Abstract
Background
Metagenomic sequencing is a well-established tool in the modern biosciences. While it promises unparalleled insights into the genetic content of the biological samples studied, ...conclusions drawn are at risk from biases inherent to the DNA sequencing methods, including inaccurate abundance estimates as a function of genomic guanine-cytosine (GC) contents.
Results
We explored such GC biases across many commonly used platforms in experiments sequencing multiple genomes (with mean GC contents ranging from 28.9% to 62.4%) and metagenomes. GC bias profiles varied among different library preparation protocols and sequencing platforms. We found that our workflows using MiSeq and NextSeq were hindered by major GC biases, with problems becoming increasingly severe outside the 45–65% GC range, leading to a falsely low coverage in GC-rich and especially GC-poor sequences, where genomic windows with 30% GC content had >10-fold less coverage than windows close to 50% GC content. We also showed that GC content correlates tightly with coverage biases. The PacBio and HiSeq platforms also evidenced similar profiles of GC biases to each other, which were distinct from those seen in the MiSeq and NextSeq workflows. The Oxford Nanopore workflow was not afflicted by GC bias.
Conclusions
These findings indicate potential sources of difficulty, arising from GC biases, in genome sequencing that could be pre-emptively addressed with methodological optimizations provided that the GC biases inherent to the relevant workflow are understood. Furthermore, it is recommended that a more critical approach be taken in quantitative abundance estimates in metagenomic studies. In the future, metagenomic studies should take steps to account for the effects of GC bias before drawing conclusions, or they should use a demonstrably unbiased workflow.
Bacteria play an important role in the degradation of bone material. However, much remains to be learnt about the structure of their communities in degrading bone, and how the depositional ...environment influences their diversity throughout the exposure period. We genetically profiled the bacterial community in an experimental series of pig bone fragments (femur and humeri) deposited at different well-defined environments in Denmark. The bacterial community in the bone fragments and surrounding depositional environment were studied over one year, and correlated with the bioerosion damage patterns observed microscopically in the bones. We observed that the bacterial communities within the bones were heavily influenced by the local microbial community, and that the general bone microbial diversity increases with time after exposure. We found the presence of several known collagenase producing bacterial groups, and also observed increases in the relative abundance of several of these in bones with tunneling. We anticipate that future analyses using shotgun metagenomics on this and similar datasets will be able to provide insights into mechanisms of microbiome driven bone degradation.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Conserving additional energy from sunlight through bacteriochlorophyll (BChl)-based reaction center or proton-pumping rhodopsin is a highly successful life strategy in environmental bacteria. BChl ...and rhodopsin-based systems display contrasting characteristics in the size of coding operon, cost of biosynthesis, ease of expression control, and efficiency of energy production. This raises an intriguing question of whether a single bacterium has evolved the ability to perform these two types of phototrophy complementarily according to energy needs and environmental conditions. Here, we report four
sp. strains (
) of monophyletic origin isolated from a high Arctic glacier in northeast Greenland (81.566° N, 16.363° W) that are at different evolutionary stages concerning phototrophy. Their >99.8% identical genomes contain footprints of horizontal operon transfer (HOT) of the complete gene clusters encoding BChl- and xanthorhodopsin (XR)-based dual phototrophy. Two strains possess only a complete XR operon, while the other two strains have both a photosynthesis gene cluster and an XR operon in their genomes. All XR operons are heavily surrounded by mobile genetic elements and are located close to a tRNA gene, strongly signaling that a HOT event of the XR operon has occurred recently. Mining public genome databases and our high Arctic glacial and soil metagenomes revealed that phylogenetically diverse bacteria have the metabolic potential of performing BChl- and rhodopsin-based dual phototrophy. Our data provide new insights on how bacteria cope with the harsh and energy-deficient environment in surface glacier, possibly by maximizing the capability of exploiting solar energy.
Over the course of evolution for billions of years, bacteria that are capable of light-driven energy production have occupied every corner of surface Earth where sunlight can reach. Only two general biological systems have evolved in bacteria to be capable of net energy conservation via light harvesting: one is based on the pigment of (bacterio-)chlorophyll and the other is based on proton-pumping rhodopsin. There is emerging genomic evidence that these two rather different systems can coexist in a single bacterium to take advantage of their contrasting characteristics in the number of genes involved, biosynthesis cost, ease of expression control, and efficiency of energy production and thus enhance the capability of exploiting solar energy. Our data provide the first clear-cut evidence that such dual phototrophy potentially exists in glacial bacteria. Further public genome mining suggests this understudied dual phototrophic mechanism is possibly more common than our data alone suggested.
