scholarly journals Correct folding of alpha-lytic protease is required for its extracellular secretion from Escherichia coli.

1992 ◽  
Vol 118 (1) ◽  
pp. 33-42 ◽  
Author(s):  
A Fujishige ◽  
K R Smith ◽  
J L Silen ◽  
D A Agard
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Outer Membrane ◽  
Temperature Sensitive ◽  
Extracellular Medium ◽  
E Coli ◽  
Extracellular Secretion ◽  
In Trans ◽  
In Cis ◽  
Pro Region

alpha-Lytic protease is a bacterial serine protease of the trypsin family that is synthesized as a 39-kD preproenzyme (Silen, J. L., C. N. McGrath, K. R. Smith, and D. A. Agard. 1988. Gene (Amst.). 69: 237-244). The 198-amino acid mature protease is secreted into the culture medium by the native host, Lysobacter enzymogenes (Whitaker, D. R. 1970. Methods Enzymol. 19:599-613). Expression experiments in Escherichia coli revealed that the 166-amino acid pro region is transiently required either in cis (Silen, J. L., D. Frank, A. Fujishige, R. Bone, and D. A. Agard. 1989. J. Bacteriol. 171:1320-1325) or in trans (Silen, J. L., and D. A. Agard. 1989. Nature (Lond.). 341:462-464) for the proper folding and extracellular accumulation of the enzyme. The maturation process is temperature sensitive in E. coli; unprocessed precursor accumulates in the cells at temperatures above 30 degrees C (Silen, J. L., D. Frank, A. Fujishige, R. Bone, and D. A. Agard. 1989. J. Bacteriol. 171:1320-1325). Here we show that full-length precursor produced at nonpermissive temperatures is tightly associated with the E. coli outer membrane. The active site mutant Ser 195----Ala (SA195), which is incapable of self-processing, also accumulates as a precursor in the outer membrane, even when expressed at permissive temperatures. When the protease domain is expressed in the absence of the pro region, the misfolded, inactive protease also cofractionates with the outer membrane. However, when the folding requirement for either wild-type or mutant protease domains is provided by expressing the pro region in trans, both are efficiently secreted into the extracellular medium. Attempts to separate folding and secretion functions by extensive deletion mutagenesis within the pro region were unsuccessful. Taken together, these results suggest that only properly folded and processed forms of alpha-lytic protease are efficiently transported to the medium.

Membrane assembly: movement of phosphatidylserine between the cytoplasmic and outer membranes of Escherichia coli

1982 ◽  
Vol 152 (3) ◽  
pp. 1033-1041
Author(s):  
K E Langley ◽  
E Hawrot ◽  
E P Kennedy
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Sucrose Gradient ◽  
Intracellular Localization ◽  
Temperature Sensitive ◽  
Inner Membrane ◽  
E Coli ◽  
Outer Membranes ◽  
Membrane Bound ◽  
Gradient Fractionation

Phosphatidylserine, normally a trace phospholipid in Escherichia coli, accumulates at high levels in temperature-sensitive phosphatidylserine decarboxylase mutants at nonpermissive temperatures. The intracellular localization of this phospholipid has now been determined. All of the accumulated phosphatidylserine is membrane bound and is distributed about equally between the inner and outer membrane fractions of E. coli as determined by isopycnic sucrose gradient fractionation. Phosphatidylserine is therefore effectively translocated from the inner to the outer membrane. Furthermore, this movement is bidirectional. Outer membrane phosphatidylserine can return to the inner membrane, as shown by the complete conversion of accumulated radioactive phosphatidylserine to phosphatidylethanolamine by inner membrane phosphatidylserine decarboxylase during chase periods. Pulse-chase experiments indicated the newly made phosphatidylserine appears first in the inner membrane and then equilibrates between the inner and outer membranes with a half-time of 12 to 13 min.

Complete signal peptide of Tk1884, an α-amylase from Thermococcus kodakarensis, is not necessary for extracellular secretion of the enzyme by Escherichia coli

Amylase ◽  
2017 ◽  
Vol 1 (1) ◽  
Author(s):  
Majida A. Muhammad ◽  
Samia Falak ◽  
Naeem Rashid ◽  
Nasir Ahmed ◽  
Qurra-Tul-Ann A. Gardner ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Signal Peptide ◽  
Amylase Activity ◽  
Signal Sequence ◽  
Ionization Mass ◽  
Extracellular Medium ◽  
E Coli ◽  
Thermococcus Kodakarensis ◽  
Extracellular Secretion

AbstractIn order to elucidate if Escherichia coli secretion system recognizes the N-terminally truncated signal sequence of an archaeal α-amylase from Thermococcus kodakarensis (Tk1884) and secretes the recombinant protein to the extracellular medium, we have cloned Tk1884 with the deletion of the sixteen N-terminal amino acids and produced the recombinant protein Tk1884Δ16 in E. coli. Analysis of the intracellular, membranous and extracellular fractions demonstrated the presence of Tk1884Δ16 in all the three fractions. The intracellular α-amylase activity, similar to the membranous fraction, increased with the passage of time till 8 h of induction and then decreased. In contrast, the extracellular α-amylase activity slowly increased with the passage of time after induction. The extracellular amylase activity was purified and determination of the molecular mass by electrospray ionization mass spectrometry demonstrated that Tk1884Δ16 was secreted to the extracellular medium without cleavage of the signal peptide. To the best of our knowledge, this is the first report on recognition of N-terminally truncated signal peptide of archaeal origin by E. coli.

