Recovering glycoside hydrolase genes from active tundra cellulolytic bacteria

2014 ◽  
Vol 60 (7) ◽  
pp. 469-476 ◽  
Author(s):  
Lee J. Pinnell ◽  
Eric Dunford ◽  
Patrick Ronan ◽  
Martina Hausner ◽  
Josh D. Neufeld
Keyword(s):  
Glycoside Hydrolase ◽  
Multiple Displacement Amplification ◽  
Arctic Tundra ◽  
Stable Isotope Probing ◽  
Glycoside Hydrolases ◽  
16S Rrna Genes ◽  
Bulk Soil ◽  
Metagenomic Analysis ◽  
Rrna Genes ◽  
Cellulolytic Bacteria

Bacteria responsible for cellulose hydrolysis in situ are poorly understood, largely because of the relatively recent development of cultivation-independent methods for their detection and characterization. This study combined DNA stable-isotope probing (DNA-SIP) and metagenomics for identifying active bacterial communities that assimilated carbon from glucose and cellulose in Arctic tundra microcosms. Following DNA-SIP, bacterial fingerprint analysis of gradient fractions confirmed isotopic enrichment. Sequenced fingerprint bands and clone library analysis of 16S rRNA genes identified active bacterial taxa associated with cellulose-associated labelled DNA, including Bacteroidetes (Sphingobacteriales), Betaproteobacteria (Burkholderiales), Alphaproteobacteria (Caulobacteraceae), and Chloroflexi (Anaerolineaceae). We also compared glycoside hydrolase metagenomic profiles from bulk soil and heavy DNA recovered from DNA-SIP incubations. Active populations consuming [13C]glucose and [13C]cellulose were distinct, based on ordinations of light and heavy DNA. Metagenomic analysis demonstrated a ∼3-fold increase in the relative abundance of glycoside hydrolases in DNA-SIP libraries over bulk-soil libraries. The data also indicate that multiple displacement amplification introduced bias into the resulting metagenomic analysis. This research identified DNA-SIP incubation conditions for glucose and cellulose that were suitable for Arctic tundra soil and confirmed that DNA-SIP enrichment can increase target gene frequencies in metagenomic libraries.

scholarly journals Multisubstrate Isotope Labeling and Metagenomic Analysis of Active Soil Bacterial Communities

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Y. Verastegui ◽  
J. Cheng ◽  
K. Engel ◽  
D. Kolczynski ◽  
S. Mortimer ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Indicator Species ◽  
Bacterial Communities ◽  
Glycoside Hydrolase ◽  
Stable Isotope Probing ◽  
Glycoside Hydrolases ◽  
Metagenomic Analysis ◽  
Functional Metagenomics ◽  
Soil Bacterial Communities ◽  
Soil Bacterial

ABSTRACTSoil microbial diversity represents the largest global reservoir of novel microorganisms and enzymes. In this study, we coupled functional metagenomics and DNA stable-isotope probing (DNA-SIP) using multiple plant-derived carbon substrates and diverse soils to characterize active soil bacterial communities and their glycoside hydrolase genes, which have value for industrial applications. We incubated samples from three disparate Canadian soils (tundra, temperate rainforest, and agricultural) with five native carbon (12C) or stable-isotope-labeled (13C) carbohydrates (glucose, cellobiose, xylose, arabinose, and cellulose). Indicator species analysis revealed high specificity and fidelity for many uncultured and unclassified bacterial taxa in the heavy DNA for all soils and substrates. Among characterized taxa,Actinomycetales(Salinibacterium),Rhizobiales(Devosia),Rhodospirillales(Telmatospirillum), andCaulobacterales(PhenylobacteriumandAsticcacaulis) were bacterial indicator species for the heavy substrates and soils tested. BothActinomycetalesandCaulobacterales(Phenylobacterium) were associated with metabolism of cellulose, andAlphaproteobacteriawere associated with the metabolism of arabinose; members of the orderRhizobialeswere strongly associated with the metabolism of xylose. Annotated metagenomic data suggested diverse glycoside hydrolase gene representation within the pooled heavy DNA. By screening 2,876 cloned fragments derived from the13C-labeled DNA isolated from soils incubated with cellulose, we demonstrate the power of combining DNA-SIP, multiple-displacement amplification (MDA), and functional metagenomics by efficiently isolating multiple clones with activity on carboxymethyl cellulose and fluorogenic proxy substrates for carbohydrate-active enzymes.IMPORTANCEThe ability to identify genes based on function, instead of sequence homology, allows the discovery of genes that would not be identified through sequence alone. This is arguably the most powerful application of metagenomics for the recovery of novel genes and a natural partner of the stable-isotope-probing approach for targeting active-yet-uncultured microorganisms. We expanded on previous efforts to combine stable-isotope probing and metagenomics, enriching microorganisms from multiple soils that were active in degrading plant-derived carbohydrates, followed by construction of a cellulose-based metagenomic library and recovery of glycoside hydrolases through functional metagenomics. The major advance of our study was the discovery of active-yet-uncultivated soil microorganisms and enrichment of their glycoside hydrolases. We recovered positive cosmid clones in a higher frequency than would be expected with direct metagenomic analysis of soil DNA. This study has generated an invaluable metagenomic resource that future research will exploit for genetic and enzymatic potential.

