Technique for Studying Arthropod and Microbial Communities within Tree Tissues

Phloem tissues of pine are habitats for many thousands of organisms. Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear young, feed, or hide from natural enemies or harsh environmental conditions outside of the tree. Organisms that persist within the phloem habitat are difficult to observe given their location under bark. We provide a technique to preserve intact phloem and prepare it for experimentation with invertebrates and microorganisms. The apparatus is called a ‘phloem sandwich’ and allows for the introduction and observation of arthropods, microbes, and other organisms. This technique has resulted in a better understanding of the feeding behaviors, life-history traits, reproduction, development, and interactions of organisms within tree phloem. The strengths of this technique include the use of inexpensive materials, variability in sandwich size, flexibility to re-open the sandwich or introduce multiple organisms through drilled holes, and the preservation and maintenance of phloem integrity. The phloem sandwich is an excellent educational tool for scientific discovery in both K-12 science courses and university research laboratories.     

UniFrac: a New Phylogenetic Method for Comparing Microbial Communities

We introduce here a new method for computing differences between microbial communities based on phylogenetic information. This method, UniFrac, measures the phylogenetic distance between sets of taxa in a phylogenetic tree as the fraction of the branch length of the tree that leads to descendants from either one environment or the other, but not both. UniFrac can be used to determine whether communities are significantly different, to compare many communities simultaneously using clustering and ordination techniques, and to measure the relative contributions of different factors, such as chemistry and geography, to similarities between samples. We demonstrate the utility of UniFrac by applying it to published 16S rRNA gene libraries from cultured isolates and environmental clones of bacteria in marine sediment, water, and ice. Our results reveal that (i) cultured isolates from ice, water, and sediment resemble each other and environmental clone sequences from sea ice, but not environmental clone sequences from sediment and water; (ii) the geographical location does not correlate strongly with bacterial community differences in ice and sediment from the Arctic and Antarctic; and (iii) bacterial communities differ between terrestrially impacted seawater (whether polar or temperate) and warm oligotrophic seawater, whereas those in individual seawater samples are not more similar to each other than to those in sediment or ice samples. These results illustrate that UniFrac provides a new way of characterizing microbial communities, using the wealth of environmental rRNA sequences, and allows quantitative insight into the factors that underlie the distribution of lineages among environments.     

A Peptide-Based Method for 13C Metabolic Flux Analysis in Microbial Communities

The study of intracellular metabolic fluxes and inter-species metabolite exchange for microbial communities is of crucial importance to understand and predict their behaviour. The most authoritative method of measuring intracellular fluxes, ¹³C Metabolic Flux Analysis (¹³C MFA), uses the labeling pattern obtained from metabolites (typically amino acids) during ¹³C labeling experiments to derive intracellular fluxes. However, these metabolite labeling patterns cannot easily be obtained for each of the members of the community. Here we propose a new type of ¹³C MFA that infers fluxes based on peptide labeling, instead of amino acid labeling. The advantage of this method resides in the fact that the peptide sequence can be used to identify the microbial species it originates from and, simultaneously, the peptide labeling can be used to infer intracellular metabolic fluxes. Peptide identity and labeling patterns can be obtained in a high-throughput manner from modern proteomics techniques. We show that, using this method, it is theoretically possible to recover intracellular metabolic fluxes in the same way as through the standard amino acid based ¹³C MFA, and quantify the amount of information lost as a consequence of using peptides instead of amino acids. We show that by using a relatively small number of peptides we can counter this information loss. We computationally tested this method with a well-characterized simple microbial community consisting of two species.     

