Bacterial 16S rDNA Sequences Research Articles

Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions.

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, we described a protocol for multiple analyses of the biological compositions in environmental PM. Five experiments are presented: (1) PM number monitoring by using a laser particle counter; (2) PM collection by using a cyclonic aerosol sampler; (3) PM collection by using a high-volume air sampler with filters; (4) culturable microbes collection by the Andersen six-stage sampler; and (5) detection of biological composition of environmental PM by bacterial 16SrDNA and fungal ITS region sequencing. We selected hazy days and a livestock farm as two typical examples of the application in this protocol. In this study, these two sampling methods, cyclonic aerosol sampler and filter sampler, showed different sampling efficiency. The cyclonic aerosol sampler performed much better in terms of collecting bacteria, while these two methods showed the same efficiency in collecting fungi. Filter samplers can work under low temperature conditions while cyclonic aerosol samplers have a sampling limitation for temperature. A solid impacting sampler, such as an Andersen six-stage sampler, can be used to sample bioaerosols directly into the culture medium, which increases the survival rate of culturable microorganisms. However, this method mainly relies on culture while more than 99% of microbes cannot be cultured. DNA extracted from the culturable bacteria collected by the Andersen six-stage sampler and samples collected by cyclonic aerosol sampler and filter sampler were detected by bacterial 16S rDNA and fungal ITS region sequencing.All the methods above may have wide application in many fields of study, such as environmental monitoring and airborne pathogen detection. From these results, we can conclude that these methods can be used under different conditions and may help other researchers further explore the health impacts of environmental bioaerosols.

Streptomyces lydicus A01 affects soil microbial diversity, improving growth and resilience in tomato.

The actinomycete Streptomyces lydicus A01 promotes tomato seedling growth; however, the underlying mechanism is unclear. In this study, we investigated whether changes in soil microbial diversity, following Streptomyces lydicus A01 treatment, were responsible for the increased tomato seedling growth. Eukaryotic 18S ribosomal DNA (rDNA) sequencing showed that S. lydicus A01-treated and untreated soil shared 193 operational taxonomic units (OTUs), whereas bacterial 16S rDNA sequencing identified 1,219 shared OTUs between the treated and untreated soil. Of the 42 dominant eukaryotic OTUs, eight were significantly increased and six were significantly decreased after A01 treatment. Of the 25 dominant bacterial OTUs, 12 were significantly increased and eight were significantly decreased after A01 treatment. Most of the eukaryotes and bacteria that increased in abundance exhibited growth promoting characteristics, which were mainly predicted to be associated with mineralization of nitrogen and phosphorus, phosphate solubilization, nutrient accumulation, and secretion of auxin, whereas some were related to plant protection, such as the degradation of toxic and hazardous substances. Soil composition tests showed that S. lydicus A01 treatment enhanced the utilization of nitrogen, phosphorus, and potassium in tomato seedlings. Thus, microbial fertilizers based on S. lydicus A01 may improve plant growth, without the detriment effects of chemical fertilizers.

Mycorrhizosphere Bacterial Communities and their Sensitivity to Antibacterial Activity of Ectomycorrhizal Fungi.

We investigated whether ectomycorrhizal (ECM) fungal species exhibit antibacterial activity towards culturable bacterial communities in mycorrhizospheres. Four hundred and thirty bacterial strains were isolated from the ECM root tips of Pinus densiflora and bulk soil, and 21 were co-cultured with six ECM fungal species. Three hundred and twenty-nine bacterial 16S rDNA sequences were identified in ECM roots (n=185) and bulk soil (n=144). Mycorrhizosphere isolates were dominated by Gram-negative Proteobacteria from 16 genera, including Burkholderia, Collimonas, Paraburkholderia, and Rhizobium. Paraburkholderia accounted for approximately 60%. In contrast, bulk soil isolates contained a high number of Gram-positive Firmicutes, particularly from Bacillus. Paraburkholderia accounted for ≤20% of the bacterial isolates from bulk soil, which was significantly lower than its percentage in ECM root tips. Co-cultures of six ECM fungal species with the 21 bacterial strains revealed that eight strains of three Gram-positive genera—Arthrobacter, Bacillus, and Lysinibacillus—were sensitive to the antibacterial activity of the fungi. In contrast, the Gram-negative strains, including five Paraburkholderia strains, two Burkholderia strains, and a Rhizobium sp., were not sensitive. The strength of fungal antibacterial activity varied in a species-dependent manner, but consistently affected Gram-positive bacteria. These results suggest that Gram-positive bacteria are excluded from the mycorrhizosphere by the antibacterial activity of ECM fungi, which develops specific soil bacterial communities in the mycorrhizosphere.