Identification of Important Amino Acid Residues That Modulate Binding of Escherichia coli GroEL to Its Various Cochaperones

Genetics ◽  
2001 ◽  
Vol 158 (2) ◽  
pp. 507-517
Author(s):  
Gracjana Klein ◽  
Costa Georgopoulos
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Temperature Sensitive ◽  
Amino Acid Residues ◽  
En Bloc ◽  
E Coli ◽  
Important Amino Acid ◽  
Suppressor Mutations ◽  
Sensitive Allele ◽  
Intragenic Suppressor

Abstract Genetic experiments have shown that the GroEL/GroES chaperone machine of Escherichia coli is absolutely essential, not only for bacterial growth but also for the propagation of many bacteriophages including λ. The virulent bacteriophages T4 and RB49 are independent of the host GroES function, because they encode their own cochaperone proteins, Gp31 and CocO, respectively. E. coli groEL44 mutant bacteria do not form colonies above 42° nor do they propagate bacteriophages λ, T4, or RB49. We found that the vast majority (40/46) of spontaneous groEL44 temperature-resistant colonies at 43° were due to the presence of an intragenic suppressor mutation. These suppressors define 21 different amino acid substitutions in GroEL, each affecting one of 13 different amino acid residues. All of these amino acid residues are located at or near the hinge, which regulates the large en bloc movements of the GroEL apical domain. All of these intragenic suppressors support bacteriophages λ, T4, and RB49 growth to various extents in the presence of the groEL44 allele. Since it is known that the GroEL44 mutant protein does not interact effectively with Gp31, the suppressor mutations should enhance cochaperone binding. Analogous intragenic suppressor studies were conducted with the groEL673 temperature-sensitive allele.

scholarly journals An Anhydro-N-Acetylmuramyl-l-Alanine Amidase with Broad Specificity Tethered to the Outer Membrane of Escherichia coli

2007 ◽  
Vol 189 (15) ◽  
pp. 5634-5641 ◽  
Author(s):  
Tsuyoshi Uehara ◽  
James T. Park
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Outer Membrane ◽  
Cell Separation ◽  
Membrane Fraction ◽  
Double Mutant ◽  
Amino Acid Sequence Homology ◽  
E Coli ◽  
Broad Specificity

ABSTRACT From its amino acid sequence homology with AmpD, we recognized YbjR, now renamed AmiD, as a possible second 1,6-anhydro-N-acetylmuramic acid (anhMurNAc)-l-alanine amidase in Escherichia coli. We have now confirmed that AmiD is an anhMurNAc-l-Ala amidase and demonstrated that AmpD and AmiD are the only enzymes present in E. coli that are able to cleave the anhMurNAc-l-Ala bond. The activity was present only in the outer membrane fraction obtained from an ampD mutant. In contrast to AmpD, which is specific for the anhMurNAc-l-alanine bond, AmiD also cleaved the bond between MurNAc and l-alanine in both muropeptides and murein sacculi. Unlike the periplasmic murein amidases, AmiD did not participate in cell separation. ampG mutants, which are unable to import GlcNAc-anhMurNAc-peptides into the cytoplasm, released mainly peptides into the medium due to AmiD activity, whereas an ampG amiD double mutant released a large amount of intact GlcNAc-anhMurNAc-peptides into the medium.

scholarly journals Substrate Specificity of the Escherichia coli Outer Membrane Protease OmpP

2006 ◽  
Vol 189 (2) ◽  
pp. 522-530 ◽  
Author(s):  
Bum-Yeol Hwang ◽  
Navin Varadarajan ◽  
Haixin Li ◽  
Sarah Rodriguez ◽  
Brent L. Iverson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Outer Membrane ◽  
Amino Acid Sequence Identity ◽  
Catalytic Efficiency ◽  
Wild Type ◽  
Peptide Substrate ◽  
Peptide Substrates ◽  
E Coli ◽  
Sequence Identity

