RNA Polymerase Beta Research Articles

Characterization and annotation of Babesia orientalis apicoplast genome.

BackgroundBabesia orientalis is an obligate intraerythrocytic protozoan parasite of the buffalo (Bubalus bubalis, Linnaeus, 1758) transmitted by the tick Rhipicephalus heamaphysaloides. It is the causative agent of water buffalo babesiosis, one of the most important pathogens of water buffalo in central and southern China. As a member of the phylum Apicomplexa, B. orientalis possesses a relatively independent and alga originated organelle the apicoplast. Apicoplasts in other apicomplexa parasites are involved in the biosynthesis of haem, fatty acids, iron-sulphur clusters and isoprenoids. Some of these metabolic pathways were shown to be essential for parasite survival, therefore can serve as potential drug targets.Methods30 pairs of primers were designed based on the full genome sequence of B. orientalis (unpublished data) and by aligning reported apicoplast genomes of Babesia bovis and Theileria parva. Conventional PCRs was performed to obtain overlapped fragments to cover the whole apicoplast genome. Then the apicoplast genome of B.orientalis was sequenced, assembled and aligned with reported apicoplast genomes of B. bovis and T. parva. The obtained apicoplast genome was annotated by using Artemis and comparing with published apicomplexan apicoplast genomes. The SSU and LSU nucleotide sequences generated were used in a phylogenetic analysis using the maximum likelihood implemented in MAGE 6.0.ResultsWe have obtained and analyzed the complete genome sequence of the B. orientalis apicoplast. It consisted of a 33.2 kb circular DNA (78.9 % A + T). The apicoplast genome unidirectionally encodes one large and one small subunit ribosomal RNAs, 24 tRNA genes, 4 DNA-dependent RNA polymerase beta subunits (rpoB, rpoC1, rpoC2a and rpoC2b), 17 ribosomal proteins, one EF-Tu elongation factor, 2 Clp protease chaperones, and 14 hypothetical proteins. In addition, it includes two copies of the clpC gene. The structure and organization of the B. orientalis apicoplast genome are most similar to those of the B. bovis apicoplast.ConclusionsThis is the first report of the complete sequence of the B. orientalis apicoplast genome. This information should be useful in the development of safe and efficient treatment against buffalo babesiosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1158-x) contains supplementary material, which is available to authorized users.

Identification of Leptospira serovars by RFLP of the RNA polymerase beta subunit gene (rpoB).

Leptospires are usually classified by methods based on DNA-DNA hybridization and the conventional cross-agglutination absorption test, which uses polyclonal antibodies against lipopolysaccharides. In this study, the amplification of the rpoB gene, which encodes the beta-subunit of RNA polymerase, was used as an alternative tool to identify Leptospira. DNA extracts from sixty-eight serovars were obtained, and the hypervariable region located between 1990 and 2500-bp in the rpoB gene was amplified by polymerase chain reaction (PCR). The 600-bp amplicons of the rpoB gene were digested with the restriction endonucleases TaqI, Tru1I, Sau3AI and MslI, and the restriction fragments were separated by 6% polyacrylamide gel electrophoresis. Thirty-five fragment patters were obtained from the combined data of restriction fragment length polymorphism (PCR-RFLP) analysis and used to infer the phylogenetic relationships among the Leptospira species and serovars. The species assignments obtained were in full agreement with the established taxonomic classifications. Twenty-two serovars were effectively identified based on differences in their molecular profiles. However, the other 46 serovars remained clustered in groups that included more than one serovar of different species. This study demonstrates the value of RFLP analysis of PCR-amplified rpoB as an initial method for identifying Leptospira species and serovars.

