An antibiotic from an uncultured bacterium binds to an immutable target.

Antimicrobial resistance is a leading mortality factor worldwide. Here, we report the discovery of clovibactin, an antibiotic isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant Gram-positive bacterial pathogens without detectable resistance. Using biochemical assays, solid-state nuclear magnetic resonance, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C55PP, lipid II, and lipid IIIWTA). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. This potent antibiotic holds the promise of enabling the design of improved therapeutics that kill bacterial pathogens without resistance development.

Secondary Metabolite Transcriptomic Pipeline (SeMa-Trap), an expression-based exploration tool for increased secondary metabolite production in bacteria.

For decades, natural products have been used as a primary resource in drug discovery pipelines to find new antibiotics, which are mainly produced as secondary metabolites by bacteria. The biosynthesis of these compounds is encoded in co-localized genes termed biosynthetic gene clusters (BGCs). However, BGCs are often not expressed under laboratory conditions. Several genetic manipulation strategies have been developed in order to activate or overexpress silent BGCs. Significant increases in production levels of secondary metabolites were indeed achieved by modifying the expression of genes encoding regulators and transporters, as well as genes involved in resistance or precursor biosynthesis. However, the abundance of genes encoding such functions within bacterial genomes requires prioritization of the most promising ones for genetic manipulation strategies. Here, we introduce the 'Secondary Metabolite Transcriptomic Pipeline' (SeMa-Trap), a user-friendly web-server, available at https://sema-trap.ziemertlab.com. SeMa-Trap facilitates RNA-Seq based transcriptome analyses, finds co-expression patterns between certain genes and BGCs of interest, and helps optimize the design of comparative transcriptomic analyses. Finally, SeMa-Trap provides interactive result pages for each BGC, allowing the easy exploration and comparison of expression patterns. In summary, SeMa-Trap allows a straightforward prioritization of genes that could be targeted via genetic engineering approaches to (over)express BGCs of interest.

Evolutionary Processes in the Emergence and Recent Spread of the Syphilis Agent, Treponema pallidum.

The incidence of syphilis has risen worldwide in the last decade in spite of being an easily treated infection. The causative agent of this sexually transmitted disease is the bacterium Treponema pallidum subspecies pallidum (TPA), very closely related to subsp. pertenue (TPE) and endemicum (TEN), responsible for the human treponematoses yaws and bejel, respectively. Although much focus has been placed on the question of the spatial and temporary origins of TPA, the processes driving the evolution and epidemiological spread of TPA since its divergence from TPE and TEN are not well understood. Here, we investigate the effects of recombination and selection as forces of genetic diversity and differentiation acting during the evolution of T. pallidum subspecies. Using a custom-tailored procedure, named phylogenetic incongruence method, with 75 complete genome sequences, we found strong evidence for recombination among the T. pallidum subspecies, involving 12 genes and 21 events. In most cases, only one recombination event per gene was detected and all but one event corresponded to intersubspecies transfers, from TPE/TEN to TPA. We found a clear signal of natural selection acting on the recombinant genes, which is more intense in their recombinant regions. The phylogenetic location of the recombination events detected and the functional role of the genes with signals of positive selection suggest that these evolutionary processes had a key role in the evolution and recent expansion of the syphilis bacteria and significant implications for the selection of vaccine candidates and the design of a broadly protective syphilis vaccine.

DamageProfiler: fast damage pattern calculation for ancient DNA.

MOTIVATION: In ancient DNA research, the authentication of ancient samples based on specific features remains a crucial step in data analysis. Because of this central importance, researchers lacking deeper programming knowledge should be able to run a basic damage authentication analysis. Such software should be user-friendly and easy to integrate into an analysis pipeline. RESULTS: DamageProfiler is a Java-based, stand-alone software to determine damage patterns in ancient DNA. The results are provided in various file formats and plots for further processing. DamageProfiler has an intuitive graphical as well as command line interface that allows the tool to be easily embedded into an analysis pipeline. AVAILABILITY AND IMPLEMENTATION: All of the source code is freely available on GitHub (https://github.com/Integrative-Transcriptomics/DamageProfiler). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Curcumin-Based Nanoformulations: A Promising Adjuvant towards Cancer Treatment.

