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Phlegenetic identification and situ detection of individual microbial cell without cultivation
Abstract
The frequent discrepancy between direct microscopic counts and numbers of culturable bacteria from environmental samples is just one of several indications that we currently know only a minor part of the diversity of microorganisms in nature. A combination of direct retrieval of rRNA sequences and whole-cell oligonucleotide probing can be used to detect specific rRNA sequences of uncultured bacteria in natural samples and to microscopically identify individual cells. Studies have been performed with microbial assemblages of various complexities ranging from simple two-component bacterial endosymbiotic associations to multispecies enrichments containing magnetotactic bacteria to highly complex marine and soil communities. Phylogenetic analysis of the retrieved rRNA sequence of an uncultured microorganism reveals its closest culturable relatives and may, together with information on the physicochemical conditions of its natural habitat, facilitate more directed cultivation attempts. For the analysis of complex communities such as multispecies biofilms and activated-sludge flocs, a different approach has proven advantageous. Sets of probes specific to different taxonomic levels are applied consecutively beginning with the more general and ending with the more specific (a hierarchical top-to-bottom approach), thereby generating increasingly precise information on the structure of the community. Not only do rRNA-targeted whole-cell hybridizations yield data on cell morphology, specific cell counts, and in situ distributions of defined phylogenetic groups, but also the strength of the hybridization signal reflects the cellular rRNA content of individual cells. From the signal strength conferred by a specific probe, in situ growth rates and activities of individual cells might be estimated for known species. In many ecosystems, low cellular rRNA content and/or limited cell permeability, combined with background fluorescence, hinders in situ identification of autochthonous populations. Approaches to circumvent these problems are discussed in detail.
... Consequently, Earth's vast microbiome represents an incredible resource for the discovery of new biochemical pathways, enzymes, or metabolites. However, exploring this genetic resource can be challenging as only 1%-15% of microorganisms can be cultivated in the laboratory (5,6). To overcome this challenge, the field of metagenomics uses culture-independent methods whereby microbial DNA is directly isolated from an ecosystem sample [referred to as environmental DNA (eDNA)] (7,8). ...
Fosmids and cosmids are vectors frequently used in functional metagenomic studies. With a large insert capacity (around 30 kb) they can encode dozens of cloned genes or in some cases, entire biochemical pathways. Fosmids with cloned inserts can be transferred to heterologous hosts and propagated to enable screening for new enzymes and metabolites. After screening, fosmids from clones with an activity of interest must be de novo sequenced, a critical step toward the identification of the gene(s) of interest. In this work, we present a new approach for rapid and high-throughput fosmid sequencing directly from Escherichia coli colonies without liquid culturing or fosmid purification. Our sample preparation involves fosmid amplification with phi29 polymerase and then direct nanopore sequencing using the Oxford Nanopore Technologies system. We also present a bioinformatics pipeline termed “phiXXer” that facilitates both de novo read assembly and vector trimming to generate a linear sequence of the fosmid insert. Finally, we demonstrate the accurate sequencing of 96 fosmids in a single run and validate the method using two fosmid libraries that contain cloned large insert (~30–40 kb) genomic or metagenomic DNA.
IMPORTANCE
Large-insert clone (fosmids or cosmids) sequencing is challenging and arguably the most limiting step of functional metagenomic screening workflows. Our study establishes a new method for high-throughput nanopore sequencing of fosmid clones directly from lysed Escherichia coli cells. It also describes a companion bioinformatic pipeline that enables de novo assembly of fosmid DNA insert sequences. The devised method widens the potential of functional metagenomic screening by providing a simple, high-throughput approach to fosmid clone sequencing that dramatically speeds the pace of discovery.
... For soil samples, algae should be cultured before microscopic observation. However, some algal groups can not be cultured due to the preference of the culture medium (Amann et al., 1995;Novakovskaya et al., 2020). Nowadays, molecular sequencing was applied to identify the algal community structure instead of microscopic observation (Satjarak et al., 2020). ...
Algae exert great impact on soil formation and biogeochemical cycling. However, there is no full understanding of the response of soil algal community structure to the seasonal fluctuations in temperature and moisture and changes of soil physicochemical properties across different forests. Here, based on 23S rRNA gene sequencing, we analyzed soil algal community structure in four different forest plantations in two seasons and examined soil physiochemical properties. The results showed the significantly seasonal variation in soil algal community structure, with the higher overall diversity in summer than in winter. In addition, there existed significant correlations between soil algae (species composition, relative abundance, diversity index) and physicochemical properties (pH, total phosphorus, organic matter and nitrate nitrogen), suggesting that edaphic characteristics are also largely responsible for the variation in soil algal community. Nevertheless, the seasonal variation in algal community structure was greater than the variation across different forest plantations. This suggest temperature and moisture are more important than soil physicochemical properties in determining soil algal community structure. The findings of the present study enhance our understanding of the algal communities in forest ecosystems and are of great significance for the management and protection of algal ecosystem.
... Traditionally, it has been difcult to obtain pure cultures of microorganisms that are in symbiotic relationships under natural conditions (for instance, extracting 5S rRNA molecules from mixed samples and analyzing the sequences to determine their phylogenetic position) [16], and enrichment and separation cultures have high selectivity [17]. It has become complicated to study the structure of fsh intestinal fora using the traditional method because it fails to refect the real situation of the microbial community in the natural state. ...
Nutrition, disease, and general wellbeing can be affected by the microbial communities associated with the digestive tracts of aquaculture species. Different sections of aquaculture species’ digestive tracts have distinctive surfaces and structures, which can change microbial communities. The present study examined the composition and distribution of bacterial species in the intestine of hybrid grouper (Epinephelus fuscoguttatus E. polyphekadion) and its aquaculture environment. Using high-throughput pyrosequencing, a 16S rRNA sequence analysis was performed on hybrid grouper foregut, midgut, and hindgut, as well as cultured water and feed. There were 610,452 sequences obtained from five components (foregut, midgut, hindgut, water, and feed). Among operational taxa (OTUs), 506 of them were detected in the foregut, 605 in the midgut, 510 in the hindgut, and 573 in aquaculture water and feed samples. A total of 113 were detected in 5 samples. A species annotation revealed that hybrid grouper intestinal tracts were dominated by Proteobacteria (67.3%–73.7%), Firmicutes (8.4%–14.0%), and Actinobacteria (6.9%–10.5%). In aquaculture culture water, Proteobacteria were predominant (36.3%), Actinobacteria (30.0%), and Planctomycetes (14.0%). Acinetobacter (1.4%–17.9%) and Photobacterium (32.0%–57.5%) dominated the intestine. Photobacterium (3.6%) and Mycobacterium (7.1%) dominated the water bacteria. The water and intestine contained five potentially pathogenic bacteria: Pseudomonas, Flavobacterium, Escherichia coli, Aeromonas bacteria, and Vibrio. The highest proportion of Vibrio was found in the water (1.7%), while Pseudomonas dominated the midgut (2.6%). Six potential probiotics were detected in the aquaculture water and intestine (Lactococcus, Streptococcus, Bdellovibrio, Lactobacillus, Bacillus, and Bacteroides). Aquaculture water and intestines contained Bacillus, Bacteroides, and Lactobacillus. According to the findings, the intestinal flora of hybrid grouper is closely correlated with its pond culture environment. Results from the study provide an experimental basis for the controlled breeding of hybrid groupers and the regulation of their microecological processes in the breeding environment deepen our understanding of the intestinal bacterial population of healthy hybrid groupers.
... The term metagenomics, coined in 1998 by Handelsman et al. [74], it has been recently redefined by Chen and Pachter as "the application of modern genomics technique without the need for isolation and laboratory cultivation of individual species" [75]. In fact, these studies revealed that usually less than 1% of microorganisms from natural sources could be cultivated under laboratory conditions [76], and that the uncultured species not only constitute a major part of the microbial communities, but they could also perform key functions in ecological processes. Therefore, despite that metagenomics is a relatively new but fast-growing field within biology, it is intended to be a priority analysis for the purpose of acquiring knowledge on genomes of environmental microbes, as well as of entire microbial communities. ...
In this review, the composting process of organic waste is discussed through an in-depth exploring of its thermophilic phase. It starts with the highlight on the thermodynamic evolution, which needs to be assessed when deciding to use reactors for composting, also in the context of energy generation. The composting process is mediated by different types of microorganisms, and the bacteria that play key roles are evaluated. The roles of the genera Bacillus and Thermus are considered, often described as the main components of the microbiota of compost. Due to their adaptation to the composting processes, they are candidates for technological purposes. Subsequentially, the focus is moved on the thermostable enzymes that can be isolated from them and their succession during the composting processes. Experimental examples of enzyme-related literature are reviewed, for example investigating proteases and ureases, which are found at the beginning of the process. In addition, cellulases, hemicellulases, lignin-modifying enzymes, and esterases have been described for their activities during the thermophilic phase, giving them great potential for biotechnological and industrial applications. Following, the composition of the microbial community is analyzed through the description of approaches of metagenomics. Despite it being a relatively new but fast-growing field within biology, it is intended to be a priority analysis to acquire knowledge on genomes of environmental microorganisms and communities. Finally, a space is dedicated to the description of the composting plant which treats olive oil wastes within the LIFE TIRSAV PLUS project (LIFE05 ENV/IT/00845). Through two plant solutions, being the Dynamic and the Static Composting, it provides a high-quality compost with an effective, flexible and economical process.
Graphical Abstract
... Real-time PCR was performed to quantify partial bacterial (16S rDNA) and fungal (18S rDNA) genes coding for ribosomal RNA, and gene phaZ (coding for polyhydroxybutyrate depolymerase) in soil DNA extracts. The primers used were 1108F (5′ ATG GYT GTC GTC AGC TCG TG 3′) and 1132R (5′ GGG TTG CGC TCG TTGC 3′) for bacteria [33], FF390 (5′ AICCA TTC AAT CGG TAIT 3′) and FR1 (5′ CGA TAA CGA ACG AGA CCT 3′) for fungi [34], PHBf (5′ CGT CTA CCG CAA CGG CAC CAAGG 3′) and ...
Background
Poly-3-hydroxybutyrate (P3HB) is a bacterial intracellular carbon and energy storage polymer, used as a thermoplastic polyester in a wide array of industrial and agricultural applications. However, how the soil microbiome and fertility are altered by exogenously applied P3HB has been relatively unexplored. This study aimed to assess the effects of P3HB addition to nutrient restricted soil: its biological properties and lettuce (Lactuca sativa L. var. capitata L.) biomass production. The experiment was designed to evaluate impacts of spatial arrangement of the relatively organic-rich (soil organic matter, P3HB particles) versus poor fractions of the matrix with confounding factors such as variable microbial biomass, inherent nutrient/energy status, different water relations (due to variable hydrophysical properties of soil augmented by sand at different ratios).
Results
The results revealed that P3HB in soils induced inconsistent to contradictory changes in the microbial abundance as well as in most enzymatic activities. The differences were conditioned by the sand content both under P3HB presence or absence. On the other hand, dehydrogenase, urease activities, basal and substrate-induced soil respirations were mostly enhanced by P3HB addition, directly with increasing sand content (several respiration types). Nevertheless, P3HB significantly inhibited lettuce biomass production.
Conclusions
P3HB introduction to soil boosts the microbial activity owing to the preferential utilization of P3HB as C source, which depletes soil N and strongly inhibits the plant growth. Enhanced microbial activity in P3HB-amended soils with high sand content (60–80%) suggested that in nutrient-impoverished soil P3HB can temporarily replace SOM as a C source for microbial communities due to the shift of their structure to preferentially P3HB-degrading microbiome.
Graphical Abstract
... In contrast, samples with a very low bacterial and archaeal fraction (e.g., S300µm, coral, and fish mucus), needed a protocol adjustment. In this case, we implemented a nested PCR performing a first full-length amplification using the 27F / 1492R 16S universal primer set25,26 in order to increase the target DNA, and a second amplification using the 515F-Y / 926R primers [Data citations #6]. -Specific primers of Symbiodiniaceae targeting the ITS2 region of the nuclear ribosomal DNA locus (SYM-VAR-5.8S2 ...
