Physiological and molecular characterization of anaerobic benzene-degrading mixed cultures

Nine distinct anaerobic benzene-degrading cultures were enriched from sediment samples from four different sites. These cultures used nitrate, sulphate or CO2 as electron acceptors. The shortest doubling times were observed in nitrate-reducing cultures, although cell yield was lowest in these cultures. The highest substrate concentration utilized and maximum absolute rates of benzene degraded (in micro M day-1) were observed in methanogenic cultures. The microbial compositions of a methanogenic and nitrate-reducing culture were determined from a clone library of 16S rRNA genes. Five Bacterial 16S rRNA sequences, one of which resembled a clone previously found in a sulphate-reducing, benzene-degrading culture and four Archaeal 16S rRNA sequences were identified in a methanogenic culture. Four Bacterial and no Archaeal 16S rRNA sequences were identified in a nitrate-reducing culture. The relative abundance of the four nitrate-reducing putative species was determined by slot blot hybridization. Two green sulphur bacteria together formed 52% of the clone library, but were found to be less than 4% of the culture by slot blot analysis. One of the cloned 16S rRNA gene sequences comprised 70% of the culture and was phylogenetically 93% similar to both Azoarcus and Dechloromonas species, which have been shown to degrade aromatic compounds, including benzene, under nitrate-reducing conditions.     

Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost for microbial community analysis

Analysis of microbial community structure in complex environmental samples using nucleic acid techniques requires efficient unbiased DNA extraction procedures; however, humic acids and other contaminants complicate the isolation of PCR-amplifiable DNA from compost and other organic-rich samples. In this study, combinations of DNA extraction and purification methods were compared based on DNA yield, humic acid contamination, PCR amplifiability, and microbial community structure assessed by terminal restriction fragment length polymorphisms (TRFLP) of amplified 16S rRNA genes. DNA yield and humic acid contamination, determined by A230, varied significantly between extraction methods. Humic acid contamination of DNA obtained from compost decreased with increasing salt concentration in the lysis buffer. DNA purified by gel permeation chromatography (Sepharose 4B columns) gave satisfactory PCR amplification with universal eubacterial 16S rRNA gene primers only when A260/A280 ratios exceeded 1.5. DNA purified with affinity chromatography (hydroxyapatite columns), and showing A260/A280 ratios as high as 1.8, did not show consistently satisfactory PCR amplification using the same 16S rRNA primers. Almost all DNA samples purified by agarose gel electrophoresis showed satisfactory PCR amplification. Principal components analysis (PCA) of TRFLP patterns differentiated compost types based on the presence/absence of peaks and on the height of the peaks, but differences in TRFLP patterns were not appreciable between extraction methods that yielded relatively pure DNA. High levels of humic acid contamination in extracted DNA resulted in TRFLP patterns that were not consistent and introduced a bias towards lower estimates of diversity.     

Phylogenetic analysis of Bacillus thuringiensis serovars based on 16S rRNA gene restriction fragment length polymorphisms

AIMS: To determine the 16S rRNA gene fingerprints of Bacillus thuringiensis strains to reveal phylogenetic relationships among them. METHODS AND RESULTS: Using 16S rRNA gene restriction fragment length polymorphisms generated by HindIII and EcoRI, 86 Bacillus thuringiensis strains were classified. This includes 80 B. thuringiensis serovars and five more strains, kurstaki HD-1, subtoxicus, dendrolimus, tenebrionis and sandiego, to assess not only interserovar DNA relatedness but also intraserovar DNA relatedness, and the non-motile strain, hence non-serotypeable, B. thuringiensis var. wuhanensis. All 86 B. thuringiensis strains tested showed distinct ribotypes. The dendrogram resulting from the numerical analysis of the distance matrix shows four distinct phylogenetic groups and two ungrouped serovars, finitimus and bolivia, at the 92.5% DNA relatedness rate. CONCLUSION: 16S rRNA gene fingerprinting cannot only be used for the classification of B. thuringiensis strains amenable or not to serotyping, but can also reveal phylogenetic relationships between strains. SIGNIFICANCE AND IMPACT OF THE STUDY: In future screening programmes, 16S rRNA gene restriction pattern analysis could be determined for novel B. thuringiensis strains, allowing them not only to be grouped but also to be positioned on the phylogenetic tree.     

