Actinobacterial 16S rRNA genes from freshwater habitats cluster in four distinct lineages

We analysed the phylogenetic relatedness of 16S rRNA genes from freshwater bacteria affiliated with the class Actinobacteria. A polymerase chain reaction assay was developed to identify reliably rare Actinobacteria-related inserts within 16S rRNA gene clone libraries. In 18 libraries constructed from seven freshwater systems, altogether 63 actinobacterial sequence types were collected from a total of > 1800 clones. Sixty of the newly obtained sequences grouped within four distinct phylogenetic lineages. They constitute approximately 75% of the nearly complete sequences within these clusters that are presently available. A comparison with > 300 sequences from various soil habitats revealed that two of these monophyletic actinobacterial clades (acI and acII) almost exclusively harbour 16S rRNA sequence types from freshwaters and estuaries. This may indicate that such bacteria are not inoculated to freshwaters from terrestrial sources, but are autochthonous components of freshwater microbial assemblages. In contrast, sequence types from freshwaters, marine sediments and soils were clearly mixed in another of the actinobacterial lineages (acIV). Sequence divergence within acIV was the highest of all four lineages (88% minimum similarity), which potentially reflects its radiation across several habitat types. Within the freshwater lineages, groups of essentially identical sequence types were retrieved from geographically distant aquatic systems with strikingly different hydrological and limnological characteristics. This points to the necessity to investigate genotypic variability, in situ abundances and activities of these Actinobacteria in freshwater plankton in greater detail by cultivation-independent techniques.     

Stable propagation of cosmid sized human DNA inserts in an F factor based vector.

Instability of complex mammalian genomic DNA inserts is commonplace in cosmid libraries constructed in conventional multicopy vectors. To develop a means to construct stable libraries, we have developed a low copy number cosmid vector based on the E. coli F factor replicon (Fosmid). We have tested relative stability of human DNA inserts in Fosmids and in two conventional multicopy vectors (Lawrist 16 and Supercos) by comparing the frequency of changes in restriction patterns of the inserts after propagating randomly picked human genomic clones based on these vectors. We found that the clones based on Fosmid vector undergo detectable changes at a greatly reduced frequency. We also observed that sequences that undergo drastic rearrangements and deletions during propagation in a conventional vector were stably propagated when recloned as Fosmids. The results indicate that Fosmid system may be useful for constructing stable libraries from complex genomes.     

Microbial Diversity of the Freshwater Sponge Spongilla lacustris

To provide insight into the phylogenetic bacterial diversity of the freshwater sponge Spongilla lacustris, a 16S rRNA gene libraries were constructed from sponge tissues and from lake water. Restriction fragment length polymorphism (RFLP) analysis of >190 freshwater sponge-derived clones resulted in six major restriction patterns, from which 45 clones were chosen for sequencing. The resulting sequences were affiliated with the Alphaproteobacteria (n = 19), the Actinobacteria (n = 15), the Betaproteobacteria (n = 2), and the Chloroflexi (n = 2) lineages. About half of the sequences belonged to previously described actinobacterial (hgc-I) and betaproteobacterial (beta-II) sequence clusters of freshwater bacteria that were also present in the lake water 16S rRNA gene library. At least two novel, deeply rooting alphaproteobacterial lineages were recovered from S. lacustris that showed <89% sequence similarity to known phylogenetic groups. Electron microscopical observations revealed that digested bacterial remnants were contained within food vacuoles of sponge archaeocytes, whereas the extracellular matrix was virtually free of bacteria. This study is the first molecular diversity study of a freshwater sponge and adds to a growing database on the diversity and community composition of sponge-associated microbial consortia.     

