Ribosomal RNA Intergenic Spacer Analysis Research Articles

Enrichment and Analysis of Stable 1,4-dioxane-Degrading Microbial Consortia Consisting of Novel Dioxane-Degraders.

Biodegradation of 1,4-dioxane, a water contaminant of emerging concern, has drawn substantial attention over the last two decades. A number of dioxane-degraders have been identified, though many of them are unable to metabolically utilize 1,4-dioxane. Moreover, it is considered more preferable to use microbial consortia rather than the pure strains, especially in conventional bioreactors for industrial wastewater treatment. In the present study, a stable 1,4-dioxane-degrading microbial consortium was enriched, namely 112, from industrial wastewater by nitrate mineral salt medium (NMSM). The consortium 112 is capable of utilizing 1,4-dioxane as a sole carbon and energy source, and can completely degrade 1,4-dioxane up to 100 mg/L. From the consortium 112, two 1,4-dioxane-degrading bacterial strains were isolated and identified, in which the Variovorax sp. TS13 was found to be a novel 1,4-dioxane-degrader that can utilize 100 mg/L of 1,4-dioxane. The efficacy of the consortium 112 was increased significantly when we cultured the consortium with mineral salt medium (MSM). The new consortium, N112, could utilize 1,4-dioxane at a rate of 1.67 mg/L·h. The results of the ribosomal RNA intergenic spacer analysis (RISA) depicted that changes in the microbial community structure of consortium 112 was the reason behind the improved degradation efficiency of consortium N112, which was exhibited as a stable and effective microbial consortium with a high potential for bioremediation of the dioxane-impacted sites and contaminated industrial wastewater.

Beware biofilm! Dry biofilms containing bacterial pathogens on multiple healthcare surfaces; a multi-centre study

Wet biofilms associated with medical devices have been widely studied and their link with healthcare-associated infections (HCAIs) is well recognized. Little attention has been paid to the presence of dry biofilms on environmental surfaces in healthcare settings. To investigate the occurrence, prevalence, and diversity of dry biofilms on hospital surfaces. Sixty-one terminally cleaned items were received from three different UK hospitals. The presence of dry biofilm was investigated using culture-based methods and scanning electron microscopy (SEM). Bacterial diversity within biofilms was investigated using ribosomal RNA intergenic spacer analysis (RISA)-polymerase chain reaction and next-generation sequencing. Multi-species dry biofilms were recovered from 95% of 61 samples. Abundance and complexity of dry biofilms were confirmed by SEM. All biofilms harboured Gram-positive bacteria including pathogens associated with HCAI; 58% of samples grew meticillin-resistant Staphylococcus aureus. Dry biofilms had similar physical composition regardless of the type of items sampled or the ward from which the samples originated. There were differences observed in the dominance of particular species: dry biofilms from two hospitals contained mostly staphylococcal DNA, whereas more Bacillus spp. DNA was found on surfaces from the third hospital. The presence of dry biofilms harbouring bacterial pathogens is virtually universal on commonly used items in healthcare settings. The role of dry biofilms in spreading HCAIs may be underestimated. The risk may be further exacerbated by inefficient cleaning and disinfection practices for hospital surfaces.

Nitrate Supply-Dependent Shifts in Communities of Root-Associated Bacteria in Arabidopsis.

