Phylogenetic differences between particle-associated and planktonic ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria in the Northwestern Mediterranean Sea

The aim of this study was to determine if there were differences between the types of ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria associated with particulate material and planktonic samples obtained from the northwestern Mediterranean Sea. A nested PCR procedure performed with ammonia oxidizer-selective primers was used to amplify 16S rRNA genes from extracted DNA. The results of partial and full-length sequence analyses of 16S rRNA genes suggested that different groups of ammonia-oxidizing bacteria were associated with the two sample types. The particle-associated sequences were predominantly related to Nitrosomonas eutropha, while the sequences obtained from the planktonic samples were related to a novel marine Nitrosospira group (cluster 1) for which there is no cultured representative yet. A number of oligonucleotide probes specific for different groups of ammonia oxidizers were used to estimate the relative abundance of sequence types in samples of clone libraries. The planktonic libraries contained lower proportions of ammonia oxidizer clones (0 to 26%) than the particulate material libraries (9 to 83%). Samples of the planktonic and particle-associated libraries showed that there were depth-related differences in the ammonia oxidizer populations, with the highest number of positive clones in the particle-associated sample occurring at a depth of 700 m. The greatest difference between planktonic and particle-associated populations occurred at a depth of 400 m, where only 4% of the clones in the planktonic library were identified as Nitrosomonas clones, while 96% of these clones were identified as clones that were related to the marine Nitrosospira species. Conversely, all ammonia oxidizer-positive clones obtained from the particle-associated library were members of the Nitrosomonas group. This is the first indication that Nitrosomonas species and Nitrosospira species may occupy at least two distinct environmental niches in marine environments. The occurrence of these groups in different niches may result from differences in physiological properties and, coupled with the different environmental conditions associated with these niches, may lead to significant differences in the nature and rates of nitrogen cycling in these environments.     

Detection of stratified microbial populations related to Chlorobium and Fibrobacter species in the Atlantic and Pacific oceans

A gene lineage (SAR406) related to Chlorobium and Fibrobacter species was found in 16S rRNA gene clone libraries prepared from samples from two oceans. The clone libraries were constructed from total picoplankton genomic DNA to assess bacterial diversity in the lower surface layer. The samples were collected by filtration from a depth of 80 m at a site in the western Sargasso Sea and from a depth of 120 m at a site in the Pacific Ocean, approximately 70 km from the Oregon coast. The PCR and primers which amplified nearly full-length 16S rRNA genes were used to prepare the clone libraries. Among the diverse gene clones in these libraries were two related clones (SAR406 and OCS307) which could not be assigned to any of the major bacterial phyla. Phylogenetic analyses demonstrated that these genes were distant relatives of the genus Fibrobacter and the green sulfur bacterial phylum, which includes the genus Chlorobium. The inclusion of SAR406 in phylogenetic trees inferred by several methods resulted in support from bootstrap replicates for the conclusion that Fibrobacter and Chlorobium species and SAR406 are a monophyletic group. An oligonucleotide probe that selectively hybridized to clone SAR406 was used to examine the distribution of this gene lineage in vertical profiles from the Atlantic and Pacific Oceans and in monthly time series at 0 and 200 m in the Atlantic Ocean. During stratified periods, the genes were most abundant slightly below the deep chlorophyll layer. Seasonal changes in the surface abundance of SAR406 rDNA were highly correlated with chlorophyll a levels (r = 0.75).     

Gamma interferon augments macrophage activation by lipopolysaccharide by two distinct mechanisms, at the signal transduction level and via an autocrine mechanism involving tumor necrosis factor alpha and interleukin-1

