Denaturing gradient gel electrophoresis (DGGE) screening of clones prior to sequencing

Whilst cloning and sequencing techniques are used ubiquitously for the identification of novel DNA sequences, the necessity to determine a consensus sequence means that this can be both labour‐intensive and costly. Here we describe a rapid and cost effective method of using denaturing gradient gel electrophoresis (DGGE) for the analysis of large numbers of clones prior to sequencing. This procedure allows for the selection of specific clones, eliminates the need to sequence multiple copies of the same clone, and reduces the likelihood of sequencing recombinant PCR product.     

Microbial communities in contrasting freshwater marsh microhabitats

Heterotrophic microorganisms are widely recognized as crucial components of ecosystems; yet information on their community structure and dynamics in benthic freshwater habitats is notably scarce. Using denaturing gradient gel electrophoresis (DGGE), we determined the composition of bacterial and fungal communities in a freshwater marsh over four seasons. DGGE revealed diverse bacterial communities in four contrasting microhabitats. The greatest compositional differences emerged between water-column and surface-associated bacteria, although communities associated with sediment also differed from those on plant litter and epiphytic biofilms. Sequences of bacterial clones derived from DGGE bands belonged to the Alphaproteobacteria (31%), Actinobacteria (19%) and Bacteriodetes (19%). Betaproteobacteria were notably absent. Fungal clones obtained from leaf litter were mainly Ascomycota , but two members of the Basidiomycota were also identified. Overall, habitat type was the most important factor explaining variation in bacterial communities among samples, whereas temporal patterns in community composition were less pronounced in spite of large seasonal variation in environmental conditions such as temperature. The observed differences among bacterial communities in different microhabitats were not caused by random variation, but rather appeared to be determined by habitat characteristics, as evidenced by largely congruent community profiles of replicate samples taken at 10–100 m distances within the marsh.     

In vitro phenotypic differentiation towards commensal and pathogenic oral biofilms

Commensal oral biofilms, defined by the absence of pathology-related phenotypes, are ubiquitously present. In contrast to pathological biofilms commensal biofilms are rarely studied. Here, the effect of the initial inoculum and subsequent growth conditions on in vitro oral biofilms was studied. Biofilms were inoculated with saliva and grown anaerobically for up to 21 days in McBain medium with or without fetal calf serum (FCS) or sucrose. Pathology-related phenotypes were quantified and the community composition was determined. Biofilms inoculated with pooled saliva or individual inocula were similar. Denaturing gradient gel electrophoresis (DGGE) analysis allowed differentiation of biofilms grown with sucrose, but not with FCS. Lactate production by biofilms was significantly increased by sucrose and protease activity by FCS. McBain grown biofilms showed low activity for both phenotypes. Three clinically relevant in vitro biofilm models were developed and could be differentiated based on pathology-related phenotypes but not DGGE analysis. These models allow analysis of health-to-disease shifts and the effectiveness of prevention measures.     

Cultivation of low-temperature (15°C), anaerobic, wastewater treatment granules

Aims: Anaerobic sludge granules underpin high‐rate waste‐to‐energy bioreactors. Granulation is a microbiological phenomenon involving the self‐immobilization of several trophic groups. Low‐temperature anaerobic digestion of wastes is of intense interest because of the economic advantages of unheated bioenergy production technologies. However, low‐temperature granulation of anaerobic sludge has not yet been demonstrated. The aims of this study were to (i) investigate the feasibility of anaerobic sludge granulation in cold (15°C) bioreactors and (ii) observe the development of methanogenic activity and microbial community structure in developing cold granules. Methods and Results: One mesophilic (R1; 37°C) and two low‐temperature (R2 and R3, 15°C) laboratory‐scale, expanded granular sludge bed bioreactors were seeded with crushed (diameter <0·4 mm) granules and were fed a glucose‐based wastewater for 194 days. Bioreactor performance was assessed by chemical oxygen demand removal, biogas production, granule growth and temporal methanogenic activity. Granulation was observed in R2 and R3 (up to 33% of the sludge). Elevated hydrogenotrophic methanogenesis was observed in psychrophilically cultivated biomass, but acetoclastic methanogenic activity was also retained. Denaturing gradient gel electrophoresis of archaeal 16S rRNA gene fragments indicated that a distinct community was associated with developing and mature granules in the low‐temperature (LT) bioreactors. Conclusions: Granulation was observed at 15°C in anaerobic bioreactors and was associated with H₂/CO₂‐mediated methanogenesis and distinct community structure development. Significance and Impact of the Study: Granulation underpins high‐rate anaerobic waste treatment bioreactors. Most LT bioreactor trials have employed mesophilic seed sludge, and granulation <20°C was not previously documented.     

