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Summary • Seed dormancy and dormancy cycling are poorly understood at the molecular level, but are central to plant community development. This study focuses on the embryonic axes of deeply dormant seeds of Prunus avium (wild cherry). • Rapid amplification of cDNA ends (RACE), differential display and quantitative PCR were used to recover and monitor expression of cDNAs. • cDNAs similar to two dormancy-imposing genes were isolated; a serine/threonine protein-phosphatase 2C homologous to ABSCISIC ACID INSENSITIVE 1 and 2 (Pa-PP2C1), and the transcription factor ABSCISIC ACID INSENSITIVE 3 (Pa-ABI3). Two germination-associated cDNAs were recovered; aconitase (Pa-ACO1) and eukaryotic translation initiation factor 3 subunit 8 (Pa-eIF3 SUBUNIT 8). Cold-treatment reduced expression of Pa-PP2C1 and Pa-ABI3, consistent with roles in establishing primary dormancy; neither was induced by imposition of secondary dormancy. Expression of these genes was distinct from expression of Pa-ACO1 and Pa-eIF3-SUBUNIT 8. • Results were consistent with a role for ABI1/ABI2 and ABI3 homologues in primary dormancy of P. avium embryonic axes, but not secondary dormancy as control of germination appeared overridden by the tissues surrounding the embryo.

Soil is an important factor that contributes to the uniqueness of a wine produced by vines grown in specific conditions. Recent data shows that the composition, diversity and function of soil microbial communities may play important roles in determining wine quality and indirectly affect its economic value. Here, we evaluated the impact of environmental variables on the soil microbiomes of 22 Barossa Valley vineyard sites based on the 16S rRNA gene hypervariable region 4. In this study, we report that environmental heterogeneity (soil plant-available P content, elevation, rainfall, temperature, spacing between row and spacing between vine) caused more microbial dissimilarity than geographic distance. Vineyards located in cooler and wetter regions showed lower beta diversity and a higher ratio of dominant taxa. Differences in microbial community composition were significantly associated with differences in fruit traits and in wine chemical and metabolomic profiles, highlighting the potential influence of microbial communities on the phenotype of grapevines. Our results suggest that environmental factors affect wine terroir, and this may be mediated by changes in microbial structure, thus providing a basic understanding of how growing conditions affect interactions between plants and their soil microbiomes.

Aboriginal Australians represent one of the longest continuous cultural complexes known. Archaeological evidence indicates that Australia and New Guinea were initially settled approximately 50 thousand years ago (ka); however, little is known about the processes underlying the enormous linguistic and phenotypic diversity within Australia. Here we report 111 mitochondrial genomes (mitogenomes) from historical Aboriginal Australian hair samples, whose origins enable us to reconstruct Australian phylogeographic history before European settlement. Marked geographic patterns and deep splits across the major mitochondrial haplogroups imply that the settlement of Australia comprised a single, rapid migration along the east and west coasts that reached southern Australia by 49–45 ka. After continent-wide colonization, strong regional patterns developed and these have survived despite substantial climatic and cultural change during the late Pleistocene and Holocene epochs. Remarkably, we find evidence for the continuous presence of populations in discrete geographic areas dating back to around 50 ka, in agreement with the notable Aboriginal Australian cultural attachment to their country. Analysis of Aboriginal Australian mitochondrial genomes shows geographic patterns and deep splits across the major haplogroups that indicate a single, rapid migration along the coasts around 49–45 ka, followed by longstanding persistence in discrete geographic areas. Aboriginal Australians preserve one of the longest continuous cultural complexes known, with archaeological evidence dating initial settlement of the continent to around 50,000 years ago. Alan Cooper and colleagues have charted the subsequent progress of humanity in and around the continent in the form of 111 mitochondrial genomes from preserved hair samples. The results show that, from landfall in the north of Australia, people spread rapidly around the east and west coasts, meeting in southern Australia as early as 49,000 years ago. Strong regional patterns of mitochondrial DNA variation suggest that when people stopped moving they stayed put, putting down cultural roots that have weathered 50,000 years of significant cultural and climatic change.

A field-scale acetate amendment experiment was performed in a contaminated aquifer at Old Rifle, CO to stimulate in situ microbial reduction of U(VI) in groundwater. To evaluate the microorganisms responsible for microbial uranium reduction during the experiment, 13C-labeled acetate was introduced into well bores via bio-traps containing porous activated carbon beads (Bio-Sep). Incorporation of the 13C from labeled acetate into cellular DNA and phospholipid fatty acid (PLFA) biomarkers was analyzed in parallel with geochemical parameters. An enrichment of active sigma-proteobacteria was demonstrated in downgradient monitoring wells: Geobacter dominated in wells closer to the acetate injection gallery, while various sulfate reducers were prominent in different downgradient wells. These results were consistent with the geochemical evidence of Fe(III), U(VI), and SO(4)2- reduction. PLFA profiling of bio-traps suspended in the monitoring wells also showed the incorporation of 13C into bacterial cellular lipids. Community composition of downgradient monitoring wells based on quinone and PLFA profiling was in general agreement with the 13C-DNA result. The direct application of 13C label to biosystems, coupled with DNA and PLFA analysis

