Bacterial strains and cell culture conditions
Bacterial cells were grown in Luria-Bertani (LB) broth or on LB agar at 37°C unless specified otherwise. Bacterial strains, most of which are derivatives of E. coli
MC4100 (Silhavy et al., 1984
), are listed in Table S1
Whole cell surface labeling
Cells were subcultured 1:1000 from overnight-grown cultures into 5 mL cultures with 0.02% (w/v) arabinose, when necessary to induce expression from plasmid constructs. Cultures were grown with aeration for 3 hours, after which time the cells were pelleted, washed 3 times in PBS, and normalized to O.D.600 = 10.0 in PBS. NHS-LC-LC-biotin (Pierce, cat. # 21343) was added to a final concentration of 2% (v/v) from a 25 mg/mL stock in DMSO and the cells were held for 20 minutes at room temperature. PEG-NHS-biotin was added to a final approximate concentration of 10% (v/v) from a 5% (w/v) stock in 50% DMSO and reacted for 20 minutes at room temperature. The reactions were quenched by the addition of Tris, pH=7.5 to a final concentration of 250 mM. Finally, the labeled cells were washed in PBS and resuspended directly in the original reaction volume of SDS/PAGE loading buffer.
To separate the soluble and insoluble subcellular fractions, a 100 mL mid-exponential culture was washed, normalized to O.D.600 = 10.0 in PBS, and labeled as above. The cells were then lysed by passage through a French pressure cell at 20,000 PSI. The lysates were spun in a microcentrifuge for 1 minute to remove intact cells and 1 mL of the supernatant was spun for 30 minutes at 100,000 rpm in a Beckman TLA 100.2 ultracentrifuge rotor. The supernatant was saved as the soluble fraction and the pellet was washed with water and saved as the insoluble fraction by solubilizing the pellet in 1 mL of 1% SDS. In order to examine whether NHS-LC-LC-biotin partitions into membranes, the same process was followed, except that cells were first washed, normalized, and mechanically lysed, and subsequently labeled and fractionated by ultracentrifugation.
Tests of the effects of NHS-LC-LC-biotin labeling on OM integrity
In the following experiments, a single mid-exponential culture of cells was washed in PBS, normalized to O.D.600 = 10.0, and split into two identical aliquots. One aliquot was labeled with NHS-LC-LC-biotin as described above, whereas the other aliquot was left untreated.
To examine the effects on viability, cells were diluted through 6, 10-fold serial dilutions in LB and approximately 1 μL of each dilution was spotted onto triplicate LB agar plates or LB agar plates containing 0.5% SDS. The highest dilution at which single colonies appeared was recorded and used to calculate the efficiency of plating. No difference was observed between the two treatments.
To examine the effects of NHS-LC-LC-biotin labeling on OM permeability, cells were plated on triplicate LB agar plates containing 0.5% SDS, and a filter disc containing 2, 5, or 10 μL of 0.5M EDTA was placed in the center of the plate. The plates were incubated overnight at 37°C and the zones of clearing were recorded. No difference in the size of the zone of clearing was observed between the labeled and unlabeled trials at any EDTA concentration.
To examine the effects of NHS-LC-LC-biotin labeling on OM permeability, the proportion of cells that were permeable was determined using Sytox Green at a final concentration of 5 mM according to the manufacturer’s instructions (Invitrogen). Over two thousand cells for each condition were examined using light and fluorescence microscopy, and the proportion of fluorescent, permeable cells was calculated.
To examine the effects of NHS-LC-LC-biotin labeling on growth rate, aliquots of labeled and unlabeled cells were diluted 100-fold into triplicate broth cultures and the optical density was monitored over several hours. The averaged treated and untreated cells grew with indistinguishable kinetics.
Fractionation of the free and bound forms of Lpp
To separate the free- and bound-form Lpp fractions, 1mL of cells were labeled, washed, resuspended in PBS + 1% SDS, and boiled for 20 minutes. The intact peptidoglycan sacculi were then pelleted by spinning for 30 minutes at 100,000 rpm in a Beckman TLA 100.2 ultracentrifuge rotor. Following this, the supernatant fraction was saved as the free-form fraction. The peptidoglycan pellets were gently washed 4 times with 10 mM Tris, pH=7.5. Next, the pellets were resuspended in 1mL of 10 mM Tris, pH=7.5 and dispersed using a brief pulse from a probe tip sonicator. Following this, the sacculi were incubated overnight at 37°C in the absence or presence of 0.2 mg/mL lysozyme to yield bound-form fraction samples. No proteins were ever observed in bound-form samples that were not treated with lysozyme, indicating that the peptidoglycan sacculi were intact and were not contaminated with free-form fraction proteins.
