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Abstract BackgroundThe increasing importance of artificial intelligence (AI) in health care has generated a growing need for health care professionals to possess a comprehensive understanding of AI technologies, requiring an adaptation in medical education. ObjectiveThis paper explores stakeholder perceptions and expectations regarding AI in medicine and examines their potential impact on the medical curriculum. This study project aims to assess the AI experiences and awareness of different stakeholders and identify essential AI-related topics in medical education to define necessary competencies for students. MethodsThe empirical data were collected as part of the TüKITZMed project between August 2022 and March 2023, using a semistructured qualitative interview. These interviews were administered to a diverse group of stakeholders to explore their experiences and perspectives of AI in medicine. A qualitative content analysis of the collected data was conducted using MAXQDA software. ResultsSemistructured interviews were conducted with 38 participants (6 lecturers, 9 clinicians, 10 students, 6 AI experts, and 7 institutional stakeholders). The qualitative content analysis revealed 6 primary categories with a total of 24 subcategories to answer the research questions. The evaluation of the stakeholders’ statements revealed several commonalities and differences regarding their understanding of AI. Crucial identified AI themes based on the main categories were as follows: possible curriculum contents, skills, and competencies; programming skills; curriculum scope; and curriculum structure. ConclusionsThe analysis emphasizes integrating AI into medical curricula to ensure students’ proficiency in clinical applications. Standardized AI comprehension is crucial for defining and teaching relevant content. Considering diverse perspectives in implementation is essential to comprehensively define AI in the medical context, addressing gaps and facilitating effective solutions for future AI use in medical studies. The results provide insights into potential curriculum content and structure, including aspects of AI in medicine.

ABSTRACT Cervimycins A–D are bis-glycosylated polyketide antibiotics produced by Streptomyces tendae HKI 0179 with bactericidal activity against Gram-positive bacteria. In this study, cervimycin C (CmC) treatment caused a spaghetti-like phenotype in Bacillus subtilis 168, with elongated curved cells, which stayed joined after cell division, and exhibited a chromosome segregation defect, resulting in ghost cells without DNA. Electron microscopy of CmC-treated Staphylococcus aureus (3 × MIC) revealed swollen cells, misshapen septa, cell wall thickening, and a rough cell wall surface. Incorporation tests in B. subtilis indicated an effect on DNA biosynthesis at high cervimycin concentrations. Indeed, artificial downregulation of the DNA gyrase subunit B gene (gyrB) increased the activity of cervimycin in agar diffusion tests, and, in high concentrations (starting at 62.5 × MIC), the antibiotic inhibited S. aureus DNA gyrase supercoiling activity in vitro. To obtain a more global view on the mode of action of CmC, transcriptomics and proteomics of cervimycin treated versus untreated S. aureus cells were performed. Interestingly, 3 × MIC of cervimycin did not induce characteristic responses, which would indicate disturbance of the DNA gyrase activity in vivo. Instead, cervimycin induced the expression of the CtsR/HrcA heat shock operon and the expression of autolysins, exhibiting similarity to the ribosome-targeting antibiotic gentamicin. In summary, we identified the DNA gyrase as a target, but at low concentrations, electron microscopy and omics data revealed a more complex mode of action of cervimycin, which comprised induction of the heat shock response, indicating protein stress in the cell.IMPORTANCEAntibiotic resistance of Gram-positive bacteria is an emerging problem in modern medicine, and new antibiotics with novel modes of action are urgently needed. Secondary metabolites from Streptomyces species are an important source of antibiotics, like the cervimycin complex produced by Streptomyces tendae HKI 0179. The phenotypic response of Bacillus subtilis and Staphylococcus aureus toward cervimycin C indicated a chromosome segregation and septum formation defect. This effect was at first attributed to an interaction between cervimycin C and the DNA gyrase. However, omics data of cervimycin treated versus untreated S. aureus cells indicated a different mode of action, because the stress response did not include the SOS response but resembled the response toward antibiotics that induce mistranslation or premature chain termination and cause protein stress. In summary, these results point toward a possibly novel mechanism that generates protein stress in the cells and subsequently leads to defects in cell and chromosome segregation.

