Colin R. Jackson

Structural and Functional Changes with Depth in Microbial Communities in a Tropical Malaysian Peat Swamp Forest

Tropical peat swamp forests are important and endangered ecosystems, although little is known of their microbial diversity and ecology. We used molecular and enzymatic techniques to examine patterns in prokaryotic community structure and overall microbial activity at 0-, 10-, 20-, and 50-cm depths in sediments in a peat swamp forest in Malaysia. Denaturing gradient gel electrophoresis profiles of amplified 16S ribosomal ribonucleic acid (rRNA) gene fragments showed that different depths harbored different bacterial assemblages and that Archaea appeared to be limited to the deeper samples. Cloning and sequencing of longer 16S rRNA gene fragments suggested reduced microbial diversity in the deeper samples compared to the surface. Bacterial clone libraries were largely dominated by ribotypes affiliated with the Acidobacteria, which accounted for at least 27–54% of the sequences obtained. All of the sequenced representatives from the archaeal clone libraries were Crenarchaeota. Activities of microbial extracellular enzymes involved in carbon, nitrogen, and phosphorus cycling declined appreciably with depth, the only exception being peroxidase. These results show that tropical peat swamp forests are unusual systems with microbial assemblages dominated by members of the Acidobacteria and Crenarchaeota. Microbial communities show clear changes with depth, and most microbial activity is likely confined to populations in the upper few centimeters, the site of new leaf litter fall, rather than the deeper, older, peat layers.

Effects of salinity and nutrients on microbial assemblages in Louisiana wetland sediments

To evaluate the effects of saltwater intrusion and nutrient enrichment on wetland microbial communities, we measured changes in sediment microbial structure and function in response to increased salinity and nutrients. Sediments were collected from a cypress-tupelo swamp near Lake Pontchartrain, Louisiana, USA, and maintained in microcosms treated with elevated salinity, nitrogen (N), or phosphorus (P). Impacts on bacterial community diversity and composition were determined via molecular techniques, while effects on function were assessed through measurement of extracellular enzyme activity. Salinity increased bacterial diversity, P had no effect, while N reduced diversity. Deltaproteobacteria dominated all treatments, although their representation, along with that of the Alphaproteobacteria and Planctomycetes, was reduced following N addition. P addition reduced the proportion of Alphaproteobacteria, while salinity increased the proportion of Betaproteobacteria. Exposure to elevated salinity also decreased phosphatase and N-acetlyglucosaminidase activity by almost 20%, with less effect on β-glucosidase. P addition had no impact on extracellular enzyme activity. Overall, exposure to elevated salinity depresses microbial function and changes the sediment microbial assemblage. These wetlands are likely N-limited, and while N additions may regenerate plant communities, they also change the structure of the sediment microbial community, decreasing diversity and impacting the mineralization of other nutrients.

Microbiome evolution along divergent branches of the vertebrate tree of life: what is known and unknown

Vertebrates harbour microbes both internally and externally, and collectively, these microorganisms (the ‘microbiome’) contain genes that outnumber the hosts genetic information 10‐fold. The majority of the microorganisms associated with vertebrates are found within the gut, where they influence host physiology, immunity and development. The development of next‐generation sequencing has led to a surge in effort to characterize the microbiomes of various vertebrate hosts, a necessary first step to determine the functional role these communities play in host evolution or ecology. This shift away from a culture‐based microbiological approach, limited in taxonomic breadth, has resulted in the emergence of patterns suggesting a core vertebrate microbiome dominated by members of the bacterial phyla Bacteroidetes, Proteobacteria and Firmicutes. Still, there is a substantial variation in the methodology used to characterize the microbiome, from differences in sample type to issues of sampling captive or wild hosts, and the majority (>90%) of studies have characterized the microbiome of mammals, which represent just 8% of described vertebrate species. Here, we review the state of microbiome studies of nonmammalian vertebrates and provide a synthesis of emerging patterns in the microbiome of those organisms. We highlight the importance of collection methods, and the need for greater taxonomic sampling of natural rather than captive hosts, a shift in approach that is needed to draw ecologically and evolutionarily relevant inferences. Finally, we recommend future directions for vertebrate microbiome research, so that attempts can be made to determine the role that microbial communities play in vertebrate biology and evolution.

