Metagenomic analysis of historical herbarium specimens reveals a postmortem microbial community

Advances in DNA extraction and next‐generation sequencing have made a vast number of historical herbarium specimens available for genomic investigation. These specimens contain not only genomic information from the individual plants themselves, but also from associated microorganisms such as bacteria and fungi. These microorganisms may have colonized the living plant (e.g., pathogens or host‐associated commensal taxa) or may result from postmortem colonization that may include decomposition processes or contamination during sample handling. Here we characterize the metagenomic profile from shotgun sequencing data from herbarium specimens of two widespread plant species (Ambrosia artemisiifolia and Arabidopsis thaliana) collected up to 180 years ago. We used blast searching in combination with megan and were able to infer the metagenomic community even from the oldest herbarium sample. Through comparison with contemporary plant collections, we identify three microbial species that are nearly exclusive to herbarium specimens, including the fungus Alternaria alternata, which can comprise up to 7% of the total sequencing reads. This species probably colonizes the herbarium specimens during preparation for mounting or during storage. By removing the probable contaminating taxa, we observe a temporal shift in the metagenomic composition of the invasive weed Am. artemisiifolia. Our findings demonstrate that it is generally possible to use herbarium specimens for metagenomic analyses, but that the results should be treated with caution, as some of the identified species may be herbarium contaminants rather than representing the natural metagenomic community of the host plant.     

Penicillin binding proteins: key players in bacterial cell cycle and drug resistance processes

Bacterial cell division and daughter cell formation are complex mechanisms whose details are orchestrated by at least a dozen different proteins. Penicillin-binding proteins (PBPs), membrane-associated macromolecules which play key roles in the cell wall synthesis process, have been exploited for over 70 years as the targets of the highly successful beta-lactam antibiotics. The increasing incidence of beta-lactam resistant microorganisms, coupled to progress made in genomics, genetics and immunofluorescence microscopy techniques, have encouraged the intensive study of PBPs from a variety of bacterial species. In addition, the recent publication of high-resolution structures of PBPs from pathogenic organisms have shed light on the complex intertwining of drug resistance and cell division processes. In this review, we discuss structural, functional and biological features of such enzymes which, albeit having initially been identified several decades ago, are now being aggressively pursued as highly attractive targets for the development of novel antibiotherapies.     

Examining the fish microbiome: vertebrate-derived bacteria as an environmental niche for the discovery of unique marine natural products

Historically, marine invertebrates have been a prolific source of unique natural products, with a diverse array of biological activities. Recent studies of invertebrate-associated microbial communities are revealing microorganisms as the true producers of many of these compounds. Inspired by the human microbiome project, which has highlighted the human intestine as a unique microenvironment in terms of microbial diversity, we elected to examine the bacterial communities of fish intestines (which we have termed the fish microbiome) as a new source of microbial and biosynthetic diversity for natural products discovery. To test the hypothesis that the fish microbiome contains microorganisms with unique capacity for biosynthesizing natural products, we examined six species of fish through a combination of dissection and culture-dependent evaluation of intestinal microbial communities. Using isolation media designed to enrich for marine Actinobacteria, we have found three main clades that show taxonomic divergence from known strains, several of which are previously uncultured. Extracts from these strains exhibit a wide range of activities against both gram-positive and gram-negative human pathogens, as well as several fish pathogens. Exploration of one of these extracts has identified the novel bioactive lipid sebastenoic acid as an anti-microbial agent, with activity against Staphylococcus aureus, Bacillus subtilis, Enterococcus faecium, and Vibrio mimicus.     