ABSTRACTArtificial intelligence (AI) in medicine and digital assistance systems such as chatbots will play an increasingly important role in future doctor – patient communication. To benefit from the potential of this technical innovation and ensure optimal patient care, future physicians should be equipped with the appropriate skills. Accordingly, a suitable place for the management and adaptation of digital assistance systems must be found in the medical education curriculum. To determine the existing levels of knowledge of medical students about AI chatbots in particular in the healthcare setting, this study surveyed medical students of the University of Luebeck and the University Hospital of Tuebingen. Using standardized quantitative questionnaires and qualitative analysis of group discussions, the attitudes of medical students toward AI and chatbots in medicine were investigated. From this, relevant requirements for the future integration of AI into the medical curriculum could be identified. The aim was to establish a basic understanding of the opportunities, limitations, and risks, as well as potential areas of application of the technology. The participants (N = 12) were able to develop an understanding of how AI and chatbots will affect their future daily work. Although basic attitudes toward the use of AI were positive, the students also expressed concerns. There were high levels of agreement regarding the use of AI in administrative settings (83.3%) and research with health-related data (91.7%). However, participants expressed concerns that data protection may be insufficiently guaranteed (33.3%) and that they might be increasingly monitored at work in the future (58.3%). The evaluations indicated that future physicians want to engage more intensively with AI in medicine. In view of future developments, AI and data competencies should be taught in a structured way during the medical curriculum and integrated into curricular teaching.

ABSTRACT Biosynthetic gene clusters (BGCs) encoding the production of bacteriocins are widespread among bacterial isolates and are important genetic determinants of competitive fitness within a given habitat. Staphylococci produce a tremendous diversity of compounds, and the corresponding BGCs are frequently associated with mobile genetic elements, suggesting gain and loss of biosynthetic capacity. Pharmaceutical biology has shown that compound production in heterologous hosts is often challenging, and many BGC recipients initially produce small amounts of compound or show reduced growth rates. To assess whether transfer of BGCs between closely related Staphylococcus aureus strains can be instantly effective or requires elaborate metabolic adaptation, we investigated the intraspecies transfer of a BGC encoding the ribosomally synthesized and posttranslationally modified peptide (RiPP) micrococcin P1 (MP1). We found that acquisition of the BGC by S. aureus RN4220 enabled immediate MP1 production but also imposed a metabolic burden, which was relieved after prolonged cultivation by adaptive mutation. We used a multiomics approach to study this phenomenon and found adaptive evolution to select for strains with increased activity of the tricarboxylic acid cycle (TCA), which enhanced metabolic fitness and levels of compound production. Metabolome analysis revealed increases of central metabolites, including citrate and α-ketoglutarate in the adapted strain, suggesting metabolic adaptation to overcome the BGC-associated growth defects. Our results indicate that BGC acquisition requires genetic and metabolic predispositions, allowing the integration of bacteriocin production into the cellular metabolism. Inappropriate metabolic characteristics of recipients can entail physiological burdens, negatively impacting the competitive fitness of recipients within natural bacterial communities. IMPORTANCE Human microbiomes are critically associated with human health and disease. Importantly, pathogenic bacteria can hide in human-associated communities and can cause disease when the composition of the community becomes unbalanced. Bacteriocin-producing commensals are able to displace pathogens from microbial communities, suggesting that their targeted introduction into human microbiomes might prevent pathogen colonization and infection. However, to develop probiotic approaches, strains are needed that produce high levels of bioactive compounds and retain cellular fitness within mixed bacterial communities. Our work offers insights into the metabolic burdens associated with the production of the bacteriocin micrococcin P1 and highlights evolutionary strategies that increase cellular fitness in the context of production. Metabolic adaptations are most likely broadly relevant for bacteriocin producers and need to be considered for the future development of effective microbiome editing strategies.

