rRNA Internal Transcribed Spacer Region Research Articles

Development of a novel PCR assay based on the 16S–23S rRNA internal transcribed spacer region for the detection of Lactococcus garvieae

Lactococcus garvieae is recognized as an emerging pathogen in fish. Several PCR-based methods have been developed for the detection and identification of L.garvieae; however, the sensitivity of these methods is still in question regarding the discrimination of this organism from other closely related species. Two primers, ITSLg30F and ITSLg319R, were designed from the sequence in the 16S-23S internal transcribed spacer (ITS) region and used for the specific detection of L.garvieae. L.garvieae strains including fish isolates were positive by this method. In contrast, previously developed PCR methods showed false-positive results with non-L.garvieae species. Our results indicate that a PCR method using the newly designed ITS primer set provides a sensitive and efficient tool for the detection of L.garvieae in fish and aquaculture environments.

Frequency and expression of ALS and HWP1 genotypes in Candida albicans strains isolated from Mexican patients suffering from vaginal candidosis

To detect the frequency and expression of eight ALS (agglutinin-like sequence) genes and the HWP1 genotype in a group of Candida albicans strains isolated from Mexican women suffering from vaginal candidosis. A group of 264 women (age 15-57 years) with vaginal infections were evaluated. C. albicans was identified by PCR amplification of the rRNA internal transcribed spacer regions ITS1 and ITS2. The ALS and HWP1 genes were identified by conventional PCR, and their expression levels were determined by real-time PCR after growing C. albicans strains in reconstituted human vaginal epithelium (RHVE). C. albicans was identified in 50 women (18.9%). The genotypic frequencies were ALS1 100%, ALS2 60%, ALS3 36%, ALS4 54%, ALS5 70%, ALS6 56%, ALS7 64%, ALS9 66% and HWP1 92%. The most frequently expressed genes in the strains harbouring all of the genes were ALS4 (100%), ALS1 (87.5%), ALS2 (87.5%), ALS3 (87.5%), ALS5 (87.5%), ALS7 (87.5%) and HWP1 (75.0%). Nineteen per cent of the vaginal infections were caused by C. albicans, and a high proportion of the strains carried genes encoding proteins involved in adhesion to epithelia. The ALS and HWP1 genes were expressed in RHVE, suggesting that the Als and Hwp1 proteins play an important role in the pathogenesis of the infection.

Microcystin producing cyanobacterial communities in Amvrakikos Gulf (Mediterranean Sea, NW Greece) and toxin accumulation in mussels (Mytilus galloprovincialis)

Various cyanobacterial species have the capacity to produce different types of toxins. Microcystins, the most prominent cyanotoxins are considered health hazards because of their potential hepatotoxic effects. They are well known to contaminate freshwater ecosystems but their presence in marine ecosystems has been reported only occasionally. We investigated seasonal changes of microcystin concentrations both in water and in the edible species of mussels Mytilus galloprovincialis collected from Amvrakikos Gulf (salinity ranging from 30‰ to 34‰), the biggest semi-enclosed basin in Greece. The microcystin concentrations in the water ranging from 0.003 to 19.8ngl−1, were below the World Health Organization (WHO) upper limit for recreational activities. In contrast, we found that microcystin concentrations in M. galloprovincialis mussels (ranging from 45±2 to 141.5±13.5ngg−1ww) exceeded the upper limit of the tolerable daily intake (TDI) of microcystin as determined by WHO.Genotype composition of the total cyanobacterial community of the Gulf was analyzed by using denaturing gradient gel electrophoresis (DGGE) profiling of the rRNA internal transcribed spacer region (rRNA-ITS). The cyanobacterial community was found to be dominated almost exclusively by the cosmopolitan species Synechococcus –Synechocystis. In order to determine genes involved in the production of microcystins, a range of both specific and degenerate molecular primers against microcystin synthetase gene cluster (mcyS) was used.To our knowledge this is the first report of the presence of the hepatotoxic microcystins in the Mediterranean Sea, the first study on the accumulation of these toxins in mussels from a Mediterranean marine ecosystem and one of the few published works suggesting a potential association of microcystins with Synechococcus and/or Synechocystis cyanobacteria.The importance of our study is strengthened by the fact that Amvrakikos Gulf is among the most productive Greek “seafood” areas and a Mediterranean wetland of international significance according to Ramsar Convention.

