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Ultramicrobacteria (UMB) that can pass through a 0.22 µm filter are attractive because of their novelty and diversity. However, isolating UMB has been difficult because of their symbiotic or parasitic lifestyles in the environment. Some UMB have extracellular electron transfer (EET)-related genes, suggesting that these symbionts may grow on an electrode surface independently. Here, we attempted to culture from soil samples bacteria that passed through a 0.22 µm filter poised with +0.2 V vs. Ag/AgCl and isolated Cellulomonas sp. strain NTE-D12 from the electrochemical reactor. A phylogenetic analysis of the 16S rRNA showed 97.9% similarity to the closest related species, Cellulomonas algicola, indicating that the strain NTE-D12 is a novel species. Electrochemical and genomic analyses showed that the strain NTE-D12 generated the highest current density compared to that in the three related species, indicating the presence of a unique electron transfer system in the strain. Therefore, the present study provides a new isolation scheme for cultivating and isolating novel UMB potentially with a symbiotic relationship associated with interspecies electron transfer. [ABSTRACT FROM AUTHOR]

Cyanobacterial blooms affect biotic interactions in aquatic ecosystems, including those involving heterotrophic bacteria. Ultra-small microbial communities are found in both surface water and groundwater and include diverse heterotrophic bacteria. Although the taxonomic composition of these communities has been described in some environments, the involvement of these small cells in the fate of environmentally relevant molecules has not been investigated. Here, we aimed to test if small-sized microbial fractions from a polluted urban lagoon were able to degrade the cyanotoxin microcystin (MC). We obtained cells after filtration through 0.45 as well as 0.22 μm membranes and characterized the morphology and taxonomic composition of bacteria before and after incubation with and without microcystin-LR (MC-LR). Communities from different size fractions (< 0.22 and < 0.45 μm) were able to remove the dissolved MC-LR. The originally small-sized cells grew during incubation, as shown by transmission electron microscopy, and changed in both cell size and morphology. The analysis of 16S rDNA sequences revealed that communities originated from < 0.22 and < 0.45 μm fractions diverged in taxonomic composition although they shared certain bacterial taxa. The presence of MC-LR shifted the structure of < 0.45 μm communities in comparison to those maintained without toxin. Actinobacteria was initially dominant and after incubation with MC-LR Proteobacteria predominated. There was a clear enhancement of taxa already known to degrade MC-LR such as Methylophilaceae. Small-sized bacteria constitute a diverse and underestimated fraction of microbial communities, which participate in the dynamics of MC-LR in natural environments. [ABSTRACT FROM AUTHOR]

Ultramicrobacteria (UMB) that can pass through a 0.22 µm filter are attractive because of their novelty and diversity. However, isolating UMB has been difficult because of their symbiotic or parasitic lifestyles in the environment. Some UMB have extracellular electron transfer (EET)-related genes, suggesting that these symbionts may grow on an electrode surface independently. Here, we attempted to culture from soil samples bacteria that passed through a 0.22 µm filter poised with +0.2 V vs. Ag/AgCl and isolated Cellulomonas sp. strain NTE-D12 from the electrochemical reactor. A phylogenetic analysis of the 16S rRNA showed 97.9% similarity to the closest related species, Cellulomonas algicola, indicating that the strain NTE-D12 is a novel species. Electrochemical and genomic analyses showed that the strain NTE-D12 generated the highest current density compared to that in the three related species, indicating the presence of a unique electron transfer system in the strain. Therefore, the present study provides a new isolation scheme for cultivating and isolating novel UMB potentially with a symbiotic relationship associated with interspecies electron transfer.

Microbial interactions play an important role in natural habitat. The long-term coevolution of various species leads to the adaptation of certain types of microorganisms as well as to the formation of a wide variety of interactions such as competitive, antagonistic, pathogenic and parasitic relationships. The aim of this work is a comprehensive study of a new ultramicrobacterium Microbacterium lacticum str. F2E, isolated from perennial oil sludge, which is characterized by high antimicrobial activity and a unique ultrastructural organization of the cell envelope, which includes globular surface ultrastructures with a high negative charge. A previously undescribed mechanism for the antagonistic action of the F2E strain against the prey bacterium is proposed. This mechanism is based on the ability to preferentially capture essential microelements, in which charge interactions and the property of phosphate accumulation may play a significant role. The revealed type of intermicrobial interaction can probably be attributed to the non-contact type antagonistic action in the absence of any diffuse factor secreted by the antagonistic bacteria.