ABSTRACT Resistance to antibiotics is an increasing problem and necessitates novel antibacterial therapies. The polyketide antibiotics cervimycin A to D are natural products of Streptomyces tendae HKI 0179 with promising activity against multidrug-resistant staphylococci and vancomycin-resistant enterococci. To initiate mode of action studies, we selected cervimycin C- and D-resistant (CmR) Staphylococcus aureus strains. Genome sequencing of CmR mutants revealed amino acid exchanges in the essential histidine kinase WalK, the Clp protease proteolytic subunit ClpP or the Clp ATPase ClpC, and the heat shock protein DnaK. Interestingly, all characterized CmR mutants harbored a combination of mutations in walK and clpP or clpC. In vitro and in vivo analyses showed that the mutations in the Clp proteins abolished ClpP or ClpC activity, and the deletion of clpP rendered S. aureus but not all Bacillus subtilis strains cervimycin-resistant. The essential gene walK was the second mutational hotspot in the CmR S. aureus strains, which decreased WalK activity in vitro and generated a vancomycin-intermediate resistant phenotype, with a thickened cell wall, a lower growth rate, and reduced cell lysis. Transcriptomic and proteomic analyses revealed massive alterations in the CmR strains compared to the parent strain S. aureus SG511, with major shifts in the heat shock regulon, the metal ion homeostasis, and the carbohydrate metabolism. Taken together, mutations in the heat shock genes clpP, clpC, and dnaK, and the walK kinase gene in CmR mutants induced a vancomycin-intermediate resistant phenotype in S. aureus, suggesting cell wall metabolism or the Clp protease system as primary target of cervimycin. IMPORTANCE Staphylococcus aureus is a frequent cause of infections in both the community and hospital setting. Resistance development of S. aureus to various antibiotics is a severe problem for the treatment of this pathogen worldwide. New powerful antimicrobial agents against Gram-positives are needed, since antibiotics like vancomycin fail to cure vancomycin-intermediate resistant S. aureus (VISA) and vancomycin-resistant enterococci (VRE) infections. One candidate substance with promising activity against these organisms is cervimycin, which is an antibiotic complex with a yet unknown mode of action. In our study, we provide first insights into the mode of action of cervimycins. By characterizing cervimycin-resistant S. aureus strains, we revealed the Clp system and the essential kinase WalK as mutational hotspots for cervimycin resistance in S. aureus. It further emerged that cervimycin-resistant S. aureus strains show a VISA phenotype, indicating a role of cervimycin in perturbing the bacterial cell envelope.

Abstract Background Recent advances in sequencing have facilitated large-scale analyses of the metagenomic composition of different samples, including the environmental microbiome of air, water, and soil, as well as the microbiome of living humans and other animals. Analyses of the microbiome of ancient human samples may provide insights into human health and disease, as well as pathogen evolution, but the field is still in its very early stages and considered highly challenging. Results The metagenomic and pathogen content of Egyptian mummified individuals from different time periods was investigated via genetic analysis of the microbial composition of various tissues. The analysis of the dental calculus’ microbiome identified Red Complex bacteria, which are correlated with periodontal diseases. From bone and soft tissue, genomes of two ancient pathogens, a 2200-year-old Mycobacterium leprae strain and a 2000-year-old human hepatitis B virus, were successfully reconstructed. Conclusions The results show the reliability of metagenomic studies on Egyptian mummified individuals and the potential to use them as a source for the extraction of ancient pathogen DNA.

Staphylococcus aureus asymptomatically colonizes the nasal cavity of mammals, but it is also a leading cause of life-threatening infections. Most human nasal isolates carry Sa3 phages, which integrate into the bacterial hlb gene encoding a sphingomyelinase. The virulence factor-encoding genes carried by the Sa3-phages are highly human-specific, and most animal strains are Sa3 negative. Thus, both insertion and excision of the prophage could potentially confer a fitness advantage to S. aureus. Here, we analyzed the phage life cycle of two Sa3 phages, Φ13 and ΦN315, in different phage-cured S. aureus strains. Based on phage transfer experiments, strains could be classified into low (8325-4, SH1000, and USA300c) and high (MW2c and Newman-c) transfer strains. High-transfer strains promoted the replication of phages, whereas phage adsorption, integration, excision, or recA transcription was not significantly different between strains. RNASeq analyses of replication-deficient lysogens revealed no strain-specific differences in the CI/Mor regulatory switch. However, lytic genes were significantly upregulated in the high transfer strain MW2c Φ13 compared to strain 8325-4 Φ13. By transcriptional start site prediction, new promoter regions within the lytic modules were identified, which are likely targeted by specific host factors. Such host-phage interaction probably accounts for the strain-specific differences in phage replication and transfer frequency. Thus, the genetic makeup of the host strains may determine the rate of phage mobilization, a feature that might impact the speed at which certain strains can achieve host adaptation.

