Terminal Restriction Fragment Length Polymorphism Results Research Articles

Irrigation frequency alters the abundance and community structure of ammonia-oxidizing archaea and bacteria in a northern Chinese upland soil

Ammonia oxidation, the first and rate-limiting step in nitrification, is a key process in biogeochemical nitrogen cycling. However, little is known about how irrigation affects ammonia oxidizing microorganisms in agroecosystems. In this study, effects of irrigation frequency on soil potential nitrification activity (PNA), the abundance and composition of ammonia-oxidizing archaea (AOA) and bacteria (AOB) were investigated using real-time PCR (qPCR), terminal restriction fragment length polymorphism (T-RFLP), and clone libraries for characterizing the ammonium monooxygenase genes (amoA) in a northern Chinese soil. Results showed that irrigation significantly increased soil PNA and archaeal amoA gene abundance but decreased bacterial amoA gene abundance. Soil PNA was positively correlated with archaeal amoA gene abundance and negatively correlated with bacterial. T-RFLP and PCA results showed that irrigation greatly changed the AOA and AOB community structures by altering the relative abundances of four T-RFs of AOA and four other T-RFs of AOB, respectively. Phylogenetic analysis revealed that all archaeal sequences fell into Group 1.1b, and the bacterial clones were dominated by Nitrosospira-like sequences within Cluster 3a.2. Irrigation induced the appearance of two archaeal and two bacterial OTUs and increased the relative abundance of another two archaeal and four bacterial OTUs, respectively. Additionally, soil moisture, soil pH, NH4+-N, and NO3−-N correlated significantly with the AOA community, and soil pH, total nitrogen (TN), and NO3−-N were significantly correlated with the AOB community. Our results demonstrate that irrigation greatly affected the abundance and community structure of AOA and AOB and that AOA appeared to play a more important role in nitrification in the study soil.

The source of inoculum drives bacterial community structure in Synechocystis sp. PCC6803-based photobioreactors

Cyanobacteria, which grow using solar energy and carbon dioxide, provide an attractive avenue for sustainable production of biomass and biofuel feedstock. Heterotrophic bacteria can colonize photobioreactor (PBR) cultures of cyanobacteria and may affect the productivity of the system. However, little is known about the types of heterotrophic bacteria associated with PBR cultures of cyanobacteria. The objective of this work was to evaluate the heterotrophic communities in PBR cultures of the cyanobacterium Synechocystis sp. PCC6803 using terminal-restriction fragment length polymorphism (T-RFLP) and high-throughput sequencing. To improve the resolution of the heterotrophic bacterial genomic signatures in the PBR communities, we utilized a targeted T-RFLP strategy to remove the T-RFLP signal of Synechocystis sp. PCC6803 from the analysis. For all experiments, the results of high-throughput sequencing were consistent with T-RFLP results. In particular, the inocula contained heterotrophic bacteria despite appearing to be pure cultures by light microscopy or non-targeted T-RFLP. Furthermore, the heterotrophic communities in the PBRs were strongly influenced by the microbial community in the inoculum used to start the PBR.

Influence of the amoxicillin concentration on organics removal and microbial community structure in an anaerobic EGSB reactor treating with antibiotic wastewater

In order to investigate the organics removal and microbial community dynamics in accordance with variable amoxicillin (AMX) concentrations, a 1.47L expanded granular sludge bed (EGSB) reactor was designed and operated for 241days treating with artificial antibiotic wastewater. Furthermore, microbiological community dynamics with increasing AMX concentrations were also investigated using clone libraries and the terminal restriction fragment length polymorphism (T-RFLP) of PCR-amplified 16S rRNA gene. AMX concentration in the reactor was controlled at 19.2mgL−1 at first and then gradually increased to around 241.7mgL−1 from the day 146 to 241. COD and AMX removal efficiencies reached to 85% and 80%, respectively. And T-RFLP results showed that the microbial community structure in the reactor changed with the increased AMX concentrations. Especially for bacteria, T-RFs of 486, 482, 166, 160 and 88bp were dominant at all stages, whereas abundance of T-RFs 91, 163 and 170bp showed remarkable changes. Besides, clone results combined with T-RFLP showed Firmicutes, Bacteroidetes, Cloacimonetes, Ignavibacteriae and Thermotogae were most dominant bacterial groups in the system and some geneses were also expected to be responsible for the degradation of AMX. For achaea, its community structure (T-RFs of 96, 122bp) kept stable in all the stages.