The composition of microbial communities on and within seeds may effect their storage and field performance, whether they are indigenous or applied as biocontrol agents. In this study, we have explored the usefulness of profiling small subunit ribosomal (SSR) gene fragments for studying the microflora associated with seeds. DNA was amplified by the polymerase chain reaction (PCR) and the amplicons separated using denaturing gradient gel electrophoresis (DGGE). Primers targeting eukaryotic SSRs were used to investigate fungal communities, and primers targeting bacterial SSRs were employed to study the eubacterial microflora. As a case study, we attempted to profile the fungi and bacteria associated with seeds of Beta vulgaris (sugar beet) to permit an insight into the varying field performance of several well-characterised commercial seed lots. Serious interference with the microbial signals was observed from the plant's own nuclear 18S rRNA genes and chloroplast 16S rRNA genes using standard PCR conditions and DNA extracted from whole seeds as template. Hot-start and touchdown PCR made no appreciable improvement to these signals. Seed imbibition and dissection into operculum and fruit wall and true seed prior to DNA extraction improved signal recovery in the fruit fraction. With primer modification, bacteria and fungi were detected in an excess of plant DNA of 100:1 and 10:1, respectively. With this method, microbial communities on seeds could be profiled, however, it is likely that targeted depletion of plant rDNA targets will be a necessary extra step before this approach can be used to screen seeds routinely.

A cDNA clone encoding a presumed full-length glycine-rich ribonucleic acid (RNA) binding protein was isolated from a lambda-ZAP Express cDNA library generated from primarily nondormant Prunus avium (wild cherry) embryonic axes. The cDNA, designated Pa-RRM-GRP1 (Prunus avium RNA recognition motif glycine-rich protein 1), contains a single N-terminal RNA recognition motif (RRM) and single C-terminal glycine-rich domain. The glycine-rich domain is unusually long at 91 amino acids, 58 of which are glycines. The 534-base pair (bp) open reading frame (ORF) of this clone encodes a 178-amino-acid polypeptide with a predicted molecular weight of 17.33 kDa and pI of 7.84. Comparative sequence alignment of Pa-RRM-GRP1 reveals extensive homology to known and presumed glycine-rich RNA binding proteins from angiosperms and gymnosperms. Genomic Southern blot analysis suggests that this gene exists as a single copy in P. avium. Expression of this gene in P. avium embryonic axes during low-temperature dormancy-breaking treatments was studied and found to be induced by cold (3 degrees C) using real-time PCR of total cDNA supported by Northern blot analysis of total RNA. Expression dropped during prolonged storage at 3 degrees C and was reduced to control levels by interruption of cold treatment by warming to 20 degrees C.

In the article 'Introducing BASE: the Biomes of Australian Soil Environments soil microbial diversity database GigaScience 2016 5:21' [1] the authorship list should have included Leon Court, who was responsible for sample collection and preparation, sampling design and sequencing method design. The authors regret this omission.

Although molecular techniques are considered to provide a more comprehensive view of species diversity of natural microbial populations, few studies have compared diversity assessed by molecular and cultivation-based approaches using the same samples. To achieve this, the diversity of natural populations of ammonia oxidising bacteria in arable soil and marine sediments was determined by analysis of 16S rDNA sequences from enrichment cultures, prepared using standard methods for this group, and from 16S rDNA cloned from DNA extracted directly from the same environmental samples. Soil and marine samples yielded 31 and 18 enrichment cultures, respectively, which were compared with 50 and 40 environmental clones. There was no evidence for selection for particular ammonia oxidizer clusters by different procedures employed for enrichment from soil samples, although no culture was obtained in medium at acid pH. In soil enrichment cultures, Nitrosospira cluster 3 sequences were most abundant, whereas clones were distributed more evenly between Nitrosospira clusters 2, 3, and 4. In marine samples, the majority of enrichment cultures contained Nitrosomonas, whereas Nitrosospira sequences were most abundant among environmental clones. Soil enrichments contained a higher proportion of identical sequences than clones, suggesting laboratory selection for particular strains, but the converse was found in marine samples. In addition, 16% of soil enrichment culture sequences were identical to those in environmental clones, but only 1 of 40 marine enrichments was found among clones, indicating poorer culturability of marine strains represented in the clone library, under the conditions employed. The study demonstrates significant differences in species composition assessed by molecular and culture-based approaches but indicates also that, employing only a limited range of cultivation conditions, 7% of the observed sequence diversity in clones of ammonia oxidizers from these environments could be obtained in laboratory enrichment culture. Further studies and experimental approaches are required to determine which approach provides better representation of the natural community.

The eutrophication of many ecosystems in recent decades has led to an increased interest in the ecology of nitrogen transformation. Chemolitho-autotrophic ammonia-oxidizing bacteria are responsible for the rate-limiting step of nitrification in a wide variety of environments, making them important in the global cycling of nitrogen. These organisms are unique in their ability to use the conversion of ammonia to nitrite as their sole energy source. Because of the importance of this functional group of bacteria, understanding of their ecology and physiology has become a subject of intense research over recent years. The monophyletic nature of these bacteria in terrestrial environments has facilitated molecular biological approaches in studying their ecology, and progress in this field has been rapid. The ammonia-oxidizing bacteria of the beta -subclass Proteobacteria have become somewhat of a model system within molecular microbial ecology, and this chapter reviews recent progress in our knowledge of their distribution, diversity, and ecology. [KEYWORDS: nitrification; ammonia oxidation; Nitrosospira; Nitrosomonas; beta-subclass Proteobacteria 16s ribosomal-rna; gradient gel-electrophoresis; in-situ hybridization; nitrifying activated-sludge; root-oxygenated sediments; nitrospira-like bacteria; fluidized-bed reactor; nitrosospira sp npav; acid forest soils; fresh-water lake]

The possibility of calculating useful microbial community diversity indices from environmental polar lipid fatty acid and 16S rDNA PCR-DGGE data was investigated. First, the behavior of the species richness, Shannon's, and Simpson's diversity indices were determined on polar lipid fatty acid profiles of 115 pure cultures, communities constructed from those profiles with different numbers of species, and constructed communities with different distributions of species. Differences in the species richness of these artificial communities was detected by all three diversity indices, but they were insensitive to the evenness of the distribution of species. Second, data from a field experiment with substrate addition to soil was used to compare the methods developed for lipid- and DNA-based diversity indices. Very good agreement was found between indices calculated from environmental polar lipid fatty acid profiles and denaturing gradient gel electrophoresis profiles from matched samples (Pearson's correlation coefficient r=0.95-0.96). A method for data pre-treatment for diversity calculations is described.

