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Ultra-small microorganisms are ubiquitous in Earth's environments. Ultramicrobacteria, which are defined as having a cell volume of

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.

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.

The stability of microbial community composition under different environmental conditions is an important part of microbial ecology, but has not been investigated in such depth before. In this study, we investigated the composition of the bacterioplankton community composition (BCC) and its stability under different temperatures (15, 25 and 35 °C, respectively) and nutrient conditions (control vs. nitrogen- and phosphorus-enriched) in aquatic microcosms. The BCC was analysed using denaturing gradient gel electrophoresis of the bacterial 16S rRNA gene, followed by cloning and sequence analysis. BCC in the microcosms significantly changed under different temperature and nutrient conditions. Based on the results from clone libraries, ecological diversification were observed within two ubiquitous and dominant genera, Methylophilus and Polynucleobacter, under different temperature and nutrient conditions. Putative ultramicrobacteria, which included Actinobacteria, Polynucleobacter sp., LD12 and LD28 clusters and bacteria affiliated with subcluster I of Methylophilus, were found to dominate in bacterioplankton communities at higher temperatures (25 and 35 °C), regardless of nutrient conditions. We also observed that the rate of BCC change increased at higher temperatures and this increase was more pronounced in nutrient-enriched microcosms. These results indicated that bacterioplankton communities become unstable and decrease in size with increased temperature and in nutrient-enriched conditions.

The use of multiple centrifuging and filtration of water suspensions from different soils and subsoil deposits allowed revealing the ultrafine forms (nanosized, nanoforms) of bacteria. In the soils studied, the number of bacteria obtained by filtration using 0.2-μm filters was 20–300 mln cells in 1 g soil; the share of these bacteria of their total population in the natural soils reached 5% and was higher (up to 15%) in the polluted urban soils. The study of bacterial nanoforms in situ by the methods of scanning and transmission microscopy has shown the presence of dividing cells, which testifies to their viability. The cells without signs of division were similar in their ultrastructural characteristics to dormant forms of nonspore-forming bacteria. They were observed in permafrost deposits. The data obtained attest that the bacterial nanoforms are widespread in soils and subsoil deposits. According to their morphological and cytological characteristics, they are represented by both active and dormant forms to survive unfavorable conditions.

The pelagic zone of large, deep freshwater lakes features pronounced horizontal and vertical gradients of physicochemical parameters, which in turn might allow for a nonuniform occurrence of specifically adapted bacterial taxa. We, therefore, studied the spatial distribution patterns of different heterotrophic bacteria, picocyanobacteria, and the dominant primary producer, the filamentous cyanobacterium Planktothrix rubescens, in a large, canyon-shaped, prealpine lake (Lake Zurich, Switzerland), in six vertical profiles along a 21.7-km longitudinal transect. Highest total densities and proportions of cells with high nucleic acid content were in the warm epilimnion and the hypoxic zone of the hypolimnion. P. rubescens formed a dense layer in the metalimnion throughout the lake, whereas picocyanobacteria populated the water layers above. The epilimnion was mainly inhabited by ultramicrobacteria related to the LD12-lineage of Alphaproteobacteria and to Actinobacteria; the latter group preferred the shallow regions. Cytophaga-Flavobacteria, in particular a population related to Fluviicola sp. were more frequent in and below the layer of maximal P. rubescens abundances. Betaproteobacteria, on the other hand, were highly abundant in the epi- and hypolimnion, but not in the P. rubescens layer. Four betaproteobacterial subpopulations with contrasting longitudinal and/or vertical habitat preferences were distinguished: putatively methylotrophic bacteria of the LD28 lineage (beta IV) preferentially inhabited the hypolimnion, Polynucleobacter acidiphobus was found throughout the epilimnion, Limnohabitans (R-BT065) more in the shallow regions of the lake, and Polynucleobacter necessarius ssp. asymbioticus only in hypoxic waters. Our results stress the potential importance of spatial niche differentiation in freshwater bacterioplankton.

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.

The abundant microbial population in a 3,043-m-deep Greenland glacier ice core was dominated by ultrasmall cells (3 ) that may represent intrinsically small organisms or starved, minute forms of normal-sized microbes. In order to examine their diversity and obtain isolates, we enriched for ultrasmall psychrophiles by filtering melted ice through filters with different pore sizes, inoculating anaerobic low-nutrient liquid media, and performing successive rounds of filtrations and recultivations at 5°C. Melted ice filtrates, cultures, and isolates were analyzed by scanning electron microscopy, flow cytometry, cultivation, and molecular methods. The results confirmed that numerous cells passed through 0.4-μm, 0.2-μm, and even 0.1-μm filters. Interestingly, filtration increased cell culturability from the melted ice, yielding many isolates related to high-G+C gram-positive bacteria. Comparisons between parallel filtered and nonfiltered cultures showed that (i) the proportion of 0.2-μm-filterable cells was higher in the filtered cultures after short incubations but this difference diminished after several months, (ii) more isolates were obtained from filtered (1,290 isolates) than from nonfiltered (447 isolates) cultures, and (iii) the filtration and liquid medium cultivation increased isolate diversity ( Proteobacteria ; Cytophaga-Flavobacteria-Bacteroides ; high-G+C gram-positive; and spore-forming, low-G+C gram-positive bacteria). Many isolates maintained their small cell sizes after recultivation and were phylogenetically novel or related to other ultramicrobacteria. Our filtration-cultivation procedure, combined with long incubations, enriched for novel ultrasmall-cell isolates, which is useful for studies of their metabolic properties and mechanisms for long-term survival under extreme conditions.

