Choline-binding protein D (CbpD) in Streptococcus pneumoniae is essential for competence-induced cell lysis

Streptococcus pneumoniae is an important human pathogen that is able to take up naked DNA from the environment by a quorum-sensing-regulated process called natural genetic transformation. This property enables members of this bacterial species to efficiently acquire new properties that may increase their ability to survive and multiply in the human host. We have previously reported that induction of the competent state in a liquid culture of Streptococcus pneumoniae triggers lysis of a subfraction of the bacterial population resulting in release of DNA. We have also proposed that such competence-induced DNA release is an integral part of natural genetic transformation that has evolved to increase the efficiency of gene transfer between pneumococci. In the present work, we have further elucidated the mechanism behind competence-induced cell lysis by identifying a putative murein hydrolase, choline-binding protein D (CbpD), as a key component of this process. By using real-time PCR to estimate the amount of extracellular DNA in competent relative to noncompetent cultures, we were able to show that competence-induced cell lysis and DNA release are strongly attenuated in a cbpD mutant. Ectopic expression of CbpD in the presence or absence of other competence proteins revealed that CbpD is essentially unable to cause cell lysis on its own but depends on at least one additional protein expressed during competence.     

Competence-induced cells of Streptococcus pneumoniae lyse competence-deficient cells of the same strain during cocultivation

Several streptococcal species are able to take up naked DNA from the environment and integrate it into their genomes by homologous recombination. This process is called natural transformation. In Streptococcus pneumoniae and related streptococcal species, competence for natural transformation is induced by a peptide pheromone through a quorum-sensing mechanism. Recently we showed that induction of the competent state initiates lysis and release of DNA from a subfraction of the bacterial population and that the efficiency of this process is influenced by cell density. Here we have further investigated the nature of this cell density-dependent release mechanism. Interestingly, we found that competence-induced pneumococci lysed competence-deficient cells of the same strain during cocultivation and that the efficiency of this heterolysis increased as the ratio of competent to noncompetent cells increased. Furthermore, our results indicate that the lysins made by competent pneumococci are not released into the growth medium. More likely, they are anchored to the surface of the competent cells by choline-binding domains and cause lysis of noncompetent pneumococci through cell-to-cell contact.     

LytF, a novel competence-regulated murein hydrolase in the genus Streptococcus

Streptococcus pneumoniae and probably most other members of the genus Streptococcus are competent for natural genetic transformation. During the competent state, S. pneumoniae produces a murein hydrolase, CbpD, that kills and lyses noncompetent pneumococci and closely related species. Previous studies have shown that CbpD is essential for efficient transfer of genomic DNA from noncompetent to competent cells in vitro. Consequently, it has been proposed that CbpD together with the cognate immunity protein ComM constitutes a DNA acquisition mechanism that enables competent pneumococci to capture homologous DNA from closely related streptococci sharing the same habitat. Although genes encoding CbpD homologs or CbpD-related proteins are present in many different streptococcal species, the genomes of a number of streptococci do not encode CbpD-type proteins. In the present study we show that the genomes of nearly all species lacking CbpD encode an unrelated competence-regulated murein hydrolase termed LytF. Using Streptococcus gordonii as a model system, we obtained evidence indicating that LytF is a functional analogue of CbpD. In sum, our results show that a murein hydrolase gene is part of the competence regulon of most or all streptococcal species, demonstrating that these muralytic enzymes constitute an essential part of the streptococcal natural transformation system.     

Co-ordinated bacteriocin production and competence development: a possible mechanism for taking up DNA from neighbouring species

It is important to ensure DNA availability when bacterial cells develop competence. Previous studies in Streptococcus pneumoniae demonstrated that the competence-stimulating peptide (CSP) induced autolysin production and cell lysis of its own non-competent cells, suggesting a possible active mechanism to secure a homologous DNA pool for uptake and recombination. In this study, we found that in Streptococcus mutans CSP induced co-ordinated expression of competence and mutacin production genes. This mutacin (mutacin IV) is a non-lantibiotic bacteriocin which kills closely related Streptococcal species such as S. gordonii. In mixed cultures of S. mutans and S. gordonii harbouring a shuttle plasmid, plasmid DNA transfer from S. gordonii to S. mutans was observed in a CSP and mutacin IV-dependent manner. Further analysis demonstrated an increased DNA release from S. gordonii upon addition of the partially purified mutacin IV extract. On the basis of these findings, we propose that Streptococcus mutans, which resides in a multispecies oral biofilm, may utilize the competence-induced bacteriocin production to acquire transforming DNA from other species living in the same ecological niche. This hypothesis is also consistent with a well-known phenomenon that a large genomic diversity exists among different S. mutans strains. This diversity may have resulted from extensive horizontal gene transfer.     

