Number Of Denaturing Gradient Gel Electrophoresis Bands Research Articles

Oral administration of kefiran exerts a bifidogenic effect on BALB/c mice intestinal microbiota.

The activity of kefiran, the exopolysaccharide present in kefir grains, was evaluated on intestinal bacterial populations in BALB/c mice. Animals were orally administered with kefiran and Eubacteria, lactobacilli and bifidobacteria populations were monitored in faeces of mice at days 0, 2, 7, 14 and 21. Profiles obtained by Denaturing Gradient Gel Electrophoresis (DGGE) with primers for Eubacteria were compared by principal component analysis and clearly defined clusters, correlating with the time of kefiran consumption, were obtained. Furthermore, profile analysis of PCR products amplified with specific oligonucleotides for bifidobacteria showed an increment in the number of DGGE bands in the groups administered with kefiran. Fluorescent In Situ Hybridisation (FISH) with specific probes for bifidobacteria showed an increment of this population in faeces, in accordance to DGGE results. The bifidobacteria population was also studied on distal colon content after 0, 2 and 7 days of kefiran administration. Analysis of PCR products by DGGE with Eubacteria primers showed an increment in the number and intensity of bands with high GC content of mice administered with kefiran. Sequencing of DGGE bands confirmed that bifidobacteria were one of the bacterial populations modified by kefiran administration. DGGE profiles of PCR amplicons obtained by using Bifidobacterium or Lactobacillus specific primers confirmed that kefiran administration enhances bifidobacteria, however no changes were observed in Lactobacillus populations. The results of the analysis of bifidobacteria populations assessed on different sampling sites in a murine model support the use of this exopolysaccharide as a bifidogenic functional ingredient.

The Application of PCR-DGGE Technique in Sheep Intestinal Microbial Diversity Research

To identify the intestinal microflora diversity of transgenic sheep and non-transgenic one with overexpression of foreign antiviral gene (VP1 & SB-SW), DNA of total bacteria were extracted from 16 sheep fecal samples (4 non-transgenic sheep, 6 VP1 transgenic sheep and 6 SB-SW transgenic sheep). And then PCR amplification with bacterial universal primers of V3 variable region of 16S rRNA were taken to get the fingerprint profile by denaturing gradient gel electrophoresis (DGGE) technology. The results showed that the DGGE profiles of the 16 fecal samples were highly polymorphic. The number of DGGE bands, considered indicative of the total species richness, did not vary predictably among the three different samples. The DNA sequences were analyzed and the dominant bacteria in sheep fecal were found to beBacteroides,Clostridium, andRuminococcus.Specially, non-transgenic sheep had moreAlistipesfinegoldiiandClostridium lentocellum, transgenic sheep with VP1 had moreClostridiumdrakeiandClostridium populetiand transgenic sheep with SB-SW had moreBarnesiellaintestinihominisandClostridium ljungdahlii. So the PCR-DGGE technique can be applied to evaluate genetically modified (GM) animal potential risks to the environment.

Response of bacterioplankton to a glucose gradient in the absence of lysis and grazing

Bacterial utilization of dissolved organic matter plays an important role in marine carbon cycling. In this study, the response of bacterioplankton to a gradient of carbon (glucose) addition was investigated experimentally in a subtropical coastal environment in the absence of top-down control by viruses and flagellates. Bacterial abundance and production were stimulated by glucose addition corresponding to a gradient of glucose. Differences in the extent of stimulation suggested different bacterial life strategies under different nutrient conditions. Bacterial community diversity as revealed by denaturing gradient gel electrophoresis (DGGE) showed a unimodal productivity-diversity (number of DGGE bands) relationship after 3-day incubation. DNA fingerprinting profiling and cluster analysis showed clear and gradual changes in bacterial community structure along the gradient of glucose concentrations, reflecting the competition for carbon supply among bacterial groups. Sequencing analysis of the DGGE bands disclosed the relative abundance of seven bacterial genotypes in the Alteromonadaceae and Roseovarius that gradually decreased with the glucose enrichment while two Vibrio genotypes showed the reverse increasing trend. This suggested that Vibrio was a more successful opportunist at high carbon availability.

