scholarly journals Molecular Cloning and Characterization of P4 Nuclease from Leishmania infantum

2011 ◽  
Vol 2011 ◽  
pp. 1-6 ◽  
Author(s):  
Safar Farajnia ◽  
Leila Rahbarnia ◽  
Bahram Maleki zanjani ◽  
Mohammad Hossein Alimohammadian ◽  
Shahin Abdoli Oskoee ◽  
...  
Keyword(s):  
Leishmania Infantum ◽  
Drug Target ◽  
Sequence Similarity ◽  
Leishmania Species ◽  
Class I ◽  
Gene Encoding ◽  
E Coli ◽  
Essential Enzyme ◽  
Sequence Similarity Analysis

Parasite of the genus Leishmania is reliant on the salvage pathway for recycling of ribonucleotides. A class I nuclease enzyme also known as P4 nuclease is involved in salvage of purines in cutaneous Leishmania species but the relevant enzymes have not been characterized in Leishmania infantum (L. infantum). The aim of this study was to clone and characterize the gene encoding class I nuclease in L. infantum. DNA extracted from L. infantum was used for amplification of P4 nuclease gene (Li-P4) by PCR. The product was cloned, sequenced, and expressed in E. coli for further characterization. Analysis of the sequence of Li-P4 revealed that the gene consists of an ORF of 951 bp. Sequence similarity analysis indicated that Li-P4 has a high homology to relevant enzymes of other kintoplastids with the highest homology (88%) to p1/s1 class I nuclease from L. donovani. Western blotting of antirecombinant Li-P4 with promastigote and amastigote stages of L. infantum showed that this nuclease is present in both stages of parasite with higher expression in amastigote stage. The highly conserved nature of this essential enzyme in Leishmania parasites suggests it as a promising drug target for leishmaniasis.

scholarly journals Characterization of Flagellotropic, Chi-Like Salmonella Phages Isolated from Thai Poultry Farms

Viruses ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 520 ◽  
Author(s):  
Preeda Phothaworn ◽  
Matthew Dunne ◽  
Rattaya Supokaivanich ◽  
Catherine Ong ◽  
Jiali Lim ◽  
...  
Keyword(s):  
Salmonella Enterica Serovar Typhimurium ◽  
Sequence Similarity ◽  
Phage Type ◽  
Pcr Amplification ◽  
Phylogenomic Analysis ◽  
Monophyletic Clade ◽  
Gene Encoding ◽  
Poultry Farms ◽  
Serovar Typhimurium

Despite a wealth of knowledge on Salmonella phages worldwide, little is known about poultry-associated Salmonella phages from Thailand. Here, we isolated 108 phages from Thai poultry farms that infect Salmonella enterica serovar Typhimurium. Phages STm101 and STm118 were identified as temperate Siphoviridae phages. Genome sequencing and analyses revealed these phages share approximately 96% nucleotide sequence similarity to phage SPN19, a member of the Chi-like virus genus. PCR amplification of the gene encoding capsid protein E of the Chi-like phage was positive for 50% of phage isolates, suggesting a predominance of this phage type among the sampled poultry farms. In addition to the flagella, two phages required the lipopolysaccharide to infect and lyse Salmonella. Furthermore, phylogenomic analysis demonstrated that phages STm101 and STm118 formed a monophyletic clade with phages isolated from Western countries, but not from closer isolated phages from Korea. However, further investigation and more phage isolates are required to investigate possible causes for this geographic distribution.

Functional characterization of the Haemophilus influenzae 4.5S RNA

1998 ◽  
Vol 44 (1) ◽  
pp. 91-94
Author(s):  
G Scott Jenkins ◽  
Mark S Chandler ◽  
Pamela S Fink
Keyword(s):  
Haemophilus Influenzae ◽  
Functional Characterization ◽  
Coli Strain ◽  
Northern Analysis ◽  
Gene Encoding ◽  
E Coli ◽  
Functional Homolog ◽  
Polymerase Chain ◽  
4.5S Rna

