rRNA Nucleotide Sequence Research Articles

Comparative evaluation of sequence analysis of 16S rRNA and rpoBgenes for identification of aquatic mycobacteria

The nucleotide sequences of partial 16S rRNA and bacterial RNA polymerase β-subunit (rpoB ) genes for 57 mycobacterial strains were determined. Compared to the 16S rRNA gene sequences, variable regions were scattered along the whole fragment sequence, indicating that the rpoB gene is more polymorphic. Unlike 16S rRNA sequences, species variation was observed within M. fortuitum strains. The topology of the rpoB-based phylogenetic tree was almost the same as that of the 16S rRNA sequence analysis. These results suggest that the rpoB gene is a highly conserved gene, and taxonomical studies based on this gene may be comparable with similar studies based on the 16S rRNA gene. The overall mean distance for rpoB -gene-based sequences was 2.5 times greater than that of the 16S rRNA gene for all 57 mycobacterial strains examined. However, some slowly growing mycobacteria could not be differentiated based on rpoB gene sequences. Moreover, a bootstrap value above 70% was observed for 13 nodes, while this value was 14 nodes in the case of 16S rRNA sequences. To the best of our knowledge, this is the first investigation evaluating the use of 16S rRNA and rpoB sequence analyses for identification of aquatic mycobacteria obtained from diverse geographical locations.

Non-contiguous-Finished Genome Sequence and Description of Paenibacillus camerounensis sp. nov.

Strain G4(T) was isolated from the stool sample of a wild gorilla (Gorilla gorilla gorilla) from Cameroon. It is a facultative anaerobic, Gram-negative, rod-shaped bacterium. This strain exhibits a 16S rRNA nucleotide sequence similarity of 97.48% with Paenibacillus typhae, the phylogenetically closest species with standing nomenclature. Moreover, the strain G4(T) presents some phenotypic differences when compared to other Paenibacillus species and shows a low MALDI-TOF Mass Spectrometry score that does not allow any identification. Thus, it is likely that this strain represents a new species. Here, we describe the characteristics of this organism, complete genome sequence, and annotation. The 6,933,847 bp size genome (1 chromosome but no plasmid) contains 5972 protein-coding genes and 54 RNAs genes, including 44 tRNA genes. In addition, digital DNA-DNA hybridization values for the genome of the strain G4(T) against the closest Paenibacillus genomes range between 19.7 and 22.1, once again confirming its new status as a new species. On the basis of these polyphasic data, consisting of phenotypic and genomic analyses, we propose the creation of Paenibacillus camerounensis sp. nov. that contains the strain G4(T).

Complete nucleotide sequence of the 16S rRNA from Lactobacillus paracasei HS-05 isolated from women's hands.

We determined the complete nucleotide sequence of the 16S rRNA from a new bacterium collected from the surfaces of women’s hands. We also compared the presence of various bacteria based on the subjects’ sex and age. The number of colonies isolated from the hand surface was larger for women than men, and the largest number of isolates was confirmed to be present for the women in their 30 s and men in their 40 s (147 and 34 isolates, respectively). The morphology of an isolated bacterial strain was found to be rod type, and the bacterium was identified as Lactobacillaceae species based on the GenBank database, through a phylogenetic analysis using the 16S rRNA sequence. Based on the results of a homology search, the isolated strain was 99 % identical to Lactobacillus paracasei, so it was designated Lactobacillus paracasei HS-05 and was registered in the Korea Culture Center of Microorganisms (KCCM) database as [KCCM11349P].

Description of a myxozoan parasite Myxobolus sp.n PKB2014 from an edible fish, with emphasis on its molecular characterization.

