Showing papers in "Fems Microbiology Letters in 2019"

Not all animals need a microbiome.

Abstract: It is often taken for granted that all animals host and depend upon a microbiome, yet this has only been shown for a small proportion of species. We propose that animals span a continuum of reliance on microbial symbionts. At one end are the famously symbiont-dependent species such as aphids, humans, corals and cows, in which microbes are abundant and important to host fitness. In the middle are species that may tolerate some microbial colonization but are only minimally or facultatively dependent. At the other end are species that lack beneficial symbionts altogether. While their existence may seem improbable, animals are capable of limiting microbial growth in and on their bodies, and a microbially independent lifestyle may be favored by selection under some circumstances. There is already evidence for several 'microbiome-free' lineages that represent distantly related branches in the animal phylogeny. We discuss why these animals have received such little attention, highlighting the potential for contaminants, transients, and parasites to masquerade as beneficial symbionts. We also suggest ways to explore microbiomes that address the limitations of DNA sequencing. We call for further research on microbiome-free taxa to provide a more complete understanding of the ecology and evolution of macrobe-microbe interactions.

Genomic diversity, lifestyles and evolutionary origins of DPANN archaea.

Abstract: Archaea-a primary domain of life besides Bacteria-have for a long time been regarded as peculiar organisms that play marginal roles in biogeochemical cycles. However, this picture changed with the discovery of a large diversity of archaea in non-extreme environments enabled by the use of cultivation-independent methods. These approaches have allowed the reconstruction of genomes of uncultivated microorganisms and revealed that archaea are diverse and broadly distributed in the biosphere and seemingly include a large diversity of putative symbiotic organisms, most of which belong to the tentative archaeal superphylum referred to as DPANN. This archaeal group encompasses at least 10 different lineages and includes organisms with extremely small cell and genome sizes and limited metabolic capabilities. Therefore, many members of DPANN may be obligately dependent on symbiotic interactions with other organisms and may even include novel parasites. In this contribution, we review the current knowledge of the gene repertoires and lifestyles of members of this group and discuss their placement in the tree of life, which is the basis for our understanding of the deep microbial roots and the role of symbiosis in the evolution of life on Earth.

Keeping crispr in check: diverse mechanisms of phage-encoded anti-crisprs.

Abstract: CRISPR-Cas represents the only adaptive immune system of prokaryotes known to date. These immune systems are widespread among bacteria and archaea, and provide protection against invasion of mobile genetic elements, such as bacteriophages and plasmids. As a result of the arms-race between phages and their prokaryotic hosts, phages have evolved inhibitors known as anti-CRISPR (Acr) proteins to evade CRISPR immunity. In the recent years, several Acr proteins have been described in both temperate and virulent phages targeting diverse CRISPR-Cas systems. Here, we describe the strategies of Acr discovery and the multiple molecular mechanisms by which these proteins operate to inhibit CRISPR immunity. We discuss the biological relevance of Acr proteins and speculate on the implications of their activity for the development of improved CRISPR-based research and biotechnological tools.

Multiplex genome editing of microorganisms using CRISPR-Cas.

Abstract: Microbial production of chemical compounds often requires highly engineered microbial cell factories. During the last years, CRISPR-Cas nucleases have been repurposed as powerful tools for genome editing. Here, we briefly review the most frequently used CRISPR-Cas tools and describe some of their applications. We describe the progress made with respect to CRISPR-based multiplex genome editing of industrial bacteria and eukaryotic microorganisms. We also review the state of the art in terms of gene expression regulation using CRISPRi and CRISPRa. Finally, we summarize the pillars for efficient multiplexed genome editing and present our view on future developments and applications of CRISPR-Cas tools for multiplex genome editing.

Decoding the ocean's microbiological secrets for marine enzyme biodiscovery.

