DNA isolation protocols affect the detection limit of PCR approaches of bacteria in samples from the human gastrointestinal tract

A major concern in molecular ecological studies is the lysis efficiency of different bacteria in a complex ecosystem. We used a PCR-based 16S rDNA approach to determine the effect of two DNA isolation protocols (i.e. the bead beating and Triton-X100 method) on the detection limit of seven feces-associated bacterial species of different genera. Glycogen was used in these protocols to improve the precipitation of small concentrations of DNA in ethanol without affecting the sequential procedures. The PCR detection limit of 16S rDNA amplicons on agarose gel from the seven strains tested varied between 8.0 (+/- 1.3) x 10(4) and 4.3 (+/- 1.6) x 10(6) cells for the bead beating method, and between 8.0 (+/- 1.3) x 10(4) and 5.4 (+/- 0.7) x 10(8) cells for the Triton X-100 method. These large differences are most like due to the difference in cell lysis efficiency, since a competitive PCR experiment did not indicate any preference for gram negative, low G+C gram positive or high G+C gram positive bacteria. Denaturing gradient gel electrophoresis (DGGE) analysis was performed to investigate the effect of both DNA isolation protocols on the lysis efficiency of bacteria in fecal samples. A higher diversity in fecal samples was observed with the bead beating method than with the Triton-X100 method. Bands in the bead beating method-derived DGGE profiles corresponding to bands of cloned sequences of the Clostridium coccoides-Eubacterium rectale group and uncultured Fusobacterium prausnitzii were absent or had low intensity in the Triton X-100 method-derived profiles. The applicability of the bead beating method was further investigated by analyzing biopsy samples from the human colon which contain approximately 10(6) cells.     

The BaeSR Two-Component Regulatory System Mediates Resistance to Condensed Tannins in Escherichia coli

The gene expression profiles of Escherichia coli strains grown anaerobically with or without Acacia mearnsii (black wattle) extract were compared to identify tannin resistance strategies. The cell envelope stress protein gene spy and the multidrug transporter-encoding operon mdtABCD, both under the control of the BaeSR two-component regulatory system, were significantly up-regulated in the presence of tannins. BaeSR mutants were more tannin sensitive than their wild-type counterparts.     

Glycoproteomic analysis of two mouse mammary cell lines during transforming growth factor (TGF)-β induced epithelial to mesenchymal transition

Background  

TGF-β acts as an antiproliferative factor in normal epithelial cells and at early stages of oncogenesis. However, later in tumor development TGF-β can become tumor promoting through mechanisms including the induction of epithelial-to-mesenchymal transition (EMT), a process that is thought to contribute to tumor progression, invasion and metastasis. To identify EMT-related breast cancer therapeutic targets and biomarkers, we have used two proteomic approaches to find proteins that change in abundance upon the induction of EMT by TGF-β in two mouse mammary epithelial cell lines, NMuMG and BRI-JM01.     

Molecular Diversity of Lactobacillus spp. and Other Lactic Acid Bacteria in the Human Intestine as Determined by Specific Amplification of 16S Ribosomal DNA

A Lactobacillus group-specific PCR primer, S-G-Lab-0677-a-A-17, was developed to selectively amplify 16S ribosomal DNA (rDNA) from lactobacilli and related lactic acid bacteria, including members of the genera Leuconostoc, Pediococcus, and Weissella. Amplicons generated by PCR from a variety of gastrointestinal (GI) tract samples, including those originating from feces and cecum, resulted predominantly in Lactobacillus-like sequences, of which ca. 28% were most similar to the 16S rDNA of Lactobacillus ruminis. Moreover, four sequences of Leuconostoc species were retrieved that, so far, have only been detected in environments other than the GI tract, such as fermented food products. The validity of the primer was further demonstrated by using Lactobacillus-specific PCR and denaturing gradient gel electrophoresis (DGGE) of the 16S rDNA amplicons of fecal and cecal origin from different age groups. The stability of the GI-tract bacterial community in different age groups over various time periods was studied. The Lactobacillus community in three adults over a 2-year period showed variation in composition and stability depending on the individual, while successional change of the Lactobacillus community was observed during the first 5 months of an infant’s life. Furthermore, the specific PCR and DGGE approach was tested to study the retention in fecal samples of a Lactobacillus strain administered during a clinical trial. In conclusion, the combination of specific PCR and DGGE analysis of 16S rDNA amplicons allows the diversity of important groups of bacteria that are present in low numbers in specific ecosystems to be characterized, such as the lactobacilli in the human GI tract.     

