What happened when Griffith injected mice with the pneumonia causing strain of bacteria that had been heat

1.1 The story of DNA

The “Griffith's Experiment,” conducted in 1928 by English bacteriologist Frederick Griffith described the conversion of a non-pathogenic pneumococcal bacteria to a virulent strain.17 In this experiment, Griffith mixed the living non-virulent bacteria with a heat inactivated virulent form. He subsequently infected mice with this mixture and much to his surprise, the mice developed pneumonia and died. Furthermore, he was able to isolate colonies of the virulent strain from these mice. Because the original virulent strain was heat inactivated, he concluded that the non-virulent strain had transformed into the virulent type. This phenomenon was observed for the first time but was confirmed a year later by Dawson and Sia who were also able to perform this transformation in vitro.18 The studies Dawson started were continued by James L. Alloway and he took the experiment one step further. He lysed the virulent strain of bacteria and filtered the intracellular substance to obtain a cell-free extract. He observed that even this extract transformed the non-virulent strain to a virulent one and he hypothesized that there was something in the cell-free extract that caused this conversion. He named this “something” the “transforming principle”.19 His studies after that focused on isolating that “something” from the cell-free extract. In 1933, he observed that it precipitates out of solution with addition of alcohol,20 but it wasn't until 1944 that Avery and McCarty were able to demonstrate that the transformation was caused by a substance known as the deoxyribonucleic acid (DNA).21 This was a time when common consensus among scientists, including Avery, was that genes are made up of proteins. The discovery of DNA caused a dramatic shift in the fundamental understanding of the molecular core of life and after that, DNA became a focus of intense examination and research.

B Methods of HGT: Transformation

Natural transformation occurs when competent recipient cells take up naked DNA from the environment (Lorenz and Wackernagel, 1994). The idea of a nonheritable exchange of genetic information was first documented in 1928 with work on the pathogen Streptococcus pneumoniae (Griffith, 1928). Griffith showed that virulence factors from a killed pathogenic strain could “transform” a nonvirulent strain to become virulent as well. Although it was unknown at the time of Griffith's experiment, DNA was determined to be the transforming principle responsible for the change from nonvirulent to virulent that Griffith had documented in his classic experiments (Avery et al., 1944).

Species such as S. pneumoniae are naturally competent; competence is mediated by cell cycle, quorum sensing via secretion of a competence stimulating peptide, and specific competence proteins (Whatmore et al., 1999). Not all bacteria are naturally competent, however, but can be forced to take up DNA using nonphysiological techniques. E. coli was the first bacterium to be forced to become competent. Forced competency was discovered stepwise; initially, helper phage-mediated transformation was used. After it was determined that calcium ions improved competency in this process, it was then established that extensive treatment with a calcium chloride solution eliminated the need for phage entirely. CaCl2-based chemical competency allowed for successful transformation using recipient and donor DNA from E. coli, Haemophilus influenza, S. pneumoniae, and Bacillus subtilis (Cohen et al., 1972; Hotchkiss and Gabor, 1970). Another breakthrough was the identification that multiple drug resistance in Shigella was found to be due to an episome, or plasmid-mediated transfer of antibiotic resistance, referred to as a resistance transfer factor (Watanabe, 1963). Due to their ease of manipulation and isolation, plasmids would become one of the most useful tools to molecular biologists. By 1989, transformation via electroporation of plasmid DNA into multiple Gram-negative species had been well established and enabled researchers to perform genetic manipulation of recipient cells with engineered plasmid DNA (Delorme, 1989). Transformation quickly became the standard for introducing DNA into microbial organisms and over 40 transformable bacterial species had been identified by 1994 (Lorenz and Wackernagel, 1994).

