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 FRI-1, a Carbapenem-Hydrolyzing Class A β-Lactamase from Enterobacter cloacae

2015 ◽  
Vol 59 (12) ◽  
pp. 7420-7425 ◽  
Author(s):  
Laurent Dortet ◽  
Laurent Poirel ◽  
Samia Abbas ◽  
Saoussen Oueslati ◽  
Patrice Nordmann
Keyword(s):  
Biochemical Characterization ◽  
Enterobacter Cloacae ◽  
Conjugative Plasmid ◽  
Lower Sensitivity ◽  
Gene Encoding ◽  
Content Type ◽  
Class A ◽  
Encoding Gene ◽  
Lactamase Gene

ABSTRACTAnEnterobacter cloacaeisolate was recovered from a rectal swab from a patient hospitalized in France with previous travel to Switzerland. It was resistant to penicillins, narrow- and broad-spectrum cephalosporins, aztreonam, and carbapenems but remained susceptible to expanded-spectrum cephalosporins. Whereas PCR-based identification of the most common carbapenemase genes failed, the biochemical Carba NP test II identified an Ambler class A carbapenemase. Cloning experiments followed by sequencing identified a gene encoding a totally novel class A carbapenemase, FRI-1, sharing 51 to 55% amino acid sequence identity with the closest carbapenemase sequences. However, it shared conserved residues as a source of carbapenemase activity. Purified β-lactamase FRI-1 hydrolyzed penicillins, aztreonam, and carbapenems but spared expanded-spectrum cephalosporins. The 50% inhibitory concentrations (IC50s) of clavulanic acid and tazobactam were 10-fold higher than those found forKlebsiella pneumoniaecarbapenemase (KPC), IMI, and SME, leading to lower sensitivity of FRI-1 activity to β-lactamase inhibitors. TheblaFRI-1gene was located on a ca. 110-kb untypeable, transferable, and non-self-conjugative plasmid. A putative LysR family regulator-encoding gene at the 5′ end of the β-lactamase gene was identified, leading to inducible expression of theblaFRI-1gene.

scholarly journals Modulation of transcription and characterization of the promoter organization of the autotransporter adhesin heptosyltransferase and the autotransporter adhesin AIDA-I

Microbiology ◽  
2010 ◽  
Vol 156 (4) ◽  
pp. 1155-1166 ◽  
Author(s):  
Inga Benz ◽  
Tessa van Alen ◽  
Julia Bolte ◽  
Mirka E. Wörmann ◽  
M. Alexander Schmidt
Keyword(s):  
Protein Translocation ◽  
Growth Conditions ◽  
Limited Attention ◽  
Gram Negative Bacteria ◽  
Regulation Of Expression ◽  
Gene Encoding ◽  
E Coli ◽  
K 12 ◽  
Transcriptional Fusions

In Gram-negative bacteria, autotransporter proteins constitute the largest family of secreted proteins, and exhibit many different functions. In recent years, research has largely focused on mechanisms of autotransporter protein translocation, where several alternative models are still being discussed. In contrast, the biogenesis of only a few autotransporters has been studied and, likewise, regulation of expression has received only very limited attention. The glycosylated autotransporter adhesin involved in diffuse adherence (AIDA)-I system consists of the aah gene, encoding a specific autotransporter adhesin heptosyltransferase (AAH), and the aidA gene, encoding the autotransporter protein (AIDA-I). In this study, we investigated the promoter organization and transcription of these two genes using reporter plasmids carrying lacZ transcriptional fusions. The two genes, aah and aidA, are transcribed as a bicistronic message. However, aidA is additionally transcribed from its own promoter. There are two distinct start sites for each of the two genes. Interestingly, transcription of both genes is enhanced in hns and rfaH mutant backgrounds. Furthermore, we addressed the influence of environmental factors and different genetic backgrounds of Escherichia coli K-12 strains on transcription activity. We found that transcription varied considerably in different E. coli K-12 laboratory strains and under different growth conditions.

scholarly journals Characterization of IMI-1 beta-lactamase, a class A carbapenem-hydrolyzing enzyme from Enterobacter cloacae.

