Identifying Low pH Active and Lactate-Utilizing Taxa within Oral Microbiome Communities from Healthy Children Using Stable Isotope Probing Techniques

<div><h3>Background</h3><p>Many human microbial infectious diseases including dental caries are polymicrobial in nature. How these complex multi-species communities evolve from a healthy to a diseased state is not well understood. Although many health- or disease-associated oral bacteria have been characterized <em>in vitro</em>, their physiology within the complex oral microbiome is difficult to determine with current approaches. In addition, about half of these species remain uncultivated to date with little known besides their 16S rRNA sequence. Lacking culture-based physiological analyses, the functional roles of uncultivated species will remain enigmatic despite their apparent disease correlation. To start addressing these knowledge gaps, we applied a combination of Magnetic Resonance Spectroscopy (MRS) with RNA and DNA based Stable Isotope Probing (SIP) to oral plaque communities from healthy children for <em>in vitro</em> temporal monitoring of metabolites and identification of metabolically active and inactive bacterial species.</p> <h3>Methodology/Principal Findings</h3><p>Supragingival plaque samples from caries-free children incubated with ¹³C-substrates under imposed healthy (buffered, pH 7) and diseased states (pH 5.5 and pH 4.5) produced lactate as the dominant organic acid from glucose metabolism. Rapid lactate utilization upon glucose depletion was observed under pH 7 conditions. SIP analyses revealed a number of genera containing cultured and uncultivated taxa with metabolic capabilities at pH 5.5. The diversity of active species decreased significantly at pH 4.5 and was dominated by <em>Lactobacillus</em> and <em>Propionibacterium</em> species, both of which have been previously found within carious lesions from children.</p> <h3>Conclusions/Significance</h3><p>Our approach allowed for identification of species that metabolize carbohydrates under different pH conditions and supports the importance of Lactobacilli and Propionibacterium in the development of childhood caries. Identification of species within healthy subjects that are active at low pH can lead to a better understanding of oral caries onset and generate appropriate targets for preventative measures in the early stages.</p> </div

Calculation of partial isotope incorporation into peptides measured by mass spectrometry

<p>Abstract</p> <p>Background</p> <p>Stable isotope probing (SIP) technique was developed to link function, structure and activity of microbial cultures metabolizing carbon and nitrogen containing substrates to synthesize their biomass. Currently, available methods are restricted solely to the estimation of fully saturated heavy stable isotope incorporation and convenient methods with sufficient accuracy are still missing. However in order to track carbon fluxes in microbial communities new methods are required that allow the calculation of partial incorporation into biomolecules.</p> <p>Results</p> <p>In this study, we use the characteristics of the so-called 'half decimal place rule' (HDPR) in order to accurately calculate the partial¹³C incorporation in peptides from enzymatic digested proteins. Due to the clade-crossing universality of proteins within bacteria, any available high-resolution mass spectrometry generated dataset consisting of tryptically-digested peptides can be used as reference.</p> <p>We used a freely available peptide mass dataset from <it>Mycobacterium tuberculosis </it>consisting of 315,579 entries. From this the error of estimated versus known heavy stable isotope incorporation from an increasing number of randomly drawn peptide sub-samples (100 times each; no repetition) was calculated. To acquire an estimated incorporation error of less than 5 atom %, about 100 peptide masses were needed. Finally, for testing the general applicability of our method, peptide masses of tryptically digested proteins from <it>Pseudomonas putida </it>ML2 grown on labeled substrate of various known concentrations were used and¹³C isotopic incorporation was successfully predicted. An easy-to-use script <abbrgrp><abbr bid="B1">1</abbr></abbrgrp> was further developed to guide users through the calculation procedure for their own data series.</p> <p>Conclusion</p> <p>Our method is valuable for estimating¹³C incorporation into peptides/proteins accurately and with high sensitivity. Generally, our method holds promise for wider applications in qualitative and especially quantitative proteomics.</p

Identification of Methanoculleus spp. as active methanogens during anoxic incubations of swine manure storage tank samples

Methane emissions represent a major environmental concern associated with manure management in the livestock industry. A more thorough understanding of how microbial communities function in manure storage tanks is a prerequisite for mitigating methane emissions. Identifying the microorganisms that are metabolically active is an important first step. Methanogenic archaea are major contributors to methanogenesis in stored swine manure, and we investigated active methanogenic populations by DNA stable isotope probing (DNA-SIP). Following a preincubation of manure samples under anoxic conditions to induce substrate starvation, [U-¹³C] acetate was added as a labeled substrate. Fingerprint analysis of density-fractionated DNA, using length-heterogeneity analysis of PCR-amplified mcrA genes (encoding the alpha subunit of methyl coenzyme M reductase), showed that the incorporation of ¹³C into DNA was detectable at in situ acetate concentrations (~7g/liter). Fingerprints of DNA retrieved from heavy fractions of the ¹³C treatment were primarily enriched in a 483-bp amplicon and, to a lesser extent, in a 481-bp amplicon. Analyses based on clone libraries of the mcrA and 16S rRNA genes revealed that both of these heavy DNA amplicons corresponded to Methanoculleus spp. Our results demonstrate that uncultivated methanogenic archaea related to Methanoculleus spp. were major contributors to acetate-C assimilation during the anoxic incubation of swine manure storage tank samples. Carbon assimilation and dissimilation rate estimations suggested that Methanoculleus spp. were also major contributors to methane emissions and that the hydrogenotrophic pathway predominated during methanogenesis

