Archaeal diversity in deep-sea sediments estimated by means of different Terminal-Restriction Fragment Length Polymorphisms (T-RFLP) protocols

Despite the increasing recognition of the quantitative importance of Archaea in all marine systems, the protocols for a rapid estimate of Archaeal diversity patterns in deep-sea sediments have been only poorly tested yet. We collected sediment samples from 11 deep-sea sites covering a wide latitudinal range (from 79°N to 36°N, at depths comprised from 469 to 5500 m) and compared the performance of two different primer sets (ARCH21f/ARCH958r and ARCH109f/ARCH 915r) and three restriction enzymes (AluI, Rsa I and HaeIII) for the fingerprinting analysis (T-RFLP) of Archaeal diversity. In silico and experimental analyses consistently indicated that different combinations of primer sets and restriction enzymes can result in different values of benthic Archaeal ribotype richness and different Archaeal assemblage compositions. The use of the ARCH109f/ARCH 915r primer set in combination with AluI provided the best results (a number ribotypes up to 4-folds higher than other combinations), suggesting that this primer set should be used in future studies dealing with the analysis of the patterns of Archaeal diversity in deep-sea sediments. Multivariate, multiple regression analysis revealed that, whatever the T-RFLP protocol utilized, latitude and temperature explained most of the variance in benthic Archaeal ribotype richness, while water depth had a negligible role

Contrasting arbuscular mycorrhizal communities colonizing different host plants show a similar response to a soil phosphorus concentration gradient

High soil phosphorus (P) concentration is frequently shown to reduce root colonization by arbuscular mycorrhizal (AM) fungi, but the influence of P on the diversity of colonizing AM fungi is uncertain. We used terminal restriction fragment length polymorphism (T-RFLP) of 18S rDNA and cloning to assess diversity of AM fungi colonizing maize (Zea mays), soybean (Glycene max) and field violet (Viola arvensis) at three time points in one season along a P gradient of 10–280 mg l−1 in the field. Percentage AM colonization changed between sampling time points but was not reduced by high soil P except in maize. There was no significant difference in AM diversity between sampling time points. Diversity was reduced at concentrations of P > 25 mg l−1, particularly in maize and soybean. Both cloning and T-RFLP indicated differences between AM communities in the different host species. Host species was more important than soil P in determining the AM community, except at the highest P concentration. Our results show that the impact of soil P on the diversity of AM fungi colonizing plants was broadly similar, despite the fact that different plants contained different communities. However, subtle differences in the response of the AM community in each host were evident

Bacterial activity in cystic fibrosis lung infections

Abstract Background Chronic lung infections are the primary cause of morbidity and mortality in Cystic Fibrosis (CF) patients. Recent molecular biological based studies have identified a surprisingly wide range of hitherto unreported bacterial species in the lungs of CF patients. The aim of this study was to determine whether the species present were active and, as such, worthy of further investigation as potential pathogens. Methods Terminal Restriction Fragment Length Polymorphism (T-RFLP) profiles were generated from PCR products amplified from 16S rDNA and Reverse Transcription Terminal Restriction Fragment Length Polymorphism (RT-T-RFLP) profiles, a marker of metabolic activity, were generated from PCR products amplified from 16S rRNA, both extracted from the same CF sputum sample. To test the level of activity of these bacteria, T-RFLP profiles were compared to RT-T-RFLP profiles. Results Samples from 17 individuals were studied. Parallel analyses identified a total of 706 individual T-RF and RT-T-RF bands in this sample set. 323 bands were detected by T-RFLP and 383 bands were detected by RT-T-RFLP (statistically significant; P ≤ 0.001). For the group as a whole, 145 bands were detected in a T-RFLP profile alone, suggesting metabolically inactive bacteria. 205 bands were detected in an RT-T-RFLP profile alone and 178 bands were detected in both, suggesting a significant degree of metabolic activity. Although Pseudomonas aeruginosa was present and active in many patients, a low occurrence of other species traditionally considered to be key CF pathogens was detected. T-RFLP profiles obtained for induced sputum samples provided by healthy individuals without CF formed a separate cluster indicating a low level of similarity to those from CF patients. Conclusion These results indicate that a high proportion of the bacterial species detected in the sputum from all of the CF patients in the study are active. The widespread activity of bacterial species in these samples emphasizes the potential importance of these previously unrecognized species within the CF lung.</p

