A fundamental question in community ecology is the role of predator-prey interactions in food-web stability and species coexistence. Although microbial microcosms offer powerful systems to investigate it, interrogating the environment is much more arduous. Here, we show in a 1-year survey that the obligate predators Bdellovibrio and like organisms (BALOs) can regulate prey populations, possibly in a density-dependent manner, in the naturally complex, species-rich environments of wastewater treatment plants. Abundant as well as rarer prey populations are affected, leading to an oscillating predatory landscape shifting at various temporal scales in which the total population remains stable. Shifts, along with differential prey range, explain co-existence of the numerous predators through niche partitioning. We validate these sequence-based findings using single-cell sorting combined with fluorescent hybridization and community sequencing. Our approach should be applicable for deciphering community interactions in other systems.

Flow cytometry has recently established itself as a tool to track short-term dynamics in microbial community assembly and link those dynamics with ecological parameters. However, instrumental configurations of commercial cytometers and variability introduced through differential handling of the cells and instruments frequently cause data set variability at the single-cell level. This is especially pronounced with microorganisms, which are in the lower range of optical resolution. Although alignment beads are valuable to generally minimize instrumental noise and align overall machine settings, an artificial microbial cytometric mock community (mCMC) is mandatory for validating lab workflows and enabling comparison of data between experiments, thus representing a necessary reference standard for the reproducible cytometric characterization of microbial communities, especially in long-term studies. In this study, the mock community consisted of two Gram-positive and two Gram-negative bacterial strains, which can be assembled with respective subsets of cells, including spores, in any selected ratio or concentration. The preparation of the four strains takes a maximum of 5 d, and the stains are storable with either PFA/ethanol fixation at –20 °C or drying at 4 °C for at least 6 months. Starting from this stock, an mCMC can be assembled within 1 h. Fluorescence staining methods are presented and representatively applied with two high-resolution cell sorters and three benchtop flow cytometers. Benchmarked data sets allow the use of bioinformatic evaluation procedures to decode community behavior or convey qualified cell sorting decisions for subsequent high-resolution sequencing or proteomic routines.

Roux-en-Y gastric bypass (RYGB) surgery is a last-resort treatment to induce substantial and sustained weight loss in cases of severe obesity. This anatomical rearrangement affects the intestinal microbiota, but so far, little information is available on how it interferes with microbial functionality and microbial-host interactions independently of weight loss. A rat model was employed where the RYGB-surgery cohort is compared to sham-operated controls which were kept at a matched body weight by food restriction. We investigated the microbial taxonomy and functional activity using 16S rRNA amplicon gene sequencing, metaproteomics, and metabolomics on samples collected from theileum, the cecum, and the colon, and separately analysed the lumen and mucus-associated microbiota. Altered gut architecture in RYGB increased the relative occurrence of Actinobacteria, especially Bifidobacteriaceae and Proteobacteria, while in general, Firmicutes were decreased although Streptococcaceae and Clostridium perfringens were observed at relative higher abundances independent of weight loss. A decrease of conjugated and secondary bile acids was observed in the RYGB-gut lumen. The arginine biosynthesis pathway in the microbiota was altered, as indicated by the changes in the abundance of upstream metabolites and enzymes, resulting in lower levels of arginine and higher levels of aspartate in the colon after RYGB. The anatomical rearrangement in RYGB affects microbiota composition and functionality as well as changes in amino acid and bile acid metabolism independently of weight loss. The shift in the taxonomic structure of the microbiota after RYGB may be mediated by the resulting change in the composition of the bile acid pool in the gut and by changes in the composition of nutrients in the gut.

Generating chemical energy carriers and bulk chemicals from solar energy by microbial metabolic capacities is a promising technology. In this long-term study of over 500 days, methane was produced by a microbial community that was fed by the mono-substrate glycolate, which was derived from engineered algae. The microbial community structure was measured on the single cell level using flow cytometry. Abiotic and operational reactor parameters were analyzed in parallel. The R-based tool flowCyBar facilitated visualization of community dynamics and indicated sub-communities involved in glycolate fermentation and methanogenesis. Cell sorting and amplicon sequencing of 16S rRNA and mcrA genes were used to identify the key organisms involved in the anaerobic conversion process. The microbial community allowed a constant fermentation, although it was sensitive to high glycolate concentrations in the feed. A linear correlation between glycolate loading rate and biogas amount was observed (R2 = 0.99) for glycolate loading rates up to 1.81 g L−1 day−1 with a maximum in biogas amount of 3635 mL day−1 encompassing 45% methane. The cytometric diversity remained high during the whole cultivation period. The dominating bacterial genera were Syntrophobotulus, Clostridia genus B55_F, Aminobacterium, and Petrimonas. Methanogenesis was almost exclusively performed by the hydrogenotrophic genus Methanobacterium.