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.

2000-year-old pathogen genomes reconstructed from metagenomic analysis of Egyptian mummified individuals.

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.

The Ancient Operational Code is Embedded in the Amino Acid Substitution Matrix and aaRS Phylogenies.

The underlying structure of the canonical amino acid substitution matrix (aaSM) is examined by considering stepwise improvements in the differential recognition of amino acids according to their chemical properties during the branching history of the two aminoacyl-tRNA synthetase (aaRS) superfamilies. The evolutionary expansion of the genetic code is described by a simple parameterization of the aaSM, in which (i) the number of distinguishable amino acid types, (ii) the matrix dimension and (iii) the number of parameters, each increases by one for each bifurcation in an aaRS phylogeny. Parameterized matrices corresponding to trees in which the size of an amino acid sidechain is the only discernible property behind its categorization as a substrate, exclusively for a Class I or II aaRS, provide a significantly better fit to empirically determined aaSM than trees with random bifurcation patterns. A second split between polar and nonpolar amino acids in each Class effects a vastly greater further improvement. The earliest Class-separated epochs in the phylogenies of the aaRS reflect these enzymes' capability to distinguish tRNAs through the recognition of acceptor stem identity elements via the minor (Class I) and major (Class II) helical grooves, which is how the ancient operational code functioned. The advent of tRNA recognition using the anticodon loop supports the evolution of the optimal map of amino acid chemistry found in the later genetic code, an essentially digital categorization, in which polarity is the major functional property, compensating for the unrefined, haphazard differentiation of amino acids achieved by the operational code.

Efficient merging of genome profile alignments.

MOTIVATION: Whole-genome alignment (WGA) methods show insufficient scalability toward the generation of large-scale WGAs. Profile alignment-based approaches revolutionized the fields of multiple sequence alignment construction methods by significantly reducing computational complexity and runtime. However, WGAs need to consider genomic rearrangements between genomes, which make the profile-based extension of several whole-genomes challenging. Currently, none of the available methods offer the possibility to align or extend WGA profiles. RESULTS: Here, we present genome profile alignment, an approach that aligns the profiles of WGAs and that is capable of producing large-scale WGAs many times faster than conventional methods. Our concept relies on already available whole-genome aligners, which are used to compute several smaller sets of aligned genomes that are combined to a full WGA with a divide and conquer approach. To align or extend WGA profiles, we make use of the SuperGenome data structure, which features a bidirectional mapping between individual sequence and alignment coordinates. This data structure is used to efficiently transfer different coordinate systems into a common one based on the principles of profiles alignments. The approach allows the computation of a WGA where alignments are subsequently merged along a guide tree. The current implementation uses progressiveMauve and offers the possibility for parallel computation of independent genome alignments. Our results based on various bacterial datasets up to several hundred genomes show that we can reduce the runtime from months to hours with a quality that is negligibly worse than the WGA computed with the conventional progressiveMauve tool. AVAILABILITY AND IMPLEMENTATION: GPA is freely available at https://lambda.informatik.uni-tuebingen.de/gitlab/ahennig/GPA. GPA is implemented in Java, uses progressiveMauve and offers a parallel computation of WGAs. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Ancient genomes reveal a high diversity of Mycobacterium leprae in medieval Europe.

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.

Pre-Columbian mycobacterial genomes reveal seals as a source of New World human tuberculosis.

Modern strains of Mycobacterium tuberculosis from the Americas are closely related to those from Europe, supporting the assumption that human tuberculosis was introduced post-contact. This notion, however, is incompatible with archaeological evidence of pre-contact tuberculosis in the New World. Comparative genomics of modern isolates suggests that M. tuberculosis attained its worldwide distribution following human dispersals out of Africa during the Pleistocene epoch, although this has yet to be confirmed with ancient calibration points. Here we present three 1,000-year-old mycobacterial genomes from Peruvian human skeletons, revealing that a member of the M. tuberculosis complex caused human disease before contact. The ancient strains are distinct from known human-adapted forms and are most closely related to those adapted to seals and sea lions. Two independent dating approaches suggest a most recent common ancestor for the M. tuberculosis complex less than 6,000 years ago, which supports a Holocene dispersal of the disease. Our results implicate sea mammals as having played a role in transmitting the disease to humans across the ocean.

