Skip to main content
NCBI home page
ACS AuthorChoice logoLink to ACS AuthorChoice
. 2026 Mar 25;65(7):1013–1024. doi: 10.1021/acs.biochem.6c00056

Stepwise Assembly of the Bacteroides fragilis Capsular Polysaccharide A Repeating Unit in Escherichia coli

Beth A Scarbrough †, Claire E Moneghan †, Sara Salamat †, Manoj K Dooda ‡, Alexis H Murray †, Jenna S Costelloe †, Matthew A Jorgenson §, Jerry M Troutman †,*
PMCID: PMC13063421  NIHMSID: NIHMS2160685  PMID: 41880207

Abstract

Bacterial surface polysaccharides are versatile structures that provide specificity to the behavior and interactions of a given bacterial strain. One surface polysaccharide displayed on the organism Bacteroides fragilis, Capsular Polysaccharide A, has been implicated as a potential therapeutic for autoimmune disorders. This polymer is composed of repeating units of the tetrasaccharide 2-acetamido-4-amino-2,4,6-trideoxygalactopyranose (AATGal), 4,6-O-pyruvate-galactopyranose (PyrGal), N-acetylgalactosamine (GalNAc), and galactofuranose (Galf). While this and other bacterial surface polysaccharides are attractive to study and apply to biomedicine, it can be difficult to acquire quick, inexpensive access to these pure materials. In this work, we developed a recombinant expression system in Escherichia coli for the stepwise production of the CPSA polymer. A series of sequential plasmids were prepared, each incorporating successive genes required for CPSA biosynthesis. Using these iterative plasmids, we were able to observe production of the CPSA repeating unit and precursors by liquid chromatography mass spectrometry (LC-MS) analysis of cell lysates. We found that it was critical to include the CPSA polymerase but not the flippase, indicating that a native E. coli flippase could support polymer production. We also provide evidence that the CPSA polymer produced by E. coli can be ligated to LPS by the E. coli WaaL ligase, and deletion of this gene led to the formation of a water-soluble polymer. Overall, this work describes the first recombinant system for CPSA production and outlines a key strategy for the production of complex glycopolymers.

graphic file with name bi6c00056_0011.jpg

graphic file with name bi6c00056_0009.jpg

Introduction

Bacteroides fragilis is one of a broad range of microbes associated with the mammalian gut microbiome. , It is a Gram-negative obligate anaerobe that is thought to play a central role in host immune system development. The role of B. fragilis in immune system modulation is dependent on its ability to produce the surface polymer capsular polysaccharide A (CPSA) (Figure ). CPSA belongs to a unique class of zwitterionic polysaccharides that, unlike the more common neutral and negatively charged glycans, can elicit a potent, T-cell dependent immune response. , CPSA also activates anti-inflammatory innate immune responses by stimulating the production of anti-inflammatory cytokines, such as IL-10. , This has led to CPSA being considered as a potential therapeutic for inflammation-associated diseases such as multiple sclerosis, ulcerative colitis, viral encephalitis, and irritable bowel syndrome. −

1.

1

Open in a new tab

Capsular polysaccharide A. (A) Repeating unit of B. fragilis Capsular Polysaccharide A. Wavy lines represent linkage points between repeat units. (B) Biosynthesis pathway for CPSA including sugar modifying enzymes UngD2 from B. fragilis and the alternative enzyme PglF from Campylobacter jejuni used in this work. Briefly: UDP-GlcNAc is converted to UDP-AATGal through the action of UngD2/PglF and WcfR. The AATGal-P is then transferred to BP by the initiating phosphoglycosyltransferase WcfS. The glycosyltransferase WcfQ then transfers galactose to AATGal and the disaccharide is pyruvylated by WcfO. The pyruvate group is donated from phosphoenolpyruvate (PEP). After pyruvylation WcfP transfers GalNAc and WcfN transfers a galactofuranose formed by WcfM which isomerizes UDP-galactopyranose. The completed repeat unit is then flipped to the periplasmic face of the inner membrane by the flippase Wzx and is polymerized by Wzy prior to transport to the cell surface.

Access to the CPSA polymer is critical for further development as a potential therapeutic. However, isolation of CPSA from B. fragilis can be difficult, due to the presence of numerous other capsular sugars on the B. fragilis surface, complex growth requirements, and laborious isolation procedures. In addition, synthetic schemes to prepare CPSA require as many as 20 chemical manipulations to build the repeating unit of the polymer, and several of these repeat units must be linked to provide a biologically active molecule. − An alternative to chemical synthesis and isolation from native organisms is the development of methods for the recombinant production of the polymer. The advantage of such systems is the ability to use easily manipulated hosts to prepare the capsule and control the expression of key genes involved in its biosynthesis. −

To design a recombinant system for polysaccharide production, it is important to understand the biosynthetic pathway that is responsible for its production in the native organism. Previous work has identified the specific roles of genes involved in CPSA production. , The CPSA repeating tetrasaccharide consists of 2-acetamido-4-amino-2,4,6-trideoxygalactopyranose (AATGal), 4,6-O-pyruvate-galactopyranose (PyrGal), N-acetylgalactosamine (GalNAc), and galactofuranose (Galf) (Figure ). The polymer is formed in a Wzx/Wzy pathway where it is assembled one sugar at a time on the lipid anchor, bactoprenyl phosphate (BP; also known as undecaprenyl phosphate or Und-P), on the cytoplasmic face of the inner membrane. CPSA biosynthesis begins with the addition of phospho-AATGal to BP by the initiating phosphoglycosyltransferase WcfS to produce BPP-AATGal (Figure ). , The remaining sugars, galactose (Gal), N-acetylgalactosamine (GalNAc), and galactofuranose (Galf) are sequentially appended to BPP-AATGal by the glycosyltransferases WcfQ, WcfP, and WcfN, respectively. In this work, we devised a recombinant system for CPSA production in E. coli by the stepwise construction of plasmids that encode the key genes required for the biosynthesis of each intermediate isoprenoid-linked glycan. Incorporation of B. fragilis wzx/wzy genes resulted in the production of a CPSA polymer. We also found that the polymer was formed as either soluble or lipid-A-linked material, and we could control the destination of the glycan through deletion of a ligase involved in conjugating glycans to lipid A. − Overall, this work represents the first recombinant production system for potentially therapeutic CPSA.

