Bacterial Persistance. Growth, Persistence and Resistance

Activity of the toxin-antitoxin system and hypothesis on the mechanism of regulation

Growth, Persistence and Resistance

Bacterial cells have the ability to regulate their growth cycles in response to changing environmental conditions. There are several mechanisms involved which include: a viable but not culturable state (VBNC), persistence which is referring to antibiotic resistance (phenotypic variants, non-heritable) by way of toxin-antitoxin pathways, and contact-dependent growth inhibition. These mechanisms are discussed below.

Bacterial Persistence

For the most part, DNA carries phenotypic (organisms outward appearance) traits over time. Phenotypic traits is referring to the genes that are expressed in that organism. There are many genes that are not expressed but still present. Organisms also have phenotypic variations that can persist for more than one generation without any direct change to the organisms DNA. This means that this phenotypic variation is not encoded in DNA but the microorganism has memory of past environments. This ability allows bacteria to influence future generations. So, genetically identical bacterial populations can respond in different ways to say antibiotic treatment. Persistence is still not well understood and there are a number of different mechanisms involved that have only been partially elucidated.

More recently, studies have been able to elucidate a persistence pathway. Keep in mind that persistent bacteria which are a subpopulation of a particular bacterial species, are tolerant to different types of antibiotics. Persistent bacteria have the same genome as their non-persistent brothers but they have entered into a dormant state which provides resistance to certain antibiotics.

A persistence locus has been identified in E. coli. The DNA locus contains a toxin-antitoxin (TA) coding region. Some of the TA nucleotide sequences code for a Long Form Filament (Lon) protease (enzyme that cleaves proteins) and an mRNA endonuclease (enzyme that cleaves RNA, mRNases). Anti-toxins happen to control the activity of mRNases which happen to be Lon substrates. It has been observed that removal of Lon generates reduced persistence and if you have overproduction of Lon you get increased levels of persistent bacteria. This is not the case if you are missing the mRNases. The mRNases encoded by the TA locus are activated in a small sub-population of growing bacteria by Lon-mediated degradation of the antitoxins. It has also been observed that activation of mRNases inhibits global cellular translation which in turn induces dormancy and persistence. A number of pathogenic bacteria that are known to enter dormant states tend to have many TA genes.

Toxin-antitoxin (TA) genes have been proposed to function as regulators of cell growth in response to environmental stress. Genes in this coding region are activated during starvation and other conditions that inhibit transcription and translation. The toxins encoded by the TA genes in bacteria are mRNA interferases (nucleases that cleave mRNA). mRNA interferases inhibit protein synthesis and rapidly arrest bacterial growth. It has been demonstrated that these mRNA interferases bring about a bacteriostatic state that can be easily reversed with antitoxin expression also encoded on the TA genes. So, environmental stress causes toxin activation which rapidly shuts down protein synthesis until better conditions return. Since TA genes are de-repressed by anti-toxin degradation, the bacterium has to produce more antitoxin to counteract toxin activity after the cell has adapted to the stress. Recovery from mRNA interferase activation is also facilitated by tmRNA quality control system, which recycles ribosomes stalled during translation of toxin-cleaved mRNAs.

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Persistence is a mechanism for growth control by phenotypic variation in bacteria. Recall that persistence is not a genetically heritable trait. Antibiotic resistance due to persistence is an entirely different mechanism from genetically inherited forms. Persistence mechanisms have been difficult to study because they are non-heritable and occur in low frequencies. One class of persister cells referred to as Type I have been studied. The generation of these persisters is associated with the TA system and expression of HipA-HipB genes. It was observed that a mutation in hipA (7) gene increases the frequency of persisters by a thousand fold. These persister cells are non-dividing, dormant cells that appear spontaneously in a population of bacteria. Another persister cell type referred to as type II has been observed. These cells behave differently from type Is. Type II cells continue to grow slowly in the presence of ampicillin for a few generations unlike type I. It is suggested that type II persister’s state may be weakly heritable. If that’s the case then this could be mediated by DNA methylation, which is heritable, but can be lost within a couple of bacterial generations.

These mechanisms are not well understood at this time and it is not known whether TA activation is a result or the cause of persistence. Some researchers suggest that persistence may be the result of cellular aging. Others suggest that persisters may be controlled by a double-negative bistable switch in which an increase in the ratio of toxin to anti-toxin could occur in a cell due to stochastically higher levels of degradation of anti-toxin, leading to a decrease in translation in that cell. That would suggest a further decrease in translation due to the higher stability of toxin compared to antitoxin.

Epigenetic phenomena have been identified, including the VBNC and persistence states, by which bacteria create phenotypic diversity. As non-heritable growth regulatory mechanisms, these are poorly understood at this time. Nevertheless, bacteria have the ability to regulate their own growth in response to changes in environmental conditions. Epigenetics involves chemical modification of specific genes or gene-associated proteins that alter how a gene is expressed and used by cells. It often involves interactions between genes and gene products that gives rise to an organisms phenotype.