Coral reef science is a fast-growing field propelled by the need to better understand coral health and resilience to devise strategies to slow reef loss resulting from environmental stresses. Key to coral resilience are the symbiotic interactions established within a complex holobiont, i.e. the multipartite assemblages comprising the host coral organism, endosymbiotic dinoflagellates, bacteria, archaea, fungi, and viruses. Tara Pacific is an ambitious project built upon the experience of previous Tara Oceans expeditions, and leveraging state-of-the-art sequencing technologies and analyses to dissect the biodiversity and biocomplexity of the coral holobiont screened across most archipelagos spread throughout the entire Pacific Ocean. Here we detail the Tara Pacific workflow for multi-omics data generation, from sample handling to nucleotide sequence data generation and deposition. This unique multidimensional framework also includes a large amount of concomitant metadata collected side-by-side that provide new assessments of coral reef biodiversity including micro-biodiversity and shape future investigations of coral reef dynamics and their fate in the Anthropocene.
... The health of the aquaculture environment is closely related to the microbial community structure in the water, as well as the levels of nutrients [3]. Microbial community structure, which reflects the biochemical reactions in the ecosystem, can be used as an indicator of the ecological health of the aquaculture environment [4]. Because aquaculture species must be fed constantly, native microorganisms cannot decompose organic matter rapidly enough to prevent water quality deterioration and the growth of harmful microorganisms [5]. ...
The study aimed to evaluate the safety of copper ion sterilization based on copper ion residues in zebrafish ( Brachydanio rerio ), as well as bacterial community structure and diversity in recirculating aquaculture systems (RASs). The copper ion content was determined using national food safety standard GB 5009.13-2017. Bacterial community structures and alpha and beta diversity indexes were examined using the 16S rRNA gene sequences produced by Illumina HiSeq sequencing. The results revealed no significant copper ion enrichment in B. rerio when the copper ion concentration was 0.15 mg/L. The relative abundances of Erythrobacter , nitrite bacteria, and Flavanobacteria were clearly higher in the treatment group than in the control and differences in bacterial species richness and diversity were obvious. In addition, there was no sharp decrease in the microflora at the outflow of the copper ion generator. In conjunction with the changes in ammonia nitrogen, nitrate, and nitrite concentrations during the experiment, the results indicated that there were no significant effects on the purification efficacy of the biological filter, but the abundances of beneficial bacteria increased significantly. This is of great relevance in order to understand the response of bacterial communities affected by changing environmental conditions, such as copper ion sterilization.
... K6 in the re-attachment experiments with purified isolates, but no indicator ASV was assigned to this genus in our results. Thus, this confirms that nemato-bacterial interactions in different soils are very complex, and the estimation that only 1% of soil bacteria are culturable (Amann et al., 1995), adds up to this complexity. ...
Nematode-suppressive soils are characterized by the ability of soil microbial communities to reduce populations of plant-parasitic nematodes (PPN) either directly or by inducing systemic resistance in plants. Various microorganisms have been recognized as antagonists of PPN in suppressive soils using culture-dependent and culture-independent methods. However, the associations that PPN form with microorganisms in nematode-conducive soils have been poorly studied. Here we drenched tomato rhizospheres with microbial suspensions from nine different soils and followed their effects on plant growth and root invasion of the infective second-stage juveniles (J2) of the northern root-knot nematode species Meloidogyne hapla. Based on the number of invaded J2, four soils were determined as nematode-suppressive, while five soils were categorized as nematode-conducive. To reveal bacteria attached to the J2 cuticle in soils with varying suppressiveness, we incubated J2 in suspensions from three suppressive and three conducive soils, and analyzed J2-attached bacteria using amplicon sequencing of the V3-V4 region of 16S rRNA gene. Our results suggest that the soil origin had a major effect on the composition of J2-attached bacteria, while the highest bacterial abundance and richness were observed on J2 from two sup-pressive soils. In addition, the highest number of indicator amplicon sequence variants (ASVs) was associated with J2 in two suppressive soils, but they had a very distinct bacterial profile. Further studies are needed to resolve the complexity of nemato-microbial interactions in soil and to determine the exact function of nematode-attached microorganisms in suppressive and conducive soils and their role in nematode suppression and protection.
... Microorganisms are smaller in sizes, but they contribute to the nutrient cycling in the ecosystem enabling specific biochemical function of the different forms of life in a community (Whitman et al., 1998). Under natural condition, microorganisms form complex metabolic network and each individual member contributes to one specific small step in the overall biochemical transformation to achieve energy conservation for each member to growth (Amann et al., 1995). Among microorganisms, they sustain their own life cycle in the ecosystem independently for the specific step and also, in doing so, support the community by association with others through a number of relationships, including symbiotic, syntrophy, commensualism, antagonism, and mutualism etc. ...
Microorganisms play an important role in cycling of elements of ecosystems, including a wide range of chemical pollutants from anthropogenic origin. These pollutants in ecosystems, particularly aquatic, and sediment and soils, are in different physical and chemical forms in association with the inorganic and organic constituents of the sediment and soils, resulting in variable availability of them to microorganisms for assimilation and transformation. A thorough and comprehensive knowledge of the physical and chemical states of them in the environments requires detailed information of both the bioavailable pollutant concentration and also the metabolic capability of the microorganisms to assess the ecological and environmental toxicity of these pollutants meaningfully. Apart from the primary role as decomposers, microorganisms are qualified to be sensitive indicators for environmental pollution, and ecological health and ecotoxicity of pollutants because of their very short generation time and quickly response to chemical pollutants than higher and large organisms. When used for testing with the same strain, different laboratories can generate high reproducible results to allow comparison of the data feasible, not mention the reduction in cost. Based on the current advances made on genomics analysis and bioinformatics, microbial genomes are easily assembled with the technologies available to providing useful transcriptomic and metabolic annotations, expression and prediction to allow advance toxicological to another level.
... Previous studies have demonstrated that Bacillus subtilis [2], Haemophilus influenza and Streptococcus pneumonia [3]could uptake exogenous DNA naturally. However, the vast majority of prokaryotes (>99%) in natural environments are not culturable [4], and therefore could not show a natural transformation ability. Even if prokaryotes can be cultured in vitro, genetic manipulation is frequently impeded because of the lack of efficient, non-invasive and simple methods for DNA delivery. ...
As a basic technique of molecular cloning, bio-transformation has been successfully used in the fields of biomedicine and food processing. In this study, we established a transformation system of exogenous DNA into E. coli cells mediated by ultrasound. Under the optimal conditions (i.e. 35 °C, 40 W, 25 s, OD600 = 0.4–0.6) optimized by RSM, the transformation efficiency reached at 1.006 × 10⁷ CFU/μg DNA. The results of membrane permeability, macromolecular substance and cell structure analysis before and after ultrasound treatment showed that the damage of host cells induced by lower (40 W) ultrasound and shorter ultrasound time (25 s) was reversible, and the transformation efficiency and cell survival rate were not significantly affected under this condition. In brief, proper changes in cell membrane and cell wall were the basic conditions for host cells to uptake exogenous DNA, while, whether exogenous DNA could be replicated and expressed in cells depends on the viability of host cells.
... mycoides, Bacillus unidentified, Klebsiella and Pseudomonas spp(Table 1). It was earlier reported by several microbiologist that the reported microbes are pathogenic and pose a threat in NICU(6,7). ...
... mycoides, Bacillus unidentified, Klebsiella and Pseudomonas spp(Table 1). It was earlier reported by several microbiologist that the reported microbes are pathogenic and pose a threat in NICU(6,7). ...
... Detection of bacterial community of rhizosphere and bulk soils at different habitats by culture-dependent method Cultivation-based methods only address the culturable bacteria [43], which are considered to represent only a small proportion (0.1-10%) of the total bacteria presented in soil and rhizosphere [44]. Although the cultivable way can't obtain all of the bacterial groups, it also fully predicts the dominant group in the bacterial community [43]. ...
Background:
Minqin is suffering from a serious desertification, whereas the knowledge about its bacterial community is limited. Herein, based on Nitraria tangutorum and Haloxylon ammodendron from Minqin, the bacterial community diversities in fixed sandy land, semi-fixed sandy land and shifting sandy land were investigated by combining with culture-dependent and culture-independent methods.
Results:
Minqin stressed with high salinity and poor nutrition is an oligotrophic environment. Bacterial community in Minqin was shaped primarily by the presence of host plants, whereas the type of plant and sandy land had no marked effect on those, which displayed a better survival in the rhizospheres of N. tangutorum and H. ammodendron. The dominant groups at phyla level were Actinobacteria, Firmicutes, Proteobacteria, Bacteroidetes, Planctomycetes, Chloroflexi, Acidobacteria and Candidate_division_TM7. The abundance of Firmicutes with ability of desiccation-tolerance was significantly higher in harsh environment, whereas Bacteroidetes were mainly distributed in areas with high nutrient content. The abundances of Proteobacteria and Bacteroidetes were relatively high in the rhizospheres of N. tangutorum and H. ammodendron, which had more plant-growth promoting rhizobacteria. A large number of Actinobacteria were detected, of which the most abundant genus was Streptomyces. The physicochemical factors related to the diversity and distribution of the bacterial community were comprehensively analyzed, such as pH, electrical conductivity, soil organic matter, C/N and sand, and the results indicated that Minqin was more suitable for the growth of N. tangutorum, which should be one of most important sand-fixing plants in Minqin.
Conclusions:
The bacterial community diversities in different types of sandy lands of Minqin were comprehensively and systematically investigated by culture-dependent and culture-independent approaches, which has a great significance in maintaining/restoring biological diversity.
... RNA quantification results also depend on the RNA structure. When targeting highly structured RNA, annealing of the ssDNA and inhibition of reverse transcriptase activity occur; thus, an accurate reverse transcription cannot be performed [26,27,28]. In addition, when targeting short RNAs, such as siRNA, it is difficult to perform an accurate RNA quantification because it is easily affected by degradation, and the reverse transcription efficiency is also reduced [29,30]. ...
Ribonucleic acid (RNA) quantification is an essential technique in biology. There has been remarkable progress in RNA quantification techniques over the recent years; however, the specificity of these techniques to quantify a very small amount of RNA is doubtful because of factors which can inhibit precise quantification. To develop a technique that leads to the most sensitive RNA quantification, these problems must be overcome. In this article, we first review the factors that inhibit precise quantification of RNA: the quality of RNA, secondary structure of RNA, efficiency of the enzyme reaction, annealing conditions, limitations of the experimental protocol and equipment, and detection sensitivity of the equipment. Next, we discuss the possible methods which contribute to these factors: RNA quality control focused on target RNA degradation, isothermal amplification, techniques for avoiding amplification errors, RNase H-dependent PCR, targeting using a fluorescent-labeled probe, targeting using a padlock probe, bridged/locked nucleic acid (BNA/LNA) and peptide nucleic acid (PNA), and the clustered regularly interspaced short palindromic repeat (CRISPR) system. One of the goals for the development of an ultrasensitive RNA quantification technique is the absolute quantification of RNA. Here, we discuss the techniques used for this type of RNA quantification.
Ambrosia gall midges (AGMs) are mostly host plant-specific. In their galls, they harbor fungal symbionts on which they feed. Therefore, they represent unique steps in the evolution of the gall-forming Cecidomyiidae (Diptera). Gall-associated fungi have been studied predominantly by cultivations, and potential larval endosymbionts have been completely neglected. Using ITS2 rRNA metabarcoding, we characterized the mycobiomes of individual gall compartments (gall surface, gall interior, and larva) of six species from two phylogenetically separated tribes (Asphondyliini and Lasiopterini). Compared to the gall surface and interior, the larvae harbored significantly higher fungal richness and taxonomic diversity, and a larger pool of indicator taxa. Larval mycobiome composition was more species-specific; however, the fungal genera Fusarium, Filobasidium, Tilletiopsis, Alternaria, and Aureobasidium were indicator taxa shared among species. Overall, the larvae harbored 29% of unique taxa that can play a functional role in the host (e.g., initiation of gall development or selection of the mycelia composition). The mycobiome of the gall interior was assembled least stochastically, and its composition was the least species-specific, being dominated by Botryosphaeria dothidea (except for Lasioptera arundinis). Therefore, the interior of ambrosia galls offers a unique environment that supports the growth of similar fungi, regardless of the host plant species and the phylogenetic distance between the AGM tribes. Our study illustrates a range of fungal microorganisms indicative of individual gall compartments, but their potential function, especially in larvae, remains to be solved.
