scholarly journals Phenotypic and Enzymatic Comparative Analysis of the Novel KPC Variant KPC-5 and Its Evolutionary Variants, KPC-2 and KPC-4

2008 ◽  
Vol 53 (2) ◽  
pp. 557-562 ◽  
Author(s):  
Daniel J. Wolter ◽  
Philip M. Kurpiel ◽  
Neil Woodford ◽  
Marie-France I. Palepou ◽  
Richard V. Goering ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Single Amino Acid ◽  
Upstream Region ◽  
The Novel ◽  
Carbapenem Resistant ◽  
Flanking Regions ◽  
Hydrolysis Of ◽  
Additional Amino Acid

ABSTRACT A novel Klebsiella pneumoniae carbapenemase (KPC) variant, designated bla KPC-5, was discovered in a carbapenem-resistant Pseudomonas aeruginosa clinical isolate from Puerto Rico. Characterization of the upstream region of bla KPC-5 showed significant differences from the flanking regions of other bla KPC variants. Comparison of amino acid sequences with those of other KPC enzymes revealed that KPC-5 was an intermediate between KPC-2 and KPC-4, differing from KPC-2 by a single amino acid substitution (Pro103→Arg), while KPC-4 contained Pro103→Arg plus an additional amino acid change (Val239→Gly). Transformation studies with an Escherichia coli recipient strain showed differences in the properties of the KPC variants. KPC-4 and KPC-5 both had pIs of 7.65, in contrast with the pI of 6.7 for KPC-2. KPC-2 transformants were less susceptible to the carbapenems than KPC-4 and KPC-5 transformants. These data correlated with higher rates of imipenem hydrolysis for KPC-2 than for KPC-4 and KPC-5. However, KPC-4 and KPC-5 transformants had higher ceftazidime MICs, and the enzymes from these transformants had slightly better hydrolysis of this drug than KPC-2. KPC-4 and KPC-5 were more sensitive than KPC-2 to inhibition by clavulanic acid in both susceptibility testing and hydrolysis assays. Thus, KPC enzymes may be evolving through stepwise mutations to alter their spectra of activity.

scholarly journals Biochemical and Molecular Characterization of Three New Variants of AmpC β-Lactamases from Morganella morganii

2006 ◽  
Vol 50 (3) ◽  
pp. 962-967 ◽  
Author(s):  
Pablo Power ◽  
Moreno Galleni ◽  
Juan A. Ayala ◽  
Gabriel Gutkind
Keyword(s):  
Amino Acid ◽  
Catalytic Properties ◽  
Amino Acid Sequences ◽  
Major Influence ◽  
Morganella Morganii ◽  
Encoding Gene ◽  
Hydrolysis Of ◽  
Similar Kinetic

ABSTRACT Morganella morganii produces an inducible, chromosomally encoded AmpC β-lactamase. We describe in this study three new variants of AmpC within this species with apparent pIs of 6.6 (M19 from M. morganii strain PP19), 7.4 (M29 from M. morganii strain PP29), and 7.8 (M37 from M. morganii strain PP37). After gene sequencing, deduced amino acid sequences displayed one to six substitutions when compared to the available Morganella AmpC sequences. An AmpR-encoding gene was also found upstream of ampC, including the LysR regulators' helix-turn-helix DNA-binding domain and the putative T-N11-A-protected region in the ampR-ampC intercistronic sequence. All three AmpC variants were purified from in vitro-generated derepressed mutants and showed overall similar kinetic parameters. None of the observed amino acid changes, occurring at the surface of the protein, appear to have a major influence in their catalytic properties. Morganella AmpCs exhibit the highest catalytic efficiencies (kcat /Km ) on classical penicillins, cefoxitin, narrow-spectrum cephalosporins, and cefotaxime. Cefotaxime was more effectively hydrolyzed than other oxyimino-cephalosporins, whereas cefepime was 3 log-fold less efficiently hydrolyzed than other cephalosporins such as cephalothin. Several differences with other AmpC β-lactamases were found. Ampicillin was more efficiently hydrolyzed than benzylpenicillin. High kcat /Km values were observed for oxacillin and piperacillin, which are usually poor substrates for AmpC. A fairly efficient hydrolysis of imipenem was detected as well. Aztreonam, carbenicillin, and tazobactam were effective transient inactivators of these variants.

