Institute For Mikrobiologie Und Biotechnologie

Bonn, Germany

Institute For Mikrobiologie Und Biotechnologie

Bonn, Germany
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Heller K.J.,Institute For Mikrobiologie Und Biotechnologie | Neve H.,Institute For Mikrobiologie Und Biotechnologie
BioSpektrum | Year: 2014

Superinfection exclusion (SIE) is a mechanism by which a prophage residing in the genome of its host bacterium prevents infection of its host by other phages. During recent years, we have studied superinfection exclusion by prophage TP-J34. Its host is Streptococcus thermophilus J34, a bacterium applied as yoghurt starter. Here we present our data on identifying the SIE protein of TP-J34, its mode of action, X-ray structure, and target protein in the superinfecting phage. © 2014 Springer-Verlag Berlin Heidelberg. Literatur:.


Zeiser J.,Institute For Mikrobiologie Und Biotechnologie | Muhlenbeck L.H.,Institute For Mikrobiologie Und Biotechnologie | Schweiger P.,Missouri State University | Deppenmeier U.,Institute For Mikrobiologie Und Biotechnologie
Applied Microbiology and Biotechnology | Year: 2014

The α-proteobacterium Sphingomonas wittichii RW1 is known for its ability to degrade dioxins and related toxic substances. Bioinformatic analysis of the genome indicated that this organism may contain the largest number of pyrroloquinoline quinone-dependent dehydrogenases of any bacteria sequenced so far. Sequence analysis also showed that one of these genes (swit-4395) encodes an enzyme that belongs to the class of periplasmic glucose dehydrogenases. This gene was fused to a pelB signal sequence and a strep-tag coding region at the 5′ and 3′ ends, respectively. The fusion product was cloned into the broad-host range expression vector pBBR1p264-Streplong and the corresponding protein was heterologously produced in Escherichia coli, purified via Strep-Tactin affinity chromatography, and characterized. The protein Swit-4395 had a subunit mass of 39.3 kDa and formed active homooctamers and homododecamers. The enzyme showed the highest activities with short- and medium-chain aldehydes (chain length C1-C6) and ketoaldehydes, such as methylglyoxal and phenylglyoxal. Butyraldehyde was the best substrate, with Vmax and apparent KM values of 3,970 U/mg protein and 12.3 mM, respectively. Pyrroloquinoline quinone was detected using UV-Vis spectroscopy and was found to be a prosthetic group of the purified enzyme. Therefore, Swit-4395 was identified as a pyrroloquinoline quinone-dependent aldehyde dehydrogenase. The enzyme could be purified from the native host when the expression vector was introduced into S. wittichii RW1, indicating homologous protein production. Overproduction of Swit-4395 in S. wittichii RW1 dramatically increased the tolerance of the bacterium toward butyraldehyde and thus might contribute to the detoxification of toxic aldehydes. © 2013 Springer-Verlag.


De Vrese M.,Institute For Mikrobiologie Und Biotechnologie | Lorenzen P.,Institute For Sicherheit Und Qualitat Bei Milch Und Fisch | Clawin-Radecker I.,Institute For Sicherheit Und Qualitat Bei Milch Und Fisch | Hammer P.,Institute For Sicherheit Und Qualitat Bei Milch Und Fisch | And 4 more authors.
Ernahrungs Umschau | Year: 2012

The MRI performed a study on 30 milk samples, including traditional fresh milk, ESL milk and UHT milk. Whatever the processing procedure, all samples fulfilled expectations with respect to adequate heating of the milk, thermal stress, content of principle nutrients and - with a single exception - microbiological safety. No evidence at all was found of decreased vitamin content or increased losses on storage of properly stored ESL milk, in comparison to traditional (pasteurised) commercial milk. Pasteurised milk may have a marginally better taste and odour than ultrapasteurised milk, although the differences were not statistically significant. Moreover, there was no deterioration in the sensory quality of ESL milk after long-term storage. In summary, ESL milk is a high quality food, as judged by the criteria of nutritional physiology, and is comparable to traditional (pasteurised) fresh milk. It is nevertheless an open question whether milk that has been stored for up to 3 weeks fulfils the consumer's conception of "freshness" or "extended freshness", or whether it would perhaps be better just to speak of "extended shelf-life milk".


