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1.
DNA sequencing has been revolutionized by the development of high-throughput sequencing technologies. Plummeting costs and the massive throughput capacities of second and third generation sequencing platforms have transformed many fields of biological research. Concurrently, new data processing pipelines made rapid de novo genome assemblies possible. However, high quality data are critically important for all investigations in the genomic era. We used chloroplast genomes of one Oryza species (O. australiensis) to compare differences in sequence quality: one genome (GU592209) was obtained through Illumina sequencing and reference-guided assembly and the other genome (KJ830774) was obtained via target enrichment libraries and shotgun sequencing. Based on the whole genome alignment, GU592209 was more similar to the reference genome (O. sativa: AY522330) with 99.2% sequence identity (SI value) compared with the 98.8% SI values in the KJ830774 genome; whereas the opposite result was obtained when the SI values in coding and noncoding regions of GU592209 and KJ830774 were compared. Additionally, the junctions of two single copies and repeat copies in the chloroplast genome exhibited differences. Phylogenetic analyses were conducted using these sequences, and the different data sets yielded dissimilar topologies: phylogenetic replacements of the two individuals were remarkably different based on whole genome sequencing or SNP data and insertions and deletions (indels) data. Thus, we concluded that the genomic composition of GU592209 was heterogeneous in coding and non-coding regions. These findings should impel biologists to carefully consider the quality of sequencing and assembly when working with next-generation data.  相似文献   

2.
We report here the complete genomic sequence of a novel duck circovirus (DuCV) strain, GX1104, isolated from Guangxi pockmark ducks in Guangxi, China. The whole nucleotide sequence had the highest homology (97.2%) with the sequence of strain TC/2002 (GenBank accession number AY394721.1) and had a low homology (76.8% to 78.6%) with the sequences of other strains isolated from China, Germany, and the United States. This report will help to understand the epidemiology and molecular characteristics of Guangxi pockmark duck circovirus in southern China.  相似文献   

3.
We analyzed the temporal and spatial diversity of the microbiota in a low-usage and a high-usage hospital tap. We identified a tap-specific colonization pattern, with potential human pathogens being overrepresented in the low-usage tap. We propose that founder effects and local adaptation caused the tap-specific colonization patterns. Our conclusion is that tap-specific colonization represents a potential challenge for water safety.Humans are exposed to and consume large amounts of tap water in their everyday life, with the tap water microbiota representing a potent reservoir for pathogens (8). Despite the potential impact, our knowledge about the ecological diversification processes of the tap water microbiota is limited (4, 11).The aim of the present work was to determine the temporal and spatial distribution patterns of the planktonic tap water microbiota. We compared the summer and winter microbiota from two hospital taps supplied from the same water source. We analyzed 16S rRNA gene clone libraries by using a novel alignment-independent approach for operational taxonomic unit (OTU) designation (6), while established OTU diversity and richness estimators were used for the ecological interpretations.Tap water samples (1 liter) from a high-usage kitchen and a low-usage toilet cold-water tap in Akershus University Hospital, Lørenskog, Norway, were collected in January and July 2006. The total DNA was isolated and the 16S rRNA gene PCR amplified and sequenced. Based on the sequences, we estimated the species richness and diversity, we calculated the distances between the communities, and trees were constructed to reflect the relatedness of the microbiota in the samples analyzed. Details about these analytical approaches are given in the materials and methods section in the supplemental material.Our initial analysis of species composition was done using the RDPII hierarchical classifier. We found that the majority of pathogen-related bacteria in our data set belonged to the class Gammaproteobacteria. The genera encompassed Legionella, Pseudomonas, and Vibrio (Table ​(Table1).1). We found a significant overrepresentation of pathogen-related bacteria in the toilet tap (P = 0.04), while there were no significant differences between summer and winter samples. Legionella showed the highest relative abundance for the pathogen-related bacteria. With respect to the total diversity, we found that Proteobacteria dominated the tap water microbiota (representing 86% of the taxa) (see Table S1 in the supplemental material). There was, however, a large portion (56%) of the taxa that could not be assigned to the genus level using this classifier.

TABLE 1.