Changes in the community structure of chemolitho-autotrophic ammonia-oxidising bacteria of the beta-subgroup Proteobacteria were monitored during nutrient-impoverishment management of slightly acidic, peaty grassland soils, which decreased in pH with succession. Specific PCR, cloning and sequence analysis. denaturing gradient gel electrophoresis (DGGE) and probe hybridisation were used to analyse rDNA sequences directly recovered from successional soils. Four previously characterised ammonia oxidiser sequence clusters were recovered from each soil, three associated with the genus Nitrosospira and one with the genus Nitrosomonas. All samples were dominated by Nitrosospira-like sequences, Nitrosospira cluster 3 was the most commonly recovered ammonia oxidiser group in all fields, but a greater representation of Nitrosospira clusters 2 and 4 was observed in older fields. Most probable number (MPN) counts were conducted using neutral and slightly acid conditions. Neutral pH (7.5) MPN's suggested a decrease in ammonia oxidiser numbers in later successional fields, but this trend was not observed using slightly acid (pH 5.8) conditions. Analysis of terminal MPN dilutions revealed a distribution of sequence clusters similar to direct soil DNA extractions. However, an increased relative recovery of Nitrosospira cluster 2 was observed for acid pH MPNs compared to neutral pH MPNs from the most acidic soil tested, in agreement with current hypotheses on the relative acid tolerance of this group. [KEYWORDS: beta-subgroup proteobacteria; soil pH; nitrification; DGGE; most probable number (MPN); oligonucleotide hybridisation; Nitrosospira; Nitrosomonas 16s ribosomal-rna; gradient gel-electrophoresis; oxidizing bacteria; nitrosomonas-europaea; gene-sequences; populations; ph; nitrification; diversity; pcr]

Cesium, cadmium, cobalt, and strontium are four contaminants frequently found in soils at biotoxic levels. Introduction of certain nongenetically modified bacteria has been frequently suggested as a method for the immobilization of heavy metal contaminants in soil, thereby reducing runoff and bioavailability. In this study, the authors have used the polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) to track the survival of the five bacterial species added to soil microcosms with and without the addition of a mixture of these metals. The PCR primers targeted conserved regions of the 165 rDNA molecular present in all bacteria. The reaction products were shown to reflect the relative abundance of the bacteria both in mixtures of pure cultures and against a background of all the eubacterial species present in the soil following inoculation. Three of the species (Pseudomonas aeruginosa FRD-1, Shewanella putrifaciens 200, and Desulfovibrio vulgaris Hildenborough) decreased rapidly following inoculation into both soils. The proportion of Alcaligenes eutrophus CH34 remained at a constant level throughout the 8-week experiment in both soil treatments. Sphingomonas aromaticivorans B0695 showed toxic metal-dependent survival in that its relative abundance dropped rapidly in pristine soil but remained at approximately inoculation levels throughout the experiment inmore » contaminated microcosms.« less

Recent advances in understanding the role and application of bacteria to the remediation of toxic metal and radionuclide contaminated terrestrial environments have come from several avenues. Novel species capable of mobilization and immobilization of metal ions have been discovered. Remediation of toxicity has been accelerated by nutrient amendment, the use of chelating agents and novel methods for phosphate amendment. Major advances in the use of natural and genetically engineered species for bioprotection and remediation of organic co-contaminants have been reported. Construction of wetland function continues to be developed for containment and decontamination of wastewaters.

The impact of pollution on soil microbial communities and subsequent bioremediation can be measured quantitatively in situ using direct, non-culture- dependent techniques. Such techniques have advantages over culture-based methods, which often account for less than 1% of the extant microbial community. In 1988, a JP-4 fuel spill contaminated the glacio-fluvial aquifer at Wurtsmith Air Force Base, Michigan, USA. In this study, lipid biomarker characterization of the bacterial and eukaryotic communities was combined with polymerase chain reaction– denaturing gradient gel electrophoresis (PCR–DGGE) analysis of the eubacterial community to evaluate correlation between contaminant (JP-4 fuel) concentration and community structure shifts. Vadose, capillary fringe and saturated zone samples were taken from cores within and up- and down-gradient from the contaminant plume. Lipid biomarker analysis indicated that samples from within the plume contained increased biomass, with large proportions of typically Gram-negative bacteria. Outside the plume, lipid profiles indicated low-biomass microbial communities compared with those within the initial spill site. 16S rDNA sequences derived from DGGE profiles from within the initial spill site suggested dominance of the eubacterial community by a limited number of phylogenetically diverse organisms. Used in tandem with pollutant quantification, these molecular techniques should facilitate significant improvements over current assessment procedures for the determination of remediation end-points.

