INTRODUCTION
The bacterial cell envelope is a barrier that protects bacteria from unpredictable and often hostile environments. In Gram-positive bacteria, such as
Staphylococcus aureus, the cell envelope comprises the cell membrane and a thick peptidoglycan (PG) layer that is decorated with a variety of proteins and polymers important for viability and virulence. Among these polymers are teichoic acids, which are negatively charged and divided into two classes based on their subcellular localization. One class, wall teichoic acids (WTA), are covalently linked to PG; the other class, lipoteichoic acids (LTA), are associated with the cell membrane through a glycolipid anchor (
1). WTA and LTA play partially redundant roles in cell envelope integrity and cannot be deleted simultaneously (
2–4).
In
S. aureus, both WTA and LTA have been implicated in the control of cell morphology and division (
3,
5,
6), virulence (
7–14), osmoregulation (
15–18), antimicrobial resistance (
6,
19–22), and spatiotemporal regulation of cell wall enzymes (
23–27). These functions may be accomplished through organization and regulation of cell envelope enzymes, binding of protons and divalent cations, and alteration of the physicochemical properties of the cell envelope. However, LTA is more important than WTA for cell viability.
S. aureus can grow under standard laboratory conditions without WTA (
28), but cells lacking lipoteichoic acid synthase (LtaS), the enzyme that assembles LTA on the cell surface, are not viable and rapidly acquire suppressor mutations (
3,
5,
16,
29,
30).
The usual glycolipid anchor and the starting unit for LTA is diglucosyl-diacylglycerol (Glc
2DAG), which is synthesized from UDP-glucose and diacylglycerol (DAG) by the glycosyltransferase UgtP (also called YpfP) (
Fig. 1A and
B) (
31,
32). Glc
2DAG is exported to the cell surface by LtaA (
10). The lipoteichoic acid polymerase is LtaS, a polytopic membrane protein with an extracellular domain that contains the active site (
4,
33). LtaS transfers phosphoglycerol units derived from phosphatidylglycerol (Ptd-Gro) to the Glc
2DAG starter unit, producing DAG as a by-product (
5,
34,
35). DAG is recycled to Ptd-Gro by a salvage pathway (
36). LTA is heavily decorated with
d-alanyl residues, a modification of teichoic acids that is important in autolysin regulation and has also been implicated in
S. aureus virulence (
8,
12,
13,
19). Notably, deleting the glycosyltransferase gene
ugtP or the genes encoding the enzymes that produce UDP-glucose, its substrate, does not result in the loss of LTA, although such mutations result in morphological and fitness defects (
10,
11,
32). Instead, LtaS uses Ptd-Gro rather than Glc
2DAG as the lipid starter unit for LTA assembly.
In
Bacillus subtilis, deleting
ugtP or the genes for the enzymes that produce UDP-glucose also results in morphological defects (
37–40). In one study,
ugtP mutant cells were shown to be shorter than wild-type cells, and it was proposed that UgtP is a nutrient sensor that negatively regulates cytokinesis in a manner that depends on UDP-glucose levels (
38). In this model, under nutrient-rich conditions, high levels of UDP-glucose localize UgtP to the cytokinetic ring and inhibit FtsZ polymerization or constriction, which delays cell division to provide cells time to grow to a larger size (
38,
41). Δ
ugtP cells are therefore small, because UgtP is not present to slow cell division. Other studies in
B. subtilis are not consistent with a role for UgtP in nutrient sensing, because mutant cells were found to be enlarged in its absence or to have shape rather than size alterations (
39,
40). Instead, loss of the glycolipids produced by UgtP was proposed to be responsible for the defects of Δ
ugtP in this organism (
42).
UgtP evidently does not act to increase cell size in
S. aureus, because Δ
ugtP mutant cells are larger, rather than smaller, than those of the wild type (
32). Like other
S. aureus cells that grow too large (
6,
43–46), the mutant cells have cell division defects, such as multiple and misplaced septa (
32). While these defects may result directly from the loss of the
ugtP gene product, the Δ
ugtP deletion is pleiotropic and causes multiple effects, including the absence of the disaccharide anchor Glc
2DAG and a reported abnormal lengthening of LTA polymers (
10). Whether increased cell size and dysregulated cell division directly result from the loss of the
ugtP gene product, from the loss of intracellular Glc
2DAG, or from the abnormally long LTA polymers has been unclear.
