16S rDNA Sequence Analysis Research Articles

16S ribosomal DNA sequence-based identification of bacteria in laboratory rodents: a practical approach in laboratory animal bacteriology diagnostics.

Correct identification of bacteria is crucial for the management of rodent colonies. Some bacteria are difficult to identify phenotypically outside reference laboratories. In this study, we evaluated the utility of 16S ribosomal DNA (rDNA) sequencing as a means of identifying a collection of 30 isolates of rodent origin which are conventionally difficult to identify. Sequence analysis of the first approximate 720 to 880 bp of the 5'- end of 16S rDNA identified 25 isolates (83.33%) with ≥ 99% similarity to a sequence of a type strain, whereas three isolates (10%) displayed a sequence similarity ≥ 97% but <99% to the type strain sequences. These similarity scores were used to define identification to species and genus levels, respectively. Two of the 30 isolates (6.67%) displayed a sequence similarity of ≥ 95 but <97% to the reference strains and were thus allocated to a family. This technique allowed us to document the association of mice with bacteria relevant for the colonies management such as Pasteurellaceae, Bordetella hinzii or Streptococcus danieliae. In addition, human potential pathogens such as Acinetobacter spp., Ochrobactrum anthropi and Paracoccus yeei or others not yet reported in mouse bacterial species such as Leucobacter chironomi, Neisseria perflava and Pantoea dispersa were observed. In conclusion, the sequence analysis of 16S rDNA proved to be a useful diagnostic tool, with higher performance characteristics than the classical phenotypic methods, for identification of laboratory animal bacteria. For the first time this method allowed us to document the association of certain bacterial species with the laboratory mouse.

Development and validation of a quantitative PCR assay for the early detection and monitoring of the invasive diatom Didymosphenia geminata

Didymosphenia geminata is a large, invasive, freshwater diatom that can produce distinctive and robust mucilaginous stalks. Over the last two decades, there has been a worldwide increase in the distribution and severity of D. geminata blooms. These dense, persistent blooms can have severe impacts on native species and ecosystem functioning. D. geminata is usually identified by microscopic methods that are time consuming, resource intensive, and dependent upon expert taxonomic identification, so the extent of surveillance programs has been limited. As an alternative, we have developed a TaqMan quantitative polymerase chain reaction (QPCR) assay for sensitive and rapid detection and enumeration of D. geminata in environmental samples. Species-specific QPCR primers and probe were designed by aligning the D. geminata 18S ribosomal DNA (rDNA) sequence with closely related diatoms. The QPCR assay was linear (R2=1.00) over a detection range of eight orders of magnitude with a lower limit of approximately two D. geminata cells. QPCR analysis of environmental samples employed the comparative cycle threshold (CT)-method with an exogenous plasmid used as an internal reference standard. The assay was evaluated using samples collected during a survey of D. geminata in three rivers in the South Island, New Zealand, and from 13 international locations where D. geminata is known to be present. Positive QPCR amplifications were confirmed as the correct amplification product through direct DNA sequencing. Phylogenetic analysis of 18S rDNA sequences suggests that D. geminata is more closely related to species in the family Cymbellaceae rather than Gomphonemataceae as currently classified.

Prevalence, morphology, and molecular analysis of Serrasentis sagittifer (Acanthocephala: Palaeacanthocephala: Rhadinorhynchidae), a parasite of the gilthead sea bream Sparus aurata (Sparidae).

