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bioRxiv2017

A phylogenetic and evolutionary analysis of antimycin biosynthesis

Streptomyces species and other Actinobacteria are ubiquitous in diverse environments worldwide and are the source of, or inspiration for, the majority of pharmaceuticals. The genomic era has enhanced biosynthetic understanding of these valuable chemical entities and has also provided a window into the diversity and distribution of natural product biosynthetic gene clusters. Antimycin is an inhibitor of mitochondrial cytochrome c reductase and more recently was shown to inhibit Bcl-2/Bcl-XLsrelated anti-apoptotic proteins commonly overproduced by cancerous cells. Here we identify 65 putative antimycin biosynthetic gene clusters (BGCs) in publicly available genome sequences of Actinobacteria and classify them based on the presence or absence of cluster-situated genes antP and antQ, which encode a kynureninase and phosphopantetheinyl transferase (PPTase), respectively. The majority of BGCs possess either both antP and antQ (L-form) or neither (S-form), while a minority of them lack either antP or antQ (IQ or IPform, respectively). We also evaluate the biogeographical distribution and phylogenetic relationships of antimycin producers and BGCs. We show that antimycin BGCs occur on five of the seven continents and are frequently isolated from plants and other higher organisms. We also provide evidence for two distinct phylogenetic clades of antimycin producers and gene clusters, which delineate S-form from L- and I-form BGCs. Finally, our findings suggest that the ancestral antimycin producer harboured an L-form gene cluster which was primarily propagated by vertical transmission and subsequently diversified into S-, IQ and IPform biosynthetic pathways.

microbiology↗

bioRxiv2017

Hepatitis B Virus Infection as a Neglected Tropical Disease

BACKGROUND BACKGROUND CURRENT STRATEGIES FOR HBV... APPLICATION OF NTD CRITERIA... RECOMMENDATIONS BASED ON NTD... CONCLUSIONS SUPPORTING INFORMATION LEGEND REFERENCES The Global Hepatitis Health Sector Strategy is aiming for elimination of viral hepatitis as a public health threat by 2030 [1], while enhanced elimination efforts for hepatitis are also promoted under the broader remit of global Sustainable Development Goals (SDGs) [2]. This is an enormous challenge for hepatitis B virus (HBV) given the estimated global burden of 260 million chronic carriers, of whom the majority are unaware of their infection [3] (Figure 1).\n\nWe here present HBV within the framework for ...

microbiology↗

bioRxiv2017

Degradation of benzene by the heavy-metal resistant bacterium Cupriavidus metallidurans CH34 reveals its catabolic potential for aromatic compounds

Benzene, toluene, ethylbenzene and the three xylene isomers are monoaromatic contaminants widely distributed on polluted sites. Some microorganisms have developed mechanisms to degrade these compounds, but their aerobic and anaerobic degradation is inhibited in presence of heavy metals, such as mercury or lead. In this report, the degradation of benzene and other aromatic compounds catalyzed by the metal resistant bacterium Cupriavidus metallidurans CH34 was characterized. A metabolic reconstruction of aromatic catabolic pathways was performed based on bioinformatics analyses. Functionality of the predicted pathways was confirmed by growing strain CH34 on benzene, toluene, o-xylene, p-cymene, 3-hydroxybenzoate, 4-hydroxybenzoate, 3-hydroxyphenylacetate, 4-hydroxyphenylacetate, homogentisate, catechol, naphthalene, and 2-aminophenol as sole carbon and energy sources. Benzene catabolic pathway was further characterized. Results showed that firstly benzene is transformed into phenol and, thereafter, into catechol. Benzene is degraded under aerobic conditions via a combined pathway catalyzed by three Bacterial Multicomponent Monooxygenases: a toluene-2-monoxygenase (TomA012345), a toluene-4-monooxygenase (TmoABCDEF) and a phenol-2-hydroxylase (PhyZABCDE). A catechol-2,3-dioxygenase (TomB) expressed at early exponential phase cleaves the catechol ring in meta-position; an ortho-cleavage of catechol is accomplished by a catechol-1,2-dioxygenase (CatA) at late exponential phase instead. This study additionally shows that C. metallidurans CH34 is capable of degrading benzene in presence of heavy metals, such as Hg(II) or Pb(II). This capability of degrading aromatic compounds in presence of heavy metals is rather unusual among environmental bacteria; therefore, C. metallidurans CH34 seems to be a promising candidate for developing novel bioremediation process for multi-contaminated environments.\n\nHIGHLIGHTSO_LIThe strain Cupriavidus metallidurans CH34 is capable to degrade benzene aerobically\nC_LIO_LIBenzene oxydation is mediated by bacterial multicomponent monoxygenases\nC_LIO_LIStrain CH34 is able to grow using a broad range of aromatic compounds as sole carbon and energy source\nC_LIO_LIBenzene degradation occurs even in presence of heavy metals such as mercury and lead\nC_LI