Materials and Methods

General

All bacterial cultures were grown in Miller Lysogeny Broth (Fisher) unless otherwise specified. Liquid chromatography mass spectrometry (LC-MS) analysis was performed on an Agilent 1260 Infinity II instrument equipped with a G6125B single quadrupole mass selective detector. All mobile phases were prepared from LC-MS grade reagents. Immunological detection of CPSA polymer utilized a polyclonal rabbit antibody serum against CPSA that is precleared by extracting nonspecific antibodies with B. fragilis that does not produce CPSA. ,

Construction of CPSA Plasmids

CPSA plasmids were constructed using the primers listed in Supporting Table S1. Briefly, the pQE-80L vector was isolated from E. coli DH5α. The vector was digested with restriction enzymes BamHI and HindIII and purified by gel electrophoresis. Gene inserts were amplified with Phusion DNA polymerase (New England Biolabs). Amplicons were purified using the Wizard SV Gel and PCR Clean-Up System (Promega). Purified, digested vector and inserts were incubated with NEBuilder HiFi DNA Assembly Mix (New England Biolabs) according to the manufacturer’s instructions and incubated at 50 °C, or overnight for constructs with more than four fragments. E. coli DH5α cells were then chemically transformed with 5 μL of the assembly reaction mixture and plated on selective media (LB/Carb100). Successful transformants were confirmed by colony PCR.

An artificial SacI site was incorporated into pBAS8 immediately following the wcfS. For construction of pBAS9–19, SacI digested pBAS8 was used as the vector backbone. For the construction of pBAS16, wcfO and wcfQ were amplified as a single insert using pBAS10 as a template. For the construction of pBAS 17–19, six genes (wcfOQP Bf, wbpP Vv, wcfMN) were amplified as a single fragment using pBAS16 as a template. The pBAS17 plasmid full sequence is provided in the Supporting Information (Supporting Figure S1).

Extraction of BPP-Linked CPSA Intermediates

Cultures for glycolipid extraction were prepared by inoculating a single colony into 5 mL of LB/Carb100 and 2% glucose. Cultures were grown overnight for 16 h at 37 °C and 220 rpm. Cell cultures were then diluted 1:1000 into 5 mL of LB/Carb100 and grown at 37 °C, 220 rpm until reaching an OD600 of approximately 0.6 before induction with 0.1 mM IPTG. After overnight induction, liquid cultures were transferred to falcon tubes and centrifugated at 5000g for 15 min at 4 °C. The supernatant was discarded, and the cell pellet was resuspended in 10 mM phosphate-buffered saline (PBS). The cell suspension was then transferred to a 15 mL glass centrifuge tube. To the cell suspension were added 1 mL of chloroform and 2 mL of methanol to create a single-phase solution of water:chloroform:methanol (0.8:1:2). Each sample was vortexed and incubated at room temperature for 20 min to ensure cell lysis. Resulting insoluble materials were clarified from the cell lysate solution by centrifugation at 2500g for 20 min at room temperature. The supernatant was then transferred to a clean glass culture tube, frozen at −80 °C, and then dried under vacuum. Dried samples were resuspended in a solution of 1 mM ammonium hydroxide:n-propanol (1:1).

LC-MS Analysis of BPP-Linked CPSA Intermediates

To evaluate the presence of CPSA intermediates in E. coli, 10 μL of cell extract was injected and separated using a Waters XBridge BEH C18 column (5 μm, 4.6 mm × 100 mm, 300 Å). For LC-MS analysis, mobile phase A consisted of 0.1% ammonium hydroxide, and mobile phase B was n-propanol. BPP-linked intermediates were separated using a 5 – 75% gradient of mobile phase B over 15 min, then 75 – 95% B for 1 min, and an isocratic hold at 95% B for 5 min at 1 mL/min for a total run time of 21 min. A 3 s needle wash was performed between injections. The column was equilibrated for 4 min at 5% B prior to the next injection. Intermediates were detected using SIM of the predicted [M-H]−1 and/or [M-H]−2 ions of each intermediate. Total ion chromatograms were collected for each injection. Controls were prepared from parent strains expressing empty plasmids and evaluated for CPSA intermediates or isobaric compounds (Supporting Figure S2).

Cell Fractionation and SDS PAGE

To evaluate CPSA glycoforms in E. coli, overnight cultures (5 mL) were prepared from single colonies, induced with 0.1 mM IPTG after reaching an OD600 of 0.8, and incubated overnight at 37 °C with shaking at 220 rpm. Cell were pelleted at 5000g, resuspended in 0.8 mL of PBS, and transferred to a glass culture tube. To the cell suspensions, 3 mL of a 1:2 solution of chloroform:methanol was added, and the mixture was incubated at room temperature for 20 min. Insoluble material containing LPS was removed from cell suspensions by centrifugation at 2500g for 20 min. The insoluble fraction was washed with a second, single-phase solution of water:chloroform:methanol (0.8:1:2) and dried under vacuum to remove any remaining solvent. The soluble fraction was converted to a two-phase Bligh and Dyer solution by the addition of water and chloroform. The aqueous and organic phases were separated into two glass tubes and then dried under a vacuum. Dried samples were resuspended in PBS and evaluated by using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and anti-CPSA blotting. Blots were prepared by transfer of SDS-PAGE separated material to nitrocellulose. The nitrocellulose was blocked with a 5% milk solution for 30 min. The nitrocellulose was then rinsed gently with deionized water (x3) and incubated at room temperature for 1 h with 1 mL of an adsorbed CPSA antiserum diluted 1:30 in sterile 10 mM PBS. After incubation with anti-CPSA serum, the nitrocellulose was washed twice for 5 min in 0.3% PBST, then placed in alkaline phosphatase conjugated antirabbit goat secondary antibody (1:10,000) for 1 h at room temperature. The nitrocellulose was then washed three times for 1 min with 0.3% PBST and developed with NBT-BCIP.

SDS-PAGE and LPS Staining

To evaluate LPS and CPS profiles of E. coli harboring pBAS16–19, overnight cultures were diluted 1:100 in LB and incubated at 37 °C with shaking until reaching an OD of ∼0.6. Cell cultures were then induced with 0.01 mM IPTG and incubated overnight at 37 °C. Cell cultures were normalized to an OD600 of 1.0, and a 0.5 mL volume of each sample was pelleted and resuspended in 100 μL of 1× SDS dye. Cell samples were then lysed at 95 °C for 10 min and cooled to room temperature before 20 μg of Pronase (Sigma) was added. The samples were incubated at room temperature for 20 min prior to 14% SDS-PAGE separation. A 5 μL sample of each lysate was separated at 40 mA for 60 min. Gels were then stained with the Pro-Q Emerald 300 kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The protein standard was CandyCane Glycoprotein Molecular Weight Standard (Thermo Fisher Scientific).

Generation of E. coli Mutants

Escherichia coli mutants MG1655 ΔwzxB:frt (BAS5), MG1655 ΔwaaL:frt (MAJ975), and MG1655 Δwza:frtΔwaaL:cam (BAS24) (Supporting Table S2) were prepared using the lambda red recombinase method of Datsenko and Wanner. Briefly, parent strains of E. coli were transformed with pKD46. Single colonies of transformants were used to inoculate 5 mL cultures containing LB/Carb100 and incubated at 30 °C. Cultures were induced with 100 mM l-arabinose and returned to the incubator for 1 h at 30 °C. Cells were then pelleted at 5000g at 4 °C and washed six times with ice-cold 10% glycerol. These cells were used immediately for transformation or stored as aliquots at −80 °C. For transformation, approximately 1–2 μg of purified amplicon was added to the cell suspensions. Cells were then electroporated at 2500 V and immediately recovered in LB at 30 °C overnight. Recovered cells were plated on LB/Cam or LB/Kan50 to select for mutants. Primers used to generate mutants are listed in Supporting Table S3. Kan resistance cassettes were removed by transforming with pCP20. To ensure that the Kan resistance cassette was successfully removed, cells were tested for sensitivity against Kan50. To cure the cells of the pCP20 plasmid, cells were cultured on LB agar and incubated at 37 °C and the colony purified twice more at 37 °C. Plasmid curing was confirmed by testing cells for sensitivity to Carb100.