Fratricide is essential for efficient gene transfer between pneumococci in biofilms

Streptococcus pneumoniae and a number of commensal streptococcal species are competent for natural genetic transformation. The natural habitat of these bacteria is multispecies biofilms in the human oral cavity and nasopharynx. Studies investigating lateral transfer of virulence and antibiotic resistance determinants among streptococci have shown that interspecies as well as intraspecies gene exchange takes place in these environments. We have previously shown that the action of a competence-specific murein hydrolase termed CbpD strongly increases the rate of gene transfer between pneumococci grown in liquid cultures. CbpD is the key component of a bacteriolytic mechanism termed the fratricide mechanism. It is secreted by competent pneumococci and mediates the release of donor DNA from sensitive streptococci present in the same environment. However, in nature, gene exchange between streptococci takes place in biofilms and not in liquid cultures. In the present study, we therefore investigated whether CbpD affects the rate of gene transfer in laboratory-grown biofilms. Our results show that the fratricide mechanism has a strong positive impact on intrabiofilm gene exchange, indicating that it is important for active acquisition of homologous donor DNA under natural conditions. Furthermore, we found that competent biofilm cells of S. pneumoniae acquire a Nov(r) marker much more efficiently from neighboring cells than from the growth medium. Efficient lysis of target cells requires that CbpD act in conjunction with the murein hydrolase LytC. In contrast, the major autolysin LytA does not seem to be important for fratricide-mediated gene exchange in a biofilm environment.     

Competence-specific autolysis in Streptococcus sanguis

Streptococcus sanguis strain Wicky activated to competence for genetic transformation is known to undergo a rapid decrease in optical density upon transfer to an alkaline buffer containing reducing agents. We studied the mechanism of this autolysis-like process and made the following observations. The process was specific because preincubation of the competence inducing factor with a specific inactivating protein prevented both cellular lysis and acquisition of competence for genetic transformation. The optical density decrease of competent bacteria involved the release of a large fraction of intracellular protein, RNA and lipid. However, no hydrolysis of phospholipid and no degradation of cell wall polymers including peptidoglycan could be detected. No peptidoglycan hydrolase activity capable of degrading radiolabelled S. sanguis cell walls was detected in unfractionated S. sanguis extracts. It is suggested that autolysis of competent S. sanguis involves the activity of a novel type of murein hydrolase that introduces only a limited number of bond breaks into the peptidoglycan.     

Conditional mutation of an essential putative glycoprotease eliminates autolysis in Staphylococcus aureus

Our previous studies demonstrated that a putative Staphylococcus aureus glycoprotease (Gcp) is essential for bacterial survival, indicating that Gcp may be a novel target for developing antibacterial agents. However, the biological function of Gcp is unclear. In order to elucidate the reason that Gcp is required for growth, we examined the role of Gcp in bacterial autolysis, which is an important biological process for bacterial growth. Using both a spacp-regulated gcp expression strain and a TetR-regulated gcp antisense expression strain, we found that the down-regulation of gcp expression can effectively inhibit Triton X-100-induced lysis, eliminate penicillin- and vancomycin-caused cell lysis, and dramatically increase tolerance to hydrolases. Moreover, we determined whether resistance to lysis is due to a defect in murein hydrolase activity by using a zymogram analysis. The results showed that the cell lysate of a down-regulated gcp expression mutant displayed several bands of decreased murein hydrolytic activity. Furthermore, we explored the potential mechanism of Gcp's involvement in autolysis and demonstrated that Gcp may function independently from several key autolysins (Atl, LytM, and LytN) and regulators (ArlRS, Mgr/Rat, and CidA). Taken together, the above results indicate that the essential Gcp is involved in the modification of substrates of murein hydrolases as well as in the regulation of expression and/or activity of some murein hydrolases, which, in turn, may play important roles in bacterial viability.     

