Contribution of Mutation at Amino Acid 45 of AcrR to acrB Expression and Ciprofloxacin Resistance in Clinical and Veterinary Escherichia coli Isolates

ABSTRACT Fluoroquinolone-resistant Escherichia coli isolates which overexpressed acrB and had a substitution at amino acid 45 of AcrR were complemented with wild-type acrR. Complementation led to increased sensitivity to ciprofloxacin and to ethidium bromide, suggesting that mutation at amino acid 45 of AcrR contributes to ciprofloxacin resistance.

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Mutations in the Escherichia coli UvrB ATPase motif compromise excision repair capacity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.86.17.6577 ◽

1989 ◽

Vol 86 (17) ◽

pp. 6577-6581 ◽

Cited By ~ 49

Author(s):

T W Seeley ◽

L Grossman

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Excision Repair ◽

Site Directed Mutagenesis ◽

Sequence Motif ◽

Amino Acid Side Chain ◽

Uv Resistance ◽

Wild Type ◽

General Applicability ◽

Repair Capacity

The Escherichia coli UvrB protein possesses an amino acid sequence motif common to many ATPases. The role of this motif in UvrB has been investigated by site-directed mutagenesis. Three UvrB mutants, with amino acid replacements at lysine-45, failed to confer UV resistance when tested in the UV-sensitive strain N364 (delta uvrB), while five other mutants constructed near this region of UvrB confer wild-type levels of UV resistance. Because even the conservative substitution of arginine for lysine-45 in UvrB results in failure to confer UV resistance, we believe we have identified an amino acid side chain in UvrB essential to nucleotide excision repair in E. coli. The properties of two purified mutant UvrB proteins, lysine-45 to alanine (K45A) and asparagine-51 to alanine (N51A), were analyzed in vitro. While the K45A mutant is fully defective in incision of UV-irradiated DNA, K45A is capable of interaction with UvrA in forming an ATP-dependent nucleoprotein complex. The K45A mutant, however, fails to activate the characteristic increase in ATPase activity observed with the wild-type UvrB in the presence of UvrA and DNA. From these results we conclude that there is a second nucleotide-dependent step in incision following initial complex formation, which is defective in the K45A mutant. This experimental approach may prove of general applicability in the study of function and mechanism of other ATPase motif proteins.

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Conformational shifts in a chemoreceptor helical hairpin control kinase signaling in Escherichia coli

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1902521116 ◽

2019 ◽

Vol 116 (31) ◽

pp. 15651-15660

Author(s):

Qun Gao ◽

Anchun Cheng ◽

John S. Parkinson

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Salt Bridge ◽

Soft Agar ◽

Side Chain ◽

Wild Type ◽

Chemical Gradients ◽

Helical Hairpin ◽

Mutant Receptors ◽

Activity Dependent

Motile Escherichia coli cells use chemoreceptor signaling arrays to track chemical gradients with exquisite precision. Highly conserved residues in the cytoplasmic hairpin tip of chemoreceptor molecules promote assembly of trimer-based signaling complexes and modulate the activity of their CheA kinase partners. To explore hairpin tip output states in the serine receptor Tsr, we characterized the signaling consequences of amino acid replacements at the salt-bridge residue pair E385-R388. All mutant receptors assembled trimers and signaling complexes, but most failed to support serine chemotaxis in soft agar assays. Small side-chain replacements at either residue produced OFF- or ON-shifted outputs that responded to serine stimuli in wild-type fashion, suggesting that these receptors, like the wild-type, operate as two-state signaling devices. Larger aliphatic or aromatic side chains caused slow or partial kinase control responses that proved dependent on the connections between core signaling units that promote array cooperativity. In a mutant lacking one of two key adapter-kinase contacts (interface 2), those mutant receptors exhibited more wild-type behaviors. Lastly, mutant receptors with charged amino acid replacements assembled signaling complexes that were locked in kinase-ON (E385K|R) or kinase-OFF (R388D|E) output. The hairpin tips of mutant receptors with these more aberrant signaling properties probably have nonnative structures or dynamic behaviors. Our results suggest that chemoeffector stimuli and adaptational modifications influence the cooperative connections between core signaling units. This array remodeling process may involve activity-dependent changes in the relative strengths of interface 1 and 2 interactions between the CheW and CheA.P5 components of receptor core signaling complexes.

