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At least 19 recordsLinked to original sources
PubMed1976

Leakage of periplasmic enzymes from lipopolysaccharide-defective mutants of Salmonella typhimurium.

Mutants of Salmonella typhimurium with defects in the heptose region of the lipopolysaccharide (LPS) molecule (heptose-deficient, chemotype Re) leak periplasmic enzymes (acid phosphatase (EC 3.1.3.2), cyclic phosphodiesterase, ribonuclease I (EC 3.1.4.22), and phosphoglucose isomerase (EC 5.3.1.9) (PGI is at least partially periplasmic in E. coli and S. typhimurium; see below)) and do not leak an internal enzyme (glucose-6-phosphate dehydrogenase) into the growth medium. The extent of this leakage is markedly increased at higher temperature (42 degrees C). Leakage of periplasmic enzymes from the strains lacking units distal to heptose I in the LPS molecule (chemotype Rd2) occurs only at 42 degrees C, and not at 30 or 37 degrees C. The extent of leakage of these enzymes from smooth strain and mutants of other LPS chemotypes (Rc, Rd1) is not significant, and is not influenced by growth temperatures. The kinetics of leakage of periplasmic enzymes after shift to 42 degrees C in nutrient broth reveal an accelerated release into the medium from heptose-deficient strains of cyclic phosphodiesterase and ribonuclease I after 30 min at 42 degrees C, and phosphoglucose isomerase after 60 min at 42 degrees C; at 30 degrees C the rate of release of cyclic phosphodiesterase and ribonuclease I is relatively slower. After 60 min at 42 degrees C in nutrient broth, growth of these strains has either slowed down or stopped. In L-broth, which permits the growth of the heptose-deficient strain (SA1377) at 42 degrees C, leakage of cyclic phosphodiesterase and phosphoglucose isomerase occurs, whereas there is no detectable leakage of these enzymes from the isogenic smooth strain (SA1355). Thus, leakage of the periplasmic enzymes from the heptose-deficient strain occurs with or without growth. Mg2+ (0.75 mM), sodium chloride (50 mM), and sucrose (100 mM) in nutrient broth at 42 degrees C prevent the leakage of these enzymes. The shedding of LPS from the heptose-deficient as well as the smooth strains is enhanced by high temperature (42 degrees C), whereas considerable leakage of protein occurs only in the heptose-deficient strain at 42 degrees C and not in the smooth strain. The smooth and heptose-deficient strains are equally sensitive to osmotic shock although a significant proportion of acid phosphatase and cyclic phosphodiesterase activities from the heptose-deficient cells grown at 42 degrees C comes off in the Tris-NaCl wash step suggesting a rather loose attachment of these enzymes onto the cell surface.

Acid Phosphatase↗
PubMed1977

Periplasmic space in Salmonella typhimurium and Escherichia coli.

The volume of the periplasmic space in Escherichia coli and Salmonella typhimurium cells was measured. This space, in cells grown and collected under conditions routinely used in work with these bacteria, was shown to comprise from 20 to 40% of the total cell volume. Further studies were conducted to determine the osmotic relationships between the periplasm, the external milieu, and the cytoplasm. Results showed that there is a Donnan equilibrium between the periplasm and the extracellular fluid, and that the periplasm and cytoplasm are isoosmotic. In minimal salts medium, the osmotic strength of the cell interior was estimated to be approximately 300 mosM, with a net pressure of approximately 3.5 atm being applied to the cell wall. A corollary of these findings was that an electrical potential exists across the outer membrane. This potential was measured by determining the distributions of Na+ and Cl- between the periplasm and the cell exterior. The potential varied with the ionic strength of the medium; for cells in minimal salts medium it was approximately 30 mV, negative inside.

Acetylglucosamine↗
PubMed2025

Repair of oxidized methionine residues in the chaperone Spy maintains periplasmic proteostasis under chlorite stress in Escherichia coli.