Seasonal and spatial variations in microbial community structure and diversity in the acid stream draining across an ongoing surface mining site

This study examined the microbial community in an acidic stream draining across the Yun-Fu pyrite mine (Guangdong, China), where extremely acidic mine water is a persistent feature due to the intensive surface mining activities. Analysis of terminal restriction fragment length polymorphism (TRFLP) of 16S rRNA gene sequences showed that microbial populations varied spatially and seasonally and correlated with geochemical and physical conditions. After the stream moves from underground to the surface, the microbial community in the acidic water rapidly evolves into a distinct community close to that in the downstream storage pond. Comparisons of TRFLP peaks with sequenced clone libraries indicated that bacteria related to the recently isolated iron-oxidizer Ferrovum myxofaciens dominated the acidophilic community throughout the year except for the samples collected in spring from the storage pond, where Ferroplasma acidiphilum -like archaea represented the most abundant group. Acidithiobacillus ferrooxidans -affiliated organisms increased along the acid stream and remained common over the year, whereas Leptospirillum ferrooxidans -like bacteria were negligible or even not detected in the analyzed samples. The data indicate that changes in environmental conditions are accompanied by significant shifts in community structure of the prokaryotic assemblages at this opencast mining site.     

Archaeal diversity in naturally occurring and impacted environments from a tropical region

AIMS: To evaluate archaeal diversity in natural and impacted habitats from Rio de Janeiro state, Brazil, a tropical region of South America. METHODS AND RESULTS: 16S rRNA gene was amplified directly by polymerase chain reaction (PCR) from genomic DNA, extracted from Guanabara Bay (GB) water, halomarine sediment (HS), municipal landfill leachate, agricultural soil and wastewater treatment (WT) system. Five archaeal 16S rDNA clone libraries were constructed. A total of 123 clones, within the five libraries analysed, were clustered into 29 operational taxonomic units, related to cultivated (24%) and uncultivated (76%) organisms. Rarefaction analysis showed that the libraries contained different levels of diversity. PCR-denaturing gradient gel electrophoresis (DGGE) of 16S-23S intergenic spacer regions confirmed the presence of a dominant phylotype, revealed by the WT system clone library. CONCLUSIONS: Archaeal communities of impacted environments seem to be confined to specific ecosystems with similar physicochemical properties, while communities from natural environments appear to be widely distributed. The presence of a high number of phylotypes related to uncultivated organisms suggests new archaeal lineages. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports, for the first time, the analysis of archaeal diversity in tropical environments from Brazil, and adds sequences from this region to the developing database of 16S rRNA clone libraries from environmental samples.     

Picoeukaryotic Plankton Diversity at the Helgoland Time Series Site as Assessed by Three Molecular Methods

We analyzed picoeukaryote assemblages in the German Bight at the Helgoland time series site by sequencing cloned eukaryotic 18S rRNA genes in six genetic libraries plus one library from the Orkney Islands from a cruise of opportunity. The libraries were constructed from environmental samples collected at different periods of the year. The same samples were also analyzed using a fingerprinting technique, single-strand conformational polymorphism (SSCP), and DNA microarrays with class-level oligonucleotide probes. One hundred unique clones were analyzed from each library, thus insuring over 85% coverage of the library. The V4 region of the 18S rRNA gene was sequenced from each of these clones, thus providing the most discrimination among the clones. The nonphotosynthetic picoeukaryotic component dominated over the photosynthetic one and was represented by the ciliates at 45% and group II alveolates at 42%. Prasinophytes dominated the photosynthetic group at 40%, but other picoplankton groups, such as bolidomonads and chrysophytes, were also present. Totally novel groups were found in the cryptomonads and in the dinoflagellates. A new algal group sister to the cryptophyte nuclear gene and the glaucocystophytes was also found. These three groups have been found in other picoeukaryotic planktonic clone libraries. SSCP analyses at closer time intervals suggest that clone libraries should be made at weekly intervals if succession in the picoeukaryotic plankton community is to be monitored accurately. A comparison of annual samples suggests thatthere appears to be an annual cycle with regard to species composition. Microarray analysis supported the clone library data and offered a faster means of community analysis, which can be performed with similar accuracy and with higher throughput for a more in-depth analysis.     

