfunctional BKD enzyme leads to a less-fluid staphylococcal cell membrane.

In membrane fluidity measurements, a statistically significant difference (P < 0.004) in the polarization value (0.390 ± 0.004) was recorded for wild-type S. aureus strain SH1000 cells compared to the polarization value of the lpd mutant cells (0.432 ± 0.011). These findings are in agreement with a higher polarization value for a less-fluid membrane (3, 4).

Decreased toluene tolerance of the lpd mutant.

It has been reported that solvent tolerance in staphylococci is associated with an increased proportion of anteiso fatty acids and hence increased membrane fluidity, in contrast to the situation for gram-negative bacteria, where solvent tolerance is associated with decreased membrane fluidity (24). Hence, it was of interest to see whether the lpd mutant showed a decreased tolerance of toluene, and the results of these experiments are shown in Fig. Fig.3.3. Strain SH1000 showed significant expanding ring-type growth at 25°C that was enhanced on BHI agar plates supplemented with 2-methylbutyrate. In comparison, growth of the lpd mutant was faint at 25°C on BHI agar, although its growth was enhanced on 2-methylbutyrate-supplemented agar. The results are consistent with decreased membrane fluidity correlating with increased solvent susceptibility.

FIG. 3.
Growth of SH1000 and the lpd mutant on toluene overlay agar plates. SH1000 and the lpd mutant were used to inoculate BHI agar with or without 2-methylbutyrate (2-MB), and cultures were incubated at 37°C and 25°C.

Response of the lpd mutant to various stress conditions.

The lpd mutant was also measured for growth defects compared to wild-type S. aureus under different stress conditions. No apparent difference in the growth kinetics of the lpd mutant was observed under acidic (BHI medium acidified to pH 5.5) or high-salt (BHI medium with an additional 1.5 M NaCl) conditions (data not shown). However, the growth of the lpd mutant was significantly slower at an alkaline pH (BHI medium, pH 9.5) than that of wild-type S. aureus strain SH1000 (Fig. (Fig.4A).4A). In addition, in BHI medium with 8.8 mM H2O2, the growth of the lpd mutant was severely retarded and no visible turbidity of the mutant was apparent by the time the wild-type S. aureus cultures reached stationary phase (Fig. (Fig.4B).4B). These growth defects of the lpd mutant at an elevated pH and in the presence of H2O2 were restored to a significant level when the mutant was complemented with the entire bkd locus on a plasmid in trans (Fig. 4A and B).

FIG. 4.
Growth kinetics of the lpd mutant and its isogenic wild-type S. aureus under stress conditions. (A) Growth in BHI medium modified to pH 9.5. (B) Growth in BHI medium containing 8.8 mM H2O2. Closed squares, wild-type S. aureus strain SH1000; open squares, ...

Inactivation of BKD decreases autolysis of S. aureus cells.

In experiments aimed to investigate Triton X-100-stimulated autolysis, the lpd mutant cells demonstrated a lower rate of autolysis than the wild-type SH1000 cells (Fig. (Fig.5A).5A). Interestingly, as is apparent from Fig. Fig.5B,5B, there was no appreciable difference between the total autolysin profiles for the lpd mutant (lanes 4) and for wild-type S. aureus strain SH1000 (lane 3). However, the zymographic pattern of the freeze-thaw extractable cell surface autolysins for the lpd mutant cells was significantly different from that for wild-type S. aureus. The number of autolysin bands was greater in wild-type S. aureus strain SH1000 (Fig. (Fig.5B,5B, lane 1) than in its derivative lpd mutant (Fig. (Fig.5B,5B, lane 2), and overall, the bands were of greater intensity in the SH1000 extract.

FIG. 5.
(A) Autolysis of the lpd mutant (open squares) compared to autolysis of the isogenic wild-type S. aureus strain SH1000 (closed squares). (B) Autolysin profiles of freeze-thaw (lanes 1 and 2) and total (lanes 3 and 4) autolysin extracts from S. aureus ...

Lack of a functional BKD impairs staphylococcal adherence to eukaryotic cells and the ability to survive in vivo.

In adherence assays, a mixture of lpd mutant and wild-type S. aureus bacteria (4.57 × 105 CFU) was added to the monolayers of A549. A total of (8.3 ± 2.2) × 104 bacterial cells adhered (18.1% adherence) to A549 cells. Although the ratio of the lpd mutant cells to wild-type SH1000 in the mixture that was used for adherence was 68:32, the ratio of these two cell types in the bound fraction was 47:53. Additionally, whereas the relative adherence rate of the wild type cells was 30.2%, only 12.51% of the mutant cells were bound to A549 cells. The experiment clearly suggests that differences in the BCFAs in the membrane affect staphylococcal adherence to eukaryotic cells.

