Testing culture purity in prokaryotes: criteria and challenges.

Reliance on pure cultures was introduced at the beginning of microbiology as a discipline and has remained significant although their adaptive properties are essentially dissimilar from those of mixed cultures and environmental populations. They are needed for (i) taxonomic identification; (ii) diagnostics of pathogens; (iii) virulence and pathogenicity studies; (iv) elucidation of metabolic properties; (v) testing sensitivity to antibiotics; (vi) full-length genome assembly; (vii) strain deposition in microbial collections; and (viii) description of new species with name validation. Depending on the specific task there are alternative claims for culture purity, i.e., when conventional criteria are satisfied or when looking deeper is necessary. Conventional proof (microscopic and plating controls) has a low resolution and depends on the observer's personal judgement. Phenotypic criteria alone cannot prove culture purity and should be complemented with genomic criteria. We consider the possible use of DNA high-throughput culture sequencing data to define criteria for only one genospecies, axenic state detection panel and only one genome. The second and third of these are preferable, although their resolving capacity (depth) is limited. Because minor contaminants may go undetected, even with deep sequencing, the reliably pure culture would be a clonal culture launched from a single cell or trichome (multicellular bacterium). Although this type of culture is associated with technical difficulties and cannot be employed on a large scale (the corresponding inoculums may have low chances of growth when transferred to solid media), it is hoped that the high-throughput culturing methods introduced by 'culturomics' will overcome this obstacle.

Evolution of prokaryotic and eukaryotic virulence effectors.

Coevolutionary interactions between plants and their bacterial and eukaryotic pathogens are mediated by virulence effectors. These effectors face the daunting challenge of carrying out virulence functions, while also potentially exposing the pathogen to host defense systems. Very strong selective pressures are imposed by these competing roles, and the subsequent genetic changes leave their footprints in the extant allelic variation. This review examines the evolutionary processes that drive pathogen-host interactions as revealed by the genetic signatures left in virulence effectors, and speculate on the different pressures imposed on bacterial versus eukaryotic pathogens. We find numerous instances of positive selection for new allelic forms, and diversifying selection for genetic variability, which results in altered host-pathogen interactions. We also describe how the genetic structure of both bacterial and eukaryotic virulence effectors may contribute to their rapid generation and turnover.

Ecological Processes Affecting Long-Term Eukaryote and Prokaryote Biofilm Persistence in Nitrogen Removal from Sewage.

The factors affecting long-term biofilm stability in sewage treatment remain largely unexplored. We therefore analyzed moving bed bioreactors (MBBRs) biofilm composition and function two years apart from four reactors in a nitrogen-removal sewage treatment plant. Multivariate ANOVA revealed a similar prokaryote microbiota composition on biofilm carriers from the same reactors, where reactor explained 84.6% of the variance, and year only explained 1.5%. Eukaryotes showed a less similar composition with reactor explaining 56.8% of the variance and year 9.4%. Downstream effects were also more pronounced for eukaryotes than prokaryotes. For prokaryotes, carbon source emerged as a potential factor for deterministic assembly. In the two reactors with methanol as a carbon source, the bacterial genus Methylotenera dominated, with M. versatilis as the most abundant species. M. versatilis showed large lineage diversity. The lineages mainly differed with respect to potential terminal electron acceptor usage (nitrogen oxides and oxygen). Searches in the Sequence Read Archive (SRA) database indicate a global distribution of the M. versatilis strains, with methane-containing sediments as the main habitat. Taken together, our results support long-term prokaryote biofilm persistence, while eukaryotes were less persistent.

Prokaryotic and eukaryotic microbiomes associated with blooms of the ichthyotoxic dinoflagellate Cochlodinium (Margalefidinium) polykrikoides in New York, USA, estuaries.