In recent years, diversity studies employing culture-independent methods have strongly expanded the knowledge about the phylogenetic composition of freshwater bacterioplankton (7, 15, 32). In a comparative study, Zwart and colleagues (32) identified 34 putative phylogenetic clusters of bacteria which seem to contain typical freshwater inhabitants. Seventy percent of these clusters were reported to contain no cultivated representatives. Each of the other clusters contained usually only a few cultivated strains, which are far from covering the whole range of diversity represented by the available environmental sequences. Zwart and coauthors also analyzed a set of 14 diversity studies for the detection of each of the 34 putative freshwater clusters. All of these analyzed studies have used random cloning of 16S rRNA genes. One of the most frequently detected putative clusters was the betaproteobacterial Polynucleobacter necessarius cluster (the ACK-1 cluster [14], or the beta II cluster [7]). This cluster was discovered by Hirons and colleagues in a study on diversity of bacterioplankton in the Adirondack mountain lakes (14). Meanwhile, more than 100 environmental 16S rRNA gene sequences affiliated with the P. necessarius cluster have been deposited in the public databases during the past eight years. Most of the sequences are partial sequences of cloned genes. The sequences have been obtained from a broad spectrum of lakes and river ecosystems widely distributed over the northern hemisphere, as well as from one lake in the southern hemisphere. This set of ecosystems included, for instance, the arctic Toolik Lake (2, 5), the antarctic Sombre Lake (19), the predominately acidic Adirondack mountain lakes (14, 18), the deep oligotrophic Lake Baikal (7, 24), and rivers ranging from oligotrophic to eutrophic (4, 22, 23, 32). Only one published P. necessarius cluster sequence has been obtained from a nonsurface freshwater system (20). This short sequence was obtained in an investigation on the bacterial diversity in boreholes along a tunnel 626 m below ground surface. No P. necessarius cluster sequences have been reported from freshwater sediments or marine or soil systems. Pernthaler and colleagues applied a cluster-specific probe for detection of P. necessarius cluster members in the acidic Lake Fuchskuhle by way of fluorescence in situ hybridization. Up to 50% of the bacterioplankton consisted of bacteria affiliated with the P. necessarius cluster (3a). No pure-culture isolates affiliated with the P. necessarius cluster have been reported thus far. Bruns and colleagues systematically studied the influence of signal compounds (e.g., cyclic AMP) and different incubation conditions on the culturability of freshwater bacterioplankton (3). They grew diluted samples of bacterioplankton in synthetic medium and analyzed the obtained mixed cultures by 16S rRNA gene fingerprinting techniques. Analysis of sequences obtained from the fingerprints demonstrated that they successfully enriched members of the P. necessarius cluster in their mixed cultures. The species after which the P. necessarius cluster was named is an obligate endosymbiont of the hypotrichous ciliate Euplotes aediculatus (13, 28). This bacterium was never grown in pure culture but was described as a species in 1987 (13). The type strain of this species is still growing as an endosymbiont in a ciliate culture. In this paper, the first isolation and cultivation of strains affiliated with the P. necessarius cluster is reported, and insights into the ecology of this group of representative freshwater bacteria are presented.

The stimulation of groundwater bacteria to form biofilms, for the remediation of polluted aquifers, is subjected to environmental regulations that include measurement of effects on microbial biodiversity. Groundwater microorganisms contain a proportion of unidentified and uncharacterized ultramicrobacteria (UMB) that might play a major role in the bioclogging of geological materials. This study aimed to assess the changes in genetic and metabolic biodiversity when a community of UMB, isolated from groundwater, is stimulated to form biofilms on a ceramic surface. UMB were stimulated with aerobic conditions and injection of molasses, in reactors reproducing groundwater composition and temperature. Concentration of planktonic viable UMB, secretion of extracellular polymeric substances (EPS), and biofilm thickness were monitored. The assessment of changes in biodiversity was achieved by comparing the initial UMB community to the biofilm community, using the single strand conformational polymorphism (SSCP) method, the cloning and sequencing of 16S rRNA gene (16S rDNA) sequences, and the Biolog microplate system. The hypothesis stating that indigenous UMB would play a significant role of in the biofilm development was corroborated. Within 13 days of stimulation, the UMB produced 700 mg L?1 of planktonic EPS and formed a biofilm up to a thickness of 1100 mm. This stimulation led to a decrease in genetic diversity and an increase in metabolic diversity. The decrease in genetic diversity was shown by a reduced number of single strand DNA fragments in the SSCP profiles. As such, 16S rDNA sequences from the biofilm revealed the predominance of four bacterial groups: Zoogloea, Bacillus/Paenibacillus, Enterobacteriaceae, and Pseudomonads. A significant increase in metabolic diversity was shown by a highest substrate richness profile and a lower substrate evenness profile of the biofilm bacterial population (p = 0.0 and p = 0.09, respectively). This higher metabolic diversity might be a consequence of the stimulation that seemed to favor the growth of bacteria having a high nutritional versatility. Stimulation of UMB, isolated from groundwater, was effective to form a biofilm having a high metabolic biodiversity. This combination of molecular-based and metabolic-based methods expanded the insight into monitoring the changes in bacterial biodiversity.