Response of soil microbial diversity to land-use conversion of natural forests to plantations in a subtropical mountainous area of southern China

Land-use conversion can affect the soil microbial community diversity, soil organic matter and nutrient cycling. In this study, soils within a representative land-use sequence were sampled in a subtropical region of China, including four natural forests, Altingia gracilipes Hemsl. (ALG), Cinnamomum chekiangense Nakai (CIC), Castanopsis fargesii Franch. (CAF), and Tsoongiodendron odorum Chun (TSO), and two plantations, Cunninghamia lanceolata (Lamb.) Hook. (CUL) and a citrus orchard (Citrus reticulata Blanco). The soil microbial diversity was investigated by phospholipid fatty acid (PLFA) analysis, denaturing gradient gel electrophoresis (DGGE) and real-time quantitative polymerase chain reaction (PCR). Results showed that microbial community diversity exhibited distinct patterns among land-use types. After conversion of natural forests to plantations, the amount of PLFA and the number of bacterial 16 S rRNA gene copies were reduced significantly, as well as the number of DGGE bands. The average quantity of PLFA was lower by 31% in the CUL plantation and 57% in the citrus orchard, respectively, than in natural forests. Simultaneously, the average copy numbers of the bacterial 16 S rRNA gene were significantly decreased from 8.1 × 1010 g−1 dry weight (DW) in natural forest to 4.9 × 1010 g−1 DW in CUL plantation, and 3.1 × 1010 g−1 DW in the citrus orchard. Such negative responses of soil microbes to conversion of natural forests to plantations could mainly result from decreases in soil organic carbon and necessary elements for growth during land-use conversion, as revealed by statistical analysis. Our results suggested that the soil microbial diversity was indirectly influenced by land-use types in the mid-subtropical mountainous area of southern China. Changes in the amount of litterfall and the soil nutrient status that resulted from land-use conversion drove these indirect changes. Furthermore, deliberate management brought negative effects on soil microbes, which is not beneficial to the sustainability of the ecosystem.

Efficiency of DNA extraction methods on the evaluation of soil microeukaryotic diversity

The evaluation of microbial molecular diversity has been mainly based on the extraction of total DNA from environmental samples. The indirect extraction methods, which have been used for prokaryotes, have never been used to recover soil microeukaryotic DNA. We evaluated the efficiency of an improved indirect DNA extraction protocol developed herein and the direct lysis (the sodium dodecyl sulfate (SDS)-based method and commercial DNA extraction kit) on estimating the molecular diversity of soil microbial eukaryotes. DNA quality and quantity as well as denaturing gradient gel electrophoresis (DGGE) profiles were determined using three soil samples from different stations. The indirect method detected the highest DGGE bands in spite of the low DNA yield. The commercial kit detected a lower number of DGGE bands than the indirect method. The SDS-based method produced the lowest DGGE bands and DNA purity but the highest yield. Using the indirect method, we further evaluated the effect of freezing and air-dried preservations on estimating the microeukaryotic diversity. In spite of the low DNA yield obtained from the air-dried preservation, no significant differences were found in either the number of DGGE bands or the DNA purity between two manners. Our results indicate that the improved indirect method could obtain a high purity of intracellular DNA and high efficiency in the estimation of molecular diversity of soil microbial eukaryotes.

Bacterial Community Dynamics during Swine In vitro Fermentation Using Starch as a Substrate with Different Feed Additives for Odor Reduction.

The experiment was conducted by in vitro fermentation and bacterial community analysis to investigate the reduction of odorous compounds in response to the use of feed additives (FA) during carbohydrate overload in growing pigs. Soluble starch at 1% (control) and various FA at 0.1% Ginseng meal (FA1); Persimmon leaf (FA2); Gingko nut (FA3) and Oregano lippia (FA4) were added to fecal slurry and incubated anaerobically for 12 and 24 h. In vitro parameters and microbial diversity of the dominant bacteria following fermentation were analyzed using Denaturing Gradient Gel Electrophoresis (DGGE), band cloning and sequencing of the V3 region. Results showed that total gas production increased with the advancement of incubation (p<0.05). pH values of FAs and control groups were decreased except the FA4 group which increased somewhat from 12 to 24 h (p<0.05). Ammonia nitrogen (NH3-N) and H2S gas concentrations were comparatively lower in both stages in FA4 treatment than in the other groups (p<0.05). Hence, NH3-N concentrations in liquid phases were increased (p<0.05) from 12 to 24 h, but the trend was lowest in FA4 than in the other groups at both stages. The total VFA production was comparatively lower and butyrate levels were moderate in FA4 group than in the the other groups during both stages (p<0.05). Indirect odor-reducing compounds such as NO2, NO3 and SO4 concentrations were higher in the FA4 and FA3 than in the other groups at 24 h (p<0.05). After fermentation, ten dominant bands appeared, six of which appeared in all samples and four in only the FA4 treated group. The total number of DGGE bands and diversity was higher in the FA4-group compared to other groups. Additionally, similarity indices were lowest (71%) in the FA4, which represented a different bacterial community compared with the other groups. These findings indicate that NH3-N, H2S and VFA production was minimal, and pH was also better in the FA4 group than in the other groups. Furthermore, the conversion of odor-reducing indirect compounds or their intermediates was higher in the FA4 group in compared to the other groups. FA4 group generated less odorous products and more indirect products by in vitro fermentation at 24 h, and their microbial pattern appeared to differ from that of the other groups. These findings suggest that this particular FA could change the microbial population, which may have a beneficial effect on odor reduction. It is recommended that the oregano lippia may be supplied to growing pigs as FA along with excess carbohydrate sources to reduce the production of odorous compounds.