Cloned sequences related to human pathogensa
Sampling placeSampling timePathogenNCBI accession no.Identity (%)
ToiletSummerEscherichia coliEF41861499
ToiletSummerEscherichia sp.EF07430799
ToiletSummerLegionella sp.AY92415595
ToiletSummerLegionella sp.AY92415395
ToiletSummerLegionella sp.AY92415396
ToiletWinterLegionella sp.AY92406196
ToiletWinterLegionella sp.AY92415897
ToiletWinterLegionella sp.AY92415897
KitchenWinterLegionella sp.AY92399697
ToiletSummerPseudomonas fluorescensEF41307398
ToiletSummerPseudomonas fluorescensEF41307398
KitchenSummerPseudomonas fluorescensDQ20773199
ToiletWinterVibrio sp.DQ40838898
ToiletWinterVibrio sp.AB27476098
KitchenWinterVibrio sp.DQ40838898
KitchenWinterVibrio lentusAY29293699
KitchenWinterVibrio sp.AM18376597
ToiletWinterStenotrophomonas maltophiliaAY83773099
KitchenWinterStenotrophomonas maltophiliaDQ42487098
ToiletWinterStreptococcus suisAF28457898
ToiletWinterStreptococcus suisAF28457898
Open in a separate windowaThe relatedness between the cloned sequences and potential pathogens was determined by BLAST searches of the NCBI database, carried out using default settings.To obtain a better resolution of the uncharacterized microbiota, we analyzed the data using a clustering approach that is not dependent on a predefined bacterial group (see the materials and methods section in the supplemental material for details). These analyses showed that there were three relatively tightly clustered groups in our data set (Fig. ​(Fig.1A).1A). The largest group (n = 590) was only distantly related to characterized betaproteobacteria within the order Rhodocyclales. We also identified another large betaproteocaterial group (n = 320) related to Polynucleobacter. Finally, a tight group (n = 145) related to the alphaproteobacterium Sphingomonas was identified.Open in a separate windowFIG. 1.Tap water microbiota diversity, determined by use of a principal component analysis coordinate system. (A) Each bacterium is classified by coordinates, with the following color code: brown squares, kitchen summer; red diamonds, toilet summer; green triangles, kitchen winter; and green circles, toilet winter. (B and C) Each square represents a 1 × 1 (B) or 5 × 5 (C) OTU. PC1, first principal component; PC2, second principal component.The tap-specific distributions of the bacterial groups were investigated using density distribution analyses. A dominant population related to Polynucleobacter was identified for the toilet summer samples, while for the winter samples there was a dominance of the Rhodocyclales-related bacteria. The kitchen summer samples revealed a dominance of Sphingomonas. The corresponding winter samples did not reveal distinct high-density bacterial populations (see Table S2 in the supplemental material).Hierarchical clustering for the 1 × 1 OTU density distribution confirmed the relatively low overlap for the microbiota in the samples analyzed (Fig. ​(Fig.2).2). We found that the microbiota clustered according to tap and not season.Open in a separate windowFIG. 2.Hierarchical clustering for the density distribution of the tap water microbiota. The density of 1 × 1 OTUs was used as a pseudospecies for hierarchical clustering. The tree for the Cord distance matrix is presented, while the distances calculated using the three distance matrices Cord, Brad Curtis, and Sneath Sokal, respectively, are shown for each branch.We have described the species diversity and richness of the microbiota in Table S3 in the supplemental material. For the low taxonomic level, these analyses showed that the diversity and species richness were greater for the winter samples than for the summer samples. Comparing the two taps, the diversity and richness were greater in the kitchen tap than in the toilet tap. In particular, the winter sample from the kitchen showed great richness and diversity. The high taxonomic level, however, did not reveal the same clear differences as did the low level, and the distributions were more even. Rarefaction analyses for the low taxonomic level confirmed the richness and diversity estimates (see Fig. S1 in the supplemental material).Our final analyses sought to fit the species rank distributions to common rank abundance curves. Generally, the rank abundance curves were best fitted to log series or truncated log normal distributions (see Table S4 in the supplemental material). The log series distribution could be fit to all of the samples except the kitchen summer samples at the low taxonomic level, while the truncated log normal distribution could not be fit to the kitchen samples at the high taxonomic level. Interestingly, however, the kitchen winter sample was best fit to a geometric curve at both the high and the low taxonomic level.Diversifying, adaptive biofilm barriers have been documented for tap water bacteria (7), and it is known that planktonic bacteria can interact with biofilms in an adaptive manner (3). On the other hand, tap usage leads to water flowthrough and replacement of the global with the local water population by stochastic founder effects (1).Therefore, we propose that parts of the local diversity observed can be explained by local adaptation (10) and parts by founder effects (9).Most prokaryote diversity measures assume log normal or log series OTU dominance density distributions (5). The kitchen winter sample, however, showed deviations from these patterns by being correlated to geometric distributions (in addition to the log series and truncated log normal distributions for the high taxonomic level). This sample also showed a much greater species richness than the other samples. A possible explanation is that the species richness of the tap water microbiota can be linked to usage and that the kitchen tap is driven toward a founder microbiota by high usage.Since our work indicates an overrepresentation of Legionella in the low-usage tap, it would be of high interest to determine whether the processes for local Legionella colonization can be related to tap usage. Understanding the ecological forces affecting Legionella and other pathogens are of great importance for human health. At the Akerhus University Hospital, this was exemplified by a Pseudomonas aeruginosa outbreak in an intensive care unit, where the outbreak could be traced back to a single tap (2).  相似文献   

