scholarly journals Antibiotic-Sensitive TolC Mutants and Their Suppressors

2004 ◽  
Vol 186 (6) ◽  
pp. 1851-1860 ◽  
Author(s):  
Anne Marie Augustus ◽  
Teresa Celaya ◽  
Fasahath Husain ◽  
Matthew Humbard ◽  
Rajeev Misra
Keyword(s):  
Efflux Pump ◽  
Regulatory Region ◽  
Single Amino Acid ◽  
Missense Mutations ◽  
Antibiotic Resistant ◽  
Isolation And Characterization ◽  
Tolc Protein ◽  
Acrab Efflux Pump ◽  
Extragenic Suppressors ◽  
Intragenic Suppressor

ABSTRACT The TolC protein of Escherichia coli, through its interaction with AcrA and AcrB, is thought to form a continuous protein channel that expels inhibitors from the cell. Consequently, tolC null mutations display a hypersensitive phenotype. Here we report the isolation and characterization of tolC missense mutations that direct the synthesis of mutant TolC proteins partially disabled in their efflux role. All alterations, consisting of single amino acid substitutions, were localized within the periplasmic α-helical domain. In two mutants carrying an I106N or S350F substitution, the hypersensitivity phenotype may be in part due to aberrant TolC assembly. However, two other alterations, R367H and R390C, disrupted efflux function by affecting interactions among the helices surrounding TolC's periplasmic tunnel. Curiously, these two TolC mutants were sensitive to a large antibiotic, vancomycin, and exhibited a Dex+ phenotype. These novel phenotypes of TolCR367H and TolCR390C were likely the result of a general influx of molecules through a constitutively open tunnel aperture, which normally widens only when TolC interacts with other proteins during substrate translocation. An intragenic suppressor alteration (T140A) was isolated from antibiotic-resistant revertants of the hypersensitive TolCR367H mutant. T140A also reversed, either fully (R390C) or partially (I106N and S350F), the hypersensitivity phenotype of other TolC mutants. Our data suggest that this global suppressor phenotype of T140A is the result of impeded antibiotic influx caused by tapering of the tunnel passage rather than by correcting individual mutational defects. Two extragenic suppressors of TolCR367H, mapping in the regulatory region of acrAB, uncoupled the AcrR-mediated repression of the acrAB genes. The resulting overexpression of AcrAB reduced the hypersensitivity phenotype of all the TolC mutants. Similar results were obtained when the chromosomal acrR gene was deleted or the acrAB genes were expressed from a plasmid. Unlike the case for the intragenic suppressor T140A, the overexpression of AcrAB diminished hypersensitivity towards only erythromycin and novobiocin, which are substrates of the TolC-AcrAB efflux pump, but not towards vancomycin, which is not a substrate of this pump. This showed that the two types of suppressors produced their effects by fundamentally different means, as the intragenic suppressor decreased the general influx while extragenic suppressors increased the efflux of TolC-AcrAB pump-specific antibiotics.

scholarly journals SSN20 is an essential gene with mutant alleles that suppress defects in SUC2 transcription in Saccharomyces cerevisiae.

1987 ◽  
Vol 7 (2) ◽  
pp. 672-678 ◽  
Author(s):  
L Neigeborn ◽  
J L Celenza ◽  
M Carlson
Keyword(s):  
Gene Dosage ◽  
Essential Gene ◽  
Regulatory Region ◽  
Upstream Region ◽  
Temperature Sensitive ◽  
Missense Mutations ◽  
Haploid Cell ◽  
Recessive Mutations ◽  
In Trans ◽  
Extragenic Suppressors

Dominant and recessive mutations at the SSN20 locus were previously isolated as extragenic suppressors of mutations in three genes (SNF2, SNF5, and SNF6) that are required in trans to derepress invertase expression. All ssn20 alleles cause recessive, temperature-sensitive lethality. In this study we cloned the SSN20 gene, identified a 4.6-kilobase poly(A)-containing RNA, and showed that disruption of the gene is lethal in a haploid cell. Genetic mapping of SSN20 to a locus on chromosome VII 10 centimorgans distal to cly8 led to the finding that SSN20 is the same gene as SPT6, which affects expression of delta insertions in the 5' noncoding region of HIS4 (F. Winston, D. T. Chaleff, B. Valent, and G. R. Fink, Genetics 107:179-197, 1984). We also showed that an ssn20 mutation restored expression of secreted invertase from deletions of the SUC2 upstream regulatory region; ssn20 restored derepression of SUC2 mRNA in strains with a SUC2 upstream region deletion or a snf2 mutation. Increased or decreased gene dosage of SSN20 also suppressed defects that are suppressed by ssn20 missense mutations. These findings suggest that SSN20 plays a role in general transcriptional processes.

