Molecular analysis of a consortium of ruminal microbes that detoxify pyrrolizidine alkaloids

2005 ◽  
Vol 51 (6) ◽  
pp. 455-465 ◽  
Author(s):  
S L Lodge-Ivey ◽  
M S Rappe ◽  
W H Johnston ◽  
R E Bohlken ◽  
A M Craig
Keyword(s):  
Metabolic Pathways ◽  
Pyrrolizidine Alkaloids ◽  
Fragment Length Polymorphism ◽  
Phylogenetic Analyses ◽  
Rflp Analysis ◽  
Molecular Method ◽  
16S Rdna Gene ◽  
Ruminal Bacteria ◽  
Banding Patterns ◽  
Tansy Ragwort

Members of a consortium of bacteria, isolated from the rumen of sheep, that degrades pyrrolizidine alkaloids (PAs) found in tansy ragwort (Senecio jacobaea) were characterized. An enrichment of ruminal bacteria was isolated from a sample of ruminal fluid using standard anaerobic techniques. The PA degradative capacity of the enrichment was tested by spiking purified PA extract from tansy ragwort. Length heterogeneity analysis by PCR (LH-PCR) and restriction fragment length polymorphism (RFLP) analysis was used to identify members of the consortium. Phylogenetic analysis of the 16S rDNA gene revealed differing results based on the molecular method used. LH-PCR identified 7 different organisms in 3 groups while RFLP identified 6 organisms with differing banding patterns in 5 groups. After the phylogenetic analyses of both methods were combined, the combined isolates represented 6 groups. The majority of the members of this consortium are <97.0% homologous with known bacteria, indicating this consortium may contain novel organisms able to detoxify PAs found in tansy ragwort. Further understanding of the metabolic pathways used by this consortium to degrade PAs could lead to the use of the consortium as a probiotic therapy for livestock and horses afflicted with tansy ragwort toxicosis.Key words: pyrrolizidine alkaloids, ruminal bacteria, tansy ragwort, RFLP, LH-PCR.

Molecular characterization of trypanosome species and subgroups within subgenus Nannomonas

Parasitology ◽  
1995 ◽  
Vol 111 (3) ◽  
pp. 301-312 ◽  
Author(s):  
L. H. Garside ◽  
W. C. Gibson
Keyword(s):  
Fragment Length Polymorphism ◽  
Genomic Library ◽  
Molecular Level ◽  
Pcr Amplification ◽  
Rflp Analysis ◽  
Rdna Locus ◽  
Kinetoplast Dna ◽  
Banding Patterns ◽  
High Level

SUMMARYRestriction fragment length polymorphism (RFLP) analysis of both genomic and kinetoplast DNA from representative stocks from 3 Trypanosoma congolense subgroups (Savannah, Forest, and Kilifi), T. simiae and T. godfreyi, was used to investigate the relatedness of the different groups within subgenus Nannomonas, DNA probes for β-tubulin and the ribosomal DNA (rDNA) locus were isolated from a T. congolense Savannah genomic library; additional probes were generated by PCR amplification of mini-exon and glutamate and alanine rich protein (GARP) gene sequences. Our results provide evidence that at the molecular level the T. congolense Savannah and Forest groups are the most closely related groups within the subgenus Nannomonas: the Savannah and the Forest groups had mini-exon gene repeats of identical size, which shared homology, had mini-circles of the same size and had a high level of similarity (63%) when the banding patterns produced with a tubulin and rDNA probe were subjected to numerical analysis. All other pairwise combinations of groups have very low percentage similarities of < 10%, suggesting that the Kilifi group trypanosomes, are as distantly related to the T. congolense Savannah and Forest groups as they are to T. simiae or T. godfreyi. The conservation of the GARP gene between the Savannah, Forest and Kilifi groups provides the only evidence linking the Kilifi trypanosomes to the other groups in T. congolense. We find no evidence for the presence of the GARP gene in the T. simiae or T. godfreyi group trypanosomes.

Localization of the Br Polymorphism on a 144 bp Exon of the GPIa Gene and Its Application in Platelet DNA Typing

1994 ◽  
Vol 71 (05) ◽  
pp. 651-654 ◽  
Author(s):  
Rainer Kalb ◽  
Sentot Santoso ◽  
Katja Unkelbach ◽  
Volker Kiefel ◽  
Christian Mueller-Eckhardt
Keyword(s):  
Genomic Organization ◽  
Fragment Length Polymorphism ◽  
Human Platelet ◽  
Dna Typing ◽  
Rflp Analysis ◽  
Serological Typing ◽  
Allele Specific ◽  
Polymerase Chain ◽  
Alloimmune Thrombocytopenia ◽  
Rflp Typing

