Several well-recognized puzzles in microbiology have remained unsolved for decades. These include latent bacterial infections, unculturable microorganisms, persister cells and biofilm multidrug tolerance. Accumulating evidence suggests that these seemingly disparate phenomena result from the ability of bacteria to enter into a dormant (non-dividing) state. The molecular mechanisms that underlie the formation of dormant persister cells are now being unravelled and are the focus of this Review.

http://www.medicinabiomolecular.com.br/biblioteca/pdfs/Cancer/ca-3102.pdf

Persister cells, dormancy and infectious disease

Although toxin-antitoxin gene cassettes were first found in plasmids, recent database mining has shown that these loci are abundant in free-living prokaryotes, including many pathogenic bacteria. For example, Mycobacterium tuberculosis has 38 chromosomal toxin-antitoxin loci, including 3 relBE and 9 mazEF loci. RelE and MazF are toxins that cleave mRNA in response to nutritional stress. RelE cleaves mRNAs that are positioned at the ribosomal A-site, between the second and third nucleotides of the A-site codon. It has been proposed that toxin-antitoxin loci function in bacterial programmed cell death, but evidence now indicates that these loci provide a control mechanism that helps free-living prokaryotes cope with nutritional stress.

Prokaryotic toxin-antitoxin stress response loci.

The biological cost of antibiotic resistance

Prokaryotic chromosomes code for toxin-antitoxin (TA) loci, often in multiple copies. In E.coli, experimental evidence indicates that TA loci are stress-response elements that help cells survive unfavorable growth conditions. The first gene in a TA operon codes for an antitoxin that combines with and neutralizes a regulatory 'toxin', encoded by the second gene. RelE and MazF toxins are regulators of translation that cleave mRNA and function, in interplay with tmRNA, in quality control of gene expression. Here, we present the results from an exhaustive search for TA loci in 126 completely sequenced prokaryotic genomes (16 archaea and 110 bacteria). We identified 671 TA loci belonging to the seven known TA gene families. Surprisingly, obligate intracellular organisms were devoid of TA loci, whereas free-living slowly growing prokaryotes had particularly many (38 in Mycobacterium tuberculosis and 43 in Nitrosomonas europaea). In many cases, TA loci were clustered and closely linked to mobile genetic elements. In the most extreme of these cases, all 13 TA loci of Vibrio cholerae were bona fide integron elements located in the V.cholerae mega-integron. These observations strongly suggest that TA loci are mobile cassettes that move frequently within and between chromosomes and also lend support to the hypothesis that TA loci function as stress-response elements beneficial to free-living prokaryotes.

Toxin–antitoxin loci are highly abundant in free-living but lost from host-associated prokaryotes

Bacterial populations produce persisters, cells that neither grow nor die in the presence of bactericidal agents, and thus exhibit multidrug tolerance (MDT). The mechanisms of MDT and the nature of persisters have remained elusive. Our previous research has shown that persisters are largely responsible for the recalcitrance of biofilm infections. A general method for isolating persisters was developed, based on lysis of regular cells by ampicillin. A gene expression profile of persisters contained toxin-antitoxin (TA) modules and other genes that can block important cellular functions such as translation. Bactericidal antibiotics kill cells by corrupting the target function (for example, aminoglycosides interrupt translation, producing toxic peptides). We reasoned that inhibition of translation will lead to a shutdown of cellular functions, preventing antibiotics from corrupting their targets, giving rise to MDT persister cells. Overproduction of the RelE toxin, an inhibitor of translation, caused a sharp increase in persisters. Functional expression of a putative HipA toxin also increased persisters, while deletion of the hipBA module caused a sharp decrease in persisters in both stationary and biofilm populations. HipA is thus the first validated persister-MDT gene. We suggest that random fluctuation in the levels of MDT proteins leads to the formation of rare persister cells. The function of these specialized dormant cells is to ensure the survival of the population in the presence of lethal factors.

Specialized persister cells and the mechanism of multidrug tolerance in Escherichia coli.

