Absence of the highly expressed small carbohydrate-binding protein Cgt improves the acarbose formation in Actinoplanes sp. SE50/110

Schaffert L, Schneiker-Bekel S, Gierhake J, et al. Absence of the highly expressed small carbohydrate-binding protein Cgt improves the acarbose formation in Actinoplanes sp. SE50/110. Applied Microbiology and Biotechnology. 2020;104:5395–5408.Actinoplanes sp. SE50/110 (ATCC 31044) is the wild type of industrial producer strains of acarbose. Acarbose has been used since the early 1990s as an inhibitor of intestinal human α-glucosidases in the medical treatment of type II diabetes mellitus. The small secreted protein Cgt, which consists of a single carbohydrate-binding module (CBM) 20-domain, was found to be highly expressed in Actinoplanes sp. SE50/110 in previous studies, but neither its function nor a possible role in the acarbose formation was explored, yet. Here, we demonstrated the starch-binding function of the Cgt protein in a binding assay. Transcription analysis showed that the cgt gene was strongly repressed in the presence of glucose or lactose. Due to this and its high abundance in the extracellular proteome of Actinoplanes, a functional role within the sugar metabolism or in the environmental stress protection was assumed. However, the gene deletion mutant ∆cgt, constructed by CRISPR/Cas9 technology, displayed no apparent phenotype in screening experiments testing for pH and osmolality stress, limited carbon source starch, and the excess of seven different sugars in liquid culture and further 97 carbon sources in the Omnilog Phenotypic Microarray System of Biolog. Therefore, a protective function as a surface protein or a function within the retainment and the utilization of carbon sources could not be experimentally validated. Remarkably, enhanced production of acarbose was determined yielding into 8–16% higher product titers when grown in maltose-containing medium

Deployment of Image Analysis Algorithms under Prevalence Shifts

Domain gaps are among the most relevant roadblocks in the clinical translation of machine learning (ML)-based solutions for medical image analysis. While current research focuses on new training paradigms and network architectures, little attention is given to the specific effect of prevalence shifts on an algorithm deployed in practice. Such discrepancies between class frequencies in the data used for a method's development/validation and that in its deployment environment(s) are of great importance, for example in the context of artificial intelligence (AI) democratization, as disease prevalences may vary widely across time and location. Our contribution is twofold. First, we empirically demonstrate the potentially severe consequences of missing prevalence handling by analyzing (i) the extent of miscalibration, (ii) the deviation of the decision threshold from the optimum, and (iii) the ability of validation metrics to reflect neural network performance on the deployment population as a function of the discrepancy between development and deployment prevalence. Second, we propose a workflow for prevalence-aware image classification that uses estimated deployment prevalences to adjust a trained classifier to a new environment, without requiring additional annotated deployment data. Comprehensive experiments based on a diverse set of 30 medical classification tasks showcase the benefit of the proposed workflow in generating better classifier decisions and more reliable performance estimates compared to current practice

Complete genome sequence of the actinomycete Actinoalloteichus hymeniacidonis type strain HPA 177T isolated from a marine sponge

Schaffert L, Albersmeier A, Winkler A, Kalinowski J, Zotchev SB, Rückert C. Complete genome sequence of the actinomycete Actinoalloteichus hymeniacidonis type strain HPA 177T isolated from a marine sponge. Standards in Genomic Sciences. 2016;11(1): 91.Actinoalloteichus hymeniacidonis HPA 177T is a Gram-positive, strictly aerobic, black pigment producing and spore-forming actinomycete, which forms branching vegetative hyphae and was isolated from the marine sponge Hymeniacidon perlevis. Actinomycete bacteria are prolific producers of secondary metabolites, some of which have been developed into anti-microbial, anti-tumor and immunosuppressive drugs currently used in human therapy. Considering this and the growing interest in natural products as sources of new drugs, actinomycete bacteria from the hitherto poorly explored marine environments may represent promising sources for drug discovery. As A. hymeniacidonis, isolated from the marine sponge, is a type strain of the recently described and rare genus Actinoalloteichus, knowledge of the complete genome sequence enables genome analyses to identify genetic loci for novel bioactive compounds. This project, describing the 6.31 Mbp long chromosome, with its 5346 protein-coding and 73 RNA genes, will aid the Genomic Encyclopedia of Bacteria and Archaea project