Diversity of methanogens in the hindgut of grower and finisher pigs

This study examined the diversity of the methanogens in the hindgut of two different weight groups of pigs and correlated it with the amount of digested organic carbon (OC) and various components of dietary fiber. Five grower (58.9 ± 1.15 kg) and five finisher (89.4 ± 0.85 kg) Duroc × Landrace × Large Yorkshire female pigs were allocated into two groups and individually housed in cages. During the experiment, feed intake and fecal output were recorded for determination of apparent digestibility of OC, crude fiber (CF), neutral detergent fiber (NDF) and acid detergent fiber (ADF). At the end of the digestibility trial, pigs were sacrificed, and the contents of five segments of hindgut were sterilely collected to determine diversity of methanogens. Total microbial DNA of the hindgut contents was used as template for amplification of the methanogen16S rRNA gene, and the PCR products were further subjected to denaturing gradient gel electrophoresis (DGGE) analysis. Results show that the number of DGGE bands and Shannon diversity index for the 90 kg pigs were higher (P<0.05) than those for the 60 kg pigs. Methanogen communities did not alter along the different segments of the hindgut for the two weight groups. In addition, the amount of OC, CF, NDF and ADF digested (g/d) for the 90 kg pigs (1018.77, 23.11, 268.86 and 99.16, respectively) was higher (P<0.05) than the respective values for the 60 kg pigs (669.27, 13.77, 222.31 and 69.07), indicating that the higher diversity of methanogens in the former group was related to the higher quantity of fiber materials fermented in the hindgut. The positive correlation (p<0.05) between number of DGGE bands and Shannon diversity index with quantity of digested OC and ADF further reaffirmed the above suggestion. Key words : Methanogen, pig, Shannon diversity index, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE).

Comparison of microbial community structures in four Black soils along a climatic gradient in northeast China

Mi, L., Wang, G., Jin, J., Sui, Y., Liu, J. and Liu, X. 2012. Comparison of microbial community structures in four Black soils along a climatic gradient in northeast China. Can. J. Soil Sci. 92: 543–549. Surveys of microorganisms across climatic gradients provide important information about their biodiversity and spatial distribution, which is linked to fundamental ecological functions. The present study investigated the bacterial communities, including total and culturable communities, and fungal communities in Black soils collected from Lishu (lat. 43°20′N, long. 124°28′E), Dehui (lat. 44°12′N, long. 125°33′E), Hailun (lat. 47°26′N, long. 126°38′E) and Beian (lat. 48°17′N, long. 127°15′E) in northeast China. Bacterial and fungal communities were evaluated by polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) banding patterns of partial 16S rDNA and fungal rDNA internal transcribed spacer regions (ITS), respectively. Bacterial and fungal diversity, based on the number of DGGE bands, were similar among the locations, but cluster analysis of banding patterns showed distinct microbial communities along the climatic gradient. A closer relationship was found among soil bacterial (total and culturable) and fungal communities in neighboring locations than those at greater distance, which suggested that the spatial distribution of microbial community existed in the Black Soil Zone. Comparison of DGGE profiles among the four locations showed that the changes of fungal community and culturable bacterial community were greater than that of bacterial community, suggesting that fungal community and culturable bacterial community are more suitable to study microbial biogeographic distribution in Black soils.