Acanthamoebae are ubiquitous soil and water bactivores which may serve as amplification vehicles for a variety of pathogenic facultative bacteria and as hosts to other, presently uncultured bacterial endosymbionts. The spectrum of uncultured endosymbionts includes gram-negative rods and gram-variable cocci, the latter recently shown to be members of the Chlamydiales. We report here the isolation from corneal scrapings of two Acanthamoeba strains that harbor gram-negative rod endosymbionts that could not be cultured by standard techniques. These bacteria were phylogenetically characterized following amplification and sequencing of the near-full-length 16S rRNA gene. We used two fluorescently labelled oligonucleotide probes targeting signature regions within the retrieved sequences to detect these organisms in situ. Phylogenetic analyses demonstrated that they displayed 99.6% sequence similarity and formed an independent and well-separated lineage within the Rickettsiales branch of the alpha subdivision of the Proteobacteria. Nearest relatives included members of the genus Rickettsia, with sequence similarities of approximately 85 to 86%, suggesting that these symbionts are representatives of a new genus and, perhaps, family. Distance matrix, parsimony, and maximum-likelihood tree-generating methods all consistently supported deep branching of the 16S rDNA sequences within the Rickettsiales. The oligonucleotide probes displayed at least three mismatches to all other available 16S rDNA sequences, and they both readily permitted the unambiguous detection of rod-shaped bacteria within intact acanthamoebae by confocal laser-scanning microscopy. Considering the long-standing relationship of most Rickettsiales with arthropods, the finding of a related lineage of endosymbionts in protozoan hosts was unexpected and may have implications for the preadaptation and/or recruitment of rickettsia-like bacteria to metazoan hosts.     

p16/INK4a and p15/INK4b gene methylation and absence of p16/INK4a mRNA and protein expression in Burkitt''s lymphoma

So-called sulfur-turf microbial mats, which are macroscopic white filaments or bundles consisting of large sausage-shaped bacteria and elemental sulfur particles, occur in sulfide-containing hot springs in Japan. However, no thermophiles from sulfur-turf mats have yet been isolated as cultivable strains. This study was undertaken to determine the phylogenetic positions of the sausage-shaped bacteria in sulfur-turf mats by direct cloning and sequencing of 16S rRNA genes amplified from the bulk DNAs of the mats. Common clones with 16S rDNA sequences with similarity levels of 94.8 to 99% were isolated from sulfur-turf mat samples from two geographically remote hot springs. Phylogenetic analysis showed that the phylotypes of the common clones formed a major cluster with members of the Aquifex-Hydrogenobacter complex, which represents the most deeply branching lineage of the domain bacteria. Furthermore, the bacteria of the sulfur-turf mat phylotypes formed a clade distinguishable from that of other members of the Aquifex-Hydrogenobacter complex at the order or subclass level. In situ hybridization with clone-specific probes for 16S rRNA revealed that the common phylotype of sulfur-turf mat bacteria is that of the predominant sausage-shaped bacteria.     

A qualitative evaluation of the published oligonucleotides specific for the 16S rRNA gene sequences of the ammonia-oxidizing bacteria

Over the past few years, there has been an increasing interest in making oligonucleotides specific for ammonia-oxidizing bacteria (AOB), in order to detect and monitor these slow growing bacteria in environmental samples, in enrichment cultures and in wastewater treatment plants. Based on 16S rDNA sequences, a broad selection of oligonucleotides have been designed, either encompassing all known AOB in the beta-subgroup of the Proteobacteria (beta AOB), or subclasses within beta AOB. Thirty different oligonucleotides have so far been published, with varying specificity. The first AOB-specific oligonucleotides published were obtained as a result of an alignment of only eleven 16S rDNA sequences from AOB. Including the present study, there are now forty nearly full length 16S rDNA sequences available from these bacteria, in addition to a number of partial sequences, so that an improved evaluation of the published oligonucleotides can be done. Two new 16S rRNA gene sequences from Nitrosospira are presented here, in a phylogenetic analysis containing every 16S rRNA gene sequences (> 1 kb) available from AOB. On the basis of an alignment of all these sequences, combined with searches in the nucleotide sequence databases, an evaluation of the thirty published oligonucleotides is presented. The analysis expose the strength and weakness of each oligonucleotide and discuss the use of oligonucleotides specific for 16S rRNA genes in future studies of AOB. The present work also identifies one new, broad range primer, specific for the AOB in the beta-subgroup of the Proteobacteria.     