Previous studies have shown that a significant part of the bacterial communities of Antarctic soils is represented by cells passing through filters with pore sizes of 0.2 µm. These results raised new research questions about the composition and diversity of the filterable forms of bacteria (FFB) in Antarctic soils and their role in the adaptation of bacteria to the extreme living conditions. To answer such questions, we analyzed the succession of bacterial communities during incubation of Antarctic soil samples from the Bunger Hills at increased humidity and positive temperatures (5 °C and 20 °C). We determined the total number of viable cells by fluorescence microscopy in all samples and assessed the taxonomic diversity of bacteria by next-generation sequencing of the 16S rRNA gene region. Our results have shown that at those checkpoints where the total number of cells reached the maximum, the FFB fraction reached its minimum, and vice versa. We did not observe significant changes in taxonomic diversity in the soil bacterial communities during succession. During our study, we found that the soil bacterial communities as a whole and the FFB fraction consist of almost the same phylogenetic groups. We suppose rapid transition of the cells of the active part of the bacterial population to small dormant forms is one of the survival strategies in extreme conditions and contributes to the stable functioning of microbial communities in Antarctic soils.

Two strains of ultrasmall gram-negative bacteria (USGNB), FM1 and FM2, were isolated from the skin of the smooth clawed frog Xenopus laevis. The cytological, physiological, biochemical, and genotypic characteristics of the isolates were studied. Based on the sequencing of their 16S rRNA genes and on their phenotypic properties, the isolates were assigned to the genus Chryseobacterium. The cells were extremely small, with cell volumes of ~0.06 and ~0.015 µm3 for developing cultures of strains FM1 and FM2, respectively. Since the USGNB cells were firmly attached to the skin surface and could not be removed by repeated washing with water, these bacteria may be classified as epibionts. Adhesive properties of the fimbria-like appendages revealed in strains FM1 and FM2 by electron microscopy could probably contribute to tight binding of USGNB cells to the skin. Localization of ultrasmall gram-negative bacteria on skin surface of the frogs may indicate their action as a protective bacterial filter; skin surface of Xenopus laevis is thus characterized for the first time as a specific habitat of ultrasmall Chryseobacterium strains. Isolation and characterization of two ultrasmall Chryseobacterium strains, FM1 and FM2, improves our understanding of diversity of the cellular structural and functional characteristics and of the ecological niches of this bacterial genus. [ABSTRACT FROM AUTHOR]

Ultramicrobacteria (UMB) are omnipresent and numerically dominate in freshwater, as microbes can present in drinking water systems, however, the UMB communities that occur and their removal behaviors remain poorly characterized in drinking water treatment plants (DWTPs). To gain insights into these issues, we profiled bacterial cell density, community structure and functions of UMB and their counterpart large bacteria (LB) using flow cytometry and filtration paired with 16S rRNA gene high-throughput sequencing in a full-scale DWTP. Contrary to the reduction of bacterial density and diversity, the proportion of UMB in the total bacteria community increased as the drinking water treatment process progressed, and biological activated carbon facilitated bacterial growth. Moreover, UMB were less diverse than LB, and their community structure and predicted functions were significantly different. In the DWTP, UMB indicator taxa were mainly affiliated with α/β/γ-Proteobacteria, Deinococcus–Thermus, Firmicutes, Acidobacteria, and Dependentiae. In particular, the exclusive clustering of UMB at the phylum level, e.g., Parcubacteria, Elusimicrobia, and Saccharibacteria, confirmed the fact that the ultra-small size of UMB is a naturally and evolutionarily conserved trait. Additionally, the streamlined genome could be connected to UMB, such as candidate phyla radiation (CPR) bacteria, following a symbiotic or parasitic lifestyle, which then leads to the observed high connectedness, i.e., non-random intra-taxa co-occurrence patterns within UMB. Functional prediction analysis revealed that environmental information processing and DNA replication and repair likely contribute to the higher resistance of UMB to drinking water treatment processes in comparison to LB. Overall, the study provides valuable insights into the occurrence and fate of UMB regarding community structure, phylogenetic characteristics and potential functions in a full-scale DWTP, and it is a useful reference for beneficial manipulation of the drinking water microbiome.

Dissolved organic matter (DOM) plays a central role in regulating productivity and nutrient cycling in freshwaters. It is therefore vital that we can representatively sample and preserve DOM in freshwaters for subsequent analysis. Here we investigated the effect of filtration, temperature (5 and 25 °C) and acidification (HCl) on the persistence of low molecular weight (MW) dissolved organic carbon (DOC), nitrogen (DON) and orthophosphate in oligotrophic and eutrophic freshwater environments. Our results showed the rapid loss of isotopically-labelled glucose and amino acids from both filtered (0.22 and 0.45 μm) and unfiltered waters. We ascribe this substrate depletion in filtered samples to the activity of ultra-small (< 0.45 μm) microorganisms (bacteria and archaea) present in the water. As expected, the rate of C, N and P loss was much greater at higher temperatures and was repressed by the addition of HCl. Based on our results and an evaluation of the protocols used in recently published studies, we conclude that current techniques used to sample water for low MW DOM characterisation are frequently inadequate and lack proper validation. In contrast to the high degree of analytical precision and rigorous statistical analysis of most studies, we argue that insufficient consideration is still given to the presence of ultra-small microorganisms and potential changes that can occur in the low MW fraction of DOM prior to analysis. [ABSTRACT FROM AUTHOR]