Molecular Analysis of Microbial Communities in Endotracheal Tube Biofilms

BackgroundVentilator-associated pneumonia is the most prevalent acquired infection of patients on intensive care units and is associated with considerable morbidity and mortality. Evidence suggests that an improved understanding of the composition of the biofilm communities that form on endotracheal tubes may result in the development of improved preventative strategies for ventilator-associated pneumonia.Methodology/Principal FindingsThe aim of this study was to characterise microbial biofilms on the inner luminal surface of extubated endotracheal tubes from ICU patients using PCR and molecular profiling. Twenty-four endotracheal tubes were obtained from twenty mechanically ventilated patients. Denaturing gradient gel electrophoresis (DGGE) profiling of 16S rRNA gene amplicons was used to assess the diversity of the bacterial population, together with species specific PCR of key marker oral microorganisms and a quantitative assessment of culturable aerobic bacteria. Analysis of culturable aerobic bacteria revealed a range of colonisation from no growth to 2.1×108 colony forming units (cfu)/cm2 of endotracheal tube (mean 1.4×107 cfu/cm2). PCR targeting of specific bacterial species detected the oral bacteria Streptococcus mutans (n = 5) and Porphyromonas gingivalis (n = 5). DGGE profiling of the endotracheal biofilms revealed complex banding patterns containing between 3 and 22 (mean 6) bands per tube, thus demonstrating the marked complexity of the constituent biofilms. Significant inter-patient diversity was evident. The number of DGGE bands detected was not related to total viable microbial counts or the duration of intubation.Conclusions/SignificanceMolecular profiling using DGGE demonstrated considerable biofilm compositional complexity and inter-patient diversity and provides a rapid method for the further study of biofilm composition in longitudinal and interventional studies. The presence of oral microorganisms in endotracheal tube biofilms suggests that these may be important in biofilm development and may provide a therapeutic target for the prevention of ventilator-associated pneumonia.

Distribution and Diversity of Bacteria in a Saline Meromictic Lake as Determined by PCR-DGGE of 16S rRNA Gene Fragments

The variations in vertical distribution and composition of bacteria in the meromictic Lake Faro (Messina, Italy) were analysed by culture-independent methods in two different mixing conditions. Water samples were collected from a central station from the surface to the bottom (30 m depth) on two different sampling dates--the first characterised by a well-mixed water mass and the second by a marked stratification. A 'red-water' layer, caused by a dense growth of photosynthetic sulphur bacteria, was present at a depth of 25 m in December 2005 and at 15 m in August 2006, defining two different zones in terms of their physicochemical properties. The vertical distribution of bacterioplankton showed that the interface zones were more densely populated than others. In both sampling periods, the highest numbers of live cells were observed within 'red water' layers. The dominant phylotypes of the bacterial community were determined by sequencing the Denaturing Gradient Gel Electrophoresis (DGGE) bands resulting from PCR amplification of 16S rRNA gene fragments. The number of DGGE bands, considered indicative of the total species richness, did not vary predictably across the two different sampling periods. Proteobacteria (α-, γ-, δ- and ε subclass members), Cytophaga-Flavobacterium-Bacteroides, green sulphur bacteria and Cyanobacteria were retrieved from Lake Faro. Most of the bands showed DNA sequences that did not match with other previously described organisms, suggesting the presence of new indigenous bacterial phylotypes.

Population variation of invasive Spartina alterniflora can differentiate bacterial diversity in its rhizosphere

While several studies have documented that invasive plants can change the microbial communities, little is known about how soil microbial communities respond to population variation of invasive plants. Here, nine populations of Spartina alterniflora were selected from the east coast of China along latitudinal gradient to compare bacterial diversity of rhizospheres among these populations. The bacterial diversity in S. alterniflora rhizospheres was valued by denaturing gradient gel electrophoresis (DGGE) analysis. Shannon–Weaver diversity index (H′) and number of DGGE bands showed that rhizosphere bacterial diversity of S. alterniflora populations increased along a latitudinal gradient when all the populations were grown in a common garden. These findings suggest that population variation of S. alterniflora can differentiate the rhizosphere bacterial diversity, and the latitudinal gradient can shape the specific plant–bacterial diversity relationship. Our results adding to the recent literature suggest that invasive plant–soil biota interactions would have clinal variation with environmental gradients and improve our understanding of the mechanisms and processes of plant invasions.