Although the practice of composting animal wastes for use as biofertilizers has increased in recent years, little is known about the microorganisms responsible for the nitrogen transformations which occur in compost and during the composting process. Ammonia is the principle available nitrogenous compound in composting material, and the conversion of this compound to nitrite in the environment by chemolithotrophic ammonia-oxidizing bacteria is an essential step in nitrogen cycling. Therefore, the distribution of ammonia-oxidizing members of the β subdivision of the class Proteobacteria in a variety of composting materials was assessed by amplifying 16S ribosomal DNA (rDNA) and 16S rRNA by PCR and reverse transcriptase PCR (RT-PCR), respectively. The PCR and RT-PCR products were separated by denaturing gradient gel electrophoresis (DGGE) and were identified by hybridization with a hierarchical set of oligonucleotide probes designed to detect ammonia oxidizer-like sequence clusters in the genera Nitrosospira and Nitrosomonas . Ammonia oxidizer-like 16S rDNA was detected in almost all of the materials tested, including industrial and experimental composts, manure, and commercial biofertilizers. A comparison of the DGGE and hybridization results after specific PCR and RT-PCR suggested that not all of the different ammonia oxidizer groups detected in compost are equally active. amoA , the gene encoding the active-site-containing subunit of ammonia monooxygenase, was also targeted by PCR, and template concentrations were estimated by competitive PCR. Detection of ammonia-oxidizing bacteria in the composts tested suggested that such materials may not be biologically inert with respect to nitrification and that the fate of nitrogen during composting and compost storage may be affected by the presence of these organisms.

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 β-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.

Contamination of soils with toxic metals is a major problem on military, industrial, and mining sites worldwide. Of particular interest to the field of bioremediation is the selection of biological markers for the end point of remediation. In this microcosm study, we focus on the effect of addition of a mixture of toxic metals (cadmium, cobalt, cesium, and strontium as chlorides) to soil on the population structure and size of the ammonia oxidizers that are members of the beta subgroup of the Proteobacteria (β-subgroup ammonia oxidizers). In a parallel experiment, the soils were also treated by the addition of five strains of metal-resistant heterotrophic bacteria. Effects on nitrogen cycling were measured by monitoring the NH 3 and NH 4 + levels in soil samples. The gene encoding the α-subunit of ammonia monooxygenase ( amo A) was selected as a functional molecular marker for the β-subgroup ammonia oxidizing bacteria. Community structure comparisons were performed with clone libraries of PCR-amplified fragments of amo A recovered from contaminated and control microcosms for 8 weeks. Analysis was performed by restriction digestion and sequence comparison. The abundance of ammonia oxidizers in these microcosms was also monitored by competitive PCR. All amo A gene fragments recovered grouped with sequences derived from cultured Nitrosospira . These comprised four novel sequence clusters and a single unique clone. Specific changes in the community structure of β-subgroup ammonia oxidizers were associated with the addition of metals. These changes were not seen in the presence of the inoculated metal-resistant bacteria. Neither treatment significantly altered the total number of β-subgroup ammonia-oxidizing cells per gram of soil compared to untreated controls. Following an initial decrease in concentration, ammonia began to accumulate in metal-treated soils toward the end of the experiment.

The rhizosphere of oxygen-releasing wetland plants provides a niche for oxygen-consuming microorganisms such as chemolithotrophic ammonia-oxidising bacteria. These bacteria are adapted to oxygen limitation with respect to their affinity for oxygen, ability to survive periods of anoxia, and immediate response to the appearance of oxygen. In this study the techniques of specific amplification of ammonia oxidiser 16S rDNA fragments by PCR, separation of mixed PCR samples by denaturing gradient gel electrophoresis (DGGE), and band identification by specific hybridisation with oligonucleotide probes were combined to allow for the comparison of the community composition of multiple samples over space and time. DGGE bands of interest were also excised for DNA isolation, reamplification, sequence determination and phylogenetic analysis. We compared monthly samples from both the root zone and the bare sediment of a shallow lake inhabited by the emergent macrophyte Glyceria maxima to determine the seasonal effects that the plant roots and the oxygen availability might have on the beta-subgroup ammonia-oxidiser populations present. Similarly, five soil or sediment samples, varying in oxygen availability, from different locations in the Netherlands were compared. Although the presence of two previously defined Nitrosospira sequence clusters could be differentially detected in the samples examined, there was no evidence for a particular group which was specific to periodically anoxic environments. [KEYWORDS: Nitrosospira; Nitrosomonas; diversity; nitrification; biogeography; oxygen limitation 16s ribosomal-rna; gradient gel-electrophoresis; polymerase chain-reaction; oxidizing bacteria; nitrifying bacteria; gene-sequences; class proteobacteria; dna fragments; plants; nitrification]

