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1.
PLoS Biol ; 22(1): e3002457, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38175839

RESUMEN

Heteroresistance (HR) is an enigmatic phenotype where, in a main population of susceptible cells, small subpopulations of resistant cells exist. This is a cause for concern, as this small subpopulation is difficult to detect by standard antibiotic susceptibility tests, and upon antibiotic exposure the resistant subpopulation may increase in frequency and potentially lead to treatment complications or failure. Here, we determined the prevalence and mechanisms of HR for 40 clinical Staphylococcus aureus isolates, against 6 clinically important antibiotics: daptomycin, gentamicin, linezolid, oxacillin, teicoplanin, and vancomycin. High frequencies of HR were observed for gentamicin (69.2%), oxacillin (27%), daptomycin (25.6%), and teicoplanin (15.4%) while none of the isolates showed HR toward linezolid or vancomycin. Point mutations in various chromosomal core genes, including those involved in membrane and peptidoglycan/teichoic acid biosynthesis and transport, tRNA charging, menaquinone and chorismite biosynthesis and cyclic-di-AMP biosynthesis, were the mechanisms responsible for generating the resistant subpopulations. This finding is in contrast to gram-negative bacteria, where increased copy number of bona fide resistance genes via tandem gene amplification is the most prevalent mechanism. This difference can be explained by the observation that S. aureus has a low content of resistance genes and absence of the repeat sequences that allow tandem gene amplification of these genes as compared to gram-negative species.


Asunto(s)
Daptomicina , Infecciones Estafilocócicas , Humanos , Staphylococcus aureus/genética , Vancomicina , Linezolid/uso terapéutico , Teicoplanina/uso terapéutico , Prevalencia , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/tratamiento farmacológico , Oxacilina/uso terapéutico , Mutación , Gentamicinas
2.
PLoS Biol ; 20(9): e3001808, 2022 09.
Artículo en Inglés | MEDLINE | ID: mdl-36170241

RESUMEN

In a collection of Escherichia coli isolates, we discovered a new mechanism leading to frequent and high-level tigecycline resistance involving tandem gene amplifications of an efflux pump encoded by the tet(A) determinant. Some isolates, despite carrying a functional tet(A), could not evolve high-level tigecycline resistance by amplification due to the presence of a deletion in the TetR(A) repressor. This mutation impaired induction of tetA(A) (encoding the TetA(A) efflux pump) in presence of tetracyclines, with the strongest effect observed for tigecycline, subsequently preventing the development of tet(A) amplification-dependent high-level tigecycline resistance. We found that this mutated tet(A) determinant was common among tet(A)-carrying E. coli isolates and analysed possible explanations for this high frequency. First, while the mutated tet(A) was found in several ST-groups, we found evidence of clonal spread among ST131 isolates, which increases its frequency within E. coli databases. Second, evolution and competition experiments revealed that the mutation in tetR(A) could be positively selected over the wild-type allele at sub-inhibitory concentrations of tetracyclines. Our work demonstrates how low concentrations of tetracyclines, such as those found in contaminated environments, can enrich and select for a mutation that generates an evolutionary dead-end that precludes the evolution towards high-level, clinically relevant tigecycline resistance.


Asunto(s)
Escherichia coli , Tetraciclinas , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Escherichia coli/genética , Pruebas de Sensibilidad Microbiana , Mutación/genética , Plásmidos , Inhibidores de la Síntesis de la Proteína/farmacología , Tetraciclinas/farmacología , Tigeciclina/farmacología
3.
J Bacteriol ; 199(12)2017 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-28373273

