scholarly journals Multiple DNA Extractions Coupled with Stable-Isotope Probing of Anthracene-Degrading Bacteria in Contaminated Soil

2011 ◽  
Vol 77 (9) ◽  
pp. 2984-2991 ◽  
Author(s):  
Maiysha D. Jones ◽  
David R. Singleton ◽  
Wei Sun ◽  
Michael D. Aitken
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Copy Number ◽  
Stable Isotope Probing ◽  
Gene Copy ◽  
Rrna Gene ◽  
Content Type ◽  
Dna Yield ◽  
Degrading Bacteria ◽  
Bacterial Groups

ABSTRACTIn many of the DNA-based stable-isotope probing (SIP) studies published to date in which soil communities were investigated, a single DNA extraction was performed on the soil sample, usually using a commercial DNA extraction kit, prior to recovering the13C-labeled (heavy) DNA by density-gradient ultracentrifugation. Recent evidence suggests, however, that a single extraction of a soil sample may not lead to representative recovery of DNA from all of the organisms in the sample. To determine whether multiple DNA extractions would affect the DNA yield, the eubacterial 16S rRNA gene copy number, or the identification of anthracene-degrading bacteria, we performed seven successive DNA extractions on the same aliquot of contaminated soil either untreated or enriched with [U-13C]anthracene. Multiple extractions were necessary to maximize the DNA yield and 16S rRNA gene copy number from both untreated and anthracene-enriched soil samples. Sequences within the orderSphingomonadales, but unrelated to any previously described genus, dominated the 16S rRNA gene clone libraries derived from13C-enriched DNA and were designated “anthracene group 1.” Sequences clustering withVariovoraxspp., which were also highly represented, and sequences related to the genusPigmentiphagawere newly associated with anthracene degradation. The bacterial groups collectively identified across all seven extracts were all recovered in the first extract, although quantitative PCR analysis of SIP-identified groups revealed quantitative differences in extraction patterns. These results suggest that performing multiple DNA extractions on soil samples improves the extractable DNA yield and the number of quantifiable eubacterial 16S rRNA gene copies but have little qualitative effect on the identification of the bacterial groups associated with the degradation of a given carbon source by SIP.

scholarly journals Development of a Real-Time PCR Assay for Detection and Quantification of Rhizobium leguminosarum Bacteria and Discrimination between Different Biovars in Zinc-Contaminated Soil

2011 ◽  
Vol 77 (13) ◽  
pp. 4626-4633 ◽  
Author(s):  
Catriona A. Macdonald ◽  
Ian M. Clark ◽  
Penny R. Hirsch ◽  
Fang-Jie Zhao ◽  
Steve P. McGrath
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Rhizobium Leguminosarum ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Most Probable Number ◽  
Rrna Gene ◽  
Probable Number ◽  
Content Type ◽  
Gene Copies

ABSTRACTPrimers were designed to target 16S rRNA andnodDgenes ofRhizobium leguminosarumfrom DNA extracted from two different soil types contaminated with Zn applied in sewage sludge. Numbers of rhizobia estimated using 16S rRNA gene copy number showed higher abundance than those estimated by bothnodDand the most-probable-number (MPN) enumeration method using a plant trap host. Both 16S rRNA gene copies and the MPN rhizobia declined with increased levels of Zn contamination, as did the abundance of the functional genenodD, providing compelling evidence of a toxic effect of Zn onR. leguminosarumpopulations in the soil. Regression analysis suggested the total Zn concentration in soil as a better predictor of rhizobial numbers than both NH4NO3-extractable and soil solution Zn.R. leguminosarumbv. viciaenodDgene copies were generally less sensitive to Zn thanR. leguminosarumbv. trifoliinodD.The latter were generally below detection limits at Zn levels of >250 mg kg−1. Although there were differences in the actual numbers estimated by each approach, the response to Zn was broadly similar across all methods. These differences were likely to result from the fact that the molecular approaches assess the potential for nodulation while the MPN approach assesses actual nodulation. The results demonstrate that the use of targeted gene probes for assessing environmental perturbations of indigenous soil rhizobial populations may be more sensitive than the conventional plant bioassay and MPN methods.

scholarly journals Polycyclovorans algicola gen. nov., sp. nov., an Aromatic-Hydrocarbon-Degrading Marine Bacterium Found Associated with Laboratory Cultures of Marine Phytoplankton