Seventy specimens of the gilthead sea bream Sparus aurata of the Red Sea were collected during the period from March to November 2013; they were dissected and examined for parasitic acanthocephalans. Only 40 (57.14%) specimens were found to be naturally infected with Serrasentis sagittifer belonging to family Rhadinorhynchidae. The infection was recorded in the intestine, pyloric ceca, and the external surfaces of some internal organs of the infected fish. Seasonally, the prevalence of infection was increased to 77.14% during summer season and decreased to 37.14% during winter. Light and scanning electron microscopic investigation revealed that the adult worm was elongated (with broad anterior and narrow posterior ends) and measured 6.9-8.6 (7.6 ± 0.2) × 0.57-0.73 (0.63 ± 0.02) mm for male and 10.2-12.1 (11.5 ± 0.2) × 0.71-0.82 (0.76 ± 0.02) mm for female. Proboscis was long and cylindrical with a length of 0.97-1.6 mm (1.2 ± 0.2) for male and 1.12-1.17 mm (1.14 ± 0.02) for female. It was covered with numerous uniform spines arranged longitudinally as 9-11 rows each equipped by 15-18 spines. Spines were triangular, arrow-shaped, strong, and covered with cuticular theca; they decreased in size from the apex to the base of the proboscis. The proboscis is followed by a short spineless neck region followed by the body proper which is supported by multiple combs of spines (16-20) on its ventral surface. Molecular analysis of 18S rDNA sequence for the parasite demonstrated a close identity (>83%) between the present acanthocephalan and other previously described species within class Palaeacanthocephala with 98% identity with the previously recorded S. sagittifer (acc. no. JX014227) which is supported by the morphological data and the presence of trunk spines arranged within rows (comb-like) and the presence of four cement glands in the males. So, according to the records of morphological and molecular analyses, the present parasite is classified as S. sagittifer belonging to class Palaeacanthocephala and family Rhadinorhynchidae with a new host record from the gilthead sea bream S. aurata of the Red Sea.

The sexual adult of Cercaria praecox Walker, 1971 (Digenea: Fellodistomidae), with the proposal of Oceroma n. g.

A sexual adult trematode that is considered to be conspecific with the distinctive larval trematode Cercaria praecox Walker, 1971 is reported from the kyphosid fish Scorpis lineolata Kner in Moreton Bay, Queensland, Australia. The sexual adult is consistent with the cercarial body of Cercaria praecox in having a single caecum with an asymmetrical appendix, symmetrical testes immediately posterior to the ventral sucker, and the ovary and vitellarium both well posterior to the testes. This combination of characters is distinct within the Fellodistomidae Nicoll, 1909 and requires the proposal of a new genus, Oceroma n. g. Analysis of 28S rDNA sequences demonstrates that this species forms a clade with Coomera Dove & Cribb, 1995 within the Fellodistomidae. The life-cycle of the species is predicted to require two hosts and to involve the direct ingestion of the cercaria.

Identification of Dickeya zeae as a Causal Agent of Bacterial Soft Rot in Banana in China.

Bacterial soft rot of banana was first noticed in 2009 in Guangzhou city, China. The disease also was observed on various banana cultivars of different genotypes in several other cities. Symptoms of the disease included leaf wilting, collapse of pseudostems, and unusual odor. Five isolated strains that fulfilled Koch's postulates were used for biochemical testing. The five strains were most similar to Dickeya dadantii or D. zeae, but were much less similar to D. paradisiaca when using several phenotype characteristics. Sequence analysis of 16S rDNA, dnaX, gryB, and recA of a reference strain revealed a similarity of 99% with the sequences of D. zeae, rather than D. paradisiaca. Phylogenic analysis of concatenated sequences of dnaX, gryB, and recA indicated that the banana strain constituted a distinguishable clade with several D. zeae strains involving rice pathogens D. zeae EC1 and ZJU1202 from Guangdong province, but the banana pathogen had several characteristics that distinguished it from the rice pathogens. Therefore, the banana pathogen was determined to be D. zeae. This is the first report of banana soft rot caused by D. zeae in China; however, the pathogen can infect other important crops.

Phylogenetic study of Japanese Dickeya spp. and development of new rapid identification methods using PCR–RFLP

Forty-one representative Japanese Dickeya spp. (Erwinia chrysanthemi) strains isolated from 24 plants in Japan were investigated using multilocus sequence analysis of recA, dnaX, rpoD, gyrB and 16S rDNA; PCR–RFLP (restriction fragment length polymorphism) of recA, rpoD and gyrB genes; PCR genomic fingerprinting; and biochemical tests. Based on the recA, dnaX, rpoD, gyrB and 16S rDNA sequences and PCR genomic fingerprinting, the strains were essentially divided into six groups (I–VI). Group I corresponded to D. chrysanthemi, group II corresponded to D. dadantii, group III to D. dianthicola and group IV to D. zeae. Meanwhile, group V and group VI could not be assigned to any existing Dickeya species, and they were deduced to be two putative new species. The PCR–RFLP analysis of gyrB, rpoD and recA clearly differentiated the six groups of Dickeya strains. From the results of the biochemical tests, the strains were assigned to biovars 1, 3, 5, 8 and 9; only one strain (SUPP 2525) was not assignable to the existing biovars. We also showed that the PCR–RFLP analysis of rpoD, gyrB and recA can be used as a rapid technique to identify Japanese Dickeya strains.

