Inactivation of pgsA gene affects biosynthesis and secretion of Escherichia coli alkaline phosphatase

Inactivation of pgsA, which is responsible for biosynthesis of anionic phospholipid phosphatidylglycerol (PG), was shown to affect biosynthesis and secretion of alkaline phosphatase (PhoA) in Escherichia coli. A decrease in PG, but not in total anionic phospholipids, correlated with reduction of PhoA secretion, suggesting the role of PG in this process. A dramatic decrease in PG (from 18 to 3, but not 8, percent of the total phospholipids) inhibited not only secretion, but also synthesis of PhoA. In addition, pgsA inactivation expedited repression of PhoA synthesis by exogenous orthophosphate.     

Membrane phospholipid composition of Escherichia coli affects secretion of periplasmic alkaline phosphatase into the medium

Secretion of alkaline phosphatase (PhoA) encoded by a gene constituent of plasmids has been studied in Escherichia coli strains with controlled synthesis of anionic phospholipids (phosphatidylglycerol and cardiolipin, strain HDL11) and zwitterionic phospholipid (phosphatidylethanolamine, strain AD93). Changing the phospholipid composition of the membrane of these strains leads to an increase in secretion of PhoA, which is usually localized in the periplasm, into the culture medium. This correlates with a higher secretion of exopolysaccharides and lower content of lipopolysaccharide in the outer membrane. The results show the possibility of coupling protein secretion into the medium with biogenesis of cell envelope components in which phospholipids are involved.     

The major phospholipid of Escherichia coli, phosphatidylethanolamine, is required for efficient production and secretion of alkaline phosphatase

The major phospholipid of the Escherichia coli membranes--the zwitterion phosphatidylethanolamine (PE)--is the only phospholipid involved in the formation of non-bilayer structure of membrane lipids, which is supposed to be necessary for efficient translocation of secreted proteins across the cytoplasmic membrane. The effect of PE on the production and secretion of alkaline phosphatase has been studied in this work using the mutant strain E. coli AD93, which is unable to synthesize PE. It was shown that this phospholipid is required for the efficient production and secretion of alkaline phosphatase. The anionic phospholipid cardiolipin in combination with divalent cations Mg2+ functionally replaces PE in these processes, participating in the regulation of lipid polymorphism.     

Proteolytic modification of Escherichia coli alkaline phosphatase

Proteolytic modification of the native alkaline phosphatase dimer is restricted to sites in the amino-terminal portion of the sequence. Complementing previous studies of the product of trypsin cleavage at the R-11, A-12 bond (Roberts, C. H., and Chlebowski, J. F. (1984) J. Biol. Chem. 259, 729-733; Roberts, C. H., and Chlebowski, J. F. (1984) J. Biol. Chem. 260, 7557-7561) circular dichroic spectroscopy indicates that cleavage at this site results in a rearrangement of secondary structure and change in tertiary structure as monitored in the far and near UV regions, respectively. Under more vigorous reaction conditions, trypsin cleaves at the R-35, D-36 bond. The deletion of an additional 24 residues yields a species whose functional and structural properties are similar to the initial product of trypsin cleavage. Treatment of the enzyme with Protease V-8 results in cleavage at the E-9, N-10 bond. In contrast to the products of trypsin treatment, this truncated enzyme is similar to the native enzyme. These results indicate that the residues at the N-10 and R-11 positions play a unique role in maintaining the structural integrity and catalytic potency of the enzyme although this locus is distant from the enzyme active centers. These observations are discussed in terms of the three-dimensional structure of the enzyme.     