Abstract: A global census of marine microbial life has been underway over the past several decades. During this period, there have been scientific breakthroughs in estimating microbial diversity and understanding microbial functioning and ecology. It is estimated that the ocean, covering 71% of the earth's surface with its estimated volume of about 2 × 1018 m3 and an average depth of 3800 m, hosts the largest population of microbes on Earth. More than 2 million eukaryotic and prokaryotic species are thought to thrive both in the ocean and on its surface. Prokaryotic cell abundances can reach densities of up to 1012 cells per millilitre, exceeding eukaryotic densities of around 106 cells per millilitre of seawater. Besides their large numbers and abundance, marine microbial assemblages and their organic catalysts (enzymes) have a largely underestimated value for their use in the development of industrial products and processes. In this perspective article, we identified critical gaps in knowledge and technology to fast-track this development. We provided a general overview of the presumptive microbial assemblages in oceans, and an estimation of what is known and the enzymes that have been currently retrieved. We also discussed recent advances made in this area by the collaborative European Horizon 2020 project 'INMARE'.

Genome editing of lactic acid bacteria: opportunities for food, feed, pharma and biotech

Abstract: This mini-review provides a perspective of traditional, emerging and future applications of lactic acid bacteria (LAB) and how genome editing tools can be used to overcome current challenges in all these applications. It also describes available tools and how these can be further developed, and takes current legislation into account. Genome editing tools are necessary for the construction of strains for new applications and products, but can also play a crucial role in traditional ones, such as food and probiotics, as a research tool for gaining mechanistic insights and discovering new properties. Traditionally, recombinant DNA techniques for LAB have strongly focused on being food-grade, but they lack speed and the number of genetically tractable strains is still rather limited. Further tool development will enable rapid construction of multiple mutants or mutant libraries on a genomic level in a wide variety of LAB strains. We also propose an iterative Design-Build-Test-Learn workflow cycle for LAB cell factory development based on systems biology, with 'cell factory' expanding beyond its traditional meaning of production strains and making use of genome editing tools to advance LAB understanding, applications and strain development.

Transmission mode is associated with environment type and taxa across bacteria-eukaryote symbioses: a systematic review and meta-analysis

Abstract: Symbiotic associations between bacteria and eukaryotes exhibit a range of transmission strategies. The rates and distributions of transmission modes have not been thoroughly investigated across associations, despite their consequences on symbiont and host evolution. To address this empirically, I compiled data from the literature on bacteria-multicellular eukaryote associations for which transmission mode data was available. Of the total 528 analyzed symbioses, 21.2% were strictly horizontally transmitted, 36.0% exhibited some form of mixed mode transmission and 42.8% were strictly vertically transmitted. Controlling for phylogenetically independent symbiosis events revealed modes were approximately equally distributed among the 113 independent associations, at 32.1%+/-0.57% horizontal, 37.8%+/-1.4% mixed mode and 31.1%+/-1.3% vertical transmission. Binning symbioses by environment revealed an abundance of vertical transmission on land and a lack of it in aquatic environments. The naturally occurring uneven distribution of taxa among environments prevented controlling for host/symbiont phylogeny. However, the results were robust over a large number of independently evolved associations, suggesting that many vertically transmitted bacteria are capable of mixed mode transmission and barriers exist that reduce the rate of horizontal transmission events. Thus, both the environment type and host/symbiont taxa influence symbiont transmission mode evolution.

A multidimensional perspective on microbial interactions

Abstract: Beyond being simply positive or negative, beneficial or inhibitory, microbial interactions can involve a diverse set of mechanisms, dependencies and dynamical properties. These more nuanced features have been described in great detail for some specific types of interactions, (e.g. pairwise metabolic cross-feeding, quorum sensing or antibiotic killing), often with the use of quantitative measurements and insight derived from modeling. With a growing understanding of the composition and dynamics of complex microbial communities for human health and other applications, we face the challenge of integrating information about these different interactions into comprehensive quantitative frameworks. Here, we review the literature on a wide set of microbial interactions, and explore the potential value of a formal categorization based on multidimensional vectors of attributes. We propose that such an encoding can facilitate systematic, direct comparisons of interaction mechanisms and dependencies, and we discuss the relevance of an atlas of interactions for future modeling and rational design efforts.

Titer regulation in arthropod-Wolbachia symbioses.