Results

Plasmid Assembly for the Formation of a BPP-Linked AATGal

The first step in CPSA biosynthesis is the addition of AATGal-P to BP to form BPP-AATGal (Figure ). The AATGal sugar is formed through the oxidation, dehydration, and reduction of UDP-GlcNAc to form UDP-2-acetamido-2,6-dideoxy-α-d-xylo-hexos-4-ulose followed by an amino transfer reaction catalyzed by WcfR to form UDP-AATGal (Figure a). Previous work had identified the gene ungD2 to encode the likely dehydrogenase that forms the 4-keto sugar substrate for WcfR. However, this gene was not encoded in the CPSA biosynthesis operon, and previous attempts to overexpress it in E. coli were unsuccessful. The C. jejuni gene pglF has been readily overexpressed in E. coli and serves the same function as the expected product of the ungD2 gene. Using the Gibson assembly, we combined pglF, wcfR, and wcfS into plasmid pQE-80L (Table , pBAS8). We chose this plasmid due to the presence of a T5 RNA polymerase promoter that could be used with a wide range of E. coli strains. We also incorporated a strong E. coli ribosome binding site (AGGAGA) before each gene. The plasmid was transformed and expressed in E. coli DH5α, and cell lysate extracts were analyzed by LC-MS in selected ion monitoring (SIM) mode for the BPP-linked monosaccharide product of WcfS (Figure b). We found that BPP-AATGal was not detected with the empty plasmid (Supporting Figure S2). With the pglF, wcfR, and wcfS genes inserted, a clear BPP-AATGal peak was observed with an m/z of 1111.5 consistent with the [M-H]−1 species and the mass spectrum corresponded to the isoprenoid-linked monosaccharide mass as the major component (Figure c).

2.

2

Open in a new tab

Formation of BPP-AATGal with pBAS8. (A) Reaction pathway for the generation of UDP-AATGal and transfer to BP. UDP-N-acetyl-glucosamine is converted to UDP-4-keto-6-deoxy-N-acetyl-glucosamine via the C. jejuni dehydratase, PglF. Then, UDP-4-keto-6-deoxy-N-acetyl-glucosamine is aminated via the B. fragilis enzyme, WcfR. Finally, AATGal-P is transferred to the BP lipid anchor by WcfS. (B) LC-MS SIM chromatogram scanning for BP (black), BPP-AATGal (blue), and the next potential product, BPP-AATGal-Gal (green) of pBAS8 expressing cells. The inset shows resolution of the two peaks with the shift in retention corresponding to the addition of AATGal-P to BP. (C) Mass spectrum of the peak corresponding to BPP-AATGal which shows a signal for the [M-H]−1 ion 1111.5.

1. Plasmids Used to Construct CPSA Repeating Unit .

      expected
 observed
plasmid geneslncorporated CPSA intermediate [M-H]−1 [M-H]−1 [M-2H]−2
pQE80L BP 845.67      
pBAS8 pglF Cj , wcfRS BPP-AATGal 1111.73 1111.5  
pBAS9 pglF Cj , wcfRSQ BPP-AATGal-Gal 1273.76 1273.6  
pBAS10 pglF Cj , wcfRSQO BPP-AATGal-PyrGal 1343.79 1343.6 671.4
pBAS11 pglF Cj , wcfRSQOP BPP-AATGal-PyrGal 1343.79 1343.6 671.4
pBAS12 pglF Cj , wcfRSQOPMN BPP-AATGal-PyrGal 1343.79 1343.6 671.4
pBAS15 pglF Cj , wbpP Vv , wcfRSQOP BPP-AATGal-PyrGal-GalNAc 1546.87 1546.7 773.3
pBAS16 pglF Cj , wbpP Vv , wcfRSQOPMN BPP-AATGal-PyrGal-GalNAc-Galf 1707.92   854.3
Open in a new tab
a

Each plasmid incorporates genes from the organisms C. jejuni (Cj), B. fragilis, and V. vulnificus (Vv) to produce each BPP-linked CPSA intermediate, including the complete BPP-linked repeating unit (pBAS16).

Formation of a Pyruvylated BPP-Linked Disaccharide

After the addition of AATGal-P to BP, the next sugar to be incorporated is pyruvylated galactose, which provides a negative charge to the zwitterionic polysaccharide. The genes wcfQ and wcfO are known to encode the galactosyltransferase and pyruvylating enzymes, respectively (Figure ). We next incorporated each of these genes into the pBAS8 plasmid (Table , pBAS9/10), then analyzed transformed DH5α for the formation of the BPP-linked disaccharide and pyruvylated disaccharide (Figure b–e). We again found that the isoprenoid-linked disaccharide and pyruvylated disaccharide were formed based on the retention time of the new products and detection of the expected ions for BPP-AATGal-Gal, [M-H]−1 1273.6 (Table , Figure b,c) and BPP-AATGal-PyrGal [M-H]−1 1343.6 and [M-2H]−2 671.4 representing the −1 and −2 ions for the pyruvylated species (Figure d,e). These products were not observed with an empty vector (Supporting Figure S2), nor were they observed without the appropriate genes incorporated into the vector.

3.

3

Open in a new tab

Formation of a BPP-linked disaccharide with pBAS9/10. (A) Reaction pathway with WcfQ transferring galactose from UDP and pyruvylation of galactose by WcfO using phosphoenolpyruvate (PEP). (B) LC-MS SIM chromatogram for BP (black), BPP-AATGal-Gal (green), and BPP-AATGal-PyrGal (red) of pBAS9 expressing cells. (C) Mass spectrum for the peak associated with BPP-AATGal-Gal (m/z 1273.6). (D) LC-MS SIM chromatogram for BP (black), BPP-AATGal-PyrGal (red), and BPP-AATGal-PyrGal-GalNAc (purple) displaying a new retention time with addition of pyruvate to galactose of pBAS10 expressing cells. (E) Mass spectrum of the BPP-AATGal-PyrGal peak showing signals for the −1 and −2 ions (1343.6 and 671.4, respectively).