Very small forms of bacteria have been reported from marine and freshwater systems as well as from soils, subsurface terrestrial environments' and more recently from samples of kidney stones. Also, such small cells could be obtained in the laboratory during starvation experiments, indicating that bacteria which survive periods of nutrient deprivation manifest a decrease of cell size. Even though the various reports brought about poorly defined designations for small bacteria, they have stimulated the discussion on how small a living bacterium could be. The information derived from the literature concerning starvation forms, ultramicrobacteria and nanobacteria is analysed in the light of own field and laboratory observations. It could be shown that despite conceptual shortcomings and problems with definitions of what is meant by "small", starvation forms and ultramicrobacteria are clearly distinguishable according to physiological characteristics, which could not be achieved for nanobacteria or nanobes. Furthermore, it is documented that ultramicrobacteria are not the smallest procaryotes and that the size of bacterial starvation forms are much closer to the calculated minimal sizes required to ensure independent viable life. A bacterial cell which is growing and dividing needs to be large enough to accommodate DNA and RNA, enzymes for replication transcription and translation, solvent for ubstances as well as a minimum set of proteins and plasamtic space to run the operations. Many authors assume that this requires a cell with a diameter not smaller than 200 nm and with a volume between 0.014 and 0.06 μm3. Most cells with diameters equal or below 0.2 μm are rods while cocci are numerically of minor importance in natural aquatic systems. It seems that the rod morphotype has more potential to produce viable cells with minimal volumes than spherical morhotypes. This supports the assumption that not only size but also cell shape is important to achieve functional minimum cell volumes. Many cell parameters can be estimated with relatively high precision, but one should remember that absolute calibration is still not possible. Even if we assume that there are errors involved in most biometric measurements, there is still a trend which indicates that rod shaped cells can function with diameters below 0.2 μm and cell volumes well below 0.02 μm3. Such dimensions could be an indication that the lower size limit of a viable bacterium may be close to the size of the smallest hypothetical living cell, indicating that there is still more to know about the minimal required cell components allowing a bacterium the remain viable.

Although the existence of 0.2 µm filterable bacteria has been known since the early 80's, they are not taken into consideration when modeling microbial food webs, due to an overall lack of information concerning this specific size class. According to physiological studies on starvation forms and investigations on small bacterial cells in marine ecosystems, a 0.2 µm filtrate may consist of different phenotypes: starvation forms of typical marine bacteria, ultramicrobacteria or bacterial cells, even larger than 0.2 µm, but flexible enough to pass the nominal filter pore-size. In this pilot study we examined three filtered seawater fractions from the Western Mediterranean Sea (Bay of Calvi, Corsica/France) - the total bacterial population, the bacterial fraction above 0.2 µm and the 0.2 µm filtrate - to investigate the bacterial community structure of each of those fractions by the molecular approach of denaturing gradient gel electrophoresis (DGGE) of 16S rDNA fragments. The analysis of the resulting DGGE profiles revealed different patterns of dominant bands for the 0.2 µm filterable and the total bacterial populations within the samples. Additionally the 0.2 µm filterable bacterial compartment exhibited obvious differences in band patterns for winter and summer samples, which were not observed for the total bacterial fraction. According to the current knowledge concerning the status of 0.2 µm filterable bacteria, DGGE patterns indicate that most of the fragments representing 0.2 µm filterable bacteria were rather starvation forms of marine bacteria than ultramicrobacteria. The sequencing of excised and cloned DNA bands of the DGGE profiles characterized the phylogenetic affiliation of the corresponding 0.2 µm filterable bacteria, clustering mainly with known, typical marine isolates of both alpha-subclass and gamma-subclass of the Proteobacteria and the Cytophaga-Flavobacterium-Bacteroides branch.

Lakes and ponds harbour a high number of diverse planktonic microorganisms that are centrally involved in biochemical cycles and aquatic food webs. Although the open water body (pelagial) seems to be a uniform and unstructured environment, ecological niche separation of coexisting microbial taxa might be triggered by limiting resources (bottom-up control) and mortality factors (top-down control), leading to distinct spatial and temporal distribution patterns of different microbes. This review gives an overview of the most abundant prokaryotic populations by grouping them in specific ecological guilds based on their life strategies. Defense specialists such as very small Actinobacteria or big filamentous bacteria mostly occur at times of highest grazing pressure by heterotrophic nanoflagellates, the main consumers of bacteria. Oligotrophic ultramicrobacteria, on the other hand, seem to be mostly adapted to nutrient depleted water layers during summer stratification, while opportunistic bacteria profit from material released during short-living algal blooms. Seasonal changes in abiotic and biotic factors may be the main causes for periodic reoccurring density maxima of different prokaryotes populations in the pelagial of temperate lakes, reflected in a distinct seasonality of the freshwater bacterioplankton.