Evaluation of the inclusion of a mixture of organic acids or lactulose into the feed of pigs experimentally challenged with Salmonella Typhimurium

A mixture of organic acids and lactulose for preventing or reducing colonization of the gut by Salmonella Typhimurium was evaluated in pigs. A total of 63 4-week-old commercial piglets were randomly distributed into three different experimental dietary groups: a plain diet without additives (PD) and the same diet supplemented with either 0.4% (w/v) formic acid and 0.4% lactic acid (w/v) (AC) or 1% (w/v) lactulose (LC). After 7 days of adaptation, two-thirds of the pigs (14 from each diet) were challenged with a 2-mL oral dose of 108CFU/mL of Salmonella Typhimurium, leaving the remaining animals unchallenged (UC). After 4 and 10 days post-challenge, pigs were euthanized and the ileum and caecum content were aseptically sampled to (a) quantify lactic, formic, and short-chain fatty acids (SCFA), (b) quantify bacterial populations and Salmonella by fluorescence in situ hybridization and (c) qualitatively analyse bacterial populations through denaturing gradient gel electrophoresis (DGGE). Modification of fermentation products and counts of some of the bacterial groups analysed in the challenged pigs receiving the treatments AC and LC were minimal. Treatments only influenced the bacterial diversity after 10 days post-challenge, with AC generating a lower number of DGGE bands than UC (P<0.05). Neither the inclusion of a mixture of 0.4% (w/v) formic and 0.4% (w/v) lactic acids nor of 1% (w/v) lactulose in the feed influenced numbers of Salmonella in the ileum and caecum of experimentally challenged pigs.

Bacterial ‘cosmopolitanism’ and importance of local environmental factors for community composition in remote high‐altitude lakes

In October 2004, plankton samples were collected from six permanent lakes located between 4960 and 5440 m a.s.l. in the Mount Everest region (Nepal) to assess how spatial and local environmental factors affect natural bacterial community composition. Fingerprinting analysis of the bacterial 16S rRNA gene fragment was done by denaturing gradient gel electrophoresis (DGGE).The number of DGGE bands (range: 12-23) was not correlated with lake area or remoteness, but there was a strong negative correlation with the ratio of catchment to lake area (r = -0.826, P < 0.05), suggesting that hydraulic retention time affects the establishment of the bacterial community in these seepage lakes.Most dominant sequences belonged to Betaproteobacteria except in two lakes where members of Bacteroidetes made the largest relative contribution. Up to 81% of the phylotypes had high similarity (>98 to 100%) in partial 16S rRNA gene sequence to those reported from other alpine lakes and glaciers around the world, suggesting the presence of 'cosmopolitan' bacteria.An analysis based on dissimilarity matrices and the Mantel test revealed the existence of dissimilarities in bacterial community composition related to geographical distance over a small spatial scale (<6 km), but determined by local environmental constraints.Our results suggest that several bacterial phylotypes are ubiquitous in the freshwater aquatic realm, but taxon sorting by local environmental constraints is important.

Bacterial communities in soybean rhizosphere in response to soil type, soybean genotype, and their growth stage

Three experiments were conducted in this study in order to investigate the impacts of soil type, soybean genotype, and the reproductive growth stage on bacterial communities in the soybean rhizosphere. Communities were evaluated by principal component analysis of denaturing gradient gel electrophoresis (DGGE) banding patterns and sequencing of partial 16S rDNA polymerase chain reaction (PCR) amplicons. A pot experiment analyzing three soybean genotypes grown in two different types of soil (Black soil and Dark Brown soil) indicated that soil type was the major factor in influencing the bacterial communities in the soybean rhizosphere, with a more significant effect observed in the Black soil samples than in the Dark Brown soil samples. A field experiment was conducted in Dark Brown soil using three soybean genotypes, and the results gleaned from both pot and field experiments indicated that bacterial communities in the soybean rhizosphere changed with growth stages, and higher number of DGGE bands observed in early reproductive growth stages, while surprisingly, a significant impact of genotype on the bacterial communities was not observed in these experiments. However, a plate culture experiment targeting the culturable bacterial communities detected a remarkable difference in the community structures of the rhizosphere between the two soybean genotypes, suggesting that a small portion of the total bacteria was influenced by genotype. Sequence analysis of DGGE bands indicated that bacterial phyla of Proteobacteria, Actinobacteria, Bacteroidetes, Nitrospirae, Firmicutes, Verrucomicrobia and Acidobacteria commonly inhabit the soybean rhizosphere.

