scholarly journals Integration Site Selection by the Bacteroides Conjugative Transposon CTnBST

2007 ◽  
Vol 189 (18) ◽  
pp. 6594-6601 ◽  
Author(s):  
Bo Song ◽  
Nadja B. Shoemaker ◽  
Jeffrey F. Gardner ◽  
Abigail A. Salyers
Keyword(s):  
Amino Acids ◽  
Integration Site ◽  
Consensus Sequence ◽  
Base Change ◽  
The Other ◽  
Tyrosine Recombinase ◽  
Crossover Region ◽  
Conjugative Transposon ◽  
Frequency Changes ◽  
Conserved Amino Acids

ABSTRACT A newly discovered Bacteroides conjugative transposon (CTn), CTnBST, integrates more site specifically than two other well-studied CTns, the Bacteroides CTn CTnDOT and the enterococcal CTn Tn916. Moreover, the integrase of CTnBST, IntBST, had the C-terminal 6-amino-acid signature that is associated with the catalytic regions of members of the tyrosine recombinase family, most of which integrate site specifically. Also, in most of these integrases, all of the conserved amino acids are required for integration. In the case of IntBST, however, we found that changing three of the six conserved amino acids in the signature, one of which was the presumed catalytic tyrosine, resulted in a 1,000-fold decrease in integration frequency. Changes in the other amino acids had little or no effect. Thus, although the CTnBST integrase still seems to be a member of the tyrosine recombinase family, it clearly differs to some extent from other members of the family in its catalytic site. We also determined the sequence requirements for CTnBST integration in the 18-bp region where the crossover occurs preferentially during integration. We found that CTnBST integrates in this preferred site about one-half of the time but can also use other sites. A consensus sequence was tentatively derived by comparison of a few secondary sites: AATCTGNNAAAT. We report here that within the consensus region, no single base change affected the frequency of integration. However, 3 bp at one end of the consensus sequence (CTG) proved to be essential for integration into the preferred site. This sequence appeared to be at one end of a 7-bp crossover region, CTGNNAA. The other bases could vary without affecting either integration frequency or specificity. Thus, in contrast to well-studied site-specific recombinases which require homology throughout the crossover region, integration of CTnBST requires homology at one end of the crossover region but not at the other end.

scholarly journals Consensus Finder web tool to predict stabilizing substitutions in proteins

2020 ◽  
Author(s):  
Bryan J. Jones ◽  
Chi Nok Enoch Kan ◽  
Christine Luo ◽  
Romas J. Kazlauskas
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Consensus Sequence ◽  
Fine Tuning ◽  
Protein Fold ◽  
Web Tool ◽  
Multiple Sequence ◽  
Homologous Sequences ◽  
The Stability ◽  
Conserved Amino Acids

AbstractThe consensus sequence approach to predicting stabilizing substitutions in proteins rests on the notion that conserved amino acids are more likely to contribute to the stability of a protein fold than non-conserved amino acids. To implement a prediction for a target protein sequence, one finds homologous sequences and aligns them in a multiple sequence alignment. The sequence of the most frequently occurring amino acid at each position is the consensus sequence. Replacement of a rarely occurring amino acid in the target with a frequently occurring amino acid is predicted to be stabilizing. Consensus Finder is an open-source web tool that automates this prediction. This chapter reviews the rationale for the consensus sequence approach and explains the options for fine-tuning this approach using Staphylococcus nuclease A as an example.

scholarly journals Integration and Excision of a Newly Discovered Bacteroides Conjugative Transposon, CTnBST

2006 ◽  
Vol 189 (3) ◽  
pp. 1072-1082 ◽  
Author(s):  
Neil A. Wesslund ◽  
Gui-Rong Wang ◽  
Bo Song ◽  
Nadja B. Shoemaker ◽  
Abigail A. Salyers
Keyword(s):  
Integration Site ◽  
Antibiotic Resistance Genes ◽  
Erythromycin Resistance ◽  
Crossover Region ◽  
Sequence Comparisons ◽  
Conjugative Transposon ◽  
Integrase Gene ◽  
Conjugative Transposons ◽  
Acid Protein ◽  
Amino Acid Protein

ABSTRACT Conjugative transposons (CTns) are major contributors to the spread of antibiotic resistance genes among Bacteroides species. CTnBST, a newly discovered Bacteroides conjugative transposon, carries an erythromycin resistance gene, ermB, and previously has been estimated to be about 100 kbp in size. We report here the locations and sequencing of both of its ends. We have also located and sequenced the gene that catalyzes the integration of CTnBST, intBST. The integrase gene encodes a 377-amino-acid protein that has the C-terminal R-K-H-R-H-Y motif that is characteristic of members of the tyrosine recombinase family of integrases. DNA sequence comparisons of the ends of CTnBST, the joined ends of the circular intermediate, and the preferred site into which the circular form of CTnBST had integrated revealed that the preferred integration site (attB1) contained an 18-bp sequence of identity to the crossover region, attBST, on CTnBST. Although this site was used in about one-half of the integration events, sequence analysis of these integration events revealed that both CTnBST and a miniature form of CTnBST (miniBST) integrated into a variety of other sites in the chromosome. All of the sites had two conserved regions, AATCTG and AAAT. These two regions flanked a 2-bp sequence, bp 10 and bp 11 of the 18-bp sequence, that varied in some of the different sites and sometimes in the attBST sequences. Our results suggest that CTnBST integrates site selectively and that the crossover appears to occur within a 12-bp region that contains the two regions of conserved sequences.

scholarly journals IntDOT Interactions with Core- and Arm-Type Sites of the Conjugative Transposon CTnDOT