The bacterial cell envelope is exposed to various stresses, including oxidative stress caused by different types of oxidants, such as reactive oxygen species (ROS) and reactive chlorine species (RCS). In Escherichia coli, the reduction of chlorate into chlorite, a toxic RCS compound, induces the expression of the MsrPQ system, which repairs periplasmic proteins oxidized at methionine residues (methionine sulfoxide, Met-O). In this study, using a proteomic-based approach, we show that chlorite stress also triggers the overproduction of the periplasmic molecular chaperone Spheroplast Protein Y (Spy). This response is mediated by the activation of the BaeSR two-component system. Furthermore, both in vivo and in vitro evidence reveal that Spy's susceptibility to oxidation is critical for its chaperone activity. We demonstrate that the MsrPQ repair system ensures Spy's functionality by reducing its Met-O, thereby safeguarding its role in periplasmic protein homeostasis. Overall, this work reveals Spy as a key target of chlorite-induced oxidative damage and underscores the essential role of MsrPQ in preserving periplasmic protein quality control.

Methionine↗
PubMed2025

Bacterial cell widening alters periplasmic size and activates envelope stress responses.

The Rcs signal transduction system is a phosphorelay responsible for sensing enterobacterial cell envelope stresses. In Escherichia coli, the Rcs system is required to survive treatment with A22 and mecillinam, antibiotics that perturb cell size. To test whether size changes are correlated with envelope damage and thereby sensed by the Rcs system, we tuned E. coli cell size via A22 treatment, mutations in the cell-shape determinant MreB, and mechanically confined growth. In all conditions, cell width was strongly correlated with Rcs activation, and RcsF, the outer-membrane-localized upstream component, was essential for responding to cell width changes. Several gene deletions that induce Rcs resulted in cells that were wider than wild-type. Cryo-electron microscopy revealed that the periplasm of a wide MreB mutant is ~3 nm thinner than in wild-type cells, bringing RcsF closer to the downstream, inner-membrane-localized components of the signaling cascade. Conversely, extending the RcsF linker region in wild-type cells by ~3 nm increased Rcs activity. Thus, we propose that the Rcs system responds to changes in cell width due to altered periplasmic thickness.

Periplasm↗
PubMed2025

Origin and Evolution of Bacterial Periplasmic Force Transducers.

In double-membraned bacteria, non-equilibrium processes that occur at the outer membrane are typically coupled to the chemiosmotically energized inner membrane. TolA and TonB are homologous proteins which energetically couple inner membrane motor proteins to the essential processes of outer membrane stabilization and substrate import, respectively. The evolutionary trajectories of these proteins have been difficult to elucidate due to low-sequence conservation, yet they are thought to transduce force similarly. Here, this problem was addressed using structural prediction approaches to identify and annotate force transduction operons to trace their distribution and evolutionary origins. In the process, we identify a novel outer membrane-tethering system and a previously unknown family of monomeric force transducers. This approach revealed putative tolA genes, and thus the core organizational principles of the tol-pal operon throughout diverse bacterial taxa. We discovered that the α-helical structure of the periplasm-spanning domain II of TolA previously thought its hallmark, is anomalous amongst most Tol-Pal systems. This structure is mainly prevalent in γ-proteobacteria, likely in adaptation to their lifestyle. Comparison of Tol-Pal and Ton system distribution suggests that TolA emerged from a TonB paralogue and co-emerged with Pal, the outer membrane-tethering lipoprotein that functionalizes the Tol-Pal system. We also determined that TolB, the Pal-mobilizing protein, likely emerged from a family of outer membrane proteins; and CpoB, a periplasmic factor that coordinates peptidoglycan remodeling with cell division, was originally a lipoprotein present in the ancestral Tol-Pal system. The extensive conservation of the Tol-Pal system throughout Gracilicutes highlights its significance in bacterial cell biology.

Evolution, Molecular↗
PubMed1977

The effect of translation and transcription inhibitors on the synthesis of periplasmic phosphatases in E. coli.