Additionally, the role of BKD in S. aureus pathogenesis was examined. Mice were injected intraperitoneally with a mixture of wild-type SH1000 and the lpd mutant bacteria. Mice were sacrificed at 4, 8, and 18 h postinfection. In these experiments, the numbers of bacterial cells (total CFU g−1 tissue) in liver and spleen samples decreased with time (data not shown). However, the fraction of lpd mutant cells recovered from either the liver (Fig. (Fig.6A)6A) or the spleen (Fig. (Fig.6B)6B) in infected mice decreased with time, and at the same time, the fraction of wild-type cells increased. An increase in the population of wild-type cells compared to lpd mutant cells suggests that a decrease in BCFAs led to a disadvantage in terms of the survival of S. aureus cells in this animal model.

FIG. 6.
Survival of the lpd mutant and the isogenic wild-type S. aureus strain SH1000 in mouse. Approximately 1.15 × 107 CFU (28% wild-type and 72% lpd mutant) were injected intraperitoneally into mice. Three mice were sacrificed at 4, ...

DISCUSSION

Bacterial membrane fatty acids, as acyl chains of phospholipids and glycolipids, determine the fluidity or viscosity of the membrane, and modulation of fatty acid composition allows bacteria to survive in a wide range of physical and chemical environments (37). The total fatty acid composition of S. aureus is comprised of a complex mixture of BCFAs and SCFAs, which are major determinants of the biophysical properties of the membrane. BCFAs in general and anteiso C15:0 fatty acid, in particular, are believed to be major determinants of the fluidity of the S. aureus membrane. In order to further study the role of BCFA in the function of the S. aureus cytoplasmic membrane, we created a mutant in the lpd gene of the bkd gene cluster that plays a major role in the biosynthesis of BCFAs. The lpd mutant was defective in BCFAs and showed decreased membrane fluidity. Besides having a negative impact on growth at low temperatures, decreased membrane fluidity also correlated with lower tolerance of a variety of stresses and possibly in the insertion and display of cell surface proteins.

Studies with the BKD-inactivated mutant provided insight into the critical roles for this enzyme complex in staphylococcal growth. Inactivation of BKD led to a less-fluid membrane and a lower growth rate for the mutant. This level of growth reduction in the mutant became more dramatic at lower temperatures, and the mutant failed to grow at 12°C. Deficiencies of BCFAs in the lpd mutant produce a more-rigid (less-fluid) membrane that impairs the ability of the mutant to perform various essential membrane-associated processes. Restoration of the growth defect in the mutant with 2-methylbutyrate further supports the lack of a functional BKD enzyme complex in the mutant cells. It is evident from the fatty acid composition analysis (Table (Table2)2) that fatty acid anteiso C15:0 fatty acid, which is derived from 2-methylbutyryl-CoA, is the major BCFA in the staphylococcal cell membrane. This probably is the reason that other short-chain fatty acid precursors, isovalerate and isobutyrate, provided much less significant improvement in the growth of the BKD-inactivated mutant. Similar results have been noted with BKD mutants of L. monocytogenes (1, 38). L. monocytogenes differs from S. aureus in that Listeria normally contains almost no SCFAs. The S. aureus lpd mutant was not completely deficient in BCFA. Probably, the BCFAs that are present, which may represent close to a minimum requirement for BCFAs in the membrane lipids (7, 38), are produced via the pyruvate dehydrogenase and, possibly, the α-keto glutarate dehydrogenase complexes, which have some activity with branched-chain α-keto acids (14, 25).

Fluorescence polarization measurements using the probe DPH indicated that the membrane of the lpd mutant was less fluid than that of the parent strain. Consistent with this was the decreased solvent tolerance of the mutant, which could be increased by growth in the presence of 2-methylbutyrate. It has been shown that increased solvent tolerance in staphylococci is associated with increased membrane anteiso fatty acid content and fluidity (24).

