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Jean-Claude Lazzaroni Raymond Portalier 《Applied microbiology and biotechnology》1982,16(2-3):146-150
Summary Col E1 hybrid plasmids carrying the phoA
+ structural gene of alkaline phosphatase, a periplasmic enzyme of Escherichia coli K-12, were identified from the Clarke and Carbon genomic bank. Wild-type (lky
+) phoA
+ plasmid-bearing strains synthesized 14 times more intracellular enzyme than the haploid lky
+ strain. Phosphate-induced repression was maintained in transformed strains.
PhoA
+ plasmids carrying the phoB and phoR regulatory genes were introduced into a periplasmic-leaky (lky) recipient strain able to release alkaline phosphatase into the extracellular medium. Transformed lky mutants excreted up to 90% of total enzyme activity which corresponded to 3.5 times the amount of intracellular alkaline phosphatase made by the haploid lky
+ strain. The protein composition analysis of periplasmic and extracellular fractions showed that: (i) wild-type phoA
+ hybrid plasmidbearing clones did not excrete alkaline phosphatase but had a modified periplasmic content; (ii) alkaline phosphatase was the major excreted protein by transformed lky mutants. The use of periplasmic-leaky phoA
+ hybrid plasmid-bearing mutants for an easier production and purification of alkaline phosphatase is discussed. 相似文献
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Bradyrhizobium japonicum mutant 132 was obtained by random TnphoA mutagenesis of strain 110spc4. A 6.5 kb BamHI kanamycin-resistance-coding DNA fragment of mutant 132 was used as a hybridization probe to clone the corresponding wild-type fragment. DNA sequence analysis of a 3213 bp BamHI—ClaI fragment revealed that three open reading frames (ORFs) were encoded in the same orientation. Based on sequence similarities to other proteins in the database, the second ORF was called sipS (signal peptidase). The TnphoA insertion in mutant 132 was found to be in frame near the 3′ end of sipS. The resulting SipS—PhoA hybrid polypeptide was shown to be expressed in free-living B. japonicum and in Escherichia coli cultures. An immunoblot analysis with a polyclonal antibody directed against the alkaline phosphatase revealed the appearance of a weak signal of a 70 kDa polypeptide both in B. Japonicum and in E. coli, in agreement with the calculated size of the hybrid polypeptide. A much stronger 52 kDa band was also detected. Mutant 132 was specifically disturbed in the interaction with soybean (Glycine max) when the bacteria were released from the infection threads. The bacteroids were not stably maintained within the symbiosome. Numerous vesicles were found in the plant cytosol, which finally resulted in the formation of large vacuoles within the infected nodule cells. Immunohistochemical analyses with antibodies directed against nodulins of the peribacteroid membrane indicated a lower expression of these proteins. The mutant phenotype was genetically complemented by 4.4 kb BamHI fragment including sipS. Subfragments thereof also complemented a temperature-sensitive E. coli lepB mutant, demonstrating that the B. japonicum fragment was functionally replacing Lepts in E. coli. Genetic studies suggested that the three genes are organized in one common operon which is expressed from a promoter upstream of the sequenced region. Inactivation of the gene downstream of sipS did not result in a detectable phenotype. 相似文献
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E. Fernández F. Lombó C. Méndez J. A. Salas 《Molecular genetics and genomics : MGG》1996,250(6):692-698
The organization of the phosphate-specific transport (pst) operon inPseudomonas aeruginosa has been determined. The gene order of thepst operon ispstC, pstA, pstB, phoU, and a well-conserved Pho box sequence (16/18 bases identical) exists in the promoter region. The most striking difference from the knownEscherichia coli pst operon is the lack of thepstS gene encoding a periplasmic phosphate (Pi)-binding protein. Even though the threepst genes were absolutely required for Pi-specific transport, expression of thepst operon at high levels did not increase Pi uptake inP. aeruginosa. DNA sequences for thepstB andphoU genes have been determined previously. The newly identifiedpstC andpstA genes encode possible integral membrane proteins of 677 amino acids (M r 73 844) and 513 amino acids (M r 56 394), respectively. The amino acid sequences of PstC and PstA predict that these proteins contain a long hydrophilic domain not seen in theirE. coli counterparts. A chromosomal deletion of the entirepst operon renderedP. aeruginosa unable to repress Pi taxis under conditions of Pi excess. ThephoU andpstB genes are essential for repressing Pi taxis. However, mutants lacking either PstC or PstA alone were able to repress Pi taxis under conditions of Pi excess. 相似文献
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The pho regulon of Shigella flexneri 总被引:1,自引:0,他引:1