The putative 4.5S RNA of Haemophilus influenzae was identified in the genome by computer analysis, amplified by the polymerase chain reaction, and cloned. We have determined that this putative 4.5S RNA will complement an Escherichia coli strain conditionally defective in 4.5S RNA production. The predicted secondary structures of the molecules were quite similar, but Northern analysis showed that the H. influenzae RNA was slightly larger than the E. coli RNA. The H. influenzae gene encoding this RNA is the functional homolog of the ffs gene in E. coli. Key words: ffs gene, complementation studies, small RNA, prokaryotic genetics.

scholarly journals Characterization of Multidrug-Resistant Escherichia coli Isolates from Animals Presenting at a University Veterinary Hospital

2011 ◽  
Vol 77 (20) ◽  
pp. 7104-7112 ◽  
Author(s):  
Maria Karczmarczyk ◽  
Yvonne Abbott ◽  
Ciara Walsh ◽  
Nola Leonard ◽  
Séamus Fanning
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanisms ◽  
Gene Array ◽  
Multidrug Resistant ◽  
Genetic Determinants ◽  
Gene Encoding ◽  
Content Type ◽  
E Coli ◽  
Class 1

ABSTRACTIn this study, we examined molecular mechanisms associated with multidrug resistance (MDR) in a collection ofEscherichia coliisolates recovered from hospitalized animals in Ireland. PCR and DNA sequencing were used to identify genes associated with resistance. Class 1 integrons were prevalent (94.6%) and contained gene cassettes recognized previously and implicated mainly in resistance to aminoglycosides, β-lactams, and trimethoprim (aadA1,dfrA1-aadA1,dfrA17-aadA5,dfrA12-orfF-aadA2,blaOXA-30-aadA1,aacC1-orf1-orf2-aadA1,dfr7). Class 2 integrons (13.5%) contained thedfrA1-sat1-aadA1gene array. The most frequently occurring phenotypes included resistance to ampicillin (97.3%), chloramphenicol (75.4%), florfenicol (40.5%), gentamicin (54%), neomycin (43.2%), streptomycin (97.3%), sulfonamide (98.6%), and tetracycline (100%). The associated resistance determinants detected includedblaTEM,cat,floR,aadB,aphA1,strA-strB,sul2, andtet(B), respectively. TheblaCTX-M-2gene, encoding an extended-spectrum β-lactamase (ESβL), andblaCMY-2, encoding an AmpC-like enzyme, were identified in 8 and 18 isolates, respectively. The mobility of the resistance genes was demonstrated using conjugation assays with a representative selection of isolates. High-molecular-weight plasmids were found to be responsible for resistance to multiple antimicrobial compounds. The study demonstrated that animal-associated commensalE. coliisolates possess a diverse repertoire of transferable genetic determinants. Emergence of ESβLs and AmpC-like enzymes is particularly significant. To our knowledge, theblaCTX-M-2gene has not previously been reported in Ireland.

scholarly journals Functional Characterization of Bacterial Oligosaccharyltransferases Involved in O-Linked Protein Glycosylation

2007 ◽  
Vol 189 (22) ◽  
pp. 8088-8098 ◽  
Author(s):  
Amirreza Faridmoayer ◽  
Messele A. Fentabil ◽  
Dominic C. Mills ◽  
John S. Klassen ◽  
Mario F. Feldman
Keyword(s):  
Sequence Similarity ◽  
Functional Characterization ◽  
Protein Glycosylation ◽  
Structural Components ◽  
En Bloc ◽  
E Coli ◽  
New Family ◽  
Type Iv ◽  
Domains Of Life

ABSTRACT Protein glycosylation is an important posttranslational modification that occurs in all domains of life. Pilins, the structural components of type IV pili, are O glycosylated in Neisseria meningitidis, Neisseria gonorrhoeae, and some strains of Pseudomonas aeruginosa. In this work, we characterized the P. aeruginosa 1244 and N. meningitidis MC58 O glycosylation systems in Escherichia coli. In both cases, sugars are transferred en bloc by an oligosaccharyltransferase (OTase) named PglL in N. meningitidis and PilO in P. aeruginosa. We show that, like PilO, PglL has relaxed glycan specificity. Both OTases are sufficient for glycosylation, but they require translocation of the undecaprenol-pyrophosphate-linked oligosaccharide substrates into the periplasm for activity. Whereas PilO activity is restricted to short oligosaccharides, PglL is able to transfer diverse oligo- and polysaccharides. This functional characterization supports the concept that despite their low sequence similarity, PilO and PglL belong to a new family of “O-OTases” that transfer oligosaccharides from lipid carriers to hydroxylated amino acids in proteins. To date, such activity has not been identified for eukaryotes. To our knowledge, this is the first report describing recombinant O glycoproteins synthesized in E. coli.

