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Friedrich Götz - One of the best experts on this subject based on the ideXlab platform.
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new insights in the coordinated amidase and glucosaminidase activity of the major Autolysin atl in staphylococcus aureus
Communications biology, 2020Co-Authors: Mulugeta Nega, Paula Maria Tribelli, Katharina Hipp, Mark Stahl, Friedrich GötzAbstract:After bacterial cell division, the daughter cells are still covalently interlinked by the peptidoglycan network which is resolved by specific hydrolases (Autolysins) to release the daughter cells. In staphylococci, the major Autolysin (Atl) with its two domain enzymes, N-acetylmuramyl-L-alanine amidase (AmiA) and β-N-acetylglucosaminidase (GlcA), resolves the peptidoglycan to release the daughter cells. Internal deletions in each of the enzyme domains revealed defined morphological alterations such as cell cluster formation in ΔamiA, ΔglcA and Δatl, and asymmetric cell division in the ΔglcA. A most important finding was that GlcA activity requires the prior removal of the stem peptide by AmiA for its activity thus the naked glycan strand is its substrate. Furthermore, GlcA is not an endo-β-N-acetylglucosaminidase but an exo-enzyme that cuts the glycan backbone to disaccharides independent of its O-acetylation modification. Our results shed new light into the sequential peptidoglycan hydrolysis by AmiA and GlcA during cell division in staphylococci.
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functional and structural analysis of the major amidase atl in staphylococcus
International Journal of Medical Microbiology, 2014Co-Authors: Friedrich Götz, Thilo Stehle, Christine HeilmannAbstract:The cytoplasmic membrane of most bacteria is surrounded by a more or less thick murein layer (peptidoglycan) that protects the protoplast from mechanical damage, osmotic rupture and lysis. When bacteria are dividing processes are initiated stepwise that involve DNA replication, constriction of the membranes, cell growth, biosynthesis of new murein, and finally the generation of two daughter cells. As the daughter cells are still covalently interlinked by the murein network they must be separated by specific peptidoglycan hydrolases, also referred to as Autolysins. In staphylococci, the major Autolysin (Atl) and its processed products N-acetylmuramoyl-l-alanine amidase (AM) and endo-β-N-acetylglucosaminidase (GL) have been in the research focus for long time. This review addresses phenotypic consequences of atl mutants, impact of Atl in virulence, the mechanism of targeting to the septum region, regulation of atl, the structure of the amidase and the repeat regions, as well as the phylogeny of Atl and its use in Staphylococcus genus and species typing.
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proton binding capacity of staphylococcus aureus wall teichoic acid and its role in controlling Autolysin activity
PLOS ONE, 2012Co-Authors: Raja Biswas, Raul E Martinez, Nadine Gohring, Martin Schlag, Michaele Josten, Guoqing Xia, Florian Hegler, Cordula Gekeler, Anne Kathrin Gleske, Friedrich GötzAbstract:Wall teichoic acid (WTA) or related polyanionic cell wall glycopolymers are produced by most gram-positive bacterial species and have been implicated in various cellular functions. WTA and the proton gradient across bacterial membranes are known to control the activity of Autolysins but the molecular details of these interactions are poorly understood. We demonstrate that WTA contributes substantially to the proton-binding capacity of Staphylococcus aureus cell walls and controls autolysis largely via the major Autolysin AtlA whose activity is known to decline at acidic pH values. Compounds that increase or decrease the activity of the respiratory chain, a main source of protons in the cell wall, modulated autolysis rates in WTA-producing cells but did not affect the augmented autolytic activity observed in a WTA-deficient mutant. We propose that WTA represents a cation-exchanger like mesh in the gram-positive cell envelopes that is required for creating a locally acidified milieu to govern the pH-dependent activity of Autolysins.