SummaryAlloimmunization against the human platelet alloantigen system Br (HPA-5) is the second most common cause of neonatal alloimmune thrombocytopenia (NAIT) in Caucasian populations. We have recently shown that a single base polymorphism at position 1648 on platelet mRNA coding for GPIa results in an aminoacid substitution at position 505 on the mature GPIa which is associated with the two serological defined Br phenotypes.Since DNA-typing of platelet alloantigens offers possibilities for useful clinical applications, we designed genomic DNA-based restriction fragment length polymorphism (RFLP) typing for Br alloantigens. To establish this technique we analyzed the genomic organization of GPIa adjacent to the polymorphic base. Using the polymerase chain reaction (PCR) of blood cell DNA we have identified two introns (approximately 1.7 and 1.9 kb) flanking a 144 bp coding sequence of the GPIa gene encompassing the polymorphic base 1648. Based on the in- tron sequence, a PCR primer was constructed to amplify a 274 bp fragment which was used for allele-specific RFLP to determine the Br genotypes. The results of RFLP analysis using Mnll endonuclease obtained from 15 donors (2 Br37*, 2 Br^ and 11 Brb/b) correlate perfectly with serological typing by monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay.

scholarly journals Helarchaeota and co-occurring sulfate-reducing bacteria in subseafloor sediments from the Costa Rica Margin

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Rui Zhao ◽  
Jennifer F. Biddle
Keyword(s):  
Costa Rica ◽  
Metabolic Pathways ◽  
Phylogenetic Analyses ◽  
Sulfate Reducing Bacteria ◽  
Sulfate Reducing ◽  
Hydrocarbon Emission ◽  
Costa Rica Margin ◽  
Reducing Bacteria ◽  
Electron Transfers ◽  
Deep Sediments

AbstractDeep sediments host many archaeal lineages, including the Asgard superphylum which contains lineages predicted to require syntrophic partnerships. Our knowledge about sedimentary archaeal diversity and their metabolic pathways and syntrophic partners is still very limited. We present here new genomes of Helarchaeota and the co-occurring sulfate-reducing bacteria (SRB) recovered from organic-rich sediments off Costa Rica Margin. Phylogenetic analyses revealed three new metagenome-assembled genomes (MAGs) affiliating with Helarchaeota, each of which has three variants of the methyl-CoM reductase-like (MCR-like) complex that may enable them to oxidize short-chain alkanes anaerobically. These Helarchaeota have no multi-heme cytochromes but have Group 3b and Group 3c [NiFe] hydrogenases, and formate dehydrogenase, and therefore have the capacity to transfer the reducing equivalents (in the forms of hydrogen and formate) generated from alkane oxidation to external partners. We also recovered five MAGs of SRB affiliated with the class of Desulfobacteria, two of which showed relative abundances (represented by genome coverages) positively correlated with those of the three Helarchaeota. Genome analysis suggested that these SRB bacteria have the capacity of H2 and formate utilization and could facilitate electron transfers from other organisms by means of these reduced substances. Their co-occurrence and metabolic features suggest that Helarchaeota may metabolize synergistically with some SRB, and together exert an important influence on the carbon cycle by mitigating the hydrocarbon emission from sediments to the overlying ocean.

scholarly journals Nephromyces represents a diverse and novel lineage of the Apicomplexa that has retained apicoplasts

2019 ◽  
Author(s):  
Sergio A Muñoz-Gómez ◽  
Keira Durnin ◽  
Laura Eme ◽  
Christopher Paight ◽  
Christopher E Lane ◽  
...  
Keyword(s):  
Life Style ◽  
Metabolic Pathways ◽  
Evolutionary History ◽  
Phylogenetic Analyses ◽  
Phylogenetic Position ◽  
Biosynthetic Pathways ◽  
History Of ◽  
Sea Squirts ◽  
Shed Light ◽  
Apicoplast Genome

Abstract A most interesting exception within the parasitic Apicomplexa is Nephromyces, an extracellular, probably mutualistic, endosymbiont found living inside molgulid ascidian tunicates (i.e., sea squirts). Even though Nephromyces is now known to be an apicomplexan, many other questions about its nature remain unanswered. To gain further insights into the biology and evolutionary history of this unusual apicomplexan, we aimed to (1) find the precise phylogenetic position of Nephromyces within the Apicomplexa, (2) search for the apicoplast genome of Nephromyces, and (3) infer the major metabolic pathways in the apicoplast of Nephromyces. To do this, we sequenced a metagenome and a metatranscriptome from the molgulid renal sac, the specialized habitat where Nephromyces thrives. Our phylogenetic analyses of conserved nucleus-encoded genes robustly suggest that Nephromyces is a novel lineage sister to the Hematozoa, which comprises both the Haemosporidia (e.g., Plasmodium) and the Piroplasmida (e.g., Babesia and Theileria). Furthermore, a survey of the renal sac metagenome revealed 13 small contigs that closely resemble the genomes of the non-photosynthetic reduced plastids, or apicoplasts, of other apicomplexans. We show that these apicoplast genomes correspond to a diverse set of most closely related but genetically divergent Nephromyces lineages that co-inhabit a single tunicate host. In addition, the apicoplast of Nephromyces appears to have retained all biosynthetic pathways inferred to have been ancestral to parasitic apicomplexans. Our results shed light on the evolutionary history of the only probably mutualistic apicomplexan known, Nephromyces, and provide context for a better understanding of its life style and intricate symbiosis.

