Metaproteomics method to determine carbon sources and assimilation pathways of species in microbial communities

Measurements of stable carbon isotope ratios (δ13C) are widely used in biology to address questions regarding food sources and metabolic pathways used by organisms. The analysis of these so-called stable isotope fingerprints (SIFs) for microbes involved in biogeochemical cycling and microbiota of plants and animals has led to major discoveries in environmental microbiology. Currently, obtaining SIFs for microbial communities is challenging as the available methods either only provide low taxonomic resolution, such as the use of lipid biomarkers, or are limited in throughput, such as nanoscale secondary ion MS imaging of single cells. Here we present “direct protein-SIF” and the Calis-p software package (https://sourceforge.net/projects/calis-p/), which enable high-throughput measurements of accurate δ13C values for individual species within a microbial community. We benchmark the method using 20 pure culture microorganisms and show that the method reproducibly provides SIF values consistent with gold-standard bulk measurements performed with an isotope ratio mass spectrometer. Using mock community samples, we demonstrate that SIF values can also be obtained for individual species within a microbial community. Finally, a case study of an obligate bacteria–animal symbiosis shows that direct protein-SIF confirms previous physiological hypotheses and can provide unexpected insights into the symbionts’ metabolism. This confirms the usefulness of this approach to accurately determine δ13C values for different species in microbial community samples.

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A metaproteomics method to determine carbon sources and assimilation pathways of species in microbial communities

10.1101/245290 ◽

2018 ◽

Author(s):

Manuel Kleiner ◽

Xiaoli Dong ◽

Tjorven Hinzke ◽

Juliane Wippler ◽

Erin Thorson ◽

...

Keyword(s):

Microbial Community ◽

Microbial Communities ◽

Carbon Isotope ◽

Isotope Ratio ◽

Single Cells ◽

Stable Carbon Isotope ◽

Carbon Sources ◽

Food Sources ◽

Individual Species ◽

Stable Carbon

AbstractMeasurements of the carbon stable isotope ratio (δ13C) are widely used in biology to address major questions regarding food sources and metabolic pathways used by organisms. Measurement of these so called stable carbon isotope fingerprints (SIFs) for microbes involved in biogeochemical cycling and microbiota of plants and animals have led to major discoveries in environmental microbiology. Currently, obtaining SIFs for microbial communities is challenging as the available methods either only provide limited taxonomic resolution, such as with the use of lipid biomarkers, or are limited in throughput, such as NanoSIMS imaging of single cells.Here we present “direct Protein-SIF” and the Calis-p software package (https://sourceforge.net/projects/calis-p/), which enable high-throughput measurements of accurate δ13C values for individual species within a microbial community. We benchmark the method using 20 pure culture microorganisms and show that the method reproducibly provides SIF values consistent with gold standard bulk measurements performed with an isotope ratio mass spectrometer. Using mock community samples, we show that SIF values can also be obtained for individual species within a microbial community. Finally, a case study of an obligate bacteria-animal symbiosis showed that direct Protein-SIF confirms previous physiological hypotheses and can provide unexpected new insights into the symbionts’ metabolism. This confirms the usefulness of this new approach to accurately determine δ13C values for different species in microbial community samples.SignificanceTo understand the roles that microorganisms play in diverse environments such as the open ocean and the human intestinal tract, we need an understanding of their metabolism and physiology. A variety of methods such as metagenomics and metaproteomics exist to assess the metabolism of environmental microorganisms based on gene content and gene expression. These methods often only provide indirect evidence for which substrates are used by a microorganism in a community. The direct Protein-SIF method that we developed allows linking microbial species in communities to the environmental carbon sources they consume by determining their stable carbon isotope signature. Direct Protein-SIF also allows assessing which carbon fixation pathway is used by autotrophic microorganisms that directly assimilate CO2.

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Microbial Communities Show Parallels at Sites with Distinct Litter and Soil Characteristics

Applied and Environmental Microbiology ◽

10.1128/aem.00527-11 ◽

2011 ◽

Vol 77 (21) ◽

pp. 7560-7567 ◽

Cited By ~ 17

Author(s):

Marketa Sagova-Mareckova ◽

Marek Omelka ◽

Ladislav Cermak ◽

Zdenek Kamenik ◽

Jana Olsovska ◽

...