4.
Three cohorts of farmed yellowtail kingfish (Seriola lalandi) from South Australia were examined for Chlamydia-like organisms associated with epitheliocystis. To characterize the bacteria, 38 gill samples were processed for histopathology, electron microscopy, and 16S rRNA amplification, sequencing, and phylogenetic analysis. Microscopically, the presence of membrane-enclosed cysts was observed within the gill lamellae. Also observed was hyperplasia of the epithelial cells with cytoplasmic vacuolization and fusion of the gill lamellae. Transmission electron microscopy revealed morphological features of the reticulate and intermediate bodies typical of members of the order Chlamydiales. A novel 1,393-bp 16S chlamydial rRNA sequence was amplified from gill DNA extracted from fish in all cohorts over a 3-year period that corresponded to the 16S rRNA sequence amplified directly from laser-dissected cysts. This sequence was only 87% similar to the reported “Candidatus Piscichlamydia salmonis” (AY462244) from Atlantic salmon and Arctic charr. Phylogenetic analysis of this sequence against 35 Chlamydia and Chlamydia-like bacteria revealed that this novel bacterium belongs to an undescribed family lineage in the order Chlamydiales. Based on these observations, we propose this bacterium of yellowtail kingfish be known as “Candidatus Parilichlamydia carangidicola” and that the new family be known as “Candidatus Parilichlamydiaceae.”  相似文献   

5.
Lactobacillus plantarum is a lactic acid bacterium (LAB) species commonly used as a probiotic. We have sequenced the genome of Lactobacillus plantarum JDM1, which is a Chinese commercial LAB with several probiotic functions, using a GS 20 system. We recommend that each commercial probiotic strain should undergo complete genome sequencing to ensure safety and stability.Lactic acid bacteria (LAB) play a prominent role in the world food supply, performing the main bioconversions in fermented food, and are also used as probiotic supplements in dairy products and other foods. Lactobacillus plantarum is a LAB species commonly used as a probiotic. We have sequenced the genome of Lactobacillus plantarum JDM1, which is a widely used Chinese commercial LAB with several probiotic functions, using a GS 20 system (454 Life Science Corporation) (11). Two hundred thirty-six thousand, five hundred sixty-three high-quality reads were assembled with the 454 assembly tool, which had an average depth of 18.6-fold coverage of the genome and yielded 367 contigs. Among these, 225 large contigs represented 99.17% of the draft sequence. In the finishing process, the order of the selected large contigs was determined by BLAST analysis with the originally published genome sequence of strain WCFS1 (GenBank accession number AL935263) (8). Physical gaps were filled through sequencing of PCR products that spanned these regions using ABI 3730 xl DNA sequencers. Sequence assembly was accomplished by using the Phred/Phrap/Consed software package (4, 7). To ensure final accuracy, the errors in homopolymer sites that arose from the pyrosequencing method were solved via comparison with the corresponding sites on WCFS1 and then resequencing of the ambiguous bases using the ABI 3730 xl DNA sequencer.The complete genome of Lactobacillus plantarum JDM1 contains a single, circular chromosome of 3,197,759 bp and two plasmids (pLP2000 [2,062 bp] and pLP9000 [9,254 bp]). The two plasmids have been sequenced and published, with GenBank accession numbers AY096004 and AY096005 (3). The overall GC content of the chromosome is 44.66%, whereas the plasmids have a GC content slightly lower than that of the chromosome. The entire genome of JDM1 contains 2,948 protein-coding genes, 62 tRNA-encoding genes, and 16 rRNA-encoding genes. Several repeated sequences, designated ISP2, were found in the chromosome which were almost the same as those in WCSF1, identified as a class of transposase-encoding regions representing mobile genetic elements. The other repeated sequence, ISP1 of WCSF1, was absent in JDM1.The entire genomic sequence of L. plantarum JDM1 was a little shorter than that of L. plantarum WCSF1 (3.3 Mb). The two genomes were highly similar (>90% by BLASTN analysis) with respect to genome structure and gene order. Intraspecies diversity may be required for successful adaptation in a complex ecological habitat (2). L. plantarum JDM1 has been grown as a probiotic in rich nutritional medium for so long that the genome may have gradually contracted. As supporting evidence, many sugar transport and metabolism genes in WCFS1 were absent in JDM1.The prophage sequences and locations of JDM1 and WCFS1 are highly variable. L. plantarum JDM1 contains three prophage elements in its genome. R-Pg1, representing a short prophage remnant, is about 14 kb in size, which is similar to R-Lp3 in WCFS1. Pg2 and Pg3 are two ∼39-kb-long prophages that are closely related to Listeria phage B025 (accession no. DQ003639) and the phage Pediococcus pentosaceus ATCC 25745 (accession no. CP000422), respectively.The genomes of LAB evolve actively to adapt to nutritionally rich environments. Even for two strains of the same species, differences obviously exist. The degradation of the genome appears to be an ongoing process not only in all species of Lactobacillus (10) but also in different strains of the same species(2).With the development of better living conditions, the biosafety of food and medicine has received more attention. Lactobacillus bacteria have been supposed to have a “generally accepted as safe” status, but they still have been associated with negative reports (1, 6, 9). More about the functional mechanisms of JDM1 and potential side effects would be explored by complete genome sequencing and data mining. Furthermore, comparative genomics analysis could be carried out with Chinese and European strains. We believe the complete genome of each probiotic strain should be sequenced to ensure safety and stability. At the end of the day, we will get what we pay for in terms of microbial genome sequencing projects (5).  相似文献   