Studies on alkaline phosphatase. Transientstate and steady-state kinetics of Escherichia coli alkaline phosphatase

1. Reduction of a 19s immunoglobulin M with 3mm-mercaptoethanol or 0.05-0.5mm-dithiothreitol followed by alkylation gave sedimentation patterns indicating products compatible with structures consisting of one, two, three, four and five 7s sub-units. This supports the concept of a five-sub-unit structure for immunoglobulin M. 2. Reduction with 0.125mm-dithiothreitol or 20mm-cysteine produced 7s sub-units that could not be dissociated into chains in m-propionic acid. 3. By labelling (with iodo[2-(14)C]acetic acid) the thiol groups liberated during reduction with 0.125mm-dithiothreitol, it was possible to identify the tryptic peptides involved in the disulphide bridges that link the 7s sub-units together (inter-sub-unit bridges). 4. By further reducing and labelling (with iodo[2-(14)C]acetic acid) the 7s sub-units produced by 0.125mm-dithiothreitol, it was possible to identify tryptic peptides derived from intra-sub-unit bridges. 5. Sub-units produced by reduction with 20mm-cysteine proved to be unsuitable for distinguishing between inter-sub-unit bridges and intra-sub-unit bridges. 6. The possible arrangement of the interchain disulphide bridges was deduced.     

Interaction of SecB and SecA with the N-terminal region of mature alkaline phosphatase on its secretion in Escherichia coli

Export-specific chaperone SecB and translocational ATPase SecA catalyze the cytoplasmic steps of Sec-dependent secretion in Escherichia coli. Their effects on secretion of periplasmic alkaline phosphatase (PhoA) were shown to depend on the N-terminal region of the mature PhoA sequence contained in the PhoA precursor. Amino acid substitutions in the vicinity of the signal peptide (positions +2, +3) not only dramatically inhibited secretion, but also reduced its dependence on SecB and SecA. Immunoprecipitation reported their impaired binding with mutant prePhoA. The results testified that SecB and SecA interact with the mature PhoA region located close to the signal peptide in prePhoA.     

Molecular asymmetry in alkaline phosphatase of Escherichia coli

Thermal inactivation of alkaline phosphatase of Escherichia coli has been studied at different temperatures (45 to 70 degrees C) and pHs (7.5, 9.0, and 10.0) for the commercial, buffer-dialyzed (pH 9.0) and EDTA-dialyzed (pH 9.0) enzymes. In each case, the inactivation exhibits biphasic kinetics consistent with the rate equation, (formula; see text) where A0 and A are activities at time zero and t, and k1 and k2 are first-order rate constants for the fast and slow phase, respectively. Values of k1 and k2 change independently with temperature, pH, and pretreatment (dialysis) of the enzyme. Time course of inactivation of the enzyme with excess EDTA and effect of Zn2+ ion concentration on the activity of EDTA-dialyzed enzyme have been investigated. The data suggest that the dimeric enzyme protein has two types of catalytic sites which have equal catalytic efficiency (or specific activity) but differ in several other properties. Structural implications of these results have been discussed.     

Signal sequence of alkaline phosphatase of Escherichia coli.

The amino acid sequence of the signal sequence of phoA was determined by DNA sequencing by using the dideoxy chain termination technique (Sanger et al., Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467, 1977). The template used was single-stranded DNA obtained from M13 on f1 phage derivatives carrying phoA, constructed by in vitro recombination. The results confirm the sequence of the first five amino acids determined by Sarthy et al. (J. Bacteriol. 139:932-939, 1979) and extend the sequence in the same reading frame into the amino terminal region of the mature alkaline phosphatase (Bradshaw et al., Proc. Natl. Acad. Sci. U.S.A., 78:3473-3477, 1981). As was predicted (Inouye and Beckwith, Proc. Natl. Acad. Sci. U.S.A. 74:1440-1444, 1977), the signal sequence was highly hydrophobic. The alteration of DNA sequence was identified for a promoter mutation that results in the expression of phoA independent of the positive control gene phoB and in insensitivity to high phosphate.     

The pro-region of Streptomyces hygroscopicus transglutaminase affects its secretion by Escherichia coli

Streptomyces transglutaminase (TGase) is secreted as a zymogen (pro-TGase) in liquid cultures and is then processed by the removal of its N-terminal region, resulting in active TGase. To date, there is no report describing TGase (or pro-TGase) secretion in Escherichia coli. In this study, the pro-TGase from Streptomyces hygroscopicus was efficiently secreted by E.?coli BL21(DE3) using the TGase signal peptide or the pelB signal peptide. The secreted pro-TGase was efficiently transformed into active TGase by adding dispase to the culture supernatant of the recombinant strains. Mutational analysis showed that deletion of the first six amino acids of the N-terminal of the pro-region reduced the secretion of pro-TGase, and removal of the next 10 amino acids resulted in the formation of insoluble pro-TGase. These results suggest that the pro-region of TGase is essential for its efficient secretion and solubility in E.?coli.     