Abstract Background Several serious vegetable-associated outbreaks of enterohemorrhagic Escherichia coli (EHEC) infections have occurred during the last decades. In this context, vegetables have been suggested to function as secondary reservoirs for EHEC strains. Increased knowledge about the interaction of EHEC with plants including gene expression patterns in response to plant-derived compounds is required. In the current study, EHEC O157:H7 strain Sakai, EHEC O157:H− strain 3072/96, and the EHEC/enteroaggregative E. coli (EAEC) hybrid O104:H4 strain C227–11φcu were grown in lamb’s lettuce medium and in M9 minimal medium to study the differential transcriptional response of these strains to plant-derived compounds with RNA-Seq technology. Results Many genes involved in carbohydrate degradation and peptide utilization were similarly upregulated in all three strains, suggesting that the lamb’s lettuce medium provides sufficient nutrients for proliferation. In particular, the genes galET and rbsAC involved in galactose metabolism and D-ribose catabolism, respectively, were uniformly upregulated in the investigated strains. The most prominent differences in shared genome transcript levels were observed for genes involved in the expression of flagella. Transcripts of all three classes of the flagellar hierarchy were highly abundant in strain C227–11φcu. Strain Sakai expressed only genes encoding the basal flagellar structure. In addition, both strains showed increased motility in presence of lamb’s lettuce extract. Moreover, strain 3072/96 showed increased transcription activity for genes encoding the type III secretion system (T3SS) including effectors, and was identified as a powerful biofilm-producer in M9 minimal medium. Conclusion The current study provides clear evidence that EHEC and EHEC/EAEC strains are able to adjust their gene expression patterns towards metabolization of plant-derived compounds, demonstrating that they may proliferate well in a plant-associated environment. Moreover, we propose that flagella and other surface structures play a fundamental role in the interaction of EHEC and EHEC/EAEC with plants.

The mechanisms behind CO2 dependency in non-autotrophic bacterial isolates are unclear. Here the authors show that the Staphylococcus aureus mpsAB operon is crucial for growth at atmospheric CO2 levels, and provide evidence indicating that MpsAB represents a bicarbonate concentrating system.

Abstract Background The ability to generate recombinant drug target proteins is important for drug discovery research as it facilitates the investigation of drug-target-interactions in vitro. To accomplish this, the target’s exact protein sequence is required. Public databases, such as Ensembl, UniProt and RefSeq, are extensive protein and nucleotide sequence repositories. However, many sequences for non-human organisms are predicted by computational pipelines and may thus be incomplete or incorrect. This could lead to misinterpreted experimental outcomes due to gaps or errors in orthologous drug target sequences. Transcriptome analysis by RNA-Seq has been established as a standard method for gene expression analysis. Apart from this common application, paired-end RNA-Seq data can also be used to obtain full coverage cDNA sequences via de novo transcriptome assembly. Methods To assess whether de novo transcriptome assemblies can be used to determine a protein’s sequence by searching the assembly for a known orthologous sequence, we generated 3 × 6 = 18 tissue specific assemblies (three organs: brain, kidney and liver; six species: human, mouse, rat, dog, pig and cynomolgus monkey). These assemblies and the manually curated human protein sequences from UniProtKB/Swiss-Prot were used in a reciprocal BLAST search to identify best matching hits. We automated and generalised our approach and present the a&o-tool, a workflow which exploits de novo assemblies of paired-end RNA-Seq data and orthology information for target sequence validation and refinement across related species. Furthermore, the a&o-tool extracts best hits’ sequences from a reciprocal BLAST search, translates them into protein sequences, computes a multiple sequence alignment and quantifies the refinement. Results For the three human assemblies we observed a hit rate greater than 60% with 100% sequence coverage and identity. For assemblies from the other species we observed similar hit rates and coverage with highest identities for cynomolgus monkey. Conclusions In summary, we show how to refine protein sequences using RNA-Seq data and sequence information from closely related species. With the a&o-tool we provide a fully automated pipeline to perform refinement including cDNA translation and multiple sequence alignment for visual inspection. The major prerequisite for applying the a&o-tool is high quality sequencing data.

