Evaluation of a novel 16S rRNA/tRNA mitochondrial marker for the identification and phylogenetic analysis of shrimp species belonging to the superfamily Penaeoidea

In this study, we infer the phylogenetic relationships within commercial shrimp using sequence data from a novel mitochondrial marker consisting of an approximately 530-bp region of the 16S ribosomal RNA (rRNA)/transfer RNA (tRNA)^Val genes compared with two other mitochondrial genes: 16S rRNA and cytochrome c oxidase I (COI). All three mitochondrial markers were considerably AT rich, exhibiting values up to 78.2% for the species Penaeus monodon in the 16S rRNA/tRNA^Val genes, notably higher than the average among other Malacostracan mitochondrial genomes. Unlike the 16S rRNA and COI genes, the 16S rRNA/tRNA^Val marker evidenced that Parapenaeus is more closely related to Metapenaeus than to Solenocera, a result that seems to be more in agreement with the taxonomic status of these genera. To our knowledge, our study using the 16S rRNA/tRNA^Val gene as a marker for phylogenetic analysis offers the first genetic evidence to confirm that Pleoticus muelleri and Solenocera agassizi constitute a separate group and that they are more related to each other than to genera belonging to the family Penaeidae. The 16S rRNA/tRNA^Val region was also found to contain more variable sites (56%) than the other two regions studied (33.4% for the 16S rRNA region and 42.7% for the COI region). The presence of more variable sites in the 16S rRNA/tRNA^Val marker allowed the interspecific differentiation of all 19 species examined. This is especially useful at the commercial level for the identification of a large number of shrimp species, particularly when the lack of morphological characteristics prevents their differentiation.     

Establishment of a mitochondrial DNA sequence database for the identification of fish species commercially available in South Africa

The limitations intrinsic to morphology-based identification systems have created an urgent need for reliable genetic methods that enable the unequivocal recognition of fish species, particularly those that are prone to overexploitation and/or market substitution. The aim of this study was to develop a comprehensive reference library of DNA sequence data to allow the explicit identification of 53 commercially available fish species in South Africa, most of which were locally caught marine species. Sequences of approximately 655 base pairs were generated for all species from the cytochrome c oxidase I (COI) gene, the region widely adopted for DNA barcoding. Specimens of the genus Thunnus were examined in further detail, employing additional mitochondrial DNA control region sequencing. Cumulative analysis of the sequences from the COI region revealed mean conspecific, congeneric and confamilial Kimura 2-parameter distances of 0.10%, 4.58% and 15.43%, respectively. The results showed that the vast majority (98%) of fish species examined could be readily differentiated by their COI barcodes, but that supplementary control region sequencing was more useful for the discrimination of three Thunnus species. Additionally, the analysis of COI data raised the prospect that Thyrsites atun (snoek) could constitute a species pair. The present study has established the necessary genetic information to permit the unambiguous identification of 53 commonly marketed fish species in South Africa, the applications of which hold a plethora of benefits relating to ecology research, fisheries management and control of commercial practices.     

Culture-dependent bacteria in commercial fishes: Qualitative assessment and molecular identification using 16S rRNA gene sequencing

Fish contamination has been extensively investigated along the Saudi coasts, but studies pertaining to bacterial pathogens are scarce. We conducted qualitative assessment and molecular identification of culture-dependent bacteria in 13 fish species from three coastal sites and a local fish market in Jeddah, Saudi Arabia. Bacterial counts of gills, skin, gut and muscle were examined on agar plates of Macconkey’s (Mac), Eosin Methylene Blue (EMB) and Thiosulfate Citrate Bile Salts (TCBS) culture media. Bacterial counts significantly differed between species, sources and feeding habits of examined fishes. Mugil cephalus exhibited higher counts on TCBS (all body parts), Mac (gills, muscle and gut) and EMB (gills and muscle). Fishes from Area I had higher bacterial loads, coinciding with those in seawater and sediment from the same site, indicating direct association between habitat conditions and the levels of bacterial contamination. By feeding habit, detritivorous fish harbored higher counts than herbivorous and carnivorous species. Bacterial counts of skin were higher in fish from market than field sites, and positively correlated with other body parts indicating the relation of surface bacterial load on the overall quality of fish. Rahnella aquatilis (Enterobacteriaceae) and Photobacterium damselae (Vibrionaceae) were among the dominant species from fish muscle based on 16S rRNA sequencing. These species are known human pathogens capable of causing foodborne illness with severe antibiotic resistance. Opportunistic pathogens, e.g. Hafnia sp. (Enterobacteriaceae) and Pseudomonas stutzeri (Pseudomonadaceae) also occurred in fish muscle. The inclusion of bacterial contamination in future monitoring efforts is thus crucial.     