Actinobacterial Diversity in Microbial Mats of Five Hot Springs in Central and Central-Eastern Tibet,China

The diversity and community composition of Actinobacteria in microbial mats of five Tibetan hot springs (temperatures 26°C to 81°C) and a sympatric soil were investigated with 16S rRNA gene phylogentic analysis. A total of 278 clones were obtained. The actinobacterial communities in the Tibetan hot springs were diverse, and most of the retrieved clones were affiliated with Actinobacteridae, Acidimicrobidae, and unclassified Actinobacteria. The Actinobacteridae sequences were distributed into seven suborders (e.g., Frankineae, Corynebacterineae, Micromonosporineae, Pseudonocardineae, Propionibacterineae, Micrococcineae, and Actinomycineae) and unclassified Actinobacteridae. The actinobacterial composition varied among different hot springs. Statistical analysis showed that the actinobacterial diversity in the investigated Tibetan hot springs was not significantly correlated with temperature, suggesting that temperature is not a key factor in shaping the actinobacterial diversity in hot springs.     

Construction and utility of a human chromosome 22-specific Fosmid library

We have previously demonstrated the capability of the Fosmid vector based on Escherichia coli F-factor replicon to stably propagate cosmid-sized human genomic DNA fragments. Using the Fosmid vector, we have constructed and arrayed a 10 × human chromosome 22-specific library, partly by picking human positive clones from a total Fosmid library constructed using DNA from human-hamster hybrid cell line containing human chromosome 22, and partly by using flow-sorted chromosomal DNA. The clones and physical contig maps of the clones in the library will serve as a valuable resource for detailed analysis of the chromosome by providing reliable materials for high resolution mapping and sequencing. In order to efficiently built physical maps for the chromosomal regions of interest spanning several hundred kilobases to a megabase, it is necessary to rapidly identify subsets of the Fosmid clones from the library that cover such regions. In this report, we describe a method of using random amplification products derived from YAC clones to rapidly identify a subset of Fosmid clones that cover a specific genomic subregion.     

Diversity of Actinobacterial community in saline sediments from Yunnan and Xinjiang, China

The diversity and community structures of actinobacteria in saline sediments collected from Yunnan and Xinjiang Provinces, China, were investigated with cultivation and 16S rRNA gene analysis. A total of 163 actinobacterial isolates were obtained, and they were affiliated with the order Actinomycetales (distributed into five suborders: Streptosporangineae, Micrococcineae, Streptomycineae, Pseudonocardineae, and Glycomycineae). A total of 748 actinobacterial 16S rRNA gene clones were examined, and they could be classified into Actinomycetales, Acidimicrobiales, and unclassified actinobacteria. The Actinomycetales sequences were distributed into nine suborders: Streptosporangineae, Glycomycineae, Micromonosporineae, Pseudonocardineae, Corynebacterineae, Frankineae, Propionibacterineae, Streptomycineae, and Micrococcineae. The unclassified actinobacteria contained three new clusters at the level of subclass or order. Our 16S rRNA gene phylogenetic data indicated that actinobacterial communities were very diverse in the investigated saline sediments (salinity 0.4–11.6%) and some actinobacterial members may be halotolerant or halophilic. The actinobacterial community structures in the saline sediments were different from those in marine and freshwater environments. Our data have implications for a better understanding of the distribution of Actinobacteria in saline environments. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Random sheared fosmid library as a new genomic tool to accelerate complete finishing of rice (Oryza sativa spp. Nipponbare) genome sequence: sequencing of gap-specific fosmid clones uncovers new euchromatic portions of the genome

The International Rice Genome Sequencing Project has recently announced the high-quality finished sequence that covers nearly 95% of the japonica rice genome representing 370 Mbp. Nevertheless, the current physical map of japonica rice contains 62 physical gaps corresponding to approximately 5% of the genome, that have not been identified/represented in the comprehensive array of publicly available BAC, PAC and other genomic library resources. Without finishing these gaps, it is impossible to identify the complete complement of genes encoded by rice genome and will also leave us ignorant of some 5% of the genome and its unknown functions. In this article, we report the construction and characterization of a tenfold redundant, 40 kbp insert fosmid library generated by random mechanical shearing. We demonstrated its utility in refining the physical map of rice by identifying and in silico mapping 22 gap-specific fosmid clones with particular emphasis on chromosomes 1, 2, 6, 7, 8, 9 and 10. Further sequencing of 12 of the gap-specific fosmid clones uncovered unique rice genome sequence that was not previously reported in the finished IRGSP sequence and emphasizes the need to complete finishing of the rice genome. Fosmid library reported here is publicly available from our web site     

