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bioRxiv2015

Shift and adapt: the costs and benefits of karyotype variations

Variation is the spice of life or, in the case of evolution, variation is the necessary material on which selection can act to enable adaptation. Karyotypic variation in ploidy (the number of homologous chromosome sets) and aneuploidy (imbalance in the number of chromosomes) are fundamentally different than other types of genomic variants. Karyotypic variation emerges through different molecular mechanisms than other mutational events, and unlike mutations that alter the genome at the base pair level, rapid reversion to the wild type chromosome number is often possible. Although karyotypic variation has long been noted and discussed by biologists, interest in the importance of karyotypic variants in evolutionary processes has spiked in recent years, and much remains to be discovered about how karyotypic variants are produced and subsequently selected.

Microbiology↗

bioRxiv2015

DNA-metabarcoding uncovers the diversity of soil-inhabiting fungi in the tropical island of Puerto Rico

Soil fungal communities in tropical regions remain poorly understood. In order to increase the knowledge of diversity of soil-inhabiting fungi, we extracted total DNA from top-organic soil collected in seven localities dominated by four major ecosystems in the tropical island of Puerto Rico. In order to comprehensively characterize the fungal community, we PCR-amplified the internal transcribed spacer 2 (ITS2) fungal barcode using newly designed degenerated primers and varying annealing temperatures to minimize primer bias. Sequencing results, obtained using Ion Torrent technology, comprised a total of 566,613 sequences after quality filtering. These sequences were clustered into 4,140 molecular operational taxonomic units (MOTUs) after removing low frequency sequences and rarefaction to account for differences in read depth between samples. Our results demonstrate that soil fungal communities in Puerto Rico are structured by ecosystem. Ascomycota, followed by Basidiomycota, dominates the diversity of fungi in soil. Amongst Ascomycota, the recently described soil-inhabiting class Archaeorhizomycetes was present in all localities, and taxa in Archaeorhizomycetes were among the most commonly observed MOTUs. The Basidiomycota community was dominated by soil decomposers and ectomycorrhizal fungi with a distribution strongly affected by local variation to a greater degree than Ascomycota.

Microbiology↗

bioRxiv2015

Cationic Peptides Facilitate Iron-induced Mutagenesis in Bacteria

Pseudomonas aeruginosa is the causative agent of chronic respiratory infections and is an important pathogen of cystic fibrosis patients. Adaptive mutations play an essential role for antimicrobial resistance and persistence. The factors that contribute to bacterial mutagenesis in this environment are not clear. Recently it has been proposed that cationic antimicrobial peptides such as LL-37 could act as a mutagen in P. aeruginosa. Here we provide experimental evidence that mutagenesis is the product of a joint action of LL-37 and free iron. By estimating mutation rate, mutant frequencies and assessing mutational spectra in P. aeruginosa treated either with LL-37, iron or a combination of both we demonstrate that mutation rate and mutant frequency were increased only when free iron and LL-37 were present simultaneously. The addition of an iron chelator completely abolished this mutagenic effect, suggesting that LL-37 enables iron to enter the cells resulting in DNA damage by Fenton reactions. This was also supported by the observation that the mutational spectrum of the bacteria under LL-37-iron regime showed one of the characteristic Fenton reaction fingerprints: C to T transitions. Free iron concentration in nature and within hosts is kept at a very low level, but the situation in infected lungs of cystic fibrosis patients is different. Intermittent bleeding and damage to the epithelial cells in lungs may contribute to the release of free iron that in turn leads to generation of reactive oxygen species and deterioration of the respiratory tract, making it more susceptible to the infection.\n\nAuthor SummaryCationic antimicrobial peptides (cAMPs) are small proteins naturally produced by the immune system to limit bacterial growth mainly through pore formation in the membrane. It has recently been suggested that sub-inhibitory concentrations of cAMPs promote bacterial mutagenesis, similarly to antibiotics. However, we previously reported that cAMPs do not increase mutation rate and do not activate bacterial stress responses. Here we resolve this contradiction. We report that free iron in the culture medium increases mutagenesis in the presence of cAMPs. We show that sub-inhibitory concentrations of cAMPs facilitate entry of free iron into bacterial cells, where it interacts with hydrogen peroxide, thereby resulting in production of DNA-damaging reactive oxygen species and increased mutagenesis. Moreover, these results may have clinically-relevant implications: while very little free iron is normally present in healthy individuals, this is not the case in patients suffering from cystic fibrosis, where elevated bacterial mutagenesis promotes antibiotic resistance and contributes to persistence and severity of infection. Thus, an intervention aimed at reduction of free iron in the lungs could reduce the cAMPs-facilitation of iron-mediated mutagenesis; hence antibiotic resistance and pathoadaptation.

