M. Ehlingschulz - Academia.edu (original) (raw)

Papers by M. Ehlingschulz

International Journal of Molecular Sciences

The emetic type of foodborne disease caused by Bacillus cereus is produced by the small peptide t... more The emetic type of foodborne disease caused by Bacillus cereus is produced by the small peptide toxin cereulide. The genetic locus encoding the Ces nonribosomal peptide synthetase (CesNRPS) multienzyme machinery is located on a 270 kb megaplasmid, designated pCER270, which shares its backbone with the Bacillus anthracis toxin plasmid pXO1. Although the ces genes are plasmid-borne, the chromosomally encoded pleiotropic transcriptional factors CodY and AbrB are key players in the control of ces transcription. Since these proteins only repress cereulide synthesis during earlier growth phases, other factors must be involved in the strict control of ces expression and its embedment in the bacterial life cycle. In silico genome analysis revealed that pCER270 carries a putative ArsR/SmtB family transcription factor showing high homology to PagR from B. anthracis. As PagR plays a crucial role in the regulation of the protective antigen gene pagA, which forms part of anthrax toxin, we used a...

Dairy Science & Technology, 2016

The ingredients used for the development and production of milk-based products pose the risk of t... more The ingredients used for the development and production of milk-based products pose the risk of the introduction of new, emerging spore-formers producing highly thermoresistant spores. Therefore, the aim of this study was to examine the heat resistance of spores isolated from dehydrated ingredients, intermediate and final products. Furthermore, the influence of the heating medium (milk or phosphate buffer) on the heat resistance was determined in order to assess which medium is best to use in the context of dairy processing. Sixteen spore-forming strains from seven different species (Bacillus amyloliquefaciens, Bacillus flexus, Bacillus subtilis, Bacillus thermoamylovorans, Bacillus smithii, Geobacillus pallidus, Geobacillus stearothermophilus) producing thermoresistant spores were selected after their isolation from diverse food products obtained from local dairies such as cocoa powder, milk powder, spices, and dessert products. Spores produced from the chosen strains were tested regarding their heat resistance at 110, 120, and 125°C for 30 min. Highly thermoresistant spores surviving a heat treatment for 30 min at 125°C in milk were produced by B. amyloliquefaciens and G. stearothermophilus. The inactivation of the spores was higher in milk than in phosphate buffer. This study highlights that not only raw milk but also dehydrated ingredients are important sources of thermoresistant spores in dairy processing. Since

Foods

In this study, the performance of four alternative selective chromogenic B. cereus agar was compa... more In this study, the performance of four alternative selective chromogenic B. cereus agar was compared to the reference mannitol-yolk polymyxin (MYP) agar (ISO 7932) using inclusion and exclusion test strains (n = 110) and by analyzing naturally contaminated milk and other food samples (n = 64). Subsequently, the panC group affiliation and toxin gene profile of Bacillus cereus senso lato (s.l.) isolates were determined. Our results corroborate that the overall best performing media CHROMagar™ B. cereus (93.6% inclusivity; 82.7% exclusivity) and BACARA® (98.2% inclusivity, 62.7% exclusivity) are more sensitive and specific compared to Brilliance™ B. cereus, MYP and ChromoSelect Bacillus Agar. Both media allow unequivocal detection of B. cereus with low risks of misidentification. Media containing ß-D-glucosidase for the detection of presumptive B. cereus may form atypical colony morphologies resulting in a false negative evaluation of the sample. Naturally contaminated samples presente...

Scientific reports, Jan 30, 2018

Surface carbohydrate moieties are essential for bacterial communication, phage-bacteria and host-... more Surface carbohydrate moieties are essential for bacterial communication, phage-bacteria and host-pathogen interaction. Most Staphylococcus aureus produce polyribitolphosphate type Wall teichoic acids (WTAs) substituted with α- and/or β-O-linked N-acetyl-glucosamine (α-/β-O-GlcNAc) residues. GlcNAc modifications have attracted particular interest, as they were shown to govern staphylococcal adhesion to host cells, to promote phage susceptibility conferring beta-lactam resistance and are an important target for antimicrobial agents and vaccines. However, there is a lack of rapid, reliable, and convenient methods to detect and quantify these sugar residues. Whole cell Fourier transform infrared (FTIR) spectroscopy could meet these demands and was employed to analyse WTAs and WTA glycosylation in S. aureus. Using S. aureus mutants, we found that a complete loss of WTA expression resulted in strong FTIR spectral perturbations mainly related to carbohydrates and phosphorus-containing mole...