Phylogenetic diversity and metagenomics of candidate division OP3

Except for environmental 16S rRNA gene sequences, no information is available for members of the candidate division OP3. These bacteria appear to thrive in anoxic environments, such as marine sediments, hypersaline deep sea, freshwater lakes, aquifers, flooded paddy soils and methanogenic bioreactors. The 16S rRNA phylogeny suggests that OP3 belongs to the Planctomycetes/Verrucomicrobia/Chlamydiae (PVC) superphylum. Metagenomic fosmid libraries were constructed from flooded paddy soil and screened for 16S rRNA gene‐containing fragments affiliated with the PVC superphylum. The screening of 63 000 clones resulted in 23 assay‐positive fosmids, of which three clones were affiliated with OP3. The 16S rRNA gene sequence divergence between the fragments OP3/1, OP3/2 and OP3/3 ranges from 18% to 25%, indicating that they belong to different OP3 subdivisions. The 23S rRNA phylogeny confirmed the membership of OP3 in the PVC superphylum. Sequencing the OP3 fragments resulted in a total of 105 kb of genomic information and 90 ORFs, of which 47 could be assigned a putative function and 11 were conserved hypothetical. Using BLASTP searches, a high proportion of ORFs had best matches to homologues from Deltaproteobacteria, rather than to those of members of the PVC superphylum. On the fragment OP3/3, a cluster of nine ORFs was predicted to encode the bacterial NADH dehydrogenase I. Given the high proportion of homologues present in deltaproteobacteria and anoxic conditions in the natural environment of OP3 bacteria, the detection of NADH dehydrogenase I may suggest an anaerobic respiration mode. Oligonucleotide frequencies calculated for OP3/1, OP3/2 and OP/3 show high intraphylum correlations. This novel sequence information could therefore be used to identify OP3‐related fragments in large metagenomic data sets using marker gene‐independent procedures in the future. In addition to the OP3 fragments, a single metagenomic fragment affiliated with the candidate division BRC1 was obtained and analysed.     

Characterization of large-insert DNA libraries from soil for environmental genomic studies of Archaea

Complex genomic libraries are increasingly being used to retrieve complete genes, operons or large genomic fragments directly from environmental samples, without the need to cultivate the respective microorganisms. We report on the construction of three large-insert fosmid libraries in total covering 3 Gbp of community DNA from two different soil samples, a sandy ecosystem and a mixed forest soil. In a fosmid end sequencing approach including 5376 sequence tags of approximately 700 bp length, we show that mostly bacterial and, to a much lesser extent, archaeal and eukaryotic genome fragments (approximately 1% each) have been captured in our libraries. The diversity of putative protein-encoding genes, as reflected by their distribution into different COG clusters, was comparable to that encoded in complete genomes of cultivated microorganisms. A huge variety of genomic fragments has been captured in our libraries, as seen by comparison with sequences in the public databases and by the large variation in G+C contents. We dissect differences between the libraries, which relate to the different ecosystems analysed and to biases introduced by different DNA preparations. Furthermore, a range of taxonomic marker genes (other than 16S rRNA) has been identified that allows the assignment of genome fragments to specific lineages. The complete sequences of two genome fragments identified as being affiliated with Archaea, based on a gene encoding a CDC48 homologue and a thermosome subunit, respectively, are presented and discussed. We thereby extend the genomic information of uncultivated crenarchaeota from soil and offer hints to specific metabolic traits present in this group.     