Impact of antibiotics on the gut microbiota of critically ill patients

We evaluated the relationship between the intestinal microbiota composition and clinical outcome in a group of 15 high-risk patients admitted for acute infection and/or surgical/accidental trauma who were treated with systemic antibiotics according to standard intensive care unit (ICU) protocols. There was a high mortality rate amongst these patients, each of whom had a considerable organ failure score at admission, respiratory assistance during the most of their ICU stay and a long length of stay. All of these individuals received sedation and enteral nutrition, and the majority also received insulin, vasoactive drugs and some stress-ulcer prophylaxis agents. The intestinal microbiota composition was assessed using denaturing gradient gel electrophoresis (DGGE), a molecular biology tool used to characterize bacterial ecosystems. As all of the patient subjects were in good health prior to their acute illness and admission to the ICU, the first faecal samples obtained from this group showed a DGGE banding pattern that was similar to that of healthy subjects. After 1 week of critical illness, coupled with intensive care treatment, including antibiotics, a very definite alteration in the overall microbiota composition was evident, as revealed by a reduction in the number of DGGE bands. Further pronounced changes to the DGGE banding profiles could be observed in patients remaining in the ICU for 2 weeks. Moreover, a dominant band, identified by sequencing as highly related to Enterococcus, was detected in the DGGE profile of some of our patient subjects. We also performed real-time PCR and obtained results that were in agreement with our qualitative evaluations using DGGE. The degree of organ failure and ICU mortality was significantly higher in patients for whom a high reduction in microbiota biodiversity was coupled with a massive presence of enterococci. A statistically significant link between these two ecological traits and the use of clindamycin was also found.

A multiphasic characterization of the impact of the herbicide acetochlor on freshwater bacterial communities

Acetochlor is the third most frequently detected herbicide in natural waters; however, it is unknown if exposure to environmentally relevant concentrations of acetochlor will impact bacterial community structure and function. This study examined the impact of acetochlor on freshwater heterotrophic bacteria number, and community structure and function using direct counting, community level physiological profiling (CLPP) and denaturing gradient gel electrophoresis (DGGE) analysis. Acetochlor concentration did not appear to correlate with the number of total (P=0.69) and viable (P=0.80) bacteria, even at concentrations up to 500 microg l(-1). However, CLPP indicated that acetochlor increased functional diversity as shown by (i) an increase in the number of carbon sources utilized by the microbial community, relative to nonexposed controls and (ii) increased functional evenness within the heterotrophic bacterial community. Conversely, DGGE fingerprints suggested that exposure to acetochlor generally decreased the community complexity, as the average number of DGGE bands in most treatments was significantly less than in the control treatment. Cluster analysis of DGGE fingerprints revealed three distinct, dose-dependent clusters (i) communities exposed to 0, 1 and 5 microg l(-1); (ii) 50 and 100 microg l(-1) and (iii) 500 microg l(-1), indicating a relationship between acetochlor concentration bacterial community changes. This study indicated that while exposure to environmentally relevant concentrations of acetochlor resulted in no significant impact to the number of freshwater bacteria, impacts to the function and structure of the community were revealed by adopting a multiphasic approach.

Viruses and flagellates sustain apparent richness and reduce biomass accumulation of bacterioplankton in coastal marine waters

To gain a better understanding of the interactions among bacteria, viruses and flagellates in coastal marine ecosystems, we investigated the effect of viral lysis and protistan bacterivory on bacterial abundance, production and diversity [determined by 16S rRNA gene polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE)] in three coastal marine sites with different nutrient supplies in Hong Kong. Six experiments were set up using filtration and dilution methods to develop virus, flagellate and virus+flagellate treatments for natural bacterial populations. All three predation treatments had significant repressing effects on bacterial abundance. Bacterial production was significantly repressed by flagellates and both predators (flagellates and viruses). Bacterial apparent species richness (indicated as the number of DGGE bands) was always significantly higher in the presence of viruses, flagellates and both predators than in the predator-free control. Cluster analysis of the DGGE patterns showed that the effects of viruses and flagellates on bacterial community structure were relatively stochastic while the co-effects of predators caused consistent trends (DGGE always showed the most similar patterns when compared with those of in situ environments) and substantially increased the apparent richness. Overall, we found strong evidence that viral lysis and protist bacterivory act additively to reduce bacterial production and to sustain diversity. This first systematic attempt to study the interactive effects of viruses and flagellates on the diversity and production of bacterial communities in coastal waters suggests that a tight control of bacterioplankton dominants results in relatively stable bacterioplankton communities.