Indigenous heterotrophic marine bacteria are of great importance to global nutrient cycling. Predominant native bacteria are of ultramicrobacterial dimensions, are not associated with aggregates and must have truly remarkable abilities for substrate capture. Agar media are unsuited for the isolation of the dominant oceanic bacterioplankton. In contrast, the dilution culture technique [Button et al. (1993) Appl. Environ. Microbiol. 59, 881–891; Schut et al. (1993) Appl. Environ. Microbiol. 59, 2150–2160] leads to a successful enrichment of the dominant cell types. Up to 50% of the indigenous bacterial population in water obtained from Resurrection Bay was able to grow in dilution tubes containing only filtered, autoclaved natural sea water (FAS). Ultramicrobacteria (UMB), very small bacteria with small genomes, predominate in such cultures. Generally, dilution factors that resulted in inocula of approximately 2 cells per tube were optimal and prevented outgrowth of atypical large bacteria. Strain RB2256 [Schut et al. (1993) Appl. Environ. Microbiol. 59, 2150–2160], one of the UMB isolated by this dilution culture technique and tentatively identified as a marine Sphingomonas sp., was investigated in more detail. Although reverted from obligately oligotrophic to facultatively oligotrophic upon isolation, this strain possessed a number of traits assigned to a ‘model oligotroph’ and some unpredicted novel properties. The cells showed no miniaturisation upon starvation but consistently exhibited low cell volumes. They had a very low DNA content, were rich in protein, and contained only one copy of the rRNA operon. The cells were well adapted to the simultaneous utilisation of mixed substrates. A constitutive, high-affinity and binding protein-dependent uptake system for mixed amino acids was found that would allow realistic in situ generation times at the prevailing amino acid concentrations. Further studies on this same organism revealed that the cells appeared to be extremely resistant to various stress-inducing agents. High survival rates were observed after high-intensity heat shocks, treatments with H2O2 or with ethanol. Moreover, no marked differences were observed between starved or actively growing cells in this respect, particularly when cells were grown in chemostat. Application of the dilution culture technique to the field of subsurface microbiology could be adopted to study the occurrence of UMB in groundwater with a comparable and stable ‘low-nutrient-conditioned’ phenotype.

Indigenous heterotrophic marine bacteria are of great importance to global nutrient cycling. Predominant native bacteria are of ultramicrobacterial dimensions, are not associated with aggregates and must have truly remarkable abilities for substrate capture. Agar media are unsuited for the isolation of the dominant oceanic bacterioplankton. In contrast, the dilution culture technique [Button et al. (1993) Appl. Environ. Microbiol. 59, 881-891; Schut et al. (1993) Appl. Environ. Microbiol. 59, 2150-2160] leads to a successful enrichment of the dominant cell types. Up to 50% of the indigenous bacterial population in water obtained from Resurrection Bay was able to grow in dilution tubes containing only filtered, autoclaved natural sea water (FAS). Ultramicrobacteria (UMB), very small bacteria with small genomes, predominate in such cultures. Generally, dilution factors that resulted in inocula of approximately 2 cells per tube were optimal and prevented outgrowth of atypical large bacteria. Strain RB2256 [Schut et al. (1993) Appl. Environ. Microbiol. 59, 2150-2160], one of the UMB isolated by this dilution culture technique and tentatively identified as a marine Sphingomonas sp., was investigated in more detail. Although reverted from obligately oligotrophic to facultatively oligotrophic upon isolation, this strain possessed a number of traits assigned to a 'model oligotroph' and some unpredicted novel properties. The cells showed no miniaturisation upon starvation but consistently exhibited low cell volumes. They had a very low DNA content, were rich in protein, and contained only one copy of the rRNA operon. The cells were well adapted to the simultaneous utilisation of mixed substrates. A constitutive, high-affinity and binding protein-dependent uptake system for mixed amino acids was found that would allow realistic in situ generation times at the prevailing amino acid concentrations. Further studies on this same organism revealed that the cells appeared to be extremely resistant to various stress-inducing agents. High survival rates were observed after high-intensity heat shocks, treatments with H2O2 or with ethanol. Moreover, no marked differences were observed between starved or actively growing cells in this respect, particularly when cells were grown in chemostat. Application of the dilution culture technique to the field of subsurface microbiology could be adopted to study the occurrence of UMB in groundwater with a comparable and stable 'low-nutrient-conditioned' phenotype.

