Cleavage of the antitoxin of the parD toxin-antitoxin system is determined by the ClpAP protease and is modulated by the relative ratio of the toxin and the antitoxin.

Differential stability of toxins and antitoxins is the key for the conditional activation and function of Toxin-Antitoxin systems. Here we report the evaluation of the action of cell proteases Lon, ClpAP, ClpXP and ClpYQ on the Kis antitoxin and the Kid toxin of the parD TA system of plasmid R1. In vitro analysis shows that Kis antitoxin, but not the Kid toxin, is cleaved specifically by the ClpAP protease. The Kid toxin is not cleaved either by this protease or by any of the others cell proteases tested but in complex with the Kis antitoxin protects the cleavage of this protein in a way that is dependent on the toxin-antitoxin ratio. We further show that this protection is correlated with the inability of the ClpA chaperone to access the Kis antitoxin when in complex with Kid toxin. The stability of the antitoxin greatly increases in vivo in a clpP- background and plasmid maintenance mediated by the parD system, which is dependent on the differential decay of the antitoxin, is reduced to the levels observed in the absence of a functional toxin. The functional implications of these data are further discussed within the frame of the regulation of the parD system and of the available information on the nature of the toxin-antitoxin complexes formed at different toxin-antitoxin ratios.

Fitness of the pMV158 replicon in Streptococcus pneumoniae.

Promiscuous, rolling-circle replication plasmid pMV158 determines tetracycline resistance to its host and can be mobilized by conjugation. Plasmid pLS1 is a deletion derivative of pMV158 that has lost its conjugative mobilization ability. Both plasmids replicate efficiently and are stably inherited in Streptococcus pneumoniae. We have analyzed the effect of pMV158 and pLS1 carriage on the bacterial growth rate. Whereas the parental plasmid does not significantly modify the cell doubling time, pLS1 slows down the growth of the bacterial host by 8-9%. The bases of the differential burden caused by pMV158 and pLS1 carriage are not yet understood. The negligible cost of the pMV158 parental natural plasmid on the host might explain the prevalence of small, multicopy, rolling-circle replication plasmids, even though they lack any selectable trait.

Kis antitoxin couples plasmid R1 replication and parD (kis,kid) maintenance modules.

The coupling between the replication and parD (kis, kid) maintenance modules of R1 has been revisited here by the isolation of a significant collection of conditional replication mutants in the pKN1562 mini-R1 plasmid, and in its derivative, pJLV01, specifically affected in the RNase activity of the Kid toxin. This new analysis aims to identify key factors in this coupling. For this purpose we have quantified and characterized the restriction introduced by parD to isolate conditional replication mutants of this plasmid, a signature of the modular coupling. This restriction depends on the RNase activity of the Kid toxin and it is relieved by either over-expression of the Kis antitoxin or by preventing its degradation by Lon and ClpAP proteases. Based on these data and on the correlation between copy numbers and parD transcriptional levels obtained in the different mutants, it is proposed that a reduction of Kis antitoxin levels in response to inefficient plasmid replication is the key factor for coupling plasmid replication and parD modules.

Anti-staphylococcal hydrogels based on bacterial cellulose and the antimicrobial biopolyester poly(3-hydroxy-acetylthioalkanoate-co-3-hydroxyalkanoate).

Polymeric hydrogels from bacterial cellulose (BC) have been widely used for the development of wound dressings due to its water holding capacity, its high tensile strength and flexibility, its permeability to gases and liquids, but lacks antibacterial activity. In this work, we have developed novel antimicrobial hydrogels composed of BC and the antimicrobial poly(3-hydroxy-acetylthioalkanoate-co-3-hydroxyalkanoate) (PHACOS). Hydrogels based on different PHACOS contents (20 and 50 wt%) were generated and analysed through different techniques (IR, DSC, TGA, rheology, SEM and EDX) and their bactericidal activity was studied against Staphylococcus aureus. PHACOS20 (BC 80%-PHACOS 20%) hydrogel shows mechanical and thermal properties in the range of human skin and anti-staphylococcal activity (kills 1.8 logs) demonstrating a huge potential for wound healing applications. Furthermore, the cytotoxicity assay using fibroblast cells showed that it keeps cell viability over 85% in all the cases after seven days.

Conditional Activation of Toxin-Antitoxin Systems: Postsegregational Killing and Beyond.

Toxin-antitoxin (TA) systems are small genetic modules formed by a stable toxin and an unstable antitoxin that are widely present in plasmids and in chromosomes of Bacteria and Archaea. Toxins can interfere with cell growth or viability, targeting a variety of key processes. Antitoxin inhibits expression of the toxin, interacts with it, and neutralizes its effect. In a plasmid context, toxins are kept silent by the continuous synthesis of the unstable antitoxins; in plasmid-free cells (segregants), toxins can be activated owing to the faster decay of the antitoxin, and this results in the elimination of these cells from the population (postsegregational killing [PSK]) and in an increase of plasmid-containing cells in a growing culture. Chromosomal TA systems can also be activated in particular circumstances, and the interference with cell growth and viability that ensues contributes in different ways to the physiology of the cell. In this article, we review the conditional activation of TAs in selected plasmidic and chromosomal TA pairs and the implications of this activation. On the whole, the analysis underscores TA interactions involved in PSK and points to the effective contribution of TA systems to the physiology of the cell.