scholarly journals Biphenyl-Metabolizing Bacteria in the Rhizosphere of Horseradish and Bulk Soil Contaminated by Polychlorinated Biphenyls as Revealed by Stable Isotope Probing

2009 ◽  
Vol 75 (20) ◽  
pp. 6471-6477 ◽  
Author(s):  
Ondrej Uhlik ◽  
Katerina Jecna ◽  
Martina Mackova ◽  
Cestmir Vlcek ◽  
Miluse Hroudova ◽  
...  
Keyword(s):  
Microbial Community ◽  
Stable Isotope ◽  
Polychlorinated Biphenyls ◽  
Stable Isotope Probing ◽  
Amino Acid Sequences ◽  
16S Rrna Genes ◽  
Bulk Soil ◽  
Rrna Genes ◽  
Soil Environment ◽  
Α Subunits

ABSTRACT DNA-based stable isotope probing in combination with terminal restriction fragment length polymorphism was used in order to identify members of the microbial community that metabolize biphenyl in the rhizosphere of horseradish (Armoracia rusticana) cultivated in soil contaminated with polychlorinated biphenyls (PCBs) compared to members of the microbial community in initial, uncultivated bulk soil. On the basis of early and recurrent detection of their 16S rRNA genes in clone libraries constructed from [13C]DNA, Hydrogenophaga spp. appeared to dominate biphenyl catabolism in the horseradish rhizosphere soil, whereas Paenibacillus spp. were the predominant biphenyl-utilizing bacteria in the initial bulk soil. Other bacteria found to derive carbon from biphenyl in this nutrient-amended microcosm-based study belonged mostly to the class Betaproteobacteria and were identified as Achromobacter spp., Variovorax spp., Methylovorus spp., or Methylophilus spp. Some bacteria that were unclassified at the genus level were also detected, and these bacteria may be members of undescribed genera. The deduced amino acid sequences of the biphenyl dioxygenase α subunits (BphA) from bacteria that incorporated [13C]into DNA in 3-day incubations of the soils with [13C]biphenyl are almost identical to that of Pseudomonas alcaligenes B-357. This suggests that the spectrum of the PCB congeners that can be degraded by these enzymes may be similar to that of strain B-357. These results demonstrate that altering the soil environment can result in the participation of different bacteria in the metabolism of biphenyl.

scholarly journals Arctic tundra soil bacterial communities active at subzero temperatures detected by stable isotope probing

2019 ◽  
Vol 96 (2) ◽  
Author(s):  
Preshita S Gadkari ◽  
Lora R McGuinness ◽  
Minna K Männistö ◽  
Lee J Kerkhof ◽  
Max M Häggblom
Keyword(s):  
Stable Isotope ◽  
16S Rrna ◽  
Arctic Tundra ◽  
Stable Isotope Probing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Subzero Temperature ◽  
Tundra Soil ◽  
Arctic Soils ◽  
Subzero Temperatures

ABSTRACT Arctic soils store vast amounts of carbon and are subject to intense climate change. While the effects of thaw on the composition and activities of Arctic tundra microorganisms has been examined extensively, little is known about the consequences of temperature fluctuations within the subzero range in seasonally frozen or permafrost soils. This study identified tundra soil bacteria active at subzero temperatures using stable isotope probing (SIP). Soils from Kilpisjärvi, Finland, were amended with 13C-cellobiose and incubated at 0, −4 and −16°C for up to 40 weeks. 16S rRNA gene sequence analysis of 13C-labelled DNA revealed distinct subzero-active bacterial taxa. The SIP experiments demonstrated that diverse bacteria, including members of Candidatus Saccharibacteria, Melioribacteraceae, Verrucomicrobiaceae, Burkholderiaceae, Acetobacteraceae, Armatimonadaceae and Planctomycetaceae, were capable of synthesising 13C-DNA at subzero temperatures. Differences in subzero temperature optima were observed, for example, with members of Oxalobacteraceae and Rhizobiaceae found to be more active at 0°C than at −4°C or −16°C, whereas Melioribacteriaceae were active at all subzero temperatures tested. Phylogeny of 13C-labelled 16S rRNA genes from the Melioribacteriaceae, Verrucomicrobiaceae and Candidatus Saccharibacteria suggested that these taxa formed subzero-active clusters closely related to members from other cryo-environments. This study demonstrates that subzero temperatures impact active bacterial community composition and activity, which may influence biogeochemical cycles.