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staphylococcal major Autolysin atl is involved in excretion of cytoplasmic proteins
Journal of Biological Chemistry, 2010Co-Authors: Linda Pasztor, Mulugeta Nega, Martin Schlag, Annekathrin Ziebandt, Sabine Haase, Mirita Franzwachtel, Johannes Madlung, Alfred Nordheim, David E Heinrichs, Friedrich GötzAbstract:Many microorganisms excrete typical cytoplasmic proteins into the culture supernatant. As none of the classical secretion systems appears to be involved, this type of secretion was referred to as "nonclassical protein secretion." Here, we demonstrate that in Staphylococcus aureus the major Autolysin plays a crucial role in release of cytoplasmic proteins. Comparative secretome analysis revealed that in the wild type S. aureus strain, 22 typical cytoplasmic proteins were excreted into the culture supernatant, although in the atl mutant they were significantly decreased. The presence or absence of prophages had little influence on the secretome pattern. In the atl mutant, secondary peptidoglycan hydrolases were increased in the secretome; the corresponding genes were transcriptionally up-regulated suggesting a compensatory mechanism for the atl mutation. Using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a cytoplasmic indicator enzyme, we showed that all clinical isolates tested excreted this protein. In the wall teichoic acid-deficient tagO mutant with its increased autolysis activity, GAPDH was excreted in even higher amounts than in the WT, confirming the importance of autolysis in excretion of cytoplasmic proteins. To answer the question of how discriminatory the excretion of cytoplasmic proteins is, we performed a two-dimensional PAGE of cytoplasmic proteins isolated from WT. Surprisingly, the most abundant proteins in the cytoplasm were not found in the secretome of the WT, suggesting that there exists a selection mechanism in the excretion of cytoplasmic proteins. As the major Autolysin binds at the septum site, we assume that the proteins are preferentially released at and during septum formation.
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structural basis of cell wall cleavage by a staphylococcal Autolysin
PLOS Pathogens, 2010Co-Authors: Sebastian Zoll, Bernhard Pätzold, Thilo Stehle, Hubert Kalbacher, Friedrich Götz, Martin SchlagAbstract:The major Autolysins (Atl) of Staphylococcus epidermidis and S. aureus play an important role in cell separation, and their mutants are also attenuated in virulence. Therefore, Autolysins represent a promising target for the development of new types of antibiotics. Here, we report the high-resolution structure of the catalytically active amidase domain AmiE (amidase S. epidermidis) from the major Autolysin of S. epidermidis. This is the first protein structure with an amidase-like fold from a bacterium with a gram-positive cell wall architecture. AmiE adopts a globular fold, with several α-helices surrounding a central β-sheet. Sequence comparison reveals a cluster of conserved amino acids that define a putative binding site with a buried zinc ion. Mutations of key residues in the putative active site result in loss of activity, enabling us to propose a catalytic mechanism. We also identified and synthesized muramyltripeptide, the minimal peptidoglycan fragment that can be used as a substrate by the enzyme. Molecular docking and digestion assays with muramyltripeptide derivatives allow us to identify key determinants of ligand binding. This results in a plausible model of interaction of this ligand not only for AmiE, but also for other PGN-hydrolases that share the same fold. As AmiE active-site mutations also show a severe growth defect, our findings provide an excellent platform for the design of specific inhibitors that target staphylococcal cell separation and can thereby prevent growth of this pathogen.
Girbe Buist - One of the best experts on this subject based on the ideXlab platform.
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Expression of prophage-encoded endolysins contributes to autolysis of Lactococcus lactis
Applied Microbiology and Biotechnology, 2016Co-Authors: Ganesh Ram R. Visweswaran, Francisco Romero Pastrana, Monika Szeliga, Dorota Kurek, Oscar P. Kuipers, Girbe BuistAbstract:Analysis of autolysis of derivatives of Lactococcus lactis subsp. cremoris MG1363 and subsp. lactis IL1403, both lacking the major Autolysin AcmA, showed that L. lactis IL1403 still lysed during growth while L. lactis MG1363 did not. Zymographic analysis revealed that a peptidoglycan hydrolase activity of around 30 kDa is present in cell extracts of L. lactis IL1403 that could not be detected in strain MG1363. A comparison of all genes encoding putative peptidoglycan hydrolases of IL1403 and MG1363 led to the assumption that one or more of the 99 % homologous 27.9-kDa endolysins encoded by the prophages bIL285, bIL286 and bIL309 could account for the autolysis phenotype of IL1403. Induced expression of the endolysins from bIL285, bIL286 or bIL309 in L. lactis MG1363 resulted in detectable lysis or lytic activity. Prophage deletion and insertion derivatives of L. lactis IL1403 had a reduced cell lysis phenotype. RT-qPCR and zymogram analysis showed that each of these strains still expressed one or more of the three phage lysins. A homologous gene and an endolysin activity were also identified in the natural starter culture L. lactis subsp. cremoris strains E8, Wg2 and HP, and the lytic activity could be detected under growth conditions that were identical as those used for IL1403. The results presented here show that these endolysins of L. lactis are expressed during normal growth and contribute to autolysis without production of (lytic) phages. Screening for natural strains expressing homologous endolysins could help in the selection of strains with enhanced autolysis and, thus, cheese ripening properties.