In India, more than 104 species of Myxobolus have so far been reported infecting freshwater as well as marine fishes. The study focuses on the description of a new myxosporean species, Myxobolus sp.n PKB 2014 from the gill lamellae of an Indian major carp Labeo rohita. The species have been described on the basis of morphological characterization of the spores, tissue architecture and 18S rDNA sequence data. The plasmodia of Myxobolus sp.n PKB 2014 were round in shape measuring, 50 to 70 μm in diameter and spores were ellipsoidal in frontal view measures 14.7 ± 0.51 μm. The 18S rRNA nucleotide sequence with 806 bp of Myxobolus sp.n PKB 2014 (Accession number KJ652226) clustered phylogenitically with other Myxobolus spp. infecting cyprinid gills with 90-99% similarity. According to the phylogenetic study we concluded that M. wulli was the closest relative having 99% similarity with the species under description but the sequence was distinct in each species which additionally exhibited different morphological features. The infection rate was low to moderate. After through comparison it can be concluded that the species being described here is new to science which is designated as Myxobolus sp.n. PKB 2014.

Combined mitochondrial DNA analysis of the Mesopotamian spiny eel, Mastacembelus mastacembelus (Banks & Solander 1794), and its phylogenetic position

Nucleotide sequences of the 12S rRNA, 16S rRNA, tRNA Phe and tRNA Val genes of mtDNA of the Mesopotamian spiny eel, M. mastacembelus was determined for the first time. The comparison of the three populations of Mesopotamian spiny eel from Turkish part of the Tigris basin based on the obtained combined mitochondrial DNA was performed. Based on the results, no differences were determined and the identity found to be 100% among three populations. Furthermore, the obtained results from molecular methods were compared with morphological findings to validate the position of the studied populations of M. mastacembelus . In addition, the phylogenetic position of the Mesopotamian spiny eel was examined among the Mastacembelidae and Synbranchioformes based on 12S rRNA and 16S rRNA. The constructed phylogenetic relationship between M. mastacembelus and some other members of Synbranchioformes order supported their taxonomic hierarchy.

Molecular identification of croaker dried swim bladders (maw) on sale in Hong Kong using 16S rRNA nucleotide sequences and implications for conservation

Hong Kong is a major import and transhipment centre for seafood inclusive of a wide range of dried products. Dried fish swim bladder (commonly referred to as ‘maw’) has long been a significant and valuable component of dried seafood sold in the city. Although the species and provenance of swim bladder are poorly documented the major target taxon involved has historically been the croakers (Sciaenidae). This study is the first to examine the possible provenance and species supplying dried fish swim bladder on sale in Hong Kong using DNA barcoding based on the partial sequence of the 16S rRNA gene. A reference sequence database was constructed from available Genbank fragments and sequences obtained from freshly identified fish specimens. Seventy-nine individual dried fish swim bladders, consisting of four commonly sold morphotypes, were examined with sequences successfully obtained from ∼46% of the samples. Five taxa were identified, four of which matched known species in the sequence database, including three sciaenids and one latid. Confidence in species identifications was high due to low within-group divergence (0.0–1.3%) and high between-group divergence (≥2.9%). None of the identified species is local to Hong Kong waters and none are cultured. Several croakers exploited for swim bladder are depleted or threatened, two of them globally endangered. Interest in fish swim bladder in Chinese markets remains high and it has recently become a replacement for sharks' fin in banquets with decline in fin use. These findings highlight the value of barcoding for species verification of processed fish swim bladders and its potential application in the monitoring of their international trade. The findings also highlight data gaps for species coverage in the available DNA sequences and the need for better trade monitoring of dried swim bladder.

Non contiguous-finished genome sequence and description of Bacillus jeddahensis sp. nov.

