Nocturnal production of endospores in natural populations of epulopiscium-like surgeonfish symbionts

Prior studies have described a morphologically diverse group of intestinal microorganisms associated with surgeonfish. Despite their diversity of form, 16S rRNA gene surveys and fluorescent in situ hybridizations indicate that these bacteria are low-G+C gram-positive bacteria related to Epulopiscium spp. Many of these bacteria exhibit an unusual mode of reproduction, developing multiple offspring intracellularly. Previous reports have suggested that some Epulopiscium-like symbionts produce dormant or phase-bright intracellular offspring. Close relatives of Epulopiscium, such as Metabacterium polyspora and Clostridium lentocellum, are endospore-forming bacteria, which raises the possibility that the phase-bright offspring are endospores. Structural evidence and the presence of dipicolinic acid demonstrate that phase-bright offspring of Epulopiscium-like bacteria are true endospores. In addition, endospores are formed as part of the normal daily life cycle of these bacteria. In the populations studied, mature endospores were seen only at night and the majority of cells in a given population produced one or two endospores per mother cell. Phylogenetic analyses confirmed the close relationship between the endospore-forming surgeonfish symbionts characterized here and previously described Epulopiscium spp. The broad distribution of endospore formation among the Epulopiscium phylogenetic group raises the possibility that sporulation is a characteristic of the group. We speculate that spore formation in Epulopiscium-like symbionts may be important for dispersal and may also enhance survival in the changing conditions of the fish intestinal tract.     

Molecular phylogeny of the genus Dactylopius (Hemiptera: Dactylopiidae) and identification of the symbiotic bacteria

Phylogenetic analyses, from polymerase chain reaction (PCR)-amplified 12S rRNA and 18S rRNA gene sequences from cochineal insects of the genus Dactylopius present in Mexico, showed that D. ceylonicus, D. confusus, and D. opuntiae are closely related. D. coccus constitutes a separate clade, and D. tomentosus is the most distantly related. Bacterial 16S rRNA sequences from all the Dactylopius species sampled showed a common β-proteobacteria, related to Azoarcus, also found in eggs and in bacteriocytes in D. coccus. We propose the name "Candidatus Dactylopiibacterium carminicum" for this endosymbiont. Other bacterial sequences recovered from the samples were close to those from soil or plant associated bacteria, like Massilia, Herbaspirillum, Acinetobacter, Mesorhizobium, and Sphingomonas, suggesting a possible horizontal transmission from Cactaceae plant sap to Dactylopius spp. during feeding. This is the first molecular analysis of Dactylopius species and of their associated bacteria.     

Genetic analyses of Dinophysis spp. support kleptoplastidy

The question of whether the toxin-producing and bloom-forming dinoflagellate genus Dinophysis contains plastids that are permanent or contains temporary so-called kleptoplastids is still unresolved. We sequenced plastid 16S rRNA gene, the complete trnA gene and the intergenic transcribed spacer region located between the trnA gene and the 23S rRNA gene, and performed diagnostic PCR on cells of the genus Dinophysis. Dinophysis spp. were collected from five different geographical regions: the Baltic Sea, the North Sea, the Greenland Sea and the Norwegian fjord Masfjorden. In most cases the sequence analysis showed that the sequences were identical to each other and to sequences from the cryptophyte Teleaulax amphioxeia SCCAP K0434, regardless of the place of sampling or the species analyzed. The exception was some cells of Dinophysis spp. from the Greenland Sea. These contained a 16S rRNA gene sequence that was more closely related to the cryptophyte Geminigera cryophila. The cells of Dinophysis contained either one of the 16S rRNA gene sequences or both in the same cell. Our results challenge the hypothesis that the plastids in Dinophysis are permanent and suggest that they are more likely to be kleptoplastids.     

