Full-length 16S rDNA Sequences Research Articles

Culturable bacterial endophytes of Aconitum carmichaelii Debx. were diverse in phylogeny, plant growth promotion, and antifungal potential.

Medicinal plants harbor tremendously diverse bacterial endophytes that maintain plant growth and health. In the present study, a total of 124 culturable bacterial endophytes were isolated from healthy Aconitum carmichaelii Debx. plants. These strains were clustered into 10 genera based on full-length 16S rDNA sequences, among which Bacillus and Pseudomonas were the dominant genera. In addition, A. carmichaelii may capture 10 potential new bacterial species based on multi-locus sequence analysis of three housekeeping genes (gyrA, rpoB, and atpD). The majority of these bacterial endophytes exhibited plant growth-promoting ability through diverse actions including the production of either indole acetic acid and siderophore or hydrolytic enzymes (glucanase, cellulose, and protease) and solubilization of phosphate or potassium. A total of 20 strains inhibited hyphal growth of fungal pathogens Sclerotium rolfsii and Fusarium oxysporum in vitro on root slices of A. carmichaelii by the dual-culture method, among which Pseudomonas sp. SWUSTb-19 showed the best antagonistic activity. Field experiment confirmed that Pseudomonas sp. SWUSTb-19 significantly reduced the occurrence of southern blight and promoted plant biomass compared with non-inoculation treatment. The possible mode of actions for Pseudomonas sp. SWUSTb-19 to antagonize against S. rolfsii involved the production of glucanase, siderophore, lipopeptides, and antimicrobial volatile compounds. Altogether, this study revealed that A. carmichaelii harbored diverse plant growth-promoting bacterial endophytes, and Pseudomonas sp. SWUSTb-19 could be served as a potential biocontrol agent against southern blight.

Alteration of gut microbiota in wild-borne long-tailed macaques after 1-year being housed in hygienic captivity

The wild-born long-tailed macaques (Macaca fascicularis) were recently recruited and used as breeders for the National Primate Research Center of Thailand, Chulalongkorn University (NPRCT-CU), and changes in their in-depth gut microbiota profiles were investigated. The Oxford Nanopore Technology (ONT) was used to explore full-length 16S rDNA sequences of gut microbiota in animals once captured in their natural habitat and 1-year following translocation and housing in a hygienic environment at NPRCT-CU. Our findings show that the gut microbiota of macaques after 1 year of hygienic housing and programmed diets feeding was altered and reshaped. The prevalent gut bacteria such as Prevotella copri and Faecalibacterium prausnitzii were enriched after translocation, causing the lower alpha diversity. The correlation analysis revealed that Prevotella copri, Phascolarctobacterium succinatutens, and Prevotella stercorea, showed a positive correlation with each other. Significantly enriched pathways in the macaques after translocation included biosynthesis of essential amino acids, fatty acids, polyamine and butanoate. The effects of microbiota change could help macaques to harvest the energy from programmed diets and adapt their gut metabolism. The novel probiotics and microbiota engineering approach could be further developed based on the current findings and should be helpful for captive animal health care management.

Integrated 16S rDNA, metabolomics, and TNF-α/NF-κB signaling pathway analyses to explain the modulatory effect of Poria cocos aqueous extract on anxiety-like behavior

BackgroundPoria cocos is an ancient medicine and modern functional food, which exerts excellent effects on anxiety, although its mechanism is unknown. PurposeTo explore the mechanisms of the aqueous extract of P. cocos (PCD) in ameliorating anxiety-like behavior caused by chronic sleep deprivation (CSD). MethodsPCD chemical composition was analyzed by UPLC-QTOF-MS/MS. A CSD rat model was established over 21 days. We examined the effects and mechanisms after 10 days of CSD using open-field tests (OFTs), enzyme-linked immunosorbent assays, 16S rDNA, non-targeted metabolomics, and Western blot analyses. ResultsSixty-two triterpenoids were identified in PCD. CSD-induced anxiety-like behavior was significantly attenuated by PCD treatment. PCD improved hypothalamic neurotransmitters, decreased proinflammatory cytokines, and depressed the proteins expression of tumor necrosis factor (TNF)-α/nuclear factor (NF)-κB signaling pathway. The full-length 16S rDNA sequence of bacterial cells was also sequenced by high-throughput analysis. CSD caused significant changes in the intestinal flora. PCD improved the species diversity and bacterial abundance in the intestines of rats with anxiety. Metabolomics analysis indicated that 12 PCD-related metabolites in serum and 32 PCD-related metabolites in feces were identified, respectively. Metabolite analysis in serum, PCD treatment affected taurine, hypotaurine, cysteine, methionine, glycine, serine, and threonine metabolism, among others. Metabolite analysis in feces showed significant effects of PCD treatment on the metabolism of vitamin B6, tyrosine, drugs, and glycerophospholipid. Additionally, the correlation analysis of heatmaps showed a tight relationship between inflammatory factors, metabolic parameters, and gut microbial phylotypes. ConclusionsPCD relieved anxiety by regulating intestinal flora, regulating metabolic disorders, and inhibiting inflammatory pathways in chronic sleep-deprived rats.