A combination of denaturing gradient gel electrophoresis (DGGE) and oligonucleotide probing was used to investigate the influence of soil pH on the compositions of natural populations of autotrophic β-subgroup proteobacterial ammonia oxidizers. PCR primers specific to this group were used to amplify 16S ribosomal DNA (rDNA) from soils maintained for 36 years at a range of pH values, and PCR products were analyzed by DGGE. Genus- and cluster-specific probes were designed to bind to sequences within the region amplified by these primers. A sequence specific to all β-subgroup ammonia oxidizers could not be identified, but probes specific for Nitrosospira clusters 1 to 4 and Nitrosomonas clusters 6 and 7 (J. R. Stephen, A. E. McCaig, Z. Smith, J. I. Prosser, and T. M. Embley, Appl. Environ. Microbiol. 62:4147–4154, 1996) were designed. Elution profiles of probes against target sequences and closely related nontarget sequences indicated a requirement for high-stringency hybridization conditions to distinguish between different clusters. DGGE banding patterns suggested the presence of Nitrosomonas cluster 6a and Nitrosospira clusters 2, 3, and 4 in all soil plots, but results were ambiguous because of overlapping banding patterns. Unambiguous band identification of the same clusters was achieved by combined DGGE and probing of blots with the cluster-specific radiolabelled probes. The relative intensities of hybridization signals provided information on the apparent selection of different Nitrosospira genotypes in samples of soil of different pHs. The signal from the Nitrosospira cluster 3 probe decreased significantly, relative to an internal control probe, with decreasing soil pH in the range of 6.6 to 3.9, while Nitrosospira cluster 2 hybridization signals increased with increasing soil acidity. Signals from Nitrosospira cluster 4 were greatest at pH 5.5, decreasing at lower and higher values, while Nitrosomonas cluster 6a signals did not vary significantly with pH. These findings are in agreement with a previous molecular study (J. R. Stephen, A. E. McCaig, Z. Smith, J. I. Prosser, and T. M. Embley, Appl. Environ. Microbiol 62:4147–4154, 1996) of the same sites, which demonstrated the presence of the same four clusters of ammonia oxidizers and indicated that selection might be occurring for clusters 2 and 3 at acid and neutral pHs, respectively. The two studies used different sets of PCR primers for amplification of 16S rDNA sequences from soil, and the similar findings suggest that PCR bias was unlikely to be a significant factor. The present study demonstrates the value of DGGE and probing for rapid analysis of natural soil communities of β-subgroup proteobacterial ammonia oxidizers, indicates significant pH-associated differences in Nitrosospira populations, and suggests that Nitrosospira cluster 2 may be of significance for ammonia-oxidizing activity in acid soils.

We have conducted a preliminary phylogenetic survey of ammonia-oxidizing beta-proteobacteria, using 16S rRNA gene libraries prepared by selective PCR and DNA from acid and neutral soils and polluted and nonpolluted marine sediments. Enrichment cultures were established from samples and analyzed by PCR. Analysis of 111 partial sequences of c. 300 bases revealed that the environmental sequences formed seven clusters, four of which are novel, within the phylogenetic radiation defined by cultured autotrophic ammonia oxidizers. Longer sequences from 13 cluster representatives support their phylogenetic positions relative to cultured taxa. These data suggest that known taxa may not be representative of the ammonia-oxidizing beta-proteobacteria in our samples. Our data provide further evidence that molecular and culture-based enrichment methods can select for different community members. Most enrichments contained novel Nitrosomonas-like sequences whereas novel Nitrosospira-like sequences were more common from gene libraries of soils and marine sediments. This is the first evidence for the occurrence of Nitrosospira-like strains in marine samples. Clear differences between the sequences of soil and marine sediment libraries were detected. Comparison of 16S rRNA sequences from polluted and nonpolluted sediments provided no strong evidence that the community composition was determined by the degree of pollution. Soil clone sequences fell into four clusters, each containing sequences from acid and neutral soils in varying proportions. Our data suggest that some related strains may be present in both samples, but further work is needed to resolve whether there is selection due to pH for particular sequence types.

We have been studying the conjugative transposon Tn5397, originally isolated from the Gram-positive pathogen Clostridium difficile. Physical analysis of this transposon demonstrated that it contained a group II intron. This is the first report of an intron in a conjugative transposon and the first report of a group II intron in Gram-positive bacteria. The intron interrupted a gene in Tn5397 that is almost identical to orf14 from Tn916. DNA hybridisation analysis showed that elements related to Tn5397, containing the group II intron, were present in five other C. difficile strains from different geographical locations suggesting that the element is likely to be widely distributed.

The introduction of molecular techniques into environmental and forensic sciences has opened up an entirely new window of observation into our world's biology and its history. However, the very sensitive nature of such methods leaves them particularly prone to error and artifact. This is especially true in the analysis of problematic samples with suboptimal nucleic acid quantity or quality, as is often the case in microbial ecological studies and ancient DNA investigations. Numerous studies have highlighted the potential sources and extent of artifact in such molecular approaches, but such cautionary messages have generally gone unheeded. Some unavoidable sources of error and artifact are inherent to molecular studies and must simply be kept in mind when interpreting the robustness of results. Other causes of error can at least be partially addressed via good laboratory practice measures, proper controls and thorough data scrutiny. Some laboratories and disciplines have been keen to adopt appropriate measures to circumvent some of the validity issues involved in molecular studies, whereas others lag behind. Here, we identify sources of error, contamination and artifact in nucleic acid-based studies, especially as they apply to problematic material, suboptimal in sample size, source, quality or purity. In evaluating these concerns, we further offer some considerations and suggestions to help minimize sources of error and artifacts in laboratories studying molecular ecology and evolution with frequently problematic, and usually rare, sample materials.

