Characterization of an amino-acid substitution leading to spectinomycin resistance in Escherichia coli

The amikacin resistance gene aac(6')-Im [corrected] from Citrobacter freundii Cf155 encoding an aminoglycoside 6'-N-acetyltransferase was characterized. The gene was identified as a coding sequence of 521 bp located down-stream from the 5' conserved segment of an integron. The sequence of this aac(6')-Im [corrected] gene corresponded to a protein of 173 amino acids which possessed 64.2% identity in a 165-amino-acid overlap with the aac(6')-Ia gene product (F.C. Tenover, D. Filpula, K.L. Phillips, and J. J. Plorde, J. Bacteriol. 170:471-473, 1988). By using PCR, the aac(6')-Im [corrected] gene could be detected in 8 of 86 gram-negative clinical isolates from two Belgian hospitals, including isolates of Citrobacter, Klebsiella spp., and Escherichia coli. PCR mapping of the aac(6')-Im [corrected] gene environment in these isolates indicated that the gene was located within a sulI-type integron; the insert region is 1,700 bases long and includes two genes cassettes, the second being ant (3")-Ib.

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Molecular characterization of long direct repeat (LDR) sequences expressing a stable mRNA encoding for a 35-amino-acid cell-killing peptide and a cis-encoded small antisense RNA in Escherichia coli

Molecular Microbiology ◽

10.1046/j.1365-2958.2002.03042.x ◽

2002 ◽

Vol 45 (2) ◽

pp. 333-349 ◽

Cited By ~ 86

Author(s):

Mitsuoki Kawano ◽

Taku Oshima ◽

Hiroaki Kasai ◽

Hirotada Mori

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Molecular Characterization ◽

Antisense Rna ◽

Direct Repeat ◽

Cell Killing

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Purification and Characterization of the Recombinant Thermus sp. Strain T2 α-Galactosidase Expressed in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.67.4.1601-1616.2001 ◽

2001 ◽

Vol 67 (4) ◽

pp. 1601-1606 ◽

Cited By ~ 31

Author(s):

Mitsunori Ishiguro ◽

Satoshi Kaneko ◽

Atsushi Kuno ◽

Yoshinori Koyama ◽

Shigeki Yoshida ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Polyacrylamide Gel Electrophoresis ◽

Active Form ◽

Gel Filtration ◽

Amino Acid Sequences ◽

Side Chain ◽

Catalytic Function ◽

Native Polyacrylamide Gel Electrophoresis

ABSTRACT The nucleotide sequence of the Thermus sp. strain T2 DNA coding for a thermostable α-galactosidase was determined. The deduced amino acid sequence of the enzyme predicts a polypeptide of 474 amino acids (M r, 53,514). The observed homology between the deduced amino acid sequences of the enzyme and α-galactosidase from Thermus brockianus was over 70%.Thermus sp. strain T2 α-galactosidase was expressed in its active form in Escherichia coli and purified. Native polyacrylamide gel electrophoresis and gel filtration chromatography data suggest that the enzyme is octameric. The enzyme was most active at 75°C forp-nitrophenyl-α-d-galactopyranoside hydrolysis, and it retained 50% of its initial activity after 1 h of incubation at 70°C. The enzyme was extremely stable over a broad range of pH (pH 6 to 13) after treatment at 40°C for 1 h. The enzyme acted on the terminal α-galactosyl residue, not on the side chain residue, of the galactomanno-oligosaccharides as well as those of yeasts and Mortierella vinacea α-galactosidase I. The enzyme has only one Cys residue in the molecule.para-Chloromercuribenzoic acid completely inhibited the enzyme but did not affect the mutant enzyme which contained Ala instead of Cys, indicating that this Cys residue is not responsible for its catalytic function.