Strain JCET was isolated from the fecal sample of a 24-year-old obese man living in Jeddah, Saudi Arabia. It is an aerobic, Gram-positive, rod-shaped bacterium. This strain exhibits a 16S rRNA nucleotide sequence similarity of 97.5 % with Bacillus niacini, the phylogenetically closest species with standing nomenclature. Moreover, the strain JCET presents many phenotypic differences, when it is compared to other Bacillus species, and shows a low MALDI-TOF Mass Spectrometry score that does not allow any identification. Thus, it is likely that this strain represents a new species. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 4,762,944 bp long genome (1 chromosome but no plasmid) contains 4,654 protein-coding and 98 RNAs genes, including 92 tRNA genes. The strain JCET differs from most of the other closely Bacillus species by more than 1 % in G + C content. In addition, digital DNA-DNA hybridization values for the genome of the strain JCET against the closest Bacillus genomes range between 19.5 to 28.1, that confirming again its new species status. On the basis of these polyphasic data made of phenotypic and genomic analyses, we propose the creation of Bacillus jeddahensis sp. nov. that contains the strain JCET.Electronic supplementary materialThe online version of this article (doi:10.1186/s40793-015-0024-y) contains supplementary material, which is available to authorized users.

Practical identification of human originated Lactobacillus species by amplified ribosomal DNA restriction analysis (ARDRA) for probiotic use.

Probiotics are gaining popularity and increasing the importance of their accurate speciation. Lactobacillus species are commonly used as probiotic strains mostly of clinical importance. Present knowledge indicates that at least 14 Lactobacillus species are associated with the human intestinal tract. Currently, researchers are interested in developing efficient techniques for screening and selecting probiotics bacteria, but unfortunately most of these methods are time-consuming, labor-intensive and costly. The aim of this study is to develop reliable, rapid and accurate method to identify 14 references Lactobacillus species that could have been found in the human alimentary tract by 16S ribosomal DNA restriction analysis. In this study, to develop an effective method for the genotype-based identification of the reference Lactobacillus species, 1.5kb of 16S rRNA nucleotide sequences of 14 Lactobacillus were collected from the Gene Bank aligned, in silico restricted and analyzed in respect to their 16S-rRNA restriction fragment polymorphism. In silico restriction profiles of 16S-rRNA indicated that FspBI, HinfI and DraI restriction enzymes (RE) are convenient for differentiation of 14 Lactobacillus species in human intestinal tract except Lb. casei and Lb. paracasei. The patterns of our experimental findings obtained from 16S PCR-ARDRA completely confirmed our in silico patterns. The present work demonstrated that 16S PCR-ARDRA method with FspBI, HinfI and DraI RE is a rapid, accurate and reliable method for the identification of Lactobacillus species from human alimentary tract, especially during the identification of large numbers of isolates and any laboratory equipped with a thermo cycler for probiotic use.

김치에서 분리한 Lactococcus lactis가 생산하는 박테리오신의 정제 및 특성

The bacterial strain isolated from Kimchi showed antibacterial activity against Micrococcus luteus IAM 1056. The selected strain was identified as Lactococcus lactis by 16S rRNA nucleotide sequence analysis and named as Lactococcus sp. KD 28. The treatment of culture supernatant with proteinase K removed antibacterial activity, indicating its proteinaceous nature, a bacteriocin. This bacteriocin was sensitive to hydrolytic enzymes such as α-chymotrypsion, trypsin, proteinase K, lipase, α-amylase and subtilisin A. The bacteriocin was highly thermostable and resistant to heating at 80℃ for up to an hour but 50 % of the total activity was remained at 100℃ for 30 min. The pH range from 2.0 to 8.0 had no effect on bacteriocin activity and it was not affected by solvents such as acetonitrile, isopropanol, methanol, chloroform and acetone up to 50% concentration. The bacteriocin showed antibacterial activity against M. luteus IAM 1056, Lactobacillus delbrueckii subsp. lactis KCTC 1058, Enterococcus faecium KCTC 3095, Bacillus cereus KCTC 1013, B. subtilis KCTC 1023, Listeria ivanovii subsp. ivanovii KCTC 3444, Staphylococcus aureus subsp. aureus KCTC 1916, B. megaterium KCTC 1098 and B. sphaericus KCTC 1184. The bacteriocin was purified through ammonium sulfate concentration, SP-Sepharose chromatography and RP-HPLC. The molecular weight was estimated to be about 3.4 kDa by tricine-SDS-PAGE analysis.