Mutants lacking
ltaA, which encodes the flippase that exports Glc
2DAG to the cell surface, provide a means to distinguish among these possibilities. Δ
ltaA mutants express UgtP and synthesize Glc
2DAG but are unable to export it efficiently; they may have higher intracellular concentrations of Glc
2DAG than wild-type cells (
10). The LTA polymers produced by Δ
ltaA mutants are longer than those of the wild type but shorter than those of Δ
ugtP mutant cells, being a heterogeneous mixture in which some polymers are assembled on Ptd-Gro and some on Glc
2DAG (which is exported to the cell surface by an alternative, unknown mechanism) (
10). Although Δ
ltaA cells have an altered distribution of glycolipids in the membrane leaflets, and Δ
ugtP cells lack glycolipids entirely, we hypothesized that if the defects observed in Δ
ugtP cells were caused by the abnormally long LTA polymers, we should also observe cell size and division defects in the Δ
ltaA mutant. Under this assumption, we predicted that the Δ
ltaA mutant would have less pronounced phenotypes than the Δ
ugtP mutant due to the intermediate polymer length in the Δ
ltaA mutant.
Here, we show that the production of long, abnormal LTA is sufficient to alter cell size and lead to cell division defects. We also report that LTA pathway mutants with these morphological defects are highly susceptible to beta-lactam antibiotics and PG hydrolases and are dependent on other cell envelope pathways that are dispensable in wild-type strains. We used an inhibitor of one of these pathways to select for suppressor mutations in ΔugtP strains and found that most of the suppressor mutations were located in the LTA polymerase gene, ltaS, and caused a reduction in LTA polymer length. Polymer abundance was frequently decreased as well, in some cases to almost undetectable levels. The ltaS suppressor mutations partially reversed the cell size and division abnormalities caused by the ΔugtP mutation. Taken together, these studies indicate that LTA length and abundance play a crucial role in controlling cell size and cell envelope integrity in S. aureus.
DISCUSSION
The principal goals of this investigation were to elucidate the roles of ugtP and LTA length in S. aureus cell growth and division and to identify mechanisms contributing to cell viability when LTA levels are low. First, by comparing multiple phenotypes of ΔltaA and ΔugtP mutants, we showed that cells producing long, abnormal LTA have cell growth and division defects and are also more susceptible to cell lysis than the wild type. Second, we showed that suppressor mutations in ltaS that reduced LTA length and abundance corrected these defects. Third, by comparing otherwise isogenic ltaS mutants that make either high or low levels of LTA, we showed that WTA levels correlated inversely with LTA levels. It is likely that the increased abundance of WTA partially compensated for low levels of LTA.
Previous work has shown that
S. aureus Δ
ugtP mutants, which make long LTA, grow larger than normal (
32). In
B. subtilis,
ugtP is also important in cell growth and division, although its absence led to decreased cell size in some studies (
37,
38).
B. subtilis UgtP is proposed to function as a nutrient sensor that, when present with sufficient UDP-glucose, slows Z-ring assembly so that cells grow to a larger size prior to division. Our results show that such a mechanism does not operate in
S. aureus. First, Δ
ugtP mutant cells spend a longer time growing before initiating septal synthesis and consequently are larger rather than smaller than the wild type. Second, Δ
ltaA mutants are also larger than the wild type even though they express
ugtP and produce intracellular Glc
2DAG. Therefore, dysregulated cell growth in LTA pathway mutants upstream of the LtaS polymerase is not due to the absence of the proteins or their products but rather is due to the production of abnormally long LTA. Consistent with this conclusion, cell growth defects were reduced in a Δ
ugtP mutant background by mutations in
ltaS that reduced LTA length.
Previous work has shown that cell division defects result when
S. aureus cells grow larger than normal (
6,
43–46). Consistent with these findings, we observed a remarkable correlation between cell size and frequency of cell division defects for the mutants studied here. For example, Δ
ltaA mutants were substantially smaller than Δ
ugtP mutants, and we found that Δ
ltaA mutants had 50% fewer cell division defects. Moreover,
ltaS mutations that reduced cell size in a Δ
ugtP background had fewer cell division defects, with the magnitudes of the reduction in size and the reduction in division defects tracking closely. We therefore concluded that the cell division defects observed in Δ
ugtP and Δ
ltaA mutants are due to dysregulated coordination between cell growth and division.
