| dc.description.abstract | In recent years, reducing the environmental impact of various industries has become increasingly important. The genetic engineering* of bacteria can help to meet this need. Engineered bacteria have many sustainable applications, including drug manufacturing, producing biofuels, and bioremediation (e.g., cleanup of oil spills). To date, researchers have most intensively studied one bacterial species, Escherichia coli (E. coli), because this bacterium is easily cultured and readily accessible. However, E. coli does not always function optimally in the harsh environments of bioreactors and other future industrial applications. Other bacteria may be much more suitable for this. One example is Pseudomonas aeruginosa, a versatile bacterium with a rich and adaptive metabolism that is much more stress-resistant than E. coli. Yet, despite its potential, the lack of genetic engineering tools makes it challenging to work with this bacterium. This report discusses a new type of tool to engineer P. aeruginosa: integrases.
This new tool is borrowed from bacteria’s biggest enemies: bacteriophages. These bacterial eaters are viruses that specifically infect bacteria to replicate themselves. This often leads to the rupture and death of the bacterium. Sometimes, however, it may be advantageous for a bacteriophage to ‘hide out’ inside their host, for example when there are not many other bacteria around to infect. To do this, the bacteriophage uses an integrase to integrate its own DNA into that of its host. This integration happens at a specific location in the genome and in a very precise, controlled manner, without the loss of DNA.
If bacteriophages can use integrases to ‘cut-and-paste’ their DNA into that of the bacterium, these integrases could also be used as engineering tools. Scientists would of course not use integrases to cut-and-paste virus-DNA, but to introduce new genes into the bacteria. This genetic engineering will let the bacteria produce industrially relevant products, such as vaccine components or enzymes that play a role in plastic degradation.
Since there is currently only one P. aeruginosa-associated integrase available, scientists are looking for more integrases in nature. A recent study found several integrases by analyzing the largely undescribed regions of bacteriophage DNA, known as ‘viral dark matter’. The next step has been to establish a suitable system for the high-throughput testing of these candidate integrases. Two already described integrases, one from P. aeruginosa and one from a different bacterium, were used as controls. If the system works for these two controls, it is good enough to be used for the testing of the new integrases.
The integrase test systems discussed in this work were based on a marker gene in the genome. This gene encodes a fluorescent protein. When the integration is successful, the fluorescence from this protein can be measured. The first systems were designed to integrate a ‘gene activator’ (promoter) in front of the gene. These were fully developed and tested, but no integration occurred. Next, two other systems were designed. In these systems, the marker gene would already be present in the genome, but it would be inverted, like a word in which the letters are upside down and in the reverse order. The integrase causes the gene to ‘flip’ and makes it readable again.
When this system will finally be established, it will be a useful method to test the efficiency of new integrases for P. aeruginosa and other promising bacterial ‘work-horses’. This would expand the toolbox for the genetic engineering of these bacteria and prime them as our partners in human health and the sustainable care of the planet. | |