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acmd a homolog of the major Autolysin acma of lactococcus lactis binds to the cell wall and contributes to cell separation and autolysis
PLOS ONE, 2013Co-Authors: Ganesh Ram R. Visweswaran, Monika Szeliga, Oscar P. Kuipers, Jan Kok, Anton Steen, Kees Leenhouts, Beata Ruban, Anne Hesselingmeinders, Bauke W Dijkstra, Girbe BuistAbstract:Lactococcus lactis expresses the homologous glucosaminidases AcmB, AcmC, AcmA and AcmD. The latter two have three C-terminal LysM repeats for peptidoglycan binding. AcmD has much shorter intervening sequences separating the LysM repeats and a lower iso-electric point (4.3) than AcmA (10.3). Under standard laboratory conditions AcmD was mainly secreted into the culture supernatant. An L. lactis acmAacmD double mutant formed longer chains than the acmA single mutant, indicating that AcmD contributes to cell separation. This phenotype could be complemented by plasmid-encoded expression of AcmD in the double mutant. No clear difference in cellular lysis and protein secretion was observed between both mutants. Nevertheless, overexpression of AcmD resulted in increased autolysis when AcmA was present (as in the wild type strain) or when AcmA was added to the culture medium of an AcmA-minus strain. Possibly, AcmD is mainly active within the cell wall, at places where proper conditions are present for its binding and catalytic activity. Various fusion proteins carrying either the three LysM repeats of AcmA or AcmD were used to study and compare their cell wall binding characteristics. Whereas binding of the LysM domain of AcmA took place at pHs ranging from 4 to 8, LysM domain of AcmD seems to bind strongest at pH 4.
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reduced lysis upon growth of lactococcus lactis on galactose is a consequence of decreased binding of the Autolysin acma
Applied and Environmental Microbiology, 2008Co-Authors: Anton Steen, Girbe Buist, Oscar P. Kuipers, Naomi E Kramer, Ruud Jalving, Germaine F J D Benus, Gerard Venema, Jan KokAbstract:When Lactococcus lactis subsp. lactis IL1403 or L. lactis subsp. cremoris MG1363 is grown in a medium with galactose as the carbon source, the culture lyses to a lesser extent in stationary phase than when the bacteria are grown in a medium containing glucose. Expression of AcmA, the major Autolysin of L. lactis, is not influenced by the carbon source. Binding studies with a fusion protein consisting of the MSA2 protein of Plasmodium falciparum and the C-terminal peptidoglycan-binding domain of AcmA revealed that cell walls of cells from both subspecies grown on galactose bind less AcmA than cell walls of cells grown on glucose. Cells grown on glucose or galactose and treated with trichloroacetic acid prior to AcmA binding bind similar amounts of AcmA. Analysis of the composition of the lipoteichoic acids (LTAs) of L. lactis IL1403 cells grown on glucose or galactose showed that the LTA composition is influenced by the carbon source: cells grown on galactose contain LTA with less galactose than cells grown on glucose. In conclusion, growth of L. lactis on galactose changes the LTA composition in the cell wall in such a way that less AcmA is able to bind to the peptidoglycan, resulting in a decrease in autolysis.