Evaluation of vector systems and promoters for overexpression of the acarbose biosynthesis gene acbC in Actinoplanes sp. SE50/110

Schaffert L, März C, Burkhardt L, et al. Evaluation of vector systems and promoters for overexpression of the acarbose biosynthesis gene acbC in Actinoplanes sp. SE50/110. Microbial Cell Factories. 2019;18(1): 114.Background Actinoplanes sp. SE50/110 is a natural producer of acarbose. It has been extensively studied in the last decades, which has led to the comprehensive analysis of the whole genome, transcriptome and proteome. First genetic and microbial techniques have been successfully established allowing targeted genome editing by CRISPR/Cas9 and conjugal transfer. Still, a suitable system for the overexpression of singular genes does not exist for Actinoplanes sp. SE50/110. Here, we discuss, test and analyze different strategies by the example of the acarbose biosynthesis gene acbC. Results The integrative φC31-based vector pSET152 was chosen for the development of an expression system, as for the replicative pSG5-based vector pKC1139 unwanted vector integration by homologous recombination was observed. Since simple gene duplication by pSET152 integration under control of native promoters appeared to be insufficient for overexpression, a promoter screening experiment was carried out. We analyzed promoter strengths of five native and seven heterologous promoters using transcriptional fusion with the gusA gene and glucuronidase assays as well as reverse transcription quantitative PCR (RT-qPCR). Additionally, we mapped transcription starts and identified the promoter sequence motifs by 5′-RNAseq experiments. Promoters with medium to strong expression were included into the pSET152-system, leading to an overexpression of the acbC gene. AcbC catalyzes the first step of acarbose biosynthesis and connects primary to secondary metabolism. By overexpression, the acarbose formation was not enhanced, but slightly reduced in case of strongest overexpression. We assume either disturbance of substrate channeling or a negative feed-back inhibition by one of the intermediates, which accumulates in the acbC-overexpression mutant. According to LC–MS-analysis, we conclude, that this intermediate is valienol-7P. This points to a bottleneck in later steps of acarbose biosynthesis. Conclusion Development of an overexpression system for Actinoplanes sp. SE50/110 is an important step for future metabolic engineering. This system will help altering transcript amounts of singular genes, that can be used to unclench metabolic bottlenecks and to redirect metabolic resources. Furthermore, an essential tool is provided, that can be transferred to other subspecies of Actinoplanes and industrially relevant derivatives

Terrigenous dissolved organic matter persists in the energy-limited deep groundwaters of the Fennoscandian Shield

The deep terrestrial biosphere encompasses the life belowthe photosynthesisfueled surface that perseveres in typically nutrient and energy depleted anoxic groundwaters. The composition and cycling of this vast dissolved organic matter (DOM) reservoir relevant to the global carbon cycle remains to be deciphered. Here we show that recent Baltic Sea-influenced to ancient preHolocene saline Fennoscandian Shield deep bedrock fracture waters carried DOM with a strong terrigenous signature and varying contributions from abiotic and biotic processes. Removal of easily degraded carbon at the surfaceto-groundwater transition and corresponding microbial community assembly processes likely resulted in the highly similar DOM signatures across the notably different water types that selected for a core microbiome. In combination with the aliphatic character, depleted d13C signatures in DOM indicated recent microbial production in the oldest, saline groundwater. Our study revealed the persistence of terrestrially-sourced carbon in severely energy limited deep continental groundwaters supporting deep microbial life

Surgörning av flytgödsel : Strukturanalyser av betong efter exponering i surgjord och icke surgjord flytgödsel

