Sequence analysis of 12S rRNA and 16S rRNA mitochondrial genes in Iranian Afshari sheep

Partial sequences of 12S and 16S rRNA genes were collected from four thoracican barnacle genera, Capitulum, Chthamalus, Megabalanus, and Tetraclita, to investigate their phylogenetic relationships. Both neighbor joining and maximum parsimony analyses and combined data showed monophyly of the four genera with high bootstrap values, congruent with classification of barnacles based on morphology. The three genera, Chthamalus, Megabalanus and Tetraclita, formed a monophylic group, and the pedunculate genus, Capitulum, formed a paraphylic group with them. The phylogenetic relationships among the three sessile barnacles based on the 12S rRNA gene differed from those based on the 16S rRNA gene or a combination of the 12S and 16S rRNA genes. Based on the 12S rRNA gene the two sessile barnacle genera, Megabalanus and Chthamalus, formed one group, whereas Tetraclita formed another group. However, based on the 16S rRNA gene and combined data of the 12S and 16S rRNA genes, Megabalanus and Tetraclita were the closest relatives among the three sessile barnacles. This was congruent with the results based on larval characters and the nuclear 18S rRNA gene reported previously, but differed from morphological classification. Thus, the 16S rRNA gene appears to be more reliable than the 12S rRNA gene in elucidating the phylogenetic relationships of these four genera within the thoracican barnacles.

The taxonomy of the genus Campylobacter has changed dramatically since its inception in 1963. At that time the genus comprised just two species. At present, taxa that were once assigned to Campylobacter may belong to one of over 50 species distributed among six genera. Most of these taxa belong to a phylogenetically distinct group referred to as either ribosomal RNA (rRNA) superfamily VI or the epsilon division of the class Proteobacteria. The taxonomic diversity of the group is matched by the diverse habitats in which they may be found, and by the wide range of diseases that they are associated with. Recognition of their clinical and economic importance has resulted in intense interest in the group, and the application of increasingly sophisticated isolation, detection and chemotaxonomic methods continues to elucidate new aspects of their biodiversity. However, despite the advances in new bacterial systematics, there remain a number of important issues concerning the classification of various campylobacterial taxa that require careful consideration. Ultimately, these issues are relevant to many working in the field of applied microbiology, including clinicians, veterinarians, epidemiologists and taxonomists. The purpose of this article is briefly to review the major developments in the taxonomy of Campylobacter from its inception to the present day; summarize the most recent changes in the field; analyse current topical issues of special relevance to applied microbiologists, including identification of the bacteria; and speculate on future prospects for campylobacterial taxonomy.

Aiming to use DNA sequence for proper identification of venomous snakes in Saudi Arabia, mitochondrial 12S rRNA gene sequences of Bitis arietans, Cerastes cerastes, C. gasperettii, and Echis coloratus were investigated. Concatenated analysis of 16S and 12S rRNA gene sequences (as mosaic) was also performed to validate the usefulness of using more than one gene marker in molecular taxonomy of venomous snakes. DNA extracted from whole blood samples and 12S rRNA was amplified using PCR. Sequences submitted to GenBank were analyzed for similarity using BLAST and aligned using Clustal X followed by manual editing with BioEdit software. Phylogenetic analysis was generated using PAUP version 4, and the evolutionary distance among different groups was calculated using MEGA5 software for both 12S and 16S rRNA genes. Contrary to the morphological classification, phylogenetic analysis revealed that the collected isolates formed three different statistically supported clusters (B. arietans, C. cerastes, and E. coloratus). Although the combined analysis of 16S and 12S rRNA sequences improved the signal, the C. cerastes and C. gasperettii intermixing have not been resolved. Both single and combined genetic distance analyses showed that Echis is more distant from Cerastes than from Bitis. Further studies are mandatory to evaluate other gene markers in molecular classification.