RESUMEN

The alternative sigma factor σE is a key component of the Escherichia coli response to cell envelope stress and is required for viability even in the absence of stress. The activity of σE increases during entry into stationary phase, suggesting an important role for σE when nutrients are limiting. Elevated σE activity has been proposed to activate a pathway leading to the lysis of nonculturable cells that accumulate during early stationary phase. To better understand σE-directed cell lysis and the role of σE in stationary phase, we investigated the effects of elevated σE activity in cultures grown for 10 days. We demonstrate that high σE activity is lethal for all cells in stationary phase, not only those that are nonculturable. Spontaneous mutants with reduced σE activity, due primarily to point mutations in the region of σE that binds the -35 promoter motif, arise and take over cultures within 5 to 6 days after entry into stationary phase. High σE activity leads to large reductions in the levels of outer membrane porins and increased membrane permeability, indicating membrane defects. These defects can be counteracted and stationary-phase lethality delayed significantly by stabilizing membranes with Mg2+ and buffering the growth medium or by deleting the σE-dependent small RNAs (sRNAs) MicA, RybB, and MicL, which inhibit the expression of porins and Lpp. Expression of these sRNAs also reverses the loss of viability following depletion of σE activity. Our results demonstrate that appropriate regulation of σE activity, ensuring that it is neither too high nor too low, is critical for envelope integrity and cell viability.IMPORTANCE The Gram-negative cell envelope and cytoplasm differ significantly, and separate responses have evolved to combat stress in each compartment. An array of cell envelope stress responses exist, each of which is focused on different parts of the envelope. The σE response is conserved in many enterobacteria and is tuned to monitor pathways for the maturation and delivery of outer membrane porins, lipoproteins, and lipopolysaccharide to the outer membrane. The activity of σE is tightly regulated to match the production of σE regulon members to the needs of the cell. In E. coli, loss of σE results in lethality. Here we demonstrate that excessive σE activity is also lethal and results in decreased membrane integrity, the very phenotype the system is designed to prevent.


Asunto(s)
Fosfatasa Ácida/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiología , Regulación Bacteriana de la Expresión Génica , Viabilidad Microbiana , Factor sigma/metabolismo , Fosfatasa Ácida/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Porinas/metabolismo , Factor sigma/genética
4.
Mol Microbiol ; 102(2): 274-289, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27381382

RESUMEN

Heteroresistance, a phenomenon where subpopulations of a bacterial isolate exhibit different susceptibilities to an antibiotic, is a growing clinical problem where the underlying genetic mechanisms in most cases remain unknown. We isolated colistin resistant mutants in Escherichia coli and Salmonella enterica serovar Typhimurium at different concentrations of colistin. Genetic analysis showed that genetically stable pmrAB point mutations were responsible for colistin resistance during selection at high drug concentrations for both species and at low concentrations for E. coli. In contrast, for S. Typhimurium mutants selected at low colistin concentrations, amplification of different large chromosomal regions conferred a heteroresistant phenotype. All amplifications included the pmrD gene, which encodes a positive regulator that up-regulates proteins that modify lipid A, and as a result increase colistin resistance. Inactivation and over-expression of the pmrD gene prevented and conferred resistance, respectively, demonstrating that the PmrD protein is required and sufficient to confer resistance. The heteroresistance phenotype is explained by the variable gene dosage of pmrD in a population, where sub-populations with different copy number of the pmrD gene show different levels of colistin resistance. We propose that variability in gene copy number of resistance genes can explain the heteroresistance observed in clinically isolated pathogenic bacteria.


Asunto(s)
Antibacterianos/farmacología , Colistina/farmacología , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana Múltiple/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Amplificación de Genes , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Mutación , Salmonella typhimurium/metabolismo , Factores de Transcripción/genética
5.
J Antimicrob Chemother ; 71(1): 100-10, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26467993

RESUMEN

OBJECTIVES: Indirect resistance (IR), the ability of an antibiotic-resistant population of bacteria to protect a susceptible population, has been previously observed for ß-lactamase-producing bacteria and associated with antimicrobial treatment failures. Here, we determined whether other resistance determinants could cause IR in the presence of five other classes of antibiotics. METHODS: A test was designed to detect IR and 14 antibiotic resistance genes were tested in the presence of 13 antibiotics from six classes. A bioassay was used to measure the ability of resistance-causing enzymes to decrease the concentration of active antibiotics in the medium. RESULTS: We confirmed IR in the presence of ß-lactam antibiotics (ampicillin and mecillinam) when TEM-1A was expressed. We found that bacteria expressing antibiotic-modifying or -degrading enzymes Ere(A), Tet(X2) or CatA1 caused IR in the presence of macrolides (erythromycin and clarithromycin), tetracyclines (tetracycline and tigecycline) and chloramphenicol, respectively. IR was not observed with resistance determinants that did not modify or destroy antibiotics or with enzymes modifying aminoglycosides or degrading fosfomycin. IR was dependent on the resistance enzymes decreasing the concentration of active antibiotics in the medium, hence allowing nearby susceptible bacteria to resume growth once the antibiotic concentration fell below their MIC. CONCLUSIONS: IR was not limited to ß-lactamase-producing bacteria, but was also caused by resistant bacteria carrying cytoplasmic antibiotic-modifying or -degrading enzymes that catalyse energy-consuming reactions requiring complex cellular cofactors. Our results suggest that IR is common and further emphasizes that coinfecting agents and the human microflora can have a negative impact during antimicrobial therapy.