Bacterial diversity associated with saline efflorescences damaging the walls of a French decorated prehistoric cave registered as a World Cultural Heritage Site

This study focused on a French prehistoric decorated cave which suffers from the development of saline efflorescences. The goal was to determine the ecology of microorganisms associated with efflorescences, and determine if the sessile microbiota could have a role in the deterioration process. Samples from 2 sampling campaigns were analyzed. Elemental, mineralogical and isotopic analyses showed gypsum containing sulfur originating mainly from the host-rock. Microscopic observations, direct cell counting, culture-dependent assays and high-throughput sequencing (HTS) of bacterial 16S rDNA sequences indicated an abundant sessile microbiota, where Actinobacteria, Alphaprotebacteria, Gammaproteobacteria, Bacteroidetes and Planctomycetes were the main phylogenetic groups. Although a relatively low abundance of genera associated with sulfur cycling were identified, the presence of dense biofilms at the interface rock/efflorescences composed of a high relative abundance of Actinobacteria may reflect previously described indirect biomineralization. An increase of microbial abundance and bacterial richness between October 2014 and April 2015 might be attributed to fluctuations in environmental factors. This study gives insight into origin and bacterial composition of efflorescences that threatened Sorcerer's cave engravings. HTS has rarely been used in the field of cultural heritage and thus represents one of the originalities of this work.

High-throughput sequencing of microbial community diversity in soil, grapes, leaves, grape juice and wine of grapevine from China.

In this study Illumina MiSeq was performed to investigate microbial diversity in soil, leaves, grape, grape juice and wine. A total of 1,043,102 fungal Internal Transcribed Spacer (ITS) reads and 2,422,188 high quality bacterial 16S rDNA sequences were used for taxonomic classification, revealed five fungal and eight bacterial phyla. At the genus level, the dominant fungi were Ascomycota, Sordariales, Tetracladium and Geomyces in soil, Aureobasidium and Pleosporaceae in grapes leaves, Aureobasidium in grape and grape juice. The dominant bacteria were Kaistobacter, Arthrobacter, Skermanella and Sphingomonas in soil, Pseudomonas, Acinetobacter and Kaistobacter in grape and grapes leaves, and Oenococcus in grape juice and wine. Principal coordinate analysis showed structural separation between the composition of fungi and bacteria in all samples. This is the first study to understand microbiome population in soil, grape, grapes leaves, grape juice and wine in Xinjiang through High-throughput Sequencing and identify microorganisms like Saccharomyces cerevisiae and Oenococcus spp. that may contribute to the quality and flavor of wine.

Antibiotic-Induced Gut Microbiota Disruption Decreases TNF-α Release by Mononuclear Cells in Healthy Adults.