microbiology↗

bioRxiv2017

Evolution within the fungal genus Verticillium is characterized by chromosomal rearrangement and gene loss

The fungal genus Verticillium contains ten species, some of which are notorious plant pathogens causing vascular wilt diseases in host plants, while others are known as saprophytes and opportunistic plant pathogens. Whereas the genome of V. dahliae, the most notorious plan pathogen of the genus, has been well characterized, evolution and speciation of other members of the genus received little attention thus far. Here, we sequenced the genomes of the nine haploid Verticillium spp. to study evolutionary trajectories of their divergence from a last common ancestor. Frequent occurrence of chromosomal rearrangement and gene family loss was identified. In addition to ~11,000 core genes that are shared among all species, only 200-600 species-specific genes occur. Intriguingly, these species-specific genes show different features than core genes.

microbiology↗

bioRxiv2017

Resilience mechanisms of small intestinal lactobacilli to the toxicity of soybean oil fatty acids

Over the past century, soybean oil (SBO) consumption in the United States increased dramatically. The main SBO fatty acid, linoleic acid (18:2), inhibits in vitro the growth of lactobacilli, beneficial members of the small intestinal microbiota. Human-associated lactobacilli have declined in prevalence in Western microbiomes, but how dietary changes may have impacted their ecology is unclear. Here, we compared the in vitro and in vivo effects of 18:2 on Lactobacillus reuteri and L. johnsonii. Directed evolution in vitro in both species led to strong 18:2 resistance with mutations in genes for lipid biosynthesis, acid stress, and the cell membrane or wall. Small-intestinal Lactobacillus populations in mice were unaffected by chronic and acute 18:2 exposure, yet harbored both 18:2- sensitive and resistant strains. This work shows that extant small intestinal lactobacilli are protected from toxic dietary components via the gut environment as well as their own capacity to evolve resistance.

microbiology↗

bioRxiv2017

Conditionally rare taxa contribute but do not account for prokaryotic community changes in soils

Conditionally rare taxa (CRT) are thought to greatly impact microbial community turnover across many environments, but little is known about their role in soils. Here, we investigate the contribution of CRT to whole community variation over space and time in a series of geographically distinct soils dedicated to three agricultural practices of differing intensities and sampled over a full seasonal cycle. We demonstrate that soil CRT do not account for observed total community changes, but that these rare taxa can be modified by spatiotemporal filters.

microbiology↗

bioRxiv2017

Isolation and characterization of BpL1, a broad acting lytic bacteriophage against Brucella

We have isolated a new broad acting lytic brucellaphage (BpL1) from the sewage of a dairy farm. The phage lysed all the 12 Brucella abortus field isolates, B. abortus strain 99 and Brucella melitensis but did not lyse any of the heterologous species tested viz. Staphylococcus aureus, Pasteurella multocida, Escherichia coli, and Salmonella species. Streaking the plaques on Brucella lawn gave clear zones along the streak lines. The plaques were circular with a diameter of 0.5- 3.0 mm. At a concentration of 10-4 the phage count was 4.5 x 106 plaques per ml. It was a tailed phage with icosahedral head (62.2 nm in diameter and 73.71 nm in length), and the head to tail length was 229.21 nm. The phage belonged to the order Caudovirales and family Siphoviridae. It was inactivated within one hour at 55{degrees}C and within 4 hours at -20{degrees}C. Treatment at pH 2 for 4 hours and at pH 4 for 12 hours inactivated it. It was inactivated after 4 hours exposure to sunlight, and within 4 minutes by UV light. Chloroform and Sodium Dodecyl Sulfate inactivated it within 15 minutes. Lysozyme inactivated it within 1 hour whereas RNase treatment did not affect its activity.