PCR-independent methods PCR-Dependent Methods Keywords: polymerase chain reaction (PCR); cloning; denaturing gradient gel electrophoresis (DGGE); fingerprinting: community fingerprinting techniques; methodology; rybotyping methods

Microbially mediated reduction and immobilization of U(VI) to U(IV) plays a role in both natural attenuation and accelerated bioremediation of uranium-contaminated sites. To realize bioremediation potential and accurately predict natural attenuation, it is important to first understand the microbial diversity of such sites. In this paper, the distribution of sulfate-reducing bacteria (SRB) in contaminated groundwater associated with a uranium mill tailings disposal site at Shiprock, N.Mex., was investigated. Two culture-independent analyses were employed: sequencing of clone libraries of PCR-amplified dissimilatory sulfite reductase (DSR) gene fragments and phospholipid fatty acid (PLFA) biomarker analysis. A remarkable diversity among the DSR sequences was revealed, including sequences from δ-Proteobacteria, gram-positive organisms, and the Nitrospira division. PLFA analysis detected at least 52 different mid-chain-branched saturate PLFA and included a high proportion of 10me16:0. Desulfotomaculum and Desulfotomaculum -like sequences were the most dominant DSR genes detected. Those belonging to SRB within δ-Proteobacteria were mainly recovered from low-uranium (≤302 ppb) samples. One Desulfotomaculum -like sequence cluster overwhelmingly dominated high-U (>1,500 ppb) sites. Logistic regression showed a significant influence of uranium concentration over the dominance of this cluster of sequences ( P = 0.0001). This strong association indicates that Desulfotomaculum has remarkable tolerance and adaptation to high levels of uranium and suggests the organism's possible involvement in natural attenuation of uranium. The in situ activity level of Desulfotomaculum in uranium-contaminated environments and its comparison to the activities of other SRB and other functional groups should be an important area for future research.

A defined template mixture of seven closely related 16S-rDNA clones was used in a PCR-cloning experiment to assess and track sources of artifactual sequence variation in 16S rDNA clone libraries. At least 14% of the recovered clones contained aberrations. Artifact sources were polymerase errors, a mutational hot spot, and cloning of heteroduplexes and chimeras. These data may partially explain the high degree of microheterogeneity typical of sequence clusters detected in environmental clone libraries. [KEYWORDS: Ribosomal-rna genes; polymerase chain-reaction; chimeric molecules; dna amplification; escherichia-coli heteroduplexes; populations; coamplification; consequence; diversity]

The community structure of ammonia-oxidizing bacteria of the beta -subclass Proteobacteria was investigated with respect to environmental gradients along the Schelde, a eutrophic estuary system. A dominance of Nitrosomonas-Like sequences was detected using molecular techniques targeting the 16S rRNA gene on 3 separate sampling dates, and different Nitrosomonas-like populations were most dominant at different locations along the estuary. The most frequently detected ammonia oxidizer-like sequences in the freshwater part of the estuary were associated with a sequence cluster previously designated as Nitrosomonas Cluster 6a. This group, which is closely affiliated with the cultured species N, ureae, has previously been detected as the dominant ammonia-oxidizer group in various freshwater systems, and was also the dominant recovered sequence cluster from a contributory, untreated sewage effluent sample. The 16S rDNA recovered from brackish locations further downstream was dominated by a group of novel Nitrosomonas-like sequences. Nitrosospira-like sequences represented only a small minority of those detected for all samples. The shift in dominant ammonia-oxidizer populations occurred in the estuarine region with the sharpest observed gradients in salinity, oxygen, and ammonia. These results provide evidence in support of the differential selection of physiologically distinct Nitrosomonas-like groups according to the environmental gradients encountered along the estuary. [KEYWORDS: ammonia-oxidizing bacteria; estuaries; DGGE; Nitrosomonas Gradient gel-electrophoresis; 16s ribosomal-rna; root-oxygenated sediments; fresh-water lake; nitrifying bacteria; community structure; dna fragments; in-situ; nitrification; diversity]

In this study, we investigated the size and structure of autotrophic ammonia oxidizer (AAO) communities in the groundwater of a contamination plume originating from a mill-tailings disposal site. The site has high levels of dissolved N from anthropogenic sources, and exhibited wide variations in the concentrations of NO3-and NH3+ NH4+. Community structures were examined by PCR-DGGE targeting 16S rDNA with band excision and sequence analysis, and by analysis of amoA fragment clone libraries. AAO population sizes were estimated by competitive PCR targeting the gene amoA, and correlated significantly with nitrate concentration. Most samples revealed novel diversity in AAO 16S rDNA and amoA gene sequences. Both 16S rDNA and amoA analyses suggested that all samples were dominated by Nitrosomonas sp., Nitrosospira sp. being detected in only 3 of 15 samples. This study indicated numerical dominance of Nitrosomonas over Nitrosospira in groundwater, and suggests that groundwater ammonia oxidizers are more similar to those dominating freshwater sediments than bulk soil.Key words: Nitrosomonas, Nitrosospira, nitrification, groundwater, amoA, 16S.

Aerobically grown enrichment cultures derived from hydrocarbon- contaminated seawater and freshwater sediments were generated by growth on crude oil as sole carbon source. Both cultures displayed a high rate of degradation for a wide range of hydrocarbon compounds. The bacterial species composition of these cultures was investigated by PCR of the 16S rDNA gene using multiple primer combinations. Near full-length 16S rDNA clone libraries were generated and screened by restriction analysis prior to sequence analysis. Polymerase chain reaction- denaturing gradient gel electrophoresis (PCR-DGGE) was carried out using two other PCR primer sets targeting either the V3 or V6-V8 regions, and sequences derived from prominent DGGE bands were compared to sequences obtained via cloning. All data sets suggested that the seawater culture was dominated by alpha- subgroup proteobacteria, whereas the freshwater culture was dominated by members of the beta- and gamma-proteobacteria. However, the V6-V8 primer pair was deficient in the recovery of Sphingomonas-like 16S rDNA due to a 3' terminal mismatch with the reverse primer. Most 16S rDNA sequences recovered from the marine enrichment were not closely related to genera containing known oil-degrading organisms, although some were detected. All methods suggested that the freshwater enrichment was dominated by genera containing known hydrocarbon-degrading species. [KEYWORDS: enrichment cultures; DGGE; hydrocarbon degradation; polymerase chain reaction; phylogenetic analysis; 16S rDNA 16s ribosomal-rna; gradient gel-electrophoresis; degrading bacteria; microbial-populations; genus sphingomonas; activated-sludge; ecology; diversity; database; probes]

