RESUMEN
Regardless of their targets and modes of action, subinhibitory concentrations of antibiotics can have an impact on cell physiology and trigger a large variety of cellular responses in different bacterial species. Subinhibitory concentrations of ß-lactam antibiotics cause reactive oxygen species production and induce PolIV-dependent mutagenesis in Escherichia coli. Here we show that subinhibitory concentrations of ß-lactam antibiotics induce the RpoS regulon. RpoS-regulon induction is required for PolIV-dependent mutagenesis because it diminishes the control of DNA-replication fidelity by depleting MutS in E. coli, Vibrio cholerae and Pseudomonas aeruginosa. We also show that in E. coli, the reduction in mismatch-repair activity is mediated by SdsR, the RpoS-controlled small RNA. In summary, we show that mutagenesis induced by subinhibitory concentrations of antibiotics is a genetically controlled process. Because this mutagenesis can generate mutations conferring antibiotic resistance, it should be taken into consideration for the development of more efficient antimicrobial therapeutic strategies.
Asunto(s)
Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Proteínas Bacterianas/fisiología , Replicación del ADN/efectos de los fármacos , Mutagénesis , Factor sigma/fisiología , beta-Lactamas/farmacología , Bacterias/genética , Replicación del ADN/fisiología , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Vibrio cholerae/efectos de los fármacos , Vibrio cholerae/genética , Vibrio cholerae/metabolismoRESUMEN
Healthcare-associated infections (HAIs) have been a hot topic for several decades. An understanding of HAIs should be based on an understanding of the organisms that cause infection and determine prevention. Although some improvements in control in hospitals have been recorded, the community setting is now implicated, and the role of microbiology in diagnosis, detection of carriers and strain typing of organisms is evident. As healthcare systems vary widely, prevention strategies must be designed accordingly. Hand hygiene, however, remains applicable in all settings, and the WHO is strongly promoting alcohol-based hand rubs to interrupt transmission. Some countries are only beginning to develop standards, whereas compliance is obligatory in others. Economics and cost factors are common to all countries, and litigation is increasingly a factor in some.
Asunto(s)
Infección Hospitalaria/epidemiología , Infección Hospitalaria/prevención & control , Control de Infecciones/métodos , Transmisión de Enfermedad Infecciosa de Profesional a Paciente/prevención & control , Alcoholes/administración & dosificación , Infección Hospitalaria/transmisión , Desinfectantes/administración & dosificación , Desinfección de las Manos/métodos , HumanosRESUMEN
Many recent Asian clinical Vibrio cholerae E1 Tor O1 and O139 isolates are resistant to the antibiotics sulfamethoxazole (Su), trimethoprim (Tm), chloramphenicol (Cm), and streptomycin (Sm). The corresponding resistance genes are located on large conjugative elements (SXT constins) that are integrated into prfC on the V. cholerae chromosome. We determined the DNA sequences of the antibiotic resistance genes in the SXT constin in MO10, an O139 isolate. In SXT(MO10), these genes are clustered within a composite transposon-like structure found near the element's 5' end. The genes conferring resistance to Cm (floR), Su (sulII), and Sm (strA and strB) correspond to previously described genes, whereas the gene conferring resistance to Tm, designated dfr18, is novel. In some other O139 isolates the antibiotic resistance gene cluster was found to be deleted from the SXT-related constin. The El Tor O1 SXT constin, SXT(ET), does not contain the same resistance genes as SXT(MO10). In this constin, the Tm resistance determinant was located nearly 70 kbp away from the other resistance genes and found in a novel type of integron that constitutes a fourth class of resistance integrons. These studies indicate that there is considerable flux in the antibiotic resistance genes found in the SXT family of constins and point to a model for the evolution of these related mobile elements.
Asunto(s)
Proteínas Bacterianas/genética , Genes Bacterianos/genética , Familia de Multigenes/genética , Vibrio cholerae/efectos de los fármacos , Vibrio cholerae/genética , Clonación Molecular , Medios de Cultivo , Cartilla de ADN , Farmacorresistencia Microbiana , Operón , Plásmidos/genéticaRESUMEN
Integrons were first identified as the primary mechanism for antibiotic resistance gene capture and dissemination among Gram-negative bacteria. More recently, their role in genome evolution has been extended with the discovery of larger integron structures, the super-integrons, as genuine components of the genomes of many species throughout the gamma-proteobacterial radiation. The functional platforms of these integrons appear to be sedentary, whereas their gene cassette contents are highly variable. Nevertheless, the gene cassettes for which an activity has been experimentally demonstrated encode proteins related to simple adaptive functions and their recruitment is seen as providing the bacterial host with a selective advantage. The widespread occurrence of the integron system among Gram-negative bacteria is discussed, with special focus on the super-integrons. Some of the adaptive functions encoded by these genes are also reviewed, and implications of integron-mediated genome evolution in the emergence of novel bacterial species are highlighted.