Throughout the United States, cancer remains the second leading cause of death. Traditional treatments induce significant medical toxic effects and unpleasant adverse reactions, making them inappropriate for long-term use. Consequently, anticancer-drug resistance and relapse are frequent in certain situations. Thus, there is an urgent necessity to find effective antitumor medications that are specific and have few adverse consequences. Curcumin is a polyphenol derivative found in the turmeric plant (Curcuma longa L.), and provides chemopreventive, antitumor, chemo-, and radio-sensitizing properties. In this paper, we summarize the new nano-based formulations of polyphenolic curcumin because of the growing interest in its application against cancers and tumors. According to recent studies, the use of nanoparticles can overcome the hydrophobic nature of curcumin, as well as improving its stability and cellular bioavailability in vitro and in vivo. Several strategies for nanocurcumin production have been developed, each with its own set of advantages and unique features. Because the majority of the curcumin-based nanoformulation evidence is still in the conceptual stage, there are still numerous issues impeding the provision of nanocurcumin as a possible therapeutic option. To support the science, further work is necessary to develop curcumin as a viable anti-cancer adjuvant. In this review, we cover the various curcumin nanoformulations and nanocurcumin implications for therapeutic uses for cancer, as well as the current state of clinical studies and patents. We further address the knowledge gaps and future research orientations required to develop curcumin as a feasible treatment candidate.

Trace amines (TA) are endogenously produced in mammals, have a low concentration in the central nervous system (CNS), but trigger a variety of neurological effects and intervene in host cell communication. It emerged that neurotransmitters and TA are produced also by the microbiota. As it has been shown that TA contribute to wound healing, we examined the skin microbiome of probands using shotgun metagenomics. The phyla Actinobacteria, Proteobacteria, Firmicutes, and Bacteroidetes were predominant. Since SadA is a highly promiscuous TA-producing decarboxylase in Firmicutes, the skin microbiome was specifically examined for the presence of sadA-homologous genes. By mapping the reads of certain genes, we found that, although there were less reads mapping to sadA than to ubiquitous housekeeping genes (arcC and mutS), normalized reads counts were still >1000 times higher than those of rare control genes (icaA, icaB, and epiA). At protein sequence level SadA homologs were found in at least 7 phyla: Firmicutes, Actinobacteria, Proteobacteria, Bacteroidetes, Acidobacteria, Chloroflexi, and Cyanobacteria, and in 23 genera of the phylum Firmicutes. A high proportion of the genera that have a SadA homolog belong to the classical skin and intestinal microbiota. The distribution of sadA in so many different phyla illustrates the importance of horizontal gene transfer (HGT). We show that the sadA gene is widely distributed in the human skin microbiome. When comparing the sadA read counts in the probands, there was no correlation between age and gender, but an enormous difference in the sadA read counts in the microbiome of the individuals. Since sadA is involved in TA synthesis, it is likely that the TA content of the skin is correlated with the amount of TA producing bacteria in the microbiome. In this way, the microbiome-generated TA could influence signal transmission in the epithelial and nervous system.

Summary: Many biosynthetic gene clusters (BGCs) require heterologous expression to realize their genetic potential, including silent and metagenomic BGCs. Although the engineered Streptomyces coelicolor M1152 is a widely used host for heterologous expression of BGCs, a systemic understanding of how its genetic modifications affect the metabolism is lacking and limiting further development. We performed a comparative analysis of M1152 and its ancestor M145, connecting information from proteomics, transcriptomics, and cultivation data into a comprehensive picture of the metabolic differences between these strains. Instrumental to this comparison was the application of an improved consensus genome-scale metabolic model (GEM) of S. coelicolor. Although many metabolic patterns are retained in M1152, we find that this strain suffers from oxidative stress, possibly caused by increased oxidative metabolism. Furthermore, precursor availability is likely not limiting polyketide production, implying that other strategies could be beneficial for further development of S. coelicolor for heterologous production of novel compounds.