6.
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7.
Containment strategies for outbreaks of invasive Neisseria meningitidis disease are informed by serogroup assays that characterize the polysaccharide capsule. We sought to uncover the genomic basis of conflicting serogroup assay results for an isolate (M16917) from a patient with acute meningococcal disease. To this end, we characterized the complete genome sequence of the M16917 isolate and performed a variety of comparative sequence analyses against N. meningitidis reference genome sequences of known serogroups. Multilocus sequence typing and whole-genome sequence comparison revealed that M16917 is a member of the ST-11 sequence group, which is most often associated with serogroup C. However, sequence similarity comparisons and phylogenetic analysis showed that the serogroup diagnostic capsule polymerase gene (synD) of M16917 belongs to serogroup B. These results suggest that a capsule-switching event occurred based on homologous recombination at or around the capsule locus of M16917. Detailed analysis of this locus uncovered the locations of recombination breakpoints in the M16917 genome sequence, which led to the introduction of an ∼2-kb serogroup B sequence cassette into the serogroup C genomic background. Since there is no currently available vaccine for serogroup B strains of N. meningitidis, this kind capsule-switching event could have public health relevance as a vaccine escape mutant.  相似文献   

8.
  相似文献   

9.
Braarudosphaera bigelowii (Prymnesiophyceae) is a coastal coccolithophore with a long fossil record, extending back to the late Cretaceous (ca. 100 Ma). A recent study revealed close phylogenetic relationships between B. bigelowii, Chrysochromulina parkeae (Prymnesiophyceae), and a prymnesiophyte that forms a symbiotic association with the nitrogen-fixing cyanobacterium UCYN-A. In order to further examine these relationships, we conducted transmission electron microscopic and molecular phylogenetic studies of B. bigelowii. TEM studies showed that, in addition to organelles, such as the nucleus, chloroplasts and mitochondria, B. bigelowii contains one or two spheroid bodies with internal lamellae. In the 18S rDNA tree of the Prymnesiophyceae, C. parkeae fell within the B. bigelowii clade, and was close to B. bigelowii Genotype III (99.89% similarity). Plastid 16S rDNA sequences obtained from B. bigelowii were close to the unidentified sequences from the oligotrophic SE Pacific Ocean (e.g. HM133411) (99.86% similarity). Bacterial16S rDNA sequences obtained from B. bigelowii were identical to the UCYN-A sequence AY621693 from Arabian Sea, and fell in the UCYN-A clade. From these results, we suggest that; 1) C. parkeae is the alternate life cycle stage of B. bigelowii sensu stricto or that of a sibling species of B. bigelowii, and 2) the spheroid body of B. bigelowii originated from endosymbiosis of the nitrogen-fixing cyanobacterium UCYN-A.  相似文献   

10.
In this study, a phenylalanine ammonia-lyase (PAL) gene was cloned from Dendrobium candidum using homology cloning and RACE. The full-length sequence and catalytic active sites that appear in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum are also found: PAL cDNA of D. candidum (designated Dc-PAL1, GenBank No. JQ765748) has 2,458 bps and contains a complete open reading frame (ORF) of 2,142 bps, which encodes 713 amino acid residues. The amino acid sequence of DcPAL1 has more than 80% sequence identity with the PAL genes of other plants, as indicated by multiple alignments. The dominant sites and catalytic active sites, which are similar to that showing in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum, are also found in DcPAL1. Phylogenetic tree analysis revealed that DcPAL is more closely related to PALs from orchidaceae plants than to those of other plants. The differential expression patterns of PAL in protocorm-like body, leaf, stem, and root, suggest that the PAL gene performs multiple physiological functions in Dendrobium candidum.  相似文献   

11.
In this short report, the genome-wide homologous recombination events were re-evaluated for classical swine fever virus (CSFV) strain AF407339. We challenged a previous study which suggested only one recombination event in AF407339 based on 25 CSFV genomes. Through our re-analysis on the 25 genomes in the previous study and the 41 genomes used in the present study, we argued that there should be possibly at least two clear recombination events happening in AF407339 through genome-wide scanning. The reasons for identifying only one recombination event in the previous study might be due to the limited number of available CSFV genome sequences at that time and the limited usage of detection methods. In contrast, as identified by most detection methods using all available CSFV genome sequences, two major recombination events were found at the starting and ending zones of the genome AF407339, respectively. The first one has two parents AF333000 (minor) and AY554397 (major) with beginning and ending breakpoints located at 19 and 607 nt of the genome respectively. The second one has two parents AF531433 (minor) and GQ902941 (major) with beginning and ending breakpoints at 8397 and 11,078 nt of the genome respectively. Phylogenetic incongruence analysis using neighbor-joining algorithm with 1000 bootstrapping replicates further supported the existence of these two recombination events. In addition, we also identified additional 18 recombination events on the available CSFV strains. Some of them may be trivial and can be ignored. In conclusion, CSFV might have relatively high frequency of homologous recombination events. Genome-wide scanning of identifying recombination events should utilize multiple detection methods so as to reduce the risk of misidentification.  相似文献   

12.
The purpose of this table is to provide the community with a citable record of publications of ongoing genome sequencing projects that have led to a publication in the scientific literature. While our goal is to make the list complete, there is no guarantee that we may have omitted one or more publications appearing in this time frame. Readers and authors who wish to have publications added to subsequent versions of this list are invited to provide the bibliographic data for such references to the SIGS editorial office.