Asunto(s)
Antibacterianos/metabolismo , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Bacterias/metabolismo , Enzimas/metabolismo , Consorcios Microbianos , Biotransformación , Medios de Cultivo/química , Humanos , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos
6.
Mol Microbiol ; 93(3): 479-93, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24946009

RESUMEN

The E. coli alternative sigma factor, σ(E) , transcribes genes required to maintain the cell envelope and is activated by conditions that destabilize the envelope. σ(E) is also activated during entry into stationary phase in the absence of envelope stress by the alarmone (p)ppGpp. (p)ppGpp controls a large regulatory network, reducing expression of σ(70) -dependent genes required for rapid growth and activating σ(70) -dependent and alternative sigma factor-dependent genes required for stress survival. The DksA protein often potentiates the effects of (p)ppGpp. Here we examine regulation of σ(E) by (p)ppGpp and DksA following starvation for nutrients. We find that (p)ppGpp is required for increased σ(E) activity under all conditions tested, but the requirement for DksA varies. DksA is required during amino acid starvation, but is dispensable during phosphate starvation. In contrast, regulation of σ(S) is (p)ppGpp- and DksA-dependent under all conditions tested, while negative regulation of σ(70) is DksA- but not (p)ppGpp-dependent during phosphate starvation, yet requires both factors during amino acid starvation. These findings suggest that the mechanism of transcriptional regulation by (p)ppGpp and/or DksA cannot yet be explained by a unifying model and is specific to individual promoters, individual holoenzymes, and specific starvation conditions.


Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/crecimiento & desarrollo , Escherichia coli/genética , Guanosina Pentafosfato/metabolismo , Guanosina Tetrafosfato/metabolismo , Holoenzimas/genética , Factor sigma/metabolismo , Proteínas Bacterianas/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Holoenzimas/metabolismo , Mutación , Regiones Promotoras Genéticas , Factor sigma/genética , Transcripción Genética
7.
Mol Microbiol ; 90(6): 1233-48, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24325250

RESUMEN

Previous work demonstrated that selection for Escherichia coli mutants with low antibiotic resistance frequently resulted in co-selection of lon mutations and that lon(-) mutants evolved higher-level resistance faster than a lon(+) strain. Here we show that lon mutation causes a very low multidrug resistance by inducing the AcrAB-TolC pump via stabilization of the acrAB transcriptional activators MarA and SoxS, which are substrates of the Lon protease. Fast evolution of lon(-) mutants towards higher resistance involves selection of frequent next-step mutations consisting of large duplications including acrAB and the mutated lon gene. Resistance results from the combined effects of acrAB duplication and lon mutation increasing dosage of efflux pump. In contrast, when acrAB duplication occurs as the first step mutation, increased Lon activity caused by lon(+) co-duplication mitigates the effect of acrAB duplication on resistance, and faster evolution towards higher resistance is not observed. As predicted, when the functional lon gene is relocated far from acrAB to prevent their co-duplication, first-step acrAB duplication confers higher resistance, which then allows selection of frequent next-step mutations and results in faster evolution towards higher resistance. Our results demonstrate how order of appearance of mutations and gene location can influence the rate of resistance evolution.