Objectives:Broad-spectrum antibiotics disrupt the intestinal microbiota. The microbiota is essential for physiological processes, such as the development of the gut immune system. Recent murine data suggest that the intestinal microbiota also modulates systemic innate immune responses; however, evidence in humans is lacking.Methods:Twelve healthy young men were given oral broad-spectrum antibiotics (ciprofloxacin 500 mg bid, vancomycin 500 mg tid and metronidazole 500 mg tid) for 7 days. At baseline, 1 day, and 6 weeks after antibiotics, blood and feces were sampled. Whole blood and isolated mononuclear cells were stimulated with selected Toll-like receptor agonists and heat-killed bacteria. Microbiota diversity and composition was determined using bacterial 16S rDNA sequencing.Results:One day after the antibiotic course, microbial diversity was significantly lower compared with baseline. After antibiotic therapy, systemic mononuclear cells produced lower levels of tumor necrosis factor (TNF)-α after ex vivo stimulation with lipopolysaccharide (LPS). This diminished capacity to produce TNF-α was restored 6 weeks after cessation of antibiotic therapy. In whole blood, a reduced capacity to release interleukin (IL)-1β and IL-6 was observed after LPS stimulation. Antibiotic treatment did not impact on differential leukocyte counts, phagocytosis, and cell surface markers of neutrophils and monocytes.Conclusions:In this proof-of-principle study of healthy subjects, microbiota disruption by broad-spectrum antibiotics is reversibly associated with decreased systemic cellular responsiveness towards LPS. The implications of these findings in a clinical setting remain to be determined.

Thinking beside the box: Should we care about the non-coding strand of the 16S rRNA gene?

The 16S rRNA gene (16S rDNA) codes for RNA that plays a fundamental role during translation in the ribosome and is used extensively as a marker gene to establish relationships among bacteria. However, the complementary non-coding 16S rDNA (nc16S rDNA) has been ignored. An idea emerged in the course of analyzing bacterial 16S rDNA sequences in search for nucleotide composition and substitution patterns: Does the nc16S rDNA code? If so, what does it code for? More importantly: Does 16S rDNA evolution reflect its own evolution or the evolution of its counterpart nc16S rDNA? The objective of this minireview is to discuss these thoughts. nc strands often encode small RNAs (sRNAs), ancient components of gene regulation. nc16S rDNA sequences from different bacterial groups were used to search for possible matches in the Bacterial Small Regulatory RNA Database. Intriguingly, the sequence of one published sRNA obtained from Legionella pneumophila (GenBank: AE0173541) showed high non-random similarity with nc16S rDNA corresponding in part to the V5 region especially from Legionella and relatives. While the target(s) of this sRNA is unclear at the moment, its mere existence might open up a new chapter in the use of the 16S rDNA to study relationships among bacteria.

Effects of Intercropping with Potato Onion on the Growth of Tomato and Rhizosphere Alkaline Phosphatase Genes Diversity.

Background and Aims: In China, excessive fertilization has resulted in phosphorus (P) accumulation in most greenhouse soils. Intercropping can improve the efficiency of nutrient utilization in crop production. In this study, pot experiments were performed to investigate the effects of intercropping with potato onion (Allium cepa L. var. aggregatum G. Don) on tomato (Solanum lycopersicum L.) seedlings growth and P uptake, the diversity of rhizosphere phosphobacteria and alkaline phosphatase (ALP) genes in phosphorus-rich soil.Methods: The experiment included three treatments, namely tomato monoculture (TM), potato onion monoculture (OM), and tomato/potato onion intercropping (TI-tomato intercropping and OI-potato onion intercropping). The growth and P uptake of tomato and potato onion seedlings were evaluated. The dilution plating method was used to determine the population of phosphate-solubilizing bacteria (PSB) and phosphate-mineralizing bacteria (PMB). The genomic DNAs of PSB and PMB in the rhizosphere of tomato and potato onions were extracted and purified, and then, with the primer set of 338f /518r, the PCR amplification of partial bacterial 16S rDNA sequence was performed and sequenced to determine the diversities of PSB and PMB. After extracting the total genomic DNAs from the rhizosphere, the copy numbers and diversities of ALP genes were investigated using real-time PCR and PCR-DGGE, respectively.Results: Intercropping with potato onion promoted the growth and P uptake of tomato seedlings, but inhibited those of potato onion. After 37 days of transplanting, compared to the rhizosphere of TM, the soil pH increased, while the electrolytic conductivity and Olsen P content decreased (p < 0.05) in the rhizosphere of TI. The populations and diversities of PSB, PMB, and ALP genes increased significantly in the rhizosphere of TI, compared to the rhizosphere of TM.Conclusion: The results indicated that intercropping with potato onion promoted the growth and P uptake of tomato in phosphorus-rich soil and affected the community structure and function of phosphobacteria in tomato rhizosphere. Intercropping with potato onion also improved soil quality by lowering levels of soil acidification and salinization.