Investigation of bacterial diversity in Brazilian tropical estuarine sediments reveals high actinobacterial diversity

Phylogenetic and statistical analyses of 16S rRNA gene libraries were used for the investigation of actinobacterial communities present in two tropical estuarine sediments (Santos-São Vicente estuary, Brazil). The libraries were constructed from samples collected at the brackish end of the estuary, highly hydrocarbon-contaminated, and at the marine end, uncontaminated. Clones from the marine end of the estuary were all related to sequences from non-cultured Actinobacteria and unidentified bacteria recovered from a wide range of environmental samples, whereas clones from the brackish end were mainly related to sequences from cultured Actinobacteria. Statistical analyses showed that the community recovered from the hydrocarbon-contaminated sediment sample, at the brackish end, was less diverse than the uncontaminated one, at the marine end, and that the communities from the two libraries were differently structured, suggesting that these may have not originated from the same community. The recognition of the spatial pattern of actinobacterial distribution in a natural environment is a first step towards understanding the way these communities are organized, providing valuable data for further investigations of their taxonomic and functional diversity.     

Involvement of Cell Surface Structures in Size-Independent Grazing Resistance of Freshwater Actinobacteria

We compared the influences of grazing by the bacterivorous nanoflagellate Poterioochromonas sp. strain DS on ultramicrobacterial Actinobacteria affiliated with the Luna-2 cluster and ultramicrobacterial Betaproteobacteria of the species Polynucleobacter cosmopolitanus. These bacteria were almost identical in size (<0.1 μm³) and shape. Predation on a Polynucleobacter strain resulted in a reduction of >86% relative to the initial bacterial cell numbers within 20 days, while in comparable predation experiments with nine actinobacterial strains, no significant decrease of cell numbers by predation was observed over the period of ≥39 days. The differences in predation mortality between the actinobacterial strains and the Polynucleobacter strain clearly demonstrated size-independent grazing resistance for the investigated Actinobacteria. Importantly, this size-independent grazing resistance is shared by all nine investigated Luna-2 strains and thus represents a group-specific trait. We investigated if an S-layer, previously observed in an ultrastructure study, was responsible for the grazing resistance of these strains. Experiments aiming for removal of the S-layer or modification of cell surface proteins of one of the grazing-resistant strains by treatment with lithium chloride, EDTA, or formaldehyde resulted in 4.2- to 5.2-fold higher grazing rates in comparison to the levels for untreated cells. These results indicate the protective role of a proteinaceous cell surface structure in the size-independent grazing resistance of the actinobacterial Luna-2 strains, which can be regarded as a group-specific trait.Predation by phagotrophic flagellates is considered (besides the effect of viruses and sedimentation) one of the major mortality factors affecting planktonic bacteria in freshwater and marine environments (13, 24, 28). A large number of investigations demonstrated that bacteria with small cell sizes are less susceptible to grazing mortality caused by flagellates than medium-sized cells (e.g., references 9 and 34). However, even bacterial cells with ultramicrobacterial (<0.1 μm³) cell sizes can be ingested and probably processed by bacterivorous flagellates (4). Interestingly, an increasing number of observations indicate that planktonic Actinobacteria indigenous to freshwater systems are less vulnerable to protistan predation than other taxa of freshwater bacterioplankton. Pernthaler and colleagues observed in a two-stage continuous cultivation system that freshwater Actinobacteria increased in relative and absolute abundance in the presence of a bacterivorous flagellate (26). Other investigations also indicate at least a low level of vulnerability of freshwater Actinobacteria to protistan grazing (17, 29). Experiments with a single actinobacterial strain affiliated with the Luna-2 cluster indicated complete grazing resistance for this freshwater strain against predation by a bacterivorous flagellate (14). Incubation of the strain in the presence of the flagellate over a period of 20 days resulted only in minor changes of bacterial cell numbers. Addition of heat-killed cells of another bacterial species serving as alternative prey resulted even in simultaneous increases of actinobacterial and flagellate numbers. Thus, even the rapidly growing flagellate population was not able to efficiently reduce the actinobacterial cell numbers. Recently, experiments with natural bacterial and protist communities demonstrated a strong negative selection of planktonic Actinobacteria by bacterivorous flagellates (18). In this investigation, the relative abundances of several phylogenetic groups of bacteria inside food vacuoles of flagellate cells and in the surrounding water have been determined. Actinobacterial cells were strongly underrepresented in the food vacuoles compared to their presence in the surrounding water. Currently, it is not known if the observed strong predation resistance of freshwater Actinobacteria is the result of postingestional protection or digestion protection taking place after ingestion of cells by a predator. Furthermore, it is unknown if this grazing protection is a common trait shared by all planktonic Actinobacteria present in freshwater systems or if only some taxa are protected.Importantly, Actinobacteria constitute large fractions (up to 60%) of bacterioplankton in freshwater systems (8) and seem to present a ubiquitous component of freshwater bacterioplankton (8, 23, 37). Almost all actinobacterial taxa identified by cultivation-independent methods so far represent indigenous taxa exclusively known to occur in freshwater habitats (8, 37, 39), and only some of these indigenous actinobacterial taxa are represented by cultivated strains (7, 14-16).The cultivated strains representing indigenous freshwater Actinobacteria provide excellent opportunities for detailed studies of the grazing resistance mechanisms of this ecologically important group of bacteria. Cultivated strains affiliated with the Luna cluster (14), also known as the acII clade (37), currently represent the best-characterized planktonic Actinobacteria indigenous to freshwater systems. Many of the cultivated Luna strains are characterized by C-shaped (selenoid) cells with ultramicrobacterial sizes of <0.07 μm³. An electron microscopy investigation revealed a so-called S-layer at the surfaces of their cells (14). S-layers are monomolecular arrays composed of identical protein or glycoprotein subunits forming crystalline layers at the cell surfaces of many Bacteria and Archaea (30, 35). We hypothesize that this S-layer is involved in the grazing protection of the previously investigated freshwater Actinobacteria.In the study presented here, we investigated (i) if grazing resistance is a common trait among strains of the Luna-2 cluster, (ii) if the grazing protection previously demonstrated for an actinobacterial strain affiliated with the Luna-2 cluster is independent of the small size and the C shape of the cells, and (iii) if the cell surface structure is involved in the grazing resistance mechanism of Luna-2 strains.     