Addition of a 4-Epimerase is Critical for CPSA Trisaccharide Formation

Trisaccharide formation requires the addition of GalNAc to BPP-AATGal-PyrGal (Figure ). Therefore, we next constructed a plasmid that added the GalNAc transferase encoding gene wcfP (Table , pBAS11). We were surprised to find that no isoprenoid-linked trisaccharide formed, and we observed only the disaccharide product of WcfO (Figure b). Closer inspection of the DH5α genome indicated that this strain does not encode a clear GlcNAc-4-epimerase that could convert UDP-GlcNAc to UDP-GalNAc. Our previous work demonstrated that WcfP could utilize UDP-GlcNAc to a limited extent as a substrate; however, since no product containing GlcNAc was observed, it was clear that this limited activity was not relevant E. coli. We have recently shown that the gene wbpP from Vibrio vulnificus encodes a UDP-GlcNAc 4-epimerase and is robustly expressed in E. coli. We incorporated wbpP into our plasmid (pBAS15) and found that indeed the lack of UDP-GalNAc was the likely reason that we could not detect BPP-trisaccharide without it. The BPP-trisaccharide was readily detected in this strain with LC-MS SIM [M-H]−1 = 1546.7 and [M-2H]−2 = 773.3 (Figure c,d). No peaks with these m/z values were formed without wbpP present or with the empty vector (Supporting Figure S2).

4.

4

Open in a new tab

Formation of BPP-linked trisaccharide with pBAS15. (A) Reaction catalyzed by WcfP. (B) LC-MS SIM chromatogram scanning for BP (black), BPP-AATGal-PyrGal (red), and BPP-AATGal-PyrGal-GalNAc (purple) in cells expressing pBAS11. No trisaccharide-linked BPP was observed. (C) Reaction scheme showing epimerization of UDP-GlcNAc to UDP-GalNAc by the V. vulnificus WbpP. (D) LC-MS SIM chromatogram of BP (black), BPP-AATGal-PyrGal-GalNAc (purple), and BPP-AATGal-PyrGal-GalNAc-Galf (brown) in pBAS15 expressing cells showing the presence of the expected product of WcfP with WbpP present. (E) Mass spectrum of the BPP-AATGal-PyrGal-GalNAc peak from panel (D) demonstrating clear signals for the [M-H]−1 and [M-2H]−2 ions.

Complete Tetrasaccharide Repeat Unit Assembly

The final step in CPSA repeat unit assembly is the addition of Galf by WcfN. The Galf sugar is initially formed through the isomerization of UDP-galactopyranose to the furanose form by WcfM (Figure ). We incorporated both the wcfM and wcfN genes to produce vectors pBAS12 and pBAS16, where pBAS12 did not include the epimerase encoding gene wbpP (Table ). Lysates of transformed DH5α were then analyzed for complete repeat unit formation. A SIM peak corresponding to the [M-2H]−2 854.3 was detected for the repeating unit (Figure b) that was also clear in the total ion mass spectrum (Figure c) and once again, only disaccharide was detected without wbpP (data not shown). We did not observe substantial product ions for the −1 charged species and noted that the intensity of the −1

5.

5

Open in a new tab

Formation of BPP-linked tetrasaccharide with pBAS16. (A) Reaction pathway for the formation of UDP-galactofuranose from UDP-galactopyranose by WcfM and transfer of Galf by WcfN. (B) LC-MS SIM chromatogram for BP (black), BPP-AATGal-PyrGal-GalNAc (purple), BPP-AATGal-PyrGal-GalNAc-Galf (brown) in extracted lysates of E. coli expressing pBAS16. (C) Mass spectrum of the BPP-tetrasaccharide from panel (B), showing a clear signal for the [M-2H]−2 BPP-AATGal-PyrGal-GalNAc-Galf. Inset shows isotopic abundance associated with the −2 charged species. (D) LC-MS SIM chromatogram scanning for the same species in B in lysates of E. coli expressing pBAS17.

charged species for each product progressively decreased with larger glycans (Figures –). We next incorporated genes encoding the predicted flippase (wzx) and polymerase (wzy) (pBAS17, Table ) associated with the CPSA biosynthesis operon. In strains expressing pBAS17, we observed BPP-linked tetrasaccharide, indicating that if there was polymer formation, it did not utilize all of the available tetrasaccharide (Figure d).

2. Plasmids Containing B. fragilis Polymerase or Flippase .
plasmid B. fragilis flippase wzx B. fragilis polymerase wzy
pBAS17 + +
pBAS18 + –
pBAS19 – +
Open in a new tab
a

Each plasmid includes pglF Cj ,wbpP Vv , wcfRSQOPMN in addition to the indicated polymerase or flippase genes.

Coexpression of wzx and wzy from B. fragilis Produce CPSA Polymers

Once we confirmed production of the CPSA BPP-linked repeat unit by our recombinant E. coli strain, we were interested in whether polymer was formed in strains that contained pBAS17 which included the B. fragilis CPSA flippase (wzx) and polymerase (wzy) genes. In addition, we switched to an MG1655 strain of E. coli due to access to a variety of mutant strains that would allow us to interrogate polysaccharide formation. To evaluate whether CPSA polymer was formed in cells expressing pBAS17, we tested cell lysates by SDS-PAGE and blotting with anti-CPSA serum cleared of nonspecific antibodies through adsorption with B. fragilis that does not produce CPSA. , We found polymers that were reactive with the anti-CPSA serum in MG1655 expressing pBAS17 at an apparent molecular weight of around 20 kDa relative to a protein standard (Figure ). The growth medium also contained anti-CPSA serum reactive polymer. However, no anti-CPSA serum reactive polymer was detected with empty vector transformed cells.

6.

6

Open in a new tab

Formation of CPSA polymer detected by anti-CPSA serum. (A) Wild-type and ΔwaaL MG1655 strains containing pQE-80L or pBAS17 were lysed and blotted with an anti-CPSA antibody serum. In the presence of pBAS17, polymer was detected both from cells and from the growth culture media supernatant from the cell culture. A small difference in molecular weight was observed between the ΔwaaL deletion mutant (red arrow) and the wild-type strain (black arrow) in the growth media which was not clear in this cell extract. (B) expected transfer of the CPSA repeat unit to Lipid A by the WaaL ligase.

WaaL Ligates CPSA Fragments to Lipid A

In E. coli, it has been shown that oligo- and polysaccharides other than O-antigens (O–Ag) can be ligated to the lipid A core of LPS, including colanic acid, enterobacterial common antigen (ECA), and peptidoglycan fragments through the activity of the ligase, WaaL (Figure B). ,, We next chose to test for the production of the CPSA polymer in a ΔwaaL strain of MG1655. Interestingly, we observed that a slightly lower molecular weight polymer formed in the absence of waaL in the growth medium supernatant (Figure A). We suspect that the difference in molecular weight is due to CPSA being linked to lipid A by the WaaL ligase, while in the absence of WaaL, the polymer is not linked to the bacterial cell surface. The decrease in molecular weight was not as apparent in this analysis of the cell lysate, but was clear in the analyses below.