Differentiation of Bartonella species using restriction endonuclease analysis of PCR-amplified 16S rRNA genes

Abstract Differentiation of the four Bartonella species which were formerly classified as Rochalimaea using restriction endonuclease analysis of PCR-amplified citrate synthase gene fragments has previously been described. However, attempts to extend this method to include all members of Bartonella were confounded when amplification of the gene fragment from strains of B. bacilliformis each yielded two products of differing sizes. An alternative differentiation scheme for Bartonella species was developed based on restriction endonuclease analysis of their 16S rRNA genes. As the complete 16S rRNA gene sequences of all extant Bartonella species are available, the usefulness of specific endonucleases could be theoretically predetermined rather than discovered empirically. The potential usefulness of the restriction enzymes Ddel and Mnll was established using this approach, and this potential was confirmed in practice as all eight species could be distinguished from each other.     

Evaluation of four methods of assigning species and genus to medically important bacteria using 16S rRNA gene sequence analysis

The four methods for assigning bacterial species are the Clinical and Laboratory Standards Institute (CLSI), modified CLSI (mCLSI), phylogenetic analysis (PA) and closest match (CM) methods, these are used to identify the genus and species using 16S rRNA gene sequence results. In this study, the results of identification by these four methods of 37 aerobic reference strains, 30 anaerobic reference strains, 15 Acinetobacter reference strains and 167 Acinetobacter clinical strains were compared. The rates of accurate identification to the species level using the CLSI, mCLSI, PA and CM methods were as follows: 24.3, 86.5, 86.5 and 89.2%, respectively, for the 37 aerobic reference strains; 73.3%, 96.7%, 90.0% and 93.3%, respectively, for the 30 anaerobic reference strains; 40.0%, 93.3%, 100% and 93.3%, respectively, for the 15 Acinetobacter reference strains; and 53.9%, 90.4%, 95.8% and 90.4%, respectively, for the 167 Acinetobacter clinical strains. The rates of accurate identification to the genus level using the CLSI, mCLSI, PA, and CM methods were as follows: 91.9%, 91.9%, 94.6% and 91.9%, respectively, for the 37 aerobic reference strains; 100%, 100%, 100% and 100%, respectively, for all of the 30 anaerobic reference strains, 15 Acinetobacter reference strains and the 167 Acinetobacter clinical strains. The mCLSI is the most practical and pragmatic method for identification of species based on 16S rRNA sequences for hospital, research or industry laboratories because it performs well and involves a simple procedure.     

Organization of the ribosomal RNA genes in Mycoplasma hyopneumoniae: the 5S rRNA gene is separated from the 16S and 23S rRNA genes

Summary In order to study the organization of the ribosomal RNA genes of Mycoplasma hyopneumoniae the rRNA genes were cloned in phage vectors EMBL3 and EMBL4. By subcloning the restriction fragments into various plasmids and analysing the resulting clones by Southern and Northern blot hybridization, a restriction map of the rRNA genes was generated and the organization of the rRNA genes was determined. The results show that the genes for the 16S and 23S rRNAs are closely spaced and occur only once in the genome, whereas the 5S rRNA gene is separated from the other two genes by more than 4 kb.     

Multiplex PCR using 16S rRNA gene-targeted primers for the identification of bifidobacteria from human origin

Three multiplex polymerase chain reactions (PCRs) targeted on Bifidobacterium and related species were designed to identify human species. The selected primers yielded amplified products of various sizes, each specific for a species. Three to four pairs were gathered in one PCR reaction and their specificity under multiplex conditions was confirmed using DNA from 26 reference strains. Using this technique on unidentified faecal strains, B. bifidum, B. longum and B. breve species were commonly recovered in infants while B. adolescentis, B. catenulatum/B. pseudocatenulatum continuum and B. longum species were predominant in adults. Thus, a single PCR can provide the assignment of a strain to one these species, reducing the number of PCR reactions and hands-on time for the identification of human isolates of bifidobacteria. Moreover, this technique is also applicable for the in situ detection of bifidobacteria in DNA extracts from human stools.     

