Neuroprotection by nicotine against hypoxia-induced apoptosis in cortical cultures involves activation of multiple nicotinic acetylcholine receptor subtypes.

Activation of neuronal nicotinic acetylcholine receptors (nAChR) by nicotine has been suggested to protect neurons against a hypoxic insult. The objective of this study was to examine the nature of cell death induced by acute hypoxia in rat primary cortical cultures and the neuroprotective potential of nicotine in ameliorating these processes. Neuronal cell death induced by a 4-h exposure to hypoxia (0.1% O(2)) was apoptotic, as shown by TUNEL staining and assays monitoring DNA strand breaks and caspase-3/7 activity. The presence of nicotine (10 microM) during the hypoxic insult protected a subpopulation of susceptible neurones against DNA damage and apoptosis induced by oxygen deprivation. This protective effect of nicotine was prevented by a 30-min pre-incubation with either 100 nM alpha-bungarotoxin or 1 microM dihydro-beta-erythroidine, but not 1 microM atropine, suggesting that activation of at least two subtypes of nAChR, alpha7 and beta2* nAChR, is involved in mediating nicotine neuroprotection.

PAH-degradation by Paenibacillus spp. and description of Paenibacillus naphthalenovorans sp. nov., a naphthalene-degrading bacterium from the rhizosphere of salt marsh plants.

Bacteria belonging to the genus Paenibacillus were isolated by enrichment from petroleum-hydrocarbon-contaminated sediment and salt marsh rhizosphere using either naphthalene or phenanthrene as the sole carbon source, and were characterized using phenotypic, morphological and molecular techniques. The isolates were grouped by their colony morphologies and polyaromatic hydrocarbon-degradation patterns. Phenanthrene-degrading isolates produced mottled colonies on solid media and were identified as P. validus by fatty acid methyl ester and 16S rRNA gene sequence analyses. In contrast, the naphthalene-degrading isolates with mucoid colony morphology were distantly related to Paenibacillus validus, according to fatty acid methyl ester and 16S rRNA gene sequence analyses. The predominant fatty acids of the mucoid isolates were 15:0 anteiso, 16:1omega11c, 16:0 and 17:0 anteiso, constituting, on average, 50.5, 12.0, 11.2 and 6.5% of the total, respectively. The G+C contents of their DNA ranged from 47 to 52 mol%. The 16S rDNA sequence analysis revealed the highest (< or = 94%) similarity to P. validus. In addition, phylogenetic analyses based on 16S rDNA sequences showed that the mucoid isolates formed a distinct cluster within Paenibacillus. DNA-DNA hybridization experiments showed only a 6% DNA similarity between the type strain of P. validus and mucoid strain PR-N1. On the basis of the morphological, phenotypic and molecular data, the naphthalene-degrading isolates merit classification as a new Paenibacillus species, for which the name Paenibacillus naphthalenovorans sp. nov. is proposed, with PR-N1T (= ATCC BAA-206T = DSM 14203T) as the type strain.

Phylogeny, ribosomal RNA gene typing and relative abundance of new Pseudomonas species (sensu stricto) isolated from two pinyon-juniper woodland soils of the arid southwest U.S.

Rhizosphere-inhabiting Pseudomonas species interact with plant roots and may be important for plant performance under stressful environmental conditions. A comparison was conducted of culturable Pseudomonas isolates associated with pinyon rhizosphere and between-tree interspace areas in a hot, dry, volcanic cinder field and an adjacent sandy loam soil, in order to identify Pseudomonas species which may be involved in pinyon pine survival under stressful conditions. From a collection of 800 isolates, eleven isolates exhibiting different colony morphology were selected for 16S ribosomal RNA gene sequencing. Phylogenetic analysis of rDNA sequences from the eleven field isolates, forty-six described Pseudomonas species, and thirty-four previously characterized environmental isolates indicated that the isolates from the cinders and sandy loam soil clustered into three groups. The field isolates were distinct from any of the named species or other environmental isolates. Oligonucleotide primer pairs that differentiated three field isolate groups were designed from the 16S rDNA sequences, and eight hundred Pseudomonas field isolates cultured from pinyon rhizospheres and interspaces in the cinders and sandy loam soils were typed into the three groups using PCR assays. The composition of Pseudomonas populations in four environments was significantly different. The relative abundance of the three rDNA-based groups appeared to be affected by both the soil type and the pinyon rhizosphere.