Keyword(s):

Microbial Community ◽

Microbial Communities ◽

Community Composition ◽

Soil Ph ◽

Soil Chemistry ◽

High Performance ◽

Bacterial Community Composition ◽

Microbial Community Composition ◽

Carbon Sources ◽

Soil Extracts

ABSTRACTPlant and microbial community composition in connection with soil chemistry determines soil nutrient cycling. The study aimed at demonstrating links between plant and microbial communities and soil chemistry occurring among and within four sites: two pine forests with contrasting soil pH and two grasslands of dissimilar soil chemistry and vegetation. Soil was characterized by C and N content, particle size, and profiles of low-molecular-weight compounds determined by high-performance liquid chromatography (HPLC) of soil extracts. Bacterial and actinobacterial community composition was assessed by terminal restriction fragment length polymorphism (T-RFLP) and cloning followed by sequencing. Abundances of bacteria, fungi, and actinobacteria were determined by quantitative PCR. In addition, a pool of secondary metabolites was estimated byermresistance genes coding for rRNA methyltransferases. The sites were characterized by a stable proportion of C/N within each site, while on a larger scale, the grasslands had a significantly lower C/N ratio than the forests. A Spearman's test showed that soil pH was correlated with bacterial community composition not only among sites but also within each site. Bacterial, actinobacterial, and fungal abundances were related to carbon sources while T-RFLP-assessed microbial community composition was correlated with the chemical environment represented by HPLC profiles. Actinobacteria community composition was the only studied microbial characteristic correlated to all measured factors. It was concluded that the microbial communities of our sites were influenced primarily not only by soil abiotic characteristics but also by dominant litter quality, particularly, by percentage of recalcitrant compounds.

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High-Resolution Phylogenetic and Population Genetic Analysis of Microbial Communities with RoC-ITS

10.1101/2020.10.16.342691 ◽

2020 ◽

Author(s):

Douglas B. Rusch ◽

Jie Huang ◽

Chris Hemmerich ◽

Matthew W. Hahn

Keyword(s):

Microbial Community ◽

Microbial Communities ◽

Single Molecule ◽

Rolling Circle Amplification ◽

Microbial Community Composition ◽

Ribosomal Gene ◽

Taxonomic Resolution ◽

Illumina Platform ◽

Population Genetic Analysis ◽

Sequencing Platform

AbstractMicrobial communities are inter-connected systems of incredible complexity and dynamism that play crucial roles in health, energy, and the environment. To better understand microbial communities and how they respond to change, it is important to know which microbes are present and their relative abundances at the greatest taxonomic resolution possible. Here, we describe a novel protocol (RoC-ITS) that uses the single-molecule Nanopore sequencing platform to assay the composition of microbial communities in unprecedented detail. This methodology produces long-read sequences including multiple copies of the same complete 16S ribosomal gene and its neighboring internally transcribed spacer (ITS) using rolling-circle amplification. The ribosomal 16S gene provides phylogenetic information down to the species-level, while the much less conserved ITS region contains strain-level information. When linked together, this combination of markers allows for the identification of individual ribosomal units within a specific organism, the assessment of their relative stoichiometry, and the ability to monitor subtle shifts in microbial community composition with a single generic assay. We applied RoC-ITS to a mock microbial community that was also sequenced using the Illumina platform, demonstrating its accuracy in quantifying the relative abundance and identity of each species.

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Composition and Physiological Profiling of Sprout-Associated Microbial Communities

Journal of Food Protection ◽

10.4315/0362-028x-65.12.1903 ◽

2002 ◽

Vol 65 (12) ◽

pp. 1903-1908 ◽

Cited By ~ 21

Author(s):

ANABELLE MATOS ◽

JAY L. GARLAND ◽

WILLIAM F. FETT

Keyword(s):