The skin colonizing coagulase-negative Staphylococcus epidermidis causes nosocomial infections and is an important opportunistic and highly adaptable pathogen. To gain more insight into this species, we sequenced the genome of the biofilm positive, methicillin susceptible S. epidermidis O47 strain (hereafter O47). This strain belongs to the most frequently isolated sequence type 2. In comparison to the RP62A strain, O47 can be transformed, which makes it a preferred strain for molecular studies. S. epidermidis O47’s genome has a single chromosome of about 2.5 million base pairs and no plasmid. Its oriC sequence has the same directionality as S. epidermidis RP62A, S. carnosus, S. haemolyticus, S. saprophyticus and is inverted in comparison to Staphylococcus aureus and S. epidermidis ATCC 12228. A phylogenetic analysis based on all S. epidermidis genomes currently available at GenBank revealed that O47 is closest related to DAR1907. The genome of O47 contains genes for the typical global regulatory systems known in staphylococci. In addition, it contains most of the genes encoding for the typical virulence factors for S. epidermidis but not for S. aureus with the exception of a putative hemolysin III. O47 has the typical S. epidermidis genetic islands and several mobile genetic elements, which include staphylococcal cassette chromosome (SCC) of about 54 kb length and two prophages φO47A and φO47B. However, its genome has no transposons and the smallest number of insertion sequence (IS) elements compared to the other known S. epidermidis genomes. By sequencing and analyzing the genome of O47, we provide the basis for its utilization in genetic and molecular studies of biofilm formation.

Abstract Objective Treponema pallidum subsp. pallidum (TPA) is the causative agent of syphilis. Genetic analyses of TPA reference strains and human clinical isolates have revealed two genetically distinct groups of syphilis-causing treponemes, called Nichols-like and SS14-like groups. So far, no genetic intermediates, i.e. strains containing a mixed pattern of Nichols-like and SS14-like genomic sequences, have been identified. Recently, Sun et al. (Oncotarget 2016. https://doi.org/10.18632/oncotarget.10154 ) described a new “phylogenetic group” (called Lineage 2) among Chinese TPA strains. This lineage exhibited a “mosaic genomic structure” of Nichols-like and SS14-like lineages. Results We reanalyzed the primary sequencing data (Project Number PRJNA305961) from the Sun et al. publication with respect to the molecular basis of Lineage 2. While Sun et al. based the analysis on several selected genomic single nucleotide variants (SNVs) and a subset of highly variable but phylogenetically poorly informative genes, which may confound the phylogenetic analysis, our reanalysis primarily focused on a complete set of whole genomic SNVs. Based on our reanalysis, only two separate TPA clusters were identified: one consisted of Nichols-like TPA strains, the other was formed by the SS14-like TPA strains, including all Chinese strains.

Archaeological and historical records had shown ancient Egypt before and after Ptolemaic and Roman periods to be a hub of human migration and exchange. Here, Schuenemann and colleagues analyse ancient mitochondrial and nuclear DNA to investigate the genetic history of Egypt.

Studying ancient DNA allows us to retrace the evolutionary history of human pathogens, such as Mycobacterium leprae, the main causative agent of leprosy. Leprosy is one of the oldest recorded and most stigmatizing diseases in human history. The disease was prevalent in Europe until the 16th century and is still endemic in many countries with over 200,000 new cases reported annually. Previous worldwide studies on modern and European medieval M. leprae genomes revealed that they cluster into several distinct branches of which two were present in medieval Northwestern Europe. In this study, we analyzed 10 new medieval M. leprae genomes including the so far oldest M. leprae genome from one of the earliest known cases of leprosy in the United Kingdom-a skeleton from the Great Chesterford cemetery with a calibrated age of 415-545 C.E. This dataset provides a genetic time transect of M. leprae diversity in Europe over the past 1500 years. We find M. leprae strains from four distinct branches to be present in the Early Medieval Period, and strains from three different branches were detected within a single cemetery from the High Medieval Period. Altogether these findings suggest a higher genetic diversity of M. leprae strains in medieval Europe at various time points than previously assumed. The resulting more complex picture of the past phylogeography of leprosy in Europe impacts current phylogeographical models of M. leprae dissemination. It suggests alternative models for the past spread of leprosy such as a wide spread prevalence of strains from different branches in Eurasia already in Antiquity or maybe even an origin in Western Eurasia. Furthermore, these results highlight how studying ancient M. leprae strains improves understanding the history of leprosy worldwide.

