RESUMO
In oval-shaped Streptococcus pneumoniae, septal and longitudinal peptidoglycan syntheses are performed by independent functional complexes: the divisome and the elongasome. Penicillin-binding proteins (PBPs) were long considered the key peptidoglycan-synthesizing enzymes in these complexes. Among these were the bifunctional class A PBPs, which are both glycosyltransferases and transpeptidases, and monofunctional class B PBPs with only transpeptidase activity. Recently, however, it was established that the monofunctional class B PBPs work together with transmembrane glycosyltransferases (FtsW and RodA) from the shape, elongation, division, and sporulation (SEDS) family to make up the core peptidoglycan-synthesizing machineries within the pneumococcal divisome (FtsW/PBP2x) and elongasome (RodA/PBP2b). The function of class A PBPs is therefore now an open question. Here we utilize the peptidoglycan hydrolase CbpD that targets the septum of S. pneumoniae cells to show that class A PBPs have an autonomous role during pneumococcal cell wall synthesis. Using assays to specifically inhibit the function of PBP2x and FtsW, we demonstrate that CbpD attacks nascent peptidoglycan synthesized by the divisome. Notably, class A PBPs could process this nascent peptidoglycan from a CbpD-sensitive to a CbpD-resistant form. The class A PBP-mediated processing was independent of divisome and elongasome activities. Class A PBPs thus constitute an autonomous functional entity which processes recently formed peptidoglycan synthesized by FtsW/PBP2×. Our results support a model in which mature pneumococcal peptidoglycan is synthesized by three functional entities, the divisome, the elongasome, and bifunctional PBPs. The latter modify existing peptidoglycan but are probably not involved in primary peptidoglycan synthesis.
Assuntos
Amidoidrolases/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano/metabolismo , Streptococcus pneumoniae/fisiologia , Amidoidrolases/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Divisão Celular , Proteínas de Membrana/metabolismo , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismoRESUMO
Staphylococcus aureus needs to control the position and timing of cell division and cell wall synthesis to maintain its spherical shape. We identified two membrane proteins, named CozEa and CozEb, which together are important for proper cell division in S. aureus. CozEa and CozEb are homologs of the cell elongation regulator CozESpn of Streptococcus pneumoniae. While cozEa and cozEb were not essential individually, the ΔcozEaΔcozEb double mutant was lethal. To study the functions of cozEa and cozEb, we constructed a CRISPR interference (CRISPRi) system for S. aureus, allowing transcriptional knockdown of essential genes. CRISPRi knockdown of cozEa in the ΔcozEb strain (and vice versa) causes cell morphological defects and aberrant nucleoid staining, showing that cozEa and cozEb have overlapping functions and are important for normal cell division. We found that CozEa and CozEb interact with and possibly influence localization of the cell division protein EzrA. Furthermore, the CozE-EzrA interaction is conserved in S. pneumoniae, and cell division is mislocalized in cozESpn -depleted S. pneumoniae cells. Together, our results show that CozE proteins mediate control of cell division in S. aureus and S. pneumoniae, likely via interactions with key cell division proteins such as EzrA.