To examine the whether bound-form Lpp could be labeled by NHS-LC-LC-biotin in vivo, mid-exponential cultures of CC01 and CC14 were washed and normalized as above and NHS-LC-LC-biotin was added to a final concentration of 5% (v/v). Cells were reacted, quenched, and fractionated into the free- and bound-form fractions, as above. Following fractionation, blots were detected with streptavidin-HRP at 1:10,000 and exposed to film for an extended length of time.
α-Lpp and α-biotin immunoblot analyses were carried out with standard protocols with nitrocellulose membranes in TBS-T. Prior to detection, blots were blocked for 1 hour with 5% milk in TBS-T. Lpp was detected with polyclonal rabbit primary α-Lpp antiserum (kindly provided by M. Inouye) at 1:400,000 and a donkey, α-rabbit, HRP-conjugated secondary antibody at 1:8,000 (GE Healthcare, cat. # NA934V). Biotin and LC-LC-biotin were detected with a streptavidin-HRP conjugate (Rockland Immunochemicals, cat. # S000-03) in TBS-T + 1% milk used at 1:20,000 from a 1 mg/mL stock. OmpA was detected with an α-LamB polyclonal rabbit primary antiserum that cross-reacts with OmpA at 1:20,000 in TBS-T. NlpB was detected with α-NlpB polyclonal rabbit primary antiserum at 1:20,000 in TBS-T (Wu et al., 2005
). TraT was detected with α-TraT polyclonal rabbit primary antiserum at 1:20,000 in TBS-T (see below).
Synthesis of PEG-NHS-biotin
PEG 3500-NHS-biotin was synthesized using tricarballylic acid, O
-benzylhydroxylamine hydrochloride, PEG 3500, and biotin (Acros) using both previously published (Schlemminger et al., 2003
) and standard chemical procedures. Briefly, tricarballylic acid was condensed onto O
-benzylhydroxylamine to yield a benzyl-protected NHS ester derivative. The free carboxylic acid of this product was esterified with PEG 3500. The product was then deprotected, activated, and biotin was linked to the activated NHS ester. A more detailed description of this synthesis is shown in Figure S2
The susceptibility of the LC-LC-biotin label attached to Lpp and OmpA was examined by treating labeled and washed whole cells with 0.5 mg/mL trypsin or proteinase K at 37°C for 1 hour in 100 mM NaCl, 20 mM Tris, pH=7.5. The proteases were inactivated by adding PMSF to 5 mM and washing the cells 4 times in 100 mM NaCl, 20 mM Tris, pH=7.5 + 1 mM PMSF. The cells were then normalized to an equivalent O.D.600 = 5.0 by resuspending the cell pellet in SDS/PAGE loading buffer + 1 mM PMSF.
To examine the protease susceptibility of Lpp-FLAG, lpp null cells (strain CC03) harboring pBAD/lpp or pBAD/lpp-FLAG plasmids were grown to mid-log phase in LB broth supplemented with 0.02% arabinose to induce expression of the Lpp constructs. Cells were washed in 100mM NaCl, 20mM Tris, pH=7.5, normalized to O.D.600 = 10.0, and incubated in the presence of 0.5 mg/mL trypsin + 2% glycerol at 37°C for 1 hour. The cells were then washed three times in PBS, normalized to O.D.600 = 10.0 in PBS, and labeled with NHS-LC-LC-biotin, as described above.
To examine the susceptibility of free- and bound-form Lpp to periplasmic proteolysis, cells were washed in 100mM NaCl, 20mM Tris, pH=7.5 and normalized to O.D.600 = 10.0. EDTA was added to a final concentration of 5 mM and the cells were split into two identical aliquots. Proteinase K was added to one aliquot to a final concentration of 0.5 mg/mL and the cells were incubated at 37°C for 1 hour. Following this, PMSF was added to a final concentration of 5 mM and the cells were separated into free- and bound-form fractions, as above, except that 1mM PMSF was added to the overnight lysozyme digestion reactions.
Purification of TraT and production of α-TraT antibodies
To purify TraT, traT was amplified using oligonucleotides TraTNsolFor and TraTSalSTOPRev, digested with NdeI and SalI, and cloned into similarly-digested pET28a. The resulting plasmid, pET/TraTNsol, was inserted into BL21(λDE3) cells. An overnight culture of the expression strain was subcultured 1:150 into 6L of LB + 20 μM IPTG and the cells were grown for 3 hours with shaking. Cells were pelleted, washed, and mechanically lysed using a pressure lysis device (Microfluidics). His-TraT was batch-purified from the cells using 12 mL Ni-NTA resin, according to the manufacturer (Qiagen). The 100 and 250 mM imidzaole eluate fractions were pooled and dialyzed against 2 large volumes of 100mM NaCl, 50mM Tris, 0.5mM EDTA, 5% glycerol, pH=7.75. Finally, the dialyzed protein was concentrated by passage through a 50 kDa MWCO Centricon filter (Millipore) and retention on a 30 kDa MWCO Centricon filter.