2012 ◽  
Vol 79 (1) ◽  
pp. 205-214 ◽  
Author(s):  
Tony Gutierrez ◽  
David H. Green ◽  
Peter D. Nichols ◽  
William B. Whitman ◽  
Kirk T. Semple ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Aromatic Hydrocarbon ◽  
Skeletonema Costatum ◽  
Marine Phytoplankton ◽  
Marine Diatom ◽  
Rrna Gene ◽  
Strictly Aerobic ◽  
Content Type ◽  
Degrading Bacteria

ABSTRACTA strictly aerobic, halotolerant, rod-shaped bacterium, designated strain TG408, was isolated from a laboratory culture of the marine diatomSkeletonema costatum(CCAP1077/1C) by enrichment with polycyclic aromatic hydrocarbons (PAHs) as the sole carbon source. 16S rRNA gene sequence analysis placed this organism within the orderXanthomonadalesof the classGammaproteobacteria. Its closest relatives included representatives of theHydrocarboniphaga-Nevskia-Sinobacterclade (<92% sequence similarity) in the familySinobacteraceae. The strain exhibited a narrow nutritional spectrum, preferring to utilize aliphatic and aromatic hydrocarbon compounds and small organic acids. Notably, it displayed versatility in degrading two- and three-ring PAHs. Moreover, catechol 2,3-dioxygenase activity was detected in lysates, indicating that this strain utilizes themeta-cleavage pathway for aromatic compound degradation. Cells produced surface blebs and contained a single polar flagellum. The predominant isoprenoid quinone of strain TG408 was Q-8, and the dominant fatty acids were C16:0, C16:1ω7c, and C18:1ω7c. The G+C content of the isolate's DNA was 64.3 mol% ± 0.34 mol%. On the basis of distinct phenotypic and genotypic characteristics, strain TG408 represents a novel genus and species in the classGammaproteobacteriafor which the namePolycyclovorans algicolagen. nov., sp. nov., is proposed. Quantitative PCR primers targeting the 16S rRNA gene of this strain were developed and used to show that this organism is found associated with other species of marine phytoplankton. Phytoplankton may be a natural biotope in the ocean where new species of hydrocarbon-degrading bacteria await discovery and which contribute significantly to natural remediation processes.

scholarly journals Stable-Isotope Probing Identifies Uncultured Planctomycetes as Primary Degraders of a Complex Heteropolysaccharide in Soil

2015 ◽  
Vol 81 (14) ◽  
pp. 4607-4615 ◽  
Author(s):  
Xiaoqing Wang ◽  
Christine E. Sharp ◽  
Gareth M. Jones ◽  
Stephen E. Grasby ◽  
Allyson L. Brady ◽  
...  
Keyword(s):  
Stable Isotope ◽  
16S Rrna ◽  
Soil Bacteria ◽  
Stable Isotope Probing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Content Type ◽  
Degrading Bacteria ◽  
Aerobic Soil

ABSTRACTThe exopolysaccharides (EPSs) produced by some bacteria are potential growth substrates for other bacteria in soil. We used stable-isotope probing (SIP) to identify aerobic soil bacteria that assimilated the cellulose produced byGluconacetobacter xylinusor the EPS produced byBeijerinckia indica. The latter is a heteropolysaccharide comprised primarily ofl-guluronic acid,d-glucose, andd-glycero-d-mannoheptose.13C-labeled EPS and13C-labeled cellulose were purified from bacterial cultures grown on [13C]glucose. Two soils were incubated with these substrates, and bacteria actively assimilating them were identified via pyrosequencing of 16S rRNA genes recovered from13C-labeled DNA. Cellulose C was assimilated primarily by soil bacteria closely related (93 to 100% 16S rRNA gene sequence identities) to known cellulose-degrading bacteria. However,B. indicaEPS was assimilated primarily by bacteria with low identities (80 to 95%) to known species, particularly by different members of the phylumPlanctomycetes. In one incubation, members of thePlanctomycetesmade up >60% of all reads in the labeled DNA and were only distantly related (<85% identity) to any described species. Although it is impossible with SIP to completely distinguish primary polysaccharide hydrolyzers from bacteria growing on produced oligo- or monosaccharides, the predominance ofPlanctomycetessuggested that they were primary degraders of EPS. Other bacteria assimilatingB. indicaEPS included members of theVerrucomicrobia, candidate division OD1, and theArmatimonadetes. The results indicate that some uncultured bacteria in soils may be adapted to using complex heteropolysaccharides for growth and suggest that the use of these substrates may provide a means for culturing new species.