We have shown that
S. aureus WTA levels increase substantially when LTA levels are low. This finding is reminiscent of findings in
Streptococcus pneumoniae showing that levels of WTA and LTA are inversely regulated (
61).
S. pneumoniae synthesizes both forms of teichoic acid through the same pathway, with the outcome being distinguished only by a final ligation step where the polymer precursor is transferred to PG or to a glycolipid (
62). In
S. pneumoniae, LTA synthesis predominates during exponential growth, but there is a switch to predominantly WTA synthesis as cells approach stationary phase.
S. aureus WTA and LTA are synthesized by different pathways, with only the
d-alanine tailoring modification as a shared feature. Nevertheless, there appears to be a mechanism to redistribute resources between the WTA and LTA pathways when one is lacking. Although LTA and WTA share some functions and act synergistically to maintain cell envelope integrity, their spatial localization is different. LTA is located between the membrane and the inner layer of PG, and WTA extends from the inner layer of PG beyond the outer layer. Consistent with this difference in localization, we have found that producing long, abundant LTA tends to promote cell lysis, whereas producing abundant WTA tends to limit cell lysis. Given this evidence that physiological roles of LTA and WTA in
S. aureus are not identical, it would not be surprising to find that regulatory mechanisms exist to control the relative abundance of these polymers.
Expression of normal LTA is critical for
S. aureus virulence. Previous work has shown that strains lacking
ltaA or
ugtP have attenuated pathogenicity (
10,
11). While the physiological basis of this attenuation is not known, in light of our data on the susceptibility of Δ
ltaA and Δ
ugtP to PG hydrolases, it may involve an increased sensitivity to host lytic enzymes. We show here that the defects caused by expression of long, abnormal LTA also result in increased susceptibility of MRSA to beta-lactam antibiotics. We therefore anticipate that inhibitors of enzymes that act upstream of LtaS will have therapeutic potential, particularly if used in combination with an appropriate beta-lactam. Future work will focus on identifying such inhibitors and elucidating the mechanism for glycolipid-dependent control of LTA polymer length.
MATERIALS AND METHODS
General information.
Chemicals and reagents were purchased from Sigma-Aldrich unless otherwise indicated. Oligonucleotides were purchased from Integrated DNA Technologies. Detergents were purchased from Anatrace. Restriction enzymes were purchased from New England Biolabs. Staphylococcus aureus strains were grown with shaking at 30°C unless otherwise indicated in tryptic soy broth (TSB; Becton Dickinson Biosciences) or cation-adjusted Mueller-Hinton broth 2 (MHB2). Escherichia coli strains were grown at 37°C with shaking in LB-Miller broth (LB; Becton Dickinson Biosciences). Growth on solid medium used the appropriate broth plus 1.5% (wt/vol) agar (Becton Dickinson Biosciences). When required, antibiotics were used at the following concentrations: 50 μg/ml kanamycin, 50 μg/ml neomycin, 10 μg/ml erythromycin, 10 μg/ml chloramphenicol, 3 μg/ml tetracycline, and 100 μg/ml carbenicillin. Anhydrotetracycline (aTc) was used at 400 nM. S. aureus genomic DNA was isolated using a Wizard genomic DNA purification kit (Promega). Genomic DNA from S. aureus RN4220 or isolated amsacrine-resistant mutants was used as a template in PCRs to amplify S. aureus genes.
Strain construction.
To construct strains with aTc-inducible, integrated copies of genes via pTP63, plasmids were electroporated into
S. aureus RN4220 containing the pTP44 plasmid. Transformants were selected on chloramphenicol at 30°C. Marked deletions, marked transposon insertions, and aTc-inducible genes were transduced via bacteriophage ϕ85 as described previously (
63).
Analysis of LTA length by Western blotting.
