Mitochondrial F-ATP Synthase Co-Migrating Proteins and Ca2+-Dependent Formation of Large Channels.

Monomers, dimers, and individual FOF1-ATP synthase subunits are, presumably, involved in the formation of the mitochondrial permeability transition pore (PTP), whose molecular structure, however, is still unknown. We hypothesized that, during the Ca2+-dependent assembly of a PTP complex, the F-ATP synthase (subunits) recruits mitochondrial proteins that do not interact or weakly interact with the F-ATP synthase under normal conditions. Therefore, we examined whether the PTP opening in mitochondria before the separation of supercomplexes via BN-PAGE will increase the channel stability and channel-forming capacity of isolated F-ATP synthase dimers and monomers in planar lipid membranes. Additionally, we studied the specific activity and the protein composition of F-ATP synthase dimers and monomers from rat liver and heart mitochondria before and after PTP opening. Against our expectations, preliminary PTP opening dramatically suppressed the high-conductance channel activity of F-ATP synthase dimers and monomers and decreased their specific "in-gel" activity. The decline in the channel-forming activity correlated with the reduced levels of as few as two proteins in the bands: methylmalonate-semialdehyde dehydrogenase and prohibitin 2. These results indicate that proteins co-migrating with the F-ATP synthase may be important players in PTP formation and stabilization.

N-acetylmuramic acid recognition by MurK kinase from the MurNAc auxotrophic oral pathogen Tannerella forsythia.

The bacterial cell wall consists of a three-dimensional peptidoglycan layer, composed of peptides linked to the sugars N-acetylmuramic acid (MurNAc) and GlcNAc. Unlike other bacteria, the pathogenic Tannerella forsythia, a member of the red complex group of bacteria associated with the late stages of periodontitis, lacks biosynthetic pathways for MurNAc production and therefore obtains MurNAc from the environment. Sugar kinases play a crucial role in the MurNAc recycling process, activating the sugar molecules by phosphorylation. In this study, we present the first crystal structures of a MurNAc kinase, called murein sugar kinase (MurK), in its unbound state as well as in complexes with the ATP analog ß-γ-methylene adenosine triphosphate (AMP-PCP) and with MurNAc. We also determined the crystal structures of K1058, a paralogous MurNAc kinase of T. forsythia, in its unbound state and in complex with MurNAc. We identified the active site and residues crucial for MurNAc specificity as the less bulky side chains of S133, P134, and L135, which enlarge the binding cavity for the lactyl ether group, unlike the glutamate or histidine residues present in structural homologs. In establishing the apparent kinetic parameters for both enzymes, we showed a comparable affinity for MurNAc (Km 180 µM and 30 µM for MurK and K1058, respectively), with MurK being over two hundred times faster than K1058 (Vmax 80 and 0.34 µmol min-1 mg-1, respectively). These data might support a structure-guided approach to development of inhibitory MurNAc analogs for pathogen MurK enzymes.

Pleiotropic effects of PipX, PipY, or RelQ overexpression on growth, cell size, photosynthesis, and polyphosphate accumulation in the cyanobacterium Synechococcus elongatus PCC7942.