scholarly journals Abundance, Distribution, and Activity of Fe(II)-Oxidizing and Fe(III)-Reducing Microorganisms in Hypersaline Sediments of Lake Kasin, Southern Russia

2012 ◽  
Vol 78 (12) ◽  
pp. 4386-4399 ◽  
Author(s):  
Maren Emmerich ◽  
Ankita Bhansali ◽  
Tina Lösekann-Behrens ◽  
Christian Schröder ◽  
Andreas Kappler ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Copy ◽  
Rrna Gene ◽  
Sediment Layer ◽  
Hypersaline Environments ◽  
Probable Number ◽  
Content Type ◽  
Southern Russia ◽  
Hypersaline Sediments

ABSTRACTThe extreme osmotic conditions prevailing in hypersaline environments result in decreasing metabolic diversity with increasing salinity. Various microbial metabolisms have been shown to occur even at high salinity, including photosynthesis as well as sulfate and nitrate reduction. However, information about anaerobic microbial iron metabolism in hypersaline environments is scarce. We studied the phylogenetic diversity, distribution, and metabolic activity of iron(II)-oxidizing and iron(III)-reducingBacteriaandArchaeain pH-neutral, iron-rich salt lake sediments (Lake Kasin, southern Russia; salinity, 348.6 g liter−1) using a combination of culture-dependent and -independent techniques. 16S rRNA gene clone libraries forBacteriaandArchaearevealed a microbial community composition typical for hypersaline sediments. Most-probable-number counts confirmed the presence of 4.26 × 102to 8.32 × 103iron(II)-oxidizingBacteriaand 4.16 × 102to 2.13 × 103iron(III)-reducing microorganisms per gram dry sediment. Microbial iron(III) reduction was detected in the presence of 5 M NaCl, extending the natural habitat boundaries for this important microbial process. Quantitative real-time PCR showed that 16S rRNA gene copy numbers of totalBacteria, totalArchaea, and species dominating the iron(III)-reducing enrichment cultures (relatives ofHalobaculum gomorrense,Desulfosporosinus lacus, and members of theBacilli) were highest in an iron oxide-rich sediment layer. Combined with the presented geochemical and mineralogical data, our findings suggest the presence of an active microbial iron cycle at salt concentrations close to the solubility limit of NaCl.

scholarly journals Stable Isotope Probing of an Algal Bloom To Identify Uncultivated Members of the Rhodobacteraceae Associated with Low-Molecular-Weight Polycyclic Aromatic Hydrocarbon Degradation

2011 ◽  
Vol 77 (21) ◽  
pp. 7856-7860 ◽  
Author(s):  
Tony Gutierrez ◽  
David R. Singleton ◽  
Michael D. Aitken ◽  
Kirk T. Semple
Keyword(s):  
Stable Isotope ◽  
Polycyclic Aromatic Hydrocarbon ◽  
Aromatic Hydrocarbon ◽  
Algal Bloom ◽  
Stable Isotope Probing ◽  
Gene Copy ◽  
Rrna Gene ◽  
Content Type ◽  
Degrading Bacteria ◽  
Polycyclic Aromatic

ABSTRACTPolycyclic aromatic hydrocarbon (PAH)-degrading bacteria associated with an algal bloom in Tampa Bay, FL, were investigated by stable isotope probing (SIP) with uniformly labeled [13C]naphthalene. The dominant sequences in clone libraries constructed from13C-enriched bacterial DNA (from naphthalene enrichments) were identified as uncharacterized members of the familyRhodobacteraceae. Quantitative PCR primers targeting the 16S rRNA gene of these uncultivated organisms were used to determine their abundance in incubations amended with unlabeled naphthalene and phenanthrene, both of which showed substantial increases in gene copy numbers during the experiments. As demonstrated by this work, the application of uniformly13C-labeled PAHs in SIP experiments can successfully be used to identify novel PAH-degrading bacteria in marine waters.

scholarly journals Quantification of the Detrimental Effect of a Single Primer-Template Mismatch by Real-Time PCR Using the 16S rRNA Gene as an Example

