Klebsiella pneumoniae peptide hijacks a Streptococcus pneumoniae permease to subvert pneumococcal growth and colonization.

Treatment of pneumococcal infections is limited by antibiotic resistance and exacerbation of disease by bacterial lysis releasing pneumolysin toxin and other inflammatory factors. We identified a previously uncharacterized peptide in the Klebsiella pneumoniae secretome, which enters Streptococcus pneumoniae via its AmiA-AliA/AliB permease. Subsequent downregulation of genes for amino acid biosynthesis and peptide uptake was associated with reduction of pneumococcal growth in defined medium and human cerebrospinal fluid, irregular cell shape, decreased chain length and decreased genetic transformation. The bacteriostatic effect was specific to S. pneumoniae and Streptococcus pseudopneumoniae with no effect on Streptococcus mitis, Haemophilus influenzae, Staphylococcus aureus or K. pneumoniae. Peptide sequence and length were crucial to growth suppression. The peptide reduced pneumococcal adherence to primary human airway epithelial cell cultures and colonization of rat nasopharynx, without toxicity. We identified a peptide with potential as a therapeutic for pneumococcal diseases suppressing growth of multiple clinical isolates, including antibiotic resistant strains, while avoiding bacterial lysis and dysbiosis.

A novel proteinaceous molecule produced by Lysinibacillus sp. OF-1 depends on the Ami oligopeptide transporter to kill Streptococcus pneumoniae.

Infections caused by antibiotic-resistant Streptococcus pneumoniae are of growing concern for healthcare systems, which need new treatment options. Screening microorganisms in terrestrial environments has proved successful for discovering antibiotics, while production of antimicrobials by marine microorganisms remains underexplored. Here we have screened microorganisms sampled from the Oslo Fjord in Norway for production of molecules that prevent the human pathogen S. pneumoniae from growing. A bacterium belonging to the genus Lysinibacillus was identified. We show that this bacterium produces a molecule that kills a wide range of streptococcal species. Genome mining in BAGEL4 and AntiSmash suggested that it was a new antimicrobial compound, and we therefore named it lysinicin OF. The compound was resistant to heat (100 °C) and polymyxin acylase but susceptible to proteinase K, showing that it is of proteinaceous nature, but most probably not a lipopeptide. S. pneumoniae became resistant to lysinicin OF by obtaining suppressor mutations in the ami locus, which encodes the AmiACDEF oligo peptide transporter. We created ΔamiC and ΔamiEF mutants to show that pneumococci expressing a compromised Ami system were resistant to lysinicin OF. Furthermore, by creating mutants expressing an intact but inactive Ami system (AmiED184A and AmiFD175A) we could conclude that the lysinicin OF activity depended on the active form (ATP-hydrolysing) of the Ami system. Microscopic imaging and fluorescent labelling of DNA showed that S. pneumoniae treated with lysinicin OF had an average reduced cell size with condensed DNA nucleoid, while the integrity of the cell membrane remained intact. The characteristics and possible mode of action of lysinicin OF are discussed.

Competence remodels the pneumococcal cell wall exposing key surface virulence factors that mediate increased host adherence.

Competence development in the human pathogen Streptococcus pneumoniae controls several features such as genetic transformation, biofilm formation, and virulence. Competent bacteria produce so-called "fratricins" such as CbpD that kill noncompetent siblings by cleaving peptidoglycan (PGN). CbpD is a choline-binding protein (CBP) that binds to phosphorylcholine residues found on wall and lipoteichoic acids (WTA and LTA) that together with PGN are major constituents of the pneumococcal cell wall. Competent pneumococci are protected against fratricide by producing the immunity protein ComM. How competence and fratricide contribute to virulence is unknown. Here, using a genome-wide CRISPRi-seq screen, we show that genes involved in teichoic acid (TA) biosynthesis are essential during competence. We demonstrate that LytR is the major enzyme mediating the final step in WTA formation, and that, together with ComM, is essential for immunity against CbpD. Importantly, we show that key virulence factors PspA and PspC become more surface-exposed at midcell during competence, in a CbpD-dependent manner. Together, our work supports a model in which activation of competence is crucial for host adherence by increased surface exposure of its various CBPs.

SmdA is a Novel Cell Morphology Determinant in Staphylococcus aureus.