Levels of bacterial community diversity in four arid soils compared by cultivation and 16S rRNA gene cloning.

Techniques based on amplification of 16S rRNA genes for comparing bacterial communities are now widely used in microbial ecology, but calibration of these techniques with traditional tools, such as cultivation, has been conspicuously absent. In this study, we compared levels of bacterial community diversity in two pinyon rhizosphere soil samples and two between-tree (interspace) soil samples by analyzing 179 cultivated bacterial isolates and 801 16S rRNA genes amplified from extracted soil DNA. Phylotypes were defined by performing a restriction fragment length polymorphism analysis of 16S rRNA gene sequences with the enzymes RsaI and BstUI. The average level of 16S rRNA gene sequence similarity of members of a phylotype was 86.6% based on an analysis of partial sequences. A total of 498 phylotypes were identified among the 16S ribosomal DNA (rDNA) clones, while 34 phylotypes occurred among the cultivated isolates. Analysis of sequences from a subset of the phylotypes showed that at least seven bacterial divisions were represented in the clone libraries, whereas the isolates represented only three. The phylotype richness, frequency distribution (evenness), and composition of the four culture collections and the four clone libraries were investigated by using a variety of diversity indices. Although cultivation and 16S rRNA cloning analyses gave contradictory descriptions of the relative phylotype richness for one of the four environments, the two methods identified qualitatively consistent relationships when levels of evenness were compared. The levels of phylotype similarity between communities were uniformly low (15 to 31%). Both methods consistently indicated that one environment was distinct from the other three. Our data illustrate that while 16S rDNA cloning and cultivation generally describe similar relationships between soil microbial communities, significant discrepancies can occur.

Wide distribution and diversity of members of the bacterial kingdom Acidobacterium in the environment.

To assess the distribution and diversity of members of the recently identified bacterial kingdom Acidobacterium, members of this kingdom present in 43 environmental samples were surveyed by PCR amplification. A primer designed to amplify rRNA gene sequences (ribosomal DNAs [rDNAs]) from most known members of the kingdom was used to interrogate bulk DNA extracted from the samples. Positive PCR results were obtained with all temperate soil and sediment samples tested, as well as some hot spring samples, indicating that members of this kingdom are very widespread in terrestrial environments. PCR primers specific for four phylogenetic subgroups within the kingdom were used in similar surveys. All four subgroups were detected in most neutral soils and some sediments, while only two of the groups were seen in most low-pH environments. The combined use of these primers allowed identification of a novel lineage within the kingdom in a hot spring environment. Phylogenetic analysis of rDNA sequences from our survey and the literature outlines at least six major subgroups within the kingdom. Taken together, these data suggest that members of the Acidobacterium kingdom are as genetically and metabolically diverse, environmentally widespread and perhaps as ecologically important as the well-known Proteobacteria and gram-positive bacterial kingdoms.

Diverse uncultivated bacterial groups from soils of the arid southwestern United States that are present in many geographic regions.

We have performed a phylogenetic survey of microbial species present in two soils from northern Arizona. Microbial DNA was purified directly from soil samples and subjected to PCR amplification with primers specific for bacterial 16S rRNA gene sequences (rDNAs). Clone libraries from the two soils were constructed, and 60 clone inserts were partially sequenced. Phylogenetic analysis of these sequences revealed extensive diversity. Most of the analyzed sequences (64%) fell into five novel clusters having no known cultured members. Extensive analysis of 10 nearly full-length rDNAs from clones representative of the novel groups indicated that four of the five groups probably cluster into a large "supergroup" which is as distinct from currently recognized bacterial divisions as the latter are from each other. From this we postulate the existence of a major bacterial lineage, previously known only from a single cultured representative, whose diversity and ecology we are only beginning to explore. Analysis of our data and that from other rDNA sequence-based studies of soils from different geographic regions shows considerable overlap of sequence types. Taken together, these groups encompass most of the novel rDNA sequences recovered in each comparable analysis reported to date, despite large differences in soil types and geographic sources. Our results indicate that members of these new groups comprise a phylogenetically diverse, geographically widespread, and perhaps numerically important component of the soil microbiota.

Comparison of the 16S ribosomal DNA sequences from the intracellular agents of proliferative enteritis in a hamster, deer, and ostrich with the sequence of a porcine isolate of Lawsonia intracellularis.