Traditional morphology-based ciliate classification is often time-consuming and inaccurate, necessitating molecular approaches. Although 18S rRNA gene sequencing is widely used for taxonomic analyses of ciliates, its high degree of conservation makes it challenging to achieve species-level resolution. This study explores the potential of internal transcribed spacers (ITS1 and ITS2) and the 28S rRNA gene to improve taxonomic resolution beyond that offered by 18S rRNA gene in free-living and host-associated ciliates. A comparative analysis of ITS, the 18S, and 28S rRNA gene sequences retrieved from public databases indicated that ITS regions exhibit greater inter- and intra-specific sequence dissimilarity compared to 18S rRNA gene, supporting existing literature. We then designed universal primers targeting the ITS and 28S rRNA gene for freshwater and rumen ciliates. These primers were rigorously evaluated for their inclusiveness, specificity, and amplification efficiency using in-silico PCR, experimental PCR, followed by sequencing and metataxonomic analyses of the ciliate communities. In-silico analyses revealed inclusiveness exceeding 80%, while experimental analyses validated their specificity. Metataxonomic analyses of ciliates demonstrated that the ITS and 28S rRNA gene captured significantly greater taxonomic diversity than 18S rRNA gene. Also, ITS1 offered superior taxonomic resolution by detecting the most ciliate species that went unnoticed by the 18S rRNA gene. These findings underscore the superiority of ITS1, and to a lesser extent ITS2, as taxonomic markers for enhancing the resolution of freshwater and rumen ciliate communities. We recommend ITS1 as an alternative marker to overcome the limitations of 18S rRNA gene-based approaches in free-living and host-associated ciliate taxonomy.

In our previous study based on hsp60 PCR-restriction fragment length polymorphism and 16S rRNA gene sequencing, we stated that the bifidobacterial strains isolated from the individual faecal samples of five baby common marmosets constituted different phylogenetically isolated groups of the genus Bifidobacterium. In that study, we also proposed that these isolated groups potentially represented novel species of the genus Bifidobacterium. Out of them, Bifidobacterium aesculapii, Bifidobacterium myosotis, Bifidobacterium tissieri and Bifidobacterium hapali, have been described recently. Another strain, designated MRM 8.19T, has been classified as member of the genus Bifidobacterium on the basis of positive results for fructose-6-phosphate phosphoketolase activity and analysis of partial 16S rRNA, hsp60, clpC, dnaJ, dnaG and rpoB gene sequences. Analysis of 16S rRNA and hsp60 gene sequences revealed that strain MRM 8.19T was related to B. tissieri DSM 100201T (95.8 %) and to Bifidobacterium bifidum ATCC 29521T (93.7 %), respectively. The DNA G+C composition was 63.7 mol% and the peptidoglycan structure was l-Orn(Lys)-l-Ser. Based on the phylogenetic, genotypic and phenotypic data reported, strain MRM 8.19T represents a novel taxon within the genus Bifidobacterium for which the name Bifidobacterium catulorum sp. nov. (type strain MRM 8.19T=DSM 103154T=JCM 31794T) is proposed.

Phytoplasmas are classified into 16Sr groups and subgroups and 'Candidatus Phytoplasma' species, largely or entirely based on analysis of 16S rRNA gene sequences. Yet, distinctions among closely related 'Ca. Phytoplasma' species and strains based on 16S rRNA genes alone have limitations imposed by the high degree of rRNA nucleotide sequence conservation across diverse phytoplasma lineages and by the presence in a phytoplasma genome of two, sometimes sequence-heterogeneous, copies of the 16S rRNA gene. Since the DNA-dependent RNA polymerase (DpRp) β-subunit gene (rpoB) exists as a single copy in the phytoplasma genome, we explored the use of rpoB for phytoplasma classification and phylogenetic analysis. We sequenced a clover phyllody (CPh) phytoplasma genetic locus containing ribosomal protein genes, a complete rpoB gene and a partial rpoC gene encoding the β'-subunit of DpRp. Primers and reaction conditions were designed for PCR-mediated amplification of rpoB gene fragments from diverse phytoplasmas. The rpoB gene sequences from phytoplasmas classified in groups 16SrI, 16SrII, 16SrIII, 16SrX and 16SrXII were subjected to sequence similarity and phylogenetic analyses. The rpoB gene sequences were more variable than 16S rRNA gene sequences, more clearly distinguishing among phytoplasma lineages. Phylogenetic trees based on 16S rRNA and rpoB gene sequences had similar topologies, and branch lengths in the rpoB tree facilitated distinctions among closely related phytoplasmas. Virtual RFLP analysis of rpoB gene sequences also improved distinctions among closely related lineages. The results indicate that the rpoB gene provides a useful additional marker for phytoplasma classification that should facilitate studies of disease aetiology and epidemiology.

