scholarly journals Functional Characterization of Escherichia coli GlpG and Additional Rhomboid Proteins Using an aarA Mutant of Providencia stuartii

2006 ◽  
Vol 188 (9) ◽  
pp. 3415-3419 ◽  
Author(s):  
Katy M. Clemmer ◽  
Gwen M. Sturgill ◽  
Alexander Veenstra ◽  
Philip N. Rather
Keyword(s):  
Escherichia Coli ◽  
Serine Proteases ◽  
Functional Characterization ◽  
Catalytic Triad ◽  
Cell Physiology ◽  
Phenotype Microarray ◽  
Cellular Functions ◽  
Extracellular Signaling ◽  
Lactam Antibiotic ◽  
Providencia Stuartii

ABSTRACT The Providencia stuartii AarA protein is a member of the rhomboid family of intramembrane serine proteases and required for the production of an extracellular signaling molecule that regulates cellular functions including peptidoglycan acetylation, methionine transport, and cysteine biosynthesis. Additional aarA-dependent phenotypes include (i) loss of an extracellular yellow pigment, (ii) inability to grow on MacConkey agar, and (iii) abnormal cell division. Since these phenotypes are easily assayed, the P. stuartii aarA mutant serves as a useful host system to investigate rhomboid function. The Escherichia coli GlpG protein was shown to be functionally similar to AarA and rescued the above aarA-dependent phenotypes in P. stuartii. GlpG proteins containing single alanine substitutions at the highly conserved catalytic triad of asparagine (N154A), serine (S201A), or histidine (H254A) residues were nonfunctional. The P. stuartii aarA mutant was also used as a biosensor to demonstrate that proteins from a variety of diverse sources exhibited rhomboid activity. In an effort to further investigate the role of a rhomboid protein in cell physiology, a glpG mutant of E. coli was constructed. In phenotype microarray experiments, the glpG mutant exhibited a slight increase in resistance to the β-lactam antibiotic cefotaxime.

scholarly journals Cloning, Sequence Analysis, and Expression in Escherichia coli of the Gene Encoding an α-Amino Acid Ester Hydrolase from Acetobacter turbidans

2002 ◽  
Vol 68 (1) ◽  
pp. 211-218 ◽  
Author(s):  
Jolanda J. Polderman-Tijmes ◽  
Peter A. Jekel ◽  
Erik J. de Vries ◽  
Annet E. J. van Merode ◽  
René Floris ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Acid Ester ◽  
Serine Proteases ◽  
Genomic Library ◽  
Sequence Similarity ◽  
Penicillin Acylase ◽  
Amino Acid Ester ◽  
Ester Hydrolase ◽  
Lactam Antibiotic

ABSTRACT The α-amino acid ester hydrolase from Acetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing β-lactam antibiotics, such as cephalexin and ampicillin. N-terminal amino acid sequencing of the purified α-amino acid ester hydrolase allowed cloning and genetic characterization of the corresponding gene from an A. turbidans genomic library. The gene, designated aehA, encodes a polypeptide with a molecular weight of 72,000. Comparison of the determined N-terminal sequence and the deduced amino acid sequence indicated the presence of an N-terminal leader sequence of 40 amino acids. The aehA gene was subcloned in the pET9 expression plasmid and expressed in Escherichia coli. The recombinant protein was purified and found to be dimeric with subunits of 70 kDa. A sequence similarity search revealed 26% identity with a glutaryl 7-ACA acylase precursor from Bacillus laterosporus, but no homology was found with other known penicillin or cephalosporin acylases. There was some similarity to serine proteases, including the conservation of the active site motif, GXSYXG. Together with database searches, this suggested that the α-amino acid ester hydrolase is a β-lactam antibiotic acylase that belongs to a class of hydrolases that is different from the Ntn hydrolase superfamily to which the well-characterized penicillin acylase from E. coli belongs. The α-amino acid ester hydrolase of A. turbidans represents a subclass of this new class of β-lactam antibiotic acylases.

scholarly journals Identification and Characterization of a New Porin Gene of Klebsiella pneumoniae: Its Role in β-Lactam Antibiotic Resistance

1999 ◽  
Vol 181 (9) ◽  
pp. 2726-2732 ◽  
Author(s):  
Antonio Doménech-Sánchez ◽  
Santiago Hernández-Allés ◽  
Luis Martínez-Martínez ◽  
Vicente J. Benedí ◽  
Sebastián Albertí
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Blot Analysis ◽  
Southern Blot Analysis ◽  
Functional Characterization ◽  
Standard Laboratory ◽  
Lactam Antibiotic ◽  
Resistant Strains ◽  
Identification And Characterization

