Ananya Bansal, Dr. D. Y. Patil Biotechnology and Bioinformatics institute
Introduction:
Lactobacilli are gram-positive, rod-shaped bacteria commonly found in humans and various animals. They produce lactic acid as their main metabolic end-product. Their ubiquitous nature is from them possessing stress-tolerant phenotypic traits which allow them to colonize a wide range of environments that comprise varying physiological parameters. In addition to their popularity as probiotics in the diet, people also use certain strains of Lactobacilli as anti-inflammatory agents, fermentation agents, biotherapeutic agents (vaccine vectors), and more. Despite their ubiquity, limitations for engineered Lactobacillus exist. Unknown aspects of the knowledge about genetic parts and certain biochemical pathways are one such limitation.
The authors of this study reported their scientific discovery related to a type of bacteria called Lactiplantibacillus plantarum. The researchers found two important parts in the genetic makeup of these bacteria that allow them to express genes at high levels. Interestingly, these genes can pass on to future generations without requiring antibiotics, bacteriocins (substances that kill bacteria), or genetic manipulations.
- The first part they discovered is a special switch, called a promoter, which comes from a different bacterium called Salmonella typhimurium. This switch can turn on genes in Lactiplantibacillus plantarum more effectively than any other previously known switches, resulting in up to five times higher gene expression.
- The second part they found is a group of toxin-antitoxin systems, which act like a self-contained mechanism for keeping the genes inside the bacteria.
These systems help the bacteria retain important genes without the need for complicated genetic modifications. This ease of modification allows scientists to adapt these bacteria for diverse healthcare applications.
The PtlpA promoter from Salmonella typhimurium:
The authors mention of important characteristics of the promoter called “PtlpA” which made it a suitable option for driving transcription. They discovered that this particular promoter doesn’t contain certain building blocks called cytosine (C) bases, which are usually present in other promoters of this bacteria. They also noticed that the spacer region between two important regions of the promoter doesn’t have adenine (A) bases. It’s worth mentioning that both cytosine and adenine bases can undergo a process called methylation in bacteria, which can influence gene regulation.
However, when the researchers analysed several different promoters, including the one they discovered, they couldn’t find a clear correlation between the strength of the promoter (how well it turns on genes) and the number of cytosine or adenine bases. This suggests that if methylation is indeed affecting promoter strength, there might be other factors involved that need to be understood. To explore this further, the researchers looked for similar DNA sequences to the discovered promoter in the L. plantarum genome. They found a similar sequence upstream of a gene that encodes a known protein. However, this sequence showed a few differences compared to the original promoter. When they tested this slightly different sequence as a promoter, they observed weak gene expression. This suggests that those differences in the sequence compared to the original promoter are crucial for achieving high-level gene expression.
These unique characteristics of the discovered promoter provide interesting clues for understanding the factors that affect promoter strength in Lactiplantibacillus plantarum. To gain a better understanding, further studies using mutant promoters and analyzing DNA methylation patterns will be necessary.
Using the Toxin/Antitoxin system to retain plasmids and transient GEMs:
TA systems work by producing both toxins and antitoxins. As long as the plasmid is present in the bacteria, enough antitoxin is produced to neutralize the toxin. However, if a daughter cell doesn’t inherit any plasmid copies during cell division, the antitoxin degrades rapidly, and the active toxin kills the cell. While TA systems have been previously studied for other purposes, their efficiency in retaining plasmids was not as good as other methods like antibiotics.
Recently, there has been renewed interest in using TA systems for therapeutic applications because of improved understanding and their ability to reduce the transfer of genes between bacteria. Some studies have shown promising results in using TA systems for engineering bacteria as live vaccines or drug delivery vehicles. In this study, the researchers selected and tested five different TA systems in lactobacilli.
They found that combining two of the best-performing systems, one from Lactiplantibacillus plantarum itself and another from a different bacterium, resulted in even better plasmid retention. The bacteria retained the plasmids containing the desired genes for a longer time without the need for antibiotics. The researchers also observed that the growth rate and expression levels of the desired genes were minimally affected by the presence of the TA systems. However, the bacteria with the best-performing TA system showed slightly lower expression levels of the desired genes compared to other TA systems.
They also introduced a new metric called G50, which represents the number of generations it takes for half of the bacterial population to lose the plasmid. By using different TA systems, they can tune the retention lifetime of the genetically modified lactobacilli. This concept allows the generation of transient genetically modified organisms (GEMs) that can be used for specific applications.
Also read: National Scientific Writing Contest- BIOXONE BIOSCIENCES
Reference:
Dey, S., Blanch‐Asensio, M., Balaji Kuttae, S., & Sankaran, S. (2023). Novel genetic modules encoding high‐level antibiotic‐free protein expression in probiotic lactobacilli. Microbial Biotechnology, 16(6), 1264–1276. https://doi.org/10.1111/1751-7915.14228
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