Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography

Podsumowanie

The bacterial cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by peptides. Ultra Performance Liquid Chromatography provides high resolution and throughput for novel discoveries of peptidoglycan composition. We present a procedure for the isolation of cell walls (sacculi) and their subsequent preparation for analysis via UPLC.

Streszczenie

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the face of turgor pressures several atmospheres in magnitude. Across the diverse shapes and sizes of the bacterial kingdom, the cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by short peptides. Peptidoglycan’s central importance to bacterial physiology underlies its use as an antibiotic target and has motivated genetic, structural, and cell biological studies of how it is robustly assembled during growth and division. Nonetheless, extensive investigations are still required to fully characterize the key enzymatic activities in peptidoglycan synthesis and the chemical composition of bacterial cell walls. High Performance Liquid Chromatography (HPLC) is a powerful analytical method for quantifying differences in the chemical composition of the walls of bacteria grown under a variety of environmental and genetic conditions, but its throughput is often limited. Here, we present a straightforward procedure for the isolation and preparation of bacterial cell walls for biological analyses of peptidoglycan via HPLC and Ultra Performance Liquid Chromatography (UPLC), an extension of HPLC that utilizes pumps to deliver ultra-high pressures of up to 15,000 psi, compared with 6,000 psi for HPLC. In combination with the preparation of bacterial cell walls presented here, the low-volume sample injectors, detectors with high sampling rates, smaller sample volumes, and shorter run times of UPLC will enable high resolution and throughput for novel discoveries of peptidoglycan composition and fundamental bacterial cell biology in most biological laboratories with access to an ultracentrifuge and UPLC.

Wprowadzenie

The goal of the method described herein is to isolate intact bacterial cell walls (sacculi) and to digest the peptidoglycan (PG) such that Ultra Performance Liquid Chromatography (UPLC) can be used to provide information such as the identity of the muropeptide components and their concentrations, the average length of glycan strands, and the fraction of material involved in crosslinks between strands. For a detailed discussion of PG biochemistry and muropeptide species, there are several excellent reviews that describe PG structure and its role in infection, resistance, morphogenesis, and growth^1-6. High Performance Liquid Chromatography (HPLC) for PG analy

Protokół

1. Grow Bacterial Cultures in 2.5 ml of Media Overnight

Back-dilute cultures 1:100 into 250 ml of fresh media and grow to OD₆₀₀ of 0.7-0.8. Prepare a solution of 6% sodium dodecyl sulfate (SDS) in water.

CAUTION: SDS powder is hazardous - avoid inhaling SDS powder; wear a mask over nose and mouth.

2. Day 1 - Lysing Bacterial Cultures is Performed over the Course of One Day and Overnight

1. While diluted cultures are growing, set up a boiling water bath on a hot plate in a 1 L beaker. Once the water is boiling, aliquot 6 ml of 6% SDS into 50 ml 

Wyniki

Using the procedure outlined in Figure 1, the final sample should consist of at least 200 µl of clear solution that has been filtered directly into a UPLC vial (step 4.4). UPLC separation of the various muropeptides in a bacterial sample relies upon their relative solubility between the liquid mobile phase and the column’s stationary phase. Reversed-phase C18 columns provide a strongly hydrophobic matrix to separate the muropeptide species based on hydrophobicity and size⁸...

Dyskusje

A critical step in this procedure is step 3.1 of the second day of sample preparation. If the SDS has precipitated overnight, or if the samples have been stored in 4% SDS for several weeks at room temperature, the samples must be reboiled for at least 1 hr to redissolve the SDS. A common cause for SDS precipitation is the use of media with potassium salts, so potassium should be avoided in media if possible. As mentioned in the Representative Results section, it is also critical to adjust the pH to within the isoelectric...

Materiały

Name	Company	Catalog Number	Comments
Pronase E	Amresco	E629
Mutanolysin from Streptomyces	Sigma-Aldrich	M9901
Sodium borohydride (NaBH4)	Sigma-Aldrich	452882	Sodium borohydride is highly reactive and dangerous to handle
Orthophosphoric acid	Sigma-Aldrich	79607	Orthophosphoric acid is corrosive and dangerous to handle
Boric acid	Sigma-Aldrich	31146
Sodium azide	Sigma-Aldrich	S2002	Sodium azide is a poison
Sodium tetraborate	Sigma-Aldrich	221732
Millex 0.22 μm syringe filters	Fisher	SLGVR04NL
pH strips (pH range 0-6)	Fisher	M95863
50 ml polypropylene Falcon tubes	VWR	21008-951
13 mm x 100 mm glass tubes	Kimble Chase	60CM13
12 mm x 32 mm screw neck glass recovery vial	Waters	186000327C
Sodium Dodecyl Sulfate	Ambion	AM9820	SDS powder is hazardous
Instrumentation
Waters Acquity UPLC H-Class system, including:
Acquity UPLC H-Class Sample Manager FTN
Acquity UPLC H-Class Quaternary Solvent Manager
Acquity UPLC BEH C18 1.7 µm column
Acquity UPLC PDA Detector
Waters Fraction Collector III
Acquity UPLC 30 cm Column Heater/Cooler