Microbiology↗

bioRxiv2015

Prediction of Hfq in actinobacteria

Hfq is the bacterial orthologue of the eukaryotic (L)Sm family of proteins found across all domains of life and potentially an ancient protein, but it has not been found in all phyletic lines. A careful search successfully identified a distant hfq orthologue in the cyanobacteria leaving the actinobacteria as the major phylum with no known hfq orthologue. A search for hfq in actinobacteria, using domain enhanced searching (DELTA-BLAST) with cyanobacterial hfq, identified a conserved actinobacterial specific protein as remotely homologous. Structural homology modelling using profile matching to fold libraries and ab initio 3D structure determination supports this prediction and suggests module shuffling in the evolution of the actinobacterial hfq. Our results provide the basis to explore this prediction, and exploit it, across diverse taxa with potentially important post-transcriptional regulatory effects in virulence, antibiotic production and interactions in human microbiomes. However, the role of hfq in gram positive bacteria has remained elusive and experimental verification will be challenging.

Microbiology↗

bioRxiv2015

Complete assembly of novel environmental bacterial genomes by MinIONTM sequencing

In this study, we adapt a protocol for the growth of previously uncultured environmental bacterial isolates, to make it compatible with whole genome sequencing. We demonstrate that in combination with the MinION sequencing device, complete assemblies can be derived, allowing genomic comparisons to be made. This approach allows rapid, inexpensive and straightforward discovery, and genomic analysis, of previously uncultured prokaryotic genomes, and brings greater ownership of all parts of the sequencing process back to individual researchers.

Microbiology↗

bioRxiv2015

Microbiome engineering could select for more virulent pathogens

Recent insights into the human microbiome offer the hope of manipulating its composition to help fight infectious diseases1-7. While this strategy has shown huge potential, its consequences for pathogen evolution have not been explored. Here we show that manipulating the microbiome to increase the competition that pathogens face could lead to the evolution of increased production of virulence factors that pathogens use to combat commensals, an evolutionary response that can increase total disease induced mortality in the long-term. However, if treatment with microbiome engineering is sufficiently aggressive this evolutionary response can be avoided and the pathogen eradicated. Furthermore, we show that using damage limitation therapies8 (e.g. antivirulence and anti-inflammatory drugs) in combination with microbiome manipulation increases the potential for pathogen eradication. While manipulating our microbiota offers a promising alternative to antibiotics, our results show that these treatments must be designed with careful consideration of the potential evolutionary responses of target pathogens.

Microbiology↗

bioRxiv2015

Habitat Fluctuations Drive Species Covariation in the Human Microbiota

Two species with similar resource requirements respond in a characteristic way to variations in their habitat - their abundances rise and fall in concert. We use this idea to learn how bacterial populations in the microbiota respond to habitat conditions that vary from person-to-person across the human population. Our mathematical framework shows that habitat fluctuations are sufficient for explaining intra-bodysite correlations in relative species abundances from the Human Microbiome Project. We explicitly show that the relative abundances of phylogenetically related species are positively correlated and can be predicted from taxonomic relationships. We identify a small set of functional pathways related to metabolism and maintenance of the cell wall that form the basis of a common resource sharing niche space of the human microbiota.

Microbiology↗

bioRxiv2015

Time-kill curve analysis and pharmacodynamic functions for in vitro evaluation of antimicrobials against Neisseria gonorrhoeae

Gonorrhea is a sexually transmitted infection caused by the Gram-negative bacterium Neisseria gonorrhoeae. Resistance to first-line empirical monotherapy has emerged, so robust methods are needed to appropriately evaluate the activity of existing and novel antimicrobials against the bacterium. Pharmacodynamic functions, which describe the relationship between the concentration of antimicrobials and the bacterial net growth rate, provide more detailed information than the MIC only. In this study, a novel standardized in vitro time-kill curve assay was developed. The assay was validated using five World Health Organization N. gonorrhoeae reference strains and various concentrations of ciprofloxacin, and then the activity of nine antimicrobials with different target mechanisms were examined against a highly susceptible clinical wild type isolate (cultured in 1964). From the time-kill curves, the bacterial net growth rates at each antimicrobial concentration were estimated. Finally, a pharmacodynamic function was fitted to the data, resulting in four parameters that describe the pharmacodynamic properties of each antimicrobial. Ciprofloxacin resistance determinants shifted the pharmacodynamic MIC (zMIC) and attenuated the bactericidal effect at antimicrobial concentrations above the zMIC. Ciprofloxacin, spectinomycin and gentamicin had the strongest bactericidal effect during the first six hours of the assay. Only tetracycline and chloramphenicol showed a purely bacteriostatic effect. The pharmacodynamic functions differed between antimicrobials, showing that the effect of the drugs at concentrations below and above the MIC vary widely. In conclusion, N. gonorrhoeae time-kill curve experiments analyzed with pharmacodynamic functions have potential for in vitro evaluation of new and existing antimicrobials and dosing strategies to treat gonorrhea.