microbiology↗

bioRxiv2017

Cellular reductase activity in uncultivated Thiomargarita spp. assayed using a redox-sensitive dye

The largest known bacteria, Thiomargarita spp., have yet to be isolated in pure culture, but their large size allows for individual cells to be followed in time course experiments, or to be individually sorted for omics-based investigations. Here we report a novel application of a tetrazolium-based dye that measures the flux of reductase production from catabolic pathways to investigate the metabolic activity of individual cells of Thiomargarita spp. When coupled to microscopy, staining of the cells with a tetrazolium-formazan dye allows for metabolic responses in Thiomargarita spp. to be to be tracked in the absence of observable cell division. Additionally, the metabolic activity of Thiomargarita spp. cells can be differentiated from the metabolism of other microbes in specimens that contain adherent bacteria. The results of our redox-dye-based assay suggests that Thiomargarita is the most metabolically versatile under anoxic conditions where it appears to express cellular reductase activity in response to the electron donors succinate, acetate, citrate, formate, thiosulfate, H2, and H2S. Under hypoxic conditions, formazan staining results suggest the metabolism of succinate, and likely acetate, citrate, and H2S. Cells incubated under oxic conditions showed the weakest formazan staining response, and then only to H2S, citrate, and perhaps succinate. These results provide experimental validation of recent genomic studies of Ca. Thiomargarita nelsonii that suggest metabolic plasticity and mixotrophic metabolism. The cellular reductase response of bacteria attached to the exteriors of Thiomargarita also supports the possibility of trophic interactions between these largest of known bacteria and attached epibionts.\n\nIMPORTANCEThe metabolic potentials of many microorganisms that cannot be grown in the laboratory are known only from genomic data. Genomes of Thiomargarita spp. suggest that these largest of known bacteria are mixotrophs, combining lithotrophic metabolisms with organic carbon degradation. Our use of a redox-sensitive tetrazolium dye to query the metabolism of these bacteria provides an independent line of evidence that corroborates the apparent metabolic plasticity of Thiomargarita observed in recently produced genomes. Finding new cultivation- independent means of testing genomic results is critical to testing genome-derived hypotheses on the metabolic potentials of uncultivated microorganisms.

microbiology↗

bioRxiv2017

Collaborative environmental DNA sampling from petal surfaces of flowering cherry Cerasus ×- yedoensis 'Somei-yoshino' across the Japanese archipelago

Recent studies have shown that environmental DNA is found almost everywhere. Flower petal surfaces are an attractive tissue to use for investigation of the dispersal of environmental DNA in nature as they are isolated from the external environment until the bud opens and only then can the petal surface accumulate environmental DNA. Here, we performed a crowdsourced experiment, the \"Ohanami Project\", to obtain environmental DNA samples from petal surfaces of Cerasus x yedoensis Somei-yoshino across the Japanese archipelago during spring 2015. C. x yedoensis is the most popular garden cherry species in Japan and clones of this cultivar bloom simultaneously every spring. Data collection spanned almost every prefecture and totaled 577 DNA samples from 149 collaborators. Preliminary amplicon-sequencing analysis showed the rapid attachment of environmental DNA onto the petal surfaces. Notably, we found DNA of other common plant species in samples obtained across a wide distribution; this DNA likely originated from pollen of the Japanese cedar. Our analysis supports our belief that petal surfaces after blossoming are a promising target to reveal the dynamics of environmental DNA in nature. The success of our experiment also shows that crowdsourced environmental DNA analyses have considerable value in ecological studies.

