William E. Holben

Soil biota and exotic plant invasion

Invasive plants are an economic problem and a threat to the conservation of natural systems. Escape from natural enemies might contribute to successful invasion, with most work emphasizing the role of insect herbivores; however, microbial pathogens are attracting increased attention. Soil biota in some invaded ecosystems may promote ‘exotic’ invasion, and plant–soil feedback processes are also important. Thus, relatively rare species native to North America consistently demonstrate negative feedbacks with soil microbes that promote biological diversity, whereas abundant exotic and native species demonstrate positive feedbacks that reduce biological diversity. Here we report that soil microbes from the home range of the invasive exotic plant Centaurea maculosa L. have stronger inhibitory effects on its growth than soil microbes from where the weed has invaded in North America. Centaurea and soil microbes participate in different plant–soil feedback processes at home compared with outside Centaureas home range. In native European soils, Centaurea cultivates soil biota with increasingly negative effects on the weeds growth, possibly leading to its control. But in soils from North America, Centaurea cultivates soil biota with increasingly positive effects on itself, which may contribute to the success of this exotic species in North America.

Phylogenetic analysis of intestinal microflora indicates a novel mycoplasma phylotype in farmed and wild salmon

All studies of the microbial community of the gastrointestinal tract of salmon to date have employed culture-based approaches, typically on pond- or tank-raised, freshwater animals. We present a phylogenetic survey of the bacterial populations present in the distal intestine of salmon from three different marine locations in Europe. This was accomplished through PCR amplification, cloning, and sequencing of partial 16S rDNA genes from microbial community DNA isolated from the contents of the GI tract distal to the pyloric ceca. Using this approach, the intestinal microbial communities of wild salmon from Scotland and pen-raised salmon from Scotland and Norway were compared. The predominating bacterial populations detected were Acinetobacter junii and a novel Mycoplasma phylotype. This Mycoplasma phylotype apparently comprised ~96% of the total microbes in the distal intestine of wild salmon. Substantial differences in intestinal microbial community composition and diversity were observed between the two groups of pen-raised salmon, which, in addition to geographical separation, were raised on different feeds. The microbial profiles found in this study were substantially different from those indicated in earlier culture-based studies for several species of fish, presumably because of the culture-independent techniques employed. Further, analysis of short-chain fatty acids in the digestive tract indicated that the decreasing redox gradient from proximal to distal reaches common to homeothermic animals was absent in salmon, and that the bacterial fermentation levels were much lower than are reported in homeothermic animals.

Percent G+C profiling accurately reveals diet-related differences in the gastrointestinal microbial community of broiler chickens.

ABSTRACT Broiler chickens from eight commercial farms in Southern Finland were analyzed for the structure of their gastrointestinal microbial community by a nonselective DNA-based method, percent G+C-based profiling. The bacteriological impact of the feed source and in-farm whole-wheat amendment of the diet was assessed by percent G+C profiling. Also, a phylogenetic 16S rRNA gene (rDNA)-based study was carried out to aid in interpretation of the percent G+C profiles. This survey showed that most of the 16S rDNA sequences found could not be assigned to any previously known bacterial genus or they represented an unknown species of one of the taxonomically heterogeneous genera, such as Ruminococcus or Clostridium. The data from bacterial community profiling were analyzed byt-test, multiple linear regression, and principal-component statistical approaches. The percent G+C profiling method with appropriate statistical analyses detected microbial community differences smaller than 10% within each 5% increment of the percent G+C profiles. Diet turned out to be the strongest determinant of the cecal bacterial community structure. Both the source of feed and local feed amendment changed the bacteriological profile significantly, whereas profiles of individual farms with identical feed regimens hardly differed from each other. This suggests that the management of typical Finnish farms is relatively uniform or that hygiene on the farm, in fact, has little impact on the structure of the cecal bacterial community. Therefore, feed compounders should have a significant role in the modulation of gut microflora and consequently in prevention of gastrointestinal disorders in farm animals.

