scholarly journals Pseudomonas aeruginosa MutL protein functions in Escherichia coli

2005 ◽  
Vol 388 (3) ◽  
pp. 879-887 ◽  
Author(s):  
Daniela K. JACQUELÍN ◽  
Adrián FILIBERTI ◽  
Carlos E. ARGARAÑA ◽  
José L. BARRA
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Amino Acid Sequences ◽  
Single Amino Acid ◽  
Repair System ◽  
E Coli ◽  
New Findings

Escherichia coli MutS, MutL and MutH proteins act sequentially in the MMRS (mismatch repair system). MutH directs the repair system to the newly synthesized strand due to its transient lack of Dam (DNA-adenine methylase) methylation. Although Pseudomonas aeruginosa does not have the corresponding E. coli MutH and Dam homologues, and consequently the MMRS seems to work differently, we show that the mutL gene from P. aeruginosa is capable of complementing a MutL-deficient strain of E. coli. MutL from P. aeruginosa has conserved 21 out of the 22 amino acids known to affect functioning of E. coli MutL. We showed, using protein affinity chromatography, that the C-terminal regions of P. aeruginosa and E. coli MutL are capable of specifically interacting with E. coli MutH and retaining the E. coli MutH. Although, the amino acid sequences of the C-terminal regions of these two proteins are only 18% identical, they are 88% identical in the predicted secondary structure. Finally, by analysing (E. coli–P. aeruginosa) chimaeric MutL proteins, we show that the N-terminal regions of E. coli and P. aeruginosa MutL proteins function similarly, in vivo and in vitro. These new findings support the hypothesis that a large surface, rather than a single amino acid, constitutes the MutL surface for interaction with MutH, and that the N- and C-terminal regions of MutL are involved in such interactions.

scholarly journals DL-α-Monofluoromethylputrescine is a potent irreversible inhibitor of Escherichia coli ornithine decarboxylase

1982 ◽  
Vol 204 (3) ◽  
pp. 771-775 ◽  
Author(s):  
A Kallio ◽  
P P McCann ◽  
P Bey
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Ornithine Decarboxylase ◽  
Half Life ◽  
Arginine Decarboxylase ◽  
Biosynthetic Enzyme ◽  
Irreversible Inhibitor ◽  
E Coli

DL-alpha-Monofluoromethylputrescine (compound R.M.I. 71864) is an enzyme-activated irreversible inhibitor of the biosynthetic enzyme ornithine decarboxylase from Escherichia coli. This compound, however, has much less effect in vitro on ornithine decarboxylase obtained from Pseudomonas aeruginosa. These findings are in contrast with those previously found with the substrate analogue DL-alpha-difluoromethylornithine (compound R.M.I. 71782). The K1 of the DL-alpha-monofluoromethylputrescine for the E. coli ornithine decarboxylase is 110 microM, and the half-life (t1/2) calculated for an infinite concentration of inhibitor is 2.1 min. When DL-alpha-monofluoromethylputrescine is used in combination with DL-alpha-difluoromethylarginine (R.M.I. 71897), an irreversible inhibitor of arginine decarboxylase, in vivo in E. coli, both decarboxylase activities are inhibited (greater than 95%) but putrescine levels are only decreased to about one-third of control values and spermidine levels are slightly increased.

scholarly journals tRNA-dependent cleavage of the ColE1 plasmid-encoded RNA I

Microbiology ◽  
2006 ◽  
Vol 152 (12) ◽  
pp. 3467-3476 ◽  
Author(s):  
Zhijun Wang ◽  
Zhenghong Yuan ◽  
Li Xiang ◽  
Junjie Shao ◽  
Grzegorz Węgrzyn
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Plasmid Dna ◽  
Copy Number ◽  
E Coli ◽  
Cole1 Plasmid ◽  
Specific Degradation ◽  
Rna I

