The primary structures of the 16s rRNAs of Bacillus anthracis, Bacillus cereus, Bacillus mycoides, and Bacillus thuringiensis were determined by using the reverse transcription-dideoxy sequencing method. All of the strains exhibited very high levels of sequence similarity (>9%) that were consistent with the close relationships shown by previous DNA hybridization studies. The species Bacillus anthracis, Bacillus cereus, Bacillus mycoides, and Bacillus thuringiensis were originally described on the basis of their habitats, their pathogenicity for mammals or insects, and their morphological and physiological characteristics. However, the taxonomic interrelationships of these species are equivocal. All four species share many phenotypic properties, and several workers have questioned their status as separate species (6, 11, 12). DNA-DNA hybridization studies on strains of B. anthracis, B. cereus, and B. thuringiensis have also shown that these organisms share relatively high levels of chromosomal base sequence similarity (7, 10, 13). However, inconsistencies in reported levels of DNA relatedness make it difficult to draw firm conclusions regarding species differentiation within this group of organisms.Small-subunit rRNA is now recognized as a powerful molecular chronometer (16). Degrees of sequence conservation, ranging from highly variable to highly conserved regions, enable systematists to measure small as well as great genealogical distances. In this study we determined partial primary 16s rRNA sequences of B. anthracis, B. cereus, B. mycoides, and B. thuringiensis in order to investigate the genealogical interrelationships of these organisms. MATERIALS AND METHODSCultures and cultivation. Details concerning the test strains which we examined are shown in Table 1. Strains were grown in shake flasks containing nutrient broth no. 2 (Oxoid) to late exponential phase at 30°C.Extraction and sequence determination of 16s rRNA. Total cellular rRNA was extracted from ca. 2 g of wet cells by mechanical disruption, using glass beads and a Braun homogenizer, and was purified as described by Embley et al. (5). Nucleotide sequences were determined by the Sanger dideoxynucleotide method (9) directly from cellular rRNA, using avian myeloblastosis virus reverse transcriptase (8). The sequences of the oligonucleotide primers were the same as those described by Embley et al. (5). In addition, the * Corresponding author.following primer was used: S'TCACCAACTAGCTAATG, which is complementary to positions 258 to 242 (Escherichia coli nomenclature). This primer was included to enable determination of the sequence between positions 100 and 150, which could not always be fully established by using the primer at position 357 described by Lane et al. (8).Nucleotide sequence accession numbers. The 16s rRNA sequences have been deposited in the EMBL Data Library (accession numbers X55059 to X55063). RESULTS AND DISCUSSIONThe 16s rRNA sequences of B. anthracis Sterne, B. cereus NCDO 1771T (T = type strain), B. cereus NCTC 11143 (emetic strain)...
On the basis of ribosomal 16S sequence comparison, Brucella abortus has been found to be a member of the alpha-2 subdivision of the class Proteobacteria (formerly named purple photosynthetic bacteria and their nonphototrophic relatives). Within the alpha-2 subgroup, brucellae are specifically related to rickettsiae, agrobacteria, and rhizobiae, organisms that also have the faculty or the obligation of living in close association to eucaryotic cells. Comparison of the Brucella lipid composition with that of the other Proteobacteria also suggests a close phylogenetical relationship with members of the alpha-2 subdivision. The genealogical grouping of Brucella species with pericellular and intracellular plant and animal pathogens as well as with intracellular plant symbionts suggests a possible evolution of Brucella species from plant-arthropod-associated bacteria.Members of the genus Brucella are gram-negative facultative intracellular pathogens that induce abortion and severe clinical symptoms in mammals (10, 30). The importance of the disease, mainly in developing countries, is recognized by the considerable economic losses due to infection of domestic animals and by the zoonotic problems caused through the ingestion or contact of contaminated secretions and products (30).Six species of Brucella have been described (10, 30): B. melitensis, B. abortus, B. suis, B. neotomae, B. ovis, and B. canis. This classification has been based mainly on the animal host specificity, susceptibility to dyes, metabolic patterns, phage typing, and serological testing (10, 30). Recently Verger et al. (72), using DNA similarity, challenged the separation of Brucella into different species and proposed a single species only: B. melitensis, containing several biovars. In addition, De-Ley et al. (14) established taxonomic affiliations of Brucella species with members of the Centers for Disease Control group Vd in the rRNA superfamily IV and close relationship of these organisms to members of the family Rhizobiaceae.In preliminary reports on the Brucella 16S rRNA sequence (18) and on lipid A analyses (42, 47; J. W. Cherwonogrodzky, G. Dubray, E. Moreno, and H. Mayer, in K. Nielsen and B. Duncan, ed., Animal Brucellosis, in press), we have suggested, that Brucella species are related to the alpha-2 subdivision of the class Proteobacteria (64), formerly named "purple photosynthetic bacteria and their * Corresponding author. nonphototrophic relatives" (77), which includes phototrophic and chemoorganotrophic organisms (77,78). In the present study we propose that species of the genus Brucella are closely related to gram-negative bacteria, such as agrobacteria, rhizobiae, and rickettsiae, which also form intimate pericellular or intracellular associations with eucaryotic cells. MATERIALS AND METHODSExtraction and purification of LPS. The characteristics and culture conditions of smooth B. abortus 1119-3, smooth B. melitensis 16M, rough B. abortus 45/20, rough B. melitensis B115, B. canis (strain obtained from R. Diaz, University of Navarra,...
