Vegetation cover of forest, shrub and pasture strongly influences soil bacterial community structure as revealed by 16S rRNA gene T-RFLP analysis

Bacterial community structure is influenced by vegetation, climate and soil chemical properties. To evaluate these influences, terminal restriction fragment length polymorphism (T-RFLP) and cloning of the 16S rRNA gene were used to analyze the soil bacterial communities in different ecosystems in southwestern China. We compared (1) broad-leaved forest, shrub and pastures in a high-plateau region, (2) three broad-leaved forests representing a climate gradient from high-plateau temperate to subtropical and tropical regions and (3) the humus and mineral soil layers of forests, shrub lands and pastures with open and restricted grazing activities, having varied soil carbon and nutrient contents. Principal component analysis of the T-RFLP patterns revealed that soil bacterial communities of the three vegetation types were distinct. The broad-leaved forests in different climates clustered together, and relatively minor differences were observed between the soil layers or the grazing regimes. Acidobacteria dominated the broad-leaved forests (comprising 62% of the total clone sequences), but exhibited lower relative abundances in the soils of shrub (31%) and pasture (23%). Betaproteobacteria was another dominant taxa of shrub land (31%), whereas Alpha- (19%) and Gammaproteobacteria (13%) and Bacteriodetes (16%) were major components of pasture. Vegetation exerted more pronounced influences than climate and soil chemical properties.     

Evaluation of terminal-restriction fragment length polymorphism analysis in contrasting marine environments

Terminal-restriction fragment length polymorphism (T-RFLP) analysis is widely used in microbial ecology studies. In the present study, T-RFLP analysis of PCR products digested by five restriction enzymes (AluI, HaeIII, MspI, Sau3AI and TaqI) was applied for 20 samples from three contrasting coastal environments to assess the biases associated with the choice of enzyme digestion and T-RF analysis. The five enzyme digestions produced highly variable species richness (in terms of number of T-RFs). Analysis of peak areas with a threshold of 0.5% of the total peak area, which recovered 92-96% of the total peak area, revealed different diversity indexes from the five enzyme digestions. Multidimensional scaling, based on matrices that were generated by scoring peak presence/absence and area, revealed similar bacterial community structure patterns among the 20 samples, regardless of the choice of restriction enzymes. Our results strongly argue that the choice of different digestion enzymes in the T-RFLP technique generated valid and consistent bacterial community structures but highly variable species richness and diversity indices. The biases associated with the choice of digestion enzymes needs to be evaluated carefully or at least to be addressed when using T-RFLP analysis.     

Diversity of aquatic prokaryotic communities in the Cuatro Cienegas basin

The Cuatro Cienegas basin (Coahuila, México) is a composite of different water systems in the middle of the desert with unusually high levels of endemism and diversity in different taxa. Although the diversity of macrobiota has been well described, little is known about the diversity and distribution of microorganisms in the oligotrophic ponds. Here we describe the extent and distribution of diversity found in aquatic prokaryotic communities by analysis of terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes and phylogenetic analysis of cloned genes. Twelve locations within the basin were sampled. Among all the samples, we found a total of 117 operational taxonomic units (OTUs) using T-RFLPs, which ranged in any single sample from four to 49. OTU richness and Shannon diversity indices for different sites varied, but none were particularly high. 16S rRNA gene sequence data showed 68 different phylotypes among 198 clones. The most abundant phylotypes were Gamma- and Betaproteobacteria, and extreme halophiles. The differences among sites were significant; 45 TRFs were found only once, and 37% of the total diversity was represented by differences between sites, suggesting high beta-diversity. Further studies are needed to test whether this is a direct consequence of environmental heterogeneity in the basin.     

Bacterial community dynamics during <Emphasis Type="Italic">in-situ</Emphasis> bioremediation of petroleum waste sludge in landfarming sites