Host specificity and genetic diversity of race 4 strains of Ralstonia solanacearum

Ralstonia solanacearum race 4 isolates were obtained from Zingiberaceae plants in India during bacterial wilt outbreaks. Polyphasic phenotypic and genotypic analysis revealed intraracial diversity and dominance of biovar 3 over biovar 4. Biovar 3 strains were isolated from very severely wilted Zingiberaceae plants in the field and found to be present across diverse geographical, host and seasonal boundaries. It was hypothesized that these isolates belong to a single, ‘fast wilting’, lineage. Using one ‘fast wilting’ isolate in controlled inoculations, rapid wilt was observed in ginger within 5–7 days. Wilting was also observed in several other closely and distantly related hosts such as turmeric (Curcuma longa), aromatic turmeric (Curcuma aromatica), black turmeric (Curcuma caesia), sand ginger (Kaempferia galanga), white turmeric (Curcuma zeodaria), awapuhi (Zingiber zerumbet), greater galangal (Alpinia galanga), globba (Globba sp.), small cardamom (Elettaria cardamomum) and large cardamom (Ammomum subulatum) of the Zingiberaceae family, and in tomato (Solanum lycopersicum). Molecular analysis, including multiplex PCR‐based phylotyping, sequence analysis of 16S rDNA, 16–23S intergenic spacer and the recN gene, and multilocus sequence typing, revealed minimal differences between fast wilting isolates, confirming that almost all belong to the same lineage. Biovar 4 was isolated from plants showing slow wilt progression and self‐limiting wilting in restricted geographical locations instead, and was identified to be genetically distinct from the fast wilting biovar 3 isolates. To the authors' knowledge, this is the first report of host range and genetic analysis of R. solanacearum race 4 in India.

Characterization of Pectobacterium carotovorum subsp. odoriferum causing soft rot of stored vegetables

Pectobacterium carotovorum subsp. odoriferum has been generally considered to have a narrow host range and has been isolated most often from chicory. Research was conducted to identify 91 Pectobacterium spp. strains isolated from different vegetables in Europe, North and South America, Asia, and Africa, and to compare their ability to cause disease in chicory and potato. Among the 91 strains, 22 strains from Europe were identified as P. c. subsp. odoriferum. Based on phylogenetic analysis of 16S rDNA, recA, and rpoS gene sequences, strains isolated from stored vegetables clustered together with the type strain of P. c. subsp. odoriferum and clustered separately from the P. c. subsp. carotovorum isolates. Eleven strains previously identified as P. c. subsp. carotovorum were reclassified as P. c. subsp. odoriferum. All P. c. subsp. odoriferum isolates were able to cause soft rot symptoms on chicory and potato. Moreover, the symptoms on potatoes were more severe at temperatures from 15 to 37 °C with P. c. subsp. odoriferum isolates than with P. atrosepticum or P. c. subsp. carotovorum isolates. Tissue maceration by P. c. subsp. odoriferum isolates was highest at 28 °C, and at that temperature tissue maceration was two-times greater for P. c. subsp. odoriferum isolates than for P. c. subsp. carotovorum isolates. Symptoms on inoculated chicory leaves were more severe with P. c. subsp. odoriferum (regardless of origin) than with other subspecies or species. To our knowledge, this is the first report that P. c. subsp. odoriferum occurs on a wide range of vegetables and has the ability to cause soft rot during potato storage.