Microbial activity and decomposition of fine particulate organic matter in a Louisiana cypress swamp

Abstract Fine particulate organic matter (FPOM) is an important source of C and energy in aquatic systems, but the decomposition of FPOM has rarely been studied. Litterbags were used to study the decomposition of particles in 2 size fractions of FPOM in surface sediments of a cypress swamp in southeastern Louisiana for slightly over 1 y. Particles in both size fractions followed linear decay models, but fine particles (0.25–1 mm) derived from the remains of coarser material decomposed more slowly than very fine particles (0.063–0.25 mm) that probably were derived from a number of sources. The activities of 6 microbial extracellular enzymes involved in the degradation of lignocellulose also were analyzed, and enzymatic decomposition models were developed to relate the decomposition of particles in each size fraction of FPOM to microbial activity. Very fine particles decomposed more rapidly than fine particles, although enzyme activities were lower, suggesting that this material was more efficiently degraded than fine particles. Enzymatic activity also was measured on particles in 2 size fractions of unconfined FPOM in sediment, and the enzymatic decomposition models were used to predict instantaneous mass-loss rates. Unconfined fine particles decomposed 1.5× faster than fine particles confined in litterbags, whereas unconfined very fine particles decomposed 2 to 4× faster than very fine particles confined in litterbags. Confining FPOM within litterbags clearly led to underestimation of decomposition rates, which might be more effectively determined using enzymatic decomposition models. The differences in microbial activity and decomposition rates between particles in different size fractions show that the concept of FPOM as uniform particles <1 mm in diameter is simplistic, and that a more accurate concept would recognize the distinction between different sizes of particles that might be derived from different sources.

Free-Living and Particle-Associated Bacterioplankton in Large Rivers of the Mississippi River Basin Demonstrate Biogeographic Patterns

ABSTRACT The different drainage basins of large rivers such as the Mississippi River represent interesting systems in which to study patterns in freshwater microbial biogeography. Spatial variability in bacterioplankton communities in six major rivers (the Upper Mississippi, Missouri, Illinois, Ohio, Tennessee, and Arkansas) of the Mississippi River Basin was characterized using Ion Torrent 16S rRNA amplicon sequencing. When all systems were combined, particle-associated (>3 μm) bacterial assemblages were found to be different from free-living bacterioplankton in terms of overall community structure, partly because of differences in the proportional abundance of sequences affiliated with major bacterial lineages (Alphaproteobacteria, Cyanobacteria, and Planctomycetes). Both particle-associated and free-living communities ordinated by river system, a pattern that was apparent even after rare sequences or those affiliated with Cyanobacteria were removed from the analyses. Ordination of samples by river system correlated with environmental characteristics of each river, such as nutrient status and turbidity. Communities in the Upper Mississippi and the Missouri and in the Ohio and the Tennessee, pairs of rivers that join each other, contained similar taxa in terms of presence-absence data but differed in the proportional abundance of major lineages. The most common sequence types detected in particle-associated communities were picocyanobacteria in the Synechococcus/Prochlorococcus/Cyanobium (Syn/Pro) clade, while free-living communities also contained a high proportion of LD12 (SAR11/Pelagibacter)-like Alphaproteobacteria. This research shows that while different tributaries of large river systems such as the Mississippi River harbor distinct bacterioplankton communities, there is also microhabitat variation such as that between free-living and particle-associated assemblages.

The Influence of Vegetation on Microbial Enzyme Activity and Bacterial Community Structure in Freshwater Constructed Wetland Sediments

Microorganisms play important roles in wetland ecosystems, but little is known about the influence of wetland plants on microbial community structure and activity. A greenhouse experiment was conducted to study the short-term influence of wetland vegetation on the sediment microbial community. Mesocosms were either planted with Juncus effusus, Carex lurida, or Dichanthelium acuminatum var. acuminatum or remained unvegetated. After eight weeks, sediment samples were taken and assayed for the activity of five microbial extracellular enzymes associated with carbon, nitrogen, and phosphorus cycling. β-1,4-glucosidase, phosphatase, and N-acetylglucosaminidase exhibited similar activity for all vegetation treatments, while the activity of the phenolic-degrading enzymes phenol oxidase and peroxidase was higher in sediments with no vegetation. Denaturing gradient gel electrophoresis and sequencing of partial 16S rRNA genes indicate differences in the sediment bacterial community associated with each plant regime. Acidobacteria, Firmicutes and Proteobacteria were the dominant phyla, although unvegetated sediments contained proportionally fewer Firmicutes and Alphaproteobacteria. This study provides insights into the structure of wetland bacterial communities and suggests that vegetation can influence both bacterial community structure and specific enzyme activity in wetland sediments. Moreover, these influences can occur over a relatively short time and could occur within just a few months of vegetation changes.