In-situ bioremediation of petroleum waste sludge in landfarming sites of Motor Oil Hellas (petroleum refinery) was studied by monitoring the changes of the petroleum composition of the waste sludge, as well as the changes in the structure of the microbial community, for a time period of 14 months. The analyses indicated an enhanced degradation of the petroleum hydrocarbons in the landfarming areas. A depletion of n-alkanes of approximately 75–100% was obtained. Marked changes of the microbial communities of the landfarms occurred concomitantly with the degradation of the petroleum hydrocarbons. The results obtained from terminal restriction fragment length polymorphism (T-RFLP) analysis of polymerase chain reaction (PCR) amplified 16S rRNA genes demonstrated that bacteria originating from the refinery waste sludge and newly selected bacteria dominated the soil bacterial community during the period of the highest degradation activity. However, the diversity of the microbial community was decreased with increased degradation of the petroleum hydrocarbons contained in the landfarms. T-RFLP fingerprints of bacteria of the genera Enterobacter and Ochrobactrum were detected in the landfarmed soil over the entire treatment period of 14 months. In contrast, the genus Alcaligenes appeared in significant numbers only within the 10 month old landfarmed soil. Genes encoding catechol 2,3-dioxygenase (subfamily I.2.A) were detected only in DNA of the untreated refinery waste sludge. However, none of the genes known to encode the enzymes alkane hydroxylase AlkB, catechol 2,3-dioxygenase (subfamily I.2.A) and naphthalene dioxygenase nahAc could be detected in DNA of the landfarmed soils.     

Endophytic and ectophytic potato-associated bacterial communities differ in structure and antagonistic function against plant pathogenic fungi

Differences between endophytic and ectophytic bacterial communities with stress on antagonistic bacteria, were studied by comparing the composition of communities isolated from the rhizosphere, phyllosphere, endorhiza and endosphere of field-grown potato plants using a multiphasic approach. Terminal restriction fragment length polymorphism analysis of 16S rDNA of the bacterial communities revealed discrete microenvironment-specific patterns. To measure the antagonistic potential of potato-associated bacteria, a total of 2648 bacteria were screened by dual testing of antagonism to the soilborne pathogens Verticillium dahliae and Rhizoctonia solani. Composition and diversity of bacterial antagonists were mainly specific for each microenvironment. The rhizosphere and endorhiza were the main reservoirs for antagonistic bacteria and showed the highest similarity in their colonisation by antagonists. The most prominent species of all microenvironments was Pseudomonas putida, and rep-PCR with BOX primers showed that these isolates showed microenvironment-specific DNA fingerprints. P. putida isolates from the rhizosphere and endorhiza gave nearly identical fingerprints confirming the high similarity of bacterial populations. The phlD gene, involved in the production of the antibiotic 2,4-diacetyl-phloroglucinol, was found only among Pseudomonas isolates from the rhizosphere and endorhiza. Evaluation of the bacterial isolates for biocontrol potential based on fungal antagonism and physiological characteristics resulted in the selection of five promising isolates from each microenvironment. The most effective isolate was Serratia plymuthica 3Re4-18 isolated from the endorhiza.     

Impacts of warming and fertilization on nitrogen-fixing microbial communities in the Canadian High Arctic

The impacts of simulated climate change (warming and fertilization treatments) on diazotroph community structure and activity were investigated at Alexandra Fiord, Ellesmere Island, Canada. Open Top Chambers, which increased growing season temperatures by 1-3 degrees C, were randomly placed in a dwarf-shrub and cushion-plant dominated mesic tundra site in 1995. In 2000 and 2001 20N:20P2O5:20K2O fertilizer was applied at a rate of 5 gm(-2) year(-1). Estimates of nitrogen fixation rates were made in the field by acetylene reduction assays (ARA). Higher rates of N fixation were observed 19-35 days post-fertilization but were otherwise unaffected by treatments. However, moss cover was significantly positively associated with ARA rate. NifH gene variants were amplified from bulk soil DNA and analyzed by terminal restriction fragment length polymorphism analysis. Non-metric multidimensional scaling was used to ordinate treatment plots in nifH genotype space. NifH gene communities were more strongly structured by the warming treatment late in the growing season, suggesting that an annual succession in diazotroph community composition occurs.     