Application of a statistically enhanced, novel, organic solvent stable lipase from Bacillus safensis DVL-43

This paper presents the molecular identification of a newly isolated bacterial strain producing a novel and organic solvent stable lipase, statistical optimization of fermentation medium, and its application in the synthesis of ethyl laurate. On the basis of nucleotide homology and phylogenetic analysis of 16S rDNA sequence, the strain was identified as Bacillus safensis DVL-43 (Gen-bank accession number KC156603). Optimization of fermentation medium using Plackett–Burman design and response surface methodology led to 11.4-fold increase in lipase production. The lipase from B. safensis DVL-43 exhibited excellent stability in various organic solvents. The enzyme retained 100% activity after 24h incubation in xylene, DMSO and toluene, each solvent being used at a concentration of 25% (v/v). The use of partially purified DVL-43 lipase as catalyst in the synthesis of ethyl laurate, an esterification product of lauric acid and ethanol, resulted in 80% esterification in 12h under optimized conditions. The formation of ethyl laurate was confirmed using TLC and 1H NMR. Organic solvent stable lipases exhibiting potential application in enzymatic esterification are in great demand in flavor, fine chemicals and pharma industries. We could not find any report on lipase production from B. safensis strain and its application in esterification.

Real-Time PCR Quantification of Protease-Producing Bacteria in Traditional Chinese Fish Sauce

This study aims to isolate, identify, and quantify protease-producing bacteria in traditional Chinese fish sauce. Fifty-five protease-producing bacteria were isolated from 10 fermented fish sauce samples in five growth media based on their morphology and caseinolytic activity. These isolates were identified through their 16S rDNA gene sequences. BLAST analysis of 16S rDNA sequences revealed that 46 and 9 strains belonged to Bacillus sp. and Virgibacillus halodenitrificans, respectively. We used EvaGreen dye in real-time PCR assay to target the partial bacterial 16S rDNA gene sequences of the 55 strains from fish sauce. Two primer pairs (A and B) were designed. Primer pair B was more suitable than primer pair A for real-time PCR assay, in which the optimum annealing temperature was 60 °C. The significance of the developed method was proven by the highly linear characteristic of the standard curve that relates lower threshold cycle (Ct) values and 16S rDNA copy numbers of the standard DNA sample. This method was used to calculate the number of protease-producing bacteria in fish sauce. The minimum level of detection (1.44 × 103 copies/μL) was also verified. The concentration of protease-producing bacteria in fish sauce was estimated to be (1.47 ± 0.73) × 103 colony-forming units (cfu)/mL by real-time PCR assay and showed a percentage of positive results correctly assigned of 91.8 % when compared to the plate culture method used as reference. The results may serve as a foundation for future studies on the microbial succession and variation of microorganisms during fish sauce fermentation.

Development of microbial properties and enzyme activities in copper mine wasteland during natural restoration

A copper mine wasteland consists of a pedogenetic substrate, and regeneration of ecosystems on mine wasteland undergoes typical primary succession. In this study, microbial biomass, basic respiration, microbial diversity and enzyme activities were investigated during the natural successional process in two copper mine wastelands with different restoration development times. As the plant ecosystem progressed from bare land with cryptogamic crusts to vascular plant communities, organic matter, total nitrogen (N), available phosphorus (P) and water holding capacity increased, whereas pH and toxicity of heavy metals decreased. Along with the natural process of soil development resulting from the amelioration of substrate condition, microbial biomass C and N, and urease and alkaline phosphatase activities in surface layers increased, and the metabolic quotient decreased. Microbial biomass gradually exhibited strong spatial heterogeneity during succession. Microbial biomass and enzyme activities had positive correlations with organic matter and nutrient conditions (N and P), and negative correlations with pH and heavy metals. The concentrations of organic matter, N, P and available Cu significantly influenced microbial biomass and activity. The analysis of 16S rDNA fragments by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) showed no obvious trend in microbial diversity during succession. Phylogenetic analysis of 16S rDNA sequences generated from DGGE gels indicated that bacteria mainly included Proteobacteria (alpha-, beta- and gamma-Proteobacteria), Firmicutes (Bacillus and Clostridia), Actinobacteria, Nitrospirae, Deinococcus-Thermus and Bacteroidetes. The natural successional series consisted of species adapted to mine wastelands. Soil microbial biomass and enzyme activities tended to be more sensitive to heavy metals.