Nghiên cứu đa dạng vi khuẩn trong trầm tích biển tại quần đảo Trường Sa bằng phương pháp truyền thống kết hợp kỹ thuật điện di biến tính

The denaturing gradient gel electrophoresis (DGGE) is an effective culture-independent technique for assessing diversity of microbial communities. In this study, the PCR-DGGE technique was applied for analyzing the diversity of bacterial communities in marine sediment in the Truong Sa archipelago (Spratly Islands), Vietnam, and a comparison with cultivable bacterial fraction was performed. Three sediment samples taken from different depths were used in the study. A total of 17 bacterial strains were isolated and divided into 6 groups based on their colony and cell morphologies. Comparative analyses of nearly full-length 16S rDNA sequences retrieved from six representative strains of these morphologically distinguished groups indicated that they were most closely related to Bacillus amyloliquefaciens, Bacillus filamentosus, Bacillus endophyticus, Bacillus licheniformis, Pseudomonas balearica, and Raoultella ornithinolytica. The PCR-DGGE analyses carried out with the three sediment samples showed relatively diverse bacterial communities with 9 distinct bands, 6 of which were related to the isolated representatives, whereas the other three were identified as Vibrio proteolyticus, Priestia filamentosa, and Clostridium halophilum. Thus, by applying the PCR-DGGE technique, more thorough information on bacterial communities in the sediment of Truong Sa archipelago could be elucidated, providing useful information for further studies on the conservation and utilization of microbial resources in this ecosystem.

First Report of Necrotic Disease Caused by Pantoea agglomerans on Plum (Prunus salicina) in China

Prunus salicina L., commonly called Chinese plum or Japanese plum, is a small deciduous tree native to China and widely distributed in the world. Since spring of 2018, an unknown disease has affected commercial plums grown in an orchard located in Dazhou, Sichuan Province, China (31°40′41″N, 107°43′57″E). Disease symptoms in plum trees include water-soaked lesions on immature fruits, brown necrotic spots, and holes with a yellow halo in leaves, as well as dark brown oily cankers on twigs. To isolate the causal agent, surface-disinfested tissues from three separate symptomatic fruits, leaves, and twigs were placed onto nutrient agar (NA) plates and were incubated at 28°C. Pale yellow colonies with smooth margins and mucoid texture appeared after 12 h of incubation. Individual colonies were transferred two times to NA plates using the conventional streak plate techniques to obtain pure cultures. Purified bacteria cells were rod shaped, 1.5 to 3.0 μm long, and 0.5 to 1.0 μm wide. Three isolates (named CdjP1, CdjP2, and CdjP3) were used for further characterization. Biochemical tests using Gram stain, MacConkey agar, and Kovacs’s indole kit (Solarbio, Beijing, China) indicated that the three isolates were gram negative, lactose positive, and indole negative. The 16s rDNA fragment was amplified for the three strains using universal primers 27F and 1492R (Lane 1991) and sequenced (GenBank accession nos. MN542182, MN542183, and MN542184). BLASTn analysis revealed the nearly full-length (about 1,400 bp) 16S rDNA sequences of CdjP1, CdjP2, and CdjP3 shared over 99% identity with Pantoea agglomerans (MH101508) (identities 1,400/1,406, 1,407/1,412, 1,405/1,413), P. vagans (CP038853) (identities 1,397/1,406, 1,403/1,412, 1,402/1,414), and P. ananatis (KC178592) (identities 1,399/1,406, 1,405/1,412, 1,404/1,414), respectively. Multilocus sequence analysis has been shown to be a powerful molecular method for delineation of Pantoea species (Deletoile et al. 2009; Tambong et al. 2014). To identify the species of CdjP1, CdjP2, and CdjP3, six housekeeping genes (fusA, gyrB, leuS, pyrG, rlpB, and rpoB) of each strain were amplified and sequenced (GenBank accession nos. MN549363 to MN549380). Then a phylogenetic tree was constructed using Bayes inference methods based on fusA-gyrB-leuS-pyrG-rlpB-rpoB concatenated fragments (2,997 bp), which showed that CdjP1, CdjP2, and CdjP3 are most closely related to P. agglomerans. To fulfill Koch’s postulates, pathogenicity tests were conducted on 3-year-old plum trees (cv. Qinba) grown in Sichuan Province, China (31°40′53″N, 107°44′2″E). Immature plum fruits, leaves, and twigs were wounded with a sterile toothpick and were inoculated with 20 µl of bacterial suspension (10⁸ CFU/ml) of each isolate. Plants inoculated with sterile water were used as a control. All treatments and the control were repeated three times. The trees were then grown under natural conditions (monthly mean maximum temperature 32°C, monthly mean minimum temperature 23°C). After 2 weeks, all test-isolate-inoculated plum fruits, leaves, and twigs developed symptoms similar to those of the natural infections observed. No lesions were observed on the controls. Bacteria reisolated from the infected plum fruits, leaves, and twigs were similar in colony appearance to the original isolates, and the 16s rDNA fragments were identical to their original isolate sequences, thereby fulfilling Koch’s postulates. Based on colony morphology, biochemical tests, 16S rRNA gene sequencing data, and phylogenetic analysis, the pathogen was identified as P. agglomerans. In China, P. agglomerans has been reported to infect cotton, walnut, Vigna angularis, and Ziziphus jujuba (Lu et al. 2015; Ren et al. 2008; She et al. 2019; Yang et al. 2011). To our knowledge, this is the first report of P. agglomerans causing bacterial necrosis of plum in China or worldwide. This report expands the host range of P. agglomerans and will help to develop effective disease control strategies, as the etiology is known.