While harmful algal blooms caused by the ichthyotoxic dinoflagellate, Cochlodinium (Margalefidinium) polykrikoides, are allelopathic and may have unique associations with bacteria, a comprehensive assessment of the planktonic communities associated with these blooms has been lacking. Here, we used high-throughput amplicon sequencing to assess size fractionated (0.2 and 5 µm) bacterial (16S) and phytoplankton assemblages (18S) associated with blooms of C. polykrikoides during recurrent blooms in NY, USA. Over a three-year period, samples were collected inside ('patch') and outside ('non-patch') dense accumulations of C. polykrikoides to assess the microbiome associated with these blooms. Eukaryotic plankton communities of blooms had significantly lower diversity than non-bloom samples, and non-bloom samples hosted 30 eukaryotic operational taxonomic units (OTUs) not found within blooms, suggesting they may have been allelopathically excluded from blooms. Differential abundance analyses revealed that C. polykrikoides blooms were significantly enriched in dinoflagellates (p<0.001) and the experimental enrichment of C. polykrikoides led to a significant increase in the relative abundance of eight genera of dinoflagellates but a significant decline in other eukaryotic plankton. Amoebophrya co-dominated both within- and near- C. polykrikoides blooms and was more abundant in bloom patches. The core bacterial microbiome of the >0.2µm fraction of blooms was dominated by an uncultured bacterium from the SAR11 clade, while the >5µm size fraction was co-dominated by an uncultured bacterium from Rhodobacteraceae and Coraliomargarita. Two bacterial lineages within the >0.2µm fraction, as well as the Gammaproteobacterium, Halioglobus, from the >5µm fraction were unique to the microbiome of blooms, while there were 154 bacterial OTUs only found in non-bloom waters. Collectively, these findings reveal the unique composition and potential function of eukaryotic and prokaryotic communities associated with C. polykrikoides blooms.

Characterization of oral microbiota in marmosets: Feasibility of using the marmoset as a human oral disease model.

With rapid aging of the world's population, the demand for research, for a better understanding of aging and aging-related disorders, is increasing. Ideally, such research should be conducted on human subjects. However, due to ethical considerations, animals such as rodents and monkeys are used as alternatives. Among these alternative models, non-human primates are preferred because of their similarities with humans. The small South American common marmoset (Callithrix jacchus) may offer several advantages over other non-human primates in terms of its smaller size, shorter life-span, and dental anatomy identical to humans. The purpose of this study was to determine the viability of using the marmoset as a human oral disease model. We collected saliva samples from eight marmosets and eight human subjects. Prokaryotic DNA was extracted from the saliva samples, and 16S bacterial rRNA gene sequencing was performed on each of the samples. Our results indicated that the types of oral microbiomes detected among human and marmoset samples were nearly indistinguishable. In contrast, the oral microbiomes of our human and marmoset subjects were distinctly different from those reported for rats and dogs, which are currently popular research animals. The oral microbiomes of marmosets showed greater diversity than those of humans. However, the oral microbiota of marmosets exhibited less variation than those of humans, which may be attributed to the fact that the marmoset subjects were kept in a controlled environment with identical lifestyles. The characteristics of its oral microbiota, combined with other technical advantages, suggest that the marmoset may provide the best animal model thus far for the study of oral health. This study characterized the oral microbes of the marmoset, thereby providing information to support future application of the marmoset as a model for age-related oral disease.

Identification of new promising plant-based lead compounds for inhibition of prokaryotic replicative DNA polymerases: combination of in silico and in vitro studies.

Emerging widespread bacterial resistance to current antibiotics with traditional targets is one of the major global concerns. Therefore, so many investigations are exploring the potential of other druggable macromolecules of bacteria such as replication machinery components that are not addressed by previous antibiotics. DNA polymerase is the major part of this machine. However, a few studies have been done on it so far. In this respect, we report the discovery of four new plant-based leads against DNA polymerase (pol) IIIC (three leads) and pol IIIE (one lead) of Gram-positive and negative bacteria by combining a sequentially constrained high-throughput virtual screenings on Traditional Chinese Medicine Database with in vitro assays. The compounds displayed relatively good levels of inhibitory effect. They were active against their designated targets at micromolar concentrations. The IC50 values for them are ranged from 25 to 111 µM. In addition, they showed minimum inhibitory concentrations in the range of 8-128 µg/mL against five representatives of pathogenic bacteria species. However, they were inactive against Pseudomonas aeruginosa. Given these results, these leads hold promise for future modification and optimization to be more effective in lower concentrations and also against most of the important bacterial species. Communicated by Ramaswamy H. Sarma.

Co-existence of multiple bacterivorous clevelandellid ciliate species in hindgut of wood-feeding cockroaches in light of their prokaryotic consortium.

