scholarly journals Ribosomal mutations promote the evolution of antibiotic resistance in a multidrug environment

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
James E Gomez ◽  
Benjamin B Kaufmann-Malaga ◽  
Carl N Wivagg ◽  
Peter B Kim ◽  
Melanie R Silvis ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Mycobacterium Smegmatis ◽  
Large Scale ◽  
Wild Type ◽  
Membrane Stress ◽  
Stepping Stones ◽  
Suppressor Mutations ◽  
Genes Encoding ◽  
High Level ◽  
Chromosomal Mutations

Antibiotic resistance arising via chromosomal mutations is typically specific to a particular antibiotic or class of antibiotics. We have identified mutations in genes encoding ribosomal components in Mycobacterium smegmatis that confer resistance to several structurally and mechanistically unrelated classes of antibiotics and enhance survival following heat shock and membrane stress. These mutations affect ribosome assembly and cause large-scale transcriptomic and proteomic changes, including the downregulation of the catalase KatG, an activating enzyme required for isoniazid sensitivity, and upregulation of WhiB7, a transcription factor involved in innate antibiotic resistance. Importantly, while these ribosomal mutations have a fitness cost in antibiotic-free medium, in a multidrug environment they promote the evolution of high-level, target-based resistance. Further, suppressor mutations can then be easily acquired to restore wild-type growth. Thus, ribosomal mutations can serve as stepping-stones in an evolutionary path leading to the emergence of high-level, multidrug resistance.

scholarly journals Arabidopsis Genes Essential for Seedling Viability: Isolation of Insertional Mutants and Molecular Cloning

Genetics ◽  
2001 ◽  
Vol 159 (4) ◽  
pp. 1765-1778
Author(s):  
Gregory J Budziszewski ◽  
Sharon Potter Lewis ◽  
Lyn Wegrich Glover ◽  
Jennifer Reineke ◽  
Gary Jones ◽  
...  
Keyword(s):  
Large Scale ◽  
Protein Translocation ◽  
Gene Families ◽  
Mutant Phenotype ◽  
Lethal Mutant ◽  
A Genome ◽  
Genes Encoding ◽  
High Level ◽  
Mutant Lines ◽  
Genome Scale

Abstract We have undertaken a large-scale genetic screen to identify genes with a seedling-lethal mutant phenotype. From screening ~38,000 insertional mutant lines, we identified >500 seedling-lethal mutants, completed cosegregation analysis of the insertion and the lethal phenotype for >200 mutants, molecularly characterized 54 mutants, and provided a detailed description for 22 of them. Most of the seedling-lethal mutants seem to affect chloroplast function because they display altered pigmentation and affect genes encoding proteins predicted to have chloroplast localization. Although a high level of functional redundancy in Arabidopsis might be expected because 65% of genes are members of gene families, we found that 41% of the essential genes found in this study are members of Arabidopsis gene families. In addition, we isolated several interesting classes of mutants and genes. We found three mutants in the recently discovered nonmevalonate isoprenoid biosynthetic pathway and mutants disrupting genes similar to Tic40 and tatC, which are likely to be involved in chloroplast protein translocation. Finally, we directly compared T-DNA and Ac/Ds transposon mutagenesis methods in Arabidopsis on a genome scale. In each population, we found only about one-third of the insertion mutations cosegregated with a mutant phenotype.

scholarly journals Overproduction of Wild-Type and Bioengineered Derivatives of the Lantibiotic Lacticin 3147

2006 ◽  
Vol 72 (6) ◽  
pp. 4492-4496 ◽  
Author(s):  
Paul D. Cotter ◽  
Lorraine A. Draper ◽  
Elaine M. Lawton ◽  
Olivia McAuliffe ◽  
Colin Hill ◽  
...  
Keyword(s):  
Broad Spectrum ◽  
Structural Genes ◽  
Wild Type ◽  
Genetic Determinants ◽  
Lacticin 3147 ◽  
Genes Encoding ◽  
High Level ◽  
Derivatives Of

ABSTRACT Lacticin 3147 is a broad-spectrum two-peptide lantibiotic whose genetic determinants are located on two divergent operons on the lactococcal plasmid pMRC01. Here we introduce each of 14 subclones, containing different combinations of lacticin 3147 genes, into MG1363 (pMRC01) and determine that a number of them can facilitate overproduction of the lantibiotic. Based on these studies it is apparent that while the provision of additional copies of genes encoding the biosynthetic/production machinery and the regulator LtnR is a requirement for high-level overproduction, the presence of additional copies of the structural genes (i.e., ltnA1A2) is not.

scholarly journals An improved Xer-cise technology for the generation of multiple unmarked mutants in Mycobacteria