Phylogeny of Korean Hornbeam (Carpinus turczaninovii) based on nuclear ribosomal ITS sequence

The genus Carpinus belonging to Coryloideae, Betulaceae, has significant economic and ornamental importance. This study was undertaken with the aim to understand the genetic diversity among eighteen isolates of Carpinus turczaninovii collected from different geographical regions of Korea, using ribosomal RNA (rRNA) internal transcribed spacer (ITS) sequences, to compare the infraspecific-phylogenetic relationships among C. turczaninovii in Korea, and some known Carpinus plants. The size variation of sequenced rRNA ITS regions was not seen, with 215, 162, 222 bp of ITS1 region, 5.8S rRNA gene, ITS2 region, respectively. However, some certain nucleotide variations resulted in genetic diversity. In the genus Carpinus, C. turczaninovii closely genetic with Castanopsis kawakamii, Carpinus orientalis,Carpinus monbeigiana, and Calyptranthes polynenra formed one monophyletic clade, while Carpinus betulus and Carpinus laxiflora, respectively formed one monophyletic clade. In the present ITS sequence analysis, Carpinus fangiana,Carpinus coreana, Carpinus japonica, and Carpinus caroliniana were considered as Out Group, compared to C. turczaninovii group. The highest intraspecific variation was found between C. caroliniana and C. japonica. The least intraspecific variation was obtained between C. turczaninovii and C. coreana. The results will help further understanding Carpinus infraspecies population and their phylogenetic analysis. Key words: Carpinus, Hornbeam, Carpinus turczaninovii, molecular identification, nuclear ribosomal RNA, internal transcribed spacer, infraspecific-phylogenetic relationship.

Specific characters of 16S rRNA gene and 16S–23S rRNA internal transcribed spacer sequences of Xylella fastidiosa pear leaf scorch strains

Pear leaf scorch, the only Xylella fastidiosa-induced disease reported from Taiwan, was found in area where the variety Hengshan (Pyrus pyrifolia) was grown. Strains of pear leaf scorch Xyl. fastidiosa (XF-PLS) shared similarities to strains of other host origins in the requirement of complex medium and the exhibition of rippled cell walls, however, recent serological and molecular biology studies showed difference among them. Five strains of XF-PLS were compared with 20 other strains originally isolated from almond, oleander, pecan, plum, peach, mulberry, grapes, citrus, coffee, and sycamore by sequence analyses of the 16S rRNA gene and 16S–23S rRNA internal transcribed spacer region (ITS). When sequences of 16S rRNA gene based on fragment size of 1,537–1,540 bp were compared, the similarity index among 5 XF-PLS strains was 99.3–99.8%, whereas it was 97.8–98.6% between XF-PLS strains and strains from other hosts. When sequences of 16S–23S rRNA ITS based on fragment size of 510–540 bp were compared, the similarity index among 5 XF-PLS strains was 99.0–100%, whereas it was 80.7–82% between XF-PLS strains and strains from other hosts. Multiple sequence alignments led to the identification of 5 polymorphic nucleotides in the 16S rRNA gene among the 25 Xyl. fastidiosa strains, and there were considerable variations in the nucleotide sequences of 16S–23S rRNA ITS between XF-PLS and the other 20 Xyl. fastidiosa strains. The phylogenetic trees revealed that XF-PLS strains were separated from strains of other hosts. Strains of other hosts were divided into four subgroups: strains from (1) oleander, (2) grape, almond M23 and mulberry, (3) citrus and coffee, and (4) pecan, peach, plum, sycamore and almond M12. Results indicate that XF-PLS strains were not closely related to the above-mentioned strains from other hosts and could possibly belong to a new subspecies of Xyl. fastidiosa.