The lpd mutant shows that intact BKD is critical for BCFA content and membrane fluidity in S. aureus, and this study provides additional interesting phenotypes associated with it compared to its wild-type counterpart. While no significant growth defect was noted when the lpd mutant was grown in the presence of high salt (1.5 M NaCl) or low pH (5.5), a much lower growth rate was observed for the mutant compared to the wild-type S. aureus at alkaline pH (9.5) or in the presence of 8.8 mM H2O2. It is speculated that the lack of BCFAs, particularly anteiso 15:0 fatty acid, in the cytoplasmic membrane of the lpd mutant leads to an increased susceptibility of the membrane to hydroxylation or peroxidation, rather than a direct role of the BKD enzyme complex in protection of S. aureus cells from these adverse conditions. Evidence for a role of BCFAs in the tolerance of alkali stress in L. monocytogenes has been presented (10).

The lpd mutant bacteria showed decreased susceptibility to the action of autolysins. This decreased autolysis of the mutant cells was observed irrespective of whether the cells were grown at 20°C (data not shown) or 37°C. The lpd mutant cells were also less susceptible than wild-type S. aureus strain SH1000 to the action of lysostaphin in a disc diffusion assay when the mutant bacteria were grown at 20°C (data not shown). However, this decreased susceptibility to lysostaphin was not apparent between the lpd mutant and the wild-type S. aureus when both types of cells were grown at 37°C (data not shown).

Another notable observation with the lpd mutant was its decreased ability to adhere to eukaryotic cell surfaces. Proper adherence to eukaryotic cell surfaces is an important stage during the colonization of a host by pathogenic bacterial species. In addition to its reduced adherence, a relative decrease in the survival of the lpd mutant was observed compared to the wild-type S. aureus in an in vivo competition assay in a murine model.

It was expected that the inactivation of the BKD enzyme complex would lead to reduced BCFAs and a decreased growth rate at lower temperatures. It is clear that the decrease in susceptibility to autolysins is not due to the downregulation of autolysins in the mutant. Similarly, a decrease in adherence of the lpd mutant to eukaryotic cells is unlikely to be due to reduced expression of surface adhesins in the mutant. It is likely that a reduction in BCFA content in the cell membrane of the lpd mutant leads to a decrease in membrane fluidity, which becomes more prominent at lower temperatures. This in turn leads to an alteration and/or positioning of autolysin and adhesion molecules displayed on the bacterial cell surface, leading to the observed phenotypes.

As alternatives to vertebrate animal models of S. aureus infection, a nematode killing assay has been developed to identify virulence genes also required for S. aureus infection of warm-blooded animals (9, 31), and Drosophila melanogaster has been used as a model host for S. aureus infection (23). The Caenorhabditis elegans and D. melanogaster assays are carried out at 25°C, whereas in vertebrate animal models of infection, the temperature is 37°C or higher. The membrane fatty acid composition of S. aureus is different at 25°C than at 37°C, and this may be a factor affecting the virulence of S. aureus at this temperature. The inclusion of 2-methylbutyrate in the growth medium markedly stimulates the growth of wild-type S. aureus at lower temperatures and boosts the amount of anteiso fatty acids in the membrane and its fluidity. It would be interesting to know whether this had any effect on the virulence of the organism for C. elegans and D. melanogaster.

A change in membrane fatty acid composition is an important strategy employed by S. aureus to adapt to changes in environmental conditions. In vitro resistance of S. aureus to thrombin-induced platelet microbicidal protein is also associated with alterations in cytoplasmic membrane fluidity (4). A decrease in membrane fluidity increased resistance to oleic acid killing in S. aureus (5). However, in another staphylococcal species, Staphylococcus haemolyticus, the organism increased its BCFA content, particularly that of anteiso C15:0, and decreased its SCFA content, particularly that of C20:0, and hence increased its membrane fluidity, when grown in the presence of the membrane active solvent toluene (24). A comparison of the fatty acid composition of methicillin-sensitive and methicillin-resistant S. aureus strains has revealed differences in the proportions of anteiso and iso fatty acids in the cytoplasmic membranes of these two types of strains (27). The lpd mutant described here provides a useful tool for further evaluation of the role of membrane fluidity in the interaction of S. aureus with a wide variety of antimicrobial agents. In addition, perturbation of cell membrane fatty acid composition may be an important target for the development of antimicrobial agents.

Acknowledgments

This work was supported in part by a Warner/Fermaturo and ATSU Board of Trustees Research Funds to V.K.S. and in part by USDA National Research Initiative Competitive Grant Program award 2006-35201-17386 to B.J.W.

Footnotes

[down-pointing small open triangle]Published ahead of print on 8 August 2008.

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