IMPORTANCE
Ambrosia gall midges are endophagous insect herbivores whose larvae live enclosed within a single gall for their entire development period. They may exhibit phytomycetophagy, a remarkable feeding mode that involves the consumption of plant biomass and mycelia of their cultivated gall symbionts. Thus, AGMs are ideal model organisms for studying the role of microorganisms in the evolution of host specificity in insects. However, compared to other fungus-farming insects, insect–fungus mutualism in AGMs has been neglected. Our study is the first to use DNA metabarcoding to characterize the complete mycobiome of the entire system of the gall-forming insects as we profiled gall surfaces, nutritive mycelia, and larvae. Interestingly, larval mycobiomes were significantly different from their nutritive mycelia, although Botryosphaeria dothidea dominated the nutritive mycelia, regardless of the evolutionary separation of the tribes studied. Therefore, we confirmed a long-time hypothesized paradigm for the important evolutionary association of this fungus with AGMs.
The bacterial community structure in biological activated carbon (BAC) process in drinking water treatment plant was investigated by Fluorescent in situ Hybridization (FISH) with rRNA-targeted oligonucleotide probe. Samples were collected at different three points in BAC process every month for one year. They were hybridized with a probe specific for the alpha, beta, gamma subclass of the class Proteobacteria, Cytophaga-Flavobacteria group and Gram-positive high G+C content (HGC) group. Total numbers of bacteria in BAC process counted by 4’,6-diamidino-2-phenylindole (DAPI) staining were (top), (middle) and cells/ml (bottom). The number of the culturable bacteria was from to cells/ml and the culturability was about 0.05%. The faction of bacteria detectable by FISH with the probe EUB338 was about 83% of DAPI counts. Gamma and alpha subclass of the class Proteobacteria were predominant in BAC process and their ratios were over 20% respectively. In top and middle, alpha, beta and gamma subclass of the class Proteobacteria competed with each other and their percentages was changed according to the season. In bottom, gamma subclass of the class Proteobacteria was predominant all through the year. It could be successfully observed the seasonal distribution of bacterial community in biological activated carbon process using FISH.
Special geographical location and abundant organic matter profiles in tidal flats have resulted in great microbial diversity, in which Bacteroidota strains are considered as one of the primary degraders of polysaccharides, playing a crucial role in the carbon cycle. In this study, we collected sediment or sand samples from 34 bare tidal flats in China and investigated the profile of culturable bacteria, selected three Bacteroidota for polyphasic taxonomic analysis and revealed their polysaccharide metabolic potential. Totally, we isolated 352 pure cultured bacteria and they mainly distributed in Bacteroidota, Pseudomonadota, Bacillota, and Actinomycetota. It is shown that the bare tidal flats contained a large number of potential novel species, mainly distributed in Flavobacteriales and Cytophagales within Bacteroidota. Three Bacteroidota strains, M17T, M82T, and M415T, isolated from mudflat were selected for polyphasic taxonomic analysis. The 16S rRNA gene sequence similarity between strain M17T and Mangrovivirga cuniculi KCTC 72349T was 99.28%, and less than 90.09% with other species; strain M82T shared the highest 16S rRNA gene sequence similarity of 97.85% with Pontibacter litorisediminis KCTC 52252T, and less than 97.43% with other species; strain M415T had higher 16S rRNA gene sequence similarities with type species of genera Eudoraea (92.62-93.68%), Zeaxanthinibacter (92.02-92.91%), and Muriicola (92.21-92.83%). Phylogenetic analysis based on 16S rRNA gene sequences and single-copy orthologous clusters showed that strains M17T and M82T represent novel species within the genus Mangrovivirga and Pontibacter, respectively, and strain M415T represents a novel species of a novel genus within the family Flavobacteriaceae. The potential in polysaccharide metabolism of all these three strains was analyzed by genomes. The analysis revealed that glycoside hydrolases and glycosyltransferases account for more than 70% of the total CAZymes. Additionally, the numbers of polysaccharide utilization loci (PULs) and annotated CAZymes in Cytophagales spp. M17T and M82T were found to be higher than those in Flavobacteriales sp. M415T. Highly specialized saccharolytic systems and the presence of numerous diversified CAZymes for obtaining energy through polysaccharide metabolism were speculated to help the three novel strains adapt to the utilization of both terrestrial and marine polysaccharides.
Biodiversity, both aboveground and belowground, is negatively affected by global changes such as drought or warming. This loss of biodiversity impacts Earth's ecosystems, as there is a positive relationship between biodiversity and ecosystem functioning (BEF). Even though soils host a large fraction of biodiversity that underlies major ecosystem functions, studies exploring the relationship between soil biodiversity and ecosystem functioning (sBEF) as influenced by global change drivers (GCDs) remain scarce. Here we highlight the need to decipher sBEF relationships under the effect of interactive GCDs that are intimately connected in a changing world. We first state that sBEF relationships depend on the type of function (e.g., C cycling or decomposition) and biodiversity facet (e.g., abundance, species richness, or biomass) considered. Then, we shed light on the impact of single and interactive GCDs on soil biodiversity and sBEF and show that results from scarce studies studying interactive effects range from antagonistic to additive to synergistic when two individual GCDs cooccur. This indicates the need for studies quantitatively accounting for the impacts of interactive GCDs on sBEF relationships. Finally, we provide guidelines for optimized methodological and experimental approaches to study sBEF in a changing world that will provide more valuable information on the real impact of (interactive) GCDs on sBEF. Together, we highlight the need to decipher the sBEF relationship in soils to better understand soil functioning under ongoing global changes, as changes in sBEF are of immediate importance for ecosystem functioning.
The use of Jiuqu as a saccharifying and fermenting starter in the production of fermented foods is a very old biotechnological process that can be traced back to ancient times. Jiuqu harbors a hub of microbial communities, in which prokaryotes and eukaryotes cohabit, interact, and communicate. However, the spontaneous fermentation based on empirical processing hardly guarantees the stable assembly of the microbiome and a standardized quality of Jiuqu. This review describes the state of the art, limitations, and challenges towards the application of traditional and omics‐based technology to study the Jiuqu microbiome and highlights the need for integrating meta‐omics data. In addition, we review the varieties of Jiuqu and their production processes, with particular attention to factors shaping the microbiota of Jiuqu. Then, the potentials of integrated omics approaches used in Jiuqu research are examined in order to understand the assembly of the microbiome and improve the quality of the products. A variety of different approaches, including molecular and mass spectrometry‐based techniques, have led to scientific advances in the analysis of the complex ecosystem of Jiuqu. To date, the extensive research on Jiuqu has mainly focused on the microbial community diversity, flavor profiles, and biochemical characteristics. An integrative approach to large‐scale omics datasets and cultivated microbiota has great potential for understanding the interrelation of the Jiuqu microbiome. Further research on the Jiuqu microbiome may explain the inherent property of compositional stability and stable performance of a complex microbiota coping with environmental perturbations and provide important insights to reconstruct synthetic microbiota and develop modern intelligent manufacturing procedures for Jiuqu.
In this study bacterial populations were identified in the rumen of zebu cattle fed various diets and classified taxonomically with metagenomic sequencing of the 16S rRNA gene. Twenty-four (24) heifers were used in a completely randomized experimental design to test the effect of the diets. Treatment 1 consisted of range grass hay. Treatment 2 was composed of the hay diet augmented with sun-dried cassava leaves. Treatment 3 comprised hay plus sun-dried azolla. Treatments 4 to 6 were similar to treatments 1 to 3. but with a basal diet of Brachiaria Mulato II hay. Rumen liquor samples were collected from the heifers, from which a total of 192 DNA samples were amplified and the resulting 16S rRNA sequences were compared with those in the National Centre for Biotechnology Information BLAST database using MetagenAssist. Bioinformatics analyses indicated that 17 operational taxonomic units (OTUs) were present at phylum level, of which 43.3% were Firmicutes, 27.2% Bacteroidetes, 22.8% Proteobacteria and 1.7% Euryarchaeota. The remaining OTUs were Cyanobacteria (1.4%) and Chloroflexi (1%) with Actinobacteria, Verrucomicrobia, Spirochaetes, Tenericutes, Planctomycetes, Elusimicrobia, Lentisphaerae, Armatimonadetes, Fibrobacteres, Synergistetes and Arthropoda all below 1% of the organisms present. Time and diet both affected (P<0.05) the abundance of microbes, but not their diversity in the rumen. Thus, these diets could affect the performance of animals.
Microorganism contamination is one of the most important factors affecting the spoilage and food safety of Manila clams. This study aimed to gain insights into bacterial composition and the dynamic change of bacterial communities on retailed Manila clam during refrigerated storage within the edible period. High-throughput sequencing was conducted to monitor the bacterial population with the prolongation of storage time of Day 0, Day 1, and Day 3. Result demonstrated that phyla of Proteobacteria, Actinobacteriota, Acidobacteriota, and Chloroflexi composed the majority of bacterial communities during the whole observation process. Furthermore, the increase of Proteobacteria showed a positive correlation with the storage time, whereas Acidobacteriota and Chloroflexi continued to decline in storage. For genus annotation, none of genus obtained dominant population in storage. From Day 0 to Day 1, the genera of Streptomyces , Bradyrhizobium , and Mycobacterium significantly increased; meanwhile, 12 genera significantly decreased. Compared with samples at Day 0, a total of 15 genera significantly decreased with the reduced proportion ranging from 0.50 to 4.40% at Day 3. At the end of the storage, the genus Crossiella became the most redundant population. Both the richness and diversity decreased at the start of storage at Day 1, and then slightly increased at Day 3 was observed. Based on the result in this study, strategy targeting the increased bacteria could be tested to improve the consumption quality and safety of refrigerated clam.
The inclusion of bioaugmented low-cost biochar in current wastewater treatment technologies is a promising way to enhance the removal and degradation of emerging contaminants. In this paper, the properties of two wood waste biochars (wood waste mix - AB, and date palm fiber wood - PDF), and coffee bean husks (COF), produced at four temperatures (350, 450, 500, 550°C) were compared, and investigated in the presence of Geobacter sulfurreducens or a mixed freshwater stream bacterial culture to understand their potential for the adsorption and biotransformation of two types of pesticides (thiacloprid, pirimicarb), and two pharmaceuticals (ibuprofen, diclofenac). Biochar yield was similar for all three biochars and ranged between 30 and 35%. The ash content of PDF and COF was significantly higher than AB. pH and electrical conductivity (EC) were initially high for COF (pH: 7.4–8; EC: 3–4.27 mS/cm) and PDF (pH: 7.7–10.1; EC: 4–6.24 mS/cm) after 24 h, but stabilized at neutral pH and <0.5 mS/cm EC after additional washes. COF and AB did not leach high concentrations of chloride (<10 mg/L), nitrate (<1 mg/L), nor sulphate (<76 mg/L), this in contrast to date palm fiber wood (PDF) with 1760 mg/L Cl⁻ (550°C), and 846 mg/L sulphate (350°C). Lower pyrolysis temperatures reduced leachable anions. The biochars were highly (ultra)microporous with little meso- and macroporosity. The adsorption experiments showed that AB and COF biochars were both suited to sorb more than 90% of the initially spiked 10 ppm pirimicarb, AB removed 50.2% of the initial diclofenac concentration compared to only 5% for the no-biochar control, and both biochars could remove about 55% of the initially spiked thiacloprid, and 40% of the ibuprofen. In the presence of a mixed culture, on average 30% more thiacloprid and ibuprofen was removed from the supernatant by AB and COF than the sterile control. This work shows that selected wood-waste feedstocks and low pyrolysis temperature can produce environmentally-safe biochars that have suitable characteristics to sorb emergent pollutants from water. These materials could be further studied in multi-pollution sorption/competition experiments, and in larger environmental wastewater treatment systems.
The search for new natural compounds for application in medicine and cosmetics is a trend in biotechnology. One of the sources of such active compounds is the snail mucus. Snail physiology and the biological activity of their fluids (especially the mucus) are still poorly studied. Only a few previous studies explored the relationship between snails and their microbiome. The present study was focused on the biodiversity of the snail mucus used in the creation of cosmetic products, therapeutics, and nutraceuticals. The commonly used cultivation techniques were applied for the determination of the number of major bacterial groups. Fluorescence in situ hybridization for key taxa was performed. The obtained images were subjected to digital image analysis. Sequencing of the 16S rRNA gene was also done. The results showed that the mucus harbors a rich bacterial community (10.78 × 1010 CFU/ml). Among the dominant bacteria, some are known for their ability to metabolize complex polysaccharides or are usually found in soil and plants (Rhizobiaceae, Shewanella, Pedobacter, Acinetobacter, Alcaligenes). The obtained data demonstrated that the snail mucus creates a unique environment for the development of the microbial community that differs from other parts of the animal and which resulted from the combined contribution of the microbiomes derived from the soil, plants, and the snails. Bacteria Acinetobacter sp. and the Rhizobiaceae family, (Pedobacter sp., Aeromonas sp., Shewanella sp., and Alcaligenes sp.) were identified as main members of the snail mucus‐associated bacterial community.