Cyanobacterial Diversity and a New Acaryochloris-Like Symbiont from Bahamian Sea-Squirts

Symbiotic interactions between ascidians (sea-squirts) and microbes are poorly understood. Here we characterized the cyanobacteria in the tissues of 8 distinct didemnid taxa from shallow-water marine habitats in the Bahamas Islands by sequencing a fragment of the cyanobacterial 16S rRNA gene and the entire 16S–23S rRNA internal transcribed spacer region (ITS) and by examining symbiont morphology with transmission electron (TEM) and confocal microscopy (CM). As described previously for other species, Trididemnum spp. mostly contained symbionts associated with the Prochloron-Synechocystis group. However, sequence analysis of the symbionts in Lissoclinum revealed two unique clades. The first contained a novel cyanobacterial clade, while the second clade was closely associated with Acaryochloris marina. CM revealed the presence of chlorophyll d (chl d) and phycobiliproteins (PBPs) within these symbiont cells, as is characteristic of Acaryochloris species. The presence of symbionts was also observed by TEM inside the tunic of both the adult and larvae of L. fragile, indicating vertical transmission to progeny. Based on molecular phylogenetic and microscopic analyses, Candidatus Acaryochloris bahamiensis nov. sp. is proposed for this symbiotic cyanobacterium. Our results support the hypothesis that photosymbiont communities in ascidians are structured by host phylogeny, but in some cases, also by sampling location.

Identification of Escherichia coli through analysis of 16S rRNA and 16S-23S rRNA internal transcribed spacer region sequences.

A bacterial strain, designated BzDS03 was isolated from water sample, collected from Dal Lake Srinagar. The strain was characterized by using 16S ribosomal RNA gene and 16S-23S rRNA internal transcribed spacer region sequences. Phylogenetic analysis showed that 16S rRNA sequence of the isolate formed a monophyletic clade with genera Escherichia. The closest phylogenetic relative was Escherichia coli with 99% 16S rRNA gene sequence similarity. The result of Ribosomal database project's classifier tool revealed that the strain BzDS03 belongs to genera Escherichia.16S rRNA sequence of isolate was deposited in GenBank with accession number FJ961336. Further analysis of 16S-23S rRNA sequence of isolate confirms that the identified strain BzDS03 be assigned as the type strain of Escherichia coli with 98% 16S-23S rRNA sequence similarity. The GenBank accession number allotted for 16S-23S rRNA intergenic spacer sequence of isolate is FJ961337.

Evaluation of the Speed-oligo Mycobacteria assay for the rapid differentiation of frequent Mycobacterium ssp - a multi-centre and multi-country survey

A new DNA line probe assay (Speed-oligo Mycobacteria, Vircell) was evaluated for the differentiation of mycobacterial species. It is based on PCR targeting the 16S-23S rRNA internal transcribed spacer region followed by hybridization to specific oligonucleotides by means of a simple dip stick technique.

Phylogeny of Korean Hornbeam ( Carpinus turczaninovii ) based on nuclear ribosomal ITS sequence

The genus Carpinus belonging to Coryloideae, Betulacea e, has significant economic and ornamental importance. This study was undertaken with the aim to understand the genetic diversity among eighteen isolates of Carpinus turczaninovii collected from different geographical regions of Korea, using ribosomal RNA (rRNA) internal transcribed spacer (ITS) sequences, to compare the infraspecificphylogenetic relationships among C. turczaninovii in Korea, and some known Carpinus plants. The size variation of sequenced rRNA ITS regions was not seen, with 215, 162, 222 bp of ITS1 region, 5.8S rRNA gene, ITS2 region, respectively. However, some certain nucleotide variations resulted in genetic diversity. In the genus Carpinus, C. turczaninovii closely genetic with Castanopsis kawakam ii, Carpinus orientalis, Carpinus monbeigiana , and Calyptranthes polynenra formed one monophyletic clade, while Carpinus betulus and Carpinus laxiflora , respectively formed one monophyletic clade. In the present ITS sequence analysis, Carpinus fangiana, Carpinus coreana, Carpinus japonica , and Carpinus caroliniana were considered as Out Group, compared to C. turczaninovii group. The highest intraspecific variation was found between C. caroliniana and C. japonica. The least intraspecific variation was obtained between C. turczaninovii and C. coreana . The results will help further understanding Carpinus infraspecies population and their phylogenetic analysis. Key words: Carpinus, Hornbeam, Carpinus turczaninovii, molecular identification, nuclear ribosomal RNA, internal transcribed spacer, infraspecific-phylogenetic relationship.