Molecular typing of isolates of the fish pathogen, Flavobacterium columnare, by single-strand conformation polymorphism analysis

Flavobacterium columnare intraspecies diversity was revealed by analyzing the 16S rRNA gene and the 16S-23S internal spacer region (ISR). Standard restriction fragment length polymorphism (RFLP) of these sequences was compared with single-strand conformation polymorphism (SSCP). Diversity indexes showed that both 16S-SSCP and ISR-SSCP improved resolution (D>or=0.9) when compared with standard RFLP. ISR-SSCP offered a simpler banding pattern than 16S-SSCP while providing high discrimination between isolates. SSCP analysis of rRNA genes proved to be a simple, rapid, and cost-effective method for routine fingerprinting of F. columnare.     

Archaeal Microbial Diversity of Hypersaline Lake Acıgöl,Denizli, Turkey

Archaeal diversity in Lake Ac?göl, a closed-basin, alkaline, hypersaline lake located at the northern edge of western Tourides in southwest Anatolia, was investigated using culture-independent methods. Microbial mat samples were collected from six different points. Archaeal 16S rRNA gene libraries were generated using domain specific oligonucleotide primers, and 16S rRNA gene sequences of clone libraries were analyzed phylogenetically. Denaturing gradient gel electrophoresis of 16S rRNA genes showed a variance in diversity with spatial differences. Archaeal diversity of Ac?göl is dominated by the members of family Halobacteriaceae which requires both high salt concentration and high pH for growth. Sequence analysis of archaeal 16s rRNA genes indicates the presence of the phylotypes affiliated with the genera Halorubrum, Halosimplex, Halorhabdus, Haloterrigena and Natronococcus in the analyzed samples.     

Phylogenetic analysis of Bacillus thuringiensis serovars based on 16S rRNA gene restriction fragment length polymorphisms

K.-B. JOUNG AND J.-C. CÔTÉ. 2001 .
Aims: To determine the 16S rRNA gene fingerprints of Bacillus thuringiensis strains to reveal phylogenetic relationships among them.
Methods and Results: Using 16S rRNA gene restriction fragment length polymorphisms generated by Hin dIII and Eco RI, 86 Bacillus thuringiensis strains were classified. This includes 80 B. thuringiensi s serovars and five more strains, kurstaki HD-1, subtoxicus , dendrolimus , tenebrionis and sandiego , to assess not only interserovar DNA relatedness but also intraserovar DNA relatedness, and the non-motile strain, hence non-serotypeable, B. thuringiensis var. wuhanensis . All 86 B. thuringiensis strains tested showed distinct ribotypes. The dendrogram resulting from the numerical analysis of the distance matrix shows four distinct phylogenetic groups and two ungrouped serovars, finitimus and bolivia , at the 92·5% DNA relatedness rate.
Conclusions: 16S rRNA gene fingerprinting cannot only be used for the classification of B. thuringiensis strains amenable or not to serotyping, but can also reveal phylogenetic relationships between strains.
Significance and Impact of the Study: In future screening programmes, 16S rRNA gene restriction pattern analysis could be determined for novel B. thuringiensis strains, allowing them not only to be grouped but also to be positioned on the phylogenetic tree.     

The use of molecular techniques based on ribosomal RNA and DNA for rumen microbial ecosystem studies: a review