Activated sludge in wastewater treatment bioreactors contains diverse bacteria, while little is known about the community structure of bacteria responsible for degradation of refractory organic compounds (ROCs). In this study, 10 ROCs frequently detected in sewage were investigated, and the potential bacteria degrading these ROCs were analyzed by DNA stable isotope probing and high-throughput sequencing. The results showed that the bacterial communities responsible for degradation of different ROCs were largely different. A total of 84 bacterial genera were found to be involved in degrading at least one of the 10 ROCs, however, only six genera (Acinetobacter, Bacteroides, Bosea, Brevundimonas, Lactobacillus and Pseudomonas) were common to all 10 ROCs. This suggests that different ROCs may have specific assimilating bacteria in the activated sludge. Our results also showed that these ROC-degrading bacteria are difficult to isolate by conventional methods and that most of them have relatively low relative abundance in municipal wastewater treatment bioreactors. Development of new technologies to increase the abundance and activity of these bacteria may significantly improve the removal efficiency of ROCs from wastewater.
Rhizoremediation is a potential technique for PAH remediation; however, the catabolic pathways of in situ rhizosphere PAH degraders and the main factors driving PAH rhizoremediation remain unclear. To address these issues, stable‐isotope‐probing coupled with metagenomics and molecular ecological network analyses were firstly used to investigate the phenanthrene rhizoremediation by three different prairie grasses in this study. All rhizospheres exhibited a significant increase in phenanthrene removal and markedly modified the diversity of phenanthrene degraders by increasing their populations and interactions with other microbes. Of all the active phenanthrene degraders, Marinobacter and Enterobacteriaceae dominated in the bare and switchgrass rhizosphere, respectively; Achromobacter was markedly enriched in ryegrass and tall fescue rhizospheres. Metagenomes of 13C‐DNA illustrated several complete pathways of phenanthrene degradation for each rhizosphere, which clearly explained their unique rhizoremediation mechanisms. Additionally, propanoate and inositol phosphate of carbohydrates were identified as the dominant factors that drove PAH rhizoremediation by strengthening the ecological networks of soil microbial communities. This was verified by the results of rhizospheric and non‐rhizospheric treatments supplemented with these two substances, further confirming their key roles in PAH removal and in situ PAH rhizoremediation. Our study offers novel insights into the mechanisms of in situ rhizoremediation at PAH‐contaminated sites. This article is protected by copyright. All rights reserved.
Extremophilic endoxylanases grabbed attention in recent years due to their applicability under harsh conditions of several industrial processes. Thermophilic, alkaliphilic, and acidophilic endoxylanases found their employability in bio-bleaching of paper pulp, bioconversion of lignocellulosic biomass into xylooligosaccharides, bioethanol production, and improving the nutritious value of bread and other bakery products. Xylanases obtained from extremophilic bacteria and archaea are considered better than fungal sources for several reasons. For example, enzymatic activity under broad pH and temperature range, low molecular weight, cellulase-free activity, and longer stability under extreme conditions of prokaryotic derived xylanases make them a good choice. In addition, a short life span, easy cultivation/harvesting methods, higher yield, and rapid DNA manipulations of bacterial and archaeal cells further reduces the overall cost of the product. This review focuses on the diversity of prokaryotic endoxylanases, their characteristics, and their functional attributes. Besides, the molecular mechanisms of their extreme behavior have also been presented here.
The soil environment determines plants’ health and performance during their life cycle. Therefore, ecological understanding on variations in soil environments, including physical, chemical, and biological properties, is crucial for managing agricultural fields. Here, we present a comprehensive and extensive blueprint of the bacterial, archaeal, and fungal communities in rice paddy soils with differing soil types and chemical properties. We discovered that natural variations of soil nutrients are important factors shaping microbial diversity. The responses of microbial diversity to soil nutrients were related to the distribution of microbial trophic lifestyles (oligotrophy and copiotrophy) in each community. The compositional changes of bacterial and archaeal communities in response to soil nutrients were mainly governed by oligotrophs, whereas copiotrophs were mainly involved in fungal compositional changes. Compositional shift of microbial communities by fertilization is linked to switching of microbial trophic lifestyles. Random forest models demonstrated that depletion of prokaryotic oligotrophs and enrichment of fungal copiotrophs are the dominant responses to fertilization in low-nutrient conditions, whereas enrichment of putative copiotrophs was important in high-nutrient conditions. Network inference also revealed that trophic lifestyle switching appertains to decreases in intra- and inter-kingdom microbial associations, diminished network connectivity, and switching of hub nodes from oligotrophs to copiotrophs. Our work provides ecological insight into how soil nutrient-driven variations in microbial communities affect soil health in modern agricultural systems.
Premise:
Within a broader study on leaf fossilization in freshwater environments, a long-term study on the development and microbiome composition of biofilms on the foliage of aquatic plants has been initiated to understand how microbes and biofilms contribute to leaf decay and preservation. Here, water lily leaves are employed as a study model to investigate the relationship between bacterial microbiomes, biodegradation, and fossilization. We compare four DNA extraction kits to reduce biases in interpretation and to identify the most suitable kit for the extraction of DNA from bacteria associated with biofilms on decaying water lily leaves for 16S rRNA amplicon analysis.
Methods:
We extracted surface-associated DNA from Nymphaea leaves in early stages of decay at two water depth levels using four commercially available kits to identify the most suitable protocol for bacterial extraction, applying a mock microbial community standard to enable a reliable comparison of the kits.
Results:
Kit 4, the FastDNA Spin Kit for Soil, resulted in high DNA concentrations with better quality and yielded the most accurate depiction of the mock community. Comparison of the leaves at two water depths showed no significant differences in community composition.
Discussion:
The success of Kit 4 may be attributed to its use of bead beating with a homogenizer, which was more efficient in the lysis of Gram-positive bacteria than the manual vortexing protocols used by the other kits. Our results show that microbial composition on leaves during early decay remains comparable and may change only in later stages of decomposition.
Stakeholders of petroleum hydrocarbon-contaminated (PHC) sites aim to meet remediation goals cost-effectively. But contaminated sites are typically complex, and when inefficient remediation strategies are implemented, time and money are wasted. Various strategies can be implemented for site remediation and almost all at some point involve a biological component. Therefore, during site characterization, seeing the complete picture by obtaining multiple lines of evidence—chemistry (concentrations of contaminants and daughter products), geochemistry (redox status, electron acceptors, electron donors), microbiology (species and their genes for catalyzing biodegradation of reactions, i.e., functional genes), and the contaminant degradation ongoing at baseline (stable isotope methods)—is critically important for selecting the best remediation strategy. Additionally, molecular biological evidence is important for monitoring treatment progress and informing decisions to retreat, change treatments, or transition to monitored natural attenuation.
Soil contaminated with used lubricant oil (ULO) has become an emerging environmental threat. Phytoremediation is a cost effective, environmentally friendly and novel technique with great potential for remediation of soils contaminated with used lubricating oil (ULO). Therefore, the aim of this study was to investigate the phytoremediation potential of Crotalaria retusa L. and Impatiens balsamina for ULO contaminated soil. A randomized block pot experiment was conducted for each species under plant house conditions in soil contaminated with ULO at a concentration of 5,000 mg kg⁻¹ (0.5% w/w) - 30,000 mg kg⁻¹ (3% w/w). The percentage biodegradation of ULO in the rhizosphere soil of both species following 90 days exposure indicated significant (p < 0.05) time dependent increases compared to the respective control carried out in contaminated soil without plants. Comparatively higher biodegradation of ULO was recorded from the rhizosphere of C. retusa L. than that of I. balsamina. The measured total microbial activity and cultivable population size of hydrocarbon utilizing bacteria (HUB) suggest that phytoremediation phytoremediation might have occurred mainly via rhizodegradation. The Allium cepa bioassay revealed the reduction of potential cytotoxicity and genotoxicity with the decrease of ULO in phytoremediated soils. The results further revealed significant (p<0.05) positive correlation of N−NO3−1, extractable P concentration and significant (p<0.05) negative correlation of soil pH with percentage ULO degradation. Therefore phytoremediation using the test plant species not only reduced ULO levels, cytotoxicity and genotoxicity but also improved overall soil quality. These results highlight the higher phytoremediation potential of C. retusa than that of I. balsamina for soil contaminated with ULO.
COnsensus DEgenerate Hybrid Oligonucleotide Primers (CODEHOP) were developed for the detection of the dszB desulfinase gene (2'-hydroxybiphenyl-2-sulfinate desulfinase; EC 3.13.1.3) by polymerase chain reaction (PCR), which allow to reveal larger diversity than traditional primers. The new developed primers were used as molecular monitoring tool to drive a procedure for the isolation of desulfurizing microorganisms. The primers revealed a large dszB gene diversity in environmental samples, particularly in diesel-contaminated soil that served as inoculum for enrichment cultures. The isolation procedure using the dibenzothiophene sulfone (DBTO2) as sole sulfur source reduced drastically the dszB gene diversity. A dszB gene closely related to that carried by Gordonia species was selected. The desulfurization activity was confirmed by the production of desulfurized 2-hydroxybiphenyl (2-HBP). Metagenomic 16S rRNA gene sequencing showed that the Gordonia genus was represented at low abundance in the initial bacterial community. Such observation highlighted that the culture medium and conditions represent the bottleneck for isolating novel desulfurizing microorganisms. The new developed primers constitute useful tool for the development of appropriate cultural-dependent procedures, including medium and culture conditions, to access novel desulfurizing microorganisms useful for the petroleum industry.
Plasmid-mediated dissemination of antibiotic resistance among fecal Enterobacteriaceae in natural ecosystems may contribute to the persistence of antibiotic resistance genes in anthropogenically impacted environments. Plasmid transfer frequencies measured under laboratory conditions might lead to overestimation of plasmid transfer potential in natural ecosystems. This study assessed differences in the conjugative transfer of an IncP-1 (pKJK5) plasmid to three natural Escherichia coli strains carrying extended-spectrum beta-lactamases, by filter mating. Matings were performed under optimal laboratory conditions (rich LB medium and 37 • C) and environmentally relevant temperatures (25, 15 and 9 • C) or nutrient regimes mimicking environmental conditions and limitations (synthetic wastewater and soil extract). Under optimal nutrient conditions and temperature, two recipients yielded high transfer frequencies (5 × 10 −1) while the conjugation frequency of the third strain was 1000-fold lower. Decreasing mating temperatures to psychrophilic ranges led to lower transfer frequencies, albeit all three strains conjugated under all the tested temperatures. Low nutritive media caused significant decreases in transconjugants (−3 logs for synthetic wastewater; −6 logs for soil extract), where only one of the strains was able to produce detectable transconjugants. Collectively, this study highlights that despite less-than-optimal conditions, fecal organisms may transfer plasmids in the environment, but the transfer of pKJK5 between microorganisms is limited mainly by low nutrient conditions.
Prostomatean ciliates play important roles in the flow of material and energy in aquatic microbial food webs, and thus have attracted wide attention for over a century. Their taxonomy and systematics are, however, still poorly understood because of their relatively few taxonomically informative morphological characters. In this study, two new prostomateans, Lagynus binucleatus sp. n. and Foissnerophrys alveolata gen. n., sp. n., collected from a freshwater pool and the intertidal zone of a sandy beach, respectively, in Qingdao, China, are investigated using living observation, protargol staining, and SSU rRNA gene sequencing methods. The genus Lagynus is redefined, and the new species L. binucleatus sp. n. is established based on significant morphological differences with similar forms. Furthermore, a new genus, Foissnerophrys gen. n., is established based on a combination of morphological and molecular data with F. alveaolata sp. n. the type species by monotypy. The identities of intracellular prokaryotes of these two new species are discussed based on fluorescence in situ hybridization (FISH) data and newly obtained 16S rRNA gene sequences.
Two related salmonids, Arctic char (Salvelinus alpinus) and lake whitefish (Coregonus clupeaformis) sampled from the high Arctic region of Nunavut, Canada are anadromous fish, migrating annually from the same ice‐covered freshwater waterbodies to spend summers in the marine waters of the Arctic Ocean. Microbiota associated with the skin‐associated mucus undergo community change coincident with migration, and irrespective of this turnover, antibiotic resistance was detected in mixed bacterial cultures initiated with mucus samples. Although as expected most bacteria were unculturable, however, 5/7 isolates showed susceptibility to a panel of five common antibiotics. The fish were sampled under severe conditions and at remote locations far from human habitation. Regardless, two isolates, ‘Carnobacterium maltaromaticum sm‐2’ and ‘Arthrobacter citreus sm’, showed multi‐resistance to two or more antibiotics including ampicillin and streptomycin indicating multiple resistance genes. It is unknown if these fish bacteria have ‘natural’ resistance phenotypes or if resistance has been acquired. As result of these observations, we urge long‐term monitoring of drug‐resistant bacteria in the region and caution the assumption of a lack of drug‐resistant organisms even in such extreme environments.