Influence of molecular resolution on sequence-based discovery of ecological diversity among Synechococcus populations in an alkaline siliceous hot spring microbial mat.

Previous research has shown that sequences of 16S rRNA genes and 16S-23S rRNA internal transcribed spacer regions may not have enough genetic resolution to define all ecologically distinct Synechococcus populations (ecotypes) inhabiting alkaline, siliceous hot spring microbial mats. To achieve higher molecular resolution, we studied sequence variation in three protein-encoding loci sampled by PCR from 60°C and 65°C sites in the Mushroom Spring mat (Yellowstone National Park, WY). Sequences were analyzed using the ecotype simulation (ES) and AdaptML algorithms to identify putative ecotypes. Between 4 and 14 times more putative ecotypes were predicted from variation in protein-encoding locus sequences than from variation in 16S rRNA and 16S-23S rRNA internal transcribed spacer sequences. The number of putative ecotypes predicted depended on the number of sequences sampled and the molecular resolution of the locus. Chao estimates of diversity indicated that few rare ecotypes were missed. Many ecotypes hypothesized by sequence analyses were different in their habitat specificities, suggesting different adaptations to temperature or other parameters that vary along the flow channel.

Novel lineages of Prochlorococcus thrive within the oxygen minimum zone of the eastern tropical South Pacific

The eastern tropical Pacific Ocean holds two of the main oceanic oxygen minimum zones of the global ocean. The presence of an oxygen-depleted layer at intermediate depths, which also impinges on the seafloor and in some cases the euphotic zone, plays a significant role in structuring both pelagic and benthic communities, and also in the vertical partitioning of microbial assemblages. Here, we assessed the genetic diversity and distribution of natural populations of the cyanobacteria Prochlorococcus and Synechococcus within oxic and suboxic waters of the eastern tropical Pacific using cloning and sequencing, and terminal restriction fragment length polymorphism (T-RFLP) analyses applied to the 16S-23S rRNA internal transcribed spacer region. With the T-RFLP approach we could discriminate 19 cyanobacterial clades, of which 18 were present in the study region. Synechococcus was more abundant in the surface oxic waters of the eastern South Pacific, while Prochlorococcus dominated the subsurface low-oxygen waters. Two of the dominant clades in the oxygen-deficient waters belong to novel and yet uncultivated lineages of low-light adapted Prochlorococcus.

Development and Application of Quantitative-PCR Tools for Subgroups of the Roseobacter Clade

Specific SYBR green-based quantitative-PCR assays targeting conserved regions in the 16S-23S rRNA internal transcribed spacer regions were developed for five subgroups of the environmentally abundant and biogeochemically active Roseobacter clade of marine bacteria. The assays were applied to field samples demonstrating their utility in investigations of abundant Roseobacter group phylotypes in the environment.

Bartonella rattaustraliani sp. nov., Bartonella queenslandensis sp. nov. and Bartonella coopersplainsensis sp. nov., identified in Australian rats

A total of 11 Bartonella isolates were recovered from the blood of Melomys, Uromys and Rattus species in Australia and were characterized using phenotypic and genotypic methods. Comparison of 16S rRNA gene, ftsZ, gltA and 16S-23S rRNA internal transcribed spacer region sequences from the isolates indicated that they formed three sequence similarity groups that were distinct from one another and from the currently recognized Bartonella species. Phylogenetic analysis based on alignment of concatenated sequences inferred distinct evolutionary lineages for each of the three groups within the genus Bartonella. On the basis of these data, we propose the isolates be accommodated in three novel Bartonella species, namely Bartonella rattaustraliani sp. nov. (type strain AUST/NH4(T) =CIP 109051(T) =CCUG 52161(T) =CSUR B609(T)), Bartonella queenslandensis sp. nov. (type strain AUST/NH12(T) =CIP 109057(T) =CCUG 52167(T) =CSUR B617(T)) and Bartonella coopersplainsensis sp. nov. (type strain AUST/NH20(T) =CIP 109064(T) =CCUG 52174(T) =CSUR B619(T)).