Temperature gradient gel electrophoresis analysis of 16S rRNA from human fecal samples reveals stable and host-specific communities of active bacteria

The diversity of the predominant bacteria in the human gastrointestinal tract was studied by using 16S rRNA-based approaches. PCR amplicons of the V6 to V8 regions of fecal 16S rRNA and ribosomal DNA (rDNA) were analyzed by temperature gradient gel electrophoresis (TGGE). TGGE of fecal 16S rDNA amplicons from 16 individuals showed different profiles, with some bands in common. Fecal samples from two individuals were monitored over time and showed remarkably stable profiles over a period of at least 6 months. TGGE profiles derived from 16S rRNA and rDNA amplicons showed similar banding patterns. However, the intensities of bands with similar mobilities differed in some cases, indicating a different contribution to the total active fraction of the prominent fecal bacteria. Most 16S rRNA amplicons in the TGGE pattern of one subject were identified by cloning and sequence analysis. Forty-five of the 78 clones matched 15 bands, and 33 clones did not match any visible band in the TGGE pattern. Nested PCR of amplified 16S rDNA indicated preferential amplification of a sequence corresponding to 12 of the 33 nonmatching clones with similar mobilities in TGGE. The sequences matching 15 bands in the TGGE pattern showed 91.5 to 98.7% homology to sequences derived from different Clostridium clusters. Most of these were related to strains derived from the human intestine. The results indicate that the combination of cloning and TGGE analysis of 16S rDNA amplicons is a reliable approach to monitoring different microbial communities in feces.     

Denaturing gradient gel electrophoresis used to monitor the enrichment culture of aerobic chemoorganotrophic bacteria from a hot spring cyanobacterial mat

Previous studies investigating microbial diversity in the Octopus Spring cyanobacterial mat community (Yellowstone National Park) have shown a discrepancy between bacterial populations observed by molecular retrieval and cultivation techniques. To investigate how selective enrichment culture techniques affect species composition, we used denaturing gradient gel electrophoresis (DGGE) separation of PCR-amplified 16S rRNA gene fragments to monitor the populations contained within enrichment cultures of aerobic chemoorganotrophic bacteria from the ca. 50 degrees C region of the mat community. By varying the degree of dilution of the inoculum, medium composition, and enrichment conditions and duration and by analyzing the cultures by DGGE, we detected 14 unique 16S rRNA sequence types. These corresponded to alpha-, beta-, gamma-, and delta-proteobacteria, Thermus relatives, and gram-positive bacteria with high G + C ratio and, at the highest inoculum dilutions, Chloroflexus aurantiacus relatives, which were estimated to still be approximately 300 times less abundant than cells of the mat primary producer, Synechococcus spp. Only three of these populations were previously cultivated on solidified medium after similar enrichment. Only two of these population have 16S rRNA sequences which were previously cloned directly from the mat. These results reveal a diversity of bacterial populations in enrichment culture which were not detected by either molecular retrieval or strain purification techniques.     

Phylogenetic characterization of ineffective Frankia in Alnus glutinosa (L.) Gaertn. nodules from wetland soil inoculants

Ineffective Frankia endophytes were retrieved from various wet soils by using Alnus glutinosa clones as trapping plants. No pure cultures could be isolated from these ineffective nodules. Therefore, the phylogenetic position of these endophytes was determined by sequence analysis of cloned PCR products of bacterial 16S rDNA, derived from nodules. The results showed that all nodule endophytes belong to a hitherto undescribed cluster of the Frankia phylogenetic tree. The position of these uncultured ineffective Frankia nodule endophytes is different from that of the ineffective Frankia isolates derived from A. glutinosa nodules, even when originating from the same geographical location. This suggests a bias in current isolation techniques.     

Deleterious mutations destabilize ribosomal RNA in endosymbiotic bacteria

In populations that are small and asexual, mutations with slight negative effects on fitness will drift to fixation more often than in large or sexual populations in which they will be eliminated by selection. If such mutations occur in substantial numbers, the combined effects of long-term asexuality and small population size may result in substantial accumulation of mildly deleterious substitutions. Prokaryotic endosymbionts of animals that are transmitted maternally for very long periods are effectively asexual and experience smaller effective population size than their free-living relatives. The contrast between such endosymbionts and related free-living bacteria allows us to test whether a population structure imposing frequent bottlenecks and asexuality does lead to an accumulation of slightly deleterious substitutions. Here we show that several independently derived insect endosymbionts, each with a long history of maternal transmission, have accumulated destabilizing base substitutions in the highly conserved 16S rRNA. Stabilities of Domain I of this subunit are 15-25% lower in endosymbionts than in closely related free-living bacteria. By mapping destabilizing substitutions onto a reconstructed phylogeny, we show that decreased ribosomal stability has evolved separately in each endosymbiont lineage. Our phylogenetic approach allows us to demonstrate statistical significance for this pattern: becoming endosymbiotic predictably results in decreased stability of rRNA secondary structure.     