Aminobacter sp. MSH1 (CIP 110285) can use the pesticide dichlobenil and its recalcitrant transformation product, 2,6-dichlorobenzamide (BAM), as sole source of carbon, nitrogen, and energy. The ...concentration of BAM in groundwater often exceeds the threshold limit for drinking water, requiring additional treatment in drinking water treatment plants or closure of the affected abstraction wells. Biological treatment with MSH1 is considered a potential sustainable alternative to remediate BAM-contamination in drinking water production. We present the complete genome of MSH1, which was determined independently in two institutes at Aarhus University and KU Leuven. Divergences were observed between the two genomes, i.e. one of them lacked four plasmids compared to the other. Besides the circular chromosome and the two previously described plasmids involved in BAM catabolism, pBAM1 and pBAM2, the genome of MSH1 contained two megaplasmids and three smaller plasmids. The MSH1 substrain from KU Leuven showed a reduced genome lacking a megaplasmid and three smaller plasmids and was designated substrain MK1, whereas the Aarhus variant with all plasmids was designated substrain DK1. A plasmid stability experiment indicate that substrain DK1 may have a polyploid chromosome when growing in R2B medium with more chromosomes than plasmids per cell. Finally, strain MSH1 is reassigned as Aminobacter niigataensis MSH1.
Abstract
Background
Riverine ecosystems are biogeochemical powerhouses driven largely by microbial communities that inhabit water columns and sediments. Because rivers are used extensively for ...anthropogenic purposes (drinking water, recreation, agriculture, and industry), it is essential to understand how these activities affect the composition of river microbial consortia. Recent studies have shown that river metagenomes vary considerably, suggesting that microbial community data should be included in broad-scale river ecosystem models. But such ecogenomic studies have not been applied on a broad “aquascape” scale, and few if any have applied the newest nanopore technology.
Results
We investigated the metagenomes of 11 rivers across 3 continents using MinION nanopore sequencing, a portable platform that could be useful for future global river monitoring. Up to 10 Gb of data per run were generated with average read lengths of 3.4 kb. Diversity and diagnosis of river function potential was accomplished with 0.5–1.0 ⋅ 106 long reads. Our observations for 7 of the 11 rivers conformed to other river-omic findings, and we exposed previously unrecognized microbial biodiversity in the other 4 rivers.
Conclusions
Deeper understanding that emerged is that river microbial consortia and the ecological functions they fulfil did not align with geographic location but instead implicated ecological responses of microbes to urban and other anthropogenic effects, and that changes in taxa manifested over a very short geographic space.
Isolating single phages using plaque assays is a laborious and time-consuming process. Whether single isolated phages are the most lyse-effective, the most abundant in viromes, or those with the ...highest ability to make plaques in solid media is not well known. With the increasing accessibility of high-throughput sequencing, metaviromics is often used to describe viruses in environmental samples. By extracting and sequencing metaviromes from organic waste with and without exposure to a host-of-interest, we show a host-related phage community's shift, as well as identify the most enriched phages. Moreover, we isolated plaque-forming single phages using the same virome-host matrix to observe how enrichments in liquid media correspond to the metaviromic data. In this study, we observed a significant shift (
= 0.015) of the 47 identified putative
phages with a minimum twofold change above zero in read abundance when adding a
DC3000 host. Surprisingly, it appears that only two out of five plaque-forming phages from the same organic waste sample, targeting the
strain, were highly abundant in the metavirome, while the other three were almost absent despite host exposure. Lastly, our sequencing results highlight how long reads from Oxford Nanopore elevates the assembly quality of metaviromes, compared to short reads alone.
Antibiosis is a key feature widely exploited to develop biofungicides based on the ability of biological control agents (BCAs) to produce fungitoxic compounds. A less recognised attribute of ...plant-associated beneficial microorganisms is their ability to stimulate the plant immune system, which may provide long-term, systemic self-protection against different types of pathogens. By using conventional antifungal in vitro screening coupled with
assays, we found antifungal and non-antifungal
strains that protected the ornamental plant Kalanchoe against the soil-borne pathogen
in experimental and commercial production settings. Further examination of one antifungal and one non-antifungal strain indicated that high protection efficacy
did not correlate with antifungal activity
. Whole-genome sequencing showed that the non-antifungal strain EC9 lacked the biosynthetic gene clusters associated with typical antimicrobial compounds. Instead, this bacterium triggers the expression of marker genes for the jasmonic and salicylic acid defence pathways, but only after pathogen challenge, indicating that this strain may protect Kalanchoe plants by priming immunity. We suggest that the stimulation of the plant immune system is a promising mode of action of BCAs for the development of novel biological crop protection products.