Species-level resolution of 16S rRNA gene amplicons sequenced through the MinION™ portable nanopore sequencer.

BackgroundThe miniaturised and portable DNA sequencer MinION™ has been released to the scientific community within the framework of an early access programme to evaluate its application for a wide variety of genetic approaches. This technology has demonstrated great potential, especially in genome-wide analyses. In this study, we tested the ability of the MinION™ system to perform amplicon sequencing in order to design new approaches to study microbial diversity using nearly full-length 16S rDNA sequences.ResultsUsing R7.3 chemistry, we generated more than 3.8 million events (nt) during a single sequencing run. These data were sufficient to reconstruct more than 90 % of the 16S rRNA gene sequences for 20 different species present in a mock reference community. After read mapping and 16S rRNA gene assembly, consensus sequences and 2d reads were recovered to assign taxonomic classification down to the species level. Additionally, we were able to measure the relative abundance of all the species present in a mock community and detected a biased species distribution originating from the PCR reaction using ‘universal’ primers.ConclusionsAlthough nanopore-based sequencing produces reads with lower per-base accuracy compared with other platforms, the MinION™ DNA sequencer is valuable for both high taxonomic resolution and microbial diversity analysis. Improvements in nanopore chemistry, such as minimising base-calling errors and the nucleotide bias reported here for 16S amplicon sequencing, will further deliver more reliable information that is useful for the specific detection of microbial species and strains in complex ecosystems.

CRYPTIC HAPLOTYPES OF “CANDIDATUS LIBERIBACTER AFRICANUS”

“Candidatus Liberibacter africanus” (Laf) has long been recognised as a causal agent of the devastating citrus disease huanglongbing (HLB) or citrus greening. This species is currently restricted to Africa, the Arabian Peninsula and some Indian Ocean islands and is vectored by the African citrus psyllid, Trioza erytreae. Blotchy mottle on citrus leaves is characteristic of the disease. Somewhat similar symptoms in the rutaceous tree Calodendrum capensis (Cape chestnut) resulted in the discovery of Laf outside commercial citrus crops in South Africa. This was classed as a subspecies of Laf (capensis, hence LafC). In subsequent surveys of commercial citrus crops and Calodendrum, both natural and ornamental specimens, LafC was not found in citrus, nor has Laf been found in C. capensis. HLB was reported from Madagascar in 1968 but no sequences from this source have so far been published. Until fairly recently, only the reference 16S rRNA gene sequences of Laf (L22533) and LafC (AF137368) had been deposited in GenBank. Both of these reference sequences contain a number of unresolved nucleotides. When these nucleotide positions are invstigated by aligning against more recently available sequences, it becomes evident that these unresolved positions represent one percentage point difference in similarity between Laf and LafC. The originally reported 97.4% similarity is therefore incorrect based on this new information. Recalculating the similarity on the full length 16S rDNA sequence results in 99.54% similarity, a value too high to justify a subspecies status. LafC should therefore be downgraded as a haplotype of Laf. Further, the six 16S rRNA gene sequences currently available in GenBank identified as Laf as a species separate into two haplotype groups. The three haplotypes of Laf are therefore: (i) LafA, designated as the first accession sequenced (L22533); (ii) LafC, i.e the former subspecies “capensis”, to recognise the priority in the use of this term; (iii) LafB, the third previously unrecognised haplotype. Thus the cryptic presence of three haplotypes is revealed by this review of the Laf 16S rDNA sequences.