First Report of a Garlic Bulb Rot Caused by Erwinia persicina in Europe

HomePlant DiseaseVol. 99, No. 5First Report of a Garlic Bulb Rot Caused by Erwinia persicina in Europe PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of a Garlic Bulb Rot Caused by Erwinia persicina in EuropeL. Gálvez, J. Gil-Serna, M. García-Díaz, and D. PalmeroL. GálvezSearch for more papers by this author, J. Gil-SernaSearch for more papers by this author, M. García-DíazSearch for more papers by this author, and D. PalmeroSearch for more papers by this authorAffiliationsAuthors and Affiliations L. Gálvez J. Gil-Serna , Universidad Politécnica de Madrid (UPM), EUIT Agrícola, 28040 Madrid, Spain M. García-Díaz , Plant Genetic Resources Centre, INIA, 28800 Alcalá de Henares, Spain D. Palmero , Universidad Politécnica de Madrid (UPM), EUIT Agrícola, 28040 Madrid, Spain. Published Online:29 May 2015https://doi.org/10.1094/PDIS-11-14-1195-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat In July 2013, symptoms of pinkish rot were observed in postharvest Chinese type garlic bulbs (Allium sativum L.) collected from Tembleque (Toledo, Spain). Fifty percent of the bulbs displayed symptoms of pink soft rot in at least one clove showing the pink color along all the flesh. Symptomatic cloves were surface disinfested for 3 min in a 2% sodium hypochlorite solution, rinsed twice with sterilized distilled water, and cultured for 2 days in trypticase soy agar (TSA). Gram-negative, oxidase negative, catalase positive, and facultatively anaerobic, motile, rod-shaped bacteria that were positive for glucose fermentation and nitrate reductase were consistently isolated from symptomatic tissues. Single colonies were light beige, circular, convex with entire margins, and pink-pigmented in PDA but not in TSA. Partial sequences of the 16S rRNA gene (1,386 bp) of two of the isolated bacterial strains were obtained using the primer pair p8FPL and p806R (McCabe et al. 1999) and deposited in EMBL database (accession nos. LM651373 and LM651372). BLAST analysis of the 16S rDNA sequences showed a 99% nucleotide sequence identity with the corresponding sequences of this gene previously available on the NCBI database of Erwinia aphidicola (accession no. NR_104724), E. persicina (NR_114078), and E. rhapontici (KF500098). Additionally, the gene encoding the RNA polymerase beta subunit was sequenced using primers ErpoBF (5′-GGACATGATCAACGCCAAGCCAA-3′) and ErpoBR (5′-GCACCCATCAATGCGCGGTTG-3′) designed in this work on the basis of different species of Erwinia genus. The sequences (581 bp, accession nos. KP100068 and KP100067) showed high identity (99%) with those of E. persicina previously available in databases (i.e. JF311799 and HQ393635) and 97% with the same gene in E. rhapontici (JF311793 and JF311794). The identification was completed with the sorbitol (positive result) and xylitol (negative) fermentation tests that confirmed the strains as E. persicina (Moretti et al. 2011). Pathogenicity tests were performed on Chinese garlic cloves using two E. persicina isolates. Ten cloves were disinfected in 5% sodium hypochlorite solution for 20 min and rinsed twice with sterile distilled water. Thereafter, cloves were wounded with a 200 µl pipette sterile tip (5 mm depth) and inoculated with 10 µl of bacterial suspension (adjusted to a concentration of 107 CFU/ml of TSB) using an automatic pipette. Subsequently, cloves were incubated in petri dishes with a paper towel that was soaked with sterilized distilled water. Ten control cloves were wounded and inoculated with sterilized TSB as negative controls. Inoculated garlic cloves were incubated at 28°C for 10 days in the dark. Pink rot symptoms were observed after 5 days on the inoculated cloves. Control cloves remained symptomless and no bacterium was reisolated. The experiment was repeated once obtaining the same results in both cases. All bacteria reisolated from the pink lesions were identified as E. persicina using the methodology described above. E. persicina has been previously isolated from lucerne, soybean, bean, and pea (González et al. 2007; Zhang and Nan 2014). To our knowledge, this is the first report of clove rot caused by E. persicina in garlic in Europe.

A taxonomic framework for emerging groups of ecologically important marine gammaproteobacteria based on the reconstruction of evolutionary relationships using genome-scale data.

In recent years a large number of isolates were obtained from saline environments that are phylogenetically related to distinct clades of oligotrophic marine gammaproteobacteria, which were originally identified in seawater samples using cultivation independent methods and are characterized by high seasonal abundances in coastal environments. To date a sound taxonomic framework for the classification of these ecologically important isolates and related species in accordance with their evolutionary relationships is missing. In this study we demonstrate that a reliable allocation of members of the oligotrophic marine gammaproteobacteria (OMG) group and related species to higher taxonomic ranks is possible by phylogenetic analyses of whole proteomes but also of the RNA polymerase beta subunit, whereas phylogenetic reconstructions based on 16S rRNA genes alone resulted in unstable tree topologies with only insignificant bootstrap support. The identified clades could be correlated with distinct phenotypic traits illustrating an adaptation to common environmental factors in their evolutionary history. Genome wide gene-content analyses revealed the existence of two distinct ecological guilds within the analyzed lineage of marine gammaproteobacteria which can be distinguished by their trophic strategies. Based on our results a novel order within the class Gammaproteobacteria is proposed, which is designated Cellvibrionales ord. nov. and comprises the five novel families Cellvibrionaceae fam. nov., Halieaceae fam. nov., Microbulbiferaceae fam. nov., Porticoccaceae fam. nov., and Spongiibacteraceae fam. nov.