Overnight cultures grown in TSB were diluted in fresh TSB and grown to an approximate optical density at 600 nm (OD
600) of 0.8. A 0.5-ml portion of each culture (normalized by OD
600) were harvested by centrifugation and suspended in 50 μl of a solution containing 50 mM Tris (pH 7.4), 150 mM NaCl, and 200 μg/ml lysostaphin (from
Staphylococcus staphylolyticus; AMBI Products). The cells were incubated at 37°C for 10 min, diluted with one volume of 4× sodium dodecyl sulfate (SDS)-PAGE loading buffer, and boiled for 30 min. Samples were centrifuged for 10 min at 16,000 ×
g to pellet any insoluble material. The supernatant was diluted with 1 vol of water and treated with 0.5 μl of 20 mg/ml proteinase K (New England Biolabs) at 50°C for 2 h. Samples were separated on 4% to 20% Mini-Protean TGX acrylamide gels (Bio-Rad) with a running buffer consisting of 5 g/liter Tris base, 15 g/liter glycine, and 1 g/liter SDS and transferred to a polyvinylidene difluoride (PVDF) membrane. Western blotting was performed as described previously (
60).
Transmission electron microscopy.
Overnight cultures grown in TSB were diluted in fresh TSB and grown to mid-log phase. Cells were treated with dimethyl sulfoxide (DMSO) or 16 μg/ml amsacrine for 4 h at 30°C, added to an equal volume of fixative solution (1.25% formaldehyde, 2.5% glutaraldehyde, and 0.03% picric acid in 100 mM sodium cacodylate [pH 7.4]), and pelleted for fixation. Samples were prepared for TEM by the Harvard Medical School Electron Microscopy Facility, and images were captured on a JEOL 1200EX instrument.
Microscopy.
Overnight cultures grown in TSB were diluted in fresh TSB and grown to an approximate OD600 of 0.5. To stain the cell membrane, FM 4-64 (Thermo Fisher Scientific) was added at a final concentration of 5 μg/ml to 1 ml of culture for 5 min at room temperature. Cells were pelleted at 4,000 × g for 1 min, washed with PBS (pH 7.4) (50 mM sodium phosphate [pH 7.4], 150 mM NaCl), and suspended in 100 μl of PBS (pH 7.4). A 1-μl aliquot of cells was spotted on top of a 2% agarose gel pad mounted on a glass slide. A 1.5-mm coverslip was placed over the cells and sealed with wax before imaging.
The cells were imaged at 30°C as described previously (
46). For each field of view, 3 images were taken: (i) phase-contrast, (ii) bright field, and (iii) fluorescence. The phase-contrast and bright-field images were acquired at a 100-ms camera exposure, while the fluorescence image was acquired at 500 ms. The bright-field images were used for cell segmentation for quantitative image analyses. Fluorescence images were used to detect division defects and sort cells, as depicted in Fig. S1C.
Image segmentation, cell volume quantification, and cellular phenotype classification were performed as described previously (
46). Cell volumes were calculated from cells lacking visible septa. For each strain, 600 to 1,000 cells were quantified. A two-tailed Mann-Whitney U nonparametric test was used to calculate the
P value for the differences in distribution of cell sizes among strains. Three hundred to 600 cells of each strain were assessed for cell division phenotypes.
Expression and purification of MBP-Sle1.
S. aureus sle1 (SAOUHSC_00427) was cloned into the NdeI and BamHI sites of pMAL-c5X (New England Biolabs) with primers oTD22 and oTD23 and transformed into E. coli NEB Express cells. An overnight culture grown at 30°C was used to inoculate fresh medium, and the culture was grown at 37°C to an approximate OD600 of 0.5. Cultures were cooled on ice and induced with 0.3 mM IPTG (isopropyl-β-d-thiogalactopyranoside) at 16°C overnight. Cells were pelleted at 3,600 × g for 15 min at 4°C, suspended in lysis buffer (20 mM Tris [pH 7.2], 200 mM NaCl, 10% glycerol) plus 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 100 μg/ml lysozyme, and 100 μg/ml DNase, and lysed with 3 passages through an EmulsiFlex-C3 cell disruptor (Avestin). All subsequent steps were performed at 4°C. Insoluble material was pelleted at 119,000 × g for 35 min, and the supernatant was bound to amylose resin (New England Biolabs). The resin was washed with lysis buffer and eluted with lysis buffer plus 10 mM maltose. The elution was concentrated with a 30-kDa-molecular-weight-cutoff spin concentrator (EMD Millipore) and further purified on a Superdex 200 Increase 10/300 GL (GE Life Sciences) equilibrated in lysis buffer. Appropriate fractions were concentrated, flash frozen with liquid nitrogen, and stored at –80°C.