ACS Combinatorial Science, 2018

Over the past decades, ionic liquids (ILs) have gained considerable attention from the scientific... more Over the past decades, ionic liquids (ILs) have gained considerable attention from the scientific 19 community due to their versatile and designable properties. As a result, there are numerous IL 20 applications, particularly in organic synthesis, catalysis or extraction, but also as active pharmaceutical ingredients or novel antimicrobials. While considerable effort has been put into 22 developing quantitative structure-activity relationship (QSAR) models for IL toxicity prediction, 23 little is known about their actual mode of action. In this study, Fourier transform infrared (FTIR) 24 spectroscopy is used to monitor IL induced molecular responses directly at the cellular level. 25 Investigation of the well-known cationic alkyl side-chain effect (increasing side chain length 26 leads to increasing toxicity) of imidazolium-and ammonium-based ILs on two bacterial 27 pathogens, enteropathogenic Escherichia coli (EPEC) and methicillin-resistant 28 Staphylococcus aureus (MRSA), surprisingly revealed two distinct modes of action. Contrary 29 to prior models, it was only for [TMC 16 A][Cl] where a molecular response in the membrane was 30 found, while ILs with shorter side-chain lengths predominantly affected bacterial proteins. The 31 results of this study highlight the importance of further direct investigations of the impact of ILs 32 at the cellular level in order to improve toxicity prediction and assess the usefulness of 33 spectroscopic methods such as FTIR spectroscopy at achieving this goal.

International Journal of Dairy Technology, 2011

Molecular Microbiology, 2002

The UV-B and desiccation-tolerant terrestrial cyanobacterium Nostoc commune was grown under defin... more The UV-B and desiccation-tolerant terrestrial cyanobacterium Nostoc commune was grown under defined UV irradiation. Proteome changes were monitored in the membrane and the cytosolic and the extracellular fractions. Tools were developed to separate stress-triggered from growth stage-dependent changes. UV-B changed the relative cellular concentration of 493 out of 1,350 protein spots at least by a factor of three, rendering the UV-B stimulon of N. commune the most complex one described so far. It comprises two different parts: an early shock response influencing 214 proteins and a late acclimation response involving 279 proteins. The shock response comprised many membrane or membrane-associated proteins, whereas the acclimation response mainly changed cytosolic proteins. Most of the shock-induced changes were transient and did not overlap with the acclimation response. In the extracellular fraction, UV irradiation induced superoxide dismutase and the water stress protein. In total, 27 intracellular, UV-B-induced proteins were partially sequenced by electrospray ionization tandem mass spectrometry. Three functional classes were identified: proteins involved in lipid metabolism, in carbohydrate metabolism and in regulatory pathways. About 50% of the sequenced proteins were homologous to cyanobacterial database entries with un-known function. Interestingly, all of these proteins belong to the UV-B acclimation response. We conclude that the UV-B shock response and the UV-B acclimation response represent two completely different and remarkably complex strategies of N. commune to protect itself against UV-B radiation in its natural environment.

Molecular Microbiology, 2012

Bacillus cereus causes gastrointestinal diseases and local and systemic infections elicited by th... more Bacillus cereus causes gastrointestinal diseases and local and systemic infections elicited by the depsipeptide cereulide, enterotoxins, phospholipases, cytolysins and proteases. The PlcR-PapR quorum sensing system activates the expression of several virulence factors, whereas the Spo0A-AbrB regulatory circuit partially controls the plasmid-borne cereulide synthetase (ces) operon. Here, we show that CodY, a nutrient-responsive regulator of Gram-positive bacteria, has a profound effect on both regulatory systems, which have been assumed to operate independently of each other. Deletion of codY resulted in downregulation of virulence genes belonging to the PlcR regulon and a concomitant upregulation of the ces genes. CodY was found to be a repressor of the ces operon, but did not interact with the promoter regions of PlcR-dependent virulence genes in vitro, suggesting an indirect regulation of the latter. Furthermore, CodY binds to the promoter of the immune inhibitor metalloprotease InhA1, demonstrating that CodY directly links B. cereus metabolism to virulence. In vivo studies using a Galleria mellonella infection model, showed that the codY mutant was substantially attenuated, highlighting the importance of CodY as a key regulator of pathogenicity. Our results demonstrate that CodY profoundly modulates the virulence of B. cereus, possibly controlling the development of pathogenic traits in suitable host environments.