Microheterogeneity in 16S ribosomal DNA-defined bacterial populations from a stratified planktonic environment is related to temporal changes and to ecological adaptations

Temporal changes of the bacterioplankton from a meromictic lake (Lake Vilar, Banyoles, Spain) were analyzed with four culture-independent techniques: epifluorescence microscopy, PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting, fluorescence in situ whole-cell hybridization and flow cytometry sorting. Microscopically, blooms of one cyanobacterium (Synechococcus sp.-like), one green sulfur bacterium (Chlorobium phaeobacteroides-like), and one purple sulfur bacterium (Thiocystis minor-like) were observed at different depths and times. DGGE retrieved these populations and, additionally, populations related to the Cytophaga-Flavobacterium-Bacteroides phylum as predominant community members. The analyses of partial 16S ribosomal DNA sequences from the DGGE fingerprints (550 bp analyzed) revealed higher genetic diversity than expected from microscopic observation for most of these groups. Thus, the sequences of two Synechococcus spp. (both had a similarity of 97% to Synechococcus sp. strain PCC6307 in 16S rRNA), two Thiocystis spp. (similarities to Thiocystis minor of 93 and 94%, respectively), and three Cytophaga spp. (similarities to Cytophaga fermentans of 88 and 89% and to Cytophaga sp. of 93%, respectively) were obtained. The two populations of Synechococcus exhibited different pigment compositions and temporal distributions and their 16S rRNA sequences were 97.3% similar. The two Thiocystis populations differed neither in pigment composition nor in morphology, but their 16S rRNA sequences were only 92.3% similar and they also showed different distributions over time. Finally, two of the Cytophaga spp. showed 96.2% similarity between the 16S rRNA sequences, but one of them was found to be mostly attached to particles and only in winter. Thus, the identity of the main populations changed over time, but the function of the microbial guilds was maintained. Our data showed that temporal shifts in the identity of the predominant population is a new explanation for the environmental 16S rRNA microdiversity retrieved from microbial assemblages and support the hypothesis that clusters of closely related 16S rRNA environmental sequences may actually represent numerous closely related, yet ecologically distinct, populations.     

Bacteria and Methanogens Differ along the Gastrointestinal Tract of Chinese Roe Deer (Capreolus pygargus)

The current study provides the insight into the bacteria in the gastrointestinal tract (GIT) and methanogens presented in the rumen and cecum of the Chinese roe deer (Capreolus pygargus). The ruminal, ileal, cecal, and colonic contents, as well as feces, were obtained from each of the three, free-range, roe deer ingesting natural pasture after euthanasia. For the bacterial community, a total of 697,031 high-quality 16S rRNA gene sequences were generated using high-throughput sequencing, and assigned to 2,223 core operational taxonomic units (OTUs) (12 bacterial phyla and 87 genera). The phyla Firmicutes (51.2%) and Bacteroidetes (39.4%) were the dominant bacteria in the GIT of roe deer. However, the bacterial community in the rumen was significantly (P<0.01) different from the other sampled regions along the GIT. Secondly, Prevotella spp., Anaerovibrio spp., and unidentified bacteria within the families Veillonellaceae and Paraprevotellaceae were more abundant in the rumen than in the other regions. Unidentified bacteria within the family Enterobacteriaceae, Succinivibrio spp., and Desulfovibrio spp. were more predominant in the colon than in other regions. Unidentified bacteria within the family Ruminococcaceae, and Bacteroides spp. were more prevalent in the ileum, cecum and fecal pellets. For methanogens in the rumen and cecum, a total of 375,647 high quality 16S rRNA gene sequences were obtained and assigned to 113 core OTUs. Methanobrevibacter millerae was the dominant species accounting for 77.3±7.4 (S.E) % and 68.9±4.4 (S.E) % of total sequences in the rumen and cecum of roe deer, respectively. However, the abundance of Methanobrevibacter smithii was higher in the rumen than in the cecum (P = 0.004). These results revealed that there was intra variation in the bacterial community composition across the GIT of roe deer, and also showed that the methanogen community in the rumen differed from that in the cecum.     

Detection of Jeotgalicoccus spp. in poultry house air

Investigations of bioaerosols collected from turkey, chicken and duck houses, as well as from a duck slaughterhouse, each in triplicate, revealed that 4–18% of 16S rRNA gene sequences in investigated 16S rRNA gene clone libraries were closely related to Jeotgalicoccus spp. J. halotolerans- and J. psychrophilus-related sequences were obtained in all investigated bioaerosol samples and formed a distinct group with sequences of both species type strains, which were collectively entitled Jeot-cluster-I. For a quantification of Jeot-cluster-I bacteria, a group specific PCR primer combination targeting the 16S rRNA genes was developed. Estimated concentrations by quantitative real-time PCR analyses revealed cell numbers between 10⁴ and 10⁶ Jeotgalicoccus cells m⁻³ air in turkey, duck, and chicken houses, respectively. These results indicated the remarkable proportion (1–39%) of total cell counts and the hitherto unknown wide distribution of Jeotgalicoccus spp. in the poultry rearing industry.     