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autolysis of lactococcus lactis is increased upon d alanine depletion of peptidoglycan and lipoteichoic acids
Journal of Bacteriology, 2005Co-Authors: Anton Steen, Girbe Buist, Oscar P. Kuipers, Jan Kok, Emmanuelle Palumbo, Marie Deghorain, Pier Sandro Cocconcelli, Jean Delcour, Pascal HolsAbstract:Mutations in the genes encoding enzymes responsible for the incorporation of D-Ala into the cell wall of Lactococcus lactis affect autolysis. An L. lactis alanine racemase (alr) mutant is strictly dependent on an external supply of D-Ala to be able to synthesize peptidoglycan and to incorporate D-Ala in the lipoteichoic acids (LTA). The mutant lyses rapidly when D-Ala is removed at mid-exponential growth. AcmA, the major lactococcal Autolysin, is partially involved in the increased lysis since an alr acmA double mutant still lyses, albeit to a lesser extent. To investigate the role of D-Ala on LTA in the increased cell lysis, a dltD mutant of L. lactis was investigated, since this mutant is only affected in the D-alanylation of LTA and not the synthesis of peptidoglycan. Mutation of dltD results in increased lysis, showing that D-alanylation of LTA also influences autolysis. Since a dltD acmA double mutant does not lyse, the lysis of the dltD mutant is totally AcmA dependent. Zymographic analysis shows that no degradation of AcmA takes place in the dltD mutant, whereas AcmA is degraded by the extracellular protease HtrA in the wild-type strain. In L. lactis, LTA has been shown to be involved in controlled (directed) binding of AcmA. LTA lacking D-Ala has been reported in other bacterial species to have an improved capacity for Autolysin binding. Mutation of dltD in L. lactis, however, does not affect peptidoglycan binding of AcmA; neither the amount of AcmA binding to the cells nor the binding to specific loci is altered. In conclusion, D-Ala depletion of the cell wall causes lysis by two distinct mechanisms. First, it results in an altered peptidoglycan that is more susceptible to lysis by AcmA and also by other factors, e.g., one or more of the other (putative) cell wall hydrolases expressed by L. lactis. Second, reduced amounts of D-Ala on LTA result in decreased degradation of AcmA by HtrA, which results in increased lytic activity.
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autolysis of lactococcus lactis caused by induced overproduction of its major Autolysin acma
Applied and Environmental Microbiology, 1997Co-Authors: Girbe Buist, Harma Karsens, Arjen Nauta, D Van Sinderen, G Venema, Jan KokAbstract:The optical density of a culture of lactococcus lactis MG1363 was reduced more than 60% during prolonged stationary phase. Reduction in optical density (autolysis) was almost absent in a culture of an isogenic mutant containing a deletion in the major Autolysin gene, acmA. An acmA mutant carrying multiple coples of a plasmid encoding AcmA lysed to a greater extent than the wild-type strain did. Intercellular action of AcmA was shown by mixing end-exponential-phase cultures of an acmA deletion mutant and a tripeptidase (pepT) deletion mutant. PepT, produced by the acmA mutant, was detected in the supernatant of the mixed culture, but no PepT was present in the culture supernatant of the acmA mutant. A plasmid was constructed in which acmA, lacking its own promoter, was placed downstream of the inducible promoter/operator region of the temperate lactococcal bacteriophage r1t. After mitomycin induction of an exponential-phase culture of L. lactis LL302 carrying this plasmid, the cells became subject to autolysis, resulting in the release of intracellular proteins.
Soottawat Benjakul - One of the best experts on this subject based on the ideXlab platform.
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compositions and yield of lipids extracted from hepatopancreas of pacific white shrimp litopenaeus vannamei as affected by prior autolysis
Food Chemistry, 2012Co-Authors: Theeraphol Senphan, Soottawat BenjakulAbstract:Abstract Compositions and yield of lipids extracted from hepatopancreas of Pacific white shrimp ( Litopenaeus vannamei ) subjected to autolysis at 60 °C for different times (0, 30, 60, 90 120 and 150 min) were investigated. Extraction yield increased from 7.4% to 8.8% as autolysis time increased from 0 to 150 min. Coincidental increase in total carotenoid content was obtained with increasing autolysis time ( p p -anisidine value (AV) of lipids were noticeable when autolysis time increased ( p p > 0.05), but subsequently increased up to 150 min. ( p n − 3)) as the most abundant fatty acid, followed by eicosapentaenoic acid (EPA; C20:5( n − 3)). Therefore, prior autolysis at 60 °C for 60 min increased the extraction yield without negative effect on lipid quality.