The structure and functions of microbial communities in coastal sediments are influenced by various environmental factors. Being the largest sink of organic carbon, coastal sediments have a pivotal role in global carbon cycling. Sedimentary organic matter may be labile or refractory based on their biochemical composition and resultant stability. The organic matter available in the coastal sediments may be produced by the phytoplankton in situ or transported from various sources into the coastal sediments. The former is referred to as autochthonous and latter as allochthonous organic matter. In the sediments, microbes degrade organic matter using a sequence of respiratory and fermentative metabolisms. The major groups of microbial communities found in coastal sediments according to their functional role in organic matter degradation are biopolymer hydrolyzers, nitrate reducers, metal reducers, sulfate reducers, and methanogens. The metabolic pathway and microbes involved are affected by the quality and degradability of the organic carbon and the availability of terminal electron acceptors. Sediments with different biogeochemical properties have microbial communities that exhibit distinct catabolic responses to a range of carbon sources. Thus the composition and activities of microbial communities are regulated by the quality and availability of carbon.
In order to clarify the effect of nanocomposite-based packaging (NP) on umami and microflora characteristics of F. filiformis during cold storage, the contents of umami amino acids and 5'-nucleotides, equivalent umami concentration (EUC), and microflora succession were investigated. Results showed that NP could delay the degradation of umami components and inhibit bacterial growth in F. filiformis. At the initial stage, the dominant bacteria were Lactobacillus, Thermus and Acinetobacter. After 15 days of storage, the bacteria count in NP reached 7.63 lg cfu/g, which was significantly (P<0.05) lower than that in control, and the major bacterial communities of packaged F. filiformis were Ewingella, Serratia and Pseudomonas. Moreover, the correlation analysis showed that Lactobacillus, Brevibacillus and Okibacterium were negatively correlated with AMP and IMP 5-nucleotides. Present work suggested that NP could enhance the umami flavor formation and improve the microbial community structure of F. filiformis, resulting in a better commercial quality. The results provided theoretical basis for large-scale applications of NP.
The fish gut is a complex ecosystem populated by an exceedingly diverse and assorted group of microbes, influenced by various factors such as habitat salinity, temperature, trophic level, taxonomy, feeding habits as well as host selective pressures within the gut. The gut microbiota is involved in several physiological functions of the host such as digestion, metabolism, reproduction, development, and immune response and is often heralded as an ‘extra organ’. Earlier studies employed culture‐dependent techniques for the study of fish gut microflora. The establishment of great plate anomaly and technological strides in culture‐independent techniques, particularly in the omics technologies such as metagenomics and next‐generation sequencing techniques have led to a better understanding of the confederation of microorganisms with their respective hosts. This article reviews the applications of and prospects of metagenomics and amplicon and shotgun sequencing approaches in fish gut microbiota research.
Studies of unculturable microbes often combine methods, such as 16S rRNA sequencing, metagenomics, and metaproteomics. To apply these techniques to the microbial community inhabiting the surfaces of marine macrophytes, it is advisable to perform a selective DNA and protein isolation prior to the analysis to avoid biases due to the host material being present in high quantities. Two protocols for DNA and protein isolation were adapted for selective extractions of DNA and proteins from epiphytic communities inhabiting the surfaces of two marine macrophytes, the seagrass Cymodocea nodosa and the macroalga Caulerpa cylindracea. Protocols showed an almost complete removal of the epiphytic community regardless of the sampling season, station, settlement, or host species. The obtained DNA was suitable for metagenomic and 16S rRNA sequencing, while isolated proteins could be identified by mass spectrometry. Low presence of host DNA and proteins in the samples indicated a high specificity of the protocols. The procedures are based on universally available laboratory chemicals making the protocols widely applicable. Taken together, the adapted protocols ensure an almost complete removal of the macrophyte epiphytic community. The procedures are selective for microbes inhabiting macrophyte surfaces and provide DNA and proteins applicable in 16S rRNA sequencing, metagenomics, and metaproteomics.
Huanglongbing (HLB) which is caused by Candidatus liberibacter asiaticus (CLas) is one of the citrus diseases that cause the largest losses on citrus orchards in Indonesia and other countries. An experiment was conducted at the HLB endemic area in Purworejo district of Central Java to study the response of biofertilizer treatment on HLB susceptible citrus plant (Citrus reticulata) against the disease. The trial was using graft inoculated and non-inoculated by inoculum HLB. Plants treated by biofertilizer A (manufactured) or biofertilizer B (farmer’s formulation), once a month during 3 years and 8 months. CLas detection was conducted in the last 9 months. The results showed that application of biofertilizer B was effective in reducing HLB disease naturally transmitted by vector (AUDPC value of 195.24 by leaf score and 218.58 by canopy score). Biofertilizer A was effective to control HLB disease on HLB-inoculated plants. Detection of CLas by PCR (Polymerase Chain Reaction) show that some plants have instability in the detection
results that were influenced by the concentration of CLas. The rhizobacteria population in plant without the HLB inoculation and applied biofertilizer A were higher than in plants with the HLB inoculation and applied biofertilizer B. The total population of rhizobacteria tended to increase except in the treatment of biofertilizer A, that were applied on HLB-inoculated plants. The application of biofertilizer to artificially infected plants and naturally transmitted through vectors were able to prolong the viability of the plant.
Les études de la diversité et de la richesse bactérienne des sols constituent un enjeu primordial pour développer des bio pesticides efficaces en lutte biologique contre les diptères phytoravageurs et vecteurs de maladie (malaria, dengue…). Dans ce manuscrit, la caractérisation de la diversité bactérienne des sols de chênes et de pins Libanais a été étudiée par comparaison aux mêmes sols d’Autriche, une zone géographique différente et de climat différent. La composition en classes et genres du phylum des Firmicutes et son abondance dans les différents sols étudiés ont été particulièrement caractérisées par des indices de diversité (alpha, bêta, OTUs…) et des méthodes moléculaires (SSCP et qRT-PCR). A partir des sols de pins et de chênes Libanais et précisément à partir de ce phylum, des souches de Bacillusthuringiensis ont été criblées pour leur activité insecticides contre les diptères,Culex pipiens, Aedes albopictus et Anopheles gambiae. Deux souches de Bt ont été sélectionnées. La souche A23 possède toxicité in vivo plus élevée que la souche de référence Bacillus thuringiensis isarelensis (Bti). Cette souche n’a cependant pas révélé de différences génétiques et moléculaires par rapport àBti. La souche H3, quant à elle, a été étudiée étant non cytolytique et présentant une activité insecticide in vivo contre les diptères mais à fortes concentrations par rapport à Bti. Cette souche a révélé un contenu génique et protéique différent de Bti. H3 a probablement un nouveau variant du gène cry4B et du gène cry40connu pour avoir une activité diptéricide. Des investigations supplémentaires sont nécessaires afin de confirmer ces résultats et de comprendre les mécanismes de toxicité de ces deux souches.
Most microorganisms resist cultivation under standard laboratory conditions. On the other hand, cultivating microbes in a membrane-bound device incubated in nature (in situ cultivation) can be an effective approach to overcome this limitation. In the present study, we applied in situ cultivation to isolate diverse previously uncultivated marine sponge-associated microbes and comparatively analyzed this method’s efficiencies with those of the conventional method. Then, we attempted to investigate the key and previously unidentified mechanism of growing uncultivated microorganisms by in situ cultivation focusing on growth triggering via growth initiation factor. Significantly more novel and diverse microbial types were isolated via in situ cultivation than by standard direct plating (SDP). We hypothesized that some of environmental microorganisms which resist cultivation are in a non-growing state and require growth initiation factors for the recovery and that these can be provided from the environment (in this study from marine sponge). According to the hypothesis, the effect of the sponge extract on recovery on agar medium was compared between strains derived from in situ and SDP cultivation. Adding small amounts of the sponge extracts to the medium elevated the colony-formation efficiencies of the in situ strains at the starvation recovery step, while it showed no positive effect on that of SDP strains. Conversely, specific growth rates or saturated cell densities of all tested strains were not positively affected. These results indicate that, (1) the sponge extract contains chemical compounds that facilitate recovery of non-growing microbes, (2) these substances worked on the in situ strains, and (3) growth initiation factor in the sponge extract did not continuously promote growth activity but worked as triggers for regrowth (resuscitation from non-growing state).
Lichens represent self-supporting symbioses, which occur in a wide range of terrestrial habitats and which contribute significantly to mineral cycling and energy flow at a global scale. Lichens usually grow much slower than higher plants. Nevertheless, lichens can contribute substantially to biomass production. This review focuses on the lichen symbiosis in general and especially on the model species Lobaria pulmonaria L. Hoffm., which is a large foliose lichen that occurs worldwide on tree trunks in undisturbed forests with long ecological continuity. In comparison to many other lichens, L . pulmonaria is less tolerant to desiccation and highly sensitive to air pollution. The name-giving mycobiont (belonging to the Ascomycota), provides a protective layer covering a layer of the green-algal photobiont ( Dictyochloropsis reticulata ) and interspersed cyanobacterial cell clusters ( Nostoc spec.). Recently performed metaproteome analyses confirm the partition of functions in lichen partnerships. The ample functional diversity of the mycobiont contrasts the predominant function of the photobiont in production (and secretion) of energy-rich carbohydrates, and the cyanobiont’s contribution by nitrogen fixation. In addition, high throughput and state-of-the-art metagenomics and community fingerprinting, metatranscriptomics, and MS-based metaproteomics identify the bacterial community present on L. pulmonaria as a surprisingly abundant and structurally integrated element of the lichen symbiosis. Comparative metaproteome analyses of lichens from different sampling sites suggest the presence of a relatively stable core microbiome and a sampling site-specific portion of the microbiome. Moreover, these studies indicate how the microbiota may contribute to the symbiotic system, to improve its health, growth and fitness.
During the winter of 2013 and 2016, several Croatian fish farms experienced mortalities in the fry of European sea bass, Dicentrarchus labrax. Affected fish showed abnormal swimming behaviour and reduced appetite, and death ensued several days after the onset of clinical signs of disease. Necropsy revealed pale liver, empty digestive tract, distended gall bladder, and hyperaemia and congestion of the meninges. Routine bacteriological examination tested negative, and virological examination ruled out nodavirus infection. Histological examination revealed multifocal necrosis and extensive inflammation in the brain with abundant cellular debris in the ventricles. Inflammatory cells displayed intra‐cytoplasmic basophilic vacuoles leading to suspicion of Piscirickettsia salmonis infection. Fluorescent in situ hybridization using an oligonucleotide probe targeting Domain Bacterium applied to tissue sections tested positive. The pathogen was identified by 16S rRNA gene sequencing of brain material, and the sequence showed 99% similarity with P. salmonis. This result enabled the design of an oligonucleotide probe specifically targeting P. salmonis. In 2016, P. salmonis was successfully isolated on CHAB from the brain of an affected specimen and identified using 16S rRNA gene sequencing and MALDI‐TOF. This study describes the first outbreak of disease caused by P. salmonis in sea bass in Croatia, while new diagnostic tools will enable further research on its epidemiology and pathogenicity.
The Gulf of Mexico Research Consortium (Consorcio de Investigación del Golfo de México (CIGoM), 2020) was founded in 2015 as a consortium of scientific research and consulting services, specializing in multidisciplinary projects related to the potential environmental impacts of natural and human-induced oil spills in marine ecosystems, to understand and act in the case of possible large-scale oil spills in the Gulf of Mexico. CIGoM comprises more than 300 specialized researchers trained at the most recognized Mexican institutions. Among the main interests of CIGoM are developing the first baseline of the bacterial community inhabiting the southern Gulf of Mexico, investigating the natural degradation of hydrocarbons by bacterial communities and microbial consortia and identifying and characterizing industrially relevant enzymes. In this review, using third-generation sequencing methodologies coupled to function screening methodologies, we report the bacterial profile found in samples of water and sediments in Mexican regions that include the Perdido Fold Belt (northwest of Mexico), Campeche Knolls (in the southeast) and Southwest region of the Gulf of Mexico. We also highlight some examples of novel lipases and dioxygenases with high biotechnological potential and some culturable hydrocarbon-degrading strains used in diverse bioremediation processes.