scholarly journals Carbapenem-Resistant Acinetobacter baumannii Isolates from Tunisia Producing the OXA-58-Like Carbapenem-Hydrolyzing Oxacillinase OXA-97

2008 ◽  
Vol 52 (5) ◽  
pp. 1613-1617 ◽  
Author(s):  
Laurent Poirel ◽  
Wejdene Mansour ◽  
Olfa Bouallegue ◽  
Patrice Nordmann
Keyword(s):  
Amino Acid ◽  
Acinetobacter Baumannii ◽  
Amino Acid Substitution ◽  
Multidrug Resistant ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
The Novel ◽  
Cloning And Sequencing ◽  
Carbapenem Resistant ◽  
Genetic Structures

ABSTRACT The basis of the β-lactam resistance of 39 multidrug-resistant Acinetobacter baumannii isolates recovered from hospitalized patients was studied. These isolates were collected from 2001 to 2005 at the Sahloul Hospital in Sousse, Tunisia. They belonged to two distinct clones. One clone that grouped 19 isolates produced a carbapenem-hydrolyzing oxacillinase, OXA-97, that differed from OXA-58 by a single amino acid substitution and conferred the same β-lactam resistance profile as OXA-58. The bla OXA-97 gene was located on plasmids that varied in size in 18 isolates and was chromosomally located in a single isolate. Cloning and sequencing identified genetic structures surrounding the bla OXA-97 gene similar to those reported to be adjacent to the bla OXA-58 gene. In addition, the novel ISAba8 element (which is of the IS21 family) was identified. This is the first report of the nosocomial spread of carbapenemase producers in A. baumannii isolates in Africa.

scholarly journals Characterization of the Gallate Dioxygenase Gene: Three Distinct Ring Cleavage Dioxygenases Are Involved in Syringate Degradation by Sphingomonas paucimobilis SYK-6

2005 ◽  
Vol 187 (15) ◽  
pp. 5067-5074 ◽  
Author(s):  
Daisuke Kasai ◽  
Eiji Masai ◽  
Keisuke Miyauchi ◽  
Yoshihiro Katayama ◽  
Masao Fukuda
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Terminal Region ◽  
Ring Cleavage ◽  
Sphingomonas Paucimobilis ◽  
Acid Protein ◽  
Demethylation Pathway ◽  
Dioxygenase Gene ◽  
Amino Acid Protein

ABSTRACT Sphingomonas paucimobilis SYK-6 converts vanillate and syringate to protocatechuate (PCA) and 3-O-methylgallate (3MGA) in reactions with the tetrahydrofolate-dependent O-demethylases LigM and DesA, respectively. PCA is further degraded via the PCA 4,5-cleavage pathway, whereas 3MGA is metabolized via three distinct pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and 3MGA O-demethylase (LigM) are involved. In the 3MGA O-demethylation pathway, LigM converts 3MGA to gallate, and the resulting gallate appears to be degraded by a dioxygenase other than LigAB or DesZ. Here, we isolated the gallate dioxygenase gene, desB, which encodes a 418-amino-acid protein with a molecular mass of 46,843 Da. The amino acid sequences of the N-terminal region (residues 1 to 285) and the C-terminal region (residues 286 to 418) of DesB exhibited ca. 40% and 27% identity with the sequences of the PCA 4,5-dioxygenase β and α subunits, respectively. DesB produced in Escherichia coli was purified and was estimated to be a homodimer (86 kDa). DesB specifically attacked gallate to generate 4-oxalomesaconate as the reaction product. The Km for gallate and the V max were determined to be 66.9 ± 9.3 μM and 42.7 ± 2.4 U/mg, respectively. On the basis of the analysis of various SYK-6 mutants lacking the genes involved in syringate degradation, we concluded that (i) all of the three-ring cleavage dioxygenases are involved in syringate catabolism, (ii) the pathway involving LigM and DesB plays an especially important role in the growth of SYK-6 on syringate, and (iii) DesB and LigAB are involved in gallate degradation.

scholarly journals Molecular Cloning and Characterization of Bifidobacterium bifidum 1,2-α-l-Fucosidase (AfcA), a Novel Inverting Glycosidase (Glycoside Hydrolase Family 95)