Molecular Study of a New Disease of Peach in Iran Associated with a Phytoplasma

In recent years, a disease has been reported to affect peach trees in Kurdistan province of Iran causing serious losses to the production. Main symptoms of disease include leaf stunting and yellowing, which lead to failure in fruit production at harvest. For diagnosis of disease and identification of the causal agent, symptomatic leaf samples were collected in Kurdistan orchards during summer 2010 and were carried to the laboratory. Total DNA was extracted from plant samples according to the standard procedures and indexed by grafting and nested PCR using phytoplasma generic primers, P1/P7 and R16F2n/R2. PCR products were characterized by RFLP technique and direct sequencing. The 16S rDNA sequences were compared with those of other phytoplasmas in GenBank. Phytoplasma rDNA was amplified from 20 out 35 samples. The 16S rDNA sequences of the phytoplasma were identified in Peach samples which showed 98% similarity to that of “Candidatus Phytoplasma phoenicium” which is considered to be the causal agent of Almond witches’ broom. Phylogenetic analysis of 16S rDNA sequences placed peach strains in Almond witches broom isolate as a member of pigeon pea witches broom (PPWB) group. Further studies on the epidemiology of “Ca. Phytoplasma phoenicium” and its vector(s) in Iran are recommended in order to identify new natural hosts and develop successful disease management programs.

Association of pigeon pea witches’ broom phytoplasma infectingCarpobrotus edulis(L.) N.E. Br in India

Leaf yellowing and little leaf symptoms with reduced size of flowers were observed in Carpobrotus edulis at Gorakhpur, Uttar Pradesh, India in the years 2012–2013. Phytoplasma presence was confirmed in symptomatic C. edulis leaves using phytoplasma universal primers P1/P7 and R16F2n/R16R2 that produced expected size amplicons of ~1.8 kb and ~1.2kb, respectively. BLAST analysis of 16S rDNA sequence of the phytoplasma detected revealed 99% similarity with those of tomato big bud, Brassica rapa phyllody, Iranian almond witches’ broom, Phlox drummondii, sesame phyllody and Iran eggplant big bud all belonging to 16SrIX group. Phylogenetic analysis of the C. edulis phytoplasma 16S rDNA sequence confirmed its closest relationship with members of the 16SrIX group. Virtual RFLP analysis of 1.25 kb sequence of 16S rDNA identified it as a new strain in the 16SrIX-C subgroup that is for the first time reported in C. edulis species worldwide.

Association of ‘Candidatusphytoplasma asteris’ (16SrI group) with chlorosis and little leaf disease ofStreblus asper

During survey in the year 2010, chlorosis and little leaf symptoms were observed on Streblus asper plants growing in different gardens and nurseries of Gorakhpur, Eastern U.P., India. The phytoplasma suspected symptomatic sample was collected and analyzed for phytoplasma association through PCR assays. The DNA extracted from symptomatic Strebulus asper sample produced amplicon of ∼1.2 kb in nested PCR assay expected for the presence of phytoplasmas. The amplicon of ∼1.2 kb was sequenced and the consensus sequence obtained was submitted to GenBank Acc. no. HM230687. BLAST analysis of 16Sr DNA sequence of S. asper revealed the highest identity (99%) with identified members of 16SrI ‘Ca. Phytoplasma asteris’ group. Phylogenetic analysis further identified that S. asper phytoplasma clustered with members of ‘Ca. P. asteris’ group. Hence, it was confirmed that the chlorosis and little leaf disease of S. asper plant in the study belonged to 16Sr I group. To our knowledge this is the first report of a ‘Ca. Phytoplasma asteris’ isolate associated with chlorosis and little leaf disease of Streblus asper plant in the world.