Asunto(s)
Elementos Transponibles de ADN , Evolución Molecular , Genoma Bacteriano , Bacterias Gramnegativas/genética , Integrasas/genética , HumanosRESUMEN
Integrons are genetic elements that acquire and exchange exogenous DNA, known as gene cassettes, by a site-specific recombination mechanism. Characterized gene cassettes consist of a target recombination sequence (attC site) usually associated with a single open reading frame coding for an antibiotic resistance determinant. The affiliation of multiresistant integrons (MRIs), which contain various combinations of antibiotic resistance gene cassettes, with transferable elements underlies the rapid evolution of multidrug resistance among diverse Gram-negative bacteria. Yet the origin of MRIs remains unknown. Recently, a chromosomal super-integron (SI) harboring hundreds of cassettes was identified in the Vibrio cholerae genome. Here, we demonstrate that the activity of its associated integrase is identical to that of the MRI integrase, IntI1. We have also identified equivalent integron superstructures in nine distinct genera throughout the gamma-proteobacterial radiation. Phylogenetic analysis revealed that the evolutionary history of the system paralleled that of the radiation, indicating that integrons are ancient structures. The attC sites of the 63 antibiotic-resistance gene cassettes identified thus far in MRIs are highly variable. Strikingly, one-fifth of these were virtually identical to the highly related yet species-specific attC sites of the SIs described here. Furthermore, antimicrobial resistance homologues were identified among the thousands of genes entrapped by these SIs. Because the gene cassettes of SIs are substrates for MRIs, these data identify SIs as the source of contemporary MRIs and their cassettes. However, our demonstration of the metabolic functions, beyond antibiotic resistance and virulence, of three distinct SI gene cassettes indicates that integrons function as a general gene-capture system for bacterial innovation.
Asunto(s)
Farmacorresistencia Microbiana/genética , Evolución Molecular , Genoma Bacteriano , Bacterias Gramnegativas/genética , Alteromonas/genética , Sitios de Ligazón Microbiológica/genética , Genes Bacterianos , Bacterias Gramnegativas/clasificación , Integrasas/genética , Integrasas/fisiología , Datos de Secuencia Molecular , Nitrosomonas/genética , Filogenia , Recombinación Genética , Shewanella/genética , Transformación Bacteriana/genética , Vibrio/genética , Vibrionaceae/genética , Xanthomonas campestris/genéticaRESUMEN
The 72 Escherichia coli strains of the ECOR collection were examined for resistance to 10 different antimicrobial agents including ampicillin, tetracycline, mercury, trimethoprim, and sulfonamides. Eighteen strains were resistant to at least one of the antibiotics tested, and nearly 20% (14 of 72) were resistant to two or more. Several of the resistance determinants were shown to be carried on conjugative elements. The collection was screened for the presence of the three classes of integrons and for the sul1 gene, which is generally associated with class 1 integrons. The four strains found to carry a class 1 integron also had Tn21-encoded mercury resistance. One of the integrons encoded a novel streptomycin resistance gene, aadA7, with an attC site (or 59-base element) nearly identical to the attC site associated with the qacF gene cassette found in In40 (M.-C. Ploy, P. Courvalin, and T. Lambert, Antimicrob. Agents Chemother. 42:2557-2563, 1998). The conservation of associated attC sites among unrelated resistance cassettes is similar to arrangements found in the Vibrio cholerae superintegrons (D. Mazel, B. Dychinco, V. A. Webb, and J. Davies, Science 280:605-608, 1998) and supports the hypothesis that resistance cassettes are picked up from superintegron pools and independently assembled from unrelated genes and related attC sites.
Asunto(s)
Proteínas Bacterianas/genética , Farmacorresistencia Microbiana/genética , Proteínas de Escherichia coli , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Genes Bacterianos , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
Integrons are the primary mechanism for antibiotic-resistance gene capture and dissemination among Gram-negative bacteria. The recent finding of super-integron structures in the genomes of several bacterial species has expanded their role in genome evolution and suggests that they are the source of mobile multi-resistant integrons.
