Functional genomics and expression analysis of the Corynebacterium glutamicum fpr2-cysIXHDNYZ gene cluster involved in assimilatory sulphate reduction

Rückert C, Koch DJ, Rey DA, et al. Functional genomics and expression analysis of the Corynebacterium glutamicum fpr2-cysIXHDNYZ gene cluster involved in assimilatory sulphate reduction. BMC Genomics. 2005;6(1): 121.Background: Corynebacterium glutamicum is a high-GC Gram-positive soil bacterium of great biotechnological importance for the production of amino acids. To facilitate the rational design of sulphur amino acid-producing strains, the pathway for assimilatory sulphate reduction providing the necessary reduced sulfur moieties has to be known. Although this pathway has been well studied in Gram-negative bacteria like Escherichia coli and low-GC Gram-positives like Bacillus subtilis, little is known for the Actinomycetales and other high-GC Gram-positive bacteria. Results: The genome sequence of C. glutamicum was searched for genes involved in the assimilatory reduction of inorganic sulphur compounds. A cluster of eight candidate genes could be identified by combining sequence similarity searches with a subsequent synteny analysis between C. glutamicum and the closely related C. efficiens. Using mutational analysis, seven of the eight candidate genes, namely cysZ, cysY, cysN, cysD, cysH, cysX, and cysI, were demonstrated to be involved in the reduction of inorganic sulphur compounds. For three of the up to now unknown genes possible functions could be proposed: CysZ is likely to be the sulphate permease, while CysX and CysY are possibly involved in electron transfer and cofactor biosynthesis, respectively. Finally, the candidate gene designated fpr2 influences sulphur utilisation only weakly and might be involved in electron transport for the reduction of sulphite. Real-time RT-PCR experiments revealed that cysIXHDNYZ form an operon and that transcription of the extended cluster fpr2 cysIXHDNYZ is strongly influenced by the availability of inorganic sulphur, as well as L-cysteine. Mapping of the fpr2 and cysIXHDNYZ promoters using RACE-PCR indicated that both promoters overlap with binding-sites of the transcriptional repressor McbR, suggesting an involvement of McbR in the observed regulation. Comparative genomics revealed that large parts of the extended cluster are conserved in 11 of 17 completely sequenced members of the Actinomycetales. Conclusion: The set of C. glutamicum genes involved in assimilatory model organisms E. coli and B. subtilis is used by members of this order, providing the basis for further biochemical studies.sulphate reduction was identified and four novel genes involved in this pathway were found. The high degree of conservation of this cluster among the Actinomycetales supports the hypothesis that a different metabolic pathway for the reduction of inorganic sulphur compounds than that known from the well-studied model organisms E. coli and B. subtilis is used by members of this order, providing the basis for further biochemical studies

Genomics of Sponge-Associated Streptomyces spp. Closely Related to Streptomyces albus J1074: Insights into Marine Adaptation and Secondary Metabolite Biosynthesis Potential

A total of 74 actinomycete isolates were cultivated from two marine sponges, Geodia barretti and Phakellia ventilabrum collected at the same spot at the bottom of the Trondheim fjord (Norway). Phylogenetic analyses of sponge-associated actinomycetes based on the 16S rRNA gene sequences demonstrated the presence of species belonging to the genera Streptomyces, Nocardiopsis, Rhodococcus, Pseudonocardia and Micromonospora. Most isolates required sea water for growth, suggesting them being adapted to the marine environment. Phylogenetic analysis of Streptomyces spp. revealed two isolates that originated from different sponges and had 99.7% identity in their 16S rRNA gene sequences, indicating that they represent very closely related strains. Sequencing, annotation, and analyses of the genomes of these Streptomyces isolates demonstrated that they are sister organisms closely related to terrestrial Streptomyces albus J1074. Unlike S. albus J1074, the two sponge streptomycetes grew and differentiated faster on the medium containing sea water. Comparative genomics revealed several genes presumably responsible for partial marine adaptation of these isolates. Genome mining targeted to secondary metabolite biosynthesis gene clusters identified several of those, which were not present in S. albus J1074, and likely to have been retained from a common ancestor, or acquired from other actinomycetes. Certain genes and gene clusters were shown to be differentially acquired or lost, supporting the hypothesis of divergent evolution of the two Streptomyces species in different sponge hosts