Native E. coli Gene Impact CPSA Polymer Formation

We next chose to inspect the minimum requirements for recombinant glycan expression in MG1655. To do this, we separated Pronase treated cell lysates of ΔwaaL (ligase), Δwza (transporter), ΔwaaLΔwza, or ΔwzxB (O-antigen flippase) MG1655 expressing either an empty vector or pBAS17and analyzed by Pro-Q Emerald 300 stain which detects sugar polymers. We found that in wild-type MG1655 expressing pBAS17, there was a high molecular weight polymer formed just above the 26 kDa protein standard that was not present with the empty vector (Figure ). We also observed this same sized polymer with the deletion of the wza and wzxB genes. However, as observed in our anti-CPSA serum blotting (Figure ), when the waaL gene was deleted, a lower molecular weight polymer was formed with an apparent molecular weight lower than the polymer formed in strains without this deletion. In addition to the bands detected at an apparent molecular weight relative to protein standards at 26 kDa and slightly smaller than 26 kDa, we also detected two unique bands with a molecular weight slightly higher than that of lipid A (approximately 15 kDa relative to protein standards) that were not present when the waaL gene was deleted or in the empty vector control strain (Figure ). These bands were unaffected by the absence of wza or wzxB. We suspect that these bands represent one or two repeat units linked to lipid A through the activity of the ligase.

7.

7

Open in a new tab

CPSA is linked to Lipid A through the activity of WaaL. Pro-Q Emerald 300 stain of LPS from Pronase treated cell lysates show production of a higher molecular weight polymer with pBAS17 in wild-type (black arrow) MG1655 compared to ΔwaaL (red arrow). Deletion of wza had no impact on materials formed relative to wild type transformed with pBAS17. Deletion of waaL resulted in loss of both lower molecular weight, presumed lipid A conjugates (white arrow) that are slightly higher molecular weight relative to lipid A (blue arrow). Deletion of the O-antigen flippase, wzxB, had no impact on glycan formations relative to wild-type MG1655 transformed with pBAS17. Empty vector transformed cells produced no high molecular weight polymers or presumed lipid A-CPSA conjugates.

Escherichia coli WzxB Promotes Formation of CPSA Polymers

It was not clear from the previous experiments whether B. fragilis flippase and polymerase were both required for CPSA polymer formation. To determine whether both B. fragilis enzymes were necessary for CPSA polymerization, we constructed plasmids containing only B. fragilis flippase (wzx) (pBAS18) or polymerase (wzy) (pBAS19, Table ). We then evaluated CPSA production in wild type (wt), ΔwaaL, and ΔwzxB MG1655 expressing pBAS16 (Table ), 18, or 19 using anti-CPSA serum or Pro-Q Emerald 300 stain. MG1655 transformed with pBAS16 or pBAS18, which did not contain the B. fragilis polymerase, led to the formation of no anti-CPSA serum reactive polymers regardless of the strain background (Figure ). However, pBAS19, which contained the polymerase but not the flippase, resulted in a polymer similar to pBAS17 in the wild type and ΔwaaL backgrounds. Interestingly, no polymer was observed in ΔwzxB MG1655 with pBAS19 suggesting that there was not another flippase in E. coli capable of transporting the repeat unit through the inner membrane. We next inspected the glycoprofile of these strains (Figure b). We found that with pBAS16 (−wzx Bf , −wzy Bf ), we observed a small molecular weight lipid A conjugate at approximately 13 kDa relative to a set of glycoprotein standards in the wild-type strain that was not present in the empty vector control. This band was also absent in the ΔwaaL and ΔwzxB backgrounds with pBAS16. This indicated that the repeat unit was likely transferred via WaaL, and without the B. fragilis flippase, required the E. coli O-antigen flippase for transport across the inner membrane. In strains transformed with pBAS18 (+wzx, −wzy), we again observed no higher molecular weight polymers but did observe presumed lipid-A modified glycan in the wild-type and ΔwzxB strains, suggesting that the B. fragilis flippase could replace the endogenous WzxB protein. Finally, with pBAS19 transformed strains (−wzx, +wzy), we observed polymer consistent with the anti-CPSA serum blot with both the wild-type and ΔwaaL strain, but without the E. coli wzxB or B. fragilis flippase, we did not observe any unique product relative to empty vector controls. It is important to note the presence of a presumed lipid-A modified material in wild-type strains transformed with pBAS16–19 indicating repeat unit transfer occurs only when waaL is present. It does not appear that the anti-CPSA serum is useful for detecting these likely single repeat units. It is also important to note that the ΔwaaL mutant consistently leads to a lower molecular weight polymer formation when the B. fragilis polymerase is present in the plasmid. The difference in the molecular weight could be related to the addition of the polymer to lipid-A.

8.

8

Open in a new tab

CPSA production in E. coli is dependent on a B. fragilis polymerase but not the flippase. (A) anti-CPSA serum blot of lysates from MG1655 strains (wt, ΔwaaL, and ΔwzxB) transformed with pBAS16 (−wzx, −wzy), pBAS18 (+wzx, −wzy), pBAS19 (−wzx, +wzy) or the empty vector (pQE-80L). A sharp CPSA polymer ladder (black arrow) is detected when the B. fragilis polymerase is expressed from plasmid pBAS19 which contains only the polymerase. The downward shift in the ΔwaaL lane confirms the ligase role in conjugating the polymer to the lipid A-core (red arrow). The complete loss of signal in the ΔwzxB lane with pBAS19 suggests that the native E. coli flippase is required if the Wzx flippase from B. fragilis is not included. (B) Pro-Q Emerald 300 stain of glycans from Pronase treated cell lysates identical to panel (A). Polymer consistent with the anti-CPSA blot was formed with a molecular weight of approximately 29 kDa (black arrow) relative to the CandyCane Glycoprotein ladder and a smaller molecular weight ladder was also detected (red arrow). Lipid A conjugates are also observed with pBAS16/18/19 in wild-type MG1655 (white arrow), but not ΔwaaL or ΔwzxB except when the B. fragilis flippase is included with pBAS18. Lipid A is shown with a blue arrow. Note that we did not detect these lower molecular weight lipid A conjugates with the anti-CPSA serum suggesting that conjugate does not have an epitope for the anti-CPSA serum or detection required multiple epitope interactions to rise above detection limits.

Discussion

A detailed understanding of the biochemical requirements for recombinant polysaccharide biosynthesis is essential for expanding access to structurally complex glycans, which are otherwise difficult to obtain. In this work, we reconstructed the biosynthetic pathway for capsular polysaccharide A (CPSA) from B. fragilis in E. coli and used this system to systematically evaluate individual steps of the Wzx/Wzy-dependent polysaccharide pathway. , Such approaches provide a practical alternative to total chemical synthesis, direct isolation from B. fragilis, or chemoenzymatic production strategies, which remain limited by the lack of a purified CPSA polymerase capable of in vitro polymerization. More broadly, this work illustrates a general framework for dissecting bottlenecks in recombinant polysaccharide production systems.