Flow cytometric sorting is a powerful tool to physically separate cells within mixed microbial communities. If combined with phylogenetic staining (fluorescence in situ hybridization, FISH) it allows to specifically sort defined genotypic microbial populations from complex natural samples. However, the targeted enrichment of freshwater ultramicrobacteria, such as members of the LD12 clade of Alphaproteobacteria (SAR11-IIIb), is still challenging. Current FISH protocols, even in combination with signal amplification by catalysed reporter deposition (CARD), are not sufficiently sensitive for the distinction of these bacteria from background noise by flow cytometry, presumably due to their low ribosome content and small cell sizes. We, therefore, modified a CARD based flow sorting protocol with the aim of increasing its sensitivity to a level sufficient for ultramicrobacteria. This was achieved by a second signal amplification step mediated by horseradish peroxidase labelled antibodies targeted to the fluorophores that were previously deposited by CARD-FISH staining. The protocol was tested on samples from an oligo-mesotrophic lake. Ultramicrobacteria affiliated with LD12 Alphaproteobacteria could be successfully sorted to high purity by flow cytometry. The ratios of median fluorescence signal to background ranged around 20, and hybridization rates determined by flow cytometry were comparable to those obtained by fluorescence microscopy. Potential downstream applications of our modified cell staining approach range from the analysis of microdiversity within 16S rRNA-defined populations to that of functional properties, such as the taxon-specific incorporation rates of organic substrates.

SAR11 bacteria are small, heterotrophic, marine alphaproteobacteria found throughout the oceans. They thrive at the low nutrient concentrations typical of open ocean conditions, although the adaptations required for life under those conditions are not well understood. To illuminate this issue, we used cryo-electron tomography to study "Candidatus Pelagibacter ubique" strain HTCC1062, a member of the SAR11 clade. Our results revealed its cellular dimensions and details of its intracellular organization. Frozen-hydrated cells, which were preserved in a life-like state, had an average cell volume (enclosed by the outer membrane) of 0.037 ± 0.011 μm3. Strikingly, the periplasmic space occupied ~20% to 50% of the total cell volume in log-phase cells and ~50% to 70% in stationary-phase cells. The nucleoid occupied the convex side of the crescent-shaped cells and the ribosomes predominantly occupied the concave side, at a relatively high concentration of 10,000 to 12,000 ribosomes/μm3. Outer membrane pore complexes, likely composed of PilQ, were frequently observed in both log-phase and stationary-phase cells. Long filaments, most likely type IV pili, were found on dividing cells. The physical dimensions, intracellular organization, and morphological changes throughout the life cycle of "Ca. Pelagibacter ubique" provide structural insights into the functional adaptions of these oligotrophic ultramicrobacteria to their habitat. [ABSTRACT FROM AUTHOR]

The effect of ultramicrobacterial epibionts of the genera Kaistia (strain NF1), Chryseobacterium (strain NF4), and Stenotrophomonas (strain FM3) on the process of sporulation of Bacillus subtilis ATCC 6633 was studied. The investigated strains of ultramicrobacteria (UMB) were found to inhibit the sporulation process of B. subtilis ATCC 6633 in binary mixed cultures, exhibiting a 3-day delay of the onset of sporulation compared to the control one, an extended period of the prospore maturation, formation of the fraction of immature spores, and development of ultrastructural defects in many endospores. Thus, investigation of binary mixed cultures of B. subtilis and UMB revealed that, apart from suppression of reproduction and lysis of host vegetative cells, inhibition of spore formation and destruction of endospores was yet another feature of intermicrobial parasitism. The UMB parasites of the studied genera are assumed to participate in the regulation of development and reproduction of B. subtilis in natural habitats of this spore-forming bacterium. [ABSTRACT FROM AUTHOR]

Microbial cells vary widely in size, specific density, shearing resistance, oxygen sensitivity and abundance so that differential harvesting and washing procedures are needed to efficiently recover cells from dilute suspensions. We here describe a mild, simple, variable and cost-efficient method to harvest cells on columns packed with silica beads. The method collects and concentrates 40–98% of the cells preserving enzymatic activity and cell viability. The method can be applied for strictly anaerobic microorganisms, is scalable to different culture volumes and can be multiplexed in standardized systems. We see major application potential in harvesting small cells leaking through 0.2 μm filters, for harvesting strictly anaerobic cells and for differential harvesting of cells according to cell size using a gradient system. [ABSTRACT FROM AUTHOR]