3.2.4 Griffith’s Decisive Experiment: The Discovery of Bacterial Transformation

Griffith noticed that while some serotypes of stable R variants of pneumococcus did not revert in the mouse into encapsulated S forms, revertants were detected when the R cells were coinoculated with heat-killed S bacteria. Expanding on these preliminary observations, he defined experimental conditions that maximized the capacity of heat-killed S cells to affect the transformation of R cells into virulent encapsulated S pneumococci. In these preparatory experiments Griffith identified combination of serotypes of the heat-killed S cells and living R bacteria that yielded significant R to S transformation in the mouse. Thus, for instance, R cells that were derived from Type SII cells were successfully transformed when they were coinjected with heat-killed SI bacteria. Griffith also defined optimal temperature, usually 60°C that effectively killed the S bacteria while allowing for significant transformation. He also found that transformation occurred only when the inoculum size of the heat-killed S cells was much larger than that of the living R cells. Importantly, using heat-killed S cells and alive R cells of two different serotypes, Griffith noticed that under proper conditions the R to S transformed cells acquired the serotype of the heat-killed S cells rather than maintaining the serotype of the original R cells.37 Next, Griffith conducted fully controlled experiments that are schematically depicted in Fig. 3.8. Multiple experiments (see Fig. 3.9B for representative original results) showed that neither R cells nor heat-killed S pneumococci by themselves could produce disease and death in mice. By contrast, injected R cells that were mixed with a large excess of heat-killed S bacteria caused death in the mice. The serotype of bacteria that were recovered from the dead animals was that of the heat-killed S cells and not the different original serotype of the R cells. Typical features of the transformation of pneumococcus in Griffith’s hands (and in the hands of researchers who later replicated his results) are shown in Fig. 3.9B.

First, whereas heat-killed S cells alone did not produce disease and death in mice, injection of their mixture with living R cells resulted in some, but not all, cases in death of the animals.

Importantly, the newly encapsulated transformed bacteria that were recovered from the hearts of infected dead mice were of serotype I of the heat-killed S cells and not of Type II from which the R cells were originally derived. Notably, Griffith also executed the reverse experiment, that is, mice that were inoculated with a mixture of heat-killed SI cells and living RII pneumococci died and produced living SI cells.

Two features of the results shown in Fig. 3.9B are noteworthy. First, transformation did not always occur. Rather, whereas it was observed in some cases (mice 645 and 650 in Fig. 3.9B), in other instances the mice remained asymptomatic and the sacrificed animals either had no detectable bacteria (mouse 651) or they bore some untransformed R pneumococci (mice 646–648 and 652). Second, to affect even this inconsistent transformation, the heat-killed S cells had to be added at an excess of about 200-fold over living R bacteria.

Although the source for the incomplete efficiency of transformation and for the need for a large excess of heat-killed S cells was unclear at the time, these features are easily understood in hindsight. As will be discussed later, transformation of pneumococci by DNA was inefficient, especially under the less than optimal conditions that were employed by Griffith and his immediate successors. It was impossible, therefore, to obtain uniformly successful transformation even when heat-killed S cells were added at a great excess over R cells. Significantly, Griffith also tried to achieve transformation in vitro by mixing in the test tube heat-killed S cells with living R bacteria. However, neither R to S conversion nor transformation of serotype was attained. This failure to achieve transformation in vitro was most likely due to experimental conditions that were suboptimal for efficient transformation and to degradation of the transforming material by enzymes that were released from autolyzed R cells.40

DNA/RNA Is Genetic Material

Transformation in Bacteria

In 1928, in an attempt to develop a vaccine against pneumonia, Frederick Griffith became the first to identify bacterial transformation, in which the form and function of a bacterium changes. Both virulent and avirulent Streptococcus pneumoniae were under his study. The virulence of the bacterium is determined by its capsular polysaccharide. Virulent strains have a capsule which is enclosed in a capsular polysaccharide, whereas avirulent strains do not. The nonencapsulated bacteria are readily engulfed and destroyed by phagocytic cells in the host animal's circulatory system. However, due to their protective outer polysaccharide capsule, virulent strains are not easily engulfed by the host's immune system, so they can multiply and cause pneumonia.