ABSTRACT Klebsiella pneumoniae porin genes were analyzed to detect mutations accounting for the porin deficiency observed in many β-lactam-resistant strains. PCR and Southern blot analysis revealed the existence of a third porin gene in addition to the OmpK36 and OmpK35 porin genes previously described. This new porin gene was designated ompK37 and is present in all of the clinical isolates tested. The OmpK37 porin gene was cloned, sequenced, and overexpressed in Escherichia coli. In contrast to that of the major porins, OmpK37 porin expression was only detectable by Western blot analysis in porin-deficient β-lactam-resistant strains, suggesting strong down regulation under standard laboratory conditions. Functional characterization suggested a narrower pore for the OmpK37 porin than for K. pneumoniae porins OmpK36 and OmpK35. This correlated with the susceptibility to certain β-lactam antibiotics, since a K. pneumoniae strain expressing porin OmpK37, but not porin OmpK36 or OmpK35, was less susceptible to β-lactam antibiotics than the same strain expressing either porin OmpK36 or OmpK35.

scholarly journals Role of CysE in Production of an Extracellular Signaling Molecule in Providencia stuartii and Escherichia coli: Loss of cysE Enhances Biofilm Formation in Escherichia coli

2004 ◽  
Vol 186 (22) ◽  
pp. 7610-7617 ◽  
Author(s):  
Gwen Sturgill ◽  
Christine M. Toutain ◽  
John Komperda ◽  
George A. O'Toole ◽  
Philip N. Rather
Keyword(s):  
Escherichia Coli ◽  
Conditioned Medium ◽  
Physiological Role ◽  
Reading Frame ◽  
E Coli ◽  
Transposon Insertion ◽  
Extracellular Signaling ◽  
Extracellular Signal ◽  
Dependent Signal ◽  
Providencia Stuartii

ABSTRACT A mini-Tn5Cm insertion has been identified that significantly reduced the amount of an extracellular activating signal for a lacZ fusion (cma37::lacZ) in Providencia stuartii. The transposon insertion was located immediately upstream of an open reading frame encoding a putative CysE ortholog. The CysE enzyme, serine acetyltransferase, catalyzes the conversion of serine to O-acetyl-l-serine (OAS). This activating signal was also produced by Escherichia coli, and production was abolished in a strain containing a null allele of cysE. Products of the CysE enzyme (OAS, N-acetyl-l-serine [NAS], O-acetyl-l-threonine, and N-acetyl-l-threonine) were individually tested for the ability to activate cma37::lacZ. Only OAS was capable of activating the cma37::lacZ fusion. The ability of OAS to activate the cma37::lacZ fusion was abolished by pretreatment at pH 8.5, which converts OAS to NAS. However, the activity of the native signal in conditioned medium was not decreased by treatment at pH 8.5. In contrast, conditioned medium prepared from cells grown at pH 8.5 exhibited a 4- to 10-fold-higher activity, relative to pH 6.0. Additional genes regulated by the CysE-dependent signal and OAS were identified in P. stuartii and E. coli. The response to the extracellular signal in E. coli was dependent on CysB, a positive activator that requires NAS as a coactivator. In E. coli, a cysE mutant formed biofilms at an accelerated rate compared to the wild type, suggesting a physiological role for this extracellular signal.

scholarly journals Amdinocillin (Mecillinam) Resistance Mutations in Clinical Isolates and Laboratory-Selected Mutants of Escherichia coli

2015 ◽  
Vol 59 (3) ◽  
pp. 1718-1727 ◽  
Author(s):  
Elisabeth Thulin ◽  
Martin Sundqvist ◽  
Dan I. Andersson
Keyword(s):  
Escherichia Coli ◽  
Clinical Isolates ◽  
Cysteine Biosynthesis ◽  
Resistance Mutations ◽  
Content Type ◽  
E Coli ◽  
Major Mechanism ◽  
Laboratory Selection ◽  
Lactam Antibiotic ◽  
Tract Infections