Studies on Transformations of Hemophilus influenzae : I. Competence

A procedure has been developed for obtaining Hemophilus influenzae of such competence that 1 to 10 per cent transform to any of several genetic factors by utilizing a period of aerobic growth followed by a non-aerobic period. Differences in levels of competence were not due to differences in genetic background. Competence was due to at least one factor intrinsic to the cell or site on the cell and was not transferable to non-competent cells. Competence was affected by salt concentration, pH, and temperature. Washing competent cells reduces their ability to transform, but not their capacity to bind DNA reversibly. The irreversible step could be restored with little or no accompanying growth. These facts suggest that reversible and irreversible binding represent separate biochemical steps. DNA initiates a reaction in cells leading to a loss of competence. In the absence of DNA the cells remain competent for at least an hour. Competence correlates quantitatively with predictability of multiple transformations. The observed and calculated values of multiple transformations are in closer agreement, the higher the frequency of transformation for single markers. The correction needed to bring the two figures into agreement is a measure of the fraction of non-competent cells.     

Murein (peptidoglycan) structure, architecture and biosynthesis in Escherichia coli

The periplasmic murein (peptidoglycan) sacculus is a giant macromolecule made of glycan strands cross-linked by short peptides completely surrounding the cytoplasmic membrane to protect the cell from lysis due to its internal osmotic pressure. More than 50 different muropeptides are released from the sacculus by treatment with a muramidase. Escherichia coli has six murein synthases which enlarge the sacculus by transglycosylation and transpeptidation of lipid II precursor. A set of twelve periplasmic murein hydrolases (autolysins) release murein fragments during cell growth and division. Recent data on the in vitro murein synthesis activities of the murein synthases and on the interactions between murein synthases, hydrolases and cell cycle related proteins are being summarized. There are different models for the architecture of murein and for the incorporation of new precursor into the sacculus. We present a model in which morphogenesis of the rod-shaped E. coli is driven by cytoskeleton elements competing for the control over the murein synthesis multi-enzyme complexes.     

Murein (peptidoglycan) structure, architecture and biosynthesis in Escherichia coli

The periplasmic murein (peptidoglycan) sacculus is a giant macromolecule made of glycan strands cross-linked by short peptides completely surrounding the cytoplasmic membrane to protect the cell from lysis due to its internal osmotic pressure. More than 50 different muropeptides are released from the sacculus by treatment with a muramidase. Escherichia coli has six murein synthases which enlarge the sacculus by transglycosylation and transpeptidation of lipid II precursor. A set of twelve periplasmic murein hydrolases (autolysins) release murein fragments during cell growth and division. Recent data on the in vitro murein synthesis activities of the murein synthases and on the interactions between murein synthases, hydrolases and cell cycle related proteins are being summarized. There are different models for the architecture of murein and for the incorporation of new precursor into the sacculus. We present a model in which morphogenesis of the rod-shaped E. coli is driven by cytoskeleton elements competing for the control over the murein synthesis multi-enzyme complexes.     

Competence related proteins in the supernatant of competent cells of Bacillus subtilis

Summary We report that centrifugation at relatively high g-forces reduces the ability of competent cells of Bacillus subtilis to bind and take up DNA, and to be transformed. The centrifugation supernatant from competent cells restores this reduction of competence; the supernatant from non-competent cells is inactive. Phosphocellulose chromatography of centrifugation supernatants from radioactive competent cultures gave rise to six sharp peaks, together, these were shown by subsequent SDS polyacrylamide gel electrophoresis to contain over 60 different polypeptide bands. Peak II, which showed competence restoring activity, produced three polypeptides. When these bands were further examined, one of these exhibited DNA binding activity and the other two each contained a different endonuclease. Competence restoring activity was not recovered from the SDS polyacrylamide gel of peak II. The three peaks from non-competent cultures produced altogether five faint bands in gel electrophoresis. None of these bands were similar to those found in peak II.This work was performed in partial fulfillment of the requirements of Georgetown University for the degree of Doctor of Philosophy     

Overproduction of inactive variants of the murein synthase PBP1B causes lysis in Escherichia coli