The presence or absence of the capsule also causes a visible difference between colonies of virulent and avirulent strains. Encapsulated bacteria form smooth, shiny-surfaced colonies (S) when grown on an agar culture plate. On the other hand, nonencapsulated strains produce rough colonies (R). As such, it is easy to identify the difference between these two strains through standard microbiological culture technique.

In Griffith's experiment, the virulent S. pneumoniae that has a smooth (S) capsule in its appearance was capable of causing lethal infections upon injection into mice (Fig. 1.1). Because of their lack of a protective coat, the R-type bacteria are destroyed by the animal after the injection, as previously described. As such, the mice are still alive after the injection of R-type bacteria. When S-type bacteria were killed by the heat, they were no longer able to cause a lethal infection upon injection into mice alone. However, when the heat-killed S-type bacteria and live R-type bacteria were injected together, neither of which causes lethal infection alone, the mice died as a result of pneumonia infection. It was found that the virulent trait that was responsible for production of the polysaccharide capsule was passed from the heat-killed S-type cells into the live R-type cells, thus converting the R-type bacteria into S-type bacteria, allowing it to become virulent and lethal by evading the host's immune response. Griffith concluded that the heat-killed bacteria somehow converted live avirulent cells to virulent cells, and he called the component of the dead S-type bacteria the “transforming principle.”

Fig. 1.1. Schematic diagram of Griffith's experiment which demonstrates bacterial transformation.

DNA Is the Genetic Material for Bacteria

Griffith's work led to further research of the transformation phenomenon. In 1944, Oswald Avery, Colin MacLeod, and Maclyn McCarty published what is now considered a classic paper in the field of molecular genetics. In this work, they demonstrated that DNA is the transforming principle. The schematic diagram of their experiment is shown in Fig. 1.2. In their experiments, they removed the protein from the transforming extract through organic solvent extraction. After this treatment, proteins were absent from the transforming extract. They found that the transforming principle was still active, which meant the heat-killed bacteria were still able to convert live avirulent cells to virulent cells. They also performed chemical, enzymatic, and serological analysis, together with the results from electrophoresis, ultracentrifugation, and ultraviolet spectroscopy. These treatments can remove carbohydrates, lipids, protein, or RNA from the extract. They found that carbohydrates, lipids, protein, and RNA were also not the transforming substance. Chemical testing of the final product gave a strong positive reaction for DNA. The final confirmation came with experiments using crude samples of the DNA-digesting enzyme deoxyribonuclease (DNase), which can degrade DNA, specifically. They demonstrated that the transforming principle can be destroyed by this enzyme. There was no loss of transforming activity after heat inactivated this enzyme. As such, their observations confirmed that DNA is the transforming substance.

Fig. 1.2. Schematic diagram of Avery, MacLeod, and McCarty's experiment which demonstrates that DNA is the transforming principle.

DNA Is the Genetic Material for Bacteriophage

The second major piece of evidence supporting DNA as the genetic material was through experiments conducted by A. D. Hershey and Martha Chase in 1952. Hershey and Chase used T2 bacteriophage in their experiment to identify whether DNA or protein is the genetic material. Bacteriophage can infect E. coli and use the host to synthesize new phage particle. The phage consists of a protein coat surrounding a core of DNA. The phage attaches to the bacterial cell, and the genetic component of the phage enters the bacterial cells. Following infection, the viral genetic component dominates the cellular machinery of the host cells and leads to viral reproduction. Subsequently, many new phages are constructed, and the bacterial cell is lysed, releasing the progeny viruses. This process is normally referred to as the lytic cycle.