The results of ecological and phylogenetic studies of femtoplankton, the smallest size fraction of plankton formed by viruses and ultramicrobacteria (UMB), are overviewed to shed light on the problem of transbiome invasions by microbes. Phylogenetic lineages of viruses and UMB are shown to be associated with particular biomes, thus indicating infrequent transbiome transitions in the microbial world. An alternative hypothesis of widespread cross-colonization events requires a deeper analysis of the factors that form the barrier between biomes and are responsible for the adaptation of microorganisms to different environments. [ABSTRACT FROM AUTHOR]

We isolated 50 strains of free-living ultrasmall bacteria with a cell volume that varies from 0.02 to 1.3 μm from a range of natural biotopes, namely permafrost soils, oil slime, soils, lake silt, thermal swamp moss, and the skin integuments of the clawed frog, Xenopus laevis. Of them, 23 isolates, characterized by a cell size of less than 0.1 μm and a genome size from 1.5 to 2.4 Mb, were subsumed to ultramicrobacteria belonging to different philogenetic groups ( Alphaproteobacteria, Bacteroidetes, Actinobacteria) and genera ( Kaistia, Chryseobacterium, Microbacterium, Leucobacter, Leifsonia, and Agrococcus) of the Bacteria domain. They are free-living mesophilic heterotrophic aerobic bacteria. The representatives of Kaistia and Chryseobacterium genera were capable of facultative parasitism on other species of chemo-organotrophic bacteria and cyanobacteria. The ultramicrobacteria differed in their morpholgy, cell ultrastructural organization, and physiological and biochemical features. According to the fine structure of their cell walls, the isolates were subdivided into two groups, namely Gram-positive and Gram-negative forms. [ABSTRACT FROM AUTHOR]

Flow cytometric sorting is a powerful tool to physically separate cells within mixed microbial communities. If combined with phylogenetic staining (fluorescence in situ hybridization, FISH) it allows to specifically sort defined genotypic microbial populations from complex natural samples. However, the targeted enrichment of freshwater ultramicrobacteria, such as members of the LD12 clade of Alphaproteobacteria (SAR11-IIIb), is still challenging. Current FISH protocols, even in combination with signal amplification by catalyzed reporter deposition (CARD), are not sufficiently sensitive for the distinction of these bacteria from background noise by flow cytometry, presumably due to their low ribosome content and small cell sizes. We, therefore, modified a CARD based flow sorting protocol with the aim of increasing its sensitivity to a level sufficient for ultramicrobacteria. This was achieved by a second signal amplification step mediated by horseradish peroxidase labeled antibodies targeted to the fluorophores that were previously deposited by CARD-FISH staining. The protocol was tested on samples from an oligo-mesotrophic lake. Ultramicrobacteria affiliated with LD12 Alphaproteobacteria could be successfully sorted to high purity by flow cytometry. The ratios of median fluorescence signal to background ranged around 20, and hybridization rates determined by flow cytometry were comparable to those obtained by fluorescence microscopy. Potential downstream applications of our modified cell staining approach range from the analysis of microdiversity within 16S rRNA-defined populations to that of functional properties, such as the taxon-specific incorporation rates of organic substrates. [ABSTRACT FROM AUTHOR]

Although the lower size limit of microorganisms was previously believed to be c. 0.2 μm, there is evidence for the existence of microorganisms that can pass through 0.2 μm-pore-size filters called ultramicrobacteria or nanobacteria. However, information on the phylogeny and biogeography of these bacteria is limited. We obtained 53 isolates of 0.2 μm-passable bacteria from 31 samples collected at 26 locations worldwide, including the Arctic Svalbard Islands, deserts, and Maritime Antarctica. Phylogenetic analysis of near full-length 16S rRNA gene sequences revealed that 18 of the 53 isolates were < 97% homologous with previously cultured isolates, representing potentially novel species. Two isolates (order Rhizobiales) (100% identical) collected from Byers Peninsula, Livingston Island in Maritime Antarctica, were closely related (99.8% similarity) to an isolate collected from intertidal sediments in East Antarctica. In addition, the sequence of this Antarctic isolate showed ≥ 97% similarity to 901 sequences derived from known isolates and samples collected at geographically disparate locations under various environmental conditions. Interestingly, among 13 sequences showing ≥ 99% similarity, ten were isolated from cryospheric habitats such as Arctic, Antarctic, and alpine environments. This implies that such Rhizobiales strains occur in the cryospheric regions, however, their abundance and biomass may be scarce depending on the geographic location. [ABSTRACT FROM PUBLISHER]

