Abstract

I propose to develop novel genetic transformation techniques for Sporosarcina pasteurii, a Gram-positive bacterium noted for its robust urease activity and potential applications in biocementation and bioremediation. Drawing inspiration from ultrasound-based sonoporation methods and protoplast transformation approaches used successfully in other recalcitrant bacterial strains, I will adapt and optimize protocols to overcome the species-specific barriers typically associated with thick cell walls and strong restriction-modification systems. First, I will identify and, if required, engineer methyltransferase systems to protect incoming DNA from host restriction enzymes. Next, I will test two transformation strategies—one based on transient protoplast formation, the other on ultrasound-mediated DNA delivery—for efficacy and reproducibility under S. pasteurii growth conditions. Ultimately, the goal is to establish a reliable method to introduce and express heterologous genes, enabling deeper exploration of this microbe’s valuable biomineralization capabilities and paving the way for new biotechnological applications.

Background and Motivation

a. Motivation

I am motivated to pursue this project because Sporosarcina pasteurii plays a key role in biomineralization processes that have promising applications in construction, bioremediation, and carbon capture. Despite its industrial potential, genetic manipulation of S. pasteurii remains challenging due to its recalcitrant cell wall and distinct restriction-modification systems. Developing effective transformation methods will allow us to insert desired genes, alter metabolic pathways, and thoroughly investigate the biochemical mechanisms driving urea hydrolysis and calcium carbonate precipitation. Such insights can significantly enhance current applications and enable new biotechnological strategies for sustainable materials production and environmental protection.

b. Current State of Knowledge

Gram-positive bacteria with thick cell envelopes often require specialized protocols for DNA uptake. Investigators have successfully employed ultrasound-mediated sonoporation in thermophilic anaerobes, demonstrating that it can significantly increase transformation efficiency without extensively damaging cells (Lin et al. 2010). Meanwhile, protoplast-based methods, including careful selection of osmotic stabilizers and the use of hard-gel regeneration media, have proven effective for other stubborn alkaliphilic Bacillus species (Gao et al. 2011). These precedents suggest that S. pasteurii can likewise benefit from targeted modifications of these techniques to overcome host-specific barriers.

c. Innovation and Significance

My project is innovative because it combines sonoporation and protoplast transformation—two methodologies typically optimized for distinct bacterial taxa—and applies them to a new host, S. pasteurii. This dual approach provides flexibility should one technique prove suboptimal. By establishing robust genetic tools, I will expand the capacity to interrogate S. pasteurii physiology and improve its biomineralization traits. The significance extends to industrial and environmental realms: reproducible methods for engineering S. pasteurii could lead to improved biocementation processes, bio-based concrete repair, and innovative solutions for environmental challenges.

In Aim 1, my goal is to establish a reproducible protoplast-based transformation protocol for Sporosarcina pasteurii, which is a Gram-positive bacterium with a thick cell wall and potential restriction-modification barriers. Below is a more detailed plan and rationale for each step:

Step 1: Optimizing Cell Growth and Pretreatment

1. Growth Conditions
   • I will initially culture S. pasteurii in a nutrient-rich medium (e.g., tryptic soy broth or a suitable complex medium) at an optimal growth temperature (often around 30 °C), ensuring cells reach the mid-logarithmic phase.
   • Achieving mid-log phase is important because cells are metabolically active and more amenable to cell-wall weakening agents.
2. Cell-Wall Stressors
   • Before harvesting the cells, I plan to supplement cultures with sublethal concentrations of cell-wall-perturbing compounds (e.g., 0.5–1.0% glycine or other S. pasteurii compatible stressors).
   • In previous work with other recalcitrant Gram-positive species, glycine enhances protoplast formation by interfering with peptidoglycan crosslinking (Gao et al. 2011, pone.0028148).
   • The choice and concentration of such additives will be optimized through small-scale pilot experiments.

Step 2: Protoplast Formation

1. Enzymatic Digestion
   • After harvesting cells by centrifugation, I will wash them in a carefully selected osmotic stabilization buffer. This buffer often contains high concentrations of sucrose or sodium succinate (0.3–0.5 M) at a neutral or slightly alkaline pH.
   • Next, I will expose the cells to lysozyme (0.1–0.5 mg/mL) at 30 °C or 37 °C for 30–60 minutes, titrating both enzyme concentration and incubation time to maximize protoplast yield.
   • I will monitor progress microscopically—if a large fraction of spherical protoplasts is observed, the digestion is considered complete.
2. Protoplast Stabilization
   • Maintaining osmotic balance is crucial to keep protoplasts intact. After enzymatic digestion, I will gently pellet the protoplasts under cold conditions (4 °C) and resuspend them in a similar osmotic buffer (often containing 0.5 M sucrose, 0.02 M maleate, and 0.02 M MgCl₂) to prevent lysis.
   • Protoplasts can then be stored briefly on ice, or in some cases at −80 °C with cryoprotectants for future use, although fresh protoplasts typically yield higher transformation efficiencies.

Step 3: DNA Preparation and in vivo Methylation (Optional)