A facultatively oligotrophic ultramicrobacterium (strain RB2256) isolated from an Alaskan fjord by extinction dilution in seawater, was grown in batch culture and under single- and dual-substrate-limitation of alanine and glucose in a chemostat. The nature of the uptake systems, and the uptake kinetics and utilization patterns of alanine and glucose were investigated. Glucose uptake was inducible, the system exhibited a narrow substrate specificity, and part of the uptake system was osmotic-shock-sensitive. Half-saturation constants for glucose were between 7 and 74 mu M during glucose limitation. The initial step in glucose metabolism was the synthesis of sugar polymers, even during glucose-limited growth. The alanine uptake system was constitutively expressed and was binding-protein-dependent. In addition to L-alanine, nine other amino acids inhibited accumulation of [C-14]L-alanine, indicating broad substrate specificity of the alanine transporter. Half-saturation constants between 1.3 and 1.8 mu M were determined for alanine uptake during alanine limitation, Simultaneous utilization of glucose and alanine occurred during substrate-limited growth in the chemostat, and during growth in batch culture at relatively high (mM) substrate concentrations. However, the half-saturation constant for alanine transport during dual-substrate-limitation, i.e. in the presence of glucose, increased almost fivefold. We conclude that mixed substrate utilization is an inherent property of this organism.

Coastal lagoons, both hypersaline and freshwater, are common, but still understudied ecosystems. We describe, for the first time, using high throughput sequencing, the extant microbiota of two large and representative Mediterranean coastal lagoons, the hypersaline Mar Menor, and the freshwater Albufera de Valencia, both located on the south eastern coast of Spain. We show there are considerable differences in the microbiota of both lagoons, in comparison to other marine and freshwater habitats. Importantly, a novel uncultured sulfur oxidizing Alphaproteobacteria was found to dominate bacterioplankton in the hypersaline Mar Menor. Also, in the latter prokaryotic cyanobacteria were almost exclusively comprised by Synechococcus and no Prochlorococcus was found. Remarkably, the microbial community in the freshwaters of the hypertrophic Albufera was completely in contrast to known freshwater systems, in that there was a near absence of well known and cosmopolitan groups of ultramicrobacteria namely Low GC Actinobacteria and the LD12 lineage of Alphaproteobacteria.

Alphaproteobacteria are common members of marine bacterioplankton assemblages, but are believed to be rare in lacustrine systems. However, uncultured Alphaproteobacteria of the freshwater LD12 lineage form a tight monophyletic sister group with the numerically dominant bacteria in marine epipelagic waters, the SAR11 clade or genus Pelagibacter. Comparative rRNA sequence analysis reveals a global occurrence of LD12 bacteria in freshwater systems. The association of genotypic subclades with single-study systems moreover suggests a regional diversification. LD12 bacteria exhibit distinct and annually recurring spatio-temporal distribution patterns in prealpine lakes, as assessed by seasonally resolved vertical profiling and high-throughput cell counting. During the summer months, these ultramicrobacteria can form cell densities in the surface (epilimnetic) water layers that are comparable to those of their marine counterparts (5 × 10(8) cells per l). LD12 bacteria had a pronounced preference for glutamine and glutamate over 7 other amino acids in situ, and they exhibited substantially higher uptake of these two substrates (and glycine) than the microbial assemblage in general. In addition, members of LD12 were also able to exploit other monomeric sources of organic carbon such as glucose, fructose or acetate. LD12 seemed to follow an oligotrophic lifestyle with slow but efficient uptake already at low substrate concentrations. Thus, LD12 bacteria do not only share phenotypic and metabolic traits with Pelagibacter, but also seem to thrive in the analogous spatiotemporal niche in freshwaters. The two groups together form one of the rare monophyletic lineages of ultramicrobacteria that have successfully traversed the barrier between marine and freshwater habitats.

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 MY14 T 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.

Summary: Changes in cell size and shape associated with bacterial starvation and resuscitation were used as a technique to enhance oil recovery. Injection of starved bacteria followed by nutrients into rock cores produced deep bacterial plugs. This work reports on an investigation of the dispersion and resuscitation of ultramicrobacteria (UMB) produced by starvation of Pseudomonas sp. FC3. Initially, the UMB were injected into one-dimensional sandcores; in other words, flow was from the inlet to the outlet in one direction. To provide closer simulation of oil well conditions, the UMB were injected into a three-dimensional reservoir simulator, 45 cm in diameter by 38 cm in length. Sample probes strategically positioned within the sandpack detected the UMB as they dispersed throughout the pack. After 24 d of nutrient stimulation, the sandpack was dismantled and analysed. Scanning electron microscopy and carbohydrate assays of the sandpack sections showed glycocalyx (polymer) production in every section. The three-dimensional model confirmed the findings of the unidirectional core models in that the UMB penetrated the sandpack, resuscitated and grew in situ to form a confluent bacterial plug when nutrient-stimulated.