Control in the Label: Self-Declared, Certified, Accredited? : On-Pack Consumer Communication About Compliance Control in Voluntary Food Schemes from a Legal Perspective

In the EU, the number of voluntary food schemes has grown rampantly over the past 20 years. From a consumer perspective, what was initially praised as a solution to the information asymmetry problem of credence goods soon became a problem of itself: consumers do not know what the various food scheme labels mean, and especially whether and how they are subject to compliance control (self-declaration, certification, and accreditation). In this contribution, we argue that it is necessary to examine how messages about compliance control are communicated to the consumer on pack and how they affect consumers, and show that on pack communication of food scheme labels offers significant opportunities for influencing consumer perceptions. Consequently, food businesses are under a legal duty not to mislead consumers and to ensure that information is sufficiently clear, including elements indicating the control a scheme is subject to

Genomics of Sponge-Associated Streptomyces spp. Closely Related to Streptomyces albus J1074: Insights into Marine Adaptation and Secondary Metabolite Biosynthesis Potential

Ian E, Malko DB, Sekurova ON, et al. Genomics of Sponge-Associated Streptomyces spp. Closely Related to Streptomyces albus J1074: Insights into Marine Adaptation and Secondary Metabolite Biosynthesis Potential. PloS one. 2014;9(5): e96719.A total of 74 actinomycete isolates were cultivated from two marine sponges, Geodia barretti and Phakellia ventilabrum collected at the same spot at the bottom of the Trondheim fjord (Norway). Phylogenetic analyses of sponge-associated actinomycetes based on the 16S rRNA gene sequences demonstrated the presence of species belonging to the genera Streptomyces, Nocardiopsis, Rhodococcus, Pseudonocardia and Micromonospora. Most isolates required sea water for growth, suggesting them being adapted to the marine environment. Phylogenetic analysis of Streptomyces spp. revealed two isolates that originated from different sponges and had 99.7% identity in their 16S rRNA gene sequences, indicating that they represent very closely related strains. Sequencing, annotation, and analyses of the genomes of these Streptomyces isolates demonstrated that they are sister organisms closely related to terrestrial Streptomyces albus J1074. Unlike S. albus J1074, the two sponge streptomycetes grew and differentiated faster on the medium containing sea water. Comparative genomics revealed several genes presumably responsible for partial marine adaptation of these isolates. Genome mining targeted to secondary metabolite biosynthesis gene clusters identified several of those, which were not present in S. albus J1074, and likely to have been retained from a common ancestor, or acquired from other actinomycetes. Certain genes and gene clusters were shown to be differentially acquired or lost, supporting the hypothesis of divergent evolution of the two Streptomyces species in different sponge hosts

Activity-Based Protein Profiling for the Identification of Novel Carbohydrate-Active Enzymes Involved in Xylan Degradation in the Hyperthermophilic Euryarchaeon Thermococcus sp. Strain 2319x1E