Phylogeny-function analysis of (meta)genomic libraries: screening for expression of ribosomal RNA genes by large-insert library fluorescent in situ hybridization (LIL-FISH)

We assessed the utility of fluorescent in situ hybridization (FISH) in the screening of clone libraries of (meta)genomic or environmental DNA for the presence and expression of bacterial ribosomal RNA (rRNA) genes. To establish proof-of-principle, we constructed a fosmid-based library in Escherichia coli of large-sized genomic DNA fragments of the mycophagous soil bacterium Collimonas fungivorans, and hybridized 768 library clones with the Collimonas-specific fluorescent probe CTE998-1015. Critical to the success of this approach (which we refer to as large-insert library FISH or LIL-FISH) was the ability to induce fosmid copy number, the exponential growth status of library clones in the FISH assay and the use of a simple pooling strategy to reduce the number of hybridizations. Twelve out of 768 E. coli clones were suspected to harbour and express Collimonas 16S rRNA genes based on their hybridization to CTE998-1015. This was confirmed by the finding that all 12 clones were also identified in an independent polymerase chain reaction-based screening of the same 768 clones using a primer set for the specific detection of Collimonas 16S ribosomal DNA (rDNA). Fosmids isolated from these clones were grouped by restriction analysis into two distinct contigs, confirming that C. fungivorans harbours at least two 16S rRNA genes. For one contig, representing 1-2% of the genome, the nucleotide sequence was determined, providing us with a narrow but informative view of Collimonas genome structure and content.     