This paper analyses the research progress in the use of molecular techniques based on ribosomal RNA and DNA (rRNA/rDNA) for rumen microbial ecosystem since first literature by Stahl et al. (1988). Because rumen microbial populations could be under-estimated by adopting the traditional techniques such as roll-tube technique or most-probable-number estimates, modern molecular techniques based on 16S/18S rRNA/rDNA can be used to more accurately provide molecular characterization, microbe populations and classification scheme than traditional methods. Phylogenetic-group-specific probes can be used to hybridize samples for detecting and quantifying of rumen microbes. But, competitive-PCR and real-time PCR can more sensitively quantify rumen microbes than hybridization. Molecular fingerprinting techniques including both denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE) and restriction fragment length polymorphisms (RFLP) can used to explore diversity of bacteria, protozoa and fungi in the rumen ecosystem. By constructing clone libraries of 16S/18S rRNA/rDNA of rumen microbes, more new microbes can be discovered and identified. For fungi, internal transcribed spacers (ITS) of fungi are better than 18S rRNA/rDNA for discriminating operational taxonomic units. In conclusion, 16S/18S rRNA/rDNA procedures have been used with success in rumen microbes and are quickly gaining acceptance for studying rumen microbial ecosystem, and will become useful methods for rumen ecology research. However, molecular techniques based on 16S/18S rRNA/rDNA don't preclude classical and traditional microbiological techniques. It should used together to acquire accurate and satisfactory results.     

Spatial heterogeneity of crenarchaeal assemblages within mesophilic soil ecosystems as revealed by PCR-single-stranded conformation polymorphism profiling

Microbial ecologists have discovered novel rRNA genes (rDNA) in mesophilic soil habitats worldwide, including sequences that affiliate phylogenetically within the division Crenarchaeota (domain Archaea). To characterize the spatial distribution of crenarchaeal assemblages in mesophilic soil habitats, we profiled amplified crenarchaeal 16S rDNA sequences from diverse soil ecosystems by using PCR-single-stranded-conformation polymorphism (PCR-SSCP) analysis. PCR-SSCP profiles provide a measure of relative microbial diversity in terms of richness (number of different phylotypes as estimated from the number of unique PCR-SSCP peaks) and evenness (abundance of each phylotype as estimated from the relative area under a peak). Crenarchaeal assemblages inhabiting prairie, forest, turf, and agricultural soils were characterized at six sampling locations in southern and central Wisconsin. Phylotype richness was found to be more stable than evenness among triplicate samples collected within 30 cm at each sampling location. Transformation of the PCR-SSCP data by principal-component analysis, followed by statistical testing (analysis of variance [P < 0.0001] and least-significant-difference analysis [alpha = 0.5]), supported the conclusion that each location exhibited a unique profile. To further characterize the spatial distribution of crenarchaeal assemblages at one location, additional soil samples (a total of 30) were collected from agricultural field plots at the Hancock Agricultural Research Station. PCR-SSCP revealed a patchy spatial distribution of crenarchaeal assemblages within and between these plots. This mosaic of crenarchaeal assemblages was characterized by differences in phylotype evenness that could not be correlated with horizontal distance (15 to 30 m) or with depth (0 to 20 cm below the surface). Crenarchaeal 16S rDNA clone libraries were produced and screened for unique SSCP peaks. Clones representing the dominant phylotypes at each location were identified, sequenced, and found to group phylogenetically with sequences in crenarchaeal clade C1b.     

Identification of unique type II polyketide synthase genes in soil

Many bacteria, particularly actinomycetes, are known to produce secondary metabolites synthesized by polyketide synthases (PKS). Bacterial polyketides are a particularly rich source of bioactive molecules, many of which are of potential pharmaceutical relevance. To directly access PKS gene diversity from soil, we developed degenerate PCR primers for actinomycete type II KS(alpha) (ketosynthase) genes. Twenty-one soil samples were collected from diverse sources in New Jersey, and their bacterial communities were compared by terminal restriction fragment length polymorphism (TRFLP) analysis of PCR products generated using bacterial 16S rRNA gene primers (27F and 1525R) as well as an actinomycete-specific forward primer. The distribution of actinomycetes was highly variable but correlated with the overall bacterial species composition as determined by TRFLP. Two samples were identified to contain a particularly rich and unique actinomycete community based on their TRFLP patterns. The same samples also contained the greatest diversity of KS(alpha) genes as determined by TRFLP analysis of KS(alpha) PCR products. KS(alpha) PCR products from these and three additional samples with interesting TRFLP pattern were cloned, and seven novel clades of KS(alpha) genes were identified. Greatest sequence diversity was observed in a sample containing a moderate number of peaks in its KS(alpha) TRFLP. The nucleotide sequences were between 74 and 81% identical to known sequences in GenBank. One cluster of sequences was most similar to the KS(alpha) involved in ardacin (glycopeptide antibiotic) production by Kibdelosporangium aridum. The remaining sequences showed greatest similarity to the KS(alpha) genes in pathways producing the angucycline-derived antibiotics simocyclinone, pradimicin, and jasomycin.     