Quantitative Real-Time PCR Assay for QPX (Thraustochytriidae), a Parasite of the Hard Clam ( Mercenaria mercenaria )

We developed a real-time quantitative PCR (qPCR) assay targeting the rRNA internal transcribed spacer region of the hard clam pathogen QPX. The qPCR assay was more sensitive than was histology in detecting clams with light QPX infections. QPX was detected in 4 of 43 sediment samples but in none of 40 seawater samples.

Investigating pleuromutilin-producingClitopilusspecies and related basidiomycetes

Pleuromutilin is a broad-spectrum antibiotic that has been used in veterinary medicine for over 20 years, but is now gaining interest as a human therapeutic. The compound is a fungal secondary metabolite, but there is some degree of confusion within the literature concerning which species may produce pleuromutilin, with several differently named fungi reported to make the compound. Here, we describe a taxonomic survey of publicly available cultures known to produce pleuromutilin, and other similar species. The pleuromutilin production of these strains was assessed and a phylogenetic assessment was carried out based on the sequence of the nuclear rRNA internal transcribed spacer region. Eleven strains were confirmed as being pleuromutilin producers and all of these isolates appear to fall within a discrete clade of the genus Clitopilus. The phylogenetic analysis also highlights the need for a revision of the taxonomic status of these fungi.

Intercistronic heterogeneity of the 16S–23S rRNA spacer region among Pseudomonas strains isolated from subterranean seeds of hog peanut (Amphicarpa bracteata)

Intercistronic heterogeneity of the 16S-23S rRNA internal transcribed spacer regions (ITS1) was investigated in 29 strains of fluorescent pseudomonads isolated from subterranean seeds of Amphicarpa bracteata (hog peanut). PCR amplification of the ITS1 region generated one or two products from the strains. Sequence analysis of the amplified fragments revealed an ITS1 fragment of about 517 bp that contained genes for tRNA(Ile) and tRNA(Ala) in all 29 strains; an additional smaller ITS1 of 279 bp without tRNA features was detected in 15 of the strains. The length difference appeared to be due to deletions of several nucleotide blocks between the 70 bp and 359 bp positions of the alignment. The end of the deletions in the variant ITS1 type coincided with the start of antiterminator box A, which is homologous to box A of other bacteria. Phylogenetic analyses using the neighbour-joining algorithm revealed two major phylogenetic clusters, one for each of the ITS1 types. Using a single specific primer set and the DNA-intercalating dye SYBR Green I for real-time PCR and melting-curve analysis produced highly informative curves with one or two recognizable melting peaks that readily distinguished between the two ITS1 types in pure cultures. The assay was used to confirm the presence of the variant ITS1 type in the Pseudomonas community in total DNA from root-zone soil and seed coats of hog peanut. Heterogeneity of the ITS1 region between species has potential for studying molecular systematics and population genetics of the genus Pseudomonas, but the presence of non-identical rRNA operons within a genome may pose problems.

Weathering of chrysotile asbestos by the serpentine rock-inhabiting fungus Verticillium leptobactrum

Verticillium leptobactrum, a rare fungal species, has repeatedly been isolated from serpentinic rocks in the Western Alps, thus suggesting that it adapts easily to this selective mineral substrate. The rRNA internal transcribed spacer region of several isolates has been sequenced to confirm their identity and taxonomic position within Verticillium, a recently revised polyphyletic entity. Isolates of V. leptobactrum have also been investigated to establish their ability to weather asbestos chrysotile, the most common mineral in the isolation sites. The results of solubilization assays on magnesium and silicon, as well as measurement of the Mg/Si ratio in the asbestos fibres after exposure to fungal mycelia, indicate a high bioweathering activity of V. leptobactrum towards chrysotile. Comparison with data on Fusarium oxysporum shows differences among species, with V. leptobactrum being more active than F. oxysporum in removing structural ions from chrysotile. Asbestos weathering by fungi could be envisaged as a bioremediation strategy for hazardous asbestos-rich soils (e.g. abandoned mines). Fungi that have adapted to live in serpentine sites could be good candidates for this purpose.