Role for Glycine Betaine Transport in Vibrio cholerae Osmoadaptation and Biofilm Formation within Microbial Communities

Vibrio cholerae is a halophilic facultative human pathogen found in marine and estuarine environments. Accumulation of compatible solutes is important for growth of V. cholerae at NaCl concentrations greater than 250 mM. We have identified and characterized two compatible solute transporters, OpuD and PutP, that are involved in uptake of glycine betaine and proline by V. cholerae. V. cholerae does not, however, possess the bet genes, suggesting that it is unable to synthesize glycine betaine. In contrast, many Vibrio species are able to synthesize glycine betaine from choline. It has been shown that many bacteria not only synthesize but also secrete glycine betaine. We hypothesized that sharing of compatible solutes might be a mechanism for cooperativity in microbial communities. In fact, we have demonstrated that, in high-osmolarity medium, V. cholerae growth and biofilm development are enhanced by supplementation with either glycine betaine or spent media from other bacterial species. Thus, we propose that compatible solutes provided by other microorganisms may contribute to survival of V. cholerae in the marine environment through facilitation of osmoadaptation and biofilm development.     

Cell Heterogeneity in Staphylococcal Communities

The human pathogen Staphylococcus aureus is a gram-positive bacterium that causes difficult-to-treat infections. One of the reasons why S. aureus is such as successful pathogen is due to the cell-to-cell physiological variability that exists within microbial communities. Many laboratories around the world study the genetic mechanisms involved in S. aureus cell heterogeneity to better understand infection mechanism of this bacterium. It was recently shown that the Agr quorum-sensing system, which antagonistically regulates biofilm-associated or acute bacteremia infections, is expressed in a subpopulation of specialized cells. In this review, we discuss the different genetic mechanism for bacterial cell differentiation and the physiological properties of the distinct cell types that are already described in S. aureus communities, as well as the role that these cell types play during an infection process.     

A Method for Assessing the Level of Microbial Contamination in Surgical Materials

S ummary . A method is described which increases the efficiency of recovery of microorganisms from solid, fibrous and polymer materials. It is suitable for routine assessment of the microbial quality of various medical products.     

Sensitive and Accurate Methodology for Measuring the Kinetics of Concentration-Dependent Hydrocarbon Metabolism Rates in Seawater by Microbial Communities

A method having sufficient sensitivity to resolve the kinetic constants for dissolved nonpolar substrate metabolism, together with the related rate constants in natural waters, is presented. The method is based on the rate of ¹⁴CO₂ recovery from radioactive dissolved substrate. Sensitivity is enhanced by using large seawater volumes, high-specific-activity isotopes, and by reducing background radioactivity. Before use, commercial isotopes are purified by mild alkaline hydrolysis followed by sublimation from base to remove ¹⁴CO₂ as well as interfering polar ¹⁴C-substrates. During sample analysis, chilled Tenax resin is used to remove volatile ¹⁴C-substrate from the nitrogen stream containing ¹⁴CO₂ recovered from substrate oxidation. Chromatographic evidence of purity, shown to be insufficient, is augmented by kinetic data from toluene utilization by mixed cultures and by rates in induced versus noninduced pure cultures. Accuracy is enhanced by using short (<10 h) incubation times and small hydrocarbon concentrations so that the metabolism rates in unamended natural water systems can be evaluated. Toluene metabolism rates in seawater as low as 1 pg/liter per h and at concentrations as low as 20 ng/liter have been determined.     

A Natural View of Microbial Biodiversity within Hot Spring Cyanobacterial Mat Communities

This review summarizes a decade of research in which we have used molecular methods, in conjunction with more traditional approaches, to study hot spring cyanobacterial mats as models for understanding principles of microbial community ecology. Molecular methods reveal that the composition of these communities is grossly oversimplified by microscopic and cultivation methods. For example, none of 31 unique 16S rRNA sequences detected in the Octopus Spring mat, Yellowstone National Park, matches that of any prokaryote previously cultivated from geothermal systems; 11 are contributed by genetically diverse cyanobacteria, even though a single cyanobacterial species was suspected based on morphologic and culture analysis. By studying the basis for the incongruity between culture and molecular samplings of community composition, we are beginning to cultivate isolates whose 16S rRNA sequences are readily detected. By placing the genetic diversity detected in context with the well-defined natural environmental gradients typical of hot spring mat systems, the relationship between gene and species diversity is clarified and ecological patterns of species occurrence emerge. By combining these ecological patterns with the evolutionary patterns inherently revealed by phylogenetic analysis of gene sequence data, we find that it may be possible to understand microbial biodiversity within these systems by using principles similar to those developed by evolutionary ecologists to understand biodiversity of larger species. We hope that such an approach guides microbial ecologists to a more realistic and predictive understanding of microbial species occurrence and responsiveness in both natural and disturbed habitats.     