Deltaproteobacteria (Pelobacter) and Methanococcoides are responsible for choline-dependent methanogenesis in a coastal saltmarsh sediment

Coastal saltmarsh sediments represent an important source of natural methane emissions, much of which originates from quaternary and methylated amines, such as choline and trimethylamine. In this study, we combine DNA stable isotope probing with high throughput sequencing of 16S rRNA genes and 13C2-choline enriched metagenomes, followed by metagenome data assembly, to identify the key microbes responsible for methanogenesis from choline. Microcosm incubation with 13C2-choline leads to the formation of trimethylamine and subsequent methane production, suggesting that choline-dependent methanogenesis is a two-step process involving trimethylamine as the key intermediate. Amplicon sequencing analysis identifies Deltaproteobacteria of the genera Pelobacter as the major choline utilizers. Methanogenic Archaea of the genera Methanococcoides become enriched in choline-amended microcosms, indicating their role in methane formation from trimethylamine. The binning of metagenomic DNA results in the identification of bins classified as Pelobacter and Methanococcoides. Analyses of these bins reveal that Pelobacter have the genetic potential to degrade choline to trimethylamine using the choline-trimethylamine lyase pathway, whereas Methanococcoides are capable of methanogenesis using the pyrrolysine-containing trimethylamine methyltransferase pathway. Together, our data provide a new insight on the diversity of choline utilizing organisms in coastal sediments and support a syntrophic relationship between Bacteria and Archaea as the dominant route for methanogenesis from choline in this environment

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

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

Microbial cycling of isoprene, the most abundantly produced biological volatile organic compound on Earth

Isoprene (2-methyl-1,3-butadiene), the most abundantly produced biogenic volatile organic compound (BVOC) on Earth, is highly reactive and can have diverse and often detrimental atmospheric effects, which impact on climate and health. Most isoprene is produced by terrestrial plants, but (micro)algal production is important in aquatic environments, and the relative bacterial contribution remains unknown. Soils are a sink for isoprene, and bacteria that can use isoprene as a carbon and energy source have been cultivated and also identified using cultivation-independent methods from soils, leaves and coastal/marine environments. Bacteria belonging to the Actinobacteria are most frequently isolated and identified, and Proteobacteria have also been shown to degrade isoprene. In the freshwater-sediment isolate, Rhodococcus strain AD45, initial oxidation of isoprene to 1,2-epoxy-isoprene is catalyzed by a multicomponent isoprene monooxygenase encoded by the genes isoABCDEF. The resultant epoxide is converted to a glutathione conjugate by a glutathione S-transferase encoded by isoI, and further degraded by enzymes encoded by isoGHJ. Genome sequence analysis of actinobacterial isolates belonging to the genera Rhodococcus, Mycobacterium and Gordonia has revealed that isoABCDEF and isoGHIJ are linked in an operon, either on a plasmid or the chromosome. In Rhodococcus strain AD45 both isoprene and epoxy-isoprene induce a high level of transcription of 22 contiguous genes, including isoABCDEF and isoGHIJ. Sequence analysis of the isoA gene, encoding the large subunit of the oxygenase component of isoprene monooxygenase, from isolates has facilitated the development of PCR primers that are proving valuable in investigating the ecology of uncultivated isoprene-degrading bacteria

Ecophysiology of syntrophic communities that degrade saturated and unsaturated long-chain fatty acids

Syntrophic relationships are the key for biodegradation in methanogenic environments. We review the ecological and physiological features of syntrophic communities involved in the degradation of saturated and unsaturated long-chain fatty acids (LCFA), as well as their potential application to convert lipids/fats containing waste to biogas. Presently, about 14 species have been described with the ability to grow on fatty acids in syntrophy with methanogens, all belonging to the families Syntrophomonadaceae and Syntrophaceae. The principle pathway of LCFA degradation is through β-oxidation, but the initial steps in the conversion of unsaturated LCFA are unclear. Communities enriched on unsaturated LCFA also degrade saturated LCFA, but the opposite generally is not the case. For efficient methane formation, the physical and inhibitory effects of LCFA on methanogenesis need to be considered. LCFA adsorbs strongly to biomass, which causes encapsulation of active syntrophic communities and hampers diffusion of substrate and products in and out of the biomass. Quantification of archaea by real-time PCR analysis suggests that potential LCFA inhibitory effect towards methanogens might be reversible. Rather, the conversion of adsorbed LCFA in batch assays was shown to result in a significant increase of archaeal cell numbers in anaerobic sludge samples.The authors thank J. Prosser for the invitation to write this minireview. We appreciated the critical reading of I.M. Head and of the anonymous reviewers, and we thank them for their constructive comments and suggestions. This work was possible through the financial support provided by the Portuguese Science Foundation (FCT) and European Social Fund (ESF) (grant SFRH/BD/8726/2002), and by the Wageningen Institute for Environmental and Climate Research (WIMEK)