Reconstitution of CPSA biosynthesis in E. coli revealed that production of the repeating unit requires supplementation of enzymatic activities not encoded within the native CPSA biosynthesis operon. Incorporation of a UDP-GlcNAc C4 epimerase and a 4,6-dehydratase, which are absent from the CPSA operon but required for precursor sugar biosynthesis, was necessary to generate detectable CPSA intermediates. , These results highlight a recurring limitation of heterologous glycan expression systems: biosynthetic gene clusters often rely on host metabolic enzymes that may not be present or sufficiently expressed in the recombinant host. The stepwise reconstruction strategy used here provides a systematic means of identifying these requirements.

This system also enabled evaluation of key steps of CPSA assembly in the recombinant host. Surprisingly, CPSA polymerization was dependent on expression of the B. fragilis Wzy polymerase, whereas the native E. coli flippase WzxB could functionally substitute for the CPSA flippase Wzx Bf . The observation that CPSA polymers were produced in the absence of Wzx Bf indicates that WzxB possesses sufficient substrate tolerance to translocate CPSA isoprenoid-linked oligosaccharides. This promiscuity is consistent with previous reports of relaxed specificity among Wzx flippases and suggests that endogenous flippases in E. coli may support the production of a broad range of heterologous glycans. , At the same time, the ability of wzx Bf to complement a ΔwzxB mutant demonstrates that the B. fragilis flippase is functionally compatible with E. coli.

Despite evidence for functional flippase and polymerase activity, accumulation of BPP-linked tetrasaccharide intermediates was observed in strains expressing both Wzx and Wzy. The persistence of these intermediates suggests that polymerization by Wzy Bf may represent a limiting step in the recombinant system. One explanation is that efficient CPSA polymerization requires additional components of the native capsular transport machinery, such as Wza and Wzz, which are thought to assemble into a complex that coordinates polymerization, export, and chain length regulation. Alternatively, inefficient expression or folding of the multipass membrane protein WzyBf in E. coli could also limit catalytic turnover and lead to accumulation of lipid-linked intermediates.

Consistent with this interpretation, the CPSA polymers produced in the recombinant system were substantially shorter than those isolated from native B. fragilis. Native CPSA typically appears as a 180 kDa polymer by SDS-PAGE, whereas the polymers produced here were markedly smaller. Previous studies have shown that CPSA fragments exhibit size-dependent biological activity: short oligomers (∼5 kDa, six repeating units) lack protective activity in mouse intra-abdominal abscess models, whereas fragments containing ∼22 repeating units (∼17 kDa) exhibit activity comparable to native CPSA. Structural studies further indicate that as few as three repeating units can form the helical conformation necessary for MHCII presentation. These observations suggest that even modest improvements in polymer length within recombinant systems could yield material suitable for functional and immunological studies.

The accumulation of lipid-linked CPSA intermediates may also impose physiological constraints on the host cell. − Cells expressing CPSA constructs exhibited irregular morphologies and altered growth profiles following induction (data not shown). Similar phenotypes have been reported in other glycan biosynthesis systems when bactoprenyl phosphate (BP) becomes sequestered in stalled intermediates, thereby limiting its availability for peptidoglycan synthesis. Detection of BPP-linked CPSA tetrasaccharide intermediates in this study is consistent with such sequestration and highlights the importance of balancing glycan assembly with host lipid carrier availability when engineering recombinant pathways. Efficient recovery of soluble CPSA further required the disruption of native glycan attachment pathways. Deletion of waaL, which encodes the O-antigen ligase responsible for transferring glycans onto lipopolysaccharide, was necessary to obtain CPSA that was not covalently associated with lipid A. Similar strategies have been used in other recombinant polysaccharide systems to prevent diversion of heterologous glycans.

Conclusions

Collectively, these results underscore the challenges associated with transferring complex polysaccharide biosynthesis loci into heterologous hosts. Even when complete gene clusters are introduced, productive glycan synthesis may depend on host metabolic enzymes, membrane transport systems, and regulatory factors not encoded within the cluster itself. Recent efforts to engineer E. coli strains optimized for recombinant glycan production including modifications that enhance precursor availability, control polymer chain length, and minimize competition from endogenous pathways provide promising avenues to address these limitations. , The modular strategy described here complements these approaches by enabling the systematic identification of bottlenecks within recombinant Wzx/Wzy-dependent pathways. Integration of these strategies should facilitate scalable production of structurally defined glycopolymers and expand access to biologically important polysaccharides, such as CPSA.

Supplementary Material

bi6c00056_si_001.pdf (225.7KB, pdf)

Acknowledgments

We thank Laurie E. Comstock (University of Chicago) for providing adsorbed anti-CPSA antiserum.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.biochem.6c00056.

  • Primers used for preparing plasmids and E. coli mutants and strain genotypes, the sequence of the full pBAS17 plasmid obtained through whole plasmid sequencing (Eurofins-Operon), and LC-MS chromatograms for blank pQE-80L with each intermediate SIM (PDF)

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding supporting this work was received through grants from the National Institutes of Health General Medical Sciences, grant numbers: R01GM123251 (J.M.T.) and R35GM154672 (M.A.J.).

The authors declare no competing financial interest.