BackgroundMolecular advances have accelerated our understanding of nematode systematics and taxonomy. However, comparative analyzes between various genetic markers have led to discrepancies in nematode phylogenies. This study aimed to evaluate the suitability of using mitochondrial 12S and 16S ribosomal RNA genes for nematode molecular systematics.MethodsTo study the suitability of mitochondrial 12S and 16S ribosomal RNA genes as genetic markers for nematode molecular systematics, we compared them with the other commonly used genetic markers, nuclear internal transcribed spacer 1 and 2 regions, nuclear 18S and 28S ribosomal RNA genes, and mitochondrial cytochrome c oxidase subunit 1 gene. After that, phylum-wide primers for mitochondrial 12S and 16S ribosomal RNA genes were designed, and parasitic nematodes of humans and animals from 75 taxa with 21 representative species were inferred through phylogenetic analyzes. Phylogenetic analyzes were carried out using maximum likelihood and Bayesian inference algorithms.ResultsThe phylogenetic relationships of nematodes based on the mitochondrial 12S rRNA gene supported the monophyly of nematodes in clades I, IV, and V, reinforcing the potential of this gene as a genetic marker for nematode systematics. In contrast, the mitochondrial 16S rRNA gene only supported the monophyly of clades I and V, providing evidence that the 12S rRNA gene is more suitable for nematode molecular systematics. In this study, subclades of clade III containing various nematode families were not monophyletic when the 16S or 12S rRNA gene was used as the genetic marker. This is similar to the phylogenetic relationship revealed by previous studies using whole mitochondrial genomes as genetic markers.ConclusionsThis study supports the use of the 12S rRNA gene as a genetic marker for studying the molecular systematics of nematodes to understand intra-phyla relationships. Phylum-wide primers for nematodes using mitochondrial ribosomal genes were prepared, which may enhance future studies. Furthermore, sufficient genetic variation in the mitochondrial 12S and 16S rRNA genes between species also allowed for accurate taxonomy to species level, revealing the potential of these two genes as genetic markers for DNA barcoding.

Bacteria in the genus Elizabethkingia have emerged as a cause of life-threatening infections in humans. However, accurate species identification of these pathogens relies on molecular techniques. We aimed to evaluate the accuracy of 16S rRNA and complete RNA polymerase β-subunit (rpoB) gene sequences in identifying Elizabethkingia species. A total of 173 Elizabethkingia strains with whole-genome sequences in GenBank were included. The 16S rRNA gene and rpoB gene sequences from the same Elizabethkingia strains were examined. Of the 41 E. meningoseptica strains, all exhibited >99.5% 16S rRNA similarity to its type strain. Only 83% of the 99 E. anophelis strains shared >99.5% 16S rRNA gene similarity with its type strain. All strains of E. meningoseptica and E. anophelis formed a cluster distinct from the other Elizabethkingia species in the 16S rRNA and rpoB gene phylogenetic trees. The polymorphisms of 16S rRNA gene sequences are not sufficient for constructing a phylogenetic tree to discriminate species in the E. miricola cluster (E. miricola, E. bruuniana, E. occulta, and E. ursingii). The complete rpoB gene phylogenetic tree clearly delineates all strains of Elizabethkingia species. The complete rpoB gene sequencing could be a useful complementary phylogenetic marker for the accurate identification of Elizabethkingia species.

