Bt Research 2025, Vol.16, No.5, 224-233 http://microbescipublisher.com/index.php/bt 227 balance resource allocation by regulating metabolic pathways, thereby changing the expression levels of certain chromosomal genes. 3.3 The role of plasmids in genetic stability and functional regulation The plasmid itself has a mechanism to maintain its own stability and regulate host function. In order to effectively pass to offspring during cell division, many Bt plasmids have evolved into special distribution systems. Large plasmids often carry distribution devices similar to tubulin TubZ and binding protein TubR, which can actively push the plasmid to two cell poles that are about to divide, thereby increasing the probability that the plasmid will be inherited by the two daughter cells during division. Experiments have shown that destroying the tubZ gene on the Bt plasmid results in a significant increase in the plasmid loss rate, indicating that such systems are crucial for stable passage of plasmids (Montabana and Agard, 2014). Plasmids often carry the toxin-antitoxin (TA) system and other descendant killing mechanisms. If the daughter cells do not inherit the plasmid, after the antitoxin degrades, the stable toxin encoded by the plasmid will accumulate in the cell and cause cell death, thus eliminating the "lost plasmid" offspring from the population. The type III toxin-antioxin system tenpIN, a small RNA encoding protein toxins and anti-toxicity, was found on some Bt plasmids, and was reported to promote plasmid retention during Bt spore formation (Short et al., 2015). These mechanisms ensure the genetic stability of the plasmid within the strain. On the other hand, plasmids can also improve their own and host adaptability by regulating host function. 4 Molecular Mechanisms of Plasmid Loss 4.1 Inducement of plasmid loss (culture conditions, metabolic pressure, etc). Loss of Bt plasmids are often closely related to specific environmental triggers and culture conditions. High temperature is a common factor in inducing plasmid instability. Early research found that culturing Bt strains at 42 °C for several generations can lead to high frequency loss of large plasmids they carry. High temperatures may interfere with the function of the plasmid replicase or distribution system, making it impossible for plasmids to be effectively delivered to offspring. Medium composition and metabolic pressure will also affect plasmid stability. In nutrient-rich media, strains may gradually tend to discard plasmids to save metabolic costs because virulence genes on plasmids are not essential for growth, which is often observed in continuous passage cultures without selective pressure (Driss et al., 2011). In addition, some chemical reagents can induce plasmid loss, such as ethidium bromide, propidium dynamodine, etc., interfere with replication by inserting plasmid DNA, and are widely used as a method for "curing" plasmids in laboratory. Ultraviolet irradiation is also one of the causes of plasmid instability. Strong UV may damage plasmid DNA, which will lead to failure to repair and be lost in subsequent replication. 4.2 Defects in plasmid replication and allocation mechanism Defects in the plasmid's own replication and allocation mechanism are one of the direct causes of plasmid loss. If the plasmid key maintenance gene is mutated or dysfunctional, the loss rate will be greatly increased. Mutations in the origin of plasmid replication (ori) will reduce the replication efficiency of plasmids. Secondly, defects in plasmid distribution system components can also cause loss. Large plasmids rely on systems such as ParA/ParB or TubZ/TubR to localize plasmids at the fission poles. If the ParA or TubZ protein mutations are inactive, the plasmid will randomly be biased on one side during cell division, resulting in the failure of the plasmid to obtain in the other daughter cell (Ni et al., 2010; Aylett and Löwe, 2012). In the long run, plasmid-free system will increase and replace plasmid-free system, manifesting as population plasmid loss. In addition, plasmid incompatibility in multiplasmid coexisting strains can also cause loss. Some plasmids share similar replication regulation mechanisms, and in the same cell they compete with each other for replication resources, often failing to coexist stably for a long time. Therefore, when the Bt strain carries multiple plasmids of the same incompatible group, one or more plasmids may be eliminated as generations are replicated. Large-scale recombination or deletion of plasmid DNA can also lead to functional loss. For example, large fragment deletion of plasmids (including the origin of replication or essential genes) may not be able to replicate passage even if they are physically present in the cell, which is equivalent to functional loss (Tang et al., 2007).
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