Exposure to bioaerosols poses important health effects on occupants. To elucidate seasonal and room variations of household airborne bacteria, this study investigated 30 residential homes during summer and winter throughout Nanjing, Southeast China, with a humid subtropical climate. Culturing and 16S rDNA sequencing methods were combined in this study. Results showed that the community structure and composition in the same season but different homes show similarity, however, they in the same home but in different seasons show a huge difference, with Sphingomonas (25.3%), Clostridium (14.8%), and Pseudomonas (7.6%) being the dominant bacteria in summer, and Pseudomonas (57.1%) was dominant bacteria in winter. Culturable concentrations of bacteria were also significantly higher in summer (854 ± 425 CFU/m3) than in winter (231 ± 175 CFU/m3), but difference by home or room was relatively minor. More than 80% of culturable bacteria (<4.7 μm) could penetrate into lower respiratory tract. The seasonal variations of bacterial community and concentrations were closely associated with seasonal variations of temperature, humidity, and PM2.5. Higher concentrations and larger sizes were observed in the bathroom and kitchen, typically with higher humidity than other rooms.
Phagotrophic protists are key players in aquatic food webs. Although sequencing-based studies have revealed their enormous diversity, ecological information on in situ abundance, feeding modes, grazing preferences, and growth rates of specific lineages can be reliably obtained only using microscopy-based molecular methods, such as Catalyzed Reporter Deposition-Fluorescence in situ Hybridization (CARD-FISH). CARD-FISH is commonly applied to study prokaryotes, but less so to microbial eukaryotes. Application of this technique revealed that Paraphysomonas or Spumella-like chrysophytes, considered to be among the most prominent members of protistan communities in pelagic environments, are omnipresent but actually less abundant than expected, in contrast to little known groups such as heterotrophic cryptophyte lineages (e.g., CRY1), cercozoans, katablepharids, or the MAST lineages. Combination of CARD-FISH with tracer techniques and application of double CARD-FISH allow visualization of food vacuole contents of specific flagellate groups, thus considerably challenging our current, simplistic view that they are predominantly bacterivores. Experimental manipulations with natural communities revealed that larger flagellates are actually omnivores ingesting both prokaryotes and other protists. These new findings justify our proposition of an updated model of microbial food webs in pelagic environments, reflecting more authentically the complex trophic interactions and specific roles of flagellated protists, with inclusion of at least two additional trophic levels in the nanoplankton size fraction. Moreover, we provide a detailed CARD-FISH protocol for protists, exemplified on mixo-and heterotrophic nanoplanktonic flagellates, together with tips on probe design, a troubleshooting guide addressing most frequent obstacles, and an exhaustive list of published probes targeting protists.
Fluorescent oligonucleotide probes can be used to identify bacterial cells by hybridizing to specific rRNA sequences that are unique to a taxon. It has been suggested that single-cell RNA content could be estimated from the resulhng cell fluorescence, which is directly proportional to rRNA content However, this possibility was never assessed quantitatively for use in field studies, where most natural bacteria, due to their small size and slow growth, do not have enough rRNA to yield sufficient fluorescence for detection. One approach for increasing the fluorescence per rRNA molecule IS the use of multiple probes targeted to independent sites in the rRNA molecule. In this study, we hybridized n~ultiple probes (3 universal and 4 bacterial) to cultured marine bacterial isolates, and to natural bacterial assemblages from coastal water. For S of the 7 probes (2 probes did not hybridize to some of the isolates), cell fluorescence increased linearly with the number of probes hybridized. The natural bacterial assemblages showed an asymptotic increase of the probe-labeled fraction from ca 20% (1 probe) to 75% (5 probes) of the total population. The estimated detection limit was 0.3 fg RNA cell-' with S probes. RNA contents estimated from the fluorescence of probe-labeled cells agreed well to those determined separately by ethidium bromide fluorometry (differed by average 16 %, n = 3). Calibration of the photometer against fluorescence standards was critical for accurate measurements and comparisons.
The primary endosymbionts of aphids are maternally inherited bacteria
that live only within specialized host cells. Phylogenetic analysis of
the 16S ribosomal DNA sequences of aphid endosymbionts reveals that they
are a monophyletic group with a phylogeny completely concordant with
that of their hosts, implying long-term cospeciation. Here we show that
rates of base substitution are similar in the 16S ribosomal DNA of
different endosymbiont lineages. In addition, we calibrate these rates
by assigning age estimates for ancestral aphid hosts to the
corresponding endosymbionts. The resulting rate estimates (1-2% per 50
Ma) are among the most reliable available for prokaryotes. They are very
near values previously conjectured by using more tenuous assumptions for
dating divergence events in eubacteria. Rates calibrated using dates
inferred from fossil aphids imply that Asian and American species of the
aphid tribe Melaphidina diverged by the early Eocene; this result
confirms an earlier hypothesis based on biogeographic evidence. Based on
these rate estimates, the minimum age of this endosymbiotic association
and the age of aphids as a whole is estimated at 160-280 Ma.
All aphids are thought to have an association with primary endosymbionts, which are located within specialized cells known as mycetocytes. The primary endosymbionts are gram-negative, spherical or slightly oval cells which have not been cultivated outside aphid hosts. Recently, it has been shown that the 16s rRNA sequences of the primary endosymbionts of 11 host species belonging to four aphid families are similar and thus that these organisms are related. Comparisons with the 16s rRNAs of other procaryotes indicated that the primary endosymbionts form a distinct lineage within the gamma-3 subgroup of the Proteobacteria. On the basis of these results we propose that the primary endosymbionts should be placed in Buchnera gen. nov. and Buchnera aphidicola sp. nov. The primary endosymbiont found in the mycetocytes of Schizaphis graminum (green bug) is designated the type strain of this species. It is generally thought that all members of the insect :superfamily Aphidoidea (aphids) have an obligatory associ- ation with gram-negative, spherical or slightly oval cells, which are commonly called primary endosymbionts (for reviews see references 2-5,7, 8, 10). The primary endosym- bionts are found in specialized organs, the mycetomes, which consist of cells (mycetocytes) containing the endo- symbionts. The endosymbionts are enclosed within vesicles which are derived from the cell membranes of the myceto- cytes. None of the endosymbionts has been successfully cultivated in bacteriological media or in tissue culture. The function of the endosymbionts has not been definitively elucidated. However, it is clear that these organisms are essential for the life of the aphids since the removal of the endosymbionts by antibiotics leads to aphid sterility. The endosymbionts are maternally transmitted, and there are complex mechanisms which assure that the progeny are infected (2, 16).
The bacterioplankton community of confined seawater at 25 degrees C changed significantly within 16 h of collection. Confinement increased CFU, total cell number (by epifluorescence microscopy), and average cell volume of bacterioplankton and increased the turnover rate of amino acids in seawater sampled at Frying Pan Shoals, N.C. The bacterioplankton community was characterized by two components: differential doubling times during confinement shifted dominance from bacteria which were nonculturable to bacteria which were culturable on a complex nutrient medium. Culturable cells (especially those of the genera Pseudomonas, Alcaligenes, and Acinetobacter) increased from 0.08% of the total cell number in the seawater immediately after collection to 13% at 16 h and 41% at 32 h of confinement. Differential filtration before confinement indicated that particles passing through a 3.9-microns-, but retained by a 0.2-micron-, pore-size Nuclepore filter may be a major source of primary amines to the confined population. The 3.0-microns filtration increased growth rate and ultimate numbers of culturable cells through the removal of bacterial predators or the release of primary amines from cells damaged during filtration or both.
Herbivorous surgeonfish (Acanthurus species) in the Red Sea harbor gut
symbionts that include bacteria, trichomonadid flagellates, and a
peculiar putative protist that attains densities of 20,000 to 100,000
cells per milliliter of gut contents. The structure, mode of
reproduction, and within-gut distribution of the latter are described.
This may be the first report of an organism of this type and the first
evidence of a consistent endosymbiosis in the gut of a herbivorous
marine fish.
In a series of microcosm and field studies, we have observed that over time V. cholerae and related human pathogens enter a viable but non-culturable state. Direct viable counts by epifluorescent microscopy are consistently higher than corresponding plate counts. Thus, the assumption that microorganisms, including pathogens, "die-off" or "decay" in the marine environment must be re-evaluated, since stressed or nutrient starved cells are unable to grow and be enumerated by standard plate count methods. Furthermore, animal passage reveals pathogenicity persisting for such "non-viable"cells. Indirect immunofluorescent microscopy offers a more sensitive detection system for environmental sampling for human pathogens and therefore, a more valid estimation of population size. One implication of these findings for the release of genetically engineered organisms is that highly specific methods of detection and monitoring are required, with direct detection by fluorescent antibody the most reliable at the present time.
Using specific deoxyoligonucleotide probes we have discovered seasonally strong (up to ≈ 100%) dominance of bacteria hybridizing to a single probe, in near shore waters off Scripps pier (32°53′N; 117°15′W). The probes were designed from partially sequenced 16S rRNA (V3 domain) of isolated marine bacteria. The results indicate that this approach may be used for studies of bacterial populations in the marine environment. We have shown that a number of genotypes that at times are dominant in the natural assemblages are culturable (and not, ‘viable-but-unculturable’). Additionally, our data suggests that the discrepancy between viable counts and direct counts in sea water samples can be explained by low plating efficiency.
A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme, isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified. Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule present only once in a sample of 10(5) cells.
The phylogenetic diversity of an oligotrophic marine picoplankton community was examined by analyzing the sequences of cloned ribosomal genes. This strategy does not rely on cultivation of the resident microorganisms. Bulk genomic DNA was isolated from picoplankton collected in the north central Pacific Ocean by tangential flow filtration. The mixed-population DNA was fragmented, size fractionated, and cloned into bacteriophage lambda. Thirty-eight clones containing 16S rRNA genes were identified in a screen of 3.2 x 10(4) recombinant phage, and portions of the rRNA gene were amplified by polymerase chain reaction and sequenced. The resulting sequences were used to establish the identities of the picoplankton by comparison with an established data base of rRNA sequences. Fifteen unique eubacterial sequences were obtained, including four from cyanobacteria and eleven from proteobacteria. A single eucaryote related to dinoflagellates was identified; no archaebacterial sequences were detected. The cyanobacterial sequences are all closely related to sequences from cultivated marine Synechococcus strains and with cyanobacterial sequences obtained from the Atlantic Ocean (Sargasso Sea). Several sequences were related to common marine isolates of the gamma subdivision of proteobacteria. In addition to sequences closely related to those of described bacteria, sequences were obtained from two phylogenetic groups of organisms that are not closely related to any known rRNA sequences from cultivated organisms. Both of these novel phylogenetic clusters are proteobacteria, one group within the alpha subdivision and the other distinct from known proteobacterial subdivisions. The rRNA sequences of the alpha-related group are nearly identical to those of some Sargasso Sea picoplankton, suggesting a global distribution of these organisms.
Examination of collections of 16S rRNA sequences revealed sequence domains that were unique to (and invariant within) the three primary lines of cellular descent: the archaebacteria, the eubacteria, and the eucaryotes. Oligodeoxynucleotides complementary to these conserved sequence domains were synthesized and used as hybridization probes. Each of the radiolabeled probes specifically hybridized to nylon membrane-bound 16S rRNA from the targeted kingdom. A probe complementary to a universally conserved sequence in 16S rRNAs was used as a positive control, while its complement provided a negative control for nonspecific binding. The abilities of the probes to bind specifically to whole, fixed cells representing a broad array of phylogenetic diversity were tested in whole-cell dot blot assays. Again, all of the probes specifically bound the targeted groups. By microautoradiography, the method was extended to permit phylogenetic identification of single cells microscopically.
This chapter deals with the genera Achromatium, Macromonas, Thiobacterium, Thiospira, and Thiovulum (see Fig. 1). They all belong to chemotrophic microbial populations generally encountered in natural habitats that are characterized by the simultaneous presence of H2S and O2, i.e., at the border between aerobic and anaerobic zones in surface waters and in the outflows of H2S-bearing springs. It will be useful to introduce this discussion with some general remarks on the special nature of this ecological niche to help explain the embarassing paucity of knowledge we possess about its inhabitants.