Previous studies by others have indicated that the synthesis of secreted enzymes is unusually sensitive to many translation inhibitors and resistant, for about 30 min, to rifampicin. We have studied the sensitivity of secreted (periplasmic) phosphatases to such inhibitors. Alkaline phosphatase synthesis is more sensitive than total protein synthesis to tetracyclin and spectinomycin, but not to sparsomycin, streptomycin, chloramphenicol, kasugamycin, blasticidin S or thiostrepton; it is slightly more resistant than total protein synthesis to the latter two antibiotics. Acid hexose-phosphatase was also preferentially sensitive to tetracyclin and spectinomycin and also to kasugamycin. beta-galactosidase was also included in the study, as an intracellular enzyme, and was found to be preferentially inhibited ("repressed"), sometimes transiently, by all eight translation inhibitors. This effect did not seem to be mediated through cyclic AMP or guanosine tetraphosphate; the "repression" was still evident in mutants with altered rho factor indicating that it may also not be related to artificial polarity. Synthesis of both periplasmic phosphatases was immediately inhibited by rifampicin. These results differ from those found in previous studies with other organisms and suggest a reappraisal of the usual interpretation of these phenomena.

Acid Phosphatase↗
PubMed1979

Use of gene fusion to study secretion of maltose-binding protein into Escherichia coli periplasm.

We have employed the technique of gene fusion to fuse the LacZ gene encoding the cytoplasmic enzyme beta-galactosidase with the malE gene encoding the periplasmic maltose binding protein (MBP). Strains were obtained which synthesize malE-lacZ hybrid proteins of various sizes. These proteins have, at their amino terminus, a portion of the MBP and at their carboxyl terminus, enzymatically active beta-galactosidase. When the hybrid protein includes only a small, amino-terminal portion of the MBP, the hybrid protein residues in the cytoplasm. When the hybrid protein contains enough of the MBP to include an intact MBP signal sequence, a significant portion of the hybrid protein is found in the cytoplasmic membrane, suggesting that secretion of the hybrid protein has been initiated. However, in no case is the hybrid protein secreted into the periplasm, even when the hybrid protein includes almost the entire MBP. In the latter case, the synthesis and attempted export of the hybrid protein interferes with the export of at least certain normal envelope proteins, which accumulate in the cell in their precursor forms, and the cell dies. These results suggest that a number of envelope proteins may be exported at a common site, and that there are only a limited number of such sites. Also, these results indicate that it is not sufficient to simply attach an amino-terminal signal sequence to a polypeptide to assure its export.

Amino Acid Sequence↗
PubMed1979

Haemophilus influenzae periplasmic protein which binds deoxyribonucleic acid: properties and possible participation in genetic transformation.

A protein which binds to either single-stranded or double-stranded deoxyribonucleic acid (DNA) but not to ribonucleic acid has been isolated by osmotic shock treatment of growing cells. This periplasmic protein differs from the principal intracellular binding protein in its greater thermolability and by the absence of salt-induced cooperativity in its interaction with single-stranded DNA. Certain mutant strains of Haemophilus influenzae defective in the DNA suptake steps of genetic transformation were found to be deficient in periplasmic DNA-binding protein, suggesting that this protein participates in the uptake of DNA in transformation.

Bacterial Proteins↗
PubMed1979

Escherichia coli pleiotropic mutant that reduces amounts of several periplasmic and outer membrane proteins.

We have isolated a mutant of Escherichia coli K-12 that is reduced from 6- to 10-fold in the amount of alkaline phosphatase found in the periplasmic space. The reduced synthesis is not due to effects at the level of transcription regulation of the phoA gene, the structural gene for the enzyme. In addition, the mutation (termed perA) responsible for this phenotype results in reduced amounts of possibly six or more other periplasmic proteins and at least three outer membrane proteins. One of the outer membrane proteins affected is protein IA (D. L. Diedrich, A. O. Summers, and C. A. Schnaitman, J. Bacteriol. 131:598-607, 1977). Although other possibilities exist, one explanation for the phenotype of the perA mutation is that it affects the cell's secretory apparatus.

Alkaline Phosphatase↗
PubMed1976

The release and characterization of some periplasm-located enzymes of Pseudomona aeruginosa.

Pseudomonas aeruginosa (ATCC 9027) releases four periplasm-located enzymes, i.e., ribonuclease (EC 3.1.4.22; EC 3.1.4.23), alkaline phosphatase (EC 3.1.3.1), cyclic-2', 3'-phosphodiesterase (EC 3.1.4.d), and 5'-nucleotidase (EC 3.1.3.5) into the medium during growth. Ribonuclease and alkaline phosphatase are classed as enzymes which are readily extracted by osmotic shock and spheroplast formation whereas cyclic-2',3'-phosphodiesterase and 5'-nucleotidase are classed as enzymes which are not readily extracted by these procedures. In view of the relative ease of extraction of the former enzymes it is suggested that the lattter enzymes, cyclic-2',3'-phosphodiesterase and 5'-nucleotidase, are bound and located in the periplasm in a manner different to ribonuclease and alkaline phosphatase.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1975

Purification and properties of glutamate binding protein from the periplasmic space of Escherichia coli K-12.