Root-associated bacterial communities are necessary for healthy plant growth. Nitrate is a signal molecule as well as a major nitrogen source for plant growth. In this study, nitrate-dependent alterations in root-associated bacterial communities and the relationship between nitrate signaling and root-associated bacteria in Arabidopsis were examined. The bacterial community was analyzed by a ribosomal RNA intergenic spacer analysis (RISA) and 16S rRNA amplicon sequencing. The Arabidopsis root-associated bacterial community shifted depending on the nitrate amount and timing of nitrate application. The relative abundance of operational taxonomic units of 25.8% was significantly changed by the amount of nitrate supplied. Moreover, at the family level, the relative abundance of several major root-associated bacteria including Burkholderiaceae, Paenibacillaceae, Bradyrhizobiaceae, and Rhizobiaceae markedly fluctuated with the application of nitrate. These results suggest that the application of nitrate strongly affects root-associated bacterial ecosystems in Arabidopsis. Bulk soil bacterial communities were also affected by the application of nitrate; however, these changes were markedly different from those in root-associated bacteria. These results also suggest that nitrate-dependent alterations in root-associated bacterial communities are mainly affected by plant-derived factors in Arabidopsis. T-DNA insertion plant lines of the genes for two transcription factors involved in nitrate signaling in Arabidopsis roots, NLP7 and TCP20, showed similar nitrate-dependent shifts in root-associated bacterial communities from the wild-type, whereas minor differences were observed in root-associated bacteria. Thus, these results indicate that NLP7 and TCP20 are not major regulators of nitrate-dependent bacterial communities in Arabidopsis roots.

Geographically conserved microbiomes of four temperate water tunicates.

Tunicates are useful models for exploring microbiomes because they have an innate immune system resembling that of chordates. Automated ribosomal RNA intergenic spacer analysis and High-Throughput Sequencing were used to compare the tunic microbiomes of Ciona robusta (formerly Ciona intestinalis type A), Ciona savignyi, Botrylloides leachi and Botryllus schlosseri sampled from three distinct locations with limited genetic connectivity. Bacterial phylotype profiles were conserved within each species, and there were no detectable differences between tunic and tunic + cuticle subsamples from an individual. Bacterial operational taxonomic unit (OTU) diversity was lowest for C. savignyi (320 ± 190 OTUs) and highest for B. schlosseri (1260 ± 190 OTUs). Each species had a distinct set of bacterial OTUs (pseudo-F = 3.0, p > 0.001), with the exception of B. leachi and B. schlosseri from one sampling location (t = 1.2, p = 0.09). Of note were OTUs assigned to Alphaproteobacteria from C. robusta plus Phyllobacteriaceae and Endozoicomonas from C. savignyi. These OTUs contributed 51, 22 and 10% of sequence reads, respectively, and are related to known bacterial symbionts. The within-species conservation of core OTUs across three distinct and co-occurring populations of tunicates provides compelling evidence that these tunicates foster defined microbiomes.

124 Ribosomal RNA intergenic spacer analysis (RISA) as a rapid and diagnostic means to profile microbial diversity in cystic fibrosis sputum

123 A shotgun metaproteomics approach to study the faecal microbiome of patients with cystic fibrosis reveals a reduction of butyrate-producing bacteria G. Debyser1, B. Mesuere2, L. Clement2, G. Duytschaever1, P. Van Hecke1, P. Dawyndt2, K. De Boeck3, P. Vandamme1, B. Devreese1. 1Ghent University, Department of Biochemistry and Microbiology, Ghent, Belgium; 2Ghent University, Department of Applied Mathematics and Computer Science, Ghent, Belgium; 3University Hospital of Leuven, Department of Pediatrics, Leuven, Belgium

Characterization of Soil Bacterial Assemblies in Brazilian Savanna-Like Vegetation Reveals Acidobacteria Dominance

The Brazilian Cerrado is the second largest biome in Brazil and is considered a biodiversity hotspot. In this work, we compared the bacterial communities in Cerrado soil associated with four types of native vegetation (Cerrado Denso, Cerrado sensu stricto, Campo Sujo, and Mata de Galeria) by ribosomal RNA intergenic spacer analysis, terminal fragment restriction length polymorphism and pyrosequencing. The fingerprinting results were very similar. The bacterial communities of Cerrado Denso and Cerrado sensu stricto grouped together and were distinct from those in Campo Sujo and Mata de Galeria. Pyrosequencing generated approximately 40,000 16S rRNA gene sequences per sample and allowed the identification of 17 phyla in soil samples under Cerrado vegetation. Acidobacteria were dominant in all areas studied with a relative frequency of 40-47%, followed closely by Proteobacteria accounting for 34-40% of the sequences. Results from all molecular techniques used suggested that the bacterial communities of Cerrado sensu stricto and Cerrado Denso are very similar to each other, while Campo Sujo forms a separate group, and Mata de Galeria is the most distinct with higher species richness. This is the first extensive study of native Cerrado soil microbiota, an important but endangered biome.