2019 ◽  
Author(s):  
Yves-Marie Boudehen ◽  
Maximillian Wallat ◽  
Philippe Rousseau ◽  
Olivier Neyrolles ◽  
Claude Gutierrez
Keyword(s):  
Antibiotic Resistance ◽  
High Frequency ◽  
Mycobacterium Smegmatis ◽  
Bacterial Species ◽  
Wild Type ◽  
Model Species ◽  
Mycobacterial Genome ◽  
Bacterial Chromosomes ◽  
Genetic Constructs ◽  
Zeocin Resistance

SummaryXer-cise is a technique using antibiotic resistance cassettes flanked by dif sites allowing spontaneous and accurate excision from bacterial chromosomes with a high frequency through the action of the cellular recombinase XerCD. Here, we report a significant improvement of Xer-cise in Mycobacteria. Zeocin-resistance cassettes flanked by variants of the natural Mycobacterium tuberculosis dif site were constructed and shown to be effective tools to construct multiple unmarked mutations in M. tuberculosis and in the model species Mycobacterium smegmatis. The dif site variants harbor mutations in the central region and can therefore not recombine with the wild type or other variants, resulting in mutants of increased genetic stability. The herein described method should be generalizable to virtually any transformable bacterial species.Method summarydif-ZeoR-dif cassettes are used to replace non-essential genes in mycobacterial genome through recombineering. Spontaneous excision of the cassette is carried out under the action of the recombinase XerCD, resulting in unmarked deletions. Subsequent rounds of mutagenesis using cassettes flanked by a range of dif site variants allow construction of multiple mutants in which the different dif sites cannot recombine which each other, yielding stable genetic constructs.

scholarly journals Altered permeability and beta-lactam resistance in a mutant of Mycobacterium smegmatis.

1997 ◽  
Vol 41 (8) ◽  
pp. 1721-1724 ◽  
Author(s):  
S Mukhopadhyay ◽  
P Chakrabarti
Keyword(s):  
Mycobacterium Smegmatis ◽  
Parent Strain ◽  
Beta Lactamase ◽  
Wild Type ◽  
Beta Lactam ◽  
Beta Lactams ◽  
Intact Cells ◽  
Glycine Uptake ◽  
Penicillin Binding Proteins ◽  
High Level

Beta-lactam resistance in mycobacteria results from an interplay between the following: (i) beta-lactamase production, (ii) affinity of the penicillin-binding proteins (PBPs) for the drugs, and (iii) permeation of the drugs. A laboratory mutant of Mycobacterium smegmatis was studied in order to evaluate the roles of these factors in beta-lactam resistance. Mutant M13 was between 7- and 78-fold more resistant than the wild type to cephaloridine, cefoxitin, cefazolin, cefamandole, and cephalothin. Increased beta-lactamase activity toward these antibiotics was not observed in the mutant. The PBP profiles of the wild type and M13 were comparable. However, the affinities of PBP 1 for the beta-lactams tested were lower for the mutant than for the wild type. The permeation of the drugs measured in intact cells was lower for M13 than for the parent strain. The liposome swelling technique, which could be used for cephaloridine, also supported this view. Reduced permeation was not restricted to the beta-lactams alone. Glycine uptake was also lower in M13. Taken together, the results suggest that decreased affinities of PBP 1 for beta-lactams, combined with the decreased permeability of the cell wall of the mutant, lead to the development of high-level acquired beta-lactam resistance.

scholarly journals The Phn system of Mycobacterium smegmatis: a second high-affinity ABC-transporter for phosphate

Microbiology ◽  
2006 ◽  
Vol 152 (11) ◽  
pp. 3453-3465 ◽  
Author(s):  
Susanne Gebhard ◽  
Sieu L. Tran ◽  
Gregory M. Cook
Keyword(s):  
Transport System ◽  
Mycobacterium Smegmatis ◽  
Single Copy ◽  
Phosphate Transport ◽  
Phosphate Limitation ◽  
Wild Type ◽  
High Affinity ◽  
Genes Encoding ◽  
Limiting Nutrient ◽  
In The Wild