Microbiology, 2009

Cereulide, a depsipeptide structurally related to the antibiotic valinomycin, is responsible for ... more Cereulide, a depsipeptide structurally related to the antibiotic valinomycin, is responsible for the emetic type of gastrointestinal disease caused by Bacillus cereus. Recently, it has been shown that cereulide is produced non-ribosomally by the plasmid-encoded peptide synthetase Ces. Using deletion mutants of the emetic reference strain B. cereus F4810/72, the influence of the well-known transcription factors PlcR, Spo0A and AbrB on cereulide production and on the transcription of the cereulide synthetase gene cluster was investigated. Our data demonstrate that cereulide synthesis is independent of the B. cereus specific virulence regulator PlcR but belongs to the Spo0A-AbrB regulon. Although cereulide production turned out to be independent of sporulation, it required the activity of the sporulation factor Spo0A. The σ A-promoted transcription of spo0A was found to be crucial for cereulide production, while the σ H-driven transcription of spo0A did not affect cereulide synthesis. ...

International Journal of Food Microbiology, 2013

Due to changes in the design of industrial food processing and increasing international trade, hi... more Due to changes in the design of industrial food processing and increasing international trade, highly thermoresistant spore-forming bacteria are an emerging problem in food production. Minimally processed foods and products with extended shelf life, such as milk products, are at special risk for contamination and subsequent product damages, but information about origin and food quality related properties of highly heat-resistant spore-formers is still limited. Therefore, the aim of this study was to determine the biodiversity, heat resistance, and food quality and safety affecting characteristics of aerobic spore-formers in the dairy sector. Thus, a comprehensive panel of strains (n=467), which originated from dairy processing environments, raw materials and processed foods, was compiled. The set included isolates associated with recent food spoilage cases and product damages as well as isolates not linked to product spoilage. Identification of the isolates by means of Fourier-transform infrared spectroscopy and molecular methods revealed a large biodiversity of spore-formers, especially among the spoilage associated isolates. These could be assigned to 43 species, representing 11 genera, with Bacillus cereus s.l. and Bacillus licheniformis being predominant. A screening for isolates forming thermoresistant spores (TRS, surviving 100°C, 20 min) showed that about one third of the tested spore-formers was heat-resistant, with Bacillus subtilis and Geobacillus stearothermophilus being the prevalent species. Strains producing highly thermoresistant spores (HTRS, surviving 125°C, 30 min) were found among mesophilic as well as among thermophilic species. B. subtilis and Bacillus amyloliquefaciens were dominating the group of mesophilic HTRS, while Bacillus smithii and Geobacillus pallidus were dominating the group of thermophilic HTRS. Analysis of spoilage-related enzymes of the TRS isolates showed that mesophilic strains, belonging to the B. subtilis and B. cereus groups, were strongly proteolytic, whereas thermophilic strains displayed generally a low enzymatic activity and thus spoilage potential. Cytotoxicity was only detected in B. cereus, suggesting that the risk of food poisoning by aerobic, thermoresistant spore-formers outside of the B. cereus group is rather low.

PROTEOMICS, 2008

Proteome analysis was combined with whole-cell metabolic fingerprinting to gain insight into the ... more Proteome analysis was combined with whole-cell metabolic fingerprinting to gain insight into the physiology of mature biofilm in Bordetella pertussis, the agent responsible for whooping cough. Recent reports indicate that B. pertussis adopts a sessile biofilm as a strategy to persistently colonize the human host. However, since research in the past mainly focused on the planktonic lifestyle of B. pertussis, knowledge on biofilm formation of this important human pathogen is still limited. Comparative studies were carried out by combining 2-DE and Fourier transform infrared (FT-IR) spectroscopy with multivariate statistical methods. These complementary approaches demonstrated that biofilm development has a distinctive impact on B. pertussis physiology. Results from MALDI-TOF/MS identification of proteins together with results from FT-IR spectroscopy revealed the biosynthesis of a putative acidic-type polysaccharide polymer as the most distinctive trait of B. pertussis life in a biofilm. Additionally, expression of proteins known to be involved in cellular regulatory circuits, cell attachment and virulence was altered in sessile cells, which strongly suggests a significant impact of biofilm development on B. pertussis pathogenesis. In summary, our work showed that the combination of proteomics and FT-IR spectroscopy with multivariate statistical analysis provides a powerful tool to gain further insight into bacterial lifestyles.

