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1.
FEBS J ; 278(18): 3508-17, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21791000

RESUMEN

A mutation in the infA gene encoding initiation factor 1 (IF1) gives rise to a cold-sensitive phenotype. An Escherichia coli strain with this mutation was used as a tool to select for second-site suppressors that compensate for the cold sensitivity and map specifically to rRNA. Several suppressor mutants with altered 16S rRNA that partially restore growth of an IF1 mutant strain in the cold were isolated and characterized. Suppressor mutations were found in helix (h)18, h32, h34 and h41 in 16S rRNA. These mutations are not clustered to any particular region in 16S rRNA and none overlap previously reported sites of interaction with IF1. While the isolated suppressors are structurally diverse, they are functionally related because all affect ribosomal subunit association in vivo. Furthermore, in vitro subunit-association experiments indicate that most of the suppressor mutations directly influence ribosomal subunit association even though none of these are confined to any of the known intersubunit bridges. These results are consistent with the model that IF1 is an rRNA chaperone that induces large-scale conformational changes in the small ribosomal subunit, and as a consequence modulates initiation of translation by affecting subunit association.


Asunto(s)
Frío/efectos adversos , Proteínas de Escherichia coli/metabolismo , Escherichia coli/crecimiento & desarrollo , Mutación , Factor 1 Procariótico de Iniciación/metabolismo , ARN Ribosómico 16S/metabolismo , Subunidades Ribosómicas/metabolismo , Sustitución de Aminoácidos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Metiltransferasas/genética , Metiltransferasas/metabolismo , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutagénesis Insercional , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/metabolismo , Conformación de Ácido Nucleico , Factor 1 Procariótico de Iniciación/genética , Multimerización de Proteína , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Ribosómico 16S/química , ARN Ribosómico 16S/genética , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Supresión Genética
2.
Plasmid ; 66(1): 26-37, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-21550364

RESUMEN

Bacteria of the genus Nocardia cause opportunistic infections of lung, brain and central nervous system, and cutaneous tissue. They are also producers of antibiotics and industrially important enzymes. As studies describing plasmids in this genus are limited, we have characterized a 4326bp cryptic plasmid pYS1 from Nocardia aobensis IFM 10795. Three open reading frames (ORFs) were predicted. Both sequence analyses and detection of single-stranded intermediates suggested a rolling-circle mechanism as the mode of replication of pYS1. Mutageneses and deletion analyses revealed both the predicted double- and single-stranded origins to be indispensable in replication, suggesting a lack of secondary signals for leading and lagging strand synthesis. The replicon of pYS1 is broad-host-range and compatible to that of pAL5000 of mycobacteria, making it potentially useful in genetic manipulation of various actinomycetes. Insertion analyses showed orf1, despite its sequence similarity to plasmid transfer genes, is involved in plasmid stability rather than conjugation and is lethal in the absence of a functional orf3. This situation is somewhat analogous to the kil/kor system of pIJ101 of Streptomyces, except that orf3 was unrelated to korA and was shown by promoter-probe assays to encode a novel transcriptional repressor negatively regulating orf1 expression.


Asunto(s)
Replicación del ADN , ADN Bacteriano/genética , Nocardia/genética , Plásmidos/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN/genética , ADN/metabolismo , Variaciones en el Número de Copia de ADN , ADN Bacteriano/metabolismo , ADN Circular/genética , ADN Circular/metabolismo , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Regulación Bacteriana de la Expresión Génica , Vectores Genéticos , Datos de Secuencia Molecular , Mutagénesis Insercional , Nocardia/metabolismo , Conformación de Ácido Nucleico , Sistemas de Lectura Abierta , Plásmidos/aislamiento & purificación , Plásmidos/metabolismo , Origen de Réplica , Replicón , Homología de Secuencia de Aminoácido , Transformación Bacteriana
3.
Bacteriophage ; 1(4): 195-197, 2011 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-23050212

RESUMEN

Bacteriophage-encoded proteins which inhibit or modify cellular components may contribute to antibacterial drug discovery by allowing the identification of novel targets. Given their abundance and diversity, phages may have various strategies in host inhibition and therefore may possess a variety of such proteins. Using Rhodococcus equi and phage YF1, we show that a single phage possesses numerous genes that inhibit the host when introduced into the host on a plasmid. These genes mostly encode proteins of unknown function, confirming the potential that this approach may have in providing new antibacterial targets.