A ruminant can be thought of as a fermentation factory (Fig. 1). The animal ingests plant polymers (in grasses, hay, corn, silage, etc.) which are the raw material for its fermentation. Preliminary processing occurs in the oral cavity and consists mainly of comminution of food by mastication. The plant material is then swallowed and transported to the ruminant’s complex stomach. The stomach, called the rumen reticulum or, more simply, rumen, is the site of fermentation. A massive community of microorganisms, bacteria and protozoa, ferments the plant material to short-chain volatile fatty acids, methane, and carbon dioxide. The acids are removed from the rumen by absorption into the bloodstream and are subsequently used as the animal’s primary sources of energy and carbon. Gases are waste products and are removed by belching. The fermentation provides nutrients and energy for the growth and division of the microbial populations participating in the fermentation. Microorganisms and undigested food are semicontinuously removed from the rumen by passage to the lower part of the ruminant’s digestive tract.
Man has long been aware of microbial attachment to solid surfaces in the sea and in freshwaters, as slime layers formed by attached microorganisms and their associated polymers are easily detected by touch. Moreover, such slime layers frequently become a nuisance, or even a serious fouling problem, when they occur on submerged man-made structures, such as ship hulls, platforms, or dams.
Methods to adapt hybridization techniques for preparations of cells and chromosomes were devised and introduced in 1969 and used successfully to localize highly reiterated or amplified genes. Improvements in both hybridization techniques and labeling of specific probes have extended the compass of in situ hybridization to detection of single genomes of viruses in cells and single genes in chromosomes. These developments herald a new era in the analysis of viral infections and the role of viruses in chronic diseases and present some exceptional opportunities to examine the expression of genes in individual cells in other disciplines, such as developmental biology. This chapter provides descriptions of methods, presents examples of the use of in situ hybridization in virology, and discusses some of the important issues of which solution will be greatly influenced by in situ hybridization. In in situ hybridization, a nucleic acid probe, labeled radioactively or with a reporter molecule, is annealed to suitably prepared cells or chromosomes, and the formation of hybrids is assessed by autoradiography, by fluorescence, or by histochemical means. The great increase in sensitivity achieved in current techniques is the result of the increased signal generated by contemporary probes and the increased efficiency of hybridization. This chapter reflects these improvements, first, in the preparation of probes and, second, in the treatment of cells and conditions for hybridization.
A new genus and species of obligate intracellular bacterial parasite of small free-living amoebae is described. This bacterium causes fatal infections in amoebae belonging to the Acanthamoeba-Naegleria group. It does not grow on any artificial substrate deprived of living amoeba cells. The entry of the bacterium into a host occurs by phagocytosis, but growth occurs in the cytoplasm, not in phagosomes. This parasite is readily distinguished from other kinds of previously recognized bacteria that live within amoeba cells on the basis of its host cell lytic activity. The bacterium is a gram-negative, short rod with tapered ends. It multiplies intracellularly by a transverse central pinching-off process. Cells are motile by means of a polar tuft of flagella. The bacterium is surrounded by a distinct multilayered cell envelope with a chemtype A1γ peptidoglycan. The peptidoglycan is unique in its high content of glucosamine residues with free amino groups. The DNA base composition of this organism is 43 mol% guanine plus cytosine. The name Sarcobium lyticum gen. nov., sp. nov. is proposed for this bacterium. The type of S. lyticum is strain L2 (= PCM 2298).
Based on comparative analyses of 16S and 23S ribosomal RNA sequences we have located sites specific for the alpha-, beta-, and gamma-subclasses of Proteobacteria. Short oligodeoxynucleotides complementary to these signature regions were evaluated as potential nucleic acid probes for the differentiation of the major subclasses of Proteobacteria. Hybridization conditions were optimized by the addition of formamide to the hybridization buffer and high stringency post-hybridization washing. Single-mismatch discrimination of probes was further improved by blocking nontarget probe binding sites with competitor oligonucleotides. Nonisotopic dot-blot hybridization to reference strains demonstrated the expected probe specificities, whole cell hybridization with fluorescent probe derivatives allowed the classification of individual microbial cells. The probes will be useful for determinative studies and for the in situ monitoring of population distribution and dynamics in microbial communities.
The 16S rDNA genes of an apparently pure culture of a psychrophilic and strict barophilic bacterium (WHB 46) were studied by PCR-mediated amplification and cloning into phage M13 mp18. Sequence analysis of five individual clones revealed the presence of two different 16S rDNA types. The homology value of 90% indicates that culture WHB 46 is actually composed of two closely related species (WHB 46-1 and 46-2). Both strains are members of the γ-subdivision of proteobacteria. Analysis of a sixth clone (WHB 46-1/2) leads to the conclusion that it represents a 16S rDNA hybrid molecule assembled during the PCR reaction. This hypothesis was confirmed by secondary structure analysis of the chimeric rDNA. The appearance of such hybrid molecules point to a potential risk in studies on the diversity of bacterial populations by analysis of rDNA pattern via PCR-mediated amplification because they suggest the existence of organisms that do not actually exist in the sample investigated.
Sequences of 23S rRNA specific for the type strains of Streptococcus thermophilus and Streptococcus salivarius were identified and complementary oligonucleotides synthesized. The specifity of the probes was checked by dot blot hybridization. The probes reacted only with strains of S. thermophilus and S. salivarius, respectively. Other lactic acid bacteria tested did not react with these oligonucleotide probes.
Gram-negative filamentous bacteria are commonly observed in activated sludge and contribute to poor settlement of activated sludge flocs in secondary sedimentation tanks, a problem referred to as activated sludge bulking. However, the standard morphological identification system is of limited value for a high resolution, rapid monitoring of these bacteria. Therefore, specific 16S rRNA-targeted oligonucleotide probes were developed for Haliscomenobacter spp., Sphaerotilus spp., Leptothrix spp., Thiothrix spp., Leucothrix mucor and bacteria of the Eikelboom type 021N. Probe specificities were evaluated by nonisotopic dot blot hybridization to 145 reference strains representing a diverse collection of taxa. In situ hybridization with fluorescent probe derivatives was combined with scanning confocal laser microscopy (SCLM) for analyzing the three dimensional localization of the filaments inside the sludge flocs. Filaments could be localized even in the center of fixed flocs at a high resolution undisturbed by problems like autofluorescence.
The genera Lactococcus, Enterococcus, and Streptococcus contain species of biotechnological and medical importance. The need for rapid and reliable identification of isolates has prompted the development of several 16S and 23S rRNA based oligonucleotide probes. Fluorescent derivatives of probes specific for Lactococcus lactis, L. lactis subsp. cremoris, Enterococcus faecalis, E. faecium, Streptococcus salivarius and S. thermophilus were used for whole cell hybridization. Optimal probe permeability of fixed cells required special pretreatment of these Gram-positive organisms. Fluorescent probing in combination with epifluorescence microscopy resulted in single cell identification. With this assay lactococci, enterococci, and streptococci can be identified in milk samples within one day.
Genetic diversity among 15 strains of Fibrobacter succinogenes and F. intestinalis was evaluated by comparative 16S rRNA sequencing, by estimation of genomic DNA similarity, and by restriction endonuclease digestion patterns. The previous division of the genus into two genetically well-resolved species (F. succinogenes and F. intestinalis) was confirmed by the additional comparative studies. All measures of genomic relatedness were consistent. The two species were related at approximately 91 to 93% 16S rRNA sequence similarity and less than 20% genomic DNA similarity. F. succinogenes subsp. elongata was found to comprise three subgroups related at ca 97% 16S rRNA sequence similarity; although subspecies status would be phylogenetically justifiable, difficulties in phenotypic distinction of these subgroups contraindicate formation of additional subspecies. Analysis of all pairwise comparisons showed that 50% DNA-DNA pairing corresponded to approximately 99% 16S rRNA sequence similarity.
The use of specific biomarkers for the identification and tracking of microorganisms in the environment is about to come of age. Exciting progress has been made in measuring microbial function and activity in environmental samples. A significant advance is that a variety of recent studies have correlated the genetic and chemical profiles of microbial communities with population structure and function.
Fluorescent markers appropriately conjugated to antibody proteins provide the basis for a method to visualize those antibodies as they participate in antigenantibody reactions. The method is referred to as the fluorescent antibody (FA) or immunofluorescence (IF) technique; it has been in widespread and successful use in medical microbiology and in pathology as a highly sensitive and specific cytochemical staining procedure for many years. Most current applications of the technique are for the localization of cellular and viral antigens in tissues and for the rapid detection and identification of infectious agents. The same features of the FA technique that make possible the detection and identification of microbial pathogens in animal tissues provide its potential for autecological application in microbial ecology. Autecology, as an approach whereby the individual microorganism is studied directly in its natural environment, has been virtually unavailable to microbial ecology because of technical difficulties imposed by the small size and nondescript morphology of microorganisms and by the physical complexities of the natural environment. Direct microscopic examination of the natural environment could become a powerful tool for autecological study if a method were available to recognize one specific microorganism among all others present in the field of view. Such a capability is inherent in the FA technique. Because extremely small amounts of fluorochrome-marked antibody may be detected against a dark background by fluo
The phylogenetic diversity of macroaggregate-attached vs. free-living marine bacteria, co-occurring in the same water mass, was compared. Bacterial diversity and phylogcnetic identity were inferred by analyzing polymerase chain reaction (PCR) amplified, cloned ribosomal RNA (rRNA) genes. Ribosomal RNA genes from macroaggregatc-associated bacteria were fundamentally different from those of free-living bacterio- plankton. Most rRNA types recovered from the free-living bacterioplankton were closely related to a phenotypically undcscribcd (Y Proteobacteria group, previously detected in surface waters of North Pacific and Atlantic central ocean gyres. The results suggest that members of this phylogenetically distinct, (Y proteobacterial group are abundant free-living bactcrioplankters in coastal, as well as open-ocean habitats. In contrast, most macroaggregate-associated rRNA clones were closely related to Cytophuga, Plancto- myce.s, or y Proteobacteria, within the domain Bacteria. These data indicate that specific bacterial pop- ulations, different from those which predominate in free-living bacterioplankton, develop on marine phytodetrital aggregates. The inferred properties of attached bacterial assemblages have significant im- plications for models of microbially mediated transformation of particulate organic material.
Advances in molecular biology are now providing the means for solving long-standing problems in microbiology. One of the best examples is the development of a rational approach to the phylogenetic classification of microorganisms, based on comparative analysis of slowly evolving molecular components, most notably ribosomal RNAs (Woese, 1987). Molecular biologists and microbiologists have been quick to recognize how rRNA sequence variation could be used to answer major questions limiting progress in microbial ecology. Only a few years after the initial rRNA-based phylogenetic observations were published (Woese and Fox, 1977), the 16S rRNA molecule was used to characterize Prochloron, an uncultivated symbiont of marine invertebrates (Seewaldt and Stackebrandt, 1982), and the smallest ribosomal RNA molecule, 5S rRNA, was used to analyze the composition of a few simple microbial communities (Stahl et al., 1984, 1985; Lane et al., 1985b). Some further ecologic work with 5S rRNA has appeared (Colwell et al., 1989), but extensive community analysis with this molecule is complicated by the difficulty of physically separating 5S rRNAs, and by the relatively small size and thus limited information content of this molecule. In the last few years, considerable emphasis has been given in both microbial phylogeny and microbial ecology to the development of methods for studying the larger and more informative rRNAs. Most of the work has been with small ribosomal subunit rRNA (SSU rRNA, 16S in prokaryotes and 18S in eukaryotes), though a limited amount of work has been done with the larger rRNAs of large ribosomal subunits (here termed LSU rRNA, 23S in prokaryotes and 28S in eukaryotes) and with internal transcribed spacer (ITS) regions separating rRNA genes.
The marine ciliate Strombidium purpureum Kahl harbours endosymbiotic purple non-sulphur bacteria. The bacteria contain bacteriochlorophyll a and the carotenoid spirilloxanthin, and they have photosynthetic membranes and cell walls. The ciliates require light for survival and growth under anaerobic conditions; in the dark the cells prefer microaerobic conditions. The ciliates show a photosensory behaviour, and they accumulate in light at wave lenghts corresponding to the absorption spectrum of the symbionts. The findings are discussed in terms of theories on the endosymbiotic origin of mitochondria.
