Polymerase Chain Reaction Denaturing Gradient Gel Electrophoresis Fingerprints Research Articles

Bacterial communities and volatile compounds in Doubanjiang, a Chinese traditional red pepper paste.

To identify core bacterial communities and volatile compounds and to investigate their relationships in Doubanjiang, a Chinese traditional red pepper paste. Two main culture-independent methods, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and MiSeq sequencing technique, were used to investigate bacterial composition. Headspace solid-phase microextraction combined with gas chromatography-mass spectrometry (HS-SPME-GC-MS) was used to isolate and identify volatile organic compounds. Partial least squares (PLS) regression and correlation coefficients analyses were used for elucidating the relationship between core bacterial community and volatile compounds. Lactic acid bacteria (LAB) and Enterobacteriaceae family were identified primarily by the PCR-DGGE fingerprinting. Furthermore, the core microbiome (>1% average relative abundance) were indentified by Miseq sequencing technique, which mainly assigned to Proteobacteria, Cyanobacteria and Firmicutes phyla. For volatile profile, a total of 29 volatile compounds were identified by HP-SPME-GC-MS, mainly alcohols, esters, aldehydes, ketones and phenols. PLS regression and Pearson's correlation coefficients analyses revealed highly covariable relationships between volatile compounds and bacterial taxa. Notably, Pseudomonas most highly significantly associated with 3-methyl butanal, 2-methyl butanal, benzeneacetaldehyde and 2-acetylpyrrole (P<0·001), and significantly correlated with 5-methylfurfural and 5-methyl-2-phenyl-2-hexenal (P<0·01). Core bacterial communities were identified and the relationship between bacterial taxa and flavour compounds was revealed, these results could be beneficial for improving the quality of Doubanjiang.

Abundance and diversity of sulfate-reducing bacteria in the sediment of the Zhou Cun drinking water reservoir in Eastern China.

Sulfate-reducing bacteria (SRB) play an important role in the sediments of bay areas, estuaries, and lakes. However, information regarding the genetic diversity of SRB in the sediments of drinking water reservoirs is scarce. In this study, we collected sediment samples from different sites in the Zhou Cun drinking water reservoir between April and June 2012. To explore the genetic diversity of SRB, we used the most-probable-number (MPN) method, polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE), and a cloning approach. The average content of acid-volatile sulfide at the deepest sampling site was 205.87 μg/g sediment. This result is often associated with a large abundance of SRB in the associated sediment. The highest MPN estimate (1.15 x 10(5) cells/g sediment) was detected in May at the deepest sampling site. The PCR-DGGE fingerprints of SRB based on the dissimilatory sulfite reductase beta subunit (dsrB) gene varied according to the different sampling sites and dates. The highest abundance of SRB in the sediments was predominantly found at the deepest sampling sites, as expected from the acid-volatile sulfide content. The dominant species were Desulfobulbus sp, Desulfobacterium sp, and uncultured sulfate-reducing bacteria. Redundancy analysis revealed that organic matter and the concentrations of nitrogen and phosphorus in the sediments were significantly correlated with the diversity of SRB communities present. The results of this study provide a better understanding of the sulfate-reducing microbial species in the sediments of the Zhou Cun drinking water reservoir.

Temporal dynamics of prokaryotic communities in the marine sponge Sarcotragus spinosulus.

In spite of their putative relevance to host functioning, in-depth knowledge of sponge microbiome stability over time is scarce. This study tackles the temporal maintenance of bacterial and archaeal assemblages in the model host Sarcotragus spinosulus along three successive years. Prokaryotic communities were profiled by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and 454-pyrosequencing of S.spinosulus-derived 16S rRNA gene amplicons. Prevailing bacterial phyla were Actinobacteria, Acidobacteria, Proteobacteria, Poribacteria, PAUC34f, Chloroflexi and Bacteroidetes, with Bacteroidetes, Chloroflexi and Poribacteria showing different abundances over the years. At the approximate species level (operational taxonomic units, OTUs, defined at 97% sequence similarity), no major changes in bacterial richness and composition were found through time. Nearly 50% of all detected bacterial symbionts (96 in 205 OTUs) were recovered from all sampling years, whereas a taxonomically equivalent community of less dominant bacteria characterized the transient sponge microbiota. Despite the evidence for temporal symbiont maintenance, an intriguing cumulative degree of variation between individuals was unravelled, with all the surveyed sponge specimens sharing only 27 bacterial OTUs. Archaeal communities were dominated by one single symbiont of the candidate genus Nitrosopumilus (Thaumarchaeota), known for its ability to aerobically oxidize ammonia to nitrite. Only few bacterial ammonia oxidizers consistently occurred in S.spinosulus across the years as documented by PCR-DGGE fingerprinting. In conclusion, prokaryotic symbionts of S.spinosulus display a state of dynamic stability shaped by the interplay between the maintenance of dominant players and turnover of less prevalent community members, in time and across host individuals, with no apparent consequences to holobiont functioning.