To define the function of the protein coat and nucleic acid in the viral reproduction process, Hersey and Chase radioactively labeled phage DNA with phosphorus-32 (32P) and labeled phage protein with sulfur-35 (35S). This is because DNA has phosphorus but not sulfur, whereas protein contains sulfur, but not phosphorus. Hershey and Chase let the labeled T2 bacteriophages infect the unlabeled bacteria and inject their genetic material into the cells (Fig. 1.3). After the attachment and genetic material entry, the empty phage coats were removed through high shear force in a blender. The force stripped off the attached phages so that the phage and the bacteria could be analyzed separately. Centrifugation separated the lighter phage particles from the heavier bacterial cells. Following this separation, the bacterial cells, which now contained viral-labeled DNA, were eventually lysed as the new phages were produced. These progeny phages contained 32P but not 35S. These results suggested that the protein of the phage coat remains outside the host cells and is not involved in directing the production of new phages. On the other hand, phage DNA enters the host cells and is directly involved in phage reproduction. Because the genetic material must first enter the infected cells, they concluded that DNA is the genetic material, and that it contains genes passed along through generations.

Fig. 1.3. Schematic diagram of Hershey-Chase experiment which demonstrates that DNA is directing reproduction of T2 phage in infected E. coli cells.

Taken together with work that had been done before, Hershey and Chase's work provided final, strong evidence to prove that DNA is the genetic material. Although these experiments demonstrated that DNA is the genetic material in bacteria and viruses, it was generally accepted that DNA is a universal substance as the genetic material in eukaryotes. This is because some indirect evidence has indicated that DNA is the genetic material in eukaryotes. For example, the genetic material should reside on the chromosome and be found in the nucleus. Both DNA and protein fit these criteria, but only DNA is enriched inside the nucleus, whereas protein is enriched in the cytoplasm. Furthermore, DNA is also found in both chloroplasts and mitochondria, which are also known for performing genetic functions. As such, DNA is only found where primary genetic functions occur. On the other hand, protein is found everywhere in the cell.

Direct evidence that DNA is the genetic material in eukaryotes comes from recombinant DNA technology. For example, a segment of a DNA fragment corresponding to a specific gene is isolated and ligated to bacterial DNA which can self-replicate inside the bacterial cell. The resulting complex is sent into a bacterial cell, and its genetic expression is examined. The subsequent production of the eukaryotic protein derived from that specific DNA segment in the bacterial cell demonstrates that DNA is the genetic material in the eukaryotic cells. This so-called gene cloning technique is now widely used in current biomedical research and pharmaceutical production (Fig. 1.4).

Fig. 1.4. Schematic diagram of a typical gene cloning process and the application of the gene cloning. The production of the specific eukaryotic protein derived from that introduced eukaryotic DNA segment proves that DNA is the genetic material in the eukaryotic cells.

Fratricide in S. pneumoniae

S. pneumoniae is not only the causative agent of pneumonia but also an important model organism in bacterial genetics. In his famous experiments, Griffith [66] demonstrated in 1928 that the ability to synthesis a capsule and hence become virulent can be transferred from heat-inactivated “smooth” strains to living but avirulent “rough” strains of S. pneumoniae. This initial observation was then taken up and painstakingly expanded by Avery et al. [67], who ultimately demonstrated for the very first time that DNA (and not proteins or lipids) is the “transforming principle.” These hallmark studies not only paved the way for identifying the crucial role of DNA in heredity but also identified the process of transformation, that is, the uptake of free DNA from the environment. S. pneumoniae is therefore the first of an ever-increasing number of bacteria that can become naturally competent for genetic transformation. Fratricide is initiated along with the development of natural competence: non-competent siblings are challenged with killing factors that ultimately lead to allolysis, cell death through cell-lysis either directly or in trans (Fig. 2) [68,69].