Phylum Crenarchaeota

Phylum Deinococcus-Thermus

Phylum Proteobacteria

Phylum Tenericutes

Phylum Firmicutes

Phylum Actinobacteria

Phylum Spirochaetes

Non-Bacterial genomes

  相似文献   

13.
The purpose of this table is to provide the community with a citable record of publications of ongoing genome sequencing projects that have led to a publication in the scientific literature. While our goal is to make the list complete, there is no guarantee that we may have omitted one or more publications appearing in this time frame. Readers and authors who wish to have publications added to subsequent versions of this list are invited to provide the bibliographic data for such references to the SIGS editorial office.

Phylum Euryarchaeota

Phylum Crenarchaeota

Phylum Deinococcus-Thermus

Phylum Proteobacteria

Phylum Tenericutes

Phylum Firmicutes

Phylum Actinobacteria

Non-Bacterial genomes

  相似文献   

14.
The purpose of this table is to provide the community with a citable record of publications of ongoing genome sequencing projects that have led to a publication in the scientific literature. While our goal is to make the list complete, there is no guarantee that we may have omitted one or more publications appearing in this time frame. Readers and authors who wish to have publications added to this subsequent versions of this list are invited to provide the bibliometric data for such references to the SIGS editorial office.

Non-Bacterial genomes

  相似文献   

15.
A novel isolate belonging to the genus Streptomyces, strain SL-4T, was isolated from soil sample collected from a sanitary landfill, New Delhi, India. The taxonomic status of this isolate was studied by polyphasic approach including morphological, physiological and chemo-taxonomic characterization. Spore chains of SL-4T were open loops, hooks or extended spirals of wide diameter (retinaculiperti). The cell wall peptidoglycan of the isolate SL-4T contained L,L-diaminopimelic acid, suggesting that the strain has a cell wall of chemotype-I. The polar lipid profile of the isolate was of Type II, with phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides. The 16SrRNA gene sequence similarity between SL-4T and its phylogenetic relatives Streptomyces atrovirens NRRLB 16357T (DQ026672), S. albogriseolus NRRLB 1305T (AJ494865), S viridodiastaticus NBRC 13106T (AB184317), S. caelestis NRRL 2418T (X80824), S. flavoviridis NBRC 12772T (AB184842), S. pilosus NBRC 12807T (AB184161) and S. longispororuber NBRC 13488T (AB184440) was 99.65, 99.65, 99.64, 99.23, 99.15, 99.14 and 99.13 % respectively. Subsequent DNA–DNA hybridization experiments with the test strain and its clade members showed 55.27, 44.27, 36.86, and 15.65 % relatedness between SL-4T and its relatives S. atrovirens,S. albogriseolus, S. viridodiastaticus and S. longispororuber respectively. The genotypic and phenotypic data was analyzed to verify possibility of the isolate SL-4T representing novel member of the genus Streptomyces, for which the name S. antibioticalis is being proposed. The type strain is SL-4T (=CCM 7434T=MTCC 8588T).  相似文献   

16.
The purpose of this table is to provide the community with a citable record of publications of ongoing genome sequencing projects that have led to a publication in the scientific literature. While our goal is to make the list complete, there is no guarantee that we may have omitted one or more publications appearing in this time frame. Readers and authors who wish to have publications added to subsequent versions of this list are invited to provide the bibliographic data for such references to the SIGS editorial office.

Non-Bacterial genomes

  相似文献   

17.
The aim of this study was to characterize a Triticum aestivum-Psathyrostachys huashanica Keng (2n = 2x = 14, NsNs) disomic addition line 2-1-6-3. Individual line 2-1-6-3 plants were analyzed using cytological, genomic in situ hybridization (GISH), EST-SSR, and EST-STS techniques. The alien addition line 2-1-6-3 was shown to have two P. huashanica chromosomes, with a meiotic configuration of 2n = 44 = 22 II. We tested 55 EST-SSR and 336 EST-STS primer pairs that mapped onto seven different wheat chromosomes using DNA from parents and the P. huashanica addition line. One EST-SSR and nine EST-STS primer pairs indicated that the additional chromosome of P. huashanica belonged to homoeologous group 7, the diagnostic fragments of five EST-STS markers (BE404955, BE591127, BE637663, BF482781 and CD452422) were cloned, sequenced and compared. The results showed that the amplified polymorphic bands of P. huashanica and disomic addition line 2-1-6-3 shared 100% sequence identity, which was designated as the 7Ns disomic addition line. Disomic addition line 2-1-6-3 was evaluated to test the leaf rust resistance of adult stages in the field. We found that one pair of the 7Ns genome chromosomes carried new leaf rust resistance gene(s). Moreover, wheat line 2-1-6-3 had a superior numbers of florets and grains per spike, which were associated with the introgression of the paired P. huashanica chromosomes. These high levels of disease resistance and stable, excellent agronomic traits suggest that this line could be utilized as a novel donor in wheat breeding programs.  相似文献   