scholarly journals Impact of plants on the diversity and activity of methylotrophs in soil

Microbiome ◽  
2020 ◽  
Vol 8 (1) ◽  
Author(s):  
Michael C. Macey ◽  
Jennifer Pratscher ◽  
Andrew T. Crombie ◽  
J. Colin Murrell
Keyword(s):  
Stable Isotope ◽  
Plant Growth ◽  
Molecular Probes ◽  
Stable Isotope Probing ◽  
16S Rrna Genes ◽  
Bulk Soil ◽  
Rrna Genes ◽  
Methylotrophic Bacteria ◽  
Volatile Organic ◽  
Plant Exudates

Abstract Background Methanol is the second most abundant volatile organic compound in the atmosphere, with the majority produced as a metabolic by-product during plant growth. There is a large disparity between the estimated amount of methanol produced by plants and the amount which escapes to the atmosphere. This may be due to utilisation of methanol by plant-associated methanol-consuming bacteria (methylotrophs). The use of molecular probes has previously been effective in characterising the diversity of methylotrophs within the environment. Here, we developed and applied molecular probes in combination with stable isotope probing to identify the diversity, abundance and activity of methylotrophs in bulk and in plant-associated soils. Results Application of probes for methanol dehydrogenase genes (mxaF, xoxF, mdh2) in bulk and plant-associated soils revealed high levels of diversity of methylotrophic bacteria within the bulk soil, including Hyphomicrobium, Methylobacterium and members of the Comamonadaceae. The community of methylotrophic bacteria captured by this sequencing approach changed following plant growth. This shift in methylotrophic diversity was corroborated by identification of the active methylotrophs present in the soils by DNA stable isotope probing using 13C-labelled methanol. Sequencing of the 16S rRNA genes and construction of metagenomes from the 13C-labelled DNA revealed members of the Methylophilaceae as highly abundant and active in all soils examined. There was greater diversity of active members of the Methylophilaceae and Comamonadaceae and of the genus Methylobacterium in plant-associated soils compared to the bulk soil. Incubating growing pea plants in a 13CO2 atmosphere revealed that several genera of methylotrophs, as well as heterotrophic genera within the Actinomycetales, assimilated plant exudates in the pea rhizosphere. Conclusion In this study, we show that plant growth has a major impact on both the diversity and the activity of methanol-utilising methylotrophs in the soil environment, and thus, the study contributes significantly to efforts to balance the terrestrial methanol and carbon cycle.

scholarly journals The Response of the Soil Microbiota to Long-Term Mineral and Organic Nitrogen Fertilization is Stronger in the Bulk Soil than in the Rhizosphere

Genes ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 456 ◽  
Author(s):  
Massimiliano Cardinale ◽  
Stefan Ratering ◽  
Aitak Sadeghi ◽  
Sushil Pokhrel ◽  
Bernd Honermeier ◽  
...  
Keyword(s):  
N Fertilization ◽  
16S Rrna Genes ◽  
Bulk Soil ◽  
Rrna Genes ◽  
Soil Microbiome ◽  
Future Research ◽  
Arable Soils ◽  
Mineral N ◽  
Bacterial Microbiota