Characterization of a defined 2,3,5, 6-tetrachlorobiphenyl-ortho-dechlorinating microbial community by comparative sequence analysis of genes coding for 16S rRNA

Defined microbial communities were developed by combining selective enrichment with molecular monitoring of total community genes coding for 16S rRNAs (16S rDNAs) to identify potential polychlorinated biphenyl (PCB)-dechlorinating anaerobes that ortho dechlorinate 2,3, 5,6-tetrachlorobiphenyl. In enrichment cultures that contained a defined estuarine medium, three fatty acids, and sterile sediment, a Clostridium sp. was predominant in the absence of added PCB, but undescribed species in the delta subgroup of the class Proteobacteria, the low-G+C gram-positive subgroup, the Thermotogales subgroup, and a single species with sequence similarity to the deeply branching species Dehalococcoides ethenogenes were more predominant during active dechlorination of the PCB. Species with high sequence similarities to Methanomicrobiales and Methanosarcinales archaeal subgroups were predominant in both dechlorinating and nondechlorinating enrichment cultures. Deletion of sediment from PCB-dechlorinating enrichment cultures reduced the rate of dechlorination and the diversity of the community. Substitution of sodium acetate for the mixture of three fatty acids increased the rate of dechlorination, further reduced the community diversity, and caused a shift in the predominant species that included restriction fragment length polymorphism patterns not previously detected. Although PCB-dechlorinating cultures were methanogenic, inhibition of methanogenesis and elimination of the archaeal community by addition of bromoethanesulfonic acid only slightly inhibited dechlorination, indicating that the archaea were not required for ortho dechlorination of the congener. Deletion of Clostridium spp. from the community profile by addition of vancomycin only slightly reduced dechlorination. However, addition of sodium molybdate, an inhibitor of sulfate reduction, inhibited dechlorination and deleted selected species from the community profiles of the class Bacteria. With the exception of one 16S rDNA sequence that had the highest sequence similarity to the obligate perchloroethylene-dechlorinating Dehalococcoides, the 16S rDNA sequences associated with PCB ortho dechlorination had high sequence similarities to the delta, low-G+C gram-positive, and Thermotogales subgroups, which all include sulfur-, sulfate-, and/or iron(III)-respiring bacterial species.     

Proposal to reclassify Zoogloea ramigera IAM 12670 (P. R. Dugan 115) as Duganella zoogloeoides gen. nov., sp. nov

The taxonomic position of a misclassified strain, Zoogloea remigera IAM 12670T (= ATCC 25925T = P. R. Dugan 115T), was reevaluated. A phylogenetic analysis based on 16S ribosomal rDNA sequences revealed that this organism was located in the beta subclass of the class Proteobacteria with members of the genus Telluria as its closest relatives. On the basis of phenotypic and phylogenetic information, we propose that this organism should be reclassified in a new taxon with the name Duganella zoogloeoides gen. nov., sp. nov.     

膜生物反应器脱氮除磷及细菌16S rDNA多样性研究

采用一体式膜生物反应器(MBR)处理富含氮磷、有机物、高悬浮物的畜禽养殖废水.在最佳的处理条件下,采用分子生物学手段,构建16S rDNA基因文库,并通过16SrDNA序列的系统发育分析,对膜生物反应器中的细菌种群多样性和群落结构进行了研究.实验结果表明,反应器中的细菌种群多样性十分丰富,系统中的优势菌群为γ-变形细菌(proteobacteria)和β-变形细菌(proteobacteria).     