Report of rpoB mutation in clinically suspected cases of drug resistant leprosy: a study from Eastern India.

The current strategy for leprosy control depends mainly on early case detection and providing the recommended multidrug therapy (MDT) dosage. Understanding the molecular mechanisms of drug resistance to each of these drugs is essential in providing effective treatment and preventing the spread of resistant strains in the community. The progress of molecular biology research provides a very efficient opportunity for the diagnosis of drug resistance by in vitro method. We aimed to investigate the point mutations within the rpoB gene region of the Mycobacterium leprae genome, which are responsible for resistance to rifampicin, in order to determine the emergence of drug resistance in leprosy in the Kolkata region of West Bengal. A total of 50 patients with a relapse of leprosy were enrolled in the study. Skin smears were obtained for estimation of bacillary index and biopsies were obtained in 70% alcohol for extraction of DNA. The extracted DNA was amplified by M. leprae-polymerase chain reaction (PCR) targeting rpoB gene region. Every single nucleotide base in the sequence is aligned to reference sequence and identity gaps were determined by NCBI - BLAST. Later in-silico analysis was done to identify the changes in the translated protein sequences. A mutation at the base pair position 2275405 where G is replaced by C in the M. leprae genome, which corresponds to the coding region of rpoB gene (279 bp - 2275228 to2275506), was observed in two patients. This missense mutation in CAC codon brings about a glutamic acid to histidine change in the amino acid sequence of RNA polymerase beta subunit at the position 442 (Glu442His), a region specific for rifampicin interaction, which might be responsible for unresponsiveness to rifampicin by manifesting a stable bacteriological index in these 2 patients even after completion of 24 months of multibacillary multi-drug therapy (MB-MDT). The major limitations of multiple-primer PCR amplification refractory mutation system (MARS) assay is that it capable of detecting mutation at codon 425 and cannot distinguish any silent amino acid changes. The study indicates the existence of rifampicin drug resistance in Eastern India.

Isolation and molecular identification of Bartonellae from wild rats (Rattus species) in Malaysia.

This study describes our investigation on the prevalence and molecular identification of bartonellae from Rattus diardii and R. norvegicus in the urban areas of Malaysia. Of 95 rats investigated, Bartonella tribocorum, B. rattimassiliensis, B. coopersplainsensis, B. elizabethae, and B. queenslandensis were isolated from kidney and spleen homogenates of four rats. Bartonellae DNA was amplified from the rat organ tissues by using primers specific for the bartonellae RNA polymerase beta subunit (rpoB) gene in nine other rats. Sequence analysis of the rpoB gene fragments shows the identification of B. queenslandensis in five rats, B. elizabethae in three rats, and B. tribocorum in one rat. Combining the results of isolation and molecular detection of bartonellae, we found that the prevalence of Bartonella infection in the Rattus spp. investigated in this study was 13.7%. Implementation of effective rat control program in the urban areas is necessary to prevent the spillover of bartonellosis from rats to humans.

Molecular characterization of bacteria from permafrost of the Taylor Valley, Antarctica

While bacterial communities from McMurdo Dry Valley soils have been studied using molecular techniques, data from permafrost are particularly scarce given the logistical difficulties of sampling. This study examined the molecular diversity and culturability of bacteria in permafrost from the Taylor Valley (TV), Antarctica. A 16S rRNA gene clone library was constructed to assess bacterial diversity, while a clone library of the RNA polymerase beta subunit (rpoB) gene was constructed to examine amino acid composition of an essential protein-coding gene. The 16S rRNA gene clone library was dominated by Acidobacteria from Gp6 and Gemmatimonadetes. The rpoB gene clone library (created with primers designed in this study) was also dominated by Acidobacteria. The ability of sequence analyses to garner additional information about organisms represented by TV sequences was explored. Specifically, optimum growth temperature was estimated from the stem GC content of the 16S rRNA gene, while potential cold adaptations within translated rpoB sequences were assessed. These analyses were benchmarked using known psychrophiles and mesophiles. Bioinformatic analyses suggested that many TV sequences could represent organisms capable of activity at low temperatures. Plate counts confirmed that c. 10(3) cells per gram permafrost remained viable and were culturable, while laboratory respiration assays demonstrated that microbial activity occurred at -5 °C and peaked at 15 °C.