A new disease named as periwinkle leaf yellowing (PLY) was first observed in a flower production farm in Dayuan Township (Taoyuan county, Taiwan) in August 2005. Sequence analysis of 16S rDNA, 16S-23S rDNA ISR, and partial 23S rDNA sequence revealed that the causative agent of PLY was closely related to the phytoplasmas of the aster yellows (AY) group (16SrI group) which cause diseases in many horticultural and vegetable crops worldwide, and can be delineated into 10 subgroups. Six cultivated plants including periwinkle plant, chrysanthemum, cosmos, torenia, Persian violet, goosegrass and cucumber, were determined to be the host plants of PLY phytolasma. Monthly PCR detection indicated that PLY phytoplasma was detected in host plants from June to October in 2007, and from July to October in 2007. However, it can be detected earlier since April in 2009. To further clarify the phylogenetic relationship of strain PLY among 16SrI phytoplasmas, six phylogenetic trees were constructed in this study. Beside the phylogenetic trees based on the independent analysis of 16S rRNA, rplV-rpsC, secY and tuf gene sequences, two other trees based on the analysis of the comprising gene sequence of 16S rRNA, rplV-rpsC and secY, and the comprising gene sequence of rplV-rpsC and secY were also constructed. The results indicate that the strain PLY was closely related to 16SrI-B and 16SrI-D subgroup, and could be a new subgroup based on the phylogenetic tree constructed by rplV-rpsC gene sequences. In addition, the phylogenetic analysis of the comprised gene sequences showed similar tree topology when compared with sequence analysis of the rplV/ rpsC gene or of the secY gene alone. The main difference is that the branch lengths were elongated in the comprised gene tree. To further confirm the subgroup affiliation of PLY phytoplasma, the 16S rRNA gene sequences of 10 closely related phytoplasma strains were digested in silico, and the similarity coefficients were then calculated. The results also support the conclusion that PLY phytoplasma might belongs to a new 16SrI subgroup. The putative restriction site analysis can also distinguish PLY phytoplasma from other close related phytoplasma strains in phylogenetic analysis.

RFLP analyses of 16S rDNA nested PCR products from 34 phytoplasma strains with 17 restriction enzymes delineated distinct pattern types. Based on similarity coefficients derived from RFLP analyses, the 34 representative phytoplasma strains were differentiated into 14 major groups (termed 16Sr groups) and 32 sub-groups. The similarity coefficients of RFLP patterns between distinct groups were 90% or below. By including additional groups and sub-groups from which RFLP analyses were not performed but for which 16S rDNA sequence data were available to predict restriction sites, a total of 14 groups and 41 sub-groups were proposed. By combined RFLP analyses of 16S rRNA and ribosomal protein gene sequences, thus far, a total of 46 subgroups have been recognized. The phytoplasma 16Sr groups were consistent with the phylogenetic groups (subclades) defined by phylogenetic analysis of near-full-length 16S rRNA gene sequences, indicating that the RFLP-based groups are phylogenetically valid. The approach using RFLP analyses of PCR-amplified 16S rDNA (and ribosomal protein gene sequences) provides a simple, reliable and rapid means for differentiation and classification of unknown phytoplasmas.

BackgroundTwo clustered clinical cases of canine babesiosis were diagnosed by veterinary practitioners in two areas of northeastern Italy close to natural parks. This study aimed to determine the seroprevalence of babesial infection in dogs, the etiological agents that cause canine babesiosis and the potential tick vector for the involved Babesia spp.MethodsThe study area was represented by two parks in northeastern Italy: Groane Regional Park (Site A) and the Ticino Valley Lombard Park (Site B). From March to May 2015 ticks were collected from the vegetation in three transects in each site. In the same period, blood samples were collected from 80 dogs randomly chosen from veterinary clinics and kennel located in the two areas. Morphological identification of the ticks was performed and six specimens were molecularly characterised by the amplification and sequencing of partial mitochondrial 12S rRNA, 16S rRNA and cox1 genes. For phylogenetic analyses, sequences herein obtained for all genes and those available from GenBank for other Dermacentor spp. were included. Dog serum samples were analysed with a commercial indirect fluorescent antibody test to detect the presence of IgG antibodies against Babesia canis. Ticks and blood samples were tested by PCR amplification using primers targeting 18S rRNA gene of Babesia spp.ResultsTicks collected (n = 34) were morphologically identified as adults of D. reticulatus. Twenty-eight ticks were found in all transects from Site A and the remaining six were collected in Site B. Blast analysis of mitochondrial sequences confirmed the morphological identification of processed tick specimens by revealing a highest nucleotide similarity (99–100 %) with those of D. reticulatus available in the GenBank database. The phylogenetic trees were concordant in clustering D. reticulatus in a monophyletic clade. Seven dogs (8.8 %) had antibodies against B. canis, most of which (n = 6) came from Site A. Analysis of nucleotide sequences obtained from one tick and from one dog identified B. canis displayed a 100 % similarity to those available in GenBank.ConclusionsThis study morphologically and molecularly confirms the presence of D. reticulatus in Italy and links it, for the first time, with the occurrence of B. canis infection in dogs in this country.