First report of Tragopogon dubius witches' broom disease associated with a subgroup 16SrI‐B phytoplasma in Iran

Tragopogon dubius (yellow salsify, Asteraceae) is a perennial herb in Iran. In a survey conducted for phytoplasma diseases in 2014, T. dubius witches' broom disease was observed in vineyards and green areas of Bajgah and Zarghan localities (Fars province, Iran). The characteristic symptoms of T. dubius witches' broom were flower virescence and phyllody, crown proliferation and witches' broom (Fig. 1). Since the symptoms were suggestive of phytoplasma infection, plants were assayed for presence of phytoplasma by PCR amplification. Total DNA was extracted from 0.3 g of midrib tissue of four witches' broom affected and two symptomless T. dubius plants (two plants per location), using the procedure of Zhang et al. (6). Total DNA samples were tested by both direct and nested PCR assays using phytoplasma universal 16S rDNA primer pairs P1/P7 (Deng & Hiruki, 1; Schneider et al., 4) and R16F2n/R16R2 (Gundersen & Lee, 2) that amplify fragments of 1800 bp and 1250 bp, respectively. Expected fragments were amplified following direct and nested PCR from all symptom-bearing but not from symptomless plants. P1/P7-primed PCR products amplified from four witches' broom-affected T. dubius plants were separately cloned and sequenced. The full-length 16S rDNA sequences displayed 100% sequence identity with each other. A consensus sequence corresponding to the Bajgah T. dubius witches' broom was deposited in GenBank (Accession No. KR262955). A Blast search showed that this sequence had 100% identity with Oenothera phytoplasma (‘Candidatus Phytoplasma asteris‘) (M30790), representative of subgroup 16SrI-B. Virtual RFLP analysis using iPhyClassifier online tool (http://www.ba.ars.usda.gov/data/mppl/iphyclassifier), carried out with 17 restriction enzymes, indicated the presence of a 16SrI-B phytoplasma in witches' broom-affected T. dubius plants (Fig. 2). Phylogenetic analysis of full length 16S rDNA sequences using MEGA version 5 (Tamura et al., 5) grouped T. dubius witches' broom phytoplasma with onion yellows phytoplasma (GenBank Accession No. D12569), a member of 16SrI-B subgroup (Fig. 3). To our knowledge this is the first report of natural occurrence and partial molecular characterisation of a phytoplasma associated with T. dubius witches' broom disease. A 16SrI-B subgroup phytoplasma has previously been reported as causal agent of rapeseed phyllody disease in Zarghan (Salehi et al., 3). The significance of diseased T. dubius as natural reservoir of rapeseed phyllody phytoplasma remains to be determined. Aster yellows phytoplasmas were reported in grapevine in several countries. However, possible transmission of T. dubius witches' broom phytoplasma to grapevine is yet to be tested.

The feline oral microbiome: A provisional 16S rRNA gene based taxonomy with full-length reference sequences

The human oral microbiome is known to play a significant role in human health and disease. While less well studied, the feline oral microbiome is thought to play a similarly important role. To determine roles oral bacteria play in health and disease, one first has to be able to accurately identify bacterial species present. 16S rRNA gene sequence information is widely used for molecular identification of bacteria and is also useful for establishing the taxonomy of novel species.The objective of this research was to obtain full 16S rRNA gene reference sequences for feline oral bacteria, place the sequences in species-level phylotypes, and create a curated 16S rRNA based taxonomy for common feline oral bacteria.Clone libraries were produced using “universal” and phylum-selective PCR primers and DNA from pooled subgingival plaque from healthy and periodontally diseased cats. Bacteria in subgingival samples were also cultivated to obtain isolates. Full-length 16S rDNA sequences were determined for clones and isolates that represent 171 feline oral taxa. A provisional curated taxonomy was developed based on the position of each taxon in 16S rRNA phylogenetic trees.The feline oral microbiome curated taxonomy and 16S rRNA gene reference set will allow investigators to refer to precisely defined bacterial taxa. A provisional name such as “Propionibacterium sp. feline oral taxon FOT-327” is an anchor to which clone, strain or GenBank names or accession numbers can point. Future next-generation-sequencing studies of feline oral bacteria will be able to map reads to taxonomically curated full-length 16S rRNA gene sequences.