Muhlig A.,TU Munich | Kabisch J.,Institute For Mikrobiologie Und Biotechnologie | Pichner R.,Institute For Mikrobiologie Und Biotechnologie | Scherer S.,TU Munich | Muller-Herbst S.,TU Munich
Food Microbiology | Year: 2014

The antimicrobial action of the curing agent sodium nitrite (NaNO2) in raw sausage fermentation is thought to mainly depend on the release of cytotoxic nitric oxide (NO) at acidic pH. Salmonella Typhimurium is capable of detoxifying NO via the flavohemoglobin HmpA, the flavorubredoxin NorV and the periplasmic cytochrome C nitrite reductase NrfA. In this study, the contribution of these systems to nitrosative stress tolerance in raw sausages was investigated. Invitro growth assays of the S. Typhimurium 14028 deletion mutants δhmpA, δnorV and δnrfA revealed a growth defect of δhmpA in the presence of acidified NaNO2. Transcriptional analysis of the genes hmpA, norV and nrfA in the wild-type showed a 41-fold increase in hmpA transcript levels in the presence of 150mg/l acidified NaNO2, whereas transcription of norV and nrfA was not enhanced. However, challenge assays performed with short-ripened spreadable sausages produced with 0 or 150mg/kg NaNO2 failed to reveal a phenotype for any of the mutants compared to the wild-type. Hence, none of the NO detoxification systems HmpA, NorV and NrfA is solely responsible for nitrosative stress tolerance of S. Typhimurium in raw sausages. Whether these systems act cooperatively, or if there are other yet undescribed mechanisms involved is currently unknown. © 2014 Elsevier Ltd.


Ziegler E.,Susanne Eckardt | Eckardt S.,Institute for Mikrobiologie und Biotechnologie | Kabisch J.,Institute for Mikrobiologie und Biotechnologie | Hechelmann H.,Institute for Mikrobiologie und Biotechnologie | Gareis M.,Ludwig Maximilians University of Munich
Fleischwirtschaft | Year: 2012

In Germany no information is available on the occurrence of Clostridium (Cl.) estertheticum on refrigerated raw meat. These psych-rophilic anaerobic sporeforming bacteria often cause blown pack spoilage of vacuum packed beef and the contamination most likely occurs by airborne spores. However, there are no specific protocols for DNA extraction from Clostridia-spores. The goal of this study was to establish a reliable DNA preparation method for PCR detection of Cl. estertheticum vegetative cells and spores. Moreover, we developed a colony hybridisation assay for the identification of Cl. estertheticum and Cl. estertheticum related organisms on solid media. These methods were applied to examine vacuum-packed beef samples from the German retail. For an optimised DNA extraction best results were obtained when the spores were disrupted in a matrix of glass and silica particles with a commercial cell disrupter. The novel method to identify Cl. estertheticum on solid media via colony hybridisation with a Digoxigenin-labelled probe produced positive results for Cl. estertheticum, Cl. estertheticum-like strains and Cl. frigoris after colony hybridisation. Analysis of the meat samples (n = 20) with this hybridisation technique identified two Cl. estertheticum and 21 Cl. estertheticum-like strains in five beef samples. The described methods allow a reliable and specific detection and isolation of Cl. estertheticum and Cl. estertheticum related organisms (Cl. estertheticum-like/Cl. frigoris) from contaminated meat samples.


Muller-Herbst S.,TU Munich | Wustner S.,TU Munich | Wustner S.,Ludwig Maximilians University of Munich | Kabisch J.,Institute For Mikrobiologie Und Biotechnologie | And 3 more authors.
International Journal of Food Microbiology | Year: 2016

Sodium nitrite (NaNO2) is added as a preservative during raw meat processing such as raw sausage production to inhibit growth of pathogenic bacteria. In the present study it was shown in challenge assays that the addition of sodium nitrite indeed inhibited growth and survival of Listeria monocytogenes in short-ripened spreadable raw sausages. Furthermore, in vitro growth analyses were performed, which took into account combinations of various parameters of the raw sausage ripening process like temperature, oxygen availability, pH, NaCl concentration, and absence or presence of NaNO2. Data based on 300 growth conditions revealed that the inhibitory effect of nitrite was most prominent in combination with acidification, a combination that is also achieved during short-ripened spreadable raw sausage production. At pH 6.0 and below, L. monocytogenes was unable to replicate in the presence of 200 mg/l NaNO2. During the adaptation of L. monocytogenes to acidified nitrite stress (pH 6.0, 200 mg/l NaNO2) in comparison to acid exposure only (pH 6.0, 0 mg/l NaNO2), a massive transcriptional adaptation was observed using microarray analyses. In total, 202 genes were up-regulated and 204 genes were down-regulated. In accordance with growth inhibition, a down-regulation of genes encoding for proteins which are involved in central cellular processes, like cell wall/membrane/envelope biogenesis, translation and ribosomal structure and biogenesis, transcription, and replication, recombination and repair, was observed. Among the up-regulated genes the most prominent group belonged to poorly characterized genes. A considerable fraction of the up-regulated genes has been shown previously to be up-regulated intracellularly in macrophages, after exposure to acid shock or to be part of the SigB regulon. These data indicate that the adaptation to acidified nitrite partly overlaps with the adaptation to stress conditions being present during host colonization. © 2016 Elsevier B.V.


