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1.
Environ Microbiol ; 21(1): 164-181, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30289191

RESUMO

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 ß-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.


Assuntos
Microbiota/fisiologia , Análise de Célula Única , Ecossistema
2.
Eur J Immunol ; 46(5): 1300-3, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26909672

RESUMO

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.


Assuntos
Bactérias/genética , Bactérias/isolamento & purificação , Colite/microbiologia , Fezes/microbiologia , Citometria de Fluxo/métodos , Microbioma Gastrointestinal , Animais , Bactérias/classificação , Bactérias/citologia , Humanos , Doenças Inflamatórias Intestinais/microbiologia , Filogenia , RNA Ribossômico 16S/genética
3.
Microb Cell Fact ; 16(1): 180, 2017 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-29084543

RESUMO

BACKGROUND: 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. RESULTS: In this study we present an autofluorescence-based, flow cytometric method for the fast low-cost quantification of methanogenic archaea in complex microbial communities and crude substrates. The method was applied to a methanogenic enrichment culture (MEC) and digester samples (DS). The methanogenic archaea were quantified using the distinct fluorescence of their cofactor F420 in a range from 3.7 × 108 (± 3.3 × 106) cells mL-1 and 1.8 x 109 (± 1.1 × 108) cells mL-1. We evaluated different fixation methods and tested the sample stability. Stable abundance and fluorescence intensity were recorded up to 26 days during aerobic storage in PBS at 6 °C. The discrimination of the whole microbial community from the ubiquitous particle noise was facilitated by SYBR Green I staining and enabled calculation of relative abundances of methanogenic archaea of up to 9.64 ± 0.23% in the MEC and up to 4.43 ± 0.74% in the DS. The metaprofiling of the mcrA gene reinforced the results. CONCLUSIONS: The presented method allows for fast and reliable quantification of methanogenic archaea in microbial communities under authentic digester conditions and can thus be useful for process monitoring and control in biogas digesters.


Assuntos
Archaea/metabolismo , Metano/metabolismo , Archaea/citologia , Archaea/genética , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Benzotiazóis , Biocombustíveis , Biomassa , Diaminas , Citometria de Fluxo , Microscopia de Fluorescência , Compostos Orgânicos/química , Quinolinas , RNA Ribossômico 16S/química , RNA Ribossômico 16S/isolamento & purificação , RNA Ribossômico 16S/metabolismo , Análise de Sequência de DNA
4.
Microb Cell Fact ; 15(1): 211, 2016 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-27993152

RESUMO

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.


Assuntos
Variações do Número de Cópias de DNA , Citometria de Fluxo/métodos , Plasmídeos/genética , Reação em Cadeia da Polimerase/métodos , Escherichia coli/virologia , Plasmídeos/metabolismo
5.
Microbiology (Reading) ; 159(Pt 10): 2180-2190, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23873782

RESUMO

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.


Assuntos
Betaproteobacteria/crescimento & desenvolvimento , Betaproteobacteria/metabolismo , Fotossíntese , terc-Butil Álcool/metabolismo , Anaerobiose , Bacterioclorofila A/análise , Betaproteobacteria/química , Betaproteobacteria/fisiologia , DNA Bacteriano/química , DNA Bacteriano/genética , Metabolismo Energético , Dados de Sequência Molecular , Análise de Sequência de DNA , Xantofilas/análise
6.
Methods ; 57(3): 338-49, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22504724

RESUMO

Analyzing natural anaerobic microbial communities is a challenge and interpretation of the respective members' performances arduous. Strict anaerobes are often slow-growing and difficult to cultivate due to their unknown physiological capacities. Additionally, abiotic micro-environmental data are difficult to assess, limiting the information on the eco-chemical background in natural environments. This review describes how qualitative and quantitative data can be obtained on anaerobic microbial communities isolated from anoxic environments and treated under laboratory conditions. It gives information on how community composition ('phylogenetic fingerprint') and community structure ('cytometric fingerprint') can be described by PCR-based and single cell-based techniques, respectively. A cell sorting step combined both approaches and enabled quantitative and more precise community resolution. The community dynamics found were swift and strong, despite low and slow changes in measured abiotic parameters. Therefore, the community structure itself mirrored variation in the constructed long term (6 years) ecosystem in a most sensitive way and can be used as sensor for the ecosystems situation. New statistical tools are presented allowing suddenly changing performances of complex communities to be detected and community (in) stabilities to be monitored and/or predicted.