Opioid-induced gut microbial disruption and bile dysregulation leads to gut barrier compromise and sustained systemic inflammation.

Morphine and its pharmacological derivatives are the most prescribed analgesics for moderate to severe pain management. However, chronic use of morphine reduces pathogen clearance and induces bacterial translocation across the gut barrier. The enteric microbiome has been shown to play a critical role in the preservation of the mucosal barrier function and metabolic homeostasis. Here, we show for the first time, using bacterial 16s rDNA sequencing, that chronic morphine treatment significantly alters the gut microbial composition and induces preferential expansion of gram-positive pathogenic and reduction in bile-deconjugating bacterial strains. A significant reduction in both primary and secondary bile acid levels was seen in the gut, but not in the liver with morphine treatment. Morphine induced microbial dysbiosis and gut barrier disruption was rescued by transplanting placebo-treated microbiota into morphine-treated animals, indicating that microbiome modulation could be exploited as a therapeutic strategy for patients using morphine for pain management.

Diversity, Bacterial Symbionts and Antibacterial Potential of Gut-Associated Fungi Isolated from the Pantala flavescens Larvae in China.

The diversity of fungi associated with the gut of Pantala flavescens larvae was investigated using a culture-dependent method and molecular identification based on an analysis of the internally transcribed spacer sequence. In total, 48 fungal isolates were obtained from P. flavescens larvae. Based on phylogenetic analyses, the fungal isolates were grouped in 5 classes and 12 different genera. Fourteen bacterial 16S rDNA sequences derived from total genomic DNA extractions of fungal mycelia were obtained. The majority of the sequences were associated with Proteobacteria (13/14), and one Bacillaceae (1/14) was included. Leclercia sp., Oceanobacillus oncorhynchi and Methylobacterium extorquens, were reported for the first time as bacterial endosymbionts in fungi. High-performance liquid chromatography (HPLC) analysis indicated that bacterial symbionts produced specific metabolites and also exerted an inhibitory effect on fungal metabolites. The biological activity of the fungal culture extracts against the pathogenic bacteria Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633) and Escherichia coli (ATCC 8739) was investigated, and 20 extracts (42%) exhibited antibacterial activity against at least one of the tested bacterial strains. This study is the first report on the diversity and antibacterial activity of symbiotic fungi residing in the gut of P. flavescens larvae, and the results show that these fungi are highly diverse and could be exploited as a potential source of bioactive compounds.

Isolation and Pathogenicity Analyses on Yersinia enterocolitica from Pelteobagrus vachelli

AbstractYersinia enterocolitica is an important zoonotic pathogen that can induce disease outbreaks in a wide host range. Strain YER6022 was isolated from Pelteobagrus vachelli and identified using bacterial morphology and 16S rDNA sequence analysis. Five virulence factors were detected, then artificial infection experiment and histopathological method were carried out. These results showed that strain YER6022 was one of Y. enterocolitica family members. In addition, ail, ystb, virF, yadA and HPIint were dectected. In artificial infection experiment, with 80% mortality and 100% morbidity, injected Pelteobagrus vachellis showed red swollen of the anus, abdomen swelling and fim bleeding. There existed serious hyperaemia and edema in kidney, spleen, intestine and liver at the light microscope. Ultrastructural observation indicated that mitochondria of the liver, kidney, spleen and intestine swelled and mitochondrial cristae broke. The data had further shed light on its pathogenicity in Pelteobagrus vachelli. It would benefit for further studies on pathogenesis of Pelteobagrus vachelli infected with Y. enterocolitica.

