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1.
Regulation of lactose-phosphoenolpyruvate-dependent phosphotransferase system and beta-D-phosphogalactoside galactohydrolase activities in Lactobacillus casei. 总被引:20,自引:12,他引:8
The lactose-phosphoenolpyruvate-dependent phosphotransferase system (lac-PTS) and beta-D-phosphogalactoside galactohydrolase (P-beta-gal) mediate the metabolism of lactose by Lactobacillus casei. Starved cells of L. casei contained a high intracellular concentration of phosphoenolpyruvate, and this endogenous energy reserve facilitated characterization of phosphotransferase system activities in physiologically intact cells. Data obtained from transport studies with whole cells and from in vitro phosphotransferase system assays with permeabilized cells revealed that the lac-PTS had a high affinity for beta-galactosides (e.g., lactose, lactulose, lactobionic acid, and arabinosyl-beta-D-galactoside). lac-PTS and P-beta-gal activities were determined in wild-type strains and strains defective in the glucose-phosphoenolpyruvate-dependent phosphotransferase system after growth on various sugars and in the presence of potential inducers. We found that (i) the lac genes (i.e., the genes coding for the lac-PTS proteins and P-beta-gal) were induced by metabolizable and non-metabolizable beta-galactosides (presumably acting as their phosphorylated derivatives), (ii) galactose 6-phosphate was not an inducer in most strains, (iii) the ratio of lac-PTS activity to P-beta-gal activity in a given strain was not constant, and (iv) inhibition of lac gene expression during growth on glucose was a consequence of glucose-phosphoenolpyruvate-dependent phosphotransferase system-mediated inducer exclusion, repressive effects of a functional glucose-phosphoenolpyruvate-dependent phosphotransferase system and glucose-derived metabolites. The expression of the lac-PTS structural genes and the expression of the P-beta-gal gene are independently regulated and may be subject to both positive control and negative control. 相似文献
2.
The Escherichia coli K-12 sad gene, which encodes an NAD-dependent succinic semialdehyde dehydrogenase, was cloned into a high-copy-number vector. Minicells carrying a sad+ plasmid produced a 55,000-dalton peptide, the probable sad gene product. 相似文献
3.
Cloning and expression of the exbB gene of Escherichia coli K-12 总被引:4,自引:0,他引:4
Katrin Eick-Helmerich Klaus Hantke Volkmar Braun 《Molecular & general genetics : MGG》1987,206(2):246-251
4.
Cloning and expression of the ilvB gene of Escherichia coli K-12 总被引:12,自引:0,他引:12
Thomas Newman Philip Friden Ann Sutton Martin Freundlich 《Molecular & general genetics : MGG》1982,186(3):378-384
Summary A plasmid containing theilvB operon, which codes for acetohydroxy acid synthase I ofEscherichia coli K-12, was isolated using a ligated mixture of DNA from plasmid pBR322 and F'ilvB4 treated with endonucleaseSalI. A shortened derivative of this plasmid was isolated by cloning a 3.4 kb bacterial fragment into plasmid pKEN005 to yield plasmid pTCN12. The orientation of theilvB operon relative to plasmid genes was determined by restriction enzyme mapping. Measurement of the level of the product of theilvB gene, acetohydroxy acid synthase I, indicated that plasmid pTCN12 contained a functionalilvB promoter and control region. The DNA from this plasmid was used as a probe to show that the rate of synthesis ofilvB mRNA was proportional to the levels of acetohydroxy acid synthase I. 相似文献
5.
An Escherichia coli heme-requiring, heme-permeable mutant had no detectable 5-aminolevulinate dehydratase or porphobilinogen deaminase activities. The gene which complemented this mutation was cloned to a high-copy-number plasmid, and porphobilinogen deaminase activity was restored to normal levels, but the synthesis of 5-aminolevulinate dehydratase increased 20- to 30-fold. A maxicell procedure confirmed that the gene cloned was hemB. 相似文献
6.