Asunto(s)
Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Elementos Transponibles de ADN , Farmacorresistencia Microbiana/genética , Genes Bacterianos , Bacterias/genética , HumanosRESUMEN
The treatment of infectious disease is compromised by the development of antibiotic-resistant strains of microbial pathogens. A variety of biochemical processes are involved that may keep antibiotics out of the cell, alter the target of the drug, or disable the antibiotic. Studies have shown that resistance determinants arise by either of two genetic mechanisms: mutation and acquisition. Antibiotic resistance genes can be disseminated among bacterial populations by several processes, but principally by conjugation. Thus the overall problem of antibiotic resistance is one of genetic ecology and a better understanding of the contributing parameters is necessary to devise rational approaches to reduce the development and spread of antibiotic resistance and so avoid a critical situation in therapy--a return to a pre-antibiotic era.
Asunto(s)
Bacterias/genética , Farmacorresistencia Microbiana/genética , Animales , Bacterias/patogenicidad , Conjugación Genética/genética , Ecología , Transferencia de Gen Horizontal , Genes Bacterianos/genética , Humanos , Mutación/genética , Filogenia , Recombinación Genética/genéticaRESUMEN
Integrons represent the primary mechanism for antibiotic resistance gene capture and dissemination among gram-negative bacteria. The recent finding of super-integron (SI) structures in the genomes of several bacterial species has expanded their role in genome evolution. The Vibrio cholerae superintegron is gathered in a single chromosomal super-structure harbouring hundreds of gene cassettes. The encoded functions, when identifiable, are linked to adaptations extending beyond antibiotic resistance and pathogenicity. Comparison of the cassette contents of super-integrons from remote Vibrio species suggests that most of their cassettes are species-specific. Many bacterial species belonging to several distinct genera of the gamma- and beta-proteobacteria undoubtedly carry or show strong evidence for the presence of chromosomal SIs. If each bacterial species harbouring a SI has its own cassette pool, the resource in terms of gene cassette availability may be immense.
Asunto(s)
Farmacorresistencia Microbiana/genética , Genes Bacterianos , Integrasas/genética , Vibrio/genética , Secuencia de Bases , Evolución Molecular , Genoma Bacteriano , Integrasas/química , Datos de Secuencia Molecular , Proteobacteria/genética , Recombinación Genética , Secuencias Repetitivas de Ácidos Nucleicos , Alineación de Secuencia , Especificidad de la Especie , Vibrio/enzimologíaRESUMEN
The ability of bacteria to acquire and disseminate heterologous genes has been a major factor in the development of multiple drug resistance. A gene, intI4, was identified that encodes a previously unknown integrase that is associated with a "gene-VCR" organization (VCRs are Vibrio cholerae repeated sequences), similar to that of the well-characterized antibiotic resistance integrons. The similarity was confirmed by IntI1-mediated recombination of a gene-VCR cassette into a class 1 integron. VCR cassettes are found in a number of Vibrio species including a strain of V. metschnikovii isolated in 1888, suggesting that this mechanism of heterologous gene acquisition predated the antibiotic era.
Asunto(s)
Genes Bacterianos , Integrasas/genética , Recombinación Genética , Secuencias Repetitivas de Ácidos Nucleicos , Vibrio cholerae/genética , Secuencia de Aminoácidos , Evolución Biológica , Codón , Conjugación Genética , Farmacorresistencia Microbiana/genética , Genoma Bacteriano , Integrasas/química , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Vibrio/genética , Vibrio cholerae/enzimología , Vibrio cholerae/patogenicidad , Virulencia/genéticaRESUMEN
N-terminal formylation of ribosome-synthesized polypeptides is assumed to be among the most conserved features that distinguish the eubacterial line of descent from other living phyla. In order to assess the ancientness of this trait, def genes encoding polypeptide deformylase were characterized from four eubacterial species, Lactococcus lactis, Bacillus subtilis, Calothrix PCC7601 and Thermotoga maritima, taking advantage of the conditional viability of the def mutants of Escherichia coli. Altogether, eight sequences of polypeptide deformylase have been obtained from all the eubacterial sources which were investigated, either through systematic genome sequence analysis or through genetic screening, yielding a highly homologous family. A gene putatively encoding Met-tRNAi formyltransferase, fmt, was found downstream of the deformylase gene except in L. lactis, Mycoplasma genitalium, Calothrix PCC7601 and T. maritima. These results argue strongly for the ancestral character of N-terminal formylation in eubacteria. Most of the wide deviations of amino acid usage observed in def- and fmt-encoded proteins among species is best accounted for by the nucleotide composition of genomes. Furthermore, the species of origin of each protein appears to be more recognizable than its function, considering only its amino acid composition.