Glutamate binding protein released from the periplasmic space of Escherichia coli K-12 by lysozyme-EDTA treatment was purified to homogeneity and its physical and chemical properties were studied. It is a basic protein with a pI of 9.1. Its molecular weight, determined in an analytical ultracentrifuge, and by gel filtration on Sephadex G-100 and dodecylsulphate acrylamide is 29 700, 27 800 and 32 000, respectively. The KD value for glutamate was 6.7 - 10- minus 6 M. L-Aspartate, reduced glutathione, G-glutamate-gamma-benzylester and L-glutamate-gamma-ethylester competitively inhibited glutamate binding with K-i; values of 7.8 - 10- minus 5, 1.1 - 10- minus 5, 1.0 - 10- minus 5 and 1.0 - 10- minus 5 M, respectively. Spheroplasts retained 40% of glutamate transport as compared to intact cells. The glutamate binding activity of a glutamate-utilizing strain (CS7), was 1.6 times as high as that of the glutamate non-utilizing parent strain (CS101). Similarly, the glutamate binding activity of a temperature conditional glutamate-utilizing mutant (CS2-TC) was 1.9 times higher when grown at the permissive temperature (42 degrees C) than when grown at the restrictive temperature (30 degrees C).

Amino Acids↗
PubMed1975

Purification and properties of a periplasmic aminoendopeptidase from Escherichia coli.

A periplasmic aminoendopeptidase from Escherichia coli has been purified to hemogeneity. It is a monomer of molecular weight 45000 and containing one -- SH group that is necessary for catalytic activity. The study of its substrate specificity indicated that the enzyme has both aminopeptidase and endopeptidase activity. The pH optimum for L-alanine p-nitroanilide hydrolysis is between 7 and 7.5 and that for 125I-labeled casein proteolysis between 7.3 and 7.6. The activation energy for the hydrolysis of L-anine p-nitroanilide was calculated to be 5.3 kcal X mol-1 (22.2 kJ X mol-1).

Amino Acids↗
PubMed1977

Purification and properties of a periplasmic protein related to sn-glycerol-3-phosphate transport in Escherichia coli.

Protein GLPT, a periplasmic protein previously recognized as closely related to the active transport of sn-glycerol-3-phosphate in Escherichia coli was isolated by the cold osmotic shock procedure. It was purified by Sephadex chromatography and isoelectric focussing. The purified protein does not exhibit any detectable binding activity toward sn-glycerol-3-phosphate. It has no activity as a glycerol phosphatase nor as a glycerol kinase. Polyacrylamide gel electrophoresis in the presence of dodecylsulfate of the protein subsequent to treatment in urea, boiling in dodecylsulfate and crosslinking indicates that it occurs as an oligomeric protein composed of four identical subunits of 40 000 molecular weight. Membrane vesicles of wild-type strains that contain protein GLPT in whole cells loose it during vesicle preparation. However, they still exhibit high transport activity toward sn-glycerol-3-phosphate. Membrane vesicles prepared from glp T mutants that may or may not contain protein GLPT do not transport sn-glycerol-3-phospahte. We conclude from these results that protein GLPT does not participate in the energy-dependent active transport through the cytoplasmic membrane but could be involved in facilitating the diffusion of sn-glycerol-3-phosphate through the outer layers of E. coli.

Biological Transport↗
PubMed1979

Procaine, a local anesthetic interacting with the cell membrane, inhibits the processing of precursor forms of periplasmic proteins in Escherichia coli.