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.

Class A PBPs: It is time to rethink traditional paradigms.

Until recently, class A penicillin-binding proteins (aPBPs) were the only enzymes known to catalyze glycan chain polymerization from lipid II in bacteria. Hence, the discovery of two novel lipid II polymerases, FtsW and RodA, raises new questions and has consequently received a lot of attention from the research community. FtsW and RodA are essential and highly conserved members of the divisome and elongasome, respectively, and work in conjunction with their cognate class B PBPs (bPBPs) to synthesize the division septum and insert new peptidoglycan into the lateral cell wall. The identification of FtsW and RodA as peptidoglycan glycosyltransferases has raised questions regarding the role of aPBPs in peptidoglycan synthesis and fundamentally changed our understanding of the process. Despite their dethronement, aPBPs are essential in most bacteria. So, what is their function? In this review, we discuss recent progress in answering this question and present our own views on the topic.

EloR interacts with the lytic transglycosylase MltG at midcell in Streptococcus pneumoniae R6.

The ellipsoid shape of Streptococcus pneumoniae is determined by the synchronized actions of the elongasome and the divisome, which have the task of creating a protective layer of peptidoglycan (PG) enveloping the cell membrane. The elongasome is necessary for expanding PG in the longitudinal direction whereas the divisome synthesizes the PG that divides one cell into two. Although there is still little knowledge about how these two modes of PG synthesis are coordinated, it was recently discovered that two RNA-binding proteins called EloR and KhpA are part of a novel regulatory pathway controlling elongation in S. pneumoniae EloR and KhpA form a complex that work closely with the Ser/Thr kinase StkP to regulate cell elongation. Here, we have further explored how this regulation occur. EloR/KhpA is found at midcell, a localization fully dependent on EloR. Using a bacterial two-hybrid assay we probed EloR against several elongasome proteins and found an interaction with the lytic transglycosylase homolog MltG. By using EloR as bait in immunoprecipitation assays, MltG was pulled down confirming that they are part of the same protein complex. Fluorescent microscopy demonstrated that the Jag domain of EloR is essential for EloR's midcell localization and its interaction with MltG. Since MltG is found at midcell independent of EloR, our results suggest that MltG is responsible for recruitment of the EloR/KhpA complex to the division zone to regulate cell elongation.Importance Bacterial cell division has been a successful target for antimicrobial agents for decades. How different pathogens regulate cell division is, however, poorly understood. To fully exploit the potential for future antibiotics targeting cell division, we need to understand the details of how the bacteria regulate and construct cell wall during this process. Here we have revealed that the newly identified EloR/KhpA complex, regulating cell elongation in S. pneumoniae, forms a complex with the essential peptidoglycan transglycosylase MltG at midcell. EloR, KhpA and MltG are conserved among many bacterial species and the EloR/KhpA/MltG regulatory pathway is most likely a common mechanism employed by many Gram-positive bacteria to coordinate cell elongation and septation.

A CozE Homolog Contributes to Cell Size Homeostasis of Streptococcus pneumoniae.

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.

Structural Characterization of the Essential Cell Division Protein FtsE and Its Interaction with FtsX in Streptococcus pneumoniae.