The community structure of beta-subclass Proteobacteria ammonia-oxidizing bacteria was determined in semi-natural chalk grassland soils at different stages of secondary succession. Both culture-mediated (most probable number; MPN) and direct nucleic acid-based approaches targeting genes encoding 16S rRNA and the AmoA subunit of ammonia monooxygenase were used. Similar shifts were detected in the composition of the ammonia oxidizer communities by both culture-dependent and independent approaches. A predominance of Nitrosospira sequence cluster 3 in early successional fields was replaced by Nitrosospira sequence cluster 4 in late successional fields. The rate of this shift differed between the two areas examined. This shift occurred in a background of relative stability in the dominant bacterial populations in the soil, as determined by domain- level polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Molecular analysis of enrichment cultures obtained using different ammonia concentrations revealed biases towards Nitrosospira sequence cluster 3 or Nitrosospira sequence cluster 4 under high- or low-ammonia conditions respectively. High-ammonia MPNs suggested a decease in ammonia oxidizer numbers with succession, but low-ammonia MPNs and competitive PCR targeting amoA failed to support such a trend. Ammonia turnover rate, not specific changes in plant diversity and species composition, is implicated as the major determinant of ammonia oxidizer community structure in successional chalk grassland soils. [KEYWORDS: 16s ribosomal-rna; gradient gel-electrophoresis; nitrosomonas-europaea; nitrifying bacteria; species composition; gene-sequences; dna fragments; populations; nitrification; soil]

Three crude oil bioremediation techniques were applied in a randomized block field experiment simulating a coastal oil spill. Four treatments (no oil control, oil alone, oil plus nutrients, and oil plus nutrients plus an indigenous inoculum) were applied. In situ microbial community structures were monitored by phospholipid fatty acid (PLFA) analysis and 16S rDNA PCR-denaturing gradient gel electrophoresis (DGGE) to (i) identify the bacterial community members responsible for the decontamination of the site and (ii) define an end point for the removal of the hydrocarbon substrate. The results of PLFA analysis demonstrated a community shift in all plots from primarily eukaryotic biomass to gram-negative bacterial biomass with time. PLFA profiles from the oiled plots suggested increased gram-negative biomass and adaptation to metabolic stress compared to unoiled controls. DGGE analysis of untreated control plots revealed a simple, dynamic dominant population structure throughout the experiment. This banding pattern disappeared in all oiled plots, indicating that the structure and diversity of the dominant bacterial community changed substantially. No consistent differences were detected between nutrient-amended and indigenous inoculum-treated plots, but both differed from the oil-only plots. Prominent bands were excised for sequence analysis and indicated that oil treatment encouraged the growth of gram-negative microorganisms within the α-proteobacteria and Flexibacter-Cytophaga-Bacteroides phylum. α-Proteobacteria were never detected in unoiled controls. PLFA analysis indicated that by week 14 the microbial community structures of the oiled plots were becoming similar to those of the unoiled controls from the same time point, but DGGE analysis suggested that major differences in the bacterial communities remained.

Contamination of soils with heavy metal ions is a major problem on industrial and defense-related sites worldwide. The bioavailability and mobility of these contaminants is partially determined by the microbial biomass present at these sites. In this study, we have assessed the effect of the addition of a mixture of toxic metal salts on the prokaryotic community of microcosms consisting of sandy-loam soil using direct molecular analysis of the recoverable eubacterial 16S rDNA molecules by polymerase chain reaction - denaturing gradient gel electrophoresis (PCR-DGGE) and limited phospholipid fatty acid analysis (PLFA). Addition of toxic metals (nonradioactive surrogates of Sr, Co, Cs, Cd) resulted in rapid (ca. 1 week) changes in the DGGE profile of the indigenous eubacterial community when compared with pristine controls. These changes were stable over the course of the experiment (8 weeks). No changes in the eubacterial population of control microcosms were detected. The major changes in community structure in metal-contaminated microcosms consisted of the appearance of four novel bands not detected in controls. Sequence analysis of these bands suggested that two organisms related to the genus Acinetobacter and two related to the genus Burkholderia carried a selective advantage over other indigenous eubacteria under heavy metal induced stress. The Burkholderia spp. were then cultured and further characterized using lipid analysis.Key words: metals, PCR-DGGE, microbial community response.

Sphingomonas spp possess unique abilities to degrade refractory contaminants and are found ubiquitously in the environment. We developed Sphingomonas genus-specific PCR primers (SPf-190 and SPr1-852) which showed specific amplification of a 627-bp 16S rDNA fragment from Sphingomonas spp. A PCR assay using these Sphingomonas specific primers was developed to detect Sphingomonas aromaticivorans B0695R in three texturally distinct soil types, showing detection limits between 1.3-2.2 x 10(3) CFU g(-1) dry soil. A sphingolipid extraction protocol was also developed to monitor Sphingomonas populations in soil quantitatively. The detection limit of the assay was 20 pmol g(-1) dry soil, equivalent to about 3 x 10(5) cells g(-1) dry soil. Survival of S. aromaticivorans B0695R was monitored in the three different soils by antibiotic selective plate counting, PCR and sphingolipid analysis. All three approaches showed that the B0695R cells persisted in the low biomass Sequatchie sub-soil at about 3-5 x 10(7)cells g(-1) dry soil. In comparison to the plate counting assay, both the PCR and sphingolipid analysis detected a significantly higher level of B0695R cells in the clay soil and Sequatchie top-soil, indicating the possibility of the presence of viable but non-culturable B0695R cells in the soils. The combination of PCR and sphingolipid analysis may provide a more realistic estimation of Sphingomonas population in the environment.