The hindgut of wood-feeding Panesthia cockroaches harbours a diverse microbial community, whose most morphologically prominent members are bacterivorous clevelandellid ciliates. Co-occurrence and correlation patterns of prokaryotes associated with these endosymbiotic ciliates were investigated. Multidimensional scaling based on taxa interaction-adjusted index showed a very clear separation of the hindgut ciliate samples from the ciliate-free hindgut samples. This division was corroborated also by SparCC analysis which revealed strong negative associations between prokaryotic taxa that were relatively more abundant in the ciliate-free hindgut samples and prokaryotic taxa that were more abundant in the ciliate samples. This very likely reflects the grazing behaviour of hindgut ciliates which prefer Proteobacteria, Firmicutes and Actinobacteria, causing their abundances to be increased in the ciliate samples at the expense of abundances of Euryarchaeota and Bacteroidetes which prevail in the hindgut content. Ciliate species do not distinctly differ in the associated prokaryotes, indicating that minute variations in the proportion of associated bacteria might be sufficient to avoid competition between bacterivorous ciliate species and hence enable their co-occurrence in the same host. The nearest free-living relatives of hindgut ciliates have a different pattern of associations with prokaryotes, i.e., alphaproteobacteria are predominantly associated with free-living ciliates while gammaproteobacteria with hindgut ciliates.

A Pseudomonas aeruginosa type VI secretion phospholipase D effector targets both prokaryotic and eukaryotic cells.

Widely found in animal and plant-associated proteobacteria, type VI secretion systems (T6SSs) are potentially capable of facilitating diverse interactions with eukaryotes and/or other bacteria. Pseudomonas aeruginosa encodes three distinct T6SS haemolysin coregulated protein (Hcp) secretion islands (H1, H2, and H3-T6SS), each involved in different aspects of the bacterium's interaction with other organisms. Here we describe the characterization of a P. aeruginosa H3-T6SS-dependent phospholipase D effector, PldB, and its three tightly linked cognate immunity proteins. PldB targets the periplasm of prokaryotic cells and exerts an antibacterial activity. Surprisingly, PldB also facilitates intracellular invasion of host eukaryotic cells by activation of the PI3K/Akt pathway, revealing it to be a trans-kingdom effector. Our findings imply a potentially widespread T6SS-mediated mechanism, which deploys a single phospholipase effector to influence both prokaryotic cells and eukaryotic hosts.

Unicellular but not asocial. Life in community of a bacterium

All living organisms have acquired the outstanding ability to overcome the limitations imposed by changeable environments through the gain of genetic traits over years of evolution and the tendency of individuals to associate in communities. The complementation of a singular weakness, the deployment of reinforcement for the good of the community, the better use of resources, or effective defense against external aggression are advantages gained by this communal behavior. Communication has been the cohesive element prompting the global responses that promote efficiency in two features of any community: specialization in differentiated labor and the spatio-temporal organization of the environment. These principles illustrate that what we call human ecology also applies to the cellular world and is exemplified in eukaryotic organisms, where sophisticated cell-to-cell communication networks coordinate cell differentiation and the specialization of multiple tissues consisting of numerous cells embedded in a multifunctional extracellular matrix. This sophisticated molecular machinery appears, however, to be invented by the ‘simple’ but still fascinating bacteria. What I will try to expand in the following sections are notions of how ‘single prokaryotic cells’ organize a multicellular community (AU)

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Ecological traits of planktonic viruses and prokaryotes along a full-salinity gradient.

Virus-prokaryote interactions were investigated in four natural sites in Senegal (West Africa) covering a salinity gradient ranging from brackish (10‰) to near salt saturation (360‰). Both the viral and the prokaryote communities exhibited remarkable differences in their physiological, ecological and morphological traits along the gradient. Above 240‰ salinity, viral and prokaryotic abundance increased considerably with the emergence of (1) highly active square haloarchaea and of (2) viral particles with pleiomorphic morphologies (predominantly spindle, spherical and linear shaped). Viral life strategies also showed some salinity-driven dependence, switching from a prevalence of lytic to lysogenic modes of infection at the highest salinities. Interestingly, the fraction of lysogenized cells was positively correlated with the proportion of square cells. Overall, the extraordinary abundance of viruses in hypersaline systems (up to 6.8 × 10(8) virus-like particles per milliliter) appears to be partly explained by their high stability and specific ability to persist and proliferate in these apparently restrictive habitats.