References

  1. Erturk-Hasdemir D., Ochoa-Reparaz J., Kasper D. L., Kasper L. H.. Exploring the Gut-Brain Axis for the Control of CNS Inflammatory Demyelination: Immunomodulation by Bacteroides fragilis’ Polysaccharide A. Front. Immunol. 2021;12:662807. doi: 10.3389/fimmu.2021.662807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Erturk-Hasdemir D., Oh S. F., Okan N. A., Stefanetti G., Gazzaniga F. S., Seeberger P. H., Plevy S. E., Kasper D. L.. Symbionts exploit complex signaling to educate the immune system. Proc. Natl. Acad. Sci. U.S.A. 2019;116:26157–26166. doi: 10.1073/pnas.1915978116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Mazmanian S. K., Kasper D. L.. The love-hate relationship between bacterial polysaccharides and the host immune system. Nat. Rev. Immunol. 2006;6:849–858. doi: 10.1038/nri1956. [DOI] [PubMed] [Google Scholar]
  4. Brubaker J. O., Li Q., Tzianabos A. O., Kasper D. L., Finberg R. W.. Mitogenic Activity of Purified Capsular Polysaccharide A from Bacteroides fragilis: Differential Stimulatory Effect on Mouse and Rat Lymphocytes In Vitro1. J. Immunol. 1999;162:2235–2242. doi: 10.4049/jimmunol.162.4.2235. [DOI] [PubMed] [Google Scholar]
  5. Tzianabos A. O., Finberg R. W., Wang Y., Chan M., Onderdonk A. B., Jennings H. J., Kasper D. L.. T Cells Activated by Zwitterionic Molecules Prevent Abscesses Induced by Pathogenic Bacteria *. J. Biol. Chem. 2000;275:6733–6740. doi: 10.1074/jbc.275.10.6733. [DOI] [PubMed] [Google Scholar]
  6. Zhou J. Y., Zhou D., Telfer K., Reynero K., Jones M. B., Hambor J., Cobb B. A.. Antigen presenting cell response to polysaccharide A is characterized by the generation of anti-inflammatory macrophages. Glycobiology. 2022;32:136–147. doi: 10.1093/glycob/cwab111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ramakrishna C., Kujawski M., Chu H., Li L., Mazmanian S. K., Cantin E. M.. Bacteroides fragilis polysaccharide A induces IL-10 secreting B and T cells that prevent viral encephalitis. Nat. Commun. 2019;10:2153. doi: 10.1038/s41467-019-09884-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Zheng L., Luo M., Kuang G., Liu Y., Liang D., Huang H., Yi X., Wang C., Wang Y., Xie Q., Zhi F.. Capsular Polysaccharide From Bacteroides fragilis Protects Against Ulcerative Colitis in an Undegraded Form. Front. Pharmacol. 2020;11:570476. doi: 10.3389/fphar.2020.570476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ochoa-Repáraz J., Mielcarz D. W., Wang Y., Begum-Haque S., Dasgupta S., Kasper D. L., Kasper L. H.. A polysaccharide from the human commensal Bacteroides fragilis protects against CNS demyelinating disease. Mucosal Immunol. 2010;3:487–495. doi: 10.1038/mi.2010.29. [DOI] [PubMed] [Google Scholar]
  10. Ochoa-Repáraz J., Mielcarz D. W., Ditrio L. E., Burroughs A. R., Begum-Haque S., Dasgupta S., Kasper D. L., Kasper L. H.. Central nervous system demyelinating disease protection by the human commensal Bacteroides fragilis depends on polysaccharide A expression. J. Immunol. 2010;185:4101–4108. doi: 10.4049/jimmunol.1001443. [DOI] [PubMed] [Google Scholar]
  11. Alvarez C. A., Cobb B. A.. Purification of Capsular Polysaccharide Complex from Gram-Negative Bacteria. Methods Mol. Biol. 2019;1954:25–35. doi: 10.1007/978-1-4939-9154-9_3. [DOI] [PubMed] [Google Scholar]
  12. Wang Z., Poveda A., Zhang Q., Unione L., Overkleeft H. S., van der Marel G. A., Jesus J. B., Codee J. D. C.. Total Synthesis and Structural Studies of Zwitterionic Bacteroides fragilis Polysaccharide A1 Fragments. J. Am. Chem. Soc. 2023;145:14052–14063. doi: 10.1021/jacs.3c03976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Pathan E. K., Ghosh B., Podilapu A. R., Kulkarni S. S.. Total Synthesis of the Repeating Unit of Bacteroides fragilis Zwitterionic Polysaccharide A1. J. Org. Chem. 2021;86:6090–6099. doi: 10.1021/acs.joc.0c02935. [DOI] [PubMed] [Google Scholar]
  14. Eradi P., Ghosh S., Andreana P. R.. Total Synthesis of Zwitterionic Tetrasaccharide Repeating Unit from Bacteroides fragilis ATCC 25285/NCTC 9343 Capsular Polysaccharide PS A1 with Alternating Charges on Adjacent Monosaccharides. Org. Lett. 2018;20:4526–4530. doi: 10.1021/acs.orglett.8b01829. [DOI] [PubMed] [Google Scholar]
  15. Pragani R., Seeberger P. H.. Total synthesis of the Bacteroides fragilis zwitterionic polysaccharide A1 repeating unit. J. Am. Chem. Soc. 2011;133:102–107. doi: 10.1021/ja1087375. [DOI] [PubMed] [Google Scholar]
  16. van den Bos L. J., Boltje T. J., Provoost T., Mazurek J., Overkleeft H. S., van der Marel G. A.. A synthetic study towards the PSA1 tetrasaccharide repeating unit. Tetrahedron Lett. 2007;48:2697–2700. doi: 10.1016/j.tetlet.2007.02.067. [DOI] [Google Scholar]
  17. Kay E. J., Yates L. E., Terra V. S., Cuccui J., Wren B. W.. Recombinant expression of Streptococcus pneumoniae capsular polysaccharides in Escherichia coli . Open Biol. 2016;6:150243. doi: 10.1098/rsob.150243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kay E. J., Mauri M., Willcocks S. J., Scott T. A., Cuccui J., Wren B. W.. Engineering a suite of E. coli strains for enhanced expression of bacterial polysaccharides and glycoconjugate vaccines. Microb. Cell Fact. 2022;21:66. doi: 10.1186/s12934-022-01792-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Nothaft H., Davis B., Lock Y. Y., Perez-Munoz M. E., Vinogradov E., Walter J., Coros C., Szymanski C. M.. Engineering the Campylobacter jejuni N-glycan to create an effective chicken vaccine. Sci. Rep. 2016;6:26511. doi: 10.1038/srep26511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Feldman M. F., Wacker M., Hernandez M., Paul G. H., Cristina L. M., Kowarik M., Howard R. M., Dell A., Miguel A. V., Aebi M.. Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coli . Proc. Natl. Acad. Sci. U.S.A. 2005;102:3016–3021. doi: 10.1073/pnas.0500044102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sharma S., Erickson K. M., Troutman J. M.. Complete Tetrasaccharide Repeat Unit Biosynthesis of the Immunomodulatory Bacteroides fragilis Capsular Polysaccharide A. ACS Chem. Biol. 2017;12:92–101. doi: 10.1021/acschembio.6b00931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Comstock L. E., Coyne M. J., Tzianabos A. O., Pantosti A., Onderdonk A. B., Kasper D. L.. Analysis of a capsular polysaccharide biosynthesis locus of Bacteroides fragilis. Infect. Immun. 1999;67:3525–3532. doi: 10.1128/IAI.67.7.3525-3532.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Baumann H., Tzianabos A. O., Brisson J. R., Kasper D. L., Jennings H. J.. Structural elucidation of two capsular polysaccharides from one strain of Bacteroides fragilis using high-resolution NMR spectroscopy. Biochemistry. 1992;31:4081–4089. doi: 10.1021/bi00131a026. [DOI] [PubMed] [Google Scholar]
  24. Mostafavi A. Z., Troutman J. M.. Biosynthetic Assembly of the Bacteroides fragilis Capsular Polysaccharide A Precursor Bactoprenyl Diphosphate-Linked Acetamido-4-amino-6-deoxygalactopyranose. Biochemistry. 2013;52:1939–1949. doi: 10.1021/bi400126w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Meredith T. C., Mamat U., Kaczynski Z., Lindner B., Holst O., Woodard R. W.. Modification of Lipopolysaccharide with Colanic Acid (M-antigen) Repeats in Escherichia coli*. J. Biol. Chem. 2007;282:7790–7798. doi: 10.1074/jbc.M611034200. [DOI] [PubMed] [Google Scholar]