Semantic segmentation of cerebrospinal fluid and brain volume with a convolutional neural network in pediatric hydrocephalus-transfer learning from existing algorithms.

BACKGROUND: For the segmentation of medical imaging data, a multitude of precise but very specific algorithms exist. In previous studies, we investigated the possibility of segmenting MRI data to determine cerebrospinal fluid and brain volume using a classical machine learning algorithm. It demonstrated good clinical usability and a very accurate correlation of the volumes to the single area determination in a reproducible axial layer. This study aims to investigate whether these established segmentation algorithms can be transferred to new, more generalizable deep learning algorithms employing an extended transfer learning procedure and whether medically meaningful segmentation is possible. METHODS: Ninety-five routinely performed true FISP MRI sequences were retrospectively analyzed in 43 patients with pediatric hydrocephalus. Using a freely available and clinically established segmentation algorithm based on a hidden Markov random field model, four classes of segmentation (brain, cerebrospinal fluid (CSF), background, and tissue) were generated. Fifty-nine randomly selected data sets (10,432 slices) were used as a training data set. Images were augmented for contrast, brightness, and random left/right and X/Y translation. A convolutional neural network (CNN) for semantic image segmentation composed of an encoder and corresponding decoder subnetwork was set up. The network was pre-initialized with layers and weights from a pre-trained VGG 16 model. Following the network was trained with the labeled image data set. A validation data set of 18 scans (3289 slices) was used to monitor the performance as the deep CNN trained. The classification results were tested on 18 randomly allocated labeled data sets (3319 slices) and on a T2-weighted BrainWeb data set with known ground truth. RESULTS: The segmentation of clinical test data provided reliable results (global accuracy 0.90, Dice coefficient 0.86), while the CNN segmentation of data from the BrainWeb data set showed comparable results (global accuracy 0.89, Dice coefficient 0.84). The segmentation of the BrainWeb data set with the classical FAST algorithm produced consistent findings (global accuracy 0.90, Dice coefficient 0.87). Likewise, the area development of brain and CSF in the long-term clinical course of three patients was presented. CONCLUSION: Using the presented methods, we showed that conventional segmentation algorithms can be transferred to new advances in deep learning with comparable accuracy, generating a large number of training data sets with relatively little effort. A clinically meaningful segmentation possibility was demonstrated.

Differential transcriptome analysis of enterohemorrhagic Escherichia coli strains reveals differences in response to plant-derived compounds.

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.

AureoWiki ̵ The repository of the Staphylococcus aureus research and annotation community.

In light of continuously accumulating data and knowledge on major human pathogens, comprehensive and up-to-date sources of easily accessible information are urgently required. The AureoWiki database (http://aureowiki.med.uni-greifswald.de) provides detailed information on the genes and proteins of clinically and experimentally relevant S. aureus strains, currently covering NCTC 8325, COL, Newman, USA300_FPR3757, and N315. By implementing a pan-genome approach, AureoWiki facilitates the transfer of knowledge gained in studies with different S. aureus strains, thus supporting functional annotation and better understanding of this organism. All data related to a given gene or gene product is compiled on a strain-specific gene page. The gene pages contain sequence-based information complemented by data on, for example, protein function and localization, transcriptional regulation, and gene expression. The information provided is connected via links to other databases and published literature. Importantly, orthologous genes of the individual strains, which are linked by a pan-genome gene identifier and a unified gene name, are presented side by side using strain-specific tabs. The respective pan-genome gene page contains an orthologue table for 32 S. aureus strains, a multiple-strain genome viewer, a protein sequence alignment as well as other comparative information. The data collected in AureoWiki is also accessible through various download options in order to support bioinformatics applications. In addition, based on two large-scale gene expression data sets, AureoWiki provides graphical representations of condition-dependent mRNA levels and protein profiles under various laboratory and infection-related conditions.