Assuntos
Bactérias Anaeróbias/crescimento & desenvolvimento , Citometria de Fluxo/métodos , Análise de Célula Única/métodos , Anaerobiose , Bactérias Anaeróbias/efeitos dos fármacos , Bactérias Anaeróbias/genética , Ácido Benzoico/farmacologia , Ecossistema , Consórcios Microbianos/efeitos dos fármacos , Consórcios Microbianos/fisiologia , Modelos Biológicos , Filogenia , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Tolueno/farmacologia
7.
Environ Sci Technol ; 46(1): 84-92, 2012 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-21805973

RESUMO

Wastewater treatment often suffers from instabilities and the failure of specific functions such as biological phosphorus removal by polyphosphate accumulating organisms. Since most of the microorganisms involved in water clarification are unknown it is challenging to operate the process accounting for the permanent varying abiotic parameters and the complex composition and unrevealed metabolic capacity of a wastewater microbial community. Fulfilling the demands for water quality irrespective of substrate inflow conditions may emit severe problems if the limited management resources of municipal wastewater treatment plants are regarded. We used flow cytometric analyses of cellular DNA and polyphosphate to create patterns mirroring dynamics in community structure. These patterns were resolved in up to 15 subclusters, the presence and abundances of which correlated with abiotic data. The study used biostatistics to determine the kind and strength of the correlation. Samples investigated were obtained from a primary clarifier and two activated sludge basins. The stability of microbial community structure was found to be high in the basins and low in the primary clarifier. Despite major abiotic changes certain subcommunities were dominantly present (up to 80% stability), whereas others emerged only sporadically (down to 3% stability, both according to equivalence testing). Additionally, subcommunities of diagnostic value were detected showing positive correlation with substrate influxes. For instance blackwater (r(s) = 0.5) and brewery inflow (both r(s) = 0.6) were mirrored by increases in cell abundances in subclusters 1 and 6 as well as 4 and 8, respectively. Phosphate accumulation was obviously positively correlated with nitrate (r(s) = 0.4) and the presence of denitrifying organisms (Rhodacyclaceae). Various other correlations between community structure and abiotic parameters were apparent. The bacterial composition of certain subcommunities was determined by cell sorting and phylogenetic tools like T-RFLP. In essence, we developed a monitoring tool which is quick, cheap and causal in its interpretation. It will make laborious PCR based technique less obligatory as it allows reliable process monitoring and control in wastewater treatment plants.


Assuntos
Bactérias/crescimento & desenvolvimento , Eliminação de Resíduos Líquidos , Purificação da Água/métodos , Bactérias/genética , DNA Bacteriano/genética , Luz , Dados de Sequência Molecular , Filogenia , Espalhamento de Radiação , Estatísticas não Paramétricas
8.
Nat Commun ; 12(1): 5481, 2021 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-34531395

RESUMO

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.


Assuntos
Bdellovibrio/genética , RNA Ribossômico 16S/genética , Análise de Sequência de DNA/métodos , Esgotos/microbiologia , Bactérias/classificação , Bactérias/genética , Bactérias/crescimento & desenvolvimento , Bdellovibrio/classificação , Bdellovibrio/fisiologia , Ecossistema , Cadeia Alimentar , Variação Genética , Filogenia , Dinâmica Populacional , Análise de Célula Única/métodos
9.
Appl Microbiol Biotechnol ; 88(2): 575-84, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20676634

RESUMO

Flow cytometry approaches are applicable to recover sub-populations of microbial cultures in a purified form. To examine the characteristics of each sorted cell population, Omics technologies can be used for comprehensively monitoring cellular physiology, adaptation reactions, and regulated processes. In this study, we combined flow cytometry and gel-free proteomic analysis to investigate an artificial mixed bacterial culture consisting of Escherichia coli K-12 and Pseudomonas putida KT2440. Therefore, a filter-based device technique and an on-membrane digestion protocol were combined in conjunction with liquid chromatography and mass spectrometry. This combination enabled us to identify 903 proteins from sorted E. coli K-12 and 867 proteins from sorted P. putida KT2440 bacteria from only 5 x 10(6) cells of each. Comparative proteomic analysis of sorted and non-sorted samples was done to prove that sorting did not significantly influence the bacterial proteome profile. We further investigated the physicochemical properties, namely M (r), pI, hydropathicity, and transmembrane helices of the proteins covered. The on-membrane digestion protocol applied did not require conventional detergents or urea, but exhibited similar recovery of all protein classes as established protocols with non-sorted bacterial samples.