Shifts in ascomycete community of bisolarizated substrate infested with Fusarium oxysporum f. sp. conglutinans and F. oxysporum f. sp. basilici by PCR-DGGE

Substrate samples were artificially infested with Fusarium oxysporum f. sp. conglutinans (FOC) and F. oxysporum f. sp. basilici (FOB) in order to evaluate the shift in fungal population by using culture dependent and culture independent methods. Solarization was carried out with transparent polyethylene film during a summer period on a greenhouse located in Northern Italy, in combination or not with Brassica carinata defatted seed meals and/or compost. Biosolarization treatment was carried out in a growth chamber by heating the substrate for 7 and 14 days at optimal (55–52°C for 6h, 50–48°C for 8h and 47–45°C for 10h/day) and sub-optimal (50–48°C for 20h, 45–43°C for 8h and 40–38°C for 10h/day) temperatures. Plate counts and polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) analyses were performed to evaluate the effect of biosolarization on the microbial population. The abundance of FOC and FOB were reduced as a consequence of biosolarization approach, while bacterial population (total aerobic mesophilic bacteria and Pseudomonas spp.) were higher compared to control samples during the experiment. PCR-DGGE fingerprints of the ascomycete community obtained from DNA directly extracted from infested substrate samples showed that the use of organic amendments increased the similarity of the fungal population.

Effect of elevated atmospheric CO2 and temperature on disease severity of Fusarium oxysporum f.sp. lactucae on lettuce plants

Climate change effects on Fusarium oxysporum f.sp. lactucae (FOL) on lettuce plants grown under simulated climate change were studied. An artificial growing substrate was infested with FOL to reach a final concentration of 1×104 CFUg−1 of substrate. A non infested tank was used as control.Subsequently, 32 pots (2l each) were prepared from the infested tank and other 32 pots were prepared from the non infested tank (control). Lettuce plants were then transplanted into the pots and grown in phytotrons under four simulated environmental conditions: (1) 800ppm CO2, 22–26°C; (2) 800ppm CO2, 18–22°C; (3) 400ppm CO2, 22–26°C and (4) 400ppm CO2, 18–22°C. Substrate samples were collected from each phytotron at 7, 14, 21 and 28 days after transplanting. Plate counts, enzymatic assays, and polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) analyses were performed to evaluate the effect of climate change on the microbial population. The abundance of Fusarium spp. and the severity of Fusarium wilt of lettuce varied significantly as a consequence of increased temperature (22–26°C). Increased CO2 levels showed no effect on the severity of Fusarium wilt of lettuce and on the abundance of Fusarium spp. On the other hand, the total bacterial abundance was reduced at elevated CO2 concentration (800ppm). PCR-DGGE fingerprints of the ascomycete community obtained from DNA directly extracted from infested substrate samples did not change as a consequence of elevated temperature and CO2.Enzymatic activities were not affected by the elevated CO2 level. Our study indicates that the CO2 concentration used in our experiment had no detectable impact on Fusarium wilt of lettuce. Only temperature influenced all observed parameters, but did not affect the fungal species diversity. Other factors, such as nutrient limitation and the effect of plant species needs further study.

Characterisation of the spoilage bacterial microbiota in oyster gills during storage at different temperatures

The spoilage bacterial community in oyster gill was investigated during storage at 4, 10 and 20 °C. Aerobic plate counts and pH values were determined. Total bacterial DNA was extracted from oyster gill and bulk cells of plate count media. The major bacterial species during fresh or different temperatures storage were determined by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The initial aerobic plate count in oyster gill reached 6.70 log CFU g(-1). PCR-DGGE fingerprinting analysis of the 16S rRNA gene V3 region revealed that most of the strains in fresh oyster gill belonged to the genera Lactococcus and Enterobacter. The major spoilage bacteria at a storage temperature of 20 °C were Leuconostoc pseudomesenteroides, an uncultured bacterium, Cytophaga fermentans, Lactococcus lactis, Pseudoalteromonas sp., Enterococcus mundtii, Clostridium difficile and an uncultured Fusobacteria; those at 10 °C were Lactococcus spp., Lactobacillus curvatus, Weissella confusa and C. difficile; those at 4 °C were Lactococcus, Weissella, Enterobacter and Aeromonas. The other minor species were L. curvatus, Pseudomonas sp. and E. mundtii. Lactococcus spp. was the most common main spoilage bacteria in oyster gill during chilled storage. PCR-DGGE revealed the complexity of the bacterial microbiota and the major bacteria species in oyster gill for fresh and storage.