1996 ◽  
Vol 40 (9) ◽  
pp. 2080-2086 ◽  
Author(s):  
B A Rasmussen ◽  
K Bush ◽  
D Keeney ◽  
Y Yang ◽  
R Hare ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Clavulanic Acid ◽  
Regulatory Protein ◽  
Enterobacter Cloacae ◽  
Amino Acid Sequences ◽  
Beta Lactamase ◽  
Gene Encoding ◽  
Class A ◽  
Lactamase Gene

In 1984, a year prior to the U.S. approval of imipenem for clinical use, a wound isolate and a bile isolate of Enterobacter cloacae were obtained from two patients in a California hospital. These isolates were resistant to imipenem, penicillins, and inhibitor combinations; early cephalosporins such as cephalothin, cefamandole, and cefoxitin; and cefoperazone. However, they were susceptible (MICs, < 4 micrograms/ml) to cefotaxime, ceftriaxone, ceftazidime, and moxalactam. Both strains produced an apparent TEM-1 beta-lactamase; an inducible NmcA-type imipenem-hydrolyzing beta-lactamase, IMI-1, with a pl of 7.05; and an inducible beta-lactamase with a pI of 8.1, typical of an E. cloacae AmpC beta-lactamase. Purified IMI-1 hydrolyzed imipenem and benzylpenicillin at modest rates, but more slowly than cephaloridine. The enzyme was inhibited by clavulanic acid and tazobactam. EDTA did not inhibit the cephaloridine-hydrolyzing activity. The beta-lactamase gene encoding IMI-1, imiA1, was cloned from E. cloacae 1413B. Sequence analysis identified the imiA1 gene as encoding a class A serine beta-lactamase. Both the imiA1 DNA and encoded amino acid sequences shared greater than 95% identity with the NmcA gene and its encoded protein. DNA sequence analysis also identified a gene upstream of imiA1 that shares > 95% identity with nmcR and that may encode a regulatory protein. In conclusion, IMI-1, a carbapenem-hydrolyzing beta-lactamase inhibited by clavulanic acid, was identified as a group 2f, class A, carbapenem-hydrolyzing cephalosporinase.

scholarly journals Crystal Structure and Activity Studies of the Mycobacterium tuberculosis β-Lactamase Reveal Its Critical Role in Resistance to β-Lactam Antibiotics

2006 ◽  
Vol 50 (8) ◽  
pp. 2762-2771 ◽  
Author(s):  
Feng Wang ◽  
Craig Cassidy ◽  
James C. Sacchettini
Keyword(s):  
Crystal Structure ◽  
Mycobacterium Tuberculosis ◽  
Critical Role ◽  
Gram Negative Bacteria ◽  
Gene Encoding ◽  
Class A ◽  
Increased Sensitivity ◽  
Distinct Features ◽  
Structure And Activity

ABSTRACT β-Lactam antibiotics are extremely effective in disrupting the synthesis of the bacterial cell wall in both gram-positive and gram-negative bacteria. However, they are ineffective against Mycobacterium tuberculosis, due to the production of a β-lactamase enzyme encoded on the chromosome of M. tuberculosis that degrades these antibiotics. Indeed, recent studies have demonstrated that deletion of the blaC gene, the only gene encoding a β-lactamase in M. tuberculosis, or inhibition of the encoded enzyme resulted in significantly increased sensitivity to β-lactam antibiotics. In this paper we present a biochemical and structural characterization of M. tuberculosis BlaC. Recombinant BlaC shows a broad range of specificity with almost equal penicillinase and cepholothinase activity. While clavulanate is a mechanism-based inhibitor to class A β-lactamase with high potency (typically Ki < 0.1 μM), it is a relatively poor inhibitor of the M. tuberculosis BlaC (Ki = 2.4 μM). The crystal structure of the enzyme, determined at a resolution of 1.7 Å, shows that the overall fold of the M. tuberculosis enzyme is similar to other class A β-lactamases. There are, however, several distinct features of the active site, such as the amino acid substitutions N132G, R164A, R244A, and R276E, that explain the broad specificity of the enzyme, relatively low penicillinase activity, and resistance to clavulanate.

Антимікробна резистентність провідних збудників інфекцій сечових шляхів у Києві (Україна)

2017 ◽  
Vol 1 (2) ◽  
pp. 48-60
Author(s):  
A.G. Salmanov ◽  
A.V. Rudenko
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Klebsiella Pneumoniae ◽  
Staphylococcus Epidermidis ◽  
Klebsiella Oxytoca ◽  
Enterobacter Aerogenes ◽  
Enterobacter Cloacae ◽  
Proteus Vulgaris ◽  
Providencia Rettgeri ◽  
E Coli