A combined sequence-based and fragment-based characterization of microbial eukaryote assemblages provides taxonomic context for the Terminal Restriction Fragment Length Polymorphism (T-RFLP) method

Microbial eukaryotes in seawater samples collected from two depths (5m and 500m) at the USC Microbial Observatory off the coast of Southern California, USA, were characterized by cloning and sequencing of 18S rRNA genes, as well as DNA fragment analysis of these genes. The sequenced genes were assigned to operational taxonomic units (OTUs), and taxonomic information for the sequence-based OTUs was obtained by comparison to public sequence databases. The sequences were then subjected to in silico digestion to predict fragment sizes, and that information was compared to the results of the T-RFLP method applied to the same samples in order to provide taxonomic context for the environmental T-RFLP fragments. A total of 663 and 678 sequences were analyzed for the 5m and 500m samples, respectively, which clustered into 157 OTUs and 183 OTUs. The sequences yielded substantially fewer taxonomic units as in silico fragment lengths (i.e., following in silico digestion), and the environmental T-RFLP resulted in the fewest unique OTUs (unique fragments). Bray–Curtis similarity analysis of protistan assemblages was greater using the T-RFLP dataset compared to the sequence-based OTU dataset, presumably due to the inability of the fragment method to differentiate some taxa and an inability to detect many rare taxa relative to the sequence-based approach. Nonetheless, fragments in our analysis generally represented the dominant sequence-based OTUs and putative identifications could be assigned to a majority of the fragments in the environmental T-RFLP results. Our empirical examination of the T-RFLP method identified limitations relative to sequence-based community analysis, but the relative ease and low cost of fragment analysis make this method a useful approach for characterizing the dominant taxa within complex assemblages of microbial eukaryotes in large datasets.

Seasonal Diversity of Planktonic Protists in Southwestern Alberta Rivers over a 1-Year Period as Revealed by Terminal Restriction Fragment Length Polymorphism and 18S rRNA Gene Library Analyses

The temporal dynamics of planktonic protists in river water have received limited attention despite their ecological significance and recent studies linking phagotrophic protists to the persistence of human-pathogenic bacteria. Using molecular-based techniques targeting the 18S rRNA gene, we studied the seasonal diversity of planktonic protists in Southwestern Alberta rivers (Oldman River Basin) over a 1-year period. Nonmetric multidimensional scaling analysis of terminal restriction fragment length polymorphism (T-RFLP) data revealed distinct shifts in protistan community profiles that corresponded to season rather than geographical location. Community structures were examined by using clone library analysis; HaeIII restriction profiles of 18S rRNA gene amplicons were used to remove prevalent solanaceous plant clones prior to sequencing. Sanger sequencing of the V1-to-V3 region of the 18S rRNA gene libraries from spring, summer, fall, and winter supported the T-RFLP results and showed marked seasonal differences in the protistan community structure. The spring library was dominated by Chloroplastidae (29.8%), Centrohelida (28.1%), and Alveolata (25.5%), while the summer and fall libraries contained primarily fungal clones (83.0% and 88.0%, respectively). Alveolata (35.6%), Euglenozoa (24.4%), Chloroplastida (15.6%), and Fungi (15.6%) dominated the winter library. These data demonstrate that planktonic protists, including protozoa, are abundant in river water in Southwestern Alberta and that conspicuous seasonal shifts occur in the community structure.

O3-S6.01 Improved diagnostics of bacterial vaginosis with molecular techniques

BackgroundBacterial vaginosis (BV) is a disturbance of the vaginal microflora. BV can cause discharge complaints and lead to pelvic inflammatory disease, ectopic pregnancy and premature birth. We evaluated a combination...