Abstract: Symbiosis between intracellular bacteria (endosymbionts) and animals are widespread. The alphaproteobacterium Wolbachia pipientis is known to maintain a variety of symbiotic associations, ranging from mutualism to parasitism, with a wide range of invertebrates. Wolbachia infection might deeply affect host fitness (e.g. reproductive manipulation and antiviral protection), which is thought to explain its high prevalence in nature. Bacterial loads significantly influence both the infection dynamics and the extent of bacteria-induced host phenotypes. Hence, fine regulation of bacterial titers is considered as a milestone in host-endosymbiont interplay. Here, we review both environmental and biological factors modulating Wolbachia titers in arthropods.

Dietary butyrate suppresses inflammation through modulating gut microbiota in high-fat diet-fed mice.

Abstract: Butyrate, a key metabolite fermented by gut microbiota mainly from undigested carbohydrates such as dietary fibers is widely used as feed additive. However, mechanisms of its contributions in maintaining host health are relatively poorly revealed. The aim of this study was to investigate how butyrate impacts gut microbiota and immunity response in high-fat diet-fed mice. Gut microbial analysis exhibited that butyrate intervention increased short-chain fatty acids (SCFAs)-producing bacteria and decreased pathogenic bacteria, such as endotoxin-secreting bacteria. Our result also demonstrated that butyrate intervention enhanced fecal SCFAs concentrations, and inhibited endotoxin levels in feces and serum. Correlation analysis indicated positive relation between endotoxin level and Desulfovibrionaceae abundance. Furthermore, butyrate intervention inhibited expressions of IL-1β, IL-6 and MCP1/CCL2 in liver, as well as TLR4 in adipose tissue. Apart from inhibiting expressions of proinflammatory cytokines, butyrate exerted anti-inflammation effect through selectively modulating gut microbiota, such as increasing SCFAs-producing bacteria and decreasing endotoxin-secreting bacteria, as well as via regulating levels of microbiota-dependent metabolites and components, such as SCFAs and endotoxin.

The seagrass holobiont: understanding seagrass-bacteria interactions and their role in seagrass ecosystem functioning.

Abstract: This review shows that the presence of seagrass microbial community is critical for the development of seagrasses; from seed germination, through to phytohormone production and enhanced nutrient availability, and defence against pathogens and saprophytes. The tight seagrass-bacterial relationship highlighted in this review supports the existence of a seagrass holobiont and adds to the growing evidence for the importance of marine eukaryotic microorganisms in sustaining vital ecosystems. Incorporating a micro-scale view on seagrass ecosystems substantially expands our understanding of ecosystem functioning and may have significant implications for future seagrass management and mitigation against human disturbance.

Influence of 16S rRNA variable region on perceived diversity of marine microbial communities of the Northern North Atlantic

Abstract: Marine microbes play essential roles in global energy and nutrient cycles. A primary method of determining their diversity and distribution is through sequencing of 16S ribosomal RNA genes from environmental samples. However, the perceived community composition may vary significantly based on differences in methodology, including choice of 16S variable region(s). This study investigated the influence of 16S variable region selection (V4-V5 or V6-V8) on perceived community composition and diversity for bacteria, Archaea and chloroplasts by tag-Illumina sequencing. We used 24 samples from the photic zone of the Scotian Shelf, northwest Atlantic, collected during a spring phytoplankton bloom. Taxonomic assignment and community composition varied greatly depending on the choice of variable regions while observed patterns of beta diversity were reproducible between variable regions. V4-V5 was considered the preferred variable region for future studies based on its superior recognition of Archaea, which has received little attention in bloom dynamics. The V6-V8 region captured more of the bacterial diversity, including the abundant SAR11 clades and, to a lesser extent, that of chloroplasts. However, the magnitude of difference between variable regions for bacteria and chloroplast was less than for Archaea.