Мета роботи — вивчити резистентність до антибіотиків бактеріальних збудників інфекцій сечових шляхів (ІСШ), виділених у пацієнтів урологічного стаціонару в м. Києві. Матеріали і методи. Досліджено 1612 штамів бактерій, виділених із сечі хворих з ІСШ (цистит, уретрит, пієлонефрит), госпіталізованих в урологічне відділення ДУ «Інститут урології НАМН України» у м. Києві протягом 2016 р. Серед пацієнтів переважали жінки — 1201 (74,5 %). Вік хворих становив від 17 до 74 років. Для збору даних використано медичну документацію лікарні. Мікробіологічні дослідження виконано у лабораторії мікробіології ДУ «Інститут урології НАМН України». Аналізували результати культурального дослідження зразків сечі, зібраних за наявності клінічних ознак ІСШ. Дослідження клінічного матеріалу та інтерпретацію отриманих результатів проводили загальноприйнятими методами. Вивчено чутливість уропатогенів до 31 антибіотика дискодифузійним методом відповідно до рекомендацій Інституту клінічних та лабораторних стандартів США (Clinical and Laboratory Standards Institute (CLSI)). Результати та обговорення. Аналіз мікробного спектра сечі виявив домінування серед уропатогенів штамів Escherichia coli (32,0 %), Enterococcus faecalis (19,5 %), Klebsiella pneumoniae (10,9 %), Staphylococcus epidermidis (8,9 %), S. haemolyticus (6,5 %) та Pseudomonas aeruginosa (6,4 %). Частка Enterococcus faecium, Enterobacter aerogenes і Streptococcus viridans становила відповідно 2,5, 2,2 і 1,6 %, Enterobacter cloacae, Klebsiella oxytoca, Acinetobacter baumannii, Proteus vulgaris та Providencia rettgeri — менше 1,0 %. У більшості випадків (69,7 %) мікроорганізми виділено у монокультурі, у решті випадків — у мікробних асоціа- ціях. Високу резистентність до тестованих антибіотиків виявили штами E. aerogenes (45,1 %), E. cloacae (45,7 %), E. faecium (40,9 %), E. faecalis (40,7 %), E. coli (39,9 %), P. aeruginosa (34,0 %), K. pneumoniae (28,6 %). Найбільш активними до уропатогенів були іміпенем (E. coli — 87,6 %, P. aeruginosa — 75,7 %, E. cloacae — 67,3 %, E. aerogenes — 72,6 %, K. pneumoniae — 93,2 %), меропенем (E. coli — 89,1 %, P. aeruginosa — 76,7 %, K. pneumoniae — 82,6 %), лефлоцин (E. coli — 74,5 %, ентерококи — 78,7 %, P. aeruginosa — 76,7 %, E. cloacae — 73,9 %, E. aerogenes — 80,4 %, K. pneumoniae — 83,5 %), амоксицилін/клавуланат (ентерококи — 84,6 %), фурагін (ентерококи — 82,6 %), цефоперазон (K. pneumoniae — 89,2 %, P. aeruginosa — 73,8 %), цефтріаксон (K. pneumoniae — 80,1 %). Висновки. Антибіотикорезистентність збудників ІСШ — важлива терапевтична проблема. Найбільшою активністю до уропатогенів характеризуються іміпенем, меропенем, лефлоцин, амоксицилін/ клавуланат, фурагін, цефоперазон, цефтріаксон, які можна розглядати як препарат вибору для призначення стартової терапії ІСШ. Необхідно здійснювати постійний моніторинг за резистентністю до дії антибіотиків. Політику використання антибіотиків у кожному стаціонарі слід визначати залежно від локальних даних щодо резистентності до протимікробних препаратів.

Protein expression and extraction of hard-to-produce proteins in the periplasmic space of Escherichia coli v1

2021 ◽  
Author(s):  
Cristina Hernandez Rollan ◽  
Kristoffer Bach Falkenberg ◽  
Maja Rennig ◽  
Andreas Birk Bertelsen ◽  
Morten Norholm
Keyword(s):  
Protein Production ◽  
Expression System ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
New Family ◽  
Lytic Polysaccharide Monooxygenases ◽  
Strain Bl21 ◽  
Polysaccharide Monooxygenases

E. coli is a gram-negative bacteria used mainly in academia and in some industrial scenarios, as a protein production workhorse. This is due to its ease of manipulation and the range of genetic tools available. This protocol describes how to express proteins in the periplasm E. coli with the strain BL21 (DE3) using a T7 expression system. Specifically, it describes a series of steps and tips to express "hard-to-express" proteins in E. coli, as for instance, LPMOs. The protocol is adapted from Hemsworth, G. R., Henrissat, B., Davies, G. J., and Walton, P. H. (2014) Discovery and characterization of a new family of lytic polysaccharide monooxygenases. Nat. Chem. Biol.10, 122–126. .

scholarly journals Novel Class A β-Lactamase Sed-1 fromCitrobacter sedlakii: Genetic Diversity of β-Lactamases within the Citrobacter Genus