Ultramicrobacteria (UMB) are species of the domain Bacteria characterized by very small sizes of proliferating cells (less than 0.1 μm in volume) and small genomes (3.2 to 0.58 Mb). Some authors use the term nanobacteria as a synonym of UMB. Several tens of UMB species have been isolated from various natural habitats: sea water, soil, silt, Greenland ice sheet, permafrost soils, and intestines of humans and insects. Under laboratory conditions, they are cultivated on different nutrient media. In the second prokaryotic domain, the Archaea, ultrasmall forms (ultramicroarchaea) have also been described, including nanoarchaea (members of the genus Nanoarchaeum) with a cell volume of less than 0.1 μm. The term nanobacteria is used in the literature also to denote ultrasmall bacterium-like particles occurring in rocks, sands, soils, deep sub-surface layers, meteorites, and clinical samples. The systematic position and the capacity for self-reproduction of these particles are still unclear. The cultured UMB forms are characterized by highly diverse morphology, ultrastructural organization, physiology, biochemistry, and ecology. UMB form three groups according to the type of cell wall structure and the reaction to Gram staining: (1) gram-negative, (2) gram-positive, and (3) cell wall-lacking. Their cells divide by constriction, septation, or budding. The unique processes performed by UMB are dehalorespiration and obligate or facultative epibiotic parasitism. The UMB that synthesize organic compounds in ocean waters with the involvement of proteorhodopsin play a great role in the biosphere. UMB have been found in seven large phylogenetic groups of prokaryotes, where their closest relatives are organisms with larger cells typical of bacteria, which is evidence of the polyphyletic origin of the currently known UMB species and the reductive mode of their evolution. [ABSTRACT FROM AUTHOR]

Two strains, NF4 and NF5, of a yellow-colored gram-negative bacterium were isolated from sediments of Lake Baikal and from old oil sludge of the Nizhnekamsk oil-processing plant. The cells of the strains are ultrasmall coccoids or short rods, measuring 0.2-0.4 × 0.2-0.5 μm; the average cell volume ranges from 0.004 to 0.04 μm. A considerable proportion (30-60%) of cells have nanometer dimensions (180-300 nm in diameter and 0.004-0.02 μm in volume). The new isolates are thus among the smallest representatives of presently known free-living ultramicrobacteria. The two studied isolates are gram-negative nonmotile cells possessing a pronounced outer membrane. The cells do not have flagella and are not capable of gliding motility. They divide by constriction, budding, and multiple septation. The multiplicity of reproduction mechanisms results in a high degree of cell polymorphism. The isolates are chemoorganotrophic, aerobic, psychrotolerant, oxidase- and catalase-positive. Their characteristic trait is the absence of extracellular hydrolytic enzymes, such as proteases, lipases, pectinases, and cellulases. Menaquinone MK-6 is the main respiratory quinone; the flexirubin pigment was not detected. The G + C contents of the DNA of strains NF4 and NF5 are 40.8 and 40.5 mol %, respectively. The DNA-DNA hybridization level of strains NF4 and NF5 was close to 100%. Analysis of the 16S rRNA gene sequences and the fatty acid compositions showed that the isolates are most closely related to certain representatives of the genus Chryseobacterium ( C. solincola, C. antarcticum, and C. jeonii). However, the differences in the 16S rRNA gene sequences, as well as in the phenotypic properties, such as formation of ultrasmall cells, the absence of extracellular hydrolases, oligotrophy, and the capacity for epibiosis on bacterial cells, suggest that the studied strains belong to a new species of the genus Chryseobacterium. The capacity for epibiosis, i.e., the ability to exist in a tightly adhered state on the surfaces of host Bacillus subtilis cells, is a peculiar trait of the studied isolates. It is assumed that adhesion of the cells of strains NF4 and NF5 (members of the phylum Bacteroidetes) occurs via by the same unique mechanism as the mechanism that we previously described for representatives of Alphaproteobacteria ( Kaistia sp., NF1, and NF3), which use polysaccharide chains equipped with sticky granules as trapping and constricting cords. [ABSTRACT FROM AUTHOR]

The number, physiological state, and taxonomic composition of ultramicrobacteria and archaea in various soils (alluvial meadow, sod-podzolic, leached chernozem, and peat) were studied. In all of the tested soil samples, a large number of ultramicrobacteria (tens and hundreds of millions of cells per 1 g of soil) was revealed by fluorescence microscopy. The portion of cells with intact membranes was larger among ultra-microbacteria than among the ordinary-size cells (95–98 and 50%, respectively). Ultramicrobacteria were characterized by high taxonomic diversity and included representatives of the main phylogenetic groups widespread in soils, such as Archaea, Actinobacteria, Cytophaga, and Proteobacteria. The results indicate that ultramicrobacteria are widespread in soils in a viable state and are involved in soil processes. [ABSTRACT FROM AUTHOR]