Evaluation of candidate reference genes for gene expression studies in Cymbidium kanran

Quantitative real-time polymerase chain reaction (RT-qPCR) is a preferred method for rapid and reliable quantification of gene expression study. However the transcription levels of target genes may be misinterpreted due to differential expression of the reference gene which was thought to express stably. Thus, a systematic evaluation on the expression stability of nine frequently used reference genes across various tissues (including leaf, root, stem, sepal, peal, lip and gynostemium) and treatments (including phytohormone and stress treatments) in Cymbidium kanran was performed in this study. The results demonstrated that expression stability of the candidate genes varied greatly across different sample pools. Based on geNorm and NormFinder analysis, Tubulin (TUB), Actin (ACT) and Ribosomal protein S3 (RPS3) were considered to be the most suitable reference genes when all 25 samples were taken together. In addition, ACT and TUB were also the suitable internal controls in C. kanran with ABA, NaCl, cold and CuSO4 treatments. RPS3 and TUB exhibited the most stable expression pattern in NAA, 6-BA and GA treatments, while ACT and RNA polymerase beta (RpoB) could be used as the reliable internal control genes under PbAC stress. In different tissues, RPS3 and RpoB could be served as the reliable internal controls, while RpoB and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were ranked top in heat treatment. On the other hand, 18S ribosomal RNA (18S), ribosomal protein S19 (RPS19) and photosystem I P700 apoprotein B (PsaB) performed poorly in expression stability analysis across all sample pools, and they could not be used as internal controls for RT-qPCR studies in C. kanran.

Eosinophilic Granuloma with Splendore-Hoeppli Material Caused by Toxigenic <i>Corynebacterium ulcerans</i> in a Heifer

ABSTRACTRaised lesions were present on the left nasal vestibule of a 20-month-old Japanese Brown heifer. The largest mass which caused partial nasal obstruction was removed surgically. Corynebacterium ulcerans was identified in the mass. 16S ribosomal RNA and RNA polymerase beta subunit genes were 100% and 98% identical to other C. ulcerans strains. Histologically, multiple foci of eosinophilic granuloma with Splendore-Hoeppli material were seen. Rod-shaped Gram-positive organisms were detected with metachromatic granules, producing diphtheria toxin with 5, 30 and 48 amino acid differences to another C. ulcerans strain, C. diphtheriae or C. pseudotuberculosis, respectively. The toxin is highly cytotoxic and may be responsible for the formation of abundant Splendore-Hoeppli material. The lesion was therefore judged to be an allergic reaction to bacterial antigens or diphtheria toxin.

Multigene sequence data reveal morphologically cryptic phylogenetic species within the genus Laccaria in southern Australia

Laccaria (Hydnangiaceae, Agaricales, Basidiomycota) is one of the more intensively studied ectomycorrhizal genera; however, species boundaries within Laccaria and the closely related Hydnangium and Podohydnangium in Australia have not yet been examined with molecular sequence data. Based on morphological characters, eight native species of Laccaria are currently recognized in Australia, as well as three Hydnangium species and the monotypic Podohydnangium australe. Sequences of the internal transcribed spacer region of nuclear rDNA (ITS), RNA polymerase beta subunit II (rpb2) and translation elongation factor 1 alpha (tef-1α) were generated from 77 collections of Laccaria, Hydnangium and Podohydnangium from Australia. Ten phylogenetic species and a further 11 potential species (represented by singletons) of Laccaria in Australia are delimited from sequence analyses. Most of the morphological species contained cryptic phylogenetic species, but these species were always nested entirely within a given morphological species, although not always as sister taxa. The rpb2 locus performed best as a species barcode with pairwise and patristic distance measures. The ITS sequence region returned the least resolved gene tree of the three regions examined and was the least useful as a barcode region. Based on the phylogenetic topology, there appears to have been multiple gains and/or losses of the ectomycorrhizal association of some species with the myrtle beech, Nothofagus cunninghamii as well as of sequestrate basidiocarps and two-spored basidia.