18.
Molecular analysis of black band disease of corals revealed that samples frozen immediately after collection yielded more proteobacterial 16S rRNA sequences, while unfrozen samples produced more cyanobacterial and sulfur-oxidizing bacterial sequences. These results suggest the need to use multiple approaches for preparation of samples to characterize this complex polymicrobial disease.Black band disease (BBD) is a polymicrobial disease that affects corals on reefs worldwide. It consists of a migrating microbial mat dominated by cyanobacteria that lyses coral tissue, leading to coral colony death, and is one of the most destructive of coral diseases. Microscopic examination of BBD samples consistently reveals an abundance of nonheterocystous, filamentous cyanobacteria and colorless gliding bacteria with internal elemental sulfur granules characteristic of the genus Beggiatoa (6, 17, 18). It is thought that these are key players in the etiology of BBD. However, with one exception (2), previous molecular studies of BBD consistently detected very low proportions of cyanobacteria (4, 8, 9, 19, 20) and only one study has detected Beggiatoa (19). Instead, all molecular BBD studies indicate a highly variable and diverse composition of heterotrophic bacteria, mostly members of the Alphaproteobacteria.It is unknown why the dominant cyanobacteria and filamentous sulfur-oxidizing bacteria observable microscopically in BBD samples are poorly or not at all detected by molecular methods. It is possible that freezing of the samples in these studies is the cause for low detection of BBD cyanobacteria and sulfur oxidizers. Freezing is the common method of sample processing to extract DNA for microbial community analysis of BBD and has been used in all previous molecular studies. However, this approach may impart a bias to detection of specific BBD bacteria. Suomalainen et al. (22) reported that freezing of samples targeting the fish pathogen Flavobacterium columnare destroyed DNA, suggested to be due to the release of DNase and other enzymes from the cell, leaving most of the F. columnare DNA undetectable by PCR. They noted that DNA from bacteria such as Escherichia coli was not affected (22). Bissett et al. (3) speculated that freezing sediments prior to DNA extraction lysed Beggiatoa filaments and caused their DNA to be lost (3). A recent report showed that algae and cyanobacteria with large cell sizes, including filamentous strains, could not be sufficiently cryopreserved (5). While the above-described studies showed or speculated that freezing of samples affects the detection of some microorganisms in environmental samples, none of these studies included detailed investigation of the mechanism responsible for the effect of freezing or of the effect of freezing on the assessment of microbial community composition.In the present study, we investigated the effect of freezing on molecular analysis of the BBD microbial community by using DNA extracts of frozen and unfrozen BBD samples from two coral hosts (Siderastrea siderea and Diploria clivosa), using both universal and cyanobacterium-specific primers targeting the 16S rRNA gene. BBD samples (i.e., the BBD microbial mat) were collected by suctioning the mat off the coral surface using individual sterile syringes while scuba diving. Samples were transferred to 2-ml cryovials (after decanting seawater) upon return to shore and either immediately frozen and stored at −20°C until DNA extraction or maintained at ambient temperature with DNA extracted within 1 h of collection. Eleven samples were collected from reefs of the Florida Keys (United States), Lee Stocking Island (Bahamas), and St. Croix (United States Virgin Islands).Genomic DNA was extracted by the bead-beating method as previously described (12, 19, 20). Frozen samples were first thawed at room temperature, and 500-μl aliquots were directly transferred into multimix lysing matrix tubes by using trimmed pipette tips, excluding any water. Unfrozen samples were transferred to multimix lysing matrix tubes in the same way. The extracted DNA was verified by gel electrophoresis, and DNA extracts from frozen samples were stored at −20°C, whereas DNA extracts from unfrozen samples were kept at 4°C until used for PCR amplification.DNA extracted from both frozen and unfrozen samples was amplified by PCR using universal bacterial primers 27F and 1492R (14) and cyanobacterium-specific primers CYA359F and CYA781R(B) (15) targeting 16S rRNA genes. The purification of PCR products, cloning, and sequencing of plasmid inserts were done as described previously (20). Primer M13F (11) or CYA359F (15) was used to obtain partial sequences, and an additional primer, 518F (13), M13R (11), or CYA781R(B) (15), was used to obtain full-length sequences. Sequence editing, BLAST (1), and phylogenetic analysis using ARB (10) were done as described previously (19, 20). Sequences that matched at similarity identity values of 97% and above were considered to be of the same operational taxonomic unit. Representative gene sequences that were closely related to cyanobacterial sequences were subjected to maximum-parsimony, neighbor-joining, and maximum-likelihood phylogenetic analyses, and