Temporal heterogeneity of plankton community in Lake Dianchi and its relation to environmental factors

The plankton community in Fubao Bay of Lake Dianchi, a large, shallow and highly polluted freshwater lake in China, was investigated on three dates. First, a total of 76 zooplankton species were identified by morphological characteristics of which 44 were protozoa, 20 rotifer, and 12 crustacean. Then, the plankton community was characterized by using random amplified polymorphic DNA (RAPD) and Polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) over the time period. Morphological composition, RAPD fingerprints, PCR-DGGE fingerprints, and unweighted pair-group method using arithmetic averages clustering analysis all showed that samples within a season were generally clustered into the same group. The results also revealed that RAPD fingerprints and PCR-DGGE fingerprints were identical to morphological composition in characterizing community similarity. Canonical correspondence analysis based on morphological composition, RAPD fingerprints, and PCR-DGGE fingerprints all suggested that the factors strongly correlated with the first two ordination axes were seasonally different, further suggesting high- temporal heterogeneity in Lake Dianchi. There were close relationships between morphological composition, RAPD and PCR-DGGE fingerprints, and: (1) total nitrogen in summer, (2) total phosphorus in autumn, and (3) BOD5 or NO2-N in winter. Results of this study suggest that molecular techniques are acceptable methods for examining plankton community composition and measuring plankton community responses to stress and perturbation.

Bacterial Communities Associated with Chenopodium album and Stellaria media Seeds from Arable Soils

The bacterial community compositions in Chenopodium album and Stellaria media seeds recovered from soil (soil weed seedbank), from bulk soil, and from seeds harvested from plants grown in the same soils were compared. It was hypothesized that bacterial communities in soil weed seedbanks are distinct from the ones present in bulk soils. For that purpose, bacterial polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) fingerprints, made from DNA extracts of different soils and seed fractions, were analyzed by principal component analysis. Bacterial fingerprints from C. album and S. media seeds differed from each other and from soil. Further, it revealed that bacterial fingerprints from soil-recovered and plant-harvested seeds from the same species clustered together. Hence, it was concluded that microbial communities associated with seeds in soil mostly originated from the mother plant and not from soil. In addition, the results indicated that the presence of a weed seedbank in arable soils can increase soil microbial diversity. Thus, a change in species composition or size of the soil weed seedbank, for instance, as a result of a change in crop management, could affect soil microbial diversity. The consequence of increased diversity is yet unknown, but by virtue of identification of dominant bands in PCR-DGGE fingerprints as Lysobacter oryzae (among four other species), it became clear that bacteria potentially antagonizing phytopathogens dominate in C. album seeds in soil. The role of these potential antagonists on weed and crop plant growth was discussed.

Cultivation and molecular monitoring of halophilic microorganisms inhabiting an extreme environment presented by a salt-attacked monument