The qualified presumption of safety assessment and its role in EFSA risk evaluations: 15 years past

Abstract: Microorganisms are intentionally added at different stages of the food and feed chain (food or feed additive, novel food or plant protection product) and are subjected to regulation and safety assessment by the European Food Safety Authority. Safety evaluation is based on application dossiers for market authorisation to the European Commission. The qualified presumption of safety (QPS) concept was developed in 20031 to provide a harmonised generic safety pre-appraisal of the above microorganisms. Unambiguously defined biological taxonomic units (TUs) are assessed for their body of knowledge, their safety and their end use. Identified safety concerns for a certain TU can be, where reasonable in number and not universally present, reflected as 'qualifications.' Strains belonging to TUs having QPS status may benefit of a fast track evaluation. The lowest TU for which the QPS status is granted is the species level for bacteria and yeasts and the family for viruses. The QPS concept is also applicable to genetically modified microorganisms used for production purposes. Based on the current body of knowledge and/or the ambiguous taxonomic position, some TUs, such as filamentous fungi, bacteriophages, Enterococcus faecium, Escherichia coli, Streptomyces spp. and Oomycetes, are not considered liable for QPS status.

A novel six-phage cocktail reduces Pectobacterium atrosepticum soft rot infection in potato tubers under simulated storage conditions.

Abstract: Pectobacterium atrosepticum is a species of plant pathogenic bacteria responsible for significant losses in potato production worldwide. Pectobacterium atrosepticum can cause blackleg disease on potato stems as well as the tuber disease termed potato soft rot. Methods for the effective control of these diseases are limited and are primarily based on good agricultural practices. Bacteriophages, viruses of bacteria, could be used as an alternative, environmentally friendly, control measure. Here, we describe the isolation and characterization of 29 phages virulent to P. atrosepticum. The phages belong to 12 different species based on a 95% sequence identity cut-off. Furthermore, based on sequence diversity and propagation results, we selected six of these phages to form a phage cocktail. The phages in the cocktail was tested on a number of P. atrosepticum strains in order to determine their host range. The phages was found to lyse 93% of the tested strains. The cocktail was subsequently tested for its effectiveness in combatting potato soft rot under simulated storage conditions. Use of the phage cocktail reduced both disease incidence and disease severity by 61% and 64%, respectively, strongly indicating that phage biocontrol has the potential to reduce the economic impact of soft rot in potato production.

Vaginal microbiota transplantation for the treatment of bacterial vaginosis: a conceptual analysis.

Abstract: Bacterial vaginosis (BV), caused by the vaginal dysbacteriosis as well as the excessive growth of pathogenic bacteria, is a pathological condition of the vagina; its treatment using the antibiotics metronidazole or clindamycin often causes high recurrence rates. Considering the similar physiological environments of the intestinal tract and vaginal tract, as well as the pathological mechanism of intestinal infection and vaginal infection, we first propose the conception of vaginal microbiota transplantation (VMT) and discuss its potential use in BV. This review focuses on the pathology of BV and the side effects caused by its standardised treatment. The extremely dynamic and diverse gut microbiota forms the most intensive microbial system and also plays a significant role in human body, and Lactobacilli dominate in the vaginal tract of women, keeping them healthy. Accordingly, we also propose the concept of VMT based on the effects of faecal microbiota transplantation (FMT) in treating intestinal infections, and list the potential hurdles for the implementation of VMT.

Application of lactic acid bacteria in green biorefineries

Abstract: Lactic acid bacteria (LAB) have extensive industrial applications as producers of lactic acid, as probiotics, as biocontrol agents and as biopreservatives. LAB play a large role in food fermentation and in silage processes, where crops such as grass, legumes, cereals or corn are fermented into high-moisture feed that is storable and can be used to feed cattle, sheep or other ruminants. LAB also have great applications within green biorefineries, with simultaneous production of protein-rich feed for monogastric animals, silage or feed pellets for ruminants and production of lactic acid or specific amino acids. In green biorefineries, fresh or ensiled wet biomass is mechanically fractionated into green juice and solid residues (press cake), where the plant juice, for example, can be used for production of lactic acid using LAB. In a process named 'ENLAC', recovery of protein and chlorophyll from silage by simultaneous lactic acid fermentation and enzyme hydrolysis has been developed. Furthermore, a process for protein recovery was recently developed by applying a specific LAB starter culture to green juice from freshly harvested crops. This paper focuses on reviewing LAB for their applications within biorefining of 'green' crops such as clover, alfalfa, grasses and other green plant materials.

Biogeography of the fish pathogen Aeromonas salmonicida inferred by vapA genotyping.