The cyanobacterial protein PipY belongs to the Pyridoxal-phosphate (PLP)-binding proteins (PLPBP/COG0325) family of pyridoxal-phosphate-binding proteins, which are represented in all three domains of life. These proteins share a high degree of sequence conservation, appear to have purely regulatory functions, and are involved in the homeostasis of vitamin B6 vitamers and amino/keto acids. Intriguingly, the genomic context of the pipY gene in cyanobacteria connects PipY with PipX, a protein involved in signaling the intracellular energy status and carbon-to-nitrogen balance. PipX regulates its cellular targets via protein-protein interactions. These targets include the PII signaling protein, the ribosome assembly GTPase EngA, and the transcriptional regulators NtcA and PlmA. PipX is thus involved in the transmission of multiple signals that are relevant for metabolic homeostasis and stress responses in cyanobacteria, but the exact function of PipY is still elusive. Preliminary data indicated that PipY might also be involved in signaling pathways related to the stringent stress response, a pathway that can be induced in the unicellular cyanobacterium Synechococcus elongatus PCC7942 by overexpression of the (p)ppGpp synthase, RelQ. To get insights into the cellular functions of PipY, we performed a comparative study of PipX, PipY, or RelQ overexpression in S. elongatus PCC7942. Overexpression of PipY or RelQ caused similar phenotypic responses, such as growth arrest, loss of photosynthetic activity and viability, increased cell size, and accumulation of large polyphosphate granules. In contrast, PipX overexpression decreased cell length, indicating that PipX and PipY play antagonistic roles on cell elongation or cell division. Since ppGpp levels were not induced by overexpression of PipY or PipX, it is apparent that the production of polyphosphate in cyanobacteria does not require induction of the stringent response.

YgfB increases ß-lactam resistance in Pseudomonas aeruginosa by counteracting AlpA-mediated ampDh3 expression.

YgfB-mediated ß-lactam resistance was recently identified in multi drug resistant Pseudomonas aeruginosa. We show that YgfB upregulates expression of the ß-lactamase AmpC by repressing the function of the regulator of the programmed cell death pathway AlpA. In response to DNA damage, the antiterminator AlpA induces expression of the alpBCDE autolysis genes and of the peptidoglycan amidase AmpDh3. YgfB interacts with AlpA and represses the ampDh3 expression. Thus, YgfB indirectly prevents AmpDh3 from reducing the levels of cell wall-derived 1,6-anhydro-N-acetylmuramyl-peptides, required to induce the transcriptional activator AmpR in promoting the ampC expression and ß-lactam resistance. Ciprofloxacin-mediated DNA damage induces AlpA-dependent production of AmpDh3 as previously shown, which should reduce ß-lactam resistance. YgfB, however, counteracts the ß-lactam enhancing activity of ciprofloxacin by repressing ampDh3 expression and lowering the benefits of this drug combination. Altogether, YgfB represents an additional player in the complex regulatory network of AmpC regulation.

Synthesis of 3-Aryl-3-(Furan-2-yl)Propanoic Acid Derivatives, and Study of Their Antimicrobial Activity.

Reactions of 3-(furan-2-yl)propenoic acids and their esters with arenes in Brønsted superacid TfOH affords products of hydroarylation of the carbon-carbon double bond, 3-aryl-3-(furan-2-yl)propenoic acid derivatives. According to NMR and DFT studies, the corresponding O,C-diprotonated forms of the starting furan acids and esters should be reactive electrophilic species in these transformations. Starting compounds and their hydroarylation products, at a concentration of 64 µg/mL, demonstrate good antimicrobial activity against yeast-like fungi Candida albicans. Apart from that, these compounds suppress Escherichia coli and Staphylococcus aureus.

Fluorogenic RNA-Based Biosensor to Sense the Glycolytic Flux in Mammalian Cells.

The visualization of metabolic flux in real time requires sensor molecules that transduce variations of metabolite concentrations into an appropriate output signal. In this regard, fluorogenic RNA-based biosensors are promising molecular tools as they fluoresce only upon binding to another molecule. However, to date no such sensor is available that enables the direct observation of key metabolites in mammalian cells. Toward this direction, we selected and characterized an RNA light-up sensor designed to respond to fructose 1,6-bisphosphate and applied it to probe glycolytic flux variation in mammal cells.

NamZ1 and NamZ2 from the Oral Pathogen Tannerella forsythia Are Peptidoglycan Processing Exo-ß-N-Acetylmuramidases with Distinct Substrate Specificities.