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PBP5, PBP6 and DacD play different roles in intrinsic β-lactam resistance of Escherichia coli

Microbiology ◽

10.1099/mic.0.046227-0 ◽

2011 ◽

Vol 157 (9) ◽

pp. 2702-2707 ◽

Cited By ~ 25

Author(s):

Sujoy Kumar Sarkar ◽

Mouparna Dutta ◽

Chiranjit Chowdhury ◽

Akash Kumar ◽

Anindya S. Ghosh

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Parent Strain ◽

Amino Acid Identity ◽

Enzymic Activity ◽

Exponential Phase ◽

Wild Type ◽

Acid Identity ◽

Penicillin Binding Proteins ◽

E Coli

Escherichia coli PBP5, PBP6 and DacD, encoded by dacA, dacC and dacD, respectively, share substantial amino acid identity and together constitute ~50 % of the total penicillin-binding proteins of E. coli. PBP5 helps maintain intrinsic β-lactam resistance within the cell. To test if PBP6 and DacD play simlar roles, we deleted dacC and dacD individually, and dacC in combination with dacA, from E. coli 2443 and compared β-lactam sensitivity of the mutants and the parent strain. β-Lactam resistance was complemented by wild-type, but not dd-carboxypeptidase-deficient PBP5, confirming that enzymic activity of PBP5 is essential for β-lactam resistance. Deletion of dacC and expression of PBP6 during exponential or stationary phase did not alter β-lactam resistance of a dacA mutant. Expression of DacD during mid-exponential phase partially restored β-lactam resistance of the dacA mutant. Therefore, PBP5 dd-carboxypeptidase activity is essential for intrinsic β-lactam resistance of E. coli and DacD can partially compensate for PBP5 in this capacity, whereas PBP6 cannot.

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TolC-Dependent Exclusion of Porphyrins in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00595-08 ◽

2008 ◽

Vol 190 (18) ◽

pp. 6228-6233 ◽

Cited By ~ 36

Author(s):

Ryoko Tatsumi ◽

Masaaki Wachi

Keyword(s):

Escherichia Coli ◽

Uv Irradiation ◽

High Performance ◽

Aminolevulinic Acid ◽

Wild Type ◽

E Coli ◽

Chromatography Analysis ◽

Increased Sensitivity ◽

Mutant Cells ◽

Reddish Brown Color

ABSTRACT We found that Escherichia coli tolC mutants showed increased sensitivity to 5-aminolevulinic acid (ALA), a precursor of porphyrins. The tolC mutant cells grown in the presence of ALA showed a reddish brown color under visible light and a strong red fluorescence under near-UV irradiation. Fluorescence spectrometry and high-performance liquid chromatography analysis showed that the tolC mutant cells grown in the presence of ALA accumulated a large amount of coproporphyrin(ogen) intracellularly. In contrast, the wild-type cells produced coproporphyrin extracellularly. The tolC mutant cells grown in the presence of ALA, which were capable of surviving in the dark, were killed by near-UV irradiation, suggesting that the intracellular coproporphyrin(ogen) renders these cells photosensitive. These results suggest that the TolC-dependent efflux system is involved in the exclusion of porphyrin(ogen)s in E. coli.

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Expression in Escherichia coli and characterization of a reconstituted recombinant 7Fe ferredoxin from Desulfovibrio africanus

Biochemical Journal ◽

10.1042/bj3140063 ◽

1996 ◽

Vol 314 (1) ◽

pp. 63-71 ◽

Cited By ~ 10

Author(s):

Johanneke L. H. BUSCH ◽

Jacques L. J. BRETON ◽

Barry M. BARTLETT ◽

Richard JAMES ◽

E. Claude HATCHIKIAN ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Amino Acid Sequence ◽

Magnetic Circular Dichroism ◽

Wild Type ◽

E Coli ◽

Excess Iron ◽

Expression In Escherichia Coli ◽

Type D

Desulfovibrio africanus ferredoxin III is a monomeric protein (molecular mass of 6585 Da) that contains one [3Fe-4S]1+/0 and one [4Fe-4S]2+/1+ cluster when isolated aerobically. The amino acid sequence consists of 61 amino acids, including seven cysteine residues that are all involved in co-ordination to the clusters. In order to isolate larger quantities of D. africanus ferredoxin III, we have overexpressed it in Escherichia coli by constructing a synthetic gene based on the amino acid sequence of the native protein. The recombinant ferredoxin was expressed in E. coli as an apoprotein. We have reconstituted the holoprotein by incubating the apoprotein with excess iron and sulphide in the presence of a reducing agent. The reconstituted recombinant ferredoxin appeared to have a lower stability than that of wild-type D. africanus ferredoxin III. We have shown by low-temperature magnetic circular dichroism and EPR spectroscopy that the recombinant ferredoxin contains a [3Fe-4S]1+/0 and a [4Fe-4S]2+/1+ cluster similar to those found in native D. africanus ferredoxin III. These results indicate that the two clusters have been correctly inserted into the recombinant ferredoxin.