Abstract: A recently described typing system based on sequence variation in the virulence array protein (vapA) gene, encoding the A-layer surface protein array, allows unambiguous subtyping of Aeromonas salmonicida. In the present study, we compile A-layer typing results from a total of 675 A. salmonicida isolates, recovered over a 59-year period from 50 different fish species in 26 countries. Nine novel A-layer types (15-23) are identified, several of which display a strong predilection towards certain fish hosts, including e.g. Cyprinidae and Pleuronectidae species. Moreover, we find indications that anthropogenic transport of live fish may have aided the near global dissemination of two cyprinid-associated A-layer types. Comparison of whole genome phylogeny and A-layer typing for a subset of strains further resulted in compatible tree topologies, indicating the utility of vapA as a phylogenetic as well as an epizootiological marker in A. salmonicida. A Microreact project (microreact.org/project/r1pcOAx9m) has been created, allowing public access to the vapA analyses and relevant metadata. In sum, the results generated provide valuable insights into the global population structure of A. salmonicida, particularly in relation to its piscine host spectrum and the geographic distribution of these hosts.

Short exposure to oxygen and sulfide alter nitrification, denitrification, and DNRA activity in seasonally hypoxic estuarine sediments.

Abstract: Increased organic loading to sediments from eutrophication often results in hypoxia, reduced nitrification and increased production of hydrogen sulfide, altering the balance between nitrogen removal and retention. We examined the effect of short-term exposure to various oxygen and sulfide concentrations on sediment nitrification, denitrification and DNRA from a chronically hypoxic basin in Roskilde Fjord, Denmark. Surprisingly, nitrification rates were highest in the hypoxic and anoxic treatments (about 5 μmol cm-3 d-1) and the high sulfide treatment was not significantly different than the oxic treatment. Denitrification in the hypoxic treatment was highest at 1.4 μmol cm-3 d-1 and significantly higher than the high sulfide treatment. For DNRA, the rate in high sulfide treatment was 2 μmol cm-3 d-1. This was significantly higher than all oxygen treatments that were near zero. In this system, nitrifiers rapidly recovered from conditions typically considered inhibiting, while denitrifiers had a more muted response. DNRA bacteria appear to depend on sulfide for nitrate reduction. Anammox was insignificant. Thus, in estuaries and coastal systems that experience short-term variations in oxygen and sulfide, capabilities of microbial communities are more diverse and tolerant of suboptimal conditions than some paradigms suggest.

The genetic diversity and evolution of diatom-diazotroph associations highlights traits favoring symbiont integration

Abstract: Diatom diazotroph associations (DDAs) are a widespread marine planktonic symbiosis between several diatom genera and di-nitrogen (N2)-fixing bacteria. Combining single cell confocal microscopy observations and molecular genetic approaches on individual field collected cells, we determined the phylogenetic diversity, distribution and evolution of the DDAs. Confocal analyses coupled with 3-D imaging re-evaluated the cellular location of DDA symbionts. DDA diversity was resolved by paired gene sequencing (18S rRNA and rbcL genes, 16S rRNA and nifH genes). A survey using the newly acquired sequences against public databases found sequences with high similarity (99-100%) to either host (18S rRNA) or symbiont (16S rRNA) in atypical regions for DDAs (high latitudes, anoxic basin and copepod gut). Concatenated phylogenies were congruent for the host and cyanobacteria sequences and implied co-evolution. Time-calibrated trees dated the appearance of N2 fixing planktonic symbiosis from 100-50Mya and were consistent with the symbiont cellular location: symbioses with internal partners are more ancient. An ancestral state reconstruction traced the evolution of traits in DDAs and highlight that the adaptive radiation to the marine environment was likely facilitated by the symbiosis. Our results present the evolutionary nature of DDAs and provide new genetic and phenotypic information for these biogeochemically relevant populations.