Microbiology↗

bioRxiv2015

CRISPR system acquisition and evolution of an obligate intracellular Chlamydia-related bacterium

Recently, a new Chlamydia-related organism, Protochlamydia naegleriophila KNic, was discovered within a Naegleria amoeba. To decipher the mechanisms at play in the modeling of genomes from the Protochlamydia genus, we sequenced de novo the full genome of Pr. naegleriophila combining the advantages of two second-generation sequencing technologies. The assembled complete genome comprises a 2,885,111 bp chromosome and a 145,285 bp megaplasmid. For the first time within the Chlamydiales order, a CRISPR system, the immune system of bacteria, was discovered on the chromosome. It is composed of a small CRISPR locus comprising eight repeats and the associated cas and cse genes of the subtype I-E. A CRISPR locus was also found within Chlamydia sp. Diamant, another Pr. naegleriophila strain whose genome was recently released, suggesting that the CRISPR system was acquired by a common ancestor of these two members of Pr. naegleriophila, after the divergence from Pr. amoebophila. The plasmid encodes an F-type conjugative system similar to that found in the Pam100G genomic island of Pr. amoebophila suggesting an acquisition of this conjugative system before the divergence of both Protochlamydia species and the integration of a putative Pr. amoebophila plasmid into its main chromosome giving rise to the Pam100G genomic island. Overall, this new Pr. naegleriophila genome sequence enables to investigate further the dynamic processes shaping the genomes of Chlamydia-related bacteria.

Microbiology↗

bioRxiv2015

Tuning microscale, retractable, membrane-breaking protein needles

The refractile (R) bodies found in Caedibacter taeniospiralis, a bacterial endosymbiont of Paramecium tetraurelia, are large, polymeric protein structures that can switch between two conformations. At cytoplasmic pH, they resemble coiled ribbons of protein 500nm in diameter. At low pH, they extend to form hollow needles up to 20 microns long. They can be expressed heterologously from an operon containing four short open reading frames and can function in vitro in diverse buffer conditions.\n\nIn this study, R bodies purified from Escherichia coli were found to be capable of undergoing many consecutive extension-contraction cycles. Furthermore, the solubility of R bodies, which can easily be interpreted by eye, was found to correlate with their extension state. This macroscopic phenotype was used to develop a quantitative, high-throughput assay for R body state, enabling a visual screen of R body mutants defective in extension. The role of specific amino acids in extension was determined, and this information was used to construct rationally-designed mutants tailored to extend at higher pH. Furthermore, R bodies were able to rupture E. coli spheroplasts to release soluble proteins across lipid bilayers. Taken together, these results show that R bodies act as tunable, pH-actuated pistons suitable for a variety of membrane-breaking applications.

Microbiology↗

bioRxiv2015

A highly diverse, desert-like microbial biocenosis on solar panels in a Mediterranean city

Microorganisms colonize a wide range of natural and artificial environments although there are hardly any data on the microbial ecology of one the most widespread man-made extreme structures: solar panels. Here we show that solar panels in a Mediterranean city (Valencia, Spain) harbor a highly diverse microbial community with more than 500 different species per panel, most of which belong to drought-, heat- and radiation-adapted bacterial genera, and sun-irradiation adapted epiphytic fungi. The taxonomic and functional profiles of this microbial community and the characterization of selected culturable bacteria reveal the existence of a diverse mesophilic microbial community on the panels surface. This biocenosis proved to be more similar to the ones inhabiting deserts than to any human or urban microbial ecosystem. This unique microbial community shows different day/night proteomic profiles; it is dominated by reddish pigment- and sphingolipid-producers, and is adapted to withstand circadian cycles of high temperatures, desiccation and solar radiation.

