Sven Halbedel

Localisation of DivIVA by targeting to negatively curved membranes

DivIVA is a conserved protein in Gram‐positive bacteria and involved in various processes related to cell growth, cell division and spore formation. DivIVA is specifically targeted to cell division sites and cell poles. In Bacillus subtilis, DivIVA helps to localise other proteins, such as the conserved cell division inhibitor proteins, MinC/MinD, and the chromosome segregation protein, RacA. Little is known about the mechanism that localises DivIVA. Here we show that DivIVA binds to liposomes, and that the N terminus harbours the membrane targeting sequence. The purified protein can stimulate binding of RacA to membranes. In mutants with aberrant cell shapes, DivIVA accumulates where the cell membrane is most strongly curved. On the basis of electron microscopic studies and other data, we propose that this is due to molecular bridging of the curvature by DivIVA multimers. This model may explain why DivIVA localises at cell division sites. A Monte‐Carlo simulation study showed that molecular bridging can be a general mechanism for binding of proteins to negatively curved membranes.

Glycerol Metabolism Is Important for Cytotoxicity of Mycoplasma pneumoniae

Glycerol is one of the few carbon sources that can be utilized by Mycoplasma pneumoniae. Glycerol metabolism involves uptake by facilitated diffusion, phosphorylation, and the oxidation of glycerol 3-phosphate to dihydroxyacetone phosphate, a glycolytic intermediate. We have analyzed the expression of the genes involved in glycerol metabolism and observed constitutive expression irrespective of the presence of glycerol or preferred carbon sources. Similarly, the enzymatic activity of glycerol kinase is not modulated by HPr-dependent phosphorylation. This lack of regulation is unique among the bacteria for which glycerol metabolism has been studied so far. Two types of enzymes catalyze the oxidation of glycerol 3-phosphate: oxidases and dehydrogenases. Here, we demonstrate that the enzyme encoded by the M. pneumoniae glpD gene is a glycerol 3-phosphate oxidase that forms hydrogen peroxide rather than NADH(2). The formation of hydrogen peroxide by GlpD is crucial for cytotoxic effects of M. pneumoniae. A glpD mutant exhibited a significantly reduced formation of hydrogen peroxide and a severely reduced cytotoxicity. Attempts to isolate mutants affected in the genes of glycerol metabolism revealed that only the glpD gene, encoding the glycerol 3-phosphate oxidase, is dispensable. In contrast, the glpF and glpK genes, encoding the glycerol facilitator and the glycerol kinase, respectively, are essential in M. pneumoniae. Thus, the enzymes of glycerol metabolism are crucial for the pathogenicity of M. pneumoniae but also for other essential, yet-to-be-identified functions in the M. pneumoniae cell.

Features Critical for Membrane Binding Revealed by Diviva Crystal Structure.

DivIVA is a conserved protein in Gram‐positive bacteria that localizes at the poles and division sites, presumably through direct sensing of membrane curvature. DivIVA functions as a scaffold and is vital for septum site selection during vegetative growth and chromosome anchoring during sporulation. DivIVA deletion causes filamentous growth in Bacillus subtilis, whereas overexpression causes hyphal branching in Streptomyces coelicolor. We have determined the crystal structure of the N‐terminal (Nt) domain of DivIVA, and show that it forms a parallel coiled‐coil. It is capped with two unique crossed and intertwined loops, exposing hydrophobic and positively charged residues that we show here are essential for membrane binding. An intragenic suppressor introducing a positive charge restores membrane binding after mutating the hydrophobic residues. We propose that the hydrophobic residues insert into the membrane and that the positively charged residues bind to the membrane surface. A low‐resolution crystal structure of the C‐terminal (Ct) domain displays a curved tetramer made from two parallel coiled‐coils. The Nt and Ct parts were then merged into a model of the full length, 30 nm long DivIVA protein.