Assuntos
Escherichia coli K12/metabolismo , Citometria de Fluxo/métodos , Proteômica/métodos , Pseudomonas putida/metabolismo , Carga Bacteriana , Proteínas de Bactérias/análise , Separação Celular , Cromatografia Líquida , Escherichia coli K12/crescimento & desenvolvimento , Escherichia coli K12/isolamento & purificação , Espectrometria de Massas , Proteoma/análise , Pseudomonas putida/crescimento & desenvolvimento , Pseudomonas putida/isolamento & purificação
10.
Nat Protoc ; 15(9): 2788-2812, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32770154

RESUMO

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.


Assuntos
Bactérias/citologia , Técnicas Citológicas/normas , Microbiota , Biologia Computacional , Padrões de Referência , Reprodutibilidade dos Testes
11.
Microbiome ; 8(1): 13, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32033593

RESUMO

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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. Video abstract.


Assuntos
Bactérias/classificação , Derivação Gástrica , Microbioma Gastrointestinal , Interações entre Hospedeiro e Microrganismos , Redução de Peso , Animais , Bactérias/metabolismo , Ácidos e Sais Biliares/metabolismo , Modelos Animais de Doenças , Fezes/microbiologia , Masculino , RNA Ribossômico 16S/genética , Ratos , Ratos Wistar
12.
Microorganisms ; 6(4)2018 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-30287755

RESUMO

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.

13.
FEBS J ; 274(8): 2088-98, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17388813

RESUMO

Bacteriophytochromes constitute a light-sensing subgroup of sensory kinases with a chromophore-binding motif in the N-terminal half and a C-terminally located histidine kinase activity. The cyanobacterium Fremyella diplosiphon (also designated Calothrix sp.) expresses two sequentially very similar bacteriophytochromes, cyanobacterial phytochrome A (CphA) and cyanobacterial phytochrome B (CphB). Cyanobacterial phytochrome A has the canonical cysteine residue, by which covalent chromophore attachment is accomplished in the same manner as in plant phytochromes; however, its paralog cyanobacterial phytochrome B carries a leucine residue at that position. On the basis of in vitro experiments that showed, for both cyanobacterial phytochrome A and cyanobacterial phytochrome B, light-induced autophosphorylation and phosphate transfer to their cognate response regulator proteins RcpA and RcpB [Hübschmann T, Jorissen HJMM, Börner T, Gärtner W & deMarsac NT (2001) Eur J Biochem268, 3383-3389], we aimed at the identification of a chromophore that is incorporated in vivo into cyanobacterial phytochrome B within the cyanobacterial cell. The approach was based on the introduction of a copy of cphB into the cyanobacterium via triparental conjugation. The His-tagged purified, recombinant protein (CphBcy) showed photoreversible absorption bands similar to those of plant and bacterial phytochromes, but with remarkably red-shifted maxima [lambda(max) 700 and 748 nm, red-absorbing (P(r)) and far red-absorbing (P(fr)) forms of phytochrome, respectively]. A comparison of the absorption maxima with those of the heterologously generated apoprotein, assembled with phycocyanobilin (lambda(max) 686 and 734 nm) or with biliverdin IXalpha (lambda(max) 700 and 750 +/- 2 nm), shows biliverdin IXalpha to be a genuine chromophore. The kinase activity of CphBcy and phosphotransfer to its cognate response regulator was found to be strictly P(r)-dependent. As an N-terminally located cysteine was found as an alternative covalent binding site for several bacteriophytochrome photoreceptors that bind biliverdin and lack the canonical cysteine residue (e.g. Agrobacterium tumefaciens and Deinococcus radiodurans), this corresponding residue in heterologously expressed cyanobacterial phytochrome B was mutated into a serine (C24S); however, there was no change in its spectral properties. On the other hand, the mutation of His267, which is located directly after the canonical cysteine, into alanine (H267A), caused complete loss of the capability of cyanobacterial phytochrome B to form a chromoprotein.


Assuntos
Biliverdina/metabolismo , Cianobactérias/metabolismo , Fitocromo B/metabolismo , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas , Dados de Sequência Molecular , Mutação , Fosforilação , Fitocromo B/análise , Fitocromo B/química , Fitocromo B/genética
14.
Sci Rep ; 6: 36390, 2016 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-27811990

RESUMO

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.