S. pneumoniae enters the competent state under favorable growth conditions during exponential growth, in a process that is governed by quorum sensing [16,70,71]. The rapid increase in cell density leads to an extracellular accumulation of the quorum sensing signal, the competence-stimulating peptide (CSP). Once it exceeds a threshold concentration, it activates the ComD histidine kinase, leading to its autophosphorylation. Upon phospho-transfer, its cognate response regulator, ComE, becomes activated and regulates about 20 early competence (com) genes [70,72,73]. Among these are two identical copies of comX, which encode alternative sigma factors that activate the transcription of the late com genes [74] (Fig. 2). In total, the CSP-responsive regulon in S. pneumoniae comprises about 120 genes, of which only 22 are necessary for developing natural competence [23,75,76]. This suggests that the majority of the com system is involved in additional cellular processes beyond the machinery required for DNA uptake and recombination [16].

Fratricide is expressed both in a ComX-dependent and -independent manner. The ComX-dependent genes encode the murein hydrolases CbpD, the autolysin LytA as well as the two peptide bacteriocin CibAB [23,68,77]. In addition, the non-CSP-regulated lysozyme LytC is suggested to contribute to fratricide [78] (Fig. 2). Experimentally, fratricide is monitored in liquid culture based on the release of chromosomal DNA and cytoplasmic β-galactosidase, as well as the ability for clumping [79–81]. Clumping was already observed more than four decades ago under mild acidic conditions and linked to competence development. Later, it was demonstrated that this behavior relies on the release of chromosomal DNA [23,82]. Fratricide is also detectable on solid blood agar plates, where lysis can be followed by the release of pneumolysin, a cytolytic virulence factor [68].

The contribution and impact of the individual fratricide toxins depends on the assay applied [16]. CbpD plays only a minor role in fratricide development on plates, but abolishes the effect of clumping and has a strong effect on DNA and β-galactosidase release in liquid cultures [23,68,77]. The opposite effects are observed for CibAB, since inactivation leads to the loss of pneumolysin release on plates, while clumping remains un-effected [23,68]. In contrast, both lytic enzymes, LytA and LytC, are absolutely required, since inactivation results in a strong and assay-independent reduction of fratricide.

The lysis of non-competent cells by the competent cells is termed allolysis and requires cell-to-cell contact [68]. In the absence of the lytic enzymes, CibAB cannot provoke allolysis alone, suggesting that CibAB only supports cell lysis by inserting into the membrane of non-competent siblings and de-energizing them, thereby increasing their susceptibility toward lysis [16,23,68]. CibAB is co-transcribed with a putative transmembrane protein, CibC, which is implicated in CibAB immunity, since its inactivation increases susceptibility to allolysis [68]. Protection against self-lysis of the competent sub-population is ensured by ComM, a CSP-responsive early com gene [23] (Fig. 2). Overexpression of ComM results in growth inhibition and severe morphological defects. During competence, accumulation of ComM is prevented through its processing—and hence inactivation—by an intra-membrane protease [83]. But the mechanism of how this membrane protein confers resistance is still unknown.

The physiological role of fratricide is still not fully understood. It seems to play a role in enhancing the genetic diversity throughout the population by providing extracellular DNA for uptake. By the targeted elimination of non-competent cells, the exchange of genetic material could be promoted [23]. Fratricide seems to be particularly important for efficient gene transfer between pneumococci in biofilms, where it is important for the active acquisition of homologous donor DNA under natural conditions [84]. An antibiotic resistance marker was transferred much more efficiently from neighboring cells than from the growth medium. Under biofilm conditions, efficient lysis of target cells requires CbpD and LytC, while the major autolysin LytA does not seem to be important for fratricide in the biofilm environment [84]. Another hypothesis along those lines suggests that S. pneumoniae triggers fratricide in order to release potential cytoplasmic virulence factors and inflammatory mediators from the non-competent cells, as part of the infection process within the host [16,23].