Effects of tRNAAla(UGC) and its derivative devoid of the 3′-ACCA motif [tRNAAla(UGC)ΔACCA] on the cleavage of the ColE1-like plasmid-derived RNA I were analysed in vivo and in vitro. In an amino-acid-starved relA mutant, in which uncharged tRNAs occur in large amounts, three products of specific cleavage of RNA I were observed, in contrast to an otherwise isogenic relA + host. Overexpression of tRNAAla(UGC), which under such conditions occurs in Escherichia coli mostly in an uncharged form, induced RNA I cleavage and resulted in an increase in ColE1-like plasmid DNA copy number. Such effects were not observed during overexpression of the 3′-ACCA-truncated tRNAAla(UGC). Moreover, tRNAAla(UGC), but not tRNAAla(UGC)ΔACCA, caused RNA I cleavage in vitro in the presence of MgCl2. These results strongly suggest that tRNA-dependent RNA I cleavage occurs in ColE1-like plasmid-bearing E. coli, and demonstrate that tRNAAla(UGC) participates in specific degradation of RNA I in vivo and in vitro. This reaction is dependent on the presence of the 3′-ACCA motif of tRNAAla(UGC).

scholarly journals The transcription factor DNR from Pseudomonas aeruginosa specifically requires nitric oxide and haem for the activation of a target promoter in Escherichia coli

Microbiology ◽  
2009 ◽  
Vol 155 (9) ◽  
pp. 2838-2844 ◽  
Author(s):  
Nicoletta Castiglione ◽  
Serena Rinaldo ◽  
Giorgio Giardina ◽  
Francesca Cutruzzolà
Keyword(s):  
Nitric Oxide ◽  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Consensus Sequence ◽  
Signal Molecules ◽  
Reporter System ◽  
Nucleotide Substitutions ◽  
E Coli

Pseudomonas aeruginosa is a well-known pathogen in chronic respiratory diseases such as cystic fibrosis. Infectivity of P. aeruginosa is related to the ability to grow under oxygen-limited conditions using the anaerobic metabolism of denitrification, in which nitrate is reduced to dinitrogen via nitric oxide (NO). Denitrification is activated by a cascade of redox-sensitive transcription factors, among which is the DNR regulator, sensitive to nitrogen oxides. To gain further insight into the mechanism of NO-sensing by DNR, we have developed an Escherichia coli-based reporter system to investigate different aspects of DNR activity. In E. coli DNR responds to NO, as shown by its ability to transactivate the P. aeruginosa norCB promoter. The direct binding of DNR to the target DNA is required, since mutations in the helix–turn–helix domain of DNR and specific nucleotide substitutions in the consensus sequence of the norCB promoter abolish the transcriptional activity. Using an E. coli strain deficient in haem biosynthesis, we have also confirmed that haem is required in vivo for the NO-dependent DNR activity, in agreement with the property of DNR to bind haem in vitro. Finally, we have shown, we believe for the first time, that DNR is able to discriminate in vivo between different diatomic signal molecules, NO and CO, both ligands of the reduced haem iron in vitro, suggesting that DNR responds specifically to NO.

scholarly journals Mutational Analysis of Escherichia coli Heat Shock Transcription Factor Sigma 32 Reveals Similarities with Sigma 70 in Recognition of the −35 Promoter Element and Differences in Promoter DNA Melting and −10 Recognition

2005 ◽  
Vol 187 (19) ◽  
pp. 6762-6769 ◽  
Author(s):  
Olga V. Kourennaia ◽  
Laura Tsujikawa ◽  
Pieter L. deHaseth
Keyword(s):  
Escherichia Coli ◽  
Transcription Factor ◽  
Amino Acid ◽  
Heat Shock ◽  
Dna Sequences ◽  
Transcription Initiation ◽  
Amino Acid Sequences ◽  
Single Amino Acid ◽  
Promoter Dna

ABSTRACT Upon the exposure of Escherichia coli to high temperature (heat shock), cellular levels of the transcription factor σ32 rise greatly, resulting in the increased formation of the σ32 holoenzyme, which is capable of transcription initiation at heat shock promoters. Higher levels of heat shock proteins render the cell better able to cope with the effects of higher temperatures. To conduct structure-function studies on σ32 in vivo, we have carried out site-directed mutagenesis and employed a previously developed system involving σ32 expression from one plasmid and a β-galactosidase reporter gene driven by the σ32-dependent groE promoter on another in order to monitor the effects of single amino acid substitutions on σ32 activity. It was found that the recognition of the −35 region involves similar amino acid residues in regions 4.2 of E. coli σ32 and σ70. Three conserved amino acids in region 2.3 of σ32 were found to be only marginally important in determining activity in vivo. Differences between σ32 and σ70 in the effects of mutation in region 2.4 on the activities of the two sigma factors are consistent with the pronounced differences between both the amino acid sequences in this region and the recognized promoter DNA sequences.

scholarly journals Amino acid sequence repertoire of the bacterial proteome and the occurrence of untranslatable sequences