Cross-fostering immediately after birth induces a permanent microbiota shift that is shaped by the nursing mother

BackgroundCurrent research has led to the appreciation that there are differences in the commensal microbiota between healthy individuals and individuals that are predisposed to disease. Treatments to reverse disease pathogenesis through the manipulation of the gastrointestinal (GI) microbiota are now being explored. Normalizing microbiota between different strains of mice in the same study is also needed to better understand disease pathogenesis. Current approaches require repeated delivery of bacteria and large numbers of animals and vary in treatment start time. A method is needed that can shift the microbiota of predisposed individuals to a healthy microbiota at an early age and sustain this shift through the lifetime of the individual.ResultsWe tested cross-fostering of pups within 48 h of birth as a means to permanently shift the microbiota from birth. Taxonomical analysis revealed that the nursing mother was the critical factor in determining bacterial colonization, instead of the birth mother. Data was evaluated using bacterial 16S rDNA sequences from fecal pellets and sequencing was performed on an Illumina Miseq using a 251 bp paired-end library.ConclusionsThe results show that cross-fostering is an effective means to induce an early and maintained shift in the commensal microbiota. This will allow for the evaluation of a prolonged microbial shift and its effects on disease pathogenesis. Cross-fostering will also eliminate variation within control models by normalizing the commensal microbiota between different strains of mice.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-015-0080-y) contains supplementary material, which is available to authorized users.

Characterization and quantification of the fungal microbiome in serial samples from individuals with cystic fibrosis.

BackgroundHuman-associated microbial communities include fungi, but we understand little about which fungal species are present, their relative and absolute abundances, and how antimicrobial therapy impacts fungal communities. The disease cystic fibrosis (CF) often involves chronic airway colonization by bacteria and fungi, and these infections cause irreversible lung damage. Fungi are detected more frequently in CF sputum samples upon initiation of antimicrobial therapy, and several studies have implicated the detection of fungi in sputum with worse outcomes. Thus, a more complete understanding of fungi in CF is required.ResultsWe characterized the fungi and bacteria in expectorated sputa from six CF subjects. Samples were collected upon admission for systemic antibacterial therapy and upon the completion of treatment and analyzed using a pyrosequencing-based analysis of fungal internal transcribed spacer 1 (ITS1) and bacterial 16S rDNA sequences. A mixture of Candida species and Malassezia dominated the mycobiome in all samples (74%–99% of fungal reads). There was not a striking trend correlating fungal and bacterial richness, and richness showed a decline after antibiotic therapy particularly for the bacteria. The fungal communities within a sputum sample resembled other samples from that subject despite the aggressive antibacterial therapy. Quantitative PCR analysis of fungal 18S rDNA sequences to assess fungal burden showed variation in fungal density in sputum before and after antibacterial therapy but no consistent directional trend. Analysis of Candida ITS1 sequences amplified from sputum or pure culture-derived genomic DNA from individual Candida species found little (<0.5%) or no variation in ITS1 sequences within or between strains, thereby validating this locus for the purpose of Candida species identification. We also report the enhancement of the publically available Visualization and Analysis of Microbial Population Structures (VAMPS) tool for the analysis of fungal communities in clinical samples.ConclusionsFungi are present in CF respiratory sputum. In CF, the use of intravenous antibiotic therapy often does not profoundly impact bacterial community structure, and we observed a similar stability in fungal species composition. Further studies are required to predict the effects of antibacterials on fungal burden in CF and fungal community stability in non-CF populations.

Analysis of endophytic bacterial community composition by 16S rDNA clone library in Achnatherum inebrians

The endophytic bacterial diversity of drunken horse grass (DHG) (Achnatherum inebrians) growing in native habitat station in Xinjiang Province, China was analyzed by 16S rDNA cloning, amplified ribosomal DNA restriction analysis (ARDRA), and sequence homology comparison. To effectively exclude the interference of chloroplast DNA and mitochondrial DNA of DHG, a pair of bacterial PCR primers was selected to specifically amplify bacterial 16S rDNA sequences directly from DHG root tissues. Among 249 positive clones in the 16S rDNA library of endophytes, 57 OTUs were identified based on the similarity of the ARDRA banding profiles. Sequence analysis revealed diverse phyla of bacteria in the 16S rDNA library, which consisted of alpha, beta, gamma, delta, and epsilon subclasses of the Proteobacteria, Flavobacteria, Actinobacteria, Sphingobacteria, and uncultured bacteria. The dominant group was Proteobacteria, whie the most numerous genus was Bacillus. More than 8.00% of the total clones showed a high similarity to uncultured bacteria, suggesting that nonculturable bacteria make a significant part of DHG endophytic bacterial community. To our knowledge, this is the first report that endophytic bacteria associated with DHG are revealed by the culture-independent approach. The results suggest that the diversity of endophytic bacteria is abundant in DHG.

