A multi-omic screening approach for the discovery of thermoactive glycoside hydrolases.

Next-generation sequencing and computational biology have facilitated the implementation of new combinatorial screening approaches to discover novel enzymes of biotechnological interest. In this study, we describe the successful establishment of a multi-omic approach for the identification of thermostable hydrolase-encoding genes by determination of gene expression levels. We applied this combinatorial approach using an anaerobic enrichment culture from an Azorean hot spring sample grown on green coffee beans as recalcitrant substrate. An in-depth analysis of the microbial community resulted in microorganisms capable of metabolizing the selected substrate, such as the genera Caloramator, Dictyoglomus and Thermoanaerobacter as active and abundant microorganisms. To discover glycoside hydrolases, 90,342 annotated genes were screened for specific reaction types. A total number of 106 genes encoding cellulases (EC 3.2.1.4), beta-glucosidases (EC 3.2.1.21) and endo-1,4-beta-mannosidases (EC 3.2.1.78) were selected. Mapping of RNA-Seq reads to the related metagenome led to expression levels for each gene. Amongst those, 14 genes, encoding glycoside hydrolases, showed highest expression values, and were used for further cloning. Four proteins were biochemically characterized and were identified as thermoactive glycoside hydrolases with a broad substrate range. This work demonstrated that a combinatory omic approach is a suitable strategy identifying unique thermoactive enzymes from environmental samples.

The RNase YbeY Is Vital for Ribosome Maturation, Stress Resistance, and Virulence of the Natural Genetic Engineer Agrobacterium tumefaciens.

Riboregulation involving regulatory RNAs, RNA chaperones, and ribonucleases is fundamental for the rapid adaptation of gene expression to changing environmental conditions. The gene coding for the RNase YbeY belongs to the minimal prokaryotic genome set and has a profound impact on physiology in a wide range of bacteria. Here, we show that the Agrobacterium tumefaciensybeY gene is not essential. Deletion of the gene in the plant pathogen reduced growth, motility, and stress tolerance. Most interestingly, YbeY is crucial for A. tumefaciens-mediated T-DNA transfer and tumor formation. Comparative proteomics by using isobaric tags for relative and absolute quantitation (iTRAQ) revealed dysregulation of 59 proteins, many of which have previously been found to be dependent on the RNA chaperone Hfq. YbeY and Hfq have opposing effects on production of these proteins. Accumulation of a 16S rRNA precursor in the ybeY mutant suggests that A. tumefaciens YbeY is involved in rRNA processing. RNA coimmunoprecipitation-sequencing (RIP-Seq) showed binding of YbeY to the region immediately upstream of the 16S rRNA. Purified YbeY is an oligomer with RNase activity. It does not physically interact with Hfq and thus plays a partially overlapping but distinct role in the riboregulatory network of the plant pathogen.IMPORTANCE Although ybeY gene belongs to the universal bacterial core genome, its biological function is incompletely understood. Here, we show that YbeY is critical for fitness and host-microbe interaction in the plant pathogen Agrobacterium tumefaciens Consistent with the reported endoribonuclease activity of YbeY, A. tumefaciens YbeY acts as a RNase involved in maturation of 16S rRNA. This report adds a worldwide plant pathogen and natural genetic engineer of plants to the growing list of bacteria that require the conserved YbeY protein for host-microbe interaction.

Enrichment of anaerobic heterotrophic thermophiles from four Azorean hot springs revealed different community composition and genera abundances using recalcitrant substrates.

DGGE analysis combined with a metagenomic approach was used to get insights into heterotrophic anoxic enrichment cultures of four hot springs of Vale das Furnas, Portugal, using the recalcitrant substrate spent coffee ground (SCG). Parallel enrichment cultures were performed using the major components of spent coffee ground, namely arabinogalactan, galactomannan, cellulose, and proteins. DGGE revealed that heterotrophic thermophilic bacteria are highly abundant in the hydrothermal springs and significant differences in community composition depending on the substrate were observed. DNA, isolated from enrichment cultures of different locations that were grown on the same substrate were pooled, and the respective metagenomes were analyzed. Results indicated that cultures grown on recalcitrant substrate SCG consists of a totally different thermophilic community, dominated by Dictyoglomus. Enrichments with galactomannan and arabinogalactan were dominated by Thermodesulfovibrio, while cultures with casein and cellulose were dominated by Thermus. This study indicates the high potential of thermophilic bacteria degrading recalcitrant substrate such as SCG and furthermore how the accessibility to complex polymers shapes the bacterial community.