Environmental Factors Affect Acidobacterial Communities below the Subgroup Level in Grassland and Forest Soils

In soil, Acidobacteria constitute on average 20% of all bacteria, are highly diverse, and are physiologically active in situ. However, their individual functions and interactions with higher taxa in soil are still unknown. Here, potential effects of land use, soil properties, plant diversity, and soil nanofauna on acidobacterial community composition were studied by cultivation-independent methods in grassland and forest soils from three different regions in Germany. The analysis of 16S rRNA gene clone libraries representing all studied soils revealed that grassland soils were dominated by subgroup Gp6 and forest soils by subgroup Gp1 Acidobacteria. The analysis of a large number of sites (n = 57) by 16S rRNA gene fingerprinting methods (terminal restriction fragment length polymorphism [T-RFLP] and denaturing gradient gel electrophoresis [DGGE]) showed that Acidobacteria diversities differed between grassland and forest soils but also among the three different regions. Edaphic properties, such as pH, organic carbon, total nitrogen, C/N ratio, phosphorus, nitrate, ammonium, soil moisture, soil temperature, and soil respiration, had an impact on community composition as assessed by fingerprinting. However, interrelations with environmental parameters among subgroup terminal restriction fragments (T-RFs) differed significantly, e.g., different Gp1 T-RFs correlated positively or negatively with nitrogen content. Novel significant correlations of Acidobacteria subpopulations (i.e., individual populations within subgroups) with soil nanofauna and vascular plant diversity were revealed only by analysis of clone sequences. Thus, for detecting novel interrelations of environmental parameters with Acidobacteria, individual populations within subgroups have to be considered.     

Comparative analysis of polychlorinated biphenyl-dechlorinating communities in enrichment cultures using three different molecular screening techniques

The catalysts for many microbially mediated environmental processes such as the dechlorination of polychlorinated biphenyls (PCBs) have been difficult to identify by traditional isolation techniques. Numerous, as yet unsuccessful, attempts have been made to isolate and culture the dechlorinating species. To overcome this limitation, amplified rDNA restriction analysis (ARDRA) of a clone library, denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (TRFLP) were used concurrently to compare their effectiveness for characterizing an enriched microbial community. These methods were applied to enrichment cultures that selectively dechlorinated double-flanked chlorines in the PCB congener 2,3,4,5 chlorinated biphenyl. The methods have different biases, which were apparent from discrepancies in the relative clone frequencies (ARDRA), band intensities (DGGE) or peak heights (TRFLP) from the same enrichment culture. However, each method was effectively qualitative and identified the same organisms: a low G + C Gram-positive eubacterium, an organism most similar to the green non-sulphur bacteria, an Aminobacterium sp. and a Desulfovibrio sp. Overall, in community fingerprinting and preliminary identification, DGGE proved to be the most rapid and effective tool for the monitoring of microorganisms within a highly enriched culture. TRFLP results corroborated DGGE fingerprint analysis; however, identification required the additional step of creating a clone library. ARDRA provided an in-depth analysis of the community and this technique detected slight intraspecies sequence variation in 16S rDNA. These molecular methods are common in environmental microbiology, but rarely are they compared with the same sample site or culture. In general, all three methods detected similar community profiles, but inherent biases resulted in different detection limits for individual OTUs (operational taxonomic units).     