1.2.1 Enhancing variability: regulation of fratricide and transformation

Studies on DNA and gene transfer were initially described in Streptococcus pneumoniae (Griffith, 1928). In 1928, Frederick Griffith proved that an avirulent pneumococcus strain was able to acquire lethal properties from a pathogenic variant from what was thought to be based on protein transfer (Griffith, 1928). In 1944, the work of Oswald Avery and others was essential to discover, that in fact, it was the DNA that was responsible for the change in pathogenicity in Griffith experiment (Avery et al., 1944). This phenomenon is known as natural bacteria transformation or natural competence. S. pneumoniae is equipped with specialized and tightly regulated proteins involved in the DNA-uptake and integration of exogenous DNA into its chromosome, annotated as transformasome (Attaiech et al., 2011; Chandler and Morrison, 1987; Kausmally et al., 2005; Laurenceau et al., 2013; Mortier-Barriere et al., 1998; Peterson et al., 2004; Takeno et al., 2011; Ween et al., 1999; Wholey et al., 2016). Pneumococci acquires survival and adaptive traits and evolves antibiotic resistance as well. The expression of pathogenicity islets (such as Pilus-1, RD-10, among others) and the troublesome serotype replacement behaviour by acquiring genes via horizontal gene transfer from different capsular loci as a consequence of vaccination and environmental pressure are other key characteristics of pneumococci (Coffey et al., 1998; Feikin et al., 2013; Geno et al., 2015; Mera et al., 2008). Several studies focusing on competence and transformation lead to a profound understanding of the mechanisms involved in the regulation of this process, identifying a TCS as the artifice of pneumococcal competence control (Fig. 3A) (Chandler and Morrison, 1987; Chen and Morrison, 1987; Havarstein et al., 1995a; Lee and Morrison, 1999; Pestova et al., 1996; Peterson et al., 2004; Ween et al., 1999). TCS12 or ComDE (competence operon) is expressed as an operon along with a small peptide ComC (comCDE), encoding two highly conserved proteins, namely a HK (ComD) and a RR (ComE) (Havarstein et al., 1996). The TCS ComDE uses a quorum sensing mechanism in tandem with the ATP binding cassette transporter ComAB to sense, activate, and regulate the pneumococcal transformation machinery under specific environmental conditions (Chandler and Morrison, 1987; Hui and Morrison, 1991; Hui et al., 1995). In brief, the competence signalling peptide ComC (CSP) functions as an autoinducer molecule, with a characteristic GG leader peptide (Havarstein et al., 1995a; Peterson et al., 2004). ComC is transcribed, cleaved and secreted to the extracellular milieu via the ComAB transporter (Havarstein et al., 1995a, 1995b; Hui and Morrison, 1991). The CSP is then extracellularly accumulated and detected by ComD, leading to a conformational change and autophosphorylation of the kinase domain in ComD (Havarstein et al., 1996; Martin et al., 2013; Pestova et al., 1996). The RR ComE is recruited and receives the phosphate group from ComD, thereby activating the expression of approx. 20 early competence genes including the comAB system and comCDE. In consequence, this enables an enhancing loop for competence (Martin et al., 2013; Ween et al., 1999). The RR ComE has been shown to bind to a specific DNA repeat sequence preceding the early competence genes (Ween et al., 1999). Early competence genes comX and comW participate in the expression of ∼80 late competence genes necessary for DNA uptake, processing and integration into the pneumococcal chromosome via homologous recombination (Campbell et al., 1998; Dagkessamanskaia et al., 2004; Laurenceau et al., 2013; Peterson et al., 2004; Sung and Morrison, 2005; Tovpeko and Morrison, 2014).

In parallel to competence, the expression of the immunity protein ComM and hydrolases CbpD, LytA and LytC is triggered by the ComX-ComW complex. This induces a targeted attack on non-competent cells, referred to as allolysis. The attacked cells, then release their DNA content and virulence factors (Guiral et al., 2005; Kausmally et al., 2005; Moscoso and Claverys, 2004; Sung and Morrison, 2005).