Unique Microbial Signatures of the Alien Hawaiian Marine Sponge Suberites zeteki

Invasive species poses a threat to the world’s oceans. Alien sponges account for the majority of introduced marine species in the isolated Hawaiian reef ecosystems. In this study, cultivation-dependent and cultivation-independent techniques were applied to investigate microbial consortia associated with the alien Hawaiian marine sponge Suberites zeteki. Its microbial communities were diverse with representatives of Actinobacteria, Firmicutes, α- and γ-Proteobacteria, Bacteroidetes, Chlamydiae, Planctomycetes, and Cyanobacteria. Specifically, the genus Chlamydia was identified for the first time from marine sponges, and two genera (Streptomyces and Rhodococcus) were added to the short list of culturable actinobacteria from sponges. Culturable microbial communities were dominated by Bacillus species (63%) and contained actinobacterial species closely affiliated with those from habitats other than marine sponges. Cyanobacterial clones were clustered with free-living cyanobacteria from water column and other environmental samples; they show no affiliation with other sponge-derived cyanobacteria. The low sequence similarity of Planctomycetes, Chlamydiae, and α-Proteobacteria clones to other previously described sequences suggested that S. zeteki may contain new lineages of these bacterial groups. The microbial diversity of S. zeteki was different from that of other studied marine sponges. This is the first report on microbial communities of alien marine invertebrate species. For the first time, it provides an insight into microbial structure within alien marine sponges in the Hawaiian marine ecosystems.     

Fosmid Library Construction and Initial Analysis of End Sequences in Zhikong Scallop (<Emphasis Type="Italic">Chlamys farreri</Emphasis>)

Zhikong scallop (Chlamys farreri Jones et Preston, 1904) is one of the most commercially important bivalves in China, but research on its genome is underdeveloped. In this study, we constructed the first Zhikong scallop fosmid library, and analyzed the fosmid end sequences to provide a preliminary assessment of the genome. The library consists of 133,851 clones with an average insert size of about 40 kb, amounting to 4.3 genome equivalents. Fosmid stability assays indicate that Zhikong scallop DNA was stable during propagation in the fosmid system. Library screening with two genes and seven microsatellite markers yielded between two and eight positive clones, and none of those tested was absent from the library. End-sequencing of 480 individual clones generated 828 sequences after trimming, with an average sequence length of 624 bp. BLASTN searches of the nr and EST databases of GenBank and BLASTX searches of the nr database resulted in 213 (25.72%) and 44 (5.31%) significant hits (E < e⁻⁵), respectively. Repetitive sequences analysis resulted in 375 repeats, accounting for 15.84% of total length, which were composed of interspersed repetitive sequences, tandem repeats, and low-complexity sequences. The fosmid library, in conjunction with the fosmid end sequences, will serve as a useful resource for physical mapping and positional cloning, and provide a better understanding of the Zhikong scallop genome.     

Phylogenetic Analysis of the Gut Bacterial Microflora of the Fungus-Growing Termite Odontotermes formosanus

We constructed a bacterial 16S rRNA gene clone library from the gut microbial community of O. formosanus and phylogenetically analyzed it in order to contribute to the evolutional study of digestive symbiosis and method development for termite control. After screening by restriction fragment length polymorphism (RFLP) analysis, 56 out of 280 clones with unique RFLP patterns were sequenced and phylogenetically analyzed. The representative phylotypes were affiliated to four phylogenetic groups, Firmicutes, the Bacteroidetes/Chlorobi group, Proteobacteria, and Actinobacteria of the domain Bacteira. No one clone affiliated with the phylum Spirochaetes was identified, in contrast to the case of wood-feeding termites. The phylogenetic analysis revealed that nearly half of the representative clones (25 phylotypes) formed monophyletic clusters with clones obtained from other termite species, especially with the sequences retrieved from fungus-growing termites. These results indicate that the presence of termite-specific bacterial lineages implies a coevolutional relationship of gut microbes and host termites.     