Background The loss of photosynthesis has occurred often in eukaryotic evolution, even more than its acquisition, which occurred at least nine times independently and which generated the evolution of the supergroups Archaeplastida, Rhizaria, Chromalveolata and Excavata. This secondary loss of autotrophic capability is essential to explain the evolution of eukaryotes and the high diversity of protists, which has been severely underestimated until recently. However, the ecological and evolutionary scenarios behind this evolutionary “step back” are still largely unknown. Methodology/Principal Findings Using a dynamic model of heterotrophic and mixotrophic flagellates and two types of prey, large bacteria and ultramicrobacteria, we examine the influence of DOC concentration, mixotroph's photosynthetic growth rate, and external limitations of photosynthesis on the coexistence of both types of flagellates. Our key premises are: large bacteria grow faster than small ones at high DOC concentrations, and vice versa; and heterotrophic flagellates are more efficient than the mixotrophs grazing small bacteria (both empirically supported). We show that differential efficiency in bacteria grazing, which strongly depends on cell size, is a key factor to explain the loss of photosynthesis in mixotrophs (which combine photosynthesis and bacterivory) leading to purely heterotrophic lineages. Further, we show in what conditions an heterotroph mutant can coexist, or even out-compete, its mixotrophic ancestor, suggesting that bacterivory and cell size reduction may have been major triggers for the diversification of eukaryotes. Conclusions/Significance Our results suggest that, provided the mixotroph's photosynthetic advantage is not too large, the (small) heterotroph will also dominate in nutrient-poor environments and will readily invade a community of mixotrophs and bacteria, due to its higher efficiency exploiting the ultramicrobacteria. As carbon-limited conditions were presumably widespread throughout Earth history, such a scenario may explain the numerous transitions from phototrophy to mixotrophy and further to heterotrophy within virtually all major algal lineages. We challenge prevailing concepts that affiliated the evolution of phagotrophy with eutrophic or strongly light-limited environments only.

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.

We investigated the survival strategies and the competitive strength of pigmented and colourless bacterivorous flagellates using 2 model organisms, the bacterivorous chrysophytes Poteri- oochromonas malhamensis and Spumella sp., fed small ultramicrobacteria (0.044 µm 3 ) and large bac- teria (0.38 µm 3 ). The numerical responses indicated that small bacteria are not such a good food source as large bacteria, even when bacterial cell volume was taken into account. The growth rates of the heterotrophic Spumella sp. exceeded those of the mixotrophic P. malhamensis at high bacter- ial concentrations of both sizes of bacteria. At very low prey abundances, P. malhamensis grew bet- ter than Spumella sp., mainly because the mortality rate of the latter was very high. At bacterial bio- volumes encountered in oligotrophic/mesotrophic lakes, the growth curves of the 2 flagellates intersected. Although P. malhamensis possesses a chloroplast, growth of cultures incubated in the light and the dark did not differ even at very low prey concentrations, indicating that photosynthesis plays a minor role for this chrysophyte. Experiments carried out with mixed flagellate cultures showed that P. malhamensis ingested Spumella sp. cells. Applying our data to natural abundances of small and large bacteria as well as pico/nanoplankton we could demonstrate that P. malhamensis is a K-strategist. Large bacteria made up the major portion of its carbon uptake, but small bacteria, pico-nanoplankton and (to a minor extent) photosynthesis also contributed to its gross carbon uptake, indicating a broad use of different resources by this species. Spumella sp. is more of an r-strategist and relies mainly on large bacteria for growth although small bacteria can contribute significantly to its diet.

