scholarly journals Conversion of Escherichia coli pyruvate oxidase to an ‘α-ketobutyrate oxidase’

2000 ◽  
Vol 352 (3) ◽  
pp. 717-724 ◽  
Author(s):  
Ying-Ying CHANG ◽  
John E. CRONAN
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Random Mutagenesis ◽  
Fold Increase ◽  
Natural Substrate ◽  
Wild Type ◽  
Pyruvate Oxidase ◽  
Mutant Proteins ◽  
Essentially Normal

Escherichia coli pyruvate oxidase (PoxB), a lipid-activated homotetrameric enzyme, is active on both pyruvate and 2-oxobutanoate (‘α-ketobutyrate’), although pyruvate is the favoured substrate. By localized random mutagenesis of residues chosen on the basis of a modelled active site, we obtained several PoxB enzymes that had a markedly decreased activity with the natural substrate, pyruvate, but retained full activity with 2-oxobutanoate. In each of these mutant proteins Val-380had been replaced with a smaller residue, namely alanine, glycine or serine. One of these, PoxB V380A/L253F, was shown to lack detectable pyruvate oxidase activity in vivo; this protein was purified, studied and found to have a 6-fold increase in Km for pyruvate and a 10-fold lower Vmax with this substrate. In contrast, the mutant had essentially normal kinetic constants with 2-oxobutanoate. The altered substrate specificity was reflected in a decreased rate of pyruvate binding to the latent conformer of the mutant protein owing to the V380A mutation. The L253F mutation alone had no effect on PoxB activity, although it increased the activity of proteins carrying substitutions at residue 380, as it did that of the wild-type protein. The properties of the V380A/L253F protein provide new insights into the mode of substrate binding and the unusual activation properties of this enzyme.

scholarly journals Analysis of ftsQ Mutant Alleles in Escherichia coli: Complementation, Septal Localization, and Recruitment of Downstream Cell Division Proteins

2002 ◽  
Vol 184 (3) ◽  
pp. 695-705 ◽  
Author(s):  
Joseph C. Chen ◽  
Michael Minev ◽  
Jon Beckwith
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Random Mutagenesis ◽  
Dominant Negative ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
Negative Effects ◽  
Mutant Proteins ◽  
Division Site

ABSTRACT FtsQ, a 276-amino-acid, bitopic membrane protein, is one of the nine proteins known to be essential for cell division in gram-negative bacterium Escherichia coli. To define residues in FtsQ critical for function, we performed random mutagenesis on the ftsQ gene and identified four alleles (ftsQ2, ftsQ6, ftsQ15, and ftsQ65) that fail to complement the ftsQ1(Ts) mutation at the restrictive temperature. Two of the mutant proteins, FtsQ6 and FtsQ15, are functional at lower temperatures but are unable to localize to the division site unless wild-type FtsQ is depleted, suggesting that they compete poorly with the wild-type protein for septal targeting. The other two mutants, FtsQ2 and FtsQ65, are nonfunctional at all temperatures tested and have dominant-negative effects when expressed in an ftsQ1(Ts) strain at the permissive temperature. FtsQ2 and FtsQ65 localize to the division site in the presence or absence of endogenous FtsQ, but they cannot recruit downstream cell division proteins, such as FtsL, to the septum. These results suggest that FtsQ2 and FtsQ65 compete efficiently for septal targeting but fail to promote the further assembly of the cell division machinery. Thus, we have separated the localization ability of FtsQ from its other functions, including recruitment of downstream cell division proteins, and are beginning to define regions of the protein responsible for these distinct capabilities.

scholarly journals Cross-subunit catalysis and a new phenomenon of recessive resurrection in Escherichia coli RNase E

2019 ◽  
Vol 48 (2) ◽  
pp. 847-861 ◽  
Author(s):  
Nida Ali ◽  
Jayaraman Gowrishankar
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Rna Binding ◽  
Initial Step ◽  
Dominant Negative ◽  
Rnase E ◽  
Wild Type ◽  
Catalytic Active Site

Abstract RNase E is a 472-kDa homo-tetrameric essential endoribonuclease involved in RNA processing and turnover in Escherichia coli. In its N-terminal half (NTH) is the catalytic active site, as also a substrate 5′-sensor pocket that renders enzyme activity maximal on 5′-monophosphorylated RNAs. The protein's non-catalytic C-terminal half (CTH) harbours RNA-binding motifs and serves as scaffold for a multiprotein degradosome complex, but is dispensable for viability. Here, we provide evidence that a full-length hetero-tetramer, composed of a mixture of wild-type and (recessive lethal) active-site mutant subunits, exhibits identical activity in vivo as the wild-type homo-tetramer itself (‘recessive resurrection’). When all of the cognate polypeptides lacked the CTH, the active-site mutant subunits were dominant negative. A pair of C-terminally truncated polypeptides, which were individually inactive because of additional mutations in their active site and 5′-sensor pocket respectively, exhibited catalytic function in combination, both in vivo and in vitro (i.e. intragenic or allelic complementation). Our results indicate that adjacent subunits within an oligomer are separately responsible for 5′-sensing and cleavage, and that RNA binding facilitates oligomerization. We propose also that the CTH mediates a rate-determining initial step for enzyme function, which is likely the binding and channelling of substrate for NTH’s endonucleolytic action.