Cloning, expression, and characterization of the Escherichia coli K-12 rfaD gene. 总被引:13,自引:8,他引:5
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The rfaD gene encodes ADP-L-glycero-D-mannoheptose-6-epimerase, an enzyme required for the biosynthesis of the lipopolysaccharide precursor ADP-L-glycerol-D-mannoheptose. The precise localization of the rfaD gene on a 1.3-kilobase SspI-HpaI fragment is reported. The rfaD gene and the flanking regions were completely sequenced. The location of the rfaD gene on the physical map of the Escherichia coli chromosome was determined. Primer extension studies were used to define the regulatory region of the rfaD gene. The cloned rfaD gene directed the synthesis of a 37,000-dalton polypeptide in several in vivo and in vitro expression systems. N-terminal analysis of purified ADP-L-glycero-D-mannoheptose-6-epimerase confirmed the first 34-amino-acid sequence deduced from the nucleotide sequence of the rfaD gene coding region. The primary structure of the rfaD protein contains the sequence fingerprint for the ADP-binding beta alpha beta fold at the N terminus. 相似文献
7.
Cloning, structure, and expression of the Escherichia coli K-12 hisC gene. 总被引:1,自引:5,他引:1
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We used an expression vector plasmid containing the Escherichia coli K-12 histidine operon regulatory region to subclone the E. coli hisC gene. Analysis of plasmid-coded proteins showed that hisC was expressed in minicells. A protein with an apparent molecular weight of 38,500 was identified as the primary product of the hisC gene. Expression was under control of the hisGp promoter and resulted in very efficient synthesis (over 100-fold above the wild-type levels) of imidazolylacetolphosphate:L-glutamate aminotransferase, the hisC gene product. The complete nucleotide sequence of the hisC gene has been determined. The gene is 1,071 nucleotides long and codes for a protein of 356 amino acids with only one histidine residue. 相似文献
8.
Lactose metabolism in Lactobacillus casei occurs via phosphoenolpyruvate-dependent phosphotransferase uptake of lactose and subsequent cleavage of lactose-6-phosphate by beta-D-phosphogalactoside galactohydrolase (P-beta Gal). The genes for lactose uptake and P-beta Gal have been shown to be plasmid-associated in L. casei 64H [Chassy et al., Curr. Microbiol. 1 (1978) 141-144]. The cloned P-beta Gal-coding gene (pbg) previously described [Lee et al., J. Bacteriol. 152 (1982) 1138-1146] was subcloned on a 2.9-kb KpnI-Bg/II fragment isolated from pLZ605. Sequence analysis of this fragment revealed an open reading frame of 1422 bp capable of coding for a protein product containing 474 amino acids and having an Mr of 53,989. The L. casei protein showed a high degree of homology to the proteins whose sequence was deduced from the nucleotide sequence of the pbg genes of Staphylococcus aureus and Streptococcus lactis. Because of the significant homologies observed, as reflected in amino acid content as well as predicted structural characteristics of the three proteins, we suggest a common origin for the P-beta Gals of these three organisms. 相似文献
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10.
Cloning, expression, and nucleotide sequence of the Escherichia coli K-12 ackA gene. 总被引:6,自引:4,他引:2
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The Escherichia coli K-12 ackA gene, which encodes an acetate kinase, was cloned. The acetate kinase activities of ackA+ plasmid-containing strains were amplified 160- to 180-fold. The complete nucleotide sequence of the ackA gene was determined. It was deduced that the ackA gene coded for a protein of 400 amino acids with an Mr of 43,297. The ackA gene was found to be located about 15 kilobases upstream of the purF-folC-hisT region of the chromosome. 相似文献
11.
Cloning and characterization of Escherichia coli K-12 regulator gene tyrR. 总被引:3,自引:3,他引:3
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The Escherichia coli K-12 regulator gene tyrR was cloned into the multicopy plasmid pBR322 from a lambda(Tn10)tyrR+ specialized transducing phage. Further subcloning localized the gene within a 2.1-kilobase region. Analysis of plasmid-coded proteins showed that the tyrR gene codes for a 63,000-dalton polypeptide. 相似文献
12.
The adenylate cyclase gene of Escherichia coli has been cloned on the plasmid vector pBR325. The hybrid plasmid pTH4 obtained has a molecular weight of 6,4 megadalton and represents pBR325 plasmid with the insertion of 2,8 megadalton in the Pst1 site. The cya mutant bacteria carrying pTH4 recover their ability to utilize mannitol, lactose and other carbohydrates as carbon sources, and lose this ability again in the case of rare spontaneous excision of the DNA insert from the Pst1 site. The phenotypical effect of pTH4 in cya mutants can be only seen in the crp+ genome. The strains carrying pTH4 are also characterized by the ability of beta-galactosidase induction under conditions of catabolite repression. Besides, the bacteria containing cya+ allele on the plasmid do not grow on glycerol, which seems to be caused by toxic concentrations of methylglyoxal formed as a result of the increased intracellular level of cyclic adenosine monophosphate. 相似文献
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16.