a consensus tree was produced based on maximum-parsimony analysis.The results for universal bacterial primers indicated that all of the frozen BBD samples except one (Fig. ​(Fig.1,1, clone library E) were dominated (44 to 87%) by Alphaproteobacteria (Fig. ​(Fig.1;1; see Tables S1 and S2 in the supplemental material). We previously (19) compared the 16S rRNA gene sequences retrieved from seven of these libraries (Fig. ​(Fig.1,1, libraries A to G), all of which were obtained from frozen BBD samples from the host S. siderea, to investigate the diversity of BBD microorganisms between BBD infections. In the present study, we focus on the differences in results obtained using frozen versus unfrozen BBD samples from S. siderea (Fig. ​(Fig.1,1, libraries G and H) and a second coral host, D. clivosa (Fig. ​(Fig.1,1, libraries I and J). The S. siderea samples (libraries G and H) were taken from different host colonies on the same reef (Butler Bay Reef site), whereas the D. clivosa clone libraries were constructed from subsamples of one BBD sample.Open in a separate windowFIG. 1.Dominant bacterial phylogenetic groups, based on 16S rRNA gene sequence types and universal primers, present in clone libraries produced from frozen and unfrozen BBD samples from the coral hosts Siderastrea siderea and Diploria clivosa. The numbers above the bars represent the numbers of sequences in the respective clone libraries. Libraries A to H, frozen (A to G) and unfrozen (H) BBD from S. siderea. Libraries I and J, frozen (I) and unfrozen (J) BBD from D. clivosa. Sampling locations and sampling dates: libraries A and B, Horseshoe Reef, Lee Stocking Island, Bahamas, 19 July 2004; C, Rainbow Garden Reef, Lee Stocking Island, Bahamas, 16 July 2004; D, Watson''s Reef, Florida Keys, 3 May 2005; E, G, and H, Butler Bay Reef site, St. Croix, U.S. Virgin Islands (USVI), 22 October 2005, 1 June 2005, and 5 June 2006, respectively; F, Frederiksted Reef site, St. Croix, USVI, 1 June 2005; I and J, Frederiksted Reef site, St. Croix, USVI, 7 August 2006. All of the sequences from clone libraries A to G have been previously published by Sekar et al. (19, 20).This approach yielded strikingly different results for the two methods. For example, the clone library produced from one frozen sample (Fig. ​(Fig.1,1, library G) from S. siderea contained only one (of 60) cyanobacterium-related sequence (see EF123584 [GenBank sequence accession no.] in Table S1 in the supplemental material), which was phylogenetically related to a sequence from an uncultured planktonic Synechococcus sp. (GenBank sequence accession no. AY172810; Fig. ​Fig.2).2). In contrast, the clone library from the corresponding unfrozen sample (Fig. ​(Fig.1,1, library H) was dominated by a cyanobacterial ribotype which represented 37% of the clones. This ribotype was closely related to an Oscillatoria ribotype (GenBank sequence accession no. AY038527/AF473936) detected in almost all reported BBD molecular studies (2, 4, 7, 23). The sequence was confirmed as the BBD Oscillatoria sequence by phylogenetic analysis using two representative clone sequences (GenBank sequence accession no. EF123639 and EF123644) (Fig. ​(Fig.2).2). The unfrozen S. siderea clone library additionally produced a dominant epsilonproteobacterial ribotype (14 of 15 clones) (see Table S1 in the supplemental material) that was not detected in the corresponding frozen sample. Phylogenetic analysis of two representative sequences (GenBank sequence accession no. EF123607 and EF123613, not shown in Fig. ​Fig.2)2) determined that the sequence was related to a sequence from the sulfur-oxidizing bacterium “Candidatus Arcobacter sulfidicus” (GenBank sequence accession no. AY035822) (24), a species known to deposit filamentous sulfur (21) and reported previously in BBD (9).Open in a separate windowFIG. 2.Phylogenetic tree derived from the 16S rRNA gene sequences closely related to Synechococcus spp., Xenococcus spp., and Oscillatoria spp. sequences detected in BBD and their neighbors. The tree topology is based on the maximum-parsimony analysis. The bar represents 10% estimated sequence divergence. Boldface type indicates sequences from this study, designated as follows. FRSSBA, UFSSBA, FRSSCY, and UFSSCY indicate sequences retrieved from frozen (FR) and unfrozen (UF) samples of S. siderea (SS) using universal bacterial primers (BA) and cyanobacterium-specific (CY) primers for 16S rRNA gene amplification. FRDCBA, UFDCBA, and UFDCCY indicate sequences retrieved from frozen and unfrozen samples of Diploria clivosa (DC), and the same primer designations are used as for S. siderea sequences. GenBank sequence accession numbers are listed for all sequences. Asterisks designate sequences corresponding to the sequence from the BBD Oscillatoria discussed in the text.Again in clone library I, from the frozen subsample of D. clivosa (see Table S2 in the supplemental material), the Alphaproteobacteria were dominant (44%) and cyanobacteria represented in low percentages (4%). These cyanobacterial sequences were phylogenetically related to sequences of Leptolyngbya spp. (not shown in Fig. ​Fig.2)2) and a planktonic cyanobacterium Xenococcus sp. (GenBank sequence accession