RESUMO
The Escherichia coli otsBA operon, located at min 42, was sequenced and shown to encode a 29.1-kDa trehalose-6-phosphate phosphatase (OtsB) and a 53.6-kDa trehalose-6-phosphate synthase (OtsA). Both proteins display sequence homology with subunits of the Saccharomyces cerevisiae trehalose-6-phosphate synthase/phosphatase complex, which is made up of the subunits TPS1, TPS2 and TPS3 (TSL1). OtsA has homology to the full-length TPS1, the N-terminal part of TPS2 and an internal region of TPS3 (TSL1). OtsB has homology to the C-terminal part of TPS2, but no homology to the other subunits. Primer extension analysis showed only one transcription start point upstream from otsB and one upstream from otsA, regardless of the growth conditions tested. The start codons of the otsB and otsA genes were established by N-terminal sequence determination of the proteins. The 3' end of the otsB coding region overlaps the 5' end of the otsA coding region by 23 nucleotides. The araH gene is located directly upstream from otsBA, and otsB may be identical to pexA.
Assuntos
Escherichia coli/genética , Glucosiltransferases/genética , Complexos Multienzimáticos/genética , Óperon , Monoéster Fosfórico Hidrolases/genética , Saccharomyces cerevisiae/genética , Trealose/biossíntese , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Bacteriano , Dados de Sequência Molecular , Saccharomyces cerevisiae/enzimologia , Homologia de Sequência de Aminoácidos , Trealose/genéticaRESUMO
Endogenously synthesized trehalose is a stress protectant in Escherichia coli. Externally supplied trehalose does not serve as a stress protectant, but it can be utilized as the sole source of carbon and energy. Mutants defective in trehalose synthesis display an impaired osmotic tolerance in minimal growth media without glycine betaine, and an impaired stationary-phase-induced heat tolerance. Mechanisms for stress protection by trehalose are discussed. The genes for trehalose-6-phosphate synthase (otsA) and anabolic trehalose-6-phosphate phosphatase (otsB) constitute an operon. Their expression is induced both by osmotic stress and by growth into the stationary phase and depend on the sigma factor encoded by rpoS (katF). rpoS is amber-mutated in E. coli K-12 and its DNA sequence varies among K-12 strains. For trehalose catabolism under osmotic stress E. coli depends on the osmotically inducible periplasmic trehalase (TreA). In the absence of osmotic stress, trehalose induces the formation of an enzyme IITre (TreB) of the group translocation system, a catabolic trehalose-6-phosphate phosphatase (TreE), and an amylotrehalase (TreC) which converts trehalose to free glucose and a glucose polymer.
Assuntos
Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Trealose/metabolismo , Sequência de Bases , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Genes Bacterianos , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Dados de Sequência Molecular , Pressão Osmótica , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Fosfotransferases/genética , Fosfotransferases/metabolismo , Trealase/metabolismoRESUMO
A Saccharomyces cerevisiae gene for trehalose-6-phosphate synthase (TPS1) was sequenced. The gene appeared to code for a protein of 495 amino acid residues, giving the protein a molecular mass of 56 kDa. The TPS1 gene was able to restore both osmotolerance and trehalose accumulation during salt stress in an Escherichia coli strain mutated in the otsA gene encoding trehalose-6-phosphate synthase. Complementation studies with E. coli galU mutants showed that the TPS1-encoded trehalose-6-phosphate synthase is UDP-glucose-dependent. Sequence analysis and data base searches showed that TPS1 is allelic to GGS1, byp1, cif1 and fdp1. A possible gene for trehalose-6-phosphate synthase in Methanobacterium thermoautotrophicum was identified.
Assuntos
Proteínas Fúngicas/genética , Genes Fúngicos , Glucosiltransferases/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Escherichia coli/enzimologia , Escherichia coli/genética , Teste de Complementação Genética , Methanobacterium/enzimologia , Methanobacterium/genética , Dados de Sequência Molecular , Peso Molecular , Saccharomyces cerevisiae/enzimologia , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
It has been shown previously that the otsA and otsB mutations block osmoregulatory trehalose synthesis in Escherichia coli. We report that the transcription of these osmoregulated ots genes is dependent on KatF (AppR), a putative sigma factor for certain stationary phase- and starvation-induced genes. The transcription of the osmoregulated bet and proU genes was not katF dependent. Our genetic analysis showed that katF carries an amber mutation in E. coli K-12 and many of its derivatives but that katF has reverted to an active form in the much-used strain MC4100. This amber mutation in katF leads to strain variations in trehalose synthesis and other katF-dependent functions of E. coli. We have performed a molecular cloning of the otsBA genes, and we present evidence that they constitute an operon encoding trehalose-6-phosphate phosphatase and trehalose-6-phosphate synthase. A cloning and restriction site analysis, performed by comparing the cloned fragments with the known physical map of the E. coli chromosome, revealed that the otsBA genes are situated on a 2.9-kb HindIII fragment located 8 to 11 kb clockwise of tar (41.6 min).