Community Structure ◽

Microbial Community ◽

Microbial Communities ◽

Microbial Community Structure ◽

Carbon Sources ◽

The Other ◽

Independent Effect ◽

Patterns Of Utilization ◽

Plate Counts ◽

Alfalfa Sprouts

The native microfloras of various types of sprouts (alfalfa, clover, sunflower, mung bean, and broccoli sprouts) were examined to assess the relative effects of sprout type and inoculum factors (i.e., sprout-growing facility, seed lot, and inoculation with sprout-derived inocula) on the microbial community structure of sprouts. Sprouts were sonicated for 7 min or hand shaken with glass beads for 2 min to recover native microfloras from the surface, and the resulting suspensions were diluted and plated. The culturable fraction was characterized by the density (log CFU/g), richness (e.g., number of types of bacteria), and diversity (e.g., microbial richness and evenness) of colonies on tryptic soy agar plates incubated for 48 h at 30°C. The relative similarity between sprout-associated microbial communities was assessed with the use of community-level physiological profiles (CLPPs) based on patterns of utilization of 95 separate carbon sources. Aerobic plate counts of 7.96 ± 0.91 log CFU/g of sprout tissue (fresh weight) were observed, with no statistically significant differences in microbial cell density, richness, or diversity due to sprout type, sprout-growing facility, or seed lot. CLPP analyses revealed that the microbial communities associated with alfalfa and clover sprouts are more similar than those associated with the other sprout types tested. Variability among sprout types was more extensive than any differences between microbial communities associated with alfalfa and clover sprouts from different sprout-growing facilities and seed lots. These results indicate that the subsequent testing of biocontrol agents should focus on similar organisms for alfalfa and clover, but alternative types may be most suitable for the other sprout types tested. The inoculation of alfalfa sprouts with communities derived from various sprout types had a significant, source-independent effect on microbial community structure, indicating that the process of inoculation alters the dynamics of community development regardless of the types of organisms involved.

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Dynamic Changes of Microbiome with the Utilization of Volatile Fatty Acids as Electron Donors for Denitrification

Water ◽

10.3390/w13111556 ◽

2021 ◽

Vol 13 (11) ◽

pp. 1556

Author(s):

Okkyoung Choi ◽

Se-jin Cha ◽

Hyunjin Kim ◽

Hyunook Kim ◽

Byoung-In Sang

Keyword(s):

Fatty Acids ◽

Microbial Community ◽

Microbial Communities ◽

Volatile Fatty Acids ◽

Carbon Sources ◽

Electron Donors ◽

Environmental Condition ◽

Dynamic Changes ◽

Nitrification And Denitrification ◽

By Products

Volatile fatty acids can be used as carbon sources for denitrification and are easily supplied as by-products from the anaerobic digestion of waste materials. Nitrification and denitrification processes were carried out in a single reactor feeding volatile fatty acids as electron donors and the changes in microbial communities in the reactor were investigated. The microbial communities in the alternating aerobic and anoxic systems were different, and their structure flexibly changed within one reactor. Bacteroidetes and Firmicutes were highly distributed during denitrification, whereas Proteobacteria was a major phylum during nitrification. In addition, in the denitrification system, the microbial community was substrate dependent. It showed the sequential nitrogen removal in one reactor and the microbial community also followed the change of environmental condition, cyclic nitrification, and denitrification.

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Unravelling the Functional Diversity of the Soil Microbial Community of Chinese Fir Plantations of Different Densities

Forests ◽

10.3390/f9090532 ◽

2018 ◽

Vol 9 (9) ◽

pp. 532 ◽

Cited By ~ 6

Author(s):

Chaoqun Wang ◽

Lin Xue ◽

Yuhong Dong ◽

Yihui Wei ◽

Ruzhen Jiao

Keyword(s):

Microbial Community ◽

Microbial Communities ◽

Functional Diversity ◽

Soil Microbial Community ◽

Diversity Index ◽

Carbon Sources ◽

Soil Microbial Communities ◽

Stand Density ◽

Chinese Fir ◽

Soil Microbial

The structure and function of forest ecosystems are directly or indirectly affected by their stand density. However, what effect the density of Chinese fir plantations has on the functional diversity of the soil microbial community remains unclear. The microbial metabolic functional diversity of soils sampled at the topsoil (0–20 cm) of 35-year-old Chinese fir plantations of five initial densities (D1: 1667 stems∙hm−2, D2: 3333 stems∙hm−2, D3: 5000 stems∙hm−2, D4: 6667 stems∙hm−2, and D5: 10,000 stems∙hm−2) was studied by using Biolog ECO technology. The results showed that the soil pH, oxidizable organic carbon (SOOC), available N (AN), available P (AP), and available K (AK) contents all showed a gradual increase from D1 to D4 and a decrease from D4 to D5, while the number of culturable bacteria and total microorganisms, the average well color development (AWCD) values for the single carbon substrate and six types of carbon sources used by the microbial community, as well as the Shannon-Wiener diversity index (H’), Pielou evenness index (J), and McIntosh Diversity Index (U), were the opposite, suggesting that low-densities favored C and N mineralization and the nutrient cycle. The density of Chinese fir plantations had a significant effect on the use of carbohydrates, amino acids, carboxylic acids, and phenolic acids by the soil microbial community, but it had no significant effect on the use of polymers (p < 0.05). Principal component analysis (PCA) revealed that carbohydrates, polymers, and phenolic acids were sensitive carbon sources that caused differences in the metabolic functions of soil microbial communities in Chinese fir plantations. Redundancy analysis (RDA) showed that physicochemical factors have a significant influence on the metabolic function of soil microbial communities (RDA1 and RDA2 explained >85% variance). The changes in density affected the soil physicochemical properties, the composition, and the metabolic functional diversity of microbial communities in Chinese fir plantations, which is certainly useful for the stand density regulation of Chinese fir plantations.