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Diversity of TEM Mutants in Proteus mirabilis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.11.2671 ◽

1999 ◽

Vol 43 (11) ◽

pp. 2671-2677 ◽

Cited By ~ 51

Author(s):

R. Bonnet ◽

C. De Champs ◽

D. Sirot ◽

C. Chanal ◽

R. Labia ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Substitution ◽

Proteus Mirabilis ◽

Clinical Isolates ◽

Amino Acid Substitutions ◽

First Report ◽

Conjugative Plasmids ◽

Extended Spectrum ◽

Nucleotide Mutation

ABSTRACT In a survey of resistance to amoxicillin among clinical isolates ofProteus mirabilis, 10 TEM-type β-lactamases were characterized: (i) the well-known penicillinases TEM-1 and TEM-2, the extended-spectrum β-lactamases (ESBLs) TEM-3 and TEM-24, and the inhibitor-resistant TEM (IRT) TEM-44 and (ii) five novel enzymes, a penicillinase TEM-57 similar to TEM-1, an ESBL TEM-66 similar to TEM-3, and three IRTs, TEM-65, TEM-73, and TEM-74. The penicillinase TEM-57 and the ESBL TEM-66 differed from TEM-1 and TEM-3, respectively, by the amino acid substitution Gly-92→Asp (nucleotide mutation G-477→A). This substitution could have accounted for the decrease in pIs (5.2 for TEM-57 and 6.0 for TEM-66) but did not necessarily affect the intrinsic activities of these enzymes. The IRT TEM-65 was an IRT-1-like IRT (Cys-244) related to TEM-2 (Lys-39). The two other IRTs, TEM-73 and TEM-74, were related to IRT-1 (Cys-244) and IRT-2 (Ser-244), respectively, and harbored the amino acid substitutions Leu-21→Phe and Thr-265→Met. In this study, the ESBLs TEM-66, TEM-24, and TEM-3 were encoded by large (170- to 180-kb) conjugative plasmids that exhibited similar patterns after digestion and hybridization with the TEM and AAC(6′)I probes. The three IRTs TEM-65, TEM-73, and TEM-74 were encoded by plasmids that ranged in size from 42 to 70 kb but for which no transfer was obtained. The characterization of five new plasmid-mediated TEM-type β-lactamases and the first report of TEM-24 in P. mirabilis are evidence of the wide diversity of β-lactamases produced in this species and of its possible role as a β-lactamase-encoding plasmid reservoir.

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A single amino acid substitution in aromatic hydroxylase (HpaB) of Escherichia coli alters substrate specificity of the structural isomers of hydroxyphenylacetate

BMC Microbiology ◽

10.1186/s12866-020-01798-4 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Hanseol Kim ◽

Sinyeon Kim ◽

Dohyeon Kim ◽

Sung Ho Yoon

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Substrate Specificity ◽

Amino Acid Substitution ◽

Single Amino Acid Substitution ◽

Single Amino Acid ◽

Structural Isomers

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Comparison of the structural and kinetic properties of the cytochrome c nitrite reductases from Escherichia coli, Wolinella succinogenes, Sulfurospirillum deleyianum and Desulfovibrio desulfuricans

Biochemical Society Transactions ◽

10.1042/bst0340143 ◽

2006 ◽

Vol 34 (1) ◽

pp. 143-145 ◽

Cited By ~ 17

Author(s):

T.A. Clarke ◽

A.M. Hemmings ◽

B. Burlat ◽

J.N. Butt ◽

J.A. Cole ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Active Site ◽

Desulfovibrio Desulfuricans ◽

Amino Acid Sequences ◽

Kinetic Properties ◽

Wolinella Succinogenes ◽

Nitrite Reductases ◽

Sulphite Reductase

The recent crystallographic characterization of NrfAs from Sulfurospirillum deleyianum, Wolinella succinogenes, Escherichia coli and Desulfovibrio desulfuricans allows structurally conserved regions to be identified. Comparison of nitrite and sulphite reductase activities from different bacteria shows that the relative activities vary according to organism. By comparison of both amino acid sequences and structures, differences can be identified in the monomer–monomer interface and the active-site channel; these differences could be responsible for the observed variance in substrate activity and indicate that subtle changes in the NrfA structure may optimize the enzyme for different roles.

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