Multiple-Mutation Reaction: a Method for Simultaneous Introduction of Multiple Mutations into the glpK Gene of Mycoplasma pneumoniae

ABSTRACT In Mycoplasma pneumoniae, the UGA opal codon specifies tryptophan rather than a translation stop site. This often makes it difficult to express Mycoplasma proteins in E. coli isolates. In this work, we developed a strategy for the one-step introduction of several mutations. This method, the multiple-mutation reaction, is used to simultaneously replace nine opal codons in the M. pneumoniae glpK gene.

In Vivo Activity of Enzymatic and Regulatory Components of the Phosphoenolpyruvate:Sugar Phosphotransferase System in Mycoplasma pneumoniae

Mycoplasma pneumoniae is a pathogenic bacterium that is highly adapted to life on mucosal surfaces. This adaptation is reflected by the very compact genome and the small number of regulatory proteins. However, M. pneumoniae possesses the HPr kinase/phosphorylase (HPrK/P), the key regulator of carbon metabolism in the Firmicutes. In contrast to the enzymes of other bacteria, the HPrK/P of M. pneumoniae is already active at very low ATP concentrations, suggesting a different mode of regulation. In this work, we studied the ability of M. pneumoniae to utilize different carbohydrates and their effects on the activity of the different phosphotransferase system (PTS) components. Glucose served as the best carbon source, with a generation time of about 30 h. Fructose and glycerol were also used but at lower rates and with lower yields. In contrast, M. pneumoniae is unable to use mannitol even though the bacterium is apparently equipped with all the genes required for mannitol catabolism. This observation is probably a reflection of the continuing and ongoing reduction of the M. pneumoniae genome. The general enzymatic and regulatory components of the PTS, i.e., enzyme I, HPr, and HPrK/P, were present under all growth conditions tested in this study. However, HPrK/P activity is strongly increased if the medium contains glycerol. Thus, the control of HPrK/P in vivo differs strongly between M. pneumoniae and the other Firmicutes. This difference may relate to the specific conditions on lipid-rich cell surfaces.

Regulatory Protein Phosphorylation in Mycoplasma pneumoniae A PP2C-TYPE PHOSPHATASE SERVES TO DEPHOSPHORYLATE HPr(Ser-P)

Among the few regulatory events in the minimal bacterium Mycoplasma pneumoniae is the phosphorylation of the HPr phosphocarrier protein of the phosphotransferase system. In the presence of glycerol, HPr is phosphorylated in an ATP-dependent manner by the HPr kinase/phosphorylase. The role of the latter enzyme was studied by constructing a M. pneumoniae hprK mutant defective in HPr kinase/phosphorylase. This mutant strain no longer exhibited HPr kinase activity but, surprisingly, still had phosphatase activity toward serine-phosphorylated HPr (HPr(Ser-P)). An inspection of the genome sequence revealed the presence of a gene (prpC) encoding a presumptive protein serine/threonine phosphatase of the PP2C family. The phosphatase PrpC was purified and its biochemical activity in HPr(Ser-P) dephosphorylation demonstrated. Moreover, a prpC mutant strain was isolated and found to be impaired in HPr(Ser-P) dephosphorylation. Homologues of PrpC are present in many bacteria possessing HPr(Ser-P), suggesting that PrpC may play an important role in adjusting the cellular HPr phosphorylation state and thus controlling the diverse regulatory functions exerted by the different forms of HPr.

DivIVA affects secretion of virulence-related autolysins in Listeria monocytogenes.