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The exchange of vitamin B1 and its biosynthesis intermediates in synthetic microbial communities shapes the community composition and reveals complexities of nutrient sharing

10.1101/2021.09.29.462401 ◽

2021 ◽

Author(s):

Rupali R. M. Sathe ◽

Ryan W. Paerl ◽

Amrita B. Hazra

Keyword(s):

Microbial Communities ◽

Large Scale ◽

General Pattern ◽

Carbon Sources ◽

Vibrio Anguillarum ◽

Vitamin B1 ◽

Individual Species ◽

Community Members ◽

E Coli ◽

The Media

AbstractMicrobial communities occupy diverse niches in nature, and exchanges of metabolites such as carbon sources, amino acids, and vitamins occur routinely among the community members. While large-scale metagenomic and metabolomic studies shed some light on these exchanges, the contribution of individual species and the molecular details of specific interactions are difficult to track. Here, we explore the molecular picture of vitamin B1 (thiamin) metabolism occurring in synthetic communities of Escherichia coli thiamin auxotrophs which engage in the exchange of thiamin and its biosynthesis intermediates. In E. coli, the two parts of thiamin – the 4-amino-5-hydroxymethyl-2-methylpyrimidine and the 4-methyl-5-(2-hydroxyethyl)thiazole – are synthesized by separate pathways using enzymes ThiC and ThiG, respectively, and are then joined by ThiE to form thiamin. We observed that even though E. coli ΔthiC, ΔthiE, and ΔthiG mutants are thiamin auxotrophs, co-cultures of ΔthiC-ΔthiE and ΔthiC-ΔthiG grow in a thiamin-deficient minimal medium, whereas the ΔthiE-ΔthiG co-culture does not. Analysis of the exchange of thiamin and its intermediates in Vibrio anguillarum co-cultures, and in mixed co-cultures of V. anguillarum and E. coli revealed that the general pattern of thiamin metabolism and exchange among microbes is conserved across species. Specifically, the microorganisms exchange HMP and thiamin easily among themselves but not THZ. Furthermore, we observe that the availability of exogenous thiamin in the media affects whether these strains interact with each other or grow independently. This underscores the importance of the exchange of essential metabolites as a defining factor in building and modulating synthetic or natural microbial communities.

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Metaproteomics: Much More than Measuring Gene Expression in Microbial Communities

mSystems ◽

10.1128/msystems.00115-19 ◽

2019 ◽

Vol 4 (3) ◽

Cited By ~ 19

Author(s):

Manuel Kleiner

Keyword(s):

Gene Expression ◽

Community Structure ◽

Microbial Community ◽

Microbial Communities ◽

Large Scale ◽

Molecular Level ◽

Carbon Sources ◽

Community Members ◽

Insight Into

ABSTRACT Metaproteomics is the large-scale identification and quantification of proteins from microbial communities and thus provides direct insight into the phenotypes of microorganisms on the molecular level. Initially, metaproteomics was mainly used to assess the “expressed” metabolism and physiology of microbial community members. However, recently developed metaproteomic tools allow quantification of per-species biomass to determine community structure, in situ carbon sources of community members, and the uptake of labeled substrates by community members. In this perspective, I provide a brief overview of the questions that we can currently address, as well as new metaproteomics-based approaches that we and others are developing to address even more questions in the study of microbial communities and plant and animal microbiota. I also highlight some areas and technologies where I anticipate developments and potentially major breakthroughs in the next 5 years and beyond.