First Report of Bacterial Wilt Caused by Ralstonia solanacearum on Ageratum conyzoides in China.

Ageratum conyzoides L. is believed to act as reservoir host for many plant diseases. In June 2011, a 30% incidence of bacterial wilt on A. conyzoides was observed in a field of Rhizoma kaempferiae in Yangchun city of Guangdong province. The initial symptoms were wilting of the apical leaves during the day, which recovered at night. After 4 to 6 days, the leaves became totally necrotic. The basal stems of the diseased plants were blackened and the vascular tissue turned brown. To investigate the disease etiology before understanding the disease link between A. conyzoides and R. kaempferiae, 10 plants with typical wilting symptoms were collected from the field. A total of 10 bacterial isolates were isolated from the vascular tissue of each diseased plant on tripheny tetrazolium chloride (TZC) medium. After incubation at 30°C for 2 days, the plates had large, irregular round, fluidal, white colonies with a pink center. Thirty healthy A. conyzoides plants at the four- to six-leaf growth stage were inoculated by injuring the roots and soaking them in a bacterial suspension (1 × 108 cfu/ml) for 20 min with the 10 bacterial isolates separately, and planted in 10-cm pots with sterile gardening soil in a glasshouse (28 to 35°C). Sterile water was used as a negative control. Five days after inoculation, a few leaves of the inoculated plants began to exhibit wilting. The inoculated plants eventually showed the same symptoms as those in the field. The same bacterium was reisolated from inoculated plants. The 30 negative control plants did not have wilt symptoms. With the same inoculation procedure, the bacterium also caused wilting on tomato (25 of 30), pepper (10 of 30), eggplant (2 of 30), ginger (11 of 15), and R. kaempferiae (8 of 15). Using the universal bacterial 16S rDNA primer set 27f/1541R (3), approximately 1,400 bp-fragments were amplified from the 10 isolates, respectively. The sequences for the 10 fragments (GenBank Accession Nos. JX294065 to JX294074) were identical and had 100% sequence identity with 16S rDNA of R. solanacearum GMI1000 (AL646052). The 10 isolates were able to oxidize disaccharides (lactose, maltose, and cellobiose) and hexose alcohols (mannitol, dulcitol, and sorbitol). According to Hayward's classification, all isolates were biovar 3 (2). Based on the pathogenicity tests, carbohydrate utilization, and near full-length 16S rDNA sequences, the bacterial isolates from the diseased A. conyzoides belonged to race 4 and biovar 3 of R. solanacearum. Furthermore, the specific 280-bp and 140-bp fragments were respectively amplified from all 10 isolates by using the multiplex PCR (1). In addition, specific 165-bp fragments were amplified from all the isolates using the specific primers AKIF/AKIR (3), which indicates the bacterium belongs to R. solanacearum Phylotype I. To our knowledge, this is the first report of a disease caused by R. solanacearum on A. conyzoides in China.

Molecular characterization of a phytoplasma of the ash yellows group occurring in strawberry (Fragaria x ananassa Duch.) plants in Argentina

Strawberry (Fragaria x ananassa Duch. cv. Camino Real) plants showing phyllody symptoms were detected in production fields in Bella Vista (Corrientes province, Argentina). DNA from all symptomatic samples analyzed yielded fragments of the expected size in PCR reactions using phytoplasma universal primers. DNA from asymptomatic plants yielded no products. The associated phytoplasma was designated as Argentinean Strawberry Phyllody (ASP). The ASP sequence of the 16S rRNA gene showed 99–98% homology with members of the 16SrVII ash yellows group. The putative RFLP profile was indistinguishable from the Argentinean alfalfa witches’ broom (ArAWB) phytoplasma. The phylogenetic analysis of nearly full-length 16S rDNA sequence and 16S-23S spacer region yielded a consensus tree wherein ASP clustered into the ash yellows group (16Sr VII) with high confidence values (95), generating a separated branch (100 bootstrap value) together with ArAWB (16Sr VII-C). In Argentina, the 16Sr VII group was also detected in alfalfa (Medicago sativa L.) crops, and weeds [Artemisa annua L. and Conyza bonariensis (L.) Cronquist]. To our knowledge, this is the first report of a phytoplasma from the 16Sr VII group affecting strawberries in Argentina and worldwide.