Sequence heterogeneity in the two 16S rRNA genes of Phormium yellow leaf phytoplasma.

Phormium yellow leaf (PYL) phytoplasma causes a lethal disease of the monocotyledon, New Zealand flax (Phormium tenax). The 16S rRNA genes of PYL phytoplasma were amplified from infected flax by PCR and cloned, and the nucleotide sequences were determined. DNA sequencing and Southern hybridization analysis of genomic DNA indicated the presence of two copies of the 16S rRNA gene. The two 16S rRNA genes exhibited sequence heterogeneity in 4 nucleotide positions and could be distinguished by the restriction enzymes BpmI and BsrI. This is the first record in which sequence heterogeneity in the 16S rRNA genes of a phytoplasma has been determined by sequence analysis. A phylogenetic tree based on 16S rRNA gene sequences showed that PYL phytoplasma is most closely related to the stolbur and German grapevine yellows phytoplasmas, which form the stolbur subgroup of the aster yellows group. This phylogenetic position of PYL phytoplasma was supported by 16S/23S spacer region sequence data.     

16S rRNA-based analysis of microbiota from the cecum of broiler chickens.

The microbiota of the intestinal tract of chickens plays an important role in inhibiting the establishment of intestinal pathogens. Earlier culturing and microscopic examinations indicated that only a fraction of the bacteria in the cecum of chickens could be grown in the laboratory. Therefore, a survey of cecal bacteria was done by retrieval of 16S rRNA gene sequences from DNA isolated from the cecal content and the cecal mucosa. The ribosomal gene sequences were amplified with universal primers and cloned or subjected to temporal temperature gradient gel electrophoresis (TTGE). Partial 16S rRNA gene sequences were determined from the clones and from the major bands in TTGE gels. A total of 1,656 partial 16S rRNA gene sequences were obtained and compared to sequences in the GenBank. The comparison indicated that 243 different sequences were present in the samples. Overall, sequences representing 50 phylogenetic groups or subgroups of bacteria were found, but approximately 89% of the sequences represented just four phylogenetic groups (Clostridium leptum, Sporomusa sp., Clostridium coccoides, and enterics). Sequences of members of the Bacteroides group, the Bifidobacterium infantis subgroup, and of Pseudomonas sp. each accounted for less than 2% of the total. Sequences related to those from the Escherichia sp. subgroup and from Lactobacillus, Pseudomonas, and Bifidobacterium spp. were generally between 98 and 100% identical to sequences already deposited in the GenBank. Sequences most closely related to those of the other bacteria were generally 97% or less identical to those in the databases and therefore might be from currently unknown species. TTGE and random cloning indicated that certain phylogenetic subgroups were common to all birds analyzed, but sequence data from random cloning also provided evidence for qualitative and quantitative differences among the cecal microbiota of individual birds reared under very similar conditions.     

Contrasting nifD and ribosomal gene relationships among Mesorhizobium from Lotus oroboides in northern Mexico

PCR screens for length variation in a 5' portion of 23S ribosomal RNA and in the 3' end of the 16S rRNA-23S rRNA internal transcribed spacer (ITS) region indicated that nodule bacteria from a Mexican population of Lotus oroboides were diverse on a local scale. Three 23S rRNA length variants and five ITS length variants were detected among the 22 isolates. Sequencing of nearly full-length 16S rRNA genes in three isolates indicated that they fell into the genus Mesorhizobium, but comprised two distinct groups. Two isolates were closely related to M. loti LMG 6125T, while the other isolate clustered with an assemblage of Mesorhizobium taxa that included M. amorphae, M. plurifarium and M. huakuii. However, a phylogenetic tree based on 715 bp of the nitrogenase alpha-subunit (nifD) gene was significantly discordant with the relationships inferred from rRNA sequences. Two isolates that were nearly identical for 16S rRNA had nifD genes that varied at 2% of sites, and one of these nifD sequences was identical to that of another isolate with a strongly divergent 16S rRNA gene. A plasmid screen followed by Southern hybridization indicated that only one of these strains harbored a plasmid-borne nifD gene. These results imply that gene transfer events have altered the distribution of nifD sequences among lineages within this natural population of Mesorhizobium strains.     