no. AF132783) (Fig. ​(Fig.2;2; see Table S2 in the supplemental material). The library from the unfrozen BBD subsample of D. clivosa (see Table S2 in the supplemental material) was dominated by Gammaproteobacteria (35%), followed by cyanobacteria (24%) which had the same cyanobacterial sequence type (BBD Oscillatoria) observed in the unfrozen S. siderea sample (see Table S2 in the supplemental material). For corroboration of these results, we constructed an additional clone library, using universal primers, from an unfrozen BBD sample from S. siderea collected during June 2007; in this sample, 47% of the sequences were also related to the sequence from BBD Oscillatoria.The use of cyanobacterium-specific primers produced results similar to the overall pattern we detected using universal primers. Frozen BBD from S. siderea produced 27 sequences, of which 24 were closely related to sequences from planktonic Synechococcus spp. and Xenococcus sp. (see Table S3 in the supplemental material). This was confirmed by phylogenetic analysis (Fig. ​(Fig.2)2) using representative sequences (GenBank sequence accession no. EU019432, EU019435, EU019439, EU019442, EU019449, and EU019455). In contrast, all of the sequences (n = 37) obtained from unfrozen S. siderea samples were closely related to the sequence from the BBD Oscillatoria (see Table S3 in the supplemental material). Representative sequences (GenBank sequence accession no. EU019460 and EU019467) confirmed this phylogenetic affiliation (Fig. ​(Fig.2).2). Similarly, each of 38 sequences obtained from the unfrozen subsample of D. clivosa with cyanobacterium-specific primers was closely related to the sequence from the BBD Oscillatoria (see Table S3 in the supplemental material), again confirmed by phylogenetic analysis using two representative sequences (GenBank sequence accession no. EU019508 and EU019515) (Fig. ​(Fig.22).There was very little overlap (6 to 10%) between sequences obtained from frozen versus unfrozen BBD samples collected from both coral hosts when considering all of the BBD bacterial sequences detected (see Tables S1 and S2 in the supplemental material). Only four sequences were common to both frozen and unfrozen clone libraries (6% of 62 sequences detected within 136 clones) for S. siderea and seven sequences (10% of 69 sequences detected within 108 clones) for D. clivosa. Statistical analysis (ANOSIM) showed that the sequence types differed significantly between frozen and unfrozen clone libraries (R = 0.987; P = 0.022). Overall, all frozen libraries (libraries A to G and I) were 69% similar to each other, while the two unfrozen libraries (libraries H and J) were 58% similar.The results of our study are significant for the ongoing investigations into the etiology of BBD. While it is well known that the BBD microbial community consists of photoautotrophs (cyanobacteria), sulfate-reducing bacteria, sulfur-oxidizing bacteria, and heterotrophs (16), we are just beginning to understand the roles of these functional groups in the disease process. A first step in this understanding is the valid and repeatable detection of specific members of the BBD consortium. In summary, we show here that unfrozen samples produce better results for detection of BBD cyanobacteria and sulfur-oxidizing bacteria, while frozen samples are best for detection of heterotrophic proteobacterial sequences. The latter is particularly important because of the consistent finding of Proteobacteria associated with toxic dinoflagellates (19, 20), as well as other marine invertebrate pathogens (4), in BBD. We have not studied the mechanism behind the freezing effect (e.g., release of DNase), which is outside the scope of this study. Though the current study was done with BBD samples, the effect of freezing on other microbial mats or biofilms cannot be ignored. Based on the results of this study, we suggest using multiple sample-processing approaches to characterize the microbial communities associated with BBD and other microbial mats.  相似文献   

19.
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20.
In this study, we have collected and screened a total of 268 stool samples from diarrheal patients admitted to an Infectious disease hospital in Kolkata for the presence of Cryptosporidium spp. The initial diagnosis was carried out by microscopy followed by genus specific polymerase chain reaction assays based on 70 kDa heat shock proteins (HSP70). DNA sequencing of the amplified locus has been employed for determination of genetic diversity of the local isolates. Out of 268 collected samples, 12 (4.48%) were positive for Cryptosporidium spp. Sequences analysis of 70 kDa heat shock proteins locus in 12 Cryptosporidium local isolates revealed that 2.24% and 1.86% of samples were showing 99% to 100% identity with C. parvum and C. hominis. Along with the other 2 major species one recently described globally distributed pathogenic species Cryptosporidium viatorum has been identified. The HSP70 locus sequence of the isolate showed 100% similarity with a previously described isolate of C. viatorum (Accession No. JX978274.1, JX978273.1, and JN846706.1) present in GenBank.  相似文献   

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