scholarly journals Reversal of the Drug Binding Pocket Defects of the AcrB Multidrug Efflux Pump Protein of Escherichia coli

2015 ◽  
Vol 197 (20) ◽  
pp. 3255-3264 ◽  
Author(s):  
Ketaki Soparkar ◽  
Alfred D. Kinana ◽  
Jon W. Weeks ◽  
Keith D. Morrison ◽  
Hiroshi Nikaido ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Efflux Pump ◽  
Conformational Dynamics ◽  
Binding Pocket ◽  
Drug Binding ◽  
Single Amino Acid ◽  
Multidrug Efflux Pump ◽  
Content Type ◽  
Isolation And Characterization ◽  
Efflux Kinetics

ABSTRACTThe AcrB protein ofEscherichia coli, together with TolC and AcrA, forms a contiguous envelope conduit for the capture and extrusion of diverse antibiotics and cellular metabolites. In this study, we sought to expand our knowledge of AcrB by conducting genetic and functional analyses. We began with an AcrB mutant bearing an F610A substitution in the drug binding pocket and obtained second-site substitutions that overcame the antibiotic hypersusceptibility phenotype conferred by the F610A mutation. Five of the seven unique single amino acid substitutions—Y49S, V127A, V127G, D153E, and G288C—mapped in the periplasmic porter domain of AcrB, with the D153E and G288C mutations mapping near and at the distal drug binding pocket, respectively. The other two substitutions—F453C and L486W—were mapped to transmembrane (TM) helices 5 and 6, respectively. The nitrocefin efflux kinetics data suggested that all periplasmic suppressors significantly restored nitrocefin binding affinity impaired by the F610A mutation. Surprisingly, despite increasing MICs of tested antibiotics and the efflux ofN-phenyl-1-naphthylamine, the TM suppressors did not improve the nitrocefin efflux kinetics. These data suggest that the periplasmic substitutions act by influencing drug binding affinities for the distal binding pocket, whereas the TM substitutions may indirectly affect the conformational dynamics of the drug binding domain.IMPORTANCEThe AcrB protein and its homologues confer multidrug resistance in many important human bacterial pathogens. A greater understanding of how these efflux pump proteins function will lead to the development of effective inhibitors against them. The research presented in this paper investigates drug binding pocket mutants of AcrB through the isolation and characterization of intragenic suppressor mutations that overcome the drug susceptibility phenotype of mutations affecting the drug binding pocket. The data reveal a remarkable structure-function plasticity of the AcrB protein pertaining to its drug efflux activity.

SSN20 is an essential gene with mutant alleles that suppress defects in SUC2 transcription in Saccharomyces cerevisiae

1987 ◽  
Vol 7 (2) ◽  
pp. 672-678
Author(s):  
L Neigeborn ◽  
J L Celenza ◽  
M Carlson
Keyword(s):  
Gene Dosage ◽  
Essential Gene ◽  
Regulatory Region ◽  
Upstream Region ◽  
Temperature Sensitive ◽  
Missense Mutations ◽  
Haploid Cell ◽  
Recessive Mutations ◽  
In Trans ◽  
Extragenic Suppressors

Dominant and recessive mutations at the SSN20 locus were previously isolated as extragenic suppressors of mutations in three genes (SNF2, SNF5, and SNF6) that are required in trans to derepress invertase expression. All ssn20 alleles cause recessive, temperature-sensitive lethality. In this study we cloned the SSN20 gene, identified a 4.6-kilobase poly(A)-containing RNA, and showed that disruption of the gene is lethal in a haploid cell. Genetic mapping of SSN20 to a locus on chromosome VII 10 centimorgans distal to cly8 led to the finding that SSN20 is the same gene as SPT6, which affects expression of delta insertions in the 5' noncoding region of HIS4 (F. Winston, D. T. Chaleff, B. Valent, and G. R. Fink, Genetics 107:179-197, 1984). We also showed that an ssn20 mutation restored expression of secreted invertase from deletions of the SUC2 upstream regulatory region; ssn20 restored derepression of SUC2 mRNA in strains with a SUC2 upstream region deletion or a snf2 mutation. Increased or decreased gene dosage of SSN20 also suppressed defects that are suppressed by ssn20 missense mutations. These findings suggest that SSN20 plays a role in general transcriptional processes.