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Cj1386, an Atypical Hemin-Binding Protein, Mediates Hemin Trafficking to KatA in Campylobacter jejuni

Journal of Bacteriology ◽

10.1128/jb.02346-14 ◽

2014 ◽

Vol 197 (5) ◽

pp. 1002-1011 ◽

Cited By ~ 6

Author(s):

Annika Flint ◽

Alain Stintzi

Keyword(s):

Escherichia Coli ◽

Campylobacter Jejuni ◽

Biochemical Properties ◽

Wild Type ◽

Visible Spectroscopy ◽

Content Type ◽

Increased Sensitivity ◽

Uv Visible

Catalase enzymes detoxify H2O2by the dismutation of H2O2into O2and H2O through the use of hemin cofactors. While the structure and biochemical properties of catalase enzymes have been well characterized over many decades of research, it remained unclear how catalases acquire hemin. We have previously reported that Cj1386 is essential for ensuring proper hemin content inCampylobacter jejunicatalase (KatA) (A. Flint, Y. Q. Sun, and A. Stintzi, J Bacteriol194:334–345, 2012). In this report, an in-depth molecular characterization of Cj1386 was performed to elucidate the mechanistic details of this association. Coimmunoprecipitation assays revealed that KatA-Cj1386 transiently interactin vivo, and UV-visible spectroscopy demonstrated that purified Cj1386 protein binds hemin. Furthermore, hemin titration experiments determined that hemin binds to Cj1386 in a 1:1 ratio with hexacoordinate hemin binding. Mutagenesis of potential hemin-coordinating residues in Cj1386 showed that tyrosine 57 was essential for hemin coordination when Cj1386 was overexpressed inEscherichia coli. The importance of tyrosine 57 in hemin traffickingin vivowas confirmed by introducing thecj1386Y57Aallele into aC. jejuniΔcj1386mutant background. Thecj1386Y57Amutation resulted in increased sensitivity toward H2O2relative to the wild type, suggesting that KatA was not functional in this strain. In support of this finding, KatA immunoprecipitated from the Δcj1386+cj1386Y57Amutant had significantly reduced hemin content compared to that of thecj1386WTbackground. Overall, these findings indicate that Cj1386 is involved in directly trafficking hemin to KatA and that tyrosine 57 plays a key role in this function.

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Lipase and Its Modulator fromPseudomonas sp. Strain KFCC 10818: Proline-to-Glutamine Substitution at Position 112 Induces Formation of Enzymatically Active Lipase in the Absence of the Modulator

Journal of Bacteriology ◽

10.1128/jb.183.20.5937-5941.2001 ◽

2001 ◽

Vol 183 (20) ◽

pp. 5937-5941 ◽

Cited By ~ 22

Author(s):

Eun Kyung Kim ◽

Won Hee Jang ◽

Jung Ho Ko ◽

Jong Seok Kang ◽

Moon Jong Noh ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Amino Acid Sequence ◽

Future Research ◽

The Novel ◽

Wild Type ◽

Functional Interaction ◽

Lipase Gene ◽

Efficient Expression ◽

Active Lipase

ABSTRACT A lipase gene, lipK, and a lipase modulator gene,limK, of Pseudomonas sp. strain KFCC 10818 have been cloned, sequenced, and expressed in Escherichia coli. The limK gene is located immediately downstream of the lipK gene. Enzymatically active lipase was produced only in the presence of the limK gene. The effect of the lipase modulator LimK on the expression of active lipase was similar to those of the Pseudomonas subfamily I.1 and I.2 lipase-specific foldases (Lifs). The deduced amino acid sequence of LimK shares low homology (17 to 19%) with the knownPseudomonas Lifs, suggesting thatPseudomonas sp. strain KFCC 10818 is only distantly related to the subfamily I.1 and I.2 Pseudomonasspecies. Surprisingly, a lipase variant that does not require LimK for its correct folding was isolated in the study to investigate the functional interaction between LipK and LimK. When expressed in the absence of LimK, the P112Q variant of LipK formed an active enzyme and displayed 63% of the activity of wild-type LipK expressed in the presence of LimK. These results suggest that the Pro112residue of LipK is involved in a key step of lipase folding. We expect that the novel finding of this study may contribute to future research on efficient expression or refolding of industrially important lipases and on the mechanism of lipase folding.

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De NovoCharacterization of Genes That Contribute to High-Level Ciprofloxacin Resistance in Escherichia coli

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00889-16 ◽

2016 ◽

Vol 60 (10) ◽

pp. 6353-6355 ◽

Cited By ~ 2

Author(s):

Thu Tran ◽

Qinghong Ran ◽

Lev Ostrer ◽

Arkady Khodursky

Keyword(s):

Escherichia Coli ◽

Antibiotic Susceptibility ◽

Relative Fitness ◽

Resistant Bacteria ◽

Wild Type ◽

Content Type ◽

Wild Type Level ◽

Transposon Insertion ◽

Ciprofloxacin Resistance ◽

High Level

ABSTRACTSensitization of resistant bacteria to existing antibiotics depends on the identification of candidate targets whose activities contribute to resistance. Using a transposon insertion library in anEscherichia colimutant that was 2,000 times less susceptible to ciprofloxacin than its parent and the relative fitness scores, we identified 19 genes that contributed to the acquired ciprofloxacin resistance and mapped the shortest genetic path that increased the antibiotic susceptibility of the resistant bacteria back to a near wild-type level.

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