Samples of three different concrete qualities were prepared and hardened, before exposure in cattle slurry without sulphuric acid (A) and with sulphuric acid added until pH<5.5 (B). The samples were exposed for two years in containers with about 45 L slurry. The boxes with slurry and concrete samples were placed in a ventilated room at 20 °C. The slurry and air temperatures were recorded continuously with temperature loggers, data being recorded every third hour. The slurry level in the boxes and the slurry pH were checked regularly during the experiment. Slurry or acid was added, if necessary, to maintain the level and pH<5.5. Before pH measurements, the slurry was stirred gently in both boxes. To restrict evaporation, the containers had non-airtight plastic covers between measurements. Half-way through exposure, the old slurry was replaced with fresh slurry (acidified and non-acidified treatments) to mimic conditions in farm storage where fresh slurry is added continuously during storage. After two years’ storage, the experiment was finalised. The concrete samples were taken out of the slurry, washed gently with water and put into labelled plastic bags. The samples were delivered to RISE CBI’s concrete laboratory, where the structural analyses were performed. These used petrographic microscopy techniques to examine the effects of exposure to two potentially aggressive environments, non-acidified and acidified cattle slurry, on concrete with three different mixes. The studied surfaces in the concrete samples were oriented vertically in the plastic containers. Polished sections were evaluated with a stereo microscope, and thin sections were evaluated using a polarising microscope and sources for visible and UV light. The results of the study show that the acidified slurry is more chemically aggressive to the cement paste in all the concrete mixes analysed. This can be explained by the solution’s lower pH. The extent of the chemical attack correlates with the initial quality of the concrete mix (water-powder ratio and type of binder). The deepest chemical attacks were observed in samples A1 and B1 consisting of “regular” concrete mix with w/c 0.59. The “long lasting quality” (LLC) concrete with a binder specially developed for low-pH environments shows markedly better resistance to chemical attack. The effects of the chemical attack on concrete after two years’ exposure can be classified as weak, consisting mainly of an increase in the capillary porosity of the cement paste in the outer layer of the concrete. The increase in porosity is considered to be due to the partial leaching of calcium hydroxide.Surgörning av flytgödsel används som en metod för att minska ammoniak­avgången från stallgödsel vid hanteringen. För att minimera emissionerna under lagringen, eftersträvas ett pH-värde av 5,5 hos gödseln. Svavelsyra är den vanligaste syran eftersom svavelsyran är stark och prisvärd. En pH-sänkning hos gödseln kan dock innebära frätskador på betongen i lager, som kan betyda kortare livslängd om inte betongkvaliteten anpassas till gödselns pH. Syftet med denna studie var att se hur surgjord gödsel (B) påverkar betongytan hos olika betongkvaliteter jämfört med icke-surgjord nötflytgödsel (A). I studien ingick betongprover av tre olika kvaliteter, som motsvarade kvaliteten hos 1) bottenplattan hos flytgödsellager, 2) prefabricerade väggelement till flytgödsellager samt 3) en betongkvalité kallad ”Long Lasting Concrete”, som utvecklats av Abetong AB för lagring av material med lågt pH, t.ex. ensilage. Betongprover (0,1 m x 0,1 m x 0,1 m) tillverkades av Abetong AB och exponerades under två år i nötflytgödsel utan syra (A) respektive surgjord nötflytgödsel (B). Behållarna med respektive gödseltyp placerades i rum med konstant temperatur ca 20°C. Under lagringen mättes regelbundet pH och vid behov tillsattes mer gödsel samt syra för att hålla pH-värdet under 5,5. Halvvägs genom studien byttes gödseln ut mot färsk gödsel för att efterlikna verkliga lager och efter två år avslutades studien. Betongproverna togs ut ur gödselbehållarna, duschades försiktigt, paketerades och fördes till RISE CBI:s betonglaboratorium.   I laboratoriet utfördes strukturanalyser av betongen, där den studerade ytan hos betongen hade varit vertikalt orienterad i gödselbehållaren. För att undersöka gödseltypernas effekt på de olika betongblandningarna användes betongpetrografiska analysmetoder. Polerade betongsnitt och tunnslip tillverkade av betongproverna utvärderades dels med hjälp av stereomikroskop, dels med polarisationsmikroskop och ljuskällor för synligt och ultraviolett ljus. Resultaten från studierna visade att den surgörande gödseln var mer kemiskt aggressiv mot cementen i alla tre betongblandningarna. Det förklaras med det lägre pH-värdet hos den surgjorda gödseln. Graden av kemiska påverkan hade samband med kvaliteten hos betongen, dvs. förhållandet vatten:cement och typ av bindemedel i betongen. Största kemiska påverkan uppmättes i betongkvalité 1 som består av ”ordinär” betong med vattencementtal 0,59 (prover A1 och B1), motsvarande den som används för bottenplattan i flytgödsellager. Betongkvalité 3, utvecklad för material med lågt pH (LLC), visade betydligt högre motståndskraft mot kemisk påverkan. Den kemiska påverkan på betongen var totalt sett svag efter två års exponering och bestod främst av en ökning av den kapillära porositeten hos bindemedlet i betongens yttre skikt. Den ökade porositeten bedöms bero på att en del av cementpastans kalciumhydroxid har brutits ner och lakats ur betongen. Vanligtvis är livslängden hos ett gödsellager minst 20 år, så det finns anledning att vara observant över tid på hur betongen påverkas eller att som förebyggande åtgärd använda en betong av högre kvalitet