Summary In completely insular microbial communities, evolution of community structure cannot be shaped by the immigration of new members. In addition, when those communities are run in steady state, the influence of environmental factors on their assembly is reduced. Therefore, one would expect similar community structures under steady‐state conditions. Yet, in parallel setups, variability does occur. To reveal ecological mechanisms behind this phenomenon, five parallel reactors were studied at the single‐cell level for about 100 generations and community structure variations were quantified by ecological measures. Whether community variability can be controlled was tested by implementing soft temperature stressors as potential synchronizers. The low slope of the lognormal rank‐order abundance curves indicated a predominance of neutral mechanisms, i.e., where species identity plays no role. Variations in abundance ranks of subcommunities and increase in inter‐community pairwise β‐diversity over time support this. Niche differentiation was also observed, as indicated by steeper geometric‐like rank‐order abundance curves and increased numbers of correlations between abiotic and biotic parameters during initial adaptation and after disturbances. Still, neutral forces dominated community assembly. Our findings suggest that complex microbial communities in insular steady‐state environments can be difficult to synchronize and maintained in their original or desired structure, as they are non‐equilibrium systems.

The widely established production of CH4 from renewable biomass in industrial scale anaerobic reactors may play a major role in the future energy supply. It relies on methanogenic archaea as key organisms which represent the bottleneck in the process. The quantitative analysis of these organisms can help to maximize process performance, uncover disturbances before failure, and may ultimately lead to community-based process control schemes. Existing qPCR and fluorescence microscopy-based methods are very attractive but can be cost-intensive and laborious.

Using high-resolution flow cytometry of bacterial shape (forward scatter) and DNA content (DAPI staining), we detected dramatic differences in the fecal microbiota composition during murine colitis that were validated using 16S rDNA sequencing. This innovative method provides a fast and inexpensive tool to interrogate the microbiota on the single-cell level.

BACKGROUND:Plasmids are widely used for molecular cloning or production of proteins in laboratory and industrial settings. Constant modification has brought forth countless plasmid vectors whose characteristics in terms of average plasmid copy number (PCN) and stability are rarely known. The crucial factor determining the PCN is the replication system; most replication systems in use today belong to a small number of different classes and are available through repositories like the Standard European Vector Architecture (SEVA).RESULTS:In this study, the PCN was determined in a set of seven SEVA-based expression plasmids only differing in the replication system. The average PCN for all constructs was determined by Droplet Digital PCR and ranged between 2 and 40 per chromosome in the host organism Escherichia coli. Furthermore, a plasmid-encoded EGFP reporter protein served as a means to assess variability in reporter gene expression on the single cell level. Only cells with one type of plasmid (RSF1010 replication system) showed a high degree of heterogeneity with a clear bimodal distribution of EGFP intensity while the others showed a normal distribution. The heterogeneous RSF1010-carrying cell population and one normally distributed population (ColE1 replication system) were further analyzed by sorting cells of sub-populations selected according to EGFP intensity. For both plasmids, low and highly fluorescent sub-populations showed a remarkable difference in PCN, ranging from 9.2 to 123.4 for ColE1 and from 0.5 to 11.8 for RSF1010, respectively.CONCLUSIONS:The average PCN determined here for a set of standardized plasmids was generally at the lower end of previously reported ranges and not related to the degree of heterogeneity. Further characterization of a heterogeneous and a homogeneous population demonstrated considerable differences in the PCN of sub-populations. We therefore present direct molecular evidence that the average PCN does not represent the true number of plasmid molecules in individual cells.

Horizontal gene transfer (HGT) is a main mechanism of bacterial evolution endowing bacteria with new genetic traits. The transfer of mobile genetic elements such as plasmids (conjugation) requires the close proximity of cells. HGT between genetically distinct bacteria largely depends on cell movement in water films, which are typically discontinuous in natural systems like soil. Using laboratory microcosms, a bacterial reporter system and flow cytometry, we here investigated if and to which degree mycelial networks facilitate contact of and HGT between spatially separated bacteria. Our study shows that the network structures of mycelia promote bacterial HGT by providing continuous liquid films in which bacterial migration and contacts are favoured. This finding was confirmed by individual-based simulations, revealing that the tendency of migrating bacteria to concentrate in the liquid film around hyphae is a key factor for improved HGT along mycelial networks. Given their ubiquity, we propose that hyphae can act as focal point for HGT and genetic adaptation in soil.