Asunto(s)
Antibacterianos/farmacología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efectos de los fármacos , Proteasa La/genética , Proteasa La/metabolismo , Cromosomas Bacterianos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Farmacorresistencia Bacteriana Múltiple/genética , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Evolución Molecular , Duplicación de Gen , Datos de Secuencia Molecular , Mutación , Transducción de Señal/genética , Resistencia a la Tetraciclina/genética , Transactivadores/genética , Transactivadores/metabolismo
8.
Nat Commun ; 15(1): 3981, 2024 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-38730266

RESUMEN

Heteroresistance is a medically relevant phenotype where small antibiotic-resistant subpopulations coexist within predominantly susceptible bacterial populations. Heteroresistance reduces treatment efficacy across diverse bacterial species and antibiotic classes, yet its genetic and physiological mechanisms remain poorly understood. Here, we investigated a multi-resistant Klebsiella pneumoniae isolate and identified three primary drivers of gene dosage-dependent heteroresistance for several antibiotic classes: tandem amplification, increased plasmid copy number, and transposition of resistance genes onto cryptic plasmids. All three mechanisms imposed fitness costs and were genetically unstable, leading to fast reversion to susceptibility in the absence of antibiotics. We used a mouse gut colonization model to show that heteroresistance due to elevated resistance-gene dosage can result in antibiotic treatment failures. Importantly, we observed that the three mechanisms are prevalent among Escherichia coli bloodstream isolates. Our findings underscore the necessity for treatment strategies that address the complex interplay between plasmids, resistance cassettes, and transposons in bacterial populations.


Asunto(s)
Antibacterianos , Variaciones en el Número de Copia de ADN , Escherichia coli , Klebsiella pneumoniae , Plásmidos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/efectos de los fármacos , Animales , Antibacterianos/farmacología , Ratones , Plásmidos/genética , Escherichia coli/genética , Escherichia coli/efectos de los fármacos , Farmacorresistencia Bacteriana Múltiple/genética , Pruebas de Sensibilidad Microbiana , Dosificación de Gen , Infecciones por Klebsiella/microbiología , Infecciones por Klebsiella/tratamiento farmacológico , Humanos , Elementos Transponibles de ADN/genética , Femenino
9.
Antimicrob Agents Chemother ; 53(11): 4944-8, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19721064

RESUMEN

Transposon inactivation of ycgE, a gene encoding a putative transcriptional regulator, led to decreased multidrug susceptibility in an Escherichia coli lon mutant. The multidrug susceptibility phenotype (e.g., to tetracycline and beta-lactam antibiotics) required the inactivation of both lon and ycgE. In this mutant, a decreased amount of OmpF porin contributes to the lowered drug susceptibility, with a greater effect at 26 degrees C than at 37 degrees C.


Asunto(s)
Proteínas de Escherichia coli/fisiología , Escherichia coli/efectos de los fármacos , Porinas/fisiología , Proteasa La/fisiología , Proteínas de Unión al ADN/fisiología , Farmacorresistencia Bacteriana Múltiple , Escherichia coli/genética , Lipoproteínas/fisiología , Proteínas de Transporte de Membrana/fisiología , Pruebas de Sensibilidad Microbiana , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/fisiología
10.
Nat Rev Microbiol ; 17(8): 479-496, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31235888

RESUMEN

Antibiotic heteroresistance is a phenotype in which a bacterial isolate contains subpopulations of cells that show a substantial reduction in antibiotic susceptibility compared with the main population. Recent work indicates that heteroresistance is very common for several different bacterial species and antibiotic classes. The resistance phenotype is often unstable, and in the absence of antibiotic pressure it rapidly reverts to susceptibility. A common mechanistic explanation for the instability is the occurrence of genetically unstable tandem amplifications of genes that cause resistance. Due to their instability, low frequency and transient character, it is challenging to detect and study these subpopulations, which often leads to difficulties in unambiguously classifying bacteria as susceptible or resistant. Finally, in vitro experiments, mathematical modelling, animal infection models and clinical studies show that the resistant subpopulations can be enriched during antibiotic exposure, and increasing evidence suggests that heteroresistance can lead to treatment failure.