Enrichment and identification of large filamentous sulfur bacteria related to Beggiatoa species from brackishwater ecosystems of Tamil Nadu along the southeast coast of India

Beggiatoa species are filamentous sulfide-oxidizing bacteria belonging to the family Beggiatoaceae that contains several largest bacteria known today. These large sulfur bacteria occur in diverse ecosystems and play an important role in the global sulfur, nitrogen and phosphorus cycle. In this study, sediment samples from brackishwater shrimp culture ponds and other brackishwater ecosystems from Tamil Nadu, southeast coast of India, were enriched for Beggiatoa species. Extracted hay medium supplemented with catalase was used and were incubated for two weeks at 28°C. Out of seven set-ups, four yielded positive growth of filamentous sulfide-oxidizing bacteria. The filaments were several millimeters long, ranged in width between 2 and 15μm and exhibited typical gliding motility. The 16S rRNA gene of four single filaments representing the four positive enrichments was subjected to PCR-DGGE followed by sequencing. All four filaments were affiliated to the Beggiatoaceae, but showed less than 89% identity with the Beggiatoa type strain Beggiatoa alba and less than 93% identity with any other sequence of the family. One of the four filaments revealed a nearly full-length 16S rDNA sequence (1411bp) and it formed a monophyletic cluster with two of the partial DGGE-16S rRNA gene sequences (99–100% identity) within the Beggiatoa species cluster. These organisms could possibly represent a novel genus within the family Beggiatoaceae. The fourth partial sequence affiliated with less than 93% sequence identity to the genera Parabeggiatoa, Thioploca and Thiopilula, and was likewise strongly delineated from any sequence published in the family.

Production of conjugated linoleic acids by Lactobacillus plantarum strains isolated from naturally fermented Chinese pickles.

Naturally fermented pickles harbour many lactic acid bacteria (LAB). Forty-three LAB strains with conjugated linoleic acid (CLA)-producing ability were isolated from three naturally fermented pickle brines. Of these isolates, lp15 identified as Lactobacillus plantarum by API 50 CHL system and full-length 16S rDNA sequence analysis exhibited the highest CLA-producing ability (26.1% conversion) at 48 h in de Man Rogosa Sharpe (MRS) broth in the presence of 100 µg/ml of linoleic acid (LA). Compared to other strains, L. plantarum strain lp15 showed the highest tolerance upon increased levels of LA in the medium, i.e., up to 600 µg/ml. This strain converted about 25% of LA into CLA isomers [predominantly cis-9, trans-11 CLA (9-CLA) and trans-10, cis-12 CLA (10-CLA)], of which 75% was 9-CLA. Interestingly, though the conversion rate of LA into CLA by lp15 remained stable between 100 to 600 µg/ml LA levels in the medium, it dropped sharply at 1000 µg/ml. Taken together, the lp15 strain displayed relatively high LA tolerance with higher conversion rate, which implies that this strain is a valuable candidate for enhancing the CLA content in food-sources like pickles.

Phylogenetic diversity of bacteria associated with the endemic freshwater sponge Lubomirskia baicalensis

In this study, for the first time the diversity of bacteria associated with the endemic freshwater sponge Lubomirskia baicalensis collected from the Sousern Basin of Lake Baikal was investigated employing cultivation-independent approaches. In total, 102 bacterial 16S rRNA clones were screened using restriction fragment length polymorphism (RFLP) and 30 were selected for sequencing. BLASTN and phylogenetic analysis based on near full length 16S rDNA sequences showed that 22 operational taxonomic units (OTUs) were clustered in six known phyla: Actinobacteria (8 OTUs), alpha-Proteobacteria (4 OTUs), beta-Proteobacteria (4 OTUs), Verrucomicrobia (4 OTUs), Nitrospiracea (1 OTU) and Bacteroidetes (1 OTU). Remarkably all phylotypes were affiliated to uncultured microorganisms, however, all alpha-Proteobacteria sequences were closely related to bacteria derived from the freshwater sponge Spongilla lacustris. Our results reveal a high diversity in the L. baicalensis bacterial community and provide an insight into microbial ecology and diversity within freshwater sponges inhabiting the ancient Lake Baikal ecosystem.