1 mg purified His-TraT was mixed with Freund’s complete adjuvant and injected into a New Zealand white rabbit (Covance). Serum was collected at 28 days and reacted with a 25 kDa protein in F+ cells, but showed no cross-reactivity with other E. coli proteins in F- cells.
Molecular biological methods
The oligonucleotides used in this study are listed in Table S1
. Chromosomal mutant lpp
alleles were constructed in CC05 using single oligo-mediated mutagenesis (Costatino & Court, 2003
) with oligos “delta17–37ultramer” and “lppdeltaK58.” CC06 (an lppΔ17–37
strain) was identified by screening candidate colonies with PCR reactions using “lppF2” and “lppseqrev” primers to identify strains with PCR amplicons that were shorter than the wild-type lpp
product. CC07 (an lppΔK58
strain) was identified by screening candidate colonies for sensitivity to 0.5% SDS + 1 mM EDTA. The sequence of both alleles was determined by PCR amplifying the lpp
gene using primers “lppF2” and “lppseqrev” and then sequencing the product with primer lpp “lppF3” at GENEWIZ, Inc. (South Plainfield, NJ) to verify the expected sequence from the gene’s promoter to the stop codon. Following this, the alleles were mobilized from CC06 and CC07 into CC01 by P1 transduction to create strains CC08 and CC09 (lppΔ17–37
Standard molecular biological methods were used for plasmid construction. Plasmids pBAD/lpp, pBAD/lppΔK58, and pBAD/lppΔ55–58 were constructed by amplifying the lpp open reading frame and ribosome binding site using primers pBADlppFor and either pBADlppRev, pBADlpp-CKRev, or pBAD/lpp-C2K rev, respectively. The correct sequence of all plasmids was determined by directly sequencing with both pBAD-Forward and pBAD-Reverse primers at GENEWIZ, Inc.
Examination of NHS-LC-LC-biotin surface-specificity using NlpB and TraT control lipoproteins
To examine the surface-specificity of NHS-LC-LC-biotin using lipoproteins with known orientations, CC11 (ompA-null, nlpB-null) cells were transformed with pBAD/nlpB or pBAD/traT. An ompA-null strain was used, as NlpB co-migrates with OmpA. NlpB expression was induced with 0.02% arabinose and TraT expression was induced with 0.001% or 0.002% arabinose, and the cells were surface-labeled with NHS-LC-LC-biotin, as described above. Known quantities of purified NlpB-His (kindly provided by J. Malinverni) or His-TraT were used as references for immunoblot quantification of each protein’s expression level in the labeled samples. Reference protein standards were varied through several, twofold dilutions, and were used in ranges that flanked the concentration of the experimental proteins. Duplicate quantifications were performed for each sample.
Mass spectrometric analyses
For mass spectrometric analyses, whole cells were surface-labeled with NHS-LC-LC-biotin and insoluble-fraction SDS/PAGE samples were prepared as described above. The samples were separated using 10–20% SDS/PAGE gels and the 10–12 kDa region of the gel was excised and diced into 1-mm3
pieces. The sample was digested with 12.5 ng/mL sequencing grade modified trypsin (Promega), and the resulting peptides were extracted on reverse-phase resin (Poros 20 R2, PerSeptive Biosystems), as previously described (Cristea et al., 2005
). The peptide mixtures were eluted directly onto a MALDI target in 50% methanol, 20% acetonitrile, 0.1% trifluoroacetic acid + 2 mg/mL α-Cyano-4-hydroxycinnamic acid matrix. MALDI MS and MS/MS analyses were performed using a MALDI LTQ Orbitrap XL, as previously described (Luo et al., 2010
). Lists of the expected m
for tryptic peptides and ion fragments were generated using Prowl (http://prowl.rockefeller.edu/
) with K +452.24571 for modification by LC-LC-biotin.
For MALDI LTQ MS2
analysis, the ion of interest (LDNMATK*YRK*, m
2144.143) was isolated in the ion trap with a 2 Da mass range window, and subjected to collision induced dissociation (CID), as described (Luo et al., 2010
), using a normalized collision energy of 35%. As expected from the primary sequence of this ion, the predominant ion was the y8
ion, resulting from the preferential cleavage at the carboxyl-terminus of the aspartic acid (Qin & Chait, 1995
). Thus, this ion (NMATK*YRK*, m
1916.11) was further subjected to MALDI LTQ CID MS3
analysis with the same settings as above (Fig. S1
in vivo crosslinking
To crosslink Lpp in vivo, cells were subcultured 1:1000 and grown for 3 hours, washed three times in PBS, and normalized to O.D.600 = 10.0. Whole cells were crosslinked with a range of concentrations of disuccinimidyl suberate (DSS) concentrations for 20 minutes at room temperature. Following this, the reactions were quenched by the addition of Tris, pH=7.5 to a final concentration of 250 mM. Finally, the crosslinked cells were washed in PBS and resuspended in SDS/PAGE loading buffer at their original concentration.