Asunto(s)
Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Variación Biológica Poblacional , Farmacorresistencia Bacteriana , Animales , Antibacterianos/uso terapéutico , Infecciones Bacterianas/tratamiento farmacológico , Modelos Animales de Enfermedad , Insuficiencia del Tratamiento
11.
Nat Microbiol ; 4(3): 504-514, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30742072

RESUMEN

When choosing antibiotics to treat bacterial infections, it is assumed that the susceptibility of the target bacteria to an antibiotic is reflected by laboratory estimates of the minimum inhibitory concentration (MIC) needed to prevent bacterial growth. A caveat of using MIC data for this purpose is heteroresistance, the presence of a resistant subpopulation in a main population of susceptible cells. We investigated the prevalence and mechanisms of heteroresistance in 41 clinical isolates of the pathogens Escherichia coli, Salmonella enterica, Klebsiella pneumoniae and Acinetobacter baumannii against 28 different antibiotics. For the 766 bacteria-antibiotic combinations tested, as much as 27.4% of the total was heteroresistant. Genetic analysis demonstrated that a majority of heteroresistance cases were unstable, with an increased resistance of the subpopulations resulting from spontaneous tandem amplifications, typically including known resistance genes. Using mathematical modelling, we show how heteroresistance in the parameter range estimated in this study can result in the failure of antibiotic treatment of infections with bacteria that are classified as antibiotic susceptible. The high prevalence of heteroresistance with the potential for treatment failure highlights the limitations of MIC as the sole criterion for susceptibility determinations. These results call for the development of facile and rapid protocols to identify heteroresistance in pathogens.


Asunto(s)
Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Farmacorresistencia Bacteriana Múltiple/genética , Amplificación de Genes , Acinetobacter/efectos de los fármacos , Acinetobacter/genética , Bacterias/genética , Colistina/farmacología , Humanos , Pruebas de Sensibilidad Microbiana , Modelos Teóricos , Prevalencia
12.
Sci Rep ; 9(1): 11261, 2019 08 02.
Artículo en Inglés | MEDLINE | ID: mdl-31375711

RESUMEN

We describe a portable epigenetic switch based on opvAB, a Salmonella enterica operon that undergoes bistable expression under DNA methylation control. A DNA fragment containing the opvAB promoter and the opvAB upstream regulatory region confers bistability to heterologous genes, yielding OFF and ON subpopulations. Bistable expression under opvAB control is reproducible in Escherichia coli, showing that the opvAB switch can be functional in a heterologous host. Subpopulations of different sizes can be produced at will using engineered opvAB variants. Controlled formation of antibiotic-resistant and antibiotic-susceptible subpopulations may allow use of the opvAB switch in the study of bacterial heteroresistance to antibiotics.


Asunto(s)
Farmacorresistencia Bacteriana/genética , Epigénesis Genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos/genética , Operón/genética , Antibacterianos/farmacología , Técnicas Biosensibles/métodos , Metilación de ADN , Pruebas Antimicrobianas de Difusión por Disco , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Mutagénesis Sitio-Dirigida , Mutación , Plásmidos/genética , Regiones Promotoras Genéticas/genética , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Biología Sintética/métodos
13.
Sci Rep ; 9(1): 17820, 2019 Nov 25.
Artículo en Inglés | MEDLINE | ID: mdl-31767950

RESUMEN

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

14.
Mol Microbiol ; 67(3): 619-32, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18086212

RESUMEN

One of the major signalling pathways responsible for intercompartmental communication between the cell envelope and cytoplasm in Escherichia coli is mediated by the alternative sigma factor, sigmaE. sigmaE has been studied primarily for its role in response to the misfolding of outer membrane porins. This response is essentially reactionary; cells are stressed, porin folding is disrupted, and the response is activated. sigmaE can also be activated following starvation for a variety of nutrients by the alarmone ppGpp. This response is proactive, as sigmaE is activated in the absence of any obvious damage to the cell envelope sensed by the stress signalling pathway. Here we examine the mechanism of regulation of sigmaE by ppGpp. ppGpp has been proposed to activate at least two alternative sigma factors, sigmaN and sigmaS, indirectly by altering the competition for core RNA polymerase between the alternative sigma factors and the housekeeping sigma factor, sigma70. In vivo experiments with sigmaE are consistent with this model. However, ppGpp and its cofactor DksA can also activate transcription by EsigmaEin vitro, suggesting that the effects of ppGpp on sigmaE activity are both direct and indirect.


Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Guanosina Tetrafosfato/metabolismo , Factor sigma/metabolismo , Factores de Transcripción/metabolismo , Fusión Artificial Génica , Escherichia coli/genética , Genes Reporteros , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismo
15.
Antimicrob Agents Chemother ; 51(4): 1293-303, 2007 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-17220404

RESUMEN

Thirteen spontaneous multiple-antibiotic-resistant (Mar) mutants of Escherichia coli AG100 were isolated on Luria-Bertani (LB) agar in the presence of tetracycline (4 microg/ml). The phenotype was linked to insertion sequence (IS) insertions in marR or acrR or unstable large tandem genomic amplifications which included acrAB and which were bordered by IS3 or IS5 sequences. Five different lon mutations, not related to the Mar phenotype, were also found in 12 of the 13 mutants. Under specific selective conditions, most drug-resistant mutants appearing late on the selective plates evolved from a subpopulation of AG100 with lon mutations. That the lon locus was involved in the evolution to low levels of multidrug resistance was supported by the following findings: (i) AG100 grown in LB broth had an important spontaneous subpopulation (about 3.7x10(-4)) of lon::IS186 mutants, (ii) new lon mutants appeared during the selection on antibiotic-containing agar plates, (iii) lon mutants could slowly grow in the presence of low amounts (about 2x MIC of the wild type) of chloramphenicol or tetracycline, and (iv) a lon mutation conferred a mutator phenotype which increased IS transposition and genome rearrangements. The association between lon mutations and mutations causing the Mar phenotype was dependent on the medium (LB versus MacConkey medium) and the antibiotic used for the selection. A previously reported unstable amplifiable high-level resistance observed after the prolonged growth of Mar mutants in a low concentration of tetracycline or chloramphenicol can be explained by genomic amplification.


Asunto(s)
Elementos Transponibles de ADN/genética , Farmacorresistencia Bacteriana Múltiple/genética , Escherichia coli/efectos de los fármacos , Amplificación de Genes/genética , Genoma Bacteriano , Inestabilidad Genómica , Antibacterianos/farmacología , ADN Bacteriano/análisis , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Mutación , Proteasa La/genética , Proteasa La/metabolismo
16.
J Bacteriol ; 188(12): 4413-23, 2006 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16740948

RESUMEN

A spontaneous mutant (M113) of Escherichia coli AG100 with an unstable multiple antibiotic resistance (Mar) phenotype was isolated in the presence of tetracycline. Two mutations were found: an insertion in the promoter of lon (lon3::IS186) that occurred first and a subsequent large tandem duplication, dupIS186, bearing the genes acrAB and extending from the lon3::IS186 to another IS186 present 149 kb away from lon. The decreased amount of Lon protease increased the amount of MarA by stabilization of the basal quantities of MarA produced, which in turn increased the amount of multidrug effux pump AcrAB-TolC. However, in a mutant carrying only a lon mutation, the overproduced pump mediated little, if any, increased multidrug resistance, indicating that the Lon protease was required for the function of the pump. This requirement was only partial since resistance was mediated when amounts of AcrAB in a lon mutant were further increased by a second mutation. In M113, amplification of acrAB on the duplication led to increased amounts of AcrAB and multidrug resistance. Spontaneous gene duplication represents a new mechanism for mediating multidrug resistance in E. coli through AcrAB-TolC.


Asunto(s)
Antibacterianos/farmacología , Proteínas de Escherichia coli/fisiología , Escherichia coli/efectos de los fármacos , Proteasa La/fisiología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/genética , Western Blotting , Medios de Cultivo , Elementos Transponibles de ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Farmacorresistencia Bacteriana Múltiple , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Amplificación de Genes , Lipoproteínas/genética , Lipoproteínas/metabolismo , Proteínas de Transporte de Membrana , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Mutación , Proteínas Represoras/genética , Tetraciclina/farmacología
17.
J Bacteriol ; 188(13): 4777-86, 2006 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-16788187