Particle-attached and free-living bacterial communities in a contrasting marine environment: Victoria Harbor, Hong Kong

Diversity of particle-attached and free-living marine bacteria in Victoria Harbor, Hong Kong, and its adjacent coastal and estuarial environments was investigated using DNA fingerprinting and clone library analysis. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes showed that bacterial communities in three stations of Victoria Harbor were similar, but differed from those in adjacent coastal and estuarine stations. Particle-attached and free-living bacterial community composition differed in the Victoria Harbor area. DNA sequencing of 28 bands from DGGE gel showed Alphaproteobacteria was the most abundant group, followed by the Bacteroidetes, and other Proteobacteria. Bacterial species richness (number of DGGE bands) differed among stations and populations (particle-attached and free-living; bottom and surface). BIOENV analysis indicated that the concentrations of suspended solids were the major contributing parameter for the spatial variation of total bacterial community structure. Samples from representative stations were selected for clone library (548 clones) construction and their phylogenetic distributions were similar to those of sequences from DGGE. Approximately 80% of clones were affiliated to Proteobacteria, Bacteroidetes and Cyanobacteria. The possible influences of dynamic pollution and hydrological conditions in the Victoria Harbor area on the particle-attached and free-living bacterial community structures were discussed.

DGGE and 16S rDNA sequencing analysis of bacterial communities in colon content and feces of pigs fed whole crop rice

The effect of feeding whole crop rice (WCR) to growing-finishing pigs at three levels 0 (Control), 10% and 20% on bacterial communities in colon content and feces was analyzed using 16S rDNA-based techniques. Amplicons of the V6–V8 variable regions of bacterial 16S rDNA were analyzed by denaturing gradient gel electrophoresis (DGGE), cloning and sequencing. The total number of DGGE bands and Shannon index of diversity for feces samples were higher in the pigs fed WCR-containing diets compared with the control, while a decrease trend was observed in these two parameters for colon content samples with the inclusion of WCR in the diets, although statistical differences were not significant. In general, the intestinal bacterial communities were prone to form the cluster for pig fed the same diet. Feeding of WCR induced the presence of special DGGE band with the sequence showing 99% similarity to that of Lactobacillus reuteri ( DSM 20016T). The sequences of seven amplicons in total nine clones showed less than 97% similarity with those of previously identified or unidentified bacteria, suggesting that most bacteria in gastrointestinal tracts have not been cultured or identified. The results suggest that the diet containing WCR did not affect the major groups of bacteria, but stimulated the growth of L. reuteri-like species.

Synergistic and antagonistic effects of viral lysis and protistan grazing on bacterial biomass, production and diversity

In a mesotrophic reservoir, we examined the effects on the bacterioplankton of distinct consumers of bacteria, viruses and heterotrophic nanoflagellates, both alone and combined in an experiment using natural populations and in situ incubations in dialysis bags. Ribosomal RNA-targeted probes were employed as well as 16S RNA gene based PCR denaturing gradient gel electrophoresis (DGGE) to enumerate bacterial groups and assess bacterial community composition. We employed probes for Actinobacteria (HGC69a probe), Cytophaga-Flavobacterium-Bacteroidetes bacteria (CF319a probe), BET42a probe (Betaproteobacteria) and a subgroup-Betaproteobacteria (R-BT065 probe). We found consumer-specific effects on bacterial activity and diversity (against a background of CF and BET dominating all treatments) suggesting distinct vulnerabilities to the two sources of mortality. For example, growth rate of Actinobacteria was only positive in the presence of flagellates, while towards the end of the experiment (T(72-96 h)) growth rate of R-BT was only positive in the viruses only treatment. More specific data on how viruses and flagellates influenced Flectobacillus are shown in the companion paper. Highest richness (number of DGGE bands) was found in the virus only treatment and lowest when both consumers were present. In addition, we found suggestions of both antagonistic and synergistic interactions between the two sources of bacterial mortality. Notably, bactivory by flagellates was associated with reductions in bacterial diversity and increases in viral production.