Microbial Communities in Pre-Columbian Coprolites

The study of coprolites from earlier cultures represents a great opportunity to study an “unaltered” composition of the intestinal microbiota. To test this, pre-Columbian coprolites from two cultures, the Huecoid and Saladoid, were evaluated for the presence of DNA, proteins and lipids by cytochemical staining, human and/or dog-specific Bacteroides spp. by PCR, as well as bacteria, fungi and archaea using Terminal Restriction Fragment analyses. DNA, proteins and lipids, and human-specific Bacteroides DNA were detected in all coprolites. Multidimensional scaling analyses resulted in spatial arrangements of microbial profiles by culture, further supported by cluster analysis and ANOSIM. Differences between the microbial communities were positively correlated with culture, and SIMPER analysis indicated 68.8% dissimilarity between the Huecoid and Saladoid. Proteobacteria, Bacteroidetes and methanogens were found in all coprolite samples. Propionebacteria, Shewanella and lactic acid bacteria dominated in the Huecoid samples, while Acidobacteria, and peptococci were dominant in Saladoid samples. Yeasts, including Candida albicans and Crypotococcus spp. were found in all samples. Basidiomycetes were the most notable fungi in Huecoid samples while Ascomycetes predominated in Saladoid samples, suggesting differences in dietary habits. Our study provides an approach for the study of the microbial communities of coprolite samples from various cultures.     

鸟类组织中稳定同位素周转率研究现状

稳定同位素分析技术被广泛应用于鸟类生态学多个方面的研究,如鸟类迁徙、营养级关系、生活史、食性等。根据不同的研究目的和要求,选择目标周期相对应的组织进行研究。在此前提下,掌握不同组织稳定同位素的周转率就显得尤为重要。目前国内有关鸟类组织稳定同位素周转率及其差异的研究较为有限,本文对国际上研究周转率的常见假说、方法和影响周转率的因素进行综述,旨在抛砖引玉,为利用稳定同位素技术开展鸟类学方面的研究提供依据。     

Aspects of Diversity Measurement for Microbial Communities

A useful measure of diversity was calculated for microbial communities collected from lake water and sediment samples using the Shannon index (H′) and rarefaction [E(S)]. Isolates were clustered by a numerical taxonomy approach in which limited (<20) tests were used so that the groups obtained represented a level of resolution other than species. The numerical value of diversity for each sample was affected by the number of tests used; however, the relative diversity compared among several sampling locations was the same whether 11 or 19 characters were examined. The number of isolates (i.e., sample size) strongly influenced the value of H′ so that unequal sized samples could not be compared. Rarefaction accounts for differences in sample size inherently so that such comparisons are made simple. Due to the type of sampling carried out by microbiologists, H′ is estimated and not determined and therefore requires a statement of error associated with it. Failure to report error provided potentially misleading results. Calculation of the variance of H′ is not a simple matter and may be impossible when handling a large number of samples. With rarefaction, the variance of E(S) is readily determined, facilitating the comparison of many samples.     

Microbial Astronauts: Assembling Microbial Communities for Advanced Life Support Systems

Extension of human habitation into space requires that humans carry with them many of the microorganisms with which they coexist on Earth. The ubiquity of microorganisms in close association with all living things and biogeochemical processes on Earth predicates that they must also play a critical role in maintaining the viability of human life in space. Even though bacterial populations exist as locally adapted ecotypes, the abundance of individuals in microbial species is so large that dispersal is unlikely to be limited by geographical barriers on Earth (i.e., for most environments everything is everywhere given enough time). This will not be true for microbial communities in space where local species richness will be relatively low because of sterilization protocols prior to launch and physical barriers between Earth and spacecraft after launch. Although community diversity will be sufficient to sustain ecosystem function at the onset, richness and evenness may decline over time such that biological systems either lose functional potential (e.g., bioreactors may fail to reduce BOD or nitrogen load) or become susceptible to invasion by human-associated microorganisms (pathogens) over time. Research at the John F. Kennedy Space Center has evaluated fundamental properties of microbial diversity and community assembly in prototype bioregenerative systems for NASA Advanced Life Support. Successional trends related to increased niche specialization, including an apparent increase in the proportion of nonculturable types of organisms, have been consistently observed. In addition, the stability of the microbial communities, as defined by their resistance to invasion by human-associated microorganisms, has been correlated to their diversity. Overall, these results reflect the significant challenges ahead for the assembly of stable, functional communities using gnotobiotic approaches, and the need to better define the basic biological principles that define ecosystem processes in the space environment.     