Effect of the Earthworms Lumbricus terrestris and Aporrectodea caliginosa on Bacterial Diversity in Soil

Earthworms ingest large amounts of soil and have the potential to radically alter the biomass, activity, and structure of the soil microbial community. In this study, the diversity of eight bacterial groups from fresh soil, gut, and casts of the earthworms Lumbricus terrestris and Aporrectodea caliginosa were studied by single-strand conformation polymorphism (SSCP) analysis using both newly designed 16S rRNA gene-specific primer sets targeting Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Bacteroidetes, Verrucomicrobia, Planctomycetes, and Firmicutes and a conventional universal primer set for SSCP, with RNA and DNA as templates. In parallel, the study of the relative abundance of these taxonomic groups in the same samples was performed using fluorescence in situ hybridization. Bacteroidetes, Alphaproteobacteria, and Betaproteobacteria were predominant in communities from the soil and worm cast samples. Representatives of classes Flavobacteria and Sphingobacteria (Bacteroidetes) and Pseudomonas spp. (low-abundant Gammaproteobacteria) were detected in soil and worm cast samples with conventional and taxon-targeting SSCP and through the sequence analysis of 16S rRNA clone libraries. Physiologically active unclassified Sphingomonadaceae (Alphaproteobacteria) and Alcaligenes spp. (Betaproteobacteria) also maintained their diversities during transit through the earthworm intestine and were found on taxon-targeting SSCP profiles from the soil and worm cast samples. In conclusion, our results suggest that some specific bacterial taxonomic groups maintain their diversity and even increase their relative numbers during transit through the gastrointestinal tract of earthworms.     

Dispersal limitation and the assembly of soil Actinobacteria communities in a long-term chronosequence

It is uncertain whether the same ecological forces that structure plant and animal communities also shape microbial communities, especially those residing in soil. We sought to uncover the relative importance of present-day environmental characteristics, climatic variation, and historical contingencies in shaping soil actinobacterial communities in a long-term chronosequence. Actinobacteria communities were characterized in surface soil samples from four replicate forest stands with nearly identical edaphic and ecological properties, which range from 9500 to 14,000 years following glacial retreat in Michigan. Terminal restriction fragment length polymorphism (TRFLP) profiles and clone libraries of the actinobacterial 16S rRNA gene were constructed in each site for phenetic and phylogenetic analysis to determine whether dispersal limitation occurred following glacial retreat, or if community composition was determined by environmental heterogeneity. At every level of examination, actinobacterial community composition most closely correlated with distance, a surrogate for time, than with biogeochemical, plant community, or climatic characteristics. Despite correlation with leaf litter C:N and annual temperature, the significant and consistent relationship of biological communities with time since glacial retreat provides evidence that dispersal limitation is an ecological force structuring actinobacterial communities in soil over long periods of time.     

Spatial Heterogeneity of Crenarchaeal Assemblages within Mesophilic Soil Ecosystems as Revealed by PCR-Single-Stranded Conformation Polymorphism Profiling

Microbial ecologists have discovered novel rRNA genes (rDNA) in mesophilic soil habitats worldwide, including sequences that affiliate phylogenetically within the division Crenarchaeota (domain Archaea). To characterize the spatial distribution of crenarchaeal assemblages in mesophilic soil habitats, we profiled amplified crenarchaeal 16S rDNA sequences from diverse soil ecosystems by using PCR-single-stranded-conformation polymorphism (PCR-SSCP) analysis. PCR-SSCP profiles provide a measure of relative microbial diversity in terms of richness (number of different phylotypes as estimated from the number of unique PCR-SSCP peaks) and evenness (abundance of each phylotype as estimated from the relative area under a peak). Crenarchaeal assemblages inhabiting prairie, forest, turf, and agricultural soils were characterized at six sampling locations in southern and central Wisconsin. Phylotype richness was found to be more stable than evenness among triplicate samples collected within 30 cm at each sampling location. Transformation of the PCR-SSCP data by principal-component analysis, followed by statistical testing (analysis of variance [P < 0.0001] and least-significant-difference analysis [α = 0.5]), supported the conclusion that each location exhibited a unique profile. To further characterize the spatial distribution of crenarchaeal assemblages at one location, additional soil samples (a total of 30) were collected from agricultural field plots at the Hancock Agricultural Research Station. PCR-SSCP revealed a patchy spatial distribution of crenarchaeal assemblages within and between these plots. This mosaic of crenarchaeal assemblages was characterized by differences in phylotype evenness that could not be correlated with horizontal distance (15 to 30 m) or with depth (0 to 20 cm below the surface). Crenarchaeal 16S rDNA clone libraries were produced and screened for unique SSCP peaks. Clones representing the dominant phylotypes at each location were identified, sequenced, and found to group phylogenetically with sequences in crenarchaeal clade C1b.     