The Gram-negative periodontal pathogen Tannerella forsythia is inherently auxotrophic for N-acetylmuramic acid (MurNAc), which is an essential carbohydrate constituent of the peptidoglycan (PGN) of the bacterial cell wall. Thus, to build up its cell wall, T. forsythia strictly depends on the salvage of exogenous MurNAc or sources of MurNAc, such as polymeric or fragmentary PGN, derived from cohabiting bacteria within the oral microbiome. In our effort to elucidate how T. forsythia satisfies its demand for MurNAc, we recognized that the organism possesses three putative orthologs of the exo-ß-N-acetylmuramidase BsNamZ from Bacillus subtilis, which cleaves nonreducing end, terminal MurNAc entities from the artificial substrate pNP-MurNAc and the naturally-occurring disaccharide substrate MurNAc-N-acetylglucosamine (MurNAc-GlcNAc). TfNamZ1 and TfNamZ2 were successfully purified as soluble, pure recombinant His6-fusions and characterized as exo-lytic ß-N-acetylmuramidases with distinct substrate specificities. The activity of TfNamZ1 was considerably lower compared to TfNamZ2 and BsNamZ, in the cleavage of MurNAc-GlcNAc. When peptide-free PGN glycans were used as substrates, we revealed striking differences in the specificity and mode of action of these enzymes, as analyzed by mass spectrometry. TfNamZ1, but not TfNamZ2 or BsNamZ, released GlcNAc-MurNAc disaccharides from these glycans. In addition, glucosamine (GlcN)-MurNAc disaccharides were generated when partially N-deacetylated PGN glycans from B. subtilis 168 were applied. This characterizes TfNamZ1 as a unique disaccharide-forming exo-lytic ß-N-acetylmuramidase (exo-disaccharidase), and, TfNamZ2 and BsNamZ as sole MurNAc monosaccharide-lytic exo-ß-N-acetylmuramidases. IMPORTANCE Two exo-N-acetylmuramidases from T. forsythia belonging to glycosidase family GH171 (www.cazy.org) were shown to differ in their activities, thus revealing a functional diversity within this family: NamZ1 releases disaccharides (GlcNAc-MurNAc/GlcN-MurNAc) from the nonreducing ends of PGN glycans, whereas NamZ2 releases terminal MurNAc monosaccharides. This work provides a better understanding of how T. forsythia may acquire the essential growth factor MurNAc by the salvage of PGN from cohabiting bacteria in the oral microbiome, which may pave avenues for the development of anti-periodontal drugs. On a broad scale, our study indicates that the utilization of PGN as a nutrient source, involving exo-lytic N-acetylmuramidases with different modes of action, appears to be a general feature of bacteria, particularly among the phylum Bacteroidetes.

The C-terminal domain of Bacillus cereus hemolysin II oligomerizes by itself in the presence of cell membranes to form ion channels.

Bacillus cereus hemolysin II, a pore-forming ß-barrel toxin (HlyII), has a C-terminal extension of 94 amino acid residues, designated as the C-terminal domain of HlyII (HlyIICTD). HlyIICTD is capable of forming oligomers in aqueous solutions. Oligomerization of HlyIICTD significantly increased in the presence of erythrocytes and liposomes. Its affinity for erythrocytes of various origins differed insignificantly but was noticeably higher for T-cells. HlyIICTD destroyed THP-1 monocytes and J774 macrophages, acted most effectively on Jurkat T-lymphocytes and had virtually no impact on B-cell lines. HlyIICTD was able to form ion-conducting channels on an artificial bilayer membrane.

Peptidoglycan Salvage Enables the Periodontal Pathogen Tannerella forsythia to Survive within the Oral Microbial Community.