Microbiology↗

bioRxiv2015

Functional analysis of the archaea, bacteria, and viruses from a halite endolithic microbial community

Halite endoliths in the Atacama Desert represent one of the most extreme microbial ecosystems on Earth. Here we sequenced and characterized a shotgun metagenome from halite nodules collected in Salar Grande, Chile. The community is dominated by archaea and functional analysis attributed most of the autotrophic CO2 fixation to a unique cyanobacterium. The assembled 1.1 Mbp genome of a novel nanohaloarchaeon, Candidatus Nanopetramus SG9, revealed a photoheterotrophic life style and a low median isoelectric point (pI) for all predicted proteins, suggesting a \"salt-in\" strategy for osmotic balance. Predicted proteins of the algae identified in the community also had pI distributions similar to \"salt-in\" strategists. The Nanopetramus genome contained a unique CRISPR/Cas system with a spacer that matched a partial viral genome from the metagenome. A combination of reference-independent methods identified over 30 complete or near complete viral or proviral genomes with diverse genome structure, genome size, gene content, and hosts. Putative hosts included Halobacteriaceae, Nanohaloarchaea, and Cyanobacteria. Despite the dependence of the halite community on deliquescence for liquid water availability, this study exposed an ecosystem spanning three phylogenetic domains, containing a large diversity of viruses, and a predominant \"salt-in\" strategy to balance the high osmotic pressure of the environment.

Microbiology↗

bioRxiv2015

Human Microbiota of the Argentine Population- A pilot study

Here we present the first dataset based on human microbiota samples of an urban middle-income population in South America. We characterized the microbiota of six different body habitats: palatine tonsils, saliva, buccal mucosa, throat, anterior nares and gut from samples of healthy individual living in a metropolitan area in Argentina. Our initial findings revealed differences in the structure and composition of the microbial communities compared to the US urban population. By sharing our data, we want to actively encourage its reuse for comparison purposes. This will hopefully result in novel biological insights on the variability of the microbiota of healthy individuals across populations worldwide. Moreover, the understanding of the human microbiota ecosystem in a health-associated state will help to answer questions related to the role of the microbiota in disease.

Microbiology↗

bioRxiv2015

Crop diversity enhances disease suppressive potential in soils

Microbiomes can aid in the protection of hosts from infection and disease, but the mechanisms underpinning these functions in complex environmental systems remain unresolved. Soils contain microbiomes that influence plant performance, including their susceptibility to disease. For example, some soil microorganisms produce antimicrobial compounds that suppress the growth of plant pathogens, which can provide benefits for sustainable agricultural management. Evidence shows that crop rotations increase soil fertility and tend to promote microbial diversity, and it has been hypothesized that crop rotations can enhance disease suppressive capacity, either through the influence of plant diversity impacting soil bacterial composition or through the increased abundance of disease suppressive microorganisms. In this study, we used a long-term field experiment to test the effects of crop diversity through time (i.e., rotations) on soil microbial diversity and disease suppressive capacity. We sampled soil from seven treatments along a crop diversity gradient (from monoculture to five crop species rotation) and a spring fallow (non-crop) treatment to examine crop diversity influence on soil microbiomes including bacteria that are capable of producing antifungal compounds. Crop diversity significantly influenced bacterial community composition, where the most diverse cropping systems with cover crops and fallow differed from bacterial communities in the 1-3 crop species diversity treatments. While soil bacterial diversity was about 4% lower in the most diverse crop rotation (corn-soy-wheat + 2 cover crops) compared to monoculture corn, crop diversity increased disease suppressive functional group prnD gene abundance in the more diverse rotation by about 9% compared to monocultures. Identifying patterns in microbial diversity and ecosystem function relationships can provide insight into microbiome management, which will require manipulating soil nutrients and resources mediated through plant diversity.

Microbiology↗

bioRxiv2015

Common and phylogenetically widespread coding for peptides by bacterial small RNAs

While eukaryotic noncoding RNAs have recently received intense scrutiny, it is becoming clear that bacterial transcription is at least as pervasive. Bacterial small RNAs and antisense RNAs (sRNAs) are often assumed to be noncoding, due to their lack of long open reading frames (ORFs). However, there are numerous examples of sRNAs encoding for small proteins, whether or not they also have a regulatory role at the RNA level. Here, we apply flexible machine learning techniques based on sequence features and comparative genomics to quantify the prevalence of sRNA ORFs under natural selection to maintain protein-coding function in phylogenetically diverse bacteria. A majority of annotated sRNAs have at least one ORF between 10 and 50 amino acids long, and we conservatively predict that 188{+/-}25.5 unannotated sRNA ORFs are under selection to maintain coding, an average of 13 per species considered here. This implies that overall at least 7.5 {+/-}0.3% of sRNAs have a coding ORF, and in some species at least 20% do. 84 {+/-} 9.8 of these novel coding ORFs have some antisense overlap to annotated ORFs. As experimental validation, many of our predictions are translated according to ribosome profiling data and are identified via mass spectrometry shotgun proteomics. B. subtilis sRNAs with coding ORFs are enriched for high expression in biofilms and confluent growth, and two S. pneumoniae sRNAs with coding ORFs are involved in virulence. sRNA coding ORFs are enriched for transmembrane domains and many are novel components of type I toxin/antitoxin systems. Our predictions for sRNA coding ORFs, including novel type I toxins, are freely available in a user-friendly format at http://disco-bac.web.pasteur.fr.

