RESUMEN
The expression of the moa locus, which encodes enzymes required for molybdopterin biosynthesis, is enhanced under anaerobiosis but repressed when the bacterium is able to synthesize active molybdenum cofactor. In addition, moa expression exhibits a strong requirement for molybdate. The molybdate enhancement of moa transcription is fully dependent upon the molybdate-binding protein, ModE, which also mediates molybdate repression of the mod operon encoding the high-affinity molybdate uptake system. Due to the repression of moa in molybdenum cofactor-sufficient strains, the positive molybdate regulation of moa is revealed only in strains unable to make the active cofactor. Transcription of moa is controlled at two sigma-70-type promoters immediately upstream of the moaA gene. Deletion mutations covering the region upstream of moaA have allowed each of the promoters to be studied in isolation. The distal promoter is the site of the anaerobic enhancement which is Fnr-dependent. The molybdate induction of moa is exerted at the proximal promoter. Molybdate-ModE binds adjacent to the -35 region of this promoter, acting as a direct positive regulator of moa. The molybdenum cofactor repression also appears to act at the proximal transcriptional start site, but the mechanism remains to be established. Tungstate in the growth medium affects moa expression in two ways. Firstly, it can act as a functional molybdate analogue for the ModE-mediated regulation. Secondly, tungstate brings about the loss of the molybdenum cofactor repression of moa. It is proposed that the tungsten derivative of the molybdenum cofactor, which is known to be formed under such conditions, is ineffective in bringing about repression of moa. The complex control of moa is discussed in relation to the synthesis of molybdoenzymes in the bacterium.
Asunto(s)
Proteínas Bacterianas , Coenzimas , Proteínas de Escherichia coli , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Metaloproteínas/genética , Molibdeno/metabolismo , Operón , Factores de Transcripción/metabolismo , Aerobiosis , Anaerobiosis , Secuencia de Bases , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Eliminación de Gen , Metaloproteínas/metabolismo , Datos de Secuencia Molecular , Cofactores de Molibdeno , Operón/genética , Operón/fisiología , Regiones Promotoras Genéticas/genética , Pteridinas/metabolismo , Transcripción Genética , Tungsteno/metabolismoRESUMEN
The mob locus of Escherichia coli encodes functions which catalyse the synthesis of active molybdenum cofactor, molybdopterin guanine dinucleotide, from molybdopterin and GTP. Reporter translational lac fusion mutations in the mobA gene have been constructed using lambda placMu9 mutagenesis. The mob locus is expressed at very low levels under both aerobic and anaerobic growth conditions. Neither additions to the growth media (nitrate, tungstate or molybdate) nor secondary mutations at the moa, mob, mod, moe or mog loci affected the level of expression. Two transcription initiation sites and their associated promoter regions have been identified upstream of mobA. Both of the promoter regions show a poor match to the -35 and -10 consensus sequences for sigma 70 promoters. A 2.2 kb chromosomal DNA fragment which complemented all available mob mutants has been sequenced. Two ORFs were identified, arranged as a single transcription unit. The encoded polypeptides have predicted molecular masses of 21642 Da and 19362 Da, respectively. The DNA has been subcloned into a T7 overexpression system and the predicted products identified. The mobA gene encodes protein FA, which has been purified to homogeneity and brings about the activation of inactive molybdoenzymes in cell extracts of mob mutants. The mobB gene encodes a polypeptide with a putative nucleotide binding site. All available mob mutations which have been selected for by their ability to grow anaerobically in the presence of chlorate are located in the mobA gene.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Nucleótidos de Guanina/metabolismo , Pterinas/metabolismo , Secuencia de Aminoácidos , Anaerobiosis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Cloratos/farmacología , Clonación Molecular , Escherichia coli/genética , Nucleótidos de Guanina/biosíntesis , Nucleótidos de Guanina/genética , Datos de Secuencia Molecular , Mutagénesis Insercional , Nitrato Reductasas/genética , Operón , Factor Tu de Elongación Peptídica/genética , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Transducción GenéticaRESUMEN
Introducing the Escherichia coli topA20::Tn10 allele to Shigella flexneri results in osmotic sensitivity, a reduced growth rate, an increase in reporter plasmid supercoiling (all common to the E. coli mutants), an inability to grow on MacConkey agar and a loss of virulence gene expression. E. coli mutants harbouring this topA allele often compensate for the loss of DNA topoisomerase I by amplifying the genes coding for topoisomerase IV. Unlike the E. coli topA mutants, derivatives of S. flexneri harbouring this topA allele did not appear to acquire any compensatory mutations. We investigated the possibility that this was due in part to an inability of the S. flexneri topoisomerase IV genes to compensate for loss of DNA topoisomerase I when overexpressed. The S. flexneri genes encoding the alpha- and beta subunits of topoisomerase IV were detected and cloned in separate multicopy plasmids. These plasmids complemented well-characterized Salmonella typhimurium temperature-sensitive topoisomerase IV mutations, showing that the S. flexneri and S. typhimurium proteins are capable of combining to form active complexes. When the S. flexneri topoisomerase IV genes were cloned in the same multicopy plasmid and introduced into a S. flexneri topA mutant, the plasmid restored osmotic tolerance, improved the growth rate, allowed growth on MacConkey indicator plates and resulted in a relaxation of reporter plasmid supercoiling. The same plasmid also partially restored S. flexneri virulence gene transcription. These data show that overexpression of the S. flexneri topoisomerase IV genes can compensate for the loss of topoisomerase I in terms of general viability of the cell, DNA supercoiling, and (partially) virulence gene expression. The fact that S. flexneri does not exploit topoisomerase IV gene amplification as E. coli does points to a significant difference in the abilities of these species to adapt to the loss of topoisomerase I.
Asunto(s)
Proteínas Bacterianas/biosíntesis , ADN-Topoisomerasas de Tipo I/biosíntesis , ADN-Topoisomerasas de Tipo I/deficiencia , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Shigella flexneri/genética , Alelos , Proteínas Bacterianas/genética , Clonación Molecular , Topoisomerasa de ADN IV , ADN-Topoisomerasas de Tipo I/genética , ADN Bacteriano/genética , ADN Superhelicoidal/genética , Inducción Enzimática , Prueba de Complementación Genética , Plásmidos , Salmonella typhimurium/genética , Shigella flexneri/patogenicidad , VirulenciaRESUMEN
A 3.2 kb chromosomal DNA fragment which complements the defects in a series of twelve moa::Mucts insertion mutants has been sequenced. Five open reading frames (ORFs) were identified and these are arranged in a manner consistent with their forming an operon. The encoded proteins (MoaA-MoaE) have predicted molecular weights of 37,346, 18,665, 17,234, 8843 and 16,981 respectively. Examination of subclones of the whole locus in an expression system demonstrated the predicted products. N-terminal amino acid sequences for the moaA, B, C and E products confirmed the translational starts. Genetic analysis distinguished four classes of moa mutants corresponding to genes moaA, C, D and E. Potential promoter sequences upstream of moaA and a possible transcription termination signal have been identified. Genetic analysis of the chlA1 and chlM mutants, which have been biochemically characterized as defective in molybdopterin biosynthesis, indicates that these carry lesions in moaA and moaD respectively. The moa locus is orientated clockwise at 17.7 minutes in the chromosome.