Tannerella forsythia is an anaerobic, fusiform Gram-negative oral pathogen strongly associated with periodontitis, a multibacterial inflammatory disease that leads to the destruction of the teeth-supporting tissue, ultimately causing tooth loss. To survive in the oral habitat, T. forsythia depends on cohabiting bacteria for the provision of nutrients. For axenic growth under laboratory conditions, it specifically relies on the external supply of N-acetylmuramic acid (MurNAc), which is an essential constituent of the peptidoglycan (PGN) of bacterial cell walls. T. forsythia comprises a typical Gram-negative PGN; however, as evidenced by genome sequence analysis, the organism lacks common enzymes required for the de novo synthesis of precursors of PGN, which rationalizes its MurNAc auxotrophy. Only recently insights were obtained into how T. forsythia gains access to MurNAc in its oral habitat, enabling synthesis of the own PGN cell wall. This report summarizes T. forsythia's strategies to survive in the oral habitat by means of PGN salvage pathways, including recovery of exogenous MurNAc and PGN-derived fragments but also polymeric PGN, which are all derived from cohabiting bacteria either via cell wall turnover or decay of cells. Salvage of polymeric PGN presumably requires the removal of peptides from PGN by an unknown amidase, concomitantly with the translocation of the polymer across the outer membrane. Two recently identified exo-lytic N-acetylmuramidases (Tf_NamZ1 and Tf_NamZ2) specifically cleave the peptide-free, exogenous (nutrition source) PGN in the periplasm and release the MurNAc and disaccharide substrates for the transporters Tf_MurT and Tf_AmpG, respectively, whereas the peptide-containing, endogenous (the self-cell wall) PGN stays unattached. This review also outlines how T. forsythia synthesises the PGN precursors UDP-MurNAc and UDP-N-acetylglucosamine (UDP-GlcNAc), involving homologs of the Pseudomonas sp. recycling enzymes AmgK/MurU and a monofunctional uridylyl transferase (named Tf_GlmU*), respectively.

The exo-ß-N-acetylmuramidase NamZ from Bacillus subtilis is the founding member of a family of exo-lytic peptidoglycan hexosaminidases.

Endo-ß-N-acetylmuramidases, commonly known as lysozymes, are well-characterized antimicrobial enzymes that catalyze an endo-lytic cleavage of peptidoglycan; i.e., they hydrolyze the ß-1,4-glycosidic bonds connecting N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc). In contrast, little is known about exo-ß-N-acetylmuramidases, which catalyze an exo-lytic cleavage of ß-1,4-MurNAc entities from the non-reducing ends of peptidoglycan chains. Such an enzyme was identified earlier in the bacterium Bacillus subtilis, but the corresponding gene has remained unknown so far. We now report that ybbC of B. subtilis, renamed namZ, encodes the reported exo-ß-N-acetylmuramidase. A ΔnamZ mutant accumulated specific cell wall fragments and showed growth defects under starvation conditions, indicating a role of NamZ in cell wall turnover and recycling. Recombinant NamZ protein specifically hydrolyzed the artificial substrate para-nitrophenyl ß-MurNAc and the peptidoglycan-derived disaccharide MurNAc-ß-1,4-GlcNAc. Together with the exo-ß-N-acetylglucosaminidase NagZ and the exo-muramoyl-l-alanine amidase AmiE, NamZ degraded intact peptidoglycan by sequential hydrolysis from the non-reducing ends. A structure model of NamZ, built on the basis of two crystal structures of putative orthologs from Bacteroides fragilis, revealed a two-domain structure including a Rossmann-fold-like domain that constitutes a unique glycosidase fold. Thus, NamZ, a member of the DUF1343 protein family of unknown function, is now classified as the founding member of a new family of glycosidases (CAZy GH171; www.cazy.org/GH171.html). NamZ-like peptidoglycan hexosaminidases are mainly present in the phylum Bacteroidetes and less frequently found in individual genomes within Firmicutes (Bacilli, Clostridia), Actinobacteria, and γ-proteobacteria.

Study of supramolecular complex of nifedipine with arabinogalactan on Wistar and ISIAH rats.

Aim: Physicochemical and pharmacological study of the supramolecular inclusion complexes of the hypotensive drug nifedipine (NF) with the larch polysaccharide arabinogalactan (AG). Materials & methods: The NF:AG complexes were obtained and their physicochemical properties were studied. Their hypotensive action and pharmacokinetic profiles were evaluated in rats with normal and elevated arterial blood pressure. Results: In both rat lines the NF:AG complex decreased the arterial blood pressure at a lower dose than free NF (1.75 mg/kg of NF in complex compared with 3.5 mg/kg of free NF) and has a better pharmacokinetic profile than free NF. Conclusion: The use of the NF:AG complex is an effective way to sufficiently enhance and hasten NF's hypotensive action.