Asunto(s)
Amidohidrolasas , Aminopeptidasas/genética , Bacterias/genética , Proteínas Bacterianas/biosíntesis , Evolución Molecular , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Aminoácidos/análisis , Bacterias/clasificación , Bacterias/enzimología , Clonación Molecular , Biblioteca Genómica , Datos de Secuencia Molecular , N-Formilmetionina/metabolismo , Iniciación de la Cadena Peptídica Traduccional , Filogenia , Mapeo Restrictivo , Selección Genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Pigment mutant strain FdR1 of the filamentous cyanobacterium Fremyella diplosiphon is characterized by constitutive synthesis of the phycobiliprotein phycoerythrin due to insertional inactivation of the rcaC regulatory gene by endogenous transposon Tn5469. Whereas the parental strain Fd33 harbors five genomic copies of Tn5469, cells of strain FdR1 harbor six genomic copies of the element; the sixth copy in FdR1 is localized to the rcaC gene. Electroporation of FdR1 cells yielded secondary pigment mutant strains FdR1E1 and FdR1E4, which identically exhibited the FdR1 phenotype with significantly reduced levels of phycoerythrin. In both FdR1E1 and FdR1E4, a seventh genomic copy of Tn5469 was localized to the cpeY gene of the sequenced but phenotypically uncharacterized cpeYZ gene set. This gene set is located downstream of the cpeBA operon which encodes the alpha and beta subunits of phycoerythrin. Complementation experiments correlated cpeYZ activity to the phenotype of strains FdR1E1 and FdR1E4. The predicted CpeY and CpeZ proteins share significant sequence identity with the products of homologous cpeY and cpeZ genes reported for Pseudanabaena sp. strain PCC 7409 and Synechococcus sp. strain WH 8020, both of which synthesize phycoerythrin. The CpeY and CpeZ proteins belong to a family of structurally related cyanobacterial proteins that includes the subunits of the CpcE/CpcF phycocyanin alpha-subunit lyase of Synechococcus sp. strain PCC 7002 and the subunits of the PecE/PecF phycoerythrocyanin alpha-subunit lyase of Anabaena sp. strain PCC 7120. Phycobilisomes isolated from mutant strains FdR1E1 and FdR1E4 contained equal amounts of chromophorylated alpha and beta subunits of phycoerythrin at 46% of the levels of the parental strain FdR1. These results suggest that the cpeYZ gene products function in phycoerythrin synthesis, possibly as a lyase involved in the attachment of phycoerythrobilin to the alpha or beta subunit.
Asunto(s)
Proteínas Bacterianas/genética , Cianobacterias/genética , Genes Bacterianos , Ficoeritrina/biosíntesis , Secuencia de Aminoácidos , Proteínas Bacterianas/análisis , Proteínas Bacterianas/química , Proteínas Bacterianas/fisiología , Cianobacterias/química , Cianobacterias/metabolismo , Elementos Transponibles de ADN , Prueba de Complementación Genética , Genotipo , Complejos de Proteína Captadores de Luz , Datos de Secuencia Molecular , Mutación , Orgánulos/química , Fenotipo , Ficobilisomas , Proteínas de Plantas/análisis , Alineación de SecuenciaRESUMEN
Deformylase performs an essential step in the maturation of proteins in eubacteria, by removing the formyl group from the N-terminal methionine residue of ribosome-synthesized polypeptides. In spite of this important role in translation, the enzyme had so far eluded characterization because of its instability. We report the isolation of the deformylase gene of Escherichia coli, def, by overexpression of a genomic library from a high-copy-number plasmid and selection for utilization of the substrate analogue formyl-leucyl-methionine as a source of methionine. The def gene encodes a 169 amino acid polypeptide that bears no obvious resemblance to other known proteins. It forms an operon with the fmt gene, that encodes the initiator methionyl-tRNA(i) transformylase, which was recently characterized (Guillon et al., J. Bacteriol., 174, 4294-4301, 1992). This operon was mapped at min 72 of the E. coli chromosome. The def gene could be inactivated if the fmt gene was also inactivated, or if biosynthesis of N10-formyl-tetrahydrofolate, the formyl donor in methionyl-tRNA(i) transformylation, was blocked by trimethoprim. These findings designate deformylase as a target for antibacterial chemotherapy.