新規外来フジツボ <i>Perforatus perforatus</i> の日本への侵入確認およびリアルタイムPCR法を用いた検出方法について

The acorn barnacle Perforatus perforatus is a common European species that is distributed from southwest England to the Mediterranean and the northwest coast of Africa. Here, we present the first record of invasion of P. perforatus in Japan. This species was found in September, 2012, at a high density on a buoy in Toga Bay in Akita Prefecture, northwest Honshu. It was identified based on the morphological characteristics of the shell and opercular valves, and by molecular analysis using a 300 bp nucleotide sequence of the mitochondrial 12S rRNA. Additionally, a detection and quantification method for P. perforatus using species-specific primer sets for quantitative real-time polymerase chain reaction was developed. Using this method, it was possible to identify and quantify the planktonic nauplius larvae of this alien species.

Molecular epidemiological characterization of poultry red mite, Dermanyssus gallinae, in Japan.

Dermanyssus gallinae, the poultry red mite, is an obligatory blood-sucking ectoparasite. The genetic diversity of D. gallinae has been examined in some countries, but so far not in Asian countries. Here, we sequenced a part of the mitochondrial cytochrome oxidase subunit I (COI) and16S rRNA genes and nuclear internal transcribed spacers (ITS) region in 239 mite samples collected from 40 prefectures throughout Japan. The COI and 16S rRNA nucleotide sequences were classified into 28 and 26 haplotypes, respectively. In phylogenetic trees, the haplotypes clustered into 2 haplogroups corresponding to haplogroups A and B, which were previously reported. Haplogroups A and B were further subdivided into sub-haplogroups AJ1 and AJ2, and BJ1 and BJ2, respectively. In both trees, the sequences of haplotypes in AJ1 and BJ2 were relatively distant from those reported in other countries, while some sequences in AJ2 and BJ1 were identical to those in Europe. In addition, the ITS sequences were classified into two sequences, and both sequences were closely related to the sequences found in European countries. These findings indicate a possibility of international oversea transmission of D. gallinae.

Carnobacterium Isolated from Caviar of Sturgeon (Acipenser ruthenus) Farmed in Korea

Sturgeon aqua-cultured in Korea is mainly Acipenser ruthenus and its culture began in the early 2000s. In this study, Carnobacterium sp. was isolated from unprocessed caviar of aqua-cultured Acipenser ruthenus. The 16s rRNA nucleotide sequence obtained from Carnobacterium sp. isolate (accession no. KM236206) was deposited with GenBank and homologous with Carnobacterium divergens DSM 20623 and NBRC15683 strain. In conclusion, this is first report of isolation of Carnobacterium sp. from caviar of Acipenser ruthenus aqua-cultured in Korea. In the future, it must be ascertained whether Carnobacterium sp. degenerate of caviar or cause diseases in sturgeon.

Most hydrocarbonoclastic bacteria in the total environment are diazotrophic, which highlights their value in the bioremediation of hydrocarbon contaminants.

Eighty-two out of the 100 hydrocarbonoclastic bacterial species that have been already isolated from oil-contaminated Kuwaiti sites, characterized by 16S rRNA nucleotide sequencing, and preserved in our private culture collection, grew successfully in a mineral medium free of any nitrogenous compounds with oil vapor as the sole carbon source. Fifteen out of these 82 species were selected for further study based on the predominance of most of the isolates in their specific sites. All of these species tested positive for nitrogenase using the acetylene reduction reaction. They belonged to the genera Agrobacterium, Sphingomonas, and Pseudomonas from oily desert soil and Nesiotobacter, Nitratireductor, Acinetobacter, Alcanivorax, Arthrobacter, Marinobacter, Pseudoalteromonas, Vibrio, Diatzia, Mycobacterium, and Microbacterium from the Arabian/Persian Gulf water body. A PCR-DGGE-based sequencing analysis of nifH genes revealed the common occurrence of the corresponding genes among all the strains tested. The tested species also grew well and consumed crude oil effectively in NaNO3 -containing medium with and without nitrogen gas in the top space. On the other hand, these bacteria only grew and consumed crude oil in the NaNO3 -free medium when the top space gas contained nitrogen. We concluded that most hydrocarbonoclastic bacteria are diazotrophic, which allows for their wide distribution in the total environment. Therefore, these bacteria are useful for the cost-effective, environmentally friendly bioremediation of hydrocarbon contaminants.