4.
Biochemistry ; 42(16): 4691-8, 2003 Apr 29.
Artículo en Inglés | MEDLINE | ID: mdl-12705832

RESUMEN

An Escherichia coli mutant, LL103, harboring a mutation (Ser15 to Phe) in ribosomal protein L7/L12 was isolated among revertants of a streptomycin-dependent strain. In the crystal structure of the L7/L12 dimer, residue 15 within the N-terminal domain contacts the C-terminal domain of the partner monomer. We tested effects of the mutation on molecular assembly by biochemical approaches. Gel electrophoretic analysis showed that the Phe15-L7/L12 variant had reduced ability in binding to L10, an effect enhanced in the presence of 0.05% of nonionic detergent. Mobility of Phe15-L7/L12 on gel containing the detergent was very low compared to the wild-type proteins, presumably because of an extended structural state of the mutant L7/L12. Ribosomes isolated from LL103 cells contained a reduced amount of L7/L12 and showed low levels (15-30% of wild-type ribosomes) of activities dependent on elongation factors and in translation of natural mRNA. The ribosomal activity was completely recovered by addition of an excess amount of Phe15-L7/L12 to the ribosomes, suggesting that the mutant L7/L12 exerts normal functions when bound on the ribosome. The interaction of Ser15 with the C-terminal domain of the partner molecule seems to contribute to formation of the compact dimer structure and its efficient assembly into the ribosomal GTPase center. We propose a model relating compact and elongated forms of L7/L12 dimers. Phe15-L7/L12 provides a new tool for studying the functional structure of the homodimer.


Asunto(s)
Proteínas de Escherichia coli/química , Proteínas Ribosómicas/química , Ribosomas/enzimología , Secuencia de Aminoácidos , Dimerización , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/fisiología , GTP Fosfohidrolasas , Modelos Moleculares , Fenilalanina/genética , Mutación Puntual , Estructura Terciaria de Proteína , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/fisiología , Ribosomas/química , Serina/genética
5.
Antimicrob Agents Chemother ; 47(4): 1476-8, 2003 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-12654698

RESUMEN

Rhodococcus equi and species of Nocardia and Gordonia may be human opportunistic pathogens. We find that these, as well as several isolates from closely related genera, are highly susceptible to the imidazoles bifonazole, clotrimazole, econazole, and miconazole, whose MICs are

Asunto(s)
Antifúngicos/farmacología , Nocardia/efectos de los fármacos , Rhodococcus/efectos de los fármacos , Clotrimazol/farmacología , Fluconazol/farmacología , Imidazoles/farmacología , Miconazol/farmacología , Pruebas de Sensibilidad Microbiana
6.
Appl Environ Microbiol ; 68(10): 4764-71, 2002 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-12324318

RESUMEN

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a high explosive which presents an environmental hazard as a major land and groundwater contaminant. Rhodococcus rhodochrous strain 11Y was isolated from explosive contaminated land and is capable of degrading RDX when provided as the sole source of nitrogen for growth. Products of RDX degradation in resting-cell incubations were analyzed and found to include nitrite, formaldehyde, and formate. No ammonium was excreted into the medium, and no dead-end metabolites were observed. The gene responsible for the degradation of RDX in strain 11Y is a constitutively expressed cytochrome P450-like gene, xplA, which is found in a gene cluster with an adrenodoxin reductase homologue, xplB. The cytochrome P450 also has a flavodoxin domain at the N terminus. This study is the first to present a gene which has been identified as being responsible for RDX biodegradation. The mechanism of action of XplA on RDX is thought to involve initial denitration followed by spontaneous ring cleavage and mineralization.


Asunto(s)
Rhodococcus/genética , Triazinas/metabolismo , Secuencia de Aminoácidos , Biodegradación Ambiental , Clonación Molecular , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Nitrógeno/metabolismo , Rhodococcus/crecimiento & desarrollo , Rhodococcus/metabolismo , Homología de Secuencia de Aminoácido , Microbiología del Suelo , Contaminantes del Suelo/metabolismo , Triazinas/química
7.
J Biol Chem ; 277(6): 3857-62, 2002 Feb 08.
Artículo en Inglés | MEDLINE | ID: mdl-11729183

RESUMEN

Ribosomal L10-L7/L12 protein complex and L11 bind to a highly conserved RNA region around position 1070 in domain II of 23 S rRNA and constitute a part of the GTPase-associated center in Escherichia coli ribosomes. We replaced these ribosomal proteins in vitro with the rat counterparts P0-P1/P2 complex and RL12, and tested them for ribosomal activities. The core 50 S subunit lacking the proteins on the 1070 RNA domain was prepared under gentle conditions from a mutant deficient in ribosomal protein L11. The rat proteins bound to the core 50 S subunit through their interactions with the 1070 RNA domain. The resultant hybrid ribosome was insensitive to thiostrepton and showed poly(U)-programmed polyphenylalanine synthesis dependent on the actions of both eukaryotic elongation factors 1alpha (eEF-1alpha) and 2 (eEF-2) but not of the prokaryotic equivalent factors EF-Tu and EF-G. The results from replacement of either the L10-L7/L12 complex or L11 with rat protein showed that the P0-P1/P2 complex, and not RL12, was responsible for the specificity of the eukaryotic ribosomes to eukaryotic elongation factors and for the accompanying GTPase activity. The presence of either E. coli L11 or rat RL12 considerably stimulated the polyphenylalanine synthesis by the hybrid ribosome, suggesting that L11/RL12 proteins play an important role in post-GTPase events of translation elongation.


Asunto(s)
Escherichia coli/metabolismo , GTP Fosfohidrolasas/metabolismo , Extensión de la Cadena Peptídica de Translación/fisiología , Ribosomas/fisiología , Animales , Ratas
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