DivIVA is a well‐conserved coiled‐coil protein present in most Gram‐positive bacteria and has been implicated in division site selection, peptidoglycan biosynthesis and sporulation. DivIVA proteins bind lipid membranes and characteristically accumulate at curved membrane areas, i.e. the cell poles and the division site, to which they recruit various interaction partners. We have studied the role of this morphogen in the human pathogen Listeria monocytogenes and our results suggest a novel mechanism by which DivIVA contributes to cell division. Contrary to expectation a ΔdivIVA mutant exhibited a pronounced chaining phenotype rather than a defect in cell division which we attributed to reduced extracellular levels of the autolytic enzymes p60 and MurA. We demonstrate that this is due to a malfunction in secretion of these autolysins and phenotypic comparison of the ΔdivIVA strain with a ΔsecA2 mutant suggests that DivIVA influences the activity of the SecA2 secretion route in L. monocytogenes. Also from the phenotypic analysis it was clear that divIVA affected swarming motility, biofilm formation, invasiveness and cell‐to‐cell spread in cell culture infection models. Thus, our experiments show that DivIVA is an important factor for various listerial traits that are essential for the pathogenicity of this organism.

Protein-Protein Interaction Domains of Bacillus subtilis DivIVA

DivIVA proteins are curvature-sensitive membrane binding proteins that recruit other proteins to the poles and the division septum. They consist of a conserved N-terminal lipid binding domain fused to a less conserved C-terminal domain. DivIVA homologues interact with different proteins involved in cell division, chromosome segregation, genetic competence, or cell wall synthesis. It is unknown how DivIVA interacts with these proteins, and we used the interaction of Bacillus subtilis DivIVA with MinJ and RacA to investigate this. MinJ is a transmembrane protein controlling division site selection, and the DNA-binding protein RacA is crucial for chromosome segregation during sporulation. Initial bacterial two-hybrid experiments revealed that the C terminus of DivIVA appears to be important for recruiting both proteins. However, the interpretation of these results is limited since it appeared that C-terminal truncations also interfere with DivIVA oligomerization. Therefore, a chimera approach was followed, making use of the fact that Listeria monocytogenes DivIVA shows normal polar localization but is not biologically active when expressed in B. subtilis. Complementation experiments with different chimeras of B. subtilis and L. monocytogenes DivIVA suggest that MinJ and RacA bind to separate DivIVA domains. Fluorescence microscopy of green fluorescent protein-tagged RacA and MinJ corroborated this conclusion and suggests that MinJ recruitment operates via the N-terminal lipid binding domain, whereas RacA interacts with the C-terminal domain. We speculate that this difference is related to the cellular compartments in which MinJ and RacA are active: the cell membrane and the cytoplasm, respectively.

Architecturally the same, but playing a different game: The diverse species-specific roles of DivIVA proteins

Homologs of the Bacillus subtilis DivIVA protein can be found in a wide variety of Gram-positive bacteria. DivIVA proteins are coiled-coil proteins that bind to the cytosolic face of the cytoplasmic membrane and accumulate at membrane regions with higher curvature. By directly interacting with downstream proteins they serve as spatial regulators of other cellular processes. Initial DivIVA studies focused on its role as a topological determinant for the MinCDJ division inhibiting complex in B. subtilis, but recent evidence suggests that DivIVA can fulfill more diverse roles in different species of the firmicutes and actinomycetes and can even be relevant for virulence.

Structure of the bacterial cell division determinant GpsB and its interaction with penicillin‐binding proteins

Each bacterium has to co‐ordinate its growth with division to ensure genetic stability of the population. Consequently, cell division and growth are tightly regulated phenomena, albeit different bacteria utilise one of several alternative regulatory mechanisms to maintain control. Here we consider GpsB, which is linked to cell growth and division in Gram‐positive bacteria. ΔgpsB mutants of the human pathogen Listeria monocytogenes show severe lysis, division and growth defects due to distortions of cell wall biosynthesis. Consistent with this premise, GpsB interacts both in vitro and in vivo with the major bi‐functional penicillin‐binding protein. We solved the crystal structure of GpsB and the interaction interfaces in both proteins are identified and validated. The inactivation of gpsB results in strongly attenuated virulence in animal experiments, comparable in degree to classical listerial virulence factor mutants. Therefore, GpsB is essential for in vitro and in vivo growth of a highly virulent food‐borne pathogen, suggesting that GpsB could be a target for the future design of novel antibacterials.