Proliferative enteritis is an enteric disease that affects a variety of animals. The causative agent in swine has been determined to be an obligate intracellular bacterium, Lawsonia intracellularis, related to the sulfate-reducing bacterium Desulfovibrio desulfuricans. The intracellular agents found in the lesions of different animal species are antigenically similar. In addition, strains from the pig, ferret, and hamster have been shown to be genetically similar. In this study we performed a partial 16S ribosomal DNA sequence analysis on the intracellular agent of proliferative enteritis from a hamster, a deer, and an ostrich and compared these sequences to that of the porcine L. intracellularis isolate. Results of this study indicate that the intracellular agents from these species with proliferative enteritis have high sequence similarity, indicating that they are all in the genus Lawsonia and that they may also be the same species, L. intracellularis.

Perspectives on archaeal diversity, thermophily and monophyly from environmental rRNA sequences.

Phylogenetic analysis of ribosomal RNA sequences obtained from uncultivated organisms of a hot spring in Yellowstone National Park reveals several novel groups of Archaea, many of which diverged from the crenarchaeal line of descent prior to previously characterized members of that kingdom. Universal phylogenetic trees constructed with the addition of these sequences indicate monophyly of Archaea, with modest bootstrap support. The data also show a specific relationship between low-temperature marine Archaea and some hot spring Archaea. Two of the environmental sequences are enigmatic: depending upon the data set and analytical method used, these sequences branch deeply within the Crenarchaeota, below the bifurcation between Crenarchaeota and Euryarchaeota, or even as the sister group to Eukaryotes. If additional data confirm either of the latter two placements, then the organisms represented by these ribosomal RNA sequences would merit recognition as a new kingdom, provisionally named "Korarchaeota."

Mineralization of 2,4,6-trinitrophenol (picric acid): characterization and phylogenetic identification of microbial strains.

Four bacterial strains that use picric acid as their sole carbon and energy source were isolated. Mineralization of 14C-UL-picric acid showed that up to 65% of the radioactivity was released as 14CO2. HPLC and UV/Vis spectral analyses indicated complete degradation of picric acid by these organisms. HPLC and LC/MS analyses showed transient formation of 2,4-dinitrophenol during picric acid degradation. Degradation of picric acid was concomitant with stoichiometric release of three moles of nitrite per mole of picric acid. The four picric acid degraders were identified as close relatives of Nocardioides simplex (ATCC 6946) based on their small subunit (16S) rRNA gene sequences.

Phylogenetic perspective on microbial life in hydrothermal ecosystems, past and present.

Understanding hydrothermal ecosystems, both past and present, requires basic information on the types of organisms present. Traditional methods, which require cultivation of microorganisms, fail to detect many taxa. We have used phylogenetic analyses of small subunit rRNA sequences obtained from microorganisms of a hot spring in Yellowstone National Park to explore the archael (archaebacterial) diversity present. Analysis of these sequences reveals several novel groups of archaea, greatly expanding our conception of the diversity of high temperature microorganisms, and demonstrating that hydrothermal systems harbour a rich variety of life. Many of these groups diverged from the archael line of descent early during evolution, and an understanding of their common properties may assist in inference of the nature of the last common ancestor of all life. The data also show a specific relationship between low-temperature marine archaea and some hot spring archaea, consistent with a thermophilic origin of life. Future use of rRNA-sequence-based techniques in exploration of hydrothermal systems should greatly facilitate study of modern thermophiles and give us insight into the activities of extinct communities as well.

Characterization of Lawsonia intracellularis gen. nov., sp. nov., the obligately intracellular bacterium of porcine proliferative enteropathy.

A novel obligately intracellular bacterium, ileal symbiont intracellularis, which was obtained from the intestines of pigs with proliferative enteropathy disease, was grown in pure cocultures with tissue cultures of rat cells. An examination of the 16S ribosomal DNA gene sequence revealed that the isolates which we obtained are members of the delta subdivision of the Proteobacteria and that the sequences of these organisms exhibit a level of similarly of 91% with the sequence of Desulfovibrio desulfuricans ATCC 27774. These isolates were homogeneous and differed in cellular morphology, acid fastness, phenotype, electrophoretic protein profile, and habitat from Desulfovibrio species. On the basis of the results of an integrated study of the phenotype and genotype of a consistent morphological entity found in particular porcine cells and associated with a well-defined clinical condition, we concluded that these bacteria belong to a previously undescribed genus and species, for which we propose the name Lawsonia intracellularis gen. nov., sp. nov. A species-specific recombinant DNA probe was cloned previously, and this probe was used to identify the bacterium in tissue culture cells and in the ileal epithelia of pigs with proliferative enteropathy disease. Coculture of the organism with a rat enterocyte cell line allowed us to designate strain NCTC 12656 the type strain and to describe the new genus and species. The organism which we cultured is pathogenic for pigs and causes proliferative enteropathy lesions in their ilea and colons, and Koch's postulates were fulfilled for this organism.