Inducible expression of (pp)pGpp synthetases in Staphylococcus aureus is associated with activation of stress response genes.

The stringent response is characterized by the synthesis of the messenger molecules pppGpp, ppGpp or pGpp (here collectively designated (pp)pGpp). The phenotypic consequences resulting from (pp)pGpp accumulation vary among species and can be mediated by different underlying mechanisms. Most genome-wide analyses have been performed under stress conditions, which often mask the immediate effects of (pp)pGpp-mediated regulatory circuits. In Staphylococcus aureus, (pp)pGpp can be synthesized via the RelA-SpoT-homolog, RelSau upon amino acid limitation or via one of the two small (pp)pGpp synthetases RelP or RelQ upon cell wall stress. We used RNA-Seq to compare the global effects in response to induction of the synthetase of rel-Syn (coding for the enzymatic region of RelSau) or relQ without the need to apply additional stress conditions. Induction of rel-Syn resulted in changes in the nucleotide pool similar to induction of the stringent response via the tRNA synthetase inhibitor mupirocin: a reduction in the GTP pool, an increase in the ATP pool and synthesis of pppGpp, ppGpp and pGpp. Induction of all three enzymes resulted in similar changes in the transcriptome. However, RelQ was less active than Rel-Syn and RelP, indicating strong restriction of its (pp)pGpp-synthesis activity in vivo. (pp)pGpp induction resulted in the downregulation of many genes involved in protein and RNA/DNA metabolism. Many of the (pp)pGpp upregulated genes are part of the GTP sensitive CodY regulon and thus likely regulated through lowering of the GTP pool. New CodY independent transcriptional changes were detected including genes involved in the SOS response, iron storage (e.g. ftnA, dps), oxidative stress response (e.g., perR, katA, sodA) and the psmα1-4 and psmß1-2 operons coding for cytotoxic, phenol soluble modulins (PSMs). Analyses of the ftnA, dps and psm genes in different regulatory mutants revealed that their (pp)pGpp-dependent regulation can occur independent of the regulators PerR, Fur, SarA or CodY. Moreover, psm expression is uncoupled from expression of the quorum sensing system Agr, the main known psm activator. The expression of central genes of the oxidative stress response protects the bacteria from anticipated ROS stress derived from PSMs or exogenous sources. Thus, we identified a new link between the stringent response and oxidative stress in S. aureus that is likely crucial for survival upon phagocytosis.

Utilization of different MurNAc sources by the oral pathogen Tannerella forsythia and role of the inner membrane transporter AmpG.