Evaluation of reference genes for reverse transcription quantitative PCR analyses of fish-pathogenic Francisella strains exposed to different growth conditions

BackgroundReverse transcription quantitative PCR has become a powerful technique to monitor mRNA transcription in response to different environmental conditions in many bacterial species. However, correct evaluation of data requires accurate and reliable use of reference genes whose transcription does not change during the course of the experiment. In the present study exposure to different growth conditions was used to validate the transcription stability of eight reference gene candidates in three strains from two subspecies of Francisella noatunensis, a pathogen causing disease in both warm and cold water fish species.ResultsRelative transcription levels for genes encoding DNA gyrase (gyrA), RNA polymerase beta subunit (rpoB), DNA polymerase I (polA), cell division protein (ftsZ), outer membrane protein (fopA), riboflavin biosynthesis protein (ribC), 16S ribosomal RNA (16S rRNA) and DNA helicases (uvrD) were quantified under exponential, stationary and iron-restricted growth conditions. The suitability of selected reference genes for reliable interpretation of gene expression data was tested using the virulence-associated intracellular growth locus subunit C (iglC) gene.ConclusionAlthough the transcription stability of the reference genes was slightly different in the three strains studied, fopA, ftsZ and polA proved to be the most stable and suitable for normalization of gene transcription in Francisella noatunensis ssp.

BartonellaPrevalence and Genetic Diversity in Small Mammals from Ethiopia

More than 500 small mammals were trapped at 3 localities in northern Ethiopia to investigate Bartonella infection prevalence and the genetic diversity of the Bartonella spp. We extracted total DNA from liver samples and performed PCR using the primers 1400F and 2300R targeting 852 bp of the Bartonella RNA polymerase beta subunit (rpoB) gene. We used a generalized linear mixed model to relate the probability of Bartonella infection to species, season, locality, habitat, sex, sexual condition, weight, and ectoparasite infestation. Overall, Bartonella infection prevalence among the small mammals was 34.0%. The probability of Bartonella infection varied significantly with species, sex, sexual condition, and some locality, but not with season, elevation, habitat type, animal weight, and ectoparasite infestation. In total, we found 18 unique Bartonella genotypes clustered into 5 clades, 1 clade exclusively Ethiopian, 2 clades clustered with genotypes from central and eastern Africa, and the remaining 2 clades clustered with genotypes and species from Africa and Asia. The close relatedness of several of our Bartonella genotypes obtained from the 3 dominant rodent species in Tigray with the pathogenic Bartonella elizabethae from Rattus spp. in Asia indicates a potential public health threat.

Description of Riemerella columbipharyngis sp. nov., isolated from the pharynx of healthy domestic pigeons (Columba livia f. domestica), and emended descriptions of the genus Riemerella , Riemerella anatipestifer and Riemerella columbina

A group of 11 bacterial strains was isolated during microbiological investigations of pharyngeal swabs collected from domestic pigeons (Columba livia f. domestica). Phenotypic properties of the isolates closely resembled those of members of the genus Riemerella within the family Flavobacteriaceae. The genus presently contains two species, Riemerella anatipestifer and Riemerella columbina. The pigeon isolates differed from R. columbina by their lack of pigment production and negative CAMP co-haemolysis reaction. They grew more slowly at 37 °C under microaerobic conditions and showed reduced viability during storage under aerobic conditions at different temperatures, compared with both Riemerella species. Comparisons of protein profiles with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS analysis allowed differentiation between the new pigeon isolates and both R. anatipestifer and R. columbina. Phylogenetic analysis based on 16S rRNA gene and rpoB gene (encoding RNA polymerase beta subunit) sequences supported the affiliation of the 11 strains to a novel species within the genus Riemerella, for which we propose the name Riemerella columbipharyngis sp. nov. The type strain is 8151(T) (=DSM 24015(T) = LMG 26094(T)). Emended descriptions of the genus Riemerella and of its species Riemerella anatipestifer and Riemerella columbina are also presented.