Whole-cell lytic assays.
Overnight cultures grown in TSB were diluted in fresh TSB and grown to an approximate OD600 of 3. A 1.5-ml portion of culture (normalized by OD600) was harvested by centrifugation, washed with PBS (pH 7.2), and suspended in 1.6 ml of PBS (pH 7.2). A 75-μl portion of cell suspension was added to 75 μl of 25 nM lysostaphin, 250 to 500 nM purified Sle1, or PBS (pH 7.2). Samples were incubated at 25°C with shaking in a SpectraMax Plus 384 microplate reader, and OD600 was monitored over time.
MIC determination.
Overnight cultures grown in TSB were diluted in fresh MHB2 (without antibiotics) and grown to an approximate OD600 of 0.8. Cultures were diluted with medium to an OD600 of 0.001, and 146 to 147 μl was added to each well of a 96-well plate. Three to four microliters of a compound dilution series was added, and cultures were grown with shaking at 37°C for 18 h. Each condition was tested in technical triplicates, and the MIC was determined as the lowest concentration that prevented growth.
Spot dilutions.
Overnight cultures of each strain grown in TSB were diluted in fresh TSB and grown to an approximate OD600 of 0.8. Cultures were normalized to an OD600 of 0.1, a 10-fold dilution series from 10−1 to 10−6 was created, and dilutions were spotted on TSB agar containing any desired compounds. Strains grown with anhydrotetracycline were washed once with TSB before diluting. Plates were incubated at 30°C and imaged the next day with a Nikon D3400 DSLR camera fitted with an AF Micro-Nikkor 60-mm f/2.8D lens.
Growth curves.
Overnight cultures of each strain grown in TSB were diluted in fresh TSB and grown to an approximate OD600 of 0.8. Cultures were diluted to an OD600 of 0.03, and amsacrine or DBI-1 (in DMSO) was added at a final concentration of 10 μg/ml. DMSO was added to untreated control cultures at a final concentration of 2%. Cultures were grown at 30°C in a 150-μl volume with shaking in a SpectraMax Plus 384 microplate reader (Molecular Devices), and OD600 was monitored over time.
Raising amsacrine-resistant mutants.
Amsacrine-resistant mutants were raised in the following strains: SEJ1
ugtP::Tn, HG003
ugtP::Tn, RN4220 Δ
ugtP, and SEJ1 Δ
ugtP::Kan. For mutants in the SEJ1
ugtP::Tn background, 50 μl of undiluted overnight cell culture was plated on TSB agar plus 6 μg/ml amsacrine at 30°C for 2 days. For mutants in the backgrounds SEJ1 Δ
ugtP::Kan, HG003
ugtP::Tn, and RN4220 Δ
ugtP, overnight cultures were diluted in TSB and grown at 30°C to an OD
600 of 1.0. One milliliter of this culture was harvested, suspended in 100 μl fresh TSB, and plated on TSB agar plus 10 μg/ml amsacrine at 30°C for 2 days. Multiple independent cultures were used to increase the diversity of mutants. Whole-genome sequencing of select mutants was performed with an Illumina MiSeq as described previously (
46).
Construction of strains with anhydrotetracycline-inducible ltaS alleles.
Genes encoding wild-type LtaS and Ins3, F93L, and L181S LtaS variants were cloned from the genomic DNA of RN4220 or their respective suppressor mutants into pTP63 with the primers iLtaS_1 and iLtaS_2 and electroporated into RN4220 bearing pTP44 for integration (
45). Each resulting strain was transduced with ϕ85 lysate from a strain with an erythromycin-marked
ltaS deletion. These strains were then optionally transduced with ϕ85 lysate from a strain with a kanamycin-marked
ugtP or
ltaA deletion.
Phosphate quantification from purified sacculi.