Genes for the ribosomal proteins S12 and S7 and elongation factors EF-G and EF-Tu of the cyanobacterium, Anacystis nidulans: structural homology between 16S rRNA and S7 mRNA

Summary A 6.5 kb region from the genome of the cyanobacterium, Anacystis nidulans 6301 was cloned using the tobacco chloroplast gene for ribosomal protein S12 as a probe. Sequence analysis revealed the presence of genes for ribosomal proteins S12 and S6 and elongation factors EF-G and EF-Tu in this DNA region. The arrangement is rps12 (124 codons)-167 bp spacer-rps7 (156 codons)-77 bp spacer-fus (694 codons)-26 bp spacer-tufA (409 codons), which is similar to that of the Escherichia coli str operon. The deduced amino acid sequences of the A. nidulans S12 and EF-Tu show high homology (72%–82%) with the E. coli and chloroplast counterparts while those of the A. nidulans S7 and EF-G give low homology (51%–59%). Striking structural homology was found between the potential S7 binding region of 16S rRNA and the beginning of S7 mRNA, suggesting that feedback regulation of rps7 expression operates in A. nidulans.     

Molecular identification of pufferfish species using PCR amplification and restriction analysis of a segment of the 16S rRNA gene

This study amplified the mitochondrial 16S rRNA gene using polymerase chain reaction (PCR) with a template of total DNA from muscle tissues of nine pufferfish species collected from the coastal area of Okinawa Islands in Japan: Pleuranacanthus sceleratus, Triodon macropterus, Chelonodon patoca, Sphoeroides pachygaster, Arothron hispidus, A. stellatus, A. manilensis, A. mappa, and A. nigropunctatus. Then nucleotide sequence encoding a partial region of the 16S rRNA gene was compared among species. The sequenced fragment was also used to select restriction enzymes, yielding species-specific restriction fragment length polymorphisms (RFLP). The sequence of the segment of the 16S rRNA gene consisted of about 615 nucleotides and showed interspecies variations in the targeted region. After calculation of corresponding RFLP-patterns of nine species investigated with suitable restriction enzymes, three restriction enzymes – BanII, DdeI, and NlaIII – were found to be sufficient for identification of all nine species. Successful testing of this methodology in frozen and heated food samples suggests its utility for pufferfish species authentication in food products.     

Genetic diversity in ribosomal 18S rRNA and mitochondrial COIII genes in Chinese cultured populations of mussel Mytilus galloprovincialis

To analyze the genetic diversity and genetic structure of cultured populations of Mytilus galloprovincialis, 44 individuals were sampled from three localities, Yantai, Rushan of Shandong province and Dongji of Zhejiang province. Sequence analysis of ribosomal (18S rRNA gene) and mitochondrial DNA (COIII gene) revealed 23 haplotypes of 18S rRNA and 30 haplotypes of COIII. Haplotype diversities (Hd), nucleotide diversities (Pi) and average nucleotide differences (K) were 0.712 (18S rRNA) and 0.946 (COIII), 0.0044 (18S rRNA) and 0.0207 (COIII), and 0.703 (18S rRNA) and 15.316 (COIII), respectively. Fixation indices (Φ_ST) of the three cultured populations showed no genetic divergence between Yantai and Rushan populations but significant genetic divergence between Dongji and the other two populations. In the present study, we found no evidence for decline in genetic polymorphism of cultural M. galloprovincialis populations. The findings of the present study will be useful in the management and conservation of M. galloprovincialis resources.     