Schweiger P.,Institute For Mikrobiologie Und Biotechnologie | Gross H.,Institute For Pharmazeutische Biologie | Deppenmeier U.,Institute For Mikrobiologie Und Biotechnologie
Applied Microbiology and Biotechnology | Year: 2010

Two cytosolic NADPH-dependent carbonyl reductases from Gluconobacter oxydans 621H, Gox0644 and Gox1615, were heterologously produced in Escherichia coli. The recombinant proteins were purified to homogeneity and characterized. Gox0644 and Gox1615 were dimers with native molecular masses of 66.1 and 74.5 kDa, respectively. The enzymes displayed broad substrate specificities and reduced α-ketocarbonyls at the keto moiety most proximal to the terminus of the alkyl chain to produce alpha-hydroxy carbonyls, as demonstrated by NMR. With respect to stereoselectivity, protein Gox0644 specifically reduced 2,3-pentanedione to 2R-hydroxy-pentane-3-one, whereas Gox1615 produced 2S-hydroxy-pentane-3-one. Both enzymes also reduced 1-phenyl-1,2-propanedione to 2-hydroxy-1-phenylpropane-1-one, which is a key intermediate in the production of numerous pharmaceuticals, such as antifungal azoles and antidepressants. Gox0644 displayed highest activities with 2,3-diones, α-ketoaldehydes, α-keto esters, and 2,5-diketogluconate. Gox1615 was less active with these substrates, but displayed a broader substrate spectrum reducing a variety of α-diketones and aldehydes. © 2010 Springer-Verlag.


PubMed | Institute For Mikrobiologie Und Biotechnologie
Type: Journal Article | Journal: Applied microbiology and biotechnology | Year: 2014

The -proteobacterium Sphingomonas wittichii RW1 is known for its ability to degrade dioxins and related toxic substances. Bioinformatic analysis of the genome indicated that this organism may contain the largest number of pyrroloquinoline quinone-dependent dehydrogenases of any bacteria sequenced so far. Sequence analysis also showed that one of these genes (swit_4395) encodes an enzyme that belongs to the class of periplasmic glucose dehydrogenases. This gene was fused to a pelB signal sequence and a strep-tag coding region at the 5 and 3 ends, respectively. The fusion product was cloned into the broad-host range expression vector pBBR1p264-Streplong and the corresponding protein was heterologously produced in Escherichia coli, purified via Strep-Tactin affinity chromatography, and characterized. The protein Swit_4395 had a subunit mass of 39.3kDa and formed active homooctamers and homododecamers. The enzyme showed the highest activities with short- and medium-chain aldehydes (chain length C1-C6) and ketoaldehydes, such as methylglyoxal and phenylglyoxal. Butyraldehyde was the best substrate, with V max and apparent K M values of 3,970U/mg protein and 12.3mM, respectively. Pyrroloquinoline quinone was detected using UV-Vis spectroscopy and was found to be a prosthetic group of the purified enzyme. Therefore, Swit_4395 was identified as a pyrroloquinoline quinone-dependent aldehyde dehydrogenase. The enzyme could be purified from the native host when the expression vector was introduced into S. wittichii RW1, indicating homologous protein production. Overproduction of Swit_4395 in S. wittichii RW1 dramatically increased the tolerance of the bacterium toward butyraldehyde and thus might contribute to the detoxification of toxic aldehydes.


PubMed | Institute For Mikrobiologie Und Biotechnologie and TU Munich
Type: | Journal: Food microbiology | Year: 2014

The antimicrobial action of the curing agent sodium nitrite (NaNO2) in raw sausage fermentation is thought to mainly depend on the release of cytotoxic nitric oxide (NO) at acidic pH. Salmonella Typhimurium is capable of detoxifying NO via the flavohemoglobin HmpA, the flavorubredoxin NorV and the periplasmic cytochrome C nitrite reductase NrfA. In this study, the contribution of these systems to nitrosative stress tolerance in raw sausages was investigated. Invitro growth assays of the S. Typhimurium 14028 deletion mutants hmpA, norV and nrfA revealed a growth defect of hmpA in the presence of acidified NaNO2. Transcriptional analysis of the genes hmpA, norV and nrfA in the wild-type showed a 41-fold increase in hmpA transcript levels in the presence of 150mg/l acidified NaNO2, whereas transcription of norV and nrfA was not enhanced. However, challenge assays performed with short-ripened spreadable sausages produced with 0 or 150mg/kg NaNO2 failed to reveal a phenotype for any of the mutants compared to the wild-type. Hence, none of the NO detoxification systems HmpA, NorV and NrfA is solely responsible for nitrosative stress tolerance of S. Typhimurium in raw sausages. Whether these systems act cooperatively, or if there are other yet undescribed mechanisms involved is currently unknown.

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