Monica Scholten Riny Janssen Corné Bogaarts Joost van Strien Jan Tommassen 《Molecular microbiology》1995,15(2):247-254
Growth of Escherichia coli K-12 in low-phosphate conditions results in the induction of the synthesis of many proteins, including the outer membrane porin PhoE, alkaline phosphatase, and the Pst system for the transport of phosphate (Pi). This response is controlled by a two-component regulatory system of which PhoB and PhoR are the response-regulator and the sensor/kinase, respectively. When Shigella flexneri was starved for Pi, neither PhoE nor alkaline phosphatase was produced. However, induction of the synthesis of the PstS protein was observed, indicating that S. flexneri contains a functional PhoB/PhoR regulatory system. Consistent with this notion, the introduction of the B. coli phoA gene in S. flexneri resulted in the induction of alkaline phosphatase synthesis under phosphate limitation. However, introduction of phoE on a plasmid did not lead to the expression of PhoE protein, indicating that S. flexneri PhoB does not recognize the phoE promoter region. The phoB gene was cloned and sequenced and in the deduced amino acid sequence two deviations from that of E. coli PhoB were detected. Site-directed mutagenesis revealed that one of these deviations, i.e. Leu-172, which is Arg in E. coli PhoB, is responsible for the lack of expression of the PhoE protein in S. flexneri. 相似文献
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Further genetic mapping of the phoA-phoR region for alkaline phosphatase synthesis in Escherichia coli K 12 总被引:9,自引:0,他引:9
Summary Genetic mapping of the E. coli chromosomal region carrying the structural gene (phoA) and the regulatory gene (phoR) for alkaline phosphatase synthesis was carried out by conjugation. Recombinant colonies were selected and the segregation frequency of outside markers was determined. The genetic order lac phoA proC phoR tsx lon is proposed. 相似文献
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Klaus Erb Monika Vogel Wilma Wagner Werner Goebel 《Molecular & general genetics : MGG》1987,208(1-2):88-93
Summary Fusion of the alkaline phosphatase gene (phoA) which lacks its own signal peptide sequence to the N-terminal region of hlyA, the structural gene for Escherichia coli haemolysin, leads to active alkaline phosphatase (AP). AP activity depends on the length of the N-terminal region of hlyA. An optimum is reached when 100–200 amino acids of HlyA are fused to PhoA but fusion of as little as 13 amino acids of HlyA to PhoA is sufficient to yield appreciable AP activity. When cells are treated with lysozyme most of the AP activity is found associated with the membrane fraction but a substantial amount is also found in the soluble fraction, most of which may represent, a periplasmic pool of AP. The soluble portion of AP activity is significantly increased when the cells are disrupted by ultrasonication, which indicates that the fusion proteins are only loosely associated with the membrane and that large parts are already located on the outside of the cytoplasmic membrane. The expected fusion proteins were identified in the soluble and the membrane fractions and their amounts in these fractions correlated well with AP activity. 相似文献
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A topological model for the haemolysin translocator protein HlyD 总被引:8,自引:0,他引:8
Ralf Schülein Ivaylo Gentschev Hans-Joachim Mollenkopf Werner Goebel 《Molecular & general genetics : MGG》1992,234(1):155-163
Summary A topological model for HlyD is proposed that is based on results obtained with gene fusions of lacZ and phoA to hlyD. Active H1yD-LacZ fusion proteins were only generated when lacZ was fused to hlyD. within the first 180 by (60 amino acids). H1yD-PhoA proteins exhibiting alkaline phosphatase (AP) activity were obtained when phoA was inserted into hlyD. between nucleotides 262 (behind amino acid position 87) and 1405 (behind amino acid position 468, only 10 amino acids away from the C-terminus of HlyD Active insertions of phoA into the middle region of hlyD. were not observed on in vivo transposition but such fusions exhibiting AP activity could be constructed by in vitro techniques. A fusion protein that carried the PhoA part close to the C-terminal end of HlyD proved to be the most stable HlyD-PhoA fusion protein. In contrast to the other, rather unstable, HlyD-PhoA+ fusions, no proteolytic degradation product of this HlyD-PhoA protein was observed and nearly all the alkaline phosphatase activity was membrane bound. Protease accessibility and cell fractionation experiments indicated that the alkaline phosphatase moiety of this fusion protein was located in the periplasm as for all other HlyD-PhoA+ proteins. These data and computer-assisted predictions suggest a topological model for HlyD with the N-terminal 60 amino acids located in the cytoplasm, a single transmembrane segment from amino acids 60 to 80 and a large periplasmic region extending from amino acid 80 to the C-terminus. Neither the HlyD fusion proteins obtained nor a mutant HlyD protein that had lost the last 10 amino acids from the C-terminus of HlyD exhibited translocator activity for HlyA or other reporter proteins carrying the HlyA signal sequence. The C-terminal 10 amino acids of HlyD showed significant similarity with the corresponding sequences of other HlyD-related proteins involved in protein secretion. 相似文献