microbiology↗

bioRxiv2017

Pseudopaline, a staphylopine-like metallophore involved in zinc and nickel uptake in Pseudomonas aeruginosa

Metal uptake is vital for all living organisms. In metal scarce conditions, a common bacterial strategy consists in the biosynthesis of metallophores, their export in the extracellular medium and the recovery of a metal-metallophore complex through dedicated membrane transporters. Staphylopine is a recently described metallophore distantly related to plant nicotianamine that contributes to the broad-spectrum metal uptake capabilities of Staphylococcus aureus. Here, we characterize a four genes operon (PA4837-PA4834) in Pseudomonas aeruginosa involved in the biosynthesis and trafficking of a staphylopine-like metallophore named pseudopaline. Pseudopaline differs from staphylopine with regard to the stereochemistry of its histidine moiety associated to an alpha ketoglutarate moiety instead of pyruvate. In vivo, the pseudopaline operon is regulated by zinc through the Zur repressor. The metal-uptake property of the pseudopaline system appears different from that of staphylopine with a predominant effect on nickel uptake, and on zinc uptake in metal scarce conditions mimicking a chelating environment, thus reconciling the regulation of the cnt operon by zinc with its function as a zinc importer under metal scarce conditions.\n\nAUTHOR SUMMARYZinc is an essential micronutrients for bacteria, particularly important at the host-pathogen interface where the host tends to sequester metals in a so called nutritional immunity framework, and the pathogenic bacterium increases its metal uptake efforts in order to keep up with its metal requirements. Here we reveal a novel metallophore, named pseudopaline, which is synthesized and exported by Pseudomonas aeruginosa and serves for the uptake of nickel in metal poor media, and for the uptake of zinc in metal scarce conditions that mimic the chelating environment that presumably prevails within a host.

microbiology↗

bioRxiv2017

The electron transport chain sensitises Staphylococcus aureus and Enterococcus faecalis to the oxidative burst

Small colony variants (SCVs) of Staphylococcus aureus typically lack a functional electron transport chain and cannot produce virulence factors such as leukocidins, hemolysins or the anti-oxidant staphyloxanthin. Despite this, SCVs are associated with persistent infections of the bloodstream, bones and prosthetic devices. The survival of SCVs in the host has been ascribed to intracellular residency, biofilm formation and resistance to antibiotics. However, the ability of SCVs to resist host defences is largely uncharacterised. To address this, we measured survival of wild-type and SCV S. aureus in whole human blood, which contains high numbers of neutrophils, the key defense against staphylococcal infection. Despite the loss of leukcocidin production and staphyloxanthin biosynthesis, SCVs defective for heme or menquinone biosynthesis were significantly more resistant to the oxidative burst than wild-type bacteria in a whole human blood model. Supplementation of the culture medium of the heme-auxotrophic SCV with heme, but not iron, restored growth, hemolysin and staphyloxanthin production, and sensitivity to the oxidative burst. Since Enterococcus faecalis is a natural heme auxotroph and cause of bloodstream infection, we explored whether restoration of the electron transport chain in this organism also affected survival in blood. Incubation of E. faecalis with heme increased growth and restored catalase activity, but resulted in decreased survival in human blood via increased sensitivity to the oxidative burst. Therefore, the lack of functional electron transport chains in SCV S. aureus and wild-type E. faecalis results in reduced growth rate but provides resistance to a key immune defence mechanism.

microbiology↗

bioRxiv2017

Context-dependent Requirements for FimH and Other Canonical Virulence Factors in Gut Colonization by Extraintestinal Pathogenic Escherichia coli