Characterization of psychrotrophic bacterial communities in modified atmosphere-packed meat with terminal restriction fragment length polymorphism

Characterization of psychrotrophic lactic acid bacteria (LAB) and Brochothrix thermosphacta communities is needed to understand the microbial ecology of spoilage of modified atmosphere-packed (MAP) meats. To overcome the limitations of the currently used methods for the characterization of psychrotrophic bacterial communities in meat, we developed a culture-independent, 16S rRNA gene-targeted terminal restriction fragment length polymorphism (T-RFLP) method. An identification library consisting of 100 Gram-positive and 30 Gram-negative meat-associated bacterial strains was set up to identify the terminal restriction fragments derived from the communities. The taxonomic resolution level of the T-RFLP method was in between genus and species within the investigated LAB strains and within family and genus within the investigated Gram-negative strains. The established library was applied to identify the members of bacterial communities in MAP minced meat at the end of the shelf life. The T-RFLP results and plate counts on Man-Rogosa-Sharpe, Violet Red Bile Glucose, and Streptomycin sulfate thallium acetate actidione agars indicated that LAB and B. thermosphacta predominated in meat. The bacterial taxa associated with the T-RFLP results were compared to those identified among plate-grown LAB isolates by numerical ribopattern analysis. Both methods agreed that Leuconostoc spp. and Carnobacterium spp. prevailed in the LAB community in minced meat followed by Lactobacillus algidus, Lactococcus spp. and Weissella spp. Colony identification revealed that Leuconostoc gasicomitatum, L. gelidum, Carnobacterium divergens and C. maltaromaticum were the predominant LAB species. The T-RFLP results were shown to correlate with viable counts of Leuconostoc spp. and B. thermosphacta. The T-RFLP method was found to be a useful tool enabling rapid and high-throughput characterization of psychrotrophic bacteria prevailing in MAP meat.

EFFECT OF POVIDONE IODINE TREATMENT ON BACTERIAL COMMUNITY ASSOCIATED WITH WHITE SHRIMP (LITOPENAEUS VANNAMEI) LARVAE

The efficacy of povidone iodine as a surface disinfectant was assessed in white shrimp (Litopenaeus vannamei) eggs and larvae. Eggs and nauplii were exposed to povidone iodine 20 ppm for 20 seconds. Culture-based method and Terminal Restriction Fragment Length Polymorphism (T-RFLP) were used to monitor the total number of bacteria and diversity of the bacterial community associated with shrimp eggs and larva at each developmental stage. Povidone iodine reduced the total culturable bacteria, especially Vibrio, on eggs and nauplii, as well as the total bacteria in the whole community as estimated by T-RFLP results. Povidone iodine also reduced the diversity of bacterial community and altered the evenness of phylotypes distribution suggesting that the use of povidone iodine as a surface disinfectant in shrimp aquaculture should be reconsidered.

Qualitative and quantitative estimation of host-specific fecal pollution using Bacteroides–Prevotella 16S rRNA genetic markers by T-RFLP and real-time PCR analyses

Rapid and reliable determination of the non-point sources of fecal pollution is a critical issue for the environmental microbiologists all over the world. In this work we evaluated the use of anaerobic bacterial group Bacteroides-Prevotella as an alternative fecal pollution indicator. Terminal restriction fragment length polymorphism (T-RFLP) and real-time polymerase chain reaction (RT-PCR) analyses were used to monitor and quantify human-, cow- and pig-specific fecal contamination in natural river waters. We also included DNA sequence analysis of the identified fecal markers revealed by T-RFLP in order to clarify the specificity of each marker. It was suggested that the most influent peaks for each fecal source could be used to identify the source of fecal pollution. Development of specific probes based on these markers permit to quantify source of contamination by quantitative RT-PCR. Therefore, we combined the T-RFLP results and RT-PCR assay to quantify fecal contamination by certain host. We can conclude that T-RFLP and RT-PCR analyses showed high reproducibility and sensitivity during analyzing real water samples and can be used to identify, track and quantify host-specific bacterial genetic markers in complex natural water environments.

