scholarly journals Site-Specific Recombination of TemperateMyxococcus xanthus Phage Mx8: Genetic Elements Required for Integration

1999 ◽  
Vol 181 (13) ◽  
pp. 4050-4061 ◽  
Author(s):  
Vincent Magrini ◽  
Chad Creighton ◽  
Philip Youderian
Keyword(s):  
Amino Acid ◽  
Phage Genome ◽  
Myxococcus Xanthus ◽  
Plasmid Vector ◽  
Amino Acid Residues ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Coding Sequence ◽  
Site Specific Integration ◽  
C Terminus

ABSTRACT Like most temperate bacteriophages, phage Mx8 integrates into a preferred locus on the genome of its host, Myxococcus xanthus, by a mechanism of site-specific recombination. The Mx8int-attP genes required for integration map within a 2.2-kilobase-pair (kb) fragment of the phage genome. When this fragment is subcloned into a plasmid vector, it facilitates the site-specific integration of the plasmid into the 3′ ends of either of two tandem tRNAAsp genes, trnD1 and trnD2, located within the attB locus of the M. xanthusgenome. Although Int-mediated site-specific recombination occurs between attP and either attB1 (withintrnD1) or attB2 (within trnD2), theattP × attB1 reaction is highly favored and often is accompanied by a deletion between attB1 andattB2. The int gene is the only Mx8 gene required in trans for attP × attBrecombination. The int promoter lies within the 106-bp region immediately upstream of one of two alternate GTG start codons, GTG-5208 (GTG at bp 5208) and GTG-5085, for integrase and likely is repressed in the prophage state. All but the C-terminal 30 amino acid residues of the Int protein are required for its ability to mediateattP × attB recombination efficiently. TheattP core lies within the int coding sequence, and the product of integration is a prophage in which the 3′ end ofint is replaced by host sequences. The prophageintX gene is predicted to encode an integrase with a different C terminus.

scholarly journals The IntP C-Terminal Segment Is Not Required for Excision of Bacteriophage Mx8 from the Myxococcus xanthus Chromosome

2003 ◽  
Vol 185 (7) ◽  
pp. 2187-2193 ◽  
Author(s):  
Nobuki Tojo ◽  
Teruya Komano
Keyword(s):  
Amino Acid ◽  
High Frequency ◽  
Myxococcus Xanthus ◽  
Terminal Segment ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Coding Sequence ◽  
Phage Dna ◽  
New Protein

ABSTRACT During lysogenization of myxophage Mx8, phage DNA can be integrated into the attB site of the Myxococcus xanthus chromosome through site-specific recombination. We previously demonstrated that the Mx8 attP site is located within the coding sequence of the Mx8 intP gene. Hence, the integration of Mx8 into the M. xanthus chromosome results in the conversion of the 112-amino-acid C-terminal segment of the IntP protein into a 13-amino-acid C-terminal segment of a new protein, IntR. To examine whether IntR is active for Mx8 excision, we have constructed a series of plasmids carrying various lengths of the intP-attP or intR-attR regions as well as the lacZ gene. The integrated Mx8 was excised at a high frequency, indicating that IntR is active for the excision. For Mx8 excision, a gene designated xis was shown to be required in addition to intR.

scholarly journals Site-Specific Recombination of TemperateMyxococcus xanthus Phage Mx8: Regulation of Integrase Activity by Reversible, Covalent Modification

1999 ◽  
Vol 181 (13) ◽  
pp. 4062-4070 ◽  
Author(s):  
Vincent Magrini ◽  
Michael L. Storms ◽  
Philip Youderian
Keyword(s):  
Specific Activity ◽  
Active Form ◽  
Attachment Site ◽  
Covalent Modification ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Site Specific Integration ◽  
C Terminus ◽  
Primary Amino ◽  
Reversible Covalent