The reconstruction of genomes using mapping-based approaches with short reads experiences difficulties when resolving repetitive regions. These repetitive regions in genomes result in low mapping qualities of the respective reads, which in turn lead to many unresolved bases. Currently, the reconstruction of these regions is often based on modified references in which the repetitive regions are masked. However, for many references, such masked genomes are not available or are based on repetitive regions of other genomes. Our idea is to identify repetitive regions in the reference genome de novo. These regions can then be used to reconstruct them separately using short read sequencing data. Afterward, the reconstructed repetitive sequence can be inserted into the reconstructed genome. We present the program detection, characterization, and reconstruction of repetitive regions, which performs these steps automatically. Our results show an increased base pair resolution of the repetitive regions in the reconstruction of Treponema pallidum samples, resulting in fewer unresolved bases.

Altitudinal gradients provide valuable information about the effects of environmental variables on changes in species richness and composition as well as the distribution of below ground fungal communities. Since most knowledge in this respect has been gathered on aboveground communities, we focused our study towards the characterization of belowground fungal communities associated with two different ages of Norway spruce (Picea abies) trees along an altitudinal gradient. By sequencing the internal transcribed spacer (ITS) region on the Illumina platform, we investigated the fungal communities in a floristically and geologically relatively well explored forest on the slope of Mt. Iseler of the Bavarian Alps. From fine roots and rhizosphere of a total of 90 of Norway spruce trees from 18 plots we detected 1285 taxa, with a range of 167 to 506 (average 377) taxa per plot. Fungal taxa are distributed over 96 different orders belonging to the phyla Ascomycota, Basidiomycota, Chrytridiomycota, Glomeromycota, and Mucoromycota. Overall the Agaricales (438 taxa) and Tremellales (81 taxa) belonging to the Basidiomycota and the Hypocreales (65 spp.) and Helotiales (61 taxa) belonging to the Ascomycota represented the taxon richest orders. The evaluation of our multivariate generalized mixed models indicate that the altitude has a significant influence on the composition of the fungal communities (p < 0.003) and that tree age determines community diversity (p < 0.05). A total of 47 ecological guilds were detected, of which the ectomycorrhizal and saprophytic guilds were the most taxon-rich. Our ITS amplicon Illumina sequencing approach allowed us to characterize a high fungal community diversity that would not be possible to capture with fruiting body surveys alone. We conclude that it is an invaluable tool for diverse monitoring tasks and inventorying biodiversity, especially in the detection of microorganisms developing very ephemeral and/or inconspicuous fruiting bodies or lacking them all together. Results suggest that the altitude mainly influences the community composition, whereas fungal diversity becomes higher in mature/older trees. Finally, we demonstrate that novel techniques from bacterial microbiome analyses are also useful for studying fungal diversity and community structure in a DNA metabarcoding approach, but that incomplete reference sequence databases so far limit effective identification.

Syphilis is an important public health problem and an increasing incidence has been noted in recent years. Characterization of strain diversity through molecular data plays a critical role in the epidemiological understanding of this re-emergence. We here propose a new high-resolution multilocus sequence typing (MLST) scheme for Treponema pallidum subsp. pallidum (TPA). We analyzed 30 complete and draft TPA genomes obtained directly from clinical samples or from rabbit propagated strains to identify suitable typing loci and tested the new scheme on 120 clinical samples collected in Switzerland and France. Our analyses yielded three loci with high discriminatory power: TP0136, TP0548, and TP0705. Together with analysis of the 23S rRNA gene mutations for macrolide resistance, we propose these loci as MLST for TPA. Among clinical samples, 23 allelic profiles as well as a high percentage (80% samples) of macrolide resistance were revealed. The new MLST has higher discriminatory power compared to previous typing schemes, enabling distinction of TPA from other treponemal bacteria, distinction between the two main TPA clades (Nichols and SS14), and differentiation of strains within these clades.