2016 ◽  
Vol 113 (26) ◽  
pp. 7166-7170 ◽  
Author(s):  
Sharon Penias Navon ◽  
Guy Kornberg ◽  
Jin Chen ◽  
Tali Schwartzman ◽  
Albert Tsai ◽  
...  
Keyword(s):  
Amino Acid ◽  
Single Molecule ◽  
Bioinformatic Analysis ◽  
Single Amino Acid ◽  
E Coli ◽  
Naturally Occurring ◽  
Exit Tunnel ◽  
Amino Acid Triplet

Bioinformatic analysis of Escherichia coli proteomes revealed that all possible amino acid triplet sequences occur at their expected frequencies, with four exceptions. Two of the four underrepresented sequences (URSs) were shown to interfere with translation in vivo and in vitro. Enlarging the URS by a single amino acid resulted in increased translational inhibition. Single-molecule methods revealed stalling of translation at the entrance of the peptide exit tunnel of the ribosome, adjacent to ribosomal nucleotides A2062 and U2585. Interaction with these same ribosomal residues is involved in regulation of translation by longer, naturally occurring protein sequences. The E. coli exit tunnel has evidently evolved to minimize interaction with the exit tunnel and maximize the sequence diversity of the proteome, although allowing some interactions for regulatory purposes. Bioinformatic analysis of the human proteome revealed no underrepresented triplet sequences, possibly reflecting an absence of regulation by interaction with the exit tunnel.

scholarly journals Antagonism of Ultraviolet-Light Mutagenesis by the Methyl-Directed Mismatch-Repair System of Escherichia coli

Genetics ◽  
2000 ◽  
Vol 154 (2) ◽  
pp. 503-512 ◽  
Author(s):  
Hongbo Liu ◽  
Stephen R Hewitt ◽  
John B Hays
Keyword(s):  
Escherichia Coli ◽  
Mismatch Repair ◽  
Excision Repair ◽  
Spontaneous Mutation ◽  
Repair System ◽  
Uv Mutagenesis ◽  
Repair Protein ◽  
Mismatch Repair System

Abstract Previous studies have demonstrated that the Escherichia coli MutHLS mismatch-repair system can process UV-irradiated DNA in vivo and that the human MSH2·MSH6 mismatch-repair protein binds more strongly in vitro to photoproduct/base mismatches than to “matched” photoproducts in DNA. We tested the hypothesis that mismatch repair directed against incorrect bases opposite photoproducts might reduce UV mutagenesis, using two alleles at E. coli lacZ codon 461, which revert, respectively, via CCC → CTC and CTT → CTC transitions. F′ lacZ targets were mated from mut+ donors into mutH, mutL, or mutS recipients, once cells were at substantial densities, to minimize spontaneous mutation prior to irradiation. In umu+ mut+ recipients, a range of UV fluences induced lac+ revertant frequencies of 4–25 × 10−8; these frequencies were consistently 2-fold higher in mutH, mutL, or mutS recipients. Since this effect on mutation frequency was unaltered by an Mfd− defect, it appears not to involve transcription-coupled excision repair. In mut+ umuC122::Tn5 bacteria, UV mutagenesis (at 60 J/m2) was very low, but mutH or mutL or mutS mutations increased reversion of both lacZ alleles roughly 25-fold, to 5–10 × 10−8. Thus, at UV doses too low to induce SOS functions, such as Umu2′D, most incorrect bases opposite occasional photoproducts may be removed by mismatch repair, whereas in heavily irradiated (SOS-induced) cells, mismatch repair may only correct some photoproduct/base mismatches, so UV mutagenesis remains substantial.

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.

scholarly journals Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

1987 ◽  
Vol 248 (1) ◽  
pp. 43-51 ◽  
Author(s):  
J Charlier ◽  
R Sanchez
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Activity Ratio ◽  
Deae Cellulose ◽  
Trna Synthetase ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

scholarly journals Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

2005 ◽  
Vol 71 (7) ◽  
pp. 3468-3474 ◽  
Author(s):  
Gyeong Tae Eom ◽  
Jae Kwang Song ◽  
Jung Hoon Ahn ◽  
Yeon Soo Seo ◽  
Joon Shick Rhee
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Abc Transporter ◽  
Microtiter Plate ◽  
Single Amino Acid ◽  
Wild Type ◽  
E Coli ◽  
Single Amino Acid Change ◽  
Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

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