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Exposing the Three-Dimensional Biogeography and Metabolic States of Pathogens in Cystic Fibrosis Sputum via Hydrogel Embedding, Clearing, and rRNA Labeling

mBio ◽

10.1128/mbio.00796-16 ◽

2016 ◽

Vol 7 (5) ◽

Cited By ~ 49

Author(s):

William H. DePas ◽

Ruth Starwalt-Lee ◽

Lindsey Van Sambeek ◽

Sripriya Ravindra Kumar ◽

Viviana Gradinaru ◽

...

Keyword(s):

Cystic Fibrosis ◽

Growth Rate ◽

Microbial Communities ◽

Spatial Organization ◽

Single Cells ◽

Host Cells ◽

Individual Species ◽

Complex Environments ◽

Host Interactions

ABSTRACT Physiological resistance to antibiotics confounds the treatment of many chronic bacterial infections, motivating researchers to identify novel therapeutic approaches. To do this effectively, an understanding of how microbes survive in vivo is needed. Though much can be inferred from bulk approaches to characterizing complex environments, essential information can be lost if spatial organization is not preserved. Here, we introduce a tissue-clearing technique, termed MiPACT, designed to retain and visualize bacteria with associated proteins and nucleic acids in situ on various spatial scales. By coupling MiPACT with hybridization chain reaction (HCR) to detect rRNA in sputum samples from cystic fibrosis (CF) patients, we demonstrate its ability to survey thousands of bacteria (or bacterial aggregates) over millimeter scales and quantify aggregation of individual species in polymicrobial communities. By analyzing aggregation patterns of four prominent CF pathogens, Staphylococcus aureus , Pseudomonas aeruginosa , Streptococcus sp., and Achromobacter xylosoxidans , we demonstrate a spectrum of aggregation states: from mostly single cells ( A. xylosoxidans ), to medium-sized clusters ( S. aureus ), to a mixture of single cells and large aggregates ( P. aeruginosa and Streptococcus sp.). Furthermore, MiPACT-HCR revealed an intimate interaction between Streptococcus sp. and specific host cells. Lastly, by comparing standard rRNA fluorescence in situ hybridization signals to those from HCR, we found that different populations of S. aureus and A. xylosoxidans grow slowly overall yet exhibit growth rate heterogeneity over hundreds of microns. These results demonstrate the utility of MiPACT-HCR to directly capture the spatial organization and metabolic activity of bacteria in complex systems, such as human sputum. IMPORTANCE The advent of metagenomic and metatranscriptomic analyses has improved our understanding of microbial communities by empowering us to identify bacteria, calculate their abundance, and profile gene expression patterns in complex environments. We are still technologically limited, however, in regards to the many questions that bulk measurements cannot answer, specifically in assessing the spatial organization of microbe-microbe and microbe-host interactions. Here, we demonstrate the power of an enhanced optical clearing method, MiPACT, to survey important aspects of bacterial physiology (aggregation, host interactions, and growth rate), in situ , with preserved spatial information when coupled to rRNA detection by HCR. Our application of MiPACT-HCR to cystic fibrosis patient sputum revealed species-specific aggregation patterns, yet slow growth characterized the vast majority of bacterial cells regardless of their cell type. More broadly, MiPACT, coupled with fluorescent labeling, promises to advance the direct study of microbial communities in diverse environments, including microbial habitats within mammalian systems.

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A simple linear relationship between resource availability and microbial community diversity

10.1101/2020.09.12.294660 ◽

2020 ◽

Author(s):

Martina Dal Bello ◽

Hyunseok Lee ◽

Akshit Goyal ◽

Jeff Gore

Keyword(s):

Microbial Community ◽

Microbial Communities ◽

Linear Relationship ◽

Resource Availability ◽

Carbon Sources ◽

Community Diversity ◽

Microbial Community Diversity ◽

Wide Range ◽

Simple Linear Relationship

AbstractMicrobial community diversity is pivotal for the functioning of our planet, but its drivers are still unclear, in particular the role of resource number and identity. To fill this gap, we studied the assembly of hundreds of soil-derived microbial communities on a wide range of well-defined resource environments, from single carbon sources to combinations of up to 16. We found a remarkable diversity in single resources but a linear one-by-one increase in the number of species with the number of additional resources. We show, both experimentally and theoretically, that both these observations could originate from generalist and specialist taxa interacting in a modular fashion within the community. Since generalists and specialists are ubiquitous in natural microbiomes, our results might apply to a variety of different ecological settings, providing a framework to predict how community diversity responds to changes in resource availability.One Sentence SummaryWhile many species coexist in single resources, community diversity only increases one-by-one as more resources are added.

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