2007 ◽  
Vol 189 (7) ◽  
pp. 2692-2701 ◽  
Author(s):  
Jeanne M. DiChiara ◽  
Aras N. Mattis ◽  
Jeffrey F. Gardner
Keyword(s):  
Integration Site ◽  
Higher Order ◽  
Conjugative Transposon ◽  
Tyrosine Recombinases ◽  
Terminal Set ◽  
Dnase I Footprinting ◽  
Protein Dna Interactions ◽  
Nucleoprotein Complexes ◽  
Conserved Amino Acids ◽  
Order Structures

ABSTRACT CTnDOT is a Bacteroides conjugative transposon (CTn) that has facilitated the spread of antibiotic resistances among bacteria in the human gut in recent years. Although the integrase encoded by CTnDOT (IntDOT) carries the C-terminal set of conserved amino acids that is characteristic of the tyrosine family of recombinases, the reaction it catalyzes involves a novel step that creates a short region of heterology at the joined ends of the element during recombination. Also, in contrast to tyrosine recombinases, IntDOT catalyzes a reaction that is not site specific. To determine what types of contacts IntDOT makes with the DNA during excision and integration, we first developed an agarose gel-based assay for CTnDOT recombination, which facilitated the purification of the native IntDOT protein. The partially purified IntDOT was then used for DNase I footprinting analysis of the integration site attDOT and the excision sites attL and attR. Our results indicate that CTnDOT has five or six arm sites that are likely to be involved in forming higher-order nucleoprotein complexes necessary for synapsis. In addition, there are four core sites that flank the sites of strand exchange during recombination. Thus, despite the fact that the reaction catalyzed by IntDOT appears to be different from that typically catalyzed by tyrosine recombinases, the protein-DNA interactions required for higher-order structures and recombination appear to be similar.

Natural Vulcanization Accelerators in Hevea Latex

1948 ◽  
Vol 21 (4) ◽  
pp. 853-859
Author(s):  
R. F. A. Altman
Keyword(s):  
Amino Acids ◽  
Experimental Results ◽  
The Other ◽  
Active Nitrogen ◽  
Nitrogen Bases ◽  
Other Hand ◽  
Decomposition Processes ◽  
Volatile Products ◽  
Nitrogenous Substances ◽  
The One

Abstract As numerous investigators have shown, some of the nonrubber components of Hevea latex have a decided accelerating action on the process of vulcanization. A survey of the literature on this subject points to the validity of certain general facts. 1. Among the nonrubber components of latex which have been investigated, certain nitrogenous bases appear to be most important for accelerating the rate of vulcanization. 2. These nitrogen bases apparently occur partly naturally in fresh latex, and partly as the result of putrefaction, heating, and other decomposition processes. 3. The nitrogen bases naturally present in fresh latex at later stages have been identified by Altman to be trigonelline, stachhydrine, betonicine, choline, methylamine, trimethylamine, and ammonia. These bases are markedly active in vulcanization, as will be seen in the section on experimental results. 4. The nitrogenous substances formed by the decomposition processes have only partly been identified, on the one hand as tetra- and pentamethylene diamine and some amino acids, on the other hand as alkaloids, proline, diamino acids, etc. 5. It has been generally accepted that these nitrogenous substances are derived from the proteins of the latex. 6. Decomposition appears to be connected with the formation of a considerable amount of acids. 7. The production of volatile nitrogen bases as a rule accompanies the decomposition processes. These volatile products have not been identified. 8. The active nitrogen bases, either already formed or derived from complex nitrogenous substances, seem to be soluble in water but only slightly soluble in acetone.

METABOLISM OF AROMATIC COMPOUNDS IN HEALTHY AND RUST-INFECTED PRIMARY LEAVES OF WHEAT: II. STUDIES WITH L-PHENYLALANINE-U-14C, L-TYROSINE-U-14C, AND FERULATE-U-14C

1967 ◽  
Vol 45 (11) ◽  
pp. 2137-2153 ◽  
Author(s):  
A. Fuchs ◽  
R. Rohringer ◽  
D. J. Samborski
Keyword(s):  
Amino Acids ◽  
Stem Rust ◽  
Aromatic Compounds ◽  
The Other ◽  
Major Portion ◽  
Insoluble Residue ◽  
Resistant Reaction ◽  
Wheat Leaves

Wheat leaves infected with stem rust, especially those of susceptible plants, contained more phenylalanine and tyrosine than healthy leaves. The utilization of phenylalanine was increased in both the susceptible and resistant reaction, but the utilization of tyrosine was increased only in the susceptible reaction. No evidence of interconversion of these amino acids was obtained.In n-butanol extracts, which contained glycosides, many constituents were labelled after feeding of L-phenylalanine-U-14C. Most of the n-butanol extractives from resistant-reacting leaves contained more label than those from susceptible-reacting leaves or from healthy leaves. However, one of the n-butanol extractives from susceptible-reacting leaves was 5–10 times as active as that isolated from the other tissues.With L-phenylalanine-U-14C and ferulate-U-14C as precursors, more activity was recovered in insoluble than in soluble esters (of ferulate and p-coumarate). With L-tyrosine-U-14C as precursor, the reverse was observed. After infection, the proportion of label in insoluble esters increased more in resistant leaves than it did in susceptible leaves, regardless of the precursor used.A major portion of the activity from these precursors was recovered in the insoluble residue that contained protein and other polymers. In the experiment with L-phenylalanine-U-14C, this residue was fractionated into protein and non-hydrolyzable material. Susceptible-reacting leaves contained equal amounts of activity in these fractions, while resistant-reacting leaves incorporated 2.5 times as much activity into the non-hydrolyzable material as into protein.

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