Biodiversity of Phototrophic Biofilms Dwelling on Monumental Fountains

Among the stone monumental assets, artistic fountains are particularly affected by microbial colonization due to constant contact with water, giving rise to biodegradation processes related with physical-chemical and aesthetical alterations. In this paper, we make an overview of reported biodiversity of the phototrophic patina developed in various fountains of Italy and Spain. The microbial composition of four fountains (two from Florence, Italy and two from Granada, Spain) was investigated using traditional and/or molecular techniques. The results indicated many common similarities with regard the phototrophic biodiversity for all the investigated fountains. Automated ribosomal RNA intergenic spacer analysis (ARISA), a molecular fingerprint tool, was used to examine the eubacterial and cyanobacterial community for two of the investigated fountains. The principal component analysis of ARISA profiles strengthens the results obtained by traditional methods and revealed separate clusters, as a consequence of the differences of micro-environmental conditions for each fountain.

Organic loading rates affect composition of soil-derived bacterial communities during continuous, fermentative biohydrogen production

Bacterial community composition during steady-state, fermentative H 2 production was compared across a range of organic loading rates (OLRs) of 0.5–19 g COD l −1 h −1 in a 2-l continuous flow reactor at 30 °C. The varied OLRs were achieved with glucose concentrations of 2.5–10 g l −1 and hydraulic retention times of 1–10 h. The synthetic wastewater feed was amended with l-cysteine and maintained at a pH of 5.5. For each run at a given glucose concentration, the reactor was inoculated with an aliquot of well-mixed agricultural topsoil that had been heat-treated to reduce numbers of vegetative cells. At OLRs less than 2 g COD l −1 h −1, DNA sequences from ribosomal RNA intergenic spacer analysis profiles revealed more diverse and variable populations ( Selenomonas, Enterobacter, and Clostridium spp.) than were observed above 2 g COD l −1 h −1 ( Clostridium spp. only). An isolate, LYH1, was cultured from a reactor sample (10 g glucose l −1 at a 10-h HRT) on medium containing l-cysteine. In confirming H 2 production by LYH1 in liquid batch culture, lag periods for H 2 production in the presence and absence of l-cysteine were 5 and 50 h, respectively. The 16S rRNA gene sequence of LYH1 indicated that the isolate was a Clostridium sp. affiliated with RNA subcluster Ic, with >99% similarity to Clostridium sp. FRB1. In fluorescent in situ hybridization tests, an oligonucleotide probe complementary to the 16S rRNA of LYH1 hybridized with 90% of cells observed at an OLR of 2 g COD h −1, compared to 26% of cells at an OLR of 0.5 g COD l −1 h −1. An OLR of 2 g COD l −1 h −1 appeared to be a critical threshold above which clostridia were better able to outcompete Enterobacteriaceae and other organisms in the mixed soil inoculum. Our results are discussed in light of other biohydrogen studies employing pure cultures and mixed inocula.

Characterization of Fe(II) oxidizing bacterial activities and communities at two acidic Appalachian coalmine drainage-impacted sites