Microbiology↗

bioRxiv2015

Evolutionary history of the global emergence of the Escherichia coli epidemic clone ST131

BackgroundEscherichia coli sequence type 131 (ST131) has emerged globally as the most predominant lineage within this clinically important species, and its association with fluoroquinolone and extended-spectrum cephalosporin resistance impacts significantly on treatment. The evolutionary histories of this lineage, and of important antimicrobial resistance elements within it, remain unclearly defined.\n\nResultsThis study of the largest worldwide collection (n = 215) of sequenced ST131 E. coli isolates to date demonstrates that clonal expansion of two previously recognized antimicrobial-resistant clades, C1/H30R and C2/H30Rx, started around 25 years ago, consistent with the widespread introduction of fluoroquinolones and extended-spectrum cephalosporins in clinical medicine. These two clades appear to have emerged in the United States, with the expansion of the C2/H30Rx clade driven by the acquisition of a blaCTX-M-15-containing IncFII-like plasmid that has subsequently undergone extensive rearrangement. Several other evolutionary processes influencing the trajectory of this drug-resistant lineage are described, including sporadic acquisitions of CTX-M resistance plasmids, and chromosomal integration of blaCTX-M within sub-clusters followed by vertical evolution. These processes are also occurring for another family of CTX-M gene variants more recently observed amongst ST131, the blaCTX-M-14/14-like group.\n\nConclusionsThe complexity of the evolutionary history of ST131 has important implications for antimicrobial resistance surveillance, epidemiological analysis, and control of emerging clinical lineages of E. coli. These data also highlight the global imperative to reduce specific antibiotic selection pressures, and demonstrate the important and varied roles played by plasmids and other mobile genetic elements in the perpetuation of antimicrobial resistance within lineages.

Genomics↗

bioRxiv2015

Cannabis microbiome sequencing reveals several mycotoxic fungi native to dispensary grade cannabis flowers

The Center for Disease Control estimates 128,000 people in the U.S. are hospitalized annually due to food borne illnesses. This has created a demand for food safety testing targeting the detection of pathogenic mold and bacteria on agricultural products. This risk extends to medical Cannabis and is of particular concern with inhaled, vaporized and even concentrated Cannabis products. As a result, third party microbial testing has become a regulatory requirement in the medical and recreational Cannabis markets, yet knowledge of the Cannabis microbiome is limited. Here we describe the first next generation sequencing survey of the microbial communities found in dispensary based Cannabis flowers and demonstrate the limitations in the culture based regulations that are being superimposed from the food industry.

Microbiology↗

bioRxiv2015

A computational model of inhibition of HIV-1 by interferon-alpha

Type 1 interferons such as interferon-alpha (IFN) inhibit replication of Human immunodeficiency virus (HIV-1) by upregulating the expression of genes that interfere with specific steps in the viral life cycle. This pathway thus represents a potential target for immune-based therapies that can alter the dynamics of host-virus interactions to benefit the host. To obtain a deeper mechanistic understanding of how IFN impacts spreading HIV-1 infection, we modeled the interaction of HIV-1 with CD4 T cells and IFN as a dynamical system. This model was then tested using experimental data from a cell culture model of spreading HIV-1 infection. We found that a model in which IFN induces reversible cellular states that block both early and late stages of HIV-1 infection, combined with a saturating rate of conversion to these states, was able to successfully fit the experimental dataset. Sensitivity analysis showed that the potency of inhibition by IFN was particularly dependent on specific network parameters and rate constants. This model will be useful for designing new therapies targeting the IFN network in HIV-1-infected individuals, as well as potentially serving as a template for understanding the interaction of IFN with other viruses.\n\nAuthor SummaryInterferon-alpha (IFN) is a key component of the host response to HIV-1, but the details of how IFN regulates infection are still incompletely understood. To provide a deeper understanding of the dynamics of how IFN inhibits HIV-1, we simulated the interaction of IFN and HIV-1 as a computational model and compared this model to an experimental dataset. We identify a model structure that is able to fit many key features of the data. Furthermore, we use the model to predict optimal strategies for targeting the IFN pathway therapeutically. We anticipate that this model will be useful for further analysis of HIV-IFN interactions and will help to guide new therapeutic strategies.

Microbiology↗