scholarly journals Specificity Mutants of the Binding Protein of the Oligopeptide Transport System of Lactococcus lactis

2000 ◽  
Vol 182 (6) ◽  
pp. 1600-1608 ◽  
Author(s):  
Antonia Picon ◽  
Edmund R. S. Kunji ◽  
Frank C. Lanfermeijer ◽  
Wil N. Konings ◽  
Bert Poolman
Keyword(s):  
Lactococcus Lactis ◽  
Fold Increase ◽  
Binding Activity ◽  
Kinetic Properties ◽  
Wild Type ◽  
Mutant Proteins ◽  
Serovar Typhimurium ◽  
Peptide Binding Site ◽  
Oligopeptide Transport

ABSTRACT The kinetic properties of wild-type and mutant oligopeptide binding proteins of Lactococcus lactis were determined. To observe the properties of the mutant proteins in vivo, the oppAgene was deleted from the chromosome of L. lactis to produce a strain that was totally defective in oligopeptide transport. Amplified expression of the oppA gene resulted in an 8- to 12-fold increase in OppA protein relative to the wild-type level. The amplified expression was paralleled by increased bradykinin binding activity, but had relatively little effect on the overall transport of bradykinin via Opp. Several site-directed mutants were constructed on the basis of a comparison of the primary sequences of OppA fromSalmonella enterica serovar Typhimurium and L. lactis, taking into account the known structure of the serovar Typhimurium protein. Putative peptide binding-site residues were mutated. All the mutant OppA proteins exhibited a decreased binding affinity for the high-affinity peptide bradykinin. Except for OppA(D471R), the mutant OppA proteins displayed highly defective bradykinin uptake, whereas the transport of the low-affinity substrate KYGK was barely affected. Cells expressing OppA(D471R) had a similarKm for transport, whereas theV max was increased more than twofold as compared to the wild-type protein. The data are discussed in the light of a kinetic model and imply that the rate of transport is determined to a large extent by the donation of the peptide from the OppA protein to the translocator complex.

Effects of amino acid substitutions at the active site in Escherichia coli beta-galactosidase.

Genetics ◽  
1988 ◽  
Vol 120 (3) ◽  
pp. 637-644
Author(s):  
C G Cupples ◽  
J H Miller
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Lacz Gene ◽  
Wild Type ◽  
Mutant Proteins ◽  
Beta Galactosidase

Abstract Forty-nine amino acid substitutions were made at four positions in the Escherichia coli enzyme beta-galactosidase; three of the four targeted amino acids are thought to be part of the active site. Many of the substitutions were made by converting the appropriate codon in lacZ to an amber codon, and using one of 12 suppressor strains to introduce the replacement amino acid. Glu-461 and Tyr-503 were replaced, independently, with 13 amino acids. All 26 of the strains containing mutant enzymes are Lac-. Enzyme activity is reduced to less than 10% of wild type by substitutions at Glu-461 and to less than 1% of wild type by substitutions at Tyr-503. Many of the mutant enzymes have less than 0.1% wild-type activity. His-464 and Met-3 were replaced with 11 and 12 amino acids, respectively. Strains containing any one of these mutant proteins are Lac+. The results support previous evidence that Glu-461 and Tyr-503 are essential for catalysis, and suggest that His-464 is not part of the active site. Site-directed mutagenesis was facilitated by construction of an f1 bacteriophage containing the complete lacZ gene on a single EcoRI fragment.

scholarly journals Human mismatch-repair protein MutL homologue 1 (MLH1) interacts with Escherichia coli MutL and MutS in vivo and in vitro: a simple genetic system to assay MLH1 function

2003 ◽  
Vol 371 (1) ◽  
pp. 183-189 ◽  
Author(s):  
Barbara QUARESIMA ◽  
Pietro ALIFANO ◽  
Pierfrancesco TASSONE ◽  
Enrico V. AVVEDIMENTO ◽  
Francesco S. COSTANZO ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mismatch Repair ◽  
Fold Increase ◽  
Genetic System ◽  
Mutation Rates ◽  
Wild Type ◽  
Mismatched Dna ◽  
Repair Protein