Assuntos
Proteínas de Bactérias/fisiologia , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/genética , Proteínas de Membrana/fisiologia , Staphylococcus aureus/fisiologia , Proteínas de Bactérias/genética , Proteínas de Ciclo Celular/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA Bacteriano/genética , Técnicas de Silenciamento de Genes , Proteínas de Membrana/genética , Mutação , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidadeRESUMO
The oval shape of pneumococci results from a combination of septal and lateral peptidoglycan synthesis. The septal cross-wall is synthesized by the divisome, while the elongasome drives cell elongation by inserting new peptidoglycan into the lateral cell wall. Each of these molecular machines contains penicillin-binding proteins (PBPs), which catalyze the final stages of peptidoglycan synthesis, plus a number of accessory proteins. Much effort has been made to identify these accessory proteins and determine their function. In the present paper we have used a novel approach to identify members of the pneumococcal elongasome that are functionally closely linked to PBP2b. We discovered that cells depleted in PBP2b, a key component of the elongasome, display several distinct phenotypic traits. We searched for proteins that, when depleted or deleted, display the same phenotypic changes. Four proteins, RodA, MreD, DivIVA and Spr0777, were identified by this approach. Together with PBP2b these proteins are essential for the normal function of the elongasome. Furthermore, our findings suggest that DivIVA, which was previously assigned as a divisomal protein, is required to correctly localize the elongasome at the negatively curved membrane region between the septal and lateral cell wall.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Sequência de Aminoácidos , Aminoaciltransferases/metabolismo , Proteínas de Transporte/metabolismo , Crescimento Celular , Parede Celular/metabolismo , Proteínas de Membrana/metabolismo , Testes de Sensibilidade Microbiana , Penicilinas , Peptidoglicano/biossíntese , Peptidil Transferases/metabolismo , Streptococcus pneumoniae/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
In a screen for mutations suppressing the lethal loss of PBP2b in Streptococcus pneumoniae we identified Spr1851 (named EloR), a cytoplasmic protein of unknown function whose inactivation removed the requirement for PBP2b as well as RodA. It follows from this that EloR and the two elongasome proteins must be part of the same functional network. This network also includes StkP, as this serine/threonine kinase phosphorylates EloR on threonine 89 (T89). We found that ΔeloR cells, and cells expressing the phosphoablative form of EloR (EloRT89A ), are significantly shorter than wild-type cells. Furthermore, the phosphomimetic form of EloR (EloRT89E ) is not tolerated unless the cell in addition acquires a truncated MreC or non-functional RodZ protein. By itself, truncation of MreC as well as inactivation of RodZ gives rise to less elongated cells, demonstrating that the stress exerted by the phosphomimetic form of EloR is relieved by suppressor mutations that reduce or abolish the activity of the elongasome. Of note, it was also found that loss of elongasome activity caused by truncation of MreC elicits increased StkP-mediated phosphorylation of EloR. Together, the results support a model in which phosphorylation of EloR stimulates cell elongation, while dephosphorylation has an inhibitory effect.
Assuntos
Streptococcus pneumoniae/genética , Aminoaciltransferases/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Divisão Celular/genética , Morfogênese/genética , Morfogênese/fisiologia , Mutação , Proteínas de Ligação às Penicilinas/antagonistas & inibidores , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Streptococcus pneumoniae/metabolismoRESUMO
The important human pathogen Streptococcus pneumoniae is a naturally transformable species. When developing the competent state, it expresses proteins involved in DNA uptake, DNA processing and homologous recombination. In addition to the proteins required for the transformation process, competent pneumococci express proteins involved in a predatory DNA acquisition mechanism termed fratricide. This is a mechanism by which the competent pneumococci secrete a muralytic fratricin termed CbpD, which lyses susceptible sister cells or closely related streptococcal species. The released DNA can then be taken up by the competent pneumococci and integrated into their genomes. To avoid committing suicide, competent pneumococci produce an integral membrane protein, ComM, which protects them against CbpD by an unknown mechanism. In the present study, we show that overexpression of ComM results in growth inhibition and development of severe morphological abnormalities, such as cell elongation, misplacement of the septum and inhibition of septal cross-wall synthesis. The toxic effect of ComM is tolerated during competence because it is not allowed to accumulate in the competent cells. We provide evidence that an intra-membrane protease called RseP is involved in the process of controlling the ComM levels, since â³rseP mutants produce higher amounts of ComM compared to wild-type cells. The data presented here indicate that ComM mediates immunity against CbpD by a mechanism that is detrimental to the pneumococcus if exaggerated.