Two strains of aerobic, non-spore-forming, Gram-negative, rod-shaped bacteria (ND5 and MY14T), previously isolated from urban soil using the membrane-filter enrichment technique, were characterized. Analysis of their 16S rRNA gene sequence grouped strains ND5 and MY14T within the family Oxalobacteraceae ( Betaproteobacteria). The highest pairwise sequence similarities for strain ND5 were found with members of the genus Herminiimonas, namely with Herminiimonas saxobsidens NS11T (99.8%) and Herminiimonas glaciei UMB49T (99.6%). Although some fatty acid profiles, physiological and biochemical differences exist between strain ND5 and the respective Herminiimonas-type strains, DNA–DNA hybridization experiments confirm that strain ND5 is a member of the H. glaciei genospecies. Taxonomical analyses revealed a wider range of variability within this genus than considered previously. The highest pairwise nucleotide similarity for strain MY14T was found with Oxalicibacterium flavum (96.8%). Phylogenetic analyses based on 16S rRNA and cpn60 gene sequences, DNA–DNA hybridization, fatty acid profiles, physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain MY14T from other Oxalicibacterium species representing a new species, for which the name Oxalicibacterium solurbis sp. nov. (type strain MY14T=NBRC 102665T,=CCM 7664T) is proposed. [ABSTRACT FROM AUTHOR]

Three novel orange, ultramicrobacterial isolates, UMB10, UMB14, and UMB34T were isolated from enrichment cultures inoculated with a melted 3,043 m deep Greenland ice core sample. Phylogenetic analysis of the 16S rRNA gene sequences indicated that the isolates belonged to a single species within the genus Chryseobacterium. They were most closely related to Chryseobacterium aquaticum (99.3%), Chryseobacterium soli (97.1%), and Chryseobacterium soldanellicola (96.9%). Genomic hybridization showed low levels of relatedness between UMB34T and C. aquaticum and C. soldanellicola (19–30%) and C. soli and Chryseobacterium jejuense (45–56%). Comparative genomic fingerprinting analysis using the enterobacterial repetitive intergenic consensus (ERIC) sequence showed nearly identical banding patterns for the three isolates and these patterns were distinct from those of C. aquaticum, C. soldanellicola, C. soli, and C. jejuense. The cells were short rods, lacked flagella, had cell volumes of <0.1 μm3, formed buds and smaller protrusions (blebs), produced copious extracellular material and a flexirubin type pigment. UMB34T produced acids from carbohydrates and utilized glucose and maltose although it did not assimilate mannose. The DNA G + C was 39.6–41.6 mol%. Based on the differences from validly named Chryseobacterium species, it was concluded that these isolates represent a new species for which the name, Chryseobacterium greenlandense is proposed. The type strain is UMB34T (=CIP 110007T = NRRL B-59357). [ABSTRACT FROM AUTHOR]

Two strains (NF1 and NF3) of free-living chemoorganotrophic bacteria have been isolated from multiyear oil slime and Pedilanthus tithymaloides rhizosphere and ascribed to the genus Kaistia of the class Alphaproteobacteria on the basis of the nucleotide sequences of 16S rRNA gene and phenotypic characteristics. These strains can be assigned to ultramicrobacteria as their populations are represented by two subpopulations: (1) ultrasmall cells, on average 200–300 nm in diameter and <0.1 μm3 in volume, of up to 60% of the total number of cells in a population, and (2) cells 400–800 nm in diameter and 0.15–0.5 μm3 in volume, of up to 40% of the total number of cells in a population. The interaction of the isolated ultramicrobacteria strains (IUMB) with different bacterial species has been studied in cocultures grown under starvation and in complete nutrient media. It has been found that IUMB can be facultative parasites on certain species of chemoorganotrophic and phototrophic bacteria. The interaction of IUMB with prey bacteria exhibits the extracellular type of parasitism and involves establishing stable cell–cell contacts between the parasites and their prey to cause destruction of host cells. [ABSTRACT FROM AUTHOR]

The morphology, ultrastructure, and quantity of bacterial nanoforms were studied in extreme biotopes: East Siberia permafrost soil (1–3 Ma old), petroleum-containing slimes (35 years old), and biofilms from subsurface oil pipelines. The morphology and ultrastructure of microbial cells in natural biotopes in situ were investigated by high-resolution transmission electron microscopy and various methods of sample preparation: ultrathin sectioning, cell replicas, and cryofractography. It was shown that the biotopes under study contained high numbers of bacterial nanoforms (29–43% of the total number of microorganisms) that could be assigned to ultramicrobacteria due to their size (diameter of ≤ 0.3 μm and volume of ≤ 0.014 μm3) and structural characteristics (the presence of the outer and cytoplasmic membranes, nucleoid, and cell wall, as well as their division patterns). Seven different morphostructural types of nanoforms of vegetative cells, as well as nanospores and cyst-like cells were described, potentially representing new species of ultramicrobacteria. In petroleum-containing slimes, a peculiar type of nanocells was discovered, gram-negative cells mostly 0.18–0.20 × 0.20–0.30 μm in size, forming in situ spherical aggregates (microcolonies) of dividing cells. The data obtained promoted the isolation of pure cultures of ultramicrobacteria from petroleum-containing slimes; they resembled the ultramicrobacterium observed in situ in their morphology and ultrastructure. [ABSTRACT FROM AUTHOR]