2004 ◽  
Vol 186 (15) ◽  
pp. 4885-4893 ◽  
Author(s):  
Takane Katayama ◽  
Akiko Sakuma ◽  
Takatoshi Kimura ◽  
Yutaka Makimura ◽  
Jun Hiratake ◽  
...  
Keyword(s):  
Amino Acid ◽  
Genomic Library ◽  
Bifidobacterium Bifidum ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Sugar Chain ◽  
Hydrolysis Of

ABSTRACT A genomic library of Bifidobacterium bifidum constructed in Escherichia coli was screened for the ability to hydrolyze the α-(1→2) linkage of 2′-fucosyllactose, and a gene encoding 1,2-α-l-fucosidase (AfcA) was isolated. The afcA gene was found to comprise 1,959 amino acid residues with a predicted molecular mass of 205 kDa and containing a signal peptide and a membrane anchor at the N and C termini, respectively. A domain responsible for fucosidase activity (the Fuc domain; amino acid residues 577 to 1474) was localized by deletion analysis and then purified as a hexahistidine-tagged protein. The recombinant Fuc domain specifically hydrolyzed the terminal α-(1→2)-fucosidic linkages of various oligosaccharides and a sugar chain of a glycoprotein. The stereochemical course of the hydrolysis of 2′-fucosyllactose was determined to be inversion by using 1H nuclear magnetic resonance. The primary structure of the Fuc domain exhibited no similarity to those of any glycoside hydrolases (GHs) but showed high similarity to those of several hypothetical proteins in a database. Thus, it was revealed that the AfcA protein constitutes a novel inverting GH family (GH family 95).

Functional and Structural Characterization of Acquired Pan-Aminoglycoside Resistance 16S rRNA Methyltransferase NpmB1

2021 ◽  
Author(s):  
Akito Kawai ◽  
Masahiro Suzuki ◽  
Kentaro Tsukamoto ◽  
Yusuke Minato ◽  
Yohei Doi
Keyword(s):  
Amino Acid ◽  
16S Rrna ◽  
Amino Acid Identity ◽  
Single Amino Acid ◽  
Aminoglycoside Resistance ◽  
E Coli ◽  
The United Kingdom ◽  
Amino Acid Variant ◽  
A Site

Post-translational methylation of the A site of 16S rRNA at position A1408 leads to pan-aminoglycoside resistance encompassing both 4,5- and 4,6-disubstituted 2-deoxystreptamine (DOS) aminoglycosides. To date, NpmA is the only acquired enzyme with such function. Here, we present function and structure of NpmB1 whose sequence was identified in Escherichia coli genomes registered from the United Kingdom. NpmB1 possesses 40% amino acid identity with NpmA1 and confers resistance to all clinically relevant aminoglycosides including 4,5-DOS agents. Phylogenetic analysis of NpmB1 and NpmB2, its single amino acid variant, revealed that the encoding gene was likely acquired by E. coli from a soil bacterium. The structure of NpmB1 suggests that it requires a structural change of the β6/7 linker in order to bind to 16S rRNA. These findings establish NpmB1 and NpmB2 as the second group of acquired pan-aminoglycoside resistance 16S rRNA methyltransferases.

Biochemical and genetic characterization of a yeast TFIID mutant that alters transcription in vivo and DNA binding in vitro

1992 ◽  
Vol 12 (5) ◽  
pp. 2372-2382
Author(s):  
K M Arndt ◽  
S L Ricupero ◽  
D M Eisenmann ◽  
F Winston
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Direct Repeat ◽  
Single Amino Acid ◽  
Wild Type ◽  
Dna Binding Specificity ◽  
Selection For

A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent spt15 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition.

scholarly journals Molecular characterization of Prunus necrotic ringspot virus isolated from rose in Brazil

2015 ◽  
Vol 45 (12) ◽  
pp. 2197-2200 ◽  
Author(s):  
Thor Vinícius Martins Fajardo ◽  
Monique Bezerra Nascimento ◽  
Marcelo Eiras ◽  
Osmar Nickel ◽  
Gilvan Pio-Ribeiro
Keyword(s):  
Amino Acid ◽  
Molecular Characterization ◽  
Molecular Analysis ◽  
Nucleotide Sequences ◽  
Amino Acid Sequences ◽  
Causal Agent ◽  
Ringspot Virus ◽  
Prunus Necrotic Ringspot Virus ◽  
First Time

ABSTRACT: There is no molecular characterization of Brazilian isolates of Prunus necrotic ringspot virus (PNRSV), except for those infecting peach. In this research, the causal agent of rose mosaic was determined and the movement (MP) and coat (CP) protein genes of a PNRSV isolate from rose were molecularly characterized for the first time in Brazil. The nucleotide and deduced amino acid sequences of MP and CP complete genes were aligned and compared with other isolates. Molecular analysis of the MP and CP nucleotide sequences of a Brazilian PNRSV isolate from rose and others from this same host showed highest identities of 96.7% and 98.6%, respectively, and Rose-Br isolate was classified in PV32 group.

scholarly journals Single amino acid exchanges in separate domains of the Drosophila serendipity delta zinc finger protein cause embryonic and sex biased lethality.