Molecular characterization of Myxobolus cuttacki (Myxozoa, Myxosporea, Bivalvulida) infecting gill lamellae of minor carp Labeo bata (Ham.).

As new pathogenic strains are emerging and threatening aquaculture development, myxosporeans (Myxozoa) are receiving much attention in recent years. Myxosporean taxonomy is traditionally based on morphology of the myxospore stage. Molecular data on Indian myxosporeans are rare. In this report, the 18S rRNA gene sequence of Myxobolus cuttacki infecting gill lamellae of minor carp Labeo bata (Ham.) and its phylogenetic relationship with other myxobolids are described for the first time. The plasmodia of M. cuttacki were 0.5-0.9 mm in size and whitish with a round to oval shape. The mean mature spore size was 16.10×7.05 μm. The 18S rRNA nucleotide sequence with 1703 bp of M. cuttacki (accession number KF465682) clustered phylogenetically with other Myxobolus spp. infecting cyprinid gills with 78-90% homogeneity. The gill lamellae infecting M. catmrigalae (KC933944) and M. orissae (KF448527) of Indian major carp Cirrhinus mrigala from India, exhibited 86% and 81% homogeneity with M. cuttacki, respectively. The infection rate was low to moderate on the gills which can have a negative impact on respiratory and physiological functions and subsequently on fish production.

Bacillus mesophilum sp. nov., strain IITR-54T, a novel 4-chlorobiphenyl dechlorinating bacterium.

The taxonomic position of a Gram-positive, endospore-forming bacterium isolated from soil sample collected from an industrial site was analyzed by a polyphasic approach. The strain designated as IITR-54T matched most of the phenotypic and chemical characteristics of the genus Bacillus and represents a novel species. It was found to biodegrade 4-chlorobiphenyl through dechlorination and was isolated through enrichment procedure from an aged polychlorinated biphenyl-contaminated soil. Both resting cell assay and growth under aerobic liquid conditions using 4-chlorobiphenyl as sole source of carbon along with 0.01% yeast extract, formation of chloride ions was measured. 16S rRNA (1,489 bases) nucleotide sequence of isolated strain was compared with those of closely related Bacillus type strains and confirmed that the strain belongs to the genus Bacillus. Strain IITR-54T differs from all other species of Bacillus by at least 2.1% at the 16S rRNA level, and the moderately related species are Bacillus oceanisediminis (97.9%) followed by Bacillus infantis (97.7%), Bacillus firmus (97.4%), Bacillus drentensis (97.3%), Bacillus circulans (97.2%), Bacillus soli (97.1%), Bacillus horneckiae (97.1%), Bacillus pocheonensis (97.1%) and Bacillus bataviensis (97.1%), respectively. The cell wall peptidoglycan contained meso-diaminopimelic acid and the major isoprenoid quinone was MK-7. Major fatty acids are iso-C15:0 (32.4%) and anteiso-C15:0 (27.4%). Predominant polar lipids are diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The results of physiological and biochemical tests allowed the genotypic and phenotypic distinctiveness of strain IITR-54T with its phylogenetic relatives and suggest that the strain IITR-54T should be recognized as a novel species, for which the name Bacillus mesophilum sp. nov. is proposed. The type strain is IITR-54T (=MTCC 11060T=JCM 19208T).