2008 ◽  
Vol 74 (5) ◽  
pp. 1660-1663 ◽  
Author(s):  
D. Bru ◽  
F. Martin-Laurent ◽  
L. Philippot
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Copy Number ◽  
Pcr Amplification ◽  
Gene Copy ◽  
Rrna Gene ◽  
Template Dna ◽  
Single Primer ◽  
The 16S Rrna Gene ◽  
Extension Sequence

ABSTRACT We investigated the effects of internal primer-template mismatches on the efficiency of PCR amplification using the 16S rRNA gene as the model template DNA. We observed that the presence of a single mismatch in the second half of the primer extension sequence can result in an underestimation of up to 1,000-fold of the gene copy number, depending on the primer and position of the mismatch.

scholarly journals Identification of a Toluene-Degrading Bacterium from a Soil Sample through H218O DNA Stable Isotope Probing

2011 ◽  
Vol 77 (17) ◽  
pp. 5995-5999 ◽  
Author(s):  
Angela Woods ◽  
Maribeth Watwood ◽  
Egbert Schwartz
Keyword(s):  
Stable Isotope ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Stable Isotope Probing ◽  
Rrna Gene ◽  
Soil Dna ◽  
Content Type ◽  
Degrading Bacterium ◽  
Rhodococcus Jostii ◽  
Oxygen Atoms

ABSTRACTDNA stable isotope probing (DNA-SIP) with H218O was used to identify a toluene-degrading bacterium in soil amended with 48 ppm toluene. After quantification of toluene degradation rates in soil, DNA was extracted from soil incubated with H218O, H216O, H216O and 48 ppm toluene, or H218O and 48 ppm toluene. A single DNA band formed along a cesium chloride gradient after isopycnic centrifugation of extracts from soils incubated with H216O. With extracts from soils to which only H218O was added, two distinct DNA bands formed, while three bands formed when DNA extracted from soil incubated with both H218O and toluene was analyzed. We suggest that this third band formed because toluene does not contain any oxygen atoms and toluene-degrading organisms had to transfer oxygen atoms from H218O into metabolic intermediates to form nucleic acidsde novo. We extracted the third DNA band and amplified a large fraction of the bacterial 16S rRNA gene. Direct sequencing of the PCR product obtained from the labeled DNA, as well as cloned 16S rRNA amplicons, identified a known toluene degrader,Rhodococcus jostiiRHA1. A toluene-degrading bacterial strain was subsequently isolated from soil and shown to beRhodococcus jostiiRHA1. Finally, quantitative real-time PCR analysis showed that the abundance of the 16S rRNA gene ofRhodococcus jostiiRHA1 increased in soil after toluene exposure but not in soils from which toluene was withheld. This study indicates that H218O DNA-SIP can be a useful method for identifying pollutant-degrading bacteria in soil.

scholarly journals Phyllobacterium loti sp. nov. isolated from nodules of Lotus corniculatus

2014 ◽  
Vol 64 (Pt_3) ◽  
pp. 781-786 ◽  
Author(s):  
Maximo Sánchez ◽  
Martha-Helena Ramírez-Bahena ◽  
Alvaro Peix ◽  
María J. Lorite ◽  
Juan Sanjuán ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Sequence Similarity ◽  
Carbon Sources ◽  
Lotus Corniculatus ◽  
Culture Conditions ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type ◽  
The 16S Rrna Gene

Strain S658T was isolated from a Lotus corniculatus nodule in a soil sample obtained in Uruguay. Phylogenetic analysis of the 16S rRNA gene and atpD gene showed that this strain clustered within the genus Phyllobacterium . The closest related species was, in both cases, Phyllobacterium trifolii PETP02T with 99.8 % sequence similarity in the 16S rRNA gene and 96.1 % in the atpD gene. The 16S rRNA gene contains an insert at the beginning of the sequence that has no similarities with other inserts present in the same gene in described rhizobial species. Ubiquinone Q-10 was the only quinone detected. Strain S658T differed from its closest relatives through its growth in diverse culture conditions and in the assimilation of several carbon sources. It was not able to reproduce nodules in Lotus corniculatus. The results of DNA–DNA hybridization, phenotypic tests and fatty acid analyses confirmed that this strain should be classified as a representative of a novel species of the genus Phyllobacterium , for which the name Phyllobacterium loti sp. nov. is proposed. The type strain is S658T( = LMG 27289T = CECT 8230T).

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