Environmental factors have a strong impact on the composition and diversity of the gut bacterial community of Chinese black honeybees

The Chinese black honeybees is an ecotype of the European honeybee that is formed by the natural hybridization of Apis mellifera mellifera and A. m. carnica. It is distributed in nature reserves in North China and has been an important breeding resource for disease resistance and other desirable traits. Compared to the areas outside of reserves, the nature reserves offer significant biodiversity benefits not only to the Chinese black honey bees but also to the other valuable plants and animals. In recent years there has been growing evidence that environmental factors including food choices play an important role in shaping the composition and activity of gut microbiota, which in turn can impact host health. In the previous studies on Chinse black honeybees, little attention has been paid to the diverse population of microbes in the gut that play a vital role in host health. In order to achieve a better understanding on the role of environmental factors in diversity and composition of gut microbiota of honey bees, in the present study, we analyzed the gut bacterial communities of Chinese black honeybees using terminal restriction fragment length polymorphism (T-RFLP). The results showed that the samples from the national nature reserves that are protected and managed so as to preserve and enrich their natural condition and resources for Chinese black honeybees had higher variety and richness of gut bacteria than that collected from unreserved regions that also harbor populations of Chinese black honeybees. The four terminal restriction fragments (T-RFs), 201, 223, 247 and 320 bp, were identified to be the dominant bacteria of Chinese black honeybees. Of which 247 and 320 bp had greater differences between bee groups sampled in different regions and therefore could be used as genetic markers to separate samples collected from the national nature reserves to samples collected from unreserved regions. The results clearly indicate that national nature reserve protects biological diversity and ecological and evolutionary processes which have had a significant influence on the diversity of gut bacteria of Chinese black honeybees. The ubiquity of gut symbiotic bacteria identified in Chinese black honeybee suggests that environmental factors could play an important role in diversity and composition of gut bacteria and warrant further investigation into the functional significance of these gut bacteria for the honeybee health.     

Bacterial community structures in honeybee intestines and their response to two insecticidal proteins

In this study, the effects of the Bt-toxin Cry1Ab and a soybean trypsin inhibitor (SBTI) on intestinal bacterial communities of adult honeybees (Apis mellifera) were investigated. It was hypothesized that changes in intestinal bacterial communities of honeybees may represent a sensitive indicator for altered intestinal physiology. Honeybees were fed in a laboratory set-up with maize pollen from the Bt-transgenic cultivar MON810 or from the non-transgenic near isoline. Purified Cry1Ab (0.0014% w/v) and SBTI (0.1% or 1% w/v) represented supplementary treatments. For comparison, free-flying honeybees from two locations in Switzerland were analysed. PCR-amplification of bacterial 16S rRNA gene fragments and terminal restriction fragment length polymorphism analyses revealed a total of 17 distinct terminal restriction fragments (T-RFs), which were highly consistent between laboratory-reared and free-flying honeybees. The T-RFs were affiliated to Alpha-, Beta-, and Gammaproteobacteria, to Firmicutes, and to Bacteriodetes. Neither Bt-maize pollen nor high concentrations of Cry1Ab significantly affected bacterial communities in honeybee intestines. Only the high concentration of SBTI significantly reduced the number of T-RFs detected in honeybee midguts, a concentration that also increases bee mortality. Therefore, total bacterial community structures may not be a sensitive indicator for providing evidence for the impact of insecticidal proteins on honeybees at sublethal levels.     

The relationship between microbiota and polyamine concentration in the human intestine: a pilot study

The fecal microbiota of 10 hospitalized elderly subjects and 14 healthy adults were analyzed by terminal-restriction fragment length polymorphism (T-RFLP) analysis using Hha I, Msp I, Hae III, and Alu I, as well as fecal polyamine (PA) concentration. The T-RFLP profiles of the fecal microbiota of the subjects were roughly divided into 2 clusters-I (9 out of 11 were derived from hospitalized elderly subjects) and II (12 out of 13 were derived from healthy adults). The average concentration of putrescine in Cluster II was 5.8 times higher than that of putrescine in Cluster I (P=0.0015). Using a phylogenetic assignment database for T-RFLP analysis of human colonic microbiota, the terminal-restriction fragments (T-RFs) characteristically detected in the case of subjects with high fecal PA concentration were predicted to be derived from bacterial species and phylotypes belonging to Clostridium subcluster XIVa, particularly including Clostridium xylanolyticum, Clostridium saccharolyticum, the uncultured human intestinal bacterium clone JW1H4 (a relative of Desulfotomaculum guttoideum), Roseburia intestinalis, the uncultured bacterium clone 41F10 (a relative of Eubacterium ramulus), Roseburia cecicola, Ruminococcus obeum and its relatives. From these results, we concluded that fecal microbiota may be linked with fecal PA concentration and that some bacterial species belonging to Clostridium subcluster XIVa may play a major role in the control of intestinal PA concentration in humans.