Bacterial Community Analysis of Beef Cattle Feedlots Reveals That Pen Surface Is Distinct from Feces

The surface of beef cattle feedlot pens is commonly conceptualized as being packed uncomposted manure. Despite the important role that the feedlot pen may play in the transmission of veterinary and zoonotic pathogens, the bacterial ecology of feedlot surface material is not well understood. Our present study characterized the bacterial communities of the beef cattle feedlot pen surface material using 3647 full-length 16S rDNA sequences, and we compared the community composition of feedlot pens to the fecal source material. The feedlot surface composite was represented by members of the phylum Actinobacteria (42%), followed by Firmicutes (24%), Bacteroidetes (24%), and Proteobacteria (9%). The feedlot pen surface material bacterial communities were clearly distinct from those of the feces from animals in the same pen. Comparisons with previously published results of feces from the animals in the same pen reveal that, of 139 genera identified, only 25 were present in both habitats. These results indicate that, microbiologically, the feedlot pen surface material is separate and distinct from the fecal source material, suggesting that bacteria that originate in cattle feces face different selection pressures and survival challenges during their tenure in the feedlot pen, as compared to their residence in the gastrointestinal tract.

Metagenomic study of the human skin microbiome associated with acne

The human microbiota contributes to our normal postnatal development and plays a significant role in defining our physiology. To understand the role of microbiota in human health and disease, we study the skin microbiome in pilosebaceous units (hair follicles) and its association with acne.Acne is one of the most common skin diseases. Although its etiology still needs to be defined, a bacterial factor has been suggested in the development of the disease. In fact, antibiotic therapy targeting Propionibacterium acnes has been a mainstay treatment for more than 30 years.Our preliminary study shows that the microcomedone, a specialized skin compartment where acne arises, has a tractable microbiome, with a single dominant species, P. acnes. This system offers a unique advantage allowing in-depth analysis of a human microbiome at the subspecies level by sequencing. Our preliminary study suggests that the microbiome associated with acne offers promise for understanding the correlation between the composition of the microbiome and human health and disease.The goal of the project is to determine whether the microbiota in the pilosebaceous units contributes to acne. We plan to investigate the microbiome associated with acne in three directions. First, we plan to investigate the strain diversity of P. acnes in a disease cohort and a normal cohort and examine whether certain strains of P. acnes are correlated with the disease. Second, we plan to investigate the non- P. acnes microbes in microcomedones and disease lesions and examine whether they correlate with acne pathogenesis. Third, we will examine the interactions between the microbes and the host by transcriptional profiling of both the microbiota and the host.During the first year of this project, two main questions were asked. 1. Are certain strains of P. acnes associated with acne, but rarely found in normal individuals? 2. If specific strains are associated with acne, what are the differences in their genetic composition compared to other P. acnes strains that are not associated with acne? We collected microcomedone samples from more than 100 subjects, including acne patients and normal individuals. Genomic DNA was extracted from each sample, and 16S rDNA was amplified using universal primers (8F and 1510R), cloned and sequenced using Sanger method. Approximately 384 near full length 16S rDNA sequences were obtained for each sample. Some of the microcomedone samples were also cultured under anaerobic condition to isolate different P. acnes strains. Sixty-eight isolates were selected for whole genome shotgun sequencing using Solexa/Illumina platform. By the end of the first year of the project, we completed the sequencing of more than 40,000 16S rDNA clones and 68 genomes of P. acnes isolates.