Genetic and functional properties of uncultivated MCG archaea assessed by metagenome and gene expression analyses

The Miscellaneous Crenarchaeota group (MCG) Archaea is one of the predominant archaeal groups in anoxic environments and may have significant roles in the global biogeochemical cycles. However, no isolate of MCG has been cultivated or characterized to date. In this study, we investigated the genetic organization, ecophysiological properties and evolutionary relationships of MCG archaea with other archaeal members using metagenome information and the result of gene expression experiments. A comparison of the gene organizations and similarities around the 16S rRNA genes from all available MCG fosmid and cosmid clones revealed no significant synteny among genomic fragments, demonstrating that there are large genetic variations within members of the MCG. Phylogenetic analyses of large-subunit+small-subunit rRNA, concatenated ribosomal protein genes and topoisomerases IB gene (TopoIB) all demonstrate that MCG constituted a sister lineage to the newly proposed archaeal phylum Aigarchaeota and Thaumarchaeota. Genes involved in protocatechuate degradation and chemotaxis were found in a MCG fosmid 75G8 genome fragment, suggesting that this MCG member may have a role in the degradation of aromatic compounds. Moreover, the expression of a putative 4-carboxymuconolactone decarboxylase was observed when the sediment was supplemented with protocatechuate, further supporting the hypothesis that this MCG member degrades aromatic compounds.     

<Emphasis Type="Italic">Escherichia coli</Emphasis> MW005: lambda Red-mediated recombineering and copy-number induction of <Emphasis Type="Italic">oriV</Emphasis>-equipped constructs in a single host

Background  

Escherichia coli strain EL350 contains chromosomally integrated phage lambda Red recombinase genes enabling this strain to be used for modifying the sequence of resident clones via recombineering. BAC and fosmid clones are highly suitable for modification by recombineering but, because they are present at low (1-2) copies per cell, the DNA is difficult to isolate in high yield and purity. To overcome this limitation vectors, e.g. pCC1FOS, have been constructed that contain the additional replication origin, oriV, which permits copy-number to be induced transiently when propagated in a suitable host strain, e.g. EPI300, that supplies the cognate trans -replication protein TrfA. Previously, we used EL350 and EPI300 sequentially to recombineer oriV -equipped fosmid genomic clones and, subsequently, to induce copy-number of the resulting recombinant clone. To eliminate these intervening DNA isolation and transformation steps we retrofitted EL350 with a P _BAD-driven trfA gene generating strain MW005 that supports, independently, both recombineering and copy-number induction.     

Characterizing microbial diversity in production water from an Alaskan mesothermic petroleum reservoir with two independent molecular methods

The phylogenetic diversity of Bacteria and Archaea within a biodegraded, mesothermic petroleum reservoir in the Schrader Bluff Formation of Alaska was examined by two culture-independent methods based on fosmid and small-subunit rRNA gene PCR clone libraries. Despite the exclusion of certain groups by each method, there was overall no significant qualitative difference in the diversity of phylotypes recovered by the two methods. The resident Bacteria belonged to at least 14 phylum-level lineages, including the polyphyletic Firmicutes , which accounted for 36.2% of all small-subunit rRNA gene-containing (SSU⁺) fosmid clones identified. Members of uncultured divisions were also numerous and made up 35.2% of the SSU⁺ fosmid clones. Clones from domain Archaea accounted for about half of all SSU⁺ fosmids, suggesting that their cell numbers were comparable to those of the Bacteria in this microbial community. In contrast to the Bacteria , however, nearly all archaeal clones recovered by both methods were related to methanogens, especially acetoclastic methanogens, while the plurality of bacterial fosmid clones was affiliated with Synergistes -like acetogenic Firmicutes that possibly degrade longer-chain carboxylic acid components in the crude oil to acetate. These data suggest that acetate may be a key intermediary metabolite in this subsurface anaerobic food chain, which leads to methane production as the primary terminal electron sink.