Horizontal gene transfer is the transmission of genetic information between different cells, and can even occur between cells of different species. It plays an important role in the adaptability of bacteria to changes in their environment. Three main modes of horizontal gene transfer are recognized today: transduction, conjugation and transformation. Transduction involves the transmission of genetic information through bacteriophages. Transduction is in large limited to
1 Introduction
4
genetic transfer between related bacteria, as the cells involved must possess the specific receptors needed for interaction with the phage (Soucy et al., 2015). Conjugation involves gene transfer through cell-to-cell contact, in which a mobile genetic element such as a plasmid or transposon is transferred from a donor cell to an acceptor cell via a conjugation pilus (Soucy et al., 2015). Transformation involves the uptake of free DNA from the surrounding environment, which can then be incorporated into the genome of the recipient through homologous recombination (Johnsborg et al., 2007).
Among streptococci, natural transformation has been most comprehensively studied in S.
pneumoniae, but evidence suggest that this ability is prevalent among all the species in the genus (Håvarstein, 2010). The bacteria can undergo natural transformation when they enter a physiological state termed competence. Competence is transiently induced under certain environmental conditions by a quorum-sensing mechanism. When cells enter the competent state, they activate expression of genes involved in the active uptake of free extracellular DNA and its integration into the host genome through homologous recombination, as well as genes involved in a predatory mechanism called fratricide (see section 1.2.2) (Johnsborg et al., 2007, Straume et al., 2015).
1.2.1 Regulation of the competent state
In S. pneumoniae, and other streptococci of the mitis and anginous phylogenetic groups, the competent state is regulated by the three-component ComCDE system (Figure 1.2) (Straume et al., 2015). The ComCDE system consists of the competence stimulating peptide (CSP), a CSP specific receptor, ComD, and the transcriptional regulator ComE. In non-competent pneumococci, ComCDE are constitutively expressed at a low level (Straume et al., 2015). As ComC is translocated across the membrane through the ABC transporter ComAB, the N-terminal leader sequence is cleaved off, and the matured CSP is released from the cell.
Extracellular CSP binds to its cognate receptor, the transmembrane histidine kinase ComD.
This results in the autophosphorylation of ComD, followed by the transfer of the phosphate group from ComD to the cytoplasmic protein ComE. Phosphorylated ComE binds to two direct repeats motifs found in the promoters of so-called early competence genes resulting in transcriptional activation. When the concentration of CSP reaches a critical level, increased transcription of the early competence genes (comABCDE) are activated, which generates a positive feedback loop resulting in the induction of the competent state. The factors allowing CSP to accumulate to a concentration required to trigger the autocatalytic loop is not completely
1 Introduction
5
clear, but it is known that different stresses and antibiotics increasing the levels or stability of CSP result in competence induction. (Straume et al., 2015). In addition to the comABCDE genes, the early competence genes include a gene encoding ComX. ComX is an alternative σ-factor, and promotes expression of the late competence genes. The ~80 late competence genes encode proteins involved in the acquisition of free extracellular DNA, and its incorporation into the host genome through homologous recombination. The late genes also encode Drpa, which mediates the termination of the competent state by binding phosphorylated ComE, effectively preventing it from activating further transcription of the early genes (Straume et al., 2015). In addition, the late genes encode CbpD, a murein hydrolase involved in the fratricide mechanism (Claverys et al., 2007).
Figure 1.2 Competence regulation in S. pneumoniae. In S. pneumoniae, competence for natural genetic transformation is regulated by the ComCDE system. Expression of the genes involved in the active uptake of free, extracellular DNA, its integration into the host genome through homologous recombination, and the fratricide mechanism, is induced by a quorum sensing mechanism, when the extracellular concentration of the competence stimulating peptide (CSP) reaches a threshold level. CSP binds its receptor, the transmembrane histidine kinase ComD, which results in the activation of ComD, which regulates transcription of the abovementioned genes. See text for details. Figure from (Berg et al., 2012).
1.2.1 Competence-induced fratricide
Fratricide is a predatory mechanism first described to be executed by S. pneumoniae during competence (Guiral et al., 2005). It involves a murein hydrolase called CbpD (choline binding protein D) that is secreted by competent pneumococci. CbpD binds to choline decorated
1 Introduction
6
teichoic acids in the cell wall of non-competent pneumococci or closely related species where it makes cuts in the peptidoglycan layer leading to cell lysis. This results in the release of nutrients and DNA that become available to the competent cells (Berg et al., 2012, Johnsborg et al., 2008). The competent pneumococci protect themselves from CbpD by expressing the early competence gene comM (Håvarstein et al., 2006). The fratricide mechanism is not unique to pneumococci, since the presence of competence induced genes encoding fratricins are found within the genomes of all streptococci known to be competent for natural transformation. This suggests that the fratricide mechanism, although not essential for DNA up-take and recombination, is an important aspect of natural transformation in streptococci (Johnsborg et al., 2008, Berg et al., 2012). It is believed to be a mechanism providing homologous DNA to the competent cells, since up-take of foreign DNA can be hazardous to the cells. (Eldholm et al., 2010). Competent pneumococci indiscriminately take up any free DNA from the surrounding environment, but a selective attack on closely related species increases the probability that the DNA taken up by the cells will be homologous (Berg et al., 2012). Bearing in mind that competent pneumococci have been shown to take up and recombine DNA stretches up to 100 kb long from lysed target cells (Cowley et al., 2018), it emphasizes the importance of this DNA being homologous to avoid killing the host. The ability to undergo natural transformation while selectively targeting closely related species through the fratricide mechanism, has been instrumental to the genomic plasticity and adaptability of S. pneumoniae (Straume et al., 2015). A good example is how S. pneumoniae acquire penicillin resistance.
Specific enzymes, called penicillin binding proteins (PBPs), taking part in constructing the bacterial cell wall are usually inhibited by penicillin (see section 1.6). However, the pneumococcus becomes resistant to penicillin by expressing a set of mutated enzymes that are not inhibited by penicillin. Genome sequencing has revealed that S. pneumoniae can acquire these penicillin resistance genes from close relatives such as Streptococcus oralis and Streptococcus mitis through horizontal gene transfer and homologous recombination (Jensen et al., 2015). Natural competence is a major driving force in spreading these resistance genes among pneumococcal strains when put under a penicillin selection pressure in the clinics.
1.3 Pneumococcal cell wall synthesis and cell division