Klaus T, Ninck S, Albersmeier A, et al. Activity-Based Protein Profiling for the Identification of Novel Carbohydrate-Active Enzymes Involved in Xylan Degradation in the Hyperthermophilic Euryarchaeon Thermococcus sp. Strain 2319x1E. Frontiers in Microbiology . 2022;12: 734039.Activity-based protein profiling (ABPP) has so far scarcely been applied in Archaea in general and, especially, in extremophilic organisms. We herein isolated a novel Thermococcus strain designated sp. strain 2319x1E derived from the same enrichment culture as the recently reported Thermococcus sp. strain 2319x1. Both strains are able to grow with xylan as the sole carbon and energy source, and for Thermococcus sp. strain 2319x1E (optimal growth at 85 degrees C, pH 6-7), the induction of xylanolytic activity in the presence of xylan was demonstrated. Since the solely sequence-based identification of xylanolytic enzymes is hardly possible, we established a complementary approach by conducting comparative full proteome analysis in combination with ABPP using alpha- or beta-glycosidase selective probes and subsequent mass spectrometry (MS)-based analysis. This complementary proteomics approach in combination with recombinant protein expression and classical enzyme characterization enabled the identification of a novel bifunctional maltose-forming alpha-amylase and deacetylase (EGDIFPOO_00674) belonging to the GH57 family and a promiscuous beta-glycosidase (EGIDFPOO_00532) with beta-xylosidase activity. We thereby further substantiated the general applicability of ABPP in archaea and expanded the ABPP repertoire for the identification of glycoside hydrolases in hyperthermophiles

Outcome and clinical course of EHEC O104 infection in hospitalized patients: A prospective single center study

<div><p>Objectives</p><p>Shiga-toxin producing O157:H7 Entero Haemorrhagic E. coli [STEC/EHEC] are the most common cause of Haemolytic Uraemic Syndrome [HUS] related to infectious haemorrhagic colitis. Nearly all recommendations on long term treatment of EHEC infections refer to this strain. The 2011 outbreak in Northern Europe was the first of this dimension to be caused by the serotype O104:H4. We report on the 3.5 year follow up of 61 patients diagnosed with symptomatic EHEC O104:H4 infection in spring 2011.</p><p>Methods</p><p>Patients with EHEC O104 infection were followed in a monocentric, prospective observational study at four time points: 4, 12, 24 and 36 months. These data include the patients’ histories, clinical findings, and complications.</p><p>Results</p><p>Sixty-one patients suffering from EHEC O104:H4 associated enterocolitis participated in the study at the time of hospital discharge. The mean age of patients was 43 ± 2 years, 37 females and 24 males. 48 patients participated in follow up 1 [FU 1], 34 patients in follow up 2 [FU 2], 23 patients in follow up 3 [FU 3] and 18 patients in follow up 4 [FU 4]. Out of 61 patients discharged from the hospital and included in the study, 54 [84%] were examined at least at one additional follow up. Serum creatinine decreased significantly between discharge and FU 1 from 1.3 ± 0.1 mg/dl to 0.7 ± 0.1 mg/dl [p = 0.0045]. From FU 1 until FU 4, no further change in creatinine levels could be observed. The patients need of antihypertensive medications decreased significantly [p = 0.0005] between discharge and FU 1 after four months. From FU 1 until FU 3, 24 months later, no further significant change in antihypertensive treatment was observed.</p><p>Conclusions</p><p>Our findings suggest that patients free of pathological findings at time of discharge do not need a specific follow up. Patients with persistent health problems at hospital discharge should be clinically monitored over four months to evaluate chronic organ damage. Progressive or new emerging renal damage could not be observed over time in any patient.</p></div

Activity-based protein profiling for the identification of novel carbohydrate-active enzymes involved in xylan degradation in the hyperthermophilic euryarchaeon thermococcus sp. Strain 2319x1E