Outcomes and characterization of chromosomal self-targeting by native CRISPR-Cas systems in Streptococcus thermophilus

Abstract: CRISPR-Cas systems provide adaptive immunity against phages in prokaryotes via DNA-encoded, RNA-mediated, nuclease-dependent targeting and cleavage. Due to inefficient and relatively limited DNA repair pathways in bacteria, CRISPR-Cas systems can be repurposed for lethal DNA targeting that selects for sequence variants. In this study, the relative killing efficiencies of endogenous Type I and Type II CRISPR-Cas systems in the model organism Streptococcus thermophilus DGCC7710 were assessed. Additionally, the genetic and phenotypic outcomes of chromosomal targeting by plasmid-programmed Type I-E or Type II-A systems were analyzed. Efficient killing was observed using both systems, in a dose-dependent manner when delivering 0.4-400 ng of plasmid DNA. Targeted PCR screening and genome sequencing were used to determine the genetic basis enabling survival, showing that evasion of Type I-E self-targeting was primarily the result of low-frequency defective plasmids that excised the targeting spacer. The most notable genotype recovered from Type II-A targeting of genomic locus, lacZ, was a 34 kb-deletion derived from homologous recombination (HR) between identical conserved sequences in two separate galE coding regions, resulting in 2% loss of the genome. Collectively, these results suggest that HR contributes to the plasticity and remodeling of bacterial genomes, leading to evasion of genome targeting by CRISPR-Cas systems.

Linking biosynthetic and chemical space to accelerate microbial secondary metabolite discovery

Abstract: Secondary metabolites can be viewed as a chemical language, facilitating communication between microorganisms. From an ecological point of view, this metabolite exchange is in constant flux due to evolutionary and environmental pressures. From a biomedical perspective, the chemistry is unsurpassed for its antibiotic properties. Genome sequencing of microorganisms has revealed a large reservoir of Biosynthetic Gene Clusters (BGCs); however, linking these to the secondary metabolites they encode is currently a major bottleneck to chemical discovery. This linking of genes to metabolites with experimental validation will aid the elicitation of silent or cryptic (not expressed under normal laboratory conditions) BGCs. As a result, this will accelerate chemical dereplication, our understanding of gene transcription and provide a comprehensive resource for synthetic biology. This will ultimately provide an improved understanding of both the biosynthetic and chemical space. In recent years, integrating these complex metabolomic and genomic data sets has been achieved using a spectrum of manual and automated approaches. In this review, we cover examples of these approaches, while addressing current challenges and future directions in linking these data sets.

Golden Gate-based metabolic engineering strategy for wild-type strains of Yarrowia lipolytica.

Abstract: The yeast Yarrowia lipolytica represents a future microbial cell factory for numerous applications in a bio-based economy. Outstanding feature of this yeast is the metabolic flexibility in utilising various substrates (sugars, fatty acids, glycerol, etc.). The potential of wild-type isolates of Y. lipolytica to convert glycerol into various value-added compounds is attracting attention of academia and industry. However, the already established tools for efficient engineering of the metabolism of Y. lipolytica are often dependent on genetic features like auxotrophic markers. With the present work we want to introduce a new set of vectors for metabolic engineering strategies, including CRISPR/Cas9 technology. The system is based on GoldenMOCS, a recently established rapid Golden Gate cloning strategy applicable in multiple organisms. We could show that our new GoldenMOCS plasmids are suitable for the extrachromosomal overexpression of the gene glycerol kinase (GUT1) in wild-type isolates of Y. lipolytica resulting in enhanced conversion of glycerol to erythritol and citric acid. Moreover, a GoldenMOCS plasmid for CRISPR/Cas9 mediated genome editing has been designed, which facilitates single gene knock-outs with efficiencies between 6% and 25% in strains with genetic wild-type background.

Low-moisture food matrices as probiotic carriers

Abstract: To exert a beneficial effect on the host, adequate doses of probiotics must be administered and maintaining their viability until consumption is thus essential. Dehydrated probiotics exhibit enhanced long-term viability and can be incorporated into low-moisture food matrices, which also possess high stability at refrigeration and ambient temperature. However, several factors associated with the desiccation process, the physicochemical properties of the matrix and the storage conditions can affect probiotic survival. In the near future, an increased demand for probiotics based on functionally dominant members of the gut microbiome ('next-generation probiotics', NGP) is expected. NGPs are very sensitive to oxygen and efficient encapsulation protocols are needed. Strategies to improve the viability of traditional probiotics and particularly of NGPs involve the selection of a suitable carrier as well as proper desiccation and protection techniques. Dehydrated probiotic microcapsules may constitute an alternative to improve the microbial viability during not only storage but also upper gastrointestinal tract passage. Here we review the main dehydration techniques that are applied in the industry as well as the potential stresses associated with the desiccation process and storage. Finally, low- or intermediate-moisture food matrices suitable as carriers of traditional as well as NGPs will be discussed.