남조세균 흔들말목(Cyanobacteria, Oscillatoriales) 해양 균주의 16S rRNA와 rpoB 유전자 변이

본 연구는 남조세균 흔들말목(Cyanobacteria, Oscillatoriales)의 16S ribosomal RNA (rRNA) 및 RNA polymerase beta subunit(rpoB) 유전자를 대상으로 염기서열 변이 및 분자계통학적 특성을 분석한 것이다. 흔들말목 rpoB 유전자는 16S rRNA보다 유전자 변이(유전거리: rpoB=0.270, 16S=0.109)가 큰 것으로 조사되었으며, 통계적으로 유의한 차이를 보였다(Student t-test, p<0.001). 흔들말목 16S rRNA와 rpoB의 계통분석에서 유사한 계통 분지형태를 보였으며, rpoB 유전자가 높은 해상도를 갖고 있어 흔들말목 분류군을 더 명확하게 구분하였다. 또한, parsimony 분석을 통해 rpoB 유전자가 16S rRNA 보다 2.40배 빠르게 진화하는 것으로 파악되었다. 본 연구결과는 rpoB 유전자가 흔들말목의 분자계통 및 종 분류 연구에 매우 유용하다는 것을 제시해 준다. In this study, we investigated molecular divergences and phylogenetic characteristics of the 16S ribosomal RNA (rRNA) and RNA polymerase beta subunit (rpoB) gene sequences from the order Oscillatoriales (Cyanobacteria). The rpoB of Oscillatoriales showed higher genetic divergence when compared with those of 16S rRNA (p-distance: rpoB=0.270, 16S=0.109), and these differences were statistically significant (Student t-test, p<0.001). Phylogenetic trees of 16S rRNA and rpoB were generally compatible; however, rpoB tree clearly separated the compared Oscillatoriales taxa, with higher phylogenetic resolution. In addition, parsimony analyses showed that rpoB gene evolved 2.40-fold faster than 16S rRNA. These results suggest that the rpoB is a useful gene for the molecular phylogenetics and species discrimination in the order Oscillatoriales.

Microbial Analysis of Bite Marks by Sequence Comparison of Streptococcal DNA

Bite mark injuries often feature in violent crimes. Conventional morphometric methods for the forensic analysis of bite marks involve elements of subjective interpretation that threaten the credibility of this field. Human DNA recovered from bite marks has the highest evidentiary value, however recovery can be compromised by salivary components. This study assessed the feasibility of matching bacterial DNA sequences amplified from experimental bite marks to those obtained from the teeth responsible, with the aim of evaluating the capability of three genomic regions of streptococcal DNA to discriminate between participant samples. Bite mark and teeth swabs were collected from 16 participants. Bacterial DNA was extracted to provide the template for PCR primers specific for streptococcal 16S ribosomal RNA (16S rRNA) gene, 16S–23S intergenic spacer (ITS) and RNA polymerase beta subunit (rpoB). High throughput sequencing (GS FLX 454), followed by stringent quality filtering, generated reads from bite marks for comparison to those generated from teeth samples. For all three regions, the greatest overlaps of identical reads were between bite mark samples and the corresponding teeth samples. The average proportions of reads identical between bite mark and corresponding teeth samples were 0.31, 0.41 and 0.31, and for non-corresponding samples were 0.11, 0.20 and 0.016, for 16S rRNA, ITS and rpoB, respectively. The probabilities of correctly distinguishing matching and non-matching teeth samples were 0.92 for ITS, 0.99 for 16S rRNA and 1.0 for rpoB. These findings strongly support the tenet that bacterial DNA amplified from bite marks and teeth can provide corroborating information in the identification of assailants.

Molecular serotyping of Salmonella enterica by complete rpoB gene sequencing

Serotyping has been the gold standard for identifying Salmonella, but it requires large amounts of standard antisera. Multilocus sequence typing (MLST) has been applied to identify Salmonella serovars, but the recombination of 4-7 housekeeping genes and multiple analytic steps diminish its applicability. In the present study, we determined the complete sequences of the RNA polymerase beta subunit gene (rpoB) and 7 housekeeping genes (aroC, dnaN, hemD, hisD, purE, sucA, and thrA) for 76 strains of 33 Salmonella enterica serovars and conducted phylogenetic analyses together with the corresponding gene sequences of 24 reference strains registered in the GenBank database. Based on the phylogenetic analyses, 100 strains from 40 serovars and 91 strains from 37 serovars were classified into 60 rpoB (RST) and 49 multilocus sequence types (ST), respectively. The nucleotide similarities were 98.8-100% and 96.9-100% for the complete rpoB gene and the seven concatenated housekeeping genes, respectively. The strains of 35 and 30 serovars formed serovar-specific branches or clusters in the rpoB and housekeeping gene phylogenetic trees, respectively. Therefore, complete rpoB gene sequencing and phylogenetic analysis may be a useful method for identifying Salmonella serovars that is a simpler, more cost-effective, and less time-consuming alternative or complementary method to MLST and conventional serotyping.

Draft Genome Sequence of Fusobacterium nucleatum ChDC F128, Isolated from a Periodontitis Lesion

Fusobacterium nucleatum is classified into five subspecies. F. nucleatum ChDC F128 was isolated from a periodontitis lesion and proposed as a new subspecies based on the comparison of the nucleotide sequences of the RNA polymerase beta subunit and zinc protease genes. Here, we report the draft genome sequence of the strain.