Phylogenetic analysis of cecal microbiota in chicken by the use of 16S rDNA clone libraries

The chicken cecum contains a great many bacteria, most of which are strict anaerobes. A strictly anaerobe culture-based method was used in the present study, in conjunction with the 16S rDNA clone library, to elucidate bacterial diversity and the phylogenetic relationship of cecal microbiota in the chicken. A comparative 16S rDNA sequence analysis of cultivated strains and retrieved clones from cecal contents was performed. Approximately 90% of the bacterial cells detected by microscopy did not form colonies on a medium 10 in plate-in-bottle. The 19 isolated strains yielded 11 distinct rDNA sequences, 58% of which were classified as low G + C gram-positive bacteria, 26% were related to Bacteroides spp., and 16% were classified as Proteobacteria. Based on the sequence analysis of 164 clones, 24% were identified to belong to 8 known species and 76% were considered to be 65 novel phylotypes. Approximately 94% of cloned sequences were classified into low G + C gram-positive bacteria, 4% were related to Bacteroides spp., and 2% were classified into Proteobacteria. Clostridium subcluster XIVa (38%), Clostridium cluster IV (13%), Lactobacillus spp. (24%), and Bacteroides spp. (4%) were the major groups constituting the cecal microbiota in chicken, in which the Clostridium subcluster XIVa was the most phylogenetically diverse group in chicken cecum. The 16S rDNA sequences of Lactobacillus acidophilus, L. crispatus, L. salivarius, and L. reuteri were the most frequently found in the Lactobacillus group in chicken cecum.     

Identification of a haemomycoplasma species in anemic reindeer (Rangifer tarandus)

During an 18-mo period (May 2002-November 2003), 10 animals in a herd of 19 reindeer (Rangifer tarandus) at the National Animal Disease Center (NADC) experienced episodes of anemia. Affected animals had histories of weight loss, unthriftiness, occasionally edema of dependent parts and moderate anemia characterized by microcytosis or macrocytosis, hypochromasia, schistocytosis, keratocytosis, acanthocytosis, and dacryocytosis. Numerous basophilic punctate to ring-shaped bodies, measuring less than 1.0 microm, were found on the surface of red blood cells and were often observed encircling the outer margins of the cells. Based on cytologic findings, DNA preparations from selected affected animals in the NADC herd and one animal from a private herd experiencing similar episodes of anemia were assayed by polymerase chain reaction (PCR) for the presence of hemotropic bacteria using primers targeting the 16S rRNA genes of Mycoplasma (Eperythrozoon) suis, Mycoplasma (Haemobartonella) haemofelis, Anaplasma marginale, Anaplasma spp., and Ehrlichia spp. Amplification products were detected from four of the affected animals using primers specific for the 16S rRNA gene of M. haemofelis and Mycoplasma haemocanis. Product from one of the animals was sequenced and internal primers were designed from the resulting sequence to perform a nested PCR assay. Samples from 10 reindeer were positive using the nested PCR reaction and products from seven animals were sequenced; BLAST searches and phylogenetic analysis were performed on the resulting sequences. Sequence data from six animals revealed homology to an organism most closely related to Mycoplasma ovis, Mycoplasma wenyonii, and Mycoplasma haemolamae; sequence from a single animal was most closely related to M. haemofelis and M. haemocanis. This represents the first identification of a haemomycoplasma species in reindeer. Although several animals were also infected with abomasal nematodes, the presence of this newly described haemomycoplasma may have contributed to the anemic syndrome.     