The effects of different agronomic practices, such as fertilization regimes, can be experimentally tested in long-term experiments (LTE). Here, we aimed to evaluate the effect of different nitrogen fertilizations on the bacterial microbiota in both rhizosphere and bulk soil of sugar beet, in the Giessen-LTE (Germany). Fertilization treatments included mineral-N, manure, mineral-N + manure and no N-amendment. Metabarcoding and co-occurrence analysis of 16S rRNA genes, qPCR of amoA, nirK, nirS, nosZ-I and nosZ-II genes and soil physico-chemical analyses were performed. The effect of the fertilization treatments was more evident in the bulk soil, involving 33.1% of the microbiota. Co-occurrence analysis showed a rhizosphere cluster, dominated by Proteobacteria, Actinobacteria and Verrucomicrobia (hub taxa: Betaproteobacteriales), and a bulk soil cluster, dominated by Acidobacteria, Gemmatominadetes and “Latescibacteria” (hub taxa: Acidobacteria). In the bulk soil, mineral N-fertilization reduced nirK, amoA, nosZ-I and nosZ-II genes. Thirteen Operational taxonomic units (OTUs) showed 23 negative correlations with gene relative abundances. These OTUs likely represent opportunistic species that profited from the amended mineral-N and outgrew the species carrying N-cycle genes. Our results indicate trajectories for future research on soil microbiome in LTE and add new experimental evidence that will be helpful for sustainable management of nitrogen fertilizations on arable soils.

scholarly journals Integrated Meta-omics Approaches To Understand the Microbiome of Spontaneous Fermentation of Traditional Chinese Pu-erh Tea

mSystems ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Ming Zhao ◽  
Xiao Q. Su ◽  
Bo Nian ◽  
Li J. Chen ◽  
Dong L. Zhang ◽  
...  
Keyword(s):  
Phenolic Compounds ◽  
Gallic Acid ◽  
Glycoside Hydrolases ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Fermented Foods ◽  
Catechol Dioxygenase ◽  
Spontaneous Fermentation ◽  
Food Fermentation

ABSTRACT The microbiome in fermentation has direct impacts on the quality of fermented foods and is of great scientific and commercial interest. Despite considerable effort to explain the microbial metabolism associated with food fermentation, the role of the microbiome in pu-erh tea fermentation remains unknown. Here, we applied integrated meta-omics approaches to characterize the microbiome in two repeated fermentations of pu-erh tea. Metabarcoding analysis of bacterial 16S rRNA genes showed a decrease in the proportion of Proteobacteria and an increase in the abundance of Firmicutes during fermentation. Metabarcoding analysis of fungal internal transcribed spacer (ITS) sequence demonstrated that Rasamsonia, Thermomyces, and Aspergillus were dominant at the intermediate stage, whereas Aspergillus was dominant at other stages in fermentation. Metaproteomics analysis assigned primary microbial metabolic activity to metabolism and identified microbial carbohydrate-active enzymes involved in the degradation of polysaccharides including cellulose, xylan, xyloglucan, pectin, starch, lignin, galactomannan, and chitin. Metabolomics and high-performance liquid chromatography analysis revealed that levels of phenolic compounds, including gallates, decreased whereas contents of gallic acid and ellagic acid significantly increased after fermentation (P < 0.05). The changes in levels of gallates and gallic acid were associated with the hydrolysis of tannase. Glycoside hydrolases, phenol 2-monooxygenase, salicylaldehyde dehydrogenase, salicylate 1-monooxygenase, catechol O-methyltransferase, catechol dioxygenase, and quercetin 2,3-dioxygenases were hypothesized to be related to oxidation, conversion, or degradation of phenolic compounds. We demonstrated microbiota in fermentation and their function in the production of enzymes related to the degradation of polysaccharides, and metabolism of phenolic compounds, resulting in changes in metabolite contents and the quality of pu-erh tea. IMPORTANCE Fermented foods play important roles in diets worldwide and account for approximately one-third of all foods and beverages consumed. To date, traditional fermentation has used spontaneous fermentation. The microbiome in fermentation has direct impacts on the quality and safety of fermented foods and contributes to the preservation of traditional methods. Here, we used an integrated meta-omics approach to study the microbiome in the fermentation of pu-erh tea, which is a well-known Chinese fermented food with a special flavor and healthful benefits. This study advanced the knowledge of microbiota, metabolites, and enzymes in the fermentation of pu-erh tea. These novel insights shed light onto the complex microbiome in pu-erh fermentation and highlight the power of integrated meta-omics approaches in understanding the microbiome in food fermentation ecosystems.

scholarly journals Microbial Iron Oxidation in the Arctic Tundra and Its Implications for Biogeochemical Cycling

2015 ◽  
Vol 81 (23) ◽  
pp. 8066-8075 ◽  
Author(s):  
David Emerson ◽  
Jarrod J. Scott ◽  
Joshua Benes ◽  
William B. Bowden
Keyword(s):  
Relative Abundance ◽  
Iron Oxidation ◽  
Arctic Tundra ◽  
16S Rrna Genes ◽  
The Arctic ◽  
Rrna Genes ◽  
Oxygenic Photosynthesis ◽  
Field Station ◽  
Content Type ◽  
Microbial Iron Oxidation