scholarly journals Identification and characterization of the gene encoding the human phosphopantetheine adenylyltransferase and dephospho-CoA kinase bifunctional enzyme (CoA synthase)

2002 ◽  
Vol 365 (1) ◽  
pp. 13-18 ◽  
Author(s):  
Suren AGHAJANIAN ◽  
D.Margaret WORRALL
Keyword(s):  
Biosynthetic Pathway ◽  
Sequence Similarity ◽  
Kinetic Properties ◽  
Sequence Information ◽  
Bifunctional Enzyme ◽  
Gene Encoding ◽  
Pig Liver ◽  
Identification And Characterization ◽  
Similar Kinetic

The final two enzymes in the CoA biosynthetic pathway, phosphopantetheine adenylyltransferase (PPAT; EC 2.7.7.3) and dephospho-CoA kinase (DPCK; EC 2.7.1.24), are separate proteins in prokaryotes, but exist as a bifunctional enzyme in pig liver. In the present study we have obtained sequence information from purified pig-liver enzyme, and identified the corresponding cDNA in a number of species. The human gene localizes to chromosome 17q12-21 and contains regions with sequence similarity to the monofunctional Escherichia coli DPCK and PPAT. The recombinant 564-amino-acid human protein confirmed the associated transferase and kinase activities, and gave similar kinetic properties to the wild-type pig enzyme.

scholarly journals Characterization of Glucose-Specific Catabolite Repression-Resistant Mutants of Bacillus subtilis: Identification of a Novel Hexose:H+ Symporter

1998 ◽  
Vol 180 (3) ◽  
pp. 498-504 ◽  
Author(s):  
Ian T. Paulsen ◽  
Sylvie Chauvaux ◽  
Peter Choi ◽  
Milton H. Saier
Keyword(s):  
Bacillus Subtilis ◽  
Catabolite Repression ◽  
Genetic Background ◽  
Insertional Mutagenesis ◽  
Sequence Similarity ◽  
Major Facilitator Superfamily ◽  
Phosphotransferase System ◽  
E Coli ◽  
Major Facilitator

ABSTRACT Insertional mutagenesis was conducted on Bacillus subtilis cells to screen for mutants resistant to catabolite repression. Three classes of mutants that were resistant to glucose-promoted but not mannitol-promoted catabolite repression were identified. Cloning and sequencing of the mutated genes revealed that the mutations occurred in the structural genes for (i) enzyme II of the phosphoenolpyruvate-glucose phosphotransferase (PtsG), (ii) antiterminator GlcT, which controls PtsG synthesis, and (iii) a previously uncharacterized carrier of the major facilitator superfamily, which we have designated GlcP. The last protein exhibits greatest sequence similarity to the fucose:H+ symporter ofEscherichia coli and the glucose/galactose:H+symporter of Brucella abortus. In a wild-type B. subtilis genetic background, theglcP::Tn10 mutation (i) partially but specifically relieved glucose- and sucrose-promoted catabolite repression, (ii) reduced the growth rate in minimal glucose medium, and (iii) reduced rates of [14C]glucose and [14C]methyl α-glucoside uptake. In a Δptsgenetic background no phenotype was observed, suggesting that expression of the glcP gene required a functional phosphotransferase system. When overproduced in a Δptsmutant of E. coli, GlcP could be shown to specifically transport glucose, mannose, 2-deoxyglucose and methyl α-glucoside with low micromolar affinities. Accumulation of the nonmetabolizable glucose analogs was demonstrated, and inhibitor studies suggested a dependency on the proton motive force. We conclude that B. subtilis possesses at least two distinct routes of glucose entry, both of which contribute to the phenomenon of catabolite repression.

