Isolation and characterization of low nucleic acid (LNA)-content bacteria

ISME Journal - Tập 3 Số 8 - Trang 889-902 - 2009
Li Wang1,2, Frederik Hammes1, Nico Boon3, Mohamed Chami4, Thomas Egli1,2
1Department of Environmental Microbiology, Eawag, Swiss Federal Institute of Aquatic Science and Technology, Dübendorf, Switzerland
2Institute of Biogeochemistry and Pollutant Dynamics, ETH Zürich, Zürich, Switzerland
3Ghent University, Faculty of Bioscience Engineering, Laboratory of Microbial Ecology and Technology (LabMET), Gent, Belgium
4ME Müller Institute for Structural Biology, Biozentrum, University of Basel , Basel , Switzerland

Tóm tắt

Abstract Most planktonic bacteria are ‘uncultivable’ with conventional methods. Flow cytometry (FCM) is one approach that has been taken to study these bacteria. In natural aquatic environments, bacteria with high nucleic acid (HNA) and low nucleic acid (LNA) content are commonly observed with FCM after staining with fluorescent dyes. Although several studies have focused on the relative abundance and in situ activities of these two groups, knowledge on the growth of particularly LNA bacteria is largely limited. In this study, typical LNA bacteria were enriched from three different freshwater sources using extinction dilution (ED) and fluorescence-activated cell sorting (FACS). We have shown for the first time that LNA bacteria can be isolated and cultivated by using sterile freshwater as a growth medium. During growth, the typical LNA characteristics (that is, low-fluorescence intensity and sideward scatter (SSC)) remained distinct from those of typical HNA bacteria. Three LNA pure cultures that are closely affiliated to the Polynucleobacter cluster according to 16S rRNA sequencing results were isolated. Owing to their small size, cells of the isolates remained intact during cryo-transmission electronic microscopy examination and showed a Gram-negative cell-wall structure. The extremely small cell volume (0.05 μm3) observed for all three isolates indicates that they are among the smallest free-living heterotrophic organisms known in culture. Their isolation and cultivation allow further detailed investigation of this group of organisms under defined laboratory conditions.

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Tài liệu tham khảo

Al-Amoudi, 2004, Cryo-electron microscopy of vitreous sections of native biological cells and tissues, J Struct Biol, 148, 131, 10.1016/j.jsb.2004.03.010

Bernard, 2000, Relationships among bacterial cell size, productivity, and genetic diversity in aquatic environments using cell sorting and flow cytometry, Microb Ecol, 40, 148, 10.1007/s002480000046

Berney, 2008, Rapid, cultivation-independent assessment of microbial viability in drinking water, Water Res, 42, 4010, 10.1016/j.watres.2008.07.017

Billen, 1990, Role of bacteria in the North Sea ecosystem, Neth J Sea Res, 26, 265, 10.1016/0077-7579(90)90093-V

Boenigk, 2004, Strain-specific differences in the grazing sensitivities of closely related ultramicrobacteria affiliated with the Polynucleobacter cluster, Appl Environ Microbiol, 70, 5787, 10.1128/AEM.70.10.5787-5793.2004

Boon, 2002, Evaluation of nested PCR-DGGE (denaturing gradient gel electrophoresis) with group-specific 16S rRNA primers for the analysis of bacterial communities from different wastewater treatment plants, FEMS Microbiol Ecol, 39, 101

Bouvier, 2007, A comparative study of the cytometric characteristics of high and low nucleic-acid bacterioplankton cells from different aquatic ecosystems, Environ Microbiol, 9, 2050, 10.1111/j.1462-2920.2007.01321.x

Bruns, 2003, Effect of signal compounds and incubation conditions on the culturability of freshwater bacterioplankton, Appl Environ Microbiol, 69, 1980, 10.1128/AEM.69.4.1980-1989.2003

Dubochet, 1988, Cryo-electron microscopy of vitrified specimens, Q Rev Biophys, 21, 129, 10.1017/S0033583500004297

Eydal, 2007, Use of an ATP assay to determine viable microbial biomass in Fennoscandian Shield groundwater from depths of 3–1000 m, J Microbiol Meth, 70, 363, 10.1016/j.mimet.2007.05.012

Felip, 2007, Suitability of flow cytometry for estimating bacterial biovolume in natural plankton samples: comparison with microscopy data, Appl Environ Microbiol, 73, 4508, 10.1128/AEM.00733-07

Gasol, 1999, Effects of filtration on bacterial activity and picoplankton community structure as assessed by flow cytometry, Aquat Microb Ecol, 16, 251, 10.3354/ame016251