Active bacterial community structure along vertical redox gradients in Baltic Sea sediment

Community structures of active bacterial populations were investigated along a vertical redox profile in coastal Baltic Sea sediments by terminal-restriction fragment length polymorphism (T-RFLP) and clone library analysis. According to correspondence analysis of T-RFLP results and sequencing of cloned 16S rRNA genes, the microbial community structures at three redox depths (179, -64 and -337 mV) differed significantly. The bacterial communities in the community DNA differed from those in bromodeoxyuridine (BrdU)-labelled DNA, indicating that the growing members of the community that incorporated BrdU were not necessarily the most dominant members. The structures of the actively growing bacterial communities were most strongly correlated to organic carbon followed by total nitrogen and redox potentials. Bacterial identification by sequencing of 16S rRNA genes from clones of BrdU-labelled DNA and DNA from reverse transcription polymerase chain reaction showed that bacterial taxa involved in nitrogen and sulfur cycling were metabolically active along the redox profiles. Several sequences had low similarities to previously detected sequences, indicating that novel lineages of bacteria are present in Baltic Sea sediments. Also, a high number of different 16S rRNA gene sequences representing different phyla were detected at all sampling depths.

Broad Distribution and Phylogeny of Anaerobic Endophytes of Cluster XIVa Clostridia in Plant Species Including Crops

Endophytic clostridia present on various plants as obligate anaerobes were surveyed by terminal restriction fragment length polymorphism (TRFLP) analysis specific to the clostridial 16S rRNA gene. Endophytic clostridia were detected in 10 plant types: sugarcane, cultivated rice, corn, tobacco, soybean, bermuda grass, tall fescue, and three mangrove species. Phylogenetically, cluster XIVa clostridia were detected more frequently than cluster I clostridia in aerial parts. Isolation of clostridia from surface-sterilized sugarcane stem validated the TRFLP results. Plant-derived clostridia occupied two unique phylogenetic positions (groups I and II) within cluster XIVa. Most of cluster XIVa clostridia from other sources (e.g., human, animal, and insect intestines) were located outside these groups. Thus two unique groups of cluster XIVa clostridia are widely distributed in plants, including crops. In field-grown soybeans, TRFLP analysis revealed clostridia only in a non-nodulating mutant. Ribosomal intergenic spacer analysis (RISA) showed that the bacterial community in soybean shoot depended partly on the soybean nodulation genotype.

Modulation of Rat Cecal Microbiota by Administration of Raffinose and Encapsulated Bifidobacterium breve

To investigate the effects of administration of raffinose and encapsulated Bifidobacterium breve JCM 1192T cells on the rat cecal microbiota, in a preclinical synbiotic study groups of male WKAH/Hkm Slc rats were fed for 3 weeks with four different test diets: basal diet (group BD), basal diet supplemented with raffinose (group RAF), basal diet supplemented with encapsulated B. breve (group CB), and basal diet supplemented with both raffinose and encapsulated B. breve (group RCB). The bacterial populations in cecal samples were determined by fluorescence in situ hybridization (FISH) and terminal restriction fragment length polymorphism (T-RFLP). B. breve cells were detected only in the RCB group and accounted for about 6.3% of the total cells as determined by FISH analysis. B. breve was also detected only in the RCB group by T-RFLP analysis. This was in contrast to the CB group, in which no B. breve signals were detected by either FISH or T-RFLP. Increases in the sizes of the populations of Bifidobacterium animalis, a Bifidobacterium indigenous to the rat, were observed in the RAF and RCB groups. Principal-component analysis of T-RFLP results revealed significant alterations in the bacterial populations of rats in the RAF and RCB groups; the population in the CB group was similar to that in the control group (group BD). To the best of our knowledge, these results provide the first clear picture of the changes in the rat cecal microbiota in response to synbiotic administration.