Bacterial 16S rRNA Gene Analysis Revealed That Bacteria Related to Arcobacter spp. Constitute an Abundant and Common Component of the Oyster Microbiota (Tiostrea chilensis)

To explore the bacterial microbiota in Chilean oyster (Tiostrea chilensis), a molecular approach that permits detection of different bacteria, independently of their capacity to grow in culture media, was used. Bacterial diversity was assessed by analysis of both the 16S rDNA and the 16S-23S intergenic region, obtained by PCR amplifications of DNA extracted from depurated oysters. RFLP of the PCR amplified 16S rDNA showed a prevailing pattern in most of the individuals analyzed, indicating that a few bacterial species were relatively abundant and common in oysters. Cloning and sequencing of the 16S rDNA with the prevailing RFLP pattern indicated that this rRNA was most closely related to Arcobacter spp. However, analysis by the size of the amplified 16S-23S rRNA intergenic regions revealed not Arcobacter spp. but Staphylococcus spp. related bacteria as a major and common component in oyster. These different results may be caused by the absence of target for one of the primers employed for amplification of the intergenic region. Neither of the two bacteria species found in large abundance was recovered after culturing under aerobic, anaerobic, or microaerophilic conditions. This result, however, is expected because the number of bacteria recovered after cultivation was less than 0.01% of the total. All together, these observations suggest that Arcobacter-related strains are probably abundant and common in the Chilean oyster bacterial microbiota.     

Non-Frankia actinomycetes isolated from surface-sterilized roots of Casuarina equisetifolia fix nitrogen

Based on partial 16S sequences, we previously described a novel group of nonsymbiotic, acetylene reduction activity-positive actinomycetes which were isolated from surface-sterilized roots of Casuarina equisetifolia growing in Mexico. An amplified rRNA restriction analysis confirmed that these actinomycetes are distinct from Frankia, a finding substantiated by a 16S rRNA gene phylogenetic analysis of two of the Mexican isolates. Further support for these actinomycetes being separate from Frankia comes from the very low DNA-DNA homology that was found. Nevertheless, the Mexican isolates may be diazotrophs based not only on their ability to grow in N-free medium and reduce acetylene to ethylene but also on the results from (15)N isotope dilution analysis and the finding that a nifH gene was PCR amplified. A comparison of the nifH sequences from the various isolates showed that they are closely related to nifH from Frankia; the similarity was 84 to 98% depending on the host specificity group. An analysis of complete 16S rRNA gene sequences demonstrated that the two strains analyzed in detail are most closely related to actinobacteria in the Thermomonosporaceae and the Micromonosporaceae.     

Microheterogeneity in 16S Ribosomal DNA-Defined Bacterial Populations from a Stratified Planktonic Environment Is Related to Temporal Changes and to Ecological Adaptations

Temporal changes of the bacterioplankton from a meromictic lake (Lake Vilar, Banyoles, Spain) were analyzed with four culture-independent techniques: epifluorescence microscopy, PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting, fluorescence in situ whole-cell hybridization and flow cytometry sorting. Microscopically, blooms of one cyanobacterium (Synechococcus sp.-like), one green sulfur bacterium (Chlorobium phaeobacteroides-like), and one purple sulfur bacterium (Thiocystis minor-like) were observed at different depths and times. DGGE retrieved these populations and, additionally, populations related to the Cytophaga-Flavobacterium-Bacteroides phylum as predominant community members. The analyses of partial 16S ribosomal DNA sequences from the DGGE fingerprints (550 bp analyzed) revealed higher genetic diversity than expected from microscopic observation for most of these groups. Thus, the sequences of two Synechococcus spp. (both had a similarity of 97% to Synechococcus sp. strain PCC6307 in 16S rRNA), two Thiocystis spp. (similarities to Thiocystis minor of 93 and 94%, respectively), and three Cytophaga spp. (similarities to Cytophaga fermentans of 88 and 89% and to Cytophaga sp. of 93%, respectively) were obtained. The two populations of Synechococcus exhibited different pigment compositions and temporal distributions and their 16S rRNA sequences were 97.3% similar. The two Thiocystis populations differed neither in pigment composition nor in morphology, but their 16S rRNA sequences were only 92.3% similar and they also showed different distributions over time. Finally, two of the Cytophaga spp. showed 96.2% similarity between the 16S rRNA sequences, but one of them was found to be mostly attached to particles and only in winter. Thus, the identity of the main populations changed over time, but the function of the microbial guilds was maintained. Our data showed that temporal shifts in the identity of the predominant population is a new explanation for the environmental 16S rRNA microdiversity retrieved from microbial assemblages and support the hypothesis that clusters of closely related 16S rRNA environmental sequences may actually represent numerous closely related, yet ecologically distinct, populations.     