Endosymbiotic associations within protists.

The establishment of an endosymbiotic relationship typically seems to be driven through complementation of the host's limited metabolic capabilities by the biochemical versatility of the endosymbiont. The most significant examples of endosymbiosis are represented by the endosymbiotic acquisition of plastids and mitochondria, introducing photosynthesis and respiration to eukaryotes. However, there are numerous other endosymbioses that evolved more recently and repeatedly across the tree of life. Recent advances in genome sequencing technology have led to a better understanding of the physiological basis of many endosymbiotic associations. This review focuses on endosymbionts in protists (unicellular eukaryotes). Selected examples illustrate the incorporation of various new biochemical functions, such as photosynthesis, nitrogen fixation and recycling, and methanogenesis, into protist hosts by prokaryotic endosymbionts. Furthermore, photosynthetic eukaryotic endosymbionts display a great diversity of modes of integration into different protist hosts. In conclusion, endosymbiosis seems to represent a general evolutionary strategy of protists to acquire novel biochemical functions and is thus an important source of genetic innovation.

Temperature influences synonymous codon and amino acid usage biases in the phages infecting extremely thermophilic prokaryotes.

To see the effect of temperature on the codon and amino acid usage in phages, codon and amino acid usage of 13 phages of extremely thermophilic prokaryotes were compared with that of 14 phages of mesophilic prokaryotes. Correspondence analysis on RSCU values of two groups of phage genomes clearly shows that phages are separated along the second major axis according to their growth temperature, whereas, they are separated along the first major axis according to their GC content. Correspondence analysis on RAAU values of two groups of phages clearly shows that protein encoding genes of the phages along the second major axis are highly correlated with the GRAVY, aromaticity and cysteine content. Moreover, correspondence analysis on the regular and irregular structures of proteins of phages infecting extremely thermophilic prokaryotes reveals that temperature is one of the factors responsible for most significant differentiation of codon and amino acid usages variation in these phages.

[Proterozoic history and present state of cyanobacteria]. / Proterosoiskaia istoriia tsianobakterii i ikh sovremennoe sostoianie.

The paper delves into the main regularities of the distribution of fossil microorganisms in Precambrian rocks, beginning from the Archean Eon about 3.5 billion years ago and ending in the Cambrian Period about 0.5 billion years ago. The paper analyzes facial peculiarities in the lateral differentiation of microfossils in Proterozoic basins and the main stages of temporal changes in fossil cyanobacterial communities, which are based on the irreversible succession of physicochemical conditions on the Earth and the evolution of eukaryotic microorganisms and their incorporation into prokaryotic ecosystems. To gain insight into Proterozoic fossil records, modern stratified cyanobacterial mats built up from layers of prokaryotes are considered. The analysis of phosphatization, carbonatization, and silification processes in modern algal-bacterial communities suggests that analogous processes took place in Proterozoic microbiotas. A comparison of modern and Precambrian living forms confirms the inference that cyanobacterial communities are very conservative and have changed insignificantly both morphologically and physiologically during the past two billion years.

Identification of individual prokaryotic cells by using enzyme-labeled, rRNA-targeted oligonucleotide probes.

A method to microscopically detect and identify individual cells of members of the domains Bacteria and Archaea is presented. rRNA-targeted oligonucleotides were 5' end labeled with the enzyme horseradish peroxidase and used for whole-cell hybridization. Specifically bound probe was visualized by the enzymatic formation of an intracellular precipitate from the substrate diaminobenzidine. Permeation of the enzyme-labeled probe into whole fixed cells of gram-negative bacteria required their pretreatment with lysozyme-EDTA, whereas permeability of some archaebacterial cells was improved by addition of detergent to the hybridization buffer. Hitherto we had not achieved penetration of enzyme-labeled probe into gram-positive bacteria and yeast cells. This method should be a valuable tool for identification of suitable prokaryotic cells in environments with elevated background fluorescence or in situations in which an epifluorescence microscope is not available.