Surgörning av flytgödsel : Strukturanalyser av betong efter exponering i surgjord och icke surgjord flytgödsel

Samples of three different concrete qualities were prepared and hardened, before exposure in cattle slurry without sulphuric acid (A) and with sulphuric acid added until pH<5.5 (B). The samples were exposed for two years in containers with about 45 L slurry. The boxes with slurry and concrete samples were placed in a ventilated room at 20 °C. The slurry and air temperatures were recorded continuously with temperature loggers, data being recorded every third hour. The slurry level in the boxes and the slurry pH were checked regularly during the experiment. Slurry or acid was added, if necessary, to maintain the level and pH<5.5. Before pH measurements, the slurry was stirred gently in both boxes. To restrict evaporation, the containers had non-airtight plastic covers between measurements. Half-way through exposure, the old slurry was replaced with fresh slurry (acidified and non-acidified treatments) to mimic conditions in farm storage where fresh slurry is added continuously during storage. After two years’ storage, the experiment was finalised. The concrete samples were taken out of the slurry, washed gently with water and put into labelled plastic bags. The samples were delivered to RISE CBI’s concrete laboratory, where the structural analyses were performed. These used petrographic microscopy techniques to examine the effects of exposure to two potentially aggressive environments, non-acidified and acidified cattle slurry, on concrete with three different mixes. The studied surfaces in the concrete samples were oriented vertically in the plastic containers. Polished sections were evaluated with a stereo microscope, and thin sections were evaluated using a polarising microscope and sources for visible and UV light. The results of the study show that the acidified slurry is more chemically aggressive to the cement paste in all the concrete mixes analysed. This can be explained by the solution’s lower pH. The extent of the chemical attack correlates with the initial quality of the concrete mix (water-powder ratio and type of binder). The deepest chemical attacks were observed in samples A1 and B1 consisting of “regular” concrete mix with w/c 0.59. The “long lasting quality” (LLC) concrete with a binder specially developed for low-pH environments shows markedly better resistance to chemical attack. The effects of the chemical attack on concrete after two years’ exposure can be classified as weak, consisting mainly of an increase in the capillary porosity of the cement paste in the outer layer of the concrete. The increase in porosity is considered to be due to the partial leaching of calcium hydroxide.Surgörning av flytgödsel används som en metod för att minska ammoniak­avgången från stallgödsel vid hanteringen. För att minimera emissionerna under lagringen, eftersträvas ett pH-värde av 5,5 hos gödseln. Svavelsyra är den vanligaste syran eftersom svavelsyran är stark