2001 ◽  
Vol 45 (8) ◽  
pp. 2287-2298 ◽  
Author(s):  
Stephanie Petrella ◽  
Dominique Clermont ◽  
Isabelle Casin ◽  
Vincent Jarlier ◽  
Wladimir Sougakoff
Keyword(s):  
Dna Hybridization ◽  
Klebsiella Oxytoca ◽  
Transcriptional Regulator ◽  
Enterobacter Cloacae ◽  
Clinical Strain ◽  
Proteus Vulgaris ◽  
Class A ◽  
Gene Coding ◽  
Quinolone Antibiotics ◽  
Reference Strains

ABSTRACT Citrobacter sedlakii 2596, a clinical strain resistant to aminopenicillins, carboxypenicillins, and early cephalosporins such as cephalothin, but remaining susceptible to acylureidopenicillins, carbapenems, and later cephalosporins such as cefotaxime, was isolated from the bile of a patient treated with β-lactam and quinolone antibiotics. The isolate produced an inducible class A β-lactamase of pI 8.6, named Sed-1, which was purified. Characterized by a molecular mass of 30 kDa, Sed-1 preferentially hydrolyzed benzylpenicillin, cephalothin, and cloxacillin. The corresponding gene,bla Sed-1, was cloned and sequenced. Its deduced amino acid sequence shared more than 60% identity with the chromosome-encoded β-lactamases from Citrobacter koseri(formerly C. diversus) (84%), Klebsiella oxytoca (74%), Serratia fonticola (67%), andProteus vulgaris (63%) and 71% identity with the plasmid-mediated enzyme MEN-1. A gene coding for a LysR transcriptional regulator was found upstream from bla Sed-1. This regulator, named SedR, displayed 90% identity with the AmpR sequence of the chromosomal β-lactamase from C. koseriand 63 and 50% identity with the AmpR sequences of P. vulgaris and Enterobacter cloacae, respectively. By using DNA-DNA hybridization, a bla Sed-1-like gene was identified in two reference strains, C. sedlakii(CIP-105037) and Citrobacter rodentium (CIP-104675), but not in the 18 strains of C. koseri studied. Two DNA fragments were amplified and sequenced from the reference strains ofC. sedlakii CIP-105037 and C. rodentiumCIP-104675 using two primers specific forbla Sed-1. They shared 98 and 80% identity withbla Sed-1, respectively, confirming the diversity of the chromosomally encoded class A β-lactamases found inCitrobacter.

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 Proteolytic interconversion and N-terminal sequences of the Citrobacter diversus major β-lactamases

1991 ◽  
Vol 275 (3) ◽  
pp. 629-633 ◽  
Author(s):  
N Franceschini ◽  
G Amicosante ◽  
M Perilli ◽  
M Maccarrone ◽  
A Oratore ◽  
...  
Keyword(s):  
Klebsiella Oxytoca ◽  
Limited Proteolysis ◽  
Major Product ◽  
Beta Lactamase ◽  
Amino Acid Residues ◽  
Beta Lactamases ◽  
Citrobacter Diversus ◽  
High Degree ◽  
Papain Digestion ◽  
Degree Of Similarity

The N-terminal sequences of the two major beta-lactamases produced by Citrobacter diversus differed only by the absence of the first residue in form II and the loss of five amino acid residues at the C-terminal end. Limited proteolysis of the homogeneous form I protein yielded a variety of enzymatically active products. In the major product obtained after the action of papain, the first three N-terminal residues of form I had been cleaved, whereas at the C-terminal end the treated enzyme lacked five residues. However, this cannot explain the different behaviours of form I, form II and papain digestion product upon chromatofocusing. Form I, which was sequenced up to position 56, exhibited a very high degree of similarity with a Klebsiella oxytoca beta-lactamase. The determined sequence, which contained the active serine residue, demonstrated that the chromosome-encoded beta-lactamase of Citrobacter diversus belong to class A.

scholarly journals Characterization of the β-lactamase specified by the resistance factor R-1818 in Escherichia coli K12 and other Gram-negative bacteria

1971 ◽  
Vol 123 (4) ◽  
pp. 501-505 ◽  
Author(s):  
J. W. Dale
Keyword(s):  
Escherichia Coli ◽  
Host Species ◽  
Host Bacterium ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
R Factor ◽  
Relationship Of ◽  
The Relationship

1. The amino acid composition of the β-lactamase from E. coli (R-1818) was determined. 2. The R-1818 β-lactamase is inhibited by formaldehyde, hydroxylamine, sodium azide, iodoacetamide, iodine and sodium chloride. 3. The Km values for benzylpenicillin, ampicillin and oxacillin have been determined by using the R-factor enzyme from different host species. The same values were obtained, irrespective of the host bacterium. 4. The molecular weight of the enzyme was found to be 44600, and was the same for all host species. 5. The relationship of R-1818 and R-GN238 β-lactamases is discussed.

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