일반 프라이머를 이용한 PCR의 식품원료 진위 판별에 적용

본 연구는 식품원료의 진위여부를 판별하기 위한 시험법으로 일반 프라이머를 이용한 DNA barcode 기법을 도입하였다. 동물성식품원료의 경우 미토콘드리아 DNA 중 cytochrome oxidase subunit I(COI) 부위 검출을 위하여 디자인된 프라이머(LCO1490/HCO2198 및 VF2/FISH R2)와 cytochrome b(cyt b) 부위 검출을 위하여 디자인된 프라이머(L14724/H15915)를 사용하였다. 상기 3 종류의 프라이머를 사용하여 가축류 6종(소, 돼지, 염소, 양, 말 및 사슴), 가금류 4종(닭, 오리, 칠면조 및 타조), 어류 7종(명태, 대구, 청대구, 청어, 송어, 다랑어 및 우럭)을 대상으로 PCR 후 전기영동하여 예상되는 PCR 산물의 생성 유무를 확인하였다. 가축류 6종에 대하여는 LCO1490/HCO2198, VF2/FISH R2 및 L14724/H15915 프라이머를 사용한 경우 COI 및 cyt b가 모두 검출되었으며, 가금류 4종은 LCO1490/HCO2198 및 VF2/FISH R2 프라이머를 사용한 경우만 COI이 검출되었다. 또한 어류 7종은 VF2/FISH R2 프라이머를 사용한 경우에만 COI 부위가 검출됨을 확인하였다. 식물의 경우 엽록체 DNA 부위를 이용하여 디자인된 3 종류의 프라이머(trnH/psbA, rpoB 1F/4R 및 rbcL 1F/724R)를 사용하였다. 각각의 프라이머를 이용하여 식물 5종(마늘, 양파, 무, 녹차 및 시금치)에 대하여 실험한 결과 3종류의 프라이머에서 PCR의 산물을 모두 확인하였으며 trnH/psbA 프라이머의 경우 식물 종마다 PCR 산물의 크기는 다르게 검출되었다. 본 연구에서는 17종의 식품원료별 일반 프라이머 및 PCR 조건을 확립하였으며, 생산된 PCR 산물을 대상으로 염기서열을 결정하고 유전자은행에 있는 염기서열과 DB 비교 분석을 통하여 식품에 사용된 원료의 진위여부 판별에 적용이 가능할 것으로 기대된다. In order to determine an authenticity of food ingredient, we used DNA barcode method by universal primers. For identification of animal food ingredients, LCO1490/HCO2198 and VF2/FISH R2 designed for amplifying cytochrome c oxidase subunit1 (CO1) region and L14724/H15915 for cytochrome b (cyt b) region on mitochondrial DNA were used. Livestock (cow, pig, goat, sheep, a horse and deer) was amplified by LCO1490/HCO 2198, VF2/FISH R2 and L14724/H15915 primers. Poultry (chicken, duck, turkey and ostrich) was amplified by LCO1490/HCO 2198 and VF2/FISH R2 primers. But, Fishes (walleye pollack, herring, codfish, blue codfish, trout, tuna and rockfish) were only amplified by VF2/FISH R2 primers. For plant food ingredients, 3 types of primers (trnH/psbA, rpoB 1F/4R and rbcL 1F/724R) have been used an intergenic spacer, a RNA polymerase beta subunit and a ribulose bisphosphate carboxylase region on plastid, respectively. Garlic, onion, radish, green tea and spinach were amplified by trnH/psbA, rpoB 1F/4R and rbcL 1F/724R. The PCR product sizes were same by rpoB 1F/4R and rbcL 1F/724R but, the PCR product size using trnH/psbA primer was different with others for plants each. We established PCR condition and universal primer selection for 17 item's raw materials for foods and determine base sequences aim to PCR products in this study. This study can apply to determine an authenticity of foods through making an comparison between databases and base sequences in gene bank. Therefore, DNA barcode method using universal primers can be a useful for species identification techniques not only raw materials but also processed foods that are difficult to analyze by chemical analysis.