A simple genetic system has been developed to test the effect of over-expression of wild-type or mutated human MutL homologue 1 (hMLH1) proteins on methyl-directed mismatch repair (MMR) in Escherichia coli. The system relies on detection of Lac+ revertants using MMR-proficient or MMR-deficient E. coli strains carrying a lac +1 frameshift mutation expressing hMLH1 proteins. We report that expression of wild-type hMLH1 protein causes an approx. 19-fold increase in mutation rates. The mutator phenotype was due to the ability of hMLH1 protein to interact with bacterial MutL and MutS proteins, thereby interfering with the formation of complexes between MMR proteins and mismatched DNA. Conversely, expression of proteins encoded by alleles deriving from hereditary-non-polyposis-colon-cancer (HNPCC) families decreases mutation rates, depending on the specific amino acid substitutions. These effects parallel the MutL-and MutS-binding and ATP-binding/hydrolysis activities of the mutated proteins.

scholarly journals Substitutions in an Active Site Loop ofEscherichia coliIscS Result in Specific Defects in Fe-S Cluster and Thionucleoside Biosynthesisin Vivo

2004 ◽  
Vol 279 (19) ◽  
pp. 19551-19558 ◽  
Author(s):  
Charles T. Lauhon ◽  
Elizabeth Skovran ◽  
Hugo D. Urbina ◽  
Diana M. Downs ◽  
Larry E. Vickery
Keyword(s):  
Active Site ◽  
Cluster Formation ◽  
Wild Type ◽  
Cysteine Desulfurase ◽  
Mutant Proteins ◽  
Active Site Cysteine ◽  
Sulfur Transfer ◽  
Amino Acid Region

IscS catalyzes the fragmentation ofl-cysteine tol-alanine and sulfane sulfur in the form of a cysteine persulfide in the active site of the enzyme. InEscherichia coliIscS, the active site cysteine Cys328resides in a flexible loop that potentially influences both the formation and stability of the cysteine persulfide as well as the specificity of sulfur transfer to protein substrates. Alanine-scanning substitution of this 14 amino acid region surrounding Cys328identified additional residues important for IscS functionin vivo. Two mutations, S326A and L333A, resulted in strains that were severely impaired in Fe-S cluster synthesisin vivo. The mutant strains were deficient in Fe-S cluster-dependent tRNA thionucleosides (s2C and ms2i6A) yet showed wild type levels of Fe-S-independent thionucleosides (s4U and mnm5s2U) that require persulfide formation and transfer.In vitro, the mutant proteins were similar to wild type in both cysteine desulfurase activity and sulfur transfer to IscU. These results indicate that residues in the active site loop can selectively affect Fe-S cluster biosynthesisin vivowithout detectably affecting persulfide delivery and suggest that additional assays may be necessary to fully represent the functions of IscS in Fe-S cluster formation.

scholarly journals degS Is Necessary for Virulence and Is among Extraintestinal Escherichia coli Genes Induced in Murine Peritonitis

2003 ◽  
Vol 71 (6) ◽  
pp. 3088-3096 ◽  
Author(s):  
Peter Redford ◽  
Paula L. Roesch ◽  
Rodney A. Welch
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Luria Bertani ◽  
Broth Culture ◽  
Wild Type ◽  
E Coli ◽  
Virulence Attenuation

ABSTRACT Extraintestinal Escherichia coli strains cause meningitis, sepsis, urinary tract infection, and other infections outside the bowel. We examined here extraintestinal E. coli strain CFT073 by differential fluorescence induction. Pools of CFT073 clones carrying a CFT073 genomic fragment library in a promoterless gfp vector were inoculated intraperitoneally into mice; bacteria were recovered by lavage 6 h later and then subjected to fluorescence-activated cell sorting. Eleven promoters were found to be active in the mouse but not in Luria-Bertani (LB) broth culture. Three are linked to genes for enterobactin, aerobactin, and yersiniabactin. Three others are linked to the metabolic genes metA, gltB, and sucA, and another was linked to iha, a possible adhesin. Three lie before open reading frames of unknown function. One promoter is associated with degS, an inner membrane protease. Mutants of the in vivo-induced loci were tested in competition with the wild type in mouse peritonitis. Of the mutants tested, only CFT073 degS was found to be attenuated in peritoneal and in urinary tract infection, with virulence restored by complementation. CFT073 degS shows growth similar to that of the wild type at 37°C but is impaired at 43°C or in 3% ethanol LB broth at 37°C. Compared to the wild type, the mutant shows similar serum survival, motility, hemolysis, erythrocyte agglutination, and tolerance to oxidative stress. It also has the same lipopolysaccharide appearance on a silver-stained gel. The basis for the virulence attenuation is unclear, but because DegS is needed for σE activity, our findings implicate σE and its regulon in E. coli extraintestinal pathogenesis.