RESUMEN

The uracil salvage pathway in Lactobacillus plantarum was demonstrated to be dependent on the upp-pyrP gene cluster. PyrP was the only high-affinity uracil transporter since a pyrP mutant no longer incorporated low concentrations of radioactively labeled uracil and had increased resistance to the toxic uracil analogue 5-fluorouracil. The upp gene encoded a uracil phosphoribosyltransferase (UPRT) enzyme catalyzing the conversion of uracil and 5-phosphoribosyl-alpha-1-pyrophosphate to UMP and pyrophosphate. Analysis of mutants revealed that UPRT is a major cell supplier of UMP synthesized from uracil provided by preformed nucleic acid degradation. In a mutant selection study, seven independent upp mutants were isolated and all were found to excrete low amounts of pyrimidines to the growth medium. Pyrimidine-dependent transcription regulation of the biosynthetic pyrimidine pyrR1-B-C-Aa1-Ab1-D-F-E operon was impaired in the upp mutants. Despite the fact that upp and pyrP are positioned next to each other on the chromosome, they are not cotranscribed. Whereas pyrP is expressed as a monocistronic message, the upp gene is part of the lp_2376-glyA-upp operon. The lp_2376 gene encodes a putative protein that belongs to the conserved protein family of translation modulators such as Sua5, YciO, and YrdC. The glyA gene encodes a putative hydroxymethyltransferase involved in C1 unit charging of tetrahydrofolate, which is required in the biosynthesis of thymidylate, pantothenate, and purines. Unlike upp transcription, pyrP transcription is regulated by exogenous pyrimidine availability, most likely by the same mechanism of transcription attenuation as that of the pyr operon.


Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Lactobacillus plantarum/genética , Lactobacillus plantarum/metabolismo , Pentosiltransferasa/genética , Proteínas Represoras/genética , Uracilo/metabolismo , Uridina Monofosfato/metabolismo , Proteínas Bacterianas/genética , Genes Bacterianos , Familia de Multigenes/genética , Operón , Pentosiltransferasa/metabolismo , Pirimidinas/metabolismo , Transcripción Genética , Uridina Monofosfato/biosíntesis
18.
Arch Microbiol ; 183(5): 307-16, 2005 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-15864550

RESUMEN

Among proteins specifically found in most gram-positive bacteria of the phylum Firmicutes, conserved proteins of the family pfam06177-DUF988-COG4708 are of unknown function. The citrulline cluster-linked (ccl) gene of Lactobacillus plantarum codes one such protein and is adjacent to the citrulline biosynthesis operon argCJBDF, a situation also found in Lactococcus lactis. This gene is well conserved among L. plantarum species, and 1 isolate out of 24 harbored two ccl copies. Northern hybridization with a ccl probe revealed two arginine-repressed transcripts with sizes corresponding to the predicted argCJBDF-ccl operon and the ccl gene alone. Transcription start sites of both transcripts were characterized. Four different 5' ends were mapped at the argF-ccl intergenic region, resulting from either regulated transcription initiation or maturation of the transcripts. Transcriptional ccl-gusA gene fusion confirmed the promoter activity of the argF-ccl intergenic region. Thus, the ccl gene is arginine-repressed and transcribed both monocistronically and polycistronically in the argCJBDF-ccl operon. The ccl gene is not essential in L. plantarum, because a ccl gene deletion was obtained in strain CCM 1904. Although no functions were found in the tested laboratory conditions, the Ccl-like proteins may play a role in environmental conditions of life.


Asunto(s)
Proteínas Bacterianas/genética , Citrulina/biosíntesis , Genes Bacterianos , Lactobacillus plantarum/genética , Arginina/farmacología , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Lactobacillus plantarum/efectos de los fármacos , Regiones Promotoras Genéticas , Transcripción Genética
19.
J Bacteriol ; 187(6): 2093-104, 2005 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-15743958