Financiadora de Estudos e Projetos Strains Lc (CCT 7277) and Lg (CCT 7137), isolated from landfill leachate-contaminated groundwater and found to produce exopolysaccharides (ESP), were characterized by polyphasic taxonomy. The growth and EPS production of Lg strain on sugarcane molasses, at concentrations between 2% and 10% and on the standard GYM medium were studied and its responses to nutrient starvation were analysed. Morphological, biochemical, chemotaxonomic and molecular tests indicated that Lc and Lg have properties typical of Gordonia polyisoprenivorans DSM 44303T. Viable cell counts revealed that GYM medium was the best for growth (µmax = 0,232 h-1). EPS production started during the exponential phase and continued during the stationary phase, in all culture media. After 24h of stationary phase, G. polyisoprenivorans CCT 7137 showed a highest total EPS production in 6% (172,86 g/l) and 10% (139,47 g/l) sugarcane molasses media, a highest EPS production for unit of cellular concentration in 10% sugarcane molasses medium (39,03 x 10- 11g/UFC) and a highest porcentage of free EPS in 2%, 6% e 10% sugarcane molasses media (88,37%, 62,4% e 64,22%, respectively). G. polyisoprenivorans CCT 7137 showed three responses to nutrient starvation: a decrease in the cell viability (13,7% and 3,25% after 7 and 56 days respectively), a reduction in cell size (from 0,55 to 0,33 µm3) and a cell shape change (from rod to coccobacillus). G. polyisoprenivorans CCT 7277 and CCT 7137 are two of six strains of G. polyisoprenivorans so far isolated and recorded in the literature, and the first strains found in a brazilian ecosystem. The data presented here suggest the potencial of G. polyisoprenivorans CCT7137 to be used as ultramicrobacteria in porous media experiments such as to aim for the study of sugarcane molasses as nutrient source for in situ EPS to biorremediate contaminated sites through the biobarrier technology. As linhagens Lc (CCT 7277) e Lg (CCT 7137), isoladas da água subterrânea contaminada pelo chorume de um antigo aterro controlado (São Paulo, Brasil) e produtoras de exopolissacarídeos (EPS), foram caracterizadas por taxonomia polifásica. A linhagem Lg teve a) o crescimento e a produção de EPS analisados em meio GYM e em diferentes concentrações de melaço-de-cana de açúcar (entre 2% e 10%) e b) o desempenho avaliado quando submetida ao processo de desnutrição. As análises morfológicas, bioquímicas, quimiotaxonômicas e moleculares mostraram que as linhagens Lc e Lg possuem propriedades típicas da linhagem tipo de Gordonia polyisoprenivorans (DSM 44302T). A análise do número de células viáveis mostrou que o meio GYM foi o que mais favoreceu o crescimento da bactéria (µmax = 0,232 h-1). A produção de EPS , em todos os meios de cultura, iniciou-se durante a fase exponencial e continuou ao longo da fase estacionária de crescimento. Após 24h de fase estacionária foi detectada uma maior produção de EPS total nos meios melaço 6% (172,86 g/l) e 10% (139,47 g/l), uma maior produção de EPS total por unidade de concentração celular no meio melaço 10% (39,03 x 10-11g/UFC) e uma maior porcentagem de EPS livre nos meios melaço 2%, 6% e 10% (88,37%, 62,4% e 64,22% do total de EPS, respectivamente). G. polyisoprenivorans CCT 7137 apresentou, como respostas de sobrevivência à desnutrição, redução da densidade de células cultiváveis (13,7% e 3,25% após 7 e 56 dias, respectivamente), redução no volume celular (de 0,55 para 0,33 µm3) e alteração da forma celular que passou de bastonete para cocobacilar. Gordonia polyisoprenivorans CCT 7277 e CCT 7137 constituem duas das seis linhagens de G. polyisoprenivorans até então isoladas e as primeiras isoladas de ecossistema brasileiro, Os dados obtidos no presente trabalho mostraram o potencial de G. polyisoprenivorans CCT7137 para ser utilizada como ultramicrobactéria em testes em meio poroso e para se estudar a utilização de melaço de cana-de-açúcar como meio nutriente para a produção de EPS in situ visando a biorremediação de ambientes impactados, utilizando a tecnologia de biobarreiras.

Ultramicrobacteria (cell volume < 0.1 μm 3 ) are the numerically dominant organisms in the plankton of marine and freshwater habitats. Flagellates and other protists are assumed to be the most important predators of these ultramicrobacteria as well as of larger planktonic bacteria. However, due to controversial observations conducted previously, it is not clear as to whether fractions of the ultramicrobacteria are resistant to flagellate predation. Furthermore, it is not known if closely related bacteria vary significantly in their sensitivity to flagellate predation. We investigated the sensitivity of ultramicrobacteria affiliated with the cosmopolitan Polynucleobacter cluster to grazing by Spumella -like nanoflagellates. Laboratory grazing experiments with four closely related (≥99.6% 16S rRNA gene sequence similarity) bacteria and three closely related (100% 18S rRNA gene sequence similarity) flagellates were performed. In comparison to larger bacteria, predation on the ultramicrobacterial Polynucleobacter strains was weak, and the growth of the predating flagellates was slow. Specific clearance rates ranged between 0.14 × 10 5 and 2.8 × 10 5 units of predator size h −1 . Feeding rates strongly depended on the flagellate and bacterial strain ( P < 0.001). Grazing mortality rates of the three flagellate strains investigated varied for the same prey strain by up to almost fourfold. We conclude that (i) ultramicrobacteria affiliated with the Polynucleobacter cluster are not protected from grazing, (ii) strain-specific variations in grazing sensitivity even between closely related bacteria are high, and (iii) strain-specific differences in predator-prey interaction could be an important factor in the evolution and maintenance of microbial microdiversity.

The majority of the bacterial cells present in the bacterioplankton of marine and freshwater habitats are small (cell length, 1.5 μm; volume, >0.3 μm3) than most planktonic bacteria. In the case of several normally large-cell-size bacterial species, it has been shown that under strong starvation conditions the cell size decreases to dimensions typical of the majority of planktonic bacteria (23, 26, 27, 31). There are only a few bacterial strains that have been isolated from marine and soil habitats whose cells are in the ultramicro size range (volume

The heterotrophic bacterial community of oceanic aggregates which mediates particle solubilization, displays features (low carbon demand and low turnover) that are difficult to reconcile with the observed high enzyme activities and cell numbers. Hypotheses are proposed to explain this discrepancy. Furthermore, production of both free and attached bacteria may have been underestimated by neglecting the ultramicrobacteria (UMB). Production of UMB may represent up to 28% of the total bacterial production.