Isolation of new bacterial species from drinking water biofilms and proof of their in situ dominance with highly specific 16S rRNA probes

A polyphasic approach involving cultivation, direct viable counts, rRNA-based phylogenetic classification, and in situ probing was applied for the characterization of the dominant microbial population in a municipal drinking water distribution system. A total of 234 bacterial strains cultivated on R2A medium were screened for bacteria affiliated with the in situ dominating beta subclass of Proteobacteria. The isolates were grouped according to common features of their cell and colony morphologies, and eight representative strains were used for 16S rRNA sequencing and the development of a suite of strain-specific oligonucleotide probes. Phylogenetic analysis indicated that all of the isolates were hitherto unknown bacteria. Three of them, strains B4, B6, and B8, formed a separate cluster of closely related organisms within the beta 1 subclass of Proteobacteria. In situ probing revealed that (i) 67 to 72% of total bacteria, corresponding to more than 80% of beta-subclass bacteria, could be encompassed with the strain-specific probes and (ii) the dominating bacterial species were culturable on R2A medium. Additionally, two-thirds of the autochthonous drinking water population could be shown to be in a viable but nonculturable (VBNC) state by using a direct viable count approach. The comparison of isolation frequencies with the in situ abundances of the eight investigated strains revealed differences in their culturability, indicating variable ratios of culturable to VBNC cells among the strains. The further characterization of biofilms throughout the distribution network demonstrated strains B6 and B8 to be dominant bacterial strains in groundwater and distribution system biofilms. The other strains could be found at various frequencies in the different parts of the distribution system; several strains appeared exclusively in drinking water biofilms obtained from a house installation system.     

PCR-ribotyping of Xenorhabdus and Photorhabdus isolates from the Caribbean region in relation to the taxonomy and geographic distribution of their nematode hosts

The genetic diversity of symbiotic Xenorhabdus and Photorhabdus bacteria associated with entomopathogenic nematodes was examined by a restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes (rDNAs). A total of 117 strains were studied, most of which were isolated from the Caribbean basin after an exhaustive soil sampling. The collection consisted of 77 isolates recovered from entomopathogenic nematodes in 14 Caribbean islands and of 40 reference strains belonging to Xenorhabdus and Photorhabdus spp. collected at various localities worldwide. Thirty distinctive 16S rDNA genotypes were identified, and cluster analysis was used to distinguish the genus Xenorhabdus from the genus Photorhabdus. The genus Xenorhabdus appears more diverse than the genus Photorhabdus, and for both genera the bacterial genotype diversity is in congruence with the host-nematode taxonomy. The occurrence of symbiotic bacterial genotypes was related to the ecological distribution of host nematodes.     

Genome evolution within the alpha Proteobacteria: why do some bacteria not possess plasmids and others exhibit more than one different chromosome?

Animal intracellular Proteobacteria of the alpha subclass without plasmids and containing one or more chromosomes are phylogenetically entwined with opportunistic, plant-associated, chemoautotrophic and photosynthetic alpha Proteobacteria possessing one or more chromosomes and plasmids. Local variations in open environments, such as soil, water, manure, gut systems and the external surfaces of plants and animals, may have selected alpha Proteobacteria with extensive metabolic alternatives, broad genetic diversity, and more flexible and larger genomes with ability for horizontal gene flux. On the contrary, the constant and isolated animal cellular milieu selected heterotrophic alpha Proteobacteria with smaller genomes without plasmids and reduced genetic diversity as compared to their plant-associated and phototrophic relatives. The characteristics and genome sizes in the extant species suggest that a second chromosome could have evolved from megaplasmids which acquired housekeeping genes. Consequently, the genomes of the animal cell-associated Proteobacteria evolved through reductions of the larger genomes of chemoautotrophic ancestors and became rich in adenosine and thymidine, as compared to the genomes of their ancestors. Genome organisation and phylogenetic ancestor-descendent relationships between extant bacteria of closely related genera and within the same monophyletic genus and species suggest that some strains have undergone transition from two chromosomes to a single replicon. It is proposed that as long as the essential information is correctly expressed, the presence of one or more chromosomes within the same genus or species is the result of contingency. Genetic drift in clonal bacteria, such as animal cell-associated alpha Proteobacteria, would depend almost exclusively on mutation and internal genetic rearrangement processes. Alternatively, genomic variations in reticulate bacteria, such as many intestinal and plant cell-associated Proteobacteria, will depend not only on these processes, but also on their genetic interactions with other bacterial strains. Common pathogenic domains necessary for the invasion and survival in association with cells have been preserved in the chromosomes of the animal and plant-associated alpha Proteobacteria. These pathogenic domains have been maintained by vertical inherence, extensively ameliorated to match the chromosome G + C content and evolved within chromosomes of alpha Proteobacteria.     