och prisvärd. En pH-sänkning hos gödseln kan dock innebära frätskador på betongen i lager, som kan betyda kortare livslängd om inte betongkvaliteten anpassas till gödselns pH. Syftet med denna studie var att se hur surgjord gödsel (B) påverkar betongytan hos olika betongkvaliteter jämfört med icke-surgjord nötflytgödsel (A). I studien ingick betongprover av tre olika kvaliteter, som motsvarade kvaliteten hos 1) bottenplattan hos flytgödsellager, 2) prefabricerade väggelement till flytgödsellager samt 3) en betongkvalité kallad ”Long Lasting Concrete”, som utvecklats av Abetong AB för lagring av material med lågt pH, t.ex. ensilage. Betongprover (0,1 m x 0,1 m x 0,1 m) tillverkades av Abetong AB och exponerades under två år i nötflytgödsel utan syra (A) respektive surgjord nötflytgödsel (B). Behållarna med respektive gödseltyp placerades i rum med konstant temperatur ca 20°C. Under lagringen mättes regelbundet pH och vid behov tillsattes mer gödsel samt syra för att hålla pH-värdet under 5,5. Halvvägs genom studien byttes gödseln ut mot färsk gödsel för att efterlikna verkliga lager och efter två år avslutades studien. Betongproverna togs ut ur gödselbehållarna, duschades försiktigt, paketerades och fördes till RISE CBI:s betonglaboratorium.   I laboratoriet utfördes strukturanalyser av betongen, där den studerade ytan hos betongen hade varit vertikalt orienterad i gödselbehållaren. För att undersöka gödseltypernas effekt på de olika betongblandningarna användes betongpetrografiska analysmetoder. Polerade betongsnitt och tunnslip tillverkade av betongproverna utvärderades dels med hjälp av stereomikroskop, dels med polarisationsmikroskop och ljuskällor för synligt och ultraviolett ljus. Resultaten från studierna visade att den surgörande gödseln var mer kemiskt aggressiv mot cementen i alla tre betongblandningarna. Det förklaras med det lägre pH-värdet hos den surgjorda gödseln. Graden av kemiska påverkan hade samband med kvaliteten hos betongen, dvs. förhållandet vatten:cement och typ av bindemedel i betongen. Största kemiska påverkan uppmättes i betongkvalité 1 som består av ”ordinär” betong med vattencementtal 0,59 (prover A1 och B1), motsvarande den som används för bottenplattan i flytgödsellager. Betongkvalité 3, utvecklad för material med lågt pH (LLC), visade betydligt högre motståndskraft mot kemisk påverkan. Den kemiska påverkan på betongen var totalt sett svag efter två års exponering och bestod främst av en ökning av den kapillära porositeten hos bindemedlet i betongens yttre skikt. Den ökade porositeten bedöms bero på att en del av cementpastans kalciumhydroxid har brutits ner och lakats ur betongen. Vanligtvis är livslängden hos ett gödsellager minst 20 år, så det finns anledning att vara observant över tid på hur betongen påverkas eller att som förebyggande åtgärd använda en betong av högre kvalitet

The roseoflavin producer Streptomyces davaonensis has a high catalytic capacity and specific genetic adaptations with regard to the biosynthesis of riboflavin.