Isolation of a hyperthermophilic archaeum predicted by in situ RNA analysis.

A variety of hyperthermophilic bacteria and archaea have been isolated from high-temperature environments by plating and serial dilutions. However, these techniques allow only the small percentage of organisms able to form colonies, or those that are predominant within environmental samples, to be obtained in pure culture. Recently, in situ 16S ribosomal RNA analyses of samples from the Obsidian hot pool at Yellowstone National Park, Wyoming, revealed a variety of archaeal sequences, which were all different from those of previously isolated species. This suggests substantial diversity of archaea with so far unknown morphological, physiological and biochemical features, which may play an important part within high-temperature ecosystems. Here we describe a procedure to obtain pure cultures of unknown organisms harbouring specific 16S rRNA sequences identified previously within the environment. It combines visual recognition of single cells by phylogenetic staining and cloning by 'optical tweezers'. Our result validates polymerase chain reaction data on the existence of large archael communities.

Remarkable archaeal diversity detected in a Yellowstone National Park hot spring environment.

Of the three primary phylogenetic domains--Archaea (archaebacteria), Bacteria (eubacteria), and Eucarya (eukaryotes)--Archaea is the least understood in terms of its diversity, physiologies, and ecological panorama. Although many species of Crenarchaeota (one of the two recognized archaeal kingdoms sensu Woese [Woese, C. R., Kandler, O. & Wheelis, M. L. (1990) Proc. Natl. Acad. Sci. USA 87, 4576-4579]) have been isolated, they constitute a relatively tight-knit cluster of lineages in phylogenetic analyses of rRNA sequences. It seemed possible that this limited diversity is merely apparent and reflects only a failure to culture organisms, not their absence. We report here phylogenetic characterization of many archaeal small subunit rRNA gene sequences obtained by polymerase chain reaction amplification of mixed population DNA extracted directly from sediment of a hot spring in Yellowstone National Park. This approach obviates the need for cultivation to identify organisms. The analyses document the existence not only of species belonging to well-characterized crenarchaeal genera or families but also of crenarchaeal species for which no close relatives have so far been found. The large number of distinct archaeal sequence types retrieved from this single hot spring was unexpected and demonstrates that Crenarchaeota is a much more diverse group than was previously suspected. The results have impact on our concepts of the phylogenetic organization of Archaea.

Ileal symbiont intracellularis, an obligate intracellular bacterium of porcine intestines showing a relationship to Desulfovibrio species.

A new genus and species of obligate intracellular-bacteria found in porcine intestines are described. Growth on any bacteriological medium deprived of living cells has not been demonstrated. The organism has been grown intracellularly in cell culture. The 16S rRNA gene sequence data, DNA probe results, and microscopic observations provide evidence that these bacteria differ from those in other described genera and that they belong to the delta subdivision of the class Proteobacteria. We have amplified and sequenced the 16S ribosomal DNA of four preparations of the intracellular bacterium from pigs. For this, intracellular organisms were released and purified from the infected cells without culture techniques. After DNA purification, the polymerase chain reaction with primers complementary to highly conserved eubacterial sequences was used to amplify regions of 16S ribosomal DNA which were subsequently cloned (in some cases) and sequenced directly by standard techniques. The sequences obtained from each preparation were identical and were most similar to that of a sulfate-reducing proteobacterium, Desulfovibrio desulfuricans ATCC 27774 (91% similarity). An oligonucleotide probe complementary to a hypervariable region of the 16S rRNA sequence of the bacterium hybridized with intracellular organisms obtained from porcine intestines. The bacterium is a gram-negative, curved rod with tapered ends. It multiplies intracellularly in the cytoplasm of ileal epithelial cells by septation. The vernacular name Ileal symbiont (IS) intracellularis is proposed for this bacterium. The type strain of IS intracellularis is strain 1482/89 grown in cell culture from a pig affected by proliferative enteropathy. It is deposited in the National Collection of Type Cultures, Colindale, London, as NCTC 12656.