Assuntos
Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Glucosiltransferases/genética , Monoéster Fosfórico Hidrolases/genética , Equilíbrio Hidroeletrolítico/genética , Mapeamento Cromossômico , Clonagem Molecular , Indução Enzimática , Mutação/genética , Óperon/genética , Fator sigma/genética , Trealose/metabolismoRESUMO
The sequence was determined of 6493 nucleotides encompassing the bet genes of Escherichia coli which encode the osmoregulatory choline-glycine betaine pathway. Four open reading frames were identified: betA encoding choline dehydrogenase, a flavoprotein of 61.9kDa; betB encoding betaine aldehyde dehydrogenase (52.8kDa); betT encoding a proton-motive-force-driven, high-affinity transport system for choline (75.8kDa); and betl, capable of encoding a protein of 21.8kDa, implicated as a repressor involved in choline regulation of the bet genes. Identification of the genes was supported by subcloning, physical mapping of lambda placMu53 insertions, amino acid sequence similarity, or N-terminal amino acid sequencing. The bet genes are tightly spaced, with betT located upstream of, and transcribed divergently to, the tandemly linked betIBA genes.
Assuntos
Oxirredutases do Álcool/genética , Aldeído Oxirredutases/genética , Proteínas de Bactérias/genética , Colina/metabolismo , Escherichia coli/genética , Genes Bacterianos , Glicina/metabolismo , Proteínas Repressoras/genética , Oxirredutases do Álcool/metabolismo , Aldeído Oxirredutases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Betaína-Aldeído Desidrogenase , Transporte Biológico , Colina Desidrogenase , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Fases de Leitura Aberta , Pressão Osmótica , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/metabolismo , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
An analysis of the bet genes governing the osmoregulatory choline-glycine betaine pathway of Escherichia coli was performed. A 9 kb BamHI fragment, located 30 to 39 kb counterclockwise of the EcoRI site of lacZ, coded for all known Bet activities. The following genes were identified: the betA gene for the choline dehydrogenase, the betB gene for the betaine aldehyde dehydrogenase, and the betT gene or operon for the high-affinity choline transport. The betB and the betT genes were named in this paper, and the clockwise gene order was shown to be betA,B,T. Subcloning gave plasmids which expressed each of the three Bet activities separately. The cloned bet genes remained osmotically regulated, indicating the existence of several osmotically regulated promoters in the bet region. Salmonella typhimurium, which carried the bet region of E. coli in the broad-host-range vector pRK293 expressed the three Bet activities and displayed increased osmotic tolerance in the presence of choline.
Assuntos
Betaína/metabolismo , Colina/metabolismo , Escherichia coli/genética , Genes Bacterianos , Mapeamento Cromossômico , Cromossomos Bacterianos , Clonagem Molecular , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Oxirredutases/metabolismo , Salmonella typhimurium/genética , Equilíbrio HidroeletrolíticoRESUMO
It has been shown previously that Escherichia coli accumulates endogenously synthesized trehalose under osmotic stress. We report here that E. coli contained an osmotically regulated trehalose-phosphate synthase which utilized UDP-glucose and glucose 6-phosphate as substrates. In the wild type, the synthase was induced by growth in glucose-mineral medium of elevated osmotic strength and the synthase itself was strongly stimulated by K+ and other monovalent cations. A laboratory strain which expressed the synthase at a high constitutive level was found. GalU mutants, defective in synthesis of UDP-glucose, did not accumulate trehalose. Two genes governing the synthase were identified and named otsA and otsB (osmoregulatory trehalose synthesis). They mapped near 42 min in the flbB-uvrC region. Mutants with an otsA-lacZ or otsB-lacZ operon fusion displayed osmotically inducible beta-galactosidase activity; i.e., the activity was increased fivefold by growth in medium of elevated osmotic strength. Mutants unable to synthesize trehalose (galU, otsA, and otsB) were osmotically sensitive in glucose-mineral medium. But an osmotically tolerant phenotype was restored in the presence of glycine betaine, which also partially repressed the synthesis of synthase in the wild type and of beta-galactosidase in ots-lacZ fusion mutants.
Assuntos
Dissacarídeos/biossíntese , Escherichia coli/genética , Trealose/biossíntese , Equilíbrio Hidroeletrolítico , Clonagem Molecular , Escherichia coli/metabolismo , Glucose-6-Fosfato , Glucofosfatos/metabolismo , Óperon , Potássio/farmacologia , Salmonella typhimurium/metabolismo , Trealase/metabolismo , Trealose/genética , Uridina Difosfato Glucose/metabolismo , beta-Galactosidase/metabolismoRESUMO
We have constructed plasmids which overproduce the tag and alkA gene products of Escherichia coli, i.e., 3-methyladenine DNA glycosylases I and II. The tag and alkA gene products were identified radiochemically in maxi- or minicells as polypeptides of 21 and 30 kilodaltons, respectively, which are consistent with the gel filtration molecular weights of the enzyme activities, thus confirming the identity of the cloned genes. High expression of the tag+-coded glycosylase almost completely suppressed the alkylation sensitivity of alkA mutants, indicating that high levels of 3-methyladenine DNA glycosylase I will eliminate the need for 3-methyladenine DNA glycosylase II in repair of alkylated DNA. Furthermore, overproduction of the alkA+-coded glycosylase greatly sensitizes wild-type cells to alkylation, suggesting that only a limited expression of this enzyme will allow efficient DNA repair.