RESUMEN

Carbamoyl phosphate is a precursor for both arginine and pyrimidine biosynthesis. In Lactobacillus plantarum, carbamoyl phosphate is synthesized from glutamine, ATP, and carbon dioxide by two sets of identified genes encoding carbamoyl phosphate synthase (CPS). The expression of the carAB operon (encoding CPS-A) responds to arginine availability, whereas pyrAaAb (encoding CPS-P) is part of the pyrR1BCAaAbDFE operon coding for the de novo pyrimidine pathway repressed by exogenous uracil. The pyr operon is regulated by transcription attenuation mediated by a trans-acting repressor that binds to the pyr mRNA attenuation site in response to intracellular UMP/phosphoribosyl pyrophosphate pools. Intracellular pyrimidine triphosphate nucleoside pools were lower in mutant FB335 (carAB deletion) harboring only CPS-P than in the wild-type strain harboring both CPS-A and CPS-P. Thus, CPS-P activity is the limiting step in pyrimidine synthesis. FB335 is unable to grow in the presence of uracil due to a lack of sufficient carbamoyl phosphate required for arginine biosynthesis. Forty independent spontaneous FB335-derived mutants that have lost regulation of the pyr operon were readily obtained by their ability to grow in the presence of uracil and absence of arginine; 26 harbored mutations in the pyrR1-pyrB loci. One was a prototroph with a deletion of both pyrR1 and the transcription attenuation site that resulted in large amounts of excreted pyrimidine nucleotides and increased intracellular UTP and CTP pools compared to wild-type levels. Low pyrimidine-independent expression of the pyr operon was obtained by antiterminator site-directed mutagenesis. The resulting AE1023 strain had reduced UTP and CTP pools and had the phenotype of a high-CO2-requiring auxotroph, since it was able to synthesize sufficient arginine and pyrimidines only in CO2-enriched air. Therefore, growth inhibition without CO2 enrichment may be due to low carbamoyl phosphate pools from lack of CPS activity.


Asunto(s)
Proteínas Bacterianas/genética , Dióxido de Carbono/metabolismo , Lactobacillus plantarum/crecimiento & desarrollo , Lactobacillus plantarum/genética , Operón/fisiología , Pentosiltransferasa/genética , Proteínas Represoras/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/metabolismo , Carbamoil Fosfato/metabolismo , Regulación Bacteriana de la Expresión Génica , Lactobacillus plantarum/metabolismo , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Nucleósidos/metabolismo , Pentosiltransferasa/metabolismo , ARN Bacteriano/química , ARN Bacteriano/genética , Proteínas Represoras/metabolismo , Transcripción Genética
20.
Appl Environ Microbiol ; 69(10): 6032-40, 2003 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-14532059

RESUMEN

We describe the first functional insertion sequence (IS) element in Lactobacillus plantarum. ISLpl1, an IS30-related element, was found on the pLp3 plasmid in strain FB335. By selection of spontaneous mutants able to grow in the presence of uracil, it was demonstrated that the IS had transposed into the uracil phosphoribosyltransferase-encoding gene upp on the FB335 chromosome. The plasmid-carried IS element was also sequenced, and a second potential IS element was found: ISLpl2, an IS150-related element adjacent to ISLpl1. When Southern hybridization was used, the copy number and genome (plasmid versus chromosome) distribution data revealed different numbers and patterns of ISLpl1-related sequences in different L. plantarum strains as well as in Pediococcus strains. The ISLpl1 pattern changed over many generations of the strain L. plantarum NCIMB 1406. This finding strongly supports our hypothesis that ISLpl1 is a mobile element in L. plantarum. Database analysis revealed five quasi-identical ISLpl1 elements in Lactobacillus, Pediococcus, and Oenococcus strains. Three of these elements may be cryptic IS, since point mutations or 1-nucleotide deletions were found in their transposase-encoding genes. In some cases, ISLpl1 was linked to genes involved in cold shock adaptation, bacteriocin production, sugar utilization, or antibiotic resistance. ISLpl1 is transferred among lactic acid bacteria (LAB) and may play a role in LAB genome plasticity and adaptation to their environment.


Asunto(s)
Elementos Transponibles de ADN , Lactobacillus/genética , Plásmidos , Proteínas Bacterianas/genética , Secuencia de Bases , Mapeo Cromosómico , Dosificación de Gen , Lactobacillaceae/genética , Datos de Secuencia Molecular , Pediococcus/genética , Análisis de Secuencia de ADN , Transposasas/genética
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