AbstractIn the last few years several investigations, based on culture-dependent and -independent techniques, have shown that salt-attacked stone surfaces present a habitat for extremely salt tolerant and moderate halophilic microorganisms. The inner walls of the Chapel of St. Virgil in Vienna (Austria) are an example of this phenomenon. Salt crusts cover most of the wall surfaces and salt crystallization in the porous space of the stone is causing decohesion of material and destruction of the original medieval paintings. The salt, together with the oligotrophic conditions, creates a very special and extreme habitat for halotolerant and halophilic microorganisms.In this study we investigate and monitor the cultivable and non-cultivable members of the microbial community present on the stonework of the medieval Chapel of St. Virgil after several severe disturbances of the microbial environment caused by desalination and disinfection treatments. With this finality, a combination of culture-dependent and -independent techniques was selected. The genetic diversity of a total of 104 bacterial strains isolated from the stone samples was analysed by denaturing gradient gel electrophoresis (DGGE), random amplified polymorphic DNA (RAPD) analysis and 16S rRNA gene sequencing. Strains were distributed over 29 groups on the basis of their RAPD patterns. Only 19 groups were differentiated by DGGE. Comparative sequence analyses showed that the isolated strains belong to related species of the generaHalobacillus(47.1%),Bacillus(35.6%),Acinetobacter(4.8%),Halomonas(3.9%),Nesterenkonia(2.9%),Paucisalibacillus(2.9%),Paenibacillus(1%),Staphylococcus(1%) andExiguobacterium(1%).In addition, polymerase chain reaction DGGE fingerprints, in combination with the creation of clone libraries and sequencing analyses, were used to monitor and identifyArchaea, the non-cultivable fraction of the microbial community. The detected archaeal sequences were closely related to different uncultured archaeons as well as to the cultured generaHalococcusandHalalkalicoccusandHalobacterium.Cultivation and molecular analyses revealed the presence of highly specialized microorganisms that were able to thrive and survive after several desalination and disinfection treatments in the extreme environment presented by the salt-attacked Chapel of St. Virgil.

Microbial community structures in a horizontal biotrickling filter degrading H 2S and NH 3

In this study, the temporal and spatial differences in the microbial community structures in a horizontal biotrickling filter (HBTF) for simultaneous biodegradation of H 2S and NH 3 was investigated by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR) amplified 16S rRNA gene fragments. Over 90% H 2S and 95% NH 3 were removed simultaneously under the loadings up to 137 g m −3 h −1 in most cases. PCR–DGGE fingerprints revealed high population diversity of the biofilm in the HBTF. The microbial community structures in the HBTF could adapt to changing operating conditions, e.g., inlet loadings and substrates starvation, while they were relatively stable under the constant loadings. In addition, the microbial community structures differed as a function of the section of the HBTF during early operational period, while they were much more similar over extended operation.

Genetic Diversity of the Plankton Community and Its Relation to Taxonomic Composition and Environmental Factors in Lake Xiliang

ABSTRACT Genetic diversity of the plankton community in Lake Xiliang was depicted by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprinting. Seventy-seven bands (33 of 16s rDNA and 44 of 18s rDNA) were detected, sixty-two planktonic taxa were identified in six sample stations in November 2007. The most common taxa were Ceratium hirundinella, Bdelloidea, Keratella cochlearis, Polyarthra trigla, and copepod nauplii. Based on environmental factors, taxonomic composition, and PCR-DGGE fingerprinting, unweighted pair-group method using arithmetic averages clustering and principal components analysis were used to analyze habitat similarities. There was distinct spatial heterogeneity in Lake Xiliang, and the genetic diversity of the plankton community was closely related to taxonomic composition and environmental factors.

Application of broad-range 16S rRNA PCR amplification and DGGE fingerprinting for detection of tick-infecting bacteria

Ticks play an important role in the transmission of arthropod-borne diseases of viral, protozoal and bacterial origin. The present article describes a molecular–biological based method, which facilitated the broad-range analyses of bacterial communities in ixodid ticks ( Ixodes ricinus). DNA was extracted both from single ticks and pooled adult ticks. Eubacterial 16S rRNA gene fragments (16S rDNA) were amplified by polymerase chain reaction (PCR) with broad-range ribosomal primers. Sequences spanning the hypervariable V3 region of the 16S rDNA and representing individual bacterial taxons were separated by denaturing gradient gel electrophoresis (DGGE). For phylogenetic identification, DGGE bands were exised, cloned and sequenced. In addition, we set up a 16S rDNA clone library which was screened by DGGE. Sequences were compared with sequences of known bacteria listed in the GenBank database. A number of bacteria were affiliated with the genera Rickettsia, Bartonella, and Borrelia, which are known to be pathogenic and transmitted by ticks. Two sequences were related to the yet to be cultivated Haemobartonella. To our knowledge, Haemobartonella has never been directly detected in I. ricinus. In addition, members of the genera Staphylococcus, Rhodococcus, Pseudomonas, and Moraxella were detected, which have not been identified in ticks so far. Two bacteria were most closely related to a rickettsial endosymbiont of an Acanthamoeba sp., and to an endosymbiont (Legionellaceae, Coxiella group) of the microarthropod Folsomia candida. The results prove that 16S rDNA genotyping in combination with DGGE analysis is a promising approach for the detection and identification of bacteria infecting ticks, regardless of whether these bacteria are fastidious, obligate intracellular or noncultivable.