Genetics ◽  
1992 ◽  
Vol 131 (4) ◽  
pp. 905-916 ◽  
Author(s):  
M Crozatier ◽  
K Kongsuwan ◽  
P Ferrer ◽  
J R Merriam ◽  
J A Lengyel ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Essential Gene ◽  
Larval Instar ◽  
Single Amino Acid ◽  
Isolation And Characterization ◽  
Finger Protein

Abstract The Drosophila serendipity (sry) delta (delta) zinc finger protein is a sequence-specific DNA binding protein, maternally inherited by the embryo and present in nuclei of transcriptionally active cells throughout fly development. We report here the isolation and characterization of four ethyl methanesulfate-induced zygotic lethal mutations of different strengths in the sry delta gene. For the stronger allele, all of the lethality occurs during late embryogenesis or the first larval instar. In the cases of the three weaker alleles, most of the lethality occurs during pupation; moreover, those adult escapers that emerge are sterile males lacking partially or completely in spermatozoa bundles. Genetic analysis of sry delta thus indicates that it is an essential gene, whose continued expression throughout the life cycle, notably during embryogenesis and pupal stage, is required for viability. Phenotypic analysis of sry delta hemizygote escaper males further suggests that sry delta may be involved in regulation of two different sets of genes: genes required for viability and genes involved in gonadal development. All four sry delta alleles are fully rescued by a wild-type copy of sry delta, but not by an additional copy of the sry beta gene, reinforcing the view that, although structurally related, these two genes exert distinct functions. Molecular characterization of the four sry delta mutations revealed that these mutations correspond to single amino acid replacements in the sry delta protein. Three of these replacements map to the same (third out of seven) zinc finger in the carboxy-terminal DNA binding domain; interestingly, none affects the zinc finger consensus residues. The fourth mutation is located in the NH2-proximal part of the protein, in a domain proposed to be involved in specific protein-protein interactions.

scholarly journals In vitro and In Silico Approach For Characterization of Antimicrobial Peptide From Probiotics Against Staphylococcus Aureus and Escherichia Coli

2021 ◽  
Author(s):  
Amrutha Bindu ◽  
Lakshmi Devi
Keyword(s):  
Amino Acid ◽  
Antimicrobial Peptide ◽  
Lactobacillus Plantarum ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Proteinase K ◽  
Kinetic Assay ◽  
Wide Range

Abstract The focus of present study was to characterize antimicrobial peptide produced by probiotic cultures, Enterococcus durans DB-1aa (MCC4243), Lactobacillus plantarum Cu2-PM7 (MCC4246) and Lactobacillus fermentum Cu3-PM8 (MCC4233) against Staphylococus aureus and E. coli. The growth kinetic assay revealed 24 h of incubation to be optimum for bacteriocin production. The partially purified compound after ion-exchange chromatography was found to be thermoresistant and stable under wide range of pH. The compound was sensitive to proteinase-K, but resistant to trypsin, a-amylase and lipase. The apparent molecular weight of bacteriocin from MCC4243 and MCC4246 was found to be 3.5 KDa. Translated partial amino acid sequence of plnA gene in MCC4246 displayed 48 amino acid sequences showing 100% similarity with plantaricin A of Lactobacillus plantarum (WP_0036419). The sequence revealed 7 β sheets, 6 α sheets, 6 predicted coils and 9 predicted turns. The functions on cytoplasm show 10.82 isoelectric point and 48.6% hydrophobicity. The molecular approach of using Geneious Prime software and protein prediction data base for characterization of bacteriocin is novel and predicts “KSSAYSLQMGATAIKQVKKLFKKWGW” as peptide responsible for antimicrobial activity. The study provides information about broad spectrum bacteriocin in native probiotic culture and paves a way towards its application in functional foods as biopreservative agents.

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