Aerobic anoxygenic phototrophic bacteria

The aerobic anoxygenic phototrophic bacteria are a relatively recently discovered bacterial group. Although taxonomically and phylogenetically heterogeneous, these bacteria share the following distinguishing features: the presence of bacteriochlorophyll a incorporated into reaction center and light-harvesting complexes, low levels of the photosynthetic unit in cells, an abundance of carotenoids, a strong inhibition by light of bacteriochlorophyll synthesis, and the inability to grow photosynthetically under anaerobic conditions. Aerobic anoxygenic phototrophic bacteria are classified in two marine (Erythrobacter and Roseobacter) and six freshwater (Acidiphilium, Erythromicrobium, Erythromonas, Porphyrobacter, Roseococcus, and Sandaracinobacter) genera, which phylogenetically belong to the alpha-1, alpha-3, and alpha-4 subclasses of the class Proteobacteria. Despite this phylogenetic information, the evolution and ancestry of their photosynthetic properties are unclear. We discuss several current proposals for the evolutionary origin of aerobic phototrophic bacteria. The closest phylogenetic relatives of aerobic phototrophic bacteria include facultatively anaerobic purple nonsulfur phototrophic bacteria. Since these two bacterial groups share many properties, yet have significant differences, we compare and contrast their physiology, with an emphasis on morphology and photosynthetic and other metabolic processes.     

Extremely barophilic bacteria isolated from the Mariana Trench, Challenger Deep, at a depth of 11,000 meters

Two strains of obligately barophilic bacteria were isolated from a sample of the world's deepest sediment, which was obtained by the unmanned deep-sea submersible Kaiko in the Mariana Trench, Challenger Deep, at a depth of 10,898 m. From the results of phylogenetic analysis based on 16S rRNA gene sequences, DNA-DNA relatedness study, and analysis of fatty acid composition, the first strain (DB21MT-2) appears to be most highly similar to Shewanella benthica and close relatives, and the second strain (DB21MT-5) appears to be closely related to the genus Moritella. The optimal pressure conditions for growth of these isolates were 70 MPa for strain DB21MT-2 and 80 MPa for strain DB21MT-5, and no growth was detected at pressures of less than 50 MPa with either strain. This is the first evidence of the existence of an extreme-barophile bacterium of the genus Moritella isolated from the deep-sea environment.     

Opportunities for mupirocin calcium cream in the emergency department

A multidisciplinary approach was used to study the effects of pollution from a marine fish farm on nitrification rates and on the community structure of ammonia-oxidizing bacteria in the underlying sediment. Organic content, ammonium concentrations, nitrification rates, and ammonia oxidizer most-probable-number counts were determined in samples of sediment collected from beneath a fish cage and on a transect at 20 and 40 m from the cage. The data suggest that nitrogen cycling was significantly disrupted directly beneath the fish cage, with inhibition of nitrification and denitrification. Although visual examination indicated some slight changes in sediment appearance at 20 m, all other measurements were similar to those obtained at 40 m, where the sediment was considered pristine. The community structures of proteobacterial beta-subgroup ammonia-oxidizing bacteria at the sampling sites were compared by PCR amplification of 16S ribosomal DNA (rDNA), using primers which target this group. PCR products were analyzed by denaturing gradient gel electrophoresis (DGGE) and with oligonucleotide hybridization probes specific for different ammonia oxidizers. A DGGE doublet observed in PCR products from the highly polluted fish cage sediment sample was present at a lower intensity in the 20-m sample but was absent from the pristine 40-m sample station. Band migration, hybridization, and sequencing demonstrated that the doublet corresponded to a marine Nitrosomonas group which was originally observed in 16S rDNA clone libraries prepared from the same sediment samples but with different PCR primers. Our data suggest that this novel Nitrosomonas subgroup was selected for within polluted fish farm sediments and that the relative abundance of this group was influenced by the extent of pollution.