On the origin of prokaryotic "species": the taxonomy of halophilic Archaea

The consistent use of the taxonomic system of binomial nomenclature (genus and species) was first popularized by Linnaeus nearly three-hundred years ago to classify mainly plants and animals. His main goal was to give labels that would ensure that biologists could agree on which organism was under investigation. One-hundred fifty years later, Darwin considered the term species as one of convenience and not essentially different from variety. In the modern era, exploration of the world's niches together with advances in genomics have expanded the number of named species to over 1.8 million, including many microorganisms. However, even this large number excludes over 90% of microorganisms that have yet to be cultured or classified. In naming new isolates in the microbial world, the challenge remains the lack of a universally held and evenly applied standard for a species. The definition of species based on the capacity to form fertile offspring is not applicable to microorganisms and 70% DNA-DNA hybridization appears rather crude in light of the many completed genome sequences. The popular phylogenetic marker, 16S rRNA, is tricky for classification since it does not provide multiple characteristics or phenotypes used classically for this purpose. Using most criteria, agreement may usually be found at the genus level, but species level distinctions are problematic. These observations lend credence to the proposal that the species concept is flawed when applied to prokaryotes. In order to address this topic, we have examined the taxonomy of extremely halophilic Archaea, where the order, family, and even a genus designation have become obsolete, and the naming and renaming of certain species has led to much confusion in the scientific community.     

Monitoring of microbial degraders in manned space stations

Samples of microorganisms from the surface of constructions of Mir Space Station (Mir SS) were taken and examined after 13 years of operation. The following microorganisms were isolated and identified: 12 fungal species belonging to the genera Penicillium, Aspergillus, Cladosporium, and Aureobasidium; 3 yeast species belonging to the genera Debaryomyces, Candida, and Rhodotorula; and 4 bacterial species belonging to the genera Bacillus, Myxococcus, and Rhodococcus. The predominant species in all samples was Penicillium chrisogenum. It was shown that the fungi isolated could damage polymers and induce corrosion of aluminum-magnesium alloys. We commenced a study of microbial degraders on constructions of the Russian section of the International Space Station (RS ISS). Twenty-six species of fungi, bacteria, yeasts, and actinomycetes, known as active biodegraders, were identified in three sample sets taken at intervals. We founded a collection of microorganisms surviving throughout space flights. This collection can be used to test spacecraft production materials, in order to determine their resistance to biodegradation.     

Repertoire of intensive care unit pneumonia microbiota

Despite the considerable number of studies reported to date, the causative agents of pneumonia are not completely identified. We comprehensively applied modern and traditional laboratory diagnostic techniques to identify microbiota in patients who were admitted to or developed pneumonia in intensive care units (ICUs). During a three-year period, we tested the bronchoalveolar lavage (BAL) of patients with ventilator-associated pneumonia, community-acquired pneumonia, non-ventilator ICU pneumonia and aspiration pneumonia, and compared the results with those from patients without pneumonia (controls). Samples were tested by amplification of 16S rDNA, 18S rDNA genes followed by cloning and sequencing and by PCR to target specific pathogens. We also included culture, amoeba co-culture, detection of antibodies to selected agents and urinary antigen tests. Based on molecular testing, we identified a wide repertoire of 160 bacterial species of which 73 have not been previously reported in pneumonia. Moreover, we found 37 putative new bacterial phylotypes with a 16S rDNA gene divergence ≥ 98% from known phylotypes. We also identified 24 fungal species of which 6 have not been previously reported in pneumonia and 7 viruses. Patients can present up to 16 different microorganisms in a single BAL (mean ± SD; 3.77 ± 2.93). Some pathogens considered to be typical for ICU pneumonia such as Pseudomonas aeruginosa and Streptococcus species can be detected as commonly in controls as in pneumonia patients which strikingly highlights the existence of a core pulmonary microbiota. Differences in the microbiota of different forms of pneumonia were documented.     