Phylogenetic Characterization of Archaea in Saltpan Sediments

A study was undertaken to investigate the presence of archaeal diversity in saltpan sediments of Goa, India by 16S rDNA-dependent molecular phylogeny. Small subunit rRNA (16S rDNA) from saltpan sediment metagenome were amplified by polymerase chain reaction (PCR) using primers specific to the domain archaea. 10 unique phylotypes were obtained by PCR based RFLP of 16S rRNA genes using endonuclease Msp 1, which was most suitable to score the genetic diversity. These phylotypes spanned a wide range within the domain archaea including both crenarchaeota and euryarcheaota. None of the retrieved crenarchaeota sequences could be grouped with previously cultured crenarchaeota however; two sequences were related with haloarchaea. Most of the sequences determined were closely related to the sequences that had been previously obtained from metagenome of a variety of marine environments. The phylogenetic study of a site investigated for the first time revealed the presence of low archaeal population but showed yet unclassified species, may specially adapted to the salt pan sediment of Goa.     

Phylogenetic diversity of bacterial symbionts of Solemya hosts based on comparative sequence analysis of 16S rRNA genes.

The bacterial endosymbionts of two species of the bivalve genus Solemya from the Pacific Ocean, Solemya terraeregina and Solemya pusilla, were characterized. Prokaryotic cells resembling gram-negative bacteria were observed in the gills of both host species by transmission electron microscopy. The ultrastructure of the symbiosis in both host species is remarkably similar to that of all previously described Solemya spp. By using sequence data from 16S rRNA, the identity and evolutionary origins of the S. terraeregina and S. pusilla symbionts were also determined. Direct sequencing of PCR-amplified products from host gill DNA with primers specific for Bacteria 16S rRNA genes gave a single, unambiguous sequence for each of the two symbiont species. In situ hybridization with symbiont-specific oligonucleotide probes confirmed that these gene sequences belong to the bacteria residing in the hosts gills. Phylogenetic analyses of the 16S rRNA gene sequences by both distance and parsimony methods identify the S. terraeregina and S. pusilla symbionts as members of the gamma subdivision of the Proteobacteria. In contrast to symbionts of other bivalve families, which appear to be monophyletic, the S. terraeregina and S. pusilla symbionts share a more recent common ancestry with bacteria associating endosymbiotically with bivalves of the superfamily Lucinacea than with other Solemya symbionts (host species S. velum, S. occidentalis, and S. reidi). Overall, the 16S rRNA gene sequence data suggest that the symbionts of Solemya hosts represent at least two distinct bacterial lineages within the gamma-Proteobacteria. While it is increasingly clear that all extant species of Solemya live in symbiosis with specific bacteria, the associations appear to have multiple evolutionary origins.     

Identification of the midgut microbiota of An. stephensi and An. maculipennis for their application as a paratransgenic tool against malaria

The midgut microbiota associated with Anopheles stephensi and Anopheles maculipennis (Diptera: Culicidae) was investigated for development of a paratransgenesis-based approach to control malaria transmission in Eastern Mediterranean Region (EMR). Here, we present the results of a polymerase chain reaction (PCR) and biochemical-based approaches to identify the female adult and larvae mosquitoe microbiota of these two major malaria vectors, originated from South Eastern and North of Iran. Plating the mosquito midgut contents from lab-reared and field-collected Anopheles spp. was used for microbiota isolation. The gram-negative and gram-positive bacterial colonies were identified by Gram staining and specific mediums. Selected colonies were identified by differential biochemical tests and 16S rRNA gene sequence analysis. A number of 10 An. stephensi and 32 An. maculipennis adult mosquitoes and 15 An. stephensi and 7 An. maculipennis larvae were analyzed and 13 sequences of 16S rRNA gene bacterial species were retrieved, that were categorized in 3 classes and 8 families. The majority of the identified bacteria were belonged to the γ-proteobacteria class, including Pseudomonas sp. and Aeromonas sp. and the others were some closely related to those found in other vector mosquitoes, including Pantoea, Acinetobacter, Brevundimonas, Bacillus, Sphingomonas, Lysinibacillus and Rahnella. The 16S rRNA sequences in the current study aligned with the reference strains available in GenBank were used for construction of the phylogenetic tree that revealed the relatedness among the bacteria identified. The presented data strongly encourage further investigations, to verify the potential role of the detected bacteria for the malaria control in Iran and neighboring countries.     