Microarray analysis and barcoded pyrosequencing provide consistent microbial profiles depending on the source of human intestinal samples

Large-scale and in-depth characterization of the intestinal microbiota necessitates application of high-throughput 16S rRNA gene-based technologies, such as barcoded pyrosequencing and phylogenetic microarray analysis. In this study, the two techniques were compared and contrasted for analysis of the bacterial composition in three fecal and three small intestinal samples from human individuals. As PCR remains a crucial step in sample preparation for both techniques, different forward primers were used for amplification to assess their impact on microbial profiling results. An average of 7,944 pyrosequences, spanning the V1 and V2 region of 16S rRNA genes, was obtained per sample. Although primer choice in barcoded pyrosequencing did not affect species richness and diversity estimates, detection of Actinobacteria strongly depended on the selected primer. Microbial profiles obtained by pyrosequencing and phylogenetic microarray analysis (HITChip) correlated strongly for fecal and ileal lumen samples but were less concordant for ileostomy effluent. Quantitative PCR was employed to investigate the deviations in profiling between pyrosequencing and HITChip analysis. Since cloning and sequencing of random 16S rRNA genes from ileostomy effluent confirmed the presence of novel intestinal phylotypes detected by pyrosequencing, especially those belonging to the Veillonella group, the divergence between pyrosequencing and the HITChip is likely due to the relatively low number of available 16S rRNA gene sequences of small intestinal origin in the DNA databases that were used for HITChip probe design. Overall, this study demonstrated that equivalent biological conclusions are obtained by high-throughput profiling of microbial communities, independent of technology or primer choice.     

Metamorphosis in the Cirripedia Rhizocephala and the homology of the kentrogon and trichogon

The Rhizocephala are considered to be monophyletic due to several synapomorphies in the ontogeny of the cndoparasitic phase. The various types of metamorphosis described in the Rhizocephala are discussed and compared to metamorphosis in the Cirripedia Thoracica and Acrothoracica. In males and females of the suborder Kentrogonida. the cyprid settles and metamorphoses into a new instar, in males the trichogen and in females the infective kentrogon. The kentrogon goes through yet another. incomplete moult associated with the development of the stylet. Within the three kentrogonidan families. the Iernaeodiscid-peltogastrid type of kentrogon differs from the sacculinid type in the mode of attachment to the host. in the complexity of internal anatomy. in the position and penetration of the stylet, and in whether or not the cyprid carapace must be shed prior to penetration of the stylet. In the Akentrogonida metamorphosis never results in a new instar. Where observed (Clistosaccidae and Thompsoniidae). both male and female cyprids settle and penetrate into their substrate (female parasite or new host) with one of the antennules. Using the antennule as a syringe. male cyprids inject spermatogonia while female cyprids injects embryonic cells developing into an endoparasite. By comparison with metamorphosis in the Cirripedia Thoracica and Acrothoracica it is concluded that the presence of a metamorphic moult leading to a post-cyprid instar is plesiomorphic and that the trichogon and kentrogon are homologous with the first metamorphosed juvenile in these outgroups. The abbreviated ontogeny in the Akentrogonida without metamorphic moult and post-cyprid larval instars is considered apomorphic. This contradicts the long-held supposition that the Akentrogonida are the most‘primitive’Rhizocephala and dovetails with new information that this suborder contains many advanced traits. Within the Kentrogonida. the lernacodiseid-peltogastrid type of kentrogon is considered more plesiomorphic than the sacculinid type, which resembles the clistosaccidthompsoniid type in having the antennules involved in the penetration process. The homologization of the kentrogon with a juvenile barnacle indicates that presence of a kentrogon is plesiomorphic within the Rhizocephala and that the Kentrogonida is paraphyletic.     

Concerted transpositions of mobile genetic elements coupled with fitness changes in Drosophila melanogaster

In an inbred low-activity (LA) strain of Drosophila melanogaster with a low level of fitness and a complex of inadaptive characters, in situ hybridization reveals an invariant pattern of distribution of three copia-like elements (mdg-1, mdg-3, and copia). Rare, spontaneous, multiple transpositions of mobile elements in the LA strain were shown to be coupled with a drastic increase of fitness. A changed pattern of various types of mobile elements was also observed on selecting the LA strain for higher fitness. High-fitness strains show transpositions of mobile elements to definite chromosomal sites ("hot spots"). Concerted changes in the location of three different mobile elements were found to be coupled with an increase of fitness. The mdg-1 distribution patterns were also examined in two low-fitness strains independently selected from the high-fitness ones. Fitness decrease was accompanied by mdg-1 excision from the hot spots of their location usually detected in the high-fitness strains. The results suggest the existence of a system of adaptive transpositions of mobile elements that takes part in fitness control.      