Disordered Microbial Communities in Asthmatic Airways

Background

A rich microbial environment in infancy protects against asthma [1], [2] and infections precipitate asthma exacerbations [3]. We compared the airway microbiota at three levels in adult patients with asthma, the related condition of COPD, and controls. We also studied bronchial lavage from asthmatic children and controls.

Principal Findings

We identified 5,054 16S rRNA bacterial sequences from 43 subjects, detecting >70% of species present. The bronchial tree was not sterile, and contained a mean of 2,000 bacterial genomes per cm² surface sampled. Pathogenic Proteobacteria, particularly Haemophilus spp., were much more frequent in bronchi of adult asthmatics or patients with COPD than controls. We found similar highly significant increases in Proteobacteria in asthmatic children. Conversely, Bacteroidetes, particularly Prevotella spp., were more frequent in controls than adult or child asthmatics or COPD patients.

Significance

The results show the bronchial tree to contain a characteristic microbiota, and suggest that this microbiota is disturbed in asthmatic airways.     

Simultaneous Rates of Ribonucleic Acid and Deoxyribonucleic Acid Syntheses for Estimating Growth and Cell Division of Aquatic Microbial Communities

A method for measuring rates of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) syntheses using a single radioactive precursor has been devised and tested using bacterial cultures and natural assemblages of marine and freshwater microorganisms. The procedure is based upon the uptake and incorporation of exogenous [³H]adenine into cellular adenosine triphosphate and deoxyadenosine triphosphate pools which serve as the immediate precursors for the adenine incorporated into RNA and DNA, respectively. It is proposed that the DNA/RNA rate ratio is correlated with the specific growth rate of microorganisms and can be used as an index for estimating and comparing the productivities of microbial assemblages in nature. This technique can also be used to detect discontinuous growth and cell division processes which frequently occur in surface plankton populations. The DNA/RNA rate ratios measured in a variety of aquatic ecosystems ranged from 3.3 to 31.8% without significant correlation to total microbial biomass.     

High Metabolic Rates in Beach Cast Communities

Metabolic hotspots at land–water interfaces are important in supporting biogeochemical processes. Here we confirm the generality of land–aquatic interfaces as biogeochemical hot spots by extending this concept to marine beach cast materials. In situ atmospheric pCO_2, from a respiration chamber (10 cm in diameter and 20 cm high) inserted into wrack deposits, was determined using a high-precision (±1 ppm) non-dispersive infrared gas analyzer (EGM-4, PP-systems) at 1 minute recording intervals. The wrack deposits supported high metabolic activities, with CO₂ fluxes averaging (±SE) 6.62 ± 0.88 μmol C m⁻² s⁻¹, compared to median value of 0.98 μmol C m⁻² s⁻¹ (mean 2.21 ± 1.25 μmol C m⁻² s⁻¹) for bare sand adjacent to deposits. Wrack metabolic rates ranged 40-fold across beaches, from a minimum of 0.57 ± 0.22 μmol C m⁻² s⁻¹ to a maximum of 20.8 ± 5.04 μmol C m⁻² s⁻¹, both derived from beaches with deposits dominated by Sargassum. Rates tended to increase significantly (F test, P < 0.05) from the shoreline to reach maximum rates at about 10 m from the shoreline, declining sharply further from the shoreline, and increased with increasing thickness of the deposits (maximum about 10 cm deep), declining for thicker deposits. Wrack differing in composition had similar metabolic rates, although deposits consisting of a mixture of seagrass and algae tended to show somewhat higher rates. Our results show a meter square of wrack deposit supports a metabolic rate equivalent to that supported by 3 m² of living seagrass or macroalgal habitat. In wrack, the marine environment provides organic material and moisture and the land environment provides oxygen to render wrack ecosystems an efficient metabolic reactor. Intense wrack metabolism should also be conducive to organismal growth by supporting the development of a cryptic, but diverse wrack-based food web.     