Assuntos
Bactérias/genética , Transferência Genética Horizontal , Micélio/fisiologia , Fenômenos Fisiológicos Bacterianos , Variação Genética , Microbiologia do Solo
15.
FEBS Lett ; 579(7): 1613-8, 2005 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-15757650

RESUMO

Cyanobacteria possess genes encoding phytochrome-related proteins. We used a DNA microarray approach to evaluate the impact of the phytochromes Cph1 and Cph2 on red light (R)- and far-red light (FR)-dependent gene expression in the unicellular cyanobacterium Synechocystis sp. PCC 6803. In cells of wild-type and phytochrome mutants, one-fourth of all 3165 annotated putative protein encoding genes was light-responsive. R predominantly enhanced the expression of genes involved in transcription, translation, and photosynthesis, whereas FR upregulated the transcript level of genes known to be inducible by stress. The absence of Cph1 and/or Cph2 altered the light-dependent expression of about 20 genes. Hence, receptor(s) different from the two phytochromes are supposed to trigger the global R/FR alterations of the expression profile.


Assuntos
Proteínas de Bactérias/fisiologia , Regulação Bacteriana da Expressão Gênica/genética , Luz , Fitocromo/fisiologia , Proteínas Quinases/fisiologia , Synechocystis/genética , Transcrição Gênica/efeitos da radiação , Proteínas de Bactérias/genética , Regulação para Baixo , Perfilação da Expressão Gênica , Mutação/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fotorreceptores Microbianos , Fitocromo/genética , Proteínas Quinases/genética , Synechocystis/metabolismo
16.
Front Microbiol ; 6: 243, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25932020

RESUMO

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 (15)N2 and (13)CO2 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 (15)N and (13)C 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.

17.
Microb Biotechnol ; 8(3): 379-91, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25185955

RESUMO

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.


Assuntos
Técnicas Bacteriológicas/métodos , Citometria de Fluxo/métodos , Shewanella putrefaciens/crescimento & desenvolvimento , Aerobiose , Anaerobiose , Biofilmes/crescimento & desenvolvimento , Biologia Computacional , Microscopia , Shewanella putrefaciens/fisiologia
18.
Microb Biotechnol ; 6(2): 157-67, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23279937

RESUMO

Phenotypic variation of microbial populations is a well-known phenomenon and may have significant impact on the success of industrial bioprocesses. Flow cytometry (FC) and the large repertoire of fluorescent dyes bring the high-throughput analysis of multiple parameters in single bacterial cells into reach. In this study, we evaluated a set of different fluorescent dyes for suitability in FC single cell analysis of the biotechnological platform organism Corynebacterium glutamicum. Already simple scattering properties of C. glutamicum cells in the flow cytometer were shown to provide valuable information on the growth activity of analysed cells. Furthermore, we used DAPI staining for a FC-based determination of the DNA content of C. glutamicum cells grown on standard minimal or complex media. Characteristic DNA patterns were observed mirroring the typical uncoupled DNA synthesis in the logarithmic (log) growth phase and are in agreement with a symmetric type of cell division of C. glutamicum. Application of the fluorescent dyes Syto 9, propidium iodide, and DiOC(2)(3) allowed the identification of subpopulations with reduced viability and membrane potential within early log and stationary phase populations. The presented data highlight the potential of FC-based analyses for online monitoring of C. glutamicum bioprocesses and provide a first reference for future applications and protocols.


Assuntos
Corynebacterium glutamicum/citologia , Corynebacterium glutamicum/crescimento & desenvolvimento , Citometria de Fluxo/métodos , Biotecnologia/métodos , Divisão Celular , Corynebacterium glutamicum/genética , DNA Bacteriano/análise , Corantes Fluorescentes/metabolismo , Indóis/metabolismo , Potenciais da Membrana , Dinâmica Populacional , Coloração e Rotulagem/métodos
19.
Nat Protoc ; 8(1): 190-202, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23288319

RESUMO

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.


Assuntos
Microambiente Celular , Citometria de Fluxo/métodos , Interações Microbianas , Águas Residuárias/microbiologia , Bactérias/citologia , Bactérias/genética , Biodiversidade , Etiópia , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição
20.
J Microbiol Methods ; 95(3): 463-9, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23680233

RESUMO

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.


Assuntos
Fungos/isolamento & purificação , Parasitologia/métodos , Solo/parasitologia , Estramenópilas/isolamento & purificação , Sobrevivência Celular , Centrifugação , Clorófitas/classificação , Clorófitas/genética , Citometria de Fluxo , Fungos/classificação , Fungos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Dados de Sequência Molecular , Estramenópilas/classificação , Estramenópilas/genética
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