Diagnostic prediction of complicated appendicitis by combined clinical and radiological appendicitis severity index (APSI).

OBJECTIVES: To develop a routinely applicable severity index for the management of acute appendicitis in adults using combined clinical and radiological parameters and retroperitoneal space planes (RSP). METHODS: Two hundred consecutive patients with histologically proven acute appendicitis and available presurgical CT scans were analysed retrospectively. Two radiologists assessed all CT scans for morphologic sings of appendicitis and six RSP. Clinical parameters were age, body temperature, C-reactive protein (CRP), white blood cell count, and duration of symptoms. Radiological parameters were appendix diameter and wall thickness, periappendiceal fat stranding and fluid, intraluminal and extraluminal air, thinning of appendiceal wall, caecal wall thickening, appendicolith and abscess formation. RESULTS: One hundred and three patients (51%) had histologically proven complicated appendicitis. Based on three clinical (age ≥52 years, body temperature ≥37.5°C, duration of symptoms ≥48 h) and four computed tomography (CT) findings (appendix diameter ≥14 mm, presence of periappendiceal fluid, extraluminal air, perityphlitic abscess), the APSI was developed using regression coefficients of multivariate logistic regression analyses with a maximum of 10 points. A score of ≥4 points predicted complicated appendicitis with a positive predictive value of 92% and a negative predictive value of 83%. Substantial to excellent interobserver agreement was found for the four radiological parameters of the APSI [intraclass correlation coefficient (ICC), 0.78-0.83]. The RSP evaluation presented no added value for the diagnosis of complicated appendicitis. CONCLUSIONS: Using APSI, an accurate and simple prediction of complicated appendicitis in adults was possible. The RSP count was not useful for the diagnosis of complicated appendicitis. KEY POINTS: • Appendicitis severity score provides an accurate and simple prediction of complicated appendicitis • Appendicitis severity score ≥4 accurately predicted complicated appendicitis (PPV 92%;NPV 83%) • Evaluation of retroperitoneal space planes was not useful in diagnosing complicated appendicitis.

Physiological data for affective computing in HRI with anthropomorphic service robots: the AFFECT-HRI data set.

In human-human and human-robot interaction, the counterpart influences the human's affective state. Contrary to humans, robots inherently cannot respond empathically, meaning non-beneficial affective reactions cannot be mitigated. Thus, to create a responsible and empathetic human-robot interaction (HRI), involving anthropomorphic service robots, the effect of robot behavior on human affect in HRI must be understood. To contribute to this understanding, we provide the new comprehensive data set AFFECT-HRI, including, for the first time, physiological data labeled with human affect (i.e., emotions and mood) gathered from a conducted HRI study. Within the study, 146 participants interacted with an anthropomorphic service robot in a realistic and complex retail scenario. The participants' questionnaire ratings regarding affect, demographics, and socio-technical ratings are provided in the data set. Five different conditions (i.e., neutral, transparency, liability, moral, and immoral) were considered during the study, eliciting different affective reactions and allowing interdisciplinary investigations (e.g., computer science, law, and psychology). Each condition includes three scenes: a consultation regarding products, a request for sensitive personal information, and a handover.