Sacculi containing covalently linked WTA were isolated in a manner similar to that described previously (
64). Two milliliters of an overnight culture grown in TSB (normalized by OD
600) was harvested, washed with buffer 1 (50 mM MES [pH 6.5]), and suspended in buffer 2 (50 mM MES [pH 6.5], 4% SDS). Cells were boiled for 1 h, and pellets were harvested at 16,000 ×
g for 10 min. The supernatant was discarded, and the pellets were washed twice with buffer 2, once with buffer 3 (50 mM MES [pH 6.5], 342 mM NaCl), and twice with buffer 1. Pellets were treated with 50 μg/ml DNase and 50 μg/ml RNase in buffer 1 at 37°C for 1 h. Pellets were harvested, washed with buffer 1, and suspended in a solution containing 20 mM Tris (pH 8), 0.5% SDS. Samples were treated with 20 μg/ml proteinase K at 50°C for 2 h with light shaking. After pellets were harvested by centrifugation, pellets were washed once with buffer 3 and then three times with water.
Purified sacculi were suspended in 1 M HCl. A dilution series of K2HPO4 in 1 M HCl was also prepared. Samples were treated at 80°C for 16 h and cooled to room temperature. Any insoluble material remaining was pelleted by centrifugation, and the supernatant was retained. An ammonium molybdate reagent was prepared by mixing, in order, equal volumes of 2 M H2SO4, 2.5% (wt/vol) ammonium molybdate, and 10% (wt/vol) ascorbic acid. One volume of ammonium molybdate reagent was added to each sample, and samples were incubated at 37°C for 1 h. Orthophosphate was quantified by absorbance at 820 nm with the K2HPO4 standard curve.
Polyacrylamide gel electrophoresis of WTA polymers.
Sacculi containing covalently linked WTA were isolated as described above but treated with 100 mM NaOH at room temperature for 16 h. Three volumes of loading buffer (50% glycerol, 100 mM Tris-Tricine, 0.02% bromophenol blue) were added to each sample.
High-resolution 20- by 20-cm polyacrylamide gels were prepared as described previously (
64), but with a stacking gel consisting of 3% acrylamide (3% T–3.3% C, where T is total acrylamide and C is the percentage of T consisting of bisacrylamide), 900 mM Tris (pH 8.5), 0.1% ammonium persulfate, and 0.01% tetramethylethylenediamine. Gels were run at 4°C in a Protean II xi Cell electrophoresis system (Bio-Rad) at 40 mA/gel with a running buffer consisting of 100 mM Tris-Tricine (pH 8.2) until the bromophenol blue loading dye was near the bottom of the gel. Gels were washed with water, stained with 1 mg/ml aqueous alcian blue for 30 min, destained with water and 40% ethanol–5% acetic acid, and rehydrated with water. Silver staining was performed with a Silver Stain Plus kit (Bio-Rad) without the fixation step. Images were taken with a Nikon D3400 DSLR camera fitted with an AF Micro-Nikkor 60-mm f/2.8D lens and converted to an 8-bit image using ImageJ.
Purification of LtaS mutants and proteoliposome analysis of DAG production.
Mutant LtaS constructs were cloned from genomic DNA isolated from the original suppressor mutants and assembled into pET28b with the primers LtaS_F and LtaS_R as previously described (
60). LtaS constructs were expressed, purified, and reconstituted into proteoliposomes as previously described (
60). Proteoliposomes were added to 9 volumes of a solution containing 20 mM succinate (pH 6.0), 50 mM NaCl, and 5% DMSO. Reaction mixtures were incubated in the presence or absence of 1 mM MnCl
2. LTA was detected by Western blotting as previously described (
60). To measure DAG production, reactions proceeded for 4 h at 30°C, and then DAG was extracted and assayed according to the instructions provided by the Cell BioLabs DAG assay kit. Reactions were performed in duplicate and plotted using GraphPad Prism. Absolute activity was calculated by subtracting activity values calculated from reactions that did not use MnCl
2 from the values from reactions that did use MnCl
2. Activity was compared between mutants by setting the activity in reactions with proteoliposomes containing wild-type LtaS to 100%.
Data availability.
Whole-genome sequencing data (accession number
PRJNA612838) can be found in the NCBI BioProject database.