Coculture with marine bacteria confers resistance to complete viral lysis of diatom cultures

Supplement. The following information supports the ‘Materials and methods’ section of the main text for testing the possibility of viral genome integration into the host genome DNA, i.e. latent or lysogenic infection, and for the effects of all bacterial isolates on the host–virus system. These results are shown in Figs. S1 & S2, respectively. Furthermore, the results of the BLAST analysis based on the bacterial 16S rDNA sequence data are shown in Table S1

16S rDNA‐based metagenomic analysis of dental plaque and lung bacteria in patients with severe acute exacerbations of chronic obstructive pulmonary disease

Acute exacerbations of chronic obstructive pulmonary disease (AE-COPD) are leading causes of mortality in hospital intensive care units. We sought to determine whether dental plaque biofilms might harbor pathogenic bacteria that can eventually cause lung infections in patients with severe AE-COPD. Paired samples of subgingival plaque biofilm and tracheal aspirate were collected from 53 patients with severe AE-COPD. Total bacterial DNA was extracted from each sample individually for polymerase chain reaction amplification and/or generation of bacterial 16S rDNA sequences and cDNA libraries. We used a metagenomic approach, based on bacterial 16S rDNA sequences, to compare the distribution of species present in dental plaque and lung. Analysis of 1060 sequences (20 clones per patient) revealed a wide range of aerobic, anaerobic, pathogenic, opportunistic, novel and uncultivable bacterial species. Species indistinguishable between the paired subgingival plaque and tracheal aspirate samples (97-100% similarity in 16S rDNA sequence) were dental plaque pathogens (Aggregatibacter actinomycetemcomitans, Capnocytophaga sputigena, Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola) and lung pathogens (Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa and Streptococcus pneumoniae). Real-time polymerase chain reaction of 16S rDNA indicated lower levels of Pseudomonas aeruginosa and Porphyromonas gingivalis colonizing the dental plaques compared with the paired tracheal aspirate samples. These results support the hypothesis that dental bacteria may contribute to the pathology of severe AE-COPD.

Diversity of endophytic bacteria in Caragana microphylla grown in the desert grassland of the Ningxia Hui autonomous region of China.

The diversity of endophytic bacteria in the sand-fixation plant Caragana microphylla was investigated by amplified rDNA restriction analysis and by sequence and phylogenetic comparisons of the 16S rRNA genes. A total of 24, 19, and 17 operational taxonomic units were identified from 16S rDNA libraries of the plant roots, stems and leaves, respectively. Homology analysis revealed a 92-100% identity of bacterial 16S rDNA sequences compared with those in the GenBank database. The bacteria identified by sequence homology fell into the following groups: α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, bacilli, and uncultured bacterium. Sequence analysis demonstrated that the roots were colonized predominantly by Bradyrhizobiaceae, while bacteria from Burkholderiaceae and Sphingomonadaceae were predominant in the stems and leaves, respectively. Additionally, the endophytic bacterial community in leaves was more diverse than those in the roots and stems. Overall, the most abundant bacteria in all three tissues analyzed were from the Sphingomonadaceae and Burkholderiaceae families, although many bacterial populations were found in only a single tissue. These results suggest that the bacterial population of C. microphylla is diverse.