FtsEX is a membrane complex widely conserved across diverse bacterial genera and involved in critical processes such as recruitment of division proteins and in spatial and temporal regulation of muralytic activity during cell division or sporulation. FtsEX is a member of the ABC transporter superfamily. The component FtsX is an integral membrane protein, whereas FtsE is an ATPase and is required for the transmission of a conformational signal from the cytosol through the membrane to regulate the activity of cell wall hydrolases in the periplasm. Both proteins are essential in the major human respiratory pathogenic bacterium Streptococcus pneumoniae, and FtsX interacts with the modular peptidoglycan hydrolase PcsB at the septum. Here, we report high-resolution structures of pneumococcal FtsE bound to different nucleotides. Structural analysis revealed that FtsE contains all the conserved structural motifs associated with ATPase activity and afforded interpretation of the in vivo dimeric arrangement in both the ADP and ATP states. Interestingly, three specific FtsE regions with high structural plasticity were identified that shape the cavity in which the cytosolic region of FtsX would be inserted. The residues corresponding to the FtsX coupling helix, responsible for contacting FtsE, were identified and validated by in vivo mutagenesis studies showing that this interaction is essential for cell growth and proper morphology.IMPORTANCE Bacterial cell division is a central process that requires exquisite orchestration of both the cell wall biosynthetic and lytic machineries. The essential membrane complex FtsEX, widely conserved across bacteria, plays a central role by recruiting proteins to the divisome apparatus and by regulating periplasmic muralytic activity from the cytosol. FtsEX is a member of the type VII family of the ABC-superfamily, but instead of being a transporter, it couples the ATP hydrolysis catalyzed by FtsE to mechanically transduce a conformational signal that provokes the activation of peptidoglycan (PG) hydrolases. So far, no structural information is available for FtsE. Here, we provide the structural characterization of FtsE, confirming its ATPase nature and revealing regions with high structural plasticity which are key for FtsE binding to FtsX. The complementary binding region in FtsX has also been identified and validated in vivo Our results provide evidence on how the difference between the ATP/ADP-bound states in FtsE would dramatically alter the interaction of FtsEX with the PG hydrolase PcsB in pneumococcal division.

Class A PBPs have a distinct and unique role in the construction of the pneumococcal cell wall.

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.

CRISPR Interference for Rapid Knockdown of Essential Cell Cycle Genes in Lactobacillus plantarum.

Studies of essential genes in bacteria are often hampered by the lack of accessible genetic tools. This is also the case for Lactobacillus plantarum, a key species in food and health applications. Here, we develop a clustered regularly interspaced short palindromic repeat interference (CRISPRi) system for knockdown of gene expression in L. plantarum The two-plasmid CRISPRi system, in which a nuclease-inactivated Cas9 (dCas9) and a gene-specific single guide RNA (sgRNA) are expressed on separate plasmids, allows efficient knockdown of expression of any gene of interest. We utilized the CRISPRi system to gain initial insights into the functions of key cell cycle genes in L. plantarum As a proof of concept, we investigated the phenotypes resulting from knockdowns of the cell wall hydrolase-encoding acm2 gene and of the DNA replication initiator gene dnaA and of ezrA, which encodes an early cell division protein. Furthermore, we studied the phenotypes of three cell division genes which have recently been functionally characterized in ovococcal bacteria but whose functions have not yet been investigated in rod-shaped bacteria. We show that the transmembrane CozE proteins do not seem to play any major role in cell division in L. plantarum On the other hand, RNA-binding proteins KhpA and EloR are critical for proper cell elongation in this bacterium.IMPORTANCEL. plantarum is an important bacterium for applications in food and health. Deep insights into the biology and physiology of this species are therefore necessary for further strain optimization and exploitation; however, the functions of essential genes in the bacterium are mainly unknown due to the lack of accessible genetic tools. The CRISPRi system developed here is ideal to quickly screen for phenotypes of both essential and nonessential genes. Our initial insights into the function of some key cell cycle genes represent the first step toward understanding the cell cycle in this bacterium.

Prevention of EloR/KhpA heterodimerization by introduction of site-specific amino acid substitutions renders the essential elongasome protein PBP2b redundant in Streptococcus pneumoniae.

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.

Structure of the Large Extracellular Loop of FtsX and Its Interaction with the Essential Peptidoglycan Hydrolase PcsB in Streptococcus pneumoniae.

Streptococcus pneumoniae is a leading killer of infants and immunocompromised adults and has become increasingly resistant to major antibiotics. Therefore, the development of new antibiotic strategies is desperately needed. Targeting bacterial cell division is one such strategy, specifically by targeting proteins that are essential for the synthesis and breakdown of peptidoglycan. One complex important to this process is FtsEX. FtsEX comprises a cell division-regulating integral membrane protein (FtsX) and a cytoplasmic ATPase (FtsE) that resembles an ATP-binding cassette (ABC) transporter. Here, we present nuclear magnetic resonance (NMR) solution structural and crystallographic models of the large extracellular domain of FtsX, denoted extracellular loop 1 (ECL1). The structure of ECL1 reveals an upper extended ß-hairpin and a lower α-helical lobe, each extending from a mixed α-ß core. The helical lobe mediates a physical interaction with the peptidoglycan hydrolase PcsB via the coiled-coil domain of PcsB (PscBCC). Characterization of S. pneumoniae strain D39-derived strains harboring mutations in the α-helical lobe shows that this subdomain is essential for cell viability and required for proper cell division of S. pneumoniaeIMPORTANCE FtsX is a ubiquitous bacterial integral membrane protein involved in cell division that regulates the activity of peptidoglycan (PG) hydrolases. FtsX is representative of a large group of ABC3 superfamily proteins that function as "mechanotransmitters," proteins that relay signals from the inside to the outside of the cell. Here, we present a structural characterization of the large extracellular loop, ECL1, of FtsX from the opportunistic human pathogen S.pneumoniae We show the molecular nature of the direct interaction between the peptidoglycan hydrolase PcsB and FtsX and demonstrate that this interaction is essential for cell viability. As such, FtsX represents an attractive, conserved target for the development of new classes of antibiotics.