Denaturing gradient gel electrophoresis (DGGE) is a powerful and convenient tool for analyzing the sequence diversity of complex natural microbial populations, DGGE was evaluated for the identification of ammonia oxidizers of the beta subdivision of the Proteobacteria based on the mobility of PCR-amplified 16S rDNA fragments and for the analysis of mixtures of PCR products from this group generated by selective PCR of DNA extracted from coastal sand dunes, Degenerate PCR primers, CTO189f-GC and CTO654r, incorporating a 5' GC clamp, were designed to amplify a 465-bp 16S rDNA region spanning the V-2 and V-3 variable domains, The primers were tested against a representative selection of clones and cultures encompassing the currently recognized P-subdivision ammonia oxidizer 16S rDNA sequence diversity, Analysis of these products by DGGE revealed that while many of the sequences could be separated, some which were known to be different migrated similarly in the denaturant system used, The CTO primer pair was used to amplify 16S rDNA sequences from DNA extracted from soil sampled from Dutch coastal dune locations differing in pH and distance from the beach, The derived DGGE patterns were reproducible across multiple DNA isolations and PCRs, Ammonia oxidizer-like sequences from different phylogenetic groupings isolated from gene libraries made from the same sand dune DNA samples but prepared with different primers gave DGGE bands which comigrated with most of the bands detected from the sand dune samples, Bands from the DGGE gels of environmental samples were excised, reamplified, and directly sequenced, revealing strong similarity or identity of the recovered products to the corresponding regions of library clones, Six of the seven recognized sequence clusters of beta-subdivision ammonia oxidizers were detected in the dune systems, and differences in community structure between some sample sites were demonstrated, The most seaward dune site contained sequences showing affinity with sequence clusters previously isolated only from marine environments and was the only site where sequences related to the Nitrosomonas genus could be detected. Nitrosospira-like sequences were present in all sites, and there was some evidence of differences between Nitrosospira populations in acid and alkaline dune soils, Such differences in community structure may reflect physiological differences within beta-subdivision ammonia oxidizers, with consequent effects on nitrification rates in response to key environmental factors. [KEYWORDS: Purple bacteria; rna; phylogeny; genes; samples; soils]

Autotrophic ammonia oxidizer (AAO) populations in soils from native, tilled, and successional treatments at the Kellogg Biological Station Long-Term Ecological Research site in southwestern Michigan were compared to assess effects of disturbance on these bacteria. N fertilization effects on AAO populations were also evaluated with soils from fertilized microplots within the successional treatments. Population structures were characterized by PCR amplification of microbial community DNA with group-specific 16S rRNA gene (rDNA) primers, cloning of PCR products and clone hybridizations with group-specific probes, phylogenetic analysis of partial 16S rDNA sequences, and denaturing gradient gel electrophoresis (DGGE) analysis. Population sizes were estimated by using most-probable-number (MPN) media containing varied concentrations of ammonium sulfate. Tilled soils contained higher numbers than did native soils of culturable AAOs that were less sensitive to different ammonium concentrations in MPN media. Compared to sequences from native soils, partial 16S rDNA sequences from tilled soils were less diverse and grouped exclusively within Nitrosospira cluster 3. Native soils yielded sequences representing three different AAO clusters. Probes for Nitrosospira cluster 3 hybridized with DGGE blots from tilled and fertilized successional soils but not with blots from native or unfertilized successional soils. Hybridization results thus suggested a positive association between the Nitrosospira cluster 3 subgroup and soils amended with inorganic N. DGGE patterns for soils sampled from replicated plots of each treatment were nearly identical for tilled and native soils in both sampling years, indicating spatial and temporal reproducibility based on treatment.

Several dinoflagellate species have been shown to produce potent neurotoxins known as paralytic shellfish toxins. Evidence is also accumulating that marine bacteria associated with dinoflagellates play a role in the accumulation of paralytic shellfish toxins. In this study, the diversity of bacteria in cultures of both toxic and non-toxic dinoflagellates, Alexandrium spp. and Scrippsiella trochoidea, were compared using colony morphology, restriction fragment length polymorphisms, denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA genes and, ultimately, sequence determination of the 16S rRNA genes. The results suggest that a number of different bacterial species are associated with dinoflagellates, some of which are common to each of the dinoflagellate cultures examined, whereas others appear to be unique to a particular dinoflagellate. The phylogenetic diversity of the bacteria observed was limited to two bacterial phyla, the Proteobacteria and the Cytophaga-Flavobacter-Bacteroides (CFB). Although phylum level diversity was limited, many distinct phylogenetic clades were recovered, including members of both the K- and Q-subclasses of the Proteobacteria. Additionally, several of the bacterial phylotypes isolated were not closely related to any published bacterial species but, rather, were identical to isolates characterised from Alexandrium cultures 4 years earlier. Finally, many of the bacteria isolated from the dinoflagellate cultures were related to microorganisms with known surface-associated life histories (e.g. the CFB phylum, Hyphomonas, Caulobacter and some members of the Roseobacter clade including Ruegeria algicola). fl 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.