Bacterial growth is often difficult to estimate beyond classical cultivation approaches. Low cell numbers, particles or coloured and dense media may disturb reliable growth assessment. Further difficulties appear when cells are attached to surfaces and detachment is incomplete. Therefore, flow cytometry was tested and used for analysis of bacterial growth on the single-cell level. Shewanella putrefaciens was cultivated as a model organism in planktonic or biofilm culture. Materials of smooth and rough surfaces were used for biofilm cultivation. Both aerobic and anaerobic as well as feast and famine conditions were applied. Visualization of growth was also done using Environmental Scanning and Phase Contrast Microscopy. Bioinformatic tools were applied for data interpretation. Cytometric proliferation patterns based on distributions of DNA contents per cell corresponded distinctly to the various lifestyles, electron acceptors and substrates tested. Therefore, cell cycling profiles of S.putrefaciens were found to mirror growth conditions. The cytometric patterns were consistently detectable with exception of some biofilm types whose resolution remained challenging. Corresponding heat maps proved to be useful for clear visualization of growth behaviour under all tested conditions. Therefore, flow cytometry in combination with bioinformatic tools proved to be powerful means to determine various growth states of S.putrefaciens, even in constrained environments. The approach is universal and will also be applicable for other bacterial species.

Populations of genetically identical microorganisms residing in the same environment can display marked variability in their phenotypic traits; this phenomenon is termed phenotypic heterogeneity. The relevance of such heterogeneity in natural habitats is unknown, because phenotypic characterization of a sufficient number of single cells of the same species in complex microbial communities is technically difficult. We report a procedure that allows to measure phenotypic heterogeneity in bacterial populations from natural environments, and use it to analyze N2 and CO2 fixation of single cells of the green sulfur bacterium Chlorobium phaeobacteroides from the meromictic lake Lago di Cadagno. We incubated lake water with 15N2 and 13CO2 under in situ conditions with and without NH4+. Subsequently, we used flow cell sorting with auto-fluorescence gating based on a pure culture isolate to concentrate C. phaeobacteroides from its natural abundance of 0.2% to now 26.5% of total bacteria. C. phaeobacteroides cells were identified using catalyzed-reporter deposition fluorescence in situ hybridization (CARD-FISH) targeting the 16S rRNA in the sorted population with a species-specific probe. In a last step, we used nanometer-scale secondary ion mass spectrometry to measure the incorporation 15N and 13C stable isotopes in more than 252 cells. We found that C. phaeobacteroides fixes N2 in the absence of NH4+, but not in the presence of NH4+ as has previously been suggested. N2 and CO2 fixation were heterogeneous among cells and positively correlated indicating that N2 and CO2 fixation activity interact and positively facilitate each other in individual cells. However, because CARD-FISH identification cannot detect genetic variability among cells of the same species, we cannot exclude genetic variability as a source for phenotypic heterogeneity in this natural population. Our study demonstrates the technical feasibility of measuring phenotypic heterogeneity in a rare bacterial species in its natural habitat, thus opening the door to study the occurrence and relevance of phenotypic heterogeneity in nature.

Functions of complex natural microbial communities are realized by single cells that contribute differently to the overall performance of a community. Usually, molecular and, more recently, deep-sequencing techniques are used for detailed but resource-consuming phylogenetic or functional analyses of microbial communities. Here we present a method for analyzing dynamic community structures that rapidly detects functional (rather than phylogenetic) coherent subcommunities by monitoring changes in cell-specific and abiotic microenvironmental parameters. The protocol involves the use of flow cytometry to analyze elastic light scattering and fluorescent cell labeling, with subsequent determination of cell gate abundance and finally the creation of a cytometric community fingerprint. Abiotic parameter analysis data are correlated with the dynamic cytometric fingerprint to obtain a time-bound functional heat map. The map facilitates the identification of activity hot spots in communities, which can be further resolved by subsequent cell sorting of key subcommunities and concurrent phylogenetic analysis (terminal restriction fragment length polymorphism, tRFLP). The cytometric fingerprint information is based on gate template settings and the functional heat maps are created using an R script. Cytometric fingerprinting and evaluation can be accomplished in 1 d, and additional subcommunity composition information can be obtained in a further 6 d.