scholarly journals Missense Mutations in the CrrB Protein Mediate Odilorhabdin Derivative Resistance in Klebsiella pneumoniae

2021 ◽  
Vol 65 (5) ◽  
Author(s):  
Lucile Pantel ◽  
Paulo Juarez ◽  
Marine Serri ◽  
Lilia Boucinha ◽  
Emilie Lessoud ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Efflux Pump ◽  
Response Regulator ◽  
Single Amino Acid ◽  
Missense Mutations ◽  
Transmembrane Region ◽  
Content Type ◽  
Resistance Pathway ◽  
Resistant Strains

ABSTRACT NOSO-502 is a preclinical antibiotic candidate of the odilorhabdin class. This compound exhibits activity against Enterobacteriaceae pathogens, including carbapenemase-producing bacteria and most of the colistin (CST)-resistant strains. Among a collection of CST-resistant Klebsiella pneumoniae strains harboring mutations in the genes pmrAB, mgrB, phoPQ, and crrB, only those bearing mutations in the gene crrB were found to be resistant to NOSO-502. CrrB is a histidine kinase which acts with the response regulator CrrA to modulate the PmrAB system, which induces the restructuring of lipopolysaccharide on the outer membrane and thus leads to CST resistance. Moreover, crrB mutations also enhance the transcription of neighboring genes, such as H239_3063, encoding an ABC transporter transmembrane region, H239_3064, encoding a putative efflux pump also known as KexD, and H239_3065, encoding an N-acetyltransferase. To elucidate the mechanism of resistance to NOSO-502 induced by CrrB missense mutations in K. pneumoniae, mutants of NCTC 13442 and ATCC BAA-2146 strains resistant to NOSO-502 and CST with single amino acid substitutions in CrrB (S8N, F33Y, Y34N, W140R, N141I, P151A, P151L, P151S, P151T, or F303Y) were selected. Full susceptibility to NOSO-502 was restored in crrA- or crrB-deleted K. pneumoniae NCTC 13442 CrrB (P151L) mutants, confirming the role of CrrAB in controlling this resistance pathway. Deletion of kexD (but not other neighboring genes) in the same mutant also restored NOSO-502-susceptibility. Upregulation of the kexD gene expression was observed for all CrrB mutants. Finally, plasmid expression of kexD in a K. pneumoniae strain missing the locus crrABC and kexD significantly increased resistance to NOSO-502.

scholarly journals Characterization of RarA, a Novel AraC Family Multidrug Resistance Regulator in Klebsiella pneumoniae

2012 ◽  
Vol 56 (8) ◽  
pp. 4450-4458 ◽  
Author(s):  
Mark Veleba ◽  
Paul G. Higgins ◽  
Gerardo Gonzalez ◽  
Harald Seifert ◽  
Thamarai Schneiders
Keyword(s):  
Multidrug Resistance ◽  
Klebsiella Pneumoniae ◽  
Efflux Pump ◽  
Content Type ◽  
Resistance Phenotype ◽  
Serratia Proteamaculans ◽  
Virulence Potential ◽  
Article Type ◽  
Acrab Efflux Pump

ABSTRACTTranscriptional regulators, such as SoxS, RamA, MarA, and Rob, which upregulate the AcrAB efflux pump, have been shown to be associated with multidrug resistance in clinically relevant Gram-negative bacteria. In addition to the multidrug resistance phenotype, these regulators have also been shown to play a role in the cellular metabolism and possibly the virulence potential of microbial cells. As such, the increased expression of these proteins is likely to cause pleiotropic phenotypes.Klebsiella pneumoniaeis a major nosocomial pathogen which can express the SoxS, MarA, Rob, and RamA proteins, and the accompanying paper shows that the increased transcription oframAis associated with tigecycline resistance (M. Veleba and T. Schneiders, Antimicrob. Agents Chemother. 56:4466–4467, 2012). Bioinformatic analyses of the availableKlebsiellagenome sequences show that an additional AraC-type regulator is encoded chromosomally. In this work, we characterize this novel AraC-type regulator, hereby called RarA (Regulator of antibiotic resistance A), which is encoded inK. pneumoniae,Enterobactersp. 638,Serratia proteamaculans568, andEnterobacter cloacae. We show that the overexpression ofrarAresults in a multidrug resistance phenotype which requires a functional AcrAB efflux pump but is independent of the other AraC regulators. Quantitative real-time PCR experiments show thatrarA(MGH 78578 KPN_02968) and its neighboring efflux pump operonoqxAB(KPN_02969_02970) are consistently upregulated in clinical isolates collected from various geographical locations (Chile, Turkey, and Germany). Our results suggest thatrarAoverexpression upregulates theoqxABefflux pump. Additionally, it appears thatoqxR, encoding a GntR-type regulator adjacent to theoqxABoperon, is able to downregulate the expression of theoqxABefflux pump, where OqxR complementation resulted in reductions to olaquindox MICs.