Uptake of inorganic phosphate, an essential but often limiting nutrient, in bacteria is usually accomplished by the high-affinity ABC-transport system Pst. Pathogenic species of mycobacteria contain several copies of the genes encoding the Pst system (pstSCAB), and two of the encoded proteins, PstS1 and PstS2, have been shown to be virulence factors in Mycobacterium tuberculosis. The fast-growing Mycobacterium smegmatis contains only a single copy of the pst operon. This study reports the biochemical and molecular characterization of a second high-affinity phosphate transport system, designated Phn. The Phn system is encoded by a three-gene operon that constitutes the components of a putative ABC-type phosphonate/phosphate transport system. Expression studies using phnD– and pstS–lacZ transcriptional fusions showed that both operons were induced when the culture entered phosphate limitation, indicating a role for both systems in phosphate uptake at low extracellular concentrations. Deletion mutants in either phnD or pstS failed to grow in minimal medium with a 10 mM phosphate concentration, while the isogenic wild-type strain mc2155 grew at micromolar phosphate concentrations. Analysis of the kinetics of phosphate transport in the wild-type and mutant strains led to the proposal that the Phn and Pst systems are both high-affinity phosphate transporters with similar affinities for phosphate (i.e. apparent K m values between 40 and 90 μM Pi). The Phn system of M. smegmatis appears to be unique in that, unlike previously identified Phn systems, it does not recognize phosphonates or phosphite as substrates.

scholarly journals A SUPPRESSOR OF snf1 MUTATIONS CAUSES CONSTITUTIVE HIGH-LEVEL INVERTASE SYNTHESIS IN YEAST

Genetics ◽  
1984 ◽  
Vol 107 (1) ◽  
pp. 19-32
Author(s):  
Marian Carlson ◽  
Barbara C Osmond ◽  
Lenore Neigeborn ◽  
David Botstein
Keyword(s):  
Genetic Characterization ◽  
Glucose Repression ◽  
Invertase Activity ◽  
Complementation Group ◽  
Regulatory Function ◽  
Wild Type ◽  
Suppressor Mutations ◽  
Complementation Groups ◽  
High Level

ABSTRACT The SNF1 gene product of Saccharomyces cerevisiae is required to derepress expression of many glucose-repressible genes, including the SUC2 structural gene for invertase. Strains carrying a recessive snf1 mutation are unable to ferment sucrose. We have isolated 30 partial phenotypic revertants of a snf1 mutant that were able to ferment sucrose. Genetic characterization of these revertants showed that the suppressor mutations were all recessive and defined eight complementation groups, designated ssn1 through ssn8 (suppressor of snf1). The revertants were assayed for secreted invertase activity, and although activity was detected in members of each complementation group, only the ssn6 strains contained wild-type levels. Synthesis of secreted invertase in ssn6 strains was found to be constitutive, that is, insensitive to glucose repression; moreover, the ssn6 mutations also conferred constitutivity in a wild-type (SNF1) genetic background and are, therefore, not merely suppressors of snf1. Pleiotropic defects were observed in ssn6 mutants. Genetic analysis suggested that the ssn6 mutations are allelic to the cyc8 mutation isolated by R. J. Rothstein and F. Sherman, which causes increased production of iso-2-cytochrome c. The data suggest a regulatory function for SSN6.

scholarly journals ABSENCE OF DETECTABLE MITOCHONDRIAL RECOMBINATION IN PARAMECIUM

Genetics ◽  
1979 ◽  
Vol 93 (4) ◽  
pp. 797-831
Author(s):  
André Adoutte ◽  
Jonathan K Knowles ◽  
Annie Sainsard-Chanet
Keyword(s):  
Antibiotic Resistance ◽  
Large Scale ◽  
Nuclear Gene ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Resistance Mutations ◽  
Mitochondrial Recombination ◽  
Mitochondrial Ribosomes ◽  
Molecular Alterations ◽  
Resistance Markers

ABSTRACT An extensive search for recombination between mitochondrial markers was carried out in Paramecium tetraurelia. Thirty-two combinations, altogether involving 24 different markers, were studied. The markers belonged to the three main categories of mitochondrial mutations presently available in this organism. (a) Spontaneous or UV-induced antibiotic resistance mutations, most probably affecting mitochondrial ribosomes, (b) nitrowguanidine-induced antibiotic resistance markers displaying thermosensitivity or slow growth, enabling easy selection of possible wild-type recombinants, and (c) mitochondrial partial suppressors of a nuclear gene, probably corresponding to molecular alterations distinct from the preceding two categories. In addition, different genetic configurations were analyzed (i e., mutant x mutant, double-mutant x wild-type, etc.).——None of the combinations yielded any evidence for the occurrence of recombined genomes despite the fact that: (1) all of them were studied on a large scale involving the screening of at least several thousand mitochondrial genomes (often several millions), (2) in many of them the detection level was sufficiently high to enable the isolation of spontaneous mutants in control cells, and (3) in several of them, reconstitution experiments carried out in parallel show that the conditions were fully adequate to detect recombinant genotypes. The results are in marked contrast with those obtained on the few other organisms in which mitochondrial recombination has been studied, particularly Saccharomyces cerevisiae, in which mitochondrial recombination is intense.——The most likely basis for the various manifestations of mitochondrial genetic autonomy in Paramecium, described in this as well as in previous publications, is that the chondriome of this organism is made up of thousands of structurally discrete, noninteracting units.