ABSTRACTThe role that neutrophilic iron-oxidizing bacteria play in the Arctic tundra is unknown. This study surveyed chemosynthetic iron-oxidizing communities at the North Slope of Alaska near Toolik Field Station (TFS) at Toolik Lake (lat 68.63, long −149.60). Microbial iron mats were common in submerged habitats with stationary or slowly flowing water, and their greatest areal extent is in coating plant stems and sediments in wet sedge meadows. Some Fe-oxidizing bacteria (FeOB) produce easily recognized sheath or stalk morphotypes that were present and dominant in all the mats we observed. The cool water temperatures (9 to 11°C) and reduced pH (5.0 to 6.6) at all sites kinetically favor microbial iron oxidation. A microbial survey of five sites based on 16S rRNA genes found a predominance ofProteobacteria, withBetaproteobacteriaand members of the familyComamonadaceaebeing the most prevalent operational taxonomic units (OTUs). In relative abundance, clades of lithotrophic FeOB composed 5 to 10% of the communities. OTUs related to cyanobacteria and chloroplasts accounted for 3 to 25% of the communities. Oxygen profiles showed evidence for oxygenic photosynthesis at the surface of some mats, indicating the coexistence of photosynthetic and FeOB populations. The relative abundance of OTUs belonging to putative Fe-reducing bacteria (FeRB) averaged around 11% in the sampled iron mats. Mats incubated anaerobically with 10 mM acetate rapidly initiated Fe reduction, indicating that active iron cycling is likely. The prevalence of iron mats on the tundra might impact the carbon cycle through lithoautotrophic chemosynthesis, anaerobic respiration of organic carbon coupled to iron reduction, and the suppression of methanogenesis, and it potentially influences phosphorus dynamics through the adsorption of phosphorus to iron oxides.

scholarly journals Active Ammonia Oxidizers in an Acidic Soil Are Phylogenetically Closely Related to Neutrophilic Archaeon

2013 ◽  
Vol 80 (5) ◽  
pp. 1684-1691 ◽  
Author(s):  
Baozhan Wang ◽  
Yan Zheng ◽  
Rong Huang ◽  
Xue Zhou ◽  
Dongmei Wang ◽  
...  
Keyword(s):  
16S Rrna ◽  
Buoyant Density ◽  
Stable Isotope Probing ◽  
Acidic Soil ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Molecular Evidence ◽  
Ammonia Oxidizing Bacteria ◽  
Content Type ◽  
Metabolic Versatility

ABSTRACTAll cultivated ammonia-oxidizing archaea (AOA) within theNitrososphaeracluster (former soil group 1.1b) are neutrophilic. Molecular surveys also indicate the existence ofNitrososphaera-like phylotypes in acidic soil, but their ecological roles are poorly understood. In this study, we present molecular evidence for the chemolithoautotrophic growth ofNitrososphaera-like AOA in an acidic soil with pH 4.92 using DNA-based stable isotope probing (SIP). Soil microcosm incubations demonstrated that nitrification was stimulated by urea fertilization and accompanied by a significant increase in the abundance of AOA rather than ammonia-oxidizing bacteria (AOB). Real-time PCR analysis ofamoAgenes as a function of the buoyant density of the DNA gradient following the ultracentrifugation of the total DNA extracted from SIP microcosms indicated a substantial growth of soil AOA during nitrification. Pyrosequencing of the total 16S rRNA genes in the “heavy” DNA fractions suggested that archaeal communities were labeled to a much greater extent than soil AOB. Acetylene inhibition further showed that13CO2assimilation by nitrifying communities depended solely on ammonia oxidation activity, suggesting a chemolithoautotrophic lifestyle. Phylogenetic analysis of both13C-labeledamoAand 16S rRNA genes revealed that most of the active AOA were phylogenetically closely related to the neutrophilic strainsNitrososphaera viennensisEN76 and JG1 within theNitrososphaeracluster. Our results provide strong evidence for the adaptive growth ofNitrososphaera-like AOA in acidic soil, suggesting a greater metabolic versatility of soil AOA than previously appreciated.

scholarly journals Ruminococcus albus 8 Mutants Defective in Cellulose Degradation Are Deficient in Two Processive Endocellulases, Cel48A and Cel9B, Both of Which Possess a Novel Modular Architecture