scholarly journals Complete Deletion of theramRGene in anIn Vitro-Selected Mutant of Klebsiella pneumoniae Overexpressing the AcrAB Efflux Pump

2012 ◽  
Vol 57 (1) ◽  
pp. 672-673 ◽  
Author(s):  
Suzanne Bialek-Davenet ◽  
Véronique Leflon-Guibout ◽  
Olivier Tran Minh ◽  
Estelle Marcon ◽  
Richard Moreau ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Efflux Pump ◽  
Acrab Efflux Pump ◽  
Complete Deletion

scholarly journals Single amino acid exchanges in separate domains of the Drosophila serendipity delta zinc finger protein cause embryonic and sex biased lethality.

Genetics ◽  
1992 ◽  
Vol 131 (4) ◽  
pp. 905-916 ◽  
Author(s):  
M Crozatier ◽  
K Kongsuwan ◽  
P Ferrer ◽  
J R Merriam ◽  
J A Lengyel ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Essential Gene ◽  
Larval Instar ◽  
Single Amino Acid ◽  
Isolation And Characterization ◽  
Finger Protein

Abstract The Drosophila serendipity (sry) delta (delta) zinc finger protein is a sequence-specific DNA binding protein, maternally inherited by the embryo and present in nuclei of transcriptionally active cells throughout fly development. We report here the isolation and characterization of four ethyl methanesulfate-induced zygotic lethal mutations of different strengths in the sry delta gene. For the stronger allele, all of the lethality occurs during late embryogenesis or the first larval instar. In the cases of the three weaker alleles, most of the lethality occurs during pupation; moreover, those adult escapers that emerge are sterile males lacking partially or completely in spermatozoa bundles. Genetic analysis of sry delta thus indicates that it is an essential gene, whose continued expression throughout the life cycle, notably during embryogenesis and pupal stage, is required for viability. Phenotypic analysis of sry delta hemizygote escaper males further suggests that sry delta may be involved in regulation of two different sets of genes: genes required for viability and genes involved in gonadal development. All four sry delta alleles are fully rescued by a wild-type copy of sry delta, but not by an additional copy of the sry beta gene, reinforcing the view that, although structurally related, these two genes exert distinct functions. Molecular characterization of the four sry delta mutations revealed that these mutations correspond to single amino acid replacements in the sry delta protein. Three of these replacements map to the same (third out of seven) zinc finger in the carboxy-terminal DNA binding domain; interestingly, none affects the zinc finger consensus residues. The fourth mutation is located in the NH2-proximal part of the protein, in a domain proposed to be involved in specific protein-protein interactions.

scholarly journals Isolation and characterization of bacteria with multiple traits: Hydrocarbon degradation, antibiotic-resistant and metal tolerant

2019 ◽  
Vol 10 (4) ◽  
pp. 3789-3795 ◽  
Author(s):  
Neeta Bhagat ◽  
Pranita Roy ◽  
Sohini Singh ◽  
Tanu Allen
Keyword(s):  
Heavy Metals ◽  
Polycyclic Aromatic Hydrocarbons ◽  
Soil Pollution ◽  
Aromatic Hydrocarbons ◽  
Well Being ◽  
Antibiotic Resistant ◽  
Morphological Studies ◽  
Isolation And Characterization ◽  
Degrading Bacteria ◽  
Polycyclic Aromatic