BACKGROUND: The Gram-negative oral pathogen Tannerella forsythia strictly depends on the external supply of the essential bacterial cell wall sugar N-acetylmuramic acid (MurNAc) for survival because of the lack of the common MurNAc biosynthesis enzymes MurA/MurB. The bacterium thrives in a polymicrobial biofilm consortium and, thus, it is plausible that it procures MurNAc from MurNAc-containing peptidoglycan (PGN) fragments (muropeptides) released from cohabiting bacteria during natural PGN turnover or cell death. There is indirect evidence that in T. forsythia, an AmpG-like permease (Tanf_08365) is involved in cytoplasmic muropeptide uptake. In E. coli, AmpG is specific for the import of N-acetylglucosamine (GlcNAc)-anhydroMurNAc(-peptides) which are common PGN turnover products, with the disaccharide portion as a minimal requirement. Currently, it is unclear which natural, complex MurNAc sources T. forsythia can utilize and which role AmpG plays therein. RESULTS: We performed a screen of various putative MurNAc sources for T. forsythia mimicking the situation in the natural habitat and compared bacterial growth and cell morphology of the wild-type and a mutant lacking AmpG (T. forsythia ΔampG). We showed that supernatants of the oral biofilm bacteria Porphyromonas gingivalis and Fusobacterium nucleatum, and of E. coli ΔampG, as well as isolated PGN and defined PGN fragments obtained after enzymatic digestion, namely GlcNAc-anhydroMurNAc(-peptides) and GlcNAc-MurNAc(-peptides), could sustain growth of T. forsythia wild-type, while T. forsythia ΔampG suffered from growth inhibition. In supernatants of T. forsythia ΔampG, the presence of GlcNAc-anhMurNAc and, unexpectedly, also GlcNAc-MurNAc was revealed by tandem mass spectrometry analysis, indicating that both disaccharides are substrates of AmpG. The importance of AmpG in the utilization of PGN fragments as MurNAc source was substantiated by a significant ampG upregulation in T. forsythia cells cultivated with PGN, as determined by quantitative real-time PCR. Further, our results indicate that PGN-degrading amidase, lytic transglycosylase and muramidase activities in a T. forsythia cell extract are involved in PGN scavenging. CONCLUSION: T. forsythia metabolizes intact PGN as well as muropeptides released from various bacteria and the bacterium's inner membrane transporter AmpG is essential for growth on these MurNAc sources, and, contrary to the situation in E. coli, imports both, GlcNAc-anhMurNAc and GlcNAc-MurNAc fragments.

The Comparative Immunotropic Activity of Carrageenan, Chitosan and Their Complexes.

The immunotropic activity of polyelectrolyte complexes (PEC) of κ-carrageenan (κ-CGN) and chitosan (CH) of various compositions was assessed in comparison with the initial polysaccharides in comparable doses. For this, two soluble forms of PEC, with an excess of CH (CH:CGN mass ratios of 10:1) and with an excess of CGN (CH: CGN mass ratios of 1:10) were prepared. The ability of PEC to scavenge NO depended on the content of the κ-CGN in the PEC. The ability of the PEC to induce the synthesis of pro-inflammatory (tumor necrosis factor-α (TNF-α)) and anti-inflammatory (interleukine-10 (IL-10)) cytokines in peripheral blood mononuclear cell was determined by the activity of the initial κ-CGN, regardless of their composition. The anti-inflammatory activity of PEC and the initial compounds was studied using test of histamine-, concanavalin A-, and sheep erythrocyte immunization-induced inflammation in mice. The highest activity of PEC, as well as the initial polysaccharides κ-CGN and CH, was observed in a histamine-induced exudative inflammation, directly related to the activation of phagocytic cells, i.e., macrophages and neutrophils.

The occurrence of dot-like micro B chromosomes in Korean field mice Apodemus peninsulae from the shore of the Teletskoye Lake (Altai Mountains).

The data on the changes in the cytogenetic structure of the geographic population of Korean field mouse Apodemus (Alsomys) peninsulae Thomas, 1907 at the southern shore of the Teletskoye Lake (Altai Republic) are presented. In 1980 no dot-like microchromosomes were found in 34 mice captured on the southern and northern coasts of the Teletskoye Lake. In 2011, a 1.6-fold (from 2.7 to 4.3) increase in the mean number of B chromosomes compared to the rate estimated there earlier in 1980 was discovered. In 11 of the 15 mice (73%) captured in 2011, the karyotypes contained 1-2 dot-like micro B chromosomes and 1-5 macro B chromosomes. The pollution of the territory by the remains of the rocket fuel components may be an appropriate explanation for the cause of the karyological changes observed in A. peninsulae in this region.

Bacteria's different ways to recycle their own cell wall.