scholarly journals Phenotypic and Biochemical Comparison of the Carbapenem-Hydrolyzing Activities of Five Plasmid-Borne AmpC β-Lactamases

2010 ◽  
Vol 54 (11) ◽  
pp. 4556-4560 ◽  
Author(s):  
Hedi Mammeri ◽  
Hélène Guillon ◽  
François Eb ◽  
Patrice Nordmann
Keyword(s):  
Biochemical Analysis ◽  
Carbapenem Resistance ◽  
Catalytic Efficiency ◽  
Wild Type ◽  
E Coli ◽  
Genes Encoding ◽  
High Level ◽  
Biochemical Comparison ◽  
Outer Membrane Permeability

ABSTRACT The CMY-2, ACT-1, DHA-1, ACC-1, and FOX-1 enzymes are representative of five plasmid-mediated AmpC (pAmpC) β-lactamase clusters. Resistance to imipenem has been reported in Enterobacteriaceae as a result of pAmpC expression combined with decreased outer membrane permeability. The aim of this study was to determine the role of different pAmpCs in carbapenem resistance and to define the structure/activity relationship supporting carbapenemase activity. The ampC genes encoding the five pAmpCs and the chromosomal AmpC of Escherichia coli EC6, which was used as a reference cephalosporinase, were cloned and introduced into wild-type E. coli TOP10 and OmpC/OmpF porin-deficient E. coli HB4 strains. The MICs of β-lactams for the recombinant strains revealed that CMY-2, ACT-1, and DHA-1 β-lactamases conferred a high level of resistance to ceftazidime and cefotaxime once expressed in E. coli TOP10 and reduced significantly the susceptibility to imipenem once expressed in E. coli HB4. In contrast, FOX-1 and ACC-1 enzymes did not confer resistance to imipenem. Biochemical analysis showed that CMY-2 β-lactamase and, to a lesser extent, ACT-1 exhibited the highest catalytic efficiency toward imipenem and showed low Km values. A modeling study revealed that the large R2 binding site of these two enzymes may support the carbapenemase activity. Therefore, CMY-2-type, ACT-1-type, and DHA-1-type β-lactamases may promote the emergence of carbapenem resistance in porin-deficient clinical isolates.

scholarly journals An improved Xer-cise technology for the generation of multiple unmarked mutants in Mycobacteria

BioTechniques ◽  
2020 ◽  
Vol 68 (2) ◽  
pp. 106-110 ◽  
Author(s):  
Yves-Marie Boudehen ◽  
Maximilian Wallat ◽  
Philippe Rousseau ◽  
Olivier Neyrolles ◽  
Claude Gutierrez
Keyword(s):  
Antibiotic Resistance ◽  
Mycobacterium Tuberculosis ◽  
Central Region ◽  
High Frequency ◽  
Mycobacterium Smegmatis ◽  
Bacterial Species ◽  
Wild Type ◽  
Model Species ◽  
Bacterial Chromosomes ◽  
Zeocin Resistance

Xer-cise is a technique using antibiotic resistance cassettes flanked by dif sites allowing spontaneous and accurate excision from bacterial chromosomes with a high frequency through the action of the cellular recombinase XerCD. Here, we report a significant improvement of Xer-cise in Mycobacteria. Zeocin resistance cassettes flanked by variants of the natural Mycobacterium tuberculosis dif site were constructed and shown to be effective tools to construct multiple unmarked mutations in M. tuberculosis and in the model species Mycobacterium smegmatis. The dif site variants harbor mutations in the central region and can therefore not recombine with the wild-type or other variants, resulting in mutants of increased genetic stability. The herein described method should be generalizable to virtually any transformable bacterial species.

scholarly journals High-Level Resistance to Colistin Mediated by Various Mutations in the crrB Gene among Carbapenemase-Producing Klebsiella pneumoniae

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Aurélie Jayol ◽  
Patrice Nordmann ◽  
Adrian Brink ◽  
Maria-Virginia Villegas ◽  
Véronique Dubois ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Wild Type ◽  
Content Type ◽  
Resistance Phenotype ◽  
Two Component ◽  
Genes Encoding ◽  
High Level ◽  
Type Strains ◽  
Level Resistance

ABSTRACT Mutations in crrAB genes encoding a two-component regulator involved in modifications of lipopolysaccharide were searched for among a collection of colistin-resistant Klebsiella pneumoniae isolates. Four isolates, respectively, producing carbapenemases NDM-1, OXA-181, or KPC-2 showed mutated CrrB proteins compared with those in wild-type strains. Complementation assays with a wild-type CrrB protein restored the susceptibility to colistin in all cases, confirming the involvement of the identified substitutions in the resistance phenotype.

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