scholarly journals Characterization of the hca Cluster Encoding the Dioxygenolytic Pathway for Initial Catabolism of 3-Phenylpropionic Acid in Escherichia coliK-12

1998 ◽  
Vol 180 (11) ◽  
pp. 2915-2923 ◽  
Author(s):  
Eduardo Díaz ◽  
Abel Ferrández ◽  
José L. García
Keyword(s):  
Sequence Similarity ◽  
Recombinant Bacteria ◽  
Sequence Comparisons ◽  
Phenylpropionic Acid ◽  
E Coli ◽  
Catabolic Genes ◽  
Dihydrodiol Dehydrogenase ◽  
Class Iib ◽  
K 12

ABSTRACT We have identified, cloned, and sequenced the hcacluster encoding the dioxygenolytic pathway for initial catabolism of 3-phenylpropionic acid (PP) in Escherichia coli K-12. This cluster maps at min 57.5 of the chromosome and is composed of five catabolic genes arranged as a putative operon (hcaA1A2CBD) and two additional genes transcribed in the opposite direction that encode a potential permease (hcaT) and a regulator (hcaR). Sequence comparisons revealed that whilehcaA1A2CD genes encode the four subunits of the 3-phenylpropionate dioxygenase, the hcaB gene codes for the corresponding cis-dihydrodiol dehydrogenase. This type of catabolic module is homologous to those encoding class IIB dioxygenases and becomes the first example of such a catabolic cluster in E. coli. The inducible expression of the hca genes requires the presence of the hcaR gene product, which acts as a transcriptional activator and shows significant sequence similarity to members of the LysR family of regulators. Interestingly, the HcaA1A2CD and HcaB enzymes are able to oxidize not only PP to 3-(2,3-dihydroxyphenyl)propionate (DHPP) but also cinnamic acid (CI) to its corresponding 2,3-dihydroxy derivative. Further catabolism of DHPP requires the mhp-encoded meta fission pathway for the mineralization of 3-hydroxyphenylpropionate (3HPP) (A. Ferrández, J. L. Garcı́a, and E. Dı́az, J. Bacteriol. 179:2573–2581, 1997). Expression in Salmonella typhimurium of the mhp genes alone or in combination with the hca cluster allowed the growth of the recombinant bacteria in 3-hydroxycinnamic acid (3HCI) and CI, respectively. Thus, the convergent mhp- and hca-encoded pathways are also functional in S. typhimurium, and they are responsible for the catabolism of different phenylpropanoid compounds (3HPP, 3HCI, PP, and CI) widely available in nature.

scholarly journals Identification of the rrmA Gene Encoding the 23S rRNA m1G745 Methyltransferase in Escherichia coli and Characterization of an m1G745-Deficient Mutant

1998 ◽  
Vol 180 (2) ◽  
pp. 359-365 ◽  
Author(s):  
Claes Gustafsson ◽  
Britt C. Persson
Keyword(s):  
Escherichia Coli ◽  
Polypeptide Chain ◽  
Chain Elongation ◽  
23S Rrna ◽  
Drastic Reduction ◽  
Gene Encoding ◽  
E Coli ◽  
Loosely Coupled ◽  
Rich Media

ABSTRACT An Escherichia coli mutant lacking the modified nucleotide m1G in rRNA has previously been isolated (G. R. Björk and L. A. Isaksson, J. Mol. Biol. 51:83–100, 1970). In this study, we localize the position of the m1G to nucleotide 745 in 23S rRNA and characterize a mutant deficient in this modification. This mutant shows a 40% decreased growth rate in rich media, a drastic reduction in loosely coupled ribosomes, a 20% decreased polypeptide chain elongation rate, and increased resistance to the ribosome binding antibiotic viomycin. TherrmA gene encoding 23S rRNA m1G745 methyltransferase was mapped to bp 1904000 on the E. colichromosome and identified to be identical to the previously sequenced gene yebH.

scholarly journals Characterization of SFO-1, a Plasmid-Mediated Inducible Class A β-Lactamase from Enterobacter cloacae