Gasol, 1999, Significance of size and nucleic acid content heterogeneity as measured by flow cytometry in natural planktonic bacteria, Appl Environ Microbiol, 65, 4475, 10.1128/AEM.65.10.4475-4483.1999

Glockner, 1999, Bacterioplankton compositions of lakes and oceans: a first comparison based on fluorescence in situ hybridization, Appl Environ Microbiol, 65, 3721, 10.1128/AEM.65.8.3721-3726.1999

Graham, 1991, Periplasmic space and the concept of the periplasm, Trends Biochem Sci, 16, 328, 10.1016/0968-0004(91)90135-I

Grossart, 2008, Top-down and bottom-up induced shifts in bacterial abundance, production and community composition in an experimentally divided humic lake, Environ Microbiol, 10, 635, 10.1111/j.1462-2920.2007.01487.x

Hahn, 2003, Isolation of strains belonging to the cosmopolitan Polynucleobacter necessarius cluster from freshwater habitats located in three climatic zones, Appl Environ Microbiol, 69, 5248, 10.1128/AEM.69.9.5248-5254.2003

Hahn, 2003, Isolation of novel ultramicrobacteria classified as actinobacteria from five freshwater habitats in Europe and Asia, Appl Environ Microbiol, 69, 1442, 10.1128/AEM.69.3.1442-1451.2003

Hahn, 2005, Low intraspecific diversity in a Polynucleobacter subcluster population numerically dominating bacterioplankton of a freshwater pond, Appl Environ Microbiol, 71, 4539, 10.1128/AEM.71.8.4539-4547.2005

Hammes, 2008, Flow-cytometric total bacterial cell counts as a descriptive microbiological parameter for drinking water treatment processes, Water Res, 42, 269, 10.1016/j.watres.2007.07.009

Hammes, 2005, New method for assimilable organic carbon determination using flow-cytometric enumeration and a natural microbial consortium as inoculum, Environ Sci Technol, 39, 3289, 10.1021/es048277c

Heckmann, 1987, Polynucleobacter necessarius gen. nov., sp. nov., an obligately endosymbiotic bacterium living in the cytoplasm of Euplotes aediculatus, Int J Syst Bacteriol, 37, 456, 10.1099/00207713-37-4-456

Jellett, 1996, Metabolic activity of bacterioplankton communities assessed by flow cytometry and single carbon substrate utilization, Mar Ecol Prog Ser, 136, 213, 10.3354/meps136213

Jochem, 2004, Growth and grazing rates of bacterial groups with different apparent DNA content in the Gulf of Mexico, Mar Biol, 145, 1213, 10.1007/s00227-004-1406-7

Kajander, 1998, Nanobacteria: an alternative mechanism for pathogenic intra- and extracellular calcification and stone formation, PNAS, 95, 8274, 10.1073/pnas.95.14.8274

Koch, 1996, What size should a bacterium be? A question of scale, Annu Rev Microbiol, 50, 317, 10.1146/annurev.micro.50.1.317

Lebaron, 2001, Does the high nucleic acid content of individual bacterial cells allow us to discriminate between active cells and inactive cells in aquatic systems?, Appl Environ Microbiol, 67, 1775, 10.1128/AEM.67.4.1775-1782.2001

Lebaron, 2002, Variations of bacterial-specific activity with cell size and nucleic acid content assessed by flow cytometry, Aquat Microb Ecol, 28, 131, 10.3354/ame028131

Li, 1995, DNA distributions in planktonic bacteria stained with TOTO or TO-PRO, Limnol Oceanogr, 40, 1485, 10.4319/lo.1995.40.8.1485

Longnecker, 2005, Activity and phylogenetic diversity of bacterial cells with high and low nucleic acid content and electron transport system activity in an upwelling ecosystem, Appl Environ Microbiol, 71, 7737, 10.1128/AEM.71.12.7737-7749.2005

Marie, 1997, Enumeration and cell cycle analysis of natural populations of marine picoplankton by flow cytometry using the stain SYBR Green I, Appl Environ Microbiol, 63, 186, 10.1128/aem.63.1.186-193.1997

Matias, 2003, Cryo-transmission electron microscopy of frozen-hydrated sections of Escherichia coli and Pseudomonas aeruginosa, J Bacteriol, 185, 6112, 10.1128/JB.185.20.6112-6118.2003

Morita, 1997, Bacteria in oligotrophic environments: starvation-survival life style, 193

Münster, 1993, Concentrations and fluxes of organic carbon substrates in the aquatic environment, Antonie Van Leeuwenhoek, 63, 243, 10.1007/BF00871222