ABSTRACT Escherichia coli OmpP is an F episome-encoded outer membrane protease that exhibits 71% amino acid sequence identity with OmpT. These two enzymes cleave substrate polypeptides primarily between pairs of basic amino acids. We found that, like OmpT, purified OmpP is active only in the presence of lipopolysaccharide. With optimal peptide substrates, OmpP exhibits high catalytic efficiency (k cat/Km = 3.0 × 106 M−1s−1). Analysis of the extended amino acid specificity of OmpP by substrate phage revealed that both Arg and Lys are strongly preferred at the P1 and P1′ sites of the enzyme. In addition, Thr, Arg, or Ala is preferred at P2; Leu, Ala, or Glu is preferred at P4; and Arg is preferred at P3′. Notable differences in OmpP and OmpT specificities include the greater ability of OmpP to accept Lys at the P1 or P1′, site as well as the prominence of Ser at P3 in OmpP substrates. Likewise, the OmpP P1 site could better accommodate Ser; as a result, OmpP was able to cleave a peptide substrate between Ser-Arg about 120 times more efficiently than was OmpT. Interestingly, OmpP and OmpT cleave peptides with three consecutive Arg residues at different sites, a difference in specificity that might be important in the inactivation of cationic antimicrobial peptides. Accordingly, we show that the presence of an F′ episome results in increased resistance to the antimicrobial peptide protamine both in ompT mutants and in wild-type E. coli cells.

scholarly journals Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

2021 ◽  
Vol 22 (3) ◽  
pp. 1018
Author(s):  
Hiroaki Yokota
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Underlying Mechanism ◽  
Amino Acid Sequences ◽  
E Coli ◽  
X Ray Crystallography ◽  
Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

Expression and partial purification of human pro-opiomelanocortin in Escherichia coli

1989 ◽  
Vol 3 (2) ◽  
pp. 105-112 ◽  
Author(s):  
T. S. Grewal ◽  
P. J. Lowry ◽  
D. Savva
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Fusion Protein ◽  
Western Blot ◽  
Blot Analysis ◽  
Expression Vectors ◽  
Partial Purification ◽  
Amino Acid Residues ◽  
E Coli ◽  
Dna Fragment

ABSTRACT A large portion of the human pro-opiomelanocortin (POMC) peptide corresponding to amino acid residues 59–241 has been cloned and expressed in Escherichia coli. A 1·0 kb DNA fragment encoding this peptide was cloned into the expression vectors pUC8 and pUR291. Plasmid pJMBG51 (a pUC8 recombinant) was found to direct the expression of a 24 kDa peptide. The recombinant pUR291 (pJMBG52) was shown to produce a β-galactosidase fusion protein of 140 kDa. Western blot analysis showed that both the 24 kDa and 140 kDa peptides are recognized by antibodies raised against POMC-derived peptides. The β-galactosidase fusion protein has been partially purified from crude E. coli cell lysates using affinity chromatography on p-aminobenzyl-1-thio-β-d-galactopyranoside agarose.

SATP (YaaH), a succinate–acetate transporter protein in Escherichia coli

2013 ◽  
Vol 454 (3) ◽  
pp. 585-595 ◽  
Author(s):  
Joana Sá-Pessoa ◽  
Sandra Paiva ◽  
David Ribas ◽  
Inês Jesus Silva ◽  
Sandra Cristina Viegas ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Acetic Acid ◽  
Succinic Acid ◽  
Critical Role ◽  
Amino Acid Residues ◽  
E Coli ◽  
Transporter Protein ◽  
Affinity Constants ◽  
In Trans

In the present paper we describe a new carboxylic acid transporter in Escherichia coli encoded by the gene yaaH. In contrast to what had been described for other YaaH family members, the E. coli transporter is highly specific for acetic acid (a monocarboxylate) and for succinic acid (a dicarboxylate), with affinity constants at pH 6.0 of 1.24±0.13 mM for acetic acid and 1.18±0.10 mM for succinic acid. In glucose-grown cells the ΔyaaH mutant is compromised for the uptake of both labelled acetic and succinic acids. YaaH, together with ActP, described previously as an acetate transporter, affect the use of acetic acid as sole carbon and energy source. Both genes have to be deleted simultaneously to abolish acetate transport. The uptake of acetate and succinate was restored when yaaH was expressed in trans in ΔyaaH ΔactP cells. We also demonstrate the critical role of YaaH amino acid residues Leu131 and Ala164 on the enhanced ability to transport lactate. Owing to its functional role in acetate and succinate uptake we propose its assignment as SatP: the Succinate–Acetate Transporter Protein.

scholarly journals Mechanisms of Resistance in Multiple-Antibiotic-Resistant Escherichia coli Strains of Human, Animal, and Food Origins

2004 ◽  
Vol 48 (10) ◽  
pp. 3996-4001 ◽  
Author(s):  
Yolanda Sáenz ◽  
Laura Briñas ◽  
Elena Domínguez ◽  
Joaquim Ruiz ◽  
Myriam Zarazaga ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Amino Acid ◽  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Mechanisms Of Resistance ◽  
Antibiotic Resistant ◽  
E Coli ◽  
Class 1 ◽  
Human Animal

ABSTRACT Seventeen multiple-antibiotic-resistant nonpathogenic Escherichia coli strains of human, animal, and food origins showed a wide variety of antibiotic resistance genes, many of them carried by class 1 and class 2 integrons. Amino acid changes in MarR and mutations in marO were identified for 15 and 14 E. coli strains, respectively.

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