Transmission electron and fluorescence microscopy was used to study the character of the interaction of free-living ultramicrobacterial (UMB) strains NF1 and NF3, affiliated with the genus Kaistia, and seven species of gram-positive and gram-negative heterotrophic bacteria. Strains NF1 and NF3 were found to exhibit parasitic activity against gram-positive Bacillus subtilis and gram-negative Acidovorax delafildii. UMB cells are tightly attached to the envelopes of the victim cells and induce their lysis, thus demonstrating the features of typical ectoparasitism. The selectivity of parasitism of the studied UMB to the victim bacteria has been shown: only two soil microorganisms of the seven test objects, B. subtilis ATCC 6633 and an aerobic gramnegative bacterium A. delafildii 39, were found to be sensitive to UMB attack. Other bacteria ( Micrococcus luteus VKM Ac-2230, Staphylococcus aureus 209-P, Pseudomonas putida BS394, Escherichia coli C 600, and Pantoea agglomerans ATCC 27155) were not attacked by UMB. It was established for the first time that free-living UMB may be facultative parasites not only of phototrophic bacteria, as we have previously demonstrated [1], but of heterotrophic bacteria as well. The UMB under study seem to play an important role in the regulation of the quantity of microorganisms and in the functioning of microbial communities in some natural ecotopes. [ABSTRACT FROM AUTHOR]

Gram-negative chemoorganotrophic soil ultramicrobacteria (UMB), strains NF1 and NF3, have been isolated. In their development cycle, the strains formed small coccoid cells of 400–800 nm and ultrasmall cells of 200–300 nm. Phylogenetically, the strains NF1 and NF3 belong to Alphaproteobacteria and are close to the type strain of the recently described species Kaistia adipata. The ultrastructure of UMB cells has been studied using ultrathin sections and freeze-fracturing. It has been shown that the structure of UMB cell walls is of the gram-negative type; the outer membrane and peptidoglycan layer are well differentiated. The cell surface has numerous protrusions (prosthecae) of conical or spherical shape filled with the contents of the periplasm. The formation of unusual cellular structures (not occurring in known free-living bacteria) is a feature of UMB; these include the following: (a) piles of rod-like subunits, ca. 30 Å in diameter and 150–250 Å in length; (b) long bunches (up to 300–400 Å) comprised of filamentous subunits; and (c) large electron-dense spherical bodies (up to 200–300 Å in diameter) localized in the periplasm. A distinctive feature of UMB is their ability to grow as facultative parasites on living cyanobacterial (CB) cells. In this case, three types of interaction between UMB and CB have been revealed: (1) adsorption of UMB cells on the surface of CB cells; (2) penetration of UMB into polysaccharide sheathes; and (3) penetration of UMB into CB cytoplasm. UMB cells have been shown to reproduce by budding, with buds (up to 2–3) located directly on the mother cell, without formation of intermediate hyphae. [ABSTRACT FROM AUTHOR]

We developed a novel method, the filtration–acclimatization method (FAM), which enables the isolation and cultivation of an important fraction of the bacterial diversity, which is not cultivable by standard methods. The method consists of a filtration step, which removes most of the readily cultivable bacteria able to overgrow slowly growing bacteria, and an acclimatization procedure that provides a slow transition from the low environmental substrate concentrations to the high concentration of standard microbial media. So far, we isolated in total 65 strains from surface freshwater habitats by utilizing FAM. The isolates are affiliated with Actinobacteria, Alpha-, Betaproteobacteria, Bacteroidetes, and Spirochaeta. All isolates are pure cultures and form visible colonies on agar plates with high substrate concentrations. For further analysis, strains sharing more than a 97% 16S rRNA gene sequence similarity were grouped into one taxon. Based on sequence similarities, 88% of the obtained taxa can be considered to be undescribed species (<97% similarity to closest species). The highest similarity value of the taxa to the respective closest related species ranged from 87.7% to 99.8%, and was on average 94.5%. For comparison we isolated, by direct plating of water samples on a rich agar medium, a similar number of taxa. Amongst these taxa the percentage of taxa, which can be considered to be undescribed species, was only half of the percentage found for the taxa isolated by FAM. More importantly, it was amongst the taxa obtained by the standard method no taxon that was closer related to an uncultured bacterium than to an isolate, while 56% of the taxa isolated by FAM were closely related to uncultured bacteria. [Copyright &y& Elsevier]