Assuntos
Amidoidrolases/metabolismo , Proteínas de Bactérias/biossíntese , Bacteriólise/fisiologia , Competência de Transformação por DNA/genética , Proteínas de Membrana/biossíntese , Peptídeo Hidrolases/metabolismo , Streptococcus pneumoniae/crescimento & desenvolvimento , Amidoidrolases/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , DNA Bacteriano/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Peptídeo Hidrolases/genética , Peptidoglicano/biossíntese , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/patogenicidade , Transformação Bacteriana/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
WalRK is the only two-component regulatory system essential for viability in Streptococcus pneumoniae. Despite its importance, the biological role of this system is not well understood. However, previous studies have shown that it has a crucial role in controlling pneumococcal cell division. Considerable efforts have been made to understand how the WalRK system is regulated, but no signal(s) sensed by the WalK histidine kinase has been identified so far. Here, we provide evidence that the serine/threonine protein kinase StkP modulates the activity of WalK through direct protein-protein interaction, suggesting that this interaction is one of the signals sensed by WalK. In most low-G+C content Gram-positive bacteria, WalK orthologues are attached to the cytoplasmic membrane via two transmembrane segments separated by a large extracellular loop believed to function as a sensor domain. In contrast, members of the genus Streptococcus have WalK histidine kinases that are anchored to the cytoplasmic membrane by a single transmembrane segment. It has been a long-standing question whether this segment only serves as a membrane anchor or if it also functions as a signal-sensing domain. Our data strongly support the latter, i.e. that the transmembrane segment senses signals that regulate the activity of WalK.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Streptococcus pneumoniae/metabolismo , Sequência de Aminoácidos/genética , Proteínas de Bactérias/genética , Composição de Bases/genética , Divisão Celular/genética , Membrana Celular/metabolismo , Domínios Proteicos/genética , Transdução de SinaisRESUMO
Coordinated membrane and cell wall synthesis is vital for maintaining cell integrity and facilitating cell division in bacteria. However, the molecular mechanisms that underpin such coordination are poorly understood. Here we uncover the pivotal roles of the staphylococcal proteins CozEa and CozEb, members of a conserved family of membrane proteins previously implicated in bacterial cell division, in the biosynthesis of lipoteichoic acids (LTA) and maintenance of membrane homeostasis in Staphylococcus aureus. We establish that there is a synthetic lethal relationship between CozE and UgtP, the enzyme synthesizing the LTA glycolipid anchor Glc2DAG. By contrast, in cells lacking LtaA, the flippase of Glc2DAG, the essentiality of CozE proteins was alleviated, suggesting that the function of CozE proteins is linked to the synthesis and flipping of the glycolipid anchor. CozE proteins were indeed found to modulate the flipping activity of LtaA in vitro. Furthermore, CozEb was shown to control LTA polymer length and stability. Together, these findings establish CozE proteins as novel players in membrane homeostasis and LTA biosynthesis in S. aureus.IMPORTANCELipoteichoic acids are major constituents of the cell wall of Gram-positive bacteria. These anionic polymers are important virulence factors and modulators of antibiotic susceptibility in the important pathogen Staphylococcus aureus. They are also critical for maintaining cell integrity and facilitating proper cell division. In this work, we discover that a family of membrane proteins named CozE is involved in the biosynthesis of lipoteichoic acids (LTAs) in S. aureus. CozE proteins have previously been shown to affect bacterial cell division, but we here show that these proteins affect LTA length and stability, as well as the flipping of glycolipids between membrane leaflets. This new mechanism of LTA control may thus have implications for the virulence and antibiotic susceptibility of S. aureus.
Assuntos
Proteínas de Bactérias , Lipopolissacarídeos , Proteínas de Membrana , Staphylococcus aureus , Ácidos Teicoicos , Ácidos Teicoicos/biossíntese , Ácidos Teicoicos/metabolismo , Staphylococcus aureus/metabolismo , Staphylococcus aureus/genética , Lipopolissacarídeos/biossíntese , Lipopolissacarídeos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Parede Celular/metabolismo , Membrana Celular/metabolismoRESUMO
Streptococcus pneumoniae produces two class B penicillin-binding proteins, PBP2x and PBP2b, both of which are essential. It is generally assumed that PBP2x is specifically involved in septum formation, while PBP2b is dedicated to peripheral cell wall synthesis. However, little experimental evidence exists to substantiate this belief. In the present study, we obtained evidence that strongly supports the view that PBP2b is essential for peripheral peptidoglycan synthesis. Depletion of PBP2b expression gave rise to long chains of cells in which individual cells were compressed in the direction of the long axis and looked lentil shaped. This morphological change is consistent with a role for pneumococcal PBP2b in the synthesis of the lateral cell wall. Depletion of PBP2x, on the other hand, resulted in lemon-shaped and some elongated cells with a thickened midcell region. Low PBP2b levels gave rise to changes in the peptidoglycan layer that made pneumococci sensitive to exogenously added LytA during logarithmic growth and refractory to chain dispersion upon addition of LytB. Interestingly, analysis of the cell wall composition of PBP2b-depleted pneumococci revealed that they had a larger proportion of branched stem peptides in their peptidoglycan than the corresponding undepleted cells. Furthermore, MurM-deficient mutants, i.e., mutants lacking the ability to synthesize branched muropeptides, were found to require much higher levels of PBP2b to sustain growth than those required by MurM-proficient strains. These findings might help to explain why increased incorporation of branched muropeptides is required for high-level beta-lactam resistance in S. pneumoniae.
Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano/química , Peptidoglicano/metabolismo , Streptococcus pneumoniae/metabolismo , Streptococcus pneumoniae/ultraestrutura , Aminoaciltransferases/genética , Forma Celular/genética , Forma Celular/fisiologia , Parede Celular/fisiologia , Regulação Bacteriana da Expressão Gênica , Mutação , Proteínas de Ligação às Penicilinas/genética , Streptococcus pneumoniae/classificação , Streptococcus pneumoniae/genéticaRESUMO
Cell division and cell wall synthesis in staphylococci need to be precisely coordinated and controlled to allow the cell to multiply while maintaining its nearly spherical shape. The mechanisms ensuring correct placement of the division plane and synthesis of new cell wall have been studied intensively. However, hitherto unknown factors and proteins are likely to play key roles in this complex interplay. Here, we identified and investigated a protein with a major influence on cell morphology in Staphylococcus aureus. The protein, named SmdA (for staphylococcal morphology determinant A), is a membrane protein with septum-enriched localization. By CRISPRi knockdown and overexpression combined with different microscopy techniques, we demonstrated that proper levels of SmdA were necessary for cell division, including septum formation and cell splitting. We also identified conserved residues in SmdA that were critical for its functionality. Pulldown and bacterial two-hybrid interaction experiments showed that SmdA interacted with several known cell division and cell wall synthesis proteins, including penicillin-binding proteins (PBPs) and EzrA. Notably, SmdA also affected susceptibility to cell wall targeting antibiotics, particularly in methicillin-resistant S. aureus (MRSA). Together, our results showed that S. aureus was dependent on balanced amounts of membrane attached SmdA to carry out proper cell division. IMPORTANCE Staphylococcus aureus is an important human and animal pathogen. Antibiotic resistance is a major problem in the treatment of staphylococcal infections, and cell division and cell wall synthesis factors have previously been shown to modulate susceptibility to antibiotics in this species. Here, we investigated the function of a protein named SmdA, which was identified based on its septal localization and knockdown phenotype resulting in defective cellular morphologies. We demonstrated that this protein was critical for normal cell division in S. aureus. Depletion of SmdA sensitized resistant staphylococci to ß-lactam antibiotics. This work revealed a new staphylococcal cell division factor and a potential future target for narrow-spectrum antimicrobials or compounds to resensitize antibiotic-resistant staphylococcal strains.
Assuntos
Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Humanos , Staphylococcus aureus Resistente à Meticilina/metabolismo , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Staphylococcus/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismoRESUMO
Control of peptidoglycan assembly is critical to maintain bacterial cell size and morphology. Penicillin-binding proteins (PBPs) are crucial enzymes for the polymerization of the glycan strand and/or their cross-linking via peptide branches. Over the last few years, it has become clear that PBP activity and localization can be regulated by specific cognate regulators. The first regulator of PBP activity in Gram-positive bacteria was discovered in the human pathogen Streptococcus pneumoniae This regulator, named CozE, controls the activity of the bifunctional PBP1a to promote cell elongation and achieve a proper cell morphology. In this work, we studied a previously undescribed CozE homolog in the pneumococcus, which we named CozEb. This protein displays the same membrane organization as CozE but is much more widely conserved among Streptococcaceae genomes. Interestingly, cozEb deletion results in cells that are smaller than their wild-type counterparts, which is the opposite effect of cozE deletion. Furthermore, double deletion of cozE and cozEb results in poor viability and exacerbated cell shape defects. Coimmunoprecipitation further showed that CozEb is part of the same complex as CozE and PBP1a. However, although we confirmed that CozE is required for septal localization of PBP1a, the absence of CozEb has no effect on PBP1a localization. Nevertheless, we found that the overexpression of CozEb can compensate for the absence of CozE in all our assays. Altogether, our results show that the interplay between PBP1a and the cell size regulators CozE and CozEb is required for the maintenance of pneumococcal cell size and shape.IMPORTANCE Penicillin-binding proteins (PBPs), the proteins catalyzing the last steps of peptidoglycan assembly, are critical for bacteria to maintain cell size, shape, and integrity. PBPs are consequently attractive targets for antibiotics. Resistance to antibiotics in Streptococcus pneumoniae (the pneumococcus) are often associated with mutations in the PBPs. In this work, we describe a new protein, CozEb, controlling the cell size of pneumococcus. CozEb is a highly conserved integral membrane protein that works together with other proteins to regulate PBPs and peptidoglycan synthesis. Deciphering the intricate mechanisms by which the pneumococcus controls peptidoglycan assembly might allow the design of innovative anti-infective strategies, for example, by resensitizing resistant strains to PBP-targeting antibiotics.