The ability to recover components of their own cell wall is a common feature of bacteria. This was initially recognized in the Gram-negative bacterium Escherichia coli, which recycles about half of the peptidoglycan of its cell wall during one cell doubling. Moreover, E. coli was shown to grow on peptidoglycan components provided as nutrients. A distinguished recycling enzyme of E. coli required for both, recovery of the cell wall sugar N-acetylmuramic acid (MurNAc) of the own cell wall and for growth on external MurNAc, is the MurNAc 6-phosphate (MurNAc 6P) lactyl ether hydrolase MurQ. We revealed however, that most Gram-negative bacteria lack a murQ ortholog and instead harbor a pathway, absent in E. coli, that channels MurNAc directly to peptidoglycan biosynthesis. This "anabolic recycling pathway" bypasses the initial steps of peptidoglycan de novo synthesis, including the target of the antibiotic fosfomycin, thus providing intrinsic resistance to the antibiotic. The Gram-negative oral pathogen Tannerella forsythia is auxotrophic for MurNAc and apparently depends on the anabolic recycling pathway to synthesize its own cell wall by scavenging cell wall debris of other bacteria. In contrast, Gram-positive bacteria lack the anabolic recycling genes, but mostly contain one or two murQ orthologs. Quantification of MurNAc 6P accumulation in murQ mutant cells by mass spectrometry allowed us to demonstrate for the first time that Gram-positive bacteria do recycle their own peptidoglycan. This had been questioned earlier, since peptidoglycan turnover products accumulate in the spent media of Gram-positives. We showed, that these fragments are recovered during nutrient limitation, which prolongs starvation survival of Bacillus subtilis and Staphylococcus aureus. Peptidoglycan recycling in these bacteria however differs, as the cell wall is either cleaved exhaustively and monosaccharide building blocks are taken up (B. subtilis) or disaccharides are released and recycled involving a novel phosphomuramidase (MupG; S.aureus). In B. subtilis also the teichoic acids, covalently bound to the peptidoglycan (wall teichoic acids; WTAs), are recycled. During phosphate limitation, the sn-glycerol-3-phosphate phosphodiesterase GlpQ specifically degrades WTAs of B. subtilis. In S. aureus, in contrast, GlpQ is used to scavenge external teichoic acid sources. Thus, although bacteria generally recover their own cell wall, they apparently apply distinct strategies for breakdown and reutilization of cell wall fragments. This review summarizes our work on this topic funded between 2011 and 2019 by the DFG within the collaborative research center SFB766.

Action of tannin on cellular membranes: Novel insights from concerted studies on lipid bilayers and native cells.

Hydrolyzable tannin (3,6-bis-O-digalloyl-1,2,4-tri-O-galloyl-ß-d-glucose) has a dual effect on the cell membrane: (1) it binds to a plasmalemmal protein of the Chara corallina cell (C50 = 2.7 ±â€¯0.3 µM) and (2) it forms ionic channels in the lipid membrane. Based on these facts, a molecular model for the interaction of tannins with the cell membrane is proposed. The model suggests that the molecules of hydrolyzable tannin bind electrostatically to the outer groups of the membrane protein responsible for the Ca2+-dependent chloride current and blocks it. Some tannin molecules penetrate into the hydrophobic region of the membrane, and when a particular concentration is reached, they form ion-conducting structures selective toward Cl-.

Recovery of the Peptidoglycan Turnover Product Released by the Autolysin Atl in Staphylococcus aureus Involves the Phosphotransferase System Transporter MurP and the Novel 6-phospho-N-acetylmuramidase MupG.