抗菌光敏剂的分类及研究进展

目前控制细菌和病毒感染性疾病的方法很多,但由于微生物菌株种类越来越多,且耐药微生物菌株不断涌现,已有的治疗手段无法取得良好疗效,因此探索新的抗微生物治疗方法迫在眉睫。光动力抗菌化学疗法是基于光动力疗法的原理,利用光敏剂在异常组织选择性聚集,在分子氧的参与下,由特定波长的光激发产生活性氧,引发一系列的光化学反应,对微生物进行选择性杀伤的一种新方法。光动力抗菌化学疗法对细菌、真菌和病毒引起的感染,特别是耐药菌感染均显示很好的疗效。本文将对光动力抗菌化学疗法中常使用的光敏剂进行分类,并对其研究进展进行综述。     

Implications of bacterial,viral and mycotic microorganisms in vultures for wildlife conservation,ecosystem services and public health

The effects that microorganisms (bacteria, viruses and fungi) have on their hosts remain unexplored for most vulture species. This is especially relevant for vultures, as their diet consists of carcasses in various stages of decomposition, which are breeding grounds for potentially pathogenic microorganisms. Here we review current knowledge of bacterial, viral and mycotic microorganisms present in wild vultures. We consider their potential to cause disease in vultures and whether this poses any population-level threats. Furthermore, we address the question of whether vultures may act as disease spreaders or mitigators. We found 76 articles concerning bacterial, viral and mycotic microorganisms present in 13 vulture species, 57 evaluating bacteria, 13 evaluating viruses and six evaluating mycotic microorganisms. These studies come from all continents where vultures are present, but mainly from Europe and North America, and the most studied species was the Griffon Vulture Gyps fulvus. We found that vultures are colonized by zoonotic pathogens, and even host-specific human pathogens. Some recorded bacteria showed multi-antibiotic resistance, especially those that can be associated with anthropogenic food subsides such as supplementary feeding stations. We found evidence that vulture health can be affected by some microorganisms, producing a wide array of clinical alterations that have the potential to influence mortality risk and fitness. We did not find clear scientific evidence that vultures play an epidemiological role spreading microorganisms to humans and other species. However, there is evidence that vultures could prevent the spread of infectious diseases through their removal of decomposing organic material. The evaluation of vulture exposure to microorganisms is of fundamental importance to design better conservation policies for this threatened group, which may serve a key role as ecosystem cleaners.     

Synergistic plant-microbes interactions in the rhizosphere: a potential headway for the remediation of hydrocarbon polluted soils

Soil pollution is an unavoidable evil; many crude-oil exploring communities have been identified to be the most ecologically impacted regions around the world due to hydrocarbon pollution and their concurrent health risks. Several clean-up technologies have been reported on the removal of hydrocarbons in polluted soils but most of them are either very expensive, require the integration of advanced mechanization and/or cannot be implemented in small scale. However, “Bioremediation” has been reported as an efficient, cost-effective and environment-friendly technology for clean-up of hydrocarbon”s contaminated soils. Here, we suggest the implementation of synergistic mechanism of bioremediation such as the use of rhizosphere mechanism which involves the actions of plant and microorganisms, which involves the exploitation of plant and microorganisms for effective and speedy remediation of hydrocarbon”s contaminated soils. In this mechanism, plant”s action is synergized with the soil microorganisms through the root rhizosphere to promote soil remediation. The microorganisms benefit from the root metabolites (exudates) and the plant in turn benefits from the microbial recycling/solubilizing of mineral nutrients. Harnessing the abilities of plants and microorganisms is a potential headway for cost-effective clean-up of hydrocarbon”s polluted sites; such technology could be very important in countries with great oil producing activities/records over many years but still developing.     

The potential of in situ hybridization and an immunogold assay to identify Legionella associations with other microorganisms.