Kissling L, Schneider C, Seibel K, et al. The roseoflavin producer Streptomyces davaonensis has a high catalytic capacity and specific genetic adaptations with regard to the biosynthesis of riboflavin. Environmental microbiology. 2020.The bacterium Streptomyces davaonensis synthesizes the antibiotic roseoflavin in the stationary phase of growth. The starting point for roseoflavin biosynthesis is riboflavin (vitamin B2 ) and four enzymes (RibC, RosB, RosA and RosC) are necessary to convert a vitamin (riboflavin) into a potent, broad-spectrum antibiotic (roseoflavin). In S. davaonensis seven enzymatic functions are required to synthesize the roseoflavin precursor riboflavin from the central building blocks GTP and ribulose 5-phosphate. When compared to other bacterial and in particular Streptomyces genomes the S. davaonensis genome contains an unusual high number (21) of putative riboflavin biosynthetic genes (rib genes), including a rib gene encoding an additional riboflavin synthase originating from an Archaeon. We show by complementation analyses and enzyme assays that 17 out of these 21 putative rib genes indeed encode for riboflavin biosynthetic enzymes. Biochemical analyses of selected enzymes support this finding. Transcriptome analyses show that all of the rib genes are expressed either in the exponential or in the stationary phase of growth and thus do not represent silent genes. We conclude that the Rib enzymes produced in the stationary phase represent a physiological adaptation to support roseoflavin biosynthesis. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved

Water chemistry of unfiltered groundwater samples from the Fennoscandian Shield deep terrestrial biosphere in 2018 and 2019

Copious amounts of organic carbon are stored for long periods of time in deep continental groundwaters. Little is known about its composition and cycling, mainly due to the difficulties in obtaining sample material. Cool fracture waters of different origins can be obtained under clean conditions at Äspö Hard Rock Laboratory (Äspö HRL, Sweden), operated by the Swedish Nuclear Fuel and Waste Management Company (SKB). We sampled groundwater from different depth (171 to 507 meter below sea level) in the bedrock fractures in November 2018 and March-April 2019. We assessed water chemistry and dissolved organic matter composition via stable carbon isotopic and molecular-formula level analysis in recent Baltic Sea-influenced to old saline fracture waters in the granitic Fennoscandian shield. Physicochemical parameters, major ions, water isotopic compositions (δ18O and δD), total nitrogen as well as dissolved organic matter concentration and stable isotopic composition were obtained for unfiltered groundwater samples from different boreholes

A maltose-regulated large genomic region is activated by the transcriptional regulator MalT in Actinoplanes sp. SE50/110

Droste J, Kulisch M, Wolf T, et al. A maltose-regulated large genomic region is activated by the transcriptional regulator MalT in Actinoplanes sp. SE50/110. Applied microbiology and biotechnology. 2020;104:9283–9294.Actinoplanes sp. SE50/110 is the industrially relevant producer of acarbose, which is used in the treatment of diabetes mellitus. Recent studies elucidated the expression dynamics in Actinoplanes sp. SE50/110 during growth. From these data, we obtained a large genomic region (ACSP50_3900 to ACSP50_3950) containing 51 genes, of which 39 are transcribed in the same manner. These co-regulated genes were found to be stronger transcribed on maltose compared with glucose as a carbon source. The transcriptional regulator MalT was identified as an activator of this maltose-regulated large genomic region (MRLGR). Since most of the genes are poorly annotated, the function of this region is farther unclear. However, comprehensive BLAST analyses indicate similarities to enzymes involved in amino acid metabolism. We determined a conserved binding motif of MalT overlapping the -35 promoter region of 17 transcription start sites inside the MRLGR. The corresponding sequence motif 5'-TCATCC-5nt-GGATGA-3' displays high similarities to reported MalT binding sites in Escherichia coli and Klebsiella pneumoniae, in which MalT is the activator of mal genes. A malT deletion and an overexpression mutant were constructed. Differential transcriptome analyses revealed an activating effect of MalT on 40 of the 51 genes. Surprisingly, no gene of the maltose metabolism is affected. In contrast to many other bacteria, MalT is not the activator of mal genes in Actinoplanes sp. SE50/110. Finally, the MRLGR was found partly in other closely related bacteria of the family Micromonosporaceae. Even the conserved MalT binding site was found upstream of several genes inside of the corresponding regions. KEY POINTS : MalT is the maltose-dependent activator of a large genomic region in ACSP50_WT. The consensus binding motif is similar to MalT binding sites in other bacteria. MalT is not the regulator of genes involved in maltose metabolism in ACSP50_WT