Fine Scale Patterns in Microbial Extracellular Enzyme Activity during Leaf Litter Decomposition in a Stream and its Floodplain

Microorganisms mediate the decomposition of leaf-litter through the release of extracellular enzymes. The surfaces of decomposing leaves are both chemically and physically heterogeneous, and spatial patterns in microbial enzyme activity on the litter surface should provide insights into fine-scale patterns of leaf-litter decomposition. Platanus occidentalis leaves were collected from the floodplain of a third-order stream in northern Mississippi, enclosed in individual litter bags, and placed in the stream channel and in the floodplain. Replicate leaves were collected approximately monthly over a 9-month period and assayed for spatial variation in microbial extracellular enzyme activity and rates of organic matter (OM) decomposition. Spatial variation in enzyme activity was measured by sampling 96 small discs (5-mm diameter) cut from each leaf. Discs were assayed for the activity of enzymes involved in lignin (oxidative enzymes) and cellulose (β-glucosidase, cellobiohydrolase) degradation. Rates of OM loss were greater in the stream than the floodplain. Activities of all enzymes displayed high variability in both environments, with severalfold differences across individual leaves, and replicate leaves varied greatly in their distribution of activities. Geostatistical analysis revealed no clear patterns in spatial distribution of activity over time or among replicates, and replicate leaves were highly variable. These results show that fine-scale spatial heterogeneity occurs on decomposing leaves, but the level of spatial variability varies among individual leaves at the measured spatial scales. This study is the first to use geostatistical analyses to analyze landscape patterns of microbial activity on decomposing leaf litter and in conjunction with studies of the microbial community composition and/or substrate characteristics, should provide key insights into the function of these processes.

Phylogenetic Analysis of Bacterial Communities in Different Regions of the Gastrointestinal Tract of Agkistrodon piscivorus, the Cottonmouth Snake

Vertebrates are metagenomic organisms in that they are composed not only of their own genes but also those of their associated microbial cells. The majority of these associated microorganisms are found in the gastrointestinal tract (GIT) and presumably assist in processes such as energy and nutrient acquisition. Few studies have investigated the associated gut bacterial communities of non-mammalian vertebrates, and most rely on captive animals and/or fecal samples only. Here we investigate the gut bacterial community composition of a squamate reptile, the cottonmouth snake, Agkistrodon piscivorus through pyrosequencing of the bacterial 16S rRNA gene. We characterize the bacterial communities present in the small intestine, large intestine and cloaca. Many bacterial lineages present have been reported by other vertebrate gut community studies, but we also recovered unexpected bacteria that may be unique to squamate gut communities. Bacterial communities were not phylogenetically clustered according to GIT region, but there were statistically significant differences in community composition between regions. Additionally we demonstrate the utility of using cloacal swabs as a method for sampling snake gut bacterial communities.

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample.

Total Bacterial Load within Echinacea purpurea, Determined Using a New PCR-based Quantification Method, is Correlated with LPS Levels and In Vitro Macrophage Activity

Our previous studies indicate that the majority of in vitro monocyte/macrophage activation exhibited by extracts of Echinacea depends on bacterial components. In the present study, total bacterial load was determined within E. purpurea samples and ranged from 6.4 × 10(6) to 3.3 × 10(8) bacteria/g of dry plant material. To estimate total bacterial load, we developed a PCR-based quantification method that circumvents the problems associated with nonviable/nonculturable cells (which precludes using plate counts) or the coamplification of mitochondrial or chloroplast DNA with the use of universal bacterial primers (which precludes the use of qPCR). Differences in total bacterial load within Echinacea samples were strongly correlated with the activity (NF-κB activation in THP-1 cells) and content of bacterial lipopolysaccharides within extracts of this plant material. These results add to the growing body of evidence that bacteria within Echinacea are the main source of components responsible for enhancing innate immune function.