RNA polymerase beta subunit (rpoB) gene and the 16S–23S rRNA intergenic transcribed spacer region (ITS) as complementary molecular markers in addition to the 16S rRNA gene for phylogenetic analysis and identification of the species of the family Mycoplasmataceae

Conventional classification of the species in the family Mycoplasmataceae is mainly based on phenotypic criteria, which are complicated, can be difficult to measure, and have the potential to be hampered by phenotypic deviations among the isolates. The number of biochemical reactions suitable for phenotypic characterization of the Mycoplasmataceae is also very limited and therefore the strategy for the final identification of the Mycoplasmataceae species is based on comparative serological results. However, serological testing of the Mycoplasmataceae species requires a performance panel of hyperimmune sera which contains anti-serum to each known species of the family, a high level of technical expertise, and can only be properly performed by mycoplasma-reference laboratories. In addition, the existence of uncultivated and fastidious Mycoplasmataceae species/isolates in clinical materials significantly complicates, or even makes impossible, the application of conventional bacteriological tests. The analysis of available genetic markers is an additional approach for the primary identification and phylogenetic classification of cultivable species and uncultivable or fastidious organisms in standard microbiological laboratories. The partial nucleotide sequences of the RNA polymerase β-subunit gene (rpoB) and the 16S–23S rRNA intergenic transcribed spacer (ITS) were determined for all known type strains and the available non-type strains of the Mycoplasmataceae species. In addition to the available 16S rRNA gene data, the ITS and rpoB sequences were used to infer phylogenetic relationships among these species and to enable identification of the Mycoplasmataceae isolates to the species level. The comparison of the ITS and rpoB phylogenetic trees with the 16S rRNA reference phylogenetic tree revealed a similar clustering patterns for the Mycoplasmataceae species, with minor discrepancies for a few species that demonstrated higher divergence of their ITS and rpoB in comparison to their neighbor species. Overall, our results demonstrated that the ITS and rpoB gene could be useful complementary phylogenetic markers to infer phylogenetic relationships among the Mycoplasmataceae species and provide useful background information for the choice of appropriate metabolic and serological tests for the final classification of isolates. In summary, three-target sequence analysis, which includes the ITS, rpoB, and 16S rRNA genes, was demonstrated to be a reliable and useful taxonomic tool for the species differentiation within the family Mycoplasmataceae based on their phylogenetic relatedness and pairwise sequence similarities. We believe that this approach might also become a valuable tool for routine analysis and primary identification of new isolates in medical and veterinary microbiological laboratories.

Granulicella arctica sp. nov., Granulicella mallensis sp. nov., Granulicella tundricola sp. nov. and Granulicella sapmiensis sp. nov., novel acidobacteria from tundra soil

Four aerobic bacteria, designated MP5ACTX2(T), MP5ACTX8(T), MP5ACTX9(T) and S6CTX5A(T), were isolated from tundra soil of north-western Finland (69° 03' N 20° 50' E). Cells of all isolates were Gram-negative, non-motile rods. Phylogenetic analysis indicated that they belonged to the genus Granulicella of subdivision 1 of the phylum Acidobacteriahttp://dx.doi.org/10.1601/nm.7918. 16S rRNA gene sequence similarity between the new isolates and the type strains of Granulicella aggregans, Granulicella paludicola, Granulicella pectinivorans and Granulicella rosea ranged from 94 to 99 %. Analysis of the RNA polymerase beta subunit (rpoB) gene sequence indicated that the isolates represented novel species of the genus Granulicella (<92 % rpoB sequence similarity between the isolates and members of the genus Granulicella). This was also confirmed by low DNA-DNA relatedness (31 %) between strain S6CTX5A(T) and the type strain of G. pectinivorans, which exhibited 99.1 % 16S rRNA gene sequence similarity and 91.7 % rpoB gene sequence similarity. The isolates grew at pH 3.5-6.5 and at 4-26 °C. Sugars were the preferred growth substrates. The major cellular fatty acids were iso-C(15 : 0), C(16 : 1)ω7c and C(16 : 0) and the major isoprenoid quinone was MK-8. The DNA G+C content was 56-60 mol%. On the basis of phylogenetic analysis and chemotaxonomic and physiological data, the isolates represent four novel species of the genus Granulicella, for which the names Granulicella arctica MP5ACTX2(T) (= ATCC BAA-1858(T) = DSM 23128(T)), Granulicella mallensis MP5ACTX8(T) (= ATCC BAA-1857(T) = DSM 23137(T)), Granulicella tundricola MP5ACTX9(T) (ATCC BAA-1859(T) = DSM 23138(T)) and Granulicella sapmiensis S6CTX5A(T) (= LMG 26174(T) = DSM 23136(T)) are proposed. An emended description of the genus Granulicella is also presented.