Nishimura, 2005, Vertical and seasonal variations of bacterioplankton subgroups with different nucleic acid contents: possible regulation by phosphorus, Appl Environ Microbiol, 71, 5828, 10.1128/AEM.71.10.5828-5836.2005

Oliver, 1993, Starvation in bacteria, 239, 10.1007/978-1-4899-2439-1_11

Page, 2004, Representative freshwater bacterioplankton isolated from Crater Lake, Oregon, Appl Environ Microbiol, 70, 6542, 10.1128/AEM.70.11.6542-6550.2004

Pernthaler, 2007, Methods in Molecular Biology, 153

Pernthaler, 2002, Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria, Appl Environ Microbiol, 68, 3094, 10.1128/AEM.68.6.3094-3101.2002

Rappé, 2002, Cultivation of the ubiquitous SAR11 marine bacterioplankton clade, Nature, 418, 630, 10.1038/nature00917

Rappé, 2003, The uncultured microbial majority, Annu Rev Microbiol, 57, 369, 10.1146/annurev.micro.57.030502.090759

Rosenberg, 2002, Microbial diseases of corals and global warming, Environ Microbiol, 4, 318, 10.1046/j.1462-2920.2002.00302.x

Sekar, 2003, An improved protocol for quantification of freshwater Actinobacteria by fluorescence in situ hybridization, Appl Environ Microbiol, 69, 2928, 10.1128/AEM.69.5.2928-2935.2003

Schut, 1997, Oligotrophy and pelagic marine bacteria: facts and fiction, Aquat Microb Ecol, 12, 177, 10.3354/ame012177

Schut, 1993, Isolation of typical marine bacteria by dilution culture: growth, maintenance, and characteristics of isolates under laboratory conditions, Appl Environ Microbiol, 59, 2150, 10.1128/aem.59.7.2150-2160.1993

Servais, 2003, Activity and diversity of bacterial cells with high and low nucleic acid content, Aquat Microb Ecol, 33, 41, 10.3354/ame033041

Tadonleke, 2005, Microbial food webs in boreal humic lakes and reservoirs: ciliates as a major factor related to the dynamics of the most active bacteria, Microb Ecol, 49, 325, 10.1007/s00248-004-0232-2

Versalovic, 1994, Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction, Methods Mol Cell Biol, 5, 25

Vital, 2007, Growth of Vibrio cholerae O1 Ogawa Eltor in freshwater, Microbiology, 153, 1993, 10.1099/mic.0.2006/005173-0

Vital, 2008, Escherichia coli O157 can grow in natural freshwater at low carbon concentrations, Environ Microbiol, 10, 2387, 10.1111/j.1462-2920.2008.01664.x

Wang, 2007, Quantification of the filterability of freshwater bacteria through 0.45, 0.22, and 0.1 μm pore size filters and shape-dependent enrichment of filterable bacterial communities, Environ Sci Technol, 41, 7080, 10.1021/es0707198

Wang, 2008, Influence of size, shape, and flexibility on bacterial passage through micropore membrane filters, Environ Sci Technol, 42, 6749, 10.1021/es800720n

Ward, 1963, Hierarchical grouping to optimize an objective function, J Am Stat Assoc, 58, 236, 10.1080/01621459.1963.10500845

Warnecke, 2005, Abundances, identity, and growth state of Actinobacteria in mountain lakes of different UV transparency, Appl Environ Microbiol, 71, 5551, 10.1128/AEM.71.9.5551-5559.2005

White, 1991, The effect of temperature and algal biomass on bacterial production and specific growth rate in freshwater and marine habitats, Microb Ecol, 21, 99, 10.1007/BF02539147

Whitman, 1998, Prokaryotes: The unseen majority, PNAS, 95, 6578, 10.1073/pnas.95.12.6578

Wick, 2002, The apparent clock-like evolution of Escherichia coli in glucose-limited chemostats is reproducible at large but not at small population sizes and can be explained with Monod kinetics, Microbiol, 148, 2889, 10.1099/00221287-148-9-2889

Zubkov, 2001, Comparison of cellular and biomass specific activities of dominant bacterioplankton groups in stratified waters of the Celtic Sea, Appl Environ Microbiol, 67, 5210, 10.1128/AEM.67.11.5210-5218.2001

Zubkov, 2002, Mesoscale distribution of dominant bacterioplankton groups in the northern North Sea in early summer, Aquat Microb Ecol, 29, 135, 10.3354/ame029135

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ISME Journal

Tập 3 Số 8

889-902

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