Use of 16S rRNA and rpoB genes as molecular markers for microbial ecology studies

Several characteristics of the 16S rRNA gene, such as its essential function, ubiquity, and evolutionary properties, have allowed it to become the most commonly used molecular marker in microbial ecology. However, one fact that has been overlooked is that multiple copies of this gene are often present in a given bacterium. These intragenomic copies can differ in sequence, leading to identification of multiple ribotypes for a single organism. To evaluate the impact of such intragenomic heterogeneity on the performance of the 16S rRNA gene as a molecular marker, we compared its phylogenetic and evolutionary characteristics to those of the single-copy gene rpoB. Full-length gene sequences and gene fragments commonly used for denaturing gradient gel electrophoresis were compared at various taxonomic levels. Heterogeneity found between intragenomic 16S rRNA gene copies was concentrated in specific regions of rRNA secondary structure. Such "heterogeneity hot spots" occurred within all gene fragments commonly used in molecular microbial ecology. This intragenomic heterogeneity influenced 16S rRNA gene tree topology, phylogenetic resolution, and operational taxonomic unit estimates at the species level or below. rpoB provided comparable phylogenetic resolution to that of the 16S rRNA gene at all taxonomic levels, except between closely related organisms (species and subspecies levels), for which it provided better resolution. This is particularly relevant in the context of a growing number of studies focusing on subspecies diversity, in which single-copy protein-encoding genes such as rpoB could complement the information provided by the 16S rRNA gene.     

Use of rpoB gene analysis for identification of nitrogen-fixing Paenibacillus species as an alternative to the 16S rRNA gene

AIM: To avoid the limitations of 16S rRNA-based phylogenetic analysis for Paenibacillus species, the usefulness of the RNA polymerase beta-subunit encoding gene (rpoB) was investigated as an alternative to the 16S rRNA gene for taxonomic studies. METHODS AND RESULTS: Partial rpoB sequences were generated for the type strains of eight nitrogen-fixing Paenibacillus species. The presence of only one copy of rpoB in the genome of P. graminis strain RSA19(T) was demonstrated by denaturing gradient gel electrophoresis and hybridization assays. A comparative analysis of the sequences of the 16S rRNA and rpoB genes was performed and the eight species showed between 91.6-99.1% (16S rRNA) and 77.9-97.3% (rpoB) similarity, allowing a more accurate discrimination between the different species using the rpoB gene. Finally, 24 isolates from the rhizosphere of different cultivars of maize previously identified as Paenibacillus spp. were assigned correctly to one of the nitrogen-fixing species. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The data obtained in this study indicate that rpoB is a powerful identification tool, which can be used for the correct discrimination of the nitrogen-fixing species of agricultural and industrial importance within the genus Paenibacillus.     

Cloning and sequence analysis of the 18 S ribosomal RNA gene of tomato and a secondary structure model for the 18 S rRNA of angiosperms

Summary The gene of a cytoplasmic 18 S ribosomal RNA (18 S rDNA) of the dicotyledonous plant tomato (ycopersicon esculentum) cv. Rentita has been cloned, and its complete primary structure has been determined. The tomato 18 S rDNA is 1805 by long with a G+C content of 49.6%. Its sequence exhibits 94%–96% positional identity when it is colinearly aligned with the previously reported sequences of the 17–18 S rDNAs of the dicot soybean and the monocots maize and rice. A model of the secondary structure of the 18 S rRNA of angiosperms is presented and its genera-specific structural features are compared with a current eukaryotic 18 S rRNA consensus model.     

Comparison of 16S rRNA sequencing with conventional and commercial phenotypic techniques for identification of enterococci from the marine environment

AIMS: To compare accuracy of genus and species level identification of presumptive enterococci isolates from the marine environment using conventional biochemical testing, four commercial identification systems and 16S rRNA sequence analysis. METHODS AND RESULTS: Ninety-seven environmental bacterial isolates identified as presumptive enterococci on mEI media were tested using conventional and Enterococcus genus screen biochemical tests, four commercial testing systems and 16S rRNA sequencing. Conventional and Enterococcus genus screen biochemical testing, 16S rRNA sequencing and two commercial test systems achieved an accuracy of > or = 94% for Enterococcus genus confirmation. Conventional biochemical testing and 16S rRNA sequencing achieved an accuracy of > or = 90% for species level identification. CONCLUSIONS: For confirmation of Enterococcus genus from mEI media, conventional or genus screen biochemical testing, 16S rRNA sequencing and the four commercial systems were correct 79-100% of the time. For speciation to an accuracy of 90% or better, either conventional biochemical testing or 16S rRNA sequencing is required. SIGNIFICANCE AND IMPACT OF THE STUDY: Accurate identification of presumptive environmental Enterococcus isolates to genus and species level is an integral part of laboratory quality assurance and further characterization of Enterococcus species from pollution incidents. This investigation determines the ability of six different methods to correctly identify environmental isolates.     