Control of the Protein Turnover Rates in Lemna minor

The control of protein turnover in Lemna minor has been examined using a method described in the previous paper for determining the rate constants of synthesis and degradation of protein. If Lemna is placed on water, there is a reduction in the rate constants of synthesis of protein and an increase (3- to 6-fold) in the rate constant of degradation. The net effect is a loss of protein from the tissue. Omission of nitrate, phosphate, sulfate, magnesium, or calcium results in increases in the rate constant of degradation of protein.     

Microbial Food Webs in Marine Sediments. I. Trophic Interactions and Grazing Rates in Two Tidal Flat Communities

Abstract The role of grazing by marine sediment flagellates, ciliates, and meiobenthic animals in controlling production of their bacterial and diatom prey was investigated. Several novel or modified techniques were used to enumerate prey (bacteria and diatoms), measure bacterial production, quantify proto- and micrometazoan predators, and evaluate rates of bacterivory and herbivory. The results indicated that, in a temperate, marine intertidal flat composed of fine sand, colorless nanoflagellates, ciliates, and nematodes were the most important bacterivores. Together, these organisms were responsible for removing up to 53% of bacterial production, by grazing. The observed rates of bacterivory were high enough to hypothesize that periods of grazing control of bacterial production might occur regularly in similar habitats. Colorless microflagellates, ciliates, and nematodes had high rates of diatom consumption. The combined small diatom consumption rate was equivalent to 132% of diatom standing stock per day. Trophic interactions between diatoms and micro- and meiobenthos might be a factor limiting growth of small (around 10 μm) diatoms. In coarse sands of an open beach, all micrograzers except pigmented nanoflagellates were rare, whereas bacterial and diatom assemblages were rather abundant and active. In this type of sediment, the micrograzers were able to consume only a marginal percentage of bacterial production (<1%) and diatom standing stock (3.8%), thus playing a minor role in controlling the dynamics of their prey. Received: 11 June 1996; Accepted: 13 August 1996     

Determining Rates of Change and Evaluating Group-Level Resiliency Differences in Hyporheic Microbial Communities in Response to Fluvial Heavy-Metal Deposition

Prior field studies by our group have demonstrated a relationship between fluvial deposition of heavy metals and hyporheic-zone microbial community structure. Here, we determined the rates of change in hyporheic microbial communities in response to heavy-metal contamination and assessed group-level differences in resiliency in response to heavy metals. A controlled laboratory study was performed using 20 flowthrough river mesocosms and a repeated-measurement factorial design. A single hyporheic microbial community was exposed to five different levels of an environmentally relevant metal treatment (0, 4, 8, 16, and 30% sterilized contaminated sediments). Community-level responses were monitored at 1, 2, 4, 8, and 12 weeks via denaturing gradient gel electrophoresis and quantitative PCR using group-specific primer sets for indigenous populations most closely related to the α-, β-, and γ-proteobacteria. There was a consistent, strong curvilinear relationship between community composition and heavy-metal contamination (R² = 0.83; P < 0.001), which was evident after only 7 days of metal exposure (i.e., short-term response). The abundance of each phylogenetic group was negatively affected by the heavy-metal treatments; however, each group recovered from the metal treatments to a different extent and at a unique rate during the course of the experiment. The structure of hyporheic microbial communities responded rapidly and at contamination levels an order of magnitude lower than those shown to elicit a response in aquatic macroinvertebrate assemblages. These studies indicate that hyporheic microbial communities are a sensitive and useful indicator of heavy-metal contamination in streams.