Bacterial gene abundances as indicators of greenhouse gas emission in soils

Nitrogen fixing and denitrifying bacteria, respectively, control bulk inputs and outputs of nitrogen in soils, thereby mediating nitrogen-based greenhouse gas emissions in an ecosystem. Molecular techniques were used to evaluate the relative abundances of nitrogen fixing, denitrifying and two numerically dominant ribotypes (based on the ⩾97% sequence similarity at the 16S rRNA gene) of bacteria in plots representing 10 agricultural and other land-use practices at the Kellogg biological station long-term ecological research site. Quantification of nitrogen-related functional genes (nitrite reductase, nirS; nitrous oxide reductase, nosZ; and nitrogenase, nifH) as well as two dominant 16S ribotypes (belonging to the phyla Acidobacteria, Thermomicrobia) allowed us to evaluate the hypothesis that microbial community differences are linked to greenhouse gas emissions under different land management practices. Our results suggest that the successional stages of the ecosystem are strongly linked to bacterial functional group abundance, and that the legacy of agricultural practices can be sustained over decades. We also link greenhouse gas emissions with specific compositional responses in the soil bacterial community and assess the use of denitrifying gene abundances as proxies for determining nitrous oxide emissions from soils.

Culture-Independent Microbial Community Analysis Reveals that Inulin in the Diet Primarily Affects Previously Unknown Bacteria in the Mouse Cecum

ABSTRACT Inulin is a well-known fructose-based prebiotic which has been shown to stimulate the growth of bifidobacteria, a bacterial group generally considered beneficial for intestinal health. In the present study, we analyzed inulin-associated shifts in the total bacterial community of wild-type mice and mice carrying a genetically inactivated adenomatous polyposis coli tumor suppressor gene by using DNA-based approaches independent of bacterial culturability. Mice were fed a high-fat, nonfiber diet with or without inulin inclusion at a 10% (wt/wt) concentration. Cecal contents were analyzed after 0, 3, and 9 weeks on the experimental diets. Inulin inclusion significantly affected the total bacterial community structure of the cecum as determined by both a nonselective percent-guanine-plus-cytosine-based profiling analysis and a more specific 16S ribosomal DNA sequence analysis. The shifts included stimulation of bifidobacteria and suppression of clostridia, but sequence comparison revealed that the major shifts were within previously unknown bacterial taxa. Concomitantly, significantly higher bacterial densities, determined by flow cytometry, were observed with the inulin-amended diet, and the metabolism of the cecal bacterial community was altered, as indicated by higher levels of residual short-chain fatty acids, particularly lactic acid. With regard to all of the microbiological parameters measured, the wild-type mice and mice carrying a genetically inactivated adenomatous polyposis coli tumor suppressor gene were essentially identical. Studies of the implications of pre- and probiotics may need to be expanded to include careful analysis of their effects on the entire microbial community, rather than just a few well-known species. Further studies are needed to increase our understanding of the possible roles of currently unknown gastrointestinal bacteria in health and disease.

Differences in Hyporheic-Zone Microbial Community Structure along a Heavy-Metal Contamination Gradient

ABSTRACT The hyporheic zone of a river is nonphotic, has steep chemical and redox gradients, and has a heterotrophic food web based on the consumption of organic carbon entrained from downwelling surface water or from upwelling groundwater. The microbial communities in the hyporheic zone are an important component of these heterotrophic food webs and perform essential functions in lotic ecosystems. Using a suite of methods (denaturing gradient gel electrophoresis, 16S rRNA phylogeny, phospholipid fatty acid analysis, direct microscopic enumeration, and quantitative PCR), we compared the microbial communities inhabiting the hyporheic zone of six different river sites that encompass a wide range of sediment metal loads resulting from large base-metal mining activity in the region. There was no correlation between sediment metal content and the total hyporheic microbial biomass present within each site. However, microbial community structure showed a significant linear relationship with the sediment metal loads. The abundances of four phylogenetic groups (groups I, II, III, and IV) most closely related to α-, β-, and γ-proteobacteria and the cyanobacteria, respectively, were determined. The sediment metal content gradient was positively correlated with group III abundance and negatively correlated with group II abundance. No correlation was apparent with regard to group I or IV abundance. This is the first documentation of a relationship between fluvially deposited heavy-metal contamination and hyporheic microbial community structure. The information presented here may be useful in predicting long-term effects of heavy-metal contamination in streams and provides a basis for further studies of metal effects on hyporheic microbial communities.