A magnetotactic spirillum (strain MSR-1) was isolated from the mud of the eutrophic river Ryck near Greifswald. Its morphological, ultrastructural and biochemical characteristics were compared with those of Aquaspirillum magnetotacticum. A cell suspension of strain MSR-1 was used to amplify in vitro 16S rRNA genes (rDNA). The amplified 16S rDNA was cloned and sequenced. For comparison 16S rDNA was also directly amplified from a stock culture of Aquaspirillum magnetotacticum DSM 3856T. It was cloned, sequenced and compared with the 16S rDNA sequences of strain MSR-1 and other bacteria. An oligonucleotide probe complementary to a 16S rRNA region unique to the two magnetic spirilla was synthesized. Specific hybridization of the fluorescent oligonucleotide to whole cells of both strains demonstrated that the 16S rDNA clones originated from strain MSR-1 and A. magnetotacticum. Aquaspirillum magnetotacticum and strain MSR-1 are related and belong to the alpha subclass of Proteobacteria, whereas Aquaspirillum serpens, the type species of the genus Aquaspirillum, is a member of the beta subclass of Proteobacteria. A new genus Magnetospirillum sp. gen. with the type species Magnetospirillum gryphiswaldense sp. nov., and the transfer of A. magnetotacticum to Magnetospirillum magnetotacticum comb. nov. are proposed. The type strain of M. gryphiswaldense is DSM 6361.
Magnetotactic bacteria show a diverse morphology and are widely distributed in nature. Currently, only a few of them can be grown in axenic culture. To identify and characterize also nonculturable magnetotactic bacteria we examined enriched populations from freshwater sediment with a combination of 16S rRNA sequence retrieval and fluorescent whole cell hybridization. Comparative sequence analyses revealed an unexpectedly high diversity within the retrieved rRNA molecules exceeding the number of observed morphotypes. Fluorescent oligonucleotides complementary to unique signature regions of distinct rRNA sequences assigned three frequently occurring sequences to three populations of magnetotactic cocci. The analysed cocci group with the alpha-subclass of the proteobacterial phylum and form there a separate lineage of descent. Hardly discriminated on a morphological basis the three genotypes also demonstrated partially different tactic behaviour. The combination of 16S rRNA sequence retrieval and fluorescent whole cell hybridization has opened a new dimension to the structural analysis of natural communities. It is for the first time that nonculturable bacteria from multispecies enrichments could be identified on the single cell level.
The colonization of biofilms of a benzoate-degrading Gram-positive water bacterium, strain B4, by a pathogenic Escherichia coli was studied in a continuous flow reactor. E. coli added to a fixed bed reactor colonized by B4, was able to grow in the biofilms and subsequently re-enter the free water phase in high numbers. Mixed biofilms of strain B4 and E. coli were also grown on glass slides for detailed examination of the spatial order of the mixed population biofilm. Individual cells as well as microcolonies of E. coli were detected in the biofilms by hybridization with a fluorescently labeled 23S rRNA-targeted oligonucleotide probe. The spatial distribution of E. coli could be analyzed in all layers of even thick biofilms.
Obligately endosymbiotic bacteria living in the cytoplasm of ciliates of the genus Euplotes constitute the new genus Polynucleobacter gen. nov. These endosymbionts are commonly known as omicron and omicronlike particles. The best-studied form, as well as the type species, is omicron from stock 15 of Euplotes aediculatus. We propose the name Polynucleobacter necessarius gen. nov., sp. nov., for this bacterium. Endosymbiotic bacteria often are seen in protozoa. How- ever, only a few of them have been studied extensively, and several of these are still referred to by Greek letters, as is customary for cytoplasmic elements. Among the endosym- bionts which are well characterized but do not have binary names are omicron and the omicronlike endosymbionts. The purpose of the present paper is to name this group of symbionts in accordance with the rules of the Bacteriological Code. The name Polynucleobacter was selected to indicate a characteristic feature of this genus: all members show mul- tiple nucleoids easily visible in these endosymbionts after staining with acetocarmin, acetoorcein, or deoxyribonucleic acid (DNA)-fluorescent dyes. The epithet necessarius was chosen to indicate the relationship of omicron and its host Euplotes aediculatus, which depend upon each other (3). Omicron. The characteristic features of omicron, orig- inally described by Heckmann (2), are summarized as fol- lows. Approximately 900 to 1,000 cells of P. necessarius inhabit the cytoplasm of their host. They have a length of 2.5 to 7.5 pm (most commonly 5 pm) and a width of about 0.3 pm, and are gram negative. If P. necessarius is stained with DNA-specific dyes, 3 to 9 and in some cases up to 12 intensely stained and regularly spaced dots become visible (Fig. 1). They are considered to be nucleoids. When studied with the electron microscope, these nucleoids differ from those of most free living bacteria by exhibiting an electron- dense central core, which resembles the chromatin of eu- caryotes (Fig. 2). Whether this core is formed by proteins associated with DNA or some other material is not clear. The symbionts are contained in vesicles to which ribosomes are often attached. P. necessarius reproduces by transverse binary fission in a typical bacterial manner. The fission products were, however, often found to differ from each other in size. It is possible to isolate P. necessarius from E. aediculatus homogenates and to obtain pure preparations of the symbionts in quantities large enough for an extraction and characterization of their DNA. The guanine-plus-cytosine (G+C) content was computed to be 48 mol% by thermal denaturation (T,J. A value 2.8% lower was found when the G+C content was calculated from the buoyant density of the DNA, which was 1.7036 g/cm3 (5). Similar differences re- ported by others are explained as caused by rare bases, but no DNA chemistry is available for P. necessarius. The average DNA content of this symbiont is 5.8 x lop3 pg per
A set of six oligonucleotides, complementary to conserved tracts of 16S rRNA from phylogenetically-defined groups of sulfate-reducing bacteria, was characterized for use as hybridization probes in determinative and environmental microbiology. Four probes were genus specific and identified Desulfobacterium spp., Desulfobacter spp., Desulfobulbus spp., or Desulfovibrio spp. The other two probes encompassed more diverse assemblages. One probe was specific for the phylogenetic lineage composed of Desulfococcus multivorans, Desulfosarcina variabilis, and Desulfobotulus sapovorans. The remaining probe was specific for Desulfobacterium spp., Desulfobacter spp., D. multivorans, D. variabilis, and D. sapovorans. Temperature of dissociation was determined for each probe and the designed specificities of each were evaluated by hybridizations against closely related nontargeted species. In addition, each probe was screened by using a 'phylogrid' membrane which consisted of nucleic acids from sixtyfour non-targeted organisms representing a diverse collection of eukarya, archaea, and bacteria. The value of these probes to studies in environmental microbiology was evaluated by hybridizations to 16S rRNAs of sulfate-reducing bacteria present in marine sediments.
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Azospirillum is a soil bacterium with plant growth-promoting potential. Strain-specific monoclonal antibodies (mAbs) which bind to different antigenic determinants of the bacterial cell surface with high specificity for A. brasilense Sp7 were used to monitor this bacterium in the rhizosphere of wheat plants. The mAbs were marked directly with the fluorochromes fluorescein (FITC) or tetramethylrhodamine (TRITC). With the labelled mAbs and confocal scanning laser microscopy, an in situ identification and localization of this bacterium in root segments was possible.
The difference between the results of viable and total counting procedures for bacteria are exemplified by vertical profiles from a deep and a shallow lake and from seasonal changes in the epilimnion and hypolimnion of a shallow eutrophic lake. The viable count was, on average, 0.25% of the total count, the greatest difference being noted in the anoxic hypolimnion, probably due to the inadequacy of the viable counting procedure for the isolation of bacteria from such samples. There was a general trend for the more nutrient-rich waters to support larger bacterial populations but such observations did not provide any further information on the factors responsible for the population changes observed. Seasonal fluctuations in the counts are studied and the qualitative and quantitative changes resulting from artificial enclosure of water are discussed. Not all the temporal changes could be explained and short-term changes resulting from nutrient additions to the experimental enclosures were not always reproducible. Horizontal variability was examined, found to be significant and could play an important role where water movement and turbulence is considerable. Results from six sites sampled between 1969 and 1974, representing total and viable bacterial population estimates and a total of eighteen independent or regressor variables were then subjected to principal components analysis. Results taken from the whole water column showed the overwhelming effect of the process of stratification on the bacterial population accounting for 30%-60% of its variability. Secondary components representing algal productivity could account for 10% to 20% of the variability. Many of the chosen regressor variables were acting as measures of the same phenomenon without providing significant information on what affected the bacterial population. To overcome this problem results from the hypolimnion and epilimnion were analysed separately. The analysis demonstrated the importance of seasonal changes in nutrient concentrations in the epilimnion and the development of anoxic conditions in the hypolimnion. Algal biomass, phosphate concentration and the interaction of pH and ammonia appeared to be important. It was concluded that most of the variability in the bacterial population estimates could have been explained by five of the regressor variables and that the factors most likely to provide more information would include some measure of predation and lake retention time.
Fluorescent bacteria were demonstrated to be abundantly spread as single cells throughout the cytoplasm of the giant amoeba Pelomyxa palustris, the sapropelic ciliate Metopus striatus and six other anaerobic protozoa examined. The endosymbionts of P. palustris and M. striatus were identified as methanogenic bacteria on the basis of the presence of the deazaflavin coenzyme F420 and the pterin compound F342. Moreover individuals of P. palustris produced methane over a long period of incubation. The number of methanogenic bacteria was above 1010 cells/ml protozoal cytoplasm. Two types of methanogenic bacteria together with unidentified thick bacteria were found in P. palustris.The physiological background of this endosymbiosis and its functioning in degradation processes in the anoxic environment are discussed.
An in situ hybridization procedure with a fluorescent RNA probe applied to yeast detection by flow cytometry is described. Saccharomyces ceerevisiae cell were permeabilized to allow the use of a biotinylated rRNA probe of about ≈ 100 nucleotides, by formaldehyde treatment, followed by a limited enzymatic hydrolysis of the cell wall. Hybridization was performed with cells in suspension and hybrids were detected with streptavidin-FITC (fluorescein isothiocyanate) by microscopy and flow cytometry. The signal obtained with yeast antisense rRNA probe was 30 times higher than the nonspecific signal obtained with a bacterial sense rRNA probe. The yeast-specific signal was not eliminated after posthybridization RNase treatment althoug it completely disappeared when cells were treated with RNase before hybirdization, confirming the rRNA was the target. This study is the first demonstration of the use of in situ hybridization for yeast detection by flow cytomertry. With appropriate probes, the procedure could be used also for detection of specific yeast populations.
Five polymerase chain reaction (PCR) primer pairs were synthesized on the basis of the aligned 16S-like rRNA sequences of eukaryotes or 16S rRNA sequences of eubacteria, Mollicutes, and intracellular organelles. These PCR primer pairs had high sequence homology to the conserved 16S rRNA genes of various culturable and nonculturable Mollicutes, but less sequence homology to the eukaryotic nuclear 16S-like rRNA or 16S rRNA genes of intracellular organelles. Full-length 16S rRNA genes and partial-length 16S rRNA genes of evolutionarily variable regions were successfully amplified when DNA preparations from culturable Mollicutes such as Mycoplasma flocculare and three Spiroplasma strains and nonculturable Mollicutes associated with various plant diseases were used as PCR templates. Amplifications were not detected when Escherichia coli genomic DNA and DNA preparations from healthy plants were used under high stringency annealing conditions in thermocycling. The results suggest the possibility that 16S rRNA genes of culturable and nonculturable Mollicutes can be amplified for detection and for a phylogenetic study using crude Mollicutes DNA preparations under appropriately controlled thermocycling conditions.
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From hot marine sediments (depth: 106 m) at the Kolbeinsey Ridge, Iceland, novel bacterial hyperthermophiles were isolated. Cells were Gram-negative highly motile rods exhibiting a complex envelope consisting of murein, an outer membrane and a surface protein layer. Growth occurred between 67 and 95 °C (opt.: 85 °C; 75 min doubling time), pH 5.4 and 7.5 (opt.: pH 6.8), and 1 to 5% NaCl (opt.: 3% NaCl). The novel isolates were strict chemolithoautotrophs. They used molecular hydrogen, thiosulfate and elemental sulfur as electron donors and oxygen (low concentrations) and nitrate as electron acceptors. During growth, sulfuric acid was formed from S° and thiosulfate. In the late logarithmic growth phase, H2S was formed from S° and H2. The core lipids consisted mainly of alkyl ethers of glycerol. The GC content of the DNA was 40 mol%. By 16S rRNA comparisons, the new isolates did not belong to any of the phyla known and represent the deepest phylogenetic branch-off within the Bacteria domain. Based on the physiological and molecular properties of the new isolates, we describe here a new genus, which we name Aquifex (the “water-maker”). The type species is Aquifex pyrophilus (type strain: Kol5a; DSM 6858).