Comparative study of different molecular methods for typing of Acinetobacter baumannii clinical isolates from University Hospitals

Acinetobacter baumannii, a rod-shape Gram-negative bacterium, is an opportunistic pathogen causing diseases in humans. This bacterium has been recognized as one of the leading causes of nosocomial infection which occurs in hospital or hospital-like setting. The antibiotic resistance of A. baumannii could result from the heavy use of antibiotics and has been recognized as a threat to human health. However, prevention against the disease caused by A. baumannii is difficult due to variable host susceptibility against their infections. We isolated 53 bacterial strains from four different university hospitals in South Korea and identified 34 out of the 53 isolates as A. baumannii, based on the nucleotide sequence of 16S rRNA and gpi genes. For the subtyping of the clinical isolates, we used enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and multilocus sequencing typing (MLST) and compared the results. The result of ERIC-PCR showed that there are 14 distinct DNA fingerprint patterns in the 34 A. baumannii clinical isolates. For MLST analysis of the isolates, we amplified and sequenced seven housekeeping genes (gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD) from each isolate. Each unique allelic profile at the concatenated nucleotide sequences of the seven genes was assigned as a sequence type (ST) and six different STs (ST92, ST105, ST138, ST169, ST262, and ST357) were detected in the 34 A. baumannii clinical isolates. Among the six STs, ST138 was the most ubiquitous in the A. baumannii clinical isolates. To examine the regional distribution of the isolates, STs were clustered into clonal complexes based on their similarity to a previously registered central genotype and the clustering was verified by network phylogenetic analysis.

플레인 요구르트와 소이 요구르트의 미생물학적, 물리화학적 및 항산화 활성

양배추 피클로부터 분리된 유산균으로 발효하여 제조한 플레인 요구르트와 소이 요구르트를 저장하는 동안 미생물학적 및 물리화학적 변화와 항산화 특성을 조사하였다. 분리 균주는 당분해능과 유전자 염기서열 분석을 통해 L. casei PC05와 L. acidophilus PC16으로 동정되었다. Lactobacillus acidophilus PC16 균주로 발효시킨 요구르트의 적정산도, 점도 및 균수는 L. casei PC05 균주에 의해 제조된 요구르트에 비해 높게 나타났으며, 특히 플레인 요구르트보다 소이 요구르트에서 세균수와 물리화학적 요인의 측정값이 유의하게 높은 것으로 확인되었다. 한편, L. acidophilus PC16 균주로 발효시킨 요구르트는 DPPH 라디칼 소거능과 철이온 킬레이팅 활성이 높았으나, L. casei PC05 균주의 요구르트는 superoxide anion 제거능과 SOD 활성이 높은 것으로 나타났다. 대부분의 항산화능은 플레인 요구르트보다 소이 요구르트에서 더 높게 나타났으며, 요구르트의 지질과산화 억제능은 항산화 활성에 기인하는 것으로 추정된다. 또한 발효직후 요구르트의 미생물학적, 물리화학적 및 항산화 활성은 <TEX>$4^{\circ}C$</TEX>에서 8일간 저장하는 동안 거의 일정한 수준으로 유지되었다. This study evaluated the physicochemical and microbiological characteristics and antioxidant properties of yogurt samples fermented with lactic acid bacteria (LAB) obtained from pickled cabbage. API 50 CHL systems and 16S rRNA nucleotide sequence analyses revealed that the isolates were Lactobacillus casei PC05 and L. acidophilus PC16. Cell counts, titratable acidity, and viscosity of the yogurt samples fermented with L. acidophilus PC16 were significantly higher than those of the samples fermented with L. casei PC05 (P<0.05). The detected cell counts and physicochemical properties were significantly lower in plain yogurt than in soy yogurt (P<0.05). Yogurt samples fermented with L. acidophilus PC16 exhibited higher antioxidant activity, measured as ability to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and chelate ferrous ions, than those fermented with L. casei PC05. However, the ability to scavenge superoxide anions and superoxide dismutase (SOD) activity were significantly (P<0.05) higher in yogurt samples fermented with L. casei PC05 compared to those in samples fermented with L. acidophilus PC16. The antioxidant activity of soy yogurt was significantly (P<0.05) higher than that of plain yogurt. The antioxidant activity of the tested strains resulted in lipid peroxidation inhibition (in vitro), which may be related to the elimination of free radicals, chelating ability, and reducing power. There were no significant differences in the physicochemical properties and antioxidant activities of the yogurt samples during cold storage.