A SARS-CoV-2 Omicron outbreak among crew members on a cruise ship in Germany in early 2022.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreaks on cruise ships have rarely been investigated. In early 2022, we were informed about a SARS-CoV-2 outbreak on a cruise ship calling Port of Hamburg after 10 infections among crew members were detected. We conducted an outbreak investigation in collaboration between ship owners, the ship physician and Hamburg's Institute for Hygiene and Environment, to identify risk factors and to achieve containment. The aim was to identify risk factors for SARS-CoV-2 infection and SARS-CoV-2 variants in a cohort of 165 crew members. MATERIALS AND METHODS: For this purpose, we collected data on age, sex, nationality, boarding-time, cabin use (single/shared), work place, and vaccination status of the study participants. Cases were defined as individuals who tested SARS-CoV-2 positive at least once in daily screenings during the outbreak period (10 days) by polymerase chain reaction or antigen test. We investigated risk factors for infection by descriptive, univariable and multivariable analysis. We performed whole genome sequencing to identify SARS-CoV-2 variants. RESULTS: We verified 103 SARS-CoV-2 positive cases (attack rate [AR] 62.4%); 39/41 sequenced samples were BA.2.3 Omicron subtype, one BA.1 and one BA.1.1. Among boostered crew members, AR was 38% vs. 65% among those vaccinated once or twice. Among those who stayed < 30 days on board, AR was 31% vs. 72% among those staying on board longer. Among Europeans, the AR was 53% vs. 71% in non- -Europeans. Adjusting for age and sex, cases were more likely to have received no booster vaccine (odds ratio [OR]: 2.66, 95% confidence interval [CI]: 0.99-7.13), to have spent more time on board (≥ 30 days, OR: 6.36, 95% CI: 2.81-14.40 vs. < 30 days) and to have a non-European nationality (OR: 2.14, 95% CI: 1.08-4.27). The outbreak stopped shortly after offboard isolation of cases. CONCLUSIONS: This investigation confirms the importance of a booster vaccine against COVID-19. Longer stays onboard could facilitate social mixing. Further studies could investigate the impact of social, cultural/ behavioural patterns and public health access on the infection risk. Physical distancing together with screening and isolation can contain SARS-CoV-2 outbreaks on cruise ships.

Digitalization in microbiology - Paving the path to sustainable circular bioeconomy.

The transition to a sustainable bio-based circular economy requires cutting edge technologies that ensure economic growth with environmentally responsible action. This transition will only be feasible when the opportunities of digitalization are also exploited. Digital methods and big data handling have already found their way into life sciences and generally offer huge potential in various research areas. While computational analyses of microbial metagenome data have become state of the art, the true potential of bioinformatics remains mostly untapped so far. In this article we present challenges and opportunities of digitalization including multi-omics approaches in discovering and exploiting the microbial diversity of the planet with the aim to identify robust biocatalysts for application in sustainable bioprocesses as part of the transition from a fossil-based to a bio-based circular economy. This will contribute to solving global challenges, including utilization of natural resources, food supply, health, energy and the environment.

Characterization of a Theme C Glycoside Hydrolase Family 9 Endo-Beta-Glucanase from a Biogas Reactor Metagenome.

From a biogas reactor metagenome an ORF (bp_cel9A) encoding a bacterial theme C glycoside hydrolase family 9 (GH9) enzyme was recombinantly produced in E. coli BL21 pQE-80L. BP_Cel9A exhibited ≤ 55% identity to annotated sequences. Subsequently, the enzyme was purified to homogeneity by affinity chromatography. The endo-beta-glucanase BP_Cel9A hydrolyzed the beta-1,3-1,4-linked barley beta-glucan with 24 U/mg at 30 °C and pH 6.0. More than 62% of activity was measured between 10 and 40 °C. Lichenan and xyloglucan were hydrolyzed with 67% and 40% of activity, respectively. The activity towards different substrates varied with different temperatures. However, the enzyme activity on CMC was extremely low (> 1%). In contrast to BP_Cel9A, most GH9 glucanases act preferably on crystalline or soluble cellulose with only side activities towards related substrates. The addition of calcium or magnesium enhanced the activity of BP_Cel9A, especially at higher temperatures. EDTA inhibited the enzyme, whereas EGTA had no effect, suggesting that Mg2+ may adopt the function of Ca2+. BP_Cel9A exhibited a unique substrate spectrum when compared to other GH9 enzymes with great potential for mixed-linked glucan or xyloglucan degrading processes at moderate temperatures.