A variable minisatellite sequence in the chloroplast genome of Sorbus L. (Rosaceae: Maloideae)

Genome ◽  
2002 ◽  
Vol 45 (3) ◽  
pp. 570-576 ◽  
Author(s):  
R Andrew King ◽  
Colin Ferris
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Chloroplast Genome ◽  
Length Polymorphism ◽  
Sequence Variation ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Chloroplast Microsatellites ◽  
Sorbus Aucuparia ◽  
Pcr Product ◽  
Restriction Fragment Length

The chloroplast genome is now known to be more variable than was once thought. Reports of RFLP (restriction fragment length polymorphism) and sequence variation, as well as variation in chloroplast microsatellites, are common. Here, data are presented on the variability of a minisatellite sequence in the chloroplast genome of Sorbus species. RFLP analysis of a PCR product comprising the region between the trnM and rbcL genes of nine Sorbus species identified seven size variants. Sequencing revealed the observed size polymorphism to be due to differences in the number of copies of an imperfect 9-bp motif. A more intensive survey of the variability of the minisatellite was undertaken in populations of Sorbus aucuparia. The potential uses of such regions in chloroplast DNA are discussed and a possible mechanism for the evolution of the minisatellite is presented.Key words: atpE, homoplasy, microsatellite, rowan, VNTR.

scholarly journals Changes in Nitrogen-Fixing and Ammonia-Oxidizing Bacterial Communities in Soil of a Mixed Conifer Forest after Wildfire

2005 ◽  
Vol 71 (5) ◽  
pp. 2713-2722 ◽  
Author(s):  
Chris M. Yeager ◽  
Diana E. Northup ◽  
Christy C. Grow ◽  
Susan M. Barns ◽  
Cheryl R. Kuske
Keyword(s):  
Bacterial Communities ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Ammonia Oxidizing Bacteria ◽  
Nitrogen Fixing ◽  
Conifer Forest ◽  
Mixed Conifer ◽  
Mixed Conifer Forest ◽  
In Fire ◽  
Sequence Types

ABSTRACT This study was undertaken to examine the effects of forest fire on two important groups of N-cycling bacteria in soil, the nitrogen-fixing and ammonia-oxidizing bacteria. Sequence and terminal restriction fragment length polymorphism (T-RFLP) analysis of nifH and amoA PCR amplicons was performed on DNA samples from unburned, moderately burned, and severely burned soils of a mixed conifer forest. PCR results indicated that the soil biomass and proportion of nitrogen-fixing and ammonia-oxidizing species was less in soil from the fire-impacted sites than from the unburned sites. The number of dominant nifH sequence types was greater in fire-impacted soils, and nifH sequences that were most closely related to those from the spore-forming taxa Clostridium and Paenibacillus were more abundant in the burned soils. In T-RFLP patterns of the ammonia-oxidizing community, terminal restriction fragments (TRFs) representing amoA cluster 1, 2, or 4 Nitrosospira spp. were dominant (80 to 90%) in unburned soils, while TRFs representing amoA cluster 3A Nitrosospira spp. dominated (65 to 95%) in fire-impacted soils. The dominance of amoA cluster 3A Nitrosospira spp. sequence types was positively correlated with soil pH (5.6 to 7.5) and NH3-N levels (0.002 to 0.976 ppm), both of which were higher in burned soils. The decreased microbial biomass and shift in nitrogen-fixing and ammonia-oxidizing communities were still evident in fire-impacted soils collected 14 months after the fire.

scholarly journals Molecular Characterization and Classification of Phytoplasmas Associated with Canola Yellows and a New Phytoplasma Strain Associated with Dandelions

Plant Disease ◽  
2001 ◽  
Vol 85 (1) ◽  
pp. 76-79 ◽  
Author(s):  
Keri Wang ◽  
Chuji Hiruki
Keyword(s):  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Universal Primers ◽  
Dna Fragments ◽  
Chain Reaction ◽  
Aster Yellows ◽  
Distinct Subgroup ◽  
16S Ribosomal Dna ◽  
Polymerase Chain

DNA isolated from symptomatic canola (Brassica napus, Brassica rapa) and dandelion (Taraxacum officinale) was used to amplify 16S ribosomal DNA fragments by polymerase chain reaction using two pairs of universal primers P1/P6 and R16F2n/R2. Restriction fragment length polymorphism (RFLP) analysis of the amplified DNA fragments using endonucleases AluI, HhaI, HpaII, MseI, RsaI, and Sau 3AI revealed two distinct types of phytoplasmas in canola with similar symptoms. One had the same RFLP profiles as the phytoplasmas in subgroup 16SrI-A, whereas the other one had RFLP profiles similar to those of phytoplasmas in subgroup 16SrI-B. Phytoplasmas were detected in symptomatic dandelion plants that were collected from canola and alfalfa fields where severe alfalfa witches'-broom occurred. Comparative studies indicated that two different phytoplasmas were associated with the dandelion plants. One was identified as a member of subgroup 16SrI-A, whereas another one was classified as a member of a distinct subgroup in the aster yellows group on the basis of the unique RFLP patterns.

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