Penicillin-binding protein 1B (PBP1B) of Escherichia coli is a bifunctional murein synthase containing both a transpeptidase domain and a transglycosylase domain. The protein is present in three forms (alpha, beta, and gamma) which differ in the length of their N-terminal cytoplasmic region. Expression plasmids allowing the production of native PBP1B or of PBP1B variants with an inactive transpeptidase or transglycosylase domain or both were constructed. The inactive domains contained a single amino acid exchange in an essential active-site residue. Overproduction of the inactive PBP1B variants, but not of the active proteins, caused lysis of wild-type cells. The cells became tolerant to lysis by inactive PBP1B at a pH of 5.0, which is similar to the known tolerance for penicillin-induced lysis under acid pH conditions. Lysis was also reduced in mutant strains lacking several murein hydrolases. In particular, a strain devoid of activity of all known lytic transglycosylases was virtually tolerant, indicating that mainly the lytic transglycosylases are responsible for the observed lysis effect. A possible structural interaction between PBP1B and murein hydrolases in vivo by the formation of a multienzyme complex is discussed.     

Localization of competence-induced proteins in Streptococcus pneumoniae.

Intracellular locations of 11 proteins associated with the development of competence in Streptococcus pneumoniae were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of subcellular fractions prepared from protoplasts. Controls showed that the competence-induced proteins were stable during the formation of protoplasts at 25 degrees C even though some had a half-life of only 8 min at 37 degrees C. Five competence-induced proteins p38, p27, p19.5, p16, and p14.5, were found in the cytoplasm. Two, p52 and p41, were associated with the membrane, and one, p10, was extracellular. Three others, p50, p36, and p29, were recovered in both cytoplasmic and membrane fractions. No competence-induced protein was detected in the periplasmic fraction except under conditions where leakage of all components was occurring, a phenomenon that was seen in many preparations. Similar fractionation of competent cells soon after uptake of [3H]DNA showed the "eclipse complex" of single-stranded DNA and p19.5 was associated approximately one-third with membranes and two-thirds with cytoplasmic fractions, with almost none in the periplasm. This result suggests strongly that at the time the donor DNA entered the cytosol it was in single-stranded form and it had not yet paired with the recipient DNA.     

Failure to trigger the autolytic enzymes in minicells of Escherichia coli

Minicells from Escherichia coli P678-54 are refractory towards procedures known to induce bacteriolysis of DNA-containing E. coli cells. Although still engaged in murein synthesis, minicells could not be lysed by penicillin G. Likewise, endogenous overproduction of the cloned soluble lytic transglycosylase, the predominant murein hydrolytic activity in E. coli, failed to lyse minicells. Furthermore, induction of the phage MS2 lysis protein, a hydrophobic protein assumed to trigger the autolytic system of the host, did not result in bacteriolysis. It is concluded that the murein hydrolases present in minicells are under a tight cellular control.     

A competence regulon in Streptococcus pneumoniae revealed by genomic analysis

Transformation in bacteria is the uptake and incorporation of exogenous DNA into a cell's genome. Several species transform naturally during a regulated state defined as competence. Genetic elements in Streptococcus pneumoniae induced during transformation were identified by combining a genetic screen with genomic analysis. Six loci were discovered that composed a competence-induced regulon. These loci shared a consensus promoter sequence and encoded proteins, some of which were similar to proteins involved in DNA processing during transformation in other bacteria. Each locus was induced during competence and essential for genetic transformation.     

Electroporation of Haemophilus influenzae is effective for transformation of plasmid but not chromosomal DNA.

Electroporation of plasmid and chromosomal DNAs were tested in Haemophilus influenzae because of an interest in introducing DNA into mutants that are deficient in competence for transformation. The initial experiments were designed to investigate and optimize conditions for electroporation of H. influenzae. Plasmid DNA was introduced into the competence proficient strain Rd and its competence-deficient uptake mutants com-52, com-59, and com-88, and the recombination deficient mutant rec1. Plasmid DNA could also be electroporated into the non-transforming strains Ra, Rc, Re and Rf. Plasmid DNA without sequences that are involved in tight binding (uptake) of DNA by competent cells of H. influenzae Rd was electroporated into both competent and non-competent cells. Competent cells were several orders of magnitude less efficient than non-competent cells for electroporation of plasmid DNAs. Electroporation of H. influenzae chromosomal DNA was not successful. Low levels of integration of chromosomal markers were observed following electroporation and these could be ascribed to transformation. The treatment of cells with DNasel following electroporation separated the effects due to electroporation from those due to transformation. The DNasel treatment did not affect the efficiency of plasmid incorporation, but severely restricted effects due to natural DNA transformation.