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autolysis of pacific white shrimp litopenaeus vannamei meat characterization and the effects of protein additives
Journal of Food Science, 2008Co-Authors: P Eakpetch, Soottawat Benjakul, Wonnop Visessanguan, Kongkarn KijroongrojanaAbstract:Autolytic activity of Pacific white shrimp (Litopenaeus vannamei) mince in the absence and in the presence of 2.5%NaCl was investigated. Pacific white shrimp mince exhibited the maximum autolytic activity at 35 and 40 degrees C in the absence and in the presence of 2.5%NaCl, respectively, as evidenced by the highest TCA-soluble peptide content and the greatest disappearance of myosin heavy chain (MHC). The autolysis was more pronounced in the acidic pH values, followed by alkaline pH ranges. Pepstatin A showed the highest inhibition toward autolysis in the acidic condition, revealing that aspartic proteinase was dominant in shrimp muscle. Nevertheless, soybean trypsin inhibitor effectively inhibited the autolysis at neutral and alkaline pH values, suggesting that serine proteinase was present in shrimp mince but contributed to autolysis at a lower extent in shrimp meat. Autolysis in shrimp meat could be inhibited partially by all protein additives, including bovine plasma protein (BPP), egg white (EW), and whey protein concentrate (WPC). The inhibition of autolysis increased when the level of protein additives increased with the concomitant increase in band intensity of MHC retained. WPC and BPP in the range of 2% to 3% exhibited the highest inhibition toward autolysis of shrimp mince.
Jan Kok - One of the best experts on this subject based on the ideXlab platform.
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acmd a homolog of the major Autolysin acma of lactococcus lactis binds to the cell wall and contributes to cell separation and autolysis
PLOS ONE, 2013Co-Authors: Ganesh Ram R. Visweswaran, Monika Szeliga, Oscar P. Kuipers, Jan Kok, Anton Steen, Kees Leenhouts, Beata Ruban, Anne Hesselingmeinders, Bauke W Dijkstra, Girbe BuistAbstract:Lactococcus lactis expresses the homologous glucosaminidases AcmB, AcmC, AcmA and AcmD. The latter two have three C-terminal LysM repeats for peptidoglycan binding. AcmD has much shorter intervening sequences separating the LysM repeats and a lower iso-electric point (4.3) than AcmA (10.3). Under standard laboratory conditions AcmD was mainly secreted into the culture supernatant. An L. lactis acmAacmD double mutant formed longer chains than the acmA single mutant, indicating that AcmD contributes to cell separation. This phenotype could be complemented by plasmid-encoded expression of AcmD in the double mutant. No clear difference in cellular lysis and protein secretion was observed between both mutants. Nevertheless, overexpression of AcmD resulted in increased autolysis when AcmA was present (as in the wild type strain) or when AcmA was added to the culture medium of an AcmA-minus strain. Possibly, AcmD is mainly active within the cell wall, at places where proper conditions are present for its binding and catalytic activity. Various fusion proteins carrying either the three LysM repeats of AcmA or AcmD were used to study and compare their cell wall binding characteristics. Whereas binding of the LysM domain of AcmA took place at pHs ranging from 4 to 8, LysM domain of AcmD seems to bind strongest at pH 4.
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reduced lysis upon growth of lactococcus lactis on galactose is a consequence of decreased binding of the Autolysin acma
Applied and Environmental Microbiology, 2008Co-Authors: Anton Steen, Girbe Buist, Oscar P. Kuipers, Naomi E Kramer, Ruud Jalving, Germaine F J D Benus, Gerard Venema, Jan KokAbstract:When Lactococcus lactis subsp. lactis IL1403 or L. lactis subsp. cremoris MG1363 is grown in a medium with galactose as the carbon source, the culture lyses to a lesser extent in stationary phase than when the bacteria are grown in a medium containing glucose. Expression of AcmA, the major Autolysin of L. lactis, is not influenced by the carbon source. Binding studies with a fusion protein consisting of the MSA2 protein of Plasmodium falciparum and the C-terminal peptidoglycan-binding domain of AcmA revealed that cell walls of cells from both subspecies grown on galactose bind less AcmA than cell walls of cells grown on glucose. Cells grown on glucose or galactose and treated with trichloroacetic acid prior to AcmA binding bind similar amounts of AcmA. Analysis of the composition of the lipoteichoic acids (LTAs) of L. lactis IL1403 cells grown on glucose or galactose showed that the LTA composition is influenced by the carbon source: cells grown on galactose contain LTA with less galactose than cells grown on glucose. In conclusion, growth of L. lactis on galactose changes the LTA composition in the cell wall in such a way that less AcmA is able to bind to the peptidoglycan, resulting in a decrease in autolysis.