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Surachet Burut-Archanai Aran Incharoensakdi Julian J. Eaton-Rye 《Biochemical and biophysical research communications》2009,378(3):383-388
A novel 47 amino acid extension at the N-terminus of the SphS histidine kinase has been identified in the cyanobacterium Synechocystis sp. PCC 6803. Here, we demonstrate this region is required for activation of the SphS-SphR phosphate-sensing two-component system under phosphate-limiting conditions and mutants lacking this extension do not show constitutive alkaline phosphatase activity when the negative regulator SphU is inactivated. We have also identified a putative membrane-associated domain within this region involved in control of the Pho regulon. In addition, there are two high-affinity ABC-type phosphate uptake systems in this organism. Our results demonstrate that the Pst1 system, but not the Pst2 system, is required for suppression of the Pho regulon under phosphate-sufficient conditions. Deletion of the pst1 operon and disruption of the membrane-spanning domain may both target the same control mechanism since constitutive alkaline phosphatase activity is similar in the double and single mutants. 相似文献
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Induction of the wild type cholera toxin operon (ctxAB) from multicopy clones inEscherichia coliinhibited growth and resulted in low yields of cholera toxin (CT). We found that production of wild type CT or its B subunit (CT-B) as a periplasmic protein was toxic forE. coli,but by replacing the native signal sequences of both CT-A and CT-B with the signal sequence from the B subunit ofE. coliheat-labile enterotoxin LTIIb we succeeded for the first time in producing CT holotoxin in high yield inE. coli.Based on these findings, we designed and constructed versatile cloning vectors that use the LTIIb-B signal sequence to direct recombinant native proteins with high efficiency to the periplasm ofE. coli.We confirmed the usefulness of these vectors by producing two other secreted recombinant proteins. First, usingphoAfromE. coli,we demonstrated that alkaline phosphatase activity was 17-fold greater when the LTIIb-B signal sequence was used than when the native leader for alkaline phosphatase was used. Second, using thepspAgene that encodes pneumococcal surface protein A fromStreptococcus pneumoniae,we produced a 299-residue amino-terminal fragment of PspA inE. coliin large amounts as a soluble periplasmic protein and showed that it was immunoreactive in Western blots with antibodies against native PspA. The vectors described here will be useful for further studies on structure–function relationships and vaccine development with CT and PspA, and they should be valuable as general tools for delivery of other secretion-competent recombinant proteins to the periplasm inE. coli. 相似文献
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Mineralization of Paraoxon and Its Use as a Sole C and P Source by a Rationally Designed Catabolic Pathway in Pseudomonas putida
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Matthew de la Pea Mattozzi Sundiep K. Tehara Thomas Hong Jay D. Keasling 《Applied microbiology》2006,72(10):6699-6706
Organophosphate compounds, which are widely used as pesticides and chemical warfare agents, are cholinesterase inhibitors. These synthetic compounds are resistant to natural degradation and threaten the environment. We constructed a strain of Pseudomonas putida that can efficiently degrade a model organophosphate, paraoxon, and use it as a carbon, energy, and phosphorus source. This strain was engineered with the pnp operon from Pseudomonas sp. strain ENV2030, which encodes enzymes that transform p-nitrophenol into β-ketoadipate, and with a synthetic operon encoding an organophosphate hydrolase (encoded by opd) from Flavobacterium sp. strain ATCC 27551, a phosphodiesterase (encoded by pde) from Delftia acidovorans, and an alkaline phosphatase (encoded by phoA) from Pseudomonas aeruginosa HN854 under control of a constitutive promoter. The engineered strain can efficiently mineralize up to 1 mM (275 mg/liter) paraoxon within 48 h, using paraoxon as the sole carbon and phosphorus source and an inoculum optical density at 600 nm of 0.03. Because the organism can utilize paraoxon as a sole carbon, energy, and phosphorus source and because one of the intermediates in the pathway (p-nitrophenol) is toxic at high concentrations, there is no need for selection pressure to maintain the heterologous pathway. 相似文献