ABSTRACT Temperate Myxococcus xanthus phage Mx8 integrates into the attB locus of the M. xanthus genome. The phage attachment site, attP, is required in cisfor integration and lies within the int (integrase) coding sequence. Site-specific integration of Mx8 alters the 3′ end ofint to generate the modified intX gene, which encodes a less active form of integrase with a different C terminus. The phage-encoded (Int) form of integrase promotes attP × attB recombination more efficiently than attR × attB, attL × attB, or attB × attB recombination. The attP and attBsites share a common core. Sequences flanking both sides of theattP core within the int gene are necessary forattP function. This information shows that the directionality of the integration reaction depends on arm sequences flanking both sides of the attP core. Expression of theuoi gene immediately upstream of int inhibits integrative (attP × attB) recombination, supporting the idea that uoi encodes the Mx8 excisionase. Integrase catalyzes a reaction that alters the primary sequence of its gene; the change in the primary amino acid sequence of Mx8 integrase resulting from the reaction that it catalyzes is a novel mechanism by which the reversible, covalent modification of an enzyme is used to regulate its specific activity. The lower specific activity of the prophage-encoded IntX integrase acts to limit excisive site-specific recombination in lysogens carrying a single Mx8 prophage, which are less immune to superinfection than lysogens carrying multiple, tandem prophages. Thus, this mechanism serves to regulate Mx8 site-specific recombination and superinfection immunity coordinately and thereby to preserve the integrity of the lysogenic state.

scholarly journals Primary- and secondary-structural analysis of a unique prokaryotic metallothionein from a Synechococcus sp. cyanobacterium

1988 ◽  
Vol 251 (3) ◽  
pp. 691-699 ◽  
Author(s):  
R W Olafson ◽  
W D McCubbin ◽  
C M Kay
Keyword(s):  
Amino Acid ◽  
Metal Binding ◽  
Helical Structure ◽  
Basic Amino Acid ◽  
Cysteine Residues ◽  
Amino Acid Residues ◽  
C Terminus ◽  
Empirical Relations ◽  
Heavy Metal Binding ◽  
Synechococcus Sp

Biochemical and physiological studies of Synechococcus cyanobacteria have indicated the presence of a low-Mr heavy-metal-binding protein with marked similarity to eukaryotic metallothioneins (MTs). We report here the characterization of a Synechococcus prokaryotic MT isolated by gel-permeation and reverse-phase chromatography. The large number of variants of this molecule found during chromatographic separation could not be attributed to the presence of major isoproteins as assessed by amino acid analysis and amino acid sequencing of isoforms. Two of the latter were shown to have identical primary structures that differed substantially from the well-described eukaryotic MTs. In addition to six long-chain aliphatic residues, two aromatic residues were found adjacent to one another near the centre of the molecule, making this the most hydrophobic MT to be described. Other unusual features included a pair of histidine residues located in repeating Gly-His-Thr-Gly sequences near the C-terminus and a complete lack of association of hydroxylated residues with cysteine residues, as is commonly found in eukaryotes. Similarly, aside from a single lysine residue, no basic amino acid residues were found adjacent to cysteine residues in the sequence. Most importantly, sequence alignment analyses with mammalian, invertebrate and fungal MT sequences showed no statistically significant homology aside from the presence of Cys-Xaa-Cys structures common to all MTs. On the other hand, like other MTs, the prokaryotic molecule appears to be free of alpha-helical structure but has a considerable amount of beta-structure, as predicted by both c.d. measurements and the Chou & Fasman empirical relations. Considered together, these data suggested that some similarity between the metal-thiolate clusters of the prokaryote and eukaryote MTs may exist.

scholarly journals Site-specific replacement of amino acid residues within the CD binding loop of rat oncomodulin.