Extraintestinal pathogenic Escherichia coli (ExPEC) act as commensals within the mammalian gut, but can induce pathology upon dissemination to other host environments such as the urinary tract and bloodstream. It is thought that ExPEC genomes are shaped in large part by evolutionary forces encountered within the gut where the bacteria spend much of their time, provoking the question of how their extraintestinal virulence traits arose. The principle of coincidental evolution, in which a gene that evolved in one niche happens to be advantageous in another, has been used to argue that ExPEC virulence factors originated in response to selective pressures within the gut ecosystem. As a test of this hypothesis, the fitness of ExPEC mutants lacking canonical virulence factors was assessed within the intact murine gut in the absence of any antibiotic treatment. We found that most of the tested factors--including CNF1, Usp, colibactin, flagella, and the plasmid pUTI89--were dispensable for gut colonization. Deletion of genes encoding the adhesin PapG or the toxin HlyA caused transient defects, but did not affect longer-term persistence. In contrast, a mutant missing the type 1 pilus-associated adhesin FimH displayed reduced persistence within the gut. However, this phenotype was variable, being dependent on the presence of specific competing strains and partially attributable to aberrant flagellin expression by the fimH mutant. These data indicate that FimH and other key ExPEC-associated factors are not strictly required for gut colonization, suggesting that selective pressures within the gut do not drive the development of all extraintestinal virulence traits.

microbiology↗

bioRxiv2017

Dramatic expansion of microbial groups that shape the global sulfur cycle

The biogeochemical cycle of sulfur on Earth is driven by microbial sulfate reduction, yet organisms from relatively few lineages have been implicated in this process. Recent studies using functional marker genes have detected abundant, novel dissimilatory sulfite reductases that confer the capacity for microbial sulfate reduction and could do not be affiliated with known organisms. Thus, the identity of a significant fraction of sulfate reducing microbes has remained elusive. Here we report the discovery of the capacity for sulfate reduction in the genomes of organisms from twelve bacterial and archaeal phyla, thereby doubling the number of microbial phyla associated with this process. Eight of the twelve newly identified groups are candidate phyla that lack isolated representatives, a finding only possible given genomes from metagenomes. Two candidate phyla, Candidatus Rokubacteria and Candidatus Hydrothermarchaeota contain the earliest evolved genes. The capacity for sulfate reduction has been laterally transferred in multiple events within some phyla, and a key gene potentially capable of switching sulfur oxidation to sulfate reduction in associated cells has been acquired by putatively symbiotic bacteria. We conclude that functional predictions based on phylogeny will significantly underestimate the extent of sulfate reduction across Earths ecosystems. Understanding the prevalence of this capacity is integral to interpreting the carbon cycle because sulfate reduction is often coupled to turnover of buried organic carbon. Our findings expand the diversity of microbial groups associated with sulfur transformations in the environment and motivate revision of biogeochemical process models based on microbial community composition.

microbiology↗

bioRxiv2017

Using MinION to characterize dog skin microbiota through full-length 16S rRNA gene sequencing approach

The most common strategy to assess microbiota is sequencing specific hypervariable regions of 16S rRNA gene using 2nd generation platforms (such as MiSeq or Ion Torrent PGM). Despite obtaining high-quality reads, many sequences fail to be classified at the genus or species levels due to their short length. This pitfall can be overcome sequencing the full-length 16S rRNA gene (1,500bp) by 3rd generation sequencers.\n\nWe aimed to assess the performance of nanopore sequencing using MinION on characterizing microbiota complex samples. First set-up step was performed using a staggered mock community (HM-783D). Then, we sequenced a pool of several dog skin microbiota samples previously sequenced by Ion Torrent PGM. Sequences obtained for full-length 16S rRNA with degenerated primers retrieved increased richness estimates at high taxonomic level (Bacteria and Archaea) that were missed with short-reads. Besides, we were able to obtain taxonomic assignments down to species level, although it was not always feasible due to: i) incomplete database; ii) primer set chosen; iii) low taxonomic resolution of 16S rRNA gene within some genera; and/or iv) sequencing errors. Nanopore sequencing of the full-length 16S rRNA gene using MinION with 1D sequencing kit allowed us inferring microbiota composition of a complex microbial community to lower taxonomic levels than short-reads from 2nd generation sequencers.

microbiology↗

bioRxiv2017

Cytosine methylation within marine sediment microbial communities: potential epigenetic adaptation to the environment