An organism's fitness is highly dependent on resource quality. The diet of saprobiotic organisms often comprises a variety of microorganisms. Saprophagous Drosophila melanogaster Meigen (Diptera: Drosophilidae) is known to feed on various yeast species, both as larva and adult. The yeasts encountered by the insects may differ in composition and quality, and thus in their influence on larval and adult performance. Our study explores life-history consequences of larval diet on selected larval and adult traits, as well as larval and adult food choice and egg-laying behaviour. The chosen yeast species could be shown to influence several life-history traits of D. melanogaster, such as survival, development time, and adult body weight. Additionally, the amount of yeast biomass initially fed to the larvae significantly influenced development time and adult body weight, whereby the effect depended on the yeast species offered. There were also yeast-specific influences on the measured fitness traits when larvae were reared at different densities. Larvae exhibited a preference for those yeast species that had shown to favour most of the measured life-history traits. Adults, on the other hand, exhibited a different preference. Contrary to our expectation based on the preference-performance hypothesis, female adult flies did not prefer to lay their eggs on substrates inoculated with yeast species that had been shown to favourably influence larval development time. Possible reasons for this seemingly 'bad mother'-behaviour are being discussed.
A fluorescence in situ hybridization (FISH) technique has been developed for the fluorescent labelling of Cryptosporidium parvum oocysts in water samples. The FISH technique employs a fluorescently labelled oligonucleotide probe (Cry1 probe) targeting a specific sequence in the 18S ribosomal RNA (rRNA) of C. parvum. Hybridization with the Cry1 probe resulted in fluorescence of sporozoites within oocysts that were capable of excystation, while oocysts that were dead prior to fixation did not fluoresce. Correlation of the FISH method with viability as measured by in vitro excystation was statistically highly significant, with a calculated correlation coefficient of 0.998. Examination of sequence data for Cryptosporidium spp. other than C. parvum suggests that the Cry1 probe is C. parvum-specific. In addition, 19 isolates of C. parvum were tested, and all fluoresced after hybridization with the Cry1 probe. Conversely, isolates of C. baileyi and C. muris were tested and found not to fluoresce after hybridization with the Cry1 probe. The fluorescence of FISH-stained oocysts was not bright enough to enable detection of oocysts in environmental water concentrates containing autofluorescent algae and mineral particles. However, in combination with immunofluorescence staining, FISH enabled species-specific detection and viability determination of C. parvum oocysts in water samples.
The inter-and intrageneric relationships of the genus Vibrio were investigated by performing a comparative analysis of the 16s rRNAs of 10 species, including four pathogenic representatives. The results of immunological and 5s rRNA studies were confirmed in that the genus is a neighboring taxon of the family Enterobacteriaceae. With regard to the intrageneric structure, Vibrio alginolyticus, Vibrio campbellii, Vibrio natriegens, Vibrio harveyi, Vibrio proteolyticus, Vibrio parahaemolyticus, and Vibrio vulnificus form the core of the genus, while Vibrio (Listonella) anguillarum, Vibrio diazotrophicus, and Vibrio hollisae are placed on the outskirts of the genus. Variable regions around positions 80, 180, and 450 could be used as target sites for genus-and species-specific oligonucleotide probes and polymerase chain reaction primers to be used in molecular identification.The relationships among members of the genus Vibrio have been thoroughly investigated by using phenotypic and genotypic analyses. Baumann and Baumann (3,4), Baumann et al. (5, 6), and Colwell (11) have compiled the relevant approaches and publications of the early era. The more recent studies dealing with the inter-and intrageneric relationships of these organisms are studies in which numerical phenetic analyses (19, 32,42,43), chemotaxonomy (39), and genetic analyses (13, 27,44,46) were used. The results of all of the genetic studies are consistent in that the genera Vibrio and Photobacterium (family Vibrionaceae [41]) are phylogenetic neighbors of the families Aeromonadaceue, Enterobacteriaceae, and Pasteurellaceae and the genera Alteromonas and Ruminobacter; these taxa form one major subline of descent within the gamma subclass of the Proteobcrcteriu (12,46,47). When a polyphasic approach was used, the genus Vibrio was subdivided, and former Vibrio species are now found in the genera Listonella (27), Colwellia (16), and "Moritella' ' (13).However, the inconsistent levels at which members of the genus Vibrio are found to be related when different phylogenetic approaches are used are unsatisfactory. Several species that have been found to be closely related by Vibrio harveyi. Vibrio campbellii, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio natriegens, and Vibrio vulnificus [13, 341 [called the Vibrio core organisms below]) also appear to be closely related when immunological approaches (la), rRNA cistron similarities (3, and 5s rRNA sequences (13, 28) are used. However, the levels at which these species are related vary. For example, as determined by DNA similarity, V . harveyi and V . alginolyticus are more closely related to each other (55%) than they are to Vibrio proteolyticus (average level of relatedness, 26%). While determinations of the imrnunological distances of the superoxide dismutases gave similar results (2,5), V . alginolyticus clustered with V . proteolyticus to the exclusion of V . harveyi when the 5s rRNAs were compared (28, 30). In a more recent 5s rRNA study, in which the authors included additional outside referenc...
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