Molecular characterization of phytoplasmas in lilies with fasciation in the Czech Republic

Lilium spp. with symptoms of severe fasciation were observed in Southern and central Bohemia during the period 1999-2003. Nucleic acids extracted from symptomatic and asymptomatic plants were used in nested-PCR assays with primers amplifying 16S-23S rRNA sequences specific for phytoplasmas. The subsequent nested-PCR with phytoplasma group-specific primers followed by RFLP analyses and the 16S ribosomal gene sequencing, allowed classification of the detected phytoplasmas in the aster yellows group, subgroups 16SrI-B and 16SrI-C alone, and in mixed infection. Samples infected by 16SrI-C phytoplasmas showed different overlapping RFLP profiles after TruI digestion of R16F2/R2 amplicons. Two of these amplicons were sequenced, one of them directly and the other after cloning; sequence analyses and blast alignment confirmed the presence of two different overlapping patterns in samples studied. The sequences obtained were closely related, respectively, to operon A and operon B ribosomal sequences of the clover phyllody phytoplasma. Direct PCR followed by RFLP analyses of the tuf gene with two restriction enzymes showed no differences from reference strain of subgroup 16SrI-C. Infection with aster yellows phytoplasmas of 16SrI-B subgroup in asymptomatic lilies cv. Sunray was also detected.     

干酪乳杆菌的近缘种及亚种部分看家基因的系统发育分析

【背景】16S rRNA基因序列分析已广泛应用于细菌的分类鉴定,但是存在一定局限性,而使用看家基因作为分子标记在近缘种及亚种间的系统发育分析中具有其独特的优势。【目的】研究16S rRNA、uvr C (核酸外切酶ABC,C亚基)和mur E (UDP-N-乙酰胞壁酰三肽合酶)基因序列对干酪乳杆菌的近缘种及亚种的区分能力。【方法】采用分离自传统发酵乳中的6株干酪乳杆菌为研究对象,选取uvr C和mur E基因片段,通过PCR扩增、测序,结合已公布的干酪乳杆菌的近缘种或亚种的相应序列计算遗传距离、构建系统发育树,并与16S rRNA基因序列分析技术进行比较。【结果】研究发现Lactobacilluscasei及相近种间的uvr C、mur E和联合基因(uvr C-mur E)构建的系统发育树拓扑结构与16S rRNA基因结果基本一致,区别在于相似性的不同,其分别为79.00%-99.16%、89.08%-99.20%、76.56%-99.69%和99.58%-100%。基于16S rRNA基因不能区分干酪乳杆菌的近缘种及亚种,而看家基因uvr C和mur E基因序列能够很好地区分干酪乳杆菌的近缘种及亚种,并且将uvr C和mur E基因串联使用后,试验菌株与参考菌株的分类关系更加清晰。【结论】联合基因(uvr C-mur E)可作为16SrRNA基因的辅助工具用于干酪乳杆菌的近缘种及亚种的快速准确鉴定。     

Bacillus DNA in fossil bees: an ancient symbiosis?

We report here the isolation of DNA from abdominal tissue of four extinct stingless bees (Proplebeia dominicana) in Dominican amber, PCR amplification of a 546-bp fragment of the 16S rRNA gene from Bacillus spp., and their corresponding nucleotide sequences. These sequences were used in basic local alignment search tool searches of nonredundant nucleic acid data bases, and the highest scores were obtained with 16S rRNA sequences from Bacillus spp. Phylogenetic inference analysis by the maximum-likelihood method revealed close phylogenetic relationships of the four presumed ancient Bacillus sequences with Bacillus pumilus, B. firmus, B. subtilis, and B. circulans. These four extant Bacillus spp. are commonly isolated from abdominal tissue of stingless bees. The close phylogenetic association of the extracted DNA sequences with these bee colonizers suggests that a similar bee-Bacillus association existed in the extinct species P. dominicana.