The peptidoglycan of the bacterial cell wall undergoes a permanent turnover during cell growth and differentiation. In the Gram-positive pathogen Staphylococcus aureus, the major peptidoglycan hydrolase Atl is required for accurate cell division, daughter cell separation and autolysis. Atl is a bifunctional N-acetylmuramoyl-L-alanine amidase/endo-ß-N-acetylglucosaminidase that releases peptides and the disaccharide N-acetylmuramic acid-ß-1,4-N-acetylglucosamine (MurNAc-GlcNAc) from the peptido-glycan. Here we revealed the recycling pathway of the cell wall turnover product MurNAc-GlcNAc in S. aureus. The latter disaccharide is internalized and concomitantly phosphorylated by the phosphotransferase system (PTS) transporter MurP, which had been implicated previously in the uptake and phosphorylation of MurNAc. Since MurP mutant cells accumulate MurNAc-GlcNAc and not MurNAc in the culture medium during growth, the disaccharide represents the physiological substrate of the PTS transporter. We further identified and characterized a novel 6-phospho-N-acetylmuramidase, named MupG, which intracellularly hydrolyses MurNAc 6-phosphate-GlcNAc, the product of MurP-uptake and phosphorylation, yielding MurNAc 6-phosphate and GlcNAc. MupG is the first characterized representative of a novel family of glycosidases containing domain of unknown function 871 (DUF871). The corresponding gene mupG (SAUSA300_0192) of S. aureus strain USA300 is the first gene within a putative operon that also includes genes encoding the MurNAc 6-phosphate etherase MurQ, MurP, and the putative transcriptional regulator MurR. Using mass spectrometry, we observed cytoplasmic accumulation of MurNAc 6-phosphate-GlcNAc in ΔmupG and ΔmupGmurQ markerless non-polar deletion mutants, but not in the wild type or in the complemented ΔmupG strain. MurNAc 6-phosphate-GlcNAc levels in the mutants increased during stationary phase, in accordance with previous observations regarding peptidoglycan recycling in S. aureus.

Regulation of the opposing (p)ppGpp synthetase and hydrolase activities in a bifunctional RelA/SpoT homologue from Staphylococcus aureus.

The stringent response is characterized by (p)ppGpp synthesis resulting in repression of translation and reprogramming of the transcriptome. In Staphylococcus aureus, (p)ppGpp is synthesized by the long RSH (RelA/SpoT homolog) enzyme, RelSau or by one of the two short synthetases (RelP, RelQ). RSH enzymes are characterized by an N-terminal enzymatic domain bearing distinct motifs for (p)ppGpp synthetase or hydrolase activity and a C-terminal regulatory domain (CTD) containing conserved motifs (TGS, DC and ACT). The intramolecular switch between synthetase and hydrolase activity of RelSau is crucial for the adaption of S. aureus to stress (stringent) or non-stress (relaxed) conditions. We elucidated the role of the CTD in the enzymatic activities of RelSau. Growth pattern, transcriptional analyses and in vitro assays yielded the following results: i) in vivo, under relaxed conditions, as well as in vitro, the CTD inhibits synthetase activity but is not required for hydrolase activity; ii) under stringent conditions, the CTD is essential for (p)ppGpp synthesis; iii) RelSau lacking the CTD exhibits net hydrolase activity when expressed in S. aureus but net (p)ppGpp synthetase activity when expressed in E. coli; iv) the TGS and DC motifs within the CTD are required for correct stringent response, whereas the ACT motif is dispensable, v) Co-immunoprecipitation indicated that the CTD interacts with the ribosome, which is largely dependent on the TGS motif. In conclusion, RelSau primarily exists in a synthetase-OFF/hydrolase-ON state, the TGS motif within the CTD is required to activate (p)ppGpp synthesis under stringent conditions.

p38-MK2 signaling axis regulates RNA metabolism after UV-light-induced DNA damage.

Ultraviolet (UV) light radiation induces the formation of bulky photoproducts in the DNA that globally affect transcription and splicing. However, the signaling pathways and mechanisms that link UV-light-induced DNA damage to changes in RNA metabolism remain poorly understood. Here we employ quantitative phosphoproteomics and protein kinase inhibition to provide a systems view on protein phosphorylation patterns induced by UV light and uncover the dependencies of phosphorylation events on the canonical DNA damage signaling by ATM/ATR and the p38 MAP kinase pathway. We identify RNA-binding proteins as primary substrates and 14-3-3 as direct readers of p38-MK2-dependent phosphorylation induced by UV light. Mechanistically, we show that MK2 phosphorylates the RNA-binding subunit of the NELF complex NELFE on Serine 115. NELFE phosphorylation promotes the recruitment of 14-3-3 and rapid dissociation of the NELF complex from chromatin, which is accompanied by RNA polymerase II elongation.