Increasing soil pollution all over the world has instigated global concerns as enormous quantities of toxic chemicals and heavy metals like cadmium, lead, mercury, petrochemicals, insecticides, polycyclic aromatic hydrocarbons (PAHs) and chlorophenols are finding their way into the environment, affecting the land and soil, causing soil pollution and thus posing a threat and menace to health and well- being of people and ecosystem. The ubiquitous dissemination, low bioavailability, high perseverance of contaminants like poly-hydrocarbon and metals in soil have the potentially destructive effects to human health, envisages to study the biodegradation of PAHs (polycyclic aromatic hydrocarbons) and PACs (polycyclic aromatic compounds). The diversity of micro-organisms that diminish the PAHs/PACs can be utilized in the advancement of bioremediation techniques. The role of metal-tolerant, (PAH)-degrading bacteria helps in the biodegradation of organic compounds at miscellaneous polluted sites. The isolation of (PAHs)-degrading bacteria from contaminated soil samples collected from garages and petrol pumps of Delhi and NCR region was carried out in the present study.  Also, the bacterial samples were tested for the tolerance towards 4 heavy metals- arsenic (As), lead (Pb), cadmium (Cd), and mercury (Hg). Morphological studies and biochemical tests were conducted to find the genera of the bacterial samples. The study indicates that hydrocarbons were degraded by the isolates P1, P2, P4, P5, P5*, G1, G3. These isolates were also found to be tolerant at a high concentration of metals (Arsenic, Cadmium, Mercury, and Lead) as minimum inhibitory concentration (MIC) was also calculated. Antibiotic susceptibility of the isolates was tested against various antibiotics. Thus the study suggests that the isolates identified as Pseudomonas aeruginosa, Acinetobacter baumanii, and Klebsiella pneumoniae are not only PAH-degrading but metal-tolerant and antibiotic-resistant too and are of immense potential for bioremediation of contaminated soils.

scholarly journals Isolation and characterization of the SPT2 gene, a negative regulator of Ty-controlled yeast gene expression.

1985 ◽  
Vol 5 (7) ◽  
pp. 1543-1553 ◽  
Author(s):  
G S Roeder ◽  
C Beard ◽  
M Smith ◽  
S Keranen
Keyword(s):  
Gene Product ◽  
Regulatory Region ◽  
Negative Regulator ◽  
Transcriptional Level ◽  
Wild Type ◽  
Isolation And Characterization ◽  
Mutant Strains ◽  
Insertion Mutations ◽  
His4 Gene

The his4-917 mutation of Saccharomyces cerevisiae results from the insertion of the Ty element Ty917 into the regulatory region of the HIS4 gene and renders the cell His-. The hist4-912 delta mutant, which carries a solo delta in the 5'-noncoding region of HIS4, is His+ at 37 degrees C but His- at 23 degrees C. Both these mutations interfere with HIS4 expression at the transcriptional level. The His- phenotype of both insertion mutations is suppressed by mutations at the SPT2 locus. The product of the wild-type SPT2 gene apparently represses HIS4 transcription in these mutant strains; this repression is relieved when the SPT2 gene is destroyed by mutation. The repression of transcription by SPT2 presumably results from an interaction between the SPT2+ gene product and Ty or delta sequences. In this paper, we report the cloning and DNA sequence analysis of the wild-type SPT2 gene and show that the gene is capable of encoding a protein of 333 amino acids in length. In addition, we show that a dominant mutation of the SPT2 gene results from the generation of an ochre codon which is presumed to lead to a shortened SPT2 gene product.

scholarly journals Identification of a Copper-Inducible Promoter for Use in Ectopic Expression in the Fungal Pathogen Histoplasma capsulatum

2006 ◽  
Vol 5 (6) ◽  
pp. 935-944 ◽  
Author(s):  
Dana Gebhart ◽  
Adam K. Bahrami ◽  
Anita Sil
Keyword(s):  
Gene Expression ◽  
Fungal Pathogen ◽  
Large Scale ◽  
Histoplasma Capsulatum ◽  
Fluorescent Protein ◽  
Efflux Pump ◽  
Regulatory Region ◽  
Ectopic Expression ◽  
Upstream Regulatory Region ◽  
Yeast Cultures

ABSTRACT Despite the existence of a number of genetic tools to study the fungal pathogen Histoplasma capsulatum, strategies for conditional gene expression have not been developed. We used microarray analysis to identify genes that are transcriptionally induced or repressed by the addition of copper sulfate (CuSO4) to H. capsulatum yeast cultures. One of these genes, CRP1, encodes a putative copper efflux pump that is significantly induced in the presence of CuSO4. The upstream regulatory region of CRP1 was sufficient to drive copper-regulated expression of two reporter genes, lacZ and the gene encoding green fluorescent protein. Microarray experiments were performed to determine a copper concentration that triggers accumulation of the CRP1 transcript without significant perturbation of global gene expression. These studies show that the CRP1 upstream regulatory region can be used for ectopic expression of heterologous genes in H. capsulatum. Furthermore, they demonstrate the strategic use of microarrays to identify conditional promoters that confer induction in the absence of large-scale shifts in gene expression.

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