Insight into the evolution and origin of leprosy bacilli from the genome sequence of Mycobacterium lepromatosis.

Mycobacterium lepromatosis is an uncultured human pathogen associated with diffuse lepromatous leprosy and a reactional state known as Lucio's phenomenon. By using deep sequencing with and without DNA enrichment, we obtained the near-complete genome sequence of M. lepromatosis present in a skin biopsy from a Mexican patient, and compared it with that of Mycobacterium leprae, which has undergone extensive reductive evolution. The genomes display extensive synteny and are similar in size (∼3.27 Mb). Protein-coding genes share 93% nucleotide sequence identity, whereas pseudogenes are only 82% identical. The events that led to pseudogenization of 50% of the genome likely occurred before divergence from their most recent common ancestor (MRCA), and both M. lepromatosis and M. leprae have since accumulated new pseudogenes or acquired specific deletions. Functional comparisons suggest that M. lepromatosis has lost several enzymes required for amino acid synthesis whereas M. leprae has a defective heme pathway. M. lepromatosis has retained all functions required to infect the Schwann cells of the peripheral nervous system and therefore may also be neuropathogenic. A phylogeographic survey of 227 leprosy biopsies by differential PCR revealed that 221 contained M. leprae whereas only six, all from Mexico, harbored M. lepromatosis. Phylogenetic comparisons indicate that M. lepromatosis is closer than M. leprae to the MRCA, and a Bayesian dating analysis suggests that they diverged from their MRCA approximately 13.9 Mya. Thus, despite their ancient separation, the two leprosy bacilli are remarkably conserved and still cause similar pathologic conditions.

dRNA-seq transcriptional profiling of the FK506 biosynthetic gene cluster in Streptomyces tsukubaensis NRRL18488 and general analysis of the transcriptome.

FK506 (tacrolimus) is a valuable immunosuppressant produced by several Streptomyces strains. In the genome of the wild type producer Streptomyces tsukubaensis NRRL18488, FK506 biosynthesis is encoded by a gene cluster that spans 83.5 (kb). A whole transcriptome differential shotgun sequencing (dRNA-seq) of S. tsukubaensis was performed to analyze transcription at 2 different time points; before and during active FK506 production. In total, 8,914 transcription start sites were identified in either condition, which enabled precise determination of the 5'-UTR length of the corresponding transcripts as well as the identification of 2 consensus sequence motifs in the promoter regions. The transcription start sites of all gene operons within the FK506 cluster were identified, including 3 examples of leaderless RNA transcripts. These data provide detailed insight into the transcription of the FK506 biosynthetic gene cluster to support future regulatory studies, genetic manipulation, and industrial production.

Differential RNA-seq (dRNA-seq) for annotation of transcriptional start sites and small RNAs in Helicobacter pylori.

The global mapping of transcription boundaries is a key step in the elucidation of the full complement of transcriptional features of an organism. It facilitates the annotation of operons and untranslated regions as well as novel transcripts, including cis- and trans-encoded small RNAs (sRNAs). So called RNA sequencing (RNA-seq) based on deep sequencing of cDNAs has greatly facilitated transcript mapping with single nucleotide resolution. However, conventional RNA-seq approaches typically cannot distinguish between primary and processed transcripts. Here we describe the recently developed differential RNA-seq (dRNA-seq) approach, which facilitates the annotation of transcriptional start sites (TSS) based on deep sequencing of two differentially treated cDNA library pairs, with one library being enriched for primary transcripts. Using the human pathogen Helicobacter pylori as a model organism, we describe the application of dRNA-seq together with an automated TSS annotation approach for generation of a genome-wide TSS map in bacteria. Besides a description of transcriptome and regulatory features that can be identified by this approach, we discuss the impact of different library preparation protocols and sequencing platforms as well as manual and automated TSS annotation. Moreover, we have set up an easily accessible online browser for visualization of the H. pylori transcriptome data from this and our previous H. pylori dRNA-seq study.