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autolysis of lactococcus lactis is increased upon d alanine depletion of peptidoglycan and lipoteichoic acids
Journal of Bacteriology, 2005Co-Authors: Anton Steen, Girbe Buist, Oscar P. Kuipers, Jan Kok, Emmanuelle Palumbo, Marie Deghorain, Pier Sandro Cocconcelli, Jean Delcour, Pascal HolsAbstract:Mutations in the genes encoding enzymes responsible for the incorporation of D-Ala into the cell wall of Lactococcus lactis affect autolysis. An L. lactis alanine racemase (alr) mutant is strictly dependent on an external supply of D-Ala to be able to synthesize peptidoglycan and to incorporate D-Ala in the lipoteichoic acids (LTA). The mutant lyses rapidly when D-Ala is removed at mid-exponential growth. AcmA, the major lactococcal Autolysin, is partially involved in the increased lysis since an alr acmA double mutant still lyses, albeit to a lesser extent. To investigate the role of D-Ala on LTA in the increased cell lysis, a dltD mutant of L. lactis was investigated, since this mutant is only affected in the D-alanylation of LTA and not the synthesis of peptidoglycan. Mutation of dltD results in increased lysis, showing that D-alanylation of LTA also influences autolysis. Since a dltD acmA double mutant does not lyse, the lysis of the dltD mutant is totally AcmA dependent. Zymographic analysis shows that no degradation of AcmA takes place in the dltD mutant, whereas AcmA is degraded by the extracellular protease HtrA in the wild-type strain. In L. lactis, LTA has been shown to be involved in controlled (directed) binding of AcmA. LTA lacking D-Ala has been reported in other bacterial species to have an improved capacity for Autolysin binding. Mutation of dltD in L. lactis, however, does not affect peptidoglycan binding of AcmA; neither the amount of AcmA binding to the cells nor the binding to specific loci is altered. In conclusion, D-Ala depletion of the cell wall causes lysis by two distinct mechanisms. First, it results in an altered peptidoglycan that is more susceptible to lysis by AcmA and also by other factors, e.g., one or more of the other (putative) cell wall hydrolases expressed by L. lactis. Second, reduced amounts of D-Ala on LTA result in decreased degradation of AcmA by HtrA, which results in increased lytic activity.
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autolysis of lactococcus lactis caused by induced overproduction of its major Autolysin acma
Applied and Environmental Microbiology, 1997Co-Authors: Girbe Buist, Harma Karsens, Arjen Nauta, D Van Sinderen, G Venema, Jan KokAbstract:The optical density of a culture of lactococcus lactis MG1363 was reduced more than 60% during prolonged stationary phase. Reduction in optical density (autolysis) was almost absent in a culture of an isogenic mutant containing a deletion in the major Autolysin gene, acmA. An acmA mutant carrying multiple coples of a plasmid encoding AcmA lysed to a greater extent than the wild-type strain did. Intercellular action of AcmA was shown by mixing end-exponential-phase cultures of an acmA deletion mutant and a tripeptidase (pepT) deletion mutant. PepT, produced by the acmA mutant, was detected in the supernatant of the mixed culture, but no PepT was present in the culture supernatant of the acmA mutant. A plasmid was constructed in which acmA, lacking its own promoter, was placed downstream of the inducible promoter/operator region of the temperate lactococcal bacteriophage r1t. After mitomycin induction of an exponential-phase culture of L. lactis LL302 carrying this plasmid, the cells became subject to autolysis, resulting in the release of intracellular proteins.