We characterized the microbiologically mediated oxidative precipitation of Fe(II) from coalmine-derived acidic mine drainage (AMD) along flow-paths at two sites in northern Pennsylvania. At the Gum Boot site, dissolved Fe(II) was efficiently removed from AMD whereas minimal Fe(II) removal occurred at the Fridays-2 site. Neither site received human intervention to treat the AMD. Culturable Fe(II) oxidizing bacteria were most abundant at sampling locations along the AMD flow path corresponding to greatest Fe(II) removal and where overlying water contained abundant dissolved O(2). Rates of Fe(II) oxidation determined in laboratory-based sediment incubations were also greatest at these sampling locations. Ribosomal RNA intergenic spacer analysis and sequencing of partial 16S rRNA genes recovered from sediment bacterial communities revealed similarities among populations at points receiving regular inputs of Fe(II)-rich AMD and provided evidence for the presence of bacterial lineages capable of Fe(II) oxidation. A notable difference between bacterial communities at the two sites was the abundance of Chloroflexi-affiliated 16S rRNA gene sequences in clone libraries derived from the Gum Boot sediments. Our results suggest that inexpensive and reliable AMD treatment strategies can be implemented by mimicking the conditions present at the Gum Boot field site.

Biogeochemistry of Metalliferous Peats: Sulfur Speciation and Depth Distributions of dsrAB Genes and Cd, Fe, Mn, S, and Zn in Soil Cores

Spatial relationships between concentrations of Cd, Fe, Mn, S, and Zn and bacterial genes for dissimilatory sulfate reduction were studied in soils of the Manning peatland region in western New York. Peat cores were collected within a field exhibiting areas of Zn phytotoxicity, and pH and elemental concentrations were determined with depth. The oxidation states of S were estimated using S-XANES spectroscopy. Soil microbial community DNA was extracted from peat soils for ribosomal RNA intergenic spacer analysis (RISA) of diversity profiles with depth. To assess the presence of sulfate-reducing microorganisms (SRM), DNA extracts were also used as templates for PCR detection of dsrAB genes coding for dissimilatory (bi)-sulfite reductase. Elemental distributions, S redox speciation, and detection of dsrAB genes varied with depth and water content. The pH of peat soils increased with depth. The highest concentrations of Zn, Cd, and S occurred at intermediate depths, whereas Mn concentrations were highest in the topmost peat layers. Iron showed a relatively uniform distribution with depth. Concentrations of redox sensitive elements, S and Mn, but not Fe, seemed to respond to variations in water content and indicated vertical redox stratification in peat cores where topmost peats were typically acidic and oxidizing and deeper peats were typically circumneutral and reducing. Even then, S-XANES analyses showed that surface peats contained >50% of the total S in reduced forms while deep peats contained generally <5% of the total S in oxidized forms. While bacterial RISA profiles of the peats were diverse, dsrAB gene detection followed redox stratification chemistry closely. For the most part, dsrAB genes were detected in deeper peats, where S accumulation was evident, while they were not detected in topmost peat layers where Mn accumulation indicated oxic conditions. Combined chemical, spectroscopic, and microbiological analyses indicated that prolonged exposure to dry-wet cycles resulted in the formation of two redox-stratified zones with distinct chemical and microbiological signatures within peat cores of the Manning peatland region. As illustrated in this study, changes in redox conditions affect bacterial community composition and downward mobility of toxic elements, which has implications for water contamination and the design of metal remediation strategies.

Structure of microbial communities in ethanol biofilters

Despite its central role in biofiltration, the ecology of the microbial community in biofilters remains largely unknown, primarily because of the difficulty of making detailed observations. Recently developed molecular techniques now allow determination of community composition from DNA extracted from the biofilter, without the need to culture the organisms. This study examined the structure of the microbial communities in ethanol biofilters through the application of the molecular fingerprinting technique, automated ribosomal RNA intergenic spacer analysis (ARISA). Three communities were tracked through acclimation to ethanol in liquid culture, and two of these were followed through growth within two biofilters packed with sand or lava rock. One of these created a successful set of biofilters while the other failed because of acidification. Analysis of the fingerprints revealed a strong decrease in species diversity towards the end of the acclimation period. Diversity indices show a rise in diversity for the biofilter with inconsistent removal of ethanol and low diversities in the successful biofilters, which removed ethanol with an efficiency near 80% for both packing materials. The results indicate that community fingerprinting shows promise as a means of assessing biofilter microbial communities.