Nitrogen removal and ammonia-oxidising bacteria in a vertical flow constructed wetland treating inorganic wastewater

Nitrogen removal performance and the ammonia-oxidising bacterial (AOB) community were assessed in the batch loaded 1.3ha saturated surface vertical flow wetland at CSBP Ltd, a fertiliser and chemical manufacturer located in Kwinana, Western Australia. From September 2008 to October 2009 water quality was monitored and sediment samples collected for bacterial analyses. During the period of study the wetland received an average inflow of 1109m3/day with NH3-N = 40mg/L and NO3-N=23mg/L. Effluent NH3-N and NO3-N were on average 31mg/L and 25mg/L respectively. The overall NH3-N removal rate for the period was 1.2g/m2/d indicating the nitrifying capacity of the wetland. The structure of the AOB community was analysed using group specific primers for the ammonia monooxygenase gene (amoA) by terminal restriction fragment length polymorphism (T-RFLP) and by clone libraries to identify key members. The majority of sequences obtained were most similar to Nitrosomonas sp. while Nitrosospira sp. was less frequent. Another two vertical flow wetlands, 0.8ha each, were commissioned at CSBP in July 2009, since then the wetland in this study has received nitrified effluent from these two new cells

Cellulosomics, a gene-centric approach to investigating the intraspecific diversity and adaptation of Ruminococcus flavefaciens within the rumen

Peer reviewedPublisher PD

Comparison of bacterioneuston and bacterioplankton dynamics during a phytoplankton bloom in a fjord mesocosm

The bacterioneuston is the community of Bacteria present in surface microlayers, the thin surface film that forms the interface between aquatic environments and the atmosphere. In this study we compared bacterial cell abundance and bacterial community structure of the bacterioneuston and the bacterioplankton (from the subsurface water column) during a phytoplankton bloom mesocosm experiment. Bacterial cell abundance, determined by flow cytometry, followed a typical bacterioplankton response to a phytoplankton bloom, with Synechococcus and high nucleic acid (HNA) bacterial cell numbers initially falling, probably due to selective protist grazing. Subsequently HNA and low nucleic acid (LNA) bacterial cells increased in abundance but Synechococcus did not. There was no significant difference between bacterioneuston and bacterioplankton cell abundances during the experiment. Conversely, distinct and consistent differences between the bacterioneuston and the bacterioplankton community structure were observed. This was monitored simultaneously by Bacteria 16S rRNA gene terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE). The conserved patterns of community structure observed in all of the mesocosms indicate that the bacterioneuston is distinctive and non-random

Microbial diversity in Baltic Sea sediments

This thesis focuses on microbial community structures and their functions in Baltic Sea sediments. First we investigated the distribution of archaea and bacteria in Baltic Sea sediments along a eutrophication gradient. Community profile analysis of 16S rRNA genes using terminal restriction length polymorphism (T-RFLP) indicated that archaeal and bacterial communities were spatially heterogeneous. By employing statistical ordination methods we observed that archaea and bacteria were structured and impacted differently by environmental parameters that were significantly linked to eutrophication. In a separate study, we analyzed bacterial communities at a different site in the Baltic Sea that was heavily contaminated with polyaromatic hydrocarbons (PAHs) and several other pollutants. Sediment samples were collected before and after remediation by dredging in two consecutive years. A polyphasic experimental approach was used to assess growing bacteria and degradation genes in the sediments. The bacterial communities were significantly different before and after dredging of the sediment. Several isolates collected from contaminated sediments showed an intrinsic capacity for degradation of phenanthrene (a PAH model compound). Quantititative real-time PCR was used to monitor the abundance of degradation genes in sediment microcosms spiked with phenanthrene. Although both xylE and phnAc genes increased in abundance in the microcosms, the isolates only carried phnAc genes. Isolates with closest 16S rRNA gene sequence matches to Exigobacterium oxidotolerans, a Pseudomonas sp. and a Gammaproteobacterium were identified by all approaches used as growing bacteria that are capable of phenanthrene degradation. These isolates were assigned species and strain designations as follows: Exiguobacterium oxidotolerans AE3, Pseudomonas fluorescens AE1 and Pseudomonas migulae AE2. We also identified and studied the distribution of actively growing bacteria along red-ox profiles in Baltic Sea sediments. Community structures were found to be significantly different at different red-ox depths. Also, according to multivariate statistical ordination analysis organic carbon, nitrogen, and red-ox potential were crucial parameters for structuring the bacterial communities on a vertical scale. Novel lineages of bacteria were obtained by sequencing 16S rRNA genes from different red-ox depths and sampling stations indicating that bacterial diversity in Baltic Sea sediments is largely unexplored