Based on in vitro studies, bacteria in the genus Legionella are believed to multiply within protozoa such as amoebae in aquatic environments. Current methods used for detection of Legionella species, however, are not designed to show this relationship. Thus the natural intimate association of Legionella with other microorganisms remains to be clearly documented and the extent to which protozoa might be infected with Legionella species remains undefined. In this report we describe methods based on the use of Legionella specific reagents that would prove useful in describing its associations with other microorganisms. An immunogold and in situ hybridization technique have the potential to demonstrate the natural occurrence of Legionella species in free-living amoebae. In preliminary observations, however, bacteria reactive with Legionella specific reagents were often not intimately associated with amoebae. Bacteria occurred as free single cells, as cell aggregates, in proximity to other cells and debris, and only occasionally in close proximity to amoebae. Although some Legionella species replicate within amoebae, these preliminary observations suggest the bacteria may be encountered most frequently as extracellular microorganisms, either free-floating or in association with other structures or microorganisms. The future use of these techniques will aid in the elucidation of any naturally occurring relationships between Legionella species and other microorganisms.     

青海两盐湖细菌多样性研究

用DGGE法和纯培养法,对青海柯柯盐湖、茶卡盐湖底泥及周边土壤样品的细菌多样性进行了研究。结果显示,两个盐湖存在大量的未知细菌, 分离到的纯培养仅占实有细菌的小部分。采用多相分类方法,鉴定了12株纯培养细菌,属5个可能的新种,另有一株菌可能成立一个新属。认为提出新思路、设计新程序,从自然极端环境取样,分离未知菌,是微生物资源开发利用的关键之一。     

Plant-microbe-soil interactions in the rhizosphere: an evolutionary perspective

Soils are the product of the activities of plants, which supply organic matter and play a pivotal role in weathering rocks and minerals. Many plant species have a distinct ecological amplitude that shows restriction to specific soil types. In the numerous interactions between plants and soil, microorganisms also play a key role. Here we review the existing literature on interactions between plants, microorganisms and soils, and include considerations of evolutionary time scales, where possible. Some of these interactions involve intricate systems of communication, which in the case of symbioses such as the arbuscular mycorrhizal symbiosis are several hundreds of millions years old; others involve the release of exudates from roots, and other products of rhizodeposition that are used as substrates for soil microorganisms. The possible reasons for the survival value of this loss of carbon over tens or hundreds of millions of years of evolution of higher plants are discussed, taking a cost-benefit approach. Co-evolution of plants and rhizosphere microorganisms is discussed, in the light of known ecological interactions between various partners in terrestrial ecosystems. Finally, the role of higher plants, especially deep-rooted plants and associated microorganisms in the weathering of rocks and minerals, ultimately contributing to pedogenesis, is addressed. We show that rhizosphere processes in the long run are central to biogeochemical cycles, soil formation and Earth history. Major anticipated discoveries will enhance our basic understanding and allow applications of new knowledge to deal with nutrient deficiencies, pests and diseases, and the challenges of increasing global food production and agroecosystem productivity in an environmentally responsible manner.     

原核微生物之杆菌和小杆菌

原核微生物的发现已有约340年,成千上万的原核微生物被发现和命名。近年来,随着分子生物学技术的发展,众多新的种属已经得到鉴定和命名,对这些微生物系统的总结并对中文命名系统适当调整已迫在眉睫。本文试图对人类发现最早、总量最多的350多属1 500多种杆菌和120多属1 200多种小杆菌进行总结和归纳,从生境、形态学(动静态、形状和尺寸)、生物化学、细菌和古菌属性等特征层次规范这类菌属的命名,期盼有助于推动国内外微生物分类学发展,有利于推动国际交流与合作。     

Lack of host specificity leads to independent assortment of dipterocarps and ectomycorrhizal fungi across a soil fertility gradient

Plants interact with a diversity of microorganisms, and there is often concordance in their community structures. Because most community‐level studies are observational, it is unclear if such concordance arises because of host specificity, in which microorganisms or plants limit each other's occurrence. Using a reciprocal transplant experiment, we tested the hypothesis that host specificity between trees and ectomycorrhizal fungi determines patterns of tree and fungal soil specialisation. Seedlings of 13 dipterocarp species with contrasting soil specialisations were seeded into plots crossing soil type and canopy openness. Ectomycorrhizal colonists were identified by DNA sequencing. After 2.5 years, we found no evidence of host specificity. Rather, soil environment was the primary determinant of ectomycorrhizal diversity and composition on seedlings. Despite their close symbiosis, our results show that ectomycorrhizal fungi and tree communities in this Bornean rain forest assemble independently of host‐specific interactions, raising questions about how mutualism shapes the realised niche.     