Sequence differences in the internal transcribed spacers of DNA among four species of hookworm (Ancylostomatoidea: Ancylostoma)

The two ribosomal DNA internal transcribed spacers (1 and 2) of the hookworms Ancylostoma caninum, A. tubaeforme, A. ceylanicum and A. duodenale were sequenced. The sequence lengths were similar among the four species, except that A. ceylanicum had slightly longer (by 5–7 bp) internal transcribed spacer 1 and 2 sequences. The predicted secondary structure of the internal transcribed spacer 2 precursor rRNA was similar for all species, despite interspecific differences in primary sequence ranging from 0.9% to 13.2%. Interspecific differences in internal transcribed spacer 1 sequence ranged from 0.9% to 7.5%. A cladistic analysis of the sequence data, using the human hookworm Necator americanus as the outgroup, provided little resolution of the phylogenetic relationships, except that A. ceylanicum occurred on a branch external to the other three species. Nonetheless, internal transcribed spacers 1 and 2 may provide useful phylogenetic information at higher taxonomic levels within the superfamily Ancylostomatoidea.     

Assessment of rpoB and 16S rRNA genes as targets for PCR-based identification of Pasteurella pneumotropica

Diagnosis of Pasteurella pneumotropica in laboratory animals relies on isolation of the organism, biochemical characterization, and, more recently, DNA-based diagnostic methods. 16S rRNA and rpoB gene sequences were examined for development of a real-time PCR assay. Partial sequencing of rpoB (456 bp) and 16S rRNA (1368 bp) of Pasteurella pneumotropica isolates identified by microbiologic and biochemical assays indicated that either gene sequence can be used to distinguish P. pneumotropica from other members of the Pasteurellaceae family. However, alignment of rpoB sequences from the Pasteurella pneumotropica Heyl (15 sequences) and Jawetz (16 sequences) biotypes with other Pasteurellaceae sequences from GenBank indicated that although rpoB DNA sequencing could be used for diagnosis, development of diagnostic primers and probes would be difficult, because the sequence variability between Heyl and Jawetz biotypes is not clustered in any particular region of the rpoB sequence. In contrast, alignment of 16S rRNA sequences revealed a region with unique and stable nucleotide motifs sufficient to permit development of a specific fluorogenic real-time PCR assay to confirm P. pneumotropica isolated by culture and to differentiate Heyl and Jawetz biotypes.     

Multiplex 16S rRNA haplotype-specific PCR, a rapid and convenient method for fish species identification: an application to West African Clupeiform larvae

A multiplex haplotype-specific polymerase chain reaction (MHS-PCR) method was developed, which identified seven Clupeiform species living in the tropical Eastern Atlantic region: Sardinella aurita, Sardinella maderensis, Ethmalosa fimbriata, Sardina pilchardus, Engraulis encrasicholus, Pellonula leonensis and Ilisha africana. 16S rRNA fragments were amplified using a species-specific set of primers, yielding species-specific size fragments, and then separated using agarose gel electrophoresis, enabling direct visual identification of targeted species. This method provides an accurate, easy and rapid tool for identifying species within large Clupeiform samples. It is suitable for investigations on early Clupeiform stages, species and identification in fishery management in the tropical Eastern Atlantic area.     

The nucleotide sequence of the entire ribosomal DNA operon and the structure of the large subunit rRNA of Giardia muris

Summary The total nucleotide sequence of the rDNA of Giardia muris, an intestinal protozoan parasite of rodents, has been determined. The repeat unit is 7668 basepairs (bp) in size and consists of a spacer of 3314 bp, a small-subunit rRNA (SSU-rRNA) gene of 1429, and a large-subunit rRNA (LSU-rRNA) gene of 2698 bp. The spacer contains long direct repeats and is heterogeneous in size. The LSU-rRNA of G. muris was compared to that of the human intestinal parasite Giardia duodenalis, to the bird parasite Giardia ardeae, and to that of Escherichia coli. The LSU-rRNA has a size comparable to the 23S rRNA of E. coli but shows structural features typical for eukaryotes. Some variable regions are typically small and account for the overall smaller size of this rRNA. The structure of the G. muris LSU-rRNA is similar to that of the other Giardia rRNA, but each rRNA has characteristic features residing in a number of variable regions.Offprint requests to: H. van Keulen