Marine sediments harbor a vast amount of Earths microbial biomass, yet little is understood regarding how cells subsist in this low-energy, presumably slow-growth environment. Cells in marine sediments may require additional methods for genetic regulation, such as epigenetic modification via DNA methylation. We investigated this potential phenomenon within a shallow estuary sediment core spanning 100 years of age across its depth. Here we provide evidence of dynamic community m5-cytosine methylation within estuarine sediment metagenomes using a methylation-sensitive Illumina assay. The methylation states of individual CpG sites were reconstructed and quantified across three depths within the sediment core. A total of 6254 CpG sites were aligned for direct comparison of methylation states between samples, with 4235 sites mapped to taxa and genes. Our results demonstrate the presence of differential methylation within environmental CpG sites across an age/depth gradient of sediment. We show that epigenetic modification can be detected within complex environmental communities. The change in methylation state of environmentally relevant genes across depths may indicate a dynamic role of DNA methylation in biogeochemical processes.

microbiology↗

bioRxiv2017

Towards a highly efficient diversity census of the prokaryotic biosphere: a group testing approach

Exploring the microbial biosphere has grown exponentially in recent years, although we are far from understanding its entirety. We present the adiversity censusa problem of exploring all bacterial species in a large cohort of specimens, and detecting a specimen that contains each species. The naive approach to this problem is to sequence each specimen, thus requiring costly sample preparation steps.\n\nWe suggest an orders of magnitude more efficient approach for diversity censusing. Specimens are pooled according to a predefined design and standard 16S rRNA sequencing is performed over each pool. For each bacterial species, from the ultra-rare to the most common, the algorithm detects a single specimen that contains the bacterial species. The approach can be applied to large cohorts of monomicrobial cultures or to complex samples containing a mixture of organisms.\n\nWe model the experimental procedure and show via in silico simulations that the approach enables censusing more than 95% of the species while taking 10 - 70 fold less resources. Simulating experiments using real samples display the utility in censusing large cohorts of samples.\n\nDiversity censusing presents a novel problem in the mathematical field of group testing that may also be applied in other biological problems and in other domains.

microbiology↗

bioRxiv2017

Conditionally Redundant Bacteriocin Targeting by Pseudomonas syringae

The widespread use of antimicrobials under clinical and agricultural settings has resulted in the evolution of resistance to these compounds. To combat the emergence of resistance, current research efforts are focusing on designing treatments to exploit combinations of antimicrobials, where the evolution of resistance confers sensitivity to alternative compounds. In this work we demonstrate that strains of Pseudomonas syringae possess a natural analogue to this strategy. Specifically, we demonstrate that a single strain produces multiple bacteriocins that can target another strain, but antimicrobial activity of the second bacteriocin is manifest only after resistance to the first emerges. The evolution of resistance also sensitizes the target strain to bacteriocins from a variety of other strains. Strains of P. syringae therefore encode multiple bacteriocins that can act in a conditionally redundant manner. It is possible that combinations of bacteriocins could be applied as a cocktail or sequentially, to potentially achieve durable pathogen control.

microbiology↗

bioRxiv2017

Cyanophages exhibit rhythmic infection patterns under light-dark cycles

Most living organisms exhibit diurnal rhythms as an adaptation to the daily light-dark (diel) cycle. However, diurnal rhythms have not been found in viruses. Here, we studied the diel infection patterns of bacteriophages infecting the unicellular cyanobacteria Prochlorococcus and Synechococcus, which are the most abundant photosynthetic organisms in the oceans. With lab cultures, we found that cyanophages used three infection strategies in the dark: no adsorption, adsorption but no replication, and replication. Interestingly, the former two exhibited rhythmic infection patterns under light-dark cycles. We further showed in the South China Sea and the Western Pacific Ocean that cyanophage abundances varied rhythmically, with a peak at night. Moreover, diel transcriptional rhythms of many cyanophage genes were found in the North Pacific Subtropical Gyre, which also peaked at night. Our results suggested that cyanophage infection of Prochlorococcus is synchronized to the light-dark cycle, which may result in a synchronized release of dissolved organic matter to the marine food web.

microbiology↗