The stability locus parB+ of plasmid R1 has been found to specify a unique type of plasmid maintenance function. Two genes, hok (host killing) and sok (suppressor of killing), are required for the stabilizing activity. The hok gene encodes a highly toxic gene product, whose overexpression causes a rapid killing and a concomitant dramatic change in morphology of the host cell. The other gene, sok, was found to encode a product that counteracts the hok gene-mediated killing. The parB+ region was inserted in a plasmid with a temperature-sensitive replication system. At nonpermissive temperature, the parB+ plasmid was maintained in the population for a significantly longer period than the corresponding parB- plasmid. Coupled to this extended maintenance, a large fraction of the population was shown to be nonviable plasmid-free cells with the characteristic hok-induced change in morphology. Based on these findings, we propose that the parB+ locus mediates plasmid stability by killing cells that have lost the parB+ plasmid during the preceding cell division, thereby ensuring that a growing bacterial culture predominantly consists of plasmid-containing cells.

https://www.pnas.org/content/pnas/83/10/3116.full.pdf

Unique type of plasmid maintenance function: postsegregational killing of plasmid-free cells.

The stability locus parB+ of plasmid Rl has been found to specify a unique type of plasmid maintenance function. Two genes, hok (host killing) and sok (suppressor of killing), are required for the stabilizing activity. The hok gene encodes a highly toxic gene product, whose overexpression causes a rapid killing and a concomitant dramatic change in morphology of the host cell. The other gene, sok, was found to encode a product that counteracts the hok gene-mediated killing. The parB+ region was inserted in a plasmid with a temperature-sensitive replication system. At nonpermissive temperature, the parB+ plasmid was maintained in the popu- lation for a significantly longer period than the corresponding parB- plasmid. Coupled to this extended maintenance, a large fraction of the population was shown to be nonviable plasmid- free cells with the characteristic hok-induced change in mor- phology. Based on these findings, we propose that the parB+ locus mediates plasmid stability by killing cells that have lost the parB+ plasmid during the preceding cell division, thereby ensuring that a growing bacterial culture predominantly con- sists of plasmid-containing cells.

Unique type of plasmid maintenance function: Postsegregational killing of plasmid-free cells (plasmid stability/parB+ of plasmid Ri/regulated killing/phage X promoter PR)

Plasmid R1 in Escherichia coli carries two loci which independently contribute to the stable maintenance of the plasmid. A genetic analysis of one of these, parB+, was carried out, and it was shown that the minimal region exerting stabilizing activity comprises at most 580 bp. The nucleotide sequence of the parB+ locus was determined, and indicated the presence of two genes, of which one probably codes for a 52 amino acid polypeptide, whereas the other gene product may be an untranslated RNA. These suggestions, based on the nucleotide sequence information, were supported by gene expression studies employing lac fusions. An incompatibility phenotype connected to parB+ was localized to that par of the 580 bp parB+ region which seems to encode the untranslated RNA.

Genetic analysis of the parB+ locus of plasmid R1.

The stable maintenance of a replicon in a population of growing cells is ensured by providing the replicon with a sequence which encodes a product capable of killing the cell harbouring the replicon or the progeny of the cell (or encodes a precursor for the product) and a sequence encoding an antagonist for the killing product (or a precursor for the antagonist). The antagonist is one which suppresses the killing product (or a precursor for the killing product) in cells harbouring the replicon, whereas the antagonist activity decays when the replicon is lost from the cell so that the antagonist (or its precursor) is no longer continuously expressed. This means that the killing product (or its precursor) present in the now replicon-free cell is no longer suppressed by the antagonist, resulting in cell death. Cells containing the thus stabilized replicon may be grown on a large scale without any significant loss of the replicon from the cell population even when no selection pressure is applied. Cells containing the thus stabilized replicon may be grown on a large scale without any significant loss of the replicon from the cell population even when no selection pressure is applied.

Poul B. Rasmussen

Papers

Unique type of plasmid maintenance function: postsegregational killing of plasmid-free cells.

Unique type of plasmid maintenance function: Postsegregational killing of plasmid-free cells (plasmid stability/parB+ of plasmid Ri/regulated killing/phage X promoter PR)

Genetic analysis of the parB+ locus of plasmid R1.

Stabilization of unstably inherited replicons