Cloning the Escherichia coli K-12 argD gene specifying acetylornithine delta-transaminase 总被引:2,自引:0,他引:2
The argD gene of Escherichia coli was shown to be present in plasmids pLC2-28 and pLC3-11 of the collection of Clarke and Carbon [Cell 9 (1976) 91-99]. The gene was cloned into pBR322 as a 6.3-kb BamHI fragment. Enzyme determination showed that the cloned DNA contains the structural gene for acetylornithine delta-transaminase. The argD DNA was used as a probe in hybridization experiments which indicated that the argM gene resides in a duplicated portion of E. coli DNA that is highly similar to the argD region. 相似文献
17.
Cloning and characterization of the gene encoding inorganic pyrophosphatase of Escherichia coli K-12. 总被引:8,自引:3,他引:5
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R Lahti T Pitkranta E Valve I Ilta E Kukko-Kalske J Heinonen 《Journal of bacteriology》1988,170(12):5901-5907
Escherichia coli K-12 gene ppa encoding inorganic pyrophosphatase (PPase) was cloned and sequenced. The 5' end of the ppa mRNA was identified by primer extension mapping. A typical E. coli sigma 70 promoter was identified immediately upstream of the mRNA 5' end. The structural gene of ppa contains 528 base pairs, from which a 175-amino-acid translation product, Mr 19,572, was deduced. The deduced amino acid composition perfectly fitted with that of PPase as previously determined (P. Burton, D. C. Hall, and J. Josse, J. Biol. Chem. 245:4346-4351, 1970). Furthermore, the partial amino acid sequence (residues 1 to 108) of E. coli PPase determined by S. A. Cohen (Ph.D. thesis, University of Chicago, 1978) was the same as that deduced from the nucleotide sequence. This is the first report of the cloning of a PPase gene. 相似文献
18.
Cloning, expression, and mapping of the Aeromonas hydrophila aerolysin gene determinant in Escherichia coli K-12. 总被引:21,自引:6,他引:15
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DNA sequences corresponding to the aerolysin gene (aer) of Aeromonas hydrophila AH2 DNA were identified by screening a cosmid gene library for hemolytic and cytotoxic activities. A plasmid containing a 5.8-kilobase EcoRI fragment of A. hydrophila DNA was required for full expression of the hemolytic and cytotoxic phenotype in Escherichia coli K-12. Deletion analysis and transposon mutagenesis allowed us to localize the gene product to 1.4 kilobases of Aeromonas DNA and define flanking DNA regions affecting aerolysin production. The reduced hemolytic activity with plasmids lacking these flanking regions is associated with a temporal delay in the appearance of hemolytic activity and is not a result of a loss of transport functions. The aerolysin gene product was detected as a 54,000-dalton protein in E. coli maxicells harboring aer plasmids and by immunoblotting E. coli whole cells carrying aer plasmids. We suggest that the gene coding aerolysin be designated aerA and that regions downstream and upstream of aerA which modulate its expression and activity be designated aerB and aerC, respectively. 相似文献
19.
Cloning and structure of the hem A gene of Escherichia coli K-12 总被引:16,自引:0,他引:16
An Escherichia coli gene, which complements two independent hemA mutants of E. coli, has been cloned onto a multi-copy plasmid and both its strands have been sequenced. Both complemented mutants produce 5-aminolevulinic acid (ALA) and display fluorescence after 24h. The cloned sequence appears to encode a 46-kDa protein, which when produced in the maxicell procedure is processed to a 41-kDa protein as determined by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. The amino acid sequence of the cloned gene product shows no significant homologies with any cloned ALA synthase, nor with any protein, in two E. coli databanks. A second cloned gene fragment, which has its coding region 34 bp away from the coding region of the gene that complements hemA, has been identified as part of protein release factor 1(RF1), thus confirming the location of hemA at min 26.7 and mapping it precisely near RF1. We have shown that E. coli utilizes the intact five-carbon chain of glutamate for the synthesis of ALA [Li et al., J Bacteriol. 171 (1989b) 2547-2552]. 相似文献
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