Structure of the essential peptidoglycan amidotransferase MurT/GatD complex from Streptococcus pneumoniae.

The universality of peptidoglycan in bacteria underlies the broad spectrum of many successful antibiotics. However, in our times of widespread resistance, the diversity of peptidoglycan modifications offers a variety of new antibacterials targets. In some Gram-positive species such as Streptococcus pneumoniae, Staphylococcus aureus, or Mycobacterium tuberculosis, the second residue of the peptidoglycan precursor, D-glutamate, is amidated into iso-D-glutamine by the essential amidotransferase MurT/GatD complex. Here, we present the structure of this complex at 3.0 Å resolution. MurT has central and C-terminal domains similar to Mur ligases with a cysteine-rich insertion, which probably binds zinc, contributing to the interface with GatD. The mechanism of amidation by MurT is likely similar to the condensation catalyzed by Mur ligases. GatD is a glutaminase providing ammonia that is likely channeled to the MurT active site through a cavity network. The structure and assay presented here constitute a knowledge base for future drug development studies.

CozEa and CozEb play overlapping and essential roles in controlling cell division in Staphylococcus aureus.

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.

Identification of EloR (Spr1851) as a regulator of cell elongation in Streptococcus pneumoniae.

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.

The Leaderless Bacteriocin Enterocin K1 Is Highly Potent against Enterococcus faecium: A Study on Structure, Target Spectrum and Receptor.

Enterocin K1 (EntK1), enterocin EJ97 (EntEJ97), and LsbB are three sequence related leaderless bacteriocins. Yet LsbB kills only lactococci while EntK1 and EntEJ97 target wider spectra with EntK1 being particularly active against Enterococcus faecium, including nosocomial multidrug resistant isolates. NMR study of EntK1 showed that it had a structure very similar to LsbB - both having an amphiphilic N-terminal α-helix and an unstructured C-terminus. The α-helix in EntK1 is, however, about 3-4 residues longer than that of LsbB. Enterococcal mutants highly resistant to EntEJ97 and EntK1 were found to have mutations within rseP, a gene encoding a stress response membrane-bound Zn-dependent protease. Heterologous expression of the enterococcal rseP rendered resistant cells of Streptococcus pneumoniae sensitive to EntK1 and EntEJ97, suggesting that RseP likely serves as the receptor for EntK1 and EntEJ97. It was also shown that the conserved proteolytic active site in E. faecalis RseP is partly required for EntK1 and EntEJ97 activity, since alanine substitutions of its conserved residues (HExxH) reduced the sensitivity of the clones to the bacteriocins. RseP is known to be involved in bacterial stress response. As expected, the growth of resistant mutants with mutations within rseP was severely affected when they were exposed to higher (stressing) growth temperatures, e.g., at 45°C, at which wild type cells still grew well. These findings allow us to design a hurdle strategy with a combination of the bacteriocin(s) and higher temperature that effectively kills bacteriocin sensitive bacteria and prevents the development of resistant cells.

Identification of pneumococcal proteins that are functionally linked to penicillin-binding protein 2b (PBP2b).

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.

Overexpression of the fratricide immunity protein ComM leads to growth inhibition and morphological abnormalities in Streptococcus pneumoniae.

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 △rseP 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.

Evidence that pneumococcal WalK is regulated by StkP through protein-protein interaction.

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.

Natural transformation and genome evolution in Streptococcus pneumoniae.

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.