Trafficking and localization studies of recombinant alpha1, 3- fucosyltransferase VI stably expressed in CHO cells

Peripheral alpha1,3-fucosylation of glycans occurs by the action of either one of five different alpha1,3-fucosyltransferases (Fuc-Ts) cloned to date. Fuc-TVI is one of the alpha1,3-fucosyltransferases which is capable to synthesize selectin ligands. The major alpha1, 3- fucosyltransferase activity in human plasma is encoded by the gene for fucosyltransferase VI, which presumably originates from liver cells. While the sequence, chromosomal localization, and kinetic properties of Fuc-TVI are known, immunocytochemical localization and trafficking studies have been impossible because of the lack of specific antibodies. Here we report on the development and characterization of a peptide-specific polyclonal antiserum monospecific to Fuc-TVI and an antiserum to purified soluble recombinant Fuc-TVI crossreactive with Fuc-TIII and Fuc-TV. Both antisera were applied for immunodetection in stably transfected CHO cells expressing the full-length form of this enzyme (CHO clone 61/11). Fuc-TVI was found to be a resident protein of the Golgi apparatus. In addition, more than 30% of cell-associated and released enzyme activity was found in the medium. Maturation and release of Fuc-TVI was analyzed in metabolically labeled CHO 61/11 cells followed by immunoprecipitation. Fuc-TVI occurred in two forms of 47 kDa and 43 kDa bands, while the secreted form was detected as a 43 kDa. These two different intracellular forms arose by posttranslational modification, as shown by pulse-chase experiments. Fuc-TVI was released to the supernatant by proteolytic cleavage as a partially endo-H resistant glycoform.      

Comparative morphology and phylogeny of the family Thompsoniidae (Cirripedia, Rhizocephala, Akentrogonida), with descriptions of three new genera and seven new species

Akentrogonid rhizocephalans morphologically resembling the genus Thompsonia are revised as a result of examination of new material. The species concerned are all obligatorily colonial and have ovoid or cylindrically shaped externae with a terminal stalk and a much reduced anatomy. A numerical cladistic analysis of all Rhizocephala Akentrogonida using the Hennig 86 program leads to a redefinition of the Thompsoniidae HOeg and Rybakov, 1992. Autapomorphies for the Thompsoniidae are primarily the morphology of the attachment to the host and the total absence of a mesentery. The cladistic analysis refutes that the Thompsoniidae should have a plesiomorphic morphology and branch off very low on the rhizocephalan phylogeny. The family now comprises four genera: Pottsia gen. n. (monotypic), Diplothylacus gen. n. with two species, Thompsonia Kossmann with five species. A revived and redefined Thylacoplethus Coutière includes eight species. The genera are distinguished by the location of the spermatogenic tissue, the site where the eggs are fertilized, the presence or absence of a mantle pore and the way it is formed, the number or absence of oviducts, and the number of cuticular annuli on the stalk. All 16 species, of which six are new to science, are described when necessary, and, if possible, illustrated. A phylogeny for the redefined family is proposed. Thylacoplethus is morphologically closest to the hypothetically ancestral thompsoniid and is likely paraphyletic. The new genus Polysaccus with two species, one of them new to science, and the monotypic genus Pirusaccus Lützen resemble thompsoniids in externa morphology and in being obligatorily colonial.     

High temporal and inter‐individual variation detected in the human ileal microbiota

The diversity and temporal stability of the predominant bacteria in the human ileum was studied with the use of ileal effluent samples of seven individuals with Brooke ileostomies. The total number of bacteria within the ileal effluent was in the range of 10⁷–10⁸ bacteria per gram (wet weight). The diversity of the bacteria in the ileal effluent showed marked differences compared with that in faecal samples from age‐matched healthy adults. The ileal effluent had a higher relative abundance of species within the orders Lactobacillales and Clostridiales, mainly Streptococcus bovis‐related species, and the Veillonella group, and a lower proportion of species related to Ruminococcus gnavus, R. obeum and Bacteroides plebeius. In addition, inter‐individual differences were found, indicative of a highly personal ileal microbiota profile. Furthermore, temporal profiles showed large fluctuations per individual over a period of 9–28 days (average similarity over a period of 9 days was as low as 44%), and differences between morning and afternoon profiles were observed. Parallel cloning and sequencing efforts revealed several phylotypes that were not identified in previous studies (12 out of 65 phylotypes showed less than 97% sequence similarity with previously reported sequences). Achaea were found to be below detection limit by quantitative PCR. Overall, the results indicate that the microbiota of the human ileum is relatively unstable, less complex and consisting of different dominating phylotypes when compared with the colonic microbiota.