1990 ◽  
Vol 265 (24) ◽  
pp. 14450-14456 ◽  
Author(s):  
W.A. Palmisano ◽  
C.L. Treviño ◽  
M.T. Henzl
Keyword(s):  
Amino Acid ◽  
Amino Acid Residues ◽  
Site Specific ◽  
Binding Loop

scholarly journals Deletion of the Actin-Associated Tropomyosin Tpm3 Leads to Reduced Cell Complexity in Cultured Hippocampal Neurons—New Insights into the Role of the C-Terminal Region of Tpm3.1

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 715
Author(s):  
Tamara Tomanić ◽  
Claire Martin ◽  
Holly Stefen ◽  
Esmeralda Parić ◽  
Peter Gunning ◽  
...  
Keyword(s):  
Amino Acid ◽  
Hippocampal Neurons ◽  
Molecular Mechanisms ◽  
Growth Cones ◽  
Amino Acid Residues ◽  
Terminal Amino Acid ◽  
C Terminus ◽  
Primary Mouse ◽  
Terminal Amino

Tropomyosins (Tpms) have been described as master regulators of actin, with Tpm3 products shown to be involved in early developmental processes, and the Tpm3 isoform Tpm3.1 controlling changes in the size of neuronal growth cones and neurite growth. Here, we used primary mouse hippocampal neurons of C57/Bl6 wild type and Bl6Tpm3flox transgenic mice to carry out morphometric analyses in response to the absence of Tpm3 products, as well as to investigate the effect of C-terminal truncation on the ability of Tpm3.1 to modulate neuronal morphogenesis. We found that the knock-out of Tpm3 leads to decreased neurite length and complexity, and that the deletion of two amino acid residues at the C-terminus of Tpm3.1 leads to more detrimental changes in neurite morphology than the deletion of six amino acid residues. We also found that Tpm3.1 that lacks the 6 C-terminal amino acid residues does not associate with stress fibres, does not segregate to the tips of neurites, and does not impact the amount of the filamentous actin pool at the axonal growth cones, as opposed to Tpm3.1, which lacks the two C-terminal amino acid residues. Our study provides further insight into the role of both Tpm3 products and the C-terminus of Tpm3.1, and it forms the basis for future studies that aim to identify the molecular mechanisms underlying Tpm3.1 targeting to different subcellular compartments.

Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase resides at the carboxy terminus

1989 ◽  
Vol 9 (1) ◽  
pp. 83-91
Author(s):  
S Miyazawa ◽  
T Osumi ◽  
T Hashimoto ◽  
K Ohno ◽  
S Miura ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rat Liver ◽  
Coenzyme A ◽  
Terminal Region ◽  
Proteinase K ◽  
Amino Acid Residues ◽  
C Terminus ◽  
Targeting Signal ◽  
Acyl Coenzyme A

To identify the topogenic signal of peroxisomal acyl-coenzyme A oxidase (AOX) of rat liver, we carried out in vitro import experiments with mutant polypeptides of the enzyme. Full-length AOX and polypeptides that were truncated at the N-terminal region were efficiently imported into peroxisomes, as determined by resistance to externally added proteinase K. Polypeptides carrying internal deletions in the C-terminal region exhibited much lower import activities. Polypeptides that were truncated or mutated at the extreme C terminus were totally import negative. When the five amino acid residues at the extreme C terminus were attached to some of the import-negative polypeptides, the import activities were rescued. Moreover, the C-terminal 199 and 70 amino acid residues of AOX directed fusion proteins with two bacterial enzymes to peroxisomes. These results are interpreted to mean that the peroxisome targeting signal of AOX residues at the C terminus and the five or fewer residues at the extreme terminus have an obligatory function in targeting. The C-terminal internal region also has an important role for efficient import, possibly through a conformational effect.

scholarly journals Construction of an Integration-Proficient Vector Based on the Site-Specific Recombination Mechanism of Enterococcal Temperate Phage φFC1

2002 ◽  
Vol 184 (7) ◽  
pp. 1859-1864 ◽  
Author(s):  
Hee-Youn Yang ◽  
Young-Woo Kim ◽  
Hyo-Ihl Chang
Keyword(s):  
Pcr Amplification ◽  
Temperate Phage ◽  
Vector System ◽  
Recombination Mechanism ◽  
Integrase Gene ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Integration Vector ◽  
Site Specific Integration ◽  
A Site