Metagenomic study of the human skin microbiome associated with acne

AbstractThe human microbiota contributes to our normal postnatal development and plays a significant role in defining our physiology. To understand the role of microbiota in human health and disease, we study the skin microbiome in pilosebaceous units (hair follicles) and its association with acne.Acne is one of the most common skin diseases. Although its etiology still needs to be defined, a bacterial factor has been suggested in the development of the disease. In fact, antibiotic therapy targeting Propionibacterium acnes has been a mainstay treatment for more than 30 years.Our preliminary study shows that the microcomedone, a specialized skin compartment where acne arises, has a tractable microbiome, with a single dominant species, P. acnes. This system offers a unique advantage allowing in-depth analysis of a human microbiome at the subspecies level by sequencing. Our preliminary study suggests that the microbiome associated with acne offers promise for understanding the correlation between the composition of the microbiome and human health and disease.The goal of the project is to determine whether the microbiota in the pilosebaceous units contributes to acne. We plan to investigate the microbiome associated with acne in three directions. First, we plan to investigate the strain diversity of P. acnes in a disease cohort and a normal cohort and examine whether certain strains of P. acnes are correlated with the disease. Second, we plan to investigate the non- P. acnes microbes in microcomedones and disease lesions and examine whether they correlate with acne pathogenesis. Third, we will examine the interactions between the microbes and the host by transcriptional profiling of both the microbiota and the host.During the first year of this project, two main questions were asked. 1. Are certain strains of P. acnes associated with acne, but rarely found in normal individuals? 2. If specific strains are associated with acne, what are the differences in their genetic composition compared to other P. acnes strains that are not associated with acne? We collected microcomedone samples from more than 100 subjects, including acne patients and normal individuals. Genomic DNA was extracted from each sample, and 16S rDNA was amplified using universal primers (8F and 1510R), cloned and sequenced using Sanger method. Approximately 384 near full length 16S rDNA sequences were obtained for each sample. Some of the microcomedone samples were also cultured under anaerobic condition to isolate different P. acnes strains. Sixty-eight isolates were selected for whole genome shotgun sequencing using Solexa/Illumina platform. By the end of the first year of the project, we completed the sequencing of more than 40,000 16S rDNA clones and 68 genomes of P. acnes isolates.

Bacterial diversity in surface sediments from the Pacific Arctic Ocean

In order to assess bacterial diversity within four surface sediment samples (0-5 cm) collected from the Pacific Arctic Ocean, 16S ribosomal DNA clone library analysis was performed. Near full length 16S rDNA sequences were obtained for 463 clones from four libraries and 13 distinct major lineages of Bacteria were identified (alpha, beta, gamma, delta and epsilon-Proteobacteria, Acidobacteria, Bacteroidetes, Chloroflexi, Actinobacteria, Firmicutes, Planctomycetes, Spirochetes, and Verrucomicrobia). alpha, gamma, and delta-Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria were common phylogenetic groups from all the sediments. The gamma-Proteobacteria were the dominant bacterial lineage, representing near or over 50% of the clones. Over 35% of gamma-Proteobacteria clones of four clone library were closely related to cultured bacterial isolates with similarity values ranging from 94 to 100%. The community composition was different among sampling sites, which potentially was related to geochemical differences.

Bacterial diversity in activated sludge from a consecutively aerated submerged membrane bioreactor treating domestic wastewater

The bacterial diversity of activated sludge from submerged membrane bioreactor (SMBR) was investigated. A 16S rDNA clone library was generated, and 150 clones were screened using restriction fragment length polymorphism (RFLP). Of the screened clones, almost full-length 16S rDNA sequences of 64 clones were sequenced. Phylogenetic tree was constructed with a database containing clone sequences from this study and bacterial rDNA sequences from NCBI for identification purposes. The 90.6% of the clones were affiliated with the two phyla Bacteroidetes (50%) and Proteobacteria (40%), and beta-, gamma-, and delta-Proteobacteria accounted for 7.8%, 28.1%, and 4.7%, respectively. Minor portions were affiliated with the Actinobacteria and Firmicutes (both 3.1%). Only 6 out of 64 16S rDNA sequences exhibited similarities of more than 97% to classified bacterial species, which indicated that a substantial fraction of the clone sequences were derived from unknown taxa. Rarefaction analysis of operational taxonomic units (OTUs) clusters demonstrated that 150 clones screened were still insufficient to describe the whole bacterial diversity. Measurement of water quality parameter demonstrated that performance of the SMBR maintained high level, and the SMBR system remained stable during this study.

Diversity of endophytic diazotrophs isolated fromBambusa blumeanain Guangdong province

AbstractEndophytic bacteria isolated from Guangdong bamboo (Bambusa blumeana) plants were obtained under aerobic and anaerobic cultural conditions. Using the acetylene reduction assay (ARA), 40 strains were identified as diazotrophs. Diazotrophic isolates were grouped into five clusters (I, II, III, IV and V) by SDS-PAGE whole-cell protein patterns. The analysis of full-length 16S rDNA sequences of the representative strains of each group indicated that the endophytic diazotrophs showed great diversity and belonged to the generaAzospirillum(α-subclass of proteobacteria),EscherichiaandPseudomonas(γ-subclass of proteobacteria), andAquaspirillum(β-subclass of proteobacteria).