High-resolution transcriptome maps reveal strain-specific regulatory features of multiple Campylobacter jejuni isolates.

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.

Global transcriptional start site mapping using differential RNA sequencing reveals novel antisense RNAs in Escherichia coli.

While the model organism Escherichia coli has been the subject of intense study for decades, the full complement of its RNAs is only now being examined. Here we describe a survey of the E. coli transcriptome carried out using a differential RNA sequencing (dRNA-seq) approach, which can distinguish between primary and processed transcripts, and an automated prediction algorithm for transcriptional start sites (TSS). With the criterion of expression under at least one of three growth conditions examined, we predicted 14,868 TSS candidates, including 5,574 internal to annotated genes (iTSS) and 5,495 TSS corresponding to potential antisense RNAs (asRNAs). We examined expression of 14 candidate asRNAs by Northern analysis using RNA from wild-type E. coli and from strains defective for RNases III and E, two RNases reported to be involved in asRNA processing. Interestingly, nine asRNAs detected as distinct bands by Northern analysis were differentially affected by the rnc and rne mutations. We also compared our asRNA candidates with previously published asRNA annotations from RNA-seq data and discuss the challenges associated with these cross-comparisons. Our global transcriptional start site map represents a valuable resource for identification of transcription start sites, promoters, and novel transcripts in E. coli and is easily accessible, together with the cDNA coverage plots, in an online genome browser.

Pan-Tetris: an interactive visualisation for Pan-genomes.

BACKGROUND: Large-scale genome projects have paved the way to microbial pan-genome analyses. Pan-genomes describe the union of all genes shared by all members of the species or taxon under investigation. They offer a framework to assess the genomic diversity of a given collection of individual genomes and moreover they help to consolidate gene predictions and annotations. The computation of pan-genomes is often a challenge, and many techniques that use a global alignment-independent approach run the risk of not separating paralogs from orthologs. Also alignment-based approaches which take the gene neighbourhood into account often need additional manual curation of the results. This is quite time consuming and so far there is no visualisation tool available that offers an interactive GUI for the pan-genome to support curating pan-genomic computations or annotations of orthologous genes. RESULTS: We introduce Pan-Tetris, a Java based interactive software tool that provides a clearly structured and suitable way for the visual inspection of gene occurrences in a pan-genome table. The main features of Pan-Tetris are a standard coordinate based presentation of multiple genomes complemented by easy to use tools compensating for algorithmic weaknesses in the pan-genome generation workflow. We demonstrate an application of Pan-Tetris to the pan-genome of Staphylococcus aureus. CONCLUSIONS: Pan-Tetris is currently the only interactive pan-genome visualisation tool. Pan-Tetris is available from http://bit.ly/1vVxYZT.

A novel point mutation promotes growth phase-dependent daptomycin tolerance in Staphylococcus aureus.

Recalcitrance of genetically susceptible bacteria to antibiotic killing is a hallmark of bacterial drug tolerance. This phenomenon is prevalent in biofilms, persisters, and also planktonic cells and is associated with chronic or relapsing infections with pathogens such as Staphylococcus aureus. Here we report the in vitro evolution of an S. aureus strain that exhibits a high degree of nonsusceptibility to daptomycin as a result of cyclic challenges with bactericidal concentrations of the drug. This phenotype was attributed to stationary growth phase-dependent drug tolerance and was clearly distinguished from resistance. The underlying genetic basis was revealed to be an adaptive point mutation in the putative inorganic phosphate (Pi) transporter gene pitA. Drug tolerance caused by this allele, termed pitA6, was abrogated when the upstream gene pitR was inactivated. Enhanced tolerance toward daptomycin, as well as the acyldepsipeptide antibiotic ADEP4 and various combinations of other drugs, was accompanied by elevated intracellular concentrations of Pi and polyphosphate, which may reversibly interfere with critical cellular functions. The evolved strain displayed increased rates of survival within human endothelial cells, demonstrating the correlation of intracellular persistence and drug tolerance. These findings will be useful for further investigations of S. aureus drug tolerance, toward the development of additional antipersister compounds and strategies.