Phylogenetic characterization of Encephalitozoon romaleae (Microsporidia) from a grasshopper host: Relationship to Encephalitozoon spp. infecting humans

Encephalitozoon species are the most common microsporidian pathogens of humans and domesticated animals. We recently discovered a new microsporidium, Encephalitozoon romaleae, infecting the eastern lubber grasshopper Romalea microptera. To understand its evolutionary relationships, we compared partial gene sequences of α- and β-tubulin and methionine aminopeptidase 2 enzyme from this and related species. We also analyzed the rRNA internal transcribed spacer. Based on tubulin and MetAP-2 gene phylogenetic analysis, E. romaleae clustered with the Encephalitzoon group with strong bootstrap support (>99%). Within the Encephalitozoon clade, E. romaleae clustered with Encephalitozoon hellem for both the β-tubulin and MetAP-2 phylogenies based on ML tree. The α-tubulin based ML tree, however, placed the new microsporidium closer to Encephalitozoon cuniculi. The rRNA internal transcribed spacer region of E. romaleae has 91% homology with E. hellem.

Description of Perkinsus beihaiensis n. sp., a new Perkinsus sp. Parasite in Oysters of Southern China

Oysters were collected from coastal locations in China from 1999-2006 for parasite analyses by molecular, culture, and histological techniques. Polymerase chain reaction-based assays targeting the internal transcribed spacer (ITS) region of the ribosomal RNA gene complex were performed to detect the presence of Perkinsus species. Sequencing and phylogenetic analysis of amplified Perkinsus sp. DNAs indicated that a novel Perkinsus sp. infects Crassostrea hongkongensis, Crassostrea ariakensis, and other bivalve hosts from Fujian to Guangxi provinces in southern China. Prevalence of this Perkinsus sp. reaches as high as 60% in affected oyster populations. Analyses of nucleotide sequences of the rRNA ITS region and of large subunit rRNA and actin genes, consistently confirmed the genus affiliation of this Perkinsus sp., but distinguished it from currently accepted Perkinsus species. Parasite cell types, such as signet ring trophozoites of 2-8 microm diameter, were observed by histology, and application of both genus Perkinsus and Perkinsus species-specific in situ hybridization probes consistently labelled the same Perkinsus sp. cells in histological sections from infected oyster tissues. Combined phylogenetic and histological results support the identity of a new parasite species, Perkinsus beihaiensis n. sp.

Phylogenetic ecology of the freshwater Actinobacteria acI lineage.

The acI lineage of freshwater Actinobacteria is a cosmopolitan and often numerically dominant member of lake bacterial communities. We conducted a survey of acI 16S rRNA genes and 16S-23S rRNA internal transcribed spacer regions from 18 Wisconsin lakes and used standard nonphylogenetic and phylogenetic statistical approaches to investigate the factors that determine acI community composition at the local scale (within lakes) and at the regional scale (across lakes). Phylogenetic reconstruction of 434 acI 16S rRNA genes revealed a well-defined and highly resolved phylogeny. Eleven previously unrecognized monophyletic clades, each with > or =97.9% within-clade 16S rRNA gene sequence identity, were identified. Clade community similarity positively correlated with lake environmental similarity but not with geographic distance, implying that the lakes represent a single biotic region containing environmental filters for communities that have similar compositions. Phylogenetically disparate clades within the acI lineage were most abundant at the regional scale, and local communities were comprised of more closely related clades. Lake pH was a strong predictor of the community composition, but only when lakes with a pH below 6 were included in the data set. In the remaining lakes (pH above 6) biogeographic patterns in the landscape were instead a predictor of the observed acI community structure. The nonrandom distribution of the newly defined acI clades suggests potential ecophysiological differences between the clades, with acI clades AI, BII, and BIII preferring acidic lakes and acI clades AII, AVI, and BI preferring more alkaline lakes.