ABSTRACTAmdinocillin (mecillinam) is a β-lactam antibiotic that is used mainly for the treatment of uncomplicated urinary tract infections. The objectives of this study were to identify mutations that confer amdinocillin resistance on laboratory-isolated mutants and clinical isolates ofEscherichia coliand to determine why amdinocillin resistance remains rare clinically even though resistance is easily selected in the laboratory. Under laboratory selection, frequencies of mutation to amdinocillin resistance varied from 8 × 10−8to 2 × 10−5per cell, depending on the concentration of amdinocillin used during selection. Several genes have been demonstrated to give amdinocillin resistance, but here eight novel genes previously unknown to be involved in amdinocillin resistance were identified. These genes encode functions involved in the respiratory chain, the ribosome, cysteine biosynthesis, tRNA synthesis, and pyrophosphate metabolism. The clinical isolates exhibited significantly greater fitness than the laboratory-isolated mutants and a different mutation spectrum. ThecysBgene was mutated (inactivated) in all of the clinical isolates, in contrast to the laboratory-isolated mutants, where mainly other types of more costly mutations were found. Our results suggest that the frequency of mutation to amdinocillin resistance is high because of the large mutational target (at least 38 genes). However, the majority of these resistant mutants have a low growth rate, reducing the probability that they are stably maintained in the bladder. Inactivation of thecysBgene and a resulting loss of cysteine biosynthesis are the major mechanism of amdinocillin resistance in clinical isolates ofE. coli.

scholarly journals Visualizing the dynamics of exported bacterial proteins with the chemogenetic fluorescent reporter FAST

2020 ◽  
Author(s):  
Yankel Chekli ◽  
Caroline Peron-Cane ◽  
Dario Dell’Arciprete ◽  
Jean-François Allemand ◽  
Chenge Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Listeria Monocytogenes ◽  
Cell Surface ◽  
Bacterial Cell ◽  
Surface Proteins ◽  
Cellular Functions ◽  
Reporter Protein ◽  
Cell Surface Proteins ◽  
Fluorescent Reporter ◽  
Bacterial Proteins

AbstractBacterial proteins exported to the cell surface play key cellular functions. However, despite the interest to study the localization of surface proteins such as adhesins, transporters or hydrolases, monitoring their dynamics in live imaging remains challenging, due to the limited availability of fluorescent probes remaining functional after secretion. In this work, we used the Escherichia coli intimin and the Listeria monocytogenes InlB invasin as surface exposed scaffolds fused with the recently developed chemogenetic fluorescent reporter protein FAST. Using both membrane permeant (HBR-3,5DM) and non-permeant (HBRAA-3E) fluorogens that fluoresce upon binding to FAST, we demonstrated that fully functional FAST can be exposed at the cell surface and specifically tagged on the external side of the bacterial envelop in both diderm and monoderm bacteria. Our work opens new avenues to study of the organization and dynamics of the bacterial cell surface proteins.

scholarly journals An Efficient System for Intestinal On-site Butyrate Production Using Novel Microbiome-Derived Esterases

2021 ◽  
Author(s):  
Dah Hyun Jung ◽  
Ji Hyun Yong ◽  
Wontae Hwang ◽  
Mi Young Yoon ◽  
Sang Sun Yoon
Keyword(s):  
Escherichia Coli ◽  
Artificial Chromosome ◽  
Short Chain Fatty Acids ◽  
Catalytic Triad ◽  
Nucleotide Sequence Analysis ◽  
Efficient System ◽  
Gastrointestinal Health ◽  
Metagenome Library ◽  
The Family ◽  
Butyrate Production

Abstract Short-chain fatty acids, especially butyrate, play beneficial roles in sustaining gastrointestinal health. However, due to limitations associated with direct consumption of butyrate, there has been interest in using prodrugs of butyrate. Tributyrin (TB), a triglyceride composed of three butyrate molecules and a glycerol, is a well-studied precursor of butyrate. We screened a metagenome library consisting of 5,760 bacterial artificial chromosome clones, with DNA inserts originating from mouse microbiomes, and identified two clones that efficiently hydrolyse TB into butyrate. Nucleotide sequence analysis indicated that inserts in these two clones are derived from unknown microbes. BLASTp analysis, however, revealed that each insert contains a gene homologous to acetylesterase or esterase genes, from Clostridium spp. and Bacteroides spp., respectively. Predicted structures of these two proteins both contain serine-histidine-aspartate catalytic triad, highly conserved in the family of esterases. Escherichia coli host expressing each of the two candidate genes invariably produced greater amounts of butyrate in the presence of TB. Importantly, administration of TB together with cloned Escherichia coli cells alleviated inflammatory symptoms in a mouse model of acute colitis. Based on these results, we established an efficient on-site and real-time butyrate production system that releases butyrate in a controlled manner inside the intestine

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