Arbuscular mycorrhizal fungi in organic systems

Arbuscular mycorrhizal fungi (AMF) are potential contributors to plant nutrition and pathogen suppression in low input agricultural systems, although individual species of AMF vary widely in their functional attributes. Recent studies at HRI and elsewhere have suggested that in some agricultural systems inoculum of AMF is substantially lower under conventional management relative to that under organic management. Further studies have suggested that conventional management selects AMF communities with limited benefits to their plant hosts relative to those in organic systems. There is a need to investigate the generality of these findings, and their implications for the productivity of organic systems, particularly during the period following conversion to organic management. The current project was designed to pull together existing understanding of the role, and potential role, of AMF in organic systems, and to identify sites and develop methods for use in a subsequent research programme. The project had three objectives: 01 To deliver a literature review covering current knowledge of the role of AMF in conventional and organic agricultural systems. The review considered the ways in which management influences the structure and functioning of AMF communities, including their contributions under conventional and organic management, and recommendations for future research needs. 02 To establish the extent of differences in AMF inoculum between organic and conventional systems, covering a range of management practices. Paired organic and conventional fields at 12 sites from across England were selected to investigate the relationships between management, AMF communities and soil chemistry. Organic and conventionally managed soils showed no significant difference in soil chemical properties (Organic C, total N, total P, extractable P, K, Mg). However, organically managed soils had greater AMF spore numbers and root colonisation potential, and therefore higher AMF inoculum potential, than conventionally managed soil. The relative difference in AMF spore numbers between organic and conventionally managed fields increased with time since conversion. Differences in AMF inoculum potential between organic and conventionally managed fields, and between farm sites, could not be related to differences in soil chemistry. 03 To develop a method suitable for characterising AM fungus communities in soil libraries, based on 18S rRNA terminal restriction fragment length polymorphism (T-RFLP) T-RFLP was shown to provide a rapid semi-quantitative method for analysis of AMF community diversity. However it was clear that primers currently used to amplify AMF are selective and do not allow diversity of the whole AMF community to be determined. Additionally these primers amplify contaminant fungi which need to be removed from the T-RFLP profile prior to analysis. However, contaminant diversity was shown to be low. The project has identified sites and techniques which could be valuable in future research to study the role of AMF under organic management. The study has also highlighted a number of key areas in which further research is needed in order to harness AMF to improve sustainability and productivity of organic and other agricultural systems. In particular, there is a need to determine the extent to which AMF diversity varies between organic and conventional management, the rate and mechanisms by which AMF diversity increases following conversion to organic production, the relationships between AMF diversity and crop nutrition/ pathogen control, and the soil factors controlling the effectiveness of AMF inoculum

Stable isotope probing: Technical considerations when resolving ¹⁵N-labeled RNA in gradients

RNA based stable isotope probing (SIP) facilitates the detection and identification of active members of microbial populations that are involved in the assimilation of an isotopically labeled compound. ¹⁵N-RNA-SIP is a new method that has been discussed in recent literature but has not yet been tested. Herein, we define the limitations to using ¹⁵N-labeled substrates for SIP and propose modifications to compensate for some of these shortcomings. We have used ¹⁵N-RNA-SIP as a tool for analysing mixed bacterial populations that use nitrogen substrates. After incubating mixed microbial communities with ¹⁵N-ammonium chloride or ¹⁵N₂ we assessed the fractionation resolution of ¹⁵N-RNA by isopycnic centrifugation in caesium trifluoroacetate (CsTFA) gradients. We found that the more isotopic label incorporated, the further the buoyant density (BD) separation between ¹⁵N- and ¹⁴N-RNA, however it was not possible to resolve the labeled from unlabeled RNA definitively through gradient fractionation. Terminal-restriction fragment length polymorphism (T-RFLP) analysis of the extracted RNA and fluorescent in situ hybridisation (FISH) analysis of the enrichment cultures provided some insight into the organisms involved in nitrogen fixation. This approach is not without its limitations and will require further developments to assess its applicability to other nitrogen-fixing environments