As mentioned previously, the pneumococcal competence state is initiated by the accumulation of extracellular CSP due to an autoinduced loop driven by the ComCDE and ComAB systems (Hui and Morrison, 1991; Hui et al., 1995; Lee and Morrison, 1999; Martin et al., 2013; Peterson et al., 2004). However, a secondary pneumococcal TCS, TCS05, has been described to be involved in pneumococcal competence (Dagkessamanskaia et al., 2004; Echenique et al., 2000; Martin et al., 2000; Zahner et al., 1996). TCS05 or CiaRH (competence induction and altered cefotaxime susceptibility) is one of the best characterized pneumococcal TCS and known to negatively affect the development of competence. Indeed, original studies on this topic have discussed a repressor effect of competence via CiaR, nevertheless no binding of this protein was detected to any of the competence gene loci (Mascher et al., 2003). Different theories have been proposed involving an indirect effect of CiaR on the expression of the competence genes. One study focused on the upregulation of the virulence factor HtrA (high temperature requirement A) by CiaR and investigated the effect of HtrA on competence by its proteolytic activity and the ribosomal translation error rate. Here, an inverse relation between number of misfolded proteins and competence by HtrA was reported (Stevens et al., 2011). HtrA has further been shown to influence pneumococcal competence activity by degrading the extracellular CSP (Cassone et al., 2012). Comparatively, 5 non-coding RNAs, designated as cia-dependent small RNAs (csRNA), were reported to modulate competence by interacting with the comC gene and disrupting its expression (Laux et al., 2015). Additionally, 25 CiaR targets were detected by using solid phase DNA binding (SPDB) assay, but no consensus sequence could be obtained due to the low level of conservation of the different regions (Mascher et al., 2003). The different mechanism and theories existing for the role of TCS CiaRH in competence suggest a counterbalance to the activity of ComDE, effectively repressing competence.

Pneumococcal TCS02 or WalRK (WalRK, VicRK, YycFG and MicAB) is also depicted to be involved in the regulation of competence in pneumococci (Echenique and Trombe, 2001). Here, the regulation is hypothesized to rely on the presence of a PAS domain in the HK WalK, suggesting an inhibition of pneumococcal natural competence under low oxygen concentrations (Echenique and Trombe, 2001). Experiments performed with a null mutation in WalK and a point mutation in WalR resulted in an upregulation of competence genes under microaerobiosis (Echenique and Trombe, 2001). The authors of this work finally suggested the possibility of WalK to sense signals related to oxygen concentration. More recent studies using a WalR deficient mutant and a WalR overexpressing strain could not detect changes in the competence operon (Mohedano et al., 2005; Ng et al., 2005). Thus, the question if the WalRK system is related to competence control is still unresolved.

Likewise, pneumococcal TCS03 or LiaRS has been shown to be involved in the competence process by responding to peptidoglycan (PGN) cleavage by LytA, CbpD and LytC murein hydrolases. These hydrolases are known to be involved in the fratricide phenomenon during competence (Eldholm et al., 2010). The proposed mechanism involves the action of a third protein annotated as LiaF. The full LiaRSF system is present in pneumococci and is assumed to behave similarly as its homologue in Bacillus subtilis (Eldholm et al., 2010). As such, the helper protein LiaF Interacts directly with LiaS hindering its functionality. Upon PGN cleavage, LiaF releases itself from LiaS, allowing its activation and transfer of the signal to LiaR and generating thereby a response in its regulated gene loci (Jordan et al., 2006). Even though the sensed signal for LiaS is not known, the binding sequence for LiaR has been determined for S. pneumoniae. Additionally, microarrays studies identified 3 genes (hrcA, grpE and spr_0810) involved in stress response to be controlled by LiaR. Finally, the authors concluded that LiaRS is activated by the activity of the PGN hydrolases CbpD, LytA and LytC and modulates the response of stress relief proteins in pneumococci (Eldholm et al., 2010). The effect on the activation of the LiaRS system via cell wall cleavage allows to hypothesize a complementary role of LiaRS to CiaRH and WalRK on pneumococcal survival and resistance to allolysis.