High throughput sequencing analysis of biogeographical distribution of bacterial communities in the black soils of northeast China

Black soils (Mollisols) are one of the most important soil resources for maintaining food security of China and are mainly distributed in northeast China. To understand which environmental factors influence the microbial communities and how the communities are distributed in the black soils, we collected 26 soil samples with different soil carbon contents across the black soil zone in northeast China, and the soil bacterial community compositions were estimated using high resolution bar-coded pyrosequencing. A total of 355,813 bacterial 16S rDNA sequences were obtained, which were classified into at least 35 bacterial groups. The dominant groups across all samples (>5% of all sequences) were Acidobacteria, Actinobacteria, Proteobacteria, Bacteroidetes, Chloroflexi, Gemmatimonadetes and Planctomycetes. The composition and diversity of the soil bacterial community were dominantly affected by both soil pH and soil total carbon content, and the effect of soil pH was stronger than that of soil carbon content. Variance partitioning analysis showed that geographic distance contributed 14.75% of the bacterial community variation, and soil environmental factors explained approximately 37.52% of the variation. Pairwise analysis showed that a relatively higher diversity of the bacterial community was observed at lower latitudes, suggesting that a latitudinal diversity gradient of the bacterial community might be present in the black soil zone. In general, our results indicated that contemporary factors, such as soil pH and soil carbon content, were more important than the historical factor of geographic distance in shaping the bacterial community in the black soil zone in northeast China.

Design and application of specific 16S rDNA-targeted primers for assessing endophytic diversity in Dendrobium officinale using nested PCR-DGGE

Novel specific 16S rDNA-targeted primers were successfully designed and applied to the characterization of endophytic diversity in Dendrobium officinale. Using the popular universal bacterial primers 27f/1492r, the fragments of chloroplast and mitochondrion 16S/18S rDNA were amplified from D. officinale. They shared high nucleotide identity with the chloroplast 16S rDNAs (99-100%) and with the mitochondrion 18S rDNAs (93-100%) from various plants, respectively, and both shared 73-86% identities with the bacterial 16S rDNA sequences in GenBank. The current bacterial universal primers, including 27f/1492r, match well with the chloroplast and mitochondrion 16S/18S rDNAs, which accordingly renders these primers not useful for endophytic diversity analysis. Novel 16S rDNA-targeted primers fM1 (5'-CCGCGTGNRBGAHGAAGGYYYT-3') and rC5 (5'-TAATCCTGTTTGCTCC CCAC-3') were designed, which show good specificity compared to the 16S/18S rDNAs of D. officinale, and perfect universality within bacteria except for Cyanobacteria. The primers fM1/rC5, together with 515f-GC/rC5, which overlaps the whole V4 region of 16S rDNA, were subjected to nested polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) to analyze the diversity of endophytic bacteria in D. officinale from three different sources in China. The results showed diversities in roots and stems of the plants from all three locations. Altogether, 29 bands were identified as bacteria, with the dominant group being Proteobacteria and the dominant genus being Burkholderia, some of which commonly has the function of nitrogen fixation and thus may play potentially important roles in D. officinale. Therefore, the nested PCR-DGGE method based on the novel primers provides a good alternative for investigating the communities and roles of endophytes in D. officinale.

Diversity and Chemical Defense Role of Culturable Non-Actinobacterial Bacteria Isolated from the South China Sea Gorgonians

The diversity of culturable non-actinobacterial (NA) bacteria associated with four species of South China Sea gorgonians was investigated using culture-dependent methods followed by analysis of the bacterial 16S rDNA sequence. A total of 76 bacterial isolates were recovered and identified, which belonged to 21 species of 7 genera, and Bacillus was the most diverse genus. Fifty-one percent of the 76 isolates displayed antibacterial activities, and most of them belonged to the Bacillus genus. From the culture broth of gorgonian-associated Bacillus methylotrophicus SCSGAB0092 isolated from gorgonian Melitodes squamata, 11 antimicrobial lipopeptides including seven surfactins and four iturins were obtained. These results imply that Bacillus strains associated with gorgonians play roles in coral defense mechanisms through producing antimicrobial substances. This study, for the first time, compares the diversity of culturable NA bacterial communities among four species of South China Sea gorgonians and investigates the secondary metabolites of gorgonian-associated B. methylotrophicus SCSGAB0092.