Evolutionary relationships within the fungi: analyses of nuclear small subunit rRNA sequences.

Nucleotide sequences of the small subunit ribosomal RNA (18S) gene were used to investigate evolutionary relationships within the Fungi. The inferred tree topologies are in general agreement with traditional classifications in the following ways: (1) the Chytridiomycota and Zygomycota appear to be basal groups within the Fungi. (2) The Ascomycota and Basidiomycota are a derived monophyletic group. (3) Relationships within the Ascomycota are concordant with traditional orders and divide the hemi- and euascomycetes into distinct lineages. (4) The Basidiomycota is divided between the holobasidiomycetes and phragmobasidiomycetes. Conflicts with traditional classification were limited to weakly supported branches of the tree. Strongly supported relationships were robust to minor changes in alignment, method of analysis, and various weighting schemes. Weighting, either of transversions or by site, did not convincingly improve the status of poorly supported portions of the tree. The rate of variation at particular sites does not appear to be independent of lineage, suggesting that covariation of sites may be an important phenomenon in these genes.

Phylogeny of cytoplasmic incompatibility micro-organisms in the parasitoid wasp genus Nasonia (Hymenoptera: Pteromalidae) based on 16S ribosomal DNA sequences.

Cytoplasmic incompatibility results in embryo mortality in diploids, or all male offspring in haplodiploids, when individuals carrying different cytoplasmic factors are crossed. Cytoplasmic factors have been identified as intracellular micro-organisms. Microbe-induced cytoplasmic incompatibility is found in many insect taxa and may play a role in reproductive isolation between populations. Such micro-organisms cause bidirectional incompatibility between species of the parasitoid wasp genus Nasonia. The phylogenetic relationship of cytoplasmic incompatibility microorganisms (CIM) of different Nasonia species was analysed using their 16S ribosomal DNA (rDNA) sequence. Two 16S rDNA operons were detected in the CIM of each Nasonia species. Sequence analysis indicates that the Nasonia CIM are closely related and belong to the alpha group of the Proteobacteria.

Evolutionary relationships among pathogenic Candida species and relatives.

Small subunit rRNA sequences have been determined for 10 of the most clinically important pathogenic species of the yeast genus Candida (including Torulopsis [Candida] glabrata and Yarrowia [Candida] lipolytica) and for Hansenula polymorpha. Phylogenetic analyses of these sequences and those of Saccharomyces cerevisiae, Kluyveromyces marxianus var. lactis, and Aspergillus fumigatus indicate that Candida albicans, C. tropicalis, C. parapsilosis, and C. viswanathii form a subgroup within the genus. The remaining significant pathogen, T. glabrata, falls into a second, distinct subgroup and is specifically related to S. cerevisiae and more distantly related to C. kefyr (psuedotropicalis) and K. marxianus var. lactis. The 18S rRNA sequence of Y. lipolytica has evolved rapidly in relation to the other Candida sequences examined and appears to be only distantly related to them. As anticipated, species of several other genera appear to bear specific relationships to members of the genus Candida.

16S ribosomal DNA amplification for phylogenetic study.

A set of oligonucleotide primers capable of initiating enzymatic amplification (polymerase chain reaction) on a phylogenetically and taxonomically wide range of bacteria is described along with methods for their use and examples. One pair of primers is capable of amplifying nearly full-length 16S ribosomal DNA (rDNA) from many bacterial genera; the additional primers are useful for various exceptional sequences. Methods for purification of amplified material, direct sequencing, cloning, sequencing, and transcription are outlined. An obligate intracellular parasite of bovine erythrocytes, Anaplasma marginale, is used as an example; its 16S rDNA was amplified, cloned, sequenced, and phylogenetically placed. Anaplasmas are related to the genera Rickettsia and Ehrlichia. In addition, 16S rDNAs from several species were readily amplified from material found in lyophilized ampoules from the American Type Culture Collection. By use of this method, the phylogenetic study of extremely fastidious or highly pathogenic bacterial species can be carried out without the need to culture them. In theory, any gene segment for which polymerase chain reaction primer design is possible can be derived from a readily obtainable lyophilized bacterial culture.