Assuntos
Proteínas de Bactérias/genética , Homeostase , Proteínas de Membrana/genética , Proteínas de Ligação às Penicilinas/genética , Streptococcus pneumoniae/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Biologia Computacional , Proteínas de Membrana/metabolismo , Testes de Sensibilidade Microbiana , Mutação , Peptidoglicano/metabolismo , Fenótipo , Streptococcus pneumoniae/efeitos dos fármacosRESUMO
Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to most ß-lactams due to the expression of an extra penicillin-binding protein, PBP2a, with low ß-lactam affinity. It has long been known that heterologous expression of the PBP2a-encoding mecA gene in methicillin-sensitive S. aureus (MSSA) provides protection towards ß-lactams, however, some reports suggest that the degree of protection can vary between different ß-lactams. To test this more systematically, we introduced an IPTG-inducible mecA into the MSSA laboratory strain RN4220. We confirm, by growth assays as well as single-cell microfluidics time-lapse microscopy experiments, that PBP2a expression protects against ß-lactams in S. aureus RN4220. By testing a panel of ten different ß-lactams, we conclude that there is also a great variation in the level of protection conferred by PBP2a. Expression of PBP2a resulted in an only fourfold increase in minimum inhibitory concentration (MIC) for imipenem, while a 32-fold increase in MIC was observed for cefaclor and cephalexin. Interestingly, in our experimental setup, PBP2a confers the highest protection against cefaclor and cephalexin-two ß-lactams that are known to have a high specific affinity toward the transpeptidase PBP3 of S. aureus. Notably, using a single-cell microfluidics setup we demonstrate a considerable phenotypic variation between cells upon ß-lactam exposure and show that mecA-expressing S. aureus can survive ß-lactam concentrations much higher than the minimal inhibitory concentrations. We discuss possible explanations and implications of these results including important aspects regarding treatment of infection.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Proteínas de Ligação às Penicilinas/genética , beta-Lactamas/farmacologia , Proteínas de Bactérias/metabolismo , Humanos , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Testes de Sensibilidade Microbiana , Microfluídica , Proteínas de Ligação às Penicilinas/metabolismoRESUMO
The RNA binding proteins EloR and KhpA are important components of the regulatory network that controls and coordinates cell elongation and division in S. pneumoniae. Loss of either protein reduces cell length, and makes the essential elongasome proteins PBP2b and RodA dispensable. It has been shown previously in formaldehyde crosslinking experiments that EloR co-precipitates with KhpA, indicating that they form a complex in vivo. In the present study, we used 3D modeling and site directed mutagenesis in combination with protein crosslinking to further study the relationship between EloR and KhpA. Protein-protein interaction studies demonstrated that KhpA forms homodimers and that KhpA in addition binds to the KH-II domain of EloR. Site directed mutagenesis identified isoleucine 61 (I61) as crucial for KhpA homodimerization. When substituting I61 with phenylalanine, KhpA lost the ability to homodimerize, while it still interacted clearly with EloR. In contrast, both homo- and heterodimerization were lost when I61 was substituted with tyrosine. By expressing these KhpA versions in S. pneumoniae, we were able to show that disruption of EloR/KhpA heterodimerization makes the elongasome redundant in S. pneumoniae. Of note, loss of KhpA homodimerization did not give rise to this phenotype, demonstrating that the EloR/KhpA complex is crucial for regulating the activity of the elongasome. In support of this conclusion, we found that localization of KhpA to the pneumococcal mid-cell region depends on its interaction with EloR. Furthermore, we found that the EloR/KhpA complex co-localizes with FtsZ throughout the cell cycle.
Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Streptococcus pneumoniae/metabolismo , Substituição de Aminoácidos , Aminoaciltransferases/genética , Proteínas de Bactérias/genética , Proteínas de Ligação às Penicilinas/genética , Domínios Proteicos , Multimerização Proteica , Streptococcus pneumoniae/citologia , Streptococcus pneumoniae/genéticaRESUMO
Streptococcus pneumoniae is a frequent colonizer of the human nasopharynx that has the potential to cause severe infections such as pneumonia, bacteremia and meningitis. Despite considerable efforts to reduce the burden of pneumococcal disease, it continues to be a major public health problem. After the Second World War, antimicrobial therapy was introduced to fight pneumococcal infections, followed by the first effective vaccines more than half a century later. These clinical interventions generated a selection pressure that drove the evolution of vaccine-escape mutants and strains that were highly resistant against antibiotics. The remarkable ability of S. pneumoniae to acquire drug resistance and evade vaccine pressure is due to its recombination-mediated genetic plasticity. S. pneumoniae is competent for natural genetic transformation, a property that enables the pneumococcus to acquire new traits by taking up naked DNA from the environment and incorporating it into its genome through homologous recombination. In the present paper, we review current knowledge on pneumococcal transformation, and discuss how the pneumococcus uses this mechanism to adapt and survive under adverse and fluctuating conditions.
Assuntos
Evolução Molecular , Genoma Bacteriano , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/fisiologia , Transformação Bacteriana , Adaptação Biológica , Competência de Transformação por DNA , Interação Gene-Ambiente , Humanos , Recombinação GenéticaRESUMO
Separation of daughter cells during bacterial cell division requires splitting of the septal cross wall by peptidoglycan hydrolases. In Streptococcus pneumoniae, PcsB is predicted to perform this operation. Recent evidence shows that PcsB is recruited to the septum by the transmembrane FtsEX complex, and that this complex is required for cell division. However, PcsB lacks detectable catalytic activity in vitro, and while it has been proposed that FtsEX activates PcsB, evidence for this is lacking. Here we demonstrate that PcsB has muralytic activity, and report the crystal structure of full-length PcsB. The protein adopts a dimeric structure in which the V-shaped coiled-coil (CC) domain of each monomer acts as a pair of molecular tweezers locking the catalytic domain of each dimeric partner in an inactive configuration. This suggests that the release of the catalytic domains likely requires an ATP-driven conformational change in the FtsEX complex, conveyed towards the catalytic domains through coordinated movements of the CC domain.
Assuntos
Proteínas de Bactérias/metabolismo , Divisão Celular/fisiologia , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Streptococcus pneumoniae/fisiologia , Proteínas de Bactérias/ultraestrutura , Domínio Catalítico/fisiologia , Divisão Celular/genética , Parede Celular/fisiologia , Cristalografia por Raios X , Streptococcus pneumoniae/genéticaRESUMO
In the present work we have constructed a new tandem affinity purification tag and used it to purify two different polypeptides, PcsB and ECL1 from Streptococcus pneumoniae. PcsB probably functions as a peptidoglycan hydrolase and is believed to be involved in splitting of the septum during cell division. ECL1 is the extracellular domain of the membrane spanning protein FtsX. Experimental evidence indicates that the ECL1 domain controls the activity of PcsB through direct interaction (Sham et al., 2011). The affinity tag consists of an N-terminal 6xHis-tag, a choline binding domain followed by a proteolytic site specific for the TEV (tobacco etch virus) endopeptidase. Based on the choline-binding His-tag combination the new 16.5 kDa tag was named CHiC. CHiC-tagged PcsB and ECL1 were expressed in Escherichia coli and sequentially purified by employing diethylaminoethyl-cellulose affinity chromatography and Ni(2+) immobilized metal affinity chromatography. After TEV digestion, the CHiC-tag, TEV-protease and undigested fusion protein were easily separated from the target protein in a single purification step. By using this method, 4-7 mg of recombinant PcsB and ECL1 were obtained from one liter of cell culture with a purity estimated to be at least 95%. In addition, we found that the tag has the potential to function as a solubilisation partner as it markedly increased the solubility of PcsB. In sum, the CHiC-tag is a versatile tool that allows purification of milligram quantities of highly purified recombinant protein in only one or two steps.