Babesia behnkei sp. nov., a novel Babesia species infecting isolated populations of Wagner¿s gerbil, Dipodillus dasyurus, from the Sinai Mountains, Egypt

Although a number of new species of Babesia/Theileria have been described recently, there are still relatively few reports of species from Africa. In this study based on the evaluation of morphology and phylogenetic relationships, we describe a novel species from Wagner’s gerbil, Babesia behnkei n. sp. Rodents (n = 1021) were sampled in four montane valleys (wadies) in 2000, 2004, 2008 and 2012 in the Sinai Mountains, Egypt. The overall prevalence of Babesia spp. was highest in the Wagner’s gerbil (Dipodillus dasyurus; 38.7%) in comparison to the prevalence in the spiny mice species, Acomys dimidiatus and A. russatus. Morphological investigations were conducted for the comparison of trophozoites of the novel species of Babesia with the B. microti King’s 67 reference strain. Thirty-two isolates derived from D. dasyurus over a 9 year period (2004-2012) from two wadies (29 isolates from Wadi Gebel and 3 from Wadi El-Arbaein) were investigated by microscopic, molecular and phylogenetic analysis. A near-full-length sequence of the 18S rRNA gene and the second internal transcribed spacer (ITS2) region were amplified, sequenced and used for the construction of phylogenetic trees. A novel species of Babesia was identified in two isolated populations of D. dasyurus. Phylogenetic analysis of 18S rDNA and ITS2 sequences revealed that B. behnkei n. sp. is most closely related to B. lengau from cheetahs from South Africa and to Nearctic species found only in North America (the pathogenic B. duncani and B. conradae) and that it is more distant to the cosmopolitan rodent parasite B. microti. Trophozoites of B. behnkei were smaller and less polymorphic than trophozoites of B. microti. Babesia behnkei n. sp. is a novel species of the ‘Duncani group’ maintained in isolated populations of Dipodillus dasyurus occurring in the Sinai Mountains of Egypt.

Erratum to: Optimal Condition for Eicosapentaenoic Acid Production and Purification from Psychrophillic Marine Baterium Shewanella sp. L93

To obtain eicosapentaenoic acid (EPA)-producing bacteria, some 600 strains of bacteria were isolated from Antarctic sediment and marine organisms during the summer expedition of 1999-2000 and 7 EPA-producing bacteria were obtained through screening with TLC and GC. A strain designated as L93 showed the highest EPA production, which was gram-negative, rod-shaped bacterium. L93 strain was identified as Shewanella sp., from the sequence analysis of 16S rDNA. Optimal conditions temperature and pH for the growth and EPA production were about and pH 7. In addition, its production was optimized by 50%(w/v) sea salt. We establish the optimal production system to produce about 320 mg per liter by using this optimal EPA production conditions. EPA-methyl ester was purified from cultured L93 strain to a purity of higher than 97% and typical purification yield is greater than 72% of the input amount via urea complexation and HPLC.

Molecular identification of 16S rII phytoplasma group in commercial pistachio cultivars in Iran

Evaluation of phytoplasmas infection was conducted in the pistachio-growing areas of Iran (Rafsanjan in Kerman province) in early autumn of 2011. A total of 30 pistachio trees collected from a pistachio orchard in Rafsanjan showing Psylla damage symptoms and 10 samples with different abnormal symptoms from miscellaneous orchards were tested for the presence of phytoplasma. By using nested PCR with primer pairs P1/P7 and internal primer sets R16F2N/R16R2 and fU3-rU5, amplified fragment of expected size was observed in some trees with deformation and yellowing symptoms. On the basis of nucleotide sequence analysis of 16S rDNA amplified by PCR, this phytoplasma was classified in group 16S rII. In addition, we observed false positive reaction in three trees of Ahmadaghaei cultivar by using primer sets R16F2N/R16R2 and sequence analysis of ~1250bp PCR product indicated that amplified fragment was related to Schinus terebinthifolius; a species of flowering plant in the Anacardiaceae family.