Bacterial degraders of chlorophenoxy herbicides have been isolated from various ecosystems, including pristine environments. Among these degraders, the sphingomonads constitute a prominent group that ...displays versatile xenobiotic-degradation capabilities. Four separate sequencing strategies were required to provide the complete sequence of the complex and plastic genome of the canonical chlorophenoxy herbicide-degrading Sphingobium herbicidovorans MH. The genome has an intricate organization of the chlorophenoxy-herbicide catabolic genes sdpA, rdpA, and cadABCD that encode the (R)- and (S)-enantiomer-specific 2,4-dichlorophenoxypropionate dioxygenases and four subunits of a Rieske non-heme iron oxygenase involved in 2-methyl-chlorophenoxyacetic acid degradation, respectively. Several major genomic rearrangements are proposed to help understand the evolution and mobility of these important genes and their genetic context. Single-strain mobilomic sequence analysis uncovered plasmids and insertion sequence-associated circular intermediates in this environmentally important bacterium and enabled the description of evolutionary models for pesticide degradation in strain MH and related organisms. The mobilome presented a complex mosaic of mobile genetic elements including four plasmids and several circular intermediate DNA molecules of insertion-sequence elements and transposons that are central to the evolution of xenobiotics degradation. Furthermore, two individual chromosomally integrated prophages were shown to excise and form free circular DNA molecules. This approach holds great potential for improving the understanding of genome plasticity, evolution, and microbial ecology.
Deciphering the mechanisms governing population genetic divergence and local adaptation across heterogeneous environments is a central theme in marine ecology and conservation. While population ...divergence and ecological adaptive potential are classically viewed at the genetic level, it has recently been argued that their microbiomes may also contribute to population genetic divergence. We explored whether this might be plausible along the well-described environmental gradient of the Baltic Sea in two species of sand lance (Ammodytes tobianus and Hyperoplus lanceolatus). Specifically, we assessed both their population genetic and gut microbial composition variation and investigated not only which environmental parameters correlate with the observed variation, but whether host genome also correlates with microbiome variation.
We found a clear genetic structure separating the high-salinity North Sea from the low-salinity Baltic Sea sand lances. The observed genetic divergence was not simply a function of isolation by distance, but correlated with environmental parameters, such as salinity, sea surface temperature, and, in the case of A. tobianus, possibly water microbiota. Furthermore, we detected two distinct genetic groups in Baltic A. tobianus that might represent sympatric spawning types. Investigation of possible drivers of gut microbiome composition variation revealed that host species identity was significantly correlated with the microbial community composition of the gut. A potential influence of host genetic factors on gut microbiome composition was further confirmed by the results of a constrained analysis of principal coordinates. The host genetic component was among the parameters that best explain observed variation in gut microbiome composition.
Our findings have relevance for the population structure of two commercial species but also provide insights into potentially relevant genomic and microbial factors with regards to sand lance adaptation across the North Sea-Baltic Sea environmental gradient. Furthermore, our findings support the hypothesis that host genetics may play a role in regulating the gut microbiome at both the interspecific and intraspecific levels. As sequencing costs continue to drop, we anticipate that future studies that include full genome and microbiome sequencing will be able to explore the full relationship and its potential adaptive implications for these species.
The 2-methyl-4-chlorophenoxyacetic (MCPA) acid-degrader Sphingomonas sp. ERG5 has recently been isolated from MCPA-degrading bacterial communities. Using Illumina-sequencing, the 5.7 Mb genome of ...this isolate was sequenced in this study, revealing the 138 kbp plasmid pCADAB1 harboring the 32.5 kbp composite transposon Tn6228 which contains genes encoding proteins for the removal of 2,4-dichlorophenoxyacetic acid (2,4-D) and MCPA, as well as the regulation of this pathway. Transposon Tn6228 was confirmed by PCR to be situated on the plasmid and also exist in a circular intermediate state - typical of IS3 elements. The canonical tfdAα-gene of group III 2,4-D degraders, encoding the first step in degradation of 2,4-D and related compounds, was not present in the chromosomal contigs. However, the alternative cadAB genes, also providing the initial degradation step, were found in Tn6228, along with the 2,4-D-degradation-associated genes tfdBCDEFKR and cadR. Putative reductase and ferredoxin genes cadCD of Rieske non-heme iron oxygenases were also present in close proximity to cadAB, suggesting that these might have an unknown role in the initial degradation reaction. Parts of the composite transposon contain sequence displaying high similarity to previously analyzed 2,4-D degradation genes, suggesting rapid dissemination and high conservation of the chlorinated-phenoxyacetic acid (PAA)-degradation genotype among the sphingomonads.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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