Raman targets bacterial cell walls

Bacterial cell walls are a key target for antibiotics but they can change structure during reproduction. Now, Raman spectroscopy and atomic force microscopy have been used to home in on these changes in a bacterium and so provide important clues about the biochemical changes that occur at the cellular level.

Gerald McEwen, Yangzhe Wu, Anhong Zhou of the Biological Engineering Program, College of Engineering, at Utah State University, in Logan, have now evaluated variations in the culture time-dependent cell wall structure of Pseudomonas putida KT2440 at both the single cell level and in colonies.

P. putida is a well-researched gram-negative rod-shaped saprotrophic soil bacterium, which can serve as a safe laboratory model for testing analytical techniques as well as a model for bioremediation as this strain can digest the volatile organic solvent, toluene. A better understanding of this microbe could lead to improved engineered variants for cleaning up contaminated industrial sites.

The team points out that Raman microspectroscopy is commonly utilized to study the vibrational properties of macromolecules and polymers, but until the early 1980s, the technique was used to focus mainly on physical and structural investigations. Today, it is becoming increasingly popular in microbiology as a tool to identify microorganisms, not only because it is non-destructive but also because it can readily identify the characteristic spectral fingerprints of bacteria.

More recently, researchers have used Raman to monitor the appearance of surface biopolymers (including DNA/RNA, lipids, proteins, and carbohydrates) in growth and antibiotic use, the team explains. This has proved particularly useful in studies of other Pseudomonas family bacterium, like Pseudomonas aeruginosa, which causes human lung bacterial infections.

The team has now combined Raman with atomic force microscopy (AFM) to get a more detailed look than ever before at changes in the bacterial cell wall. AFM, which evolved from scanning tunnelling microscopy in the 1980s, scans the surface topography of a sample by detecting dynamic variation of the force between the AFM tip and the sample surface. It has several unique advantages over other microscopy techniques, the team says, not least that it can be performed in situ under near-physiological conditions, there is little of the complex sample preparation required of other techniques and it gives nanoscale resolution.

The cell wall of Gram-negative bacteria comprises an outer membrane, a periplasmic space, and an inner membrane. The membranes provide the first line of defence against attacking white blood cells as well as against antibiotics, enzymes, detergents, heavy metals, bile salts, and certain dye molecules.

The team recorded Raman spectra for P. putida KT2440 growing on agar gel over a thirty-hour period using a Renishaw inVia Raman microscope (controlled by WiRE 3.0 software) equipped with a 785 nm near-IR laser. A Picoplus contact mode AFM (controlled by Picoscan 5.4) was used to measure the growth time-dependent cell surface topography features and nanomechanic properties of the bacterial cells at room temperature in air.

""The Raman spectra indicate that the appearance of DNA/RNA, protein, lipid, and carbohydrates occurs up to six hours of cultivation time under our experimental conditions," the team explains. "AFM characterization reveals the changes of the cellular surface ultrastructures over the culture time period, which is a gradual increase in surface roughness during the time between the first two and eight hours cultivation time." Zhou adds, "Our next step is to use a combined AFM/Raman instrument setup to measure the dynamic changes of biomechanical (by AFM) biochemical (by Raman) of bacterial cells during the period of the growth in one platform. This new instrumental analysis provides an exciting application of the study of the bacterium-drug interaction at nanoscale resolution and single cell level."

They also conclude that it is entirely feasible to combined Raman spectroscopy and AFM for the investigation of bacterial cell surface biopolymers at the level of the single cell. Such studies should provide insights into creating engineered soil microbes for bioremediation applications as well as potentially offering new clues for finding targets for novel antibiotics.