Vertical stratification of bacteria and archaea in sediments of a small boreal humic lake.

Abstract: Although sediments of small boreal humic lakes are important carbon stores and greenhouse gas sources, the composition and structuring mechanisms of their microbial communities have remained understudied. We analyzed the vertical profiles of microbial biomass indicators (PLFAs, DNA and RNA) and the bacterial and archaeal community composition (sequencing of 16S rRNA gene amplicons and qPCR of mcrA) in sediment cores collected from a typical small boreal lake. While microbial biomass decreased with sediment depth, viable microbes (RNA and PLFA) were present all through the profiles. The vertical stratification patterns of the bacterial and archaeal communities resembled those in marine sediments with well-characterized groups (e.g. Methanomicrobia, Proteobacteria, Cyanobacteria, Bacteroidetes) dominating in the surface sediment and being replaced by poorly-known groups (e.g. Bathyarchaeota, Aminicenantes and Caldiserica) in the deeper layers. The results also suggested that, similar to marine systems, the deep bacterial and archaeal communities were predominantly assembled by selective survival of taxa able to persist in the low energy conditions. Methanotrophs were rare, further corroborating the role of these methanogen-rich sediments as important methane emitters. Based on their taxonomy, the deep-dwelling groups were putatively organo-heterotrophic, organo-autotrophic and/or acetogenic and thus may contribute to changes in the lake sediment carbon storage.

The role of exploitation in the establishment of mutualistic microbial symbioses.

Abstract: Evolutionary theory suggests that the conditions required for the establishment of mutualistic symbioses through mutualism alone are highly restrictive, often requiring the evolution of complex stabilising mechanisms. Exploitation, whereby initially the host benefits at the expense of its symbiotic partner and mutual benefits evolve subsequently through trade-offs, offers an arguably simpler route to the establishment of mutualistic symbiosis. In this review, we discuss the theoretical and experimental evidence supporting a role for host exploitation in the establishment and evolution of mutualistic microbial symbioses, including data from both extant and experimentally evolved symbioses. We conclude that exploitation rather than mutualism may often explain the origin of mutualistic microbial symbioses.

New CRISPR-Cas9 vectors for genetic modifications of Bacillus species.

Abstract: Genetic manipulation is a fundamental procedure for the study of gene and operon functions and new characteristics acquisition. Modern CRISPR-Cas technology allows genome editing more precisely and increases the efficiency of transferring mutations in a variety of hard to manipulate organisms. Here, we describe new CRISPR-Cas vectors for genetic modifications in bacillary species. Our plasmids are single CRISPR-Cas plasmids comprising all components for genome editing and should be functional in a broad host range. They are highly efficient (up to 97%) and precise. The employment and delivery of these plasmids to bacillary strains can be easily achieved by conjugation from Escherichia coli. During our research we also demonstrated the absence of compatibility between CRISPR-Cas system and non-homologous end joining in Bacillus subtilis.

Molecular host mimicry and manipulation in bacterial symbionts

Abstract: It is common among intracellular bacterial pathogens to use eukaryotic-like proteins that mimic and manipulate host cellular processes to promote colonization and intracellular survival. Eukaryotic-like proteins are bacterial proteins with domains that are rare in bacteria, and known to function in the context of a eukaryotic cell. Such proteins can originate through horizontal gene transfer from eukaryotes or, in the case of simple repeat proteins, through convergent evolution. Recent studies of microbiomes associated with several eukaryotic hosts suggest that similar molecular strategies are deployed by cooperative bacteria that interact closely with eukaryotic cells. Some mimics, like ankyrin repeats, leucine rich repeats and tetratricopeptide repeats are shared across diverse symbiotic systems ranging from amoebae to plants, and may have originated early, or evolved independently in multiple systems. Others, like plant-mimicking domains in members of the plant microbiome are likely to be more recent innovations resulting from horizontal gene transfer from the host, or from microbial eukaryotes occupying the same host. Host protein mimics have only been described in a limited set of symbiotic systems, but are likely to be more widespread. Systematic searches for eukaryote-like proteins in symbiont genomes could lead to the discovery of novel mechanisms underlying host-symbiont interactions.