The great screen anomaly—a new frontier in product discovery through functional metagenomics

Functional metagenomics, the study of the collective genome of a microbial community by expressing it in a foreign host, is an emerging field in biotechnology. Over the past years, the possibility of novel product discovery through metagenomics has developed rapidly. Thus, metagenomics has been heralded as a promising mining strategy of resources for the biotechnological and pharmaceutical industry. However, in spite of innovative work in the field of functional genomics in recent years, yields from function-based metagenomics studies still fall short of producing significant amounts of new products that are valuable for biotechnological processes. Thus, a new set of strategies is required with respect to fostering gene expression in comparison to the traditional work. These new strategies should address a major issue, that is, how to successfully express a set of unknown genes of unknown origin in a foreign host in high throughput. This article is an opinionating review of functional metagenomic screening of natural microbial communities, with a focus on the optimization of new product discovery. It first summarizes current major bottlenecks in functional metagenomics and then provides an overview of the general metagenomic assessment strategies, with a focus on the challenges that are met in the screening for, and selection of, target genes in metagenomic libraries. To identify possible screening limitations, strategies to achieve optimal gene expression are reviewed, examining the molecular events all the way from the transcription level through to the secretion of the target gene product

Proteomic and Physiological Responses of Kineococcus radiotolerans to Copper

Copper is a highly reactive, toxic metal; consequently, transport of this metal within the cell is tightly regulated. Intriguingly, the actinobacterium Kineococcus radiotolerans has been shown to not only accumulate soluble copper to high levels within the cytoplasm, but the phenotype also correlated with enhanced cell growth during chronic exposure to ionizing radiation. This study offers a first glimpse into the physiological and proteomic responses of K. radiotolerans to copper at increasing concentration and distinct growth phases. Aerobic growth rates and biomass yields were similar over a range of Cu(II) concentrations (0–1.5 mM) in complex medium. Copper uptake coincided with active cell growth and intracellular accumulation was positively correlated with Cu(II) concentration in the growth medium (R2 = 0.7). Approximately 40% of protein coding ORFs on the K. radiotolerans genome were differentially expressed in response to the copper treatments imposed. Copper accumulation coincided with increased abundance of proteins involved in oxidative stress and defense, DNA stabilization and repair, and protein turnover. Interestingly, the specific activity of superoxide dismutase was repressed by low to moderate concentrations of copper during exponential growth, and activity was unresponsive to perturbation with paraquot. The biochemical response pathways invoked by sub-lethal copper concentrations are exceptionally complex; though integral cellular functions are preserved, in part, through the coordination of defense enzymes, chaperones, antioxidants and protective osmolytes that likely help maintain cellular redox. This study extends our understanding of the ecology and physiology of this unique actinobacterium that could potentially inspire new biotechnologies in metal recovery and sequestration, and environmental restoration

Finding the Needles in the Metagenome Haystack

In the collective genomes (the metagenome) of the microorganisms inhabiting the Earth’s diverse environments is written the history of life on this planet. New molecular tools developed and used for the past 15 years by microbial ecologists are facilitating the extraction, cloning, screening, and sequencing of these genomes. This approach allows microbial ecologists to access and study the full range of microbial diversity, regardless of our ability to culture organisms, and provides an unprecedented access to the breadth of natural products that these genomes encode. However, there is no way that the mere collection of sequences, no matter how expansive, can provide full coverage of the complex world of microbial metagenomes within the foreseeable future. Furthermore, although it is possible to fish out highly informative and useful genes from the sea of gene diversity in the environment, this can be a highly tedious and inefficient procedure. Microbial ecologists must be clever in their pursuit of ecologically relevant, valuable, and niche-defining genomic information within the vast haystack of microbial diversity. In this report, we seek to describe advances and prospects that will help microbial ecologists glean more knowledge from investigations into metagenomes. These include technological advances in sequencing and cloning methodologies, as well as improvements in annotation and comparative sequence analysis. More significant, however, will be ways to focus in on various subsets of the metagenome that may be of particular relevance, either by limiting the target community under study or improving the focus or speed of screening procedures. Lastly, given the cost and infrastructure necessary for large metagenome projects, and the almost inexhaustible amount of data they can produce, trends toward broader use of metagenome data across the research community coupled with the needed investment in bioinformatics infrastructure devoted to metagenomics will no doubt further increase the value of metagenomic studies in various environments