All together now: experimental multispecies biofilm model systems

Studies of microorganisms have traditionally focused on single species populations, which have greatly facilitated our understanding of the genetics and physiology that underpin microbial growth, adaptation and biofilm development. However, given that most microorganisms exist as multispecies consortia, the field is increasingly exploring microbial communities using a range of technologies traditionally limited to populations, including meta‐omics based approaches and high resolution imaging. The experimental communities currently being explored range from relatively low diversity, for example, two to four species, to significantly more complex systems, comprised of several hundred species. Results from both defined and undefined communities have revealed a number of emergent properties, including improved stress tolerance, increased biomass production, community level signalling and metabolic cooperation. Based on results published to date, we submit that community‐based studies are timely and increasingly reveal new properties associated with multispecies consortia that could not be predicted by studies of the individual component species. Here, we review a range of defined and undefined experimental systems used to study microbial community interactions.     

天山北坡甜菜内生菌分离鉴定及其动态变化

对新疆昌吉和石河子两地种植的甜菜内生菌进行了分离、鉴定和分析,结果表明甜菜内生菌多属于细菌,其中假单胞菌 (Pseudomonas sp. )和芽孢菌类(Bacillus sp.)的分离频率分别在33.2%～59.2%和12.7%～28.1%,是甜菜植株中的优势内生菌群.16S rDNA 和 ITS 序列同源性比较和系统发育分析表明内生菌具有丰富的多样性.根中内生菌的多样性高于茎、叶,昌吉地区种植的甜菜中分离出的内生菌种类较多.从感病品种及生长不良甜菜植株中分离出的内生菌种类比较丰富.通过回接分离及利用扫描电镜观察内生菌在植物体内分布发现,内生菌能够定殖于甜菜块根.     

Wolbachia endosymbionts responsible for various alterations of sexuality in arthropods.

Rickettsia-like maternally inherited bacteria have been shown to be involved in a variety of alterations of arthropod sexuality, such as female-biased sex ratios, parthenogenesis, and sterility of crosses either between infected males and uninfected females or between infected individuals (cytoplasmic incompatibility). We have characterized several of these microorganisms through partial sequences of the small (16S) and large (23S) subunit ribosomal DNA. All the symbionts identified, which include several cytoplasmic incompatibility microorganisms, several endosymbionts of terrestrial isopods, and symbionts of two thelytokous Trichogramma wasp species, belong to a monophyletic group of related symbionts, some of which have previously been detected in several insects exhibiting cytoplasmic incompatibility. Three molecular lineages can be identified on the basis of 16S as well as 23S sequences. Although they are only known as endocellular symbionts, Wolbachia spread by horizontal transfer across host lineages as evidenced by their diversification which occurred long after that of their hosts, and by the non-congruence of the phylogenetic relationships of symbionts and their hosts. Indeed, symbionts of two different lineages have been found in the same host species, whereas closely related endosymbionts are found in distinct insect orders. Isopod endosymbionts form a separate lineage, and they can determine feminization as well as cytoplasmic incompatibility. The ability to determine cytoplasmic incompatibility, found in all lineages, is probably ancestral to this group.     