Heterogeneity of surface attached microbial communities from Sydney Harbour, Australia

There is limited information on bacterial communities attached to marine surfaces. These surface attached bacterial communities can vary at a micro scale and these differences may be due to surface characteristics in marine environments. The current study investigates the heterogeneity of bacterial communities on five different marine invertebrates (Heliocidaris erythrogramma, Austrocochlea concamerata, Crassostrea gigas, Dendrilla rosea, and Actinia tenebrosa), the alga, Lobophora variegata and marine gravel from a 20 m × 20 m quadrant in Camp Cove, Sydney Harbour, Australia. Terminal restriction fragment length polymorphism (TRFLP) of 16S sequences showed that each surface contained unique combinations of TRFLP fragment lengths. Phylogenetic analysis of random clones picked from clone libraries constructed from the amplified 16S sequences revealed that 16S sequences from the communities on different surfaces clustered into distinct clades. None of the bacteria identified by 16S sequencing of the whole (uncultured) microbial communities was detected after cultivation. Overall, the study shows surface type plays a major role in shaping microbial communities in marine environments.     

Detection of exon polymorphisms in the human lactoferrin gene.

We previously demonstrated that lactoferrin gene polymorphisms occur in cancer cells of patients with leukemia and breast cancer. In this study, we established a non-radioactive polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis, one of the most sensitive and simplest methods to detect polymorphisms and mutations of the human lactoferrin gene. We optimized the PCR conditions for nine different DNA templates and 16 pairs of exon primers for SSCP analysis. The DNA templates used in the analyses were prepared from a cosmid clone (CT6-1) that contains the human lactoferrin gene, human placental tissue, leukocytes from 10 normal volunteers, leukemic cells of two patients, and previously established three breast and two leukemic cell lines. With the appropriate exon-primer sets, PCR products from exon I to exon 16 of the lactoferrin gene were generated from the DNA templates and analyzed by SSCP. Compared with the homogenous cloned DNA, lactoferrin gene polymorphisms were detected within exons 2, 5, 7, 9, 13, 14, and 15 of the normal placental and leukocyte DNA. In addition, abnormal migration patterns of the lactoferrin gene in cancer cells were detected in exons 4, 5, 13, 14, and 15. The PCR-SSCP band migration patterns can be attributed either to gene polymorphism in normal cells or to DNA mutations in cancer cells and the employed method cannot distinguish between them. Nonetheless, the present analysis suggests that genetic polymorphisms of the lactoferrin gene exist in selected exons and additional mutations of the lactoferrin gene do occur in the cancer cells.     

Microbial biodiversity of thermophilic communities in hot mineral soils of Tramway Ridge, Mount Erebus, Antarctica

Tramway Ridge, located near the summit of Mount Erebus in Antarctica, is probably the most remote geothermal soil habitat on Earth. Steam fumaroles maintain moist, hot soil environments creating extreme local physicochemical differentials. In this study a culture-independent approach combining automated rRNA intergenic spacer analysis (ARISA) and a 16S rRNA gene library was used to characterize soil microbial (Bacterial and Archaeal) diversity along intense physicochemical gradients. Statistical analysis of ARISA data showed a clear delineation between bacterial community structure at sites close to fumaroles and all other sites. Temperature and pH were identified as the primary drivers of this demarcation. A clone library constructed from a high-temperature site led to the identification of 18 novel bacterial operational taxonomic units (OTUs). All 16S rRNA gene sequences were deep branching and distantly (85–93%) related to other environmental clones. Five of the signatures branched with an unknown group between candidate division OP10 and Chloroflexi . Within this clade, sequence similarity was low, suggesting it contains several yet-to-be described bacterial groups. Five archaeal OTUs were obtained and exhibited high levels of sequence similarity (95–97%) with Crenarchaeota sourced from deep-subsurface environments on two distant continents. The novel bacterial assemblage coupled with the unique archaeal affinities reinvigorates the hypotheses that Tramway Ridge organisms are relics of archaic microbial lineages specifically adapted to survive in this harsh environment and that this site may provide a portal to the deep-subsurface biosphere.