Activity-based protein profiling (ABPP) has so far scarcely been applied in Archaea in general and, especially, in extremophilic organisms. We herein isolated a novel Thermococcus strain designated sp. strain 2319x1E derived from the same enrichment culture as the recently reported Thermococcus sp. strain 2319x1. Both strains are able to grow with xylan as the sole carbon and energy source, and for Thermococcus sp. strain 2319x1E (optimal growth at 85°C, pH 6-7), the induction of xylanolytic activity in the presence of xylan was demonstrated. Since the solely sequence-based identification of xylanolytic enzymes is hardly possible, we established a complementary approach by conducting comparative full proteome analysis in combination with ABPP using α- or β-glycosidase selective probes and subsequent mass spectrometry (MS)-based analysis. This complementary proteomics approach in combination with recombinant protein expression and classical enzyme characterization enabled the identification of a novel bifunctional maltose-forming α-amylase and deacetylase (EGDIFPOO_00674) belonging to the GH57 family and a promiscuous β-glycosidase (EGIDFPOO_00532) with β-xylosidase activity. We thereby further substantiated the general applicability of ABPP in archaea and expanded the ABPP repertoire for the identification of glycoside hydrolases in hyperthermophiles.</p

Response of the thermoacidophilic Archaeon Sulfolobus acidocaldarius to solvent stress exemplified by 1-butanol exposure

Benninghoff JC, Kuschmierz L, Zhou X, et al. Response of the thermoacidophilic Archaeon Sulfolobus acidocaldarius to solvent stress exemplified by 1-butanol exposure. Applied and environmental microbiology. 2021.Sulfolobus acidocaldarius is a thermoacidophilic crenarchaeon with optimal growth at 80 °C and pH 2 - 3. Due to its unique physiological properties allowing life at environmental extremes and recent availability of genetic tools, this extremophile receives increasing interest for biotechnological application. In order to elucidate the potential of tolerating process-related stress conditions, we investigated the response of S. acidocaldarius towards the industrially relevant organic solvent 1-butanol.In response to butanol exposure, biofilm formation of S. acidocaldarius was enhanced and occurred up to 1.5% (v/v) 1-butanol, while planktonic growth was only observed up to 1% (v/v) 1-butanol. Confocal laser scanning microscopy revealed that biofilm architecture changed with the formation of denser and higher tower-like structures. Concomitantly, changes in the extracellular polymeric substances with enhanced carbohydrate and protein content were determined in 1-butanol-exposed biofilms. Using scanning electron microscopy three different cell morphotypes were observed in response to 1-butanol.Transcriptome and proteome analyses were performed comparing the response of planktonic and biofilm cells in absence and presence of 1-butanol. In response to 1% (v/v) 1-butanol transcript levels of genes encoding motility and cell envelope structures as well as membrane proteins were reduced. Cell division and/or vesicle formation was upregulated. Furthermore, changes of immune and defence systems, as well as metabolism and general stress response were observed. Our findings show that the extreme lifestyle of S. acidocaldarius coincided with a high tolerance to organic solvents. This study provides first insights into biofilm formation and membrane/cell stress caused by organic solvents in S. acidocaldarius ImportanceArchaea are unique in terms of metabolic and cellular processes as well as the adaptation to extreme environments. In the past few years, the development of genetic systems and biochemical, genetic and poly-omics studies have provided deep insights into the physiology of some archaeal model organisms. In this study, we used S. acidocaldarius adapted to two extremes, low pH and high temperature, to study its tolerance and robustness as well as its global cellular response towards organic solvents exemplified by 1-butanol. We were able to identify biofilm formation as primary cellular response to 1-butanol. Furthermore, the triggered cell/membrane stress led to significant changes in culture heterogeneity accompanied by changes in central cellular processes such as cell division and cellular defense systems, thus suggesting a global response for the protection at population level. Copyright © 2021 Benninghoff et al

Trend of systolic and diastolic blood pressure.

<p>Patients that suffered from EHEC infection with or without HUS, at the time of their hospital discharge. FU 1 (4 month, n = 48), FU 2 (12 month, n = 34), FU 3 (24 month, n = 23) and FU 4 (42 month, n = 18) (mean ± SD).</p

Trend of serum creatinine, urine albumin and antihypertensive drugs.

<p>Patient case of severe renal impairment at the time of hospital discharge. FU 1 (4 month, n = 48), FU 2 (12 month, n = 34), FU 3 (24 month, n = 23) and FU 4 (42 month, n = 18).</p