Metagenomic profiling: microarray analysis of an environmental genomic library

Genomic libraries derived from environmental DNA (metagenomic libraries) are useful for characterizing uncultured microorganisms. However, conventional library-screening techniques permit characterization of relatively few environmental clones. Here we describe a novel approach for characterization of a metagenomic library by hybridizing the library with DNA from a set of groundwater isolates, reference strains, and communities. A cosmid library derived from a microcosm of groundwater microorganisms was used to construct a microarray (COSMO) containing approximately 1-kb PCR products amplified from the inserts of 672 cosmids plus a set of 16S ribosomal DNA controls. COSMO was hybridized with Cy5-labeled genomic DNA from each bacterial strain, and the results were compared with the results for a common Cy3-labeled reference DNA sample consisting of a composite of genomic DNA from multiple species. The accuracy of the results was confirmed by the preferential hybridization of each strain to its corresponding rDNA probe. Cosmid clones were identified that hybridized specifically to each of 10 microcosm isolates, and other clones produced positive results with multiple related species, which is indicative of conserved genes. Many clones did not hybridize to any microcosm isolate; however, some of these clones hybridized to community genomic DNA, suggesting that they were derived from microbes that we failed to isolate in pure culture. Based on identification of genes by end sequencing of 17 such clones, DNA could be assigned to functions that have potential ecological importance, including hydrogen oxidation, nitrate reduction, and transposition. Metagenomic profiling offers an effective approach for rapidly characterizing many clones and identifying the clones corresponding to unidentified species of microorganisms.     

Metagenomic Profiling: Microarray Analysis of an Environmental Genomic Library

Genomic libraries derived from environmental DNA (metagenomic libraries) are useful for characterizing uncultured microorganisms. However, conventional library-screening techniques permit characterization of relatively few environmental clones. Here we describe a novel approach for characterization of a metagenomic library by hybridizing the library with DNA from a set of groundwater isolates, reference strains, and communities. A cosmid library derived from a microcosm of groundwater microorganisms was used to construct a microarray (COSMO) containing ~1-kb PCR products amplified from the inserts of 672 cosmids plus a set of 16S ribosomal DNA controls. COSMO was hybridized with Cy5-labeled genomic DNA from each bacterial strain, and the results were compared with the results for a common Cy3-labeled reference DNA sample consisting of a composite of genomic DNA from multiple species. The accuracy of the results was confirmed by the preferential hybridization of each strain to its corresponding rDNA probe. Cosmid clones were identified that hybridized specifically to each of 10 microcosm isolates, and other clones produced positive results with multiple related species, which is indicative of conserved genes. Many clones did not hybridize to any microcosm isolate; however, some of these clones hybridized to community genomic DNA, suggesting that they were derived from microbes that we failed to isolate in pure culture. Based on identification of genes by end sequencing of 17 such clones, DNA could be assigned to functions that have potential ecological importance, including hydrogen oxidation, nitrate reduction, and transposition. Metagenomic profiling offers an effective approach for rapidly characterizing many clones and identifying the clones corresponding to unidentified species of microorganisms.     

A Novel Nutritional Predictor Links Microbial Fastidiousness with Lowered Ubiquity,Growth Rate,and Cooperativeness

Understanding microbial nutritional requirements is a key challenge in microbiology. Here we leverage the recent availability of thousands of automatically generated genome-scale metabolic models to develop a predictor of microbial minimal medium requirements, which we apply to thousands of species to study the relationship between their nutritional requirements and their ecological and genomic traits. We first show that nutritional requirements are more similar among species that co-habit many ecological niches. We then reveal three fundamental characteristics of microbial fastidiousness (i.e., complex and specific nutritional requirements): (1) more fastidious microorganisms tend to be more ecologically limited; (2) fastidiousness is positively associated with smaller genomes and smaller metabolic networks; and (3) more fastidious species grow more slowly and have less ability to cooperate with other species than more metabolically versatile organisms. These associations reflect the adaptation of fastidious microorganisms to unique niches with few cohabitating species. They also explain how non-fastidious species inhabit many ecological niches with high abundance rates. Taken together, these results advance our understanding microbial nutrition on a large scale, by presenting new nutrition-related associations that govern the distribution of microorganisms in nature.