The purpose of this study was to observe the effects of different starvation conditions on the surface characteristics and adhesive properties of bacteria. Three pure culture xenobiotic degrading bacteria were used in this study: Pseudomonas sp. strain A, Pseudomonas sp. strain D and Rhodococcus corallinus strain 11. These microorganisms were selected for to their ability to degrade cyanuric acid, which is a derivative of s-Triazine, a common herbicide. These microorganisms were separately starved for carbon (glucose) and nitrogen (cyanuric acid) and their physiological responses to starvation were measured. Results indicated that there was a remarkable shrinkage in size of Pseudomonas strain A starved for carbon for 16 days. The surface properties of these three microorganisms changed significantly on starvation. Surface hydrophobicities for all the microorganisms stayed more or less constant during carbon starvation conditions, whereas, there was a significant decrease in hydrophobicity when all three cultures were starved for nitrogen. In parallel to these findings, it was observed that when the hydrophobicity of the cultures decreased, the attachment capabilities of these microorganisms decreased. This is believed to be due to the production of highly hydrated extracellular polysaccharides when carbon is present in the medium. Results of this study have important implications on the transport and attachment of microorganisms during soil remediation processes. [Copyright &y& Elsevier]

Microbial interactions play an important role in natural habitat. The long-term coevolution of various species leads to the adaptation of certain types of microorganisms as well as to the formation of a wide variety of interactions such as competitive, antagonistic, pathogenic and parasitic relationships. The aim of this work is a comprehensive study of a new ultramicrobacterium Microbacterium lacticum str. F2E, isolated from perennial oil sludge, which is characterized by high antimicrobial activity and a unique ultrastructural organization of the cell envelope, which includes globular surface ultrastructures with a high negative charge. A previously undescribed mechanism for the antagonistic action of the F2E strain against the prey bacterium is proposed. This mechanism is based on the ability to preferentially capture essential microelements, in which charge interactions and the property of phosphate accumulation may play a significant role. The revealed type of intermicrobial interaction can probably be attributed to the non-contact type antagonistic action in the absence of any diffuse factor secreted by the antagonistic bacteria. [ABSTRACT FROM AUTHOR]

The soil and subsurface strata are low nutrient environments and their bacterial inhabitants must adopt starvation responses to survive. These responses include the formation of dormant, viable cells which, although reduced in cell size and volume, are able to respond to any improvement in nutrient availability. Starved bacteria are able to survive for extended periods without nutrients and their reduced size allows them to disperse deeply within rocks and soils greatly improving their penetration. These combined factors may increase opportunities for bacteria to reach a deep waste disposal site. [ABSTRACT FROM AUTHOR]

Previous studies have shown that a significant part of the bacterial communities of Antarctic soils is represented by cells passing through filters with pore sizes of 0.2 µm. These results raised new research questions about the composition and diversity of the filterable forms of bacteria (FFB) in Antarctic soils and their role in the adaptation of bacteria to the extreme living conditions. To answer such questions, we analyzed the succession of bacterial communities during incubation of Antarctic soil samples from the Bunger Hills at increased humidity and positive temperatures (5 °C and 20 °C). We determined the total number of viable cells by fluorescence microscopy in all samples and assessed the taxonomic diversity of bacteria by next-generation sequencing of the 16S rRNA gene region. Our results have shown that at those checkpoints where the total number of cells reached the maximum, the FFB fraction reached its minimum, and vice versa. We did not observe significant changes in taxonomic diversity in the soil bacterial communities during succession. During our study, we found that the soil bacterial communities as a whole and the FFB fraction consist of almost the same phylogenetic groups. We suppose rapid transition of the cells of the active part of the bacterial population to small dormant forms is one of the survival strategies in extreme conditions and contributes to the stable functioning of microbial communities in Antarctic soils. [ABSTRACT FROM AUTHOR]

The goal of the present work was to investigate the physicochemical and radiochemical conditions and the microbial diversity in groundwater collected near the Lake Karachai (Russia), which was formerly used for the disposal of liquid radioactive waste, to isolate the dominant bacteria, and to determine their taxonomy and the physiological characteristics responsible for their adaptation to this environment. Groundwater samples contained high concentrations of acetate, oxalate, nitrate, and sulfate, as well as radionuclides. High-throughput sequencing and analysis of the clone libraries revealed lower microbial diversity in the most strongly contaminated groundwater and a predominance of bacteria of the genera Polynucleobacter, Pusillimonas, Candidatus Pelagibacter, and of the candidate phylum Parcubacteria; these groups include species with an ultra small cell size. Archaeal sequences in the libraries belonged to ammonium oxidizers of the phylum Thaumarchaeota and methanogens of the phylum Euryarchaeota. Pure cultures of obligate and facultative ultramicrobacteria belonging to the genera Chryseobacterium, Microbacterium, Salinibacterium, Pusillimonas, Roseomonas, and Janibacter were isolated from water samples. In genomes of Pusillimonas and Roseomonas strains the genes associated with nitrate reduction, resistance to heavy metals and metalloids were revealed. Several isolates are able to participate in the geochemical process of nitrate conversion to N2 using acetate; this results in decreasing redox potential, which in turn may stimulate radionuclide reduction and decrease radionuclide migration in groundwater. [ABSTRACT FROM AUTHOR]