Detection of Francisella-like endosymbiont in Hyalomma rufipes from Ethiopia

Molecular and symbiotic characterization of peanut bradyrhizobia from the semi-arid region of Brazil

Phylogenies of housekeeping gene and 16S rRNA gene sequences were compared to improve the classification of the bacterial family Pasteurellaceae and knowledge of the evolutionary relationships of its members. Deduced partial protein sequences of the housekeeping genes atpD, infB and rpoB were compared in 28, 36 and 28 representative taxa of the Pasteurellaceae, respectively. The monophyly of representatives of the genus Gallibacterium was recognized by analysis of all housekeeping genes, while members of Mannheimia, Actinobacillus sensu stricto and the core group of Pasteurella sensu stricto formed monophyletic groups with two out of three housekeeping genes. Representatives of Mannheimia, Actinobacillus sensu stricto, [Haemophilus] ducreyi and [Pasteurella] trehalosi formed a monophyletic unit by analysis of all three housekeeping genes, which was in contrast to the 16S rRNA gene-derived phylogeny, where these taxa occurred at separate positions in the phylogenetic tree. Representatives of the Rodent, Avian and Aphrophilus-Haemophilus 16S rRNA gene groups were weakly supported by phylogenetic analysis of housekeeping genes. Phylogenies derived by comparison of the housekeeping genes diverged significantly from the 16S rRNA gene-derived phylogeny as evaluated by the likelihood ratio test. A low degree of congruence was also observed between the individual housekeeping gene-derived phylogenies. Estimates on speciation derived from 16S rRNA and housekeeping gene sequence comparisons resulted in quite different evolutionary scenarios for members of the Pasteurellaceae. The phylogeny based on the housekeeping genes supported observed host associations between Mannheimia, Actinobacillus sensu stricto and [Pasteurella] trehalosi and animals with paired hooves.

There has been an increasing emphasis on the need to exploit un- and underexplored environments especially the marine environments for microbial and chemical diversity. Previous in-depth exploration of Chilean marine sediments have led to the isolation of members of the Micromonosporaceae, which require de-replication and characterization to establish taxonomic status along with screening of the isolates for the ability to produce bioactive compounds. This study was, therefore, embarked on with the aim of assessing phylogenetic relationship of the isolates and screening for novel polyketide synthases type I (PKS-I), non-ribosomal peptide synthases (NRPS) biosynthetic genes (BGC). This involved culture, de-replication by the traditional colour grouping to select representative strains, amplification of 16S rRNA, PKS-I and NRPS genes, sequencing and phylogenetic analysis. Thirty-four representative strains were selected from 66 Micromonospora species. Following the 16S rRNA gene sequence analysis, 11 belonged to the genus Micromonospora, 7 strains residing in the genera Exiguobacterium and Bacillus. The phylogenetic analysis of the 16S rRNA gene sequences of the strains inferred that these strains are novel members of this sub-order. The partial sequences of PKS-I and NRPS genes amplified from eight Micromonospora strains, produced matches with a variety of BGCs including Streptomyces noursei, S. neyagawaensis concanamycin A and Streptomyces sp. heptaene macrolide complex synthesis gene cluster. There exists an untapped microbial diversity in the Chilean marine sediments with great potential of been exploited for novel bioactive compounds as the search for newer and more potent natural products deepens.