1999 ◽  
Vol 43 (2) ◽  
pp. 307-313 ◽  
Author(s):  
Yoshimi Matsumoto ◽  
Matsuhisa Inoue
Keyword(s):  
Klebsiella Oxytoca ◽  
Enterobacter Cloacae ◽  
Gram Negative Bacteria ◽  
Phenotypic Characteristics ◽  
Gene Encoding ◽  
E Coli ◽  
Class A ◽  
Citrobacter Diversus ◽  
High Degree

ABSTRACT Enterobacter cloacae 8009 produced an inducible class A β-lactamase which hydrolyzed cefotaxime efficiently. It also hydrolyzed other β-lactams except cephamycins and carbapenems. The activity was inhibited by clavulanic acid and imipenem. Thebla gene was transferable to Escherichia coliby electroporation of plasmid DNA. The molecular mass of the β-lactamase was 29 kDa and its pI was 7.3. All of these phenotypic characteristics of the enzyme except for inducible production resemble those of some extended-spectrum class A β-lactamases like FEC-1. The gene encoding this β-lactamase was cloned and sequenced. The deduced amino acid sequence of the β-lactamase was homologous to the AmpA sequences of the Serratia fonticola chromosomal enzyme (96%), MEN-1 (78%), Klebsiella oxytoca chromosomal enzymes (77%), TOHO-1 (75%), and FEC-1 (72%). The conserved sequences of class A β-lactamases, including the S-X(T)-X(S)-K motif, in the active site were all conserved in this enzyme. On the basis of the high degree of homology to the β-lactamase of S. fonticola, the enzyme was named SFO-1. The ampR gene was located upstream of the ampA gene, and the AmpR sequence of SFO-1 had homology with the AmpR sequences of the chromosomal β-lactamases from Citrobacter diversus(80%), Proteus vulgaris (68%), and Pseudomonas aeruginosa (60%). SFO-1 was also inducible in E. coli. However, a transformant harboring plasmid without intactampR produced a small amount of β-lactamase constitutively, suggesting that AmpR works as an activator ofampA of SFO-1. This is the first report from Japan describing an inducible plasmid-mediated class A β-lactamase in gram-negative bacteria.

scholarly journals Genetic and Biochemical Characterization of Salmonella enterica Serovar Typhi Deoxyribokinase

2000 ◽  
Vol 182 (4) ◽  
pp. 869-873 ◽  
Author(s):  
Lise Tourneux ◽  
Nadia Bucurenci ◽  
Cosmin Saveanu ◽  
Pierre Alexandre Kaminski ◽  
Madeleine Bouzon ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Biochemical Characterization ◽  
Site Directed Mutagenesis ◽  
Gene Encoding ◽  
E Coli ◽  
Salmonella Enterica Serovar Typhi ◽  
Acid Protein ◽  
Serovar Typhimurium ◽  
Amino Acid Protein

ABSTRACT We identified in the genome of Salmonella entericaserovar Typhi the gene encoding deoxyribokinase, deoK. Two other genes, vicinal to deoK, were determined to encode the putative deoxyribose transporter (deoP) and a repressor protein (deoQ). This locus, located between theuhpA and ilvN genes, is absent inEscherichia coli. The deoK gene inserted on a plasmid provides a selectable marker in E. coli for growth on deoxyribose-containing medium. Deoxyribokinase is a 306-amino-acid protein which exhibits about 35% identity with ribokinase from serovar Typhi, S. enterica serovar Typhimurium, or E. coli. The catalytic properties of the recombinant deoxyribokinase overproduced in E. colicorrespond to those previously described for the enzyme isolated from serovar Typhimurium. From a sequence comparison between serovar Typhi deoxyribokinase and E. coliribokinase, whose crystal structure was recently solved, we deduced that a key residue differentiating ribose and deoxyribose is Met10, which in ribokinase is replaced by Asn14. Replacement by site-directed mutagenesis of Met10 with Asn decreased theV max of deoxyribokinase by a factor of 2.5 and increased the K m for deoxyribose by a factor of 70, compared to the parent enzyme.

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