Background

Horizontal Gene Transfer

     Horizontal gene transfer (HGT) is the process by which organisms transfer genetic information without reproducing, hence being horizontal across generations. It can occur through three methods: transformation, or the uptake of environmental DNA, transduction, or the transfer of DNA through a bacteriophage, and conjugation, where cells exchange DNA through physical contact. Our model organism, Acinetobacter baylyi, performs HGT through natural competence, or or natural DNA transformation. 

Studying HGT

     Currently traces of past HGT can be quantified using computation-heavy techniques, such as comparing the genomes of unrelated organisms in databases, and finding sequence similarities through data processing. Within the organism studied for HGT, one can then group these transferred genes based on their relation to the host. One can also use phylogenetic analysis, where homologous coding sequences have their phylogenies determined, and compared to their ancestor. Any conflicts can thus be reconciled with HGT as an explanation.

     Studying the process of horizontal gene transfer rather than its traces as with the last paragraph's methods is performed using the above experimental procedure. First, two strains of bacteria each carrying different antibiotic resistance genes are mixed on a plate. Following this, the bacteria are grown, and the resulting colonies are collected and resuspended in liquid culture. These cultures are then serially diluted onto plates containing either the individual antibiotics or both. By comparing the number of colonies grown on the “both” plate with the number on the individual plates, the rate of horizontal gene transfer can thus be determined.

      This method for the study of ongoing HGT, however, is still flawed. It lacks continuity due to ending the growth of the bacteria after picking it up for serial dilution and is not very accurate to nature due to not including any motion that bacteria may experience on its stationary plate. 

Importance

       HGT plays a notable role in evolution, allowing for unrelated species to exchange genetic information. While mostly occurring between bacteria, this also occurs in eukaryotes. The presence of HGT effectively increases the gene pool of organisms capable of it, allowing them to be more resilient in the face of environmental changes or threats with their access to additional potentially helpful genes. 

       The most significant  evolutionary consequence of HGT to human society is its ability to transfer antibiotic resistance between different bacteria. Antibiotics are ubiquitous in today's world, being found in pharmacies, hospitals, and especially in industrial agriculture. All of these sources provide plenty of avenues for any bacteria that resist antibiotics to transfer this resistance gene to other bacteria through HGT. This poses a significant public health risk, as the rate at which new antibiotics are created is exceeded by the rate at which bacteria can evolve, potentially creating diseases faster than they can be cured. As of 2019, antibiotic resistant bacteria have caused at least 1.27 million deaths that year alone, with the death toll likely to be higher as of this year. 

Acinetobacter baylyi

The Acinetobacter genus is a group of gram-negative coccobacilli that are aerobic, saprophytic, catalase-positive, oxidase-negative, and use a variety of energy and carbon sources. Acinetobacter baylyi, the model organism this project uses to study HGT, is an effective model organism for this principle because it has a small genome that is easy to study and quantify mutations in, it exhibits efficient natural transformation, and because its genome is easily modifiable due to its ADP1 gene. As such, A. baylyi can be modified to suit different experimental conditions, such as having various forms of antibiotic resistance to increase selectivity in an antibiotic-laced growth medium like in our project. 

Bioreactors and Chemostats

Bioreactors are systems that maintain the proper environment for cells or enzymatic reactions to occur to yield either cells or cellular products. Since they can run continuously, they can provide a continually renewing supply of nutrients and media, unlike traditional plating techniques that have a finite supply of both. This benefits the study of HGT, since it provides a continuous supply of nutrients and a predictable, controlled environment during experimentation, allowing for the long term active transfer of genes between bacteria as a population progresses.

Composition and Types

      Physically, such a bioreactor system is typically composed of a nutrient reservoir attached to a stirred-tank reactor containing growth media and a system to maintain reactor volume. Experimental and process goals can then be met through variations of the following aspects of the continuous culture design: Mixing time, Power Input, the Volumetric Mass Transfer Coefficient (in cases media is replenished), the amount of inoculant cells, and the concentration of the limiting factor. There are four main types of continuous culture:

• 1) Batch, where a culture is supplied with nutrients and stabilized without any addition of nutrients during the process. 

• 2) Fed Batch: A batch is started and nutrient-rich media is added, changing volume over time. 

• 3) Perfusion: A batch starts and the old media is recycled with new media, while cells are retained and kept in the batch. 

• 4) Continuous Fermentation: Similar to Perfusion, however cells are not retained and are output of the system

Continuous fermentation closely aligns with the more conventional continuous culture model used widely by microbiologists and bio-manufacturers, the Chemostat. The process continuously dispenses and replenishes media containing the cultured cells while also inputting necessary power and reagents, resulting in a steady growth rate of the cells. By employing Michaelis-Menten differential equations with substrate and metabolized products being the balanced variables, the Chemostat effectively allows for the growth rate of cells and the expense of nutrients to be calculated and predicted according to the limitation of a particular variable of interest. As such, our bioreactor design is modeled on the Chemostat.

Written by Miguel De Guzman