Frontiers in Microbiology

While the relevance of Bacillus (B.) cereus as a major cause of gastroenteritis is undisputed, th... more While the relevance of Bacillus (B.) cereus as a major cause of gastroenteritis is undisputed, the role of the closely related B. thuringiensis in foodborne disease is unclear. B. thuringiensis strains frequently harbor enterotoxin genes. However, the organism has only very rarely been associated with foodborne outbreaks, possibly due to the fact that during outbreak investigations, B. cereus is routinely not differentiated from B. thuringiensis. A recent EFSA scientific opinion stresses the urgent need for further data allowing for improved risk assessment, in particular as B. thuringiensis is a commonly used biopesticide. Therefore, the aim of this study was to gain further insights into the hazardous potential of B. thuringiensis. To this end, 39 B. thuringiensis isolates obtained from commercially used biopesticides, various food sources, as well as from foodborne outbreaks were characterized by panC typing, panC-based SplitsTree analysis, toxin gene profiling, FTIR spectroscopic analysis, a cytotoxicity assay screening for enterotoxic activity, and a sphingomyelinase assay. The majority of the tested B. thuringiensis isolates exhibited low (23%, n = 9) or mid level enterotoxicity (74%, n = 29), and produced either no (59%, n = 23) or low levels (33%, n = 13) of sphingomyelinase, which is reported to act synergistically with enterotoxins Nhe and Hbl. One strain isolated from rosemary was however classified as highly enterotoxic surpassing the cytotoxic activity of the high-level reference strain by a factor of 1.5. This strain also produced vast amounts of sphingomyelinase. Combining all results obtained in this study into a fingerprint pattern, several enterotoxic biopesticide strains were indistinguishable from those of isolates from foods or collected in association with outbreaks. Our study shows that many B. thuringiensis biopesticide strains exhibit mid-level cytotoxicity in a Vero cell assay and that some of these strains cannot be differentiated from isolates collected from foods or in association with outbreaks. Thus, we demonstrate that the use of B. thuringiensis strains as biopesticides can represent a food safety risk, underpinning the importance of assessing the hazardous potential of each strain and formulation used.

Food Microbiology, 2011

In light of the increasing number of serious food borne outbreaks caused by emetic Bacillus cereu... more In light of the increasing number of serious food borne outbreaks caused by emetic Bacillus cereus, a better understanding of the cereulide synthetase (ces) gene expression and toxin synthesis is required. Here, the relative expression levels of three ces genes (cesP, cesA and cesB) were investigated using quantitative real-time reverse transcription PCR in relation to growth, degree of sporulation and toxin production of the emetic reference strain B. cereus F4810/72 and the weakly emetic strain IH41385. The strict co-transcription of all three genes confirmed the operon structure of the ces gene cluster responsible for cereulide formation. ces transcription turned out to be highly temporal and tightly regulated; ces mRNA was only detectable during mid to late exponential growth in both strains. The low toxigenic potential of the weakly emetic strain IH41385 correlated well with its respective ces transcripts, showing reduced activity at a transcriptional level. Two non-sporulating mutants (F4810/72Dspo0A and F4810/72INsigH) demonstrated that cereulide synthesis is part of the Spo0A regulon but independent of later sporulation processes. Besides strain specific intrinsic factors, ces transcription was found to be significantly influenced by the cellular growth state as well as by extrinsic abiotic factors, like salt. An increase of sodium chloride in the media resulted in lower ces transcription and coincided with lower cereulide toxin levels. Interestingly, at 25 g l À1 NaCl, toxin levels were already reduced without strongly affecting the growth of B. cereus, indicating an inhibitory effect of NaCl on cereulide biosynthesis independent of growth. This illustrates that ces gene expression and toxicity cannot be predicted solely from growth rates or cell numbers, but is influenced by complex interactions of various intrinsic as well as extrinsic factors, which remain to be clarified in detail.