2004 ◽  
Vol 186 (1) ◽  
pp. 136-145 ◽  
Author(s):  
Estelle Devillard ◽  
Dara B. Goodheart ◽  
Sanjay K. R. Karnati ◽  
Edward A. Bayer ◽  
Raphael Lamed ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Sequence Data ◽  
Cellulose Degradation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Glycoside Hydrolases ◽  
Cellulolytic Bacterium ◽  
Cellulolytic Bacteria ◽  
Modular Architecture ◽  
Ruminococcus Albus ◽  
Mutant Strains

ABSTRACT The cellulolytic bacterium Ruminococcus albus 8 adheres tightly to cellulose, but the molecular biology underpinning this process is not well characterized. Subtractive enrichment procedures were used to isolate mutants of R. albus 8 that are defective in adhesion to cellulose. Adhesion of the mutant strains was reduced 50% compared to that observed with the wild-type strain, and cellulose solubilization was also shown to be slower in these mutant strains, suggesting that bacterial adhesion and cellulose solubilization are inextricably linked. Two-dimensional polyacrylamide gel electrophoresis showed that all three mutants studied were impaired in the production of two high-molecular-mass, cell-bound polypeptides when they were cultured with either cellobiose or cellulose. The identities of these proteins were determined by a combination of mass spectrometry methods and genome sequence data for R. albus 8. One of the polypeptides is a family 9 glycoside hydrolase (Cel9B), and the other is a family 48 glycoside hydrolase (Cel48A). Both Cel9B and Cel48A possess a modular architecture, Cel9B possesses features characteristic of the B2 (or theme D) group of family 9 glycoside hydrolases, and Cel48A is structurally similar to the processive endocellulases CelF and CelS from Clostridium cellulolyticum and Clostridium thermocellum, respectively. Both Cel9B and Cel48A could be recovered by cellulose affinity procedures, but neither Cel9B nor Cel48A contains a dockerin, suggesting that these polypeptides are retained on the bacterial cell surface, and recovery by cellulose affinity procedures did not involve a clostridium-like cellulosome complex. Instead, both proteins possess a single copy of a novel X module with an unknown function at the C terminus. Such X modules are also present in several other R. albus glycoside hydrolases and are phylogentically distinct from the fibronectin III-like and X modules identified so far in other cellulolytic bacteria.

scholarly journals Origin and Diversity of Metabolically Active Gut Bacteria from Laboratory-Bred Larvae of Manduca sexta (Sphingidae, Lepidoptera, Insecta)

2008 ◽  
Vol 74 (23) ◽  
pp. 7189-7196 ◽  
Author(s):  
Nicole Brinkmann ◽  
Rainer Martens ◽  
Christoph C. Tebbe
Keyword(s):  
16S Rrna ◽  
Manduca Sexta ◽  
Metabolic Activity ◽  
Stable Isotope Probing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Bacterial Cells ◽  
Rt Pcr ◽  
Tobacco Leaves ◽  
Active Bacteria

ABSTRACT Cultivation-independent analyses based on genetic profiling of partial bacterial 16S rRNA genes by PCR-single-strand conformation polymorphism (PCR-SSCP), reverse transcriptase (RT)-PCR-SSCP of the 16S rRNA itself, and stable isotope probing (SIP), followed by RT-PCR-SSCP, were applied to characterize the diversity of metabolically active bacteria in the larval gut of Manduca sexta bred on tobacco leaves under greenhouse conditions. For SIP, hatching larvae were fed with leaves from tobacco plants grown in a 13CO2-enriched atmosphere. Dominant SSCP bands were sequenced and phylogenetically analyzed. Only one major gut colonizer, an Enterococcus relative, was detected; it occurred in the heavy RNA fraction, demonstrating its metabolic activity, and it originated from eggs, where its metabolic activity was also indicated by rRNA-based SSCP profiles. In contrast, a Citrobacter sedlakii relative was detected on eggs by DNA-SSCP, but rRNA-SSCP and SIP-rRNA-SSCP were negative, suggesting that these bacterial cells were inactive. A Burkholderia relative was dominant and metabolically active on the tobacco leaves but inactive inside the gut, where it was also quantitatively reduced, as suggested by lower band intensities in the DNA-based SSCP profiles. SIP-RNA-SSCP detected another metabolically active gut bacterium (Enterobacter sp.) and more bacteria in the light RNA fraction, indicating low or no metabolic activity of the latter inside the gut. We conclude that the larval gut supported only a low diversity of metabolically active bacteria.

Sign in / Sign up

Export Citation Format

Share Document