  26. Maciejewska A., Kaszowska M., Jachymek W., Lugowski C., Lukasiewicz J.. Lipopolysaccharide-Linked Enterobacterial Common Antigen (ECA­(LPS)) Occurs in Rough Strains of Escherichia coli R1, R2, and R4. Int. J. Mol. Sci. 2020;21:6038. doi: 10.3390/ijms21176038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schmidt G., Mannel D., Mayer H., Whang H. Y., Neter E.. Role of a lipopolysaccharide gene for immunogenicity of the enterobacterial common antigen. J. Bacteriol. 1976;126:579–586. doi: 10.1128/jb.126.2.579-586.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Coyne M. J., Tzianabos A. O., Mallory B. C., Carey V. J., Kasper D. L., Comstock L. E.. Polysaccharide biosynthesis locus required for virulence of Bacteroides fragilis. Infect. Immun. 2001;69:4342–4350. doi: 10.1128/IAI.69.7.4342-4350.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Henderson J. C., O’Brien J. P., Brodbelt J. S., Trent M. S.. Isolation and chemical characterization of lipid A from gram-negative bacteria. J. Visualized Exp. 2013;79:50623. doi: 10.3791/50623-v. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Bligh E. G., Dyer W. J.. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 1959;37:911–917. doi: 10.1139/y59-099. [DOI] [PubMed] [Google Scholar]
  31. Datsenko K. A., Wanner B. L.. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 2000;97:6640–6645. doi: 10.1073/pnas.120163297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ranjit D. K., Young K. D.. Colanic Acid Intermediates Prevent De Novo Shape Recovery of Escherichia coli Spheroplasts, Calling into Question Biological Roles Previously Attributed to Colanic Acid. J. Bacteriol. 2016;198:1230–1240. doi: 10.1128/JB.01034-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Coyne M. J., Chatzidaki-Livanis M., Paoletti L. C., Comstock L. E.. Role of glycan synthesis in colonization of the mammalian gut by the bacterial symbiont Bacteroides fragilis. Proc. Natl. Acad. Sci. U.S.A. 2008;105:13099–13104. doi: 10.1073/pnas.0804220105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Olivier N. B., Chen M. M., Behr J. R., Imperiali B.. In vitro biosynthesis of UDP-N,N’-diacetylbacillosamine by enzymes of the Campylobacter jejuni general protein glycosylation system. Biochemistry. 2006;45:13659–13669. doi: 10.1021/bi061456h. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Troutman J. M., Sharma S., Erickson K. M., Martinez C. D.. Functional identification of a galactosyltransferase critical to Bacteroides fragilis Capsular Polysaccharide A biosynthesis. Carbohydr. Res. 2014;395:19–28. doi: 10.1016/j.carres.2014.06.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Reid A. J., Erickson K. M., Hazel J. M., Lukose V., Troutman J. M.. Chemoenzymatic Preparation of a Campylobacter jejuni Lipid-Linked Heptasaccharide on an Azide-Linked Polyisoprenoid. ACS Omega. 2023;8:15790–15798. doi: 10.1021/acsomega.3c01657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Marolda C. L., Tatar L. D., Alaimo C., Aebi M., Valvano M. A.. Interplay of the Wzx translocase and the corresponding polymerase and chain length regulator proteins in the translocation and periplasmic assembly of lipopolysaccharide o antigen. J. Bacteriol. 2006;188:5124–5135. doi: 10.1128/JB.00461-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Ma, Z. ; Wang, P. G. . RecET Direct Cloning of Polysaccharide Gene Cluster from Gram-Negative Bacteria. In Bacterial Polysaccharides: Methods and Protocols; Brockhausen, I. , Ed.; Springer New York,: New York, NY, 2019; pp 15–23. [DOI] [PubMed] [Google Scholar]
  39. Wacker M., Linton D., Hitchen P. G., Nita-Lazar M., Haslam S. M., North S. J., Panico M., Morris H. R., Dell A., Wren B. W., Aebi M.. N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli . Science. 2002;298:1790–1793. doi: 10.1126/science.298.5599.1790. [DOI] [PubMed] [Google Scholar]
  40. Kreisman L. S. C., Friedman J. H., Neaga A., Cobb B. A.. Structure and function relations with a T-cell-activating polysaccharide antigen using circular dichroism. Glycobiology. 2007;17:46–55. doi: 10.1093/glycob/cwl056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Merino S., Gonzalez V., Tomás J. M.. The first sugar of the repeat units is essential for the Wzy polymerase activity and elongation of the O-antigen lipopolysaccharide. Future Microbiol. 2016;11:903–918. doi: 10.2217/fmb-2015-0028. [DOI] [PubMed] [Google Scholar]
  42. Marolda C. L., Vicarioli J., Valvano M. A.. Wzx proteins involved in biosynthesis of O antigen function in association with the first sugar of the O-specific lipopolysaccharide subunit. Microbiology. 2004;150:4095–4105. doi: 10.1099/mic.0.27456-0. [DOI] [PubMed] [Google Scholar]
  43. Cuthbertson L., Mainprize I. L., Naismith J. H., Whitfield C.. Pivotal roles of the outer membrane polysaccharide export and polysaccharide copolymerase protein families in export of extracellular polysaccharides in gram-negative bacteria. Microbiol. Mol. Biol. Rev. 2009;73:155–177. doi: 10.1128/MMBR.00024-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Kalka-Moll W. M., Tzianabos A. O., Wang Y., Carey V. J., Finberg R. W., Onderdonk A. B., Kasper D. L.. Effect of molecular size on the ability of zwitterionic polysaccharides to stimulate cellular immunity. J. Immunol. 2000;164:719–724. doi: 10.4049/jimmunol.164.2.719. [DOI] [PubMed] [Google Scholar]
  45. Kay E. J., Dooda M. K., Bryant J. C., Reid A. J., Wren B. W., Troutman J. M., Jorgenson M. A.. Engineering Escherichia coli for increased Und-P availability leads to material improvements in glycan expression technology. Microb. Cell Fact. 2024;23:72. doi: 10.1186/s12934-024-02339-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Jorgenson M. A.. Pardon the interruption: how Und-P sequestration has reshaped our understanding of the bacterial cell envelope. J. Bacteriol. 2025:e0045125. doi: 10.1128/jb.00451-25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Jorgenson M. A., Kannan S., Laubacher M. E., Young K. D.. Dead-end intermediates in the enterobacterial common antigen pathway induce morphological defects in Escherichia coli by competing for undecaprenyl phosphate. Mol. Microbiol. 2016;100:1–14. doi: 10.1111/mmi.13284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Jorgenson M. A., MacCain W. J., Meberg B. M., Kannan S., Bryant J. C., Young K. D.. Simultaneously inhibiting undecaprenyl phosphate production and peptidoglycan synthases promotes rapid lysis in Escherichia coli . Mol. Microbiol. 2019;112:233–248. doi: 10.1111/mmi.14265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Jorgenson M. A., Young K. D.. Interrupting Biosynthesis of O Antigen or the Lipopolysaccharide Core Produces Morphological Defects in Escherichia coli by Sequestering Undecaprenyl Phosphate. J. Bacteriol. 2016;198:3070–3079. doi: 10.1128/JB.00550-16. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

bi6c00056_si_001.pdf (225.7KB, pdf)

Articles from Biochemistry are provided here courtesy of American Chemical Society

RESOURCES