PCR primers for assessing community structure of aquatic fungi including Chytridiomycota and Cryptomycota

Fungi are diverse and have the potential for material cycling in freshwater ecosystems. Species composition of aquatic fungi and their seasonal dynamics are often key to their role in the functioning of the ecosystems. However, community structure of aquatic fungi, especially of Chytridiomycota (Chytrids) and Cryptomycota, is still limited because few primer sets are available to examine species composition. In this study, we validated six primer sets for the detection of aquatic fungi by denaturing gradient gel electrophoresis (DGGE) analysis and found that FF390W/EF3r showed the highest specificity among the primer sets tested. We detected both Chytrids and Cryptomycota from Lake Inba by DGGE analysis using the FF390W/EF3r-GC primer set. Further study with our established analysis revealed community structures of aquatic fungi and their seasonal succession patterns in the lake. Results of our study are useful for selection of the primer set for studying community structures of aquatic fungi and their seasonal succession.     

Evaluation of five antibiotics on larval gut bacterial diversity of Plutella xylostella (Lepidoptera: Plutellidae)

Larvae of the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae), have rich microbial communities inhabiting the gut, and these bacteria contribute to the fitness of the pest. In this study we evaluated the effects of five antibiotics (rifampicin, ampicillin, tetracycline, streptomycin sulfate and chloramphenicol) on the gut bacterial diversity of P. xylostella larvae. We screened five different concentrations for each antibiotic in a leaf disc assay, and found that rifampicin and streptomycin sulfate at 3 mg/mL significantly reduced the diversity of the bacterial community, and some bacterial species could be rapidly eliminated. The number of gut bacteria in the rifampicin group and streptomycin sulfate group decreased more rapidly than the others. With the increase of antibiotic concentration, the removal efficiency was improved, whereas toxic effects became more apparent. All antibiotics reduced larval growth and development, and eventually caused high mortality, malformation of the prepupae, and hindered pupation and adult emergence. Among the five antibiotics, tetracycline was the most toxic and streptomycin sulfate was a relatively mild one. Some dominant bacteria were not affected by feeding antibiotics alone. Denaturing gradient gel electrophoresis graph showed that the most abundant and diverse bacteria in P. xylostella larval gut appeared in the cabbage feeding group, and diet change and antibiotics intake influenced gut flora abundance. Species diversity was significantly reduced in the artificial diet and antibiotics treatment groups. After feeding on the artificial diet with rifampicin, streptomycin sulfate and their mixture for 10 days, larval gut bacteria could not be completely removed as detected with the agarose gel electrophoresis method.     

Analysis of effects induced by a pollock protein hydrolysate on early development, innate immunity and the bacterial community structure of first feeding of Atlantic halibut (Hippoglossus hippoglossus L.) larvae

A pollock protein hydrolysate was used for enrichment of the live feed offered to halibut larvae from the onset of exogenous feeding and the effects of treatment on selected innate immune parameters studied. The effects of treatment on the bacterial community structure of larvae were furthermore studied using the PCR–DGGE method. C3 and lysozyme were identified in larvae already at the onset of first feeding and low concentrations of IgM detected at this stage indicate maternal origin. Endogenous production of IgM was validated in the gastrointestinal tract of larvae from 29 days post onset of first feeding, with similar concentrations found in both groups. Feeding the peptide-enriched live feed stimulated production of lysozyme and affected the distribution of C3 in larval tissue but survival and normal development of halibut larvae were not affected by the treatment. Vibrio sp. and Pseudoalteromonas sp. dominated the bacterial community of larvae from both groups and peptide enrichment of the live feed was not found to affect the bacterial community structure associated with surface sterilized larvae.