Treatment of Escherichia coli cells with procaine (0.55%, w/v) results in the accumulation of precursor in addition to mature forms of two periplasmic proteins, alkaline phosphatase and glutamine-binding protein. The precursor form of alkaline phosphatase has a higher molecular weight than the mature form by about 2600. An experimental technique is described to isolate and purify precursor forms of any presumably exported protein. After the membrane solubilization step in the presence of nonionic detergent, a peptidase is stimulated, resulting in partial cleavage of the precursors. The products of this cleavage have been identified as the mature protein and presumably the signal peptide in the case of alkaline phosphatase. The amino acid composition of this peptide, which is comprised of 25 residues, has been determined. Procaine (0.55%, w/v) causes an increase in molecular packing of lipid molecules in the membrane which might result in an alteration of membrane fluidity sufficient for selective inhibition of processing of precursors of exported proteins.

Alkaline Phosphatase↗
PubMed1977

Mutants of Escherichia coli "cryptic" for certain periplasmic enzymes: evidence for an alteration of the outer membrane.

Mutants in which the expression of periplasmic enzymes by whole cells is reduced (termed "cryptic") are also found to show greatly reduced uptake of labeled adenosine 5'-monophosphate (5'-AMP), providing a rapid assay for crypticity. The crypticity of 3'- and 5'-nucleotidase has been examined as a function of substrate concentration. The Km for 3'- or 5'-AMP increases in the cryptic mutants when whole cells are used as the enzyme source. The Vmax is not altered. Electrophoretic analysis of protein prepared from cell envelopes showed that three cryptic mutants have a polypeptide absent from the outer membrane and a relatively high proportion of a polypeptide in the inner membrane. Analysis of the molar ratios of constituent sugars of the lipopolysaccharides showed no differences between three cryptic mutants and the parent strain. One cryptic mutant (3--41), however, has altered sensitivity to phage T4. By selection for phage resistance, derivatives of the cryptic mutants that are deoxycholate sensitive have been obtained. These mutants are no longer cryptic. We suggest that cryptic mutants have an altered outer membrane, with decreased permeability to 3'- and 5'-AMP, as a result of an altered polypeptide.

Adenosine Monophosphate↗
PubMed1977

Resolution and purification of three periplasmic phosphatases of Salmonella typhimurium.

A survey of Salmonella typhimurium enzymes possessing phosphatase or phosphodiesterase activity was made using several different growth conditions. These studies revealed the presence of three major enzymes, all of which were subsequently purified: a cyclic 2' ,3'-nucleotide phosphodiesterase (EC 3.1.4.d), an acid hexose phosphatase (EC 3.1.3.2), and a nonspecific acid phosphatase (EC 3.1.3.2). A fourth enzyme hydrolyzed bis-(p-nitrophenyl)phosphate but none of the other substrates tested. No evidence was found for the existence of an alkaline phosphatase (EC 3.1.3.1) or a specific 5'-nucleotidase (EC 3.1.3.5) in S. typhimurium LT2. All three phosphatases could be measured efficiently in intact cells, which suggested a periplasmic location; however, they were not readily released by osmotic shock procedures. The nonspecific acid phosphatase, which was purified to apparent homogeneity, yielded a single polypeptide band on both sodium dodecyl sulfate and acidic urea gel electrophoretic systems.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1977

Purification and properties of the periplasmic glucose-binding protein of Pseudomonas aeruginosa.

A glucose-binding glycoprotein (GBP) from the periplasm of Pseudomonas aeruginosa was purified to homogeneity as judged by polyacrylamide gel electrophoresis, molecular sieve chromatography, and double-diffusion gel precipitation. It had an average molecular weight of 44,500 and an isoelectric point of 4.7. One mole of glucose was bound per mole of GBP with a dissociation constant of 0.35 muM. The binding of radioactive glucose by GBP was not significantly inhibited by 10-fold-higher concentrations of other carbohydrates; however, a number of related compounds were found to compete at 100-fold-higher concentrations. Amino acid analyses revealed predominant amounts of alanine, glutamate, and glycine and a low content of sulfur-containing amino acids. The carbohydrate moiety of GBP, comprising nearly 16% of the total weight, contained galactosamine, glucosamine, fucose, galactose, glucose, and mannose. A GBP-deficient mutant, strain MB723, was found to be defective in both membrane transport and glucose chemotaxis. Strain MB724, a revertant to GBP-positive phenotype, simultaneously recovered normal levels of both membrane functions.

Bacterial Proteins↗