International Journal of Food Microbiology, 2013

Microbiology, 2009

Cereulide, a depsipeptide structurally related to the antibiotic valinomycin, is responsible for ... more Cereulide, a depsipeptide structurally related to the antibiotic valinomycin, is responsible for the emetic type of gastrointestinal disease caused by Bacillus cereus. Recently, it has been shown that cereulide is produced non-ribosomally by the plasmid-encoded peptide synthetase Ces. Using deletion mutants of the emetic reference strain B. cereus F4810/72, the influence of the well-known transcription factors PlcR, Spo0A and AbrB on cereulide production and on the transcription of the cereulide synthetase gene cluster was investigated. Our data demonstrate that cereulide synthesis is independent of the B. cereus specific virulence regulator PlcR but belongs to the Spo0A-AbrB regulon. Although cereulide production turned out to be independent of sporulation, it required the activity of the sporulation factor Spo0A. The s A -promoted transcription of spo0A was found to be crucial for cereulide production, while the s H -driven transcription of spo0A did not affect cereulide synthesis. Overexpression of the transition state factor AbrB in B. cereus F4810/72 resulted in a non-toxic phenotype. Moreover, AbrB was shown to bind efficiently to the main promoter region of the ces operon, indicating that AbrB acts as a repressor of cereulide production by negatively affecting ces transcription.

Molecular Microbiology, 2012

local and systemic infections elicited by the depsipeptide cereulide, enterotoxins, phospholipase... more local and systemic infections elicited by the depsipeptide cereulide, enterotoxins, phospholipases, cytolysins and proteases. The PlcR-PapR quorum sensing system activates the expression of several virulence factors, whereas the Spo0A-AbrB regulatory circuit partially controls the plasmid-borne cereulide synthetase (ces) operon. Here, we show that CodY, a nutrient-responsive regulator of Gram-positive bacteria, has a profound effect on both regulatory systems, which have been assumed to operate independently of each other. Deletion of codY resulted in downregulation of virulence genes belonging to the PlcR regulon and a concomitant upregulation of the ces genes. CodY was found to be a repressor of the ces operon, but did not interact with the promoter regions of PlcRdependent virulence genes in vitro, suggesting an indirect regulation of the latter. Furthermore, CodY binds to the promoter of the immune inhibitor metalloprotease InhA1, demonstrating that CodY directly links B. cereus metabolism to virulence. In vivo studies using a Galleria mellonella infection model, showed that the codY mutant was substantially attenuated, highlighting the importance of CodY as a key regulator of pathogenicity. Our results demonstrate that CodY profoundly modulates the virulence of B. cereus, possibly controlling the development of pathogenic traits in suitable host environments.

International Journal of Molecular Sciences

Dairy Science & Technology, 2016

Foods

Scientific reports, Jan 30, 2018

ACS Combinatorial Science, 2018

International Journal of Dairy Technology, 2011

Molecular Microbiology, 2002

Molecular Microbiology, 2012

Microbiology, 2009

International Journal of Food Microbiology, 2013

PROTEOMICS, 2008

Frontiers in Microbiology

Food Microbiology, 2011

International Journal of Food Microbiology, 2013

Microbiology, 2009

Cereulide, a depsipeptide structurally related to the antibiotic valinomycin, is responsible for ... more Cereulide, a depsipeptide structurally related to the antibiotic valinomycin, is responsible for the emetic type of gastrointestinal disease caused by Bacillus cereus. Recently, it has been shown that cereulide is produced non-ribosomally by the plasmid-encoded peptide synthetase Ces. Using deletion mutants of the emetic reference strain B. cereus F4810/72, the influence of the well-known transcription factors PlcR, Spo0A and AbrB on cereulide production and on the transcription of the cereulide synthetase gene cluster was investigated. Our data demonstrate that cereulide synthesis is independent of the B. cereus specific virulence regulator PlcR but belongs to the Spo0A-AbrB regulon. Although cereulide production turned out to be independent of sporulation, it required the activity of the sporulation factor Spo0A. The s A -promoted transcription of spo0A was found to be crucial for cereulide production, while the s H -driven transcription of spo0A did not affect cereulide synthesis. Overexpression of the transition state factor AbrB in B. cereus F4810/72 resulted in a non-toxic phenotype. Moreover, AbrB was shown to bind efficiently to the main promoter region of the ces operon, indicating that AbrB acts as a repressor of cereulide production by negatively affecting ces transcription.

Molecular Microbiology, 2012