scholarly journals Engineering Desiccation Tolerance inEscherichia coli

2000 ◽  
Vol 66 (4) ◽  
pp. 1680-1684 ◽  
Author(s):  
Daniela Billi ◽  
Deborah J. Wright ◽  
Richard F. Helm ◽  
Todd Prickett ◽  
Malcolm Potts ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Phase Transition ◽  
Freeze Drying ◽  
Fold Increase ◽  
Inescherichia Coli ◽  
Vibration Frequencies ◽  
Wild Type ◽  
Air Drying ◽  
Phase Transition Temperatures ◽  
Cell Dehydration

ABSTRACT Recombinant sucrose-6-phosphate synthase (SpsA) was synthesized inEscherichia coli BL21DE3 by using the spsA gene of the cyanobacterium Synechocystis sp. strain PCC 6803. Transformants exhibited a 10,000-fold increase in survival compared to wild-type cells following either freeze-drying, air drying, or desiccation over phosphorus pentoxide. The phase transition temperatures and vibration frequencies (PO stretch) in phospholipids suggested that sucrose maintained membrane fluidity during cell dehydration.

scholarly journals OmpR and LeuO Positively Regulate the Salmonella enterica Serovar Typhi ompS2 Porin Gene

2004 ◽  
Vol 186 (10) ◽  
pp. 2909-2920 ◽  
Author(s):  
Marcos Fernández-Mora ◽  
José Luis Puente ◽  
Edmundo Calva
Keyword(s):  
Salmonella Enterica ◽  
Type Strain ◽  
Wild Type Strain ◽  
Phosphorylation Site ◽  
Regulatory Region ◽  
Random Mutagenesis ◽  
Fold Increase ◽  
Upstream Regulatory Region ◽  
Wild Type ◽  
Transposon Insertion

ABSTRACT The Salmonella enterica serovar Typhi ompS2 gene codes for a 362-amino-acid outer membrane protein that contains motifs common to the porin superfamily. It is expressed at very low levels compared to the major OmpC and OmpF porins, as observed for S. enterica serovar Typhi OmpS1, Escherichia coli OmpN, and Klebsiella pneumoniae OmpK37 quiescent porins. A region of 316 bp, between nucleotides −413 and −97 upstream of the transcriptional start point, is involved in negative regulation, as its removal resulted in a 10-fold increase in ompS2 expression in an S. enterica serovar Typhi wild-type strain. This enhancement in expression was not observed in isogenic mutant strains, which had specific deletions of the regulatory ompB (ompR envZ) operon. Furthermore, ompS2 expression was substantially reduced in the presence of the OmpR D55A mutant, altered in the major phosphorylation site. Upon random mutagenesis, a mutant where the transposon had inserted into the upstream regulatory region of the gene coding for the LeuO regulator, showed an increased level of ompS2 expression. Augmented expression of ompS2 was also obtained upon addition of cloned leuO to the wild-type strain, but not in an ompR isogenic derivative, consistent with the notion that the transposon insertion had increased the cellular levels of LeuO and with the observed dependence on OmpR. Moreover, LeuO and OmpR bound in close proximity, but independently, to the 5′ upstream regulatory region. Thus, the OmpR and LeuO regulators positively regulate ompS2.

scholarly journals Zebrafish nephrosin helps host defence against Escherichia coli infection

Open Biology ◽  
2017 ◽  
Vol 7 (8) ◽  
pp. 170040 ◽  
Author(s):  
Qianqian Di ◽  
Qing Lin ◽  
Zhibin Huang ◽  
Yali Chi ◽  
Xiaohui Chen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Innate Immunity ◽  
Host Defence ◽  
Bacterial Burden ◽  
Wild Type ◽  
Lower Survival Rate ◽  
E Coli ◽  
Escherichia Coli Infection ◽  
Effective Models

Neutrophils play important roles in innate immunity and are mainly dependent on various enzyme-containing granules to kill engulfed microorganisms. Zebrafish nephrosin ( npsn ) is specifically expressed in neutrophils; however, its function is largely unknown. Here, we generated an npsn mutant ( npsn smu5 ) via CRISPR/Cas9 to investigate the in vivo function of Npsn. The overall development and number of neutrophils remained unchanged in npsn -deficient mutants, whereas neutrophil antibacterial function was defective. Upon infection with Escherichia coli , the npsn smu5 mutants exhibited a lower survival rate and more severe bacterial burden, as well as augmented inflammatory response to challenge with infection when compared with wild-type embryos, whereas npsn -overexpressing zebrafish exhibited enhanced host defence against E. coli infection. These findings demonstrated that zebrafish Npsn promotes host defence against bacterial infection. Furthermore, our findings suggested that npsn -deficient and -overexpressing zebrafish might serve as effective models of in vivo innate immunity.

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