Detection of Meat Origin (Species) Using Polymerase Chain Reaction

A quick and reliable method for identifying meat origin is developed to ensure species origin of livestock products for consumers. The present study examined the identification of meat sources (duck, chicken, goat, deer, pig, cattle, sheep, and horse) using PCR by exploiting the mitochondrial 12S rRNA and mitochondrial cytochrome b genes. Species-specific primers were designed for some or all mitochondrial 12S rRNA nucleotide sequences to identify meat samples from duck, chicken, goat, and deer. Mitochondrial cytochrome b genes from pig, cattle, sheep, and horse were used to construct species-specific primers, which were used to amplify DNA from different meat samples. Primer sets developed in this study were found to be superior for detecting meat origin when compared to other available methods, for which the discrimination of meat origin was not equally applicable in some cases. Our new development of species-specific primer sets could be multiplexed in a single PCR reaction to significantly reduce the time and labor required for determining meat samples of unknown origin from the 8 species. Therefore, the technique developed in this study can be used efficiently to trace the meat origin in a commercial venture and help consumers to preserve their rights knowing origin of meat products for social, religious or health consciousness.

GENOTYPIC ANALYSIS OF NODULE BACTERIA NODULATING SOYBEAN IN UKRAINE SOILS

Background. Distribution of root nodule bacteria of soybean in soils of Ukraine is the result of intensive cultivation of soybeans over the last 20 years. During the observation the structure of soybean rhizobia populations for the first time we have determined the strains which significantly differ in phenotypic properties from typical slow-growing bacteria of B. japonicum species previously described. These strains are characterized by high speed growth and we tentatively called them “stains with intensive growth”. The aim of our work was to investigate the genotypic properties of microsymbionts of soybeans with different rates of growth spreading in soils of Ukraine. Materials and methods. The 16S rRNA gene and intergenic 16S-23S rRNA region of six strains - typical representatives of soybean nodule bacteria with slow- and intensive growth-rates was carried out. The strains were picked up from different Ukrainian soils. Results. Analysis of the 16S rRNA nucleotide sequences showed the 100% similarity of slow-growing strains to B. japonicum USDA 6T and USDA 4 ones. This analysis proved propinquity of strains with intensive growth to the strain B. japonicum USDA 127 (USDA 123 group). Representatives of this group possessed increased saprophytic competence so as the examined strains. With use of restriction analysis of ITS intergenic region soybean rhizobia were divided among two ITS types: 1st ITS type - strains with intensive growth, 2nd ITS type - slow- growing strains. According to results of ITS-region sequencing soybean rhizobia form 3 reliably different clusters: two of which include slow-growing strains (group USDA 6 and USDA 4), and a third include soybean microsymbionts with intensive growth (USDA 123 group). Conclusion. On the basis of phenotypic and genotypic (16S rRNA and 16S-23S rRNA) analysis all of the investigated soybean strains of root nodule bacteria were related to the Bradyrhizobium japonicum species. The division of strains by the structure of the ITS-region into two genotypes corresponds to the division of strains into two physiological groups: the strains of an intense and slow growth.

Evaluation of the DNA-dependent RNA polymerase β-subunit gene (rpoB) for phytoplasma classification and phylogeny

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.