Most reconstruction methods for genomes of ancient origin that are used today require a closely related reference. In order to identify genomic rearrangements or the deletion of whole genes, de novo assembly has to be used. However, because of inherent problems with ancient DNA, its de novo assembly is highly complicated. In order to tackle the diversity in the length of the input reads, we propose a two-layer approach, where multiple assemblies are generated in the first layer, which are then combined in the second layer. We used this two-layer assembly to generate assemblies for two different ancient samples and compared the results to current de novo assembly approaches. We are able to improve the assembly with respect to the length of the contigs and can resolve more repetitive regions.

Campylobacter jejuni is currently the leading cause of bacterial gastroenteritis in humans. Comparison of multiple Campylobacter strains revealed a high genetic and phenotypic diversity. However, little is known about differences in transcriptome organization, gene expression, and small RNA (sRNA) repertoires. Here we present the first comparative primary transcriptome analysis based on the differential RNA-seq (dRNA-seq) of four C. jejuni isolates. Our approach includes a novel, generic method for the automated annotation of transcriptional start sites (TSS), which allowed us to provide genome-wide promoter maps in the analyzed strains. These global TSS maps are refined through the integration of a SuperGenome approach that allows for a comparative TSS annotation by mapping RNA-seq data of multiple strains into a common coordinate system derived from a whole-genome alignment. Considering the steadily increasing amount of RNA-seq studies, our automated TSS annotation will not only facilitate transcriptome annotation for a wider range of pro- and eukaryotes but can also be adapted for the analysis among different growth or stress conditions. Our comparative dRNA-seq analysis revealed conservation of most TSS, but also single-nucleotide-polymorphisms (SNP) in promoter regions, which lead to strain-specific transcriptional output. Furthermore, we identified strain-specific sRNA repertoires that could contribute to differential gene regulation among strains. In addition, we identified a novel minimal CRISPR-system in Campylobacter of the type-II CRISPR subtype, which relies on the host factor RNase III and a trans-encoded sRNA for maturation of crRNAs. This minimal system of Campylobacter, which seems active in only some strains, employs a unique maturation pathway, since the crRNAs are transcribed from individual promoters in the upstream repeats and thereby minimize the requirements for the maturation machinery. Overall, our study provides new insights into strain-specific transcriptome organization and sRNAs, and reveals genes that could modulate phenotypic variation among strains despite high conservation at the DNA level.

The successful reconstruction of an ancient bacterial genome from archaeological material presents an important methodological advancement for infectious disease research. The reliability of evolutionary histories inferred by the incorporation of ancient data, however, are highly contingent upon the level of genetic diversity represented in modern genomic sequences that are publicly accessible, and the paucity of available complete genomes restricts the level of phylogenetic resolution that can be obtained. Here we add to our original analysis of the Yersinia pestis strain implicated in the Black Death by consolidating our dataset for 18 modern genomes with single nucleotide polymorphism (SNP) data for an additional 289 strains at over 600 positions. The inclusion of this additional data reveals a cluster of Y. pestis strains that diverge at a time significantly in advance of the Black Death, with divergence dates roughly coincident with the Plague of Justinian (6(th) to 8(th) century AD). In addition, the analysis reveals further clues regarding potential radiation events that occurred immediately preceding the Black Death, and the legacy it may have left in modern Y. pestis populations. This work reiterates the need for more publicly available complete genomes, both modern and ancient, to achieve an accurate understanding of the history of this bacterium.

Recently emerged deep sequencing technologies offer new high-throughput methods to quantify gene expression, epigenetic modifications and DNA-protein binding. From a computational point of view, the data is very different from that produced by the already established microarray technology, providing a new perspective on the samples under study and complementing microarray gene expression data. Software offering the integrated analysis of data from different technologies is of growing importance as new data emerge in systems biology studies. Mayday is an extensible platform for visual data exploration and interactive analysis and provides many methods for dissecting complex transcriptome datasets. We present Mayday SeaSight, an extension that allows to integrate data from different platforms such as deep sequencing and microarrays. It offers methods for computing expression values from mapped reads and raw microarray data, background correction and normalization and linking microarray probes to genomic coordinates. It is now possible to use Mayday's wealth of methods to analyze sequencing data and to combine data from different technologies in one analysis.