Low virus to prokaryote ratios in the cold: benthic viruses and prokaryotes in a subpolar marine ecosystem (Hornsund, Svalbard)

The density and spatial distribution of benthic viruses and prokaryotes in relation to biotic and abiotic factors were investigated in sediment cores collected in Hornsund, a permanently cold fjord on the West coast of Svalbard, Norway. The cores were obtained from the mouth of the fjord to the central basin, along a longitudinal transect. The results of our analyses showed lower densities of viruses (0.2 x 10(8) to 5.4 x 10(8) virus-like particles/g) and lower virus-to-prokaryote ratios (0.2-0.6, with the exception of the uppermost layer in the central basin, where the ratio was about 1.2) at the study site than generally found in the temperate areas, despite the relatively high organic matter content in subpolar sediments. Variations in benthic viral and prokaryote abundances along gradients of particle sedimentation rates, phytopigment concentrations, and macrobenthic species composition together suggested the influence of particle sedimentation and macrobenthic bioturbation on the abundance and spatial distribution ofprokaryotes and viruses in cold habitats (AU)

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Microbial community composition of anoxic marine sediments in the Bay of Cádiz (Spain)

The composition of the microbial community inhabiting the anoxic coastal sediments of the Bay of Cádiz (southern Spain) was investigated using a molecular approach consisting of PCR cloning and denaturing gradient gel electrophoresis (DGGE), based on 16S rRNA sequences. The total cell count was 1-5 × 10⁸ cells/g sediment and, as determined by catalyzed reporter deposition-fluorescent in situ hybridization (CARD-FISH), the proportion of Bacteria to Archaea was about 70:30. The analysis of 16S-rRNA gene sequences revealed a wide spectrum of microorganisms, which could be grouped into 111 operational taxonomic units (OTUs). Many of the OTUs showed high phylogenetic similarity to microorganisms living in marine sediments of diverse geographic origin. The phylogenetic groups that were predominantly detected were Firmicutes, Deltaproteobacteria, and Gammaproteobacteria, accounting for 23, 15, and 14% of the clones, respectively. Diversity in the domain Archaea was significantly lower than in the domain Bacteria. The majority of the archaeal OTUs belonged to the Crenarchaeota phylum. Since most of the sequences could not be identified precisely at the genus/species level, the functional roles of the microorganisms in the ecosystem could not be inferred. However, seven OTUs affiliated with the Delta- and Epsilonproteobacteria were identified down to the genus level, with all of the identified genera known to occur in sulfate-rich marine environments (AU)

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Salinity dependence of the distribution of multicellular magnetotactic prokaryotes in a hypersaline lagoon

Candidatus Magnetoglobus multicellularis is an unusual magnetotactic multicellular microorganism composed of a highly organized assemblage of gram-negative bacterial cells. In this work, the salinity dependence of Ca. M. multicellularis and its abundance in the hypersaline Araruama Lagoon, Brazil were studied. Viability experiments showed that Ca. M. multicellularis died in salinities >55 per-mille and <40 per-mille. Low salinities were also observed to modify the cellular assemblage. In microcosms prepared with different salinities, the microorganism grew better at intermediate salinities whereas in high or low salinities, the size of the population did not increase over time. The concentrations of Ca. M. multicellularis in the lagoon were related to salinity; sites with lower and higher salinities than the lagoon average contained less Ca. M. multicellularis. These results demonstrate the influence of salinity on the survival and distribution of Ca. M. multicellularis in the environment. In sediments, the abundance of Ca. M. multicellularis ranged from 0 to 103 microorganisms/ml, which represented 0.001% of the counts of total bacteria. The ability of Ca. M. multicellularis to accumulate iron and sulfur in high numbers of magnetosomes (up to 905 per microorganism) suggests that its impact on the sequestration of these elements (0.1% for biogenic bacterial iron) is not proportional to its abundance in the lagoon (AU)

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Osmoadaptation mechanisms in prokaryotes: distribution of compatible solutes

Microorganisms respond to osmotic stress mostly by accumulating compatible solutes, either by uptake from the medium or by de novo synthesis. These osmotically active molecules preserve the positive turgor pressure required for cell division. The diversity of compatible solutes is large but falls into a few major chemical categories; they are usually small organic molecules such as amino acids or their derivatives, and carbohydrates or their derivatives. Some are widely distributed in nature while others seem to be exclusively present in specific groups of organisms. This review discusses the diversity and distribution of known classes of compatible solutes found in prokaryotes as well as the increasing knowledge of the genes and pathways involved in their synthesis. The alternative roles of some archetypal compatible solutes not subject to osmoregulatory constraints are also discussed (AU)

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