ABSTRACT The genome of temperate phage φFC1 integrates into the chromosome of Enterococcus faecalis KBL 703 via site-specific recombination. In this study, an integration vector containing the attP site and putative integrase gene mj1 of phage φFC1 was constructed. A 2,744-bp fragment which included the attP site and mj1 was inserted into a pUC19 derivative containing the cat gene to construct pEMJ1-1. E. faecalis KBL 707, which does not contain the bacteriophage but which has a putative attB site within its genome, could be transformed by pEMJ1-1. Southern hybridization, PCR amplification, and DNA sequencing revealed that pEMJ1-1 was integrated specifically at the putative attB site within the E. faecalis KBL 707 chromosome. This observation suggested that the 2,744-bp fragment carrying mj1 and the attP site of phage φFC1 was sufficient for site-specific recombination and that pEMJ1-1 could be used as a site-specific integration vector. The transformation efficiency of pEMJ1-1 was as high as 6 × 103 transformants/μg of DNA. In addition, a vector (pATTB1) containing the 290-bp attB region was constructed. pATTB1 was transformed into Escherichia coli containing a derivative of the pET14b vector carrying attP and mj1. This resulted in the formation of chimeric plasmids by site-specific recombination between the cloned attB and attP sequences. The results indicate that the integration vector system based on the site-specific recombination mechanism of phage φFC1 can be used for genetic engineering in E. faecalis and in other hosts.

scholarly journals Differentiation of propeptide residues regulating the compartmentalization, maturation and activity of the broad-range phospholipase C of Listeria monocytogenes

2010 ◽  
Vol 432 (3) ◽  
pp. 557-566 ◽  
Author(s):  
Emily R. Slepkov ◽  
Alan Pavinski Bitar ◽  
Hélène Marquis
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Amino Acid ◽  
Enzymatic Activity ◽  
Phospholipase C ◽  
Bacterial Pathogen ◽  
Amino Acid Residues ◽  
C Terminus ◽  
N Terminus ◽  
Vacuolar Acidification

The intracellular bacterial pathogen Listeria monocytogenes secretes a broad-range phospholipase C enzyme called PC-PLC (phosphatidylcholine phospholipase C) whose compartmentalization and enzymatic activity is regulated by a 24-amino-acid propeptide (Cys28–Ser51). During intracytosolic multiplication, bacteria accumulate the proform of PC-PLC at their membrane–cell-wall interface, whereas during cell-to-cell spread vacuolar acidification leads to maturation and rapid translocation of PC-PLC across the cell wall in a manner that is dependent on Mpl, the metalloprotease of Listeria. In the present study, we generated a series of propeptide mutants to determine the minimal requirement to prevent PC-PLC enzymatic activity and to identify residues regulating compartmentalization and maturation. We found that a single residue at position P1 (Ser51) of the cleavage site is sufficient to prevent enzymatic activity, which is consistent with P1′ (Trp52) being located within the active-site pocket. We observed that mutants with deletions at the N-terminus, but not the C-terminus, of the propeptide are translocated across the cell wall more effectively than wild-type PC-PLC at a physiological pH, and that individual amino acid residues within the N-terminus influence Mpl-mediated maturation of PC-PLC at acidic pH. However, deletion of more than 75% of the propeptide was required to completely prevent Mpl-mediated maturation of PC-PLC. These results indicate that the N-terminus of the propeptide regulates PC-PLC compartmentalization and that specific residues within the N-terminus influence the ability of Mpl to mediate PC-PLC maturation, although a six-residue propeptide is sufficient for Mpl to mediate PC-PLC maturation.

scholarly journals Noncharged amino acid residues at the solvent-exposed positions in the middle and at the C terminus of the α-helix have the same helical propensity

2003 ◽  
Vol 12 (6) ◽  
pp. 1169-1176 ◽  
Author(s):  
Dmitri N. Ermolenko ◽  
John M. Richardson ◽  
George I. Makhatadze
Keyword(s):  
Amino Acid ◽  
Amino Acid Residues ◽  
C Terminus ◽  
Α Helix
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