Free-living, aerobic, copiotrophic ultramicrobacteria (UMB) that passed through a 0.45mgr;m membrane filter and had a cell volume of less than 0.3mgr;m(3) were isolated from polluted urban soil by using both the direct plating method and the membrane-filter enrichment technique. The efficiency of recovering UMB from the soil was much higher in the latter method than in the former. All of the UMB isolates grew well with a doubling time of less than 6 h either in a complex nutrient medium or a chemically defined medium. The average cell volumes of the UMB isolates, as measured by scanning electron microscopy and epifluorescent microscopy with an image analysis, ranged from 0.07 to 0.22mgr;m(3). The cell size was larger at the exponential phase of growth than at the stationary growth stage in general. Ultrathin-section electron microscopy of representatives of the UMB isolates showed that they had complete cell wall structures like typical Gram-negative or -positive bacteria. Phenotypic studies and phylogenetic analyses on the basis of 16S rDNA sequences showed that the UMB isolates were classified into three major groups, the beta and gamma subdivisions of the Proteobacteria and the Actinobacteria (the high G+C DNA group of Gram-positives). However, none of these isolates were assigned to any previously known species. These results demonstrate that free-living, relatively fast-growing, copiotrophic UMB strains undescribed so far are widely distributed in terrestrial environments, including urban soil.

Bacteria survive periods of low-nutrient conditions by adopting a series of starvation-survival responses to produce ultramicrobacteria. These small cells are better able to penetrate many different environments than vegetative cells, which helps them reach subterranean environments. They may therefore be used to enhance oil recovery or for in situ bioremedies.

Sphingomonas sp. strain RB2256 is a representative of the dominant class of ultramicrobacteria that are present in marine oligotrophic waters. In this study we examined the rRNA copy number and ribosome content of RB2256 to identify factors that may be associated with the relatively low rate of growth exhibited by the organism. It was found that RB2256 contains a single copy of the rRNA operon, in contrast to Vibrio spp., which contain more than eight copies. The maximum number of ribosomes per cell was observed during mid-log phase; however, this maximum content was low compared to those of faster-growing, heterotrophic bacteria (approximately 8% of the maximum ribosome content of Escherichia coli with a growth rate of 1.5 h −1 ). The low number of ribosomes per cell appears to correlate with the low rate of growth (0.16 to 0.18 h −1 ) and the presence of a single copy of the rRNA operon. However, on the basis of cell volume, RB2256 appears to have a higher concentration of ribosomes than E. coli (approximately double that of E. coli with a growth rate of 1.5 h −1 ). Ribosome numbers reached maximum levels during mid-log-phase growth but decreased rapidly to 10% of maximum during late log phase through 7 days of starvation. The cells in late log phase and at the onset of starvation displayed an immediate response to a sudden addition of excess glucose (3 mM). This result demonstrates that a ribosome content 10% of maximum is sufficient to allow cells to immediately respond to nutrient upshift and achieve maximum rates of growth. These data indicate that the bulk of the ribosome pool is not required for protein synthesis and that ribosomes are not the limiting factor contributing to a low rate of growth. Our findings show that the regulation of ribosome content, the number of ribosomes per cell, and growth rate responses in RB2256 are fundamentally different from those characteristics in fast-growing heterotrophs like E. coli and that they may be characteristics typical of oligotrophic ultramicrobacteria.

Indigenous heterotrophic marine bacteria are of great importance to global nutrient cycling. Predominant native bacteria are of ultramicrobacterial dimensions, are not associated with aggregates and must have truly remarkable abilities for substrate capture. Agar media are unsuited for the isolation of the dominant oceanic bacterioplankton. In contrast, the dilution culture technique [Button et al. (1993) Appl. Environ. Microbiol. 59, 881-891; Schut et al. (1993) Appl. Environ. Microbiol. 59, 2150-2160] leads to a successful enrichment of the dominant cell types. Up to 50% of the indigenous bacterial population in water obtained from Resurrection Bay was able to grow in dilution tubes containing only filtered, autoclaved natural sea water (FAS). Ultramicrobacteria (UMB), very small bacteria with small genomes, predominate in such cultures. Generally, dilution factors that resulted in inocula of approximately 2 cells per tube were optimal and prevented outgrowth of atypical large bacteria. Strain RB2256 [Schut et al. (1993) Appl. Environ. Microbiol. 59, 2150-2160], one of the UMB isolated by this dilution culture technique and tentatively identified as a marine Sphingomonas sp., was investigated in more detail. Although reverted from obligately oligotrophic to facultatively oligotrophic upon isolation, this strain possessed a number of traits assigned to a 'model oligotroph' and some unpredicted novel properties. The cells showed no miniaturisation upon starvation but consistently exhibited low cell volumes. They had a very low DNA content, were rich in protein, and contained only one copy of the rRNA operon. The cells were well adapted to the simultaneous utilisation of mixed substrates. A constitutive, high-affinity and binding protein-dependent uptake system for mixed amino acids was found that would allow realistic in situ generation times at the prevailing amino acid concentrations. Further studies on this same organism revealed that the cells appeared to be extremely resistant to various stress-inducing agents. High survival rates were observed after high-intensity heat shocks, treatments with H2O2 or with ethanol. Moreover, no marked differences were observed between starved or actively growing cells in this respect, particularly when cells were grown in chemostat. Application of the dilution culture technique to the field of subsurface microbiology could be adopted to study the occurrence of UMB in groundwater with a comparable and stable 'low-nutrient-conditioned' phenotype.