Neighboring plant influences on arbuscular mycorrhizal fungal community composition as assessed by T-RFLP analysis

Controls on root colonization by arbuscular mycorrhizal fungi (AMF) include host nutrient status, identity of symbionts and soil physico-chemical properties. Here we show, in the field, that the subset of the AMF community colonizing the roots of a common grass species, Dactylis glomerata, was strongly controlled by neighboring roots of a different plant species, Centaurea maculosa, an invasive forb, thus adding a biological spatial component to controls on root colonization. Using an AMF-specific, 18s rDNA-based terminal restriction fragment length polymorphism (T-RFLP) analysis method, significant differences were found between AMF community fingerprints of samples derived from roots of grasses with (GCm) and without (G0) neighboring C. maculosa. There were also significant differences between samples derived from C. maculosa roots (Cmac) and both GCm and G0 roots. Sample ordination indicated three generally distinct groups consisting of Cmac, GCm and G0, with GCm samples being of intermediate distance between G0and Cmac. Our results indicate that, with the presence of C. maculosa, AMF communities of D. glomerata shift to reflect community composition associated with C. maculosa roots. These results highlight the importance of complex spatial distributions of AMF communities at the scale of a root system. An additional dimension to our study is that C. maculosa is an aggressively invasive plant in the intermountain West. Viewed in this light, these results suggest that pervasive influences of this plant on AMF communities, specifically in roots of its competitors, may represent a mechanism contributing to its invasive success. However, further work is clearly required to determine the extent to which AMF genotypic alteration by neighboring plants influences competitive relationships.

DNA‐based monitoring of total bacterial community structure in environmental samples

Determining the structure of bacterial communities and their response to stimuli is key to understanding community function and the interactions that occur between microorganisms and the environment. However, bacterial communities often comprise complex assemblages of large numbers of different bacterial populations. An approach is presented which allows bacterial community structure to be determined by fractionation of the complex mixture of total bacterial community DNA using the DNA‐binding dye bisbenzimidazole which imposes G + C‐dependent changes in the buoyant density of DNA. Bacterial community structure presented as percentage of total DNA vs. percentage G + C content of DNA is an indication of the relative abundance of phylogenetic groups of bacteria. Changes in the composition of a soil bacterial community in response to perturbations in the form of carbon amendment and altered water status were monitored.

Spatial Stratification of Soil Bacterial Populations in Aggregates of Diverse Soils

Most soil microbial community studies to date have focused on homogenized bulk soil samples. However, it is likely that many important microbial processes occur in spatially segregated microenvironments in the soil leading to a microscale biogeography. This study attempts to localize specific microbial populations to different fractions or compartments within the soil matrix. Microbial populations associated with macroaggregates and inner- versus total-microaggregates of three diverse soils were characterized using culture-independent, molecular methods. Despite their relative paucity in most surveys of soil diversity, representatives of Gemmatimonadetes and Actinobacteria subdivision Rubrobacteridae were found to be highly abundant in inner-microaggregates of most soils analyzed. By contrast, clones affiliated with Acidobacteria were found to be relatively enriched in libraries derived from macroaggregate fractions of nearly all soils, but poorly represented in inner-microaggregate fractions. Based upon analysis of 16S rRNA, active community members within microaggregates of a Georgian Ultisol were comprised largely of Gemmatimonadetes and Rubrobacteridae, while within microaggregates of a Nebraska Mollisol, Rubrobacteridae and Alphaproteobacteria were the predominant active bacterial lineages. This work suggests that microaggregates represent a unique microenvironment that selects for specific microbial lineages across disparate soils.

GC Fractionation Enhances Microbial Community Diversity Assessment and Detection of Minority Populations of Bacteria by Denaturing Gradient Gel Electrophoresis

ABSTRACT Effectively and accurately assessing total microbial community diversity is one of the primary challenges in modern microbial ecology. This is particularly true with regard to the detection and characterization of unculturable populations and those present only in low abundance. We report a novel strategy, GC fractionation combined with denaturing gradient gel electrophoresis (GC-DGGE), which combines mechanistically different community analysis approaches to enhance assessment of microbial community diversity and detection of minority populations of microbes. This approach employs GC fractionation as an initial step to reduce the complexity of the community in each fraction. This reduced complexity facilitates subsequent detection of diversity in individual fractions. DGGE analysis of individual fractions revealed bands that were undetected or only poorly represented when total bacterial community DNA was analyzed. Also, directed cloning and sequencing of individual bands from DGGE lanes corresponding to individual G+C fractions allowed detection of numerous phylotypes that were not recovered using a traditional random cloning and sequencing approach.