Thilo Stehle - One of the best experts on this subject based on the ideXlab platform.
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functional and structural analysis of the major amidase atl in staphylococcus
International Journal of Medical Microbiology, 2014Co-Authors: Friedrich Götz, Thilo Stehle, Christine HeilmannAbstract:The cytoplasmic membrane of most bacteria is surrounded by a more or less thick murein layer (peptidoglycan) that protects the protoplast from mechanical damage, osmotic rupture and lysis. When bacteria are dividing processes are initiated stepwise that involve DNA replication, constriction of the membranes, cell growth, biosynthesis of new murein, and finally the generation of two daughter cells. As the daughter cells are still covalently interlinked by the murein network they must be separated by specific peptidoglycan hydrolases, also referred to as Autolysins. In staphylococci, the major Autolysin (Atl) and its processed products N-acetylmuramoyl-l-alanine amidase (AM) and endo-β-N-acetylglucosaminidase (GL) have been in the research focus for long time. This review addresses phenotypic consequences of atl mutants, impact of Atl in virulence, the mechanism of targeting to the septum region, regulation of atl, the structure of the amidase and the repeat regions, as well as the phylogeny of Atl and its use in Staphylococcus genus and species typing.
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structural basis of cell wall cleavage by a staphylococcal Autolysin
PLOS Pathogens, 2010Co-Authors: Sebastian Zoll, Bernhard Pätzold, Thilo Stehle, Hubert Kalbacher, Friedrich Götz, Martin SchlagAbstract:The major Autolysins (Atl) of Staphylococcus epidermidis and S. aureus play an important role in cell separation, and their mutants are also attenuated in virulence. Therefore, Autolysins represent a promising target for the development of new types of antibiotics. Here, we report the high-resolution structure of the catalytically active amidase domain AmiE (amidase S. epidermidis) from the major Autolysin of S. epidermidis. This is the first protein structure with an amidase-like fold from a bacterium with a gram-positive cell wall architecture. AmiE adopts a globular fold, with several α-helices surrounding a central β-sheet. Sequence comparison reveals a cluster of conserved amino acids that define a putative binding site with a buried zinc ion. Mutations of key residues in the putative active site result in loss of activity, enabling us to propose a catalytic mechanism. We also identified and synthesized muramyltripeptide, the minimal peptidoglycan fragment that can be used as a substrate by the enzyme. Molecular docking and digestion assays with muramyltripeptide derivatives allow us to identify key determinants of ligand binding. This results in a plausible model of interaction of this ligand not only for AmiE, but also for other PGN-hydrolases that share the same fold. As AmiE active-site mutations also show a severe growth defect, our findings provide an excellent platform for the design of specific inhibitors that target staphylococcal cell separation and can thereby prevent growth of this pathogen.
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Development of a novel fluorescent substrate for Autolysin E, a bacterial type II amidase.
Biochemical and Biophysical Research Communications, 2009Co-Authors: Nicolas Lützner, Bernhard Pätzold, Sebastian Zoll, Thilo Stehle, Hubert KalbacherAbstract:Abstract The bifunctional Autolysin E from Staphylococcus epidermidis, contains a Zn2+-dependent N-acetylmuramoyl- l -alanine amidase AmiE (EC 3.5.1.28). This enzyme hydrolyzes the amide bond between the carbohydrate chain and the peptide stem of bacterial peptidoglycan. Since peptidoglycan is the mayor component of bacterial cell walls, type II amidases like Autolysin E play an essential role in the bacterial life cycle. Therefore bacterial amidases are appropriate drug targets in the development of antibiotics. The drug discovery process relies on sensitive enzyme assay systems to test possible lead candidates for enzyme inhibition. However, specific determination of bacterial amidase activity is complicated because a simple and accurate enzyme assay is currently unavailable. In this study we developed a sensitive fluorescent substrate for the type II amidase Autolysin E from S. epidermidis, which is suitable for quantifying amidase activity in a high throughput compatible fashion. Using derivatives of the substrate Mca-Ala- d -isoGln-Lys(Dnp)- d -Ala-Arg-OH, we were further able to characterize the amidase substrate specificity of Autolysin E.