Co-metabolic biodegradation of acetamiprid by Pseudoxanthomonas sp. AAP-7 isolated from a long-term acetamiprid-polluted soil

An AAP-degrading bacterium, AAP-7, was isolated from AAP-polluted soil. AAP-7 was identified as Pseudoxanthomonas sp. on the basis of the comparative analysis of 16S rDNA sequences. The strain was able to transformate more than 80% AAP by means of co-metabolism and degraded AAP via hydrolysis or demethylation to form (E)-3-(((6-chloropyridin-3yl)methyl)(methyl)amino)acrylonitrile and N-((6-chloropyridin-3yl)methyl)-N-methylprop-1-en-2-amine, both of which transformed into ultimate product, which was 1-(6-chloropyridin-3yl)-N-methylmethanamine. A novel degradation pathway was proposed based on these metabolites. AAP could be transformed with a maximum specific degradation rate, half-saturation constant and inhibit constant of 1.775/36 h, 175.3 mg L(-1), and 396.5 mg L(-1), respectively, which proved that the degradation rate of AAP could be restrained at high AAP concentration. This paper highlights a significant potential use of co-metabolic cultures of microbial cells for the cleanup of AAP-contaminated soil.

A review on the characterization of a novel oral Veillonella species, V. tobetsuensis, and its role in oral biofilm formation

Abstract The genus Veillonella consists of small, strictly anaerobic, gram-negative cocci that lack flagella, spores, and a capsule. Although 11 species have been established in the genus Veillonella , five species, namely V. atypica , V. denticariosi , V. dispar , V. parvula , and V. rogosae , are recognized as oral Veillonella . The main habitats of oral Veillonella are the tongue, dental biofilm, and the buccal mucosa, and correspondingly it has been suggested that oral Veillonella contributes to oral biofilm formation and may lead to the formation of dental disease such as periodontitis. Recently, unknown strains displaying all phenotypic characteristics of the genus Veillonella were isolated from human tongue biofilm. The production of a major cellular fatty acid (C 13:0 and C 17:1 ω8) was consistent with that of other members of the genus Veillonella . Based on the comparative analysis of 16S rDNA, dnaK , and rpoB gene sequences, the unknown strains represent a novel species, and were named Veillonella tobetsuensis . V. tobetsuensis was detected by PCR using specific primers based on the dnaK gene sequence in 5 out of 27 subjects with other oral Veillonella species. The prevalence of V. tobetsuensis ranged from 7.6 to 20.0% in these subjects. Using the wire method, all six oral Veillonella species stimulated the formation of biofilm by Streptococcus gordonii . The greatest amount of biofilm was formed by S. gordonii in the presence of V. tobetsuensis. These results suggest that among the oral Veillonella species, V. tobetsuensis plays an important role in biofilm formation by S. gordonii .

Near-full length sequencing of 16S rDNA and RFLP indicates that Rhizobium etli is the dominant species nodulating Egyptian winter Berseem clover (Trifolium alexandrinum L.)

Egyptian winter Berseem clover (EWBC) is one of the main important forage legume crops in Egypt that is used for animal feeding in winter and it occupies about 2.5 million feddans (Feddan=4200m2) in winter agricultural rotation systems. Forty-eight rhizobial isolates that nodulated this legume host from different geographical regions within Egypt were isolated. RFLP analyses of 16S rDNA (1.5kb) and whole ribosomal DNA (5kb), the sequencing of 16S rDNA, and the sequencing of nodC, nifH and house keeping genes were used to identify these isolates. The RFLP analysis of 16S rDNA (1.5kb) among 15 representative strains with three enzymes generated two genotypes. The largest genotype was similar to Rhizobium etli CFN42T (93.33%) except for strain 902 that failed to re-nodulate EWBC. RFLP analysis of complete ribosomal DNA (5kb) produced five genotypes. The majority of tested strains shared the genotype with R. etli CFN42T (53.33%). Only one strain (1002) shared the genotype with Rhizobium leguminosarum sv. trifolii 3023. The other four strains were comprised of two unique genotypes. Phylogenetic analysis of 16S rDNA sequences revealed that seven representative strains could be divided into two genetic clusters sharing the ancestral clad with R. etli CFN42T. A phylogenetic tree based on nodC gene sequence confirmed that all the examined strains shared the genetic lineage with R. leguminosarum sv. trifolii WSM1325. The phylogenetic trees of house keeping genes are supported strongly the identification of majority of strains as a novel symbiovar of R. etli with new lineages.