1. Introduction Cell sampling during long-term experiments usually requires reliable storage of cells for later analysis. In this study, we evaluated three differ-ent preservation strategies (sodium azide fixation, deep freezing and vacuum drying) with regard to their effects on bacterial cells. Pseudomo-nas putida was used as a model organism and stored for shorter (2 d) and longer periods (28 d). The impact of the treatments (preservation method, duration) was evaluated on the level of single cells using flow cytometry and on the population level using protein mass spectrome-try. On the single cell level, the effec t of sodium azide fixation was found to be small (1.01 ≤ s d ≤ 1.76) for short term and larger for long term storage (1.59 ≤ s d ≤ 2.33), as determined by FlowFP fingerprinting . In contrast, deep frozen and vacuum dried cells showed properties highly similar to fresh reference cells, even after extended storage (0.5 ≤ sd ≤ 1.2). On the population level, the mass spectrometric analysis revealed about 800 proteins for each sample and storage condition. The proteome profiles evaluated by label-free quantification showed that variation within functional groups was least for deep frozen and vacuum dried cell samples after 2 d (sd log2 relative protein quantity < 1) and margin-ally increased after 28 d. In contrast, sodium azide fixation caused higher variations between functional groups although the number of de-tected proteins and the respective peptide coverage excluded protein degradation. In conclusion, deep freezing was found to be the method of choice, but simple vacuum drying of cells with storage at 4°C can be a convenient alternative.

Aerobic anoxygenic photosynthesis (AAP) is found in an increasing number of proteobacterial strains thriving in ecosystems ranging from extremely oligotrophic to eutrophic. Here, we have investigated whether the fuel oxygenate-degrading betaproteobacterium Aquincola tertiaricarbonis L108 can use AAP to compensate kinetic limitations at low heterotrophic substrate fluxes. In a fermenter experiment with complete biomass retention and also during chemostat cultivation, strain L108 was challenged with extremely low substrate feeding rates of tert-butyl alcohol (TBA), an intermediate of methyl tert-butyl ether (MTBE). Interestingly, formation of photosynthetic pigments, identified as bacteriochlorophyll a and spirilloxanthin, was only induced in growing cells at TBA feeding rates less than or equal to maintenance requirements observed under energy excess conditions. Growth continued at rates between 0.001 and 0.002 h(-1) even when the TBA feed was decreased to values close to 30 % of this maintenance rate. Partial sequencing of genomic DNA of strain L108 revealed a bacteriochlorophyll synthesis gene cluster (bchFNBHL) and photosynthesis regulator genes (ppsR and ppaA) typically found in AAP and other photosynthetic proteobacteria. The usage of light as auxiliary energy source enabling evolution of efficient degradation pathways for kinetically limited heterotrophic substrates and for lowering the threshold substrate concentration Smin at which growth becomes zero is discussed.

Eukaryotic unicellular organisms are an important part of the soil microbial community, but they are often neglected in soil functional microbial diversity analysis, principally due to the absence of specific investigation methods in the special soil environment. In this study we used a method based on high-density centrifugation to specifically isolate intact algal and yeast cells, with the aim to analyze them with flow cytometry and sort them for further molecular analysis such as deep sequencing. Recovery efficiency was tested at low abundance levels that fit those in natural environments (10(4) to 10(6) cells per g soil). Five algae and five yeast morphospecies isolated from soil were used for the testing. Recovery efficiency was between 1.5 to 43.16% and 2 to 30.2%, respectively, and was dependent on soil type for three of the algae. Control treatments without soil showed that the majority of cells were lost due to the method itself (58% and 55.8% respectively). However, the cell extraction technique did not much compromise cell vitality because a fluorescein di-acetate assay indicated high viability percentages (73.3% and 97.2% of cells, respectively). The low abundant algae and yeast morphospecies recovered from soil were cytometrically analyzed and sorted. Following, their DNA was isolated and amplified using specific primers. The developed workflow enables isolation and enrichment of intact autotrophic and heterotrophic soil unicellular eukaryotes from natural environments for subsequent application of deep sequencing technologies.

Quantitative Analysis of Spatial Association between Discrete Events Represented by Small Foci in Multicolor 3D Confocal Microscopy Krzysztof Berniak1, Tytus Bernas2, Hong Zhao3, Paulina Rybak4, Miroslaw Zarebski4, Zbigniew Darzynkiewicz5, Jerzy Dobrucki4 1Jagiellonian University, Cracow, Poland, Poland, 2Jagiellonian University in Krakow, 3New York Medical College, 4Jagiellonian University, Cracow, Poland, 5Brander Cancer Research Institute, New York, Medical College