Functional interrelationships in the alkaline phosphatase superfamily: phosphodiesterase activity of Escherichia coli alkaline phosphatase

Escherichia coli alkaline phosphatase (AP) is a proficient phosphomonoesterase with two Zn(2+) ions in its active site. Sequence homology suggests a distant evolutionary relationship between AP and alkaline phosphodiesterase/nucleotide pyrophosphatase, with conservation of the catalytic metal ions. Furthermore, many other phosphodiesterases, although not evolutionarily related, have a similar active site configuration of divalent metal ions in their active sites. These observations led us to test whether AP could also catalyze the hydrolysis of phosphate diesters. The results described herein demonstrate that AP does have phosphodiesterase activity: the phosphatase and phosphodiesterase activities copurify over several steps; inorganic phosphate, a strong competitive inhibitor of AP, inhibits the phosphodiesterase and phosphatase activities with the same inhibition constant; a point mutation that weakens phosphate binding to AP correspondingly weakens phosphate inhibition of the phosphodiesterase activity; and mutation of active site residues substantially reduces both the mono- and diesterase activities. AP accelerates the rate of phosphate diester hydrolysis by 10(11)-fold relative to the rate of the uncatalyzed reaction [(k(cat)/K(m))/k(w)]. Although this rate enhancement is substantial, it is at least 10(6)-fold less than the rate enhancement for AP-catalyzed phosphate monoester hydrolysis. Mutational analysis suggests that common active site features contribute to hydrolysis of both phosphate monoesters and phosphate diesters. However, mutation of the active site arginine to serine, R166S, decreases the monoesterase activity but not the diesterase activity, suggesting that the interaction of this arginine with the nonbridging oxygen(s) of the phosphate monoester substrate provides a substantial amount of the preferential hydrolysis of phosphate monoesters. The observation of phosphodiesterase activity extends the previous observation that AP has a low level of sulfatase activity, further establishing the functional interrelationships among the sulfatases, phosphatases, and phosphodiesterases within the evolutionarily related AP superfamily. The catalytic promiscuity of AP could have facilitated divergent evolution via gene duplication by providing a selective advantage upon which natural selection could have acted.     

Derepression of alkaline phosphatase in Escherichia coli by p-fluorophenylalanine

p-Fluorophenylalanine (FPA) causes a 100-fold increase in alkaline phosphatase in Escherichia coli B, strain PR1 at 30 C in minimal medium that contains excess inorganic phosphate (1.92 x 10(-3)m). Little increase in alkaline phosphatase synthesis occurs under these conditions at 22 C. [This strain is known to have a mutation in a regulator gene (R(2)) that, in the absence of FPA, permits derepression of alkaline phosphatase synthesis at 37 C, but not at 30 C or below.] In contrast, E. coli B3 (the strain from which E. coli B strain PR1 was derived) is not derepressed at 30 C by FPA. (14)C-FPA is incorporated into bacterial proteins. Temperature-shift experiments (30 Cright harpoon over left harpoon22 C) in the presence of FPA are consistent with the following mechanism. FPA is incorporated into the genetically altered R(2) protein at 30 and 22 C. This further alteration due to the incorporation of analogue makes the R(2) protein inactive at 30 C, but active at 22 C.     

Effect of export-specific cytoplasmic chaperone, protein SecB, on secretion of Escherichia coli alkaline phosphatase

The efficiency of secretion of Escherichia coli alkaline phosphatase depends on the presence in cells of a cytoplasmic chaperone—protein SecB. Secretion increases in the presence of this chaperone at 30°C, which is the most favorable for the interaction of SecB with the export-initiation domain found previously in the N-terminal region of the mature enzyme. This interaction most likely occurs in the region of the export domain, which is located close to the signal peptide and in complex with a translocational ATPase—protein SecA.