Evidence for the natural occurrence of Wolbachia in Aedes aegypti mosquitoes.

Abstract: Wolbachia, a Gram-negative bacterium, is widely known to be present in arthropods and nematodes. Of late, great impetus is given to employ this intracellular bacterium, as an alternative to conventional biocontrol agents for the control of mosquitoes because of its inherent ability to induce sperm-egg incompatibility, feminisation etc. By employing molecular tools, we have shown the presence of Wolbachia from Aedes aegypti mosquito population collected from Coimbatore, India by PCR amplifying the Ae. aegypti mosquito genome with Wolbachia specific 16S rRNA, wsp and ftsZ gene primers. The phylogenetic analysis of these gene sequences incorporating MLST and GenBank reference sequences has confirmed the occurrence of Wolbachia supergroup B in Ae. aegypti. In addition, qRT-PCR results have shown the dynamics of Wolbachia across the developmental stages of mosquito. The absence of Wolbachia in tetracycline-treated Ae. aegypti mosquitoes evidenced by transmission electron microscopy reinforced our finding conclusively. After confirming their persistence through generations, we have designated Wolbachia from Ae. aegypti as wAegB. In our considered view, wAegB could play a dynamic role in impeding mosquito multiplication and consequently impinging transmission of the dreadful dengue.

Dark chocolate as a stable carrier of microencapsulated Akkermansia muciniphila and Lactobacillus casei.

Abstract: The viability of probiotics is affected by several factors during manufacturing, storage and gastrointestinal tract passage. Protecting the probiotics from harmful conditions is particularly critical for oxygen sensitive species like Akkermansia muciniphila, a bacterium which recently has been proposed as a next-generation probiotic candidate. Previously, we have developed a protocol for microencapsulating A. muciniphila in a xanthan/gellan gum matrix. Here, we report the enhanced survival during storage and in vitro gastric passage of microencapsulated A. muciniphila embedded in dark chocolate. Lactobacillus casei, as a representative species of traditional probiotics, was included in order to compare its behavior with that of A. muciniphila. For A. muciniphila we observed a 0.63 and 0.87 log CFU g-1 reduction during 60 days storage at 4°C or 15°C, respectively. The viability of L. casei remained stable during the same period. During simulated gastric transit (pH 3), microencapsulated A. muciniphila embedded in chocolate showed 1.80 log CFU mL-1 better survival than naked cells, while for L. casei survival was improved with 0.8 log CFU mL-1. In a hedonic sensory test, dark chocolate containing microcapsules were not significantly different from two commercially available chocolates. The developed protocol constitutes a promising approach for A. muciniphila dosage.

An IS26 variant with enhanced activity.

Abstract: The insertion sequence IS26 plays a major role in the mobilization, expression and dissemination of antibiotic resistance genes in Gram-negative bacteria. Though IS26 is abundant in sequenced genomes and in plasmids that harbour antibiotic resistance genes, only a few minor variations in the IS26 sequence have been recorded. The most common variant, IS26* (also known as IS15Δ1), encodes a Tnp26 transposase with a single amino acid substitution, G184N in the catalytic domain. Using computational modelling, this substitution was predicted to increase the length of the helix that includes the E173 residue of the catalytic DDE triad, and its effect on activity was tested. An IS26 mutant generated in vitro producing Tnp26-G184N formed cointegrates in a standard untargeted reaction at 5-fold higher frequency than IS26 producing Tnp26. When the target included a single copy of IS26, the G184N substitution increased the cointegration frequency 10-fold and the reaction was targeted and conservative. Hence, the substitution increased Tnp26 activity. The longer helix may stabilise the position of the E173 of the DDE for the catalysis reaction and the specific G184N substitution may also enhance activity by increasing binding to the terminal inverted repeats.