Asunto(s)
Amidohidrolasas , Aminopeptidasas/genética , Bacterias/enzimología , Transferasas de Hidroximetilo y Formilo , Biosíntesis de Proteínas , Aciltransferasas/genética , Secuencia de Aminoácidos , Aminopeptidasas/biosíntesis , Aminopeptidasas/metabolismo , Bacterias/genética , Secuencia de Bases , Clonación Molecular , ADN Bacteriano , Escherichia coli/enzimología , Escherichia coli/genética , Datos de Secuencia Molecular , Operón , Eliminación de Secuencia , Especificidad por SustratoRESUMEN
The phycobilisome of the eukaryotic unicellular red alga Rhodella violacea presents in some respects an organization that is intermediate between those of the homologous counterparts found in cyanobacteria (the putative chloroplast progenitor) and more advanced, pluricellular red algae. This suggests evolutionary relationships that we investigated at the genome level. The present work describes the sequences of two rhodophytan phycobilisome genes, rpeA and rpeB. These chloroplast genes encode the alpha and beta subunits of phycoerythrin, the major component of the light-harvesting antennae and one of the most abundant cellular proteins in these algae. The amino acid sequences deduced from both rpeA and rpeB present strong homologies with those previously reported for phycoerythrin subunits of cyanobacteria, rhodophyta, and cryptomonads. The main difference with the corresponding cyanobacterial genes was the unexpected occurrence of an intervening sequence that split rpeB into two exons. This intervening sequence presents characteristics of group II introns but lacks several structural domains. Transcriptional analyses showed that the two rpe genes are cotranscribed and that the major RNA species detected corresponds to a mature mRNA lacking the intron. As the phycobiliproteins form a group of closely related polypeptides in cyanobacteria and rhodophyta, the molecular events affecting the corresponding genes, such as the rpeB intron, may be a clue to elucidate some aspects of the molecular processes involved in the evolution of plastid genes.
Asunto(s)
Genes , Ficoeritrina/genética , Rhodophyta/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Expresión Génica , Intrones , Datos de Secuencia Molecular , Ficobilisomas , ARN Mensajero/genética , Mapeo Restrictivo , Alineación de SecuenciaRESUMEN
We describe the characterization of two insertion elements, IS701 and IS702, isolated from Calothrix species PCC 7601. These insertion elements were cloned from spontaneous pigmentation mutants. Both show the characteristics of typical bacterial insertion sequences, i.e. they present long terminal inverted repeats and they duplicate target DNA upon insertion. These elements share no homology with the only other cyanobacterial insertion sequence described so far, IS891. At least 15 copies of IS701 and 9 copies of IS702 were detected by hybridization experiments in the Calothrix 7601 genome. Their occurrence in several cyanobacterial strains is also reported.
Asunto(s)
Cianobacterias/genética , Elementos Transponibles de ADN/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Biblioteca de Genes , Datos de Secuencia Molecular , Familia de Multigenes , Mutación , Ficocianina/genética , Ficoeritrina/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
We characterized three distinct families of repeated sequences in the genome of the cyanobacterium Calothrix sp. strain PCC 7601. These repeated sequences were present at a level of about 100 copies per Calothrix genome and consisted of tandemly amplified heptanucleotides. These elements were named short tandemly repeated repetitive (STRR) sequences. We used the three different Calothrix STRR sequences as probes to perform Southern hybridization experiments with DNAs extracted from various cyanobacterial strains, Bacillus subtilis, and Escherichia coli. The three different STRR sequences were found as repetitive genomic DNA components specific to the heterocystous strains tested. The role of the STRR sequences, as well as their possible use in taxonomic studies, is discussed.
Asunto(s)
Cianobacterias/genética , Genes , Secuencias Repetitivas de Ácidos Nucleicos , Bacillus subtilis/genética , Secuencia de Bases , Southern Blotting , Clonación Molecular , Sondas de ADN , Escherichia coli/genética , Amplificación de Genes , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Operón , Mapeo RestrictivoRESUMEN
Sulphur is unique among the main elements of living cells in that it is covalently bound to biopolymers but does not occur in the biopolymer backbone. Indeed, most of the bacterial sulphur content resides in the methionine and cysteine side-chains of proteins. The growth yield of an organism under conditions of sulphur limitation could therefore be greatly enhanced by mutations that substitute Met and Cys in the organism's proteins for sulphur-free amino acids. Because the saving in sulphur would increase with such accumulating mutations, Met and Cys changes could be progressively selected. Abundant proteins should be the prime targets of such a selection. A few published observations give credence to this scenario. Sulphate permease, which is abundantly produced by sulphur-starved Salmonella typhimurium, lacks Met and Cys residues. Also, two species of marine purple bacteria synthesize more protein than can be expected from a limited sulphate supply. We now report that the cyanobacterium Calothrix sp. PCC 7601 (referred to here as Calothrix) encodes sulphur-depleted versions of its most abundant proteins--phycocyanin and its auxiliary polypeptides--which it specifically expresses under conditions of sulphur limitation. Although these proteins do not take part in the fixation of sulphur, their elevated synthesis affects the sulphur budget of cyanobacterial cells. Direct evidence is thus provided that the structure of macromolecules can be subject to metabolic optimization.